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1

Molecular cloned SHIV-CN97001: a Replication-Competent, R5 Simian-Human Immunodeficiency Virus Containing Env of a Primary Chinese HIV-1 Clade C isolate  

PubMed Central

The increasing prevalence of human immunodeficiency virus type 1 (HIV-1) subtype C infection worldwide calls for efforts to develop a relevant animal model for evaluating AIDS candidate vaccines. In China, the prevalent HIV strains comprise a circulating recombinant form, BC (CRF07_BC), in which the envelope belongs to subtype C. To evaluate potential AIDS vaccines targeting Chinese viral strains in nonhuman primate models, we constructed a simian-human immunodeficiency virus (SHIV) carrying most of the envelope sequence of a primary HIV-1 clade C strain isolated from an HIV-positive intravenous drug user from YunNan province in China. Infection of six Chinese rhesus macaques with SHIV-CN97001 resulted in a low level of viremia and no significant alteration in CD4+ T-lymphocyte counts. To determine whether in vivo adaptation would enhance the infectivity of SHIV-CN97001, the parental infectious strain was serially passaged through eight Chinese rhesus macaques. The hallmarks of SHIV infectivity developed gradually, as shown by the increasingly elevated peak viremia with each passage. These findings establish that the R5-tropic SHIV-CN97001/Chinese rhesus macaque model should be very useful for evaluation of HIV-1 subtype C vaccines in China.

Liu, Qiang; Li, Yue; Yang, GuiBo; Dai, JieJie; Ruprecht, Ruth M; Shao, YiMing

2012-01-01

2

Suppression of Acute Viremia by Short-Term Postexposure Prophylaxis of Simian\\/Human Immunodeficiency Virus SHIV-RT-Infected Monkeys with a Novel Reverse Transcriptase Inhibitor (GW420867) Allows for Development of Potent Antiviral Immune Responses Resulting in Efficient Containment of Infection  

Microsoft Academic Search

A nonnucleoside reverse transcriptase (RT) inhibitor, GW420867, was tested for postexposure prophylaxis (PEP) in rhesus macaques experimentally infected with 100 50% tissue culture infective doses of a chimeric simian\\/human immunodeficiency virus (SHIV) containing the RT gene of HIV-1 (SHIV-RT). Animals were ei- ther mock treated, or treated for 4 weeks starting at 8 or 24 h postinfection (p.i.) with GW420867.

KAZUYASU MORI; YASUHIRO YASUTOMI; SHUZO SAWADA; FRANCOIS VILLINGER; KAZUSHIGE SUGAMA; BRIGITTE ROSENWITH; JONATHAN L. HEENEY; KLAUS UBERLA; SHUDO YAMAZAKI; AFTAB A. ANSARI; HELGA RUBSAMEN-WAIGMANN

2000-01-01

3

Incorporation of CD40 Ligand into SHIV Virus-Like Particles (VLP) Enhances SHIV-VLP-Induced Dendritic Cell Activation and Boosts Immune Responses against HIV  

PubMed Central

Engagement of CD40 with CD40L induces dendritic cell (DC) maturation and activation, thereby promoting immune responses. The objective of this study was to investigate whether immunization with chimeric CD40L/SHIV virus-like particles (CD40L/SHIV-VLP) could enhance immune responses to SIV Gag and HIV Env proteins by directly activating DCs. We found that CD83, CD40, and CD86 were significantly up-regulated and significantly increased cytokines production were observed after hCD40L/SHIV-VLP treatment in human CD14+ monocyte-derived DCs as compared to SHIV-VLP treatment. Mice immunized with mCD40L/SHIV-VLP showed more than a two-fold increase in HIV Env-specific IgG antibody production, an increase in SIV Gag and HIV Env-specific IFN-? and IL-4 producing cells, and an increase in HIV Env-specific cytotoxic activity compared to that in SHIV-VLP immunized mice. Furthermore, multifunctional CD4+ Th1 cells, which simultaneously produce IFN-?, IL-2 and TNF-? triple cytokines, and CD8+ T-cells, which produce IFN-? were elevated in the mCD40L/SHIV-VLP immunized group. These data demonstrate that chimeric CD40L/SHIV-VLP potently induce DC activation and enhance the magnitude of both humoral and cellular immune responses to the SIV Gag and HIV Env proteins in the mouse model. Therefore, incorporation of CD40L into VLP may represent a novel strategy to develop effective HIV vaccines.

Zhang, Rongxin; Zhang, Sheng; Li, Min; Chen, Changyi; Yao, Qizhi

2010-01-01

4

Development of sensitive single-round pol or env RT-PCR assays to screen for XMRV in multiple sample types.  

PubMed

The potential association between xenotropic murine leukemia virus-related virus (XMRV) and prostate cancer and chronic fatigue syndrome (CFS) has been much debated. To help resolve the potential role of XMRV in human disease, it is critical to develop sensitive and accurate reverse transcriptase (RT)-PCR assays to screen for the virus. Single-round RT-PCR assays were developed on the automated m2000™ system for detection of the pol or env regions of XMRV in whole blood, plasma, urine cell pellets and urogenital swab samples. Assay performance was assessed by testing two blinded panels, one comprised of whole blood and the other of plasma spiked with serial dilutions of XMRV-infected tissue culture cells and supernatant, respectively, prepared by the Blood XMRV Scientific Research Working Group (SRWG). For both whole blood and plasma panel testing, the assays showed excellent specificity and sensitivity as compared to the other tests included in the SRWG phase I study. Analytical specificity of the assays was also evaluated. Neither pol nor env PCR assays detected a panel of potential cross-reactive microorganisms, although some cross-reaction was observed with mouse genomic DNA. Screening of 196 normal human blood donor plasma, 214 HIV-1 seropositive plasma, 20 formalin-fixed paraffin-embedded (FFPE) prostate cancer specimens, 4 FFPE benign prostate specimens, 400 urine pellets from prostate cancer patients, 166 urine pellets from non-prostate cancer patients, and 135 cervical swab specimens, detected no samples as unequivocally XMRV positive. PMID:22057262

Tang, Ning; Frank, Andrea; Leckie, Gregor; Hackett, John; Simmons, Graham; Busch, Michael; Abravaya, Klara

2011-10-25

5

Long-term vaccine protection from AIDS and clearance of viral DNA following SHIV89.6P challenge  

Microsoft Academic Search

In an earlier study, our group vaccinated rhesus macaques with vesicular stomatitis virus (VSV) vectors expressing Gag, Pol, and Env proteins from a hybrid simian\\/human immunodeficiency virus (SHIV). This was followed by a single boost with modified vaccinia virus Ankara (MVA) vectors expressing the same proteins. Following challenge with SHIV89.6P, vaccinated animals cleared challenge virus RNA from the blood by

John Schell; Nina F. Rose; Nicole Fazo; Preston A. Marx; Meredith Hunter; Elizabeth Ramsburg; David Montefiori; Patricia Earl; Bernard Moss; John K. Rose

2009-01-01

6

SIVagm containing the SHIV89.6P Envelope gene replicates poorly and is non-pathogenic  

Microsoft Academic Search

SIVagm does not induce disease in its African green monkey (AGM) host. In comparison, the hybrid simian-human immunodeficiency virus SHIV89.6P that carries the HIV env gene induces disease in rhesus macaques more rapidly than the SIVmac parent virus. To address the possibility that this enhancement of disease by HIV env would also occur when present in SIVagm, a full-length SIVagm\\/89.6Penv

Mario Perkovi?; Stephen Norley; Ralf Sanzenbacher; Marion Battenberg; Sylvia Panitz; Cheik Coulibaly; Egbert Flory; Christine Siegismund; Carsten Münk; Klaus Cichutek

2010-01-01

7

Immunization with Wild-Type or CD4-Binding-Defective HIV-1 Env Trimers Reduces Viremia Equivalently following Heterologous Challenge with Simian-Human Immunodeficiency Virus?  

PubMed Central

We recently reported that rhesus macaques inoculated with CD4-binding-competent and CD4-binding-defective soluble YU2-derived HIV-1 envelope glycoprotein (Env) trimers in adjuvant generate comparable levels of Env-specific binding antibodies (Abs) and T cell responses. We also showed that Abs directed against the Env coreceptor binding site (CoRbs) were elicited only in animals immunized with CD4-binding-competent trimers and not in animals immunized with CD4-binding-defective trimers, indicating that a direct interaction between Env and CD4 occurs in vivo. To investigate both the overall consequences of in vivo Env-CD4 interactions and the elicitation of CoRbs-directed Abs for protection against heterologous simian-human immunodeficiency virus (SHIV) challenge, we exposed rhesus macaques immunized with CD4-binding-competent and CD4-binding-defective trimers to the CCR5-tropic SHIV-SF162P4 challenge virus. Compared to unvaccinated controls, all vaccinated animals displayed improved control of plasma viremia, independent of the presence or absence of CoRbs-directed Abs prior to challenge. Immunization resulted in plasma responses that neutralized the heterologous SHIV challenge stock in vitro, with similar neutralizing Ab titers elicited by the CD4-binding-competent and CD4-binding-defective trimers. The neutralizing responses against both the SHIV-SF162P4 stock and a recombinant virus pseudotyped with a cloned SHIV-SF162P4-derived Env were significantly boosted by the SHIV challenge. Collectively, these results suggest that the capacity of soluble Env trimers to interact with primate CD4 in vivo and to stimulate the production of moderate titers of CoRbs-directed Abs did not influence the magnitude of the neutralizing Ab recall response after viral challenge or the subsequent control of viremia in this heterologous SHIV challenge model.

Sundling, Christopher; O'Dell, Sijy; Douagi, Iyadh; Forsell, Mattias N.; Morner, Andreas; Lore, Karin; Mascola, John R.; Wyatt, Richard T.; Karlsson Hedestam, Gunilla B.

2010-01-01

8

Long-term vaccine protection from AIDS and clearance of viral DNA following SHIV89.6P challenge.  

PubMed

In an earlier study, our group vaccinated rhesus macaques with vesicular stomatitis virus (VSV) vectors expressing Gag, Pol, and Env proteins from a hybrid simian/human immunodeficiency virus (SHIV). This was followed by a single boost with modified vaccinia virus Ankara (MVA) vectors expressing the same proteins. Following challenge with SHIV89.6P, vaccinated animals cleared challenge virus RNA from the blood by day 150 and maintained normal CD4 T cell counts for 8 months. Here we report on the long-term (>5-year post-challenge) status of these animals and the immunological correlates of long-term protection. Using real-time PCR, we found that viral DNA in peripheral blood mononuclear cells (PBMCs) of the vaccinees declined continuously and fell to below detection (<5copies/10(5)cells) by approximately 3 years post-challenge. SHIV DNA was also below the limit of detection in the lymph nodes of two of the four animals at 5 years post-challenge. We detected long-term persistence of multi-functional Gag-specific CD8(+) T cells in both PBMCs and lymph nodes of the two protected animals with the Mamu A01(+) MHC I allele. All animals also maintained SHIV89.6P neutralizing antibody titers for 5 years. Our results show that this vaccine approach generates solid, long-term control of SHIV infection, and suggest that it is mediated by both cytotoxic T lymphocytes and neutralizing antibody. PMID:19135115

Schell, John; Rose, Nina F; Fazo, Nicole; Marx, Preston A; Hunter, Meredith; Ramsburg, Elizabeth; Montefiori, David; Earl, Patricia; Moss, Bernard; Rose, John K

2009-01-07

9

Impact of antibody quality and anamnestic response on viremia control post-challenge in a combined Tat/Env vaccine regimen in rhesus macaques.  

PubMed

Previously, priming rhesus macaques with Adenovirus type 5 host range mutant-recombinants encoding Tat and Env and boosting with Tat and Env protein in MPL-SE controlled chronic viremia by 4 logs following homologous intravenous SHIV89.6P challenge. Here we evaluated Tat, Env, and Tat/Env regimens for immunogenicity and protective efficacy using clade C Env, alum adjuvant, and a heterologous intrarectal SHIV1157ipd3N4 challenge. Despite induction of strong cellular and humoral immunity, Tat/Env group T and B-cell memory responses were not significantly enhanced over Tat- or Env-only groups. Lack of viremia control post-challenge was attributed to lower avidity Env antibodies and no anamnestic ADCC response or SHIV1157ipd3N4 neutralizing antibody development post-challenge. Poor biologic activity of the Tat immunogen may have impaired Tat immunity. In the absence of sterilizing immunity, strong anamnestic responses to heterologous virus can help control viremia. Both antibody breadth and optimal adjuvanticity are needed to elicit high-quality antibody for protective efficacy. PMID:23528732

Demberg, Thorsten; Brocca-Cofano, Egidio; Kuate, Seraphin; Aladi, Stanley; Vargas-Inchaustegui, Diego A; Venzon, David; Kalisz, Irene; Kalyanaraman, V S; Lee, Eun Mi; Pal, Ranajit; DiPasquale, Janet; Ruprecht, Ruth M; Montefiori, David C; Srivastava, Indresh; Barnett, Susan W; Robert-Guroff, Marjorie

2013-03-22

10

Comparison of Systemic and Mucosal Immunization with Helper-Dependent Adenoviruses for Vaccination against Mucosal Challenge with SHIV  

PubMed Central

Most HIV-1 infections are thought to occur at mucosal surfaces during sexual contact. It has been hypothesized that vaccines delivered at mucosal surfaces may mediate better protection against HIV-1 than vaccines that are delivered systemically. To test this, rhesus macaques were vaccinated by intramuscular (i.m.) or intravaginal (ivag.) routes with helper-dependent adenoviral (HD-Ad) vectors expressing HIV-1 envelope. Macaques were first immunized intranasally with species C Ad serotype 5 (Ad5) prior to serotype-switching with species C HD-Ad6, Ad1, Ad5, and Ad2 vectors expressing env followed by rectal challenge with CCR5-tropic SHIV-SF162P3. Vaccination by the systemic route generated stronger systemic CD8 T cell responses in PBMC, but weaker mucosal responses. Conversely, mucosal immunization generated stronger CD4 T cell central memory (Tcm) responses in the colon. Intramuscular immunization generated higher levels of env-binding antibodies, but neither produced neutralizing or cytotoxic antibodies. After mucosal SHIV challenge, both groups controlled SHIV better than control animals. However, more animals in the ivag. group had lower viral set points than in in the i.m. group. These data suggest mucosal vaccination may have improve protection against sexually-transmitted HIV. These data also demonstrate that helper-dependent Ad vaccines can mediate robust vaccine responses in the face of prior immunity to Ad5 and during four rounds of adenovirus vaccination.

Nehete, Bharti P.; Yang, Guojun; Buchl, Stephanie J.; Hanley, Patrick W.; Palmer, Donna; Montefiori, David C.; Ferrari, Guido; Ng, Philip; Sastry, K. Jagannadha; Barry, Michael A.

2013-01-01

11

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

PubMed

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

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

2012-09-01

12

Higher levels of IL18 circulate during primary infection of monkeys with a pathogenic SHIV than with a nonpathogenic SHIV  

Microsoft Academic Search

We have monitored kinetics of peripheral blood Interleukin (IL)-18 level, viral RNA load, and CD4+ T cell counts in cynomolgus and rhesus macaques following infections of various simian\\/human immunodeficiency viruses (SHIVs) causing differential pathogenicity. Infections of cynomolgus and rhesus macaques with pathogenic SHIVs-C2\\/1 and -89.6PD, respectively, induced high levels of plasma IL-18 (0.1–1 ng\\/ml) and enhanced apoptosis of peripheral blood

Masahiko Kaizu; Yasushi Ami; Tadashi Nakasone; Yuko Sasaki; Yasuyuki Izumi; Hironori Sato; Eiji Takahashi; Koji Sakai; Katsuaki Shinohara; Kenji Nakanishi; Mitsuo Honda

2003-01-01

13

Inclusion of a CRF01_AE HIV envelope protein boost with a DNA/MVA prime-boost vaccine; impact on humoral and cellular immunogenicity and viral load reduction after SHIV-E challenge  

PubMed Central

The current study assessed the immunogenicity and protective efficacy of various prime-boost vaccine regimens in rhesus macaques using combinations of recombinant DNA (rDNA), recombinant MVA (rMVA), and subunit gp140 protein. The rDNA and rMVA vectors were constructed to express Env from HIV-1 subtype CRF01_AE and Gag-Pol from CRF01_AE or SIVmac 239. One of the rMVAs, MVA/CMDR, has been recently tested in humans. Immunizations were administered at months 0 and 1 (prime) and months 3 and 6 (boost). After priming, HIV env-specific serum IgG was detected in monkeys receiving gp140 alone or rMVA but not in those receiving rDNA. Titers were enhanced in these groups after boosting either with gp140 alone or with rMVA plus gp140. The groups that received the rDNA prime developed env-specific IgG after boosting with rMVA with or without gp140. HIV Env-specific serum IgG binding antibodies were elicited more frequently and of higher titer, and breadth of neutralizing antibodies was increased with the inclusion of the subunit Env boost. T cell responses were measured by tetramer binding to Gag p11c in Mamu-A*01 macaques, and by IFN-gamma ELISPOT assay to SIV-Gag. T cell responses were induced after vaccination with the highest responses seen in macaques immunized with rDNA and rMVA. Macaques were challenged intravenously with a novel SHIV-E virus (SIVmac239 Gag-Pol with an HIV-1 subtype E-Env CAR402). Post challenge with SHIV-E, antibody titers were boosted in all groups and peaked at 4 weeks. Robust T cell responses were seen in all groups post challenge and in macaques immunized with rDNA and rMVA a clear boosting of responses was seen. A greater than 2 log drop in RNA copies/ml at peak viremia and earlier set point was achieved in macaques primed with rDNA, and boosted with rMVA/SHIV-AE plus gp140. Post challenge viremia in macaques immunized with other regimens was not significantly different to that of controls. These results demonstrate that a gp140 subunit and inclusion of SIV Gag-Pol may be critical for control of SHIV post challenge.

Cox, Josephine H.; Ferrari, Maria G.; Earl, Patricia; Lane, James R.; Jagodzinski, Linda L.; Polonis, Victoria R.; Kuta, Ellen G.; Boyer, Jean D.; Ratto-Kim, Silvia; Eller, Leigh-Anne; Pham, Doan-Trang; Hart, Lydia; Montefiori, David; Ferrari, Guido; Parrish, Stephanie; Weiner, David B.; Moss, Bernard; Kim, Jerome H.; Birx, Deborah; VanCott, Thomas C.

2012-01-01

14

Polyvalent DNA prime and envelope protein boost HIV-1 vaccine elicits humoral and cellular responses and controls plasma viremia in rhesus macaques following rectal challenge with an R5 SHIV isolate  

PubMed Central

Immunization of macaques with multivalent DNA encoding gp120 genes from HIV-1 subtypes A, B, C and E and a gag gene followed by boosting with homologous gp120 proteins elicited strong anti-gp120 antibodies capable of neutralizing homologous and to a lesser degree heterologous HIV-1 isolates. Both Env- and Gag-specific cell mediated immune (CMI) responses were detected in the immunized animals. Following rectal challenge with an SHIV isolate encoding HIV-1Ba-L env, plasma viremia in the infected immunized animals was significantly lower than that observed in the naïve animals. Further, one of six immunized animals was completely protected whereas all six naïve animals were infected. These results demonstrate that a vaccine based on priming with a polyvalent DNA vaccine from multiple HIV-1 subtypes followed by boosting with homologous Env proteins elicits anti-HIV-1 immune responses capable of controlling rectal transmission of SHIVBa-L.

Pal, Ranajit; Wang, Shixia; Kalyanaraman, V.S.; Nair, B.C.; Whitney, Stephen; Keen, Timothy; Hocker, Lindsey; Hudacik, Lauren; Rose, Nicolas; Cristillo, Anthony; Mboudjeka, Innocent; Shen, Siyuan; Wu-Chou, Te-Hui; Montefiori, David; Mascola, John; Lu, Shan; Markham, Phillip

2008-01-01

15

Functional Assessment of EnvZ/OmpR Two-Component System in Shewanella oneidensis  

PubMed Central

EnvZ and OmpR constitute the bacterial two-component signal transduction system known to mediate osmotic stress response in a number of Gram-negative bacteria. In an effort to understand the mechanism through which Shewanella oneidensis senses and responds to environmental osmolarity changes, structure of the ompR-envZ operon was determined with Northern blotting assay and roles of the EnvZ/OmpR two-component system in response to various stresses were investigated with mutational analysis, quantitative reverse transcriptase PCR (qRT-PCR), and phenotype microarrays. Results from the mutational analysis and qRT-PCR suggested that the EnvZ/OmpR system contributed to osmotic stress response of S. oneidensis and very likely engaged a similar strategy employed by E. coli, which involved reciprocal regulation of two major porin coding genes. Additionally, the ompR-envZ system was also found related to cell motility. We further showed that the ompR-envZ dependent regulation of porin genes and motility resided almost completely on ompR and only partially on envZ, indicating additional mechanisms for OmpR phosphorylation. In contrast to E. coli lacking ompR-envZ, however, growth of S. oneidensis did not show a significant dependence on ompR-envZ even under osmotic stress. Further analysis with phenotype microarrays revealed that the S. oneidensis strains lacking a complete ompR-envZ system displayed hypersensitivities to a number of agents, especially in alkaline environment. Taken together, our results suggest that the function of the ompR-envZ system in S. oneidensis, although still connected with osmoregulation, has diverged considerably from that of E. coli. Additional mechanism must exist to support growth of S. oneidensis under osmotic stress.

Yuan, Jie; Wei, Buyun; Shi, Miaomiao; Gao, Haichun

2011-01-01

16

EFOM-ENV/GAMS interface. User's guide.  

National Technical Information Service (NTIS)

EFOM-ENV (Energy Flow and Optimization Model - ENVironment) is a linear programming model which models national energy systems. It improves the flexibility the Netherlands Energy Research Foundation specified EFOM-ENV in GAMS. GAMS (General Algebraic Mode...

A. L. Roos

1992-01-01

17

Comparison of the Effects of Pathogenic Simian Human Immunodeficiency Virus Strains SHIV-89.6P and SHIV-KU2 in Cynomolgus Macaques  

PubMed Central

Factors explaining why human immunodeficiency virus (HIV) enhances the risk of reactivated tuberculosis (TB) are poorly understood. Unfortunately, experimental models of HIV-induced reactivated TB are lacking. We examined whether cynomolgus macaques, which accurately model latent TB in humans, could be used to model pathogenesis of HIV infection in the lungs and associated lymph nodes. These experiments precede studies modeling the effects of HIV infection on latent TB. We infected two groups of macaques with chimeric simian–human immunodeficiency viruses (SHIV-89.6P and SHIV-KU2) and followed viral titers and immunologic parameters including lymphocytes numbers and phenotype in the blood, bronchoalveolar lavage cells, and lymph nodes over the course of infection. Tissues from the lungs, liver, kidney, spleen, and lymph nodes were similarly examined at necropsy. Both strains produced dramatic CD4+ T cell depletion. Plasma titers were not different between viruses, but we found more SHIV-89.6P in the lungs. Both viruses induced similar patterns of cell activation markers. SHIV-89.6P induced more IFN-? expression than SHIV-KU2. These results indicate SHIV-89.6P and SHIV-KU2 infect cynomolgus macaques and may be used to accurately model effects of HIV infection on latent TB.

PAWAR, SANTOSH N.; MATTILA, JOSHUA T.; STURGEON, TIMOTHY J.; LIN, PHILANA LING; NARAYAN, OPENDRA; MONTELARO, RONALD C.; FLYNN, JOANNE L.

2012-01-01

18

SHIV infection protects against heterologous pathogenic SHIV challenge in macaques: A gold-standard for HIV-1 vaccine development?  

PubMed Central

A current debate in the HIV-1 vaccine field concerns the ability of an immunodeficiency virus to elicit a protective response. One argument is that HIV-1 superinfections are frequent in healthy individuals, because virus evades conventional immune surveillance, a serious obstacle to vaccine design. The opposing argument is that protection from superinfection is significant, reflecting a robust immune response that might be harnessed by vaccination to prevent disease. In an experiment designed to address the debate, two macaques received an I.V. inoculation with SHIV KU-1-d (a derivative of SHIV KU-1) and were rested for ?10 months. Infection elicited diverse neutralizing antibody activities in both animals. Animals were then exposed to SHIV 89.6P (I.V.), a virus carrying a heterologous envelope protein relative to the vaccine strain. Infection was monitored by viral load and CD4+ T-cell measurements. All control animals were infected and most succumbed to disease. In contrast, protection from superinfection was statistically significant in test monkeys; one animal showed no evidence of superinfection at any time point and the second showed evidence of virus at only one time point over a 6-month observation period. Neither animal showed signs of disease. Perhaps this protective state may serve as a ‘gold-standard’ for HIV-1 vaccine development, as a similar degree of protection against immunodeficiency virus infections in humans would be much desired.

Sealy, Robert; Zhan, Xiaoyan; Lockey, Timothy D.; Martin, Louis; Blanchard, James; Traina-Dorge, Vicki; Hurwitz, Julia L.

2009-01-01

19

Incorporation of chimeric HIV-SIV-Env and modified HIV-Env proteins into HIV pseudovirions  

SciTech Connect

Low level incorporation of the viral glycoprotein (Env) into human immunodeficiency virus (HIV) particles is a major drawback for vaccine strategies against HIV/AIDS in which HIV particles are used as immunogen. Within this study, we have examined two strategies aimed at achieving higher levels of Env incorporation into non-infectious pseudovirions (PVs). First, we have generated chimeric HIV/SIV Env proteins containing the truncated C-terminal tail region of simian immunodeficiency virus (SIV)mac239-Env767{sup stop}, which mediates strongly increased incorporation of SIV-Env into SIV particles. In a second strategy, we have employed a truncated HIV-Env protein (Env-Tr752{sup N750K}) which we have previously demonstrated to be incorporated into HIV virions, generated in infected T-cells, to a higher level than that of Wt-HIV-Env. Although the chimeric HIV/SIV Env proteins were expressed at the cell surface and induced increased levels of cell-cell fusion in comparison to Wt-HIV-Env, they did not exhibit increased incorporation into either HIV-PVs or SIV-PVs. Only Env-Tr752{sup N750K} exhibited significantly higher (threefold) levels of incorporation into HIV-PVs, an improvement, which, although not dramatic, is worthwhile for the large-scale preparation of non-infectious PVs for vaccine studies aimed at inducing Env humoral responses.

Devitt, Gerard [Forschungsschwerpunkt Infektion und Krebs, F020, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg (Germany); Emerson, Vanessa [Forschungsschwerpunkt Infektion und Krebs, F020, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg (Germany); Holtkotte, Denise [Forschungsschwerpunkt Infektion und Krebs, F020, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg (Germany); Pfeiffer, Tanya [Forschungsschwerpunkt Infektion und Krebs, F020, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg (Germany); Pisch, Thorsten [Forschungsschwerpunkt Infektion und Krebs, F020, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg (Germany); Bosch, Valerie [Forschungsschwerpunkt Infektion und Krebs, F020, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg (Germany)]. E-mail: v.bosch@dkfz.de

2007-05-10

20

Reduced Inflammation and CD4 Loss in Acute SHIV Infection During Oral Pre-Exposure Prophylaxis  

PubMed Central

Background.The impact of pre-exposure prophylaxis (PrEP) with antiretrovirals on breakthrough HIV or SHIV infection is not fully documented. We addressed the hypothesis that SHIVSF162P3 infection despite active PrEP results in altered early immune parameters, compared with untreated infection. Methods.Eleven rhesus macaques were infected during repeated, rectal, low-dose SHIVSF162P3 exposures while receiving concurrent oral PrEP (Truvada [n = 2] or GS7340 [n = 4]) or as untreated controls (n = 5). We measured SHIV RNA, inflammatory cytokines, CD4 cells, and SHIV-specific and memory T cells until 20 weeks after peak viremia. Results.SHIV infection during PrEP resulted in 100-fold lower peak viremia and lower IL-15, IL-18, and IL-1Ra levels, compared with controls (P < .05; Wilcoxon rank-sum test). Unlike controls, PrEP-treated macaques showed no significant CD4 cell count reduction during acute infection and developed more SHIV-specific central memory T cells, relative to controls. After in vivo CD8 cell depletion, viral load increased to similar levels, indicating that CD8 cells were critical for viral control in both groups. Conclusions.PrEP with antiretrovirals has beneficial effects on early SHIV infection even when infection is not prevented. Although long-term immune control could not be examined in this SHIV infection model, our results suggest that PrEP results in improved early disease parameters in breakthrough infections.

Kersh, Ellen N.; Luo, Wei; Zheng, Qi; Adams, Debra R.; Hanson, Debra; Youngpairoj, Ae S.; Cong, Mian-er; Butler, Katherine; Hendry, R. Michael; McNicholl, Janet M.; Heneine, Walid; Garcia-Lerma, J. Gerardo

2012-01-01

21

Special Challenges of Rearing Infant Macaques Infected with Lentivirus (SIV, HIV, SHIV)  

Microsoft Academic Search

\\u000a The nonhuman primate lentivirus model is perhaps the best animal model of human immunodeficiency virus (HIV) infection. The\\u000a lentiviruses (lentivirinae), a subfamily of retroviruses (family Retroviridae), include, in addition to HIV-1 and HIV-2, simian\\u000a immunodeficiency virus (SIV) and a variety of simian\\/human immunodeficiency viruses (SHIVs). SHIVs are humanly engineered,\\u000a chimeric viruses made by inserting HIV genes into an SIV. Lentivirus-infected

Julie M. Worlein; James C. Ha; Christy Harris; Jennifer Leigh; Kelsey Stratton; Rodney J. R. Ho

22

An Antiretroviral/Zinc Combination Gel Provides 24 Hours of Complete Protection against Vaginal SHIV Infection in Macaques  

PubMed Central

Background Repeated use, coitus-independent microbicide gels that do not contain antiretroviral agents also used as first line HIV therapy are urgently needed to curb HIV spread. Current formulations require high doses (millimolar range) of antiretroviral drugs and typically only provide short-term protection in macaques. We used the macaque model to test the efficacy of a novel combination microbicide gel containing zinc acetate and micromolar doses of the novel non-nucleoside reverse transcriptase inhibitor MIV-150 for up to 24 h after repeated gel application. Methods and Findings Rhesus macaques were vaginally challenged with SHIV-RT up to 24 h after repeated administration of microbicide versus placebo gels. Infection status was determined by measuring virologic and immunologic parameters. Combination microbicide gels containing 14 mM zinc acetate dihydrate and 50 µM MIV-150 afforded full protection (21 of 21 animals) for up to 24 h after 2 weeks of daily application. Partial protection was achieved with the MIV-150 gel (56% of control at 8 h after last application, 11% at 24 h), while the zinc acetate gel afforded more pronounced protection (67% at 8–24 h). Marked protection persisted when the zinc acetate or MIV-150/zinc acetate gels were applied every other day for 4 weeks prior to challenge 24 h after the last gel was administered (11 of 14 protected). More MIV-150 was associated with cervical tissue 8 h after daily dosing of MIV-150/zinc acetate versus MIV-150, while comparable MIV-150 levels were associated with vaginal tissues and at 24 h. Conclusions A combination MIV-150/zinc acetate gel and a zinc acetate gel provide significant protection against SHIV-RT infection for up to 24 h. This represents a novel advancement, identifying microbicides that do not contain anti-viral agents used to treat HIV infection and which can be used repeatedly and independently of coitus, and underscores the need for future clinical testing of their safety and ability to prevent HIV transmission in humans.

Singer, Rachel; Hsu, Mayla; Rodriguez, Aixa; Kizima, Larisa; Abraham, Ciby J.; Menon, Radhika; Seidor, Samantha; Chudolij, Anne; Gettie, Agegnehu; Blanchard, James; Lifson, Jeffrey D.; Piatak, Michael; Fernandez-Romero, Jose A.; Zydowsky, Thomas M.; Robbiani, Melissa

2011-01-01

23

Prime-boost vaccination with heterologous live vectors encoding SIV gag and multimeric HIV-1 gp160 protein: efficacy against repeated mucosal R5 clade C SHIV challenges.  

PubMed

We sought to induce primate immunodeficiency virus-specific cellular and neutralizing antibody (nAb) responses in rhesus macaques (RM) through a bimodal vaccine approach. RM were immunized intragastrically (i.g.) with the live-attenuated Listeria monocytogenes (Lm) vector Lmdd-BdopSIVgag encoding SIVmac239 gag. SIV Gag-specific cellular responses were boosted by intranasal and intratracheal administration of replication-competent adenovirus (Ad5hr-SIVgag) encoding the same gag. To broaden antiviral immunity, the RM were immunized with multimeric HIV clade C (HIV-C) gp160 and HIV Tat. SIV Gag-specific cellular immune responses and HIV-1 nAb developed in some RM. The animals were challenged intrarectally with five low doses of R5 SHIV-1157ipEL-p, encoding a heterologous HIV-C Env (22.1% divergent to the Env immunogen). All five controls became viremic. One out of ten vaccinees was completely protected and another had low peak viremia. Sera from the completely and partially protected RM neutralized the challenge virus > 90%; these RM also had strong SIV Gag-specific proliferation of CD8? T cells. Peak and area under the curve of plasma viremia (during acute phase) among vaccinees was lower than for controls, but did not attain significance. The completely protected RM showed persistently low numbers of the ?4?7-expressing CD4? T cells; the latter have been implicated as preferential virus targets in vivo. Thus, vaccine-induced immune responses and relatively lower numbers of potential target cells were associated with protection. PMID:21693155

Lakhashe, Samir K; Velu, Vijayakumar; Sciaranghella, Gaia; Siddappa, Nagadenahalli B; Dipasquale, Janet M; Hemashettar, Girish; Yoon, John K; Rasmussen, Robert A; Yang, Feng; Lee, Sandra J; Montefiori, David C; Novembre, Francis J; Villinger, François; Amara, Rama Rao; Kahn, Maria; Hu, Shiu-Lok; Li, Sufen; Li, Zhongxia; Frankel, Fred R; Robert-Guroff, Marjorie; Johnson, Welkin E; Lieberman, Judy; Ruprecht, Ruth M

2011-07-14

24

Efficient priming of simian\\/human immunodeficiency virus (SHIV)-specific T-cell responses with DNA encoding hybrid SHIV\\/hepatitis B surface antigen particles  

Microsoft Academic Search

Recent efforts to design an human immunodeficiency virus type 1 (HIV-1) vaccine candidate have focused on means of eliciting anti-viral T-cell responses. We tried to improve the immunogenicity of DNA vaccines by designing hybrid DNA constructs encoding hepatitis B surface antigen (HBsAg) fused to antigenic domains of simian\\/human immunodeficiency virus (SHIV 89.6P). Immunisation with hybrid DNA induced both effector and

Anne-Laure Puaux; Delphine Marsac; Stéphane Prost; Mandal K Singh; Patricia Earl; Bernard Moss; Roger Le Grand; Yves Riviere; Marie-Louise Michel

2004-01-01

25

Protection of macaques against AIDS with a live attenuated SHIV vaccine is of finite duration  

Microsoft Academic Search

Using background data that live vaccines against several viral pathogens are effective in inducing life-long protection against disease, we undertook studies in macaques to determine the duration of protection that two live SHIV vaccines could induce against AIDS. Earlier studies had established that macaques immunized once with a live vaccine and challenged 6 months later were protected, and that other macaques

Anil Kumar; Zhenqian Liu; Darlene Sheffer; Marilyn Smith; Dinesh K. Singh; Shilpa Buch; Opendra Narayan

2008-01-01

26

Retroviral Env Glycoprotein Trafficking and Incorporation into Virions  

PubMed Central

Together with the Gag protein, the Env glycoprotein is a major retroviral structural protein and is essential for forming infectious virus particles. Env is synthesized, processed, and transported to certain microdomains at the plasma membrane and takes advantage of the same host machinery for its trafficking as that used by cellular glycoproteins. Incorporation of Env into progeny virions is probably mediated by the interaction between Env and Gag, in some cases with the additional involvement of certain host factors. Although several general models have been proposed to explain the incorporation of retroviral Env glycoproteins into virions, the actual mechanism for this process is still unclear, partly because structural data on the Env protein cytoplasmic tail is lacking. This paper presents the current understanding of the synthesis, trafficking, and virion incorporation of retroviral Env proteins.

