Characteristics of a group of nonnucleoside reverse transcriptase inhibitors with structural diversity and potent anti-human immunodeficiency virus activity.

PubMed

Yang, S S; Fliakas-Boltz, V; Bader, J P; Buckheit, R W

1995-10-01

Current thrust in controlling the Acquired Immune Deficiency Syndrome (AIDS) focuses on antiviral drug development targeting the infection and replication of the human immunodeficiency virus (HIV), the causative agent of AIDS. To date, treatment of AIDS has relied on nucleoside reverse transcriptase inhibitors such as AZT, ddI, and ddC, which eventually become ineffective upon the emergence of resistant mutants bearing specific nucleotide substitutions. The Anti-AIDS Drug Screening Program of the NCI conducts and coordinates a high-capacity semi-robotic in vitro screening of synthetic or natural compounds submitted by academic, research and pharmaceutical institutions world-wide. About 10,000 synthetic compounds are screened annually for anti-HIV activity. Confirmed active agents are subjected to in-depth studies on range and mechanism of action. Emerging from this intense screening activity were a number of potentially promising categories of nonnucleoside reverse transcriptase inhibitors (NNRTI) with structural diversity but strong and reproducible anti-HIV activity. Over 2500 active compounds were evaluated for their inhibitory activity against a panel of both laboratory and clinical virus isolates in the appropriate established cell line or fresh human peripheral blood leukocyte and macrophage preparations. Out of these, 40 agents could be placed structurally in nine categories with an additional 16 unique compounds that share the characteristics of NNRTI. These NNRTIs were shown to inhibit reverse transcriptase enzymatically using homopolymeric or ribosomal RNA as templates. NNRTIs demonstrated similarity in their inhibitory pattern against the HIV-1 laboratory strains IIIB and RF, and an AZT-resistant strain; all were inactive against HIV-2. These compounds were further tested against NNRTI-resistant HIV-1 isolates. NNRTI-resistant HIV-1 isolates were selected and characterized with respect to the change(s) in the viral reverse transcriptase nucleotide sequence. Also, differential cross-resistance or sensitivity patterns to NNRTIs were studied in detail among NNRTI-resistant mutants. When tested in combination with AZT, all of the NNRTI's uniformly exhibited synergistic inhibition of HIV-1, suggesting that combination antiviral therapy of NNRTIs with AZT may be therapeutically promising for AIDS treatment.

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

PubMed

Huang, Chun; Li, Runqin; Zhang, Yinglin; Gong, Jianping

2017-10-01

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. 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. 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. 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.

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

Telomerase reverse transcriptase (TERT) - enhancer of zeste homolog 2 (EZH2) network regulates lipid metabolism and DNA damage responses in glioblastoma.

PubMed

Ahmad, Fahim; Patrick, Shruti; Sheikh, Touseef; Sharma, Vikas; Pathak, Pankaj; Malgulwar, Prit Benny; Kumar, Anupam; Joshi, Shanker Datt; Sarkar, Chitra; Sen, Ellora

2017-12-01

Elevated expression of enhancer of zeste homolog 2 (EZH2), a histone H3K27 methyltransferase, was observed in gliomas harboring telomerase reverse transcriptase (TERT) promoter mutations. Given the known involvement of TERT and EZH2 in glioma progression, the correlation between the two and subsequently its involvement in metabolic programming was investigated. Inhibition of human telomerase reverse transcriptase either pharmacologically or through genetic manipulation not only decreased EZH2 expression, but also (i) abrogated FASN levels, (ii) decreased de novo fatty acid accumulation, and (iii) increased ataxia-telangiectasia-mutated (ATM) phosphorylation levels. Conversely, diminished TERT and FASN levels upon siRNA-mediated EZH2 knockdown indicated a positive correlation between TERT and EZH2. Interestingly, ATM kinase inhibitor rescued TERT inhibition-mediated decrease in FASN and EZH2 levels. Importantly, TERT promoter mutant tumors exhibited greater microsatellite instability, heightened FASN levels and lipid accumulation. Coherent with in vitro findings, pharmacological inhibition of TERT by costunolide decreased lipid accumulation and elevated ATM expression in heterotypic xenograft glioma mouse model. By bringing TERT-EZH2 network at the forefront as driver of dysregulated metabolism, our findings highlight the non-canonical but distinct role of TERT in metabolic reprogramming and DNA damage responses in glioblastoma. © 2017 International Society for Neurochemistry.

Application of 3D-QSAR, Pharmacophore, and Molecular Docking in the Molecular Design of Diarylpyrimidine Derivatives as HIV-1 Nonnucleoside Reverse Transcriptase Inhibitors.

PubMed

Liu, Genyan; Wang, Wenjie; Wan, Youlan; Ju, Xiulian; Gu, Shuangxi

2018-05-11

Diarylpyrimidines (DAPYs), acting as HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs), have been considered to be one of the most potent drug families in the fight against acquired immunodeficiency syndrome (AIDS). To better understand the structural requirements of HIV-1 NNRTIs, three-dimensional quantitative structure⁻activity relationship (3D-QSAR), pharmacophore, and molecular docking studies were performed on 52 DAPY analogues that were synthesized in our previous studies. The internal and external validation parameters indicated that the generated 3D-QSAR models, including comparative molecular field analysis (CoMFA, q 2 = 0.679, R 2 = 0.983, and r pred 2 = 0.884) and comparative molecular similarity indices analysis (CoMSIA, q 2 = 0.734, R 2 = 0.985, and r pred 2 = 0.891), exhibited good predictive abilities and significant statistical reliability. The docking results demonstrated that the phenyl ring at the C₄-position of the pyrimidine ring was better than the cycloalkanes for the activity, as the phenyl group was able to participate in π⁻π stacking interactions with the aromatic residues of the binding site, whereas the cycloalkanes were not. The pharmacophore model and 3D-QSAR contour maps provided significant insights into the key structural features of DAPYs that were responsible for the activity. On the basis of the obtained information, a series of novel DAPY analogues of HIV-1 NNRTIs with potentially higher predicted activity was designed. This work might provide useful information for guiding the rational design of potential HIV-1 NNRTI DAPYs.

[Thyroid dysfunction in adults infected by human immunodeficiency virus].

PubMed

Abelleira, Erika; De Cross, Graciela A; Pitoia, Fabián

2014-01-01

Patients infected with human immunodeficiency virus (HIV) have a higher prevalence of thyroid dysfunction when compared with the general population. The most frequently observed manifestations are euthyroid sick syndrome, Graves' disease and subclinical hypothyroidism. The relationship between the use of highly active antiretroviral therapy and the increased prevalence of thyroid dysfunction has been demonstrated in several series of patients. Grave's disease is recognized as a consequence of immune restitution syndrome. Besides, several studies have suggested an association between hypothyroidism and the use of nucleoside reverse transcriptase inhibitors, particularly stavudine and non-nucleoside reverse transcriptase inhibitors such as efavirenz. Further studies could provide additional evidence of the need for routine assessment of thyroid function in HIV-infected patients.

Expanded-spectrum nonnucleoside reverse transcriptase inhibitors inhibit clinically relevant mutant variants of human immunodeficiency virus type 1.

PubMed

Corbett, J W; Ko, S S; Rodgers, J D; Jeffrey, S; Bacheler, L T; Klabe, R M; Diamond, S; Lai, C M; Rabel, S R; Saye, J A; Adams, S P; Trainor, G L; Anderson, P S; Erickson-Viitanen, S K

1999-12-01

A research program targeted toward the identification of expanded-spectrum nonnucleoside reverse transcriptase inhibitors which possess increased potency toward K103N-containing mutant human immunodeficiency virus (HIV) and which maintain pharmacokinetics consistent with once-a-day dosing has resulted in the identification of the 4-cyclopropylalkynyl-4-trifluoromethyl-3, 4-dihydro-2(1H)quinazolinones DPC 961 and DPC 963 and the 4-cyclopropylalkenyl-4-trifluoromethyl-3, 4-dihydro-2(1H)quinazolinones DPC 082 and DPC 083 for clinical development. DPC 961, DPC 963, DPC 082, and DPC 083 all exhibit low-nanomolar potency toward wild-type virus, K103N and L100I single-mutation variants, and many multiply amino acid-substituted HIV type 1 mutants. This high degree of potency is combined with a high degree of oral bioavailability, as demonstrated in rhesus monkeys and chimpanzees, and with plasma serum protein binding that can result in significant free levels of drug.

Dual HIV-1 reverse transcriptase and integrase inhibitors from Limonium morisianum Arrigoni, an endemic species of Sardinia (Italy).

PubMed

Sanna, Cinzia; Rigano, Daniela; Corona, Angela; Piano, Dario; Formisano, Carmen; Farci, Domenica; Franzini, Genni; Ballero, Mauro; Chianese, Giuseppina; Tramontano, Enzo; Taglialatela-Scafati, Orazio; Esposito, Francesca

2018-02-04

During our search for potential templates of HIV-1 reverse transcriptase (RT) and integrase (IN) dual inhibitors, the methanolic extract obtained from aerial parts of Limonium morisianum was investigated. Repeated bioassay-guided chromatographic purifications led to the isolation of the following secondary metabolites: myricetin, myricetin 3-O-rutinoside, myricetin-3-O-(6″-O-galloyl)-β-d-galactopyranoside, (-)-epigallocatechin 3-O-gallate, tryptamine, ferulic and phloretic acids. The isolated compounds were tested on both HIV-1 RT-associated RNase H and IN activities. Interestingly, (-)-epigallocatechin-3-O-gallate and myricetin-3-O-(6″-O-galloyl)-β-d-galactopyranoside potently inhibited both enzyme activities with IC 50 values ranging from 0.21 to 10.9 μM. Differently, tryptamine and ferulic acid exhibited a significant inhibition only on the IN strand transfer reaction, showing a selectivity for this viral enzyme. Taken together these results strongly support the potential of this plant as a valuable anti HIV-1 drugs source worthy of further investigations.

Pharmacokinetics of antiretroviral drugs in anatomical sanctuary sites: the male and female genital tract.

PubMed

Else, Laura J; Taylor, Stephen; Back, David J; Khoo, Saye H

2011-01-01

HIV resides within anatomical 'sanctuary sites', where local drug exposure and viral dynamics may differ significantly from the systemic compartment. Suboptimal antiretroviral concentrations in the genital tract may result in compartmentalized viral replication, selection of resistant mutations and possible re-entry of wild-type/resistant virus into the systemic circulation. Therefore, achieving adequate antiretroviral exposure in the genital tract has implications for the prevention of sexual and vertical transmission of HIV. Penetration of antiretrovirals in the genital tract is expressed by accumulation ratios derived from the measurement of drug concentrations in time-matched seminal plasma/cervicovaginal fluid and plasma samples. Penetration varies by gender and may be drug (as opposed to class) specific with high interindividual variability. Concentrations in seminal plasma are highest for nucleoside analogues and lowest for protease inhibitors and efavirenz. Seminal accumulation of newer agents, raltegravir and maraviroc, is moderate (rank order of accumulation is nucleoside/nucleotide reverse transcriptase inhibitors [lamivudine/zidovudine/tenofovir/didanosine > stavudine/abacavir] > raltegravir > indinavir/maraviroc/nevirapine > efavirenz/protease inhibitors [amprenavir/atazanavir/darunavir > lopinavir/ritonavir > saquinavir] > enfuvirtide). In the female genital tract, the nucleoside analogues exhibit high accumulation ratios, whereas protease inhibitors have limited penetration; however, substantial variability exists between individuals and study centres. Second generation non-nucleoside reverse transcriptase inhibitor etravirine, and maraviroc and raltegravir, demonstrate effective accumulation in cervicovaginal secretions (rank order of accumulation is nucleoside/nucleotide reverse transcriptase inhibitor [zidovudine/lamivudine/didanosine > emtricitabine/tenofovir] > indinavir > maraviroc/raltegravir/darunavir/etravirine > nevirapine/abacavir > protease inhibitors [amprenavir/atazanavir/ritonavir] > lopinavir/stavudine/efavirenz > saquinavir).

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

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

Deregulation of the telomerase reverse transcriptase (TERT) gene by chromosomal translocations in B-cell malignancies.

PubMed

Nagel, Inga; Szczepanowski, Monika; Martín-Subero, José I; Harder, Lana; Akasaka, Takashi; Ammerpohl, Ole; Callet-Bauchu, Evelyne; Gascoyne, Randy D; Gesk, Stefan; Horsman, Doug; Klapper, Wolfram; Majid, Aneela; Martinez-Climent, José A; Stilgenbauer, Stephan; Tönnies, Holger; Dyer, Martin J S; Siebert, Reiner

2010-08-26

Sequence variants at the TERT-CLPTM1L locus in chromosome 5p have been recently associated with disposition for various cancers. Here we show that this locus including the gene encoding the telomerase reverse-transcriptase TERT at 5p13.33 is rarely but recurrently targeted by somatic chromosomal translocations to IGH and non-IG loci in B-cell neoplasms, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma and splenic marginal zone lymphoma. In addition, cases with genomic amplification of TERT locus were identified. Tumors bearing chromosomal aberrations involving TERT showed higher TERT transcriptional expression and increased telomerase activity. These data suggest that deregulation of TERT gene by chromosomal abnormalities leading to increased telomerase activity might contribute to B-cell lymphomagenesis.

A novel Met-to-Thr mutation in the YMDD motif of reverse transcriptase from feline immunodeficiency virus confers resistance to oxathiolane nucleosides.

PubMed Central

Smith, R A; Remington, K M; Lloyd, R M; Schinazi, R F; North, T W

1997-01-01

Variants of feline immunodeficiency virus (FIV) that possess a unique methionine-to-threonine mutation within the YMDD motif of reverse transcriptase (RT) were selected by culturing virus in the presence of inhibitory concentrations of (-)-beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine [(-)-FTC]. The mutants were resistant to (-)-FTC and (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (3TC) and additionally exhibited low-level resistance to 2',3'-dideoxycytidine (ddC). DNA sequence analysis of the RT-encoding region of the pol gene amplified from resistant viruses consistently identified a Met-to-Thr mutation in the YMDD motif. Purified RT from the mutants was also resistant to the 5'-triphosphate forms of 3TC, (-)-FTC, and ddC. Site-directed mutants of FIV were engineered which contain either the novel Met-to-Thr mutation or the Met-to-Val mutation seen in oxathiolane nucleoside-resistant HIV-1. Both site-directed mutants displayed resistance to 3TC, thus confirming the role of these mutations in the resistance of FIV to beta-L-3'-thianucleosides. PMID:9032372

Five Antiretroviral Drug Class-Resistant HIV-1 in a Treatment-Naïve Patient Successfully Suppressed with Optimized Antiretroviral Drug Selection.

PubMed

Volpe, Joseph M; Ward, Douglas J; Napolitano, Laura; Phung, Pham; Toma, Jonathan; Solberg, Owen; Petropoulos, Christos J; Walworth, Charles M

2015-01-01

Transmitted HIV-1 exhibiting reduced susceptibility to protease and reverse transcriptase inhibitors is well documented but limited for integrase inhibitors and enfuvirtide. We describe here a case of transmitted 5 drug class-resistance in an antiretroviral (ARV)-naïve patient who was successfully treated based on the optimized selection of an active ARV drug regimen. The value of baseline resistance testing to determine an optimal ARV treatment regimen is highlighted in this case report. © The Author(s) 2015.

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.

High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases

PubMed Central

Qin, Yidan; Yao, Jun; Wu, Douglas C.; Nottingham, Ryan M.; Mohr, Sabine; Hunicke-Smith, Scott; Lambowitz, Alan M.

2016-01-01

Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from <1 ng of plasma RNA in <5 h. TGIRT-seq of RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling. PMID:26554030

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

Drug Susceptibility and Resistance Mutations After First-Line Failure in Resource Limited Settings

PubMed Central

Wallis, Carole L.; Aga, Evgenia; Ribaudo, Heather; Saravanan, Shanmugam; Norton, Michael; Stevens, Wendy; Kumarasamy, Nagalingeswaran; Bartlett, John; Katzenstein, David

2014-01-01

Background. The development of drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) has been associated with baseline human immunodeficiency virus (HIV)-1 RNA level (VL), CD4 cell counts (CD4), subtype, and treatment failure duration. This study describes drug resistance and levels of susceptibility after first-line virologic failure in individuals from Thailand, South Africa, India, Malawi, Tanzania. Methods. CD4 and VL were captured at AIDs Clinical Trial Group (ACTG) A5230 study entry, a study of lopinavir/ritonavir (LPV/r) monotherapy after first-line virologic failure on an NNRTI regimen. HIV drug-resistance mutation associations with subtype, site, study entry VL, and CD4 were evaluated using Fisher exact and Kruskall–Wallis tests. Results. Of the 207 individuals who were screened for A5230, sequence data were available for 148 individuals. Subtypes observed: subtype C (n = 97, 66%) AE (n = 27, 18%), A1 (n = 12, 8%), and D (n = 10, 7%). Of the 148 individuals, 93% (n = 138) and 96% (n = 142) had at least 1 reverse transcriptase (RT) mutation associated with NRTI and NNRTI resistance, respectively. The number of NRTI mutations was significantly associated with a higher study screening VL and lower study screening CD4 (P < .001). Differences in drug-resistance patterns in both NRTI and NNRTI were observed by site. Conclusions. The degree of NNRTI and NRTI resistance after first-line virologic failure was associated with higher VL at study entry. Thirty-two percent of individuals remained fully susceptible to etravirine and rilpivirine, protease inhibitor resistance was rare. Some level of susceptibility to NRTI remained; however, VL monitoring and earlier virologic failure detection may result in lower NRTI resistance. PMID:24795328

Protecting the fetus against HIV infection: a systematic review of placental transfer of antiretrovirals.

PubMed

McCormack, Shelley A; Best, Brookie M

2014-11-01

Maternal-to-fetal transfer of antiretroviral drugs contributes to prevention of vertical transmission of HIV. This systematic review discusses published studies containing data pertaining to the pharmacokinetics of placental transfer of antiretrovirals in humans, including paired cord and maternal plasma samples collected at the time of delivery as well as ex vivo placental perfusion models. Articles pertaining to placental transfer of antiretrovirals were identified from PubMed, from references of included articles, and from US Department of Health and Human Services Panel on Treatment of HIV-infected Pregnant Women and Prevention of Perinatal Transmission guidelines. Articles from non-human animal models or that had no original maternal-to-fetal transfer data were excluded. PRISMA guidelines were followed. A total of 103 published studies were identified. Data across studies appeared relatively consistent for the nucleoside reverse transcriptase inhibitors (NRTIs) and the non-nucleotide reverse transcriptase inhibitors (NNRTIs), with cord to maternal ratios approaching 1 for many of these agents. The protease inhibitors atazanavir and lopinavir exhibited consistent maternal-to-fetal transfer across studies, although the transfer may be influenced by variations in drug-binding proteins. The protease inhibitors indinavir, nelfinavir, and saquinavir exhibited unreliable placental transport, with cord blood concentrations that were frequently undetectable. Limited data, primarily from case reports, indicate that darunavir and raltegravir provide detectable placental transfer. These findings appear consistent with current guidelines of using two NRTIs plus an NNRTI, atazanavir/ritonavir, or lopinavir/ritonavir to maximize placental transfer as well as to optimally suppress maternal viral load. Darunavir/ritonavir and raltegravir may reasonably serve as second-line agents.

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.

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.

Simultaneous determination of the HIV nucleoside analogue reverse transcriptase inhibitors lamivudine, didanosine, stavudine, zidovudine and abacavir in human plasma by reversed phase high performance liquid chromatography.

PubMed

Verweij-van Wissen, C P W G M; Aarnoutse, R E; Burger, D M

2005-02-25

A reversed phase high performance liquid chromatography method was developed for the simultaneous quantitative determination of the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine, didanosine, stavudine, zidovudine and abacavir in plasma. The method involved solid-phase extraction with Oasis MAX cartridges from plasma, followed by high performance liquid chromatography with a SymmetryShield RP 18 column and ultraviolet detection set at a wavelength of 260 nm. The assay was validated over the concentration range of 0.015-5 mg/l for all five NRTIs. The average accuracies for the assay were 92-102%, inter- and intra-day coefficients of variation (CV) were <2.5% and extraction recoveries were higher than 97%. This method proved to be simple, accurate and precise, and is currently in use in our laboratory for the quantitative analysis of NRTIs in plasma.

A Laccase with Antiproliferative and HIV-I Reverse Transcriptase Inhibitory Activities from the Mycorrhizal Fungus Agaricus placomyces

PubMed Central

Sun, Jian; Chen, Qing-Jun; Cao, Qing-Qin; Wu, Ying-Ying; Xu, Li-Jing; Zhu, Meng-Juan; Ng, Tzi-Bun; Wang, He-Xiang; Zhang, Guo-Qing

2012-01-01

A novel 68 kDa laccase was purified from the mycorrhizal fungus Agaricus placomyces by utilizing a procedure that comprised three successive steps of ion exchange chromatography and gel filtration as the final step. The monomeric enzyme exhibited the N-terminal amino acid sequence of DVIGPQAQVTLANQD, which showed only a low extent of homology to sequences of other fungal laccases. The optimal temperature for A. placomyces laccase was 30°C, and optimal pH values for laccase activity towards the substrates 2,7′-azinobis[3-ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS) and hydroquinone were 5.2 and 6.8, respectively. The laccase displayed, at 30°C and pH 5.2, Km values of 0.392 mM towards hydroquinone and 0.775 mM towards ABTS. It potently suppressed proliferation of MCF 7 human breast cancer cells and Hep G2 hepatoma cells and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity with an IC50 of 1.8 μM, 1.7 μM, and 1.25 μM, respectively, signifying that it is an antipathogenic protein. PMID:23093860

Anti-HIV and cytotoxic biphenyls, benzophenones and xanthones from stems, leaves and twigs of Garcinia speciosa.

PubMed

Pailee, Phanruethai; Kuhakarn, Chutima; Sangsuwan, Chanyapat; Hongthong, Sakchai; Piyachaturawat, Pawinee; Suksen, Kanoknetr; Jariyawat, Surawat; Akkarawongsapat, Radeekorn; Limthongkul, Jitra; Napaswad, Chanita; Kongsaeree, Palangpon; Prabpai, Samran; Jaipetch, Thaworn; Pohmakotr, Manat; Tuchinda, Patoomratana; Reutrakul, Vichai

2018-03-01

Eleven previously undescribed compounds, including four benzophenones (garciosones A-D), four xanthones (garciosones E-H) and three biphenyls (garciosines A-C), along with eighteen known compounds were isolated from the stems, leaves and twigs of Garcinia speciosa Wall. (Clusiaceae). Their structures were established by extensive spectroscopic analysis. For garciosines A-C, the structures were confirmed by single crystal X-ray diffraction analysis. Most of the isolated compounds were evaluated for their cytotoxic activity and anti-HIV-1 activity using the syncytium inhibition assay and HIV-1 reverse transcriptase (RT) assay. The known compounds, 4,6,3',4'-tetrahydroxy-2-methoxybenzophenone and macluraxanthone, displayed significant cytotoxic activity with the ED 50 in the range of 1.85-11.76 μM. 1,5-Dihydroxyxanthone exhibited the most potent anti-HIV activity against syncytium formation with EC 50  < 17.13 μM (SI > 25.28) and 2-(3,3-dimethylallyl)-1,3,7-trihydroxyxanthone was the most active compound in the HIV-1 reverse transcriptase assay with IC 50 value of 58.24 μM. Structure-activity relationship of some isolated compounds were also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Exploiting Drug-Resistant Enzymes as Tools to Identify Thienopyrimidinone Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease H

PubMed Central

Masaoka, Takashi; Chung, Suhman; Caboni, Pierluigi; Rausch, Jason W.; Wilson, Jennifer A.; Taskent-Sezgin, Humeyra; Beutler, John A.; Tocco, Graziella; Le Grice, Stuart F. J.

2013-01-01

The thienopyrimidinone 5,6-dimethyl-2-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4(3H)-one (DNTP) occupies the interface between the p66 ribonuclease H (RNase H) domain and p51 thumb of human immunodeficiency virus reverse transcriptase (HIV RT), thereby inducing a conformational change incompatible with catalysis. Here, we combined biochemical characterization of 39 DNTP derivatives with antiviral testing of selected compounds. In addition to wild-type HIV-1 RT, derivatives were evaluated with rationally-designed, p66/p51 heterodimers exhibiting high-level DNTP sensitivity or resistance. This strategy identified 3′,4′-dihydroxyphenyl (catechol)-substituted thienopyrimidinones with sub-micromolar in vitro activity against both wild type HIV-1 RT and drug-resistant variants. Thermal shift analysis indicates that, in contrast to active site RNase H inhibitors, these thienopyrimidinones destabilize the enzyme, in some instances reducing the Tm by 5°C. Importantly, catechol-containing thienopyrimidinones also inhibit HIV-1 replication in cells. Our data strengthens the case for allosteric inhibition of HIV RNase H activity, providing a platform for designing improved antagonists for use in combination antiviral therapy. PMID:23631411

Functional analysis of the interactions between reovirus particles and various proteases in vitro

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

Sargent, M.D.; Long, D.G.; Borsa, J.

1977-01-01

The digestion of purified reovirus particles by various proteases including chymotrypsin, trypsin, pronase, papain, bromelain, proteinase K, and fibrinolysin has been examined as it relates to virion transcriptase activation and alteration of infectivity. In every case uncoating to the level of active transcriptase proceeds via two mechanistically distinct steps. All the proteases tested serve to mediate only the first of the two steps, converting intact virions to intermediate subviral particles (ISVP) in which the transcriptase is retained in a latent state. The second step of the uncoating process is mediated by a K/sup +/ ion-triggered, endogenous mechanism and results inmore » conversion of ISVP to cores, concomitant with transcriptase activation and loss of infectivity. All of the tested enzymes, except trypsin, reversibly block the second step of uncoating. These results indicate the generality, with respect to protease employed, of the two-step process for reovirus uncoating and transcriptase activation demonstrated previously with chymotrypsin.« less

Mutations in the S gene and in the overlapping reverse transcriptase region in chronic hepatitis B Chinese patients with coexistence of HBsAg and anti-HBs.

PubMed

Ding, Feng; Miao, Xi-Li; Li, Yan-Xia; Dai, Jin-Fen; Yu, Hong-Gang

2016-01-01

The mechanism underlying the coexistence of hepatitis B surface antigen and antibodies to HBsAg in chronic hepatitis B patients remains unknown. This research aimed to determine the clinical and virological features of the rare pattern. A total of 32 chronic hepatitis B patients infected by HBV genotype C were included: 15 carrying both HBsAg and anti-HBs (group I) and 17 solely positive for HBsAg (group II). S gene and reverse transcriptase region sequences were amplified, sequenced and compared with the reference sequences. The amino acid variability within major hydrophilic region, especially the "a" determinant region, and within reverse transcriptase for regions overlapping the major hydrophilic region in group I is significantly higher than those in group II. Mutation sI126S/T within the "a" determinant was the most frequent change, and only patients from group I had the sQ129R, sG130N, sF134I, sG145R amino acid changes, which are known to alter immunogenicity. In chronic patients, the concurrent HBsAg/anti-HBs serological profile is associated with an increased aa variability in several key areas of HBV genome. Additional research on these genetic mutants are needed to clarify their biological significance for viral persistence. Copyright © 2015 Elsevier Editora Ltda. All rights reserved.

Pathology and genomics of pediatric melanoma: A critical reexamination and new insights.

PubMed

Bahrami, Armita; Barnhill, Raymond L

2018-02-01

The clinicopathologic features of pediatric melanoma are distinct from those of the adult counterpart. For example, most childhood melanomas exhibit a uniquely favorable biologic behavior, save for those arising in large/giant congenital nevi. Recent studies suggest that the characteristically favorable biologic behavior of childhood melanoma may be related to extreme telomere shortening and dysfunction in the cancer cells. Herein, we review the genomic profiles that have been defined for the different subtypes of pediatric melanoma and particularly emphasize the potential prognostic value of telomerase reverse transcriptase alterations for these tumors. © 2017 Wiley Periodicals, Inc.

Structural and Preclinical Studies of Computationally Designed Non-Nucleoside Reverse Transcriptase Inhibitors for Treating HIV infection

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

Kudalkar, Shalley N.; Beloor, Jagadish; Chan, Albert H.

The clinical benefits of HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are hindered by their unsatisfactory pharmacokinetic (PK) properties along with the rapid development of drug-resistant variants. However, the clinical efficacy of these inhibitors can be improved by developing compounds with enhanced pharmacological profiles and heightened antiviral activity. We used computational and structure-guided design to develop two next-generation NNRTI drug candidates, compounds I and II, which are members of a class of catechol diethers. We evaluated the preclinical potential of these compounds in BALB/c mice because of their high solubility (510 µg/ml for compound I and 82.9 µg/ml for compoundmore » II), low cytotoxicity, and enhanced antiviral activity against wild-type (WT) HIV-1 RT and resistant variants. Additionally, crystal structures of compounds I and II with WT RT suggested an optimal binding to the NNRTI binding pocket favoring the high anti-viral potency. A single intraperitoneal dose of compounds I and II exhibited a prolonged serum residence time of 48 hours and concentration maximum (Cmax) of 4000- to 15,000-fold higher than their therapeutic/effective concentrations. These Cmax values were 4- to 15-fold lower than their cytotoxic concentrations observed in MT-2 cells. Compound II showed an enhanced area under the curve (0–last) and decreased plasma clearance over compound I and efavirenz, the standard of care NNRTI. Hence, the overall (PK) profile of compound II was excellent compared with that of compound I and efavirenz. Furthermore, both compounds were very well tolerated in BALB/c mice without any detectable acute toxicity. Taken together, these data suggest that compounds I and II possess improved anti-HIV-1 potency, remarkable in vivo safety, and prolonged in vivo circulation time, suggesting strong potential for further development as new NNRTIs for the potential treatment of HIV infection.« less

(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.

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.

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.

The discovery of novel diarylpyri(mi)dine derivatives with high level activity against a wide variety of HIV-1 strains as well as against HIV-2.

PubMed

Lu, Xueyi; Yang, Jiapei; Kang, Dongwei; Gao, Ping; Daelemans, Dirk; De Clercq, Erik; Pannecouque, Christophe; Zhan, Peng; Liu, Xinyong

2018-05-01

By means of structure-based molecular hybridization strategy, a series of novel diarylpyri(mi)dine derivatives targeting the entrance channel of HIV-1 reverse transcriptase (RT) were designed, synthesized and evaluated as potent non-nucleoside reverse transcriptase inhibitors (NNRTIs). Encouragingly, all the tested compounds showed good activities against wild-type (WT) HIV-1 (IIIB) with EC 50 in the range of 1.36 nM-29 nM, which is much better than those of nevirapine (NVP, EC 50  = 125.42 nM) and azidothymidine (AZT, EC 50  = 11.36 nM). Remarkably, these compounds also displayed effective activity against the most of the single and double-mutated HIV-1 strains with low EC 50 values, which is comparable to the control drugs. Besides, these compounds were also exhibited favorable enzymatic inhibitory activity. Moreover, preliminary structure-activity relationships (SARs) and molecular modeling study were investigated and discussed in detail. Unexpectedly, four diarylpyrimidines yielded moderate anti-HIV-2 activities. To our knowledge, this is rarely reported that diarylpyrimidine-based NNRTIs have potent activity against both HIV-1 and HIV-2 in cell culture. Copyright © 2018 Elsevier Ltd. All rights reserved.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine, MK-8591: a novel HIV-1 reverse transcriptase translocation inhibitor.

PubMed

Markowitz, Martin; Sarafianos, Stefan G

2018-07-01

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a nucleoside reverse transcriptase inhibitor (NRTI) with a novel mechanism of action, unique structure, and amongst NRTIs, unparalleled anti-HIV-1 activity. We will summarize its structure and function, antiviral activity, resistance profile, and potential as an antiretroviral for use in the treatment and preexposure prophylaxis of HIV-1 infection. EFdA is active against wild-type (EC50 as low as 50 pmol/l) and most highly NRTI-resistant viruses. The active metabolite, EFdA-triphosphate, has been shown to have a prolonged intracellular half-life in human and rhesus (Rh) blood cells. As a result, single drug doses tested in simian immunodeficiency virus mac251-infected Rh macaques and HIV-1-infected individuals exhibited robust antiviral activity of 7-10 days duration. Preclinical studies of EFdA as preexposure prophylaxis in the Rh macaque/simian/human immunodeficiency virus low-dose intrarectal challenge model have shown complete protection when given in clinically relevant doses. EFdA is a novel antiretroviral with activity against both wild-type and NRTI-resistant viruses. As a result of the prolonged intracellular half-life of its active moiety, it is amenable to flexibility in dosing of at least daily to weekly and perhaps longer.

HIV drug resistance in infants increases with changing prevention of mother-to-child transmission regimens.

PubMed

Poppe, Lisa K; Chunda-Liyoka, Catherine; Kwon, Eun H; Gondwe, Clement; West, John T; Kankasa, Chipepo; Ndongmo, Clement B; Wood, Charles

2017-08-24

The objectives of this study were to determine HIV drug resistance (HIVDR) prevalence in Zambian infants upon diagnosis, and to determine how changing prevention of mother-to-child transmission (PMTCT) drug regimens affect drug resistance. Dried blood spot (DBS) samples from infants in the Lusaka District of Zambia, obtained during routine diagnostic screening, were collected during four different years representing three different PMTCT drug treatment regimens. DNA extracted from dried blood spot samples was used to sequence a 1493 bp region of the reverse transcriptase gene. Sequences were analyzed via the Stanford HIVDRdatabase (http://hivdb.standford.edu) to screen for resistance mutations. HIVDR in infants increased from 21.5 in 2007/2009 to 40.2% in 2014. Nonnucleoside reverse transcriptase inhibitor resistance increased steadily over the sampling period, whereas nucleoside reverse transcriptase inhibitor resistance and dual class resistance both increased more than threefold in 2014. Analysis of drug resistance scores in each group revealed increasing strength of resistance over time. In 2014, children with reported PMTCT exposure, defined as infant prophylaxis and/or maternal treatment, showed a higher prevalence and strength of resistance compared to those with no reported exposure. HIVDR is on the rise in Zambia and presents a serious problem for the successful lifelong treatment of HIV-infected children. PMTCT affects both the prevalence and strength of resistance and further research is needed to determine how to mitigate its role leading to resistance.

Plastid, nuclear and reverse transcriptase sequences in the mitochondrial genome of Oenothera: is genetic information transferred between organelles via RNA?

PubMed Central

Schuster, W; Brennicke, A

1987-01-01

We describe an open reading frame (ORF) with high homology to reverse transcriptase in the mitochondrial genome of Oenothera. This ORF displays all the characteristics of an active plant mitochondrial gene with a possible ribosome binding site and 39% T in the third codon position. It is located between a sequence fragment from the plastid genome and one of nuclear origin downstream from the gene encoding subunit 5 of the NADH dehydrogenase. The nuclear derived sequence consists of 528 nucleotides from the small ribosomal RNA and contains an expansion segment unique to nuclear rRNAs. The plastid sequence contains part of the ribosomal protein S4 and the complete tRNA(Ser). The observation that only transcribed sequences have been found i more than one subcellular compartment in higher plants suggests that interorganellar transfer of genetic information may occur via RNA and subsequent local reverse transcription and genomic integration. PMID:14650433

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

Reverse transcriptase genes are highly abundant and transcriptionally active in marine plankton assemblages

PubMed Central

Lescot, Magali; Hingamp, Pascal; Kojima, Kenji K; Villar, Emilie; Romac, Sarah; Veluchamy, Alaguraj; Boccara, Martine; Jaillon, Olivier; Iudicone, Daniele; Bowler, Chris; Wincker, Patrick; Claverie, Jean-Michel; Ogata, Hiroyuki

2016-01-01

Genes encoding reverse transcriptases (RTs) are found in most eukaryotes, often as a component of retrotransposons, as well as in retroviruses and in prokaryotic retroelements. We investigated the abundance, classification and transcriptional status of RTs based on Tara Oceans marine metagenomes and metatranscriptomes encompassing a wide organism size range. Our analyses revealed that RTs predominate large-size fraction metagenomes (>5 μm), where they reached a maximum of 13.5% of the total gene abundance. Metagenomic RTs were widely distributed across the phylogeny of known RTs, but many belonged to previously uncharacterized clades. Metatranscriptomic RTs showed distinct abundance patterns across samples compared with metagenomic RTs. The relative abundances of viral and bacterial RTs among identified RT sequences were higher in metatranscriptomes than in metagenomes and these sequences were detected in all metatranscriptome size fractions. Overall, these observations suggest an active proliferation of various RT-assisted elements, which could be involved in genome evolution or adaptive processes of plankton assemblage. PMID:26613339

Snapshot of the equilibrium dynamics of a drug bound to HIV-1 reverse transcriptase

NASA Astrophysics Data System (ADS)

Kuroda, Daniel G.; Bauman, Joseph D.; Challa, J. Reddy; Patel, Disha; Troxler, Thomas; Das, Kalyan; Arnold, Eddy; Hochstrasser, Robin M.

2013-03-01

The anti-AIDS drug rilpivirine undergoes conformational changes to bind HIV-1 reverse transcriptase (RT), which is an essential enzyme for the replication of HIV. These changes allow it to retain potency against mutations that otherwise would render the enzyme resistant. Here we report that water molecules play an essential role in this binding process. Femtosecond experiments and theory expose the molecular level dynamics of rilpivirine bound to HIV-1 RT. Two nitrile substituents, one on each arm of the drug, are used as vibrational probes of the structural dynamics within the binding pocket. Two-dimensional vibrational echo spectroscopy reveals that one nitrile group is unexpectedly hydrogen-bonded to a mobile water molecule, not identified in previous X-ray structures. Ultrafast nitrile-water dynamics are confirmed by simulations. A higher (1.51 Å) resolution X-ray structure also reveals a water-drug interaction network. Maintenance of a crucial anchoring hydrogen bond may help retain the potency of rilpivirine against pocket mutations despite the structural variations they cause.

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

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).

The chromosomal distributions of Ty1-copia group retrotransposable elements in higher plants and their implications for genome evolution

Treesearch

J.S. (Pat) Heslop-Harrison; Andrea Brandes; Shin Taketa; Thomas Schmidt; Alexander V. Vershinin; Elena G. Alkhimova; Anette Kamm; Robert L. Doudrick; [and others

1997-01-01

Retrotransposons make up a major fraction - sometimes more than 40% - of all plant genomes investigated so far. We have isolated the reverse transcriptase domains of theTyl-copia group elements from several species, ranging in genome size from some 100 Mbp to 23,000 Mbp, and determined the distribution patterns of these retrotransposons on metaphase chromosomes and...

Identification and characterization of jute LTR retrotransposons:

PubMed Central

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

2011-01-01

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

In Vitro Characterization of MK-1439, a Novel HIV-1 Nonnucleoside Reverse Transcriptase Inhibitor

PubMed Central

Feng, Meizhen; Falgueyret, Jean-Pierre; Tawa, Paul; Witmer, Marc; DiStefano, Daniel; Li, Yuan; Burch, Jason; Sachs, Nancy; Lu, Meiqing; Cauchon, Elizabeth; Campeau, Louis-Charles; Grobler, Jay; Yan, Youwei; Ducharme, Yves; Côté, Bernard; Asante-Appiah, Ernest; Hazuda, Daria J.; Miller, Michael D.

2014-01-01

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are a mainstay of therapy for treating human immunodeficiency type 1 virus (HIV-1)-infected patients. MK-1439 is a novel NNRTI with a 50% inhibitory concentration (IC50) of 12, 9.7, and 9.7 nM against the wild type (WT) and K103N and Y181C reverse transcriptase (RT) mutants, respectively, in a biochemical assay. Selectivity and cytotoxicity studies confirmed that MK-1439 is a highly specific NNRTI with minimum off-target activities. In the presence of 50% normal human serum (NHS), MK-1439 showed excellent potency in suppressing the replication of WT virus, with a 95% effective concentration (EC95) of 20 nM, as well as K103N, Y181C, and K103N/Y181C mutant viruses with EC95 of 43, 27, and 55 nM, respectively. MK-1439 exhibited similar antiviral activities against 10 different HIV-1 subtype viruses (a total of 93 viruses). In addition, the susceptibility of a broader array of clinical NNRTI-associated mutant viruses (a total of 96 viruses) to MK-1439 and other benchmark NNRTIs was investigated. The results showed that the mutant profile of MK-1439 was superior overall to that of efavirenz (EFV) and comparable to that of etravirine (ETR) and rilpivirine (RPV). Furthermore, E138K, Y181C, and K101E mutant viruses that are associated with ETR and RPV were susceptible to MK-1439 with a fold change (FC) of <3. A two-drug in vitro combination study indicated that MK-1439 acts nonantagonistically in the antiviral activity with each of 18 FDA-licensed drugs for HIV infection. Taken together, these in vitro data suggest that MK-1439 possesses the desired properties for further development as a new antiviral agent. PMID:24379202

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

Analytical validation of a reverse transcriptase droplet digital PCR (RT-ddPCR) for quantitative detection of infectious hematopoietic necrosis virus

USGS Publications Warehouse

Jia, Peng; Purcell, Maureen; Pan, Guang; Wang, Jinjin; Kan, Shifu; Liu, Yin; Zheng, Xiaocong; SHi, Xiujie; He, Junqiang; Yu, Li; Hua, Qunyi; Lu, Tikang; Lan, Wensheng; Winton, James; Jin, Ningyi; Liu, Hong

2017-01-01

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/μl while the LOD for the RT-qPCR was 0.2 PFU/μl. Good agreement (69.4–100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout (Oncorhynchus mykiss) and arctic char (Salvelinus alpinus). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.

Vaginal Microbicide Film Combinations of Two Reverse Transcriptase Inhibitors, EFdA and CSIC, for the Prevention of HIV-1 Sexual Transmission.

PubMed

Zhang, Wei; Hu, Minlu; Shi, Yuan; Gong, Tiantian; Dezzutti, Charlene S; Moncla, Bernard; Sarafianos, Stefan G; Parniak, Michael A; Rohan, Lisa C

2015-09-01

EFdA is a potent nucleoside reverse transcriptase inhibitor (NRTI) with activity against a wide spectrum of wild-type and drug resistant HIV-1 variants. CSIC is a tight-binding non-nucleoside reverse transcriptase inhibitor (NNRTI) with demonstrated anti-HIV properties important for use in topical prevention of HIV transmission. The objective of this study was to develop and characterize film-formulated EFdA and CSIC for use as a female-controlled vaginal microbicide to prevent sexual transmission of HIV. Assessments of EFdA- and CSIC-loaded films included physicochemical characteristics, in vitro cytotoxicity, epithelia integrity studies, compatibility with the normal vaginal Lactobacillus flora and anti-HIV bioactivity evaluations. No significant change in physicochemical properties or biological activity of the combination films were noted during 3 months storage. In vitro cytotoxicity and bioactivity testing showed that 50% cytotoxic concentration (CC50) of either EFdA or CSIC was several orders of magnitude higher than the 50% effective concentration (EC50) values. Film-formulated EFdA and CSIC combination showed additive inhibitory activity against wild type and drug-resistant variants of HIV. Epithelial integrity studies demonstrated that the combination vaginal film had a much lower toxicity to HEC-1A monolayers compared to that of VCF®, a commercial vaginal film product containing nonoxynol-9. Polarized ectocervical explants showed films with drug alone or in combination were effective at preventing HIV infection. Our data suggest that vaginal microbicide films containing a combination of the NRTI EFdA and the NNRTI CSIC have potential to prevent HIV-1 sexual transmission.

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.

ONIOM DFT/PM3 calculations on the interaction between dapivirine and HIV-1 reverse transcriptase, a theoretical study.

PubMed

Liang, Y H; Chen, F E

2007-08-01

Theoretical investigations of the interaction between dapivirine and the HIV-1 RT binding site have been performed by the ONIOM2 (B3LYP/6-31G (d,p): PM3) and B3LYP/6-31G (d,p) methods. The results derived from this study indicate that this inhibitor dapivirine forms two hydrogen bonds with Lys101 and exhibits strong π-π stacking or H…π interaction with Tyr181 and Tyr188. These interactions play a vital role in stabilizing the NNIBP/dapivirine complex. Additionally, the predicted binding energy of the BBF optimized structure for this complex system is -18.20 kcal/mol.

The isolation and characterization of a telomerase immortalized goat trophoblast cell line.

PubMed

Dong, F; Huang, Y; Li, W; Zhao, X; Zhang, W; Du, Q; Zhang, H; Song, X; Tong, D

2013-12-01

Trophoblast cells play vital roles in the processes of embryonic implantation and placentation. Many toxicological compounds can induce the malfunction of trophoblast cells, resulting in implantation failure or early embryonic loss. The finite lifespan of primary trophoblast cells limits investigation of the long-term effects of some toxicological compounds on trophoblast cells in vitro. In this study, primary goat trophoblast cells were purified by density gradient centrifugation and specific immuno-affinity purification. Then, the purified cells were immortalized through transfection of a plasmid containing the human telomerase reverse transcriptase (hTERT) gene. hTERT-transfected goat trophoblast cells (hTERT-GTCs) could steadily express hTERT gene and exhibit higher telomerase activity, and persistently proliferate without any signs of senescence up to 50 passages. The immortalized goat trophoblast cells still possessed the basic and key properties of normal primary goat trophoblast cells to express the specific intracellular marker cytokeratin 7 (CK-7) and secrete chorionic gonadotrophin β-subunit (CG-β) and placental lactogen (PL). Further studies showed that the immortalized goat trophoblast cells expressed vimentin and non-classical MHC class I antigen and exhibited invasive phenotype, suggesting that the immortalized goat trophoblasts resembled human extravillous trophoblasts. In addition, this cell line did not show neoplastic transformation either in vivo or in vitro. We concluded the immortalized goat trophoblast cells by hTERT transfection retained the basic and key characteristics of primary trophoblast cells and may provide a useful model to study the effects of some toxicological compounds on trophoblast cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

Unique case of oligoastrocytoma with recurrence and grade progression: Exhibiting differential expression of high mobility group-A1 and human telomerase reverse transcriptase

PubMed Central

Gandhi, Puneet; Khare, Richa; Niraj, Kavita; Garg, Nitin; Sorte, Sandeep K; Gulwani, Hanni

2016-01-01

Mixed gliomas, primarily oligoastrocytomas, account for about 5%-10% of all gliomas. Distinguishing oligoastrocytoma based on histological features alone has limitations in predicting the exact biological behavior, necessitating ancillary markers for greater specificity. In this case report, human telomerase reverse transcriptase (hTERT) and high mobility group-A1 (HMGA1); markers of proliferation and stemness, have been quantitatively analyzed in formalin-fixed paraffin-embedded tissue samples of a 34 years old patient with oligoastrocytoma. Customized florescence-based immunohistochemistry protocol with enhanced sensitivity and specificity is used in the study. The patient presented with a history of generalized seizures and his magnetic resonance imaging scans revealed infiltrative ill-defined mass lesion with calcified foci within the left frontal white matter, suggestive of glioma. He was surgically treated at our center for four consecutive clinical events. Histopathologically, the tumor was identified as oligoastrocytoma-grade II followed by two recurrence events and final progression to grade III. Overall survival of the patient without adjuvant therapy was more than 9 years. Glial fibrillary acidic protein, p53, Ki-67, nuclear atypia index, pre-operative neutrophil-lymphocyte ratio, are the other parameters assessed. Findings suggest that hTERT and HMGA1 are linked to tumor recurrence and progression. Established markers can assist in defining precise histopathological grade in conjuction with conventional markers in clinical setup. PMID:27672647

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

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

Maintenance of tumor initiating cells of defined genetic composition by nucleostemin.

PubMed

Okamoto, Naoko; Yasukawa, Mami; Nguyen, Christine; Kasim, Vivi; Maida, Yoshiko; Possemato, Richard; Shibata, Tatsuhiro; Ligon, Keith L; Fukami, Kiyoko; Hahn, William C; Masutomi, Kenkichi

2011-12-20

Recent work has identified a subset of cells resident in tumors that exhibit properties similar to those found in normal stem cells. Such cells are highly tumorigenic and may be involved in resistance to treatment. However, the genes that regulate the tumor initiating cell (TIC) state are unknown. Here, we show that overexpression of either of the nucleolar GTP-binding proteins nucleostemin (NS) or GNL3L drives the fraction of genetically defined tumor cells that exhibit markers and tumorigenic properties of TICs. Specifically, cells that constitutively express elevated levels of NS or GNL3L exhibit increased TWIST expression, phosphorylation of STAT3, expression of genes that induce pluripotent stem cells, and enhanced radioresistance; in addition, they form tumors even when small numbers of cells are implanted and exhibit an increased propensity to metastasize. GNL3L/NS forms a complex with the telomerase catalytic subunit [human telomerase reverse transcriptase (hTERT)] and the SWItch-Sucrose NonFermentable (SWI-SNF) complex protein brahma-related gene 1 (BRG1), and the expression of each of these components is necessary to facilitate the cancer stem cell state. Together, these observations define a complex composed of TERT, BRG1, and NS/GNL3L that maintains the function of TICs.

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.

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:...

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.

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

Vaginal microbicide film combinations of two reverse transcriptase inhibitors, EFdA and CSIC, for the prevention of HIV-1 sexual transmission

PubMed Central

Zhang, Wei; Hu, Minlu; Shi, Yuan; Gong, Tiantian; Dezzutti, Charlene S.; Moncla, Bernard; Sarafianos, Stefan G.; Parniak, Michael A.; Rohan, Lisa C.

2015-01-01

Purpose EFdA is a potent nucleoside reverse transcriptase inhibitor (NRTI) with activity against a wide spectrum of wild-type and drug resistant HIV-1 variants. CSIC is a tight-binding non-nucleoside reverse transcriptase inhibitor (NNRTI) with demonstrated anti-HIV properties important for use in topical prevention of HIV transmission. The objective of this study was to develop and characterize film-formulated EFdA and CSIC for use as a female-controlled vaginal microbicide to prevent sexual transmission of HIV. Methods Assessments of EFdA- and CSIC-loaded films included physicochemical characteristics, in vitro cytotoxicity, epithelia integrity studies, compatibility with the normal vaginal Lactobacillus flora and anti-HIV bioactivity evaluations. Results No significant change in physicochemical properties or biological activity of the combination films were noted during 3 months storage. In vitro cytotoxicity and bioactivity testing showed that 50% cytotoxic concentration (CC50) of either EFdA or CSIC was several orders of magnitude higher than the 50% effective concentration (EC50) values. Film-formulated EFdA and CSIC combination showed additive inhibitory activity against wild type and drug-resistant variants of HIV. Epithelial integrity studies demonstrated that the combination vaginal film had a much lower toxicity to HEC-1A monolayers compared to that of VCF®, a commercial vaginal film product containing nonoxynol-9. Polarized ectocervical explants showed films with drug alone or in combination were effective at preventing HIV infection. Conclusions Our data suggest that vaginal microbicide films containing a combination of the NRTI EFdA and the NNRTI CSIC have potential to prevent HIV-1 sexual transmission. PMID:25794967

Transmitted drug resistance is still low in newly diagnosed human immunodeficiency virus type 1 CRF06_cpx-infected patients in Estonia in 2010.

PubMed

Avi, Radko; Huik, Kristi; Pauskar, Merit; Ustina, Valentina; Karki, Tõnis; Kallas, Eveli; Jõgeda, Ene-Ly; Krispin, Tõnu; Lutsar, Irja

2014-03-01

The presence of transmitted drug resistance (TDR) in treatment-naive HIV-1-positive subjects is of concern, especially in the countries of the former Soviet Union in which the number of subjects exposed to antiretrovirals (ARV) has exponentially increased during the past decade. We assessed the rate of TDR among newly diagnosed subjects in Estonia in 2010 and compared it to that in 2008. The study included 325 subjects (87% of all subjects tested HIV positive from January 1 to December 31, 2010). Of the 244 sequenced viral genomic RNA in the reverse transcriptase (RT) region 214 were CRF06_cpx, nine were subtype A1, three (one each) were subtype B and subtype C, CRF02_AG, and CRF03_AB; 15 viruses remained unclassified as putative recombinant forms between CRF06_cpx and subtype A1. HIV-1 TDR mutations in 2010 and 2008 (n=145) occurred at similar frequency in 4.5% (95% CI 2.45; 7.98) and 5.5% (95% CI 1.8; 9.24) of the patients, respectively. In 2010, 2.5% (6/244) of the sequences harbored nonnucleoside reverse transcriptase inhibitor (NNRTI) (K103N and K101E), 1.6% (4/244) nucleoside reverse transcriptase inhibitor (NRTI) (M41L, M184I, and K219E), and 0.4% (1/244) protease inhibitor (PI) (V82A) mutations. Our findings indicate that in spite of the increased consumption of ARVs the rate of TDR in Estonia has remained unchanged over the past 3 years. Similar stabilizing or even decreasing trends have been described in Western Europe and North America albeit at higher levels and in different socioeconomic backgrounds.

A decade of HIV-1 drug resistance in the United States: trends and characteristics in a large protease/reverse transcriptase and co-receptor tropism database from 2003 to 2012.

PubMed

Paquet, Agnes C; Solberg, Owen D; Napolitano, Laura A; Volpe, Joseph M; Walworth, Charles; Whitcomb, Jeannette M; Petropoulos, Christos J; Haddad, Mojgan

2014-01-01

Drug resistance testing and co-receptor tropism determination are key components of the management of antiretroviral therapy for HIV-1-infected individuals. The purpose of this study was to examine trends of HIV-1 resistance and viral evolution in the past decade by surveying a large commercial patient testing database. Temporal trends of drug resistance, viral fitness and co-receptor usage among samples submitted for routine phenotypic and genotypic resistance testing to protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), as well as for tropism determination were investigated. Within 62,397 resistant viruses reported from 2003 to 2012, we observed a decreasing trend in the prevalence of three-class resistance (from 25% to 9%) driven by decreased resistance to PIs (43% to 21%) and NRTIs (79% to 57%), while observing a slight increase in NNRTI resistance (68% to 75%). The prevalence of CXCR4-mediated entry among tropism testing samples (n=52,945) declined over time from 47% in 2007 to 40% in 2012. A higher proportion of CXCR4-tropic viruses was observed within samples with three-class resistance (50%) compared with the group with no resistance (36%). Decreased prevalence of three-class resistance and increased prevalence of one-class resistance was observed within samples reported between 2003 and 2012. The fraction of CXCR4-tropic viruses has decreased over time; however, CXCR4 usage was more prevalent among multi-class-resistant samples, which may be due to the more advanced disease stage of treatment-experienced patients. These trends have important implications for clinical practice and future drug discovery and development.

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...

Interaction of aurintricarboxylic acid (ATA) with four nucleic acid binding proteins DNase I, RNase A, reverse transcriptase and Taq polymerase

NASA Astrophysics Data System (ADS)

Ghosh, Utpal; Giri, Kalyan; Bhattacharyya, Nitai P.

2009-12-01

In the investigation of interaction of aurintricarboxylic acid (ATA) with four biologically important proteins we observed inhibition of enzymatic activity of DNase I, RNase A, M-MLV reverse transcriptase and Taq polymerase by ATA in vitro assay. As the telomerase reverse transcriptase (TERT) is the main catalytic subunit of telomerase holoenzyme, we also monitored effect of ATA on telomerase activity in vivo and observed dose-dependent inhibition of telomerase activity in Chinese hamster V79 cells treated with ATA. Direct association of ATA with DNase I ( Kd = 9.019 μM)), RNase A ( Kd = 2.33 μM) reverse transcriptase ( Kd = 0.255 μM) and Taq polymerase ( Kd = 81.97 μM) was further shown by tryptophan fluorescence quenching studies. Such association altered the three-dimensional conformation of DNase I, RNase A and Taq polymerase as detected by circular dichroism. We propose ATA inhibits enzymatic activity of the four proteins through interfering with DNA or RNA binding to the respective proteins either competitively or allosterically, i.e. by perturbing three-dimensional structure of enzymes.

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

Base modifications affecting RNA polymerase and reverse transcriptase fidelity.

PubMed

Potapov, Vladimir; Fu, Xiaoqing; Dai, Nan; Corrêa, Ivan R; Tanner, Nathan A; Ong, Jennifer L

2018-06-20

Ribonucleic acid (RNA) is capable of hosting a variety of chemically diverse modifications, in both naturally-occurring post-transcriptional modifications and artificial chemical modifications used to expand the functionality of RNA. However, few studies have addressed how base modifications affect RNA polymerase and reverse transcriptase activity and fidelity. Here, we describe the fidelity of RNA synthesis and reverse transcription of modified ribonucleotides using an assay based on Pacific Biosciences Single Molecule Real-Time sequencing. Several modified bases, including methylated (m6A, m5C and m5U), hydroxymethylated (hm5U) and isomeric bases (pseudouridine), were examined. By comparing each modified base to the equivalent unmodified RNA base, we can determine how the modification affected cumulative RNA polymerase and reverse transcriptase fidelity. 5-hydroxymethyluridine and N6-methyladenosine both increased the combined error rate of T7 RNA polymerase and reverse transcriptases, while pseudouridine specifically increased the error rate of RNA synthesis by T7 RNA polymerase. In addition, we examined the frequency, mutational spectrum and sequence context of reverse transcription errors on DNA templates from an analysis of second strand DNA synthesis.

Effects of water extract of Curcuma longa (L.) roots on immunity and telomerase function.

PubMed

Pan, Min-Hsiung; Wu, Jia-Ching; Ho, Chi-Tang; Badmaev, Vladimir

2017-05-12

Background Immunity and Longevity Methods A water extract of Curcuma longa (L.) [vern. Turmeric] roots (TurmericImmune™) standardized for a minimum 20 % of turmeric polysaccharides ukonan A, B, C and D was evaluated for its biological properties in in vitro tissue culture studies. Results The water extract of turmeric (TurP) exhibited induced-nitric oxide (NO) production in RAW264.7 macrophages. These results suggested the immunomodulatory effects of TurP. In addition, the polysaccharides up-regulated function of telomerase reverse transcriptase (TERT) equally to the phenolic compound from turmeric, curcumin. Conclusions The ukonan family of polysaccharides may assist in promoting cellular immune responses, tissue repair and lifespan by enhancing immune response and telomere function.

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

Homodimerization of the p51 Subunit of HIV-1 Reverse Transcriptase

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

Zheng, X.; Mueller, G; Cuneo, M

2010-01-01

The dimerization of HIV reverse transcriptase (RT), required to obtain the active form of the enzyme, is influenced by mutations, non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleotide substrates, Mg ions, temperature, and specifically designed dimerization inhibitors. In this study, we have utilized nuclear magnetic resonance (NMR) spectroscopy of the [methyl-{sup 13}C]methionine-labeled enzyme and small-angle X-ray scattering (SAXS) to investigate how several of these factors influence the dimerization behavior of the p51 subunit. The {sup 1}H-{sup 13}C HSQC spectrum of p51 obtained at micromolar concentrations indicates that a significant fraction of the p51 adopts a 'p66-like' conformation. SAXS data obtained for p51more » samples were used to determine the fractions of monomer and dimer in the sample and to evaluate the conformation of the fingers/thumb subdomain. All of the p51 monomer observed was found to adopt the compact, 'p51C' conformation observed for the p51 subunit in the RT heterodimer. The NMR and SAXS data indicate that the p51 homodimer adopts a structure that is similar to the p66/p51 heterodimer, with one p51C subunit and a second p51 subunit in an extended, 'p51E' conformation that resembles the p66 subunit of the heterodimer. The fractional dimer concentration and the fingers/thumb orientation are found to depend strongly on the experimental conditions and exhibit a qualitative dependence on nevirapine and ionic strength (KCl) that is similar to the behavior reported for the heterodimer and the p66 homodimer. The L289K mutation interferes with p51 homodimer formation as it does with formation of the heterodimer, despite its location far from the dimer interface. This effect is readily interpreted in terms of a conformational selection model, in which p51{sub L289K} has a much greater preference for the compact, p51C conformation. A reduced level of dimer formation then results from the reduced ratio of the p51E{sub L289K} to p51C{sub L289K} monomers.« less

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...

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

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.

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.

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

PubMed

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

2005-01-01

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

In Vitro Virology Profile of Tenofovir Alafenamide, a Novel Oral Prodrug of Tenofovir with Improved Antiviral Activity Compared to That of Tenofovir Disoproxil Fumarate

PubMed Central

Stepan, George; Tian, Yang; Miller, Michael D.

2015-01-01

Tenofovir alafenamide (TAF) is an investigational oral prodrug of the HIV-1 nucleotide reverse transcriptase inhibitor tenofovir (TFV). Tenofovir disoproxil fumarate (TDF) is another TFV prodrug, widely used for the treatment of HIV-1 infection. TAF is converted mostly intracellularly to TFV and, in comparison to TDF, achieves higher tenofovir diphosphate (TFV-DP) levels in peripheral blood mononuclear cells. As a result, TAF has demonstrated potent anti-HIV-1 activity at lower doses than TDF in monotherapy studies. Here, the in vitro virology profile of TAF was evaluated and compared to that of TDF. TAF displayed potent antiviral activity against all HIV-1 groups/subtypes, as well as HIV-2. TAF exhibited minimal changes in the drug concentration needed to inhibit 50% of viral spread (EC50) upon removal of the prodrug, similar to TDF, demonstrating intracellular antiviral persistence. While TAF and TDF exhibited comparable potencies in the absence of serum pretreatment, TAF maintained activity in the presence of human serum, whereas TDF activity was significantly reduced. This result demonstrates TAF's improved plasma stability over TDF, which is driven by the different metabolic pathways of the two prodrugs and is key to TAF's improved in vivo antiviral activity. The activity of TAF is specific for HIV, as TAF lacked activity against a large panel of human viruses, with the exception of herpes simplex virus 2, where weak TAF antiviral activity was observed, as previously observed with TFV. Finally, in vitro combination studies with antiretroviral drugs from different classes showed additive to synergistic interactions with TAF, consistent with ongoing clinical studies with TAF in fixed-dose combinations with multiple other antiretroviral drugs for the treatment of HIV. PMID:26149992

Osteoporosis and osteopenia are not associated with T-cell activation in older cART-treated HIV-infected patients.

