An integrated target sequence and signal amplification assay, reverse transcriptase-PCR-enzyme-linked immunosorbent assay, to detect and characterize flaviviruses.

PubMed

Chang, G J; Trent, D W; Vorndam, A V; Vergne, E; Kinney, R M; Mitchell, C J

1994-02-01

We previously described a reverse transcriptase-PCR using flavivirus genus-conserved and virus species-specific amplimers (D. W. Trent and G. J. Chang, p. 355-371, in Y. Becker and C. Darai; ed., Frontiers of Virology, vol. 1, 1992). Target amplification was improved by redesigning the amplimers, and a sensitive enzyme-linked immunosorbent assay (ELISA) technique has been developed to detect amplified digoxigenin (DIG)-modified DNA. A single biotin motif and multiple DIG motifs were incorporated into each amplicon, which permitted amplicon capture by a biotin-streptavidin interaction and detection with DIG-specific antiserum in a colorimetric ELISA. We evaluated the utility of this assay for detecting St. Louis encephalitis (SLE) viral RNA in infected mosquitoes and dengue viral RNA in human serum specimens. The reverse transcriptase-PCR-ELISA was as sensitive as isolation of SLE virus by cell culture in detecting SLE viral RNA in infected mosquitoes. The test was 89% specific and 95 to 100% sensitive for identification of dengue viral RNA in serum specimens compared with isolation of virus by Aedes albopictus C6/36 cell culture and identification by the indirect immunofluorescence assay.

Isolation and characterization of a FLOWERING LOCUS T homolog from pineapple (Ananas comosus (L.) Merr).

PubMed

Lv, LingLing; Duan, Jun; Xie, JiangHui; Wei, ChangBin; Liu, YuGe; Liu, ShengHui; Sun, GuangMing

2012-09-01

FLOWERING LOCUS T (FT)-like genes are crucial regulators of flowering in angiosperms. A homolog of FT, designated as AcFT (GenBank ID: HQ343233), was isolated from pineapple cultivar Comte de Paris by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA sequence of AcFT is 915 bp in length and contains an ORF of 534 bp, which encodes a protein of 177 aa. Molecular weight was 19.9 kDa and isoelectric point was 6.96. The deduced protein sequence of AcFT was 84% and 82% identical to homologs encoded by CgFT in Cymbidium goeringii and OgFT in Oncidium Gower Ramsey respectively. Quantitative real-time PCR (qRT-PCR) analyses showed that the expression of AcFT was high in flesh and none in leaves. qRT-PCR analyses in different stages indicated that the expression of AcFT reached the highest level on 40 d after flower inducing, when the multiple fruit and floral organs were forming. The 35S::AcFT transgenic Arabidopsis plants flowered earlier and had more inflorescences or branches than wild type plants. Copyright © 2012 Elsevier B.V. All rights reserved.

Comparison of reverse transcriptase PCR, reverse transcriptase loop-mediated isothermal amplification, and culture-based assays for Salmonella detection from pork processing environments.

PubMed

Techathuvanan, Chayapa; Draughon, Frances Ann; D'Souza, Doris Helen

2011-02-01

Novel rapid Salmonella detection assays without the need for sophisticated equipment or labor remain in high demand. Real-time reverse transcriptase PCR (RT-PCR) assays, though rapid and sensitive, require expensive thermocyclers, while a novel RT loop-mediated isothermal amplification (RT-LAMP) method requires only a simple water bath. Our objective was to compare the detection sensitivity of Salmonella Typhimurium from the pork processing environment by RT-LAMP, RT-PCR, and culture-based assays. Carcass and surface swabs and carcass rinses were obtained from a local processing plant. Autoclaved carcass rinses (500 ml) were spiked with Salmonella Typhimurium and filtered. Filters were placed in stomacher bags containing tetrathionate broth (TTB) and analyzed with or without 10-h enrichment at 37 °C. Natural swabs were stomached with buffered peptone water, and natural carcass rinses were filtered, preenriched, and further enriched in TTB. Serially-diluted enriched samples were enumerated by spread plating on xylose lysine Tergitol 4 agar. RNA was extracted from 5 ml of enriched TTB with TRIzol. RT-LAMP assay using previously described invA primers was conducted at 62 °C for 90 min in a water bath with visual detection and by gel electrophoresis. SYBR Green I-based-real-time RT-PCR was carried out with invA primers followed by melt temperature analysis. The results of RT-LAMP detection for spiked carcass rinses were comparable to those of RT-PCR and cultural plating, with detection limits of 1 log CFU/ml, although they were obtained significantly faster, within 24 h including preenrichment and enrichment. RT-LAMP showed 4 of 12 rinse samples positive, while RT-PCR showed 1 of 12 rinse samples positive. For swabs, 6 of 27 samples positive by RT-LAMP and 5 of 27 by RT-PCR were obtained. This 1-day RT-LAMP assay shows promise for routine Salmonella screening by the pork industry. Copyright ©, International Association for Food Protection

Functional analysis of a viroid RNA motif mediating cell-to-cell movement in Nicotiana benthamiana.

PubMed

Jiang, Dongmei; Wang, Meng; Li, Shifang

2017-01-01

Cell-to-cell trafficking through different cellular layers is a key process for various RNAs including those of plant viruses and viroids, but the regulatory mechanisms involved are still not fully elucidated and good model systems are important. Here, we analyse the function of a simple RNA motif (termed 'loop19') in potato spindle tuber viroid (PSTVd) which is required for trafficking in Nicotiana benthamiana leaves. Northern blotting, reverse transcriptase PCR (RT-PCR) and in situ hybridization analyses demonstrated that unlike wild-type PSTVd, which was present in the nuclei in all cell types, the trafficking-defective loop19 mutants were visible only in the nuclei of upper epidermal and palisade mesophyll cells, which shows that PSTVd loop19 plays a role in mediating RNA trafficking from palisade to spongy mesophyll cells in N.benthamiana leaves. Our findings and approaches have broad implications for studying the RNA motifs mediating trafficking of RNAs across specific cellular boundaries in other biological systems.

[Difference of three standard curves of real-time reverse-transcriptase PCR in viable Vibrio parahaemolyticus quantification].

PubMed

Jin, Mengtong; Sun, Wenshuo; Li, Qin; Sun, Xiaohong; Pan, Yingjie; Zhao, Yong

2014-04-04

We evaluated the difference of three standard curves in quantifying viable Vibrio parahaemolyticus in samples by real-time reverse-transcriptase PCR (Real-time RT-PCR). The standard curve A was established by 10-fold diluted cDNA. The cDNA was reverse transcripted after RNA synthesized in vitro. The standard curve B and C were established by 10-fold diluted cDNA. The cDNA was synthesized after RNA isolated from Vibrio parahaemolyticus in pure cultures (10(8) CFU/mL) and shrimp samples (10(6) CFU/g) (Standard curve A and C were proposed for the first time). Three standard curves were performed to quantitatively detect V. parahaemolyticus in six samples, respectively (Two pure cultured V. parahaemolyticus samples, two artificially contaminated cooked Litopenaeus vannamei samples and two artificially contaminated Litopenaeus vannamei samples). Then we evaluated the quantitative results of standard curve and the plate counting results and then analysed the differences. The three standard curves all show a strong linear relationship between the fractional cycle number and V. parahaemolyticus concentration (R2 > 0.99); The quantitative results of Real-time PCR were significantly (p < 0.05) lower than the results of plate counting. The relative errors compared with the results of plate counting ranked standard curve A (30.0%) > standard curve C (18.8%) > standard curve B (6.9%); The average differences between standard curve A and standard curve B and C were - 2.25 Lg CFU/mL and - 0.75 Lg CFU/mL, respectively, and the mean relative errors were 48.2% and 15.9%, respectively; The average difference between standard curve B and C was among (1.47 -1.53) Lg CFU/mL and the average relative errors were among 19.0% - 23.8%. Standard curve B could be applied to Real-time RT-PCR when quantify the number of viable microorganisms in samples.

Reverse Transcriptase Activity in Mature Spermatozoa of Mouse

PubMed Central

Giordano, Roberto; Magnano, Anna Rosa; Zaccagnini, Germana; Pittoggi, Carmine; Moscufo, Nicola; Lorenzini, Rodolfo; Spadafora, Corrado

2000-01-01

We show here that a reverse transcriptase (RT) activity is present in murine epididymal spermatozoa. Sperm cells incubated with human poliovirus RNA can take up exogenous RNA molecules and internalize them in nuclei. Direct PCR amplification of DNA extracted from RNA-incubated spermatozoa indicate that poliovirus RNA is reverse-transcribed in cDNA fragments. PCR analysis of two-cell embryos shows that poliovirus RNA-challenged spermatozoa transfer retrotranscribed cDNA molecules into eggs during in vitro fertilization. Finally, RT molecules can be visualized on sperm nuclear scaffolds by immunogold electron microscopy. These results, therefore, reveal a novel metabolic function in spermatozoa, which may play a role during early embryonic development. PMID:10725323

Universal reverse-transcriptase real-time PCR for infectious hematopoietic necrosis virus (IHNV)

USGS Publications Warehouse

Purcell, Maureen K.; Thompson, Rachel L.; Garver, Kyle A.; Hawley, Laura M.; Batts, William N.; Sprague, Laura; Sampson, Corie; Winton, James R.

2013-01-01

Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonid fishes in North America, Europe and Asia and is reportable to the World Organization for Animal Health (OIE). Phylogenetic analysis has identified 5 major virus genogroups of IHNV worldwide, designated U, M, L, E and J; multiple subtypes also exist within those genogroups. Here, we report the development and validation of a universal IHNV reverse-transcriptase real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) gene. Properties of diagnostic sensitivity (DSe) and specificity (DSp) were defined using laboratory-challenged steelhead trout Oncorhynchus mykiss, and the new assay was compared to the OIE-accepted conventional PCR test and virus isolation in cell culture. The IHNV N gene RT-rPCR had 100% DSp and DSe and a higher estimated diagnostic odds ratio (DOR) than virus culture or conventional PCR. The RT-rPCR assay was highly repeatable within a laboratory and highly reproducible between laboratories. Field testing of the assay was conducted on a random sample of juvenile steelhead collected from a hatchery raceway experiencing an IHN epizootic. The RT-rPCR detected a greater number of positive samples than cell culture and there was 40% agreement between the 2 tests. Overall, the RT-rPCR assay was highly sensitive, specific, repeatable and reproducible and is suitable for use in a diagnostic setting.

Application of Reverse Transcriptase -PCR (RT-PCR) for rapid detection of viable Escherichia coli in drinking water samples.

PubMed

Molaee, Neda; Abtahi, Hamid; Ghannadzadeh, Mohammad Javad; Karimi, Masoude; Ghaznavi-Rad, Ehsanollah

2015-01-01

Polymerase chain reaction (PCR) is preferred to other methods for detecting Escherichia coli (E. coli) in water in terms of speed, accuracy and efficiency. False positive result is considered as the major disadvantages of PCR. For this reason, reverse transcriptase-polymerase chain reaction (RT-PCR) can be used to solve this problem. The aim of present study was to determine the efficiency of RT-PCR for rapid detection of viable Escherichia coli in drinking water samples and enhance its sensitivity through application of different filter membranes. Specific primers were designed for 16S rRNA and elongation Factor II genes. Different concentrations of bacteria were passed through FHLP and HAWP filters. Then, RT-PCR was performed using 16srRNA and EF -Tu primers. Contamination of 10 wells was determined by RT-PCR in Arak city. To evaluate RT-PCR efficiency, the results were compared with most probable number (MPN) method. RT-PCR is able to detect bacteria in different concentrations. Application of EF II primers reduced false positive results compared to 16S rRNA primers. The FHLP hydrophobic filters have higher ability to absorb bacteria compared with HAWB hydrophilic filters. So the use of hydrophobic filters will increase the sensitivity of RT-PCR. RT-PCR shows a higher sensitivity compared to conventional water contamination detection method. Unlike PCR, RT-PCR does not lead to false positive results. The use of EF-Tu primers can reduce the incidence of false positive results. Furthermore, hydrophobic filters have a higher ability to absorb bacteria compared to hydrophilic filters.

Nucleic Acid Research Group (NARG) 2009-2010 Study : Optimal Priming Strategies for cDNA Synthesis in Real-Time RT-qPCR

PubMed Central

Hunter, T.C.; Knudtson, K.L.; Nadella, V.; Sol-Church, K.; Taylor, W.L.; Tighe, S.; Yueng, A.T.; Chittur, S.

2010-01-01

r1-1 Real-time reverse transcriptase quantitative PCR (RT-qPCR) is a widely used technique for measuring transcript levels. Priming strategy and reverse transcriptase enzyme are key elements that affect sensitivity and variability of RT-qPCR and microarray results. Previously, the Nucleic Acid Research Group (NARG) had conducted preliminary studies within the group to examine the effects of priming strategy on generating cDNA for use with qPCR. This year's study was an open study in which the qPCR community was invited to participate. Participants received the RT primers and RNA template and were asked to perform the RT reaction using their preferred reaction conditions. Each participating laboratory was provided at least two RNA templates of varying quality. The RT products were returned to the NARG and all RT reactions were used in a qPCR reaction. The qPCR assays looked at three genes of varying abundance, b-actin (high copy), b-glucuronidase (medium copy) and TATA binding protein (low copy) as well as varying distance from the 3? end for each transcript. Results from participating laboratories will be evaluated to determine the impact of priming strategy, assay chemistry and experimental setup on the RT step. Additionally, we will address the impact of RNA integrity on cDNA synthesis.

Microwave or autoclave treatments destroy the infectivity of infectious bronchitis virus and avian pneumovirus but allow detection by reverse transcriptase-polymerase chain reaction.

PubMed

Elhafi, G; Naylor, C J; Savage, C E; Jones, R C

2004-06-01

A method is described for enabling safe transit of denatured virus samples for polymerase chain reaction (PCR) identification without the risk of unwanted viable viruses. Cotton swabs dipped in avian infectious bronchitis virus (IBV) or avian pneumovirus (APV) were allowed to dry. Newcastle disease virus and avian influenza viruses were used as controls. Autoclaving and microwave treatment for as little as 20 sec destroyed the infectivity of all four viruses. However, both IBV and APV could be detected by reverse transcriptase (RT)-PCR after autoclaving and as long as 5 min microwave treatment (Newcastle disease virus and avian influenza viruses were not tested). Double microwave treatment of IBV and APV with an interval of 2 to 7 days between was tested. After the second treatment, RT-PCR products were readily detected in all samples. Swabs from the tracheas and cloacas of chicks infected with IBV shown to contain infectious virus were microwaved. Swabs from both sources were positive by RT-PCR. Microwave treatment appears to be a satisfactory method of inactivating virus while preserving nucleic acid for PCR identification.

Validation of a real-time reverse transcriptase-PCR assay for the detection of H7 avian influenza virus

USGS Publications Warehouse

Pedersen, J.; Killian, M.L.; Hines, N.; Senne, D.; Panigrahy, B.; Ip, Hon S.; Spackman, Erica

2010-01-01

This report describes the validation of an avian influenza virus (AIV) H7 subtype-specific real-time reverse transcriptasePCR (rRT-PCR) assay developed at the Southeast Poultry Research Laboratory (SEPRL) for the detection of H7 AI in North and South American wild aquatic birds and poultry. The validation was a collaborative effort by the SEPRL and the National Veterinary Services Laboratories. The 2008 H7 rRT-PCR assay detects 101 50% embryo infectious doses per reaction, or 103104 copies of transcribed H7 RNA. Diagnostic sensitivity and specificity were estimated to be 97.5% and 82.4%, respectively; the assay was shown to be specific for H7 AI when tested with >270 wild birds and poultry viruses. Following validation, the 2008 H7 rRT-PCR procedure was adopted as an official U.S. Department of Agriculture procedure for the detection of H7 AIV. The 2008 H7 assay replaced the previously used (2002) assay, which does not detect H7 viruses currently circulating in wild birds in North and South America. ?? 2010 American Association of Avian Pathologists.

Reverse transcriptase polymerase chain reaction on fine needle aspirates for rapid detection of translocations in synovial sarcoma.

PubMed

Nilsson, G; Wang, M; Wejde, J; Kanter, L; Karlén, J; Tani, E; Kreicbergs, A; Larsson, O

1998-01-01

To evaluate the utilization of fine needle aspiration (FNA) biopsy to obtain material for reverse-transcriptase polymerase chain reaction (RT-PCR) in the detection of the t(X;18)(p11.2;q11.2) translocation in synovial sarcomas. We applied RT-PCR to detection of synovial sarcoma fusion gene transcripts on fine needle aspirates. Five clinical samples were first analyzed: one was a tumor previously diagnosed as malignant hemangiopericytoma, one was a poorly defined tumor, and three were suspected synovial sarcomas. FNA material was transferred directly to the RT-PCR reaction tube without RNA extraction. The t(X;18) translocation could be detected on the limited amount of material that FNA provides. In each of the cases studied the representivity of the tumor samples was confirmed microscopically. Our protocol permits analysis directly on representative samples without extraction of RNA. The results imply that RT-PCR offers reliable detection of sarcoma fusion gene transcripts on fine needle aspirates. The procedure, apart from being applicable to outpatients, is rapid and sensitive.

Effect of saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus in oral fluid by quantitative reverse transcriptase real-time PCR.

PubMed

Decorte, Inge; Van der Stede, Yves; Nauwynck, Hans; De Regge, Nick; Cay, Ann Brigitte

2013-08-01

This study evaluated the effect of extraction-amplification methods, storage temperature and saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus (PRRSV) RNA by quantitative reverse transcriptase real-time PCR (qRT-PCR) in porcine oral fluid. The diagnostic performance of different extraction-amplification methods was examined using a dilution series of oral fluid spiked with PRRSV. To determine RNA stability, porcine oral fluid, with or without commercially available saliva stabilisers, was spiked with PRRSV, stored at 4°C or room temperature and tested for the presence of PRRSV RNA by qRT-PCR. PRRSV RNA could be detected in oral fluid using all extraction-amplification combinations, but the limit of detection varied amongst different combinations. Storage temperature and saliva stabilisers had an effect on the stability of PRRSV RNA, which could only be detected for 7 days when PRRSV spiked oral fluid was kept at 4°C or stabilised at room temperature with a commercial mRNA stabiliser. Copyright © 2013 Elsevier Ltd. All rights reserved.

Improved serotype-specific dengue virus detection in Trinidad and Tobago using a multiplex, real-time RT-PCR.

PubMed

Waggoner, Jesse J; Sahadeo, Nikita S D; Brown, Arianne; Mohamed-Hadley, Alisha; Hadley, Dexter; Carrington, Leslie; Carrington, Christine V F; Pinsky, Benjamin A

2015-02-01

Dengue virus (DENV) transmission occurs throughout the Caribbean, though laboratory confirmation and epidemiologic surveillance are limited by the availability of serotype-specific molecular diagnostics. In this study, we show that a serotype-specific DENV multiplex, real-time reverse transcriptase-PCR (RT-PCR) detected DENV RNA in significantly more samples (82/182) than a reference hemi-nested RT-PCR (57/182; P=0.01). Copyright © 2015 Elsevier Inc. All rights reserved.

Detection of Anti-Hepatitis B Virus Drug Resistance Mutations Based on Multicolor Melting Curve Analysis.

PubMed

Mou, Yi; Athar, Muhammad Ammar; Wu, Yuzhen; Xu, Ye; Wu, Jianhua; Xu, Zhenxing; Hayder, Zulfiqar; Khan, Saeed; Idrees, Muhammad; Nasir, Muhammad Israr; Liao, Yiqun; Li, Qingge

2016-11-01

Detection of anti-hepatitis B virus (HBV) drug resistance mutations is critical for therapeutic decisions for chronic hepatitis B virus infection. We describe a real-time PCR-based assay using multicolor melting curve analysis (MMCA) that could accurately detect 24 HBV nucleotide mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The two-reaction assay had a limit of detection of 5 copies per reaction and could detect a minor mutant population (5% of the total population) with the reverse transcriptase M204V amino acid mutation in the presence of the major wild-type population when the overall concentration was 10 4 copies/μl. The assay could be finished within 3 h, and the cost of materials for each sample was less than $10. Clinical validation studies using three groups of samples from both nucleos(t)ide analog-treated and -untreated patients showed that the results for 99.3% (840/846) of the samples and 99.9% (8,454/8,460) of the amino acids were concordant with those of Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (PCR Sanger sequencing). HBV DNA in six samples with mixed infections consisting of minor mutant subpopulations was undetected by the PCR Sanger sequencing method but was detected by MMCA, and the results were confirmed by coamplification at a lower denaturation temperature-PCR Sanger sequencing. Among the treated patients, 48.6% (103/212) harbored viruses that displayed lamivudine monoresistance, adefovir monoresistance, entecavir resistance, or lamivudine and adefovir resistance. Among the untreated patients, the Chinese group had more mutation-containing samples than did the Pakistani group (3.3% versus 0.56%). Because of its accuracy, rapidness, wide-range coverage, and cost-effectiveness, the real-time PCR assay could be a robust tool for the detection if anti-HBV drug resistance mutations in resource-limited countries. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Evaluation of primer and probe mismatches in sensitivity of select RRT-PCR tests for avian influenza

USDA-ARS?s Scientific Manuscript database

The recent outbreak of pH1N1 in animals highlighted an imperfection of the matrix real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) that has become the primary screening test for avian and swine influenza viruses. Four mismatches in one primer resulted in an important loss of sens...

Comparison of Real-Time PCR, Reverse Transcriptase Real-Time PCR, Loop-Mediated Isothermal Amplification, and the FDA Conventional Microbiological Method for the Detection of Salmonella spp. in Produce ▿ †

PubMed Central

Zhang, Guodong; Brown, Eric W.; González-Escalona, Narjol

2011-01-01

Contamination of foods, especially produce, with Salmonella spp. is a major concern for public health. Several methods are available for the detection of Salmonella in produce, but their relative efficiency for detecting Salmonella in commonly consumed vegetables, often associated with outbreaks of food poisoning, needs to be confirmed. In this study, the effectiveness of three molecular methods for detection of Salmonella in six produce matrices was evaluated and compared to the FDA microbiological detection method. Samples of cilantro (coriander leaves), lettuce, parsley, spinach, tomato, and jalapeno pepper were inoculated with Salmonella serovars at two different levels (105 and <101 CFU/25 g of produce). The inoculated produce was assayed by the FDA Salmonella culture method (Bacteriological Analytical Manual) and by three molecular methods: quantitative real-time PCR (qPCR), quantitative reverse transcriptase real-time PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP). Comparable results were obtained by these four methods, which all detected as little as 2 CFU of Salmonella cells/25 g of produce. All control samples (not inoculated) were negative by the four methods. RT-qPCR detects only live Salmonella cells, obviating the danger of false-positive results from nonviable cells. False negatives (inhibition of either qPCR or RT-qPCR) were avoided by the use of either a DNA or an RNA amplification internal control (IAC). Compared to the conventional culture method, the qPCR, RT-qPCR, and LAMP assays allowed faster and equally accurate detection of Salmonella spp. in six high-risk produce commodities. PMID:21803916

Strand-specific real-time RT-PCR quantitation of Maize fine streak virus genomic and positive-sense RNAs using high temperature reverse transcription

USDA-ARS?s Scientific Manuscript database

Efforts to analyze the replicative RNA produced by Maize fine streak virus (MVSF) within maize tissue was complicated by the lack of specificity during cDNA generation using standard reverse transcriptase protocols. Real-time qRT-PCR using cDNA generated by priming with random hexamers does not dist...

Identification of sexually dimorphic gene expression in brain tissue of the fish Leporinus macrocephalus through mRNA differential display and real time PCR analyses.

PubMed

Alves-Costa, Fernanda A; Wasko, A P

2010-03-01

Differentially expressed genes in males and females of vertebrate species generally have been investigated in gonads and, to a lesser extent, in other tissues. Therefore, we attempted to identify sexually dimorphic gene expression in the brains of adult males and females of Leporinus macrocephalus, a gonochoristic fish species that presents a ZZ/ZW sex determination system, throughout a comparative analysis using differential display reverse transcriptase-PCR and real-time PCR. Four cDNA fragments were characterized, representing candidate genes with differential expression between the samples. Two of these fragments presented no significant identity with previously reported gene sequences. The other two fragments, isolated from male specimens, were associated to the gene that codes for the protein APBA2 (amyloid beta (A4) precursor protein-binding, family A, member 2) and to the Rab 37 gene, a member of the Ras oncogene family. The overexpression of these genes has been associated to a greater production of the beta-amyloid protein which, in turns, is the major factor that leads to Alzheimer's disease, and to the development of brain-tumors, respectively. Quantitative RT-PCR analyses revealed a higher Apba2 gene expression in males, thus validating the previous data on differential display. L. macrocephalus may represent an interesting animal model to the understanding of the function of several vertebrate genes, including those involved in neurodegenerative and cancer diseases.

Analysis of heterogeneity of Copia-like retrotransposons in the genome of cassava (Manihot esculenta Crantz).

PubMed

Gbadegesin, Micheal A; Beeching, John R

2011-12-20

Retrotransposons are ubiquitous in eukaryotic genomes and now proving to be useful genetic tools for genetic diversity and phylogenetic analyses, especially in plants. In order to assess the diversity of Ty1/Copia-like retrotransposons of cassava, we used PCR primers anchored on the conserved domains of reverse transcriptases (RTs) to amplify cassava Ty1/Copia-like RT. The PCR product was cloned and sequenced. Sequences analysis of the clones revealed the presence of 69 families of Ty1/Copia-like retrotransposon in the genome of cassava. Comparative analyses of the predicted amino acid sequences of these clones with those of other plants showed that retroelements of this class are very heterogeneous in cassava. Cassava is widely grown for its edible roots in the tropical and subtropical regions of the world. Cassava roots, though poor in protein, are rich in starch (makes up about 80% of the dry matter), vitamin C, carotenes, calcium and potassium. It has a great commercial importance as a source of starch and starch based products. Realizing the importance of cassava, it stands out as a crop to benefit from biotechnology development. Heterogeneity of Mecops (Manihot esculenta copia-like Retrotransposons) showed that they may be useful for genetic diversity and phylogenetic analyses of cassava germplasm.

Validation of housekeeping genes as an internal control for gene expression studies in Giardia lamblia using quantitative real-time PCR.

PubMed

Marcial-Quino, Jaime; Fierro, Francisco; De la Mora-De la Mora, Ignacio; Enríquez-Flores, Sergio; Gómez-Manzo, Saúl; Vanoye-Carlo, America; Garcia-Torres, Itzhel; Sierra-Palacios, Edgar; Reyes-Vivas, Horacio

2016-04-25

The analysis of transcript levels of specific genes is important for understanding transcriptional regulation and for the characterization of gene function. Real-time quantitative reverse transcriptase PCR (RT-qPCR) has become a powerful tool to quantify gene expression. The objective of this study was to identify reliable housekeeping genes in Giardia lamblia. Twelve genes were selected for this purpose, and their expression was analyzed in the wild type WB strain and in two strains with resistance to nitazoxanide (NTZ) and metronidazole (MTZ), respectively. RefFinder software analysis showed that the expression of the genes is different in the three strains. The integrated data from the four analyses showed that the NADH oxidase (NADH) and aldolase (ALD) genes were the most steadily expressed genes, whereas the glyceraldehyde-3-phosphate dehydrogenase gene was the most unstable. Additionally, the relative expression of seven genes were quantified in the NTZ- and MTZ-resistant strains by RT-qPCR, using the aldolase gene as the internal control, and the results showed a consistent differential pattern of expression in both strains. The housekeeping genes found in this work will facilitate the analysis of mRNA expression levels of other genes of interest in G. lamblia. Copyright © 2016 Elsevier B.V. All rights reserved.

Regulation of Glycan Structures in Animal Tissues

PubMed Central

Nairn, Alison V.; York, William S.; Harris, Kyle; Hall, Erica M.; Pierce, J. Michael; Moremen, Kelley W.

2008-01-01

Glycan structures covalently attached to proteins and lipids play numerous roles in mammalian cells, including protein folding, targeting, recognition, and adhesion at the molecular or cellular level. Regulating the abundance of glycan structures on cellular glycoproteins and glycolipids is a complex process that depends on numerous factors. Most models for glycan regulation hypothesize that transcriptional control of the enzymes involved in glycan synthesis, modification, and catabolism determines glycan abundance and diversity. However, few broad-based studies have examined correlations between glycan structures and transcripts encoding the relevant biosynthetic and catabolic enzymes. Low transcript abundance for many glycan-related genes has hampered broad-based transcript profiling for comparison with glycan structural data. In an effort to facilitate comparison with glycan structural data and to identify the molecular basis of alterations in glycan structures, we have developed a medium-throughput quantitative real time reverse transcriptase-PCR platform for the analysis of transcripts encoding glycan-related enzymes and proteins in mouse tissues and cells. The method employs a comprehensive list of >700 genes, including enzymes involved in sugar-nucleotide biosynthesis, transporters, glycan extension, modification, recognition, catabolism, and numerous glycosylated core proteins. Comparison with parallel microarray analyses indicates a significantly greater sensitivity and dynamic range for our quantitative real time reverse transcriptase-PCR approach, particularly for the numerous low abundance glycan-related enzymes. Mapping of the genes and transcript levels to their respective biosynthetic pathway steps allowed a comparison with glycan structural data and provides support for a model where many, but not all, changes in glycan abundance result from alterations in transcript expression of corresponding biosynthetic enzymes. PMID:18411279

Use of a novel virus inactivation method for a multicenter avian influenza real-time reverse transcriptase-polymerase chain reaction proficiency study.

PubMed

Spackman, Erica; Suarez, David L

2005-01-01

Proficiency assessments are important elements in quality control for diagnostic laboratories. Traditionally, proficiency testing for polymerase chain reaction (PCR)-based assays has involved the use of clinical samples, samples "spiked" with live agents or DNA plasmids. Because of government regulations and biosecurity concerns, distribution of live high-consequence pathogens of livestock and poultry, such as avian influenza, is not possible, and DNA plasmids are not technically suitable for evaluating RNA virus detection. Therefore, a proficiency testing panel using whole avian influenza in a diluent containing a phenolic disinfectant that inactivates the virus while preserving the RNA for at least 8 weeks at -70 C was developed and used in a multicenter proficiency assessment for a type A influenza real-time reverse transcriptase (RT)-PCR test. The test, which was highly standardized, except for variation in the real-time RT-PCR equipment used, was shown to be highly reproducible by proficiency testing in 12 laboratories in the United States, Canada, and Hong Kong. Variation in cycle threshold values among 35 data sets and 490 samples was minimal (CV = 5.19%), and sample identifications were highly accurate (96.7% correct identifications) regardless of real-time PCR instrumentation.

Design and performance of the CDC real-time reverse transcriptase PCR swine flu panel for detection of 2009 A (H1N1) pandemic influenza virus.

PubMed

Shu, Bo; Wu, Kai-Hui; Emery, Shannon; Villanueva, Julie; Johnson, Roy; Guthrie, Erica; Berman, LaShondra; Warnes, Christine; Barnes, Nathelia; Klimov, Alexander; Lindstrom, Stephen

2011-07-01

Swine influenza viruses (SIV) have been shown to sporadically infect humans and are infrequently identified by the Influenza Division of the Centers for Disease Control and Prevention (CDC) after being received as unsubtypeable influenza A virus samples. Real-time reverse transcriptase PCR (rRT-PCR) procedures for detection and characterization of North American lineage (N. Am) SIV were developed and implemented at CDC for rapid identification of specimens from cases of suspected infections with SIV. These procedures were utilized in April 2009 for detection of human cases of 2009 A (H1N1) pandemic (pdm) influenza virus infection. Based on genetic sequence data derived from the first two viruses investigated, the previously developed rRT-PCR procedures were optimized to create the CDC rRT-PCR Swine Flu Panel for detection of the 2009 A (H1N1) pdm influenza virus. The analytical sensitivity of the CDC rRT-PCR Swine Flu Panel was shown to be 5 copies of RNA per reaction and 10(-1.3 - -0.7) 50% infectious doses (ID(50)) per reaction for cultured viruses. Cross-reactivity was not observed when testing human clinical specimens or cultured viruses that were positive for human seasonal A (H1N1, H3N2) and B influenza viruses. The CDC rRT-PCR Swine Flu Panel was distributed to public health laboratories in the United States and internationally from April 2009 until June 2010. The CDC rRT-PCR Swine Flu Panel served as an effective tool for timely and specific detection of 2009 A (H1N1) pdm influenza viruses and facilitated subsequent public health response implementation.

Design and Performance of the CDC Real-Time Reverse Transcriptase PCR Swine Flu Panel for Detection of 2009 A (H1N1) Pandemic Influenza Virus▿†‡

PubMed Central

Shu, Bo; Wu, Kai-Hui; Emery, Shannon; Villanueva, Julie; Johnson, Roy; Guthrie, Erica; Berman, LaShondra; Warnes, Christine; Barnes, Nathelia; Klimov, Alexander; Lindstrom, Stephen

2011-01-01

Swine influenza viruses (SIV) have been shown to sporadically infect humans and are infrequently identified by the Influenza Division of the Centers for Disease Control and Prevention (CDC) after being received as unsubtypeable influenza A virus samples. Real-time reverse transcriptase PCR (rRT-PCR) procedures for detection and characterization of North American lineage (N. Am) SIV were developed and implemented at CDC for rapid identification of specimens from cases of suspected infections with SIV. These procedures were utilized in April 2009 for detection of human cases of 2009 A (H1N1) pandemic (pdm) influenza virus infection. Based on genetic sequence data derived from the first two viruses investigated, the previously developed rRT-PCR procedures were optimized to create the CDC rRT-PCR Swine Flu Panel for detection of the 2009 A (H1N1) pdm influenza virus. The analytical sensitivity of the CDC rRT-PCR Swine Flu Panel was shown to be 5 copies of RNA per reaction and 10−1.3∼−0.7 50% infectious doses (ID50) per reaction for cultured viruses. Cross-reactivity was not observed when testing human clinical specimens or cultured viruses that were positive for human seasonal A (H1N1, H3N2) and B influenza viruses. The CDC rRT-PCR Swine Flu Panel was distributed to public health laboratories in the United States and internationally from April 2009 until June 2010. The CDC rRT-PCR Swine Flu Panel served as an effective tool for timely and specific detection of 2009 A (H1N1) pdm influenza viruses and facilitated subsequent public health response implementation. PMID:21593260

Floral organ MADS-box genes in Cercidiphyllum japonicum (Cercidiphyllaceae): Implications for systematic evolution and bracts definition.

PubMed

Jin, Yupei; Wang, Yubing; Zhang, Dechun; Shen, Xiangling; Liu, Wen; Chen, Faju

2017-01-01

The dioecious relic Cercidiphyllum japonicum is one of two species of the sole genus Cercidiphyllum, with a tight inflorescence lacking an apparent perianth structure. In addition, its systematic place has been much debated and, so far researches have mainly focused on its morphology and chloroplast genes. In our investigation, we identified 10 floral organ identity genes, including four A-class, three B-class, two C-class and one D-class. Phylogenetic analyses showed that all ten genes are grouped with Saxifragales plants, which confirmed the phylogenetic place of C. japonicum. Expression patterns of those genes were examined by quantitative reverse transcriptase PCR, with some variations that did not completely coincide with the ABCDE model, suggesting some subfunctionalization. As well, our research supported the idea that thebract actually is perianth according to our morphological and molecular analyses in Cercidiphyllum japonicum.

Floral organ MADS-box genes in Cercidiphyllum japonicum (Cercidiphyllaceae): Implications for systematic evolution and bracts definition

PubMed Central

Zhang, Dechun; Shen, Xiangling; Chen, Faju

2017-01-01

The dioecious relic Cercidiphyllum japonicum is one of two species of the sole genus Cercidiphyllum, with a tight inflorescence lacking an apparent perianth structure. In addition, its systematic place has been much debated and, so far researches have mainly focused on its morphology and chloroplast genes. In our investigation, we identified 10 floral organ identity genes, including four A-class, three B-class, two C-class and one D-class. Phylogenetic analyses showed that all ten genes are grouped with Saxifragales plants, which confirmed the phylogenetic place of C. japonicum. Expression patterns of those genes were examined by quantitative reverse transcriptase PCR, with some variations that did not completely coincide with the ABCDE model, suggesting some subfunctionalization. As well, our research supported the idea that thebract actually is perianth according to our morphological and molecular analyses in Cercidiphyllum japonicum. PMID:28562649

Quantitative expression and localization of cysteine and aspartic proteases in human abdominal aortic aneurysms

PubMed Central

Lohoefer, Fabian; Reeps, Christian; Lipp, Christina; Rudelius, Martina; Haertl, Felix; Matevossian, Edouard; Zernecke, Alma; Eckstein, Hans-Henning; Pelisek, Jaroslav

2014-01-01

Cysteine and aspartic proteases possess high elastolytic activity and might contribute to the degradation of the abdominal aortic aneurysm (AAA) wall. The aim of this study was to analyze, in detail, the proteases (cathepsins B, D, K, L and S, and inhibitor cystatin C) found in human AAA and healthy aortic tissue samples. The vessel walls from AAA patients (n=36) and nonaneurysmal aortae (n=10) were retrieved using conventional surgical repair and autopsy methods. Serum samples from the same AAA patients and 10 healthy volunteers were also collected. Quantitative expression analyses were performed at the mRNA level using real-time reverse transcriptase-PCR (RT–PCR). Furthermore, analyses at the protein level included western blot and immunoprecipitation analyses. Cellular sources of cysteine/aspartic proteases and cystatin C were identified by immunohistochemistry (IHC). All cysteine/aspartic proteases and cystatin C were detected in the AAA and control samples. Using quantitative RT–PCR, a significant increase in expression was observed for cathepsins B (P=0.021) and L (P=0.018), compared with the controls. Cathepsin B and cystatin C were also detected in the serum of AAA patients. Using IHC, smooth muscle cells (SMCs) and macrophages were positive for all of the tested cathepsins, as well as cystatin C; in addition, the lymphocytes were mainly positive for cathepsin B, followed by cathepsins D and S. All cysteine/aspartic proteases analyzed in our study were detected in the AAA and healthy aorta. The highest expression was found in macrophages and SMCs. Consequently, cysteine/aspartic proteases might play a substantial role in AAA. PMID:24833013

Comparison of ELISA and RT-PCR for the detection of Prunus necrotic ring spot virus and prune dwarf virus in almond (Prunus dulcis).

PubMed

Mekuria, Genet; Ramesh, Sunita A; Alberts, Evita; Bertozzi, Terry; Wirthensohn, Michelle; Collins, Graham; Sedgley, Margaret

2003-12-01

A technique based on the reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to detect the presence of Prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) simultaneously in almond. This paper presents the results of a 3-year study comparing both enzyme-linked immunosorbent assay (ELISA) and RT-PCR for the detection of PNRSV and PDV using 175 almond leaf samples. Multiplex RT-PCR was found to be more sensitive than ELISA, especially when followed by nested PCR for the detection of PDV. The RT-PCR technique has the added advantage that plant material can be tested at any time throughout the growing season.

Consensus criteria for sensitive detection of minimal neuroblastoma cells in bone marrow, blood and stem cell preparations by immunocytology and QRT-PCR: recommendations by the International Neuroblastoma Risk Group Task Force

PubMed Central

Beiske, K; Burchill, S A; Cheung, I Y; Hiyama, E; Seeger, R C; Cohn, S L; Pearson, A D J; Matthay, K K

2009-01-01

Disseminating disease is a predictive and prognostic indicator of poor outcome in children with neuroblastoma. Its accurate and sensitive assessment can facilitate optimal treatment decisions. The International Neuroblastoma Risk Group (INRG) Task Force has defined standardised methods for the determination of minimal disease (MD) by immunocytology (IC) and quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) using disialoganglioside GD2 and tyrosine hydroxylase mRNA respectively. The INRG standard operating procedures (SOPs) define methods for collecting, processing and evaluating bone marrow (BM), peripheral blood (PB) and peripheral blood stem cell harvest by IC and QRT-PCR. Sampling PB and BM is recommended at diagnosis, before and after myeloablative therapy and at the end of treatment. Peripheral blood stem cell products should be analysed at the time of harvest. Performing MD detection according to INRG SOPs will enable laboratories throughout the world to compare their results and thus facilitate quality-controlled multi-centre prospective trials to assess the clinical significance of MD and minimal residual disease in heterogeneous patient groups. PMID:19401690

The use of quantitative real-time reverse transcriptase PCR for 5' and 3' portions of ALK transcripts to detect ALK rearrangements in lung cancers.

PubMed

Wang, Rui; Pan, Yunjian; Li, Chenguang; Hu, Haichuan; Zhang, Yang; Li, Hang; Luo, Xiaoyang; Zhang, Jie; Fang, Zhaoyuan; Li, Yuan; Shen, Lei; Ji, Hongbin; Garfield, David; Sun, Yihua; Chen, Haiquan

2012-09-01

Approximately 3% to 7% of non-small cell lung cancers (NSCLC) harbor an ALK fusion gene, thus defining a tumor group that may be responsive to targeted therapy. The breakpoint in ALK consistently occurs at exon 20 and EML4 or other fusion partners, thus driving a strong expression of ALK kinase domain and resulting in an unbalanced expression in 5' and 3' portions of ALK transcripts. We have developed a rapid and accurate method by simultaneously detecting the expression in 5' and 3' portions of ALK mRNA. Quantitative real-time reverse transcriptase PCR (qRT-PCR) was used to examine expression levels of the 5' and 3' portions of ALK transcripts in177 NSCLCs, in which EGFR, KRAS, HER2, and BRAF mutations were absent. If unbalanced ALK mRNA expression was seen, ALK rearrangement was assumed to exist. ALK FISH was used to confirm the accuracy of qRT-PCR. RT-PCR and 5' RACE coupling sequencing identified the fusion variants. Real-time RT-PCR showed excellent sensitivity and specificity (100% and 100%, respectively) for detection of ALK rearrangements in resected specimens. In addition, six novel ALK fusion variants were identified, including one KIF5B-ALK (E17;A20) and five EML4-ALK variants (E6a;A19, E6a/b ins 18;A20, E17b ins 39;A20, E10a/b, E13;A20, and E17 ins 65;A20). Real-time RT-PCR is a rapid and accurate method for diagnosing ALK-rearranged lung cancers. Coupling of 5' RACE to this method should further facilitate rapid identification of novel ALK fusion genes. ©2012 AACR.

High sensitive RNA detection by one-step RT-PCR using the genetically engineered variant of DNA polymerase with reverse transcriptase activity from hyperthermophilies.

PubMed

Okano, Hiroyuki; Baba, Misato; Kawato, Katsuhiro; Hidese, Ryota; Yanagihara, Itaru; Kojima, Kenji; Takita, Teisuke; Fujiwara, Shinsuke; Yasukawa, Kiyoshi

2018-03-01

One-step RT-PCR has not been widely used even though some thermostable DNA polymerases with reverse transcriptase (RT) activity were developed from bacterial and archaeal polymerases, which is owing to low cDNA synthesis activity from RNA. In the present study, we developed highly-sensitive one-step RT-PCR using the single variant of family A DNA polymerase with RT activity, K4pol L329A (L329A), from the hyperthermophilic bacterium Thermotoga petrophila K4 or the 16-tuple variant of family B DNA polymerase with RT activity, RTX, from the hyperthermophilic archaeon Thermococcus kodakarensis. Optimization of reaction condition revealed that the activities for cDNA synthesis and PCR of K4pol L329A and RTX were highly affected by the concentrations of MgCl 2 and Mn(OCOCH 3 ) 2 as well as those of K4pol L329A or RTX. Under the optimized condition, 300 copies/μl of target RNA in 10 μl reaction volumes were successfully detected by the one-step RT-PCR with K4pol L329A or RTX, which was almost equally sensitive enough compared with the current RT-PCR condition using retroviral RT and thermostable DNA polymerase. Considering that K4pol L329A and RTX are stable even at 90-100°C, our results suggest that the one-step RT-PCR with K4pol L329A or RTX is more advantageous than the current one. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Literature Reference for Influenza H5N1 (Emerging Infectious Diseases. 2005. 11(8): 1303–1305)

EPA Pesticide Factsheets

Procedures are described for analysis of clinical samples and may be adapted for assessment of solid, particulate, aerosol, liquid and water samples. This is a two-step, real-time reverse transcriptase-PCR multiplex assay.

Glycosylation-related genes in NS0 cells are insensitive to moderately elevated ammonium concentrations

PubMed Central

Brodsky, Arthur Nathan; Caldwell, Mary; Bae, Sooneon; Harcum, Sarah W.

2014-01-01

NS0 and Chinese hamster ovary (CHO) cell lines are used to produce recombinant proteins for human therapeutics; however, ammonium accumulation can negatively impact cell growth, recombinant protein production, and protein glycosylation. To improve product quality and decrease costs, the relationship between ammonium and protein glycosylation needs to be elucidated. While ammonium has been shown to adversely affect glycosylation-related gene expression in CHO cells, NS0 studies have not been performed. Therefore, this study sought to determine if glycosylation in NS0 cells were ammonium-sensitive at the gene expression level. Using a DNA microarray that contained mouse glycosylation-related and housekeeping genes, the of these genes was analysed in response to various culture conditions – elevated ammonium, elevated salt, and elevated ammonium with proline. Surprisingly, no significant differences in gene expression levels were observed between the control and these conditions. Further, the elevated ammonium cultures were analysed using real-time quantitative reverse transcriptase PCR (qRT-PCR) for key glycosylation genes, and the qRT-PCR results corroborated the DNA microarray results, demonstrating that NS0 cells are ammonium-insensitive at the gene expression level. Since NS0 are known to have elevated nucleotide sugar pools under ammonium stress, and none of the genes directly responsible for these metabolic pools were changed, consequently cellular control at the translational or substrate-level must be responsible for the universally observed decreased glycosylation quality under elevated ammonium. PMID:25062658

Molecular diagnosis of lyssaviruses and sequence comparison of Australian bat lyssavirus samples.

PubMed

Foord, A J; Heine, H G; Pritchard, L I; Lunt, R A; Newberry, K M; Rootes, C L; Boyle, D B

2006-07-01

To evaluate and implement molecular diagnostic tests for the detection of lyssaviruses in Australia. A published hemi-nested reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of all lyssavirus genotypes was modified to a fully nested RT-PCR format and compared with the original assay. TaqMan assays for the detection of Australian bat lyssavirus (ABLV) were compared with both the nested and hemi-nested RT-PCR assays. The sequences of RT-PCR products were determined to assess sequence variations of the target region (nucleocapsid gene) in samples of ABLV originating from different regions. The nested RT-PCR assay was highly analytically specific, and at least as analytically sensitive as the hemi-nested assay. The TaqMan assays were highly analytically specific and more analytically sensitive than either RT-PCR assay, with a detection level of approximately 10 genome equivalents per microl. Sequence of the first 544 nucleotides of the nucleocapsid protein coding sequence was obtained from all samples of ABLV received at Australian Animal Health Laboratory during the study period. The nested RT-PCR provided a means for molecular diagnosis of all tested genotypes of lyssavirus including classical rabies virus and Australian bat lyssavirus. The published TaqMan assay proved to be superior to the RT-PCR assays for the detection of ABLV in terms of analytical sensitivity. The TaqMan assay would also be faster and cross contamination is less likely. Nucleotide sequence analyses of samples of ABLV from a wide geographical range in Australia demonstrated the conserved nature of this region of the genome and therefore the suitability of this region for molecular diagnosis.

Disappearance of AML1-MTG8 transcript by reverse transcriptase polymerase chain reaction in a patient in remission of acute myeloid leukemia (M2) after low-dose cytosine arabinoside.

PubMed

Sawada, H; Serino, Y; Wake, A; Yamasaki, Y; Izumi, Y

1998-09-01

It is well-known that low dose cytosine arabinoside (LDAC) has activity in elderly patients with acute myeloid leukemia (AML). Several studies have shown that AML patients with t(8;21) in long term complete remission (CR) following intensive chemotherapy or allogeneic bone marrow transplantation (BMT) still have persistence of AML1-MTG8 transcripts by reverse transcriptase polymerase chain reaction (RT-PCR) method. We report here a patient who has no evidence of residual disease detectable by RT-PCR after LDAC. A 69-year-old patient did not obtain CR after two courses of intensive chemotherapy with behenoyl-ara-C, daunorubicin, 6-mercaptopurine and prednisolone. He received subcutaneous LDAC 10 mg every 12 h and granulocyte colony-stimulating factor (G-CSF) for 29 days and achieved CR. He continued on a 21 to 28-day course of LDAC without G-CSF every 2 or 3 months and has remained well and in CR for 5 years without chimeric AMLI-MTG8 transcript by RT-PCR. LDAC therapy seems to be effective in eradicating the leukemic clone as post-induction or maintenance therapy in this patient. This is the first case report of the disappearance of AML1-MTG8 transcript by RT-PCR in a patient with t(8;21) in long-term remission after LDAC.

Clinical comparison of branched DNA and reverse transcriptase-PCR and nucleic acid sequence-based amplification assay for the quantitation of circulating recombinant form_BC HIV-1 RNA in plasma.

PubMed

Pan, Pinliang; Tao, Xiaoxia; Zhang, Qi; Xing, Wenge; Sun, Xianguang; Pei, Lijian; Jiang, Yan

2007-12-01

To investigate the correlation between three viral load assays for circulating recombinant form (CRF)_BC. Recent studies in HIV-1 molecular epidemiology, reveals that CRF_BC is the dominant subtype of HIV-1 virus in mainland China, representing over 45% of the HIV-1 infected population. The performances of nucleic acid sequence-based amplification (NASBA), branched DNA (bDNA) and reverse transcriptase polymerase chain reaction (RT-PCR) were compared for the HIV-1 viral load detection and quantitation of CRF_BC in China. Sixteen HIV-1 positive and three HIV-1 negative samples were collected. Sequencing of the positive samples in the gp41 region was conducted. The HIV-1 viral load values were determined using bDNA, RT-PCR and NASBA assays. Deming regression analysis with SPSS 12.0 (SPS Inc., Chicago, Illinois, USA) was performed for data analysis. Sequencing and phylogenetic analysis of env gene (gp41) region of the 16 HIV-1 positive clinical specimens from Guizhou Province in southwest China revealed the dominance of the subtype CRF_BC in that region. A good correlation of their viral load values was observed among three assays. Pearson's correlation between RT-PCR and bDNA is 0.969, Lg(VL)RT-PCR = 0.969 * Lg(VL)bDNA + 0.55; Pearson's correlation between RT-PCR and NASBA is 0.968, Lg(VL)RT-PCR = 0.968 * Lg(VL)NASBA + 0.937; Pearson's correlation between NASBA and bDNA is 0.980, Lg(VL)NASBA = 0.980 * Lg(VL)bDNA - 0.318. When testing with 3 different assays, RT-PCR, bDNA and NASBA, the group of 16 HIV-1 positive samples showed the viral load value was highest for RT-PCR, followed by bDNA then NASBA, which is consistent with the former results in subtype B. The three viral load assays are highly correlative for CRF_BC in China.

Molecular Epidemiology of Norovirus Outbreaks in Norway during 2000 to 2005 and Comparison of Four Norovirus Real-Time Reverse Transcriptase PCR Assays

PubMed Central

Vainio, Kirsti; Myrmel, Mette

2006-01-01

During the period from January 2000 to August 2005 a total of 204 outbreaks of norovirus gastroenteritis were diagnosed at the Norwegian Institute of Public Health. A clear increase in the norovirus activity was seen in healthcare institutions during the winter seasons. Polymerase sequence analysis of norovirus strains from 122 outbreaks showed that 112 were caused by GII strains (91.8%). Two norovirus variants seen during the study period—GIIb and GII.4—were predominant between January 2000 and September 2002, whereas GII.4 was predominant from September 2002 onward. The highest norovirus activity was seen during the 2002-2003 and 2004-2005 seasons with the emergence of new GII.4 variants. This study describes the molecular epidemiology of norovirus strains circulating in Norway during the five previous seasons and compares four norovirus real-time reverse transcriptase PCR assays. A suitable assay for routine diagnostics is suggested. PMID:17021099

Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay.

PubMed

Cronin, Maureen; Pho, Mylan; Dutta, Debjani; Stephans, James C; Shak, Steven; Kiefer, Michael C; Esteban, Jose M; Baker, Joffre B

2004-01-01

Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10- micro m FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests.

Evaluation of Cytokine Synthesis in Human Whole Blood by Enzyme Linked Immunoassay (ELISA), Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), and Flow Cytometry

DTIC Science & Technology

2007-05-08

deoxynucleotide triphosphates, from Sigma. Sequences for glyceraldehyde-3-phosphate dehydrogenase ( G3PDH ), IL-8,and TNF-a were amplified with primer...This was accomplished by normalizing all samples to the mRNA for the moderately expressed housekeeping function glyceraldehyde-3 -phosphate...without and with isolation of cells before reverse transcription and PCR. G3PDH mRNA target amplifies at 983 base pairs. The 630 base pair band is the

What assay is optimal for the diagnosis of measles virus infection? An evaluation of the performance of a measles virus real-time reverse transcriptase PCR using the Cepheid SmartCycler(®) and antigen detection by immunofluorescence.

PubMed

Chua, Kyra Y L; Thapa, Kiran; Yapa, Chaturangi M; Somerville, Lucy K; Chen, Sharon C-A; Dwyer, Dominic E; Sheppeard, Vicky; Kok, Jen

2015-09-01

Despite the World Health Organization (WHO)-reported elimination of measles in Australia, importation of cases especially in travellers from Asia continues in Sydney, Australia's largest city. Laboratory confirmation supports clinico-epidemiological evidence of measles virus infection, and is needed to establish elimination. To evaluate the performance of a random access real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay using the moderate complexity SmartCycler(®) platform, and measles antigen detection by immunofluorescence (IFA), for the detection of measles virus in patient samples. One hundred samples comprising nose and throat swabs, nasopharyngeal aspirates and urine, collected from patients with suspected measles were tested in parallel using IFA and nucleic acid testing using the SmartCycler(®) and LightCycler(®) RT-PCR platforms. The LightCycler(®) RT-PCR was used as the reference assay against which the SmartCycler(®) RT-PCR and IFA were compared. Using the LightCycler(®) RT-PCR, measles virus was detected in 35 clinical samples. There was 100% concordance between the results of the SmartCycler(®) and the LightCycler(®)-based RT-PCR. Measles genotypes detected included B3, D8, and D9. Testing urine in addition to NTS did not improve diagnostic yield. In contrast, the sensitivity and specificity of IFA compared to the reference LightCycler(®) RT-PCR was 34.3% and 96.7%, respectively. The performance of the SmartCycler(®) is comparable to the LightCycler(®) for the detection of measles virus. However, IFA had poor sensitivity and should not be used to confirm measles virus infection where nucleic acid testing is available. Copyright © 2015 Elsevier B.V. All rights reserved.

Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis.

PubMed

Enyeart, Peter J; Mohr, Georg; Ellington, Andrew D; Lambowitz, Alan M

2014-01-13

Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into 'targetrons.' Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and 'cut-and-pastes' (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The high processivity and fidelity of group II intron reverse transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology.

Susceptibility Testing by Polymerase Chain Reaction DNA Quantitation: A Method to Measure Drug Resistance of Human Immunodeficiency Virus Type 1 Isolates

NASA Astrophysics Data System (ADS)

Eron, Joseph J.; Gorczyca, Paul; Kaplan, Joan C.; D'Aquila, Richard T.

1992-04-01

Polymerase chain reaction (PCR) DNA quantitation (PDQ) susceptibility testing rapidly and directly measures nucleoside sensitivity of human immunodeficiency virus type 1 (HIV-1) isolates. PCR is used to quantitate the amount of HIV-1 DNA synthesized after in vitro infection of peripheral blood mononuclear cells. The relative amounts of HIV-1 DNA in cell lysates from cultures maintained at different drug concentrations reflect drug inhibition of virus replication. The results of PDQ susceptibility testing of 2- or 3-day cultures are supported by assays measuring HIV-1 p24 antigen production in supernatants of 7- or 10-day cultures. DNA sequence analyses to identify mutations in the reverse transcriptase gene that cause resistance to 3'-azido-3'-deoxythymidine also support the PDQ results. With the PDQ method, both infectivity titration and susceptibility testing can be performed on supernatants from primary cultures of peripheral blood mononuclear cells. PDQ susceptibility testing should facilitate epidemiologic studies of the clinical significance of drug-resistant HIV-1 isolates.

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction.

PubMed

Patel, Palak G; Selvarajah, Shamini; Boursalie, Suzanne; How, Nathan E; Ejdelman, Joshua; Guerard, Karl-Philippe; Bartlett, John M; Lapointe, Jacques; Park, Paul C; Okello, John B A; Berman, David M

2016-08-21

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

First report of Cocksfoot mottle virus infecting wheat (Triticum aestivum) in Ohio

USDA-ARS?s Scientific Manuscript database

Cocksfoot mottle virus (CfMV) was discovered in Ohio wheat during a 2016 field survey utilizing RNA-Seq to identify virus-like sequences. Virus sequences were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Sanger sequencing, and CfMV was transmitted to orchardgrass and pas...

RNA Extraction Methods for Reverse Transcriptase Real-Time PCR and Microarray Analysis of Cryptosporidium and Toxoplasma gondii Oocysts

EPA Science Inventory

The ability of infectious oocyst forms of Toxoplasma gondii and Cryptosporidium spp. to resist disinfection treatments and cause disease may have significant public health implications. Currently, little is known about oocyst-specific factors involved during host cell invasion p...

In-vitro Cell Culture and Real-time Reverse Transcriptase PCR-based Assays to Detect Infective Toxoplas gondii Oocysts

EPA Science Inventory

Toxoplasma gondii is an obligate intracellular, apicomplexan parasite that infects humans. It is ubiquitous in nature and seroprevalence in the United States and in Europe ranges from 25->70%. Although typically associated with causing foodborne outbreaks, recent studies in Canad...

Enzyme engineering through evolution: thermostable recombinant group II intron reverse transcriptases provide new tools for RNA research and biotechnology.

PubMed

Collins, Kathleen; Nilsen, Timothy W

2013-08-01

Current investigation of RNA transcriptomes relies heavily on the use of retroviral reverse transcriptases. It is well known that these enzymes have many limitations because of their intrinsic properties. This commentary highlights the recent biochemical characterization of a new family of reverse transcriptases, those encoded by group II intron retrohoming elements. The novel properties of these enzymes endow them with the potential to revolutionize how we approach RNA analyses.

Investigation of PAX3/7-FKHR fusion genes and IGF2 gene expression in rhabdomyosarcoma tumors.

PubMed

de Souza, Robson Ramos; Oliveira, Indhira Dias; Caran, Eliana Maria Monteiro; Alves, Maria Teresa de Seixas; Abib, Simone; Toledo, Silvia Regina Caminada

2012-12-01

The purpose of our study was to investigate the prevalence of the PAX3/7-FKHR fusion genes and quantify the IGF2 gene expression in rhabdomyosarcoma (RMS) samples. Soft tissue sarcomas account 5% of childhood cancers and 50% of them are RMS. Morphological evaluation of pediatric RMS has defined two histological subtypes, embryonal (ERMS) and alveolar (ARMS). Chromosomal analyses have demonstrated two translocations associated with ARMS, resulting in the PAX3/7-FKHR rearrangements. Reverse transcriptase-polymerase chain reaction (RT-PCR) is extremely useful in the diagnosis of ARMS positive for these rearrangements. Additionally, several studies have shown a significant involvement of IGF pathway in the pathogenesis of RMS. The presence of PAX3/7-FKHR gene fusions was studied in 25 RMS samples from patients attending the IOP-GRAACC/UNIFESP and three RMS cell lines by RT-PCR. IGF2 gene expression was quantified by qPCR and related with clinic pathological parameters. Of the 25 samples, nine (36%) were ARMS and 16 (64%) were ERMS. PAX3/7-FKHR gene fusions expression was detected in 56% of ARMS tumor samples. IGF2 overexpression was observed in 80% of samples and could indicate an important role of this pathway in RMS biology. Copyright © 2012 Elsevier Ltd. All rights reserved.

Touch-down reverse transcriptase-PCR detection of IgV(H) rearrangement and Sybr-Green-based real-time RT-PCR quantitation of minimal residual disease in patients with chronic lymphocytic leukemia.

PubMed

Peková, Sona; Marková, Jana; Pajer, Petr; Dvorák, Michal; Cetkovský, Petr; Schwarz, Jirí

2005-01-01

Patients with chronic lymphocytic leukemia (CLL) can relapse even after aggressive therapy and autografts. It is commonly assumed that to prevent relapse the level of minimal residual disease (MRD) should be as low as possible. To evaluate MRD, highly sensitive quantitative assays are needed. The aim of the study was to develop a robust and sensitive method for detection of the clonal immunoglobulin heavy-chain variable (IgV(H)) rearrangement in CLL and to introduce a highly sensitive and specific methodology for MRD monitoring in patients with CLL who undergo intensive treatment. As a prerequisite for MRD detection, touch-down reverse transcriptase (RT)-PCR using degenerate primers were used for the diagnostic identification of (H) gene rearrangement(s). For quantitative MRD detection in 18 patients, we employed a real-time RT-PCR assay (RQ-PCR) making use of patient-specific primers and the cost-saving Sybr-Green reporter dye (SG). For precise calibration of RQ-PCR, patient-specific IgV(H) sequences were cloned. Touch-down RT-PCR with degenerate primers allowed the successful detection of IgV(H) clonal rearrangement(s) in 252 of 257 (98.1%) diagnostic samples. Biallelic rearrangements were found in 27 of 252 (10.7%) cases. Degenerate primers used for the identification of clonal expansion at diagnosis were not sensitive enough for MRD detection. In contrast, our RQ-PCR assay using patient-specific primers and SG reached the sensitivity of 10(-)(6). We demonstrated MRD in each patient tested, including four of four patients in complete remission following autologous hematopoietic stem cell transplantation (HSCT) and three of three following allogeneic 'mini'-HSCT. Increments in MRD might herald relapse; aggressive chemotherapy could induce molecular remission. Our touch-down RT-PCR has higher efficiency to detect clonal IgV(H) rearrangements including the biallelic ones. MRD quantitation of IgV(H) expression using SG-based RQ-PCR represents a highly specific, sensitive, and economic alternative to the current quantitative methods.

Identification of Suitable Reference Genes for Gene Expression Normalization in qRT-PCR Analysis in Watermelon

PubMed Central

Gao, Lingyun; Zhao, Shuang; Jiang, Wei; Huang, Yuan; Bie, Zhilong

2014-01-01

Watermelon is one of the major Cucurbitaceae crops and the recent availability of genome sequence greatly facilitates the fundamental researches on it. Quantitative real-time reverse transcriptase PCR (qRT–PCR) is the preferred method for gene expression analyses, and using validated reference genes for normalization is crucial to ensure the accuracy of this method. However, a systematic validation of reference genes has not been conducted on watermelon. In this study, transcripts of 15 candidate reference genes were quantified in watermelon using qRT–PCR, and the stability of these genes was compared using geNorm and NormFinder. geNorm identified ClTUA and ClACT, ClEF1α and ClACT, and ClCAC and ClTUA as the best pairs of reference genes in watermelon organs and tissues under normal growth conditions, abiotic stress, and biotic stress, respectively. NormFinder identified ClYLS8, ClUBCP, and ClCAC as the best single reference genes under the above experimental conditions, respectively. ClYLS8 and ClPP2A were identified as the best reference genes across all samples. Two to nine reference genes were required for more reliable normalization depending on the experimental conditions. The widely used watermelon reference gene 18SrRNA was less stable than the other reference genes under the experimental conditions. Catalase family genes were identified in watermelon genome, and used to validate the reliability of the identified reference genes. ClCAT1and ClCAT2 were induced and upregulated in the first 24 h, whereas ClCAT3 was downregulated in the leaves under low temperature stress. However, the expression levels of these genes were significantly overestimated and misinterpreted when 18SrRNA was used as a reference gene. These results provide a good starting point for reference gene selection in qRT–PCR analyses involving watermelon. PMID:24587403

Identification of suitable reference genes for gene expression normalization in qRT-PCR analysis in watermelon.

PubMed

Kong, Qiusheng; Yuan, Jingxian; Gao, Lingyun; Zhao, Shuang; Jiang, Wei; Huang, Yuan; Bie, Zhilong

2014-01-01

Watermelon is one of the major Cucurbitaceae crops and the recent availability of genome sequence greatly facilitates the fundamental researches on it. Quantitative real-time reverse transcriptase PCR (qRT-PCR) is the preferred method for gene expression analyses, and using validated reference genes for normalization is crucial to ensure the accuracy of this method. However, a systematic validation of reference genes has not been conducted on watermelon. In this study, transcripts of 15 candidate reference genes were quantified in watermelon using qRT-PCR, and the stability of these genes was compared using geNorm and NormFinder. geNorm identified ClTUA and ClACT, ClEF1α and ClACT, and ClCAC and ClTUA as the best pairs of reference genes in watermelon organs and tissues under normal growth conditions, abiotic stress, and biotic stress, respectively. NormFinder identified ClYLS8, ClUBCP, and ClCAC as the best single reference genes under the above experimental conditions, respectively. ClYLS8 and ClPP2A were identified as the best reference genes across all samples. Two to nine reference genes were required for more reliable normalization depending on the experimental conditions. The widely used watermelon reference gene 18SrRNA was less stable than the other reference genes under the experimental conditions. Catalase family genes were identified in watermelon genome, and used to validate the reliability of the identified reference genes. ClCAT1and ClCAT2 were induced and upregulated in the first 24 h, whereas ClCAT3 was downregulated in the leaves under low temperature stress. However, the expression levels of these genes were significantly overestimated and misinterpreted when 18SrRNA was used as a reference gene. These results provide a good starting point for reference gene selection in qRT-PCR analyses involving watermelon.

Reference Gene Selection for Quantitative Real-Time Reverse-Transcriptase PCR in Annual Ryegrass (Lolium multiflorum) Subjected to Various Abiotic Stresses.

PubMed

Liu, Qiuxu; Qi, Xiao; Yan, Haidong; Huang, Linkai; Nie, Gang; Zhang, Xinquan

2018-01-16

To select the most stable reference genes in annual ryegrass ( Lolium multiflorum ), we studied annual ryegrass leaf tissues exposed to various abiotic stresses by qRT-PCR and selected 11 candidate reference genes, i.e., 18S rRNA, E2, GAPDH, eIF4A, HIS3, SAMDC, TBP-1, Unigene71, Unigene77, Unigene755, and Unigene14912. We then used GeNorm, NormFinder, and BestKeeper to analyze the expression stability of these 11 genes, and used RefFinder to comprehensively rank genes according to stability. Under different stress conditions, the most suitable reference genes for studies of leaf tissues of annual ryegrass were different. The expression of the eIF4A gene was the most stable under drought stress. Under saline-alkali stress, Unigene14912 has the highest expression stability. Under acidic aluminum stress, SAMDC expression stability was highest. Under heavy metal stress, Unigene71 expression had the highest stability. According to the software analyses, Unigene14912, HIS3, and eIF4A were the most suitable for analyses of abiotic stress in tissues of annual ryegrass. GAPDH was the least suitable reference gene. In conclusion, selecting appropriate reference genes under abiotic stress not only improves the accuracy of annual ryegrass gene expression analyses, but also provides a theoretical reference for the development of reference genes in plants of the genus Lolium .

Lack of detection of a putative retrovirus associated with haemic neoplasia in the soft shell clam Mya arenaria.

PubMed

AboElkhair, M; Iwamoto, T; Clark, K F; McKenna, P; Siah, A; Greenwood, S J; Berthe, F C J; Casey, J W; Cepica, A

2012-01-01

Haemic neoplasia (HN) is a leukemia-like disease that affects at least 20 species of marine bivalves including soft shell clam, Mya arenaria. Since the disease was discovered in 1969, the etiology remains unknown. A retroviral etiology has been suggested based on the detection of reverse transcriptase activity and electron microscopic observation of retroviral-like particles using negative staining. To date, however no virus isolate and no retroviral sequence from HN has been obtained. Moreover, transmission of the disease by cell-free filtrate from affected clams has not been reproduced. In the current study, we reinvestigated the association of HN with a putative retrovirus. Sucrose gradient centrifugation followed by assessment of reverse transcriptase activity, electrophoretic analysis of protein and RNA, and electron microscopic examinations of fractions corresponding to retroviral density were employed. Detection of retroviral pol sequences using degenerate RT-PCR approaches was also attempted. Our results showed visible bands at the expected density of retrovirus in HN-positive and HN-negative clam tissues and both with reverse transcriptase activity. Electron microscopy, RNA analysis, protein analysis, and PCR systems targeting the pol gene of retroviruses did not however provide clear evidence supporting presence of a retrovirus. We point out that the retrovirus etiology of HN of Mya arenaria proposed some 25 years ago should be reconsidered in the absence of a virus isolate or virus sequences. Copyright © 2011 Elsevier Inc. All rights reserved.

Cloning and Expression Analysis of a PISTILLATA Homologous Gene from Pineapple (Ananas comosus L. Merr)

PubMed Central

Lv, Ling-Ling; Duan, Jun; Xie, Jiang-Hui; Liu, Yu-Ge; Wei, Chang-Bin; Liu, Sheng-Hui; Zhang, Jian-Xia; Sun, Guang-Ming

2012-01-01

PISTILLATA (PI)-like genes are crucial regulators of flowering in angiosperms. A homologue of PI, designated as AcPI (Genbank accession number HQ717796), was isolated from pineapple cultivar Comte de Paris by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA sequence of AcPI is 907 bp in length and contains an open reading frame of 594 bp, which encodes a protein of 197 amino acids. The molecular weight was 2.29 kDa and the isoelectric point was 9.28. The alignment showed that AcPI had a high identity with CsPIC2 (78.6%), AoPI (77.4%), OrcPI (75.7%) and HPI2 (72.4%). Quantitative real-time polymerase chain reaction (qRT-PCR) analyses in different tissues showed that the expression pattern of AcPI was different from the B-class genes in eudicots. AcPI was expressed in all the tissues investigated. The expression level was very low in fruit stems, bracts, leaves and sepals, high in petals and carpels, and moderate in apical meristems, flesh and stamens. The qRT-PCR analyses in different stages indicated that the expression of AcPI reached the highest level at 40 days after flower inducement, when the multiple fruit and floral organs were forming. It proved the important role of AcPI in floral organs and fruit development. The 35S::AcPI transgenic Arabidopsis plants flowered earlier and had more inflorescences or branches than wild type plants. PMID:22312303

Cloning and expression analysis of a PISTILLATA homologous gene from pineapple (Ananas comosus L. Merr).

PubMed

Lv, Ling-Ling; Duan, Jun; Xie, Jiang-Hui; Liu, Yu-Ge; Wei, Chang-Bin; Liu, Sheng-Hui; Zhang, Jian-Xia; Sun, Guang-Ming

2012-01-01

PISTILLATA (PI)-like genes are crucial regulators of flowering in angiosperms. A homologue of PI, designated as AcPI (Genbank accession number HQ717796), was isolated from pineapple cultivar Comte de Paris by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA sequence of AcPI is 907 bp in length and contains an open reading frame of 594 bp, which encodes a protein of 197 amino acids. The molecular weight was 2.29 kDa and the isoelectric point was 9.28. The alignment showed that AcPI had a high identity with CsPIC2 (78.6%), AoPI (77.4%), OrcPI (75.7%) and HPI2 (72.4%). Quantitative real-time polymerase chain reaction (qRT-PCR) analyses in different tissues showed that the expression pattern of AcPI was different from the B-class genes in eudicots. AcPI was expressed in all the tissues investigated. The expression level was very low in fruit stems, bracts, leaves and sepals, high in petals and carpels, and moderate in apical meristems, flesh and stamens. The qRT-PCR analyses in different stages indicated that the expression of AcPI reached the highest level at 40 days after flower inducement, when the multiple fruit and floral organs were forming. It proved the important role of AcPI in floral organs and fruit development. The 35S::AcPI transgenic Arabidopsis plants flowered earlier and had more inflorescences or branches than wild type plants.

Analysis of plasma membrane Ca(2+)-ATPase expression in control and SV40-transformed human fibroblasts.

PubMed

Reisner, P D; Brandt, P C; Vanaman, T C

1997-01-01

It has been long known that neoplastic transformation is accompanied by a lowered requirement for extracellular Ca2+ for growth. The studies presented here demonstrate that human fibroblastic cell lines produce the two commonly found 'housekeeping' isoforms of the plasma membrane Ca(2+)-ATPase (PMCA), PMCA1b and 4b, and at the expression of both is demonstrably lower in cell lines neoplastically transformed by SV40 than in the corresponding parental cell lines. Western blot analyses of lysates from control (GM00037) and SV40-transformed (GM00637) skin fibroblasts revealed a 138 kDa PMCA whose level was significantly lower in the SV40-transformed cells relative to either total cellular protein or alpha-tubulin. Similar analyses of plasma membrane preparations from control WI-38) and SV40-transformed (WI-38VA13) lung fibroblasts revealed 3-4-fold lower levels of PMCA in the SV40-transformed cells. Competitive ELISAs performed on detergent solubilized plasma membrane preparations indicated at least 3-4-fold lower levels of PMCA in the SV40-transformed cell lines compared to controls. Reverse transcriptase coupled-PCR analyses showed that PMCA1b and PMCA4b were the only isoforms expressed in all four cell lines. The PMCA4b mRNA level detected by Northern analysis also was substantially lower in SV40 transformed skin fibroblasts than in non-transformed fibroblasts. Quantitative RT-PCR analyses showed levels of PMCA1b and 4b mRNAs to be 5 and 10-fold lower, respectively, in GM00637 than in GM00037 when the levels of PCR products were normalized to glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA. These results demonstrate that the expression of these distinct PMCA genes is substantially lower in SV40 transformed human skin and lung fibroblasts and may be coordinately regulated in these cells.

Prognostic role of a multigene reverse transcriptase-PCR assay in patients with node-negative breast cancer not receiving adjuvant systemic therapy.

PubMed

Esteva, Francisco J; Sahin, Aysegul A; Cristofanilli, Massimo; Coombes, Kevin; Lee, Sang-Joon; Baker, Joffre; Cronin, Maureen; Walker, Michael; Watson, Drew; Shak, Steven; Hortobagyi, Gabriel N

2005-05-01

To test the ability of a reverse transcriptase-PCR (RT-PCR) assay, based on gene expression profiles, to accurately determine the risk of recurrence in patients with node-negative breast cancer who did not receive systemic therapy using formalin-fixed, paraffin-embedded tissue. A secondary objective was to determine whether the quantitative RT-PCR data correlated with immunohistochemistry assay data regarding estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 status. We obtained archival paraffin-embedded tissue from patients with invasive breast cancer but no axillary lymph node involvement who had received no adjuvant systemic therapy and been followed for at least 5 years. RNA was extracted from three 10-microm-thick sections. The expression of 16 cancer-related genes and 5 reference genes was quantified using RT-PCR. A gene expression algorithm was used to calculate a recurrence score for each patient. We then assessed the ability of the test to accurately predict distant recurrence-free survival in this population. We identified 149 eligible patients. Median age at diagnosis was 59 years; mean tumor diameter was 2 cm; and 69% of tumors were estrogen receptor positive. Median follow-up was 18 years. The 5-year disease-free survival rate for the group was 80%. The 21 gene-based recurrence score was not predictive of distant disease recurrence. However, a high concordance between RT-PCR and immunohistochemical assays for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 status was noted. RT-PCR can be done on paraffin-embedded tissue to validate the large numbers of genes associated with breast cancer recurrence. However, further work needs to be done to develop an assay to identify the likelihood of recurrent disease in patients with node-negative breast cancer who do not receive adjuvant tamoxifen or chemotherapy.

Comparison of FilmArray and Quantitative Real-Time Reverse Transcriptase PCR for Detection of Zaire Ebolavirus from Contrived and Clinical Specimens

PubMed Central

Southern, Timothy R.; Racsa, Lori D.; Albariño, César G.; Fey, Paul D.; Hinrichs, Steven H.; Murphy, Caitlin N.; Herrera, Vicki L.; Sambol, Anthony R.; Hill, Charles E.; Ryan, Emily L.; Kraft, Colleen S.; Campbell, Shelley; Sealy, Tara K.; Schuh, Amy; Ritchie, James C.; Lyon, G. Marshall; Mehta, Aneesh K.; Varkey, Jay B.; Ribner, Bruce S.; Brantly, Kent P.; Ströher, Ute; Iwen, Peter C.

2015-01-01

Rapid, reliable, and easy-to-use diagnostic assays for detection of Zaire ebolavirus (ZEBOV) are urgently needed. The goal of this study was to examine the agreement among emergency use authorization (EUA) tests for the detection of ZEBOV nucleic acids, including the BioFire FilmArray BioThreat (BT) panel, the FilmArray BT-E panel, and the NP2 and VP40 quantitative real-time reverse transcriptase (qRT) PCR assays from the Centers for Disease Control and Prevention (CDC). Specimens used in this study included whole blood spiked with inactivated ZEBOV at known titers and whole-blood, plasma, and urine clinical specimens collected from persons diagnosed with Ebola virus disease (EVD). The agreement for FilmArray and qRT-PCR results using contrived whole-blood specimens was 100% (6/6 specimens) for each ZEBOV dilution from 4 × 107 to 4 × 102 50% tissue culture infective dose (TCID50)/ml, as well as the no-virus negative-control sample. The limit of detection for FilmArray and qRT-PCR assays with inactivated ZEBOV, based on duplicate positive results, was determined to be 4 × 102 TCID50/ml. Rates of agreement between FilmArray and qRT-PCR results for clinical specimens from patients with EVD were 85% (23/27 specimens) for whole-blood specimens, 90% (18/20 specimens) for whole-blood specimens tested by FilmArray testing and matched plasma specimens tested by qRT-PCR testing, and 85% (11/13 specimens) for urine specimens. Among 60 specimens, eight discordant results were noted, with ZEBOV nucleic acids being detected only by FilmArray testing in four specimens and only by qRT-PCR testing in the remaining four specimens. These findings demonstrate that the rapid and easy-to-use FilmArray panels are effective tests for evaluating patients with EVD. PMID:26157148

Increased yield of PCR products by addition of T4 gene 32 protein to the SMART PCR cDNA synthesis system.

PubMed

Villalva, C; Touriol, C; Seurat, P; Trempat, P; Delsol, G; Brousset, P

2001-07-01

Under certain conditions, T4 gene 32 protein is known to increase the efficiency of different enzymes, such as Taq DNA polymerase, reverse transcriptase, and telomerase. In this study, we compared the efficiency of the SMART PCR cDNA synthesis kit with and without the T4 gene 32 protein. The use of this cDNA synthesis procedure, in combination with T4 gene 32 protein, increases the yield of RT-PCR products from approximately 90% to 150%. This effect is even observed for long mRNA templates and low concentrations of total RNA (25 ng). Therefore, we suggest the addition of T4 gene 32 protein in the RT-PCR mixture to increase the efficiency of cDNA synthesis, particularly in cases when low amounts of tissue are used.

Development of Reverse Transcription Thermostable Helicase-Dependent DNA Amplification for the Detection of Tomato Spotted Wilt Virus.

PubMed

Wu, Xinghai; Chen, Chanfa; Xiao, Xizhi; Deng, Ming Jun

2016-11-01

A protocol for the reverse transcription-helicase-dependent amplification (RT-HDA) of isothermal DNA was developed for the detection of tomato spotted wilt virus (TSWV). Specific primers, which were based on the highly conserved region of the N gene sequence in TSWV, were used for the amplification of virus's RNA. The LOD of RT-HDA, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP), and reverse transcriptase-polymerase chain reaction (RT-PCR) assays were conducted using 10-fold serial dilution of RNA eluates. TSWV sensitivity in RT-HDA and RT-LAMP was 4 pg RNA compared with 40 pg RNA in RT-PCR. The specificity of RT-HDA for TSWV was high, showing no cross-reactivity with other tomato and Tospovirus viruses including cucumber mosaic virus (CMV), tomato black ring virus (TBRV), tomato mosaic virus (ToMV), or impatiens necrotic spot virus (INSV). The RT-HDA method is effective for the detection of TSWV in plant samples and is a potential tool for early and rapid detection of TSWV.

Detection of EML4-ALK fusion gene in Chinese non-small cell lung cancer by using a sensitive quantitative real-time reverse transcriptase PCR technique.

PubMed

Fu, Sha; Wang, Fang; Shao, Qiong; Zhang, Xu; Duan, Li-Ping; Zhang, Xiao; Zhang, Li; Shao, Jian-Yong

2015-04-01

Anaplastic lymphoma kinase (ALK) rearrangement is present in approximately 5% of lung adenocarcinoma. Clinical trials on ALK inhibitor phase I to III have shown an interesting disease control rate and acceptable tolerability in ALK rearrangement patients. In clinical application, the precise diagnostic strategy for identifying ALK rearrangements remains to be determined. In this study, ALK rearrangement was screened by using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), direct sequencing, 2 fluorescence in situ hybridization (FISH) assays, and immunohistochemistry in 173 lung adenocarcinomas. We identified 18 cases (10.4%) with EML4-ALK fusion-positive by qRT-PCR, and all were positive for EML4-ALK fusion gene validated by direct sequencing. The result was consistent with that of other methods. Furthermore, of the 18 EML4-ALK fusion-positive cases, 16 (9.2%) were positive by using EML4-ALK fusion probe FISH, and 15 (8.7%) were positive by using ALK break-apart probe FISH and immunohistochemistry staining. Of the 18 ALK fusion-positive lung adenocarcinomas, 8 cases (44.4%) were histologically diagnosed as subtypes of cribriform adenocarcinoma, 7 cases (38.9%) as cribriform adenocarcinoma mixed with papillary and/or mucinous pattern, 2 cases (11.1%) as papillary adenocarcinoma, and 1 case (5.6%) as mucinous adenocarcinoma. In the present study, the ALK rearrangement frequency detected by qRT-PCR in Chinese NSCLC patients was higher than that in the western populations. QRT-PCR is a rapid, sensitive technology that could be used as a screening tool for identifying EML4-ALK fusion-positive NSCLC patients who would be sensitive for receiving ALK inhibitor therapy.

Nipah virus in the fruit bat Pteropus vampyrus in Sumatera, Indonesia.

PubMed

Sendow, Indrawati; Ratnawati, Atik; Taylor, Trevor; Adjid, R M Abdul; Saepulloh, Muharam; Barr, Jennifer; Wong, Frank; Daniels, Peter; Field, Hume

2013-01-01

Nipah virus causes periodic livestock and human disease with high case fatality rate, and consequent major economic, social and psychological impacts. Fruit bats of the genus Pteropus are the natural reservoir. In this study, we used real time PCR to screen the saliva and urine of P. vampyrus from North Sumatera for Nipah virus genome. A conventional reverse transcriptase (RT-PCR) assay was used on provisionally positive samples to corroborate findings. This is the first report of Nipah virus detection in P. vampyrus in Sumatera, Indonesia.

fbpABC gene cluster in Neisseria meningitidis is transcribed as an operon.

PubMed

Khun, H H; Deved, V; Wong, H; Lee, B C

2000-12-01

The neisserial fbpABC locus has been proposed to constitute a single transcriptional unit. To confirm this operonic arrangement, transcription assays using reverse transcriptase PCR amplification were conducted with Neisseria meningitidis. The presence of fbpAB and fbpBC transcripts obtained by priming cDNA synthesis with an fbpC-sequence-specific oligonucleotide indicates that fbpABC is organized as a single expression unit. The ratio of fbpA to fbpABC mRNA was approximately between 10- to 20-fold, as determined by real-time quantitative PCR.

fbpABC Gene Cluster in Neisseria meningitidis Is Transcribed as an Operon

PubMed Central

Khun, Heng H.; Deved, Vinay; Wong, Howard; Lee, B. Craig

2000-01-01

The neisserial fbpABC locus has been proposed to constitute a single transcriptional unit. To confirm this operonic arrangement, transcription assays using reverse transcriptase PCR amplification were conducted with Neisseria meningitidis. The presence of fbpAB and fbpBC transcripts obtained by priming cDNA synthesis with an fbpC-sequence-specific oligonucleotide indicates that fbpABC is organized as a single expression unit. The ratio of fbpA to fbpABC mRNA was approximately between 10- to 20-fold, as determined by real-time quantitative PCR. PMID:11083849

Quantitative Assessment of the Sensitivity of Various Commercial Reverse Transcriptases Based on Armored HIV RNA

PubMed Central

Okello, John B. A.; Rodriguez, Linda; Poinar, Debi; Bos, Kirsten; Okwi, Andrew L.; Bimenya, Gabriel S.; Sewankambo, Nelson K.; Henry, Kenneth R.; Kuch, Melanie; Poinar, Hendrik N.

2010-01-01

Background The in-vitro reverse transcription of RNA to its complementary DNA, catalyzed by the enzyme reverse transcriptase, is the most fundamental step in the quantitative RNA detection in genomic studies. As such, this step should be as analytically sensitive, efficient and reproducible as possible, especially when dealing with degraded or low copy RNA samples. While there are many reverse transcriptases in the market, all claiming to be highly sensitive, there is need for a systematic independent comparison of their applicability in quantification of rare RNA transcripts or low copy RNA, such as those obtained from archival tissues. Methodology/Principal Findings We performed RT-qPCR to assess the sensitivity and reproducibility of 11 commercially available reverse transcriptases in cDNA synthesis from low copy number RNA levels. As target RNA, we used a serially known number of Armored HIV RNA molecules, and observed that 9 enzymes we tested were consistently sensitive to ∼1,000 copies, seven of which were sensitive to ∼100 copies, while only 5 were sensitive to ∼10 RNA template copies across all replicates tested. Despite their demonstrated sensitivity, these five best performing enzymes (Accuscript, HIV-RT, M-MLV, Superscript III and Thermoscript) showed considerable variation in their reproducibility as well as their overall amplification efficiency. Accuscript and Superscript III were the most sensitive and consistent within runs, with Accuscript and Superscript II ranking as the most reproducible enzymes between assays. Conclusions/Significance We therefore recommend the use of Accuscript or Superscript III when dealing with low copy number RNA levels, and suggest purification of the RT reactions prior to downstream applications (eg qPCR) to augment detection. Although the results presented in this study were based on a viral RNA surrogate, and applied to nucleic acid lysates derived from archival formalin-fixed paraffin embedded tissue, their relative performance on RNA obtained from other tissue types may vary, and needs future evaluation. PMID:21085668

Regulation of expression and biochemical characterization of a beta-class carbonic anhydrase from the plant growth-promoting rhizobacterium, Azospirillum brasilense Sp7.

PubMed

Kaur, Simarjot; Mishra, Mukti Nath; Tripathi, Anil K

2009-10-01

Carbonic anhydrase (CA; [EC 4.2.1.1]) is a ubiquitous enzyme catalysing the reversible hydration of CO(2) to bicarbonate, a reaction that supports various biochemical and physiological functions. Genome analysis of Azospirillum brasilense, a nonphotosynthetic, nitrogen-fixing, rhizobacterium, revealed an ORF with homology to beta-class carbonic anhydrases (CAs). Biochemical characteristics of the beta-class CA of A. brasilense, analysed after cloning the gene (designated as bca), overexpressing in Escherichia coli and purifying the protein by affinity purification, revealed that the native recombinant enzyme is a homotetramer, inhibited by the known CA inhibitors. CA activity in A. brasilense cell extracts, reverse transcriptase (RT)-PCR and Western blot analyses showed that bca was constitutively expressed under aerobic conditions. Lower beta-galactosidase activity in A. brasilense cells harbouring bca promoter: lacZ fusion during the stationary phase or during growth on 3% CO(2) enriched air or at acidic pH indicated that the transcription of bca was downregulated by the stationary phase, elevated CO(2) levels and acidic pH conditions. These observations were also supported by RT-PCR analysis. Thus, beta-CA in A. brasilense seems to be required for scavenging CO(2) from the ambient air and the requirement of CO(2) hydration seems to be higher for the cultures growing exponentially at neutral to alkaline pH.

Simultaneous quantification of alternatively spliced transcripts in a single droplet digital PCR reaction.

PubMed

Sun, Bing; Tao, Lian; Zheng, Yun-Ling

2014-06-01

Human telomerase reverse transcriptase (hTERT) is an essential component required for telomerase activity and telomere maintenance. Several alternatively spliced forms of hTERT mRNA have been reported in human primary and tumor cells. Currently, however, there is no sensitive and accurate method for the simultaneous quantification of multiple alternatively spliced RNA transcripts, such as in the case of hTERT. Here we show droplet digital PCR (ddPCR) provides sensitive, simultaneous digital quantification in a single reaction of two alternatively spliced single deletion hTERT transcripts (α-/β+ and α+/β-) as well as the opportunity to manually quantify non-deletion (α+/β+) and double deletion (α-/β-) transcripts. Our ddPCR method enables direct comparison among four alternatively spliced mRNAs without the need for internal standards or multiple primer pairs specific for each variant as real-time PCR (qPCR) requires, thus eliminating potential variation due to differences in PCR amplification efficiency.

Molecular cloning and expression analysis of annexin A2 gene in sika deer antler tip.