Murakami, Tsutomu

2012-01-01

27

Lymphocyte Activation during Acute Simian\\/Human Immunodeficiency Virus SHIV89.6PD Infection in Macaques  

Microsoft Academic Search

Host-virus interactions control disease progression in human immunodeficiency virus-infected human beings and in nonhuman primates infected with simian or simian\\/human immunodeficiency viruses (SHIV). These interactions evolve rapidly during acute infection and are key to the mechanisms of viral persistence and AIDS. SHIV89.6PD infection in rhesus macaques can deplete CD4 1 T cells from the peripheral blood, spleen, and lymph nodes

MARIANNE WALLACE; PAUL M. WATERMAN; JACQUE L. MITCHEN; MAHMOUD DJAVANI; CHARLES BROWN; PARUL TRIVEDI; DOUGLAS HOREJSH; MARTA DYKHUIZEN; MOIZ KITABWALLA; C. DAVID PAUZA

1999-01-01

28

AquaEnv : An Aqua tic Acid–Base Modelling Env ironment in R  

Microsoft Academic Search

AquaEnv\\u000a is an integrated software package for aquatic chemical model generation focused on ocean acidification and antropogenic CO2 uptake. However, the package is not restricted to the carbon cycle or the oceans: it calculates, converts, and visualizes\\u000a information necessary to describe pH, related CO2 air–water exchange, as well as aquatic acid–base chemistry in general for marine, estuarine or freshwater systems.

Andreas F. HofmannKarline; Karline Soetaert; Jack J. Middelburg; Filip J. R. Meysman

2010-01-01

29

Protection of macaques against AIDS with a live attenuated SHIV vaccine is of finite duration.  

PubMed

Using background data that live vaccines against several viral pathogens are effective in inducing life-long protection against disease, we undertook studies in macaques to determine the duration of protection that two live SHIV vaccines could induce against AIDS. Earlier studies had established that macaques immunized once with a live vaccine and challenged 6 months later were protected, and that other macaques given two sequential inoculations of live vaccines were protected for at least 1 year. Protection was associated with persistence of the vaccine viruses. In this study, we sought to determine whether the duration of protection in macaques given a single inoculation of replication competent live vaccines would extend beyond 3 years. Two groups of four rhesus macaques were inoculated with two live SHIV vaccines, respectively. The viruses replicated transiently in all animals but at the 3-year time point, PCR analysis of PBMC did not detect DNA of either virus in any of the animals, and all were negative for CMI responses in the blood. All 8 animals succumbed to disease when challenged with pathogenic viruses. PMID:17988702

Kumar, Anil; Liu, Zhenqian; Sheffer, Darlene; Smith, Marilyn; Singh, Dinesh K; Buch, Shilpa; Narayan, Opendra

2007-11-07

30

Highly pathogenic SHIVs and SIVs target different CD4+ T cell subsets in rhesus monkeys, explaining their divergent clinical courses  

PubMed Central

In contrast to simian immunodeficiency viruses (SIVs), which induce immunodeficiency over a 1- to 3-year period, highly pathogenic simian-human immunodeficiency viruses (SHIVs) cause a complete, irreversible, and systemic depletion of CD4+ T lymphocytes in rhesus monkeys within weeks of infection. By using small-molecule competitors specific for CCR5 and CXCR4 in ex vivo assays, we found that highly pathogenic SHIVDH12R exclusively uses CXCR4 for infection of rhesus peripheral blood mononuclear cells, whereas SIVmac239 and SIVsmE543 use CCR5 for entry into the same cells. During the period of peak virus production in SHIVDH12R- or SHIV89.6P-infected rhesus monkeys, massive elimination of CXCR4+ naïve CD4+ T cells occurred. In contrast, circulating CCR5+ memory CD4+ T cells were selectively depleted in rapidly progressing SIV-infected monkeys. At the time of their death, two SIV rapid progressors had experienced a nearly complete loss of the memory CD4+ T cell subset from the blood and mesenteric lymph nodes. Thus, pathogenic SHIVs and SIVs target different subsets of CD4+ T cells in vivo, with the pattern of CD4+ T lymphocyte depletion being inextricably linked to chemokine receptor use. In the context of developing an effective prophylactic vaccine, which must potently control virus replication during the primary infection, regimens that suppress SHIVs might not protect monkeys against SIV or humans against HIV-1.

Nishimura, Yoshiaki; Igarashi, Tatsuhiko; Donau, Olivia K.; Buckler-White, Alicia; Buckler, Charles; Lafont, Bernard A. P.; Goeken, Robert M.; Goldstein, Simoy; Hirsch, Vanessa M.; Martin, Malcolm A.

2004-01-01

31

Analysis of transcribed human endogenous retrovirus W env loci clarifies the origin of multiple sclerosis-associated retrovirus env sequences  

Microsoft Academic Search

BACKGROUND: Multiple sclerosis-associated retrovirus (MSRV) RNA sequences have been detected in patients with multiple sclerosis (MS) and are related to the multi-copy human endogenous retrovirus family type W (HERV-W). Only one HERV-W locus (ERVWE1) codes for a complete HERV-W Env protein (Syncytin-1). Syncytin-1 and the putative MSRV Env protein have been involved in the pathogenesis of MS. The origin of

Georg Laufer; Jens Mayer; Benedikt F Mueller; Nikolaus Mueller-Lantzsch; Klemens Ruprecht

2009-01-01

32

Infection of macaques with an R5-tropic SHIV bearing a chimeric envelope carrying subtype E V3 loop among subtype B framework  

Microsoft Academic Search

Summary.  ?To establish simian\\/human immunodeficiency virus (SHIV) clones bearing a chimeric envelope carrying subtype E V3 loop among\\u000a subtype B envelope, four subtype E V3 sequences were substituted into SHIVMD14, a SHIV clone bearing an envelope derived from a CXCR4 (X4)\\/CCR5 (R5)-dual tropic subtype B HIV-1 strain. SHIV-TH09V3, an\\u000a only V3-chimera clone capable of replicating in human and macaque peripheral blood

M. Kaizu; H. Sato; Y. Ami; Y. Izumi; T. Nakasone; Y. Tomita; K. Someya; Y. Takebe; K. Kitamura; O. Tochikubo; M. Honda

2003-01-01

33

Aaron Shepard's RT Page  

NSDL National Science Digital Library

Aaron Shepard, author of Stories on Stage: Scripts for Reader's Theater (H.W. Wilson, 1993), has provided this web site as a way to broaden interest in the reader's theater (RT) genre. Using minimal (or no) sets, props, and costumes, RT performances are opportunities for students to participate in short dramatic adaptations of prose or dramatic works. Of the RT scripts available on the site, several are original and several are adaptations of well-known short stories. There are twelve scripts for grade, middle, and high school, and five for college classes.

Shepard, Aaron.

1996-01-01

34

Schistosoma mansoni Enhances Host Susceptibility to Mucosal but Not Intravenous Challenge by R5 Clade C SHIV  

PubMed Central

Background The high prevalence of HIV-1/AIDS in areas endemic for schistosomiasis and other helminthic infections has led to the hypothesis that parasites increase host susceptibility to immunodeficiency virus infection. We previously showed that rhesus macaques (RM) with active schistosomiasis were significantly more likely to become systemically infected after intrarectal (i.r.) exposure to an R5-tropic clade C simian-human immunodeficiency virus (SHIV-C) than were parasite-free controls. However, we could not address whether this was due to systemic or mucosal effects. If systemic immunoactivation resulted in increased susceptibility to SHIV-C acquisition, a similarly large difference in host susceptibility would be seen after intravenous (i.v.) SHIV-C challenge. Conversely, if increased host susceptibility was due to parasite-induced immunoactivation at the mucosal level, i.v. SHIV-C challenge would not result in significant differences between parasitized and parasite-free monkeys. Methods and Findings We enrolled two groups of RM and infected one group with Schistosoma mansoni; the other group was left parasite-free. Both groups were challenged i.v. with decreasing doses of SHIV-C. No statistically significant differences in 50% animal infectious doses (AID50) or peak viremia were seen between the two groups. These data strongly contrast the earlier i.r. SHIV-C challenge (using the same virus stock) in the presence/absence of parasites, where we noted a 17-fold difference in AID50 and one log higher peak viremia in parasitized monkeys (P<0.001 for both). The lack of significant differences after the i.v. challenge implies that the increased host susceptibility is predominantly due to parasite-mediated mucosal upregulation of virus replication and spread, rather than systemic effects. Conclusions The major impact of schistosome-induced increased host susceptibility is at the mucosal level. Given that >90% of all new HIV-1 infections worldwide are acquired through mucosal contact, parasitic infections that inflame mucosae may play an important role in the spread of HIV-1.

Siddappa, Nagadenahalli B.; Hemashettar, Girish; Shanmuganathan, Vivekanandan; Semenya, Amma A.; Sweeney, Elizabeth D.; Paul, Katherine S.; Lee, Sandra J.; Secor, W. Evan; Ruprecht, Ruth M.

2011-01-01

35

Biodegradation of Ether Pollutants by Pseudonocardia sp. Strain ENV478  

PubMed Central

A bacterium designated Pseudonocardia sp. strain ENV478 was isolated by enrichment culturing on tetrahydrofuran (THF) and was screened to determine its ability to degrade a range of ether pollutants. After growth on THF, strain ENV478 degraded THF (63 mg/h/g total suspended solids [TSS]), 1,4-dioxane (21 mg/h/g TSS), 1,3-dioxolane (19 mg/h/g TSS), bis-2-chloroethylether (BCEE) (12 mg/h/g TSS), and methyl tert-butyl ether (MTBE) (9.1 mg/h/g TSS). Although the highest rates of 1,4-dioxane degradation occurred after growth on THF, strain ENV478 also degraded 1,4-dioxane after growth on sucrose, lactate, yeast extract, 2-propanol, and propane, indicating that there was some level of constitutive degradative activity. The BCEE degradation rates were about threefold higher after growth on propane (32 mg/h/g TSS) than after growth on THF, and MTBE degradation resulted in accumulation of tert-butyl alcohol. Degradation of 1,4-dioxane resulted in accumulation of 2-hydroxyethoxyacetic acid (2HEAA). Despite its inability to grow on 1,4-dioxane, strain ENV478 degraded this compound for >80 days in aquifer microcosms. Our results suggest that the inability of strain ENV478 and possibly other THF-degrading bacteria to grow on 1,4-dioxane is related to their inability to efficiently metabolize the 1,4-dioxane degradation product 2HEAA but that strain ENV478 may nonetheless be useful as a biocatalyst for remediating 1,4-dioxane-contaminated aquifers.

Vainberg, Simon; McClay, Kevin; Masuda, Hisako; Root, Duane; Condee, Charles; Zylstra, Gerben J.; Steffan, Robert J.

2006-01-01

36

Appreciating HIV-1 diversity: subtypic differences in ENV  

SciTech Connect

Human immunodeficiency virus type 1 (HIV-1) group M is responsible for the current AIDS pandemic and exhibits exceedingly high levels of viral genetic diversity around the world, necessitating categorization of viruses into distinct lineages, or subtypes. These subtypes can differ by around 35% in the envelope (Env) glycoproteins of the virus, which are displayed on the surface of the virion and are targets for both neutralizing antibody and cell-mediated immune responses. This diversity reflects the remarkable ability of the virus to adapt to selective pressures, the bulk of which is applied by the host immune response, and represents a serious obstacle for developing an effective vaccine with broad coverage. Thus, it is important to understand the underlying biological consequences of inter-subtype diversity. Recent studies have revealed that the HIV-1 subtypes exhibit phenotypic differences that result from subtle differences in Env structure, particularly within the highly immunogenic V3 domain, which participates directly in viral entry. This review will therefore explore current research that describes subtypic differences in Env at the genetic and phenotypic level, focusing in particular on V3, and highlighting recent discoveries about the unique features of subtype C Env, which is the most prevalent subtype globally.

Gnanakaran, S [Los Alamos National Laboratory; Shen, Tongye [Los Alamos National Laboratory; Lynch, Rebecca M [NON LANL; Derdeyn, Cynthia A [NON LANL

2008-01-01

37

Most rhesus macaques infected with the CCR5-tropic SHIV(AD8) generate cross-reactive antibodies that neutralize multiple HIV-1 strains.  

PubMed

The induction of broadly reacting neutralizing antibodies has been a major goal of HIV vaccine research. Characterization of a pathogenic CCR5 (R5)-tropic SIV/HIV chimeric virus (SHIV) molecular clone (SHIV(AD8-EO)) revealed that eight of eight infected animals developed cross-reactive neutralizing antibodies (NAbs) directed against an envelope glycoprotein derived from the heterologous HIV-1(DH12) strain. A panel of plasmas, collected from monkeys inoculated with either molecularly cloned or uncloned SHIV(AD8) stocks, exhibited cross-neutralization against multiple tier 1 and tier 2 HIV-1 clade B isolates. One SHIV(AD8)-infected animal also developed NAbs against clades A and C HIV-1 strains. In this particular infected macaque, the cross-reacting anti-HIV-1 NAbs produced between weeks 7 and 13 were directed against a neutralization-sensitive virus strain, whereas neutralizing activities emerging at weeks 41-51 targeted more neutralization-resistant HIV-1 isolates. These results indicate that the SHIV(AD8) macaque model represents a potentially valuable experimental system for investigating B-cell maturation and the induction of cross-reactive NAbs directed against multiple HIV-1 strains. PMID:23129652

Shingai, Masashi; Donau, Olivia K; Schmidt, Stephen D; Gautam, Rajeev; Plishka, Ronald J; Buckler-White, Alicia; Sadjadpour, Reza; Lee, Wendy R; LaBranche, Celia C; Montefiori, David C; Mascola, John R; Nishimura, Yoshiaki; Martin, Malcolm A

2012-11-05

38

Anti-HIV env immunities elicited by nucleic acid vaccines  

Microsoft Academic Search

Plasmid DNA vaccines encoding HIV-1 env were used to immunize mice and nonhuman primates. Plasmids were prepared that produced either secreted gp120 or full-length gp160. Mice immunized with gp120 DNA developed strong antigen-specific antibody responses, CD8+ cytotoxic T lymphocytes (CTL) (following in vitro restimulation with gp120-derived peptide), and showed in vitro proliferation and Th1-like cytokine secretion [?-interferon, interleukin (IL)-2 with

John W. Shiver; Mary-Ellen Davies; Yasuhiro Yasutomi; Helen C. Perry; Daniel C. Freed; Norman L. Letvin; Margaret A. Liu

1997-01-01

39

Env-less endogenous retroviruses are genomic superspreaders  

PubMed Central

Endogenous retroviruses (ERVs) differ from typical retroviruses in being inherited through the host germline and therefore are a unique combination of pathogen and selfish genetic element. Some ERV lineages proliferate by infecting germline cells, as do typical retroviruses, whereas others lack the env gene required for virions to enter cells and thus behave like retrotransposons. We wished to know what factors determined the relative abundance of different ERV lineages, so we analyzed ERV loci recovered from 38 mammal genomes by in silico screening. By modeling the relationship between proliferation and replication mechanism in detail within one group, the intracisternal A-type particles (IAPs), and performing simple correlations across all ERV lineages, we show that when ERVs lose the env gene their proliferation within that genome is boosted by a factor of ?30. We also show that ERV abundance follows the Pareto principle or 20/80 rule, with ?20% of lineages containing 80% of the loci. This rule is observed in many biological systems, including infectious disease epidemics, where commonly ?20% of the infected individuals are responsible for 80% of onward infection. We thus borrow simple epidemiological and ecological models and show that retrotransposition and loss of env is the trait that leads endogenous retroviruses to becoming genomic superspreaders that take over a significant proportion of their host's genome.

Magiorkinis, Gkikas; Gifford, Robert J.; Katzourakis, Aris; De Ranter, Joris; Belshaw, Robert

2012-01-01

40

Estimating the impact of vaccination in acute SHIV-SIV infection  

SciTech Connect

Human Immunodeficiency Virus (HIV) infects approxmately 0.5% of the world population, and is a major cause of morbidity and mortality worldwide. A vaccine for HIV is urgently required, and a variety of vaccine modalities have been tested in animal models of infection. A number of these studies have shown protection in monkey models of infection, although the ability of the vaccine to protect appears to vary with the viral strain and animal model used. The recent failure of a large vaccine study in humans suggests that further understanding of the basic dynamics of infection and impact of vaccination are required, in order to understand the variable efficacy of vaccination in different infections. The dynamics of HIV infection have been studied in humans and in a variety of animal models. The standard model of infection has been used to estimate the basic reproductive ratio (R{sub 0}) of the virus, calculated from the growth rate of virus in acute infection. This method has not been useful in studying the effects of vaccination, since, in the vaccines developed so far, early growth rates of virus do not differ between control and vaccinated animals. Here, we use the standard model of viral dynamics to derive the reproductive ratio from the peak viral load and nadir of target cell numbers in acute infection. We apply this method to data from studies of vaccination in Simian Human Immunodeficiency Virus (SHIV) and Simian Immunodeficiency Virus (SIV) infection and demonstrate that vaccination can reduce the reproductive ratio by 2.3 and 2 fold respectively. This method allows the comparison of vaccination efficacy amongst different viral strains and animal models in vivo.

Ribeiro, Ruy [Los Alamos National Laboratory

2008-01-01

41

The HIV-1 Env Protein: A Coat of Many Colors  

PubMed Central

Human immunodeficiency virus type 1 (HIV-1) is completely dependent upon the Env protein to enter cells. The virus typically replicates in activated CD4+ T cells due to viral entry requirements for the CCR5 coreceptor and for high surface levels of the CD4 receptor. This is the case for the transmitted virus and for most of the virus sampled in the blood. Over the course of infection, the env gene can evolve to encode a protein with altered receptor and coreceptor usage allowing the virus to enter alternative host cells. In about 50% of HIV-1 infections, the viral population undergoes coreceptor switching, usually late in disease, allowing the virus to use CXCR4 to enter a different subset of CD4+ T cells. Neurocognitive disorders occur in about 10% of infections, also usually late in disease, but caused (ultimately) by viral replication in the brain either in CD4+ T cells or macrophage and/or microglia. Expanded host range is significantly intertwined with pathogenesis. Identification and characterization of such HIV-1 variants may be useful for early detection which would allow intervention to reduce viral pathogenesis in these alternative cell types.

Arrildt, Kathryn Twigg; Joseph, Sarah Beth; Swanstrom, Ronald

2013-01-01

42

Prevention of Rectal SHIV Transmission in Macaques by Daily or Intermittent Prophylaxis with Emtricitabine and Tenofovir  

PubMed Central

Background In the absence of an effective vaccine, HIV continues to spread globally, emphasizing the need for novel strategies to limit its transmission. Pre-exposure prophylaxis (PrEP) with antiretroviral drugs could prove to be an effective intervention strategy if highly efficacious and cost-effective PrEP modalities are identified. We evaluated daily and intermittent PrEP regimens of increasing antiviral activity in a macaque model that closely resembles human transmission. Methods and Findings We used a repeat-exposure macaque model with 14 weekly rectal virus challenges. Three drug treatments were given once daily, each to a different group of six rhesus macaques. Group 1 was treated subcutaneously with a human-equivalent dose of emtricitabine (FTC), group 2 received orally the human-equivalent dosing of both FTC and tenofovir-disoproxil fumarate (TDF), and group 3 received subcutaneously a similar dosing of FTC and a higher dose of tenofovir. A fourth group of six rhesus macaques (group 4) received intermittently a PrEP regimen similar to group 3 only 2 h before and 24 h after each weekly virus challenge. Results were compared to 18 control macaques that did not receive any drug treatment. The risk of infection in macaques treated in groups 1 and 2 was 3.8- and 7.8-fold lower than in untreated macaques (p = 0.02 and p = 0.008, respectively). All six macaques in group 3 were protected. Breakthrough infections had blunted acute viremias; drug resistance was seen in two of six animals. All six animals in group 4 that received intermittent PrEP were protected. Conclusions This model suggests that single drugs for daily PrEP can be protective but a combination of antiretroviral drugs may be required to increase the level of protection. Short but potent intermittent PrEP can provide protection comparable to that of daily PrEP in this SHIV/macaque model. These findings support PrEP trials for HIV prevention in humans and identify promising PrEP modalities.

Garcia-Lerma, J. Gerardo; Otten, Ron A; Qari, Shoukat H; Jackson, Eddie; Cong, Mian-er; Masciotra, Silvina; Luo, Wei; Kim, Caryn; Adams, Debra R; Monsour, Michael; Lipscomb, Jonathan; Johnson, Jeffrey A; Delinsky, David; Schinazi, Raymond F; Janssen, Robert; Folks, Thomas M; Heneine, Walid

2008-01-01

43

Dynamics of envelope evolution in clade C SHIV-infected pig-tailed macaques during disease progression analyzed by ultra-deep pyrosequencing.  

PubMed

Understanding the evolution of the human immunodeficiency virus type 1 (HIV-1) envelope during disease progression can provide tremendous insights for vaccine development, and simian-human immunodeficiency virus (SHIV) infection of non-human primate provides an ideal platform for such studies. A newly developed clade C SHIV, SHIV-1157ipd3N4, which was able to infect rhesus macaques, closely resembled primary HIV-1 in transmission and pathogenesis, was used to infect several pig-tailed macaques. One of the infected animals subsequently progressed to AIDS, whereas one remained a non-progressor. The viral envelope evolution in the infected animals during disease progression was analyzed by a bioinformatics approach using ultra-deep pyrosequencing. Our results showed substantial envelope variations emerging in the progressor animal after the onset of AIDS. These envelope variations impacted the length of the variable loops and charges of different envelope regions. Additionally, multiple mutations were located at the CD4 and CCR5 binding sites, potentially affecting receptor binding affinity, viral fitness and they might be selected at late stages of disease. More importantly, these envelope mutations are not random since they had repeatedly been observed in a rhesus macaque and a human infant infected by either SHIV or HIV-1, respectively, carrying the parental envelope of the infectious molecular clone SHIV-1157ipd3N4. Moreover, similar mutations were also observed from other studies on different clades of envelopes regardless of the host species. These recurring mutations in different envelopes suggest that there may be a common evolutionary pattern and selection pathway for the HIV-1 envelope during disease progression. PMID:22427893

Tso, For Yue; Tully, Damien C; Gonzalez, Sandra; Quince, Christopher; Ho, On; Polacino, Patricia; Ruprecht, Ruth M; Hu, Shiu-Lok; Wood, Charles

2012-03-12

44

NMobTec-EnvEdu: M-Learning System for Environmental Education  

ERIC Educational Resources Information Center

This paper introduced the implementation of a New Mobile Technologies and Environmental Education System (NMobTec-EnvEdu) designed for m-learning environments. The NMobTec-EnvEdu system has been developed to provide environmental education in a collaborative framework to undergraduate students through the Internet using mobile phones. The study…

Cavus, Nadire

2008-01-01

45

The Fusion-Controlling Disulfide Bond Isomerase in Retrovirus Env Is Triggered by Protein Destabilization  

Microsoft Academic Search

The membrane fusion function of murine leukemia virus (MLV) is carried by the Env protein. This protein is composed of three SU-TM subunit complexes. The fusion activity is loaded into the transmembrane TM subunit and controlled by the peripheral, receptor-binding SU subunit. It is assumed that TM adopts a metastable conformation in the native Env and that fusion activation involves

Michael Wallin; Maria Ekstrom; Henrik Garoff

2005-01-01

46

The role of dynamin in HIV type 1 Env-mediated cell-cell fusion.  

PubMed

HIV-1 envelope glycoproteins are the key viral proteins that mediate HIV-1 entry and cell-cell fusion. In contrast to HIV-1 entry, the mechanism of HIV-1 Env-mediated cell-cell fusion is relatively unclear. This study demonstrated that dynasore, a dynamin inhibitor, suppressed HIV-1 Env-mediated cell-cell fusion. Dynasore sensitivity of HIV-1 Env-mediated cell-cell fusion varied depending on the viral strains. Results from testing a panel of gp41 cytoplasmic tail truncation mutants suggested that the gp41 cytoplasmic tail might play a role in dynasore sensitivity. HIV-1 Env-mediated cell-cell fusion could also be suppressed by a dynamin dominant-negative mutant DNM2(K44A). In summary, these results suggested that dynamin 2 might play a role in HIV-1 Env-mediated cell-cell fusion. PMID:21338326

Lai, Weihong; Huang, Li; Ho, Phong; Montefiori, David; Chen, Chin-Ho

2011-03-23

47

Chimeras between the human immunodeficiency virus (HIV1) Env and vaccinia virus immunogenic proteins p14 and p39 generate in mice broadly reactive antibodies and specific activation of CD8+ T cell responses to Env  

Microsoft Academic Search

A vaccine based on the envelope protein (Env) of the human immunodeficiency virus type 1 (HIV-1) that triggers widely reactive antibodies might be a desirable approach to control virus infection. To expose epitopes which could induce broadly reactive antibodies against HIV-1 Env, we have generated vaccinia virus (VV) recombinants that express Env fused at its N- or C-terminus with two

Manuel Collado; Dolores Rodr??guez; Juan Ramón Rodr??guez; Isabel Vázquez; Rosa M. Gonzalo; Mariano Esteban

2000-01-01

48

A single amino acid substitution within the transmembrane domain of the human immunodeficiency virus type 1 Vpu protein renders simian-human immunodeficiency virus (SHIV{sub KU-1bMC33}) susceptible to rimantadine  

SciTech Connect

Previous studies from our laboratory have shown that the transmembrane domain (TM) of the Vpu protein of human immunodeficiency virus type 1 (HIV-1) contributes to the pathogenesis of SHIV{sub KU-1bMC33} in macaques and that the TM domain of Vpu could be replaced with the M2 protein viroporin from influenza A virus. Recently, we showed that the replacement of the TM domain of Vpu with that of the M2 protein of influenza A virus resulted in a virus (SHIV{sub M2}) that was sensitive to rimantadine [Hout, D.R., Gomez, M.L., Pacyniak, E., Gomez, L.M., Inbody, S.H., Mulcahy, E.R., Culley, N., Pinson, D.M., Powers, M.F., Wong, S.W., Stephens, E.B., 2006. Substitution of the transmembrane domain of Vpu in simian human immunodeficiency virus (SHIV{sub KU-1bMC33}) with that of M2 of influenza A results in a virus that is sensitive to inhibitors of the M2 ion channel and is pathogenic for pig-tailed macaques. Virology 344, 541-558]. Based on previous studies of the M2 protein which have shown that the His-X-X-X-Trp motif within the M2 is essential to the function of the M2 proton channel, we have constructed a novel SHIV in which the alanine at position 19 of the TM domain was replaced with a histidine residue resulting in the motif His-Ile-Leu-Val-Trp. The SHIV{sub VpuA19H} replicated with similar kinetics as the parental SHIV{sub KU-1bMC33} and pulse-chase analysis revealed that the processing of viral proteins was similar to SHIV{sub KU-1bMC33}. This SHIV{sub VpuA19H} virus was found to be more sensitive to the M2 ion channel blocker rimantadine than SHIV{sub M2}. Electron microscopic examination of SHIV{sub VpuA19H}-infected cells treated with rimantadine revealed an accumulation of viral particles at the cell surface and within intracellular vesicles, which was similar to that previously observed to SHIV{sub M2}-infected cells treated with rimantadine. These data indicate that the Vpu protein of HIV-1 can be converted into a rimantadine-sensitive ion channel with the alteration of one amino acid and provide additional evidence that drugs targeting the Vpu TM/ion channel can be effective anti-HIV-1 drugs.

Hout, David R. [Department of Anatomy and Cell Biology, University of Kansas Medical Center 3901 Rainbow Blvd., Kansas City, KS 66160 (United States); Gomez, Lisa M. [Department of Anatomy and Cell Biology, University of Kansas Medical Center 3901 Rainbow Blvd., Kansas City, KS 66160 (United States); Pacyniak, Erik [Department of Anatomy and Cell Biology, University of Kansas Medical Center 3901 Rainbow Blvd., Kansas City, KS 66160 (United States); Miller, Jean-Marie [Department of Anatomy and Cell Biology, University of Kansas Medical Center 3901 Rainbow Blvd., Kansas City, KS 66160 (United States); Hill, M. Sarah [Department of Anatomy and Cell Biology, University of Kansas Medical Center 3901 Rainbow Blvd., Kansas City, KS 66160 (United States); Stephens, Edward B. [Department of Anatomy and Cell Biology, University of Kansas Medical Center 3901 Rainbow Blvd., Kansas City, KS 66160 (United States)]. E-mail: estephen@kumc.edu

2006-05-10

49

Induction of potent local cellular immunity with low dose X4 SHIV{sub SF33A} vaginal exposure  

SciTech Connect

Intravaginal inoculation of rhesus macaques with varying doses of the CXCR4 (X4)-tropic SHIV{sub SF33A} isolate revealed a threshold inoculum for establishment of systemic virus infection and a dose dependency in overall viral burden and CD4+ T cell depletion. While exposure to inoculum size of 1000 or greater 50% tissue infectious dose (TCID{sub 50}) resulted in high viremia and precipitous CD4+ T cell loss, occult infection was observed in seven of eight macaques exposed to 500 TCID{sub 50} of the same virus. The latter was characterized by intermittent detection of low level virus with no evidence of seroconversion or CD4+ T cell decline, but with signs of an ongoing antiviral T cell immune response. Upon vaginal re-challenge with the same limiting dose 11-12 weeks after the first, classic pathogenic X4 SHIV{sub SF33A} infection was established in four of the seven previously exposed seronegative macaques, implying enhanced susceptibility to systemic infection with prior exposure. Pre-existing peripheral SIV gag-specific CD4+ T cells were more readily demonstrable in macaques that became systemically infected following re-exposure than those that were not. In contrast, early presence of circulating polyfunctional cytokine secreting CD8+ T cells or strong virus-specific proliferative responses in draining lymph nodes and in the gut associated lymphoid tissue (GALT) following the first exposure was associated with protection from systemic re-infection. These studies identify the gut and lymphoid tissues proximal to the genital tract as sites of robust CD8 T lymphocyte responses that contribute to containment of virus spread following vaginal transmission.

Tasca, Silvana; Tsai, Lily; Trunova, Nataliya; Gettie, Agegnehu [Aaron Diamond AIDS Research Center, Rockefeller University, 455 First Ave., 7th Floor, New York, NY 10016 (United States); Saifuddin, Mohammed [CONRAD, Eastern Virginia Medical School, 1611 North Kent Street Suite 806, Arlington, VA 22209 (United States); Bohm, Rudolf [Tulane National Primate Research Center, Tulane University Medical Center, 18702 Three Rivers Road, Covington, LA 70433 (United States); Chakrabarti, Lisa [Institut Pasteur, Unite d'Immunologie Virale, 28 rue du Dr roux, 75724 Paris Cedex 15 (France); Cheng-Mayer, Cecilia [Aaron Diamond AIDS Research Center, Rockefeller University, 455 First Ave., 7th Floor, New York, NY 10016 (United States)], E-mail: cmayer@adarc.org

2007-10-10

50

Coreceptor-dependent inhibition of the cell fusion activity of simian immunodeficiency virus Env proteins.  

PubMed

The cytoplasmic tail (R peptide) sequence is able to regulate the fusion activity of the murine leukemia virus (MuLV) envelope (Env) protein. We have previously shown that this sequence exerts a profound inhibitory effect on the fusion activity of simian immunodeficiency virus (SIV)-MuLV chimeric Env proteins which contain the extracellular and transmembrane domains of the SIV Env protein. Recent studies have shown that SIV can utilize several alternative cellular coreceptors for its fusion and entry into the cell. We have investigated the fusion activity of SIV and SIV-MuLV chimeric Env proteins using cells that express different coreceptors. HeLa cells were transfected with plasmid constructs that carry the SIV or SIV-MuLV chimeric Env protein genes and were overlaid with either CEMx174 cells or Ghost Gpr15 cells, which express the Gpr15 coreceptor for SIV, or Ghost CCR5 cells, which express CCR5, an alternate coreceptor for SIV. The R-peptide sequence in the SIV-MuLV chimeric proteins was found to inhibit the fusion with CEMx174 cells or Ghost Gpr15 cells. However, a significant level of fusion was still observed when HeLa cells expressing the chimeric Env proteins were cocultivated with Ghost CCR5 cells. These results show that the R-peptide sequence exerts differential effects on the fusion activity of SIV Env proteins using target cells that express alternative coreceptors. PMID:10846110

Yang, C; Yang, Q; Compans, R W

2000-07-01

51

Multiple Gag Domains Contribute to Selective Recruitment of Murine Leukemia Virus (MLV) Env to MLV Virions  

PubMed Central

Retroviruses, like all enveloped viruses, must incorporate viral glycoproteins to form infectious particles. Interactions between the glycoprotein cytoplasmic tail and the matrix domain of Gag are thought to direct recruitment of glycoproteins to native virions for many retroviruses. However, retroviruses can also incorporate glycoproteins from other viruses to form infectious virions known as pseudotyped particles. The glycoprotein murine leukemia virus (MLV) Env can readily form pseudotyped particles with many retroviruses, suggesting a generic mechanism for recruitment. Here, we sought to identify which components of Gag, particularly the matrix domain, contribute to recruitment of MLV Env into retroviral particles. Unexpectedly, we discovered that the matrix domain of HIV-1 Gag is dispensable for generic recruitment, since it could be replaced with a nonviral membrane-binding domain without blocking active incorporation of MLV Env into HIV virions. However, MLV Env preferentially assembles with MLV virions. When MLV and HIV particles are produced from the same cell, MLV Env is packaged almost exclusively by MLV particles, thus preventing incorporation into HIV particles. Surprisingly, the matrix domain of MLV Gag is not required for this selectivity, since MLV Gag containing the matrix domain from HIV is still able to outcompete HIV particles for MLV Env. Although MLV Gag is sufficient for selective incorporation to occur, no single Gag domain dictates the selectivity. Our findings indicate that Env recruitment is more complex than previously believed and that Gag assembly/budding sites have fundamental properties that affect glycoprotein incorporation.

Gregory, Devon A.; Lyddon, Terri D.

2013-01-01

52

Antibodies to CD4-induced sites in HIV gp120 correlate with the control of SHIV challenge in macaques vaccinated with subunit immunogens  

PubMed Central

Epitopes located in and around the coreceptor binding site of HIV-1 envelope glycoprotein (gp120) exhibit enhanced exposure after attachment to the CD4 receptor and comprise some of the most conserved and functionally important residues on the viral envelope. Therefore, antibody responses to these epitopes [designated as CD4-induced (CD4i)] should be highly cross-reactive and potentially useful for HIV vaccine development. To address this question, rhesus macaques were vaccinated with subunit immunogens designed to raise humoral responses against CD4i epitopes and challenged rectally with SHIV162P3, which encodes a heterologous envelope versus the immunogen. We found that animals vaccinated with a rhesus full-length single-chain (rhFLSC) complex exhibited significantly accelerated clearance of plasma viremia and an absence of long-term tissue viremia compared with unvaccinated control animals. Such control of infection correlated with stronger responses to CD4i epitopes in the rhFLSC-vaccinated animals, compared with macaques immunized with gp120, cross-linked gp120–CD4 complexes, or soluble CD4 alone. These responses were strongly boosted in the rhFLSC-vaccinated animals by SHIV162P3 infection. The control of infection was not associated with anti-CD4 responses, overall anti-gp120-binding titers, or neutralizing activity measured in conventional assays. Vaccine-naive animals also developed anti-CD4i epitope responses after simian/ human immunodeficiency virus (SHIV) challenge, which appeared later than the overall anti-gp120 responses and in concert with the decline of viremia to a low set point. Collectively, these data suggest that antibodies to CD4i epitopes may play a role in controlling SHIV infection and provide insights for HIV vaccine development.

