Amarogentin Induces Apoptosis of Liver Cancer Cells via Upregulation of p53 and Downregulation of Human Telomerase Reverse Transcriptase in Mice

PubMed Central

Li, Runqin; Zhang, Yinglin

2016-01-01

Background and Objective: Amarogentin has been reported to have a preventive effect on liver cancer via inducing cancer cell apoptosis. We attempted to elucidate the roles of p53-associated apoptosis pathways in the chemopreventive mechanism of amarogentin. The findings of this study will facilitate the development of a novel supplementary strategy for the treatment of liver cancer. Materials and Methods: The purity of amarogentin was assessed by high-performance liquid chromatography. The inhibitory ratios of the liver cell lines were determined using a Cell Counting Kit-8 following treatment with a gradient concentration of amarogentin. Cell apoptosis was detected by flow cytometry using annexin V-fluorescein isothiocyanate/propidium iodide kits. The gene and protein expression of p53-associated molecules, such as Akt, human telomerase reverse transcriptase, RelA, and p38, was detected by real-time quantitative polymerase chain reaction, Western blotting, and immunohistochemical staining in liver cancer cells and mouse tumor tissues after treatment with amarogentin. Results: The inhibitory effect of amarogentin on cell proliferation was more obvious in liver cancer cells, and amarogentin was more likely to induce the apoptosis of liver cancer cells than that of normal liver cells. The gene and protein expression levels of Akt, RelA, and human telomerase reverse transcriptase were markedly higher in the control group than in the preventive group and treatment groups. Only the expression of human telomerase reverse transcriptase was downregulated, accompanied by the upregulation of p53. Conclusion: The results of our study suggest that amarogentin promotes apoptosis of liver cancer cells by the upregulation of p53 and downregulation of human telomerase reverse transcriptase and prevents the malignant transformation of these cells. PMID:27402632

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.

The origin and early evolution of nucleic acid polymerases

NASA Technical Reports Server (NTRS)

Lazcano, A.; Cappello, R.; Valverde, V.; Llaca, V.; Oro, J.

1992-01-01

The hypothesis that vestiges of the ancestral RNA-dependent RNA polymerase involved in the replication of RNA genomes of Archean cells are present in the eubacterial RNA-polymerase beta-prime subunit and its homologues is discussed. It is shown that, in the DNA-dependent RNA polymerases from three cellular lineages, a very conserved sequence of eight amino acids, also found in a small RNA-binding site previously described for the E. coli polynucleotide phosphorylase and the S1 ribosomal protein, is present. The optimal conditions for the replicase activity of the avian-myeloblastosis-virus reverse transcriptase are presented. The evolutionary significance of the in vitro modifications of substrate and template specificities of RNA polymerases and reverse transcriptases is discussed.

Biofilms and Antifungal Susceptibility Testing.

PubMed

Simitsopoulou, Maria; Chatzimoschou, Athanasios; Roilides, Emmanuel

2016-01-01

Yeasts and filamentous fungi both exist as single cells and hyphal forms, two morphologies used by most fungal organisms to create a complex multilayered biofilm structure. In this chapter we describe the most widely used assays for the determination of biofilm production and assessment of susceptibility of biofilms to antifungal agents or host phagocytes as various methods, the most frequent of which are staining, confocal laser scanning microscopy, quantification of extracellular DNA and protein associated with extracellular matrix and XTT metabolic reduction assay. Pathway-focused biofilm gene expression profiling is assessed by real-time reverse transcriptase polymerase chain reaction.

[Application of transcription mediated amplification and real-time reverse transcription polymerase chain reaction in detection of human immunodeficiency virus RNA].

PubMed

Wu, Daxian; Tao, Shuhui; Liu, Shuiping; Zhou, Jiebin; Tan, Deming; Hou, Zhouhua

2017-07-28

To observe the sensitivity of transcription mediated amplification (TMA), and to compare its performance with real-time reverse transcription polymerase chain reaction (real-time RT-PCR) in detecting human immunodeficiency virus RNA (HIV RNA).
 Methods: TMA system was established with TaqMan probes, specific primers, moloney murine leukemia virus (MMLV) reverse transcriptase, T7 RNA polymerase, and reaction substrates. The sensitivity of TMA was evaluated by amplifying a group of 10-fold diluted HIV RNA standards which were transcribed in vitro. A total of 60 plasma of HIV infected patients were measured by TMA and Cobas Amplicor HIV-1 Monitor test to observe the positive rate. The correlation and concordance of the above two technologies were investigated by linear regression and Bland-Altman analysis.
 Results: TMA system was established successfully and HIV RNA transcribed standards at concentration of equal or more than 10 copies/mL could be detected by TMA technology. Among 60 samples of plasma from HIV infected patients, 46 were positively detected and 12 were negatively amplified by both TMA and Cobas reagents; 2 samples were positively tested by Cobas reagent but negatively tested by TMA system. The concordance rate of the two methods was 97.1% and the difference of positive detection rate between the two methods was not statistically significant (P>0.05). Linear regression was used for 46 samples which were positively detected by both TMA and Cobas reagents and showed an excellent correlation between the two reagents (r=0.997, P<0.001). Bland-Altma analysis revealed that the mean different value of HIV RNA levels for denary logarithm was 0.02. Forty-four samples were included in 95% of credibility interval of concordance.
 Conclusion: TMA system has the potential of high sensitivity. TMA and real-time RT-PCR keep an excellent correlation and consistency in detecting HIV RNA.

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

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.

Novel Structure of Ty3 Reverse Transcriptase | Center for Cancer Research

Cancer.gov

Retrotransposons are mobile genetic elements that self amplify via a single-stranded RNA intermediate, which is converted to double-stranded DNA by an encoded reverse transcriptase (RT) with both DNA polymerase (pol) and ribonuclease H (RNase) activities. Categorized by whether they contain flanking long terminal repeat (LTR) sequences, retrotransposons play a critical role in

A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication.

PubMed

Herzig, Eytan; Voronin, Nickolay; Kucherenko, Nataly; Hizi, Amnon

2015-08-01

The process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting the in vitro data. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis. Reverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting the in vitro data. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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.

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.

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.

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.

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.

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

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.

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

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...

Reverse transcriptase activity and particles of retroviral density in cultured canine lymphosarcoma supernatants.

PubMed Central

Tomley, F. M.; Armstrong, S. J.; Mahy, B. W.; Owen, L. N.

1983-01-01

Lymphoid tissue from 43 cases of canine lymphosarcoma and from 40 clinically normal dogs have been examined for markers of retrovirus infection. From 69-76% of culture supernatants from lymphosarcomas were shown to contain particles of retroviral density and to possess poly rC-oligo dG templated polymerase (reverse transcriptase) activity compared with 17-24% of culture supernatants from normal canine lymphoid cells. In 6 culture supernatants from cases of lymphosarcoma, high molecular weight 60-70S RNA was detected and shown to be found in association with this particulate reverse transcriptase activity. No such RNA was detected in 6 culture supernatants from normal canine lymphoid cells. PMID:6186265

Novel Structure of Ty3 Reverse Transcriptase | Center for Cancer Research

Cancer.gov

Retrotransposons are mobile genetic elements that self amplify via a single-stranded RNA intermediate, which is converted to double-stranded DNA by an encoded reverse transcriptase (RT) with both DNA polymerase (pol) and ribonuclease H (RNase) activities. Categorized by whether they contain flanking long terminal repeat (LTR) sequences, retrotransposons play a critical role in the architecture of eukaryotic genomes and are the evolutionary origin of retroviruses, including human immunodeficiency virus (HIV).

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

Synthesis, Activity and Structural Analysis of Novel α-Hydroxytropolone Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease H

PubMed Central

Chung, Suhman; Himmel, Daniel M.; Jiang, Jian-Kang; Wojtak, Krzysztof; Bauman, Joseph D.; Rausch, Jason W.; Wilson, Jennifer A.; Beutler, John A.; Thomas, Craig J.; Arnold, Eddy; Le Grice, Stuart F.J.

2011-01-01

The α-hydroxytroplone, manicol (5,7-dihydroxy-2-isopropenyl-9-methyl-1,2,3,4-tetrahydro-benzocyclohepten-6-one) potently and specifically inhibits ribonuclease H (RNase H) activity of human immunodeficiency virus reverse transcriptase (HIV RT) in vitro. However, manicol was ineffective in reducing virus replication in culture. Ongoing efforts to improve the potency and specificity over the lead compound led us to synthesize 14 manicol derivatives that retain the divalent metal-chelating α-hydroxytropolone pharmacophore. These efforts were augmented by a high resolution structure of p66/p51 HIV-1 RT containing the nonnucleoside reverse transcriptase inhibitor (NNRTI), TMC278 and manicol in the DNA polymerase and RNase H active sites, respectively. We demonstrate here that several modified α-hydroxytropolones exhibit antiviral activity at non-cytotoxic concentrations. Inclusion of RNase H active site mutants indicated that manicol analogs can occupy an additional site in or around the DNA polymerase catalytic center. Collectively, our studies will promote future structure-based design of improved α-hydroxytropolones to complement the NRTI and NNRTI currently in clinical use. PMID:21568335

A modified single-tube one-step product-enhanced reverse transcriptase (mSTOS-PERT) assay with heparin as DNA polymerase inhibitor for specific detection of RTase activity.

PubMed

Fan, Xiao-Yong; Lü, Guo-Zhen; Wu, Li-Na; Chen, Jing-Hua; Xu, Wen-Qing; Zhao, Chun-Nü; Guo, Sheng-Qi

2006-12-01

Current regulations and recommendations proposed for the production of vaccines in continuous cell lines of any origin demand that these be free of exogenous viruses, particularly retroviruses. Recently, the ultra-sensitive product-enhanced reverse transcriptase (PERT) assay can be used to detect minute of reverse transcriptase (RTase) in single retroviral particle and is 10(6) times more sensitive than the conventional RTase assays. However, coincidental with this increase in sensitivity is an increase in false-positive reactions derived from contaminating cellular DNA polymerases, which are known to have RTase-like activities. To develop a modified single-tube one-step PERT (mSTOS-PERT) assay with improvements on decreasing significantly the level of false-positive reactions, and to evaluate the mSTOS-PERT assay for sensitivity and specificity. Ampliwaxtrade mark was used to compartmentalize the reverse transcription (RT) and PCR step in the same micro-tube with more efficiency and reproducibility, while maintaining the high sensitivity. The DNA amplification products were separated by 2% agarose gel electrophoresis, and then analyzed by non-isotopic Southern blot hybridization. A wide variety of cell lines used in biologicals production were detected to validate the improved mSTOS-PERT assay. The detection limit for the mSTOS-PERT assay was at least 10(-9)U, when using AMV-RTase as a positive control. Furthermore, heparin involvement in the RT step can eliminate completely the false-positive PERT signals which are exhibited by cellular polymerases such as DNA-dependent DNA polymerase alpha, gamma released by cell death. Most mammalian cells (MRC-5, Vero, WISH, 2BS, RK-13, MDCK, etc.) are PERT-negative in cell supernatants. Some PERT-positive signals in cell lysates were found to be introduced by the cellular DNA polymerases and could be inhibited specifically by heparin. Chick cells derived from either chick embryo fibroblasts (CEF) or allantoic fluid from SPF embryonated eggs, murine hybridoma cell SP2/0, etc., contained authentic RTase activities, which could not be inactivated by heparin. The improved mSTOS-PERT assay described here may distinguish the genuine RTase activity from cellular polymerases with high sensitivity and specificity, and is rapid and easy to perform to screen for the possible contamination of minute retroviruses in the cell substrates used in vaccine production.

Synthesis, biological evaluation and molecular modeling of 2-Hydroxyisoquinoline-1,3-dione analogues as inhibitors of HIV reverse transcriptase associated ribonuclease H and polymerase.

PubMed

Tang, Jing; Vernekar, Sanjeev Kumar V; Chen, Yue-Lei; Miller, Lena; Huber, Andrew D; Myshakina, Nataliya; Sarafianos, Stefan G; Parniak, Michael A; Wang, Zhengqiang

2017-06-16

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not clinically validated as an antiviral target. 2-Hydroxyisoquinoline-1,3-dione (HID) is known to confer active site directed inhibition of divalent metal-dependent enzymatic functions, such as HIV RNase H, integrase (IN) and hepatitis C virus (HCV) NS5B polymerase. We report herein the synthesis and biochemical evaluation of a few C-5, C-6 or C-7 substituted HID subtypes as HIV RNase H inhibitors. Our data indicate that while some of these subtypes inhibited both the RNase H and polymerase (pol) functions of RT, potent and selective RNase H inhibition was achieved with subtypes 8-9 as exemplified with compounds 8c and 9c. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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.

Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

PubMed Central

Euler, Milena; Wang, Yongjie; Heidenreich, Doris; Patel, Pranav; Strohmeier, Oliver; Hakenberg, Sydney; Niedrig, Matthias; Hufert, Frank T.

2013-01-01

Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and Marburg virus. Their analytical sensitivities ranged from 16 to 21 molecules detected (probit analysis) for the majority of RPA and RT-RPA assays. A magnetic bead-based total nucleic acid extraction method was combined with the RPAs and tested using inactivated whole organisms spiked into plasma. The RPA showed comparable sensitivities to real-time RCR assays in these extracts. The run times of the assays at 42°C ranged from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome. The RPAs therefore seem suitable for the implementation of syndromic panels onto microfluidic platforms. PMID:23345286

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

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.

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.

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

Emergence of a replicating species from an in vitro RNA evolution reaction

NASA Technical Reports Server (NTRS)

Breaker, R. R.; Joyce, G. F.

1994-01-01

The technique of self-sustained sequence replication allows isothermal amplification of DNA and RNA molecules in vitro. This method relies on the activities of a reverse transcriptase and a DNA-dependent RNA polymerase to amplify specific nucleic acid sequences. We have modified this protocol to allow selective amplification of RNAs that catalyze a particular chemical reaction. During an in vitro RNA evolution experiment employing this modified system, a unique class of "selfish" RNAs emerged and replicated to the exclusion of the intended RNAs. Members of this class of selfish molecules, termed RNA Z, amplify efficiently despite their inability to catalyze the target chemical reaction. Their amplification requires the action of both reverse transcriptase and RNA polymerase and involves the synthesis of both DNA and RNA replication intermediates. The proposed amplification mechanism for RNA Z involves the formation of a DNA hairpin that functions as a template for transcription by RNA polymerase. This arrangement links the two strands of the DNA, resulting in the production of RNA transcripts that contain an embedded RNA polymerase promoter sequence.

Evidence for retrovirus infections in green turtles Chelonia mydas from the Hawaiian islands

USGS Publications Warehouse

Casey, R.N.; Quackenbush, S.L.; Work, Thierry M.; Balazs, G.H.; Bowser, P.R.; Casey, J.W.

1997-01-01

Apparently normal Hawaiian green turtles Chelonia mydas and those displaying fibropapillomas were analyzed for infection by retroviruses. Strikingly, all samples were positive for polymerase enhanced reverse transcriptase (PERT) with levels high enough to quantitate by the conventional reverse transcriptase (RT) assay. However, samples of skin, even from asymptomatic turtles, were RT positive, although the levels of enzyme activity in healthy turtles hatched and raised in captivity were much lower than those observed in asymptomatic free-ranging turtles. Turtles with fibropapillomas displayed a broad range of reverse transcriptase activity. Skin and eye fibropapillomas and a heart tumor were further analyzed and shown to have reverse transcriptase activity that banded in a sucrose gradient at 1.17 g ml-1. The reverse transcriptase activity purified from the heart tumor displayed a temperature optimum of 37??C and showed a preference for Mn2+ over Mg2+. Sucrose gradient fractions of this sample displaying elevated reverse transcriptase activity contained primarily retrovitalsized particles with prominent envelope spikes, when negatively stained and examined by electron microscopy. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of gradient-purified virions revealed a conserved profile among 4 independent tumors and showed 7 prominent proteins having molecular weights of 116, 83, 51, 43, 40, 20 and 14 kDa. The data suggest that retroviral infections are widespread in Hawaiian green turtles and a comprehensive investigation is warranted to address the possibility that these agents cause green turtle fibropapillomatosis (GTFP).

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.

Expression of TGF-beta1, osteonectin, and BMP-4 in mandibular distraction osteogenesis with compression stimulation: reverse transcriptase-polymerase chain reaction study and biomechanical test.

PubMed

Kim, Uk-Kyu; Park, Seong-Jin; Seong, Wook-Jin; Heo, Jun; Hwang, Dae-Seok; Kim, Yong-Deok; Shin, Sang-Hun; Kim, Gyoo-Cheon

2010-09-01

This study compared the levels of transforming growth factor-beta1 (TGF-beta1), osteonectin, and bone morphogenetic protein-4 (BMP-4) expression in regenerated bone in a rabbit mandible that had undergone conventional distraction osteogenesis (DO) with those in regenerated bone from a modified DO technique with compression stimulation. A total of 42 rabbits were used in this reverse transcriptase-polymerase chain reaction study. In the control group, distraction was performed at 1 mm/day for 8 days. In the experimental group, overdistraction was performed for 10 days, followed by a 3-day latency period and 2 days of compression to achieve the same amount of DO. Three rabbits per subgroup were killed at 0, 5, 13, 20, 27, 34, and 41 days after the initial osteotomy. The levels of TGF-beta1, osteonectin, and BMP-4 in the bone regenerates were measured by reverse transcriptase-polymerase chain reaction. A biomechanical microhardness test was also performed in 8 rabbits as a separate experiment. Reverse transcriptase-polymerase chain reaction revealed a greater level of TGF-beta1 in the experimental group immediately after applying the compression force that continued for 2 weeks. The level then decreased to that of the control group at 3 weeks. The greater level of osteonectin in the experimental group after compression than that in the control group continued for 3 weeks. In the experimental group, the level of BMP-4 increased immediately after compression. However, the level in the control group decreased. The microhardness ratio of distracted bone to normal bone on the cortex was statistically different at 0.47 in the control group and 0.80 in the experimental group (P = .049) at 55 days after osteotomy. The effectiveness of the new DO technique with compression stimulation was confirmed by the gene expression study and the biomechanical test findings. Copyright 2010 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

miR-128 modulates chemosensitivity and invasion of prostate cancer cells through targeting ZEB1.

PubMed

Sun, Xianglun; Li, Youkong; Yu, Jie; Pei, Hong; Luo, Pengcheng; Zhang, Jie

2015-05-01

Recent reports strongly suggest the profound role of miRNAs in cancer therapeutic response and progression, including invasion and metastasis. The sensitivity to therapy and invasion is the major obstacle for successful treatment in prostate cancer. We aimed to investigate the regulative effect of miR-128/zinc-finger E-box-binding homeobox 1 axis on prostate cancer cell chemosensitivity and invasion. The miR-128 expression pattern of prostate cancer cell lines and tissues was detected by real-time reverse transcriptase-polymerase chain reaction, while the mRNA and protein expression levels of zinc-finger E-box-binding homeobox 1 were measured by real-time reverse transcriptase-polymerase chain reaction and western blot assay, respectively. Dual-luciferase reporter gene assay was used to find the direct target of miR-128. Furthermore, prostate cancer cells were treated with miR-128 mimic or zinc-finger E-box-binding homeobox 1-siRNA, and then the cells' chemosensitivity and invasion were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and transwell assay, respectively. We found miR-128 expression obviously decreased in prostate cancer tissues compared with paired normal tissues. Restored miR-128 expression sensitized prostate cancer cells to cisplatin and inhibited the invasion. Furthermore, there was an inverse expression pattern between miR-128 and zinc-finger E-box-binding homeobox 1 in prostate cancer cells and tissues, and zinc-finger E-box-binding homeobox 1 was identified as a direct target of miR-128 in prostate cancer. Knockdown of zinc-finger E-box-binding homeobox 1 expression efficiently sensitized prostate cancer cells to cisplatin and inhibited the invasion. However, ectopic zinc-finger E-box-binding homeobox 1 expression impaired the effects of miR-128 on chemosensitivity and invasion in prostate cancer cells. miR-128 functions as a potential cancer suppressor in prostate cancer progression and rational therapeutic strategies for prostate cancer would be developed based on miR-128/zinc-finger E-box-binding homeobox 1 axis. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Effects of Trichostatin A on drug uptake transporters in primary rat hepatocyte cultures

PubMed Central

Ramboer, Eva; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

2015-01-01

The present study was set up to investigate the effects of Trichostatin A (TSA), a prototypical epigenetic modifier, on the expression and activity of hepatic drug uptake transporters in primary cultured rat hepatocytes. To this end, the expression of the sinusoidal transporters sodium-dependent taurocholate cotransporting polypeptide (Ntcp) and organic anion transporting polypeptide 4 (Oatp4) was monitored by real-time quantitative reverse transcriptase polymerase chain reaction analysis and immunoblotting. The activity of the uptake transporters was analyzed using radiolabeled substrates and chemical inhibitors. Downregulation of the expression and activity of Oatp4 and Ntcp was observed as a function of the cultivation time and could not be counteracted by TSA. In conclusion, the epigenetic modifier TSA does not seem to exert a positive effect on the expression and activity of the investigated uptake transporters in primary rat hepatocyte cultures. PMID:26648816

Vascular endothelial growth factor and platelet-derived growth factor are potential angiogenic and metastatic factors in human breast cancer.

PubMed

Anan, K; Morisaki, T; Katano, M; Ikubo, A; Kitsuki, H; Uchiyama, A; Kuroki, S; Tanaka, M; Torisu, M

1996-03-01

Angiogenesis is a prerequisite for tumor growth and metastasis. Tumor angiogenesis may be mediated by several angiogenic factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), transforming growth factor-alpha, and basic fibroblast growth factor. Differential mRNA expressions of VEGF, PDGF (A chain), transforming growth factor-alpha and basic fibroblast growth factor in 32 primary invasive breast tumors were examined by reverse transcriptase-polymerase chain reaction. We analyzed relationships between mRNA expressions of these angiogenic factors and the degree of angiogenesis, tumor size, and metastasis. Quantification of angiogenesis was achieved by the immunohistochemical staining of endothelial cells with antibody to CD31. VEGF and PDGF-A mRNAs were expressed more frequently in breast tumors than in nontumor breast tissues, whereas no difference was found in expression frequency of either transforming growth factor-alpha or basic fibroblast growth factor mRNA. Vascular counts in tumors correlated with each expression frequency of VEGF and PDGF-A mRNA. PDGF-A mRNA was expressed more frequently in tumors with lymph node metastasis than in those without metastasis. Expression of VEGF and PDGF mRNAs detected by reverse transcriptase-polymerase chain reaction in breast tumors correlates with tumor-related characteristics of angiogenesis and metastatic potential. Analysis of these mRNAs by reverse transcriptase-polymerase chain reaction may be useful for assessing the biologic behavior of a breast tumor before surgical treatment.

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.

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.

A full-coordinate model of the polymerase domain of HIV-1 reverse transcriptase and its interaction with a nucleic acid substrate

NASA Technical Reports Server (NTRS)

Setlik, R. F.; Meyer, D. J.; Shibata, M.; Roskwitalski, R.; Ornstein, R. L.; Rein, R.

1994-01-01

We present a full-coordinate model of residues 1-319 of the polymerase domain of HIV-I reverse transcriptase. This model was constructed from the x-ray crystallographic structure of Jacobo-Molina et al. (Jacobo-Molina et al., P.N.A.S. USA 90, 6320-6324 (1993)) which is currently available to the degree of C-coordinates. The backbone and side-chain atoms were constructed using the MAXSPROUT suite of programs (L. Holm and C. Sander, J. Mol. Biol. 218, 183-194 (1991)) and refined through molecular modeling. A seven base pair A-form dsDNA was positioned in the nucleic acid binding cleft to represent the template-primer complex. The orientation of the template-primer complex in the nucleic acid binding cleft was guided by the positions of phosphorus atoms in the crystal structure.

The molecular genetic basis of mitochondrial malfunction in bladder tissue following outlet obstruction.

PubMed

Levin, Robert M; Hudson, Alan P

2004-08-01

Bladder dysfunction following partial outlet obstruction is a frequent consequence of benign prostatic hyperplasia and an increasingly common problem given the aging of the general population. Recent studies from this and other groups have begun to elucidate the molecular bases for the well described physiological malfunctions that characterize this clinical entity. We summarized and synthesized that information. Using modern methods of molecular genetics, including real-time polymerase chain reaction, real-time reverse transcriptase-polymerase chain reaction and others, as well as traditional experimental techniques such as electron microscopy we and others examined the transcriptional profile, morphology, etc of bladder smooth muscle mitochondria in experimental models of outlet obstruction. Data from many studies have demonstrated that aberrant gene expression in the mitochondrial and mitochondria related nuclear genetic systems underlies the loss of compliance and other attributes of bladder dysfunction following outlet obstruction. Such aberrant transcriptional characteristics engender loss of function in the electron transport and oxidative phosphorylation systems. Morphological studies of mitochondria in the animal model systems support this conclusion. In large part the loss of function in bladder smooth muscle following outlet obstruction results from the attenuation of mitochondrial energy production. In this article we reviewed and synthesized all available experimental observations relevant to this problem and we suggest future lines of inquiry that should prove fruitful in developing new strategies to treat the condition.

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.

Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time, reverse transcription polymerase chain reaction (rRT-PCR) and virus isolation

USDA-ARS?s Scientific Manuscript database

The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 real-time, reverse transcription polymerase chain reaction (rRT-PCR) and 7 virus isolation (VI) assays. To conduct the study, OF was inoculated with H1N1 or H3N2 IAV and serially 10-fold d...

Incorporation of deoxyribonucleotides and ribonucleotides by a dNTP-binding cleft mutated reverse transcriptase in hepatitis B virus core particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Hee-Young; Kim, Hye-Young; Jung, Jaesung

2008-01-05

Our recent observation that hepatitis B virus (HBV) DNA polymerase (P) might initiate minus-strand DNA synthesis without primer [Kim et al., (2004) Virology 322, 22-30], raised a possibility that HBV P protein may have the potential to function as an RNA polymerase. Thus, we mutated Phe 436, a bulky amino acid with aromatic side chain, at the putative dNTP-binding cleft in reverse transcriptase (RT) domain of P protein to smaller amino acids (Gly or Val), and examined RNA polymerase activity. HBV core particles containing RT dNTP-binding cleft mutant P protein were able to incorporate {sup 32}P-ribonucleotides, but not HBV coremore » particles containing wild type (wt), priming-deficient mutant, or RT-deficient mutant P proteins. Since all the experiments were conducted with core particles isolated from transfected cells, our results indicate that the HBV RT mutant core particles containing RT dNTP-binding cleft mutant P protein could incorporate both deoxyribonucleotides and ribonucleotides in replicating systems.« less

Direct and quantitative detection of HIV-1 RNA in human plasma with a branched DNA signal amplification assay.

PubMed

Urdea, M S; Wilber, J C; Yeghiazarian, T; Todd, J A; Kern, D G; Fong, S J; Besemer, D; Hoo, B; Sheridan, P J; Kokka, R

1993-11-01

To determine the relative effect of sample matrix on the quantitation of HIV RNA in plasma. Two HIV-positive specimens were diluted into five and 10 different HIV-negative plasma samples, respectively. Branched DNA signal amplification technology and reverse-transcriptase polymerase chain reaction were used to measure the viral load. In one sample the viral load by polymerase chain reaction ranged from undetectable to 1.9 x 10(5) copies/ml, and the branched DNA results ranged from 2.6 x 10(4) to 4.2 x 10(4) HIV RNA equivalent/ml. In the other sample the corresponding figures were 6.3 x 10(4) to 5.5 x 10(5) copies/ml and 5.7 x 10(4) to 7.5 x 10(4) HIV RNA equivalents/ml. In contrast to reverse-transcriptase polymerase chain reaction the branched DNA signal amplification assay does not require a separate extraction step or enzymatic amplification of the target. Therefore this measurement is less affected by the sample matrix and the signal generated is directly proportional to the viral load.

Carbocyclic nucleoside analogues: classification, target enzymes, mechanisms of action and synthesis

NASA Astrophysics Data System (ADS)

Matyugina, E. S.; Khandazhinskaya, A. P.; Kochetkov, Sergei N.

2012-08-01

Key biological targets (S-adenosyl-L-homocysteine hydrolase, telomerase, human immunodeficiency virus reverse transcriptase, herpes virus DNA polymerase and hepatitis B virus DNA polymerase) and the mechanisms of action of carbocyclic nucleoside analogues are considered. Structural types of analogues are discussed. Methods of synthesis for the most promising compounds and the spectrum of their biological activities are described. The bibliography includes 126 references.

Failure to Detect Borna Disease Virus Antibody and RNA from Peripheral Blood Mononuclear Cells of Psychiatric Patients

PubMed Central

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

2009-01-01

Objective Borna disease virus (BDV) is a highly neurotropic agent causing various neuropsychiatric symptoms in animals. Over the past two decades, it has been suggested that BDV might be associated with human psychiatric diseases. We aimed to investigate whether BDV is associated with psychiatric patients in Korea. Methods We recruited 60 normal controls and 198 psychiatric patients (98 patients with depressive disorder, 60 with schizophrenia, and 40 with bipolar disorder). We used an indirect immunofluorescence antibody (IFA) test for the BDV antibody and a real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay for p24 and p40 RNA from peripheral blood mononuclear cells (PBMCs). Results Neither the BDV antibody nor p24, p40 RNA was detected in controls and patients groups. Conclusion Our results suggest that BDV might not be associated with psychiatric patients in Korea. PMID:20140130

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.

Problem-Solving Test: Catalytic Activities of a Human Nuclear Enzyme

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2011-01-01

Terms to be familiar with before you start to solve the test: ion exchange chromatography, polynucleotides, oligonucleotides, radioactive labeling, template, primer, DNA polymerase, reverse transcriptase, helicase, nucleoside triphosphates, nucleoside diphosphates, nucleoside monophosphates, nucleosides, 5'-end and 3'-end, bacteriophage,…

Structural basis for the D-stereoselectivity of human DNA polymerase β

PubMed Central

Vyas, Rajan; Reed, Andrew J.; Raper, Austin T.; Zahurancik, Walter J.; Wallenmeyer, Petra C.

2017-01-01

Abstract Nucleoside reverse transcriptase inhibitors (NRTIs) with L-stereochemistry have long been an effective treatment for viral infections because of the strong D-stereoselectivity exhibited by human DNA polymerases relative to viral reverse transcriptases. The D-stereoselectivity of DNA polymerases has only recently been explored structurally and all three DNA polymerases studied to date have demonstrated unique stereochemical selection mechanisms. Here, we have solved structures of human DNA polymerase β (hPolβ), in complex with single-nucleotide gapped DNA and L-nucleotides and performed pre-steady-state kinetic analysis to determine the D-stereoselectivity mechanism of hPolβ. Beyond a similar 180° rotation of the L-nucleotide ribose ring seen in other studies, the pre-catalytic ternary crystal structures of hPolβ, DNA and L-dCTP or the triphosphate forms of antiviral drugs lamivudine ((-)3TC-TP) and emtricitabine ((-)FTC-TP) provide little structural evidence to suggest that hPolβ follows the previously characterized mechanisms of D-stereoselectivity. Instead, hPolβ discriminates against L-stereochemistry through accumulation of several active site rearrangements that lead to a decreased nucleotide binding affinity and incorporation rate. The two NRTIs escape some of the active site selection through the base and sugar modifications but are selected against through the inability of hPolβ to complete thumb domain closure. PMID:28402499

Gene expression in the rectus abdominus muscle of patients with and without pelvic organ prolapse.

PubMed

Hundley, Andrew F; Yuan, Lingwen; Visco, Anthony G

2008-02-01

The objective of the study was to compare gene expression in a group of actin and myosin-related proteins in the rectus muscle of 15 patients with pelvic organ prolapse and 13 controls. Six genes previously identified by microarray GeneChip analysis were examined using real-time quantitative reverse transcriptase-polymerase chain reaction analysis, including 2 genes showing differential expression in pubococcygeus muscle. Samples and controls were run in triplicate in multiplexed wells, and levels of gene expression were analyzed using the comparative critical threshold method. One gene, MYH3, was 3.2 times overexpressed in patients with prolapse (P = .032), but no significant differences in expression were seen for the other genes examined. An age-matched subset of 9 patients and controls showed that MYH3 gene expression was no longer significantly different (P = .058). Differential messenger ribonucleic acid levels of actin and myosin-related genes in patients with pelvic organ prolapse and controls may be limited to skeletal muscle from the pelvic floor.

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.

Expression of Connexin 43 in Synovial Tissue of Patients With Rheumatoid Arthritis

PubMed Central

MATSUKI, Tomohiro; TSUCHIDA, Shinji; TERAUCHI, Ryu; ODA, Ryo; FUJIWARA, Hiroyoshi; MAZDA, Osam; KUBO, Toshikazu

2016-01-01

Objectives This study aims to identify the distribution and expression level of connexin 43 (Cx43) in synovial tissue in patients with rheumatoid arthritis (RA). Patients and methods The expression of Cx43 in synovial tissue from eight patients with RA (2 males, 6 females; mean age 59.5±2.7 years; range 52 to 71 years), five patients with osteoarthritis (2 males, 3 females; mean age 68.4±2.7 years; range 61 to 81 years), and one normal female subject (mean age 61 year) was analyzed by quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry of tissue sections. Induction of Cx43 following stimulation of human RA synovial fibroblasts with tumor necrosis factor-alpha (TNF-a) cultures was examined by quantitative reverse transcriptase polymerase chain reaction. The effect of small interfering ribonucleic acid targeting Cx43 (siCx43) on the expression of TNF-a and interleukin-6 was examined using quantitative reverse transcriptase polymerase chain reaction and enzyme-linked immunosorbent assays. Results Connexin 43 was highly expressed in RA synovial tissue, which also expressed TNF-a, but was expressed lower in osteoarthritis and normal synovial tissue. Expression of Cx43 was markedly up-regulated in RA synovial fibroblasts after stimulation with TNF-a. The over-expression of pro- inflammatory cytokines was suppressed by transfection of siCx43. Conclusion This study shows that Cx43 is expressed in RA synovial tissue and that its expression is induced by stimulation with TNF-a. The expression of the pro-inflammatory cytokines was inhibited by transfection of siCx43. Cx43 may be a novel marker of inflammation in RA synovial tissue. PMID:29900991

Approved Antiviral Drugs over the Past 50 Years

PubMed Central

2016-01-01

SUMMARY Since the first antiviral drug, idoxuridine, was approved in 1963, 90 antiviral drugs categorized into 13 functional groups have been formally approved for the treatment of the following 9 human infectious diseases: (i) HIV infections (protease inhibitors, integrase inhibitors, entry inhibitors, nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and acyclic nucleoside phosphonate analogues), (ii) hepatitis B virus (HBV) infections (lamivudine, interferons, nucleoside analogues, and acyclic nucleoside phosphonate analogues), (iii) hepatitis C virus (HCV) infections (ribavirin, interferons, NS3/4A protease inhibitors, NS5A inhibitors, and NS5B polymerase inhibitors), (iv) herpesvirus infections (5-substituted 2′-deoxyuridine analogues, entry inhibitors, nucleoside analogues, pyrophosphate analogues, and acyclic guanosine analogues), (v) influenza virus infections (ribavirin, matrix 2 protein inhibitors, RNA polymerase inhibitors, and neuraminidase inhibitors), (vi) human cytomegalovirus infections (acyclic guanosine analogues, acyclic nucleoside phosphonate analogues, pyrophosphate analogues, and oligonucleotides), (vii) varicella-zoster virus infections (acyclic guanosine analogues, nucleoside analogues, 5-substituted 2′-deoxyuridine analogues, and antibodies), (viii) respiratory syncytial virus infections (ribavirin and antibodies), and (ix) external anogenital warts caused by human papillomavirus infections (imiquimod, sinecatechins, and podofilox). Here, we present for the first time a comprehensive overview of antiviral drugs approved over the past 50 years, shedding light on the development of effective antiviral treatments against current and emerging infectious diseases worldwide. PMID:27281742

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.

Optimization of the elution buffer and concentration method for detecting hepatitis E virus in swine liver using a nested reverse transcription-polymerase chain reaction and real-time reverse transcription-polymerase chain reaction.

PubMed

Son, Na Ry; Seo, Dong Joo; Lee, Min Hwa; Seo, Sheungwoo; Wang, Xiaoyu; Lee, Bog-Hieu; Lee, Jeong-Su; Joo, In-Sun; Hwang, In-Gyun; Choi, Changsun

2014-09-01

The aim of this study was to develop an optimal technique for detecting hepatitis E virus (HEV) in swine livers. Here, three elution buffers and two concentration methods were compared with respect to enhancing recovery of HEV from swine liver samples. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and nested RT-PCR were performed to detect HEV RNA. When phosphate-buffered saline (PBS, pH 7.4) was used to concentrate HEV in swine liver samples using ultrafiltration, real-time RT-PCR detected HEV in 6 of the 26 samples. When threonine buffer was used to concentrate HEV using polyethylene glycol (PEG) precipitation and ultrafiltration, real-time RT-PCR detected HEV in 1 and 3 of the 26 samples, respectively. When glycine buffer was used to concentrate HEV using ultrafiltration and PEG precipitation, real-time RT-PCR detected HEV in 1 and 3 samples of the 26 samples, respectively. When nested RT-PCR was used to detect HEV, all samples tested negative regardless of the type of elution buffer or concentration method used. Therefore, the combination of real-time RT-PCR and ultrafiltration with PBS buffer was the most sensitive and reliable method for detecting HEV in swine livers. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

Expression and characterization of a novel reverse transcriptase of the LTR retrotransposon Tf1.

PubMed

Kirshenboim, Noa; Hayouka, Zvi; Friedler, Assaf; Hizi, Amnon

2007-09-30

The LTR retrotransposon of Schizosacharomyces pombe, Tf1, has several distinctive properties that can be related to the unique properties of its reverse transcriptase (RT). Consequently, we expressed, purified and studied the recombinant Tf1 RT. This monomeric protein possesses all activities typical to RTs: DNA and RNA-dependent DNA polymerase as well as an inherent ribonuclease H. The DNA polymerase activity shows preference to Mn(+)(2) or Mg(+)(2), depending on the substrate used, whereas the ribonuclease H strongly prefers Mn(+)(2). The most outstanding feature of Tf1 RT is its capacity to add non-templated nucleotides to the 3'-ends of the nascent DNA. This is mainly apparent in the presence of Mn(+)(2), as is the noticeable low fidelity of DNA synthesis. In all, Tf1 RT has a marked infidelity in synthesizing DNA at template ends, a phenomenon that can explain, as discussed herein, some of the features of Tf1 replication in the host cells.

Hsp90 is required for the activity of a hepatitis B virus reverse transcriptase.

PubMed Central

Hu, J; Seeger, C

1996-01-01

The heat shock protein Hsp90 is known as an essential component of several signal transduction pathways and has now been identified as an essential host factor for hepatitis B virus replication. Hsp90 interacts with the viral reverse transcriptase to facilitate the formation of a ribonucleoprotein (RNP) complex between the polymerase and an RNA ligand. This RNP complex is required early in replication for viral assembly and initiation of DNA synthesis through a protein-priming mechanism. These results thus invoke a role for the Hsp90 pathway in the formation of an RNP. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8577714

Real-Time Reverse Transcription–Polymerase Chain Reaction Assay for SARS-associated Coronavirus

PubMed Central

Emery, Shannon L.; Bowen, Michael D.; Newton, Bruce R.; Winchell, Jonas M.; Meyer, Richard F.; Tong, Suxiang; Cook, Byron T.; Holloway, Brian P.; McCaustland, Karen A.; Rota, Paul A.; Bankamp, Bettina; Lowe, Luis E.; Ksiazek, Tom G.; Bellini, William J.; Anderson, Larry J.

2004-01-01

A real-time reverse transcription–polymerase chain reaction (RT-PCR) assay was developed to rapidly detect the severe acute respiratory syndrome–associated coronavirus (SARS-CoV). The assay, based on multiple primer and probe sets located in different regions of the SARS-CoV genome, could discriminate SARS-CoV from other human and animal coronaviruses with a potential detection limit of <10 genomic copies per reaction. The real-time RT-PCR assay was more sensitive than a conventional RT-PCR assay or culture isolation and proved suitable to detect SARS-CoV in clinical specimens. Application of this assay will aid in diagnosing SARS-CoV infection. PMID:15030703

Dimethylarsinic acid in drinking water changed the morphology of urinary bladder but not the expression of DNA repair genes of bladder transitional epithelium in F344 rats.

PubMed

Wang, Amy; Wolf, Douglas C; Sen, Banalata; Knapp, Geremy W; Holladay, Steven D; Huckle, William R; Caceci, Thomas; Robertson, John L

2009-06-01

Inorganic arsenic increases urinary bladder transitional cell carcinoma in humans. In F344 rats, dimethylarsinic acid (DMA[V]) increases transitional cell carcinoma. Arsenic-induced inhibition of DNA repair has been reported in cultured cell lines and in lymphocytes of arsenic-exposed humans, but it has not been studied in urinary bladder. Should inhibition of DNA damage repair in transitional epithelium occur, it may contribute to carcinogenesis or cocarcinogenesis. We investigated morphology and expression of DNA repair genes in F344 rat transitional cells following up to 100 ppm DMA(V) in drinking water for four weeks. Mitochondria were very sensitive to DMA(V), and swollen mitochondria appeared to be the main source of vacuoles in the transitional epithelium. Real-time reverse transcriptase polymerase chain reaction (Real-Time RT PCR) showed the mRNA levels of tested DNA repair genes, ataxia telangectasia mutant (ATM), X-ray repair cross-complementing group 1 (XRCC1), excision repair cross-complementing group 3/xeroderma pigmentosum B (ERCC3/XPB), and DNA polymerase beta (Polbeta), were not altered by DMA(V). These data suggested that either DMA(V) does not affect DNA repair in the bladder or DMA(V) affects DNA repair without affecting baseline mRNA levels of repair genes. The possibility remains that DMA(V) may lower damage-induced increases in repair gene expression or cause post-translational modification of repair enzymes.

Differentiation of human-induced pluripotent stem cells into insulin-producing clusters.

PubMed

Shaer, Anahita; Azarpira, Negar; Vahdati, Akbar; Karimi, Mohammad Hosein; Shariati, Mehrdad

2015-02-01

In diabetes mellitus type 1, beta cells are mostly destroyed; while in diabetes mellitus type 2, beta cells are reduced by 40% to 60%. We hope that soon, stem cells can be used in diabetes therapy via pancreatic beta cell replacement. Induced pluripotent stem cells are a kind of stem cell taken from an adult somatic cell by "stimulating" certain genes. These induced pluripotent stem cells may be a promising source of cell therapy. This study sought to produce isletlike clusters of insulin-producing cells taken from induced pluripotent stem cells. A human-induced pluripotent stem cell line was induced into isletlike clusters via a 4-step protocol, by adding insulin, transferrin, and selenium (ITS), N2, B27, fibroblast growth factor, and nicotinamide. During differentiation, expression of pancreatic β-cell genes was evaluated by reverse transcriptase-polymerase chain reaction; the morphologic changes of induced pluripotent stem cells toward isletlike clusters were observed by a light microscope. Dithizone staining was used to stain these isletlike clusters. Insulin produced by these clusters was evaluated by radio immunosorbent assay, and the secretion capacity was analyzed with a glucose challenge test. Differentiation was evaluated by analyzing the morphology, dithizone staining, real-time quantitative polymerase chain reaction, and immunocytochemistry. Gene expression of insulin, glucagon, PDX1, NGN3, PAX4, PAX6, NKX6.1, KIR6.2, and GLUT2 were documented by analyzing real-time quantitative polymerase chain reaction. Dithizone-stained cellular clusters were observed after 23 days. The isletlike clusters significantly produced insulin. The isletlike clusters could increase insulin secretion after a glucose challenge test. This work provides a model for studying the differentiation of human-induced pluripotent stem cells to insulin-producing cells.

Structure of the HIV-1 reverse transcriptase Q151M mutant: insights into the inhibitor resistance of HIV-1 reverse transcriptase and the structure of the nucleotide-binding pocket of Hepatitis B virus polymerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakamura, Akiyoshi; Tamura, Noriko; Yasutake, Yoshiaki, E-mail: y-yasutake@aist.go.jp

The structure of the HIV-1 reverse transcriptase Q151M mutant was determined at a resolution of 2.6 Å in space group P321. Hepatitis B virus polymerase (HBV Pol) is an important target for anti-HBV drug development; however, its low solubility and stability in vitro has hindered detailed structural studies. Certain nucleotide reverse transcriptase (RT) inhibitors (NRTIs) such as tenofovir and lamivudine can inhibit both HBV Pol and Human immunodeficiency virus 1 (HIV-1) RT, leading to speculation on structural and mechanistic analogies between the deoxynucleotide triphosphate (dNTP)-binding sites of these enzymes. The Q151M mutation in HIV-1 RT, located at the dNTP-binding site,more » confers resistance to various NRTIs, while maintaining sensitivity to tenofovir and lamivudine. The residue corresponding to Gln151 is strictly conserved as a methionine in HBV Pol. Therefore, the structure of the dNTP-binding pocket of the HIV-1 RT Q151M mutant may reflect that of HBV Pol. Here, the crystal structure of HIV-1 RT Q151M, determined at 2.6 Å resolution, in a new crystal form with space group P321 is presented. Although the structure of HIV-1 RT Q151M superimposes well onto that of HIV-1 RT in a closed conformation, a slight movement of the β-strands (β2–β3) that partially create the dNTP-binding pocket was observed. This movement might be caused by the introduction of the bulky thioether group of Met151. The structure also highlighted the possibility that the hydrogen-bonding network among amino acids and NRTIs is rearranged by the Q151M mutation, leading to a difference in the affinity of NRTIs for HIV-1 RT and HBV Pol.« less

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...

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...

Isolation and characterization of an AGAMOUS homolog from Fraxinus pennsylvanica

Treesearch

Ningxia Du; Paula M. Pijut

2010-01-01

An AGAMOUS homolog (FpAG) was isolated from green ash (Fraxinus pennsylvanica) using a reverse transcriptase polymerase chain reaction method. Southern blot analysis indicated that FpAG was present as a single-copy sequence in the genome of green ash. RNA accumulated in the reproductive tissues (female...

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

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.

Two assays for measuring fibrosis: reverse transcriptase-polymerase chain reaction of collagen alpha(1) (III) mRNA is an early predictor of subsequent collagen deposition while a novel serum N-terminal procollagen (III) propeptide assay reflects manifest fibrosis in carbon tetrachloride-treated rats.

PubMed

Kauschke, S G; Knorr, A; Heke, M; Kohlmeyer, J; Schauer, M; Theiss, G; Waehler, R; Burchardt, E R

1999-11-15

Using a novel quantitative reverse transcriptase-polymerase chain reaction assay, we have determined the amount of specific mRNA for procollagen alpha(1) (III) (PIIIP) in the carbon tetrachloride (CCl(4)) model of liver fibrosis in rats. After a single week of CCl(4) application, the amount of PIIIP mRNA was increased approximately 10 times over the untreated control group and continued to increase to approximately 30 times after 7 weeks of intoxication. In this model substantial fibrosis was demonstrated by computer-aided morphometry after 5 to 7 weeks of treatment. Using recombinant murine N-terminal procollagen alpha(1) (III) propeptide (PIIINP), a novel sensitive immunoassay for the measurement of circulating PIIINP in rodent sera was established. An increase in PIIINP serum levels was observed after 5 to 7 weeks of CCl(4) intoxication. Our results suggest PIIIP gene expression is an early marker of tissue fibrosis. Early PIIIP gene expression is correlated with the extent of the subsequent fibrosis. PIIIP mRNA levels increase much earlier than conventional histological examination or PIIINP levels. PIIINP measurements with our new serum assay, on the other hand, are a good noninvasive marker of manifest fibrosis but are a poor marker of fibrogenesis. Copyright 1999 Academic Press.

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.

Development and evaluation of a simple and effective RT-qPCR inhibitory assay for detection of the efficacy of compounds towards HIV reverse transcriptase.

PubMed

Marino-Merlo, Francesca; Frezza, Caterina; Papaianni, Emanuela; Valletta, Elena; Mastino, Antonio; Macchi, Beatrice

2017-11-01

Assessing the actual efficacy of compounds to directly inhibit HIV reverse transcriptase (RT) activity is a main goal in preclinical antiretroviral studies. Our previous studies demonstrated that the effects of inhibitor compounds towards HIV-RT could be efficiently assessed through a simple cell-free assay based on conventional reverse transcription PCR. In the present study, we describe a modified variant of our assay, termed RT real-time quantitative PCR inhibitory assay (RT-qPCR-IA), in which the ability of compounds to restrict the complementary DNA (cDNA) generation by HIV-RT using a specific RNA template is performed by the real-time technique, in order to improve both accuracy and sensitivity of the method. As specific RNA template, RNA extracted from stable transfectants ectopically expressing the herpes simplex virus 1 glycoprotein D gene was utilized. HIV-RT, of both commercial or house-made viral lysate origin, was employed for the assay. To assess the reliability of RT-qPCR-IA, we performed a comparative, quantitative analysis of the dose-dependent effect exerted by known nucleotide and non-nucleotide reverse-transcriptase inhibitors, using the SYBR Green dye chemistry as detection system. The results obtained with RT-qPCR-IA were compared to that obtained using a one-step PicoGreen technology-based commercial kit. The outcome of our study indicates that the development of the novel RT-qPCR-IA will provide rapid and accurate evaluation of the inhibitory efficacy of compounds towards HIV-RT activity. This evaluation could be very useful for large-scale screening of potential new anti-HIV drugs.

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.

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.

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

The limitations of point of care testing for pandemic influenza: what clinicians and public health professionals need to know.

PubMed

Hatchette, Todd F; Bastien, Nathalie; Berry, Jody; Booth, Tim F; Chernesky, Max; Couillard, Michel; Drews, Steven; Ebsworth, Anthony; Fearon, Margaret; Fonseca, Kevin; Fox, Julie; Gagnon, Jean-Nicolas; Guercio, Steven; Horsman, Greg; Jorowski, Cathy; Kuschak, Theodore; Li, Yan; Majury, Anna; Petric, Martin; Ratnam, Sam; Smieja, Marek; Van Caeseele, Paul

2009-01-01

As the world prepares for the next influenza pandemic, governments have made significant funding commitments to vaccine development and antiviral stockpiling. While these are essential components to pandemic response, rapid and accurate diagnostic testing remains an often neglected cornerstone of pandemic influenza preparedness. Clinicians and Public Health Practitioners need to understand the benefits and drawbacks of different influenza tests in both seasonal and pandemic settings. Culture has been the traditional gold standard for influenza diagnosis but requires from 1-10 days to generate a positive result, compared to nucleic acid detection methods such as real time reverse transcriptase polymerase chain reaction (RT-PCR). Although the currently available rapid antigen detection kits can generate results in less than 30 minutes, their sensitivity is suboptimal and they are not recommended for the detection of novel influenza viruses. Until point-of-care (POC) tests are improved, PILPN recommends that the best option for pandemic influenza preparation is the enhancement of nucleic acid-based testing capabilities across Canada.

The role of oxidative stress in the development of cisplatin resistance in epithelial ovarian cancer.

PubMed

Belotte, Jimmy; Fletcher, Nicole M; Awonuga, Awoniyi O; Alexis, Mitchell; Abu-Soud, Husam M; Saed, Mohammed G; Diamond, Michael P; Saed, Ghassan M

2014-04-01

To investigate the role of oxidative stress in the development of cisplatin resistance in epithelial ovarian cancer (EOC). Two parent EOC cell lines (MDAH-2774 and SKOV-3) and their chemoresistant counterparts (cisplatin, 50 µmol/L) were used. Total RNA was extracted and subjected to real-time reverse transcriptase polymerase chain reaction to evaluate the expression of glutathione reductase (GSR) and inducible nitric oxide synthase (iNOS), as well as nitrate/nitrite levels. Analysis of variance was used for main effects and Tukey for post hoc analysis at P < .05 for statistical significance. Both cisplatin resistant cell lines displayed a significant decrease in GSR messenger RNA (mRNA) levels and activity (P < .01). As compared to sensitive controls, nitrate/nitrite levels were significantly higher in SKOV-3 cisplatin resistant cells while iNOS mRNA levels were significantly higher in MDAH-2774 cisplatin resistant cells (P < .05). Our data suggest that the development of cisplatin resistance tilts the balance toward a pro-oxidant state in EOC.

Interleukin-4 induces expression of eotaxin in endometriotic stromal cells.

PubMed

Ouyang, Zhuo; Osuga, Yutaka; Hirota, Yasushi; Hirata, Tetsuya; Yoshino, Osamu; Koga, Kaori; Yano, Tetsu; Taketani, Yuji

2010-06-01

To study the relationship between eotaxin and interleukin-4 (IL-4) in the pathophysiology of endometriosis. Comparative and laboratory study. University teaching hospital reproductive endocrinology and infertility practice. Ectopic endometrial tissues were collected from women with endometriosis. Ectopic endometrial stromal cells (ESCs) were isolated and cultured with IL-4. Ectopic endometriotic tissues were immunostained for eotaxin and IL-4. Gene expression of eotaxin was determined by standard and real-time reverse-transcriptase polymerase chain reaction. Secretion of eotaxin from ESC was measured using specific ELISA. The immunostained sections were examined. Interleukin-4 (IL-4) increased mRNA expression and protein secretion of eotaxin from ESC in a dose-dependent manner. Immunohistochemical analysis showed that eotaxin-positive cells colocalized with IL-4-positive cells and accumulated around the blood vessels in the stroma of endometriotic tissue. IL-4 induces eotaxin in ESCs, which might promote angiogenesis and the subsequent development of endometriosis. Copyright (c) 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Ependymin, a gene involved in regeneration and neuroplasticity in vertebrates, is overexpressed during regeneration in the echinoderm Holothuria glaberrima.

PubMed

Suárez-Castillo, Edna C; Medina-Ortíz, Wanda E; Roig-López, José L; García-Arrarás, José E

2004-06-09

We report the characterization of an ependymin-related gene (EpenHg) from a regenerating intestine cDNA library of the sea cucumber Holothuria glaberrima. This finding is remarkable because no ependymin sequence has ever been reported from invertebrates. Database comparisons of the conceptual translation of the EpenHg gene reveal 63% similarity (47% identity) with mammalian ependymin-related proteins (MERPs) and close relationship with the frog and piscine ependymins. We also report the partial sequences of ependymin representatives from another species of sea cucumber and from a sea urchin species. Conventional and real-time reverse transcriptase polymerase chain reaction (RT-PCRs) show that the gene is expressed in several echinoderm tissues, including esophagus, mesenteries, gonads, respiratory trees, hemal system, tentacles and body wall. Moreover, the ependymin product in the intestine is overexpressed during sea cucumber intestinal regeneration. The discovery of ependymins in echinoderms, a group well known for their regenerative capacities, can give us an insight on the evolution and roles of ependymin molecules.

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.

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.

Periodontal therapy alters gene expression of peripheral blood monocytes

PubMed Central

Papapanou, Panos N.; Sedaghatfar, Michael H.; Demmer, Ryan T.; Wolf, Dana L.; Yang, Jun; Roth, Georg A.; Celenti, Romanita; Belusko, Paul B.; Lalla, Evanthia; Pavlidis, Paul

2009-01-01

Aims We investigated the effects of periodontal therapy on gene expression of peripheral blood monocytes. Methods Fifteen patients with periodontitis gave blood samples at four time points: 1 week before periodontal treatment (#1), at treatment initiation (baseline, #2), 6-week (#3) and 10-week post-baseline (#4). At baseline and 10 weeks, periodontal status was recorded and subgingival plaque samples were obtained. Periodontal therapy (periodontal surgery and extractions without adjunctive antibiotics) was completed within 6 weeks. At each time point, serum concentrations of 19 biomarkers were determined. Peripheral blood monocytes were purified, RNA was extracted, reverse-transcribed, labelled and hybridized with AffymetrixU133Plus2.0 chips. Expression profiles were analysed using linear random-effects models. Further analysis of gene ontology terms summarized the expression patterns into biologically relevant categories. Differential expression of selected genes was confirmed by real-time reverse transcriptase-polymerase chain reaction in a subset of patients. Results Treatment resulted in a substantial improvement in clinical periodontal status and reduction in the levels of several periodontal pathogens. Expression profiling over time revealed more than 11,000 probe sets differentially expressed at a false discovery rate of <0.05. Approximately 1/3 of the patients showed substantial changes in expression in genes relevant to innate immunity, apoptosis and cell signalling. Conclusions The data suggest that periodontal therapy may alter monocytic gene expression in a manner consistent with a systemic anti-inflammatory effect. PMID:17716309

Detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction.

PubMed

Corman, V M; Eckerle, I; Bleicker, T; Zaki, A; Landt, O; Eschbach-Bludau, M; van Boheemen, S; Gopal, R; Ballhause, M; Bestebroer, T M; Muth, D; Müller, M A; Drexler, J F; Zambon, M; Osterhaus, A D; Fouchier, R M; Drosten, C

2012-09-27

We present two real-time reverse-transcription polymerase chain reaction assays for a novel human coronavirus (CoV), targeting regions upstream of the E gene (upE) or within open reading frame (ORF)1b, respectively. Sensitivity for upE is 3.4 copies per reaction (95% confidence interval (CI): 2.5–6.9 copies) or 291 copies/mL of sample. No cross-reactivity was observed with coronaviruses OC43, NL63, 229E, SARS-CoV, nor with 92 clinical specimens containing common human respiratory viruses. We recommend using upE for screening and ORF1b for confirmation.

Downregulation of telomerase activity in human promyelocytic cell line using RNA interference.

PubMed

Miri-Moghaddam, E; Deezagi, A; Soheili, Z S

2009-12-01

Telomerase is a ribonucleoprotein complex. It consists of two main components, human telomerase reverse transcriptase (hTERT) and human telomerase RNA. High telomerase activity is present in most malignant cells, but it is barely detectable in majority of somatic cells. The direct correlation between telomerase reactivation and carcinogens has made hTERT a key target for anticancer therapeutic studies. In this study, for the first time, we evaluated the ability of the new generation of short interfering RNA (siRNA) to regulate telomerase activity in the human promyelocytic leukemia cell line (HL-60). Transient transfection cell line by hTERT siRNAs resulted in statistically significant suppression of hTERT messenger RNAs which were detected by quantitative real-time polymerase chain reaction, while the expressed hTERT protein levels were measured by flow cytometry. The results of telomeric repeat amplification protocol showed that telomerase activity was significantly reduced upon transfection of the HL-60 cell line with hTERT siRNAs. The results of this study showed that telomerase activity and cell proliferation were efficiently inhibited in the hTERT siRNA-treated leukemic cell line.

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.

Hepatotoxicity of nucleoside reverse transcriptase inhibitors.

PubMed

Montessori, Valentina; Harris, Marianne; Montaner, Julio S G

2003-05-01

Hepatotoxicity is an adverse effect of all available classes of antiretrovirals, including nucleoside reverse transcriptase inhibitors (NRTI). A syndrome of hepatic steatosis and lactic acidosis has been recognized as a rare, potentially fatal complication since the advent of NRTI monotherapy in the early 1990s. Today, NRTI remain the backbone of antiretroviral combination regimens, and, with the success of current treatment strategies, exposure to two or more of these agents may occur over a number of years. Hepatic steatosis and lactic acidosis are accordingly being observed more frequently, along with a more recently recognized syndrome of chronic hyperlactatemia. These as well as other adverse effects of NRTI are mediated by inhibition of human DNA polymerase gamma, resulting in mitochondrial dysfunction in the liver and other tissues. Early recognition and intervention are essential to avert serious outcomes.

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

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.

[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.