PubMed

Xia, Yanling; Qu, Haomiao; Lu, Binshan; Zhang, Qiang; Li, Heping

2018-04-01

Molecular cloning and bioinformatics analysis of annexin A2 ( ANXA2 ) gene in sika deer antler tip were conducted. The role of ANXA2 gene in the growth and development of the antler were analyzed initially. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to clone the cDNA sequence of the ANXA2 gene from antler tip of sika deer ( Cervus Nippon hortulorum ) and the bioinformatics methods were applied to analyze the amino acid sequence of Anxa2 protein. The mRNA expression levels of the ANXA2 gene in different growth stages were examined by real time reverse transcriptase polymerase chain reaction (real time RT-PCR). The nucleotide sequence analysis revealed an open reading frame of 1,020 bp encoding 339 amino acids long protein of calculated molecular weight 38.6 kDa and isoelectric point 6.09. Homologous sequence alignment and phylogenetic analysis indicated that the Anxa2 mature protein of sika deer had the closest genetic distance with Cervus elaphus and Bos mutus . Real time RT-PCR results showed that the gene had differential expression levels in different growth stages, and the expression level of the ANXA2 gene was the highest at metaphase (rapid growing period). ANXA2 gene may promote the cell proliferation, and the finding suggested Anxa2 as an important candidate for regulating the growth and development of deer antler.

Clinical validation of 3 commercial real-time reverse transcriptase polymerase chain reaction assays for the detection of Middle East respiratory syndrome coronavirus from upper respiratory tract specimens.

PubMed

Mohamed, Deqa H; AlHetheel, AbdulKarim F; Mohamud, Hanat S; Aldosari, Kamel; Alzamil, Fahad A; Somily, Ali M

2017-04-01

Since discovery of Middle East respiratory syndrome coronavirus (MERS-CoV), a novel betacoronavirus first isolated and characterized in 2012, MERS-CoV real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assays represent one of the most rapidly expanding commercial tests. However, in the absence of extensive evaluations of these assays on positive clinical material of different sources, evaluating their diagnostic effectiveness remains challenging. We describe the diagnostic performance evaluation of 3 common commercial MERS-CoV rRT-PCR assays on a large panel (n = 234) of upper respiratory tract specimens collected during an outbreak episode in Saudi Arabia. Assays were compared to the RealStar® MERS-CoV RT-PCR (Alton Diagnostics, Hamburg, Germany) assay as the gold standard. Results showed i) the TIB MolBiol® LightMix UpE and Orf1a assays (TIB MolBiol, Berlin, Germany) to be the most sensitive, followed by ii) the Anyplex™ Seegene MERS-CoV assay (Seegene, Seoul, Korea), and finally iii) the PrimerDesign™ Genesig® HCoV_2012 assay (PrimerDesign, England, United Kingdom). We also evaluate a modified protocol for the PrimerDesign™ Genesig® HCoV_2012 assay. Copyright © 2017 Elsevier Inc. All rights reserved.

Bioinformatic analysis of variability of Newcastle disease virus diagnostic primers and probes and the potential for false negative detection

USDA-ARS?s Scientific Manuscript database

The use of reverse transcriptase polymerase chain reaction (RT-PCR) or other molecular diagnostic methods is commonly used for the primary diagnosis of Newcastle disease virus (NDV). However, NDV in nature has a range of virulence, and the low virulence viruses must be differentiated from virulent ...

Two Novel Real-Time Reverse Transcriptase PCR Assays for Rapid Detection of Bacterial Contamination in Platelet Concentrates

PubMed Central

Dreier, Jens; Störmer, Melanie; Kleesiek, Knut

2004-01-01

The incidence of platelet bacterial contamination is approximately 1 per 2,000 units and has been acknowledged as the most frequent infectious risk from transfusion. In preliminary studies, the sterility of platelet concentrates (PCs) was tested with an automated bacterial blood culturing system and molecular genetic assays. Two real-time reverse transcriptase PCR (RT-PCR) assays performed in a LightCycler instrument were developed and compared regarding specificity and sensitivity by the use of different templates to detect the majority of the clinically important bacterial species in platelets. Primers and probes specific for the conserved regions of the eubacterial 23S rRNA gene or the groEL gene (encoding the 60-kDa heat shock protein Hsp60) were designed. During the development of the 23S rRNA RT-PCR, problems caused by the contamination of reagents with bacterial DNA were noted. Treatment with 8-methoxypsoralen and UV irradiation reduced the level of contaminating DNA. The sensitivity of the assays was greatly influenced by the enzyme system which was used. With rTth DNA polymerase in a one-enzyme system, we detected 500 CFU of Escherichia coli or Staphylococcus epidermidis/ml. With a two-enzyme system consisting of Moloney murine leukemia virus RT and Taq DNA polymerase, we detected 16 CFU/ml. With groEL mRNA as the target of RT-PCR under optimized conditions, we detected 125 CFU of E. coli/ml, and no problems with false-positive results caused by reagent contamination or a cross-reaction with human nucleic acids were found. Furthermore, the use of mRNA as an indicator of viability was demonstrated. Here we report the application of novel real-time RT-PCR assays for the detection of bacterial contamination of PCs that are appropriate for transfusion services. PMID:15472337

Teschoviruses as Indicators of Porcine Fecal Contamination of Surface Water

PubMed Central

Jiménez-Clavero, Miguel Angel; Fernández, Carlos; Ortiz, José Antonio; Pro, Javier; Carbonell, Gregoria; Tarazona, José Vicente; Roblas, Neftalí; Ley, Victoria

2003-01-01

Teschoviruses specifically infect pigs and are shed in pig feces. Hence, their presence in water should indicate contamination with pig fecal residues. To assess this hypothesis, we have developed a real-time reverse transcriptase PCR (RT-PCR) method that allows the quantitative detection of pig teschovirus (PTV) RNA. The method is able to detect 92 fg of PTV RNA per ml of sample. Using this method, we have detected the presence of PTV RNA in water and fecal samples from all pig farms examined (n = 5). Feces from other animal species (cattle, sheep, and goats) were negative in this test. To compare the PTV RNA detection method with conventional chemical determinations currently in use for evaluation of water contamination, we analyzed water samples collected downstream from a pig slurry spillage site. We have found a positive correlation within both types of determinations. The sensitivity of the PTV detection assay was similar to that achieved by unspecific organic matter determination and superior to all other conventional chemical analyses performed. Furthermore, the new method is highly specific, revealing the porcine origin of the contamination, a feature that is lacking in currently available methods for the assessment of water contamination. PMID:14532098

Progress towards Rapid Detection of Measles Vaccine Strains: a Tool To Inform Public Health Interventions

PubMed Central

2016-01-01

ABSTRACT Rapid differentiation of vaccine from wild-type strains in suspect measles cases is a valuable epidemiological tool that informs the public health response to this highly infectious disease. Few public health laboratories sequence measles virus-positive specimens to determine genotype, and the vaccine-specific real-time reverse transcriptase PCR (rRT-PCR) assay described by F. Roy et al. (J. Clin. Microbiol. 55:735–743, 2017, https://doi.org/10.1128/JCM.01879-16) offers a rapid, easily adoptable method to identify measles vaccine strains in suspect cases. PMID:28003421

Progress towards Rapid Detection of Measles Vaccine Strains: a Tool To Inform Public Health Interventions.

PubMed

Hacker, Jill K

2017-03-01

Rapid differentiation of vaccine from wild-type strains in suspect measles cases is a valuable epidemiological tool that informs the public health response to this highly infectious disease. Few public health laboratories sequence measles virus-positive specimens to determine genotype, and the vaccine-specific real-time reverse transcriptase PCR (rRT-PCR) assay described by F. Roy et al. (J. Clin. Microbiol. 55:735-743, 2017, https://doi.org/10.1128/JCM.01879-16) offers a rapid, easily adoptable method to identify measles vaccine strains in suspect cases. Copyright © 2017 American Society for Microbiology.

Influenza and other respiratory virus infections in outpatients with medically attended acute respiratory infection during the 2011-12 influenza season.

PubMed

Zimmerman, Richard K; Rinaldo, Charles R; Nowalk, Mary Patricia; Gk, Balasubramani; Thompson, Mark G; Moehling, Krissy K; Bullotta, Arlene; Wisniewski, Stephen

2014-07-01

Respiratory tract infections are a major cause of outpatient visits, yet only a portion is tested to determine the etiologic organism. Multiplex reverse transcriptase polymerase chain reaction (MRT-PCR) assays for detection of multiple viruses are being used increasingly in clinical settings. During January-April 2012, outpatients with acute respiratory illness (≤ 7 days) were tested for influenza using singleplex RT-PCR (SRT-PCR). A subset was assayed for 18 viruses using MRT-PCR to compare detection of influenza and examine the distribution of viruses and characteristics of patients using multinomial logistic regression. Among 662 participants (6 months-82 years), detection of influenza was similar between the MRT-PCR and SRT-PCR (κ = 0.83). No virus was identified in 267 (40.3%) samples. Commonly detected viruses were human rhinovirus (HRV, 15.4%), coronavirus (CoV, 10.4%), respiratory syncytial virus (RSV, 8.4%), human metapneumovirus (hMPV, 8.3%), and influenza (6%). Co-detections were infrequent (6.9%) and most commonly occurred among those <18 years old. In regression analyses, compared with non-viral illnesses, RSV and hMPV were significantly more frequent in children and less frequent in 18- to 49-year-olds than in those ≥ 50 years (P = 0.01), fever was more common in hMPV and influenza infections (P = 0.008), nasal congestion was more frequent in CoV, HRV, hMPV, influenza and RSV infections (P = 0.001), and body mass index was higher among those with influenza (P = 0.036). Using MRT-PCR, a viral etiology was found in three-fifths of patients with medically attended outpatient visits for acute respiratory illness during the influenza season; co-detected viruses were infrequent. Symptoms varied by viral etiology. © 2014 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

Evaluation of different embryonating bird eggs and cell cultures for isolation efficiency of avian influenza A virus and avian paramyxovirus serotype 1 from real-time reverse transcription polymerase chain reaction--positive

USDA-ARS?s Scientific Manuscript database

Two hundred samples collected from Anseriformes, Charadriiformes, Gruiformes, and Galliformes were assayed using real-time reverse transcriptase polymerase chain reaction (RRT-PCR) for presence of avian influenza virus and avian paramyxovirus-1. Virus isolation using embryonating chicken eggs, embr...

Characterization of constitutive and putative differentially expressed mRNAs by means of expressed sequence tags, differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR from the sand fly vector Lutzomyia longipalpis.

PubMed

Ramalho-Ortigão, J M; Temporal, P; de Oliveira , S M; Barbosa, A F; Vilela, M L; Rangel, E F; Brazil, R P; Traub-Cseko, Y M

2001-01-01

Molecular studies of insect disease vectors are of paramount importance for understanding parasite-vector relationship. Advances in this area have led to important findings regarding changes in vectors' physiology upon blood feeding and parasite infection. Mechanisms for interfering with the vectorial capacity of insects responsible for the transmission of diseases such as malaria, Chagas disease and dengue fever are being devised with the ultimate goal of developing transgenic insects. A primary necessity for this goal is information on gene expression and control in the target insect. Our group is investigating molecular aspects of the interaction between Leishmania parasites and Lutzomyia sand flies. As an initial step in our studies we have used random sequencing of cDNA clones from two expression libraries made from head/thorax and abdomen of sugar fed L. longipalpis for the identification of expressed sequence tags (EST). We applied differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR to characterize differentially expressed mRNA from sugar and blood fed insects, and, in one case, from a L. (V.) braziliensis-infected L. longipalpis. We identified 37 cDNAs that have shown homology to known sequences from GeneBank. Of these, 32 cDNAs code for constitutive proteins such as zinc finger protein, glutamine synthetase, G binding protein, ubiquitin conjugating enzyme. Three are putative differentially expressed cDNAs from blood fed and Leishmania-infected midgut, a chitinase, a V-ATPase and a MAP kinase. Finally, two sequences are homologous to Drosophila melanogaster gene products recently discovered through the Drosophila genome initiative.

A novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis C viral RNA with armored RNA as internal control

PubMed Central

2010-01-01

Background The hepatitis C virus (HCV) genome is extremely heterogeneous. Several HCV infections can not be detected using currently available commercial assays, probably because of mismatches between the template and primers/probes. By aligning the HCV sequences, we developed a duplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay using 2 sets of primers/probes and a specific armored RNA as internal control. The 2 detection probes were labelled with the same fluorophore, namely, 6-carboxyfluorescein (FAM), at the 5' end; these probes could mutually combine, improving the power of the test. Results The limit of detection of the duplex primer/probe assay was 38.99 IU/ml. The sensitivity of the assay improved significantly, while the specificity was not affected. All HCV genotypes in the HCV RNA Genotype Panel for Nucleic Acid Amplification Techniques could be detected. In the testing of 109 serum samples, the performance of the duplex real-time RT-PCR assay was identical to that of the COBAS AmpliPrep (CAP)/COBAS TaqMan (CTM) assay and superior to 2 commercial HCV assay kits. Conclusions The duplex real-time RT-PCR assay is an efficient and effective viral assay. It is comparable with the CAP/CTM assay with regard to the power of the test and is appropriate for blood-donor screening and laboratory diagnosis of HCV infection. PMID:20529244

Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types.

PubMed

Kilian, A; Bowtell, D D; Abud, H E; Hime, G R; Venter, D J; Keese, P K; Duncan, E L; Reddel, R R; Jefferson, R A

1997-11-01

Telomerase is a multicomponent reverse transcriptase enzyme that adds DNA repeats to the ends of chromosomes using its RNA component as a template for synthesis. Telomerase activity is detected in the germline as well as the majority of tumors and immortal cell lines, and at low levels in several types of normal cells. We have cloned a human gene homologous to a protein from Saccharomyces cerevisiae and Euplotes aediculatus that has reverse transcriptase motifs and is thought to be the catalytic subunit of telomerase in those species. This gene is present in the human genome as a single copy sequence with a dominant transcript of approximately 4 kb in a human colon cancer cell line, LIM1215. The cDNA sequence was determined using clones from a LIM1215 cDNA library and by RT-PCR, cRACE and 3'RACE on mRNA from the same source. We show that the gene is expressed in several normal tissues, telomerase-positive post-crisis (immortal) cell lines and various tumors but is not expressed in the majority of normal tissues analyzed, pre-crisis (non-immortal) cells and telomerase-negative immortal (ALT) cell lines. Multiple products were identified by RT-PCR using primers within the reverse transcriptase domain. Sequencing of these products suggests that they arise by alternative splicing. Strikingly, various tumors, cell lines and even normal tissues (colonic crypt and testis) showed considerable differences in the splicing patterns. Alternative splicing of the telomerase catalytic subunit transcript may be important for the regulation of telomerase activity and may give rise to proteins with different biochemical functions.

Combined use of reverse transcriptase polymerase chain reaction and flow cytometry to study minimal residual disease in Philadelphia positive acute lymphoblastic leukemia.

PubMed

Muñoz, L; López, O; Martino, R; Brunet, S; Bellido, M; Rubiol, E; Sierra, J; Nomdedéu, J F

2000-07-01

The Philadelphia chromosome in acute lymphoblastic leukemia (Ph+ ALL) is associated with a poor prognosis given the high frequency of chemoresistance and leukemia relapse. Minimal residual disease (MRD) detection before cytogenetic and hematologic relapse could be useful in early therapy. The most suitable methods for detecting MRD in Ph+ ALL are flow cytometry (FC) and reverse transcriptase polymerase chain reaction (RT-PCR). However, since both techniques carry the risk of false-negative results the combined use of these two techniques could overcome this problem. We report our experience using this approach in 47 bone marrow samples obtained from 10 Ph+ ALL patients. Twenty-seven marrow aspirates were taken from patients in clinical remission (CR). The samples were considered positive for MRD by FC when two conditions were met: 1) detection of an abnormal B-cell differentiation pattern and 2) presence of more than 1x10(-3) cells coexpressing CD22/CD34/CD45 or CD66/CD34/CD10. After FC analysis, RNA was purified using standard methods. FC was positive in 23/27 samples in CR (sensitivity 85%). RT-PCR was successfully performed in 23 samples in CR. RT-PCR was positive in 18/23 samples (sensitivity 78%). There were 5 samples with discordant results. FC was positive in 3 samples with a negative RT-PCR and FC was negative in 2 samples with a positive RT. All the 10 patients relapsed and only 1 is currently alive after an allogeneic stem cell transplantation (alloSCT). The median (range) time from MRD detection to relapse in patients treated with chemotherapy was 42 (39-71) days. These data suggest that RT-PCR may be negative despite the presence of neoplastic cells identified by their immunophenotypic traits. We conclude that immunologic and molecular techniques can be used in tandem for monitoring MRD in Ph+ ALL.

Detection of PAX3-FKHR and PAX7-FKHR fusion transcripts in rhabdomyosarcoma by reverse transcriptase-polymerase chain reaction using paraffin-embedded tissue.

PubMed

Chen, B F; Chen, M L; Liang, D C; Huang, Y W; Liu, H C; Chen, S H

1999-02-01

Alveolar rhabdomyosarcoma (RMS) is associated with a characteristic chromosomal translocation t(2;13)(q35;q14). The genes involved in this translocation are paired box (PAX)3 on chromosome 2 and forkhead in RMS (FKHR) on chromosome 13. An occasional variant translocation t(1;13)(p36;q14) affecting PAX7 and FKHR on chromosomes 1 and 13, respectively, has also been described. Chromosomal translocations in RMS are detected using conventional cytogenetic analysis, fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction (RT-PCR) on fresh or frozen tissue samples. We describe the results of RT-PCR analysis of PAX3-FKHR and PAX7-FKHR chimeric messages in formalin-fixed, paraffin-embedded tissue samples from 17 RMS cases. RNA was extracted from formalin-fixed, paraffin-embedded RMS tissue. Oligonucleotide primers corresponding to the regions of PAX3, PAX7 and FKHR were used for the detection of PAX3-FKHR and PAX7-FKHR chimeric messages. A seminested PCR of the PCR products was used to increase the sensitivity of detection. The amplified fragments were purified and directly sequenced to confirm the specificity of the methods. The PAX3-FKHR chimeric message was detected in all three cases of alveolar RMS but not in any of the 12 embryonal and two pleomorphic RMS cases. The PAX7-FKHR fusion transcript was detected in one case of embryonal RMS. The results indicate that the RT-PCR assay is a reliable method for the detection of the PAX3-FKHR fusion transcript of alveolar RMS in formalin-fixed, paraffin-embedded tissue. This simple method enables pathologists to identify chromosomal rearrangements in RMS as a diagnostic aid in cases where fresh or frozen tissue is not available.

Detection of Anaplasma marginale and A. phagocytophilum in Bovine Peripheral Blood Samples by Duplex Real-Time Reverse Transcriptase PCR Assay ▿

PubMed Central

Reinbold, James B.; Coetzee, Johann F.; Sirigireddy, Kamesh R.; Ganta, Roman R.

2010-01-01

Insufficient diagnostic sensitivity and specificity coupled with the potential for cross-reactivity among closely related Anaplasma species has made the accurate determination of infection status problematic. A method for the development of simplex and duplex real-time quantitative reverse transcriptase PCR (qRT-PCR) assays for the detection of A. marginale and A. phagocytophilum 16S rRNA in plasma-free bovine peripheral blood samples is described. The duplex assay was able to detect as few as 100 copies of 16S rRNA of both A. marginale and A. phagocytophilum in the same reaction. The ratio of 16S rRNA to 16S DNA copies for A. marginale was determined to be 117.9:1 (95% confidence interval [95% CI], 100.7:1, 135.2:1). Therefore, the detection limit is the minimum infective unit of one A. marginale bacterium. The duplex assay detected nonequivalent molar ratios as high as 100-fold. Additionally, the duplex assay and a competitive enzyme-linked immunosorbent assay (cELISA) were used to screen 237 samples collected from herds in which anaplasmosis was endemic. When the cELISA was evaluated by the results of the qRT-PCR, its sensitivity and specificity for the detection of A. marginale infection were found to be 65.2% (95% CI, 55.3%, 75.1%) and 97.3% (95% CI, 94.7%, 99.9%), respectively. A. phagocytophilum infection was not detected in the samples analyzed. One- and two-way receiver operator characteristic curves were constructed in order to recommend the optimum negative cutoff value for the cELISA. Percentages of inhibition of 20 and 15.3% were recommended for the one- and two-way curves, respectively. In conclusion, the duplex real-time qRT-PCR assay is a highly sensitive and specific diagnostic tool for the accurate and precise detection of A. marginale and A. phagocytophilum infections in cattle. PMID:20463162

VIRUS ISOLATION AND MOLECULAR DETECTION OF BLUETONGUE AND EPIZOOTIC HEMORRHAGIC DISEASE VIRUSES FROM NATURALLY INFECTED WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS).

PubMed

Kienzle, Clara; Poulson, Rebecca L; Ruder, Mark G; Stallknecht, David E

2017-10-01

Hemorrhagic disease in North America is caused by multiple serotypes of epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV). Diagnostic tests for detection of EHDV and BTV include virus isolation (VI), reverse transcriptase (RT)-PCR, and real-time RT-PCR (rRT-PCR). Our objective was to compare the diagnostic capabilities of three rRT-PCR protocols for detection of EHDV and BTV from naturally infected white-tailed deer (Odocoileus virginianus). We compared the effectiveness of these assays to traditional viral detection methods (e.g., VI) for historic and current clinical cases. Because of the variable nature of tissue collection and storage before diagnostic testing, an evaluation of viral persistence on multiple freeze-thaw events was also conducted. Two of the rRT-PCR assays provided for reliable detection of EHDV and BTV from 100% of clinically affected and VI-confirmed infected animals. Additionally, no significant change in viral titer was observed on multiple freeze-thaw events.

Differential regulation of mnp2, a new manganese peroxidase-encoding gene from the ligninolytic fungus Trametes versicolor PRL 572

Treesearch

Tomas Johansson; Per Olof Nyman; Daniel Cullen

2002-01-01

A peroxidase-encoding gene, mnp2, and its corresponding cDNA were characterized from the white-rot basidiomycete Trametes versicolor PRL 572. We used quantitative reverse transcriptase-mediated PCR to identify mnp2 transcripts in nutrient-limited stationary cultures. Although mnp2 lacks upstream metal response elements (MREs), addition of MnSO4 to cultures increased...

Capsicum annum, a new host of watermelon mosaic virus.

PubMed

Hajizadeh, Mohammad; Mohammadi, Kazhal

2016-03-01

The occurrence of Watermelon mosaic virus (WMV) in sweet pepper (Capsicum annuum L.) in Kurdistan province, Iran was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and partial characterization of coat protein. To the best of our knowledge, this is the first report of WMV infecting C. annuum, adding a new host to list of more than 170 species infected by this virus.

copia-like retrotransposons are ubiquitous among plants.

PubMed Central

Voytas, D F; Cummings, M P; Koniczny, A; Ausubel, F M; Rodermel, S R

1992-01-01

Transposable genetic elements are assumed to be a feature of all eukaryotic genomes. Their identification, however, has largely been haphazard, limited principally to organisms subjected to molecular or genetic scrutiny. We assessed the phylogenetic distribution of copia-like retrotransposons, a class of transposable element that proliferates by reverse transcription, using a polymerase chain reaction assay designed to detect copia-like element reverse transcriptase sequences. copia-like retrotransposons were identified in 64 plant species as well as the photosynthetic protist Volvox carteri. The plant species included representatives from 9 of 10 plant divisions, including bryophytes, lycopods, ferns, gymnosperms, and angiosperms. DNA sequence analysis of 29 cloned PCR products and of a maize retrotransposon cDNA confirmed the identity of these sequences as copia-like reverse transcriptase sequences, thereby demonstrating that this class of retrotransposons is a ubiquitous component of plant genomes. Images PMID:1379734

The presence of p53 influences the expression of multiple human cytomegalovirus genes at early times postinfection.

PubMed

Hannemann, Holger; Rosenke, Kyle; O'Dowd, John M; Fortunato, Elizabeth A

2009-05-01

Human cytomegalovirus (HCMV) is a common cause of morbidity and mortality in immunocompromised and immunosuppressed individuals. During infection, HCMV is known to employ host transcription factors to facilitate viral gene expression. To further understand the previously observed delay in viral replication and protein expression in p53 knockout cells, we conducted microarray analyses of p53(+/+) and p53(-/-) immortalized fibroblast cell lines. At a multiplicity of infection (MOI) of 1 at 24 h postinfection (p.i.), the expression of 22 viral genes was affected by the absence of p53. Eleven of these 22 genes (group 1) were examined by real-time reverse transcriptase, or quantitative, PCR (q-PCR). Additionally, five genes previously determined to have p53 bound to their nearest p53-responsive elements (group 2) and three control genes without p53 binding sites in their upstream sequences (group 3) were also examined. At an MOI of 1, >3-fold regulation was found for five group 1 genes. The expression of group 2 and 3 genes was not changed. At an MOI of 5, all genes from group 1 and four of five genes from group 2 were found to be regulated. The expression of control genes from group 3 remained unchanged. A q-PCR time course of four genes revealed that p53 influences viral gene expression most at immediate-early and early times p.i., suggesting a mechanism for the reduced and delayed production of virions in p53(-/-) cells.

A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus.

PubMed

Kang, Xiao-ping; Jiang, Tao; Li, Yong-qiang; Lin, Fang; Liu, Hong; Chang, Guo-hui; Zhu, Qing-yu; Qin, E-de; Qin, Cheng-feng; Yang, Yin-hui

2010-06-02

A duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was improved for simultaneous detection of highly pathogenic H5N1 avian influenza virus and pandemic H1N1 (2009) influenza virus, which is suitable for early diagnosis of influenza-like patients and for epidemiological surveillance. The sensitivity of this duplex real-time RT-PCR assay was 0.02 TCID50 (50% tissue culture infective dose) for H5N1 and 0.2 TCID50 for the pandemic H1N1, which was the same as that of each single-target RT-PCR for pandemic H1N1 and even more sensitive for H5N1 with the same primers and probes. No cross reactivity of detecting other subtype influenza viruses or respiratory tract viruses was observed. Two hundred and thirty-six clinical specimens were tested by comparing with single real-time RT-PCR and result from the duplex assay was 100% consistent with the results of single real-time RT-PCR and sequence analysis.

An epidemiological study of enteric viruses in sewage with molecular characterization by RT-PCR and sequence analysis.

PubMed

Arraj, A; Bohatier, J; Aumeran, C; Bailly, J L; Laveran, H; Traoré, O

2008-09-01

The aim of this study was to assess the presence and seasonal frequency of various enteric viruses in wastewater treatment. The detection of astrovirus, norovirus, enterovirus, hepatitis A virus (HAV) and rotavirus was carried out by molecular analyses in concentrated water samples collected over 18 months at the entrance and exit of an activated sludge sewage treatment plant. The reverse transcriptase-polymerase chain reaction (RT-PCR) results were confirmed by sequencing, and comparative phylogenetic analysis was performed on the isolated strains. Genomes of human astrovirus and human rotavirus were identified in 26/29 and 11/29 samples of raw sewage, respectively, and in 12/29 and 13/29 treated effluent samples, respectively. Some rotavirus sequences detected in environmental samples were very close to those of clinical strains. Noroviruses, enteroviruses and HAV were not detected during the study period. This could be related to the small sample volume, to the sensitivity of the detection methods or to local epidemiological situations. Frequent detection of viral RNA, whether infectious or not, in the exit effluent of sewage treatment indicates wide dispersion of enteric viruses in the environment. Consequently, viral contamination resulting from the use of these treated waters is a risk that needs to be addressed.

Adenovirus-mediated p53 gene delivery inhibits 9L glioma growth in rats.

PubMed

Badie, B; Drazan, K E; Kramar, M H; Shaked, A; Black, K L

1995-06-01

Adenoviral vectors have recently been shown to effectively deliver genes into a variety of tissues. Since these vectors have some advantages over the more extensively investigated retroviruses, we studied the effect of two replication-defective adenovectors bearing human wild type tumor suppressor gene p53 (Adp53) and Escherichia coli beta-galactosidase gene (AdLacZ) on 9L glioma cells. Successful in vitro gene transfer was shown by DNA polymerase chain reaction (PCR), and expression was confirmed by reverse transcriptase RNA PCR and Western blot analyses. Transduction of 9L cells with the Adp53 inhibited cell growth and induced phenotypic changes consistent with cell death at low titers, while AdLacZ caused cytopathic changes only at high titers. Stereotactic injection of AdLacZ (10(7) plaque forming units) into tumor bed stained 25 to 30% of tumor cells at the site of vector delivery. Injection of Adp53 (10(7) plaque forming units), but not AdLacZ (controls), into established 4-day old 9L glioma brain tumors decreased tumor volume by 40% after 14 days. As a step toward gene therapy of brain tumors using replication-defective adenoviruses, these data support the use of tumor suppressor gene transfer for in vivo treatment of whole animal brain tumor models.

A conserved segmental duplication within ELA.

PubMed

Brinkmeyer-Langford, C L; Murphy, W J; Childers, C P; Skow, L C

2010-12-01

The assembled genomic sequence of the horse major histocompatibility complex (MHC) (equine lymphocyte antigen, ELA) is very similar to the homologous human HLA, with the notable exception of a large segmental duplication at the boundary of ELA class I and class III that is absent in HLA. The segmental duplication consists of a ∼ 710 kb region of at least 11 repeated blocks: 10 blocks each contain an MHC class I-like sequence and the helicase domain portion of a BAT1-like sequence, and the remaining unit contains the full-length BAT1 gene. Similar genomic features were found in other Perissodactyls, indicating an ancient origin, which is consistent with phylogenetic analyses. Reverse-transcriptase PCR (RT-PCR) of mRNA from peripheral white blood cells of healthy and chronically or acutely infected horses detected transcription from predicted open reading frames in several of the duplicated blocks. This duplication is not present in the sequenced MHCs of most other mammals, although a similar feature at the same relative position is present in the feline MHC (FLA). Striking sequence conservation throughout Perissodactyl evolution is consistent with a functional role for at least some of the genes included within this segmental duplication. © 2010 The Authors, Journal compilation © 2010 Stichting International Foundation for Animal Genetics.

Screening, isolation and optimization of anti–white spot syndrome virus drug derived from terrestrial plants

PubMed Central

Ghosh, Upasana; Chakraborty, Somnath; Balasubramanian, Thangavel; Das, Punyabrata

2014-01-01

Objective To screen, isolate and optimize anti-white spot syndrome virus (WSSV) drug derived from various terrestrial plants and to evaluate the efficacy of the same in host–pathogen interaction model. Methods Thirty plants were subjected to Soxhlet extraction using water, ethanol, methanol and hexane as solvents. The 120 plant isolates thus obtained were screened for their in vivo anti–WSSV property in Litopenaeus vannamei. The best anti–WSSV plant isolate, TP22C was isolated and further analyzed. The drug was optimized at various concentrations. Viral and immune genes were analysed using reverse transcriptase PCR to confirm the potency of the drug. Results Seven plant isolates exhibited significant survivability in host. The drug TP22C thus formulated showed 86% survivability in host. The surviving shrimps were nested PCR negative at the end of the 15 d experimentation. The lowest concentration of TP22C required intramuscularly for virucidal property was 10 mg/mL. The oral dosage of 750 mg/kg body weight/day survived at the rate of 86%. Neither VP28 nor ie 1 was expressed in the test samples at 42nd hour and 84th hour post viral infection. Conclusions The drug TP22C derived from Momordica charantia is a potent anti-white spot syndrome virus drug. PMID:25183066

Handmade Cloned Transgenic Piglets Expressing the Nematode Fat-1 Gene

PubMed Central

Zhang, Peng; Zhang, Yidi; Dou, Hongwei; Yin, Jingdong; Chen, Yu; Pang, Xinzhi; Vajta, Gabor; Bolund, Lars

2012-01-01

Abstract Production of transgenic animals via somatic cell nuclear transfer (SCNT) has been adapted worldwide, but this application is somewhat limited by its relatively low efficiency. In this study, we used handmade cloning (HMC) established previously to produce transgenic pigs that express the functional nematode fat-1 gene. Codon-optimized mfat-1 was inserted into eukaryotic expression vectors, which were transferred into primary swine donor cells. Reverse transcriptase PCR (RT-PCR), gas chromatography, and chromosome analyses were performed to select donor clones capable of converting n-6 into n-3 fatty acids. Blastocysts derived from the clones that lowered the n-6/n-3 ratio to approximately 1:1 were transferred surgically into the uteri of recipients for transgenic piglets. By HMC, 37% (n=558) of reconstructed embryos developed to the blastocyst stage after 7 days of culture in vitro, with an average cell number of 81±36 (n=14). Three recipients became pregnant after 408 day-6 blastocysts were transferred into four naturally cycling females, and a total of 14 live offspring were produced. The nematode mfat-1 effectively lowered the n-6/n-3 ratio in muscle and major organs of the transgenic pig. Our results will help to establish a reliable procedure and an efficient option in the production of transgenic animals. PMID:22686479

Up-regulation of proproliferative genes and the ligand/receptor pair placental growth factor and vascular endothelial growth factor receptor 1 in hepatitis C cirrhosis.

PubMed

Huang, Xiao X; McCaughan, Geoffrey W; Shackel, Nicholas A; Gorrell, Mark D

2007-09-01

Cirrhosis can lead to hepatocellular carcinoma (HCC). Non-diseased liver and hepatitis C virus (HCV)-associated cirrhosis with or without HCC were compared. Proliferation pathway genes, immune response genes and oncogenes were analysed by a quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunostaining. Real-time RT-PCR showed up-regulation of genes in HCV cirrhosis including the proliferation-associated genes bone morphogenetic protein 3 (BMP3), placental growth factor 3 (PGF3), vascular endothelial growth factor receptor 1 (VEGFR1) and soluble VEGFR1, the oncogene FYN, and the immune response-associated genes toll-like receptor 9 (TLR9) and natural killer cell transcript 4 (NK4). Expressions of TLR2 and the oncogenes B-cell CLL/lymphoma 9 (BCL9) and PIM2 were decreased in HCV cirrhosis. In addition, PIM2 and TLR2 were increased in HCV cirrhosis with HCC compared with HCV cirrhosis. The ligand/receptor pair PGF and VEGFR1 was intensely expressed by the portal tract vascular endothelium. VEGFR1 was expressed in reactive biliary epithelial structures in fibrotic septum and in some stellate cells and macrophages. PGF and VEGFR1 may have an important role in the pathogenesis of the neovascular response in cirrhosis.

Development of a highly sensitive screen for influenza A in guano and its application in the search for ancient RNA preserved under Antarctic Adelie penguin colonies.

PubMed

Briggs, L C; Ashton, R M; Metcalf, P

2003-01-01

We have developed a reverse transcriptase polymerase chain reaction (RT-PCR)-based assay to detect influenza A in guano samples as part of our program to investigate ancient viral RNA from under Antarctic Adelie penguin (Pygoscelis adeliae) colonies. Of five extraction protocols tested (RNeasy, GTC TRIZOL, GTC Silica, Rnaid, and AGPC), AGPC proved to be the most consistent and sensitive to low concentrations of influenza, but further purification with commercial viral nucleic acid spin filter system was still required to remove remaining PCR inhibitors. RT-PCR was then performed on the eluent and 650 bases of the M1 gene were amplified. The assay was found to be able to detect as little as 100 microl of 0.1 hemagglutination units (HU)/ml influenza.

Detection of infectious bronchitis virus with the use of real-time quantitative reverse transcriptase-PCR and correlation with virus detection in embryonated eggs.

PubMed

Roh, Ha-Jung; Hilt, Deborah A; Jackwood, Mark W

2014-09-01

Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assays have been used to detect the presence of challenge virus when the efficacy of infectious bronchitis virus (IBV) vaccine against field viruses is being experimentally evaluated. However, federal guidelines for licensing IBV vaccines indicate that challenge-virus detection following vaccination is to be conducted in embryonated eggs. In this study, we examined qRT-PCR data with the use of universal and type-specific primers and probe sets for IBV detection and compared those data with challenge-virus detection in embryonated eggs to determine if the two methods of evaluating vaccine efficacy are comparable. In addition, we tested the qRT-PCR assays on thermocyclers from two different manufacturers. We found the universal IBV primers and probe set to be comparable to challenge-virus detection in embryonated eggs. However, for some IBV types (Mass41 and Conn on the SmartCycler II and Ark, Mass41, Conn, and GA98 on the ABI 7500) the qRT-PCR assay was more sensitive than virus detection in embryonated eggs. This may simply be due to the universal IBV qRT-PCR assay being more sensitive than virus detection in eggs or to the assay detecting nucleic acid from nonviable virus. This finding is important and needs to be considered when evaluating challenge-virus detection for vaccination and challenge studies, because qRT-PCR could potentially identify positive birds that would otherwise be negative by virus detection in embryonated eggs; thus it could lead to a more stringent measure of vaccine efficacy. We also found that the IBV type-specific primers and probe sets designed in this study were in general less sensitive than the universal IBV primers and probe set. Only the Ark-DPI-spedcific assay on the SmartCycler II and the Ark-DPI-, Mass41-, and DE072/GA98- (for detection of GA98 virus only) specific assays on the ABI 7500 were comparable in sensitivity to virus detection in eggs. We found that a number of variables, including the virus type examined, primers and probe efficiency and stability, and assay conditions, including thermocycler platform, can affect the data obtained from qRT-PCR assays. These results indicate that qRT-PCR assays can be used to detect IBV challenge virus, but each assay, including the assay conditions and thermocycler, should be individually evaluated if those data are expected to be comparable to virus detection in embryonated eggs.

Effects of anticoagulant, processing delay, and assay method (branched DNA versus reverse transcriptase PCR) on measurement of human immunodeficiency virus type 1 RNA levels in plasma.

PubMed

Kirstein, L M; Mellors, J W; Rinaldo, C R; Margolick, J B; Giorgi, J V; Phair, J P; Dietz, E; Gupta, P; Sherlock, C H; Hogg, R; Montaner, J S; Muñoz, A

1999-08-01

We conducted two studies to determine the potential influence of delays in blood processing, type of anticoagulant, and assay method on human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma. The first was an experimental study in which heparin- and EDTA-anticoagulated blood samples were collected from 101 HIV-positive individuals and processed to plasma after delays of 2, 6, and 18 h. HIV-1 RNA levels in each sample were then measured by both branched-DNA (bDNA) and reverse transcriptase PCR (RT-PCR) assays. Compared to samples processed within 2 h, the loss (decay) of HIV-1 RNA in heparinized blood was significant (P < 0.05) but small after 6 h (bDNA assay, -0.12 log(10) copies/ml; RT-PCR, -0.05 log(10) copies/ml) and after 18 h (bDNA assay, -0.27 log(10) copies/ml; RT-PCR, -0.15 log(10) copies/ml). Decay in EDTA-anticoagulated blood was not significant after 6 h (bDNA assay, -0.002 log(10) copies/ml; RT-PCR, -0.02 log(10) copies/ml), but it was after 18 h (bDNA assay, -0.09 log(10) copies/ml; RT-PCR, -0.09 log(10) copies/ml). Only 4% of samples processed after 6 h lost more than 50% (>/=0.3 log(10) copies/ml) of the HIV-1 RNA, regardless of the anticoagulant or the assay that was used. The second study compared HIV-1 RNA levels in samples from the Multicenter AIDS Cohort Study (MACS; samples were collected in heparin-containing tubes in 1985, had a 6-h average processing delay, and were assayed by bDNA assay) and the British Columbia Drug Treatment Program (BCDTP) (collected in EDTA- or acid citrate dextrose-containing tubes in 1996 and 1997, had a 2-h maximum processing delay, and were assayed by RT-PCR). HIV-1 RNA levels in samples from the two cohorts were not significantly different after adjusting for CD4(+)-cell count and converting bDNA assay values to those corresponding to the RT-PCR results. In summary, the decay of HIV-1 RNA measured in heparinized blood after 6 h was small (-0.05 to -0.12 log(10) copies/ml), and the minor impact of this decay on HIV-1 RNA concentrations in archived plasma samples of the MACS was confirmed by the similarity of CD4(+)-cell counts and assay-adjusted HIV-1 RNA concentrations in the MACS and BCDTP.

An immortalized goat mammary epithelial cell line induced with human telomerase reverse transcriptase (hTERT) gene transfer.

PubMed

He, Y L; Wu, Y H; He, X N; Liu, F J; He, X Y; Zhang, Y

2009-06-01

Although mammary epithelial cell lines can provide a rapid and reliable indicator of gene expression efficiency of transgenic animals, their short lifespan greatly limits this application. To provide stable and long lifespan cells, goat mammary epithelial cells (GMECs) were transduced with pLNCX2-hTERT by retrovirus-mediated gene transfer. Transduced GMECs were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR), proliferation assays, karyotype analysis, telomerase activity assay, western blotting, soft agar assay, and injection into nude mice. Non-transduced GMECs were used as a control. The hTERT-GMECs had higher telomerase activity and extended proliferative lifespan compared to non-transfected GMECs; even after Passage 50, hTERT-GMECs had a near diploid complement of chromosomes. Furthermore, they did not gain the anchorage-independent growth property and were not associated with a malignant phenotype in vitro or in vivo.

Human telomerase reverse transcriptase is a promising target for cancer inhibition in squamous cell carcinomas.

PubMed

Park, Young-Jin; Kim, Eun-Kyoung; Moon, Sook; Hong, Doo-Pyo; Bae, Jung Yoon; Kim, Jin

2014-11-01

The present study aimed to investigate whether the down-regulation of human telomerase reverse transcriptase (hTERT) may induce an anti-invasive effect in oral squamous cell cancer cell lines. A genetically-engineered squamous carcinoma cell line overexpressing hTERT in immortalized oral keratinocytes transfected by human papilloma virus (HPV)-16 E6/E7 (IHOK) was used. In vivo tumorigenicity was examined using an orthotopic xenograft model of nude mice. For evaluating anti-invasive activity by knockdown of hTERT expression, transwell invasion assay and real-time polymerase chain reaction (PCR) for matrix metalloproteinases (MMP) were employed. The down-regulation of hTERT expression reduced the invasive activity and MMP expression. This result was re-confirmed in the HSC3 oral squamous carcinoma cell line. Targeting hTERT may lead to novel therapeutic approaches. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Comparison of IHC, FISH and RT-PCR methods for detection of ALK rearrangements in 312 non-small cell lung cancer patients in Taiwan.

PubMed

Wu, Yi-Cheng; Chang, Il-Chi; Wang, Chi-Liang; Chen, Tai-Di; Chen, Ya-Ting; Liu, Hui-Ping; Chu, Yen; Chiu, Yu-Ting; Wu, Tzu-Hua; Chou, Li-Hui; Chen, Yi-Rong; Huang, Shiu-Feng

2013-01-01

Recently Echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase (EML4-ALK) fusion gene has become an important biomarker for ALK tyrosine kinase inhibitor (crizotinib) treatment in NSCLC. However, the best detection method and the significance of EML4-ALK variant types remain uncertain. Reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence in Situ hybridization (FISH) and Immunohistochemical (IHC) stain were performed on tumor tissues of 312 NSCLC patients for detection of ALK rearrangements. Mutation analyses for EGFR and KRAS genes were also performed. Thirteen of the 312 patients (4.17%) had ALK rearrangements detected by RT-PCR. If RT-PCR data was used as the gold standard, FISH tests had a low sensitivity (58.33%), but very good specificity (99.32%). IHC stain had better sensitivity (91.67%) than FISH, but lower specificity (79.52%), when the cut off was IHC2+. All of the 8 patients with high abundance of EML4-ALK positive cells in tumor tissues (assessed by the signal intensities of the RT-PCR product), were also have high expression of ALK protein (IHC3+), and positive for FISH, except one failed in FISH. Variants 3a+3b (4/5, 80%) of EML4-ALK fusion gene were more common to have high abundance of EML4-ALK positive cells in tumor tissues than variant 1 (1/3, 33.3%). Meta-analysis of the published data of 2273 NSCLC patients revealed that variant 3 (23/44, 52.3%) was the most common type in Chinese population, while variant 1 (28/37, 75.7%) was most common in Caucasian. Among the three detection methods, RT-PCR could detect not only the presence of EML4-ALK fusion gene and their variant types, but also the abundance of EML4-ALK positive cells in NSCLC tumor tissues. The latter two factors might affect the treatment response to anti-ALK inhibitor. Including RT-PCR as a diagnostic test for ALK inhibitor treatment in the prospective clinical trials is recommended.

Comparison of IHC, FISH and RT-PCR Methods for Detection of ALK Rearrangements in 312 Non-Small Cell Lung Cancer Patients in Taiwan

PubMed Central

Wang, Chi-Liang; Chen, Tai-Di; Chen, Ya-Ting; Liu, Hui-Ping; Chu, Yen; Chiu, Yu-Ting; Wu, Tzu-Hua; Chou, Li-Hui; Chen, Yi-Rong; Huang, Shiu-Feng

2013-01-01

Background Recently Echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase (EML4-ALK) fusion gene has become an important biomarker for ALK tyrosine kinase inhibitor (crizotinib) treatment in NSCLC. However, the best detection method and the significance of EML4-ALK variant types remain uncertain. Methods Reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence in Situ hybridization (FISH) and Immunohistochemical (IHC) stain were performed on tumor tissues of 312 NSCLC patients for detection of ALK rearrangements. Mutation analyses for EGFR and KRAS genes were also performed. Results Thirteen of the 312 patients (4.17%) had ALK rearrangements detected by RT-PCR. If RT-PCR data was used as the gold standard, FISH tests had a low sensitivity (58.33%), but very good specificity (99.32%). IHC stain had better sensitivity (91.67%) than FISH, but lower specificity (79.52%), when the cut off was IHC2+. All of the 8 patients with high abundance of EML4-ALK positive cells in tumor tissues (assessed by the signal intensities of the RT-PCR product), were also have high expression of ALK protein (IHC3+), and positive for FISH, except one failed in FISH. Variants 3a+3b (4/5, 80%) of EML4-ALK fusion gene were more common to have high abundance of EML4-ALK positive cells in tumor tissues than variant 1 (1/3, 33.3%). Meta-analysis of the published data of 2273 NSCLC patients revealed that variant 3 (23/44, 52.3%) was the most common type in Chinese population, while variant 1 (28/37, 75.7%) was most common in Caucasian. Conclusions Among the three detection methods, RT-PCR could detect not only the presence of EML4-ALK fusion gene and their variant types, but also the abundance of EML4-ALK positive cells in NSCLC tumor tissues. The latter two factors might affect the treatment response to anti-ALK inhibitor. Including RT-PCR as a diagnostic test for ALK inhibitor treatment in the prospective clinical trials is recommended. PMID:23951022

Identification of reference genes for RT-qPCR analysis in peach genotypes with contrasting chilling requirements.

PubMed

Marini, N; Bevilacqua, C B; Büttow, M V; Raseira, M C B; Bonow, S

2017-05-25

Selecting and validating reference genes are the first steps in studying gene expression by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). The present study aimed to evaluate the stability of five reference genes for the purpose of normalization when studying gene expression in various cultivars of Prunus persica with different chilling requirements. Flower bud tissues of nine peach genotypes from Embrapa's peach breeding program with different chilling requirements were used, and five candidate reference genes based on the RT-qPCR that were useful for studying the relative quantitative gene expression and stability were evaluated using geNorm, NormFinder, and bestKeeper software packages. The results indicated that among the genes tested, the most stable genes to be used as reference genes are Act and UBQ10. This study is the first survey of the stability of reference genes in peaches under chilling stress and provides guidelines for more accurate RT-qPCR results.

Development of a real-time RT-PCR assay for a novel influenza A (H1N1) virus.

PubMed

Jiang, Tao; Kang, Xiaoping; Deng, Yongqiang; Zhao, Hui; Li, Xiaofeng; Yu, Xuedong; Yu, Man; Qin, Ede; Zhu, Qingyu; Yang, Yinhui; Qin, Chengfeng

2010-02-01

A pandemic caused by a novel influenza A virus (H1N1) poses a serious public health threat. In this study, a real-time reverse transcriptase PCR (RT-PCR) assay based on the hemagglutinin gene was developed that discriminates the novel H1N1 from swine influenza virus, seasonal H1N1/H3N2 virus and the highly pathogenic H5N1 avian influenza virus. The sensitivity of this assay was 0.2 50% tissue culture infective dose of virus and 200 copies of in vitro-transcribed target RNA. Three hundred and forty-eight clinical specimens from suspected H1N1 patients were tested using this assay, and forty-two (12.07%) were found to be positive. Tests using the real-time PCR assay recommended by WHO and virus isolation gave identical results. This sensitive and specific real-time RT-PCR assay will contribute to the early diagnosis and control of the emerging H1N1 influenza pandemic. 2009 Elsevier B.V. All rights reserved.

Non-nucleoside reverse transcriptase inhibitor efavirenz increases monolayer permeability of human coronary artery endothelial cells

PubMed Central

Jamaluddin, Md Saha; Lin, Peter H.; Yao, Qizhi; Chen, Changyi

2009-01-01

Highly active antiretroviral therapy (HAART) is often associated with endothelial dysfunction and cardiovascular complications. In this study, we determined whether HIV non-nucleoside reverse transcriptase inhibitor efavirenz (EFV) could increase endothelial permeability. Human coronary artery endothelial cells (HCAECs) were treated with EFV (1, 5 and 10 µg/ml) and endothelial permeability was determined by a transwell system with a fluorescence-labeled dextran tracer. HCAECs treated with EFV showed a significant increase of endothelial permeability in a concentration-dependent manner. With real time PCR analysis, EFV significantly reduced the mRNA levels of tight junction proteins claudin-1, occludin, zonula occluden-1 and junctional adhesion molecule-1 compared with controls (P < 0.05). Protein levels of these tight junction molecules were also reduced substantially in the EFV-treated cells by western blot and flow cytometry analyses. In addition, EFV also increased superoxide anion production with dihydroethidium and cellular glutathione assays, while it decreased mitochondrial membrane potential with JC-staining. Antioxidants (ginkgolide B and MnTBAP) effectively blocked EFV-induced endothelial permeability and mitochondrial dysfunction. Furthermore, EFV increased the phosphorylation of MAPK JNK and IκBα, thereby increasing NFκB translocation to the nucleus. Chemical JNK inhibitor and dominant negative mutant JNK and IkBa adenoviruses effectively blocked the effects of EFV on HCAECs. Thus, EFV increases endothelial permeability which may be due to the decrease of tight junction proteins and the increase of superoxide anion. JNK and NFκB activation may be directly involved in the signal transduction pathway of EFV action in HCAECs. PMID:19674747

Application of Coamplification at Lower Denaturation Temperature-PCR Sequencing for Early Detection of Antiviral Drug Resistance Mutations of Hepatitis B Virus

PubMed Central

Wong, Danny Ka-Ho; Tsoi, Ottilia; Huang, Fung-Yu; Seto, Wai-Kay; Fung, James; Lai, Ching-Lung

2014-01-01

Nucleoside/nucleotide analogue for the treatment of chronic hepatitis B virus (HBV) infection is hampered by the emergence of drug resistance mutations. Conventional PCR sequencing cannot detect minor variants of <20%. We developed a modified co-amplification at lower denaturation temperature-PCR (COLD-PCR) method for the detection of HBV minority drug resistance mutations. The critical denaturation temperature for COLD-PCR was determined to be 78°C. Sensitivity of COLD-PCR sequencing was determined using serially diluted plasmids containing mixed proportions of HBV reverse transcriptase (rt) wild-type and mutant sequences. Conventional PCR sequencing detected mutations only if they existed in ≥25%, whereas COLD-PCR sequencing detected mutations when they existed in 5 to 10% of the viral population. The performance of COLD-PCR was compared to conventional PCR sequencing and a line probe assay (LiPA) using 215 samples obtained from 136 lamivudine- or telbivudine-treated patients with virological breakthrough. Among these 215 samples, drug resistance mutations were detected in 155 (72%), 148 (69%), and 113 samples (53%) by LiPA, COLD-PCR, and conventional PCR sequencing, respectively. Nineteen (9%) samples had mutations detectable by COLD-PCR but not LiPA, while 26 (12%) samples had mutations detectable by LiPA but not COLD-PCR, indicating both methods were comparable (P = 0.371). COLD-PCR was more sensitive than conventional PCR sequencing. Thirty-five (16%) samples had mutations detectable by COLD-PCR but not conventional PCR sequencing, while none had mutations detected by conventional PCR sequencing but not COLD-PCR (P < 0.0001). COLD-PCR sequencing is a simple method which is comparable to LiPA and superior to conventional PCR sequencing in detecting minor lamivudine/telbivudine resistance mutations. PMID:24951803

Modified concentration method for the detection of enteric viruses on fruits and vegetables by reverse transcriptase-polymerase chain reaction or cell culture.

PubMed

Dubois, Eric; Agier, Cécilia; Traoré, Ousmane; Hennechart, Catherine; Merle, Ghislaine; Crucière, Catherine; Laveran, Henri

2002-12-01

Fruits and vegetables may act as a vehicle of human enteric virus if they are irrigated with sewage-contaminated water or prepared by infected food handlers. An elution-concentration method was modified to efficiently detect, by reverse transcriptase-polymerase chain reaction (RT-PCR) or by cell culture, contamination by poliovirus, hepatitis A virus (HAV), and Norwalk-like virus (NLV) of various fresh and frozen berries and fresh vegetables. The protocol included washing the fruit or vegetable surface with 100 mM Tris-HCl, 50 mM glycine, and 3% beef extract, pH 9.5 buffer, which favors viral elution from acid-releasing berries, supplemented with 50 mM MgCl2 to reduce the decrease in viral infectivity during the process. The viral concentration method was based on polyethylene glycol precipitation. Copurified RT-PCR inhibitors and cytotoxic compounds were removed from viral concentrates by chloroform-butanol extraction. Viruses from 100 g of vegetal products could be recovered in volumes of 3 to 5 ml. Viral RNAs were isolated by a spin column method before molecular detection or concentrates were filtered (0.22-microm porosity) and inoculated on cell culture for infectious virus detection. About 15% of infectious poliovirus and 20% of infectious HAV were recovered from frozen raspberry surfaces. The percentage of viral RNA recovery was estimated by RT-PCR to be about 13% for NLV, 17% for HAV, and 45 to 100% for poliovirus. By this method, poliovirus and HAV RNA were detected on products inoculated with a titer of about 5 x 10(1) 50% tissue culture infectious dose per 100 g. NLV RNA was detected at an initial inoculum of 1.2 x 10(3) RT-PCR amplifiable units. This method would be useful for the viral analysis of fruits or vegetables during an epidemiological investigation of foodborne diseases.

Retrotransposon expression and incorporation of cloned human and mouse retroelements in human spermatozoa.

PubMed

Lazaros, Leandros; Kitsou, Chrysoula; Kostoulas, Charilaos; Bellou, Sofia; Hatzi, Elissavet; Ladias, Paris; Stefos, Theodoros; Markoula, Sofia; Galani, Vasiliki; Vartholomatos, Georgios; Tzavaras, Theodore; Georgiou, Ioannis

2017-03-01

To investigate the expression of long interspersed element (LINE) 1, human endogenous retrovirus (HERV) K10, and short interspersed element-VNTR-Alu element (SVA) retrotransposons in ejaculated human spermatozoa by means of reverse-transcription (RT) polymerase chain reaction (PCR) analysis as well as the potential incorporation of cloned human and mouse active retroelements in human sperm cell genome. Laboratory study. University research laboratories and academic hospital. Normozoospermic and oligozoospermic white men. RT-PCR analysis was performed to confirm the retrotransposon expression in human spermatozoa. Exogenous retroelements were tagged with a plasmid containing a green fluorescence (EGFP) retrotransposition cassette, and the de novo retrotransposition events were tested with the use of PCR, fluorescence-activated cell sorting analysis, and confocal microscopy. Retroelement expression in human spermatozoa, incorporation of cloned human and mouse active retroelements in human sperm genome, and de novo retrotransposition events in human spermatozoa. RT-PCR products of expressed human LINE-1, HERV-K10, and SVA retrotransposons were observed in ejaculated human sperm samples. The incubation of human spermatozoa with either retrotransposition-active human LINE-1 and HERV-K10 or mouse reverse transcriptase-deficient VL30 retrotransposons tagged with an EGFP-based retrotransposition cassette led to EGFP-positive spermatozo; 16.67% of the samples were positive for retrotransposition. The respective retrotransposition frequencies for the LINE-1, HERV-K10, and VL30 retrotransposons in the positive samples were 0.34 ± 0.13%, 0.37 ± 0.17%, and 0.30 ± 0.14% per sample of 10,000 spermatozoa. Our results show that: 1) LINE-1, HERV-K10, and SVA retrotransposons are transcriptionally expressed in human spermatozoa; 2) cloned active retroelements of human and mammalian origin can be incorporated in human sperm genome; 3) active reverse transcriptases exist in human spermatozoa; and 4) de novo retrotransposition events occur in human spermatozoa. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Biodegradation of trichloroethylene (TCE) by methanotrophic community.

PubMed

Shukla, Awadhesh K; Vishwakarma, Pranjali; Upadhyay, S N; Tripathi, Anil K; Prasana, H C; Dubey, Suresh K

2009-05-01

Laboratory incubation experiments were carried out to assess the potential of methanotrophic culture for degrading TCE. Measurements of the growth rate and TCE degradation showed that the methanotrophs not only grew in presence of TCE but also degraded TCE. The rate of TCE degradation was found to be 0.19 ppm h(-1). The reverse transcriptase-PCR test was conducted to quantify expression of pmoA and mmoX genes. RT-PCR revealed expression of pmoA gene only. This observation provides evidence that the pmoA gene was functionally active for pMMO enzyme during the study. The diversity of the methanotrophs involved in TCE degradation was assessed by PCR amplification, cloning, restriction fragment length polymorphism and phylogenetic analysis of pmoA genes. Results suggested the occurrence of nine different phylotypes belonging to Type II methanotrophs in the enriched cultures. Out of the nine, five clustered with, genera Methylocystis and rest got clustered in to a separate group.

Incidence of Enteric Viruses in Groundwater from Household Wells in Wisconsin

PubMed Central

Borchardt, Mark A.; Bertz, Phil D.; Spencer, Susan K.; Battigelli, David A.

2003-01-01

Recent studies on the contamination of groundwater with human enteric viruses have focused on public water systems, whereas little is known about the occurrence of viruses in private household wells. The objective of the present study was to estimate the incidence of viruses in Wisconsin household wells located near septage land application sites or in rural subdivisions served by septic systems. Fifty wells in seven hydrogeologic districts were sampled four times over a year, once each season. Reverse transcriptase PCR (RT-PCR), followed by Southern hybridization, was used to detect enteroviruses, rotavirus, hepatitis A virus (HAV), and Norwalk-like viruses (NLVs). In addition, cell culture was used to detect culturable enteroviruses. Companion water samples were collected for total coliforms, Escherichia coli, fecal enterococci, F-specific RNA coliphages, nitrate, and chloride analyses. Among the 50 wells, four (8%) were positive for viruses by RT-PCR. Three wells were positive for HAV, and the fourth well was positive for both rotavirus and NLV in one sample and an enterovirus in another sample. Contamination was transient, since none of the wells was virus positive for two sequential samples. Culturable enteroviruses were not detected in any of the wells. Water quality indicators were not statistically associated with virus occurrence, although some concordance was noted for chloride. The present study is the first in the United States to systematically monitor private household wells for virus contamination and, combined with data for public wells, provides further insight on the extent of groundwater contamination with human enteric viruses. PMID:12571044

A rapid single-tube protocol for HAV detection by nested real-time PCR.

PubMed

Hu, Yuan; Arsov, Ivica

2014-09-01

Infections by food-borne viruses such as hepatitis A virus (HAV) and norovirus are significant public health concerns worldwide. Since food-borne viruses are rarely confirmed through direct isolation from contaminated samples, highly sensitive molecular techniques remain the methods of choice for the detection of viral genetic material. Our group has previously developed a specific nested real-time PCR (NRT-PCR) assay for HAV detection that improved overall sensitivity. Furthermore in this study, we have developed a single-tube NRT-PCR approach for HAV detection in food samples that reduces the likelihood of cross contamination between tubes during sample manipulation. HAV RNA was isolated from HAV-spiked food samples and HAV-infected cell cultures. All reactions following HAV RNA isolation, including conventional reverse transcriptase PCR, nested-PCR, and RT-PCR were performed in a single tube. Our results demonstrated that all the samples tested positive by RT-PCR and nested-PCR were also positive by a single-tube NRT-PCR. The detection limits observed for HAV-infected cell cultures and HAV-spiked green onions were 0.1 and 1 PFU, respectively. This novel method retained the specificity and robustness of the original NRT-PCR method, while greatly reducing sample manipulation, turnaround time, and the risk of carry-over contamination. Single-tube NRT-PCR thus represents a promising new tool that can potentially facilitate the detection of HAV in foods thereby improving food safety and public health.

Digital-Direct-RT-PCR: a sensitive and specific method for quantification of CTC in patients with cervical carcinoma

PubMed Central

Pfitzner, Claudia; Schröder, Isabel; Scheungraber, Cornelia; Dogan, Askin; Runnebaum, Ingo Bernhard; Dürst, Matthias; Häfner, Norman

2014-01-01

The detection of circulating tumour cells (CTC) in cancer patients may be useful for therapy monitoring and prediction of relapse. A sensitive assay based on HPV-oncogene transcripts which are highly specific for cervical cancer cells was established. The Digital-Direct-RT-PCR (DD-RT-PCR) combines Ficoll-separation, ThinPrep-fixation and one-step RT-PCR in a low-throughput digital-PCR format enabling the direct analysis and detection of individual CTC without RNA isolation. Experimental samples demonstrated a sensitivity of one HPV-positive cell in 500,000 HPV-negative cells. Spike-in experiments with down to 5 HPV-positive cells per millilitre EDTA-blood resulted in concordant positive results by PCR and immunocytochemistry. Blood samples from 3 of 10 CxCa patients each contained a single HPV-oncogene transcript expressing CTC among 5 to 15*105 MNBC. Only 1 of 7 patients with local but 2 of 3 women with systemic disease had CTC. This highly sensitive DD-RT-PCR for the detection of CTC may also be applied to other tumour entities which express tumour-specific transcripts. Abbreviations: CTC – circulating tumour cells, CxCa – cervical cancer, DD-RT-PCR – Digital-Direct Reverse Transcriptase PCR, HPV – Human Papilloma Virus, MNBC – mononuclear blood cells, ICC – immunocytochemistry. PMID:24496006

Sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction detecting feline coronavirus mutations in effusion and serum/plasma of cats to diagnose feline infectious peritonitis.

PubMed

Felten, Sandra; Leutenegger, Christian M; Balzer, Hans-Joerg; Pantchev, Nikola; Matiasek, Kaspar; Wess, Gerhard; Egberink, Herman; Hartmann, Katrin

2017-08-02

Feline coronavirus (FCoV) exists as two pathotypes, and FCoV spike gene mutations are considered responsible for the pathotypic switch in feline infectious peritonitis (FIP) pathogenesis. The aim of this study was to evaluate sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) specifically designed to detect FCoV spike gene mutations at two nucleotide positions. It was hypothesized that this test would correctly discriminate feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV). The study included 63 cats with signs consistent with FIP. FIP was confirmed in 38 cats. Twenty-five control cats were definitively diagnosed with a disease other than FIP. Effusion and/or serum/plasma samples were examined by real-time RT-PCR targeting the two FCoV spike gene fusion peptide mutations M1058 L and S1060A using an allelic discrimination approach. Sensitivity, specificity, negative and positive predictive values including 95% confidence intervals (95% CI) were calculated. FIPV was detected in the effusion of 25/59 cats, one of them being a control cat with chronic kidney disease. A mixed population of FIPV/FECV was detected in the effusion of 2/59 cats; all of them had FIP. RT-PCR was negative or the pathotype could not be determined in 34/59 effusion samples. In effusion, sensitivity was 68.6% (95% CI 50.7-83.2), specificity was 95.8% (95% CI 78.9-99.9). No serum/plasma samples were positive for FIPV. Although specificity of the test in effusions was high, one false positive result occurred. The use of serum/plasma cannot be recommended due to a low viral load in blood.

Isolation, cDNA cloning and gene expression of an antibacterial protein from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros.

PubMed

Yang, J; Yamamoto, M; Ishibashi, J; Taniai, K; Yamakawa, M

1998-08-01

An antibacterial protein, designated rhinocerosin, was purified to homogeneity from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros immunized with Escherichia coli. Based on the amino acid sequence of the N-terminal region, a degenerate primer was synthesized and reverse-transcriptase PCR was performed to clone rhinocerosin cDNA. As a result, a 279-bp fragment was obtained. The complete nucleotide sequence was determined by sequencing the extended rhinocerosin cDNA clone by 5' rapid amplification of cDNA ends. The deduced amino acid sequence of the mature portion of rhinocerosin was composed of 72 amino acids without cystein residues and was shown to be rich in glycine (11.1%) and proline (11.1%) residues. Comparison of the deduced amino acid sequence of rhinocerosin with those of other antibacterial proteins indicated that it has 77.8% and 44.6% identity with holotricin 2 and coleoptrecin, respectively. Rhinocerosin had strong antibacterial activity against E. coli, Streptococcus pyogenes, Staphylococcus aureus but not against Pseudomonas aeruginosa. Results of reverse-transcriptase PCR analysis of gene expression in different tissues indicated that the rhinocerosin gene is strongly expressed in the fat body and the Malpighian tubule, and weakly expressed in hemocytes and midgut. In addition, gene expression was inducible by bacteria in the fat body, the Malpighian tubule and hemocyte but constitutive expression was observed in the midgut.

Glutathione Transferase as a Potential Marker for Gut Epithelial Injury versus the Protective Role of Breast Milk sIgA in Infants with Rota Virus Gastroenteritis

PubMed Central

Sherif, Lobna S.; Raouf, Randaa K. Abdel; Sayede, Rokaya M. El; Wakkadd, Amany S. El; Shoaib, Ashraf R.; Ali, Hanan M.; Refay, Amira S. El

2015-01-01

BACKGROUND: Secretory immunoglobulin A (SIgA) plays an important protective role in the recognition and clearance of enteric pathogens. AIM: This study was designed to assess if mucosal integrity “measured by secretory IgA (SIgA)” is a protective factor from more epithelial alteration “measured by glutathione transferase” in infants with Rota gastroenteritis and its relation to infants’ feeding pattern. PATIENTS AND METHODS: This study was conducted on 79 infants aged 6 months and less from those diagnosed as having gastroenteritis and admitted to Gastroenteritis Department in Abo El Rish Pediatric Hospital, Cairo University. Plasma glutathione s-transferases and Stool SIgA were measured using ELISA technique. Rota virus detection was done by Reverse transcriptase PCR. RESULTS: SIgA was found to be significantly positive in exclusive breast fed infants, Glutathione transferase was significantly more frequently positive in Rota positive cases than Rota negative cases by Reverse transcriptase PCR. A significant negative correlation between Glutathione transferase and Secretory IgA was found, (p < 0.05). CONCLUSION: Breast feeding should be encouraged and highly recommended in the first two years of life as it provides Secretory IgA to breast fed infants who in turn protect them against epithelial damage caused by Rota viral gastroenteritis. PMID:27275307

Screening, isolation and optimization of anti–white spot syndrome virus drug derived from marine plants

PubMed Central

Chakraborty, Somnath; Ghosh, Upasana; Balasubramanian, Thangavel; Das, Punyabrata

2014-01-01

Objective To screen, isolate and optimize anti-white spot syndrome virus (WSSV) drug derived from various marine floral ecosystems and to evaluate the efficacy of the same in host–pathogen interaction model. Methods Thirty species of marine plants were subjected to Soxhlet extraction using water, ethanol, methanol and hexane as solvents. The 120 plant isolates thus obtained were screened for their in vivo anti-WSSV property in Litopenaeus vannamei. By means of chemical processes, the purified anti-WSSV plant isolate, MP07X was derived. The drug was optimized at various concentrations. Viral and immune genes were analysed using reverse transcriptase PCR to confirm the potency of the drug. Results Nine plant isolates exhibited significant survivability in host. The drug MP07X thus formulated showing 85% survivability in host. The surviving shrimps were nested PCR negative at the end of the 15 d experimentation. The lowest concentration of MP07X required intramuscularly for virucidal property was 10 mg/mL. The oral dosage of 1 000 mg/kg body weight/day survived at the rate of 85%. Neither VP28 nor ie 1 was expressed in the test samples at 42nd hour and 84th hour post viral infection. Conclusions The drug MP07X derived from Rhizophora mucronata is a potent anti-WSSV drug. PMID:25183065

Expression of c-Jun and Bcl-2 family proteins in apoptotic photoreceptors of RCS rats.

PubMed

Katai, Naomichi; Yanagidaira, Tomoko; Senda, Nami; Murata, Toshinori; Yoshimura, Nagahisa

2006-01-01

To determine if c-Jun and Bcl-2 family proteins play a role in photoreceptor apoptosis in Royal College of Surgeons (RCS) rats. RCS and Sprague-Dawley rats were used. Cryosections of retinas harvested at various postnatal periods were immunostained with antibodies against c-Jun, Bcl-2, and Bax. Double staining with TdT-dUTP nick-end labeling (TUNEL) or propidium iodide (PI) and antibodies was also done. To study the time course of gene and protein expression, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting analyses were carried out. TUNEL-positive photoreceptors of RCS rats were stained strongly with antibodies against c-Jun and Bax. The number of immunoreactive cells increased on days 21 and 28 after birth (P21 and P28) and decreased on P45. Semiquantitative RT-PCR analysis showed that mRNAs for c-Jun and Bax were upregulated at P21 and P28, but those for Bcl-2 were unchanged. On immunoblotting, a 43-kDa band was revealed by the anti-c-Jun antibody and a 21-kDa band, by the anti-Bax antibody. Protein expression of c-Jun and Bax were increased at both P21 and P28. The temporal profiles of immunoreactivity, protein expression, and mRNA expression were similar. c-Jun and Bax may play a role in photoreceptor apoptosis in RCS rats.

Human epidermal growth factor receptor 2 assessment in a case-control study: comparison of fluorescence in situ hybridization and quantitative reverse transcription polymerase chain reaction performed by central laboratories.

PubMed

Baehner, Frederick L; Achacoso, Ninah; Maddala, Tara; Shak, Steve; Quesenberry, Charles P; Goldstein, Lynn C; Gown, Allen M; Habel, Laurel A

2010-10-01

The optimal method to assess human epidermal growth factor receptor 2 (HER2) status remains highly controversial. Before reporting patient HER2 results, American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines mandate that laboratories demonstrate ≥ 95% concordance to another approved laboratory or methodology. Here, we compare central laboratory HER2 assessed by fluorescence in situ hybridization (FISH) and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) using Oncotype DX in lymph node-negative, chemotherapy-untreated patients from a large Kaiser Permanente case-control study. Breast cancer specimens from the Kaiser-Genomic Health study were examined. Central FISH assessment of HER2 amplification and polysomy 17 was conducted by PhenoPath Laboratories (ratios > 2.2, 1.8 to 2.2, and < 1.8 define HER2 positive, HER2 equivocal, and HER2 negative, respectively). HER2 expression by RT-PCR was conducted using Oncotype DX by Genomic Health (normalized expression units ≥ 11.5, 10.7 to < 11.5, and < 10.7 define HER2 positive, HER2 equivocal, and HER2 negative, respectively). Concordance analyses followed ASCO/CAP guidelines. HER2 concordance by central FISH and central RT-PCR was 97% (95% CI, 96% to 99%). Twelve percent (67 of 568 patients) and 11% (60 of 568 patients) of patients were HER2 positive by RT-PCR and FISH, respectively. HER2-positive patients had increased odds of dying from breast cancer compared with HER2-negative patients. Polysomy 17 was demonstrated in 12.5% of all patients and 33% of FISH-positive patients. Nineteen of 20 FISH-positive patients with polysomy 17 were also RT-PCR HER2 positive. Although not statistically significantly different, HER2-positive/polysomy 17 patients tended to have the worst prognosis, followed by HER2-positive/eusomic, HER2-negative/polysomy 17, and HER2-negative/eusomic patients. There is a high degree of concordance between central FISH and quantitative RT-PCR using Oncotype DX for HER2 status, and the assay warrants additional study in a trastuzumab-treated population.

Effects of Anticoagulant, Processing Delay, and Assay Method (Branched DNA versus Reverse Transcriptase PCR) on Measurement of Human Immunodeficiency Virus Type 1 RNA Levels in Plasma

PubMed Central

Kirstein, Lynn M.; Mellors, John W.; Rinaldo, Charles R.; Margolick, Joseph B.; Giorgi, Janis V.; Phair, John P.; Dietz, Edith; Gupta, Phalguni; Sherlock, Christopher H.; Hogg, Robert; Montaner, J. S. G.; Muñoz, Alvaro

1999-01-01

We conducted two studies to determine the potential influence of delays in blood processing, type of anticoagulant, and assay method on human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma. The first was an experimental study in which heparin- and EDTA-anticoagulated blood samples were collected from 101 HIV-positive individuals and processed to plasma after delays of 2, 6, and 18 h. HIV-1 RNA levels in each sample were then measured by both branched-DNA (bDNA) and reverse transcriptase PCR (RT-PCR) assays. Compared to samples processed within 2 h, the loss (decay) of HIV-1 RNA in heparinized blood was significant (P < 0.05) but small after 6 h (bDNA assay, −0.12 log10 copies/ml; RT-PCR, −0.05 log10 copies/ml) and after 18 h (bDNA assay, −0.27 log10 copies/ml; RT-PCR, −0.15 log10 copies/ml). Decay in EDTA-anticoagulated blood was not significant after 6 h (bDNA assay, −0.002 log10 copies/ml; RT-PCR, −0.02 log10 copies/ml), but it was after 18 h (bDNA assay, −0.09 log10 copies/ml; RT-PCR, −0.09 log10 copies/ml). Only 4% of samples processed after 6 h lost more than 50% (≥0.3 log10 copies/ml) of the HIV-1 RNA, regardless of the anticoagulant or the assay that was used. The second study compared HIV-1 RNA levels in samples from the Multicenter AIDS Cohort Study (MACS; samples were collected in heparin-containing tubes in 1985, had a 6-h average processing delay, and were assayed by bDNA assay) and the British Columbia Drug Treatment Program (BCDTP) (collected in EDTA- or acid citrate dextrose-containing tubes in 1996 and 1997, had a 2-h maximum processing delay, and were assayed by RT-PCR). HIV-1 RNA levels in samples from the two cohorts were not significantly different after adjusting for CD4+-cell count and converting bDNA assay values to those corresponding to the RT-PCR results. In summary, the decay of HIV-1 RNA measured in heparinized blood after 6 h was small (−0.05 to −0.12 log10 copies/ml), and the minor impact of this decay on HIV-1 RNA concentrations in archived plasma samples of the MACS was confirmed by the similarity of CD4+-cell counts and assay-adjusted HIV-1 RNA concentrations in the MACS and BCDTP. PMID:10405379

Strong Inverse Correlation Between MicroRNA-125b and Human Papillomavirus DNA in Productive Infection

PubMed Central

Nuovo, Gerard J.; Wu, Xin; Volinia, Stefano; Yan, Fengting; di Leva, Gianpiero; Chin, Nena; Nicol, Alcina F.; Jiang, Jinmai; Otterson, Gregory; Schmittgen, Thomas D.; Croce, Carlo

2014-01-01

Infection by the human papillomavirus (HPV) is a cause of cervical intraepithelial neoplasia (CIN) and cancer. microRNA (miRNA) in situ analysis of the transformation zone epithelia, the site of initial cervical HPV infection, showed that miRNAs let-7c, — 99a, 26a, and 125b were the most abundantly expressed. In situ testing of CIN 1 showed a dramatic reduction in miR-125b expression in the koilocytes, the cytologic marker of productive HPV infection. A marked reduction in miR-125b was likewise observed in the HPV-infected cells of the condyloma acuminatum, verruca vulgaris, and epidermodysplasia verruciformis. Reverse transcriptase in situ polymerase chain reaction (PCR) showed that the pre-miRNA 125b was present in the koilocyte, suggesting direct inactivation of the mature miRNA. HEK cells transfected with only the antimiR-125b showed perinuclear halos equivalent to HPV-infected koilocytes. NIH 3T3 cells transfected with the HPV 16 full-length genome and mimetic miR-125b showed a marked reduction in viral DNA and protein synthesis by quantitative PCR and in situ-based analyses, respectively (P=0.002). Alternatively, cotransfection with anti-miR-125b and HPV 16 markedly increased HPV DNA (P=0.002). Sequence analyses showed strong homology between L2 of different HPV genotypes and miR-125b. Transfection with HPV 16 L2 resulted in a marked reduction in miR-125b levels in the NIH 3T3 cells. HPV L2-induced inactivation of miR-125b is associated with the classic cytologic changes of the koilocyte, and the exogenous application of mimetic miR-125b markedly inhibits HPV DNA synthesis. PMID:20736742

Strong inverse correlation between microRNA-125b and human papillomavirus DNA in productive infection.

PubMed

Nuovo, Gerard J; Wu, Xin; Volinia, Stefano; Yan, Fengting; di Leva, Gianpiero; Chin, Nena; Nicol, Alcina F; Jiang, Jinmai; Otterson, Gregory; Schmittgen, Thomas D; Croce, Carlo

2010-09-01

Infection by the human papillomavirus (HPV) is a cause of cervical intraepithelial neoplasia (CIN) and cancer. microRNA (miRNA) in situ analysis of the transformation zone epithelia, the site of initial cervical HPV infection, showed that miRNAs let-7c, -99a, 26a, and 125b were the most abundantly expressed. In situ testing of CIN 1 showed a dramatic reduction in miR-125b expression in the koilocytes, the cytologic marker of productive HPV infection. A marked reduction in miR-125b was likewise observed in the HPV-infected cells of the condyloma acuminatum, verruca vulgaris, and epidermodysplasia verruciformis. Reverse transcriptase in situ polymerase chain reaction (PCR) showed that the pre-miRNA 125b was present in the koilocyte, suggesting direct inactivation of the mature miRNA. HEK cells transfected with only the antimiR-125b showed perinuclear halos equivalent to HPV-infected koilocytes. NIH 3T3 cells transfected with the HPV 16 full-length genome and mimetic miR-125b showed a marked reduction in viral DNA and protein synthesis by quantitative PCR and in situ-based analyses, respectively (P=0.002). Alternatively, cotransfection with anti-miR-125b and HPV 16 markedly increased HPV DNA (P=0.002). Sequence analyses showed strong homology between L2 of different HPV genotypes and miR-125b. Transfection with HPV 16 L2 resulted in a marked reduction in miR-125b levels in the NIH 3T3 cells. HPV L2-induced inactivation of miR-125b is associated with the classic cytologic changes of the koilocyte, and the exogenous application of mimetic miR-125b markedly inhibits HPV DNA synthesis.

Development of Real-Time Reverse Transcriptase PCR Assays for the Detection of Punta Toro Virus and Pichinde Virus

DTIC Science & Technology

2016-09-09

Gowen et al., 2006c; Smee et al., 1993) and guinea pigs (Jahrling et al., 1981; Lucia et al., 1989) 91 as LASV infection in humans. Both PICV and...Moe, J.B., 1981. Pathogenesis of a pichinde virus 281 strain adapted to produce lethal infections in guinea pigs . Infect Immun 32, 872-880. 282... guinea pig model: antiviral 286 therapy with recombinant interferon-alpha, the immunomodulator CL246,738 and ribavirin. Antiviral 287 Res 12, 279-292

Simultaneous Quantification of Multiple Alternatively Spliced mRNA Transcripts Using Droplet Digital PCR.

PubMed

Sun, Bing; Zheng, Yun-Ling

2018-01-01

Currently there is no sensitive, precise, and reproducible method to quantitate alternative splicing of mRNA transcripts. Droplet digital™ PCR (ddPCR™) analysis allows for accurate digital counting for quantification of gene expression. Human telomerase reverse transcriptase (hTERT) is one of the essential components required for telomerase activity and for the maintenance of telomeres. Several alternatively spliced forms of hTERT mRNA in human primary and tumor cells have been reported in the literature. Using one pair of primers and two probes for hTERT, four alternatively spliced forms of hTERT (α-/β+, α+/β- single deletions, α-/β- double deletion, and nondeletion α+/β+) were accurately quantified through a novel analysis method via data collected from a single ddPCR reaction. In this chapter, we describe this ddPCR method that enables direct quantitative comparison of four alternatively spliced forms of the hTERT messenger RNA without the need for internal standards or multiple pairs of primers specific for each variant, eliminating the technical variation due to differential PCR amplification efficiency for different amplicons and the challenges of quantification using standard curves. This simple and straightforward method should have general utility for quantifying alternatively spliced gene transcripts.

Transfer and targeted overexpression of γ-tocopherol methyltransferase (γ-TMT) gene using seed-specific promoter improves tocopherol composition in Indian soybean cultivars.

PubMed

Arun, Muthukrishnan; Subramanyam, Kondeti; Theboral, Jeevaraj; Sivanandhan, Ganeshan; Rajesh, Manoharan; Kapil Dev, Gnanajothi; Jaganath, Balusamy; Manickavasagam, Markandan; Girija, Shanmugam; Ganapathi, Andy

2014-02-01

Soybean oil contains high levels of tocopherols which are an important source of vitamin E in human diet. The conversion of γ- to α-tocopherol catalyzed by γ-tocopherol methyltransferase (γ-TMT) is found to be the rate limiting factor in soybean which influences the tocopherol composition. Using Agrobacterium-mediated transformation, we overexpressed the γ-TMT gene of Perilla frutescens under the control of the seed-specific promoter vicillin in cultivar Pusa 16. Transgene integration and expression was confirmed in five independently transformed GUS positive soybean plants by polymerase chain reaction (PCR), Southern hybridization, and reverse transcriptase-PCR (RT-PCR). High-performance liquid chromatography (HPLC) analysis showed that overexpression of Pf-γ-TMT resulted in efficient conversion of γ-tocopherol to α-tocopherol and concomitant increase in seed α-tocopherol content in RT-PCR positive plants. The protocol was successfully applied to three more cultivars PK 416, Gujarat soybean 1, and VL soya 1 in which seeds of transformed plants showed elevated level of α-tocopherol than wild-type seeds.

Detection of betanodaviruses in apparently healthy aquarium fishes and invertebrates.

PubMed

Gomez, Dennis Kaw; Lim, Dong Joo; Baeck, Gun Wook; Youn, Hee Jeong; Shin, Nam Shik; Youn, Hwa Young; Hwang, Cheol Yong; Park, Jun Hong; Park, Se Chang

2006-12-01

Betanodaviruses are the causative agents of viral nervous necrosis (VNN) in cultured marine fish. A total of 237 apparently healthy aquarium fish, marine (65 species) and freshwater (12 species) fishes and marine invertebrates (4 species), which were stocked in a commercial aquarium in Seoul, South Korea, were collected from November 2005 to February 2006. The brains of the fish and other tissues of the invertebrates were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR to detect betanodavirus. Positive nested PCR results were obtained from the brains of 8 marine fish species (shrimp fish Aeoliscus strigatus, milkfish Chanos chanos, three spot damsel Dascyllus trimaculatus, Japanese anchovy Engraulis japonicus, pinecone fish Monocentris japonica, blue ribbon eel Rhinomuraena quaesita, look down fish Selene vomer, yellow tang Zebrasoma flavesenes), 1 marine invertebrate species (spiny lobster Pamulirus versicolor), and 2 freshwater fish species (South American leaf fish Monocirrhus polyacanthus and red piranha Pygocentrus nattereri). The detection rate in nested PCR was 11/237 (4.64%). These subclinically infected aquarium fish and invertebrates may constitute an inoculum source of betanodaviruses for cultured fishes in the Korean Peninsula.

Detection of betanodaviruses in apparently healthy aquarium fishes and invertebrates

PubMed Central

Gomez, Dennis Kaw; Lim, Dong Joo; Baeck, Gun Wook; Youn, Hee Jeong; Shin, Nam Shik; Youn, Hwa Young; Hwang, Cheol Yong; Park, Jun Hong

2006-01-01

Betanodaviruses are the causative agents of viral nervous necrosis (VNN) in cultured marine fish. A total of 237 apparently healthy aquarium fish, marine (65 species) and freshwater (12 species) fishes and marine invertebrates (4 species), which were stocked in a commercial aquarium in Seoul, South Korea, were collected from November 2005 to February 2006. The brains of the fish and other tissues of the invertebrates were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR to detect betanodavirus. Positive nested PCR results were obtained from the brains of 8 marine fish species (shrimp fish Aeoliscus strigatus, milkfish Chanos chanos, three spot damsel Dascyllus trimaculatus, Japanese anchovy Engraulis japonicus, pinecone fish Monocentris japonica, blue ribbon eel Rhinomuraena quaesita, look down fish Selene vomer, yellow tang Zebrasoma flavesenes), 1 marine invertebrate species (spiny lobster Pamulirus versicolor), and 2 freshwater fish species (South American leaf fish Monocirrhus polyacanthus and red piranha Pygocentrus nattereri). The detection rate in nested PCR was 11/237 (4.64%). These subclinically infected aquarium fish and invertebrates may constitute an inoculum source of betanodaviruses for cultured fishes in the Korean Peninsula. PMID:17106229

A rapid method of accurate detection and differentiation of Newcastle disease virus pathotypes by demonstrating multiple bands in degenerate primer based nested RT-PCR.

PubMed

Desingu, P A; Singh, S D; Dhama, K; Kumar, O R Vinodh; Singh, R; Singh, R K

2015-02-01

A rapid and accurate method of detection and differentiation of virulent and avirulent Newcastle disease virus (NDV) pathotypes was developed. The NDV detection was carried out for different domestic avian field isolates and pigeon paramyxo virus-1 (25 field isolates and 9 vaccine strains) by using APMV-I "fusion" (F) gene Class II specific external primer A and B (535bp), internal primer C and D (238bp) based reverses transcriptase PCR (RT-PCR). The internal degenerative reverse primer D is specific for F gene cleavage position of virulent strain of NDV. The nested RT-PCR products of avirulent strains showed two bands (535bp and 424bp) while virulent strains showed four bands (535bp, 424bp, 349bp and 238bp) on agar gel electrophoresis. This is the first report regarding development and use of degenerate primer based nested RT-PCR for accurate detection and differentiation of NDV pathotypes by demonstrating multiple PCR band patterns. Being a rapid, simple, and economical test, the developed method could serve as a valuable alternate diagnostic tool for characterizing NDV isolates and carrying out molecular epidemiological surveillance studies for this important pathogen of poultry. Copyright © 2014 Elsevier B.V. All rights reserved.

Gene I mutants of peanut chlorotic streak virus, a caulimovirus, replicate in plants but do not move from cell to cell.

PubMed Central

Ducasse, D A; Mushegian, A R; Shepherd, R J

1995-01-01

Gene I of peanut chlorotic streak virus (PCISV), a caulimovirus, is homologous to gene I of other caulimoviruses and may encode a protein for virus movement. To evaluate the function of gene I, several mutations were created in this gene of an infectious, partially redundant clone of PCISV. Constructs with an in-frame deletion and a single amino acid substitution in gene I were not infectious. To test for replication of these mutants in primarily infected cells, an immunosorbent PCR technique was devised. Virus particles formed by mutants in plants were recovered by binding to antivirus antibodies on a solid matrix and DNase treated to discriminate against residual inoculum, and DNA of trapped virions was subjected to PCR amplification. Gene I mutants were shown to direct formation of encapsidated DNA as revealed by a PCR product. Control gene V mutants (reverse transcriptase essential for replication) did not yield a PCR product. Quantitative PCR allowed estimation of the proportion of cells initially infected by gene I mutants and the amount of extractable virus per cell. It is concluded that PCISV gene I encodes a movement protein and that the immunoselection-PCR technique is useful in studying subliminal virus infection in plants. PMID:7543587

Keratin 23 promotes telomerase reverse transcriptase expression and human colorectal cancer growth.

PubMed

Zhang, Ningning; Zhang, Rui; Zou, Kun; Yu, Wendan; Guo, Wei; Gao, Yingying; Li, Jia; Li, Mei; Tai, Yidi; Huang, Wenlin; Song, Chun; Deng, Wuguo; Cui, Xiaonan

2017-07-27

The overexpression of human telomerase reverse transcriptase (hTERT) has been associated with the proliferation and migration of colorectal cancer (CRC) cells. We investigated the roles of KRT23 and hTERT in promoting CRC cell proliferation and migration. We verified the relationship between KRT23 and hTERT in CRC using streptavidin-agarose pulldown and chromatin immunoprecipitation (ChIP) assays. One hundred and fifty-four human CRC specimens were analyzed using immunohistochemistry. The roles of KRT23 and hTERT in cell growth and migration were studied using siRNA and lentiviruses in vivo and in vitro. Western blot and wound scratch analyses were used to determine the signaling pathway for KRT23-mediated activation of CRC growth and migration. Telomerase activity was measured by using the TeloTAGGG Telomerase PCR ELISA PLUS Kit. We identified KRT23 as a new hTERT promoter-binding protein. Patients with high KRT23 and hTERT expression had markedly shorter overall survival. Overexpression of KRT23 upregulated the expression of hTERT protein, hTERT promoter-driven luciferase and telomerase activity in CRC. Conversely, inhibition of KRT23 by a KRT23-specific siRNA repressed the endogenous hTERT protein, the expression of hTERT promoter-driven luciferase and telomerase activity. Overexpression of KRT23 also promoted CRC proliferation and migration. By contrast, KRT23 inhibition significantly inhibited tumor cell growth in vitro and in vivo. KRT23 promoted cancer stem cell properties and increased the expression of CD133 and CD44. These results demonstrate that KRT23 is an important cellular factor that promotes CRC growth by activating hTERT expression and that KRT23 is a potential novel therapeutic target for CRC.