PubMed

Krikke, M; Klomberg, R C W; van der Veer, E; Tesselaar, K; Verhaar, H J J; Hoepelman, A I M; Arends, J E

2017-05-01

A higher risk of developing osteopenia/ osteoporosis has been seen in HIV-infected patients. We compared HIV-infected patients, all treated with combination antiretroviral therapy (cART), with a low bone mineral density (BMD) (T-score < -1) to those with a normal BMD (T-score > -1), examining the relation with T-cell activation and bone turnover markers (c-terminal telopeptide (CTX) and procollagen type 1 amino-terminal propeptide (P1NP)). In this single visit pilot study, bone turnover markers, T-cell activation (CD38 + HLA - DR +) and senescence (CD57+) of T cells were measured in patients who had previously undergone dual energy X-ray absorptiometry scanning. All study participants (n = 16) were male, on cART, with a median age of 61 years (IQR 56-66). Nine patients had osteopenia/osteoporosis. When comparing the patients with osteopenia/osteoporosis with those with a normal BMD, no differences in activation and senescence were found. A relation was seen between higher bone formation (P1NP) and patients who were on cART for longer. The median length of cART use was 5.5 years (IQR 4.5-7.8), with all patients on nucleoside reverse transcriptase inhibitors, 88% on tenofovir, 63% on non-nucleoside reverse transcriptase inhibitors (NNRTIs) and 38% on protease inhibitors. Osteopenia/osteoporosis was seen in 100% of the patients on protease inhibitors versus 30% of those on NNRTIs. This study did not find an association between activated T cells and BMD, thus did not explain the higher prevalence of osteopenia/osteoporosis in HIV-infected patients. Interestingly, this small pilot showed that cART might influence BMD, with a possible negative effect for protease inhibitors and a possible protective effect for NNRTIs. These results warrant further investigation.

High level of APOBEC3F/3G editing in HIV-2 DNA vif and pol sequences from antiretroviral-naive patients.

PubMed

Bertine, Mélanie; Charpentier, Charlotte; Visseaux, Benoit; Storto, Alexandre; Collin, Gilles; Larrouy, Lucile; Damond, Florence; Matheron, Sophie; Brun-Vézinet, Françoise; Descamps, Diane

2015-04-24

In HIV-1, hypermutation introduced by APOBEC3F/3G cytidine deaminase activity leads to defective viruses. In-vivo impact of APOBEC3F/3G editing on HIV-2 sequences remains unknown. The objective of this study was to assess the level of APOBEC3F/3G editing in HIV-2-infected antiretroviral-naive patients. Direct sequencing of vif and pol regions was performed on HIV-2 proviral DNA from antiretroviral-naive patients included in the French Agence Nationale de Recherches sur le SIDA et les hépatites virales CO5 HIV-2 cohort. Hypermutated sequences were identified using Hypermut2.0 program. HIV-1 proviral sequences from Genbank were also assessed. Among 82 antiretroviral-naive HIV-2-infected patients assessed, 15 (28.8%) and five (16.7%) displayed Vif proviral defective sequences in HIV-2 groups A and B, respectively. A lower proportion of defective sequences was observed in protease-reverse transcriptase region. A higher median number of G-to-A mutations was observed in HIV-2 group B than in group A, both in Vif and protease-reverse transcriptase regions (P = 0.02 and P = 0.006, respectively). Compared with HIV-1 Vif sequences, a higher number of Vif defective sequences was observed in HIV-2 group A (P = 0.00001) and group B sequences (P = 0.013). We showed for the first time a high level of APOBEC3F/3G editing in HIV-2 sequences from antiretroviral-naive patients. Our study reported a group effect with a significantly higher level of APOBEC3F/3G editing in HIV-2 group B than in group A sequences.

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.

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.

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.

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.

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.

Bauhinia variegata var. variegata trypsin inhibitor: From isolation to potential medicinal applications

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

Fang, Evandro Fei; Wong, Jack Ho; Bah, Clara Shui Fern

Here we report for the first time of a new Kunitz-type trypsin inhibitor (termed BvvTI) from seeds of the Camel's foot tree, Bauhinia variegata var. variegata. BvvTI shares the same reactive site residues (Arg, Ser) and exhibits a homology of N-terminal amino acid sequence to other Bauhinia protease inhibitors. The trypsin inhibitory activity (K{sub i}, 0.1 x 10{sup -9} M) of BvvTI ranks the highest among them. Besides anti-HIV-1 reverse transcriptase activity, BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way. This may partially be contributed by its induction of cytokines and apoptotic bodies.more » These results unveil potential medicinal applications of BvvTI.« less

Bauhinia variegata var. variegata trypsin inhibitor: from isolation to potential medicinal applications.

PubMed

Fang, Evandro Fei; Wong, Jack Ho; Bah, Clara Shui Fern; Lin, Peng; Tsao, Sai Wah; Ng, Tzi Bun

2010-06-11

Here we report for the first time of a new Kunitz-type trypsin inhibitor (termed BvvTI) from seeds of the Camel's foot tree, Bauhinia variegata var. variegata. BvvTI shares the same reactive site residues (Arg, Ser) and exhibits a homology of N-terminal amino acid sequence to other Bauhinia protease inhibitors. The trypsin inhibitory activity (K(i), 0.1 x 10(-9)M) of BvvTI ranks the highest among them. Besides anti-HIV-1 reverse transcriptase activity, BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way. This may partially be contributed by its induction of cytokines and apoptotic bodies. These results unveil potential medicinal applications of BvvTI. (c) 2010 Elsevier Inc. All rights reserved.

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.

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

A novel hemagglutinin with antiproliferative activity against tumor cells from the hallucinogenic mushroom Boletus speciosus.

PubMed

Sun, Jian; Ng, Tzi-Bun; Wang, Hexiang; Zhang, Guoqing

2014-01-01

Little was known about bioactive compounds from the hallucinogenic mushroom Boletus speciosus. In the present study, a hemagglutinin (BSH, B. speciosus hemagglutinin) was isolated from its fruiting bodies and enzymatic properties were also tested. The chromatographic procedure utilized comprised anion exchange chromatography on Q-Sepharose, cation exchange chromatography on CM-Cellulose, cation exchange chromatography on SP-Sepharose, and gel filtration by FPLC on Superdex 75. The hemagglutinin was a homodimer which was estimated to be approximately 31 kDa in size. The activity of BSH was stable up to 60°C, while there was a precipitous drop in activity when the temperature was elevated to 70°C. BSH retained 25% hemagglutinating activity when exposed to 100 mM NaOH and 25 mM HCl. The activity was potently inhibited by 1.25 mM Hg(2+) and slightly inhibited by Fe(2+), Ca(2+), and Pb(2+). None of the sugars tested showed inhibition towards BSH. Its hemagglutinating activity towards human erythrocytes type A, type B, and type AB was higher than type O. The hemagglutinin showed antiproliferative activity towards hepatoma Hep G2 cells and mouse lymphocytic leukemia cells (L1210) in vitro, with IC50 of 4.7 μ M and 7.0 μ M, respectively. It also exhibited HIV-1 reverse transcriptase inhibitory activity with an IC50 of 7.1 μ M.

Three Dimensional Neuronal Cell Cultures More Accurately Model Voltage Gated Calcium Channel Functionality in Freshly Dissected Nerve Tissue

PubMed Central

Kisaalita, William

2012-01-01

It has been demonstrated that neuronal cells cultured on traditional flat surfaces may exhibit exaggerated voltage gated calcium channel (VGCC) functionality. To gain a better understanding of this phenomenon, primary neuronal cells harvested from mice superior cervical ganglion (SCG) were cultured on two dimensional (2D) flat surfaces and in three dimensional (3D) synthetic poly-L-lactic acid (PLLA) and polystyrene (PS) polymer scaffolds. These 2D- and 3D-cultured cells were compared to cells in freshly dissected SCG tissues, with respect to intracellular calcium increase in response to high K+ depolarization. The calcium increases were identical for 3D-cultured and freshly dissected, but significantly higher for 2D-cultured cells. This finding established the physiological relevance of 3D-cultured cells. To shed light on the mechanism behind the exaggerated 2D-cultured cells’ functionality, transcriptase expression and related membrane protein distributions (caveolin-1) were obtained. Our results support the view that exaggerated VGCC functionality from 2D cultured SCG cells is possibly due to differences in membrane architecture, characterized by uniquely organized caveolar lipid rafts. The practical implication of use of 3D-cultured cells in preclinical drug discovery studies is that such platforms would be more effective in eliminating false positive hits and as such improve the overall yield from screening campaigns. PMID:23049767

Evidence for a relief of repression mechanism for activation of the human telomerase reverse transcriptase promoter.

PubMed

Wang, Shuwen; Zhu, Jiyue

2003-05-23

The transcriptional activation of human telomerase reverse transcriptase (hTERT) is an important step during cellular immortalization and tumorigenesis. To study how this activation occurs during immortalization, we have established a set of genetically related pre-crisis cells and their immortal progeny. As expected, hTERT mRNA was detected in our telomerase-positive immortal cells but not in pre-crisis cells or telomerase-negative immortal cells. However, transiently transfected luciferase reporters controlled by hTERT promoter sequences exhibited similar levels of luciferase activity in both telomerase-positive and -negative cells, suggesting that the endogenous chromatin context is likely required for hTERT regulation. Analysis of chromatin susceptibility to DNase I digestion consistently identified a DNase I hypersensitivity site (DHS) near the hTERT transcription initiation site in telomerase-positive cells. In addition, the histone deacetylase inhibitor trichostatin A (TSA) induced hTERT transcription and also a general increase in chromatin sensitivity to DNase treatment in telomerase-negative cells. The TSA-induced hTERT transcription in pre-crisis cells was accompanied by the formation of a DHS at the hTERT promoter. Furthermore, the TSA-induced hTERT transcription and chromatin alterations were not blocked by cycloheximide, suggesting that this induction does not require de novo protein synthesis and that TSA induces hTERT expression through the inhibition of histone deacetylation at the hTERT promoter. Taken together, our results suggest that the endogenous chromatin environment plays a critical role in the regulation of hTERT expression during cellular immortalization.

The site-specific ribosomal DNA insertion element R1Bm belongs to a class of non-long-terminal-repeat retrotransposons.

PubMed Central

Xiong, Y; Eickbush, T H

1988-01-01

Two types of insertion elements, R1 and R2 (previously called type I and type II), are known to interrupt the 28S ribosomal genes of several insect species. In the silkmoth, Bombyx mori, each element occupies approximately 10% of the estimated 240 ribosomal DNA units, while at most only a few copies are located outside the ribosomal DNA units. We present here the complete nucleotide sequence of an R1 insertion from B. mori (R1Bm). This 5.1-kilobase element contains two overlapping open reading frames (ORFs) which together occupy 88% of its length. ORF1 is 461 amino acids in length and exhibits characteristics of retroviral gag genes. ORF2 is 1,051 amino acids in length and contains homology to reverse transcriptase-like enzymes. The analysis of 3' and 5' ends of independent isolates from the ribosomal locus supports the suggestion that R1 is still functioning as a transposable element. The precise location of the element within the genome implies that its transposition must occur with remarkable insertion sequence specificity. Comparison of the deduced amino acid sequences from six retrotransposons, R1 and R2 of B. mori, I factor and F element of Drosophila melanogaster, L1 of Mus domesticus, and Ingi of Trypanosoma brucei, reveals a relatively high level of sequence homology in the reverse transcriptase region. Like R1, these elements lack long terminal repeats. We have therefore named this class of related elements the non-long-terminal-repeat (non-LTR) retrotransposons. Images PMID:2447482

Efficacy and safety of TMC278 in antiretroviral-naive HIV-1 patients: week 96 results of a phase IIb randomized trial.

PubMed

Pozniak, Anton L; Morales-Ramirez, Javier; Katabira, Elly; Steyn, Dewald; Lupo, Sergio H; Santoscoy, Mario; Grinsztejn, Beatriz; Ruxrungtham, Kiat; Rimsky, Laurence T; Vanveggel, Simon; Boven, Katia

2010-01-02

TMC278 is a next-generation nonnucleoside reverse transcriptase inhibitor highly active against wild-type and nonnucleoside reverse transcriptase inhibitor-resistant HIV-1 in vitro. The week 96 analysis of TMC278-C204, a large dose-ranging study of TMC278 in treatment-naive HIV-1-infected patients, is presented. Phase IIb randomized trial. Three hundred sixty-eight patients were randomized and treated with three blinded once-daily TMC278 doses 25, 75 or 150 mg, or an open-label, active control, efavirenz 600 mg once daily, all with two nucleoside reverse transcriptase inhibitors. The primary analysis was at week 48. No TMC278 dose-response relationship for efficacy and safety was observed. TMC278 demonstrated potent antiviral efficacy comparable with efavirenz over 48 weeks that was sustained to week 96 (76.9-80.0% and 71.4-76.3% of TMC278-treated patients with confirmed viral load <50 copies/ml, respectively; time-to-loss of virological-response algorithm). Median increases from baseline in CD4 cell count with TMC278 at week 96 (138.0-149.0 cells/microl) were higher than at week 48 (108.0-123.0 cells/microl). All TMC278 doses were well tolerated. The incidences of the most commonly reported grade 2-4 adverse events at least possibly related to study medication, including nausea, dizziness, abnormal dreams/nightmare, dyspepsia, asthenia, rash, somnolence and vertigo, were low and lower with TMC278 than with efavirenz. Incidences of serious adverse events, grade 3 or 4 adverse events and discontinuations due to adverse events were similar among groups. All TMC278 doses demonstrated potent and sustained efficacy comparable with efavirenz in treatment-naive patients over 96 weeks. TMC278 was well tolerated with lower incidences of neurological and psychiatric adverse events, rash and lower lipid elevations than those with efavirenz. TMC278 25 mg once daily was selected for further clinical development.

Nelfinavir and non-nucleoside reverse transcriptase inhibitor-based salvage regimens in heavily HIV pretreated patients

PubMed Central

Baril, Jean-Guy; Lefebvre, Eric A; Lalonde, Richard G; Shafran, Stephen D; Conway, Brian

2003-01-01

OBJECTIVE: To assess the efficacy of nelfinavir mesylate (NFV) in combination with delavirdine mesylate (DLV) or efavirenz (EFV) and other antiretroviral agents following virological failure on other protease inhibitor (PI)-based regimens. DESIGN: Multicentre, retrospective chart review. METHODS: One hundred-one patients who were naive to both NFV and non-nucleoside reverse transcriptase inhibitors (NNRTIs) and who initiated NFV plus DLV or EFV-based salvage regimens were reviewed. Response to treatment was defined as a reduction in HIV ribonucleic acid (RNA) levels to unquantifiable levels (less than 50 copies/mL, less than 400 copies/mL, less than 500 copies/mL) on at least one occasion after the initiation of salvage therapy. Baseline correlates of response, including prior duration of HIV infection, prior number of regimens, viral load and CD4 cell counts were also evaluated. RESULTS: Patients had a mean duration of HIV infection of 10 years, a mean duration of prior therapy of four years, a median of four prior nucleoside reverse transcriptase inhibitors and a median of two prior PIs. At the time of review the mean duration of salvage therapy was 63.4 weeks. Virological suppression was achieved in 59 (58.4%) patients within a mean of eight weeks and maintained for a mean of 44.9 weeks (the mean follow-up was 78 weeks). Of the non-responders, 16 (38%) achieved a less than 1 log10 decrease in HIV RNA levels. Although there was no association between baseline correlates, response rate (75.7%) was significantly higher in patients with HIV RNA levels of 50,000 copies/mL or lower and CD4 counts greater than 200 cells/mm3. CONCLUSION: NFV/NNRTI-based highly active antiretroviral therapy regimens are an effective therapy in many patients who have experienced virological breakthroughs on at least one prior PI-based regimen. PMID:18159457

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).

The Role of eIF4E Activity in Breast Cancer

DTIC Science & Technology

2010-08-01

ORF, open reading frame; qPCR, quantitative PCR; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase ; uORF, upstream ORF; UTR...were also performed using template lacking RT ( reverse transcriptase ): products were either undetectable or greatly reduced (>30000-fold less product...have previously shown that a 5’UTR expressed from the human AXIN2 gene contains a sixty nucleotide sequence that is predicted to form a stable stem

Crystal structures of HIV-1 nonnucleoside reverse transcriptase inhibitors: N-benzyl-4-methyl-benzimidazoles

NASA Astrophysics Data System (ADS)

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

2009-07-01

HIV-1 nonnucleoside reverse transcriptase inhibitors are potentially specific and effective drugs in AIDS therapy. The presence of two aromatic systems with an angled orientation in the molecule of the inhibitor is crucial for interactions with HIV-1 RT. The inhibitor drives like a wedge into the cluster of aromatic residues of RT HIV-1 and restrains the enzyme in a conformation that blocks the chemical step of nucleotide incorporation. Structural studies provide useful information for designing new, more active inhibitors. The crystal structures of four NNRTIs are presented here. The investigated compounds are derivatives of N-benzyl-4-methyl-benzimidazole with various aliphatic and aromatic substituents at carbon 2 positions and a 2,6-dihalogeno-substituted N-benzyl moiety. Structural data reported here show that the conformation of the investigated compounds is relatively rigid. Such feature is important for the nonnucleoside inhibitor binding to HIV-1 reverse transcriptase.

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.

The history of antiretrovirals: key discoveries over the past 25 years.

PubMed

De Clercq, Erik

2009-09-01

Within 25 years after zidovudine (3'-azido-2',3'-dideoxythymidine, AZT) was first described as an inhibitor of HIV replication, 25 anti-HIV drugs have been formally approved for clinical use in the treatment of HIV infections: seven nucleoside reverse transcriptase inhibitors (NRTIs): zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir and emtricitabine; one nucleotide reverse transcriptase inhibitor (NtRTI): tenofovir [in its oral prodrug form: tenofovir disoproxil fumarate (TDF)]; four non-nucleoside reverse transcriptase inhibitors (NNRTIs): nevirapine, delavirdine, efavirenz and etravirine; ten protease inhibitors (PIs): saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, atazanavir, fosamprenavir, tipranavir and darunavir; one fusion inhibitor (FI): enfuvirtide; one co-receptor inhibitor (CRI): maraviroc and one integrase inhibitor (INI): raltegravir. These compounds are used in various drug combination (some at fixed dose) regimens so as to achieve the highest possible benefit and tolerability, and to diminish the risk of virus-drug resistance development. (c) 2009 John Wiley & Sons, Ltd.

Substrate-induced stable enzyme-inhibitor complex formation allows tight binding of novel 2-aminopyrimidin-4(3H)-ones to drug-resistant HIV-1 reverse transcriptase mutants.

PubMed

Samuele, Alberta; Facchini, Marcella; Rotili, Dante; Mai, Antonello; Artico, Marino; Armand-Ugón, Mercedes; Esté, José A; Maga, Giovanni

2008-09-01

We recently reported the synthesis and biological evaluation of a novel series of 5-alkyl-2-(N,N-disubstituted)amino-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H)-ones (F(2)-N,N-DABOs). These compounds are highly active against both wild-type HIV-1 and the K103N, Y181C, and Y188L mutant strains. Herein we present novel 6-(2-chloro-6-fluorophenylalkyl)-N,N-DABO (2-Cl-6-F-N,N-DABO) derivatives and investigate the molecular basis for their high-affinity binding to HIV-1 reverse transcriptase (RT). Our results show that the new compounds display higher association rates than the difluoro derivatives toward wild-type HIV-1 RT or drug-resistant RT mutant forms. We also show that they preferentially associate to either the free enzyme or the enzyme-nucleic acid binary complex, and that this binding is stabilized upon formation of the ternary complex between HIV-1 RT and both the nucleic acid and nucleotide substrates. Interestingly, one compound showed dissociation rates from the ternary complex with RT mutants K103N and Y181I 10-20-fold slower than from the corresponding complex with wild-type RT.

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

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.

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.

The public health approach to identify antiretroviral therapy failure: high-level nucleoside reverse transcriptase inhibitor resistance among Malawians failing first-line antiretroviral therapy

PubMed Central

Hosseinipour, Mina C.; van Oosterhout, Joep J.G.; Weigel, Ralf; Phiri, Sam; Kamwendo, Debbie; Parkin, Neil; Fiscus, Susan A.; Nelson, Julie A.E.; Eron, Joseph J.; Kumwenda, Johnstone

2010-01-01

Background Over 150 000 Malawians have started antiretroviral therapy (ART), in which first-line therapy is stavudine/lamivudine/nevirapine. We evaluated drug resistance patterns among patients failing first-line ART on the basis of clinical or immunological criteria in Lilongwe and Blantyre, Malawi. Methods Patients meeting the definition of ART failure (new or progressive stage 4 condition, CD4 cell count decline more than 30%, CD4 cell count less than that before treatment) from January 2006 to July 2007 were evaluated. Among those with HIV RNA of more than 1000 copies/ml, genotyping was performed. For complex genotype patterns, phenotyping was performed. Results Ninety-six confirmed ART failure patients were identified. Median (interquartile range) CD4 cell count, log10 HIV-1 RNA, and duration on ART were 68 cells/μl (23–174), 4.72 copies/ml (4.26–5.16), and 36.5 months (26.6–49.8), respectively. Ninety-three percent of samples had nonnucleoside reverse transcriptase inhibitor mutations, and 81% had the M184V mutation. The most frequent pattern included M184V and nonnucleoside reverse transcriptase inhibitor mutations along with at least one thymidine analog mutation (56%). Twenty-three percent of patients acquired the K70E or K65R mutations associated with tenofovir resistance; 17% of the patients had pan-nucleoside resistance that corresponded to K65R or K70E and additional resistance mutations, most commonly the 151 complex. Emergence of the K65R and K70E mutations was associated with CD4 cell count of less than 100 cells/μl (odds ratio 6.1) and inversely with the use of zidovudine (odds ratio 0.18). Phenotypic susceptibility data indicated that the nucleoside reverse transcriptase inhibitor backbone with the highest activity for subsequent therapy was zidovudine/lamivudine/tenofovir, followed by lamivudine/tenofovir, and then abacavir/didanosine. Conclusion When clinical and CD4 cell count criteria are used to monitor first-line ART failure, extensive nucleoside reverse transcriptase inhibitor and nonnucleoside reverse transcriptase inhibitor resistance emerges, with most patients having resistance profiles that markedly compromise the activity of second-line ART. PMID:19417582

The Role of elF4E Activity in Breast Cancer

DTIC Science & Technology

2011-08-01

protein; ORF, open reading frame; qPCR, quantitative PCR; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase ; uORF, upstream ORF; UTR...Reactions were also performed using template lacking RT ( reverse transcriptase ): products were either undetectable or greatly reduced (>30000-fold less...that a 5’UTR expressed from the human AXIN2 gene contains a sixty nucleotide sequence that is predicted to form a stable stem-loop structure6. This

Molecular docking and 3D-QSAR studies on triazolinone and pyridazinone, non-nucleoside inhibitor of HIV-1 reverse transcriptase.

PubMed

Sivan, Sree Kanth; Manga, Vijjulatha

2010-06-01

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are allosteric inhibitors of the HIV-1 reverse transcriptase. Recently a series of Triazolinone and Pyridazinone were reported as potent inhibitors of HIV-1 wild type reverse transcriptase. In the present study, docking and 3D quantitative structure activity relationship (3D QSAR) studies involving comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed on 31 molecules. Ligands were built and minimized using Tripos force field and applying Gasteiger-Hückel charges. These ligands were docked into protein active site using GLIDE 4.0. The docked poses were analyzed; the best docked poses were selected and aligned. CoMFA and CoMSIA fields were calculated using SYBYL6.9. The molecules were divided into training set and test set, a PLS analysis was performed and QSAR models were generated. The model showed good statistical reliability which is evident from the r2 nv, q2 loo and r2 pred values. The CoMFA model provides the most significant correlation of steric and electrostatic fields with biological activities. The CoMSIA model provides a correlation of steric, electrostatic, acceptor and hydrophobic fields with biological activities. The information rendered by 3D QSAR model initiated us to optimize the lead and design new potential inhibitors.

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.

Novel Codon Insert in HIV Type 1 Clade B Reverse Transcriptase Associated with Low-Level Viremia During Antiretroviral Therapy

PubMed Central

Gianella, Sara; Vazquez, Homero; Ignacio, Caroline; Zweig, Adam C.; Richman, Douglas D.; Smith, Davey M.

2014-01-01

Abstract We investigated the pol genotype in two phylogenetically and epidemiologically linked partners, who were both experiencing persistent low-level viremia during antiretroviral therapy. In one partner we identified a new residue insertion between codon 248 and 249 of the HIV-1 RNA reverse transcriptase (RT) coding region (HXB2 numbering). We then investigated the potential impact of identified mutations in RT and antiretroviral binding affinity using a novel computational approach. PMID:24020934

Transmitted drug resistance and type of infection in newly diagnosed HIV-1 individuals in Honduras.

PubMed

Murillo, Wendy; Paz-Bailey, Gabriela; Morales, Sonia; Monterroso, Edgar; Paredes, Mayte; Dobbs, Trudy; Parekh, Bharat S; Albert, Jan; Rivera, Ivette Lorenzana de

2010-12-01

Transmitted drug resistance (TDR) reduces the efficacy of antiretroviral treatment and is a public health concern. To gain insight in the epidemiology of TDR in Honduras by evaluating the amount of TDR in a representative sample of newly diagnosed individuals and by determining whether these are recent or established infections. Two hundred treatment-naïve, newly diagnosed HIV-positive individuals representing different population groups (general population, Garifunas ethnic group, female sex workers and men who have sex with men) and different geographic regions were enrolled during April 2004-April 2007. The HIV-1 pol gene was sequenced to identify drug-resistant mutations and TDR was scored as recommended by the WHO. Specimens were classified as recent or established infections using the BED assay. Among 200 samples analyzed from Honduran patients the prevalence of TDR was 7% (95% CI: 3.9-11.5%), 5% for non-nucleoside reverse transcriptase inhibitors (NNRTIs), 3% for nucleoside reverse transcriptase inhibitors (NRTIs) and 0.5% for protease inhibitors (PIs). Testing of these samples with the BED assay revealed that 12% of the specimens were associated with recent infections. TDR was significantly more common in specimens with recent infection (21%) than established infection (5%) (p=0.016). The prevalence of TDR in Honduras was moderate (7%). The percentage of specimens who were recently infected was low (12%), suggesting that late HIV diagnosis is common. The TDR prevalence was higher in recent than in established infections, which may indicate that TDR is increasing over time. The higher prevalence of NNRTI and NRTI mutations as compared to PI mutations is probably due to a broader and longer use of these drugs in Honduras. Copyright © 2010 Elsevier B.V. All rights reserved.

Prevalence and patterns of HIV transmitted drug resistance in Guatemala.

PubMed

Avila-Ríos, Santiago; Mejía-Villatoro, Carlos R; García-Morales, Claudia; Soto-Nava, Maribel; Escobar, Ingrid; Mendizabal, Ricardo; Girón, Amalia; García, Leticia; Reyes-Terán, Gustavo

2011-12-01

To assess human immunodeficiency virus (HIV) diversity and the prevalence of transmitted drug resistance (TDR) in Guatemala. One hundred forty-five antiretroviral treatment-naïve patients referred to the Roosevelt Hospital in Guatemala City were enrolled from October 2010 to March 2011. Plasma HIV pol sequences were obtained and TDR was assessed with the Stanford algorithm and the World Health Organization (WHO) TDR surveillance mutation list. HIV subtype B was highly prevalent in Guatemala (96.6%, 140/145), and a 2.8% (4/145) prevalence of BF1 recombinants and 0.7% (1/145) prevalence of subtype C viruses were found. TDR prevalence for the study period was 8.3% (12/145) with the Stanford database algorithm (score > 15) and the WHO TDR surveillance mutation list. Most TDR cases were associated with non-nucleoside reverse transcriptase inhibitors (NNRTIs) (83.3%, 10/12); a low prevalence of nucleoside reverse transcriptase inhibitors and protease inhibitors was observed in the cohort (< 1% for both families). Low selection of antiretroviral drug resistance mutations was found, except for NNRTI-associated mutations. Major NNRTI mutations such as K101E, K103N, and E138K showed higher frequencies than expected in ART-naïve populations. Higher literacy was associated with a greater risk of TDR (odds ratio 4.14, P = 0.0264). This study represents one of the first efforts to describe HIV diversity and TDR prevalence and trends in Guatemala. TDR prevalence in Guatemala was at the intermediate level. Most TDR cases were associated with NNRTIs. Further and continuous TDR surveillance is necessary to gain more indepth knowledge about TDR spread and trends in Guatemala and to optimize treatment outcomes in the country.

Murine Leukemia Virus Reverse Transcriptase: Structural Comparison with HIV-1 Reverse Transcriptase

PubMed Central

Coté, Marie L.; Roth, Monica J.

2008-01-01

Recent X-ray crystal structure determinations of Moloney murine leukemia virus reverse transcriptase (MoMLV RT) have allowed for more accurate structure/function comparisons to HIV-1 RT than were formerly possible. Previous biochemical studies of MoMLV RT in conjunction with knowledge of sequence homologies to HIV-1 RT and overall fold similarities to RTs in general, provided a foundation upon which to build. In addition, numerous crystal structures of the MoMLV RT fingers/palm subdomain had also shed light on one of the critical functions of the enzyme, specifically polymerization. Now in the advent of new structural information, more intricate examination of MoMLV RT in its entirety can be realized, and thus the comparisons with HIV-1 RT may be more critically elucidated. Here, we will review the similarities and differences between MoMLV RT and HIV-1 RT via structural analysis, and propose working models for the MoMLV RT based upon that information. PMID:18294720

HIV type 1 genotypic variation in an antiretroviral treatment-naive population in southern India.

PubMed

Balakrishnan, Pachamuthu; Kumarasamy, Nagalingeswaran; Kantor, Rami; Solomon, Suniti; Vidya, Sundararajan; Mayer, Kenneth H; Newstein, Michael; Thyagarajan, Sadras P; Katzenstein, David; Ramratnam, Bharat

2005-04-01

Most studies of HIV-1 drug resistance have examined subtype B viruses; fewer data are available from developing countries, where non-B subtypes predominate. We determined the prevalence of mutations at protease and reverse transcriptase drug resistance positions in antiretroviral drug-naive individuals in southern India. The pol region of the genome was amplified from plasma HIV-1 RNA in 50 patients. All sequences clustered with HIV-1 subtype C. All patients had at least one protease and/or RT mutation at a known subtype B drug resistance position. Twenty percent of patients had mutations at major protease inhibitor resistance positions and 100% had mutations at minor protease inhibitor resistance positions. Six percent and 14% of patients had mutations at nucleoside reverse transcriptase inhibitor and/or nonnucleoside reverse transcriptase inhibitor resistance positions, respectively. Larger scale studies need to be undertaken to better define the genotypic variation of circulating Indian subtype C viruses and their potential impact on drug susceptibility and clinical outcome in treated individuals.

Discovery of genetic variants of the kinases that activate tenofovir among individuals in the United States, Thailand, and South Africa: HPTN067.

PubMed

Figueroa, Dominique B; Tillotson, Joseph; Li, Maoji; Piwowar-Manning, Estelle; Hendrix, Craig W; Holtz, Timothy H; Bokoch, Kevin; Bekker, Linda-Gail; van Griensven, Frits; Mannheimer, Sharon; Hughes, James P; Grant, Robert M; Bumpus, Namandjé N

2018-01-01

Tenofovir (TFV), a nucleotide reverse transcriptase inhibitor, requires two phosphorylation steps to form a competitive inhibitor of HIV reverse transcriptase. Adenylate kinase 2 (AK2) has been previously demonstrated to phosphorylate tenofovir to tenofovir-monophosphate, while creatine kinase, muscle (CKM), pyruvate kinase, muscle (PKM) and pyruvate kinase, liver and red blood cell (PKLR) each have been found to phosphorylate tenofovir-monophosphate to the pharmacologically active tenofovir-diphosphate. In the present study, genomic DNA isolated from dried blood spots collected from 505 participants from Bangkok, Thailand; Cape Town, South Africa; and New York City, USA were examined for variants in AK2, CKM, PKM, and PKLR using next-generation sequencing. The bioinformatics tools SIFT and PolyPhen predicted that 19 of the 505 individuals (3.7% frequency) carried variants in at least one kinase that would result in a decrease or loss of enzymatic activity. To functionally test these predictions, AK2 and AK2 variants were expressed in and purified from E. coli, followed by investigation of their activities towards tenofovir. Interestingly, we found that purified AK2 had the ability to phosphorylate tenofovir-monophosphate to tenofovir-diphosphate in addition to phosphorylating tenofovir to tenofovir-monophosphate. Further, four of the six AK2 variants predicted to result in a loss or decrease of enzyme function exhibited a ≥30% decrease in activity towards tenofovir in our in vitro assays. Of note, an AK2 K28R variant resulted in a 72% and 81% decrease in the formation of tenofovir-monophosphate and tenofovir-diphosphate, respectively. These data suggest that there are naturally occurring genetic variants that could potentially impact TFV activation.

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

Transgenic rat model of childhood-onset dermatitis by overexpressing telomerase reverse transcriptase (TERT).

PubMed

Kaneko, Ryosuke; Sato, Atsuko; Hamada, Shun; Yagi, Takeshi; Ohsawa, Ichiro; Ohtsuki, Mamitaro; Kobayashi, Eiji; Hirabayashi, Masumi; Murakami, Takashi

2016-08-01

Childhood-onset dermatitis is one of the most common skin disorders in children. Although various mouse models that mirror aspects of dermatitis have become available, there is still a need for an animal model that develops dermatitis in childhood and is more suitable for performing tissue transplantation experiments. There is emerging evidence that peripheral blood T lymphocytes from patients with dermatitis have significantly increased telomerase activity. Here, we developed telomerase reverse transcriptase (TERT)-expressing transgenic (Tg) rats that spontaneously developed eczematous skin inflammation in childhood. Newborn TERT-Tg rats developed visible dermatitis in 56 % of cases, and the skin lesions microscopically showed spongiosis and acanthosis with infiltration of lymphocytes, eosinophils and mast cells. TERT-Tg rats with dermatitis exhibited increased CD4 (2.5-fold) and CD8 (fivefold) T cell numbers compared with dermatitis-free TERT-Tg rats. Stronger TERT activity was observed in the peripheral lymphocytes of dermatitis-positive TERT-Tg rats than those of dermatitis-free TERT-Tg rats. RT-PCR analysis revealed that IL-4 was markedly elevated in the spleen of dermatitis-positive TERT-Tg rats, and that interferon-gamma was increased in the dermatitis lesions. Moreover, skin grafting of TERT-Tg rats with dermatitis onto T cell-deficient nude rats demonstrated that the inflamed skin lesions could not be maintained. Taken together, the results suggest that TERT activation in T lymphocytes is one of the potential predisposing factors for dermatitis. Moreover, our results demonstrated that the TERT-Tg rats mirror aspects of human childhood-onset dermatitis and that these animals represent a potential animal model system for studying childhood-onset dermatitis.

5-Hydroxypyrido[2,3-b]pyrazin-6(5H)-one derivatives as novel dual inhibitors of HIV-1 reverse transcriptase-associated ribonuclease H and integrase.

PubMed

Sun, Lin; Gao, Ping; Dong, Guanyu; Zhang, Xujie; Cheng, Xiqiang; Ding, Xiao; Wang, Xueshun; Daelemans, Dirk; De Clercq, Erik; Pannecouque, Christophe; Menéndez-Arias, Luis; Zhan, Peng; Liu, Xinyong

2018-06-18

We reported herein the design, synthesis and biological evaluation of a series of 5-hydroxypyrido[2,3-b]pyrazin-6(5H)-one derivatives as HIV-1 reverse transcriptase (RT) ribonuclease H (RNase H) inhibitors using a privileged structure-guided scaffold refining strategy. In view of the similarities between the pharmacophore model of RNase H and integrase (IN) inhibitors as well as their catalytic sites, we also performed IN inhibition assays. Notably, the majority of these derivatives inhibited RNase H and IN at micromolar concentrations. Among them, compound 7a exhibited similar inhibitory activity against RNase H and IN (IC 50 RNase H  = 1.77 μM, IC 50 IN  = 1.18 μM, ratio = 1.50). To the best of our knowledge, this is the first reported dual HIV-1 RNase H-IN inhibitor based on a 5-hydroxypyrido[2,3-b]pyrazin-6(5H)-one structure. Molecular modeling has been used to predict the binding mode of 7a in complex with the catalytic cores of HIV-1 RNase H and IN. Taken together these results strongly support the feasibility of developing HIV-1 dual inhibitors from analog-based optimization of divalent metal ion chelators. Recently, the identification of dual inhibitors proved to be a highly effective strategy for novel antivirals discovery. Therefore, these compounds appear to be useful leads that can be further modified to develop more valuable anti-HIV-1 molecules with suitable drug profiles. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Substrate mimicry: HIV-1 reverse transcriptase recognizes 6-modified-3′-azido-2′,3′-dideoxyguanosine-5′-triphosphates as adenosine analogs

PubMed Central

Herman, Brian D.; Schinazi, Raymond F.; Zhang, Hong-wang; Nettles, James H.; Stanton, Richard; Detorio, Mervi; Obikhod, Aleksandr; Pradère, Ugo; Coats, Steven J.; Mellors, John W.; Sluis-Cremer, Nicolas

2012-01-01

β-D-3′-Azido-2′,3′-dideoxyguanosine (3′-azido-ddG) is a potent inhibitor of HIV-1 replication with a superior resistance profile to zidovudine. Recently, we identified five novel 6-modified-3′-azido-ddG analogs that exhibit similar or superior anti-HIV-1 activity compared to 3′-azido-ddG in primary cells. To gain insight into their structure–activity–resistance relationships, we synthesized their triphosphate (TP) forms and assessed their ability to inhibit HIV-1 reverse transcriptase (RT). Steady-state and pre-steady-state kinetic experiments show that the 6-modified-3′-azido-ddGTP analogs act as adenosine rather than guanosine mimetics in DNA synthesis reactions. The order of potency of the TP analogs against wild-type RT was: 3′-azido-2,6-diaminopurine >3′-azido-6-chloropurine; 3′-azido-6-N-allylaminopurine > 2-amino-6-N,N-dimethylaminopurine; 2-amino-6-methoxypurine. Molecular modeling studies reveal unique hydrogen-bonding interactions between the nucleotide analogs and the template thymine base in the active site of RT. Surprisingly, the structure–activity relationship of the analogs differed in HIV-1 RT ATP-mediated excision assays of their monophosphate forms, suggesting that it may be possible to rationally design a modified base analog that is efficiently incorporated by RT but serves as a poor substrate for ATP-mediated excision reactions. Overall, these studies identify a promising strategy to design novel nucleoside analogs that exert profound antiviral activity against both WT and drug-resistant HIV-1. PMID:21914723

Autocatalytic caspase-3 driven by human telomerase reverse transcriptase promoter suppresses human ovarian carcinoma growth in vitro and in mice.

PubMed

Song, Yue; Xia, Zhijun; Shen, Keng; Zhai, Xingyue

2013-05-01

To construct recombinant adenoviruses AdHT-rev-casp3 and Ad-rev-casp3, which express autocatalysis caspase-3 driven by human telomerase reverse transcriptase promoter and cytomegalovirus promoter, respectively; and to investigate their antitumor effects on ovarian cancer in vitro and in vivo. Cell viabilities were determined using the cell counting kit 8 and flow cytometry. Reverse transcriptase polymerase chain reaction and immunoblotting assays were used to detect cellular apoptotic activities after treatments. Tumor growth and survival of mice bearing AO cells were studied. AdHT-rev-casp3 significantly suppressed the survival of AO cells in a dose-dependent modality with a viability rate of 60.45% ± 7.8% at an multiplicity of infection (MOI) of 70 and 42.18 ± 5.3% at an MOI of 100, which was somewhat lower than that of the AO cells treated with Ad-rev-casp3 (32.28% ± 5.3% and 21.84% ± 3.4%, respectively). In contrast, AdHT-rev-casp3 induced little human umbilical vein epithelial cell (HUVEC) death with a viability rate of 98.52% ± 6.9% at an MOI of 70, whereas Ad-rev-casp3 induced significant cell death in HUVEC with a viability rate of 27.14% ± 5.4%. Additionally, AdHT-rev-casp3 (MOI = 70) caused significant apoptosis in AO cells with an apoptotic rate of 25.97%, whereas it caused undetectable apoptosis in HUVECs with the rate of only 1.75%. Ad-rev-casp3 (MOI = 70) caused strong apoptosis in both AO and HUVECs, with the rate of 35.82% and 38.12%, respectively. AdHT-rev-casp3 caused markedly higher levels of active caspase-3, causing no detectable active caspase-3 expression in HUVECs. The tumor growth suppression rate of AdHT-rev-casp3 was 54.94%, significantly higher than that of phosphate-buffered saline at the end point of the study. AdHT-rev-casp3 significantly improved the survival of mice receiving intraperitoneal inoculation of AO cells with little liver damage, with the mean survival of 177 ± 12 days. AdHT-rev-casp3 causes effective apoptosis with significant tumor selectivity, suppresses tumor growth, and improves the mouse survival with little liver toxicity. It can be a potent therapeutic agent for the tumor-targeting treatment of ovarian cancer.

Evidence that acidic fibroblast growth factor promotes maturation of rat satellite-cell-derived myotubes in vitro.

PubMed

Düsterhöft, S; Pette, D

1999-11-01

Satellite cells isolated from fast tibialis anterior (TA) and slow soleus (SOL) rat muscles were cultivated on matrigel, and treated with acidic fibroblast growth factor (aFGF). The following observations were made: 1) aFGF-treated cultures exhibited enhanced proliferation as mirrored by a twofold increase in DNA content. 2) Compared to the untreated cultures, myotubes in the aFGF cultures were larger; 3) Using reverse transcriptase polymerase chain reaction (RT-PCR) and northern blot analyses, we observed enhanced expression of all adult myosin heavy chain (MHC) isoforms, as well as of myogenin. These findings indicate that, under the culture conditions used, aFGF has a stimulatory effect on proliferation but also on maturation and differentiation of satellite cells. Furthermore, transcript levels of FGF receptor 1 (FGFR1) and 4 (FGFR4) isoforms, as well as of aFGF and bFGF were assessed by RT-PCR. aFGF-treated myotubes displayed increased expression of aFGF and bFGF, suggesting a paracrine effect of exogenous aFGF. In this regard, SOL-derived cultures responded more strongly than TA-derived cultures. The effects of aFGF treatment on the two receptors consisted of a decrease in FGFR1 and an increase in FGFR4 mRNA levels in 5-day-old cultures. In 8-day-old TA cultures, effects of FGF were similar to those in 5-day-old cultures. 8-day FGF-treated SOL cultures treated with FGF for 8 days exhibited higher FGFR1 and FGFR4 mRNA levels than the respective untreated cultures. Compared to 5 day-treated cultures, FGFR1 increased and FGFR4 decreased. This led to a shift in the ratio of FGFR1 to FGFR4 in the FGF-treated cultures which may explain the ability of satellite cells to differentiate under the influence of aFGF.

Down-regulation of telomerase activity in DLD-1 human colorectal adenocarcinoma cells by tocotrienol

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

Eitsuka, Takahiro; Nakagawa, Kiyotaka; Miyazawa, Teruo

2006-09-15

As high telomerase activity is detected in most cancer cells, inhibition of telomerase by drug or dietary food components is a new strategy for cancer prevention. Here, we investigated the inhibitory effect of vitamin E, with particular emphasis on tocotrienol (unsaturated vitamin E), on human telomerase in cell-culture study. As results, tocotrienol inhibited telomerase activity of DLD-1 human colorectal adenocarcinoma cells in time- and dose-dependent manner, interestingly, with {delta}-tocotrienol exhibiting the highest inhibitory activity. Tocotrienol inhibited protein kinase C activity, resulting in down-regulation of c-myc and human telomerase reverse transcriptase (hTERT) expression, thereby reducing telomerase activity. In contrast to tocotrienol,more » tocopherol showed very weak telomerase inhibition. These results provide novel evidence for First time indicating that tocotrienol acts as a potent candidate regulator of telomerase and supporting the anti-proliferative function of tocotrienol.« less

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

PubMed

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

2018-04-17

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

Coordinated DNA dynamics during the human telomerase catalytic cycle

NASA Astrophysics Data System (ADS)

Parks, Joseph W.; Stone, Michael D.

2014-06-01

The human telomerase reverse transcriptase (hTERT) utilizes a template within the integral RNA subunit (hTR) to direct extension of telomeres. Telomerase exhibits repeat addition processivity (RAP) and must therefore translocate the nascent DNA product into a new RNA:DNA hybrid register to prime each round of telomere repeat synthesis. Here, we use single-molecule FRET and nuclease protection assays to monitor telomere DNA structure and dynamics during the telomerase catalytic cycle. DNA translocation during RAP proceeds through a previously uncharacterized kinetic substep during which the 3‧-end of the DNA substrate base pairs downstream within the hTR template. The rate constant for DNA primer realignment reveals this step is not rate limiting for RAP, suggesting a second slow conformational change repositions the RNA:DNA hybrid into the telomerase active site and drives the extrusion of the 5‧-end of the DNA primer out of the enzyme complex.

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 Novel Hemagglutinin with Antiproliferative Activity against Tumor Cells from the Hallucinogenic Mushroom Boletus speciosus

PubMed Central

Ng, Tzi-Bun; Wang, Hexiang; Zhang, Guoqing

2014-01-01

Little was known about bioactive compounds from the hallucinogenic mushroom Boletus speciosus. In the present study, a hemagglutinin (BSH, B. speciosus hemagglutinin) was isolated from its fruiting bodies and enzymatic properties were also tested. The chromatographic procedure utilized comprised anion exchange chromatography on Q-Sepharose, cation exchange chromatography on CM-Cellulose, cation exchange chromatography on SP-Sepharose, and gel filtration by FPLC on Superdex 75. The hemagglutinin was a homodimer which was estimated to be approximately 31 kDa in size. The activity of BSH was stable up to 60°C, while there was a precipitous drop in activity when the temperature was elevated to 70°C. BSH retained 25% hemagglutinating activity when exposed to 100 mM NaOH and 25 mM HCl. The activity was potently inhibited by 1.25 mM Hg2+ and slightly inhibited by Fe2+, Ca2+, and Pb2+. None of the sugars tested showed inhibition towards BSH. Its hemagglutinating activity towards human erythrocytes type A, type B, and type AB was higher than type O. The hemagglutinin showed antiproliferative activity towards hepatoma Hep G2 cells and mouse lymphocytic leukemia cells (L1210) in vitro, with IC50 of 4.7 μM and 7.0 μM, respectively. It also exhibited HIV-1 reverse transcriptase inhibitory activity with an IC50 of 7.1 μM. PMID:24977148

Establishment and evaluation of a stable steroidogenic caprine luteal cell line.

PubMed

Li, Wei; Xu, Xingang; Huang, Yong; Li, Zhaocai; Yu, Gaoshui; Wang, Zhisheng; Ding, Li; Tong, Dewen

2012-07-15

Many physiological, biological, pharmacologic, and toxicologic events and compounds affect the function of Saanen dairy goat luteal cells, resulting in implantation failure or early embryonic loss. Although primary luteal cell cultures have been used, their finite lifespan precludes assessment of long-term effects. In the present study, primary caprine luteal cells (CLCs) were immortalized through transfection of a plasmid containing the human telomerase reverse transcriptase (hTERT) gene. The expression of hTERT and telomerase activity were evaluated in transduced CLCs (hTERT-CLCs). In this study, these cells steadily expressed hTERT gene and exhibited higher telomerase activity at Passages 30 and 50. The hTERT-CLCs at Passages 30 and 50 expressed genes encoding key proteins, enzymes and receptors inherent to normal luteal cells, e.g., steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and LH-receptor (LH-R). In addition, immortalized caprine luteal cells produced detectable quantities of progesterone in response to 8-bromo-cAMP (8-Br-cAMP) or 22(R)-hydroxycholesterol (22R-HC) stimulation. Furthermore, this cell line appeared to proliferate more quickly than control cells, although no neoplastic transformation occurred either in vivo or in vitro. We concluded the immortalized CLCs by hTERT retained their original characteristics and may provide a useful model to study luteal cell functions. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

Purification and Characterization of a Lectin from Green Split Peas (Pisum sativum).

PubMed

Ng, Tzi Bun; Chan, Yau Sang; Ng, Charlene Cheuk Wing; Wong, Jack Ho

2015-11-01

Lectins have captured the attention of a large number of researchers on account of their various exploitable activities, including antitumor, immunomodulatory, antifungal, as well as HIV reverse transcriptase inhibitory activities. A mannose/glucose-specific lectin was isolated from green split peas (a variety of Pisum sativum) and characterized. The purification step involved anion-exchange chromatography on a DEAE-cellulose column, cation-exchange chromatography on an SP-Sepharose column, and gel filtration by fast protein liquid chromatography (FPLC) on Superdex 200. The purified lectin had a native molecular mass of around 50 kDa as determined by size exclusion chromatography. It appeared as a heterotetramer, composed of two distinct polypeptide bands with a molecular mass of 6 and 19 kDa, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The N-terminal sequence of green split pea lectin shows some degree of homology compared to lectins from other legume species. Its hemagglutinating activity was inhibited by glucose, mannose, and sucrose, and attenuated at pH values higher than 12 or lower than 3. Hemagglutinating activity was preserved at temperatures lower than 80 °C. The lectin did not show antifungal activity toward fungi including Fusarium oxysporum, Botrytis cinerea, and Mycosphaerella arachidicola. Green split pea lectin showed a mitogenic effect toward murine splenocytes and could inhibit the activity of HIV-1 reverse transcriptase.

Unconventional plasticity of HIV-1 reverse transcriptase: how inhibitors could open a connection "gate" between allosteric and catalytic sites.

PubMed

Bellucci, Luca; Angeli, Lucilla; Tafi, Andrea; Radi, Marco; Botta, Maurizio

2013-12-23

Targeted molecular dynamics (TMD) simulations allowed for identifying the chemical/structural features of the nucleotide-competitive HIV-1 inhibitor DAVP-1, which is responsible for the disruption of the T-shape motif between Try183 and Trp229 of the reverse transcriptase (RT). DAVP-1 promoted the opening of a connection "gate" between allosteric and catalytic sites of HIV-1 RT, thus explaining its peculiar mechanism of action and providing useful insights to develop novel nucleotide-competitive RT inhibitors.

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

PubMed

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

2014-01-13

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

High Potency of Indolyl Aryl Sulfone Nonnucleoside Inhibitors towards Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutants Is Due to Selective Targeting of Different Mechanistic Forms of the Enzyme

PubMed Central

Cancio, Reynel; Silvestri, Romano; Ragno, Rino; Artico, Marino; De Martino, Gabriella; La Regina, Giuseppe; Crespan, Emmanuele; Zanoli, Samantha; Hübscher, Ulrich; Spadari, Silvio; Maga, Giovanni

2005-01-01

Indolyl aryl sulfone (IAS) nonnucleoside inhibitors have been shown to potently inhibit the growth of wild-type and drug-resistant human immunodeficiency virus type 1 (HIV-1), but their exact mechanism of action has not been elucidated yet. Here, we describe the mechanism of inhibition of HIV-1 reverse transcriptase (RT) by selected IAS derivatives. Our results showed that, depending on the substitutions introduced in the IAS common pharmacophore, these compounds can be made selective for different enzyme-substrate complexes. Moreover, we showed that the molecular basis for this selectivity was a different association rate of the drug to a particular enzymatic form along the reaction pathway. By comparing the activities of the different compounds against wild-type RT and the nonnucleoside reverse transcriptase inhibitor-resistant mutant Lys103Asn, it was possible to hypothesize, on the basis of their mechanism of action, a rationale for the design of drugs which could overcome the steric barrier imposed by the Lys103Asn mutation. PMID:16251294

Indolylarylsulfones carrying a heterocyclic tail as very potent and broad spectrum HIV-1 non-nucleoside reverse transcriptase inhibitors.

PubMed

Famiglini, Valeria; La Regina, Giuseppe; Coluccia, Antonio; Pelliccia, Sveva; Brancale, Andrea; Maga, Giovanni; Crespan, Emmanuele; Badia, Roger; Riveira-Muñoz, Eva; Esté, José A; Ferretti, Rosella; Cirilli, Roberto; Zamperini, Claudio; Botta, Maurizio; Schols, Dominique; Limongelli, Vittorio; Agostino, Bruno; Novellino, Ettore; Silvestri, Romano

2014-12-11

We synthesized new indolylarylsulfone (IAS) derivatives carrying a heterocyclic tail at the indole-2-carboxamide nitrogen as potential anti-HIV/AIDS agents. Several new IASs yielded EC50 values <1.0 nM against HIV-1 WT and mutant strains in MT-4 cells. The (R)-11 enantiomer proved to be exceptionally potent against the whole viral panel; in the reverse transcriptase (RT) screening assay, it was remarkably superior to NVP and EFV and comparable to ETV. The binding poses were consistent with the one previously described for the IAS non-nucleoside reverse transcriptase inhibitors. Docking studies showed that the methyl group of (R)-11 points toward the cleft created by the K103N mutation, different from the corresponding group of (S)-11. By calculating the solvent-accessible surface, we observed that the exposed area of RT in complex with (S)-11 was larger than the area of the (R)-11 complex. Compounds 6 and 16 and enantiomer (R)-11 represent novel robust lead compounds of the IAS class.

Structure-based virtual screening efforts against HIV-1 reverse transcriptase to introduce the new potent non-nucleoside reverse transcriptase inhibitor

NASA Astrophysics Data System (ADS)

Hosseini, Yaser; Mollica, Adriano; Mirzaie, Sako

2016-12-01

The human immunodeficiency virus (HIV) which is strictly related to the development of AIDS, is treated by a cocktail of drugs, but due its high propensity gain drug resistance, the rational development of new medicine is highly desired. Among the different mechanism of action we selected the reverse transcriptase (RT) inhibition, for our studies. With the aim to identify new chemical entities to be used for further rational drug design, a set of 3000 molecules from the Zinc Database have been screened by docking experiments using AutoDock Vina software. The best ranked compounds with respect of the crystallographic inhibitor MK-4965 resulted to be five compounds, and the best among them was further tested by molecular dynamics (MD) simulation. Our results indicate that comp1 has a stronger interaction with the subsite p66 of RT than MK-4965 and that both are able to stabilize specific conformational changes of the RT 3D structure, which may explain their activity as inhibitors. Therefore comp1 could be a good candidate for biological tests and further development.

Recent advances in the development of next generation non-nucleoside reverse transcriptase inhibitors.

PubMed

Tarby, Christine M

2004-01-01

Since their discovery, non-nucleoside reverse transcriptase inhibitors (NNRTIs) have become one of the cornerstones of highly active anti-retroviral therapy (HAART). Currently, three NNRTI agents, efavirenz, nevirapine and delavirdine are commercially available. Efavirenz and nevirapine, used in combination with nucleoside reverse transcriptase inhibitors (NRTIs), provide durable regimens with efficacy comparable to protease inhibitor (PI) containing therapies. When virological failure occurs following treatment with an NNRTI, the resistance mutations can confer reduced sensitivity to the entire agent class. Therefore, the strategy for the development of next generation NNRTIs has been to focus on compounds which have improved potencies against the clinically relevant viral mutants. Agents with improved virological profiles and which maintain the ease of administration and favorable safety profiles of the current agents should find use in anti-retroviral naïve patients as well as in components of salvage regimens in the anti-retroviral experienced patient. This review summarizes the recent developments with compounds in clinical trials as of January 2002 as well as to summarize information on new agents appearing in the primary and patent literature between January 2001 and December 2002.

Studies on the inhibition of Moloney murine leukemia virus reverse transcriptase by N-tritylamino acids and N-tritylamino acid-nucleotide compounds.

PubMed

Hawtrey, Arthur; Pieterse, Anton; van Zyl, Johann; Van der Bijl, Pieter; Van der Merwe, Marichen; Nel, William; Ariatti, Mario

2008-09-01

N-Acylated derivatives of 8-(6-aminohexyl) amino-adenosine-5 '-phosphate were prepared and studied with regard to their effect on DNA synthesis by the Moloney leukemia virus reverse transcriptase. N-palmitoyl and N-nicotinyl derivatives and bis-8-(6-aminohexyl) amino-5'-AMP inhibited the enzyme partially using poly (rA).oligo d(pT)(16-18) as template-primer with [(3)H]dTTP. In order to increase hydrophobicity in the acyl component tethered to the 8-(6-aminohexyl) amino group on the adenine nucleotide, N-trityl-L-phenylalanine and the N-trityl derivatives of the o, m, and p-fluoro-DL-phenylalanine were initially examined for inhibition of the enzyme using the above template-primer system. The compounds all inhibited the reverse transcriptase with IC(50) values of approximately 60-80 microM. However, when N-trityl-m-fluoro-DL-phenylalanine was coupled to the nucleotide 8-(6-aminohexyl) amino-adenosine-5'-phosphate, the inhibitory activity of this compound increased significantly (IC(50) = 5 microM).

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

Negligible risk associated with the movement of processed rainbow trout, Oncorhynchus mykiss (Walbaum), from an infectious haematopoietic necrosis virus (IHNV) endemic area

USGS Publications Warehouse

LaPatra, S.E.; Batts, W.N.; Overturf, K.; Jones, G.N.; Shewmaker, W.D.; Winton, J.R.

2001-01-01

To assess the risk of transmission of infectious haematopoietic necrosis virus (IHNV) associated with the movement of processed rainbow trout, Oncorhynchus mykiss, from an area where the virus is endemic, 240 freshly eviscerated fish (225-500 g) exhibiting spinal curvature or spinal compression types of deformities were tested for IHNV by virus isolation and polymerase chain reaction (PCR) techniques. Commercially produced rainbow trout, approximately 1-year-old, that exhibited spinal deformities were considered to have had a high likelihood of having survived an outbreak of IHN. Serological analysis of fish exhibiting spinal curvature or spinal compression types of deformities for anti-IHNV antibodies resulted, in 71 and 50% of the serum samples, respectively, with detectable neutralization activity suggesting previous infection with IHNV. A portion of the skin and muscle in the area of the deformity was collected, as well as brain tissue from each commercially processed fish. Tissue homogenates were tested for IHNV using the epithelioma papulosum cyprini (EPC) cell line pretreated with polyethylene glycol and the chinook salmon embryo (CHSE-214) cell line using standard methods. Nested, reverse transcriptase (RT)-PCR for the detection of IHNV used the central 1231 bp portion of the glycoprotein (G) challenge studies and is suggested as a mechanism responsible for virus clearance. These results provide scientific information that can be used to assess the risk associated with the movement of processed rainbow trout from an IHNV endemic area.

Tenofovir-related nephrotoxicity: case report and review of the literature.

PubMed

James, Christopher W; Steinhaus, Mary C; Szabo, Susan; Dressier, Robert M

2004-03-01

Tenofovir is a nucleotide reverse transcriptase inhibitor for treatment of human immunodeficiency virus (HIV) infection. Several cases of renal failure associated with tenofovir therapy recently have been reported. A 54-year-old man with HIV experienced decreasing renal function and Fanconi's syndrome secondary to tenofovir therapy. His condition gradually improved after discontinuation of the drug. The available medical literature for reported cases of tenofovir-related nephrotoxicity indicates that this complication is apparently rare. However, our case report and literature review underscore the importance of monitoring renal function when treating patients with any nucleotide reverse transcriptase inhibitor.

Homologous recombination occurs in a distinct retroviral subpopulation and exhibits high negative interference.

PubMed Central

Hu, W S; Bowman, E H; Delviks, K A; Pathak, V K

1997-01-01

Homologous recombination and deletions occur during retroviral replication when reverse transcriptase switches templates. While recombination occurs solely by intermolecular template switching (between copackaged RNAs), deletions can occur by an intermolecular or an intramolecular template switch (within the same RNA). To directly compare the rates of intramolecular and intermolecular template switching, two spleen necrosis virus-based vectors were constructed. Each vector contained a 110-bp direct repeat that was previously shown to delete at a high rate. The 110-bp direct repeat was flanked by two different sets of restriction site markers. These vectors were used to form heterozygotic virions containing RNAs of each parental vector, from which recombinant viruses were generated. By analyses of the markers flanking the direct repeats in recombinant and nonrecombinant proviruses, the rates of intramolecular and intermolecular template switching were determined. The results of these analyses indicate that intramolecular template switching is much more efficient than intermolecular template switching and that direct repeat deletions occur primarily through intramolecular template switching events. These studies also indicate that retroviral recombination occurs within a distinct viral subpopulation and exhibits high negative interference, whereby the selection of one recombination event increases the probability that a second recombination event will be observed. PMID:9223494

Cerebrospinal fluid abacavir concentrations in HIV-positive patients following once-daily administration.

PubMed

Calcagno, A; Pinnetti, C; De Nicolò, A; Scarvaglieri, E; Gisslen, M; Tempestilli, M; D'Avolio, A; Fedele, V; Di Perri, G; Antinori, A; Bonora, S

2018-06-01

Abacavir is a widely used nucleotide reverse transcriptase inhibitor, for which cerebrospinal fluid (CSF) exposure has been previously assessed in twice-daily recipients. We studied abacavir CSF concentrations in 61 and nine HIV-positive patients taking abacavir once daily and twice daily, respectively. Patients on once-daily abacavir had higher plasma and CSF concentrations (96 vs. 22 ng ml -1 , P = 0.038 and 123 vs. 49 ng ml -1 , P = 0.038) but similar CSF-to-plasma ratios (0.8 vs. 0.5, P = 0.500). CSF abacavir concentrations were adequate in patients receiving once-daily treatment. © 2018 The British Pharmacological Society.