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

The Reverse Transcriptase of the Tf1 Retrotransposon Has a Specific Novel Activity for Generating the RNA Self-Primer That Is Functional in cDNA Synthesis▿

PubMed Central

Hizi, Amnon

2008-01-01

The Tf1 retrotransposon of Schizosaccharomyces pombe represents a group of eukaryotic long terminal repeat (LTR) retroelements that, based on their sequences, were predicted to use an RNA self-primer for initiating reverse transcription while synthesizing the negative-sense DNA strand. This feature is substantially different from the one typical to retroviruses and other LTR retrotransposons that all exhibit a tRNA-dependent priming mechanism. Genetic studies have suggested that the self-primer of Tf1 can be generated by a cleavage between the 11th and 12th bases of the Tf1 RNA transcript. The in vitro data presented here show that recombinant Tf1 reverse transcriptase indeed introduces a nick at the end of a duplexed region at the 5′ end of Tf1 genomic RNA, substantiating the prediction that this enzyme is responsible for generating this RNA self-primer. The 3′ end of the primer, generated in this manner, can then be extended upon the addition of deoxynucleoside triphosphates by the DNA polymerase activity of the same enzyme, synthesizing the negative-sense DNA strand. This functional primer must have been generated by the RNase H activity of Tf1 reverse transcriptase, since a mutant enzyme lacking this activity has lost its ability to generate the self-primer. It was also found here that the reverse transcriptases of human immunodeficiency virus type 1 and of murine leukemia virus do not exhibit this specific cleavage activity. In all, it is likely that the observed unique mechanism of self-priming in Tf1 represents an early advantageous form of initiating reverse transcription in LTR retroelements without involving cellular tRNAs. PMID:18753200

The reverse transcriptase of the Tf1 retrotransposon has a specific novel activity for generating the RNA self-primer that is functional in cDNA synthesis.

PubMed

Hizi, Amnon

2008-11-01

The Tf1 retrotransposon of Schizosaccharomyces pombe represents a group of eukaryotic long terminal repeat (LTR) retroelements that, based on their sequences, were predicted to use an RNA self-primer for initiating reverse transcription while synthesizing the negative-sense DNA strand. This feature is substantially different from the one typical to retroviruses and other LTR retrotransposons that all exhibit a tRNA-dependent priming mechanism. Genetic studies have suggested that the self-primer of Tf1 can be generated by a cleavage between the 11th and 12th bases of the Tf1 RNA transcript. The in vitro data presented here show that recombinant Tf1 reverse transcriptase indeed introduces a nick at the end of a duplexed region at the 5' end of Tf1 genomic RNA, substantiating the prediction that this enzyme is responsible for generating this RNA self-primer. The 3' end of the primer, generated in this manner, can then be extended upon the addition of deoxynucleoside triphosphates by the DNA polymerase activity of the same enzyme, synthesizing the negative-sense DNA strand. This functional primer must have been generated by the RNase H activity of Tf1 reverse transcriptase, since a mutant enzyme lacking this activity has lost its ability to generate the self-primer. It was also found here that the reverse transcriptases of human immunodeficiency virus type 1 and of murine leukemia virus do not exhibit this specific cleavage activity. In all, it is likely that the observed unique mechanism of self-priming in Tf1 represents an early advantageous form of initiating reverse transcription in LTR retroelements without involving cellular tRNAs.

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.

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 ...

Gene expression analysis of wood decay fungus Fibroporia Radiculosa grown In ACQ-treated wood

Treesearch

Ayfer Akgul; Ali Akgul; Juliet D. Diehl Tang

2018-01-01

Copper-tolerant brown-rot fungi are able todegrade wood treated with copper or copper-based wood preservatives. This research used quantitative reverse transcriptase polymerase chain reaction to explore what genes of the brown-rot fungus, Fibroporia radiculosa, were expressed when the fungus was overcoming the wood preservatives and decaying the...

Problem-solving test: catalytic activities of a human nuclear enzyme.

PubMed

Szeberényi, József

2011-01-01

Terms to be familiar with before you start to solve the test: ion exchange chromatography, polynucleotides, oligonucleotides, radioactive labeling, template, primer, DNA polymerase, reverse transcriptase, helicase, nucleoside triphosphates, nucleoside diphosphates, nucleoside monophosphates, nucleosides, 5′-end and 3′-end, bacteriophage, polyacrylamide gel electrophoresis, urea, autoradiography, proofreading, telomerase, endonucleases, exonucleases, primase, topoisomerases, and excinuclease.

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

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

Probing the structural and molecular basis of nucleotide selectivity by human mitochondrial DNA polymerase γ

PubMed Central

Sohl, Christal D.; Szymanski, Michal R.; Mislak, Andrea C.; Shumate, Christie K.; Amiralaei, Sheida; Schinazi, Raymond F.; Anderson, Karen S.; Yin, Y. Whitney

2015-01-01

Nucleoside analog reverse transcriptase inhibitors (NRTIs) are the essential components of highly active antiretroviral (HAART) therapy targeting HIV reverse transcriptase (RT). NRTI triphosphates (NRTI-TP), the biologically active forms, act as chain terminators of viral DNA synthesis. Unfortunately, NRTIs also inhibit human mitochondrial DNA polymerase (Pol γ), causing unwanted mitochondrial toxicity. Understanding the structural and mechanistic differences between Pol γ and RT in response to NRTIs will provide invaluable insight to aid in designing more effective drugs with lower toxicity. The NRTIs emtricitabine [(-)-2,3′-dideoxy-5-fluoro-3′-thiacytidine, (-)-FTC] and lamivudine, [(-)-2,3′-dideoxy-3′-thiacytidine, (-)-3TC] are both potent RT inhibitors, but Pol γ discriminates against (-)-FTC-TP by two orders of magnitude better than (-)-3TC-TP. Furthermore, although (-)-FTC-TP is only slightly more potent against HIV RT than its enantiomer (+)-FTC-TP, it is discriminated by human Pol γ four orders of magnitude more efficiently than (+)-FTC-TP. As a result, (-)-FTC is a much less toxic NRTI. Here, we present the structural and kinetic basis for this striking difference by identifying the discriminator residues of drug selectivity in both viral and human enzymes responsible for substrate selection and inhibitor specificity. For the first time, to our knowledge, this work illuminates the mechanism of (-)-FTC-TP differential selectivity and provides a structural scaffold for development of novel NRTIs with lower toxicity. PMID:26124101

Identification and expression analysis of genes involved in early ovary development in diploid gynogenetic hybrids of red crucian carp x common carp.

PubMed

Liu, Dong; Liu, Shaojun; You, Cuiping; Chen, Lin; Liu, Zhen; Liu, Liangguo; Wang, Jing; Liu, Yun

2010-04-01

Diploid eggs of allotetraploid hybrids (red crucian carp female symbol x common carp male symbol), when activated by UV-irradiated sperm of scatter scale carp, can develop into diploid progenies without chromosome duplication treatment. Diploid progenies produce diploid eggs, which develop into diploid population by the same way. To understand the molecular mechanism underlying the production of diploid eggs by the diploid fish, we constructed a forward suppression subtractive hybridization complementary DNA (cDNA) library. The cDNAs from the ovary in proliferation phase were employed as the "tester," and those in growth phase were used as the "driver." Seventy-three cDNA clones that are specifically expressed in proliferation phase were detected by dot-blot hybridization. Sequencing analyses revealed that several of these cDNAs have high homologies to the known sequences in the NCBI database. Their encoded proteins include the protein preventing mitosis catastrophe (PMC), the signal recognition particle 9, the ATP-binding cassette transporter, the glucanase-xylanase fusion protein, and others. These genes were confirmed by reverse transcriptase-polymerase chain reaction. The expression profile of the PMC gene at different time points was analyzed by quantitative real-time polymerase chain reaction. The results indicated that the expression of this suppression subtractive hybridization-identified gene changed during the time course, corresponding with the cellular phenomenon in the ovary development. Our studies provide insights into the molecular mechanism underlying the ovary development of diploid gynogenetic fish.

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.

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.

Rapid detection of highly pathogenic porcine reproductive and respiratory syndrome virus by a fluorescent probe-based isothermal recombinase polymerase amplification assay.

PubMed

Yang, Yang; Qin, Xiaodong; Sun, Yingjun; Chen, Ting; Zhang, Zhidong

2016-12-01

A novel fluorescent probe-based real-time reverse transcription recombinase polymerase amplification (real-time RT-RPA) assay was developed for rapid detection of highly pathogenic type 2 porcine reproductive and respiratory syndrome virus (HP-PRRSV). The sensitivity analysis showed that the detection limit of RPA was 70 copies of HP-PRRSV RNA/reaction. The real-time RT-RPA highly specific amplified HP-PRRSV with no cross-reaction with classic PRRSV, classic swine fever virus, pseudorabies virus, and foot-and-mouth disease virus. Assessment with 125 clinical samples showed that the developed real-time RT-RPA assay was well correlated with real-time RT-qPCR assays for detection of HP-PRRSV. These results suggest that the developed real-time RT-RPA assay is suitable for rapid detection of HP-PRRSV.

Off-Target Effects of Drugs that Disrupt Human Mitochondrial DNA Maintenance

PubMed Central

Young, Matthew J.

2017-01-01

Nucleoside reverse transcriptase inhibitors (NRTIs) were the first drugs used to treat human immunodeficiency virus (HIV) the cause of acquired immunodeficiency syndrome. Development of severe mitochondrial toxicity has been well documented in patients infected with HIV and administered NRTIs. In vitro biochemical experiments have demonstrated that the replicative mitochondrial DNA (mtDNA) polymerase gamma, Polg, is a sensitive target for inhibition by metabolically active forms of NRTIs, nucleotide reverse transcriptase inhibitors (NtRTIs). Once incorporated into newly synthesized daughter strands NtRTIs block further DNA polymerization reactions. Human cell culture and animal studies have demonstrated that cell lines and mice exposed to NRTIs display mtDNA depletion. Further complicating NRTI off-target effects on mtDNA maintenance, two additional DNA polymerases, Pol beta and PrimPol, were recently reported to localize to mitochondria as well as the nucleus. Similar to Polg, in vitro work has demonstrated both Pol beta and PrimPol incorporate NtRTIs into nascent DNA. Cell culture and biochemical experiments have also demonstrated that antiviral ribonucleoside drugs developed to treat hepatitis C infection act as off-target substrates for POLRMT, the mitochondrial RNA polymerase and primase. Accompanying the above-mentioned topics, this review examines: (1) mtDNA maintenance in human health and disease, (2) reports of DNA polymerases theta and zeta (Rev3) localizing to mitochondria, and (3) additional drugs with off-target effects on mitochondrial function. Lastly, mtDNA damage may induce cell death; therefore, the possibility of utilizing compounds that disrupt mtDNA maintenance to kill cancer cells is discussed. PMID:29214156

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.

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.

A novel duplex real time quantitative reverse transcription polymerase chain reaction for rubella virus with armored RNA as a noncompetitive internal positive control.

PubMed

Zhao, Lihong; Li, Ruiying; Liu, Aihua; Zhao, Shuping

2015-07-01

The objective of this study was to build and apply a duplex real time quantitative reverse transcription-polymerase chain reaction (RT-PCR) for rubella virus. Firstly, a 60-bp-long armored RV RNA was constructed in the laboratory. Secondly, a duplex real time RT-PCR assay was established. Thirdly, the 60-bp-long armored RV RNA was used as an internal positive control (IPC) for the duplex real time RT-PCR. And finally the duplex real time RT-PCR assay was applied to detect RV RNA in clinical specimens. The in-house assay has a high amplification efficiency (0.99), a high analytical sensitivity (200 copies/mL), and a good reproducibility. The diagnostic specificity and sensitivity of the in-house assay were both 100%, due to the monitoring of the armored RV RNA IPC. Therefore, the in-house duplex real time quantitative RT-PCR assay is a specific, sensitive, reproducible and accurate assay for quantitation of RV RNA in clinical specimens. And noncompetitive armored RV RNA IPC can monitor RT-PCR inhibition and prevent false-negative and inaccurate results in the real time detection system. Copyright © 2015 Elsevier B.V. All rights reserved.

Ferrate oxidation of murine leukemia virus reverse transcriptase: identification of the template-primer binding domain.

PubMed

Reddy, G; Nanduri, V B; Basu, A; Modak, M J

1991-08-20

Treatment of murine leukemia virus reverse transcriptase (MuLV RT) with potassium ferrate, an oxidizing agent known to oxidize amino acids involved in phosphate binding domains of proteins, results in the irreversible inactivation of both the DNA polymerase and the RNase H activities. Significant protection from ferrate-mediated inactivation is observed in the presence of template-primer but not in the presence of substrate deoxynucleoside triphosphates. Furthermore, ferrate-treated enzyme loses template-primer binding activity as judged by UV-mediated cross-linking of radiolabeled DNA. Comparative tryptic peptide mapping by reverse-phase HPLC of native and ferrate-oxidized enzyme indicated the presence of two new peptides eluting at 38 and 57 min and a significant loss of a peptide eluting at 74 min. Purification, amino acid composition, and sequencing of these affected peptides revealed that they correspond to amino acid residues 285-295, 630-640, and 586-599, respectively, in the primary amino acid sequence of MuLV RT. These results indicate that the domains constituted by the above peptides are important for the template-primer binding function in MuLV RT. Peptide I is located in the polymerase domain whereas peptides II and III are located in the RNase H domain. Amino acid sequence analysis of peptides I and II suggested Lys-285 and Cys-635 as the probable sites of ferrate action.

A novel mechanism of sugar selection utilized by a human X-family DNA polymerase.

PubMed

Brown, Jessica A; Fiala, Kevin A; Fowler, Jason D; Sherrer, Shanen M; Newmister, Sean A; Duym, Wade W; Suo, Zucai

2010-01-15

During DNA synthesis, most DNA polymerases and reverse transcriptases select against ribonucleotides via a steric clash between the ribose 2'-hydroxyl group and the bulky side chain of an active-site residue. In this study, we demonstrated that human DNA polymerase lambda used a novel sugar selection mechanism to discriminate against ribonucleotides, whereby the ribose 2'-hydroxyl group was excluded mostly by a backbone segment and slightly by the side chain of Y505. Such steric clash was further demonstrated to be dependent on the size and orientation of the substituent covalently attached at the ribonucleotide C2'-position. Copyright 2009 Elsevier Ltd. All rights reserved.

Prospective and retrospective evaluation of the Cepheid Xpert® Flu/RSV XC assay for rapid detection of influenza A, influenza B, and respiratory syncytial virus.

PubMed

Salez, Nicolas; Nougairede, Antoine; Ninove, Laetitia; Zandotti, Christine; de Lamballerie, Xavier; Charrel, Remi N

2015-04-01

A total of 281 clinical specimens (nasal swabs and nasopharyngeal aspirates) were tested with the Xpert® Flu/RSV XC. The results were compared to those obtained with the real-time retro transcriptase-polymerase chain reaction assays routinely used in our laboratory. The Xpert® Flu/RSV XC showed sensitivity/specificity of 97.8%/100% and 97.9%/100% for flu and respiratory syncytial virus, respectively. Copyright © 2015 Elsevier Inc. All rights reserved.

A competitive chemiluminescence enzyme immunoassay method for β-defensin-2 detection in transgenic mice.

PubMed

Yang, Xi; Zhou, Tao; Yu, Lei; Tan, Wenwen; Zhou, Rui; Hu, Yonggang

2015-03-01

A competitive chemiluminescence enzyme immunoassay (CLEIA) method for porcine β-defensin-2 (pBD-2) detection in transgenic mice was established. Several factors that affect detection, including luminol, p-iodophenol and hydrogen peroxide concentrations, as well as pH, were studied and optimized. The linear range of the proposed method for pBD-2 detection under optimal conditions was 0.05-80 ng/mL with a correlation coefficient of 0.9960. Eleven detections of a 30 ng/mL pBD-2 standard sample were performed. Reproducible results were obtained with a relative standard deviation of 3.94%. The limit of detection of the method for pBD-2 was 3.5 pg/mL (3σ). The proposed method was applied to determine pBD-2 expression levels in the tissues of pBD-2 transgenic mice, and compared with LC-MS/MS and quantitative real-time reverse-transcriptase polymerase chain reaction. This suggests that the CLEIA can be used as a valuable method to detect and quantify pBD-2. Copyright © 2014 John Wiley & Sons, Ltd.

Expression of glutathione S-transferases in poplar trees (Populus trichocarpa) exposed to 2,4,6-trinitrotoluene (TNT).

PubMed

Brentner, Laura B; Mukherji, Sachiyo T; Merchie, Kate M; Yoon, Jong Moon; Schnoor, Jerald L; Van Aken, Benoit

2008-10-01

Twelve Populus genes were identified from Arabidopsis thaliana sequences previously shown to be induced by exposure to 2,4,6-trinitrotoluene (TNT). Using the resources of the Poplar Genome Project and National Center for Biotechnology Information databases, Populus conserved domains were identified and used to design gene specific primers. RNA extracted from root tissues of TNT-exposed hydroponic poplar plants was used to quantify the expression of genes by reverse-transcriptase real-time polymerase chain reaction. Cyclophilin and 18S ribosomal DNA genes were used as internal standards. Exposure to TNT resulted in a significant increase of gene expression of two glutathione S-transferases (GST), peaking at levels of 25.0 +/- 13.1 and 10 +/- 0.7 fold the expression level of non-exposed plants after 24 h for each of the GST genes, respectively. This paper demonstrates the use of functional genomics information from the model plant species, Arabidopsis, to identify genes which may be important in detoxification of TNT in the model phytoremediation species, Populus trichocarpa.

Analysis of differential gene expression by bead-based fiber-optic array in nonfunctioning pituitary adenomas.

PubMed

Jiang, Z; Gui, S; Zhang, Y

2011-05-01

Nonfunctioning pituitary adenomas (NFPAs) are relatively common, accounting for 30% of all pituitary adenomas; however, their pathogenesis remains enigmatic. To explore the possible pathogenesis of NFPAs, we used fiber-optic BeadArray to examine gene expression in 5 NFPAs compared with 3 normal pituitaries. 4 differentially expressed genes were chosen randomly for validation by reverse transcriptase-real time quantitative polymerase chain reaction (RT-qPCR). We then analyzed the differentially expressed gene profile with Kyoto Encyclopedia of Genes and Genomes (KEGG). The array analysis indentified significant increases in the expression of 1,402 genes and 383 expressed sequence tags (ESTs), and decreases in 1,697 genes and 113 ESTs in the NFPAs. Bioinformatic and pathway analysis showed that the genes HIGD1B, FAM5C, PMAIP1 and the pathway cell-cycle regulation may play an important role in tumorigenesis and progression of NFPAs. Our data suggest fiber-optic BeadArray combined with pathway analysis of differential gene expression profile appears to be a valid approach for investigating the pathogenesis of tumors. © Georg Thieme Verlag KG Stuttgart · New York.

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.

Molecular cloning and expression profile analysis of porcine TCAP gene.

PubMed

Cheng, Hunjun; Xu, Xuewen; Zhao, Shuhong; Liu, Bang; Yu, Mei; Fan, Bin

2010-03-01

The gradually discovered sarcomeric proteins play important roles for structural integrity and signal transduction of sarcomere during myofibril genesis. TCAP (also described as telethonin, T-cap), one of the sarcomeric protein genes, is regulated developmentally. In this study, we reported the molecular characteristics of porcine TCAP gene. A 979 bp TCAP cDNA nucleotide sequence was obtained in pig and the deduced amino acid sequence had 92 and 91% identity to those of human and mouse homologous genes, respectively. One SNP was discovered and the allele frequency analysis showed that G allele frequency was low among 221 unrelated pigs from seven breeds. The tissue distribution patterns revealed that TCAP mRNA was expressed abundantly in skeletal and heart muscle tissue. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) results displayed TCAP mRNA was up-regulated in both Tongcheng and Landrace pigs during prenatal skeletal muscle development stages. This study suggested that TCAP gene might be a prospective candidate gene affecting muscle mass and meat quality traits in the pig, and also implicated the possible significance of TCAP on sarcomere assembly.

Occurrence of virus-induced COPD exacerbations during four seasons.

PubMed

Djamin, Remco S; Uzun, Sevim; Snelders, Eveline; Kluytmans, Jan J W; Hoogsteden, Henk C; Aerts, Joachim G J V; Van Der Eerden, Menno M

2015-02-01

In this study, we investigated the occurrence of viral infections in acute exacerbations of chronic obstructive pulmonary disease (COPD) during four seasons. Viral infections were detected by the use of real-time reverse transcriptase polymerase chain reaction on pharyngeal swabs. During a 12-month period pharyngeal swabs were obtained in 136 exacerbations of 63 patients. In 35 exacerbations (25.7%) a viral infection was detected. Most viral infections occurred in the winter (n = 14, 40.0%), followed by summer (n = 9, 25.7%), autumn (n = 6, 17.1%), and spring (n = 6, 17.1%). Rhinovirus was the most frequently isolated virus (n = 19, 51.4%), followed by respiratory syncytial virus (n = 6, 16.2%), human metapneumovirus (n = 5, 13.5%), influenza A (n = 4, 10.8%), parainfluenza 4 (n = 2, 5.4%), and parainfluenza 3 (n = 1, 2.7%). This study showed that virus-induced COPD exacerbations occur in all four seasons with a peak in the winter months. However, the distribution of rhinovirus infections showed a different pattern, with most infections occurring in July.

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.

Placental expression of D6 decoy receptor in preeclampsia

PubMed Central

Cho, Geum Joon; Lee, Eun Sung; Jin, Hye Mi; Lee, Ji Hye; Kim, Yeun Sun; Seol, Hyun-Joo; Hong, Soon-Cheol; Kim, Hai-Joong

2015-01-01

Objective The purpose of this study was to investigate the expression of the D6 decoy receptor that can bind chemokines and target them for degradation, resulting in inhibition of inflammation in placentas from preeclamptic and normal pregnancies. Methods The current study was carried out in 35 pregnant women (23 patients with preeclampsia and 12 healthy, normotensive pregnant women) during the third trimester of pregnancy. The expressions of D6 decoy receptor in the placenta were determined with real time reverse transcriptase polymerase chain reaction and western blotting. Results The mRNA and protein of D6 decoy receptor were detected in all of placentas from preeclamptic and normal pregnancies. Placental D6 decoy receptor mRNA expression was significantly lower in patients with preeclampsia than in patients with normal pregnancies. Western blot analyses revealed decreased protein expression in cases of preeclampsia. Conclusion The expression of the D6 decoy receptor in preeclamptic placentas was significantly lower than in normal placentas. Further studies are needed to clarify the underlying mechanisms that link decreased expression of placental D6 decoy receptor and preeclampsia. PMID:26430656

West Nile virus in overwintering Culex mosquitoes, New York City, 2000.

PubMed Central

Nasci, R. S.; Savage, H. M.; White, D. J.; Miller, J. R.; Cropp, B. C.; Godsey, M. S.; Kerst, A. J.; Bennett, P.; Gottfried, K.; Lanciotti, R. S.

2001-01-01

After the 1999 West Nile (WN) encephalitis outbreak in New York, 2,300 overwintering adult mosquitoes were tested for WN virus by cell culture and reverse transcriptase-polymerase chain reaction. WN viral RNA and live virus were found in pools of Culex mosquitoes. Persistence in overwintering Cx. pipiens may be important in the maintenance of WN virus in the northeastern United States. PMID:11585542

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.

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.

Telomerase Mechanism of Telomere Synthesis

PubMed Central

Wu, R. Alex; Upton, Heather E.; Vogan, Jacob M.; Collins, Kathleen

2017-01-01

Telomerase is the essential reverse transcriptase required for linear chromosome maintenance in most eukaryotes. Telomerase supplements the tandem array of simple-sequence repeats at chromosome ends to compensate for the DNA erosion inherent in genome replication. The template for telomerase reverse transcriptase is within the RNA subunit of the ribonucleoprotein complex, which in cells contains additional telomerase holoenzyme proteins that assemble the active ribonucleoprotein and promote its function at telomeres. Telomerase is distinct among polymerases in its reiterative reuse of an internal template. The template is precisely defined, processively copied, and regenerated by release of single-stranded product DNA. New specificities of nucleic acid handling that underlie the catalytic cycle of repeat synthesis derive from both active site specialization and new motif elaborations in protein and RNA subunits. Studies of telomerase provide unique insights into cellular requirements for genome stability, tissue renewal, and tumorigenesis as well as new perspectives on dynamic ribonucleoprotein machines. PMID:28141967

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

Laboratory Evaluation of Commercially Available Platforms to Detect West Nile and Zika Viruses From Honey Cards.

PubMed

Burkhalter, Kristen L; Wiggins, Keenan; Burkett-Cadena, Nathan; Alto, Barry W

2018-05-04

Commercially available assays utilizing antigen or nucleic acid detection chemistries provide options for mosquito control districts to screen their mosquito populations for arboviruses and make timely operational decisions regarding vector control. These assays may be utilized even more advantageously when combined with honey-soaked nucleic acid preservation substrate ('honey card') testing by reducing or replacing the time- and labor-intensive efforts of identifying and processing mosquito pools. We tested artificially inoculated honey cards and cards fed upon individually by West Nile virus (WNV) and Zika virus (ZIKV)-infected mosquitoes with three assays to compare detection rates and the limit of detection for each platform with respect to virus detection of a single infected mosquito and quantify the time interval of virus preservation on the cards. Assays evaluated included CDC protocols for real-time reverse transcriptase polymerase chain reaction (RT-PCR) for WNV and ZIKV, Pro-Lab Diagnostics ProAmpRT WNV loop-mediated amplification (LAMP) and ZIKV LAMP assays, and the Rapid Analyte Measurement Platform (RAMP) WNV assay. Real-time RT-PCR was the most sensitive assay and the most robust to viral RNA degradation over time. To maximize the detection of virus, honey cards should be left in the traps ≤1 d if using LAMP assays and ≤3 d if using real-time RT-PCR to detect viruses from field samples. The WNV RAMP assay, although effective for pool screening, lacks sensitivity required for honey card surveillance. Future studies may determine the minimum number of infectious mosquitoes required to feed on a honey card that would be reliably detected by the LAMP or RAMP assays.

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.

HOXB2 as a novel prognostic indicator for stage I lung adenocarcinomas.

PubMed

Inamura, Kentauro; Togashi, Yuki; Okui, Michiyo; Ninomiya, Hironori; Hiramatsu, Miyako; Satoh, Yukitoshi; Okumura, Sakae; Nakagawa, Ken; Shimoji, Takashi; Noda, Tetsuo; Ishikawa, Yuichi

2007-09-01

Outcomes of patients with lung adenocarcinomas can be predicted to some extent from the pathologic stage (p-stage). Although all attempts are made to fully remove cancer lesions, still a number of p-stage I patients without metastatic disease at the time of surgery develop recurrences and die of cancer. It is thus very important to identify p-stage I patients who are at risk of recurrence. Previously, using microdissected samples, we identified metastasis-related genes. Using real-time reverse-transcriptase polymerase chain reaction analysis, we investigated the transcriptional levels of the top metastasis-related genes using 96 independent test lung adenocarcinoma samples and investigated their correlations with the prognosis. We document evidence that p-stage I patients with HOXB2 up-regulation have a worse prognosis than those with HOXB2 down-regulation (p = 0.0065), whereas the HOXB2 status has no prognostic significance for p-stage II-IV patients. Comparing tumors and corresponding normal lung tissue, we confirmed HOXB2 up-regulated lesions to have much higher HOXB2 expression than the corresponding normal tissue. Confirmation with a larger number of samples is needed, with further research to clarify the molecular functions of HOXB2.

Real-Time PCR (qPCR) Primer Design Using Free Online Software

ERIC Educational Resources Information Center

Thornton, Brenda; Basu, Chhandak

2011-01-01

Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

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.

Mosquito species involved in the circulation of West Nile and Usutu viruses in Italy.

PubMed

Mancini, Giuseppe; Montarsi, Fabrizio; Calzolari, Mattia; Capelli, Gioia; Dottori, Michele; Ravagnan, Silvia; Lelli, Davide; Chiari, Mario; Santilli, Adriana; Quaglia, Michela; Quaglia, Michela; Federici, Valentina; Monaco, Federica; Goffredo, Maria; Savini, Giovanni

2017-06-30

Usutu (USUV) and West Nile (WNV) are mosquito-borne Flavivirus emerged in Italy in 1996 and 1998, respectively, and reappeared 10 years later. The aim of this work is to review the Italian mosquito species found positive for WNV and USUV between 2008 and 2014. Moreover, the role of mosquitoes in promoting the overwintering of these viruses is discussed, as a result of the mosquito collections performed in Molise region between September 2010 and April 2011. Overall 99,000 mosquitoes were collected: 337 and 457 mosquito pools tested positive by real time reverse transcriptase polymerase chain reaction (real time RT-PCR) for WNV and USUV, respectively. West Nile virus was detected in pools of Culex pipiens s.l. (329), Ochlerotatus caspius (4), Culex modestus (2), and Culex spp. (2). Positive USUV pools were from Cx. pipiens s.l. (435), Aedes albopictus (12), Oc. caspius (5), Culex spp. (2), Anopheles maculipennis s.l. (1), Culiseta annulata (1), and Ochlerotatus detritus (1). In Molise region, 1,694 mosquitoes were collected, and USUV was identi ed in Cx. pipiens s.l., Cs. annulata, and Oc. detritus pools. This paper shows that Cx. pipiens s.l. is the mosquito species most involved in the WNV and USUV circulation in Italy, although other species would also support the spread of both the viruses during Winter.

Matrix metalloprotease-3 expression in the medial plica and pannus-like tissue in knees from patients with medial compartment osteoarthritis.

PubMed

Wang, Hwai-Shi; Kuo, Pei-Yin; Yang, Chih-Chang; Lyu, Shaw-Ruey

2011-03-01

The severity of cartilage degeneration is positively correlated with the severity of the pathologic change of medial plica. However, knowledge of the pathogenic mechanisms and the impact of plica on cartilage destruction is limited. The aim of the present study was therefore to investigate matrix metalloprotease-3 (MMP-3) expression in the plica isolated from patients with medial compartment osteoarthritis of the knee. Immunohistochemistry showed that MMP-3 was highly expressed in pannus-like tissue and the plica. Western blotting of culture supernatants showed that interleukin-1β (IL-1β) treatment induced MMP-3 release by cells isolated from pannus tissue or the plica. Furthermore, reverse transcriptase polymerase chain reaction and real-time polymerase chain reaction analysis showed that MMP-3 mRNA levels were increased after IL-1β treatment of the cultured cells. MMP-3 and IL-1β mRNAs were expressed in the plica and pannus-like tissue, with MMP-3 mRNA being expressed at significantly higher levels in the plica than in normal synovial membrane and highly expressed in the plica at different stages in osteoarthritis (OA) patients. Pannus-like tissue and the plica express IL-1β and MMP-3. Moreover, MMP-3 mRNA and protein expression in the plica may contribute to the pathogenesis of OA. © 2011 Blackwell Publishing Limited.

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

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.

In vivo cartilage formation using chondrogenic-differentiated human adipose-derived mesenchymal stem cells mixed with fibrin glue.

PubMed

Jung, Sung-No; Rhie, Jong Won; Kwon, Ho; Jun, Young Joon; Seo, Je-Won; Yoo, Gyeol; Oh, Deuk Young; Ahn, Sang Tae; Woo, Jihyoun; Oh, Jieun

2010-03-01

Human adipose-derived mesenchymal stem cells (MSCs) were differentiated into chondrogenic MSCs, and fibrin glue was used together to explore the feasibility of whether cartilages can be generated in vivo by injecting the differentiated cells. Mesenchymal stem cells extracted from human adipose were differentiated into chondrogenic MSCs, and such differentiated cells mixed with fibrin glue were injected subcutaneously into the back of the nude mouse. In addition to visual evaluation of the tissues formed after 4, 8, and 12 weeks, hematoxylin-eosin staining, Masson trichrome staining, measurement of glycosaminoglycan concentration using dimethylmethylene blue, agreecan through reverse transcriptase-polymerase chain reaction, type II collagen, and expression of SOX-9 were verified. Moreover, the results were compared with 2 groups of controls: 1 control group that received only injection of chondrogenic-differentiated MSC and the supporting control group that received only fibrin glue injection. For the experimental group, cartilage-like tissues were formed after 4, 8, and 12 weeks. Formation of cartilage tissues was not observed in any of 4, 8, and 12 weeks of the control group. The supporting control group had only a small structure formation after 4 weeks, but the formed structure was completely decomposed by the 8th and 12th weeks. The range of staining dramatically increased with time at 4, 8, and 12 weeks in Masson trichrome staining. The concentration of glycosaminoglycan also increased with time. The increased level was statistically significant with more than 3 times more after 8 weeks compared with 4 weeks and more than 2 times more after 12 weeks compared with 8 weeks. Also, in reverse transcriptase-polymerase chain reaction at 4, 8, and 12 weeks, all results expressed a cartilage-specific gene called aggrecan, type II collagen, and SOX-9. The study verified that the chondrogenic-differentiated MSCs derived from human adipose tissues with fibrin glue can proliferate and form new cartilage. Our findings suggest that formation of cartilages in vivo is possible.

Pandemic influenza A(H1N1)pdm09: An unrecognized cause of mortality in children in Pakistan

PubMed Central

ALI, SYED ASAD; AZIZ, FATIMA; AKHTAR, NIDA; QURESHI, SHAHIDA; EDWARDS, KATHRYN; ZAIDI, ANITA

2016-01-01

The role of influenza virus as a cause of child mortality in South Asia is under-recognized. We aimed to determine the incidence and case fatality rate of influenza A(H1N1)pdm09 infections in hospitalized children in Karachi, Pakistan. Children less than 5 y old admitted with respiratory illnesses to the Aga Khan University Hospital, Karachi, from 17 August 2009 to 16 September 2011, were tested for influenza A(H1N1)pdm09 using a real-time reverse transcriptase polymerase chain reaction. Out of 2650 children less than 5 y old admitted with a respiratory illness during the study period, 812 (31%) were enrolled. Influenza A(H1N1)pdm09 virus was detected in 27 (3.3%) children. There were 4 deaths in children who tested positive for influenza A(H1N1)pdm09 (case fatality rate of 15%). Children with influenza A(H1N1)pdm09 were 5 times more likely to be admitted or transferred to the intensive care unit, 5.5 times more likely to be intubated, and 12.9 times more likely to die as compared to children testing negative for influenza A(H1N1)pdm09. PMID:23826795

The structure of unliganded reverse transcriptase from the human immunodeficiency virus type 1.

PubMed Central

Rodgers, D W; Gamblin, S J; Harris, B A; Ray, S; Culp, J S; Hellmig, B; Woolf, D J; Debouck, C; Harrison, S C

1995-01-01

The crystal structure of the reverse transcriptase (RT) from the type 1 human immunodeficiency virus has been determined at 3.2-A resolution. Comparison with complexes between RT and the polymerase inhibitor Nevirapine [Kohlstaedt, L.A., Wang, J., Friedman, J.M., Rice, P.A. & Steitz, T.A. (1992) Science 256, 1783-1790] and between RT and an oligonucleotide [Jacobo-Molina, A., Ding, J., Nanni, R., Clark, A. D., Lu, X., Tantillo, C., Williams, R. L., Kamer, G., Ferris, A. L., Clark, P., Hizi, A., Hughes, S. H. & Arnold, E. (1993) Proc. Natl. Acad. Sci. USA 90, 6320-6324] reveals changes associated with ligand binding. The enzyme is a heterodimer (p66/p51), with domains labeled "fingers," "thumb," "palm," and "connection" in both subunits, and a ribonuclease H domain in the larger subunit only. The most striking difference between RT and both complex structures is the change in orientation of the p66 thumb (approximately 33 degrees rotation). Smaller shifts relative to the core of the molecule were also found in other domains, including the p66 fingers and palm, which contain the polymerase active site. Within the polymerase catalytic region itself, there are no rearrangements between RT and the RT/DNA complex. In RT/Nevirapine, the drug binds in the p66 palm near the polymerase active site, a region that is well-packed hydrophobic core in the unliganded enzyme. Room for the drug is provided by movement of a small beta-sheet within the palm domain of the Nevirapine complex. The rearrangement within the palm and thumb, as well as domain shifts relative to the enzyme core, may prevent correct placement of the oligonucleotide substrate when the drug is bound. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7532306

Development of field-based real-time reverse transcription-polymerase chain reaction assays for detection of Chikungunya and O'nyong-nyong viruses in mosquitoes.

PubMed

Smith, Darci R; Lee, John S; Jahrling, Jordan; Kulesh, David A; Turell, Michael J; Groebner, Jennifer L; O'Guinn, Monica L

2009-10-01

Chikungunya (CHIK) and O'nyong-nyong (ONN) are important emerging arthropod-borne diseases. Molecular diagnosis of these two viruses in mosquitoes has not been evaluated, and the effects of extraneous mosquito tissue on assay performance have not been tested. Additionally, no real-time reverse transcription-polymerase chain reaction (RT-PCR) assay exists for detecting ONN virus (ONNV) RNA. We describe the development of sensitive and specific real-time RT-PCR assays for detecting CHIK and ONN viral RNA in mosquitoes, which have application for field use. In addition, we compared three methods for primer/probe design for assay development by evaluating their sensitivity and specificity. This comparison resulted in development of virus-specific assays that could detect less than one plaque-forming unit equivalent of each of the viruses in mosquitoes. The use of these assays will aid in arthropod-borne disease surveillance and in the control of the associated diseases.

Possible Application of Biotechnology to the Development of Biological Agents by Potential Enemies

DTIC Science & Technology

1987-06-01

of enzyme catalyzed reactions. Although cloning techniques are directly applicable to the manipulation of proteinaceous toxins, they would be less...useful for nonproteinaceous toxins because the corresponding gene for each enzyme must be cloned and expressed in a coordinated manner. Effective...to produce a synthetic DNA. The enzyme reverse transcriptase (RNA dependent DNA polymerase), which is obtained from retroviruses, is the only enzyme

HOX genes in human lung: altered expression in primary pulmonary hypertension and emphysema.

PubMed

Golpon, H A; Geraci, M W; Moore, M D; Miller, H L; Miller, G J; Tuder, R M; Voelkel, N F

2001-03-01

HOX genes belong to the large family of homeodomain genes that function as transcription factors. Animal studies indicate that they play an essential role in lung development. We investigated the expression pattern of HOX genes in human lung tissue by using microarray and degenerate reverse transcriptase-polymerase chain reaction survey techniques. HOX genes predominantly from the 3' end of clusters A and B were expressed in normal human adult lung and among them HOXA5 was the most abundant, followed by HOXB2 and HOXB6. In fetal (12 weeks old) and diseased lung specimens (emphysema, primary pulmonary hypertension) additional HOX genes from clusters C and D were expressed. Using in situ hybridization, transcripts for HOXA5 were predominantly found in alveolar septal and epithelial cells, both in normal and diseased lungs. A 2.5-fold increase in HOXA5 mRNA expression was demonstrated by quantitative reverse transcriptase-polymerase chain reaction in primary pulmonary hypertension lung specimens when compared to normal lung tissue. In conclusion, we demonstrate that HOX genes are selectively expressed in the human lung. Differences in the pattern of HOX gene expression exist among fetal, adult, and diseased lung specimens. The altered pattern of HOX gene expression may contribute to the development of pulmonary diseases.

HOX Genes in Human Lung

PubMed Central

Golpon, Heiko A.; Geraci, Mark W.; Moore, Mark D.; Miller, Heidi L.; Miller, Gary J.; Tuder, Rubin M.; Voelkel, Norbert F.

2001-01-01

HOX genes belong to the large family of homeodomain genes that function as transcription factors. Animal studies indicate that they play an essential role in lung development. We investigated the expression pattern of HOX genes in human lung tissue by using microarray and degenerate reverse transcriptase-polymerase chain reaction survey techniques. HOX genes predominantly from the 3′ end of clusters A and B were expressed in normal human adult lung and among them HOXA5 was the most abundant, followed by HOXB2 and HOXB6. In fetal (12 weeks old) and diseased lung specimens (emphysema, primary pulmonary hypertension) additional HOX genes from clusters C and D were expressed. Using in situ hybridization, transcripts for HOXA5 were predominantly found in alveolar septal and epithelial cells, both in normal and diseased lungs. A 2.5-fold increase in HOXA5 mRNA expression was demonstrated by quantitative reverse transcriptase-polymerase chain reaction in primary pulmonary hypertension lung specimens when compared to normal lung tissue. In conclusion, we demonstrate that HOX genes are selectively expressed in the human lung. Differences in the pattern of HOX gene expression exist among fetal, adult, and diseased lung specimens. The altered pattern of HOX gene expression may contribute to the development of pulmonary diseases. PMID:11238043

Microwave-Assisted Rapid Enzymatic Synthesis of Nucleic Acids.

PubMed

Hari Das, Rakha; Ahirwar, Rajesh; Kumar, Saroj; Nahar, Pradip

2016-07-02

Herein we report microwave-induced enhancement of the reactions catalyzed by Escherichia coli DNA polymerase I and avian myeloblastosis virus-reverse transcriptase. The reactions induced by microwaves result in a highly selective synthesis of nucleic acids in 10-50 seconds. In contrast, same reactions failed to give desired reaction products when carried out in the same time periods, but without microwave irradiation. Each of the reactions was carried out for different duration of microwave exposure time to find the optimum reaction time. The products produced by the respective enzyme upon microwave irradiation of the reaction mixtures were identical to that produced by the conventional procedures. As the microwave-assisted reactions are rapid, microwave could be a useful alternative to the conventional and time consuming procedures of enzymatic synthesis of nucleic acids.

Inhibition of P-glycoprotein and glutathione S-transferase-pi mediated resistance by fluoxetine in MCF-7/ADM cells.

PubMed

Zhang, Ye; Zhou, Ting; Duan, Jingjing; Xiao, Zhijun; Li, Guihua; Xu, Feng

2013-10-01

Chemotherapy is important in the systematic treatment of breast cancer. While multidrug resistance (MDR) is the main obstacle in chemotherapy, a reversal reagent with high reversal effect but low toxicity is the hotspot issue at present to overcome MDR. Antidepressant fluoxetine (FLX) is a potential new highly effective chemosensitizer, however, the possible mechanism is unclear. In this study, the effect of FLX on multidrug resistance mediated by P-glycoprotein (P-gp) and glutathione S-transferase-pi (GST-π) were researched in resistant/sensitive breast cancer cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to determine the cells viability after being incubated with FLX/Adriamycin (ADM)/Paclitaxel (PTX) alone or FLX-ADM, FLX-PTX combination. Western blot was performed to assay the expression of P-gp and GST-π proteins. Reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) were performed to assay the level of MDR1 mRNA. The results showed that pre-treatment with FLX enhance cytotoxicity significantly both on resistant and sensitive cells, downregulated the expression of P-gp and GST-π proteins in resistance cells, decreased the MDR1 mRNA by FLX-PTX combination only. No P-gp and GST-π were detected in sensitive cells. Our research thus indicated that FLX reverse the breast cancer cell's resistance and enhance the chemosensitivity by regulating P-gp and GST-π levels. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Crystallographic Study of a Novel Sub-Nanomolar Inhibitor Provides Insight on the Binding Interactions of Alkenyldiarylmethanes with Human Immunodeficiency Virus-1 (HIV-1) Reverse Transcriptase†

PubMed Central

Cullen, Matthew D.; Ho, William C.; Bauman, Joseph D.; Das, Kalyan; Arnold, Eddy; Hartman, Tracy L.; Watson, Karen M.; Buckheit, Robert W.; Pannecouque, Christophe; De Clercq, Erik; Cushman, Mark

2009-01-01

Two crystal structures have been solved for separate complexes of alkenyldiarylmethane (ADAM) non-nucleoside reverse transcriptase inhibitors (NNRTI) 3 and 4 with HIV-1 reverse transcriptase (RT). The structures reveal inhibitor binding is exclusively hydrophobic in nature and the shape of the inhibitor-bound NNRTI binding pocket is unique among other reported inhibitor-RT crystal structures. Primarily, ADAMs 3 and 4 protrude from a large gap in the backside of the binding pocket, placing portions of the inhibitors unusually close to the polymerase active site and allowing 3 to form a weak hydrogen bond with Lys223. The lack of additional stabilizing interactions, beyond the observed hydrophobic surface contacts, between 4 and RT is quite perplexing given the extreme potency of the compound (IC50 ≤ nM). ADAM 4 was designed to be hydrolytically stable in blood plasma, and an investigation of its hydrolysis in rat plasma demonstrated it has a significantly prolonged half-life in comparison to ADAM lead compounds 1 and 2. PMID:19775161

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

Reverse transcription of phage RNA and its fragment directed by synthetic heteropolymeric primers

PubMed Central

Frolova, L. Yu.; Metelyev, V. G.; Ratmanova, K. I.; Smirnov, V. D.; Shabarova, Z. A.; Prokofyev, M. A.; Berzin, V. M.; Jansone, I. V.; Gren, E. J.; Kisselev, L. L.

1977-01-01

DNA synthesis catalysed by RNA-directed DNA-polymerase (reverse transcriptase) was found to proceed on the RNA template of an MS2 phage in the presence of heteropolymeric synthetic octa- and nonadeoxyribonucleotide primers complementary to the intercistronic region (coat protein binding site) and the region of the coat protein cistron, respectively. The product of synthesis consists of discrete DNA fractions of different length, including transcripts longer than 1,000 nucleotides. The coat protein inhibits DNA synthesis if it is initiated at its binding site, but has no effect on DNA synthesis initiated at the coat protein cistron. It has been suggested that, in this system, the initiation of DNA synthesis by synthetic primers is topographically specific. The MS2 coat protein binding site (an RNA fragment of 59 nucleotides) serves as a template for polydeoxyribonucleotide synthesis in the presence of octanucleotide primer and reverse transcriptase. The product of synthesis is homogenous and its length corresponds to the length of the template. The effective and complete copying of the fragment having a distinct secondary structure proves that the secondary structure does not interfere, in principle, with RNA being a template in the system of reverse transcription. PMID:71713

Simultaneous detection of hemagglutinin and neuraminidase genes of novel influenza A (H7N9) by duplex real-time reverse transcription polymerase chain reaction.

PubMed

Li, Yan; Wu, Tao; Qi, Xian; Ge, Yiyue; Guo, Xiling; Wu, Bin; Yu, Huiyan; Zhu, Yefei; Shi, Zhiyang; Wang, Hua; Cui, Lunbiao; Zhou, Minghao

2013-12-01

A novel reassortant influenza A (H7N9) virus emerged recently in China. In this study, a duplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay was developed for the simultaneous detection of hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 influenza viruses. The sensitivity of the assay was determined to be 10 RNA copies per reaction for both HA and NA genes. No cross-reactivity was observed with other influenza virus subtypes or respiratory tract viruses. One hundred and forty-six clinical and environmental specimens were tested and compared with reference methods and were found to be consistent. The assay is suitable for large-scale screening due to short turnaround times and high specificity, sensitivity, and reproducibility. Copyright © 2013 Elsevier B.V. All rights reserved.

Trends of drug-resistance-associated mutations in the reverse transcriptase gene of HIV type 1 isolates from North India.

PubMed

Azam, Mohd; Malik, Abida; Rizvi, Meher; Rai, Arvind

2014-04-01

A major cause of failure of antiretroviral therapy (ART) is the presence of drug-resistance-associated mutations in the polymerase gene of HIV-1. The paucity of data regarding potential drug resistance to reverse transcriptase inhibitors (RTIs) prompted us to carry out this study. This information will shed light on the extent of drug resistance already present in HIV strains and will give future directions in patient treatment and in drug design. Drug resistance genotyping of a partial reverse transcriptase gene was done in 103 HIV-1-infected patients, including the ART-naive and ART-experienced population. The drug resistance pattern was analyzed using the Stanford HIV-DR database, the IAS-USA mutation list and the REGA algorithm-v8.0. Subtyping was done using the REGA HIV-1 subtyping tool-v2.01. The majority of our sequences (96 %) were found to be subtype C, and four (3.8 %) were subtype A1. Significant prevalence of DR mutations (28 %) was observed in the RT gene. Major amino acid substitutions were seen at positions 41, 90, 98, 103, 106, 108, 138, 181, 184, 190, 215, and 219, which confer high/intermediate levels of resistance to most RTIs, independently or together. Our results show that there is an urgent need to tailor ART drug regimens to the individual to achieve optimum therapeutic outcome in North India.

A Novel Mechanism of Sugar Selection Utilized by a Human X-family DNA Polymerase†

PubMed Central

Brown, Jessica A.; Fiala, Kevin A.; Fowler, Jason D.; Sherrer, Shanen M.; Newmister, Sean A.; Dyum, Wade W.; Suo, Zucai

2009-01-01

During DNA synthesis, most DNA polymerases and reverse transcriptases select against ribonucleotides via a steric clash between the ribose 2′-hydroxyl group and the bulky side chain of an active site residue. Here, we demonstrated that human DNA polymerase λ used a novel sugar selection mechanism to discriminate against ribonucleotides, whereby the ribose 2′-hydroxyl group was excluded mostly by a backbone segment and slightly by the side chain of Y505. Such a steric clash was further demonstrated to be dependent on the size and orientation of the substituent covalently attached at the ribonucleotide C2′ position. PMID:19900463

On the early emergence of reverse transcription: theoretical basis and experimental evidence

NASA Technical Reports Server (NTRS)

Lazcano, A.; Valverde, V.; Hernandez, G.; Gariglio, P.; Fox, G. E.; Oro, J.

1992-01-01

Reverse transcriptase (RT) was first discovered as an essential catalyst in the biological cycle of retroviruses. However, in the past years evidence has accumulated showing that RTs are involved in a surprisingly large number of RNA-mediated transpositional events that include both viral and nonviral genetic entities. Although it is probable that some RT-bearing genetic elements like the different types of AIDS viruses and the mammalian LINE family have arisen in recent geological times, the possibility that reverse transcription first took place in the early Archean is supported by (1) the hypothesis that RNA preceded DNA as cellular genetic material; (2) the existence of homologous regions of the subunit tau of the E. coli DNA polymerase III with the simian immunodeficiency virus RT, the hepatitis B virus RT, and the beta' subunit of the E. coli RNA polymerase (McHenry et al. 1988); (3) the presence of several conserved motifs, including a 14-amino-acid segment that consists of an Asp-Asp pair flanked by hydrophobic amino acids, which are found in all RTs and in most cellular and viral RNA polymerases. However, whether extant RTs descend from the primitive polymerase involved in the RNA-to-DNA transition remains unproven. Substrate specificity of the AMV and HIV-1 RTs can be modified in the presence of Mn2+, a cation which allows them to add ribonucleotides to an oligo (dG) primer in a template-dependent reaction. This change in specificity is comparable to that observed under similar conditions in other nucleic acid polymerases. This experimentally induced change in RT substrate specificity may explain previous observations on the misincorporation of ribonucleotides by the Maloney murine sarcoma virus RT in the minus and plus DNA of this retrovirus (Chen and Temin 1980). Our results also suggest that HIV-infected macrophages and T-cell cells may contain mixed polynucleotides containing both ribo- and deoxyribonucleotides. The evolutionary significance of these changes in substrate specificities of nucleic acid polymerases is also discussed.

Variation in Bluetongue virus real-time reverse transcription polymerase chain reaction assay results in blood samples of sheep, cattle, and alpaca.

PubMed

Brito, Barbara P; Gardner, Ian A; Hietala, Sharon K; Crossley, Beate M

2011-07-01

Bluetongue is a vector-borne viral disease that affects domestic and wild ruminants. The epidemiology of this disease has recently changed, with occurrence in new geographic areas. Various real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) assays are used to detect Bluetongue virus (BTV); however, the impact of biologic differences between New World camelids and domestic ruminant samples on PCR efficiency, for which the BTV real-time qRT-PCR was initially validated are unknown. New world camelids are known to have important biologic differences in whole blood composition, including hemoglobin concentration, which can alter PCR performance. In the present study, sheep, cattle, and alpaca blood were spiked with BTV serotypes 10, 11, 13, and 17 and analyzed in 10-fold dilutions by real-time qRT-PCR to determine if species affected nucleic acid recovery and assay performance. A separate experiment was performed using spiked alpaca blood subsequently diluted in 10-fold series in sheep blood to assess the influence of alpaca blood on performance efficiency of the BTV real-time qRT-PCR assay. Results showed that BTV-specific nucleic acid detection from alpaca blood was consistently 1-2 logs lower than from sheep and cattle blood, and results were similar for each of the 4 BTV serotypes analyzed.

Serial detection of circulating tumour cells by reverse transcriptase-polymerase chain reaction assays is a marker for poor outcome in patients with malignant melanoma

PubMed Central

Palmieri, Giuseppe; Satriano, Sabrina MR; Budroni, Mario; Cossu, Antonio; Tanda, Francesco; Canzanella, Sergio; Caracò, Corrado; Simeone, Ester; Daponte, Antonio; Mozzillo, Nicola; Comella, Giuseppe; Castello, Giuseppe; Ascierto, Paolo A

2006-01-01

Background Detection of circulating malignant cells (CMCs) through a reverse transcriptase-polymerase chain reaction (RT-PCR) assay seems to be a demonstration of systemic disease. We here evaluated the prognostic role of RT-PCR assays in serially-taken peripheral blood samples from patients with malignant melanoma (MM). Methods One hundred forty-nine melanoma patients with disease stage ranging from I to III were consecutively collected in 1997. A multi-marker RT-PCR assay was used on peripheral blood samples obtained at time of diagnosis and every 6 months during the first two years of follow-up (total: 5 samples). Univariate and multivariate analyses were performed after 83 months of median follow-up. Results Detection of at least one circulating mRNA marker was considered a signal of the presence of CMC (referred to as PCR-positive assay). A significant correlation was found between the rate of recurrences and the increasing number of PCR-positive assays (P = 0.007). Presence of CMC in a high number (≥2) of analysed blood samples was significantly correlated with a poor clinical outcome (disease-free survival: P = 0.019; overall survival: P = 0.034). Multivariate analysis revealed that presence of a PCR-positive status does play a role as independent prognostic factors for overall survival in melanoma patients, adding precision to the predictive power of the disease stage. Conclusion Our findings indicated that serial RT-PCR assay may identify a high risk subset of melanoma patients with occult cancer cells constantly detected in blood circulation. Prolonged presence of CMCs seems to act as a surrogate marker of disease progression or a sign of more aggressive disease. PMID:17107608

Serial detection of circulating tumour cells by reverse transcriptase-polymerase chain reaction assays is a marker for poor outcome in patients with malignant melanoma.

PubMed

Palmieri, Giuseppe; Satriano, Sabrina M R; Budroni, Mario; Cossu, Antonio; Tanda, Francesco; Canzanella, Sergio; Caracò, Corrado; Simeone, Ester; Daponte, Antonio; Mozzillo, Nicola; Comella, Giuseppe; Castello, Giuseppe; Ascierto, Paolo A

2006-11-15

Detection of circulating malignant cells (CMCs) through a reverse transcriptase-polymerase chain reaction (RT-PCR) assay seems to be a demonstration of systemic disease. We here evaluated the prognostic role of RT-PCR assays in serially-taken peripheral blood samples from patients with malignant melanoma (MM). One hundred forty-nine melanoma patients with disease stage ranging from I to III were consecutively collected in 1997. A multi-marker RT-PCR assay was used on peripheral blood samples obtained at time of diagnosis and every 6 months during the first two years of follow-up (total: 5 samples). Univariate and multivariate analyses were performed after 83 months of median follow-up. Detection of at least one circulating mRNA marker was considered a signal of the presence of CMC (referred to as PCR-positive assay). A significant correlation was found between the rate of recurrences and the increasing number of PCR-positive assays (P = 0.007). Presence of CMC in a high number (> or =2) of analysed blood samples was significantly correlated with a poor clinical outcome (disease-free survival: P = 0.019; overall survival: P = 0.034). Multivariate analysis revealed that presence of a PCR-positive status does play a role as independent prognostic factors for overall survival in melanoma patients, adding precision to the predictive power of the disease stage. Our findings indicated that serial RT-PCR assay may identify a high risk subset of melanoma patients with occult cancer cells constantly detected in blood circulation. Prolonged presence of CMCs seems to act as a surrogate marker of disease progression or a sign of more aggressive disease.

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.

TOPICAL TENOFOVIR, A MICROBICIDE EFFECTIVE AGAINST HIV, INHIBITS HERPES SIMPLEX VIRUS-2 REPLICATION

PubMed Central

Andrei, Graciela; Lisco, Andrea; Vanpouille, Christophe; Introini, Andrea; Balestra, Emanuela; van den Oord, Joost; Cihlar, Tomas; Perno, Carlo-Federico; Snoeck, Robert; Margolis, Leonid; Balzarini, Jan

2011-01-01

SUMMARY The HIV reverse transcriptase inhibitor tenofovir, was recently formulated into a vaginal gel for use as a microbicide. In human trials, a 1% tenofovir gel inhibited HIV sexual transmission by 39% and surprisingly herpes simplex virus-2 (HSV-2) transmission by 51%. We demonstrate that the concentration achieved intravaginally with a 1% tenofovir topical gel has direct anti-herpetic activity. Tenofovir inhibits the replication of HSV clinical isolates in human embryonic fibroblasts, keratinocytes, and organotypic epithelial 3D-rafts, decreases HSV replication in human lymphoid and cervical tissues ex vivo, and delays HSV-induced lesions and death of topically treated HSV-infected mice. The active tenofovir metabolite inhibits HSV DNA-polymerase and HIV reverse transcriptase. Tenofovir must be topically administered to achieve concentrations, which are higher than systemic levels after oral treatment, that exert these dual antiviral effects. These findings indicate that a single topical treatment, like tenofovir, can inhibit the transmission of HIV and its co-pathogens. PMID:22018238

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.

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.

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).

Detection of carcinoembryonic antigen messenger RNA in blood using quantitative real-time reverse transcriptase-polymerase chain reaction to predict recurrence of gastric adenocarcinoma

PubMed Central

2010-01-01

Background The existence of circulating tumor cells (CTCs) in peripheral blood as an indicator of tumor recurrence has not been clearly established, particularly for gastric cancer patients. We conducted a retrospective analysis of the relationship between CTCs in peripheral blood at initial diagnosis and clinicopathologic findings in patients with gastric carcinoma. Methods Blood samples were obtained from 123 gastric carcinoma patients at initial diagnosis. mRNA was extracted and amplified for carcinoembryonic antigen (CEA) mRNA detection using real-time RT-PCR. Periodic 3-month follow-up examinations included serum CEA measurements and imaging. Results The minimum threshold for corrected CEA mRNA score [(CEA mRNA/GAPDH mRNA) × 106] was set at 100. Forty-five of 123 patients (36.6%) were positive for CEA mRNA expression. CEA mRNA expression significantly correlated with T stage and postoperative recurrence status (P = 0.001). Recurrent disease was found in 44 of 123 cases (35.8%), and 25 of these (56.8%) were positive for CEA mRNA. Of these patients, CEA mRNA was more sensitive than serum CEA in indicating recurrence. Three-year disease-free survival of patients positive for CEA mRNA was significantly poorer than of patients negative for CEA mRNA (P < 0.001). Only histological grade and CEA mRNA positivity were independent factors for disease-free survival using multivariate analysis. Conclusions CEA mRNA copy number in peripheral blood at initial diagnosis was significantly associated with disease recurrence in gastric adenocarcinoma patients. Real-time RT-PCR detection of CEA mRNA levels at initial diagnosis appears to be a promising predictor for disease recurrence in gastric adenocarcinoma patients. PMID:21040522

Detection of carcinoembryonic antigen messenger RNA in blood using quantitative real-time reverse transcriptase-polymerase chain reaction to predict recurrence of gastric adenocarcinoma.