New Subtypes and Genetic Recombination in HIV Type 1-Infecting Patients with Highly Active Antiretroviral Therapy in Peru (2008–2010)

PubMed Central

Acuña, Maribel; Gazzo, Cecilia; Salinas, Gabriela; Cárdenas, Fanny; Valverde, Ada; Romero, Soledad

2012-01-01

Abstract HIV-1 subtype B is the most frequent strain in Peru. However, there is no available data about the genetic diversity of HIV-infected patients receiving highly active antiretroviral therapy (HAART) here. A group of 267 patients in the Peruvian National Treatment Program with virologic failure were tested for genotypic evidence of HIV drug resistance at the Instituto Nacional de Salud (INS) of Peru between March 2008 and December 2010. Viral RNA was extracted from plasma and the segments of the protease (PR) and reverse transcriptase (RT) genes were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), purified, and fully sequenced. Consensus sequences were submitted to the HIVdb Genotypic Resistance Interpretation Algorithm Database from Stanford University, and then aligned using Clustal X v.2.0 to generate a phylogenetic tree using the maximum likelihood method. Intrasubtype and intersubtype recombination analyses were performed using the SCUEAL program (Subtype Classification by Evolutionary ALgo-rithms). A total of 245 samples (91%) were successfully genotyped. The analysis obtained from the HIVdb program showed 81.5% resistance cases (n=198). The phylogenetic analysis revealed that subtype B was predominant in the population (98.8%), except for new cases of A, C, and H subtypes (n=4). Of these cases, only subtype C was imported. Likewise, recombination analysis revealed nine intersubtype and 20 intrasubtype recombinant cases. This is the first report of the presence of HIV-1 subtypes C and H in Peru. The introduction of new subtypes and circulating recombinants forms can make it difficult to distinguish resistance profiles in patients and consequently affect future treatment strategies against HIV in this country. PMID:22559065

Recurrent TERT promoter mutations identified in a large-scale study of multiple tumour types are associated with increased TERT expression and telomerase activation.

PubMed

Huang, Dong-Sheng; Wang, Zhaohui; He, Xu-Jun; Diplas, Bill H; Yang, Rui; Killela, Patrick J; Meng, Qun; Ye, Zai-Yuan; Wang, Wei; Jiang, Xiao-Ting; Xu, Li; He, Xiang-Lei; Zhao, Zhong-Sheng; Xu, Wen-Juan; Wang, Hui-Ju; Ma, Ying-Yu; Xia, Ying-Jie; Li, Li; Zhang, Ru-Xuan; Jin, Tao; Zhao, Zhong-Kuo; Xu, Ji; Yu, Sheng; Wu, Fang; Liang, Junbo; Wang, Sizhen; Jiao, Yuchen; Yan, Hai; Tao, Hou-Quan

2015-05-01

Several somatic mutation hotspots were recently identified in the telomerase reverse transcriptase (TERT) promoter region in human cancers. Large scale studies of these mutations in multiple tumour types are limited, in particular in Asian populations. This study aimed to: analyse TERT promoter mutations in multiple tumour types in a large Chinese patient cohort, investigate novel tumour types and assess the functional significance of the mutations. TERT promoter mutation status was assessed by Sanger sequencing for 13 different tumour types and 799 tumour tissues from Chinese cancer patients. Thymic epithelial tumours, gastrointestinal leiomyoma, and gastric schwannoma were included, for which the TERT promoter has not been previously sequenced. Functional studies included TERT expression by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), telomerase activity by the telomeric repeat amplification protocol (TRAP) assay and promoter activity by the luciferase reporter assay. TERT promoter mutations were highly frequent in glioblastoma (83.9%), urothelial carcinoma (64.5%), oligodendroglioma (70.0%), medulloblastoma (33.3%) and hepatocellular carcinoma (31.4%). C228T and C250T were the most common mutations. In urothelial carcinoma, several novel rare mutations were identified. TERT promoter mutations were absent in gastrointestinal stromal tumour (GIST), thymic epithelial tumours, gastrointestinal leiomyoma, gastric schwannoma, cholangiocarcinoma, gastric and pancreatic cancer. TERT promoter mutations highly correlated with upregulated TERT mRNA expression and telomerase activity in adult gliomas. These mutations differentially enhanced the transcriptional activity of the TERT core promoter. TERT promoter mutations are frequent in multiple tumour types and have similar distributions in Chinese cancer patients. The functional significance of these mutations reflect the importance to telomere maintenance and hence tumourigenesis, making them potential therapeutic targets. Copyright © 2015 Elsevier Ltd. All rights reserved.

New subtypes and genetic recombination in HIV type 1-infecting patients with highly active antiretroviral therapy in Peru (2008-2010).

PubMed

Yabar, Carlos Augusto; Acuña, Maribel; Gazzo, Cecilia; Salinas, Gabriela; Cárdenas, Fanny; Valverde, Ada; Romero, Soledad

2012-12-01

HIV-1 subtype B is the most frequent strain in Peru. However, there is no available data about the genetic diversity of HIV-infected patients receiving highly active antiretroviral therapy (HAART) here. A group of 267 patients in the Peruvian National Treatment Program with virologic failure were tested for genotypic evidence of HIV drug resistance at the Instituto Nacional de Salud (INS) of Peru between March 2008 and December 2010. Viral RNA was extracted from plasma and the segments of the protease (PR) and reverse transcriptase (RT) genes were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), purified, and fully sequenced. Consensus sequences were submitted to the HIVdb Genotypic Resistance Interpretation Algorithm Database from Stanford University, and then aligned using Clustal X v.2.0 to generate a phylogenetic tree using the maximum likelihood method. Intrasubtype and intersubtype recombination analyses were performed using the SCUEAL program (Subtype Classification by Evolutionary ALgo-rithms). A total of 245 samples (91%) were successfully genotyped. The analysis obtained from the HIVdb program showed 81.5% resistance cases (n=198). The phylogenetic analysis revealed that subtype B was predominant in the population (98.8%), except for new cases of A, C, and H subtypes (n=4). Of these cases, only subtype C was imported. Likewise, recombination analysis revealed nine intersubtype and 20 intrasubtype recombinant cases. This is the first report of the presence of HIV-1 subtypes C and H in Peru. The introduction of new subtypes and circulating recombinants forms can make it difficult to distinguish resistance profiles in patients and consequently affect future treatment strategies against HIV in this country.

Comparison of methods for in-house screening of HLA-B*57:01 to prevent abacavir hypersensitivity in HIV-1 care.

PubMed

De Spiegelaere, Ward; Philippé, Jan; Vervisch, Karen; Verhofstede, Chris; Malatinkova, Eva; Kiselinova, Maja; Trypsteen, Wim; Bonczkowski, Pawel; Vogelaers, Dirk; Callens, Steven; Ruelle, Jean; Kabeya, Kabamba; De Wit, Stephane; Van Acker, Petra; Van Sandt, Vicky; Emonds, Marie-Paule; Coucke, Paul; Sermijn, Erica; Vandekerckhove, Linos

2015-01-01

Abacavir is a nucleoside reverse transcriptase inhibitor used as part of combination antiretroviral therapy in HIV-1-infected patients. Because this drug can cause a hypersensitivity reaction that is correlated with the presence of the HLA-B*57:01 allotype, screening for the presence of HLA-B*57:01 is recommended before abacavir initiation. Different genetic assays have been developed for HLA-B*57:01 screening, each with specific sensitivity, turnaround time and assay costs. Here, a new real-time PCR (qPCR) based analysis is described and compared to sequence specific primer PCR with capillary electrophoresis (SSP PCR CE) on 149 patient-derived samples, using sequence specific oligonucleotide hybridization combined with high resolution SSP PCR as gold standard. In addition to these PCR based methods, a complementary approach was developed using flow cytometry with an HLA-B17 specific monoclonal antibody as a pre-screening assay to diminish the number of samples for genetic testing. All three assays had a maximum sensitivity of >99. However, differences in specificity were recorded, i.e. 84.3%, 97.2% and >99% for flow cytometry, qPCR and SSP PCR CE respectively. Our data indicate that the most specific and sensitive of the compared methods is the SSP PCR CE. Flow cytometry pre-screening can substantially decrease the number of genetic tests for HLA-B*57:01 typing in a clinical setting.

P-gp expression levels in the erythrocytes of brown trout: a new tool for aquatic sentinel biomarker development.

PubMed

Valton, Emeline; Wawrzyniak, Ivan; Amblard, Christian; Combourieu, Bruno; Bayle, Marie-Laure; Desmolles, François; Kwiatkowski, Fabrice; Penault-Llorca, Frédérique; Bamdad, Mahchid

2017-09-01

P-glycoprotein (P-gp) is a ubiquitous membrane detoxification pump involved in cellular defence against xenobiotics. Blood is a hub for the trade and transport of physiological molecules and xenobiotics. Our recent studies have highlighted the expression of a 140-kDa P-gp in brown trout erythrocytes in primary cell culture and its dose-dependent response to Benzo[a]pyrene pollutant. The purpose of this study was focused on using P-gp expression in brown trout erythrocytes as a biomarker for detecting the degree of river pollution. abcb1 gene and P-gp expression level were analysed by reverse transcriptase-PCR and Western blot, in the erythrocytes of brown trouts. The latter were collected in upstream and downstream of four rivers in which 17 polycyclic aromatic hydrocarbons and 348 varieties of pesticides micro-residues were analysed by liquid chromatography and mass spectrometry. The abcb1 gene and the 140-kDa P-gp were not expressed in trout erythrocytes from uncontaminated river. In contrast, they are clearly expressed in contaminated rivers, in correlation with the river pollution degree and the nature of the pollutants. This biological tool may offer considerable advantages since it provides an effective response to the increasing need for an early biomarker.

Clinicopathological characteristics of TERT promoter mutation and telomere length in hepatocellular carcinoma.

PubMed

Lee, Hye Won; Park, Tae In; Jang, Se Young; Park, Soo Young; Park, Won-Jin; Jung, Soo-Jung; Lee, Jae-Ho

2017-02-01

Promoter mutations in telomerase reverse transcriptase (TERT) and telomere length have been studied in various tumors. In the present study, the frequency and clinical characteristics of TERT promoter mutation and telomere length were studied in hepatocellular carcinoma (HCC). TERT promoter mutation and telomere length were analyzed in 162 tumor samples of the patients with HCC by sequencing and real-time PCR, respectively. The TERT promoter mutation rate was 28.8% (46/160) in HCC and was associated with males (P = 0.027). The telomere length was not significantly different in the presence of a TERT promoter mutation but was shorter in high-grade tumor stages (P = 0.048). Survival analyses showed that poor overall survival was associated with longer telomere length (P = 0.013). However, the TERT promoter mutation did not have a prognostic value for HCC. Multivariate survival analyses demonstrated that the telomere length was an independent prognostic marker for poor overall survival (hazard ratio = 1.75, 95% confidence interval: 1.046-2.913, P = 0.033). These data demonstrated that TERT promoter mutation is a frequent event in HCC; however, telomere length, but not the presence of a TERT promoter mutation, might have potential value as a prognostic indicator of HCC.

[The effect of retrovirus-mediated hTRT transfection into cultured oral keratinocytes].

PubMed

Huang, Ji-yan; Liu, Wei; Zhou, Zeng-tong; Zhou, Hai-wen

2014-06-01

Human telomerase reverse transcriptase (hTRT) was transfected into cultured oral keratinocytes (OKC) mediated by pBABE-tert recombined retrovirus to investigate the effect on OKC lifespan. pBABE-tert recombined retrovirus loaded with hTRT gene was amplified by transfected PT67 cells, and then transfected into cultured OKC in vitro. The positive clones of OKC were separated by puromycin and subcultured. Telomerase activity was analyzed by telomerase PCR-ELISA and PCR-PAGE. The hTRT positive clones of OKC showed telomerase expression, with extending lifespan to 8-9 passages. The hTRT transfected OKC can prolong doubly lifespan but not be immortalized, which indicates that cellular immortality mechanism is complicated and multi-controled. Telomerase activity is the key for cell immortalization but not the only impact factor.

Methods for molecular surveillance of influenza.

PubMed

Wang, Ruixue; Taubenberger, Jeffery K

2010-05-01

Molecular-based techniques for detecting influenza viruses have become an integral component of human and animal surveillance programs in the last two decades. The recent pandemic of the swine-origin influenza A virus (H1N1) and the continuing circulation of highly pathogenic avian influenza A virus (H5N1) further stress the need for rapid and accurate identification and subtyping of influenza viruses for surveillance, outbreak management, diagnosis and treatment. There has been remarkable progress on the detection and molecular characterization of influenza virus infections in clinical, mammalian, domestic poultry and wild bird samples in recent years. The application of these techniques, including reverse transcriptase-PCR, real-time PCR, microarrays and other nucleic acid sequencing-based amplifications, have greatly enhanced the capability for surveillance and characterization of influenza viruses.

Investigation of the presence of human or bovine respiratory syncytial virus in the lungs of mink (Neovison vison) with hemorrhagic pneumonia due to Pseudomonas aeruginosa.

PubMed

Salomonsen, Charlotte M; Breum, Solvej Ø; Larsen, Lars E; Jakobsen, Jeanette; Høiby, Niels; Hammer, Anne S

2012-11-26

Hemorrhagic pneumonia is a disease of farmed mink (Neovison vison) caused by Pseudomonas aeruginosa. The disease is highly seasonal in Danish mink with outbreaks occurring almost exclusively in the autumn. Human respiratory syncytial virus (RSV) has been shown to augment infection with P. aeruginosa in mice and to promote adhesion of P. aeruginosa to human respiratory cells. We tested 50 lung specimens from mink with hemorrhagic pneumonia for bovine RSV by reverse transcriptase polymerase chain reaction (PCR) and for human RSV by a commercial real-time PCR. RSV was not found. This study indicates that human and bovine RSV is not a major co-factor for development of hemorrhagic pneumonia in Danish mink.

Characterization of neuronal cell death in the spiral ganglia of a mouse model of endolymphatic hydrops.

PubMed

Semaan, Maroun T; Zheng, Qing Y; Han, Fengchan; Zheng, Yuxi; Yu, Heping; Heaphy, John C; Megerian, Cliff A

2013-04-01

Spiral ganglion neurons (SGN) in the Phex male mouse, a murine model of postnatal endolymphatic hydrops (ELH) undergo progressive deterioration reminiscent of human and other animal models of ELH with features suggesting apoptosis as an important mechanism. Histologic analysis of the mutant's cochlea demonstrates ELH by postnatal Day (P) 21 and SGN loss by P90. The SGN loss seems to occur in a consistent topographic pattern beginning at the cochlear apex. SGN were counted at P60, P90, and P120. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative PCR, and immunohistochemical analyses of activated caspase-3, caspase-8, and caspase-9 were performed on cochlear sections obtained from mutants and controls. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling assay (TUNEL) was carried out on 2 mutants and 2 controls. Corrected SGN counts in control mice were greater in the apical turn of the cochleae at P90 and P120, respectively (p < 0.01). Increased expression of activated caspase-3, caspase-8, and caspase-9 was seen in the mutant. At later time points, activated caspase expression gradually declined in the apical turns and increased in basal turns of the cochlea. Quantitative and semiquantitative PCR analysis confirmed increased expression of caspase-3, caspase-8, and caspase-9 at P21 and P40. TUNEL staining demonstrated apoptosis at P90 in the apical and basal turns of the mutant cochleae. SGN degeneration in the Phex /Y mouse seems to mimic patterns observed in other animals with ELH. Apoptosis plays an important role in the degeneration of the SGN in the Phex male mouse.

Novel chromosomal rearrangements and break points at the t(6;9) in salivary adenoid cystic carcinoma: association with MYB-NFIB chimeric fusion, MYB expression, and clinical outcome.

PubMed

Mitani, Yoshitsugu; Rao, Pulivarthi H; Futreal, P Andrew; Roberts, Dianna B; Stephens, Philip J; Zhao, Yi-Jue; Zhang, Li; Mitani, Mutsumi; Weber, Randal S; Lippman, Scott M; Caulin, Carlos; El-Naggar, Adel K

2011-11-15

To investigate the molecular genetic heterogeneity associated with the t(6:9) in adenoid cystic carcinoma (ACC) and correlate the findings with patient clinical outcome. Multimolecular and genetic techniques complemented with massive pair-ended sequencing and single-nucleotide polymorphism array analyses were used on tumor specimens from 30 new and 52 previously analyzed fusion transcript-negative ACCs by reverse transcriptase PCR (RT-PCR). MYB mRNA expression level was determined by quantitative RT-PCR. The results of 102 tumors (30 new and 72 previously reported cases) were correlated with the clinicopathologic factors and patients' survival. The FISH analysis showed 34 of 82 (41.5%) fusion-positive tumors and molecular techniques identified fusion transcripts in 21 of the 82 (25.6%) tumors. Detailed FISH analysis of 11 out the 15 tumors with gene fusion without transcript formation showed translocation of NFIB sequences to proximal or distal sites of the MYB gene. Massive pair-end sequencing of a subset of tumors confirmed the proximal translocation to an NFIB sequence and led to the identification of a new fusion gene (NFIB-AIG1) in one of the tumors. Overall, MYB-NFIB gene fusion rate by FISH was in 52.9% whereas fusion transcript forming incidence was 38.2%. Significant statistical association between the 5' MYB transcript expression and patient survival was found. We conclude that: (i) t(6;9) results in complex genetic and molecular alterations in ACC, (ii) MYB-NFIB gene fusion may not always be associated with chimeric transcript formation, (iii) noncanonical MYB-NFIB gene fusions occur in a subset of tumors, (iv) high MYB expression correlates with worse patient survival.

Prospective surveillance and molecular characterization of seasonal influenza in a university cohort in Singapore.

PubMed

Virk, Ramandeep Kaur; Tambyah, Paul Anantharajah; Inoue, Masafumi; Lim, Elizabeth Ai-Sim; Chan, Ka-Wei; Chua, Catherine; Tan, Boon-Huan

2014-01-01

Southeast Asia is believed to be a potential locus for the emergence of novel influenza strains, and therefore accurate sentinel surveillance in the region is critical. Limited information exists on sentinel surveillance of influenza-like illness (ILI) in young adults in Singapore in a University campus setting. The objective of the present study was to determine the proportion of ILI caused by influenza A and B viruses in a university cohort in Singapore. We conducted a prospective surveillance study from May through October 2007, at the National University of Singapore (NUS). Basic demographic information and nasopharyngeal swabs were collected from students and staff with ILI. Reverse-transcriptase PCR (RT-PCR) and viral isolation were employed to detect influenza viruses. Sequencing of hemagglutinin (HA) and neuraminidase (NA) genes of some representative isolates was also performed. Overall proportions of influenza A and B virus infections were 47/266 (18%) and 9/266 (3%) respectively. The predominant subtype was A/H3N2 (55%) and the rest were A/H1N1 (45%). The overall sensitivity difference for detection of influenza A viruses using RT-PCR and viral isolation was 53%. Phylogenetic analyses of HA and NA gene sequences of Singapore strains showed identities higher than 98% within both the genes. The strains were more similar to strains included in the WHO vaccine recommendation for the following year (2008). Genetic markers of oseltamivir resistance were not detected in any of the sequenced Singapore isolates. HA and NA gene sequences of Singapore strains were similar to vaccine strains for the upcoming influenza season. No drug resistance was found. Sentinel surveillance on university campuses should make use of molecular methods to better detect emerging and re-emerging influenza viral threats.

Strain-specific reverse transcriptase PCR assay: means to distinguish candidate vaccine from wild-type strains of respiratory syncytial virus.

PubMed Central

Zheng, H; Peret, T C; Randolph, V B; Crowley, J C; Anderson, L J

1996-01-01

Candidate live-virus vaccines for respiratory syncytial virus are being developed and are beginning to be evaluated in clinical trials. To distinguish candidate vaccine strains from wild-type strains isolated during these trials, we developed PCR assays specific to two sets of candidate vaccine strains. The two sets were a group A strain (3A), its three attenuated, temperature-sensitive variant strains, a group B strain (2B), and its four attenuated, temperature-sensitive variant strains. The PCR assays were evaluated by testing 18 group A wild-type strains, the 3A strains, 9 group B wild-type strains, and the 2B strains. PCR specific to group A wild-type strains amplified only group A wild-type strains, and 3A-specific PCR amplified only 3A strains. PCR specific to group B wild-type strains amplified all group A and group B strains but gave a 688-bp product for group B wild-type strains, a 279-bp product for 2B strains, a 547-bp product for all group A strains, and an additional 688-bp product for some group A strains, including 3A strains. These types of PCR assays can, in conjunction with other methods, be used to efficiently distinguish candidate vaccine strains from other respiratory syncytial virus strains. PMID:8789010

Molecular pathological analysis for determining the possible mechanism of piperonyl butoxide-induced hepatocarcinogenesis in mice.

PubMed

Muguruma, Masako; Nishimura, Jihei; Jin, Meilan; Kashida, Yoko; Moto, Mitsuyoshi; Takahashi, Miwa; Yokouchi, Yusuke; Mitsumori, Kunitoshi

2006-12-07

Piperonyl butoxide (PBO), alpha-[2-(2-butoxyethoxy)ethoxy]-4,5-methylene-dioxy-2-propyltoluene, is widely used as a synergist for pyrethrins. In order to clarify the possible mechanism of non-genotoxic hepatocarcinogenesis induced by PBO, molecular pathological analyses consisting of low-density microarray analysis and real-time reverse transcriptase (RT)-PCR were performed in male ICR mice fed a basal powdered diet containing 6000 or 0 ppm PBO for 1, 4, or 8 weeks. The animals were sacrificed at weeks 1, 4, and 8, and the livers were histopathologically examined and analyzed for gene expression using the microarray at weeks 1 and 4 followed by real-time RT-PCR at each time point. Reactive oxygen species (ROS) products were also measured using liver microsomes. At each time point, the hepatocytes of PBO-treated mice showed centrilobular hypertrophy and increased lipofuscin deposition in Schmorl staining. The ROS products were significantly increased in the liver microsomes of PBO-treated mice. In the microarray analysis, the expression of oxidative and metabolic stress-related genes--cytochrome P450 (Cyp) 1A1, Cyp2A5 (week 1 only), Cyp2B9, Cyp2B10, and NADPH-cytochrome P450 oxidoreductase (Por) was over-expressed in mice given PBO at weeks 1 and 4. Fluctuations of these genes were confirmed by real-time RT-PCR in PBO-treated mice at each time point. In additional real-time RT-PCR, the expression of Cyclin D1 gene, key regulator of cell-cycle progression, and Xrcc5 gene, DNA damage repair-related gene, was significantly increased at each time point and at week 8, respectively. These results suggest the possibility that PBO has the potential to generate ROS via the metabolic pathway and to induce oxidative stress, including oxidative DNA damage, resulting in the induction of hepatocellular tumors in mice.

Gammaretrovirus-Specific Antibodies in Free-Ranging and Captive Namibian Cheetahs

PubMed Central

Krengel, Annika; Cattori, Valentino; Meli, Marina L.; Wachter, Bettina; Böni, Jürg; Bisset, Leslie R.; Thalwitzer, Susanne; Melzheimer, Jörg; Jago, Mark; Hofmann-Lehmann, Regina; Hofer, Heribert

2015-01-01

The cheetah population in Namibia is the largest free-ranging population in the world and a key population for research regarding the health status of this species. We used serological methods and quantitative real-time PCR to test free-ranging and captive Namibian cheetahs for the presence of feline leukemia virus (FeLV), a gammaretrovirus that can be highly aggressive in populations with low genetic diversity, such as cheetahs. We also assessed the presence of antibodies to other gammaretroviruses and the responses to a FeLV vaccine developed for domestic cats. Up to 19% of the free-ranging cheetahs, 27% of the captive nonvaccinated cheetahs, and 86% of the captive vaccinated cheetahs tested positive for FeLV antibodies. FeLV-antibody-positive free-ranging cheetahs also tested positive for Rauscher murine leukemia virus antibodies. Nevertheless, FeLV was not detectable by quantitative real-time PCR and no reverse transcriptase activity was detectable by product-enhanced reverse transcriptase assay in the plasma of cheetahs or the supernatants from cultures of peripheral blood mononuclear cells. The presence of antibodies to gammaretroviruses in clinically healthy specimens may be caused either by infection with a low-pathogenic retrovirus or by the expression of endogenous retroviral sequences. The strong humoral immune responses to FeLV vaccination demonstrate that cheetahs can respond to the vaccine and that vaccination against FeLV infection may be beneficial should FeLV infection ever become a threat, as was seen in Iberian lynx and Florida panthers. PMID:25809630

Expression of bitter taste receptors of the T2R family in the gastrointestinal tract and enteroendocrine STC-1 cells.

PubMed

Wu, S Vincent; Rozengurt, Nora; Yang, Moon; Young, Steven H; Sinnett-Smith, James; Rozengurt, Enrique

2002-02-19

Although a role for the gastric and intestinal mucosa in molecular sensing has been known for decades, the initial molecular recognition events that sense the chemical composition of the luminal contents has remained elusive. Here we identified putative taste receptor gene transcripts in the gastrointestinal tract. Our results, using reverse transcriptase-PCR, demonstrate the presence of transcripts corresponding to multiple members of the T2R family of bitter taste receptors in the antral and fundic gastric mucosa as well as in the lining of the duodenum. In addition, cDNA clones of T2R receptors were detected in a rat gastric endocrine cell cDNA library, suggesting that these receptors are expressed, at least partly, in enteroendocrine cells. Accordingly, expression of multiple T2R receptors also was found in STC-1 cells, an enteroendocrine cell line. The expression of alpha subunits of G proteins implicated in intracellular taste signal transduction, namely Galpha(gust), and Galpha(t)-(2), also was demonstrated in the gastrointestinal mucosa as well as in STC-1 cells, as revealed by reverse transcriptase-PCR and DNA sequencing, immunohistochemistry, and Western blotting. Furthermore, addition of compounds widely used in bitter taste signaling (e.g., denatonium, phenylthiocarbamide, 6-n-propil-2-thiouracil, and cycloheximide) to STC-1 cells promoted a rapid increase in intracellular Ca(2+) concentration. These results demonstrate the expression of bitter taste receptors of the T2R family in the mouse and rat gastrointestinal tract.

Immunological characteristics of patients infected with common intestinal helminths: results of a study based on reverse-transcriptase PCR.

PubMed

Lertanekawattana, S; Wichatrong, T; Chaisari, K; Uchikawa, R; Arizono, N

2005-01-01

To determine whether common helminth infections could modify the intestinal immunopathological status of the host, the expression in the human duodenal mucosa of cytokines, eosinophil- and mast-cell-specific molecules and monosaccharide transporters of the glucose-transporter (GLUT) family was explored. The 31 subjects were all patients at the gastro-intestinal disease unit of Nongkhai Hospital, Thailand. Four of the 10 patients who presented with eosinophilia (> or = 6.0% of their leucocytes were eosinophils), and five of the other 21 patients, had intestinal infections with helminths when they presented or within the previous 3 months. Studies based on semi-quantitative, reverse-transcriptase PCR revealed that the interleukin-5/interferon-gamma ratio was significantly higher in the noneosinophilic, helminth-infected patients than in the non-eosinophilic, uninfected patients, whereas the IgE receptor type I (Fc epsilon RI)/mast-cell tryptase ratio was significantly higher in the eosinophilic, helminth-infected patients than in the eosinophilic, uninfected patients. Expression of Charcot-Leyden-crystal protein, GLUT-1 and GLUT-5, however, showed no significant inter-group differences. Principal-components analysis of the data on eosinophils, interleukin-5, interferon-gamma, Fc epsilon RI and mast-cell tryptase revealed that one principal component could discriminate the patients who had helminth infection from the non-eosinophilic, uninfected patients, but not from the eosinophilic, uninfected patients. These results indicate that, whatever the intestinal pathology, patients infected with common intestinal helminths tend to develop a mucosal immunological response of the Th2 type.

Use of FTA filter paper for the molecular detection of Newcastle disease virus.

PubMed

Perozo, Francisco; Villegas, Pedro; Estevez, Carlos; Alvarado, Iván; Purvis, Linda B

2006-04-01

The feasibility of using Flinders Technology Associates filter papers (FTA cards) to collect allantoic fluid and chicken tissue samples for Newcastle disease virus (NDV) molecular detection was evaluated. Trizol RNA extraction and one-step reverse transcriptase-polymerase chain reaction (RT-PCR) were used. FTA cards allowed NDV identification from allantoic fluid with a titre of 10(5.8) median embryo lethal doses/ml. The inactivated virus remained stable on the cards for 15 days. NDV was detected from FTA imprints of the trachea, lung, caecal tonsil and cloacal faeces of experimentally infected birds. RT-PCR detection from FTA cards was confirmed by homologous frozen-tissue RT-PCR and virus isolation. Direct nucleotide sequence of the amplified F gene allowed prediction of NDV virulence. No virus isolation was possible from the FTA inactivated samples, indicating viral inactivation upon contact. The FTA cards are suitable for collecting and transporting NDV-positive samples, providing a reliable source of RNA for molecular characterization and a hazard-free sample.

Dengue virus in bats from southeastern Mexico.

PubMed

Sotomayor-Bonilla, Jesús; Chaves, Andrea; Rico-Chávez, Oscar; Rostal, Melinda K; Ojeda-Flores, Rafael; Salas-Rojas, Mónica; Aguilar-Setien, Álvaro; Ibáñez-Bernal, Sergio; Barbachano-Guerrero, Arturo; Gutiérrez-Espeleta, Gustavo; Aguilar-Faisal, J Leopoldo; Aguirre, A Alonso; Daszak, Peter; Suzán, Gerardo

2014-07-01

To identify the relationship between landscape use and dengue virus (DENV) occurrence in bats, we investigated the presence of DENV from anthropogenically changed and unaltered landscapes in two Biosphere Reserves: Calakmul (Campeche) and Montes Azules (Chiapas) in southern Mexico. Spleen samples of 146 bats, belonging to 16 species, were tested for four DENV serotypes with standard reverse transcriptase polymerase chain reaction (RT-PCR) protocols. Six bats (4.1%) tested positive for DENV-2: four bats in Calakmul (two Glossophaga soricina, one Artibeus jamaicensis, and one A. lituratus) and two bats in Montes Azules (both A. lituratus). No effect of anthropogenic disturbance on the occurrence of DENV was detected; however, all three RT-PCR-positive bat species are considered abundant species in the Neotropics and well-adapted to disturbed habitats. To our knowledge, this study is the first study conducted in southeastern Mexico to identify DENV-2 in bats by a widely accepted RT-PCR protocol. The role that bats play on DENV's ecology remains undetermined. © The American Society of Tropical Medicine and Hygiene.

Survey of feline leukemia virus and feline coronaviruses in captive neotropical wild felids from Southern Brazil.

PubMed

Guimaraes, Ana M S; Brandão, Paulo E; de Moraes, Wanderlei; Cubas, Zalmir S; Santos, Leonilda C; Villarreal, Laura Y B; Robes, Rogério R; Coelho, Fabiana M; Resende, Mauricio; Santos, Renata C F; Oliveira, Rosangela C; Yamaguti, Mauricio; Marques, Lucas M; Neto, Renata L; Buzinhani, Melissa; Marques, Regina; Messick, Joanne B; Biondo, Alexander W; Timenetsky, Jorge

2009-06-01

A total of 57 captive neotropical felids (one Leopardus geoffroyi, 14 Leopardus pardalis, 17 Leopardus wiedii, 22 Leopardus tigrinus, and three Puma yagouaroundi) from the Itaipu Binacional Wildlife Research Center (Refúgio Bela Vista, Southern Brazil) were anesthetized for blood collection. Feces samples were available for 44 animals, including one L. geoffroyi, eight L. pardalis, 14 L. wiedii, 20 L. tigrinus, and one P. yagouaroundi. Total DNA and RNA were extracted from blood and feces, respectively, using commercial kits. Blood DNA samples were evaluated by polymerase chain reaction (PCR) for feline leukemia virus (FeLV) proviral DNA, whereas reverse transcriptase-PCR was run on fecal samples for detection of coronavirus RNA. None of the samples were positive for coronaviruses. A male L. pardalis and a female L. tigrinus were positive for FeLV proviral DNA, and identities of PCR products were confirmed by sequencing. This is the first evidence of FeLV proviral DNA in these species in Southern Brazil.

Development of a multiplex probe combination-based one-step real-time reverse transcription-PCR for NA subtype typing of avian influenza virus.

PubMed

Sun, Zhihao; Qin, Tao; Meng, Feifei; Chen, Sujuan; Peng, Daxin; Liu, Xiufan

2017-10-18

Nine influenza virus neuraminidase (NA) subtypes have been identified in poultry and wild birds. Few methods are available for rapid and simple NA subtyping. Here we developed a multiplex probe combination-based one-step real-time reverse transcriptase PCR (rRT-PCR) to detect nine avian influenza virus NA subtypes. Nine primer-probe pairs were assigned to three groups based on the different fluorescent dyes of the probes (FAM, HEX, or Texas Red). Each probe detected only one NA subtype, without cross reactivity. The detection limit was less than 100 EID 50 or 100 copies of cDNA per reaction. Data obtained using this method with allantoic fluid samples isolated from live bird markets and H9N2-infected chickens correlated well with data obtained using virus isolation and sequencing, but was more sensitive. This new method provides a specific and sensitive alternative to conventional NA-subtyping methods.

Proteomic analysis of early phase of conidia germination in Aspergillus nidulans.

PubMed

Oh, Young Taek; Ahn, Chun-Seob; Kim, Jeong Geun; Ro, Hyeon-Su; Lee, Chang-Won; Kim, Jae Won

2010-03-01

In order to investigate proteins involved in early phase of conidia germination, proteomic analysis was performed using two-dimensional gel electrophoresis (2D-GE) in conjunction with MALDI-TOF mass spectrometry (MS). The expression levels of 241 proteins varied quantitatively with statistical significance (P<0.05) at the early phase of the germination stage. Out of these 57 were identified by MALDI-TOF MS. Through classification of physiological functions from Conserved Domain Database analysis, among the identified proteins, 21, 13, and 6 proteins were associated with energy metabolism, protein synthesis, and protein folding process, respectively. Interestingly, eight proteins, which are involved in detoxification of reactive oxygen species (ROS) including catalase A, thioredoxin reductase, and mitochondrial peroxiredoxin, were also identified. The expression levels of the genes were further confirmed using Northern blot and reverse transcriptase (RT)-PCR analyses. This study represents the first proteomic analysis of early phase of conidia germination and will contribute to a better understanding of the molecular events involved in conidia germination process. Copyright (c) 2009 Elsevier Inc. All rights reserved.

Use of alternative alkali chlorides in RT and PCR of polynucleotides containing G quadruplex structures.

PubMed

Ramos-Alemán, Fabiola; González-Jasso, Eva; Pless, Reynaldo C

2018-02-15

Several alkali chlorides were compared for their use in reverse transcription (RT) and PCR of different types of nucleic acid templates. On a test region of biological DNA incapable of forming G quadruplex (G4) structures, Taq DNA polymerase showed similar PCR performance with 50 mM KCl, CsCl, LiCl, and NaCl. In contrast, on a synthetic model polydeoxyribonucleotide prone to G4 formation, good PCR amplification was obtained with 50 mM CsCl, but little or none with LiCl or KCl. Similarly, in RT of a G4-prone model polyribonucleotide, MMLV reverse transcriptase produced a good yield with 50 mM CsCl, mediocre yields with LiCl or without added alkali chloride, and a poor yield with 50 mM KCl. The full RT-PCR assay starting from the G4-prone polyribonucleotide, showed good results with CsCl in both stages, poor results with LiCl, and no product formation with KCl. The model polynucleotides showed fast G quadruplex formation under PCR or RT conditions with 50 mM KCl, but not with CsCl or LiCl. The results argue for the use of CsCl instead of KCl for RT and PCR of G4-prone sequences. No advantage was observed when using the 7-deaza type nucleotide analog c 7 dGTP in PCR amplification of the G4-prone polydeoxyribonucleotide. Copyright © 2017 Elsevier Inc. All rights reserved.

Performance characteristics of a reverse transcriptase-polymerase chain reaction assay for the detection of tumor-specific fusion transcripts from archival tissue.

PubMed

Fritsch, Michael K; Bridge, Julia A; Schuster, Amy E; Perlman, Elizabeth J; Argani, Pedram

2003-01-01

Pediatric small round cell tumors still pose tremendous diagnostic problems. In difficult cases, the ability to detect tumor-specific gene fusion transcripts for several of these neoplasms, including Ewing sarcoma/peripheral primitive neuroectodermal tumor (ES/PNET), synovial sarcoma (SS), alveolar rhabdomyosarcoma (ARMS), and desmoplastic small round cell tumor (DSRCT) using reverse transcriptase-polymerase chain reaction (RT-PCR), can be extremely helpful. Few studies to date, however, have systematically examined several different tumor types for the presence of multiple different fusion transcripts in order to determine the specificity and sensitivity of the RT-PCR method, and no study has addressed this issue for formalin-fixed material. The objectives of this study were to address the specificity, sensitivity, and practicality of such an assay applied strictly to formalin-fixed tissue blocks. Our results demonstrate that, for these tumors, the overall sensitivity for detecting each fusion transcript is similar to that reported in the literature for RT-PCR on fresh or formalin-fixed tissues. The specificity of the assay is very high, being essentially 100% for each primer pair when interpreting the results from visual inspection of agarose gels. However, when these same agarose gels were examined using Southern blotting, a small number of tumors also yielded reproducibly detectable weak signals for unexpected fusion products, in addition to a strong signal for the expected fusion product. Fluorescence in situ hybridization (FISH) studies in one such case indicated that a rearrangement that would account for the unexpected fusion was not present, while another case was equivocal. The overall specificity for each primer pair used in this assay ranged from 94 to 100%. Therefore, RT-PCR using formalin-fixed paraffin-embedded tissue sections can be used to detect chimeric transcripts as a reliable, highly sensitive, and highly specific diagnostic assay. However, we strongly suggest that the final interpretation of the results from this assay be viewed in light of the other features of the case, including clinical history, histology, and immunohistochemistry, by the diagnostic pathologist. Additional studies such as FISH may be useful in clarifying the nature of equivocal or unexpected results.

Use of a novel FISH assay on paraffin-embedded tissues as an adjunct to diagnosis of alveolar rhabdomyosarcoma.

PubMed

Nishio, Jun; Althof, Pamela A; Bailey, Jacqueline M; Zhou, Ming; Neff, James R; Barr, Frederic G; Parham, David M; Teot, Lisa; Qualman, Stephen J; Bridge, Julia A

2006-06-01

A valuable diagnostic adjunct and important prognostic parameter in alveolar rhabdomyosarcoma (ARMS) is the identification of translocations t(2;13)(q35;q14) and t(1;13)(p36;q14), and the associated PAX3-FKHR and PAX7-FKHR fusion transcripts, respectively. Most RMS fusion gene type studies have been based on reverse transcriptase-polymerase chain reaction (RT-PCR) detection of the fusion transcript, a technique limited by RNA quality and failure of devised primer sets to detect unusual variants. As an alternative approach, we developed a fluorescence in situ hybridization (FISH) assay that can: (1) distinguish between the two most common ARMS-associated fusion genes; (2) identify potential unusual variant translocations; (3) assess histologic components in mixed alveolar/embryonal RMS; and (4) be performed on paraffinized tissue. FISH analyses of 75 specimens (40 ARMS, 16 ERMS, 8 mixed ARMS/ERMS, and 11 non-RMS tumors) using selected cosmid clone, bacterial, P1-derived, and yeast artificial chromosome probe sets were successful in all but two cases. Among specimens with informative results for both FISH and RT-PCR or standard karyotyping, PAX/FKHR classification results were concordant in 94.6% (53/56). The three discordant cases included one exhibiting a t(2;13) by FISH that was subsequently confirmed by repeat RT-PCR, a second showing a rearrangement of the PAX3 locus only (consistent with the presence of a PAX3 variant translocation), and a third revealing a t(2;13) by FISH that lacked this translocation cytogenetically. Both alveolar and embryonal components of the mixed ARMS/ERMS subtype were negative for PAX3, PAX7, and FKHR rearrangements, a surprising finding confirmed by RT-PCR and/or conventional karyotyping. These data demonstrate that FISH with newly designed probe sets is a reliable and highly specific method of detecting t(1;13) and t(2;13) in routinely processed tissue and may be useful in differentiating ARMS from other small round cell tumors. The findings also suggest that FISH may be a more sensitive assay than RT-PCR in some settings, capable of revealing variant translocations.

Supervised Risk Predictor of Breast Cancer Based on Intrinsic Subtypes

PubMed Central

Parker, Joel S.; Mullins, Michael; Cheang, Maggie C.U.; Leung, Samuel; Voduc, David; Vickery, Tammi; Davies, Sherri; Fauron, Christiane; He, Xiaping; Hu, Zhiyuan; Quackenbush, John F.; Stijleman, Inge J.; Palazzo, Juan; Marron, J.S.; Nobel, Andrew B.; Mardis, Elaine; Nielsen, Torsten O.; Ellis, Matthew J.; Perou, Charles M.; Bernard, Philip S.

2009-01-01

Purpose To improve on current standards for breast cancer prognosis and prediction of chemotherapy benefit by developing a risk model that incorporates the gene expression–based “intrinsic” subtypes luminal A, luminal B, HER2-enriched, and basal-like. Methods A 50-gene subtype predictor was developed using microarray and quantitative reverse transcriptase polymerase chain reaction data from 189 prototype samples. Test sets from 761 patients (no systemic therapy) were evaluated for prognosis, and 133 patients were evaluated for prediction of pathologic complete response (pCR) to a taxane and anthracycline regimen. Results The intrinsic subtypes as discrete entities showed prognostic significance (P = 2.26E-12) and remained significant in multivariable analyses that incorporated standard parameters (estrogen receptor status, histologic grade, tumor size, and node status). A prognostic model for node-negative breast cancer was built using intrinsic subtype and clinical information. The C-index estimate for the combined model (subtype and tumor size) was a significant improvement on either the clinicopathologic model or subtype model alone. The intrinsic subtype model predicted neoadjuvant chemotherapy efficacy with a negative predictive value for pCR of 97%. Conclusion Diagnosis by intrinsic subtype adds significant prognostic and predictive information to standard parameters for patients with breast cancer. The prognostic properties of the continuous risk score will be of value for the management of node-negative breast cancers. The subtypes and risk score can also be used to assess the likelihood of efficacy from neoadjuvant chemotherapy. PMID:19204204

Evaluation of Immunomagnetic Separation for Recovery of Infectious Cryptosporidium parvum Oocysts from Environmental Samples

PubMed Central

Rochelle, Paul A.; De Leon, Ricardo; Johnson, Anne; Stewart, Mic H.; Wolfe, Roy L.

1999-01-01

Two commercial immunomagnetic separation (IMS) kits for Cryptosporidium were compared for recovery of oocysts from environmental samples. Oocyst recovery efficiencies with the Dynal and Crypto-Scan kits ranged from 62 to 100% and 34 to 74%, respectively, for seeded environmental water concentrates (turbidity of 210 to 11,480 nephelometric turbidity units). Recovery efficiencies were dependent on the mechanism of agitation during the magnetic capture procedure. An assay combining in vitro cell culture and reverse transcriptase PCR demonstrated that oocysts recovered by IMS retained their infectivity. PMID:9925626

Ostertagia circumcincta: isolation of a partial cDNA encoding an unusual member of the mitochondrial processing peptidase subfamily of M16 metallopeptidases.

PubMed

Walker, J; Tait, A

1997-11-01

A reverse-transcriptase polymerase chain reaction (PCR) procedure was used to isolate an Ostertagia circumcincta partial cDNA encoding a protein with general primary sequence features characteristic of members of the mitochondrial processing peptidase (MPP) subfamily of M16 metallopeptidases. The structural relationships of the predicted protein (Oc MPPX) with MPP subfamily proteins from other species (including the model free-living nematode Caenorhabditis elegans) were examined, and Northern analysis confirmed the expression of the Oc mppx gene in adult nematodes.

Epigenetic Characterization of Ovarian Cancer

DTIC Science & Technology

2008-12-01

Gusberg, A. H., Whitaker, R. S., Gray , J. W., Fujii, S., Berchuck, A. and S. K. Murphy. YY1/E2F3 modulates antimicrotubule drug response in epithelial... GTG GGT TTT TGG TGT TGG GTA TT-3’; and a shared reverse primer that does not anneal to CpGs, 5’-AAC CCC ACT CCC ACC CTA CTC C-3’. PCR was performed...Superscript II RNase H- reverse transcriptase (Invitrogen). Forward primer: 5’-GCG ACA TCG GTG ACT TCA T-3’ and reverse primer 5’-ATA CAT GTC CGC CAG CTT

Distinguishing between cancer driver and passenger gene alteration candidates via cross-species comparison: a pilot study.

PubMed

Ji, Xinglai; Tang, Jie; Halberg, Richard; Busam, Dana; Ferriera, Steve; Peña, Maria Marjorette O; Venkataramu, Chinnambally; Yeatman, Timothy J; Zhao, Shaying

2010-08-13

We are developing a cross-species comparison strategy to distinguish between cancer driver- and passenger gene alteration candidates, by utilizing the difference in genomic location of orthologous genes between the human and other mammals. As an initial test of this strategy, we conducted a pilot study with human colorectal cancer (CRC) and its mouse model C57BL/6J ApcMin/+, focusing on human 5q22.2 and 18q21.1-q21.2. We first performed bioinformatics analysis on the evolution of 5q22.2 and 18q21.1-q21.2 regions. Then, we performed exon-targeted sequencing, real time quantitative polymerase chain reaction (qPCR), and real time quantitative reverse transcriptase PCR (qRT-PCR) analyses on a number of genes of both regions with both human and mouse colon tumors. These two regions (5q22.2 and 18q21.1-q21.2) are frequently deleted in human CRCs and encode genuine colorectal tumor suppressors APC and SMAD4. They also encode genes such as MCC (mutated in colorectal cancer) with their role in CRC etiology unknown. We have discovered that both regions are evolutionarily unstable, resulting in genes that are clustered in each human region being found scattered at several distinct loci in the genome of many other species. For instance, APC and MCC are within 200 kb apart in human 5q22.2 but are 10 Mb apart in the mouse genome. Importantly, our analyses revealed that, while known CRC driver genes APC and SMAD4 were disrupted in both human colorectal tumors and tumors from ApcMin/+ mice, the questionable MCC gene was disrupted in human tumors but appeared to be intact in mouse tumors. These results indicate that MCC may not actually play any causative role in early colorectal tumorigenesis. We also hypothesize that its disruption in human CRCs is likely a mere result of its close proximity to APC in the human genome. Expanding this pilot study to the entire genome may identify more questionable genes like MCC, facilitating the discovery of new CRC driver gene candidates.

Failure to detect Xenotropic murine leukaemia virus-related virus in Chinese patients with chronic fatigue syndrome

PubMed Central

2010-01-01

Background Recent controversy has surrounded the question of whether xenotropic murine leukaemia virus-related virus (XMRV) contributes to the pathogenesis of chronic fatigue syndrome (CFS). To investigate the question in a Chinese population, 65 CFS patients and 85 blood donor controls were enrolled and multiplex real-time PCR or reverse transcriptase PCR (RT-PCR) was developed to analyze the XMRV infection status of the study participants. The assay was standardized by constructing plasmid DNAs and armored RNAs as XMRV standards and competitive internal controls (CICs), respectively. Results The sensitivities of the multiplex real-time PCR and RT-PCR assays were 20 copies/reaction and 10 IU/ml, respectively, with 100% specificity. The within-run precision coefficient of variation (CV) ranged from 1.76% to 2.80% and 1.70% to 2.59%, while the between-run CV ranged from 1.07% to 2.56% and 1.06% to 2.74%. XMRV was not detected in the 65 CFS patients and 65 normal individuals out of 85 controls. Conclusions This study failed to show XMRV in peripheral blood mononuclear cells (PBMCs) and plasma of Chinese patients with CFS. The absence of XMRV nucleic acids does not support an association between XMRV infection and the development of CFS in Chinese. PMID:20836869

Validation of Reference Genes for Real-Time Quantitative PCR (qPCR) Analysis of Avibacterium paragallinarum.

PubMed

Wen, Shuxiang; Chen, Xiaoling; Xu, Fuzhou; Sun, Huiling

2016-01-01

Real-time quantitative reverse transcription PCR (qRT-PCR) offers a robust method for measurement of gene expression levels. Selection of reliable reference gene(s) for gene expression study is conducive to reduce variations derived from different amounts of RNA and cDNA, the efficiency of the reverse transcriptase or polymerase enzymes. Until now reference genes identified for other members of the family Pasteurellaceae have not been validated for Avibacterium paragallinarum. The aim of this study was to validate nine reference genes of serovars A, B, and C strains of A. paragallinarum in different growth phase by qRT-PCR. Three of the most widely used statistical algorithms, geNorm, NormFinder and ΔCT method were used to evaluate the expression stability of reference genes. Data analyzed by overall rankings showed that in exponential and stationary phase of serovar A, the most stable reference genes were gyrA and atpD respectively; in exponential and stationary phase of serovar B, the most stable reference genes were atpD and recN respectively; in exponential and stationary phase of serovar C, the most stable reference genes were rpoB and recN respectively. This study provides recommendations for stable endogenous control genes for use in further studies involving measurement of gene expression levels.

Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution

PubMed Central

Ludlow, Andrew T.; Robin, Jerome D.; Sayed, Mohammed; Litterst, Claudia M.; Shelton, Dawne N.; Shay, Jerry W.; Wright, Woodring E.

2014-01-01

The telomere repeat amplification protocol (TRAP) for the human reverse transcriptase, telomerase, is a PCR-based assay developed two decades ago and is still used for routine determination of telomerase activity. The TRAP assay can only reproducibly detect ∼2-fold differences and is only quantitative when compared to internal standards and reference cell lines. The method generally involves laborious radioactive gel electrophoresis and is not conducive to high-throughput analyzes. Recently droplet digital PCR (ddPCR) technologies have become available that allow for absolute quantification of input deoxyribonucleic acid molecules following PCR. We describe the reproducibility and provide several examples of a droplet digital TRAP (ddTRAP) assay for telomerase activity, including quantitation of telomerase activity in single cells, telomerase activity across several common telomerase positive cancer cells lines and in human primary peripheral blood mononuclear cells following mitogen stimulation. Adaptation of the TRAP assay to digital format allows accurate and reproducible quantification of the number of telomerase-extended products (i.e. telomerase activity; 57.8 ± 7.5) in a single HeLa cell. The tools developed in this study allow changes in telomerase enzyme activity to be monitored on a single cell basis and may have utility in designing novel therapeutic approaches that target telomerase. PMID:24861623

[Disappearance of residual disease confirmed by RT-PCR following induction chemotherapy in two hypoplastic leukemia patients with t(8;21)].

PubMed

Sawada, M; Tsurumi, H; Yamada, T; Hara, T; Oyama, M; Moriwaki, H

1999-04-01

Reverse transcriptase-polymerase chain reaction (RT-PCR) methods often detect the AML1/MTG8 fusion transcript even in acute myelogenous leukemia (AML) patients with t(8;21) who have been in long-term remission. We encountered 2 hypoplastic leukemia patients with t(8;21) who achieved cytogenetic remission with short-term conventional chemotherapy. Patient 1 was a 42-year-old woman. Chromosomal analysis detected t(8;21) (q22;q22) and PCR analysis (35 cycles PCR amplification; detection limit 1 x 10(-5) cells) detected the AML1/MTG8 fusion transcript. Complete remission was obtained with 1 course of chemotherapy consisting of low-dose cytarabine (20 mg x 14 days) and etoposide (50 mg x 14 days). After 2 courses of consolidation chemotherapy consisting of conventional-dose cytarabine and mitoxantrone, the RT-PCR findings were negative for the AML1/MTG8 fusion transcript. Patient 2 was a 67-year-old man. Cytogenetic analysis detected t(8;21) (q22;q22), and was positive for the AML1/MTG8 fusion transcript. After 2 courses of induction chemotherapy comprising low-dose cytarabine (20 mg x 14 days) and etoposide (50 mg x 14 days), and 3 courses of conventional consolidation chemotherapy, RT-PCR analysis confirmed the disappearance of the AML1/MTG8 fusion transcript.

The use of short and long PCR products for improved detection of prunus necrotic ringspot virus in woody plants.

PubMed

Rosner, A; Maslenin, L; Spiegel, S

1997-09-01

The reverse transcriptase-polymerase chain reaction (RT-PCR) was used for detection of prunus necrotic ringspot virus (PNRSV) in dormant peach and almond trees by the application of two different pairs of primers yielding a short and a long product, respectively. The relative amount of the short (200 base pair, bp) product was higher than the longer (785 bp) product. PNRSV was detected better in plant tissues with a low virus concentration (e.g. dormant trees) by amplification of the short PCR product, whereas the long product was product was produced at higher virus titers. Simultaneous amplification of both short and long products was demonstrated using a three-primer mixture in a single reaction tube. In this assay, amplification of either PCR product indicated the presence of PNRSV-specific sequences in the plant tissue examined, thus covering a wide range of virus concentrations in a single test. Dilution of the RNA extracted from infected plant material resulted in a steep decline in the amplification of both short and long PCR products. In contrast, serial dilutions of the intermediate cDNA template differentially affected the amplification patterns: the relative amount of the short product increased whereas that of the long product decreased. These results may explain the preferential amplification of the short PCR product observed in samples containing low virus concentrations.

Multiplex hydrolysis probe real-time PCR for simultaneous detection of hepatitis A virus and hepatitis E virus.

PubMed

Qiu, Feng; Cao, Jingyuan; Su, Qiudong; Yi, Yao; Bi, Shengli

2014-05-30

Detection of hepatitis viral infections has traditionally relied on the circulating antibody test using the enzyme-linked immunosorbent assay. However, multiplex real-time PCR has been increasingly used for a variety of viral nucleic acid detections and has proven to be superior to traditional methods. Hepatitis A virus (HAV) and hepatitis E virus (HEV) are the major causes of acute hepatitis worldwide; both HAV and HEV infection are a main public health problem. In the present study, a one-step multiplex reverse transcriptase quantitative polymerase chain reaction assay using hydrolysis probes was developed for simultaneously detecting HAV and HEV. This novel detection system proved specific to the target viruses, to be highly sensitive and to be applicable to clinical sera samples, making it useful for rapid, accurate and feasible identification of HAV and HEV.

[Expression of cell adhesion molecules in acute leukemia cell].

PubMed

Ju, Xiaoping; Peng, Min; Xu, Xiaoping; Lu, Shuqing; Li, Yao; Ying, Kang; Xie, Yi; Mao, Yumin; Xia, Fang

2002-11-01

To investigate the role of cell adhesion molecule in the development and extramedullary infiltration (EI) of acute leukemia. The expressions of neural cell adhesion molecule (NCAM) gene, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1) genes in 25 acute leukemia patients bone marrow cells were detected by microarray and reverse transcriptase-polymerase chain reaction (RT-PCR). The expressions of NCAM, ICAM-1 and VCAM-1 gene were significantly higher in acute leukemia cells and leukemia cells with EI than in normal tissues and leukemia cells without EI, respectively, both by cDNA microarray and by RT-PCR. The cDNA microarray is a powerful technique in analysis of acute leukemia cells associated genes. High expressions of cell adhesion molecule genes might be correlated with leukemia pathogenesis and infiltration of acute leukemia cell.

Comparison of four molecular assays for the detection of Tembusu virus.

PubMed

Tang, Yi; Yeh, Yin-Ting; Chen, Hao; Yu, Chunmei; Gao, Xuhui; Diao, Youxiang

2015-10-01

Tembusu virus (TMUV) belongs to the genus Flavivirus that may cause severe egg drop in ducks. In order to evaluate the most efficient TMUV detection method, the performances of a conventional RT-PCR (C-RT-PCR), a semi-nested PCR (SN-RT-PCR), a reverse-transcriptase real-time quantitative PCR (Q-RT-PCR), and a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) targeting the TMUV virus-specific NS5 gene were examined. In order to compare the sensitivity of these four techniques, two templates were used: (1) plasmid DNA that contained a partial region of the NS5 gene and (2) genomic RNA from TMUV-positive cell culture supernatants. The sensitivities using plasmid DNA detection by C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were 2 × 10(4) copies/μL, 20 copies/μL, 2 copies/μL, and 20 copies/μL, respectively. The sensitivities using genomic RNA for the C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were 100 pg/tube, 100, 10, and 100 fg/tube, respectively. All evaluated assays were specific for TMUV detection. The TMUV-specific RNA was detected in cloacal swabs from experimentally infected ducks using these four methods with different rates (52-92%), but not in the control (non-inoculated) samples. The sensitivities of RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP performed with cloacal swabs collected from suspected TMUV infected ducks within 2 weeks of severe egg-drop were 38/69 (55.1%), 52/69 (75.4%), 57/69 (82.6%), and 55/69 (79.7%), respectively. In conclusion, both RT-LAMP and Q-RT-PCR can provide a rapid diagnosis of TMUV infection, but RT-LAMP is more useful in TMUV field situations or poorly equipped laboratories.

Unique clinical and imaging findings in a first ever documented PCR positive rabies survival patient: A case report.

PubMed

M, Netravathi; V, Udani; Rs, Mani; V, Gadad; M A, Ashwini; M, Bhat; S, Mehta; A, Chowdhary; P K, Pal; S N, Madhusudana; P, Satishchandra

2015-09-01

Rabies is fatal encephalitis which is a major public health problem in Asian and African countries. Till date, only 12 cases have been reported who have survived after rabies. In this communication we report a patient who is unique as the first documented long term rabies survivor with PCR positivity even after 4½ years of illness. Child sustained dog bite following which he received adequate prophylaxis. Within two weeks, child developed encephalopathy requiring evaluation. Child continues to have persistent myoclonic jerks, seizures, is dependent on all activities with severe neurological deficits. Nested reverse transcriptase PCR (RT-PCR) specific for rabies nucleoprotein gene in CSF and nuchal skin biopsy were positive for rabies viral RNA. The nuchal skin biopsy was also positive for rabies nucleoprotein antigen by fluorescent antibody test (FAT). We describe the clinical evolution and sequential MRI brain changes in this child. Despite the uniformly dismal prognosis of human rabies, these unusual reports of survival of rabies patients may provide an impetus to explore newer therapeutic strategies for this otherwise fatal disease. Copyright © 2015 Elsevier B.V. All rights reserved.

A simplified strategy for studying the etiology of viral diseases: Apple stem grooving virus as a case study.

PubMed

Dhir, Sunny; Walia, Yashika; Zaidi, A A; Hallan, Vipin

2015-03-01

A simple method to amplify infective, complete genomes of single stranded RNA viruses by long distance PCR (LD PCR) from woody plant tissues is described in detail. The present protocol eliminates partial purification of viral particles and the amplification is achieved in three steps: (i) easy preparation of template RNA by incorporating a pre processing step before loading onto the column (ii) reverse transcription by AMV or Superscript reverse transcriptase and (iii) amplification of cDNA by LD PCR using LA or Protoscript Taq DNA polymerase. Incorporation of a preprocessing step helped to isolate consistent quality RNA from recalcitrant woody tissues such as apple, which was critical for efficient amplification of the complete genomes of Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV). Complete genome of ASGV was cloned under T7 RNA polymerase promoter and was confirmed to be infectious through transcript inoculation producing symptoms similar to the wild type virus. This is the first report for the largest RNA virus genome amplified by PCR from total nucleic acid extracts of woody plant tissues. Copyright © 2014 Elsevier B.V. All rights reserved.

RHEB expression in fibroadenomas of the breast.

PubMed

Eom, Minseob; Han, Airi; Yi, Sang Yeop; Shin, John Junghun; Cui, Ying; Park, Kwang Hwa

2008-04-01

Although fibroadenoma is one of the most common types of benign breast tumor, genes specific to the tumor have not been identified. Microarrays were used to identify differentially expressed genes between fibroadenoma and infiltrating ductal carcinoma. The comparative expression of one of the identified genes, RAS homolog enriched in the brain (RHEB), was further explored using reverse transcriptase-polymerase chain reaction (RT-PCR). Microarray analysis was performed on tissue samples from five patients with fibroadenoma. In the fibroadenoma samples, the genes HDAC1, ROS1, TNFRSF10A, WASP2, TYRP1, WEE1, and RHEB were expressed at levels more than twofold higher than in the normal tissues. RT-PCR for RHEB indicated increased expression of RHEB in fibroadenoma compared to breast cancer. When studied with real-time PCR, the average RHEB/beta-actin ratio in fibroadenoma samples was 1.99, 2.46-fold greater than the average RHEB/beta-actin ratio in breast carcinoma of 0.81 (P < 0.01). Immunohistochemistry and PCR followed by microdissection shows increased expression of RHEB in epithelial cells compared to the stromal cells of fibroadenoma. Therefore, RHEB could be used cytopathologically to distinguish fibroadenoma from malignant breast carcinomas as a secondary diagnostic tool.

Development of a duplex real-time RT-PCR for the simultaneous detection and differentiation of Theiler's murine encephalomyelitis virus and rat theilovirus.

PubMed

Yuan, Wen; Wang, Jing; Xu, Fengjiao; Huang, Bihong; Lian, Yuexiao; Rao, Dan; Yin, Xueqin; Wu, Miaoli; Zhu, Yujun; Zhang, Yu; Huang, Ren; Guo, Pengju

2016-10-01

Theiler's murine encephalomyelitis virus (TMEV) and rat theilovirus (RTV), the member of the genus Cardiovirus, are widespread in laboratory mice and rats, and are potential contaminants of biological materials. Cardioviruses infection may cause serious complications in biomedical research. To improve the efficiency of routine screening for Cardioviruses infection, a duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection and differentiation of TMEV and RTV. The duplex assay was specific for reference strains of TMEV and RTV, and no cross-reaction was found with seven other rodent viruses. The limits of detection of both TMEV and RTV were 4×10(1) copies RNA/reaction. Reproducibility was estimated using standard dilutions, with coefficients of variation <3.1%. 439 clinical samples were evaluated by both duplex real-time RT-PCR and conventional RT-PCR. For 439 clinical samples，95 samples were positive for TMEV and 72 samples were positive for RTV using duplex real-time RT-PCR approach, whereas only 77 samples were positive for TMEV and 66 samples were positive for RTV when conventional RT-PCR was applied. Mixed infections were found in 20 samples when analyzed by conventional RT-PCR whereas 30 samples were found to be mixed infection when duplex real-time RT-PCR was applied. This duplex assay provides a useful tool for routine health monitoring and screening of contaminated biological materials of these two viruses. Copyright © 2016 Elsevier B.V. All rights reserved.

TaqMan real-time RT-PCR detection of infectious salmon anaemia virus (ISAV) from formalin-fixed paraffin-embedded Atlantic salmon Salmo salar tissues.

PubMed

Godoy, M G; Kibenge, F S; Kibenge, M J; Olmos, P; Ovalle, L; Yañez, A J; Avendaño-Herrera, R

2010-05-18

The objective of this study was to evaluate the application of a TaqMan real-time reverse transcriptase PCR (RT-PCR) assay for the detection of infectious salmon anaemia virus (ISAV) in formalin-fixed paraffin-embedded (FFPE) fish tissues from Atlantic salmon Salmo salar with and without clinical signs of infection, and to compare it with histological and immunohistochemical (IHC) techniques. Sixteen fish samples obtained in 2007 and 2008 from 4 different farms in Chile were examined. The real-time RT-PCR allowed the detection of ISAV in FFPE samples from 9 of 16 fish, regardless of the organs analyzed, whereas 4 of the real-time RT-PCR negative fish were positive as indicated by histological examination and 3 of the real-time RT-PCR positive fish were negative as indicated by immunohistochemistry evaluation. The presence of ISAV in RT-PCR positive samples was confirmed by amplicon sequencing. This work constitutes the first report on the use of real-time RT-PCR for the detection of ISAV in FFPE sections. The assay is very useful for the examination of archival wax-embedded tissues, and allows for both prospective and retrospective evaluation of tissue samples for the presence of ISAV. However, the method only confirms the presence of the pathogen and should be used in combination with histopathology, which is a more precise tool. The combination of both techniques would be invaluable for confirmatory diagnosis of infectious salmon anaemia (ISA), which is essential for solving salmon farm problems.

Gammaretrovirus-specific antibodies in free-ranging and captive Namibian cheetahs.

PubMed

Krengel, Annika; Cattori, Valentino; Meli, Marina L; Wachter, Bettina; Böni, Jürg; Bisset, Leslie R; Thalwitzer, Susanne; Melzheimer, Jörg; Jago, Mark; Hofmann-Lehmann, Regina; Hofer, Heribert; Lutz, Hans

2015-06-01

The cheetah population in Namibia is the largest free-ranging population in the world and a key population for research regarding the health status of this species. We used serological methods and quantitative real-time PCR to test free-ranging and captive Namibian cheetahs for the presence of feline leukemia virus (FeLV), a gammaretrovirus that can be highly aggressive in populations with low genetic diversity, such as cheetahs. We also assessed the presence of antibodies to other gammaretroviruses and the responses to a FeLV vaccine developed for domestic cats. Up to 19% of the free-ranging cheetahs, 27% of the captive nonvaccinated cheetahs, and 86% of the captive vaccinated cheetahs tested positive for FeLV antibodies. FeLV-antibody-positive free-ranging cheetahs also tested positive for Rauscher murine leukemia virus antibodies. Nevertheless, FeLV was not detectable by quantitative real-time PCR and no reverse transcriptase activity was detectable by product-enhanced reverse transcriptase assay in the plasma of cheetahs or the supernatants from cultures of peripheral blood mononuclear cells. The presence of antibodies to gammaretroviruses in clinically healthy specimens may be caused either by infection with a low-pathogenic retrovirus or by the expression of endogenous retroviral sequences. The strong humoral immune responses to FeLV vaccination demonstrate that cheetahs can respond to the vaccine and that vaccination against FeLV infection may be beneficial should FeLV infection ever become a threat, as was seen in Iberian lynx and Florida panthers. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The prognostic role of E2A-PBX1 expression detected by real-time quantitative reverse transcriptase polymerase chain reaction (RQ-PCR) in B cell acute lymphoblastic leukemia after allogeneic hematopoietic stem cell transplantation.

PubMed

Hong, Yan; Zhao, Xiaosu; Qin, Yazhen; Zhou, Songhai; Chang, Yingjun; Wang, Yu; Zhang, Xiaohui; Xu, Lanping; Huang, Xiaojun

2018-04-28

The E2A-PBX1 rearrangement is common in B cell acute lymphoblastic leukemia (B-ALL). However, whether this fusion gene can be used as a reliable marker for minimal residual disease (MRD) following allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains unknown. In this study, clinical data were collected from 28 consecutive B-ALL patients who received allo-HSCT. Their MRD was evaluated by E2A-PBX1 and leukemia-associated immunophenotype (LAIP). The median follow-up was 374 days (55-2342 days). Of the enrolled patients, seven (25%) patients died of leukemia relapse. A total of nine (32.1%) patients experienced relapse at a median of 164 days (75-559 days) after transplantation. The median expression level in the first positive sample was 0.14% (0.0071-902.4%). The duration from E2A-PBX1-positive results to hematological relapse was 74 days (30-469 days). E2A-PBX1 expression generally became positive prior to flow cytometry. Patients with positive E2A-PBX1 gene expression pre-transplantation were more likely to have positive E2A-PBX1 expression after transplantation. Taken all together, E2A-PBX1 expression determined by real-time quantitative reverse transcriptase polymerase chain reaction (RQ-PCR) could be used to evaluate MRD status after allo-HSCT. Patients with positive E2A-PBX1 expression after transplant will have a poor prognosis.

Mapping of RNA accessible sites by extension of random oligonucleotide libraries with reverse transcriptase.

PubMed Central

Allawi, H T; Dong, F; Ip, H S; Neri, B P; Lyamichev, V I

2001-01-01

A rapid and simple method for determining accessible sites in RNA that is independent of the length of target RNA and does not require RNA labeling is described. In this method, target RNA is allowed to hybridize with sequence-randomized libraries of DNA oligonucleotides linked to a common tag sequence at their 5'-end. Annealed oligonucleotides are extended with reverse transcriptase and the extended products are then amplified by using PCR with a primer corresponding to the tag sequence and a second primer specific to the target RNA sequence. We used the combination of both the lengths of the RT-PCR products and the location of the binding site of the RNA-specific primer to determine which regions of the RNA molecules were RNA extendible sites, that is, sites available for oligonucleotide binding and extension. We then employed this reverse transcription with the random oligonucleotide libraries (RT-ROL) method to determine the accessible sites on four mRNA targets, human activated ras (ha-ras), human intercellular adhesion molecule-1 (ICAM-1), rabbit beta-globin, and human interferon-gamma (IFN-gamma). Our results were concordant with those of other researchers who had used RNase H cleavage or hybridization with arrays of oligonucleotides to identify accessible sites on some of these targets. Further, we found good correlation between sites when we compared the location of extendible sites identified by RT-ROL with hybridization sites of effective antisense oligonucleotides on ICAM-1 mRNA in antisense inhibition studies. Finally, we discuss the relationship between RNA extendible sites and RNA accessibility. PMID:11233988

Evaluation of Four RNA Extraction Methods for Gene Expression Analyses of Cryptosporidium parvum and Toxoplasma gondii Oocys

EPA Science Inventory

Cryptosporidium spp. and Toxoplasma gondii are important coccidian parasites that have caused waterborne and foodborne disease outbreaks worldwide. Techniques like subtractive hybridization, microarrays, and quantitative reverse transcriptase real-time polymerase chain reaction (...

Molecular Analysis of Ammonia-Oxidizing Bacteria of the β Subdivision of the Class Proteobacteria in Compost and Composted Materials

PubMed Central

Kowalchuk, George A.; Naoumenko, Zinaida S.; Derikx, Piet J. L.; Felske, Andreas; Stephen, John R.; Arkhipchenko, Irina A.

1999-01-01

Although the practice of composting animal wastes for use as biofertilizers has increased in recent years, little is known about the microorganisms responsible for the nitrogen transformations which occur in compost and during the composting process. Ammonia is the principle available nitrogenous compound in composting material, and the conversion of this compound to nitrite in the environment by chemolithotrophic ammonia-oxidizing bacteria is an essential step in nitrogen cycling. Therefore, the distribution of ammonia-oxidizing members of the β subdivision of the class Proteobacteria in a variety of composting materials was assessed by amplifying 16S ribosomal DNA (rDNA) and 16S rRNA by PCR and reverse transcriptase PCR (RT-PCR), respectively. The PCR and RT-PCR products were separated by denaturing gradient gel electrophoresis (DGGE) and were identified by hybridization with a hierarchical set of oligonucleotide probes designed to detect ammonia oxidizer-like sequence clusters in the genera Nitrosospira and Nitrosomonas. Ammonia oxidizer-like 16S rDNA was detected in almost all of the materials tested, including industrial and experimental composts, manure, and commercial biofertilizers. A comparison of the DGGE and hybridization results after specific PCR and RT-PCR suggested that not all of the different ammonia oxidizer groups detected in compost are equally active. amoA, the gene encoding the active-site-containing subunit of ammonia monooxygenase, was also targeted by PCR, and template concentrations were estimated by competitive PCR. Detection of ammonia-oxidizing bacteria in the composts tested suggested that such materials may not be biologically inert with respect to nitrification and that the fate of nitrogen during composting and compost storage may be affected by the presence of these organisms. PMID:9925559

MAEWEST expression in flower development of two petunia species.

PubMed

Segatto, Ana Lúcia A; Turchetto-Zolet, Andreia Carina; Aizza, Lilian Cristina B; Monte-Bello, Carolina C; Dornelas, Marcelo C; Margis, Rogerio; Freitas, Loreta B

2013-07-03

Changes in flower morphology may influence the frequency and specificity of animal visitors. In Petunia (Solanaceae), adaptation to different pollinators is one of the factors leading to species diversification within the genus. This study provides evidence that differential expression patterns of MAWEWEST (MAW) homologs in different Petunia species may be associated with adaptive changes in floral morphology. The Petunia × hybrida MAW gene belongs to the WOX (WUSCHEL-related homeobox) transcription factor family and has been identified as a controller of petal fusion during corolla formation. We analyzed the expression patterns of P. inflata and P. axillaris MAW orthologs (PiMAW and PaMAW, respectively) by reverse transcriptase polymerase chain reaction (RT-PCR), reverse transcription-quantitative PCR (qRT-PCR) and in situ hybridization in different tissues and different developmental stages of flowers in both species. The spatial expression patterns of PiMAW and PaMAW were similar in P. inflata and P. axillaris. Nevertheless, PaMAW expression level in P. axillaris was higher during the late bud development stage as compared to PiMAW in P. inflata. This work represents an expansion of petunia developmental research to wild accessions.

Prevalence of Ambler class A β-lactamases and ampC expression in cephalosporin-resistant isolates of Acinetobacter baumannii.

PubMed

Rezaee, Mohammad Ahangarzadeh; Pajand, Omid; Nahaei, Mohammad Reza; Mahdian, Reza; Aghazadeh, Mohammad; Ghojazadeh, Morteza; Hojabri, Zoya

2013-07-01

We examined the prevalence of various cephalosporins' resistance mechanisms in Acinetobacter baumannii clinical isolates. Phenotypic and molecular detection of Ambler classes A, B and D β-lactamases was performed on 75 isolates. Clonal relatedness was defined using Repetitive Extragenic Palindromic PCR. PCR mapping was used to examine the linkage of insertion sequences and the ampC gene, and ampC expression was analyzed by TaqMan reverse transcriptase-PCR. Twenty-six (37%) isolates carried at least one of the blaPER-1 or blaTEM-1. Sixty-nine (98.5%) out of 70 cephalosporin-resistant isolates had insertions upstream of the ampC gene, of which 48 (69%) and 6 (8%) were identified as ISAba1and ISAba125, respectively. Higher level of expression was obtained in resistant isolates lacking ISAba1/ampC combination in comparison with that in positive ones. The ability to up-regulate the expression of ampC gene in association with different insertion elements has become an important factor in A. baumannii resistance to cephalosporins. Copyright © 2013 Elsevier Inc. All rights reserved.

Generation of non-genomic oligonucleotide tag sequences for RNA template-specific PCR

PubMed Central

Pinto, Fernando Lopes; Svensson, Håkan; Lindblad, Peter

2006-01-01

Background In order to overcome genomic DNA contamination in transcriptional studies, reverse template-specific polymerase chain reaction, a modification of reverse transcriptase polymerase chain reaction, is used. The possibility of using tags whose sequences are not found in the genome further improves reverse specific polymerase chain reaction experiments. Given the absence of software available to produce genome suitable tags, a simple tool to fulfill such need was developed. Results The program was developed in Perl, with separate use of the basic local alignment search tool, making the tool platform independent (known to run on Windows XP and Linux). In order to test the performance of the generated tags, several molecular experiments were performed. The results show that Tagenerator is capable of generating tags with good priming properties, which will deliberately not result in PCR amplification of genomic DNA. Conclusion The program Tagenerator is capable of generating tag sequences that combine genome absence with good priming properties for RT-PCR based experiments, circumventing the effects of genomic DNA contamination in an RNA sample. PMID:16820068

Evidence for airborne transmission of Norwalk-like virus (NLV) in a hotel restaurant.

PubMed

Marks, P J; Vipond, I B; Carlisle, D; Deakin, D; Fey, R E; Caul, E O

2000-06-01

An outbreak of gastroenteritis followed a meal in a large hotel during which one of the diners vomited. The clinical features of the illness suggested Norwalk-like virus (NLV, small round structured virus) infection, and this was confirmed by electron microscopy and reverse transcriptase polymerase chain reaction (RT-PCR) of stool samples. Further characterization of the virus by nucleotide sequence analysis of the PCR amplicons revealed identical strains in all the affected individuals. The foods served at the meal could not be demonstrated to be the cause of the outbreak. Analysis of attack rates by dining table showed an inverse relationship with the distance from the person who vomited. No one eating in a separate restaurant reported illness. Transmission from person-to-person or direct contamination of food seems unlikely in this outbreak. However, the findings are consistent with airborne spread of NLV with infection by inhalation with subsequent ingestion of virus particles.

Borna Disease Virus Antibody and RNA from Peripheral Blood Mononuclear Cells of Race Horses and Jockeys in Korea

PubMed Central

Song, Jin-Won; Na, Kyoung-Sae; Tae, Seong-Ho

2011-01-01

Objective During the last two decades, Borna disease virus (BDV) has received much attention as a possible zoonotic agent, particularly as a cause of psychiatric disease. Although several studies have shown that BDV is present in Asia, BDV has not been detected in Korea. This study was designed to further investigate the presence of BDV infection in Korea. Methods Blood samples were taken from 39 race horses and 48 jockeys. Antibody to BDV was detected by indirect immunofluorescence antibody test and RNA of BDV by real time reverse transcriptase PCR (rRT-PCR). Results No evidence of BDV was detected in either the horses or the jockeys group. Conclusion Our results suggest that BDV infection may not be endemic in Korea. Further studies with novel diagnostic tools are required to clarify the prevalence of BDV infection in Korea. PMID:21519538

Relationship Between Ebola Virus Real-Time Quantitative Polymerase Chain Reaction-Based Threshold Cycle Value and Virus Isolation From Human Plasma.

PubMed

Spengler, Jessica R; McElroy, Anita K; Harmon, Jessica R; Ströher, Ute; Nichol, Stuart T; Spiropoulou, Christina F

2015-10-01

We performed a longitudinal analysis of plasma samples obtained from 4 patients with Ebola virus (EBOV) disease (EVD) to determine the relationship between the real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)-based threshold cycle (Ct) value and the presence of infectious EBOV. EBOV was not isolated from plasma samples with a Ct value of >35.5 or >12 days after onset of symptoms. EBOV was not isolated from plasma samples in which anti-EBOV nucleoprotein immunoglobulin G was detected. These data demonstrate the utility of interpreting qRT-PCR results in the context of the course of EBOV infection and associated serological responses for patient-management decisions. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

[Expression of gamma interferon during HPV and Chlamydia trachomatis infection in cervical samples].

PubMed

Colín-Ferreyra, María Del Carmen; Mendieta-Zerón, Hugo; Romero-Figueroa, María Del Socorro; Martínez-Madrigal, Migdania; Martínez-Pérez, Sergio; Domínguez-García, María Victoria

2015-02-01

The aim of this study was to mesure the expression of gamma interferon in HPV and Chlamydia trachomatis infection in squamous intraepithelial lesions. Samples from 100 patients diagnosed by colposcopy with or without squamous intraepithelial lesions were used in the present study. Each patient was found to be infected by HPV and C.trachomatis. Relative gamma interferon mRNA expression was assessed using a real-time reverse transcriptase PCR assay (RT-PCR). The relative units of expression of gamma interferon mRNA were 13, 1.8 and 0.3, for HPV and C.trachomatis co-infection, or HPV or C.trachomatis infection, respectively. HPV and C.trachomatis could overstimulate the expression of gamma interferon. Copyright © 2014 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Multiplex Hydrolysis Probe Real-Time PCR for Simultaneous Detection of Hepatitis A Virus and Hepatitis E Virus

PubMed Central

Qiu, Feng; Cao, Jingyuan; Su, Qiudong; Yi, Yao; Bi, Shengli

2014-01-01

Detection of hepatitis viral infections has traditionally relied on the circulating antibody test using the enzyme-linked immunosorbent assay. However, multiplex real-time PCR has been increasingly used for a variety of viral nucleic acid detections and has proven to be superior to traditional methods. Hepatitis A virus (HAV) and hepatitis E virus (HEV) are the major causes of acute hepatitis worldwide; both HAV and HEV infection are a main public health problem. In the present study, a one-step multiplex reverse transcriptase quantitative polymerase chain reaction assay using hydrolysis probes was developed for simultaneously detecting HAV and HEV. This novel detection system proved specific to the target viruses, to be highly sensitive and to be applicable to clinical sera samples, making it useful for rapid, accurate and feasible identification of HAV and HEV. PMID:24886818

Transcription factors WRKY11 and WRKY17 are involved in abiotic stress responses in Arabidopsis.

PubMed

Ali, Muhammad Amjad; Azeem, Farrukh; Nawaz, Muhammad Amjad; Acet, Tuba; Abbas, Amjad; Imran, Qari Muhammad; Shah, Kausar Hussain; Rehman, Hafiz Mamoon; Chung, Gyuhwa; Yang, Seung Hwan; Bohlmann, Holger

2018-04-17

Plant WRKY transcription factors play a vital role in abiotic stress tolerance and regulation of plant defense responses. This study examined AtWRKY11 and AtWRKY17 expression under ABA, salt, and osmotic stress at different developmental stages in Arabidopsis. We used reverse transcriptase PCR, quantitative real-time PCR, and promoter:GUS lines to analyze expression. Both genes were upregulated in response to abiotic stress. Next, we applied the same stressors to seedlings of T-DNA insertion wrky11 and 17 knock-out mutants (single and double). Under stress, the mutants exhibited slower germination and compromised root growth compared with the wild type. In most cases, double-mutant seedlings were more affected than single mutants. These results suggest that wrky11 and wrky17 are not strictly limited to plant defense responses but are also involved in conferring stress tolerance. Copyright © 2018 Elsevier GmbH. All rights reserved.

Comparison of Assays for Sensitive and Reproducible Detection of Cell Culture-Infectious Cryptosporidium parvum and Cryptosporidium hominis in Drinking Water

PubMed Central

Di Giovanni, George D.; Rochelle, Paul A.

2012-01-01

This study compared the three most commonly used assays for detecting Cryptosporidium sp. infections in cell culture: immunofluorescent antibody and microscopy assay (IFA), PCR targeting Cryptosporidium sp.-specific DNA, and reverse transcriptase PCR (RT-PCR) targeting Cryptosporidium sp.-specific mRNA. Monolayers of HCT-8 cells, grown in 8-well chamber slides or 96-well plates, were inoculated with a variety of viable and inactivated oocysts to assess assay performance. All assays detected infection with low doses of flow cytometry-enumerated Cryptosporidium parvum oocysts, including infection with one oocyst and three oocysts. All methods also detected infection with Cryptosporidium hominis. The RT-PCR assay, IFA, and PCR assay detected infection in 23%, 25%, and 51% of monolayers inoculated with three C. parvum oocysts and 10%, 9%, and 16% of monolayers inoculated with one oocyst, respectively. The PCR assay was the most sensitive, but it had the highest frequency of false positives with mock-infected cells and inactivated oocysts. IFA was the only infection detection assay that did not produce false positives with mock-infected monolayers. IFA was also the only assay that detected infections in all experiments with spiked oocysts recovered from Envirochek capsules following filtration of 1,000 liters of treated water. Consequently, cell culture with IFA detection is the most appropriate method for routine and sensitive detection of infectious Cryptosporidium parvum and Cryptosporidium hominis in drinking water. PMID:22038611

Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution.

PubMed

Ludlow, Andrew T; Robin, Jerome D; Sayed, Mohammed; Litterst, Claudia M; Shelton, Dawne N; Shay, Jerry W; Wright, Woodring E

2014-07-01

The telomere repeat amplification protocol (TRAP) for the human reverse transcriptase, telomerase, is a PCR-based assay developed two decades ago and is still used for routine determination of telomerase activity. The TRAP assay can only reproducibly detect ∼ 2-fold differences and is only quantitative when compared to internal standards and reference cell lines. The method generally involves laborious radioactive gel electrophoresis and is not conducive to high-throughput analyzes. Recently droplet digital PCR (ddPCR) technologies have become available that allow for absolute quantification of input deoxyribonucleic acid molecules following PCR. We describe the reproducibility and provide several examples of a droplet digital TRAP (ddTRAP) assay for telomerase activity, including quantitation of telomerase activity in single cells, telomerase activity across several common telomerase positive cancer cells lines and in human primary peripheral blood mononuclear cells following mitogen stimulation. Adaptation of the TRAP assay to digital format allows accurate and reproducible quantification of the number of telomerase-extended products (i.e. telomerase activity; 57.8 ± 7.5) in a single HeLa cell. The tools developed in this study allow changes in telomerase enzyme activity to be monitored on a single cell basis and may have utility in designing novel therapeutic approaches that target telomerase. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Development of a reverse transcription polymerase chain reaction method for yellow fever virus detection.

PubMed

Méndez, María C; Domingo, Cristina; Tenorio, Antonio; Pardo, Lissethe C; Rey, Gloria J; Méndez, Jairo A

2013-09-01

Yellow fever is considered a re-emerging disease and is endemic in tropical regions of Africa and South America. At present, there are no standardized or commercialized kits available for yellow fever virus detection. Therefore, diagnosis must be made by time-consuming routine techniques, and sometimes, the virus or its proteins are not detected. Furthermore, co-circulation with other flaviviruses, including dengue virus, increases the difficulty of diagnosis. To develop a specific reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR-based assay to improve the detection and diagnosis of yellow fever virus using both serum and fresh tissue samples. RT-PCR primers were designed to amplify a short fragment of all yellow fever virus genotypes reported. A second set of primers was used in a nested PCR to increase sensitivity. Thirty-three clinical samples were tested with the standardized reaction. The expected amplicon was obtained in 25 out of 33 samples analyzed using this approach, and 2 more samples tested positive after a subsequent nested PCR approach. This improved technique not only ensures the specific detection of a wide range of yellow fever virus genotypes but also may increase the sensitivity of detection by introducing a second round of amplification, allowing a rapid differential diagnosis between dengue and yellow fever infection, which is required for effective surveillance and opportune epidemiologic measures.

Field detection of avian influenza virus in wild birds: evaluation of a portable rRT-PCR system and freeze-dried reagents

USGS Publications Warehouse

Takekawa, John Y.; Iverson, Samuel A.; Schultz, Annie K.; Hill, Nichola J.; Cardona, Carol J.; Boyce, Walter M.; Dudley, Joseph P.

2010-01-01

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAIV) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds is often conducted in remote regions, but results are often delayed because of limited local analytical capabilities, difficulties with sample transportation and permitting, or problems keeping samples cold in the field. In response to these challenges, the performance of a portable real-time, reverse transcriptase-polymerase chain reaction (rRT-PCR) unit (RAPID(Registered), Idaho Technologies, Salt Lake City, UT) that employed lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies) was compared to virus isolation combined with real-time RT-PCR conducted in a laboratory. This study included both field and experimental-based sampling. Field samples were collected from migratory shorebirds captured in northern California, while experimental samples were prepared by spiking fecal material with an H6N2 AIV isolate. Results indicated that the portable rRT-PCR unit had equivalent specificity to virus isolation with no false positives, but sensitivity was compromised at low viral titers. Use of portable rRT-PCR with lyophilized reagents may expedite surveillance results, paving the way to a better understanding of wild bird involvement in HPAIV H5N1 transmission.

Screening for diverse PDGFRA or PDGFRB fusion genes is facilitated by generic quantitative reverse transcriptase polymerase chain reaction analysis

PubMed Central

Erben, Philipp; Gosenca, Darko; Müller, Martin C.; Reinhard, Jelena; Score, Joannah; del Valle, Francesco; Walz, Christoph; Mix, Jürgen; Metzgeroth, Georgia; Ernst, Thomas; Haferlach, Claudia; Cross, Nicholas C.P.; Hochhaus, Andreas; Reiter, Andreas

2010-01-01

Background Rapid identification of diverse fusion genes with involvement of PDGFRA or PDGFRB in eosinophilia-associated myeloproliferative neoplasms is essential for adequate clinical management but is complicated by the multitude and heterogeneity of partner genes and breakpoints. Design and Methods We established a generic quantitative reverse transcriptase polymerase chain reaction to detect overexpression of the 3′-regions of PDGFRA or PDGFRB as a possible indicator of an underlying fusion. Results At diagnosis, all patients with known fusion genes involving PDGFRA (n=5; 51 patients) or PDGFRB (n=5; 7 patients) showed significantly increased normalized expression levels compared to 191 patients with fusion gene-negative eosinophilia or healthy individuals (PDGFRA/ABL: 0.73 versus 0.0066 versus 0.0064, P<0.0001; PDGFRB/ABL: 196 versus 3.8 versus 5.85, P<0.0001). The sensitivity and specificity of the activation screening test were, respectively, 100% and 88.4% for PDGFRA and 100% and 94% for PDGFRB. Furthermore, significant overexpression of PDGFRB was found in a patient with an eosinophilia-associated myeloproliferative neoplasm with uninformative cytogenetics and an excellent response to imatinib. Subsequently, a new SART3-PDGFRB fusion gene was identified by 5′-rapid amplification of cDNA ends polymerase chain reaction (5′-RACE-PCR). Conclusions Quantitative reverse transcriptase polymerase chain reaction analysis is a simple and useful adjunct to standard diagnostic assays to detect clinically significant overexpression of PDGFRA and PDGFRB in eosinophilia-associated myeloproliferative neoplasms or related disorders. PMID:20107158

The role of pleural fluid MAGE RT-nested PCR in the diagnosis of malignant pleural effusion.

PubMed

Jeon, Eun Ju; Park, Hye Kyeong; Jeon, Kyeongman; Koh, Won-Jung; Suh, Gee Young; Chung, Man Pyo; Kim, Hojoong; Kwon, O Jung; Ki, Chang-Seok; Kim, Jong-Won; Shim, Young Mog; Um, Sang-Won

2012-11-01

  Melanoma antigen (MAGE) genes are expressed in tumor cells, the testis and the placenta. The purpose of this prospective study was to investigate the sensitivity, specificity, and accuracy of the carcinoembryonic antigen (CEA), MAGE reverse transcriptase-nested polymerase chain reaction (RT-nested PCR), and cytology of pleural fluid in the diagnosis of malignant pleural effusion.   Patients in whom unilateral pleural effusion was identified on chest radiography from January to December 2009 were included in the study. MAGE genes were analyzed by RT-nested PCR using MAGE A1-6 common primers.   Of 81 enrolled patients, 46 were diagnosed as malignant pleural effusion, and 24 were diagnosed as benign pleural effusion. The diagnoses of 11 patients were not confirmed in this study. The diagnostic sensitivity, specificity, and accuracy of MAGE RT-nested PCR were 61.4%, 95.7%, and 73.1%, respectively. The diagnostic sensitivities of cytology and CEA (>5 ng/mL) were 61.4% and 75.0%, respectively. Among 17 patients with negative cytology who had malignant pleural effusion, 12 and 10 patients were positive for CEA (>5.0 ng/mL) and MAGE RT-nested PCR, respectively. However, of five patients with malignant pleural effusion that was not recognized by cytology and CEA, MAGE RT-nested PCR correctly predicted a malignant etiology in only one additional patient (20%).   MAGE RT-nested PCR seems to add little on the combination of conventional methods in the diagnosis of malignant effusion. © 2012 Tianjin Lung Cancer Institute and Wiley Publishing Asia Pty. Ltd.