DeVico, Anthony; Fouts, Timothy; Lewis, George K.; Gallo, Robert C.; Godfrey, Karla; Charurat, Manhattan; Harris, Ilia; Galmin, Lindsey; Pal, Ranajit

2007-01-01

53

Immune Responses but No Protection against SHIV by Gene-Gun Delivery of HIV1 DNA Followed by Recombinant Subunit Protein Boosts  

Microsoft Academic Search

The efficacy of combining immunization with human immunodeficiency vitus type 1 (HIV-1) DNA and HIV-1 recombinant proteins to obtain protection from chimeric simian\\/human immunodeficiency virus (SHIV) was determined. Four cynomolgus monkeys received four gene-gun immunizations intraepidermally of plasmid DNA encoding HIV-1laienv(gp160),gag, tat, nef,andrevproteins. Ten micrograms of DNA was used per immunization. The animals were boosted twice intramuscularly with 50 ?g

Per Putkonen; Marlene Quesada-Rolander; Ann-Charlotte Leandersson; Stefan Schwartz; Rigmor Thorstensson; Kenji Okuda; Britta Wahren; Jorma Hinkula

1998-01-01

54

High susceptibility to repeated, low-dose, vaginal SHIV exposure late in the luteal phase of the menstrual cycle of pigtail macaques.  

PubMed

Fluctuations in susceptibility to HIV or SHIV during the menstrual cycle are currently not fully documented. To address this, the time point of infection was determined in 19 adult female pigtail macaques vaginally challenged during their undisturbed menstrual cycles with repeated, low-dose SHIV(SF162P3) exposures. Eighteen macaques (95%) first displayed viremia in the follicular phase, as compared with 1 macaque (5%) in the luteal phase (P < 0.0001). Due to a viral eclipse phase, we estimated a window of most frequent virus transmission between days 24 and 31 of the menstrual cycle, in the late luteal phase. Thus, susceptibility to vaginal SHIV infection is significantly elevated in the second half of the menstrual cycle when progesterone levels are high and when local immunity may be low. Such susceptibility windows have been postulated before but not definitively documented. Our data support the findings of higher susceptibility to HIV in women during progesterone-dominated periods including pregnancy and contraceptive use. PMID:21546848

Vishwanathan, Sundaram A; Guenthner, Patricia C; Lin, Carol Y; Dobard, Charles; Sharma, Sunita; Adams, Debra R; Otten, Ron A; Heneine, Walid; Hendry, R Michael; McNicholl, Janet M; Kersh, Ellen N

2011-08-01

55

Repressive Effect of Primary Virus Replication on Superinfection Correlated with Gut-Derived Central Memory CD4(+) T Cells in SHIV-Infected Chinese Rhesus Macaques.  

PubMed

A possible mechanism of susceptibility to superinfection with simian-human immunodeficiency virus (SHIV)-1157ipd3N4 was explored in twelve SHIVSF162P3-infected Chinese rhesus macaques. Based on the kinetics of viral replication for the second infecting virus following SHIV-1157ipd3N4 inoculation, the monkeys were divided into two groups: those relatively resistant to superinfection (SIR) and those relatively sensitive to superinfection (SIS). We found that superinfection-resistant macaques had high primary viremia, whereas superinfection-sensitive macaques had low primary viremia, suggesting that primary SHIVSF162P3 infection with a high viral-replication level would repress superinfection with a heterologous SHIV-1157ipd3N4. Although no correlation of protection against superinfection with virus-specific CD4(+) T cell or CD8(+) T cell immune responses from gut was observed prior to superinfection, superinfection susceptibility was strongly correlated with CD4(+) Tcm cells from gut both prior to the second infecting virus inoculation and on day 7 after superinfection, but not with CD4(+) Tem cells from gut or with CD4(+) Tcm cells from peripheral blood and lymph node. These results point to the important roles of gut-derived CD4(+) Tcm cells for the study of the mechanisms of protection against superinfection and the evaluation of the safety and efficacy of vaccines and therapies against acquired immune deficiency syndrome (AIDS). PMID:24023734

Xue, Jing; Cong, Zhe; Xiong, Jing; Wang, Wei; Jiang, Hong; Chen, Ting; Wu, Fangxin; Liu, Kejian; Su, Aihua; Ju, Bin; Chen, Zhiwei; Couto, Marcelo A; Wei, Qiang; Qin, Chuan

2013-09-02

56

Antitumor Efficacy of 34.5ENVE: A Transcriptionally Retargeted and "Vstat120"-expressing Oncolytic Virus  

PubMed Central

Here, we describe the construction and testing of a novel herpes simplex virus type 1 (HSV-1) derived oncolytic virus (OV): 34.5ENVE (viral ICP34.5 Expressed by Nestin promotor and Vstat120 Expressing), for the treatment of cancer. This virus showed significant glioma-specific killing and antiangiogenic effects in vitro and in vivo. Treatment of subcutaneous and intracranial glioma-bearing mice with 34.5ENVE showed a significant increase in median survival of mice in four different glioma models. Histology and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) revealed reduced microvessel density (MVD) and increased tumoral necrosis in 34.5ENVE-treated tumor tissue compared to control OV-treated tumor tissue. Collectively, these results describe the construction, efficacy, and impact on tumor microenvironment of a transcriptionally driven OV armed with Vstat120 gene expression. These preclinical results will facilitate future clinical testing of 34.5ENVE.

Yoo, Ji Young; Haseley, Amy; Bratasz, Anna; Chiocca, E Antonio; Zhang, Jianying; Powell, Kimerly; Kaur, Balveen

2012-01-01

57

Quantifying Selection against Synonymous Mutations in HIV-1 env Evolution.  

PubMed

Intrapatient evolution of human immunodeficiency virus type 1 (HIV-1) is driven by the adaptive immune system resulting in rapid change of HIV-1 proteins. When cytotoxic CD8(+) T cells or neutralizing antibodies target a new epitope, the virus often escapes via nonsynonymous mutations that impair recognition. Synonymous mutations do not affect this interplay and are often assumed to be neutral. We test this assumption by tracking synonymous mutations in longitudinal intrapatient data from the C2-V5 part of the env gene. We find that most synonymous variants are lost even though they often reach high frequencies in the viral population, suggesting a cost to the virus. Using published data from SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) assays, we find that synonymous mutations that disrupt base pairs in RNA stems flanking the variable loops of gp120 are more likely to be lost than other synonymous changes: these RNA hairpins might be important for HIV-1. Computational modeling indicates that, to be consistent with the data, a large fraction of synonymous mutations in this genomic region need to be deleterious with a cost on the order of 0.002 per day. This weak selection against synonymous substitutions does not result in a strong pattern of conservation in cross-sectional data but slows down the rate of evolution considerably. Our findings are consistent with the notion that large-scale patterns of RNA structure are functionally relevant, whereas the precise base pairing pattern is not. PMID:23986591

Zanini, Fabio; Neher, Richard A

2013-08-28

58

Role of oligomerization of the S13 Env-Sea oncoprotein in cell transformation.  

PubMed Central

The env-sea oncogene is a fusion of the S13 viral envelope gene, env, and cell-derived sequences encoding a tyrosine kinase domain, termed sea. The Env-Sea oncoprotein is synthesized as a precursor of 155 kDa which undergoes proteolytic processing to generate a disulfide-linked complex of the proteins gp85 and gp70. We analyzed the oligomeric state of the Env-Sea oncoprotein in S13-transformed cells and demonstrate that both gp155 and the gp85-gp70 complex can oligomerize. To address the relevance of these oligomers in transformation by S13, a mutant that is temperature sensitive for the transformed phenotype was used. The tyrosine-phosphorylated oligomers of gp155 were found at the nonpermissive temperature, and thus oligomerization per se appears to be insufficient to elicit a transformed phenotype. Efficient intracellular transport of gp155 appears to be required to generate a tyrosine-phosphorylated oligomer of the gp85-gp70 complex, the presence of which correlates with the transformed phenotype. This gp85-gp70 complex appeared to have a higher level of kinase activity than the other forms of the Env-Sea protein. These results suggest that oligomerization, transport, and intracellular localization represent levels at which the oncogenic activity of the Env-Sea oncoprotein may be regulated. Images

Morimoto, A M; Hayman, M J

1994-01-01

59

Identification of ENV determinants in V3 that influence the molecular anatomy of CCR5 utilization.  

PubMed

The V3 loop of the ENV glycoprotein exerts a dominant influence on the interaction of gp120 with coreceptors. Primary env genes cloned from sequential isolates from two seroconverters revealed Pro-->Ala conversion in the conserved GPG motif of the V3 crown in seven of 17 R5 ENV. ENV containing the GPG motif in the V3 crown had fusogenic activity with chimeric receptors containing either the N terminus or loops of CCR5, whereas those with the GAG variant utilized only the former. Site-directed mutagenesis of multiple primary and prototypic R5 env genes demonstrated that the GPG motif was necessary for dual utilization of the N terminus and body of CCR5 in both gain and loss-of-function experiments. All ENV containing the GPG V3 crown showed CCR5 binding in the presence of soluble CD4, whereas it was not detected with the GAG variants. Molecular dynamic simulations of a V3 peptide predicts that the Pro-->Ala substitution results in a conformational change with loss of the crown structure. These studies demonstrate that sequences in the third hypervariable region determine the specificity of coreceptor utilization for fusion, and that a conserved motif in the crown directly influences the molecular anatomy of the interaction between gp120 and CCR5. PMID:10970739

Hu, Q; Trent, J O; Tomaras, G D; Wang, Z; Murray, J L; Conolly, S M; Navenot, J M; Barry, A P; Greenberg, M L; Peiper, S C

2000-09-15

60

Relations among Manual RT, Visual RT and IQ.  

ERIC Educational Resources Information Center

As part of a study to determine whether visual and manual response systems are correlated, 26 children between 40 and 51 months of age took part in visual and manual reaction time (RT) tasks. Subjects, whose RTs had previously been tested at 3 months of age, were tested in 1 of 2 conditions. In the first condition, subjects viewed pictures only…

Dougherty, Thomas M.; Haith, Marshall M.

61

With Minimal Systemic T-Cell Expansion, CD8+ T Cells Mediate Protection of Rhesus Macaques Immunized with Attenuated Simian-Human Immunodeficiency Virus SHIV89.6 from Vaginal Challenge with Simian Immunodeficiency Virus?  

PubMed Central

The presence, at the time of challenge, of antiviral effector T cells in the vaginal mucosa of female rhesus macaques immunized with live-attenuated simian-human immunodeficiency virus 89.6 (SHIV89.6) is associated with consistent and reproducible protection from pathogenic simian immunodeficiency virus (SIV) vaginal challenge (18). Here, we definitively demonstrate the protective role of the SIV-specific CD8+ T-cell response in SHIV-immunized monkeys by CD8+ lymphocyte depletion, an intervention that abrogated SHIV-mediated control of challenge virus replication and largely eliminated the SIV-specific T-cell responses in blood, lymph nodes, and genital mucosa. While in the T-cell-intact SHIV-immunized animals, polyfunctional and degranulating SIV-specific CD8+ T cells were present in the genital tract and lymphoid tissues from the day of challenge until day 14 postchallenge, strikingly, expansion of SIV-specific CD8+ T cells in the immunized monkeys was minimal and limited to the vagina. Thus, protection from uncontrolled SIV replication in animals immunized with attenuated SHIV89.6 is primarily mediated by CD8+ T cells that do not undergo dramatic systemic expansion after SIV challenge. These findings demonstrate that despite, and perhaps because of, minimal systemic expansion of T cells at the time of challenge, a stable population of effector-cytotoxic CD8+ T cells can provide significant protection from vaginal SIV challenge.

Genesca, Meritxell; Skinner, Pamela J.; Hong, Jung Joo; Li, Jun; Lu, Ding; McChesney, Michael B.; Miller, Christopher J.

2008-01-01

62

Immunization with HIV-1 gp41 subunit virosomes induces mucosal antibodies protecting nonhuman primates against vaginal SHIV challenges.  

PubMed

Human immunodeficiency virus (HIV)-1 is mainly transmitted mucosally during sexual intercourse. We therefore evaluated the protective efficacy of a vaccine active at mucosal sites. Macaca mulatta monkeys were immunized via both the intramuscular and intranasal routes with an HIV-1 vaccine made of gp41-subunit antigens grafted on virosomes, a safe delivery carrier approved in humans with self-adjuvant properties. Six months after 13 vaginal challenges with simian-HIV (SHIV)-SF162P3, four out of five vaccinated animals remained virus-negative, and the fifth was only transiently infected. None of the five animals seroconverted to p27gag-SIV. In contrast, all 6 placebo-vaccinated animals became infected and seroconverted. All protected animals showed gp41-specific vaginal IgAs with HIV-1 transcytosis-blocking properties and vaginal IgGs with neutralizing and/or antibody-dependent cellular-cytotoxicity activities. In contrast, plasma IgGs totally lacked virus-neutralizing activity. The protection observed challenges the paradigm whereby circulating antiviral antibodies are required for protection against HIV-1 infection and may serve in designing a human vaccine against HIV-1-AIDS. PMID:21315623

Bomsel, Morgane; Tudor, Daniela; Drillet, Anne-Sophie; Alfsen, Annette; Ganor, Yonatan; Roger, Marie-Gaëlle; Mouz, Nicolas; Amacker, Mario; Chalifour, Anick; Diomede, Lorenzo; Devillier, Gilles; Cong, Zhe; Wei, Qiang; Gao, Hong; Qin, Chuan; Yang, Gui-Bo; Zurbriggen, Rinaldo; Lopalco, Lucia; Fleury, Sylvain

2011-02-25

63

Models of HTLV-I-induced diseases. Infectious transmission of HTLV-I in inbred rats and HTVL-I env-pX transgenic rats.  

PubMed

To examine the pathogenic roles of HTLV-I in HTLV-I-induced diseases, we developed two models; namely HTLV-I carrier rats and HTLV-I env-pX transgenic rats. Among life long HTLV-I carriers in seven rat strains, only WKAH rats with the RT1k haplotype developed chronic progressive myeloneuropathy, resembling HAM/TSP clinically and histologically in humans, designated as HAM rat disease and after long incubation periods. Apoptosis of myelin forming cells, oligodendrocytes and Schwann cells associated with HTLV-I infection appears to be the primary cause of HAM rat disease. Local activation of the pX gene and TNF alpha gene was evident in these rats. WKAH rats transgenic for HTLV-I env-pX gene were established and at age 5 weeks, swelling of the bilateral ankle joints began to develop and histological features of the affected joints resembled findings in cases of rheumatoid arthritis (RA): high-titers of rheumatoid factors were present in these rats. A series of vascular collagen diseases such as polyarteritis nodosa-like angiitis, polymyositis, myocarditis, and Sjögren's syndrome-like sialodenitis together with RA were present, even in one individual animal. These transgenic rats as well as HAM rats appear to be suitable animal models for elucidating pathogenic mechanisms implicated in HTLV-I-induced diseases and also various demyelinating vascular collagen diseases of unknown etiology. PMID:9209354

Yoshiki, T; Ikeda, H; Tomaru, U; Ohya, O; Kasai, T; Yamashita, I; Morita, K; Yamazaki, H; Ishizu, A; Nakamaru, Y; Kikuchi, K; Tanaka, S; Wakisaka, A

1997-04-01

64

Molecular structures of trimeric HIV-1 Env in complex with small antibody derivatives  

PubMed Central

The extensive carbohydrate coat, the variability of protein structural features on HIV-1 envelope glycoproteins (Env), and the steric constraints of the virus-cell interface during infection, present challenges to the elicitation of effective full-length (?150 kDa), neutralizing antibodies against HIV. These hurdles have motivated the engineering of smaller antibody derivatives that can bind Env and neutralize the virus. To further understand the mechanisms by which these proteins neutralize HIV-1, we carried out cryoelectron tomography of native HIV-1 BaL virions complexed separately to two small (?15 kDa) HIV-neutralizing proteins: A12, which binds the CD4-binding site on Env, and m36, whose binding to Env is enhanced by CD4 binding. We show that despite their small size, the presence of these proteins and their effects on the quaternary conformation of trimeric Env can be visualized in molecular structures derived by cryoelectron tomography combined with subvolume averaging. Binding of Env to A12 results in a conformational change that is comparable to changes observed upon its binding to the CD4-binding site antibody, b12. In contrast, binding of Env to m36 results in an “open” quaternary conformation similar to that seen with binding of soluble CD4 or the CD4i antibody, 17b. Because these small neutralizing proteins are less sterically hindered than full-length antibodies at zones of virus-cell contact, the finding that their binding has the same structural consequences as that of other broadly neutralizing antibodies highlights their potential for use in therapeutic applications.

Meyerson, Joel R.; Tran, Erin E. H.; Kuybeda, Oleg; Chen, Weizao; Dimitrov, Dimiter S.; Gorlani, Andrea; Verrips, Theo; Lifson, Jeffrey D.; Subramaniam, Sriram

2013-01-01

65

The nonnucleoside reverse transcription inhibitor MIV-160 delivered from an intravaginal ring, but not from a carrageenan gel, protects against simian/human immunodeficiency virus-RT Infection.  

PubMed

We previously showed that a carrageenan (CG) gel containing 50??M MIV-150 (MIV-150/CG) reduced vaginal simian/human immunodeficiency virus (SHIV)-RT infection of macaques (56%, p>0.05) when administered daily for 2 weeks with the last dose given 8?h before challenge. Additionally, when 100?mg of MIV-150 was loaded into an intravaginal ring (IVR) inserted 24?h before challenge and removed 2 weeks after challenge, >80% protection was observed (p<0.03). MIV-160 is a related NNRTI with a similar IC(50), greater aqueous solubility, and a shorter synthesis. To objectively compare MIV-160 with MIV-150, herein we evaluated the antiviral effects of unformulated MIV-160 in vitro as well as the in vivo protection afforded by MIV-160 delivered in CG (MIV-160/CG gel) and in an IVR under regimens used with MIV-150 in earlier studies. Like MIV-150, MIV-160 exhibited potent antiviral activity against SHIV-RT in macaque vaginal explants. However, formulated MIV-160 exhibited divergent effects in vivo. The MIV-160/CG gel offered no protection compared to CG alone, whereas the MIV-160 IVRs protected significantly. Importantly, the results of in vitro release studies of the MIV-160/CG gel and the MIV-160 IVR suggested that in vivo efficacy paralleled the amount of MIV-160 released in vitro. Hundreds of micrograms of MIV-160 were released daily from IVRs while undetectable amounts of MIV-160 were released from the CG gel. Our findings highlight the importance of testing different modalities of microbicide delivery to identify the optimal formulation for efficacy in vivo. PMID:22816564

Aravantinou, Meropi; Singer, Rachel; Derby, Nina; Calenda, Giulia; Mawson, Paul; Abraham, Ciby J; Menon, Radhika; Seidor, Samantha; Goldman, Daniel; Kenney, Jessica; Villegas, Guillermo; Gettie, Agegnehu; Blanchard, James; Lifson, Jeffrey D; Piatak, Michael; Fernández-Romero, José A; Zydowsky, Thomas M; Teleshova, Natalia; Robbiani, Melissa

2012-08-27

66

The Nonnucleoside Reverse Transcription Inhibitor MIV-160 Delivered from an Intravaginal Ring, But Not from a Carrageenan Gel, Protects Against Simian/Human Immunodeficiency Virus-RT Infection  

PubMed Central

Abstract We previously showed that a carrageenan (CG) gel containing 50??M MIV-150 (MIV-150/CG) reduced vaginal simian/human immunodeficiency virus (SHIV)-RT infection of macaques (56%, p>0.05) when administered daily for 2 weeks with the last dose given 8?h before challenge. Additionally, when 100?mg of MIV-150 was loaded into an intravaginal ring (IVR) inserted 24?h before challenge and removed 2 weeks after challenge, >80% protection was observed (p<0.03). MIV-160 is a related NNRTI with a similar IC50, greater aqueous solubility, and a shorter synthesis. To objectively compare MIV-160 with MIV-150, herein we evaluated the antiviral effects of unformulated MIV-160 in vitro as well as the in vivo protection afforded by MIV-160 delivered in CG (MIV-160/CG gel) and in an IVR under regimens used with MIV-150 in earlier studies. Like MIV-150, MIV-160 exhibited potent antiviral activity against SHIV-RT in macaque vaginal explants. However, formulated MIV-160 exhibited divergent effects in vivo. The MIV-160/CG gel offered no protection compared to CG alone, whereas the MIV-160 IVRs protected significantly. Importantly, the results of in vitro release studies of the MIV-160/CG gel and the MIV-160 IVR suggested that in vivo efficacy paralleled the amount of MIV-160 released in vitro. Hundreds of micrograms of MIV-160 were released daily from IVRs while undetectable amounts of MIV-160 were released from the CG gel. Our findings highlight the importance of testing different modalities of microbicide delivery to identify the optimal formulation for efficacy in vivo.

Aravantinou, Meropi; Singer, Rachel; Derby, Nina; Calenda, Giulia; Mawson, Paul; Abraham, Ciby J.; Menon, Radhika; Seidor, Samantha; Goldman, Daniel; Kenney, Jessica; Villegas, Guillermo; Gettie, Agegnehu; Blanchard, James; Lifson, Jeffrey D.; Piatak, Michael; Fernandez-Romero, Jose A.; Zydowsky, Thomas M.; Teleshova, Natalia

2012-01-01

67

Biodegradation of Bis(2-Chloroethyl) Ether by Xanthobacter sp. Strain ENV481?  

PubMed Central

Degradation of bis(2-chloroethyl) ether (BCEE) was observed to occur in two bacterial strains. Strain ENV481, a Xanthobacter sp. strain, was isolated by enrichment culturing of samples from a Superfund site located in the northeastern United States. The strain was able to grow on BCEE or 2-chloroethylethyl ether as the sole source of carbon and energy. BCEE degradation in strain ENV481 was facilitated by sequential dehalogenation reactions resulting in the formation of 2-(2-chloroethoxy)ethanol and diethylene glycol (DEG), respectively. 2-Hydroxyethoxyacetic acid was detected as a product of DEG catabolism by the strain. Degradation of BCEE by strain ENV481 was independent of oxygen, and the strain was not able to grow on a mixture of benzene, ethylbenzene, toluene, and xylenes, other prevalent contaminants at the site. Another bacterial isolate, Pseudonocardia sp. strain ENV478 (S. Vainberg et al., Appl. Environ. Microbiol. 72:5218-5224, 2006), degraded BCEE after growth on tetrahydrofuran or propane but was not able to grow on BCEE as a sole carbon source. BCEE degradation by strain ENV478 appeared to be facilitated by a monooxygenase-mediated O-dealkylation mechanism, and it resulted in the accumulation of 2-chloroacetic acid that was not readily degraded by the strain.

McClay, Kevin; Schaefer, Charles E.; Vainberg, Simon; Steffan, Robert J.

2007-01-01

68

The Role of Innate APOBEC3G and Adaptive AID Immune Responses in HLA-HIV/SIV Immunized SHIV Infected Macaques  

PubMed Central

The AID/APOBEC family (activation induced deaminase/apolipoprotein B mRNA editing cytokine deaminase) in B cells play important roles in adaptive and innate immunity. Whereas APOBEC3G has been studied in CD4+ T cells and myeloid cells its functional potential in B cells has received little attention. AID combines two critical functions of antibodies, class switching and affinity maturation and may serve as a functional surrogate of protection. These functions were studied following systemic immunization of rhesus macaques with recombinant HLA constructs, linked with HIV and SIV antigens and HSP70 to dextran. The results showed significant upregulation of AID in CD20+ B cells, APOBEC 3G in CD27+ memory B cells and CD4+ effector memory T cells. After immunization the upregulated APOBEC 3G and AID were directly correlated in B cells (p<0.0001). Following challenge with SHIV SF162.P4 the viral load was inversely correlated with AID in B cells and APOBEC 3G in B and T cells, suggesting that both deaminases may have protective functions. Investigation of major interactions between DC, T cells and B cells showed significant increase in membrane associated IL-15 in DC and CD40L in CD4+ T cells. IL-15 binds the IL-15 receptor complex in CD4+ T and B cells, which may reactivate the DC, T and B cell interactions. The overall results are consistent with AID inhibiting pre-entry SHIV by eliciting IgG and IgA antibodies, whereas APOBEC 3G may contribute to the post-entry control of SHIV replication and cellular spread.

Wang, Yufei; Whittall, Trevor; Rahman, Durdana; Bunnik, Evelien M.; Vaughan, Robert; Sch?ller, J?rgen; Bergmeier, Lesley A.; Montefiori, David; Singh, Mahavir; Schuitemaker, Hanneke; Lehner, Thomas

2012-01-01

69

Immunogenicity of HIV-1 IIIB and SHIV 89.6P Tat and Tat toxoids in rhesus macaques: induction of humoral and cellular immune responses.  

PubMed

This study compared immune responses in rhesus macaques immunized with unmodified HIV-1 IIIB Tat, SHIV89.6P Tat, and carboxymethylated IIIB and 89.6P Tat toxoids. Immunization with either IIIB or 89.6P preparation induced high titer and broadly crossreactive serum anti-Tat IgG that recognized HIV-1 subtype-E and SIVmac251 Tat. However, the response was delayed, and titers were lower in 89.6P vaccination groups. Serum anti-Tat IgG recognized peptides corresponding to the amino-terminus, basic domain, and carboxy-terminal region. Cellular proliferative responses to Tat toxoids corresponding to the immunogen were evident in vitro in both IIIB and 89.6P groups. Crossreactive proliferative responses were observed in IIIB groups in response to stimulation with 89.6P or SIVmac251 Tat toxoids, but were much less prevalent in 89.6P groups. The truncated 86 amino acid IIIB Tat appears to be more immunogenic than the 102 amino acid 89.6P Tat with respect to both humoral and cellular immune responses, and may be a better vaccine component. Despite induction of robust humoral and cellular immune responses (including both CD4+ and CD8+ T-cell responses) to Tat, all animals were infected upon intravenous challenge with 30 MID(50) of SHIV89.6P and outcome of vaccine groups was not different from controls. Sequencing both Tat exons from serum viral RNA revealed no evidence of escape mutants. These results suggest that with intravenous SHIV89.6P challenge in rhesus macaques, precipitous CD4+ T-cell decline overwhelms potentially protective immune responses. Alternatively, Tat specific CD8+ T-cell responses may not appropriately recognize infected cells in vivo in this model. In view of evidence demonstrating Tat specific CTLs in the SIV model and in humans infected with HIV-1, results in this pathogenic SHIV model may not apparently predict the efficacy of this approach in human studies. The potency and cross-reactivity of these immune responses confirm Tat toxoid as an excellent candidate vaccine component. PMID:12396606

Richardson, Max W; Mirchandani, Jyotika; Silvera, Peter; Régulier, Emmanuel G; Capini, Christelle; Bojczuk, Paul M; Hu, Jason; Gracely, Edward J; Boyer, Jean D; Khalili, Kamel; Zagury, Jean-François; Lewis, Mark G; Rappaport, Jay

2002-09-01

70

Highly potent HIV-specific antibody neutralization in vitro translates into effective protection against mucosal SHIV challenge in vivo  

PubMed Central

Most animal studies using passive administration of HIV broadly neutralizing monoclonal antibodies (bnMAbs) have associated protection against high-dose mucosal viral challenge with relatively high serum concentrations of antibody. We recently identified several bnMAbs remarkable for their in vitro potency against HIV. Of these bnMAbs, PGT121 is one of the most broad and potent antibodies isolated to date and shows 10- to 100-fold higher neutralizing activity than previously characterized bnMAbs. To evaluate the protective potency of PGT121 in vivo, we performed a protection study in rhesus macaques. Animals were i.v. administered 5 mg/kg, 1 mg/kg, or 0.2 mg/kg PGT121 24 h before being vaginally challenged with a single high dose of chimeric simian-human immunodeficiency virus (SHIV)SF162P3. Sterilizing immunity was achieved in all animals administered 5 mg/kg and 1 mg/kg and three of five animals administered 0.2 mg/kg PGT121, with corresponding average antibody serum concentrations of 95 µg/mL, 15 µg/mL, and 1.8 µg/mL, respectively. The results suggest that a protective serum concentration for PGT121 is in the single-digit µg/mL for SHIVSF162P3, showing that PGT121 can mediate sterilizing immunity at serum concentrations that are significantly lower than those observed in previous studies and that may be achievable through vaccination with the development of a suitable immunogen.

Moldt, Brian; Rakasz, Eva G.; Schultz, Niccole; Chan-Hui, Po-Ying; Swiderek, Kristine; Weisgrau, Kimberly L.; Piaskowski, Shari M.; Bergman, Zachary; Watkins, David I.; Poignard, Pascal; Burton, Dennis R.

2012-01-01

71

Molecular Evolution of HIV-1 CRF01_AE Env in Thai Patients  

PubMed Central

Background The envelope glycoproteins (Env), gp120 and gp41, are the most variable proteins of human immunodeficiency virus type 1 (HIV-1), and are the major targets of humoral immune responses against HIV-1. A circulating recombinant form of HIV-1, CRF01_AE, is prevalent throughout Southeast Asia; however, only limited information regarding the immunological characteristics of CRF01_AE Env is currently available. In this study, we attempted to examine the evolutionary pattern of CRF01_AE Env under the selection pressure of host immune responses. Methodology/Principal Findings Peripheral blood samples were collected periodically over 3 years from 15 HIV-1-infected individuals residing in northern Thailand, and amplified env genes from the samples were subjected to computational analysis. The V5 region of gp120 showed highest variability in several samples over 3 years, whereas the V1/V2 and/or V4 regions of gp120 also showed high variability in many samples. In addition, the N-terminal part of the C3 region of gp120 showed highest amino acid diversity among the conserved regions of gp120. Chronological changes in the numbers of amino acid residues in gp120 variable regions and potential N-linked glycosylation (PNLG) sites are involved in increasing the variability of Env gp120. Furthermore, the C3 region contained several amino acid residues potentially under positive selection, and APOBEC3 family protein-mediated G to A mutations were frequently detected in such residues. Conclusions/Significance Several factors, including amino acid substitutions particularly in gp120 C3 and V5 regions as well as changes in the number of PNLG sites and in the length of gp120 variable regions, were revealed to be involved in the molecular evolution of CRF01_AE Env. In addition, a similar tendency was observed between CRF01_AE and subtype C Env with regard to the amino acid variation of gp120 V3 and C3 regions. These results may provide important information for understanding the immunological characteristics of CRF01_AE Env.

Uttiyoung, Jiraporn; Yowang, Amara; Krathong, Nongkran; Chautrakul, Sununta; Yamashita, Akifumi; Ikuta, Kazuyoshi; Roobsoong, Amornsak; Kanitvittaya, Sangkom; Sawanpanyalert, Pathom; Kameoka, Masanori

2011-01-01

72

EC energy and environment model EFOM-ENV specified in GAMS. The case of the Netherlands.  

National Technical Information Service (NTIS)

EFOM-ENV (Energy Flow and Optimization Model - ENVironment) is a linear-programming (LP) model which covers the complete energy system of a country. It is originally programmed in FORTRAN. At the moment the model is operational for all the European Commun...

M. Van den Broek F. Van Oostvoorn T. Van Harmelen W. Van Arkel

1992-01-01

73

Ensemble/Variational Estimation (EnVE) and its application to canonical turbulent flow realizations  

NASA Astrophysics Data System (ADS)

The recently-developed hybrid EnVE method for data assimilation incorporates successive adjoint optimizations to update the initial conditions of a flow model, over various horizons of interest, in order to reconcile this model with recent measurements. Such adjoint optimizations typically require the trajectory to be saved over the entire interval over which the optimization is performed; in high-dimensional systems, this can lead to significant storage problems, which can be partially alleviated via checkpointing. In the EnVE framework, this requirement is eliminated, and supplanted by a requirement to march the state of the system backward in time simultaneously with the adjoint. If the system is derived from a PDE with a diffusive component, this backward-in-time state march is ill conditioned, and requires regularization/smoothing to prevent errors from accumulating rapidly at the small scales. The present talk focuses on this peculiar requirement of the EnVE algorithm. As the forecasting problem may itself be considered as a smoothing problem, it is, in fact, expected to find a ``smoothing'' ingredient at the heart of an algorithm of this sort. Various strategies are proposed and tested for accomplishing the required smoothing in the EnVE setting, and are tested on both a chaotic 1D PDE (the Kuramoto-Sivashinsky equation) as well as our in-house spectral 3D DNS/LES code, diablo.

Colburn, Christopher; Cessna, Joseph; Bewley, Thomas

2008-11-01

74

Aerobic Biodegradation of NNitrosodimethylamine by the Propanotroph Rhodococcus ruber ENV425  

Microsoft Academic Search

The propanotroph Rhodococcus ruber ENV425 was observed to rapidly biodegrade N-nitrosodimethylamine (NDMA) after growth on propane, tryptic soy broth, or glucose. The key degradation intermediates were methylamine, nitric oxide, nitrite, nitrate, and formate. Small quantities of formaldehyde and dimethylamine were also detected. A denitrosation reaction, initiated by hydrogen atom abstraction from one of the two methyl groups, is hypothesized to

Diane Fournier; Jalal Hawari; Annamaria Halasz; Sheryl H. Streger; Kevin R. McClay; Hisako Masuda; Paul B. Hatzinger

2009-01-01

75

Avian Hemangioma Retrovirus Induces Cell Proliferation via the Envelope ( env) Gene  

Microsoft Academic Search

Several years ago, a field strain retrovirus, avian hemangioma virus (AHV), was isolated from hemangioma tumors in layer hens. Sequence analysis indicated that the AHV genome contains the three prototypic retroviral genes, gag, pol, and env, and is devoid of an oncogene. In cultured endothelial cells, however, AHV induced a significant cytopathic effect through a typical apoptotic cascade. We now

Akram Alian; Dalit Sela-Donenfeld; Amos Panet; Amiram Eldor

2000-01-01

76

Both Linear and Discontinuous Ribosome Scanning Are Used for Translation Initiation from Bicistronic Human Immunodeficiency Virus Type 1 env mRNAs? †  

PubMed Central

Human immunodeficiency virus type 1 (HIV-1) generates 16 alternatively spliced isoforms of env mRNA that contain the same overlapping open reading frames for Vpu and Env proteins but differ in their 5? untranslated regions (UTR). A subset of env mRNAs carry the extra upstream Rev initiation codon in the 5? UTR. We explored the effect of the alternative UTR on the translation of Vpu and Env proteins from authentic env mRNAs expressed from cDNA constructs. Vpu expression from the subset of env mRNA isoforms with exons containing an upstream Rev AUG codon was minimal. However, every env mRNA isoform expressed similar levels of Env protein. Mutations that removed, altered the strength of, or introduced upstream AUG codons dramatically altered Vpu expression but had little impact on the consistent expression of Env. These data show that the different isoforms of env mRNA are not redundant but instead regulate Vpu production in HIV-1-infected cells. Furthermore, while the initiation of Vpu translation conforms to the leaky ribosome-scanning model, the consistent Env synthesis infers a novel, discontinuous ribosome-scanning mechanism to translate Env.

Anderson, Jenny L.; Johnson, Adam T.; Howard, Jane L.; Purcell, Damian F. J.

2007-01-01

77

Antigenic properties of a transport-competent influenza HA/HIV Env chimeric protein  

SciTech Connect

The transmembrane subunit (gp41) of the HIV Env glycoprotein contains conserved neutralizing epitopes which are not well-exposed in wild-type HIV Env proteins. To enhance the exposure of these epitopes, a chimeric protein, HA/gp41, in which the gp41 of HIV-1 89.6 envelope protein was fused to the C-terminus of the HA1 subunit of the influenza HA protein, was constructed. Characterization of protein expression showed that the HA/gp41 chimeric proteins were expressed on cell surfaces and formed trimeric oligomers, as found in the HIV Env as well as influenza HA proteins. In addition, the HA/gp41 chimeric protein expressed on the cell surface can also be cleaved into 2 subunits by trypsin treatment, similar to the influenza HA. Moreover, the HA/gp41 chimeric protein was found to maintain a pre-fusion conformation. Interestingly, the HA/gp41 chimeric proteins on cell surfaces exhibited increased reactivity to monoclonal antibodies against the HIV Env gp41 subunit compared with the HIV-1 envelope protein, including the two broadly neutralizing monoclonal antibodies 2F5 and 4E10. Immunization of mice with a DNA vaccine expressing the HA/gp41 chimeric protein induced antibodies against the HIV gp41 protein and these antibodies exhibit neutralizing activity against infection by an HIV SF162 pseudovirus. These results demonstrate that the construction of such chimeric proteins can provide enhanced exposure of conserved epitopes in the HIV Env gp41 and may represent a novel vaccine design strategy for inducing broadly neutralizing antibodies against HIV.