PubMed

Qiu, Miao-Zhen; Li, Zhuang-Hua; Zhou, Zhi-Wei; Li, Yu-Hong; Wang, Zhi-Qiang; Wang, Feng-Hua; Huang, Peng; Aziz, Fahad; Wang, Dao-Yuan; Xu, Rui-Hua

2010-10-31

The existence of circulating tumor cells (CTCs) in peripheral blood as an indicator of tumor recurrence has not been clearly established, particularly for gastric cancer patients. We conducted a retrospective analysis of the relationship between CTCs in peripheral blood at initial diagnosis and clinicopathologic findings in patients with gastric carcinoma. Blood samples were obtained from 123 gastric carcinoma patients at initial diagnosis. mRNA was extracted and amplified for carcinoembryonic antigen (CEA) mRNA detection using real-time RT-PCR. Periodic 3-month follow-up examinations included serum CEA measurements and imaging. The minimum threshold for corrected CEA mRNA score [(CEA mRNA/GAPDH mRNA) × 106] was set at 100. Forty-five of 123 patients (36.6%) were positive for CEA mRNA expression. CEA mRNA expression significantly correlated with T stage and postoperative recurrence status (P = 0.001). Recurrent disease was found in 44 of 123 cases (35.8%), and 25 of these (56.8%) were positive for CEA mRNA. Of these patients, CEA mRNA was more sensitive than serum CEA in indicating recurrence. Three-year disease-free survival of patients positive for CEA mRNA was significantly poorer than of patients negative for CEA mRNA (P < 0.001). Only histological grade and CEA mRNA positivity were independent factors for disease-free survival using multivariate analysis. CEA mRNA copy number in peripheral blood at initial diagnosis was significantly associated with disease recurrence in gastric adenocarcinoma patients. Real-time RT-PCR detection of CEA mRNA levels at initial diagnosis appears to be a promising predictor for disease recurrence in gastric adenocarcinoma patients.

Activation of RAS family genes in urothelial carcinoma.

PubMed

Boulalas, I; Zaravinos, A; Karyotis, I; Delakas, D; Spandidos, D A

2009-05-01

Bladder cancer is the fifth most common malignancy in men in Western society. We determined RAS codon 12 and 13 point mutations and evaluated mRNA expression levels in transitional cell carcinoma cases. Samples from 30 human bladder cancers and 30 normal tissues were analyzed by polymerase chain reaction/restriction fragment length polymorphism and direct sequencing to determine the occurrence of mutations in codons 12 and 13 of RAS family genes. Moreover, we used real-time reverse transcriptase-polymerase chain reaction to evaluate the expression profile of RAS genes in bladder cancer specimens compared to that in adjacent normal tissues. Overall H-RAS mutations in codon 12 were observed in 9 tumor samples (30%). Two of the 9 patients (22%) had invasive bladder cancer and 7 (77%) had noninvasive bladder cancer. One H-RAS mutation (11%) was homozygous and the remaining 89% were heterozygous. All samples were WT for K and N-RAS oncogenes. Moreover, 23 of 30 samples (77%) showed over expression in at least 1 RAS family gene compared to adjacent normal tissue. K and N-RAS had the highest levels of over expression in bladder cancer specimens (50%), whereas 27% of transitional cell carcinomas demonstrated H-RAS over expression relative to paired normal tissues. Our results underline the importance of H-RAS activation in human bladder cancer by codon 12 mutations. Moreover, they provide evidence that increased expression of all 3 RAS genes is a common event in bladder cancer that is associated with disease development.

Development of a pan-Simbu real-time reverse transcriptase PCR for the detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR systems.

PubMed

Fischer, Melina; Schirrmeier, Horst; Wernike, Kerstin; Wegelt, Anne; Beer, Martin; Hoffmann, Bernd

2013-11-05

Schmallenberg virus (SBV), a novel orthobunyavirus of the Simbu serogroup, was first identified in October 2011 in dairy cattle in Germany, where it caused fever, diarrhea and a drop in milk yield. Since then, SBV additionally has been detected in adult sheep and goats. Although symptoms of acute infection were not observed, infection during a vulnerable phase of pregnancy caused congenital malformations and stillbirths. In view of the current situation and the possible emergence of further Simbu serogroup members, a pan-Simbu real-time reverse transcriptase (RT) PCR system for the reliable detection of Simbu serogroup viruses should be developed. In this study a pan-Simbu real-time RT-PCR system was established and compared to several SBV real-time RT-PCR assays. All PCR-systems were tested using a panel of different Simbu serogroup viruses as well as several field samples from diseased cattle, sheep and goats originating from all over Germany. Several pan-Simbu real-time RT-PCR products were sequenced via Sanger sequencing. Furthermore, in silico analyses were performed to investigate suitability for the detection of further orthobunyaviruses. All tested members of the Simbu serogroup (n = 14) as well as most of the field samples were successfully detected by the pan-Simbu real-time RT-PCR system. The comparison of this intercalating dye assay with different TaqMan probe-based assays developed for SBV diagnostics confirmed the functionality of the pan-Simbu assay for screening purposes. However, the SBV-TaqMan-assay SBV-S3 delivered the highest analytical sensitivity of less than ten copies per reaction for duplex systems including an internal control. In addition, for confirmation of SBV-genome detection the highly specific SBV-M1 assay was established. The pan-Simbu real-time RT-PCR system was able to detect all tested members of the Simbu serogroup, most of the SBV field samples as well as three tested Bunyamwera serogroup viruses with a suitable sensitivity. According to in silico analyses, this system seems to be able to detect a broad orthobunyavirus spectrum. As an additional feature of the pan-Simbu real-time RT-PCR system, subsequent species classification via sequencing is feasible. Regarding SBV diagnostics, the performance of the S-segment targeting SBV-S3 assay was superior with respect to the analytical sensitivity.

Distemper outbreak and its effect on African wild dog conservation.

PubMed

van de Bildt, Marco W G; Kuiken, Thijs; Visee, Aart M; Lema, Sangito; Fitzjohn, Tony R; Osterhaus, Albert D M E

2002-02-01

In December 2000, an infectious disease spread through a captive breeding group of African wild dogs (Lycaon pictus) in Tanzania, killing 49 of 52 animals within 2 months. The causative agent was identified as Canine distemper virus (CDV) by means of histologic examination, virus isolation, reverse transcriptase-polymerase chain reaction analysis, and nucleotide sequencing. This report emphasizes the importance of adequate protection against infectious diseases for the successful outcome of captive breeding programs of endangered species.

Determination of Clinical and Demographic Predictors of Laboratory-confirmed Influenza with Subtype Analysis

DTIC Science & Technology

2012-06-07

Advisory Committee on Immunization Practicies (ACIP). vol. 57th edition. Atlanta, GA: MMWR; 2008:1–59. 3. Cox NJ, Subbarao K : Influenza. Lancet 1999...to April 2008. Reverse-transcriptase polymerase chain reaction testing and viral culture for influenza A and B with subtyping were performed on all...REPORT unclassified b . ABSTRACT unclassified c. THIS PAGE unclassified Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 RESEARCH

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.

Cardiomyogenic Differentiation in Cardiac Myxoma Expressing Lineage-Specific Transcription Factors

PubMed Central

Kodama, Hiroaki; Hirotani, Takashi; Suzuki, Yusuke; Ogawa, Satoshi; Yamazaki, Kazuto

2002-01-01

We investigated five cases of cardiac myxoma and one case of cardiac undifferentiated sarcoma by light and electron microscopy, in situ hybridization, immunohistochemical staining, and reverse transcriptase-polymerase chain reaction for cardiomyocyte-specific transcription factors, Nkx2.5/Csx, GATA-4, MEF2, and eHAND. Conventional light microscopy revealed that cardiac myxoma and sarcoma cells presented variable cellular arrangements and different histological characteristics. Ultrastructurally, some of the myxoma cells exhibited endothelium-like or immature mesenchymal cell differentiation. Immunohistochemistry for Nkx2.5/Csx, GATA-4, and eHAND was slightly to intensely positive in all myxoma cases. MEF2 immunoreactivity was observed in all cases including the case of sarcoma, thus suggesting myogenic differentiation of myxoma or sarcoma cells. In situ hybridization for Nkx2.5/Csx also revealed that all myxoma cells, but not sarcoma cells, expressed mRNA of the cardiac homeobox gene, Nkx2.5/Csx. Furthermore, nested reverse transcriptase-polymerase chain reaction from formalin-fixed, paraffin-embedded tissue was performed and demonstrated that the Nkx2.5/Csx and eHAND gene product to be detected in all cases, and in three of six cases, respectively. In conclusion, cardiac myxoma cells were found to express various amounts of cardiomyocyte-specific transcription factor gene products at the mRNA and protein levels, thus suggesting cardiomyogenic differentiation. These results support the concept that cardiac myxoma might arise from mesenchymal cardiomyocyte progenitor cells. PMID:12163362

Acute localized exanthem due to Coxsackievirus A4.

PubMed

Drago, Francesco; Ciccarese, Giulia; Gariazzo, Lodovica; Cioni, Margherita; Parodi, Aurora

2017-09-01

Enteroviruses are the leading cause of exanthems in children, especially during summer and autumn. Enterovirus infections may occur in epidemics or small outbreaks. A 30-year-old woman presented with a three-day history of an erythematous maculopapular skin rash with petechiae localized exclusively under the nipple of the right breast. The skin eruption was associated with an erythematous-petechial enanthem. The patient complained of low-grade fever, headache, asthenia, sore throat and arthromyalgias. IgM (1:128) and IgG (1:640) antibodies against Coxsackievirus A4 were detected by the virus neutralization test. Reverse transcriptase real time polymerase chain reaction (PCR) assay detected enterovirus RNA in the patient's plasma and faeces. Diagnosis of an acute localized exanthem due to Coxsachievirus A4 was performed. Skin lesions improved in seven days and completely cleared in two weeks without any systemic or topical treatment. Physicians should be aware of the possibility that enteroviruses may determine localized skin eruptions in addition to hand-foot-mouth disease and atypical exanthems. Viral infections should be considered in the differential diagnosis of localized dermatitis especially when the skin eruption is associated with enanthems and with systemic symptoms.

G protein-coupled estrogen receptor (GPER) expression in normal and abnormal endometrium.

PubMed

Plante, Beth J; Lessey, Bruce A; Taylor, Robert N; Wang, Wei; Bagchi, Milan K; Yuan, Lingwen; Scotchie, Jessica; Fritz, Marc A; Young, Steven L

2012-07-01

Rapid estrogen effects are mediated by membrane receptors, and evidence suggests a role for both a membrane-associated form of estrogen receptor alpha (ESR1; ERα) and G-protein coupled receptor 30 (GPER; GPR30). Considering estrogen's importance in endometrial physiology and endometriosis pathophysiology, we hypothesized that GPER could be involved in both cyclic changes in endometrial estrogen action and that aberrant expression might be seen in the eutopic endometrium of women with endometriosis. Using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical analysis of normal endometrium, endometrial samples demonstrated cycle-regulated expression of GPER, with maximal expression in the proliferative phase. Eutopic and ectopic endometrium from women with endometriosis overexpressed GPER as compared to eutopic endometrium of normal participants. Ishikawa cells, an adenocarcinoma cell line, expressed GPER, with increased expression upon treatment with estrogen or an ESR1 agonist, but not with a GPER-specific agonist. Decreased expression was seen in Ishikawa cells stably transfected with progesterone receptor A. Together, these data suggest that normal endometrial GPER expression is cyclic and regulated by nuclear estrogen and progesterone receptors, while expression is dysregulated in endometriosis.

Tripartite motif-containing 29 (TRIM29) is a novel marker for lymph node metastasis in gastric cancer.

PubMed

Kosaka, Yoshimasa; Inoue, Hiroshi; Ohmachi, Takahiro; Yokoe, Takeshi; Matsumoto, Toshifumi; Mimori, Koshi; Tanaka, Fumiaki; Watanabe, Masahiko; Mori, Masaki

2007-09-01

Tripartite motif-containing 29 (TRIM29) belongs to the TRIM protein family, which has unique structural characteristics, including multiple zinc finger motifs and a leucine zipper motif. TRIM29, also known as ataxia telangiectasia group D complementing gene, possesses radiosensitivity suppressor functions. Although TRIM29 has been reported to be underexpressed in prostate and breast cancer, its expression in gastrointestinal cancer has not been studied. By use of real-time reverse transcriptase-polymerase chain reaction, we analyzed TRIM29 mRNA expression status with respect to various clinicopathological parameters in 124 patients with gastric cancer. An immunohistochemical study was also conducted. The expression of TRIM29 was far higher in gastric cancer tumor tissue. Increased TRIM29 mRNA expression was markedly associated with such parameters as histological grade, large tumor size, extent of tumor invasion, and lymph node metastasis. In the TRIM29 high-expression group, it was an independent predictor for lymph node metastasis. Furthermore, patients with high TRIM29 mRNA expression showed a far poorer survival rate than those with low TRIM29 mRNA expression. TRIM29 expression may serve as a good marker of lymph node metastasis in gastric cancer.

In vivo hair growth-promoting effect of rice bran extract prepared by supercritical carbon dioxide fluid.

PubMed

Choi, Jae-Suk; Jeon, Min-Hee; Moon, Woi-Sook; Moon, Jin-Nam; Cheon, Eun Jin; Kim, Joo-Wan; Jung, Sung Kyu; Ji, Yi-Hwa; Son, Sang Wook; Kim, Mi-Ryung

2014-01-01

The potential hair growth-promoting activity of rice bran supercritical CO2 extract (RB-SCE) and major components of RB-SCE, linoleic acid, policosanol, γ-oryzanol, and γ-tocotrienol, were evaluated with the histological morphology and mRNA expression levels of cell growth factors using real-time reverse transcriptase-polymerase chain reaction (PCR) in C57BL/6 mice. RB-SCE showed hair growth-promoting potential to a similar extent as 3% minoxidil, showing that the hair follicles were induced to be in the anagen stage. The numbers of the hair follicles were significantly increased. In addition, mRNA expression levels of vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), and keratinocyte growth factor (KGF) were also significantly increased and that of transforming growth factor-β (TGF-β) decreased in RB-SCE-treated groups. Among the major components of RB-SCE, linoleic acid and γ-oryzanol induced the formation of hair follicles according to examination of histological morphology and mRNA expression levels of cell growth factors. In conclusion, our results demonstrate that RB-SCE, particularly linoleic acid and γ-oryzanol, promotes hair growth and suggests RB-SCE can be applied as hair loss treatment.

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.

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.

Comparative effects of conjugated linoleic acid (CLA) and linoleic acid (LA) on the oxidoreduction status in THP-1 macrophages.

PubMed

Rybicka, Marta; Stachowska, Ewa; Gutowska, Izabela; Parczewski, Miłosz; Baśkiewicz, Magdalena; Machaliński, Bogusław; Boroń-Kaczmarska, Anna; Chlubek, Dariusz

2011-04-27

The aim of this study was to investigate the effect of conjugated linoleic acids (CLAs) on macrophage reactive oxygen species synthesis and the activity and expression of antioxidant enzymes, catalase (Cat), glutathione peroxidase (GPx), and superoxide dismutase (SOD). The macrophages were obtained from the THP-1 monocytic cell line. Cells were incubated with the addition of cis-9,trans-11 CLA or trans-10,cis-12 CLA or linoleic acid. Reactive oxygen species (ROS) formation was estimated by flow cytometry. Enzymes activity was measured spectrophotometrically. The antioxidant enzyme mRNA expression was estimated by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Statistical analysis was based on nonparametric statistical tests [Friedman analysis of variation (ANOVA) and Wilcoxon signed-rank test]. cis-9,trans-11 CLA significantly increased the activity of Cat, while trans-10,cis-12 CLA notably influenced GPx activity. Both isomers significantly decreased mRNA expression for Cat. Only trans-10,cis-12 significantly influenced mRNA for SOD-2 expression. The CLAs activate processes of the ROS formation in macrophages. Adverse metabolic effects of each isomer action were observed.

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.

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

Telomerase and its extracurricular activities.

PubMed

Jaiswal, Rishi Kumar; Kumar, Pramod; Yadava, Pramod Kumar

2013-12-01

The classical activity of telomerase is to synthesize telomeric repeats and thus maintain telomere length, which in turn ensures chromosome stability and cellular proliferation. However, there is growing evidence that implicates telomerase in many other functions that are independent of TERC being used as its template. Telomerase has an RNA-dependent RNA polymerase (RdRP) activity in the mitochondria. Other than viral RdRPs, it is the only RNA-dependent RNA polymerase that has been identified in mammals. It also plays a role in the Wnt signaling pathway by acting as a transcriptional modulator. Telomerase acts as a reverse transcriptase independent of its core subunit, TERC. Studies indicate that telomerase is also involved in apoptosis and DNA repair.

Multiple nucleotide preferences determine cleavage-site recognition by the HIV-1 and M-MuLV RNases H.

PubMed

Schultz, Sharon J; Zhang, Miaohua; Champoux, James J

2010-03-19

The RNase H activity of reverse transcriptase is required during retroviral replication and represents a potential target in antiviral drug therapies. Sequence features flanking a cleavage site influence the three types of retroviral RNase H activity: internal, DNA 3'-end-directed, and RNA 5'-end-directed. Using the reverse transcriptases of HIV-1 (human immunodeficiency virus type 1) and Moloney murine leukemia virus (M-MuLV), we evaluated how individual base preferences at a cleavage site direct retroviral RNase H specificity. Strong test cleavage sites (designated as between nucleotide positions -1 and +1) for the HIV-1 and M-MuLV enzymes were introduced into model hybrid substrates designed to assay internal or DNA 3'-end-directed cleavage, and base substitutions were tested at specific nucleotide positions. For internal cleavage, positions +1, -2, -4, -5, -10, and -14 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV significantly affected RNase H cleavage efficiency, while positions -7 and -12 for HIV-1 and positions -4, -9, and -11 for M-MuLV had more modest effects. DNA 3'-end-directed cleavage was influenced substantially by positions +1, -2, -4, and -5 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV. Cleavage-site distance from the recessed end did not affect sequence preferences for M-MuLV reverse transcriptase. Based on the identified sequence preferences, a cleavage site recognized by both HIV-1 and M-MuLV enzymes was introduced into a sequence that was otherwise resistant to RNase H. The isolated RNase H domain of M-MuLV reverse transcriptase retained sequence preferences at positions +1 and -2 despite prolific cleavage in the absence of the polymerase domain. The sequence preferences of retroviral RNase H likely reflect structural features in the substrate that favor cleavage and represent a novel specificity determinant to consider in drug design. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Soft shell clams Mya arenaria with disseminated neoplasia demonstrate reverse transcriptase activity

USGS Publications Warehouse

House, M.L.; Kim, C.H.; Reno, P.W.

1998-01-01

Disseminated neoplasia (DN), a proliferative cell disorder of the circulatory system of bivalves, was first reported in oysters in 1969. Since that time, the disease has been determined to be transmissible through water-borne exposure, but the etiological agent has not been unequivocally identified. In order to determine if a viral agent, possibly a retrovirus, could be the causative agent of DN, transmission experiments were performed, using both a cell-free filtrate and a sucrose gradient-purified preparation of a cell-free filtrate of DN positive materials. Additionally, a PCR-enhanced reverse transcriptase assay was used to determine if reverse transcriptase was present in tissues or hemolymph from DN positive soft shell clams Mya arenaria. DN was transmitted to healthy clams by injection with whole DN cells, but not with cell-free flitrates prepared from either tissues from DN positive clams, or DN cells. The cell-free preparations from DN-positive tissues and hemolymph having high levels of DN cells in circulation exhibited positive reactions in the PCR-enhanced reverse transcriptase assay. Cell-free preparations of hemolymph from clams having low levels of DN (<0.1% of cells abnormal), hemocytes from normal soft shell clams, and normal soft shell clam tissues did not produce a positive reaction in the PCR enhanced reverse transcriptase assay.

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

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

Development of real-time and lateral flow strip reverse transcription recombinase polymerase Amplification assays for rapid detection of peste des petits ruminants virus.

PubMed

Yang, Yang; Qin, Xiaodong; Song, Yiming; Zhang, Wei; Hu, Gaowei; Dou, Yongxi; Li, Yanmin; Zhang, Zhidong

2017-02-07

Peste des petits ruminants (PPR) is an economically important, Office International des Epizooties (OIE) notifiable, transboundary viral disease of small ruminants such as sheep and goat. PPR virus (PPRV), a negative-sense single-stranded RNA virus, is the causal agent of PPR. Therefore, sensitive, specific and rapid diagnostic assay for the detection of PPRV are necessary to accurately and promptly diagnose suspected case of PPR. In this study, reverse transcription recombinase polymerase amplification assays using real-time fluorescent detection (real-time RT-RPA assay) and lateral flow strip detection (LFS RT-RPA assay) were developed targeting the N gene of PPRV. The sensitivity of the developed real-time RT-RPA assay was as low as 100 copies per reaction within 7 min at 40 °C with 95% reliability; while the sensitivity of the developed LFS RT-RPA assay was as low as 150 copies per reaction at 39 °C in less than 25 min. In both assays, there were no cross-reactions with sheep and goat pox viruses, foot-and-mouth disease virus and Orf virus. These features make RPA assay promising candidates either in field use or as a point of care diagnostic technique.

Fatal hemorrhagic fever caused by West Nile virus in the United States.

PubMed

Paddock, Christopher D; Nicholson, William L; Bhatnagar, Julu; Goldsmith, Cynthia S; Greer, Patricia W; Hayes, Edward B; Risko, Joseph A; Henderson, Corey; Blackmore, Carina G; Lanciotti, Robert S; Campbell, Grant L; Zaki, Sherif R

2006-06-01

Most West Nile virus (WNV) infections in humans are asymptomatic; severe disease occurs in relatively few patients and typically manifests as encephalitis, meningitis, or acute flaccid paralysis. A few cases of life-threatening disease with diffuse hemorrhagic manifestations have been reported in Africa; however, this clinical presentation has not been documented for any of the >16,700 cases of WNV disease reported in the United States during 1999-2004. We describe a case of fulminant WNV infection in a 59-year-old Florida man who died following a brief illness that resembled hemorrhagic disease caused by Rickettsia reckettsii, dengue virus or yellow fever virus. Traditional and contemporary diagnostic assays, including culture isolation, electron microscopic examination, reverse-transcriptase polymerase chain reaction amplification, and immunohistochemical stains, were used to confirm systemic WNV infection in the patient. WNV was isolated in a cell culture from a skin biopsy specimen obtained from the patient shortly prior to death. Electron microscopic examination identified the isolate as a flavivirus, and reverse-transcriptase polymerase chain reaction amplified specific WNV sequences from the isolate and patient tissue. Quantitative polymerase chain reaction identified approximately 1x10(7) viral copies/mL in the patient's serum. WNV antigens were detected by immunohistochemical stains in intravascular mononuclear cells and endothelium in skin, lung, liver, kidney, spleen, bone marrow, and central nervous system; no viral antigens were identified in neurons or glial cells of the central nervous system. Although hemorrhagic disease is a rare manifestation of WNV infection, the findings provided by this report may offer new insights regarding the clinical spectrum and pathogenesis of WNV disease in humans.

Familial 46,XY sex reversal without campomelic dysplasia caused by a deletion upstream of the SOX9 gene

PubMed Central

Layman, Lawrence C.; Ullmann, Reinhard; Shen, Yiping; Ha, Kyungsoo; Rehman, Khurram; Looney, Stephen; McDonough, Paul G.; Kim, Hyung-Goo; Carr, Bruce R.

2014-01-01

Background 46,XY sex reversal is a rare disorder and familial cases are even more rare. The purpose of the present study was to determine the molecular basis for a family with three affected siblings who had 46,XY sex reversal. Methods DNA was extracted from three females with 46,XY sex reversal, two normal sisters, and both unaffected parents. All protein coding exons of the SRY and NR5A1 genes were subjected to PCR-based DNA sequencing. In addition, array comparative genomic hybridization was performed on DNA from all seven family members. A deletion was confirmed using quantitative polymerase chain reaction. Expression of SOX9 gene was quantified using reverse transcriptase polymerase chain reaction. Results A 349kb heterozygous deletion located 353kb upstream of the SOX9 gene on the long arm of chromosome 17 was discovered in the father and three affected siblings, but not in the mother. The expression of SOX9 was significantly decreased in the affected siblings. Two of three affected sisters had gonadoblastomas. Conclusion This is the first report of 46,XY sex reversal in three siblings who have a paternally inherited deletion upstream of SOX9 associated with reduced SOX9 mRNA expression. PMID:24907458

Elucidating the Role of cAbl and the Abi-Family of cAbl Target Proteins in Cancer Development and Progression

DTIC Science & Technology

1999-07-01

patients with Ph’-positive leukemias also revealed loss of Abi proteins. We determined by RNase protection assay and reverse transcriptase polymerase...myelogenous leukemia . Abi protein levels also appeared unaltered by Western blot analysis of human lung, liver, colon, and breast carcinoma tissues as...generated in the presence of Bcr-Abl • Abi protein degradation was observed in Ph’+ leukemia -derived cells, but not in Ph1- leukemias or in human breast

Distemper Outbreak and Its Effect on African Wild Dog Conservation

PubMed Central

van de Bildt, Marco W.G.; Kuiken, Thijs; Visee, Aart M.; Lema, Sangito; Fitzjohn, Tony R.

2002-01-01

In December 2000, an infectious disease spread through a captive breeding group of African wild dogs (Lycaon pictus) in Tanzania, killing 49 of 52 animals within 2 months. The causative agent was identified as Canine distemper virus (CDV) by means of histologic examination, virus isolation, reverse transcriptase-polymerase chain reaction analysis, and nucleotide sequencing. This report emphasizes the importance of adequate protection against infectious diseases for the successful outcome of captive breeding programs of endangered species. PMID:11897078

Development of polymerase chain reaction-based diagnostic tests for detection of Malsoor virus & adenovirus isolated from Rousettus species of bats in Maharashtra, India.

PubMed

Shete, Anita M; Yadav, Pragya; Kumar, Vimal; Nikam, Tushar; Mehershahi, Kurosh; Kokate, Prasad; Patil, Deepak; Mourya, Devendra T

2017-01-01

Bats are recognized as important reservoirs for emerging infectious disease and some unknown viral diseases. Two novel viruses, Malsoor virus (family Bunyaviridae, genus, Phlebovirus) and a novel adenovirus (AdV) (family, Adenoviridae genus, Mastadenovirus), were identified from Rousettus bats in the Maharashtra State of India. This study was done to develop and optimize real time reverse transcription - polymerase chain reaction (RT-PCR) assays for Malsoor virus and real time and nested PCR for adenovirus from Rousettus bats. For rapid and accurate screening of Malsoor virus and adenovirus a nested polymerase chain reaction and TaqMan-based real-time PCR were developed. Highly conserved region of nucleoprotein gene of phleboviruses and polymerase gene sequence from the Indian bat AdV isolate polyprotein gene were selected respectively for diagnostic assay development of Malsoor virus and AdV. Sensitivity and specificity of assays were calculated and optimized assays were used to screen bat samples. Molecular diagnostic assays were developed for screening of Malsoor virus and AdV and those were found to be specific. Based on the experiments performed with different parameters, nested PCR was found to be more sensitive than real-time PCR; however, for rapid screening, real-time PCR can be used and further nested PCR can be used for final confirmation or in those laboratories where real-time facility/expertise is not existing. This study reports the development and optimization of nested RT-PCR and a TaqMan-based real-time PCR for Malsoor virus and AdV. The diagnostic assays can be used for rapid detection of these novel viruses to understand their prevalence among bat population.

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.

Telomerase reverse transcriptase (TERT) expression and role of vincristine sulfate in mouse model of malignancy related peritoneal ascites: an experimental metastatic condition.

PubMed

Chaklader, M; Das, P; Pereira, J A; Chatterjee, S; Basak, P; Law, A; Banerjee, T; Chauhan, S; Law, S

2011-06-01

To evaluate the efficacy of intraperitoneal vincristine administration into ascitic sarcoma-180 bearing mice as a model of human malignant ascites regarding various peritoneal/retroperitoneal sarcomatosis, and to evaluate the flowcytometric telomerase reverse transcriptase expression for the diagnostic and prognostic purposes. Present study included disease induction by intraperitoneal homologous ascitic sarcoma-180 transplantation followed by in vivo intraperitoneal drug administration to study mitotic index, flowcytometric cell cycle and telomerase reverse transcriptase expression pattern, erythrosin-B dye exclusion study for malignant cell viability assessment. Besides, in vitro malignant ascite culture in presence and absence of vincristine sulfate and survival study were also taken into consideration. Intraperitoneal vincristine administration (concentration 0.5 mg/kg body weight) significantly diminished the mitotic index in diseased subjects in comparison to untreated control subjects. Treated group of animals showed increased life span and median survival time. Cell viability assessment during the course of drug administration also revealed gradual depression on cell viability over time. Flowcytometric cell cycle analysis showed a good prognostic feature of chemotherapeutic administration schedule by representing high G2/M phase blocked cells along with reduced telomerase reverse transcriptase positive cells in treated animals. We conclude that long term administration of vincristine sulfate in small doses could be a good pharmacological intervention in case of malignant peritoneal ascites due to sarcomatosis as it indirectly reduced the level of telomerase reverse transcriptase expression in malignant cells by directly regulating cell cycle and simultaneously increased the life expectancy of the diseased subjects.

Prevalence and Diversity of Low Pathogenicity Avian Influenza Viruses in Wild Birds in Guatemala, 2010-2013.

PubMed

Gonzalez-Reiche, Ana S; Müller, Maria L; Ortiz, Lucía; Cordón-Rosales, Celia; Perez, Daniel R

2016-05-01

Waterfowl species are known to harbor the greatest diversity of low pathogenicity influenza A virus (LPAIV) subtypes and are recognized as their main natural reservoir. In Guatemala there is evidence of circulation of LPAIV in wild ducks; however, the bird species contributing to viral diversity during the winter migration in Central America are unknown. In this study, samples obtained from 1250 hunter-killed birds from 22 different species were collected on the Pacific coast of Guatemala during three winter migration seasons between 2010 and 2013. Prevalence of LPAIV detected by real-time reverse-transcriptase polymerase chain reaction was 38.2%, 23.5%, and 24.7% in the 2010-11, 2011-12, and 2012-13 seasons, respectively. The highest virus prevalence was detected in the northern shoveler (Anas clypeata), followed by the blue-winged teal (Anas discors). The majority of positive samples and viral isolates were obtained from the blue-winged teal. Analysis of LPAIV prevalence over time in this species indicated a decreasing trend in monthly prevalence within a migration season. Sixty-eight viruses were isolated, and nine HA and seven NA subtypes were identified in 19 subtype combinations. In 2012-13 the most prevalent subtype was H14, a subtype identified for the first time in the Western Hemisphere in 2010. The results from this study represent the most detailed description available to date of LPAIV circulation in Central America.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes.

PubMed

Balasubramaniam, Muthukumar; Davids, Benem; Addai, Amma B; Pandhare, Jui; Dash, Chandravanu

2017-02-22

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3' ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

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

A Portable Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of Foot-and-Mouth Disease Virus

PubMed Central

Abd El Wahed, Ahmed; El-Deeb, Ayman; El-Tholoth, Mohamed; Abd El Kader, Hanaa; Ahmed, Abeer; Hassan, Sayed; Hoffmann, Bernd; Haas, Bernd; Shalaby, Mohamed A.; Hufert, Frank T.; Weidmann, Manfred

2013-01-01

Foot-and-mouth disease (FMD) is a trans-boundary viral disease of livestock, which causes huge economic losses and constitutes a serious infectious threat for livestock farming worldwide. Early diagnosis of FMD helps to diminish its impact by adequate outbreak management. In this study, we describe the development of a real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of FMD virus (FMDV). The FMDV RT-RPA design targeted the 3D gene of FMDV and a 260 nt molecular RNA standard was used for assay validation. The RT-RPA assay was fast (4–10 minutes) and the analytical sensitivity was determined at 1436 RNA molecules detected by probit regression analysis. The FMDV RT-RPA assay detected RNA prepared from all seven FMDV serotypes but did not detect classical swine fever virus or swine vesicular disease virus. The FMDV RT-RPA assay was used in the field during the recent FMD outbreak in Egypt. In clinical samples, reverse transcription polymerase chain reaction (RT-PCR) and RT-RPA showed a diagnostic sensitivity of 100% and 98%, respectively. In conclusion, FMDV RT-RPA was quicker and much easier to handle in the field than real-time RT-PCR. Thus RT-RPA could be easily implemented to perform diagnostics at quarantine stations or farms for rapid spot-of-infection detection. PMID:23977101

A portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus.

PubMed

Abd El Wahed, Ahmed; El-Deeb, Ayman; El-Tholoth, Mohamed; Abd El Kader, Hanaa; Ahmed, Abeer; Hassan, Sayed; Hoffmann, Bernd; Haas, Bernd; Shalaby, Mohamed A; Hufert, Frank T; Weidmann, Manfred

2013-01-01

Foot-and-mouth disease (FMD) is a trans-boundary viral disease of livestock, which causes huge economic losses and constitutes a serious infectious threat for livestock farming worldwide. Early diagnosis of FMD helps to diminish its impact by adequate outbreak management. In this study, we describe the development of a real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of FMD virus (FMDV). The FMDV RT-RPA design targeted the 3D gene of FMDV and a 260 nt molecular RNA standard was used for assay validation. The RT-RPA assay was fast (4-10 minutes) and the analytical sensitivity was determined at 1436 RNA molecules detected by probit regression analysis. The FMDV RT-RPA assay detected RNA prepared from all seven FMDV serotypes but did not detect classical swine fever virus or swine vesicular disease virus. The FMDV RT-RPA assay was used in the field during the recent FMD outbreak in Egypt. In clinical samples, reverse transcription polymerase chain reaction (RT-PCR) and RT-RPA showed a diagnostic sensitivity of 100% and 98%, respectively. In conclusion, FMDV RT-RPA was quicker and much easier to handle in the field than real-time RT-PCR. Thus RT-RPA could be easily implemented to perform diagnostics at quarantine stations or farms for rapid spot-of-infection detection.

Photoaffinity labeling of the primer binding domain in murine leukemia virus reverse transcriptase.

PubMed

Tirumalai, R S; Modak, M J

1991-07-02

We have labeled the primer binding domain of murine leukemia virus reverse transcriptase (MuLV RT) by covalently cross-linking 5' end labeled d(T)8 to MuLV RT, using ultraviolet light energy. The specificity and the functional significance of the primer cross-linking reaction were demonstrated by the fact that (i) other oligomeric primers, tRNAs, and also template-primers readily compete with radiolabeled d(T)8 for the cross-linking reaction, (ii) under similar conditions, the competing primers and template-primer also inhibit the DNA polymerase activity of MuLV RT to a similar extent, (iii) substrate deoxynucleotides have no effect, and (iv) the reaction is sensitive to high ionic strength. In order to identify the primer binding domains/sites in MuLV RT; tryptic digests prepared from the covalently cross-linked MuLV RT and [32P]d(T)8 complexes were resolved on C-18 columns by reverse-phase HPLC. Three distinct radiolabeled peptides were found to contain the majority of the bound primer. Of these, peptide I contained approximately 65% radioactivity, while the remainder was associated with peptides II and III. Amino acid composition and sequence analyses of the individual peptides revealed that peptide I spans amino acid residues 72-80 in the primary amino acid sequence of MuLV RT and is located in the polymerase domain. The primer cross-linking site appears to be at or near Pro-76. Peptides II and III span amino acid residues 602-609 and 615-622, respectively, and are located in the RNase H domain. The probable cross-linking sites in peptides II and III are suggested to be at or near Leu-604 and Leu-618, respectively.

Lower expressions of the human bitter taste receptor TAS2R in smokers: reverse transcriptase-polymerase chain reaction analysis.

PubMed

Aoki, Mieko; Takao, Tetsuya; Takao, Kyoichi; Koike, Fumihiko; Suganuma, Narufumi

2014-01-01

Despite the fact that smokers have deficit in detecting taste, particularly bitter taste, no study has investigated its biological correlate. In this context, we compared the expression of the bitter taste receptor gene, taste 2 receptor (TAS2R) in the tongues of smokers and non-smokers. Tissue samples were collected from the lateral portion of the tongues of 22 smokers and 22 age- and gender-matched healthy volunteers (19 males and three females) with no history of smoking. Reverse transcriptase-polymerase chain reaction was used to examine the expression of TAS2R in the two groups, and the effect of aging on TAS2R expression was also assessed. TAS2R expression was significantly lower among smokers than non-smokers (t = 6.525, P < .0001, 11.36 ± 6.0 vs. 2.09 ± 2.8, mean ± SD, non-smokers vs. smokers). Further, a positive correlation between age and expression of TAS2R was observed in non-smokers (r = .642, P = .001), but not smokers (r = .124, P = .584). This correlation difference was significant (Z = 1.96, P = .0496). Smokers showed a significantly lower expression of the bitter taste receptor gene than non-smokers, which is potentially caused by their inability to acquire such receptors with age because of cigarette smoking, in contrast to non-smokers.

A widespread class of reverse transcriptase-related cellular genes.

PubMed

Gladyshev, Eugene A; Arkhipova, Irina R

2011-12-20

Reverse transcriptases (RTs) polymerize DNA on RNA templates. They fall into several structurally related but distinct classes and form an assemblage of RT-like enzymes that, in addition to RTs, also includes certain viral RNA-dependent RNA polymerases (RdRP) synthesizing RNA on RNA templates. It is generally believed that most RT-like enzymes originate from retrotransposons or viruses and have no specific function in the host cell, with telomerases being the only notable exception. Here we report on the discovery and properties of a unique class of RT-related cellular genes collectively named rvt. We present evidence that rvts are not components of retrotransposons or viruses, but single-copy genes with a characteristic domain structure that may contain introns in evolutionarily conserved positions, occur in syntenic regions, and evolve under purifying selection. These genes can be found in all major taxonomic groups including protists, fungi, animals, plants, and even bacteria, although they exhibit patchy phylogenetic distribution in each kingdom. We also show that the RVT protein purified from one of its natural hosts, Neurospora crassa, exists in a multimeric form and has the ability to polymerize NTPs as well as dNTPs in vitro, with a strong preference for NTPs, using Mn(2+) as a cofactor. The existence of a previously unknown class of single-copy RT-related genes calls for reevaluation of the current views on evolution and functional roles of RNA-dependent polymerases in living cells.

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.

Cykotine mRNA expression in mouse retina after laser injury by reverse transcriptase-polymerase chain reaction (RT-PCR)

NASA Astrophysics Data System (ADS)

Schuschereba, Steven T.; Bowman, Phillip D.; Ujimore, Veronica; Hoxie, Stephen W.; Pizarro, Jose M.; Cross, Michael E.; Lund, David J.

1996-04-01

The purpose of this study was to identify cytokines produced by the retina after laser injury. With the aid of a scanning laser ophthalmoscope (SLO), right eyes of mice received lesions from a continuous wave argon laser. Left eyes served as unirradiated controls. At 2, 4, 6, 12, 24, and 48 hr after laser irradiation groups of 3 mice were euthanized and retinas fixed for histology or isolated for RNA. Messenger RNA (mRNA) was reverse-transcribed into complementary DNA (cDNA) and subjected to polymerase chain reaction for the following cytokines: tumor necrosis factor-(alpha) (TNF-(alpha) ), interleukin-1(alpha) /(Beta) (IL- 1(alpha) /(Beta) ), interleukin-6 (IL-6), transforming growth factor-(Beta) 1 (TGF- (Beta) 1), macrophage colony stimulating factor (M-CSF), inducible nitric oxide synthase (iNOS), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH). Histologically, lesions were confined to the photoreceptors, retinal pigment epithelium, and choroid. In laser-injured retinas, mRNA levels were elevated for IL-1(alpha) , TGF-(Beta) 1, iNOS, and G3PDH, but not TNF-(alpha) , IL-1(Beta) , or IL-6. It appears that the retina, in response to laser injury, upregulates a select number of cytokines in a time-course dependent fashion.

Modulation expression of tumor necrosis factor α in the radiation-induced lung injury by glycyrrhizic acid.

PubMed

Refahi, Soheila; Pourissa, Masoud; Zirak, Mohammad Reza; Hadadi, GholamHassan

2015-01-01

To evaluate the ability of glycyrrhizic acid (GLA) to reduce the tumor necrosis factor α (TNF-α), release on messenger ribonucleic acid (mRNA) and protein production in the lungs using GLA in response to irradiation were studied. The animals were divided into four groups: No treatment (NT group), GLA treatment only (GLA group), irradiation only (XRT group), and GLA treatment plus irradiation (GLA/XRT group). Rats were killed at different time points. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to evaluate the mRNA expression of TNF-α in the lungs (compared with non-irradiated lungs). An enzyme-linked immunosorbant assay (ELISA) assay was used to measure the TNF-α protein level. The TNF-α mRNA expression in the lungs of the XRT rats was clearly higher at all-time points compared to the NT rats. The TNF-α mRNA expression in the lungs of the GLA/XRT rats was lower at all-time points compared to the XRT rats. Release of the TNF-α on protein level in the lungs of the XRT rats increased at all-time points compared to the NT rats. In contrast to the XRT rats, the lungs of the GLA/XRT rats revealed a reduction on TNF-α protein level at 6 h after irradiation. This study has clearly showed the immediate down-regulation of the TNF-α mRNA and protein production in the lungs using GLA in response to irradiation.

Modulation expression of tumor necrosis factor α in the radiation-induced lung injury by glycyrrhizic acid

PubMed Central

Refahi, Soheila; Pourissa, Masoud; Zirak, Mohammad Reza; Hadadi, GholamHassan

2015-01-01

To evaluate the ability of glycyrrhizic acid (GLA) to reduce the tumor necrosis factor α (TNF-α), release on messenger ribonucleic acid (mRNA) and protein production in the lungs using GLA in response to irradiation were studied. The animals were divided into four groups: No treatment (NT group), GLA treatment only (GLA group), irradiation only (XRT group), and GLA treatment plus irradiation (GLA/XRT group). Rats were killed at different time points. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to evaluate the mRNA expression of TNF-α in the lungs (compared with non-irradiated lungs). An enzyme-linked immunosorbant assay (ELISA) assay was used to measure the TNF-α protein level. The TNF-α mRNA expression in the lungs of the XRT rats was clearly higher at all-time points compared to the NT rats. The TNF-α mRNA expression in the lungs of the GLA/XRT rats was lower at all-time points compared to the XRT rats. Release of the TNF-α on protein level in the lungs of the XRT rats increased at all-time points compared to the NT rats. In contrast to the XRT rats, the lungs of the GLA/XRT rats revealed a reduction on TNF-α protein level at 6 h after irradiation. This study has clearly showed the immediate down-regulation of the TNF-α mRNA and protein production in the lungs using GLA in response to irradiation. PMID:26170556

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.

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.

Point-of-care diagnostic assay for the detection of Zika virus using the recombinase polymerase amplification method.

PubMed

Vasileva Wand, Nadina I; Bonney, Laura C; Watson, Robert J; Graham, Victoria; Hewson, Roger

2018-06-13

The sudden and explosive expansion of Zika virus (ZIKV) from the African continent through Oceania and culminating in the outbreak in South America has highlighted the importance of new rapid point-of-care diagnostic tools for the control and prevention of transmission. ZIKV infection has devastating consequences, such as neurological congenital malformations in infants born to infected mothers and Guillain-Barré syndrome in adults. Additionally, its potential for transmission through vector bites, as well as from person to person through blood transfusions and sexual contact, are important considerations for prompt diagnosis. Recombinase polymerase amplification (RPA), an isothermal method, was developed as an alternative field-applicable assay to PCR. Here we report the development of a novel ZIKV real-time reverse transcriptase RPA (RT-RPA) assay capable of detecting a range of different ZIKV strains from a variety of geographical locations. The ZIKV RT-RPA was shown to be highly sensitive, being capable of detecting as few as five copies of target nucleic acid per reaction, and suitable for use with a battery-operated portable device. The ZIKV RT-RPA demonstrated 100 % specificity and 83 % sensitivity in clinical samples. Furthermore, we determined that the ZIKV RT-RPA is a versatile assay that can be applied to crude samples, such as saliva and serum, and can be used as a vector surveillance tool on crude mosquito homogenates. Therefore, the developed ZIKV RT-RPA is a useful diagnostic tool that can be transferred to a resource-limited location, eliminating the need for a specialized and sophisticated laboratory environment and highly trained staff.

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.

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.

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

Structural Maturation of HIV-1 Reverse Transcriptase—A Metamorphic Solution to Genomic Instability

PubMed Central

London, Robert E.

2016-01-01

Human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT)—a critical enzyme of the viral life cycle—undergoes a complex maturation process, required so that a pair of p66 precursor proteins can develop conformationally along different pathways, one evolving to form active polymerase and ribonuclease H (RH) domains, while the second forms a non-functional polymerase and a proteolyzed RH domain. These parallel maturation pathways rely on the structural ambiguity of a metamorphic polymerase domain, for which the sequence–structure relationship is not unique. Recent nuclear magnetic resonance (NMR) studies utilizing selective labeling techniques, and structural characterization of the p66 monomer precursor have provided important insights into the details of this maturation pathway, revealing many aspects of the three major steps involved: (1) domain rearrangement; (2) dimerization; and (3) subunit-selective RH domain proteolysis. This review summarizes the major structural changes that occur during the maturation process. We also highlight how mutations, often viewed within the context of the mature RT heterodimer, can exert a major influence on maturation and dimerization. It is further suggested that several steps in the RT maturation pathway may provide attractive targets for drug development. PMID:27690082

Advances in Developing HIV-1 Viral Load Assays for Resource-Limited Settings

PubMed Central

Wang, ShuQi; Xu, Feng; Demirci, Utkan

2010-01-01

Commercial HIV-1 RNA viral load assays have been routinely used in developed countries to monitor antiretroviral treatment (ART). However, these assays require expensive equipment and reagents, well-trained operators, and established laboratory infrastructure. These requirements restrict their use in resource-limited settings where people are most afflicted with the HIV-1 epidemic. Inexpensive alternatives such as the Ultrasensitive p24 assay, the Reverse Transcriptase (RT) assay and in-house reverse transcription quantitative polymerase chain reaction (RT-qPCR) have been developed. However, they are still time-consuming, technologically complex and inappropriate for decentralized laboratories as point-of-care (POC) tests. Recent advances in microfluidics and nanotechnology offer new strategies to develop low-cost, rapid, robust and simple HIV-1 viral load monitoring systems. We review state-of-the-art technologies used for HIV-1 viral load monitoring in both developed and developing settings. Emerging approaches based on microfluidics and nanotechnology, which have potential to be integrated into POC HIV-1 viral load assays, are also discussed. PMID:20600784

An Outbreak of 2009 Pandemic Influenza A (H1N1) Virus Infection in an Elementary School in Pennsylvania

PubMed Central

Bhattarai, Achuyt; Fagan, Ryan P.; Ostroff, Stephen; Sodha, Samir V.; Moll, Mària E.; Lee, Bruce Y.; Chang, Chung-Chou H.; Ennis, Brent; Britz, Phyllis; Fiore, Anthony; Nguyen, Michael; Palekar, Rakhee; Archer, W. Roodly; Gift, Thomas L.; Leap, Rebecca; Nygren, Benjamin L.; Cauchemez, Simon; Angulo, Frederick J.; Swerdlow, David

2011-01-01

In May 2009, one of the earliest outbreaks of 2009 pandemic influenza A virus (pH1N1) infection resulted in the closure of a semi-rural Pennsylvania elementary school. Two sequential telephone surveys were administered to 1345 students (85% of the students enrolled in the school) and household members in 313 households to collect data on influenza-like illness (ILI). A total of 167 persons (12.4%) among those in the surveyed households, including 93 (24.0%) of the School A students, reported ILI. Students were 3.1 times more likely than were other household members to develop ILI (95% confidence interval [CI], 2.3–4.1). Fourth-grade students were more likely to be affected than were students in other grades (relative risk, 2.2; 95% CI, 1.2–3.9). pH1N1 was confirmed in 26 (72.2%) of the individuals tested by real-time reverse-transcriptase polymerase chain reaction. The outbreak did not resume upon the reopening of the school after the 7-day closure. This investigation found that pH1N1 outbreaks at schools can have substantial attack rates; however, grades and classrooms are affected variably. Additioanl study is warranted to determine the effectiveness of school closure during outbreaks. PMID:21342888

Direct quantitative comparison of molecular responses in photodamaged human skin to fractionated and fully ablative carbon dioxide laser resurfacing.

PubMed

Orringer, Jeffrey S; Sachs, Dana L; Shao, Yuan; Hammerberg, Craig; Cui, Yilei; Voorhees, John J; Fisher, Gary J

2012-10-01

Fractionated ablative laser resurfacing has become a widely used treatment modality. Its clinical results are often found to approach those of traditional fully ablative laser resurfacing. To directly compare the molecular changes that result from fractionated and fully ablative carbon dioxide (CO(2)) laser resurfacing in photodamaged human skin. Photodamaged skin of 34 adult volunteers was focally treated at distinct sites with a fully ablative CO(2) laser and a fractionated CO(2) laser. Serial skin samples were obtained at baseline and several time points after treatment. Real-time reverse transcriptase polymerase chain reaction technology and immunohistochemistry were used to quantify molecular responses to each type of laser treatment. Fully ablative and fractionated CO(2) laser resurfacing induced significant dermal remodeling and collagen induction. After a single treatment, fractionated ablative laser resurfacing resulted in collagen induction that was approximately 40% to 50% as pronounced as that induced by fully ablative laser resurfacing. The fundamental cutaneous responses that result from fully ablative and fractionated carbon dioxide laser resurfacing are similar but differ in magnitude and duration, with the fully ablative procedure inducing relatively greater changes including more pronounced collagen induction. However, the molecular data reported here provide substantial support for fractionated ablative resurfacing as an effective treatment modality for improving skin texture. © 2012 by the American Society for Dermatologic Surgery, Inc. Published by Wiley Periodicals, Inc.

Pinhole Micro-SPECT/CT for Noninvasive Monitoring and Quantitation of Oncolytic Virus Dispersion and Percent Infection in Solid Tumors

PubMed Central

Penheiter, Alan R.; Griesmann, Guy E.; Federspiel, Mark J.; Dingli, David; Russell, Stephen J.; Carlson, Stephanie K.

2011-01-01

The purpose of our study was to validate the ability of pinhole micro-single-photon emission computed tomography/computed tomography (SPECT/CT) to 1) accurately resolve the intratumoral dispersion pattern and 2) quantify the infection percentage in solid tumors of an oncolytic measles virus encoding the human sodium iodide symporter (MV-NIS). NIS RNA level and dispersion pattern were determined in control and MV-NIS infected BxPC-3 pancreatic tumor cells and mouse xenografts using quantitative, real-time, reverse transcriptase, polymerase chain reaction, autoradiography, and immunohistochemistry (IHC). Mice with BxPC-3 xenografts were imaged with 123I or 99TcO4 micro-SPECT/CT. Tumor dimensions and radionuclide localization were determined with imaging software. Linear regression and correlation analyses were performed to determine the relationship between tumor infection percentage and radionuclide uptake (% injected dose per gram) above background and a highly significant correlation was observed (r2 = 0.947). A detection threshold of 1.5-fold above the control tumor uptake (background) yielded a sensitivity of 2.7% MV-NIS infected tumor cells. We reliably resolved multiple distinct intratumoral zones of infection from noninfected regions. Pinhole micro-SPECT/CT imaging using the NIS reporter demonstrated precise localization and quantitation of oncolytic MV-NIS infection and can replace more time-consuming and expensive analyses (eg, autoradiography and IHC) that require animal sacrifice. PMID:21753796

Blastic natural killer cell leukaemia in a dog--a case report.

PubMed

Bonkobara, Makoto; Saito, Taro; Yamashita, Masahiro; Tamura, Kyoichi; Yagihara, Hiroko; Isotani, Mayu; Sato, Takashi; Washizu, Tsukimi

2007-11-01

A case of canine non-T, non-B lymphoid leukaemia was determined to be of natural killer (NK) cell lineage by detecting specific expression of canine CD56 mRNA by reverse transcriptase polymerase chain reaction analysis. Although NK cells are usually considered to be morphologically large granular lymphocytes, the malignant NK cells in this case were agranular and blast-like, resembling human blastic NK cell leukaemia. The prognosis of human NK cell leukaemia is usually poor. In this case, the dog died 10 days after initial presentation, despite chemotherapy.

Nucleotide Sequence Analysis of RNA Synthesized from Rabbit Globin Complementary DNA

PubMed Central

Poon, Raymond; Paddock, Gary V.; Heindell, Howard; Whitcome, Philip; Salser, Winston; Kacian, Dan; Bank, Arthur; Gambino, Roberto; Ramirez, Francesco

1974-01-01

Rabbit globin complementary DNA made with RNA-dependent DNA polymerase (reverse transcriptase) was used as template for in vitro synthesis of 32P-labeled RNA. The sequences of the nucleotides in most of the fragments resulting from combined ribonuclease T1 and alkaline phosphatase digestion have been determined. Several fragments were long enough to fit uniquely with the α or β globin amino-acid sequences. These data demonstrate that the cDNA was copied from globin mRNA and contained no detectable contaminants. Images PMID:4139714

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.

Effect of subinhibitory concentrations of chlorogenic acid on reducing the virulence factor production by Staphylococcus aureus.

PubMed

Li, Guanghui; Qiao, Mingyu; Guo, Yan; Wang, Xin; Xu, Yunfeng; Xia, Xiaodong

2014-09-01

Chlorogenic acid (CA) has been reported to inhibit several pathogens, but the influence of subinhibitory concentrations of CA on virulence expression of pathogens has not been fully elucidated. The aim of this study was to explore the effect of CA on the virulence factor production of Staphylococcus aureus. The minimum inhibitory concentration (MIC) of CA against S. aureus was determined using a broth microdilution method. Hemolysin assays, coagulase titer assays, adherence to solid-phase fibrinogen assays, Western blot, and real-time reverse transcriptase-polymerase chain reaction were performed to evaluate the effect of subinhibitory concentrations of CA on the virulence factors of S. aureus. MIC of CA against S. aureus ATCC29213 was found to be 2.56 mg/mL. At subinhibitory concentrations, CA significantly inhibited the hemolysis and dose-dependently decreased coagulase titer. Reduced binding to fibrinogen and decreased production of SEA were observed with treatment of CA at concentrations ranging from 1/16MIC to 1/2MIC. CA markedly inhibited the expression of hla, sea, and agr genes in S. aureus. These data demonstrate that the virulence expression of S. aureus could be reduced by CA and suggest that CA could be potentially developed as a supplemental strategy to control S. aureus infection and to prevent staphylococcal food poisoning.

Differentially expressed genes in nonsmall cell lung cancer: expression profiling of cancer-related genes in squamous cell lung cancer.

PubMed

Kettunen, Eeva; Anttila, Sisko; Seppänen, Jouni K; Karjalainen, Antti; Edgren, Henrik; Lindström, Irmeli; Salovaara, Reijo; Nissén, Anna-Maria; Salo, Jarmo; Mattson, Karin; Hollmén, Jaakko; Knuutila, Sakari; Wikman, Harriet

2004-03-01

The expression patterns of cancer-related genes in 13 cases of squamous cell lung cancer (SCC) were characterized and compared with those in normal lung tissue and 13 adenocarcinomas (AC), the other major type of nonsmall cell lung cancer (NSCLC). cDNA array was used to screen the gene expression levels and the array results were verified using a real-time reverse-transcriptase-polymerase chain reaction (RT-PCR). Thirty-nine percent of the 25 most upregulated and the 25 most downregulated genes were common to SCC and AC. Of these genes, DSP, HMGA1 (alias HMGIY), TIMP1, MIF, CCNB1, TN, MMP11, and MMP12 were upregulated and COPEB (alias CPBP), TYROBP, BENE, BMPR2, SOCS3, TIMP3, CAV1, and CAV2 were downregulated. The expression levels of several genes from distinct protein families (cytokeratins and hemidesmosomal proteins) were markedly increased in SCC compared with AC and normal lung. In addition, several genes, overexpressed in SCC, such as HMGA1, CDK4, IGFBP3, MMP9, MMP11, MMP12, and MMP14, fell into distinct chromosomal loci, which we have detected as gained regions on the basis of comparative genomic hybridization data. Our study revealed new candidate genes involved in NSCLC.

Underexpression of mineralocorticoid receptor in colorectal carcinomas and association with VEGFR-2 overexpression.

PubMed

Di Fabio, Francesco; Alvarado, Carlos; Majdan, Agnieszka; Gologan, Adrian; Voda, Linda; Mitmaker, Elliot; Beitel, Lenore K; Gordon, Philip H; Trifiro, Mark

2007-11-01

The human mineralocorticoid receptor (MR) is a steroid receptor widely expressed in colorectal mucosa. A significant role for the MR in the reduction of vascular endothelial growth factor receptor-2 (VEGFR-2) mRNA levels has been demonstrated in vitro. To evaluate a potential contribution of MR to colorectal carcinoma progression, we analyzed the expression of MR in relation to VEGFR-2. Fresh human colorectal cancer tissue and adjacent normal mucosa were harvested from 48 consecutive patients. MR and VEGFR-2 mRNA expression levels were determined by real-time reverse transcriptase-polymerase chain reaction and correlated with clinicopathological parameters. A decline of MR expression was observed in all carcinomas compared to normal mucosa. Expression of MR was a median of 11-fold lower in carcinoma compared to the normal mucosa, irrespective of the location, size, stage, and differentiation. MR was a median of 20-fold underexpressed in carcinomas with VEGFR-2 overexpression vs only 9-fold in carcinomas with VEGFR-2 underexpression (p = 0.035, Mann-Whitney test). These findings support the hypothesis that reduction of MR expression may be one of the early events involved in colorectal carcinoma progression. The inverse association between MR and VEGFR-2 expression in carcinoma suggests a potential tumor-suppressive function for MR.

Elevation of macrophage-derived chemokine in eosinophilic pneumonia: a role of alveolar macrophages.

PubMed

Manabe, Kazuyoshi; Nishioka, Yasuhiko; Kishi, Jun; Inayama, Mami; Aono, Yoshinori; Nakamura, Yoichi; Ogushi, Fumitaka; Bando, Hiroyasu; Tani, Kenji; Sone, Saburo

2005-02-01

Macrophage-derived chemokine (MDC/CCL22) and thymus-and activation-regulated chemokine (TARC/CCL17) are ligands for CC chemokine receptor 4. Recently, TARC has been reported to play a role in the pathogenesis of idiopathic eosinophilic pneumonia (IEP). The purpose of this study was to evaluate the role of MDC in IEP and other interstitial lung diseases (ILDs). MDC and TARC in the bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay in patients with ILDs and healthy volunteers (HV). We also examined the expression of MDC mRNA in alveolar macrophages (AM) by real-time quantitative reverse transcriptase-polymerase chain reaction. Both MDC and TARC were detected only in BALF obtained from IEP patients. The concentration of MDC was higher than that of TARC in all cases. The level of MDC in IEP correlated with that of TARC. AM from IEP patients expressed a significantly higher amount of MDC than that from HV at the levels of protein and mRNA. MDC in BALF from IEP dramatically decreased when patients achieved remission. These findings suggest that MDC, in addition to TARC, might be involved in the pathogenesis of IEP, and AM play a role in the elevation of MDC in IEP.

Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes*

PubMed Central

Hu, Qing-bi; He, Yu; Zhou, Xun

2015-01-01

Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China. PMID:26642182

Induction of CYP1A mRNA in Carp (Cyprinus carpio) from the Kalamazoo River polychlorinated biphenyl-contaminated superfund site and in a laboratory study.

PubMed

Fisher, M A; Mehne, C; Means, J C; Ide, C F

2006-01-01

The Kalamazoo River Superfund site in Michigan is contaminated with polychlorinated biphenyls (PCBs), which were heavily discharged into the river from several paper companies as part of the deinking process in the 1950s through 1970s. We characterized biomarkers of chronic PCB exposure in a resident fish population using real-time reverse transcriptase-polymerase chain reaction to examine mRNA expression levels of multiple genes in carp (Cyprinus carpio) liver from PCB contaminated and reference sites in the Kalamazoo River. We also measured these same genes in juvenile carp exposed to dietary PCBs for 4 months. Kalamazoo River carp had significantly increased levels of cytochrome P450 1A (CYP1A) mRNA as did carp fed PCBs in the laboratory. No significant mRNA upregulation occurred in the specific oxidative stress genes (gamma-glutamylcysteine synthetase and magnesium superoxide dismutase) and metabolic genes (phosphoenolpyruvate carboxykinase and nucleolin) examined. These data are consistent with the idea that carp from the Kalamazoo River Superfund Site are responding to PCB exposure via upregulation of CYP1A independent of activation of the oxidative stress response genes normally thought to be co-regulated with CYP1A.