Germ cell-specific heat shock protein 70-2 is expressed in cervical carcinoma and is involved in the growth, migration, and invasion of cervical cells.

PubMed

Garg, Manoj; Kanojia, Deepika; Saini, Shikha; Suri, Sushma; Gupta, Anju; Surolia, Avadhesha; Suri, Anil

2010-08-15

Cervical cancer is a major cause of death among women worldwide, and the most cases are reported in the least developed countries. Recently, a study on DNA microarray gene expression analysis demonstrated the overexpression of heat shock protein 70-2 (HSP70-2) in cervical carcinoma cells (HeLa). The objective of the current study was to evaluate the association between HSP70-2 expression in cervical carcinogenesis and its potential role in various malignant properties that result in disease progression. HSP70-2 expression was examined in various cervical cancer cell lines with different origins and in clinical cervical cancer specimens by reverse transcriptase-polymerase chain reaction (RT-PCR), flow cytometry, and immunohistochemistry (IHC) analyses. A plasmid-based, short-hairpin RNA approach was used specifically to knock down the expression of HSP70-2 in cervical tumor cells in vitro and in vivo to examine the role of HSP70-2 on various malignant properties. RT-PCR and IHC analyses revealed HSP70-2 expression in 86% of cervical cancer specimens. Furthermore, knockdown of HSP70-2 expression significantly reduced cellular growth, colony formation, migration, and invasion in vitro and reduced tumor growth in vivo. A significant association of HSP70-2 gene and protein expression was observed among the various tumor stages (P=.046) and different grades (P=.006), suggesting that HSP70-2 expression may be an indicator of disease progression. The current findings suggested that HSP70-2 may play an important role in disease progression in cervical carcinogenesis. Patients who had early stage disease and low-grade tumors had HSP70-2 expression, supporting its potential role in early detection and aggressive treatment modalities for cervical cancer management. Copyright (c) 2010 American Cancer Society.

Anti-chikungunya activity of luteolin and apigenin rich fraction from Cynodon dactylon.

PubMed

Murali, Krishnan Saravana; Sivasubramanian, Srinivasan; Vincent, Savariar; Murugan, Shanmugaraj Bala; Giridaran, Bupesh; Dinesh, Sundaram; Gunasekaran, Palani; Krishnasamy, Kaveri; Sathishkumar, Ramalingam

2015-05-01

To obtain luteolin and apigenin rich fraction from the ethanolic extract of Cynodon dactylon (L.) (C. dactylon) Pers and evaluate the fraction's cytotoxicity and anti-Chikungunya potential using Vero cells. The ethanolic extract of C. dactylon was subjected to silica gel column chromatography to obtain anti-chikungunya virus (CHIKV) fraction. Reverse phase-HPLC and GC-MS studies were carried out to identify the major phytochemicals in the fraction using phytochemical standards. Cytotoxicity and the potential of the fraction against CHIKV were evaluated in vitro using Vero cells. Reduction in viral replication was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) after treating the viral infected Vero cells with the fraction. Reverse Phase-HPLC and GC-MS studies confirmed the presence of flavonoids, luteolin and apigenin as major phytochemicals in the anti-CHIKV ethanolic fraction of C. dactylon. The fraction was found to exhibit potent viral inhibitory activity (about 98%) at the concentration of 50 µg/mL as observed by reduction in cytopathic effect, and the cytotoxic concentration of the fraction was found to be 250 µg/mL. RT-PCR analyses indicated that the reduction in viral mRNA synthesis in fraction treated infected cells was much higher than the viral infected control cells. Luteolin and apigenin rich ethanolic fraction from C. dactylon can be utilized as a potential therapeutic agent against CHIKV infection as the fraction does not show cytotoxicity while inhibiting the virus. Copyright © 2015 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

Microarray and Real-Time PCR Analyses of the Responses of High-Arctic Soil Bacteria to Hydrocarbon Pollution and Bioremediation Treatments▿

PubMed Central

Yergeau, Etienne; Arbour, Mélanie; Brousseau, Roland; Juck, David; Lawrence, John R.; Masson, Luke; Whyte, Lyle G.; Greer, Charles W.

2009-01-01

High-Arctic soils have low nutrient availability, low moisture content, and very low temperatures and, as such, they pose a particular problem in terms of hydrocarbon bioremediation. An in-depth knowledge of the microbiology involved in this process is likely to be crucial to understand and optimize the factors most influencing bioremediation. Here, we compared two distinct large-scale field bioremediation experiments, located at the Canadian high-Arctic stations of Alert (ex situ approach) and Eureka (in situ approach). Bacterial community structure and function were assessed using microarrays targeting the 16S rRNA genes of bacteria found in cold environments and hydrocarbon degradation genes as well as quantitative reverse transcriptase PCR targeting key functional genes. The results indicated a large difference between sampling sites in terms of both soil microbiology and decontamination rates. A rapid reorganization of the bacterial community structure and functional potential as well as rapid increases in the expression of alkane monooxygenases and polyaromatic hydrocarbon-ring-hydroxylating dioxygenases were observed 1 month after the bioremediation treatment commenced in the Alert soils. In contrast, no clear changes in community structure were observed in Eureka soils, while key gene expression increased after a relatively long lag period (1 year). Such discrepancies are likely caused by differences in bioremediation treatments (i.e., ex situ versus in situ), weathering of the hydrocarbons, indigenous microbial communities, and environmental factors such as soil humidity and temperature. In addition, this study demonstrates the value of molecular tools for the monitoring of polar bacteria and their associated functions during bioremediation. PMID:19684169

Identification of the heme acquisition system in Vibrio vulnificus M2799.

PubMed

Kawano, Hiroaki; Miyamoto, Katsushiro; Yasunobe, Megumi; Murata, Masahiro; Yamahata, Eri; Yamaguchi, Ryo; Miyaki, Yuta; Tsuchiya, Takahiro; Tanabe, Tomotaka; Funahashi, Tatsuya; Tsujibo, Hiroshi

2018-04-01

Vibrio vulnificus, the causative agent of serious, often fatal, infections in humans, requires iron for its pathogenesis. As such, it obtains iron via both vulnibactin and heme-mediated iron-uptake systems. In this study, we identified the heme acquisition system in V. vulnificus M2799. The nucleotide sequences of the genes encoding heme receptors HupA and HvtA and the ATP-binding cassette (ABC) transport system proteins HupB, HupC, and HupD were determined, and then used in the construction of deletion mutants developed from a Δics strain, which could not synthesize vulnibactin. Growth experiments using these mutants indicated that HupA and HvtA are major and minor heme receptors, respectively. The expressions of two proteins were analyzed by the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Furthermore, complementation analyses confirmed that the HupBCD proteins are the only ABC transport system shared by both the HupA and HvtA receptors. This is the first genetic evidence that the HupBCD proteins are essential for heme acquisition by V. vulnificus. Further investigation showed that hupA, hvtA, and hupBCD are regulated by Fur. The qRT-PCR analysis of the heme receptor genes revealed that HupR, a LysR-family positive transcriptional activator, upregulates the expression of hupA, but not hvtA. In addition, ptrB was co-transcribed with hvtA, and PtrB had no influence on growth in low-iron CM9 medium supplemented with hemin, hemoglobin, or cytochrome C. Copyright © 2018 Elsevier Ltd. All rights reserved.

Gata6 is required for complete acinar differentiation and maintenance of the exocrine pancreas in adult mice.

PubMed

Martinelli, Paola; Cañamero, Marta; del Pozo, Natalia; Madriles, Francesc; Zapata, Agustín; Real, Francisco X

2013-10-01

Previous studies have suggested an important role of the transcription factor Gata6 in endocrine pancreas, while GATA6 haploinsufficient inactivating mutations cause pancreatic agenesis in humans. We aimed to analyse the effects of Gata6 inactivation on pancreas development and function. We deleted Gata6 in all epithelial cells in the murine pancreas at the onset of its development. Acinar proliferation, apoptosis, differentiation and exocrine functions were assessed using reverse transcriptase quantitative PCR (RT-qPCR), chromatin immunoprecipitation, immunohistochemistry and enzyme assays. Adipocyte transdifferentiation was assessed using electron microscopy and genetic lineage tracing. Gata6 is expressed in all epithelial cells in the adult mouse pancreas but it is only essential for exocrine pancreas homeostasis: while dispensable for pancreatic development after e10.5, it is required for complete acinar differentiation, for establishment of polarity and for the maintenance of acinar cells in the adult. Gata6 regulates directly the promoter of genes coding for digestive enzymes and the transcription factors Rbpjl and Mist1. Upon pancreas-selective Gata6 inactivation, massive loss of acinar cells and fat replacement take place. This is accompanied by increased acinar apoptosis and proliferation, acinar-to-ductal metaplasia and adipocyte transdifferentiation. By contrast, the endocrine pancreas is spared. Our data show that Gata6 is required for the complete differentiation of acinar cells through multiple transcriptional regulatory mechanisms. In addition, it is required for the maintenance of the adult acinar cell compartment. Our studies suggest that GATA6 alterations may contribute to diseases of the human adult exocrine pancreas.

Mutations in a novel gene with transmembrane domains underlie Usher syndrome type 3.

PubMed

Joensuu, T; Hämäläinen, R; Yuan, B; Johnson, C; Tegelberg, S; Gasparini, P; Zelante, L; Pirvola, U; Pakarinen, L; Lehesjoki, A E; de la Chapelle, A; Sankila, E M

2001-10-01

Usher syndrome type 3 (USH3) is an autosomal recessive disorder characterized by progressive hearing loss, severe retinal degeneration, and variably present vestibular dysfunction, assigned to 3q21-q25. Here, we report on the positional cloning of the USH3 gene. By haplotype and linkage-disequilibrium analyses in Finnish carriers of a putative founder mutation, the critical region was narrowed to 250 kb, of which we sequenced, assembled, and annotated 207 kb. Two novel genes-NOPAR and UCRP-and one previously identified gene-H963-were excluded as USH3, on the basis of mutational analysis. USH3, the candidate gene that we identified, encodes a 120-amino-acid protein. Fifty-two Finnish patients were homozygous for a termination mutation, Y100X; patients in two Finnish families were compound heterozygous for Y100X and for a missense mutation, M44K, whereas patients in an Italian family were homozygous for a 3-bp deletion leading to an amino acid deletion and substitution. USH3 has two predicted transmembrane domains, and it shows no homology to known genes. As revealed by northern blotting and reverse-transcriptase PCR, it is expressed in many tissues, including the retina.

Isolation and characterization of the pea cytochrome c oxidase Vb gene.

PubMed

Kubo, Nakao; Arimura, Shin-Ichi; Tsutsumi, Nobuhiro; Kadowaki, Koh-Ichi; Hirai, Masashi

2006-11-01

Three copies of the gene that encodes cytochrome c oxidase subunit Vb were isolated from the pea (PscoxVb-1, PscoxVb-2, and PscoxVb-3). Northern Blot and reverse transcriptase-PCR analyses suggest that all 3 genes are transcribed in the pea. Each pea coxVb gene has an N-terminal extended sequence that can encode a mitochondrial targeting signal, called a presequence. The localization of green fluorescent proteins fused with the presequence strongly suggests the targeting of pea COXVb proteins to mitochondria. Each pea coxVb gene has 5 intron sites within the coding region. These are similar to Arabidopsis and rice, although the intron lengths vary greatly. A phylogenetic analysis of coxVb suggests the occurrence of gene duplication events during angiosperm evolution. In particular, 2 duplication events might have occurred in legumes, grasses, and Solanaceae. A comparison of amino acid sequences in COXVb or its counterpart shows the conservation of several amino acids within a zinc finger motif. Interestingly, a homology search analysis showed that bacterial protein COG4391 and a mitochondrial complex I 13 kDa subunit also have similar amino acid compositions around this motif. Such similarity might reflect evolutionary relationships among the 3 proteins.

Antiviral activity of the exopolysaccharide produced by Serratia sp. strain Gsm01 against Cucumber mosaic virus.

PubMed

Ipper, Nagesh S; Cho, Saeyoull; Lee, Seon Hwa; Cho, Jun Mo; Hur, Jang Hyun; Lim, Chun Keun

2008-01-01

The potential of the exopolysaccharide (EPS) from a Serratia sp. strain Gsm01 as an antiviral agent against a yellow strain of Cucumber mosaic virus (CMV-Y) was evaluated in tobacco plants (Nicotiana tabacum cv. Xanthi-nc). The spray treatment of plants using an EPS preparation, 72 before CMV-Y inoculation, protected them against symptom appearance. Fifteen days after challenge inoculation with CMVY, 33.33% of plants showed mosaic symptoms in EPS-treated plants compared with 100% in the control plants. The EPS-treated plants, which showed mosaic symptoms, appeared three days later than the controls. The enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR) analyses of the leaves of the protected plants revealed that the EPS treatment affected virus accumulation in those plants. Analysis of phenylalanine ammonia lyase, peroxidase, and phenols in protected plants revealed enhanced accumulation of these substances. The pathogenesis-related (PR) genes expression represented by PR-1b was increased in EPS-treated plants. This is the first report of a systemic induction of protection triggered by EPS produced by Serratia sp. against CMV-Y.

Comparative study of enteric viruses, coliphages and indicator bacteria for evaluating water quality in a tropical high-altitude system.

PubMed

Espinosa, Ana C; Arias, Carlos F; Sánchez-Colón, Salvador; Mazari-Hiriart, Marisa

2009-10-27

Bacteria used as indicators for pathogenic microorganisms in water are not considered adequate as enteric virus indicators. Surface water from a tropical high-altitude system located in Mexico City that receives rainwater, treated and non-treated wastewater used for irrigation, and groundwater used for drinking, was studied. The presence of enterovirus, rotavirus, astrovirus, coliphage, coliform bacteria, and enterococci was determined during annual cycles in 2001 and 2002. Enteric viruses in concentrated water samples were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Coliphages were detected using the double agar layer method. Bacteria analyses of the water samples were carried out by membrane filtration. The presence of viruses and bacteria in the water used for irrigation showed no relationship between current bacterial indicator detection and viral presence. Coliphages showed strong association with indicator bacteria and enterovirus, but weak association with other enteric viruses. Enterovirus and rotavirus showed significant seasonal differences in water used for irrigation, although this was not clear for astrovirus. Coliphages proved to be adequate faecal pollution indicators for the irrigation water studied. Viral presence in this tropical high-altitude system showed a similar trend to data previously reported for temperate zones.

Improved Detection of Norovirus and Hepatitis A Virus in Surface Water by Applying Pre-PCR Processing.

PubMed

Borgmästars, Emmy; Jazi, Mehrdad Mousavi; Persson, Sofia; Jansson, Linda; Rådström, Peter; Simonsson, Magnus; Hedman, Johannes; Eriksson, Ronnie

2017-12-01

Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) detection of waterborne RNA viruses generally requires concentration of large water volumes due to low virus levels. A common approach is to use dead-end ultrafiltration followed by precipitation with polyethylene glycol. However, this procedure often leads to the co-concentration of PCR inhibitors that impairs the limit of detection and causes false-negative results. Here, we applied the concept of pre-PCR processing to optimize RT-qPCR detection of norovirus genogroup I (GI), genogroup II (GII), and hepatitis A virus (HAV) in challenging water matrices. The RT-qPCR assay was improved by screening for an inhibitor-tolerant master mix and modifying the primers with twisted intercalating nucleic acid molecules. Additionally, a modified protocol based on chaotropic lysis buffer and magnetic silica bead nucleic acid extraction was developed for complex water matrices. A validation of the modified extraction protocol on surface and drinking waters was performed. At least a 26-fold improvement was seen in the most complex surface water studied. The modified protocol resulted in average recoveries of 33, 13, 8, and 4% for mengovirus, norovirus GI, GII, and HAV, respectively. The modified protocol also improved the limit of detection for norovirus GI and HAV. RT-qPCR inhibition with C q shifts of 1.6, 2.8, and 3.5 for norovirus GI, GII, and HAV, respectively, obtained for the standard nucleic acid extraction were completely eliminated by the modified protocol. The standard nucleic acid extraction method worked well on drinking water with no RT-qPCR inhibition observed and average recoveries of 80, 124, 89, and 32% for mengovirus, norovirus GI, GII, and HAV, respectively.

Gene expression profiles in paraffin-embedded core biopsy tissue predict response to chemotherapy in women with locally advanced breast cancer.

PubMed

Gianni, Luca; Zambetti, Milvia; Clark, Kim; Baker, Joffre; Cronin, Maureen; Wu, Jenny; Mariani, Gabriella; Rodriguez, Jaime; Carcangiu, Marialuisa; Watson, Drew; Valagussa, Pinuccia; Rouzier, Roman; Symmans, W Fraser; Ross, Jeffrey S; Hortobagyi, Gabriel N; Pusztai, Lajos; Shak, Steven

2005-10-10

We sought to identify gene expression markers that predict the likelihood of chemotherapy response. We also tested whether chemotherapy response is correlated with the 21-gene Recurrence Score assay that quantifies recurrence risk. Patients with locally advanced breast cancer received neoadjuvant paclitaxel and doxorubicin. RNA was extracted from the pretreatment formalin-fixed paraffin-embedded core biopsies. The expression of 384 genes was quantified using reverse transcriptase polymerase chain reaction and correlated with pathologic complete response (pCR). The performance of genes predicting for pCR was tested in patients from an independent neoadjuvant study where gene expression was obtained using DNA microarrays. Of 89 assessable patients (mean age, 49.9 years; mean tumor size, 6.4 cm), 11 (12%) had a pCR. Eighty-six genes correlated with pCR (unadjusted P < .05); pCR was more likely with higher expression of proliferation-related genes and immune-related genes, and with lower expression of estrogen receptor (ER) -related genes. In 82 independent patients treated with neoadjuvant paclitaxel and doxorubicin, DNA microarray data were available for 79 of the 86 genes. In univariate analysis, 24 genes correlated with pCR with P < .05 (false discovery, four genes) and 32 genes showed correlation with P < .1 (false discovery, eight genes). The Recurrence Score was positively associated with the likelihood of pCR (P = .005), suggesting that the patients who are at greatest recurrence risk are more likely to have chemotherapy benefit. Quantitative expression of ER-related genes, proliferation genes, and immune-related genes are strong predictors of pCR in women with locally advanced breast cancer receiving neoadjuvant anthracyclines and paclitaxel.

Limited clinical benefit of minority K103N and Y181C-variant detection in addition to routine genotypic resistance testing in antiretroviral therapy-naive patients.

PubMed

Metzner, Karin J; Scherrer, Alexandra U; von Wyl, Viktor; Böni, Jürg; Yerly, Sabine; Klimkait, Thomas; Aubert, Vincent; Furrer, Hansjakob; Hirsch, Hans H; Vernazza, Pietro L; Cavassini, Matthias; Calmy, Alexandra; Bernasconi, Enos; Weber, Rainer; Günthard, Huldrych F

2014-09-24

The presence of minority nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 variants prior to antiretroviral therapy (ART) has been linked to virologic failure in treatment-naive patients. We performed a large retrospective study to determine the number of treatment failures that could have been prevented by implementing minority drug-resistant HIV-1 variant analyses in ART-naïve patients in whom no NNRTI resistance mutations were detected by routine resistance testing. Of 1608 patients in the Swiss HIV Cohort Study, who have initiated first-line ART with two nucleoside reverse transcriptase inhibitors (NRTIs) and one NNRTI before July 2008, 519 patients were eligible by means of HIV-1 subtype, viral load and sample availability. Key NNRTI drug resistance mutations K103N and Y181C were measured by allele-specific PCR in 208 of 519 randomly chosen patients. Minority K103N and Y181C drug resistance mutations were detected in five out of 190 (2.6%) and 10 out of 201 (5%) patients, respectively. Focusing on 183 patients for whom virologic success or failure could be examined, virologic failure occurred in seven out of 183 (3.8%) patients; minority K103N and/or Y181C variants were present prior to ART initiation in only two of those patients. The NNRTI-containing, first-line ART was effective in 10 patients with preexisting minority NNRTI-resistant HIV-1 variant. As revealed in settings of case-control studies, minority NNRTI-resistant HIV-1 variants can have an impact on ART. However, the implementation of minority NNRTI-resistant HIV-1 variant analysis in addition to genotypic resistance testing (GRT) cannot be recommended in routine clinical settings. Additional associated risk factors need to be discovered.

Selection of reference genes for transcriptional analysis of edible tubers of potato (Solanum tuberosum L.).

PubMed

Mariot, Roberta Fogliatto; de Oliveira, Luisa Abruzzi; Voorhuijzen, Marleen M; Staats, Martijn; Hutten, Ronald C B; Van Dijk, Jeroen P; Kok, Esther; Frazzon, Jeverson

2015-01-01

Potato (Solanum tuberosum) yield has increased dramatically over the last 50 years and this has been achieved by a combination of improved agronomy and biotechnology efforts. Gene studies are taking place to improve new qualities and develop new cultivars. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a bench-marking analytical tool for gene expression analysis, but its accuracy is highly dependent on a reliable normalization strategy of an invariant reference genes. For this reason, the goal of this work was to select and validate reference genes for transcriptional analysis of edible tubers of potato. To do so, RT-qPCR primers were designed for ten genes with relatively stable expression in potato tubers as observed in RNA-Seq experiments. Primers were designed across exon boundaries to avoid genomic DNA contamination. Differences were observed in the ranking of candidate genes identified by geNorm, NormFinder and BestKeeper algorithms. The ranks determined by geNorm and NormFinder were very similar and for all samples the most stable candidates were C2, exocyst complex component sec3 (SEC3) and ATCUL3/ATCUL3A/CUL3/CUL3A (CUL3A). According to BestKeeper, the importin alpha and ubiquitin-associated/ts-n genes were the most stable. Three genes were selected as reference genes for potato edible tubers in RT-qPCR studies. The first one, called C2, was selected in common by NormFinder and geNorm, the second one is SEC3, selected by NormFinder, and the third one is CUL3A, selected by geNorm. Appropriate reference genes identified in this work will help to improve the accuracy of gene expression quantification analyses by taking into account differences that may be observed in RNA quality or reverse transcription efficiency across the samples.

Aberrant methylation and associated transcriptional mobilization of Alu elements contributes to genomic instability in hypoxia.

PubMed

Pal, Arnab; Srivastava, Tapasya; Sharma, Manish K; Mehndiratta, Mohit; Das, Prerna; Sinha, Subrata; Chattopadhyay, Parthaprasad

2010-11-01

Hypoxia is an integral part of tumorigenesis and contributes extensively to the neoplastic phenotype including drug resistance and genomic instability. It has also been reported that hypoxia results in global demethylation. Because a majority of the cytosine-phosphate-guanine (CpG) islands are found within the repeat elements of DNA, and are usually methylated under normoxic conditions, we suggested that retrotransposable Alu or short interspersed nuclear elements (SINEs) which show altered methylation and associated changes of gene expression during hypoxia, could be associated with genomic instability. U87MG glioblastoma cells were cultured in 0.1% O₂ for 6 weeks and compared with cells cultured in 21% O₂ for the same duration. Real-time PCR analysis showed a significant increase in SINE and reverse transcriptase coding long interspersed nuclear element (LINE) transcripts during hypoxia. Sequencing of bisulphite treated DNA as well as the Combined Bisulfite Restriction Analysis (COBRA) assay showed that the SINE loci studied underwent significant hypomethylation though there was patchy hypermethylation at a few sites. The inter-alu PCR profile of DNA from cells cultured under 6-week hypoxia, its 4-week revert back to normoxia and 6-week normoxia showed several changes in the band pattern indicating increased alu mediated genomic alteration. Our results show that aberrant methylation leading to increased transcription of SINE and reverse transcriptase associated LINE elements could lead to increased genomic instability in hypoxia. This might be a cause of genetic heterogeneity in tumours especially in variegated hypoxic environment and lead to a development of foci of more aggressive tumour cells. © 2009 The Authors Journal compilation © 2010 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

MicroRNAs for Detection of Pancreatic Neoplasia

PubMed Central

Vila-Navarro, Elena; Vila-Casadesús, Maria; Moreira, Leticia; Duran-Sanchon, Saray; Sinha, Rupal; Ginés, Àngels; Fernández-Esparrach, Glòria; Miquel, Rosa; Cuatrecasas, Miriam; Castells, Antoni; Lozano, Juan José; Gironella, Meritxell

2017-01-01

Objective: The aim of our study was to analyze the miRNome of pancreatic ductal adenocarcinoma (PDAC) and its preneoplastic lesion intraductal papillary mucinous neoplasm (IPMN), to find new microRNA (miRNA)-based biomarkers for early detection of pancreatic neoplasia. Objective: Effective early detection methods for PDAC are needed. miRNAs are good biomarker candidates. Methods: Pancreatic tissues (n = 165) were obtained from patients with PDAC, IPMN, or from control individuals (C), from Hospital Clínic of Barcelona. Biomarker discovery was done using next-generation sequencing in a discovery set of 18 surgical samples (11 PDAC, 4 IPMN, 3 C). MiRNA validation was carried out by quantitative reverse transcriptase PCR in 2 different set of samples. Set 1—52 surgical samples (24 PDAC, 7 IPMN, 6 chronic pancreatitis, 15 C), and set 2—95 endoscopic ultrasound-guided fine-needle aspirations (60 PDAC, 9 IPMN, 26 C). Results: In all, 607 and 396 miRNAs were significantly deregulated in PDAC and IPMN versus C. Of them, 40 miRNAs commonly overexpressed in both PDAC and IPMN were selected for further validation. Among them, significant up-regulation of 31 and 30 miRNAs was confirmed by quantitative reverse transcriptase PCR in samples from set 1 and set 2, respectively. Conclusions: miRNome analysis shows that PDAC and IPMN have differential miRNA profiles with respect to C, with a large number of deregulated miRNAs shared by both neoplastic lesions. Indeed, we have identified and validated 30 miRNAs whose expression is significantly increased in PDAC and IPMN lesions. The feasibility of detecting these miRNAs in endoscopic ultrasound-guided fine-needle aspiration samples makes them good biomarker candidates for early detection of pancreatic cancer. PMID:27232245

Assessment Effects of Resveratrol on Human Telomerase Reverse Transcriptase Messenger Ribonucleic Acid Transcript in Human Glioblastoma.

PubMed

Mirzazadeh, Azin; Kheirollahi, Majid; Farashahi, Ehsan; Sadeghian-Nodoushan, Fatemeh; Sheikhha, Mohammad Hasan; Aflatoonian, Behrouz

2017-01-01

Glioblastoma (GBM) is the most common and aggressive brain tumor, which has a poor prognosis despite the advent of different therapeutic strategies. There are numerous molecular biomarkers to contribute diagnosis, prognosis, and prediction of response to the current therapy in GBM. One of the most important markers that are potentially valuable is immortalization-specific or immortalization-associated marker named "hTERT messenger ribonucleic acid (mRNA)" the key subunit of telomerase enzyme, which is expressed in more than 85% of cancer cells, in spite of the majority of normal somatic cells. In this study, we investigated the effects of resveratrol (RSV) on this mRNA marker level, leading to cancer progression. U-87MG cell line was obtained from Pasteur Institute of Iran and treated with various concentrations of 0-160 μg/mL of RSV and at different time points (24, 48, and 72 h). To evaluate viability of U-87MG cells, standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed. Real-time polymerase chain reaction (RT-PCR) was used for comparative and quantitative assessment of human telomerase reverse transcriptase (hTERT) mRNA copy number versus control-untreated group. The results of our investigation suggested that RSV effectively inhibited cell growth and caused cell death in dose-dependent ( P < 0.05) and not in time-dependent manner ( P > 0.05), in vitro . Interestingly, quantitative RT-PCR analysis demonstrated that at half inhibition concentration, RSV dramatically decreased mRNA expression of hTERT, the catalytic subunit of telomerase enzyme, which leads to prevention of cell division and tumor progression. With regard to downregulation of this immortalization-associated marker, RSV may potentially be used as a therapeutic agent against GBM.

Detection of Viral Pathogens by Reverse Transcriptase PCR and of Microbial Indicators by Standard Methods in the Canals of the Florida Keys

PubMed Central

Griffin, Dale W.; Gibson, Charles J.; Lipp, Erin K.; Riley, Kelley; Paul, John H.; Rose, Joan B.

1999-01-01

In order to assess the microbial water quality in canal waters throughout the Florida Keys, a survey was conducted to determine the concentration of microbial fecal indicators and the presence of human pathogenic microorganisms. A total of 19 sites, including 17 canal sites and 2 nearshore water sites, were assayed for total coliforms, fecal coliforms, Escherichia coli, Clostridium perfringens, enterococci, coliphages, F-specific (F+) RNA coliphages, Giardia lamblia, Cryptosporidium parvum, and human enteric viruses (polioviruses, coxsackie A and B viruses, echoviruses, hepatitis A viruses, Norwalk viruses, and small round-structured viruses). Numbers of coliforms ranged from <1 to 1,410, E. coli organisms from <1 to 130, Clostridium spp. from <1 to 520, and enterococci from <1 to 800 CFU/100 ml of sample. Two sites were positive for coliphages, but no F+ phages were identified. The sites were ranked according to microbial water quality and compared to various water quality standards and guidelines. Seventy-nine percent of the sites were positive for the presence of enteroviruses by reverse transcriptase PCR (polioviruses, coxsackie A and B viruses, and echoviruses). Sixty-three percent of the sites were positive for the presence of hepatitis A viruses. Ten percent of the sites were positive for the presence of Norwalk viruses. Ninety-five percent of the sites were positive for at least one of the virus groups. These results indicate that the canals and nearshore waters throughout the Florida Keys are being impacted by human fecal material carrying human enteric viruses through current wastewater treatment strategies such as septic tanks. Exposure to canal waters through recreation and work may be contributing to human health risks. PMID:10473424

Persistence of Viral Reservoirs in Multiple Tissues after Antiretroviral Therapy Suppression in a Macaque RT-SHIV Model

PubMed Central

Franks, Tamera; Kiser, Rebecca; Coalter, Vicky; Smedley, Jeremy; Piatak, Michael; Mellors, John W.; Lifson, Jeffrey D.; Ambrose, Zandrea

2013-01-01

Although antiretroviral therapy (ART) can suppress HIV-1 replication sufficiently to eliminate measurable plasma viremia, infected cells remain and ensure viral recrudescence after discontinuation of ART. We used a macaque model of HIV-1/AIDS to evaluate the location of infected cells during ART. Twelve macaques were infected with RT-SHIVmne, a SIV containing HIV-1 reverse transcriptase, conferring sensitivity to non-nucleoside reverse transcriptase inhibitors (NNRTIs). Ten to fourteen weeks post-infection, 6 animals were treated with 3 or 4 antiretroviral drugs for 17-20 weeks; 6 control animals remained untreated. Viral DNA (vDNA) and RNA (vRNA) were measured in peripheral blood mononuclear cells (PBMC) and at necropsy in multiple tissues by quantitative PCR and RT-PCR. The majority of virally infected cells were located in lymphoid tissues with variable levels in the gastrointestinal tract of both treated and untreated animals. Tissue viral DNA levels correlated with week 1 plasma viremia, suggesting that tissues that harbor proviral DNA are established within the first week of infection. PBMC vDNA levels did not correlate with plasma viremia or tissue levels of vDNA. vRNA levels were high in lymphoid and gastrointestinal tissues of the untreated animals; animals on ART had little vRNA expressed in tissues and virus could not be cultured from lymph node resting CD4+ cells after 17-20 weeks on ART, indicating little or no ongoing viral replication. Strategies for eradication of HIV-1 will need to target residual virus in ART suppressed individuals, which may not be accurately reflected by frequencies of infected cells in blood. PMID:24367650

Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR.

PubMed Central

Huang, M M; Arnheim, N; Goodman, M F

1992-01-01

Thermus aquaticus (Taq) DNA polymerase was used to measure the extension efficiency for all configurations of matched and mismatched base pairs at template-primer 3'-termini. The transition mispairs, A(primer).C, C.A, G.T, and T.G were extended 10(-3) to 10(-4)-fold less efficiently than their correctly paired counterparts. Relative efficiencies for extending transversion mispairs were 10(-4) to 10(-5) for T.C and T.T, about 10(-6) for A.A, and less than 10(-6) for G.A, A.G, G.G and C.C. The transversion mispair C(primer).T was extended with high efficiency, about 10(-2) compared to a correct A.T basepair. The unexpected ease of extending the C.T mismatch was not likely to have been caused by primer-template misalignment. Taq polymerase was observed to bind with similar affinities to each of the correctly paired and mispaired primer-template 3'-ends. Thus, the failure of Taq polymerase to extend mismatches efficiently appears to be an intrinsic property of the enzyme and not due to an inability to bind to 3'-terminal mispairs. For almost all of the mispairs, C.T being the exception, Taq polymerase exhibits about 100 to 1000-fold greater discrimination against mismatch extension compared to avian myeloblastosis reverse transcriptase and HIV-1 reverse transcriptase which extend most mismatched basepairs permissively. Relative mismatch extension efficiencies for Taq polymerase were measured at 45 degrees C, 55 degrees C and 70 degrees C and found to be independent of temperature. The mispair extension data should be important in designing experiments using PCR to distinguish between sequences that vary by a single nucleotide. Images PMID:1408758

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing.

PubMed

Mohr, Sabine; Ghanem, Eman; Smith, Whitney; Sheeter, Dennis; Qin, Yidan; King, Olga; Polioudakis, Damon; Iyer, Vishwanath R; Hunicke-Smith, Scott; Swamy, Sajani; Kuersten, Scott; Lambowitz, Alan M

2013-07-01

Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3' ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.

Newly designed break-apart and ASPL-TFE3 dual-fusion FISH assay are useful in diagnosing Xp11.2 translocation renal cell carcinoma and ASPL-TFE3 renal cell carcinoma: a STARD-compliant article.

PubMed

Chen, Xiancheng; Yang, Yang; Gan, Weidong; Xu, Linfeng; Ye, Qing; Guo, Hongqian

2015-05-01

The diagnosis of Xp11.2 translocation renal cell carcinoma (tRCC), which relies on morphology and immunohistochemistry (IHC), is often either missed in the diagnosis or misdiagnosed. To improve the accuracy of diagnosis of Xp11.2 tRCC and ASPL-TFE3 renal cell carcinoma (RCC), we investigated newly designed fluorescence in situ hybridization (FISH) probes (diagnostic accuracy study).Based on the genetic characteristics of Xp11.2 tRCC and the ASPL-TFE3 RCC, a new break-apart TFE3 FISH probe and an ASPL-TFE3 dual-fusion FISH probe were designed and applied to 65 patients with RCC who were <45 years old or showed suspicious microscopic features of Xp11.2 tRCC in our hospital. To test the accuracy of the probes, we further performed reverse transcriptase-polymerase chain reaction (PCR) on 8 cases for which frozen tissues were available.Among the 65 cases diagnosed with RCC, TFE3 IHC was positive in 24 cases. Twenty-two cases were confirmed as Xp11.2 tRCC by break-apart TFE3 FISH, and 6 of these cases were further diagnosed as ASPL-TFE3 RCC by ASPL-TFE3 dual-fusion FISH detection. Importantly, reverse transcriptase-PCR showed concordant results with the results of FISH assay in the 8 available frozen cases.The break-apart and ASPL-TFE3 dual-fusion FISH assay can accurately detect the translocation of the TFE3 gene and ASPL-TFE3 fusion gene and can thus serve as a valid complementary method for diagnosing Xp11.2 tRCC and ASPL-TFE3 RCC.

Detection of Respiratory Viruses in Sputum from Adults by Use of Automated Multiplex PCR

PubMed Central

Walsh, Edward E.; Formica, Maria A.; Falsey, Ann R.

2014-01-01

Respiratory tract infections (RTI) frequently cause hospital admissions among adults. Diagnostic viral reverse transcriptase PCR (RT-PCR) of nose and throat swabs (NTS) is useful for patient care by informing antiviral use and appropriate isolation. However, automated RT-PCR systems are not amenable to utilizing sputum due to its viscosity. We evaluated a simple method of processing sputum samples in a fully automated respiratory viral panel RT-PCR assay (FilmArray). Archived sputum and NTS samples collected in 2008-2012 from hospitalized adults with RTI were evaluated. A subset of sputum samples positive for 10 common viruses by a uniplex RT-PCR was selected. A sterile cotton-tip swab was dunked in sputum, swirled in 700 μL of sterile water (dunk and swirl method) and tested by the FilmArray assay. Quantitative RT-PCR was performed on “dunked” sputum and NTS samples for influenza A (Flu A), respiratory syncytial virus (RSV), coronavirus OC43 (OC43), and human metapneumovirus (HMPV). Viruses were identified in 31% of 965 illnesses using a uniplex RT-PCR. The sputum sample was the only sample positive for 105 subjects, including 35% (22/64) of influenza cases and significantly increased the diagnostic yield of NTS alone (302/965 [31%] versus 197/965 [20%]; P = 0.0001). Of 108 sputum samples evaluated by the FilmArray assay using the dunk and swirl method, 99 (92%) were positive. Quantitative RT-PCR revealed higher mean viral loads in dunked sputum samples compared to NTS samples for Flu A, RSV, and HMPV (P = 0.0001, P = 0.006, and P = 0.011, respectively). The dunk and swirl method is a simple and practical method for reliably processing sputum samples in a fully automated PCR system. The higher viral loads in sputa may increase detection over NTS testing alone. PMID:25056335

Impact of the Central Polypurine Tract on the Kinetics of Human Immunodeficiency Virus Type 1 Vector Transduction

PubMed Central

Van Maele, Bénédicte; De Rijck, Jan; De Clercq, Erik; Debyser, Zeger

2003-01-01

Lentiviral vectors derived from human immunodeficiency virus type 1 (HIV-1) show great promise as gene carriers for future gene therapy. Insertion of a fragment containing the central polypurine tract (cPPT) in HIV-1 vector constructs is known to enhance transduction efficiency drastically, reportedly by facilitating the nuclear import of HIV-1 cDNA through a central DNA flap. We have studied the impact of the cPPT on the kinetics of HIV-1 vector transduction by real-time PCR. The kinetics of total HIV-1 DNA, two-long-terminal-repeat (2-LTR) circles, and, by an Alu-PCR, integrated proviral DNA were monitored. About 6 to 12 h after transduction, the total HIV-1 DNA reached a maximum level, followed by a steep decrease. The 2-LTR circles peaked after 24 to 48 h and were diluted upon cell division. Integration of HIV-1 DNA was first detected at 12 h postinfection. When HIV-1 vectors that contained the cPPT were used, DNA synthesis was similar but a threefold higher amount of 2-LTR circles was detected, confirming the impact on nuclear import. Moreover, a 10-fold increase in the amount of integrated DNA was observed in the presence of the cPPT. Only in the absence of the cPPT was a saturation in 2-LTR circle formation seen at a high multiplicity of infection, suggesting a role for the cPPT in overcoming a barrier to the nuclear import of HIV-1 DNA. A major effect of the central DNA flap on the juxtaposition of both LTRs is unlikely, since transduction with HIV-1 vectors containing ectopic cPPT fragments resulted in increased amounts of 2-LTR circles as well as integrated DNA. Inhibitors of transduction by cPPT-containing HIV vectors were also studied by real-time PCR. The reverse transcriptase inhibitor azidothymidine (AZT) and the nonnucleoside reverse transcriptase inhibitor α-APA clearly inhibited viral DNA synthesis, whereas integrase inhibitors such as the diketo acid L-708,906 and the pyranodipyrimidine V-165 specifically inhibited integration. PMID:12663775

Development and customization of a color-coded microbeads-based assay for drug resistance in HIV-1 reverse transcriptase.

PubMed

Gu, Lijun; Kawana-Tachikawa, Ai; Shiino, Teiichiro; Nakamura, Hitomi; Koga, Michiko; Kikuchi, Tadashi; Adachi, Eisuke; Koibuchi, Tomohiko; Ishida, Takaomi; Gao, George F; Matsushita, Masaki; Sugiura, Wataru; Iwamoto, Aikichi; Hosoya, Noriaki

2014-01-01

Drug resistance (DR) of HIV-1 can be examined genotypically or phenotypically. Although sequencing is the gold standard of the genotypic resistance testing (GRT), high-throughput GRT targeted to the codons responsible for DR may be more appropriate for epidemiological studies and public health research. We used a Japanese database to design and synthesize sequence-specific oligonucleotide probes (SSOP) for the detection of wild-type sequences and 6 DR mutations in the clade B HIV-1 reverse transcriptase region. We coupled SSOP to microbeads of the Luminex 100 xMAP system and developed a GRT based on the polymerase chain reaction (PCR)-SSOP-Luminex method. Sixteen oligoprobes for discriminating DR mutations from wild-type sequences at 6 loci were designed and synthesized, and their sensitivity and specificity were confirmed using isogenic plasmids. The PCR-SSOP-Luminex DR assay was then compared to direct sequencing using 74 plasma specimens from treatment-naïve patients or those on failing treatment. In the majority of specimens, the results of the PCR-SSOP-Luminex DR assay were concordant with sequencing results: 62/74 (83.8%) for M41, 43/74 (58.1%) for K65, 70/74 (94.6%) for K70, 55/73 (75.3%) for K103, 63/73 (86.3%) for M184 and 68/73 (93.2%) for T215. There were a number of specimens without any positive signals, especially for K65. The nucleotide position of A2723G, A2747G and C2750T were frequent polymorphisms for the wild-type amino acids K65, K66 and D67, respectively, and 14 specimens had the D67N mutation encoded by G2748A. We synthesized 14 additional oligoprobes for K65, and the sensitivity for K65 loci improved from 43/74 (58.1%) to 68/74 (91.9%). We developed a rapid high-throughput assay for clade B HIV-1 DR mutations, which could be customized by synthesizing oligoprobes suitable for the circulating viruses. The assay could be a useful tool especially for public health research in both resource-rich and resource-limited settings.

Factors Affecting Detection of Hepatitis E Virus on Canadian Retail Pork Chops and Pork Livers Assayed Using Real-Time RT-PCR.

PubMed

Wilhelm, B J; Leblanc, D; Avery, B; Pearl, D L; Houde, A; Rajić, A; McEwen, S A

2016-03-01

We collected 599 Canadian retail pork chops and 283 pork livers routinely (usually weekly) from April 2011 to March 2012 using the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) retail sampling platform. Samples were assayed using validated real-time (q) reverse transcriptase polymerase chain reaction (RT-PCR) and nested classical RT-PCR for the detection of hepatitis E virus (HEV), porcine enteric calicivirus (PEC) and rotavirus (RV). The presence of Escherichia coli, Salmonella spp. and Campylobacter spp. was measured on a subset of our samples. Exact logistic regression models were fitted for predictors for HEV detection, for each assay. For both assays, sample type (pork chop versus liver) was a significant predictor for HEV RNA detection. For nested classical RT-PCR but not qRT-PCR, region of sample collection was a significant predictor (P = 0.008) of HEV detection. Odds of HEV detection were greatest in spring relative to other seasons. E. coli was a significant predictor for HEV RNA detection using the qRT-PCR (P = 0.03). Overall, the prevalence of E. coli, Salmonella spp. and Campylobacter spp. was significantly greater than HEV, PEC or RV on our retail pork samples. Our sparse data set for the detection of PEC and RV precluded modelling of risk factors for the detection of these viruses. © 2015 Zoonoses and Public Health © 2015 Her Majesty the Queen in Right of Canada Reproduced with the permission of the Minister of the Public Health Agency of Canada.

Measurement of Gene Expression in Archival Paraffin-Embedded Tissues

PubMed Central

Cronin, Maureen; Pho, Mylan; Dutta, Debjani; Stephans, James C.; Shak, Steven; Kiefer, Michael C.; Esteban, Jose M.; Baker, Joffre B.

2004-01-01

Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10-μm FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests. PMID:14695316

Malaria prevalence defined by microscopy, antigen detection, DNA amplification and total nucleic acid amplification in a malaria-endemic region during the peak malaria transmission season.

PubMed

Waitumbi, John N; Gerlach, Jay; Afonina, Irina; Anyona, Samuel B; Koros, Joseph N; Siangla, Joram; Ankoudinova, Irina; Singhal, Mitra; Watts, Kate; Polhemus, Mark E; Vermeulen, Nicolaas M; Mahoney, Walt; Steele, Matt; Domingo, Gonzalo J

2011-07-01

To determine the malaria prevalence by microscopy, antigen detection and nucleic acid detection in a defined subpopulation in a Plasmodium falciparum-endemic region during the peak transmission season. Blood specimens were collected in a cross-sectional study involving children aged 5-10 years (n = 195) presenting with acute fever to two clinics in Western Kenya. All specimens underwent microscopy, HRP2 and aldolase antigen detection by enzyme immunoassay (EIA), parasite-specific DNA and total nucleic acid (RNA and DNA) by real-time PCR (qPCR) and reverse-transcriptase PCR (qRT-PCR). Microscopy detected 65/195 cases of malaria infection [95% confidence interval (CI) 52-78]. HRP2 and aldolase EIA had similar sensitivity levels detecting antigen in 65/195 (95% CI, 52-78) and 57/195 (95% CI, 45-70) cases. Discordants in antigen detection vs. microscopy occurred at <470 parasites/μl and <4900 parasites/μl for HRP2 and aldolase, respectively. Detection of total nucleic acid allowed a 3 log lower limit of detection than just DNA detection by real-time PCR in vitro. In clinical specimens, 114/195 (95% CI, 100-127) were qPCR positive (DNA), and 187/195 (95% CI, 179-191) were qRT-PCR positive (DNA plus RNA). The prevalence of submicroscopic malaria infection was significantly higher when detecting total nucleic acid than just DNA in this outpatient population during the high transmission season. Defining standards for submicroscopic infection will be important for control programmes, diagnostics development efforts and molecular epidemiology studies. © 2011 Blackwell Publishing Ltd.

Transcriptional expression of type-I interferon response genes and stability of housekeeping genes in the human endometrium and endometriosis.

PubMed

Vestergaard, Anna L; Knudsen, Ulla B; Munk, Torben; Rosbach, Hanne; Martensen, Pia M

2011-04-01

Endometriosis is a painful chronic female disease defined by the presence of endometrial tissue implants in ectopic (Ec) locations. The pathogenesis is much debated, and type-I interferons (IFNs) could be involved. The expression of genes of the type-I IFN response were profiled by a specific PCR array of RNA obtained from Ec and eutopic (Eu) endometrium collected from nine endometriosis patients and nine healthy control women. Transcriptional expression levels of selected IFN-regulated and housekeeping genes (HKGs) were investigated by real-time quantitative reverse transcriptase PCR (qRT-PCR). Stably expressed HKGs for valid normalization of transcriptional studies of endometrium and endometriosis have not yet been published. Here, seven HKGs were evaluated for stability using the GeNorm and NormFinder software. A normalization factor based on HMBS, TBP and YWHAZ expression was suitable for normalization of qRT-PCR studies of Eu versus Ec endometrium. In the endometrial cell lines HEC1A, HEC1B, Ishikawa and RL95-2, HMBS and HPRT1 were the most stably expressed. The IFN-specific PCR array indicated significantly different expression of the genes BST2, COL16A1, HOXB2 and ISG20 between the endometrial tissue types. However, by correctly normalized qRT-PCR, levels of BST2, COL16A1 and the highly type-I IFN-stimulated genes ISG12A and 6-16 displayed insignificant variations. Conversely, HOXB2 and ISG20 transcriptions were significantly reduced in endometriosis lesions compared with endometrium from endometriosis patients and healthy controls. In conclusion, appropriate HKGs for normalization of qRT-PCR studies of endometrium and endometriosis have been identified here. Abolished expression of ISG20 and HOX genes could be important in endometriosis.

Incidence of enteroviruses in Mamala Bay, Hawaii using cell culture and direct polymerase chain reaction methodologies.

PubMed

Reynolds, K A; Roll, K; Fujioka, R S; Gerba, C P; Pepper, I L

1998-06-01

The consequence of point and nonpoint pollution sources, discharged into marine waters, on public recreational beaches in Mamala Bay, Hawaii was evaluated using virus cell culture and direct reverse transcriptase-polymerase chain reaction (RT-PCR). Twelve sites, nine marine, two freshwater (one stream and one canal), and one sewage, were assessed either quarterly or monthly for 1 year to detect the presence of human enteric viruses. Water samples were concentrated from initial volumes of 400 L to final volumes of 30 mL using Filterite electronegative cartridge filters and a modified beef extract elution procedure. Cell culture was applied using the Buffalo Green Monkey kidney cell line to analyze samples for enteroviruses. Positive samples were also evaluated by RT-PCR, using enterovirus-specific primers. Levels of RT-PCR inhibition varied with each concentrated sample. Resin column purification increased PCR detection sensitivity by at least one order of magnitude in a variety of sewage outfall and recreational marine water samples but not in the freshwater canal samples. Using cell culture, viable enteroviruses were found in 50 and 17% of all outfall and canal samples, respectively. Samples were positive at beaches 8% of the time. These data illustrate the potential public health hazard associated with recreational waters. Using direct PCR, viruses were detected at the outfall but were not found in any beach or canal samples, in part, owing to substances that inhibit PCR. Therefore, conventional cell culture is the most effective means of detecting low levels of infectious enteroviruses in environmental waters, whereas direct RT-PCR is rendered less effective by inhibitory compounds and low equivalent reaction volumes.

Challenges for accurate and prompt molecular diagnosis of clades of highly pathogenic avian influenza H5N1 viruses emerging in Vietnam.

PubMed

Slomka, Marek J; To, Thanh L; Tong, Hien H; Coward, Vivien J; Hanna, Amanda; Shell, Wendy; Pavlidis, Theo; Densham, Anstice L E; Kargiolakis, Georgios; Arnold, Mark E; Banks, Jill; Brown, Ian H

2012-01-01

Forty-six chickens and 48 ducks were sampled from four Vietnamese poultry premises in 2009 infected with H5N1 highly pathogenic avian influenza (HPAI) clade 2.3.2 and 2.3.4 viruses, which also differed by cleavage site (CS) sequences in their haemagglutinin (HA) genes. All clinical specimens (n=282), namely tracheal and cloacal swabs plus feathers, were tested by five Eurasian reverse-transcriptase AI RealTime polymerase chain reaction (RRT-PCR) methods. Bayesian modelling showed similar high sensitivity for the validated H5 HA2 RRT-PCR and a new modified M-gene RRT-PCR that utilizes lyophilized reagents. Both were more sensitive than the validated "wet" M-gene RRT-PCR. Another RRT-PCR, which targeted the H5-gene CS region, was effective for clade 2.3.4 detection, but severely compromised for clade 2.3.2 viruses. Reduced sensitivity of the H5 CS and "wet" M-gene RRT-PCRs correlated with mismatches between the target and the primer and/or probe sequences. However, the H5 HA2 RRT-PCR sensitively detected both clade 2.3.2 and 2.3.4 viruses, and agreed with N1 RRT-PCR results. Feather testing from diseased chicken and duck flocks by AI RRT-PCRs resulted in the most sensitive identification of H5N1 HPAI-infected birds. Evolution of new H5N1 HPAI clades remains a concern for currently affected Asian countries, but also for more distant regions where it is important to be prepared for new incursions of H5N1 HPAI viruses. Genetic evidence for adamantane resistance and sensitivity was also observed in isolates from both clades.

A comparative kinetic RT/-PCR strategy for the quantitation of mRNAs in microdissected human renal biopsy specimens.

PubMed

Del Prete, D; Forino, M; Gambaro, G; D'Angelo, A; Baggio, B; Anglani, F

1998-01-01

Molecular biology techniques, to be applicable to a diagnostic renal biopsy specimen, should (1) be highly sensitive to be performed on a very small quantity of tissue; (2) be quantitative because they have to analyze genes normally expressed in the tissue and (3) allow the analysis of as large a number of genes as possible. Among different methods, only the reverse-transcriptase polymerase chain reaction (RT/-PCR) might comply with previous requisites, but the few RT/-PCR examples on renal biopsies in the literature do not allow starting RNA quantification and quality control; furthermore they have the drawback of analyzing only few genes. In an ongoing study to assess the expression of a number of genes in glomeruli and in tubulointerstitium of patients with different nephropathies, we developed a comparative RT/-PCR kinetic strategy based on the purification and quantification of total glomerular and tubulointerstitial RNA and on the use of an internal standard, the housekeeping gene G3PDH. We demonstrate that in microdissected diagnostic renal biopsies (1) glomerular and interstitial starting RNA can be quantified; (2) the G3PDH gene may be used both as an internal standard and as an indirect marker of RNA integrity; (3) as low as 28 ng of total RNA is sufficient to obtain PCR products of eight genes, and (4) it is worth to operate on microdissected biopsy specimens because of the different expression of genes in the two renal compartments.

Sensitive and specific miRNA detection method using SplintR Ligase

PubMed Central

Jin, Jingmin; Vaud, Sophie; Zhelkovsky, Alexander M.; Posfai, Janos; McReynolds, Larry A.

2016-01-01

We describe a simple, specific and sensitive microRNA (miRNA) detection method that utilizes Chlorella virus DNA ligase (SplintR® Ligase). This two-step method involves ligation of adjacent DNA oligonucleotides hybridized to a miRNA followed by real-time quantitative PCR (qPCR). SplintR Ligase is 100X faster than either T4 DNA Ligase or T4 RNA Ligase 2 for RNA splinted DNA ligation. Only a 4–6 bp overlap between a DNA probe and miRNA was required for efficient ligation by SplintR Ligase. This property allows more flexibility in designing miRNA-specific ligation probes than methods that use reverse transcriptase for cDNA synthesis of miRNA. The qPCR SplintR ligation assay is sensitive; it can detect a few thousand molecules of miR-122. For miR-122 detection the SplintR qPCR assay, using a FAM labeled double quenched DNA probe, was at least 40× more sensitive than the TaqMan assay. The SplintR method, when coupled with NextGen sequencing, allowed multiplex detection of miRNAs from brain, kidney, testis and liver. The SplintR qPCR assay is specific; individual let-7 miRNAs that differ by one nucleotide are detected. The rapid kinetics and ability to ligate DNA probes hybridized to RNA with short complementary sequences makes SplintR Ligase a useful enzyme for miRNA detection. PMID:27154271

Blood-Stage Parasitaemia and Age Determine Plasmodium falciparum and P. vivax Gametocytaemia in Papua New Guinea.

PubMed

Koepfli, Cristian; Robinson, Leanne J; Rarau, Patricia; Salib, Mary; Sambale, Naomi; Wampfler, Rahel; Betuela, Inoni; Nuitragool, Wang; Barry, Alyssa E; Siba, Peter; Felger, Ingrid; Mueller, Ivo

2015-01-01

A better understanding of human-to-mosquito transmission is crucial to control malaria. In order to assess factors associated with gametocyte carriage, 2083 samples were collected in a cross-sectional survey in Papua New Guinea. Plasmodium species were detected by light microscopy and qPCR and gametocytes by detection of pfs25 and pvs25 mRNA transcripts by reverse-transcriptase PCR (qRT-PCR). The parasite prevalence by PCR was 18.5% for Plasmodium falciparum and 13.0% for P. vivax. 52.5% of all infections were submicroscopic. Gametocytes were detected in 60% of P. falciparum-positive and 51% of P. vivax-positive samples. Each 10-fold increase in parasite density led to a 1.8-fold and 3.3-fold increase in the odds of carrying P. falciparum and P. vivax gametocytes. Thus the proportion of gametocyte positive and gametocyte densities was highest in young children carrying high asexual parasite densities and in symptomatic individuals. Dilution series of gametocytes allowed absolute quantification of gametocyte densities by qRT-PCR and showed that pvs25 expression is 10-20 fold lower than pfs25 expression. Between 2006 and 2010 parasite prevalence in the study site has decreased by half. 90% of the remaining infections were asymptomatic and likely constitute an important reservoir of transmission. However, mean gametocyte densities were low (approx. 1-2 gametocyte/μL) and it remains to be determined to what extent low-density gametocyte positive individuals are infective to mosquitos.

Opposing actions of Arx and Pax4 in endocrine pancreas development

PubMed Central

Collombat, Patrick; Mansouri, Ahmed; Hecksher-Sørensen, Jacob; Serup, Palle; Krull, Jens; Gradwohl, Gerard; Gruss, Peter

2003-01-01

Genes encoding homeodomain-containing proteins potentially involved in endocrine pancreas development were isolated by combined in silico and nested-PCR approaches. One such transcription factor, Arx, exhibits Ngn3-dependent expression throughout endocrine pancreas development in α, β-precursor, and δ cells. We have used gene targeting in mouse embryonic stem cells to generate Arx loss-of-function mice. Arx-deficient animals are born at the expected Mendelian frequency, but develop early-onset hypoglycemia, dehydration, and weakness, and die 2 d after birth. Immunohistological analysis of pancreas from Arx mutants reveals an early-onset loss of mature endocrine α cells with a concomitant increase in β-and δ-cell numbers, whereas islet morphology remains intact. Our study indicates a requirement of Arx for α-cell fate acquisition and a repressive action on β-and δ-cell destiny, which is exactly the opposite of the action of Pax4 in endocrine commitment. Using multiplex reverse transcriptase PCR (RT-PCR), we demonstrate an accumulation of Pax4 and Arx transcripts in Arx and Pax4 mutant mice, respectively. We propose that the antagonistic functions of Arx and Pax4 for proper islet cell specification are related to the pancreatic levels of the respective transcripts. PMID:14561778

Opposing actions of Arx and Pax4 in endocrine pancreas development.

PubMed

Collombat, Patrick; Mansouri, Ahmed; Hecksher-Sorensen, Jacob; Serup, Palle; Krull, Jens; Gradwohl, Gerard; Gruss, Peter

2003-10-15

Genes encoding homeodomain-containing proteins potentially involved in endocrine pancreas development were isolated by combined in silico and nested-PCR approaches. One such transcription factor, Arx, exhibits Ngn3-dependent expression throughout endocrine pancreas development in alpha, beta-precursor, and delta cells. We have used gene targeting in mouse embryonic stem cells to generate Arx loss-of-function mice. Arx-deficient animals are born at the expected Mendelian frequency, but develop early-onset hypoglycemia, dehydration, and weakness, and die 2 d after birth. Immunohistological analysis of pancreas from Arx mutants reveals an early-onset loss of mature endocrine alpha cells with a concomitant increase in beta-and delta-cell numbers, whereas islet morphology remains intact. Our study indicates a requirement of Arx for alpha-cell fate acquisition and a repressive action on beta-and delta-cell destiny, which is exactly the opposite of the action of Pax4 in endocrine commitment. Using multiplex reverse transcriptase PCR (RT-PCR), we demonstrate an accumulation of Pax4 and Arx transcripts in Arx and Pax4 mutant mice, respectively. We propose that the antagonistic functions of Arx and Pax4 for proper islet cell specification are related to the pancreatic levels of the respective transcripts.

A two-step lyssavirus real-time polymerase chain reaction using degenerate primers with superior sensitivity to the fluorescent antigen test.

PubMed

Suin, Vanessa; Nazé, Florence; Francart, Aurélie; Lamoral, Sophie; De Craeye, Stéphane; Kalai, Michael; Van Gucht, Steven

2014-01-01

A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤ 1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible.

A Two-Step Lyssavirus Real-Time Polymerase Chain Reaction Using Degenerate Primers with Superior Sensitivity to the Fluorescent Antigen Test

PubMed Central

Nazé, Florence; Francart, Aurélie; Lamoral, Sophie; De Craeye, Stéphane; Kalai, Michael

2014-01-01

A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible. PMID:24822188

Disappearance of Ph1 chromosome with intensive chemotherapy and detection of minimal residual disease by polymerase chain reaction in a patient with blast crisis of chronic myelogenous leukemia.

PubMed

Honda, H; Miyagawa, K; Endo, M; Takaku, F; Yazaki, Y; Hirai, H

1993-06-01

We diagnosed a patient with chronic myelogenous leukemia (CML) in chronic phase (CP) on the basis of clinical findings, Ph1 chromosome detected by cytogenetic analysis, and bcr-abl fusion mRNA detected by reverse transcriptase-dependent polymerase chain reaction (RT-PCR). One month after diagnosis, the patient developed extramedullary blast crisis in the lymph nodes, and then medullary blast crisis in the bone marrow, in which different surface markers were shown. Combination chemotherapy with BH-AC, VP16, and mitoxantrone was administered; this resulted in rapid disappearance of the lymphadenopathy, restoration of normal hematopoiesis, and no Ph1 chromosome being detected by cytogenetic analysis. RT-PCR performed to detect the residual Ph1 clone revealed that although the Ph1 clone was preferentially suppressed, it was still residual. The intensive chemotherapy regimen preferentially suppressed the Ph1-positive clone and led to both clinical and cytogenetic remission in this patient with BC of CML; we suggest that RT-PCR is a sensitive and useful method for detecting minimal residual disease during the clinical course of this disease.

Coinfection and vertical transmission of Brucella and Morbillivirus in a neonatal sperm whale (Physeter macrocephalus) in Hawaii, USA.

PubMed

West, Kristi L; Levine, Gregg; Jacob, Jessica; Jensen, Brenda; Sanchez, Susan; Colegrove, Kathleen; Rotstein, David

2015-01-01

The viral genus Morbillivirus and the bacterial genus Brucella have emerged as important groups of pathogens that are known to affect cetacean health on a global scale, but neither pathogen has previously been reported from endangered sperm whales (Physeter macrocephalus). A female neonate sperm whale stranded alive and died near Laie on the island of Oahu, Hawaii, US, in May of 2011. Congestion of the cerebrum and enlarged lymph nodes were noted on the gross necropsy. Microscopic findings included lymphoid depletion, chronic meningitis, and pneumonia, suggesting an in utero infection. Cerebrum, lung, umbilicus, and select lymph nodes (tracheobronchial and mediastinal) were positive for Brucella by PCR. Brucella sp. was also cultured from the cerebrum and from mediastinal and tracheobronchial lymph nodes. Twelve different tissues were screened for Morbillivirus by reverse-transcriptase (RT)-PCR and select tissues by immunohistochemistry, but only the tracheobronchial lymph node and spleen were positive by RT-PCR. Pathologic findings observed were likely a result of Brucella, but Morbillivirus may have played a key role in immune suppression of the mother and calf. The in utero infection in this individual strongly supports vertical transmission of both pathogens.

MAEWEST Expression in Flower Development of Two Petunia Species

PubMed Central

Segatto, Ana Lúcia A.; Turchetto-Zolet, Andreia Carina; Aizza, Lilian Cristina B.; Monte-Bello, Carolina C.; Dornelas, Marcelo C.; Margis, Rogerio; Freitas, Loreta B.

2013-01-01

Changes in flower morphology may influence the frequency and specificity of animal visitors. In Petunia (Solanaceae), adaptation to different pollinators is one of the factors leading to species diversification within the genus. This study provides evidence that differential expression patterns of MAWEWEST (MAW) homologs in different Petunia species may be associated with adaptive changes in floral morphology. The Petunia × hybrida MAW gene belongs to the WOX (WUSCHEL-related homeobox) transcription factor family and has been identified as a controller of petal fusion during corolla formation. We analyzed the expression patterns of P. inflata and P. axillaris MAW orthologs (PiMAW and PaMAW, respectively) by reverse transcriptase polymerase chain reaction (RT-PCR), reverse transcription–quantitative PCR (qRT-PCR) and in situ hybridization in different tissues and different developmental stages of flowers in both species. The spatial expression patterns of PiMAW and PaMAW were similar in P. inflata and P. axillaris. Nevertheless, PaMAW expression level in P. axillaris was higher during the late bud development stage as compared to PiMAW in P. inflata. This work represents an expansion of petunia developmental research to wild accessions. PMID:23823801

AtDMC1, the Arabidopsis homologue of the yeast DMC1 gene: characterization, transposon-induced allelic variation and meiosis-associated expression.

PubMed

Klimyuk, V I; Jones, J D

1997-01-01

Based on homologies between the yeast DMC1 and the lily LIM15 meiosis-specific genes, degenerate PCR primers were designed that amplified the Arabidopsis DMC1 gene (AtDMC1). AtDMC1 genomic DNA (8 kb) was sequenced, and the transcript was characterized by reverse transcriptase-polymerase chain reaction (RT-PCR) and by 5' and 3' RACE (rapid amplification of cDNA ends). The AtDMC1 gene contains 15 exons and 14 introns. RNA in situ hybridization analysis showed that expression of the AtDMC1 is restricted to pollen mother cells in anthers and to megaspore mother cells in ovules. The AtDMC1 promoter was fused to the GUS reporter gene, and conferred meiosis-associated expression in both male and female floral lineages. Comparison of AtDMC1 isolated from Landsberg erecta ecotype to its Columbia allele ArLIM15, revealed the presence of a 1874 bp transposon-like element within the promoter region of ArLIM15. RT-PCR analysis showed that the expression levels of AtDMC1 and ArLIM15 are similar. Possible uses for the AtDMC1 promoter are discussed.

Impact of human immunodeficiency virus type 1 reverse transcriptase inhibitor drug resistance mutation interactions on phenotypic susceptibility.

PubMed

Trivedi, Vinod; Von Lindern, Jana; Montes-Walters, Miguel; Rojo, Daniel R; Shell, Elisabeth J; Parkin, Neil; O'Brien, William A; Ferguson, Monique R

2008-10-01

The role specific reverse transcriptase (RT) drug resistance mutations play in influencing phenotypic susceptibility to RT inhibitors in virus strains with complex resistance interaction patterns was assessed using recombinant viruses that consisted of RT-PCR-amplified pol fragments derived from plasma HIV-1 RNA from two treatment-experienced patients. Specific modifications of key RT amino acids were performed by site-directed mutagenesis. A panel of viruses with defined genotypic resistance mutations was assessed for phenotypic drug resistance. Introduction of M184V into several different clones expressing various RT resistance mutations uniformly decreased susceptibility to abacavir, lamivudine, and didanosine, and increased susceptibility to zidovudine, stavudine, and tenofovir; replication capacity was decreased. The L74V mutation had similar but slightly different effects, contributing to decreased susceptibility to abacavir, lamivudine, and didanosine and increased susceptibility to zidovudine and tenofovir, but in contrast to M184V, L74V contributed to decreased susceptibility to stavudine. In virus strains with the nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations K101E and G190S, the L74V mutation increased replication capacity, consistent with published observations, but replication capacity was decreased in strains without NNRTI resistance mutations. K101E and G190S together tend to decrease susceptibility to all nucleoside RT inhibitors, but the K103N mutation had little effect on nucleoside RT inhibitor susceptibility. Mutational interactions can have a substantial impact on drug resistance phenotype and replication capacity, and this has been exploited in clinical practice with the development of fixed-dose combination pills. However, we are the first to report these mutational interactions using molecularly cloned recombinant strains derived from viruses that occur naturally in HIV-infected individuals.

Impact of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Inhibitor Drug Resistance Mutation Interactions on Phenotypic Susceptibility

PubMed Central

Trivedi, Vinod; Von Lindern, Jana; Montes-Walters, Miguel; Rojo, Daniel R.; Shell, Elisabeth J.; Parkin, Neil; O'Brien, William A.

2008-01-01

Abstract The role specific reverse transcriptase (RT) drug resistance mutations play in influencing phenotypic susceptibility to RT inhibitors in virus strains with complex resistance interaction patterns was assessed using recombinant viruses that consisted of RT-PCR-amplified pol fragments derived from plasma HIV-1 RNA from two treatment-experienced patients. Specific modifications of key RT amino acids were performed by site-directed mutagenesis. A panel of viruses with defined genotypic resistance mutations was assessed for phenotypic drug resistance. Introduction of M184V into several different clones expressing various RT resistance mutations uniformly decreased susceptibility to abacavir, lamivudine, and didanosine, and increased susceptibility to zidovudine, stavudine, and tenofovir; replication capacity was decreased. The L74V mutation had similar but slightly different effects, contributing to decreased susceptibility to abacavir, lamivudine, and didanosine and increased susceptibility to zidovudine and tenofovir, but in contrast to M184V, L74V contributed to decreased susceptibility to stavudine. In virus strains with the nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations K101E and G190S, the L74V mutation increased replication capacity, consistent with published observations, but replication capacity was decreased in strains without NNRTI resistance mutations. K101E and G190S together tend to decrease susceptibility to all nucleoside RT inhibitors, but the K103N mutation had little effect on nucleoside RT inhibitor susceptibility. Mutational interactions can have a substantial impact on drug resistance phenotype and replication capacity, and this has been exploited in clinical practice with the development of fixed-dose combination pills. However, we are the first to report these mutational interactions using molecularly cloned recombinant strains derived from viruses that occur naturally in HIV-infected individuals. PMID:18844463

Evidence for airborne transmission of Norwalk-like virus (NLV) in a hotel restaurant.

PubMed Central

Marks, P. J.; Vipond, I. B.; Carlisle, D.; Deakin, D.; Fey, R. E.; Caul, E. O.

2000-01-01

An outbreak of gastroenteritis followed a meal in a large hotel during which one of the diners vomited. The clinical features of the illness suggested Norwalk-like virus (NLV, small round structured virus) infection, and this was confirmed by electron microscopy and reverse transcriptase polymerase chain reaction (RT-PCR) of stool samples. Further characterization of the virus by nucleotide sequence analysis of the PCR amplicons revealed identical strains in all the affected individuals. The foods served at the meal could not be demonstrated to be the cause of the outbreak. Analysis of attack rates by dining table showed an inverse relationship with the distance from the person who vomited. No one eating in a separate restaurant reported illness. Transmission from person-to-person or direct contamination of food seems unlikely in this outbreak. However, the findings are consistent with airborne spread of NLV with infection by inhalation with subsequent ingestion of virus particles. PMID:10982072

Canine distemper of vaccine origin in European mink, Mustela lutreola--a case report.

PubMed

Ek-Kommonen, C; Rudbäck, E; Anttila, M; Aho, M; Huovilainen, A

2003-04-02

Cases of canine distemper (CD) related to vaccination of exotic carnivores extend over three decades and have been described in at least nine different species. Our report describes a case of acute CD in a European mink, Mustela lutreola, vaccinated with live attenuated CD vaccine licensed for use in fur-farmed mink. The male mink died of an acute grey matter disease with an unusually long incubation period. A female vaccinated at the same time showed no obvious signs of illness. The diagnosis was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and by subsequent sequencing of the PCR products. The sequenced products of the virus isolated from the mink and of the vaccine batch showed 100% identity. This is the first report in which molecular methods were used to confirm that the disease was caused by the vaccine strain. Based on our findings, it is clearly evident that current CD vaccines cannot be safely used in exotic species.

Dengue Virus in Bats from Southeastern Mexico

PubMed Central

Sotomayor-Bonilla, Jesús; Chaves, Andrea; Rico-Chávez, Oscar; Rostal, Melinda K.; Ojeda-Flores, Rafael; Salas-Rojas, Mónica; Aguilar-Setien, Álvaro; Ibáñez-Bernal, Sergio; Barbachano-Guerrero, Arturo; Gutiérrez-Espeleta, Gustavo; Aguilar-Faisal, J. Leopoldo; Aguirre, A. Alonso; Daszak, Peter; Suzán, Gerardo

2014-01-01

To identify the relationship between landscape use and dengue virus (DENV) occurrence in bats, we investigated the presence of DENV from anthropogenically changed and unaltered landscapes in two Biosphere Reserves: Calakmul (Campeche) and Montes Azules (Chiapas) in southern Mexico. Spleen samples of 146 bats, belonging to 16 species, were tested for four DENV serotypes with standard reverse transcriptase polymerase chain reaction (RT-PCR) protocols. Six bats (4.1%) tested positive for DENV-2: four bats in Calakmul (two Glossophaga soricina, one Artibeus jamaicensis, and one A. lituratus) and two bats in Montes Azules (both A. lituratus). No effect of anthropogenic disturbance on the occurrence of DENV was detected; however, all three RT-PCR–positive bat species are considered abundant species in the Neotropics and well-adapted to disturbed habitats. To our knowledge, this study is the first study conducted in southeastern Mexico to identify DENV-2 in bats by a widely accepted RT-PCR protocol. The role that bats play on DENV's ecology remains undetermined. PMID:24752688

Absence of Measles Virus Detection from Stapes of Patients with Otosclerosis.

PubMed

Flores-García, María de Lourdes; Colín-Castro, Claudia Adriana; Hernández-Palestina, Mario Sabas; Sánchez-Larios, Roberto; Franco-Cendejas, Rafael

2018-01-01

Objective To determine molecularly the presence of measles virus genetic material in the stapes of patients with otosclerosis. Study Design A cross-sectional study. Setting A tertiary referral hospital. Subjects and Methods Genetic material was extracted from the stapes of patients with otosclerosis (n = 93) during the period from March 2011 to April 2012. The presence of viral measles sequences was evaluated by the real-time reverse transcriptase polymerase chain reaction (RT-PCR). The expression of the CD46 gene was determined. Results Ninety-three patients were included in the study. No sample was positive for any of 3 measles virus genes (H, N, and F). Measles virus RNA was not detected in any sample by real-time RT-PCR. CD46 levels were positive in 3.3% (n = 3) and negative in 96.7% (n = 90). Conclusion This study does not support the theory of measles virus as the cause of otosclerosis. It is necessary to do more research about other causal theories to clarify its etiology and prevention.

Identification of genotypes of Influenza A virus in Malaysia

PubMed Central

MM, Rahman; KK, Wong; I, Isahak; ZZ, Rashid; H, Alfizah

2014-01-01

Objective: Influenza is considered as an emerging disease until today. The present study was undertaken to determine the prevalent genotypes of Influenza A virus in Malaysia. Methods: Influenza A virus was identified from respiratory specimens by real-time reverse transcriptase polymerase chain reaction (rRT-PCR). Phylogenetic analysis of the identified isolates was performed and genotypes were detected. Results: A total number of 505 throat swabs and nasopharyngeal aspirates were examined by rRT-PCR at Universiti Kebangsaan Malaysia Medical Centre (UKMMC) in which 65(12.87%) were positive for influenza A. The identified isolates were successfully genotyped by phylogenetic analysis. The identified influenza A genotypes were: H1N1 (42), H3N2 (20) and H5N1 (3). Conclusion: The findings indicated that 3 genotypes were circulating in Malaysia during 2011 in which H1N1 was the predominant. Results added new genotype (H5N1) identification record in Malaysia that may be added in data base of WHO and CDC. PMID:25225528

Identification of genotypes of Influenza A virus in Malaysia.

PubMed

Mm, Rahman; Kk, Wong; I, Isahak; Zz, Rashid; H, Alfizah

2014-09-01

Influenza is considered as an emerging disease until today. The present study was undertaken to determine the prevalent genotypes of Influenza A virus in Malaysia. Influenza A virus was identified from respiratory specimens by real-time reverse transcriptase polymerase chain reaction (rRT-PCR). Phylogenetic analysis of the identified isolates was performed and genotypes were detected. A total number of 505 throat swabs and nasopharyngeal aspirates were examined by rRT-PCR at Universiti Kebangsaan Malaysia Medical Centre (UKMMC) in which 65(12.87%) were positive for influenza A. The identified isolates were successfully genotyped by phylogenetic analysis. The identified influenza A genotypes were: H1N1 (42), H3N2 (20) and H5N1 (3). The findings indicated that 3 genotypes were circulating in Malaysia during 2011 in which H1N1 was the predominant. RESULTS added new genotype (H5N1) identification record in Malaysia that may be added in data base of WHO and CDC.

Expression of very low density lipoprotein receptor mRNA in circulating human monocytes: its up-regulation by hypoxia.

PubMed

Nakazato, K; Ishibashi, T; Nagata, K; Seino, Y; Wada, Y; Sakamoto, T; Matsuoka, R; Teramoto, T; Sekimata, M; Homma, Y; Maruyama, Y

2001-04-01

Although very low density lipoprotein (VLDL) receptor expression by macrophages has been shown in the vascular wall, it is not clear whether or not circulating monocytes express the VLDL receptor. We investigated the expression of VLDL receptor mRNA in human peripheral blood monocytes and monocyte-derived macrophages by reverse transcriptase polymerase chain reaction (RT-PCR) and nucleotide sequencing after subcloning of PCR product. VLDL receptor mRNA was detected both in peripheral blood monocytes and monocyte-derived macrophages. Expression of VLDL receptor mRNA was upregulated by hypoxia in monocytes, whereas treatment with oxidized LDL, interleukin-1beta or monocyte chemoattractant protein-1 did not affect the levels of VLDL receptor mRNA in monocytes and macrophages. The present study shows a novel response of VLDL receptor mRNA to hypoxia, suggesting a role for VLDL receptor in the metabolism of lipoproteins in the vascular wall and the development of atherosclerosis.

Rapid detection of duck hepatitis A virus genotype C using reverse transcription loop-mediated isothermal amplification.

PubMed

Li, Chuanfeng; Chen, Zongyan; Meng, Chunchun; Liu, Guangqing

2014-02-01

A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was used and optimized to develop a rapid and sensitive detection system for duck hepatitis A virus genotype C (DHAV-C) RNA. A set of four specific primers was designed against highly conserved sequences located within the 3D gene from DHAV (strain GX1201). Under optimal reaction conditions, the sensitivity of DHAV-C-specific RT-LAMP was 100-fold higher than that of reverse transcriptase-polymerase chain reaction (RT-PCR), with a detection limit of 0.3pg (6.59×10(4) copies) per reaction. No cross-reactivity was observed from the samples of other duck viruses, which is in good accordance with RT-PCR. Furthermore, a positive reaction can be visually inspected by observing turbidity or color change after the addition of SYBR green I dye. The DHAV-C-specific RT-LAMP assay was applied to the samples and compared with RT-PCR. The positive-sample ratios were 26.7% (12 of 45) by RT-LAMP and 20% (9 of 45) by RT-PCR. Therefore, the newly developed RT-LAMP assay is a rapid, specific, sensitive, and cost-effective method of DHAV-C detection. This assay has potential applications in both clinical diagnosis and field surveillance of DHAV-C infection. Copyright © 2013. Published by Elsevier B.V.

Specific, sensitive, and rapid assay for human immunodeficiency virus type 1 pol mutations associated with resistance to zidovudine and didanosine.

PubMed Central

Frenkel, L M; Wagner, L E; Atwood, S M; Cummins, T J; Dewhurst, S

1995-01-01

The effectiveness of antiretroviral therapy may be limited by the development of human immunodeficiency virus type 1 (HIV-1) resistance. Monitoring for resistance will perhaps allow changes in therapy prior to deterioration in the patient's clinical or immunologic status. Our objective was to develop a rapid, specific, and sensitive genotypic assay for HIV-1 resistance to zidovudine (ZDV) and didanosine (ddI) which is simple to perform. In our assay the DNA of HIV-1 pol was amplified by PCR using two sets of nested oligonucleotide primers. Mutations of reverse transcriptase (RT) encoding amino acids (aa) 74 and 41, 70, and 215 which have been associated with HIV-1 resistance to ddI and ZDV, respectively, were detected with a ligase detection reaction (LDR) and indicated colorimetrically. The RT genotypes of 35 patient specimens (140 codons) blindly assessed for these mutations were in agreement by PCR-LDR and by dideoxynucleotide sequencing. To evaluate the limits of the assay, other specimens with mutations close to the ligation site were evaluated by PCR-LDR. The assay was sensitive and specific for all specimens except when mutations occurred within 2 bases on either side of the ligation site. In summary, this PCR-LDR assay specifically, sensitively, and rapidly detected pol mutations (RT aa 74, 41, 70, and 215) associated with HIV-1 resistance to ddI and ZDV. PMID:7714190

Molecular characters of melon (Cucumismelo L. "Tacapa") in response to karst critical land

NASA Astrophysics Data System (ADS)

Rachmawati, Yuanita; Daryono, Budi Setiadi; Aristya, Ganies Riza

2017-06-01

Yogyakarta district has 158.600 ha critical land and spread off in three Agro Ecosystem zones. Two of them are karsts critical land. Critical lands which contain calcium carbonate in high concentration and water dehydration in upper surface give abiotic stress in wide range of plant. Melon cultivar TACAPA has superior characteristic derived from parental crossing, ♀ Action 434 and ♂ PI 371795 and potential to be developed in karsts critical land. Abscicic acid (ABA) is a phytohormone expressed by plant in abiotic stress condition. CmBG1 is a gene which regulate ABA hormone in melon. The purposes of this research were examining the molecular character of melon cultivar TACAPA in response to karsts critical land in order to study molecular characterization of CmBG1 gene. Analysis was done qualitatively by using Reverse Transcriptase-PCR (RT-PCR) and Electrophoresis, while quantitative analysis was conducted by observing absorbance score in spectrophotometer. CmBG1 gene expression is examined by using Real time PCR (qPCR). Molecular characters obtained are CmBG1 detected in size ±1258 bp, CmBG1 gene concentrations in melon which planted in control media are lower than melon in critical lands media. These results are similar with the real time quantitative analysis method. It also be revealed that melon TACAPA is more potential plant compared to another cultivar that can be developed in karst critical land area.

Depletion of Unwanted Nucleic Acid Templates by Selective Cleavage: LNAzymes, Catalytically Active Oligonucleotides Containing Locked Nucleic Acids, Open a New Window for Detecting Rare Microbial Community Members

PubMed Central

Dolinšek, Jan; Dorninger, Christiane; Lagkouvardos, Ilias; Wagner, Michael

2013-01-01

Many studies of molecular microbial ecology rely on the characterization of microbial communities by PCR amplification, cloning, sequencing, and phylogenetic analysis of genes encoding rRNAs or functional marker enzymes. However, if the established clone libraries are dominated by one or a few sequence types, the cloned diversity is difficult to analyze by random clone sequencing. Here we present a novel approach to deplete unwanted sequence types from complex nucleic acid mixtures prior to cloning and downstream analyses. It employs catalytically active oligonucleotides containing locked nucleic acids (LNAzymes) for the specific cleavage of selected RNA targets. When combined with in vitro transcription and reverse transcriptase PCR, this LNAzyme-based technique can be used with DNA or RNA extracts from microbial communities. The simultaneous application of more than one specific LNAzyme allows the concurrent depletion of different sequence types from the same nucleic acid preparation. This new method was evaluated with defined mixtures of cloned 16S rRNA genes and then used to identify accompanying bacteria in an enrichment culture dominated by the nitrite oxidizer “Candidatus Nitrospira defluvii.” In silico analysis revealed that the majority of publicly deposited rRNA-targeted oligonucleotide probes may be used as specific LNAzymes with no or only minor sequence modifications. This efficient and cost-effective approach will greatly facilitate tasks such as the identification of microbial symbionts in nucleic acid preparations dominated by plastid or mitochondrial rRNA genes from eukaryotic hosts, the detection of contaminants in microbial cultures, and the analysis of rare organisms in microbial communities of highly uneven composition. PMID:23263968

Endometrial stem cell differentiation into smooth muscle cell: a novel approach for bladder tissue engineering in women.

PubMed

Shoae-Hassani, Alireza; Sharif, Shiva; Seifalian, Alexander M; Mortazavi-Tabatabaei, Seyed Abdolreza; Rezaie, Sassan; Verdi, Javad

2013-10-01

To investigate manufacturing smooth muscle cells (SMCs) for regenerative bladder reconstruction from differentiation of endometrial stem cells (EnSCs), as the recent discovery of EnSCs from the lining of women's uteri, opens up the possibility of using these cells for tissue engineering applications, such as building up natural tissue to repair prolapsed pelvic floors as well as building urinary bladder wall. Human EnSCs that were positive for cluster of differentiation 146 (CD146), CD105 and CD90 were isolated and cultured in Dulbecco's modified Eagle/F12 medium supplemented with myogenic growth factors. The myogenic factors included: transforming growth factor β, platelet-derived growth factor, hepatocyte growth factor and vascular endothelial growth factor. Differentiated SMCs on bioabsorbable polyethylene-glycol and collagen hydrogels were checked for SMC markers by real-time reverse-transcriptase polymerase chain reaction (RT-PCR), western blot (WB) and immunocytochemistry (ICC) analyses. Histology confirmed the growth of SMCs in the hydrogel matrices. The myogenic growth factors decreased the proliferation rate of EnSCs, but they differentiated the human EnSCs into SMCs more efficiently on hydrogel matrices and expressed specific SMC markers including α-smooth muscle actin, desmin, vinculin and calponin in RT-PCR, WB and ICC experiments. The survival rate of cultures on the hydrogel-coated matrices was significantly higher than uncoated cultures. Human EnSCs were successfully differentiated into SMCs, using hydrogels as scaffold. EnSCs may be used for autologous bladder wall regeneration without any immunological complications in women. Currently work is in progress using bioabsorbable nanocomposite materials as EnSC scaffolds for developing urinary bladder wall tissue. © 2013 The Authors. BJU International © 2013 BJU International.

Genome-Wide Mapping of Cystitis Due to Streptococcus agalactiae and Escherichia coli in Mice Identifies a Unique Bladder Transcriptome That Signifies Pathogen-Specific Antimicrobial Defense against Urinary Tract Infection

PubMed Central

Tan, Chee K.; Carey, Alison J.; Cui, Xiangqin; Webb, Richard I.; Ipe, Deepak; Crowley, Michael; Cripps, Allan W.; Benjamin, William H.; Ulett, Kimberly B.; Schembri, Mark A.

2012-01-01

The most common causes of urinary tract infections (UTIs) are Gram-negative pathogens such as Escherichia coli; however, Gram-positive organisms, including Streptococcus agalactiae, or group B streptococcus (GBS), also cause UTI. In GBS infection, UTI progresses to cystitis once the bacteria colonize the bladder, but the host responses triggered in the bladder immediately following infection are largely unknown. Here, we used genome-wide expression profiling to map the bladder transcriptome of GBS UTI in mice infected transurethrally with uropathogenic GBS that was cultured from a 35-year-old women with cystitis. RNA from bladders was applied to Affymetrix Gene-1.0ST microarrays; quantitative reverse transcriptase PCR (qRT-PCR) was used to analyze selected gene responses identified in array data sets. A surprisingly small significant-gene list of 172 genes was identified at 24 h; this compared to 2,507 genes identified in a side-by-side comparison with uropathogenic E. coli (UPEC). No genes exhibited significantly altered expression at 2 h in GBS-infected mice according to arrays despite high bladder bacterial loads at this early time point. The absence of a marked early host response to GBS juxtaposed with broad-based bladder responses activated by UPEC at 2 h. Bioinformatics analyses, including integrative system-level network mapping, revealed multiple activated biological pathways in the GBS bladder transcriptome that regulate leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. These findings define a novel, minimalistic type of bladder host response triggered by GBS UTI, which comprises collective antimicrobial pathways that differ dramatically from those activated by UPEC. Overall, this study emphasizes the unique nature of bladder immune activation mechanisms triggered by distinct uropathogens. PMID:22733575

Expression analysis of Drosophila doublesex, transformer-2, intersex, fruitless-like, and vitellogenin homologs in the parahaploid predator Metaseiulus occidentalis (Chelicerata: Acari: Phytoseiidae).

PubMed

Pomerantz, Aaron F; Hoy, Marjorie A

2015-01-01

Characterization and expression analyses are essential to gain insight into sex-determination pathways in members of the Acari. Little is known about sex determination at the molecular level in the western orchard predatory mite Metaseiulus occidentalis (Arthropoda: Chelicerata: Arachnida: Acari: Phytoseiidae), a parahaploid species. In this study, eight genes previously identified as putative homologs to genes involved in the sex-determination pathway in Drosophila melanogaster were evaluated for sex-specific alternative splicing and sex-biased expression using reverse-transcriptase PCR and quantitative real-time PCR techniques, respectively. The homologs evaluated in M. occidentalis included two doublesex-like genes (Moccdsx1 and Moccdsx2), transformer-2 (Mocctra-2), intersex (Moccix), two fruitless-like genes (MoccBTB1 and MoccBTB2), as well as two vitellogenin-like genes (Moccvg1 and Moccvg2). Single transcripts of equal size were detected in males and females for Moccdsx1, Moccdsx2, Mocctra-2, Moccix, and MoccBTB2, suggesting that their pre-mRNAs do not undergo alternative splicing in a sex-specific manner. Three genes, Moccdsx1, Moccdsx2 and MoccBTB2, displayed male-biased expression relative to females. One gene, Moccix, displayed female-biased expression relative to males. Two genes, Mocctra-2 and MoccBTB1, did not display detectable differences in transcript abundance in males and females. Expression of Moccvg1 and Moccvg2 were detected in females only, and transcript levels were up-regulated in mated females relative to unmated females. To our knowledge, this represents the first attempt to elucidate expression patterns of putative sex-determination genes in an acarine. This study is an initial step towards understanding the sex-determination pathway in the parahaploid M. occidentalis.

The link between the microbial ecology, gene expression, and biokinetics of denitrifying polyphosphate-accumulating systems under different electron acceptor combinations.