The role of the glycosyl moiety of myricetin derivatives in anti-HIV-1 activity in vitro.

PubMed

Ortega, Joseph T; Suárez, Alirica I; Serrano, Maria L; Baptista, Jani; Pujol, Flor H; Rangel, Hector R

2017-10-12

Plant extracts are sources of valuable compounds with biological activity, especially for the anti-proliferative activity against pathogens or tumor cells. Myricetin is a flavonoid found in several plants that has been described as an inhibitor of Human immunodeficiency virus type 1 (HIV-1) through its action against the HIV reverse transcriptase, but myricetin derivatives have not been fully studied. The aim of this study was to evaluate the anti-HIV-1 activity of glycosylated metabolites obtained from Marcetia taxifolia and derived from myricetin: myricetin rhamnoside and myricetin 3-(6-rhamnosylgalactoside). Compounds were obtained from organic extracts by maceration of aerial parts of M. taxifolia. All biological assays were performed in the MT4 cell line. Antiviral activity was measured as inhibition of p24 and reverse transcriptase with a fluorescent assay. Both flavonoids have antiviral activity in vitro, with an EC50 of 120 µM for myricetin 3-rhamnoside (MR) and 45 µM for myricetin 3-(6-rhamnosylgalactoside) (MRG), both significantly lower than the EC50 of myricetin (230 µM). Although both compounds inhibited the reverse transcriptase activity, with an IC50 of 10.6 µM for MR and 13.8 µM for MRG, myricetin was the most potent, with an IC50 of 7.6 µM, and an inhibition greater than 80%. Molecular docking approach showed correlation between the free energy of binding with the assays of enzyme inhibition. The results suggest that glycosylated moiety might enhance the anti-HIV-1 activity of myricetin, probably by favoring the internalization of the flavonoid into the cell. The inhibition of the HIV-1 reverse transcriptase is likely responsible for the antiviral activity.

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.

Update on HIV-1 acquired and transmitted drug resistance in Africa.

PubMed

Ssemwanga, Deogratius; Lihana, Raphael W; Ugoji, Chinenye; Abimiku, Alash'le; Nkengasong, John; Dakum, Patrick; Ndembi, Nicaise

2015-01-01

The last ten years have witnessed a significant scale-up and access to antiretroviral therapy in Africa, which has improved patient quality of life and survival. One major challenge associated with increased access to antiretroviral therapy is the development of antiretroviral resistance due to inconsistent drug supply and/or poor patient adherence. We review the current state of both acquired and transmitted drug resistance in Africa over the past ten years (2001-2011) to identify drug resistance associated with the different drug regimens used on the continent and to help guide affordable strategies for drug resistance surveillance. A total of 161 references (153 articles, six reports and two conference abstracts) were reviewed. Antiretroviral resistance data was available for 40 of 53 African countries. A total of 5,541 adult patients from 99 studies in Africa were included in this analysis. The pooled prevalence of drug resistance mutations in Africa was 10.6%, and Central Africa had the highest prevalence of 54.9%. The highest prevalence of nucleoside reverse transcriptase inhibitor mutations was in the west (55.3%) and central (54.8%) areas; nonnucleoside reverse transcriptase inhibitor mutations were highest in East Africa (57.0%) and protease inhibitors mutations highest in Southern Africa (16.3%). The major nucleoside reverse transcriptase inhibitor mutation in all four African regions was M184V. Major nonnucleoside reverse transcriptase inhibitor as well as protease inhibitor mutations varied by region. The prevalence of drug resistance has remained low in several African countries although the emergence of drug resistance mutations varied across countries. Continued surveillance of antiretroviral therapy resistance remains crucial in gauging the effectiveness of country antiretroviral therapy programs and strategizing on effective and affordable strategies for successful treatment.

Detection of SYT-SSX mutant transcripts in formalin-fixed paraffin-embedded sarcoma tissues using one-step reverse transcriptase real-time PCR.

PubMed

Norlelawati, A T; Mohd Danial, G; Nora, H; Nadia, O; Zatur Rawihah, K; Nor Zamzila, A; Naznin, M

2016-04-01

Synovial sarcoma (SS) is a rare cancer and accounts for 5-10% of adult soft tissue sarcomas. Making an accurate diagnosis is difficult due to the overlapping histological features of SS with other types of sarcomas and the non-specific immunohistochemistry profile findings. Molecular testing is thus considered necessary to confirm the diagnosis since more than 90% of SS cases carry the transcript of t(X;18)(p11.2;q11.2). The purpose of this study is to diagnose SS at molecular level by testing for t(X;18) fusion-transcript expression through One-step reverse transcriptase real-time Polymerase Chain Reaction (PCR). Formalin-fixed paraffin-embedded tissue blocks of 23 cases of soft tissue sarcomas, which included 5 and 8 cases reported as SS as the primary diagnosis and differential diagnosis respectively, were retrieved from the Department of Pathology, Tengku Ampuan Afzan Hospital, Kuantan, Pahang. RNA was purified from the tissue block sections and then subjected to One-step reverse transcriptase real-time PCR using sequence specific hydrolysis probes for simultaneous detection of either SYT-SSX1 or SYT-SSX2 fusion transcript. Of the 23 cases, 4 cases were found to be positive for SYT-SSX fusion transcript in which 2 were diagnosed as SS whereas in the 2 other cases, SS was the differential diagnosis. Three cases were excluded due to failure of both amplification assays SYT-SSX and control β-2-microglobulin. The remaining 16 cases were negative for the fusion transcript. This study has shown that the application of One-Step reverse transcriptase real time PCR for the detection SYT-SSX transcript is feasible as an aid in confirming the diagnosis of synovial sarcoma.

Antiviral Activity of MK-4965, a Novel Nonnucleoside Reverse Transcriptase Inhibitor▿

PubMed Central

Lai, Ming-Tain; Munshi, Vandna; Touch, Sinoeun; Tynebor, Robert M.; Tucker, Thomas J.; McKenna, Philip M.; Williams, Theresa M.; DiStefano, Daniel J.; Hazuda, Daria J.; Miller, Michael D.

2009-01-01

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are the mainstays of therapy for the treatment of human immunodeficiency virus type 1 (HIV-1) infections. However, the effectiveness of NNRTIs can be hampered by the development of resistance mutations which confer cross-resistance to drugs in the same class. Extensive efforts have been made to identify new NNRTIs that can suppress the replication of the prevalent NNRTI-resistant viruses. MK-4965 is a novel NNRTI that possesses both diaryl ether and indazole moieties. The compound displays potency at subnanomolar concentrations against wild-type (WT), K103N, and Y181C reverse transcriptase (RT) in biochemical assays. MK-4965 is also highly potent against the WT virus and two most prevalent NNRTI-resistant viruses (viruses that harbor the K103N or the Y181C mutation), against which it had 95% effective concentrations (EC95s) of <30 nM in the presence of 10% fetal bovine serum. The antiviral EC95 of MK-4965 was reduced approximately four- to sixfold when it was tested in 50% human serum. Moreover, MK-4965 was evaluated with a panel of 15 viruses with NNRTI resistance-associated mutations and showed a superior mutant profile to that of efavirenz but not to that of etravirine. MK-4965 was similarly effective against various HIV-1 subtypes and viruses containing nucleoside reverse transcriptase inhibitor or protease inhibitor resistance-conferring mutations. A two-drug combination study showed that the antiviral activity of MK-4965 was nonantagonistic with each of the 18 FDA-licensed drugs tested vice versa in the present study. Taken together, these in vitro data show that MK-4965 possesses the desired properties for further development as a new NNRTI for the treatment of HIV-1 infection. PMID:19289522

An overview of the molecular and epidemiological features of HIV-1 infection in two major cities of Bahia state, Brazil.

PubMed

Amaral, Amanda Gm; Oliveira, Isabele B; Carneiro, Diego C; Alcantara, Luiz Cj; Monteiro-Cunha, Joana P

2017-06-01

The high mutation rate of the human immunodeficiency virus (HIV) has created a public health challenge because the use of antiretroviral drugs can generate selective pressure that drives resistance in these viruses. The aim of this work was to characterise the molecular and epidemiological profile of HIV in Bahia, Brazil. DNA sequences from regions of HIV gag, pol, and env genes were obtained from previous studies performed in this area between 2002 and 2012. Their genotype and drug-resistance mutations were identified using bioinformatics tools. Clinical and epidemiological data were analysed. Among 263 individuals (46.4% male), 97.5% were asymptomatic and 49.1% were receiving treatment. Most of the individuals were 31 to 40 years old (36.9%) and infected through heterosexual contact (40.7%). The predominant genotype was B (68.1%) followed by BF recombinants (18.6%). Among the individuals infected with either F or BF genotypes, 68.4% were women and 76.8% were infected through heterosexual transmission. The prevalence of associated mutations conferring antiretroviral resistance was 14.2%, with 3.8% of all mutations conferring resistance to protease inhibitors, 9.43% to nucleoside reverse transcriptase inhibitors, and 8.5% to non-nucleoside reverse transcriptase inhibitors. Drug resistance was higher in individuals receiving treatment (26.1%) than in the drug-naïve (4.3%) individuals. This study will contribute to the understanding and monitoring of HIV epidemic in this Brazilian region.

Arginine deiminase expressed in vivo, driven by human telomerase reverse transcriptase promoter, displays high hepatoma targeting and oncolytic efficiency.

PubMed

Jiang, Hui; Guo, Song; Xiao, Dan; Bian, Xuzhao; Wang, Jie; Wang, Ying; Zhou, Huiting; Cai, Jun; Zheng, Zhongliang

2017-06-06

Arginine starvation has the potential to selectively treat both primary tumor and (micro) metastatic tissue with very low side effects. Arginine deiminase (ADI; EC 3.5.3.6), an arginine-degrading enzyme, has been studied as a potential anti-tumor drug for the treatment of arginine-auxotrophic tumors. Though ADI-PEG20 (pegylated ADI by PEG 20,000) already passed the phase I/II clinical trials [1], it is just used as adjuvant therapy because of its low efficiency and less targeting. Then, this paper discussed the efficiency of arginine starvation mediated by ADI expressed in cytoplasm for liver cancers. In order to guarantee the tumor targeting, human telomerase reverse transcriptase (hTERT) promoter was used to drive the expression of ADI in vivo. To access the anti-tumor efficiency of ADI, p53 gene was used as the positive control. Thus, ADI displayed obvious cytotoxicity to BEL7402 and HUH7 cell lines in cytoplasm. The apoptosis rates rose from 15% to nearly 60% after changing the expression vectors from pcDNA4 plasmid to adenovirus. Compared with p53-adenovirus, ADI-adenovirus showed the higher oncolytic activity in the intratumoral injection model of mice. Tumor disappeared after the treatment of ADI-adenovirus for two weeks, and the mice pulled through all. Therefore, ADI is an ideal anti-tumor gene for caner targeting therapy with the help of hTERT promoter.

Implication of human telomerase reverse transcriptase in cervical carcinogenesis and cancer recurrence.

PubMed

Wang, P-H; Ko, J-L

2006-01-01

The objective of this study was to evaluate the implication of human telomerase reverse transcriptase (hTERT) in cervical carcinogenesis and cancer recurrence. One hundred three cases of uterine cervix, including 20 normal, 13 low-grade squamous intraepithelial lesion (LSIL), 30 high-grade squamous intraepithelial lesion (HSIL), and 40 squamous cell carcinoma (SCC) tissues, were evaluated for hTERT immunoreactivity. The expressions of hTERT in normal, LSIL, HSIL, and SCC tissues were compared by Fisher exact or Chi-square test. The relationships between hTERT and clinicopathologic variables of SCC were also assessed. Furthermore, SCC patients were subdivided into negative and positive hTERT expression subgroups, and Kaplan-Meier curves were used to plot the cumulative recurrence hazard for 5 years. There was a significant difference for hTERT expression between LSIL and HSIL subgroups (P < 0.001) but no significant difference between normal and LSIL as well as HSIL and SCC subgroups. For SCC patients, hTERT expression was positive in lymph nodes, vagina, and parametrium metastastic cases. However, it did not reach a significant difference. The cumulative recurrence hazard for 5 years was about 29% in positive hTERT expression subgroup compared to 0% in negative hTERT subgroup (P = 0.2866). In conclusion, a point stage of HSIL exists in the progression of cervical carcinogenesis when the hTERT expression increases significantly. Moreover, SCC patients with positive hTERT expression may have higher cumulative recurrence hazard.

Different degree of immune recovery using antiretroviral regimens with Vonavir or Zidovudine/Lamivudine and Efavirenz in Patients receiving first line treatment in Iran.

PubMed

Sadeghi, Leila; Moallemi, Samaneh; Tabatabai, Reza Adl; Esmaeilzadeh, Ali; Ahsani-Nasab, Sara; Ahmadi, Niloofar Eghbal; Bayanolhagh, Saeed; Lolaie, Mahin; Narouei, Arsalan; Alinaghi, Ahmad; Mohraz, Minoo

2018-01-07

The initial ART regimens used by most national treatment guidelines in resource-limited settings include 2 nucleoside reverse-transcriptase inhibitors (NRTIs) and 1 non-nucleoside reverse-transcriptase inhibitor (NNRTI). The NRTIshave consistedof Zidovudine (AZT) or Stavudine (d4T) with Lamivudine (3TC); the NNRTI component hasbeen Nevirapine (NVP) or Efavirenz (EFV). There exists few data indicating if Vonavir is more effective in increasing CD4+ T cell counts or other first-line drugs of ART.Immunological outcomes of 134 individuals who just started antiretroviral therapy with Vonavir or Zidovudine/Lamivudine and Efavirenzanalyzed. Immunological response was then assessed during 28 weeks and indicated significant CD4+ T cell increase in both groups. The increase in CD4+ T cell counts was greater in Zidovudine/Lamivudine and Efavirenz treated group. A rapid CD4+ T cell increase occurred after the first 12 weeks of therapy in Vonavir treated group with initial CD4+ T cell counts ≤100 despite the individuals with higher CD4+ T cell counts. As previous studies, we observed the noticeable increase rate of CD4+ Tcells is in the first three months of therapy. A rapid CD4+ Tcell increase occurred shortly after beginning anti retroviraltherapy using either Vonaviror Zidovudine/Lamivudine and Efavirenz. Late increases in CD4+ T cell counts are more pronounced in therapy using Zidovudine/Lamivudine and Efavirenz. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

Lentin, a novel and potent antifungal protein from shitake mushroom with inhibitory effects on activity of human immunodeficiency virus-1 reverse transcriptase and proliferation of leukemia cells.

PubMed

Ngai, Patrick H K; Ng, T B

2003-11-14

From the fruiting bodies of the edible mushroom Lentinus edodes, a novel protein designated lentin with potent antifungal activity was isolated. Lentin was unadsorbed on DEAE-cellulose, and adsorbed on Affi-gel blue gel and Mono S. The N-terminal sequence of lentin manifested similarity to endoglucanase. Lentin, which had a molecular mass of 27.5 kDa, inhibited mycelial growth in a variety of fungal species including Physalospora piricola, Botrytis cinerea and Mycosphaerella arachidicola. Lentin also exerted an inhibitory activity on HIV-1 reverse transcriptase and proliferation of leukemia cells.

Structure-Based Design of Novel Dihydroalkoxybenzyloxopyrimidine Derivatives as Potent Nonnucleoside Inhibitors of the Human Immunodeficiency Virus Reverse Transcriptase

PubMed Central

Sudbeck, Elise A.; Mao, Chen; Vig, Rakesh; Venkatachalam, T. K.; Tuel-Ahlgren, Lisa; Uckun, Fatih M.

1998-01-01

Two highly potent dihydroalkoxybenzyloxopyrimidine (DABO) derivatives targeting the nonnucleoside inhibitor (NNI) binding site of human immunodeficiency virus (HIV) reverse transcriptase (RT) have been designed based on the structure of the NNI binding pocket and tested for anti-HIV activity. Our lead DABO derivative, 5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one, elicited potent inhibitory activity against purified recombinant HIV RT and abrogated HIV replication in peripheral blood mononuclear cells at nanomolar concentrations (50% inhibitory concentration, <1 nM) but showed no detectable cytotoxicity at concentrations as high as 100 μM. PMID:9835518

HIV Resistance Prediction to Reverse Transcriptase Inhibitors: Focus on Open Data.

PubMed

Tarasova, Olga; Poroikov, Vladimir

2018-04-19

Research and development of new antiretroviral agents are in great demand due to issues with safety and efficacy of the antiretroviral drugs. HIV reverse transcriptase (RT) is an important target for HIV treatment. RT inhibitors targeting early stages of the virus-host interaction are of great interest for researchers. There are a lot of clinical and biochemical data on relationships between the occurring of the single point mutations and their combinations in the pol gene of HIV and resistance of the particular variants of HIV to nucleoside and non-nucleoside reverse transcriptase inhibitors. The experimental data stored in the databases of HIV sequences can be used for development of methods that are able to predict HIV resistance based on amino acid or nucleotide sequences. The data on HIV sequences resistance can be further used for (1) development of new antiretroviral agents with high potential for HIV inhibition and elimination and (2) optimization of antiretroviral therapy. In our communication, we focus on the data on the RT sequences and HIV resistance, which are available on the Internet. The experimental methods, which are applied to produce the data on HIV-1 resistance, the known data on their concordance, are also discussed.

Chemical system biology based molecular interactions to identify inhibitors against Q151M mutant of HIV-1 reverse transcriptase.

PubMed

Pandey, Rajan Kumar; Sharma, Drista; Ojha, Rupal; Bhatt, Tarun Kumar; Prajapati, Vijay Kumar

2018-05-09

The emergence of mutations leading to drug resistance is the main cause of therapeutic failure in the human HIV infection. Chemical system biology approach has drawn great attention to discover new antiretroviral hits with high efficacy and negligible toxicity, which can be used as a prerequisite for HIV drug resistance global action plan 2017-21. To discover potential hits, we docked 49 antiretroviral analogs (n = 6294) against HIV-1 reverse transcriptase Q151M mutant & its wild-type form and narrow downed their number in three sequential modes of docking using Schrödinger suite. Later on, 80 ligands having better docking score than reference ligands (tenofovir and lamivudine) were screened for ADME, toxicity prediction, and binding energy estimation. Simultaneously, the area under the curve (AUC) was estimated using receiver operating characteristics (ROC) curve analysis to validate docking protocols. Finally, single point energy and molecular dynamics simulation approaches were performed for best two ligands (L3 and L14). This study reveals the antiretroviral efficacy of obtained two best ligands and delivers the hits against HIV-1 reverse transcriptase Q151M mutant. Copyright © 2018 Elsevier B.V. All rights reserved.

Antitumor effect of combination of the inhibitors of two new oncotargets: proton pumps and reverse transcriptase.

PubMed

Lugini, Luana; Sciamanna, Ilaria; Federici, Cristina; Iessi, Elisabetta; Spugnini, Enrico Pierluigi; Fais, Stefano

2017-01-17

Tumor therapy needs new approaches in order to improve efficacy and reduce toxicity of the current treatments. The acidic microenvironment and the expression of high levels of endogenous non-telomerase reverse transcriptase (RT) are common features of malignant tumor cells. The anti-acidic proton pump inhibitor Lansoprazole (LAN) and the non-nucleoside RT inhibitor Efavirenz (EFV) have shown independent antitumor efficacy. LAN has shown to counteract drug tumor resistance. We tested the hypothesis that combination of LAN and EFV may improve the overall antitumor effects. We thus pretreated human metastatic melanoma cells with LAN and then with EFV, both in 2D and 3D spheroid models. We evaluated the treatment effect by proliferation and cell death/apoptosis assays in classical and in pulse administration experiments. The action of EFV was negatively affected by the tumor microenvironmental acidity, and LAN pretreatment overcame the problem. LAN potentiated the cytotoxicity of EFV to melanoma cells and, when administered during the drug interruption period, prevented the replacement of tumor cell growth.This study supports the implementation of the current therapies with combination of Proton Pumps and Reverse Transcriptase inhibitors.

Antitumor effect of combination of the inhibitors of two new oncotargets: proton pumps and reverse transcriptase

PubMed Central

Lugini, Luana; Sciamanna, Ilaria; Federici, Cristina; Iessi, Elisabetta; Spugnini, Enrico Pierluigi; Fais, Stefano

2017-01-01

Tumor therapy needs new approaches in order to improve efficacy and reduce toxicity of the current treatments. The acidic microenvironment and the expression of high levels of endogenous non-telomerase reverse transcriptase (RT) are common features of malignant tumor cells. The anti-acidic proton pump inhibitor Lansoprazole (LAN) and the non-nucleoside RT inhibitor Efavirenz (EFV) have shown independent antitumor efficacy. LAN has shown to counteract drug tumor resistance. We tested the hypothesis that combination of LAN and EFV may improve the overall antitumor effects. We thus pretreated human metastatic melanoma cells with LAN and then with EFV, both in 2D and 3D spheroid models. We evaluated the treatment effect by proliferation and cell death/apoptosis assays in classical and in pulse administration experiments. The action of EFV was negatively affected by the tumor microenvironmental acidity, and LAN pretreatment overcame the problem. LAN potentiated the cytotoxicity of EFV to melanoma cells and, when administered during the drug interruption period, prevented the replacement of tumor cell growth. This study supports the implementation of the current therapies with combination of Proton Pumps and Reverse Transcriptase inhibitors. PMID:27926505

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

PubMed Central

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

1994-01-01

We previously described a reverse transcriptase-PCR using flavivirus genus-conserved and virus species-specific amplimers (D. W. Trent and G. J. Chang, p. 355-371, in Y. Becker and C. Darai; ed., Frontiers of Virology, vol. 1, 1992). Target amplification was improved by redesigning the amplimers, and a sensitive enzyme-linked immunosorbent assay (ELISA) technique has been developed to detect amplified digoxigenin (DIG)-modified DNA. A single biotin motif and multiple DIG motifs were incorporated into each amplicon, which permitted amplicon capture by a biotin-streptavidin interaction and detection with DIG-specific antiserum in a colorimetric ELISA. We evaluated the utility of this assay for detecting St. Louis encephalitis (SLE) viral RNA in infected mosquitoes and dengue viral RNA in human serum specimens. The reverse transcriptase-PCR-ELISA was as sensitive as isolation of SLE virus by cell culture in detecting SLE viral RNA in infected mosquitoes. The test was 89% specific and 95 to 100% sensitive for identification of dengue viral RNA in serum specimens compared with isolation of virus by Aedes albopictus C6/36 cell culture and identification by the indirect immunofluorescence assay. PMID:7512096

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

PubMed

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

1994-02-01

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

Do non-nucleoside reverse transcriptase inhibitors contribute to lipodystrophy?

PubMed

Nolan, David

2005-01-01

Lipodystrophy complications, including lipoatrophy (pathological fat loss) and metabolic complications, have emerged as important long-term toxicities associated with antiretroviral therapy in the current era. The wealth of data that has accumulated over the past 6 years has now clarified the contribution of specific antiretroviral drugs to the risk of these clinical endpoints, with evidence that lipoatrophy is strongly associated with the choice of nucleoside reverse transcriptase inhibitor therapy (specifically, stavudine and to a lesser extent zidovudine). The aetiological basis of metabolic complications of antiretroviral therapy has proven to be complex, in that the risk appears to be modulated by a number of lifestyle factors that have made the metabolic syndrome highly prevalent in the general population, with additional contributions from HIV disease status itself, as well as from individual drugs within the HIV protease inhibitor class. The currently licensed non-nucleoside reverse transcriptase inhibitor (NNRTI) drugs, efavirenz and nevirapine, have been proven to have a favourable safety profile in terms of lipodystrophy complications. However, it must be noted that NNRTI drugs also have individual toxicity profiles that must be accounted for when considering and/or monitoring their use in the treatment of HIV infection.

Prevalence of HIV-1 Subtypes and Drug Resistance-Associated Mutations in HIV-1-Positive Treatment-Naive Pregnant Women in Pointe Noire, Republic of the Congo (Kento-Mwana Project).

PubMed

Bruzzone, Bianca; Saladini, Francesco; Sticchi, Laura; Mayinda Mboungou, Franc A; Barresi, Renata; Caligiuri, Patrizia; Calzi, Anna; Zazzi, Maurizio; Icardi, Giancarlo; Viscoli, Claudio; Bisio, Francesca

2015-08-01

The Kento-Mwana project was carried out in Pointe Noire, Republic of the Congo, to prevent mother-to-child HIV-1 transmission. To determine the prevalence of different subtypes and transmitted drug resistance-associated mutations, 95 plasma samples were collected at baseline from HIV-1-positive naive pregnant women enrolled in the project during the years 2005-2008. Full protease and partial reverse transcriptase sequencing was performed and 68/95 (71.6%) samples were successfully sequenced. Major mutations to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors were detected in 4/68 (5.9%), 3/68 (4.4%), and 2/68 (2.9%) samples, respectively. Phylogenetic analysis of HIV-1 isolates showed a high prevalence of unique recombinant forms (24/68, 35%), followed by CRF45_cpx (7/68, 10.3%) and subsubtype A3 and subtype G (6/68 each, 8.8%). Although the prevalence of transmitted drug resistance mutations appears to be currently limited, baseline HIV-1 genotyping is highly advisable in conjunction with antiretroviral therapy scale-up in resource-limited settings to optimize treatment and prevent perinatal transmission.

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

PubMed

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

2015-05-01

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

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

Efficacy and safety of rilpivirine in treatment-naive, HIV-1-infected patients with hepatitis B virus/hepatitis C virus coinfection enrolled in the Phase III randomized, double-blind ECHO and THRIVE trials.

PubMed

Nelson, Mark; Amaya, Gerardo; Clumeck, Nathan; Arns da Cunha, Clovis; Jayaweera, Dushyantha; Junod, Patrice; Li, Taisheng; Tebas, Pablo; Stevens, Marita; Buelens, Annemie; Vanveggel, Simon; Boven, Katia

2012-08-01

The efficacy and hepatic safety of the non-nucleoside reverse transcriptase inhibitors rilpivirine (TMC278) and efavirenz were compared in treatment-naive, HIV-infected adults with concurrent hepatitis B virus (HBV) and/or hepatitis C virus (HCV) infection in the pooled week 48 analysis of the Phase III, double-blind, randomized ECHO (NCT00540449) and THRIVE (NCT00543725) trials. Patients received 25 mg of rilpivirine once daily or 600 mg of efavirenz once daily, plus two nucleoside/nucleotide reverse transcriptase inhibitors. At screening, patients had alanine aminotransferase/aspartate aminotransferase levels ≤5× the upper limit of normal. HBV and HCV status was determined at baseline by HBV surface antigen, HCV antibody and HCV RNA testing. HBV/HCV coinfection status was known for 670 patients in the rilpivirine group and 665 in the efavirenz group. At baseline, 49 rilpivirine and 63 efavirenz patients [112/1335 (8.4%)] were coinfected with either HBV [55/1357 (4.1%)] or HCV [57/1333 (4.3%)]. The safety analysis included all available data, including beyond week 48. Eight patients seroconverted during the study (rilpivirine: five; efavirenz: three). A higher proportion of patients achieved viral load <50 copies/mL (intent to treat, time to loss of virological response) in the subgroup without HBV/HCV coinfection (rilpivirine: 85.0%; efavirenz: 82.6%) than in the coinfected subgroup (rilpivirine: 73.5%; efavirenz: 79.4%) (rilpivirine, P = 0.04 and efavirenz, P = 0.49, Fisher's exact test). The incidence of hepatic adverse events (AEs) was low in both groups in the overall population (rilpivirine: 5.5% versus efavirenz: 6.6%) and was higher in HBV/HCV-coinfected patients than in those not coinfected (26.7% versus 4.1%, respectively). Hepatic AEs were more common and response rates lower in HBV/HCV-coinfected patients treated with rilpivirine or efavirenz than in those who were not coinfected.

Novel HBV recombinants between genotypes B and C in 3'-terminal reverse transcriptase (RT) sequences are associated with enhanced viral DNA load, higher RT point mutation rates and place of birth among Chinese patients.

PubMed

Liu, Baoming; Yang, Jing-Xian; Yan, Ling; Zhuang, Hui; Li, Tong

2018-01-01

As one of the major global public health concerns, hepatitis B virus (HBV) can be divided into at least eight genotypes, which may be related to disease severity and treatment response. We previously demonstrated that genotypes B and C HBV, with distinct geographical distribution in China, had divergent genotype-dependent amino acid polymorphisms and variations in reverse transcriptase (RT) gene region, a target of antiviral therapy using nucleos(t)ide analogues. Recently recombination between HBV genotypes B and C was reported to occur in the RT region. However, their frequency and clinical significance is poorly understood. Here full-length HBV RT sequences from 201 Chinese chronic hepatitis B (CHB) patients were amplified and sequenced, among which 31.34% (63/201) were genotype B whereas 68.66% (138/201) genotype C. Although no intergenotypic recombination was detected among C-genotype HBV, 38.10% (24/63) of B-genotype HBV had recombination with genotype C in the 3'-terminal RT sequences. The patients with B/C intergenotypic recombinants had significantly (P<0.05) higher serum HBV DNA level than the "pure" B-genotype cohort did. Moreover, the B/C intergenotypic recombinants were prone to more substitutions at several specific residues in the RT region than genotype B or C. Besides, unlike their parental genotypes, the recombinant HBV appeared to display an altered geographic distribution feature in China. Our findings provide novel insight into the virological, clinical and epidemiological features of new HBV B/C intergenotypic recombinants at the 3' end of RT sequences among Chinese CHB patients. The highly complex genetic background of the novel recombinant HBV carrying new mutations affecting RT protein may contribute to an enhanced heterogeneity in treatment response or prognosis among CHB patients. Published by Elsevier B.V.

Template-independent DNA synthesis activity associated with the reverse transcriptase of the long terminal repeat retrotransposon Tf1.

PubMed

Oz-Gleenberg, Iris; Herzig, Eytan; Hizi, Amnon

2012-01-01

Reverse transcriptases (RTs) possess a non-templated addition (NTA) activity while synthesizing DNA with blunt-ended DNA primer/templates. Interestingly, the RT of the long terminal repeat retrotransposon Tf1 has an NTA activity that is substantially higher than that of HIV-1 or murine leukemia virus RTs. By performing steady state kinetics, we found that the differences between the NTA activities of Tf1 and HIV-1 RTs can be explained by the substantially lower K(M) value for the incoming dNTP of Tf1 RT (while the differences between the apparent k(cat) values of these two RTs are relatively small). Furthermore, the K(M) values, calculated for both RTs with the same dNTP, are much lower for the template-dependent synthesis (TDS) than those of NTA. However, TDS of HIV-1 RT is higher than that of Tf1 RT. The overall relative order of the apparent k(cat)/K(M) values for dATP is: HIV-1 RT (TDS) > Tf1 RT (TDS) > Tf1 RT (NTA) > HIV-1 RT (NTA). Under the employed conditions, Tf1 RT can add up to seven nucleotides to the blunt-ended substrate, while the other RTs add mostly a single nucleotide. The NTA activity of Tf1 RT is restricted to DNA primers. Furthermore, the NTA activity of Tf1 and HIV-1 RTs is suppressed by ATP, as it competes with the incoming dATP (although ATP is not incorporated by the NTA activity of the RTs). The unusually high NTA activity of Tf1 RT can explain why, after completing cDNA synthesis, the in vivo generated Tf1 cDNA has relatively long extra sequences beyond the highly conserved CA at its 3'-ends. © 2011 The Authors Journal compilation © 2011 FEBS.

Endurance training blocks uncoupling protein 1 up-regulation in brown adipose tissue while increasing uncoupling protein 3 in the muscle tissue of rats fed with a high-sugar diet.

PubMed

de Queiroz, Karina Barbosa; Rodovalho, Gisele Vieira; Guimarães, Juliana Bohnen; de Lima, Daniel Carvalho; Coimbra, Cândido Celso; Evangelista, Elísio Alberto; Guerra-Sá, Renata

2012-09-01

The mitochondrial uncoupling proteins (UCPs) of interscapular brown adipose tissue (iBAT) and of muscles play important roles in energy balance. For instance, the expression of UCP1 and UCP3 are modulated by free fatty acid gradients induced by high-sugar diets and acute exercise that is dependent on sympathetic stimulation. However, the effects of endurance training in animals fed with high-sugar diets are unknown. This study aims to evaluate the long-term effects of diet and exercise on UCP1 and UCP3 levels and energy balance efficiency. Rats fed with standard or high-sugar (HSD) diets were simultaneously subjected to running training over an 8-week period. After the training period, the rats were decapitated, and the iBAT and gastrocnemius muscle tissues were removed for evaluation of the β₃-receptor, Ucp1, and Ucp3 mRNA and protein expression, which were analyzed by quantitative reverse transcriptase polymerase chain reaction and Western blot, respectively. Groups fed with an HSD displayed a higher adiposity index and iBAT weight (P < .05), whereas exhibited an up-regulation of Ucp1 mRNA and protein levels (P < .05). Training increased β₃-receptor mRNA in iBAT and reduced the Ucp3 mRNA in muscle tissues. In association with an HSD, training restored the increasing β₃-receptor mRNA and greatly up-regulated the levels of Ucp3 mRNA. Therefore, training blocked the HSD-induced up-regulation of UCP1 expression in iBAT, whereas it up-regulated the expression of Ucp3 mRNA in muscle. These results suggest that training enhances the relationship between Ucp1/Ucp3 mRNA levels, which could result in higher energy efficiency, but not when HSD-induced elevated sympathetic activity is maintained. Copyright © 2012. Published by Elsevier Inc.

A novel 53-kDa nodulin of the symbiosome membrane of soybean nodules, controlled by Bradyrhizobium japonicum.

PubMed

Winzer, T; Bairl, A; Linder, M; Linder, D; Werner, D; Müller, P

1999-03-01

A nodule-specific 53-kDa protein (GmNOD53b) of the symbiosome membrane from soybean was isolated and its LysC digestion products were microsequenced. cDNA clones of this novel nodulin, obtained from cDNA library screening with an RT-PCR (reverse-transcriptase polymerase chain reaction)-generated hybridization probe exhibited no homology to proteins identified so far. The expression of GmNOD53b coincides with the onset of nitrogen fixation. Therefore, it is a late nodulin. Among other changes, the GmNOD53b is significantly reduced in nodules infected with the Bradyrhizobium japonicum mutant 184 on the protein level as well as on the level of mRNA expression, compared with the wild-type infected nodules. The reduction of GmNOD53b mRNA is related to an inactivation of the sipF gene in B. japonicum 184, coding for a functionally active signal peptidase.

Evidence from molecular dynamics simulations of conformational preorganization in the ribonuclease H active site

PubMed Central

Stafford, Kate A.; Palmer III, Arthur G.

2014-01-01

Ribonuclease H1 (RNase H) enzymes are well-conserved endonucleases that are present in all domains of life and are particularly important in the life cycle of retroviruses as domains within reverse transcriptase. Despite extensive study, especially of the E. coli homolog, the interaction of the highly negatively charged active site with catalytically required magnesium ions remains poorly understood. In this work, we describe molecular dynamics simulations of the E. coli homolog in complex with magnesium ions, as well as simulations of other homologs in their apo states. Collectively, these results suggest that the active site is highly rigid in the apo state of all homologs studied and is conformationally preorganized to favor the binding of a magnesium ion. Notably, representatives of bacterial, eukaryotic, and retroviral RNases H all exhibit similar active-site rigidity, suggesting that this dynamic feature is only subtly modulated by amino acid sequence and is primarily imposed by the distinctive RNase H protein fold. PMID:25075292

Splicing of a group II intron involved in the conjugative transfer of pRS01 in lactococci.

PubMed

Mills, D A; McKay, L L; Dunny, G M

1996-06-01

Analysis of a region involved in the conjugative transfer of the lactococcal conjugative element pRS01 has revealed a bacteria] group II intron. Splicing of this lactococcal intron (designated Ll.ltrB) in vivo resulted in the ligation of two exon messages (ltrBE1 and ltrBE2) which encoded a putative conjugative relaxase essential for the transfer of pRS01. Like many group II introns, the Ll.ltrB intron possessed an open reading frame (ltrA) with homology to reverse transcriptases. Remarkably, sequence analysis of ltrA suggested a greater similarity to open reading frames encoded by eukaryotic mitochondrial group II introns than to those identified to date from other bacteria. Several insertional mutations within ltrA resulted in plasmids exhibiting a conjugative transfer-deficient phenotype. These results provide the first direct evidence for splicing of a prokaryotic group II intron in vivo and suggest that conjugative transfer is a mechanism for group II intron dissemination in bacteria.

Inconspicuous Insertion 22;12 in Myxoid/Round Cell Liposarcoma Accompanied by the Secondary Structural Abnormality der(16)t(1;16)

PubMed Central

Birch, Nathan C.; Antonescu, Cristina R.; Nelson, Marilu; Sarran, Lisa; Neff, James R.; Seemayer, Thomas; Bridge, Julia A.

2003-01-01

In myxoid/round cell liposarcoma, the t(12;16)(q13;p11) and its associated fusion transcript, FUS-CHOP, characterize greater than 95% of cases. The variant translocation t(12;22)(q13;q12) and associated EWS-CHOP fusion transcript are rare. A second non-random aberration observed in roughly 20% of Ewing’s sarcomas, and to a lesser extent other select sarcomas, is the unbalanced 1;16 translocation. Recognition of this secondary aberration in the absence of an obvious primary karyotypic abnormality strongly suggests that the use of other genetic approaches will be informative in uncovering a clinically suspected primary anomaly. The following case illustrates the utility of molecular cytogenetic and reverse transcriptase-polymerase chain reaction techniques in diagnosing an ins(22;12)(q12;q13q14) and associated EWS-CHOP fusion transcript in a myxoid/round cell liposarcoma exhibiting a der(16)t(1;16)(q11;q11). PMID:12876210

Inconspicuous insertion 22;12 in myxoid/round cell liposarcoma accompanied by the secondary structural abnormality der(16)t(1;16).

PubMed

Birch, Nathan C; Antonescu, Cristina R; Nelson, Marilu; Sarran, Lisa; Neff, James R; Seemayer, Thomas; Bridge, Julia A

2003-08-01

In myxoid/round cell liposarcoma, the t(12;16)(q13;p11) and its associated fusion transcript, FUS-CHOP, characterize greater than 95% of cases. The variant translocation t(12;22)(q13;q12) and associated EWS-CHOP fusion transcript are rare. A second non-random aberration observed in roughly 20% of Ewing's sarcomas, and to a lesser extent other select sarcomas, is the unbalanced 1;16 translocation. Recognition of this secondary aberration in the absence of an obvious primary karyotypic abnormality strongly suggests that the use of other genetic approaches will be informative in uncovering a clinically suspected primary anomaly. The following case illustrates the utility of molecular cytogenetic and reverse transcriptase-polymerase chain reaction techniques in diagnosing an ins(22;12)(q12;q13q14) and associated EWS-CHOP fusion transcript in a myxoid/round cell liposarcoma exhibiting a der(16)t(1;16)(q11;q11).

Substrate promiscuity of a rosmarinic acid synthase from lavender (Lavandula angustifolia L.).

PubMed

Landmann, Christian; Hücherig, Stefanie; Fink, Barbara; Hoffmann, Thomas; Dittlein, Daniela; Coiner, Heather A; Schwab, Wilfried

2011-08-01

One of the most common types of modification of secondary metabolites is the acylation of oxygen- and nitrogen-containing substrates to produce esters and amides, respectively. Among the known acyltransferases, the members of the plant BAHD family are capable of acylating a wide variety of substrates. Two full-length acyltransferase cDNAs (LaAT1 and 2) were isolated from lavender flowers (Lavandula angustifolia L.) by reverse transcriptase-PCR using degenerate primers based on BAHD sequences. Recombinant LaAT1 exhibited a broad substrate tolerance accepting (hydroxy)cinnamoyl-CoAs as acyl donors and not only tyramine, tryptamine, phenylethylamine and anthranilic acid but also shikimic acid and 4-hydroxyphenyllactic acid as acceptors. Thus, LaLT1 forms esters and amides like its phylogenetic neighbors. In planta LaAT1 might be involved in the biosynthesis of rosmarinic acid, the ester of caffeic acid and 3,4-dihydroxyphenyllactic acid, a major constituent of lavender flowers. LaAT2 is one of three members of clade VI with unknown function.

Establishment and characterization of immortalized bovine endometrial epithelial cells

PubMed Central

Bai, Hanako; Sakurai, Toshihiro; Bai, Rulan; Yamakoshi, Sachiko; Aoki, Etsunari; Kuse, Mariko; Okuda, Kiyoshi; Imakawa, Kazuhiko

2014-01-01

Bovine primary uterine endometrial epithelial cells (EECs) are not ideal for long-term studies, because primary EECs lose hormone responsiveness quickly, and/or they tend to have a short life span. The aims of this study were to establish immortalized bovine EECs and to characterize these cells following long-term cultures. Immortalized bovine EECs were established by transfecting retroviral vectors encoding human papillomavirus (HPV) E6 and E7, and human telomerase reverse transcriptase (hTERT) genes. Established bovine immortalized EECs (imEECs) showed the same morphology as primary EECs, and could be grown without any apparent changes for over 60 passages. In addition, imEECs have maintained the features as EECs, exhibiting oxytocin (OT) and interferon tau (IFNT) responsiveness. Therefore, these imEECs, even after numbers of passages, could be used as an in vitro model to investigate cellular and molecular mechanisms, by which the uterine epithelium responds to IFNT stimulation, the event required for the maternal recognition of pregnancy in the bovine species. PMID:24735401

Regulation of the cnr Cobalt and Nickel Resistance Determinant from Ralstonia sp. Strain CH34†

PubMed Central

Grass, Gregor; Große, Cornelia; Nies, Dietrich H.

2000-01-01

Ralstonia sp. strain CH34 is resistant to nickel and cobalt cations. Resistance is mediated by the cnr determinant located on plasmid pMOL28. The cnr genes are organized in two clusters, cnrYXH and cnrCBA. As revealed by reverse transcriptase PCR and primer extension, transcription from these operons is initiated from promoters located upstream of the cnrY and cnrC genes. These two promoters exhibit conserved sequences at the −10 (CCGTATA) and −35 (CRAGGGGRAG) regions. The CnrH gene product, which is required for expression of both operons, is a sigma factor belonging to the sigma L family, whose activity seems to be governed by the membrane-bound CnrY and CnrX gene products in response to Ni2+. Half-maximal activation from the cnrCBA operon was determined by using appropriate lacZ gene fusions and was shown to occur at an Ni2+ concentration of about 50 μM. PMID:10671463

Bioinformatics approach of salt tolerance gene in mangrove plant Rhizophora stylosa

NASA Astrophysics Data System (ADS)

Basyuni, M.; Sumardi

2017-01-01

This study descibes bioinformatics approach on the analyze of the salt tolerance genes in mangrove plant, Rhizophora stylosa on DDBJ/EMBL/GenBank as well as similarity, phylogenetic, potential peptide, and subcellular localization. The DNA sequence between salt tolerance gene from R. stylosa exhibited 42-11% between themselves The target peptide value of mitochondria varied from 0.163 to 0.430, indicated it was possible to exist. These results suggested the importance of understanding the diversity and functional of properties of the different amino acids in mangrove OSC genes. To clarify the relationship among the salt-tolerant genes in R. stylosa, a phylogenetic tree was constructed. The phylogenetic tree shows that there are three clusters, first branch of Cu/Zn SOD and reverse transcriptase genes, the second branch consists of the majority genes and the last group was MAP3K alpha protein kinase only. The present study, therefore, suggested that salt tolerance genes form distinct clusters in the tree.

Efficient Single Tobamoviral Vector-Based Bioproduction of Broadly Neutralizing Anti-HIV-1 Monoclonal Antibody VRC01 in Nicotiana benthamiana Plants and Utility of VRC01 in Combination Microbicides

PubMed Central

Hamorsky, Krystal Teasley; Grooms-Williams, Tiffany W.; Husk, Adam S.; Bennett, Lauren J.; Palmer, Kenneth E.

2013-01-01

Broadly neutralizing monoclonal antibodies (bnMAbs) may offer powerful tools for HIV-1 preexposure prophylaxis, such as topical microbicides. However, this option is hampered due to expensive MAb biomanufacturing based on mammalian cell culture. To address this issue, we developed a new production system for bnMAb VRC01 in Nicotiana benthamiana plants using a tobamovirus replicon vector. Unlike conventional two-vector-based expression, this system was designed to overexpress full-length IgG1 from a single polypeptide by means of kex2p-like enzyme recognition sites introduced between the heavy and light chains. An enzyme-linked immunosorbent assay (ELISA) revealed that gp120-binding VRC01 IgG1 was maximally accumulated on 5 to 7 days following vector inoculation, yielding ∼150 mg of the bnMAb per kg of fresh leaf material. The plant-made VRC01 (VRC01p) was efficiently purified by protein A affinity followed by hydrophobic-interaction chromatography. ELISA, surface plasmon resonance, and an HIV-1 neutralization assay demonstrated that VRC01p has gp120-binding affinity and HIV-1-neutralization capacity virtually identical to the human-cell-produced counterpart. To advance VRC01p's use in topical microbicides, we analyzed combinations of the bnMAb with other microbicide candidates holding distinct antiviral mechanisms in an HIV-1 neutralization assay. VRC01p exhibited clear synergy with the antiviral lectin griffithsin, the CCR5 antagonist maraviroc, and the reverse transcriptase inhibitor tenofovir in multiple CCR5-tropic HIV-1 strains from clades A, B, and C. In summary, VRC01p is amenable to robust, rapid, and large-scale production and may be developed as an active component in combination microbicides with other anti-HIV agents such as antiviral lectins, CCR5 antagonists, and reverse transcriptase inhibitors. PMID:23403432

Alteration of Substrate and Inhibitor Specificity of Feline Immunodeficiency Virus Protease

PubMed Central

Lin, Ying-Chuan; Beck, Zachary; Lee, Taekyu; Le, Van-Duc; Morris, Garrett M.; Olson, Arthur J.; Wong, Chi-Huey; Elder, John H.

2000-01-01

Feline immunodeficiency virus (FIV) protease is structurally very similar to human immunodeficiency virus (HIV) protease but exhibits distinct substrate and inhibitor specificities. We performed mutagenesis of subsite residues of FIV protease in order to define interactions that dictate this specificity. The I37V, N55M, M56I, V59I, and Q99V mutants yielded full activity. The I37V, N55M, V59I, and Q99V mutants showed a significant increase in activity against the HIV-1 reverse transcriptase/integrase and P2/nucleocapsid junction peptides compared with wild-type (wt) FIV protease. The I37V, V59I, and Q99V mutants also showed an increase in activity against two rapidly cleaved peptides selected by cleavage of a phage display library with HIV-1 protease. Mutations at Q54K, I98P, and L101I dramatically reduced activity. Mutants containing a I35D or I57G substitution showed no activity against either FIV or HIV substrates. FIV proteases all failed to cut HIV-1 matrix/capsid, P1/P6, P6/protease, and protease/reverse transcriptase junctions, indicating that none of the substitutions were sufficient to change the specificity completely. The I37V, N55M, M56I, V59I, and Q99V mutants, compared with wt FIV protease, all showed inhibitor specificity more similar to that of HIV-1 protease. The data also suggest that FIV protease prefers a hydrophobic P2/P2′ residue like Val over Asn or Glu, which are utilized by HIV-1 protease, and that S2/S2′ might play a critical role in distinguishing FIV and HIV-1 protease by specificity. The findings extend our observations regarding the interactions involved in substrate binding and aid in the development of broad-based inhibitors. PMID:10775609

Frequent Cross-Resistance to Dapivirine in HIV-1 Subtype C-Infected Individuals after First-Line Antiretroviral Therapy Failure in South Africa.

PubMed

Penrose, Kerri J; Wallis, Carole L; Brumme, Chanson J; Hamanishi, Kristen A; Gordon, Kelley C; Viana, Raquel V; Harrigan, P Richard; Mellors, John W; Parikh, Urvi M

2017-02-01

A vaginal ring containing dapivirine (DPV) has shown moderate protective efficacy against HIV-1 acquisition, but the activity of DPV against efavirenz (EFV)- and nevirapine (NVP)-resistant viruses that could be transmitted is not well defined. We investigated DPV cross-resistance of subtype C HIV-1 from individuals on failing NVP- or EFV-containing antiretroviral therapy (ART) in South Africa. Plasma samples were obtained from individuals with >10,000 copies of HIV RNA/ml and with HIV-1 containing at least one non-nucleoside reverse transcriptase (NNRTI) mutation. Susceptibility to NVP, EFV, and DPV in TZM-bl cells was determined for recombinant HIV-1 LAI containing bulk-amplified, plasma-derived, full-length reverse transcriptase sequences. Fold change (FC) values were calculated compared with a composite 50% inhibitory concentration (IC 50 ) from 12 recombinant subtype C HIV-1 LAI plasma-derived viruses from treatment-naive individuals in South Africa. A total of 25/100 (25%) samples showed >500-FCs to DPV compared to treatment-naive samples with IC 50 s exceeding the maximum DPV concentration tested (132 ng/ml). A total of 66/100 (66%) samples displayed 3- to 306-FCs, with a median IC 50 of 17.6 ng/ml. Only 9/100 (9%) samples were susceptible to DPV (FC < 3). Mutations L100I and K103N were significantly more frequent in samples with >500-fold resistance to DPV compared to samples with a ≤500-fold resistance. A total of 91% of samples with NNRTI-resistant HIV-1 from individuals on failing first-line ART in South Africa exhibited ≥3-fold cross-resistance to DPV. This level of resistance exceeds expected plasma concentrations, but very high genital tract DPV concentrations from DPV ring use could block viral replication. It is critically important to assess the frequency of transmitted and selected DPV resistance in individuals using the DPV ring. Copyright © 2017 American Society for Microbiology.

Frequent Cross-Resistance to Dapivirine in HIV-1 Subtype C-Infected Individuals after First-Line Antiretroviral Therapy Failure in South Africa

PubMed Central

Penrose, Kerri J.; Wallis, Carole L.; Brumme, Chanson J.; Hamanishi, Kristen A.; Gordon, Kelley C.; Viana, Raquel V.; Harrigan, P. Richard; Mellors, John W.

2016-01-01

ABSTRACT A vaginal ring containing dapivirine (DPV) has shown moderate protective efficacy against HIV-1 acquisition, but the activity of DPV against efavirenz (EFV)- and nevirapine (NVP)-resistant viruses that could be transmitted is not well defined. We investigated DPV cross-resistance of subtype C HIV-1 from individuals on failing NVP- or EFV-containing antiretroviral therapy (ART) in South Africa. Plasma samples were obtained from individuals with >10,000 copies of HIV RNA/ml and with HIV-1 containing at least one non-nucleoside reverse transcriptase (NNRTI) mutation. Susceptibility to NVP, EFV, and DPV in TZM-bl cells was determined for recombinant HIV-1LAI containing bulk-amplified, plasma-derived, full-length reverse transcriptase sequences. Fold change (FC) values were calculated compared with a composite 50% inhibitory concentration (IC50) from 12 recombinant subtype C HIV-1LAI plasma-derived viruses from treatment-naive individuals in South Africa. A total of 25/100 (25%) samples showed >500-FCs to DPV compared to treatment-naive samples with IC50s exceeding the maximum DPV concentration tested (132 ng/ml). A total of 66/100 (66%) samples displayed 3- to 306-FCs, with a median IC50 of 17.6 ng/ml. Only 9/100 (9%) samples were susceptible to DPV (FC < 3). Mutations L100I and K103N were significantly more frequent in samples with >500-fold resistance to DPV compared to samples with a ≤500-fold resistance. A total of 91% of samples with NNRTI-resistant HIV-1 from individuals on failing first-line ART in South Africa exhibited ≥3-fold cross-resistance to DPV. This level of resistance exceeds expected plasma concentrations, but very high genital tract DPV concentrations from DPV ring use could block viral replication. It is critically important to assess the frequency of transmitted and selected DPV resistance in individuals using the DPV ring. PMID:27895013

Reverse Transcriptase Activity in Mature Spermatozoa of Mouse

PubMed Central

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

2000-01-01

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

In search of a treatment for HIV--current therapies and the role of non-nucleoside reverse transcriptase inhibitors (NNRTIs).

PubMed

Reynolds, Chevonne; de Koning, Charles B; Pelly, Stephen C; van Otterlo, Willem A L; Bode, Moira L

2012-07-07

The human immunodeficiency virus (HIV) causes AIDS (acquired immune deficiency syndrome), a disease in which the immune system progressively deteriorates, making sufferers vulnerable to all manner of opportunistic infections. Currently, world-wide there are estimated to be 34 million people living with HIV, with the vast majority of these living in sub-Saharan Africa. Therefore, an important research focus is development of new drugs that can be used in the treatment of HIV/AIDS. This review gives an overview of the disease and addresses the drugs currently used for treatment, with specific emphasis on new developments within the class of allosteric non-nucleoside reverse transcriptase inhibitors (NNRTIs).

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

In Vitro Cross-Resistance Profiles of Rilpivirine, Dapivirine, and MIV-150, Nonnucleoside Reverse Transcriptase Inhibitor Microbicides in Clinical Development for the Prevention of HIV-1 Infection

PubMed Central

Giacobbi, Nicholas S.

2017-01-01

ABSTRACT Rilpivirine (RPV), dapivirine (DPV), and MIV-150 are in development as microbicides. It is not known whether they will block infection of circulating nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant human immunodeficiency virus type 1 (HIV-1) variants. Here, we demonstrate that the activity of DPV and MIV-150 is compromised by many resistant viruses containing single or double substitutions. High DPV genital tract concentrations from DPV ring use may block replication of resistant viruses. However, MIV-150 genital tract concentrations may be insufficient to inhibit many resistant viruses, including those harboring K103N or Y181C. PMID:28507107

An integrated molecular dynamics, principal component analysis and residue interaction network approach reveals the impact of M184V mutation on HIV reverse transcriptase resistance to lamivudine.

PubMed

Bhakat, Soumendranath; Martin, Alberto J M; Soliman, Mahmoud E S

2014-08-01

The emergence of different drug resistant strains of HIV-1 reverse transcriptase (HIV RT) remains of prime interest in relation to viral pathogenesis as well as drug development. Amongst those mutations, M184V was found to cause a complete loss of ligand fitness. In this study, we report the first account of the molecular impact of M184V mutation on HIV RT resistance to 3TC (lamivudine) using an integrated computational approach. This involved molecular dynamics simulation, binding free energy analysis, principle component analysis (PCA) and residue interaction networks (RINs). Results clearly confirmed that M184V mutation leads to steric conflict between 3TC and the beta branched side chain of valine, decreases the ligand (3TC) binding affinity by ∼7 kcal mol(-1) when compared to the wild type, changes the overall conformational landscape of the protein and distorts the native enzyme residue-residue interaction network. The comprehensive molecular insight gained from this study should be of great importance in understanding drug resistance against HIV RT as well as assisting in the design of novel reverse transcriptase inhibitors with high ligand efficacy on resistant strains.

Dipyridodiazepinone analogs as human immunodeficiency virus type 1-specific non-nucleoside reverse transcriptase inhibitors: an overview.

PubMed

Lv, M; Xu, H

2010-01-01

According to World Health Organization (WHO)/Joint United Nations Programme on human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) (UNAIDS) Report in 2007, 33.2 million people are living with HIV, 2.5 million ones have been newly infected with HIV, and 2.1 million ones died from AIDS, including 330,000 children. Therefore, HIV/AIDS still remains a public health emergency and a leading cause of mortality worldwide. It is believed that reverse transcriptase (RT) is a crucial enzyme in the life cycle of HIV-1, and thereby RT has been the important drug target for antiretroviral (ARV) chemotherapy against AIDS. To our knowledge, dipyridodiazepinone analogs have been considered as one class of potential non-nucleoside reverse transcriptase inhibitors (NNRTIs), especially the structurally and chemically related nevirapine (Viramune(R)), which was the first NNRTI approved by the U. S. Food and Drug Administration (FDA) for the treatment of HIV-1 infection for adults in 1996 and for children in 1998. This review mainly highlights the progress of synthesis and structure-activity relationship (SAR) of dipyridodiazepinone analogs; in the meantime, the mechanism of action is also presented. It will pave the way for the design and development of novel dipyridodiazepinone analogs as NNRTIs in AIDS chemotherapy in the future.

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.

Occurrence of transmitted HIV-1 drug resistance among Drug-naïve pregnant women in selected HIV-care centres in Ghana.

PubMed

Martin-Odoom, Alexander; Adiku, Theophilus; Delgado, Elena; Lartey, Margaret; Ampofo, William K

2017-03-01

Access to antiretroviral therapy in Ghana has been scaled up across the country over the last decade. This study sought to determine the occurrence of transmitted HIV-1 drug resistance in pregnant HIV-1 positive women yet to initiate antiretroviral therapy at selected HIV Care Centres in Ghana. Plasma specimens from twenty-six (26) HIV seropositive pregnant women who were less than 28weeks pregnant with their first pregnancy and ART naïve were collected from selected HIV care centres in three (3) regions in Ghana. Genotypic testing was done for the reverse transcriptase gene and the sequences generated were analyzed for HIV-1 drug resistance mutations using the Stanford University HIV Drug Resistance Database. Resistance mutations associated with the reverse transcriptase gene were detected in 4 (15.4%) of the participants. At least one major drug resistance mutation in the reverse transcriptase gene was found in 3 (11.5%) of the women. The detection of transmitted HIV-1 drug resistance in this drug-naïve group in two regional HIV care sites is an indication of the need for renewed action in monitoring the emergence of transmitted HIV-1 drug resistance in Ghana. None declared.

Base Preferences in Non-Templated Nucleotide Incorporation by MMLV-Derived Reverse Transcriptases

PubMed Central

Zajac, Pawel; Islam, Saiful; Hochgerner, Hannah; Lönnerberg, Peter; Linnarsson, Sten

2013-01-01

Reverse transcriptases derived from Moloney Murine Leukemia Virus (MMLV) have an intrinsic terminal transferase activity, which causes the addition of a few non-templated nucleotides at the 3´ end of cDNA, with a preference for cytosine. This mechanism can be exploited to make the reverse transcriptase switch template from the RNA molecule to a secondary oligonucleotide during first-strand cDNA synthesis, and thereby to introduce arbitrary barcode or adaptor sequences in the cDNA. Because the mechanism is relatively efficient and occurs in a single reaction, it has recently found use in several protocols for single-cell RNA sequencing. However, the base preference of the terminal transferase activity is not known in detail, which may lead to inefficiencies in template switching when starting from tiny amounts of mRNA. Here, we used fully degenerate oligos to determine the exact base preference at the template switching site up to a distance of ten nucleotides. We found a strong preference for guanosine at the first non-templated nucleotide, with a greatly reduced bias at progressively more distant positions. Based on this result, and a number of careful optimizations, we report conditions for efficient template switching for cDNA amplification from single cells. PMID:24392002

[The use of complex interval models for predicting activity of non-nucleoside reverse transcriptase activity].

PubMed

Burliaeva, E V; Tarkhov, A E; Burliaev, V V; Iurkevich, A M; Shvets, V I

2002-01-01

Searching of new anti-HIV agents is still crucial now. In general, researches are looking for inhibitors of certain HIV's vital enzymes, especially for reverse transcriptase (RT) inhibitors. Modern generation of anti-HIV agents represents non-nucleoside reverse transcriptase inhibitors (NNRTIs). They are much less toxic than nucleoside analogues and more chemically stable, thus being slower metabolized and emitted from the human body. Thus, search of new NNRTIs is actual today. Synthesis and study of new anti-HIV drugs is very expensive. So employment of the activity prediction techniques for such a search is very beneficial. This technique allows predicting the activities for newly proposed structures. It is based on the property model built by investigation of a series of known compounds with measured activity. This paper presents an approach of activity prediction based on "structure-activity" models designed to form a hypothesis about probably activity interval estimate. This hypothesis formed is based on structure descriptor domains, calculated for all energetically allowed conformers for each compound in the studied sef. Tetrahydroimidazobenzodiazipenone (TIBO) derivatives and phenylethyltiazolyltiourea (PETT) derivatives illustrated the predictive power of this method. The results are consistent with experimental data and allow to predict inhibitory activity of compounds, which were not included into the training set.

A Novel Nonnucleoside Analogue That Inhibits Human Immunodeficiency Virus Type 1 Isolates Resistant to Current Nonnucleoside Reverse Transcriptase Inhibitors▿

PubMed Central

Zhang, Zhijun; Xu, Wen; Koh, Yung-Hyo; Shim, Jae Hoon; Girardet, Jean-Luc; Yeh, Li-Tain; Hamatake, Robert K.; Hong, Zhi

2007-01-01

Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are important components of current combination therapies for human immunodeficiency virus type 1 (HIV-1) infection. However, their low genetic barriers against resistance development, cross-resistance, and serious side effects can compromise the benefits of the two current drugs in this class (efavirenz and nevirapine). In this study, we report a novel and potent NNRTI, VRX-480773, that inhibits viruses from efavirenz-resistant molecular clones and most NNRTI-resistant clinical HIV-1 isolates tested. In vitro mutation selection experiments revealed that longer times were required for viruses to develop resistance to VRX-480773 than to efavirenz. RT mutations selected by VRX-480773 after 3 months of cell culture in the presence of 1 nM VRX-480773 carried the Y181C mutation, resulting in a less-than-twofold increase in resistance to the compound. A virus containing the double mutation V106I-Y181C emerged after 4 months, causing a sixfold increase in resistance. Viruses containing additional mutations of D123G, F227L, and T369I emerged when the cultures were incubated with increasing concentrations of VRX-480773. Most of the resistant viruses selected by VRX-480773 are susceptible to efavirenz. Oral administration of VRX-480773 to dogs resulted in plasma concentrations that were significantly higher than those required for the inhibition of wild-type and mutant viruses. These results warrant further clinical development of VRX-480773 for the treatment of HIV infection in both NNRTI-naive and -experienced patients. PMID:17116677

A novel nonnucleoside analogue that inhibits human immunodeficiency virus type 1 isolates resistant to current nonnucleoside reverse transcriptase inhibitors.