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

Replication of 2 subtypes of low-pathogenicity avian influenza virus of duck and gull origins in experimentally infected Mallard ducks.

PubMed

Daoust, P-Y; van de Bildt, M; van Riel, D; van Amerongen, G; Bestebroer, T; Vanderstichel, R; Fouchier, R A M; Kuiken, T

2013-05-01

Many subtypes of low-pathogenicity avian influenza (LPAI) virus circulate in wild bird reservoirs, but their prevalence may vary among species. We aimed to compare by real-time reverse-transcriptase polymerase chain reaction, virus isolation, histology, and immunohistochemistry the distribution and pathogenicity of 2 such subtypes of markedly different origins in Mallard ducks (Anas platyrhynchos): H2N3 isolated from a Mallard duck and H13N6 isolated from a Ring-billed Gull (Larus delawarensis). Following intratracheal and intraesophageal inoculation, neither virus caused detectable clinical signs, although H2N3 virus infection was associated with a significantly decreased body weight gain during the period of virus shedding. Both viruses replicated in the lungs and air sacs until approximately day 3 after inoculation and were associated with a locally extensive interstitial, exudative, and proliferative pneumonia. Subtype H2N3, but not subtype H13N6, went on to infect the epithelia of the intestinal mucosa and cloacal bursa, where it replicated without causing lesions until approximately day 5 after inoculation. Larger quantities of subtype H2N3 virus were detected in cloacal swabs than in pharyngeal swabs. The possible clinical significance of LPAI virus-associated pulmonary lesions and intestinal tract infection in ducks deserves further evaluation.

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.

Increased Levels of Cell-Free Human Placental Lactogen mRNA at 28-32 Gestational Weeks in Plasma of Pregnant Women With Placenta Previa and Invasive Placenta

PubMed Central

Sekizawa, Akihiko; Ventura, Walter; Koide, Keiko; Hori, Kyouko; Okai, Takashi; Masashi, Yoshida; Furuya, Kenichi; Mizumoto, Yoshifumi

2014-01-01

We compared the levels of cell-free human placental lactogen (hPL) messenger RNA (mRNA) in maternal plasma at 28 to 32 weeks of gestation between women with diagnosis of placenta previa or invasive placenta and women with an uneventful pregnancy. Sensitivity and specificity of hPL mRNA for the prediction of invasive placenta were further explored. Plasma hPL mRNA were quantified by real-time reverse-transcriptase polymerase chain reaction in women with placenta previa (n = 13), invasive placenta (n = 5), and normal pregnancies (n = 92). Median (range) hPL mRNA was significantly higher in women with placenta previa, 782 (10-2301) copies/mL of plasma, and in those with invasive placenta, 615 (522-2102) copies/mL of plasma, when compared to normal pregnancies, 90 (4-4407) copies/mL of plasma, P < .01 and P < .05, respectively. We found a sensitivity of 100% and a specificity of 61.5% for the prediction of invasive placenta among women with placenta previa. In conclusion, expression of hPL mRNA is increased in plasma of women with placenta previa and invasive placenta at 28 to 32 weeks of gestation. PMID:23744883

Increased levels of cell-free human placental lactogen mRNA at 28-32 gestational weeks in plasma of pregnant women with placenta previa and invasive placenta.

PubMed

Kawashima, Akihiro; Sekizawa, Akihiko; Ventura, Walter; Koide, Keiko; Hori, Kyouko; Okai, Takashi; Masashi, Yoshida; Furuya, Kenichi; Mizumoto, Yoshifumi

2014-02-01

We compared the levels of cell-free human placental lactogen (hPL) messenger RNA (mRNA) in maternal plasma at 28 to 32 weeks of gestation between women with diagnosis of placenta previa or invasive placenta and women with an uneventful pregnancy. Sensitivity and specificity of hPL mRNA for the prediction of invasive placenta were further explored. Plasma hPL mRNA were quantified by real-time reverse-transcriptase polymerase chain reaction in women with placenta previa (n = 13), invasive placenta (n = 5), and normal pregnancies (n = 92). Median (range) hPL mRNA was significantly higher in women with placenta previa, 782 (10-2301) copies/mL of plasma, and in those with invasive placenta, 615 (522-2102) copies/mL of plasma, when compared to normal pregnancies, 90 (4-4407) copies/mL of plasma, P < .01 and P < .05, respectively. We found a sensitivity of 100% and a specificity of 61.5% for the prediction of invasive placenta among women with placenta previa. In conclusion, expression of hPL mRNA is increased in plasma of women with placenta previa and invasive placenta at 28 to 32 weeks of gestation.

Seasonal and Interseasonal Dynamics of Bluetongue Virus Infection of Dairy Cattle and Culicoides sonorensis Midges in Northern California – Implications for Virus Overwintering in Temperate Zones

PubMed Central

Mayo, Christie E.; Mullens, Bradley A.; Reisen, William K.; Osborne, Cameron J.; Gibbs, E. Paul J.; Gardner, Ian A.; MacLachlan, N. James

2014-01-01

Bluetongue virus (BTV) is the cause of an economically important arboviral disease of domestic and wild ruminants. The occurrence of BTV infection of livestock is distinctly seasonal in temperate regions of the world, thus we determined the dynamics of BTV infection (using BTV-specific real time reverse transcriptase polymerase chain reaction) among sentinel cattle and vector Culicoides sonorensis (C. sonorensis) midges on a dairy farm in northern California throughout both the seasonal and interseasonal (overwintering) periods of BTV activity from August 2012 until March 2014. The data confirmed widespread infection of both sentinel cattle and vector midges during the August – November period of seasonal BTV transmission, however BTV infection of parous female midges captured in traps set during daylight hours also was detected in February of both 2013 and 2014, during the interseasonal period. The finding of BTV-infected vector midges during mid-winter suggests that BTV may overwinter in northern California by infection of long-lived female C. sonorensis midges that were infected during the prior seasonal period of virus transmission, and reemerged sporadically during the overwintering period; however the data do not definitively preclude other potential mechanisms of BTV overwintering that are also discussed. PMID:25215598

Seasonal and interseasonal dynamics of bluetongue virus infection of dairy cattle and Culicoides sonorensis midges in northern California--implications for virus overwintering in temperate zones.

PubMed

Mayo, Christie E; Mullens, Bradley A; Reisen, William K; Osborne, Cameron J; Gibbs, E Paul J; Gardner, Ian A; MacLachlan, N James

2014-01-01

Bluetongue virus (BTV) is the cause of an economically important arboviral disease of domestic and wild ruminants. The occurrence of BTV infection of livestock is distinctly seasonal in temperate regions of the world, thus we determined the dynamics of BTV infection (using BTV-specific real time reverse transcriptase polymerase chain reaction) among sentinel cattle and vector Culicoides sonorensis (C. sonorensis) midges on a dairy farm in northern California throughout both the seasonal and interseasonal (overwintering) periods of BTV activity from August 2012 until March 2014. The data confirmed widespread infection of both sentinel cattle and vector midges during the August-November period of seasonal BTV transmission, however BTV infection of parous female midges captured in traps set during daylight hours also was detected in February of both 2013 and 2014, during the interseasonal period. The finding of BTV-infected vector midges during mid-winter suggests that BTV may overwinter in northern California by infection of long-lived female C. sonorensis midges that were infected during the prior seasonal period of virus transmission, and reemerged sporadically during the overwintering period; however the data do not definitively preclude other potential mechanisms of BTV overwintering that are also discussed.

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.

Reverse transcription-polymerase chain reaction molecular testing of cytology specimens: Pre-analytic and analytic factors.

PubMed

Bridge, Julia A

2017-01-01

The introduction of molecular testing into cytopathology laboratory practice has expanded the types of samples considered feasible for identifying genetic alterations that play an essential role in cancer diagnosis and treatment. Reverse transcription-polymerase chain reaction (RT-PCR), a sensitive and specific technical approach for amplifying a defined segment of RNA after it has been reverse-transcribed into its DNA complement, is commonly used in clinical practice for the identification of recurrent or tumor-specific fusion gene events. Real-time RT-PCR (quantitative RT-PCR), a technical variation, also permits the quantitation of products generated during each cycle of the polymerase chain reaction process. This review addresses qualitative and quantitative pre-analytic and analytic considerations of RT-PCR as they relate to various cytologic specimens. An understanding of these aspects of genetic testing is central to attaining optimal results in the face of the challenges that cytology specimens may present. Cancer Cytopathol 2017;125:11-19. © 2016 American Cancer Society. © 2016 American Cancer Society.

Real-time reverse transcription polymerase chain reaction method for detection of Canine distemper virus modified live vaccine shedding for differentiation from infection with wild-type strains.

PubMed

Wilkes, Rebecca P; Sanchez, Elena; Riley, Matthew C; Kennedy, Melissa A

2014-01-01

Canine distemper virus (CDV) remains a common cause of infectious disease in dogs, particularly in high-density housing situations such as shelters. Vaccination of all dogs against CDV is recommended at the time of admission to animal shelters and many use a modified live virus (MLV) vaccine. From a diagnostic standpoint for dogs with suspected CDV infection, this is problematic because highly sensitive diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) tests are able to detect MLV virus in clinical samples. Real-time PCR can be used to quantitate amount of virus shedding and can differentiate vaccine strains from wild-type strains when shedding is high. However, differentiation by quantitation is not possible in vaccinated animals during acute infection, when shedding is low and could be mistaken for low level vaccine virus shedding. While there are gel-based RT-PCR assays for differentiation of vaccine strains from field strains based on sequence differences, the sensitivity of these assays is unable to match that of the real-time RT-PCR assay currently used in the authors' laboratory. Therefore, a real-time RT-PCR assay was developed that detects CDV MLV vaccine strains and distinguishes them from wild-type strains based on nucleotide sequence differences, rather than the amount of viral RNA in the sample. The test is highly sensitive, with detection of as few as 5 virus genomic copies (corresponding to 10(-1) TCID(50)). Sequencing of the DNA real-time products also allows phylogenetic differentiation of the wild-type strains. This test will aid diagnosis during outbreaks of CDV in recently vaccinated animals.

Rapid and sensitive detection of canine distemper virus by real-time reverse transcription recombinase polymerase amplification.

PubMed

Wang, Jianchang; Wang, Jinfeng; Li, Ruiwen; Liu, Libing; Yuan, Wanzhe

2017-08-15

Canine distemper, caused by Canine distemper virus (CDV), is a highly contagious and fatal systemic disease in free-living and captive carnivores worldwide. Recombinase polymerase amplification (RPA), as an isothermal gene amplification technique, has been explored for the molecular detection of diverse pathogens. A real-time reverse transcription RPA (RT-RPA) assay for the detection of canine distemper virus (CDV) using primers and exo probe targeting the CDV nucleocapsid protein gene was developed. A series of other viruses were tested by the RT-RPA.Thirty-two field samples were further tested by RT-RPA, and the resuts were compared with those obtained by the real-time RT-PCR. The RT-RPA assay was performed successfully at 40 °C, and the results were obtained within 3 min-12 min. The assay could detect CDV, but did not show cross-detection of canine parvovirus-2 (CPV-2), canine coronavirus (CCoV), canine parainfluenza virus (CPIV), pseudorabies virus (PRV) or Newcastle disease virus (NDV), demonstrating high specificity. The analytical sensitivity of RT-RPA was 31.8 copies in vitro transcribed CDV RNA, which is 10 times lower than the real-time RT-PCR. The assay performance was validated by testing 32 field samples and compared to real-time RT-PCR. The results indicated an excellent correlation between RT-RPA and a reference real-time RT-PCR method. Both assays provided the same results, and R 2 value of the positive results was 0.947. The results demonstrated that the RT-RPA assay offers an alternative tool for simple, rapid, and reliable detection of CDV both in the laboratory and point-of-care facility, especially in the resource-limited settings.

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.

One-Step Reverse Transcription-Polymerase Chain Reaction for Ebola and Marburg Viruses.

PubMed

Park, Sun-Whan; Lee, Ye-Ji; Lee, Won-Ja; Jee, Youngmee; Choi, WooYoung

2016-06-01

Ebola and Marburg viruses (EBOVs and MARVs, respectively) are causative agents of severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. In 2014, there was a major Ebola outbreak in various countries in West Africa, including Guinea, Liberia, Republic of Sierra Leone, and Nigeria. EBOV and MARV are clinically difficult to diagnose and distinguish from other African epidemic diseases. Therefore, in this study, we aimed to develop a method for rapid identification of the virus to prevent the spread of infection. We established a conventional one-step reverse transcription-polymerase chain reaction (RT-PCR) assay for these pathogens based on the Superscript Reverse Transcriptase-Platinum Taq polymerase enzyme mixture. All assays were thoroughly optimized using in vitro-transcribed RNA. We designed seven primer sets of nucleocapsid protein (NP) genes based on sequences from seven filoviruses, including five EBOVs and two MARVs. To evaluate the sensitivity of the RT-PCR assay for each filovirus, 10-fold serial dilutions of synthetic viral RNA transcripts of EBOV or MARV NP genes were used to assess detection limits of viral RNA copies. The potential for these primers to cross react with other filoviruses was also examined. The results showed that the primers were specific for individual genotype detection in the examined filoviruses. The assay established in this study may facilitate rapid, reliable laboratory diagnosis in suspected cases of Ebola and Marburg hemorrhagic fevers.

Sensitivity and specificity of real-time reverse transcription polymerase chain reaction, histopathology, and immunohistochemical labeling for the detection of Rift Valley fever virus in naturally infected cattle and sheep.

PubMed

Odendaal, Lieza; Fosgate, Geoffrey T; Romito, Marco; Coetzer, Jacobus A W; Clift, Sarah J

2014-01-01

Real-time reverse transcription polymerase chain reaction (real-time RT-PCR), histopathology, and immunohistochemical labeling (IHC) were performed on liver specimens from 380 naturally infected cattle and sheep necropsied during the 2010 Rift Valley fever (RVF) epidemic in South Africa. Sensitivity (Se) and specificity (Sp) of real-time RT-PCR, histopathology, and IHC were estimated in a latent-class model using a Bayesian framework. The Se and Sp of real-time RT-PCR were estimated as 97.4% (95% confidence interval [CI] = 95.2-98.8%) and 71.7% (95% CI = 65-77.9%) respectively. The Se and Sp of histopathology were estimated as 94.6% (95% CI = 91-97.2%) and 92.3% (95% CI = 87.6-95.8%), respectively. The Se and Sp of IHC were estimated as 97.6% (95% CI = 93.9-99.8%) and 99.4% (95% CI = 96.9-100%), respectively. Decreased Sp of real-time RT-PCR was ascribed to cross-contamination of samples. Stratified analysis of the data suggested variations in test accuracy with fetuses and severely autolyzed specimens. The Sp of histopathology in fetuses (83%) was 9.3% lower than the sample population (92.3%). The Se of IHC decreased from 97.6% to 81.5% in the presence of severe autolysis. The diagnostic Se and Sp of histopathology was higher than expected, confirming the value of routine postmortem examinations and histopathology of liver specimens. Aborted fetuses, however, should be screened using a variety of tests in areas endemic for RVF, and results from severely autolyzed specimens should be interpreted with caution. The most feasible testing option for countries lacking suitably equipped laboratories seems to be routine histology in combination with IHC.

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.

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

Reverse Transcription Recombinase Polymerase Amplification Assay for the Detection of Middle East Respiratory Syndrome Coronavirus

PubMed Central

Abd El Wahed, Ahmed; Patel, Pranav; Heidenreich, Doris; Hufert, Frank T.; Weidmann, Manfred

2013-01-01

The emergence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in the eastern Mediterranean and imported cases to Europe has alerted public health authorities. Currently, detection of MERS-CoV in patient samples is done by real-time RT-PCR. Samples collected from suspected cases are sent to highly-equipped centralized laboratories for screening. A rapid point-of-care test is needed to allow more widespread mobile detection of the virus directly from patient material. In this study, we describe the development of a reverse transcription isothermal Recombinase Polymerase Amplification (RT-RPA) assay for the identification of MERS-CoV. A partial nucleocapsid gene RNA molecular standard of MERS-coronavirus was used to determine the assay sensitivity. The isothermal (42°C) MERS-CoV RT-RPA was as sensitive as real-time RT-PCR (10 RNA molecules), rapid (3-7 minutes) and mobile (using tubescanner weighing 1kg). The MERS-CoV RT-RPA showed cross-detection neither of any of the RNAs of several coronaviruses and respiratory viruses affecting humans nor of the human genome. The developed isothermal real-time RT-RPA is ideal for rapid mobile molecular MERS-CoV monitoring in acute patients and may also facilitate the search for the animal reservoir of MERS-CoV. PMID:24459611

Effects of zoledronic acid and geranylgeraniol on the cellular behaviour and gene expression of primary human alveolar osteoblasts.

PubMed

Zafar, S; Coates, D E; Cullinan, M P; Drummond, B K; Milne, T; Seymour, G J

2016-11-01

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a serious complication of bisphosphonate therapy. The mechanism underlying BRONJ pathogenesis is poorly understood. To determine the effects of zoledronic acid (ZA) and geranylgeraniol (GGOH) on the mevalonate pathway (MVP) in osteoblasts generated from the human mandibular alveolar bone in terms of cell viability/proliferation, migration, apoptosis and gene expression. Primary human osteoblasts (HOBs) isolated from the mandibular alveolar bone were phenotyped. HOBs were cultured with or without ZA and GGOH for up to 72 h. Cellular behaviour was examined using a CellTiter-Blue® viability assay, an Ibidi culture-insert migration assay, an Apo-ONE® Homogeneous Caspase-3/7 apoptosis assay and transmission electron microscopy (TEM). Quantitative real-time reverse transcriptase polymerase chain reaction (qRT 2 -PCR) was used to determine the simultaneous expression of 168 osteogenic and angiogenic genes modulated in the presence of ZA and GGOH. ZA decreased cell viability and migration and induced apoptosis in HOBs. TEM revealed signs of apoptosis in ZA-treated HOBs. However, the co-addition of GGOH ameliorated the effect of ZA and partially restored the cells to the control state. Twenty-eight genes in the osteogenic array and 27 genes in the angiogenic array were significantly regulated in the presence of ZA compared with those in the controls at one or more time points. The cytotoxic effect of ZA on HOBs and its reversal by the addition of GGOH suggests that the effect of ZA on HOBs is mediated via the MVP. The results suggest that GGOH could be used as a possible therapeutic/preventive strategy for BRONJ.

Microchip capillary electrophoresis with laser-induced fluorescence combined with one-step duplex reverse-transcription polymerase chain reaction for the rapid detection of Enterovirus 71 and Coxsackievirus A16 in throat swab specimens.

PubMed

Jia, Ruan; Chengjun, Sun; Heng, Chen; Chen, Zhou; Yuanqian, Li; Yongxin, Li

2015-07-01

Enterovirus 71 and Coxsackievirus A16 are the main pathogens causing hand-foot-mouth disease. In this paper, microchip capillary electrophoresis with laser-induced fluorescence combined with one-step duplex reverse transcript-polymerase chain reaction has been developed for the detection of Enterovirus 71 and Coxsackievirus A16 in throat swab specimens. The specific reverse transcription-polymerase chain reaction amplicons labeled with SYBR Orange were separated by microchip capillary electrophoresis and detected by laser induced fluorescence detector within 7 min. The intraday and interday relative standard deviation of migration time for DNA Marker was in the range of 1.36-2.94 and 2.78-3.96%, respectively. The detection limits were as low as 2.06 × 10(3) copies/mL for Enterovirus 71 and 5 × 10(3) copies/mL for Coxsackievirus A16. No cross-reactivity was observed with rotavirus, astrovirus, norovirus, and adenovirus, which showed good specificity of the method. This assay was validated using 100 throat swab specimens that were detected by real-time reverse-transcript polymerase chain reaction in parallel and the two methods produced the same results. This study provided a rapid, sensitive and specific method for the detection of Enterovirus 71 and Coxsackievirus A16, which make a contribution to significant time and cost saving for the identification and treatment of patients. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

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.

The role of phenylalanine-119 of the reverse transcriptase of mouse mammary tumour virus in DNA synthesis, ribose selection and drug resistance.

PubMed

Entin-Meer, Michal; Sevilya, Ziv; Hizi, Amnon

2002-10-15

Phe-119 in the reverse transcriptase (RT) of mouse mammary tumour virus (MMTV) is homologous with Tyr-115 in HIV type 1 (HIV-1) RT and to Phe-155 in murine leukaemia virus (MLV) RT. By mutating these residues in HIV-1 and MLV RTs (which are strict DNA polymerases) the enzymes were shown to function also as RNA polymerases. Owing to the uniqueness of MMTV as a type B retrovirus, we have generated a Phe-119-Val mutant of MMTV RT to study the involvement of this residue in affecting the catalytic features of this RT. The data presented here show that the mutant MMTV RT can incorporate both deoxyribonucleosides and ribonucleosides while copying either RNA or DNA. In addition, this mutant RT shows resistance to nucleoside analogues and an enhanced fidelity of DNA synthesis; all relative to the wild-type enzyme. The Phe-119-Val mutant is also different from the wild-type enzyme in its preference for most template primers tested and in its ability to synthesize DNA under non-processive and processive conditions. Overall, it is likely that the aromatic side chain of Phe-119 is located at the dNTP-binding site of MMTV RT and thus might be part of a putative "steric gate" that prevents the incorporation of nucleoside triphosphates. Since the only three-dimensional structures of RTs published so far are those of HIV-1 and MLV, it is likely that MMTV RT folds quite similarly to these RTs.

Use of Scented Sugar Bait Stations to Track Mosquito-Borne Arbovirus Transmission in California

PubMed Central

LOTHROP, HUGH D.; WHEELER, SARAH S.; FANG, YING; REISEN, WILLIAM K.

2012-01-01

Laboratory and field research was conducted to determine if Culex tarsalis Coquillett expectorated West Nile virus (WNV) during sugar feeding and if a lure or bait station could be developed to exploit this behavior for WNV surveillance. Experimentally infected Cx. tarsalis repeatedly expectorated WNV onto filter paper strips and into vials with wicks containing sucrose that was readily detectable by a quantitative reverse transcriptase-polymerase chain reaction assay. Few females (33%, n = 27) became infected by imbibing sugar solutions spiked with high concentrations (107 plaque forming units/ml) of WNV, indicating sugar feeding stations probably would not be a source of WNV infection. In nature, sugar bait stations scented with the floral attractant phenyl acetaldehyde tracked WNV transmission activity in desert but not urban or agricultural landscapes in California. When deployed in areas of the Coachella Valley with WNV activity during the summer of 2011, 27 of 400 weekly sugar samples (6.8%) tested positive for WNV RNA by reverse transcriptase-polymerase chain reaction. Prevalence of positives varied spatially, but positive sugar stations were detected before concurrent surveillance measures of infection (mosquito pools) or transmission (sentinel chicken seroconversions). In contrast, sugar bait stations deployed in urban settings in Los Angeles or agricultural habits near Bakersfield in Kern County supporting WNV activity produced 1 of 90 and 0 of 60 positive weekly sugar samples, respectively. These results with sugar bait stations will require additional research to enhance bait attractancy and to understand the relationship between positive sugar stations and standard metrics of arbovirus surveillance. PMID:23270177

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.

Prevalence and diversity of low pathogenicity avian influenza viruses in wild birds in Guatemala, 2010-2013

PubMed Central

Gonzalez-Reiche, Ana S.; Müller, Maria L.; Ortiz, Lucía; Cordón-Rosales, Celia; Perez, Daniel R.

2016-01-01

Summary Waterfowl species are known to harbor the greatest diversity of low pathogenicity influenza A virus (LPAIV) subtypes and are recognized as their main natural reservoir. In Guatemala there is evidence of circulation of LPAIV in wild ducks, however the bird species contributing to viral diversity during the winter migration in Central America are unknown. In this study, samples obtained from 1,250 hunter-killed birds from 22 different species were collected on the Pacific coast of Guatemala during three winter migration seasons between 2010 and 2013. Prevalence of LPAIV detected by real-time reverse-transcriptase polymerase chain reaction was 38.2%, 23.5% and 24.7% in the 2010-11, 2011-12, and 2012-13 seasons respectively. The highest virus prevalence was detected in the northern shoveler (Anas clypeata), followed by the blue-winged teal (Anas discors). The majority of positive samples and viral isolates were obtained from the blue-winged teal. Analysis of LPAIV prevalence over time in this species indicated a decreasing trend in monthly prevalence within a migration season. Sixty-eight viruses were isolated and 9 HA and 7 NA subtypes were identified in 19 subtype combinations. In 2012-13, the most prevalent subtype was H14, a subtype identified for the first time in the western hemisphere in 2010. The results from this study represent the most detailed description available to date of LPAIV circulation in Central America. PMID:27309080

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

[Investigation of West Nile virus RNA in blood donors by real-time RT-PCR].

PubMed

Sahiner, Fatih; Avcı, Ismail Yaşar; Bedir, Orhan; Koru, Ozgür; Sener, Kenan; Yapar, Mehmet; Kubar, Ayhan

2012-07-01

West Nile virus (WNV), a member of Flaviviridae family, is an enveloped, icosahedral symmetric RNA virus. Primary reservoir hosts of WNV are birds, but the virus can cause various infections in humans and other mammals. The most common and natural transmission way of WNV infections is mosquito bites, however, humans can be infected by different routes. The most important non-mosquito transmission route is contaminated blood and blood products. In this study, we aimed to investigate the risk of WNV transmission through blood and blood products in Ankara, Turkey. The presence of WNV RNA was investigated by in house real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in serum samples obtained from 729 healthy blood donors (mean age: 27.7 years; 711 were male), regardless of the donor's seropositivity status since the virus can be transmitted at the early stages of infection when seroconversion has not yet developed. Serum samples were collected in August-September 2009, the period when these infections are more frequent due to mosquito activity. The vast majority of donors (n= 702, 96.3%) have been inhabiting in Ankara and 569 (78%) of donors have had risk factors for arboviral infections (e.g. outdoor activity, mosquito and tick bites). WNV RNA was not detected by real-time RT-PCR analysis in any serum sample included in this study. According to the results of our study, it can be said that the risk of WNV transmission through blood and blood products is low in Ankara. However, WNV seropositivity was detected within the range of 0.56 to 2.4% among blood donors in previous studies and probable and confirmed WNV infections have been reported in our region. In addition, WNV outbreaks have emerged in some countries neighbouring Turkey recently. Thus, the risk of WNV transmission through blood and blood products should not be ignored and blood donor questionnaires should be evaluated in detail.

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.

Design, synthesis and biological evaluations of N-Hydroxy thienopyrimidine-2,4-diones as inhibitors of HIV reverse transcriptase-associated RNase H.

PubMed

Kankanala, Jayakanth; Kirby, Karen A; Huber, Andrew D; Casey, Mary C; Wilson, Daniel J; Sarafianos, Stefan G; Wang, Zhengqiang

2017-12-01

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) is the only HIV enzymatic function not targeted by current antiviral drugs. Although various chemotypes have been reported to inhibit HIV RNase H, few have shown significant antiviral activities. We report herein the design, synthesis and biological evaluation of a novel N-hydroxy thienopyrimidine-2,3-dione chemotype (11) which potently and selectively inhibited RNase H with considerable potency against HIV-1 in cell culture. Current structure-activity-relationship (SAR) identified analogue 11d as a nanomolar inhibitor of RNase H (IC 50  = 0.04 μM) with decent antiviral potency (EC 50  = 7.4 μM) and no cytotoxicity (CC 50  > 100 μM). In extended biochemical assays compound 11d did not inhibit RT polymerase (pol) while inhibiting integrase strand transfer (INST) with 53 fold lower potency (IC 50  = 2.1 μM) than RNase H inhibition. Crystallographic and molecular modeling studies confirmed the RNase H active site binding mode. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Detection of the Single Nucleotide Polymorphism at Position rs2735940 in the Human Telomerase Reverse Transcriptase Gene by the Introduction of a New Restriction Enzyme Site for the PCR-RFLP Assay.

PubMed

Wang, Sihua; Ding, Mingcui; Duan, Xiaoran; Wang, Tuanwei; Feng, Xiaolei; Wang, Pengpeng; Yao, Wu; Wu, Yongjun; Yan, Zhen; Feng, Feifei; Yu, Songcheng; Wang, Wei

2017-09-01

It has been shown that the single nucleotide polymorphism (SNP) of the rs2735940 site in the human telomerase reverse transcriptase ( hTERT ) gene is associated with increased cancer risk. The traditional method to detect SNP genotypes is polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). However, there is a limitation to utilizing PCR-RFLP due to a lack of proper restriction enzyme sites at many polymorphic loci. This study used an improved PCR-RFLP method with a mismatched base for detection of the SNP rs2735940. A new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and in addition, the restriction enzyme Msp I for CRS-PCR was cheaper than other enzymes. We used this novel assay to determine the allele frequencies in 552 healthy Chinese Han individuals, and found the allele frequencies to be 63% for allele C and 37% for allele T In summary, the modified PCR-RFLP can be used to detect the SNP of rs2735940 with low cost and high efficiency. © 2017 by the Association of Clinical Scientists, Inc.

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

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.

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.

Analytical and clinical evaluation of the Abbott RealTime hepatitis B sequencing assay.

PubMed

Huh, Hee Jae; Kim, Ji-Youn; Lee, Myoung-Keun; Lee, Nam Yong; Kim, Jong-Won; Ki, Chang-Seok

2016-12-01

Long-term nucleoside analogue (NA) treatment leads to selection for drug-resistant mutations in patients undergoing hepatitis B virus (HBV) therapy. The Abbott RealTime HBV Sequencing assay (Abbott assay; Abbott Molecular Inc., Des Plaines, IL, USA) targets the reverse transcriptase region of the polymerase gene and as such has the ability to detect NA resistance-associated mutations in HBV. We evaluated the analytical performance of the Abbott assay and compared its diagnostic performance to that of a laboratory-developed nested-PCR and sequencing method. The analytical sensitivity of the Abbott assay was determined using a serially-diluted WHO International Standard. To validate the clinical performances of the Abbott assay and the laboratory-developed assay, 89 clinical plasma samples with various levels of HBV DNA were tested using both assays. The limit of detection of the Abbott assay, was 210IU/ml and it successfully detected mutations when the mutant types were present at levels ≥20%. Among 89 clinical specimens, 43 and 42 were amplification positive in the Abbott and laboratory-developed assays, respectively, with 87.6% overall agreement (78/89; 95% confidence interval [CI], 78.6-93.4). The Abbott assay failed to detect the minor mutant populations in two specimens, and therefore overall concordance was 85.3% (76/89), and the kappa value was 0.79 (95% CI, 0.67-0.90). The Abbott assay showed comparable diagnostic performance to laboratory-developed nested PCR followed by direct sequencing, and may be useful as a routine method for detecting HBV NA resistance-associated mutations in clinical laboratory settings. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Prevalence and molecular characterization of avian infectious bronchitis virus in poultry flocks in Morocco from 2010 to 2014 and first detection of Italy 02 in Africa.

PubMed

Fellahi, Siham; Ducatez, Mariette; El Harrak, Mehdi; Guérin, Jean-Luc; Touil, Nadia; Sebbar, Ghizlane; Bouaiti, El Arbi; Khataby, Khadija; Ennaji, My Mustapha; El-Houadfi, Mohammed

2015-01-01

The aim of this study was to investigate the prevalence and diversity of infectious bronchitis virus (IBV) genotypes in poultry flocks in 16 areas of Morocco between 2010 and 2014. A total of 360 chicken flocks suspected of being infected by IBV were screened for the IBV N gene using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Flocks were classified into four groups according to their IBV vaccination programme. Group 1 contained unvaccinated birds. Group 2 received a single application of live H120 vaccine. Groups 3 and 4 birds received one or two booster vaccination(s), respectively, mostly using the H120 vaccine. The real-time RT-PCR results showed that 51.7% of the flocks were positive for the IBV genome with geographical disparities. Molecular characterization of IBV was performed on 50 RT-PCR positive samples by partially sequencing the S1 gene, including the hypervariable regions (nucleotides 705-1097). Two predominant genotypes were detected, with the Massachusetts type dominating (66%), among which 25% of the samples were identical to the H120 vaccine. The second most common genotype (present in 32% of the flocks) was surprisingly Italy 02, revealing the first detection of this genotype in Morocco and also in Africa. 793B, the predominant genotype in the late 1990s in Morocco, was only detected on one occasion and was identical to the 4/91 vaccine strain. This study highlights the high prevalence of IBV in poultry farms in Morocco and confirms its continuous dynamic changes and evolution.

Gene expression of runx2, Osterix, c-fos, DLX-3, DLX-5, and MSX-2 in dental follicle cells during osteogenic differentiation in vitro.

PubMed

Morsczeck, C

2006-02-01

Recently, osteogenic precursor cells were isolated from human dental follicles, which differentiate into cementoblast- or osteoblast- like cells under in vitro conditions. However, mechanisms for osteogenic differentiation are not known in detail. Dental follicle cell long-term cultures supplemented with dexamethasone or with insulin resulted in mineralized nodules, whereas no mineralization or alkaline phosphatase activity was detected in the control culture without an osteogenic stimulus. A real-time reverse-transcriptase polymerase chain reaction (PCR) analysis was developed to investigate gene expression during osteogenic differentiation in vitro. Expression of the alkaline phosphatase (ALP) gene was detected during differentiation in the control culture and was similar to that in cultures with dexamethasone and insulin. DLX-3, DLX-5, runx2, and MSX-2 are differentially expressed during osteogenic differentiation in bone marrow mesenchymal stem cells. In dental follicle cells, gene expression of runx2, DLX-5, and MSX-2 was unaffected during osteogenic differentiation in vitro. Osteogenic differentiation appeared to be independent of MSX-2 expression; the same was true of runx2 and DLX-5, which were protagonists of osteogenic differentiation and osteocalcin promoter activity in bone marrow mesenchymal stem cells. Like in bone marrow-derived stem cells, DLX-3 gene expression was increased in dental follicle cells during osteogenic differentiation but similar to control cultures. However, gene expression of osterix was not detected in dental follicle cells during osteogenic differentiation; this gene is expressed during osteogenic differentiation in bone marrow stem cells. These real-time PCR results display molecular mechanisms in dental follicle precursor cells during osteogenic differentiation that are different from those in bone marrow-derived mesenchymal stem cells.

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.

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.

Spliced RNA of woodchuck hepatitis virus.

PubMed

Ogston, C W; Razman, D G

1992-07-01

Polymerase chain reaction was used to investigate RNA splicing in liver of woodchucks infected with woodchuck hepatitis virus (WHV). Two spliced species were detected, and the splice junctions were sequenced. The larger spliced RNA has an intron of 1300 nucleotides, and the smaller spliced sequence shows an additional downstream intron of 1104 nucleotides. We did not detect singly spliced sequences from which the smaller intron alone was removed. Control experiments showed that spliced sequences are present in both RNA and DNA in infected liver, showing that the viral reverse transcriptase can use spliced RNA as template. Spliced sequences were detected also in virion DNA prepared from serum. The upstream intron produces a reading frame that fuses the core to the polymerase polypeptide, while the downstream intron causes an inframe deletion in the polymerase open reading frame. Whereas the splicing patterns in WHV are superficially similar to those reported recently in hepatitis B virus, we detected no obvious homology in the coding capacity of spliced RNAs from these two viruses.

Impact of Antiretroviral Treatment Containing Tenofovir Difumarate on the Telomere Length of Aviremic HIV-Infected Patients.

PubMed

Montejano, Rocio; Stella-Ascariz, Natalia; Monge, Susana; Bernardino, José I; Pérez-Valero, Ignacio; Montes, María L; Valencia, Eulalia; Martín-Carbonero, Luz; Moreno, Victoria; González-García, Juan; Arnalich, Francisco; Mingorance, Jesús; Pintado Berniches, Laura; Perona, Rosario; Arribas, José R

2017-09-01

To evaluate the in vivo relevance of the inhibitory effect of tenofovir on telomerase activity observed in vitro. Cross-sectional study of HIV-infected patients with suppressed virological replication (HIV RNA <50 copies/mL for more than 1 year). Telomere length in whole blood was measured by quantitative real-time polymerase chain reaction. We performed a multivariate analysis to elucidate variables associated with telomere length and also evaluated the association between telomere length and use of tenofovir difumarate (TDF) adjusted by significant confounders. 200 patients included, 72% men, median age 49 (IQR 45-54.5), 103 with exposure to a TDF containing antiretroviral treatment (ART) regimen (69.9% for more than 5 years) and 97 never exposed to a TDF containing ART regimen. In the multivariate analysis, significant predictors of shorter telomere length were older age (P = 0.008), parental age at birth (P = 0.038), white race (P = 0.048), and longer time of known HIV infection (10-20 and ≥20 years compared with <10 years, P = 0.003 and P = 0.056, respectively). There was no association between TDF exposure and telomere length after adjusting for possible confounding factors (age, parental age at birth, race, and time of HIV infection). Total time receiving ART and duration of treatment with nucleoside reverse transcriptase inhibitors were associated with shorter telomere length, but these associations were explained by time of known HIV infection. Our data do not suggest that telomerase activity inhibition caused by TDF in vitro leads to telomere shortening in peripheral blood of HIV-infected patients.

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.

Detection and management of minimal residual disease in acute lymphoblastic leukemia.

PubMed

Schrappe, Martin

2014-12-05

The detection of minimal residual disease (MRD) has become part of the state-of-the-art diagnostics to guide treatment both in pediatric and adult acute lymphoblastic leukemia (ALL). This applies to the treatment of de novo and recurrent ALL. In high-risk ALL, MRD detection is considered an important tool to adjust therapy before and after hematopoietic stem cell transplantation. Precise quantification and quality control is instrumental to avoid false treatment assignment. A new methodological approach to analyzing MRD has become available and is based on next-generation sequencing. In principle, this technique will be able to detect a large number of leukemic subclones at a much higher speed than before. Carefully designed prospective studies need to demonstrate concordance or even superiority compared with those techniques in use right now: detection of aberrant expression of leukemia-specific antigens by flow cytometry of blood or bone marrow, or detection of specific rearrangements of the T-cell receptor or immunoglobulin genes by real-time quantitative polymerase chain reaction using DNA of leukemic cells. In some cases with known fusion genes, such as BCR/ABL, reverse transcriptase-polymerase chain reaction has been used as additional method to identify leukemic cells by analyzing RNA in patient samples. MRD detection may be used to modulate treatment intensity once it has been demonstrated at well-defined informative checkpoints that certain levels of MRD can reliably predict the risk of relapse. In addition, MRD is used as end point to determine the activity of a given agent or treatment protocol. If activity translates into antileukemic efficacy, MRD may be considered a surrogate clinical end point. © 2014 by The American Society of Hematology. All rights reserved.

Determining Resistance of Toxoplasma gondii Oocysts to UV Disinfection Using Cell Culture and a Mouse Bioassay

USDA-ARS?s Scientific Manuscript database

The effect of UV exposure on Toxoplasma gondii oocysts has not been completely defined for use in water disinfection. This study evaluated irradiated oocysts by three assays: a SCID mouse biassay, an in vitro T. gondii oocyst plaque assay (TOP-assay), and a quantitative reverse-transcriptase real-t...

Isolation, nucleotide sequence and expression of a cDNA encoding feline granulocyte colony-stimulating factor.

PubMed

Dunham, S P; Onions, D E

2001-06-21

A cDNA encoding feline granulocyte colony stimulating factor (fG-CSF) was cloned from alveolar macrophages using the reverse transcriptase-polymerase chain reaction. The cDNA is 949 bp in length and encodes a predicted mature protein of 174 amino acids. Recombinant fG-CSF was expressed as a glutathione S-transferase fusion and purified by affinity chromatography. Biological activity of the recombinant protein was demonstrated using the murine myeloblastic cell line GNFS-60, which showed an ED50 for fG-CSF of approximately 2 ng/ml. Copyright 2001 Academic Press.

Polyurethane intravaginal ring for controlled delivery of dapivirine, a nonnucleoside reverse transcriptase inhibitor of HIV-1.

PubMed

Gupta, Kavita M; Pearce, Serena M; Poursaid, Azadeh E; Aliyar, Hyder A; Tresco, Patrick A; Mitchnik, Mark A; Kiser, Patrick F

2008-10-01

Women-controlled methods for prevention of male-to-female sexual transmission of HIV-1 are urgently needed. Providing inhibitory concentrations of HIV-1 reverse transcriptase inhibitors to impede the replication of the virus in the female genital tissue offers a mechanism for prophylaxis of HIV-1. To this end, an intravaginal ring device that can provide long duration delivery of dapivirine, a nonnucleoside reverse transcriptase inhibitor of HIV-1, was developed utilizing a medical-grade polyether urethane. Monolithic intravaginal rings were fabricated and sustained release with cumulative flux linear with time was demonstrated under sink conditions for a period of 30 days. The release rate was directly proportional to the amount of drug loaded. Another release study conducted for a week utilizing liposome dispersions as sink conditions, to mimic the partitioning of dapivirine into vaginal tissue, also demonstrated release rates constant with time. These results qualify polyether urethanes for development of intravaginal rings for sustained delivery of microbicidal agents. (c) 2008 Wiley-Liss, Inc. and the American Pharmacists Association

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

Modelling Hepatitis B Virus Antiviral Therapy and Drug Resistant Mutant Strains

NASA Astrophysics Data System (ADS)

Bernal, Julie; Dix, Trevor; Allison, Lloyd; Bartholomeusz, Angeline; Yuen, Lilly

Despite the existence of vaccines, the Hepatitis B virus (HBV) is still a serious global health concern. HBV targets liver cells. It has an unusual replication process involving an RNA pre-genome that the reverse transcriptase domain of the viral polymerase protein translates into viral DNA. The reverse transcription process is error prone and together with the high replication rates of the virus, allows the virus to exist as a heterogeneous population of mutants, known as a quasispecies, that can adapt and become resistant to antiviral therapy. This study presents an individual-based model of HBV inside an artificial liver, and associated blood serum, undergoing antiviral therapy. This model aims to provide insights into the evolution of the HBV quasispecies and the individual contribution of HBV mutations in the outcome of therapy.

A Transient Kinetic Approach to Investigate Nucleoside Inhibitors of Mitochondrial DNA polymerase γ

PubMed Central

Anderson, Karen S.

2010-01-01

Nucleoside analogs play an essential role in treating human immunodeficiency virus (HIV) infection since the beginning of the AIDS epidemic and work by inhibition of HIV-1 reverse transcriptase (RT), a viral polymerase essential for DNA replication. Today, over 90% of all regimens for HIV treatment contain at least one nucleoside. Long-term use of nucleoside analogs has been associated with adverse effects including mitochondrial toxicity due to inhibition of the mitochondrial polymerase, DNA polymerase gamma (mtDNA pol ©). In this review, we describe our efforts to delineate the molecular mechanism of nucleoside inhibition of HIV-1 RT and mtDNA pol © based upon a transient kinetic approach using rapid chemical quench methodology. Using transient kinetic methods, the maximum rate of polymerization (kpol), the dissociation constant for the ground state binding (Kd), and the incorporation efficiency (kpol/Kd) can be determined for the nucleoside analogs and their natural substrates. This analysis allowed us to develop an understanding of the structure activity relationships that allow correlation between the structural and stereochemical features of the nucleoside analog drugs with their mechanistic behavior toward the viral polymerase, RT, and the host cell polymerase, mtDNA pol γ. An in-depth understanding of the mechanisms of inhibition of these enzymes is imperative in overcoming problems associated with toxicity. PMID:20573564

Evaluation of RealStar Reverse Transcription–Polymerase Chain Reaction Kits for Filovirus Detection in the Laboratory and Field

PubMed Central

Rieger, Toni; Kerber, Romy; El Halas, Hussein; Pallasch, Elisa; Duraffour, Sophie; Günther, Stephan; Ölschläger, Stephan

2016-01-01

Background. Diagnosis of Ebola virus (EBOV) disease (EVD) requires laboratory testing. Methods. The RealStar Filovirus Screen reverse transcription–polymerase chain reaction (RT-PCR) kit and the derived RealStar Zaire Ebolavirus RT-PCR kit were validated using in vitro transcripts, supernatant of infected cell cultures, and clinical specimens from patients with EVD. Results. The Filovirus Screen kit detected EBOV, Sudan virus, Taï Forest virus, Bundibugyo virus, Reston virus, and Marburg virus and differentiated between the genera Ebolavirus and Marburgvirus. The amount of filovirus RNA that could be detected with a probability of 95% ranged from 11 to 67 RNA copies/reaction on a LightCycler 480 II. The Zaire Ebolavirus kit is based on the Filovirus Screen kit but was optimized for detection of EBOV. It has an improved signal-to-noise ratio at low EBOV RNA concentrations and is somewhat more sensitive than the Filovirus kit. Both kits show significantly lower analytical sensitivity on a SmartCycler II. Clinical evaluation revealed that the SmartCycler II, compared with other real-time PCR platforms, decreases the clinical sensitivity of the Filovirus Screen kit to diagnose EVD at an early stage. Conclusions. The Filovirus Screen kit detects all human-pathogenic filoviruses with good analytical sensitivity if performed on an appropriate real-time PCR platform. High analytical sensitivity is important for early diagnosis of EVD. PMID:27549586

Rapid and sensitive detection of canine distemper virus by one-tube reverse transcription-insulated isothermal polymerase chain reaction.

PubMed

Wilkes, Rebecca P; Tsai, Yun-Long; Lee, Pei-Yu; Lee, Fu-Chun; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

2014-09-09

Canine distemper virus (CDV) has been associated with outbreaks of canine infectious respiratory disease in shelters and boarding kennel environments. POCKITTM Nucleic Acid Analyzer is a field-deployable device capable of generating automatically interpreted insulated isothermal polymerase chain reaction (iiPCR) results from extracted nucleic acid within one hour. In this study, reverse transcription iiPCR (RT-iiPCR) was developed to facilitate point-of-need diagnosis of CDV infection. Analytical sensitivity (limit of detection 95%) of the established CDV RT-iiPCR was about 11 copies of in vitro transcribed RNA per reaction. CDV RT-iiPCR generated positive signals from CDV, but not Bordetella bronchiseptica, canine parvovirus, canine herpesvirus, canine adenovirus 2, canine influenza virus (subtype H3N8), canine parainfluenza virus, and canine respiratory coronavirus. To evaluate accuracy of the established reaction in canine distemper clinical diagnosis, 110 specimens from dogs, raccoons, and foxes suspected with CDV infection were tested simultaneously by CDV RT-iiPCR and real-time RT-PCR. CDV RT-iiPCR demonstrated excellent sensitivity (100%) and specificity (100%), compared to real-time RT-PCR. The results indicated an excellent correlation between RT-iiPCR and a reference real time RT-PCR method. Working in a lyophilized format, the established method has great potential to be used for point-of-care diagnosis of canine distemper in animals, especially in resource-limited facilities.

A new approach for diagnosis of bovine coronavirus using a reverse transcription recombinase polymerase amplification assay.

PubMed

Amer, H M; Abd El Wahed, A; Shalaby, M A; Almajhdi, F N; Hufert, F T; Weidmann, M

2013-11-01

Bovine coronavirus (BCoV) is an economically significant cause of calf scours and winter dysentery of adult cattle, and may induce respiratory tract infections in cattle of all ages. Early diagnosis of BCoV helps to diminish its burden on the dairy and beef industry. Real-time RT-PCR assay for the detection of BCoV has been described, but it is relatively expensive, requires well-equipped laboratories and is not suitable for on-site screening. A novel assay, using reverse transcription recombinase polymerase amplification (RT-RPA), for the detection of BCoV is developed. The BCoV RT-RPA was rapid (10-20 min) and has an analytical sensitivity of 19 molecules. No cross-reactivity with other viruses causing bovine gastrointestinal and/or respiratory infections was observed. The assay performance on clinical samples was validated by testing 16 fecal and 14 nasal swab specimens and compared to real-time RT-PCR. Both assays provided comparable results. The RT-RPA assay was significantly more rapid than the real-time RT-PCR assay. The BCoV RT-RPA constitutes a suitable accurate, sensitive and rapid alternative to the common measures used for BCoV diagnosis. In addition, the use of a portable fluorescence reading device extends its application potential to use in the field and point-of-care diagnosis. Copyright © 2013 Elsevier B.V. All rights reserved.

Assessment of the U937 cell line for the detection of contact allergens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Python, Francois; Goebel, Carsten; Aeby, Pierre

2007-04-15

The human myeloid cell line U937 was evaluated as an in vitro test system to identify contact sensitizers in order to develop alternatives to animal tests for the cosmetic industry. Specific culture conditions (i.e., presence of interleukin-4, IL-4) were applied to obtain a dendritic cell-like phenotype. In the described test protocol, these cells were exposed to test chemicals and then analyzed by flow cytometry for CD86 expression and by quantitative real-time reverse transcriptase-polymerase chain reaction for IL-1{beta} and IL-8 gene expressions. Eight sensitizers, three non-sensitizers and five oxidative hair dye precursors were examined after 24-, 48- and 72-h exposure times.more » Test item-specific modulations of the chosen activation markers (CD86, IL-1{beta} and IL-8) suggest that this U937 activation test could discriminate test items classified as contact sensitizers or non-sensitizers in the local lymph node assay in mice (LLNA). More specifically, a test item can be considered as a potential sensitizer when it significantly induced the upregulation of the expression of at least two markers. Using this approach, we could correctly evaluate the dendritic cell (DC) activation potential for 15 out of 16 tested chemicals. We conclude that the U937 activation test may represent an useful tool in a future in vitro test battery for predicting sensitizing properties of chemicals.« less

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.

(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.

Development of an on-site rapid real-time polymerase chain reaction system and the characterization of suitable DNA polymerases for TaqMan probe technology.

PubMed

Furutani, Shunsuke; Naruishi, Nahoko; Hagihara, Yoshihisa; Nagai, Hidenori

2016-08-01

On-site quantitative analyses of microorganisms (including viruses) by the polymerase chain reaction (PCR) system are significantly influencing medical and biological research. We have developed a remarkably rapid and portable real-time PCR system that is based on microfluidic approaches. Real-time PCR using TaqMan probes consists of a complex reaction. Therefore, in a rapid real-time PCR, the optimum DNA polymerase must be estimated by using actual real-time PCR conditions. In this study, we compared the performance of three DNA polymerases in actual PCR conditions using our rapid real-time PCR system. Although KAPA2G Fast HS DNA Polymerase has the highest enzymatic activity among them, SpeedSTAR HS DNA Polymerase exhibited better performance to rapidly increase the fluorescence signal in an actual real-time PCR using TaqMan probes. Furthermore, we achieved rapid detection of Escherichia coli in 7 min by using SpeedSTAR HS DNA Polymerase with the same sensitivity as that of a conventional thermal cycler.

Low-grade sinonasal sarcoma with neural and myogenic features: a clinicopathologic analysis of 28 cases.

PubMed

Lewis, Jason T; Oliveira, Andre M; Nascimento, Antonio G; Schembri-Wismayer, David; Moore, Eric A; Olsen, Kerry D; Garcia, Joaquin G; Lonzo, Melissa L; Lewis, Jean E

2012-04-01

Sarcomas of the sinonasal region are rare. We describe a distinct spindle cell sarcoma of the sinonasal region characterized by concomitant neural and myogenic differentiation. Consultation files and surgical cases from Mayo Clinic were reviewed. Twenty-eight cases were identified that met the inclusion criteria. Clinical data were collected from medical records, consultation letters, and referring pathologists. Reverse transcriptase-polymerase chain reaction for synovial sarcoma fusion transcripts was performed on 18 cases. Cytogenetic studies were performed on 2 cases. The 21 female and 7 male patients ranged in age from 24 to 85 years (mean, 52 y). All cases showed a characteristic histology, which included a cellular spindle cell neoplasm with uniform, elongate nuclei and an infiltrative growth pattern. All tumors demonstrated expression of S-100 with actin positivity in 96% of cases tested. Reverse transcriptase-polymerase chain reaction for synovial sarcoma fusion transcripts was negative in all cases tested. Cytogenetic studies conducted on 2 cases demonstrated the chromosomal translocation t(2;4). The nasal cavity (54%) and ethmoid sinus (57%) were the most commonly involved areas, singly or in combination. Follow-up information was available for 57% (16/28) of cases, with a mean of 8.3 years. Of these, 44% (7/16) experienced at least 1 recurrence. No patient has developed metastases or died of disease. We describe a unique tumor with a characteristic morphologic, immunophenotypic, and cytogenetic profile. On the basis of the locally aggressive nature of this lesion we believe it is best considered a low-grade sarcoma and suggest the term low-grade sinonasal sarcoma with neural and myogenic features.

Transcription of G-protein coupled receptors in corporeal smooth muscle is regulated by the endogenous neutral endopeptidase inhibitor sialorphin.

PubMed

Tong, Yuehong; Tiplitsky, Scott I; Tar, Moses; Melman, Arnold; Davies, Kelvin P

2008-08-01

Several reports suggest that the rat Vcsa1 gene is down-regulated in models of erectile dysfunction. The Vcsa protein product sialorphin is an endogenous neutral endopeptidase inhibitor and its down-regulation could result in prolonged activation of G-protein activated signaling pathways by their peptide agonists. We investigated whether Vcsa1 down-regulation could result in an adaptive change in GPCR (G-protein coupled receptor) expression. Gene expression in cultured rat corporeal smooth muscle cells following treatment with siRNA directed against Vcsa1 or the neutral endopeptidase gene was analyzed using microarray and quantitative reverse transcriptase-polymerase chain reaction. In rats Vcsa1 is one of the most down-regulated genes following bilateral transection of the cavernous nerves. In that animal model we also investigated whether Vcsa1 down-regulation was accompanied by similar changes in gene expression in corporeal smooth muscle cells in which Vcsa1 was knocked down in vitro. Microarray analysis and quantitative reverse transcriptase-polymerase chain reaction demonstrated that corporeal smooth muscle cells treated in vitro with siRNA against Vcsa1 resulted in GPCR up-regulation as a functional group. In contrast, treatment of corporeal smooth muscle cells that lowered neutral endopeptidase activity resulted in decreased GPCR expression. These results suggest that the peptide product of Vcsa1, sialorphin, can effect GPCR expression by acting on neutral endopeptidase. In animals with bilaterally transected cavernous nerves the decreased Vcsa1 expression is accompanied by increased GPCR expression in cavernous tissue. These experiments suggest that the mechanism by which Vcsa1 modulates erectile function is partly mediated through changes in GPCR expression.

Analysis by rotavirus gene 6 reverse transcriptase-polymerase chain reaction assay of rotavirus-positive gastroenteritis cases observed during the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST).

PubMed

Matson, David O; Vesikari, Timo; Dennehy, Penelope; Dallas, Michael D; Goveia, Michelle G; Itzler, Robbin F; Ciarlet, Max

2014-01-01

During the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST), the period between the administration of dose 1 through 13 days after the administration of dose 3, there were more wild-type rotavirus gastroenteritis (RVGE) cases among vaccine recipients compared with placebo recipients using the protocol-specified microbiological plaque assay in the clinical-efficacy cohort, a subset of subjects where vaccine efficacy against RVGE of any severity was assessed. In this study, a rotavirus genome segment 6-based reverse transcriptase-polymerase chain reaction assay was applied post hoc to clarify the accuracy of type categorization of all these RVGE cases in vaccine recipients during the vaccination phase of REST. The assay characterized 147 (90%) of 163 re-assayed RVGE cases or rotavirus-associated health care contacts as type-determinable: either wild-type or vaccine-type rotavirus strains. In the clinical-efficacy cohort (N = 5673), 19 (18.8%) of 101 samples from RVGE cases contained wild-type rotavirus, 70 (69.3%) vaccine virus, and 12 (11.9%) were indeterminable. In the large-scale cohort (N = 68,038), 10 (34.5%) of 29 samples from RVGE-related health care contacts contained wild-type rotavirus strains, 15 (51.7%) vaccine-type rotavirus strains, and 4 (13.8%) were indeterminable. Of the 33 samples from RVGE cases in placebo recipients, all were confirmed to contain wild-type rotaviruses. Altogether, this post-hoc re-evaluation showed that the majority (75%) of type-determinable RVGE cases or health care contacts that occurred during the vaccination phase of REST in vaccine recipients were associated with vaccine-type rotavirus strains rather than wild-type rotavirus strains.

HIP1-ALK, a novel fusion protein identified in lung adenocarcinoma.

PubMed

Hong, Mineui; Kim, Ryong Nam; Song, Ji-Young; Choi, So-Jung; Oh, Ensel; Lira, Maruja E; Mao, Mao; Takeuchi, Kengo; Han, Joungho; Kim, Jhingook; Choi, Yoon-La

2014-03-01

The most common mechanism underlying overexpression and activation of anaplastic lymphoma kinase (ALK) in non-small-cell lung carcinoma could be attributed to the formation of a fusion protein. To date, five fusion partners of ALK have been reported, namely, echinoderm microtubule associated protein like 4, tropomyosin-related kinase-fused gene, kinesin family member 5B, kinesin light chain 1, and protein tyrosine phosphatase, nonreceptor type 3. In this article, we report a novel fusion gene huntingtin interacting protein 1 (HIP1)-ALK, which is conjoined between the huntingtin-interacting protein 1 gene HIP1 and ALK. Reverse-transcriptase polymerase chain reaction and immunohistochemical analysis were used to detect this fusion gene's transcript and protein expression, respectively. We had amplified the full-length cDNA sequence of this novel fusion gene by using 5'-rapid amplification of cDNA ends. The causative genomic translocation t(2;7)(p23;q11.23) for generating this novel fusion gene was verified by using genomic sequencing. The examined adenocarcinoma showed predominant acinar pattern, and ALK immunostaining was localized to the cytoplasm, with intense staining in the submembrane region. In break-apart, fluorescence in situ hybridization analysis for ALK, split of the 5' and 3' probe signals, and isolated 3' signals were observed. Reverse-transcriptase polymerase chain reaction revealed that the tumor harbored a novel fusion transcript in which exon 21 of HIP1 was fused to exon 20 of ALK in-frame. The novel fusion gene and its protein HIP1-ALK harboring epsin N-terminal homology, coiled-coil, juxtamembrane, and kinase domains, which could play a role in carcinogenesis, could become diagnostic and therapeutic target of the lung adenocarcinoma and deserve a further study in the future.

RCL2, a New Fixative, Preserves Morphology and Nucleic Acid Integrity in Paraffin-Embedded Breast Carcinoma and Microdissected Breast Tumor Cells

PubMed Central

Delfour, Christophe; Roger, Pascal; Bret, Caroline; Berthe, Marie-Laurence; Rochaix, Philippe; Kalfa, Nicolas; Raynaud, Pierre; Bibeau, Frédéric; Maudelonde, Thierry; Boulle, Nathalie

2006-01-01

Methacarn and RCL2, a new noncrosslinking fixative, were compared to formalin-fixed or frozen tissue samples of the same invasive breast carcinoma and were evaluated for their effects on tissue morphology and immunohistochemistry as well as DNA and RNA integrity. The histomorphology of methacarn- or RCL2-fixed paraffin-embedded tumors was similar to that observed with the matched formalin-fixed tissues. Immunohistochemistry using various antibodies showed comparable results with either fixative, leading to accurate breast tumor diagnosis and determination of estrogen and progesterone receptors, and HER2 status. Methacarn and RCL2 fixation preserved DNA integrity as demonstrated by successful amplification and sequencing of large DNA amplicons. Similarly, high-quality RNA could be extracted from methacarn- or RCL2-fixed paraffin-embedded MCF-7 cells, whole breast tumor tissues, or microdissected breast tumor cells, as assessed by electropherogram profiles and real-time reverse transcriptase-polymerase chain reaction quantification of various genes. Moreover, tissue morphology and RNA integrity were preserved after 8 months of storage. Altogether, these results indicate that methacarn, as previously shown, and RCL2, a promising new fixative, have great potential for performing both morphological and molecular analyses on the same fixed tissue sample, even after laser-capture microdissection, and can open new doors for investigating small target lesions such as premalignant breast lesions. PMID:16645201

Aggressive melanoma cells escape from BMP7-mediated autocrine growth inhibition through coordinated Noggin upregulation

PubMed Central

Hsu, Mei-Yu; Rovinsky, Sherry; Lai, Chiou-Yan; Qasem, Shadi; Liu, Xiaoming; How, Joan; Engelhardt, John F.; Murphy, George F.