Ye Ling [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States); Sun Yuliang [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States); Lin Jianguo [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States); Bu Zhigao [Harbin Veterinary Research Institute, CAAS, 427 Maduan Street, Nangang District, Harbin 150001 (China); Wu Qingyang [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States); Jiang, Shibo [New York Blood Center, 310 E. 67 Street, New York, NY 10021 (United States); Steinhauer, David A. [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States); Compans, Richard W. [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States); Yang Chinglai [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States)]. E-mail: chyang@emory.edu

2006-08-15

78

How Can HIV-Type-1-Env Immunogenicity Be Improved to Facilitate Antibody-Based Vaccine Development?  

PubMed Central

Abstract No vaccine candidate has induced antibodies (Abs) that efficiently neutralize multiple primary isolates of HIV-1. Preexisting high titers of neutralizing antibodies (NAbs) are essential, because the virus establishes infection before anamnestic responses could take effect. HIV-1 infection elicits Abs against Env, Gag, and other viral proteins, but of these only a subset of the anti-Env Abs can neutralize the virus. Whereas the corresponding proteins from other viruses form the basis of successful vaccines, multiple large doses of HIV-1 Env elicit low, transient titers of Abs that are not protective in humans. The inaccessibility of neutralization epitopes hinders NAb induction, but Env may also subvert the immune response by interacting with receptors on T cells, B cells, monocytes, macrophages, and dendritic cells. Here, we discuss evidence from immunizations of different species with various modified Env constructs. We also suggest how the divergent Ab responses to Gag and Env during infection may reflect differences in B cell regulation. Drawing on these analyses, we outline strategies for improving Env as a component of a vaccine aimed at inducing strong and sustained NAb responses.

Sanders, Rogier W.; Cerutti, Andrea; Moore, John P.

2012-01-01

79

HIV-1 Virus-Like Particles Bearing Pure Env Trimers Expose Neutralizing Epitopes but Occlude Nonneutralizing Epitopes  

PubMed Central

Hypothetically, since native HIV-1 Env trimers are exclusively recognized by neutralizing antibodies, they might induce the neutralizing antibodies in a vaccine setting. This idea has not been evaluated due to the difficulty of separating trimers from nonfunctional Env (uncleaved gp160 and gp41 stumps). The latter are immunodominant and induce nonneutralizing antibodies. We previously showed that nonfunctional Env can be selectively cleared from virus-like particle (VLP) surfaces by enzyme digests (E. T. Crooks, T. Tong, K. Osawa, and J. M. Binley, J.Virol. 85:5825, 2011). Here, we investigated the effects of these digests on the antigenicity of VLPs and their sensitivity to neutralization. Before digestion, WT VLPs (bearing wild-type Env) and UNC VLPs (bearing uncleaved gp160) were recognized by various Env-specific monoclonal antibodies (MAbs), irrespective of their neutralizing activity, a result which is consistent with the presence of nonfunctional Env. After digestion, only neutralizing MAbs recognized WT VLPs, consistent with selective removal of nonfunctional Env (i.e., “trimer VLPs”). Digests eliminated the binding of all MAbs to UNC VLPs, again consistent with removal of nonfunctional Env. An exception was MAb 2F5, which weakly bound to digested UNC VLPs and bald VLPs (bearing no Env), perhaps due to lipid cross-reactivity. Trimer VLPs were infectious, and their neutralization sensitivity was largely comparable to that of undigested WT VLPs. However, they were ?100-fold more sensitive to the MAbs 4E10 and Z13e1, suggesting increased exposure of the gp41 base. Importantly, a scatterplot analysis revealed a strong correlation between MAb binding and neutralization of trimer VLPs. This suggests that trimer VLPs bear essentially pure native trimer that should allow its unfettered evaluation in a vaccine setting.

Tong, Tommy; Crooks, Ema T.; Osawa, Keiko

2012-01-01

80

Selected HIV-1 Env Trimeric Formulations Act as Potent Immunogens in a Rabbit Vaccination Model  

PubMed Central

Background Ten to 30% of HIV-1 infected subjects develop broadly neutralizing antibodies (bNAbs) during chronic infection. We hypothesized that immunizing rabbits with viral envelope glycoproteins (Envs) from these patients may induce bNAbs, when formulated as a trimeric protein and in the presence of an adjuvant. Methods Based on in vitro neutralizing activity in serum, patients with bNAbs were selected for cloning of their HIV-1 Env. Seven stable soluble trimeric gp140 proteins were generated from sequences derived from four adults and two children infected with either clade A or B HIV-1. From one of the clade A Envs both the monomeric and trimeric Env were produced for comparison. Rabbits were immunized with soluble gp120 or trimeric gp140 proteins in combination with the adjuvant dimethyl dioctadecyl ammonium/trehalose dibehenate (CAF01). Env binding in rabbit immune serum was determined using ELISAs based on gp120-IIIB protein. Neutralizing activity of IgG purified from rabbit immune sera was measured with the pseudovirus-TZMbl assay and a PBMC-based neutralization assay for selected experiments. Results It was initially established that gp140 trimers induce better antibody responses over gp120 monomers and that the adjuvant CAF01 was necessary for such strong responses. Gp140 trimers, based on HIV-1 variants from patients with bNAbs, were able to elicit both gp120IIIB specific IgG and NAbs to Tier 1 viruses of different subtypes. Potency of NAbs closely correlated with titers, and an gp120-binding IgG titer above a threshold of 100,000 was predictive of neutralization capability. Finally, peptide inhibition experiments showed that a large fraction of the neutralizing IgG was directed against the gp120 V3 region. Conclusions Our results indicate that the strategy of reverse immunology based on selected Env sequences is promising when immunogens are delivered as stabilized trimers in CAF01 adjuvant and that the rabbit is a valuable model for HIV vaccine studies.

Heyndrickx, Leo; Stewart-Jones, Guillaume; Jansson, Marianne; Schuitemaker, Hanneke; Bowles, Emma; Buonaguro, Luigi; Grevstad, Berit; Vinner, Lasse; Vereecken, Katleen; Parker, Joe; Ramaswamy, Meghna; Biswas, Priscilla; Vanham, Guido; Scarlatti, Gabriella; Fomsgaard, Anders

2013-01-01

81

HIV ENV Glycoprotein-mediated Bystander Apoptosis Depends on Expression of the CCR5 Co-receptor at the Cell Surface and ENV Fusogenic Activity*  

PubMed Central

HIV-1 infections lead to a progressive depletion of CD4 cells culminating in AIDS. The coreceptor usage by HIV varies from CCR5 (R5) tropic early in infection to CXCR4 (X4) tropic in later infections. Although the coreceptor switch from R5 to X4 tropic HIV is well associated with progression to AIDS, the role of CCR5 in disease progression especially in patients infected exclusively with R5 isolates throughout the disease remains enigmatic. To better understand the role of CCR5 and R5 tropic HIV envelope in AIDS pathogenesis, we asked whether the levels of CCR5 and/or HIV Env-mediated fusion determine apoptosis of bystander cells. We generated CD4+ T cell lines expressing varying levels of CCR5 on the cell surface to show that CCR5 expression levels correlate with bystander apoptosis induction. The mechanism of apoptosis involved caspase-3 activation and mitochondrial depolarization and was dependent on gp41 fusion activity as confirmed by fusion-restricted gp41 point mutants and use of the fusion inhibitor T20. Interestingly, lower levels of CCR5 were able to support virus replication in the absence of bystander apoptosis. Our findings suggest that R5 HIV-1-mediated bystander apoptosis is dependent on both CCR5 expression levels as well as fusogenic activity of the Env glycoprotein.

Joshi, Anjali; Nyakeriga, Alice M.; Ravi, Revathi; Garg, Himanshu

2011-01-01

82

Characterization of lipoprotein EnvA in Chlamydia psittaci 6BC.  

PubMed Central

The primary sequence of the small cysteine-rich protein (EnvA) of Chlamydia psittaci 6BC has been shown to possess a potential lipid modification/signal peptidase II-processing site, and the mature protein was labeled by a [3H]palmitic acid precursor. We further characterized the mature EnvA, showing that it lacks the N-terminal methionine of the primary peptide, is hydrophobic despite a peptide sequence that is predicted to be hydrophilic, and appears to be lipid modified at an N-terminal cysteine in a manner analogous to that of murein lipoproteins of gram-negative bacteria. We also report the fatty acid content of the small cysteine-rich proteins of C. psittaci and Chlamydia trachomatis L2 as determined by combined gas chromatography-mass spectrometry. Images

Everett, K D; Desiderio, D M; Hatch, T P

1994-01-01

83

Ensemble/Variational Estimation (EnVE) and its application to turbulent flows in complex geometries  

NASA Astrophysics Data System (ADS)

A new algorithm, Ensemble/Variational Estimation (EnVE), has been developed as a consistent hybrid data assimilation method that combines the nonlinear statistical propagation properties of the Ensemble Kalman Filter (EnKF) and the retrospective analysis capabilities of 4DVar/Moving Horizon Estimation (MHE). A sophisticated C++ object-oriented framework has been developed that implements the EnVE algorithm to facilitate its application to any complex (multiscale/multiphysics) flow code of interest in a highly parallel fashion with minimal changes to the existing flow solver. In the present work, this framework has been applied to the flagship unstructured LES code (CDP) developed at the Center for Integrated Turbulence Simulations (CITS) at Stanford University.

Cessna, Joseph; Colburn, Christopher; Bewley, Thomas; Ham, Frank; Wang, Qiqi; Iaccarino, Gianluca

2008-11-01

84

Period Variations of RT Persei  

NASA Astrophysics Data System (ADS)

RT Per has been known as a close binary of which the orbital period has unpredictably varied so far. Although there are no agreements with the working mechanism for the changes of the period, two interpretations have been suggested and waiting for to be tested: 1)light-time effects due to the unseen 3rd and 4th bodies(Panchatsaram 1981), 2)Abrupt period-change due to internal variations of the system(e.q. mass transfer or mass loss) superimposing to the light-times effect by a 3rd body(Frieboes-Conde&Herczeg 1973). In the point of view that the former interprepation models could predict the behavior of the changes of the orbital period theoretically, we checked whether the recent observed times of minimum lights follow the predictions by the first model or not. We confirmed that the observed times of minimum lights have followed the variations calculated by the light-times effects due to 3rd and 4th bodies suggested by Panchatsaram. In this paper a total of 626 times of minimum lights were reanalyzed in terms of the light-time effects by the 3rd and 4th bodies. We concluded that the eclipsing pair in SV Cam system moves in an elliptic orbit about center of mass of the triple system with a period of about 42.y2, while the mass center of the triplet is in light-time orbit about the center of mass of the quadruple system with a period of 120y. The mean masses deduced for the 3rd and 4th bodies were 0.89m and 0.82m , respectively.

Kim, Chun-Hwey

1995-12-01

85

Hepatitis C Virus (HCV) Reverse Transcription (RT) ...  

Center for Biologics Evaluation and Research (CBER)

... Resources for You. Hepatitis C. -. Hepatitis C Virus (HCV) Reverse Transcription (RT) Polymerase Chain Reaction (PCR) Assay. ... More results from www.fda.gov/biologicsbloodvaccines/bloodbloodproducts/approvedproducts

86

Molecular characterization of the env gene from Brazilian field isolates of Bovine Leukemia Virus  

Microsoft Academic Search

Molecular characterization of Bovine leukemia virus (BLV) isolates from Brazil using the env gene sequences revealed a high conservation of this gene. In most cases the substitutions corresponded to silent transitions.\\u000a In addition, cystein residues, potential glycosylation sites, neutralization domains and other critical residues involved\\u000a with the envelope structural domains and viral infectivity were conserved. Most of the substitutions found

Marcelo Fernandes Camargos; Ariel Pereda; Daniel Stancek; Maurílio Andrade Rocha; Jenner Karlisson Pimenta dos Reis; Irene Greiser-Wilke; Rômulo Cerqueira Leite

2007-01-01

87

High prevalence of MMTV-like env gene sequences in gestational breast cancer  

Microsoft Academic Search

Gestational breast cancer (BC) is generally associated with rapid growth and increased mortality. Because the presence of\\u000a MMTV-like sequences in BC has been associated with laminin receptor expression, a marker of poor prognosis, gestational BCs\\u000a were analyzed for MMTV env gene-like sequences to explore whether MMTV-like sequences were also associated with its adverse outcome. Whereas 30–38%\\u000a of sporadic BC have

Y. Wang; S. M. Melana; B. Baker; I. Bleiweiss; M. Fernandez-Cobo; J. F. Mandeli; J. F. Holland; B. G. T. Pogo

2003-01-01

88

Evolution of the HIV-1 env Gene in the Rag2?/? ?C?/? Humanized Mouse Model ?  

PubMed Central

Rag2?/? ?C?/? mice transplanted with human hematopoietic stem cells (DKO-hu-HSC mice) mimic aspects of human infection with human immunodeficiency virus type 1 (HIV-1), including sustained viral replication and CD4+ T-cell decline. However, the extent of HIV-1 evolution during long-term infection in these humanized mice, a key feature of the natural infection, has not been assessed fully. In this study, we examined the types of genotypic and phenotypic changes in the viral env gene that occur in the viral populations of DKO-hu-HSC mice infected with the CCR5-tropic isolate HIV-1JRCSF for up to 44 weeks. The mean rate of divergence of viral populations in mice was similar to that observed in a cohort of humans during a similar period of infection. Many amino acid substitutions were common across mice, including losses of N-linked glycosylation sites and substitutions in the CD4 binding site and in CD4-induced epitopes, indicating common selective pressures between mice. In addition, env variants evolved sensitivity to antibodies directed at V3, suggesting a more open conformation for Env. This phenotypic change was associated with increased CD4 binding efficiency and was attributed to specific amino acid substitutions. In one mouse, env variants emerged that exhibited a CXCR4-tropic phenotype. These sequences were compartmentalized in the mesenteric lymph node. In summary, viral populations in these mice exhibited dynamic behavior that included sequence evolution, compartmentalization, and the appearance of distinct phenotypic changes. Thus, humanized mice offer a useful model for studying evolutionary processes of HIV-1 in a complex host environment.

Ince, William L.; Zhang, Liguo; Jiang, Qi; Arrildt, Kathryn; Su, Lishan; Swanstrom, Ronald

2010-01-01

89

Quaternary structures of HIV Env immunogen exhibit conformational vicissitudes and interface diminution elicited by ligand binding  

PubMed Central

The human immunodeficiency virus envelope protein is the key element mediating entry into host cells. Conformational rearrangement of Env upon binding to the host CD4 receptor and chemokine coreceptor drives membrane fusion. We elucidated the quaternary arrangement of the soluble Env trimeric immunogen o-gp140?V2TV1, in both its native (unliganded) and CD4-induced (liganded) states by cryoelectron microscopy and molecular modeling. The liganded conformation was elicited by binding gp140 to the synthetic CD4-mimicking miniprotein CD4m. Upon CD4m binding, an outward domain shift of the three gp120 subunits diminishes gp120–gp41 interactions, whereas a “flat open” concave trimer apex is observed consequent to gp120 tilting away from threefold axis, likely juxtaposing the fusion peptide with the host membrane. Additional features observed in the liganded conformation include rotations of individual gp120 subunits that may release gp41 for N- and C-helix refolding and also may lead to optimal exposure of the elicited coreceptor binding site. Such quaternary arrangements of gp140 lead to the metastable liganded conformation, with putative locations of exposed epitopes contributing to a description of sequential events occurring prior to membrane fusion. Our observations imply a mechanism whereby a soluble Env trimeric construct, as opposed to trimers extracted from virions, may better expose crucial epitopes such as the CD4 binding site and V3, as well as epitopes in the vicinity of gp41, subsequent to conjugation with CD4m. Structural features gleaned from our studies should aid the design of Env-based immunogens for inducement of potent broadly neutralizing antibodies against exposed conformational epitopes.

Moscoso, Carlos G.; Sun, Yide; Poon, Selina; Xing, Li; Kan, Elaine; Martin, Loic; Green, Dominik; Lin, Frank; Vahlne, Anders G.; Barnett, Susan; Srivastava, Indresh; Cheng, R. Holland

2011-01-01

90

HIV p24 as Scaffold for Presenting Conformational HIV Env Antigens  

PubMed Central

Heterologous protein scaffolds engrafted with structurally defined HIV Env epitopes recognized by broadly neutralizing monoclonal antibodies (MAbs) represent a promising strategy to elicit broad neutralizing antibodies. In such regards, a protein scaffold based on the HIV p24 CA protein is a highly attractive approach, providing also Gag epitopes for eliciting HIV non-neutralizing protective antibodies and specific CD4+ and CD8+ T cell responses. In the present study, computational techniques were employed to verify the presence of acceptor sites for conformational HIV Env epitopes and, as proof of concept, the analysis of HIV p24 CA-based scaffolds using a complete V3 loop in a MAb-bound conformation is presented. The V3-p24 epitope-scaffold proteins show the formation of capsomers made of hexamers similarly to the p24 wild type protein. Moreover, the conformational V3 loop presented on p24 scaffold is recognized by a panel of anti-V3 MAbs. The results suggest that HIV p24 CA protein has suitable acceptor sites for engrafting foreign epitopes, without disrupting the formation of capsomer hexamer structures, and that the V3 epitope does retain its antibody-bound conformation. This strongly support the feasibility of developing a scaffolding strategy based on p24 CA proteins displaying conformational minimal structural, antigenic HIV Env epitopes.

Tagliamonte, Maria; Marasco, Daniela; Ruggiero, Alessia; De Stradis, Angelo; Tornesello, Maria Lina; Totrov, Maxim; Buonaguro, Franco Maria; Buonaguro, Luigi

2012-01-01

91

Aerobic biodegradation of N-nitrosodimethylamine by the propanotroph Rhodococcus ruber ENV425.  

PubMed

The propanotroph Rhodococcus ruber ENV425 was observed to rapidly biodegrade N-nitrosodimethylamine (NDMA) after growth on propane, tryptic soy broth, or glucose. The key degradation intermediates were methylamine, nitric oxide, nitrite, nitrate, and formate. Small quantities of formaldehyde and dimethylamine were also detected. A denitrosation reaction, initiated by hydrogen atom abstraction from one of the two methyl groups, is hypothesized to result in the formation of n-methylformaldimine and nitric oxide, the former of which decomposes in water to methylamine and formaldehyde and the latter of which is then oxidized further to nitrite and then nitrate. Although the strain mineralized more than 60% of the carbon in [(14)C]NDMA to (14)CO(2), growth of strain ENV425 on NDMA as a sole carbon and energy source could not be confirmed. The bacterium was capable of utilizing NDMA, as well as the degradation intermediates methylamine and nitrate, as sources of nitrogen during growth on propane. In addition, ENV425 reduced environmentally relevant microgram/liter concentrations of NDMA to <2 ng/liter in batch cultures, suggesting that the bacterium may have applications for groundwater remediation. PMID:19542346

Fournier, Diane; Hawari, Jalal; Halasz, Annamaria; Streger, Sheryl H; McClay, Kevin R; Masuda, Hisako; Hatzinger, Paul B

2009-06-19

92

Antigenic variants of J subgroup avian leukosis virus: Sequence analysis reveals multiple changes in the env gene  

Microsoft Academic Search

HPRS-103, the prototype of avian leukosis virus (ALV) subgroup J, was isolated in 1989 from meat- type chickens from commercial flocks where it induces myelocytic myeloid leukosis (ML). The HPRS-103 env gene differs considerably from other ALV subgroups but shows high identity (75-97%) to env-like sequences of the different members of the EAV family of endogenous avian retroviruses. Recently, we

K. Venugopal; L. M. Smith; K. Howes; L. N. Payne

1998-01-01

93

Specificity of Antibodies Directed against Env Protein of Human Endogenous Retroviruses in Patients with Germ Cell Tumors1  

Microsoft Academic Search

We report here that 85% of the patients with germ cell tumors (GCTs) produce antibodies directed against Env protein or human endogenous retroviruses. Individuals that received antitumor treatment showed a decrease with time in their antibody titers. Importantly, of the rare cases of non-GCT individuals with Env-antibodies (n = 15, 0.8%), none pro duced antibodies directed against the transmembrane domain

Marlies Sauter; Klaus Roemer; Barbara Best; Matthias Afting; Stefanie Schommer; Gerhard Seitz; Michael Hartmann; Nikolaus Mueller-Lantzsch

94

RT3D tutorials for GMS users  

SciTech Connect

RT3D (Reactive Transport in 3-Dimensions) is a computer code that solves coupled partial differential equations that describe reactive-flow and transport of multiple mobile and/or immobile species in a three dimensional saturated porous media. RT3D was developed from the single-species transport code, MT3D (DoD-1.5, 1997 version). As with MT3D, RT3D also uses the USGS groundwater flow model MODFLOW for computing spatial and temporal variations in groundwater head distribution. This report presents a set of tutorial problems that are designed to illustrate how RT3D simulations can be performed within the Department of Defense Groundwater Modeling System (GMS). GMS serves as a pre- and post-processing interface for RT3D. GMS can be used to define all the input files needed by RT3D code, and later the code can be launched from within GMS and run as a separate application. Once the RT3D simulation is completed, the solution can be imported to GMS for graphical post-processing. RT3D v1.0 supports several reaction packages that can be used for simulating different types of reactive contaminants. Each of the tutorials, described below, provides training on a different RT3D reaction package. Each reaction package has different input requirements, and the tutorials are designed to describe these differences. Furthermore, the tutorials illustrate the various options available in GMS for graphical post-processing of RT3D results. Users are strongly encouraged to complete the tutorials before attempting to use RT3D and GMS on a routine basis.

Clement, T.P. [Pacific Northwest National Lab., Richland, WA (United States); Jones, N.L. [Brigham Young Univ., Provo, UT (United States)

1998-02-01

95

Study of HIV type 1 gag/env variability in The Gambia, using a multiplex DNA polymerase chain reaction.  

PubMed

A multiplex DNA PCR assay was developed for the simultaneous first-round amplification of HIV-1 gag and env fragments for the heteroduplex mobility assay (HMA). This assay was compared with the conventional amplification assay, using DNA extracted from PBMC samples from 30 HIV-1-seropositive individuals from The Gambia, who were enrolled between 1992 and 1997. From 27 of 30 (90%) samples both gag and env HMA fragments were amplified simultaneously. In one sample only the gag HMA fragment could be amplified by multiplex DNA PCR, and in two samples amplification was negative for both gag and env HMA in multiplex as well as the mono-DNA PCR. Of the 28 Gambian isolates subtyped by gag/env HMA or by sequencing and phylogenetic analysis, the majority (19 of 28; 68%) were intersubtype recombinant. Fifteen of 28 (53%) samples were circulating recombinant form (CRF) CRF02.AG variants. Two isolates clustering with the previously documented Gambian isolate GM4 (previously described as an env GC recombinant) are classified as gag A/env J recombinants. PMID:11118077

Cham, F; Heyndrickx, L; Janssens, W; Van der Auwera, G; Vereecken, K; De Houwer, K; Coppens, S; Whittle, H; van der Groen, G

2000-11-20

96

Analysis of the env gene of a molecularly cloned and biologically active Moloney mink cell focus-forming proviral DNA.  

PubMed Central

A biologically active molecular clone of BALB/Moloney mink cell focus-forming (Mo-MCF) proviral DNA has been reconstructed in vitro. It contains the 5' half of BALB/Moloney murine leukemia virus (Mo-MuLV) DNA and the 3' half of BALB/Mo-MCF DNA. The complete nucleotide sequence of the env gene and the 3' long terminal repeat (LTR) of the cloned Mo-MCF DNA has been determined and compared with the sequence of the corresponding region of parental Mo-MuLV DNA. The substitution in the Mo-MCF DNA encompasses 1,159 base pairs, beginning in the carboxyl terminus of the pol gene and extending to the middle of the env gene. The Mo-MCF env gene product is predicted to be 29 amino acids shorter than the parental Mo-MuLV env gene product. The portion of the env gene encoding the p15E peptide is identical in both viral DNAs. There is an additional A residue in the Mo-MCF viral DNA in a region just preceding the 3' LTR. The nucleotide sequence of the 3' LTR of Mo-MCF DNA is similar to that of the 5' LTR of BALB/Mo-MuLV DNA with the exception of two single base substitutions. We conclude that the sequence substitution in the env gene is responsible for the dual-tropic properties of Mo-MCF viruses. Images

Bosselman, R A; van Straaten, F; Van Beveren, C; Verma, I M; Vogt, M

1982-01-01

97

HIV-1 Tat Promotes Integrin-Mediated HIV Transmission to Dendritic Cells by Binding Env Spikes and Competes Neutralization by Anti-HIV Antibodies  

PubMed Central

Use of Env in HIV vaccine development has been disappointing. Here we show that, in the presence of a biologically active Tat subunit vaccine, a trimeric Env protein prevents in monkeys virus spread from the portal of entry to regional lymph nodes. This appears to be due to specific interactions between Tat and Env spikes that form a novel virus entry complex favoring R5 or X4 virus entry and productive infection of dendritic cells (DCs) via an integrin-mediated pathway. These Tat effects do not require Tat-transactivation activity and are blocked by anti-integrin antibodies (Abs). Productive DC infection promoted by Tat is associated with a highly efficient virus transmission to T cells. In the Tat/Env complex the cysteine-rich region of Tat engages the Env V3 loop, whereas the Tat RGD sequence remains free and directs the virus to integrins present on DCs. V2 loop deletion, which unshields the CCR5 binding region of Env, increases Tat/Env complex stability. Of note, binding of Tat to Env abolishes neutralization of Env entry or infection of DCs by anti-HIV sera lacking anti-Tat Abs, which are seldom present in natural infection. This is reversed, and neutralization further enhanced, by HIV sera containing anti-Tat Abs such as those from asymptomatic or Tat-vaccinated patients, or by sera from the Tat/Env vaccinated monkeys. Thus, both anti-Tat and anti-Env Abs are required for efficient HIV neutralization. These data suggest that the Tat/Env interaction increases HIV acquisition and spreading, as a mechanism evolved by the virus to escape anti-Env neutralizing Abs. This may explain the low effectiveness of Env-based vaccines, which are also unlikely to elicit Abs against new Env epitopes exposed by the Tat/Env interaction. As Tat also binds Envs from different clades, new vaccine strategies should exploit the Tat/Env interaction for both preventative and therapeutic interventions.

Monini, Paolo; Cafaro, Aurelio; Srivastava, Indresh K.; Moretti, Sonia; Sharma, Victoria A.; Andreini, Claudia; Chiozzini, Chiara; Ferrantelli, Flavia; Cossut, Maria R. Pavone.; Tripiciano, Antonella; Nappi, Filomena; Longo, Olimpia; Bellino, Stefania; Picconi, Orietta; Fanales-Belasio, Emanuele; Borsetti, Alessandra; Toschi, Elena; Schiavoni, Ilaria; Bacigalupo, Ilaria; Kan, Elaine; Sernicola, Leonardo; Maggiorella, Maria T.; Montin, Katy; Porcu, Marco; Leone, Patrizia; Leone, Pasqualina; Collacchi, Barbara; Palladino, Clelia; Ridolfi, Barbara; Falchi, Mario; Macchia, Iole; Ulmer, Jeffrey B.; Butto, Stefano; Sgadari, Cecilia; Magnani, Mauro; Federico, Maurizio P. M.; Titti, Fausto; Banci, Lucia; Dallocchio, Franco; Rappuoli, Rino; Ensoli, Fabrizio; Barnett, Susan W.; Garaci, Enrico; Ensoli, Barbara

2012-01-01

98

Elicitation of Both Anti HIV-1 Env Humoral and Cellular Immunities by Replicating Vaccinia Prime Sendai Virus Boost Regimen and Boosting by CD40Lm  

PubMed Central

For protection from HIV-1 infection, a vaccine should elicit both humoral and cell-mediated immune responses. A novel vaccine regimen and adjuvant that induce high levels of HIV-1 Env-specific T cell and antibody (Ab) responses was developed in this study. The prime-boost regimen that used combinations of replication-competent vaccinia LC16m8? (m8?) and Sendai virus (SeV) vectors expressing HIV-1 Env efficiently produced both Env-specific CD8+ T cells and anti-Env antibodies, including neutralizing antibodies (nAbs). These results sharply contrast with vaccine regimens that prime with an Env expressing plasmid and boost with the m8? or SeV vector that mainly elicited cellular immunities. Moreover, co-priming with combinations of m8?s expressing Env or a membrane-bound human CD40 ligand mutant (CD40Lm) enhanced Env-specific CD8+ T cell production, but not anti-Env antibody production. In contrast, priming with an m8? that coexpresses CD40Lm and Env elicited more anti-Env Abs with higher avidity, but did not promote T cell responses. These results suggest that the m8? prime/SeV boost regimen in conjunction with CD40Lm expression could be used as an immunization platform for driving both potent cellular and humoral immunities against pathogens such as HIV-1.

Zhang, Xianfeng; Sobue, Tomoyoshi; Isshiki, Mao; Makino, Shun-ichi; Inoue, Makoto; Kato, Kazunori; Shioda, Tatsuo; Ohashi, Takashi; Sato, Hirotaka; Komano, Jun; Hanabusa, Hideji; Shida, Hisatoshi

2012-01-01

99

Interviews as RtI Tools  

ERIC Educational Resources Information Center

With the implementation of Response to Intervention (RtI) throughout the United States and the strong evidence that validates the use of RtI as a way of supporting struggling students, teachers need ways to understand and reach tier 2 students within their classrooms. One approach to gaining a better understanding of students' misconceptions is…

Hodges, Thomas E.; Rose, Terry D.; Hicks, April D.

2012-01-01

100

Interinstrument Reliability of the RT3 Accelerometer  

ERIC Educational Resources Information Center

The objective of this study was to assess the interinstrument reliability of six RT3 accelerometers for measuring physical activities. Each of the six healthy participants, mean age 36.1 years (SD 9.4), carried six RT3 accelerometers (same type and same producer) simultaneously placed ventrally at the waist belt. The participants performed three…

Reneman, Michiel

2010-01-01

101

Sequential Immunization of Macaques with Two Differentially Attenuated Vaccines Induced Long-Term Virus-Specific Immune Responses and Conferred Protection against AIDS Caused by Heterologous Simian Human Immunodeficiency Virus (SHIV 89.6P)  

Microsoft Academic Search

Four rhesus macaques were sequentially immunized with live vaccines ?vpu?nefSHIV-4 (vaccine-I) and ?vpu SHIVPPC (vaccine-II). The vaccine viruses did not replicate productively in the peripheral blood mononuclear cells (PBMCs) of the vaccinated animals. All four animals developed binding antibodies against both the vaccine-I and -II envelope glycoproteins but neutralizing antibodies only against vaccine-I. They developed vaccine virus-specific CTLs that also

Anil Kumar; Jeffrey D. Lifson; Zhuang Li; Fenglan Jia; Sampa Mukherjee; Istvan Adany; Zhenqian Liu; Mike Piatak; Darlene Sheffer; Harold M. McClure; Opendra Narayan

2001-01-01

102

Genetic and Functional Analysis of Full-Length Human Immunodeficiency Virus Type 1 env Genes Derived from Brain and Blood of Patients with AIDS  

PubMed Central

The genetic evolution of human immunodeficiency virus type 1 (HIV-1) in the brain is distinct from that in lymphoid tissues, indicating tissue-specific compartmentalization of the virus. Few primary HIV-1 envelope glycoproteins (Envs) from uncultured brain tissues have been biologically well characterized. In this study, we analyzed 37 full-length env genes from uncultured brain biopsy and blood samples from four patients with AIDS. Phylogenetic analysis of intrapatient sequence sets showed distinct clustering of brain relative to blood env sequences. However, no brain-specific signature sequence was identified. Furthermore, there was no significant difference in the number or positions of N-linked glycosylation sites between brain and blood env sequences. The patterns of coreceptor usage were heterogeneous, with no clear distinction between brain and blood env clones. Nine Envs used CCR5 as a coreceptor, one used CXCR4, and two used both CCR5 and CXCR4 in cell-to-cell fusion assays. Eight Envs could also use CCR3, CCR8, GPR15, STRL33, Apj, and/or GPR1, but these coreceptors did not play a major role in virus entry into microglia. Recognition of epitopes by the 2F5, T30, AG10H9, F105, 17b, and C11 monoclonal antibodies varied among env clones, reflecting genetic and conformational heterogeneity. Envs from two patients contained 28 to 32 N-glycosylation sites in gp120, compared to around 25 in lab strains and well-characterized primary isolates. These results suggest that HIV-1 Envs in brain cannot be distinguished from those in blood on the basis of coreceptor usage or the number or positions of N-glycosylation sites, indicating that other properties underlie neurotropism. The study also demonstrates characteristics of primary HIV-1 Envs from uncultured tissues and implies that Env variants that are glycosylated more extensively than lab strains and well-characterized primary isolates should be considered during development of vaccines and neutralizing antibodies.

Ohagen, Asa; Devitt, Amy; Kunstman, Kevin J.; Gorry, Paul R.; Rose, Patrick P.; Korber, Bette; Taylor, Joann; Levy, Robert; Murphy, Robert L.; Wolinsky, Steven M.; Gabuzda, Dana

2003-01-01

103

Phylogenetic and structural diversity in the feline leukemia virus env gene.  

PubMed

Feline leukemia virus (FeLV) belongs to the genus Gammaretrovirus, and causes a variety of neoplastic and non-neoplastic diseases in cats. Alteration of viral env sequences is thought to be associated with disease specificity, but the way in which genetic diversity of FeLV contributes to the generation of such variants in nature is poorly understood. We isolated FeLV env genes from naturally infected cats in Japan and analyzed the evolutionary dynamics of these genes. Phylogenetic reconstructions separated our FeLV samples into three distinct genetic clusters, termed Genotypes I, II, and III. Genotype I is a major genetic cluster and can be further classified into Clades 1-7 in Japan. Genotypes were correlated with geographical distribution; Genotypes I and II were distributed within Japan, whilst FeLV samples from outside Japan belonged to Genotype III. These results may be due to geographical isolation of FeLVs in Japan. The observed structural diversity of the FeLV env gene appears to be caused primarily by mutation, deletion, insertion and recombination, and these variants may be generated de novo in individual cats. FeLV interference assay revealed that FeLV genotypes did not correlate with known FeLV receptor subgroups. We have identified the genotypes which we consider to be reliable for evaluating phylogenetic relationships of FeLV, which embrace the high structural diversity observed in our sample. Overall, these findings extend our understanding of Gammaretrovirus evolutionary patterns in the field, and may provide a useful basis for assessing the emergence of novel strains and understanding the molecular mechanisms of FeLV transmission in cats. PMID:23593376

Watanabe, Shinya; Kawamura, Maki; Odahara, Yuka; Anai, Yukari; Ochi, Haruyo; Nakagawa, So; Endo, Yasuyuki; Tsujimoto, Hajime; Nishigaki, Kazuo

2013-04-11

104

Phylogenetic and Structural Diversity in the Feline Leukemia Virus Env Gene  

PubMed Central

Feline leukemia virus (FeLV) belongs to the genus Gammaretrovirus, and causes a variety of neoplastic and non-neoplastic diseases in cats. Alteration of viral env sequences is thought to be associated with disease specificity, but the way in which genetic diversity of FeLV contributes to the generation of such variants in nature is poorly understood. We isolated FeLV env genes from naturally infected cats in Japan and analyzed the evolutionary dynamics of these genes. Phylogenetic reconstructions separated our FeLV samples into three distinct genetic clusters, termed Genotypes I, II, and III. Genotype I is a major genetic cluster and can be further classified into Clades 1–7 in Japan. Genotypes were correlated with geographical distribution; Genotypes I and II were distributed within Japan, whilst FeLV samples from outside Japan belonged to Genotype III. These results may be due to geographical isolation of FeLVs in Japan. The observed structural diversity of the FeLV env gene appears to be caused primarily by mutation, deletion, insertion and recombination, and these variants may be generated de novo in individual cats. FeLV interference assay revealed that FeLV genotypes did not correlate with known FeLV receptor subgroups. We have identified the genotypes which we consider to be reliable for evaluating phylogenetic relationships of FeLV, which embrace the high structural diversity observed in our sample. Overall, these findings extend our understanding of Gammaretrovirus evolutionary patterns in the field, and may provide a useful basis for assessing the emergence of novel strains and understanding the molecular mechanisms of FeLV transmission in cats.