PubMed

Zhang, Zhijun; Xu, Wen; Koh, Yung-Hyo; Shim, Jae Hoon; Girardet, Jean-Luc; Yeh, Li-Tain; Hamatake, Robert K; Hong, Zhi

2007-02-01

Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are important components of current combination therapies for human immunodeficiency virus type 1 (HIV-1) infection. However, their low genetic barriers against resistance development, cross-resistance, and serious side effects can compromise the benefits of the two current drugs in this class (efavirenz and nevirapine). In this study, we report a novel and potent NNRTI, VRX-480773, that inhibits viruses from efavirenz-resistant molecular clones and most NNRTI-resistant clinical HIV-1 isolates tested. In vitro mutation selection experiments revealed that longer times were required for viruses to develop resistance to VRX-480773 than to efavirenz. RT mutations selected by VRX-480773 after 3 months of cell culture in the presence of 1 nM VRX-480773 carried the Y181C mutation, resulting in a less-than-twofold increase in resistance to the compound. A virus containing the double mutation V106I-Y181C emerged after 4 months, causing a sixfold increase in resistance. Viruses containing additional mutations of D123G, F227L, and T369I emerged when the cultures were incubated with increasing concentrations of VRX-480773. Most of the resistant viruses selected by VRX-480773 are susceptible to efavirenz. Oral administration of VRX-480773 to dogs resulted in plasma concentrations that were significantly higher than those required for the inhibition of wild-type and mutant viruses. These results warrant further clinical development of VRX-480773 for the treatment of HIV infection in both NNRTI-naive and -experienced patients.

Drug resistance-related mutations T369V/I in the connection subdomain of HIV-1 reverse transcriptase severely impair viral fitness.

PubMed

Wang, Zheng; Zhang, Junli; Li, Fan; Ji, Xiaolin; Liao, Lingjie; Ma, Liying; Xing, Hui; Feng, Yi; Li, Dan; Shao, Yiming

2017-04-02

Fitness is a key parameter in the measurement of transmission capacity of individual drug-resistant HIV. Drug-resistance related mutations (DRMs) T369V/I and A371V in the connection subdomain (CN) of reverse transcriptase (RT) occur at higher frequencies in the individuals experiencing antiretroviral therapy failure. Here, we evaluated the effects of T369V/I and A371V on viral fitness, in the presence or in the absence of thymidine analogue resistance-associated mutations (TAMs) and assessed the effect of potential RT structure-related mechanism on change in viral fitness. Mutations T369V/I, A371V, alone or in combination with TAMs were introduced into a modified HIV-1 infectious clone AT1 by site-directed mutagenesis. Then, experiments on mutant and wild-type virus AT2 were performed separately using a growth-competition assay, and then the relative fitness was calculated. Structural analysis of RT was conducted using Pymol software. Results showed that T369V/I severely impaired the relative virus fitness, and A371V compensated for the viral fitness reduction caused by TAMs. Structural modeling of RT suggests that T369V/I substitutions disrupt powerful hydrogen bonds formed by T369 and V365 in p51 and p66. This study indicates that the secondary DRMs within CN might efficiently damage viral fitness, and provides valuable information for clinical surveillance and prevention of HIV-1 strains carrying these DRMs. Copyright © 2017 Elsevier B.V. All rights reserved.

Prevalence of antiretroviral drug resistance and resistance-associated mutations in antiretroviral therapy-naïve HIV-infected individuals from 40 United States cities.

PubMed

Ross, Lisa; Lim, Michael L; Liao, Qiming; Wine, Brian; Rodriguez, Allan E; Weinberg, Winkler; Shaefer, Mark

2007-01-01

Transmission of drug-resistant HIV strains to antiretroviral therapy (ART)-naïve subjects can negatively impact therapy response. As treatment strategies and utilization of antiretroviral drugs evolve, patterns of transmitted mutations may shift. Paired genotypic and phenotypic susceptibility data were retrospectively analyzed for 317 ART-naïve, HIV-infected subjects from 40 small and major metropolitan cities in the Northeastern, Midwestern, Southern, Southwestern, and Northwestern United States during 2003. Using current (January 2007) PhenoSense cutoffs, HIV-from 8% of subjects had reduced susceptibility to > or = 1 drug. By class, < 1% had reduced susceptibility to protease inhibitors (PIs), and 1% had reduced susceptibility to nucleoside reverse transcriptase inhibitors (NRTIs); reduced susceptibility to > or = 1 non-nucleoside reverse transcriptase inhibitor (NNRTIs) was seen in 7% of subjects, with 4% of all subjects having reduced susceptibility to all NNRTIs. IAS-USA-defined NRTI, NNRTI, and/or major PI HIV-drug resistance-associated mutations were detected for 0% of the subjects. HIV risk factors included homosexual contact (74%), heterosexual contact (28%), and injectable drug use/transfusion/other (7%). Reduced susceptibility to > or = 1 drug was significantly higher (p = .034) for white subjects than African Americans and Hispanics/others. The high prevalence of drug resistance in these ART-naïve subjects suggests that transmitted resistance is occurring widely within the United States. HIV genotyping and/or phenotyping for antiretroviral-naïve patients seeking treatment should be considered, especially if the therapy will include an NNRTI.

The reverse transcriptase encoded by LINE-1 retrotransposons in the genesis, progression and therapy of cancer

NASA Astrophysics Data System (ADS)

Sciamanna, Ilaria; De Luca, Chiara; Spadafora, Corrado

2016-02-01

In higher eukaryotic genomes, Long Interspersed Nuclear Element 1 (LINE-1) retrotransposons represent a large family of repeated genomic elements. They transpose using a reverse transcriptase (RT), which they encode as part of the ORF2p product. RT inhibition in cancer cells, either via RNA interference-dependent silencing of active LINE-1 elements, or using RT inhibitory drugs, reduces cancer cell proliferation, promotes their differentiation and antagonizes tumor progression in animal models. Indeed, the nonnucleoside RT inhibitor efavirenz has recently been tested in a phase II clinical trial with metastatic prostate cancer patients. An in-depth analysis of ORF2p in a mouse model of breast cancer showed ORF2p to be precociously expressed in precancerous lesions and highly abundant in advanced cancer stages, while being barely detectable in normal breast tissue, providing a rationale for the finding that RT-expressing tumours are therapeutically sensitive to RT inhibitors. We summarise mechanistic and gene profiling studies indicating that highly abundant LINE-1-derived RT can “sequester” RNA substrates for reverse transcription in tumor cells, entailing the formation of RNA:DNA hybrid molecules and impairing the overall production of regulatory miRNAs, with a global impact on the cell transcriptome. Based on these data, LINE-1-ORF2 encoded RT has a tumor-promoting potential that is exerted at an epigenetic level. We propose a model whereby LINE1-RT drives a previously unrecognized global regulatory process, the deregulation of which drives cell transformation and tumorigenesis and possibly implicated in cancer cell heterogeneity.

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

PubMed Central

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

2010-01-01

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

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.

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

Role of the K101E Substitution in HIV-1 Reverse Transcriptase in Resistance to Rilpivirine and Other Nonnucleoside Reverse Transcriptase Inhibitors

PubMed Central

Xu, Hong-Tao; Colby-Germinario, Susan P.; Huang, Wei; Oliveira, Maureen; Han, Yingshan; Quan, Yudong; Petropoulos, Christos J.

2013-01-01

Resistance to the recently approved nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV) commonly involves substitutions at positions E138K and K101E in HIV-1 reverse transcriptase (RT), together with an M184I substitution that is associated with resistance to coutilized emtricitabine (FTC). Previous biochemical and virological studies have shown that compensatory interactions between substitutions E138K and M184I can restore enzyme processivity and the viral replication capacity. Structural modeling studies have also shown that disruption of the salt bridge between K101 and E138 can affect RPV binding. The current study was designed to investigate the impact of K101E, alone or in combination with E138K and/or M184I, on drug susceptibility, viral replication capacity, and enzyme function. We show here that K101E can be selected in cell culture by the NNRTIs etravirine (ETR), efavirenz (EFV), and dapivirine (DPV) as well as by RPV. Recombinant RT enzymes and viruses containing K101E, but not E138K, were highly resistant to nevirapine (NVP) and delavirdine (DLV) as well as ETR and RPV, but not EFV. The addition of K101E to E138K slightly enhanced ETR and RPV resistance compared to that obtained with E138K alone but restored susceptibility to NVP and DLV. The K101E substitution can compensate for deficits in viral replication capacity and enzyme processivity associated with M184I, while M184I can compensate for the diminished efficiency of DNA polymerization associated with K101E. The coexistence of K101E and E138K does not impair either viral replication or enzyme fitness. We conclude that K101E can play a significant role in resistance to RPV. PMID:24002090

Role of the K101E substitution in HIV-1 reverse transcriptase in resistance to rilpivirine and other nonnucleoside reverse transcriptase inhibitors.

PubMed

Xu, Hong-Tao; Colby-Germinario, Susan P; Huang, Wei; Oliveira, Maureen; Han, Yingshan; Quan, Yudong; Petropoulos, Christos J; Wainberg, Mark A

2013-11-01

Resistance to the recently approved nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV) commonly involves substitutions at positions E138K and K101E in HIV-1 reverse transcriptase (RT), together with an M184I substitution that is associated with resistance to coutilized emtricitabine (FTC). Previous biochemical and virological studies have shown that compensatory interactions between substitutions E138K and M184I can restore enzyme processivity and the viral replication capacity. Structural modeling studies have also shown that disruption of the salt bridge between K101 and E138 can affect RPV binding. The current study was designed to investigate the impact of K101E, alone or in combination with E138K and/or M184I, on drug susceptibility, viral replication capacity, and enzyme function. We show here that K101E can be selected in cell culture by the NNRTIs etravirine (ETR), efavirenz (EFV), and dapivirine (DPV) as well as by RPV. Recombinant RT enzymes and viruses containing K101E, but not E138K, were highly resistant to nevirapine (NVP) and delavirdine (DLV) as well as ETR and RPV, but not EFV. The addition of K101E to E138K slightly enhanced ETR and RPV resistance compared to that obtained with E138K alone but restored susceptibility to NVP and DLV. The K101E substitution can compensate for deficits in viral replication capacity and enzyme processivity associated with M184I, while M184I can compensate for the diminished efficiency of DNA polymerization associated with K101E. The coexistence of K101E and E138K does not impair either viral replication or enzyme fitness. We conclude that K101E can play a significant role in resistance to RPV.

Particulate matter from indoor environments of classroom induced higher cytotoxicity and leakiness in human microvascular endothelial cells in comparison with those collected from corridor.

PubMed

Chua, M L; Setyawati, M I; Li, H; Fang, C H Y; Gurusamy, S; Teoh, F T L; Leong, D T; George, S

2017-05-01

We investigated the physicochemical properties (size, shape, elemental composition, and endotoxin) of size resolved particulate matter (PM) collected from the indoor and corridor environments of classrooms. A comparative hazard profiling of these PM was conducted using human microvascular endothelial cells (HMVEC). Oxidative stress-dependent cytotoxicity responses were assessed using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and high content screening (HCS), and disruption of monolayer cell integrity was assessed using fluorescence microscopy and transwell assay. Scanning electron microscopy (SEM) coupled with energy-dispersive X-ray spectroscopy (EDX) analysis showed differences in the morphology and elemental composition of PM of different sizes and origins. While the total mass of PM collected from indoor environment was lower in comparison with those collected from the corridor, the endotoxin content was substantially higher in indoor PM (e.g., ninefold higher endotoxin level in indoor PM 8.1-20 ). The ability to induce oxidative stress-mediated cytotoxicity and leakiness in cell monolayer were higher for indoor PM compared to those collected from the corridor. In conclusion, this comparative analysis suggested that indoor PM is relatively more hazardous to the endothelial system possibly because of higher endotoxin content. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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.

Probing the molecular mechanism of action of the HIV-1 reverse transcriptase inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) using pre-steady-state kinetics.

PubMed

Muftuoglu, Yagmur; Sohl, Christal D; Mislak, Andrea C; Mitsuya, Hiroaki; Sarafianos, Stefan G; Anderson, Karen S

2014-06-01

The novel antiretroviral 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a potent nucleoside HIV-1 reverse transcriptase (RT) inhibitor (NRTI). Unlike other FDA-approved NRTIs, EFdA contains a 3'-hydroxyl. Pre-steady-state kinetics showed RT preferred incorporating EFdA-TP over native dATP. Moreover, RT slowly inserted nucleotides past an EFdA-terminated primer, resulting in delayed chain termination with unaffected fidelity. This is distinct from KP1212, another 3'-hydroxyl-containing RT inhibitor considered to promote viral lethal mutagenesis. New mechanistic features of RT inhibition by EFdA are revealed. Copyright © 2014 Elsevier B.V. All rights reserved.

Probing the molecular mechanism of action of the HIV-1 reverse transcriptase inhibitor 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) using pre-steady-state kinetics

PubMed Central

Muftuoglu, Yagmur; Sohl, Christal D.; Mislak, Andrea C.; Mitsuya, Hiroaki; Sarafianos, Stefan G.; Anderson, Karen S.

2014-01-01

The novel antiretroviral 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a potent nucleoside HIV-1 reverse transcriptase (RT) inhibitor (NRTI). Unlike other FDA-approved NRTIs, EFdA contains a 3′-hydroxyl. Pre-steady-state kinetics showed RT preferred incorporating EFdA-TP over native dATP. Moreover, RT slowly inserted nucleotides past an EFdA-terminated primer, resulting in delayed chain termination with unaffected fidelity. This is distinct from KP1212, another 3′-hydroxyl-containing RT inhibitor considered to promote viral lethal mutagenesis. New mechanistic features of RT inhibition by EFdA are revealed. PMID:24632447

Leptin upregulates telomerase activity and transcription of human telomerase reverse transcriptase in MCF-7 breast cancer cells

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

Ren, He, E-mail: herenrh@yahoo.com.cn; Zhao, Tiansuo; Wang, Xiuchao

2010-03-26

The aim was to analyze the mechanism of leptin-induced activity of telomerase in MCF-7 breast cancer cells. We found that leptin activated telomerase in a dose-dependent manner; leptin upregulated the expression of Human Telomerase Reverse Transcriptase (hTERT) at mRNA and protein levels; blockade of signal transducer and activator of transcription 3 (STAT3) phosphorylation significantly counteracted leptin-induced hTERT transcription and protein expression; chromatin immunoprecipitation analysis showed that leptin enhanced the binding of STAT3 to the hTERT promoter. This study uncovers a new mechanism of the proliferative effect of leptin on breast cancer cells and provides a new explanation of obesity-related breastmore » cancer.« less

Oligonucleotide microarray analysis of gene expression profiles followed by real-time reverse-transcriptase polymerase chain reaction assay in chronic active Epstein-Barr virus infection.

PubMed

Ito, Yoshinori; Shibata-Watanabe, Yukiko; Ushijima, Yoko; Kawada, Jun-Ichi; Nishiyama, Yukihiro; Kojima, Seiji; Kimura, Hiroshi

2008-03-01

Chronic active Epstein-Barr virus infection (CAEBV) is characterized by recurrent infectious mononucleosis-like symptoms and has high mortality and morbidity. To clarify the mechanisms of CAEBV, the gene-expression profiles of peripheral blood obtained from patients with CAEBV were investigated. Twenty genes were differentially expressed in 4 patients with CAEBV. This microarray result was verified using a real-time reverse-transcriptase polymerase chain reaction assay in a larger group of patients with CAEBV. Eventually, 3 genes were found to be significantly upregulated: guanylate binding protein 1, tumor necrosis factor-induced protein 6, and guanylate binding protein 5. These genes may be associated with the inflammatory reaction or with cell proliferation.

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.

In Vitro Cross-Resistance Profiles of Rilpivirine, Dapivirine, and MIV-150, Nonnucleoside Reverse Transcriptase Inhibitor Microbicides in Clinical Development for the Prevention of HIV-1 Infection.

PubMed

Giacobbi, Nicholas S; Sluis-Cremer, Nicolas

2017-07-01

Rilpivirine (RPV), dapivirine (DPV), and MIV-150 are in development as microbicides. It is not known whether they will block infection of circulating nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant human immunodeficiency virus type 1 (HIV-1) variants. Here, we demonstrate that the activity of DPV and MIV-150 is compromised by many resistant viruses containing single or double substitutions. High DPV genital tract concentrations from DPV ring use may block replication of resistant viruses. However, MIV-150 genital tract concentrations may be insufficient to inhibit many resistant viruses, including those harboring K103N or Y181C. Copyright © 2017 American Society for Microbiology.

Elucidation of the TMab-6 Monoclonal Antibody Epitope Against Telomerase Reverse Transcriptase.

PubMed

Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Chang, Yao-Wen; Nakamura, Takuro; Yanaka, Miyuki; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kato, Yukinari

2018-05-03

Telomerase reverse transcriptase (TERT) and mutations of the TERT promoter are significant in the pathogenesis of 1p/19q-codeleted oligodendrogliomas and isocitrate dehydrogenase gene wild-type glioblastomas, as well as melanomas and squamous cell carcinomas. We previously developed an antihuman TERT monoclonal antibody (mAb), TMab-6, which is applicable in immunohistochemistry for human tissues. However, the binding epitope of TMab-6 against TERT is yet to be elucidated. In this study, enzyme-linked immunosorbent assay and immunohistochemistry were utilized for investigating the epitope of TMab-6. The findings revealed that the critical epitope of TMab-6 is the TERT sequence PSTSRPPRPWD; Thr310 and Ser311 of TERT are especially significant amino acids for TMab-6 recognition.

Time Lapse to Colorectal Cancer: Telomere Dynamics Define the Malignant Potential of Polyps.

PubMed

Druliner, Brooke R; Ruan, Xiaoyang; Johnson, Ruth; Grill, Diane; O'Brien, Daniel; Lai, Tsung-Po; Rashtak, Shahrooz; Felmlee-Devine, Donna; Washechek-Aletto, Jill; Malykh, Andrei; Smyrk, Thomas; Oberg, Ann; Liu, Hongfang; Shay, Jerry W; Ahlquist, David A; Boardman, Lisa A

2016-09-01

Whereas few adenomas become cancer, most colorectal cancers arise from adenomas. Telomere length is a recognized biomarker in multiple cancers, and telomere maintenance mechanisms (TMM) are exploited by malignant cells. We sought to determine whether telomere length and TMM distinguish cancer-associated adenomas from those that are cancer-free. Tissues were identified as cancer-adjacent polyp (CAP)-residual adenoma contiguous with cancer-and cancer-free polyp (CFP)-adenomas without malignancy. Telomere length, TMM, and expression were measured in 102 tissues including peripheral blood leukocytes (PBLs), normal colon epithelium, adenoma, and cancer (in CAP cases) from 31 patients. Telomere length was measured in a separate cohort of 342 PBL from CAP and CFP patients. The mean differences in telomere length between normal and adenoma were greater in CAP than in CFP cases, P=0.001; telomere length in PBL was 91.7 bp greater in CAP than in CFP, P=0.007. Each 100 bp telomere increase was associated with a 1.14 (1.04-1.26) increased odds of being a CAP, P=0.0063. The polyp tissue from CAP patients had shorter telomeres and higher Telomerase reverse transcriptase (hTERT) expression compared with polyps from CFP patients, P=0.05. There was a greater degree of alternative lengthening of telomere (ALT) level difference in CFP polyps than in CAP polyps. The polyp telomere lengths of aggressive CAPs were significantly different from the polyps of non-aggressive CAPs, P=0.01. Adenomas that progress to cancer exhibit distinct telomere length and TMM profiles. We report for the first time that PBL telomeres differ in patients with polyps that become malignant, and therefore may have clinical value in adenoma risk assessment and management.

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.

Rift Valley fever virus infections in Egyptian cattle and their prevention.

PubMed

Mroz, C; Gwida, M; El-Ashker, M; Ziegler, U; Homeier-Bachmann, T; Eiden, M; Groschup, M H

2017-12-01

Rift Valley fever virus (RVFV) causes consistently severe outbreaks with high public health impacts and economic losses in livestock in many African countries and has also been introduced to Saudi Arabia and Yemen. Egypt with its four large outbreaks in the last 40 years represents the northernmost endemic area of RVFV. The purpose of this study was to provide an insight into the current anti-RVFV antibody status in immunized as well as non-immunized dairy cattle from the Nile Delta of Egypt. During 2013-2015, a total of 4,167 dairy cattle from four governorates including Dakahlia, Damietta, Gharbia and Port Said were investigated. All cattle were born after 2007 and therewith after the last reported Egyptian RVFV outbreak in 2003. The samples derived from vaccinated animals from 26 different dairy farms as well as non-immunized cattle from 27 different smallholding flocks. All samples were examined following a three-part analysis including a commercially available competition ELISA, an in-house immunofluorescence assay and a virus neutralization test. Additionally, a subset of samples was analysed for acute infections using IgM ELISA and real-time reverse transcriptase PCR. The results indicated that the RVFV is still circulating in Egypt as about 10% of the non-immunized animals exhibited RVFV-specific antibodies. Surprisingly, the antibody prevalence in immunized animals was not significantly higher than that in non-vaccinated animals which points out the need for further evaluation of the vaccination programme. Due to the substantial role of livestock in the amplification and transmission of RVFV, further recurrent monitoring of the antibody prevalence in susceptible species is highly warranted. © 2017 Blackwell Verlag GmbH.

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion

PubMed Central

Kusuma, Gina D.; Brennecke, Shaun P.; O’Connor, Andrea J.; Kalionis, Bill

2017-01-01

Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies. PMID:28152107

Coordinated maturational regulation of PHEX and renal phosphate transport inhibitory activity: evidence for the pathophysiological role of PHEX in X-linked hypophosphatemia.

PubMed

Nesbitt, T; Fujiwara, I; Thomas, R; Xiao, Z S; Quarles, L D; Drezner, M K

1999-12-01

The mechanism by which inactivating mutations of PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) cause X-linked hypophosphatemia remains unknown. However, recent reports suggest errant PHEX activity in osteoblasts may fail to inactivate a phosphaturic factor produced by these cells. To test this possibility, we examined coordinated maturational expression of PHEX and production of phosphate transport inhibitory activity in osteoblasts from normal and hyp-mice. We assessed the inhibitory activity in conditioned medium by examining the effects on opossum kidney cell phosphate transport and osteoblast PHEX expression by reverse transcriptase-polymerase chain reaction during a 17-day maturational period. Inhibitory activity increased as a function of osteoblast maturational stage, with no activity after 3 days and persistent activity by 6 days of culture. More significantly, equal phosphate transport inhibitory activity in conditioned medium from normal and hyp-mouse osteoblasts (control 1.90 +/- 0.12, normal 1.48 +/- 0.10, hyp 1.45 +/- 0.04 nmol/mg of protein/minute) was observed at 6 days. However, by 10 days hyp-mouse osteoblasts exhibited greater inhibitory activity than controls, and by 17 days the difference in phosphate transport inhibition maximized (control 2.08 +/- 0.09, normal 1.88 +/- 0.06, hyp 1.58 +/- 0.06 nmol/mg of protein/minute). Concurrently, we observed absent PHEX expression in normal osteoblasts after 3 days, limited production at 6 days, and significant production by day 10 of culture, while hyp-mouse osteoblasts exhibited limited PHEX activity secondary to an inactivating mutation. The data suggest that the presence of inactivating PHEX mutations results in the enhanced renal phosphate transport inhibitory activity exhibited by hyp-mouse osteoblasts.

Differential chemokine, chemokine receptor and cytokine expression in Epstein-Barr virus-associated lymphoproliferative diseases.

PubMed

Ohshima, Koichi; Karube, Kennosuke; Hamasaki, Makoto; Tutiya, Takeshi; Yamaguchi, Takahiro; Suefuji, Hiroaki; Suzuki, Keiko; Suzumiya, Junji; Ohga, Shouichi; Kikuchi, Masahiro

2003-08-01

T cell immunity plays an important role in the clinicopathology of Epstein-Barr virus (EBV)-associated diseases. Acute EBV-induced infectious mononucleosis (IM) is a common self-limiting disease, however, other EBV-associated diseases, including chronic active EBV infection (CAEBV), NK cell lymphoma (NKL), and Hodgkin's lymphoma (HL), exhibit distinct clinical features. Chemokines are members of a family of small-secreted proteins. The relationships between chemokines and the chemokine receptor (R) are thought to be important for selectivity of local immunity. Some chemokines, chemokine R and cytokines closely associate with the T cell subtypes, Th1 and Th2 T cells and cytotoxic cells. To clarify the role of T cell immunity in EBV-associated diseases, we conducted gene expression profiling, using chemokine, chemokine R and cytokine DNA chips. Compared to EBV negative non-specific lymphadenitis, CAEBV and NKL exhibited diffuse down- and up-regulation, respectively, of these gene profiles. IM had a predominantly Th1-type profile, whereas HL had a mixed Th1/Th2-type profile. Reduction of the Th1-type cytokine interferon gamma (IFN-gamma) in CAEBV was confirmed by Reverse transcriptase-polymerase chain reaction, whereas IFN-gamma expression was markedly enhanced in NKL, and moderately enhanced in IM. Compared to IM, CAEBV showed slight elevation of "regulated upon activation, normal T expressed and secreted" (RANTES), but almost all other genes assayed were down-regulated. NKL exhibited elevated expression of numerous genes, particularly IFN-gamma-inducible-10 (IP-10) and monokine induced by IFN-gamma (MIG). HL showed variable elevated and reduced expression of various genes, with increased expression of IL-13 receptor and MIG. Our study demonstrated the enormous potential of gene expression profiling for clarifying the pathogenesis of EBV-associated diseases.

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion.

PubMed

Kusuma, Gina D; Brennecke, Shaun P; O'Connor, Andrea J; Kalionis, Bill; Heath, Daniel E

2017-01-01

Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies.

Higher rates of triple-class virological failure in perinatally HIV-infected teenagers compared with heterosexually infected young adults in Europe.

PubMed

Judd, A; Lodwick, R; Noguera-Julian, A; Gibb, D M; Butler, K; Costagliola, D; Sabin, C; van Sighem, A; Ledergerber, B; Torti, C; Mocroft, A; Podzamczer, D; Dorrucci, M; De Wit, S; Obel, N; Dabis, F; Cozzi-Lepri, A; García, F; Brockmeyer, N H; Warszawski, J; Gonzalez-Tome, M I; Mussini, C; Touloumi, G; Zangerle, R; Ghosn, J; Castagna, A; Fätkenheuer, G; Stephan, C; Meyer, L; Campbell, M A; Chene, G; Phillips, A

2017-03-01

The aim of the study was to determine the time to, and risk factors for, triple-class virological failure (TCVF) across age groups for children and adolescents with perinatally acquired HIV infection and older adolescents and adults with heterosexually acquired HIV infection. We analysed individual patient data from cohorts in the Collaboration of Observational HIV Epidemiological Research Europe (COHERE). A total of 5972 participants starting antiretroviral therapy (ART) from 1998, aged < 20 years at the start of ART for those with perinatal infection and 15-29 years for those with heterosexual infection, with ART containing at least two nucleoside reverse transcriptase inhibitors (NRTIs) and a nonnucleoside reverse transcriptase inhibitor (NNRTI) or a boosted protease inhibitor (bPI), were followed from ART initiation until the most recent viral load (VL) measurement. Virological failure of a drug was defined as VL > 500 HIV-1 RNA copies/mL despite ≥ 4 months of use. TCVF was defined as cumulative failure of two NRTIs, an NNRTI and a bPI. The median number of weeks between diagnosis and the start of ART was higher in participants with perinatal HIV infection compared with participants with heterosexually acquired HIV infection overall [17 (interquartile range (IQR) 4-111) vs. 8 (IQR 2-38) weeks, respectively], and highest in perinatally infected participants aged 10-14 years [49 (IQR 9-267) weeks]. The cumulative proportion with TCVF 5 years after starting ART was 9.6% [95% confidence interval (CI) 7.0-12.3%] in participants with perinatally acquired infection and 4.7% (95% CI 3.9-5.5%) in participants with heterosexually acquired infection, and highest in perinatally infected participants aged 10-14 years when starting ART (27.7%; 95% CI 13.2-42.1%). Across all participants, significant predictors of TCVF were those with perinatal HIV aged 10-14 years, African origin, pre-ART AIDS, NNRTI-based initial regimens, higher pre-ART viral load and lower pre-ART CD4. The results suggest a beneficial effect of starting ART before adolescence, and starting young people on boosted PIs, to maximize treatment response during this transitional stage of development. © 2016 The Authors. HIV Medicine published by John Wiley & Sons Ltd on behalf of British HIV Association.

Expression of Estrogen Receptors Alpha (ER-α), Beta (ER-β), and G Protein-Coupled Receptor 30 (GPR30) in Testicular Tissue of Men with Klinefelter Syndrome.

PubMed

Bernardino, R L; Alves, M G; Silva, J; Barros, A; Ferraz, L; Sousa, M; Sá, R; Oliveira, P F

2016-06-01

Men with Klinefelter syndrome (KS) present severe hormonal dysregulation, particularly elevated serum estradiol concentration. Estrogens act through specific receptors and regulate testes development and spermatogenesis. Herein, we evaluated GPR30, ERα, and ERβ mRNA expression in testis of KS men and men with 46XY karyotype by reverse transcriptase and quantitative PCR. ERβ transcripts are the most abundant in testicular tissue of 46XY men. Notably, testicular GPR30 transcription in KS men was approximately 12 times higher. Since GPR30 is essential to mediate estrogen effects over steroidogenesis, our data illustrate that GPR30 may underpin the testicular alterations observed in KS men. © Georg Thieme Verlag KG Stuttgart · New York.

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.

Lower genetic variability of HIV-1 and antiretroviral drug resistance in pregnant women from the state of Pará, Brazil.

PubMed

Machado, Luiz Fernando Almeida; Costa, Iran Barros; Folha, Maria Nazaré; da Luz, Anderson Levy Bessa; Vallinoto, Antonio Carlos Rosário; Ishak, Ricardo; Ishak, Marluisa Oliveira Guimarães

2017-04-12

The present study aimed to describe the genetic diversity of HIV-1, as well as the resistance profile of the viruses identified in HIV-1 infected pregnant women under antiretroviral therapy in the state of Pará, Northern Brazil. Blood samples were collected from 45 HIV-1 infected pregnant to determine the virus subtypes according to the HIV-1 protease (PR) gene and part of the HIV-1 reverse transcriptase (RT) gene by sequencing the nucleotides of these regions. Drug resistance mutations and susceptibility to antiretroviral drugs were analyzed by the Stanford HIV Drug Resistance Database. Out of 45 samples, only 34 could be amplified for PR and 30 for RT. Regarding the PR gene, subtypes B (97.1%) and C (2.9%) were identified; for the RT gene, subtypes B (90.0%), F (6.7%), and C (3.3%) were detected. Resistance to protease inhibitors (PI) was identified in 5.8% of the pregnant, and mutations conferring resistance to nucleoside reverse transcriptase inhibitors were found in 3.3%, while mutations conferring resistance to non-nucleoside reverse transcriptase inhibitors were found in 3.3%. These results showed a low frequency of strains resistant to antiretroviral drugs, the prevalence of subtypes B and F, and the persistent low transmission of subtype C in pregnant of the state of Pará, Brazil.

Comprehensive phylogenetic analysis of bacterial reverse transcriptases.

PubMed

Toro, Nicolás; Nisa-Martínez, Rafael

2014-01-01

Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology.

Comprehensive Phylogenetic Analysis of Bacterial Reverse Transcriptases

PubMed Central

Toro, Nicolás; Nisa-Martínez, Rafael

2014-01-01

Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology. PMID:25423096

Structure-based methods to predict mutational resistance to diarylpyrimidine non-nucleoside reverse transcriptase inhibitors.

PubMed

Azeem, Syeda Maryam; Muwonge, Alecia N; Thakkar, Nehaben; Lam, Kristina W; Frey, Kathleen M

2018-01-01

Resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) is a leading cause of HIV treatment failure. Often included in antiviral therapy, NNRTIs are chemically diverse compounds that bind an allosteric pocket of enzyme target reverse transcriptase (RT). Several new NNRTIs incorporate flexibility in order to compensate for lost interactions with amino acid conferring mutations in RT. Unfortunately, even successful inhibitors such as diarylpyrimidine (DAPY) inhibitor rilpivirine are affected by mutations in RT that confer resistance. In order to aid drug design efforts, it would be efficient and cost effective to pre-evaluate NNRTI compounds in development using a structure-based computational approach. As proof of concept, we applied a residue scan and molecular dynamics strategy using RT crystal structures to predict mutations that confer resistance to DAPYs rilpivirine, etravirine, and investigational microbicide dapivirine. Our predictive values, changes in affinity and stability, are correlative with fold-resistance data for several RT mutants. Consistent with previous studies, mutation K101P is predicted to confer high-level resistance to DAPYs. These findings were further validated using structural analysis, molecular dynamics, and an enzymatic reverse transcription assay. Our results confirm that changes in affinity and stability for mutant complexes are predictive parameters of resistance as validated by experimental and clinical data. In future work, we believe that this computational approach may be useful to predict resistance mutations for inhibitors in development. Published by Elsevier Inc.

Differential Regulation of Telomerase Reverse Transcriptase Promoter Activation and Protein Degradation by Histone Deacetylase Inhibition.

PubMed

Qing, Hua; Aono, Jun; Findeisen, Hannes M; Jones, Karrie L; Heywood, Elizabeth B; Bruemmer, Dennis

2016-06-01

Telomerase reverse transcriptase (TERT) maintains telomeres and is rate limiting for replicative life span. While most somatic tissues silence TERT transcription resulting in telomere shortening, cells derived from cancer or cardiovascular diseases express TERT and activate telomerase. In the present study, we demonstrate that histone deacetylase (HDAC) inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Finally, we demonstrate that HDAC inhibition decreases TERT expression during vascular remodeling in vivo. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition and suggest that TERT may constitute an important target for the anti-proliferative efficacy of HDAC inhibitors. © 2015 Wiley Periodicals, Inc.

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.

Herpes Simplex Virus Type 2 Suppressive Therapy with Acyclovir or Valacyclovir Does Not Select for Specific HIV-1 Resistance in HIV-1/HSV-2 Dually Infected Persons

PubMed Central

Lingappa, Jairam; Beck, Ingrid; Frenkel, Lisa M.; Pepper, Gregory; Celum, Connie; Wald, Anna; Fife, Kenneth H.; Were, Edwin; Mugo, Nelly; Sanchez, Jorge; Essex, Myron; Makhema, Joseph; Kiarie, James; Farquhar, Carey; Corey, Lawrence

2011-01-01

Recent in vitro studies suggest that acyclovir may directly inhibit HIV-1 replication and can select for a specific HIV-1 reverse transcriptase mutation (V75I) with concomitant loss of an anti-HIV-1 effect. We tested for HIV-1 genotypic resistance at reverse transcriptase codon 75 in plasma from 168 HIV-1–infected persons from Botswana, Kenya, Peru, and the United States taking daily acyclovir or valacyclovir for between 8 weeks and 24 months. No V75I cases were detected (95% confidence interval, 0%–2.2%). These prospective in vivo studies suggest that standard-dose acyclovir or valacyclovir does not select for HIV-1 resistance. PMID:21148504

Extraordinary effects of specific monovalent cations on activation of reovirus transcriptase by chymotrypsin in vitro.

PubMed

Borsa, J; Sargent, M D; Long, D G; Chapman, J D

1973-02-01

Activation of reovirus transcriptase activity, latent in intact virions, by digestion of purified virions with chymotrypsin (CHT) in vitro shows a stringent requirement for specific monovalent cations. Cs(+), Rb(+), or K(+) ions are capable of facilitating activation by chymotryptic digestion. Na(+), Li(+), or NH(4) (+) ions are not capable of facilitating the CHT activation of polymerase activity and are antagonistic towards the effects of the facilitating ions. The data indicate that the effect of the cations is exerted on activation of the polymerase activity by CHT as opposed to an effect on polymerization per se. This effect may be important biologically in that it provides a mechanism whereby the virion can sense whether it is in an intracellular or an extracellular environment and thereby can avoid premature uncoating.

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.

Prediction of Gut Wall Integrity Loss in Viral Gastroenteritis by Non-Invasive Marker

PubMed Central

Elnady, Hala G.; Sherif, Lobna S.; Saleh, Maysa T.; El-Alameey, Inas R.; Youssef, Mai M.; El Shafie, Amal I.; Helwa, Iman; Raouf, Haiam Abdel; EL-Taweel, Ahmed N.

2015-01-01

BACKGROUND: Intestinal fatty acid binding proteins (I-FABPs) are mainly expressed in the intestinal villi, which are the initial site of destruction in viral gastroenteritis. AIM: This study was designed to assess serum I-FABPs as a predictor of gut wall integrity loss in viral gastroenteritis. PATIENTS AND METHODS: This case-control cross-sectional study was conducted on 93 cases of acute viral gastroenteritis. Twenty-eight healthy children matching in age were recruited as control group. Serum I-FABPs were measured using ELISA technique. Viral detection and typing were done by PCR for adenovirus, and by Reverse transcriptase PCR for rotavirus, astrovirus and norovirus. RESULTS: Serum I-FABPs level was significantly higher in the cases compared to the controls and was also higher in the 46 rotavirus gastroenteritis cases compared to other viral gastroenteritis cases. Serum I- FABPs level was significantly higher in severely dehydrated cases as compared to mildly dehydrated ones (P=0.037). CONCLUSION: Serum I-FABPs could be used as an early and sensitive predictor marker of gut wall integrity loss in children with viral gastroenteritis and its level can indicate case severity. PMID:27275194

Probing the mechanistic consequences of 5-fluorine substitution on cytidine nucleotide analogue incorporation by HIV-1 reverse transcriptase.

PubMed

Ray, Adrian S; Schinazi, Raymond F; Murakami, Eisuke; Basavapathruni, Aravind; Shi, Junxing; Zorca, Suzana M; Chu, Chung K; Anderson, Karen S

2003-05-01

Beta-D and beta-L-enantiomers of 2',3'-dideoxycytidine analogues are potent chain-terminators and antimetabolites for viral and cellular replication. Seemingly small modifications markedly alter their antiviral and toxicity patterns. This review discusses previously published and recently obtained data on the effects of 5- and 2'-fluorine substitution on the pre-steady state incorporation of 2'-deoxycytidine-5'-monophosphate analogues by HIV-1 reverse transcriptase (RT) in light of their biological activity. The addition of fluorine at the 5-position of the pyrimidine ring altered the kinetic parameters for all nucleotides tested. Only the 5-fluorine substitution of the clinically relevant nucleosides (-)-beta-L-2',3'-dideoxy-3'-thia-5-fluorocytidine (L-FTC, Emtriva), and (+)-beta-D-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (D-D4FC, Reverset), caused a higher overall efficiency of nucleotide incorporation during both DNA- and RNA-directed synthesis. Enhanced incorporation by RT may in part explain the potency of these nucleosides against HIV-1. In other cases, a lack of correlation between RT incorporation in enzymatic assays and antiviral activity in cell culture illustrates the importance of other cellular factors in defining antiviral potency. The substitution of fluorine at the 2' position of the deoxyribose ring negatively affects incorporation by RT indicating the steric gate of RT can detect electrostatic perturbations. Intriguing results pertaining to drug resistance have led to a better understanding of HIV-1 RT resistance mechanisms. These insights serve as a basis for understanding the mechanism of action for nucleoside analogues and, coupled with studies on other key enzymes, may lead to the more effective use of fluorine to enhance the potency and selectivity of antiviral agents.

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.

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

PubMed

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

2013-07-01

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

The correlations between DNA methylation and polymorphisms in the promoter region of the human telomerase reverse transcriptase (hTERT) gene with postoperative recurrence in patients with thyroid carcinoma (TC).

PubMed

Li, Jian-Jun; Zheng, Ping Chen Jue-Ru; Wang, Yao-Zong

2017-06-06

This study aims at exploring the correlations between DNA methylation and polymorphisms in the promoter region of the human telomerase reverse transcriptase (hTERT) gene and postoperative recurrence in patients with thyroid carcinoma (TC). A total of 312 patients diagnosed with TC were chosen for the study and categorized into recurrence (n = 75) and non-recurrence (n = 237) groups. The hTERT rs2736100 and rs2736098 polymorphisms were detected by performing polymerase chain reaction-restriction fragment length polymorphism. DNA methylation in the promoter region of hTERT gene was evaluated by pyrosequencing. A telephonic and/or outpatient follow-up was conducted for all patients. The correlations of DNA methylation and polymorphisms in the promoter region of hTERT with postoperative recurrence of TC patients underwent analysis. The patient in the recurrence group showed evidently different pathological types and tumor stages in comparison to the non-recurrence group. The GG genotype of hTERT rs2736100 might increase the recurrence risk of TC patients. No correlations between hTERT rs2736098 polymorphisms and recurrence risk were observed. Compared to the TT + TG genotype frequency, the rs2736100 GG genotype frequency increased in patients without multicentricity, patients with extrathyroidal invasion, patients with lymph node metastasis, patients with undifferentiated carcinoma, and patients in the III + IV stage. The recurrence group showed significantly higher DNA methylation level compared to the non-recurrence group. The DNA methylation level was closely associated to tumor stage and lymph node metastasis of TC patients in the recurrence group. The DNA methylation and rs2736100 polymorphisms in the promoter region of hTERT gene might be in correlation to postoperative recurrence of TC patients.

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.

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.

Efficacy and safety of rilpivirine in treatment-naive, HIV-1-infected patients with hepatitis B virus/hepatitis C virus coinfection enrolled in the Phase III randomized, double-blind ECHO and THRIVE trials

PubMed Central

Nelson, Mark; Amaya, Gerardo; Clumeck, Nathan; Arns da Cunha, Clovis; Jayaweera, Dushyantha; Junod, Patrice; Li, Taisheng; Tebas, Pablo; Stevens, Marita; Buelens, Annemie; Vanveggel, Simon; Boven, Katia

2012-01-01

Objectives The efficacy and hepatic safety of the non-nucleoside reverse transcriptase inhibitors rilpivirine (TMC278) and efavirenz were compared in treatment-naive, HIV-infected adults with concurrent hepatitis B virus (HBV) and/or hepatitis C virus (HCV) infection in the pooled week 48 analysis of the Phase III, double-blind, randomized ECHO (NCT00540449) and THRIVE (NCT00543725) trials. Methods Patients received 25 mg of rilpivirine once daily or 600 mg of efavirenz once daily, plus two nucleoside/nucleotide reverse transcriptase inhibitors. At screening, patients had alanine aminotransferase/aspartate aminotransferase levels ≤5× the upper limit of normal. HBV and HCV status was determined at baseline by HBV surface antigen, HCV antibody and HCV RNA testing. Results HBV/HCV coinfection status was known for 670 patients in the rilpivirine group and 665 in the efavirenz group. At baseline, 49 rilpivirine and 63 efavirenz patients [112/1335 (8.4%)] were coinfected with either HBV [55/1357 (4.1%)] or HCV [57/1333 (4.3%)]. The safety analysis included all available data, including beyond week 48. Eight patients seroconverted during the study (rilpivirine: five; efavirenz: three). A higher proportion of patients achieved viral load <50 copies/mL (intent to treat, time to loss of virological response) in the subgroup without HBV/HCV coinfection (rilpivirine: 85.0%; efavirenz: 82.6%) than in the coinfected subgroup (rilpivirine: 73.5%; efavirenz: 79.4%) (rilpivirine, P = 0.04 and efavirenz, P = 0.49, Fisher's exact test). The incidence of hepatic adverse events (AEs) was low in both groups in the overall population (rilpivirine: 5.5% versus efavirenz: 6.6%) and was higher in HBV/HCV-coinfected patients than in those not coinfected (26.7% versus 4.1%, respectively). Conclusions Hepatic AEs were more common and response rates lower in HBV/HCV-coinfected patients treated with rilpivirine or efavirenz than in those who were not coinfected. PMID:22532465

Impact of lopinavir-ritonavir exposure in HIV-1 infected children and adolescents in Madrid, Spain during 2000-2014.

PubMed

Rojas Sánchez, Patricia; Prieto, Luis; Jiménez De Ory, Santiago; Fernández Cooke, Elisa; Navarro, Maria Luisa; Ramos, José Tomas; Holguín, África

2017-01-01

The most-used protease-inhibitor in children is Lopinavir-ritonavir (LPV/r), which provides durable suppression of viral load and increases CD4+T-counts. This study describes the virological outcome of the HIV-1-infected paediatric population exposed to LPV/r during 15 years in Spain. Patients from the Madrid Cohort of HIV-1-infected-children and adolescents exposed to LPV/r as different line therapy during 2000-2014 were selected. The baseline epidemiological-clinical features, viral suppression, changes in CD4+T-CD8+T cell counts and drug susceptibility were recorded before and during LPV/r exposure. Drug resistance mutations (DRM) were identified in viruses from samples collected until 2011. We predicted drug susceptibility to 19 antiretrovirals among those carrying DRM using the Stanford's HIVdb Algorithm. A total of 199 (37.3%) of the 534 patients from the cohort were exposed to LPV/r during 2000-2014 in first (group 1), second (group 2) or more line-therapies (group 3). Patients were mainly Spaniards (81.9%), perinatally infected (96.5%) with subtype-B (65.3%) and HIV-diagnosed before year 2000 (67.8%). The mean age at first LPV/r exposure was 9.7 years. After protease-inhibitor exposure, viral suppression was higher in groups 1 and 2 than in group 3. Viral suppression occurred in 87.5%, 68.6% and 64.8% patients from groups 1, 2 and 3, respectively. Among the 64 patients with available resistance data during LPV/r treatment, 27(42.3%) carried DRM to protease-inhibitor, 28 (58.3%) to reverse-transcriptase-inhibitors and 21 (43.7%) to non-reverse-transcriptase-inhibitors. Darunavir/ritonavir, atazanavir-ritonavir and tipranavir/ritonavir presented the highest susceptibility and nelfinavir the lowest. A better lymphocyte recovering occurred when protease-inhibitor was taken as part of a first-line regimen and a higher number of patients reached viral suppression. The least compromised antiretrovirals for rescue antiretroviral regimens, according to DRM in the LPV/r-exposed-paediatric cohort, were mainly the new protease inhibitors.

HIV-1 Drug Resistance and Second-Line Treatment in Children Randomized to Switch at Low Versus Higher RNA Thresholds.

PubMed

Harrison, Linda; Melvin, Ann; Fiscus, Susan; Saidi, Yacine; Nastouli, Eleni; Harper, Lynda; Compagnucci, Alexandra; Babiker, Abdel; McKinney, Ross; Gibb, Diana; Tudor-Williams, Gareth

2015-09-01

The PENPACT-1 trial compared virologic thresholds to determine when to switch to second-line antiretroviral therapy (ART). Using PENPACT-1 data, we aimed to describe HIV-1 drug resistance accumulation on first-line ART by virologic threshold. PENPACT-1 had a 2 × 2 factorial design, randomizing HIV-infected children to start protease inhibitor (PI) versus nonnucleoside reverse transcriptase inhibitor (NNRTI)-based ART, and switch at a 1000 copies/mL versus 30,000 copies/mL threshold. Switch criteria were not achieving the threshold by week 24, confirmed rebound above the threshold thereafter, or Center for Disease Control and Prevention stage C event. Resistance tests were performed on samples ≥1000 copies/mL before switch, resuppression, and at 4-years/trial end. Sixty-seven children started PI-based ART and were randomized to switch at 1000 copies/mL (PI-1000), 64 PIs and 30,000 copies/mL (PI-30,000), 67 NNRTIs and 1000 copies/mL (NNRTI-1000), and 65 NNRTI and 30,000 copies/mL (NNRTI-30,000). Ninety-four (36%) children reached the 1000 copies/mL switch criteria during 5-year follow-up. In 30,000 copies/mL threshold arms, median time from 1000 to 30,000 copies/mL switch criteria was 58 (PI) versus 80 (NNRTI) weeks (P = 0.81). In NNRTI-30,000, more nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations accumulated than other groups. NNRTI mutations were selected before switching at 1000 copies/mL (23% NNRTI-1000, 27% NNRTI-30,000). Sixty-two children started abacavir + lamivudine, 166 lamivudine + zidovudine or stavudine, and 35 other NRTIs. The abacavir + lamivudine group acquired fewest NRTI mutations. Of 60 switched to second-line, 79% PI-1000, 63% PI-30,000, 64% NNRTI-1000, and 100% NNRTI-30,000 were <400 copies/mL 24 weeks later. Children on first-line NNRTI-based ART who were randomized to switch at a higher virologic threshold developed the most resistance, yet resuppressed on second-line. An abacavir + lamivudine NRTI combination seemed protective against development of NRTI resistance.

HIV/AIDS Medicines

MedlinePlus

... few years. But today, there are many effective medicines to fight the infection, and people with HIV ... healthier lives. There are five major types of medicines: Reverse transcriptase (RT) inhibitors - interfere with a critical ...

Novel Bifunctional Quinolonyl Diketo Acid Derivatives as HIV-1 Integrase Inhibitors: Design, Synthesis, Biological Activities and Mechanism of Action

PubMed Central

Di Santo, Roberto; Costi, Roberta; Roux, Alessandra; Artico, Marino; Lavecchia, Antonio; Marinelli, Luciana; Novellino, Ettore; Palmisano, Lucia; Andreotti, Mauro; Amici, Roberta; Galluzzo, Clementina Maria; Nencioni, Lucia; Palamara, Anna Teresa; Pommier, Yves; Marchand, Christophe

2008-01-01

The virally encoded integrase protein is an essential enzyme in the life cycle of the HIV-1 virus and represents an attractive and validated target in the development of therapeutics against HIV infection. Drugs that selectively inhibit this enzyme, when used in combination with inhibitors of reverse transcriptase and protease, are believed to be highly effective in suppressing the viral replication. Among the HIV-1 integrase inhibitors, the β-diketo acids (DKAs) represent a major lead for anti-HIV-1drug development. In this study, novel bifunctional quinolonyl diketo acid derivatives were designed, synthesized and tested for their inhibitory ability against HIV-1 integrase. The compounds are potent inhibitors of integrase activity. Particularly, derivative 8 is a potent IN inhibitor for both steps of the reaction (3′-processing and strand transfer) and exhibits both high antiviral activity against HIV-1 infected cells and low cytotoxicity. Molecular modeling studies provide a plausible mechanism of action, which is consistent with ligand SARs and enzyme photo-crosslinking experiments. PMID:16539381

An Activity Switch in Human Telomerase Based on RNA Conformation and Shaped by TCAB1.

PubMed

Chen, Lu; Roake, Caitlin M; Freund, Adam; Batista, Pedro J; Tian, Siqi; Yin, Yi A; Gajera, Chandresh R; Lin, Shengda; Lee, Byron; Pech, Matthew F; Venteicher, Andrew S; Das, Rhiju; Chang, Howard Y; Artandi, Steven E

2018-05-18

Ribonucleoprotein enzymes require dynamic conformations of their RNA constituents for regulated catalysis. Human telomerase employs a non-coding RNA (hTR) with a bipartite arrangement of domains-a template-containing core and a distal three-way junction (CR4/5) that stimulates catalysis through unknown means. Here, we show that telomerase activity unexpectedly depends upon the holoenzyme protein TCAB1, which in turn controls conformation of CR4/5. Cells lacking TCAB1 exhibit a marked reduction in telomerase catalysis without affecting enzyme assembly. Instead, TCAB1 inactivation causes unfolding of CR4/5 helices that are required for catalysis and for association with the telomerase reverse-transcriptase (TERT). CR4/5 mutations derived from patients with telomere biology disorders provoke defects in catalysis and TERT binding similar to TCAB1 inactivation. These findings reveal a conformational "activity switch" in human telomerase RNA controlling catalysis and TERT engagement. The identification of two discrete catalytic states for telomerase suggests an intramolecular means for controlling telomerase in cancers and progenitor cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Modeling of TREX1-Dependent Autoimmune Disease using Human Stem Cells Highlights L1 Accumulation as a Source of Neuroinflammation.

PubMed

Thomas, Charles A; Tejwani, Leon; Trujillo, Cleber A; Negraes, Priscilla D; Herai, Roberto H; Mesci, Pinar; Macia, Angela; Crow, Yanick J; Muotri, Alysson R

2017-09-07

Three-prime repair exonuclease 1 (TREX1) is an anti-viral enzyme that cleaves nucleic acids in the cytosol, preventing accumulation and a subsequent type I interferon-associated inflammatory response. Autoimmune diseases, including Aicardi-Goutières syndrome (AGS) and systemic lupus erythematosus, can arise when TREX1 function is compromised. AGS is a neuroinflammatory disorder with severe and persistent intellectual and physical problems. Here we generated a human AGS model that recapitulates disease-relevant phenotypes using pluripotent stem cells lacking TREX1. We observed abundant extrachromosomal DNA in TREX1-deficient neural cells, of which endogenous Long Interspersed Element-1 retrotransposons were a major source. TREX1-deficient neurons also exhibited increased apoptosis and formed three-dimensional cortical organoids of reduced size. TREX1-deficient astrocytes further contributed to the observed neurotoxicity through increased type I interferon secretion. In this model, reverse-transcriptase inhibitors rescued the neurotoxicity of AGS neurons and organoids, highlighting their potential utility in therapeutic regimens for AGS and related disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

Oligomerization transforms human APOBEC3G from an efficient enzyme to a slowly dissociating nucleic acid-binding protein

NASA Astrophysics Data System (ADS)

Chaurasiya, Kathy R.; McCauley, Micah J.; Wang, Wei; Qualley, Dominic F.; Wu, Tiyun; Kitamura, Shingo; Geertsema, Hylkje; Chan, Denise S. B.; Hertz, Amber; Iwatani, Yasumasa; Levin, Judith G.; Musier-Forsyth, Karin; Rouzina, Ioulia; Williams, Mark C.

2014-01-01

The human APOBEC3 proteins are a family of DNA-editing enzymes that play an important role in the innate immune response against retroviruses and retrotransposons. APOBEC3G is a member of this family that inhibits HIV-1 replication in the absence of the viral infectivity factor Vif. Inhibition of HIV replication occurs by both deamination of viral single-stranded DNA and a deamination-independent mechanism. Efficient deamination requires rapid binding to and dissociation from ssDNA. However, a relatively slow dissociation rate is required for the proposed deaminase-independent roadblock mechanism in which APOBEC3G binds the viral template strand and blocks reverse transcriptase-catalysed DNA elongation. Here, we show that APOBEC3G initially binds ssDNA with rapid on-off rates and subsequently converts to a slowly dissociating mode. In contrast, an oligomerization-deficient APOBEC3G mutant did not exhibit a slow off rate. We propose that catalytically active monomers or dimers slowly oligomerize on the viral genome and inhibit reverse transcription.

Tumor-specific expression of shVEGF and suicide gene as a novel strategy for esophageal cancer therapy.

PubMed

Liu, Ting; Wu, Hai-Jun; Liang, Yu; Liang, Xu-Jun; Huang, Hui-Chao; Zhao, Yan-Zhong; Liao, Qing-Chuan; Chen, Ya-Qi; Leng, Ai-Min; Yuan, Wei-Jian; Zhang, Gui-Ying; Peng, Jie; Chen, Yong-Heng

2016-06-21

To develop a potent and safe gene therapy for esophageal cancer. An expression vector carrying fusion suicide gene (yCDglyTK) and shRNA against vascular endothelial growth factor (VEGF) was constructed and delivered into EC9706 esophageal cancer cells by calcium phosphate nanoparticles (CPNP). To achieve tumor selectivity, expression of the fusion suicide gene was driven by a tumor-specific human telomerase reverse transcriptase (hTERT) promoter. The biologic properties and therapeutic efficiency of the vector, in the presence of prodrug 5-fluorocytosine (5-FC), were evaluated in vitro and in vivo. Both in vitro and in vivo testing showed that the expression vector was efficiently introduced by CPNP into tumor cells, leading to cellular expression of yCDglyTK and decreased VEGF level. With exposure to 5-FC, it exhibited strong anti-tumor effects against esophageal cancer. Combination of VEGF shRNA with the fusion suicide gene demonstrated strong anti-tumor activity. The shVEGF-hTERT-yCDglyTK/5-FC system provided a novel approach for esophageal cancer-targeted gene therapy.

A new antifungal protein and a chitinase with prominent macrophage-stimulating activity from seeds of Phaseolus vulgaris cv. pinto.

PubMed

Ye, X Y; Ng, T B

2002-01-18

From the seeds of the pinto bean (Phaseolus vulgaris cv. pinto), a chitinase and a novel antifungal protein, both with the ability of markedly augmenting nitrite production by murine peritoneal macrophages, were isolated. The antifungal proteins, designated phasein A and phasein B, exhibited molecular weights of 28 and 32 kDa, respectively. Phaseins A and B were adsorbed on Affi-gel blue gel and CM-Sepharose and were eluted as adjacent peaks from CM-Sepharose. Phasein A demonstrated potent antifungal activity toward Fusarium oxysporum and Physalospora piricola. Phasein B was more potent than phasein A toward P. piricola but less potent than phasein A toward F. oxysporum and Rhizoctonia solani. Both antifungal proteins inhibited the activity of HIV-1 reverse transcriptase and translation in a rabbit reticulocyte lysate system, with phasein B being more potent. Nitrite production by mouse macrophages was greatly boosted in the presence of both phaseins A and B, although the effect of phasein A was more prominent. The bioactivities of phaseins were in general potent compared with those of other antifungal proteins.

Inactivation of CUG-BP1/CELF1 causes growth, viability, and spermatogenesis defects in mice.

PubMed

Kress, Chantal; Gautier-Courteille, Carole; Osborne, H Beverley; Babinet, Charles; Paillard, Luc

2007-02-01

CUG-BP1/CELF1 is a multifunctional RNA-binding protein involved in the regulation of alternative splicing and translation. To elucidate its role in mammalian development, we produced mice in which the Cugbp1 gene was inactivated by homologous recombination. These Cugbp1(-/-) mice were viable, although a significant portion of them did not survive after the first few days of life. They displayed growth retardation, and most Cugbp1(-/-) males and females exhibited impaired fertility. Male infertility was more thoroughly investigated. Histological examination of testes from Cugbp1(-/-) males showed an arrest of spermatogenesis that occurred at step 7 of spermiogenesis, before spermatid elongation begins, and an increased apoptosis. A quantitative reverse transcriptase PCR analysis showed a decrease of all the germ cell markers tested but not of Sertoli and Leydig markers, suggesting a general decrease in germ cell number. In wild-type testes, CUG-BP1 is expressed in germ cells from spermatogonia to round spermatids and also in Sertoli and Leydig cells. These findings demonstrate that CUG-BP1 is required for completion of spermatogenesis.

Differences in resistance mutations among HIV-1 non-subtype B infections: a systematic review of evidence (1996–2008)

PubMed Central

2009-01-01

Ninety percent of HIV-1-infected people worldwide harbour non-subtype B variants of HIV-1. Yet knowledge of resistance mutations in non-B HIV-1 and their clinical relevance is limited. Although a few reviews, editorials and perspectives have been published alluding to this lack of data among non-B subtypes, no systematic review has been performed to date. With this in mind, we conducted a systematic review (1996–2008) of all published studies performed on the basis of non-subtype B HIV-1 infections treated with antiretroviral drugs that reported genotype resistance tests. Using an established search string, 50 studies were deemed relevant for this review. These studies reported genotyping data from non-B HIV-1 infections that had been treated with either reverse transcriptase inhibitors or protease inhibitors. While most major resistance mutations in subtype B were also found in non-B subtypes, a few novel mutations in non-B subtypes were recognized. The main differences are reflected in the discoveries that: (i) the non-nucleoside reverse transcriptase inhibitor resistance mutation, V106M, has been seen in subtype C and CRF01_AE, but not in subtype B, (ii) the protease inhibitor mutations L89I/V have been reported in C, F and G subtypes, but not in B, (iii) a nelfinavir selected non-D30N containing pathway predominated in CRF01_AE and CRF02_AG, while the emergence of D30N is favoured in subtypes B and D, (iv) studies on thymidine analog-treated subtype C infections from South Africa, Botswana and Malawi have reported a higher frequency of the K65R resistance mutation than that typically seen with subtype B. Additionally, some substitutions that seem to impact non-B viruses differentially are: reverse transcriptase mutations G196E, A98G/S, and V75M; and protease mutations M89I/V and I93L. Polymorphisms that were common in non-B subtypes and that may contribute to resistance tended to persist or become more frequent after drug exposure. Some, but not all, are recognized as minor resistance mutations in B subtypes. These observed differences in resistance pathways may impact cross-resistance and the selection of second-line regimens with protease inhibitors. Attention to newer drug combinations, as well as baseline genotyping of non-B isolates, in well-designed longitudinal studies with long duration of follow up are needed. PMID:19566959

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.

Molecular Diagnostics in Colorectal Carcinoma: Advances and Applications for 2018.

PubMed

Bhalla, Amarpreet; Zulfiqar, Muhammad; Bluth, Martin H

2018-06-01

The molecular pathogenesis and classification of colorectal carcinoma are based on the traditional adenomaecarcinoma sequence, serrated polyp pathway, and microsatellite instability (MSI). The genetic basis for hereditary nonpolyposis colorectal cancer is the detection of mutations in the MLH1, MSH2, MSH6, PMS2, and EPCAM genes. Genetic testing for Lynch syndrome includes MSI testing, methylator phenotype testing, BRAF mutation testing, and molecular testing for germline mutations in MMR genes. Molecular makers with predictive and prognostic implications include quantitative multigene reverse transcriptase polymerase chain reaction assay and KRAS and BRAF mutation analysis. Mismatch repair-deficient tumors have higher rates of programmed death-ligand 1 expression. Cell-free DNA analysis in fluids are proving beneficial for diagnosis and prognosis in these disease states towards effective patient management. Copyright © 2018 Elsevier Inc. All rights reserved.