2009-01-01

Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily responsible for mediating a diverse array of cellular functions both during embryogenesis and in adult life. Previously, we reported that upregulation of BMP7 in human melanoma correlates with tumor progression. However, melanoma cells are either inhibited by or become resistant to BMP7 as a function of tumor progression, with normal melanocytes being most susceptible. Herein, real-time quantitative reverse transcriptase-polymerase chain reactions and Western blotting revealed that the expression of BMP antagonist, Noggin, correlates with resistance to BMP7 in advanced melanoma cells. To test the hypothesis that coordinated upregulation of Noggin protects advanced melanoma cells from autocrine inhibition by BMP7, functional expression of Noggin in susceptible melanoma cells was achieved by adenoviral gene transfer. The Noggin-overexpressing cells exhibited a growth advantage in response to subsequent BMP7 transduction in vitro under anchorage-dependent and -independent conditions, in three-dimensional skin reconstructs, as well as in vivo in severe combined immune-deficiency mice. In concordance, Noggin knockdown by lentiviral shRNA confers sensitivity to BMP7-induced growth inhibition in advanced melanoma cells. Our findings suggest that, like TGF-β, BMP7 acts as an autocrine growth inhibitor in melanocytic cells, and that advanced melanoma cells may escape from BMP7-induced inhibition through concomitant aberrant expression of Noggin. PMID:18560367

Detection of bacteria in platelet concentrates prepared from spiked single donations using cultural and molecular genetic methods.

PubMed

Störmer, M; Cassens, U; Kleesiek, K; Dreier, J

2007-02-01

Bacteria show differences in their growth kinetics depending on the type of blood component. On to storage at 22 degrees C, platelet concentrates (PCs) seem to be more prone to bacterial multiplication than red cell concentrates. Knowledge of the potential for bacterial proliferation in blood components, which are stored at a range of temperatures, is essential before considering implementation of a detection strategy. The efficacy of bacterial detection was determined, using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), following bacterial growth in blood components obtained from a deliberately contaminated whole-blood (WB) unit. Cultivation was used as the reference method. WB was spiked with 2 colony-forming units mL(-1)Staphylococcus epidermidis or Klebsiella pneumoniae, kept for 15 h at room temperature and component preparation was processed. Samples were drawn, at intervals throughout the whole separation process, from each blood component. Nucleic acids were extracted using an automated high-volume extraction method. The 15-h storage revealed an insignificant increase in bacterial titre. No bacterial growth was detected in red blood cell or plasma units. K. pneumoniae showed rapid growth in the pooled PC and could be detected immediately after preparation using RT-PCR. S. epidermidis grew slowly and was detected 24 h after separation. These experiments show that sampling is indicative at 24 h after preparation of PCs at the earliest to minimize the sampling error.

An investigation into the effect of surface roughness of stainless steel on human umbilical vein endothelial cell gene expression.

PubMed

McLucas, E; Moran, M T; Rochev, Y; Carroll, W M; Smith, T J

2006-01-01

The surface properties of vascular devices dictate the initial postimplantation reactions that occur and thus the efficacy of the implantation procedure. Over the last number of years, a number of different stent designs have emerged and stents are generally polished to a mirror finish during the manufacturing procedure. This study sought to investigate the effect of stainless steel surface roughness on endothelial cell gene expression using an appropriate cell culture in vitro assay system. Stainless steel discs were roughened by shot blasting or polished by mechanical polishing. The surface roughness of the treated and untreated discs was determined by atomic force microscopy (AFM). Cells were seeded on collagen type 1 gels and left to attach for 24 h. Stainless steel discs of varying roughness were then placed in contact with the cells and incubated for 24 h. RNA extractions and quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was then performed to determine the expression levels of candidate genes in the treated cells compared to suitable control cells. E-selectin and vascular cellular adhesion molecule (VCAM-1) were found to be significantly up-regulated in cells incubated with polished and roughened samples, indicating endothelial cell activation and inflammation. This study indicates that the surface roughness of stainless steel is an important surface property in the development of vascular stents.

RITA inhibits growth of human hepatocellular carcinoma through induction of apoptosis.

PubMed

Wang, Haihe; Chen, Guofu; Wang, Hongzhi; Liu, Chunbo

2013-01-01

RBP-J-interacting and tubulin-associated (RITA) is a novel RBP-J-interacting protein that downregulates Notch-mediated transcription. The current study focuses on the antitumor effect of RITA in human hepatocellular carcinoma (HCC) and aims to explore its molecular mechanism. Thirty paired HCC and adjacent non-tumoral liver samples were analyzed by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). RITA overexpression was induced by transfection of a pcDNA3.1-Flag-RITA plasmid into HepG2 cells. RITA knockdown was achieved by siRNA transfection. mRNA and protein expression of target genes were quantified by qRT-PCR and Western blotting, respectively. Cell proliferation and apoptosis were measured using MTT assay and flow cytometry. Our results demonstrate that adjacent nontumoral liver samples exhibited increased RITA expression compared to HCC tissues (p < 0.05); RITA levels were associated with tumor differentiation status. Overexpression of RITA suppressed cell proliferation and promoted early apoptosis, while its silencing promoted cell growth dramatically (p < 0.05). RITA overexpression upregulated p53 and reduced cyclin E levels, whereas silencing of RITA had the opposite effect on p53 and cyclin E expression. Our in vitro results represent the first evidence that RITA might suppress tumor growth and induce apoptosis in HCCs, and may be a potent antitumoral agent for HCC treatment that deserves further exploration.

Decreased expression of the APOA1-APOC3-APOA4 gene cluster is associated with risk of Alzheimer's disease.

PubMed

Lin, Qiao; Cao, Yunpeng; Gao, Jie

2015-01-01

Apolipoprotein is genetically associated with the risk of Alzheimer's disease (AD). The APOA1, APOC3, and APOA4 genes are closely linked and located on human chromosome 11. Therefore, this gene cluster may be related to the risk of AD. A total of 147 AD patients and 160 healthy controls were randomly recruited from June 2013 to August 2014. APOA1, APOC3, and APOA4 levels were measured using real-time quantitative reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. APOA1, APOC3 and APOA4 levels were significantly lower in AD patients than controls (P<0.01). APOA1, APOC3, and APOA4 levels were negatively related with the severities of AD determined by Clinical Dementia Rating scores (P<0.01). APOA1, APOC3, and APOA4 levels showed a negative relation with Montgomery-Åsberg Depression Rating Scale scores and a positive relation with RAND 36-item health-survey scores (P<0.01). There was a decreased trend for levels of APOA1, APOC3, and APOA4 in AD patients. Low levels of APOA1, APOC3, and APOA4 are associated with risk of AD. APOA1, APOC3, and APOA4 should be developed as combined drugs for the therapy of AD.

Decreased expression of the APOA1–APOC3–APOA4 gene cluster is associated with risk of Alzheimer’s disease

PubMed Central

Lin, Qiao; Cao, Yunpeng; Gao, Jie

2015-01-01

Background Apolipoprotein is genetically associated with the risk of Alzheimer’s disease (AD). The APOA1, APOC3, and APOA4 genes are closely linked and located on human chromosome 11. Therefore, this gene cluster may be related to the risk of AD. Patients and methods A total of 147 AD patients and 160 healthy controls were randomly recruited from June 2013 to August 2014. APOA1, APOC3, and APOA4 levels were measured using real-time quantitative reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. Results APOA1, APOC3 and APOA4 levels were significantly lower in AD patients than controls (P<0.01). APOA1, APOC3, and APOA4 levels were negatively related with the severities of AD determined by Clinical Dementia Rating scores (P<0.01). APOA1, APOC3, and APOA4 levels showed a negative relation with Montgomery–Åsberg Depression Rating Scale scores and a positive relation with RAND 36-item health-survey scores (P<0.01). There was a decreased trend for levels of APOA1, APOC3, and APOA4 in AD patients. Conclusion Low levels of APOA1, APOC3, and APOA4 are associated with risk of AD. APOA1, APOC3, and APOA4 should be developed as combined drugs for the therapy of AD. PMID:26491253

Local Synthesis of Pepsin in Barrett's Esophagus and the Role of Pepsin in Esophageal Adenocarcinoma.

PubMed

Samuels, Tina; Hoekzema, Craig; Gould, Jon; Goldblatt, Matthew; Frelich, Matthew; Bosler, Matthew; Lee, Sang-Hyuk; Johnston, Nikki

2015-11-01

Despite widespread use of proton pump inhibitors (PPIs), the incidence of esophageal adenocarcinoma (EAC) continues to rise. PPIs reduce reflux acidity, but only transiently inactivate gastric enzymes. Nonacid reflux, specifically nonacid pepsin, contributes to carcinogenesis in the larynx. Given the carcinogenic potential of pepsin and inefficacy of PPIs to prevent EAC, the presence and effect of pepsin in the esophagus should be investigated. Normal and Barrett's biopsies from 8 Barrett's esophagus patients were collected for pepsin analysis via Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR). Human esophageal cells cultured from healthy patients were treated with pepsin (0.01-1 mg/mL; 1-20 hours), acid (pH 4)±pepsin (5 minutes); real-time RT-PCR, ELISA, and cell migration were assayed. Pepsin was detected in all 8 Barrett's and 4 of 8 adjacent normal specimens. Pepsinogen mRNA was observed in 22 Barrett's, but not in normal adjacent samples. Pepsin induced PTSG2 (COX-2) and IL-1β expression and cell migration in vitro. Pepsin is synthesized by metaplastic, Barrett's esophageal mucosa. Nonacid pepsin increases metrics of tumorigenicity in esophageal epithelial cells in vitro. These findings implicate refluxed and locally synthesized pepsin in development and progression of EAC and, in part, explain the inefficacy of PPIs in the prevention of EAC. © The Author(s) 2015.

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.

Difference in drug resistance patterns between minor HIV-1 populations in cerebrospinal fluid and plasma.

PubMed

Bergroth, T; Ekici, H; Gisslén, M; Hagberg, L; Sönnerborg, A

2009-02-01

The aim of the study was to determine to what extent unique drug resistance patterns appear in minor and major HIV-1 quasispecies in cerebrospinal fluid (CSF) as compared with blood. Forty-four plasma and CSF samples from 13 multi-treatment-experienced patients, seven of whom provided longitudinal samples, were included in the study. The subjects had failed antiretroviral therapy including lamivudine. The reverse transcriptase (RT) gene was examined by selective real-time polymerase chain reaction (SPCR), which can detect M184I/V mutants down to 0.2% of the viral population. SPCR revealed differences at amino acid position 184 in the plasma/CSF populations in 12 paired samples from eight patients. One plasma sample was positive by SPCR where direct sequencing showed wild-type M184. The other 11 paired samples showed quantitative differences in the mixed populations of the mutant or wild-type M184 quasispecies. Differences in other resistance-associated mutations between plasma and CSF viruses were also found by direct sequencing. In multi-treatment-experienced patients with therapy failure, differences in drug resistance patterns were found frequently between plasma and CSF in both minor and major viral populations. To what extent this was a true biological phenomenon remains to be established, and the clinical relevance of these findings is yet to be determined.

Nickel (Ni) allergic patients with complications to Ni containing joint replacement show preferential IL-17 type reactivity to Ni.

PubMed

Summer, Burkhard; Paul, Carina; Mazoochian, Farhad; Rau, Christoph; Thomsen, Marc; Banke, Ingo; Gollwitzer, Hans; Dietrich, Karin-Almut; Mayer-Wagner, Susanne; Ruzicka, Thomas; Thomas, Peter

2010-07-01

Some nickel (Ni) allergic patients develop complications following Ni-containing arthroplasty. In the peri-implant tissue of such patients, we had observed lymphocyte dominated inflammation together with IFN-gamma and IL-17 expression. To determine whether Ni stimulation of peripheral blood mononuclear cells (PBMCs) of such patients would lead to a different cytokine pattern as compared to Ni-allergic patients with symptom-free arthroplasty. Based on history and patch testing in 15 Ni-allergic patients (five without implant, five with symptom-free arthroplasty, five with complicated arthroplasty) and five non-allergic individuals, lymphocyte transformation test (LTT) was performed using PBMC. In parallel in vitro cytokine response to Ni was assessed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). All 15 Ni-allergic individuals showed enhanced LTT reactivity to Ni (mean SI = 8.42 +/- 1.8) compared to the non-allergic control group. Predominant IFN-gamma expression to Ni was found both in the five allergic patients without arthroplasty and also in the five allergic, symptom-free arthroplasty patients. In contrast, in the five Ni-allergic patients with arthroplasty-linked complications a predominant, significant IL-17 expression to Ni was seen but not in patients with symptom-free arthroplasty. The predominant IL-17 type response to Ni may characterize a subgroup of Ni-allergic patients prone to develop lymphocytic peri-implant hyper-reactivity.

Effect of chitosan-N-acetylcysteine conjugate in a mouse model of botulinum toxin B-induced dry eye.

PubMed

Hongyok, Teeravee; Chae, Jemin J; Shin, Young Joo; Na, Daero; Li, Li; Chuck, Roy S

2009-04-01

To evaluate the effect of a thiolated polymer lubricant, chitosan-N-acetylcysteine conjugate (C-NAC), in a mouse model of dry eye. Eye drops containing 0.5% C-NAC, 0.3% C-NAC, a vehicle (control group), artificial tears, or fluorometholone were applied in a masked fashion in a mouse model of induced dry eye from 3 days to 4 weeks after botulinum toxin B injection. Corneal fluorescein staining was periodically recorded. Real-time reverse transcriptase-polymerase chain reaction and immunofluorescence staining were performed at the end of the study to evaluate inflammatory cytokine expressions. Mice treated with C-NAC, 0.5%, and fluorometholone showed a downward trend that was not statistically significant in corneal staining compared with the other groups. Chitosan-NAC formulations, fluorometholone, and artificial tears significantly decreased IL-1beta (interleukin 1beta), IL-10, IL-12alpha, and tumor necrosis factor alpha expression in ocular surface tissues. The botulinum toxin B-induced dry eye mouse model is potentially useful in evaluating new dry eye treatment. Evaluation of important molecular biomarkers suggests that C-NAC may impart some protective ocular surface properties. However, clinical data did not indicate statistically significant improvement of tear production and corneal staining in any of the groups tested. Topically applied C-NAC might protect the ocular surface in dry eye syndrome, as evidenced by decreased inflammatory cytokine expression.

Expanding severe acute respiratory infection (SARI) surveillance beyond influenza: The process and data from 1 year of implementation in Vietnam.

PubMed

Alroy, Karen A; Do, Trang Thuy; Tran, Phu Dac; Dang, Tan Quang; Vu, Long Ngoc; Le, Nga Thi Hang; Dang, Anh Duc; Ngu, Nghia Duy; Ngo, Tu Huy; Hoang, Phuong Vu Mai; Phan, Lan Trong; Nguyen, Thuong Vu; Nguyen, Long Thanh; Nguyen, Thinh Viet; Vien, Mai Quang; Le, Huy Xuan; Dao, Anh The; Nguyen, Trieu Bao; Pham, Duoc Tho; Nguyen, Van Thi Tuyet; Pham, Thanh Ngoc; Phan, Binh Hai; Whitaker, Brett; Do, Thuy Thi Thu; Dao, Phuong Anh; Balajee, S Arunmozhi; Mounts, Anthony W

2018-05-13

In 2016, as a component of the Global Health Security Agenda, the Vietnam Ministry of Health expanded its existing influenza sentinel surveillance for severe acute respiratory infections (SARI) to include testing for 7 additional viral respiratory pathogens. This article describes the steps taken to implement expanded SARI surveillance in Vietnam and reports data from 1 year of expanded surveillance. The process of expanding the suite of pathogens for routine testing by real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) included laboratory trainings, procurement/distribution of reagents, and strengthening and aligning SARI surveillance epidemiology practices at sentinel sites and regional institutes (RI). Surveillance data showed that of 4003 specimens tested by the RI laboratories, 20.2% (n = 810) were positive for influenza virus. Of the 3193 influenza-negative specimens, 41.8% (n = 1337) were positive for at least 1 non-influenza respiratory virus, of which 16.2% (n = 518), 13.4% (n = 428), and 9.6% (n = 308) tested positive for respiratory syncytial virus, rhinovirus, and adenovirus, respectively. The Government of Vietnam has demonstrated that expanding respiratory viral surveillance by strengthening and building upon an influenza platform is feasible, efficient, and practical. © 2018 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

The NLP toxin family in Phytophthora sojae includes rapidly evolving groups that lack necrosis-inducing activity.

PubMed

Dong, Suomeng; Kong, Guanghui; Qutob, Dinah; Yu, Xiaoli; Tang, Junli; Kang, Jixiong; Dai, Tingting; Wang, Hai; Gijzen, Mark; Wang, Yuanchao

2012-07-01

Necrosis- and ethylene-inducing-like proteins (NLP) are widely distributed in eukaryotic and prokaryotic plant pathogens and are considered to be important virulence factors. We identified, in total, 70 potential Phytophthora sojae NLP genes but 37 were designated as pseudogenes. Sequence alignment of the remaining 33 NLP delineated six groups. Three of these groups include proteins with an intact heptapeptide (Gly-His-Arg-His-Asp-Trp-Glu) motif, which is important for necrosis-inducing activity, whereas the motif is not conserved in the other groups. In total, 19 representative NLP genes were assessed for necrosis-inducing activity by heterologous expression in Nicotiana benthamiana. Surprisingly, only eight genes triggered cell death. The expression of the NLP genes in P. sojae was examined, distinguishing 20 expressed and 13 nonexpressed NLP genes. Real-time reverse-transcriptase polymerase chain reaction results indicate that most NLP are highly expressed during cyst germination and infection stages. Amino acid substitution ratios (Ka/Ks) of 33 NLP sequences from four different P. sojae strains resulted in identification of positive selection sites in a distinct NLP group. Overall, our study indicates that expansion and pseudogenization of the P. sojae NLP family results from an ongoing birth-and-death process, and that varying patterns of expression, necrosis-inducing activity, and positive selection suggest that NLP have diversified in function.

Investigation of avian influenza infection in wild birds in Ismailia and Damietta cities, Egypt

PubMed Central

Fadel, Hanaa Mohamed; Afifi, Rabab

2017-01-01

Aim: This study was carried out to monitor avian influenza (AI) infection in wild birds in Egypt. Materials and Methods: A total of 135 wild birds were examined for the presence of H5, H7, and H9 hemagglutination inhibition antibodies. Organs and swab samples of 75 birds were screened by multiplex real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) to detect AI subtypes H5, H7, and H9 matrix genes. Results: The highest seropositive result was recorded in cattle egrets (90.9%) followed by crows (88.6%), semi-captive pigeons (44.8%), and moorhens (39.1%). In cattle egrets, semi-captive pigeons and moorhens, H5 antibodies predominated. In crows, H9 antibodies predominated. Multiple infections with two or three virus subtypes were highest in crows (6/39, 15.4%) followed by cattle egrets (3/30, 10%) and moorhens’ (1/9, 11.1%) positive samples. Multiplex RRT-PCR results revealed two positive samples in cattle egrets and moorhens. Conclusion: The results indicated high seropositive rates against AI virus subtypes H5 and H9 in the examined wild birds. Multiple infections with more than one AI virus (AIV) subtypes were detected in some birds. This requires a collaboration of efforts to monitor AIV infection in wild birds and implement suitable early intervention measures. PMID:28717324

Twenty-Five Years of Lamivudine: Current and Future Use for the Treatment of HIV-1 Infection.

PubMed

Quercia, Romina; Perno, Carlo-Federico; Koteff, Justin; Moore, Katy; McCoig, Cynthia; St Clair, Marty; Kuritzkes, Daniel

2018-06-01

Innovation in medicine is a dynamic, complex, and continuous process that cannot be isolated to a single moment in time. Anniversaries offer opportunities to commemorate crucial discoveries of modern medicine, such as penicillin (1928), polio vaccination (inactivated, 1955; oral, 1961), the surface antigen of the hepatitis B virus (1967), monoclonal antibodies (1975), and the first HIV antiretroviral drugs (zidovudine, 1987). The advent of antiretroviral drugs has had a profound effect on the progress of the epidemiology of HIV infection, transforming a terminal, irreversible disease that caused a global health crisis into a treatable but chronic disease. This result has been driven by the success of antiretroviral drug combinations that include nucleoside reverse transcriptase inhibitors such as lamivudine. Lamivudine, an L-enantiomeric analog of cytosine, potently affects HIV replication by inhibiting viral reverse transcriptase enzymes at concentrations without toxicity against human polymerases. Although lamivudine was approved more than 2 decades ago, it remains a key component of first-line therapy for HIV because of its virological efficacy and ability to be partnered with other antiretroviral agents in traditional and novel combination therapies. The prominence of lamivudine in HIV therapy is highlighted by its incorporation in recent innovative treatment strategies, such as single-tablet regimens that address challenges associated with regimen complexity and treatment adherence and 2-drug regimens being developed to mitigate cumulative drug exposure and toxicities. This review summarizes how the pharmacologic and virologic properties of lamivudine have solidified its role in contemporary HIV therapy and continue to support its use in emerging therapies.

Germline V repertoires: Origin, maintenance, diversification.

PubMed

Steele, E J; Lindley, R A

2018-06-01

In our view, Melvin Cohn (Scand J Immunol. 2018;87:e12640) has set out the logical guidelines towards a resolution of the very real enigma of the selectability of vertebrate germline Ig V repertoires under the current evolutionary paradigm…" A somatically derived repertoire scrambles this (germline VL + VH) substrate so that its specificities are lost, making it un-selectable in the germline. Consequently, evolution faced an incompatibility." It is argued here in Reply that a reverse transcriptase-based soma-to-germline process (S->G) targeting germline V segment arrays goes some considerable way to resolving fundamental contradictions on the origin, maintenance and then real-time adaptive diversification of these limited sets of V segments encoded within various V repertoire arrays. © 2018 The Foundation for the Scandinavian Journal of Immunology.

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

High-throughput real-time quantitative reverse transcription PCR.

PubMed

Bookout, Angie L; Cummins, Carolyn L; Mangelsdorf, David J; Pesola, Jean M; Kramer, Martha F

2006-02-01

Extensive detail on the application of the real-time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high-throughput, 384-well-format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real-time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency-corrected DeltaCt method, and the comparative cycle time, or DeltaDeltaCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT-PCR in the same manner as the experimental samples, thus accounting for the reaction efficiencies of both procedures. This protocol describes the production and quantitation of synthetic RNA molecules for real-time and non-real-time RT-PCR applications.

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.

Imprint cytology of clear cell sarcoma-like tumor of the gastrointestinal tract in the small intestine: A case report.

PubMed

Kato, Takashi; Ichihara, Shin; Gotoda, Hiroko; Muraoka, Shunji; Kubo, Terufumi; Sugita, Shintaro; Hasegawa, Tadashi

2017-12-01

Clear cell sarcoma-like tumor of the gastrointestinal tract (CCSLGT) is an extremely rare malignant neoplasm in the digestive tract. Its cytomorphologic features have never previously been reported. Here, we describe a case of CCSLGT, including its cytologic examination findings. A 47-year-old woman presented with a mass in the small intestine, which was resected and sent for imprint cytology. Imprint smears revealed tumor cells with light eosinophilic or clear cytoplasm in a necrotic background. Many of the tumor cells were arranged in a perivascular growth with a pseudopapillary formation, and there were some non-neoplastic osteoclast-like giant cells. Histological examination revealed solid nests and a pseudopapillary pattern of the tumor cells with clear or pale eosinophilic cytoplasm and large nuclei with small nucleoli. Immunohistochemistry showed positive for vimentin, S-100, and SOX-10, and negative for SMA, c-KIT, cytokeratin, HMB-45, and MelanA. The EWSR1 gene split signal was detected by reverse transcriptase fluorescence in situ hybridization, and EWSR1-CREB1 gene fusion was indicated by reverse transcriptase polymerase chain reaction analysis. From these findings, we diagnosed the tumor as CCSLGT. To best of our knowledge, this is the first description of the imprint cytology features of CCSLGT. © 2017 Wiley Periodicals, Inc.

Hypersusceptibility to substrate analogs conferred by mutations in human immunodeficiency virus type 1 reverse transcriptase.

PubMed

Smith, Robert A; Anderson, Donovan J; Preston, Bradley D

2006-07-01

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) contains four structural motifs (A, B, C, and D) that are conserved in polymerases from diverse organisms. Motif B interacts with the incoming nucleotide, the template strand, and key active-site residues from other motifs, suggesting that motif B is an important determinant of substrate specificity. To examine the functional role of this region, we performed "random scanning mutagenesis" of 11 motif B residues and screened replication-competent mutants for altered substrate analog sensitivity in culture. Single amino acid replacements throughout the targeted region conferred resistance to lamivudine and/or hypersusceptibility to zidovudine (AZT). Substitutions at residue Q151 increased the sensitivity of HIV-1 to multiple nucleoside analogs, and a subset of these Q151 variants was also hypersusceptible to the pyrophosphate analog phosphonoformic acid (PFA). Other AZT-hypersusceptible mutants were resistant to PFA and are therefore phenotypically similar to PFA-resistant variants selected in vitro and in infected patients. Collectively, these data show that specific amino acid replacements in motif B confer broad-spectrum hypersusceptibility to substrate analog inhibitors. Our results suggest that motif B influences RT-deoxynucleoside triphosphate interactions at multiple steps in the catalytic cycle of polymerization.

Lessons from the epidemiological surveillance program, during the influenza A (H1N1) virus epidemic, in a reference university hospital of Southeastern Brazil.

PubMed

Moretti, Maria Luiza; Sinkoc, Verônica; Cardoso, Luis Gustavo de Oliveira; Camargo, Gema Jesus de; Bachur, Luis Felipe; Hofling, Christian Cruz; Angerami, Rodrigo; Trabasso, Plínio; Garcia, Márcia Teixeira; Resende, Mariângela Ribeiro

2011-01-01

The case definition of influenza-like illness (ILI) is a powerful epidemiological tool during influenza epidemics. A prospective cohort study was conducted to evaluate the impact of two definitions used as epidemiological tools, in adults and children, during the influenza A H1N1 epidemic. Patients were included if they had upper respiratory samples tested for influenza by real-time reverse transcriptase polymerase chain reaction during two periods, using the ILI definition (coughing + temperature ≤ 38ºC) in period 1, and the definition of severe acute respiratory infection (ARS) (coughing + temperature ≤ 38ºC and dyspnoea) in period 2. The study included 366 adults and 147 children, covering 243 cases of ILI and 270 cases of ARS. Laboratory confirmed cases of influenza were higher in adults (50%) than in children (21.6%) ( p < 0.0001) and influenza infection was more prevalent in the ILI definition (53%) than ARS (24.4%) (p < 0.0001). Adults reported more chills and myalgia than children (p = 0.0001). Oseltamivir was administered in 58% and 46% of adults and children with influenza A H1N1, respectively. The influenza A H1N1 case fatality rate was 7% in adults and 8.3% in children. The mean time from onset of illness until antiviral administration was 4 days. The modification of ILI to ARS definition resulted in less accuracy in influenza diagnosis and did not improve the appropriate time and use of antiviral medication.

Interactions between meniscal cells and a self assembled biomimetic surface composed of hyaluronic acid, chitosan and meniscal extracellular matrix molecules.

PubMed

Tan, Guak-Kim; Dinnes, Donna L M; Butler, Lauren N; Cooper-White, Justin J

2010-08-01

Menisci are one of the most commonly injured parts of the knee with a limited healing potential. This study focuses on fabrication and characterization of biomimetic surfaces for meniscal tissue engineering. To achieve this, a combination of hyaluronic acid/chitosan (HA/CH) mutilayers with covalently immobilized major extracellular matrix (ECM) components of native meniscus, namely collagen I/II (COL.I/II) and chondroitin-6-sulfate (C6S) was employed. Adsorption of the biomolecules was monitored using a quartz crystal microbalance with dissipation (QCM-D) and fourier transform-surface plasmon resonance (FT-SPR). Immobilization of the biomolecules onto HA/CH mutilayers was achieved by sequential adsorption, with optimum binding at a molar ratio of 1.4:1 (COL.I/II: C6S). After adding COL.I/II, the layers became relatively more rigid and large aggregates were evident. The effects of the modified surfaces on cell proliferation, gene expression and proteoglycan production of rat meniscal cells were examined. Quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) analysis showed that primary meniscal cells dedifferentiated rapidly after one passage in monolayer culture. This process could be reversed by culturing the cells on C6S surfaces, as indicated by increases in both collagen II and aggrecan gene expression, as well as proteoglycan production. Cells with abundant lipid vacuoles were evident on all the surfaces over an extended culture period. The results demonstrate the feasibility of C6S surfaces to avoid the dedifferentiation that normally occurs during monolayer expansion of meniscal cells. Crown Copyright 2010. Published by Elsevier Ltd. All rights reserved.

Correlation of Cyfra 21-1 levels in saliva and serum with CK19 mRNA expression in oral squamous cell carcinoma.

PubMed

Malhotra, Rewa; Urs, Aadithya B; Chakravarti, Anita; Kumar, Suman; Gupta, V K; Mahajan, Bhawna

2016-07-01

Oral squamous cell carcinoma (OSCC) accounts for 90 % of malignant lesions of oral cavity. The study assessed the potential of Cyfra 21-1 as a tumor marker in OSCC. The study included 50 patients of OSCC to evaluate levels of Cyfra 21-1 in serum and saliva by electrochemiluminescent immunoassay (ECLIA) and CK19 messenger RNA (mRNA) expression in tissue by florescent quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) along with healthy individuals as control. The salivary and serum Cyfra 21-1 levels in patients of OSCC were significantly higher compared to controls (p value < 0.01). There was a 2.75-fold increase in CK19 mRNA expression in OSCC cases compared to controls. A significant positive correlation was found between serum and salivary Cyfra 21-1, serum Cyfra 21-1, and CK19 mRNA expression and between salivary Cyfra 21-1 and CK19 mRNA expression. Among these, correlation between serum and salivary Cyfra 21-1 was highly significant. Salivary and serum Cyfra 21-1 showed significantly elevated levels in grade II OSCC compared to grade I histopathologically. Elevated levels of salivary Cyfra 21-1 were associated with recurrence in OSCC patients. Reverse operating curve constructed using 3 ng/ml as a cutoff for serum Cyfra 21-1 revealed the sensitivity and specificity to be 88 and 78.2 %, respectively. Using a cutoff value of 8.5 ng/ml for salivary Cyfra 21-1, the sensitivity was found to be 93.8 % and specificity 84.3 %. We advocate salivary Cyfra 21-1 as a better diagnostic marker over serum Cyfra 21-1 as well as a potential marker in the prognosis of OSCC.

Persistent fibrosis in the liver of choline-deficient and iron-supplemented L-amino acid-defined diet-induced nonalcoholic steatohepatitis rat due to continuing oxidative stress after choline supplementation.

PubMed

Takeuchi-Yorimoto, Ayano; Noto, Takahisa; Yamada, Atsushi; Miyamae, Yoichi; Oishi, Yuji; Matsumoto, Masahiro

2013-05-01

Nonalcoholic steatohepatitis (NASH) is characterized by combined pathology of steatosis, lobular inflammation, fibrosis, and hepatocellular degeneration, with systemic symptoms of diabetes or hyperlipidemia, all in the absence of alcohol abuse. Given the therapeutic importance and conflicting findings regarding the potential for healing the histopathologic features of NASH in humans, particularly fibrosis, we investigated the reversibility of NASH-related findings in Wistar rats fed a choline-deficient and iron-supplemented l-amino acid-defined (CDAA) diet for 12weeks, with a recovery period of 7weeks, during which the diets were switched to a choline-sufficient and iron-supplemented l-amino acid-defined (CSAA) one. Analysis showed that steatosis and inflammation were significantly resolved by the end of the recovery period, along with decreases in AST and ALT activities within 4weeks. In contrast, fibrosis remained even after the recovery period, to an extent similar to that in continuously CDAA-fed animals. Real-time reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemical investigations revealed that expression of some factors indicating oxidative stress (CYP2E1, 4-HNE, and iNOS) were elevated, whereas catalase and SOD1 were decreased, and a hypoxic state and CD34-positive neovascularization were evident even after the recovery period, although the fibrogenesis pathway by activated α-SMA-positive hepatic stellate cells via TGF-β and TIMPs decreased to the CSAA group level. In conclusion, persistent fibrosis was noted after the recovery period of 7weeks, possibly due to sustained hypoxia and oxidative stress supposedly caused by capillarization. Otherwise, histopathological features of steatosis and inflammation, as well as serum AST and ALT activities, were recovered. Copyright © 2013 Elsevier Inc. All rights reserved.

Molecular diagnostics of periodontitis.

PubMed

Korona-Głowniak, Izabela; Siwiec, Radosław; Berger, Marcin; Malm, Anna; Szymańska, Jolanta

2017-01-28

The microorganisms that form dental plaque are the main cause of periodontitis. Their identification and the understanding of the complex relationships and interactions that involve these microorganisms, environmental factors and the host's health status enable improvement in diagnostics and targeted therapy in patients with periodontitis. To this end, molecular diagnostics techniques (both techniques based on the polymerase chain reaction and those involving nucleic acid analysis via hybridization) come increasingly into use. On the basis of a literature review, the following methods are presented: polymerase chain reaction (PCR), real-time polymerase chain reaction (real-time PCR), 16S rRNA-encoding gene sequencing, checkerboard and reverse-capture checkerboard hybridization, microarrays, denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), as well as terminal restriction fragment length polymorphism (TRFLP) and next generation sequencing (NGS). The advantages and drawbacks of each method in the examination of periopathogens are indicated. The techniques listed above allow fast detection of even small quantities of pathogen present in diagnostic material and prove particularly useful to detect microorganisms that are difficult or impossible to grow in a laboratory.

Nucleoside reverse transcriptase inhibitors possess intrinsic anti-inflammatory activity

PubMed Central

Fowler, Benjamin J.; Gelfand, Bradley D.; Kim, Younghee; Kerur, Nagaraj; Tarallo, Valeria; Hirano, Yoshio; Amarnath, Shoba; Fowler, Daniel H.; Radwan, Marta; Young, Mark T.; Pittman, Keir; Kubes, Paul; Agarwal, Hitesh K.; Parang, Keykavous A.; Hinton, David R.; Bastos-Carvalho, Ana; Li, Shengjian; Yasuma, Tetsuhiro; Mizutani, Takeshi; Yasuma, Reo; Wright, Charles; Ambati, Jayakrishna

2014-01-01

Nucleoside reverse transcriptase inhibitors (NRTIs) are mainstay therapeutics for HIV that block retrovirus replication. Alu (an endogenous retroelement that also requires reverse transcriptase for its life cycle)-derived RNAs activate P2X7 and the NLRP3 inflammasome to cause cell death of the retinal pigment epithelium (RPE) in geographic atrophy, a type of age-related macular degeneration. We found that NRTIs inhibit P2X7-mediated NLRP3 inflammasome activation independent of reverse transcriptase inhibition. Multiple approved and clinically relevant NRTIs prevented caspase-1 activation, the effector of the NLRP3 inflammasome, induced by Alu RNA. NRTIs were efficacious in mouse models of geographic atrophy, choroidal neovascularization, graft-versus-host disease (GVHD), and sterile liver inflammation. Our findings suggest that NRTIs are ripe for drug repurposing in P2X7-driven diseases. PMID:25414314

Development and evaluation of a rapid recombinase polymerase amplification assay for detection of coxsackievirus A6.

PubMed

Wang, Kaifeng; Wu, Yue; Yin, Dan; Tang, Shixing; Hu, Guifang; He, Yaqing

2017-01-01

Coxsackievirus A6 (CV-A6) is an important pathogen causing hand, foot and mouth disease (HFMD). The aim of this study was to develop and evaluate a rapid real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for detection of CV-A6. The sensitivity of this assay was 202 copies/reaction, with 100 % specificity. Furthermore, this assay yielded consistent results comparable with a commercial qRT-PCR diagnostic kit. This assay is therefore potentially useful for surveillance of CV-A6 infections and outbreak control.

Development of recombinase polymerase amplification assays for the rapid detection of peste des petits ruminants virus.

PubMed

Zhang, Yongning; Wang, Jianchang; Zhang, Zhou; Mei, Lin; Wang, Jinfeng; Wu, Shaoqiang; Lin, Xiangmei

2018-04-01

Peste des petits ruminants (PPR) is a severe infectious disease of small ruminants caused by PPR virus (PPRV). Rapid and sensitive detection of PPRV is critical for controlling PPR. This report describes the development and evaluation of a conventional reverse transcription recombinase polymerase amplification (RT-RPA) assay and a real-time RT-RPA assay, targeting the PPRV N gene. Sensitivity analysis revealed that the conventional RT-RPA assay could detect 852 copies of standard PPRV RNA per reaction at 95% probability within 20 min at 41 °C, and the real-time RT-RPA assay could detect 103 copies of RNA molecules per reaction at 95% probability. Specificity analysis showed that both assays have no cross-reactivity with nucleic acid templates prepared from other selected viruses or common pathogens. Clinical evaluation using 162 ovine and hircine serum and nasal swab samples showed that the performance of both the real-time RT-RPA assay and the conventional RT-RPA assay were comparable to that of real-time RT-PCR. The overall agreements between real-time RT-PCR and real-time RT-RPA, and conventional RT-RPA were 99.4% (161/162) and 98.8% (160/162), respectively. The R 2 value of real-time RT-RPA and real-time RT-PCR was 0.900 by linear regression analysis. Our results suggest that both RT-RPA assays have a potential application in the rapid, sensitive and specific detection of PPRV. Copyright © 2018 Elsevier B.V. All rights reserved.

Continuous in vitro evolution of bacteriophage RNA polymerase promoters

NASA Technical Reports Server (NTRS)

Breaker, R. R.; Banerji, A.; Joyce, G. F.

1994-01-01

Rapid in vitro evolution of bacteriophage T7, T3, and SP6 RNA polymerase promoters was achieved by a method that allows continuous enrichment of DNAs that contain functional promoter elements. This method exploits the ability of a special class of nucleic acid molecules to replicate continuously in the presence of both a reverse transcriptase and a DNA-dependent RNA polymerase. Replication involves the synthesis of both RNA and cDNA intermediates. The cDNA strand contains an embedded promoter sequence, which becomes converted to a functional double-stranded promoter element, leading to the production of RNA transcripts. Synthetic cDNAs, including those that contain randomized promoter sequences, can be used to initiate the amplification cycle. However, only those cDNAs that contain functional promoter sequences are able to produce RNA transcripts. Furthermore, each RNA transcript encodes the RNA polymerase promoter sequence that was responsible for initiation of its own transcription. Thus, the population of amplifying molecules quickly becomes enriched for those templates that encode functional promoters. Optimal promoter sequences for phage T7, T3, and SP6 RNA polymerase were identified after a 2-h amplification reaction, initiated in each case with a pool of synthetic cDNAs encoding greater than 10(10) promoter sequence variants.

A general method to eliminate laboratory induced recombinants during massive, parallel sequencing of cDNA library.

PubMed

Waugh, Caryll; Cromer, Deborah; Grimm, Andrew; Chopra, Abha; Mallal, Simon; Davenport, Miles; Mak, Johnson

2015-04-09

Massive, parallel sequencing is a potent tool for dissecting the regulation of biological processes by revealing the dynamics of the cellular RNA profile under different conditions. Similarly, massive, parallel sequencing can be used to reveal the complexity of viral quasispecies that are often found in the RNA virus infected host. However, the production of cDNA libraries for next-generation sequencing (NGS) necessitates the reverse transcription of RNA into cDNA and the amplification of the cDNA template using PCR, which may introduce artefact in the form of phantom nucleic acids species that can bias the composition and interpretation of original RNA profiles. Using HIV as a model we have characterised the major sources of error during the conversion of viral RNA to cDNA, namely excess RNA template and the RNaseH activity of the polymerase enzyme, reverse transcriptase. In addition we have analysed the effect of PCR cycle on detection of recombinants and assessed the contribution of transfection of highly similar plasmid DNA to the formation of recombinant species during the production of our control viruses. We have identified RNA template concentrations, RNaseH activity of reverse transcriptase, and PCR conditions as key parameters that must be carefully optimised to minimise chimeric artefacts. Using our optimised RT-PCR conditions, in combination with our modified PCR amplification procedure, we have developed a reliable technique for accurate determination of RNA species using NGS technology.

Dietary fat intake promotes the development of hepatic steatosis independently from excess caloric consumption in a murine model.

PubMed

de Meijer, Vincent E; Le, Hau D; Meisel, Jonathan A; Akhavan Sharif, M Reza; Pan, Amy; Nosé, Vânia; Puder, Mark

2010-08-01

Nonalcoholic fatty liver disease results from overconsumption and is a significant and increasing cause of liver failure. The type of diet that is conducive to the development of this disease has not been established, and evidence-based treatment options are currently lacking. We hypothesized that the onset of hepatic steatosis is linked to the consumption of a diet with a high fat content, rather than related to excess caloric intake. In addition, we also hypothesized that fully manifested hepatic steatosis could be reversed by reducing the fat percentage in the diet of obese mice. C57BL/6J male mice were fed either a purified rodent diet containing 10% fat or a diet with 60% of calories derived from fat. A pair-feeding design was used to distinguish the effects of dietary fat content and caloric intake on dietary-induced hepatic lipid accumulation and associated injury. Livers were analyzed by quantitative reverse transcriptase polymerase chain reaction for lipid metabolism-related gene expression. After 9 weeks, mice on the 60%-fat diet exhibited more weight gain, insulin resistance, and hepatic steatosis compared with mice on a 10%-fat diet with equal caloric intake. Furthermore, mice with established metabolic syndrome at 9 weeks showed reversal of hepatic steatosis, insulin resistance, and obesity when switched to a 10%-fat diet for an additional 9 weeks, independent of caloric intake. Quantitative reverse transcriptase polymerase chain reaction revealed that transcripts related to both de novo lipogenesis and increased uptake of free fatty acids were significantly up-regulated in mice pair-fed a 60%-fat diet compared with 10%-fat-fed animals. Dietary fat content, independent from caloric intake, is a crucial factor in the development of hepatic steatosis, obesity, and insulin resistance in the C57BL/6J diet-induced obesity model caused by increased uptake of free fatty acids and de novo lipogenesis. In addition, once established, all these features of the metabolic syndrome can be successfully reversed after switching obese mice to a diet low in fat. Low-fat diets deserve attention in the investigation of a potential treatment of patients with nonalcoholic fatty liver disease. Copyright 2010 Elsevier Inc. All rights reserved.

Solid-Phase Nucleic Acid Sequence-Based Amplification and Length-Scale Effects during RNA Amplification.

PubMed

Ma, Youlong; Teng, Feiyue; Libera, Matthew

2018-06-05

Solid-phase oligonucleotide amplification is of interest because of possible applications to next-generation sequencing, multiplexed microarray-based detection, and cell-free synthetic biology. Its efficiency is, however, less than that of traditional liquid-phase amplification involving unconstrained primers and enzymes, and understanding how to optimize the solid-phase amplification process remains challenging. Here, we demonstrate the concept of solid-phase nucleic acid sequence-based amplification (SP-NASBA) and use it to study the effect of tethering density on amplification efficiency. SP-NASBA involves two enzymes, avian myeloblastosis virus reverse transcriptase (AMV-RT) and RNase H, to convert tethered forward and reverse primers into tethered double-stranded DNA (ds-DNA) bridges from which RNA - amplicons can be generated by a third enzyme, T7 RNA polymerase. We create microgels on silicon surfaces using electron-beam patterning of thin-film blends of hydroxyl-terminated and biotin-terminated poly(ethylene glycol) (PEG-OH, PEG-B). The tethering density is linearly related to the PEG-B concentration, and biotinylated primers and molecular beacon detection probes are tethered to streptavidin-activated microgels. While SP-NASBA is very efficient at low tethering densities, the efficiency decreases dramatically with increasing tethering density due to three effects: (a) a reduced hybridization efficiency of tethered molecular beacon detection probes; (b) a decrease in T7 RNA polymerase efficiency; (c) inhibition of T7 RNA polymerase activity by AMV-RT.

An Evolutionary/Biochemical Connection Between Promoter- and Primer-Dependent Polymerases Revealed by Selective Evolution of Ligands by Exponential Enrichment (SELEX).

PubMed

Fenstermacher, Katherine J; Achuthan, Vasudevan; Schneider, Thomas D; DeStefano, Jeffrey J

2018-01-16

DNA polymerases (DNAPs) recognize 3' recessed termini on duplex DNA and carry out nucleotide catalysis. Unlike promoter-specific RNA polymerases (RNAPs), no sequence specificity is required for binding or initiation of catalysis. Despite this, previous results indicate that viral reverse transcriptases bind much more tightly to DNA primers that mimic the polypurine tract. In the current report, primer sequences that bind with high affinity to Taq and Klenow polymerases were identified using a modified Selective Evolution of Ligands by Exponential Enrichment (SELEX) approach. Two Taq -specific primers that bound ∼10 (Taq1) and over 100 (Taq2) times more stably than controls to Taq were identified. Taq1 contained 8 nucleotides (5' -CACTAAAG-3') that matched the phage T3 RNAP "core" promoter. Both primers dramatically outcompeted primers with similar binding thermodynamics in PCR reactions. Similarly, exonuclease minus Klenow polymerase also selected a high affinity primer that contained a related core promoter sequence from phage T7 RNAP (5' -ACTATAG-3'). For both Taq and Klenow, even small modifications to the sequence resulted in large losses in binding affinity suggesting that binding was highly sequence-specific. The results are discussed in the context of possible effects on multi-primer (multiplex) PCR assays, molecular information theory, and the evolution of RNAPs and DNAPs. Importance This work further demonstrates that primer-dependent DNA polymerases can have strong sequence biases leading to dramatically tighter binding to specific sequences. These may be related to biological function, or be a consequences of the structural architecture of the enzyme. New sequence specificity for Taq and Klenow polymerases were uncovered and among them were sequences that contained the core promoter elements from T3 and T7 phage RNA polymerase promoters. This suggests the intriguing possibility that phage RNA polymerases exploited intrinsic binding affinities of ancestral DNA polymerases to develop their promotors. Conversely, DNA polymerases could have evolved from related RNA polymerases and retained the intrinsic binding preference despite there being no clear function for such a preference in DNA biology. Copyright © 2018 American Society for Microbiology.

In vitro stimulation with a strongly pulsed electromagnetic field on rat basophilic leukemia cells

NASA Astrophysics Data System (ADS)

Choi, J. W.; Shin, S. C.; Kim, S.; Chung, E. R.; Bang, J. H.; Cho, G. I.; Choi, S. D.; Park, Y. S.; Jang, T. S.; Yoo, Y. M.; Lee, S. S.; Hwang, D. G.

2010-05-01

In this study, the effects of pulsed electromagnetic field stimulation with a strong magnetic field on rat basophilic leukemia (RBL-2H3) cells were investigated to confirm the efficacy of the magnetic stimulator for biomedical applications. The maximum intensity of the magnetic field generated from the stimulation coil was 0.203 T, and the transition time was 126 μs. The oscillation time and frequency of the pulsed field were almost 0.1 ms and 8 kHz, respectively. The cell count as well as the mRNA expression and DNA sequence of the cytokine genes, such as the tumor necrosis factor-α (TNF-α) and interleukin-4 (IL-4), of the stimulated RBL-2H3 cells were analyzed with a hemocytometer and via reverse transcriptase polymerase chain reaction to determine the physiological response under a strong pulse field. After 12 h stimulation, cell death was observed at an increasing scale with the increase in the stimulation time. On the other hand, the cells that were stimulated for 10 min almost doubled as the interval time between the stimulations was extended.

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

Development and validation of a SYBR Green I-based real-time polymerase chain reaction method for detection of haptoglobin gene deletion in clinical materials.

PubMed

Soejima, Mikiko; Tsuchiya, Yuji; Egashira, Kouichi; Kawano, Hiroyuki; Sagawa, Kimitaka; Koda, Yoshiro

2010-06-01

Anhaptoglobinemic patients run the risk of severe anaphylactic transfusion reaction because they produce serum haptoglobin (Hp) antibodies. Being homozygous for the Hp gene deletion (HP(del)) is the only known cause of congenital anhaptoglobinemia, and clinical diagnosis of HP(del) before transfusion is important to prevent anaphylactic shock. We recently developed a 5'-nuclease (TaqMan) real-time polymerase chain reaction (PCR) method. A SYBR Green I-based duplex real-time PCR assay using two forward primers and a common reverse primer followed by melting curve analysis was developed to determine HP(del) zygosity in a single tube. In addition, to obviate initial DNA extraction, we examined serially diluted blood samples as PCR templates. Allelic discrimination of HP(del) yielded optimal results at blood sample dilutions of 1:64 to 1:1024. The results from 2231 blood samples were fully concordant with those obtained by the TaqMan-based real-time PCR method. The detection rate of the HP(del) allele by the SYBR Green I-based method is comparable with that using the TaqMan-based method. This method is readily applicable due to its low initial cost and analyzability using economical real-time PCR machines and is suitable for high-throughput analysis as an alternative method for allelic discrimination of HP(del).

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.

Comparison of the Diagnostic Value Between Real-Time Reverse Transcription-Polymerase Chain Reaction Assay and Histopathologic Examination in Sentinel Lymph Nodes for Patients With Gastric Carcinoma.

PubMed

Kwak, Yoonjin; Nam, Soo Kyung; Shin, Eun; Ahn, Sang-Hoon; Lee, Hee Eun; Park, Do Joong; Kim, Woo Ho; Kim, Hyung-Ho; Lee, Hye Seung

2016-05-01

Sentinel lymph node (SLN)-based diagnosis in gastric cancers has shown varied sensitivities and false-negative rates in several studies. Application of the reverse transcription-polymerase chain reaction (RT-PCR) in SLN diagnosis has recently been proposed. A total of 155 SLNs from 65 patients with cT1-2, N0 gastric cancer were examined. The histopathologic results were compared with results obtained by real-time RT-PCR for detecting molecular RNA (mRNA) of cytokeratin (CK)19, carcinoembryonic antigen (CEA), and CK20. The sensitivity and specificity of the multiple marker RT-PCR assay standardized against the results of the postoperative histological examination were 0.778 (95% confidence interval [CI], 0.577-0.914) and 0.781 (95% CI, 0.700-0.850), respectively. In comparison, the sensitivity and specificity of intraoperative diagnosis were 0.819 (95% CI, 0.619-0.937) and 1.000 (95% CI, 0.972-1.000), respectively. The positive predictive value of the multiple-marker RT-PCR assay was 0.355 (95% CI, 0.192-0.546) for predicting non-SLN metastasis, which was lower than that of intraoperative diagnosis (0.813, 95% CI, 0.544-0.960). The real-time RT-PCR assay could detect SLN metastasis in gastric cancer. However, the predictive value of the real-time RT-PCR assay was lower than that of precise histopathologic examination and did not outweigh that of our intraoperative SLN diagnosis. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Evaluation of the PrimerDesign™ genesig real-time reverse transcription-polymerase chain reaction assay and the INFINITI® Respiratory Viral Panel Plus assay for the detection of human metapneumovirus in Kuwait.

PubMed

Al-Turab, Mariam; Chehadeh, Wassim; Al-Mulla, Fahd; Al-Nakib, Widad

2012-04-01

Human metapneumovirus (hMPV) is a respiratory pathogen that was discovered in 2001 and is considered a major cause of both upper and lower respiratory tract infections. A sensitive, fast, and high-throughput diagnostic test is needed for the detection of hMPV that may assist in the clinical management as well as in the reduction of inappropriate therapy. Therefore, a comparison assessment was performed in this study between the PrimerDesign™ genesig real-time reverse transcription-polymerase chain reaction (RT-PCR) Assay and the INFINITI(®) Respiratory Viral Panel Plus Assay (RVP-Plus) for the detection of hMPV infection in patients with respiratory tract infections. A total of 200 respiratory samples were collected from 185 hospitalized patients, during the winter season in Kuwait. Of 185 patients, 10 (5.4%) were positive for hMPV RNA by the in-house RT-PCR assay, while 7 (4%) were positive for hMPV RNA by the real-time RT-PCR assay and 9 (5%) were positive for hMPV RNA by the INFINITI(®) RVP-Plus assay. The high incidence rate (60%) of hMPV infection was in January 2011. The sensitivity of the real-time RT-PCR and INFINITI(®) RVP-Plus assays was 70% and 90%, respectively, with specificity of 100% for both assays. hMPV types A and B could be identified in this study; however, discordant genotyping results were found between the direct sequencing method and the INFINITI(®) RVP-Plus assay in 33% of hMPV-positive patients. Copyright © 2012 Elsevier Inc. All rights reserved.

Proteomic identification of potential biomarkers for cervical squamous cell carcinoma and human papillomavirus infection.

PubMed

Qing, Song; Tulake, Wuniqiemu; Ru, Mingfang; Li, Xiaohong; Yuemaier, Reziwanguli; Lidifu, Dilare; Rouzibilali, Aierken; Hasimu, Axiangu; Yang, Yun; Rouziahong, Reziya; Upur, Halmurat; Abudula, Abulizi

2017-04-01

It is known that high-risk human papillomavirus infection is the main etiological factor in cervical carcinogenesis. However, human papillomavirus screening is not sufficient for early diagnosis. In this study, we aimed to identify potential biomarkers common to cervical carcinoma and human papillomavirus infection by proteomics for human papillomavirus-based early diagnosis and prognosis. To this end, we collected 76 cases of fresh cervical tissues and 116 cases of paraffin-embedded tissue slices, diagnosed as cervical squamous cell carcinoma, cervical intraepithelial neoplasia II-III, or normal cervix from ethnic Uighur and Han women. Human papillomavirus infection by eight oncogenic human papillomavirus types was detected in tissue DNA samples using a quantitative polymerase chain reaction. The protein profile of cervical specimens from human papillomavirus 16-positive squamous cell carcinoma and human papillomavirus-negative normal controls was analyzed by proteomics and bioinformatics. The expression of candidate proteins was further determined by quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry. We identified 67 proteins that were differentially expressed in human papillomavirus 16-positive squamous cell carcinoma compared to normal cervix. The quantitative reverse transcriptase-polymerase chain reaction analysis verified the upregulation of ASAH1, PCBP2, DDX5, MCM5, TAGLN2, hnRNPA1, ENO1, TYPH, CYC, and MCM4 in squamous cell carcinoma compared to normal cervix ( p < 0.05). In addition, the transcription of PCBP2, MCM5, hnRNPA1, TYPH, and CYC was also significantly increased in cervical intraepithelial neoplasia II-III compared to normal cervix. Immunohistochemistry staining further confirmed the overexpression of PCBP2, hnRNPA1, ASAH1, and DDX5 in squamous cell carcinoma and cervical intraepithelial neoplasia II-III compared to normal controls ( p < 0.05). Our data suggest that the expression of ASAH1, PCBP2, DDX5, and hnRNPA1, and possibly MCM4, MCM5, CYC, ENO1, and TYPH, is upregulated during cervical carcinogenesis and potentially associated with human papillomavirus infection. Further validation studies of the profile will contribute to establishing auxiliary diagnostic markers for human papillomavirus-based cancer prognosis.

The impact of HIV-1 reverse transcriptase polymorphisms on responses to first-line nonnucleoside reverse transcriptase inhibitor-based therapy in HIV-1-infected adults.

PubMed

Mackie, Nicola E; Dunn, David T; Dolling, David; Garvey, Lucy; Harrison, Linda; Fearnhill, Esther; Tilston, Peter; Sabin, Caroline; Geretti, Anna M

2013-09-10

HIV-1 genetic variability may influence antiretroviral therapy (ART) outcomes. The study aim was to determine the impact of polymorphisms in regions known to harbor major nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations (codons 90-108, 135-138, 179-190, 225-348) on virologic responses to first-line NNRTI-based ART. Reverse transcriptase sequences from ART-naive individuals who commenced efavirenz (EFV) or nevirapine (NVP) with at least two nucleos(t)ide reverse transcriptase inhibitors (NRTIs) without major drug resistance mutations were analyzed. The impact of polymorphisms on week 4 viral load decrease and time to virologic failure was measured over a median 97 weeks. Among 4528 patients, most were infected with HIV-1 subtype B (67%) and commenced EFV-based ART (84%). Overall, 2598 (57%) had at least one polymorphism, most frequently at codons 90, 98, 101, 103, 106, 135, 138, 179, and 238. Virologic failure rates were increased in patients with two (n = 597) or more than two (n = 72) polymorphisms [adjusted hazard ratio 1.43; 95% confidence interval (CI) 1.07-1.92; P = 0.016]. Polymorphisms associated with virologic failure occurred at codons 90 (mostly V90I), 98 (mostly A98S), and 103 (mostly K103R), with adjusted hazard ratios of 1.78 (1.15-2.73; P = 0.009), 1.55 (1.16-2.08; P = 0.003), and 1.75 (1.00-3.05: P = 0.049), respectively. Polymorphisms at codon 179, especially V179D/E/T, predicted reduced week 4 responses (P = 0.001) but not virologic failure. The occurrence of multiple polymorphisms, though uncommon, was associated with a small increase in the risk of NNRTI treatment failure; significant effects were seen with polymorphisms at codon 90, 98, and 103. The mechanisms underlying the slower suppression seen with V179D/E/T deserve further investigation.

Cytotoxic Effects of Nonionic Iodinated Contrast Agent on Human Adipose-derived Mesenchymal Stem Cells.

PubMed

Wu, Tao; Nie, Hai; Dietz, Allan B; Salek, David R; Smith, Jay; van Wijnen, Andre J; Qu, Wenchun

2018-05-31

Transplantation of mesenchymal stem cells (MSCs) is a promising therapy for degenerative spine conditions. However, cell therapy for painful spine degeneration presently requires use of contrast agents during fluoroscopy-guided injections and the effects of these agents on MSCs represents a gap in knowledge. To investigate the biological effects of contrast media that are co-injected with MSCs. Prospective observational study. Academic medical center. Patient-derived clinical-grade culture expanded MSCs. Iohexol(Omnipaque300) was reduced to 12.5%, 25%, 50% and 100% of the stock solution and incubated with MSCs for 30 minutes, 4 hours and 48 hours. We also used complete media and 12.5%, 25%, 50%, 100% of phosphate buffered saline (PBS) as control group. We examined cytotoxicity of Iohexol at different concentrations and exposure duration, as well as the potential for recovery over time. Cell counts, mitochondrial activity, and quantitative real time reverse-transcriptase polymerase chain reaction (qRT-PCR) of related genes were analyzed immediately after exposure (day 0) and after two days of exposure (day 2). Human MSCs exhibit a time- and concentration-dependent cytotoxic response to iodinated CM. A brief 30min exposure did not affect MSCs function and viability. However, extended treatment with iohexol for 4 hours at 50% or higher concentration had a significant impact on both viability and gene expression in MSCs. CM (Omnipaque300) is cytotoxic to MSCs in a time-and concentration-dependent manner. Hence, the concentration of CM that accompanies MSC injections should be carefully considered during mesenchymal stem cell therapy for disc degenerative diseases. Copyright © 2018 American Academy of Physical Medicine and Rehabilitation. Published by Elsevier Inc. All rights reserved.