Watanabe, Shinya; Kawamura, Maki; Odahara, Yuka; Anai, Yukari; Ochi, Haruyo; Nakagawa, So; Endo, Yasuyuki; Tsujimoto, Hajime; Nishigaki, Kazuo

2013-01-01

105

BIOCHEMISTRY: RT Slides Home⦠ 

NSDL National Science Digital Library

Access to the article is free, however registration and sign-in are required. To access its target sites, HIV reverse transcriptase (RT) slides and flips on nucleic acid substrates. Although 20 years of crystallographic and biochemical studies have illuminated the molecular details of the chemistry of DNA synthesis, there have been relatively few insights into how RT finds the end of the nucleic acid substrate where it begins DNA synthesis, how it displaces nucleic acid fragments, or where and how it executes masterful leaps when transferring DNA between templates. On page 1092 of this issue, Liu et al. (2) describe elegant single-molecule fluorescence resonance energy transfer (FRET) experiments that provide a view of RT at work. They show that RT has a remarkable ability to slide on nucleic acid duplexes, rapidly shuttling between the two ends and flipping into the polymerase-competent binding mode when needed.

Stefan G. Sarafianos (University of Missouri;Christopher S. Bond Life Sciences Center, Department of Molecular Microbiology and Immunology); Eddy Arnold (Rutgers University;Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology)

2008-11-14

106

Early Induction and Maintenance of Env-Specific T-Helper Cells following Human Immunodeficiency Virus Type 1 Infection  

PubMed Central

Mounting evidence points to a role for CD4+ T-helper (Th) cell activities in controlling human immunodeficiency virus type 1 (HIV-1) infection. To determine the induction and evolution of Th responses following acute infection, we prospectively analyzed Env- and Gag-specific Th responses longitudinally for 92 patients with acute (n = 28) or early (n = 64) HIV-1 infection (median, 55 days postinfection [DPI]). The probability of detecting HIV-1-specific lymphoproliferative responses was remarkably low, and when present, the responses were more likely to be Gag specific than Env specific (16 versus 5%). Env-specific responses were significantly more common in patients presenting at <30 DPI than in those presenting at 30 to 365 DPI (21 versus 0.5%, P = 0.001). By contrast, Gag-specific responses occurred with similar frequencies among subjects presenting at <30 DPI and 30 to 365 DPI (13 versus 17%, P = 0.6). After treatment, and regardless of the duration of infection before therapy, Gag-specific Th responses predominated. Furthermore, some acutely infected subjects lost detectable Env-specific Th proliferative responses, which failed to reemerge upon treatment. Detailed analysis for one such subject revealed Env-specific lymphoproliferation at 11 DPI but no detectable Env-specific lymphoproliferation or ex vivo gamma interferon (IFN-?) secretion at multiple subsequent time points. Env-specific CD4+ T-cell clones from 11 DPI recognized six epitopes in both conserved and variable regions within gp120 and gp41, exhibited major histocompatibility complex-restricted cytotoxicity, and secreted high levels of antiviral cytokines. T-cell receptor clonal transcript analyses and autologous virus sequencing revealed that Th cells induced during acute infection were maintained and there were no Th escape mutations. Subsequent analysis for this subject and six of seven others revealed detectable IFN-?-secreting cells, but only following in vitro gp160 stimulation. In summary, we conclude that Env-specific Th responses are elicited very early in acute infection and may precede Gag-specific responses. The inability to detect Env-specific Th responses over time and despite antiretroviral therapy may reflect low frequencies and impaired proliferative capacity, and viral escape is not necessary for this to occur.

Malhotra, Uma; Holte, Sarah; Zhu, Tuofu; Delpit, Elizabeth; Huntsberry, Claire; Sette, Alessandro; Shankarappa, Raj; Maenza, Janine; Corey, Lawrence; McElrath, M. Juliana

2003-01-01

107

Detailed Topology Mapping Reveals Substantial Exposure of the "Cytoplasmic" C-Terminal Tail (CTT) Sequences in HIV-1 Env Proteins at the Cell Surface  

PubMed Central

Substantial controversy surrounds the membrane topology of the HIV-1 gp41 C-terminal tail (CTT). While few studies have been designed to directly address the topology of the CTT, results from envelope (Env) protein trafficking studies suggest that the CTT sequence is cytoplasmically localized, as interactions with intracellular binding partners are required for proper Env targeting. However, previous studies from our lab demonstrate the exposure of a short CTT sequence, the Kennedy epitope, at the plasma membrane of intact Env-expressing cells, the exposure of which is not observed on viral particles. To address the topology of the entire CTT sequence, we serially replaced CTT sequences with a VSV-G epitope tag sequence and examined reactivity of cell- and virion-surface Env to an anti-VSV-G monoclonal antibody. Our results demonstrate that the majority of the CTT sequence is accessible to antibody binding on the surface of Env expressing cells, and that the CTT-exposed Env constitutes 20–50% of the cell-surface Env. Cell surface CTT exposure was also apparent in virus-infected cells. Passive transfer of Env through cell culture media to Env negative (non-transfected) cells was not responsible for the apparent cell surface CTT exposure. In contrast to the cell surface results, CTT-exposed Env was not detected on infectious pseudoviral particles containing VSV-G-substituted Env. Finally, a monoclonal antibody directed to the Kennedy epitope neutralized virus in a temperature-dependent manner in a post-attachment neutralization assay. Collectively, these results suggest that the membrane topology of the HIV gp41 CTT is more complex than the widely accepted intracytoplasmic model.

Steckbeck, Jonathan D.; Sun, Chengqun; Sturgeon, Timothy J.; Montelaro, Ronald C.

2013-01-01

108

Detailed topology mapping reveals substantial exposure of the "cytoplasmic" C-terminal tail (CTT) sequences in HIV-1 Env proteins at the cell surface.  

PubMed

Substantial controversy surrounds the membrane topology of the HIV-1 gp41 C-terminal tail (CTT). While few studies have been designed to directly address the topology of the CTT, results from envelope (Env) protein trafficking studies suggest that the CTT sequence is cytoplasmically localized, as interactions with intracellular binding partners are required for proper Env targeting. However, previous studies from our lab demonstrate the exposure of a short CTT sequence, the Kennedy epitope, at the plasma membrane of intact Env-expressing cells, the exposure of which is not observed on viral particles. To address the topology of the entire CTT sequence, we serially replaced CTT sequences with a VSV-G epitope tag sequence and examined reactivity of cell- and virion-surface Env to an anti-VSV-G monoclonal antibody. Our results demonstrate that the majority of the CTT sequence is accessible to antibody binding on the surface of Env expressing cells, and that the CTT-exposed Env constitutes 20-50% of the cell-surface Env. Cell surface CTT exposure was also apparent in virus-infected cells. Passive transfer of Env through cell culture media to Env negative (non-transfected) cells was not responsible for the apparent cell surface CTT exposure. In contrast to the cell surface results, CTT-exposed Env was not detected on infectious pseudoviral particles containing VSV-G-substituted Env. Finally, a monoclonal antibody directed to the Kennedy epitope neutralized virus in a temperature-dependent manner in a post-attachment neutralization assay. Collectively, these results suggest that the membrane topology of the HIV gp41 CTT is more complex than the widely accepted intracytoplasmic model. PMID:23724133

Steckbeck, Jonathan D; Sun, Chengqun; Sturgeon, Timothy J; Montelaro, Ronald C

2013-05-27

109

The Fusion-Controlling Disulfide Bond Isomerase in Retrovirus Env Is Triggered by Protein Destabilization  

PubMed Central

The membrane fusion function of murine leukemia virus (MLV) is carried by the Env protein. This protein is composed of three SU-TM subunit complexes. The fusion activity is loaded into the transmembrane TM subunit and controlled by the peripheral, receptor-binding SU subunit. It is assumed that TM adopts a metastable conformation in the native Env and that fusion activation involves the folding of TM into a stable form. Activation is suppressed by the associated SU and triggered by its dissociation, which follows receptor binding. Recently we showed that the two subunits are disulfide linked and that SU dissociation and triggering of the fusion function are caused by a switch of the intersubunit disulfide into an intrasubunit disulfide isomer using an isomerization-active CWLC motif in SU (M. Wallin, M. Ekstrom, and H. Garoff, EMBO J. 23:54-65, 2004). In the present work we address how the SU disulfide isomerase is activated. Using Moloney MLV, we show that isomerization of the SU-TM disulfide bond can be triggered by heat, urea, or guanidinium hydrochloride. Such protein perturbation treatments also significantly increase the kinetics and efficiency of viral fusion. The threshold conditions for the effects on isomerization and fusion are virtually the same. This finding indicates that destabilization of interactions in the SU oligomer induces the disulfide bond isomerase and the subsequent activation of the fusion function in TM.

Wallin, Michael; Ekstrom, Maria; Garoff, Henrik

2005-01-01

110

envB mutations confer UV-sensitivity to Salmonella typhimurium and UV-resistance to Escherichia coli.  

PubMed

An envB mutation isolated in Salmonella typhimurium LT2 was transferred by conjugation to Escherichia coli K-12. The mutation produced the same alterations in E. coli as in S. typhimurium concerning cell shape, sensitivity to drugs, autolysis, and fermentation of carbohydrates. However, although the mutation conferred sensitivity to UV irradiation in Salmonella, in E. coli it behaved as a genuine envB mutation producing resistance to UV inactivation. The fact that the mutation produced opposite effects in the survival of UV-irradiated S. typhimurium and E. coli discloses an intriguing difference between these closely related species. PMID:7012547

Antón, D N

1981-01-01

111

Evaluation of Heterologous Vaginal SHIV SF162p4 Infection Following Vaccination with a Polyvalent Clade B Virus-Like Particle Vaccine  

PubMed Central

Abstract The vast diversity of HIV-1 infections has greatly impeded the development of a successful HIV-1/AIDS vaccine. Previous vaccine work has demonstrated limited levels of protection against SHIV/SIV infection, but protection was observed only when the challenge virus was directly matched to the vaccine strain. As it is likely impossible to directly match the vaccine strain to all infecting strains in nature, it is necessary to develop an HIV-1 vaccine that can protect against a heterologous viral challenge. In this study we investigated the ability of polyvalent and consensus vaccines to protect against a heterologous clade B challenge. Rhesus macaques were vaccinated with ConB or PolyB virus-like particle vaccines. All vaccines were highly immunogenic with high titers of antibody found in all vaccinated groups against SIV Gag. Antibody responses were also observed against a diverse panel of clade B envelopes. Following vaccination nonhuman primates (NHPs) were challenged via the vaginal route with SHIVSF162p4. The PolyB vaccine induced a 66.7% reduction in the rate of infection as well as causing a two log reduction in viral burden if infection was not blocked. ConB vaccination had no effect on either the infection rate or viral burden. These results indicate that a polyvalent clade-matched vaccine is better able to protect against a heterologous challenge as compared to a consensus vaccine.

McBurney, Sean P.; Landucci, Gary; Forthal, Donald N.

2012-01-01

112

Efficient repeated low-dose intravaginal infection with X4 and R5 SHIVs in rhesus macaque: Implications for HIV-1 transmission in humans  

PubMed Central

We examined the effect of inoculum dose on SHIV transmission and infection. We found that repeated low-dose intravaginal exposure with either R5-SHIVSF162P3 or X4-SHIVSF33A results in infections that are blunted and rapidly controlled. Interestingly, although the transmission rate after all repeated exposures is comparable for the two viruses, the probability of low-dose vaginal transmission is greater for the X4 than R5 virus. Furthermore, X4-SHIVSF33A replication predominates in low-dose dually-exposed macaques, suggesting that it is better at establishing a systemic infection following transmission. However, X4-SHIVSF33A advantage in transmission and infection is not observed in macaques inoculated intravenously with low-dose mixed inoculum. The finding that although matched in tissue culture infectious dose, the X4 inoculum is more complex leads us to hypothesize that the greater genetic heterogeneity of the X4 virus population may have rendered it less susceptible to the severe bottleneck effects imposed by IVAG inoculation with small doses, allowing for greater probability of transmission and establishment of a generalized infection. These data have implications for HIV-1 transmission and infection in humans.

Tsai, Lily; Trunova, Nataliya; Gettie, Agegnehu; Mohri, Hiroshi; Bohm, Rudolf; Saifuddin, Mohammed; Cheng-Mayer, Cecilia

2007-01-01

113

Protection against Rectal Transmission of an Emtricitabine-Resistant Simian/Human Immunodeficiency Virus SHIV162p3M184V Mutant by Intermittent Prophylaxis with Truvada ?  

PubMed Central

Daily preexposure prophylaxis (PrEP) with Truvada (emtricitabine [FTC] and tenofovir disoproxil fumarate [TDF]) is a novel HIV prevention strategy recently found to reduce HIV incidence among men who have sex with men. We used a macaque model of HIV transmission to investigate if Truvada maintains prophylactic efficacy against an FTC-resistant isolate containing the M184V mutation. Five macaques received a dose of Truvada 3 days before exposing them rectally to the simian/human immunodeficiency virus mutant SHIV162p3M184V, followed by a second dose 2 h after exposure. Five untreated animals were used as controls. Virus exposures were done weekly for up to 14 weeks. Despite the high (>100-fold) level of FTC resistance conferred by M184V, all five treated animals were protected from infection, while the five untreated macaques were infected (P = 0.0008). Our results show that Truvada maintains high prophylactic efficacy against an FTC-resistant isolate. Increased susceptibility to tenofovir due to M184V and other factors, including residual antiviral activity by FTC and/or reduced virus fitness due to M184V, may all have contributed to the observed protection.

Cong, Mian-er; Youngpairoj, Ae S.; Zheng, Qi; Aung, Wutyi; Mitchell, James; Sweeney, Elizabeth; Hanson, Debra L.; Hendry, R. Michael; Dobard, Charles; Heneine, Walid; Garcia-Lerma, J. Gerardo

2011-01-01

114

Inhibition of HIV Env binding to cellular receptors by monoclonal antibody 2G12 as probed by Fc-tagged gp120  

PubMed Central

During natural HIV infection, an array of host receptors are thought to influence virus attachment and the kinetics of infection. In this study, to probe the interactions of HIV envelope (Env) with various receptors, we assessed the inhibitory properties of various anti-Env monoclonal antibodies (mAbs) in binding assays. To assist in detecting Env in attachment assays, we generated Fc fusions of full-length wild-type gp120 and several variable loop-deleted gp120s. Through investigation of the inhibition of Env binding to cell lines expressing CD4, CCR5, DC-SIGN, syndecans or combinations thereof, we found that the broadly neutralizing mAb, 2G12, directed to a unique carbohydrate epitope of gp120, inhibited Env-CCR5 binding, partially inhibited Env-DC-SIGN binding, but had no effect on Env-syndecan association. Furthermore, 2G12 inhibited Env attachment to primary monocyte-derived dendritic cells, that expressed CD4 and CCR5 primary HIV receptors, as well as DC-SIGN, and suggested that the dual activities of 2G12 could be valuable in vivo for inhibiting initial virus dissemination and propagation.

Binley, James M; Ngo-Abdalla, Stacie; Moore, Penny; Bobardt, Michael; Chatterji, Udayan; Gallay, Philippe; Burton, Dennis R; Wilson, Ian A; Elder, John H; de Parseval, Aymeric

2006-01-01

115

Dense display of HIV-1 envelope spikes on the lambda phage scaffold does not result in the generation of improved antibody responses to HIV-1 Env.  

PubMed

The generation of strong, virus-neutralizing antibody responses to the HIV-1 envelope spike (Env) is a major goal in HIV-1 vaccine research. To try to enhance the Env-specific response, we displayed oligomeric gp140 on a virus-like scaffold provided by the lambda phage capsid. To do this, an in vitro complementation system was used to "decorate" phage particles with glycosylated, mammalian cell-derived envelope oligomers. We compared the immune response to lambda phage particles displaying HIV-1 Env to that elicited by soluble oligomeric gp140 in rabbits. Env-binding antibody titers were higher in animals that received oligomeric gp140 as compared to Env decorated phage particles, as were virus neutralizing antibody responses. The Env decorated phage particles were, however, able to efficiently boost a protein-primed humoral response to levels equivalent to those elicited by high-dose adjuvanted Env oligomers. These results show that display of HIV-1 envelope spikes on the bacteriophage lambda capsid does not result in an improved, Env-specific humoral immune response. PMID:21310193

Mattiacio, Jonelle; Walter, Scott; Brewer, Matt; Domm, William; Friedman, Alan E; Dewhurst, Stephen

2011-02-18

116

Silencing of endogenous envelope genes in human choriocarcinoma cells shows that envPb1 is involved in heterotypic cell fusions.  

PubMed

Syncytin-1 and envPb1 are two conserved envelope genes in the human genome encoded by single loci from the HERV-W and -Pb families, respectively. To characterize the role of these envelope proteins in cell-cell fusion, we have developed lentiviral vectors that express short hairpin RNAs for stable knockdown of syncytin-1 and envPb1. Analysis of heterotypic fusion activity between trophoblast-derived choriocarcinoma BeWo cells, in which syncytin-1 and envPb1 are specifically silenced, and endothelial cells demonstrated that both syncytin-1 and envPb1 are important to fusion. The ability to fuse cells makes syncytin-1 and envPb1 attractive candidate molecules in therapy against cancer. Our available vectors may help eventually to decipher roles for these genes in human health and/or disease. PMID:22573740

Aagaard, Lars; Bjerregaard, Bolette; Kjeldbjerg, Anders L; Pedersen, Finn Skou; Larsson, Lars-Inge; Rossi, John J

2012-05-09

117

Differential regulation of the antibody responses to Gag and Env proteins of human immunodeficiency virus type 1.  

PubMed Central

We have studied the antibody responses to Env and Gag antigens of human immunodeficiency virus type 1 (HIV-1) in several cohorts of HIV-1-infected individuals: long-term nonprogressors, progressors to disease, acute seroconvertors, and recipients of HIV-1 protease inhibitors. We conclude that the antibody responses to Env and Gag antigens are differentially regulated and that changes in the plasma viral load in the measurable range (500 to 10(8) RNA copies per ml) do not directly affect the antibody responses to these HIV-1 proteins. We provide quantitative estimates of HIV-1-specific immunoglobulin G concentrations in plasma, which can be in excess of 1 mg/ml for both anti-gp120 and anti-p24 once the immune response to HIV-1 has stabilized after seroconversion. We discuss the apparent paradox that the absence of anti-Gag antibodies (which have, at best, limited antiviral activity) is indicative of disease progression, while the retention of anti-Env antibodies (which do have antiviral activity) is of limited (or no) prognostic value. We show that the disappearance of anti-Gag antibodies during disease progression is highly unlikely to be due to immune complexing; instead, we believe that it reflects the loss of T-cell help that is more necessary for the anti-Gag than the anti-Env response.

Binley, J M; Klasse, P J; Cao, Y; Jones, I; Markowitz, M; Ho, D D; Moore, J P

1997-01-01

118

MicroRT - Small animal conformal irradiator  

SciTech Connect

A novel small animal conformal radiation therapy system has been designed and prototyped: MicroRT. The microRT system integrates multimodality imaging, radiation treatment planning, and conformal radiation therapy that utilizes a clinical {sup 192}Ir isotope high dose rate source as the radiation source (teletherapy). A multiparameter dose calculation algorithm based on Monte Carlo dose distribution simulations is used to efficiently and accurately calculate doses for treatment planning purposes. A series of precisely machined tungsten collimators mounted onto a cylindrical collimator assembly is used to provide the radiation beam portals. The current design allows a source-to-target distance range of 1-8 cm at four beam angles: 0 deg. (beam oriented down), 90 deg., 180 deg., and 270 deg. The animal is anesthetized and placed in an immobilization device with built-in fiducial markers and scanned using a computed tomography, magnetic resonance, or positron emission tomography scanner prior to irradiation. Treatment plans using up to four beam orientations are created utilizing a custom treatment planning system--microRTP. A three-axis computer-controlled stage that supports and accurately positions the animals is programmed to place the animal relative to the radiation beams according to the microRTP plan. The microRT system positioning accuracy was found to be submillimeter. The radiation source is guided through one of four catheter channels and placed in line with the tungsten collimators to deliver the conformal radiation treatment. The microRT hardware specifications, the accuracy of the treatment planning and positioning systems, and some typical procedures for radiobiological experiments that can be performed with the microRT device are presented.

Stojadinovic, S.; Low, D. A.; Hope, A. J.; Vicic, M.; Deasy, J. O.; Cui, J.; Khullar, D.; Parikh, P. J.; Malinowski, K. T.; Izaguirre, E. W.; Mutic, S.; Grigsby, P. W. [Washington University School of Medicine, Saint Louis, Missouri 63110 (United States)

2007-12-15

119

MicroRT-small animal conformal irradiator.  

PubMed

A novel small animal conformal radiation therapy system has been designed and prototyped: MicroRT. The microRT system integrates multimodality imaging, radiation treatment planning, and conformal radiation therapy that utilizes a clinical 192Ir isotope high dose rate source as the radiation source (teletherapy). A multiparameter dose calculation algorithm based on Monte Carlo dose distribution simulations is used to efficiently and accurately calculate doses for treatment planning purposes. A series of precisely machined tungsten collimators mounted onto a cylindrical collimator assembly is used to provide the radiation beam portals. The current design allows a source-to-target distance range of 1-8 cm at four beam angles: 0 degrees (beam oriented down), 90 degrees, 180 degrees, and 270 degrees. The animal is anesthetized and placed in an immobilization device with built-in fiducial markers and scanned using a computed tomography, magnetic resonance, or positron emission tomography scanner prior to irradiation. Treatment plans using up to four beam orientations are created utilizing a custom treatment planning system-microRTP. A three-axis computer-controlled stage that supports and accurately positions the animals is programmed to place the animal relative to the radiation beams according to the microRTP plan. The microRT system positioning accuracy was found to be submillimeter. The radiation source is guided through one of four catheter channels and placed in line with the tungsten collimators to deliver the conformal radiation treatment. The microRT hardware specifications, the accuracy of the treatment planning and positioning systems, and some typical procedures for radiobiological experiments that can be performed with the microRT device are presented. PMID:18196798

Stojadinovic, S; Low, D A; Hope, A J; Vicic, M; Deasy, J O; Cui, J; Khullar, D; Parikh, P J; Malinowski, K T; Izaguirre, E W; Mutic, S; Grigsby, P W

2007-12-01

120

The avian retrovirus env gene family: molecular analysis of host range and antigenic variants.  

PubMed Central

The nucleotide sequence of the env gp85-coding domain from two avian sarcoma and leukosis retrovirus isolates was determined to identify host range and antigenic determinants. The predicted amino acid sequence of gp85 from a subgroup D virus isolate of the Schmidt-Ruppin strain of Rous sarcoma virus was compared with the previously reported sequences of subgroup A, B, C, and E avian sarcoma and leukosis retroviruses. Subgroup D viruses are closely related to the subgroup B viruses but have an extended host range that includes the ability to penetrate certain mammalian cells. There are 27 amino acid differences shared between the subgroup D sequence and three subgroup B sequences. At 16 of these sites, the subgroup D sequence is identical to the sequence of one or more of the other subgroup viruses (A, C, and E). The remaining 11 sites are specific to subgroup D and show some clustering in the two large variable regions that are thought to be major determinants of host range. Biological analysis of recombinant viruses containing a dominant selectable marker confirmed the role of the gp85-coding domain in determining the host range of the subgroup D virus in the infection of mammalian cells. We also compared the sequence of the gp85-coding domain from two subgroup A viruses, Rous-associated virus type 1 and a subgroup A virus of the Schmidt-Ruppin strain of Rous sarcoma virus. The comparison revealed 24 nonconservative amino acid changes, of which 6 result in changes in potential glycosylation sites. The positions of 10 amino acid differences are coincident with the positions of 10 differences found between two subgroup B virus env gene sequences. These 10 sites identify seven domains in the sequence which may constitute determinants of type-specific antigenicity. Using a molecular recombinant, we demonstrated that type-specific neutralization of two subgroup A viruses was associated with the gp85-coding domain of the virus.

Bova, C A; Olsen, J C; Swanstrom, R

1988-01-01

121

Broadly Neutralizing Human Anti-HIV Antibody 2G12 Is Effective in Protection against Mucosal SHIV Challenge Even at Low Serum Neutralizing Titers  

PubMed Central

Developing an immunogen that elicits broadly neutralizing antibodies (bNAbs) is an elusive but important goal of HIV vaccine research, especially after the recent failure of the leading T cell based HIV vaccine in human efficacy trials. Even if such an immunogen can be developed, most animal model studies indicate that high serum neutralizing concentrations of bNAbs are required to provide significant benefit in typical protection experiments. One possible exception is provided by the anti-glycan bNAb 2G12, which has been reported to protect macaques against CXCR4-using SHIV challenge at relatively low serum neutralizing titers. Here, we investigated the ability of 2G12 administered intravenously (i.v.) to protect against vaginal challenge of rhesus macaques with the CCR5-using SHIVSF162P3. The results show that, at 2G12 serum neutralizing titers of the order of 1?1 (IC90), 3/5 antibody-treated animals were protected with sterilizing immunity, i.e. no detectable virus replication following challenge; one animal showed a delayed and lowered primary viremia and the other animal showed a course of infection similar to 4 control animals. This result contrasts strongly with the typically high titers observed for protection by other neutralizing antibodies, including the bNAb b12. We compared b12 and 2G12 for characteristics that might explain the differences in protective ability relative to neutralizing activity. We found no evidence to suggest that 2G12 transudation to the vaginal surface was significantly superior to b12. We also observed that the ability of 2G12 to inhibit virus replication in target cells through antibody-mediated effector cell activity in vitro was equivalent or inferior to b12. The results raise the possibility that some epitopes on HIV may be better vaccine targets than others and support targeting the glycan shield of the envelope.

Hessell, Ann J.; Rakasz, Eva G.; Poignard, Pascal; Hangartner, Lars; Landucci, Gary; Forthal, Donald N.; Koff, Wayne C.; Watkins, David I.; Burton, Dennis R.

2009-01-01

122

Nature of the Penetration Barrier in Escherichia coli K-12: Effect of Macromolecular Inhibition on Penetrability in Strains Containing the envA Gene  

PubMed Central

The envA mutation in Escherichia coli K-12, which maps at 1.5 min, was previously shown to mediate sensitivity to gentian violet as well as to several antibiotics. Moreover, strains containing the envA gene were recently found to be lysed by lysozyme in the absence of ethylenediaminetetraacetate. It is here reported that the envA mutation mediates an increased uptake of gentian violet. The uptake of the dye was markedly affected by growth with different antibiotics interfering with macromolecular synthesis. Amino acid starvation of a strain containing envA with a stringent control of ribonucleic acid (RNA) synthesis resulted in a decreased uptake of gentian violet. However, no decrease in dye uptake was found during starvation in an envA transductant with a relaxed control of RNA synthesis. Inhibition of deoxyribonucleic acid (DNA) synthesis by nalidixic acid decreased the uptake of gentian violet of envA cells and, in addition, rendered the cells insensitive to the lytic action of lysozyme. Chloramphenicol treatment increased penetrability in wild-type and starved envA cells. In most instances, this effect of chloramphenicol was prevented by selectively interfering with DNA or RNA synthesis. A coordinate regulation of nucleic acid synthesis and penetrability is suggested.

Normark, Staffan; Westling, Britta

1971-01-01

123

Preparation of purified tuberculin RT 23  

PubMed Central

The technical procedure used in the preparation of a batch of more than 500 g of purified tuberculin (PPD) is described. This batch is designated RT 23, and it is estimated that the quantity now prepared will cover the global demand for purified tuberculin for human use for several years. RT 23 has been prepared by mixing 77 smaller lots of tuberculin selected from a total of 95 lots. The method of preparing the individual lots is described and the experimental data, i.e., the yield and the biological activity ascertained by skin tests in BCG-vaccinated guinea-pigs, are given for all lots. The possible causes of variations in the yield and biological activity of the individual lots are discussed.

Magnusson, Mogens; Bentzon, M. Weis

1958-01-01

124

Thermophysical properties of reversed TALSPEAK (RT) solvent  

Microsoft Academic Search

Thermophysical properties of reversed TALSPEAK extractant (0.3 M D2EPHA\\/0.2 M TBP\\/n-dodecane) were not available in literature. Authors have experimentally measured and correlated several thermophysical properties\\u000a of RT solvent like density, viscosity, refractive index, acid uptake and flash point. In this paper, results of these studies\\u000a will be discussed in detail.

Shekhar KumarS; S. Balasubramonian; Pranay Kumar Sinha; D. Sivakumar; U. Kamachi Mudali; R. Natarajan

125

Genetic and functional analysis of full-length human immunodeficiency virus type 1 env genes derived from brain and blood of patients with AIDS.  

PubMed

The genetic evolution of human immunodeficiency virus type 1 (HIV-1) in the brain is distinct from that in lymphoid tissues, indicating tissue-specific compartmentalization of the virus. Few primary HIV-1 envelope glycoproteins (Envs) from uncultured brain tissues have been biologically well characterized. In this study, we analyzed 37 full-length env genes from uncultured brain biopsy and blood samples from four patients with AIDS. Phylogenetic analysis of intrapatient sequence sets showed distinct clustering of brain relative to blood env sequences. However, no brain-specific signature sequence was identified. Furthermore, there was no significant difference in the number or positions of N-linked glycosylation sites between brain and blood env sequences. The patterns of coreceptor usage were heterogeneous, with no clear distinction between brain and blood env clones. Nine Envs used CCR5 as a coreceptor, one used CXCR4, and two used both CCR5 and CXCR4 in cell-to-cell fusion assays. Eight Envs could also use CCR3, CCR8, GPR15, STRL33, Apj, and/or GPR1, but these coreceptors did not play a major role in virus entry into microglia. Recognition of epitopes by the 2F5, T30, AG10H9, F105, 17b, and C11 monoclonal antibodies varied among env clones, reflecting genetic and conformational heterogeneity. Envs from two patients contained 28 to 32 N-glycosylation sites in gp120, compared to around 25 in lab strains and well-characterized primary isolates. These results suggest that HIV-1 Envs in brain cannot be distinguished from those in blood on the basis of coreceptor usage or the number or positions of N-glycosylation sites, indicating that other properties underlie neurotropism. The study also demonstrates characteristics of primary HIV-1 Envs from uncultured tissues and implies that Env variants that are glycosylated more extensively than lab strains and well-characterized primary isolates should be considered during development of vaccines and neutralizing antibodies. PMID:14581570

Ohagen, Asa; Devitt, Amy; Kunstman, Kevin J; Gorry, Paul R; Rose, Patrick P; Korber, Bette; Taylor, Joann; Levy, Robert; Murphy, Robert L; Wolinsky, Steven M; Gabuzda, Dana

2003-11-01

126

A rev1-vpu polymorphism unique to HIV-1 subtype A and C strains impairs envelope glycoprotein expression from rev-vpu-env cassettes and reduces virion infectivity in pseudotyping assays  

SciTech Connect

Functional studies of HIV-1 envelope glycoproteins (Envs) commonly include the generation of pseudoviruses, which are produced by co-transfection of rev-vpu-env cassettes with an env-deficient provirus. Here, we describe six Env constructs from transmitted/founder HIV-1 that were defective in the pseudotyping assay, although two produced infectious virions when expressed from their cognate proviruses. All of these constructs exhibited an unusual gene arrangement in which the first exon of rev (rev1) and vpu were in the same reading frame without an intervening stop codon. Disruption of the rev1-vpu fusion gene by frameshift mutation, stop codon, or abrogation of the rev initiation codon restored pseudovirion infectivity. Introduction of the fusion gene into wildtype Env cassettes severely compromised their function. The defect was not due to altered env and rev transcription or a dominant negative effect of the expressed fusion protein, but seemed to be caused by inefficient translation at the env initiation codon. Although the rev1-vpu polymorphism affects Env expression only in vitro, it can cause problems in studies requiring Env complementation, such as analyses of co-receptor usage and neutralization properties, since 3% of subtype A, 20% of subtype C and 5% of CRF01{sub A}/E viruses encode the fusion gene. A solution is to eliminate the rev initiation codon when amplifying rev-vpu-env cassettes since this increases Env expression irrespective of the presence of the polymorphism.

Kraus, Matthias H.; Parrish, Nicholas F.; Shaw, Katharina S.; Decker, Julie M.; Keele, Brandon F.; Salazar-Gonzalez, Jesus F.; Grayson, Truman; McPherson, David T. [Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Ping, Li-Hua; Anderson, Jeffrey A.; Swanstrom, Ronald [University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (United States); Williamson, Carolyn [Institute of Infectious Disease and Molecular Medicine, University of Cape Town (South Africa); Shaw, George M. [Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Hahn, Beatrice H., E-mail: bhahn@uab.ed [Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States)

2010-02-20

127

Quantitative RT-PCR Detection of Hepatitis A Virus, Rotaviruses and Enteroviruses in the Buffalo River and Source Water Dams in the Eastern Cape Province of South Africa  

PubMed Central

Human enteric viruses (HEntVs) are a major cause of water-related diseases. The prevalence of hepatitis A virus (HAV), rotaviruses (RoV) and enteroviruses (EnV) in Buffalo River waters was assessed quantitatively over a period of 12 months (August 2010 to July 2011). Seventy-two samples were collected from six sites, including three dams, and concentrated using the adsorption-elution method. Viral RNA was extracted using a commercial kit, and the viruses were quantified by real-time quantitative reverse transcriptase PCR (RT-qPCR). Two or more viruses were detected in 12.5% of the samples. HAV was detected in 43.1% of the samples and in significantly (p < 0.05) varying concentrations of 1.5 × 101–1.9 × 105 genome copies/L compared to RoV and EnV, while RoVs were detected in 13.9% of samples, with concentrations ranging from 2.5 × 101–2.1 × 103 genome copies/L, and EnV were detected in 9.7% of the samples, with concentrations ranging from 1.3 × 101–8.6 × 101 genome copies/L. Only HAV was detected at all the sites, with the Bridle Drift Dam recording significantly higher (p < 0.05) concentrations. The presence of enteric viruses in Buffalo River may constitute public health risks and the incidence of HAV at all the sites could reflect both the epidemiological status of hepatitis A and HAV persistence in the water environments.

Chigor, Vincent Nnamdigadi; Okoh, Anthony Ifeanyi

2012-01-01

128

Quantitative RT-PCR detection of hepatitis A virus, rotaviruses and enteroviruses in the Buffalo River and source water dams in the Eastern Cape Province of South Africa.  