Initiating therapy: when to start, what to use.

PubMed

Hirsch, Martin S

2008-05-15

Decisions regarding whether to start combination antiretroviral therapy (cART) during primary infection and when to initiate treatment during chronic infection continue to evolve. Although current data suggest that there may be a benefit to therapy during primary infection, results are inconclusive. Once begun, treatment probably should be continued indefinitely, since its potential advantages disappear over time if treatment is stopped. Recent studies suggest that cART may be useful at higher CD4 cell count thresholds than are currently recommended in several guidelines. Several regimens are acceptable as initial therapy, with tenofovir/emtricitabine/efavirenz favored by many because of potency and ease of administration. Other favored regimens include combinations of 2 nucleoside (or nucleotide) reverse-transcriptase inhibitors and a ritonavir-boosted protease inhibitor. Some new antiretroviral drugs under study, particularly integrase inhibitors, may prove useful in treatment-naive patients.

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

PubMed

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

2002-11-01

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

Extraordinary Effects of Specific Monovalent Cations on Activation of Reovirus Transcriptase by Chymotrypsin In Vitro

PubMed Central

Borsa, J.; Sargent, M. D.; Long, D. G.; Chapman, J. D.

1973-01-01

Activation of reovirus transcriptase activity, latent in intact virions, by digestion of purified virions with chymotrypsin (CHT) in vitro shows a stringent requirement for specific monovalent cations. Cs+, Rb+, or K+ ions are capable of facilitating activation by chymotryptic digestion. Na+, Li+, or NH4+ ions are not capable of facilitating the CHT activation of polymerase activity and are antagonistic towards the effects of the facilitating ions. The data indicate that the effect of the cations is exerted on activation of the polymerase activity by CHT as opposed to an effect on polymerization per se. This effect may be important biologically in that it provides a mechanism whereby the virion can sense whether it is in an intracellular or an extracellular environment and thereby can avoid premature uncoating. PMID:4347424

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

Complete inactivation of HIV-1 using photo-labeled non-nucleoside reverse transcriptase inhibitors.

PubMed

Rios, Adan; Quesada, Jorge; Anderson, Dallas; Goldstein, Allan; Fossum, Theresa; Colby-Germinario, Susan; Wainberg, Mark A

2011-01-01

We demonstrate that a photo-labeled derivative of the non-nucleoside reverse transcriptase inhibitor (NNRTI) dapivirine termed DAPY, when used together with exposure to ultraviolet light, was able to completely and irreversibly inactivate both HIV-1 RT activity as well as infectiousness in each of a T cell line and peripheral blood mononuclear cells. Control experiments using various concentrations of DAPY revealed that a combination of exposure to ultraviolet light together with use of the specific, high affinity photo-labeled compound was necessary for complete inactivation to occur. This method of HIV RT inactivation may have applicability toward preservation of an intact viral structure and warrants further investigation in regard to the potential of this approach to elicit a durable, broad protective immune response. Copyright © 2010 Elsevier B.V. All rights reserved.

Etravirine and Rilpivirine Drug Resistance Among HIV-1 Subtype C Infected Children Failing Non-Nucleoside Reverse Transcriptase Inhibitor-Based Regimens in South India.

PubMed

Saravanan, Shanmugam; Kausalya, Bagavathi; Gomathi, Selvamurthi; Sivamalar, Sathasivam; Pachamuthu, Balakrishnan; Selvamuthu, Poongulali; Pradeep, Amrose; Sunil, Solomon; Mothi, Sarvode N; Smith, Davey M; Kantor, Rami

2017-06-01

We have analyzed reverse transcriptase (RT) region of HIV-1 pol gene from 97 HIV-infected children who were identified as failing first-line therapy that included first-generation non-nucleoside RT inhibitors (Nevirapine and Efavirenz) for at least 6 months. We found that 54% and 65% of the children had genotypically predicted resistance to second-generation non-nucleoside RT inhibitors drugs Etravirine (ETR) and Rilpivirine, respectively. These cross-resistance mutations may compromise future NNRTI-based regimens, especially in resource-limited settings. To complement these investigations, we also analyzed the sequences in Stanford database, Monogram weighted score, and DUET weighted score algorithms for ETR susceptibility and found almost perfect agreement between the three algorithms in predicting ETR susceptibility from genotypic data.

Structural optimization of N1-aryl-benzimidazoles for the discovery of new non-nucleoside reverse transcriptase inhibitors active against wild-type and mutant HIV-1 strains.

PubMed

Monforte, Anna Maria; De Luca, Laura; Buemi, Maria Rosa; Agharbaoui, Fatima E; Pannecouque, Christophe; Ferro, Stefania

2018-02-01

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are recommended components of preferred combination antiretroviral therapies used for the treatment of human immunodeficiency virus (HIV) infection. These regimens are extremely effective in suppressing virus replication. Recently, our research group identified some N 1 -aryl-2-arylthioacetamido-benzimidazoles as a novel class of NNRTIs. In this research work we report the design, the synthesis and the structure-activity relationship studies of new compounds (20-34) in which some structural modifications have been introduced in order to investigate their effects on reverse transcriptase (RT) inhibition and to better define the features needed to increase the antiviral activity. Most of the new compounds proved to be highly effective in inhibiting both RT enzyme at nanomolar concentrations and HIV-1 replication in MT4 cells with minimal cytotoxicity. Among them, the most promising N 1 -aryl-2-arylthioacetamido-benzimidazoles and N 1 -aryl-2-aryloxyacetamido-benzimidazoles were also tested toward a panel of single- and double-mutants strain responsible for resistance to NNRTIs, showing in vitro antiviral activity toward single mutants L100I, K103N, Y181C, Y188L and E138K. The best results were observed for derivatives 29 and 33 active also against the double mutants F227L and V106A. Computational approaches were applied in order to rationalize the potency of the new synthesized inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synthesis and evaluation of "AZT-HEPT", "AZT-pyridinone", and "ddC-HEPT" conjugates as inhibitors of HIV reverse transcriptase.

PubMed

Pontikis, R; Dollé, V; Guillaumel, J; Dechaux, E; Note, R; Nguyen, C H; Legraverend, M; Bisagni, E; Aubertin, A M; Grierson, D S; Monneret, C

2000-05-18

To test the concept that HIV reverse transcriptase could be effectively inhibited by "mixed site inhibitors", a series of seven conjugates containing both a nucleoside analogue component (AZT 1, ddC 2) and a nonnucleoside type inhibitor (HEPT analogue 12, pyridinone 27) were synthesized and evaluated for their ability to block HIV replication. The (N-3 and C-5)AZT-HEPT conjugates 15, 22, and 23 displayed 2-5 microM anti-HIV activity, but they had no effect on the replication of HIV-2 or the HIV-1 strain with the Y181C mutation. The (C-5)AZT-pyridinone conjugates 34-37 were found to be inactive. In marked contrast, the ddC-HEPT molecule 26 displayed the same potency (EC(50) = 0.45 microM) against HIV-1 (wild type and the Y181C nevirapine-resistant strain) and HIV-2 in cell culture. No synergistic effect was observed for these bis-substrate inhibitors, suggesting that the two individual inhibitor components in these molecules do not bind simultaneously in their respective sites. Interestingly, however, the results indicate that the AZT-HEPT conjugates and the ddC-HEPT derivative 26 inhibit reverse transcriptase (RT) in an opposite manner. One explanation for this difference is that the former compounds interact preferentially with the hydrophobic pocket in RT, whereas 26 (after supposed triphosphorylation) inhibits RT through binding in the catalytic site.

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

PubMed

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

2012-01-01

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

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.

The baculovirus-integrated retrotransposon TED encodes gag and pol proteins that assemble into viruslike particles with reverse transcriptase.

PubMed Central

Lerch, R A; Friesen, P D

1992-01-01

TED is a lepidopteran retrotransposon found inserted within the DNA genome of the Autographa californica nuclear polyhedrosis virus mutant, FP-D. To examine the proteins and functions encoded by this representative of the gypsy family of retrotransposons, the gag- and pol-like open reading frames (ORFs 1 and 2) were expressed in homologous lepidopteran cells by using recombinant baculovirus vectors. Expression of ORF 1 resulted in synthesis of an abundant TED-specific protein (Pr55gag) that assembled into viruslike particles with a diameter of 55 to 60 nm. Expression of ORF 2, requiring a -1 translational frameshift, resulted in synthesis of a protease that mediated cleavage of Pr55gag to generate p37, the major protein component of the resulting particles. Expression of ORF 2 also produced reverse transcriptase that associated with these particles. Both protease and reverse transcriptase activities mapped to domains within ORF 2 that contain sequence similarities with the corresponding functional domains of the pol gene of the vertebrate retroviruses. These results indicated that TED ORFs 1 and 2 functionally resemble the retrovirus gag and pol genes and demonstrated for the first time that an invertebrate member of the gypsy family of elements encodes active forms of the structural and enzymatic functions necessary for transposition via an RNA intermediate. TED integration within the baculovirus genome thus represents one of the first examples of transposon-mediated transfer of host-derived genes to an eukaryotic virus. Images PMID:1371168

In Vitro Resistance Profile of the Candidate HIV-1 Microbicide Drug Dapivirine

PubMed Central

Schader, Susan M.; Oliveira, Maureen; Ibanescu, Ruxandra-Ilinca; Moisi, Daniela; Colby-Germinario, Susan P.

2012-01-01

Antiretroviral-based microbicides may offer a means to reduce the sexual transmission of HIV-1. Suboptimal use of a microbicide may, however, lead to the development of drug resistance in users that are already, or become, infected with HIV-1. In such cases, the efficacy of treatments may be compromised since the same (or similar) antiretrovirals used in treatments are being developed as microbicides. To help predict which drug resistance mutations may develop in the context of suboptimal use, HIV-1 primary isolates of different subtypes and different baseline resistance profiles were used to infect primary cells in vitro in the presence of increasing suboptimal concentrations of the two candidate microbicide antiretrovirals dapivirine (DAP) and tenofovir (TFV) alone or in combination. Infections were ongoing for 25 weeks, after which reverse transcriptase genotypes were determined and scrutinized for the presence of any clinically recognized reverse transcriptase drug resistance mutations. Results indicated that suboptimal concentrations of DAP alone facilitated the emergence of common nonnucleoside reverse transcriptase inhibitor resistance mutations, while suboptimal concentrations of DAP plus TFV gave rise to fewer mutations. Suboptimal concentrations of TFV alone did not frequently result in the development of resistance mutations. Sensitivity evaluations for stavudine (d4T), nevirapine (NVP), and lamivudine (3TC) revealed that the selection of resistance as a consequence of suboptimal concentrations of DAP may compromise the potential for NVP to be used in treatment, a finding of potential relevance in developing countries. PMID:22123692

In vitro resistance profile of the candidate HIV-1 microbicide drug dapivirine.

PubMed

Schader, Susan M; Oliveira, Maureen; Ibanescu, Ruxandra-Ilinca; Moisi, Daniela; Colby-Germinario, Susan P; Wainberg, Mark A

2012-02-01

Antiretroviral-based microbicides may offer a means to reduce the sexual transmission of HIV-1. Suboptimal use of a microbicide may, however, lead to the development of drug resistance in users that are already, or become, infected with HIV-1. In such cases, the efficacy of treatments may be compromised since the same (or similar) antiretrovirals used in treatments are being developed as microbicides. To help predict which drug resistance mutations may develop in the context of suboptimal use, HIV-1 primary isolates of different subtypes and different baseline resistance profiles were used to infect primary cells in vitro in the presence of increasing suboptimal concentrations of the two candidate microbicide antiretrovirals dapivirine (DAP) and tenofovir (TFV) alone or in combination. Infections were ongoing for 25 weeks, after which reverse transcriptase genotypes were determined and scrutinized for the presence of any clinically recognized reverse transcriptase drug resistance mutations. Results indicated that suboptimal concentrations of DAP alone facilitated the emergence of common nonnucleoside reverse transcriptase inhibitor resistance mutations, while suboptimal concentrations of DAP plus TFV gave rise to fewer mutations. Suboptimal concentrations of TFV alone did not frequently result in the development of resistance mutations. Sensitivity evaluations for stavudine (d4T), nevirapine (NVP), and lamivudine (3TC) revealed that the selection of resistance as a consequence of suboptimal concentrations of DAP may compromise the potential for NVP to be used in treatment, a finding of potential relevance in developing countries.

Prevalence and Mutation Patterns of HIV Drug Resistance from 2010 to 2011 among ART-Failure Individuals in the Yunnan Province, China

PubMed Central

Guo, Wei; Zhuang, Daomin; Li, Lin; Liu, Yongjian; Bao, Zuoyi; Liu, Siyang; Wang, Xiaolin; Li, Tianyi; Yang, Shaomin; Li, Jingyun

2013-01-01

Background Assessing the prevalence of HIV-1 drug-resistance and the mutation patterns associated with resistance in the geographical regions implementing free antiretroviral therapy (ART) in China is necessary for preventing the spread of resistant strains and designing the regimens for the subsequent therapies with limited resources. Methods Plasma samples in different cities/prefectures were collected at Yunnan Provincial Hospital of Infectious Disease from January 2010 to December 2011. Genotyping of drug-resistant individuals was conducted using an in-house assay on plasma samples. Viral load, CD4 T cell counts and demographic data were obtained from medical records and an administered questionnaire. Results A total of 609 pol sequences (515 ART-failure and 94 therapy-naïve individuals) derived from 664 samples were obtained. The prevalence of drug-resistance was 45.1% in the ART-failure individuals. Of these, 26.8% harbored HIV strains dually resistant to nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors, and 14.8% harbored HIV strains resistant to only one drug category. Mutations such as M184V/I, K103N, V106A, Y181C and G190A were common among the ART-failure individuals, and the frequencies of M184V/I, K103N and V106A were 28.2%, 19.2%, and 22.1%, respectively. The percentages of individuals exhibiting intermediate or high-level resistance to 3TC, FTC, EFV and NVP drugs were 28.4%, 28.2%, 37.3%, and 37.5%, respectively. Factors such as ethnicity, transmission route, CD4 counts, viral load and the duration of ART were significantly correlated with development of drug resistance in the ART-failure individuals. Conclusions The high prevalence of HIV drug-resistance observed among the ART-failure individuals from 2010 to 2011 in Yunnan province should be of increasing concern in regions where the implementation of ART is widespread. Education about the risk factors associated with HIV drug resistance is important for preventing and controlling the spread of HIV drug-resistant strains. PMID:24009694

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

PubMed

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

2014-09-01

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

Pathological and immunological responses associated with differential survival of Chinook salmon following Renibacterium salmoninarum challenge

USGS Publications Warehouse

Metzger, David C.; Elliott, Diane G.; Wargo, Andrew; Park, Linda K.; Purcell, Maureen K.

2010-01-01

Chinook salmon Oncorhynchus tshawytscha are highly susceptible to Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD). Previously we demonstrated that introduced Chinook salmon from Lake Michigan, Wisconsin (WI), USA, have higher survival following R. salmoninarum challenge relative to the progenitor stock from Green River, Washington, USA. In the present study, we investigated the pathological and immunological responses that are associated with differential survival in the 2 Chinook salmon stocks following intra-peritoneal R. salmoninarum challenge of 2 different cohort years (2003 and 2005). Histological evaluation revealed delayed appearance of severe granulomatous lesions in the kidney and lower overall prevalence of membranous glomerulopathy in the higher surviving WI stock. The higher survival WI stock had a lower bacterial load at 28 d post-infection, as measured by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). However, at all other time points, bacterial load levels were similar despite higher mortality in the more susceptible Green River stock, suggesting the possibility that the stocks may differ in their tolerance to infection by the bacterium. Interferon-γ, inducible nitric oxide synthase (iNOS), Mx-1, and transferrin gene expression were up-regulated in both stocks following challenge. A trend of higher iNOS gene expression at later time points (≥28 d post-infection) was observed in the lower surviving Green River stock, suggesting the possibility that higher iNOS expression may contribute to greater pathology in that stock.

Accumulation of catechins in tea in relation to accumulation of mRNA from genes involved in catechin biosynthesis.

PubMed

Eungwanichayapant, P D; Popluechai, S

2009-02-01

Catechins are a group of polyphenols found in tea (Camellia sinensis var. sinensis) at high levels. They are beneficial for health. From the study on accumulation of catechins in shoots and mature leaves of a tea cultivar, Oolong No. 17, using high-performance liquid chromatography (HPLC), it was found that the amounts of most catechins in the shoots were higher than those in the mature leaves, with an exception of catechins gallate (CG) that was found in trace amounts in both the shoots and mature leaves. mRNA accumulation of genes involved in catechin synthesis was studied using reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that the mRNA accumulation of the genes were higher in the shoots than in the mature leaves. These genes included genes of phenylalanine ammonia-lyase 1 (PAL1; EC 4.3.1.5), chalcone synthase (CHS; EC 2.3.1.74), dihydroflavonol 4-reductase (DFR; EC 1.1.1.219), leucoanthocyanidin reductase (LCR; EC 1.17.1.3), and flavanone 3-hydroxylase (F3H; EC 1.14.11.9).

Bacterial Group II Introns: Identification and Mobility Assay.

PubMed

Toro, Nicolás; Molina-Sánchez, María Dolores; Nisa-Martínez, Rafael; Martínez-Abarca, Francisco; García-Rodríguez, Fernando Manuel

2016-01-01

Group II introns are large catalytic RNAs and mobile retroelements that encode a reverse transcriptase. Here, we provide methods for their identification in bacterial genomes and further analysis of their splicing and mobility capacities.

Mitochondrial DNA replication, nucleoside reverse-transcriptase inhibitors, and AIDS cardiomyopathy.

PubMed

Lewis, William

2003-01-01

Nucleoside reverse-transcriptase inhibitors (NRTIs) in combination with other antiretrovirals (HAART) are the cornerstones of current AIDS therapy, but extensive use brought mitochondrial side effects to light. Clinical experience, pharmacological, cell, and molecular biological evidence links altered mitochondrial (mt-) DNA replication to the toxicity of NRTIs in many tissues, and conversely, mtDNA replication defects and mtDNA depletion in target tissues are observed. Organ-specific pathological changes or diverse systemic effects result from and are frequently attributed to HAART in which NRTIs are included. The shared features of mtDNA depletion and energy depletion became key observations and related the clinical and in vivo experimental findings to inhibition of mtDNA replication by NRTI triphosphates in vitro. Subsequent to those findings, other observations suggested that mitochondrial energy deprivation is concomitant with or the result of mitochondrial oxidative stress in AIDS (from HIV, for example) or from NRTI therapy itself. Copyright 2003, Elsevier Science (USA)

Towards novel therapeutics for HIV through fragment-based screening and drug design.

PubMed

Tiefendbrunn, Theresa; Stout, C David

2014-01-01

Fragment-based drug discovery has been applied with varying levels of success to a number of proteins involved in the HIV (Human Immunodeficiency Virus) life cycle. Fragment-based approaches have led to the discovery of novel binding sites within protease, reverse transcriptase, integrase, and gp41. Novel compounds that bind to known pockets within CCR5 have also been identified via fragment screening, and a fragment-based approach to target the TAR-Tat interaction was explored. In the context of HIV-1 reverse transcriptase (RT), fragment-based approaches have yielded fragment hits with mid-μM activity in an in vitro activity assay, as well as fragment hits that are active against drug-resistant variants of RT. Fragment-based drug discovery is a powerful method to elucidate novel binding sites within proteins, and the method has had significant success in the context of HIV proteins.

Susceptibility of recombinant porcine endogenous retrovirus reverse transcriptase to nucleoside and non-nucleoside inhibitors.

PubMed

Wilhelm, M; Fishman, J A; Pontikis, R; Aubertin, A M; Wilhelm, F X

2002-12-01

Transplantation of organs, tissues or cells from pigs to humans could be a potential solution to the shortage of human organs for transplantation. Porcine endogenous retroviruses (PERVs) remain a major safety concern for porcine xenotransplantation. Thus, finding drugs that could be used as virological prophylaxis (or therapy) against PERV replication would be desirable. One of the most effective ways to block retroviral multiplication is to inhibit the enzyme reverse transcriptase (RT) which catalyzes the reverse transcription of viral RNA to proviral double-stranded DNA. We report here the cloning and expression of PERV RT and its susceptibility to several inhibitors. Our data demonstrate PERV susceptibility in vitro to the triphosphorylated nucleoside analog of zidovudine (AZT) and to ddGTP and to a lesser extent to ddTTP but almost no susceptibility to the non-nucleoside RT inhibitors tested.

Structure of a group II intron in complex with its reverse transcriptase.

PubMed

Qu, Guosheng; Kaushal, Prem Singh; Wang, Jia; Shigematsu, Hideki; Piazza, Carol Lyn; Agrawal, Rajendra Kumar; Belfort, Marlene; Wang, Hong-Wei

2016-06-01

Bacterial group II introns are large catalytic RNAs related to nuclear spliceosomal introns and eukaryotic retrotransposons. They self-splice, yielding mature RNA, and integrate into DNA as retroelements. A fully active group II intron forms a ribonucleoprotein complex comprising the intron ribozyme and an intron-encoded protein that performs multiple activities including reverse transcription, in which intron RNA is copied into the DNA target. Here we report cryo-EM structures of an endogenously spliced Lactococcus lactis group IIA intron in its ribonucleoprotein complex form at 3.8-Å resolution and in its protein-depleted form at 4.5-Å resolution, revealing functional coordination of the intron RNA with the protein. Remarkably, the protein structure reveals a close relationship between the reverse transcriptase catalytic domain and telomerase, whereas the active splicing center resembles the spliceosomal Prp8 protein. These extraordinary similarities hint at intricate ancestral relationships and provide new insights into splicing and retromobility.

Fragment Screening and HIV Therapeutics

PubMed Central

Bauman, Joseph D.; Patel, Disha; Arnold, Eddy

2013-01-01

Fragment screening has proven to be a powerful alternative to traditional methods for drug discovery. Biophysical methods, such as X-ray crystallography, NMR spectroscopy, and surface plasmon resonance, are used to screen a diverse library of small molecule compounds. Although compounds identified via this approach have relatively weak affinity, they provide a good platform for lead development and are highly efficient binders with respect to their size. Fragment screening has been utilized for a wide-range of targets, including HIV-1 proteins. Here, we review the fragment screening studies targeting HIV-1 proteins using X-ray crystallography or surface plasmon resonance. These studies have successfully detected binding of novel fragments to either previously established or new sites on HIV-1 protease and reverse transcriptase. In addition, fragment screening against HIV-1 reverse transcriptase has been used as a tool to better understand the complex nature of ligand binding to a flexible target. PMID:21972022

Direct CRISPR spacer acquisition from RNA by a natural reverse-transcriptase-Cas1 fusion protein

PubMed Central

Sidote, David J.; Markham, Laura M.; Sanchez-Amat, Antonio; Bhaya, Devaki; Lambowitz, Alan M.; Fire, Andrew Z.

2016-01-01

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in Type I and II CRISPR systems by the acquisition of short segments of DNA (“spacers”) from invasive elements. In several Type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we show that an RT-Cas1 fusion enables the acquisition of RNA spacers in vivo in an RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze ligation of RNA segments into the CRISPR array, followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA. PMID:26917774

An Intravaginal Ring That Releases the NNRTI MIV-150 Reduces SHIV Transmission in Macaques

PubMed Central

Rodriguez, Aixa; Kizima, Larisa; Menon, Radhika; Goldman, Daniel; Kenney, Jessica; Aravantinou, Meropi; Seidor, Samantha; Gettie, Agegnehu; Blanchard, James; Piatak, Michael; Lifson, Jeffrey D.; Fernández-Romero, José A.; Robbiani, Melissa; Zydowsky, Thomas M.

2015-01-01

Microbicides may prevent HIV and sexually transmitted infections (STIs) in women; however, determining the optimal means of delivery of active pharmaceutical ingredients remains a major challenge. We previously demonstrated that a vaginal gel containing the non-nucleoside reverse transcriptase inhibitor MIV-150 partially protected macaques from SHIV-RT (simian/HIV reverse transcriptase) infection, and the addition of zinc acetate rendered the gel significantly protective. We test the activity of MIV-150 without the addition of zinc acetate when delivered from either ethylene vinyl acetate (EVA) or silicone intravaginal rings (IVRs). MIV-150 was successfully delivered, because it was detected in vaginal fluids and tissues by radioimmunoassay in pharmacokinetic studies. Moreover, EVA IVRs significantly protected macaques from SHIV-RT infection. Our results demonstrate that MIV-150–containing IVRs have the potential to prevent HIV infection and highlight the possible use of IVRs for delivering drugs that block HIV and other STIs. PMID:22956201

An intravaginal ring that releases the NNRTI MIV-150 reduces SHIV transmission in macaques.

PubMed

Singer, Rachel; Mawson, Paul; Derby, Nina; Rodriguez, Aixa; Kizima, Larisa; Menon, Radhika; Goldman, Daniel; Kenney, Jessica; Aravantinou, Meropi; Seidor, Samantha; Gettie, Agegnehu; Blanchard, James; Piatak, Michael; Lifson, Jeffrey D; Fernández-Romero, José A; Robbiani, Melissa; Zydowsky, Thomas M

2012-09-05

Microbicides may prevent HIV and sexually transmitted infections (STIs) in women; however, determining the optimal means of delivery of active pharmaceutical ingredients remains a major challenge. We previously demonstrated that a vaginal gel containing the non-nucleoside reverse transcriptase inhibitor MIV-150 partially protected macaques from SHIV-RT (simian/HIV reverse transcriptase) infection, and the addition of zinc acetate rendered the gel significantly protective. We test the activity of MIV-150 without the addition of zinc acetate when delivered from either ethylene vinyl acetate (EVA) or silicone intravaginal rings (IVRs). MIV-150 was successfully delivered, because it was detected in vaginal fluids and tissues by radioimmunoassay in pharmacokinetic studies. Moreover, EVA IVRs significantly protected macaques from SHIV-RT infection. Our results demonstrate that MIV-150-containing IVRs have the potential to prevent HIV infection and highlight the possible use of IVRs for delivering drugs that block HIV and other STIs.

Practical diagnostic testing for human immunodeficiency virus.

PubMed Central

Jackson, J B; Balfour, H H

1988-01-01

Since the discovery of human immunodeficiency virus (HIV) as the causative agent of acquired immunodeficiency syndrome in 1983, there has been a proliferation of diagnostic tests. These assays can be used to detect the presence of HIV antibody, HIV antigen, HIV ribonucleic and deoxyribonucleic acids, and HIV reverse transcriptase. Enzyme-linked immunosorbent assays, Western blot, radioimmunoprecipitation assays, indirect immunofluorescence assays, reverse transcriptase assays, and several molecular hybridization techniques are currently available. Enzyme-linked immunosorbent, Western blot, and indirect immunofluorescence assays for HIV antibody are very sensitive, specific, and adaptable to most laboratories. An enzyme-linked immunosorbent assay for HIV antigen is also readily adaptable to most laboratories and will be commercially available soon. While the other assays are more tedious, they are valuable confirmatory tests and are suitable for reference laboratories. The biohazards of performing HIV testing can be minimized with proper biosafety measures. Images PMID:3060241

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

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

PubMed

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

2014-11-01

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

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.

Occurrence of etravirine/rilpivirine-specific resistance mutations selected by efavirenz and nevirapine in Kenyan patients with non-B HIV-1 subtypes failing antiretroviral therapy.

PubMed

Crawford, Keith W; Njeru, Dorothy; Maswai, Jonah; Omondi, Milton; Apollo, Duncan; Kimetto, Jane; Gitonga, Lawrence; Munyao, James; Langat, Raphael; Aoko, Appolonia; Tarus, Jemutai; Khamadi, Samoel; Hamm, Tiffany E

2014-01-28

Resistance to efavirenz and nevirapine has not been associated with mutations at position 138 of reverse transcriptase. In an evaluation of virologic suppression rates in PEPFAR (President's Emergency Plan For AIDS Relief) clinics in Kenya among patients on first-line therapy (RV288), 63% (617/975) of randomly selected patients on antiretroviral therapy were suppressed (HIV RNA<400 copies/ml). Among those with non-nucleoside reverse transcriptase inhibitor resistance (n = 101), 14 (13.8%) had substitutions at 138 (A, G, K or Q), mutations selected only by etravirine and rilpivirine in subtype B viruses. All 14 patients received efavirenz or nevirapine, not etravirine or rilpivirine, and were predominantly subtype A1. This may be the first report of efavirenz and nevirapine selecting these mutations in these subtypes.

HIV type 1 diversity in the Seychelles.

PubMed

Razafindratsimandresy, Richter; Hollanda, Justina; Soares, Jean-Louis; Rousset, Dominique; Chetty, Agnes P; Reynes, Jean-Marc

2007-06-01

Subtype determination and drug resistance-associated mutations (DRM) detection were performed on 40 HIV-1 Western blot-positive sera detected, obtained from consecutive patients resident in the Seychelles and consulting the Communicable Disease Control Unit, HIV reference center, in Victoria Hospital (Mahe) from October 2005 to June 2006. Amplification and sequencing of at least two of the partial reverse transcriptase, protease, and partial envelope genes were successful for all strains. All three genes sequences were obtained for 39 strains. A high degree of subtype or circulating recombinant forms (CRF) was observed for these 39 strains: A-A1 (17 cases), C (10 cases), B (8 cases), CRF02_AG (2 cases), D (1 case) and CRF01_AE (1 case). According to the ANRS 2006 DRM list and algorithm, none of the 40 isolates was found to be resistant to any protease or reverse transcriptase inhibitors.

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.

Transposable elements in sexual and ancient asexual taxa

PubMed Central

Arkhipova, Irina; Meselson, Matthew

2000-01-01

Sexual reproduction allows deleterious transposable elements to proliferate in populations, whereas the loss of sex, by preventing their spread, has been predicted eventually to result in a population free of such elements [Hickey, D. A. (1982) Genetics 101, 519–531]. We tested this expectation by screening representatives of a majority of animal phyla for LINE-like and gypsy-like reverse transcriptases and mariner/Tc1-like transposases. All species tested positive for reverse transcriptases except rotifers of the class Bdelloidea, the largest eukaryotic taxon in which males, hermaphrodites, and meiosis are unknown and for which ancient asexuality is supported by molecular genetic evidence. Mariner-like transposases are distributed sporadically among species and are present in bdelloid rotifers. The remarkable lack of LINE-like and gypsy-like retrotransposons in bdelloids and their ubiquitous presence in other taxa support the view that eukaryotic retrotransposons are sexually transmitted nuclear parasites and that bdelloid rotifers evolved asexually. PMID:11121049

Effects of extracellular magnesium extract on the proliferation and differentiation of human osteoblasts and osteoclasts in coculture.

PubMed

Wu, Lili; Feyerabend, Frank; Schilling, Arndt F; Willumeit-Römer, Regine; Luthringer, Bérengère J C

2015-11-01

Coculture of osteoblasts and osteoclasts is a subject of interest in the understanding of how magnesium (Mg)-based implants influence the bone metabolism and remodeling upon degradation. Human telomerase reverse transcriptase (hTERT) transduced mesenchymal stem cells (SCP-1) were first differentiated into osteoblasts with osteogenic supplements and then further cocultured with peripheral blood mononucleated cells (PBMC) without the addition of osteoclastogenesis promoting factors. Concomitantly, the cultures were exposed to variable Mg extract dilutions (0, 30×, 10×, 5×, 3×, 2× and 1×). Phenotype characterization documented that while 2× dilution of Mg extract was extremely toxic to osteoclast monoculture, monocytes in coculture with osteoblasts exhibited a greater tolerance to higher Mg extract concentration. The dense growth of osteoblasts in cultures with 1× dilution of Mg extract suggested that high concentration of Mg extract promoted osteoblast proliferation/differentiation behavior. The results of intracellular alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) activities as well as protein and gene expressions of receptor activator of nuclear factor kappa-B ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and osteoclast-associated receptor (OSCAR) revealed significantly enhanced formation of osteoblasts whereas decreased osteoclastogenesis in the cultures with high concentrations of Mg extract (2× and 1× dilutions). In conclusion, while an increased osteoinductivity has been demonstrated, the impact of potentially decreased osteoclastogenesis around the Mg-based implants should be also taken into account. Cocultures containing both bone-forming osteoblasts and bone-resorbing osteoclasts should be preferentially performed for in vitro cytocompatibility assessment of Mg-based implants as they more closely mimic the in vivo environment. An attractive human osteoblasts and osteoclasts cocultivation regime was developed as an in vitro cytocompatibility model for magnesium implants. Parameters in terms of cellular proliferation and differentiation behaviors were investigated and we conclude that high concentration of magnesium extract could lead to a promotion in osteoblastogenesis but an inhibition in osteoclastogenesis. It could contribute to the repeated observations of enhanced bone growth adjacent to degradable magnesium alloys. More interestingly, it demonstrates that compared to monoculture, osteoclasts in cocultures with osteoblasts exhibited higher tolerance to the culture environment with high magnesium extract. It might attribute to the neutralization process of the alkaline medium by acid generated by increased amount of osteoblasts in the condition with high concentration of Mg extract. The submitted work could be of significant importance to other researchers working in the related field(s), thus appealing to the readership of Acta Biomaterialia. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Fragment Based Strategies for Discovery of Novel HIV-1 Reverse Transcriptase and Integrase Inhibitors.

PubMed

Latham, Catherine F; La, Jennifer; Tinetti, Ricky N; Chalmers, David K; Tachedjian, Gilda

2016-01-01

Human immunodeficiency virus (HIV) remains a global health problem. While combined antiretroviral therapy has been successful in controlling the virus in patients, HIV can develop resistance to drugs used for treatment, rendering available drugs less effective and limiting treatment options. Initiatives to find novel drugs for HIV treatment are ongoing, although traditional drug design approaches often focus on known binding sites for inhibition of established drug targets like reverse transcriptase and integrase. These approaches tend towards generating more inhibitors in the same drug classes already used in the clinic. Lack of diversity in antiretroviral drug classes can result in limited treatment options, as cross-resistance can emerge to a whole drug class in patients treated with only one drug from that class. A fresh approach in the search for new HIV-1 drugs is fragment-based drug discovery (FBDD), a validated strategy for drug discovery based on using smaller libraries of low molecular weight molecules (<300 Da) screened using primarily biophysical assays. FBDD is aimed at not only finding novel drug scaffolds, but also probing the target protein to find new, often allosteric, inhibitory binding sites. Several fragment-based strategies have been successful in identifying novel inhibitory sites or scaffolds for two proven drug targets for HIV-1, reverse transcriptase and integrase. While any FBDD-generated HIV-1 drugs have yet to enter the clinic, recent FBDD initiatives against these two well-characterised HIV-1 targets have reinvigorated antiretroviral drug discovery and the search for novel classes of HIV-1 drugs.

Molecular docking of (5E)-3-(2-aminoethyl)-5-(2- thienylmethylene)-1, 3-thiazolidine-2, 4-dione on HIV-1 reverse transcriptase: novel drug acting on enzyme.

PubMed

Seniya, Chandrabhan; Yadav, Ajay; Uchadia, Kuldeep; Kumar, Sanjay; Sagar, Nitin; Shrivastava, Priyanka; Shrivastava, Shilpi; Wadhwa, Gulshan

2012-01-01

The study of Human immunodeficiency virus (HIV) in humans and animal models in last 31 years suggested that it is a causative agent of AIDS. This causes serious pandemic public health concern globally. It was reported that the HIV-1 reverse transcriptase (RT) played a critical role in the life cycle of HIV. Therefore, inhibition of HIV-1RT enzyme is one of the major and potential targets in the treatment of AIDS. The enzyme (HIV-1RT) was successfully targeted by non nucleotide reverse transcriptase inhibitors (NNRTIs). But frequent application of NNRTIs led drug resistance mutation on HIV infections. Therefore, there is a need to search new NNRTIs with appropriate pharmacophores. For the purpose, a virtually screened 3D model of unliganded HIV-1RT (1DLO) was explored. The unliganded HIV-1RT (1DLO) was docked with 4-thiazolidinone and its derivatives (ChemBank Database) by using AutoDock4. The best seven docking solutions complex were selected and analyzed by Ligplot. The analysis showed that derivative (5E)-3-(2- aminoethyl)-5-(2- thienylmethylene)-1, 3-thiazolidine-2, 4-dione (CID 3087795) has maximum potential against unliganded HIV-1RT (1DLO). The analysis was done on the basis of scoring and binding ability. The derivative (5E)-3-(2- aminoethyl)-5-(2- thienylmethylene)-1, 3-thiazolidine-2, 4-dione (CID 3087795) indicated minimum energy score and highest number of interactions with active site residue and could be a promising inhibitor for HIV-1 RT as Drug target.

Antiretroviral therapy in children: recent advances.

PubMed

Lodha, Rakesh; Manglani, Mamta

2012-12-01

Availability and successful use of various antiretroviral drugs has transformed HIV/AIDS from an incurable to a treatable chronic condition. The antiretroviral therapy can successfully suppress viral replication and preserve the immune system for many years. The implementation of antiretroviral therapy program in resource limited settings using the 'public health approach' of the World Health Organization has had a dramatic impact on the lives of millions of HIV infected individuals. Antiretroviral therapy (ART) in children has many challenges: use of appropriate formulations, regular need for modification of doses as the child grows, adherence issues, etc. To reduce the high morbidity and mortality in HIV infected children, it is currently recommended that all HIV infected children less than 24 mo should receive ART; in older children the indications are based on clinical and/or immunological criteria. Highly active antiretroviral therapy regimens include at least 3 antiretroviral drugs. The first line therapy recommended for children is a combination of two nucleoside reverse transcriptase inhibitors and a non-nucleoside reverse transcriptase inhibitor. Infants who have had exposure to nevirapine should receive a combination of two nucleoside reverse transcriptase inhibitors and a protease inhibitor; the protease inhibitor of choice is ritonavir boosted lopinavir. The success of therapy is dependent on >95 % adherence. The second line regimen, used when the first line therapy fails, is based on a protease inhibitor. The ongoing research focuses on simplification of regimen, discovery of more potent drugs, availability of more pediatric formulations, treatment of drug resistant strains etc. The optimal indications for initiation of therapy in children, are also being studied.

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

Binding of Nickel to Testicular Glutamate–Ammonia Ligase Inhibits Its Enzymatic Activity

PubMed Central

SUN, YINGBIAO; OU, YOUNG; CHENG, MIN; RUAN, YIBING; VAN DER HOORN, FRANS A.

2016-01-01

SUMMARY Exposure to nickel has been shown to cause damage to the testis in several animal models. It is not known if the testis expresses protein(s) that can bind nickel. To test this, we used a nickel-binding assay to isolate testicular nickel-binding proteins. We identified glutamate–ammonia ligase (GLUL) as a prominent nickel-binding protein by mass spectrometry. Protein analysis and reverse transcriptase polymerase chain reaction showed that GLUL is expressed in the testis, predominantly in interstitial cells. We determined that GLUL has a higher affinity for nickel than for its regular co-factor manganese. We produced an enzymatically active, recombinant GLUL protein. Upon binding, nickel interferes with the manganese-catalyzed enzymatic activity of recombinant GLUL protein. We also determined that GLUL activity in testes of animals exposed to nickel sulfate is reduced. Our results identify testicular GLUL as the first testicular protein shown to be affected by nickel exposure. PMID:21254280

Evidence of human hantavirus infection and zoonotic investigation of hantavirus prevalence in rodents in western Java, Indonesia.

PubMed

Kosasih, Herman; Ibrahim, Ima Nurisa; Wicaksana, Rudi; Alisjahbana, Bachti; Hoo, Yumilia; Yo, Iing H; Antonjaya, Ungke; Widjaja, Susana; Winoto, Imelda; Williams, Maya; Blair, Patrick J

2011-06-01

During febrile surveillance in the western Java City of Bandung, Indonesia, a patient with clinical symptoms consistent with hantavirus infection was found to have elevated titers of hantavirus-specific immunoglobulin M (IgM) and IgG antibodies. A subsequent epizoological investigation demonstrated a higher prevalence of hantavirus IgG antibodies in rodents trapped in the vicinity of the patient's home compared with rodents from a control area (13.2% vs. 4.7%, p = 0.036). The Old World Seoul hantavirus was detected by reverse transcriptase-polymerase chain reaction in the organs of 71% of the seropositive rodents tested. This is the first report of a Seoul virus infection in Indonesia supported by clinical, serological, and epizoological evidences. These findings suggest that hantavirus infection should be on the clinical differential diagnosis when acutely ill febrile patients report for care in western Java.

Optimization of the expression of a laccase gene from Trametes versicolor in Pichia methanolica.

PubMed

Guo, Mei; Lu, Fuping; Du, Lianxiang; Pu, Jun; Bai, Dongqing

2006-08-01

A cDNA encoding for laccase (Lcc1) was isolated from the ligninolytic fungus Trametes versicolor by reverse transcriptase polymerase chain reaction. The Lcc1 gene was subcloned into the Pichia methanolica expression vector pMETalphaA and transformed into the P. methanolica strains PMAD11 and PMAD16. The extracellular laccase activity of the PMAD11 recombinants was found to be 1.3-fold higher than that of the PMAD16 recombinants. The identity of the recombinant protein was further confirmed by immunodetection using the Western blot analysis. As expected, the molecular mass of the mature laccase was 64.0 kDa, similar to that of the native form. The effects of copper concentration, cultivation temperature, pH and methanol concentration in the BMMY on laccase expression were investigated. The laccase activity in the PMAD11 recombinant was up to 12.6 U ml(-1) by optimization.

[Relationship between the methylenetetrahydrofolate reductase gene polymorphism and adverse reactions of high-dose methotrexate in children with acute lymphocytic leukemia].

PubMed

Zheng, Miao-Miao; Yue, Li-Jie; Chen, Xiao-Wen; Wen, Fei-Qiu; Li, Chang-Gang; Yang, Chun-Lan; Xie, Cai; Ding, Hui

2013-03-01

To study the association between methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms and toxicities after high-dose methotrexate (HD-MTX) infusion in children with acute lymphocytic leukemia (ALL). MTHFR variants in 52 children with ALL were determined by reverse transcriptase-polymerase chain reaction-denaturing gradient gel electrophoresis and sequencing. Toxicities of children who received HD-MTX chemotherapy were evaluated according to the National Cancer Institute-Common Toxicity Criteria (NCI-CTC). The children carrying MTHFR 1298AC had a higher risk of developing thrombocytopenia compared with the carriers of the 1298 AA genotype (OR=13.7, 95%CI=1.18-159.36, P=0.036). There was no significant difference in HD-MTX chemotherapy-related adverse effects between the patients with different MTHFR C677T or G1793A genotypes. MTHFR A1298C polymorohism may associate with the toxicity of HD-MTX chemotherapy in children with ALL.

TELOMERASE AND CHRONIC ARSENIC EXPOSURE IN HUMANS

EPA Science Inventory

Arsenic exposure has been associated with increased risk of skin, lung and bladder cancer in humans. The mechanisms of carcinogenesis are not well understood. Telomerase, a ribonucleoprotein containing human telomerase reverse transcriptase (hTERT), can extend telomeres of eukary...

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

PubMed Central

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

2014-01-01

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

Eicosapentaenoic acid-enriched phosphatidylcholine isolated from Cucumaria frondosa exhibits anti-hyperglycemic effects via activating phosphoinositide 3-kinase/protein kinase B signal pathway.

PubMed

Hu, Shiwei; Xu, Leilei; Shi, Di; Wang, Jingfeng; Wang, Yuming; Lou, Qiaoming; Xue, Changhu

2014-04-01

Eicosapentaenoic acid-enriched phosphatidylcholine was isolated from the sea cucumber Cucumaria frondosa (Cucumaria-PC) and its effects on streptozotocin (STZ)-induced hyperglycemic rats were investigated. Male Sprague-Dawley rats were randomly divided into normal control, model control (STZ), low- and high-dose Cucumaria-PC groups (STZ + Cucumaria-PC at 25 and 75 mg/Kg·b·wt, intragastrically, respectively). Blood glucose, insulin, glycogen in liver and gastrocnemius were determined over 60 days. Insulin signaling in the rats' gastrocnemius was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The results showed that Cucumaria-PC significantly decreased blood glucose level, increased insulin secretion and glycogen synthesis in diabetic rats. RT-PCR analysis revealed that Cucumaria-PC significantly promoted the expressions of glycometabolism-related genes of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphoinositide 3-kinase (PI3K), protein kinase B (PKB), and glucose transporter 4 (GLUT4) in gastrocnemius. Western blotting assay demonstrated that Cucumaria-PC remarkably enhanced the proteins abundance of IR-β, PI3K, PKB, GLUT4, as well as phosphorylation of Tyr-IR-β, p85-PI3K, Ser473-PKB (P < 0.05 and P < 0.01). These findings suggested that Cucumaria-PC exhibited significant anti-hyperglycemic activities through up-regulating PI3K/PKB signal pathway mediated by insulin. Nutritional supplementation with Cucumaria-PC, if validated for human studies, may offer an adjunctive therapy for diabetes mellitus. Copyright © 2013 The Society for Biotechnology, Japan. All rights reserved.

Acid-induced off-response of PKD2L1 channel in Xenopus oocytes and its regulation by Ca2+

PubMed Central

Hussein, Shaimaa; Zheng, Wang; Dyte, Chris; Wang, Qian; Yang, JungWoo; Zhang, Fan; Tang, Jingfeng; Cao, Ying; Chen, Xing-Zhen

2015-01-01

Polycystic kidney disease (PKD) protein 2 Like 1 (PKD2L1), also called transient receptor potential polycystin-3 (TRPP3), regulates Ca2+-dependent hedgehog signalling in primary cilia, intestinal development and sour tasting but with an unclear mechanism. PKD2L1 is a Ca2+-permeable cation channel that is activated by extracellular Ca2+ (on-response) in Xenopus oocytes. PKD2L1 co-expressed with PKD protein 1 Like 3 (PKD1L3) exhibits extracellular acid-induced activation (off-response, i.e., activation following acid removal) but whether PKD1L3 participates in acid sensing remains unclear. Here we used the two-microelectrode voltage-clamp, site directed mutagenesis, Western blotting, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence, and showed that PKD2L1 expressed in oocytes exhibits sustained off-response currents in the absence of PKD1L3. PKD1L3 co-expression augmented the PKD2L1 plasma membrane localization but did not alter the observed properties of the off-response. PKD2L1 off-response was inhibited by an increase in intracellular Ca2+. We also identified two intra-membrane residues aspartic acid 349 (D349) and glutamic acid 356 (E356) in the third transmembrane domain that are critical for PKD2L1 channel function. Our study suggests that PKD2L1 may itself sense acids and defines off-response properties in the absence of PKD1L3. PMID:26502994

The Streptococcus mutans Serine/Threonine Kinase, PknB, Regulates Competence Development, Bacteriocin Production, and Cell Wall Metabolism ▿

PubMed Central

Banu, Liliana Danusia; Conrads, Georg; Rehrauer, Hubert; Hussain, Haitham; Allan, Elaine; van der Ploeg, Jan R.

2010-01-01

Bacteria can detect, transmit, and react to signals from the outside world by using two-component systems (TCS) and serine-threonine kinases and phosphatases. Streptococcus mutans contains one serine-threonine kinase, encoded by pknB. A gene encoding a serine-threonine phosphatase, pppL, is located upstream of pknB. In this study, the phenotypes of pknB and pppL single mutants and a pknB pppL double mutant were characterized. All mutants exhibited a reduction in genetic transformability and biofilm formation, showed abnormal cell shapes, grew slower than the wild-type strain in several complex media, and exhibited reduced acid tolerance. The mutants had reduced cariogenic capacity but no significant defects in colonization in a rat caries model. Whole-genome transcriptome analysis revealed that a pknB mutant showed reduced expression of genes involved in bacteriocin production and genetic competence. Among the genes that were differentially regulated in the pknB mutant, several were likely to be involved in cell wall metabolism. One such gene, SMU.2146c, and two genes encoding bacteriocins were shown to also be downregulated in a vicK mutant, which encodes a sensor kinase involved in the response to oxidative stress. Collectively, the results lead us to speculate that PknB may modulate the activity of the two-component signal transduction systems VicKR and ComDE. Real-time reverse transcriptase PCR (RT-PCR) showed that genes downregulated in the pknB mutant were upregulated in the pppL mutant, indicating that PppL serves to counteract PknB. PMID:20231406

The Streptococcus mutans serine/threonine kinase, PknB, regulates competence development, bacteriocin production, and cell wall metabolism.

PubMed

Banu, Liliana Danusia; Conrads, Georg; Rehrauer, Hubert; Hussain, Haitham; Allan, Elaine; van der Ploeg, Jan R

2010-05-01

Bacteria can detect, transmit, and react to signals from the outside world by using two-component systems (TCS) and serine-threonine kinases and phosphatases. Streptococcus mutans contains one serine-threonine kinase, encoded by pknB. A gene encoding a serine-threonine phosphatase, pppL, is located upstream of pknB. In this study, the phenotypes of pknB and pppL single mutants and a pknB pppL double mutant were characterized. All mutants exhibited a reduction in genetic transformability and biofilm formation, showed abnormal cell shapes, grew slower than the wild-type strain in several complex media, and exhibited reduced acid tolerance. The mutants had reduced cariogenic capacity but no significant defects in colonization in a rat caries model. Whole-genome transcriptome analysis revealed that a pknB mutant showed reduced expression of genes involved in bacteriocin production and genetic competence. Among the genes that were differentially regulated in the pknB mutant, several were likely to be involved in cell wall metabolism. One such gene, SMU.2146c, and two genes encoding bacteriocins were shown to also be downregulated in a vicK mutant, which encodes a sensor kinase involved in the response to oxidative stress. Collectively, the results lead us to speculate that PknB may modulate the activity of the two-component signal transduction systems VicKR and ComDE. Real-time reverse transcriptase PCR (RT-PCR) showed that genes downregulated in the pknB mutant were upregulated in the pppL mutant, indicating that PppL serves to counteract PknB.

Malignant Transformation Potentials of Human Umbilical Cord Mesenchymal Stem Cells Both Spontaneously and via 3-Methycholanthrene Induction

PubMed Central

Lai, Xiulan; Liu, Sizheng; Chen, Yezeng; Zheng, Zexin; Xie, Qingdong; Maldonado, Martin; Cai, Zhiwei; Qin, Shan; Ho, Guyu; Ma, Lian

2013-01-01

Human umbilical cord mesenchymal stem cells (HUMSCs) are highly proliferative and can be induced to differentiate into advanced derivatives of all three germ layers. Thus, HUMSCs are considered to be a promising source for cell-targeted therapies and tissue engineering. However there are reports on spontaneous transformation of mesenchymal stem cells (MSCs) derived from human bone marrows. The capacity for HUMSCs to undergo malignant transform spontaneously or via induction by chemical carcinogens is presently unknown. Therefore, we isolated HUMSCs from 10 donors and assessed their transformation potential either spontaneously or by treating them with 3-methycholanthrene (3-MCA), a DNA-damaging carcinogen. The malignant transformation of HUMSCs in vitro was evaluated by morphological changes, proliferation rates, ability to enter cell senescence, the telomerase activity, chromosomal abnormality, and the ability to form tumors in vivo. Our studies showed that HUMSCs from all 10 donors ultimately entered senescence and did not undergo spontaneous malignant transformation. However, HUMSCs from two of the 10 donors treated with 3-MCA displayed an increased proliferation rate, failed to enter senescence, and exhibited an altered cell morphology. When these cells (tHUMSCs) were injected into immunodeficient mice, they gave rise to sarcoma-like or poorly differentiated tumors. Moreover, in contrast to HUMSCs, tHUMSCs showed a positive expression of human telomerase reverse transcriptase (hTERT) and did not exhibit a shortening of the relative telomere length during the long-term culture in vitro. Our studies demonstrate that HUMSCs are not susceptible to spontaneous malignant transformation. However, the malignant transformation could be induced by chemical carcinogen 3-MCA. PMID:24339974

Efavirenz or nevirapine in three-drug combination therapy with two nucleoside-reverse transcriptase inhibitors for initial treatment of HIV infection in antiretroviral-naïve individuals.

PubMed

Mbuagbaw, Lawrence Ce; Irlam, James H; Spaulding, Alicen; Rutherford, George W; Siegfried, Nandi

2010-12-08

The advent of highly active antiretroviral therapy (HAART) has reduced the morbidity and mortality due to HIV. The World Health Organisation (WHO) antiretroviral treatment (ART) guidelines focus on three classes of antiretroviral drugs, namely: nucleoside/nucleotide reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI) and protease inhibitors (PI). Two of the most common medications given in first-line treatment are the NNRTIs, efavirenz (EFV) and nevirapine (NVP). It is unclear which NNRTI is more efficacious for initial therapy. To determine which NNRTI, EFV or NVP, is more efficacious when given in combination with two NRTIs as part of initial ART for HIV infection in adults and children. We used a comprehensive and exhaustive strategy in an attempt to identify all relevant studies, regardless of language or publication status, in electronic databases and conference proceedings from 1996 to 2009. All randomised controlled trials comparing EFV to NVP in HIV-infected individuals without prior exposure to ART, irrespective of the dosage or NRTI backbone.The primary outcome of interest was virologic response to ART. Other primary outcomes included mortality, clinical progression, severe adverse events, and discontinuation of therapy for any reason. Secondary outcomes were immunologic response to ART, treatment failure, development of ART drug resistance, and prevention of sexual transmission of HIV. Two authors assessed each reference for inclusion and exclusion criteria established a priori. Data were abstracted independently using a standardised abstraction form. Data were analysed on an intention-to-treat basis and reported as per dosage of NVP. We identified seven randomised controlled trials that met our inclusion criteria.The trials were pooled as per dosage of NVP. None of these trials included children.The seven trials enrolled 1,688 participants and found no critical differences between EFV and NVP, except for different toxicity profiles. EFV is more likely to cause central nervous system side-effects, while NVP is more likely to result in raised transaminases and neutropoenia. There was a higher mortality rate in the NVP 400mg once daily arm.The quality of literature to support these conclusions is moderate to high. Drug resistance was slightly less common with EFV than NVP, but the quality of this literature is low since only one of the seven studies reported on this outcome. No studies reported on sexual transmission of HIV. The length of follow-up time, study settings, and NRTI backbone varied greatly. Both drugs have equivalent efficacies in initial treatment of HIV infection when combined with two NRTIs, but different side effects.

Applications of 2D IR spectroscopy to peptides, proteins, and hydrogen-bond dynamics

PubMed Central

Kim, Yung Sam; Hochstrasser, Robin M.

2010-01-01

Following a survey of 2D IR principles this Feature Article describes recent experiments on the hydrogen-bond dynamics of small ions, amide-I modes, nitrile probes, peptides, reverse transcriptase inhibitors, and amyloid fibrils. PMID:19351162

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

EPA Science Inventory

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

Opposing actions of Arx and Pax4 in endocrine pancreas development

PubMed Central

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

2003-01-01

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

Opposing actions of Arx and Pax4 in endocrine pancreas development.

PubMed

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

2003-10-15

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

A novel and exploitable antifungal peptide from kale (Brassica alboglabra) seeds.

PubMed

Lin, Peng; Ng, Tzi Bun

2008-10-01

The aim of this study was to purify and characterize antifungal peptides from kale seeds in view of the paucity of information on antifungal peptides from the family Brassicaceae, and to compare its characteristics with those of published Brassica antifungal peptides. A 5907-Da antifungal peptide was isolated from kale seeds. The isolation procedure comprised affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose and Mono S, and gel filtration on Superdex Peptide. The peptide was adsorbed on the first three chromatographic media. It inhibited mycelial growth in a number of fungal species including Fusarium oxysporum, Helminthosporium maydis, Mycosphaerella arachidicola and Valsa mali, with an IC(50) of 4.3microM, 2.1microM, 2.4microM, and 0.15microM, respectively and exhibited pronounced thermostability and pH stability. It inhibited proliferation of hepatoma (HepG2) and breast cancer (MCF7) cells with an IC(50) of 2.7microM and 3.4microM, and the activity of HIV-1 reverse transcriptase with an IC(50) of 4.9microM. Its N-terminal sequence differed from those of antifungal proteins which have been reported to date.

In vitro anti-viral effect of β-santalol against influenza viral replication.

PubMed

Paulpandi, Manickam; Kannan, Soundarapandian; Thangam, Ramar; Kaveri, Krishnasamy; Gunasekaran, Palani; Rejeeth, Chandrababu

2012-02-15

The anti-influenza A/HK (H3N2) virus activity of β-santalol was evaluated in MDCK cells and investigated the effect of β-santalol on synthesis of viral mRNAs. β-Santalol was investigated for its antiviral activity against influenza A/HK (H3N2) virus using a cytopathic effect (CPE) reduction method. β-Santalol exhibited anti-influenza A/HK (H3N2) virus activity of 86% with no cytotoxicity at the concentration of 100 μg/ml reducing the formation of a visible CPE. Oseltamivir also showed moderate antiviral activity of about 83% against influenza A/HK (H3N2) virus at the concentration of 100 μg/ml. Furthermore, the mechanism of anti-influenza virus action in the inhibition of viral mRNA synthesis was analyzed by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), and the data indicated an inhibitory effect in late viral RNA synthesis compared with oseltamivir in the presence of 100 μg/ml of β-santalol. β-Santalol should be further studied for therapeutic and prophylactic potential especially for influenza epidemics and pandemics. Copyright © 2011 Elsevier GmbH. All rights reserved.

Isolation of viral haemorrhagic septicaemia virus from muskellunge, Esox masquinongy (Mitchill), in Lake St Clair, Michigan, USA reveals a new sublineage of the North American genotype

USGS Publications Warehouse

Elsayed, E.; Faisal, M.; Thomas, M.; Whelan, G.; Batts, W.; Winton, J.

2006-01-01

Viral haemorrhagic septicaemia virus (VHSV) was isolated from muskellunge, Esox masquinongy (Mitchill), caught from the NW portion of Lake St Clair, Michigan, USA in 2003. Affected fish exhibited congestion of internal organs; the inner wall of the swim bladder was thickened and contained numerous budding, fluid-filled vesicles. A virus was isolated using fish cell lines inoculated with a homogenate of kidney and spleen tissues from affected fish. Focal areas of cell rounding and granulation appeared as early as 24 h post-inoculation and expanded rapidly to destroy the entire cell sheet by 96 h. Electron microscopy revealed virions that were 170-180 nm in length by 60-70 nm in width having a bullet-shaped morphology typical of rhabdoviruses. The virus was confirmed as VHSV by reverse transcriptase-polymerase chain reaction. Sequence analysis of the entire nucleoprotein and glycoprotein genes revealed the virus was a member of the North American genotype of VHSV; however, the isolate was sufficiently distinct to be considered a separate sublineage, suggesting its origin may have been from marine species inhabiting the eastern coastal areas of the USA or Canada.

An integrated chemical biology approach reveals the mechanism of action of HIV replication inhibitors.

PubMed

Pagano, Nicholas; Teriete, Peter; Mattmann, Margrith E; Yang, Li; Snyder, Beth A; Cai, Zhaohui; Heil, Marintha L; Cosford, Nicholas D P

2017-12-01

Continuous flow (microfluidic) chemistry was employed to prepare a small focused library of dihydropyrimidinone (DHPM) derivatives. Compounds in this class have been reported to exhibit activity against the human immunodeficiency virus (HIV), but their molecular target had not been identified. We tested the initial set of DHPMs in phenotypic assays providing a hit (1i) that inhibited the replication of the human immunodeficiency virus HIV in cells. Flow chemistry-driven optimization of 1i led to the identification of HIV replication inhibitors such as 1l with cellular potency comparable with the clinical drug nevirapine (NVP). Mechanism of action (MOA) studies using cellular and biochemical assays coupled with 3D fingerprinting and in silico modeling demonstrated that these drug-like probe compounds exert their effects by inhibiting the viral reverse transcriptase polymerase (RT). This led to the design and synthesis of the novel DHPM 1at that inhibits the replication of drug resistant strains of HIV. Our work demonstrates that combining flow chemistry-driven analogue refinement with phenotypic assays, in silico modeling and MOA studies is a highly effective strategy for hit-to-lead optimization applicable to the discovery of future therapeutic agents. Copyright © 2017. Published by Elsevier Ltd.

Inactivation of CUG-BP1/CELF1 Causes Growth, Viability, and Spermatogenesis Defects in Mice▿

PubMed Central

Kress, Chantal; Gautier-Courteille, Carole; Osborne, H. Beverley; Babinet, Charles; Paillard, Luc

2007-01-01

CUG-BP1/CELF1 is a multifunctional RNA-binding protein involved in the regulation of alternative splicing and translation. To elucidate its role in mammalian development, we produced mice in which the Cugbp1 gene was inactivated by homologous recombination. These Cugbp1−/− mice were viable, although a significant portion of them did not survive after the first few days of life. They displayed growth retardation, and most Cugbp1−/− males and females exhibited impaired fertility. Male infertility was more thoroughly investigated. Histological examination of testes from Cugbp1−/− males showed an arrest of spermatogenesis that occurred at step 7 of spermiogenesis, before spermatid elongation begins, and an increased apoptosis. A quantitative reverse transcriptase PCR analysis showed a decrease of all the germ cell markers tested but not of Sertoli and Leydig markers, suggesting a general decrease in germ cell number. In wild-type testes, CUG-BP1 is expressed in germ cells from spermatogonia to round spermatids and also in Sertoli and Leydig cells. These findings demonstrate that CUG-BP1 is required for completion of spermatogenesis. PMID:17130239

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,…

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.

Dynamics of drug resistance-associated mutations in HIV-1 DNA reverse transcriptase sequence during effective ART.