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 Need for Development of New HIV-1 Reverse Transcriptase and Integrase Inhibitors in the Aftermath of Antiviral Drug Resistance

PubMed Central

Wainberg, Mark A.

2012-01-01

The use of highly active antiretroviral therapy (HAART) involves combinations of drugs to achieve maximal virological response and reduce the potential for the emergence of antiviral resistance. There are two broad classes of reverse transcriptase inhibitors, the nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs). Since the first classes of such compounds were developed, viral resistance against them has necessitated the continuous development of novel compounds within each class. This paper considers the NRTIs and NNRTIs currently in both preclinical and clinical development or approved for second line therapy and describes the patterns of resistance associated with their use, as well as the underlying mechanisms that have been described. Due to reasons of both affordability and availability, some reverse transcriptase inhibitors with low genetic barrier are more commonly used in resource-limited settings. Their use results to the emergence of specific patterns of antiviral resistance and so may require specific actions to preserve therapeutic options for patients in such settings. More recently, the advent of integrase strand transfer inhibitors represents another major step forward toward control of HIV infection, but these compounds are also susceptible to problems of HIV drug resistance. PMID:24278679

Template properties of mutagenic cytosine analogues in reverse transcription

PubMed Central

Suzuki, Tetsuya; Moriyama, Kei; Otsuka, Chie; Loakes, David; Negishi, Kazuo

2006-01-01

We have studied the mutagenic properties of ribonucleotide analogues by reverse transcription to understand their potential as antiretroviral agents by mutagenesis of the viral genome. The templating properties of nucleotide analogues including 6-(β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c](1,2)oxazin-7-one, N4-hydroxycytidine, N4-methoxycytidine, N4-methylcytidine and 4-semicarbazidocytidine, which have been reported to exhibit ambiguous base pairing properties, were examined. We have synthesized RNA templates using T3 RNA polymerase, and investigated the specificity of the incorporation of deoxyribonucleoside triphosphates opposite these cytidine analogues in RNA by HIV and AMV reverse transcriptases. Except for N4-methylcytidine, both enzymes incorporated both dAMP and dGMP opposite these analogues in RNA. This indicates that they would be highly mutagenic if present in viral RNA. To study the basis of the differences among the analogues in the incorporation ratios of dAMP to dGMP, we have carried out kinetic analysis of incorporation opposite the analogues at a defined position in RNA templates. In addition, we examined whether the triphosphates of these analogues were incorporated competitively into RNA by human RNA polymerase II. Our present data supports the view that these cytidine analogues are mutagenic when incorporated into RNA, and that they may therefore be considered as candidates for antiviral agents by causing mutations to the retroviral genome. PMID:17130163

Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays.

PubMed

Choudhary, Nandlal; Wei, G; Govindarajulu, A; Roy, Avijit; Li, Wenbin; Picton, Deric D; Nakhla, M K; Levy, L; Brlansky, R H

2015-11-01

Citrus leprosis virus C (CiLV-C), a causal agent of the leprosis disease in citrus, is mostly present in the South and Central America and spreading toward the North America. To enable better diagnosis and inhibit the further spread of this re-emerging virus a quantitative (q) real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is needed for early detection of CiLV-C when the virus is present in low titer in citrus leprosis samples. Using the genomic sequence of CiLV-C, specific primers and probe were designed and synthesized to amplify a 73 nt amplicon from the movement protein (MP) gene. A standard curve of the 73 nt amplicon MP gene was developed using known 10(10)-10(1) copies of in vitro synthesized RNA transcript to estimate the copy number of RNA transcript in the citrus leprosis samples. The one-step qRT-PCR detection assays for CiLV-C were determined to be 1000 times more sensitive when compared to the one-step conventional reverse transcription polymerase chain reaction (RT-PCR) CiLV-C detection method. To evaluate the quality of the total RNA extracts, NADH dehydrogenase gene specific primers (nad5) and probe were included in reactions as an internal control. The one-step qRT-PCR specificity was successfully validated by testing for the presence of CiLV-C in the total RNA extracts of the citrus leprosis samples collected from Belize, Costa Rica, Mexico and Panama. Implementation of the one-step qRT-PCR assays for CiLV-C diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of CiLV-C in countries where it is present and to prevent further dissemination into citrus growing countries where there is no report of CiLV-C presence. Published by Elsevier B.V.

Competitive RT-PCR Strategy for Quantitative Evaluation of the Expression of Tilapia (Oreochromis niloticus) Growth Hormone Receptor Type I

PubMed Central

2009-01-01

Quantization of gene expression requires that an accurate measurement of a specific transcript is made. In this paper, a quantitative reverse transcription-polymerase chain reaction (RT-PCR) by competition for tilapia growth hormone receptor type I is designed and validated. This experimental procedure was used to determine the abundance of growth hormone receptor type I transcript in different tilapia tissues. The results obtained with this developed competitive RT-PCR were similar to real-time PCR results reported recently. This protocol provides a reliable alternative, but less expensive than real-time PCR to quantify specific genes. PMID:19495916

Survival of influenza virus on hands and fomites in community and laboratory settings.

PubMed

Mukherjee, Dhritiman V; Cohen, Bevin; Bovino, Mary Ellen; Desai, Shailesh; Whittier, Susan; Larson, Elaine L

2012-09-01

Transmission dynamics modeling provides a practical method for virtual evaluation of the impact of public health interventions in response to prospective influenza pandemics and also may help determine the relative contribution of different modes of transmission to overall infection rates. Accurate estimates of longevity for all forms of viral particles are needed for such models to be useful. We conducted a time course study to determine the viability and longevity of H1N1 virus on naturally contaminated hands and household surfaces of 20 individuals with laboratory-confirmed infection. Participants coughed or sneezed into their hands, which were sampled immediately and again after 5, 10, and 30 minutes. Samples also were obtained from household surfaces handled by the participants immediately after coughing/sneezing. Clinically obtained H1N1 isolates were used to assess the viability and longevity of the virus on various artificially inoculated common household surfaces and human hands in a controlled laboratory setting. Viral detection was achieved by culture and real-time reverse-transcriptase polymerase chain reaction. The results suggest that H1N1 does not survive long on naturally contaminated skin and fomites, and that secretions deposited on hands by coughing or sneezing have a concentration of <2.15 × 10 to 2.94 × 10 TCID(50)/mL. These data can be used to estimate the relative contribution of direct and indirect contact transmission on overall infection rates. Copyright © 2012 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

Mechanisms involved in enhancement of the expression and function of aggrecanases by hyaluronan oligosaccharides

PubMed Central

Ariyoshi, Wataru; Takahashi, Nobunori; Hida, Daisuke; Knudson, Cheryl B.; Knudson, Warren

2011-01-01

Objective Small hyaluronan (HA) oligosaccharides serve as competitive receptor antagonists to displace HA from the cell surface and induce cell signaling events. In articular chondrocytes this cell signaling is mediated by the HA receptor CD44 and induces stimulation of genes involved in matrix degradation such as matrix metalloproteinases as well as matrix repair genes including collagen type II, aggrecan and HA synthase-2. The objective of this study was to determine changes in the expression and function of aggrecanases after disruption of chondrocyte CD44-HA interactions. Methods Bovine articular chondrocytes or bovine cartilage tissue were pre-treated with a variety of inhibitors of major signaling pathways prior to the addition of HA oligosaccharides. Changes in aggrecanase were monitored by real time reverse transcriptase-polymerase chain reaction and western blot analysis of ADAMTS4, ADAMTS5 and aggrecan proteolytic fragments. To test the interactions between ADAMTS4 and MT4-MMP, protein lysates purified from stimulated chondrocytes were subjected to co-immunoprecipitation. Results Disruption of chondrocyte CD44-HA interactions with HA oligosaccharides induced the transcription of ADAMTS4 and ADAMTS5 in time- and dose-dependent manner. The association of GPI-anchored MT4-MMP with ADAMTS4 was also induced in articular chondrocytes by HA oligosaccharides. Inhibition of the NF-κB pathway blocked HA oligosaccharides-mediated stimulation of aggrecanases. Conclusions Disruptive changes in chondrocyte-matrix interactions by HA oligosaccharides induce matrix degradation and elevate aggrecanases via the activation of the NF-κB signaling pathway. PMID:21905012

High false-negative rate of anti-HCV among Egyptian patients on regular hemodialysis.

PubMed

El-Sherif, Assem; Elbahrawy, Ashraf; Aboelfotoh, Atef; Abdelkarim, Magdy; Saied Mohammad, Abdel-Gawad; Abdallah, Abdallah Mahmoud; Mostafa, Sadek; Elmestikawy, Amr; Elwassief, Ahmed; Salah, Mohamed; Abdelbaseer, Mohamed Ali; Abdelwahab, Kouka Saadeldin

2012-07-01

Routine serological testing for hepatitis C virus (HCV) infection among hemodialysis (HD) patients is currently recommended. A dilemma existed on the value of serology because some investigators reported a high rate of false-negative serologic testing. In this study, we aimed to detect the false-negative rate of anti-HCV among Egyptian HD patients. Seventy-eight HD patients, negative for anti-HCV, anti-HIV, and hepatitis B surface antigen, were tested for HCV RNA by reverse transcriptase polymerase chain reaction (RT-PCR). In the next step, the viral load was quantified by real-time PCR in RT-PCR-positive patients. Risk factors for HCV infection, as well as clinical and biochemical indicators of liver disease, were compared between false-negative and true-negative anti-HCV HD patients. The frequency of false-negative anti-HCV was 17.9%. Frequency of blood transfusion, duration of HD, dialysis at multiple centers, and diabetes mellitus were not identified as risk factors for HCV infection. The frequency of false-negative results had a linear relation to the prevalence of HCV infection in the HD units. Timely identification of HCV within dialysis units is needed in order to lower the risk of HCV spread within the HD units. The high false-negative rate of anti-HCV among HD patients in our study justifies testing of a large scale of patients for precious assessment of effectiveness of nucleic acid amplification technology testing in screening HD patient. © 2012 The Authors. Hemodialysis International © 2012 International Society for Hemodialysis.

Relationships between infection, dissemination, and transmission of West Nile virus RNA in Culex pipiens quinquefasciatus (Diptera: Culicidae).

PubMed

Richards, Stephanie L; Anderson, Sheri L; Lord, Cynthia C; Smartt, Chelsea T; Tabachnick, Walter J

2012-01-01

Culex pipiens quinquefasciatus Say fed blood containing 6.8 +/- 0.3 logs (mean +/- SE) plaque-forming units of West Nile virus (WNV)/ml were maintained at 28 degrees C for incubation periods (IP) of 7, 14, or 21 d. Several attributes of vector competence were determined at each IP using quantitative real-time reverse transcriptase polymerase chain reaction to estimate plaque forming unit equivalents including: infection rate (WNV-positive abdomens), dissemination rate (WNV-positive legs or thoraces), combined dissemination rate (WNV-positive legs and thoraces), transmission rate (WNV-positive saliva), and WNV titers in abdomens, legs, thoraces, and saliva. Each rate increased or was equivalent with increasing IP. Mosquitoes transmitting WNV in saliva also had significantly higher IP-dependent WNV titers in abdomens, legs, and thoraces. Titers of WNV in abdomens were significantly correlated with titers in legs and thoraces, but the degree of association changed with IP. However, titers of abdomens, legs, and thoraces were not correlated with WNV presence or titer in the saliva. The results show that WNV presence or titer in the saliva of infected Cx. p. quinquefasciatus was not directly influenced by processes involved in WNV replication in other tissues. The processes controlling midgut infection and escape are, in part, independent from the infection processes in other tissues. The relationship between infection, dissemination, and transmission varied over time. The infection and replication of WNV in different tissues is likely influenced by different barriers encountered during the extrinsic incubation period. The significance of these observations for understanding vector competence is discussed.

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.

Point-of-care testing system enabling 30 min detection of influenza genes.

PubMed

Abe, Tomoteru; Segawa, Yuji; Watanabe, Hidetoshi; Yotoriyama, Tasuku; Kai, Shinichi; Yasuda, Akio; Shimizu, Norio; Tojo, Naoko

2011-03-21

We developed a portable and easy-to-use nucleic acid amplification test (NAT) system for use in point-of-care testing (POCT). The system shows sensitivity that is sufficiently higher than that of the currently available rapid diagnostic kit and is comparable to that of real-time reverse transcription polymerase chain reaction (RT-PCR) for influenza testing. This journal is © The Royal Society of Chemistry 2011

HuR interacts with human immunodeficiency virus type 1 reverse transcriptase, and modulates reverse transcription in infected cells

PubMed Central

Lemay, Julie; Maidou-Peindara, Priscilla; Bader, Thomas; Ennifar, Eric; Rain, Jean-Christophe; Benarous, Richard; Liu, Lang Xia

2008-01-01

Reverse transcription of the genetic material of human immunodeficiency virus type 1 (HIV-1) is a critical step in the replication cycle of this virus. This process, catalyzed by reverse transcriptase (RT), is well characterized at the biochemical level. However, in infected cells, reverse transcription occurs in a multiprotein complex – the reverse transcription complex (RTC) – consisting of viral genomic RNA associated with viral proteins (including RT) and, presumably, as yet uncharacterized cellular proteins. Very little is known about the cellular proteins interacting with the RTC, and with reverse transcriptase in particular. We report here that HIV-1 reverse transcription is affected by the levels of a nucleocytoplasmic shuttling protein – the RNA-binding protein HuR. A direct protein-protein interaction between RT and HuR was observed in a yeast two-hybrid screen and confirmed in vitro by homogenous time-resolved fluorescence (HTRF). We mapped the domain interacting with HuR to the RNAse H domain of RT, and the binding domain for RT to the C-terminus of HuR, partially overlapping the third RRM RNA-binding domain of HuR. HuR silencing with specific siRNAs greatly impaired early and late steps of reverse transcription, significantly inhibiting HIV-1 infection. Moreover, by mutagenesis and immunoprecipitation studies, we could not detect the binding of HuR to the viral RNA. These results suggest that HuR may be involved in and may modulate the reverse transcription reaction of HIV-1, by an as yet unknown mechanism involving a protein-protein interaction with HIV-1 RT. PMID:18544151

Isolation of Ganjam virus from ticks collected off domestic animals around Pune, Maharashtra, India.

PubMed

Joshi, M V; Geevarghese, G; Joshi, G D; Ghodke, Y S; Mourya, D T; Mishra, A C

2005-03-01

Studies on viruses of zoonotic importance in certain villages around Pune were undertaken between December 2000 and January 2002. A total of 1,138 adult ticks belonging to six different species were collected off domestic animals and processed for virus isolation. Six virus isolates were obtained. All six isolates were identified as Ganjam virus by Quick Complement Fixation test and reverse transcriptase-polymerase chain reaction using RNA nucleocapsid gene amplification. Five isolates were from the pools of adult Hemaphysalis intermedia ticks, and one isolate was from a pool of adult Rhipecephalus hemaphysaloides. This is the first report of isolation of Ganjam virus from Maharashtra state of India.

Microaspiration of esophageal gland cells and cDNA library construction for identifying parasitism genes of plant-parasitic nematodes.

PubMed

Hussey, Richard S; Huang, Guozhong; Allen, Rex

2011-01-01

Identifying parasitism genes encoding proteins secreted from a plant-parasitic nematode's esophageal gland cells and injected through its stylet into plant tissue is the key to understanding the molecular basis of nematode parasitism of plants. Parasitism genes have been cloned by directly microaspirating the cytoplasm from the esophageal gland cells of different parasitic stages of cyst or root-knot nematodes to provide mRNA to create a gland cell-specific cDNA library by long-distance reverse-transcriptase polymerase chain reaction. cDNA clones are sequenced and deduced protein sequences with a signal peptide for secretion are identified for high-throughput in situ hybridization to confirm gland-specific expression.

Coronavirus 229E-related pneumonia in immunocompromised patients.

PubMed

Pene, Frédéric; Merlat, Annabelle; Vabret, Astrid; Rozenberg, Flore; Buzyn, Agnès; Dreyfus, François; Cariou, Alain; Freymuth, François; Lebon, Pierre

2003-10-01

Coronaviruses strains 229E and OC43 have been associated with various respiratory illnesses ranging from the self-resolving common cold to severe pneumonia. Although chronic underlying conditions are major determinants of severe respiratory virus infections, few data about coronavirus-related pneumonia in immunocompromised patients are available. Here we report 2 well-documented cases of pneumonia related to coronavirus 229E, each with a different clinical presentation. Diagnosis was made on the basis of viral culture and electron microscopy findings that exhibited typical crown-like particles and through amplification of the viral genome by reverse transcriptase-polymerase chain reaction. On the basis of this report, coronaviruses should be considered as potential causative microorganisms of pneumonia in immunocompromised patients.

West Nile Flavivirus Polioencephalomyelitis in a harbor seal (Phoca vitulina).

PubMed

Del Piero, F; Stremme, D W; Habecker, P L; Cantile, C

2006-01-01

A 12-year-old male harbor seal presented with progressive signs of neurologic dysfunction including head tremors, muzzle twitching, clonic spasms, and weakness. Lesions included polioencephalomyelitis with glial nodules, spheroids, neuronophagia, ring hemorrhages, and a few neutrophils. Neurons, fibers, and glial nodules were multifocally colonized with intracytoplasmic West Nile flavivirus antigens that were demonstrated using indirect immunohistochemical analysis. Flavivirus on cultured cells also was isolated and was identified by use of monoclonal antibodies and reverse transcriptase-polymerase chain reaction analysis. Clinical signs of disease and lesion morphology and distribution were similar to those of equine West Nile virus infection. Similar to horses, alpacas, humans, dogs, and reptiles, seals can be dead-end hosts of West Nile virus.

BIRC6/Apollon gene expression in childhood acute leukemia: impact on therapeutic response and prognosis.

PubMed

Ismail, Eman Abdel Rahman; Mahmoud, Hanan Mohamed; Tawfik, Lamis Mohamed; Habashy, Deena Mohamed; Adly, Amira Abdel Moneam; El-Sherif, Nayera Hazaa; Abdelwahab, Mahmoud Ahmed

2012-02-01

Although BIRC6/Apollon seems to play a critical role as an antiapoptotic regulator, its clinical relevance in acute leukemia remains largely elusive. Therefore, we aimed to investigate BIRC6 gene expression in childhood acute leukemia in relation to clinicopathological characteristics at presentation, therapeutic response, and prognosis. BIRC6 expression level was assessed in 75 children with acute leukemia; 30 patients with acute myeloblastic leukemia (AML) and 45 patients with acute lymphoblastic leukemia (ALL) using real-time quantitative reverse transcriptase-polymerase chain reaction. The median level of BIRC6 expression did not differ significantly between AML and ALL patients. BIRC6 expression level was higher in patients with AML and ALL with extramedullary involvement, white blood cell (WBC) count ≥ 10 × 10(9) /L, and unfavorable cytogenetics at diagnosis. BIRC6 gene expression was higher in patients with unfavorable response to therapy at day 14, those who developed relapse or died in both leukemic groups. The best cutoff value of BIRC6 to predict therapeutic response and disease outcome was determined. AML and ALL patients with BIRC6 overexpression had significantly shorter overall and disease free survivals. This is the first report to study BIRC6 gene in pediatric ALL. Our results suggested that BIRC6 gene expression could be considered as an adverse risk factor in childhood acute leukemia and, hence, could be used to guide therapeutic regimens. © 2012 John Wiley & Sons A/S.

Liarozole inhibits transforming growth factor-β3–mediated extracellular matrix formation in human three-dimensional leiomyoma cultures

PubMed Central

Levy, Gary; Malik, Minnie; Britten, Joy; Gilden, Melissa; Segars, James; Catherino, William H.

2014-01-01

Objective To investigate the impact of liarozole on transforming growth factor-β3 (TGF-β3) expression, TGF-β3 controlled profibrotic cytokines, and extracellular matrix formation in a three-dimensional (3D) leiomyoma model system. Design Molecular and immunohistochemical analysis in a cell line evaluated in a three-dimensional culture. Setting Laboratory study. Patient(s) None. Intervention(s) Treatment of leiomyoma and myometrial cells with liarozole and TGF-β3 in a three-dimensional culture system. Main Outcome Measure(s) Quantitative real-time reverse-transcriptase polymerase chain reaction and Western blotting to assess fold gene and protein expression of TGF-β3 and TGF-β3 regulated fibrotic cytokines: collagen 1A1 (COL1A1), fibronectin, and versican before and after treatment with liarozole, and confirmatory immunohistochemical stains of treated three-dimensional cultures. Result(s) Both TGF-β3 gene and protein expression were elevated in leiomyoma cells compared with myometrium in two-dimensional and 3D cultures. Treatment with liarozole decreased TGF-β3 gene and protein expression. Extracellular matrix components versican, COL1A1, and fibronectin were also decreased by liarozole treatment in 3D cultures. Treatment of 3D cultures with TGF-β3 increased gene expression and protein production of COL1A1, fibronectin, and versican. Conclusion(s) Liarozole decreased TGF-β3 and TGF-β3–mediated extracellular matrix expression in a 3D uterine leiomyoma culture system. PMID:24825427

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.

Three gastroenteritis outbreaks in South Korea caused by the consumption of kimchi tainted by norovirus GI.4.

PubMed

Park, Ji-Hyuk; Jung, Sunyoung; Shin, Jaeseung; Lee, Jeong Su; Joo, In Sun; Lee, Deog-Yong

2015-03-01

In April 2013, outbreaks of acute gastroenteritis were reported at three schools in Jeonju, South Korea. Epidemiological investigations were performed to characterize the outbreaks and implement appropriate control measures. Retrospective cohort studies were performed at these schools. Stool and environmental samples were collected for bacterial and viral assessment. A food supplier of the schools, food company X, was inspected, and samples of cabbage kimchi and groundwater were tested for norovirus by real-time reverse-transcriptase polymerase chain reaction. The relatedness of the detected norovirus strains was evaluated by phylogenetic analysis. Of the 3347 questionnaires distributed, 631 (attack rate: 18.9%) met the case definition. Among the consumed food items, kimchi products (i.e., cabbage and fresh kimchi) were significantly associated with illness. The kimchi products were supplied by food company X. Among stool samples from 95 students and 34 food handlers at the 3 schools, 39 (41.1%) and 14 (41.2%) samples, respectively, were positive for norovirus. The samples of groundwater and cabbage kimchi at food company X were positive for norovirus. The predominant genotype of norovirus detected in the patient, groundwater, and cabbage kimchi samples, GI.4, shared high nucleotide identity. Kimchi products tainted with norovirus GI.4 from contaminated groundwater were linked to the acute gastroenteritis outbreaks. Therefore, kimchi manufacturers in South Korea should apply chlorine disinfection when using groundwater. Moreover, more stringent sanitation requirements and strict regulations for food companies are recommended.

Pluronic-based micelle encapsulation potentiates myricetin-induced cytotoxicity in human glioblastoma cells

PubMed Central

Tang, Xiang-Jun; Huang, Kuan-Ming; Gui, Hui; Wang, Jun-Jie; Lu, Jun-Ti; Dai, Long-Jun; Zhang, Li; Wang, Gang

2016-01-01

As one of the natural herbal flavonoids, myricetin has attracted much research interest, mainly owing to its remarkable anticancer properties and negligible side effects. It holds great potential to be developed as an ideal anticancer drug through improving its bioavailability. This study was performed to investigate the effects of Pluronic-based micelle encapsulation on myricetin-induced cytotoxicity and the mechanisms underlying its anticancer properties in human glioblastoma cells. Cell viability was assessed using a methylthiazol tetrazolium assay and a real-time cell analyzer. Immunoblotting and quantitative reverse transcriptase polymerase chain reaction techniques were used for determining the expression levels of related molecules in protein and mRNA. The results indicated that myricetin-induced cytotoxicity was highly potentiated by the encapsulation of myricetin. Mitochondrial apoptotic pathway was demonstrated to be involved in myricetin-induced glioblastoma cell death. The epidermal growth factor receptor (EGFR)/PI3K/Akt pathway located in the plasma membrane and cytosol and the RAS-ERK pathway located in mitochondria served as upstream and downstream targets, respectively, in myricetin-induced apoptosis. MiR-21 inhibitors interrupted the expression of EGFR, p-Akt, and K-Ras in the same fashion as myricetin-loaded mixed micelles (MYR-MCs) and miR-21 expression were dose-dependently inhibited by MYR-MCs, indicating the interaction of miR-21 with MYR-MCs. This study provided evidence supportive of further development of MYR-MC formulation for preferentially targeting mitochondria of glioblastoma cells. PMID:27757032

Gene expression profiling analysis of the effects of low-intensity pulsed ultrasound on induced pluripotent stem cell-derived neural crest stem cells.

PubMed

Xia, Bin; Zou, Yang; Xu, Zhiling; Lv, Yonggang

2017-11-01

Low-intensity pulsed ultrasound (LIPUS) is a noninvasive technique that has been shown to affect cell proliferation, migration, and differentiation and promote the regeneration of damaged peripheral nerve. Our previous studies had proved that LIPUS can significantly promote the neural differentiation of induced pluripotent stem cell-derived neural crest stem cells (iPSCs-NCSCs) and enhance the repair of rat-transected sciatic nerve. To further explore the underlying mechanisms of LIPUS treatment of iPSCs-NCSCs, this study reported the gene expression profiling analysis of iPSCs-NCSCs before and after LIPUS treatment using the RNA-sequencing (RNA-Seq) method. It was found that expression of 76 genes of iPSCs-NCSCs cultured in a serum-free neural induction medium and expression of 21 genes of iPSCs-NCSCs cultured in a neuronal differentiation medium were significantly changed by LIPUS treatment. The differentially expressed genes are related to angiogenesis, nervous system activity and functions, cell activities, and so on. The RNA-seq results were further verified by a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). High correlation was observed between the results obtained from qRT-PCR and RNA-Seq. This study presented new information on the global gene expression patterns of iPSCs-NCSCs after LIPUS treatment and may expand the understanding of the complex molecular mechanism of LIPUS treatment of iPSCs-NCSCs. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

Characterization of xanthine dehydrogenase and aldehyde oxidase of Marsupenaeus japonicus and their response to microbial pathogen.

PubMed

Okamura, Yo; Inada, Mari; Elshopakey, Gehad Elsaid; Itami, Toshiaki

2018-05-16

Reactive oxygen species (ROS) play key roles in many physiological processes. In particular, the sterilization mechanism of bacteria using ROS in macrophages is a very important function for biological defense. Xanthine dehydrogenase (XDH) and aldehyde oxidase (AOX), members of the molybdo-flavoenzyme subfamily, are known to generate ROS. Although these enzymes occur in many vertebrates, some insects, and plants, little research has been conducted on XDHs and AOXs in crustaceans. Here, we cloned the entire cDNA sequences of XDH (MjXDH: 4328 bp) and AOX (MjAOX: 4425 bp) from Marsupenaeus japonicus (kuruma shrimp) using reverse transcriptase-polymerase chain reaction (RT-PCR) and random amplification of cDNA ends (RACE). Quantitative real-time RT-PCR transcriptional analysis revealed that MjXDH mRNA is highly expressed in heart and stomach tissues, whereas MjAOX mRNA is highly expressed in the lymphoid organ and intestinal tissues. Furthermore, expression of MjAOX was determined to be up-regulated in the lymphoid organ in response to Vibrio penaeicida at 48 and 72 h after injection; in contrast, hydrogen peroxide (H 2 O 2 ) concentrations increased significantly at 6, 12, 48, and 72 h after injection with white spot syndrome virus (WSSV) and at 72 h after injection with V. penaeicida. To the best of our knowledge, this study is the first to have identified and cloned XDH and AOX from a crustacean species.

Angiogenesis and Vascular Architecture in Pheochromocytomas

PubMed Central

Favier, Judith; Plouin, Pierre-François; Corvol, Pierre; Gasc, Jean-Marie

2002-01-01

Angiogenesis is a critical step in tumor growth and metastatic invasion. We here report the study of the vascular status of 10 benign and 9 malignant pheochromocytomas. We examined the vascular architecture after immunostaining endothelial cells (CD34) and vascular smooth muscle cells (α-actin) and identified a vascular pattern characteristic of malignant lesions. To define a gene expression profile indicative of the invasive phenotype, we studied by in situ hybridization the expression of genes encoding several pro- and anti-angiogenic factors [hypoxia-inducible factor (HIF-1α), EPAS1, vascular endothelial growth factor (VEGF), VEGF receptors, angiopoietins and their receptor Tie2, five genes of the endothelin system, and thrombospondin 1]. A semiquantitative evaluation of the labeling revealed an induction of genes encoding EPAS1, VEGF, VEGFR-1, VEGFR-2, endothelin receptor, type B (ETB) and endothelin receptor, type A (ETA) in malignant pheochromocytomas as compared to benign tumors. These differences were observed in tumor cells, in endothelial cells, or in both. Quantification by real-time reverse-transcriptase polymerase chain reaction showed an increase of EPAS1, VEGF, and ETB transcripts of 4.5-, 3.5-, and 10-fold, respectively, in malignant versus benign tumors. Furthermore, we observed a strong correlation between the expression of EPAS1 and VEGF in tumoral tissue and between EPAS1 and ETB in endothelial cells. Altogether, our observations show that analysis of angiogenesis provides promising new criteria for the diagnosis of malignant pheochromocytomas. PMID:12368197

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

[A retrospective study of one case of human infection by the highly pathogenic avian influenza A (H5N1)].

PubMed

Zhou, Chao; Fang, Ping; Liu, You-ning; Hu, Bin; Ding, Hong-mei; Xu, Xiao-ling; Wu, Hao; Wang, Jin; Lin, Lin; Pan, Hua; Wu, Tong-sheng; Song, You-liang

2006-01-01

To describe the clinical features of the infection caused by the highly pathogenic avian influenza A (H(5)N(1)). A previously healthy 24 year old woman presented to our hospital on November 7, 2005. She was confirmed to be an H(5)N(1) infected case after death. The clinical, radiological and epidemiological data were analyzed. The patient had a history of direct contact with diseased and dead poultry (chicken and duck). The disease course was 10 days from onset of illness to death, and fever preceded dyspnea by 5 days. On admission, the striking characteristics were acute community-acquired pneumonia (CAP) and acute respiratory distress syndrome (ARDS), and the major radiographic abnormalities included extensive infiltration bilaterally, focal consolidation and air bronchograms. The radiographic and clinical deterioration was rapid, and the patient died in less than 3 days after hospitalization. The diagnosis of influenza A (H(5)N(1)) was confirmed by means of reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR on specimens of the lower respiratory tract, performed by Chinese Center for Disease Control. The postmortem examination showed bronchial hyperemia, extensive consolidation, serous cavity effusions, disseminated intravascular coagulation (DIC) and multiple organ failure (MOF). Human infection by the highly pathogenic avian influenza A (H(5)N(1)) is a fatal communicable disease. Information of avian influenza A (H(5)N(1)) virus, more attention to the epidemiologic data, and early intervention are critical in reducing the mortality.

Gender-related difference in altered gene expression of a sterol regulatory element binding protein, SREBP-2, by lead nitrate in rats: correlation with development of hypercholesterolemia.

PubMed

Kojima, Misaki; Degawa, Masakuni

2006-01-01

Changes in gene expression levels of hepatic sterol regulatory element binding protein-2 (SREBP-2) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) after a single i.v. injection of lead nitrate (LN, 100 micromol kg(-1) body weight) were examined comparatively by real time reverse transcriptase-polymerase chain reaction (RT-PCR) in male and female rats. Significant increases in the gene expression level of SREBP-2, a transcription factor for the HMGR gene, occurred at 6-12 h in male and at 24-36 h in female rats after LN-treatment. The gene expression level of HMGR, a rate-limiting enzyme for cholesterol biosynthesis, significantly increased at 3-48 h in male rats and 12-48 h in female rats. Subsequently, significant increases in the amount of hepatic total cholesterol in male and female rats were also observed at 3-48 h and 24-48 h, respectively. The present findings demonstrate that increases in gene expressions of hepatic SREBP-2 and HMGR and the amount of hepatic total cholesterol by LN occur earlier in male rats than in the females, and that increases in the gene expression level of HMGR and the amount of hepatic total cholesterol occur prior to the increase in the gene expression level of SREBP-2 in either sex of rats. Copyright (c) 2006 John Wiley & Sons, Ltd.

Effects of virus dose and extrinsic incubation temperature on vector competence of Culex nigripalpus (Diptera: Culicidae) for St. Louis encephalitis virus.

PubMed

Richards, Stephanie L; Anderson, Sheri L; Lord, Cynthia C; Tabachnick, Walter J

2012-11-01

Culex nigripalpus Theobald is a primary vector of St. Louis encephalitis virus in the southeastern United States. Cx. nigripalpus females were fed blood containing a low (4.0 +/- 0.01 log10 plaque-forming unit equivalents (PFUeq) /ml) or high (4.7 +/- 0.1 log10 PFUeq/ml) St. Louis encephalitis virus dose and maintained at extrinsic incubation temperatures (EIT) of 25 or 28 degrees C for 12 d. Vector competence was measured via quantitative real-time reverse transcriptase polymerase chain reaction to estimate PFUeq using rates of infection, dissemination, and transmission. There were no differences in infection rates between the two EITs at either dose. The low dose had higher infection rates at both EITs. Dissemination rates were significantly higher at 28 degrees C compared with 25 degrees C at both doses. Virus transmission was observed (<7%) only at 28 degrees C for both doses. The virus titer in body tissues was greater at 28 degrees C compared with 25 degrees C at both doses. The difference between the EITs was greater at the low dose, resulting in a higher titer for the low dose than the high dose at 28 degrees C. Virus titers in leg tissues were greater in mosquitoes fed the high versus low dose, but were not influenced by EIT. Further investigations using a variety of environmental and biological factors would be useful in exploring the complexity of vector competence.

Effects of Virus Dose and Extrinsic Incubation Temperature on Vector Competence of Culex nigripalpus (Diptera: Culicidae) for St. Louis Encephalitis Virus

PubMed Central

RICHARDS, STEPHANIE L.; ANDERSON, SHERI L.; LORD, CYNTHIA C.; TABACHNICK, WALTER J.

2013-01-01

Culex nigripalpus Theobald is a primary vector of St. Louis encephalitis virus in the southeastern United States. Cx. nigripalpus females were fed blood containing a low (4.0 ± 0.01 log10 plaque-forming unit equivalents (PFUeq)/ml) or high (4.7 ± 0.1 log10 PFUeq/ml) St. Louis encephalitis virus dose and maintained at extrinsic incubation temperatures (EIT) of 25 or 28°C for 12 d. Vector competence was measured via quantitative real-time reverse transcriptase polymerase chain reaction to estimate PFUeq using rates of infection, dissemination, and transmission. There were no differences in infection rates between the two EITs at either dose. The low dose had higher infection rates at both EITs. Dissemination rates were significantly higher at 28°C compared with 25°C at both doses. Virus transmission was observed (<7%) only at 28°C for both doses. The virus titer in body tissues was greater at 28°C compared with 25°C at both doses. The difference between the EITs was greater at the low dose, resulting in a higher titer for the low dose than the high dose at 28°C. Virus titers in leg tissues were greater in mosquitoes fed the high versus low dose, but were not influenced by EIT. Further investigations using a variety of environmental and biological factors would be useful in exploring the complexity of vector competence. PMID:23270182

Thromboembolic events in patients with severe pandemic influenza A/H1N1.

PubMed

Avnon, Lone Sølling; Munteanu, Daniela; Smoliakov, Alexander; Jotkowitz, Alan; Barski, Leonid

2015-10-01

The 2009 pandemic influenza A/H1N1 developed as a novel swine influenza which caused more diseases among younger age groups than in the elderly. Severe hypoxemic respiratory failure from A/H1N1 pneumonia resulted in an increased need for ICU beds. Several risk groups were identified that were at a higher risk for adverse outcomes. Pregnant women were a particularly vulnerable group of patients The CDC reported on the first ten patients with severe illness and acute hypoxemic respiratory failure associated with A/H1N1 infection, none of whom were pregnant, but they noticed that half of the patients had a pulmonary embolism. During a four-month period from September to December 2009, 252 patients were admitted to our hospital with confirmed pandemic influenza H1N1 by real-time reverse transcriptase-polymerase chain reaction test (rRT-PCR). We cared for twenty patients (7.9%) admitted to MICU with severe A/H1N1. Results on Thrombotic events were identified in five (25%) of our critically ill patients. We recommend that patients with severe influenza A/H1N1 pneumonitis and respiratory failure be administered DVT prophylaxis in particular if there are additional risk factors for TVE. Further prospective studies on the relationship of influenza A/H1N1 and VTE are needed. Copyright © 2015 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.

Expectoration of Flaviviruses during sugar feeding by mosquitoes (Diptera: Culicidae).

PubMed

van den Hurk, Andrew F; Johnson, Petrina H; Hall-Mendelin, Sonja; Northill, Judy A; Simmons, Russell J; Jansen, Cassie C; Frances, Stephen P; Smith, Greg A; Ritchie, Scott A

2007-09-01

Biological transmission of arboviruses to a vertebrate host occurs when virions are expelled along with saliva during blood feeding by a hematophagous arthropod. We undertook experiments to determine whether mosquitoes expectorate flaviviruses in their saliva while sugar feeding. Batches of Culex annulirostris Skuse and Culex gelidus Theobald (Diptera: Culicidae) were orally infected with Japanese encephalitis (family Flaviviridae, genus Flavivirus, JEV), Kunjin (family Flaviviridae, genus Flavivirus, KUNV; a subtype of West Nile virus), and Murray Valley encephalitis (family Flaviviridae, genus Flavivirus, MVEV) viruses. After a 7-d extrinsic incubation, these mosquitoes were offered sucrose meals via cotton pledgets, which were removed daily and processed for viral RNA by using real-time TaqMan reverse transcriptase-polymerase chain reaction (RT-PCR) assays. JEV, MVEV, and KUNV RNA was detected in all pledgets removed from batches of Cx. gelidus on days 7-14 postexposure. In contrast, detection rates were variable for Cx. annulirostris, with KUNV detected in 0.3 M sucrose pledgets on all days postexposure, and JEV and MVEV detected on 57 and 50% of days postexposure, respectively. Higher concentrations of sucrose in the pledget did not increase virus detection rates. When individual JEV-infected Cx. gelidus were exposed to the sucrose pledget, 73% of mosquitoes expectorated virus with titers that were detectable by TaqMan RT-PCR. These results clearly show that flaviviruses are expectorated by infected mosquitoes during the process of sugar feeding on artificial pledgets. Potential applications of the method for arboviral bioassays and field surveillance are discussed.

Serum and synovial fluid levels of tumor necrosis factor-like ligand 1A and decoy receptor 3 in rheumatoid arthritis.

PubMed

Xiu, Zijuan; Shen, Hui; Tian, Ye; Xia, Liping; Lu, Jing

2015-04-01

To measure the levels of Tumor necrosis factor (TNF)-like ligand 1A (TL1A) and decoy receptor 3 (DcR3) in serum and synovial fluid (SF) of patients with rheumatoid arthritis (RA). To evaluate the effect of recombinant human (rh) TL1A on interleukin (IL)-17 production and IL-17mRNA expression. The serum and SF levels of TL1A and DcR3, and the production of IL-17 by rhTL1A-treated PBMC were measured by enzyme-linked immunosorbent assay (ELISA). The expression of IL-17 mRNA by rhTL1A-treated PBMC was measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR). We also tested the change of TL1A and DcR3 level following TNF-α blockade therapy. Serum TL1A and DcR3 levels were higher in RA patients. This increase was more significant in RF and anti-CCP positive patients. TL1A and DcR3 levels were higher in SF samples than in paired sera. TL1A and DcR3 decreased after anti-TNF treatment. rhTL1A increased the production of IL-17 protein and the expression of IL-17mRNA. TL1A and DcR3 may be of pathogenic and potentially of therapeutic importance in RA patients. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular detection and analysis of a novel metalloprotease gene of entomopathogenic Serratia marcescens strains in infected Galleria mellonella.

PubMed

Tambong, J T; Xu, R; Sadiku, A; Chen, Q; Badiss, A; Yu, Q

2014-04-01

Serratia marcescens strains isolated from entomopathogenic nematodes (Rhabditis sp.) were examined for their pathogenicity and establishment in wax moth (Galleria mellonella) larvae. All the Serratia strains were potently pathogenic to G. mellonella larvae, leading to death within 48 h. The strains were shown to possess a metalloprotease gene encoding for a novel serralysin-like protein. Rapid establishment of the bacteria in infected larvae was confirmed by specific polymerase chain reaction (PCR) detection of a DNA fragment encoding for this protein. Detection of the viable Serratia strains in infected larvae was validated using the SYBR Green reverse transcriptase real-time PCR assay targeting the metalloprotease gene. Nucleotide sequences of the metalloprotease gene obtained in our study showed 72 single nucleotide polymorphisms (SNP) and 3 insertions compared with the metalloprotease gene of S. marcescens E-15. The metalloprotease gene had 60 synonymous and 8 nonsynonymous substitutions relative to the closest GenBank entry, S. marcescens E-15. A comparison of the amino acid composition of the new serralysin-like protein with that of the serralysin protein of S. marcescens E-15 revealed differences at 11 positions and a new aspartic acid residue. Analysis of the effect of protein variation suggests that a new aspartic acid residue resulting from nonsynonymous nucleotide mutations in the protein structure could have the most significant effect on its biological function. The new metalloprotease gene and (or) its product could have applications in plant agricultural biotechnology.

Myofibroblasts in interstitial lung diseases show diverse electron microscopic and invasive features.

PubMed

Karvonen, Henna M; Lehtonen, Siri T; Sormunen, Raija T; Harju, Terttu H; Lappi-Blanco, Elisa; Bloigu, Risto S; Kaarteenaho, Riitta L

2012-09-01

The characteristic features of myofibroblasts in various lung disorders are poorly understood. We have evaluated the ultrastructure and invasive capacities of myofibroblasts cultured from small volumes of diagnostic bronchoalveolar lavage (BAL) fluid samples from patients with different types of lung diseases. Cells were cultured from samples of BAL fluid collected from 51 patients that had undergone bronchoscopy and BAL for diagnostic purposes. The cells were visualized by transmission electron microscopy and immunoelectron microscopy to achieve ultrastructural localization of alpha-smooth muscle actin (α-SMA) and fibronectin. The levels of α-SMA protein and mRNA and fibronectin mRNA were measured by western blot and quantitative real-time reverse transcriptase polymerase chain reaction. The invasive capacities of the cells were evaluated. The cultured cells were either fibroblasts or myofibroblasts. The structure of the fibronexus, and the amounts of intracellular actin, extracellular fibronectin and cell junctions of myofibroblasts varied in different diseases. In electron and immunoelectron microscopy, cells cultured from interstitial lung diseases (ILDs) expressed more actin filaments and α-SMA than normal lung. The invasive capacity of the cells obtained from patients with idiopathic pulmonary fibrosis was higher than that from patients with other type of ILDs. Cells expressing more actin filaments had a higher invasion capacity. It is concluded that electron and immunoelectron microscopic studies of myofibroblasts can reveal differential features in various diseases. An analysis of myofibroblasts cultured from diagnostic BAL fluid samples may represent a new kind of tool for diagnostics and research into lung diseases.

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics.

PubMed

Zambenedetti, Miriam Ribas; Pavoni, Daniela Parada; Dallabona, Andreia Cristine; Dominguez, Alejandro Correa; Poersch, Celina de Oliveira; Fragoso, Stenio Perdigão; Krieger, Marco Aurélio

2017-05-01

Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

PubMed Central

Zambenedetti, Miriam Ribas; Pavoni, Daniela Parada; Dallabona, Andreia Cristine; Dominguez, Alejandro Correa; Poersch, Celina de Oliveira; Fragoso, Stenio Perdigão; Krieger, Marco Aurélio

2017-01-01

BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses. PMID:28403327

Targeted transgenic overexpression of mitochondrial thymidine kinase (TK2) alters mitochondrial DNA (mtDNA) and mitochondrial polypeptide abundance: transgenic TK2, mtDNA, and antiretrovirals.

PubMed

Hosseini, Seyed H; Kohler, James J; Haase, Chad P; Tioleco, Nina; Stuart, Tami; Keebaugh, Erin; Ludaway, Tomika; Russ, Rodney; Green, Elgin; Long, Robert; Wang, Liya; Eriksson, Staffan; Lewis, William

2007-03-01

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-gamma. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity.

Targeted Transgenic Overexpression of Mitochondrial Thymidine Kinase (TK2) Alters Mitochondrial DNA (mtDNA) and Mitochondrial Polypeptide Abundance

PubMed Central

Hosseini, Seyed H.; Kohler, James J.; Haase, Chad P.; Tioleco, Nina; Stuart, Tami; Keebaugh, Erin; Ludaway, Tomika; Russ, Rodney; Green, Elgin; Long, Robert; Wang, Liya; Eriksson, Staffan; Lewis, William

2007-01-01

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-γ. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity. PMID:17322372

Human immunodeficiency virus type 1 pol gene mutations which cause decreased susceptibility to 2',3'-dideoxycytidine.

PubMed Central

Fitzgibbon, J E; Howell, R M; Haberzettl, C A; Sperber, S J; Gocke, D J; Dubin, D T

1992-01-01

To investigate whether human immunodeficiency virus type 1 pol gene mutations are selected during prolonged 2',3'-dideoxycytidine (ddC) therapy, we used the polymerase chain reaction to amplify a portion of the reverse transcriptase segment of the pol gene from the peripheral blood mononuclear cell DNA of a patient with AIDS before and after an 80-week course of ddC therapy. The consensus sequence from the second sample contained a unique double mutation (ACT to GAT) in the codon for reverse transcriptase amino acid 69, causing substitution of aspartic acid (Asp) for the wild-type threonine (Thr). A mutation (ACA to ATA) also occurred in the codon for position 165, causing substitution of isoleucine (Ile) for Thr. The GAT (Asp) codon was introduced into the pol gene of a molecular clone of human immunodeficiency virus via site-directed mutagenesis. Following transfection, mutant and wild-type viruses were tested for susceptibility to ddC by a plaque reduction assay. The mutant virus was fivefold less susceptible to ddC than the wild type; cross-resistance to 3'-azido-3'-deoxythymidine or 2'3'-dideoxyinosine was not found. The Ile-165 mutation did not confer additional ddC resistance. The Asp-69 substitution may have contributed to the generation of resistant virus in this patient. Images PMID:1317143

Genetic characterization and antiretroviral resistance mutations among treatment-naive HIV-infected individuals in Jiaxing, China.

PubMed

Guo, Jinlei; Yan, Yong; Zhang, Jiafeng; Ji, Jimei; Ge, Zhijian; Ge, Rui; Zhang, Xiaofei; Wang, Henghui; Chen, Zhongwen; Luo, Jianyong

2017-03-14

The aim of this study was to characterize HIV-1 genotypes and antiretroviral resistance mutations among treatment-naive HIV-infected individuals in Jiaxing, China. The HIV-1 partial polymerase (pol) genes in 93 of the 99 plasma samples were successfully amplified and analyzed. Phylogenetic analysis revealed the existence of five HIV-1 genotypes, of which the most prevalent genotype was CRF01_AE (38.7%), followed by CRF07_BC (34.4%), CRF08_BC (16.1%), subtype B/B' (5.4%), and CRF55_01B (2.1%). Besides, three types of unique recombination forms (URFs) were also observed, including C/F2/A1, CRF01_AE/B, and CRF08_BC/CRF07_BC. Among 93 amplicons, 46.2% had drug resistance-associated mutations, including 23.7% for protease inhibitors (PIs) mutations, 1.1% for nucleoside reverse transcriptase inhibitors (NRTIs) mutations, and 20.4% for non-nucleoside reverse transcriptase inhibitors (NNRTIs) mutations. Six (6.5%) out of 93 treatment-naive subjects were identified to be resistant to one or more NNRTIs, while resistance to NRTIs or PIs was not observed. Our study showed the genetic diversity of HIV-1 strains circulating in Jiaxing and a relative high proportion of antiretroviral resistance mutations among treatment-naive patients, indicating a serious challenge for HIV prevention and treatment program.

Endogenous New World primate type C viruses isolated from owl monkey (Aotus trivirgatus) kidney cell line.

PubMed Central

Todaro, G J; Sherr, C J; Sen, A; King, N; Daniel, M D; Fleckenstein, B

1978-01-01

A type C virus (OMC-1) detected in a culture of owl monkey kidney cells resembled typical type C viruses morphologically, but was slightly larger than previously characterized mammalian type C viruses. OMC-1 can be transmitted to bat lung cells and cat embryo fibroblasts. The virions band at a density of 1.16 g/ml in isopycnic sucrose density gradients and contain reverse transcriptase and a 60-65S RNA genome composed of approximately 32S subunits. The reverse transcriptase is immunologically and biochemically distinct from the polymerases of othe retroviruses. Radioimmunoassays directed to the interspecies antigenic determinants of the major structure proteins of other type C viruses do not detect a related antigen in OMC-1. Nucleic acid hybridization experiments using labeled viral genomic RNA or proviral cDNA transcripts to normal cellular DNA of different species show that OMC-1 is an endogenous virus with multiple virogene copies (20-50 per haploid genome) present in normal owl monkey cells and is distinct from previously isolated type C and D viruses. Sequences related to the OMC-1 genome can be detected in other New World monkeys. Thus, similar to the Old World primates (e.g., baboons as a prototype), the New World monkeys contain endogenous type C viral genes that appear to have been transmitted in the primate germ line. Images PMID:76312

Unfolding the HIV-1 reverse transcriptase RNase H domain – how to lose a molecular tug-of-war

DOE PAGES

Zheng, Xunhai; Pedersen, Lars C.; Gabel, Scott A.; ...

2016-01-14

Formation of the mature HIV-1 reverse transcriptase (RT) p66/p51 heterodimer requires subunit-specific processing of the p66/p66' homodimer precursor. Since the ribonuclease H (RH) domain contains an occult cleavage site located near its center, cleavage must occur either prior to folding or subsequent to unfolding. Recent NMR studies have identified a slow, subunit-specific RH domain unfolding process proposed to result from a residue tug-of-war between the polymerase and RH domains on the functionally inactive, p66' subunit. Here, we describe a structural comparison of the isolated RH domain with a domain swapped RH dimer that reveals several intrinsically destabilizing characteristics of themore » isolated domain that facilitate excursions of Tyr427 from its binding pocket and separation of helices B and D. These studies provide independent support for the subunit-selective RH domain unfolding pathway in which instability of the Tyr427 binding pocket facilitates its release followed by domain transfer, acting as a trigger for further RH domain destabilization and subsequent unfolding. As further support for this pathway, NMR studies demonstrate that addition of an RH active site-directed isoquinolone ligand retards the subunit-selective RH' domain unfolding behavior of the p66/p66' homodimer. As a result, this study demonstrates the feasibility of directly targeting RT maturation with therapeutics.« less

Unfolding the HIV-1 reverse transcriptase RNase H domain – how to lose a molecular tug-of-war

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Xunhai; Pedersen, Lars C.; Gabel, Scott A.

Formation of the mature HIV-1 reverse transcriptase (RT) p66/p51 heterodimer requires subunit-specific processing of the p66/p66' homodimer precursor. Since the ribonuclease H (RH) domain contains an occult cleavage site located near its center, cleavage must occur either prior to folding or subsequent to unfolding. Recent NMR studies have identified a slow, subunit-specific RH domain unfolding process proposed to result from a residue tug-of-war between the polymerase and RH domains on the functionally inactive, p66' subunit. Here, we describe a structural comparison of the isolated RH domain with a domain swapped RH dimer that reveals several intrinsically destabilizing characteristics of themore » isolated domain that facilitate excursions of Tyr427 from its binding pocket and separation of helices B and D. These studies provide independent support for the subunit-selective RH domain unfolding pathway in which instability of the Tyr427 binding pocket facilitates its release followed by domain transfer, acting as a trigger for further RH domain destabilization and subsequent unfolding. As further support for this pathway, NMR studies demonstrate that addition of an RH active site-directed isoquinolone ligand retards the subunit-selective RH' domain unfolding behavior of the p66/p66' homodimer. As a result, this study demonstrates the feasibility of directly targeting RT maturation with therapeutics.« less

Coexistence of BRAF V600E and TERT Promoter Mutations in Low-grade Serous Carcinoma of Ovary Recurring as Carcinosarcoma in a Lymph Node: Report of a Case.

PubMed

Tavallaee, Mahkam; Steiner, David F; Zehnder, James L; Folkins, Ann K; Karam, Amer K

2018-04-03

Low-grade serous carcinomas only rarely coexist with or progress to high-grade tumors. We present a case of low-grade serous carcinoma with transformation to carcinosarcoma on recurrence in the lymph node. Identical BRAF V600E and telomerase reverse transcriptase promoter mutations were identified in both the original and recurrent tumor. Given that telomerase reverse transcriptase promotor mutations are thought to play a role in progression of other tumor types, the function of telomerase reverse transcriptase mutations in BRAF mutated low-grade serous carcinoma deserves investigation.

DNA synthesis arrest sites at the right terminus of rat long interspersed repeated (LINE or L1Rn) DNA family members.

PubMed Central

d'Ambrosio, E; Furano, A V

1987-01-01

An approximately equal to 150-bp GC-rich (approximately equal to 60%) region is at the right end of rat long interspersed repeated DNA (LINE or L1Rn) family members. We report here that one of the DNA strands from this region contains several non-palindromic sites that strongly arrest DNA synthesis in vitro by the prokaryotic Klenow and T4 DNA polymerases, the eukaryotic alpha polymerase, and AMV reverse transcriptase. The strongest arrest sites are G-rich (approximately equal to 70%) homopurine stretches of 18 or more residues. Shorter homopurine stretches (12 residues or fewer) did not arrest DNA synthesis even if the stretch contains 11/12 G residues. Arrest of the prokaryotic polymerases was not affected by their respective single strand binding proteins or polymerase accessory proteins. The region of duplex DNA which contains DNA synthesis arrest sites reacts with bromoacetaldehyde when present in negatively supercoiled molecules. By contrast, homopurine stretches that do not arrest DNA synthesis do not react with bromoacetaldehyde. The presence of bromoacetaldehyde-reactive bases in a G-rich homopurine-containing duplex under torsional stress is thought to be caused by base stacking in the homopurine strand. Therefore, we suggest that base-stacked regions of the template arrest DNA synthesis. Images PMID:2436148

A biosensor assay for the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples

PubMed Central

Kumanan, Vijayarani; Nugen, Sam R.; Baeumner, Antje J.

2009-01-01

A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with a reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of a small number (ten) of viable Mycobacterium (M.) avium subsp. paratuberculosis (MAP) cells in fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with a known quantity of (101 to 106) MAP cells and amplified using RT-PCR and identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min of assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye, sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer, or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria, such as M. avium complex, M. ulcerans, M. marium, M. kansasii, M. abscessus, M. asiaticum, M. phlei, M. fortuitum, M. scrofulaceum, M. intracellulare, M. smegmatis, and M. bovis. The overall assay for the detection of live MAP organisms is comparatively less expensive and quick, especially in comparison to standard MAP detection using a culture method requiring 6-8 weeks of incubation time, and is significantly less expensive than real-time PCR. PMID:19255522

Elevated Human telomerase reverse transcriptase gene expression in blood cells associated with chronic and arsenic exposure in Inner Mongolia, China

EPA Science Inventory

BACKGROUND: Arsenic exposure is associated with human cancer. Telomerase containing the catalytic subunit, human telomerase reverse transcriptase (hTERT), can extend telomeres of chromosomes, delay senescence and promoting cell proliferation leading to tumorigenesis. OBJECTIVE:...

Detection of Zaire Ebola virus by real-time reverse transcription-polymerase chain reaction, Sierra Leone, 2014.

PubMed

Liu, Licheng; Sun, Yang; Kargbo, Brima; Zhang, Chuntao; Feng, Huahua; Lu, Huijun; Liu, Wenseng; Wang, Chengyu; Hu, Yi; Deng, Yongqiang; Jiang, Jiafu; Kang, Xiaoping; Yang, Honglei; Jiang, Yongqiang; Yang, Yinhui; Kargbo, David; Qian, Jun; Chen, Weijun

2015-09-15

During the 2014 Ebola virus disease (EVD) outbreak, a real-time quantitative polymerase chain reaction was established to detect and identify the Zaire Ebola virus. We describe the use of this assay to screen 315 clinical samples from EVD suspected person in Sierra Leone. The detection rate in blood samples was 77.81% (207/266), and there were relatively higher detection rate (79.32% and 81.42%, respectively) during the first two weeks after onset of symptoms. In the two weeks that followed, the detection rate declined to 66.67% and 25.00%, respectively. There was the highest virus load at the first week and then decreased. The detection rate in swab samples was 89.79% (44/49). This may be benefit from the included patients. 46 of 49 swab samples were collected from died patients. Taken together, the results presented here indicate that the assay specifically and sensitively detects Zaire Ebola virus. Copyright © 2015 Elsevier B.V. All rights reserved.

Sustained high-throughput polymerase chain reaction diagnostics during the European epidemic of Bluetongue virus serotype 8.

PubMed

van Rijn, Piet A; Heutink, René G; Boonstra, Jan; Kramps, Hans A; van Gennip, René G P

2012-05-01

A real-time reverse transcription polymerase chain reaction assay (PCR test) based on genome segment 10 of Bluetongue virus (BTV) was developed. The PCR test consists of robotized viral RNA isolation from blood samples and an all-in-one method including initial denaturation of genomic double-stranded RNA, reverse transcription polymerase chain reaction (RT-PCR), and real-time detection and analysis. Reference strains of the 24 recognized BTV serotypes, isolates from different years, and geographic origins were detected. Other orbiviruses such as African horse sickness virus, Epizootic hemorrhagic disease virus, and Equine encephalosis virus were not detected. Experimentally infected animals were PCR positive from 2 days postinoculation, which was earlier than fever, other clinical signs, or seroconversion. The diagnostic sensitivity and specificity were very close to or even 100%. The PCR test played a key role in the detection of BTV serotype 8 in August 2006 in The Netherlands. The outbreak in a completely naive ruminant population allowed for further evaluation of the PCR test with field samples. In 2006, the correlation between enzyme-linked immunosorbent assay and PCR results was estimated to be 95%. In the following years, the PCR test was used for diagnosis of diseased animals, for testing of healthy animals for trade purposes, and for detection of BTV RNA in different species of the insect vector, Culicoides. In the autumn of 2008, BTV serotype 6 unexpectedly emerged in northwest Europe and was also detected with the PCR test developed in the current study. The performance in routine use over 5 years has been recorded and evaluated.

9 CFR 147.31 - Laboratory procedures recommended for the real-time polymerase chain reaction test for Mycoplasma...

Code of Federal Regulations, 2014 CFR

2014-01-01

... the real-time polymerase chain reaction test for Mycoplasma gallisepticum (MGLP ReTi). 147.31 Section... Examination Procedures § 147.31 Laboratory procedures recommended for the real-time polymerase chain reaction.... Following incubation, 100 µl of 100 percent ethanol is added to lysate. Wash and centrifuge following...

9 CFR 147.31 - Laboratory procedures recommended for the real-time polymerase chain reaction test for Mycoplasma...

Code of Federal Regulations, 2013 CFR

2013-01-01

... the real-time polymerase chain reaction test for Mycoplasma gallisepticum (MGLP ReTi). 147.31 Section... Examination Procedures § 147.31 Laboratory procedures recommended for the real-time polymerase chain reaction.... Following incubation, 100 µl of 100 percent ethanol is added to lysate. Wash and centrifuge following...