PubMed

Human enteric viruses (HEntVs) are a major cause of water-related diseases. The prevalence of hepatitis A virus (HAV), rotaviruses (RoV) and enteroviruses (EnV) in Buffalo River waters was assessed quantitatively over a period of 12 months (August 2010 to July 2011). Seventy-two samples were collected from six sites, including three dams, and concentrated using the adsorption-elution method. Viral RNA was extracted using a commercial kit, and the viruses were quantified by real-time quantitative reverse transcriptase PCR (RT-qPCR). Two or more viruses were detected in 12.5% of the samples. HAV was detected in 43.1% of the samples and in significantly (p < 0.05) varying concentrations of 1.5 × 10(1)–1.9 × 10(5) genome copies/L compared to RoV and EnV, while RoVs were detected in 13.9% of samples, with concentrations ranging from 2.5 × 10(1)–2.1 × 10(3) genome copies/L, and EnV were detected in 9.7% of the samples, with concentrations ranging from 1.3 × 10(1)–8.6 × 10(1) genome copies/L. Only HAV was detected at all the sites, with the Bridle Drift Dam recording significantly higher (p < 0.05) concentrations. The presence of enteric viruses in Buffalo River may constitute public health risks and the incidence of HAV at all the sites could reflect both the epidemiological status of hepatitis A and HAV persistence in the water environments. PMID:23202829

Chigor, Vincent Nnamdigadi; Okoh, Anthony Ifeanyi

2012-11-05

129

Obacunone Represses Salmonella Pathogenicity Islands 1 and 2 in an envZ-Dependent Fashion  

PubMed Central

Obacunone belongs to a class of unique triterpenoids called limonoids, present in Citrus species. Previous studies from our laboratory suggested that obacunone possesses antivirulence activity and demonstrates inhibition of cell-cell signaling in Vibrio harveyi and Escherichia coli O157:H7. The present work sought to determine the effect of obacunone on the food-borne pathogen Salmonella enterica serovar Typhimurium LT2 by using a cDNA microarray. Transcriptomic studies indicated that obacunone represses Salmonella pathogenicity island 1 (SPI1), the maltose transporter, and the hydrogenase operon. Furthermore, phenotypic data for the Caco-2 infection assay and maltose utilization were in agreement with microarray data suggesting repression of SPI1 and maltose transport. Further studies demonstrated that repression of SPI1 was plausibly mediated through hilA. Additionally, obacunone seems to repress SPI2 under SPI2-inducing conditions as well as in Caco-2 infection models. Furthermore, obacunone seems to repress hilA in an EnvZ-dependent fashion. Altogether, the results of the study seems to suggest that obacunone exerts an antivirulence effect on S. Typhimurium and may serve as a lead compound for development of antivirulence strategies for S. Typhimurium.

Vikram, Amit; Jayaprakasha, Guddadarangavvanahally K.; Jesudhasan, Palmy R.

2012-01-01

130

Proviral amplification of the Gypsy endogenous retrovirus of Drosophila melanogaster involves env-independent invasion of the female germline.  

PubMed Central

Gypsy is an infectious endogenous retrovirus of Drosophila melanogaster. The gypsy proviruses replicate very efficiently in the genome of the progeny of females homozygous for permissive alleles of the flamenco gene. This replicative transposition is correlated with derepression of gypsy expression, specifically in the somatic cells of the ovaries of the permissive mothers. The determinism of this amplification was studied further by making chimeric mothers containing different permissive/restrictive and somatic/germinal lineages. We show here that the derepression of active proviruses in the permissive soma is necessary and sufficient to induce proviral insertions in the progeny, even if the F1 flies derive from restrictive germ cells devoid of active proviruses. Therefore, gypsy endogenous multiplication results from the transfer of some gypsy-encoded genetic material from the soma towards the germen of the mother and its subsequent insertion into the chromosomes of the progeny. This transfer, however, is not likely to result from retroviral infection of the germline. Indeed, we also show here that the insertion of a tagged gypsy element, mutant for the env gene, occurs at high frequency, independently of the production of gypsy Env proteins by any transcomplementing helper. The possible role of the env gene for horizontal transfer to new hosts is discussed.

Chalvet, F; Teysset, L; Terzian, C; Prud'homme, N; Santamaria, P; Bucheton, A; Pelisson, A

1999-01-01

131

An activation-dependent, T-lymphocyte-specific transcriptional activator in the mouse mammary tumor virus env gene.  

PubMed Central

Transcription of the complete mouse mammary tumor virus (MMTV) proviral genome in mouse cells is controlled by a strong promoter in its long terminal repeat. In the mouse T lymphoma EL4, there is a second, activation-dependent transcriptional initiation site within the envelope (env) gene, from which a short mRNA is generated, encoding the open reading frame of the long terminal repeat. We now report the isolation of a segment of the MMTV env gene (called META, for MMTV env transcriptional activator) which has the expected transcription-activating properties seen in EL4.E1 cells. Namely, it induces activation-dependent, T-lymphocyte-specific transcription of a chloramphenicol acetyltransferase reporter gene. It is active in mouse or human T-helper lymphocyte lines when they are stimulated to transcribe lymphokine genes but is inactive in unstimulated T-helper cells, fibroblasts, a cytotoxic T-lymphocyte line, and a mastocytoma cell line. Its activity is inhibited by cyclosporin A, a specific inhibitor of lymphokine transcription. Several forms of the META have been isolated from EL4.E1 cells, a mouse T-helper cell hybridoma, and from BALB/c spleen cells. Linked to the heterologous thymidine kinase promoter, a 400-bp portion of it is an inducible, orientation-independent, and cyclosporin A-sensitive transcriptional activator in T-helper cells. Images

Miller, C L; Garner, R; Paetkau, V

1992-01-01

132

A novel human immunodeficiency virus type 1 protein, tev, shares sequences with tat, env, and rev proteins.  

PubMed Central

We have characterized a novel 28-kilodalton protein, p28tev, detected in human immunodeficiency virus type 1-infected cells. tev is recognized by both tat and rev monospecific antibodies. tev is initiated at the tat AUG and contains the first exon of tat at its amino terminus, a small portion of env in the middle, and the second exon of rev at its carboxy terminus. A cDNA clone producing tev was cloned and expressed in human cells. Sequence analysis revealed that the tev mRNA is generated by splicing to a novel exon located in the env region. This identifies a fourth class of multiply spliced human immunodeficiency virus mRNAs, produced in infected and transfected cells. tev is regulated during the virus life cycle similarly to the other regulatory proteins, tat, rev, and nef, and displays both tat and rev activities in functional assays. Since tev contains important functional domains of tat and rev and is produced very early after transfection, it may be an important regulator in the initial phase of virus expression. Another rev-related protein, p18(6)Drev, containing env and rev sequences, was characterized and was found not to have detectable rev activity. Images

Benko, D M; Schwartz, S; Pavlakis, G N; Felber, B K

1990-01-01

133

Human immunodeficiency virus-like, nonreplicating, gag-env particles assemble in a recombinant vaccinia virus expression system.  

PubMed Central

We report the assembly of human immunodeficiency virus (HIV)-like particles in African green monkey kidney cells coinfected with two recombinant vaccinia viruses, one carrying the HIV-1 gag and protease genes and the other the env gene. Biochemical analysis of particles sedimented from culture supernatants of doubly infected cells revealed that they were composed of gag proteins, primarily p24, as well as the env proteins gp120 and gp41. Thin-section immunoelectron microscopy showed that these particles were 100 to 120 nm in diameter, were characterized by the presence of cylindrical core structures, and displayed the mature gp120-gp41 complexes on their surfaces. Furthermore, thin-section immunoelectron microscopy analysis of infected cells showed that particle assembly and budding occurred at the plasma membrane. Nucleic acid hybridization suggested that the particles packaged only the gag mRNA but not the env mRNA. Therefore, the system we present is well suited for studies of HIV virion maturation. In addition, the HIV-like particles provide a novel and attractive approach for vaccine development. Images

Haffar, O; Garrigues, J; Travis, B; Moran, P; Zarling, J; Hu, S L

1990-01-01

134

A non-radioactive method for identifying enzyme-amplified products of the reticuloendotheliosis proviral env and LTR genes using psoralen-biotin labelled probes  

Microsoft Academic Search

A novel polymerase chain reaction (PCR) system based on the env gene of reticuloendotheliosis virus (REV) strain REV-A for the detection of proviral DNA is described. The designed PCR product of 807 bp was identified using an internal probe of 278 bp produced by nested PCR from REV-infected DNA CEF. The env-gene PCR was then compared with the previously described

Irit Davidson; M. Malkinson

1996-01-01

135

Cross-Subtype T-Cell Immune Responses Induced by a Human Immunodeficiency Virus Type 1 Group M Consensus Env Immunogen  

Microsoft Academic Search

The genetic diversity among globally circulating human immunodeficiency virus type 1 (HIV-1) strains is a serious challenge for HIV-1 vaccine design. We have generated a synthetic group M consensus env gene (CON6) for induction of cross-subtype immune responses and report here a comparative study of T-cell responses to this and natural strain env immunogens in a murine model. Three different

Eric A. Weaver; Zhongjing Lu; Zenaido T. Camacho; Fatiha Moukdar; Hua-Xin Liao; Ben-Jiang Ma; Mark Muldoon; James Theiler; Gary J. Nabel; Norman L. Letvin; Bette T. Korber; Beatrice H. Hahn; Barton F. Haynes; Feng Gao

2006-01-01

136

Role of Mason-Pfizer Monkey Virus (MPMV) Constitutive Transport Element (CTE) in the Propagation of MPMV Vectors by Genetic Complementation Using Homologous\\/Heterologous envGenes  

Microsoft Academic Search

To study Mason-Pfizer monkey virus (MPMV) replication over a single round, virus particles were generated that contain a replication-defective vector encoding a dominant selectable marker, thehygromycin B phosphotransferase (hygr)gene. Genetic complementation with a homologous MPMV envelope glycoprotein (Env-gp) or pseudotyping by several heterologous Env-gps from a variety of viruses resulted in infectious MPMV particles containing the replication-defective RNA. Recently, it

Tahir A. Rizvi; Kathy A. Lew; Russell D. Schmidt

1996-01-01

137

Dense display of HIV1 envelope spikes on the lambda phage scaffold does not result in the generation of improved antibody responses to HIV1 Env  

Microsoft Academic Search

The generation of strong, virus-neutralizing antibody responses to the HIV-1 envelope spike (Env) is a major goal in HIV-1 vaccine research. To try to enhance the Env-specific response, we displayed oligomeric gp140 on a virus-like scaffold provided by the lambda phage capsid. To do this, an in vitro complementation system was used to “decorate” phage particles with glycosylated, mammalian cell-derived

Jonelle Mattiacio; Scott Walter; Matt Brewer; William Domm; Alan E. Friedman; Stephen Dewhurst

2011-01-01

138

Tiered Categorization of a Diverse Panel of HIV-1 Env Pseudoviruses for Assessment of Neutralizing Antibodies ?  

PubMed Central

The restricted neutralization breadth of vaccine-elicited antibodies is a major limitation of current human immunodeficiency virus-1 (HIV-1) candidate vaccines. In order to permit the efficient identification of vaccines with enhanced capacity for eliciting cross-reactive neutralizing antibodies (NAbs) and to assess the overall breadth and potency of vaccine-elicited NAb reactivity, we assembled a panel of 109 molecularly cloned HIV-1 Env pseudoviruses representing a broad range of genetic and geographic diversity. Viral isolates from all major circulating genetic subtypes were included, as were viruses derived shortly after transmission and during the early and chronic stages of infection. We assembled a panel of genetically diverse HIV-1-positive (HIV-1+) plasma pools to assess the neutralization sensitivities of the entire virus panel. When the viruses were rank ordered according to the average sensitivity to neutralization by the HIV-1+ plasmas, a continuum of average sensitivity was observed. Clustering analysis of the patterns of sensitivity defined four subgroups of viruses: those having very high (tier 1A), above-average (tier 1B), moderate (tier 2), or low (tier 3) sensitivity to antibody-mediated neutralization. We also investigated potential associations between characteristics of the viral isolates (clade, stage of infection, and source of virus) and sensitivity to NAb. In particular, higher levels of NAb activity were observed when the virus and plasma pool were matched in clade. These data provide the first systematic assessment of the overall neutralization sensitivities of a genetically and geographically diverse panel of circulating HIV-1 strains. These reference viruses can facilitate the systematic characterization of NAb responses elicited by candidate vaccine immunogens.

Seaman, Michael S.; Janes, Holly; Hawkins, Natalie; Grandpre, Lauren E.; Devoy, Colleen; Giri, Ayush; Coffey, Rory T.; Harris, Linda; Wood, Blake; Daniels, Marcus G.; Bhattacharya, Tanmoy; Lapedes, Alan; Polonis, Victoria R.; McCutchan, Francine E.; Gilbert, Peter B.; Self, Steve G.; Korber, Bette T.; Montefiori, David C.; Mascola, John R.

2010-01-01

139

An Automated HIV-1 Env-Pseudotyped Virus Production for Global HIV Vaccine Trials  

PubMed Central

Background Infections with HIV still represent a major human health problem worldwide and a vaccine is the only long-term option to fight efficiently against this virus. Standardized assessments of HIV-specific immune responses in vaccine trials are essential for prioritizing vaccine candidates in preclinical and clinical stages of development. With respect to neutralizing antibodies, assays with HIV-1 Env-pseudotyped viruses are a high priority. To cover the increasing demands of HIV pseudoviruses, a complete cell culture and transfection automation system has been developed. Methodology/Principal Findings The automation system for HIV pseudovirus production comprises a modified Tecan-based Cellerity system. It covers an area of 5×3 meters and includes a robot platform, a cell counting machine, a CO2 incubator for cell cultivation and a media refrigerator. The processes for cell handling, transfection and pseudovirus production have been implemented according to manual standard operating procedures and are controlled and scheduled autonomously by the system. The system is housed in a biosafety level II cabinet that guarantees protection of personnel, environment and the product. HIV pseudovirus stocks in a scale from 140 ml to 1000 ml have been produced on the automated system. Parallel manual production of HIV pseudoviruses and comparisons (bridging assays) confirmed that the automated produced pseudoviruses were of equivalent quality as those produced manually. In addition, the automated method was fully validated according to Good Clinical Laboratory Practice (GCLP) guidelines, including the validation parameters accuracy, precision, robustness and specificity. Conclusions An automated HIV pseudovirus production system has been successfully established. It allows the high quality production of HIV pseudoviruses under GCLP conditions. In its present form, the installed module enables the production of 1000 ml of virus-containing cell culture supernatant per week. Thus, this novel automation facilitates standardized large-scale productions of HIV pseudoviruses for ongoing and upcoming HIV vaccine trials.

Fuss, Martina; Mazzotta, Angela S.; Sarzotti-Kelsoe, Marcella; Ozaki, Daniel A.; Montefiori, David C.; von Briesen, Hagen; Zimmermann, Heiko; Meyerhans, Andreas

2012-01-01

140

The Rous Sarcoma Virus Env Glycoprotein Contains a Highly Conserved Motif Homologous to Tyrosine-Based Endocytosis Signals and Displays an Unusual Internalization Phenotype  

PubMed Central

The cytoplasmic domains of retroviral transmembrane (TM) glycoproteins contain conserved sequence motifs that resemble tyrosine-based (YXXØ-type) endocytosis signals. We have previously described a mutant Rous sarcoma virus (RSV) Env protein, Env-?26, with an L165R mutation in the membrane-spanning domain (MSD) of TM, that exhibited dramatically decreased steady-state surface expression (G. L. Davis and E. Hunter, J. Cell Biol. 105:1191–1203, 1987; P. B. Johnston, J. Y. Dong, and E. Hunter, Virology 206:353–361, 1995). We now demonstrate that the tyrosine of the Y190RKM motif in the RSV TM cytoplasmic domain is crucial for the ?26 phenotype and is part of an efficient internalization signal in the context of a mutant MSD. In contrast, despite the presence of the Y190RKM motif, wild-type RSV Env is constitutively internalized at a slow rate (1.1%/min) more characteristic of bulk uptake during membrane turnover than of active clustering into endocytic vesicles. The ?26 mutation and two MSD mutations that abrogate palmitoylation of TM resulted in enhanced Env endocytosis indicative of active concentration into coated pits. Surprisingly, an Env-Y190A mutant was apparently excluded from coated pits since its uptake rate of 0.3%/min was significantly below that expected for the bulk rate. We suggest that in RSV Env an inherently functional endocytosis motif is silenced by a counteracting determinant in the MSD that acts to prevent clustering of Env into endocytic vesicles. Mutations in either the cytoplasmic tail or the MSD that inactivate one of the two counteracting signals would thus render the remaining determinant dominant.

Ochsenbauer, Christina; Dubay, Susan R.; Hunter, Eric

2000-01-01

141

Enhanced cell surface expression, immunogenicity and genetic stability resulting from a spontaneous truncation of HIV Env expressed by a recombinant MVA  

SciTech Connect

During propagation of modified vaccinia virus Ankara (MVA) encoding HIV 89.6 Env, a few viral foci stained very prominently. Virus cloned from such foci replicated to higher titers than the parent and displayed enhanced genetic stability on passage. Sequence analysis showed a single nucleotide deletion in the 89.6 env gene of the mutant that caused a frame shift and truncation of 115 amino acids from the cytoplasmic domain. The truncated Env was more highly expressed on the cell surface, induced higher antibody responses than the full-length Env, reacted with HIV neutralizing monoclonal antibodies and mediated CD4/co-receptor-dependent fusion. Intramuscular (IM), intradermal (ID) needleless, and intrarectal (IR) catheter inoculations gave comparable serum IgG responses. However, intraoral (IO) needleless injector route gave the highest IgA in lung washings and IR gave the highest IgA and IgG responses in fecal extracts. Induction of CTL responses in the spleens of individual mice as assayed by intracellular cytokine staining was similar with both the full-length and truncated Env constructs. Induction of acute and memory CTL in the spleens of mice immunized with the truncated Env construct by ID, IO, and IR routes was comparable and higher than by the IM route, but only the IR route induced CTL in the gut-associated lymphoid tissue. Thus, truncation of Env enhanced genetic stability as well as serum and mucosal antibody responses, suggesting the desirability of a similar modification in MVA-based candidate HIV vaccines.

Wyatt, Linda S. [Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (United States)], E-mail: lwyatt@niaid.nih.gov; Belyakov, Igor M. [Vaccine Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Earl, Patricia L. [Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (United States); Berzofsky, Jay A. [Vaccine Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Moss, Bernard [Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (United States)

2008-03-15

142

Altering an Artificial Gagpolnef Polyprotein and Mode of ENV Co-Administration Affects the Immunogenicity of a Clade C HIV DNA Vaccine  

PubMed Central

HIV-1 candidate vaccines expressing an artificial polyprotein comprising Gag, Pol and Nef (GPN) and a secreted envelope protein (Env) were shown in recent Phase I/II clinical trials to induce high levels of polyfunctional T cell responses; however, Env-specific responses clearly exceeded those against Gag. Here, we assess the impact of the GPN immunogen design and variations in the formulation and vaccination regimen of a combined GPN/Env DNA vaccine on the T cell responses against the various HIV proteins. Subtle modifications were introduced into the GPN gene to increase Gag expression, modify the expression ratio of Gag to PolNef and support budding of virus-like particles. I.m. administration of the various DNA constructs into BALB/c mice resulted in an up to 10-fold increase in Gag- and Pol-specific IFN?+ CD8+ T cells compared to GPN. Co-administering Env with Gag or GPN derivatives largely abrogated Gag-specific responses. Alterations in the molar ratio of the DNA vaccines and spatially or temporally separated administration induced more balanced T cell responses. Whereas forced co-expression of Gag and Env from one plasmid induced predominantly Env-specific T cells responses, deletion of the only H-2d T cell epitope in Env allowed increased levels of Gag-specific T cells, suggesting competition at an epitope level. Our data demonstrate that the biochemical properties of an artificial polyprotein clearly influence the levels of antigen-specific T cells, and variations in formulation and schedule can overcome competition for the induction of these responses. These results are guiding the design of ongoing pre-clinical and clinical trials.

Bredl, Simon; Kindsmuller, Kathrin; Kostler, Josef; Wagner, Ralf

2012-01-01

143

Reverse Transcriptase (RT) Inhibition of PCR at Low Concentrations of Template and Its Implications for Quantitative RT-PCR  

PubMed Central

Numerous instances of reverse transcriptase (RT) inhibition of the PCR were observed while developing nonquantitative uncoupled RT-PCR techniques for detecting nitrogenase and ammonia monooxygenase gene expression in situ. The inhibitory effect of RT on the PCR was removed with increasing template concentrations beyond 105 to 106 copies. Including T4 gene 32 protein during the reverse transcription phase of the RT-PCR reaction increased the RT-PCR product yield by as much as 483%; if gene 32 protein was introduced after reverse transcription but prior to the PCR phase, no improvement in product yield was observed. Addition of 1 ?g of exogenous calf thymus DNA or yeast tRNA did little to relieve RT inhibition of the PCR on both genomic DNA and mRNA templates. These results suggest that RT inhibition of the PCR is mediated through direct interaction with the specific primer-template combination (DNA and RNA) and point to specific assay modifications for estimating the extent of RT inhibition and counteracting some of the inhibitory effect. Furthermore, the working hypothesis of RT inhibition below a 105 to 106 copy threshold has important implications for quantitative RT-PCR studies. In particular, competitive, quantitative RT-PCR systems will consistently underestimate the actual RNA concentration. Hence, enumerations of RNA templates below 105 to 106 copies will be relative to an internal standard and will not be an absolute measure of RNA abundance in situ.

Chandler, Darrell P.; Wagnon, Christina A.; Bolton, Harvey

1998-01-01

144

Prime-Boost Immunization Schedules Based on Influenza Virus and Vaccinia Virus Vectors Potentiate Cellular Immune Responses against Human Immunodeficiency Virus Env Protein Systemically and in the Genitorectal Draining Lymph Nodes  

Microsoft Academic Search

Vaccines that elicit systemic and mucosal immune responses should be the choice to control human immunodeficiency virus (HIV) infections. We have previously shown that prime-boost immunizations with influenza virus Env and vaccinia virus (VV) WR Env recombinants induced an enhanced systemic CD8 T-cell response against HIV-1 Env antigen. In this report, we analyzed in BALB\\/c mice after priming with influenza

M. Magdalena Gherardi; JoseLuis Najera; Eva Perez-Jimenez; Susana Guerra; A. Garcia-Sastre; Mariano Esteban

2003-01-01

145

An anti-HIV-1 V3 loop antibody fully protects cross-clade and elicits T-cell immunity in macaques mucosally challenged with an R5 clade C SHIV.  

PubMed

Neutralizing antibodies have been shown to protect macaques against SHIV challenge. However, genetically diverse HIV-1 clades have evolved, and a key question left unanswered is whether neutralizing antibodies can confer cross-clade protection in vivo. The novel human monoclonal antibody HGN194 was isolated from an individual infected with an HIV-1 clade AG recombinant circulating recombinant form (CRF). HGN194 targets an epitope in the third hypervariable loop (V3) of HIV-1 gp120 and neutralizes a range of relatively neutralization-sensitive and resistant viruses. We evaluated the potential of HGN194 to protect infant rhesus monkeys against a SHIV encoding a primary CCR5-tropic HIV-1 clade C envelope. After high-dose mucosal challenge, all untreated controls became highly viremic while all HGN194-treated animals (50 mg/kg) were completely protected. When HGN194 was given at 1 mg/kg, one out of two monkeys remained aviremic, whereas the other had delayed, lower peak viremia. Interestingly, all protected monkeys given high-dose HGN194 developed Gag-specific proliferative responses of both CD4+ and CD8+ T cells. To test whether generation of the latter involved cryptic infection, we ablated CD8+ cells after HGN194 clearance. No viremia was detected in any protected monkeys, thus ruling out virus reservoirs. Thus, induction of CD8 T-cell immunity may have resulted from transient "Hit and Run" infection or cross priming via Ag-Ab-mediated cross-presentation. Together, our data identified the HGN194 epitope as protective and provide proof-of-concept that this anti-V3 loop mAb can prevent infection with sterilizing immunity after challenge with virus of a different clade, implying that V3 is a potential vaccine target. PMID:21483815

Watkins, Jennifer D; Siddappa, Nagadenahalli B; Lakhashe, Samir K; Humbert, Michael; Sholukh, Anton; Hemashettar, Girish; Wong, Yin Ling; Yoon, John K; Wang, Wendy; Novembre, Francis J; Villinger, Francois; Ibegbu, Chris; Patel, Kalpana; Corti, Davide; Agatic, Gloria; Vanzetta, Fabrizia; Bianchi, Siro; Heeney, Jonathan L; Sallusto, Federica; Lanzavecchia, Antonio; Ruprecht, Ruth M

2011-03-31

146

Selective induction of cell-mediated immunity and protection of rhesus macaques from chronic SHIV{sub KU2} infection by prophylactic vaccination with a conserved HIV-1 envelope peptide-cocktail  

SciTech Connect

Infection of Indian-origin rhesus macaques by the simian human immunodeficiency virus (SHIV) is considered to be a suitable preclinical model for directly testing efficacy of vaccine candidates based on the HIV-1 envelope. We used this model for prophylactic vaccination with a peptide-cocktail comprised of highly conserved HIV-1 envelope sequences immunogenic/antigenic in macaques and humans. Separate groups of macaques were immunized with the peptide-cocktail by intravenous and subcutaneous routes using autologous dendritic cells (DC) and Freund's adjuvant, respectively. The vaccine elicited antigen specific IFN-{gamma}-producing cells and T-cell proliferation, but not HIV-neutralizing antibodies. The vaccinated animals also exhibited efficient cross-clade cytolytic activity against target cells expressing envelope proteins corresponding to HIV-1 strains representative of multiple clades that increased after intravenous challenge with pathogenic SHIV{sub KU2}. Virus-neutralizing antibodies were either undetectable or present only transiently at low levels in the control as well as vaccinated monkeys after infection. Significant control of plasma viremia leading to undetectable levels was achieved in majority of vaccinated monkeys compared to mock-vaccinated controls. Monkeys vaccinated with the peptide-cocktail using autologous DC, compared to Freund's adjuvant, and the mock-vaccinated animals, showed significantly higher IFN-{gamma} production, higher levels of vaccine-specific IFN-{gamma} producing CD4{sup +} cells and significant control of plasma viremia. These results support DC-based vaccine delivery and the utility of the conserved HIV-1 envelope peptide-cocktail, capable of priming strong cell-mediated immunity, for potential inclusion in HIV vaccination strategies.

Nehete, Pramod N.; Nehete, Bharti P.; Hill, Lori [Department of Veterinary Sciences, University of Texas M. D. Anderson Cancer Center, Bastrop, TX 78602 (United States); Manuri, Pallavi R. [Department of Immunology, University of Texas M. D. Anderson Cancer Center, 7455 Fannin, Houston, TX 77030 (United States); Baladandayuthapani, Veerabhadran; Feng Lei [Department of Biostatistics, University of Texas M. D. Anderson Cancer Center, 7455 Fannin, Houston, TX 77030 (United States); Simmons, Johnny [Department of Veterinary Sciences, University of Texas M. D. Anderson Cancer Center, Bastrop, TX 78602 (United States); Sastry, K. Jagannadha [Department of Veterinary Sciences, University of Texas M. D. Anderson Cancer Center, Bastrop, TX 78602 (United States); Department of Immunology, University of Texas M. D. Anderson Cancer Center, 7455 Fannin, Houston, TX 77030 (United States)], E-mail: jsastry@mdanderson.org

2008-01-05

147

A Nonfucosylated Variant of the anti-HIV-1 Monoclonal Antibody b12 Has Enhanced Fc?RIIIa-Mediated Antiviral Activity In Vitro but Does Not Improve Protection against Mucosal SHIV Challenge in Macaques  

PubMed Central

Eliciting neutralizing antibodies is thought to be a key activity of a vaccine against human immunodeficiency virus (HIV). However, a number of studies have suggested that in addition to neutralization, interaction of IgG with Fc gamma receptors (Fc?R) may play an important role in antibody-mediated protection. We have previously obtained evidence that the protective activity of the broadly neutralizing human IgG1 anti-HIV monoclonal antibody (MAb) b12 in macaques is diminished in the absence of Fc?R binding capacity. To investigate antibody-dependent cellular cytotoxicity (ADCC) as a contributor to Fc?R-associated protection, we developed a nonfucosylated variant of b12 (NFb12). We showed that, compared to fully fucosylated (referred to as wild-type in the text) b12, NFb12 had higher affinity for human and rhesus macaque Fc?RIIIa and was more efficient in inhibiting viral replication and more effective in killing HIV-infected cells in an ADCC assay. Despite these more potent in vitro antiviral activities, NFb12 did not enhance protection in vivo against repeated low-dose vaginal challenge in the simian-human immunodeficiency virus (SHIV)/macaque model compared to wild-type b12. No difference in protection, viral load, or infection susceptibility was observed between animals given NFb12 and those given fully fucosylated b12, indicating that Fc?R-mediated activities distinct from Fc?RIIIa-mediated ADCC may be important in the observed protection against SHIV challenge.

Moldt, Brian; Shibata-Koyama, Mami; Rakasz, Eva G.; Schultz, Niccole; Kanda, Yutaka; Dunlop, D. Cameron; Finstad, Samantha L.; Jin, Chenggang; Landucci, Gary; Alpert, Michael D.; Dugast, Anne-Sophie; Parren, Paul W. H. I.; Nimmerjahn, Falk; Evans, David T.; Alter, Galit; Forthal, Donald N.; Schmitz, Jorn E.; Iida, Shigeru; Poignard, Pascal; Watkins, David I.

2012-01-01

148

qRT-PCR of Small RNAs.  

PubMed

Plant small RNAs are a class of 19- to 25-nucleotide (nt) RNA molecules that are essential for genome stability, development and differentiation, disease, cellular communication, signaling, and adaptive responses to biotic and abiotic stress. Small RNAs comprise two major RNA classes, short interfering RNAs (siRNAs) and microRNAs (miRNAs). Efficient and reliable detection and quantification of small RNA expression has become an essential step in understanding their roles in specific cells and tissues. Here we provide protocols for the detection of miRNAs by stem-loop RT-PCR. This method enables fast and reliable miRNA expression profiling from as little as 20 pg of total RNA extracted from plant tissue and is suitable for high-throughput miRNA expression analysis. In addition, this method can be used to detect other classes of small RNAs, provided the sequence is known and their GC contents are similar to those specific for miRNAs. PMID:20204872

Varkonyi-Gasic, Erika; Hellens, Roger P

2010-01-01

149

Enhancing Transport of Hydrogenophagaflava ENV735 for Bioaugmentation of Aquifers Contaminated with Methyl tert-Butyl Ether  

PubMed Central

The gasoline oxygenate methyl tert-butyl ether (MTBE) has become a widespread contaminant in groundwater throughout the United States. Bioaugmentation of aquifers with MTBE-degrading cultures may be necessary to enhance degradation of the oxygenate in some locations. However, poor cell transport has sometimes limited bioaugmentation efforts in the past. The objective of this study was to evaluate the transport characteristics of Hydrogenophaga flava ENV735, a pure culture capable of growth on MTBE, and to improve movement of the strain through aquifer solids. The wild-type culture moved only a few centimeters in columns of aquifer sediment. An adhesion-deficient variant (H. flava ENV735:24) of the wild-type strain that moved more readily through sediments was obtained by sequential passage of cells through columns of sterile sediment. Hydrophobic and electrostatic interaction chromatography revealed that the wild-type strain is much more hydrophobic than the adhesion-deficient variant. Electrophoretic mobility assays and transmission electron microscopy showed that the wild-type bacterium contains two distinct subpopulations, whereas the adhesion-deficient strain has only a single, homogeneous population. Both the wild-type strain and adhesion-deficient variant degraded MTBE, and both were identified by 16S rRNA analysis as pure cultures of H. flava. The effectiveness of surfactants for enhancing transport of the wild-type strain was also evaluated. Many of the surfactants tested were toxic to ENV735; however, one nonionic surfactant, Tween 20, enhanced cell transport in sand columns. Improving microbial transport may lead to a more effective bioaugmentation strategy for MTBE-contaminated sites where indigenous oxygenate degraders are absent.

Streger, Sheryl H.; Vainberg, Simon; Dong, Hailiang; Hatzinger, Paul B.

2002-01-01

150

Interaction between two regulatory proteins in osmoregulatory expression of ompF and ompC genes in Escherichia coli: a novel ompR mutation suppresses pleiotropic defects caused by an envZ mutation.  

PubMed Central

The ompR and envZ genes, which together constitute the ompB operon, are involved in osmoregulatory expression of the OmpF and OmpC proteins, major outer membrane proteins of Escherichia coli. The envZ11 mutation results in the OmpF- OmpC-constitutive phenotype. A mutant which suppressed defects caused by the envZ11 mutation was isolated. The suppressor mutation also suppressed the LamB- PhoA- phenotype caused by the envZ11 mutation. The mutation occurred in the ompR gene and hence was termed ompR77. The ompR77 mutation alone produced no obvious phenotype. Functioning of the ompR77 allele remained envZ gene dependent. Although the ompR77 mutation suppressed the envZ11 mutation, it did not suppress a mutation that occurred in another position within the envZ gene (envZ160). These results indicate that OmpR and EnvZ, two regulatory proteins, functionally interact with each other. Images

Matsuyama, S; Mizuno, T; Mizushima, S

1986-01-01

151

The conserved His8 of the Moloney murine leukemia virus Env SU subunit directs the activity of the SU-TM disulphide bond isomerase  

SciTech Connect

Murine leukemia virus (MLV) fusion is controlled by isomerization of the disulphide bond between the receptor-binding surface (SU) and fusion-active transmembrane subunits of the Env-complex. The bond is in SU linked to a CXXC motif. This carries a free thiol that upon receptor binding can be activated (ionized) to attack the disulphide and rearrange it into a disulphide isomer within the motif. To find out whether His8 in the conserved SPHQ sequence of Env directs thiol activation, we analyzed its ionization in MLV vectors with wtEnv and Env with His8 deleted or substituted for Tyr or Arg, which partially or completely arrests fusion. The ionization was monitored by following the pH effect on isomerization in vitro by Ca{sup 2+} depletion or in vivo by receptor binding. We found that wtEnv isomerized optimally at slightly basic pH whereas the partially active mutant required higher and the inactive mutants still higher pH. This suggests that His8 directs the ionization of the CXXC thiol.

Li Kejun [Department of Biosciences and Nutrition, Karolinska Institute, S-141 57 HUDDINGE (Sweden); Zhang, Shujing [Department of Biosciences and Nutrition, Karolinska Institute, S-141 57 HUDDINGE (Sweden); Kronqvist, Malin [Department of Biosciences and Nutrition, Karolinska Institute, S-141 57 HUDDINGE (Sweden); Ekstroem, Maria [Department of Biosciences and Nutrition, Karolinska Institute, S-141 57 HUDDINGE (Sweden); Wallin, Michael [Department of Biosciences and Nutrition, Karolinska Institute, S-141 57 HUDDINGE (Sweden); Garoff, Henrik [Department of Biosciences and Nutrition, Karolinska Institute, S-141 57 HUDDINGE (Sweden)]. E-mail: henrik.garoff@cbt.ki.se

2007-04-25

152

HIV1 reverse transcriptase mutations that confer decreased in vitro susceptibility to anti-RT DNA aptamer RT1t49 confer cross resistance to other anti-RT aptamers but not to standard RT inhibitors  

Microsoft Academic Search

RNA and DNA aptamers specific for HIV-1 reverse transcriptase (RT) can inhibit reverse transcription in vitro. RNA aptamers have been shown to potently block HIV-1 replication in culture. We previously reported mutants of HIV-1 RT with substitutions N255D or N265D that display resistance to the DNA aptamer RT1t49. Variant viruses bearing these mutations singly or in combination were compromised for

Timothy S Fisher; Pheroze Joshi; Vinayaka R Prasad

2005-01-01

153

Variability of the env gene in cynomolgus macaques persistently infected with human immunodeficiency virus type 2 strain ben.  