PubMed

Vieira, A; Ribera-Guardia, A; Marques, R; Barreto Crespo, M T; Oehmen, A; Carvalho, G

2018-06-02

The emission of the greenhouse gas nitrous oxide (N 2 O) can occur during biological nutrient removal. Denitrifying enhanced biological phosphorus removal (d-EBPR) systems are an efficient means of removing phosphate and nitrogen, performed by denitrifying polyphosphate-accumulating organisms (d-PAOs). The aim of this work was to study the effect of various combinations of electron acceptors, nitrate (NO 3 - ), nitrite (NO 2 - ), and N 2 O, on the denitrification pathway of a d-EBPR system. Batch tests were performed with different electron acceptor combinations, to explore the denitrification pathway. Reverse transcriptase-qPCR (RT-qPCR) and high-throughput sequencing, combined with chemical analysis, were used to study gene expression, microbial diversity, and denitrification kinetics. The potential for N 2 O production was greater than the potential for its reduction in most tests. A strong correlation was observed between the N 2 O reduction rate and the relative gene expression of nitrous oxide reductase per nitrite reductase (nosZ/(nirS + nirK)), suggesting that the expression of denitrifying marker genes is a strong predictor of the N 2 O reduction rate. The d-EBPR community maintained a core population with low variations throughout the study. Furthermore, phylogenetic analyses of the studied marker genes revealed that the organisms actively involved in denitrification were closely related to Thauera sp., Candidatus Accumulibacter phosphatis, and Candidatus Competibacter denitrificans. Moreover, Competibacter-related OTUs seem to be important contributors to the N 2 O reduction capacity of the system, likely scavenging the N 2 O produced by other organisms. Overall, this study contributes to a better understanding of the microbial biochemistry and the genetics involving biological denitrification removal, important to minimize N 2 O emissions in wastewater treatment plants.

[Significant increase of glucose transport activity in breast cancer].

PubMed

Li, Juan; Yang, Shou-jing; Zhao, Xi-long; Zhang, Ya-qing; Li, Kai-nan; Cui, Ji-hong; Li, Jing

2008-02-01

To study the expression level and significance of glucose transporter 1 (Glut-1) in normal breast tissue, adenosis, adenoma and breast carcinoma. A total of 147 cases of female breast tissue samples, including 92 cases of invasive ductal carcinoma, 26 cases of breast fibroadenoma, 24 cases of breast adenosis and 5 cases of normal breast tissues, were collected for quantitative detection of the expression of Glut-1 protein by immunohistochemistry (EnVision method) and Western blot, and its mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). In normal breast tissue and benign lesions of the breast, Glut-1 was undetectable or only weakly detectable in cytoplasm of ductal and acinar epithelia. In contrast, the intensity of Glut-1 staining was significantly higher in invasive ductal carcinomas (P = 0.0002) with protein expression predominantly in cellular membrane and lesser in cytoplasm. Western blot and RT-PCR analyses showed that the expression of Glut-1 protein and mRNA were significantly increased in invasive ductal carcinoma than fibroadenoma (P =0.001 for protein; P <0.05 for mRNA) and adenosis (P =0.001 for protein; P < 0.05 for mRNA). There was a significant difference among groups (P = 0.0002 for protein; P = 0.0001 for mRNA). Glucose transport activity, as indicated by Glut-1 protein and its mRNA expression, significantly increases in breast carcinoma than non-cancerous lesions. The over-expression of Glut-1 in breast carcinoma is tightly coupled with tumor cell proliferation, invasion and metastasis, implying that Glut-1 may serve as a new marker in the early diagnosis and prognostication of breast malignancy as well as a new therapeutic target.

Characterization of the Small RNA Transcriptome of the Marine Coccolithophorid, Emiliania huxleyi.

PubMed

Zhang, Xiaoyu; Gamarra, Jaime; Castro, Steven; Carrasco, Estela; Hernandez, Aaron; Mock, Thomas; Hadaegh, Ahmad R; Read, Betsy A

2016-01-01

Small RNAs (smRNAs) control a variety of cellular processes by silencing target genes at the transcriptional or post-transcription level. While extensively studied in plants, relatively little is known about smRNAs and their targets in marine phytoplankton, such as Emiliania huxleyi (E. huxleyi). Deep sequencing was performed of smRNAs extracted at different time points as E. huxleyi cells transition from logarithmic to stationary phase growth in batch culture. Computational analyses predicted 18 E. huxleyi specific miRNAs. The 18 miRNA candidates and their precursors vary in length (18-24 nt and 71-252 nt, respectively), genome copy number (3-1,459), and the number of genes targeted (2-107). Stem-loop real time reverse transcriptase (RT) PCR was used to validate miRNA expression which varied by nearly three orders of magnitude when growth slows and cells enter stationary phase. Stem-loop RT PCR was also used to examine the expression profiles of miRNA in calcifying and non-calcifying cultures, and a small subset was found to be differentially expressed when nutrients become limiting and calcification is enhanced. In addition to miRNAs, endogenous small RNAs such as ra-siRNAs, ta-siRNAs, nat-siRNAs, and piwiRNAs were predicted along with the machinery for the biogenesis and processing of si-RNAs. This study is the first genome-wide investigation smRNAs pathways in E. huxleyi. Results provide new insights into the importance of smRNAs in regulating aspects of physiological growth and adaptation in marine phytoplankton and further challenge the notion that smRNAs evolved with multicellularity, expanding our perspective of these ancient regulatory pathways.

Characterization of the Small RNA Transcriptome of the Marine Coccolithophorid, Emiliania huxleyi

PubMed Central

Zhang, Xiaoyu; Gamarra, Jaime; Castro, Steven; Carrasco, Estela; Hernandez, Aaron; Mock, Thomas; Hadaegh, Ahmad R.; Read, Betsy A.

2016-01-01

Small RNAs (smRNAs) control a variety of cellular processes by silencing target genes at the transcriptional or post-transcription level. While extensively studied in plants, relatively little is known about smRNAs and their targets in marine phytoplankton, such as Emiliania huxleyi (E. huxleyi). Deep sequencing was performed of smRNAs extracted at different time points as E. huxleyi cells transition from logarithmic to stationary phase growth in batch culture. Computational analyses predicted 18 E. huxleyi specific miRNAs. The 18 miRNA candidates and their precursors vary in length (18–24 nt and 71–252 nt, respectively), genome copy number (3–1,459), and the number of genes targeted (2–107). Stem-loop real time reverse transcriptase (RT) PCR was used to validate miRNA expression which varied by nearly three orders of magnitude when growth slows and cells enter stationary phase. Stem-loop RT PCR was also used to examine the expression profiles of miRNA in calcifying and non-calcifying cultures, and a small subset was found to be differentially expressed when nutrients become limiting and calcification is enhanced. In addition to miRNAs, endogenous small RNAs such as ra-siRNAs, ta-siRNAs, nat-siRNAs, and piwiRNAs were predicted along with the machinery for the biogenesis and processing of si-RNAs. This study is the first genome-wide investigation smRNAs pathways in E. huxleyi. Results provide new insights into the importance of smRNAs in regulating aspects of physiological growth and adaptation in marine phytoplankton and further challenge the notion that smRNAs evolved with multicellularity, expanding our perspective of these ancient regulatory pathways. PMID:27101007

Identification and comprehensive analyses of the CBL and CIPK gene families in wheat (Triticum aestivum L.).

PubMed

Sun, Tao; Wang, Yan; Wang, Meng; Li, Tingting; Zhou, Yi; Wang, Xiatian; Wei, Shuya; He, Guangyuan; Yang, Guangxiao

2015-11-04

Calcineurin B-like (CBL) proteins belong to a unique group of calcium sensors in plant that decode the Ca(2+) signature by interacting with CBL-interacting protein kinases (CIPKs). Although CBL-CIPK complexes have been shown to play important roles in the responses to various stresses in plants, little is known about their functions in wheat. A total of seven TaCBL and 20 TaCIPK genes were amplified from bread wheat, Triticum aestivum cv. Chinese Spring. Reverse-transcriptase-polymerase chain reaction (RT-PCR) and in silico expression analyses showed that TaCBL and TaCIPK genes were expressed at different levels in different tissues, or maintained at nearly constant expression levels during the whole life cycle of the wheat plant. Some TaCBL and TaCIPK genes showed up- or down-regulated expressions during seed germination. Preferential interactions between TaCBLs and TaCIPKs were observed in yeast two-hybrid and bimolecular fluorescence complementation experiments. Analyses of a deletion series of TaCIPK proteins with amino acid variations at the C-terminus provided new insights into the specificity of the interactions between TaCIPKs and TaCBLs, and indicated that the TaCBL-TaCIPK signaling pathway is very complex in wheat because of its hexaploid genome. The expressions of many TaCBLs and TaCIPKs were responsive to abiotic stresses (salt, cold, and simulated drought) and abscisic acid treatment. Transgenic Arabidopsis plants overexpressing TaCIPK24 exhibited improved salt tolerance through increased Na(+) efflux and an enhanced reactive oxygen species scavenging capacity. These results contribute to our understanding of the functions of CBL-CIPK complexes and provide the basis for selecting appropriate genes for in-depth functional studies of CBL-CIPK in wheat.

The relationship between quantitative human epidermal growth factor receptor 2 gene expression by the 21-gene reverse transcriptase polymerase chain reaction assay and adjuvant trastuzumab benefit in Alliance N9831.

PubMed

Perez, Edith A; Baehner, Frederick L; Butler, Steven M; Thompson, E Aubrey; Dueck, Amylou C; Jamshidian, Farid; Cherbavaz, Diana; Yoshizawa, Carl; Shak, Steven; Kaufman, Peter A; Davidson, Nancy E; Gralow, Julie; Asmann, Yan W; Ballman, Karla V

2015-10-01

The N9831 trial demonstrated the efficacy of adjuvant trastuzumab for patients with human epidermal growth factor receptor 2 (HER2) locally positive tumors by protein or gene analysis. We used the 21-gene assay to examine the association of quantitative HER2 messenger RNA (mRNA) gene expression and benefit from trastuzumab. N9831 tested the addition of trastuzumab to chemotherapy in stage I-III HER2-positive breast cancer. For two of the arms of the trial, doxorubicin and cyclophosphamide followed by paclitaxel (AC-T) and doxorubicin and cyclophosphamide followed by paclitaxel and trastuzumab concurrent chemotherapy-trastuzumab (AC-TH), recurrence score (RS) and HER2 mRNA expression were determined by the 21-gene assay (Oncotype DX®) (negative <10.7, equivocal 10.7 to <11.5, and positive ≥11.5 log2 expression units). Cox regression was used to assess the association of HER2 expression with trastuzumab benefit in preventing distant recurrence. Median follow-up was 7.4 years. Of 1,940 total patients, 901 had consent and sufficient tissue. HER2 by reverse transcriptase polymerase chain reaction (RT-PCR) was negative in 130 (14 %), equivocal in 85 (9 %), and positive in 686 (76 %) patients. Concordance between HER2 assessments was 95 % for RT-PCR versus central immunohistochemistry (IHC) (>10 % positive cells = positive), 91 % for RT-PCR versus central fluorescence in situ hybridization (FISH) (≥2.0 = positive) and 94 % for central IHC versus central FISH. In the primary analysis, the association of HER2 expression by 21-gene assay with trastuzumab benefit was marginally nonsignificant (nonlinear p = 0.057). In hormone receptor-positive patients (local IHC) the association was significant (p = 0.002). The association was nonlinear with the greatest estimated benefit at lower and higher HER2 expression levels. Concordance among HER2 assessments by central IHC, FISH, and RT-PCR were similar and high. Association of HER2 mRNA expression with trastuzumab benefit as measured by time to distant recurrence was nonsignificant. A consistent benefit of trastuzumab irrespective of mHER2 levels was observed in patients with either IHC-positive or FISH-positive tumors. Trend for benefit was observed also for the small groups of patients with negative results by any or all of the central assays. Clinicaltrials.gov NCT00005970 . Registered 5 July 2000.

Diagnostic performance of influenza viruses and RSV rapid antigen detection tests in children in tertiary care.

PubMed

Moesker, F M; van Kampen, J J A; Aron, G; Schutten, M; van de Vijver, D A M C; Koopmans, M P G; Osterhaus, A D M E; Fraaij, P L A

2016-06-01

Rapid antigen detection tests (RADTs) are increasingly used to detect influenza viruses and respiratory syncytial virus (RSV). However, their sensitivity and specificity are a matter of debate, challenging their clinical usefulness. Comparing diagnostic performances of BinaxNow Influenza AB(®) (BNI) and BinaxNow RSV(®) (BNR), to those of real-time reverse transcriptase PCR (RT-PCR), virus isolation and direct immunofluorescence (D-IF) in paediatric patients. Between November 2005 and September 2013, 521 nasal washings from symptomatic children (age <5 years) attending our tertiary care centre were tested, with a combination of the respective assays using RT-PCR as gold standard. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of BNI were 69% (confidence interval [CI] [51-83]), 96% [94-97], 55% [39-70] and 98% [96-99] respectively. Of eleven false-negative samples, RT-PCR Ct-values were higher than all RT-PCR positive test results (27 vs 22, p=0.012). Of twenty false-positive samples, none were culture positive and two tested positive in D-IF. Sensitivity, specificity, PPV and NPV for BNR were 79% [73-85], 98% [96-99], 97% [93-99] and 88% [84-91]. Of the 42 false-negative samples the median Ct-value was higher than that of all RT-PCR positive samples (31 vs 23, p<0.0001). Five false-positive samples were detected. Three of these tested positive for RSV in virus isolation and D-IF. RADTs have a high specificity with BNR being superior to BNI. However, their relative low sensitivity limits their usefulness for clinical decision making in a tertiary care paediatric hospital. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Diagnostic Yield of Laboratory Methods and Value of Viral Genotyping during an Outbreak of Mumps in a Partially Vaccinated Population in British Columbia, Canada.

PubMed

Nunn, Alexandra; Masud, Shazia; Krajden, Mel; Naus, Monika; Jassem, Agatha N

2018-05-01

Mumps remains endemic in North America despite routine use of the measles, mumps, and rubella (MMR) vaccine. In 2016, an outbreak of mumps in British Columbia, Canada, provided an opportunity to determine the diagnostic utility of laboratory testing methods. Specimens from patients with clinical mumps were tested for infection using a commercial enzyme-linked immunosorbent assay (ELISA) for antibody detection and an in-house reverse transcriptase PCR (RT-PCR) targeting viral fusion and small hydrophobic (SH) genes. Viral genotyping was performed by SH gene sequencing. Laboratory data was linked with epidemiologic case data. Of the 139 confirmed cases, 94 (68%) had reported or documented history of MMR vaccination. Specimens were typically collected 1 day (for buccal and IgM tests) or 2 days (for urine tests) after symptom onset. Most confirmed cases (69%) were confirmed by buccal swab RT-PCR. Among cases tested by multiple methods, the percent positivity for buccal swab RT-PCR was 90% (96/107) compared to 43% (30/69) for both IgM ELISA and urine RT-PCR. Mumps IgM detection was higher in confirmed cases with no history of vaccination than in those with history (64% versus 34%, P = 0.02). The outbreak strain was identified as genotype G related to MuVi/Sheffield.GBR/1.05 but with conserved variations in five nucleotides within the SH gene that allowed linkage of geographically distinct cases. In conclusion, RT-PCR of buccal specimens had the highest diagnostic yield during a mumps outbreak in a partially vaccinated population. To optimize mumps diagnostic potential, clinicians should collect specimens depending on when the patient presents for care and their immunization history. © Crown copyright 2018.

Defining the Enterovirus Diversity Landscape of a Fecal Sample: A Methodological Challenge?

PubMed

Faleye, Temitope Oluwasegun Cephas; Adewumi, Moses Olubusuyi; Adeniji, Johnson Adekunle

2016-01-12

Enteroviruses are a group of over 250 naked icosahedral virus serotypes that have been associated with clinical conditions that range from intrauterine enterovirus transmission withfataloutcome through encephalitis and meningitis, to paralysis. Classically, enterovirus detection was done by assaying for the development of the classic enterovirus-specific cytopathic effect in cell culture. Subsequently, the isolates were historically identified by a neutralization assay. More recently, identification has been done by reverse transcriptase-polymerase chain reaction (RT-PCR). However, in recent times, there is a move towards direct detection and identification of enteroviruses from clinical samples using the cell culture-independent RT semi-nested PCR (RT-snPCR) assay. This RT-snPCR procedure amplifies the VP1 gene, which is then sequenced and used for identification. However, while cell culture-based strategies tend to show a preponderance of certain enterovirus species depending on the cell lines included in the isolation protocol, the RT-snPCR strategies tilt in a different direction. Consequently, it is becoming apparent that the diversity observed in certain enterovirus species, e.g., enterovirus species B(EV-B), might not be because they are the most evolutionarily successful. Rather, it might stem from cell line-specific bias accumulated over several years of use of the cell culture-dependent isolation protocols. Furthermore, it might also be a reflection of the impact of the relative genome concentration on the result of pan-enterovirus VP1 RT-snPCR screens used during the identification of cell culture isolates. This review highlights the impact of these two processes on the current diversity landscape of enteroviruses and the need to re-assess enterovirus detection and identification algorithms in a bid to better balance our understanding of the enterovirus diversity landscape.

Defining the Enterovirus Diversity Landscape of a Fecal Sample: A Methodological Challenge?

PubMed Central

Faleye, Temitope Oluwasegun Cephas; Adewumi, Moses Olubusuyi; Adeniji, Johnson Adekunle

2016-01-01

Enteroviruses are a group of over 250 naked icosahedral virus serotypes that have been associated with clinical conditions that range from intrauterine enterovirus transmission withfataloutcome through encephalitis and meningitis, to paralysis. Classically, enterovirus detection was done by assaying for the development of the classic enterovirus-specific cytopathic effect in cell culture. Subsequently, the isolates were historically identified by a neutralization assay. More recently, identification has been done by reverse transcriptase-polymerase chain reaction (RT-PCR). However, in recent times, there is a move towards direct detection and identification of enteroviruses from clinical samples using the cell culture-independent RT semi-nested PCR (RT-snPCR) assay. This RT-snPCR procedure amplifies the VP1 gene, which is then sequenced and used for identification. However, while cell culture-based strategies tend to show a preponderance of certain enterovirus species depending on the cell lines included in the isolation protocol, the RT-snPCR strategies tilt in a different direction. Consequently, it is becoming apparent that the diversity observed in certain enterovirus species, e.g., enterovirus species B(EV-B), might not be because they are the most evolutionarily successful. Rather, it might stem from cell line-specific bias accumulated over several years of use of the cell culture-dependent isolation protocols. Furthermore, it might also be a reflection of the impact of the relative genome concentration on the result of pan-enterovirus VP1 RT-snPCR screens used during the identification of cell culture isolates. This review highlights the impact of these two processes on the current diversity landscape of enteroviruses and the need to re-assess enterovirus detection and identification algorithms in a bid to better balance our understanding of the enterovirus diversity landscape. PMID:26771630

Monocytes inhibit NK activity via TGF-β in patients with obstructive sleep apnoea.

PubMed

Hernández-Jiménez, Enrique; Cubillos-Zapata, Carolina; Toledano, Victor; Pérez de Diego, Rebeca; Fernández-Navarro, Isabel; Casitas, Raquel; Carpio, Carlos; Casas-Martín, Jose; Valentín, Jaime; Varela-Serrano, Anibal; Avendaño-Ortiz, Jose; Alvarez, Enrique; Aguirre, Luis A; Pérez-Martínez, Antonio; De Miguel, Maria P; Belda-Iniesta, Cristobal; García-Río, Francisco; López-Collazo, Eduardo

2017-06-01

Obstructive sleep apnoea (OSA) is associated with cancer incidence and mortality. The contribution of the immune system appears to be crucial; however, the potential role of monocytes and natural killer (NK) cells remains unclear.Quantitative reverse transcriptase PCR, flow cytometry and in vitro assays were used to analyse the phenotype and immune response activity in 92 patients with OSA (60 recently diagnosed untreated patients and 32 patients after 6 months of treatment with continuous positive airway pressure (CPAP)) and 29 healthy volunteers (HV).We determined that monocytes in patients with OSA exhibit an immunosuppressive phenotype, including surface expression of glycoprotein-A repetitions predominant protein (GARP) and transforming growth factor-β (TGF-β), in contrast to those from the HV and CPAP groups. High levels of TGF-β were detected in OSA sera. TGF-β release by GARP + monocytes impaired NK cytotoxicity and maturation. This altered phenotype correlated with the hypoxic severity clinical score (CT90). Reoxygenation eventually restored the altered phenotypes and cytotoxicity.This study demonstrates that GARP + monocytes from untreated patients with OSA have an NK-suppressing role through their release of TGF-β. Our findings show that monocyte plasticity immunomodulates NK activity in this pathology, suggesting a potential role in cancer incidence. Copyright ©ERS 2017.

Comparative study of enteric viruses, coliphages and indicator bacteria for evaluating water quality in a tropical high-altitude system

PubMed Central

2009-01-01

Background Bacteria used as indicators for pathogenic microorganisms in water are not considered adequate as enteric virus indicators. Surface water from a tropical high-altitude system located in Mexico City that receives rainwater, treated and non-treated wastewater used for irrigation, and groundwater used for drinking, was studied. Methods The presence of enterovirus, rotavirus, astrovirus, coliphage, coliform bacteria, and enterococci was determined during annual cycles in 2001 and 2002. Enteric viruses in concentrated water samples were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Coliphages were detected using the double agar layer method. Bacteria analyses of the water samples were carried out by membrane filtration. Results The presence of viruses and bacteria in the water used for irrigation showed no relationship between current bacterial indicator detection and viral presence. Coliphages showed strong association with indicator bacteria and enterovirus, but weak association with other enteric viruses. Enterovirus and rotavirus showed significant seasonal differences in water used for irrigation, although this was not clear for astrovirus. Conclusion Coliphages proved to be adequate faecal pollution indicators for the irrigation water studied. Viral presence in this tropical high-altitude system showed a similar trend to data previously reported for temperate zones. PMID:19860917

Mutational analysis of the reverse transcriptase and ribonuclease H domains of the human foamy virus.

PubMed Central

Kögel, D; Aboud, M; Flügel, R M

1995-01-01

Human foamy or spuma virus (HFV) codes for a distinct set of pol gen products. To determine the minimal requirements for the HFV enzymatic activities, defined residues of the reverse transcriptase (RT) and ribo-nuclease H (RNase H) domain of the HFV pol gene were mutated by site-specific PCR mutagenesis. The mutant gene products were bacterially expressed, purified by Ni2+ chelate affinity chromatography and characterised by Western blotting. The enzymatic activities of the individual recombinant HFV pol mutant proteins were characterised by the situ RT, RNase H and RNase H assays. Two substitution mutants reached RT activity levels higher than that of the intact recombinant HFV RT-RH-His. When the catalytically essential D508 was substituted by A508, 5% of RNase H activity was retained while DNA polymerase activity increased 2-fold. A deletion of 11 amino acid residues in the hinge region completely abolished DNA polymerase while RNase H activity decreased 2-fold. A deletion mutant in the C-terminal RH domain showed no RNase H but retained RNase H activity indicating that the activities are genetically separable. The combined data reveal that the HFV DNA polymerase and RNase H activities are interdependent. Images PMID:7544460

High Degree of Interlaboratory Reproducibility of Human Immunodeficiency Virus Type 1 Protease and Reverse Transcriptase Sequencing of Plasma Samples from Heavily Treated Patients

PubMed Central

Shafer, Robert W.; Hertogs, Kurt; Zolopa, Andrew R.; Warford, Ann; Bloor, Stuart; Betts, Bradley J.; Merigan, Thomas C.; Harrigan, Richard; Larder, Brendon A.

2001-01-01

We assessed the reproducibility of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and protease sequencing using cryopreserved plasma aliquots obtained from 46 heavily treated HIV-1-infected individuals in two laboratories using dideoxynucleotide sequencing. The rates of complete sequence concordance between the two laboratories were 99.1% for the protease sequence and 99.0% for the RT sequence. Approximately 90% of the discordances were partial, defined as one laboratory detecting a mixture and the second laboratory detecting only one of the mixture's components. Only 0.1% of the nucleotides were completely discordant between the two laboratories, and these were significantly more likely to occur in plasma samples with lower plasma HIV-1 RNA levels. Nucleotide mixtures were detected at approximately 1% of the nucleotide positions, and in every case in which one laboratory detected a mixture, the second laboratory either detected the same mixture or detected one of the mixture's components. The high rate of concordance in detecting mixtures and the fact that most discordances between the two laboratories were partial suggest that most discordances were caused by variation in sampling of the HIV-1 quasispecies by PCR rather than by technical errors in the sequencing process itself. PMID:11283081

Course of c-myc mRNA expression in the regenerating mouse testis determined by competitive reverse transcriptase polymerase chain reaction.

PubMed

Amendola, R

1994-11-01

The c-myc proto-oncogene is a reliable marker of the "G0-early G1" transition, and its down-regulation is believed to be necessary to obtain cellular differentiation. In murine spermatogenesis, the level of c-myc transcripts does not correlate with the rate of cellular division. Proliferation of supposed staminal spermatogonia to reproduce themselves is induced with a local 5 Gy X-ray dose in 90-day-old C57Bl/6 mice. c-myc quantification by a newly developed competitive reverse transcriptase polymerase chain reaction (RT-PCR) was carried out to follow the expression course of this proto-oncogene. Damage and restoration of spermatogenesis were analyzed at days 3, 6, 9, 10, 13, 30, and 60 after injury by relative testes/body weight determination and histological examination. Proliferative status was determined by histone H3 Northern blot analysis. c-myc mRNA level was 10 times higher after 3 days in the irradiated animals compared to the controls. An increasing number of copies were noted up to 10 days, but promptly decreased to the base level found for irradiated mice from 13 to 60 days. Interestingly, the expression of histone H3 detected S phase only in testes at 60 days from damage.

The conserved N-terminal basic residues and zinc-finger motifs of HIV-1 nucleocapsid restrict the viral cDNA synthesis during virus formation and maturation

PubMed Central

Didierlaurent, Ludovic; Houzet, Laurent; Morichaud, Zakia; Darlix, Jean-Luc; Mougel, Marylène

2008-01-01

Reverse transcription of the genomic RNA by reverse transcriptase occurs soon after HIV-1 infection of target cells. The viral nucleocapsid (NC) protein chaperones this process via its nucleic acid annealing activities and its interactions with the reverse transcriptase enzyme. To function, NC needs its two conserved zinc fingers and flanking basic residues. We recently reported a new role for NC, whereby it negatively controls reverse transcription in the course of virus formation. Indeed, deleting its zinc fingers causes reverse transcription activation in virus producer cells. To investigate this new NC function, we used viruses with subtle mutations in the conserved zinc fingers and its flanking domains. We monitored by quantitative PCR the HIV-1 DNA content in producer cells and in produced virions. Results showed that the two intact zinc-finger structures are required for the temporal control of reverse transcription by NC throughout the virus replication cycle. The N-terminal basic residues also contributed to this new role of NC, while Pro-31 residue between the zinc fingers and Lys-59 in the C-terminal region did not. These findings further highlight the importance of NC as a major target for anti-HIV-1 drugs. PMID:18641038

Identification and characterization of jute LTR retrotransposons:

PubMed Central

Ahmed, Salim; Shafiuddin, MD; Azam, Muhammad Shafiul; Islam, Md. Shahidul; Ghosh, Ajit

2011-01-01

Long Terminal Repeat (LTR) retrotransposons constitute a significant part of eukaryotic genomes and play an important role in genome evolution especially in plants. Jute is an important fiber crop with a large genome of 1,250 Mbps. This genome is still mostly unexplored. In this study we aimed at identifying and characterizing the LTR retrotransposons of jute with a view to understanding the jute genome better. In this study, the Reverse Transcriptase domain of Ty1-copia and Ty3-gypsy LTR retrotransposons of jute were amplified by degenerate primers and their expressions were examined by reverse transcription PCR. Copy numbers of reverse transcriptase (RT) genes of Ty1-copia and Ty3-gypsy elements were determined by dot blot analysis. Sequence analysis revealed higher heterogeneity among Ty1-copia retrotransposons than Ty3-gypsy and clustered each of them in three groups. Copy number of RT genes in Ty1-copia was found to be higher than that of Ty3-gypsy elements from dot blot hybridization. Cumulatively Ty1-copia and Ty3-gypsy may constitute around 19% of the jute genome where two groups of Ty1-copia were found to be transcriptionally active. Since the LTR retrotransposons constitute a large portion of jute genome, these findings imply the importance of these elements in the evolution of jute genome. PMID:22016842

Expression of chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) in bladder transitional cell carcinoma.

PubMed

Ham, Won Sik; Lee, Joo Hyoung; Yu, Ho Song; Choi, Young Deuk

2008-10-01

An analysis of differentially expressed genes (DEGs) between bladder transitional cell carcinoma (TCC) and the surrounding urothelium to help identify what lies behind the mechanism of multifocal tumor development has not yet been performed. We sought to find a new DEG related to the development of bladder TCC. Thirty-nine bladder TCC tissues paired with normal-appearing urothelium tissues obtained from the same patient were used as subjects. Initially, we compared the messenger RNA (mRNA) profiles between normal-appearing urothelium and TCC tissue of 1 patient by using annealing control primer (ACP)-based GeneFishing polymerase chain reaction (PCR) and selective amplification of family members (SAFM) PCR to identify potential DEGs. To validate the results of the ACP data, reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on those of all 39 patients. Among the several DEGs discovered in the ACP data, 1 DEG was chosen as the candidate for the RT-PCR, that is present or markedly upregulated in normal-appearing urothelial tissue compared with TCC tissue. Gene sequence searching revealed that this DEG is chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI). Downregulation of COUP-TFI mRNA expression in TCC tissue compared to normal-appearing urothelium tissue of the same patient, irrespective of tumor stage and grade, was confirmed by RT-PCR in 39 patients. Our results suggest that the loss of COUP-TFI may play a role in the transition from normal epithelium to TCC. Further characterization of the COUP-TFI gene is expected to give us informations about bladder TCC tumorigenesis.

Poly(adenylic acid) complementary DNA real-time polymerase chain reaction in pancreatic ductal juice in patients undergoing pancreaticoduodenectomy.

PubMed

Oliveira-Cunha, Melissa; Byers, Richard J; Siriwardena, Ajith K

2010-03-01

There is a need to develop methods of early diagnosis for pancreatic cancer. Pancreatic juice is easily collected by endoscopic retrograde cholangiopancreatography and may facilitate diagnosis using molecular markers. The aim of this work was to explore the feasibility of measurement of gene expression in RNA isolated from ductal juice. Intraoperative sampling of pancreatic juice was undertaken in 27 patients undergoing pancreaticoduodenectomy for suspected tumor. Total RNA was extracted and used as template for poly(adenylic acid) (poly[A]) polymerase chain reaction (PCR) to generate a globally amplified complementary DNA pool representative of all expressed messenger RNAs. Real-time PCR was performed for trefoil factor 2 (TFF2), carboxypeptidase B1 (CPB1), and kallikrein-related peptidase 3 (KLK3) in a subset of samples; all samples were normalized for 3 reference genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], PSMB6, and beta-2-microglobulin [B2M]). The median volume of the pancreatic juice obtained was 1245 microL (range, 50-5000 microL). The RNA integrity number ranged from 1.9 to 10. Reverse transcriptase PCR was positive for pancreas-specific genes (TFF2 and CPB1) and negative for prostatic-specific antigen in all samples. These results demonstrate that RNA analysis of pancreatic juice is feasible using a combination of poly(A) PCR and real-time PCR. In addition, the poly(A) complementary DNA generated can be probed for multiple genes and is indefinitely renewable, thereby representing a molecular block of importance for future research.

Detection and Serotyping of Dengue Virus in Serum Samples by Multiplex Reverse Transcriptase PCR-Ligase Detection Reaction Assay▿ †

PubMed Central

Das, S.; Pingle, M. R.; Muñoz-Jordán, J.; Rundell, M. S.; Rondini, S.; Granger, K.; Chang, G.-J. J.; Kelly, E.; Spier, E. G.; Larone, D.; Spitzer, E.; Barany, F.; Golightly, L. M.

2008-01-01

The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV. PMID:18685000

Prostate stem cell antigen (PSCA) mRNA expression in peripheral blood in patients with benign prostatic hyperplasia and/or prostate cancer.

PubMed

Fawzy, Mohamed S; Mohamed, Randa H; Elfayoumi, Abdel-Rahman R

2015-03-01

The aim of this study was to determine whether detection of prostate stem cell antigen (PSCA) expression in BPH might be associated with the subsequent presence of Prostate cancer (PCa) and also to determine whether detection of PSCA expression has potential for prognosis in PCa. This study was comprised of 112 PCa patients, 111 BPH patients and 120 control subjects. We employed reverse-transcriptase polymerase chain reaction (RT-PCR) to detect PSCA mRNA-bearing cells in peripheral blood. PSCA mRNA was detected in the peripheral blood of 71.4% PCa patients and in 13.5% of patients with BPH by RT-PCR. PSCA was positive in 80% of high-grade diseases compared with 20% of low-grade diseases (P = 0.01). Whereas only 38.8% of prostate-confined diseases were PSCA positive, 61.2% of extraprostatic diseases were PSCA positive (P < 0.001). Patients with a lymphovascular invasion of tumor emboli tended to be PSCA positive (P = 0.02). BPH patients with RT-PCR PSCA positive were significantly more likely to develop prostate cancer (OR = 16, 95% CI = 8.1-31.6, P < 0.001). In conclusion, RT-PCR PSCA positivity is significantly associated with the Gleason score, LV tumor emboli and whether or not the tumor was organ confined. In this study, RT-PCR PSCA detection may be a promising tumor marker of diagnostic and metastasis detection for patients with prostate cancer. Also, it may be an important test for predicting BPH patients who are at high risk of subsequent cancer development.

Evaluation of Immunomagnetic Separation Method for the Recovery of Hepatitis A Virus and GI.1 and GII.4 Norovirus Strains Seeded on Oyster and Mussel.

PubMed

Ha, Ji-Hyoung; Choi, Changsun; Ha, Sang-Do

2014-12-01

Outbreaks of viral diseases are frequently associated with the consumption of minimally processed shellfish. Among the viruses in these outbreaks, hepatitis A virus (HAV) and human norovirus (NoV) have been increasingly reported as the most common food-borne pathogens. These viruses must be concentrated in tested samples in order to be detected. In this study, a method for the detection of NoV and HAV in shellfish using an immuno-magnetic separation (IMS) procedure combined with reverse transcriptase (RT)-PCR was developed. The IMS/RT-PCR method was applied to investigate the recovery rates of HAV, NoV GI.1, and GII.4 from oyster and mussel. Based on IMS/RT-PCR results, recovery rates for HAV from oyster and mussel test samples were 2.4 and 1.1%, respectively. The NoV GI.1 recovery rates from oyster and mussel samples were 4.9-9.2% (mean 6.9%) and 4.3-8.6% (mean 6.2%), respectively, and the NoV GII.4 recovery rates were 8.8 and 8.5%, respectively. These results verified that HAV, NoV GI.1, and GII.4 can be detected in all the test samples using the IMS/RT-PCR method, although the three inoculated viruses were recovered with low efficiency. In conclusion, the IMS/RT-PCR method can be used to efficiently and rapidly detect viruses such as HAV and NoV in shellfish such as oyster and mussel.

Three cases of imported dengue virus infection from Madeira to Belgium, 2012.

PubMed

Cnops, Lieselotte; Franco, Leticia; Van Meensel, Britt; Van den Ende, Jef; Paz Sanchez-Seco, Maria; Van Esbroeck, Marjan

2014-01-01

We report three laboratory-confirmed dengue virus (DENV) infections imported to Belgium by travelers returning from Madeira (Portugal). Despite the use of a mosquito-repellent spray as reported by two patients, the infection could not be prevented. Diagnosis was made by antigen detection and real-time reverse transcriptase polymerase chain reaction (RT-PCR) in two cases and by serology 1 month after onset of symptoms in a third one. The responsible virus was identified as DENV serotype 1, American/African genotype (genotype V). The close relationship to isolates from Colombia supports the previous findings that a South American strain originated the outbreak in Madeira in 2012. © 2014 International Society of Travel Medicine.

Detection of Usutu virus in a bullfinch (Pyrrhula pyrrhula) and a great spotted woodpecker (Dendrocopos major) in north-west Europe.

PubMed

Garigliany, Mutien-Marie; Marlier, Didier; Tenner-Racz, Klara; Eiden, Martin; Cassart, Dominique; Gandar, Frédéric; Beer, Martin; Schmidt-Chanasit, Jonas; Desmecht, Daniel

2014-01-01

In October 2012, a 3-year-old bullfinch (Pyrrhula pyrrhula) held in captivity for its entire lifespan and a wild adult great spotted woodpecker (Dendrocopos major), both with neurological signs, were found 4 km from each other and 5 days apart in the Meuse Valley, Belgium. Non-suppurative encephalitis and mild degeneration and necrosis were identified in the brain and cerebellum, and Usutu virus antigen and RNA were detected by immunohistochemistry and real-time reverse transcriptase PCR, respectively. The two cases reported here represent the most western distribution of clinical disease in birds due to Usutu virus in Europe. Copyright © 2013 Elsevier Ltd. All rights reserved.

Reverse transcription polymerase chain reaction protocols for cloning small circular RNAs.

PubMed

Navarro, B; Daròs, J A; Flores, R

1998-07-01

A protocol is described for general application for cloning small circular RNAs which requires only minimal amounts of template (approximately 50 ng) of unknown sequence. Both cDNA strands are synthesized with a 26-mer primer whose six 3'-terminal positions are totally degenerate in two consecutive reactions catalyzed by reverse transcriptase and DNA polymerase, respectively. The cDNAs are then PCR-amplified, using a 20-mer primer with the non-degenerate sequence of the previous primer, cloned and sequenced. This information permits the synthesis of one or more pairs of specific and adjacent primers for obtaining full-length cDNA clones by a protocol which is also described.

Adult fulminant subacute sclerosing panencephalitis: pathological and molecular studies--a case report.

PubMed

Souraud, J B; Faivre, A; Waku-Kouomou, D; Gaillard, T; Aouad, N; Meaudre, E; Wild, F T; Fouet, B; Soulard, R

2009-01-01

Subacute sclerosing panencephalitis is an uncommon progressive neurological disorder caused by a persistent defective measles virus, typically affecting children. We describe a case of fulminant subacute sclerosing panencephalitis in a 25-year-old male. Brain tissue biopsy showed histologic evidence of encephalitis with eosinophilic intranuclear inclusion bodies (Cowdry Type A and B), intracytoplasmic inclusion bodies, perivascular lymphoplasmacytic infiltration and gliosis. Immunohistochemical studies were positive using an anti-measles antibody. Reverse transcriptase-PCR detected measles virus RNA and phylogenetic analysis indicated a C2 genotype. The rare adult-onset form is often atypical and difficult to diagnose and should be included in the differential diagnosis of subacute "unexplained" neurological diseases and uncommon infectious disorders.

Use of quantitative molecular diagnostic methods to identify causes of diarrhoea in children: a reanalysis of the GEMS case-control study.

PubMed

Liu, Jie; Platts-Mills, James A; Juma, Jane; Kabir, Furqan; Nkeze, Joseph; Okoi, Catherine; Operario, Darwin J; Uddin, Jashim; Ahmed, Shahnawaz; Alonso, Pedro L; Antonio, Martin; Becker, Stephen M; Blackwelder, William C; Breiman, Robert F; Faruque, Abu S G; Fields, Barry; Gratz, Jean; Haque, Rashidul; Hossain, Anowar; Hossain, M Jahangir; Jarju, Sheikh; Qamar, Farah; Iqbal, Najeeha Talat; Kwambana, Brenda; Mandomando, Inacio; McMurry, Timothy L; Ochieng, Caroline; Ochieng, John B; Ochieng, Melvin; Onyango, Clayton; Panchalingam, Sandra; Kalam, Adil; Aziz, Fatima; Qureshi, Shahida; Ramamurthy, Thandavarayan; Roberts, James H; Saha, Debasish; Sow, Samba O; Stroup, Suzanne E; Sur, Dipika; Tamboura, Boubou; Taniuchi, Mami; Tennant, Sharon M; Toema, Deanna; Wu, Yukun; Zaidi, Anita; Nataro, James P; Kotloff, Karen L; Levine, Myron M; Houpt, Eric R

2016-09-24

Diarrhoea is the second leading cause of mortality in children worldwide, but establishing the cause can be complicated by diverse diagnostic approaches and varying test characteristics. We used quantitative molecular diagnostic methods to reassess causes of diarrhoea in the Global Enteric Multicenter Study (GEMS). GEMS was a study of moderate to severe diarrhoea in children younger than 5 years in Africa and Asia. We used quantitative real-time PCR (qPCR) to test for 32 enteropathogens in stool samples from cases and matched asymptomatic controls from GEMS, and compared pathogen-specific attributable incidences with those found with the original GEMS microbiological methods, including culture, EIA, and reverse-transcriptase PCR. We calculated revised pathogen-specific burdens of disease and assessed causes in individual children. We analysed 5304 sample pairs. For most pathogens, incidence was greater with qPCR than with the original methods, particularly for adenovirus 40/41 (around five times), Shigella spp or enteroinvasive Escherichia coli (EIEC) and Campylobactor jejuni o C coli (around two times), and heat-stable enterotoxin-producing E coli ([ST-ETEC] around 1·5 times). The six most attributable pathogens became, in descending order, Shigella spp, rotavirus, adenovirus 40/41, ST-ETEC, Cryptosporidium spp, and Campylobacter spp. Pathogen-attributable diarrhoeal burden was 89·3% (95% CI 83·2-96·0) at the population level, compared with 51·5% (48·0-55·0) in the original GEMS analysis. The top six pathogens accounted for 77·8% (74·6-80·9) of all attributable diarrhoea. With use of model-derived quantitative cutoffs to assess individual diarrhoeal cases, 2254 (42·5%) of 5304 cases had one diarrhoea-associated pathogen detected and 2063 (38·9%) had two or more, with Shigella spp and rotavirus being the pathogens most strongly associated with diarrhoea in children with mixed infections. A quantitative molecular diagnostic approach improved population-level and case-level characterisation of the causes of diarrhoea and indicated a high burden of disease associated with six pathogens, for which targeted treatment should be prioritised. Bill & Melinda Gates Foundation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Estrogen- and progesterone-receptor status in ECOG 2197: comparison of immunohistochemistry by local and central laboratories and quantitative reverse transcription polymerase chain reaction by central laboratory.

PubMed

Badve, Sunil S; Baehner, Frederick L; Gray, Robert P; Childs, Barrett H; Maddala, Tara; Liu, Mei-Lan; Rowley, Steve C; Shak, Steven; Perez, Edith A; Perez, Edith D; Shulman, Lawrence J; Martino, Silvana; Davidson, Nancy E; Sledge, George W; Goldstein, Lori J; Sparano, Joseph A

2008-05-20

Central and local laboratory concordance for hormone receptor measurement is therapeutically important. This study compares estrogen receptor (ER) and progesterone receptor (PR) measured by local laboratory immunohistochemistry (IHC), central IHC, and central reverse-transcriptase polymerase chain reaction (RT-PCR) using a proprietary 21-gene assay. A case-control sample of 776 breast cancer patients from Eastern Cooperative Oncology Group (ECOG) study E2197 was evaluated. Central IHC Allred score for ER and PR was obtained using tissue microarrays and 1D5 ER antibody and 636 PR antibody. Quantitative RT-PCR for ER and PR in whole sections was performed using the 21-gene assay. For ER, the concordance between local and central IHC was 90% (95% CI, 88% to 92%), between local IHC and central RT-PCR was 91% (95% CI, 89% to 93%), and between central IHC and central RT-PCR was 93% (95% CI, 91% to 95%). For PR, the concordance between local IHC and central IHC was 84% (95% CI, 82% to 87%), between local IHC and central RT-PCR was 88% (95% CI, 85% to 90%), and between central IHC and central RT-PCR was 90% (95% CI, 88% to 92%). Although concordance was high, IHC ER-negative cases that were RT-PCR positive were more common than IHC ER-positive cases that were RT-PCR negative. In ER-positive patients, ER expression by central IHC Allred score was marginally associated with recurrence (P = .091), and ER expression by central RT-PCR was significantly associated with recurrence (P = .014). However, recurrence score, which incorporates additional genes/pathways, was a highly significant predictor of recurrence (P < .0001). There is a high degree of concordance among local IHC, central IHC, and central RT-PCR by the proprietary gene assay for ER and PR status. Although ER expression is marginally associated with relapse in ER-positive patients treated with chemohormonal therapy, recurrence score is a highly significant predictor of recurrence.

Selection of reference genes for RT-qPCR analysis in a predatory biological control agent, Coleomegilla maculata (Coleoptera: Coccinellidae).

PubMed

Yang, Chunxiao; Pan, Huipeng; Noland, Jeffrey Edward; Zhang, Deyong; Zhang, Zhanhong; Liu, Yong; Zhou, Xuguo

2015-12-10

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for quantifying gene expression across various biological processes, of which requires a set of suited reference genes to normalize the expression data. Coleomegilla maculata (Coleoptera: Coccinellidae), is one of the most extensively used biological control agents in the field to manage arthropod pest species. In this study, expression profiles of 16 housekeeping genes selected from C. maculata were cloned and investigated. The performance of these candidates as endogenous controls under specific experimental conditions was evaluated by dedicated algorithms, including geNorm, Normfinder, BestKeeper, and ΔCt method. In addition, RefFinder, a comprehensive platform integrating all the above-mentioned algorithms, ranked the overall stability of these candidate genes. As a result, various sets of suitable reference genes were recommended specifically for experiments involving different tissues, developmental stages, sex, and C. maculate larvae treated with dietary double stranded RNA. This study represents the critical first step to establish a standardized RT-qPCR protocol for the functional genomics research in a ladybeetle C. maculate. Furthermore, it lays the foundation for conducting ecological risk assessment of RNAi-based gene silencing biotechnologies on non-target organisms; in this case, a key predatory biological control agent.

[Typing and subtyping avian influenza virus using DNA microarrays].

PubMed

Yang, Zhongping; Wang, Xiurong; Tian, Lina; Wang, Yu; Chen, Hualan

2008-07-01

Outbreaks of highly pathogenic avian influenza (HPAI) virus has caused great economic loss to the poultry industry and resulted in human deaths in Thailand and Vietnam since 2004. Rapid typing and subtyping of viruses, especially HPAI from clinical specimens, are desirable for taking prompt control measures to prevent spreading of the disease. We described a simultaneous approach using microarray to detect and subtype avian influenza virus (AIV). We designed primers of probe genes and used reverse transcriptase PCR to prepare cDNAs of AIV M gene, H5, H7, H9 subtypes haemagglutinin genes and N1, N2 subtypes neuraminidase genes. They were cloned, sequenced, reamplified and spotted to form a glass-bound microarrays. We labeled samples using Cy3-dUTP by RT-PCR, hybridized and scanned the microarrays to typing and subtyping AIV. The hybridization pattern agreed perfectly with the known grid location of each probe, no cross hybridization could be detected. Examinating of HA subtypes 1 through 15, 30 infected samples and 21 field samples revealed the DNA microarray assay was more sensitive and specific than RT-PCR test and chicken embryo inoculation. It can simultaneously detect and differentiate the main epidemic AIV. The results show that DNA microarray technology is a useful diagnostic method.

Molecular cloning of allelopathy related genes and their relation to HHO in Eupatorium adenophorum.

PubMed

Guo, Huiming; Pei, Xixiang; Wan, Fanghao; Cheng, Hongmei

2011-10-01

In this study, conserved sequence regions of HMGR, DXR, and CHS (encoding 3-hydroxy-3-methylglutaryl-CoA reductase, 1-deoxyxylulose-5-phosphate reductoisomerase and chalcone synthase, respectively) were amplified by reverse transcriptase (RT)-PCR from Eupatorium adenophorum. Quantitative real-time PCR showed that the expression of CHS was related to the level of HHO, an allelochemical isolated from E. adenophorum. Semi-quantitative RT-PCR showed that there was no significant difference in expression of genes among three different tissues, except for CHS. Southern blotting indicated that at least three CHS genes are present in the E. adenophorum genome. A full-length cDNA from CHS genes (named EaCHS1, GenBank ID: FJ913888) was cloned. The 1,455 bp cDNA contained an open reading frame (1,206 bp) encoding a protein of 401 amino acids. Preliminary bioinformatics analysis of EaCHS1 revealed that EaCHS1 was a member of CHS family, the subcellular localization predicted that EaCHS1 was a cytoplasmic protein. To the best of our knowledge, this is the first report of conserved sequences of these genes and of a full-length EaCHS1 gene in E. adenophorum. The results indicated that CHS gene is related to allelopathy of E. adenophorum.

Elimination of endogenous aberrant kappa chain transcripts from sp2/0-derived hybridoma cells by specific ribozyme cleavage: utility in genetic therapy of HIV-1 infections.

PubMed Central

Duan, L; Pomerantz, R J

1994-01-01

The pooled degenerate-primer polymerase chain reaction (PCR) technology is now widely used in the amplification and cloning of murine hybridoma-specific immunoglobulin gene cDNAs. The design of primers is mainly based on the highly conserved 5' terminus of immunoglobulin gene variable regions and the constant region in the 3' terminus. Of note, most murine hybridoma cell lines are derived from the Sp2/0 cell line, which is demonstrated to express endogenous aberrant kappa chains (abV kappa). This high-level endogenous abV kappa mixes with specific kappa chains in the hybridomas and interferes with the efficiency of the reverse transcriptase (RT)-PCR cloning strategy. In this report, during the cloning of murine anti-human immunodeficiency virus type I (HIV-1) hybridoma immunoglobulin cDNAs, a specific primer-PCR screening system was developed, based on the abV kappa complementarity-defining region (CDR), to eliminate abV kappa-carrying plasmids. Furthermore, an abV kappa sequence-specific derived ribozyme was developed and packaged in a retroviral expression vector system. This abV kappa ribozyme can be transduced into different murine hybridomas, and expressed intracellularly to potently eliminate endogenous abV kappa RNA. Images PMID:7816635

Identification and validation of reference genes for qRT-PCR studies of the obligate aphid pathogenic fungus Pandora neoaphidis during different developmental stages.

PubMed

Zhang, Shutao; Chen, Chun; Xie, Tingna; Ye, Sudan

2017-01-01

The selection of stable reference genes is a critical step for the accurate quantification of gene expression. To identify and validate the reference genes in Pandora neoaphidis-an obligate aphid pathogenic fungus-the expression of 13classical candidate reference genes were evaluated by quantitative real-time reverse transcriptase polymerase chain reaction(qPCR) at four developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae). Four statistical algorithms, including geNorm, NormFinder, BestKeeper and Delta Ct method were used to rank putative reference genes according to their expression stability and indicate the best reference gene or combination of reference genes for accurate normalization. The analysis of comprehensive ranking revealed that ACT1and 18Swas the most stably expressed genes throughout the developmental stages. To further validate the suitability of the reference genes identified in this study, the expression of cell division control protein 25 (CDC25) and Chitinase 1(CHI1) genes were used to further confirm the validated candidate reference genes. Our study presented the first systematic study of reference gene(s) selection for P. neoaphidis study and provided guidelines to obtain more accurate qPCR results for future developmental efforts.

Methylation and expression patterns of tropomyosin-related kinase genes in different grades of glioma.

PubMed

Palani, Mahalakshmi; Arunkumar, R; Vanisree, Arrambakam Janardhanam

2014-09-01

Tropomyosin-related kinase family (NTRK1, NTRK2 and NTRK3) is well known to play an important role in the pathogenesis of brain tumour, which exhibit heterogeneity in its biological and clinical behaviour. However, the mechanism that regulates NTRKs in glioma is not well understood. The present study investigates the epigenetic status (methylation) of NTRKs and their expression in different grades of glioma. Promoter methylation and structural relationship of NTRKs was assessed using methylation-specific PCR followed by chromatin immunoprecipitation in brain tissue samples from 220 subjects with different grades of glioma. Control brain samples were also assessed similarly. Reverse transcriptase PCR was performed to analyse the expressions of NTRK mRNAs in the grades of glioma. In addition, the expression level of p75(NTR) protein was analysed using immunofluorescent technique in all of the samples. The overall percentage of NTRK3 gene methylation frequency with subsequent loss of mRNA expression was significantly higher in glioma compared with control samples (p < 0.05). No such significance was observed in other NTRK1 and NTRK2 genes. Further, mRNA expression pattern of NTRK1 and NTRK2 genes was found to be significantly higher in low grades as compared with high grades (HG) and control samples (p < 0.05). Survival rate of HG patients with negative expressions of NTRK1 and NTRK2 was poor than those with the positive expressions of both NTRK1 and NTRK2. Further, a significant correlation was observed with reduced expression of p75(NTR) and the expression pattern of NTRK family in glioma as compared with the control samples (p < 0.05). There exists a correlation between the expression of NTRK family and different grades of glioma with a significant suggestion that the promoter methylation does not play role in the regulation of these genes in glioma. Further, poor survival could be associated with NTRK mRNAs 1 and 2. Hence, NTRKs are potential probes for assessing the behaviour of different grades of glioma, which could also function as significant prognostic factors and thus deserve wider attention for an effective management of the grades.

Interaction of HIV-1 reverse transcriptase ribonuclease H with an acylhydrazone inhibitor.

PubMed

Gong, Qingguo; Menon, Lakshmi; Ilina, Tatiana; Miller, Lena G; Ahn, Jinwoo; Parniak, Michael A; Ishima, Rieko

2011-01-01

HIV-1 reverse transcriptase is a bifunctional enzyme, having both DNA polymerase (RNA- and DNA-dependent) and ribonuclease H activities. HIV-1 reverse transcriptase has been an exceptionally important target for antiretroviral therapeutic development, and nearly half of the current clinically used antiretrovirals target reverse transcriptase DNA polymerase. However, no inhibitors of reverse transcriptase ribonuclease H are on the market or in preclinical development. Several drug-like small molecule inhibitors of reverse transcriptase ribonuclease H have been described, but little structural information is available about the interactions between reverse transcriptase ribonuclease H and inhibitors that exhibit antiviral activity. In this report, we describe NMR studies of the interaction of a new ribonuclease H inhibitor, BHMP07, with a catalytically active HIV-1 reverse transcriptase ribonuclease H domain fragment. We carried out solution NMR experiments to identify the interaction interface of BHMP07 with the ribonuclease H domain fragment. Chemical shift changes of backbone amide signals at different BHMP07 concentrations clearly demonstrate that BHMP07 mainly recognizes the substrate handle region in the ribonuclease H fragment. Using ribonuclease H inhibition assays and reverse transcriptase mutants, the binding specificity of BHMP07 was compared with another inhibitor, dihydroxy benzoyl naphthyl hydrazone. Our results provide a structural characterization of the ribonuclease H inhibitor interaction and are likely to be useful for further improvements of the inhibitors. © 2010 John Wiley & Sons A/S.

Niche specialization of novel Thaumarchaeota to oxic and hypoxic acidic geothermal springs of Yellowstone National Park

PubMed Central

Beam, Jacob P; Jay, Zackary J; Kozubal, Mark A; Inskeep, William P

2014-01-01

Novel lineages of the phylum Thaumarchaeota are endemic to thermal habitats, and may exhibit physiological capabilities that are not yet observed in members of this phylum. The primary goals of this study were to conduct detailed phylogenetic and functional analyses of metagenome sequence assemblies of two different thaumarchaeal populations found in high-temperature (65–72 °C), acidic (pH∼3) iron oxide and sulfur sediment environments of Yellowstone National Park (YNP). Metabolic reconstruction was coupled with detailed geochemical measurements of each geothermal habitat and reverse-transcriptase PCR to confirm the in situ activity of these populations. Phylogenetic analyses of ribosomal and housekeeping proteins place these archaea near the root of the thaumarchaeal branch. Metabolic reconstruction suggests that these populations are chemoorganotrophic and couple growth with the reduction of oxygen or nitrate in iron oxide habitats, or sulfur in hypoxic sulfur sediments. The iron oxide population has the potential for growth via the oxidation of sulfide to sulfate using a novel reverse sulfate reduction pathway. Possible carbon sources include aromatic compounds (for example, 4-hydroxyphenylacetate), complex carbohydrates (for example, starch), oligopeptides and amino acids. Both populations contain a type III ribulose bisphosphate carboxylase/oxygenase used for carbon dioxide fixation or adenosine monophosphate salvage. No evidence for the oxidation of ammonia was obtained from de novo sequence assemblies. Our results show that thermoacidophilic Thaumarchaeota from oxic iron mats and hypoxic sulfur sediments exhibit different respiratory machinery depending on the presence of oxygen versus sulfide, represent deeply rooted lineages within the phylum Thaumarchaeota and are endemic to numerous sites in YNP. PMID:24196321

A candidate gene for autoimmune myasthenia gravis

PubMed Central

Landouré, Guida; Knight, Melanie A.; Stanescu, Horia; Taye, Addis A.; Shi, Yijun; Diallo, Oumarou; Johnson, Janel O.; Hernandez, Dena; Traynor, Bryan J.; Biesecker, Leslie G.; Elkahloun, Abdel; Rinaldi, Carlo; Vincent, Angela; Willcox, Nick; Kleta, Robert; Fischbeck, Kenneth H.

2012-01-01

Objective: We sought to identify a causative mutation in a previously reported kindred with parental consanguinity and 5 of 10 siblings with adult-onset autoimmune myasthenia gravis. Methods: We performed genome-wide homozygosity mapping, and sequenced all known genes in the one region of extended homozygosity. Quantitative and allele-specific reverse transcriptase PCR (RT-PCR) were performed on a candidate gene to determine the RNA expression level in affected siblings and controls and the relative abundance of the wild-type and mutant alleles in a heterozygote. Results: A region of shared homozygosity at chromosome 13q13.3–13q14.11 was found in 4 affected siblings and 1 unaffected sibling. A homozygous single nucleotide variant was found in the 3′-untranslated region of the ecto-NADH oxidase 1 gene (ENOX1). No other variants likely to be pathogenic were found in genes in this region or elsewhere. The ENOX1 sequence variant was not found in 764 controls. Quantitative RT-PCR showed that expression of ENOX1 decreased to about 20% of normal levels in lymphoblastoid cells from individuals homozygous for the variant and to about 50% in 2 unaffected heterozygotes. Allele-specific RT-PCR showed a 55%–60% reduction in the level of the variant transcript in heterozygous cells due to reduced mRNA stability. Conclusion: These results indicate that this sequence variant in ENOX1 may contribute to the familial autoimmune myasthenia in these patients. PMID:22744667

Cloning of gene-encoded stem bromelain on system coming from Pichia pastoris as therapeutic protein candidate

NASA Astrophysics Data System (ADS)

Yusuf, Y.; Hidayati, W.

2018-01-01

The process of identifying bacterial recombination using PCR, and restriction, and then sequencing process was done after identifying the bacteria. This research aimed to get a yeast cell of Pichia pastoris which has an encoder gene of stem bromelain enzyme. The production of recombinant stem bromelain enzymes using yeast cells of P. pastoris can produce pure bromelain rod enzymes and have the same conformation with the enzyme’s conformation in pineapple plants. This recombinant stem bromelain enzyme can be used as a therapeutic protein in inflammatory, cancer and degenerative diseases. This study was an early stage of a step series to obtain bromelain rod protein derived from pineapple made with genetic engineering techniques. This research was started by isolating the RNA of pineapple stem which was continued with constructing cDNA using reserve transcriptase-PCR technique (RT-PCR), doing the amplification of bromelain enzyme encoder gene with PCR technique using a specific premiere couple which was designed. The process was continued by cloning into bacterium cells of Escherichia coli. A vector which brought the encoder gene of stem bromelain enzyme was inserted into the yeast cell of P. pastoris and was continued by identifying the yeast cell of P. pastoris which brought the encoder gene of stem bromelain enzyme. The research has not found enzyme gene of stem bromelain in yeast cell of P. pastoris yet. The next step is repeating the process by buying new reagent; RNase inhibitor, and buying liquid nitrogen.

Utility of dengue NS1 antigen rapid diagnostic test for use in difficult to reach areas and its comparison with dengue NS1 ELISA and qRT-PCR.

PubMed

Shukla, Mohan K; Singh, Neeru; Sharma, Ravendra K; Barde, Pradip V

2017-07-01

The objective of this study was to demonstrate the utility of dengue virus (DENV) non structural protein 1 (NS1) based rapid diagnostic test (RDT) for use in tribal and difficult to reach areas for early dengue (DEN) diagnosis in acute phase patients and evaluate its sensitivity and specificity against DENV NS1 enzyme linked immune sorbent assay (ELISA) and real time reverse transcriptase polymerase chain reaction (qRT-PCR). The DENV NS1 RDT was used for preliminary diagnosis during outbreaks in difficult to reach rural and tribal areas. The diagnosis was confirmed by DENV NS1 ELISA in the laboratory. The samples were also tested and serotyped by qRT-PCR. The results were evaluated using statistical tests. The DENV NS1 RDT showed 99.2% sensitivity and 96.0% specificity when analyzed using DENV NS1 ELISA as standard. The specificity and sensitivity of the RDT when compared with qRT-PCR was 93.6% and 91.1%, respectively. The serotype specific evaluation showed more than 90% sensitivity and specificity for DENV-1, 2, and 3. The RDT proved a good diagnostic tool in difficult to reach rural and tribal areas. Further evaluation studies with different commercially available RDTs in different field conditions are essential, that will help clinicians and patients for treatment and programme managers for timely intervention. © 2017 Wiley Periodicals, Inc.

Pemphigus vulgaris antigen mRNA quantification for the staging of sentinel lymph nodes in head and neck cancer

PubMed Central

Solassol, J; Burcia, V; Costes, V; Lacombe, J; Mange, A; Barbotte, E; de Verbizier, D; Cartier, C; Makeieff, M; Crampette, L; Boulle, N; Maudelonde, T; Guerrier, B; Garrel, R

2009-01-01

Background: Molecular diagnosis has been proposed to enhance the intra-operative diagnosis of sentinel lymph node (SLN) invasion in head and neck squamous cell carcinoma (HNSCC). Although cytokeratin (CK) mRNA quantification with real-time reverse transcriptase-PCR (QRT–PCR) has produced encouraging results, the more discriminating markers remain to be identified. Methods: Pemphigus vulgaris antigen (PVA), squamous cell carcinoma antigen (SCCA), and CK17 mRNA were quantified using QRT–PCR, and the results were compared with an extensive histopathological examination of the entire SLNs on 78 SLNs harvested from 22 patients with HNSCC. Results: SCCA and CK17 quantification showed significantly higher mRNA values for macrometastases (MAs) than for either negative or isolated tumour cell (ITC) SLNs (P<0.01). Pemphigus vulgaris antigen allowed the discrimination of all MAs and micrometastases from both negative and ITC SLNs (P<0.001). For the neck staging of patients, considering metastatic vs non-metastatic status, receiver-operating characteristic curve analysis found areas under the curve of 93.8, 97.9, and 100% for CK17, SCCA, and PVA, respectively. With PVA, a cutoff value of 562 copies per 100 ng of cDNA permitted the correct distinction between patients with positive as opposed to negative neck nodes in all cases. Conclusion: PVA seems to be a highly promising marker for accurate intra-operative SLN staging in HNSCC by QRT–PCR. PMID:19997107

Prevalence and clinicopathological characteristics of ALK fusion subtypes in lung adenocarcinomas from Chinese populations.

PubMed

Zheng, Difan; Wang, Rui; Zhang, Yang; Pan, Yunjian; Cheng, Xinghua; Cheng, Chao; Zheng, Shanbo; Li, Hang; Gong, Ranxia; Li, Yuan; Shen, Xuxia; Sun, Yihua; Chen, Haiquan

2016-04-01

We performed this retrospective study to have a comprehensive investigation of the clinicopathological characteristics of ALK fusion-positive lung adenocarcinoma in Chinese populations. We screened 1407 patients with primary lung adenocarcinoma from October 2007 to May 2013. Quantitative real-time PCR (qRT-PCR), reverse transcriptase PCR (RT-PCR), and fluorescence in situ hybridization were performed to detect ALK fusion genes, with validation of positive results using immunohistochemistry. Clinicopathological characteristics were collected to assess prognosis in ALK fusion-positive patients. Of 1407 patients with lung adenocarcinoma, there were 74 (5.3 %) ALK fusion-positive patients. Patients harboring ALK fusion were significantly younger (56.0 years vs. 59.8 years p = 0.002) and were more likely to have advanced stages (stage III or stage IV) (OR 1.761; 95 % CI 1.10-2.82, p = 0.017). Lepidic predominant adenocarcinoma was rarely found in ALK fusion patients (2.7 vs. 13.5 % p = 0.025), while IMA (invasive mucinous adenocarcinoma) predominant adenocarcinoma was more frequently found (21.6 vs. 5.0 % p < 0.001). ALK fusion was neither a risk factor nor protective factor in relapse-free survival and overall survival. Male, current smoker, and EML4-ALK variant 3 indicated poor prognosis among ALK fusion-positive lung adenocarcinomas. ALK fusion was detected in 5.3 % (74/1407) of the Chinese patients with lung adenocarcinoma. ALK fusion defines a molecular subset of lung adenocarcinoma with unique clinicopathological characteristics. Different ALK fusion variants determine distinct prognoses.

Assessment Effects of Resveratrol on Human Telomerase Reverse Transcriptase Messenger Ribonucleic Acid Transcript in Human Glioblastoma

PubMed Central

Mirzazadeh, Azin; Kheirollahi, Majid; Farashahi, Ehsan; Sadeghian-Nodoushan, Fatemeh; Sheikhha, Mohammad Hasan; Aflatoonian, Behrouz

2017-01-01

Background: Glioblastoma (GBM) is the most common and aggressive brain tumor, which has a poor prognosis despite the advent of different therapeutic strategies. There are numerous molecular biomarkers to contribute diagnosis, prognosis, and prediction of response to the current therapy in GBM. One of the most important markers that are potentially valuable is immortalization-specific or immortalization-associated marker named “hTERT messenger ribonucleic acid (mRNA)” the key subunit of telomerase enzyme, which is expressed in more than 85% of cancer cells, in spite of the majority of normal somatic cells. In this study, we investigated the effects of resveratrol (RSV) on this mRNA marker level, leading to cancer progression. Materials and Methods: U-87MG cell line was obtained from Pasteur Institute of Iran and treated with various concentrations of 0–160 μg/mL of RSV and at different time points (24, 48, and 72 h). To evaluate viability of U-87MG cells, standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed. Real-time polymerase chain reaction (RT-PCR) was used for comparative and quantitative assessment of human telomerase reverse transcriptase (hTERT) mRNA copy number versus control–untreated group. Results: The results of our investigation suggested that RSV effectively inhibited cell growth and caused cell death in dose-dependent (P < 0.05) and not in time-dependent manner (P > 0.05), in vitro. Interestingly, quantitative RT-PCR analysis demonstrated that at half inhibition concentration, RSV dramatically decreased mRNA expression of hTERT, the catalytic subunit of telomerase enzyme, which leads to prevention of cell division and tumor progression. Conclusion: With regard to downregulation of this immortalization-associated marker, RSV may potentially be used as a therapeutic agent against GBM. PMID:28706881

Guanidinylated 3-gluconamidopropyl methacrylamide-s-3-aminopropyl methacrylamide copolymer as siRNA carriers for inhibiting human telomerase reverse transcriptase expression.

PubMed

Wu, Yang; Ji, Jinkai; Yang, Ran; Zhang, Xiaoqiang; Li, Yuanhui; Pu, Yuepu; Li, Xinsong

2013-01-01

In this report, a series of well-defined glucose- and guanidine-based cationic copolymers as gene carriers were developed to inhibit human telomerase reverse transcriptase (hTERT) gene expression. First of all, guandinylated 3-gluconamidopropyl methacrylamide-s-3-aminopropyl methacrylamide copolymers (guanidinylated GAPMA-s-APMA, abbreviated as GGA) were prepared via aqueous reversible addition--fragmentation chain transfer polymerization (RAFT). Then, three target hTERT siRNA TERT-1, TERT-2 and TERT-3 were designed and combined with GGA copolymers to form siRNA/GGA polyplexes. The polyplexes were examined by dynamic light scattering and agarose gel electrophoresis. The results indicated that GGA copolymers can condense siRNA effectively to form particles with the diameter from 157 nm to 411 nm and zeta potential values in the range from +3.7 to +15.8 mV at various charge ratios (N/P). The MTT assay data of siRNA/GGA polyplexes on human hepatocellular liver carcinoma cells (HepG2) indicated that GGA copolymer had better cell viabilities than polyethylenimine (PEI). Furthermore, the transfection of siRNA/GGA polyplexes was detected by real-time quantitative PCR (RT-qPCR) in HepG2. It was found that siRNA/GGA polyplexes could effectively silence hTERT mRNA expression in serum-free media (p<0.01). In the presence of serum, the hTERT mRNA expression in HepG2 cells have significant difference (p<0.01) between siRNA/GGA3 polyplexes and blank. The results showed that the GAPMA component can reduce the aggregation of protein in serum media. Therefore, the enhancement of transfection may be attributed to the combination of guadino groups and glucose component. And, the guandinylated 3-gluconamidopropyl methacrylamide-s-3-aminopropyl methacrylamide copolymers might be promise in gene delivery.

Novel mutation in the human immunodeficiency virus type 1 reverse transcriptase gene that encodes cross-resistance to 2',3'-dideoxyinosine and 2',3'-dideoxycytidine.

PubMed Central

Gu, Z; Gao, Q; Li, X; Parniak, M A; Wainberg, M A

1992-01-01

We have used the technique of in vitro selection to generate variants of human immunodeficiency virus type 1 (HIV-1) that are resistant to 2',3'-dideoxyinosine (ddI) and cross-resistant to 2',3'-dideoxycytidine (ddC). The complete reverse transcriptase (RT)-coding regions, plus portions of flanking sequences, of viruses possessing a ddI-resistant phenotype were cloned and sequenced by polymerase chain reaction (PCR)-based methods. We observed that several of these viruses possessed mutations at amino acid sites 184 (Met-->Val; ATG-->GTG) and 294 (Pro-->Ser; CCA-->TCA). These mutations were introduced in the pol gene of infectious, cloned HXB2-D DNA by site-directed mutagenesis. Viral replication assays confirmed the importance of site 184 with regard to resistance to ddI. The recombinant viruses thus generated displayed more than fivefold-greater resistance to ddI than parental HXB2-D did. Moreover, more than fivefold-greater resistance to ddC was also documented; however, the recombinant viruses continued to be inhibited by zidovudine (AZT). No resistance to ddI, ddC, or AZT was introduced by inclusion of mutation site 294 in the pol gene of HXB2-D. PCR analysis performed on viral samples obtained from patients receiving long-term ddI therapy confirmed the presence of mutation site 184 in five of seven cases tested. In three of these five positive cases, the wild-type codon was also detected, indicating that mixtures of viral quasispecies were apparently present. Viruses possessing a ddI resistance phenotype were isolated from both subjects whose viruses contained only the mutated rather than wild-type codon at position 184 as well as from a third individual, whose viruses appeared to be mostly of the mutated variety. Images PMID:1279198

Directed evolution of DNA polymerase, RNA polymerase and reverse transcriptase activity in a single polypeptide.

PubMed

Ong, Jennifer L; Loakes, David; Jaroslawski, Szymon; Too, Kathleen; Holliger, Philipp

2006-08-18

DNA polymerases enable key technologies in modern biology but for many applications, native polymerases are limited by their stringent substrate recognition. Here we describe short-patch compartmentalized self-replication (spCSR), a novel strategy to expand the substrate spectrum of polymerases in a targeted way. spCSR is based on the previously described CSR, but unlike CSR only a short region (a "patch") of the gene under investigation is diversified and replicated. This allows the selection of polymerases under conditions where catalytic activity and processivity are compromised to the extent that full self-replication is inefficient. We targeted two specific motifs involved in substrate recognition in the active site of DNA polymerase I from Thermus aquaticus (Taq) and selected for incorporation of both ribonucleotide- (NTP) and deoxyribonucleotide-triphosphates (dNTPs) using spCSR. This allowed the isolation of multiple variants of Taq with apparent dual substrate specificity. They were able to synthesize RNA, while still retaining essentially wild-type (wt) DNA polymerase activity as judged by PCR. One such mutant (AA40: E602V, A608V, I614M, E615G) was able to incorporate both NTPs and dNTPs with the same catalytic efficiency as the wt enzyme incorporates dNTPs. AA40 allowed the generation of mixed RNA-DNA amplification products in PCR demonstrating DNA polymerase, RNA polymerase as well as reverse transcriptase activity within the same polypeptide. Furthermore, AA40 displayed an expanded substrate spectrum towards other 2'-substituted nucleotides and was able to synthesize nucleic acid polymers in which each base bore a different 2'-substituent. Our results suggest that spCSR will be a powerful strategy for the generation of polymerases with altered substrate specificity for applications in nano- and biotechnology and in the enzymatic synthesis of antisense and RNAi probes.

Molecular cloning of Kuruma shrimp Marsupenaeus japonicus endonuclease-reverse transcriptase and its positive role in white spot syndrome virus and Vibrio alginolyticus infection.

PubMed

Ma, Xiongchao; Sun, Baozhen; Zhu, Fei

2018-02-01

This study investigated the function of endonuclease-reverse transcriptase (mjERT) in Marsupenaeus japonicus. The 1129 bp cDNA sequence of mjERT was cloned from M. japonicus using rapid amplification of cDNA ends (RACE) PCR, and RT-qPCR analysis indicated that mjERT was highly expressed in the gills and hepatopancreas of M. japonicus. We also found that white spot syndrome virus (WSSV) or Vibrio alginolyticus challenge could enhance the expression of mjERT. When mjERT was inhibited, immune genes such as toll, p53, hemocyanin and tumor necrosis factor-α (TNF-α) were significantly down-regulated (P < .01) in the hemocytes of shrimp, while myosin was significantly up-regulated (P < .01). We demonstrated that mjERT is very important for the progression of WSSV infection and that the cumulative mortality of WSSV-infected and V. alginolyticus-infected shrimps was significantly increased following mjERT RNA interfere (RNAi). Apoptosis data provided information to suggest that mjERT-dsRNA challenge caused less apoptosis in hemocytes in both the disease-free and viral group. We also revealed that mjERT-dsRNA treatment resulted in a lower phagocytosis rate in the hemocytes of V. alginolyticus-challenged shrimp. Finally, we found that the absence of mjERT had an significantly negative impact upon shrimp phenoloxidase (PO) activity, superoxide dismutase (SOD) activity and total hemocyte count (THC) following WSSV or V. alginolyticus infection, indicating a regulative role for mjERT in the innate immunity of shrimp in response to pathogenic infection. In summary, we concluded that mjERT might promote the anti-WSSV immune response of shrimp by regulating apoptosis, PO activity, THC and SOD activity, and also exert a positive role in the immune response against V. alginolyticus by regulating phagocytosis, SOD activity, PO activity and THC. Copyright © 2017 Elsevier Ltd. All rights reserved.

Nine year trends of dengue virus infection in Mumbai, Western India.

PubMed

Shastri, Jayanthi; Williamson, Manita; Vaidya, Nilima; Agrawal, Sachee; Shrivastav, Om

2017-01-01

Dengue virus (DENV) causes a wide range of diseases in humans, from acute febrile illness Dengue fever (DF) to life-threatening Dengue hemorrhagic fever (DHF) or Dengue shock syndrome (DSS). Factors believed to be responsible for spread of Dengue virus infection include explosive population growth, unplanned urban overpopulation with inadequate public health systems, poor standing water and vector control, climate changes and increased international recreational, business, military travel to endemic areas. All of these factors must be addressed to control the spread of Dengue and other mosquito-borne infections. The detection of Dengue virus RNA by reverse transcriptase PCR (RT-PCR) in human serum or plasma samples is highly indicative of acute Dengue fever. Moreover, the method is able to identify the Dengue virus serotype by demonstrating defined sequence homologies in the viral genomic RNA. During the nine year period of this study analysis, 6767 strongly suspected cases were tested by RT-PCR. 1685 (24.9%) were Dengue PCR positive and confirmed as Dengue cases. Observations on the seasonality were based on the nine year's data as the intensity of sampling was at its maximum during monsoon season. Dengue typing was done on 100 positive samples after storage of Dengue RNA at - 80°C. Dengue serotypes were detected in 69 samples of which Dengue 2 was most predominant. 576 samples were processed for NS1 antigen and PCR simultaneously. 19/576 were positive (3.3 %) for NS1 as well as by PCR. 23/576 samples were negative for NS1 antigen, but were positive by RT-PCR. The remaining 534 samples which were negative for NS1 antigen were also negative by Dengue RT-PCR. In this study we sought to standardize rapid, sensitive, and specific fluorogenic probe-based RT-PCR assay to screen and serotype a representative range of Dengue viruses that are found in and around Mumbai. Qualitative Dengue virus TaqMan assays could have tremendous utility for the epidemiological investigation of Dengue illness and especially for the study of the viremic response with candidate live-attenuated dengue virus vaccines.

Spore-forming organisms in platelet concentrates: a challenge in transfusion bacterial safety.

PubMed

Störmer, M; Vollmer, T; Kleesiek, K; Dreier, J

2008-12-01

Bacterial detection and pathogen reduction are widely used methods of minimizing the risk of transfusion-transmitted bacterial infection. But, bacterial spores are highly resistant to chemical and physical agents. In this study, we assessed the bacterial proliferation of spore-forming organisms seeded into platelet concentrates (PCs) to demonstrate that spores can enter the vegetative state in PCs during storage. In the in vitro study, PCs were inoculated with 1-10 spores mL(-1)of Bacillus cereus (n = 1), Bacillus subtilis (n = 2) and Clostridium sporogenes (n = 2). Sampling was performed during 6-day aerobic storage at 22 degrees C. The presence of bacteria was assessed by plating culture, automated culture and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Spores of the C. sporogenes do not enter the vegetative phase under PC storage conditions, whereas B. subtilis and B. cereus showed growth in the PC and could be detected using RT-PCR and automated culture. Depending on the species and inoculums, bacterial spores may enter the vegetative phase during PC storage and can be detected by bacterial detection methods.

Discovery and analysis of an active long terminal repeat-retrotransposable element in Aspergillus oryzae.

PubMed

Jie Jin, Feng; Hara, Seiichi; Sato, Atsushi; Koyama, Yasuji

2014-01-01

Wild-type Aspergillus oryzae RIB40 contains two copies of the AO090005001597 gene. We previously constructed A. oryzae RIB40 strain, RKuAF8B, with multiple chromosomal deletions, in which the AO090005001597 copy number was found to be increased significantly. Sequence analysis indicated that AO090005001597 is part of a putative 6,000-bp retrotransposable element, flanked by two long terminal repeats (LTRs) of 669 bp, with characteristics of retroviruses and retrotransposons, and thus designated AoLTR (A. oryzae LTR-retrotransposable element). AoLTR comprised putative reverse transcriptase, RNase H, and integrase domains. The deduced amino acid sequence alignment of AoLTR showed 94% overall identity with AFLAV, an A. flavus Tf1/sushi retrotransposon. Quantitative real-time RT-PCR showed that AoLTR gene expression was significantly increased in the RKuAF8B, in accordance with the increased copy number. Inverse PCR indicated that the full-length retrotransposable element was randomly integrated into multiple genomic locations. However, no obvious phenotypic changes were associated with the increased AoLTR gene copy number.

The cDNA-derived amino acid sequence of hemoglobin II from Lucina pectinata.

PubMed

Torres-Mercado, Elineth; Renta, Jessicca Y; Rodríguez, Yolanda; López-Garriga, Juan; Cadilla, Carmen L

2003-11-01

Hemoglobin II from the clam Lucina pectinata is an oxygen-reactive protein with a unique structural organization in the heme pocket involving residues Gln65 (E7), Tyr30 (B10), Phe44 (CD1), and Phe69 (E11). We employed the reverse transcriptase-polymerase chain reaction (RT-PCR) and methods to synthesize various cDNA(HbII). An initial 300-bp cDNA clone was amplified from total RNA by RT-PCR using degenerate oligonucleotides. Gene-specific primers derived from the HbII-partial cDNA sequence were used to obtain the 5' and 3' ends of the cDNA by RACE. The length of the HbII cDNA, estimated from overlapping clones, was approximately 2114 bases. Northern blot analysis revealed that the mRNA size of HbII agrees with the estimated size using cDNA data. The coding region of the full-length HbII cDNA codes for 151 amino acids. The calculated molecular weight of HbII, including the heme group and acetylated N-terminal residue, is 17,654.07 Da.

Evaluation of Flinders Technology Associates cards for storage and molecular detection of avian metapneumoviruses.

PubMed

Awad, Faez; Baylis, Matthew; Jones, Richard C; Ganapathy, Kannan

2014-01-01

The feasibility of using Flinders Technology Associates (FTA) cards for the molecular detection of avian metapneumovirus (aMPV) by reverse transcriptase-polymerase chain reaction (RT-PCR) was investigated. Findings showed that no virus isolation was possible from aMPV-inoculated FTA cards, confirming viral inactivation upon contact with the cards. The detection limits of aMPV from the FTA card and tracheal organ culture medium were 10(1.5) median ciliostatic doses/ml and 10(0.75) median ciliostatic doses/ml respectively. It was possible to perform molecular characterization of both subtypes A and B aMPV using inoculated FTA cards stored for up to 60 days at 4 to 6°C. Tissues of the turbinate, trachea and lung of aMPV-infected chicks sampled either by direct impression smears or by inoculation of the tissue homogenate supernatants onto the FTA cards were positive by RT-PCR. However, the latter yielded more detections. FTA cards are suitable for collecting and transporting aMPV-positive samples, providing a reliable and hazard-free source of RNA for molecular characterization.

Involvement of a banana MADS-box transcription factor gene in ethylene-induced fruit ripening.

PubMed

Liu, Juhua; Xu, Biyu; Hu, Lifang; Li, Meiying; Su, Wei; Wu, Jing; Yang, Jinghao; Jin, Zhiqiang

2009-01-01

To investigate the regulation of MADS-box genes in banana (Musa acuminata L. AAA group cv. Brazilian) fruit development and postharvest ripening, we isolated from banana fruit a MADS-box gene designated MuMADS1. Amino acid alignment indicated MuMADS1 belongs to the AGAMOUS subfamily, and phylogenetic analysis indicates that this gene is most similar to class D MADS-box genes. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that MuMADS1 is expressed in the stamen and pistil of male and female flowers and in the rhizome, the vegetative reproductive organ of the banana plant. In preharvest banana fruit, MuMADS1 is likely expressed throughout banana fruit development. In postharvest banana ripening, MuMADS1 is associated with ethylene biosynthesis. Expression patterns of MuMADS1 during postharvest ripening as determined by real-time RT-PCR suggest that differential expression of MuMADS1 may not only be induced by ethylene biosynthesis associated with postharvest banana ripening, but also may be induced by exogenous ethylene.

A sugar gustatory receptor identified from the foregut of cotton bollworm Helicoverpa armigera.

PubMed

Xu, Wei; Zhang, Hui-Jie; Anderson, Alisha

2012-12-01

Helicoverpa armigera (Hübner) is one of the most polyphagous and cosmopolitan pest species, the larvae of which feed on numerous important crops. The gustatory system is critical in guiding insect feeding behavior. Here, we identified a gustatory receptor from H. armigera, HaGR9, which shows high levels of identity to DmGR43a from Drosophila melanogaster and BmGR9 from Bombyx mori. Reverse transcriptase PCR (RT-PCR) revealed HaGR9 is highly expressed in larval foregut, with little or no expression in other chemosensory tissues. Membrane topology studies indicated that, like two previously studied B. mori GRs, BmGR8 and BmGR53, HaGR9 has an inverted topology relative to G protein-coupled receptors (GPCRs), an intracellular N-terminus and an extracellular C-terminus. Calcium imaging studies confirmed HaGR9 is a sugar receptor showing dose-dependent responses to D-galactose, D-maltose, and D-fructose. This highly-expressed foregut-specific gustatory receptor may contribute to the regulation of larval feeding behavior.

Modified telomeric repeat amplification protocol: a quantitative radioactive assay for telomerase without using electrophoresis.

PubMed

Szatmari, I; Tókés, S; Dunn, C B; Bardos, T J; Aradi, J

2000-06-15

A polymerase chain reaction (PCR)-based radioactive telomerase assay was developed in our laboratory which is quantitative and does not require electrophoretic evaluation (designated as TP-TRAP; it utilizes two reverse primers). The main steps of the assay include (1) extension of a 20-mer oligonucleotide substrate (MTS) by telomerase, (2) amplification of the telomerase products in the presence of [(3)H]dTTP using the substrate oligonucleotide and two reverse primers (RPC3, 38 mer; RP, 20 mer), (3) isolation of the amplified radioactive dsDNA by precipitation and filtration, (4) determination of the radioactivity of the acid-insoluble DNA. The length of the telomerase products does not increase on amplification. This valuable feature of the assay is achieved by utilization of the two reverse primers and a highly specific PCR protocol. The assay is linear, accurate, and suitable for cell-biological studies where slight quantitative differences in telomerase activity must be detected. The assay is also suitable for screening and characterization of telomerase inhibitors, as shown with a chemically modified oligonucleotide reverse transcriptase inhibitor [(s(4)dU)(35)]. Copyright 2000 Academic Press.

The mechano-chemistry of a monomeric reverse transcriptase

PubMed Central

Malik, Omri; Khamis, Hadeel; Rudnizky, Sergei

2017-01-01

Abstract Retroviral reverse transcriptase catalyses the synthesis of an integration-competent dsDNA molecule, using as a substrate the viral RNA. Using optical tweezers, we follow the Murine Leukemia Virus reverse transcriptase as it performs strand-displacement polymerization on a template under mechanical force. Our results indicate that reverse transcriptase functions as a Brownian ratchet, with dNTP binding as the rectifying reaction of the ratchet. We also found that reverse transcriptase is a relatively passive enzyme, able to polymerize on structured templates by exploiting their thermal breathing. Finally, our results indicate that the enzyme enters the recently characterized backtracking state from the pre-translocation complex. PMID:29165701

Novel Method for Simultaneous Quantification of Phenotypic Resistance to Maturation, Protease, Reverse Transcriptase, and Integrase HIV Inhibitors Based on 3′Gag(p2/p7/p1/p6)/PR/RT/INT-Recombinant Viruses: a Useful Tool in the Multitarget Era of Antiretroviral Therapy▿†

PubMed Central

Weber, Jan; Vazquez, Ana C.; Winner, Dane; Rose, Justine D.; Wylie, Doug; Rhea, Ariel M.; Henry, Kenneth; Pappas, Jennifer; Wright, Alison; Mohamed, Nizar; Gibson, Richard; Rodriguez, Benigno; Soriano, Vicente; King, Kevin; Arts, Eric J.; Olivo, Paul D.; Quiñones-Mateu, Miguel E.

2011-01-01

Twenty-six antiretroviral drugs (ARVs), targeting five different steps in the life cycle of the human immunodeficiency virus type 1 (HIV-1), have been approved for the treatment of HIV-1 infection. Accordingly, HIV-1 phenotypic assays based on common cloning technology currently employ three, or possibly four, different recombinant viruses. Here, we describe a system to assess HIV-1 resistance to all drugs targeting the three viral enzymes as well as viral assembly using a single patient-derived, chimeric virus. Patient-derived p2-INT (gag-p2/NCp7/p1/p6/pol-PR/RT/IN) products were PCR amplified as a single fragment (3,428 bp) or two overlapping fragments (1,657 bp and 2,002 bp) and then recombined into a vector containing a near-full-length HIV-1 genome with the Saccharomyces cerevisiae uracil biosynthesis gene (URA3) replacing the 3,428 bp p2-INT segment (Dudley et al., Biotechniques 46:458–467, 2009). P2-INT-recombinant viruses were employed in drug susceptibility assays to test the activity of protease (PI), nucleoside/nucleotide reverse transcriptase (NRTI), nonnucleoside reverse transcriptase (NNRTI), and integrase strand-transfer (INSTI) inhibitors. Using a single standardized test (ViralARTS HIV), this new technology permits the rapid and automated quantification of phenotypic resistance for all known and candidate antiretroviral drugs targeting all viral enzymes (PR, RT, including polymerase and RNase H activities, and IN), some of the current and potential assembly inhibitors, and any drug targeting Pol or Gag precursor cleavage sites (relevant for PI and maturation inhibitors) This novel assay may be instrumental (i) in the development and clinical assessment of novel ARV drugs and (ii) to monitor patients failing prior complex treatment regimens. PMID:21628544

Genes indicative of zoonotic and swine pathogens are persistent in stream water and sediment following a swine manure spill

USGS Publications Warehouse

Haack, Sheridan K.; Duris, Joseph W.; Kolpin, Dana W.; Fogarty, Lisa R.; Johnson, Heather E.; Gibson, Kristen E.; Focazio, Michael J.; Schwab, Kellogg J.; Hubbard, Laura E.; Foreman, William T.