PubMed

Nouchi, A; Nguyen, T; Valantin, M A; Simon, A; Sayon, S; Agher, R; Calvez, V; Katlama, C; Marcelin, A G; Soulie, C

2018-05-29

To investigate the dynamics of HIV-1 variants archived in cells harbouring drug resistance-associated mutations (DRAMs) to lamivudine/emtricitabine, etravirine and rilpivirine in patients under effective ART free from selective pressure on these DRAMs, in order to assess the possibility of recycling molecules with resistance history. We studied 25 patients with at least one DRAM to lamivudine/emtricitabine, etravirine and/or rilpivirine identified on an RNA sequence in their history and with virological control for at least 5 years under a regimen excluding all drugs from the resistant class. Longitudinal ultra-deep sequencing (UDS) and Sanger sequencing of the reverse transcriptase region were performed on cell-associated HIV-1 DNA samples taken over the 5 years of follow-up. Viral variants harbouring the analysed DRAMs were no longer detected by UDS over the 5 years in 72% of patients, with viruses susceptible to the molecules of interest found after 5 years in 80% of patients with UDS and in 88% of patients with Sanger. Residual viraemia with <50 copies/mL was detected in 52% of patients. The median HIV DNA level remained stable (2.4 at baseline versus 2.1 log10 copies/106 cells 5 years later). These results show a clear trend towards clearance of archived DRAMs to reverse transcriptase inhibitors in cell-associated HIV-1 DNA after a long period of virological control, free from therapeutic selective pressure on these DRAMs, reflecting probable residual replication in some reservoirs of the fittest viruses and leading to persistent evolution of the archived HIV-1 DNA resistance profile.

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

PubMed

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

2008-10-01

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

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

PubMed Central

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

2008-01-01

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

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

PubMed

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

1997-11-01

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

The site-specific ribosomal insertion element type II of Bombyx mori (R2Bm) contains the coding sequence for a reverse transcriptase-like enzyme.

PubMed Central

Burke, W D; Calalang, C C; Eickbush, T H

1987-01-01

Two classes of DNA elements interrupt a fraction of the rRNA repeats of Bombyx mori. We have analyzed by genomic blotting and sequence analysis one class of these elements which we have named R2. These elements occupy approximately 9% of the rDNA units of B. mori and appear to be homologous to the type II rDNA insertions detected in Drosophila melanogaster. Approximately 25 copies of R2 exist within the B. mori genome, of which at least 20 are located at a precise location within otherwise typical rDNA units. Nucleotide sequence analysis has revealed that the 4.2-kilobase-pair R2 element has a single large open reading frame, occupying over 82% of the total length of the element. The central region of this 1,151-amino-acid open reading frame shows homology to the reverse transcriptase enzymes found in retroviruses and certain transposable elements. Amino acid homology of this region is highest to the mobile line 1 elements of mammals, followed by the mitochondrial type II introns of fungi, and the pol gene of retroviruses. Less homology exists with transposable elements of D. melanogaster and Saccharomyces cerevisiae. Two additional regions of sequence homology between L1 and R2 elements were also found outside the reverse transcriptase region. We suggest that the R2 elements are retrotransposons that are site specific in their insertion into the genome. Such mobility would enable these elements to occupy a small fraction of the rDNA units of B. mori despite their continual elimination from the rDNA locus by sequence turnover. Images PMID:2439905

Giant Reverse Transcriptase-Encoding Transposable Elements at Telomeres.

PubMed

Arkhipova, Irina R; Yushenova, Irina A; Rodriguez, Fernando

2017-09-01

Transposable elements are omnipresent in eukaryotic genomes and have a profound impact on chromosome structure, function and evolution. Their structural and functional diversity is thought to be reasonably well-understood, especially in retroelements, which transpose via an RNA intermediate copied into cDNA by the element-encoded reverse transcriptase, and are characterized by a compact structure. Here, we report a novel type of expandable eukaryotic retroelements, which we call Terminons. These elements can attach to G-rich telomeric repeat overhangs at the chromosome ends, in a process apparently facilitated by complementary C-rich repeats at the 3'-end of the RNA template immediately adjacent to a hammerhead ribozyme motif. Terminon units, which can exceed 40 kb in length, display an unusually complex and diverse structure, and can form very long chains, with host genes often captured between units. As the principal polymerizing component, Terminons contain Athena reverse transcriptases previously described in bdelloid rotifers and belonging to the enigmatic group of Penelope-like elements, but can additionally accumulate multiple cooriented ORFs, including DEDDy 3'-exonucleases, GDSL esterases/lipases, GIY-YIG-like endonucleases, rolling-circle replication initiator (Rep) proteins, and putatively structural ORFs with coiled-coil motifs and transmembrane domains. The extraordinary length and complexity of Terminons and the high degree of interfamily variability in their ORF content challenge the current views on the structural organization of eukaryotic retroelements, and highlight their possible connections with the viral world and the implications for the elevated frequency of gene transfer. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

Antioxidants inhibit nuclear export of telomerase reverse transcriptase and delay replicative senescence of endothelial cells.

PubMed

Haendeler, Judith; Hoffmann, Jörg; Diehl, J Florian; Vasa, Mariuca; Spyridopoulos, Ioakim; Zeiher, Andreas M; Dimmeler, Stefanie

2004-04-02

Aging is associated with a rise in intracellular reactive oxygen species (ROS) and a loss of telomerase reverse transcriptase activity. Incubation with H2O2 induced the nuclear export of telomerase reverse transcriptase (TERT) into the cytosol in a Src-family kinase-dependent manner. Therefore, we investigated the hypothesis that age-related increase in reactive oxygen species (ROS) may induce the nuclear export of TERT and contribute to endothelial cell senescence. Continuous cultivation of endothelial cells resulted in an increased endogenous formation of ROS starting after 29 population doublings (PDL). This increase was accompanied by mitochondrial DNA damage and preceded the onset of replicative senescence at PDL 37. Along with the enhanced formation of ROS, we detected an export of nuclear TERT protein from the nucleus into the cytoplasm and an activation of the Src-kinase. Moreover, the induction of premature senescence by low concentrations of H2O2 was completely blocked with the Src-family kinase inhibitor PP2, suggesting a crucial role for Src-family kinases in the induction of endothelial cell aging. Incubation with the antioxidant N-acetylcysteine, from PDL 26, reduced the intracellular ROS formation and prevented mitochondrial DNA damage. Likewise, nuclear export of TERT protein, loss in the overall TERT activity, and the onset of replicative senescence were delayed by incubation with N-acetylcysteine. Low doses of the statin, atorvastatin (0.1 micromol/L), had also effects similar to those of N-acetylcysteine. We conclude that both antioxidants and statins can delay the onset of replicative senescence by counteracting the increased ROS production linked to aging of endothelial cells.

Potent NLRP3 Inflammasome Activation by the HIV Reverse Transcriptase Inhibitor Abacavir.

PubMed

Toksoy, Atiye; Sennefelder, Helga; Adam, Christian; Hofmann, Sonja; Trautmann, Axel; Goebeler, Matthias; Schmidt, Marc

2017-02-17

There is experimental and clinical evidence that some exanthematous allergic drug hypersensitivity reactions are mediated by drug-specific T cells. We hypothesized that the capacity of certain drugs to directly stimulate the innate immune system may contribute to generate drug-specific T cells. Here we analyzed whether abacavir, an HIV-1 reverse transcriptase inhibitor often inducing severe delayed-type drug hypersensitivity, can trigger innate immune activation that may contribute to its allergic potential. We show that abacavir fails to generate direct innate immune activation in human monocytes but potently triggers IL-1β release upon pro-inflammatory priming with phorbol ester or Toll-like receptor stimulation. IL-1β processing and secretion were sensitive to Caspase-1 inhibition, NLRP3 knockdown, and K + efflux inhibition and were not observed with other non-allergenic nucleoside reverse transcriptase inhibitors, identifying abacavir as a specific inflammasome activator. It further correlated with dose-dependent mitochondrial reactive oxygen species production and cytotoxicity, indicating that inflammasome activation resulted from mitochondrial damage. However, both NLRP3 depletion and inhibition of K + efflux mitigated abacavir-induced mitochondrial reactive oxygen species production and cytotoxicity, suggesting that these processes were secondary to NLRP3 activation. Instead, depletion of cardiolipin synthase 1 abolished abacavir-induced IL-1β secretion, suggesting that mitochondrial cardiolipin release may trigger abacavir-induced inflammasome activation. Our data identify abacavir as a novel inflammasome-stimulating drug allergen. They implicate a potential contribution of innate immune activation to medication-induced delayed-type hypersensitivity, which may stimulate new concepts for treatment and prevention of drug allergies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Comparative analysis of drug resistance mutations in the human immunodeficiency virus reverse transcriptase gene in patients who are non-responsive, responsive and naive to antiretroviral therapy.

PubMed

Misbah, Mohammad; Roy, Gaurav; Shahid, Mudassar; Nag, Nalin; Kumar, Suresh; Husain, Mohammad

2016-05-01

Drug resistance mutations in the Pol gene of human immunodeficiency virus 1 (HIV-1) are one of the critical factors associated with antiretroviral therapy (ART) failure in HIV-1 patients. The issue of resistance to reverse transcriptase inhibitors (RTIs) in HIV infection has not been adequately addressed in the Indian subcontinent. We compared HIV-1 reverse transcriptase (RT) gene sequences to identify mutations present in HIV-1 patients who were ART non-responders, ART responders and drug naive. Genotypic drug resistance testing was performed by sequencing a 655-bp region of the RT gene from 102 HIV-1 patients, consisting of 30 ART-non-responding, 35 ART-responding and 37 drug-naive patients. The Stanford HIV Resistance Database (HIVDBv 6.2), IAS-USA mutation list, ANRS_09/2012 algorithm, and Rega v8.02 algorithm were used to interpret the pattern of drug resistance. The majority of the sequences (96 %) belonged to subtype C, and a few of them (3.9 %) to subtype A1. The frequency of drug resistance mutations observed in ART-non-responding, ART-responding and drug-naive patients was 40.1 %, 10.7 % and 20.58 %, respectively. It was observed that in non-responders, multiple mutations were present in the same patient, while in responders, a single mutation was found. Some of the drug-naive patients had more than one mutation. Thymidine analogue mutations (TAMs), however, were found in non-responders and naive patients but not in responders. Although drug resistance mutations were widely distributed among ART non-responders, the presence of resistance mutations in the viruses of drug-naive patients poses a big concern in the absence of a genotyping resistance test.

RNA–protein binding interface in the telomerase ribonucleoprotein

PubMed Central

Bley, Christopher J.; Qi, Xiaodong; Rand, Dustin P.; Borges, Chad R.; Nelson, Randall W.; Chen, Julian J.-L.

2011-01-01

Telomerase is a specialized reverse transcriptase containing an intrinsic telomerase RNA (TR) which provides the template for telomeric DNA synthesis. Distinct from conventional reverse transcriptases, telomerase has evolved a unique TR-binding domain (TRBD) in the catalytic telomerase reverse transcriptase (TERT) protein, integral for ribonucleoprotein assembly. Two structural elements in the vertebrate TR, the pseudoknot and CR4/5, bind TERT independently and are essential for telomerase enzymatic activity. However, the details of the TR–TERT interaction have remained elusive. In this study, we employed a photoaffinity cross-linking approach to map the CR4/5-TRBD RNA–protein binding interface by identifying RNA and protein residues in close proximity. Photoreactive 5-iodouridines were incorporated into the medaka CR4/5 RNA fragment and UV cross-linked to the medaka TRBD protein fragment. The cross-linking RNA residues were identified by alkaline partial hydrolysis and cross-linked protein residues were identified by mass spectrometry. Three CR4/5 RNA residues (U182, U187, and U205) were found cross-linking to TRBD amino acids Tyr503, Phe355, and Trp477, respectively. This CR4/5 binding pocket is distinct and separate from the previously proposed T pocket in the Tetrahymena TRBD. Based on homologous structural models, our cross-linking data position the essential loop L6.1 adjacent to the TERT C-terminal extension domain. We thus propose that stem-loop 6.1 facilitates proper TERT folding by interacting with both TRBD and C-terminal extension. Revealing the telomerase CR4/5-TRBD binding interface with single-residue resolution provides important insights into telomerase ribonucleoprotein architecture and the function of the essential CR4/5 domain. PMID:22123986

Antiretroviral drug susceptibility among drug-naive adults with recent HIV infection in Rakai, Uganda

PubMed Central

Eshleman, Susan H.; Laeyendecker, Oliver; Parkin, Neil; Huang, Wei; Chappey, Colombe; Paquet, Agnes C.; Serwadda, David; Reynolds, Steven J.; Kiwanuka, Noah; Quinn, Thomas C.; Gray, Ronald; Wawer, Maria

2009-01-01

Objective To analyze antiretroviral drug susceptibility in HIV from recently infected adults in Rakai, Uganda, prior to the availability of antiretroviral drug treatment. Methods Samples obtained at the time of HIV seroconversion (1998–2003) were analyzed using the GeneSeq HIV and PhenoSense HIV assays (Monogram Biosciences, Inc., South San Francisco, California, USA). Results Test results were obtained for 104 samples (subtypes: 26A, 1C, 66D, 9A/D, 1C/D, 1 intersubtype recombinant). Mutations used for genotypic surveillance of transmitted antiretroviral drug resistance were identified in six samples: three had nucleoside reverse transcriptase inhibitor (NRTI) surveillance mutations (two had M41L, one had K219R), and three had protease inhibitor surveillance mutations (I47V, F53L, N88D); none had nonnucleoside reverse transcriptase inhibitor (NNRTI) surveillance mutations. Other resistance-associated mutations were identified in some samples. However, none of the samples had a sufficient number of mutations to predict reduced antiretroviral drug susceptibility. Ten (9.6%) of the samples had reduced phenotypic susceptibility to at least one drug (one had partial susceptibility to didanosine, one had nevirapine resistance, and eight had resistance or partial susceptibility to at least one protease inhibitor). Fifty-three (51%) of the samples had hypersusceptibility to at least one drug (seven had zidovudine hypersusceptibility, 28 had NNRTI hypersusceptibility, 34 had protease inhibitor hypersusceptibility). Delavirdine hyper-susceptibility was more frequent in subtype A than D. In subtype D, efavirenz hypersusceptibility was associated with substitutions at codon 11 in HIV-reverse transcriptase. Conclusion Phenotyping detected reduced antiretroviral drug susceptibility and hypersusceptibility in HIV from some antiretroviral-naive Ugandan adults that was not predicted by genotyping. Phenotyping may complement genotyping for analysis of antiretroviral drug susceptibility in populations with nonsubtype B HIV infection. PMID:19276794

Antiretroviral drug susceptibility among drug-naive adults with recent HIV infection in Rakai, Uganda.

PubMed

Eshleman, Susan H; Laeyendecker, Oliver; Parkin, Neil; Huang, Wei; Chappey, Colombe; Paquet, Agnes C; Serwadda, David; Reynolds, Steven J; Kiwanuka, Noah; Quinn, Thomas C; Gray, Ronald; Wawer, Maria

2009-04-27

To analyze antiretroviral drug susceptibility in HIV from recently infected adults in Rakai, Uganda, prior to the availability of antiretroviral drug treatment. Samples obtained at the time of HIV seroconversion (1998-2003) were analyzed using the GeneSeq HIV and PhenoSense HIV assays (Monogram Biosciences, Inc., South San Francisco, California, USA). Test results were obtained for 104 samples (subtypes: 26A, 1C, 66D, 9A/D, 1C/D, 1 intersubtype recombinant). Mutations used for genotypic surveillance of transmitted antiretroviral drug resistance were identified in six samples: three had nucleoside reverse transcriptase inhibitor (NRTI) surveillance mutations (two had M41L, one had K219R), and three had protease inhibitor surveillance mutations (I47V, F53L, N88D); none had nonnucleoside reverse transcriptase inhibitor (NNRTI) surveillance mutations. Other resistance-associated mutations were identified in some samples. However, none of the samples had a sufficient number of mutations to predict reduced antiretroviral drug susceptibility. Ten (9.6%) of the samples had reduced phenotypic susceptibility to at least one drug (one had partial susceptibility to didanosine, one had nevirapine resistance, and eight had resistance or partial susceptibility to at least one protease inhibitor). Fifty-three (51%) of the samples had hypersusceptibility to at least one drug (seven had zidovudine hypersusceptibility, 28 had NNRTI hypersusceptibility, 34 had protease inhibitor hypersusceptibility). Delavirdine hypersusceptibility was more frequent in subtype A than D. In subtype D, efavirenz hypersusceptibility was associated with substitutions at codon 11 in HIV-reverse transcriptase. Phenotyping detected reduced antiretroviral drug susceptibility and hypersusceptibility in HIV from some antiretroviral-naive Ugandan adults that was not predicted by genotyping. Phenotyping may complement genotyping for analysis of antiretroviral drug susceptibility in populations with nonsubtype B HIV infection.

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.

Evolutionarily Conserved Epitopes on Human Immunodeficiency Virus Type 1 (HIV-1) and Feline Immunodeficiency Virus Reverse Transcriptases Detected by HIV-1-Infected Subjects

PubMed Central

Sanou, Missa P.; Roff, Shannon R.; Mennella, Antony; Sleasman, John W.; Rathore, Mobeen H.; Levy, Jay A.

2013-01-01

Anti-human immunodeficiency virus (HIV) cytotoxic T lymphocyte (CTL)-associated epitopes, evolutionarily conserved on both HIV type 1 (HIV-1) and feline immunodeficiency virus (FIV) reverse transcriptases (RT), were identified using gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) and carboxyfluorescein diacetate succinimide ester (CFSE) proliferation assays followed by CTL-associated cytotoxin analysis. The peripheral blood mononuclear cells (PBMC) or T cells from HIV-1-seropositive (HIV+) subjects were stimulated with overlapping RT peptide pools. The PBMC from the HIV+ subjects had more robust IFN-γ responses to the HIV-1 peptide pools than to the FIV peptide pools, except for peptide-pool F3. In contrast, much higher and more frequent CD8+ T-cell proliferation responses were observed with the FIV peptide pools than with the HIV peptide pools. HIV-1-seronegative subjects had no proliferation or IFN-γ responses to the HIV and FIV peptide pools. A total of 24% (40 of 166) of the IFN-γ responses to HIV pools and 43% (23 of 53) of the CD8+ T-cell proliferation responses also correlated to responses to their counterpart FIV pools. Thus, more evolutionarily conserved functional epitopes were identified by T-cell proliferation than by IFN-γ responses. In the HIV+ subjects, peptide-pool F3, but not the HIV H3 counterpart, induced the most IFN-γ and proliferation responses. These reactions to peptide-pool F3 were highly reproducible and persisted over the 1 to 2 years of testing. All five individual peptides and epitopes of peptide-pool F3 induced IFN-γ and/or proliferation responses in addition to inducing CTL-associated cytotoxin responses (perforin, granzyme A, granzyme B). The epitopes inducing polyfunctional T-cell activities were highly conserved among human, simian, feline, and ungulate lentiviruses, which indicated that these epitopes are evolutionarily conserved. These results suggest that FIV peptides could be used in an HIV-1 vaccine. PMID:23824804

Trends in Drug Resistance Prevalence, HIV-1 Variants and Clinical Status in HIV-1-infected Pediatric Population in Madrid: 1993 to 2015 Analysis.

PubMed

Rojas Sánchez, Patricia; Domínguez, Sara; Jiménez De Ory, Santiago; Prieto, Luis; Rojo, Pablo; Mellado, Pepa; Navarro, Marisa; Delgado, Rafael; Ramos, José Tomas; Holguín, África

2018-03-01

The expanded use of long-term antiretroviral treatments in infected children may exacerbate the problem of drug resistance mutations selection, which can compromise treatment efficiency. We describe the temporal trends of HIV drug resistance mutations and the HIV-1 variants during 23 years (1993 to March 2016) in the Madrid cohort of HIV-infected children and adolescents. We selected patients with at least one available HIV-1 pol sequence/genotypic resistance profile, establishing different groups according to the sampling year of first resistance data. We determined the prevalence of transmitted drug resistance mutations or acquired drug resistance mutations (DRM), the drug susceptibility among resistant viruses and HIV-1 variants characterized by phylogeny across time. A total of 245 pediatric patients were selected, being mainly female, Spanish native, perinatally infected and carrying HIV-1 subtype B. At first sampling, most pediatric patients were on antiretroviral therapy and heavily pretreated. During 1993 to 2016, transmitted drug resistance mutations was found in 13 (26%) of 50 naive children [non-nucleoside reverse transcriptase inhibitors (NNRTI), 14.6%; nucleoside reverse transcriptase inhibitors (NRTI), 10.4%; protease inhibitors, 8.7%]. DRM appeared in 139 (73.2%) of 190 pretreated patients (NRTI, 64.5%; NNRTI, 36%; protease inhibitors, 35.1%). DRM to NNRTI was higher in last 5 years. Non-B variants infected 14.5% of children and adolescents of the Madrid Cohort, being mainly intersubtype recombinants (76.5%), including complex unique recombinant strains. They caused 3.4% infections before 2000, rising to 85.7% during 2011 to 2016. Periodic surveillance resistance and molecular epidemiology studies in long-term pretreated HIV-infected pediatric populations are required to optimize treatment regimens. Results will permit a better understanding of long-time dynamics of viral resistance and HIV-1 variants in Spain.

Time to Switch to Second-line Antiretroviral Therapy in Children With Human Immunodeficiency Virus in Europe and Thailand.

PubMed

2018-02-01

Data on durability of first-line antiretroviral therapy (ART) in children with human immunodeficiency virus (HIV) are limited. We assessed time to switch to second-line therapy in 16 European countries and Thailand. Children aged <18 years initiating combination ART (≥2 nucleoside reverse transcriptase inhibitors [NRTIs] plus nonnucleoside reverse transcriptase inhibitor [NNRTI] or boosted protease inhibitor [PI]) were included. Switch to second-line was defined as (i) change across drug class (PI to NNRTI or vice versa) or within PI class plus change of ≥1 NRTI; (ii) change from single to dual PI; or (iii) addition of a new drug class. Cumulative incidence of switch was calculated with death and loss to follow-up as competing risks. Of 3668 children included, median age at ART initiation was 6.1 (interquartile range (IQR), 1.7-10.5) years. Initial regimens were 32% PI based, 34% nevirapine (NVP) based, and 33% efavirenz based. Median duration of follow-up was 5.4 (IQR, 2.9-8.3) years. Cumulative incidence of switch at 5 years was 21% (95% confidence interval, 20%-23%), with significant regional variations. Median time to switch was 30 (IQR, 16-58) months; two-thirds of switches were related to treatment failure. In multivariable analysis, older age, severe immunosuppression and higher viral load (VL) at ART start, and NVP-based initial regimens were associated with increased risk of switch. One in 5 children switched to a second-line regimen by 5 years of ART, with two-thirds failure related. Advanced HIV, older age, and NVP-based regimens were associated with increased risk of switch. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Recent trends and patterns in HIV-1 transmitted drug resistance in the United Kingdom.

PubMed

Tostevin, A; White, E; Dunn, D; Croxford, S; Delpech, V; Williams, I; Asboe, D; Pozniak, A; Churchill, D; Geretti, A M; Pillay, D; Sabin, C; Leigh-Brown, A; Smit, E

2017-03-01

Transmission of drug-resistant HIV-1 has decreased in the UK since the early 2000s. This analysis reports recent trends and characteristics of transmitted drug resistance (TDR) in the UK from 2010 to 2013. Resistance tests conducted in antiretroviral treatment (ART)-naïve individuals between 2010 and 2013 were analysed for the presence of transmitted drug resistance mutations (TDRMs), defined as any mutations from a modified 2009 World Health Organization surveillance list, or a modified 2013 International Antiviral Society-USA list for integrase tests. Logistic regression was used to examine associations between demographics and the prevalence of TDRMs. TDRMs were observed in 1223 (7.5%) of 16 425 individuals; prevalence declined from 8.1% in 2010 to 6.6% in 2013 (P = 0.02). The prevalence of TDRMs was higher among men who have sex with men (MSM) compared with heterosexual men and women (8.7% versus 6.4%, respectively) with a trend for decreasing TDRMs among MSM (P = 0.008) driven by a reduction in nucleoside reverse transcriptase inhibitor (NRTI)-related mutations. The most frequently detected TDRMs were K103N (2.2%), T215 revertants (1.6%), M41L (0.9%) and L90M (0.7%). Predicted phenotypic resistance to first-line ART was highest to the nonnucleoside reverse transcriptase inhibitors (NNRTIs) rilpivirine and efavirenz (6.2% and 3.4%, respectively) but minimal to NRTIs, including tenofovir, and protease inhibitors (PIs). No major integrase TDRMs were detected among 101 individuals tested while ART-naïve. We observed a decrease in TDRMs in recent years. However, this was confined to the MSM population and rates remained stable in those with heterosexually acquired HIV infection. Resistance to currently recommended first-line ART, including integrase inhibitors, remained reassuringly low. © 2016 The Authors. HIV Medicine published by John Wiley & Sons Ltd on behalf of British HIV Association.

Viro-immunological response of drug-naive HIV-1-infected patients starting a first-line regimen with viraemia >500,000 copies/ml in clinical practice.

PubMed

Santoro, Maria Mercedes; Di Carlo, Domenico; Armenia, Daniele; Zaccarelli, Mauro; Pinnetti, Carmela; Colafigli, Manuela; Prati, Francesca; Boschi, Andrea; Antoni, Anna Maria Degli; Lagi, Filippo; Sighinolfi, Laura; Gervasoni, Cristina; Andreoni, Massimo; Antinori, Andrea; Mussini, Cristina; Perno, Carlo Federico; Borghi, Vanni; Sterrantino, Gaetana

2017-09-22

Virological success (VS) and immunological reconstitution (IR) of antiretroviral-naive HIV-1-infected patients with pre-therapy viral load (VL) >500,000 copies/ml was assessed after 12 months of treatment according to initial drug-class regimens. An observational multicentre retrospective study was performed. VS was defined as the first VL <50 copies/ml from treatment start. IR was defined as an increase of at least 150 CD4 + T-lymphocytes from treatment start. Survival analysis was used to estimate the probability and predictors of VS and IR by 12 months of therapy. 428 HIV-1-infected patients were analysed. Patients were grouped according to the different first-line drug-classes used: a non-nucleoside reverse transcriptase inhibitor (NNRTI) plus two nucleoside reverse transcriptase inhibitors (NRTIs; NNRTI-group; n=105 [24.5%]); a protease inhibitor (PI) plus two NRTIs (PI-group; n=260 [60.8%]); a four-drug regimen containing a PI-regimen plus an integrase inhibitor (PI+INI-group; n=63 [14.7%]). Patients in the PI-group showed the lowest probability of VS (PI-group: 72.4%; NNRTI-group: 75.5%; PI+INI-group: 81.0%; P<0.0001). By Cox regression, patients in PI+INI and NNRTI-groups showed a higher adjusted hazard ratio (95% CI) of VS compared to those in the PI-group (PI+INI-group: 1.48 [1.08, 2.03]; P=0.014; NNRTI-group: 1.37 [1.06-1.78]; P=0.015). The probability of IR was 76.2%, and was similar among groups. Patients with AIDS showed a lower adjusted hazard ratio (95% CI) of IR compared to non-AIDS presenters (0.70 [0.54, 0.90]; P=0.005). In this multicentre retrospective study, patients with viraemia >500,000 copies/ml who start a first-line regimen containing PI+INI or NNRTI yield a better VS compared to those receiving a PI-based regimen.

Neurocognitive Function at the First-Line Failure and on the Second-Line Antiretroviral Therapy in Africa: Analyses From the EARNEST Trial.

PubMed

Kambugu, Andrew; Thompson, Jennifer; Hakim, James; Tumukunde, Dinah; van Oosterhout, Joep J; Mwebaze, Raymond; Hoppe, Anne; Abach, James; Kwobah, Charles; Arenas-Pinto, Alejandro; Walker, Sarah A; Paton, Nicholas I

2016-04-15

To assess neurocognitive function at the first-line antiretroviral therapy failure and change on the second-line therapy. Randomized controlled trial was conducted in 5 sub-Saharan African countries. Patients failing the first-line therapy according to WHO criteria after >12 months on non-nucleoside reverse transcriptase inhibitors-based regimens were randomized to the second-line therapy (open-label) with lopinavir/ritonavir (400 mg/100 mg twice daily) plus either 2-3 clinician-selected nucleoside reverse transcriptase inhibitors, raltegravir, or as monotherapy after 12-week induction with raltegravir. Neurocognitive function was tested at baseline, weeks 48 and 96 using color trails tests 1 and 2, and the Grooved Pegboard test. Test results were converted to an average of the 3 individual test z-scores. A total of 1036 patients (90% of those >18 years enrolled at 13 evaluable sites) had valid baseline tests (58% women, median: 38 years, viral load: 65,000 copies per milliliter, CD4 count: 73 cells per cubic millimeter). Mean (SD) baseline z-score was -2.96 (1.74); lower baseline z-scores were independently associated with older age, lower body weight, higher viral load, lower hemoglobin, less education, fewer weekly working hours, previous central nervous system disease, and taking fluconazole (P < 0.05 in multivariable model). Z-score was increased by mean (SE) of +1.23 (0.04) after 96 weeks on the second-line therapy (P < 0.001; n = 915 evaluable), with no evidence of difference between the treatment arms (P = 0.35). Patients in sub-Saharan Africa failing the first-line therapy had low neurocognitive function test scores, but performance improved on the second-line therapy. Regimens with more central nervous system-penetrating drugs did not enhance neurocognitive recovery indicating this need not be a primary consideration in choosing a second-line regimen.

High expression of S100A8 gene is associated with drug resistance to etoposide and poor prognosis in acute myeloid leukemia through influencing the apoptosis pathway.

PubMed

Yang, Xiao-Yan; Zhang, Ming-Ying; Zhou, Qi; Wu, Shui-Yan; Zhao, Ye; Gu, Wei-Ying; Pan, Jian; Cen, Jian-Nong; Chen, Zi-Xing; Guo, Wen-Ge; Chen, Chien-Shing; Yan, Wen-Hua; Hu, Shao-Yan

2016-01-01

S100A8 has been increasingly recognized as a biomarker in multiple solid tumors and has played pivotal roles in hematological malignancies. S100A8 is potentially an indicator for poor survival in acute myeloid leukemia (AML) in retrospective studies. However, the mechanisms of S100A8 are diverse in cancers. In this study, we investigated the correlation of S100A8 at the transcription level with clinical parameters in 91 de novo AML patients and explored its mechanisms of chemoresistance to etoposide in vitro. The transcription level of S100A8 was significantly lower at initial and relapse stages of AML samples than at complete remission (P<0.001) and than in the control group (P=0.0078), while no significant difference could be found between initial and relapse stages (P=0.257). Patients with high transcription levels of S100A8 exhibited a shorter overall survival (P=0.0012). HL-60 cells transfected with S100A8 showed resistance to etoposide with a higher level IC50 value and lower apoptosis rate compared with HL-60 cells transfected with empty vector. Thirty-six genes were significantly downregulated and 12 genes were significantly upregulated in S100A8 overexpression group compared with control group in which 360 genes involved in apoptotic genes array were performed by real-time reverse transcriptase polymerase chain reaction. Among them, the caspase-3, Bcl-2, and Bax were verified by Western blot analysis which indicated that the role of S100A8 in resistance to chemotherapy was closely related with antiapoptosis. In conclusion, critical S100A8 provided useful clinical information in predicting the outcome of AML. The main mechanism of S100A8 which promoted chemoresistance was antiapoptosis.

High expression of S100A8 gene is associated with drug resistance to etoposide and poor prognosis in acute myeloid leukemia through influencing the apoptosis pathway

PubMed Central

Yang, Xiao-yan; Zhang, Ming-ying; Zhou, Qi; Wu, Shui-yan; Zhao, Ye; Gu, Wei-ying; Pan, Jian; Cen, Jian-nong; Chen, Zi-xing; Guo, Wen-ge; Chen, Chien-shing; Yan, Wen-hua; Hu, Shao-yan

2016-01-01

S100A8 has been increasingly recognized as a biomarker in multiple solid tumors and has played pivotal roles in hematological malignancies. S100A8 is potentially an indicator for poor survival in acute myeloid leukemia (AML) in retrospective studies. However, the mechanisms of S100A8 are diverse in cancers. In this study, we investigated the correlation of S100A8 at the transcription level with clinical parameters in 91 de novo AML patients and explored its mechanisms of chemoresistance to etoposide in vitro. The transcription level of S100A8 was significantly lower at initial and relapse stages of AML samples than at complete remission (P<0.001) and than in the control group (P=0.0078), while no significant difference could be found between initial and relapse stages (P=0.257). Patients with high transcription levels of S100A8 exhibited a shorter overall survival (P=0.0012). HL-60 cells transfected with S100A8 showed resistance to etoposide with a higher level IC50 value and lower apoptosis rate compared with HL-60 cells transfected with empty vector. Thirty-six genes were significantly downregulated and 12 genes were significantly upregulated in S100A8 overexpression group compared with control group in which 360 genes involved in apoptotic genes array were performed by real-time reverse transcriptase polymerase chain reaction. Among them, the caspase-3, Bcl-2, and Bax were verified by Western blot analysis which indicated that the role of S100A8 in resistance to chemotherapy was closely related with antiapoptosis. In conclusion, critical S100A8 provided useful clinical information in predicting the outcome of AML. The main mechanism of S100A8 which promoted chemoresistance was antiapoptosis. PMID:27540302

Mitochondrial Toxicity Studied with the PBMC of Children from the Chinese National Pediatric Highly Active Antiretroviral Therapy Cohort

PubMed Central

Liu, Daojie; Yin, Jiming; Qiao, Luxin; Shi, Ying; Dong, Yaowu; Li, Ning; Zhang, Fujie; Chen, Dexi

2013-01-01

As the backbone of highly active antiretroviral therapy (HAART), nucleoside reverse transcriptase inhibitors (NRTIs) have effectively improved outcomes for HIV-infected patients. However, long-term treatment with NRTIs can cause a series of pathologies associated with mitochondrial toxicity. To date, the status and mechanism of mitochondrial toxicity induced by NRTIs are still not clear, especially in HIV-infected children. As part of the national pediatric HAART program in China, our study focused on mitochondrial toxicity and its potential mechanism in HIV-1-infected children who were divided into two groups based on their duration of treatment with NRTIs: one group received treatment for less than 36 months and one group was treated for 36 to 72 months. The control group comprised age-matched non-HIV-infected children. Blood lactic acid and ATP levels in peripheral blood mononuclear cells (PBMCs) were measured to evaluate mitochondrial function, and mtDNA copies and mutations in PBMCs were determined for detecting mtDNA lesions. Simultaneously, TK2 and P53R2 gene expression in PBMC was measured. As compared with the control group, blood lactic acid levels in both NRTI treatment groups were significantly higher, whereas ATP levels and mtDNA mutation rates in PBMCs did not differ between the control and the two NRTI treatment groups. Both NRTI treatment groups exhibited significant mtDNA loss. N Moreover, we found that P53R2 mRNA expression and protein levels were significantly reduced in both treatment groups and that TK2 mRNA expression and protein levels were induced in the long-term NRTI treatment group. These results suggest that mitochondrial toxicity occurs in long-term HAART patients and that P53R2 and TK2 levels in PBMCs are useful biomarkers for detecting mitochondrial toxicity in patients on long-term treatment with NRTIs. PMID:23468942

A cross-sectional study to evaluate the association of hyperbilirubinaemia on markers of cardiovascular disease, neurocognitive function, bone mineral density and renal markers in HIV-1 infected subjects on protease inhibitors.

PubMed

Barber, T J; Moyle, G; Hill, A; Jagjit Singh, G; Scourfield, A; Yapa, H M; Waters, L; Asboe, D; Boffito, M; Nelson, M

2016-05-01

Ongoing inflammation in controlled HIV infection contributes to non-AIDS comorbidities. High bilirubin appears to exhibit an anti-inflammatory effect in vivo. We therefore examined whether increased bilirubin in persons with HIV was associated with differences in markers of inflammation and cardiovascular, bone, renal disease, and neurocognitive (NC) impairment. This cross-sectional study examined inflammatory markers in individuals with stable HIV infection treated with two nucleoside reverse transcriptase inhibitors and a boosted protease inhibitor. Individuals recruited were those with a normal bilirubin (NBR; 0-17 μmol/L) or high bilirubin (>2.5 × upper limit of normal). Demographic and anthropological data were recorded. Blood and urine samples were taken for analyses. Pulse wave velocity (PWV) measurement, carotid intimal thickness (CIT), and calcaneal stiffness (CSI) were measured. Males were asked to answer a questionnaire about sexual function; NC testing was performed using CogState. 101 patients were screened, 78 enrolled (43 NBR and 35 HBR). Atazanavir use was significantly higher in HBR. Whilst a trend for lower CIT was seen in those with HBR, no significant differences were seen in PWV, bone markers, calculated cardiovascular risk (Framingham), or erectile dysfunction score. VCAM-1 levels were significantly lower in the HBR group. HBR was associated with lower LDL and triglyceride levels. NBR was associated with a calculated FRAX significantly lower than HBR although no associations were found after adjusting for tenofovir use. No difference in renal markers was observed. Component tests of NC testing revealed differences favouring HBR but overall composite scores were similar. High bilirubin in the context of boosted PI therapy was found not to be associated with differences in with the markers examined in this study. Some trends were noted and, on the basis of these, a larger, clinical end point study is warranted.

Synthesis and biological properties of novel 2-aminopyrimidin-4(3H)-ones highly potent against HIV-1 mutant strains.

PubMed

Mai, Antonello; Artico, Marino; Rotili, Dante; Tarantino, Domenico; Clotet-Codina, Imma; Armand-Ugón, Mercedes; Ragno, Rino; Simeoni, Silvia; Sbardella, Gianluca; Nawrozkij, Maxim B; Samuele, Alberta; Maga, Giovanni; Esté, José A

2007-11-01

Following the disclosure of dihydro-alkoxy-, dihydro-alkylthio-, and dihydro-alkylamino-benzyl-oxopyrimidines (DABOs, S-DABOs, and NH-DABOs) as potent and selective anti-HIV-1 agents belonging to the non-nucleoside reverse transcriptase inhibitor (NNRTI) class, we report here the synthesis and biological evaluation of a novel series of DABOs bearing a N,N-disubstituted amino group or a cyclic amine at the pyrimidine-C2 position, a hydrogen atom or a small alkyl group at C5 and/or at the benzylic position, and the favorable 2,6-difluorobenzyl moiety at the C6 position (F2-N,N-DABOs). The new compounds were highly active up to the subnanomolar level against both wt HIV-1 and the Y181C mutant and at the submicromolar to nanomolar range against the K103N and Y188L mutant strains. Such derivatives were more potent than S-DABOs, NH-DABOs, and nevirapine and efavirenz were chosen as reference drugs. The higher inhibitor adaptability to the HIV-1 RT non-nucleoside binding site (NNBS) may account for the higher inhibitory effect exerted by the new molecules against the mutated RTs.

Antiretroviral therapy-induced mitochondrial toxicity: potential mechanisms beyond polymerase-γ inhibition.

PubMed

Selvaraj, S; Ghebremichael, M; Li, M; Foli, Y; Langs-Barlow, A; Ogbuagu, A; Barakat, L; Tubridy, E; Edifor, R; Lam, W; Cheng, Y-C; Paintsil, E

2014-07-01

We hypothesized that competition between nucleotide reverse-transcriptase inhibitor triphosphate and endogenous deoxyribonucleotide triphosphate (dNTP) may lead to depletion of dNTP pools and mitochondrial dysfunction independent of polymerase-γ (pol-γ) inhibition. We collected peripheral blood mononuclear cells from 75 adults (25 cases: HIV-infected patients with mitochondrial toxicity, 25 HIV-infected positive controls, and 25 HIV-negative controls). We observed statistically significant individual and group differences in ribonucleotide (RN) and deoxyribonucleotide (dRN) pools. The median values for the RN pools were 10,062 (interquartile range (IQR): 7,090-12,590), 4,360 (IQR: 3,058-6,838), and 2,968 (IQR: 2,538-4,436) pmol/10(6) cells for negative controls, positive controls, and cases, respectively. Cases had significantly higher absolute mitochondrial DNA copy number as compared with negative controls (P < 0.05). Moreover, cases had significantly higher expression levels of pol-γ, nucleotide transporters, cellular kinases, and adenosine triphosphate (ATP)-binding cassette (ABC) proteins as compared with controls. Antiretroviral therapy (ART) perturbs RN and dRN pools. Depletion of RN and dRN pools may be associated with ART-induced mitochondrial toxicity independent of pol-γ inhibition.

DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo.

PubMed

Zubradt, Meghan; Gupta, Paromita; Persad, Sitara; Lambowitz, Alan M; Weissman, Jonathan S; Rouskin, Silvi

2017-01-01

Coupling of structure-specific in vivo chemical modification to next-generation sequencing is transforming RNA secondary structure studies in living cells. The dominant strategy for detecting in vivo chemical modifications uses reverse transcriptase truncation products, which introduce biases and necessitate population-average assessments of RNA structure. Here we present dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase. DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features per molecule, and allows both genome-wide studies and focused in vivo investigations of even low-abundance RNAs. We apply DMS-MaPseq for the first analysis of RNA structure within an animal tissue and to identify a functional structure involved in noncanonical translation initiation. Additionally, we use DMS-MaPseq to compare the in vivo structure of pre-mRNAs with their mature isoforms. These applications illustrate DMS-MaPseq's capacity to dramatically expand in vivo analysis of RNA structure.

A trypsin inhibitor from rambutan seeds with antitumor, anti-HIV-1 reverse transcriptase, and nitric oxide-inducing properties.

PubMed

Fang, Evandro Fei; Ng, Tzi Bun

2015-04-01

Nephelium lappaceum L., commonly known as "rambutan," is a typical tropical tree and is well known for its juicy and sweet fruit which has an exotic flavor. Chemical studies on rambutan have led to the identification of various components such as monoterpene lactones and volatile compounds. Here, a 22.5-kDa trypsin inhibitor (N . lappaceum trypsin inhibitor (NLTI)) was isolated from fresh rambutan seeds using liquid chromatographical techniques. NLTI reduced the proteolytic activities of both trypsin and α-chymotrypsin. Dithiothreitol reduced the trypsin inhibitory activity of NLTI at a concentration of 1 mM, indicating that an intact disulfide bond is essential to the activity. NLTI inhibited HIV-1 reverse transcriptase with an IC50 of 0.73 μM. In addition, NLTI manifested a time- and dose-dependent inhibitory effect on growth in many tumor cells. NLTI is one of the few trypsin inhibitors with nitric oxide-inducing activity and may find application in tumor therapy.

The unusually large Plasmodium telomerase reverse-transcriptase localizes in a discrete compartment associated with the nucleolus

PubMed Central

Figueiredo, Luisa M.; Rocha, Eduardo P. C.; Mancio-Silva, Liliana; Prevost, Christine; Hernandez-Verdun, Danièle; Scherf, Artur

2005-01-01

Telomerase replicates chromosome ends, a function necessary for maintaining genome integrity. We have identified the gene that encodes the catalytic reverse transcriptase (RT) component of this enzyme in the malaria parasite Plasmodium falciparum (PfTERT) as well as the orthologous genes from two rodent and one simian malaria species. PfTERT is predicted to encode a basic protein that contains the major sequence motifs previously identified in known telomerase RTs (TERTs). At ∼2500 amino acids, PfTERT is three times larger than other characterized TERTs. We observed remarkable sequence diversity between TERT proteins of different Plasmodial species, with conserved domains alternating with hypervariable regions. Immunofluorescence analysis revealed that PfTERT is expressed in asexual blood stage parasites that have begun DNA synthesis. Surprisingly, rather than at telomere clusters, PfTERT typically localizes into a discrete nuclear compartment. We further demonstrate that this compartment is associated with the nucleolus, hereby defined for the first time in P.falciparum. PMID:15722485

Structural investigation of HIV-1 nonnucleoside reverse transcriptase inhibitors: 2-Aryl-substituted benzimidazoles

NASA Astrophysics Data System (ADS)

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

2009-11-01

Acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) is one of the most destructive epidemics in history. Inhibitors of HIV enzymes are the main targets to develop drugs against that disease. Nonnucleoside reverse transcriptase inhibitors of HIV-1 (NNRTIs) are potentially effective and nontoxic. Structural studies provide information necessary to design more active compounds. The crystal structures of four NNRTI derivatives of 2-aryl-substituted N-benzyl-benzimidazole are presented here. Analysis of the geometrical parameters shows that the structures of the investigated inhibitors are rigid. The important geometrical parameter is the dihedral angle between the planes of the π-electron systems of the benzymidazole and benzyl moieties. The values of these dihedral angles are in a narrow range for all investigated inhibitors. There is no significant difference between the structure of the free inhibitor and the inhibitor in the complex with RT HIV-1. X-ray structures of the investigated inhibitors are a good basis for modeling enzyme-inhibitor interactions in rational drug design.

Intracellular studies of the nucleoside reverse transcriptase inhibitor active metabolites: a review.

PubMed

Rodriguez Orengo, J F; Santana, J; Febo, I; Diaz, C; Rodriguez, J L; Garcia, R; Font, E; Rosario, O

2000-03-01

Nucleoside reverse transcriptase inhibitors (NRTIs) plasma concentrations do not correlate with clinical efficacy or toxicity. These agents need to be phosphorylated to become active against HIV-infection. Thus, the characterization of the NRTIs intracellular metabolite pharmacological parameters will provide a better understanding that could lead to the development of more rational dose regimens in the HIV-infected population. Furthermore, intracellular measurements of NRTIs may provide a better marker with respect to clinical efficacy and toxicity than plasma concentrations. Thus, in this article we review the latest information regarding the intracellular pharmacological parameters of zidovudine (ZDV) and lamivudine (3TC) active metabolites in HIV-infected patients including the results from our recent clinical studies. We will start the discussion with ZDV and 3TC clinical efficacy, followed by systemic pharmacokinetics studies. We will then discuss the in vitro and in vivo intracellular studies with particular emphasis in the method development to measure these metabolites and we will conclude with the most current data from our clinical trials.

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

PubMed Central

Vainio, Kirsti; Myrmel, Mette

2006-01-01

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

Gene 2 of the sigma rhabdovirus genome encodes the P protein, and gene 3 encodes a protein related to the reverse transcriptase of retroelements.

PubMed

Landès-Devauchelle, C; Bras, F; Dezélée, S; Teninges, D

1995-11-10

The nucleotide sequence of the genes 2 and 3 of the Drosophila rhabdovirus sigma was determined from cDNAs to viral genome and poly(A)+ mRNAs. Gene 2 comprises 1032 nucleotides and contains a long ORF encoding a molecular weight 35,208 polypeptide present in infected cells and in virions which migrates in SDS-PAGE as a doublet of M(r) about 60 kDa. The distribution of acidic charges as well as the electrophoretic properties of the protein are characteristic of the rhabdovirus P proteins. Gene 3 comprises 923 nucleotides and contains a long ORF capable of coding a polypeptide of 298 amino acids of MW 33,790. The putative protein (PP3) is similar in size to a minor component of the virions. Computer analysis shows that the sequence of PP3 contains three motifs related to the conserved motifs of reverse transcriptases.

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.

The future of pre-exposure prophylaxis (PrEP) for human immunodeficiency virus (HIV) infection.

PubMed

Özdener, Ayşe Elif; Park, Tae Eun; Kalabalik, Julie; Gupta, Rachna

2017-05-01

People at high risk for HIV acquisition should be offered pre-exposure prophylaxis (PrEP). Tenofovir disoproxil fumarate (TDF)/emtricitabine (FTC) is currently the only medication recommended for pre-exposure prophylaxis (PrEP) by the Centers for Disease Control and Prevention (CDC) in people at high risk for HIV acquisition. This article will review medications currently under investigation and the future landscape of PrEP therapy. Areas covered: This article will review clinical trials that have investigated nontraditional regimens of TDF/FTC, antiretroviral agents from different drug classes such as integrase strand transfer inhibitors (INSTI), nucleoside reverse transcriptase inhibitors (NRTI), and non-nucleoside reverse transcriptase inhibitors (NNRTI) as potential PrEP therapies. Expert commentary: Currently, there are several investigational drugs in the pipeline for PrEP against HIV infection. Increased utilization of PrEP therapy depends on provider identification of people at high risk for HIV transmission. Advances in PrEP development will expand options and access for people and reduce the risk of HIV acquisition.

Pausing kinetics dominates strand-displacement polymerization by reverse transcriptase

PubMed Central

Malik, Omri; Khamis, Hadeel; Rudnizky, Sergei; Marx, Ailie

2017-01-01

Abstract Reverse transcriptase (RT) catalyzes the conversion of the viral RNA into an integration-competent double-stranded DNA, with a variety of enzymatic activities that include the ability to displace a non-template strand concomitantly with polymerization. Here, using high-resolution optical tweezers to follow the activity of the murine leukemia Virus RT, we show that strand-displacement polymerization is frequently interrupted. Abundant pauses are modulated by the strength of the DNA duplex ∼8 bp ahead, indicating the existence of uncharacterized RT/DNA interactions, and correspond to backtracking of the enzyme, whose recovery is also modulated by the duplex strength. Dissociation and reinitiation events, which induce long periods of inactivity and are likely the rate-limiting step in the synthesis of the genome in vivo, are modulated by the template structure and the viral nucleocapsid protein. Our results emphasize the potential regulatory role of conserved structural motifs, and may provide useful information for the development of potent and specific inhibitors. PMID:28973474

Antiretroviral therapy for human immunodeficiency virus infection in 1997.

PubMed Central

Katzenstein, D A

1997-01-01

It has become clear that the acquired immunodeficiency syndrome follows continuous replication of the human immunodeficiency virus (HIV) and a decrease in immune capability, most obviously a decline in the number of CD4 lymphocytes. An understanding of key elements in the infectious life cycle of HIV has led to the development of potent antiretroviral drugs selectively targeting unique reverse transcriptase and protease enzymes of the virus. Completed clinical trials have shown that antiretroviral therapy for HIV infection, begun early, reduces viral replication and reverses the decline in CD4 lymphocyte numbers. Recent studies of combination therapies have shown that decreases in plasma HIV viremia to low levels and sustained increases in CD4 cell numbers are associated with longer survival. Potent combination regimens including protease inhibitors and non-nucleoside reverse transcriptase inhibitors suppress detectable viral replication and have demonstrated clinical benefits in patients with advanced disease. Progress in antiretroviral therapy and methods to monitor responses to treatment are providing new hope in the treatment of HIV infection. PMID:9217434

Reverse transcriptase inhibitors as microbicides.

PubMed

Lewi, Paul; Heeres, Jan; Ariën, Kevin; Venkatraj, Muthusamy; Joossens, Jurgen; Van der Veken, Pieter; Augustyns, Koen; Vanham, Guido

2012-01-01

The CAPRISA 004 study in South Africa has accelerated the development of vaginal and rectal microbicides containing antiretrovirals that target specific enzymes in the reproduction cycle of HIV, especially reverse transcriptase inhibitors (RTI). In this review we discuss the potential relevance of HIV-1 RTIs as microbicides, focusing in the nucleotide RTI tenofovir and six classes of nonnucleoside RTIs (including dapivirine, UC781, urea and thiourea PETTs, DABOs and a pyrimidinedione). Although tenofovir and dapivirine appear to be most advanced in clinical trials as potential microbicides, several issues remain unresolved, e.g., the importance of nonhuman primates as a "gatekeeper" for clinical trials, the emergence and spread of drug-resistant mutants, the combination of microbicides that target different phases of viral reproduction and the accessibility to microbicides in low-income countries. Thus, here we discuss the latest research on RTI as microbicides in the light of the continuing spread of the HIV pandemic from the point of view of medicinal chemistry, virological, and pharmaceutical studies.

The ORF1 Protein Encoded by LINE-1: Structure and Function During L1 Retrotransposition

PubMed Central

Martin, Sandra L.

2006-01-01

LINE-1, or L1 is an autonomous non-LTR retrotransposon in mammals. Retrotransposition requires the function of the two, L1-encoded polypeptides, ORF1p and ORF2p. Early recognition of regions of homology between the predicted amino acid sequence of ORF2 and known endonuclease and reverse transcriptase enzymes led to testable hypotheses regarding the function of ORF2p in retrotransposition. As predicted, ORF2p has been demonstrated to have both endonuclease and reverse transcriptase activities. In contrast, no homologs of known function have contributed to our understanding of the function of ORF1p during retrotransposition. Nevertheless, significant advances have been made such that we now know that ORF1p is a high affinity RNA binding protein that forms a ribonucleoprotein particle together with L1 RNA. Furthermore, ORF1p is a nucleic acid chaperone and this nucleic acid chaperone activity is required for L1 retrotransposition. PMID:16877816

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.

Structural Insights into HIV Reverse Transcriptase Mutations Q151M and Q151M Complex That Confer Multinucleoside Drug Resistance

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

Das, Kalyan; Martinez, Sergio E.; Arnold, Eddy

HIV-1 reverse transcriptase (RT) is targeted by multiple drugs. RT mutations that confer resistance to nucleoside RT inhibitors (NRTIs) emerge during clinical use. Q151M and four associated mutations, A62V, V75I, F77L, and F116Y, were detected in patients failing therapies with dideoxynucleosides (didanosine [ddI], zalcitabine [ddC]) and/or zidovudine (AZT). The cluster of the five mutations is referred to as the Q151M complex (Q151Mc), and an RT or virus containing Q151Mc exhibits resistance to multiple NRTIs. To understand the structural basis for Q151M and Q151Mc resistance, we systematically determined the crystal structures of the wild-type RT/double-stranded DNA (dsDNA)/dATP (complex I), wild-type RT/dsDNA/ddATPmore » (complex II), Q151M RT/dsDNA/dATP (complex III), Q151Mc RT/dsDNA/dATP (complex IV), and Q151Mc RT/dsDNA/ddATP (complex V) ternary complexes. The structures revealed that the deoxyribose rings of dATP and ddATP have 3'-endo and 3'-exo conformations, respectively. The single mutation Q151M introduces conformational perturbation at the deoxynucleoside triphosphate (dNTP)-binding pocket, and the mutated pocket may exist in multiple conformations. The compensatory set of mutations in Q151Mc, particularly F116Y, restricts the side chain flexibility of M151 and helps restore the DNA polymerization efficiency of the enzyme. The altered dNTP-binding pocket in Q151Mc RT has the Q151-R72 hydrogen bond removed and has a switched conformation for the key conserved residue R72 compared to that in wild-type RT. On the basis of a modeled structure of hepatitis B virus (HBV) polymerase, the residues R72, Y116, M151, and M184 in Q151Mc HIV-1 RT are conserved in wild-type HBV polymerase as residues R41, Y89, M171, and M204, respectively; functionally, both Q151Mc HIV-1 and wild-type HBV are resistant to dideoxynucleoside analogs.« less

Sub-fertile sperm cells exemplify telomere dysfunction.

PubMed

Biron-Shental, Tal; Wiser, Amir; Hershko-Klement, Anat; Markovitch, Ofer; Amiel, Aliza; Berkovitch, Arie

2018-01-01

The purpose of this study was to evaluate telomere homeostasis in sub-fertile compared to fertile human sperm. This observational, comparative study included 16 sub-fertile men who required intracytoplasmic sperm injection and 10 fertile men. At least 100 sperm cells from each participant were assessed. Main outcome measures were telomere length and telomere aggregates. Telomerase RNA component (TERC) copy number and telomere capture were assessed using fluorescence in situ hybridization technique and human telomerase reverse transcriptase (hTERT) using immunohistochemistry. Clinical backgrounds were similar. The percentage of sperm cells with shorter telomeres was higher among the sub-fertile compared to the fertile participants (3.3 ± 3.1 vs. 0.6 ± 1.2%, respectively; P < 0.005). The percentage of cells with telomere aggregates was significantly higher in the sub-fertile group (15.12 ± 3.73 vs. 4.73 ± 3.73%; P < 0.005). TERC gene copy number was similar between groups. The percentage of cells that were positive for hTERT was lower in the sub-fertile group (3.81 ± 1.27 vs. 8.42 ± 1.80%; P < 0.005). Telomere capture rates were higher among the sub-fertile sperm cells (P < 0.005). Sub-fertile sperm cells have short telomeres that are elongated by the alternative pathway of telomere capture. Dysfunctional telomeres may affect sperm fertilizability.

WVD2 and WDL1 modulate helical organ growth and anisotropic cell expansion in Arabidopsis

NASA Technical Reports Server (NTRS)

Yuen, Christen Y L.; Pearlman, Rebecca S.; Silo-Suh, Laura; Hilson, Pierre; Carroll, Kathleen L.; Masson, Patrick H.

2003-01-01

Wild-type Arabidopsis roots develop a wavy pattern of growth on tilted agar surfaces. For many Arabidopsis ecotypes, roots also grow askew on such surfaces, typically slanting to the right of the gravity vector. We identified a mutant, wvd2-1, that displays suppressed root waving and leftward root slanting under these conditions. These phenotypes arise from transcriptional activation of the novel WAVE-DAMPENED2 (WVD2) gene by the cauliflower mosaic virus 35S promoter in mutant plants. Seedlings overexpressing WVD2 exhibit constitutive right-handed helical growth in both roots and etiolated hypocotyls, whereas the petioles of WVD2-overexpressing rosette leaves exhibit left-handed twisting. Moreover, the anisotropic expansion of cells is impaired, resulting in the formation of shorter and stockier organs. In roots, the phenotype is accompanied by a change in the arrangement of cortical microtubules within peripheral cap cells and cells at the basal end of the elongation zone. WVD2 transcripts are detectable by reverse transcriptase-polymerase chain reaction in multiple organs of wild-type plants. Its predicted gene product contains a conserved region named "KLEEK," which is found only in plant proteins. The Arabidopsis genome possesses seven other genes predicted to encode KLEEK-containing products. Overexpression of one of these genes, WVD2-LIKE 1, which encodes a protein with regions of similarity to WVD2 extending beyond the KLEEK domain, results in phenotypes that are highly similar to wvd2-1. Silencing of WVD2 and its paralogs results in enhanced root skewing in the wild-type direction. Our observations suggest that at least two members of this gene family may modulate both rotational polarity and anisotropic cell expansion during organ growth.

[New strategy for RNA vectorization in mammalian cells. Use of a peptide vector].

PubMed

Vidal, P; Morris, M C; Chaloin, L; Heitz, F; Divita, G

1997-04-01

A major barrier for gene delivery is the low permeability of nucleic acids to cellular membranes. The development of antisenses and gene therapy has focused mainly on improving methods of oligonucleotide or gene delivery to the cell. In this report we described a new strategy for RNA cell delivery, based on a short single peptide. This peptide vector is derived from both the fusion domain of the gp41 protein of HIV and the nuclear localization sequence of the SV40 large T antigen. This peptide vector localizes rapidly to the cytoplasm then to the nucleus of human fibroblasts (HS-68) within a few minutes and exhibits a high affinity for a single-stranded mRNA encoding the p66 subunit of the HIV-1 reverse transcriptase (in a 100 nM range). The peptide/RNA complex formation involves mainly electrostatic interactions between the basic residues of the peptide and the charges on the phosphate group of the RNA. In the presence of the peptide-vector fluorescently-labelled mRNA is delivered into the cytoplasm of mammalian cells (HS68 human fibroblasts) in less than 1 h with a relatively high efficiency (80%). This new concept based on a peptide-derived vector offers several advantages compared to other compounds commonly used in gene delivery. This vector is highly soluble and exhibits no cytotoxicity at the concentrations used for optimal gene delivery. This result clearly supports the fact that this peptide vector is a powerful tool and that it can be used widely, as much for laboratory research as for new applications and development in gene and/or antisense therapy.

Establishment and characterization of a telomerase immortalized porcine luteal cells.

PubMed

Zhang, Liang; Huang, Yong; Wang, Zhenyu; Luo, Xiaomao; Zhang, Hongling; Du, Qian; Chang, Lingling; Zhao, Xiaomin; Tong, Dewen

2017-05-01

Luteal cells play a crucial role in pregnancy through secreting progesterone to maintain pregnancy and support of fetus. However, low cellular yields and inability to passage primary porcine luteal cells (PLCs) in vitro limit the luteal cell study. Therefore, developing an immortalized porcine luteal cell line is necessary for studying luteal cells activity and function in different diseases. In this study, primary PLCs were obtained from gilts at day 30 to day 50 of gestation and immortalized by human telomerase reverse transcriptase (hTERT). The porcine corpus luteal cell line (hTERT-PLCs) expressed hTERT gene steady, maintained high hTERT activity and normal karyotype. The phase contrast microscope and transmission electron microscope observation showed primary PLCs and hTERT-PLCs were polygonal and exhibited abundant mitochondria, smooth endoplasmic reticulum and lipid droplets. 3β hydroxysteroid dehydrogenase (3βHSD) and Oil-Red-O staining showed that hTERT-PLCs at passage 30 and 50 were similar to primary PLCs. The hTERT-PLCs expressed steroidogenesis-related proteins, enzymes and receptors, such as steroidogenic acute regulatory protein, P450 cholesterol side-chain cleavage, 3βHSD, 20αHSD, luteinizing hormone receptor, progesterone receptor, prolactin receptor, estrogen receptorα/β, as well as primary PLCs. Consequently, hTERT-PLCs could secret progesterone and exhibited similar responses to luteinizing hormone and prostaglandin F2α as primary PLCs. In addition, the hTERT-PLCs did not show neoplastic transformation or anchorage independent growth. In summary, we developed an immortalized porcine luteal cell line which maintained its originally morphological, biological and functional characteristics. Copyright © 2017 Elsevier Inc. All rights reserved.

In vitro and in vivo anti-tumour activities of echinoside A and ds-echinoside A from Pearsonothuria graeffei.