PubMed Central

The sequence variability of distinct regions of the proviral env gene of human immunodeficiency virus type 2 strain ben (HIV-2ben) isolated sequentially over 3 to 4 years from six experimentally infected macaques was studied. The regions investigated were homologous to the V1, V2, V3, V4, V5, and V7 hypervariable regions identified in the env genes of HIV-1 and simian immunodeficiency virus SIVmac, respectively. In contrast to findings with HIV-1 and SIVmac, the V1- and V2-homologous regions were found to be highly conserved during the course of the HIV-2ben infection in macaques. The V3-homologous region showed a degree of variation comparable to that of HIV-1 but not of SIV. In the V4-, V5-, and V7-homologous regions, mutation hot spots were detected in most reisolates of the infected monkeys. Most of these mutations occurred during the first 10 weeks after infection. After 50 weeks, new mutations were rarely detected. At most mutation sites, a dynamic equilibrium between the mutated viral isotype and the infecting predominant wild type was present. This equilibrium might prevent an accumulation of mutations in isolates later in the course of infection.

Tolle, T; Petry, H; Bachmann, B; Hunsmann, G; Luke, W

1994-01-01

154

N-terminally myristoylated feline foamy virus Gag allows Env-independent budding of sub-viral particles.  

PubMed

Foamy viruses (FVs) are distinct retroviruses classified as Spumaretrovirinae in contrast to the other retroviruses, the Orthoretrovirinae. As a unique feature of FVs, Gag is not sufficient for sub-viral particle (SVP) release. In primate and feline FVs (PFV and FFV), particle budding completely depends on the cognate FV Env glycoproteins. It was recently shown that an artificially added N-terminal Gag myristoylation signal (myr-signal) overcomes this restriction in PFV inducing an Orthoretrovirus-like budding phenotype. Here we show that engineered, heterologous N-terminal myr-signals also induce budding of the distantly related FFV Gag. The budding efficiency depends on the myr-signal and its location relative to the N-terminus of Gag. When the first nine amino acid residues of FFV Gag were replaced by known myr-signals, the budding efficiency as determined by the detection of extracellular SVPs was low. In contrast, adding myr-signals to the intact N-terminus of FFV Gag resulted in a more efficient SVP release. Importantly, budding of myr-Gag proteins was sensitive towards inhibition of cellular N-myristoyltransferases. As expected, the addition or insertion of myr-signals that allowed Env-independent budding of FFV SVPs also retargeted Gag to plasma membrane-proximal sites and other intracellular membrane compartments. The data confirm that membrane-targeted FV Gag has the capacity of SVP formation. PMID:22163342

Liu, Yang; Kim, Yong-Boum; Löchelt, Martin

2011-11-14

155

Detection and Quantitation of HTLV-1 and HTLV-2 mRNA Species by Real-time RT-PCR  

PubMed Central

Summary HTLV-1 and HTLV-2 are highly related delta-retroviruses that infect and transform T-lymphocytes, but have distinct pathogenic properties. HTLV replication and survival requires the expression of multiple gene products from an unspliced and a series of highly related alternatively spliced mRNA species. To date, the comparative levels of all known HTLV-1 and HTLV-2 viral mRNAs in different transformed cell lines and at different stages of virus infection have not been assessed. In this study, we compiled a series of oligonucleotide primer pairs and probes to quantify both HTLV-1 and HTLV-2 mRNA species using real-time RT-PCR. The optimized reaction for detection of each mRNA had amplification efficiency greater than 90% with a linear range spanning 25 to 2.5 × 107 copies. The R2s of all standard curves were greater than 0.97. Quantitation of HTLV mRNAs between different cell lines showed variability (gag/pol ? tax/rex > env ? accessory proteins), but the overall levels of each mRNA relative to each other within a cell line were similar. These results provide a method to quantify all specific mRNAs from both HTLV-1 and HTLV-2, which can be used to evaluate further viral gene expression and correlate transcript levels to key stages of the virus life cycle and ultimately, pathogenesis.

Li, Min; Green, Patrick L.

2007-01-01

156

T-Cell Immune Responses Against Env from CRF12_BF and Subtype B HIV-1 Show High Clade-Specificity that Can Be Overridden by Multiclade Immunizations  

PubMed Central

Background The extreme genetic diversity of the human immunodeficiency virus type 1 (HIV-1) poses a daunting challenge to the generation of an effective AIDS vaccine. In Argentina, the epidemic is characterized by the high prevalence of infections caused by subtype B and BF variants. The aim of this study was to characterize in mice the immunogenic and antigenic properties of the Env protein from CRF12_BF in comparison with clade B, employing prime-boost schemes with the combination of recombinant DNA and vaccinia virus (VV) vectors. Methodology/Principal Findings As determined by ELISPOT from splenocytes of animals immunized with either EnvBF or EnvB antigens, the majority of the cellular responses to Env were found to be clade-specific. A detailed peptide mapping of the responses reveal that when there is cross-reactivity, there are no amino acid changes in the peptide sequence or were minimal and located at the peptide ends. In those cases, analysis of T cell polifunctionality and affinity indicated no differences with respect to the cellular responses found against the original homologous sequence. Significantly, application of a mixed immunization combining both clades (B and BF) induced a broader cellular response, in which the majority of the peptides targeted after the single clade vaccinations generated a positive response. In this group we could also find significant cellular and humoral responses against the whole gp120 protein from subtype B. Conclusions/Significance This work has characterized for the first time the immunogenic peptides of certain EnvBF regions, involved in T cell responses. It provides evidence that to improve immune responses to HIV there is a need to combine Env antigens from different clades, highlighting the convenience of the inclusion of BF antigens in future vaccines for geographic regions where these HIV variants circulate.

Monaco, Daniela C.; Rodriguez, Ana M.; Pascutti, Maria F.; Carobene, Mauricio; Falivene, Juliana; Gomez, Alejandro; Maeto, Cynthia; Turk, Gabriela; Najera, Jose L.; Esteban, Mariano; Gherardi, M. Magdalena

2011-01-01

157

A Study of Low pH-Induced Refolding of Env of Avian Sarcoma and Leukosis Virus into a Six-Helix Bundle  

PubMed Central

The fusion protein of avian sarcoma and leukosis virus is likely to fold into a six-helix bundle as part of its final configuration. A peptide, R99, inhibits fusion, probably by binding into the grooves of the triple-stranded coiled coil that becomes the central core of the six-helix bundle. The stages at which the envelope protein (Env) of avian sarcoma and leukosis virus subgroup A folds into a bundle during low pH-induced fusion were determined. Effector cells expressing Env were bound to target cells expressing the cognate receptor Tva, and intermediates of fusion were created. R99 was added and the extent of fusion inhibition was used to distinguish between a prebundle state with exposed grooves and a state in which the grooves were no longer exposed. The native conformation of Env was not sensitive to R99. But adding a soluble form of Tva to effector cells conferred sensitivity. Acidic pH applied at low temperature created an intermediate state of local hemifusion. Surprisingly, R99 caused these locally hemifused membranes to separate. This indicates that the grooves of Env were still exposed, that prebundle configurations of Env stabilized hemifused states, and that binding of R99 altered the conformation of Env. In the presence of an inhibitory lipid that blocks fusion before hemifusion, applying low pH at 37°C created an intermediate in which R99 was without effect. This suggests that the six-helix bundle can form before hemifusion and that subsequent conformational changes, such as formation of the trimeric hairpin, are responsible for pore formation and/or growth.

Markosyan, R. M.; Bates, P.; Cohen, F. S.; Melikyan, G. B.

2004-01-01

158

Role of the histidine kinase, EnvZ, in the production of outer membrane proteins in the symbiotic-pathogenic bacterium Xenorhabdus nematophilus.  

PubMed Central

We show that inactivation of envZ, the gene encoding the histidine kinase sensor protein, EnvZ, of Xenorhabdus nematophilus, affected the production of several outer membrane proteins (Opns). X. nematophilus produced five major Opns during exponential growth. Insertional inactivation of envZ led to a decrease in the production of OpnP, the OmpF-like pore-forming protein which constitutes approximately 50% of the total outer membrane protein in X. nematophilus. OpnA production was also reduced, while the remaining Opns were produced normally. During the transition to stationary phase, three new outer membrane proteins, OpnB, OpnS, and OpnX, were induced in the wild-type strain. The envZ-minus strain, ANT1, did not produce OpnB and OpnX, while OpnS was induced at markedly reduced levels. These results suggest that EnvZ was required for the high-level production of OpnP during exponential growth and may be involved in the production of OpnB, OpnS, and OpnX during stationary-phase growth. We also show that ANT1 was more pathogenic than the wild-type strain when as few as five cells were injected into the hemolymph of the larval stage of the tobacco hornworm (Manduca sexta). The larvae died before significant numbers of bacteria were detectable in the hemolymph. These results are discussed in relation to the role of EnvZ in the life cycle of X. nematophilus.

Forst, S A; Tabatabai, N

1997-01-01

159

Estimating Energy Expenditure with the RT3 Triaxial Accelerometer  

ERIC Educational Resources Information Center

|The RT3 is a relatively new triaxial accelerometer that has replaced the TriTrac. The aim of this study was to validate the RT3 against doubly labeled water (DLW) in a free-living, mixed weight sample of adults. Total energy expenditure (TEE) was measured over a 15-day period using DLW. Activity-related energy expenditure (AEE) was estimated by…

Maddison, Ralph; Jiang, Yannan; Vander Hoorn, Stephen; Mhurchu, Cliona Ni; Lawes, Carlene M. M.; Rodgers, Anthony; Rush, Elaine

2009-01-01

160

Response to Intervention (RtI) in Secondary Schools: A Comparison of the RtI Service Delivery Model  

ERIC Educational Resources Information Center

This qualitative, collective case study researched how the Response to Intervention (RtI) service delivery model was used within the secondary educational environment in two Ohio schools. Areas researched included the type of professional development used to introduce and sustain RtI, the amount of administrative support, the use of universal…

Epler-Brooks, Pam L.

2011-01-01

161

An M?-Containing Heterologous RNA, but Not env mRNA, Is Efficiently Packaged into Avian Retroviral Particles  

PubMed Central

Retroviruses preferentially package full-length genomic RNA over spliced viral messages. For most retroviruses, this preference is likely due to the absence of all or part of the packaging signal on subgenomic RNAs. In avian leukosis-sarcoma virus, however, we have shown that the minimal packaging signal, M?, is located upstream of the 5? splice site and therefore is present on both genomic and spliced RNAs. We now show that an M?-containing heterologous RNA is packaged only 2.6-fold less efficiently than genomic Rous sarcoma virus RNA. Thus, few additional packaging sequences and/or structures exist outside of M?. In contrast, we found that env mRNA is not efficiently packaged. These results indicate that either M? is not functional on this RNA or the RNA is somehow segregated from the packaging machinery. Finally, deletion of sequences from the 3? end of M? was found to reduce the packaging efficiency of heterologous RNAs.

Banks, Jennifer D.; Kealoha, Bonnie O.; Linial, Maxine L.

1999-01-01

162

UltraQual HCV RT-PCR Assay  

Center for Biologics Evaluation and Research (CBER)

The NGI UltraQual™ Hepatitis C Virus (HCV) Reverse Transcription (RT) Polymerase Chain Reaction (PCR) assay, when used in combination with ... More results from www.fda.gov/biologicsbloodvaccines/bloodbloodproducts/approvedproducts

163

Mutations in the env gene of friend spleen focus-forming virus overcome Fv-2r-mediated resistance to Friend virus-induced erythroleukemia.  

PubMed Central

Although Fv-2r homozygous mice are resistant to leukemias induced either by an erythropoietin-encoding virus or by wild-type Friend virus (FV) (M. E. Hoatlin, S. L. Kozak, F. Lilly, A. Chakraborti, C. A. Kozak, and D. Kabat, Proc. Natl. Acad. Sci. USA 87:9985-9989, 1990), they are susceptible to some variants of FV (R. A. Steeves, E. A. Mirand, A. Bulba, and P. J. Trudel, Int. J. Cancer 5:349-356, 1970; R. W. Geib, M. B. Seaward, M. L. Stevens, C.-L. Cho, and M. Majumdar, Virus Res. 14:161-174, 1989). To localize the virus gene involved in influencing the host range, we cloned and sequenced the env gene of the BB6 variant of FV (Steeves et al., Int. J. Cancer 5:349-356, 1970). In comparison with the wild-type env gene, the BB6 variant contains a 159-bp deletion that eliminates the membrane-proximal portion of the extracellular domain and 58 point mutations resulting in 13 amino acid changes. Substitution of the variant env gene for the wild-type env gene resulted in a recombinant virus that produced a Friend virus-like disease in Fv-2r homozygotes. Our results identify the spleen focus-forming virus env gene as the viral gene involved in this virus-host interaction. Additionally, they suggest that the product of the Fv-2r gene modifies the interaction between the spleen focus-forming virus envelope protein and the erythropoietin receptor. Images

Majumdar, M K; Cho, C L; Fox, M T; Eckner, K L; Kozak, S; Kabat, D; Geib, R W

1992-01-01

164

HIV-1 Env C2-V4 Diversification in a Slow-Progressor Infant Reveals a Flat but Rugged Fitness Landscape  

PubMed Central

Human immunodeficiency virus type-1 (HIV-1) fitness has been associated with virus entry, a process mediated by the envelope glycoprotein (Env). We previously described Env genetic diversification in a Zambian, subtype C infected, slow-progressor child (1157i) in parallel with an evolving neutralizing antibody response. Because of the role the Variable-3 loop (V3) plays in transmission, cell tropism, neutralization sensitivity, and fitness, longitudinally isolated 1157i C2-V4 alleles were cloned into HIV-1NL4-3-eGFP and -DsRed2 infectious molecular clones. The fluorescent reporters allowed for dual-infection competitions between all patient-derived C2-V4 chimeras to quantify the effect of V3 diversification and selection on fitness. ‘Winners’ and ‘losers’ were readily discriminated among the C2-V4 alleles. Exceptional sensitivity for detection of subtle fitness differences was revealed through analysis of two alleles differing in a single synonymous amino acid. However, when the outcomes of N?=?33 competitions were averaged for each chimera, the aggregate analysis showed that despite increasing diversification and divergence with time, natural selection of C2-V4 sequences in this individual did not appear to be producing a ‘survival of the fittest’ evolutionary pattern. Rather, we detected a relatively flat fitness landscape consistent with mutational robustness. Fitness outcomes were then correlated with individual components of the entry process. Env incorporation into particles correlated best with fitness, suggesting a role for Env avidity, as opposed to receptor/coreceptor affinity, in defining fitness. Nevertheless, biochemical analyses did not identify any step in HIV-1 entry as a dominant determinant of fitness. Our results lead us to conclude that multiple aspects of entry contribute to maintaining adequate HIV-1 fitness, and there is no surrogate analysis for determining fitness. The capacity for subtle polymorphisms in Env to nevertheless significantly impact viral fitness suggests fitness is best defined by head-to-head competition.

Smith, S. Abigail; Wood, Charles; West, John T.

2013-01-01

165

Priming with Recombinant Auxotrophic BCG Expressing HIV-1 Gag, RT and Gp120 and Boosting with Recombinant MVA Induces a Robust T Cell Response in Mice.  

PubMed

In previous studies we have shown that a pantothenate auxotroph of Myocbacterium bovis BCG (BCG?panCD) expressing HIV-1 subtype C Gag induced Gag-specific immune responses in mice and Chacma baboons after prime-boost immunization in combination with matched rMVA and VLP vaccines respectively. In this study recombinant BCG (rBCG) expressing HIV-1 subtype C reverse transcriptase and a truncated envelope were constructed using both the wild type BCG Pasteur strain as a vector and the pantothenate auxotroph. Mice were primed with rBCG expressing Gag and RT and boosted with a recombinant MVA, expressing a polyprotein of Gag, RT, Tat and Nef (SAAVI MVA-C). Priming with rBCG?panCD expressing Gag or RT rather than the wild type rBCG expressing Gag or RT resulted in higher frequencies of total HIV-specific CD8(+) T cells and increased numbers of T cells specific to the subdominant Gag and RT epitopes. Increasing the dose of rBCG from 10(5) cfu to 10(7) cfu also led to an increase in the frequency of responses to subdominant HIV epitopes. A mix of the individual rBCG?panCD vaccines expressing either Gag, RT or the truncated Env primed the immune system for a boost with SAAVI MVA-C and generated five-fold higher numbers of HIV-specific IFN-?-spot forming cells than mice primed with rBCG?panCD containing an empty vector control. Priming with the individual rBCG?panCD vaccines or the mix and boosting with SAAVI MVA-C also resulted in the generation of HIV-specific CD4(+) and CD8(+) T cells producing IFN-? and TNF-? and CD4(+) cells producing IL-2. The rBCG vaccines tested in this study were able to prime the immune system for a boost with rMVA expressing matching antigens, inducing robust, HIV-specific T cell responses to both dominant and subdominant epitopes in the individual proteins when used as individual vaccines or in a mix. PMID:23977084

Chapman, Rosamund; Stutz, Helen; Jacobs, William; Shephard, Enid; Williamson, Anna-Lise

2013-08-20

166

Modeling and real-time diagnostics in TEAMS-RT  

Microsoft Academic Search

This article presents an overview of the architectural and algorithmic features of TEAMS-RT, a real-time fault-isolation tool in Qualtech Systems' suite of software tools for integrated diagnostics, system and maintenance. Following an introduction to the fault-modeling methodology underlying the algorithms, the approach used by TEAMS-RT to isolate faults based on received test results is presented. An overview of a data-preprocessing

Amit Mathur; Somnath Deb; Krishna R. Pattipati

1998-01-01

167

Towards Compositional Verification in MEDISTAM-RT Methodological Framework  

Microsoft Academic Search

In this paper, we present results ensuring the correct compositionality of the components (named capsules in UML-RT terminology)\\u000a of a real-time system at different specification stages using the methodological framework called MEDISTAM-RT, which guarantees\\u000a the temporal consistency and safe (deadlock free) communication between capsules. This allows the compositional verification\\u000a of systems designed with this methodology, in such a way that

Kawtar Benghazi Akhlaki; Miguel J. Hornos; Manuel Noguera

2009-01-01

168

f(R,T,RT) gravity phenomenology and ?CDM universe  

NASA Astrophysics Data System (ADS)

We propose general f(R,T,RT) theory as generalization of covariant Ho?ava-like gravity with dynamical Lorentz symmetry breaking. FRLW cosmological dynamics for several versions of such theory is considered. The reconstruction of the above action is explicitly done, including the numerical reconstruction for the occurrence of ?CDM universe. De Sitter universe solutions in the presence of non-constant fluid are also presented. The problem of matter instability in f(R,T,RT) gravity is discussed.

Odintsov, Sergei D.; Sáez-Gómez, Diego

2013-10-01

169

Measurement of RT amplitudes and wavelengths of laser driven plates  

SciTech Connect

A laser drive plate, that is a dense solid plate drive by a laser heated, lower density plasma, is inherently Raleigh-Taylor (R-T) unstable, We have previously indicated that observed surface perturbation on the plate are probably R-T instabilities, initiated by the mode structure of the driving laser beam. Using a semi- transparent impact target viewed with a polarized Epi-Illuminated Confocal Streak Microscope, has allowed us to measure the amplitude and growth of the instability.

Frank, A.M.; Gillespie, C.H.

1997-10-16

170

Towards a Calculus for UML-RT Specifications  

Microsoft Academic Search

The unified modeling language (UML) developed under the coordinationof the Object Management Group (OMG) is one of the most important standardsfor the specification and design of object-oriented systems. This standardis currently tuned for real-time applications in the form of a new proposal,UML for Real-Time (UML-RT), by Rational Software Corporation and ObjecTimeLimited. Because of the importance of UML-RT we are investigatingits

R. Grosu; M. Broy; B. Selic; Gh. Stefanescu

1998-01-01

171

Hyperthermic Fibrinolysis with rt-PA: In Vitro Results  

SciTech Connect

Purpose: To investigate the influence of hyperthermia up to 45 deg. C on fibrinolysis with recombinant tissue-type plasminogen activator (rt-PA). Methods: Standardized fibrin clots were incubated in a water bath for 5 hr with either rt-PA (test group) or 0.9% sodium chloride (control group) and blood plasma at temperatures of 30-45 deg. C. Concentrations of D-dimer and time to complete clot lysis were measured.Results: The activity of fibrinolysis with rt-PA rose with increasing temperature: time to lysis approximately halved from 30 deg. C to 40 deg. C and the concentration of D-dimer tripled. In the control group clot size did not change.Conclusions: Activity of rt-PA-induced fibrinolysis rises distinctly with higher temperatures. Since even healthy subjects show a physiologic decline in body temperature in the extremities, in patients with occlusive arterial disease decreased activity of fibrinolysis with rt-PA can be expected. Controlled hyperthermia may improve fibrinolysis with rt-PA and should be investigated in vivo.

Schwarzenberg, Helmut; Mueller-Huelsbeck, Stefan; Brossman, Joachim; Christian Glueer, Claus [Klinik fuer Radiologische Diagnostik, Christian-Albrechts-Universitaet zu Kiel, Arnold-Heller-Strasse 9, D-24105 Kiel (Germany); Bruhn, Hans Dieter [Klinik fuer Allgemeine Innere Medizin, Christian-Albrechts-Universitaet zu Kiel, Schittenhelmstrasse 12, D-24105 Kiel (Germany); Heller, Martin [Klinik fuer Radiologische Diagnostik, Christian-Albrechts-Universitaet zu Kiel, Arnold-Heller-Strasse 9, D-24105 Kiel (Germany)

1998-03-15

172

Monitoring of the Symbiotic Variable RT Cru Requested  

NASA Astrophysics Data System (ADS)

The symbiotic variable RT Cru brightened in hard x-rays. Dr. Jeno Sokoloski, Columbia University, requested AAVSO assistance in monitoring RT Cru both now and in the future to see if it is doing anything unusual in the optical. The Swift/BAT hard X-ray light curve shows RT Cru has apparently been gradually brightening over the past few years. Dr. Sokoloski writes: "The hard X-ray emission from RT Cru suggests that the accreting white dwarf is close to the Chandrasekhar limit (e.g., Luna and Sokoloski 2007, "The Nature of the Hard X-Ray-Emitting Symbiotic Star RT Cru") and that it is therefore a candidate supernova Type-Ia progenitor. Also, since it is a massive white dwarf accreting at a reasonably high rate, it is similar to T CrB - so why isn't it a recurrent nova?? Fast photometry (to look for CV-like flickering from the accretion disk) and optical spectroscopy would also be very interesting and could potentially help interpret the current hard X-ray brightening." Dr. Sokoloski requests time series photometry now for a few days, and then weekly or monthly observations for the forseeable future. Visual observations are also welcome. Finder charts with sequence may be created using the AAVSO Variable Star Plotter (http://www.aavso.org/vsp). Observations should be submitted to the AAVSO International Database. See full Alert Notice for more details and magnitudes.

Waagen, Elizabeth O.

2012-01-01

173

RT-PCR detection of HIV in Republic of Macedonia.  

PubMed

The aim of the study was to detect HIV RNA in seropositive patients using RT-PCR method and thus, to establish PCR methodology in the routine laboratory works. The total of 33 examined persons were divided in two groups: 1) 13 persons seropositive for HIV; and 2) 20 healthy persons - randomly selected blood donors that made the case control group. The subjects age was between 25 and 52 years (average 38,5). ELFA test for combined detection of HIV p24 antigen and anti HIV-1+2 IgG and ELISA test for detection of antibodies against HIV-1 and HIV-2, were performed for each examined person. RNA from the whole blood was extracted using a commercial kit based on salt precipitation. Detection of HIV RNA was performed using RT-PCR kit. Following nested PCR, the product was separated by electrophoresis in 1,5 % agarose gel. The result was scored positive if the band of 210bp was visible regardless of intensity. Measures of precaution were taken during all the steps of the work and HIV infected materials were disposed of accordingly. In the group of blood donors ELFA, ELISA and RT-PCR were negative. Assuming that prevalence of HIV infection is zero, the clinical specificity of RT-PCR is 100 %. The analytical specificity of RT-PCR method was tested against Hepatitis C and B, Human Papiloma Virus, Cytomegalovirus, Herpes Simplex Virus, Rubella Virus, Mycobacterium tuberculosis, Chlamydia trachomatis. None of these templates yielded amplicon. In the group of 13 seropositive persons, 33 samples were analyzed. HIV RNA was detected in 15 samples. ELISA and ELFA test were positive in all samples. Different aliquots of the samples were tested independently and showed the same results. After different periods of storing the RNA samples at -70 masculineC, RT-PCR reaction was identical to the one performed initially. The obtained amplicons were maintained frozen at -20 masculineC for a week and the subsequently performed electrophoresis was identical to the previous one. The reaction is fast, simple for manipulation; with low detection level of 60 IU/ml. RT-PCR needs a small amount of RNA, as well as a small volume of sample. HIV RNA was detected in different periods of time with different clinical presentations in patients, with or without antiretroviral therapy. RT-PCR method gives the opportunity for reliable determination of HIV-1 RNA with border of detection of 60 IU/ml. The test is reproducible and has high analytical and clinical specificity. PMID:19125707

Bosevska, Golubinka; Panovski, Nikola; Doki?, Eleni; Grunevska, Violeta

2008-11-01

174

Regional spread of HIV-1 M subtype B in middle-aged patients by random env-C2V4 region sequencing  

PubMed Central

A transmission cluster of HIV-1 M:B was identified in 11 patients with a median age of 52 (range 26–65) in North-East Germany by C2V4 region sequencing of the env gene of HIV-1, who—except of one—were not aware of any risky behaviour. The 10 male and 1 female patients deteriorated immunologically, according to their information made available, within 4 years after a putative HIV acquisition. Nucleic acid sequence analysis showed a R5 virus in all patients and in 7 of 11 a crown motif of the V3 loop, GPGSALFTT, which is found rarely. Analysis of formation of this cluster showed that there is still a huge discrepancy between awareness and behaviour regarding HIV transmission in middle-aged patients, and that a local outbreak can be detected by nucleic acid analysis of the hypervariable env region.

Sturmer, Martin; Zimmermann, Katrin; Fritzsche, Carlos; Reisinger, Emil; Doelken, Gottfried; Berger, Annemarie; Doerr, Hans W.; Eberle, Josef

2010-01-01

175

In vivo tumorigenesis by Jaagsiekte sheep retrovirus (JSRV) requires Y590 in Env TM, but not full length orfX open reading frame  

PubMed Central

Jaagsiekte retrovirus (JSRV) causes ovine pulmonary adenocarcinoma (OPA), a transmissible lung cancer of sheep. The envelope (Env) glycoprotein protein of JSRV functions as a dominant oncoprotein in vitro and in vivo. An SH2 binding domain (YXXM) in the cytoplasmic tail of the JSRV Env is one of the main determinants of viral transformation at least in vitro. In these studies, we report the first in vivo tests of site-specific mutants of JSRV in their natural host, the sheep. We show that, in vivo, JSRV21 with the cytoplasmic tail YXXM mutated to DXXM did not cause disease nor detectable infection, indicating that this motif is absolutely required for virus replication and possibly transformation in vivo. In contrast, mutation of the JSRV open reading frame orfX, for which no function has yet been attributed, did not alter the disease induced by JSRV21.

Cousens, Chris; Maeda, Naoyoshi; Murgia, Claudio; Dagleish, Mark P.; Palmarini, Massimo; Fan, Hung

2007-01-01

176

Simian-Human Immunodeficiency Virus Containing a Human Immunodeficiency Virus Type 1 Subtype-E Envelope Gene: Persistent Infection, CD4+ T-Cell Depletion, and Mucosal Membrane Transmission in Macaques  

PubMed Central

The envelope (env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) determines several viral properties (e.g., coreceptor usage, cell tropism, and cytopathicity) and is a major target of antiviral immune responses. Most investigations on env have been conducted on subtype-B viral strains, prevalent in North America and Europe. Our study aimed to analyze env genes of subtype-E viral strains, prevalent in Asia and Africa, with a nonhuman primate model for lentivirus infection and AIDS. To this end, we constructed a simian immunodeficiency virus/HIV-1 subtype-E (SHIV) recombinant clone by replacing the env ectodomain of the SHIV-33 clone with the env ectodomain from the subtype-E strain HIV-1CAR402, which was isolated from an individual in the Central African Republic. Virus from this recombinant clone, designated SHIV-E-CAR, replicated efficiently in macaque peripheral blood mononuclear cells. Accordingly, juvenile macaques were inoculated with cell-free SHIV-E-CAR by the intravenous or intravaginal route; virus replicated in these animals but did not produce hematological abnormalities. In an attempt to elicit the pathogenic potential of the recombinant clone, we serially passaged this viral clone via transfusion of blood and bone marrow through juvenile macaques to produce SHIV-E-P4 (fourth-passage virus). The serially passaged virus established productive infection and CD4+ T-cell depletion in juvenile macaques inoculated by either the intravenous or the intravaginal route. Determination of the coreceptor usage of SHIV-E-CAR and serially passaged SHIV-E-P4 indicated that both of these viruses utilized CXCR4 as a coreceptor. In summary, the serially passaged SHIV subtype-E chimeric virus will be important for studies aimed at developing a nonhuman primate model for analyzing the functions of subtype-E env genes in viral transmission and pathogenesis and for vaccine challenge experiments with macaques immunized with HIV-1 env antigens.

Himathongkham, Sunee; Halpin, Nancy S.; Li, Jinling; Stout, Michael W.; Miller, Christopher J.; Luciw, Paul A.

2000-01-01

177

Construction of Infectious Feline Foamy Virus Genomes: Cat Antisera Do Not Cross-Neutralize Feline Foamy Virus Chimera with Serotype-Specific Env Sequences  

Microsoft Academic Search

Full-length genomes of the feline foamy virus (FFV or FeFV) isolate FUV were constructed. DNA clone pFeFV-7 stably directed the expression of infectious FFV progeny virus indistinguishable from wild-type, uncloned FFV isolate FUV. The env and bel 1 genes of pFeFV-7 were substituted for by corresponding sequences of the FFV serotype 951 since previous studies implicated a defined part of

Motomi Zemba; Alexandra Alke; Jochen Bodem; Ingrid G. Winkler; Robert L. P. Flower; K.-I. Pfrepper; Hajo Delius; Rolf M. Flügel; Martin Löchelt

2000-01-01

178

Contribution of Vpu, Env, and Nef to CD4 Down-Modulation and Resistance of Human Immunodeficiency Virus Type 1Infected T Cells to Superinfection  

Microsoft Academic Search

Human immunodeficiency virus type 1 (HIV-1) utilizes Vpu, Env, and Nef to down-modulate its primary CD4 receptor from the cell surface, and this function seems to be critical for the pathogenesis of AIDS. The physiological relevance of CD4 down-modulation, however, is currently not well understood. In the present study, we analyzed the kinetics of CD4 down-modulation and the susceptibility of

Steffen Wildum; Michael Schindler; Jan Munch; Frank Kirchhoff

2006-01-01

179

Comparative Efficacy of Recombinant Modified Vaccinia Virus Ankara Expressing Simian Immunodeficiency Virus (SIV) Gag-Pol and\\/or Env in Macaques Challenged with Pathogenic SIV  

Microsoft Academic Search

Prior studies demonstrated that immunization of macaques with simian immunodeficiency virus (SIV) Gag-Pol and Env recombinants of the attenuated poxvirus modified vaccinia virus Ankara (MVA) provided protection from high levels of viremia and AIDS following challenge with a pathogenic strain of SIV (V. M. Hirsch et al., J. Virol. 70:3741-3752, 1996). This MVA-SIV recombinant expressed relatively low levels of the

ILNOUR OURMANOV; CHARLES R. BROWN; BERNARD MOSS; MILES CARROLL; LINDA WYATT; LIUOBOV PLETNEVA; SIMOY GOLDSTEIN; DAVID VENZON; VANESSA M. HIRSCH

2000-01-01

180

Human endogenous retrovirus (HERV)-W ENV and GAG proteins: Physiological expression in human brain and pathophysiological modulation in multiple sclerosis lesions  

Microsoft Academic Search

Antigen expression of a human endogenous retrovirus family, HERV-W, in normal human brain and multiple sclerosis lesions was\\u000a studied by immunohistochemistry by three independent groups. The HERV-W multicopy family was identified in human DNA from\\u000a the previously characterized multiple sclerosis-associated retroviral element (MSRV). A panel of antibodies against envelope\\u000a (ENV) and capsid (GAG) antigens was tested. A physiological expression of

Hervé Perron; Françoise Lazarini; Klemens Ruprecht; Christine Péchoux-Longin; Alain Créange; Nicole Battail-Poirot; Michel Jolivet; Jean-Luc Darlix; Peter Rieckmann; Thomas Arzberger; Jean-Jacques Hauw; Hans Lassmann

2005-01-01

181

HIV1 Neutralization Profile and Plant-Based Recombinant Expression of Actinohivin, an Env Glycan-Specific Lectin Devoid of T-Cell Mitogenic Activity  

Microsoft Academic Search

The development of a topical microbicide blocking the sexual transmission of HIV-1 is urgently needed to control the global HIV\\/AIDS pandemic. The actinomycete-derived lectin actinohivin (AH) is highly specific to a cluster of high-mannose-type glycans uniquely found on the viral envelope (Env). Here, we evaluated AH's candidacy toward a microbicide in terms of in vitro anti-HIV-1 activity, potential side effects,

Nobuyuki Matoba; Adam S. Husk; Brian W. Barnett; Michelle M. Pickel; Charles J. Arntzen; David C. Montefiori; Atsushi Takahashi; Kazunobu Tanno; Satoshi Omura; Huyen Cao; Jason P. Mooney; Carl V. Hanson; Haruo Tanaka; Cheryl A. Stoddart

2010-01-01

182

Time-resolved Imaging of HIV1 Env-mediated Lipid and Content Mixing between a Single Virion and Cell Membrane  

Microsoft Academic Search

A method has been developed to follow fusion of individual pseudotyped virus expressing HIV-1 Env to cells by time-resolved fluorescence microscopy. Viral envelopes were labeled with a fluorescent lipid dye (DiD) and virus content was rendered visible by incorporating a Gag-GFP chimera. The Gag-GFP is naturally cleaved to the much smaller NC-GFP fragment in the mature virions. NC-GFP was readily

Ruben M. Markosyan; Fredric S. Cohen; Grigory B. Melikyan

2005-01-01

183

Identification of gp120 Binding Sites on CXCR4 by Using CD4-Independent Human Immunodeficiency Virus Type 2 Env Proteins  

PubMed Central

Human immunodeficiency virus (HIV) and simian (SIV) immunodeficiency virus entry is mediated by binding of the viral envelope glycoprotein (Env) to CD4 and chemokine receptors, CCR5 and/or CXCR4. CD4 induces extensive conformational changes that expose and/or induce formation of a chemokine receptor binding site on gp120. CD4-independent Env's of HIV type 1 (HIV-1), HIV-2, and SIV have been identified that exhibit exposed chemokine receptor binding sites and can bind directly to CCR5 or CXCR4 in the absence of CD4. While many studies have examined determinants for gp120-CCR5 binding, analysis of gp120-CXCR4 binding has been hindered by the apparently lower affinity of this interaction for X4-tropic HIV-1 isolates. We show here that gp120 proteins from two CD4-independent HIV-2 Env's, VCP and ROD/B, bind directly to CXCR4 with an apparently high affinity. By use of CXCR4 N-terminal deletion constructs, CXCR4-CXCR2 chimeras, and human-rat CXCR4 chimeras, binding determinants were shown to reside in the amino (N) terminus, extracellular loop 2 (ECL2), and ECL3. Alanine-scanning mutagenesis of charged residues, tyrosines, and phenylalanines in extracellular CXCR4 domains implicated multiple amino acids in the N terminus (E14/E15, D20, Y21, and D22), ECL2 (D187, R188, F189, Y190, and D193), and ECL3 (D262, E268, E277, and E282) in binding, although minor differences were noted between VCP and ROD/B. However, mutations in CXCR4 that markedly reduced binding did not necessarily hinder cell-cell fusion by VCP or ROD/B, especially in the presence of CD4. These gp120 proteins will be useful in dissecting determinants for CXCR4 binding and Env triggering and in evaluating pharmacologic inhibitors of the gp120-CXCR4 interaction.