2015-01-01

Manure spills to streams are relatively frequent, but no studies have characterized stream contamination with zoonotic and veterinary pathogens, or fecal chemicals, following a spill. We tested stream water and sediment over 25 days and downstream for 7.6 km for: fecal indicator bacteria (FIB); the fecal indicator chemicals cholesterol and coprostanol; 20 genes for zoonotic and swine-specific bacterial pathogens by presence/absence polymerase chain reaction (PCR) for viable cells; one swine-specific Escherichia coli toxin gene (STII) by quantitative PCR (qPCR); and nine human and animal viruses by qPCR, or reverse-transcriptase qPCR. Twelve days post-spill, and 4.2 km downstream, water concentrations of FIB, cholesterol, and coprostanol were 1-2 orders of magnitude greater than those detected before, or above, the spill, and genes indicating viable zoonotic or swine-infectious Escherichia coli, were detected in water or sediment. STII increased from undetectable before, or above the spill, to 105 copies/100 mL water 12 days post-spill. Thirteen of 14 water (8/9 sediment) samples had viable STII-carrying cells post-spill. Eighteen days post-spill porcine adenovirus and teschovirus were detected 5.6 km downstream. Sediment FIB concentrations (per gram wet weight) were greater than in water, and sediment was a continuous reservoir of genes and chemicals post-spill. Constituent concentrations were much lower, and detections less frequent, in a runoff event (200 days post-spill) following manure application, although the swine-associated STII and stx2e genes were detected. Manure spills are an underappreciated pathway for livestock-derived contaminants to enter streams, with persistent environmental outcomes, and the potential for human and veterinary health consequences.

The sensitivity and specificity of Lassa virus IgM by ELISA as screening tool at early phase of Lassa fever infection

PubMed Central

Ibekwe, Titus S.; Nwegbu, Maxwell M.; Asogun, Daniel; Adomeh, Donatus I.; Okokhere, Peter O.

2012-01-01

Background: Early diagnosis, prompt treatment, and disease containment are vital measures in the management of Lassa fever (LF), a lethal and contagious arenaviral hemorrhagic disease prevalent in West Africa. Lassa Virus (LAV)-specific Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) test, the gold standard for diagnosis, is unavailable in most centers. Serologic detection of LAV IgM is a more accessible tool and this work was to investigate its adequacy as an early marker for LF. Patients and Methods: A prospective case–control study conducted July 2007-March 2011 in a tertiary referral health center in Nigeria. Blood samples for test and control were evaluated for Lassa specific antigens and IgM using RT-PCR (primers S36+ and LVS 339) and indirect ELISA (Lassa Nucleo-protein (NP)-Antigen) respectively. RT-PCR outcome was used as standard to test for the sensitivity and specificity of IgM. Results: Of the 37 confirmed cases of LF infection by RT-PCR, 21 (57%) were IgM positive. Amongst the 35 confirmed negative cases (control group), eight were IgM positive. The diagnostic sensitivity and specificity of the IgM assay were 57% and 77% respectively. The negative and positive predictive values of the IgM serological assay were 63% and 72%, respectively, while the efficiency of the test was 67%. Conclusion: The specificity and sensitivity of IgM as a screening tool for early detection of LF appear weak and, hence, the need for a reliable LF “rapid screening kit” since RT-PCR is unavailable in most centers. In the interim, “high clinical index of suspicion,” irrespective of IgM status, requires urgent referral to confirmatory centers. PMID:23661877

The sensitivity and specificity of Lassa virus IgM by ELISA as screening tool at early phase of Lassa fever infection.

PubMed

Ibekwe, Titus S; Nwegbu, Maxwell M; Asogun, Daniel; Adomeh, Donatus I; Okokhere, Peter O

2012-10-01

Early diagnosis, prompt treatment, and disease containment are vital measures in the management of Lassa fever (LF), a lethal and contagious arenaviral hemorrhagic disease prevalent in West Africa. Lassa Virus (LAV)-specific Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) test, the gold standard for diagnosis, is unavailable in most centers. Serologic detection of LAV IgM is a more accessible tool and this work was to investigate its adequacy as an early marker for LF. A prospective case-control study conducted July 2007-March 2011 in a tertiary referral health center in Nigeria. Blood samples for test and control were evaluated for Lassa specific antigens and IgM using RT-PCR (primers S36+ and LVS 339) and indirect ELISA (Lassa Nucleo-protein (NP)-Antigen) respectively. RT-PCR outcome was used as standard to test for the sensitivity and specificity of IgM. Of the 37 confirmed cases of LF infection by RT-PCR, 21 (57%) were IgM positive. Amongst the 35 confirmed negative cases (control group), eight were IgM positive. The diagnostic sensitivity and specificity of the IgM assay were 57% and 77% respectively. The negative and positive predictive values of the IgM serological assay were 63% and 72%, respectively, while the efficiency of the test was 67%. The specificity and sensitivity of IgM as a screening tool for early detection of LF appear weak and, hence, the need for a reliable LF "rapid screening kit" since RT-PCR is unavailable in most centers. In the interim, "high clinical index of suspicion," irrespective of IgM status, requires urgent referral to confirmatory centers.

Use of molecular and milk production information for the cost-effective diagnosis of bovine viral diarrhoea infection in New Zealand dairy cattle.

PubMed

Hill, F I; Reichel, M P; Tisdall, D J

2010-04-21

An increase in veterinary and farmer interest in bovine viral diarrhoea (BVD) in New Zealand over recent years led to requests for cost-effective identification of BVD virus (BVDV) infected herds and individuals. This study was undertaken to determine if the use of real-time reverse transcriptase polymerase chain reaction (RT-PCR) technology and dairy cow production data could identify persistently infected (PI) animals in milking herds. Milk samples were collected from the vats of dairy herds and tested for the presence of BVDV by RT-PCR till four herds were found containing PI animals. Individual serum samples were then collected from every cow in the herd and tested by both RT-PCR and antigen capture enzyme-linked immunosorbent assays (ACE) to identify the PI animals. Individual animal testing found 1/223, 1/130, 2/800 and 1/275 PI's respectively in the four herds. Based on these results a maximum pool size of 400 cows contributing to the bulk tank milk was selected. After removal of the PI from the herds, further bulk milk samples were shown to be BVDV negative by RT-PCR. All the PI animals identified by this method were found in the lowest producing 10-20% of herd. This approach of targeted testing of dairy herds using PCR technology, in conjunction with animal production information, markedly reduced the cost of diagnostic testing for BVDV in dairy herds in New Zealand. Questionnaire follow-up on 81 BVDV-positive herds (15% of those tested) indicated the stratification approach identified milking PIs successfully over 90% of the time and reduced the number of individual tests to 12% of the milking herd. Copyright 2009 Elsevier B.V. All rights reserved.

Armored RNA as Virus Surrogate in a Real-Time Reverse Transcriptase PCR Assay Proficiency Panel

PubMed Central

Hietala, S. K.; Crossley, B. M.

2006-01-01

In recent years testing responsibilities for high-consequence pathogens have been expanded from national reference laboratories into networks of local and regional laboratories in order to support enhanced disease surveillance and to test for surge capacity. This movement of testing of select agents and high-consequence pathogens beyond reference laboratories introduces a critical need for standardized, noninfectious surrogates of disease agents for use as training and proficiency test samples. In this study, reverse transcription-PCR assay RNA targets were developed and packaged as armored RNA for use as a noninfectious, quantifiable synthetic substitute for four high-consequence animal pathogens: classical swine fever virus; foot-and-mouth disease virus; vesicular stomatitis virus, New Jersey serogroup; and vesicular stomatitis virus, Indiana serogroup. Armored RNA spiked into oral swab fluid specimens mimicked virus-positive clinical material through all stages of the reverse transcription-PCR testing process, including RNA recovery by four different commercial extraction procedures, reverse transcription, PCR amplification, and real-time detection at target concentrations consistent with the dynamic ranges of the existing real-time PCR assays. The armored RNA concentrations spiked into the oral swab fluid specimens were stable under storage conditions selected to approximate the extremes of time and temperature expected for shipping and handling of proficiency panel samples, including 24 h at 37°C and 2 weeks at temperatures ranging from ambient room temperature to −70°C. The analytic test performance, including the reproducibility over the dynamic range of the assays, indicates that armored RNA can provide a noninfectious, quantifiable, and stable virus surrogate for specific assay training and proficiency test purposes. PMID:16390950

Kaposi's Sarcoma-Associated Herpesvirus Can Productively Infect Primary Human Keratinocytes and Alter Their Growth Properties

PubMed Central

Cerimele, Francesca; Curreli, Francesca; Ely, Scott; Friedman-Kien, Alvin E.; Cesarman, Ethel; Flore, Ornella

2001-01-01

Previous studies have shown the presence of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) DNA in endothelial cells, in keratinocytes in the basal layer of the epidermis overlying plaque-stage nodular lesions of cutaneous Kaposi's sarcoma (KS), and in the epithelial cells of eccrine glands within KS lesions. We infected primary cell cultures of human keratinocytes with KSHV/HHV8. At 6 days post infection, transcription of viral genes was detected by reverse transcriptase PCR (RT-PCR), and protein expression was documented by an immunofluorescence assay with an anti-LANA monoclonal antibody. To determine whether the viral lytic cycle was inducible by chemical treatment, KSHV/HHV8-infected keratinocytes were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and RT-PCR was performed to confirm the transcription of lytic genes such as open reading frame 26, (which encodes a capsid protein). Finally, to assess infectious viral production, other primary human cells (human umbilical vein endothelial cells), were infected with concentrated supernatant of KSHV-infected, TPA-induced keratinocytes and the presence of viral transcripts was confirmed by RT-PCR. The uninfected keratinocytes senesced 3 to 5 weeks after mock infection, while the KSHV/HHV8-infected keratinocytes continued to proliferate and to date are still in culture. However, 8 weeks after infection, viral genomes were no longer detectable by nested PCR. Although the previously KSHV/HHV8-infected keratinocytes still expressed epithelial markers, they acquired new characteristics such as contact inhibition loss, telomerase activity, anchorage-independent growth, and changes in cytokine production. These results show that KSHV/HHV8, like other herpesviruses, can infect and replicate in epithelial cells in vitro and suggest that in vivo these cells may play a significant role in the establishment of KSHV/HHV8 infection and viral transmission. PMID:11160746

SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all hemagglutinin and neuraminidase genes of avian influenza viruses and comparison to standard serological subtyping tests.

PubMed

Tsukamoto, Kenji; Panei, Carlos Javier; Javier, Panei Carlos; Shishido, Makiko; Noguchi, Daigo; Pearce, John; Kang, Hyun-Mi; Jeong, Ok Mi; Lee, Youn-Jeong; Nakanishi, Koji; Ashizawa, Takayoshi

2012-01-01

Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 10(1.5), 10(2.3), and 10(3.1) 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples.

Swine Influenza Virus and Association with the Porcine Respiratory Disease Complex in Pig Farms in Southern Brazil.

PubMed

Schmidt, C; Cibulski, S P; Andrade, C P; Teixeira, T F; Varela, A P M; Scheffer, C M; Franco, A C; de Almeida, L L; Roehe, P M

2016-05-01

Despite the putative endemic status of swine influenza A virus (swIAV) infections, data on the occurrence of swine influenza outbreaks are scarce in Brazil. The aim of this study was to detect and subtype swIAVs from six outbreaks of porcine respiratory disease complex (PRDC) in southern Brazil. Nasal swabs were collected from 66 piglets with signs of respiratory disease in six herds. Lung tissue samples were collected from six necropsied animals. Virus detection was performed by PCR screening and confirmed by virus isolation and hemagglutination (HA). Influenza A subtyping was performed by a real-time reverse transcriptase PCR (rRT-PCR) to detect the A(H1N1)pdm09; other swIAV subtypes were determined by multiplex RT-PCR. In lung tissues, the major bacterial and viral pathogens associated with PRDC (Pasteurella multocida, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Haemophilus parasuis and PCV2) were investigated. In some affected pigs, clinico-pathological evaluations were conducted. Influenza A was detected by screening PCR in 46 of 66 swab samples and from five of six lungs. Virus was recovered from pigs of all six herds. Subtype A(H1N1)pdm09 was detected in four of six herds and H1N2 in the other two herds. In lung tissues, further agents involved in PRDC were detected in all cases; Pasteurella multocida was identified in five of six samples and Mycoplasma hyopneumoniae in three of six. Actinobacillus pleuropneumoniae (1/6), Haemophilus parasuis (1/6) and PCV2 (1/6) were also detected. These findings indicate that subtypes A(H1N1)pdm09 and H1N2 were present in pigs in southern Brazil and were associated with PRDC outbreaks. © 2015 Blackwell Verlag GmbH.

Yellow Fever Vaccination of a Primary Vaccinee During Adalimumab Therapy.

PubMed

Nash, Esther R; Brand, Myron; Chalkias, Spyridon

2015-01-01

In this case report, we describe a 63-year-old female with Crohn's disease since age 16 years, and on adalimumab therapy, who inadvertently received a yellow fever vaccine (YFV) 4 days before her next dose of adalimumab. She had never received YFV. Her next dose of tumor necrosis factor (TNF) antagonist was held. She did not report any adverse effects referable to the vaccine. Reverse transcriptase-polymerase chain reaction (RT-PCR) for yellow fever (YF) viral RNA on days 12 and 18 postvaccination was negative. Neutralizing antibody to YF virus vaccine was immunoprotective on day 18 following vaccination, which further increased by day 26. A neutralizing antibody obtained 2 years following vaccination also remained immunoprotective. © 2015 International Society of Travel Medicine.

[Prevalence of transmission of zidovudine-resistant viruses in Switzerland. l'Etude suisse de cohorte VIH].

PubMed

Yerly, S; Rakik, A; Kinloch-de-Loes, S; Erb, P; Vernazza, P; Hirschel, B; Perrin, L

1996-10-26

Zidovudine (ZDV) was the most widely used anti-HIV drug between 1987 and 1995, and, as already reported, transmission of ZDV-resistant viruses occurs. Several mutations of the reverse transcriptase gene have been identified; one of them affects the 215 codon and is associated with a high degree of resistance. We have determined, using selective PCR, the prevalence of transmission of 215 mutant isolates in 134 patients with primary HIV infection (PHI) and have identified 8 patients with 215 mutant virus between 1989 and 1995 in Switzerland. Mutant resistant viruses have been isolated from patients treated with most antiviral drugs. A systematic search for mutant viruses may provide useful information for the adaptation of treatment strategies.

A de novo transcriptome and valid reference genes for quantitative real-time PCR in Colaphellus bowringi.

PubMed

Tan, Qian-Qian; Zhu, Li; Li, Yi; Liu, Wen; Ma, Wei-Hua; Lei, Chao-Liang; Wang, Xiao-Ping

2015-01-01

The cabbage beetle Colaphellus bowringi Baly is a serious insect pest of crucifers and undergoes reproductive diapause in soil. An understanding of the molecular mechanisms of diapause regulation, insecticide resistance, and other physiological processes is helpful for developing new management strategies for this beetle. However, the lack of genomic information and valid reference genes limits knowledge on the molecular bases of these physiological processes in this species. Using Illumina sequencing, we obtained more than 57 million sequence reads derived from C. bowringi, which were assembled into 39,390 unique sequences. A Clusters of Orthologous Groups classification was obtained for 9,048 of these sequences, covering 25 categories, and 16,951 were assigned to 255 Kyoto Encyclopedia of Genes and Genomes pathways. Eleven candidate reference gene sequences from the transcriptome were then identified through reverse transcriptase polymerase chain reaction. Among these candidate genes, EF1α, ACT1, and RPL19 proved to be the most stable reference genes for different reverse transcriptase quantitative polymerase chain reaction experiments in C. bowringi. Conversely, aTUB and GAPDH were the least stable reference genes. The abundant putative C. bowringi transcript sequences reported enrich the genomic resources of this beetle. Importantly, the larger number of gene sequences and valid reference genes provide a valuable platform for future gene expression studies, especially with regard to exploring the molecular mechanisms of different physiological processes in this species.

Comprehensive phylogenetic analysis of bacterial reverse transcriptases.

PubMed

Toro, Nicolás; Nisa-Martínez, Rafael

2014-01-01

Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology.

Comprehensive Phylogenetic Analysis of Bacterial Reverse Transcriptases

PubMed Central

Toro, Nicolás; Nisa-Martínez, Rafael

2014-01-01

Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology. PMID:25423096

Notch signalling in cardiovasculogenesis: insight into their role in early cardiovascular development.

PubMed

Saravanakumar, Marimuthu; Devaraj, Halagowder

2013-05-01

The role of Notch signalling in congenital cardiovascular disease is evident by the identification of human mutations in several Notch signalling components, which also indicates the importance of activated Notch pathway in cardiovascular biology. Therefore, the aim of the present study is to investigate the expression pattern of the components of Notch signalling molecules and their role in mice embryonic heart and vascular development. Group A: normal control pregnant mice, group B: pregnant mice were injected with DMSO, group C: DAPT were subcutaneously injected to pregnant mice. The morphological and molecular changes of trabeculation-defective phenotype were analysed using histological, scanning electron microscope, immunoblot, immunolocalization and reverse transcriptase-PCR. E15.5 DAPT-treated mice revealed that there was a major reduction in the formation of septal walls between the ventricular chambers compared with normal control pregnant mice. VEGF expression was found in the DAPT treated and wild-type embryonic artery, whereas notch target genes GATA4, Hey1 expression were not found in the DAPT treated mice embryo. The role of Notch in ventricular development is supported by the trabeculation-defective phenotype seen in standard and endocardial-specific inhibition of Notch targets. The present study reveals the significant role of Notch signalling during the formation of ventricular septum and proper development of endothelial cell lineage and its precursor in mice cardiogenesis.

Interleukin-9 enhances interleukin-5 receptor expression, differentiation, and survival of human eosinophils.

PubMed

Gounni, A S; Gregory, B; Nutku, E; Aris, F; Latifa, K; Minshall, E; North, J; Tavernier, J; Levit, R; Nicolaides, N; Robinson, D; Hamid, Q

2000-09-15

Interleukin-9 (IL-9) has been implicated in the pathogenesis of allergic disorders. To examine the interaction between IL-9 and eosinophils, we evaluated mature peripheral blood eosinophils for their expression of the specific alpha-subunit of the IL-9 receptor (IL-9R-alpha). The expression of IL-9R-alpha by human eosinophils was detected at the messenger RNA (mRNA) and protein levels by reverse transcriptase-polymerase chain reaction (RT-PCR), flow cytometry, and immunocytochemical analysis, respectively. Functional analyses demonstrated that recombinant human (rh)IL-9 inhibited in vitro peripheral blood human eosinophil apoptosis in a concentration-dependent manner. We then examined the role of IL-9 in eosinophil differentiation using the human cord blood CD34(+) cells and human promyelocytic leukemia cells (HL-60). The addition of IL-9 to CD34(+) cells cultured in IL-3 and IL-5 enhanced eosinophil development, and IL-9 alone induced the expression of IL-5R-alpha. IL-9 also up-regulated the IL-5R-alpha chain cell surface expression during terminal eosinophil differentiation of the HL-60 cell line. Our findings suggest that IL-9 may potentiate in vivo eosinophil function by increasing their survival and IL-5-mediated differentiation and maturation. Taken together, these results suggest a mechanism by which IL-9 potentiates airway and tissue eosinophilia.

SGK is a primary glucocorticoid-induced gene in the human.

PubMed

Náray-Fejes-Tóth, A; Fejes-Tóth, G; Volk, K A; Stokes, J B

2000-12-01

Serum- and glucocorticoid-induced kinase (sgk) is transcriptionally regulated by corticosteroids in several cell types. Recent findings suggest that sgk is an important gene in the early action of corticosteroids on epithelial sodium reabsorption. Surprisingly, the human sgk was reported not to be transcriptionally regulated by corticosteroids in a hepatoma cell line, and thus far no glucocorticoid response element has been identified in the human SGK gene. Since humans clearly respond to both aldosterone and glucocorticoids in cells where sgk action seems to be important, in this study we determined sgk mRNA levels following dexamethasone treatment for various duration in five human cell lines. These cell lines included epithelial cells (H441, T84 and HT29) and lymphoid/monocyte (U937 and THP-1) lines. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we found that sgk mRNA levels are markedly induced by glucocorticoids in all of the five cell lines studied. Time course analyses revealed that sgk mRNA levels are elevated as early as 30 min after addition of the glucocorticoid, and remain elevated for several hours. Northern analysis in H441 cells confirmed that sgk is an early induced gene. The induction of sgk by dexamethasone was unaffected by cycloheximide, indicating that it does not require de novo protein synthesis. These results indicate that the human sgk, just like its counterparts in other species, is a primary glucocorticoid-induced gene.

Occurrence of Cucumber mosaic virus on vanilla (Vanilla planifolia Andrews) in India.

PubMed

Madhubala, R; Bhadramurthy, V; Bhat, A I; Hareesh, P S; Retheesh, S T; Bhai, R S

2005-06-01

Cucumber mosaic virus (CMV) causing mosaic, leaf distortion and stunting of vanilla (Vanilla planifolia Andrews) in India was characterized on the basis of biological and coat protein (CP) nucleotide sequence properties. In mechanical inoculation tests, the virus was found to infect members of Chenopodiaceae, Cucurbitaceae, Fabaceae and Solanaceae. Nicotiana benthamiana was found to be a suitable host for the propagation of CMV. The virus was purified from inoculated N. benthamiana plants and negatively stained purified preparations contained isometric particles of about 28 nm in diameter. The molecular weight of the viral coat protein subunits was found to be 25.0 kDa. Polyclonal antiserum was produced in New Zealand white rabbit, immunoglobulin G (IgG) was purified and conjugated with alkaline phosphatase enzyme. Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) method was standardized for the detection of CMV infection in vanilla plants. CP gene of the virus was amplified using reverse transcriptase-polymerase chain reaction (RT-PCR), cloned and sequenced. Sequenced region contained a single open reading frame of 657 nucleotides potentially coding for 218 amino acids. Sequence analyses with other CMV isolates revealed the greatest identity with black pepper isolate of CMV (99%) and the phylogram clearly showed that CMV infecting vanilla belongs to subgroup IB. This is the first report of occurrence of CMV on V. planifolia from India.

Canopy light cues affect emission of constitutive and methyl jasmonate-induced volatile organic compounds in Arabidopsis thaliana.

PubMed

Kegge, Wouter; Weldegergis, Berhane T; Soler, Roxina; Vergeer-Van Eijk, Marleen; Dicke, Marcel; Voesenek, Laurentius A C J; Pierik, Ronald

2013-11-01

The effects of plant competition for light on the emission of plant volatile organic compounds (VOCs) were studied by investigating how different light qualities that occur in dense vegetation affect the emission of constitutive and methyl-jasmonate-induced VOCs. Arabidopsis thaliana Columbia (Col-0) plants and Pieris brassicae caterpillars were used as a biological system to study the effects of light quality manipulations on VOC emissions and attraction of herbivores. VOCs were analysed using gas chromatography-mass spectrometry and the effects of light quality, notably the red : far red light ratio (R : FR), on expression of genes associated with VOC production were studied using reverse transcriptase-quantitative PCR. The emissions of both constitutive and methyl-jasmonate-induced green leaf volatiles and terpenoids were partially suppressed under low R : FR and severe shading conditions. Accordingly, the VOC-based preference of neonates of the specialist lepidopteran herbivore P. brassicae was significantly affected by the R : FR ratio. We conclude that VOC-mediated interactions among plants and between plants and organisms at higher trophic levels probably depend on light alterations caused by nearby vegetation. Studies on plant-plant and plant-insect interactions through VOCs should take into account the light quality within dense stands when extrapolating to natural and agricultural field conditions. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Adult-onset hyperthyroidism impairs spatial learning: possible involvement of mitogen-activated protein kinase signaling pathways.

PubMed

Bitiktaş, Soner; Kandemir, Başak; Tan, Burak; Kavraal, Şehrazat; Liman, Narin; Dursun, Nurcan; Dönmez-Altuntaş, Hamiyet; Aksan-Kurnaz, Işil; Suer, Cem

2016-08-03

Given evidence that mitogen-activated protein kinase (MAPK) activation is part of the nongenomic actions of thyroid hormones, we investigated the possible consequences of hyperthyroidism for the cognitive functioning of adult rats. Young adult rats were treated with L-thyroxine or saline. Twenty rats in each group were exposed to Morris water maze testing, measuring their performance in a hidden-platform spatial task. In a separate set of rats not exposed to Morris water maze testing (untrained rats), the expression and phosphorylated levels of p38-MAPK and of its two downstream effectors, Elk-1 and cAMP response element-binding protein, were evaluated using quantitative reverse transcriptase-PCR and western blotting. Rats with hyperthyroidism showed delayed acquisition of learning compared with their wild-type counterparts, as shown by increased escape latencies and distance moved on the last two trials of daily training in the water maze. The hyperthyroid rats, however, showed no difference during probe trials. Western blot analyses of the hippocampus showed that hyperthyroidism increased phosphorylated p38-MAPK levels in untrained rats. Although our study is correlative in nature and does not exclude the contribution of other molecular targets, our findings suggest that the observed impairments in acquisition during actual learning in rats with hyperthyroidism may result from the increased phosphorylation of p38-MAPK.

Induction of anti-viral genes during acute infection with Viral hemorrhagic septicemia virus (VHSV) genogroup IVa in Pacific herring (Clupea pallasii)

USGS Publications Warehouse

Hansen, John D.; Woodson, James C.; Hershberger, Paul K.; Grady, Courtney; Gregg, Jacob L.; Purcell, Maureen K.

2012-01-01

Infection with the aquatic rhabdovirus Viral hemorrhagic septicemia virus (VHSV) genogroup IVa results in high mortality in Pacific herring (Clupea pallasii) and is hypothesized to be a potential limiting factor for herring recovery. To investigate anti-viral immunity in the Pacific herring, four immune response genes were identified: the myxovirus resistance (Clpa-Mx), a major histocompatibility complex IB (named Clpa-UAA.001), the inducible immunoproteosome subunit 9 (Clpa-PSMB9) and the neutrophil chemotactic factor (Clpa-LECT2). Reverse transcriptase quantitative PCR (RT-qPCR) assays were developed based on these gene sequences to investigate the host immune response to acute VHSV infection following both injection and immersion challenge. Virus levels were measured by both plaque assay and RT-qPCR and peaked at day 6 during the 10-day exposure period for both groups of fish. The interferon stimulated genes (Clpa-Mx, −UAA.001, and −PSMB9) were significantly up-regulated in response to VHSV infection at both 6 and 10 days post-infection in both spleen and fin. Results from this study indicate that Pacific herring mount a robust, early antiviral response in both fin and spleen tissues. The immunological tools developed in this study will be useful for future studies to investigate antiviral immunity in Pacific herring.

Plastic Polymers for Efficient DNA Microarray Hybridization: Application to Microbiological Diagnostics▿

PubMed Central

Zhao, Zhengshan; Peytavi, Régis; Diaz-Quijada, Gerardo A.; Picard, Francois J.; Huletsky, Ann; Leblanc, Éric; Frenette, Johanne; Boivin, Guy; Veres, Teodor; Dumoulin, Michel M.; Bergeron, Michel G.

2008-01-01

Fabrication of microarray devices using traditional glass slides is not easily adaptable to integration into microfluidic systems. There is thus a need for the development of polymeric materials showing a high hybridization signal-to-background ratio, enabling sensitive detection of microbial pathogens. We have developed such plastic supports suitable for highly sensitive DNA microarray hybridizations. The proof of concept of this microarray technology was done through the detection of four human respiratory viruses that were amplified and labeled with a fluorescent dye via a sensitive reverse transcriptase PCR (RT-PCR) assay. The performance of the microarray hybridization with plastic supports made of PMMA [poly(methylmethacrylate)]-VSUVT or Zeonor 1060R was compared to that with high-quality glass slide microarrays by using both passive and microfluidic hybridization systems. Specific hybridization signal-to-background ratios comparable to that obtained with high-quality commercial glass slides were achieved with both polymeric substrates. Microarray hybridizations demonstrated an analytical sensitivity equivalent to approximately 100 viral genome copies per RT-PCR, which is at least 100-fold higher than the sensitivities of previously reported DNA hybridizations on plastic supports. Testing of these plastic polymers using a microfluidic microarray hybridization platform also showed results that were comparable to those with glass supports. In conclusion, PMMA-VSUVT and Zeonor 1060R are both suitable for highly sensitive microarray hybridizations. PMID:18784318

Serologic evaluation, efficacy, and safety of a commerical modified-live canine distemper vaccine in domestic ferrets.

PubMed

Wimsatt, J; Jay, M T; Innes, K E; Jessen, M; Collins, J K

2001-05-01

To determine efficacy and safety of a commercial modified-live canine distemper virus (CDV) vaccine used for prophylaxis in domestic ferrets. Sixteen 16-week-old neutered male ferrets. Equal groups of ferrets were inoculated subcutaneously at 16 and 20 weeks of age with saline (0.9% NaCl) solution or a vaccine derived from the Onderstepoort CDV strain and attenuated in a primate cell line. Live virulent CDV was administered to all ferrets intranasally and orally 3 weeks after the second inoculation. Clinical signs and body weights were monitored regularly during the study. Blood samples for serologic examination were drawn prior to each inoculation, before challenge exposure, and 10, 15, and 21 days after exposure. Blood samples for reverse transcriptase polymerase chain reaction (RT-PCR) were obtained 5 days after the first vaccination, and 5, 10, 15, and 21 days after challenge exposure. After challenge exposure, control ferrets had significantly more clinical signs and weight loss, compared with vaccinates. All vaccinated ferrets survived, whereas all control ferrets died. The RT-PCR assay was successful in detecting CDV in blood and fresh or formalin-fixed tissues from infected ferrets. Findings suggest that the vaccine when given SC to domestic ferrets as directed is safe and protective against challenge exposure with virulent CDV. The RT-PCR assay may simplify detection of CDV in fresh and fixed tissues.

Proof of Concept Study to Assess Fetal Gene Expression in Amniotic Fluid by NanoArray PCR

PubMed Central

Massingham, Lauren J.; Johnson, Kirby L.; Bianchi, Diana W.; Pei, Shermin; Peter, Inga; Cowan, Janet M.; Tantravahi, Umadevi; Morrison, Tom B.

2011-01-01

Microarray analysis of cell-free RNA in amniotic fluid (AF) supernatant has revealed differential fetal gene expression as a function of gestational age and karyotype. Once informative genes are identified, research moves to a more focused platform such as quantitative reverse transcriptase-PCR. Standardized NanoArray PCR (SNAP) is a recently developed gene profiling technology that enables the measurement of transcripts from samples containing reduced quantities or degraded nucleic acids. We used a previously developed SNAP gene panel as proof of concept to determine whether fetal functional gene expression could be ascertained from AF supernatant. RNA was extracted and converted to cDNA from 19 AF supernatant samples of euploid fetuses between 15 to 20 weeks of gestation, and transcript abundance of 21 genes was measured. Statistically significant differences in expression, as a function of advancing gestational age, were observed for 5 of 21 genes. ANXA5, GUSB, and PPIA showed decreasing gene expression over time, whereas CASC3 and ZNF264 showed increasing gene expression over time. Statistically significantly increased expression of MTOR and STAT2 was seen in female compared with male fetuses. This study demonstrates the feasibility of focused fetal gene expression analysis using SNAP technology. In the future, this technique could be optimized to examine specific genes instrumental in fetal organ system function, which could be a useful addition to prenatal care. PMID:21827969

Presence of Human Herpesvirus 6B in the Pancreas of Subjects With and Without Type 1 Diabetes.

PubMed

Ericsson, Maja; Skog, Oskar

The aims of this study were to investigate the presence of human herpesvirus 6 (HHV6) A and B in human pancreata and to search for signs of active infection in this organ of subjects with and without type 1 diabetes (T1D). Pancreata from brain-dead organ donors with and without T1D were examined for the presence of HHV6 genomic sequences by polymerase chain reaction (PCR), transcripts by reverse transcriptase-PCR, and protein by immunohistochemistry. Quantitative PCR of isolated pancreatic islets and exocrine cell clusters was used to determine the intrapancreatic location of HHV6 DNA. Human herpesvirus 6B genomic sequences were present in 1 of 2 donors who died of acute-onset T1D, 4 of 6 donors with long-standing T1D, and 9 of 12 nondiabetic donors. Higher copy numbers of HHV6B DNA were present in isolated islets than in exocrine tissue from the same donors. No signs of active HHV6 transcription were found. Human herpesvirus 6A was not present in any tested pancreas. The herein presented data demonstrate, for the first time, the presence of a latent HHV6B infection in the pancreas and islets of Langerhans. Whether this virus can contribute to disease in the pancreas remains to be determined.

Identification of the origin of faecal contamination in estuarine oysters using Bacteroidales and F-specific RNA bacteriophage markers.

PubMed

Mieszkin, S; Caprais, M P; Le Mennec, C; Le Goff, M; Edge, T A; Gourmelon, M

2013-09-01

The aim of this study was to identify the origin of faecal pollution impacting the Elorn estuary (Brittany, France) by applying microbial source tracking (MST) markers in both oysters and estuarine waters. The MST markers used were as follows: (i) human-, ruminant- and pig-associated Bacteroidales markers by real-time PCR and (ii) human genogroup II and animal genogroup I of F-specific RNA bacteriophages (FRNAPH) by culture/genotyping and by direct real-time reverse-transcriptase PCR. The higher occurrence of the human genogroup II of F-specific RNA bacteriophages using a culture/genotyping method, and human-associated Bacteroidales marker by real-time PCR, allowed the identification of human faecal contamination as the predominant source of contamination in oysters (total of 18 oyster batches tested) and waters (total of 24 water samples tested). The importance of using the intravalvular liquids instead of digestive tissues, when applying host-associated Bacteroidales markers in oysters, was also revealed. This study has shown that the application of a MST toolbox of diverse bacterial and viral methods can provide multiple lines of evidence to identify the predominant source of faecal contamination in shellfish from an estuarine environment. Application of this MST toolbox is a useful approach to understand the origin of faecal contamination in shellfish harvesting areas in an estuarine setting. © 2013 The Society for Applied Microbiology.

Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT.

PubMed

Roundhill, E; Burchill, S

2013-07-09

Primary Ewing's sarcoma family of tumours (ESFTs) may respond to chemotherapy, although many patients experience subsequent disease recurrence and relapse. The survival of ESFT cells following chemotherapy has been attributed to the development of resistant disease, possibly through the expression of ABC transporter proteins. MRP-1 and Pgp mRNA and protein expression in primary ESFTs was determined by quantitative reverse-transcriptase PCR (RT-qPCR) and immunohistochemistry, respectively, and alternative splicing of MRP-1 by RT-PCR. We observed MRP-1 protein expression in 92% (43 out of 47) of primary ESFTs, and cell membrane MRP-1 was highly predictive of both overall survival (P<0.0001) and event-free survival (P<0.0001). Alternative splicing of MRP-1 was detected in primary ESFTs, although the pattern of splicing variants was not predictive of patient outcome, with the exception of loss of exon 9 in six patients, which predicted relapse (P=0.041). Pgp protein was detected in 6% (38 out of 44) of primary ESFTs and was not associated with patient survival. For the first time we have established that cell membrane expression of MRP-1 or loss of exon 9 is predictive of outcome but not the number of splicing events or expression of Pgp, and both may be valuable factors for the stratification of patients for more intensive therapy.

Presence and Expression of Microbial Genes Regulating Soil Nitrogen Dynamics Along the Tanana River Successional Sequence

NASA Astrophysics Data System (ADS)

Boone, R. D.; Rogers, S. L.

2004-12-01

We report on work to assess the functional gene sequences for soil microbiota that control nitrogen cycle pathways along the successional sequence (willow, alder, poplar, white spruce, black spruce) on the Tanana River floodplain, Interior Alaska. Microbial DNA and mRNA were extracted from soils (0-10 cm depth) for amoA (ammonium monooxygenase), nifH (nitrogenase reductase), napA (nitrate reductase), and nirS and nirK (nitrite reductase) genes. Gene presence was determined by amplification of a conserved sequence of each gene employing sequence specific oligonucleotide primers and Polymerase Chain Reaction (PCR). Expression of the genes was measured via nested reverse transcriptase PCR amplification of the extracted mRNA. Amplified PCR products were visualized on agarose electrophoresis gels. All five successional stages show evidence for the presence and expression of microbial genes that regulate N fixation (free-living), nitrification, and nitrate reduction. We detected (1) nifH, napA, and nirK presence and amoA expression (mRNA production) for all five successional stages and (2) nirS and amoA presence and nifH, nirK, and napA expression for early successional stages (willow, alder, poplar). The results highlight that the existing body of previous process-level work has not sufficiently considered the microbial potential for a nitrate economy and free-living N fixation along the complete floodplain successional sequence.

Contamination of infectious RD-114 virus in vaccines produced using non-feline cell lines.

PubMed

Yoshikawa, Rokusuke; Sato, Eiji; Miyazawa, Takayuki

2011-01-01

All domestic cats have a replication-competent endogenous retrovirus, termed RD-114 virus, in their genome and several feline cell lines produce RD-114 viruses. Recently, we found that a portion of live attenuated feline and canine vaccines produced using feline cell lines was contaminated with infectious RD-114 viruses. In this study, we expanded our survey and examined canine vaccines produced using 'non-feline' cell lines. Consequently, we found two vaccines containing RD-114 viral RNA by reverse transcriptase (RT)-polymerase chain reaction (PCR) and real-time RT-PCR. We also confirmed the presence of infectious RD-114 virus in the vaccines by the LacZ marker rescue assay and PCR to detect proviral DNA in TE671 cells (human rhabdomyosarcoma cells) inoculated with the vaccines. It is impossible to investigate the definitive cause of contamination with RD-114 virus; however, we suspect that a seed canine parvovirus type 2 was contaminated with RD-114 virus, because many canine parvoviruses have been isolated and attenuated using feline cell lines. To exclude RD-114 virus from live attenuated vaccines, we must pay attention to the contamination of seed viruses with RD-114 virus in addition to avoiding feline cell lines producing RD-114 virus when manufacturing vaccines. Copyright © 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

3'-terminal sequence of a small round structured virus (SRSV) in Japan.

PubMed

Utagawa, E T; Takeda, N; Inouye, S; Kasuga, K; Yamazaki, S

1994-01-01

We determined the nucleotide sequence of about 1,000 bases from the 3'-terminus of a small round structured virus (SRSV), which caused a gastroenteritis outbreak in Chiba Prefecture, Japan, in 1987. The sequence was compared with the corresponding sequence region of Norwalk virus; it consisted of a part of the open reading frame 2 (ORF2), whole ORF3, and 3'-noncoding region (NCR). The 624-base-long ORF3 had sequence homology of 68% with the corresponding region of Norwalk virus. (The amino acid sequence homology was 74%.) The 94-base-long NCR had 65% homology with Norwalk virus. We then selected two consensus-sequence portions in the above sequence between Chiba and Norwalk viruses for primers in the reverse transcriptase-polymerase chain reaction (RT-PCR). Using this primer set, we detected 669-bp bands in agarose gel electrophoresis of RT-PCR products from feces containing Chiba or Norwalk viruses. Furthermore, in Southern hybridization with Chiba probes which were labeled with digoxigenin-dUTP in PCR, the bands of the two viruses were clearly stained under a low stringency condition. Since both Chiba and Norwalk viruses were detected by the above primer set although they are geographically and chronologically different viruses, our primer-pair may be useful for detection of a broad range of SRSVs which cause gastroenteritis in different areas.

Molecular detection and serotyping of infectious bronchitis virus from FTA filter paper.

PubMed

Moscoso, Hugo; Raybon, Erine O; Thayer, Stephan G; Hofacre, Charles L

2005-03-01

We investigated the feasibility of using Flinders Technology Associates (FTA) filter cards for the storage of allantoic fluid containing an infectious bronchitis virus (IBV), such as Arkansas-DPI, Connecticut, and Massachusetts, and for their identification by reverse transcriptase (RT)-polymerase chain reaction (PCR) and characterization by restriction fragment length polymorphism (RFLP) or nucleotide sequencing. FTA paper is a cotton-based cellulose membrane containing lyophilized chemicals that lyses many types of bacteria and viruses. IBV was inactivated upon contact with the FTA, as shown by the inability of the virus to be propagated in embryonating chicken eggs. RT-PCR of the S1 gene showed that viral RNA in allantoic fluid remained stable after storage on FTA filter cards and that the stability was time and temperature sensitive for the large (1700 base pair [bp]) but not the small (383 bp) PCR products. Analysis of the amplified products showed that molecular characterization is feasible in allantoic fluid stored on FTA under nonfavorable environmental conditions (41 C) for at least 15 days. The use of FTA cards for the collection, transport, and storage of IBV-containing samples is safe, inexpensive, and adequate for molecular diagnosis. We propose that specimens coming from overseas on FTA cards would be first analyzed by RT-PCR with primers yielding a 1700-bp product followed by RFLP of the positive cases. Negative cases would be analyzed with primers yielding a 383-bp product (to exdude detrimental effect of the storage conditions) followed by nucleotide sequencing of the positive cases.

The development of a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay using TaqMan technology for the pan detection of bluetongue virus (BTV).

PubMed

Mulholland, Catherine; McMenamy, Michael J; Hoffmann, Bernd; Earley, Bernadette; Markey, Bryan; Cassidy, Joseph; Allan, Gordon; Welsh, Michael D; McKillen, John

2017-07-01

Bluetongue virus (BTV) is an infectious, non-contagious viral disease of domestic and wild ruminants that is transmitted by adult females of certain Culicoides species. Since 2006, several serotypes including BTV-1, 2, 4, 6, 8, 9 and 16, have spread from the Mediterranean basin into Northern Europe for the first time. BTV-8 in particular, caused a major epidemic in northern Europe. As a result, it is evident that most European countries are at risk of BTV infection. The objective of this study was to develop and validate a real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay based on TaqMan technology for the detection of representative strains of all BTV serotypes. Primers and probes were based on genome segment 10 of the virus, the NS3 gene. The assay was tested for sensitivity, and specificity. The analytical sensitivity of the rRT-PCR assay was 200 copies of RNA per reaction. The assay did not amplify the closely related orbivirus epizootic hemorrhagic disease virus (EHDV) but successfully detected all BTV reference strains including clinical samples from animals experimentally infected with BTV-8. This real time RT-PCR assay offers a sensitive, specific and rapid alternative assay for the pan detection of BTV that could be used as part of a panel of diagnostic assays for the detection of all serotypes of BTV. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Mycophenolate mofetil prevents the delayed T cell response after pilocarpine-induced status epilepticus in mice

PubMed Central

Engelmann, Robby; Sellmann, Tina; Köhling, Rüdiger; Müller-Hilke, Brigitte

2017-01-01

Growing clinical and laboratory evidence corroborates a role for the immune system in the pathophysiology of epilepsy. In order to delineate the immune response following pilocarpine-induced status epilepticus (SE) in the mouse, we monitored the kinetics of leukocyte presence in the hippocampus over the period of four weeks. SE was induced following a ramping protocol of pilocarpine injection into 4–5 weeks old C57BL/6 mice. Brains were removed at days 1–4, 14 or 28 after SE, and the hippocampi were analyzed via flow cytometry, via quantitative reverse transcriptase PCR (qRT-PCR) and via immunohistochemistry. Epileptogenesis was confirmed by Timm staining of mossy fiber sprouting in the inner molecular layer of the dentate gyrus. The flow cytometry data revealed a biphasic immune response following pilocarpine-induced SE with a transient increase in activated CD11b+ and F4/80+ macrophages within the first four days replaced by an increase in CD3+ T-lymphocytes around day 28. This delayed T cell response was confirmed via qRT-PCR and via immunohistochemistry. In addition, qRT-PCR data could show that the delayed T cell response was associated with an increased CD8/CD4 ratio indicating a cytotoxic T cell response after SE. Intriguingly, early intervention with mycophenolate mofetil administration on days 0–3 after SE prevented this delayed T cell response. These results show an orchestrated immunological sequela and provide evidence that the delayed T cell response is sensitive to early immunomodulatory intervention. PMID:29182639

Analysis of myosin heavy chain mRNA expression by RT-PCR

NASA Technical Reports Server (NTRS)

Wright, C.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

1997-01-01

An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC) mRNA expression in rodent skeletal muscle. Only 2 microg of total RNA were necessary for the simultaneous analysis of relative mRNA expression of six different MHC genes. We designed synthetic DNA fragments as internal standards, which contained the relevant primer sequences for the adult MHC mRNAs type I, IIa, IIx, IIb as well as the embryonic and neonatal MHC mRNAs. A known amount of the synthetic fragment was added to each polymerase chain reaction (PCR) and yielded a product of different size than the amplified MHC mRNA fragment. The ratio of amplified MHC fragment to synthetic fragment allowed us to calculate percentages of the gene expression of the different MHC genes in a given muscle sample. Comparison with the traditional Northern blot analysis demonstrated that our reverse transcriptase-PCR-based assay was reliable, fast, and quantitative over a wide range of relative MHC mRNA expression in a spectrum of adult and neonatal rat skeletal muscles. Furthermore, the high sensitivity of the assay made it very useful when only small quantities of tissue were available. Statistical analysis of the signals for each MHC isoform across the analyzed samples showed a highly significant correlation between the PCR and the Northern signals as Pearson correlation coefficients ranged between 0.77 and 0.96 (P < 0.005). This assay has potential use in analyzing small muscle samples such as biopsies and samples from pre- and/or neonatal stages of development.

Comprehensive Multiplex One-Step Real-Time TaqMan qRT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses

PubMed Central

Li, Jiandong; Qu, Jing; He, Chengcheng; Zhang, Shuo; Li, Chuan; Zhang, Quanfu; Liang, Mifang; Li, Dexin

2014-01-01

Background Viral hemorrhagic fevers (VHFs) are a group of animal and human illnesses that are mostly caused by several distinct families of viruses including bunyaviruses, flaviviruses, filoviruses and arenaviruses. Although specific signs and symptoms vary by the type of VHF, initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of hemorrhagic fever viruses (HFVs) are important for effective case management and control of the spread of VHFs. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay is one of the reliable and desirable methods for specific detection and quantification of virus load. Multiplex PCR assay has the potential to produce considerable savings in time and resources in the laboratory detection. Results Primers/probe sets were designed based on appropriate specific genes for each of 28 HFVs which nearly covered all the HFVs, and identified with good specificity and sensitivity using monoplex assays. Seven groups of multiplex one-step real-time qRT-PCR assays in a universal experimental system were then developed by combining all primers/probe sets into 4-plex reactions and evaluated with serial dilutions of synthesized viral RNAs. For all the multiplex assays, no cross-reactivity with other HFVs was observed, and the limits of detection were mainly between 45 and 150 copies/PCR. The reproducibility was satisfactory, since the coefficient of variation of Ct values were all less than 5% in each dilution of synthesized viral RNAs for both intra-assays and inter-assays. Evaluation of the method with available clinical serum samples collected from HFRS patients, SFTS patients and Dengue fever patients showed high sensitivity and specificity of the related multiplex assays on the clinical specimens. Conclusions Overall, the comprehensive multiplex one-step real-time qRT-PCR assays were established in this study, and proved to be specific, sensitive, stable and easy to serve as a useful tool for rapid detection of HFVs. PMID:24752452

Detection of bovine central nervous system tissues in rendered animal by-products by one-step real-time reverse transcription PCR assay.

PubMed

Andrievskaia, Olga; Tangorra, Erin

2014-12-01

Contamination of rendered animal byproducts with central nervous system tissues (CNST) from animals with bovine spongiform encephalopathy is considered one of the vehicles of disease transmission. Removal from the animal feed chain of CNST originated from cattle of a specified age category, species-labeling of rendered meat products, and testing of rendered products for bovine CNST are tasks associated with the epidemiological control of bovine spongiform encephalopathy. A single-step TaqMan real-time reverse transcriptase (RRT) PCR assay was developed and evaluated for specific detection of bovine glial fibrillary acidic protein (GFAP) mRNA, a biomarker of bovine CNST, in rendered animal by-products. An internal amplification control, mammalian b -actin mRNA, was coamplified in the duplex RRT-PCR assay to monitor amplification efficiency, normalize amplification signals, and avoid false-negative results. The functionality of the GFAP mRNA RRT-PCR was assessed through analysis of laboratory-generated binary mixtures of bovine central nervous system (CNS) and muscle tissues treated under various thermal settings imitating industrial conditions. The assay was able to detect as low as 0.05 % (wt/wt) bovine brain tissue in binary mixtures heat treated at 110 to 130°C for 20 to 60 min. Further evaluation of the GFAP mRNA RRT-PCR assay involved samples of industrial rendered products of various species origin and composition obtained from commercial sources and rendering plants. Low amounts of bovine GFAP mRNA were detected in several bovine-rendered products, which was in agreement with declared species composition. An accurate estimation of CNS tissue content in industrial-rendered products was complicated due to a wide range of temperature and time settings in rendering protocols. Nevertheless, the GFAP mRNA RRT-PCR assay may be considered for bovine CNS tissue detection in rendered products in combination with other available tools (for example, animal age verification) in inspection programs.

Successful Validation of Sample Processing and Quantitative Real-Time PCR Capabilities on the International Space Station

NASA Technical Reports Server (NTRS)

Parra, Macarena; Jung, Jimmy; Almeida, Eduardo; Boone, Travis; Schonfeld, Julie; Tran, Luan

2016-01-01

The WetLab-2 system was developed by NASA Ames Research Center to offer new capabilities to researchers. The system can lyse cells and extract RNA (Ribonucleic Acid) on-orbit from different sample types ranging from microbial cultures to animal tissues. The purified RNA can then either be stabilized for return to Earth or can be used to conduct on-orbit quantitative Reverse Transcriptase PCR (Polymerase Chain Reaction) (qRT-PCR) analysis without the need for sample return. The qRT-PCR results can be downlinked to the ground a few hours after the completion of the run. The validation flight of the WetLab-2 system launched on SpaceX-8 on April 8, 2016. On orbit operations started on April 15th with system setup and was followed by three quantitative PCR runs using an E. coli genomic DNA template pre-loaded at three different concentrations. These runs were designed to discern if quantitative PCR functions correctly in microgravity and if the data is comparable to that from the ground control runs. The flight data showed no significant differences compared to the ground data though there was more variability in the values, this was likely due to the numerous small bubbles observed. The capability of the system to process samples and purify RNA was then validated using frozen samples prepared on the ground. The flight data for both E. coli and mouse liver clearly shows that RNA was successfully purified by our system. The E. coli qRT-PCR run showed successful singleplex, duplex and triplex capability. Data showed high variability in the resulting Cts (Cycle Thresholds [for the PCR]) likely due to bubble formation and insufficient mixing during the procedure run. The mouse liver qRT-PCR run had successful singleplex and duplex reactions and the variability was slightly better as the mixing operation was improved. The ability to purify and stabilize RNA and to conduct qRT-PCR on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. The ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. The WetLab-2 Project is supported by the Research Integration Office in the ISS Program.

Handmade Cloned Transgenic Sheep Rich in Omega-3 Fatty Acids

PubMed Central

Dou, Hongwei; Chen, Lei; Chen, Longxin; Lin, Lin; Tan, Pingping; Vajta, Gabor; Gao, Jianfeng; Du, Yutao; Ma, Runlin Z.

2013-01-01

Technology of somatic cell nuclear transfer (SCNT) has been adapted worldwide to generate transgenic animals, although the traditional procedure relies largely on instrumental micromanipulation. In this study, we used the modified handmade cloning (HMC) established in cattle and pig to produce transgenic sheep with elevated levels of omega-3 (n−3) fatty acids. Codon-optimized nematode mfat-1 was inserted into a eukaryotic expression vector and was transferred into the genome of primary ovine fibroblast cells from a male Chinese merino sheep. Reverse transcriptase PCR, gas chromatography, and chromosome analyses were performed to select nuclear donor cells capable of converting omega-6 (n−6) into n−3 fatty acids. Blastocysts developed after 7 days of in vitro culture were surgically transplanted into the uterus of female ovine recipients of a local sheep breed in Xinjiang. For the HMC, approximately 8.9% (n  = 925) of reconstructed embryos developed to the blastocyst stage. Four recipients became pregnant after 53 blastocysts were transplanted into 29 naturally cycling females, and a total of 3 live transgenic lambs were produced. Detailed analyses on one of the transgenic lambs revealed a single integration of the modified nematode mfat-1 gene at sheep chromosome 5. The transgenic sheep expressed functional n−3 fatty acid desaturase, accompanied by more than 2-folds reduction of n−6/n−3 ratio in the muscle (p<0.01) and other major organs/tissues (p<0.05). To our knowledge, this is the first report of transgenic sheep produced by the HMC. Compared to the traditional SCNT method, HMC showed an equivalent efficiency but proved cheaper and easier in operation. PMID:23437077

mRNA expression levels of hypoxia-induced and stem cell-associated genes in human glioblastoma.

PubMed

Bache, Matthias; Rot, Swetlana; Keßler, Jacqueline; Güttler, Antje; Wichmann, Henri; Greither, Thomas; Wach, Sven; Taubert, Helge; Söling, Ariane; Bilkenroth, Udo; Kappler, Matthias; Vordermark, Dirk

2015-06-01

The roles of hypoxia-induced and stem cell-associated genes in the development of malignancy and tumour progression are well known. However, there are a limited number of studies analysing the impact of mRNA expression levels of hypoxia-induced and stem cell-associated genes in the tissues of brain tumours and glioblastoma patients. In this study, tumour tissues from patients with glioblastoma multiforme and tumour adjacent tissues were analysed. We investigated mRNA expression levels of hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), carbonic anhydrase 9 (CA9), vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT-1) and osteopontin (OPN), and stem cell-associated genes survivin, epidermal growth factor receptor (EGFR), human telomerase reverse transcriptase (hTERT), Nanog and octamer binding transcription factor 4 (OCT4) using quantitative real-time polymerase chain reaction (qRT-PCR). Our data revealed higher mRNA expression levels of hypoxia-induced and stem cell-associated genes in tumour tissue than levels in the tumour adjacent tissues in patients with glioblastoma multiforme. A strong positive correlation between the mRNA expression levels of HIF-2α, CA9, VEGF, GLUT-1 and OPN suggests a specific hypoxia-associated profile of mRNA expression in glioblastoma multiforme. Additionally, the results indicate the role of stem-cell-related genes in tumour hypoxia. Kaplan-Maier analysis revealed that high mRNA expression levels of hypoxia-induced markers showed a trend towards shorter overall survival in glioblastoma patients (P=0.061). Our data suggest that mRNA expression levels of hypoxia-induced genes are important tumour markers in patients with glioblastoma multiforme.

HIV type-1 genotypic resistance profiles in vertically infected patients from Argentina reveal an association between K103N+L100I and L74V mutations.

PubMed

Aulicino, Paula C; Rocco, Carlos A; Mecikovsky, Debora; Bologna, Rosa; Mangano, Andrea; Sen, Luisa

2010-01-01

Patterns and pathways of HIV type-1 (HIV-1) antiretroviral (ARV) drug resistance-associated mutations in clinical isolates are conditioned by ARV history and factors such as viral subtype and fitness. Our aim was to analyse the frequency and association of ARV drug resistance mutations in a group of long-term vertically infected patients from Argentina. Plasma samples from 71 patients (38 children and 33 adolescents) were collected for genotypic HIV-1 ARV resistance testing during the period between February 2006 and October 2008. Statistically significant pairwise associations between ARV resistance mutations in pol, as well as associations between mutations and drug exposure, were identified using Fisher's exact tests with Bonferroni and false discovery rate corrections. Phylogenetic analyses were performed for subtype assignment. In protease (PR), resistance-associated mutations M46I/L, I54M/L/V/A/S and V82A/F/T/S/M/I were associated with each other and with minor mutations at codons 10, 24 and 71. Mutations V82A/F/T/S/M/I were primarily selected by the administration of ritonavir (RTV) in an historical ARV regimen. In reverse transcriptase, thymidine analogue mutation (TAM)1 profile was more common than TAM2. The non-nucleoside K103N+L100I mutations were observed at high frequency (15.5%) and were significantly associated with the nucleoside mutation L74V in BF recombinants. Associations of mutations at PR sites reflect the frequent use of RTV at an early time in this group of patients and convergent resistance mechanisms driven by the high exposure to protease inhibitors, as well as local HIV-1 diversity. The results provide clinical evidence of a molecular interaction between K103N+L100I and L74V mutations at the reverse transcriptase gene in vivo, limiting the future use of second-generation non-nucleoside reverse transcriptase inhibitors such as etravirine.

Molecular detection of mixed infections with multiple dengue virus serotypes in suspected dengue samples in Tamaulipas, Mexico.

PubMed

Requena-Castro, Rocío; Reyes-López, Miguel Ángel; Rodríguez-Reyna, Rosa Eminé; Palma-Nicolás, Prisco; Bocanegra-García, Virgilio

2017-07-01

This study aimed to detect dengue virus (DENV) serotypes in serum samples obtained in Matamoros Tamaulipas, Mexico, and to determine the concordance of conventional nested reverse transcriptase polymerase chain reaction (RT-PCR) and a serological test [enzyme-linked immunosorbent assay (ELISA NS1)]. Here, we detected mixed infections consisting of four serotypes of DENV. The most prevalent serotype was DENV-1, followed by DENV-4. This is the first report of DENV-4 in our region. Mixed infections were also detected in 21.5% of samples, and the predominant coinfection consisted of DENV-1 and DENV-2. Therefore, continuous epidemiological surveillance of DENV in this area is required to predict future forms of dengue heterologous infections and the effect of this on health care.

Expression of Estrogen Receptors Alpha (ER-α), Beta (ER-β), and G Protein-Coupled Receptor 30 (GPR30) in Testicular Tissue of Men with Klinefelter Syndrome.

PubMed

Bernardino, R L; Alves, M G; Silva, J; Barros, A; Ferraz, L; Sousa, M; Sá, R; Oliveira, P F

2016-06-01

Men with Klinefelter syndrome (KS) present severe hormonal dysregulation, particularly elevated serum estradiol concentration. Estrogens act through specific receptors and regulate testes development and spermatogenesis. Herein, we evaluated GPR30, ERα, and ERβ mRNA expression in testis of KS men and men with 46XY karyotype by reverse transcriptase and quantitative PCR. ERβ transcripts are the most abundant in testicular tissue of 46XY men. Notably, testicular GPR30 transcription in KS men was approximately 12 times higher. Since GPR30 is essential to mediate estrogen effects over steroidogenesis, our data illustrate that GPR30 may underpin the testicular alterations observed in KS men. © Georg Thieme Verlag KG Stuttgart · New York.

Prolonged excretion of a low-pathogenicity H5N2 avian influenza virus strain in the Pekin duck

PubMed Central

Carranza-Flores, José Manuel; Padilla-Noriega, Luis; Loza-Rubio, Elizabeth

2013-01-01

H5N2 strains of low-pathogenicity avian influenza virus (LPAIV) have been circulating for at least 17 years in some Mexican chicken farms. We measured the rate and duration of viral excretion from Pekin ducks that were experimentally inoculated with an H5N2 LPAIV that causes death in embryonated chicken eggs (A/chicken/Mexico/2007). Leghorn chickens were used as susceptible host controls. The degree of viral excretion was evaluated with real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) using samples from oropharyngeal and cloacal swabs. We observed prolonged excretion from both species of birds lasting for at least 21 days. Prolonged excretion of LPAIV A/chicken/Mexico/2007 is atypical. PMID:23820212

Cultivability of Streptococcus thermophilus in Grana Padano cheese whey starters.

PubMed

Fornasari, Maria Emanuela; Rossetti, Lia; Carminati, Domenico; Giraffa, Giorgio

2006-04-01

The application of a culture-independent approach, that of reverse transcriptase-length heterogeneity-PCR coupled with epifluorescence microscopy, allowed us to observe that Streptococcus thermophilus is metabolically active, but only partially cultivable in Grana Padano cheese whey starters. A short preincubation of the starters in sterile skimmed whey was followed by cultivation in sterile skimmed whey-enriched M17. This procedure restored the cultivability of S. thermophilus and enabled us to detect S. thermophilus at ranges (10(7)-10(8) CFU mL(-1)) which have rarely been reported in these cultures. The use of cheese whey as a cultivation-revitalization substrate can be useful to obtain an unbiased picture of the microbial composition of whey starters for Grana Padano cheese, thus avoiding an underestimation of S. thermophilus in these cultures.

An inducible HSP70 gene from the midge Chironomus dilutus: Characterization and transcription profile under environmental stress

USGS Publications Warehouse

Karouna-Renier, N. K.; Rao, K.R.

2009-01-01

In the present study, we identified and characterized an inducible heat shock protein 70 (HSP70) from the midge Chironomus dilutus and investigated the transcriptional profile of the gene under baseline and environmentally stressful conditions. Using real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), we observed increased expression of CD-HSP70-1 in response to both heat shock and copper stress. We also investigated the expression of this gene during midge development. All C. dilutus developmental stages expressed CD-HSP70-1 under normal conditions, although at extremely low levels. Phylogenetic analysis of the amino acid sequence demonstrated distinct clustering of this gene with inducible HSP70s from other insect species. ?? 2008 The Authors.

18β-Glycyrrhetinic Acid Derivatives Possessing a Trihydroxylated A Ring Are Potent Gram-Positive Antibacterial Agents.

PubMed

Huang, Li-Rong; Hao, Xiao-Jiang; Li, Qi-Ji; Wang, Dao-Ping; Zhang, Jian-Xin; Luo, Heng; Yang, Xiao-Sheng

2016-04-22

The oleanane-type triterpene 18β-glycyrrhetinic acid (1) was modified chemically through the introduction of a trihydroxylated A ring and an ester moiety at C-20 to enhance its antibacterial activity. Compounds 22, 23, 25, 28, 29, 31, and 32 showed more potent inhibitory activity against Streptomyces scabies than the positive control, streptomycin. Additionally, the inhibitory activity of the most potent compound, 29, against Bacillus subtilis, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus was greater than that of the positive controls. The antibacterial mode of action of the active derivatives involved the regulation of the expression of genes associated with peptidoglycans, the respiratory metabolism, and the inherent virulence factors found in bacteria, as determined through a quantitative real-time reverse transcriptase PCR assay.

First Imported Case of Zika Virus Infection into Korea.

PubMed

Jang, Hee-Chang; Park, Wan Beom; Kim, Uh Jin; Chun, June Young; Choi, Su-Jin; Choe, Pyoeng Gyun; Jung, Sook-In; Jee, Youngmee; Kim, Nam-Joong; Choi, Eun Hwa; Oh, Myoung-Don

2016-07-01

Since Zika virus has been spreading rapidly in the Americas from 2015, the outbreak of Zika virus infection becomes a global health emergency because it can cause neurological complications and adverse fetal outcome including microcephaly. Here, we report clinical manifestations and virus isolation findings from a case of Zika virus infection imported from Brazil. The patient, 43-year-old Korean man, developed fever, myalgia, eyeball pain, and maculopapular rash, but not neurological manifestations. Zika virus was isolated from his semen, and reverse-transcriptase PCR was positive for the virus in the blood, urine, and saliva on the 7th day of the illness but was negative on the 21st day. He recovered spontaneously without any neurological complications. He is the first case of Zika virus infection in Korea imported from Brazil.

Evaluating Fumonisin Gene Expression in Fusarium verticillioides.

PubMed

Scala, Valeria; Visentin, Ivan; Cardinale, Francesca

2017-01-01

Transcript levels of key genes in a biosynthetic pathway are often taken as a proxy for metabolite production. This is the case of FUM1, encoding the first dedicated enzyme in the metabolic pathway leading to the production of the mycotoxins Fumonisins by fungal species belonging to the genus Fusarium. FUM1 expression can be quantified by different methods; here, we detail a protocol based on quantitative reverse transcriptase polymerase chain reaction (RT-qPCR), by which relative or absolute transcript abundance can be estimated in Fusaria grown in vitro or in planta. As very seldom commercial kits for RNA extraction and cDNA synthesis are optimized for fungal samples, we developed a protocol tailored for these organisms, which stands alone but can be also easily integrated with specific reagents and kits commercially available.

Trends and advances in food analysis by real-time polymerase chain reaction.

PubMed

Salihah, Nur Thaqifah; Hossain, Mohammad Mosharraf; Lubis, Hamadah; Ahmed, Minhaz Uddin

2016-05-01

Analyses to ensure food safety and quality are more relevant now because of rapid changes in the quantity, diversity and mobility of food. Food-contamination must be determined to maintain health and up-hold laws, as well as for ethical and cultural concerns. Real-time polymerase chain reaction (RT-PCR), a rapid and inexpensive quantitative method to detect the presence of targeted DNA-segments in samples, helps in determining both accidental and intentional adulterations of foods by biological contaminants. This review presents recent developments in theory, techniques, and applications of RT-PCR in food analyses, RT-PCR addresses the limitations of traditional food analyses in terms of sensitivity, range of analytes, multiplexing ability, cost, time, and point-of-care applications. A range of targets, including species of plants or animals which are used as food ingredients, food-borne bacteria or viruses, genetically modified organisms, and allergens, even in highly processed foods can be identified by RT-PCR, even at very low concentrations. Microfluidic RT-PCR eliminates the separate sample-processing step to create opportunities for point-of-care analyses. We also cover the challenges related to using RT-PCR for food analyses, such as the need to further improve sample handling.

Tick-borne encephalitis in a naturally infected sheep.

PubMed

Böhm, Brigitte; Schade, Benjamin; Bauer, Benjamin; Hoffmann, Bernd; Hoffmann, Donata; Ziegler, Ute; Beer, Martin; Klaus, Christine; Weissenböck, Herbert; Böttcher, Jens

2017-08-22

Tick-borne encephalitis (TBE) is the most important viral tick borne zoonosis in Europe. In Germany, about 250 human cases are registered annually, with the highest incidence reported in the last years coming from the federal states Bavaria and Baden-Wuerttemberg. In veterinary medicine, only sporadic cases in wild and domestic animals have been reported; however, a high number of wild and domestic animals have tested positive for the tick-borne encephalitis virus (TBEV) antibody. In May 2015, a five-month-old lamb from a farm with 15 Merino Land sheep and offspring in Nersingen/Bavaria, a TBEV risk area, showed impaired general health with pyrexia and acute neurological signs. The sheep suffered from ataxia, torticollis, tremor, nystagmus, salivation and finally somnolence with inappetence and recumbency. After euthanasia, pathological, histopathological, immunohistochemical, bacteriological, parasitological and virological analyses were performed. Additionally, blood samples from the remaining, healthy sheep in the herd were taken for detection of TBEV antibody titres. At necropsy and accompanying parasitology, the sheep showed a moderate to severe infection with Trichostrongylids, Moniezia and Eimeria species. Histopathology revealed mild to moderate necrotising, lymphohistiocytic and granulocytic meningoencephalitis with gliosis and neuronophagia. Immunohistochemistry for TBEV was negative. RNA of a TBEV strain, closely related to the Kumlinge A52 strain, was detected in the brain by quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) and subsequent PCR product sequencing. A phylogenetic analysis revealed a close relationship to the TBEV of central Europe. TBEV was cultured from brain tissue. Serologically, one of blood samples from the other sheep in the herd was positive for TBEV in an enzyme-linked immunosorbent assay (ELISA) and in a serum neutralisation test (SNT), and one was borderline in an ELISA. To the authors' knowledge this is the first report of a natural TBEV infection in a sheep in Europe with clinical manifestation, which describes the clinical presentation and the histopathology of TBEV infection.

Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression studies.

PubMed

Yang, R; Elankumaran, Y; Hijjawi, N; Ryan, U

2015-06-01

A cell-free culture system for Cryptosporidium parvum was analysed using scanning electron microscopy (SEM) to characterise life cycle stages and compare gene expression in cell-free culture and cell culture using HCT-8 cells. Cryptosporidium parvum samples were harvested at 2 h, 8 h, 14 h, 26 h, 50 h, 74 h, 98 h, 122 h and 170 h, chemically fixed and specimens were observed using a Zeiss 1555 scanning electron microscope. The presence of sporozoites, trophozoites and type I merozoites were identified by SEM. Gene expression in cell culture and cell-free culture was studied using reverse transcriptase quantitative PCR (RT-qPCR) of the sporozoite surface antigen protein (cp15), the glycoprotein 900 (gp900), the Cryptosporidium oocyst wall protein (COWP) and 18S ribosomal RNA (rRNA) genes in both cell free and conventional cell culture. In cell culture, cp15 expression peaked at 74 h, gp900 expression peaked at 74 h and 98 h and COWP expression peaked at 50 h. In cell-free culture, CP15 expression peaked at 98 h, gp900 expression peaked at 74 h and COWP expression peaked at 122 h. The present study is the first to compare gene expression of C. parvum in cell culture and cell-free culture and to characterise life cycle stages of C. parvum in cell-free culture using SEM. Findings from this study showed that gene expression patterns in cell culture and cell-free culture were similar but in cell-free culture, gene expression was delayed for CP15 and COWP in cell free culture compared with the cell culture system and was lower. Although three life cycle stageswere conclusively identified, improvements in SEM methodology should lead to the detection of more life cycle stages. Copyright © 2015 Elsevier Inc. All rights reserved.

Porcine MuRF2 and MuRF3: molecular cloning, expression and association analysis with muscle production traits.

PubMed

Shen, H; Zhao, S H; Cao, J H; Li, X Y; Fan, B

2011-11-01

Muscle specific RING finger protein2 (MuRF2) and Muscle specific RING finger protein3(MuRF3) are two important members of the muscle specific RING finger protein family, which are especially expressed in cardiac and skeletal muscle tissues and play critical roles during the myocyte differentiation, development and morphogenesis. In this study, the molecular characteristics of porcine MuRF2 and MuRF3 gene were reported, and furthermore two variants of MuRF2 were identified. The tissue distribution pattern analyses revealed that MuRF2-b and MuRF3 mRNA was exclusively expressed in striated muscle tissues while MuRF2-a had a low-level expression in liver tissue. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) results displayed MuRF2 mRNA expression levels were significantly varied at three stages of fetal skeletal muscle in Landrace pigs, and the expression of MuRF2-a was lower than that of MuRF2-b in all stages. An essencial region of -396 to -22 for transcription was identified at the 5'UTR of porcine MuRF2 gene, while no active regulatory fragment found in the 5'UTR of mouse MuRF2. A single nucleotide polymorphism (SNP), c.915G > A was identified in MuRF2 exon 5. A HinfI PCR-RFLP was developed for SNP genotyping in two different pig populations. Association of the genotypes with growth and carcass traits showed that different genotypes of MuRF2 were significantly (P < 0.05) associated with average daily gain on test, carcass weight and carcass length. The study suggested that the porcine MuRF2 and MuRF3 genes are involved in the muscle growth and development, and can be considered as potential candidate genes affecting muscle production traits in the pig.

Levels of oxidative stress biomarkers and bone resorption regulators in apical periodontitis lesions infected by Epstein-Barr virus.

PubMed

Jakovljevic, A; Andric, M; Nikolic, N; Coric, V; Krezovic, S; Carkic, J; Knezevic, A; Beljic-Ivanovic, K; Pljesa-Ercegovac, M; Miletic, M; Soldatovic, I; Radosavljevic, T; Jovanovic, T; Simic, T; Ivanovic, V; Milasin, J

2018-06-01

To investigate whether apical periodontitis lesions infected by Epstein-Barr virus (EBV) exhibit higher levels of oxidative stress biomarkers [8-hydroxydeoxyguanosine (8-OHdG) and oxidized glutathione (GSSG)] and bone resorption regulators [receptor activator of nuclear factor (NF-κB) ligand (RANKL) and osteoprotegerin (OPG)] compared to EBV-negative periapical lesions and healthy pulp tissues. The experimental group consisted of 30 EBV-positive and 30 EBV-negative periapical lesions collected in conjunction with apicoectomy. The pulp tissues of 20 impacted third molars were used as healthy controls. The qualitative and quantitative analysis of EBV was performed by nested and real-time polymerase chain reaction (PCR), respectively. The levels of RANKL and OPG were analysed by reverse transcriptase real-time PCR. The levels of 8-OHdG and GSSG were determined by enzyme-linked immunosorbent assay (ELISA). Mann-Whitney U-test and Spearman's correlation were used for statistical analysis. The levels of RANKL, OPG, 8-OHdG and GSSG were significantly higher in apical periodontitis lesions compared to healthy pulp controls (P = 0.001, P < 0.001, P < 0.001 and P < 0.05, respectively). RANKL and OPG mRNA expression was significantly higher in EBV-positive compared to EBV-negative periapical lesions (P < 0.05). There was no significant correlation between EBV copy numbers and levels of RANKL, OPG, 8OH-dG and GSSG in apical periodontitis. Levels of bone resorption regulators and oxidative stress biomarkers were increased in apical periodontitis compared to healthy pulp tissues. EBV-positive periapical lesions exhibited higher levels of RANKL and OPG compared to EBV-negative periapical lesions. EBV may contribute to progression of apical periodontitis via enhanced production of bone resorption regulators. © 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Cloning of cDNAs for H1F0, TOP1, CLTA and CDK1 and the effects of cryopreservation on the expression of their mRNA transcripts in yak (Bos grunniens) oocytes.

PubMed

Niu, Hui-Ran; Zi, Xiang-Dong; Xiao, Xiao; Xiong, Xian-Rong; Zhong, Jin-Cheng; Li, Jian; Wang, Li; Wang, Yong

2014-08-01

We cloned and sequenced four pivotal cDNAs involved in DNA structural maintenance (H1F0 and TOP1) and the cell cycle (CLTA and CDK1) from yak oocytes. In addition, we studied the consequences of freezing-thawing (F/T) processes on the expression of their mRNA transcripts in yak immature and in vitro matured (MII) oocytes. H1F0, TOP1, CLTA and CDK1 cDNAs were cloned from yak oocytes by reverse transcriptase-polymerase chain reaction (RT-PCR) strategy. The expression of their mRNA transcript analyses were performed upon fresh and frozen-thawed immature germinal vesicle (GV) and MII yak oocytes following normalization of transcripts with GAPDH by real-time PCR. The yak H1F0, TOP1, CLTA and CDK1 cDNA sequences were found to consist of CDK1 585, 2539, 740, and 894 bp, respectively. Their coding regions encoded 195, 768, 244, and 298 amino acids, respectively. The homology with that of cattle was very high (95.2%, 98.8%, 93.6%, and 89.5%, respectively nucleotide sequence level, and 94.3%, 98.2%, 87.7%, and 90.9%, respectively at the deduced amino acid level). The overall mRNA expression levels of these four transcripts were reduced by F/T process, albeit at different levels. TOP1 in GV-oocytes, and H1F0 and CDK1 in MII-oocytes of the yak were significantly down-regulated (P<0.05). This is the first isolation and characterization of H1F0, TOP1, CLTA, and CDK1 cDNAs from yak oocytes. The lower fertility and developmental ability of yak oocytes following fertilization after cryopreservation may be explained by the alterations to their gene expression profiles. Copyright © 2014 Elsevier Inc. All rights reserved.

Evidence that acidic fibroblast growth factor promotes maturation of rat satellite-cell-derived myotubes in vitro.

PubMed

Düsterhöft, S; Pette, D

1999-11-01

Satellite cells isolated from fast tibialis anterior (TA) and slow soleus (SOL) rat muscles were cultivated on matrigel, and treated with acidic fibroblast growth factor (aFGF). The following observations were made: 1) aFGF-treated cultures exhibited enhanced proliferation as mirrored by a twofold increase in DNA content. 2) Compared to the untreated cultures, myotubes in the aFGF cultures were larger; 3) Using reverse transcriptase polymerase chain reaction (RT-PCR) and northern blot analyses, we observed enhanced expression of all adult myosin heavy chain (MHC) isoforms, as well as of myogenin. These findings indicate that, under the culture conditions used, aFGF has a stimulatory effect on proliferation but also on maturation and differentiation of satellite cells. Furthermore, transcript levels of FGF receptor 1 (FGFR1) and 4 (FGFR4) isoforms, as well as of aFGF and bFGF were assessed by RT-PCR. aFGF-treated myotubes displayed increased expression of aFGF and bFGF, suggesting a paracrine effect of exogenous aFGF. In this regard, SOL-derived cultures responded more strongly than TA-derived cultures. The effects of aFGF treatment on the two receptors consisted of a decrease in FGFR1 and an increase in FGFR4 mRNA levels in 5-day-old cultures. In 8-day-old TA cultures, effects of FGF were similar to those in 5-day-old cultures. 8-day FGF-treated SOL cultures treated with FGF for 8 days exhibited higher FGFR1 and FGFR4 mRNA levels than the respective untreated cultures. Compared to 5 day-treated cultures, FGFR1 increased and FGFR4 decreased. This led to a shift in the ratio of FGFR1 to FGFR4 in the FGF-treated cultures which may explain the ability of satellite cells to differentiate under the influence of aFGF.

Apparent intermediate K conductance channel hyposmotic activation in human lens epithelial cells.

PubMed

Lauf, Peter K; Misri, Sandeep; Chimote, Ameet A; Adragna, Norma C

2008-03-01

This study explores the nature of K fluxes in human lens epithelial cells (LECs) in hyposmotic solutions. Total ion fluxes, Na-K pump, Cl-dependent Na-K-2Cl (NKCC), K-Cl (KCC) cotransport, and K channels were determined by 85Rb uptake and cell K (Kc) by atomic absorption spectrophotometry, and cell water gravimetrically after exposure to ouabain +/- bumetanide (Na-K pump and NKCC inhibitors), and ion channel inhibitors in varying osmolalities with Na, K, or methyl-d-glucamine and Cl, sulfamate, or nitrate. Reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analyses, and immunochemistry were also performed. In isosmotic (300 mosM) media approximately 90% of the total Rb influx occurred through the Na-K pump and NKCC and approximately 10% through KCC and a residual leak. Hyposmotic media (150 mosM) decreased K(c) by a 16-fold higher K permeability and cell water, but failed to inactivate NKCC and activate KCC. Sucrose replacement or extracellular K to >57 mM, but not Rb or Cs, in hyposmotic media prevented Kc and water loss. Rb influx equaled Kc loss, both blocked by clotrimazole (IC50 approximately 25 microM) and partially by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) inhibitors of the IK channel KCa3.1 but not by other K channel or connexin hemichannel blockers. Of several anion channel blockers (dihydro-indenyl)oxy]alkanoic acid (DIOA), 4-2(butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), and phloretin totally or partially inhibited Kc loss and Rb influx, respectively. RT-PCR and immunochemistry confirmed the presence of KCa3.1 channels, aside of the KCC1, KCC2, KCC3 and KCC4 isoforms. Apparently, IK channels, possibly in parallel with volume-sensitive outwardly rectifying Cl channels, effect regulatory volume decrease in LECs.

Vector-Host Interactions Governing Epidemiology of West Nile Virus in Southern California

PubMed Central

Molaei, Goudarz; Cummings, Robert F.; Su, Tianyun; Armstrong, Philip M.; Williams, Greg A.; Cheng, Min-Lee; Webb, James P.; Andreadis, Theodore G.

2010-01-01

Southern California remains an important focus of West Nile virus (WNV) activity, with persistently elevated incidence after invasion by the virus in 2003 and subsequent amplification to epidemic levels in 2004. Eco-epidemiological studies of vectors-hosts-pathogen interactions are of paramount importance for better understanding of the transmission dynamics of WNV and other emerging mosquito-borne arboviruses. We investigated vector-host interactions and host-feeding patterns of 531 blood-engorged mosquitoes in four competent mosquito vectors by using a polymerase chain reaction (PCR) method targeting mitochondrial DNA to identify vertebrate hosts of blood-fed mosquitoes. Diagnostic testing by cell culture, real-time reverse transcriptase-PCR, and immunoassays were used to examine WNV infection in blood-fed mosquitoes, mosquito pools, dead birds, and mammals. Prevalence of WNV antibodies among wild birds was estimated by using a blocking enzyme-linked immunosorbent assay. Analyses of engorged Culex quinquefasciatus revealed that this mosquito species acquired 88.4% of the blood meals from avian and 11.6% from mammalian hosts, including humans. Similarly, Culex tarsalis fed 82% on birds and 18% on mammals. Culex erythrothorax fed on both birds (59%) and mammals (41%). In contrast, Culex stigmatosoma acquired all blood meals from avian hosts. House finches and a few other mostly passeriform birds served as the main hosts for the blood-seeking mosquitoes. Evidence of WNV infection was detected in mosquito pools, wild birds, dead birds, and mammals, including human fatalities during the study period. Our results emphasize the important role of house finches and several other passeriform birds in the maintenance and amplification of WNV in southern California, with Cx. quinquefasciatus acting as both the principal enzootic and “bridge vector” responsible for the spillover of WNV to humans. Other mosquito species, such as Cx. tarsalis and Cx. stigmatosoma, are important but less widely distributed, and also contribute to spatial and temporal transmission of WNV in southern California. PMID:21118934

Molecular detection of influenza A(H1N1)pdm09 viruses with M genes from human pandemic strains among Nigerian pigs, 2013-2015: implications and associated risk factors.

PubMed

Adeola, O A; Olugasa, B O; Emikpe, B O

2017-12-01

In the post-pandemic period, influenza A(H1N1)pdm09 virus has been detected in swine populations in different parts of the world. This study was conducted to determine the presence and spatial patterns of this human pandemic virus among Nigerian pigs and identify associated risk factors. Using a two-stage stratified random sampling method, nasal swab specimens were obtained from pigs in Ibadan, Nigeria during the 2013-2014 and 2014-2015 influenza seasons, and the virus was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Purified RT-PCR products were sequenced in both directions, and sequences were aligned using MUSCLE. Phylogenetic analysis was conducted in MEGA6. Purely spatial scan statistics and a spatial lag regression model were used to identify spatial clusters and associated risk factors. The virus was detected in both seasons, with an overall prevalence of 8·7%. Phylogenetic analyses revealed that the M genes were similar to those of pandemic strains which circulated in humans prior to and during the study. Cluster analysis revealed a significant primary spatial cluster (RR = 4·71, LLR = 5·66, P = 0·0046), while 'hours spent with pigs (R 2 = 0·90, P = 0·0018)' and 'hours spent with pigs from different farms (R 2 = 0·91, P = 0·0001)' were identified as significant risk factors (P < 0·05). These findings reveal that there is considerable risk of transmission of the pandemic virus, either directly from pig handlers or through fomites, to swine herds in Ibadan, Nigeria. Active circulation of the virus among Nigerian pigs could enhance its reassortment with endemic swine influenza viruses. Campaigns for adoption of biosecurity measures in West African piggeries and abattoirs should be introduced and sustained in order to prevent the emergence of a new influenza epicentre in the sub-region.

Reverse transcriptase genes are highly abundant and transcriptionally active in marine plankton assemblages

PubMed Central

Lescot, Magali; Hingamp, Pascal; Kojima, Kenji K; Villar, Emilie; Romac, Sarah; Veluchamy, Alaguraj; Boccara, Martine; Jaillon, Olivier; Iudicone, Daniele; Bowler, Chris; Wincker, Patrick; Claverie, Jean-Michel; Ogata, Hiroyuki

2016-01-01

Genes encoding reverse transcriptases (RTs) are found in most eukaryotes, often as a component of retrotransposons, as well as in retroviruses and in prokaryotic retroelements. We investigated the abundance, classification and transcriptional status of RTs based on Tara Oceans marine metagenomes and metatranscriptomes encompassing a wide organism size range. Our analyses revealed that RTs predominate large-size fraction metagenomes (>5 μm), where they reached a maximum of 13.5% of the total gene abundance. Metagenomic RTs were widely distributed across the phylogeny of known RTs, but many belonged to previously uncharacterized clades. Metatranscriptomic RTs showed distinct abundance patterns across samples compared with metagenomic RTs. The relative abundances of viral and bacterial RTs among identified RT sequences were higher in metatranscriptomes than in metagenomes and these sequences were detected in all metatranscriptome size fractions. Overall, these observations suggest an active proliferation of various RT-assisted elements, which could be involved in genome evolution or adaptive processes of plankton assemblage. PMID:26613339

Cost-Effectiveness of the Third-Agent Class in Treatment-Naive Human Immunodeficiency Virus-Infected Patients in Portugal

PubMed Central

Aragão, Filipa; Vera, José; Vaz Pinto, Inês

2012-01-01

Introduction Current Portuguese HIV treatment guidelines recommend initiating antiretroviral therapy with a regimen composed of two Nucleoside Reverse Transcriptase Inhibitors plus one Non-nucleoside Reverse Transcriptase Inhibitor (2NRTI+NNRTI) or two Nucleoside Reverse Transcriptase Inhibitors plus one boosted protease inhibitor (2NRTI+PI/r). Given the lower daily cost of NNRTI as the third agent when compared to the average daily costs of PI/r, it is relevant to estimate the long term impact of each treatment option in the Portuguese context. Methods We developed a microsimulation discrete events model for cost-effectiveness analysis of HIV treatment, simulating individual paths from ART initiation to death. Four driving forces determine the course of events: CD4+ cell count, viral load, resistance and adherence. Distributions of time to event are conditional to individuals’ characteristics and past history. Time to event was modeled using parametric survival analysis using Stata 11®. Disease progression was structured according to therapy lines and the model was parameterized with cohort Portuguese observational data. All resources were valued at 2009 prices. The National Health Service’s perspective was assumed considering a lifetime horizon and a 5% annual discount rate. Results In this analysis, initiating therapy with two Nucleoside Reverse Transcriptase Inhibitors plus one Non-nucleoside Reverse Transcriptase Inhibitor reduces the average number of switches by 17%, saves 19.573€ per individual and increases life expectancy by 1.7 months showing to be a dominant strategy in 57% of the simulations when compared to two Nucleoside Reverse Transcriptase Inhibitors plus one boosted protease inhibitor. Conclusion This study suggests that, when clinically valid, initiating therapy with two Nucleoside Reverse Transcriptase Inhibitors plus one Non-nucleoside Reverse Transcriptase Inhibitor is a cost-saving strategy and equally effective when compared to two Nucleoside Reverse Transcriptase Inhibitors plus one boosted protease inhibitor as the first regimen. PMID:23028618

Microgravity validation of a novel system for RNA isolation and multiplex quantitative real time PCR analysis of gene expression on the International Space Station.

PubMed

Parra, Macarena; Jung, Jimmy; Boone, Travis D; Tran, Luan; Blaber, Elizabeth A; Brown, Mark; Chin, Matthew; Chinn, Tori; Cohen, Jacob; Doebler, Robert; Hoang, Dzung; Hyde, Elizabeth; Lera, Matthew; Luzod, Louie T; Mallinson, Mark; Marcu, Oana; Mohamedaly, Youssef; Ricco, Antonio J; Rubins, Kathleen; Sgarlato, Gregory D; Talavera, Rafael O; Tong, Peter; Uribe, Eddie; Williams, Jeffrey; Wu, Diana; Yousuf, Rukhsana; Richey, Charles S; Schonfeld, Julie; Almeida, Eduardo A C

2017-01-01

The International Space Station (ISS) National Laboratory is dedicated to studying the effects of space on life and physical systems, and to developing new science and technologies for space exploration. A key aspect of achieving these goals is to operate the ISS National Lab more like an Earth-based laboratory, conducting complex end-to-end experimentation, not limited to simple microgravity exposure. Towards that end NASA developed a novel suite of molecular biology laboratory tools, reagents, and methods, named WetLab-2, uniquely designed to operate in microgravity, and to process biological samples for real-time gene expression analysis on-orbit. This includes a novel fluidic RNA Sample Preparation Module and fluid transfer devices, all-in-one lyophilized PCR assays, centrifuge, and a real-time PCR thermal cycler. Here we describe the results from the WetLab-2 validation experiments conducted in microgravity during ISS increment 47/SPX-8. Specifically, quantitative PCR was performed on a concentration series of DNA calibration standards, and Reverse Transcriptase-quantitative PCR was conducted on RNA extracted and purified on-orbit from frozen Escherichia coli and mouse liver tissue. Cycle threshold (Ct) values and PCR efficiencies obtained on-orbit from DNA standards were similar to Earth (1 g) controls. Also, on-orbit multiplex analysis of gene expression from bacterial cells and mammalian tissue RNA samples was successfully conducted in about 3 h, with data transmitted within 2 h of experiment completion. Thermal cycling in microgravity resulted in the trapping of gas bubbles inside septa cap assay tubes, causing small but measurable increases in Ct curve noise and variability. Bubble formation was successfully suppressed in a rapid follow-up on-orbit experiment using standard caps to pressurize PCR tubes and reduce gas release during heating cycles. The WetLab-2 facility now provides a novel operational on-orbit research capability for molecular biology and demonstrates the feasibility of more complex wet bench experiments in the ISS National Lab environment.

Longitudinal Detection and Persistence of Minority Drug-Resistant Populations and Their Effect on Salvage Therapy

PubMed Central

Nishizawa, Masako; Matsuda, Masakazu; Hattori, Junko; Shiino, Teiichiro; Matano, Tetsuro; Heneine, Walid; Johnson, Jeffrey A.; Sugiura, Wataru

2015-01-01

Background Drug-resistant HIV are more prevalent and persist longer than previously demonstrated by bulk sequencing due to the ability to detect low-frequency variants. To clarify a clinical benefit to monitoring minority-level drug resistance populations as a guide to select active drugs for salvage therapy, we retrospectively analyzed the dynamics of low-frequency drug-resistant population in antiretroviral (ARV)-exposed drug resistant individuals. Materials and Methods Six HIV-infected individuals treated with ARV for more than five years were analyzed. These individuals had difficulty in controlling viremia, and treatment regimens were switched multiple times guided by standard drug resistance testing using bulk sequencing. To detect minority variant populations with drug resistance, we used a highly sensitive allele-specific PCR (AS-PCR) with detection thresholds of 0.3–2%. According to ARV used in these individuals, we focused on the following seven reverse transcriptase inhibitor-resistant mutations: M41L, K65R, K70R, K103N, Y181C, M184V, and T215F/Y. Results of AS-PCR were compared with bulk sequencing data for concordance and presence of additional mutations. To clarify the genetic relationship between low-frequency and high-frequency populations, AS-PCR amplicon sequences were compared with bulk sequences in phylogenetic analysis. Results The use of AS-PCR enabled detection of the drug-resistant mutations, M41L, K103N, Y181C, M184V and T215Y, present as low-frequency populations in five of the six individuals. These drug resistant variants persisted for several years without ARV pressure. Phylogenetic analysis indicated that pre-existing K103N and T215I variants had close genetic relationships with high-frequency K103N and T215I observed during treatment. Discussion and Conclusion Our results demonstrate the long-term persistence of drug-resistant viruses in the absence of drug pressure. The rapid virologic failures with pre-existing mutant viruses detectable by AS-PCR highlight the clinical importance of low-frequency drug-resistant viruses. Thus, our results highlight the usefulness of AS-PCR and support its expanded evaluation in ART clinical management. PMID:26360259

Internalization of Murine Norovirus 1 by Lactuca sativa during Irrigation ▿

PubMed Central

Wei, Jie; Jin, Yan; Sims, Tom; Kniel, Kalmia E.