PubMed

Zhao, Qin; Xue, Yong; Wang, Jing-feng; Li, Hui; Long, Teng-teng; Li, Zhaojie; Wang, Yu-ming; Dong, Ping; Xue, Chang-hu

2012-03-15

Echinoside A (EA) and ds-echinoside A (DSEA) are triterpene glycosides isolated from the sea cucumber Pearsonothuria graeffei. DSEA, the desulfurisation product of EA, has the following structure: β-D-xylopyranosyl-holost-8(9),11(12)-diene-3β,17α-diol. In the present study, we examined the anti-tumour activities-in particular, the structure-activity relationships-of EA and DSEA in vitro and in vivo. Both EA and DSEA exhibited an inhibitory effect on cell proliferation, along with apoptosis-inducing activity, in HepG2 cells. Moreover, they significantly arrested the cell cycle in the G₀/G₁ phase. A reverse transcriptase-polymerase chain reaction assay revealed that EA and DSEA significantly increased the expression of the cell-cycle-related genes, namely, p16, p21 and c-myc, and decreased that of cyclin D₁. Western blotting analysis demonstrated that they down-regulated the expression of Bcl-2, and enhanced mitochondria cytochrome c release, caspase-3 activation, and poly(adenosine diphosphate ribose) polymerase, cleavage. Nuclear factor kappa B (NF-κB) expression was significantly decreased by DSEA, but was unaffected by EA. EA and DSEA (2.5 mg kg⁻¹) treatment of mice bearing H22 hepatocarcinoma tumours reduced the tumour weight by 49.8% and 55.0%, respectively. EA and DSEA exhibit marked anti-cancer activity in HepG2 cells, by blocking cell-cycle progression and inducing apoptosis through the mitochondrial pathway. DSEA-induced apoptosis was more potent than EA-induced apoptosis. Furthermore, the two triterpene glycosides derived from P. graeffei may induce apoptosis of HepG2 cells in an NF-κB-dependent or NF-κB-independent manner, depending on their structure. Copyright © 2011 Society of Chemical Industry.

Potential pharmacokinetic interactions between antiretrovirals and medicinal plants used as complementary and African traditional medicines.

PubMed

Müller, Adrienne C; Kanfer, Isadore

2011-11-01

The use of traditional/complementary/alternate medicines (TCAMs) in HIV/AIDS patients who reside in Southern Africa is quite common. Those who use TCAMs in addition to antiretroviral (ARV) treatment may be at risk of experiencing clinically significant pharmacokinetic (PK) interactions, particularly between the TCAMs and the protease inhibitors (PIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). Mechanisms of PK interactions include alterations to the normal functioning of drug efflux transporters, such as P-gp and/or CYP isoenzymes, such a CYP3A4 that mediate the absorption and elimination of drugs in the small intestine and liver. Specific mechanisms include inhibition and activation of these proteins and induction via the pregnane X receptor (PXR). Several clinical studies and case reports involving ARV-herb PK interactions have been reported. St John's Wort, Garlic and Cat's Claw exhibited potentially significant interactions, each with a PI or NNRTI. The potential for these herbs to induce PK interactions with drugs was first identified in reports of in vitro studies. Other in vitro studies have shown that several African traditional medicinal (ATM) plants and extracts may also demonstrate PK interactions with ARVs, through effects on CYP3A4, P-gp and PXR. The most complex effects were exhibited by Hypoxis hemerocallidea, Sutherlandia frutescens, Cyphostemma hildebrandtii, Acacia nilotica, Agauria salicifolia and Elaeodendron buchananii. Despite a high incidence of HIV/AIDs in the African region, only one clinical study, between efavirenz and Hypoxis hemerocallidea has been conducted. However, several issues/concerns still remain to be addressed and thus more studies on ATMs are warranted in order for more meaningful data to be generated and the true potential for such interactions to be determined. Copyright © 2011 John Wiley & Sons, Ltd.

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

USDA-ARS?s Scientific Manuscript database

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

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

EPA Science Inventory

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

Problem-Solving Test: Expression Cloning of the Erythropoietin Receptor

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2008-01-01

Terms to be familiar with before you start to solve the test: cytokines, cytokine receptors, cDNA library, cDNA synthesis, poly(A)[superscript +] RNA, primer, template, reverse transcriptase, restriction endonucleases, cohesive ends, expression vector, promoter, Shine-Dalgarno sequence, poly(A) signal, DNA helicase, DNA ligase, topoisomerases,…

Influenza B virus: alpha-amanitin sensitivity of replication and primer-dependence of in vitro transcription.

PubMed

Mowshowitz, S L; Deval, J

1980-01-01

The replication of influenza B/Lee/40 virus in MDCK (canine kidney) cells was sensitive to alpha-amanitin and actinomycin D. In vitro, virion transcriptase activity was stimulated by dinucleotide primers such as ApG. The above characteristics are shared by A/WSN virus.

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...

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...

Quantification of HTLV-1 reverse transcriptase activity in ATL patients treated with zidovudine and interferon-α.

PubMed

Macchi, Beatrice; Balestrieri, Emanuela; Frezza, Caterina; Grelli, Sandro; Valletta, Elena; Marçais, Ambroise; Marino-Merlo, Francesca; Turpin, Jocelyn; Bangham, Charles R; Hermine, Olivier; Mastino, Antonio; Bazarbachi, Ali

2017-05-09

The therapeutic efficacy of the AZT and IFN combination in ATL presumably reflects the inhibition of RT-related functions.HTLV-1-RT activity from short-term cultured PBMCs may represent a predictive correlate of clinical response to AZT/IFN in ATL patients.

Variation of M3 muscarinic receptor expression in different prostate tissues and its significance.

PubMed

Song, Wei; Yuan, Mingzhen; Zhao, Shengtian

2009-08-01

To detect the expression of the muscarinic receptor (M receptor) in different prostate tissues and analyze the role of its subtype in prostatic oncogenesis. Thirty-six cases of normal prostate and benign prostatic hyperplasia, and 8 cases of prostatic tumor, were used in this study from the Shandong University, Shandong, China, between 2003-2006. The protein expressions of M1, M2, and M3 receptors in each group were determined by Western-blotting. The gene expressions of the M3 receptor and vascular endothelial growth factors (VEGF) in each group were determined by reverse transcriptase-polymerase chain reaction. The protein and gene expressions of the M3 receptor in the prostatic carcinoma group were higher than that of benign prostatic hyperplasia group (p=0.0001) and normal prostate group (p=0.0001). The M3 receptor and VEGF showed positive straight-line correlations of gene expressions with the 3 groups (r=0.4999, p=0.0001). The M3 receptor may have a close relationship with prostatic oncogenesis.

Construction of a functional silk-based biomaterial complex with immortalized chondrocytes in vivo.

PubMed

Ni, Yusu; Jiang, Yi; Wen, Jianchuan; Shao, Zhenzhong; Chen, Xin; Sun, Shan; Yu, Huiqian; Li, Wen

2014-04-01

To explore the feasibility of constructing a functional biomaterial complex with regenerated silk fibroin membrane and immortalized chondrocytes in vivo. Rat auricular chondrocytes (RACs) were transfected with the lentivirus vector pGC-FU-hTERT-3FLAG or pGC-FU-GFP-3FLAG, encoding the human telomerase reverse transcriptase (hTERT) or GFP gene. The effects of regenerated silk fibroin film on the adhesion, growth of immortalized chondrocytes and expression of collagen II in vitro were analyzed with immunofluorescent histochemistry. Immortalized RACs were transformed. Induction by nutrient medium promoted higher expression levels of collagen II in transformed chondrocytes. The regenerated silk fibroin film was not cytotoxic to immortalized chondrocytes and had no adverse influence on their adhesion. Collagen II expression was good in the immortalized chondrocytes in vivo. The construction of a silk-based biomaterial complex with immortalized chondrocytes may provide a feasible kind of functional biomaterial for the repair of cartilage defects in clinical applications. Copyright © 2013 Wiley Periodicals, Inc.

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

PubMed

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

2017-06-01

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

Comparative proteomic analysis of outer membrane protein 43 (omp43)-deficient Bartonella henselae.

PubMed

Kang, Jun-Gu; Lee, Hee-Woo; Ko, Sungjin; Chae, Joon-Seok

2018-01-31

Outer membrane proteins (OMPs) of Gram-negative bacteria constitute the first line of defense protecting cells against environmental stresses including chemical, biophysical, and biological attacks. Although the 43-kDa OMP (OMP43) is major porin protein among Bartonella henselae -derived OMPs, its function remains unreported. In this study, OMP43-deficient mutant B. henselae (Δomp43) was generated to investigate OMP43 function. Interestingly, Δ omp 43 exhibited weaker proliferative ability than that of wild-type (WT) B. henselae . To study the differences in proteomic expression between WT and Δ omp 43, two-dimensional gel electrophoresis-based proteomic analysis was performed. Based on Clusters of Orthologus Groups functional assignments, 12 proteins were associated with metabolism, 7 proteins associated with information storage and processing, and 3 proteins associated with cellular processing and signaling. By semi-quantitative reverse transcriptase polymerase chain reaction, increases in tld D, efp, ntr X, pdh A, pur B, and ATPA mRNA expression and decreases in Rho and yfe A mRNA expression were confirmed in Δ omp 43. In conclusion, this is the first report showing that a loss of OMP43 expression in B. henselae leads to retarded proliferation. Furthermore, our proteomic data provide useful information for the further investigation of mechanisms related to the growth of B. henselae.

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

PubMed Central

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

2014-01-01

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

Molecular Cloning and Tissue-Specific Expression of an Anionic Peroxidase in Zucchini1

PubMed Central

Carpin, Sabine; Crèvecoeur, Michèle; Greppin, Hubert; Penel, Claude

1999-01-01

A calcium-pectate-binding anionic isoperoxidase (APRX) from zucchini (Cucurbita pepo) was purified and subjected to N-terminal amino acid microsequencing. The cDNA encoding this enzyme was obtained by reverse transcriptase polymerase chain reaction from a cDNA library. It encoded a mature protein of 309 amino acids exhibiting all of the sequence characteristics of a plant peroxidase. Despite the presence of a C-terminal propeptide, APRX was found in the apoplast. APRX protein and mRNA were found in the root, hypocotyls, and cotyledons. In situ hybridization showed that the APRX-encoding gene was expressed in many different tissues. The strongest expression was observed in root epidermis and in some cells of the stele, in differentiating tracheary elements of hypocotyl, in the lower and upper epidermis, in the palisade parenchyma of cotyledons, and in lateral and adventitious root primordia. In the hypocotyl hook there was an asymmetric expression, with the inner part containing more transcripts than the outer part. Treatment with 2,3,5-triiodobenzoic acid reduced the expression of the APRX-encoding gene in the lower part of the hypocotyl. Our observations suggest that APRX could be involved in lignin formation and that the transcription of its gene was related to auxin level. PMID:10398715

Heterozygous RTEL1 mutations are associated with familial pulmonary fibrosis.

PubMed

Kannengiesser, Caroline; Borie, Raphael; Ménard, Christelle; Réocreux, Marion; Nitschké, Patrick; Gazal, Steven; Mal, Hervé; Taillé, Camille; Cadranel, Jacques; Nunes, Hilario; Valeyre, Dominique; Cordier, Jean François; Callebaut, Isabelle; Boileau, Catherine; Cottin, Vincent; Grandchamp, Bernard; Revy, Patrick; Crestani, Bruno

2015-08-01

Pulmonary fibrosis is a fatal disease with progressive loss of respiratory function. Defective telomere maintenance leading to telomere shortening is a cause of pulmonary fibrosis, as mutations in the telomerase component genes TERT (reverse transcriptase) and TERC (RNA component) are found in 15% of familial pulmonary fibrosis (FPF) cases. However, so far, about 85% of FPF remain genetically uncharacterised.Here, in order to identify new genetic causes of FPF, we performed whole-exome sequencing, with a candidate-gene approach, of 47 affected subjects from 35 families with FPF without TERT and TERC mutations.We identified heterozygous mutations in regulator of telomere elongation helicase 1 (RTEL1) in four families. RTEL1 is a DNA helicase with roles in DNA replication, genome stability, DNA repair and telomere maintenance. The heterozygous RTEL1 mutations segregated as an autosomal dominant trait in FPF, and were predicted by structural analyses to severely affect the function and/or stability of RTEL1. In agreement with this, RTEL1-mutated patients exhibited short telomeres in comparison with age-matched controls.Our results provide evidence that heterozygous RTEL1 mutations are responsible for FPF and, thereby, extend the clinical spectrum of RTEL1 deficiency. Thus, RTEL1 enlarges the number of telomere-associated genes implicated in FPF. Copyright ©ERS 2015.

Proteomic and transcriptional analysis of Lactobacillus johnsonii PF01 during bile salt exposure by iTRAQ shotgun proteomics and quantitative RT-PCR.

PubMed

Lee, Ji Yoon; Pajarillo, Edward Alain B; Kim, Min Jeong; Chae, Jong Pyo; Kang, Dae-Kyung

2013-01-04

Lactobacillus johnsonii PF01 has been reported to be highly resistant to bile, a key property of probiotic microorganisms. Here, we examine the nature of the bile-salt tolerance of L. johnsonii PF01. Growth inhibition and surface morphology and physiology aberrations were observed after overnight exposure to bile stress. Quantitative proteomic profiles using iTRAQ-LC-MS/MS technology identified 8307 peptides from both untreated PF01 cells and those exposed to 0.1%, 0.2%, and 0.3% bile salts. Some 215 proteins exhibited changed levels in response to bile stress; of these, levels of 94 peptides increased while those of 121 decreased. These were classified into the following categories: stress responses, cell division, transcription, translation, nucleotide metabolism, carbohydrate transport and metabolism, cell wall biosynthesis, and amino acid biosynthesis, and 16 of unidentified function. Analysis of the mRNA expression of selected genes by quantitative reverse transcriptase-PCR verified the proteomic data. Both proteomic and mRNA data provided evidence for increased phosphotransferase activity and cell wall biosynthesis. In addition, three bile salt hydrolases were significantly upregulated by bile exposure. These findings provide a basis for future evaluations of the tolerance of potential probiotic strains toward the various gastrointestinal challenges, including bile stress.

Overexpression of Telomerase Reverse Transcriptase Induces Autism-like Excitatory Phenotypes in Mice.

PubMed

Kim, Ki Chan; Rhee, Jeehae; Park, Jong-Eun; Lee, Dong-Keun; Choi, Chang Soon; Kim, Ji-Woon; Lee, Han-Woong; Song, Mi-Ryoung; Yoo, Hee Jeong; Chung, ChiHye; Shin, Chan Young

2016-12-01

In addition to its classical role as a regulator of telomere length, recent reports suggest that telomerase reverse transcriptase (TERT) plays a role in the transcriptional regulation of gene expression such as β-catenin-responsive pathways. Silencing or over-expression of TERT in cultured NPCs demonstrated that TERT induced glutamatergic neuronal differentiation. During embryonic brain development, expression of transcription factors involved in glutamatergic neuronal differentiation was increased in mice over-expressing TERT (TERT-tg mice). We observed increased expression of NMDA receptor subunits and phosphorylation of α-CaMKII in TERT-tg mice. TERT-tg mice showed autism spectrum disorder (ASD)-like behavioral phenotypes as well as lowered threshold against electrically induced seizure. Interestingly, the NMDA receptor antagonist memantine restored behavioral abnormalities in TERT-tg mice. Consistent with the alteration in excitatory/inhibitory (E/I) ratio, TERT-tg mice showed autism-like behaviors, abnormal synaptic organization, and function in mPFC suggesting the role of altered TERT activity in the manifestation of ASD, which is further supported by the significant association of certain SNPs in Korean ASD patients.

Synthesis, Biological Activity, and Crystal Structure of Potent Nonnucleoside Inhibitors of HIV-1 Reverse Transcriptase That Retain Activity against Mutant Forms of the Enzyme†

PubMed Central

Morningstar, Marshall L.; Roth, Thomas; Farnsworth, David W.; Smith, Marilyn Kroeger; Watson, Karen; Buckheit, Robert W.; Das, Kalyan; Zhang, Wanyi; Arnold, Eddy; Julias, John G.; Hughes, Stephen H.; Michejda, Christopher J.

2010-01-01

In an ongoing effort to develop novel and potent nonnucleoside HIV-1 reverse transcriptase (RT) inhibitors that are effective against the wild type (WT) virus and clinically observed mutants, 1,2-bis-substituted benzimidazoles were synthesized and tested. Optimization of the N1 and C2 positions of benzimidazole led to the development of 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-4-methylbenzimidazole (1) (IC50 = 0.2 μM, EC50 = 0.44 μM, and TC50 ≥ 100 against WT). This paper describes how substitution on the benzimidazole ring profoundly affects activity. Substituents at the benzimidazole C4 dramatically enhanced potency, while at C5 or C6 substituents were generally detrimental or neutral to activity, respectively. A 7-methyl analogue did not inhibit HIV-1 RT. Determination of the crystal structure of 1 bound to RT provided the basis for accurate modeling of additional analogues, which were synthesized and tested. Several derivatives were nanomolar inhibitors of wild-type virus and were effective against clinically relevant HIV-1 mutants. PMID:17663538

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

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.

Appearance of drug resistance-associated mutations in human immunodeficiency virus type 1 protease and reverse transcriptase derived from drug-treated Indonesian patients.

PubMed

Khairunisa, Siti Qamariyah; Kotaki, Tomohiro; Witaningrum, Adiana Mutamsari; Yunifiar M, Muhammad Qushai; Sukartiningrum, Septhia Dwi; Nasronudin; Kameoka, Masanori

2015-02-01

Although HIV-1 drug resistance is a major obstacle in Indonesia, information on drug resistance is limited. In this study, the viral subtype and appearance of drug resistance mutations in the HIV-1 protease (PR) and reverse transcriptase (RT) genes were determined among drug-treated, HIV-1-infected patients in Surabaya. HIV-1 patients who received antiretroviral therapy (ART) more than 2 years were randomly recruited regardless of the viral load or ART failure. Fifty-eight HIV-1 PR genes and 53 RT genes were sequenced. CRF01_AE viruses were identified as the predominant strain. Major drug resistance mutations were not detected in the PR genes. In contrast, 37.7% (20/53) of the participants had one or more major drug resistance mutations in the RT genes, predominantly M184V (28.3%), K103N (11.3%), and thymidine analogue mutations (TAMs) (20.8%). The high prevalence of drug resistance mutations in RT genes indicated the necessity of monitoring the effectiveness of ART in Indonesia.

Molecular Characterization of the Human Immunodeficiency Virus Type 1 in Women and Their Vertically Infected Children.

PubMed

Vaz, Sara Nunes; Giovanetti, Marta; Rego, Filipe Ferreira de Almeida; Oliveira, Tulio de; Danaviah, Siva; Gonçalves, Maria Luiza Freire; Alcantara, Luiz Carlos Junior; Brites, Carlos

2015-10-01

Approximately 35 million people worldwide are infected with human immunodeficiency virus (HIV) around 3.2 million of whom are children under 15 years. Mother-to-child-transmission (MTCT) of HIV-1 accounts for 90% of all infections in children. Despite great advances in the prevention of MTCT in Brazil, children are still becoming infected. Samples from 19 HIV-1-infected families were collected. DNA was extracted and fragments from gag, pol, and env were amplified and sequenced directly. Phylogenetic reconstruction was performed. Drug resistance analyses were performed in pol and env sequences. We found 82.1% of subtype B and 17.9% of BF recombinants. A prevalence of 43.9% drug resistance-associated mutations in pol sequences was identified. Of the drug-naive children 33.3% presented at least one mutation related to protease inhibitor/nucleoside reverse transcriptase inhibitor/nonnucleoside reverse transcriptase inhibitor (PI/NRTI/NNRTI) resistance. The prevalence of transmitted drug resistance mutations was 4.9%. On env we found a low prevalence of HR1 (4.9%) and HR2 (14.6%) mutations.

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.

TNF α is involved in neuropathic pain induced by nucleoside reverse transcriptase inhibitor in rats

PubMed Central

Zheng, Xuexing; Ouyang, Handong; Liu, Shue; Mata, Marina; Fink, David J.; Hao, Shuanglin

2011-01-01

In patients with HIV/AIDS, neuropathic pain is a common neurological complication. Infection with the HIV itself may lead to neuropathic pain, and painful symptoms are enhanced when patients are treated with nucleoside reverse transcriptase inhibitors (NRTI). The mechanisms by which NRTIs contribute to the development of neuropathic pain are not known. In the current studies, we tested the role of TNFα in antiretroviral drug-induced neuropathic pain. We administered 2′,3′-dideoxycytidine (ddC, one of the NRTIs) systemically to induce mechanical allodynia. We found that ddC induced overexpression of both mRNA and proteins of GFAP and TNFα in the spinal dorsal horn. TNFα was colocalized with GFAP in the spinal dorsal horn and with NeuN in the DRG. Knockdown of TNFα with siRNA blocked the mechanical allodynia induced by ddC. Intrathecal administration of glial inhibitor or recombinant TNF soluble receptor, reversed mechanical allodynia induced by ddC. These results suggest that TNFα is involved in NRTI-induced neuropathic pain. PMID:21741472

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.

Crystal structures of the reverse transcriptase-associated ribonuclease H domain of xenotropic murine leukemia-virus related virus

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

Zhou, Dongwen; Chung, Suhman; Miller, Maria

2012-06-19

The ribonuclease H (RNase H) domain of retroviral reverse transcriptase (RT) plays a critical role in the life cycle by degrading the RNA strands of DNA/RNA hybrids. In addition, RNase H activity is required to precisely remove the RNA primers from nascent (-) and (+) strand DNA. We report here three crystal structures of the RNase H domain of xenotropic murine leukemia virus-related virus (XMRV) RT, namely (i) the previously identified construct from which helix C was deleted, (ii) the intact domain, and (iii) the intact domain complexed with an active site {alpha}-hydroxytropolone inhibitor. Enzymatic assays showed that the intactmore » RNase H domain retained catalytic activity, whereas the variant lacking helix C was only marginally active, corroborating the importance of this helix for enzymatic activity. Modeling of the enzyme-substrate complex elucidated the essential role of helix C in binding a DNA/RNA hybrid and its likely mode of recognition. The crystal structure of the RNase H domain complexed with {beta}-thujaplicinol clearly showed that coordination by two divalent cations mediates recognition of the inhibitor.« less

Apoptosis and reduced cell proliferation of HL-60 cell line caused by human telomerase reverse transcriptase inhibition by siRNA.

PubMed

Miri-Moghaddam, Ebrahim; Deezagi, Abdolkhaleg; Soheili, Zahra Sohaila; Shariati, Parvin

2010-01-01

The close correlation between telomerase activity and human telomerase reverse transcriptase (hTERT) expression has made hTERT to be considered as a selective molecular target for human cancer therapy. In this study, the ability of short-interfering RNA (siRNA) to downregulate hTERT expression and its correlation with cell growth and apoptosis in the promyelocytic cell line HL-60 was evaluated. hTERT siRNA was designed and transfected to HL-60. hTERT mRNA expression, cell proliferation and apoptotic cells were measured. The results indicated that hTERT siRNA resulted in 97.2 ± 0.6% downregulation of the hTERT mRNA content; inhibition of the cell proliferation rate was about 52.8 ± 2.3% and the apoptotic index of cells was 30.5 ± 1.5%. hTERT plays an essential role in cell proliferation and control of the viability of leukemic cells, thus promising the development of drugs for leukemia. Copyright © 2010 S. Karger AG, Basel.

Schizosaccharomyces pombe Retrotransposon Tf2 Mobilizes Primarily through Homologous cDNA Recombination

PubMed Central

Hoff, Eleanor F.; Levin, Henry L.; Boeke, Jef D.

1998-01-01

The Tf2 retrotransposon, found in the fission yeast Schizosaccharomyces pombe, is nearly identical to its sister element, Tf1, in its reverse transcriptase-RNase H and integrase domains but is very divergent in the gag domain, the protease, the 5′ untranslated region, and the U3 domain of the long terminal repeats. It has now been demonstrated that a neo-marked copy of Tf2 overexpressed from a heterologous promoter can mobilize into the S. pombe genome and produce true transposition events. However, the Tf2-neo mobilization frequency is 10- to 20-fold lower than that of Tf1-neo, and 70% of the Tf2-neo events are homologous recombination events generated independently of a functional Tf2 integrase. Thus, the Tf2 element is primarily dependent on homologous recombination with preexisting copies of Tf2 for its propagation. Finally, production of Tf2-neo proteins and cDNA was also analyzed; surprisingly, Tf2 was found to produce its reverse transcriptase as a single species in which it is fused to protease, unlike all other retroviruses and retrotransposons. PMID:9774697

A Laccase with HIV-1 Reverse Transcriptase Inhibitory Activity from the Broth of Mycelial Culture of the Mushroom Lentinus tigrinus

PubMed Central

Xu, LiJing; Wang, HeXiang; Ng, TziBun

2012-01-01

A 59 kDa laccase with inhibitory activity against HIV-1 reverse transcriptase (IC50 = 2.4 μM) was isolated from the broth of mycelial culture of the mushroom Lentinus tigrinus. The isolation procedure involved ion exchange chromatography on DEAE-cellulose and CM-cellulose, and gel filtration by fast protein liquid chromatography on Superdex 75. The laccase was adsorbed on both types of ion exchangers. About 95-fold purification was achieved with a 25.9% yield of the enzyme. The procedure resulted in a specific enzyme activity of 76.6 U/mg. Its N-terminal amino acid sequence was GIPDLHDLTV, which showed little similarity to other mushroom laccase and other Lentinus tigrinus strain laccase. Its characteristics were different from previously reported laccase of other Lentinus tigrinus strain. Maximal laccase activity was observed at a pH of 4 and at a temperature of 60°C, respectively. This study yielded the information about the potentially exploitable activities of Lentinus tigrinus laccase. PMID:22536022

The Reverse Transcriptases Associated with CRISPR-Cas Systems.

PubMed

Toro, Nicolás; Martínez-Abarca, Francisco; González-Delgado, Alejandro

2017-08-02

CRISPR (clustered regularly interspaced short palindromic repeats) and associated proteins (Cas) act as adaptive immune systems in bacteria and archaea. Some CRISPR-Cas systems have been found to be associated with putative reverse transcriptases (RT), and an RT-Cas1 fusion associated with a type III-B system has been shown to acquire RNA spacers in vivo. Nevertheless, the origin and evolutionary relationships of these RTs and associated CRISPR-Cas systems remain largely unknown. We performed a comprehensive phylogenetic analysis of these RTs and associated Cas1 proteins, and classified their CRISPR-Cas modules. These systems were found predominantly in bacteria, and their presence in archaea may be due to a horizontal gene transfer event. These RTs cluster into 12 major clades essentially restricted to particular phyla, suggesting host-dependent functioning. The RTs and associated Cas1 proteins may have largely coevolved. They are, therefore, subject to the same selection pressures, which may have led to coadaptation within particular protein complexes. Furthermore, our results indicate that the association of an RT with a CRISPR-Cas system has occurred on multiple occasions during evolution.

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.

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

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.

DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo

PubMed Central

Zubradt, Meghan; Gupta, Paromita; Persad, Sitara; Lambowitz, Alan M.; Weissman, Jonathan S.; Rouskin, Silvi

2017-01-01

Coupling structure-specific in vivo chemical modification to next-generation sequencing is transforming RNA secondary structural studies in living cells. The dominant strategy for detecting in vivo chemical modifications uses reverse transcriptase truncation products, which introduces biases and necessitates population-average assessments of RNA structure. Here we present dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase (TGIRT). DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features per molecule, and allows both genome-wide studies and focused in vivo investigations of even low abundance RNAs. We apply DMS-MaPseq for the first analysis of RNA structure within an animal tissue and to identify a functional structure involved in non-canonical translation initiation. Additionally, we use DMS-MaPseq to compare the in vivo structure of pre-mRNAs to their mature isoforms. These applications illustrate DMS-MaPseq’s capacity to dramatically expand in vivo analysis of RNA structure. PMID:27819661

Micronuclei induced by reverse transcriptase inhibitors in mononucleated and binucleated cells as assessed by the cytokinesis-block micronucleus assay

PubMed Central

2010-01-01

This study evaluated the clastogenic and/or aneugenic potential of three nucleoside reverse transcriptase inhibitors (zidovudine - AZT, lamivudine - 3TC and stavudine - d4T) using the cytokinesis-block micronucleus (CBMN) assay in human lymphocyte cultures. All three inhibitors produced a positive response when tested in binucleated cells. The genotoxicity of AZT and 3TC was restricted to binucleated cells since there was no significant increase in the frequency of micronuclei in mononucleated cells. This finding indicated that AZT and 3TC caused chromosomal breakage and that their genotoxicity was related to a clastogenic action. In addition to the positive response observed with d4T in binucleated cells, this drug also increased the frequency of micronuclei in mononucleated cells, indicating clastogenic and aneugenic actions. Since the structural differences between AZT and 3TC and AZT and d4T involve the 3' position in the 2'-deoxyribonucleoside and in an unsaturated 2',3',dideoxyribose, respectively, we suggest that an unsaturated 2', 3', dideoxyribose is responsible for the clastogenic and aneugenic actions of d4T. PMID:21637587

Characterization of UC781-Tenofovir Combination Gel Products for HIV-1 Infection Prevention in an Ex Vivo Ectocervical Model

PubMed Central

Cost, Marilyn; Dezzutti, Charlene S.; Clark, Meredith R.; Friend, David R.; Akil, Ayman

2012-01-01

HIV continues to be a problem worldwide. Topical vaginal microbicides represent one option being evaluated to stop the spread of HIV. With drug candidates that have a specific action against HIV now being studied, it is important that, when appropriate and based on the mechanism of action, the drug permeates the tissue so that it can be delivered to specific targets which reside there. Novel formulations of the nucleotide reverse transcriptase inhibitor tenofovir (TFV) and the nonnucleoside reverse transcriptase inhibitor UC781 have been developed and evaluated here. Gels with three distinct rheological properties were prepared. The three gels released both UC781 and TFV under in vitro conditions at concentrations equal to or above the reported 50% effective concentrations (EC50s). The drug concentrations in ectocervical tissues were well in excess of the reported EC50s. The gels maintain ectocervical viability and prevent infection of ectocervical explants after a HIV-1 challenge. This study successfully demonstrates the feasibility of using this novel combination of antiretroviral agents in an aqueous gel as an HIV infection preventative. PMID:22430977

Association between Antioxidant Enzyme Activities and Enterovirus-Infected Type 1 Diabetic Children.

PubMed

Abdel-Moneim, Adel; El-Senousy, Waled M; Abdel-Latif, Mahmoud; Khalil, Rehab G

2018-01-01

To examine the effect of infection with Enterovirus (EV) in children with type 1 diabetes (T1D) on the activities of serum antioxidant enzymes in diabetic and nondiabetic controls. Three hundred and eighty-two diabetic and 100 nondiabetic children were tested for EV RNA using reverse transcriptase (RT)-PCR. The activities of serum superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were also estimated in diabetic patients infected with EV (T1D-EV+), those not infected with EV (T1D-EV-), and in nondiabetic controls. The frequency of EV was higher in diabetic children (100/382; 26.2%) than in healthy controls (0/100). Levels of fasting blood glucose (FBG), glycosylated hemoglobin (HbA1c) and C-reactive protein (CRP) were significantly higher but C-peptide was significantly lower in diabetic children than in controls. CRP levels were higher in the T1D-EV+ group than in the T1D-EV- group, and higher in all diabetic children than in nondiabetic controls. The activities of the antioxidant enzymes GPx, SOD, and CAT decreased significantly in diabetic children compared to in controls. Moreover, the activities of the enzymes tested were significantly reduced in the T1D-EV+ group compared to in the T1D-EV- group. Our data indicate that EV infection correlated with a decrease in the activity of antioxidant enzymes in the T1D-EV+ group compared to in the T1D-EV- group; this may contribute to β cell damage and increased inflammation. © 2018 The Author(s) Published by S. Karger AG, Basel.

CD4 responses in the setting or suboptimal virological responses to antiretroviral therapy: features, outcomes, and associated factors.

PubMed

Collazos, Julio; Asensi, Víctor; Cartón, José Antonio

2009-07-01

The factors associated with discordant viroimmunological responses following antiretroviral therapy are unclear. We studied 1380 patients who initiated a protease inhibitor (PI)-based antiretroviral regimen and who fulfilled the criteria for inclusion. Of them, 255 (18.5%) had CD4 increases > or =100 cells/microl after 1 year of therapy despite detectable viral load (immunological responders); they were compared with 669 patients (48.5%) who had CD4 increases <100 cells/microl regardless of their final viral load (immunological nonresponders). Immunological responders had higher rates of sexual acquisition of HIV (p = 0.03), lower rates of clinical progression (p = 0.02), higher probabilities of being naive to antiretroviral therapy (p = 0.006) or to PI if antiretroviral experienced (p = 0.03), higher rates of receiving only nucleoside reverse transcriptase inhibitors in addition to the PI (p = 0.04), and lower baseline CD4 counts (p = 0.007) and higher viral loads (p = 0.009), as compared with nonresponders. Multivariate analysis revealed that sexual transmission of HIV (homosexual p = 0.004, heterosexual p = 0.03), no prior PI experience (p = 0.005), absence of clinical progression (p = 0.02), and lower baseline CD4 counts (p = 0.03) were independently associated with immunological response. However, these factors differed according to the patients' prior antiretroviral status, as higher baseline viral load was also associated with immunological response in antiretroviral-experienced patients (p = 0.02), whereas baseline CD4 count (p = 0.007) was the only predictive parameter in antiretroviral-naive patients. We conclude that immunological responses despite suboptimal viral suppression are common. Prior PI experience, HIV transmission category, baseline CD4 counts, and clinical progression were independently predictive of this condition, although the associated factors were different depending on the patient's prior antiretroviral history.

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.

Ghrelin and cholecystokinin in term and preterm human breast milk.

PubMed

Kierson, Jennifer A; Dimatteo, Darlise M; Locke, Robert G; Mackley, Amy B; Spear, Michael L

2006-08-01

To determine whether ghrelin and cholecystokinin (CCK) are present in significant quantities in term and preterm human breast milk, and to identify their source. Samples were collected from 10 mothers who delivered term infants and 10 mothers who delivered preterm infants. Estimated fat content was measured. Ghrelin and CCK levels were measured in whole and skim breast milk samples using radioimmunoassays (RIA). Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using RNA from human mammary epithelial cells (hMECs) and mammary gland with primers specific to ghrelin. The median ghrelin level in whole breast milk was 2125 pg/ml, which is significantly higher than normal plasma levels. There was a direct correlation between whole milk ghrelin levels and estimated milk fat content (r=0.84, p<0.001). Both the mammary gland and hMECs produced ghrelin. While CCK was detected in some samples, levels were insignificant. Infant gestational age, birthweight, maternal age, and maternal pre-pregnancy body mass index did not significantly affect the results. Ghrelin, but not CCK, is present in breast milk. Since the mammary gland produces ghrelin message, and ghrelin levels in breast milk are higher than those found in plasma, we conclude that ghrelin is produced and secreted by the breast.

Cloning, Developmental, and Tissue-Specific Expression of Sucrose:Sucrose 1-Fructosyl Transferase from Taraxacum officinale. Fructan Localization in Roots1

PubMed Central

Van den Ende, Wim; Michiels, An; Van Wonterghem, Dominik; Vergauwen, Rudy; Van Laere, André

2000-01-01

Sucrose:sucrose 1-fructosyl transferase (1-SST) is the key enzyme initiating fructan synthesis in Asteraceae. Using reverse transcriptase-PCR, we isolated the cDNA for 1-SST from Taraxacum officinale. The cDNA-derived amino acid sequence showed very high homology to other Asteracean 1-SSTs (Cichorium intybus 86%, Cynara scolymus 82%, Helianthus tuberosus 80%), but homology to 1-SST from Allium cepa (46%) and Aspergillus foetidus (18%) was much lower. Fructan concentrations, 1-SST activities, 1-SST protein, and mRNA concentrations were compared in different organs during vegetative and generative development of T. officinale plants. Expression of 1-SST was abundant in young roots but very low in leaves. 1-SST was also expressed at the flowering stages in roots, stalks, and receptacles. A good correlation was found between northern and western blots showing transcriptional regulation of 1-SST. At the pre-flowering stage, 1-SST mRNA concentrations and 1-SST activities were higher in the root phloem than in the xylem, resulting in the higher fructan concentrations in the phloem. Fructan localization studies indicated that fructan is preferentially stored in phloem parenchyma cells in the vicinity of the secondary sieve tube elements. However, inulin-like crystals occasionally appeared in xylem vessels. PMID:10806226

Long-term effectiveness of initiating non-nucleoside reverse transcriptase inhibitor- versus ritonavir-boosted protease inhibitor-based antiretroviral therapy: implications for first-line therapy choice in resource-limited settings

PubMed Central

Lima, Viviane D; Hull, Mark; McVea, David; Chau, William; Harrigan, P Richard; Montaner, Julio SG

2016-01-01

Introduction In many resource-limited settings, combination antiretroviral therapy (cART) failure is diagnosed clinically or immunologically. As such, there is a high likelihood that patients may stay on a virologically failing regimen for a substantial period of time. Here, we compared the long-term impact of initiating non-nucleoside reverse transcriptase inhibitor (NNRTI)- versus boosted protease inhibitor (bPI)-based cART in British Columbia (BC), Canada. Methods We followed prospectively 3925 ART-naïve patients who started NNRTIs (N=1963, 50%) or bPIs (N=1962; 50%) from 1 January 2000 until 30 June 2013 in BC. At six months, we assessed whether patients virologically failed therapy (a plasma viral load (pVL) >50 copies/mL), and we stratified them based on the pVL at the time of failure ≤500 versus >500 copies/mL. We then followed these patients for another six months and calculated their probability of achieving subsequent viral suppression (pVL <50 copies/mL twice consecutively) and of developing drug resistance. These probabilities were adjusted for fixed and time-varying factors, including cART adherence. Results At six months, virologic failure rates were 9.5 and 14.3 cases per 100 person-months for NNRTI and bPI initiators, respectively. NNRTI initiators who failed with a pVL ≤500 copies/mL had a 16% higher probability of achieving subsequent suppression at 12 months than bPI initiators (0.81 (25th–75th percentile 0.75–0.83) vs. 0.72 (0.61–0.75)). However, if failing NNRTI initiators had a pVL >500 copies/mL, they had a 20% lower probability of suppressing at 12 months than pVL-matched bPI initiators (0.37 (0.29–0.45) vs. 0.46 (0.38–0.54)). In terms of evolving HIV drug resistance, those who failed on NNRTI performed worse than bPI in all scenarios, especially if they failed with a viral load >500 copies/mL. Conclusions Our results show that patients who virologically failed at six months on NNRTI and continued on the same regimen had a lower probability of subsequently achieving viral suppression and a higher chance of evolving HIV drug resistance. These results suggest that improving access to regular virologic monitoring is critically important, especially if NNRTI-based cART is to remain a preferred choice for first-line therapy in resource-limited settings. PMID:27499064

Emergence of uncommon HIV-1 non-B subtypes and circulating recombinant forms and trends in transmission of antiretroviral drug resistance in patients with primary infection during the 2013-2015 period in Marseille, Southeastern France.

PubMed

Tamalet, Catherine; Tissot-Dupont, Hervé; Motte, Anne; Tourrès, Christian; Dhiver, Catherine; Ravaux, Isabelle; Poizot-Martin, Isabelle; Dieng, Thérèse; Tomei, Christelle; Bregigeon, Sylvie; Zaegel-Faucher, Olivia; Laroche, Hélène; Aherfi, Sarah; Mokhtari, Saadia; Chaudet, Hervé; Ménard, Amelie; Brouqui, Philippe; Stein, Andreas; Colson, Philippe

2018-05-24

Primary HIV-1 infections (PHI) with non-B subtypes are increasing in developed countries while transmission of HIV-1 harboring antiretroviral resistance-associated mutations (RAMs) remains a concern. This study assessed non-B HIV-1 subtypes and RAMs prevalence among patients with PHI in university hospitals of Marseille, Southeastern France, in 2005-2015 (11 years). HIV-1 sequences were obtained by in-house protocols from 115 patients with PHI, including 38 for the 2013-2015 period. On the basis of the phylogenetic analysis of the reverse transcriptase region, non-B subtypes were identified in 31% of these patients. They included 3 different subtypes (3A, 1C, 4F), 23 circulating recombinant forms (CRFs) (CRF02_AG, best BLAST hits being CRF 36_cpx and CRF30 in 7 and 1 cases, respectively), and 5 unclassified sequences (U). Non-B subtypes proportion increased significantly, particularly in 2011-2013 vs in 2005-2010 (P = .03). CRF02_AG viruses largely predominated in 2005-2013 whereas atypical strains more difficult to classify and undetermined recombinants emerged recently (2014-2015). The prevalence of protease, nucleos(t)ide reverse transcriptase, and first-generation nonnucleoside reverse transcriptase inhibitors-associated RAMs were 1.7% (World Health Organization [WHO] list, 2009/2.6% International AIDS Society [IAS] list, 2017), 5.2%/4.3%, and 5.2%/5.2%, respectively. Etravirine/rilpivirine-associated RAM (IAS) prevalence was 4.3%. Men who have sex with men (MSM) were more frequently infected with drug-resistant viruses than other patients (26% vs 7%; P = .011). The recent increase of these rare HIV-1 strains and the spread of drug-resistant HIV-1 among MSM in Southeastern France might be considered when implementing prevention strategies and starting therapies. © 2018 Wiley Periodicals, Inc.

Deep sequencing analysis of HIV-1 reverse transcriptase at baseline and time of failure in patients receiving rilpivirine in the phase III studies ECHO and THRIVE.

PubMed

Van Eygen, Veerle; Thys, Kim; Van Hove, Carl; Rimsky, Laurence T; De Meyer, Sandra; Aerssens, Jeroen; Picchio, Gaston; Vingerhoets, Johan

2016-05-01

Minority variants (1.0-25.0%) were evaluated by deep sequencing (DS) at baseline and virological failure (VF) in a selection of antiretroviral treatment-naïve, HIV-1-infected patients from the rilpivirine ECHO/THRIVE phase III studies. Linkage between frequently emerging resistance-associated mutations (RAMs) was determined. DS (llIumina®) and population sequencing (PS) results were available at baseline for 47 VFs and time of failure for 48 VFs; and at baseline for 49 responders matched for baseline characteristics. Minority mutations were accurately detected at frequencies down to 1.2% of the HIV-1 quasispecies. No baseline minority rilpivirine RAMs were detected in VFs; one responder carried 1.9% F227C. Baseline minority mutations associated with resistance to other non-nucleoside reverse transcriptase inhibitors (NNRTIs) were detected in 8/47 VFs (17.0%) and 7/49 responders (14.3%). Baseline minority nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) RAMs M184V and L210W were each detected in one VF (none in responders). At failure, two patients without NNRTI RAMs by PS carried minority rilpivirine RAMs K101E and/or E138K; and five additional patients carried other minority NNRTI RAMs V90I, V106I, V179I, V189I, and Y188H. Overall at failure, minority NNRTI RAMs and NRTI RAMs were found in 29/48 (60.4%) and 16/48 VFs (33.3%), respectively. Linkage analysis showed that E138K and K101E were usually not observed on the same viral genome. In conclusion, baseline minority rilpivirine RAMs and other NNRTI/NRTI RAMs were uncommon in the rilpivirine arm of the ECHO and THRIVE studies. DS at failure showed emerging NNRTI resistant minority variants in seven rilpivirine VFs who had no detectable NNRTI RAMs by PS. © 2015 Wiley Periodicals, Inc.

A Combination Microbicide Gel Protects Macaques Against Vaginal Simian Human Immunodeficiency Virus-Reverse Transcriptase Infection, But Only Partially Reduces Herpes Simplex Virus-2 Infection After a Single High-Dose Cochallenge

PubMed Central

Hsu, Mayla; Aravantinou, Meropi; Menon, Radhika; Seidor, Samantha; Goldman, Daniel; Kenney, Jessica; Derby, Nina; Gettie, Agegnehu; Blanchard, James; Piatak, Michael; Lifson, Jeffrey D.; Fernández-Romero, Jose A.; Zydowsky, Thomas M.

2014-01-01

Abstract Herpes simplex virus-2 (HSV-2) infection increases HIV susceptibility. We previously established a rhesus macaque model of vaginal HSV-2 preexposure followed by cochallenge with HSV-2 and simian/human immunodeficiency virus-reverse transcriptase (SHIV-RT). Using this model, we showed that a gel containing the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 in carrageenan (CG) reduced SHIV-RT infection. To evaluate the efficacy of new generation microbicides against both viruses, we first established dual infection after single vaginal cochallenge with SHIV-RT and HSV-2 in HSV-2-naive macaques. All animals (6/6) became HSV-2 infected, with 4/6 coinfected with SHIV-RT. In a control group cochallenged with SHIV-RT and UV-inactivated HSV-2, 2/4 became SHIV-RT infected, and none had detectable HSV-2. Low-level HSV-2-specific antibody and T cell responses were detected in some HSV-2-infected animals. To test a CG gel containing MIV-150 and zinc acetate (MZC), which provided naive animals full protection from SHIV-RT for at least 8 h, MZC (vs. CG) was applied daily for 14 days followed by cochallenge 8 h later. MZC prevented SHIV-RT infection (0/9 infected, p=0.04 vs. 3/6 in CG controls), but only reduced HSV-2 infection by 20% (6/9 infected vs. 5/6 in CG, p=0.6). In HSV-2-infected animals, none of the gel-treated animals seroconverted, and only the CG controls had measurable HSV-2-specific T cell responses. This study shows the promise of MZC to prevent immunodeficiency virus infection (even in the presence of HSV-2) and reduce HSV-2 infection after exposure to a high-dose inoculum. Additionally, it demonstrates the potential of a macaque coinfection model to evaluate broad-spectrum microbicides. PMID:24117013

A combination microbicide gel protects macaques against vaginal simian human immunodeficiency virus-reverse transcriptase infection, but only partially reduces herpes simplex virus-2 infection after a single high-dose cochallenge.

PubMed

Hsu, Mayla; Aravantinou, Meropi; Menon, Radhika; Seidor, Samantha; Goldman, Daniel; Kenney, Jessica; Derby, Nina; Gettie, Agegnehu; Blanchard, James; Piatak, Michael; Lifson, Jeffrey D; Fernández-Romero, Jose A; Zydowsky, Thomas M; Robbiani, Melissa

2014-02-01

Herpes simplex virus-2 (HSV-2) infection increases HIV susceptibility. We previously established a rhesus macaque model of vaginal HSV-2 preexposure followed by cochallenge with HSV-2 and simian/human immunodeficiency virus-reverse transcriptase (SHIV-RT). Using this model, we showed that a gel containing the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 in carrageenan (CG) reduced SHIV-RT infection. To evaluate the efficacy of new generation microbicides against both viruses, we first established dual infection after single vaginal cochallenge with SHIV-RT and HSV-2 in HSV-2-naive macaques. All animals (6/6) became HSV-2 infected, with 4/6 coinfected with SHIV-RT. In a control group cochallenged with SHIV-RT and UV-inactivated HSV-2, 2/4 became SHIV-RT infected, and none had detectable HSV-2. Low-level HSV-2-specific antibody and T cell responses were detected in some HSV-2-infected animals. To test a CG gel containing MIV-150 and zinc acetate (MZC), which provided naive animals full protection from SHIV-RT for at least 8 h, MZC (vs. CG) was applied daily for 14 days followed by cochallenge 8 h later. MZC prevented SHIV-RT infection (0/9 infected, p=0.04 vs. 3/6 in CG controls), but only reduced HSV-2 infection by 20% (6/9 infected vs. 5/6 in CG, p=0.6). In HSV-2-infected animals, none of the gel-treated animals seroconverted, and only the CG controls had measurable HSV-2-specific T cell responses. This study shows the promise of MZC to prevent immunodeficiency virus infection (even in the presence of HSV-2) and reduce HSV-2 infection after exposure to a high-dose inoculum. Additionally, it demonstrates the potential of a macaque coinfection model to evaluate broad-spectrum microbicides.

The HEPT Analogue WPR-6 Is Active against a Broad Spectrum of Nonnucleoside Reverse Transcriptase Drug-Resistant HIV-1 Strains of Different Serotypes.

PubMed

Xu, Weisi; Zhao, Jianxiong; Sun, Jianping; Yin, Qianqian; Wang, Yan; Jiao, Yang; Liu, Junyi; Jiang, Shibo; Shao, Yiming; Wang, Xiaowei; Ma, Liying

2015-08-01

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are important components of the highly active antiretroviral therapy (HAART) used to treat human immunodeficiency type 1 virus (HIV-1). However, because of the emergence of drug resistance and the adverse effects of current anti-HIV drugs, it is essential to develop novel NNRTIs with an excellent safety profile, improved activity against NNRTI-resistant viruses, and enhanced activity against clinical isolates of different subtypes. Here, we have identified 1-[(benzyloxy)methyl]-6-(3,5-dimethylbenzyl)-5-iodopyrimidine-2,4(1H,3H)-dione (WPR-6), a novel NNRTI with a 50% effective concentration (EC50) of 2 to 4 nM against laboratory-adapted HIV-1 strain SF33 and an EC50 of 7 to 14 nM against nucleoside reverse transcriptase inhibitor-resistant HIV-1 strain 7391 with a therapeutic index of >1 × 10(4). A panel of five representative clinical virus isolates of different subtypes circulating predominantly in China was highly sensitive to WPR-6, with EC50s ranging from 1 to 6 nM. In addition, WPR-6 showed excellent antiviral potency against the most prevalent NNRTI-resistant viruses containing the K103N and Y181C mutations. To determine whether WPR-6 selects for novel resistant mutants, in vitro resistance selection was conducted with laboratory-adapted HIV-1 strain SF33 on MT-4 cells. The results demonstrated that V106I and Y188L were the two dominant NNRTI-associated resistance mutations detected in the breakthrough viruses. Taken together, these in vitro data indicate that WPR-6 has greater efficacy than the reference HEPT analogue TNK651 and the marketed drug nevirapine against HIV-1. However, to develop it as a new NNRTI, further improvement of its pharmacological properties is warranted. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

HIV type-1 genotypic resistance profiles in vertically infected patients from Argentina reveal an association between K103N+L100I and L74V mutations.

PubMed

Aulicino, Paula C; Rocco, Carlos A; Mecikovsky, Debora; Bologna, Rosa; Mangano, Andrea; Sen, Luisa

2010-01-01

Patterns and pathways of HIV type-1 (HIV-1) antiretroviral (ARV) drug resistance-associated mutations in clinical isolates are conditioned by ARV history and factors such as viral subtype and fitness. Our aim was to analyse the frequency and association of ARV drug resistance mutations in a group of long-term vertically infected patients from Argentina. Plasma samples from 71 patients (38 children and 33 adolescents) were collected for genotypic HIV-1 ARV resistance testing during the period between February 2006 and October 2008. Statistically significant pairwise associations between ARV resistance mutations in pol, as well as associations between mutations and drug exposure, were identified using Fisher's exact tests with Bonferroni and false discovery rate corrections. Phylogenetic analyses were performed for subtype assignment. In protease (PR), resistance-associated mutations M46I/L, I54M/L/V/A/S and V82A/F/T/S/M/I were associated with each other and with minor mutations at codons 10, 24 and 71. Mutations V82A/F/T/S/M/I were primarily selected by the administration of ritonavir (RTV) in an historical ARV regimen. In reverse transcriptase, thymidine analogue mutation (TAM)1 profile was more common than TAM2. The non-nucleoside K103N+L100I mutations were observed at high frequency (15.5%) and were significantly associated with the nucleoside mutation L74V in BF recombinants. Associations of mutations at PR sites reflect the frequent use of RTV at an early time in this group of patients and convergent resistance mechanisms driven by the high exposure to protease inhibitors, as well as local HIV-1 diversity. The results provide clinical evidence of a molecular interaction between K103N+L100I and L74V mutations at the reverse transcriptase gene in vivo, limiting the future use of second-generation non-nucleoside reverse transcriptase inhibitors such as etravirine.

Clinical and virologic follow-up in perinatally HIV-1-infected children and adolescents in Madrid with triple-class antiretroviral drug-resistant viruses.

PubMed

Rojas Sánchez, P; de Mulder, M; Fernandez-Cooke, E; Prieto, L; Rojo, P; Jiménez de Ory, S; José Mellado, M; Navarro, M; Tomas Ramos, J; Holguín, Á

2015-06-01

Drug resistance mutations compromise the success of antiretroviral treatment in human immunodeficiency virus type 1 (HIV-1)-infected children. We report the virologic and clinical follow-up of the Madrid cohort of perinatally HIV-infected children and adolescents after the selection of triple-class drug-resistant mutations (TC-DRM). We identified patients from the cohort carrying HIV-1 variants with TC-DRM to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors and protease inhibitors according to IAS-USA-2013. We recovered pol sequences or resistance profiles from 2000 to 2011 and clinical-immunologic-virologic data from the moment of TC-DRM detection until December 2013. Viruses harbouring TC-DRM were observed in 48 (9%) of the 534 children and adolescents from 2000 to 2011, rising to 24.4% among those 197 with resistance data. Among them, 95.8% were diagnosed before 2003, 91.7% were Spaniards, 89.6% carried HIV-1-subtype B and 75% received mono/dual therapy as first regimen. The most common TC-DRM present in ≥50% of them were D67NME, T215FVY, M41L and K103N (retrotranscriptase) and L90M (protease). The susceptibility to darunavir, tipranavir, etravirine and rilpivirine was 67.7%, 43.7%, 33.3% and 33.3%, respectively, and all reported high resistance to didanosine, abacavir and nelfinavir. Despite the presence of HIV-1 resistance mutations to the three main antiretroviral families in our paediatric cohort, some drugs maintained their susceptibility, mainly the new protease inhibitors (tipranavir and darunavir) and nonnucleoside reverse transcriptase inhibitors (etravirine and rilpivirine). These data will help to improve the clinical management of HIV-infected children with triple resistance in Spain. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

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

PubMed

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

2016-11-01

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

Protein-mediated antagonism between HIV reverse transcriptase ligands nevirapine and MgATP.

PubMed

Zheng, Xunhai; Mueller, Geoffrey A; DeRose, Eugene F; London, Robert E

2013-06-18

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) play a central role in the treatment of AIDS, but their mechanisms of action are incompletely understood. The interaction of the NNRTI nevirapine (NVP) with HIV-1 reverse transcriptase (RT) is characterized by a preference for the open conformation of the fingers/thumb subdomains, and a reported variation of three orders of magnitude between the binding affinity of NVP for RT in the presence or absence of primer/template DNA. To investigate the relationship between conformation and ligand binding, we evaluated the use of methionine NMR probes positioned near the tip of the fingers or thumb subdomains. Such probes would be expected to be sensitive to changes in the local environment depending on the fractions of open and closed RT. Comparisons of the NMR spectra of three conservative mutations, I63M, L74M, and L289M, indicated that M63 showed the greatest shift sensitivity to the addition of NVP. The exchange kinetics of the M63 resonance are fast on the chemical shift timescale, but become slow in the presence of NVP due to the slow binding of RT with the inhibitor. The simplest model consistent with this behavior involves a rapid open/closed equilibrium coupled with a slow interaction of the inhibitor with the open conformation. Studies of RT in the presence of both NVP and MgATP indicate a strong negative cooperativity. Binding of MgATP reduces the fraction of RT bound to NVP, as indicated by the intensity of the NVP-perturbed M230 resonance, and enhances the dissociation rate constant of the NVP, resulting in an increase of the open/closed interconversion rate, so that the M63 resonance moves into the fast/intermediate-exchange regime. Protein-mediated interactions appear to explain most of the affinity variation of NVP for RT. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

High Rates of Baseline Drug Resistance and Virologic Failure Among ART-naive HIV-infected Children in Mali.

PubMed

Crowell, Claudia S; Maiga, Almoustapha I; Sylla, Mariam; Taiwo, Babafemi; Kone, Niaboula; Oron, Assaf P; Murphy, Robert L; Marcelin, Anne-Geneviève; Traore, Ban; Fofana, Djeneba B; Peytavin, Gilles; Chadwick, Ellen G

2017-11-01

Limited data exist on drug resistance and antiretroviral treatment (ART) outcomes in HIV-1-infected children in West Africa. We determined the prevalence of baseline resistance and correlates of virologic failure (VF) in a cohort of ART-naive HIV-1-infected children <10 years of age initiating ART in Mali. Reverse transcriptase and protease genes were sequenced at baseline (before ART) and at 6 months. Resistance was defined according to the Stanford HIV Genotypic Resistance database. VF was defined as viral load ≥1000 copies/mL after 6 months of ART. Logistic regression was used to evaluate factors associated with VF or death >1 month after enrollment. Post hoc, antiretroviral concentrations were assayed on baseline samples of participants with baseline resistance. One-hundred twenty children with a median age 2.6 years (interquartile range: 1.6-5.0) were included. Eighty-eight percent reported no prevention of mother-to-child transmission exposure. At baseline, 27 (23%), 4 (3%) and none had non-nucleoside reverse transcriptase inhibitor (NNRTI), nucleoside reverse transcriptase inhibitor or protease inhibitor resistance, respectively. Thirty-nine (33%) developed VF and 4 died >1 month post-ART initiation. In multivariable analyses, poor adherence [odds ratio (OR): 6.1, P = 0.001], baseline NNRTI resistance among children receiving NNRTI-based ART (OR: 22.9, P < 0.001) and protease inhibitor-based ART initiation among children without baseline NNRTI resistance (OR: 5.8, P = 0.018) were significantly associated with VF/death. Ten (38%) with baseline resistance had detectable levels of nevirapine or efavirenz at baseline; 7 were currently breastfeeding, but only 2 reported maternal antiretroviral use. Baseline NNRTI resistance was common in children without reported NNRTI exposure and was associated with increased risk of treatment failure. Detectable NNRTI concentrations were present despite few reports of maternal/infant antiretroviral use.

HIV-1 transmitted drug resistance and genetic diversity among patients from Piauí State, Northeast Brazil.

PubMed

Moura, Maria Edileuza Soares; da Guarda Reis, Mônica Nogueira; Lima, Yanna Andressa Ramos; Eulálio, Kelsen Dantas; Cardoso, Ludimila Paula Vaz; Stefani, Mariane Martins Araújo

2015-05-01

HIV-1 transmitted-drug-resistance and genetic diversity are dynamic and may differ in distinct locations/risk groups. In Brazil, increased AIDS incidence and related mortality have been detected in the Northeast region, differently from the epicenter in the Southeast. This cross-sectional study describes transmitted-dru- resistance and HIV-1 subtypes in protease/PR and reverse transcriptase/RT regions among antiretroviral naïve patients from Piauí State, Northeast Brazil. Among 96 patients recruited 89 (92.7%) had HIV-1 PR/RT regions sequenced: 44 females and 45 males, 22 self-declared as men who have sex with men. Transmitted-drug-resistance was investigated by CPR tool (Stanford HIV-1 Drug Resistance/SDRM). HIV-1 subtypes were assigned by REGA and phylogenetic inference. Overall, transmitted-drug-resistance rate was 11.2% (10/89; CI 95%: 5.8-19.1%); 22.7% among men who have sex with men (5/22; CI 95%: 8.8-43.4%), 10% in heterosexual men (2/20; CI 95%: 1.7-29.3%) and 6.8% in women (3/44; CI 95%: 1.8-17.4%). Singleton mutations to protease-inhibitor/PI, nucleoside-reverse-transcriptase-inhibitor/NRTI or non-nucleoside-reverse-transcriptase-inhibitor/NNRTI predominated (8/10): PI mutations (M46L, V82F, L90M); NRTI mutations (M41L, D67N) and NNRTI mutations (K103N/S). Dual class resistance mutations to NRTI and NNRTI were observed: T215L (NRTI), Y188L (NNRTI) and T215N (NRTI), F227L (NNRTI). Subtype B prevailed (86.6%; 77/89), followed by subtype F1 (1.1%, 1/89) and subtype C (1.1%, 1/89). B/F1 and B/C intersubtype recombinants represented 11.2% (10/89). In Piauí State extensive testing of incidence and transmitted-drug-resistance in all populations with risk behaviors may help control AIDS epidemic locally. © 2015 Wiley Periodicals, Inc.

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

High Levels of Transmitted HIV Drug Resistance in a Study in Papua New Guinea.

PubMed

Lavu, Evelyn; Kave, Ellan; Mosoro, Euodia; Markby, Jessica; Aleksic, Eman; Gare, Janet; Elsum, Imogen A; Nano, Gideon; Kaima, Petronia; Dala, Nick; Gurung, Anup; Bertagnolio, Silvia; Crowe, Suzanne M; Myatt, Mark; Hearps, Anna C; Jordan, Michael R

2017-01-01

Papua New Guinea is a Pacific Island nation of 7.3 million people with an estimated HIV prevalence of 0.8%. ART initiation and monitoring are guided by clinical staging and CD4 cell counts, when available. Little is known about levels of transmitted HIV drug resistance in recently infected individuals in Papua New Guinea. Surveillance of transmitted HIV drug resistance in a total of 123 individuals recently infected with HIV and aged less than 30 years was implemented in Port Moresby (n = 62) and Mount Hagen (n = 61) during the period May 2013-April 2014. HIV drug resistance testing was performed using dried blood spots. Transmitted HIV drug resistance was defined by the presence of one or more drug resistance mutations as defined by the World Health Organization surveillance drug resistance mutations list. The prevalence of non-nucleoside reverse transcriptase inhibitor transmitted HIV drug resistance was 16.1% (95% CI 8.8%-27.4%) and 8.2% (95% CI 3.2%-18.2%) in Port Moresby and Mount Hagen, respectively. The prevalence of nucleoside reverse transcriptase inhibitor transmitted HIV drug resistance was 3.2% (95% CI 0.2%-11.7%) and 3.3% (95% CI 0.2%-11.8%) in Port Moresby and Mount Hagen, respectively. No protease inhibitor transmitted HIV drug resistance was observed. The level of non-nucleoside reverse transcriptase inhibitor drug resistance in antiretroviral drug naïve individuals recently infected with HIV in Port Moresby is amongst the highest reported globally. This alarming level of transmitted HIV drug resistance in a young sexually active population threatens to limit the on-going effective use of NNRTIs as a component of first-line ART in Papua New Guinea. To support the choice of nationally recommended first-line antiretroviral therapy, representative surveillance of HIV drug resistance among antiretroviral therapy initiators in Papua New Guinea should be urgently implemented.