9 CFR 147.31 - Laboratory procedures recommended for the real-time polymerase chain reaction test for Mycoplasma...

Code of Federal Regulations, 2012 CFR

2012-01-01

... the real-time polymerase chain reaction test for Mycoplasma gallisepticum (MGLP ReTi). 147.31 Section... Examination Procedures § 147.31 Laboratory procedures recommended for the real-time polymerase chain reaction.... Following incubation, 100 µl of 100 percent ethanol is added to lysate. Wash and centrifuge following...

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

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.

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.

The Effects of Resveratrol on Inflammation and Oxidative Stress in a Rat Model of Chronic Obstructive Pulmonary Disease.

PubMed

Wang, Xiao-Li; Li, Ting; Li, Ji-Hong; Miao, Shu-Ying; Xiao, Xian-Zhong

2017-09-12

Oxidative stress and inflammation are hypothesized to contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD). Resveratrol (trans-3,5,4'-trihydroxystilbene) is known for its antioxidant and anti-inflammatory properties. The study aimed to investigate the effects of resveratrol in a rat model with COPD on the regulation of oxidative stress and inflammation via the activation of Sirtuin1 (SIRTl) and proliferator-activated receptor-γ coactivator-1α (PGC-1α). Thirty Wistar rats were randomly divided into three groups: control group, COPD group and resveratrol intervention group. The COPD model was established by instilling with lipopolysaccharide (LPS) and challenging with cigarette smoke (CS). The levels of interleukin-6 (IL-6) and interleukin-8 (IL-8) in serum were measured. The levels of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were measured. The expression levels of SIRT1 and PGC-1α in the lung tissues were examined by immunohistochemistry as well as real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) and western blotting analysis. After the treatment with resveratrol (50 mg/kg), compared with the COPD group, alleviation of inflammation and reconstruction in the small airways of the lungs were seen. Resveratrol might be correlated not only with the lower level of MDA and the higher activity of SOD, but also with the upregulation of SIRT1 and PGC-1α expression. Resveratrol treatment decreased serum levels of IL-6 and IL-8. Our findings indicate that resveratrol had a therapeutic effect in our rat COPD model, which is related to the inhibition of oxidative stress and inflammatory response. The mechanism may be related to the activation and upgrading of the SIRT1/PGC-1α signaling pathways. Thus resveratrol might be a therapeutic modality in COPD.

Up-regulation of IL-23 expression in human dental pulp fibroblasts by IL-17 via activation of the NF-κB and MAPK pathways.

PubMed

Wei, L; Liu, M; Xiong, H; Peng, B

2017-11-06

To investigate the effects of the pro-inflammatory and Th17-polarizing mediator IL-17 on HDPFs-mediated IL-23 production and the molecular mechanism involved. Interleukin (IL)-17R expression was determined by semi-quantitative reverse transcriptase-polymerase chain reaction and Western blot in cultured human dental pulp fibroblasts (HDPFs). Quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay were used to determine IL-23 mRNA and protein levels in IL-17-stimulated HDPFs, respectively. The nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) signalling pathways that mediate the IL-17-stimulated production of IL-23 was investigated using Western blot and specific signalling inhibitor analyses. Statistical analyses were performed using Kruskal-Wallis tests followed by the Mann-Whitney U-test. Statistical significance was considered when the P value < 0.05. Primary HDPFs steadily expressed IL-17R mRNA and surface-bound protein. IL-17 stimulated the expression of IL-23 mRNA and protein in cultured human dental pulp fibroblasts, which was attenuated by IL-17 or IL-17R neutralizing antibodies. In accordance with the enhanced expression of IL-23, IL-17 stimulation resulted in rapid activation of p38 MAPK, extracellular signal-regulated kinase (ERK) 1/2, c-Jun-N-terminal kinase (JNK) and NF-κB in HDPFs. Inhibitors of p38 MAPK, ERK 1/2 or NF-κB significantly suppressed, whereas blocking JNK substantially augmented IL-23 production from IL-17-stimulated HDPFs. HDPFs expressed IL-17R and responded to IL-17 to produce IL-23 via the activation of the NF-κB and MAPK signalling pathways. The findings provide insights into the cellular mechanisms of the participation of IL-17 in the activation of HDPFs in inflamed pulp tissue. © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Secretion metabolites of probiotic yeast, Pichia kudriavzevii AS-12, induces apoptosis pathways in human colorectal cancer cell lines.

PubMed

Saber, Amir; Alipour, Beitollah; Faghfoori, Zeinab; Mousavi Jam, Ali; Yari Khosroushahi, Ahmad

2017-05-01

There is a common agreement on the important role of the gastrointestinal microbiota in the etiology of cancer. Benign probiotic yeast strains are able to ameliorate intestinal microbiota and regulate the host metabolism, physiology, and immune system through anti-inflammatory, antiproliferative, and anticancer effects. We hypothesized that Pichia kudriavzevii AS-12 secretion metabolites possess anticancer activity on human colorectal cancer cells (HT-29, Caco-2) via inhibiting growth and inducing apoptosis. This study aimed to assess the anticancer effect of P. kudriavzevii AS-12 secretion metabolites and the underlying mechanisms. The cytotoxicity evaluations were performed via 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay; 4',6-diamidino-2-phenylindole staining; and FACS-flow cytometry tests. Also, the effects of P. kudriavzevii AS-12 secretion metabolites on the expression level of 6 important genes (BAD, Bcl-2, Caspase-3, Caspase-8, Caspase-9 and Fas-R) involved in the extrinsic and intrinsic apoptosis pathways were studied by real-time polymerase chain reaction method. P. kudriavzevii AS-12 secretion metabolites showed significant (P < .0001) cytotoxic effects on HT-29 cells (57.5%) and Caco-2 (32.5%) compared to KDR/293 normal cells (25%). Moreover, the cytotoxic effects of examined yeast supernatant on HT-29 cells were comparable with 5-fluorouracil, as a positive control (57.5% versus 62.2% respectively). Flow cytometric results showed that the induction of apoptosis is the main mechanism of the anticancer effects. Also, according to the reverse transcriptase polymerase chain reaction results, the expression level of proapoptotic genes (BAD, Caspase-3, Caspase-8, Caspase-9, and Fas-R) in treated HT-29 and Caco-2 cells was higher than untreated and normal cells, whereas the antiapoptotic gene (Bcl-2) was downregulated. P. kudriavzevii AS-12 secretion metabolites exert its anticancer effects by inhibiting cell proliferation and inducing intrinsic and extrinsic apoptosis in colon cancer cells. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

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.

Recommendations for the standardization of bone marrow disease assessment and reporting in children with neuroblastoma on behalf of the International Neuroblastoma Response Criteria Bone Marrow Working Group.

PubMed

Burchill, Susan A; Beiske, Klaus; Shimada, Hiroyuki; Ambros, Peter F; Seeger, Robert; Tytgat, Godelieve A M; Brock, Penelope R; Haber, Michelle; Park, Julie R; Berthold, Frank

2017-04-01

The current study was conducted to expedite international standardized reporting of bone marrow disease in children with neuroblastoma and to improve equivalence of care. A multidisciplinary International Neuroblastoma Response Criteria Bone Marrow Working Group was convened by the US National Cancer Institute in January 2012 with representation from Europe, North America, and Australia. Practical transferable recommendations to standardize the reporting of bone marrow disease were developed. To the authors' knowledge, the current study is the first to comprehensively present consensus criteria for the collection, analysis, and reporting of the percentage area of bone marrow parenchyma occupied by tumor cells in trephine-biopsies. The quantitative analysis of neuroblastoma content in bone marrow aspirates by immunocytology and reverse transcriptase-quantitative polymerase chain reaction are revised. The inclusion of paired-like homeobox 2b (PHOX2B) for immunohistochemistry and reverse transcriptase-quantitative polymerase chain reaction is recommended. Recommendations for recording bone marrow response are provided. The authors endorse the quantitative assessment of neuroblastoma cell content in bilateral core needle biopsies-trephines and aspirates in all children with neuroblastoma, with the exception of infants, in whom the evaluation of aspirates alone is advised. It is interesting to note that 5% disease is accepted as an internationally achievable level for disease assessment. The quantitative assessment of neuroblastoma cells is recommended to provide data from which evidence-based numerical criteria for the reporting of bone marrow response can be realized. This is particularly important in the minimal disease setting and when neuroblastoma detection in bone marrow is intermittent, where clinical impact has yet to be validated. The wide adoption of these harmonized criteria will enhance the ability to compare outcomes from different trials and facilitate collaborative trial design. Cancer 2017;123:1095-1105. © 2016 American Cancer Society. © 2016 American Cancer Society.

Reduction in the incidence of influenza A but not influenza B associated with use of hand sanitizer and cough hygiene in schools: a randomized controlled trial.

PubMed

Stebbins, Samuel; Cummings, Derek A T; Stark, James H; Vukotich, Chuck; Mitruka, Kiren; Thompson, William; Rinaldo, Charles; Roth, Loren; Wagner, Michael; Wisniewski, Stephen R; Dato, Virginia; Eng, Heather; Burke, Donald S

2011-11-01

Laboratory-based evidence is lacking regarding the efficacy of nonpharmaceutical interventions (NPIs) such as alcohol-based hand sanitizer and respiratory hygiene to reduce the spread of influenza. The Pittsburgh Influenza Prevention Project was a cluster-randomized trial conducted in 10 elementary schools in Pittsburgh, PA, during the 2007 to 2008 influenza season. Children in 5 intervention schools received training in hand and respiratory hygiene, and were provided and encouraged to use hand sanitizer regularly. Children in 5 schools acted as controls. Children with influenza-like illness were tested for influenza A and B by reverse-transcriptase polymerase chain reaction. A total of 3360 children participated in this study. Using reverse-transcriptase polymerase chain reaction, 54 cases of influenza A and 50 cases of influenza B were detected. We found no significant effect of the intervention on the primary study outcome of all laboratory-confirmed influenza cases (incidence rate ratio [IRR]: 0.81; 95% confidence interval [CI]: 0.54, 1.23). However, we did find statistically significant differences in protocol-specified ancillary outcomes. Children in intervention schools had significantly fewer laboratory-confirmed influenza A infections than children in control schools, with an adjusted IRR of 0.48 (95% CI: 0.26, 0.87). Total absent episodes were also significantly lower among the intervention group than among the control group; adjusted IRR 0.74 (95% CI: 0.56, 0.97). NPIs (respiratory hygiene education and the regular use of hand sanitizer) did not reduce total laboratory-confirmed influenza. However, the interventions did reduce school total absence episodes by 26% and laboratory-confirmed influenza A infections by 52%. Our results suggest that NPIs can be an important adjunct to influenza vaccination programs to reduce the number of influenza A infections among children.

Human Mitochondrial RNA Polymerase: Evaluation of the Single-Nucleotide-Addition Cycle on Synthetic RNA/DNA Scaffolds

PubMed Central

Smidansky, Eric D.; Arnold, Jamie J.; Reynolds, Shelley L.; Cameron, Craig E.

2013-01-01

The human mitochondrial RNA polymerase (h-mtRNAP) serves as both the transcriptase for expression and the primase for replication of mitochondrial DNA. As such, the enzyme is of fundamental importance to cellular energy metabolism, and defects in its function may be related to human disease states. Here we describe in vitro analysis of the h-mtRNAP kinetic mechanism for single, correct nucleotide incorporation. This was made possible by the development of efficient methods for expression and purification of h-mtRNAP using a bacterial system and by utilization of assays that rely on simple, synthetic RNA/DNA scaffolds without the need for mitochondrial transcription accessory proteins. We find that h-mtRNAP accomplishes single-nucleotide incorporation by using the same core steps, including conformational change steps before and after chemistry, that are prototypical for most types of nucleic acid polymerases. The polymerase binds to scaffolds via a two-step mechanism consisting of a fast initial-encounter step followed by a much slower isomerization that leads to catalytic competence. A substantial solvent deuterium kinetic isotope effect was observed for the forward reaction, but none was detectable for the reverse reaction, suggesting that chemistry is at least partially rate-limiting in the forward direction but not in the reverse. h-mtRNAP appears to exercise much more stringent surveillance over base than over sugar in determining the correctness of a nucleotide. The utility of developing the robust in vitro assays described here and of establishing a baseline of kinetic performance for the wild-type enzyme is that biological questions concerning h-mtRNAP may now begin to be addressed. PMID:21548588

Chemical crosslinking of the subunits of HIV-1 reverse transcriptase.

PubMed Central

Debyser, Z.; De Clercq, E.

1996-01-01

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is composed of two subunits of 66 and 51 kDa in a 1 to 1 ratio. Because dimerization is a prerequisite for enzymatic activity, interference with the dimerization process could constitute an alternative antiviral strategy for RT inhibition. Here we describe an in vitro assay for the study of the dimerization state of HIV-1 reverse transcriptase based on chemical crosslinking of the subunits with dimethylsuberimidate. Crosslinking results in the formation of covalent bonds between the subunits, so that the crosslinked species can be resolved by denaturing gel electrophoresis. Crosslinked RT species with molecular weight greater than that of the dimeric form accumulate during a 1-15-min time course. Initial evidence suggests that those high molecular weight species represent trimers and tetramers and may be the result of intramolecular crosslinking of the subunits of a higher-order RT oligomer. A peptide that corresponds to part of the tryptophan repeat motif in the connection domain of HIV-1 RT inhibits crosslink formation as well as enzymatic activity. The crosslinking assay thus allows the investigation of the effect of inhibitors on the dimerization of HIV-1 RT. PMID:8745406

9 CFR 147.31 - Laboratory procedures recommended for the real-time polymerase chain reaction test for Mycoplasma...

Code of Federal Regulations, 2010 CFR

2010-01-01

... the real-time polymerase chain reaction test for Mycoplasma gallisepticum (MGLP ReTi). 147.31 Section... Examination Procedures § 147.31 Laboratory procedures recommended for the real-time polymerase chain reaction... lp gene. (c) MGLP ReTi. Primers and probe should be utilized in a 25 µl reaction containing 12.5 µl...

9 CFR 147.31 - Laboratory procedures recommended for the real-time polymerase chain reaction test for Mycoplasma...

Code of Federal Regulations, 2011 CFR

2011-01-01

... the real-time polymerase chain reaction test for Mycoplasma gallisepticum (MGLP ReTi). 147.31 Section... Examination Procedures § 147.31 Laboratory procedures recommended for the real-time polymerase chain reaction... lp gene. (c) MGLP ReTi. Primers and probe should be utilized in a 25 µl reaction containing 12.5 µl...

T-cell acute lymphoblastic leukemia associated with complex karyotype and SET-NUP214 rearrangement: a case study and review of the literature.

PubMed

Lee, Sang-Guk; Park, Tae Sung; Cho, Sun Young; Lim, Gayoung; Park, Gwang Jin; Oh, Seung Hwan; Cho, Eun Hae; Chong, So Young; Huh, Ji Young

2011-01-01

SET-NUP214 rearrangements have been rarely reported in T-cell acute lymphoblastic leukemia (T-ALL), acute undifferentiated leukemia, and acute myeloid leukemia, and most documented cases have been associated with normal karyotypes in conventional cytogenetic analyses. Here, we describe a novel case of T-ALL associated with a mediastinal mass and a SET-NUP214 rearrangement, which was masked by a complex karyotype at the time of initial diagnosis. Using multiplex reverse transcriptase-polymerase chain reaction analysis, we detected a cryptic SET-NUP214 rearrangement in our patient. As only 11 cases (including the present study) of T-ALL with SET-NUP214 rearrangement have been reported, the clinical features and treatment outcomes have not been fully determined. Further studies are necessary to evaluate the incidence of SET-NUP214 rearrangement in T-ALL patients and the treatment responses as well as prognosis of these patients.

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.

Garlic (Allium sativum) stimulates lipopolysaccharide-induced tumor necrosis factor-alpha production from J774A.1 murine macrophages.

PubMed

Sung, Jessica; Harfouche, Youssef; De La Cruz, Melissa; Zamora, Martha P; Liu, Yan; Rego, James A; Buckley, Nancy E

2015-02-01

Garlic (Allium sativum) is known to have many beneficial attributes such as antimicrobial, antiatherosclerotic, antitumorigenetic, and immunomodulatory properties. In the present study, we investigated the effects of an aqueous garlic extract on macrophage cytokine production by challenging the macrophage J774A.1 cell line with the garlic extract in the absence or presence of lipopolysaccharide (LPS) under different conditions. The effect of allicin, the major component of crushed garlic, was also investigated. Using enzyme-linked immunosorbent assay and reverse transcriptase-quantitative polymerase chain reaction, it was found that garlic and synthetic allicin greatly stimulated tumor necrosis factor-alpha (TNF-α) production in macrophages treated with LPS. The TNF-α secretion levels peaked earlier and were sustained for a longer time in cells treated with garlic and LPS compared with cells treated with LPS alone. Garlic acted in a time-dependent manner. We suggest that garlic, at least partially via its allicin component, acts downstream from LPS to stimulate macrophage TNF-α secretion. © 2014 The Authors. Phytotherapy Research published by John Wiley & Sons, Ltd.

Inter-laboratory quality control for hormone-dependent gene expression in human breast tumors using real-time reverse transcription-polymerase chain reaction.

PubMed

de Cremoux, P; Bieche, I; Tran-Perennou, C; Vignaud, S; Boudou, E; Asselain, B; Lidereau, R; Magdelénat, H; Becette, V; Sigal-Zafrani, B; Spyratos, F

2004-09-01

Quantitative reverse transcription-polymerase chain reaction (RT-PCR) used to detect minor changes in specific mRNA concentrations may be associated with poor reproducibility. Stringent quality control is therefore essential at each step of the protocol, including the PCR procedure. We performed inter-laboratory quality control of quantitative PCR between two independent laboratories, using in-house RT-PCR assays on a series of hormone-related target genes in a retrospective consecutive series of 79 breast tumors. Total RNA was reverse transcribed in a single center. Calibration curves were performed for five target genes (estrogen receptor (ER)alpha, ERbeta, progesterone receptor (PR), CYP19 (aromatase) and Ki 67) and for two reference genes (human acidic ribosomal phosphoprotein PO (RPLPO) and TATA box-binding protein (TBP)). Amplification efficiencies of the calibrator were determined for each run and used to calculate mRNA expression. Correlation coefficients were evaluated for each target and each reference gene. A good correlation was observed for all target and reference genes in both centers using their own protocols and kits (P < 0.0001). The correlation coefficients ranged from 0.90 to 0.98 for the various target genes in the two centers. A good correlation was observed between the level of expression of the ERalpha and the PR transcripts (P < 0.001). A weak inverse correlation was observed in both centers between ERalpha and ERbeta levels, but only when TBP was the reference gene. No other correlation was observed with other parameters. Real-time PCR assays allow convenient quantification of target mRNA transcripts and quantification of target-derived nucleic acids in clinical specimens. This study addresses the importance of inter-laboratory quality controls for the use of a panel of real-time PCR assays devoted to clinical samples and protocols and to ensure their appropriate accuracy. This can also facilitate exchanges and multicenter comparison of data.

Use of propidium monoazide in reverse transcriptase PCR to distinguish between infectious and noninfectious enteric viruses in water samples

EPA Science Inventory

Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since o...

Selection and characterization of a mutant of feline immunodeficiency virus resistant to 2',3'-dideoxycytidine.

PubMed Central

Medlin, H K; Zhu, Y Q; Remington, K M; Phillips, T R; North, T W

1996-01-01

We have selected and plaque purified a mutant of feline immunodeficiency virus (FIV) that is resistant to 2',3'-dideoxycytidine (ddC). This mutant was selected in cultured cells in the continuous presence of 25 microM ddC. The mutant, designated DCR-5c, was fourfold resistant to ddC, threefold resistant to 2',3'-dideoxyinosine, and more than fourfold resistant to phosphonoformic acid. DCR-5c displayed little or no resistance to (-)-beta-2',3'-dideoxy-3'-thiacytidine, 3'-azido-3'-deoxythymidine, or 9-(2-phosphonylmethoxyethyl) adenine. Reverse transcriptase purified from DCR-5c was less susceptible to inhibition by ddCTP, phosphonoformic acid, ddATP, or azido-dTTP than the wild-type FIV reverse transcriptase. Sequence analysis of DCR-5c revealed a single base change (G to C at nucleotide 2342) in the reverse transcriptase-encoding region of FIV. This mutation results in substitution of His for Asp at codon 3 of FIV reverse transcriptase. The role of this mutation in ddC resistance was confirmed by site-directed mutagenesis. PMID:8849258

The retrotransposon Tf1 assembles virus-like particles that contain excess Gag relative to integrase because of a regulated degradation process.

PubMed

Atwood, A; Lin, J H; Levin, H L

1996-01-01

The retrotransposon Tf1, isolated from Schizosaccharomyces pombe, contains a single open reading frame with sequences encoding Gag, protease, reverse transcriptase, and integrase (IN). Tf1 has previously been shown to possess significant transposition activity. Although Tf1 proteins do assemble into virus-like particles, the assembly does not require readthrough of a translational reading frame shift or stop codon, common mechanisms used by retroelements to express Gag in molar excess of the polymerase proteins. This study was designed to determine if Tf1 particles contain equal amounts of Gag and polymerase proteins or whether they contain the typical molar excess of Gag. After using two separate methods to calibrate the strength of our antibodies, we found that both S. pombe extracts and partially purified Tf1 particles contained a 26-fold molar excess of Gag relative to IN. Knowing that Gag and IN are derived from the same Tf1 primary translation product, we concluded that the excess Gag most likely resulted from specific degradation of IN. We obtained evidence of regulated IN degradation in comparisons of Tf1 protein extracted from log-phase cells and that extracted from stationary-phase cells. The log-phase cells contained equal molar amounts of Gag and IN, whereas cells approaching stationary phase rapidly degraded IN, leaving an excess of Gag. Analysis of the reverse transcripts indicated that the bulk of reverse transcription occurred within the particles that possess a molar excess of Gag.

Orbital Angiogenesis and Lymphangiogenesis in Thyroid Eye Disease: An Analysis of Vascular Growth Factors with Clinical Correlation.

PubMed

Wong, Lindsay L; Lee, Nahyoung Grace; Amarnani, Dhanesh; Choi, Catherine J; Bielenberg, Diane R; Freitag, Suzanne K; D'Amore, Patricia A; Kim, Leo A

2016-09-01

The human orbit is an environment that is vulnerable to inflammation and edema in the setting of autoimmune thyroid disease. Our study investigated the tenet that orbital adipose tissue lacks lymphatic vessels and analyzed the clinicopathologic differences between patients with acute and chronic thyroid eye disease (TED). The underlying molecular mediators of blood and lymphatic vessel formation within the orbital fat also were evaluated. Retrospective cohort study. The study included fat specimens from 26 orbits of 15 patients with TED undergoing orbital decompression. Orbital fat specimens from patients without TED as well as cadaveric orbital fat served as controls. Tissue specimens were processed as formalin-fixed, paraffin-embedded sections or frozen cryosections for immunohistochemistry. Total RNA was extracted and analyzed via quantitative (real-time) reverse-transcription polymerase chain reaction. Clinicopathologic correlation was made by determining the clinical activity score (CAS) of each patient with TED. Samples were examined for vascular and lymphatic markers including podoplanin, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and cluster of differentiation 31 (CD31) by immunohistochemistry, as well as for mRNA levels of vascular endothelial growth factor (VEGF), VEGF receptors, semaphorin 3F, neuropilin 1, neuropilin 2, podoplanin, and LYVE-1 by quantitative (real-time) reverse-transcription polymerase chain reaction. Clinicopathologic correlation revealed increased staining of CD31-positive blood vessels in patients with acute TED with a CAS more than 4, as well as rare staining of podoplanin-positive lymphatic vessels within acutely inflamed orbital fat tissue. Additionally, quantitative (real-time) reverse-transcription polymerase chain reaction analysis demonstrated increased expression of VEGF receptor (VEGFR) 2 as well as VEGF signaling molecules VEGF-A, VEGF-C, and VEGF-D. In acute TED, compared with chronic TED and control orbital fat, there is increased blood vessel density, suggesting neovascularization and rare lymphatic vessels suggestive of limited lymphangiogenesis. This proangiogenic and prolymphangiogenic microenvironment is likely the result of the increased expression of VEGFR-2, VEGF-A, VEGF-C, and VEGF-D. These findings imply that orbital edema in acute TED may be mediated, in part, by both the formation of new, immature blood vessels and the formation of lymphatic capillaries that are functionally incapable of draining interstitial fluid. Copyright © 2016 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

Effect of human vascular endothelial growth factor gene transfer on endogenous vascular endothelial growth factor mRNA expression in a rat fibroblast and osteoblast culture model.

PubMed

Li, Ru; Li, Claire H; Nauth, Aaron; McKee, Michael D; Schemitsch, Emil H

2010-09-01

Vascular endothelial growth factor (VEGF) plays an important role in promoting angiogenesis and osteogenesis during fracture repair. Our previous studies have shown that cell-based VEGF gene therapy enhances bone healing of a rabbit tibia segmental bone defect in vivo. The aim of this project was to examine the effect of exogenous human VEGF on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model. Rat fibroblasts and osteoblasts were harvested from the dermal tissue and periosteum, respectively, of Fisher 344 rats. The cells were then cultured and transfected with pcDNA-human VEGF using Superfect reagent (Qiagen). Four experimental groups were created: 1) fibroblast-VEGF; 2) osteoblast-VEGF; 3) nontransfected fibroblast controls; and 4) nontransfected osteoblast controls. The cultured cells were harvested at 1, 3, and 7 days after the gene transfection. The total mRNA was extracted (Trizol; Invitrogen); both human VEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction and quantified by VisionWorksLS. The human VEGF165 mRNA was detected by reverse transcriptase-polymerase chain reaction from transfected fibroblasts and osteoblasts at 1, 3, and 7 days after gene transfection. The human VEGF165 levels peaked at Day 1 and then gradually reduced expression in both transfected fibroblasts and osteoblasts. Two endogenous rat VEGF isoforms were detected in this cell culture model: rat VEGF120 and rat VEGF164. We compared the rat VEGF120 and rat VEGF164 expression level of the fibroblasts or osteoblasts that were transfected with human VEGF165, with nontransfected control cells. Both the transfected fibroblasts and osteoblasts showed greater expression of rat VEGF164 than nontransfected controls at Day 1 (peak level) and Day 3, but not at Day 7. The expression of rat VEGF120 was lower in transfected fibroblasts, but higher in transfected osteoblasts, than the relevant control groups at any time point after transfection. In addition, human VEGF gene transfection increased osteoblast cell proliferation after 3 days. These in vitro results suggest that cell-based human VEGF gene therapy is not only effective at causing human VEGF expression, but also enhances endogenous rat VEGF mRNA expression in both fibroblasts and osteoblasts, particularly the rat VEGF164 isoform.

Real-time quantification of antibody-short interfering RNA conjugate in serum by antigen capture reverse transcription-polymerase chain reaction.

PubMed

Tan, Martha; Vernes, Jean-Michel; Chan, Joyce; Cuellar, Trinna L; Asundi, Aarati; Nelson, Christopher; Yip, Victor; Shen, Ben; Vandlen, Richard; Siebel, Christian; Meng, Y Gloria

2012-11-15

Short interfering RNA (siRNA) has therapeutic potential. However, efficient delivery is a formidable task. To facilitate delivery of siRNA into cells, we covalently conjugated siRNA to antibodies that bind to cell surface proteins and internalize. Understanding how these antibody-siRNA conjugates function in vivo requires pharmacokinetic analysis. Thus, we developed a simple real-time antigen capture reverse transcription-polymerase chain reaction (RT-PCR) assay to detect intact antibody-siRNA conjugates. Biotinylated antigen bound to streptavidin-coated PCR tubes was used to capture antibody-siRNA conjugate. The captured antibody-siRNA conjugate was then reverse-transcribed in the same tube, avoiding a sample transfer step. This reproducible assay had a wide standard curve range of 0.029 to 480ng/ml and could detect as low as 0.58ng/ml antibody-siRNA conjugates in mouse serum. The presence of unconjugated antibody that could be generated from siRNA degradation in vivo did not affect the assay as long as the total antibody concentration in the antigen capture step did not exceed 480ng/ml. Using this assay, we observed a more rapid decrease in serum antibody-siRNA conjugate concentrations than the total antibody concentrations in mice dosed with antibody-siRNA conjugates, suggesting loss of siRNA from the antibody. This assay is useful for optimizing antibody-siRNA and likely aptamer-siRNA conjugates to improve pharmacokinetics and aid siRNA delivery. Copyright © 2012 Elsevier Inc. All rights reserved.

Antiretroviral Drug Use in a Cross-Sectional Population Survey in Africa: NIMH Project Accept (HPTN 043).

PubMed

Fogel, Jessica M; Clarke, William; Kulich, Michal; Piwowar-Manning, Estelle; Breaud, Autumn; Olson, Matthew T; Marzinke, Mark A; Laeyendecker, Oliver; Fiamma, Agnès; Donnell, Deborah; Mbwambo, Jessie K K; Richter, Linda; Gray, Glenda; Sweat, Michael; Coates, Thomas J; Eshleman, Susan H

2017-02-01

Antiretroviral (ARV) drug treatment benefits the treated individual and can prevent HIV transmission. We assessed ARV drug use in a community-randomized trial that evaluated the impact of behavioral interventions on HIV incidence. Samples were collected in a cross-sectional survey after a 3-year intervention period. ARV drug testing was performed using samples from HIV-infected adults at 4 study sites (Zimbabwe; Tanzania; KwaZulu-Natal and Soweto, South Africa; survey period 2009-2011) using an assay that detects 20 ARV drugs (6 nucleoside/nucleotide reverse transcriptase inhibitors, 3 nonnucleoside reverse transcriptase inhibitors, and 9 protease inhibitors; maraviroc; raltegravir). ARV drugs were detected in 2011 (27.4%) of 7347 samples; 88.1% had 1 nonnucleoside reverse transcriptase inhibitors ± 1-2 nucleoside/nucleotide reverse transcriptase inhibitors. ARV drug detection was associated with sex (women>men), pregnancy, older age (>24 years), and study site (P < 0.0001 for all 4 variables). ARV drugs were also more frequently detected in adults who were widowed (P = 0.006) or unemployed (P = 0.02). ARV drug use was more frequent in intervention versus control communities early in the survey (P = 0.01), with a significant increase in control (P = 0.004) but not in intervention communities during the survey period. In KwaZulu-Natal, a 1% increase in ARV drug use was associated with a 0.14% absolute decrease in HIV incidence (P = 0.018). This study used an objective, biomedical approach to assess ARV drug use on a population level. This analysis identified factors associated with ARV drug use and provided information on ARV drug use over time. ARV drug use was associated with lower HIV incidence at 1 study site.

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.

In vitro and in vivo characterization of the anticancer activity of Thai stingless bee (Tetragonula laeviceps) cerumen

PubMed Central

Nugitrangson, Pongvit; Puthong, Songchan; Iempridee, Tawin; Pimtong, Wittaya; Chanchao, Chanpen

2015-01-01

Tetragonula laeviceps cerumen was sequentially extracted with 80% (v/v) methanol, dichloromethane, and hexane and also in the reverse order. By the MTT assay and the respective 50% inhibition concentration value, the most active fraction was further purified to apparent homogeneity by bioassay-guided silica gel column chromatography. α-Mangostin was identified by high-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance analyses. It had a potent cytotoxicity against the BT474, Chago, Hep-G2, KATO-III, and SW620 cell lines (IC50 values of 1.22 ± 0.03, 2.25 ± 0.20, 0.94 ± 0.01, 0.88 ± 0.16, and 1.50 ± 0.39 µmol/L, respectively). The in vitro cytotoxicity of α-mangostin against the five human cancer cell lines and primary fibroblasts was further characterized by real-time impedance-based analysis. Interestingly, α-mangostin was more cytotoxic against the cancer-derived cell lines than against the primary fibroblasts. Later, the migration assay was performed by continuously measuring the attachment of cells to the plate electrodes at the bottom of the transwell membrane. The combined caspase-3 and -7 activities were assayed by the Caspase-Glo® 3/7 kit. It showed that the cytotoxic mechanism involved caspase-independent apoptosis, while at low (non-toxic) concentrations α-mangostin did not significantly alter cell migration. Furthermore, the in vivo cytotoxicity and angiogenesis were determined by alkaline phosphatase staining in zebrafish embryos along with monitoring changes in the transcript expression level of two genes involved in angiogenesis (vegfaa and vegfr2) by quantitative real-time reverse transcriptase- polymerase chain reaction. It was found that the in vivo cytotoxicity of α-mangostin against zebrafish embryos had a 50% lethal concentration of 9.4 µM, but no anti-angiogenic properties were observed in zebrafish embryos at 9 and 12 µM even though it downregulated the expression of vegfaa and vegfr2 transcripts. Thus, α-mangostin is a major active compound with a potential anticancer activity in T. laeviceps cerumen in Thailand. PMID:26290139

In vitro and in vivo characterization of the anticancer activity of Thai stingless bee (Tetragonula laeviceps) cerumen.

PubMed

Nugitrangson, Pongvit; Puthong, Songchan; Iempridee, Tawin; Pimtong, Wittaya; Pornpakakul, Surachai; Chanchao, Chanpen

2016-01-01

Tetragonula laeviceps cerumen was sequentially extracted with 80% (v/v) methanol, dichloromethane, and hexane and also in the reverse order. By the MTT assay and the respective 50% inhibition concentration value, the most active fraction was further purified to apparent homogeneity by bioassay-guided silica gel column chromatography. α-Mangostin was identified by high-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance analyses. It had a potent cytotoxicity against the BT474, Chago, Hep-G2, KATO-III, and SW620 cell lines (IC50 values of 1.22 ± 0.03, 2.25 ± 0.20, 0.94 ± 0.01, 0.88 ± 0.16, and 1.50 ± 0.39 µmol/L, respectively). The in vitro cytotoxicity of α-mangostin against the five human cancer cell lines and primary fibroblasts was further characterized by real-time impedance-based analysis. Interestingly, α-mangostin was more cytotoxic against the cancer-derived cell lines than against the primary fibroblasts. Later, the migration assay was performed by continuously measuring the attachment of cells to the plate electrodes at the bottom of the transwell membrane. The combined caspase-3 and -7 activities were assayed by the Caspase-Glo® 3/7 kit. It showed that the cytotoxic mechanism involved caspase-independent apoptosis, while at low (non-toxic) concentrations α-mangostin did not significantly alter cell migration. Furthermore, the in vivo cytotoxicity and angiogenesis were determined by alkaline phosphatase staining in zebrafish embryos along with monitoring changes in the transcript expression level of two genes involved in angiogenesis (vegfaa and vegfr2) by quantitative real-time reverse transcriptase- polymerase chain reaction. It was found that the in vivo cytotoxicity of α-mangostin against zebrafish embryos had a 50% lethal concentration of 9.4 µM, but no anti-angiogenic properties were observed in zebrafish embryos at 9 and 12 µM even though it downregulated the expression of vegfaa and vegfr2 transcripts. Thus, α-mangostin is a major active compound with a potential anticancer activity in T. laeviceps cerumen in Thailand. © 2015 by the Society for Experimental Biology and Medicine.

Balancing Antiviral Potency and Host Toxicity: Identifying a Nucleotide Inhibitor with an Optimal Kinetic Phenotype for HIV-1 Reverse Transcriptase

PubMed Central

Sohl, Christal D.; Kasiviswanathan, Rajesh; Kim, Jiae; Pradere, Ugo; Schinazi, Raymond F.; Copeland, William C.; Mitsuya, Hiroaki; Baba, Masanori

2012-01-01

Two novel thymidine analogs, 3′-fluoro-3′-deoxythymidine (FLT) and 2′,3′-didehydro-3′-deoxy-4′-ethynylthymidine (Ed4T), have been investigated as nucleoside reverse transcriptase inhibitors (NRTIs) for treatment of HIV infection. Ed4T seems very promising in phase II clinical trials, whereas toxicity halted FLT development during this phase. To understand these different molecular mechanisms of toxicity, pre–steady-state kinetic studies were used to examine the interactions of FLT and Ed4T with wild-type (WT) human mitochondrial DNA polymerase γ (pol γ), which is often associated with NRTI toxicity, as well as the viral target protein, WT HIV-1 reverse transcriptase (RT). We report that Ed4T-triphosphate (TP) is the first analog to be preferred over native nucleotides by RT but to experience negligible incorporation by WT pol γ, with an ideal balance between high antiretroviral efficacy and minimal host toxicity. WT pol γ could discriminate Ed4T-TP from dTTP 12,000-fold better than RT, with only an 8.3-fold difference in discrimination being seen for FLT-TP. A structurally related NRTI, 2′,3′-didehydro-2′,3′-dideoxythymidine, is the only other analog favored by RT over native nucleotides, but it exhibits only a 13-fold difference (compared with 12,000-fold for Ed4T) in discrimination between the two enzymes. We propose that the 4′-ethynyl group of Ed4T serves as an enzyme selectivity moiety, critical for discernment between RT and WT pol γ. We also show that the pol γ mutation R964C, which predisposes patients to mitochondrial toxicity when receiving 2′,3′-didehydro-2′,3′-dideoxythymidine to treat HIV, produced some loss of discrimination for FLT-TP and Ed4T-TP. These molecular mechanisms of analog incorporation, which are critical for understanding pol γ-related toxicity, shed light on the unique toxicity profiles observed during clinical trials. PMID:22513406

Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences.

EPA Science Inventory

A complete method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for determining interferences to phage recoveries from water sample concentrates and for detecting interferences to their analysis, was developed for the direct...

Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences

EPA Science Inventory

A complete method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for determining interferences to phage recoveries from water sample concentrates and for detecting interferences to their analysis, was developed for the direct...

Structure of HIV-1 nonnucleoside reverse transcriptase inhibitors derivatives of N-benzyl-benzimidazole with different substituents in position 4

NASA Astrophysics Data System (ADS)

Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

2010-01-01

The constant development of new drugs against HIV-1 is necessary due to global expansion of AIDS and HIV-1 drug resistance. Nonnucleoside reverse transcriptase inhibitors of HIV-1 (NNRTIs) are potentially effective and nontoxic drugs in AIDS therapy. The crystal structures of six nonnucleoside inhibitors of HIV-1 reverse transcriptase (RT) derivatives of N-benzyl-benzimidazole are reported here. The investigated compounds belong to the group of so called "butterfly like" inhibitors with characteristic two π-electron moieties with an angled orientation. The structural data show the influence of the substituents of the benzimidazole ring on the geometry of the molecule and correlation between the structure of the inhibitor and its biological activity.

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.

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.

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.

Methylprednisolone therapy in deceased donors reduces inflammation in the donor liver and improves outcome after liver transplantation: a prospective randomized controlled trial.

PubMed

Kotsch, Katja; Ulrich, Frank; Reutzel-Selke, Anja; Pascher, Andreas; Faber, W; Warnick, P; Hoffman, S; Francuski, M; Kunert, C; Kuecuek, O; Schumacher, G; Wesslau, C; Lun, A; Kohler, S; Weiss, S; Tullius, S G; Neuhaus, P; Pratschke, Johann

2008-12-01

To investigate potential beneficial effects of donor treatment with methylprednisolone on organ function and outcome after liver transplantation. It is proven experimentally and clinically that the brain death of the donor leads to increased levels of inflammatory cytokines and is followed by an intensified ischemia/reperfusion injury after organ transplantation. In experiments, donor treatment with steroids successfully diminished these effects and led to better organ function after transplantation. To investigate whether methylprednisolone treatment of the deceased donor is applicable to attenuate brain death-associated damage in clinical liver transplantation we conducted a prospective randomized treatment-versus-control study in 100 deceased donors. Donor treatment (n = 50) consisted of 250 mg methylprednisolone at the time of consent for organ donation and a subsequent infusion of 100 mg/h until recovery of organs. A liver biopsy was taken immediately after laparotomy and blood samples were obtained after brain death diagnosis and before organ recovery. Cytokines were assessed by real-time reverse transcriptase-polymerase chain reaction. Soluble serum cytokines were measured by cytometric bead array system. After methylprednisolone treatment, steroid plasma levels were significantly higher (P < 0.05), and a significant decrease in soluble interleukins, monocyte chemotactic protein-1, interleukin-2, interleukin-6, tumor necrosis factor-alpha, and inducible protein-10 was observed. Methylprednisolone treatment resulted in a significant downregulation of intercellular adhesion molecule-1, tumor necrosis factor-alpha, major histocompatibility complex class II, Fas-ligand, inducible protein-10, and CD68 intragraft mRNA expression. Significantly ameliorated ischemia/reperfusion injury in the posttransplant course was accompanied by a decreased incidence of acute rejection. Our present study verifies the protective effect of methylprednisolone treatment in deceased donor liver transplantation, suggesting it as a potential therapeutical approach.

Characteristics and outcome of mechanically ventilated patients with 2009 H1N1 influenza in Bosnia and Herzegovina and Serbia: impact of newly established multidisciplinary intensive care units.

PubMed

Kojicić, Marija; Kovacević, Pedja; Bajramović, Nermina; Batranović, Uros; Vidović, Jadranka; Aganović, Kenana; Gavrilović, Srdjan; Zlojutro, Biljana; Thiery, Guillaume

2012-12-01

To describe characteristics and outcome of mechanically ventilated patients admitted to three newly established intensive care units (ICU) in Bosnia-Herzegovina and Serbia for 2009 H1N1 influenza infection. The retrospective observational study included all mechanically ventilated adult patients of three university-affiliated hospitals between November 1, 2009 and March 1 2010 who had 2009 H1N1 influenza infection confirmed by real-time reverse transcriptase-polymerase-chain-reaction (RT-PCR) from nasopharyngeal swab specimens and respiratory secretions. The study included 50 patients, 31 male (62%), aged 43±13 years. Median time from hospital to ICU admission was 1 day (range 1-2). Sixteen patients (30%) presented with one or more chronic medical condition: 8 (16%) with chronic lung disease, 5 (10%) with chronic heart failure, and 3 (6%) with diabetes mellitus. Thirty-two (64%) were obese. Forty-eight patients (96%) experienced acute respiratory distress syndrome (ARDS), 28 (56%) septic shock, and 27 (54%) multiorgan failure. Forty-five patients (90%) were intubated and mechanically ventilated, 5 received non-invasive mechanical ventilation, 7 (14%) high-frequency oscillatory ventilation, and 7 (14%) renal replacement therapy. The median duration of mechanical ventilation was 7 (4-14) days. Hospital mortality was 52%. Influenza 2009 H1N1 infection in three southeast European ICUs affected predominantly healthy young patients and was associated with rapid deterioration after hospital admission and severe respiratory and multiorgan failure. These emerging ICUs provided contemporary ICU services, resulting in case-fatality rate comparable to reports from well-established ICU settings.

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.

Irradiation Alters MMP-2/TIMP-2 System and Collagen Type IV Degradation in Brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Won Hee; Warrington, Junie P.; Sonntag, William E.

Purpose: Blood-brain barrier (BBB) disruption is one of the major consequences of radiation-induced normal tissue injury in the central nervous system. We examined the effects of whole-brain irradiation on matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) and extracellular matrix (ECM) degradation in the brain. Methods and Materials: Animals received either whole-brain irradiation (a single dose of 10 Gy {gamma}-rays or a fractionated dose of 40 Gy {gamma}-rays, total) or sham-irradiation and were maintained for 4, 8, and 24 h following irradiation. mRNA expression levels of MMPs and TIMPs in the brain were analyzed by real-time reverse transcriptase-polymerase chain reaction (PCR).more » The functional activity of MMPs was measured by in situ zymography, and degradation of ECM was visualized by collagen type IV immunofluorescent staining. Results: A significant increase in mRNA expression levels of MMP-2, MMP-9, and TIMP-1 was observed in irradiated brains compared to that in sham-irradiated controls. In situ zymography revealed a strong gelatinolytic activity in the brain 24 h postirradiation, and the enhanced gelatinolytic activity mediated by irradiation was significantly attenuated in the presence of anti-MMP-2 antibody. A significant reduction in collagen type IV immunoreactivity was also detected in the brain at 24 h after irradiation. In contrast, the levels of collagen type IV were not significantly changed at 4 and 8 h after irradiation compared with the sham-irradiated controls. Conclusions: The present study demonstrates for the first time that radiation induces an imbalance between MMP-2 and TIMP-2 levels and suggests that degradation of collagen type IV, a major ECM component of BBB basement membrane, may have a role in the pathogenesis of brain injury.« less

Characterization of the small heat shock protein Hsp27 gene in Chironomus riparius (Diptera) and its expression profile in response to temperature changes and xenobiotic exposures.

PubMed

Martínez-Paz, Pedro; Morales, Mónica; Martín, Raquel; Martínez-Guitarte, José Luis; Morcillo, Gloria

2014-07-01

Small heat shock proteins constitute the most diverse and least conserved group within the large family of heat shock proteins, which play a crucial role in cell response to environmental insults. Chironomus riparius larvae are widely used in environmental research for testing pollutant toxicity in sediments and freshwater environments. Different genes, such as Hsp70, Hsc70, Hsp90, and Hsp40, have been identified in this species as sensitive biomarkers for xenobiotics, but small Hsps genes remain largely unknown. In this study, the Hsp27 has been characterized in C. riparius and its transcriptional response evaluated under several environmental stimuli. The Hsp27 gene was mapped by FISH on polytene chromosomes at region I-C4 and was found to encode a 195 aa protein, which contains an α-crystallin domain bounded by three conserved regions. This protein shows homology with Drosophila melanogaster HSP27, Ceratitis capitata HSP27, and Sarcophaga crassipalpis HSP25. Real-time reverse transcriptase-polymerase chain reaction analysis showed that heat shock (35 °C) and cadmium dramatically upregulate this gene. Moreover, exposures to triclosan and bisphenol A were able to significantly increase mRNA levels. However, neither nonylphenol nor tributyltin altered Hsp27 gene expression. The transcriptional activity of Hsp27 gene was modulated during cold stress. Interestingly, cold shock (4 °C) significantly reduced Hsp27 transcripts, but this gene was significantly overexpressed during the recovery time at the normal growing temperature. These results show that the Hsp27 gene is sensitive to different environmental stimuli, including endocrine-disrupting pollutants, suggesting its potential as a suitable biomarker for ecotoxicological studies in aquatic systems.

Mechanisms of Laser-Induced Dissection and Transport of Histologic Specimens

PubMed Central

Vogel, Alfred; Lorenz, Kathrin; Horneffer, Verena; Hüttmann, Gereon; von Smolinski, Dorthe; Gebert, Andreas

2007-01-01

Rapid contact- and contamination-free procurement of histologic material for proteomic and genomic analysis can be achieved by laser microdissection of the sample of interest followed by laser-induced transport (laser pressure catapulting). The dynamics of laser microdissection and laser pressure catapulting of histologic samples of 80 μm diameter was investigated by means of time-resolved photography. The working mechanism of microdissection was found to be plasma-mediated ablation initiated by linear absorption. Catapulting was driven by plasma formation when tightly focused pulses were used, and by photothermal ablation at the bottom of the sample when defocused pulses producing laser spot diameters larger than 35 μm were used. With focused pulses, driving pressures of several hundred MPa accelerated the specimen to initial velocities of 100–300 m/s before they were rapidly slowed down by air friction. When the laser spot was increased to a size comparable to or larger than the sample diameter, both driving pressure and flight velocity decreased considerably. Based on a characterization of the thermal and optical properties of the histologic specimens and supporting materials used, we calculated the evolution of the heat distribution in the sample. Selected catapulted samples were examined by scanning electron microscopy or analyzed by real-time reverse-transcriptase polymerase chain reaction. We found that catapulting of dissected samples results in little collateral damage when the laser pulses are either tightly focused or when the laser spot size is comparable to the specimen size. By contrast, moderate defocusing with spot sizes up to one-third of the specimen diameter may involve significant heat and ultraviolet exposure. Potential side effects are maximal when samples are catapulted directly from a glass slide without a supporting polymer foil. PMID:17766336

Development and deployment of a rapid recombinase polymerase amplification Ebola virus detection assay in Guinea in 2015.

PubMed

Faye, Oumar; Faye, Ousmane; Soropogui, Barré; Patel, Pranav; El Wahed, Ahmed Abd; Loucoubar, Cheikh; Fall, Gamou; Kiory, Davy; Magassouba, N'Faly; Keita, Sakoba; Kondé, Mandy Kader; Diallo, Alpha Amadou; Koivogui, Lamine; Karlberg, Helen; Mirazimi, Ali; Nentwich, Oliver; Piepenburg, Olaf; Niedrig, Matthias; Weidmann, Manfred; Sall, Amadou Alpha

2015-01-01

In the absence of a vaccine or specific treatments for Ebola virus disease (EVD), early identification of cases is crucial for the control of EVD epidemics. We evaluated a new extraction kit (SpeedXtract (SE), Qiagen) on sera and swabs in combination with an improved diagnostic reverse transcription recombinase polymerase amplification assay for the detection of Ebola virus (EBOV-RT-RPA). The performance of combined extraction and detection was best for swabs. Sensitivity and specificity of the combined SE and EBOV-RT-RPA were tested in a mobile laboratory consisting of a mobile glovebox and a Diagnostics-in-a-Suitcase powered by a battery and solar panel, deployed to Matoto Conakry, Guinea as part of the reinforced surveillance strategy in April 2015 to reach the goal of zero cases. The EBOV-RT-RPA was evaluated in comparison to two real-time PCR assays. Of 928 post-mortem swabs, 120 tested positive, and the combined SE and EBOV-RT-RPA yielded a sensitivity and specificity of 100% in reference to one real-time RT-PCR assay. Another widely used real-time RT-PCR was much less sensitive than expected. Results were provided very fast within 30 to 60 min, and the field deployment of the mobile laboratory helped improve burial management and community engagement.

Real-time RT-PCR, a necessary tool to support the diagnosis and surveillance of rotavirus in Mexico.

PubMed

De La Cruz Hernández, Sergio Isaac; Anaya Molina, Yazmin; Gómez Santiago, Fabián; Terán Vega, Heidi Lizbeth; Monroy Leyva, Elda; Méndez Pérez, Héctor; García Lozano, Herlinda

2018-04-01

Rotavirus produces diarrhea in children under 5 years old. Most of those conventional methods such as polyacrylamide gel electrophoresis (PAGE) and reverse transcription-polymerase chain reaction (RT-PCR) have been used for rotavirus detection. However, these techniques need a multi-step process to get the results. In comparison with conventional methods, the real-time RT-PCR is a highly sensitive method, which allows getting the results in only one day. In this study a real-time RT-PCR assay was tested using a panel of 440 samples from patients with acute gastroenteritis, and characterized by PAGE and RT-PCR. The results show that the real-time RT-PCR detected rotavirus from 73% of rotavirus-negative samples analyzed by PAGE and RT-PCR; thus, the percentage of rotavirus-positive samples increased to 81%. The results indicate that this real-time RT-PCR should be part of a routine analysis, and as a support of the diagnosis of rotavirus in Mexico. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantification of simian immunodeficiency virus cytotoxic T lymphocyte escape mutant viruses.

PubMed

Loh, Liyen; Kent, Stephen J

2008-08-01

Escape from cytotoxic T-lymphocyte (CTL) pressure is common in HIV-1 infection of humans and simian immunodeficiency virus (SIV) infections of macaques. CTL escape typically incurs a fitness cost as reversion back to wild-type can occur upon transmission. We utilized sequence-specific primers and DNA probes with real-time polymerase chain reaction (PCR) to sensitively and specifically track wild-type and escape mutant viremia at the Mane-A*17-restricted SIV Gag(371379) epitope AF9 in pigtail macaques. The generation of minor escape mutant populations is detected by the real-time PCR 2 weeks earlier than observed using standard sequencing techniques. We passaged the AF9 CTL escape mutant virus into two naïve Mane-A*17-negative pigtail macaques and showed that reversion to wild-type was rapid during acute infection and then slowed considerably at later stages of the infection. These data help refine our understanding of how CTL escape mutant viruses evolve.

The group II intron maturase: a reverse transcriptase and splicing factor go hand in hand.

PubMed

Zhao, Chen; Pyle, Anna Marie

2017-12-01

The splicing of group II introns in vivo requires the assistance of a multifunctional intron encoded protein (IEP, or maturase). Each IEP is also a reverse-transcriptase enzyme that enables group II introns to behave as mobile genetic elements. During splicing or retro-transposition, each group II intron forms a tight, specific complex with its own encoded IEP, resulting in a highly reactive holoenzyme. This review focuses on the structural basis for IEP function, as revealed by recent crystal structures of an IEP reverse transcriptase domain and cryo-EM structures of an IEP-intron complex. These structures explain how the same IEP scaffold is utilized for intron recognition, splicing and reverse transcription, while providing a physical basis for understanding the evolutionary transformation of the IEP into the eukaryotic splicing factor Prp8. Copyright © 2017 Elsevier Ltd. All rights reserved.

Predicting human immunodeficiency virus inhibitors using multi-dimensional Bayesian network classifiers.

PubMed

Borchani, Hanen; Bielza, Concha; Toro, Carlos; Larrañaga, Pedro

2013-03-01

Our aim is to use multi-dimensional Bayesian network classifiers in order to predict the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and protease inhibitors given an input set of respective resistance mutations that an HIV patient carries. Multi-dimensional Bayesian network classifiers (MBCs) are probabilistic graphical models especially designed to solve multi-dimensional classification problems, where each input instance in the data set has to be assigned simultaneously to multiple output class variables that are not necessarily binary. In this paper, we introduce a new method, named MB-MBC, for learning MBCs from data by determining the Markov blanket around each class variable using the HITON algorithm. Our method is applied to both reverse transcriptase and protease data sets obtained from the Stanford HIV-1 database. Regarding the prediction of antiretroviral combination therapies, the experimental study shows promising results in terms of classification accuracy compared with state-of-the-art MBC learning algorithms. For reverse transcriptase inhibitors, we get 71% and 11% in mean and global accuracy, respectively; while for protease inhibitors, we get more than 84% and 31% in mean and global accuracy, respectively. In addition, the analysis of MBC graphical structures lets us gain insight into both known and novel interactions between reverse transcriptase and protease inhibitors and their respective resistance mutations. MB-MBC algorithm is a valuable tool to analyze the HIV-1 reverse transcriptase and protease inhibitors prediction problem and to discover interactions within and between these two classes of inhibitors. Copyright © 2012 Elsevier B.V. All rights reserved.

Nevirapine resistance mutation at codon 181 of the HIV-1 reverse transcriptase confers stavudine resistance by increasing nucleotide substrate discrimination and phosphorolytic activity.

PubMed

Blanca, Giuseppina; Baldanti, Fausto; Paolucci, Stefania; Skoblov, Alexander Yu; Victorova, Lyubov; Hübscher, Ulrich; Gerna, Giuseppe; Spadari, Silvio; Maga, Giovanni

2003-05-02

Recombinant HIV-1 reverse transcriptase (RT) carrying non-nucleoside inhibitors (NNRTIs) resistance mutation at codon 181 showed reduced incorporation and high efficiency of phosphorolytic removal of stavudine, a nucleoside RT inhibitor. These results reveal a new mechanism for cross-resistance between different classes of HIV-1 RT inhibitors.

Applicability of integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) for the simultaneous detection of the four human enteric enterovirus species in disinfection studies

EPA Science Inventory

A newly developed integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method and its applicability in UV disinfection studies is described. This method utilizes a singular cell culture system coupled with four RTqPCR assays to detect infectious serotypes t...

Nonnucleoside reverse transcriptase inhibitor phenotypic hypersusceptibility can be demonstrated in different assays.

PubMed

Shulman, Nancy S; Delgado, Jamael; Bosch, Ronald J; Winters, Mark A; Johnston, Elizabeth; Shafer, Robert W; Katzenstein, David A; Merigan, Thomas C

2005-05-01

HIV-1 isolates harboring multiple nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations are more susceptible ("hypersusceptible") to the nonnucleoside reverse transcriptase inhibitors (NNRTIs) than isolates lacking NRTI resistance mutations, but this has only been reported with a single-cycle replication phenotypic assay. In fact, there was a report that a commercial multicycle assay did not readily detect hypersusceptibility. To see whether NNRTI hypersusceptibility can be demonstrated in other types of phenotypic assays, including multicycle assays and enzyme inhibition assays. The susceptibility of HIV-1 clones derived from different patients in multicycle assays was tested in peripheral blood mononuclear cells (PBMCs) and in an established cell line. In addition, the reverse transcriptase (RT) of many of these clones was expressed and their susceptibility tested in an RT inhibition assay. Nevirapine and efavirenz susceptibilities were tested and compared with a control wild-type virus or RT. Hypersusceptibility to nevirapine and efavirenz was detected using each of the methods described above. R values correlating the other methods with single-cycle assay values were between 0.66 and 0.96. In addition to the high correlations, the different methods gave similar numeric results. NNRTI hypersusceptibility is readily seen in multicycle susceptibility assays and in enzyme inhibition assays.

Immortalization of pig fibroblast cells by transposon-mediated ectopic expression of porcine telomerase reverse transcriptase.

PubMed

He, Shan; Li, Yangyang; Chen, Yang; Zhu, Yue; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

2016-08-01

Pigs are the most economically important livestock, but pig cell lines useful for physiological studies and/or vaccine development are limited. Although several pig cell lines have been generated by oncogene transformation or human telomerase reverse transcriptase (TERT) immortalization, these cell lines contain viral sequences and/or antibiotic resistance genes. In this study, we established a new method for generating pig cell lines using the Sleeping Beauty (SB) transposon-mediated ectopic expression of porcine telomerase reverse transcriptase (pTERT). The performance of the new method was confirmed by generating a pig fibroblast cell (PFC) line. After transfection of primary PFCs with the SB transposon system, one cell clone containing the pTERT expression cassette was selected by dilution cloning and passed for different generations. After passage for more than 40 generations, the cell line retained stable expression of ectopic pTERT and continuous growth potential. Further characterization showed that the cell line kept the fibroblast morphology, growth curve, population doubling time, cloning efficiency, marker gene expression pattern, cell cycle distribution and anchorage-dependent growth property of the primary cells. These data suggest that the new method established is useful for generating pig cell lines without viral sequence and antibiotic resistant gene.

Quantitative gene expression deregulation in mantle-cell lymphoma: correlation with clinical and biologic factors.

PubMed

Kienle, Dirk; Katzenberger, Tiemo; Ott, German; Saupe, Doreen; Benner, Axel; Kohlhammer, Holger; Barth, Thomas F E; Höller, Sylvia; Kalla, Jörg; Rosenwald, Andreas; Müller-Hermelink, Hans Konrad; Möller, Peter; Lichter, Peter; Döhner, Hartmut; Stilgenbauer, Stephan

2007-07-01

There is evidence for a direct role of quantitative gene expression deregulation in mantle-cell lymphoma (MCL) pathogenesis. Our aim was to investigate gene expression associations with other pathogenic factors and the significance of gene expression in a multivariate survival analysis. Quantitative expression of 20 genes of potential relevance for MCL prognosis and pathogenesis were analyzed using real-time reverse transcriptase polymerase chain reaction and correlated with clinical and genetic factors, tumor morphology, and Ki-67 index in 65 MCL samples. Genomic losses at the loci of TP53, RB1, and P16 were associated with reduced transcript levels of the respective genes, indicating a gene-dosage effect as the pathomechanism. Analysis of gene expression correlations between the candidate genes revealed a separation into two clusters, one dominated by proliferation activators, another by proliferation inhibitors and regulators of apoptosis. Whereas only weak associations were identified between gene expression and clinical parameters or blastoid morphology, several genes were correlated closely with the Ki-67 index, including the short CCND1 variant (positive correlation) and RB1, ATM, P27, and BMI (negative correlation). In multivariate survival analysis, expression levels of MYC, MDM2, EZH2, and CCND1 were the strongest prognostic factors independently of tumor proliferation and clinical factors. These results indicate a pathogenic contribution of several gene transcript levels to the biology and clinical course of MCL. Genes can be differentiated into factors contributing to proliferation deregulation, either by enhancement or loss of inhibition, and proliferation-independent factors potentially contributing to MCL pathogenesis by apoptosis impairment.