2011-01-01

Romaine lettuce (Lactuca sativa) was grown hydroponically or in soil and challenged with murine norovirus 1 (MNV) under two conditions: one mimicking a severe one-time contamination event and another mimicking a lower level of contamination occurring over time. In each condition, lettuce was challenged with MNV delivered at the roots. In the first case, contamination occurred on day one with 5 × 108 reverse transcriptase quantitative PCR (RT-qPCR) U/ml MNV in nutrient buffer, and irrigation water was replaced with virus-free buffer every day for another 4 days. In the second case, contamination with 5 × 105 RT-qPCR U/ml MNV (freshly prepared) occurred every day for 5 days. Virus had a tendency to adsorb to soil particles, with a small portion suspended in nutrient buffer; e.g., ∼8 log RT-qPCR U/g MNV was detected in soil during 5 days of challenge with virus inoculums of 5 × 108 RT-qPCR U/ml at day one, but <6 log was found in nutrient buffer on days 3 and 5. For hydroponically grown lettuce, ∼3.4 log RT-qPCR U of viral RNA/50 mg of plant tissue was detected in some lettuce leaf samples after 5 days at high MNV inoculums, significantly higher than the internalized virus concentration (∼2.6 log) at low inoculums (P < 0.05). For lettuce grown in soil, approximately 2 log RT-qPCR U of viral RNA/50 mg of plant tissue was detected in lettuce with both high and low inoculums, showing no significant difference. For viral infectivity, infectious MNV was found in lettuce samples challenged with high virus inoculums grown hydroponically and in soil but not in lettuce grown with low virus inoculums. Lettuce grown hydroponically was further incubated in 99% and 70% relative humidities (RH) to evaluate plant transpiration relative to virus uptake. More lettuce samples were found positive for MNV at a significantly higher transpiration rate at 70% RH, indicating that transpiration might play an important role in virus internalization into L. sativa. PMID:21296944

An outbreak of West Nile Virus infection in the region of Monastir, Tunisia, 2003

PubMed Central

Riabi, Samira; Gaaloul, Imed; Mastouri, Maha; Hassine, Mohsen; Aouni, Mahjoub

2014-01-01

Background A West Nile (WN) fever epidemic occurred in the region of Monastir, Tunisia, between August and October 2003. Aim of the study We attempt to describe the epidemiology, clinical presentation, and outcome of patients with confirmed West Nile virus (WNV) infection. Methods Three groups of specimens were prepared. One was made up of serum only (n  =  43), the other of cerebrospinal fluid (CSF) only (n  =  30), and the third group was made up of both (n  =  40). These specimens were obtained from 113 patients. A serological diagnosis and evidence of WNV genome by nested reverse-transcriptase polymerase chain reaction (nRT-PCR) and TaqMan reverse transcription-polymerase chain reaction (RT-PCR) were carried out. Results Thirty-eight cases (33.6%) were serologically positive. Results of nRT-PCR showed a total of 10 positive cases of WNV (8.8%) detected in group 1 (n  =  1/43), group 2 (n  =  5/30), and group 3 (n  =  4/40) whereas the PCR TaqMan showed 18 positive samples (15.9%) found in group 1 (n  =  3/43), group 2 (n  =  9/30), and group 3 (n  =  6/40). All TaqMan PCR positive cases were nRT-PCR positive. In addition, four serologically probable cases were confirmed by TaqMan PCR. The attempts to isolate WNV by cell culture were unsuccessful. Considering the results of TaqMan assay and the serological diagnosis, WNV infection was confirmed in a total of 42 patients. The main clinical presentations were meningoencephalitis (40%), febrile disease (95%), and meningitis (36%). Eight patients (19%) died. The highest case-fatality rates occurred among patients aged ≧55 years. The phylogenetic analysis revealed that isolates of WNV were closely related to the Tunisian strain 1997 (PAH001) and the Israeli one (Is-98). Conclusions West Nile virus is a reemerging global pathogen that remains an important public health challenge in the next decade. PMID:24766339

Microgravity validation of a novel system for RNA isolation and multiplex quantitative real time PCR analysis of gene expression on the International Space Station

PubMed Central

Boone, Travis D.; Tran, Luan; Blaber, Elizabeth A.; Brown, Mark; Chin, Matthew; Chinn, Tori; Cohen, Jacob; Doebler, Robert; Hoang, Dzung; Hyde, Elizabeth; Lera, Matthew; Luzod, Louie T.; Mallinson, Mark; Marcu, Oana; Mohamedaly, Youssef; Ricco, Antonio J.; Rubins, Kathleen; Sgarlato, Gregory D.; Talavera, Rafael O.; Tong, Peter; Uribe, Eddie; Williams, Jeffrey; Wu, Diana; Yousuf, Rukhsana; Richey, Charles S.; Schonfeld, Julie

2017-01-01

The International Space Station (ISS) National Laboratory is dedicated to studying the effects of space on life and physical systems, and to developing new science and technologies for space exploration. A key aspect of achieving these goals is to operate the ISS National Lab more like an Earth-based laboratory, conducting complex end-to-end experimentation, not limited to simple microgravity exposure. Towards that end NASA developed a novel suite of molecular biology laboratory tools, reagents, and methods, named WetLab-2, uniquely designed to operate in microgravity, and to process biological samples for real-time gene expression analysis on-orbit. This includes a novel fluidic RNA Sample Preparation Module and fluid transfer devices, all-in-one lyophilized PCR assays, centrifuge, and a real-time PCR thermal cycler. Here we describe the results from the WetLab-2 validation experiments conducted in microgravity during ISS increment 47/SPX-8. Specifically, quantitative PCR was performed on a concentration series of DNA calibration standards, and Reverse Transcriptase-quantitative PCR was conducted on RNA extracted and purified on-orbit from frozen Escherichia coli and mouse liver tissue. Cycle threshold (Ct) values and PCR efficiencies obtained on-orbit from DNA standards were similar to Earth (1 g) controls. Also, on-orbit multiplex analysis of gene expression from bacterial cells and mammalian tissue RNA samples was successfully conducted in about 3 h, with data transmitted within 2 h of experiment completion. Thermal cycling in microgravity resulted in the trapping of gas bubbles inside septa cap assay tubes, causing small but measurable increases in Ct curve noise and variability. Bubble formation was successfully suppressed in a rapid follow-up on-orbit experiment using standard caps to pressurize PCR tubes and reduce gas release during heating cycles. The WetLab-2 facility now provides a novel operational on-orbit research capability for molecular biology and demonstrates the feasibility of more complex wet bench experiments in the ISS National Lab environment. PMID:28877184

Coal-burning endemic fluorosis is associated with reduced activity in antioxidative enzymes and Cu/Zn-SOD gene expression.

PubMed

Wang, Qi; Cui, Kang-ping; Xu, Yuan-yuan; Gao, Yan-ling; Zhao, Jing; Li, Da-sheng; Li, Xiao-lei; Huang, Hou-jin

2014-02-01

To study the effect of fluorine on the oxidative stress in coal-burning fluorosis, we investigated the environmental characteristics of coal-burning endemic fluorosis combined with fluorine content surveillance in air, water, food, briquette, and clay binder samples from Bijie region, Guizhou Province, southwest of China. The activities of antioxidant enzymes including copper/zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and level of lipid peroxidation such as malondialdehyde (MDA) were measured in serum samples obtained from subjects residing in the Bijie region. Expression of the Cu/Zn-SOD gene was assessed by quantitative reverse transcriptase PCR (qRT-PCR). Our results showed that people suffering from endemic fluorosis (the high and low exposure groups) had much higher MDA level. Their antioxidant enzyme activities and Cu/Zn-SOD gene expression levels were lower when compared to healthy people (the control group). Fluorosis can decrease the activities of antioxidant enzymes, which was associated with exposure level of fluorine. Down-regulation of Cu/Zn-SOD expression may play an important role in the aggravation of oxidative stress in endemic fluorosis.

Transmission of porcine reproductive and respiratory syndrome virus by houseflies (Musca domestica).

PubMed

Otake, S; Dee, S A; Rossow, K D; Moon, R D; Trincado, C; Pijoan, C

2003-01-18

Three hundred houseflies were allowed to feed on donor pigs viraemic with porcine reproductive and respiratory syndrome virus (PRRSV) on the fifth, sixth and seventh days after the pigs had been inoculated with the virus. After 60 seconds, the flies' feeding was interrupted, and they were transferred manually to feed to repletion on a naive recipient pig housed in a separate room. To enhance the chance of the flies obtaining the pigs' blood, the back of each pig was scarified with sandpaper until a slight haemorrhage was visible. The PRRSV was transmitted from the donor to the recipient pigs, and PRRSV RNA was detected by reverse transcriptase-PCR from homogenates of the flies. In a second experiment, 210 houseflies were allowed to feed to repletion on a PRRSV-infected pig on the sixth day after it had been inoculated, and were then maintained under laboratory conditions. Groups of 30 flies were collected immediately after they had fed and six, 12, 24, 48, 72 and 96 hours later, and were tested for PRRSV. Homogenates of the flies collected up to six hours after feeding were PCR- and pig bioassay-positive, but the others were negative by both tests.

Identification and Characterization of a Porcine Torovirus

PubMed Central

Kroneman, A.; Cornelissen, L. A. H. M.; Horzinek, M. C.; de Groot, R. J.; Egberink, H. F.

1998-01-01

A porcine torovirus (PoTV) was identified and characterized; it is a novel member of the genus Torovirus (family Coronaviridae, order Nidovirales), closely related to but clearly distinct from the already recognized equine torovirus (ETV) and bovine torovirus (BoTV) representatives. Immunoelectron microscopy of feces from piglets revealed elongated, 120- by 55-nm particles which were recognized by a torovirus-specific antiserum. Amplification by reverse transcriptase (RT) PCR with primers designed to detect conserved regions (on the basis of the genomes of BoTV strain Breda and ETV strain Berne) resulted in the identification of the 489-bp nucleocapsid gene, encoding a 18.7-kDa protein. The sequence identity in this region between PoTV and both ETV and BoTV was only about 68%, whereas the latter two show 81% identity. Neutralizing antibodies directed against ETV were found in sera of adult and young pigs. In all 10 herds sampled, seropositive animals were present, and 81% of randomly selected adult sows possessed antibodies. A longitudinal study with RT PCR showed that piglets shed virus in the feces for 1 or more days, starting 4 to 14 days after weaning. PMID:9557628

Detection of Puumala virus in the tissue of infected naturally rodent hosts in the area of central Dinarides.

PubMed

Dervović, Edina; Hukić, Mirsada

2016-04-01

Hantaviruses are the causative agents of haemorrhagic fever with renal syndrome (HFRS) in Euroasia and of hantavirus cardiopulmonary syndrome (HCPS) in the North, Central and South America. HFRS is endemic in the Balkan Peninsula, where sporadic cases or outbreaks have been reported. Puumala virus (PUUV) is the causative agent of nephropathia epidemica (NE), a mild form of HFRS. PUUV is carried by the bank voles (Myodes glareolus). In this study, we investigated viral RNA from 76 tissues samples (lung n=30, heart n=6, liver n=18 and kidney n=22) of infected naturally rodent hosts in the area of Central Dinarides caught in live traps. Puumala virus was extracted from 34,7% (16/46) rodents by nested reverse transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR. Overall, 18 (21,4%) specimens of internal organs (kidney n=8, liver n=6, heart n=2 and lung n=2) were positive for PUUV. It was shown a high rodent infestation rate in a relatively low number of rodent and their organs, although mice were not caught during the time of high density population of host rodents. Copyright © 2016 Elsevier B.V. All rights reserved.

Tangeritin inhibits adipogenesis by down-regulating C/EBPα, C/EBPβ, and PPARγ expression in 3T3-L1 fat cells.

PubMed

He, Y F; Liu, F Y; Zhang, W X

2015-10-29

The treatment of obese patients is a topic investigated by an increasing number of researchers. This study aimed to elucidate the possible inhibitory effect of tangeritin on the development and function of fat cells. 3T3-L1 fat cells were grown to confluence and subjected to different concentrations of tangeritin. The most effective tangeritin inhibition concentration was determined by the MTT assay. The treated cells were subjected to real-time reverse transcriptase PCR and western blot analysis, to detect changes in the CCAAT/enhancer binding protein (C/EBP)α, C/EBPβ, and peroxisome proliferator activated receptor (PPAR)γ expression levels. The MTT assay revealed that the fat cell growth was inhibited at a 20 ng/mL concentration of tangeritin. The results of real-time PCR revealed a significant decrease in the expression of C/EBPα, C/EBPβ, and PPARγ mRNA, following the treatment with tangeritin. Western blot analysis also presented similar results at a protein level. Therefore, we concluded that tangeritin inhibits adipogenesis via the down-regulation of C/EBPα, C/EBPβ, and PPARγ mRNA and protein expression in 3T3-L1 cells.

Primer design for a prokaryotic differential display RT-PCR.

PubMed Central

Fislage, R; Berceanu, M; Humboldt, Y; Wendt, M; Oberender, H

1997-01-01

We have developed a primer set for a prokaryotic differential display of mRNA in the Enterobacteriaceae group. Each combination of ten 10mer and ten 11mer primers generates up to 85 bands from total Escherichia coli RNA, thus covering expressed sequences of a complete bacterial genome. Due to the lack of polyadenylation in prokaryotic RNA the type T11VN anchored oligonucleotides for the reverse transcriptase reaction had to be replaced with respect to the original method described by Liang and Pardee [ Science , 257, 967-971 (1992)]. Therefore, the sequences of both the 10mer and the new 11mer oligonucleotides were determined by a statistical evaluation of species-specific coding regions extracted from the EMBL database. The 11mer primers used for reverse transcription were selected for localization in the 3'-region of the bacterial RNA. The 10mer primers preferentially bind to the 5'-end of the RNA. None of the primers show homology to rRNA or other abundant small RNA species. Randomly sampled cDNA bands were checked for their bacterial origin either by re-amplification, cloning and sequencing or by re-amplification and direct sequencing with 10mer and 11mer primers after asymmetric PCR. PMID:9108168

Primer design for a prokaryotic differential display RT-PCR.

PubMed

Fislage, R; Berceanu, M; Humboldt, Y; Wendt, M; Oberender, H

1997-05-01

We have developed a primer set for a prokaryotic differential display of mRNA in the Enterobacteriaceae group. Each combination of ten 10mer and ten 11mer primers generates up to 85 bands from total Escherichia coli RNA, thus covering expressed sequences of a complete bacterial genome. Due to the lack of polyadenylation in prokaryotic RNA the type T11VN anchored oligonucleotides for the reverse transcriptase reaction had to be replaced with respect to the original method described by Liang and Pardee [ Science , 257, 967-971 (1992)]. Therefore, the sequences of both the 10mer and the new 11mer oligonucleotides were determined by a statistical evaluation of species-specific coding regions extracted from the EMBL database. The 11mer primers used for reverse transcription were selected for localization in the 3'-region of the bacterial RNA. The 10mer primers preferentially bind to the 5'-end of the RNA. None of the primers show homology to rRNA or other abundant small RNA species. Randomly sampled cDNA bands were checked for their bacterial origin either by re-amplification, cloning and sequencing or by re-amplification and direct sequencing with 10mer and 11mer primers after asymmetric PCR.

Prediction of Gut Wall Integrity Loss in Viral Gastroenteritis by Non-Invasive Marker

PubMed Central

Elnady, Hala G.; Sherif, Lobna S.; Saleh, Maysa T.; El-Alameey, Inas R.; Youssef, Mai M.; El Shafie, Amal I.; Helwa, Iman; Raouf, Haiam Abdel; EL-Taweel, Ahmed N.

2015-01-01

BACKGROUND: Intestinal fatty acid binding proteins (I-FABPs) are mainly expressed in the intestinal villi, which are the initial site of destruction in viral gastroenteritis. AIM: This study was designed to assess serum I-FABPs as a predictor of gut wall integrity loss in viral gastroenteritis. PATIENTS AND METHODS: This case-control cross-sectional study was conducted on 93 cases of acute viral gastroenteritis. Twenty-eight healthy children matching in age were recruited as control group. Serum I-FABPs were measured using ELISA technique. Viral detection and typing were done by PCR for adenovirus, and by Reverse transcriptase PCR for rotavirus, astrovirus and norovirus. RESULTS: Serum I-FABPs level was significantly higher in the cases compared to the controls and was also higher in the 46 rotavirus gastroenteritis cases compared to other viral gastroenteritis cases. Serum I- FABPs level was significantly higher in severely dehydrated cases as compared to mildly dehydrated ones (P=0.037). CONCLUSION: Serum I-FABPs could be used as an early and sensitive predictor marker of gut wall integrity loss in children with viral gastroenteritis and its level can indicate case severity. PMID:27275194

Coronavirus infection in intensively managed cattle with respiratory disease.

PubMed

Hick, P M; Read, A J; Lugton, I; Busfield, F; Dawood, K E; Gabor, L; Hornitzky, M; Kirkland, P D

2012-10-01

A detailed laboratory investigation identified bovine coronavirus (BCoV) as the aetiological agent in an outbreak of respiratory disease at a semi-intensive beef cattle feedlot in south-east Australia. The outbreak caused 30% morbidity in the resident population and also affected two cohorts of cattle that were newly introduced to the property. At slaughter, pulmonary consolidation and inflammatory lesions in the trachea were identified in 15 of 49 animals. Pasteurella multocida or Histophilus somni was cultured from 3 of 7 animals with lesions. Histopathological examination revealed multifocal non-suppurative bronchointerstitial pneumonia with formation of epithelial syncytial cells, sometimes associated with suppurative bronchopneumonia. BCoV was detected in nasal swabs and pulmonary lesions using real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) assay and virus isolation. There was serological evidence of previous exposure to bovine viral diarrhoea virus, bovine respiratory syncytial virus and bovine parainfluenza virus type 3, but not to bovine herpesvirus type 1. None of these viral pathogens or Mycoplasma bovis was identified by qRT-PCR. This is believed to be the first report of BCoV in association with bovine respiratory disease complex in Australia. © 2012 The Authors. Australian Veterinary Journal © 2012 Australian Veterinary Association.

Delayed vaccine virus replication in chickens vaccinated subcutaneously with an immune complex infectious bursal disease vaccine: Quantification of vaccine virus by real-time polymerase chain reaction

PubMed Central

2005-01-01

Abstract The distribution of the immune complex vaccine virus for infectious bursal disease (IBD) in tissue was examined and the viral loads of the organs were quantitatively compared. One-day-old specific pathogen free (SPF) and maternally immune broiler chickens were injected subcutaneously with the vaccine. Lymphoid and non-lymphoid tissues were collected at various time intervals during the experiment to test for infectious bursal disease virus (IBDV)-RNA by using reverse transcriptase-polymerase chain reaction (RT-PCR). Only the bursa of Fabricius was found to be positive with unusually long viral persistence in the broiler group. The positive bursa samples were further investigated by using real-time PCR coupled with a TaqMan probe. The highest amounts of the virus were detected at its first appearance in the bursa: on day 14 post vaccination (PV) in the SPF chickens and on day 17 and day 21 PV in the maternally immune broiler group. The virus then gradually cleared, most likely due to the parallel appearance of the active immune response indicated by seroconversion. PMID:15971678

The association of circulating miR-30a, miR-29 and miR-133 with white-coat hypertension.

PubMed

Huang, Yu-Qing; Huang, Cheng; Chen, Ji-Yan; Li, Jie; Feng, Ying-Qing

2016-12-01

The aim of the present study was to investigate the association of circulating miRNAs with white-coat hypertension (WCH) and further analyze whether miRNAs could be as potential biomarkers for WCH. Quantitative reverse transcriptase PCR (qRT-PCR) was used to evaluate the expression of selected miRNAs. The area under the receiver-operating characteristic curve was used to evaluate diagnostic accuracy. MiR-30a yielded an AUC of 0.984 (95% CI: 0.001-1.00; p < 0.001) and 0.816 (95% CI: 0.718-0.915; p < 0.001); miR-29 yielded an AUC of 0.955 (95% CI: 0.913-0.998; p < 0.001) and 0.799 (95% CI: 0.697-0.902; p < 0.001); miR-133 yielded an AUC of 0.949 (95% CI: 0.900-0.999; p < 0.001) and 0.713 (95% CI: 0.593-0.834; p < 0.001), respectively. The study suggested that miR-30a, miR-29 and miR-133 have great potential to be noninvasive screening tools for WCH detection.

La Crosse Encephalitis Virus Infection in Field-Collected Aedes albopictus, Aedes japonicus, and Aedes triseriatus in Tennessee.

PubMed

Westby, Katie M; Fritzen, Charissa; Paulsen, Dave; Poindexter, Stephanie; Moncayo, Abelardo C

2015-09-01

La Crosse virus (LACV) is a mosquito-borne virus and a major cause of pediatric encephalitis in the USA. La Crosse virus emerged in Tennessee and other states in the Appalachian region in 1997. We investigated LACV infection rates and seasonal abundances of the native mosquito vector, Aedes triseriatus, and 2 recently introduced mosquito species, Ae. albopictus and Ae. japonicus, in an emerging disease focus in Tennessee. Mosquitoes were collected using multiple trapping methods specific for Aedes mosquitoes at recent human case sites. Mosquito pools were tested via reverse transcriptase-polymerase chain reaction (RT-PCR) of the S segment to detect multiple Bunyamwera and California serogroup viruses, including LACV, as well as real-time RT-PCR of the M segment. A total of 54 mosquito pools were positive, including wild-caught adult females and laboratory-reared adults, demonstrating transovarial transmission in all 3 species. Maximum likelihood estimates (per 1,000 mosquitoes) were 2.72 for Ae. triseriatus, 3.01 for Ae. albopictus, and 0.63 for Ae. japonicus. We conclude that Ae. triseriatus and Ae. albopictus are important LACV vectors and that Ae. japonicus also may be involved in virus maintenance and transmission.

Stable Reference Gene Selection for RT-qPCR Analysis in Nonviruliferous and Viruliferous Frankliniella occidentalis.

PubMed

Yang, Chunxiao; Li, Hui; Pan, Huipeng; Ma, Yabin; Zhang, Deyong; Liu, Yong; Zhang, Zhanhong; Zheng, Changying; Chu, Dong

2015-01-01

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for measuring and evaluating gene expression during variable biological processes. To facilitate gene expression studies, normalization of genes of interest relative to stable reference genes is crucial. The western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), the main vector of tomato spotted wilt virus (TSWV), is a destructive invasive species. In this study, the expression profiles of 11 candidate reference genes from nonviruliferous and viruliferous F. occidentalis were investigated. Five distinct algorithms, geNorm, NormFinder, BestKeeper, the ΔCt method, and RefFinder, were used to determine the performance of these genes. geNorm, NormFinder, BestKeeper, and RefFinder identified heat shock protein 70 (HSP70), heat shock protein 60 (HSP60), elongation factor 1 α, and ribosomal protein l32 (RPL32) as the most stable reference genes, and the ΔCt method identified HSP60, HSP70, RPL32, and heat shock protein 90 as the most stable reference genes. Additionally, two reference genes were sufficient for reliable normalization in nonviruliferous and viruliferous F. occidentalis. This work provides a foundation for investigating the molecular mechanisms of TSWV and F. occidentalis interactions.

The predictive and prognostic potential of plasma telomerase reverse transcriptase (TERT) RNA in rectal cancer patients

PubMed Central

Rampazzo, Enrica; Del Bianco, Paola; Bertorelle, Roberta; Boso, Caterina; Perin, Alessandro; Spiro, Giovanna; Bergamo, Francesca; Belluco, Claudio; Buonadonna, Angela; Palazzari, Elisa; Leonardi, Sara; De Paoli, Antonino; Pucciarelli, Salvatore; De Rossi, Anita

2018-01-01

Background: Preoperative chemoradiotherapy (CRT) followed by surgery is the standard care for locally advanced rectal cancer, but tumour response to CRT and disease outcome are variable. The current study aimed to investigate the effectiveness of plasma telomerase reverse transcriptase (TERT) levels in predicting tumour response and clinical outcome. Methods: 176 rectal cancer patients were included. Plasma samples were collected at baseline (before CRT=T0), 2 weeks after CRT was initiated (T1), post-CRT and before surgery (T2), and 4–8 months after surgery (T3) time points. Plasma TERT mRNA levels and total cell-free RNA were determined using real-time PCR. Results: Plasma levels of TERT were significantly lower at T2 (P<0.0001) in responders than in non-responders. Post-CRT TERT levels and the differences between pre- and post-CRT TERT levels independently predicted tumour response, and the prediction model had an area under curve of 0.80 (95% confidence interval (CI) 0.73–0.87). Multiple analysis demonstrated that patients with detectable TERT levels at T2 and T3 time points had a risk of disease progression 2.13 (95% CI 1.10–4.11)-fold and 4.55 (95% CI 1.48–13.95)-fold higher, respectively, than those with undetectable plasma TERT levels. Conclusions: Plasma TERT levels are independent markers of tumour response and are prognostic of disease progression in rectal cancer patients who undergo neoadjuvant therapy. PMID:29449673

Zidovudine, abacavir and lamivudine increase the radiosensitivity of human esophageal squamous cancer cell lines.

PubMed

Chen, Xuan; Wang, Cong; Guan, Shanghui; Liu, Yuan; Han, Lihui; Cheng, Yufeng

2016-07-01

Telomerase is a type of reverse transcriptase that is overexpressed in almost all human tumor cells, but not in normal tissues, which provides an opportunity for radiosensitization targeting telomerase. Zidovudine, abacavir and lamivudine are reverse transcriptase inhibitors that have been applied in clinical practice for several years. We sought to explore the radiosensitization effect of these three drugs on human esophageal cancer cell lines. Eca109 and Eca9706 cells were treated with zidovudine, abacavir and lamivudine for 48 h before irradiation was administered. Samples were collected 1 h after irradiation. Clonal efficiency assay was used to evaluate the effect of the combination of these drugs with radiation doses of 2, 4, 6 and 8 Gy. DNA damage was measured by comet assay. Telomerase activity (TA) and relative telomere length (TL) were detected and evaluated by real-time PCR. Apoptosis rates were assessed by flow cytometric analysis. The results showed that all the drugs tested sensitized the esophageal squamous cell carcinoma (ESCC) cell lines to radiation through an increase in radiation-induced DNA damage and cell apoptosis, deregulation of TA and decreasing the shortened TL caused by radiation. Each of the drugs investigated (zidovudine, abacavir and lamivudine) could be used for sensitizing human esophageal cancer cell lines to radiation. Consequently, the present study supports the potential of these three drugs as therapeutic agents for the radiosensitization of esophageal squamous cell cancer.

The Reverse Transcription Inhibitor Abacavir Shows Anticancer Activity in Prostate Cancer Cell Lines

PubMed Central

Molinari, Agnese; Parisi, Chiara; Bozzuto, Giuseppina; Toccacieli, Laura; Formisano, Giuseppe; De Orsi, Daniela; Paradisi, Silvia; Grober, OlÌ Maria Victoria; Ravo, Maria; Weisz, Alessandro; Arcieri, Romano; Vella, Stefano; Gaudi, Simona

2010-01-01

Background Transposable Elements (TEs) comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1) and Human Endogenous Retroviruses (HERVs) that code for their own endogenous reverse transcriptase (RT). Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs) induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC), a nucleoside reverse transcription inhibitor (NRTI), on PC3 and LNCaP prostate cancer cell lines. Principal Findings ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. Conclusions Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications. PMID:21151977

[Mutations of resistance of HIV-1 in previously untreated patients at penitentiary centers of the Autonomous Community of Valencia, Spain. REPRICOVA study].

PubMed

García-Guerrero, Julio; Herrero, Agustín; Vera, Enrique; Almenara, José M; Araújo, Rosa; Saurí, Vicente V; Castellano, Juan C; Fernández-Clemente, Luis; Bedia, Miguel; Llorente, María I; González-Morán, Francisco

2002-03-02

Our purpose was to determine the prevalence of mutations of resistance to nucleoside inhibitors of reverse transcriptase (NIRT) and protease inhibitors (PI) in the HIV-1 genotype of naïve infected subjects in the prisons of the Autonomous Community of Valencia, Spain. Multicentric, descriptive, cross-sectional study of prevalence including a systematic stratified and randomised sampling by centres. Demographic, clinical, virological and immunological data were collected. The HIV gene of protease and transcriptase was studied in peripheral blood plasma samples by means of double PCR amplification and subsequent automatic sequence. Reference: wild strain HXB2. Plasma was obtained from 133 individuals (119 men and 14 women). 117 samples were selected and the rest did not have enough copies for transcription. With regard to NIRT, 7 samples (5.2% of total) showed some mutation of resistance: M41L, D67N, L210W and K219Q, all them secondary to and associated with resistance to zidovudine, abacavir as well as group B multinucleoside-resistance. With regard to PI, only one sample showed a primary mutation, M46I, which was associated with resistance to indinavir. Moreover, a further 41 samples were found to express some secondary mutation. In our series, there was a low number of primary mutations of resistance. These results allow us to exclude the systematic use of resistance tests before an initiation antiretroviral therapy.

WHO Collaborating Centre for Acquired Immunodeficiency Syndrome for the Eastern Mediterranean Regional Office, Faculty of Medicine, Kuwait University, Kuwait.

PubMed

Altawalah, Haya; Al-Nakib, Widad

2014-01-01

In the early 1980s, the World Health Organization (WHO) designated the Virology Unit of the Faculty of Medicine, Health Sciences Centre, Kuwait University, Kuwait, a collaborating centre for AIDS for the Eastern Mediterranean Regional Office (EMRO), recognizing it to be in compliance with WHO guidelines. In this centre, research integral to the efforts of WHO to combat AIDS is conducted. In addition to annual workshops and symposia, the centre is constantly updating and renewing its facilities and capabilities in keeping with current and latest advances in virology. As an example of the activities of the centre, the HIV-1 RNA viral load in plasma samples of HIV-1 patients is determined by real-time PCR using the AmpliPrep TaqMan HIV-1 test v2.0. HIV-1 drug resistance is determined by sequencing the reverse transcriptase and protease regions on the HIV-1 pol gene, using the TRUGENE HIV-1 Genotyping Assay on the OpenGene® DNA Sequencing System. HIV-1 subtypes are determined by sequencing the reverse transcriptase and protease regions on the HIV-1 pol gene using the genotyping assays described above. A fundamental program of Kuwait's WHO AIDS collaboration centre is the national project on the surveillance of drug resistance in human deficiency virus in Kuwait, which illustrates how the centre and its activities in Kuwait can serve the EMRO region of WHO. © 2014 S. Karger AG, Basel.

A new fluorescence/PET probe for targeting intracellular human telomerase reverse transcriptase (hTERT) using Tat peptide-conjugated IgM

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jung, Kyung oh; Biomedical Sciences, Seoul National University College of Medicine; Cancer Research Institute, Seoul National University College of Medicine

Despite an increasing need for methods to visualize intracellular proteins in vivo, the majority of antibody-based imaging methods available can only detect membrane proteins. The human telomerase reverse transcriptase (hTERT) is an intracellular target of great interest because of its high expression in several types of cancer. In this study, we developed a new probe for hTERT using the Tat peptide. An hTERT antibody (IgG or IgM) was conjugated with the Tat peptide, a fluorescence dye and {sup 64}Cu. HT29 (hTERT+) and U2OS (hTERT−) were used to visualize the intracellular hTERT. The hTERT was detected by RT-PCR and western blot. Fluorescencemore » signals for hTERT were obtained by confocal microscopy, live cell imaging, and analyzed by Tissue-FAXS. In nude mice, tumors were visualized using the fluorescence imaging devices Maestro™ and PETBOX. In RT-PCR and western blot, the expression of hTERT was detected in HT29 cells, but not in U2OS cells. Fluorescence signals were clearly observed in HT29 cells and in U2OS cells after 1 h of treatment, but signals were only detected in HT29 cells after 24 h. Confocal microscopy showed that 9.65% of U2OS and 78.54% of HT29 cells had positive hTERT signals. 3D animation images showed that the probe could target intranuclear hTERT in the nucleus. In mice models, fluorescence and PET imaging showed that hTERT in HT29 tumors could be efficiently visualized. In summary, we developed a new method to visualize intracellular and intranuclear proteins both in vitro and in vivo. - Highlights: • We developed new probes for imaging hTERT using Tat-conjugated IgM antibodies labeled with a fluorescent dye and radioisotope. • This probes could be used to overcome limitation of conventional antibody imaging system in live cell imaging. • This system could be applicable to monitor intracellular and intranuclear proteins in vitro and in vivo.« less

Transcription analysis of stx1, marA, and eaeA genes in Escherichia coli O157:H7 treated with sodium benzoate.

PubMed

Critzer, Faith J; Dsouza, Doris H; Golden, David A

2008-07-01

Expression of the multiple antibiotic resistance (mar) operon causes increased antimicrobial resistance in bacterial pathogens. The activator of this operon, MarA, can alter expression of >60 genes in Escherichia coli K-12. However, data on the expression of virulence and resistance genes when foodborne pathogens are exposed to antimicrobial agents are lacking. This study was conducted to determine transcription of marA (mar activator), stx1 (Shiga toxin 1), and eaeA (intimin) genes of E. coli O157:H7 EDL933 as affected by sodium benzoate. E. coli O157:H7 was grown in Luria-Bertani broth containing 0 (control) and 1% sodium benzoate at 37 degrees C for 24 h, and total RNA was extracted. Primers were designed for hemX (209 bp; housekeeping gene), marA (261 bp), and eaeA (223 bp) genes; previously reported primers were used for stx1. Tenfold dilutions of RNA were used in a real-time one-step reverse transcriptase PCR to determine transcription levels. All experiments were conducted in triplicate, and product detection was validated by gel electrophoresis. For marA and stx1, real-time one-step reverse transcriptase PCR products were detected at a 1-log-greater dilution in sodium benzoate-treated cells than in control cells, although cell numbers for each were similar (7.28 and 7.57 log CFU/ml, respectively). This indicates a greater (albeit slight) level of their transcription in treated cells than in control cells. No difference in expression of eaeA was observed. HemX is a putative uroporphyrinogen III methylase. The hemX gene was expressed at the same level in control and treated cells, validating hemX as an appropriate housekeeping marker. These data indicate that stx1 and marA genes could play a role in pathogen virulence and survival when treated with sodium benzoate, whereas eaeA expression is not altered. Understanding adaptations of E. coli O157:H7 during antimicrobial exposure is essential to better understand and implement methods to inhibit or control survival of this pathogen in foods.

Studying bacteria in respiratory specimens by using conventional and molecular microbiological approaches

PubMed Central

Rogers, Geraint B; Daniels, Thomas WV; Tuck, Andrew; Carroll, Mary P; Connett, Gary J; David, Gondi JP; Bruce, Kenneth D

2009-01-01

Background Drawing from previous studies, the traditional routine diagnostic microbiology evaluation of samples from chronic respiratory conditions may provide an incomplete picture of the bacteria present in airways disease. Here, the aim was to determine the extent to which routine diagnostic microbiology gave a different assessment of the species present in sputa when analysed by using culture-independent assessment. Methods Six different media used in routine diagnostic microbiology were inoculated with sputum from twelve patients. Bacterial growth on these plates was harvested and both RNA and DNA extracted. DNA and RNA were also extracted directly from the same sample of sputum. All nucleic acids served as templates for PCR and reverse transcriptase-PCR amplification of "broad range" bacterial 16S rRNA gene regions. The regions amplified were separated by Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiling and compared to assess the degree of overlap between approaches. Results A mean of 16.3 (SD 10.0) separate T-RF band lengths in the profiles from each sputum sample by Direct Molecular Analysis, with a mean of 8.8 (SD 5.8) resolved by DNA profiling and 13.3 (SD 8.0) resolved by RNA profiling. In comparison, 8.8 (SD 4.4) T-RF bands were resolved in profiles generated by Culture-derived Molecular Analysis. There were a total of 184 instances of T-RF bands detected in the direct sputum profiles but not in the corresponding culture-derived profiles, representing 83 different T-RF band lengths. Amongst these were fifteen instances where the T-RF band represented more than 10% of the total band volume (with a mean value of 23.6%). Eight different T-RF band lengths were resolved as the dominant band in profiles generated directly from sputum. Of these, only three were detected in profiles generated from the corresponding set of cultures. Conclusion Due to their focus on isolation of a small group of recognised pathogens, the use of culture-dependent methods to analyse samples from chronic respiratory infections can provide a restricted understanding of the bacterial species present. The use of a culture-independent molecular approach here identifies that there are many bacterial species in samples from CF and COPD patients that may be clinically relevant. PMID:19368727

Inactivation of human norovirus and Tulane virus in simple media and fresh whole strawberries by ionizing radiation.

PubMed

DiCaprio, Erin; Phantkankum, Nuttapong; Culbertson, Doug; Ma, Yuanmei; Hughes, John H; Kingsley, David; Uribe, Roberto M; Li, Jianrong

2016-09-02

Human norovirus (NoV) is a major cause of fresh produce-associated outbreaks and human NoV in irrigation water can potentially lead to viral internalization in fresh produce. Therefore, there is a need to develop novel intervention strategies to target internalized viral pathogens while maintaining fresh produce quality. In this study electron beam (E-beam) and gamma radiation were evaluated for efficacy against a human NoV GII.4 strain and Tulane virus (TV). Virus survival following ionizing radiation treatments was determined using direct quantitative reverse transcriptase PCR (RT-qPCR), the porcine gastric mucin magnetic bead (PGM-MB) binding assay followed by RT-qPCR, and plaque assay. In simple media, a high dose of E-beam treatment was required to completely abolish the receptor binding ability of human NoV (35.3kGy) and TV (19.5-24.1kGy), as assessed using the PGM-MB binding assay. Both human NoV and TV were more susceptible to gamma irradiation than E-beam, requiring 22.4kGy to achieve complete inactivation. In whole strawberries, no human NoV or TV RNA was detected following 28.7kGy of E-beam treatment using the PGM-MB binding assay. Overall, human NoV and TV are highly resistant to ionizing radiation and therefore the technology may not be suitable to eliminate viruses in fresh produce at the currently approved levels. In addition, the PGM-MB binding assay is an improved method to detect viral infectivity compared to direct RT-qPCR. Copyright © 2016. Published by Elsevier B.V.

Dynamic Changes of Smooth Muscle and Endothelial Markers in the Early Healing Process of Dacron Vascular Grafts in the Dog, Using RT-PCR.

PubMed

Ishida; Wu; Shi; Fujita; Sauvage; Hammond; Wijelath

2000-03-01

Previous studies of neointima formation on Dacron vascular grafts mainly focused on the late stages using immunohistochemistry staining for von Willebrand factor (vWF) and smooth muscle (SM) alpha-actin. However, it is impossible to use immunohistochemistry to study the early events of neointima formation, because graft samples lack sufficient cellular material. Therefore, we used reverse transcriptase-polymerase chain reaction (RT-PCR) to demonstrate dynamic changes of SM and endothelial markers during the early stages of neointima formation. Preclotted Dacron grafts were implanted in the descending thoracic aorta of 14 mongrel dogs. Specimens were retrieved at 1-4 weeks. Total RNAs were extracted from mid-portion of graft flow surfaces, and RT-PCR for vWF, SM myosin heavy chain (MHC), and SM alpha-actin were performed and expressed as a ratio to the ribosome s17 signal. SM MHC and vWF mRNA expression was low at 1-2 weeks but elevated at 3-4 weeks (P < 0.05). However, SM alpha-actin mRNA levels were expressed consistently throughout the study period. At 3-4 weeks, vWF mRNA expression was inversely correlated to thrombus formation on the graft flow surface. Increased expressions of SM MHC and vWF mRNA corresponded to the formation of neointima and an endothelial layer at the later stages. However, SM alpha-actin mRNA expression did not vary during the healing process. The application of RT-PCR should permit further studies of gene regulation in the early vascular graft healing process in vivo. This model can also be used to study the molecular events that are involved in SM cell differentiation.

A mammalian melanopsin in the retina of a fresh water turtle, the red-eared slider (Trachemys scripta elegans).

PubMed

Dearworth, James R; Selvarajah, Brian P; Kalman, Ross A; Lanzone, Andrew J; Goch, Abraham M; Boyd, Alison B; Goldberg, Laura A; Cooper, Lori J

2011-01-28

A mammalian-like melanopsin (Opn4m) has been found in all major vertebrate classes except reptile. Since the pupillary light reflex (PLR) of the fresh water turtle takes between 5 and 10 min to achieve maximum constriction, and since photosensitive retinal ganglion cells (ipRGCs) in mammals use Opn4m to control their slow sustained pupil responses, we hypothesized that a Opn4m homolog exists in the retina of the turtle. To identify its presence, retinal tissue was dissected from seven turtles, and total RNA extracted. Reverse transcriptase-polymerase chain reactions (RT-PCRs) were carried out to amplify gene sequences using primers targeting the highly conserved core region of Opn4m, and PCR products were analyzed by gel electrophoresis and sequenced. Sequences derived from a 1004-bp PCR product were compared to those stored in GenBank by the basic local alignment search tool (BLAST) algorithm and returned significant matches to several Opn4ms from other vertebrates including chicken. Quantitative real-time PCR (qPCR) was also carried out to compare expression levels of Opn4m in different tissues. The normalized expression level of Opn4m in the retina was higher in comparison to other tissue types: iris, liver, lung, and skeletal muscle. The results suggest that Opn4m exists in the retina of the turtle and provides a possible explanation for the presence of a slow PLR. The turtle is likely to be a useful model for further understanding the photoreceptive mechanisms in the retina which control the dynamics of the PLR. Copyright © 2010 Elsevier Ltd. All rights reserved.

Antigenicity of mesenchymal stem cells in an inflamed joint environment.

PubMed

Hill, Jacqueline A; Cassano, Jennifer M; Goodale, Margaret B; Fortier, Lisa A

2017-07-01

OBJECTIVE To determine whether major histocompatability complex (MHC) class II expression in equine mesenchymal stem cells (MSCs) changes with exposure to a proinflammatory environment reflective of an inflamed joint. SAMPLE Cryopreserved bone marrow-derived MSCs from 12 horses and cartilage and synovium samples from 1 horse euthanized for reasons other than lameness. PROCEDURES In part 1 of a 3-part study, the suitability of a quantitative reverse transcriptase PCR (qRT-PCR) assay for measurement of MHC class II expression in MSCs following stimulation with interferon (IFN)-γ was assessed. In part 2, synoviocyte-cartilage cocultures were or were not stimulated with interleukin (IL)-1β (10 ng/mL) to generate conditioned media that did and did not (control) mimic an inflamed joint environment. In part 3, a qRT-PCR assay was used to measure MSC MHC class II expression after 96 hours of incubation with 1 of 6 treatments (control-conditioned medium, IL-1β-conditioned medium, and MSC medium alone [untreated control] or with IL-1β [10 ng/mL], tumor necrosis factor-α [10 ng/mL], or IFN-γ [100 ng/mL]). RESULTS The qRT-PCR assay accurately measured MHC class II expression. Compared with MHC class II expression for MSCs exposed to the untreated control medium, that for MSCs exposed to IL-1β was decreased, whereas that for MSCs exposed to IFN-γ was increased. Neither the control-conditioned nor tumor necrosis factor-α medium altered MHC class II expression. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that MSC exposure to proinflammatory cytokine IL-1β decreased MHC class II expression and antigenicity. Treatment of inflamed joints with allogeneic MSCs might not be contraindicated, but further investigation is warranted.

Diversity, dynamics, and activity of bacterial communities during production of an artisanal Sicilian cheese as evaluated by 16S rRNA analysis.

PubMed

Randazzo, Cinzia L; Torriani, Sandra; Akkermans, Antoon D L; de Vos, Willem M; Vaughan, Elaine E

2002-04-01

The diversity and dynamics of the microbial communities during the manufacturing of Ragusano cheese, an artisanal cheese produced in Sicily (Italy), were investigated by a combination of classical and culture-independent approaches. The latter included PCR, reverse transcriptase-PCR (RT-PCR), and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes (rDNA). Bacterial and Lactobacillus group-specific primers were used to amplify the V6 to V8 and V1 to V3 regions of the 16S rRNA gene, respectively. DGGE profiles from samples taken during cheese production indicated dramatic shifts in the microbial community structure. Cloning and sequencing of rDNA amplicons revealed that mesophilic lactic acid bacteria (LAB), including species of Leuconostoc, Lactococcus lactis, and Macrococcus caseolyticus were dominant in the raw milk, while Streptococcus thermophilus prevailed during lactic fermentation. Other thermophilic LAB, especially Lactobacillus delbrueckii and Lactobacillus fermentum, also flourished during ripening. Comparison of the rRNA-derived patterns obtained by RT-PCR to the rDNA DGGE patterns indicated a substantially different degree of metabolic activity for the microbial groups detected. Identification of cultivated LAB isolates by phenotypic characterization and 16S rDNA analysis indicated a variety of species, reflecting to a large extent the results obtained from the 16S rDNA clone libraries, with the significant exception of the Lactobacillus delbrueckii species, which dominated in the ripening cheese but was not detected by cultivation. The present molecular approaches combined with culture can effectively describe the complex ecosystem of natural fermented dairy products, giving useful information for starter culture design and preservation of artisanal fermented food technology.

Molecular epidemiology of human metapneumovirus in Ireland.

PubMed

Carr, Michael J; Waters, Allison; Fenwick, Fiona; Toms, Geoffrey L; Hall, William W; O'Kelly, Edwin

2008-03-01

Human metapneumovirus (hMPV) is a cause of respiratory illness ranging from wheezing to bronchiolitis and pneumonia in children. A quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay was developed for the detection of all four main genetic lineages of hMPV and employed to validate an indirect immunofluorescence (IF) assay to detect hMPV positive specimens. The IF assay detected 24 positives from a screen of 625 randomly selected pediatric respiratory specimens collected (3.8% prevalence). From this cohort of 625 specimens, 229 were also tested by real-time RT-PCR assay. This included the 24 IF positive specimens and 205 randomly selected specimens from both study periods. In addition to confirming all the IF positives, the real-time assay detected an additional six hMPV positive specimens giving rise to a combined prevalence of 4.8%. Phylogenetic analysis showed that hMPV subtypes A2b and B2 to be the most prevalent genotypes circulating in our population and surprisingly no hMPV subgroups A1 or B1 were detected during this study period. Based on this phylogenetic analysis, we propose the existence of sub-clusters of hMPV genotype B2 present in our population which we term subtypes B2a and B2b. The mean log 10 copies/ml of quantitative RT-PCR determinations from these 30 hMPV positive respiratory specimens was 6.35 (range = 4.44-8.15). Statistical analysis of quantitative RT-PCR determinations of viral load from these 30 respiratory specimens suggests that hMPV genotype B specimens have a higher viral load than hMPV genotype A isolates (P < 0.03).

Presence and distribution of leptin and leptin receptor in the canine gallbladder.

PubMed

Lee, Sungin; Lee, Aeri; Kweon, Oh-Kyeong; Kim, Wan Hee

2016-09-01

The hormone leptin is produced by mature adipocytes and plays an important role in regulating food intake and energy metabolism through its interaction with the leptin receptor. In addition to roles in obesity and obesity-related diseases, leptin has been reported to affect the components and secretion of bile in leptin-deficient mice. Furthermore, gallbladder diseases such as cholelithiasis are known to be associated with serum leptin concentrations in humans. We hypothesized that the canine gallbladder is a source of leptin and that the leptin receptor may be localized in the gallbladder, where it plays a role in regulating the function of this organ. The aim of this study was to demonstrate the presence and expression patterns of leptin and its receptors in normal canine gallbladders using reverse transcriptase-PCR (RT-PCR) and immunohistochemistry. Clinically normal gallbladder tissue samples were obtained from four healthy beagle dogs with similar body condition scores. RT-PCR and sequencing of the amplified PCR products revealed the presence of leptin mRNA and its receptors in the gallbladder. Immunohistochemical investigations demonstrated the expression of leptin and its receptors in the luminal single columnar and tubuloalveolar glandular epithelial cells. In conclusion, the results of this study demonstrated the presence of leptin and its receptors in the gallbladders of dogs. Leptin and its receptor were both localized throughout the cytoplasm of luminal and glandular epithelial cells. These results suggested that the gallbladder is not only a source of leptin, but also a target of leptin though autocrine/paracrine mechanisms. The results of this study could increase the understanding of both the normal physiological functions of the gallbladder and the pathophysiological mechanisms of gallbladder diseases characterized by leptin system dysfunction. Copyright © 2016 The Authors. Published by Elsevier GmbH.. All rights reserved.

Nitrate reduction mechanisms and rates in an unconfined eogenetic karst aquifer in two sites with different redox potential

NASA Astrophysics Data System (ADS)

Henson, W. R.; Huang, L.; Graham, W. D.; Ogram, A.

2017-05-01

This study integrates push-pull tracer tests (PPTT) with microbial characterization of extracted water via quantitative polymerase chain reaction (qPCR) and reverse transcriptase qPCR (RT-qPCR) of selected functional N transformation genes to quantify nitrate reduction mechanisms and rates in sites with different redox potential in a karst aquifer. PPTT treatments with nitrate (AN) and nitrate-fumarate (ANC) were executed in two wells representing anoxic and oxic geochemical end-members. Oxic aquifer zero-order nitrate loss rates (mmol L-1 h-1) were similar for AN and ANC treatment, ranging from 0.03 ± 0.01 to 0.05 ± 0.01. Anoxic aquifer zero-order nitrate loss rates ranged from 0.03 ± 0.02 (AN) to 0.13 ± 0.02 (ANC). Microbial characterization indicates mechanisms influencing these rates were dissimilatory nitrate reduction to ammonium (DNRA) at the anoxic site with AN treatment, assimilatory reduction of nitrate to ammonium (ANRA) with ANC treatment in the water column at both sites, and additional documented nitrate reduction that occurred in unsampled biofilms. With carbon treatment, total numbers of microbes (16S rRNA genes) significantly increased (fourteenfold to thirtyfold), supporting stimulated growth with resulting ANRA. Decreased DNRA gene concentrations (nrfA DNA) and increased DNRA activity ratio (nrfA-cDNA/DNA) supported the assertion that DNRA occurred in the anoxic zone with AN and ANC treatment. Furthermore, decreased DNRA gene copy numbers at the anoxic site with ANC treatment suggests that DNRA microbes in the anoxic site are chemolithoautotrophic. Increased RT-qPCR denitrification gene expression (nirK and nirS) was not observed in water samples, supporting that any observed NO3-N loss due to denitrification may be occurring in unsampled microbial biofilms.

Measurement of indicator genes using global complementary DNA (cDNA) amplification, by polyadenylic acid reverse transcriptase polymerase chain reaction (poly A RT-PCR): A feasibility study using paired samples from tissue and ductal juice in patients undergoing pancreatoduodenectomy.

PubMed

Sanyal, Sudip; Siriwardena, Ajith K; Byers, Richard

2018-06-01

The aim of this study is to compare gene expression profiles in RNA isolated from pancreatic ductal juice with the RNA expression profiles of the same genes from matched intra-operative tissue samples from pancreatic tumours. Intra-operative sampling of pancreatic juice and collection of matched tissue samples was undertaken in patients undergoing pancreatoduodenectomy for clinically suspected pancreatic cancer and a precursor lesion, main-duct intraductal papillary mucinous neoplasm. RNA was isolated and Poly A PCR was used to globally amplify the RNA. Real-time polymerase chain reaction (RT-PCR) was used to measure expression levels of 17 genes selected from microarray studies. Spearman's rank correlation test was used to examine the relationship of gene expression between pancreatic juice and tissue. The study was approved by Regional Ethics Committee. Mesothelin (MSLN) showed significant correlation (p < 0.008) in expression levels between paired pancreatic juice and tissue samples in pancreas cancer. In intraductal papillary mucinous neoplasms (IPMN), Matrix Metalloproteinase 7 (MMP7), showed significant correlation (p < 0.01) in the expression levels between paired pancreatic juice and tissue samples. This study confirms that RNA analysis of paired pancreatic juice and tissue samples and establishment of cDNA using poly A PCR is technically feasible. Application of the technique to non-invasively obtained pancreatic juice during endoscopic assessment of tumours and the use of gene arrays of cancer indicator genes are the next steps in development of this technique. Copyright © 2018 IAP and EPC. Published by Elsevier B.V. All rights reserved.

Epidemiological and virological characteristics of seasonal and pandemic influenza in Lao PDR, 2008–2010

PubMed Central

Khamphaphongphane, Bouaphanh; Ketmayoon, Pakapak; Lewis, Hannah C.; Phonekeo, Darouny; Sisouk, Thongchanh; Xayadeth, Sinakhone; Ongkhammy, Somvay; Vongphrachanh, Phengta; Tsuyuoka, Reiko; Moen, Ann; Corwin, Andrew

2012-01-01

Background  Information on influenza virology and epidemiology from Lao PDR is limited and the seasonal patterns of influenza have not been previously described. Objectives  To describe epidemiological and virologic characteristics of influenza in Lao PDR to recommend public health interventions, including improvements in surveillance and response. Patients/Methods  We performed a descriptive analysis of samples taken from patients with influenza‐like‐illness (ILI) (fever >38°C with cough and/or sore throat) presenting at seven sentinel hospitals in three regions of Lao PDR, January 2008–December 2010. A nasopharyngeal (NP) swab or combined nasal with oropharyngeal swab was collected from patients with ILI. Samples were tested for influenza by either Luminex RVP, conventional reverse transcriptase PCR (RT‐PCR) (January 2008–2009), or by real‐time PCR (rRT‐PCR) using US CDC reagents (February 2009 onward). Results  Of 2346 samples tested from patients with ILI, 523 (22%) were positive for influenza. The median age of those positive was 12 years (range, <1–60 year). The percentage of samples that were influenza positive was similar over the 3 years (20–23%). Each year 3–4 types/subtypes cocirculated with differing predominant type/subtype. Influenza was detected year‐round with the highest proportion of positive specimens in the 3rd and 4th quarter. Conclusions  Similar to other countries in the region, we found that influenza is present year‐round and has a peak activity from July to December. Dominant types or subtypes vary by year. A large proportion of patients with ILI are not influenza positive. ILI surveillance is critical for weighing disease burden, both morbidity and mortality, against the costs of advancing influenza vaccine delivery strategy. PMID:22716289

Comprehensive analysis of the MYB-NFIB gene fusion in salivary adenoid cystic carcinoma: Incidence, variability, and clinicopathologic significance.

PubMed

Mitani, Yoshitsugu; Li, Jie; Rao, Pulivarthi H; Zhao, Yi-Jue; Bell, Diana; Lippman, Scott M; Weber, Randal S; Caulin, Carlos; El-Naggar, Adel K

2010-10-01

The objectives of this study were to determine the incidence of the MYB-NFIB fusion in salivary adenoid cystic carcinoma (ACC), to establish the clinicopathologic significance of the fusion, and to analyze the expression of MYB in ACCs in the context of the MYB-NFIB fusion. We did an extensive analysis involving 123 cancers of the salivary gland, including primary and metastatic ACCs, and non-ACC salivary carcinomas. MYB-NFIB fusions were identified by reverse transcriptase-PCR (RT-PCR) and sequencing of the RT-PCR products, and confirmed by fluorescence in situ hybridization. MYB RNA expression was determined by quantitative RT-PCR and protein expression was analyzed by immunohistochemistry. The MYB-NFIB fusion was detected in 28% primary and 35% metastatic ACCs, but not in any of the non-ACC salivary carcinomas analyzed. Different exons in both the MYB and NFIB genes were involved in the fusions, resulting in expression of multiple chimeric variants. Notably, MYB was overexpressed in the vast majority of the ACCs, although MYB expression was significantly higher in tumors carrying the MYB-NFIB fusion. The presence of the MYB-NFIB fusion was significantly associated (P = 0.03) with patients older than 50 years of age. No correlation with other clinicopathologic markers, factors, and survival was found. We conclude that the MYB-NFIB fusion characterizes a subset of ACCs and contributes to MYB overexpression. Additional mechanisms may be involved in MYB overexpression in ACCs lacking the MYB-NFIB fusion. These findings suggest that MYB may be a specific novel target for tumor intervention in patients with ACC. ©2010 AACR.

Clinical characterization of acute and convalescent illness of confirmed chikungunya cases from Chiapas, S. Mexico: A cross sectional study

PubMed Central

Trujillo-Murillo, Karina del Carmen; Caballero-Sosa, Sandra; Sepúlveda-Delgado, Jesús; Malo-García, Iliana Rosalía; Canseco-Ávila, Luis Miguel; Salgado-Corsantes, Luis Manuel; Domínguez-Arrevillaga, Sergio; Torres-Zapata, Raúl; Gómez-Cruz, Omar; Fernández-Salas, Ildefonso

2017-01-01

Background The emerging chikungunya virus (CHIKV), is an arbovirus causing intense outbreaks in North America. The situation in Mexico is alarming, and CHIKV threatens to spread further throughout North America. Clinical and biological features of CHIKF outbreaks in Mexico have not been well described; thus, we conducted a cross sectional study of a CHIKV outbreak in Chiapas, Southern Mexico to further characterize these features. Methodology/Principal findings We collected blood samples from patients suspected of having chikungunya fever (CHIKF) who presented to Clinical Hospital ISSSTE Dr. Roberto Nettel in Tapachula, Chiapas, Mexico. In addition to the clinical examination, real-time polymerase chain reaction (PCR) standardized for the Asian Chikungunya lineage and/or enzyme-linked immunosorbent assay for immunoglobulin M (IgM) were used to confirm CHIKV diagnosis. Of a total of 850 patients who presented with probably CHIKV at Hospital “Dr. Roberto Nettel”, 112 probable CHIKF cases were enrolled in this study from November 2014- June 2015, of which 95 patients (84.8%) were CHIKV positive and 17 were negative (15.2%). Of these 95 CHIKV positive patients, 62 were positive by real-time reverse transcriptase PCR (+qRT-PCR); and 33 were seropositive to +IgM with a negative qRT-PCR. The most frequent symptoms reported were fever (100%), headache (82.3%), polyarthralgia (72.1%), and exanthem (82.3%). Biological abnormalities observed during CHIKV infection were lymphopenia (41.1%), leukopenia (51.6%), elevated transaminases (30.5%-46.3%) and high LDH (46.3%) and CRP (60.0%). Conclusion Clinical and biological data obtained from this study is providing more useful information for benchmarking purposes with outbreaks from different parts of the world and would be helpful for better patient care and treatment. PMID:29065182

RT-PCR standardization and bone mineralization after low-level laser therapy on adult osteoblast cells

NASA Astrophysics Data System (ADS)

do Bomfim, Fernando R. C.; Sella, Valéria R. G.; Zanaga, Jéssica Q.; Pereira, Nayara S.; Nouailhetas, Viviane L. A.; Plapler, Hélio

2014-03-01

Purpose: Osteoblasts are capable to produce different compounds directly connected to bone mineralization process. This study aims to standardize the reverse transcriptase polymerase chain reaction (RT-PCR) for adult osteoblasts to observe the effect of low level laser therapy on bone mineralization. Methods: Five-millimeter long fragments obtained from the mead femoral region of male Wistar rats were assigned into group A (n=10, laser) and group B (n=10, no laser), submitted to mechanic and enzymatic digestion. After 7 days, cultures of group A were irradiated daily on a single spot with a GaInAs laser, λ=808nm, 200mW/cm2, 2J/cm2, bean diameter of 0,02mm, 5 seconds for 6 days. Group B was manipulated but received no laser irradiation. After 13 days the cells were trypsinized for 15 minute and stabilized with RNA later® for RNA extraction with Trizol®. cDNA synthesis used 10μg of RNA and M-MLV® enzyme. PCR was accomplished using the β-actin gene as a control. Another aliquot was fixed for Hematoxylin-Eosin and Von Kossa staining to visualize bone mineralization areas. Results: Under UV light we observed clearly the amplification of β-actin gene around 400bp. HE and Von Kossa staining showed osteoblast clusters, a higher number of bone cells and well defined mineralization areas in group A. Conclusion: The cell culture, RNA extraction and RT-PCR method for adult osteoblasts was effective, allowing to use these methods for bone mineralization studies. Laser improved bone mineralization and further studies are needed involving osteogenesis, calcium release mechanisms and calcium related channels.

RT-PCR amplification of RNA extracted from formalin-fixed, paraffin-embedded oral cancer sections: analysis of p53 pathway.

PubMed

Tachibana, Masatsugu; Shinagawa, Yasuhiro; Kawamata, Hitoshi; Omotehara, Fumie; Horiuchi, Hideki; Ohkura, Yasuo; Kubota, Keiichi; Imai, Yutaka; Fujibayashi, Takashi; Fujimori, Takahiro

2003-01-01

We present a new approach towards the detection of the mRNAs in formalin-fixed, paraffin-embedded samples using a reverse transcriptase (RT)-polymerase chain reaction (PCR). The total RNAs were extracted from 10-micron-thick sections and were reverse-transcribed, then the RT-products were subjected to PCR amplification of GAPDH mRNA for screening the mRNA degradation. Next, nested PCR was performed for examining the expression of p53-related genes, p21WAF1, MDM2, p33ING1 and p14ARF. GAPDH mRNA expression was detectable in 12 out of 21 oral squamous cell carcinoma (SCC) samples. p21WAF1 mRNA expression was detectable in 5 out of 12 SCC samples, MDM2 mRNA expression was detectable in 5 our of 12 SCC samples and p33ING1 mRNA expression was detectable in 6 out of 12 SCC samples. However, the expression of p14ARF mRNA was not detectable in any of the samples. Seven out of 12 oral SCC samples showed abnormal nuclear accumulation of p53 protein by immunohistochemical staining, whereas 5 out of 12 oral SCCs showed negative staining for p53 protein. Of of p33ING1 mRNA. One of these was a verrucous carcinoma in which the p53 gene products might be inactivated by the oncoprotein E6 of human papilloma virus. Thus, the p53 tumor suppressor pathway was disrupted in most oral SCCs at the cellular levels, due to either an abnormality in p53 itself or loss of expression of p53 regulatory factors. This method would assist in making diagnosis, determining therapeutic strategy and predicting the prognosis of various cancers including oral SCCs.

Evaluation of reference genes in mouse preimplantation embryos for gene expression studies using real-time quantitative RT-PCR (RT-qPCR).

PubMed

Jeong, Jae-Kyo; Kang, Min-Hee; Gurunathan, Sangiliyandi; Cho, Ssang-Goo; Park, Chankyu; Seo, Han Geuk; Kim, Jin-Hoi

2014-09-25

Real-time quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) is the most sensitive, and valuable technique for rare mRNA detection. However, the expression profiles of reference genes under different experimental conditions, such as different mouse strains, developmental stage, and culture conditions have been poorly studied. mRNA stability of the actb, gapdh, sdha, ablim, ywhaz, sptbn, h2afz, tgfb1, 18 s and wrnip genes was analyzed. Using the NormFinder program, the most stable genes are as follows: h2afz for the B6D2F-1 and C57BL/6 strains; sptbn for ICR; h2afz for KOSOM and CZB cultures of B6D2F-1 and C57BL/6 strain-derived embryos; wrnip for M16 culture of B6D2F-1 and C57BL/6 strain-derived embryos; ywhaz, tgfb1, 18 s, 18 s, ywhaz, and h2afz for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst embryonic stages cultured in KSOM medium, respectively; h2afz, wrnip, wrnip, h2afz, ywhaz, and ablim for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst stage embryos cultured in CZB medium, respectively; 18 s, h2afz, h2afz, actb, h2afz, and wrnip for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst stage embryos cultured in M16 medium, respectively. These results demonstrated that candidate reference genes for normalization of target gene expression using RT-qPCR should be selected according to mouse strains, developmental stage, and culture conditions.

T lymphocytes among HIV-infected and -uninfected infants: CD4/CD8 ratio as a potential tool in diagnosis of infection in infants under the age of 2 years.

PubMed

Zijenah, Lynn S; Katzenstein, David A; Nathoo, Kusum J; Rusakaniko, Simbarashe; Tobaiwa, Ocean; Gwanzura, Christine; Bikoue, Arsene; Nhembe, Margaret; Matibe, Petronella; Janossy, George

2005-02-01

BACKGROUND: Serologic tests for HIV infection in infants less than 18 months do not differentiate exposure and infection since maternally acquired IgG antibodies may be detected in infants. Thus, the gold standard for diagnosis of HIV-1 infection in infants under the age of 2 years is DNA or reverse transcriptase polymerase chain reaction. There is an urgent need to evaluate alternative and cost effective laboratory methods for early diagnosis of infant HIV-1 infection as well as identifying infected infants who may benefit from cotrimoxazole prophylaxis and/or initiation of highly active antiretroviral therapy. METHODS: Whole blood was collected in EDTA from 137 infants aged 0 to 18 months. DNA polymerase chain reaction was used as the reference standard for diagnosis of HIV-1 infection. T-cell subset profiles were determined by flow cytometry. RESULTS: Seventy-six infants were DNA PCR positive while 61 were negative. The median CD4 counts of PCR negative infants were significantly higher than those of the PCR positive infants, p < 0.001. The median CD4/CD8 ratio and the %CD4 of the PCR positive infants were both significantly lower than those of the negative infants, p < 0.001. The CD4/CD8 ratio had a >98% sensitivity for diagnosis of HIV-1 infection and a specificity of >98%. CONCLUSION: The CD4/CD8 ratio appears useful in identifying HIV-infected infants. The development of lower cost and more robust flow cytometric methods that provide both CD4/CD8 ratio and %CD4 may be cost-effective for HIV-1 diagnosis and identification of infants for cotrimoxazole prophylaxis and/or highly active antiretroviral therapy.

T lymphocytes among HIV-infected and -uninfected infants: CD4/CD8 ratio as a potential tool in diagnosis of infection in infants under the age of 2 years

PubMed Central

Zijenah, Lynn S; Katzenstein, David A; Nathoo, Kusum J; Rusakaniko, Simbarashe; Tobaiwa, Ocean; Gwanzura, Christine; Bikoue, Arsene; Nhembe, Margaret; Matibe, Petronella; Janossy, George

2005-01-01

Background Serologic tests for HIV infection in infants less than 18 months do not differentiate exposure and infection since maternally acquired IgG antibodies may be detected in infants. Thus, the gold standard for diagnosis of HIV-1 infection in infants under the age of 2 years is DNA or reverse transcriptase polymerase chain reaction. There is an urgent need to evaluate alternative and cost effective laboratory methods for early diagnosis of infant HIV-1 infection as well as identifying infected infants who may benefit from cotrimoxazole prophylaxis and/or initiation of highly active antiretroviral therapy. Methods Whole blood was collected in EDTA from 137 infants aged 0 to 18 months. DNA polymerase chain reaction was used as the reference standard for diagnosis of HIV-1 infection. T-cell subset profiles were determined by flow cytometry. Results Seventy-six infants were DNA PCR positive while 61 were negative. The median CD4 counts of PCR negative infants were significantly higher than those of the PCR positive infants, p < 0.001. The median CD4/CD8 ratio and the %CD4 of the PCR positive infants were both significantly lower than those of the negative infants, p < 0.001. The CD4/CD8 ratio had a >98% sensitivity for diagnosis of HIV-1 infection and a specificity of >98%. Conclusion The CD4/CD8 ratio appears useful in identifying HIV-infected infants. The development of lower cost and more robust flow cytometric methods that provide both CD4/CD8 ratio and %CD4 may be cost-effective for HIV-1 diagnosis and identification of infants for cotrimoxazole prophylaxis and/or highly active antiretroviral therapy. PMID:15683549

Clinical characterization of acute and convalescent illness of confirmed chikungunya cases from Chiapas, S. Mexico: A cross sectional study.

PubMed

Danis-Lozano, Rogelio; Díaz-González, Esteban Eduardo; Trujillo-Murillo, Karina Del Carmen; Caballero-Sosa, Sandra; Sepúlveda-Delgado, Jesús; Malo-García, Iliana Rosalía; Canseco-Ávila, Luis Miguel; Salgado-Corsantes, Luis Manuel; Domínguez-Arrevillaga, Sergio; Torres-Zapata, Raúl; Gómez-Cruz, Omar; Fernández-Salas, Ildefonso

2017-01-01

The emerging chikungunya virus (CHIKV), is an arbovirus causing intense outbreaks in North America. The situation in Mexico is alarming, and CHIKV threatens to spread further throughout North America. Clinical and biological features of CHIKF outbreaks in Mexico have not been well described; thus, we conducted a cross sectional study of a CHIKV outbreak in Chiapas, Southern Mexico to further characterize these features. We collected blood samples from patients suspected of having chikungunya fever (CHIKF) who presented to Clinical Hospital ISSSTE Dr. Roberto Nettel in Tapachula, Chiapas, Mexico. In addition to the clinical examination, real-time polymerase chain reaction (PCR) standardized for the Asian Chikungunya lineage and/or enzyme-linked immunosorbent assay for immunoglobulin M (IgM) were used to confirm CHIKV diagnosis. Of a total of 850 patients who presented with probably CHIKV at Hospital "Dr. Roberto Nettel", 112 probable CHIKF cases were enrolled in this study from November 2014- June 2015, of which 95 patients (84.8%) were CHIKV positive and 17 were negative (15.2%). Of these 95 CHIKV positive patients, 62 were positive by real-time reverse transcriptase PCR (+qRT-PCR); and 33 were seropositive to +IgM with a negative qRT-PCR. The most frequent symptoms reported were fever (100%), headache (82.3%), polyarthralgia (72.1%), and exanthem (82.3%). Biological abnormalities observed during CHIKV infection were lymphopenia (41.1%), leukopenia (51.6%), elevated transaminases (30.5%-46.3%) and high LDH (46.3%) and CRP (60.0%). Clinical and biological data obtained from this study is providing more useful information for benchmarking purposes with outbreaks from different parts of the world and would be helpful for better patient care and treatment.

Site-directed mutagenesis of the conserved Asp-443 and Asp-498 carboxy-terminal residues of HIV-1 reverse transcriptase.

PubMed Central

Mizrahi, V; Usdin, M T; Harington, A; Dudding, L R

1990-01-01

Substitution of the conserved Asp-443 residue of HIV-1 reverse transcriptase by asparagine specifically suppressed the ribonuclease H activity of the enzyme without affecting the reverse transcriptase activity, suggesting involvement of this ionizable residue at the ribonuclease H active site. An analogous asparagine substitution of the Asp-498 residue yielded an unstable enzyme that was difficult to enzymatically characterize. However, the instability caused by the Asn-498 mutation was relieved by the introduction of a second distal Asn-443 substitution, yielding an enzyme with wild type reverse transcriptase activity, but lacking ribonuclease H activity. Images PMID:1699202

Synthesis, structure-activity relationship and molecular docking of cyclohexenone based analogous as potent non-nucleoside reverse-transcriptase inhibitors

NASA Astrophysics Data System (ADS)

Nazar, Muhammad Faizan; Abdullah, Muhammad Imran; Badshah, Amir; Mahmood, Asif; Rana, Usman Ali; Khan, Salah Ud-Din

2015-04-01

The chalcones core in compounds is advantageously chosen effective synthons, which offer exciting perspectives in biological and pharmacological research. The present study reports the successful development of eight new cyclohexenone based anti-reverse transcriptase analogous using rational drug design synthesis principles. These new cyclohexenone derivatives (CDs) were synthesized by following a convenient route of Robinson annulation, and the molecular structure of these CDs were later confirmed by various analytical techniques such as 1H NMR, 13C NMR, FT-IR, UV-Vis spectroscopy and mass spectrometry. All the synthesized compounds were screened theoretically and experimentally against reverse transcriptase (RT) and found potentially active reverse transcriptase (RT) inhibitors. Of the compounds studied, the compound 2FC4 showed high interaction with RT at non-nucleoside binding site, contributing high free binding energy (ΔG -8.01 Kcal) and IC50 (0.207 μg/ml), respectively. Further results revealed that the compounds bearing more halogen groups, with additional hydrophobic character, offered superior anti-reverse transcriptase activity as compared to rest of compounds. It is anticipate that the present study would be very useful for the selection of potential reverse transcriptase inhibitors featuring inclusive pharmacological profiles.

Determining the 95% limit of detection for waterborne pathogen analyses from primary concentration to qPCR

USDA-ARS?s Scientific Manuscript database

The limit of detection (LOD) for qPCR-based analyses is not consistently defined or determined in studies on waterborne pathogens. Moreover, the LODs reported often reflect the qPCR assay rather than the entire sample process. Our objective was to develop a method to determine the 95% LOD (lowest co...

Microbiology of aquatic environments: Characterizations of the microbiotas of municipal water supplies, the International Space Station Internal Active Thermal Control System's heat transport fluid, and US Space Shuttle drinking water

NASA Astrophysics Data System (ADS)

Bernardini, James Nicholas, III

An understanding of the microbiota within life support systems is essential for the prolonged presence of humans in space. This is because microbes may cause disease or induce biofouling and/or corrosion within spacecraft water systems. It is imperative that we develop effective high-throughput technologies for characterizing microbial populations that can eventually be used in the space environment. This dissertation describes testing and development of such methodologies, targeting both bacteria and viruses in water, and examines the bacterial and viral diversity within two spacecraft life support systems. The bacterial community of the International Space Station Internal Active Thermal Control System (IATCS) was examined using conventional culture-based and advanced molecular techniques including adenosine triphosphate (ATP) and Limulus Amebocyte Lysate (LAL) assays, direct microscopic examination, and analyses of 16S rRNA gene libraries from the community metagenome. The cultivable heterotrophs of the IATCS fluids ranged from below detection limit to 1.1x10 5/100 ml, and viable cells, measured by ATP, ranged from 1.4x10 3/100 ml to 7.7x105/100 ml. DNA extraction, cloning, sequencing, and bioinformatic analysis of the clones from 16S RNA gene libraries showed members of the firmicutes, alpha, beta, and gamma-proteobacteria to be present in the fluids. This persistent microbial bioburden and the presence of probable metal reducers, biofilm formers, and opportunistic pathogens illustrate the need for better characterization of bacterial communities present within spacecraft fluids. A new methodology was developed for detection of viruses in water using microarrays. Samples were concentrated by lyophilization, resuspended and filtered (0.22microm). Viral nucleic acids were then extracted, amplified, fluorescently labeled and hybridized onto a custom microarray with probes for ˜1000 known viruses. Numerous virus signatures were observed. Human Adenovirus C and Influenza A viruses were used to verify positive microarray hybridizations by quantitative polymerase chain reaction (PCR), reverse transcriptase PCR, and conventional PCR. Experiments were performed using municipal drinking water, IATCS fluids, and Shuttle drinking water. Thus, this dissertation describes what we believe is the first molecular analysis of the IATCS bacterial ecology and the first use and validation of a microarray-based assay for the detection of viral genetic signatures within drinking waters.

Differential expression of genes encoding anti-oxidant enzymes in Sydney rock oysters, Saccostrea glomerata (Gould) selected for disease resistance.

PubMed

Green, Timothy J; Dixon, Tom J; Devic, Emilie; Adlard, Robert D; Barnes, Andrew C

2009-05-01

Sydney rock oysters (Saccostrea glomerata) selectively bred for disease resistance (R) and wild-caught control oysters (W) were exposed to a field infection of disseminating neoplasia. Cumulative mortality of W oysters (31.7%) was significantly greater than R oysters (0.0%) over the 118 days of the experiment. In an attempt to understand the biochemical and molecular pathways involved in disease resistance, differentially expressed sequence tags (ESTs) between R and W S. glomerata hemocytes were identified using the PCR technique, suppression subtractive hybridisation (SSH). Sequencing of 300 clones from two SSH libraries revealed 183 distinct sequences of which 113 shared high similarity to sequences in the public databases. Putative function could be assigned to 64 of the sequences. Expression of nine ESTs homologous to genes previously shown to be involved in bivalve immunity was further studied using quantitative reverse-transcriptase PCR (qRT-PCR). The base-line expression of an extracellular superoxide dismutase (ecSOD) and a small heat shock protein (sHsP) were significantly increased, whilst peroxiredoxin 6 (Prx6) and interferon inhibiting cytokine factor (IK) were significantly decreased in R oysters. From these results it was hypothesised that R oysters would be able to generate the anti-parasitic compound, hydrogen peroxide (H(2)O(2)) faster and to higher concentrations during respiratory burst due to the differential expression of genes for the two anti-oxidant enzymes of ecSOD and Prx6. To investigate this hypothesis, protein extracts from hemolymph were analysed for oxidative burst enzyme activity. Analysis of the cell free hemolymph proteins separated by native-polyacrylamide gel electrophoresis (PAGE) failed to detect true superoxide dismutase (SOD) activity by assaying dismutation of superoxide anion in zymograms. However, the ecSOD enzyme appears to generate hydrogen peroxide, presumably via another process, which is yet to be elucidated. This corroborates our hypothesis, whilst phylogenetic analysis of the complete coding sequence (CDS) of the S. glomerata ecSOD gene is supportive of the atypical nature of the ecSOD enzyme. Results obtained from this work further the current understanding of the molecular mechanisms involved in resistance to disease in this economically important bivalve, and shed further light on the anomalous oxidative processes involved.

Plasmodium falciparum ookinete expression of plasmepsin VII and plasmepsin X.

PubMed

Li, Fengwu; Bounkeua, Viengngeun; Pettersen, Kenneth; Vinetz, Joseph M

2016-02-24

Plasmodium invasion of the mosquito midgut is a population bottleneck in the parasite lifecycle. Interference with molecular mechanisms by which the ookinete invades the mosquito midgut is one potential approach to developing malaria transmission-blocking strategies. Plasmodium aspartic proteases are one such class of potential targets: plasmepsin IV (known to be present in the asexual stage food vacuole) was previously shown to be involved in Plasmodium gallinaceum infection of the mosquito midgut, and plasmepsins VII and plasmepsin X (not known to be present in the asexual stage food vacuole) are upregulated in Plasmodium falciparum mosquito stages. These (and other) parasite-derived enzymes that play essential roles during ookinete midgut invasion are prime candidates for transmission-blocking vaccines. Reverse transcriptase PCR (RT-PCR) was used to determine timing of P. falciparum plasmepsin VII (PfPM VII) and plasmepsin X (PfPM X) mRNA transcripts in parasite mosquito midgut stages. Protein expression was confirmed by western immunoblot and immunofluorescence assays (IFA) using anti-peptide monoclonal antibodies (mAbs) against immunogenic regions of PfPM VII and PfPM X. These antibodies were also used in standard membrane feeding assays (SMFA) to determine whether inhibition of these proteases would affect parasite transmission to mosquitoes. The Mann-Whitney U test was used to analyse mosquito transmission assay results. RT-PCR, western immunoblot and immunofluorescence assay confirmed expression of PfPM VII and PfPM X in mosquito stages. Whereas PfPM VII was expressed in zygotes and ookinetes, PfPM X was expressed in gametes, zygotes, and ookinetes. Antibodies against PfPM VII and PfPM X decreased P. falciparum invasion of the mosquito midgut when used at high concentrations, indicating that these proteases play a role in Plasmodium mosquito midgut invasion. Failure to generate genetic knockouts of these genes limited determination of the precise role of these proteases in parasite transmission but suggests that they are essential during the intraerythrocytic life cycle. PfPM VII and PfPM X are present in the mosquito-infective stages of P. falciparum. Standard membrane feeding assays demonstrate that antibodies against these proteins reduce the infectivity of P. falciparum for mosquitoes, suggesting their viability as transmission-blocking vaccine candidates. Further study of the role of these plasmepsins in P. falciparum biology is warranted.

Presence of T3SS2β genes in trh⁺ Vibrio parahaemolyticus isolated from seafood harvested along Mangalore coast, India.

PubMed

Kumar, B K; Deekshit, V K; Rai, P; Shekar, M; Karunasagar, I; Karunasagar, I

2014-05-01

Vibrio parahaemolyticus is a seafood-borne pathogen autochthonous to the marine and estuarine ecosystem, responsible for gastroenteritis when contaminated raw seafood is consumed. The pathogenicity has been associated with thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH). Of late, the presence of T3SS2α and T3SS2β gene clusters has been well documented in clinical isolates of Vibrio parahaemolyticus and known to play an essential role in pathogenesis. However, reports on the presence of T3SSβ genes in V. parahaemolyticus isolated from the seafood and/or environmental samples are scanty. In this study, we have identified and analysed the distribution of the T3SS2β genes in V. parahaemolyticus isolated from seafood harvested along southwest coast of India. Results showed that T3SS2β genes are solely associated with trh⁺ and tdh⁺ /trh⁺ strains of V. parahaemolyticus. Reverse transcriptase PCR (RT-PCR) showed that the T3SS2β genes identified in trh⁺ V. parahaemolyticus were transcriptionally active. To our knowledge, this study appears to be the first description on the presence of T3SS2β-positive V. parahaemolyticus isolated from seafood in India. The study of T3SS2 along with other virulence factors will help in better understanding of the risk of seafood-borne illness due to V. parahaemolyticus. T3SSs (α or β) are the important virulence factors of Vibrio parahaemolyticus that contribute to their pathogenicity in humans. This study demonstrated the presence of T3SS2β genes in V. parahaemolyticus isolated from the seafood harvested along Mangalore coast. RT-PCR showed that the T3SS2β genes identified in seafood isolates of V. parahaemolyticus were found to be functional. To the best of our knowledge, this is the first description of T3SS2β genes in trh⁺ V. parahaemolyticus isolated from seafood in India. The presence of T3SS2 along with other virulence factors such as TDH and/or TRH highlights a potential health risk for seafood consumers. © 2013 The Society for Applied Microbiology.

Comprehensive evaluation of candidate reference genes for gene expression studies in Lysiphlebia japonica (Hymenoptera: Aphidiidae) using RT-qPCR.

PubMed

Gao, Xue-Ke; Zhang, Shuai; Luo, Jun-Yu; Wang, Chun-Yi; Lü, Li-Min; Zhang, Li-Juan; Zhu, Xiang-Zhen; Wang, Li; Lu, Hui; Cui, Jin-Jie

2017-12-30

Lysiphlebia japonica (Ashmead) is a predominant parasitoid of cotton-melon aphids in the fields of northern China with a proven ability to effectively control cotton aphid populations in early summer. For accurate normalization of gene expression in L. japonica using quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), reference genes with stable gene expression patterns are essential. However, no appropriate reference genes is L. japonica have been investigated to date. In the present study, 12 selected housekeeping genes from L. japonica were cloned. We evaluated the stability of these genes under various experimental treatments by RT-qPCR using four independent (geNorm, NormFinder, BestKeeper and Delta Ct) and one comparative (RefFinder) algorithm. We identified genes showing the most stable levels of expression: DIMT, 18S rRNA, and RPL13 during different stages; AK, RPL13, and TBP among sexes; EF1A, PPI, and RPL27 in different tissues, and EF1A, RPL13, and PPI in adults fed on different diets. Moreover, the expression profile of a target gene (odorant receptor 1, OR1) studied during the developmental stages confirms the reliability of the chosen selected reference genes. This study provides for the first time a comprehensive list of suitable reference genes for gene expression studies in L. japonica and will benefit subsequent genomics and functional genomics research on this natural enemy. Copyright © 2017. Published by Elsevier B.V.

Selection of reference genes for gene expression studies in heart failure for left and right ventricles.

PubMed

Li, Mengmeng; Rao, Man; Chen, Kai; Zhou, Jianye; Song, Jiangping

2017-07-15

Real-time quantitative reverse transcriptase-PCR (qRT-PCR) is a feasible tool for determining gene expression profiles, but the accuracy and reliability of the results depends on the stable expression of selected housekeeping genes in different samples. By far, researches on stable housekeeping genes in human heart failure samples are rare. Moreover the effect of heart failure on the expression of housekeeping genes in right and left ventricles is yet to be studied. Therefore we aim to provide stable housekeeping genes for both ventricles in heart failure and normal heart samples. In this study, we selected seven commonly used housekeeping genes as candidates. By using the qRT-PCR, the expression levels of ACTB, RAB7A, GAPDH, REEP5, RPL5, PSMB4 and VCP in eight heart failure and four normal heart samples were assessed. The stability of candidate housekeeping genes was evaluated by geNorm and Normfinder softwares. GAPDH showed the least variation in all heart samples. Results also indicated the difference of gene expression existed in heart failure left and right ventricles. GAPDH had the highest expression stability in both heart failure and normal heart samples. We also propose using different sets of housekeeping genes for left and right ventricles respectively. The combination of RPL5, GAPDH and PSMB4 is suitable for the right ventricle and the combination of GAPDH, REEP5 and RAB7A is suitable for the left ventricle. Copyright © 2017 Elsevier B.V. All rights reserved.

A putative peroxidase cDNA from turnip and analysis of the encoded protein sequence.

PubMed

Romero-Gómez, S; Duarte-Vázquez, M A; García-Almendárez, B E; Mayorga-Martínez, L; Cervantes-Avilés, O; Regalado, C

2008-12-01

A putative peroxidase cDNA was isolated from turnip roots (Brassica napus L. var. purple top white globe) by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Total RNA extracted from mature turnip roots was used as a template for RT-PCR, using a degenerated primer designed to amplify the highly conserved distal motif of plant peroxidases. The resulting partial sequence was used to design the rest of the specific primers for 5' and 3' RACE. Two cDNA fragments were purified, sequenced, and aligned with the partial sequence from RT-PCR, and a complete overlapping sequence was obtained and labeled as BbPA (Genbank Accession No. AY423440, named as podC). The full length cDNA is 1167bp long and contains a 1077bp open reading frame (ORF) encoding a 358 deduced amino acid peroxidase polypeptide. The putative peroxidase (BnPA) showed a calculated Mr of 34kDa, and isoelectric point (pI) of 4.5, with no significant identity with other reported turnip peroxidases. Sequence alignment showed that only three peroxidases have a significant identity with BnPA namely AtP29a (84%), and AtPA2 (81%) from Arabidopsis thaliana, and HRPA2 (82%) from horseradish (Armoracia rusticana). Work is in progress to clone this gene into an adequate host to study the specific role and possible biotechnological applications of this alternative peroxidase source.

Equivalency of Buffalo (Bubalus Bubalis) Embryonic Stem Cells Derived From Fertilized, Parthenogenetic, and Hand-Made Cloned Embryos

PubMed Central

Muzaffar, Musharifa; Selokar, Naresh L.; Singh, Karn P.; Zandi, Mohammad; Singh, Manoj K.; Shah, Riaz A.; Chauhan, Manmohan S.; Singla, Suresh K.; Palta, Prabhat

2012-01-01

Abstract This study was aimed at establishing buffalo embryonic stem cells (ESCs) from in vitro fertilized (IVF), parthenogenetic, and hand-made cloned (HMC) embryos and to check their equivalency in terms of stem cell marker expression, longevity, proliferation, and differentiation pattern. ESCs derived from all three sources were found by immunofluorescence to express the pluripotency markers SSEA-4, TRA-1-60, TRA-1-81, OCT4, and SOX2 and were able to form embryoid bodies containing cells expressing genes specific to endoderm (AFP, HNF4, and GATA4), mesoderm (MSX1, BMP4, and ASA), and ectoderm (cytokeratin 8 and NF68). Reverse transcriptase PCR (RT-PCR) showed cells from all sources to be positive for pluripotency markers OCT4, SOX2, NANOG, STAT3, REX1, FOXD3, NUCLEOSTEMIN, and TELOMERASE. Pluripotency markers OCT4, SOX2, NANOG, and c-MYC were also analyzed by real-time PCR. No significant differences were observed among ESCs from all three sources for all these genes except NANOG, whose expression was higher (p<0.05) in HMC-derived ESCs (6.897±2.3) compared to that in parthenogenesis- and IVF-derived cells (1.603±0.315 and 1±0, respectively). Pluripotent, stable buffalo ESC lines derived from IVF, parthenogenesis, and HMC embryos may be genetically manipulated to provide a powerful tool for studies involving embryonic development, genomic imprinting, gene targeting, cloning, chimera formation, and transgenic animal production. PMID:22582863

The role of environmental contamination with small round structured viruses in a hospital outbreak investigated by reverse-transcriptase polymerase chain reaction assay.

PubMed

Green, J; Wright, P A; Gallimore, C I; Mitchell, O; Morgan-Capner, P; Brown, D W

1998-05-01

In May 1994 an outbreak of vomiting and diarrhoea occurred in a 28-bed long-stay ward for the mentally infirm. The predominant symptoms were vomiting, diarrhoea, malaise and abdominal pain lasting for approximately 12 h in most cases. The attack rate was 62% (13/21) for patients and 46% (16/35) for staff members. Infection control measures were implemented (containment of infectious individuals, hand hygiene among staff and environmental decontamination) and the ward was closed to admissions. Affected staff were excluded from contact with patients and their food until asymptomatic for 72 h. The outbreak lasted for 17 days. Faecal samples from nine symptomatic persons were negative for bacterial enteric pathogens, Giardia, Cryptosporidium and group A rotavirus. Electron microscopy of 12 faecal samples and one sample of vomitus revealed small round structured virus (SRSV) particles in one faecal sample. A further 30 faecal samples and seven vomitus samples were tested by reverse transcription polymerase chain reaction (RT-PCR) for SRSV of which 12 (40%) and 1 (14%) were positive respectively. Twenty-eight throat swabs from symptomatic and asymptomatic patients were collected, three (9.5%) of which were positive for SRSV by RT-PCR. Thirty-six environmental swabs were collected on the affected ward, and 11 (30%) were positive by RT-PCR. Positive swabs were from lockers, curtains and commodes and confined to the immediate environment of symptomatic patients. The distribution of contamination supports the rationale of cohorting sick patients.