A deletion mutation in the 5' part of the pol gene of Moloney murine leukemia virus blocks proteolytic processing of the gag and pol polyproteins.

PubMed Central

Crawford, S; Goff, S P

1985-01-01

Deletion mutations in the 5' part of the pol gene of Moloney murine leukemia virus were generated by restriction enzyme site-directed mutagenesis of cloned proviral DNA. DNA sequence analysis indicated that one such deletion was localized entirely within the 5' part of the pol gene, did not affect the region encoding reverse transcriptase, and preserved the translational reading frame downstream of the mutation. The major viral precursor polyproteins (Pr65gag, Pr200gag-pol, and gPr80env) were synthesized at wild-type levels in cell lines carrying the mutant genome. These cell lines assembled and released wild-type levels of virion particles into the medium. Cleavage of both Pr65gag and Pr200gag-pol precursors to the mature proteins was completely blocked in the mutant virions. Surprisingly, these virions contained high levels of active reverse transcriptase; examination of the endogenous reverse transcription products synthesized by the mutant virions revealed normal amounts of minus-strand strong-stop DNA, indicating that the RNA genome was packaged and that reverse transcription in detergent-permeabilized virions was not significantly impaired. Processing of gPr80env to gP70env and P15E was not affected by the mutation, but cleavage of P15E to P12E was not observed. The mutant particles were poorly infectious; analysis indicated that infection was blocked at an early stage. The data are consistent with the idea that the 5' part of the pol gene encodes a protease directly responsible for processing Pr65gag, and possibly Pr200gag-pol, to the structural virion proteins. It appears that cleavage of the gag gene product is not required for budding and release of virions and that complete processing of the pol gene product to the mature form of reverse transcriptase is not required for its functional activation. Images PMID:3882995

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

Transmitted drug resistance in patients with acute/recent HIV infection in Brazil.

PubMed

Ferreira, Ana Cristina G; Coelho, Lara E; Grinsztejn, Eduarda; Jesus, Carlos S de; Guimarães, Monick L; Veloso, Valdiléa G; Grinsztejn, Beatriz; Cardoso, Sandra W

The widespread use of antiretroviral therapy increased the transmission of antiretroviral resistant HIV strains. Antiretroviral therapy initiation during acute/recent HIV infection limits HIV reservoirs and improves immune response in HIV infected individuals. Transmitted drug resistance may jeopardize the early goals of early antiretroviral treatment among acute/recent HIV infected patients. Patients with acute/recent HIV infection who underwent resistance test before antiretroviral treatment initiation were included in this analysis. HIV-1 sequences were obtained using an in house protease/reverse transcriptase genotyping assay. Transmitted drug resistance was identified according to the Stanford HIV Database for Transmitted Drug Resistance Mutations, based on WHO 2009 surveillance list, and HIV-1 subtyping according to Rega HIV-1 subtyping tool. Comparison between patients with and without transmitted drug resistance was made using Kruskal-Wallis and Chi-square tests. Forty-three patients were included, 13 with acute HIV infection and 30 with recent HIV infection. The overall transmitted drug resistance prevalence was 16.3% (95% confidence interval [CI]: 8.1-30.0%). The highest prevalence of resistance (11.6%, 95% CI: 8.1-24.5) was against non-nucleoside reverse transcriptase inhibitors, and K103N was the most frequently identified mutation. The high prevalence of nonnucleoside reverse transcriptase inhibitors resistance indicates that efavirenz-based regimen without prior resistance testing is not ideal for acutely/recently HIV-infected individuals in our setting. In this context, the recent proposal of including integrase inhibitors as a first line regimen in Brazil could be an advantage for the treatment of newly HIV infected individuals. However, it also poses a new challenge, since integrase resistance test is not routinely performed for antiretroviral naive individuals. Further studies on transmitted drug resistance among acutely/recently HIV-infected are needed to inform the predictors of transmitted resistance and the antiretroviral therapy outcomes among these population. Copyright © 2017 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.

Simple and simultaneous determination of the hiv-protease inhibitors amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir plus M8 nelfinavir metabolite and the nonnucleoside reverse transcriptase inhibitors efavirenz and nevirapine in human plasma by reversed-phase liquid chromatography.

PubMed

Poirier, Jean-Marie; Robidou, Pascal; Jaillon, Patrice

2005-04-01

Several studies suggest that therapeutic drug monitoring of protease inhibitors and nonnucleoside reverse transcriptase inhibitors may contribute to the clinical outcome of HIV-infected patients. Because of the growing number of antiretroviral drugs and of drug combinations than can be administered to these patients, an accurate high-performance liquid chromatographic (HPLC) method allowing the simultaneous determination of these drugs may be useful. To date, the authors present the first simultaneous HPLC determination of the new protease inhibitor atazanavir with all the others currently in use (M8 nelfinavir metabolite included) and the 2 widely used nonnucleoside reverse transcriptase inhibitors efavirenz and nevirapine. This simple HPLC method allows the analysis all these drugs at a single ultraviolet wavelength following a 1-step liquid-liquid extraction procedure. A 500-muL plasma sample was spiked with internal standard and subjected to liquid-liquid extraction using by diethyl ether at pH 10. HPLC was performed using a Symmetry Shield RP18 and gradient elution. All the drugs of interest and internal standard were detected with ultraviolet detection at 210 nm. Calibration curves were linear in the range 50-10,000 ng/mL. The observed concentrations of the quality controls at plasma concentrations ranging from 50 to 5000 ng/mL for these drugs showed that the overall accuracy varied from 92% to 104% and 92% to 106% for intraday and day-to-day analysis, respectively. No metabolites of the assayed compounds or other drugs commonly coadministered to HIV-positive patients were found to coelute with the drugs of interest or with the internal standard. This assay was developed for the purpose of therapeutic monitoring (TDM) in HIV-infected patients.

Efficacy and durability of nevirapine in antiretroviral drug näive patients.

PubMed

Lange, Joep M A

2003-09-01

Nevirapine is a non-nucleoside reverse transcriptase inhibitor (NNRTI) that was first reported in the scientific literature in 1990. Varying doses of nevirapine (NVP) and a number of regimens containing this NNRTI have been studied in antiretroviral (ARV) näive patients. Four key studies have compared the efficacy and safety of triple drug regimens containing NVP in ARV näive, HIV-1 infected patients. The INCAS study was the first demonstration of how to use NVP in an effective and durable manner: as a component of a triple drug regimen. The COMBINE Study was a comparison of protease inhibitor (PI)-based and NVP-based triple regimens. The Atlantic Study is comparing the safety and efficacy of three triple drug regimens in ARV näive patients. In this study, treatment consists of a divergent drug regimen (PI and nucleoside reverse transcriptase inhibitors, NRTIs) targeting both HIV-1 protease and reverse transcriptase or a convergent regimen targeting reverse transcriptase alone (three NRTIs or two NRTIs plus a NNRTI). A clinical endpoint study (BI 1090) compared the efficacy and durability of multi-drug regimens in ARV näive patients with high baseline plasma HIV-1 RNA levels (pVLs) and low peripheral blood CD4+ lymphocyte counts. Data from these studies confirm that triple regimens containing NVP suppressed viral replication for up to one year, even when the ARV näive patients had low CD4+ cell counts at baseline. Nevirapine-containing regimens suppressed pVLs to < 50 copies/ mL in approximately 50% of patients in the studies discussed (Intent to Treat analyses). Data from 96 weeks of follow up in the Atlantic Study demonstrates that the regimens containing didanosine and stavudine plus indinavir or NVP were significantly more successful in suppressing pVLs to < 50 copies/mL during this period than a regimen composed of these NRTIs and lamivudine (p < or = 0.001). As with other ARV drugs, NVP should always be used as part of a fully suppressive ARV regimen. When used in this way, it is an effective ARV drug, which contributes to durable virological and immunological responses in approximately half of all treated patients. Nevirapine-containing regimens are effective in patients with advanced HIV-1 infection, i.e., low CD4+ cell counts. Data will soon be available from the 2NN Study that compares the efficacy and safety of four different regimens using NVP once daily, NVP twice daily, efavirenz once daily or a combination of NVP and efavirenz. All four arms of the study include a backbone of stavudine and lamivudine.

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

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...

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...

1-Benzyl-2-(1H-indol-3-yl)-5-oxo-pyrrolidine-2-carbonitrile.

PubMed

Tamazyan, Rafael; Armen, Ayvazyan; Ashot, Martirosyan; Sahak, Gasparyan; Schinazi, Raymond

2008-01-04

In the title compound, C(20)H(17)N(3)O, a potential anti-human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse-transcriptase inhibitor, the pyrrolidine ring has an envelope conformation. In the crystal structure, adjacent mol-ecules are connected into infinite chains via an N-H⋯O hydrogen bond.

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

USDA-ARS?s Scientific Manuscript database

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

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

PubMed

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

2005-01-01

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

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

PubMed Central

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

2003-01-01

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

Connection Domain Mutations in HIV-1 Reverse Transcriptase Do Not Impact Etravirine Susceptibility and Virologic Responses to Etravirine-Containing Regimens▿†

PubMed Central

Gupta, Soumi; Vingerhoets, Johan; Fransen, Signe; Tambuyzer, Lotke; Azijn, Hilde; Frantzell, Arne; Paredes, Roger; Coakley, Eoin; Nijs, Steven; Clotet, Bonaventura; Petropoulos, Christos J.; Schapiro, Jonathan; Huang, Wei; Picchio, Gaston

2011-01-01

Connection domain mutations (CDMs) in HIV-1 reverse transcriptase (RT) alter susceptibility to some nucleoside/nonnucleoside RT inhibitors (NRTIs/NNRTIs). Their effects on susceptibility and virologic responses to etravirine were analyzed. Seventeen CDMs were evaluated: L283I, E312Q, G333D, G333E, G335C, G335D, N348I, A360I, A360T, A360V, V365I, T369I, A371V, A376S, I393L, E399D, and E399G. CDM prevalence and effects on virologic responses were analyzed retrospectively using clinical data. The effects on etravirine susceptibility were assessed in clinical samples and confirmed using site-directed mutants. The most prevalent CDMs (>10%) were A371V, E399D, A376S, N348I, A360T, G333E, and L283I. CDM presence was positively correlated with thymidine analogue-associated mutations, but not with NNRTI resistance-associated mutations (RAMs). The presence or number of CDMs did not significantly reduce etravirine susceptibility, although small reductions were seen in samples with G333D, N348I, A360V, T369I, and A376S. N348I, E399G, and N348I/T369I were associated with reduced etravirine susceptibility when present with K103N, L100I, or Y181C. N348I or T369I was associated with reduced etravirine susceptibility when present with K101P or K103R/V179D. Virologic responses to an etravirine-containing regimen were slightly diminished when G333D, G335D, or A376S was present, but this was not confirmed in subgroups with higher baseline resistance or without etravirine RAMs. CDMs alone do not confer substantial reductions in etravirine susceptibility but can further reduce etravirine susceptibility in combination with certain NNRTI mutations. Since virologic responses to etravirine were not affected by CDMs, the clinical impacts of these mutations on etravirine susceptibility appear to be minimal. PMID:21464253

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

USDA-ARS?s Scientific Manuscript database

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

1-Benzyl-2-(1H-indol-3-yl)-5-oxo­pyrrolidine-2-carbonitrile

PubMed Central

Tamazyan, Rafael; Armen, Ayvazyan; Ashot, Martirosyan; Sahak, Gasparyan; Schinazi, Raymond

2008-01-01

In the title compound, C20H17N3O, a potential anti-human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse-transcriptase inhibitor, the pyrrolidine ring has an envelope conformation. In the crystal structure, adjacent mol­ecules are connected into infinite chains via an N—H⋯O hydrogen bond. PMID:21201400

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...

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.

Probing the communication of deoxythymidine triphosphate in HIV-1 reverse transcriptase by communication maps and interaction energy studies.

PubMed

Gnanasekaran, Ramachandran

2017-11-08

We calculate communication maps for HIV-1 Reverse Transcriptase (RT) to elucidate energy transfer pathways between deoxythymidine triphosphate (dTTP) and other parts of the protein. This approach locates energy transport channels from the dTTP to remote regions of the protein via residues and water molecules. We examine the water dynamics near the catalytic site of HIV-1 RT by molecular dynamics (MD) simulations. We find that, within the catalytic site, the relaxation of water molecules is similar to that of the hydration water molecules present in other proteins and the relaxation time scale is fast enough to transport energy and helps in communication between dTTP and other residues in the system. To quantify energy transfer, we also calculate the interaction energies of dTTP, 2Mg 2+ , doxy-guanosine nucleotide (DG22) with their surrounding residues by using the B3LYP-D3 method. The results, from classical vibrational energy diffusivity and QM interaction energy, are complementary to identify the important residues involved in the process of polymerization. The positive and negative interactions by dTTP with different types of residues in the catalytic region make the residues transfer energy through vibrational communication.

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

Adipocytes impair efficacy of antiretroviral therapy.

PubMed

Couturier, Jacob; Winchester, Lee C; Suliburk, James W; Wilkerson, Gregory K; Podany, Anthony T; Agarwal, Neeti; Xuan Chua, Corrine Ying; Nehete, Pramod N; Nehete, Bharti P; Grattoni, Alessandro; Sastry, K Jagannadha; Fletcher, Courtney V; Lake, Jordan E; Balasubramanyam, Ashok; Lewis, Dorothy E

2018-06-01

Adequate distribution of antiretroviral drugs to infected cells in HIV patients is critical for viral suppression. In humans and primates, HIV- and SIV-infected CD4 T cells in adipose tissues have recently been identified as reservoirs for infectious virus. To better characterize adipose tissue as a pharmacological sanctuary for HIV-infected cells, in vitro experiments were conducted to assess antiretroviral drug efficacy in the presence of adipocytes, and drug penetration in adipose tissue cells (stromal-vascular-fraction cells and mature adipocytes) was examined in treated humans and monkeys. Co-culture experiments between HIV-1-infected CD4 T cells and primary human adipocytes showed that adipocytes consistently reduced the antiviral efficacy of the nucleotide reverse transcriptase inhibitor tenofovir and its prodrug forms tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF). In HIV-infected persons, LC-MS/MS analysis of intracellular lysates derived from adipose tissue stromal-vascular-fraction cells or mature adipocytes suggested that integrase inhibitors penetrate adipose tissue, whereas penetration of nucleoside/nucleotide reverse transcriptase inhibitors such as TDF, emtricitabine, abacavir, and lamivudine is restricted. The limited distribution and functions of key antiretroviral drugs within fat depots may contribute to viral persistence in adipose tissue. Copyright © 2018 Elsevier B.V. All rights reserved.

Executive summary of the GeSIDA/National AIDS Plan consensus document on antiretroviral therapy in adults infected by the human immunodeficiency virus (updated January 2014).

PubMed

Berenguer, Juan; Polo, Rosa; Lozano, Fernando; López Aldeguer, José; Antela, Antonio; Arribas, José Ramón; Asensi, Víctor; Blanco, José Ramón; Clotet, Bonaventura; Domingo, Pere; Galindo, María José; Gatell, José María; González-García, Juan; Iribarren, José Antonio; Locutura, Jaime; López, Juan Carlos; Mallolas, Josep; Martínez, Esteban; Miralles, Celia; Miró, José M; Moreno, Santiago; Palacios, Rosario; Pérez Elías, María Jesús; Pineda, Juan Antonio; Podzamczer, Daniel; Portilla, Joaquín; Pulido, Federico; Ribera, Esteban; Riera, Melchor; Rubio, Rafael; Santos, Jesús; Sanz, Jesús; Tuset, Montserrat; Vidal, Francesc; Rivero, Antonio

2014-01-01

In this update, antiretroviral therapy (ART) is recommended for all patients infected by type 1 human immunodeficiency virus (HIV-1). The strength and grade of the recommendation varies with clinical circumstances, number of CD4 cells, comorbid conditions and prevention of transmission of HIV. The objective of ART is to achieve an undetectable plasma viral load. Initial ART should always comprise a combination of 3 drugs, including 2 nucleoside reverse transcriptase inhibitors and a third drug from a different family (non-nucleoside reverse transcriptase inhibitor, protease inhibitor, or integrase inhibitor). This update presents the causes and criteria for switching ART in patients with undetectable plasma viral load and in cases of virological failure. An update is also provided for the specific criteria for ART in special situations (acute infection, HIV-2 infection, and pregnancy) and with comorbid conditions (tuberculosis or other opportunistic infections, kidney disease, liver disease, and cancer). Copyright © 2014 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

hTERT gene immortalized human adipose-derived stem cells and its multiple differentiations: a preliminary investigation.

PubMed

Wang, L; Song, K; Qu, X; Wang, H; Zhu, H; Xu, X; Zhang, M; Tang, Y; Yang, X

2013-03-01

Human adipose-derived adult stem cells (hADSCs) can express human telomerase reverse transcriptase phenotypes under an appropriate culture condition. Because adipose tissue is abundant and easily accessible, hADSCs offer a promising source of stem cells for tissue engineering application and other cell-based therapies. However, the shortage of cells number and the difficulty to proliferate, known as the "Hayflick limit" in vitro, limit their further clinical application. Here, hADSCs were transfected with human telomerase reverse transcriptase (hTERT) gene by the lentiviral vector to prolong the lifespan of stem cells and even immortalize them. Following to this, the cellular properties and functionalities of the transfected cell lines were assayed. The results demonstrated that hADSCs had been successfully transfected with hTERT gene (hTERT-ADSCs). Then, hTERT-ADSCs were initially selected by G418 and subsequently expanded over 20 passages in vitro. Moreover, the qualitative and quantitative differentiation criteria for 20 passages of hTERT-ADSCs also demonstrated that hTERT-ADSCs could differentiate into osteogenesis, chondrogenesis, and adipogenesis phenotypes in lineage-specific differentiation media. These findings confirmed that this transfection could prolong the lifespan of hADSCs.

A second chance for telomerase reverse transcriptase in anticancer immunotherapy.

PubMed

Zanetti, Maurizio

2017-02-01

Telomerase reverse transcriptase (TERT) is a self-antigen that is expressed constitutively in many tumours, and is, therefore, an important target for anticancer immunotherapy. In the past 10 years, trials of immunotherapy with TERT-based vaccines have demonstrated only modest benefits. In this Perspectives, I discuss the possible immunological reasons for this limited antitumour efficacy, and propose that advances in our understanding of the genetics and biology of the involvement of TERT in cancer provides the basis for renewed interest in TERT- based immunotherapy. Telomerase and TERT are expressed in cancer cells at every stage of tumour evolution, from the cancer stem cell to circulating tumour cells and tumour metastases. Many cancer types also harbour cells with mutations in the TERT promoter region, which increase transcriptional activation of this gene. These new findings should spur new interest in the development of TERT-based immunotherapies that are redesigned in line with established immunological considerations and working principles, and are tailored to patients stratified on the basis of TERT-promoter mutations and other underlying tumour characteristics. Thus, despite the disappointment of previous clinical trials, TERT offers the potential for personalized immunotherapy, perhaps in combination with immune-checkpoint inhibition.

Inhibitory Effect of 2,3,5,6-Tetrafluoro-4-[4-(aryl)-1H-1,2,3-triazol-1-yl]benzenesulfonamide Derivatives on HIV Reverse Transcriptase Associated RNase H Activities

PubMed Central

Pala, Nicolino; Esposito, Francesca; Rogolino, Dominga; Carcelli, Mauro; Sanna, Vanna; Palomba, Michele; Naesens, Lieve; Corona, Angela; Grandi, Nicole; Tramontano, Enzo; Sechi, Mario

2016-01-01

The HIV-1 ribonuclease H (RNase H) function of the reverse transcriptase (RT) enzyme catalyzes the selective hydrolysis of the RNA strand of the RNA:DNA heteroduplex replication intermediate, and represents a suitable target for drug development. A particularly attractive approach is constituted by the interference with the RNase H metal-dependent catalytic activity, which resides in the active site located at the C-terminus p66 subunit of RT. Herein, we report results of an in-house screening campaign that allowed us to identify 4-[4-(aryl)-1H-1,2,3-triazol-1-yl]benzenesulfonamides, prepared by the “click chemistry” approach, as novel potential HIV-1 RNase H inhibitors. Three compounds (9d, 10c, and 10d) demonstrated a selective inhibitory activity against the HIV-1 RNase H enzyme at micromolar concentrations. Drug-likeness, predicted by the calculation of a panel of physicochemical and ADME properties, putative binding modes for the active compounds, assessed by computational molecular docking, as well as a mechanistic hypothesis for this novel chemotype are reported. PMID:27556447

The development of HEPT-type HIV non-nucleoside reverse transcriptase inhibitors and its implications for DABO family.

PubMed

Chen, Wenmin; Zhan, Peng; Wu, Jingde; Li, Zhenyu; Liu, Xinyong

2012-01-01

1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) was discovered as the first HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) in 1989. The research on HEPT derivatives (HEPTs) has been lasted for more than 20 years and HEPT family is probably the most investigated NNRTI. Extensive molecular modifications on HEPT have led to many highly potent compounds with broad-resistance spectrum and optimal pharmacokinetic profiles. Moreover, X-crystallographic studies of HEPTs/RT complexes revealed the binding mode of HEPTs and the action mechanism of NNRTI, which has greatly facilitated the design of novel NNRTIs. Recently, the development of HEPTs was accelerated by the application of the "follow-on"-based chemical evolution strategies, such as designed multiple ligands (DMLs) and molecular hybridization (MH). Herein, this article will provide an insight into the development of HEPTs, including structural modifications, crystal structure of RT complexed with HEPTs and its structure-activity relationship (SAR). Additionally, this review also covers the emerging HEPT related dual inhibitors and HEPT-pyridinone hybrids, as well as the contributions of HEPTs to the development of dihydro-alkoxy-benzyl-oxopyrimidine (DABO) family, thus highlighting the importance of HEPTs on the development of NNRTIs.

Secondary structure model of the RNA recognized by the reverse transcriptase from the R2 retrotransposable element.

PubMed Central

Mathews, D H; Banerjee, A R; Luan, D D; Eickbush, T H; Turner, D H

1997-01-01

RNA transcripts corresponding to the 250-nt 3' untranslated region of the R2 non-LTR retrotransposable element are recognized by the R2 reverse transcriptase and are sufficient to serve as templates in the target DNA-primed reverse transcription (TPRT) reaction. The R2 protein encoded by the Bombyx mori R2 can recognize this region from both the B. mori and Drosophila melanogaster R2 elements even though these regions show little nucleotide sequence identity. A model for the RNA secondary structure of the 3' untranslated region of the D. melanogaster R2 retrotransposon was developed by sequence comparison of 10 species aided by free energy minimization. Chemical modification experiments are consistent with this prediction. A secondary structure model for the 3' untranslated region of R2 RNA from the R2 element from B. mori was obtained by a combination of chemical modification data and free energy minimization. These two secondary structure models, found independently, share several common sites. This study shows the utility of combining free energy minimization, sequence comparison, and chemical modification to model an RNA secondary structure. PMID:8990394

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.

Creation of a Long-Acting Nanoformulated 2′,3′-Dideoxy-3′-Thiacytidine

PubMed Central

Guo, Dongwei; Zhou, Tian; Araínga, Mariluz; Palandri, Diana; Gautam, Nagsen; Bronich, Tatiana; Alnouti, Yazen; McMillan, JoEllyn; Edagwa, Benson

2017-01-01

Background: Antiretroviral drug discovery and formulation design will facilitate viral clearance in infectious reservoirs. Although progress has been realized for selected hydrophobic integrase and nonnucleoside reverse transcriptase inhibitors, limited success has been seen to date with hydrophilic nucleosides. To overcome these limitations, hydrophobic long-acting drug nanoparticles were created for the commonly used nucleoside reverse transcriptase inhibitor, lamivudine (2′,3′-dideoxy-3′-thiacytidine, 3TC). Methods: A 2-step synthesis created a slow-release long-acting hydrophobic 3TC. Conjugation of 3TC to a fatty acid created a myristoylated prodrug which was encased into a folate-decorated poloxamer 407. Both in vitro antiretroviral efficacy in human monocyte-derived macrophages and pharmacokinetic profiles in mice were evaluated for the decorated nanoformulated drug. Results: A stable drug formulation was produced by poloxamer encasement that improved monocyte–macrophage uptake, antiretroviral activities, and drug pharmacokinetic profiles over native drug formulations. Conclusions: Sustained release of long-acting antiretroviral therapy is a new therapeutic frontier for HIV/AIDS. 3TC depot formation in monocyte-derived macrophages can be facilitated through stable subcellular internalization and slow drug release. PMID:27559685

The emerging profile of cross-resistance among the nonnucleoside HIV-1 reverse transcriptase inhibitors.

PubMed

Sluis-Cremer, Nicolas

2014-07-31

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are widely used to treat HIV-1-infected individuals; indeed most first-line antiretroviral therapies typically include one NNRTI in combination with two nucleoside analogs. In 2008, the next-generation NNRTI etravirine was approved for the treatment of HIV-infected antiretroviral therapy-experienced individuals, including those with prior NNRTI exposure. NNRTIs are also increasingly being included in strategies to prevent HIV-1 infection. For example: (1) nevirapine is used to prevent mother-to-child transmission; (2) the ASPIRE (MTN 020) study will test whether a vaginal ring containing dapivirine can prevent HIV-1 infection in women; (3) a microbicide gel formulation containing the urea-PETT derivative MIV-150 is in a phase I study to evaluate safety, pharmacokinetics, pharmacodynamics and acceptability; and (4) a long acting rilpivirine formulation is under-development for pre-exposure prophylaxis. Given their widespread use, particularly in resource-limited settings, as well as their low genetic barriers to resistance, there are concerns about overlapping resistance between the different NNRTIs. Consequently, a better understanding of the resistance and cross-resistance profiles among the NNRTI class is important for predicting response to treatment, and surveillance of transmitted drug-resistance.

Pharmacophore Identification, Molecular Docking, Virtual Screening, and In Silico ADME Studies of Non-Nucleoside Reverse Transcriptase Inhibitors.

PubMed

Pirhadi, Somayeh; Ghasemi, Jahan B

2012-12-01

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have gained a definitive place due to their unique antiviral potency, high specificity and low toxicity in antiretroviral combination therapies used to treat HIV. In this study, chemical feature based pharmacophore models of different classes of NNRT inhibitors of HIV-1 have been developed. The best HypoRefine pharmacophore model, Hypo 1, which has the best correlation coefficient (0.95) and the lowest RMS (0.97), contains two hydrogen bond acceptors, one hydrophobic and one ring aromatic feature, as well as four excluded volumes. Hypo 1 was further validated by test set and Fischer validation method. The best pharmacophore model was then utilized as a 3D search query to perform a virtual screening to retrieve potential inhibitors. The hit compounds were subsequently subjected to filtering by Lipinski's rule of five and docking studies by Libdock and Gold methods to refine the retrieved hits. Finally, 7 top ranked compounds based on Gold score fitness function were subjected to in silico ADME studies to investigate for compliance with the standard ranges. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Single Active Site Mutation Causes Serious Resistance of HIV Reverse Transcriptase to Lamivudine: Insight from Multiple Molecular Dynamics Simulations.

PubMed

Moonsamy, Suri; Bhakat, Soumendranath; Walker, Ross C; Soliman, Mahmoud E S

2016-03-01

Molecular dynamics simulations, binding free energy calculations, principle component analysis (PCA), and residue interaction network analysis were employed in order to investigate the molecular mechanism of M184I single mutation which played pivotal role in making the HIV-1 reverse transcriptase (RT) totally resistant to lamivudine. Results showed that single mutations at residue 184 of RT caused (1) distortion of the orientation of lamivudine in the active site due to the steric conflict between the oxathiolane ring of lamivudine and the side chain of beta-branched amino acids Ile at position 184 which, in turn, perturbs inhibitor binding, (2) decrease in the binding affinity by (~8 kcal/mol) when compared to the wild-type, (3) variation in the overall enzyme motion as evident from the PCA for both systems, and (4) distortion of the hydrogen bonding network and atomic interactions with the inhibitor. The comprehensive analysis presented in this report can provide useful information for understanding the drug resistance mechanism against lamivudine. The results can also provide some potential clues for further design of novel inhibitors that are less susceptible to drug resistance.

Expression of an Mg2+-Dependent HIV-1 RNase H Construct for Drug Screening▿†

PubMed Central

Farias, Richard V.; Vargas, Deborah A.; Castillo, Andres E.; Valenzuela, Beatriz; Coté, Marie L.; Roth, Monica J.; Leon, Oscar

2011-01-01

A single polypeptide of the HIV-1 reverse transcriptase that reconstituted Mg2+-dependent RNase H activity has been made. Using molecular modeling, the construct was designed to encode the p51 subunit joined by a linker to the thumb (T), connection (C), and RNase H (R) domains of p66. This p51-G-TCR construct was purified from the soluble fraction of an Escherichia coli strain, MIC2067(DE3), lacking endogenous RNase HI and HII. The p51-G-TCR RNase H construct displayed Mg2+-dependent activity using a fluorescent nonspecific assay and showed the same cleavage pattern as HIV-1 reverse transcriptase (RT) on substrates that mimic the tRNA removal required for second-strand transfer reactions. The mutant E706Q (E478Q in RT) was purified under similar conditions and was not active. The RNase H of the p51-G-TCR RNase H construct and wild type HIV-1 RT had similar Kms for an RNA-DNA hybrid substrate and showed similar inhibition kinetics to two known inhibitors of the HIV-1 RT RNase H. PMID:21768506

Discovery of 3-{5-[(6-Amino-1H-pyrazolo[3,4-b]pyridine-3-yl)methoxy]-2-chlorophenoxy}-5-chlorobenzonitrile (MK-4965): A Potent, Orally Bioavailable HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitor with Improved Potency against Key Mutant Viruses

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

Tucker, Thomas J.; Sisko, John T.; Tynebor, Robert M.

2009-07-10

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have been shown to be a key component of highly active antiretroviral therapy (HAART). The use of NNRTIs has become part of standard combination antiviral therapies producing clinical outcomes with efficacy comparable to other antiviral regimens. There is, however, a critical issue with the emergence of clinical resistance, and a need has arisen for novel NNRTIs with a broad spectrum of activity against key HIV-1 RT mutations. Using a combination of traditional medicinal chemistry/SAR analyses, crystallography, and molecular modeling, we have designed and synthesized a series of novel, highly potent NNRTIs that possess broad spectrummore » antiviral activity and good pharmacokinetic profiles. Further refinement of key compounds in this series to optimize physical properties and pharmacokinetics has resulted in the identification of 8e (MK-4965), which has high levels of potency against wild-type and key mutant viruses, excellent oral bioavailability and overall pharmacokinetics, and a clean ancillary profile.« less

[Use of Nadis(®) software to improve adverse drug reaction reporting of antiretroviral drugs: experience in south west of France (midi-pyrénées)].

PubMed

Pochard, Liselotte; Hauviller, Laurent; Cuzin, Lise; Eyvrard, Fréderic; Sommet, Agnès; Montastruc, Jean-Louis; Bagheri, Haleh

2014-01-01

To study the value of the module of pharmacovigilance in Nadis® to improve the antiretroviral (ARV) drugs-induced adverse drug reactions (ADRs) reporting. We collected the ADRs reported for 17 months from November 2010 until April 2012. Following data were recorded: characteristics of patients, ADRs, ARV drugs. The number of ADRs was compared to those collected in the same period (17 months) before use of Nadis®. The 119 ADRs reported (an increase of 183%) for 109 patients ADRs were mainly gastrointestinal (21.8%) followed by renal (20.2%), neuro-psychiatric (16.8%), hepatic (13.5%), cutaneous (8.4%), metabolic (6.7%) and others (12.6%). The repartition of ARV drugs was: nucleoside (31.8%), nucleotide (13.6%) reverse transcriptase inhibitors respectively, non-nucleoside reverse transcriptase inhibitors (13.1%), protease inhibitors (36.4%), and integrase inhibitors (5.1%). Our results show the improvement of ARV-induced ADRs reporting by Nadis® which could be used to reduce the rate of under-reporting in patients exposed to these drugs. © 2014 Société Française de Pharmacologie et de Thérapeutique.

Similarities between long interspersed element-1 (LINE-1) reverse transcriptase and telomerase

PubMed Central

Kopera, Huira C.; Moldovan, John B.; Morrish, Tammy A.; Moran, John V.

2011-01-01

Long interspersed element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that contain activities required for conventional retrotransposition by a mechanism termed target-site primed reverse transcription. Previous experiments in XRCC4 or DNA protein kinase catalytic subunit-deficient CHO cell lines, which are defective for the nonhomologous end-joining DNA repair pathway, revealed an alternative endonuclease-independent (ENi) pathway for L1 retrotransposition. Interestingly, some ENi retrotransposition events in DNA protein kinase catalytic subunit-deficient cells are targeted to dysfunctional telomeres. Here we used an in vitro assay to detect L1 reverse transcriptase activity to demonstrate that wild-type or endonuclease-defective L1 ribonucleoprotein particles can use oligonucleotide adapters that mimic telomeric ends as primers to initiate the reverse transcription of L1 mRNA. Importantly, these ribonucleoprotein particles also contain a nuclease activity that can process the oligonucleotide adapters before the initiation of reverse transcription. Finally, we demonstrate that ORF1p is not strictly required for ENi retrotransposition at dysfunctional telomeres. Thus, these data further highlight similarities between the mechanism of ENi L1 retrotransposition and telomerase. PMID:21940498

Similarities between long interspersed element-1 (LINE-1) reverse transcriptase and telomerase.

PubMed

Kopera, Huira C; Moldovan, John B; Morrish, Tammy A; Garcia-Perez, Jose Luis; Moran, John V

2011-12-20

Long interspersed element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that contain activities required for conventional retrotransposition by a mechanism termed target-site primed reverse transcription. Previous experiments in XRCC4 or DNA protein kinase catalytic subunit-deficient CHO cell lines, which are defective for the nonhomologous end-joining DNA repair pathway, revealed an alternative endonuclease-independent (ENi) pathway for L1 retrotransposition. Interestingly, some ENi retrotransposition events in DNA protein kinase catalytic subunit-deficient cells are targeted to dysfunctional telomeres. Here we used an in vitro assay to detect L1 reverse transcriptase activity to demonstrate that wild-type or endonuclease-defective L1 ribonucleoprotein particles can use oligonucleotide adapters that mimic telomeric ends as primers to initiate the reverse transcription of L1 mRNA. Importantly, these ribonucleoprotein particles also contain a nuclease activity that can process the oligonucleotide adapters before the initiation of reverse transcription. Finally, we demonstrate that ORF1p is not strictly required for ENi retrotransposition at dysfunctional telomeres. Thus, these data further highlight similarities between the mechanism of ENi L1 retrotransposition and telomerase.

Reverse Transcriptase Inhibitors as Potential Colorectal Microbicides▿ †

PubMed Central

Herrera, Carolina; Cranage, Martin; McGowan, Ian; Anton, Peter; Shattock, Robin J.

2009-01-01

We investigated whether reverse transcriptase (RT) inhibitors (RTI) can be combined to inhibit human immunodeficiency virus type 1 (HIV-1) infection of colorectal tissue ex vivo as part of a strategy to develop an effective rectal microbicide. The nucleotide RTI (NRTI) PMPA (tenofovir) and two nonnucleoside RTI (NNRTI), UC-781 and TMC120 (dapivirine), were evaluated. Each compound inhibited the replication of the HIV isolates tested in TZM-bl cells, peripheral blood mononuclear cells, and colorectal explants. Dual combinations of the three compounds, either NRTI-NNRTI or NNRTI-NNRTI combinations, were more active than any of the individual compounds in both cellular and tissue models. Combinations were key to inhibiting infection by NRTI- and NNRTI-resistant isolates in all models tested. Moreover, we found that the replication capacities of HIV-1 isolates in colorectal explants were affected by single point mutations in RT that confer resistance to RTI. These data demonstrate that colorectal explants can be used to screen compounds for potential efficacy as part of a combination microbicide and to determine the mucosal fitness of RTI-resistant isolates. These findings may have important implications for the rational design of effective rectal microbicides. PMID:19258271

Reverse transcriptase inhibitors as potential colorectal microbicides.

PubMed

Herrera, Carolina; Cranage, Martin; McGowan, Ian; Anton, Peter; Shattock, Robin J

2009-05-01

We investigated whether reverse transcriptase (RT) inhibitors (RTI) can be combined to inhibit human immunodeficiency virus type 1 (HIV-1) infection of colorectal tissue ex vivo as part of a strategy to develop an effective rectal microbicide. The nucleotide RTI (NRTI) PMPA (tenofovir) and two nonnucleoside RTI (NNRTI), UC-781 and TMC120 (dapivirine), were evaluated. Each compound inhibited the replication of the HIV isolates tested in TZM-bl cells, peripheral blood mononuclear cells, and colorectal explants. Dual combinations of the three compounds, either NRTI-NNRTI or NNRTI-NNRTI combinations, were more active than any of the individual compounds in both cellular and tissue models. Combinations were key to inhibiting infection by NRTI- and NNRTI-resistant isolates in all models tested. Moreover, we found that the replication capacities of HIV-1 isolates in colorectal explants were affected by single point mutations in RT that confer resistance to RTI. These data demonstrate that colorectal explants can be used to screen compounds for potential efficacy as part of a combination microbicide and to determine the mucosal fitness of RTI-resistant isolates. These findings may have important implications for the rational design of effective rectal microbicides.

Discovery of dapivirine, a nonnucleoside HIV-1 reverse transcriptase inhibitor, as a broad-spectrum antiviral against both influenza A and B viruses.

PubMed

Hu, Yanmei; Zhang, Jiantao; Musharrafieh, Rami Ghassan; Ma, Chunlong; Hau, Raymond; Wang, Jun

2017-09-01

The emergence of multidrug-resistant influenza viruses poses a persistent threat to public health. The current prophylaxis and therapeutic interventions for influenza virus infection have limited efficacy due to the continuous antigenic drift and antigenic shift of influenza viruses. As part of our ongoing effort to develop the next generation of influenza antivirals with broad-spectrum antiviral activity and a high genetic barrier to drug resistance, in this study we report the discovery of dapivirine, an FDA-approved HIV nonnucleoside reverse transcriptase inhibitor, as a broad-spectrum antiviral against multiple strains of influenza A and B viruses with low micromolar efficacy. Mechanistic studies revealed that dapivirine inhibits the nuclear entry of viral ribonucleoproteins at the early stage of viral replication. As a result, viral RNA and protein synthesis were inhibited. Furthermore, dapivirine has a high in vitro genetic barrier to drug resistance, and its antiviral activity is synergistic with oseltamivir carboxylate. In summary, the in vitro antiviral results of dapivirine suggest it is a promising candidate for the development of the next generation of dual influenza and HIV antivirals. Copyright © 2017 Elsevier B.V. All rights reserved.

Diaryltriazine non-nucleoside reverse transcriptase inhibitors are potent candidates for pre-exposure prophylaxis in the prevention of sexual HIV transmission.

PubMed

Ariën, Kevin K; Venkatraj, Muthusamy; Michiels, Johan; Joossens, Jurgen; Vereecken, Katleen; Van der Veken, Pieter; Abdellati, Saïd; Cuylaerts, Vicky; Crucitti, Tania; Heyndrickx, Leo; Heeres, Jan; Augustyns, Koen; Lewi, Paul J; Vanham, Guido

2013-09-01

Pre-exposure prophylaxis and topical microbicides are important strategies in the prevention of sexual HIV transmission, especially since partial protection has been shown in proof-of-concept studies. In search of new candidate drugs with an improved toxicity profile and with activity against common non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV, we have synthesized and investigated a library of 60 new diaryltriazine analogues. From this library, 15 compounds were evaluated in depth using a broad armamentarium of in vitro assays that are part of a preclinical testing algorithm for microbicide development. Antiviral activity was assessed in a cell line, and in primary human cells, against both subtype B and subtype C HIV-1 and against viruses resistant to therapeutic NNRTIs and the candidate NNRTI microbicide dapivirine. Toxicity towards primary blood-derived cells, cell lines originating from the female reproductive tract and female genital microflora was also studied. We identified several compounds with highly potent antiviral activity and toxicity profiles that are superior to that of dapivirine. In particular, compound UAMC01398 is an interesting new candidate that warrants further investigation because of its superior toxicity profile and potent activity against dapivirine-resistant viruses.

The Emerging Profile of Cross-Resistance among the Nonnucleoside HIV-1 Reverse Transcriptase Inhibitors

PubMed Central

Sluis-Cremer, Nicolas

2014-01-01

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are widely used to treat HIV-1-infected individuals; indeed most first-line antiretroviral therapies typically include one NNRTI in combination with two nucleoside analogs. In 2008, the next-generation NNRTI etravirine was approved for the treatment of HIV-infected antiretroviral therapy-experienced individuals, including those with prior NNRTI exposure. NNRTIs are also increasingly being included in strategies to prevent HIV-1 infection. For example: (1) nevirapine is used to prevent mother-to-child transmission; (2) the ASPIRE (MTN 020) study will test whether a vaginal ring containing dapivirine can prevent HIV-1 infection in women; (3) a microbicide gel formulation containing the urea-PETT derivative MIV-150 is in a phase I study to evaluate safety, pharmacokinetics, pharmacodynamics and acceptability; and (4) a long acting rilpivirine formulation is under-development for pre-exposure prophylaxis. Given their widespread use, particularly in resource-limited settings, as well as their low genetic barriers to resistance, there are concerns about overlapping resistance between the different NNRTIs. Consequently, a better understanding of the resistance and cross-resistance profiles among the NNRTI class is important for predicting response to treatment, and surveillance of transmitted drug-resistance. PMID:25089538

Emtricitabine: a once-daily nucleoside reverse transcriptase inhibitor.

PubMed

Modrzejewski, Krysten A; Herman, Ronald A

2004-06-01

To review the pharmacology, virology, pharmacokinetics, safety, and efficacy of the nucleoside reverse transcriptase inhibitor (NRTI) emtricitabine. English-language reports were accessed using MEDLINE (1966-June 2003) and the Iowa Drug Information Service database (1966-June 2003) using emtricitabine and Coviracil as key words. (Coviracil was the proposed trade name for the product prior to approval.) The Internet was also searched using the terms HIV/AIDS conferences, then emtricitabine within the conference proceedings. Abstracts, posters, and oral presentations from scientific conferences, both published and unpublished, were included. Preference was given to published controlled trials. Studies providing a description of the pharmacology, virology, effectiveness, safety, or pharmacokinetics of emtricitabine were used in this review. Emtricitabine is an NRTI used to treat HIV-1 infection. Once-daily administration can decrease pill burden and potentially increase adherence to multidrug HIV therapy. Further, emtricitabine has shown equivalent or improved outcomes compared with lamivudine and stavudine. Emtricitabine is a safe and effective option for HIV-1 infection in adults as part of a multidrug regimen. It may be a better alternative than lamivudine for once-daily therapy because of its extended intracellular half-life and better than lamivudine and stavudine because of a possibly decreased potential for drug resistance.

The C-terminal domain of Tetrahymena thermophila telomerase holoenzyme protein p65 induces multiple structural changes in telomerase RNA

PubMed Central

Akiyama, Benjamin M.; Loper, John; Najarro, Kevin; Stone, Michael D.

2012-01-01

The unique cellular activity of the telomerase reverse transcriptase ribonucleoprotein (RNP) requires proper assembly of protein and RNA components into a functional complex. In the ciliate model organism Tetrahymena thermophila, the La-domain protein p65 is required for in vivo assembly of telomerase. Single-molecule and biochemical studies have shown that p65 promotes efficient RNA assembly with the telomerase reverse transcriptase (TERT) protein, in part by inducing a bend in the conserved stem IV region of telomerase RNA (TER). The domain architecture of p65 consists of an N-terminal domain, a La-RRM motif, and a C-terminal domain (CTD). Using single-molecule Förster resonance energy transfer (smFRET), we demonstrate the p65CTD is necessary for the RNA remodeling activity of the protein and is sufficient to induce a substantial conformational change in stem IV of TER. Moreover, nuclease protection assays directly map the site of p65CTD interaction to stem IV and reveal that, in addition to bending stem IV, p65 binding reorganizes nucleotides that comprise the low-affinity TERT binding site within stem–loop IV. PMID:22315458

Adipocytes Impair Efficacy of Antiretroviral Therapy

PubMed Central

Couturier, Jacob; Winchester, Lee C.; Suliburk, James W.; Wilkerson, Gregory K.; Podany, Anthony T.; Agarwal, Neeti; Chua, Corrine Ying Xuan; Nehete, Pramod N.; Nehete, Bharti P.; Grattoni, Alessandro; Sastry, K. Jagannadha; Fletcher, Courtney V.; Lake, Jordan E.; Balasubramanyan, Ashok; Lewis, Dorothy E.

2018-01-01

Adequate distribution of antiretroviral drugs to infected cells in HIV patients is critical for viral suppression. In humans and primates, HIV- and SIV-infected CD4 T cells in adipose tissues have recently been identified as reservoirs for infectious virus. To better characterize adipose tissue as a pharmacological sanctuary for HIV-infected cells, in vitro experiments were conducted to assess antiretroviral drug efficacy in the presence of adipocytes, and drug penetration in adipose tissue cells (stromal-vascular-fraction cells and mature adipocytes) was examined in treated humans and monkeys. Co-culture experiments between HIV-1-infected CD4 T cells and primary human adipocytes showed that adipocytes consistently reduced the antiviral efficacy of the nucleotide reverse transcriptase inhibitor tenofovir and its prodrug forms tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF). In HIV-infected persons, LC-MS/MS analysis of intracellular lysates derived from adipose tissue stromal-vascular-fraction cells or mature adipocytes suggested that integrase inhibitors penetrate adipose tissue, whereas penetration of nucleoside/nucleotide reverse transcriptase inhibitors such as TDF, emtricitabine, abacavir, and lamivudine is restricted. The limited distribution and functions of key antiretroviral drugs within fat depots may contribute to viral persistence in adipose tissue. PMID:29630975

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.

Revealing the drug-resistant mechanism for diarylpyrimidine analogue inhibitors of HIV-1 reverse transcriptase.

PubMed

Zhang, Hao; Qin, Fang; Ye, Wei; Li, Zeng; Ma, Songyao; Xia, Yan; Jiang, Yi; Zhu, Jiayi; Li, Yixue; Zhang, Jian; Chen, Hai-Feng

2011-09-01

Diaryltriazine (DATA) and diarylpyrimidine (DAPY) were two category inhibitors with highly potent activity for wild type (wt) and four principal mutant types (L100I, K103N, Y181C and Y188L) of HIV-1 reverse transcriptase (RT). We had revealed the drug-resistant mechanism of DATA analogue inhibitors with molecular dynamics simulation and three-dimensional quantitative structure-activity relationship (3D-QSAR) methods. In this work, we investigated the drug-resistant mechanism of DAPY analogue inhibitors. It was found that DAPY analogue inhibitors form more hydrogen bonds and hydrophobic contacts with wild type and mutants of HIV-1 RT than DATA inhibitors. This could explain that DAPY analogue inhibitors are more potent than DATA for the wild type and mutants of HIV-1 RT. Then, 3D-QSAR models were constructed for these inhibitors of wild type and four principal mutant types HIV-1 RT and evaluated by test set compounds. These combined models can be used to design new chemical entities and make quantitative prediction of the bioactivities for HIV-1 RT inhibitors before resorting to in vitro and in vivo experiment. © 2011 John Wiley & Sons A/S.

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.

Telomerase deficiency in bone marrow-derived cells attenuates angiotensin II-induced abdominal aortic aneurysm formation.

PubMed

Findeisen, Hannes M; Gizard, Florence; Zhao, Yue; Cohn, Dianne; Heywood, Elizabeth B; Jones, Karrie L; Lovett, David H; Howatt, Deborah A; Daugherty, Alan; Bruemmer, Dennis

2011-02-01

Abdominal aortic aneurysms (AAA) are an age-related vascular disease and an important cause of morbidity and mortality. In this study, we sought to determine whether the catalytic component of telomerase, telomerase reverse transcriptase (TERT), modulates angiotensin (Ang) II-induced AAA formation. Low-density lipoprotein receptor-deficient (LDLr-/-) mice were lethally irradiated and reconstituted with bone marrow-derived cells from TERT-deficient (TERT-/-) mice or littermate wild-type mice. Mice were placed on a diet enriched in cholesterol, and AAA formation was quantified after 4 weeks of Ang II infusion. Repopulation of LDLr-/- mice with TERT-/- bone marrow-derived cells attenuated Ang II-induced AAA formation. TERT-deficient recipient mice revealed modest telomere attrition in circulating leukocytes at the study end point without any overt effect of the donor genotype on white blood cell counts. In mice repopulated with TERT-/- bone marrow, aortic matrix metalloproteinase-2 (MMP-2) activity was reduced, and TERT-/- macrophages exhibited decreased expression and activity of MMP-2 in response to stimulation with Ang II. Finally, we demonstrated in transient transfection studies that TERT overexpression activates the MMP-2 promoter in macrophages. TERT deficiency in bone marrow-derived macrophages attenuates Ang II-induced AAA formation in LDLr-/- mice and decreases MMP-2 expression. These results point to a previously unrecognized role of TERT in the pathogenesis of AAA.

Characterization of tight junction proteins in cultured human urothelial cells.

PubMed

Rickard, Alice; Dorokhov, Nikolay; Ryerse, Jan; Klumpp, David J; McHowat, Jane

2008-01-01

Tight junctions (TJs) are essential for normal function of epithelia, restricting paracellular diffusion and contributing to the maintenance of cell surface polarity. Superficial cells of the urothelium develop TJs, the basis for the paracellular permeability barrier of the bladder against diffusion of urinary solutes. Focusing on the superficial cell layer of stratified cell cultures of an immortalized human ureteral cell line, TEU-2 cells, we have examined the presence of TJ and TJ-associated proteins. TEU-2 cells were treated with calcium chloride and fetal bovine serum culture conditions used to induce stratification that resembles the normal transitional epithelial phenotype. Cultures were examined for TJ and TJ-associated proteins by confocal immunofluorescence microscopy and evaluated for TJ mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). TEU-2 cultures exhibited immunoreactivity at intercellular margins for claudins 1, 4, 5, 7, 14, and 16 whereas claudins 2, 8, and 12 were intracellular. RT-PCR corroborated the presence of these claudins at the mRNA level. The TJ-associated proteins occludin, JAM-1, and zonula occludens (ZO-1, ZO-2, and ZO-3) were localized at cell margins. We have found that numerous TJs and TJ-associated proteins are expressed in stratified TEU-2 cultures. Further, we propose TEU-2s provide a useful ureteral model for future studies on the involvement of TJs proteins in the normal and pathological physiology of the human urinary system.

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

Overexpression or ectopic expression of MUC2 is the common property of mucinous carcinomas of the colon, pancreas, breast, and ovary.

PubMed

Hanski, C; Hofmeier, M; Schmitt-Gräff, A; Riede, E; Hanski, M L; Borchard, F; Sieber, E; Niedobitek, F; Foss, H D; Stein, H; Riecken, E O

1997-08-01

Mucinous carcinomas of the colorectum have been reported to overexpress the intestinal mucin MUC2. The purpose of this study was to determine whether this alteration is shared by mucinous tumours of the ovary, breast, and pancreas. A total of 40 breast carcinomas (22 of mucinous and 18 of ductal invasive type), 39 ovarian adenocarcinomas (16 mucinous, 23 serous), 47 colorectal carcinomas (25 mucinous and 22 non-mucinous), and 41 pancreatic adenocarcinomas (14 mucinous, 27 non-mucinous) were investigated by immunohistochemistry with the anti-MUC2 monoclonal antibody 4F1 and the expression pattern was ranked. MUC2 mucin is expressed in the normal colonic epithelium; in the normal epithelium of the breast, ovary, and pancreas, it was not detectable by immunohistochemistry or by reverse transcriptase-polymerase chain reaction (RT-PCR). In agreement with previous reports, the colonic mucinous carcinomas differed significantly from the non-mucinous carcinomas by strong MUC2 expression. In all mucinous carcinomas of the ovary, breast, and pancreas, de novo expression of the MUC2 gene was observed, which differentiated mucinous and non-mucinous carcinomas of these tissues (P < 0.001). The overexpression or ectopic expression of the MUC2 gene exhibited by mucinous carcinomas of four organs indicates a common genetic lesion associated with the mucinous tumour phenotype.

Endogenous axon guiding chemorepulsant semaphorin-3F inhibits the growth and metastasis of colorectal carcinoma.

PubMed

Wu, Feng; Zhou, Qi; Yang, Jing; Duan, Guang-jie; Ou, Juan-juan; Zhang, Rong; Pan, Feng; Peng, Qiu-ping; Tan, Hong; Ping, Yi-fang; Cui, You-hong; Qian, Cheng; Yan, Xiao-chu; Bian, Xiu-wu

2011-05-01

To elucidate the role of Semaphorin-3F (SEMA3F), originally described as an axon guiding chemorepulsant implicated in nerve development, in the progression of colorectal carcinoma. SEMA3F and its receptor NRP2 were examined in 72 cases of human colorectal carcinoma specimens and cell lines LoVo, SW480, and SW620 with immunohistochemistry and Western blotting. SEMA3F mRNA expression in the frozen tissue specimens and cell lines was examined with quantitative reverse transcriptase-PCR. Confocal laser scanning microscopy was used for detection of cellular localization of the proteins by immunofluorescent staining. MTT assay, flow cytometry, cell adhesion and migration, and xenografts were used to evaluate biological significance of SEMA3F. SEMA3F was significantly reduced in colorectal carcinoma tissues and cell lines. Overexpression of SEMA3F resulted in reduced proliferation, adhesion to fibronectin, and migratory capability as well as reduced S-phase population and integrin αvβ3 expression of SW480 colon cancer cells. In addition, SEMA3F-overexpressing cells exhibited diminished tumorigenesis when transplanted orthotopically in nude mice and reduced liver metastases. Moreover, transfection of siRNA targeting SEMA3F in colon cancer cells increased their tumorigenicity in vivo. Endogenous SEMA3F acts as a suppressor of the growth and metastasis of human colorectal cancer cells. ©2011 AACR.

Repression of hTERT transcription by the introduction of chromosome 3 into human oral squamous cell carcinoma

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

Nishio, Sachiyo; Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Tottori, 683-8503; Ohira, Takahito

Telomerase is a ribonucleoprotein enzyme that maintains telomere length. Telomerase activity is primarily attributed to the expression of telomerase reverse transcriptase (TERT). It has been reported that introduction of an intact human chromosome 3 into the human oral squamous cell carcinoma cell line HSC3 suppresses the tumorigenicity of these cells. However, the mechanisms that regulate tumorigenicity have not been elucidated. To determine whether this reduction in tumorigenicity was accompanied by a reduction in telomerase activity, we investigated the transcriptional activation of TERT in HSC3 microcell hybrid clones with an introduced human chromosome 3 (HSC3#3). HSC#3 cells showed inhibition of hTERT transcriptionmore » compared to that of the parental HSC3 cells. Furthermore, cell fusion experiments showed that hybrids of HSC3 cells and cells of the RCC23 renal carcinoma cell line, which also exhibits suppression of TERT transcription by the introduction of human chromosome 3, also displayed suppressed TERT transcription. These results suggested that human chromosome 3 may carry functionally distinct, additional TERT repressor genes. - Highlights: • hTERT mRNA expression level decreased in the chromosome 3 introduced HSC3 clones. • hTERT mRNA expression level was tend to suppressed in HSC3 and RCC23 hybrid cells. • We provide evidence that human chromosome 3 carries at least two distinct hTERT regulatory factors.« less

Specific insertions of zinc finger domains into Gag-Pol yield engineered retroviral vectors with selective integration properties

PubMed Central

Lim, Kwang-il; Klimczak, Ryan; Yu, Julie H.; Schaffer, David V.

2010-01-01

Retroviral vectors offer benefits of efficient delivery and stable gene expression; however, their clinical use raises the concerns of insertional mutagenesis and potential oncogenesis due to genomic integration preferences in transcriptional start sites (TSS). We have shifted the integration preferences of retroviral vectors by generating a library of viral variants with a DNA-binding domain inserted at random positions throughout murine leukemia virus Gag-Pol, then selecting for variants that are viable and exhibit altered integration properties. We found seven permissive zinc finger domain (ZFD) insertion sites throughout Gag-Pol, including within p12, reverse transcriptase, and integrase. Comprehensive genome integration analysis showed that several ZFD insertions yielded retroviral vector variants with shifted integration patterns that did not favor TSS. Furthermore, integration site analysis revealed selective integration for numerous mutants. For example, two retroviral variants with a given ZFD at appropriate positions in Gag-Pol strikingly integrated primarily into four common sites out of 3.1 × 109 possible human genome locations (P = 4.6 × 10-29). Our findings demonstrate that insertion of DNA-binding motifs into multiple locations in Gag-Pol can make considerable progress toward engineering safer retroviral vectors that integrate into a significantly narrowed pool of sites on human genome and overcome the preference for TSS. PMID:20616052

Perivascular Epithelioid Cell Tumor of Gastrointestinal Tract

PubMed Central

Lu, Biyan; Wang, Chenliang; Zhang, Junxiao; Kuiper, Roland P.; Song, Minmin; Zhang, Xiaoli; Song, Shunxin; van Kessel, Ad Geurts; Iwamoto, Aikichi; Wang, Jianping; Liu, Huanliang

2015-01-01

Perivascular epithelioid cell tumors of gastrointestinal tract (GI PEComas) are exceedingly rare, with only a limited number of published reports worldwide. Given the scarcity of GI PEComas and their relatively short follow-up periods, our current knowledge of their biologic behavior, molecular genetic alterations, diagnostic criteria, and prognostic factors continues to be very limited. We present 2 cases of GI PEComas, one of which showed an aggressive histologic behavior that underwent multiple combined chemotherapies. We also review the available English-language medical literature on GI PEComas-not otherwise specified (PEComas-NOS) and discuss their clinicopathological and molecular genetic features. Pathologic analyses including histomorphologic, immunohistochemical, and ultrastructural studies were performed to evaluate the clinicopathological features of GI PEComas, their diagnosis, and differential diagnosis. Immunohistochemistry, semiquantitative reverse transcriptase polymerase chain reaction, and DNA sequencing assays were carried out to detect the potential molecular genetic alterations in our cases Microscopically, the tumors showed distinctive histologic features of PEComas-NOS, including fascicular or nested architecture, epithelioid or spindled cell type, and clear to eosinophilic cytoplasm. The tumor cells were immunohistochemically positive for melanocytic markers. Molecular pathological assays confirmed a PSF-TFE3 gene fusion in one of our cases. Furthermore, in this case microphthalmia-associated transcription factor and its downstream genes were found to exhibit elevated transcript levels. Knowledge about the molecular genetic alterations in GI PEComas is still limited and warrants further study. PMID:25621681

Incorporation of aptamers in the terminal loop of shRNAs yields an effective and novel combinatorial targeting strategy.

PubMed

Pang, Ka Ming; Castanotto, Daniela; Li, Haitang; Scherer, Lisa; Rossi, John J

2018-01-09

Gene therapy by engineering patient's own blood cells to confer HIV resistance can potentially lead to a functional cure for AIDS. Toward this goal, we have previously developed an anti-HIV lentivirus vector that deploys a combination of shRNA, ribozyme and RNA decoy. To further improve this therapeutic vector against viral escape, we sought an additional reagent to target HIV integrase. Here, we report the development of a new strategy for selection and expression of aptamer for gene therapy. We developed a SELEX protocol (multi-tag SELEX) for selecting RNA aptamers against proteins with low solubility or stability, such as integrase. More importantly, we expressed these aptamers in vivo by incorporating them in the terminal loop of shRNAs. This novel strategy allowed efficient expression of the shRNA-aptamer fusions that targeted RNAs and proteins simultaneously. Expressed shRNA-aptamer fusions targeting HIV integrase or reverse transcriptase inhibited HIV replication in cell cultures. Viral inhibition was further enhanced by combining an anti-integrase aptamer with an anti-HIV Tat-Rev shRNA. This construct exhibited efficacy comparable to that of integrase inhibitor Raltegravir. Our strategy for the selection and expression of RNA aptamers can potentially extend to other gene therapy applications. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Characterisation of a haemagglutinin from Hokkaido red bean (Phaseolus vulgaris cv. Hokkaido red bean).

PubMed

Wong, Jack H; Wan, Chung T; Ng, Tzi B

2010-01-15

A haemagglutinin was purified from Japanese Hokkaido red beans (Phaseolus vulgaris cv. Hokkaido red bean) with a procedure that included three chromatographic media. Haemagglutinating activity was adsorbed on DEAE cellulose, Affi-gel blue gel and Mono S. The pure haemagglutinin was a homodimer and each subunit was around 30 kDa in molecular mass. The haemagglutinating activity of this agglutinin could not be inhibited by a variety of simple sugars at 200 mmol L(-1) concentration including alpha-L-fucose, D(+)-galactose, D(+)-glucose, D(+)-glucosamine, D(-)galactosamine, galacturonic acid, (+)-lactose, D(+)-melibose, L(-)-mannose, D(+)-mannose, D-mannosamine, D(+)-raffinose, L-rhamnose, (+)-xylose and galacturonic acid. The haemagglutinating activity was fully retained at pH 4-11 and at 0-80 degrees C, but was completely lost at extreme pH values (0-2 and 13-14) and at very high temperatures (90 degrees C and 100 degrees C). The haemagglutinin exhibited a weak mitogenic activity toward mouse splenocytes, a stronger anti-proliferative activity than Con A toward HepG2 (human hepatoma) cells and inhibited >80% of HIV-1 reverse transcriptase inhibitory activity at 3.3 micromol L(-1). It was devoid of anti-fungal activity. Hokkaido red bean haemagglutinin possesses a potent anti-proliferative effect on HepG2 cells. Copyright (c) 2009 Society of Chemical Industry.