Mechanical tensile stress effects on the expression of bone sialoprotein in bovine cementoblasts.

PubMed

Yu, Hongyou; Ren, Yijin; Sandham, Andrew; Ren, Aishu; Huang, Lan; Bai, Ding

2009-03-01

To develop a new cementoblast culture method and to detect bone sialoprotein (BSP) expression in response to high and low mechanical tensile stress in cementoblast in vitro. Cementoblasts were collected from the roots of newborn bovine teeth and were identified with cementum-derived attachment protein (CAP) antibody 3G9. Cell proliferation was evaluated by MTT [3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, and mineralization was confirmed by von Kossa staining. Mechanical tensile stress was applied in vitro to the cementoblast with the use of a uniaxial four-point bending system with 2000 or 4000 microstrains, at a frequency of 0.5 Hz for 3, 6, 12, 24, or 36 hours. BSP mRNA level was quantified by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). A large amount of cementoblast was observed to be expressing CAP. Cementoblasts had a proliferation tendency similar to that of osteoblasts but different from that of periodontal ligament (PDL) cells. Cementoblasts had the ability to become mineralized between osteoblasts and PDL cells. The mechanical tensile stress significantly up-regulated BSP mRNA expression, which reached a peak at 24 hours in both 2000 and 4000 microstrain groups (P < .01) and was tenfold and sixfold higher than that of controls, respectively. BSP expression dropped toward baseline levels at 36 hours in both groups. Mechanical tensile stress up-regulated the expression of BSP. Low mechanical tensile stress induced earlier and more intensive up-regulation of BSP mRNA; this might represent the optimal stimuli for cementoblast activity.

Human Platelet Lysate as a Replacement for Fetal Bovine Serum in Limbal Stem Cell Therapy.

PubMed

Suri, Kunal; Gong, Hwee K; Yuan, Ching; Kaufman, Stephen C

2016-10-01

To evaluate the use of human platelet lysate (HPL) as an alternative supplement for limbal explant culture. Culture media were prepared using either 10% pooled HPL (PHPL), single donor HPL, or fetal bovine serum (FBS). Limbal tissues, obtained from the Minnesota Lions Eye Bank, were cultured in each medium on plastic plates or on denuded amniotic membrane (AM). Immunofluorescence staining was performed for ABCG2, tumor protein p63α, and cytokeratin 3 (K3). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to evaluate the expression of ABCG2 and p63. Limbal explants grown in each medium were labeled with bromodeoxyuridine (BrdU) to assess the proliferative capacity in each medium. Concentration of growth factors including epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and platelet derived growth factor (PDGF) in HPL and PHPL was compared to that in human serum (HS). Immunofluorescence staining on AM showed prominent expression of ABCG2, p63α but sparse expression of K3 in HPL and PHPL supplemented medium. Real time-PCR showed 1.7 fold higher expression of ABCG2 in PHPL supplemented medium (p = 0.03), and similar expression of p63 in HPL and PHPL supplemented medium compared to FBS medium. The proliferation assay showed that LSCs retained their proliferative potential in HPL supplemented medium. Higher concentration of growth factors were found in HPL, compared to HS. Human platelet lysate has higher concentration of grown factors and is effective in maintaining growth and stem cell phenotype of corneal limbal explant cultures.

Local matrix metalloproteinase 2 gene knockdown in balloon-injured rabbit carotid arteries using nonviral-small interfering RNA transfection.

PubMed

Hlawaty, Hanna; San Juan, Aurélie; Jacob, Marie-Paule; Vranckx, Roger; Letourneur, Didier; Feldman, Laurent J

2009-01-01

Small interfering RNA (siRNA) delivery is a promising approach for the treatment of cardiovascular diseases. Matrix metalloproteinase (MMP) 2 over-expression in the arterial wall has been implicated in restenosis after percutaneous coronary intervention, as well as in spontaneous atherosclerotic plaque rupture. We hypothesized that in vivo local delivery of siRNA targeted at MMP2 (MMP2-siRNA) in the balloon-injured carotid artery of hypercholesterolemic rabbits may lead to inhibition of MMP2 expression. Two weeks after balloon injury, 5 micromol/l of Tamra-tagged MMP2-siRNA, scramble siRNA or saline was locally injected in the carotid artery and incubated for 1 h. Fluorescent microscopy studies showed the circumferential uptake of siRNA in the superficial layers of neointimal cells. MMP2 mRNA levels, measured by the real-time reverse transcriptase-polymerase chain reaction, was decreased by 79 +/- 25% in MMP2-siRNA- versus scramble siRNA-transfected arteries (p < 0.05). MMP2 activity, measured by gelatin zymography performed on the conditioned media of MMP2-siRNA versus scramble siRNA transfected arteries, decreased by 53 +/- 29%, 50 +/- 24% and 46 +/- 14% at 24, 48 and 72 h, respectively (p < 0.005 for all). No effect was observed on MMP9, pro-MMP9 and TIMP-2 levels. The results obtained in the present study suggest that significant inhibition of gene expression can be achieved with local delivery of siRNA in the arterial wall in vivo. (c) 2008 John Wiley & Sons, Ltd.

Improving statistical inference on pathogen densities estimated by quantitative molecular methods: malaria gametocytaemia as a case study.

PubMed

Walker, Martin; Basáñez, María-Gloria; Ouédraogo, André Lin; Hermsen, Cornelus; Bousema, Teun; Churcher, Thomas S

2015-01-16

Quantitative molecular methods (QMMs) such as quantitative real-time polymerase chain reaction (q-PCR), reverse-transcriptase PCR (qRT-PCR) and quantitative nucleic acid sequence-based amplification (QT-NASBA) are increasingly used to estimate pathogen density in a variety of clinical and epidemiological contexts. These methods are often classified as semi-quantitative, yet estimates of reliability or sensitivity are seldom reported. Here, a statistical framework is developed for assessing the reliability (uncertainty) of pathogen densities estimated using QMMs and the associated diagnostic sensitivity. The method is illustrated with quantification of Plasmodium falciparum gametocytaemia by QT-NASBA. The reliability of pathogen (e.g. gametocyte) densities, and the accompanying diagnostic sensitivity, estimated by two contrasting statistical calibration techniques, are compared; a traditional method and a mixed model Bayesian approach. The latter accounts for statistical dependence of QMM assays run under identical laboratory protocols and permits structural modelling of experimental measurements, allowing precision to vary with pathogen density. Traditional calibration cannot account for inter-assay variability arising from imperfect QMMs and generates estimates of pathogen density that have poor reliability, are variable among assays and inaccurately reflect diagnostic sensitivity. The Bayesian mixed model approach assimilates information from replica QMM assays, improving reliability and inter-assay homogeneity, providing an accurate appraisal of quantitative and diagnostic performance. Bayesian mixed model statistical calibration supersedes traditional techniques in the context of QMM-derived estimates of pathogen density, offering the potential to improve substantially the depth and quality of clinical and epidemiological inference for a wide variety of pathogens.

Correlations between A/H1N1 influenza and acute cellular rejection in liver transplantation patients.

PubMed

Stucchi, R S B; Boin, I F S F; Angerami, R Nogueira; Sinckoc, V; Sa, F Cesar; Seva-Pereira, T; Escanhoela, C A Fazzio

2010-12-01

Influenza is a common cause of respiratory infection in transplant recipients. It is expected that A/H1N1 influenza virus causes more severe disease in solid-organ recipients. Our goal was to describe two A/H1N1 infections that occurred after Orthotopic liver transplantation followed by acute allograft rejection episodes. From March 2009 to March 2010 we observe two liver transplant patients with symptoms suggestive of A/H1N1 infection. The diagnosis was out based on a temperature of 37.8°C (100°F) or higher and the presence of a cough or using materials from anasopharyngeal and oropharyngeal swabs a sore throat. The diagnosis was confirmed by viral RNA detection by real-time reverse-transcriptase-polymerase-chain-reaction assay (RT-PCR) using materials from nasopharyngeal and oropharyngeal swabs. We performed the RT-PCR assay for A/H1N1 detection in a liver biopsy from one patient. Both patients were treated with usual doses of oseltamivir (75 mg twice daily for 5 days). One patient developed acute bacterial sinusitis requiring antibiotic therapy. Thereafter the liver enzymes increased and transplant biopsies showed moderate-to-severe acute cellular rejection. They were treated with corticosteroids. The liver enzymes normalized after 3 months. A/H1N1 influenza can lead to a severe acute cellular rejection episode with corticosteroid resistant treatment in liver transplant patients. Transplant centers should be aware of a possible relationship between A/H1N1 infections and acute allograft rejection episodes. Copyright © 2010 Elsevier Inc. All rights reserved.

Proteus vulgaris and Proteus mirabilis Decrease Candida albicans Biofilm Formation by Suppressing Morphological Transition to Its Hyphal Form.

PubMed

Lee, Kyoung Ho; Park, Su Jung; Choi, Sun Ju; Park, Joo Young

2017-11-01

Candida albicans (C. albicans) and Proteus species are causative agents in a variety of opportunistic nosocomial infections, and their ability to form biofilms is known to be a virulence factor. In this study, the influence of co-cultivation with Proteus vulgaris (P. vulgaris) and Proteus mirabilis (P. mirabilis) on C. albicans biofilm formation and its underlying mechanisms were examined. XTT reduction assays were adopted to measure biofilm formation, and viable colony counts were performed to quantify yeast growth. Real-time reverse transcriptase polymerase chain reaction was used to evaluate the expression of yeast-specific genes (rhd1 and rbe1), filament formation inhibiting genes (tup1 and nrg1), and hyphae-related genes (als3, ece1, hwp1, and sap5). Candida biofilm formation was markedly inhibited by treatment with either living or heat-killed P. vulgaris and P. mirabilis. Proteus-cultured supernatant also inhibited Candida biofilm formation. Likewise, treatment with live P. vulgaris or P. mirabilis or with Proteus-cultured supernatant decreased expression of hyphae-related C. albicans genes, while the expression of yeast-specific genes and the filament formation inhibiting genes of C. albicans were increased. Heat-killed P. vulgaris and P. mirabilis treatment, however, did not affect the expression of C. albicans morphology-related genes. These results suggest that secretory products from P. vulgaris and P. mirabilis regulate the expression of genes related to morphologic changes in C. albicans such that transition from the yeast form to the hyphal form can be inhibited. © Copyright: Yonsei University College of Medicine 2017

Regulatory and activated effector T cells in chronic hepatitis C virus: Relation to autoimmunity

PubMed Central

Fouad, Hanan; El Raziky, Maissa; Hassan, Eman Medhat; Aziz, Ghada Mahmoud Abdel; Darweesh, Samar K; Sayed, Ahmed Reda

2016-01-01

AIM To investigate how Tregs are regulated in chronic hepatitis C virus (HCV) patients via assessment of Tregs markers (granzyme 2, CD69 and FoxP3), Teffs markers [TNFRSF4 (OX40), INFG] and CD4, CD25 genes. METHODS A prospective study was conducted on 120 subjects divided into 4 groups: Group I (n = 30) treatment naïve chronic HCV patients; Group II (n = 30) chronic HCV treated with Peg/Riba; Group III (n = 30) chronic HCV associated with non-organ specific autoantibody and Group IV (n = 30) healthy persons as a control group. Tregs and Teffs markers were assessed in peripheral blood mononuclear cells by quantitative real time reverse transcriptase-polymerase chain reaction. RESULTS Chronic HCV patients exhibited significant higher levels of both Teffs and Tregs in comparison to healthy control group. Tregs markers were significantly decreased in Peg/Riba treated HCV patients in comparison to treatment naïve HCV group. In HCV patients with antinuclear antibody (ANA) +ve, Tregs markers were significantly decreased in comparison to all other studied groups. Teffs markers were significantly elevated in all HCV groups in comparison to control and in HCV group with ANA +ve in comparison to treatment naïve HCV group. CONCLUSION Elevated Tregs cells in chronic HCV patients dampen both CD4+ and CD8+ autologous T cell immune response. Interferon-α and ribavirin therapy suppress proliferation of Tregs. More significant suppression of Tregs was observed in HCV patients with autoantibodies favoring pathological autoimmune response. PMID:27843539

[A case of human infection with highly pathogenic avian influenza A (H7N9) virus through poultry processing without protection measure].

PubMed

Ma, Y; Zhang, Z B; Cao, L; Lu, J Y; Li, K B; Su, W Z; Li, T G; Yang, Z C; Wang, M

2018-06-10

Objective: To investigate the infection pattern and etiological characteristics of a case of human infection with highly pathogenic avian influenza A (H7N9) virus and provide evidence for the prevention and control of human infection with highly pathogenic avian influenza virus. Methods: Epidemiological investigation was conducted to explore the case's exposure history, infection route and disease progression. Samples collected from the patient, environments and poultry were tested by using real time reverse transcriptase-polymerase chain reaction (RT-PCR). Virus isolation, genome sequencing and phylogenetic analysis were conducted for positive samples. Results: The case had no live poultry contact history, but had a history of pulled chicken processing without taking protection measure in an unventilated kitchen before the onset. Samples collected from the patient's lower respiratory tract, the remaining frozen chicken meat and the live poultry market were all influenza A (H7N9) virus positive. The isolated viruses from these positive samples were highly homogenous. An insertion which lead to the addition of multiple basic amino acid residues (PEVPKRKRTAR/GL) was found at the HA cleavage site, suggesting that this virus might be highly pathogenic. Conclusions: Live poultry processing without protection measure is an important infection mode of "poultry to human" transmission of avian influenza viruses. Due to the limitation of protection measures in live poultry markets in Guangzhou, it is necessary to promote the standardized large scale poultry farming, the complete restriction of live poultry sales and centralized poultry slaughtering as well as ice fresh sale.

Molecular characterization, mRNA expression of prolactin receptor (PRLR) gene during pregnancy, nonpregnancy in the yak (Bos grunniens).

PubMed

Zi, Xiang-Dong; Chen, Da-Wen; Wang, Hong-Mei

2012-02-01

Prolactin (PRL) plays central roles in a wide range of body functions in mammals, and the actions are mediated by the specific cell surface receptor, the prolactin receptor (PRLR). To better understand the role of PRL in the yak (Bos grunniens), in the present study, we first cloned yak PRLR cDNA, and compared its mRNA expression in several tissues with cattle (Bos taurus). By reverse transcriptase-polymerase chain reaction (RT-PCR) strategy, we obtained full-length of yak PRLR cDNA sequence comprised of an open reading frame of 1746bp encoding a 581 amino acid protein, and contained a signal sequence and a transmembrane region. The intracellular domain had two pairs of cysteine residues and a WSXWS motif. The cytoplasmic domain comprised 323 residues and contained box 1 sequence. The yak PRLR shared 66.0-98.5% protein sequence identity with mammalian homologs. Real-time PCR analysis revealed that PRLR mRNA was higher in mammary tissue than in ovary and endometrium (P<0.01). During pregnancy, the ovary and mammary PRLR mRNA expression increased by 33- and 2.9-fold in yak, respectively, and increased by 46- and 3.8-fold in cattle, respectively. PRLR mRNA expression was higher (P<0.05) in mammary tissue and ovary of pregnant cow than that of pregnant yak. It is proposed that the increased ovarian and mammary PRLR mRNA expression during pregnancy may be associated with corpus luteum function for maintenance of pregnancy and mammary development for subsequent lactation. Copyright © 2011 Elsevier Inc. All rights reserved.

[Comparison of detection sensitivity in rapid-diagnosis influenza virus kits].

PubMed

Tokuno, Osamu; Fujiwara, Miki; Nakajoh, Yoshimi; Yamanouchi, Sumika; Adachi, Masayo; Ikeda, Akiko; Kitayama, Shigeo; Takahashi, Toshio; Kase, Tetsuo; Kinoshita, Shouhiro; Kumagai, Shunichi

2009-09-01

Rapid-diagnosis kits able to detect influenza A and B virus by immunochromatography developed by different manufacturers, while useful in early diagnosis, may vary widely in detection sensitivity. We compared sensitivity results for eight virus-detection kits in current use--Quick Chaser FluA, B (Mizuho Medy), Espline Influenza A & B-N (Fujirebio), Capilia Flu A + B (Nippon Beckton Dickinson & Alfesa Pharma), Poctem Influenza A/B (Otsuka Pharma & Sysmex), BD Flu Examan (Nippon Beckton Dickinson), Quick Ex-Flu "Seiken" (Denka Seiken), Quick Vue Rapid SP Influ (DP Pharma Biomedical), and Rapid Testa FLU stick (Daiichi Pure Chemicals)--against influenza virus stocks, contained five vaccination strains (one A/H1N1, two A/H3N2, and two B) and six clinical strains (two A/H1N1, two A/H3N2, and two B). Minimum detection concentrations giving immunologically positive signals in serial dilution and RNA copies in positive dilution in real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were assayed for all kits and virus stock combinations. RNA log10 copy numbers/mL in dilutions within detection limits yielded 5.68-7.02, 6.37-7,17, and 6.5-8.13 for A/H1N1, A/H3N2, and B. Statistically significant differences in sensitivity were observed between some kit combinations. Detection sensitivity tended to be relatively higher for influenza A than B virus. This is assumed due to different principles in kit methods, such as monoclonal antibodies, specimen-extraction conditions, and other unknown factors.

The natural furanone (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone disrupts quorum sensing-regulated gene expression in Vibrio harveyi by decreasing the DNA-binding activity of the transcriptional regulator protein luxR.

PubMed

Defoirdt, Tom; Miyamoto, Carol M; Wood, Thomas K; Meighen, Edward A; Sorgeloos, Patrick; Verstraete, Willy; Bossier, Peter

2007-10-01

This study aimed at getting a deeper insight in the molecular mechanism by which the natural furanone (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone disrupts quorum sensing in Vibrio harveyi. Bioluminescence experiments with signal molecule receptor double mutants revealed that the furanone blocks all three channels of the V. harveyi quorum sensing system. In further experiments using mutants with mutations in the quorum sensing signal transduction pathway, the compound was found to block quorum sensing-regulated bioluminescence by interacting with a component located downstream of the Hfq protein. Furthermore, reverse transcriptase real-time polymerase chain reaction with specific primers showed that there was no effect of the furanone on luxR(Vh) mRNA levels in wild-type V. harveyi cells. In contrast, mobility shift assays showed that in the presence of the furanone, significantly lower levels of the LuxR(Vh) response regulator protein were able to bind to its target promoter sequences in wild-type V. harveyi. Finally, tests with purified LuxR(Vh) protein also showed less shifts with furanone-treated LuxR(Vh), whereas the LuxR(Vh) concentration was found not to be altered by the furanone (as determined by SDS-PAGE). Therefore, our data indicate that the furanone blocks quorum sensing in V. harveyi by rendering the quorum sensing master regulator protein LuxR(Vh) unable to bind to the promoter sequences of quorum sensing-regulated genes.

Quantitative estimation of Nipah virus replication kinetics in vitro

PubMed Central

Chang, Li-Yen; Ali, AR Mohd; Hassan, Sharifah Syed; AbuBakar, Sazaly

2006-01-01

Background Nipah virus is a zoonotic virus isolated from an outbreak in Malaysia in 1998. The virus causes infections in humans, pigs, and several other domestic animals. It has also been isolated from fruit bats. The pathogenesis of Nipah virus infection is still not well described. In the present study, Nipah virus replication kinetics were estimated from infection of African green monkey kidney cells (Vero) using the one-step SYBR® Green I-based quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) assay. Results The qRT-PCR had a dynamic range of at least seven orders of magnitude and can detect Nipah virus from as low as one PFU/μL. Following initiation of infection, it was estimated that Nipah virus RNA doubles at every ~40 minutes and attained peak intracellular virus RNA level of ~8.4 log PFU/μL at about 32 hours post-infection (PI). Significant extracellular Nipah virus RNA release occurred only after 8 hours PI and the level peaked at ~7.9 log PFU/μL at 64 hours PI. The estimated rate of Nipah virus RNA released into the cell culture medium was ~0.07 log PFU/μL per hour and less than 10% of the released Nipah virus RNA was infectious. Conclusion The SYBR® Green I-based qRT-PCR assay enabled quantitative assessment of Nipah virus RNA synthesis in Vero cells. A low rate of Nipah virus extracellular RNA release and low infectious virus yield together with extensive syncytial formation during the infection support a cell-to-cell spread mechanism for Nipah virus infection. PMID:16784519

Prevalence of influenza virus among the paediatric population in Mumbai during 2007-2009.

PubMed

Roy, S; Patil, D; Dahake, R; Mukherjee, S; Athlekar, S V; Deshmukh, R A; Chowdhary, A

2012-01-01

Influenza has a major impact on public heath, annually affecting 15-20% of the global population. Information on the activity of influenza virus in Mumbai is limited. The present study was carried out to determine the prevalence of influenza viruses causing acute respiratory infections in children by molecular methods. To study the prevalence of influenza viruses among the paediatric population in Mumbai by real-time reverse-transcriptase polymerase chain reaction (rRT-PCR). From July 2007 to July 2009, 100 respiratory samples (nasal and throat swabs) were collected from paediatric patients with acute respiratory symptoms. attending out patients department, and admitted to the paediatric wards of B. J. Wadia Hospital for Children, Mumbai. The samples were collected and processed as per World Health Organization (WHO) guidelines. Viral RNA was extracted and one-step rRT-PCR was performed to detect influenza type A (H1 and H3) and influenza type B virus. Out of 100 samples processed by rRT-PCR, a total of 11 samples (11%) were positive for influenza virus. The typing for influenza A subtypes showed 1% (1) positivity for H1 and 5% (5) positivity for H3 subtypes and 5% (5) samples tested positive for influenza type B virus. It was observed that both influenza type A and B viruses were prevalent in Mumbai during the study period. Such surveillance data are important in the early detection of any antigenic variants that may be helpful in global influenza vaccine preparation and for any pandemic preparedness activity.

Immunomodulatory Effects of Balneotherapy with Hae-Un-Dae Thermal Water on Imiquimod-Induced Psoriasis-Like Murine Model

PubMed Central

Lee, Young Bok; Lee, Jun Young; Lee, Hye Jin; Yun, Seong Taek; Lee, Jong Tae; Kim, Hong Jig; Yu, Dong Soo

2014-01-01

Background Balneotherapy, although not a well-established dermatological treatment, is thought to have therapeutic properties for psoriasis and is used as an alternative treatment modality throughout the world. Objective To evaluate the mechanism underlying the therapeutic immunologic effects of thermomineral water. Methods A murine model of imiquimod-induced psoriasis-like skin inflammation was used for evaluating the therapeutic effects of balneotherapy with Hae-Un-Dae hot spring mineral water. The clinical improvements were evaluated by a dermatologist. Lesional cytokines, including interleukin (IL)-17A, IL-23, and IL-22, were quantitatively measured by real-time reverse transcriptase polymerase chain reaction. Serum levels of interferon-γ, IL-4, IL-5, and IL-17A were measured by enzyme-linked immunosorbent assay. T cell proportions in the spleen were evaluated by flow cytometry, and histopathological evaluation of the skin was also performed. Results The mineral water balneotherapy group showed faster improvement in skin erythema and scales than the distilled water bathing group. A substantial reduction was observed in the lesional mRNA levels of IL-17A and IL-23 in the mineral water group. Serum levels of IL-4 and IL-5 were significantly decreased in the mineral water group but not in the distilled water group. Normalized T cell proportions were observed after bathing. Conclusion Balneotherapy showed immunomodulatory effects in a psoriasis-like murine model. Balneotherapy suppressed lesional IL-23 and IL-17A, which are important cytokines in the pathogenesis of psoriasis. These results suggest that balneotherapy can be used as an effective and safe treatment for psoriasis. PMID:24882978

Immunomodulatory effects of balneotherapy with hae-un-dae thermal water on imiquimod-induced psoriasis-like murine model.

PubMed

Lee, Young Bok; Lee, Jun Young; Lee, Hye Jin; Yun, Seong Taek; Lee, Jong Tae; Kim, Hong Jig; Yu, Dong Soo; Woo, So Youn; Kim, Jin-Wou

2014-04-01

Balneotherapy, although not a well-established dermatological treatment, is thought to have therapeutic properties for psoriasis and is used as an alternative treatment modality throughout the world. To evaluate the mechanism underlying the therapeutic immunologic effects of thermomineral water. A murine model of imiquimod-induced psoriasis-like skin inflammation was used for evaluating the therapeutic effects of balneotherapy with Hae-Un-Dae hot spring mineral water. The clinical improvements were evaluated by a dermatologist. Lesional cytokines, including interleukin (IL)-17A, IL-23, and IL-22, were quantitatively measured by real-time reverse transcriptase polymerase chain reaction. Serum levels of interferon-γ, IL-4, IL-5, and IL-17A were measured by enzyme-linked immunosorbent assay. T cell proportions in the spleen were evaluated by flow cytometry, and histopathological evaluation of the skin was also performed. The mineral water balneotherapy group showed faster improvement in skin erythema and scales than the distilled water bathing group. A substantial reduction was observed in the lesional mRNA levels of IL-17A and IL-23 in the mineral water group. Serum levels of IL-4 and IL-5 were significantly decreased in the mineral water group but not in the distilled water group. Normalized T cell proportions were observed after bathing. Balneotherapy showed immunomodulatory effects in a psoriasis-like murine model. Balneotherapy suppressed lesional IL-23 and IL-17A, which are important cytokines in the pathogenesis of psoriasis. These results suggest that balneotherapy can be used as an effective and safe treatment for psoriasis.

Blast induces oxidative stress, inflammation, neuronal loss and subsequent short-term memory impairment in rats.

PubMed

Cho, H J; Sajja, V S S S; Vandevord, P J; Lee, Y W

2013-12-03

Molecular and cellular mechanisms of brain injury after exposure to blast overpressure (BOP) are not clearly known. The present study hypothesizes that pro-oxidative and pro-inflammatory pathways in the brain may be responsible for neuronal loss and behavioral deficits following BOP exposure. Male Sprague-Dawley rats were anesthetized and exposed to calibrated BOP of 129.23±3.01kPa while controls received only anesthesia. In situ dihydroethidium fluorescence staining revealed that BOP significantly increased the production of reactive oxygen species in the brain. In addition, real-time reverse transcriptase-polymerase chain reaction, immunofluorescence staining and enzyme-linked immunosorbent assay demonstrated a significant up-regulation of mRNA and protein expressions of pro-inflammatory mediators, such as interferon-γ and monocyte chemoattractant protein-1, in brains collected from BOP-exposed animals compared with the controls. Furthermore, immunoreactivity of neuronal nuclei in brains indicated that fewer neurons were present following BOP exposure. Moreover, novel object recognition paradigm showed a significant impairment in the short-term memory at 2weeks following BOP exposure. These results suggest that pro-oxidative and pro-inflammatory environments in the brain could play a potential role in BOP-induced neuronal loss and behavioral deficits. It may provide a foundation for defining a molecular and cellular basis of the pathophysiology of blast-induced neurotrauma (BINT). It will also contribute to the development of new therapeutic approaches selectively targeting these pathways, which have great potential in the diagnosis and therapy of BINT. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Tissue Phthalate Levels Correlate With Changes in Immune Gene Expression in a Population of Juvenile Wild Salmon.

PubMed

Martins, Kelly; Hagedorn, Birgit; Ali, Shareen; Kennish, John; Applegate, Ben; Leu, Matthias; Epp, Lidia; Pallister, Chris; Zwollo, Patty

2016-07-01

Phthalates have detrimental effects on health and have been shown to dysregulate the immune system of mammals, birds, and fish. We recently reported that di(2-ethylhexyl) phthalate exposure reduces the abundance and inhibits the proliferation of rainbow trout (Oncorhynchus mykiss) IgM(+) B lymphocytes and expression of secreted immunoglobulin heavy-chain mu transcripts in an in vitro culture system. We proposed that phthalates act as immunomodulators by modifying the normal B cell-activation pathways by accelerating B cell differentiation while suppressing plasmablast expansion, thus resulting in fewer IgM-secreting plasma cells. This hypothesis was tested here in an in vivo field study of juvenile Dolly Varden (Salvelinus malma) from a plastic-polluted lake in the Gulf of Alaska. Fish tissues were analyzed for both phthalate levels using liquid chromatography-coupled tandem mass spectrometry and for changes in immune gene expression using reverse transcriptase-real time polymerase chain reaction. Results showed that fish with higher tissue levels of di(2-ethylhexyl) phthalate, di(n-butyl) phthalate, and/or dimethyl phthalate expressed significantly fewer secreted and membrane-bound immunoglobulin heavy-chain mu and Blimp1 transcripts in their hematopoietic tissue. This suggests that in vivo uptake of phthalates in fish changes the expression of B cell-specific genes. Chronic exposure to phthalates likely dysregulates normal B-lymphoid development and antibody responses in salmonids and may increase susceptibility to infection. Given the conserved nature of B-lineage cells in vertebrate animals, other marine species may be similarly affected by chronic phthalate exposure.

Pannus invasion and cartilage degradation in rheumatoid arthritis: involvement of MMP-3 and interleukin-1beta.

PubMed

Ainola, M M; Mandelin, J A; Liljeström, M P; Li, T F; Hukkanen, M V J; Konttinen, Y T

2005-01-01

Synovial inflammation in rheumatoid arthritis (RA) leads to pannus tissue invasion and destruction of cartilage/bone matrix by proteinases. Our intention was to analyze some of the key matrix metalloproteinases (MMPs) in pannus tissue overlying evolving cartilage erosions in RA. Frozen tissue samples of pannus and synovium from advanced RA and synovium from osteoarthritic patients were used for immunohistochemical, western blotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis of MMP-1, -3, -13 and -14. Synovial fibroblast cultures, stimulated with tumour necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta), were analyzed with enzyme-linked immunosorbent assays (ELISA) and quantitative RT-PCR. MMP-3 was highly expressed in pannus tissue compared with significantly lower expression levels of MMP-1, -13 and -14. In fibroblast cultures IL-1beta was a potent stimulus for MMP-3, whereas TNF-alpha was more potent for MMP-1. This is the first study to demonstrate quantitatively in real time that MMP-3 mRNA expression is clearly higher in advanced RA pannus tissue compared to parallel RA or osteoarthritic synovium. MMP-3 mRNA levels were also clearly overexpressed in RA pannus compared to MMP-1, -13 and -14. Advanced RA has previously been found to overexpress IL-1beta. The high expression of MMP-3 in pannus and IL-1beta, mediated stimulation of MMP-3 suggest that MMP-3 plays a significant role in the progression of erosions through the proteoglycan-rich cartilage matrix.

Downregulation of connective tissue growth factor by three-dimensional matrix enhances ovarian carcinoma cell invasion.

PubMed

Barbolina, Maria V; Adley, Brian P; Kelly, David L; Shepard, Jaclyn; Fought, Angela J; Scholtens, Denise; Penzes, Peter; Shea, Lonnie D; Stack, M Sharon

2009-08-15

Epithelial ovarian carcinoma (EOC) is a leading cause of death from gynecologic malignancies, due mainly to the prevalence of undetected metastatic disease. The process of cell invasion during intraperitoneal anchoring of metastatic lesions requires concerted regulation of many processes, including modulation of adhesion to the extracellular matrix and localized invasion. Exploratory cDNA microarray analysis of early response genes (altered after 4 hr of 3D collagen culture) coupled with confirmatory real-time reverse-transcriptase polymerase chain reaction, multiple 3D cell culture matrices, Western blot, immunostaining, adhesion, migration and invasion assays were used to identify modulators of adhesion pertinent to EOC progression and metastasis. cDNA microarray analysis indicated a dramatic downregulation of connective tissue growth factor (CTGF) in EOC cells placed in invasion- mimicking conditions (3D Type I collagen). Examination of human EOC specimens revealed that CTGF expression was absent in 46% of the tested samples (n = 41), but was present in 100% of normal ovarian epithelium samples (n = 7). Reduced CTGF expression occurs in many types of cells and may be a general phenomenon displayed by cells encountering a 3D environment. CTGF levels were inversely correlated with invasion such that downregulation of CTGF increased, while its upregulation reduced collagen invasion. Cells adhered preferentially to a surface comprised of both collagen I and CTGF relative to either component alone using alpha6beta1 and alpha3beta1 integrins. Together these data suggest that downregulation of CTGF in EOC cells may be important for cell invasion through modulation of cell-matrix adhesion.

Viral etiologies of influenza-like illness and severe acute respiratory infections in Thailand.

PubMed

Chittaganpitch, Malinee; Waicharoen, Sunthareeya; Yingyong, Thitipong; Praphasiri, Prabda; Sangkitporn, Somchai; Olsen, Sonja J; Lindblade, Kim A

2018-07-01

Information on the burden, characteristics and seasonality of non-influenza respiratory viruses is limited in tropical countries. Describe the epidemiology of selected non-influenza respiratory viruses in Thailand between June 2010 and May 2014 using a sentinel surveillance platform established for influenza. Patients with influenza-like illness (ILI; history of fever or documented temperature ≥38°C, cough, not requiring hospitalization) or severe acute respiratory infection (SARI; history of fever or documented temperature ≥38°C, cough, onset <10 days, requiring hospitalization) were enrolled from 10 sites. Throat swabs were tested for influenza viruses, respiratory syncytial virus (RSV), metapneumovirus (MPV), parainfluenza viruses (PIV) 1-3, and adenoviruses by polymerase chain reaction (PCR) or real-time reverse transcriptase-PCR. We screened 15 369 persons with acute respiratory infections and enrolled 8106 cases of ILI (5069 cases <15 years old) and 1754 cases of SARI (1404 cases <15 years old). Among ILI cases <15 years old, influenza viruses (1173, 23%), RSV (447, 9%), and adenoviruses (430, 8%) were the most frequently identified respiratory viruses tested, while for SARI cases <15 years old, RSV (196, 14%) influenza (157, 11%) and adenoviruses (90, 6%) were the most common. The RSV season significantly overlapped the larger influenza season from July to November in Thailand. The global expansion of influenza sentinel surveillance provides an opportunity to gather information on the characteristics of cases positive for non-influenza respiratory viruses, particularly seasonality, although adjustments to case definitions may be required. © 2018 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

Low prevalence of avian influenza virus in shorebirds on the Pacific coast of North America

USGS Publications Warehouse

Iverson, Samuel A.; Takekawa, John Y.; Schwarzbach, Steven; Cardona, Carol J.; Warnock, Nils; Bishop, Mary Anne; Schirato, Greg A.; Paroulek, Sara; Ackerman, Joshua T.; Ip, Hon S.; Boyce, Walter M.

2008-01-01

The emergence of highly pathogenic avian influenza (HPAI) H5N1 has elevated concerns about wild birds as virus hosts; however, little is known about the ecological and epidemiological factors of transmission by shorebirds. Here we summarize results for 2,773 shorebirds that were live-trapped on the Pacific coast of the United States during 2006-2007 and tested for avian influenza virus using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and virus isolation. As was the case throughout North America, HPAI H5N1 was not detected in shorebirds during this interval. Contrary to other wild bird groups, most notably waterfowl, the prevalence of even low pathogenicity virus among shorebirds in our study areas in California, Washington, and Alaska was extremely low (0.5%). Virus was detected by RT-PCR from four different species, including, Dunlin (Calidris alpina; N = 3), Western Sandpiper (C. mauri; N = 8), Long-billed Dowitcher (Limnodromus scolopaceus; N = 1), and American Avocet (Recurvirostra americana; N = 1), with the detections in the latter three constituting the first published records for these birds. Based on studies in the eastern United States, we expected, but did not detect (H1 = 1.6, P = 0.21) elevated avian influenza prevalence among shorebirds during spring migration. Diagnostic tests, which were designed to evaluate testing and sampling methods, indicated poor functioning of traditional virus isolation methods and no improvement in detection likelihood by collecting oropharyngeal swabs in addition to cloacal swab samples for low pathogenicity viruses (Z1 = 0.7, P = 0.48).

Prevalence and risk factors for Campylobacter spp., Salmonella spp., Coxiella burnetii, and Newcastle disease virus in feral pigeons (Columba livia) in public areas of Montreal, Canada

PubMed Central

Gabriele-Rivet, Vanessa; Fairbrother, Julie-Hélène; Tremblay, Donald; Harel, Josée; Côté, Nathalie; Arsenault, Julie

2016-01-01

Feral pigeons (Columbia livia) can harbor a range of zoonotic pathogens. A transversal study was undertaken to estimate the prevalence of feral pigeons infected by various pathogens in public areas in Montreal, Quebec. Cloacal swabs from captured birds were cultured for Salmonella spp. and Campylobacter spp. and tested by real-time polymerase chain reaction (RT-PCR) for the detection of Coxiella burnetii. An oropharyngeal swab was also submitted to real-time reverse-transcription polymerase chain reaction (RRT-PCR) for the detection of Newcastle disease virus. Among the 187 pigeons tested from 10 public areas, 9.1% (95% CI: 3.0 to 15.2) were positive for Campylobacter spp. with all strains identified as Campylobacter jejuni. The Campylobacter status of birds was not associated with individual characteristics of birds, with the exception of body score. None of the pigeons tested positive for the other pathogens. Direct or indirect contacts with feral pigeons may constitute a potential risk for Campylobacter infection in humans. PMID:26733736

Development of Conventional and Real-Time Reverse Transcription Polymerase Chain Reaction Assays to Detect Tembusu Virus in Culex tarsalis Mosquitoes

DTIC Science & Technology

2014-08-11

methods for determining potential human health hazards , especially in field environment set- tings. We described the development of sensitive and specific...Smith, Department of Microbiology , Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort...2012. Adapted Tembusu-like virus in chickens and geese in China. J Clin Microbiol 50: 2807–2809. 7. Yun T, Ye W, Ni Z, Zhang D, Zhang C, 2012

Major groove binding track residues of the connection subdomain of human immunodeficiency virus type 1 reverse transcriptase enhance cDNA synthesis at high temperatures.

PubMed

Matamoros, Tania; Barrioluengo, Verónica; Abia, David; Menéndez-Arias, Luis

2013-12-23

At high temperatures, RNA denaturation can improve the efficiency and specificity of reverse transcription. Refined structures and molecular models of HIV-1 reverse transcriptases (RTs) from phylogenetically distant clades (i.e., group M subtype B and group O) revealed a major interaction between the template-primer and the Arg³⁵⁸-Gly³⁵⁹-Ala³⁶⁰ triad in the large subunit of HIV-1M/B RT. However, fewer contacts were predicted for the equivalent Lys³⁵⁸-Ala³⁵⁹-Ser³⁶⁰ triad of HIV-1O RT and the nucleic acid. An engineered HIV-1O K358R/A359G/S360A RT showed increased cDNA synthesis efficiency above 68 °C, as determined by qualitative and quantitative reverse transcription polymerase chain reactions. In comparison with wild-type HIV-1O RT, the mutant enzyme showed higher thermal stability but retained wild-type RNase H activity. Mutations that increased the accuracy of HIV-1M/B RTs were tested in combination with the K358R/A359G/S360A triple mutation. Some of them (e.g., F61A, K65R, K65R/V75I, and V148I) had a negative effect on reverse transcription efficiency above 65 °C. RTs with improved DNA binding affinities also showed higher cDNA synthesis efficiencies at elevated temperatures. Two of the most thermostable RTs (i.e., mutants T69SSG/K358R/A359G/S360A and K358R/A359G/S360A/E478Q) showed moderately increased fidelity in forward mutation assays. Our results demonstrate that the triad of Arg³⁵⁸, Gly³⁵⁹, and Ala³⁶⁰ in the major groove binding track of HIV-1 RT is a major target for RT stabilization, and most relevant for improving reverse transcription efficiency at high temperatures.

Discovery of piperidin-4-yl-aminopyrimidine derivatives as potent non-nucleoside HIV-1 reverse transcriptase inhibitors.

PubMed

Wan, Zheng-Yong; Yao, Jin; Tao, Yuan; Mao, Tian-Qi; Wang, Xin-Long; Lu, Yi-Pei; Wang, Hai-Feng; Yin, Hong; Wu, Yan; Chen, Fen-Er; De Clercq, Erik; Daelemans, Dirk; Pannecouque, Christophe

2015-06-05

A novel series of piperidin-4-yl-aminopyrimidine derivatives were designed fusing the pharmacophore templates of etravirine-VRX-480773 hybrids our group previously described and piperidine-linked aminopyrimidines. Most compounds displayed significantly improved activity against wild-type HIV-1 with EC50 values in single-digit nanomolar concentrations compared to etravirine-VRX-480773 hybrids. Selected compounds were also evaluated for activity against reverse transcriptase, and had lower IC50 values than that of nevirapine. The improved potency observed in this in vitro model of HIV RNA replication partly validates the mechanism by which this class of allosteric pyrimidine derivatives inhibits reverse transcriptase, and represents a remarkable step forward in the development of AIDS therapeutics. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Perinatal exposure of patas monkeys to antiretroviral nucleoside reverse-transcriptase inhibitors induces genotoxicity persistent for up to 3 years of age.

PubMed

Olivero, Ofelia A; Torres, Lorangelly Rivera; Gorjifard, Sayeh; Momot, Dariya; Marrogi, Eryney; Divi, Rao L; Liu, Yongmin; Woodward, Ruth A; Sowers, Marsha J; Poirier, Miriam C

2013-07-15

Erythrocebus patas (patas) monkeys were used to model antiretroviral (ARV) drug in human immunodeficiency virus type 1-infected pregnant women. Pregnant patas dams were given human-equivalent doses of ARVs daily during 50% of gestation. Mesenchymal cells, cultured from bone marrow of patas offspring obtained at birth and at 1 and 3 years of age, were examined for genotoxicity, including centrosomal amplification, micronuclei, and micronuclei containing whole chromosomes. Compared with controls, statistically significant increases (P < .05) in centrosomal amplification, micronuclei, and micronuclei containing whole chromosomes were found in mesenchymal cells from most groups of offspring at the 3 time points. Transplacental nucleoside reverse-transcriptase inhibitor exposures induced fetal genotoxicity that was persistent for 3 years.

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.

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.

Phylogenetic analysis of HIV-1 reverse transcriptase sequences from 382 patients recruited in JJ Hospital of Mumbai, India, between 2002 and 2008.

PubMed

Deshpande, Alaka; Jauvin, Valerie; Pinson, Patricia; Jeannot, Anne Cecile; Fleury, Herve J

2009-06-01

Analysis of reverse transcriptase (RT) sequences of 382 HIV-1 isolates from untreated and treated patients recruited in JJ Hospital (Mumbai, India) between 2002 and 2008 shows that subtype C is largely predominant (98%) and that non-C sequences cluster with A1, B, CRF01_AE, and CRF06_cpx.

Application of Reverse Transcriptase-PCR-DGGE as a rapid method for routine determination of Vibrio spp. in foods.

PubMed

Chahorm, Kanchana; Prakitchaiwattana, Cheunjit

2018-01-02

The aim of this research was to evaluate the feasibility of PCR-DGGE and Reverse Transcriptase-PCR-DGGE techniques for rapid detection of Vibrio species in foods. Primers GC567F and 680R were initially evaluated for amplifying DNA and cDNA of ten references Vibrio species by PCR method. The GC-clamp PCR amplicons were separated according to their sequences by the DGGE using 10% (w/v) polyacrylamide gel containing 45-70% urea and formamide denaturants. Two pair of Vibrio species, which could not be differentiated on the gel, was Vibrio fluvialis - Vibrio furnissii and Vibrio parahaemolyticus - Vibrio harveyi. To determine the detection limit, in the community of 10 reference strains containing the same viable population, distinct DNA bands of 3 species; Vibrio cholerae, Vibrio mimicus and Vibrio alginolyticus were consistently observed by PCR-DGGE technique. In fact, 5 species; Vibrio cholerae, Vibrio mimicus, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio fluvialis consistently observed by Reverse Transcriptase-PCR-DGGE. In the community containing different viable population increasing from 10 2 to 10 5 CFU/mL, PCR-DGGE analysis only detected the two most prevalent species, while RT-PCR-DGGE detected the five most prevalent species. Therefore, Reverse Transcriptase-PCR-DGGE was also selected for detection of various Vibrio cell conditions, including viable cell (VC), injured cells from frozen cultures (IVC) and injured cells from frozen cultures with pre-enrichment (PIVC). It was found that cDNA band of all cell conditions gave the same migratory patterns, except that multiple cDNA bands of Plesiomonas shigelloides under IVC and PIVC conditions were found. When Reverse Transcriptase-PCR-DGGE was used for detecting Vibrio parahaemolyticus in the pathogen-spiked food samples, Vibrio parahaemolyticus could be detected in the spiked samples containing at least 10 2 CFU/g of this pathogen. The results obtained also corresponded to standard method (USFDA, 2004). In comparison with the detection of the Vibrio profiles in fourteen food samples using standard method, Reverse Transcriptase-PCR-DGGE resulted in 100%, 75% and 50% similarity in 3, 1 and 6 food samples, respectively. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

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.

Induction of vascular endothelial growth factor expression in human pulp fibroblasts stimulated with black-pigmented Bacteroides.

PubMed

Yang, L-C; Tsai, C-H; Huang, F-M; Su, Y-F; Lai, C-C; Liu, C-M; Chang, Y-C

2004-09-01

To investigate the effect of black-pigmented Bacteroides on the expression of vascular endothelial growth factor (VEGF) gene in human pulp fibroblasts. The supernatants of Porphyromonas endodontalis, Porphyromonas gingivalis and Prevotella intermedia were used to evaluate VEGF gene expression in human pulp fibroblasts. The levels of mRNAs were measured by the quantitative reverse-transcriptase polymerase chain reaction analysis. Black-pigmented Bacteroides induced significantly high levels of VEGF mRNA gene expression in human pulp fibroblasts (P < 0.05). In addition, the expression of VEGF depended on the bacteria tested. Black-pigmented Bacteroides may be involved in developing pulpal disease through the stimulation of VEGF production that would lead to the expansion of the vascular network coincident to progression of the inflammation.

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.

Inhibitory effect of aqueous dandelion extract on HIV-1 replication and reverse transcriptase activity

PubMed Central

2011-01-01

Background Acquired immunodeficiency syndrome (AIDS), which is caused by the human immunodeficiency virus (HIV), is an immunosuppressive disease that results in life-threatening opportunistic infections. The general problems in current therapy include the constant emergence of drug-resistant HIV strains, adverse side effects and the unavailability of treatments in developing countries. Natural products from herbs with the abilities to inhibit HIV-1 life cycle at different stages, have served as excellent sources of new anti-HIV-1 drugs. In this study, we aimed to investigate the anti-HIV-1 activity of aqueous dandelion extract. Methods The pseudotyped HIV-1 virus has been utilized to explore the anti-HIV-1 activity of dandelion, the level of HIV-1 replication was assessed by the percentage of GFP-positive cells. The inhibitory effect of the dandelion extract on reverse transcriptase activity was assessed by the reverse transcriptase assay kit. Results Compared to control values obtained from cells infected without treatment, the level of HIV-1 replication and reverse transcriptase activity were decreased in a dose-dependent manner. The data suggest that dandelion extract has a potent inhibitory activity against HIV-1 replication and reverse transcriptase activity. The identification of HIV-1 antiviral compounds from Taraxacum officinale should be pursued. Conclusions The dandelion extract showed strong activity against HIV-1 RT and inhibited both the HIV-1 vector and the hybrid-MoMuLV/MoMuSV retrovirus replication. These findings provide additional support for the potential therapeutic efficacy of Taraxacum officinale. Extracts from this plant may be regarded as another starting point for the development of an antiretroviral therapy with fewer side effects. PMID:22078030

Structural studies of series HIV-1 nonnucleoside reverse transcriptase inhibitors 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-benzimidazoles with different 4-substituents

NASA Astrophysics Data System (ADS)

Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

2010-03-01

Over the past 10 years, several anti-viral drugs have become available to fight the HIV infection. Antiretroviral treatment reduces the mortality of AIDS. Nonnucleoside inhibitors of HIV-1 reverse transcriptase are specific and potentially nontoxic drugs against AIDS. The crystal structures of five nonnucleoside inhibitors of HIV-1 reverse transcriptase are presented here. The structural parameters, especially those describing the angular orientation of the π-electron systems and influencing biological activity, were determined for all of the investigated inhibitors. The chemical character and orientation of the substituent at C4 position of the benzimidazole moiety substantially influences the anti-viral activity. The structural data of the investigated inhibitors is a good basis for modeling enzyme-inhibitor interactions for structure-assisted drug design.

Anti-HIV drugs: 25 compounds approved within 25 years after the discovery of HIV.

PubMed

De Clercq, Erik

2009-04-01

In 2008, 25 years after the human immunodeficiency virus (HIV) was discovered as the then tentative aetiological agent of acquired immune deficiency syndrome (AIDS), exactly 25 anti-HIV compounds have been formally approved for clinical use in the treatment of AIDS. These compounds fall into six categories: nucleoside reverse transcriptase inhibitors (NRTIs: zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir and emtricitabine); nucleotide reverse transcriptase inhibitors (NtRTIs: tenofovir); non-nucleoside reverse transcriptase inhibitors (NNRTIs: nevirapine, delavirdine, efavirenz and etravirine); protease inhibitors (PIs: saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, atazanavir, fosamprenavir, tipranavir and darunavir); cell entry inhibitors [fusion inhibitors (FIs: enfuvirtide) and co-receptor inhibitors (CRIs: maraviroc)]; and integrase inhibitors (INIs: raltegravir). These compounds should be used in drug combination regimens to achieve the highest possible benefit, tolerability and compliance and to diminish the risk of resistance development.

Virtual screening studies on HIV-1 reverse transcriptase inhibitors to design potent leads.

PubMed

Vadivelan, S; Deeksha, T N; Arun, S; Machiraju, Pavan Kumar; Gundla, Rambabu; Sinha, Barij Nayan; Jagarlapudi, Sarma A R P

2011-03-01

The purpose of this study is to identify novel and potent inhibitors against HIV-1 reverse transcriptase (RT). The crystal structure of the most active ligand was converted into a feature-shaped query. This query was used to align molecules to generate statistically valid 3D-QSAR (r(2) = 0.873) and Pharmacophore models (HypoGen). The best HypoGen model consists of three Pharmacophore features (one hydrogen bond acceptor, one hydrophobic aliphatic and one ring aromatic) and further validated using known RT inhibitors. The designed novel inhibitors are further subjected to docking studies to reduce the number of false positives. We have identified and proposed some novel and potential lead molecules as reverse transcriptase inhibitors using analog and structure based studies. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Carbon nanotube-based substrates promote cardiogenesis in brown adipose-derived stem cells via β1-integrin-dependent TGF-β1 signaling pathway

PubMed Central

Sun, Hongyu; Mou, Yongchao; Li, Yi; Li, Xia; Chen, Zi; Duval, Kayla; Huang, Zhu; Dai, Ruiwu; Tang, Lijun; Tian, Fuzhou

2016-01-01

Stem cell-based therapy remains one of the promising approaches for cardiac repair and regeneration. However, its applications are restricted by the limited efficacy of cardiac differentiation. To address this issue, we examined whether carbon nanotubes (CNTs) would provide an instructive extracellular microenvironment to facilitate cardiogenesis in brown adipose-derived stem cells (BASCs) and to elucidate the underlying signaling pathways. In this study, we systematically investigated a series of cellular responses of BASCs due to the incorporation of CNTs into collagen (CNT-Col) substrates that promoted cell adhesion, spreading, and growth. Moreover, we found that CNT-Col substrates remarkably improved the efficiency of BASCs cardiogenesis by using fluorescence staining and quantitative real-time reverse transcription-polymerase chain reaction. Critically, CNTs in the substrates accelerated the maturation of BASCs-derived cardiomyocytes. Furthermore, the underlying mechanism for promotion of BASCs cardiac differentiation by CNTs was determined by immunostaining, quantitative real-time reverse transcription-polymerase chain reaction, and Western blotting assay. It is notable that β1-integrin-dependent TGF-β1 signaling pathway modulates the facilitative effect of CNTs in cardiac differentiation of BASCs. Therefore, it is an efficient approach to regulate cardiac differentiation of BASCs by the incorporation of CNTs into the native matrix. Importantly, our findings can not only facilitate the mechanistic understanding of molecular events initiating cardiac differentiation in stem cells, but also offer a potentially safer source for cardiac regenerative medicine. PMID:27660434

Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time reverse-transcription polymerase chain reaction and virus isolation

PubMed Central

Goodell, Christa K.; Zhang, Jianqiang; Strait, Erin; Harmon, Karen; Patnayak, Devi; Otterson, Tracy; Culhane, Marie; Christopher-Hennings, Jane; Clement, Travis; Leslie-Steen, Pamela; Hesse, Richard; Anderson, Joe; Skarbek, Kevin; Vincent, Amy; Kitikoon, Pravina; Swenson, Sabrina; Jenkins-Moore, Melinda; McGill, Jodi; Rauh, Rolf; Nelson, William; O’Connell, Catherine; Shah, Rohan; Wang, Chong; Main, Rodger; Zimmerman, Jeffrey J.

2016-01-01

The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 assays based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) and 7 assays based on virus isolation (VI). The OF specimens were inoculated with H1N1 or H3N2 IAV and serially diluted 10-fold (10−1 to 10−8). Eight participating laboratories received 180 randomized OF samples (10 replicates × 8 dilutions × 2 IAV subtypes plus 20 IAV-negative samples) and performed the rRT-PCR and VI procedure(s) of their choice. Analysis of the results with a mixed-effect logistic-regression model identified dilution and assay as variables significant (P < 0.0001) for IAV detection in OF by rRT-PCR or VI. Virus subtype was not significant for IAV detection by either rRT-PCR (P = 0.457) or VI (P = 0.101). For rRT-PCR the cycle threshold (Ct) values increased consistently with dilution but varied widely. Therefore, it was not possible to predict VI success on the basis of Ct values. The success of VI was inversely related to the dilution of the sample; the assay was generally unsuccessful at lower virus concentrations. Successful swine health monitoring and disease surveillance require assays with consistent performance, but significant differences in reproducibility were observed among the assays evaluated. PMID:26733728

Analytical validation of a real-time reverse transcription polymerase chain reaction test for Pan-American lineage H7 subtype Avian influenza viruses

USGS Publications Warehouse

Spackman, Erica; Ip, Hon S.; Suarez, D.L.; Slemons, R.D.; Stallknecht, D.E.

2008-01-01

A real-time reverse transcription polymerase chain reaction test for the identification of the H7 subtype in North American Avian influenza viruses (AIVs) was first reported in 2002; however, recent AIV surveillance efforts in wild birds and H7 outbreaks in poultry demonstrated that the 2002 test did not detect all H7 AIVs present in North and South America. Therefore, a new test, the 2008 Pan-American H7 test, was developed by using recently available H7 nucleotide sequences. The analytical specificity of the new assay was characterized with an RNA panel composed of 19 H7 viruses from around the world and RNA from all hemagglutinin subtypes except H16. Specificity for North and South American lineage H7 viruses was observed. Assay limits of detection were determined to be between 103 and 104 gene copies per reaction with in vitro transcribed RNA, and 100.0 and 10 0.8 50% egg infectious doses per reaction. The 2008 Pan-American H7 test also was shown to perform similarly to the 2002 test with specimens from chickens experimentally exposed to A/Chicken/BritishColumbia/314514-2/04 H7N3 highly pathogenic AIV. Furthermore, the 2008 test was able to detect 100% (n = 27) of the H7 AIV isolates recovered from North American wild birds in a 2006-2007 sample set (none of which were detected by the 2002 H7 test).

Simultaneous Detection of Rift Valley Fever, Bluetongue, Rinderpest, and Peste des Petits Ruminants Viruses by a Single-Tube Multiplex Reverse Transcriptase-PCR Assay Using a Dual-Priming Oligonucleotide System▿

PubMed Central

Yeh, Jung-Yong; Lee, Ji-Hye; Seo, Hyun-Ji; Park, Jee-Yong; Moon, Jin-San; Cho, In-Soo; Choi, In-Soo; Park, Seung-Yong; Song, Chang-Seon; Lee, Joong-Bok

2011-01-01

The aim of this study was to develop a highly sensitive and specific one-step multiplex reverse transcriptase PCR assay for the simultaneous and differential detection of Rift Valley Fever virus (RVFV), bluetongue virus (BTV), rinderpest virus (RPV), and Peste des petits ruminants virus (PPRV). These viruses cause mucosal lesions in cattle, sheep, and goats, and they are difficult to differentiate from one another based solely on their clinical presentation in suspected disease cases. In this study, we developed a multiplex reverse transcriptase PCR to detect these viruses using a novel dual-priming oligonucleotide (DPO). The DPO contains two separate priming regions joined by a polydeoxyinosine linker, which blocks extension of nonspecifically primed templates and consistently allows high PCR specificity even under less-than-optimal PCR conditions. A total of 19 DPO primers were designed to detect and discriminate between RVFV, BTV, RPV, and PPRV by the generation of 205-, 440-, 115-, and 243-bp cDNA products, respectively. The multiplex reverse transcriptase PCR described here enables the early diagnosis of these four viruses and may also be useful as part of a testing regime for cattle, sheep, or goats exhibiting similar clinical signs, including mucosal lesions. PMID:21307219

Combination nucleoside/nucleotide reverse transcriptase inhibitors for treatment of HIV infection.

PubMed

Akanbi, Maxwell O; Scarsi, Kimberly K; Scarci, Kimberly; Taiwo, Babafemi; Murphy, Robert L

2012-01-01

The combination of two nucleoside/nucleotide reverse transcriptase inhibitors (N(t)RTIs) and a third agent from another antiretroviral class is currently recommended for initial antiretroviral therapy. In general, N(t)RTIs remain relevant in subsequent regimens. There are currently six nucleoside reverse transcriptase inhibitors and one nucleotide reverse transcriptase inhibitor drug entities available, and several formulations that include two or more N(t)RTIs in a fixed-dose combination. These entities have heterogeneous pharmacological and clinical properties. Accordingly, toxicity, pill burden, dosing frequency, potential drug-drug interaction, preexisting antiretroviral drug resistance and comorbid conditions should be considered when constructing a regimen. This approach is critical in order to optimize virologic efficacy and clinical outcomes. This article reviews N(t)RTI combinations used in the treatment of HIV-infected adults. The pharmacological properties of each N(t)RTI, and the clinical trials that have influenced treatment guidelines are discussed. It is likely that N(t)RTIs will continue to dominate the global landscape of HIV treatment and prevention, despite emerging interest in N(t)RTI-free combination therapy. Clinical domains where only few alternatives to N(t)RTIs exist include treatment of HIV/HBV coinfection and HIV-2. There is a need for novel N(t)RTIs with enhanced safety and resistance profiles compared with current N(t)RTIs.

Reverse Transcriptase-Containing Particles Induced in Rous Sarcoma Virus-Transformed Rat Cells by Arginine Deprivation

PubMed Central

Kotler, Moshe; Weinberg, Eynat; Haspel, Osnat; Becker, Yechiel

1972-01-01

Incubation of rat cells transformed by Rous sarcoma virus (RSV) in an arginine-deficient medium resulted in accumulation of particles in the culture medium. Such particles did not appear when the transformed rat cells were incubated in a complete medium nor in the medium of primary rat cells which were incubated either in arginine-deficient or complete media. The particles which were released from the arginine-deprived transformed rat cells resemble C-type particles in their properties. These particles band in sucrose gradients at a density of 1.16 g/ml and contain 35S ribonucleic acid (RNA) molecules and a reverse transcriptase activity. Analysis of the cytoplasm of transformed and primary rat cells, deprived and undeprived of arginine, revealed the presence of reverse transcriptase-containing particles which banded in sucrose gradients at a density of 1.14 g/ml. These particles differed from the particles released into the medium by the arginine-deprived RSV-transformed rat cells. The deoxyribonucleic acid (DNA) molecules synthesized in vitro by the reverse transcriptase present in the particles isolated from the medium of arginine-deprived cells hybridized to RSV RNA, whereas the DNA synthesized by the cell-bound enzyme had no homology to RSV RNA. PMID:4116137

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.