Mumps outbreak and laboratory diagnosis.

PubMed

Maillet, Mylène; Bouvat, Eric; Robert, Nicole; Baccard-Longère, Monique; Morel-Baccard, Christine; Morand, Patrice; Vabret, Astrid; Stahl, Jean-Paul

2015-01-01

Several mumps outbreaks have been reported in Europe and in the United States among highly vaccinated populations. Biological diagnosis is classically based on the detection of mumps-specific IgM, but the ability of serological tests to confirm mumps infection seems to be limited among vaccinated patients. We aim to report a mumps outbreak in an engineering school in Grenoble, France, from February to June 2013 and results of the biological testing. WHO definitions were used to define cases. Mumps--specific IgM and IgG were assessed by a commercially available EIA. Mumps RNA detection by real time reverse transcriptase polymerase chain reaction tests (RT-PCR) and mumps genotyping were performed by the French National Reference Centre for Paramyxoviridae. Sixty two mumps patient-cases were identified using WHO case definitions, 20 being biologically explored, of which 17 were confirmed by biological tests. Vaccination status was documented for 27 patients/62: 4 (14.8%) patients had received one dose of MMR vaccine, and 23 (85.2) two doses of MMR vaccine. Among the biologically explored patients, 83% had a positive RT PCR at the first sampling whereas only 45% had positive or equivocal IgM. All the genotyped strains were genotype G. Mumps laboratory diagnosis in a highly vaccinated population is challenging. Serological tests among vaccinated patients should be interpreted cautiously and confirmed by RT-PCR tests at the beginning of a mumps outbreak. Copyright © 2014 Elsevier B.V. All rights reserved.

Field-based evaluation of a male-specific (F+) RNA coliphage concentration method.

PubMed

Chandler, J C; Pérez-Méndez, A; Paar, J; Doolittle, M M; Bisha, B; Goodridge, L D

2017-01-01

Fecal contamination of water poses a significant risk to public health due to the potential presence of pathogens, including enteric viruses. Therefore, sensitive, reliable and easy to use methods for the concentration, detection and quantification of microorganisms associated with the safety and quality of water are needed. In this study, we performed a field evaluation of an anion exchange resin-based method to concentrate male-specific (F+) RNA coliphages (FRNA), fecal indicator organisms, from diverse environmental waters that were suspected to be contaminated with feces. In this system, FRNA coliphages are adsorbed to anion exchange resin and direct nucleic acid isolation is performed, yielding a sample amenable to real-time reverse transcriptase (RT)-PCR detection. Matrix-dependent inhibition of this method was evaluated using known quantities of spiked FRNA coliphages belonging to four genogroups (GI, GII, GII and GIV). RT-PCR-based detection was successful in 97%, 72%, 85% and 98% of the samples spiked (10 6 pfu/l) with GI, GII, GIII and GIV, respectively. Differential FRNA coliphage genogroup detection was linked to inhibitors that altered RT-PCR assay efficiency. No association between inhibition and the physicochemical properties of the water samples was apparent. Additionally, the anion exchange resin method facilitated detection of naturally present FRNA coliphages in 40 of 65 environmental water samples (61.5%), demonstrating the viability of this system to concentrate FRNA coliphages from water. Copyright © 2016 Elsevier B.V. All rights reserved.

Cloning of an ADP-ribosylation factor gene from banana (Musa acuminata) and its expression patterns in postharvest ripening fruit.

PubMed

Wang, Yuan; Wu, Jing; Xu, Bi-Yu; Liu, Ju-Hua; Zhang, Jian-Bin; Jia, Cai-Hong; Jin, Zhi-Qiang

2010-08-15

A full-length cDNA encoding an ADP-ribosylation factor (ARF) from banana (Musa acuminata) fruit was cloned and named MaArf. It contains an open reading frame encoding a 181-amino-acid polypeptide. Sequence analysis showed that MaArf shared high similarity with ARF of other plant species. The genomic sequence of MaArf was also obtained using polymerase chain reaction (PCR). Sequence analysis showed that MaArf was a split gene containing five exons and four introns in genomic DNA. Reverse-transcriptase PCR was used to analyze the spatial expression of MaArf. The results showed that MaArf was expressed in all the organs examined: root, rhizome, leaf, flower and fruit. Real-time quantitative PCR was used to explore expression patterns of MaArf in postharvest banana. There was differential expression of MaArf associated with ethylene biosynthesis. In naturally ripened banana, expression of MaArf was in accordance with ethylene biosynthesis. However, in 1-methylcyclopropene-treated banana, the expression of MaArf was inhibited and changed little. When treated with ethylene, MaArf expression in banana fruit significantly increased in accordance with ethylene biosynthesis; the peak of MaArf was 3 d after harvest, 11 d earlier than for naturally ripened banana fruits. These results suggest that MaArf is induced by ethylene in regulating postharvest banana ripening. Finally, subcellular localization assays showed the MaArf protein in the cytoplasm. Copyright 2010 Elsevier GmbH. All rights reserved.

Expression Patterns and Identified Protein-Protein Interactions Suggest That Cassava CBL-CIPK Signal Networks Function in Responses to Abiotic Stresses.

PubMed

Mo, Chunyan; Wan, Shumin; Xia, Youquan; Ren, Ning; Zhou, Yang; Jiang, Xingyu

2018-01-01

Cassava is an energy crop that is tolerant of multiple abiotic stresses. It has been reported that the interaction between Calcineurin B-like (CBL) protein and CBL-interacting protein kinase (CIPK) is implicated in plant development and responses to various stresses. However, little is known about their functions in cassava. Herein, 8 CBL ( MeCBL ) and 26 CIPK ( MeCIPK ) genes were isolated from cassava by genome searching and cloning of cDNA sequences of Arabidopsis CBL s and CIPK s. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression levels of MeCBL and MeCIPK genes were different in different tissues throughout the life cycle. The expression patterns of 7 CBL and 26 CIPK genes in response to NaCl, PEG, heat and cold stresses were analyzed by quantitative real-time PCR (qRT-PCR), and it was found that the expression of each was induced by multiple stimuli. Furthermore, we found that many pairs of CBLs and CIPKs could interact with each other via investigating the interactions between 8 CBL and 25 CIPK proteins using a yeast two-hybrid system. Yeast cells co-transformed with cassava MeCIPK24, MeCBL10 , and Na + /H + antiporter MeSOS1 genes exhibited higher salt tolerance compared to those with one or two genes. These results suggest that the cassava CBL-CIPK signal network might play key roles in response to abiotic stresses.

Expression Patterns and Identified Protein-Protein Interactions Suggest That Cassava CBL-CIPK Signal Networks Function in Responses to Abiotic Stresses

PubMed Central

Mo, Chunyan; Wan, Shumin; Xia, Youquan; Ren, Ning; Zhou, Yang; Jiang, Xingyu

2018-01-01

Cassava is an energy crop that is tolerant of multiple abiotic stresses. It has been reported that the interaction between Calcineurin B-like (CBL) protein and CBL-interacting protein kinase (CIPK) is implicated in plant development and responses to various stresses. However, little is known about their functions in cassava. Herein, 8 CBL (MeCBL) and 26 CIPK (MeCIPK) genes were isolated from cassava by genome searching and cloning of cDNA sequences of Arabidopsis CBLs and CIPKs. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression levels of MeCBL and MeCIPK genes were different in different tissues throughout the life cycle. The expression patterns of 7 CBL and 26 CIPK genes in response to NaCl, PEG, heat and cold stresses were analyzed by quantitative real-time PCR (qRT-PCR), and it was found that the expression of each was induced by multiple stimuli. Furthermore, we found that many pairs of CBLs and CIPKs could interact with each other via investigating the interactions between 8 CBL and 25 CIPK proteins using a yeast two-hybrid system. Yeast cells co-transformed with cassava MeCIPK24, MeCBL10, and Na+/H+ antiporter MeSOS1 genes exhibited higher salt tolerance compared to those with one or two genes. These results suggest that the cassava CBL-CIPK signal network might play key roles in response to abiotic stresses. PMID:29552024

Identification of Avian and Hemoparasite DNA in Blood-Engorged Abdomens of Culex pipiens (Diptera; Culicidae) from a West Nile Virus Epidemic region in Suburban Chicago, Illinois.

PubMed

Boothe, Emily; Medeiros, Matthew C I; Kitron, Uriel D; Brawn, Jeffrey D; Ruiz, Marilyn O; Goldberg, Tony L; Walker, Edward D; Hamer, Gabriel L

2015-05-01

Multiple mosquito-borne parasites cocirculate in nature and potentially interact. To understand the community of parasites cocirculating with West Nile virus (WNV), we screened the bloodmeal content of Culex pipiens L. mosquitoes for three common types of hemoparasites. Blood-fed Cx. pipiens were collected from a WNV-epidemic area in suburban Chicago, IL, from May to September 2005 through 2010. DNA was extracted from dissected abdomens and subject to PCR and direct sequencing to identify the vertebrate host. RNA was extracted from the head or thorax and screened for WNV using quantitative reverse transcriptase PCR. Seventy-nine engorged females with avian host origin were screened using PCR and amplicon sequencing for filarioid nematodes, Haemosporida, and trypanosomatids. Filarioid nematodes were identified in 3.8% of the blooded abdomens, Plasmodium sp. in 8.9%, Haemoproteus in 31.6%, and Trypanosoma sp. in 6.3%. The sequences from these hemoparasite lineages were highly similar to sequences from birds in prior studies in suburban Chicago. Overall, 50.6% of blood-fed Culex pipiens contained hemoparasite DNA in their abdomen, presumably from current or prior bloodmeals. Additionally, we detected hemoparasite DNA in the blooded abdomen of three of 10 Cx. pipiens infected with WNV. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT

PubMed Central

Roundhill, E; Burchill, S

2013-01-01

Background: Primary Ewing's sarcoma family of tumours (ESFTs) may respond to chemotherapy, although many patients experience subsequent disease recurrence and relapse. The survival of ESFT cells following chemotherapy has been attributed to the development of resistant disease, possibly through the expression of ABC transporter proteins. Methods: MRP-1 and Pgp mRNA and protein expression in primary ESFTs was determined by quantitative reverse-transcriptase PCR (RT-qPCR) and immunohistochemistry, respectively, and alternative splicing of MRP-1 by RT-PCR. Results: We observed MRP-1 protein expression in 92% (43 out of 47) of primary ESFTs, and cell membrane MRP-1 was highly predictive of both overall survival (P<0.0001) and event-free survival (P<0.0001). Alternative splicing of MRP-1 was detected in primary ESFTs, although the pattern of splicing variants was not predictive of patient outcome, with the exception of loss of exon 9 in six patients, which predicted relapse (P=0.041). Pgp protein was detected in 6% (38 out of 44) of primary ESFTs and was not associated with patient survival. Conclusion: For the first time we have established that cell membrane expression of MRP-1 or loss of exon 9 is predictive of outcome but not the number of splicing events or expression of Pgp, and both may be valuable factors for the stratification of patients for more intensive therapy. PMID:23799853

Equivalency of buffalo (Bubalus bubalis) embryonic stem cells derived from fertilized, parthenogenetic, and hand-made cloned embryos.

PubMed

Muzaffar, Musharifa; Selokar, Naresh L; Singh, Karn P; Zandi, Mohammad; Singh, Manoj K; Shah, Riaz A; Chauhan, Manmohan S; Singla, Suresh K; Palta, Prabhat; Manik, Radheysham

2012-06-01

This study was aimed at establishing buffalo embryonic stem cells (ESCs) from in vitro fertilized (IVF), parthenogenetic, and hand-made cloned (HMC) embryos and to check their equivalency in terms of stem cell marker expression, longevity, proliferation, and differentiation pattern. ESCs derived from all three sources were found by immunofluorescence to express the pluripotency markers SSEA-4, TRA-1-60, TRA-1-81, OCT4, and SOX2 and were able to form embryoid bodies containing cells expressing genes specific to endoderm (AFP, HNF4, and GATA4), mesoderm (MSX1, BMP4, and ASA), and ectoderm (cytokeratin 8 and NF68). Reverse transcriptase PCR (RT-PCR) showed cells from all sources to be positive for pluripotency markers OCT4, SOX2, NANOG, STAT3, REX1, FOXD3, NUCLEOSTEMIN, and TELOMERASE. Pluripotency markers OCT4, SOX2, NANOG, and c-MYC were also analyzed by real-time PCR. No significant differences were observed among ESCs from all three sources for all these genes except NANOG, whose expression was higher (p<0.05) in HMC-derived ESCs (6.897±2.3) compared to that in parthenogenesis- and IVF-derived cells (1.603±0.315 and 1±0, respectively). Pluripotent, stable buffalo ESC lines derived from IVF, parthenogenesis, and HMC embryos may be genetically manipulated to provide a powerful tool for studies involving embryonic development, genomic imprinting, gene targeting, cloning, chimera formation, and transgenic animal production.

Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology.

PubMed

Smith, Cindy J; Osborn, A Mark

2009-01-01

Quantitative PCR (Q-PCR or real-time PCR) approaches are now widely applied in microbial ecology to quantify the abundance and expression of taxonomic and functional gene markers within the environment. Q-PCR-based analyses combine 'traditional' end-point detection PCR with fluorescent detection technologies to record the accumulation of amplicons in 'real time' during each cycle of the PCR amplification. By detection of amplicons during the early exponential phase of the PCR, this enables the quantification of gene (or transcript) numbers when these are proportional to the starting template concentration. When Q-PCR is coupled with a preceding reverse transcription reaction, it can be used to quantify gene expression (RT-Q-PCR). This review firstly addresses the theoretical and practical implementation of Q-PCR and RT-Q-PCR protocols in microbial ecology, highlighting key experimental considerations. Secondly, we review the applications of (RT)-Q-PCR analyses in environmental microbiology and evaluate the contribution and advances gained from such approaches. Finally, we conclude by offering future perspectives on the application of (RT)-Q-PCR in furthering understanding in microbial ecology, in particular, when coupled with other molecular approaches and more traditional investigations of environmental systems.

Characterization of the interferon genes in homozygous rainbow trout reveals two novel genes, alternate splicing and differential regulation of duplicated genes

USGS Publications Warehouse

Purcell, M.K.; Laing, K.J.; Woodson, J.C.; Thorgaard, G.H.; Hansen, J.D.

2009-01-01

The genes encoding the type I and type II interferons (IFNs) have previously been identified in rainbow trout and their proteins partially characterized. These previous studies reported a single type II IFN (rtIFN-??) and three rainbow trout type I IFN genes that are classified into either group I (rtIFN1, rtIFN2) or group II (rtIFN3). In this present study, we report the identification of a novel IFN-?? gene (rtIFN-??2) and a novel type I group II IFN (rtIFN4) in homozygous rainbow trout and predict that additional IFN genes or pseudogenes exist in the rainbow trout genome. Additionally, we provide evidence that short and long forms of rtIFN1 are actively and differentially transcribed in homozygous trout, and likely arose due to alternate splicing of the first exon. Quantitative reverse transcriptase PCR (qRT-PCR) assays were developed to systematically profile all of the rainbow trout IFN transcripts, with high specificity at an individual gene level, in na??ve fish and after stimulation with virus or viral-related molecules. Cloned PCR products were used to ensure the specificity of the qRT-PCR assays and as absolute standards to assess transcript abundance of each gene. All IFN genes were modulated in response to Infectious hematopoietic necrosis virus (IHNV), a DNA vaccine based on the IHNV glycoprotein, and poly I:C. The most inducible of the type I IFN genes, by all stimuli tested, were rtIFN3 and the short transcript form of rtIFN1. Gene expression of rtIFN-??1 and rtIFN-??2 was highly up-regulated by IHNV infection and DNA vaccination but rtIFN-??2 was induced to a greater magnitude. The specificity of the qRT-PCR assays reported here will be useful for future studies aimed at identifying which cells produce IFNs at early time points after infection. ?? 2008 Elsevier Ltd.

Grana Padano cheese whey starters: microbial composition and strain distribution.

PubMed

Rossetti, Lia; Fornasari, Maria Emanuela; Gatti, Monica; Lazzi, Camilla; Neviani, Erasmo; Giraffa, Giorgio

2008-09-30

The aim of this work was to evaluate the species composition and the genotypic strain heterogeneity of dominant lactic acid bacteria (LAB) isolated from whey starter cultures used to manufacture Grana Padano cheese. Twenty-four Grana Padano cheese whey starters collected from dairies located over a wide geographic production area in the north of Italy were analyzed. Total thermophilic LAB streptococci and lactobacilli were quantified by agar plate counting. Population structure of the dominant and metabolically active LAB species present in the starters was profiled by reverse transcriptase, length heterogeneity-PCR (RT-LH-PCR), a culture-independent technique successfully applied to study whey starter ecosystems. The dominant bacterial species were Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis, Streptococcus thermophilus, and Lactobacillus fermentum. Diversity in the species composition allowed the whey cultures to be grouped into four main typologies, the one containing L. helveticus, L. delbrueckii subsp. lactis, and S. thermophilus being the most frequent one (45% of the cultures analyzed), followed by that containing only the two lactobacilli (40%). Only a minor fraction of the cultures contained L. helveticus alone (4%) or all the four LAB species (11%). Five hundred and twelve strains were isolated from the 24 cultures and identified by M13-PCR fingerprinting coupled with 16S rRNA gene sequencing. Most of the strains were L. helveticus (190 strains; 37% of the total), L delbrueckii subsp. lactis (90 strains; 18%) and S. thermophilus (215 strains; 42%). This result was in good agreement with the qualitative whey starter composition observed by RT-LH-PCR. M13-PCR fingerprinting indicated a markedly low infra-species diversity, i.e. the same biotypes were often found in more than one culture. The distribution of the biotypes into the different cultures was mainly dairy plant-specific rather than correlated with the different production areas.

SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all hemagglutinin and neuraminidase genes of avian influenza viruses and comparison to standard serological subtyping tests

USGS Publications Warehouse

Tsukamoto, K.; Javier, P.C.; Shishido, M.; Noguchi, D.; Pearce, J.; Kang, H.-M.; Jeong, O.M.; Lee, Y.-J.; Nakanishi, K.; Ashizawa, T.

2012-01-01

Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 10 1.5, 10 2.3, and 10 3.1 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples. Copyright ?? 2012, American Society for Microbiology. All Rights Reserved.

Comparison of RNA extraction kits for the purification and detection of an enteric virus surrogate on green onions via RT-PCR.

PubMed

Xu, Ruoyang; Shieh, Y Carol; Stewart, Diana S

2017-01-01

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) offers a rapid and sensitive molecular method for detection of enteric viruses. Unfortunately, these assays are often hampered by the low virus titre found in foods and PCR inhibition due to matrix carryover during RNA extraction. Four commercial RNA extraction kits (Qiagen's QIAamp Viral RNA Mini and UltraSens Virus kits, MoBio UltraClean Tissue & Cells RNA Isolation kit, and Ambion MagMAX Viral RNA Isolation kit) were evaluated for their ability to extract and purify MS2 bacteriophage RNA, an enteric virus surrogate, from inoculated green onions, a food which has been associated with viral gastroenteritis outbreaks. Inoculated green onion wash concentrates and green onion pieces with and without Qiagen QIAshredder homogenization were assayed in the kit comparison. MS2 detection and PCR inhibition were evaluated using a duplex real-time RT-PCR for MS2 and an exogenous internal amplification control (IAC) assay. Without homogenization, MS2 inoculated at 40pfu/g was detected in at least 4 lots of green onion wash concentrates using the silica-membrane spin-column kits. Inhibition was a factor for the magnetic silica-based MagMAX kit, which resulted in detection of MS2 in 1 of 5. Addition of QIAshredder homogenization prior to extraction did not adversely affect the silica-membrane kit results but improved the MS2 detection by MagMAX to 5 of 5 lots. Use of a 1:10 dilution of primary RNA extracts also improved detection. The QIAamp Viral RNA Mini and MagMAX kits were further compared for detection of MS2 from green onion pieces inoculated at 20 and 5pfu/g. Using homogenization, the MagMAX kit detected 20pfu/g in only 1 of 2 green onion lots, whereas the QIAamp Viral RNA kit detected 2 of 2 lots at 5 pfu/g without homogenization. Published by Elsevier B.V.

Evaluation of NGS and RT-PCR Methods for ALK Rearrangement in European NSCLC Patients: Results from the European Thoracic Oncology Platform Lungscape Project.

PubMed

Letovanec, Igor; Finn, Stephen; Zygoura, Panagiota; Smyth, Paul; Soltermann, Alex; Bubendorf, Lukas; Speel, Ernst-Jan; Marchetti, Antonio; Nonaka, Daisuke; Monkhorst, Kim; Hager, Henrik; Martorell, Miguel; Sejda, Aleksandra; Cheney, Richard; Hernandez-Losa, Javier; Verbeken, Eric; Weder, Walter; Savic, Spasenija; Di Lorito, Alessia; Navarro, Atilio; Felip, Enriqueta; Warth, Arne; Baas, Paul; Meldgaard, Peter; Blackhall, Fiona; Dingemans, Anne-Marie; Dienemann, Hendrik; Dziadziuszko, Rafal; Vansteenkiste, Johan; O'Brien, Cathal; Geiger, Thomas; Sherlock, Jon; Schageman, Jeoffrey; Dafni, Urania; Kammler, Roswitha; Kerr, Keith; Thunnissen, Erik; Stahel, Rolf; Peters, Solange

2018-03-01

The reported prevalence of ALK receptor tyrosine kinase gene (ALK) rearrangement in NSCLC ranges from 2% to 7%. The primary standard diagnostic method is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has also proved to be a reproducible and sensitive technique. Reverse-transcriptase polymerase chain reaction (RT-PCR) has also been advocated, and most recently, the advent of targeted next-generation sequencing (NGS) for ALK and other fusions has become possible. This study compares anaplastic lymphoma kinase (ALK) evaluation with all four techniques in resected NSCLC from the large European Thoracic Oncology Platform Lungscape cohort. A total of 96 cases from the European Thoracic Oncology Platform Lungscape iBiobank, with any ALK immunoreactivity were examined by FISH, central RT-PCR, and NGS. An H-score higher than 120 defines IHC positivity. RNA was extracted from the same formalin-fixed, paraffin-embedded tissues. For RT-PCR, primers covered the most frequent ALK translocations. For NGS, the Oncomine Solid Tumour Fusion Transcript Kit (Thermo Fisher Scientific, Waltham, MA) was used. The concordance was assessed using the Cohen κ coefficient (two-sided α ≤ 5%). NGS provided results for 77 of the 95 cases tested (81.1%), whereas RT-PCR provided results for 77 of 96 (80.2%). Concordance occurred in 55 cases of the 60 cases tested with all four methods (43 ALK negative and 12 ALK positive). Using ALK copositivity for IHC and FISH as the criterion standard, we derived a sensitivity for RT-PCR/NGS of 70.0%/85.0%, with a specificity of 87.1%/79.0%. When either RT-PCR or NGS was combined with IHC, the sensitivity remained the same, whereas the specificity increased to 88.7% and 83.9% respectively. NGS evaluation with the Oncomine Solid Tumour Fusion transcript kit and RT-PCR proved to have high sensitivity and specificity, advocating their use in routine practice. For maximal sensitivity and specificity, ALK status should be assessed by using two techniques and a third one in discordant cases. We therefore propose a customizable testing algorithm. These findings significantly influence existing testing paradigms and have clear clinical and economic impact. Copyright © 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Supporting business continuity during a highly pathogenic avian influenza outbreak: a collaboration of industry, academia, and government.

PubMed

Hennessey, Morgan; Lee, Brendan; Goldsmith, Timothy; Halvorson, Dave; Hueston, William; McElroy, Kristina; Waters, Katherine

2010-03-01

Since 2006, a collaborative group of egg industry, state, federal, and academia representatives have worked to enhance preparedness in highly pathogenic avian influenza (HPAI) planning. The collaborative group has created a draft egg product movement protocol, which calls for realistic, science-based contingency plans, biosecurity assessments, commodity risk assessments, and real-time reverse transcriptase-PCR testing to support the continuity of egg operations while also preventing and eradicating an HPAI outbreak. The work done by this group serves as an example of how industry, government, and academia can work together to achieve better preparedness in the event of an animal health emergency. In addition, in the event of an HPAI outbreak in domestic poultry, U.S. consumers will be assured that their egg products come from healthy chickens.

Salinity inhibits post transcriptional processing of chloroplast 16S rRNA in shoot cultures of jojoba (Simmondsia chinesis).

PubMed

Mizrahi-Aviv, Ela; Mills, David; Benzioni, Aliza; Bar-Zvi, Dudy

2005-03-01

Chloroplast metabolism is rapidly affected by salt stress. Photosynthesis is one of the first processes known to be affected by salinity. Here, we report that salinity inhibits chloroplast post-transcriptional RNA processing. A differentially expressed 680-bp cDNA, containing the 3' sequence of 16S rRNA, transcribed intergenic spacer, exon 1 and intron of tRNA(Ile), was isolated by differential display reverse transcriptase PCR from salt-grown jojoba (Simmondsia chinesis) shoot cultures. Northern blot analysis indicated that although most rRNA appears to be fully processed, partially processed chloroplast 16S rRNA accumulates in salt-grown cultures. Thus, salinity appears to decrease the processing of the rrn transcript. The possible effect of this decreased processing on physiological processes is, as yet, unknown.

An evaluation of new and established methods to determine T‐DNA copy number and homozygosity in transgenic plants.

PubMed Central

Głowacka, Katarzyna; Kromdijk, Johannes; Leonelli, Lauriebeth; Niyogi, Krishna K.; Clemente, Tom E.

2016-01-01

Abstract Stable transformation of plants is a powerful tool for hypothesis testing. A rapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations. Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL‐)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T‐DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL‐PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher variability than ddPCR. Comparison of segregation analyses and ddPCR of T1 progeny from 26 T0 plants showed that at least 19% of the lines carried multiple T‐DNA insertions per locus, which can lead to unstable transgene expression. Segregation analyses failed to detect these multiple copies, presumably because of their close linkage. This shows the importance of routine T‐DNA copy number estimation. Based on our results, ddPCR is the most suitable method, because it is as reliable as Southern blot analysis yet much faster. A protocol for this application of ddPCR to large plant genomes is provided. PMID:26670088

Crosslinked type II collagen matrices: preparation, characterization, and potential for cartilage engineering.

PubMed

Pieper, J S; van der Kraan, P M; Hafmans, T; Kamp, J; Buma, P; van Susante, J L C; van den Berg, W B; Veerkamp, J H; van Kuppevelt, T H

2002-08-01

The limited intrinsic repair capacity of articular cartilage has stimulated continuing efforts to develop tissue engineered analogues. Matrices composed of type II collagen and chondroitin sulfate (CS), the major constituents of hyaline cartilage, may create an appropriate environment for the generation of cartilage-like tissue. In this study, we prepared, characterized, and evaluated type 11 collagen matrices with and without CS. Type II collagen matrices were prepared using purified, pepsin-treated, type II collagen. Techniques applied to prepare type I collagen matrices were found unsuitable for type II collagen. Crosslinking of collagen and covalent attachment of CS was performed using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide. Porous matrices were prepared by freezing and lyophilization, and their physico-chemical characteristics (degree of crosslinking, denaturing temperature, collagenase-resistance, amount of CS incorporated) established. Matrices were evaluated for their capacity to sustain chondrocyte proliferation and differentiation in vitro. After 7 d of culture, chondrocytes were mainly located at the periphery of the matrices. In contrast to type I collagen, type II collagen supported the distribution of cells throughout the matrix. After 14 d of culture, matrices were surfaced with a cartilagenous-like layer, and occasionally clusters of chondrocytes were present inside the matrix. Chondrocytes proliferated and differentiated as indicated by biochemical analyses, ultrastructural observations, and reverse transcriptase PCR for collagen types I, II and X. No major differences were observed with respect to the presence or absence of CS in the matrices.

Evolutionary History of LINE-1 in the Major Clades of Placental Mammals

PubMed Central

Waters, Paul D.; Dobigny, Gauthier; Waddell, Peter J.; Robinson, Terence J.

2007-01-01

Background LINE-1 constitutes an important component of mammalian genomes. It has a dynamic evolutionary history characterized by the rise, fall and replacement of subfamilies. Most data concerning LINE-1 biology and evolution are derived from the human and mouse genomes and are often assumed to hold for all placentals. Methodology To examine LINE-1 relationships, sequences from the 3′ region of the reverse transcriptase from 21 species (representing 13 orders across Afrotheria, Xenarthra, Supraprimates and Laurasiatheria) were obtained from whole genome sequence assemblies, or by PCR with degenerate primers. These sequences were aligned and analysed. Principal Findings Our analysis reflects accepted placental relationships suggesting mostly lineage-specific LINE-1 families. The data provide clear support for several clades including Glires, Supraprimates, Laurasiatheria, Boreoeutheria, Xenarthra and Afrotheria. Within the afrotherian LINE-1 (AfroLINE) clade, our tree supports Paenungulata, Afroinsectivora and Afroinsectiphillia. Xenarthran LINE-1 (XenaLINE) falls sister to AfroLINE, providing some support for the Atlantogenata (Xenarthra+Afrotheria) hypothesis. Significance LINEs and SINEs make up approximately half of all placental genomes, so understanding their dynamics is an essential aspect of comparative genomics. Importantly, a tree of LINE-1 offers a different view of the root, as long edges (branches) such as that to marsupials are shortened and/or broken up. Additionally, a robust phylogeny of diverse LINE-1 is essential in testing that site-specific LINE-1 insertions, often regarded as homoplasy-free phylogenetic markers, are indeed unique and not convergent. PMID:17225861

Latent Herpes Simplex Virus Infection of Sensory Neurons Alters Neuronal Gene Expression

PubMed Central

Kramer, Martha F.; Cook, W. James; Roth, Frederick P.; Zhu, Jia; Holman, Holly; Knipe, David M.; Coen, Donald M.

2003-01-01

The persistence of herpes simplex virus (HSV) and the diseases that it causes in the human population can be attributed to the maintenance of a latent infection within neurons in sensory ganglia. Little is known about the effects of latent infection on the host neuron. We have addressed the question of whether latent HSV infection affects neuronal gene expression by using microarray transcript profiling of host gene expression in ganglia from latently infected versus mock-infected mouse trigeminal ganglia. 33P-labeled cDNA probes from pooled ganglia harvested at 30 days postinfection or post-mock infection were hybridized to nylon arrays printed with 2,556 mouse genes. Signal intensities were acquired by phosphorimager. Mean intensities (n = 4 replicates in each of three independent experiments) of signals from mock-infected versus latently infected ganglia were compared by using a variant of Student's t test. We identified significant changes in the expression of mouse neuronal genes, including several with roles in gene expression, such as the Clk2 gene, and neurotransmission, such as genes encoding potassium voltage-gated channels and a muscarinic acetylcholine receptor. We confirmed the neuronal localization of some of these transcripts by using in situ hybridization. To validate the microarray results, we performed real-time reverse transcriptase PCR analyses for a selection of the genes. These studies demonstrate that latent HSV infection can alter neuronal gene expression and might provide a new mechanism for how persistent viral infection can cause chronic disease. PMID:12915567

Resveratrol suppresses TPA-induced matrix metalloproteinase-9 expression through the inhibition of MAPK pathways in oral cancer cells.

PubMed

Lin, Feng-Yan; Hsieh, Yi-Hsien; Yang, Shun-Fa; Chen, Chang-Tai; Tang, Chih-Hsin; Chou, Ming-Yung; Chuang, Yi-Ting; Lin, Chiao-Wen; Chen, Mu-Kuan

2015-10-01

Naturally occurring agents, such as resveratrol, have been determined to benefit health. Numerous studies have demonstrated that resveratrol has antioxidative, cardioprotective, and neuroprotective properties. However, the effect of resveratrol exerts on the metastasis of oral cancer cells remains unclear. In this study, we investigated the effect the anti-invasive activity of resveratrol on a human oral cancer cell line (SCC-9) in vitro and the underlying mechanisms. Cell viability was examined by MTT assay, whereas cell motility was measured by migration and wound-healing assays. Zymography, reverse-transcriptase polymerase chain reaction (PCR), and promoter assays confirmed the inhibitory effects of resveratrol on matrix metalloproteinase-9 (MMP-9) expression in oral cancer cells. We established that various concentrations (0-100 μM) of resveratrol inhibited the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced migration capacities of SCC-9 cells and caused no cytotoxic effects. Zymography and Western blot analyses suggested that resveratrol inhibited TPA-induced MMP-9 gelatinolytic activity and protein expression. In addition, the results indicated that resveratrol inhibited the phosphorylation of c-Jun N-terminal kinase (JNK)1/2 and extracellular-signal-regulated kinase (ERK)1/2 involved in downregulating protein expression and the transcription of MMP-9. In summary, resveratrol inhibited MMP-9 expression and oral cancer cell metastasis by downregulating JNK1/2 and ERK1/2 signals pathways and, thus, exerts beneficial effects in chemoprevention. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Skeletal muscle myoblasts possess a stretch-responsive local angiotensin signalling system.

PubMed

Johnston, Adam P W; Baker, Jeff; De Lisio, Michael; Parise, Gianni

2011-06-01

A paucity of information exists regarding the presence of local renin-angiotensin systems (RASs) in skeletal muscle and associated muscle stem cells. Skeletal muscle and muscle stem cells were isolated from C57BL/6 mice and examined for the presence of a local RAS using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC), Western blotting and liquid chromatography-mass spectrometry (LC-MS). Furthermore, the effect of mechanical stimulation on RAS member gene expression was analysed. Whole skeletal muscle, primary myoblasts and C2C12 derived myoblasts and myotubes differentially expressed members of the RAS including angiotensinogen, angiotensin-converting enzyme (ACE), angiotensin II (Ang II) type 1 (AT(1)) and type 2 (AT(2)). Renin transcripts were never detected, however, mRNA for the 'renin-like' enzyme cathepsin D was observed and Ang I and Ang II were identified in cell culture supernatants from proliferating myoblasts. AT(1) appeared to co-localise with polymerised actin filaments in proliferating myoblasts and was primarily found in the nucleus of terminally differentiated myotubes. Furthermore, mechanical stretch of proliferating and differentiating C2C12 cells differentially induced mRNA expression of angiotensinogen, AT(1) and AT(2). Proliferating and differentiated muscle stem cells possess a local stress-responsive RAS in vitro. The precise function of a local RAS in myoblasts remains unknown. However, evidence presented here suggests that Ang II may be a regulator of skeletal muscle myoblasts.

Ethylene Regulates Monomeric GTP-Binding Protein Gene Expression and Activity in Arabidopsis1

PubMed Central

Moshkov, Igor E.; Mur, Luis A.J.; Novikova, Galina V.; Smith, Aileen R.; Hall, Michael A.

2003-01-01

Ethylene rapidly and transiently up-regulates the activity of several monomeric GTP-binding proteins (monomeric G proteins) in leaves of Arabidopsis as determined by two-dimensional gel electrophoresis and autoradiographic analyses. The activation is suppressed by the receptor-directed inhibitor 1-methylcyclopropene. In the etr1-1 mutant, constitutive activity of all the monomeric G proteins activated by ethylene is down-regulated relative to wild type, and ethylene treatment has no effect on the levels of activity. Conversely, in the ctr1-1 mutant, several of the monomeric G proteins activated by ethylene are constitutively up-regulated. However, the activation profile of ctr1-1 does not exactly mimic that of ethylene-treated wild type. Biochemical and molecular evidence suggested that some of these monomeric G proteins are of the Rab class. Expression of the genes for a number of monomeric G proteins in response to ethylene was investigated by reverse transcriptase-PCR. Rab8 and Ara3 expression was increased within 10 min of ethylene treatment, although levels fell back significantly by 40 min. In the etr1-1 mutant, expression of Rab8 was lower than wild type and unaffected by ethylene; in ctr1-1, expression of Rab8 was much higher than wild type and comparable with that seen in ethylene treatments. Expression in ctr1-1 was also unaffected by ethylene. Thus, the data indicate a role for monomeric G proteins in ethylene signal transduction. PMID:12692329

Functional analysis of the interactions between reovirus particles and various proteases in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sargent, M.D.; Long, D.G.; Borsa, J.

1977-01-01

The digestion of purified reovirus particles by various proteases including chymotrypsin, trypsin, pronase, papain, bromelain, proteinase K, and fibrinolysin has been examined as it relates to virion transcriptase activation and alteration of infectivity. In every case uncoating to the level of active transcriptase proceeds via two mechanistically distinct steps. All the proteases tested serve to mediate only the first of the two steps, converting intact virions to intermediate subviral particles (ISVP) in which the transcriptase is retained in a latent state. The second step of the uncoating process is mediated by a K/sup +/ ion-triggered, endogenous mechanism and results inmore » conversion of ISVP to cores, concomitant with transcriptase activation and loss of infectivity. All of the tested enzymes, except trypsin, reversibly block the second step of uncoating. These results indicate the generality, with respect to protease employed, of the two-step process for reovirus uncoating and transcriptase activation demonstrated previously with chymotrypsin.« less

Molecular genotyping of Colletotrichum species based on arbitrarily primed PCR, A + T-Rich DNA, and nuclear DNA analyses

USGS Publications Warehouse

Freeman, S.; Pham, M.; Rodriguez, R.J.

1993-01-01

Molecular genotyping of Colletotrichum species based on arbitrarily primed PCR, A + T-rich DNA, and nuclear DNA analyses. Experimental Mycology 17, 309-322. Isolates of Colletotrichum were grouped into 10 separate species based on arbitrarily primed PCR (ap-PCR), A + T-rich DNA (AT-DNA) and nuclear DNA banding patterns. In general, the grouping of Colletotrichum isolates by these molecular approaches corresponded to that done by classical taxonomic identification, however, some exceptions were observed. PCR amplification of genomic DNA using four different primers allowed for reliable differentiation between isolates of the 10 species. HaeIII digestion patterns of AT-DNA also distinguished between species of Colletotrichum by generating species-specific band patterns. In addition, hybridization of the repetitive DNA element (GcpR1) to genomic DNA identified a unique set of Pst 1-digested nuclear DNA fragments in each of the 10 species of Colletotrichum tested. Multiple isolates of C. acutatum, C. coccodes, C. fragariae, C. lindemuthianum, C. magna, C. orbiculare, C. graminicola from maize, and C. graminicola from sorghum showed 86-100% intraspecies similarity based on ap-PCR and AT-DNA analyses. Interspecies similarity determined by ap-PCR and AT-DNA analyses varied between 0 and 33%. Three distinct banding patterns were detected in isolates of C. gloeosporioides from strawberry. Similarly, three different banding patterns were observed among isolates of C. musae from diseased banana.

A multicenter study of viable PCR using propidium monoazide to detect Legionella in water samples.

PubMed

Scaturro, Maria; Fontana, Stefano; Dell'eva, Italo; Helfer, Fabrizia; Marchio, Michele; Stefanetti, Maria Vittoria; Cavallaro, Mario; Miglietta, Marilena; Montagna, Maria Teresa; De Giglio, Osvalda; Cuna, Teresa; Chetti, Leonarda; Sabattini, Maria Antonietta Bucci; Carlotti, Michela; Viggiani, Mariagabriella; Stenico, Alberta; Romanin, Elisa; Bonanni, Emma; Ottaviano, Claudio; Franzin, Laura; Avanzini, Claudio; Demarie, Valerio; Corbella, Marta; Cambieri, Patrizia; Marone, Piero; Rota, Maria Cristina; Bella, Antonino; Ricci, Maria Luisa

2016-07-01

Legionella quantification in environmental samples is overestimated by qPCR. Combination with a viable dye, such as Propidium monoazide (PMA), could make qPCR (named then vPCR) very reliable. In this multicentre study 717 artificial water samples, spiked with fixed concentrations of Legionella and interfering bacterial flora, were analysed by qPCR, vPCR and culture and data were compared by statistical analysis. A heat-treatment at 55 °C for 10 minutes was also performed to obtain viable and not-viable bacteria. When data of vPCR were compared with those of culture and qPCR, statistical analysis showed significant differences (P < 0.001). However, although the heat-treatment caused an abatement of CFU/mL ≤1 to 1 log10 unit, the comparison between untreated and heat-treated samples analysed by vPCR highlighted non-significant differences (P > 0.05). Overall this study provided a good experimental reproducibility of vPCR but also highlighted limits of PMA in the discriminating capability of dead and live bacteria, making vPCR not completely reliable. Copyright © 2016 Elsevier Inc. All rights reserved.

Microbiota of an Italian Grana-Like Cheese during Manufacture and Ripening, Unraveled by 16S rRNA-Based Approaches.

PubMed

Alessandria, Valentina; Ferrocino, Ilario; De Filippis, Francesca; Fontana, Mauro; Rantsiou, Kalliopi; Ercolini, Danilo; Cocolin, Luca

2016-07-01

The microbial ecology of cheese involves a rich and complex interaction between starter lactic acid bacteria and nonstarter lactic acid bacteria (NSLAB), mainly originating from raw milk and/or from the environment, that can contribute to the final characteristics of cheese. The aim of the present research was the exploration of the active microbiota by RNA-based approaches during the manufacturing and ripening of a Grana-like cheese. Reverse transcriptase PCR (RT-PCR)-denaturing gradient gel electrophoresis (DGGE) and RNA-based high-throughput sequencing were applied to profile microbial populations, while the enumeration of active bacteria was carried out by using quantitative PCR (qPCR). Three different cheese productions (named D, E, and F) collected in the same month from the same dairy plant were analyzed. The application of the qPCR protocol revealed the presence of 7 log CFU/ml of bacterial load in raw milk, while, during ripening, active bacterial populations ranged from <4 to 8 log CFU/ml. The natural whey starters used in the three productions showed the same microbiota composition, characterized by the presence of Lactobacillus helveticus and Lactobacillus delbrueckii Nevertheless, beta-diversity analysis of the 16S rRNA sequencing data and RT-PCR-DGGE showed a clear clustering of the samples according to the three productions, probably driven by the different milks used. Milk samples were found to be characterized by the presence of several contaminants, such as Propionibacterium acnes, Acidovorax, Acinetobacter, Pseudomonas, and NSLAB. The core genera of the starter tended to limit the development of the spoilage bacteria only in two of the three batches. This study underlines the influence of different factors that can affect the final microbiota composition of the artisanal cheese. This study highlights the importance of the quality of the raw milk in the production of a hard cheese. Independent from the use of a starter culture, raw milk with low microbiological quality can negatively affect the populations of lactic acid bacteria and, as a consequence, impact the quality of the final product due to metabolic processes associated with spoilage bacteria. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Determination of internal controls for quantitative real time RT-PCR analysis of the effect of Edwardsiella tarda infection on gene expression in turbot (Scophthalmus maximus).

PubMed

Dang, Wei; Sun, Li

2011-02-01

In recent years, quantitative real time reverse transcriptase-PCR (qRT-PCR) has been used frequently in the study of gene expression in turbot (Scophthalmus maximus) in relation to bacterial infection. However, no investigations on appropriate qRT-PCR reference genes have been documented. In this report, we determined the potential of eight housekeeping genes, i.e. β-actin (ACTB), ribosomal protein L17 (RPL17), α-tubulin (TUBA), elongation factor-1-α(EF1A), β-2-Microglobulin (B2M), RNA polymerase II subunit D (RPSD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S ribosomal RNA (18S rRNA), as internal standards for qRT-PCR analysis of gene expression in turbot as a function of bacterial infection. For this purpose, the expression of the eight housekeeping genes in seven turbot tissues was determined by qRT-PCR before and after bacterial challenge, and the data were analyzed with the geNorm and NormFinder algorisms. The results showed that the expression of all the examined genes exhibited tissue-dependent variations both before and after bacterial challenge. Before bacterial challenge, geNorm and NormFinder identified RPSD as the gene that showed least tissue specific expression. At 12 h post-bacterial infection, geNorm ranked ACTB/GAPDH, 18S rRNA/ACTB, ACTB/GAPDH, 18S rRNA/ACTB, RPL17/TUBA, RPSD/GAPDH, and RPSD/B2M, respectively, as the most stably expressed genes in liver, spleen, kidney, gill, heart, muscle, and brain. Comparable ranking orders were produced by NormFinder. Similar results were obtained at 24 h post-bacterial infection. Taken together, these results indicate that RPSD is the most stable gene across tissue types under normal physiological conditions and that, during bacterial infection, ACTB might be used as an internal standard for the normalization of gene expression in immune relevant organs; however, no single gene or single pair of genes in the examined set of housekeeping genes can serve as a universal reference across all tissue types under the condition of bacterial infection. Copyright © 2011 Elsevier Ltd. All rights reserved.

Genetic diversity of Babesia bovis in virulent and attenuated strains.

PubMed

Mazuz, M L; Molad, T; Fish, L; Leibovitz, B; Wolkomirsky, R; Fleiderovitz, L; Shkap, V

2012-03-01

The aim of this study was to compare the genetic diversity of the single copy Bv80 gene sequences of Babesia bovis in populations of attenuated and virulent parasites. PCR/ RT-PCR followed by cloning and sequence analyses of 4 attenuated and 4 virulent strains were performed. Multiple fragments in the range of 420 to 744 bp were amplified by PCR or RT-PCR. Cloning of the PCR fragments and sequence analyses revealed the presence of mixed subpopulations in either virulent or attenuated parasites with a total of 19 variants with 12 different sequences that differed in number and type of tandem repeats. High levels of intra- and inter-strain diversity of the Bv80 gene, with the presence of mixed populations of parasites were found in both the virulent field isolates and the attenuated vaccine strains. In addition, during the attenuation process, sequence analyses showed changes in the pattern of the parasite subpopulations. Despite high polymorphism found by sequence analyses, the patterns observed and the number of repeats, order, or motifs found could not discriminate between virulent field isolates and attenuated vaccine strains of the parasite.

Molecular Characterization of the Human Immunodeficiency Virus Type 1 in Women and Their Vertically Infected Children.

PubMed

Vaz, Sara Nunes; Giovanetti, Marta; Rego, Filipe Ferreira de Almeida; Oliveira, Tulio de; Danaviah, Siva; Gonçalves, Maria Luiza Freire; Alcantara, Luiz Carlos Junior; Brites, Carlos

2015-10-01

Approximately 35 million people worldwide are infected with human immunodeficiency virus (HIV) around 3.2 million of whom are children under 15 years. Mother-to-child-transmission (MTCT) of HIV-1 accounts for 90% of all infections in children. Despite great advances in the prevention of MTCT in Brazil, children are still becoming infected. Samples from 19 HIV-1-infected families were collected. DNA was extracted and fragments from gag, pol, and env were amplified and sequenced directly. Phylogenetic reconstruction was performed. Drug resistance analyses were performed in pol and env sequences. We found 82.1% of subtype B and 17.9% of BF recombinants. A prevalence of 43.9% drug resistance-associated mutations in pol sequences was identified. Of the drug-naive children 33.3% presented at least one mutation related to protease inhibitor/nucleoside reverse transcriptase inhibitor/nonnucleoside reverse transcriptase inhibitor (PI/NRTI/NNRTI) resistance. The prevalence of transmitted drug resistance mutations was 4.9%. On env we found a low prevalence of HR1 (4.9%) and HR2 (14.6%) mutations.

Selection Pressure in CD8+ T-cell Epitopes in the pol Gene of HIV-1 Infected Individuals in Colombia. A Bioinformatic Approach

PubMed Central

Acevedo-Sáenz, Liliana; Ochoa, Rodrigo; Rugeles, Maria Teresa; Olaya-García, Patricia; Velilla-Hernández, Paula Andrea; Diaz, Francisco J.

2015-01-01

One of the main characteristics of the human immunodeficiency virus is its genetic variability and rapid adaptation to changing environmental conditions. This variability, resulting from the lack of proofreading activity of the viral reverse transcriptase, generates mutations that could be fixed either by random genetic drift or by positive selection. Among the forces driving positive selection are antiretroviral therapy and CD8+ T-cells, the most important immune mechanism involved in viral control. Here, we describe mutations induced by these selective forces acting on the pol gene of HIV in a group of infected individuals. We used Maximum Likelihood analyses of the ratio of non-synonymous to synonymous mutations per site (dN/dS) to study the extent of positive selection in the protease and the reverse transcriptase, using 614 viral sequences from Colombian patients. We also performed computational approaches, docking and algorithmic analyses, to assess whether the positively selected mutations affected binding to the HLA molecules. We found 19 positively-selected codons in drug resistance-associated sites and 22 located within CD8+ T-cell epitopes. A high percentage of mutations in these epitopes has not been previously reported. According to the docking analyses only one of those mutations affected HLA binding. However, algorithmic methods predicted a decrease in the affinity for the HLA molecule in seven mutated peptides. The bioinformatics strategies described here are useful to identify putative positively selected mutations associated with immune escape but should be complemented with an experimental approach to define the impact of these mutations on the functional profile of the CD8+ T-cells. PMID:25803098

(PCG) Protein Crystal Growth HIV Reverse Transcriptase

NASA Technical Reports Server (NTRS)

1992-01-01

HIV Reverse Transcriptase crystals grown during the USML-1 (STS-50) mission using Commercial Refrigerator/Incubator Module (CR/IM) at 4 degrees C and the Vapor Diffusion Apparatus (VDA). Reverse transcriptase is an enzyme responsible for copying the nucleic acid genome of the AIDS virus from RNA to DNA. Studies indicated that the space-grown crystals were larger and better ordered (beyond 4 angstroms) than were comparable Earth-grown crystals. Principal Investigators were Charles Bugg and Larry DeLucas.

Anti-adenoviral effect of anti-HIV agents in vitro in serotypes inducing keratoconjunctivitis.

PubMed

Uchio, Eiichi; Fuchigami, Aki; Kadonosono, Kazuaki; Hayashi, Akio; Ishiko, Hiroaki; Aoki, Koki; Ohno, Shigeaki

2007-09-01

Around one million people are affected by adenoviral keratoconjunctivitis a year in Japan, and it is recognized as one of the major pathogens of ophthalmological nosocomial infection worldwide. Although cidofovir can be used systemically for immunocompromised patients with disseminated adenoviral infection, no specific anti-adenoviral agent has been established for the treatment of adenoviral infection. We evaluated the anti-adenoviral effect of anti-HIV (human immunodeficiency virus) agents in this study. Five anti-HIV agents (zalcitabine, stavudine, nevirapine, indinavir and amprenavir) were subjected to in vitro evaluation. A549 cells were used for viral cell culture, and adenovirus serotypes 3, 4, 8, 19 and 37 were used. After calculating CC(50) (50% cytotoxic concentration) of each agent by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) method, we cultured adenovirus with the agents for seven days and quantitatively measured extracted adenoviral DNA by real-time PCR. Among the anti-HIV drugs, zalcitabine and stavudine, both nucleoside reverse transcriptase inhibitors, showed significant anti-adenoviral activity. In contrast, nevirapine, a non-nucleoside reverse transcriptase inhibitor, and indinavir and amprenavir, which are both protease inhibitors, were ineffective against adenovirus. These results indicate that zalcitabine and stavudine are possible candidates for the local and systemic treatment of adenoviral infection, and the anti-adenoviral effect might depend on the pharmacological properties of anti-HIV agents. The chemical properties on the clinical safety for systemic and local application need to be determined in order to for these drugs to be accepted for the treatment of adenovirus in clinical settings.

Establishment of an immortalized cell line derived from the prairie vole via lentivirus-mediated transduction of mutant cyclin-dependent kinase 4, cyclin D, and telomerase reverse transcriptase

PubMed Central

Katayama, Masafumi; Kiyono, Tohru; Horie, Kengo; Hirayama, Takashi; Eitsuka, Takahiro; Kuroda, Kengo; Donai, Kenichiro; Hidema, Shizu; Nishimori, Katsuhiko; Fukuda, Tomokazu

2015-01-01

The prairie vole (Microtus ochrogaster) shows social behaviors such as monogamy and parenting of infants with pair bonding. These social behaviors are specific to the prairie vole and have not been observed in other types of voles, such as mountain voles. Although the prairie vole has several unique characteristics, an in vitro cell culture system has not been established for this species. Furthermore, establishment of cultured cells derived from the prairie vole may be beneficial based on the three Rs (i.e., Replacement, Reduction, and Refinement) concept. Therefore, in this study, we attempted to establish an immortalized cell line derived from the prairie vole. Our previous research has shown that transduction with mutant forms of cyclin-dependent kinase 4 (CDK4), cyclin D, and telomerase reverse transcriptase (TERT) could efficiently immortalize cells from multiple species, including humans, cattle, pigs, and monkeys. Here, we introduced these three genes into prairie vole-derived muscle fibroblasts. The expression of mutant CDK4 and cyclin D proteins was confirmed by western blotting, and telomerase activity was detected in immortalized vole muscle-derived fibroblasts (VMF-K4DT cells or VMFs) by stretch PCR. Population doubling analysis showed that the introduction of mutant CDK4, cyclin D, and TERT extended the lifespan of VMFs. To the best of our knowledge, this is the first report describing the establishment of an immortalized cell line derived from the prairie vole through the expression of mutant CDK4, cyclin D, and human TERT. PMID:26496927

Establishment of an immortalized cell line derived from the prairie vole via lentivirus-mediated transduction of mutant cyclin-dependent kinase 4, cyclin D, and telomerase reverse transcriptase.

PubMed

Katayama, Masafumi; Kiyono, Tohru; Horie, Kengo; Hirayama, Takashi; Eitsuka, Takahiro; Kuroda, Kengo; Donai, Kenichiro; Hidema, Shizu; Nishimori, Katsuhiko; Fukuda, Tomokazu

2016-01-01

The prairie vole (Microtus ochrogaster) shows social behaviors such as monogamy and parenting of infants with pair bonding. These social behaviors are specific to the prairie vole and have not been observed in other types of voles, such as mountain voles. Although the prairie vole has several unique characteristics, an in vitro cell culture system has not been established for this species. Furthermore, establishment of cultured cells derived from the prairie vole may be beneficial based on the three Rs (i.e., Replacement, Reduction, and Refinement) concept. Therefore, in this study, we attempted to establish an immortalized cell line derived from the prairie vole. Our previous research has shown that transduction with mutant forms of cyclin-dependent kinase 4 (CDK4), cyclin D, and telomerase reverse transcriptase (TERT) could efficiently immortalize cells from multiple species, including humans, cattle, pigs, and monkeys. Here, we introduced these three genes into prairie vole-derived muscle fibroblasts. The expression of mutant CDK4 and cyclin D proteins was confirmed by western blotting, and telomerase activity was detected in immortalized vole muscle-derived fibroblasts (VMF-K4DT cells or VMFs) by stretch PCR. Population doubling analysis showed that the introduction of mutant CDK4, cyclin D, and TERT extended the lifespan of VMFs. To the best of our knowledge, this is the first report describing the establishment of an immortalized cell line derived from the prairie vole through the expression of mutant CDK4, cyclin D, and human TERT.

Efficacy of Inactivation of Human Enteroviruses by Multiple ...

EPA Pesticide Factsheets

Background: Ultraviolet (UV) light has been successfully used for treating a broad suite of pathogens without the concomitant formation of carcinogenic disinfection by-products (DBPs). However, conventional mercury UV lamps have some practical limitations in water treatment applications, such as the inefficiency of energy consumption and more importantly potential mercury contamination upon disposal of the lamps. The recent invention of a novel light-emitting-diodes (LED) device generating germicidal UV wavelengths could eliminate the aforementioned limitations. In this study, we investigated the efficacy of multiple-wavelength UV LEDs for inactivating USEPA contaminant candidate list (CCL) RNA enteroviruses.Methods: Of 12 enterovirus species, serotype representatives of the four human enteric species (enterovirus A-D) such as coxsackievirus A10 (CVA10), echovirus 30 (Echo30), poliovirus 1 (PV1), and enterovirus 70 (EV70) respectively were selected as testing RNA viruses. Bench-scale performance evaluation was conducted using a collimated beam (CB) apparatus with LEDs emitting at 260 nm, 280 nm, and the combination of 260|280 nm together, as well as a monochromatic low-pressure (LP) UV lamp at 254 nm for comparison. The CB tests were performed with mixed stocks of four viruses. Infectious virus concentrations were determined using an integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR).Results: The 260 nm LED was most effective at inactiva

Efficacy of Inactivation of Human Enteroviruses by Multiple ...

EPA Pesticide Factsheets

Ultraviolet (UV) light has been successfully used for treating a broad suite of pathogens without the concomitant formation of carcinogenic disinfection by-products (DBPs). However, conventional mercury UV lamps have some practical limitations in water treatment applications, such as the inefficiency of energy consumption and more importantly potential mercury contamination upon disposal of the lamps. The recent invention of a novel light-emitting-diodes (LED) device generating germicidal UV wavelengths could eliminate the aforementioned limitations. In this study, we investigated the efficacy of multiple-wavelength UV LEDs for inactivating USEPA contaminant candidate list (CCL) RNA enteroviruses. Of 12 enterovirus species, serotype representatives of the four human enteric species (enterovirus A-D) such as coxsackievirus A10 (CVA10), echovirus 30 (Echo30), poliovirus 1 (PV1), and enterovirus 70 (EV70) respectively were selected as testing RNA viruses. Bench-scale performance evaluation was conducted using a collimated beam (CB) apparatus with LEDs emitting at 260 nm, 280 nm, and the combination of 260|280 nm together, as well as a monochromatic low-pressure (LP) UV lamp at 254 nm for comparison. The CB tests were performed with mixed stocks of four viruses. Infectious virus concentrations were determined using an integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR). The 260 nm LED was most effective at inactivating all enteroviruses teste

Growth hormone mRNA in mammary gland tumors of dogs and cats.

PubMed Central

Mol, J A; van Garderen, E; Selman, P J; Wolfswinkel, J; Rijinberk, A; Rutteman, G R

1995-01-01

We have shown recently that in the dog progestin administration results in mammary production of immunoreactive growth hormone (GH). At present we demonstrate the expression of the gene encoding GH in the mammary gland of dogs and cats using reverse-transcriptase PCR. GH mRNA was found in the great majority of normal mammary tissues as well as benign and malignant mammary tumors of the dog and was associated with the presence of immunoreactive GH in cryostat sections. The mammary PCR product proved to be identical to that of the pituitary. The highest expression levels were found after prolonged treatment with progestins. In carcinomas GH mRNA was also found in progesterone receptor-negative tissue samples, indicating that after malignant transformation GH gene expression may become progestin independent. GH mRNA was also present in mammary tissues of cats with progestin-induced fibroadenomatous changes. It is concluded that GH gene expression occurs in normal, hyperplastic, and neoplastic mammary tissue of the dog. The expression in normal tissue is stimulated by progestins and might mediate the progestin-stimulated development of canine mammary tumors. The demonstration of progestin-stimulated GH expression in mammary tissue of cats indicates that the phenomenon is more generalized among mammals. Images PMID:7738169

Detection of Viral Hemorrhagic Septicemia virus (VHSV) from the leech Myzobdella lugubris Leidy, 1851

PubMed Central

Faisal, Mohamed; Schulz, Carolyn A

2009-01-01

The leech Myzobdella lugubris is widespread in the Lake Erie Watershed, especially Lake St. Clair. However, its role in pathogen transmission is not fully understood. In this same watershed, several widespread fish mortalities associated with the Viral Hemorrhagic Septicemia virus (VHSV) were recorded. Viral Hemorrhagic Septicemia is an emerging disease in the Great Lakes Basin that is deadly to the fish population, yet little is known about its mode of transmission. To assess the potential role of M. lugubris in VHSV transmission, leeches were collected from Lake St. Clair and Lake Erie and pooled into samples of five. Cell culture and reverse transcriptase polymerase chain reaction (RT-PCR) were used to determine the presence of the virus and its identity. Results showed that 57 of the 91 pooled leech samples were positive by cell culture for VHSV and 66 of the 91 pooled leech samples were positive by RT-PCR for the VHSV. Two representative virus isolates were sequenced for further genetic confirmation and genotype classification. VHSV detected within M. lugubris was homologous to the Great Lakes strain of VHSV genotype IVb. This is the first record of the VHSV being detected from within a leech, specifically M. lugubris, and suggests the potential of M. lugubris being involved in VHSV transmission. PMID:19785752

Evaluation of Energy Balance on Human Telomerase Reverse Transcriptase (hTERT) Alternative Splicing by Semi-quantitative RT-PCR in Human Umbilical Vein Endothelial Cells.

PubMed

Behjati, Mohaddeseh; Hashemi, Mohammad; Kazemi, Mohammad; Salehi, Mansoor; Javanmard, Shaghayegh Haghjooy

2017-01-01

Decreased high-energy phosphate level is involved in endothelial cell injury and dysfunction. Reduced telomerase activity in endothelial cells in parallel with reduced energy levels might be due to altered direction of alternative splicing machine as a complication of depleted energy during the process of atherosclerosis. Isolated human umbilical vein endothelial cells (HUVECs) were treated for 24 hours by oligomycine (OM) and 2-deoxy glucose (2-DG). After 24 hours, the effect of energy depletion on telomerase splicing pattern was evaluated using RT-PCR. Indeed, in both treated and untargeted cells, nitric oxide (NO) and von Willebrand factor (vWF) were measured. ATP was depleted in treated cells by 43.9% compared with control group. We observed a slight decrease in NO levels ( P = 0.09) and vWF ( P = 0.395) in the setting of 49.36% ATP depletion. In both groups, no telomerase gene expression was seen. Telomerase and housekeeping gene expression were found in positive control group (colon cancer tissue) and sample tissue. The absence of telomerase gene expression in HUVECs might be due to the mortality of these cells or the low level of telomerase gene expression in these cells under normal circumstances.

Transovarial transmission of dengue 1 virus in Aedes aegypti larvae: real-time PCR analysis in a Brazilian city with high mosquito population density.

PubMed

Moraes, Alexsander; Cortelli, Filipe C; Miranda, Taís B; Aquino, Davi R; Cortelli, José R; Guimarães, Maria Isabel A; Costa, Fernando O; Cortelli, Sheila C

2018-06-01

Transovarial transmission is among the reported factors able to influence environmental maintenance of dengue virus (DENV). Endemic areas with active transmission of dengue are suitable for studying transovarial transmission. Brazil is a country where dengue is endemic and where DENV-1 is the most common disease-related virus serotype. This study aimed to identify transovarial transmission of DENV-1 in Aedes aegypti larvae by reverse-transcriptase nested real-time polymerase chain reaction. Between March and October 2016, Culicidae larvae were collected using traps in 3 locations in Taubaté, São Paulo, Brazil, which has a high occurrence of dengue. The collected larvae were sacrificed in the 3rd or 4th larval stage, classified, and stored at -20 °C. The A. aegypti larvae samples (n = 910) were separated into 91 pools of 10 specimens each from which RNA was extracted, reverse transcribed into cDNA, and analyzed by nested qPCR. None of the pools tested positive for DENV-1. Due to the absence of detectable virus in the evaluated samples, we concluded that transovarial transmission may not be the primary mechanism for maintenance of DENV-1 in this particular environment.

Challenges in Interpretation of Diagnostic Test Results in a Mumps Outbreak in a Highly Vaccinated Population.

PubMed

Trotz-Williams, L A; Mercer, N J; Paphitis, K; Walters, J M; Wallace, D; Kristjanson, E; Gubbay, J; Mazzulli, T

2017-02-01

In spite of a greatly reduced incidence rate due to vaccination, mumps outbreaks continue to occur in several areas of the world, sometimes in vaccinated populations. This article describes an outbreak in a highly vaccinated population in southwestern Ontario, Canada, and the challenges encountered in interpreting the results of diagnostic tests used in the outbreak. During the outbreak, patients were interviewed and classified according to the outbreak case definition, and specimens were collected for diagnostic testing according to Ontario guidelines. Twenty-seven individuals were classified as confirmed cases (n = 19) or suspect cases (n = 8) according to the case definition, only 9 of which were laboratory-confirmed cases: 7 confirmed by reverse transcriptase PCR (RT-PCR) and 2 by IgM serology. All 19 confirmed cases represented patients who were associated with secondary schools in the local area and had been vaccinated against mumps with one (n = 2) or two (n = 17) doses of the measles-mumps-rubella (MMR) vaccine. This is the first published report of an outbreak of mumps in Ontario in which all confirmed cases had been vaccinated against the disease. It highlights the limitations of and difficulties in interpreting current mumps diagnostic tests when used in vaccinated individuals. Copyright © 2017 American Society for Microbiology.

Fast preparation of a long chimeric armored RNA as controls for external quality assessment for molecular detection of Zika virus.

PubMed

Lin, Guigao; Zhang, Kuo; Zhang, Dong; Han, Yanxi; Xie, Jiehong; Li, Jinming

2017-03-01

The emergence of Zika virus demands accurate laboratory diagnostics. Nucleic acid testing is currently the definitive method for diagnosis of Zika infection. In 2016, an external quality assurance (EQA) for assessing the quality of molecular testing of Zika virus was carried out in China. A single armored RNA encapsulating a 4942-nucleotides (nt) long specific RNA sequence of Zika virus was prepared and used as positive samples. A pre-tested EQA panel, consisting of 4 negative and 6 positive samples with different concentrations of armored RNA, was distributed to 38 laboratories that perform molecular detection of Zika virus. A total of 39 data sets (1 laboratory used two test kits in parallel), produced by using commercial (n=38) or laboratory developed (n=1) quantitative reverse-transcriptase PCR (qRT-PCR) kits, were received. Of these, 35 (89.7%) had correct results for all 10 samples, and 4 (10.3%) reported at least 1 error (11 in total). The testing errors were all false-negatives, highlighting the need of improvements in detecting sensitivity. The EQA reveals that the majority of participating laboratories are proficient in molecular testing of Zika virus. Copyright © 2017 Elsevier B.V. All rights reserved.

Reverse transcription strand invasion based amplification (RT-SIBA): a method for rapid detection of influenza A and B.

PubMed

Eboigbodin, Kevin; Filén, Sanna; Ojalehto, Tuomas; Brummer, Mirko; Elf, Sonja; Pousi, Kirsi; Hoser, Mark

2016-06-01

Rapid and accurate diagnosis of influenza viruses plays an important role in infection control, as well as in preventing the misuse of antibiotics. Isothermal nucleic acid amplification methods offer significant advantages over the polymerase chain reaction (PCR), since they are more rapid and do not require the sophisticated instruments needed for thermal cycling. We previously described a novel isothermal nucleic acid amplification method, 'Strand Invasion Based Amplification' (SIBA®), with high analytical sensitivity and specificity, for the detection of DNA. In this study, we describe the development of a variant of the SIBA method, namely, reverse transcription SIBA (RT-SIBA), for the rapid detection of viral RNA targets. The RT-SIBA method includes a reverse transcriptase enzyme that allows one-step reverse transcription of RNA to complementary DNA (cDNA) and simultaneous amplification and detection of the cDNA by SIBA under isothermal reaction conditions. The RT-SIBA method was found to be more sensitive than PCR for the detection of influenza A and B and could detect 100 copies of influenza RNA within 15 min. The development of RT-SIBA will enable rapid and accurate diagnosis of viral RNA targets within point-of-care or central laboratory settings.

Analysis of Acquisition and Titer of Maize Mosaic Rhabdovirus in Its Vector, Peregrinus maidis (Hemiptera: Delphacidae)

PubMed Central

Barandoc-Alviar, Karen; Ramirez, Girly M.; Rotenberg, Dorith; Whitfield, Anna E.

2016-01-01

The corn planthopper, Peregrinus maidis (Ashmead) (Hemiptera: Delphacidae), transmits Maize mosaic rhabdovirus (MMV), an important pathogen of maize and sorghum, in a persistent propagative manner. To better understand the vectorial capacity of P. maidis, we determined the efficiency of MMV acquisition by nymphal and adult stages, and characterized MMV titer through development. Acquisition efficiency, i.e., proportion of insects that acquired the virus, was determined by reverse transcriptase polymerase chain reaction (RT-PCR) and virus titer of individual insects was estimated by quantitative RT-PCR. Acquisition efficiency of MMV differed significantly between nymphs and adults. MMV titer increased significantly over time and throughout insect development from nymphal to adult stage, indication of virus replication in the vector during development. There was a positive association between the vector developmental stage and virus titer. Also, the average titer in male insects was threefold higher than female titers, and this difference persisted up to 30 d post adult eclosion. Overall, our findings indicate that nymphs are more efficient than adults at acquiring MMV and virus accumulated in the vector over the course of nymphal development. Furthermore, sustained infection over the lifespan of P. maidis indicates a potentially high capacity of this vector to transmit MMV. PMID:28076276

Natural environmental water sources in endemic regions of northeastern Brazil are potential reservoirs of viable Mycobacterium leprae.

PubMed

Arraes, Maria Luisa Bezerra de Macedo; Holanda, Maísa Viana de; Lima, Luana Nepomuceno Gondim Costa; Sabadia, José Antônio Beltrão; Duarte, Cynthia Romariz; Almeida, Rosa Livia Freitas; Kendall, Carl; Kerr, Ligia Regina Sansigolo; Frota, Cristiane Cunha

2017-12-01

The detection of live Mycobacterium leprae in soil and animals other than humans suggests that the environment plays a role in the transmission of leprosy. The objective of this study was to investigate the presence of viable M. leprae in natural water sources used by the local population in five municipalities in the state of Ceará, northeastern Brazil. Samples were collected from 30 different sources. Viable bacilli were identified by reverse transcriptase polymerase chain reaction (PCR) of the M. leprae gyrA gene and sequencing of the PCR products. Physicochemical properties of each water source were also assessed. M. leprae gyrA mRNA was found in 23 (76.7%) of the water sources. No association was found between depth of the water and sample positivity, nor was there any association between the type of water used by the population and sample positivity. An association between viable M. leprae and temperature and pH was found. Georeferencing showed a relation between the residences of leprosy cases and water source containing the bacterium. The finding of viable M. leprae in natural water sources associated with human contact suggests that the environment plays an important role in maintaining endemic leprosy in the study region.

Syncytial Hepatitis of Tilapia ( Oreochromis niloticus L.) is Associated With Orthomyxovirus-Like Virions in Hepatocytes.

PubMed

Del-Pozo, J; Mishra, N; Kabuusu, R; Cheetham, S; Eldar, A; Bacharach, E; Lipkin, W I; Ferguson, H W

2017-01-01

Using transmission electron microscopy (TEM), the presented work expands on the ultrastructural findings of an earlier report on "syncytial hepatitis," a novel disease of tilapia (SHT). Briefly, TEM confirmed the presence of an orthomyxovirus-like virus within the diseased hepatocytes but not within the endothelium. This was supported by observing extracellular and intracellular (mostly intraendosomal), 60-100 nm round virions with a trilaminar capsid containing up to 7 electron-dense aggregates. Other patterns noted included enveloped or filamentous virions and virion-containing cytoplasmic membrane folds, suggestive of endocytosis. Patterns atypical for orthymyxovirus included the formation of syncytia and the presence of virions within the perinuclear cisternae (suspected to be the Golgi apparatus). The ultrastructural morphology of SHT-associated virions is similar to that previously reported for tilapia lake virus (TiLV). A genetic homology was investigated using the available reverse transcriptase polymerase chain reaction (RT-PCR) probes for TiLV and comparing clinically sick with clinically normal fish and negative controls. By RT-PCR analysis, viral nucleic acid was detected only in diseased fish. Taken together, these findings strongly suggest that a virus is causally associated with SHT, that this virus shares ultrastructural features with orthomyxoviruses, and it presents with partial genetic homology with TiLV (190 nucleotides).

Virus-Induced Gene Silencing in Cultivated Cotton (Gossypium spp.) Using Tobacco Rattle Virus.

PubMed

Mustafa, Roma; Shafiq, Muhammad; Mansoor, Shahid; Briddon, Rob W; Scheffler, Brian E; Scheffler, Jodi; Amin, Imran

2016-01-01

The study described here has optimized the conditions for virus-induced gene silencing (VIGS) in three cultivated cotton species (Gossypium hirsutum, G. arboreum, and G. herbaceum) using a Tobacco rattle virus (TRV) vector. The system was used to silence the homolog of the Arabidopsis thaliana chloroplastos alterados 1 (AtCLA1) gene, involved in chloroplast development, in G. herbaceum, G. arboreum, and six commercial G. hirsutum cultivars. All plants inoculated with the TRV vector to silence CLA1 developed a typical albino phenotype indicative of silencing this gene. Although silencing in G. herbaceum and G. arboreum was complete, silencing efficiency differed for each G. hirsutum cultivar. Reverse transcriptase polymerase chain reaction (PCR) and real-time quantitative PCR showed a reduction in mRNA levels of the CLA1 homolog in all three species, with the highest efficiency (lowest CLA1 mRNA levels) in G. arboreum followed by G. herbaceum and G. hirsutum. The results indicate that TRV is a useful vector for VIGS in Gossypium species. However, selection of host cultivar is important. With the genome sequences of several cotton species recently becoming publicly available, this system has the potential to provide a very powerful tool for the rapid, large-scale reverse-genetic analysis of genes in Gossypium spp.

Challenges in Interpretation of Diagnostic Test Results in a Mumps Outbreak in a Highly Vaccinated Population

PubMed Central

Mercer, N. J.; Paphitis, K.; Walters, J. M.; Wallace, D.; Kristjanson, E.; Gubbay, J.; Mazzulli, T.

2016-01-01

ABSTRACT In spite of a greatly reduced incidence rate due to vaccination, mumps outbreaks continue to occur in several areas of the world, sometimes in vaccinated populations. This article describes an outbreak in a highly vaccinated population in southwestern Ontario, Canada, and the challenges encountered in interpreting the results of diagnostic tests used in the outbreak. During the outbreak, patients were interviewed and classified according to the outbreak case definition, and specimens were collected for diagnostic testing according to Ontario guidelines. Twenty-seven individuals were classified as confirmed cases (n = 19) or suspect cases (n = 8) according to the case definition, only 9 of which were laboratory-confirmed cases: 7 confirmed by reverse transcriptase PCR (RT-PCR) and 2 by IgM serology. All 19 confirmed cases represented patients who were associated with secondary schools in the local area and had been vaccinated against mumps with one (n = 2) or two (n = 17) doses of the measles-mumps-rubella (MMR) vaccine. This is the first published report of an outbreak of mumps in Ontario in which all confirmed cases had been vaccinated against the disease. It highlights the limitations of and difficulties in interpreting current mumps diagnostic tests when used in vaccinated individuals. PMID:28003216

Submicroscopic malaria infections in pregnant women from six departments in Haiti.

PubMed

Elbadry, Maha A; Tagliamonte, Massimiliano S; Raccurt, Christian P; Lemoine, Jean F; Existe, Alexandre; Boncy, Jacques; Weppelmann, Thomas A; Dame, John B; Okech, Bernard A

2017-08-01

To describe the epidemiology of malaria in pregnancy in Haiti. Cross-sectional study among pregnant women in six departments of Haiti. After obtaining informed consent, whole blood samples and demographic surveys were collected to investigate malaria prevalence, anaemia and socio-behavioural risk factors for infection, respectively. A total of 311 pregnant women were screened for Plasmodium falciparum infection using a rapid diagnostic test (RDT), microscopy and a novel, quantitative reverse transcriptase polymerase chain reaction method (qRT-PCR). Overall, 1.2% (4/311) of pregnant women were tested positive for malaria infection by both microscopy and RDT. However, using the qRT-PCR, 16.4% (51/311) of pregnant women were positive. The prevalence of malaria infection varied with geographical locations ranging between 0% and 46.4%. Additionally, 53% of pregnant women had some form of anaemia; however, no significant association was found between anaemia and submicroscopic malaria infection. The socio-behavioural risk factors identified to be protective of malaria infection were marital status (P < 0.05) and travel within one month prior to screening (P < 0.05). This study is the first to document the high prevalence of submicroscopic malaria infections among pregnant women in Haiti and identify social and behavioural risk factors for disease transmission. © 2017 John Wiley & Sons Ltd.

Integration of high-risk human papillomavirus into cellular cancer-related genes in head and neck cancer cell lines.

PubMed

Walline, Heather M; Goudsmit, Christine M; McHugh, Jonathan B; Tang, Alice L; Owen, John H; Teh, Bin T; McKean, Erin; Glover, Thomas W; Graham, Martin P; Prince, Mark E; Chepeha, Douglas B; Chinn, Steven B; Ferris, Robert L; Gollin, Susanne M; Hoffmann, Thomas K; Bier, Henning; Brakenhoff, Ruud; Bradford, Carol R; Carey, Thomas E

2017-05-01

Human papillomavirus (HPV)-positive oropharyngeal cancer is generally associated with excellent response to therapy, but some HPV-positive tumors progress despite aggressive therapy. The purpose of this study was to evaluate viral oncogene expression and viral integration sites in HPV16- and HPV18-positive squamous cell carcinoma lines. E6/E7 alternate transcripts were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Detection of integrated papillomavirus sequences (DIPS-PCR) and sequencing identified viral insertion sites and affected host genes. Cellular gene expression was assessed across viral integration sites. All HPV-positive cell lines expressed alternate HPVE6/E7 splicing indicative of active viral oncogenesis. HPV integration occurred within cancer-related genes TP63, DCC, JAK1, TERT, ATR, ETV6, PGR, PTPRN2, and TMEM237 in 8 head and neck squamous cell carcinoma (HNSCC) lines but UM-SCC-105 and UM-GCC-1 had only intergenic integration. HPV integration into cancer-related genes occurred in 7 of 9 HPV-positive cell lines and of these 6 were from tumors that progressed. HPV integration into cancer-related genes may be a secondary carcinogenic driver in HPV-driven tumors. © 2017 Wiley Periodicals, Inc. Head Neck 39: 840-852, 2017. © 2017 Wiley Periodicals, Inc.

Leptin influences estrogen metabolism and accelerates prostate cell proliferation.

PubMed

Habib, Christine N; Al-Abd, Ahmed M; Tolba, Mai F; Khalifa, Amani E; Khedr, Alaa; Mosli, Hisham A; Abdel-Naim, Ashraf B

2015-01-15

The present study was designed to investigate the effect of leptin on estrogen metabolism in prostatic cells. Malignant (PC-3) and benign (BPH-1) human prostate cells were treated with 17-β-hydroxyestradiol (1 μM) alone or in combination with leptin (0.4, 4, 40 ng/ml) for 72 h. Cell proliferation assay, immunocytochemical staining of estrogen receptor (ER), liquid chromatography-tandem mass spectrometry method (LC-MS) and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) were used. Cell proliferation assay demonstrated that leptin caused significant growth potentiation in both cells. Immunocytochemical staining showed that leptin significantly increased the expression of ER-α and decreased that of ER-β in PC-3 cells. LC-MS method revealed that leptin increased the concentration 4-hydroxyestrone and/or decreased that of 2-methoxyestradiol, 4-methoxyestradiol and 2-methoxyestrone. Interestingly, RT-PCR showed that leptin significantly up-regulated the expression of aromatase and cytochrome P450 1B1 (CYP1B1) enzymes; however down-regulated the expression of catechol-o-methyltransferase (COMT) enzyme. These data indicate that leptin-induced proliferative effect in prostate cells might be partly attributed to estrogen metabolism. Thus, leptin might be a novel target for therapeutic intervention in prostatic disorders. Copyright © 2014 Elsevier Inc. All rights reserved.

A multigene family related to chitin synthase genes of yeast in the opportunistic pathogen Aspergillus fumigatus.

PubMed

Mellado, E; Aufauvre-Brown, A; Specht, C A; Robbins, P W; Holden, D W

1995-02-06

Two approaches were used to isolate fragments of chitin synthase genes from the opportunistic human pathogen Aspergillus fumigatus. Firstly, regions of amino acid conservation in chitin synthases of Saccharomyces cerevisiae were used to design degenerate primers for amplification of portions of related genes, and secondly, a segment of the S. cerevisiae CSD2 gene was used to screen an A. fumigatus lambda genomic DNA library. the polymerase chain reaction (PCR)-based approach led to the identification of five different genes, designated chsA, chsB, chsC, chsD and chsE. chsA, chsB, and chsC fall into Classes I, II and III of the 'zymogen type' chitin synthases, respectively. The chsD fragment has approximately 35% amino acid sequence identity to both the zymogen type genes and the non-zymogen type CSD2 gene. chsF appears to be a homologue of CSD2, being 80% identical to CSD2 over 100 amino acids. An unexpected finding was the isolation by heterologous hybridization of another gene (chsE), which also has strong sequence similarity (54% identity at the amino acid level over the same region as chsF) to CSD2. Reverse transcriptase-PCR was used to show that each gene is expressed during hyphal growth in submerged cultures.

Transcriptomic study of 39 ostreid herpesvirus 1 genes during an experimental infection.

PubMed

Segarra, Amélie; Faury, Nicole; Pépin, Jean-François; Renault, Tristan

2014-06-01

Massive mortality outbreaks have been reported in France since 2008 among Pacific oysters, Crassostrea gigas, with the detection of a particular OsHV-1 variant called μVar. Virus infection can be induced in healthy spat in experimental conditions allowing to better understand the disease process, including viral gene expression. Although gene expression of other herpesviruses has been widely studied, we provide the first study following viral gene expression of OsHV-1 over time. In this context, an in vivo transcriptomic study targeting 39 OsHV-1 genes was carried out during an experimental infection of Pacific oyster spat. For the first time, several OsHV-1 mRNAs were detected by real-time PCR at 0 h, 2 h, 4 h, 18 h, 26 h and 42 h post-injection. Several transcripts were detected at 2h post-infection and at 18 h post-infection for all selected ORFs. Quantification of virus gene expression at different times of infection was also carried out using an oyster housekeeping gene, Elongation factor. Developing an OsHV-1-specific reverse transcriptase real time PCR targeting 39 viral gene appears a new tool in terms of diagnosis and can be used to complement viral DNA detection in order to monitor viral replication. Copyright © 2014. Published by Elsevier Inc.

Degradation of foot-and-mouth disease virus during composting of infected pig carcasses

PubMed Central

Guan, J.; Chan, M.; Grenier, C.; Brooks, B.W.; Spencer, J.L.; Kranendonk, C.; Copps, J.; Clavijo, A.

2010-01-01

The objective of this study was to investigate the inactivation and degradation of foot-and-mouth disease (FMD) virus during composting of infected pig carcasses as measured by virus isolation in tissue culture and by real-time reverse transcriptase polymerase chain reaction (RRT-PCR). Three FMD-infected pig carcasses were composted in a mixture of chicken manure and wood shavings in a biocontainment level 3 facility. Compost temperatures had reached 50°C and 70°C by days 10 and 19, respectively. Under these conditions, FMD virus was inactivated in specimens in compost by day 10 and the viral RNA was degraded in skin and internal organ tissues by day 21. In comparison, at ambient temperatures close to 20°C, FMD virus survived to day 10 in the skin tissue specimen from the pig that had the highest initial level of viral RNA in its tissues and the viral RNA persisted to day 21. Similarly, beta-actin mRNA, tested as a PCR control, persisted to day 21 in specimens held at ambient temperatures, but it was degraded in the remnants of tissues recovered from compost on day 21. Results from this study provide evidence that composting could be used for safe disposal of pig carcasses infected with FMD virus. PMID:20357957

Stable Reference Gene Selection for RT-qPCR Analysis in Nonviruliferous and Viruliferous Frankliniella occidentalis

PubMed Central

Pan, Huipeng; Ma, Yabin; Zhang, Deyong; Liu, Yong; Zhang, Zhanhong; Zheng, Changying; Chu, Dong

2015-01-01

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for measuring and evaluating gene expression during variable biological processes. To facilitate gene expression studies, normalization of genes of interest relative to stable reference genes is crucial. The western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), the main vector of tomato spotted wilt virus (TSWV), is a destructive invasive species. In this study, the expression profiles of 11 candidate reference genes from nonviruliferous and viruliferous F. occidentalis were investigated. Five distinct algorithms, geNorm, NormFinder, BestKeeper, the ΔC t method, and RefFinder, were used to determine the performance of these genes. geNorm, NormFinder, BestKeeper, and RefFinder identified heat shock protein 70 (HSP70), heat shock protein 60 (HSP60), elongation factor 1 α, and ribosomal protein l32 (RPL32) as the most stable reference genes, and the ΔC t method identified HSP60, HSP70, RPL32, and heat shock protein 90 as the most stable reference genes. Additionally, two reference genes were sufficient for reliable normalization in nonviruliferous and viruliferous F. occidentalis. This work provides a foundation for investigating the molecular mechanisms of TSWV and F. occidentalis interactions. PMID:26244556

Proteomic and transcriptional analysis of Lactobacillus johnsonii PF01 during bile salt exposure by iTRAQ shotgun proteomics and quantitative RT-PCR.

PubMed

Lee, Ji Yoon; Pajarillo, Edward Alain B; Kim, Min Jeong; Chae, Jong Pyo; Kang, Dae-Kyung

2013-01-04

Lactobacillus johnsonii PF01 has been reported to be highly resistant to bile, a key property of probiotic microorganisms. Here, we examine the nature of the bile-salt tolerance of L. johnsonii PF01. Growth inhibition and surface morphology and physiology aberrations were observed after overnight exposure to bile stress. Quantitative proteomic profiles using iTRAQ-LC-MS/MS technology identified 8307 peptides from both untreated PF01 cells and those exposed to 0.1%, 0.2%, and 0.3% bile salts. Some 215 proteins exhibited changed levels in response to bile stress; of these, levels of 94 peptides increased while those of 121 decreased. These were classified into the following categories: stress responses, cell division, transcription, translation, nucleotide metabolism, carbohydrate transport and metabolism, cell wall biosynthesis, and amino acid biosynthesis, and 16 of unidentified function. Analysis of the mRNA expression of selected genes by quantitative reverse transcriptase-PCR verified the proteomic data. Both proteomic and mRNA data provided evidence for increased phosphotransferase activity and cell wall biosynthesis. In addition, three bile salt hydrolases were significantly upregulated by bile exposure. These findings provide a basis for future evaluations of the tolerance of potential probiotic strains toward the various gastrointestinal challenges, including bile stress.

The Discovery of Reverse Transcriptase.

PubMed

Coffin, John M; Fan, Hung

2016-09-29

In 1970 the independent and simultaneous discovery of reverse transcriptase in retroviruses (then RNA tumor viruses) by David Baltimore and Howard Temin revolutionized molecular biology and laid the foundations for retrovirology and cancer biology. In this historical review we describe the formulation of the controversial provirus hypothesis by Temin, which ultimately was proven by his discovery of reverse transcriptase in Rous sarcoma virus virions. Baltimore arrived at the same discovery through his studies on replication of RNA-containing viruses, starting with poliovirus and then moving to vesicular stomatitis virus, where he discovered a virion RNA polymerase. Subsequent studies of reverse transcriptase led to the elucidation of the mechanism of retrovirus replication, the discovery of oncogenes, the advent of molecular cloning, the search for human cancer viruses, and the discovery and treatment of HIV/AIDS.

Microfluidics-based digital quantitative PCR for single-cell small RNA quantification.

PubMed

Yu, Tian; Tang, Chong; Zhang, Ying; Zhang, Ruirui; Yan, Wei

2017-09-01

Quantitative analyses of small RNAs at the single-cell level have been challenging because of limited sensitivity and specificity of conventional real-time quantitative PCR methods. A digital quantitative PCR (dqPCR) method for miRNA quantification has been developed, but it requires the use of proprietary stem-loop primers and only applies to miRNA quantification. Here, we report a microfluidics-based dqPCR (mdqPCR) method, which takes advantage of the Fluidigm BioMark HD system for both template partition and the subsequent high-throughput dqPCR. Our mdqPCR method demonstrated excellent sensitivity and reproducibility suitable for quantitative analyses of not only miRNAs but also all other small RNA species at the single-cell level. Using this method, we discovered that each sperm has a unique miRNA profile. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus).

PubMed

Yang, Chang Geng; Wang, Xian Li; Tian, Juan; Liu, Wei; Wu, Fan; Jiang, Ming; Wen, Hua

2013-09-15

Quantitative real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) has been used frequently to study gene expression related to fish immunology. In such studies, a stable reference gene should be selected to correct the expression of the target gene. In this study, seven candidate reference genes (glyceraldehyde-3-phosphate dehydrogenase (GADPH), ubiquitin-conjugating enzyme (UBCE), 18S ribosomal RNA (18S rRNA), beta-2-microglobulin (B2M), elongation factor 1 alpha (EF1A), tubulin alpha chain-like (TUBA) and beta actin (ACTB)), were selected to analyze their stability and normalization in seven tissues (liver, spleen, kidney, brain, heart, muscle and intestine) of Nile tilapia (Oreochromis niloticus) challenged with Streptococcus agalactiae or Streptococcus iniae, respectively. The results showed that all the candidate reference genes exhibited tissue-dependent transcriptional variations. With PBS injection as a control, UBCE was the most stable and suitable single reference gene in the intestine, liver, brain, kidney, and spleen after S. iniae infection, and in the liver, kidney, and spleen after S. agalactiae infection. EF1A was the most suitable in heart and muscle after S. iniae or S. agalactiae infection. GADPH was the most suitable gene in intestine and brain after S. agalactiae infection. In normal conditions, UBCE and 18S rRNA were the most stably expressed genes across the various tissues. These results showed that for RT-qPCR analysis of tilapia, selecting two or more reference genes may be more suitable for cross-tissue analysis of gene expression. Copyright © 2013 Elsevier B.V. All rights reserved.