Lin, George; Baribaud, Frederic; Romano, Josephine; Doms, Robert W.; Hoxie, James A.

2003-01-01

184

Endogenous Retroviral env Genes after TV-Methyl-W-nitro-TV-nitrosoguanidine Treatment of Mouse Tumor Cells: Stable DNA Amplification and Rearrangement1  

Microsoft Academic Search

Immunogenic tumor variant clones derived by yV-methyl-\\/V'-nitro-yV- nitrosoguanidine treatment of Eb lymphoma cells showed structurally altered gp70 env proteins at the cell surface. To further investigate this observation we screened for complementary DNA clones encoding gp70 antigens from a Xgtll expression library constructed from niRNA of a mutant cell clone. Using gp70-specific antibodies, a total of 10 comple mentary DNA

Doris Apt; Jeanette Schreck; Peter Altevogt

185

OmpR and EnvZ are pleiotropic regulatory proteins: Positive regulation of the tripeptide permease ( tppB ) of Salmonella typhimurium  

Microsoft Academic Search

The tppB locus of Salmonella typhimurium encodes the anaerobically-induced tripeptide permease. We have demonstrated that expression of tppB requires the function of the ompR and envZ gene products, originally identified as positive regulatory proteins required for the osmotic regulation of porin expression. Significantly, tppB expression is not osmotically regulated. We have also identified three additional genes whose expression depends on

M. M. Gibson; E. M. Ellis; K. A. Graeme-Cook; C. F. Higgins

1987-01-01

186

Cell-surface expression of CD4 reduces HIV1 infectivity by blocking Env incorporation in a Nef and Vpu-inhibitable manner  

Microsoft Academic Search

Background: Human immunodeficiency virus-1 (HIV-1) infection decreases the cell-surface expression of its cellular receptor, CD4, through the combined actions of Nef, Env and Vpu. Such functional convergence strongly suggests that CD4 downregulation is critical for optimal viral replication, yet the significance of this phenomenon has so far remained a puzzle.Results: We show that high levels of CD4 on the surface

Juan Lama; Aram Mangasarian; Didier Trono

1999-01-01

187

A Mouse Mammary Tumor Virus env-Like Exogenous Sequence Is Strictly Related to Progression of Human Sporadic Breast Carcinoma  

PubMed Central

A viral etiology of human breast cancer (HBC) has been postulated for decades since the identification of mouse mammary tumor virus (MMTV). The detection of MMTV env-like exogenous sequences (MMTVels) in 30% to 40% of invasive HBCs increased attention to this hypothesis. Looking for MMTVels during cancer progression may contribute to a better understanding of their role in HBC. Herein, we analyzed HBC preinvasive lesions for the presence of MMTVels. Samples were obtained by laser microdissection of FFPE tissues: 20 usual-type ductal hyperplasias, 22 atypical ductal hyperplasias (ADHs), 49 ductal carcinomas in situ (DCISs), 20 infiltrating ductal carcinomas (IDCs), and 26 normal epithelial cells collateral to a DCIS or an IDC. Controls included reductive mammoplastic tissue, thyroid and colon carcinoma, and blood samples from healthy donors. MMTVels were detected by fluorescence-nested PCR. DNA samples from the tissues of nine patients were analyzed by real-time quantitative PCR, revealing a different viral load correlated with stage of progression. Furthermore, as never previously described, the presence of MMTVels was investigated by chromogenic in situ hybridization. MMTVels were found in 19% of normal epithelial cells collateral to a DCIS or an IDC, 27% of ADHs, 82% of DCISs, and 35% of IDCs. No MMTVels were found in the control samples. Quantitative PCR and chromogenic in situ hybridization confirmed these results. These data could contribute to our understanding of the role of MMTVels in HBC.

Mazzanti, Chiara Maria; Al Hamad, Mohammad; Fanelli, Giovanni; Scatena, Cristian; Zammarchi, Francesca; Zavaglia, Katia; Lessi, Francesca; Pistello, Mauro; Naccarato, Antonio Giuseppe; Bevilacqua, Generoso

2011-01-01

188

The 17 Nucleotides Downstream from the env Gene Stop Codon Are Important for Murine Leukemia Virus Packaging  

PubMed Central

We have identified a previously unknown nucleotide sequence important for the packaging of murine leukemia virus. This nucleotide sequence is located downstream from the stop codon of the env gene but does not overlap the polypurine tract. Deletion of 17 bp from this region resulted in a more than 10-fold decrease in viral titer. Consistent with this result, the deletion mutant showed a 20- to 30-fold drop in the amount of virion RNA in the culture supernatant. The total amount of virion protein in the culture supernatant was comparable for the deletion mutant and the parental virus, suggesting that the mutant construct could release the empty viral particles. These results suggested that the packaging signal sequence might be present at the two extreme sites of the viral genome, one in the region around the splice donor sequence downstream from the 5? long terminal repeat (LTR) and the other immediately upstream from the 3? LTR. Implications for gene therapy, especially in regard to construction of retroviral vectors and packaging constructs, are discussed.

Shin Yu, Seung; Kim, Jong-Mook; Kim, Sunyoung

2000-01-01

189

Expression of a modified form of CD4 results in the release of an anti-HIV factor derived from the Env sequence.  

PubMed

We have studied the inhibitory effect of a CD4 chimera (CD4epsilon15) on HIV replication. This chimera is retained in the endoplasmic reticulum and traps the HIV envelope precursor gp160, preventing its maturation. Retroviral expression of the chimera strongly inhibited HIV replication even when it is expressed by only a minority of the T cell population. This protective effect on bystander nontransduced cells is mediated by a soluble factor that we identified as a fragment of HIV gp120 envelope protein and accordingly, we named this factor Env-derived antiviral factor (EDAF). Biochemical and immunoreactivity data show that EDAF is comprised of the gp120 C3-C5 regions and indeed, a recombinant protein bearing this sequence reproduces the anti-HIV properties of EDAF. Surprisingly, three tryptic peptides derived from EDAF are homologous but not identical with the corresponding sequences of the HIV isolate used to generate EDAF. We propose that EDAF results from an alternative intracellular processing of the Env protein provoked by its association to CD4epsilon15 and the selection of the best fitted Env protein sequences contained within the HIV isolate. The presence of EDAF improves the therapeutic potential of the CD4epsilon15 gene and it opens new possibilities for antiviral treatment and vaccine development. PMID:19553524

Zaldívar, Irene; Muñoz-Fernández, María Angeles; Alarcón, Balbino; San José, Ester

2009-06-24

190

Mutations of Conserved Glycine Residues within the Membrane-Spanning Domain of Human Immunodeficiency Virus Type 1 gp41 Can Inhibit Membrane Fusion and Incorporation of Env onto Virions  

Microsoft Academic Search

SUMMARY: The membrane-spanning domain (MSD) of HIV-1 envelope protein (Env) has an additional gly- cine residue within a well-conserved putative transmembrane helix-helix interaction motif, GXXXG, and forms a G690G691XXG694 sequence (G, glycine; X, any residues; the numbering indicates the position within the Env of an infectious molecular clone, HXB2). Different from vesicular stomatitis virus G (VSV-G), the glycine residues of

Kosuke Miyauchi; Rachael Curran; Erin Matthews; Jun Komano; Tyuji Hoshino; Don M. Engelman; Zene Matsuda

191

Testability Analysis and ATPG on Behavioral RT-Level VHDL  

Microsoft Academic Search

This paper proposes an environment to address Test- ability Analysis and Test Pattern Generation on VHDL descriptions at t he RT-level. The proposed approach, based on a suitable fault model and an ATPG algo- rithm, is experimentally shown to p rovide a good esti- mate of the final gate-level f ault coverage, and to g ive test patterns with excellent

Fulvio Corno; Paolo Prinetto; Matteo Sonza Reorda

1997-01-01

192

Daily Sampling of an HIV-1 Patient with Slowly Progressing Disease Displays Persistence of Multiple env Subpopulations Consistent with Neutrality  

PubMed Central

The molecular evolution of HIV-1 is characterized by frequent substitutions, indels and recombination events. In addition, a HIV-1 population may adapt through frequency changes of its variants. To reveal such population dynamics we analyzed HIV-1 subpopulation frequencies in an untreated patient with stable, low plasma HIV-1 RNA levels and close to normal CD4+ T-cell levels. The patient was intensively sampled during a 32-day period as well as approximately 1.5 years before and after this period (days ?664, 1, 2, 3, 11, 18, 25, 32 and 522). 77 sequences of HIV-1 env (approximately 3100 nucleotides) were obtained from plasma by limiting dilution with 7–11 sequences per time point, except day ?664. Phylogenetic analysis using maximum likelihood methods showed that the sequences clustered in six distinct subpopulations. We devised a method that took into account the relatively coarse sampling of the population. Data from days 1 through 32 were consistent with constant within-patient subpopulation frequencies. However, over longer time periods, i.e. between days 1…32 and 522, there were significant changes in subpopulation frequencies, which were consistent with evolutionarily neutral fluctuations. We found no clear signal of natural selection within the subpopulations over the study period, but positive selection was evident on the long branches that connected the subpopulations, which corresponds to >3 years as the subpopulations already were established when we started the study. Thus, selective forces may have been involved when the subpopulations were established. Genetic drift within subpopulations caused by de novo substitutions could be resolved after approximately one month. Overall, we conclude that subpopulation frequencies within this patient changed significantly over a time period of 1.5 years, but that this does not imply directional or balancing selection. We show that the short-term evolution we study here is likely representative for many patients of slow and normal disease progression.

Wilbe Ramsay, Karin; Alaeus, Annette; Albert, Jan; Leitner, Thomas

2011-01-01

193

Detection of naturally occurring enteroviruses in waters using direct RT-PCR and integrated cell culture-RT-PCR  

Microsoft Academic Search

Viruses detected by rapid molecular assays are not always infectious. In this study we compared enterovirus levels in natural waters using culture and reverse transcription-polymerase chain reaction (RT-PCR) techniques to determine whether molecular units of naturally occurring enteroviruses can be utilized to predict viral infectivity. Viruses were concentrated from 12 river water and effluent samples using 1MDS filter–filtration and beef

Y. C. Shieh; C. I. Wong; J. A. Krantz; F. C. Hsu

2008-01-01

194

Standardized Comparison of the Relative Impacts of HIV-1 Reverse Transcriptase (RT) Mutations on Nucleoside RT Inhibitor Susceptibility  

PubMed Central

Determining the phenotypic impacts of reverse transcriptase (RT) mutations on individual nucleoside RT inhibitors (NRTIs) has remained a statistical challenge because clinical NRTI-resistant HIV-1 isolates usually contain multiple mutations, often in complex patterns, complicating the task of determining the relative contribution of each mutation to HIV drug resistance. Furthermore, the NRTIs have highly variable dynamic susceptibility ranges, making it difficult to determine the relative effect of an RT mutation on susceptibility to different NRTIs. In this study, we analyzed 1,273 genotyped HIV-1 isolates for which phenotypic results were obtained using the PhenoSense assay (Monogram, South San Francisco, CA). We used a parsimonious feature selection algorithm, LASSO, to assess the possible contributions of 177 mutations that occurred in 10 or more isolates in our data set. We then used least-squares regression to quantify the impact of each LASSO-selected mutation on each NRTI. Our study provides a comprehensive view of the most common NRTI resistance mutations. Because our results were standardized, the study provides the first analysis that quantifies the relative phenotypic effects of NRTI resistance mutations on each of the NRTIs. In addition, the study contains new findings on the relative impacts of thymidine analog mutations (TAMs) on susceptibility to abacavir and tenofovir; the impacts of several known but incompletely characterized mutations, including E40F, V75T, Y115F, and K219R; and a tentative role in reduced NRTI susceptibility for K64H, a novel NRTI resistance mutation.

Rhee, Soo-Yon; Taylor, Jonathan; Fessel, W. Jeffrey; Kaufman, David; Towner, William; Troia-Cancio, Paolo V.; Zolopa, Andrew; Robbins, Gregory K.; Kagan, Ron; Israelski, Dennis; Shafer, Robert W.

2012-01-01

195

An adenovirus-simian immunodeficiency virus env vaccine elicits humoral, cellular, and mucosal immune responses in rhesus macaques and decreases viral burden following vaginal challenge.  

PubMed

Six female rhesus macaques were immunized orally and intranasally at 0 weeks and intratracheally at 12 weeks with an adenovirus type 5 host range mutant (Ad5hr)-simian immunodeficiency virus SIVsm env recombinant and at 24 and 36 weeks with native SIVmac251 gp120 in Syntex adjuvant. Four macaques received the Ad5hr vector and adjuvant alone; two additional controls were naive. In vivo replication of the Ad5hr wild-type and recombinant vectors occurred with detection of Ad5 DNA in stool samples and/or nasal secretions in all macaques and increases in Ad5 neutralizing antibody in 9 of 10 macaques following Ad administrations. SIV-specific neutralizing antibodies appeared after the second recombinant immunization and rose to titers > 10,000 following the second subunit boost. Immunoglobulin G (IgG) and IgA antibodies able to bind gp120 developed in nasal and rectal secretions, and SIV-specific IgGs were also observed in vaginal secretions and saliva. T-cell proliferative responses to SIV gp140 and T-helper epitopes were sporadically detected in all immunized macaques. Following vaginal challenge with SIVmac251, transient or persistent infection resulted in both immunized and control monkeys. The mean viral burden in persistently infected immunized macaques was significantly decreased in the primary infection period compared to that of control macaques. These results establish in vivo use of the Ad5hr vector, which overcomes the host range restriction of human Ads for rhesus macaques, thereby providing a new model for evaluation of Ad-based vaccines. In addition, they show that a vaccine regimen using the Ad5hr-SIV env recombinant and gp120 subunit induces strong humoral, cellular, and mucosal immunity in rhesus macaques. The reduced viral burden achieved solely with an env-based vaccine supports further development of Ad-based vaccines comprising additional viral components for immune therapy and AIDS vaccine development. PMID:9343211

Buge, S L; Richardson, E; Alipanah, S; Markham, P; Cheng, S; Kalyan, N; Miller, C J; Lubeck, M; Udem, S; Eldridge, J; Robert-Guroff, M

1997-11-01

196

A Next-Generation Cleaved, Soluble HIV-1 Env Trimer, BG505 SOSIP.664 gp140, Expresses Multiple Epitopes for Broadly Neutralizing but Not Non-Neutralizing Antibodies.  

PubMed

A desirable but as yet unachieved property of a human immunodeficiency virus type 1 (HIV-1) vaccine candidate is the ability to induce broadly neutralizing antibodies (bNAbs). One approach to the problem is to create trimeric mimics of the native envelope glycoprotein (Env) spike that expose as many bNAb epitopes as possible, while occluding those for non-neutralizing antibodies (non-NAbs). Here, we describe the design and properties of soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A transmitted/founder strain, BG505. These trimers are highly stable, more so even than the corresponding gp120 monomer, as judged by differential scanning calorimetry. They are also homogenous and closely resemble native virus spikes when visualized by negative stain electron microscopy (EM). We used several techniques, including ELISA and surface plasmon resonance (SPR), to determine the relationship between the ability of monoclonal antibodies (MAbs) to bind the soluble trimers and neutralize the corresponding virus. In general, the concordance was excellent, in that virtually all bNAbs against multiple neutralizing epitopes on HIV-1 Env were highly reactive with the BG505 SOSIP.664 gp140 trimers, including quaternary epitopes (CH01, PG9, PG16 and PGT145). Conversely, non-NAbs to the CD4-binding site, CD4-induced epitopes or gp41ECTO did not react with the trimers, even when their epitopes were present on simpler forms of Env (e.g. gp120 monomers or dissociated gp41 subunits). Three non-neutralizing MAbs to V3 epitopes did, however, react strongly with the trimers but only by ELISA, and not at all by SPR and to only a limited extent by EM. These new soluble trimers are useful for structural studies and are being assessed for their performance as immunogens. PMID:24068931

Sanders, Rogier W; Derking, Ronald; Cupo, Albert; Julien, Jean-Philippe; Yasmeen, Anila; de Val, Natalia; Kim, Helen J; Blattner, Claudia; de la Peña, Alba Torrents; Korzun, Jacob; Golabek, Michael; de Los Reyes, Kevin; Ketas, Thomas J; van Gils, Marit J; King, C Richter; Wilson, Ian A; Ward, Andrew B; Klasse, P J; Moore, John P

2013-09-19

197

A Next-Generation Cleaved, Soluble HIV-1 Env Trimer, BG505 SOSIP.664 gp140, Expresses Multiple Epitopes for Broadly Neutralizing but Not Non-Neutralizing Antibodies  

PubMed Central

A desirable but as yet unachieved property of a human immunodeficiency virus type 1 (HIV-1) vaccine candidate is the ability to induce broadly neutralizing antibodies (bNAbs). One approach to the problem is to create trimeric mimics of the native envelope glycoprotein (Env) spike that expose as many bNAb epitopes as possible, while occluding those for non-neutralizing antibodies (non-NAbs). Here, we describe the design and properties of soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A transmitted/founder strain, BG505. These trimers are highly stable, more so even than the corresponding gp120 monomer, as judged by differential scanning calorimetry. They are also homogenous and closely resemble native virus spikes when visualized by negative stain electron microscopy (EM). We used several techniques, including ELISA and surface plasmon resonance (SPR), to determine the relationship between the ability of monoclonal antibodies (MAbs) to bind the soluble trimers and neutralize the corresponding virus. In general, the concordance was excellent, in that virtually all bNAbs against multiple neutralizing epitopes on HIV-1 Env were highly reactive with the BG505 SOSIP.664 gp140 trimers, including quaternary epitopes (CH01, PG9, PG16 and PGT145). Conversely, non-NAbs to the CD4-binding site, CD4-induced epitopes or gp41ECTO did not react with the trimers, even when their epitopes were present on simpler forms of Env (e.g. gp120 monomers or dissociated gp41 subunits). Three non-neutralizing MAbs to V3 epitopes did, however, react strongly with the trimers but only by ELISA, and not at all by SPR and to only a limited extent by EM. These new soluble trimers are useful for structural studies and are being assessed for their performance as immunogens.

Sanders, Rogier W.; Derking, Ronald; Cupo, Albert; Julien, Jean-Philippe; Yasmeen, Anila; de Val, Natalia; Kim, Helen J.; Blattner, Claudia; de la Pena, Alba Torrents; Korzun, Jacob; Golabek, Michael; de los Reyes, Kevin; Ketas, Thomas J.; van Gils, Marit J.; King, C. Richter; Wilson, Ian A.; Ward, Andrew B.; Klasse, P. J.; Moore, John P.

2013-01-01

198

Generation and immunogenicity of novel HIV/AIDS vaccine candidates targeting HIV-1 Env/Gag-Pol-Nef antigens of clade C.  

PubMed

Recombinants based on the attenuated vaccinia virus strains MVA and NYVAC are considered candidate vectors against different human diseases. In this study we have generated and characterized in BALB/c and in transgenic HHD mice the immunogenicity of two attenuated poxvirus vectors expressing in a single locus (TK) the codon optimized HIV-1 genes encoding gp120 and Gag-Pol-Nef (GPN) polyprotein of clade C (referred as MVA-C and NYVAC-C). In HHD mice primed with either MVA-C or NYVAC-C, or primed with DNA-C and boosted with the poxvirus vectors, the splenic T cell responses against clade C peptides spanning gp120/GPN was broad and mainly directed against Gag-1, Env-1 and Env-2 peptide pools. In BALB/c mice immunized with the homologous or the heterologous combination of poxvirus vectors or with Semliki forest virus (SFV) vectors expressing gp120/GPN, the immune response was also broad but the most immunogenic peptides were Env-1, GPN-1 and GPN-2. Differences in the magnitude of the cellular immune responses were observed between the poxvirus vectors depending on the protocol used. The specific cellular immune response triggered by the poxvirus vectors was Th1 type. The cellular response against the vectors was higher for NYVAC than for MVA in both HHD and BALB/c mice, but differences in viral antigen recognition between the vectors was observed in sera from the poxvirus-immunized animals. These results demonstrate the immunogenic potential of MVA-C and NYVAC-C as novel vaccine candidates against clade C of HIV-1. PMID:17224219

Gómez, Carmen Elena; Nájera, Jose Luis; Jiménez, Victoria; Bieler, Kurt; Wild, Jens; Kostic, Linda; Heidari, Shirin; Chen, Margaret; Frachette, Marie-Joelle; Pantaleo, Giuseppe; Wolf, Hans; Liljeström, Peter; Wagner, Ralf; Esteban, Mariano

2006-12-06

199

Novel translation products from simian immunodeficiency virus SIVmac239 Env-encoding mRNA contain both Rev and cryptic T-cell epitopes.  

PubMed

Understanding the correlates of immune protection against human immunodeficiency virus and simian immunodeficiency virus (SIV) will require defining the entire cellular immune response against the viruses. Here, we define two novel translation products from the SIV env mRNA that are targeted by the T-cell response in SIV-infected rhesus macaques. The shorter product is a subset of the larger product, which contains both the first exon of the Rev protein and a translated portion of the rev intron. Our data suggest that the translation of viral alternate reading frames may be an important source of T-cell epitopes, including epitopes normally derived from functional proteins. PMID:19605480

Maness, Nicholas J; Sacha, Jonah B; Piaskowski, Shari M; Weisgrau, Kimberly L; Rakasz, Eva G; May, Gemma E; Buechler, Matthew B; Walsh, Andrew D; Wilson, Nancy A; Watkins, David I

2009-07-15

200

Genetic analysis of the Rous sarcoma virus subgroup D env gene: mammal tropism correlates with temperature sensitivity of gp85.  

PubMed Central

Subgroup D avian sarcoma and leukosis viruses can penetrate a variety of mammalian cells in addition to cells from their natural host, chickens. Sequences derived from the gp85-coding domain within the env gene of a mammal-tropic subgroup D virus (Schmidt-Ruppin D strain of Rous sarcoma virus [SR-D RSV]) and a non-mammal-tropic subgroup B virus (Rous-associated virus type 2) were recombined to map genetic determinants that allow penetration of mammalian cells. The following conclusions were based on host range analysis of the recombinant viruses. (i) The determinants of gp85 that result in the mammal tropism phenotype of SR-D RSV are encoded within the 160 codons that lie 3' of codon 121 from the corresponding amino terminus of the gp85 protein. (ii) Small linear domains of the SR-D RSV gp85-coding domain placed in the subgroup B background did not yield viruses with titers equal to that of the subgroup D virus in a human cell line. (iii) Recombinant viruses that contained subgroup D sequences within the hr1 variable domain of gp85 showed modest-to-significant increases in infectivity on human cells relative to chicken cells. A recombinant virus that contained three fortuitous amino acid substitutions in the gp85-coding domain was found to penetrate the human cell line and give a titer similar to that of the subgroup D virus. In addition, we found that the subgroup D virus, the mutant virus, and recombinant viruses with an increased mammal tropism phenotype were unstable at 42 degrees C. These results suggest that the mammal tropism of the SR-D strain is not related to altered receptor specificity but rather to an unstable and fusogenic viral glycoprotein. A temperature sensitivity phenotype for infectivity of mammalian cells was also observed for another mammal-tropic avian retrovirus, the Bratislava 77 strain of RSV, a subgroup C virus, but was not seen for any other avian retrovirus tested, strengthening the correlation between mammal tropism and temperature sensitivity.

Bova-Hill, C; Olsen, J C; Swanstrom, R

1991-01-01

201

Assessment of pulmonary fibrosis after radiotherapy (RT) in breast conserving surgery: comparison between conventional external beam RT (EBRT) and intraoperative RT with electrons (ELIOT).  

PubMed

The aim of this study was to assess the frequency and the grade of RT-induced pulmonary fibrosis in patients who underwent EBRT compared to patients who underwent ELIOT. One-hundred-seventy-eight patients enrolled in a prospective randomized phase III trial to compare the efficacy of ELIOT (a single dose of 21 Gy prescribed at the 90% isodose) versus EBRT (50 Gy to the whole breast plus a 10 Gy boost to the tumour bed), underwent a spiral 16-detector row Computed Tomography (CT) examination to assess RT-induced pulmonary fibrosis: 83 patients in the EBRT arm and 95 in the ELIOT arm. All patients (age range 48-75 years) were affected by unicentric infiltrating carcinoma of the breast with diameter < 2.5 cm. This study was approved by our Institutional Ethical Committee and informed consent was obtained from each patient. Two observers, blinded to patient's randomization, independently evaluated each CT examination and assigned a fibrosis score (Grades 0 to 3). Inter-observer agreement for the fibrosis score was evaluated and a consensus between observers was obtained. Differences in fibrosis score between the two arms were evaluated by Chi Square test and Odds Ratio (OR) with 95% Confidence Intervals (CI). Pulmonary fibrosis was diagnosed in 42 patients (23.6%): 38 (90%) were in the EBRT arm and 4 (10%) in the ELIOT arm (p < 0.0001); twenty-six of them were Grade 1 (one ELIOT), fifteen were Grade 2 (three ELIOT) and one was Grade 3. The post-radiotherapy risk in the EBRT arm to develop at least Grade 1 fibrosis was 19 times higher than in the ELIOT one (OR: 19.20; 95%CI: 6.46-57.14) and 6 times higher to develop at least Grade 2 (OR: 5.70; 95%CI: 1.56-20.76). In conclusion, CT detected pulmonary fibrosis in patients treated with ELIOT is significantly less frequent compared to patients treated with EBRT. PMID:21728389

Rampinelli, C; Bellomi, M; Ivaldi, G B; Intra, M; Raimondi, S; Meroni, S; Orecchia, R; Veronesi, U

2011-08-01

202

Combination Treatment with rt-PA is More Effective than rt-PA Alone in an in Vitro Human Clot Model  

PubMed Central

Incidence of intra-cranial hemorrhage linked to treatment of ischemic stroke with recombinant tissue plasminogen activator (rt-PA) has led to interest in adjuvant therapies such as ultrasound (US) or plasminogen, to enhance rt-PA efficacy and improve patient safety. High-frequency US (~MHz) such as 2-MHz transcranial Doppler (TCD) has demonstrated increased recanalization in situ. Low-frequency US (~kHz) enhanced thrombolysis (UET) has demonstrated higher lytic capabilities but has been associated with incidence of intracerebral hemorrhage in some clinical trials. In vitro studies using plasminogen have shown enhancement of lysis. This study compared rt-PA-induced lysis using adjuvant therapies, with 120-kHz or 2-MHz pulsed US, or plasminogen, in an in vitro human whole blood clot model. Blood was drawn from 30 subjects after local institutional approval. Clots were exposed to rt-PA at concentrations of 0 to 3.15 ?g/ml. Clots were exposed to rt-PA alone (rt-PA) or in combination with plasminogen (Plg), 120-kHz US (120-kHz), or 2-MHz US (2-MHz). Thrombolytic efficacy was determined by assessing the percent fractional clot loss (FCL) at 30 minutes using microscopic imaging. There was no enhancement of lysis for combination therapy with [rt-PA]=0 ?g/ml. Adding rt-PA increased lysis for all groups. As [rt-PA] increased, lysis tended to increase for 120-kHz and Plg (FCL: from 50% to 70%, 120-kHz; 65% to 83%, Plg) but not for 2-MHz (58% to 52%). Lytic efficacy in combination therapy depends on rt-PA concentration and the adjuvant therapy type. For non-zero rt-PA concentrations, all combination therapies produced more lysis than rt-PA alone.

Meunier, Jason M.; Holland, Christy K.; Porter, Tyrone M.; Lindsell, Christopher J.; Shaw, George J.

2013-01-01

203

RAMSES-RT: radiation hydrodynamics in the cosmological context  

NASA Astrophysics Data System (ADS)

We present a new implementation of radiation hydrodynamics (RHD) in the adaptive mesh refinement (AMR) code RAMSES. The multigroup radiative transfer (RT) is performed on the AMR grid with a first-order Godunov method using the M1 closure for the Eddington tensor, and is coupled to the hydrodynamics via non-equilibrium thermochemistry of hydrogen and helium. This moment-based approach has the great advantage that the computational cost is independent of the number of radiative sources - it can even deal with continuous regions of emission such as bound-free emission from gas. As it is built directly into RAMSES, the RT takes natural advantage of the refinement and parallelization strategies already in place. Since we use an explicit advection solver for the radiative transport, the time-step is restricted by the speed of light - a severe limitation that can be alleviated using the so-called reduced speed of light approximation. We propose a rigorous framework to assess the validity of this approximation in various conditions encountered in cosmology and galaxy formation. We finally perform with our newly developed code a complete suite of RHD tests, comparing our results to other RHD codes. The tests demonstrate that our code performs very well and is ideally suited for exploring the effect of radiation on current scenarios of structure and galaxy formation.

Rosdahl, J.; Blaizot, J.; Aubert, D.; Stranex, T.; Teyssier, R.

2013-10-01

204

NASA/SPoRt: GOES-R Activities in Support of Product Development, Management, and Training.  

National Technical Information Service (NTIS)

SPoRT is using current capabilities of MODIS and VIIRS, combined with current GOES (i.e. Hybrid Imagery) to demonstrate mesoscale capabilities of future ABI instrument. SPoRT is transitioning RGBs from EUMETSAT standard "recipes" to demonstrate ...

A. Molthan G. Jedlovec G. Stano K. Fuell

2012-01-01

205

Cross-talk Suppression between the CpxA-CpxR and EnvZ-OmpR Two-Component Systems in E. coli  

PubMed Central

Many bacteria possess large numbers of two-component signaling systems, which are composed of histidine kinase-response regulator pairs. The high level of sequence similarity between some systems raises the possibility of undesired cross-talk between a histidine kinase and a non-cognate response regulator. Although molecular specificity ensures that phospho-transfer occurs primarily between correct partners, even a low level of inappropriate cross-talk could lead to unacceptable levels of noise or interference in signal transduction. To explore mechanisms that provide insulation against such interference, we have examined cross-talk between the histidine kinase CpxA and non-cognate response regulator OmpR in Escherichia coli. Our results show that there are two mechanisms that suppress cross-talk between these two proteins, which depend on the corresponding cognate partners CpxR and EnvZ and on the bifunctional nature of the histidine kinases CpxA and EnvZ. When cross-talk is detectable, we find it is independent of CpxA stimulus. We also show that cross-talk suppression leads to mutational robustness, i.e. it masks the effects of mutations that would otherwise lead to increased cross-talk. The mechanisms that provide insulation against interference described here may be applicable to many other two-component systems.

Siryaporn, Albert; Goulian, Mark

2009-01-01

206

Time-resolved Imaging of HIV-1 Env-mediated Lipid and Content Mixing between a Single Virion and Cell Membrane  

PubMed Central

A method has been developed to follow fusion of individual pseudotyped virus expressing HIV-1 Env to cells by time-resolved fluorescence microscopy. Viral envelopes were labeled with a fluorescent lipid dye (DiD) and virus content was rendered visible by incorporating a Gag-GFP chimera. The Gag-GFP is naturally cleaved to the much smaller NC-GFP fragment in the mature virions. NC-GFP was readily released upon permeabilization of the viral envelope, whereas the capsid was retained. The NC-GFP thus provides a relatively small and mobile aqueous marker to follow viral content transfer. In fusion experiments, virions were bound to cells at low temperature, and fusion was synchronously triggered by a temperature jump. DiD transferred from virions to cells without a significant lag after the temperature jump. Some virions released DiD but retained NC-GFP. Surprisingly, the fraction of lipid mixing events yielding NC-GFP transfer was dependent on the type of target cell: of three infectable cell lines, only one permitted NC-GFP transfer within minutes of raising temperature. NC-GFP release did not correlate with the level of CD4 or coreceptor expression in the target cells. The data indicate that fusion pores formed by HIV-1 Env can remain small for a relatively long time before they enlarge.

Markosyan, Ruben M.; Cohen, Fredric S.; Melikyan, Grigory B.

2005-01-01

207

Formaldehyde-Treated, Heat-Inactivated Virions with Increased Human Immunodeficiency Virus Type 1 Env Can Be Used To Induce High-Titer Neutralizing Antibody Responses  

PubMed Central

The lack of success of subunit human immunodeficiency virus (HIV) type 1 vaccines to date suggests that multiple components or a complex virion structure may be required. We hypothesized that the failure of current vaccine strategies to induce protective antibodies is linked to the inability of native envelope structures to readily elicit these types of antibodies. We have previously reported on the ability of a formaldehyde-treated, heat-inactivated vaccine to induce modest antibody responses in animal vaccine models. We investigated here whether immunization for HIV with an envelope-modified, formaldehyde-stabilized, heat-inactivated virion vaccine could produce higher-titer and/or broader neutralizing antibody responses. Thus, a clade B vaccine which contains a single point mutation in gp41 (Y706C) that results in increased incorporation of oligomeric Env into virions was constructed. This vaccine was capable of inducing high-titer antibodies that could neutralize heterologous viruses, including those of clades A and C. These results further support the development of HIV vaccines with modifications in native Env structures for the induction of neutralizing antibody responses.

Poon, B.; Hsu, J. F.; Gudeman, V.; Chen, I. S. Y.; Grovit-Ferbas, K.

2005-01-01

208

A replication-competent, endogenous retrovirus from an aged DBA/2 mouse contains the complete env from Emv-3 and a novel gag partially related to AKT-8.  

PubMed Central

We previously described an endogenous murine retrovirus, rv-DBA/2aged, isolated from an aged DBA/2 mouse. The previous report showed that a recombination which resulted in the replacement of Emv-3 gag sequences with gag sequences homologous to those found in the AKT-8 virus had taken place. This recombination allowed production of a competent virus from the defective Emv-3 locus. However, the extent of replacement of Emv-3 gag was not known. We report here the entire sequence for the gag gene of rv-DBA/2aged as well as the previously unsequenced 3' end of the Emv-3 gag gene. These data demonstrate that while sequences homologous to the entire gag gene fragment found in AKT-8 are represented in rv-DBA/2aged, the remainder of rv-DBA/2aged gag is not derived from Emv-3 but is a unique gag sequence. Furthermore, a complete comparison of env sequences shows that the env of rv-DBA/2aged is derived entirely from Emv-3. Additional data suggest that the recombination which led to production of the rv-DBA/2aged virus may be a common event in aging DBA/2 mice. Finally, comparison of the new sequences of Emv-3 with those of the Akv virus (also designated AKR-623 and Emv-11) and Emv-1 shows that this endogenous virus locus is very closely related to the other Emv loci at the nucleotide sequence level.

Bartman, T; Murasko, D M; Blank, K J

1995-01-01

209

Increased Level and Longevity of Protective Immune Responses Induced by DNA Vaccine Expressing the HIV-1 Env Glycoprotein when Combined with IL-21 and IL-15 Gene Delivery1  

PubMed Central

We investigated the ability of a plasmid-derived IL-21 delivered alone or in combination with the IL-15 gene to regulate immune responses to the HIV-1 envelope (Env) glycoprotein induced by DNA vaccination. Mice were injected with the gp140?CFIHXB2/89.6 vector expressing a modified Env glycoprotein with C-terminal mutations intended to mimic a fusion intermediate, in which the most divergent region encoding the variable V1, V2, and V3 domains of CXCR4-tropic HxB2 virus was replaced with the dual-tropic 89.6 viral strain. Using a recombinant vaccinia virus expressing 89.6 Env glycoprotein (vBD3) in a mouse challenge model, we observed that IL-21 plasmid produced sustained resistance to viral transmission when injected 5 days after DNA vaccination. Moreover, IL-21 in a synergistic manner with IL-15 expression vector augmented the vaccine-induced recall responses to the vBD3 challenge compared with those elicited by immunization in the presence of either cytokine alone. The synergistic combination of IL-21 and IL-15 plasmids promoted expansion of CD8+CD127+ memory T cell pools specific for a subdominant HLA-A2-restricted Env121–129 epitope (KLTPLCVTL). Our results also show that coimmunization with IL-21 and IL-15 plasmid combination resulted in enhanced CD8+ T cell function that was partially independent of CD4+ T cell help in mediating protection against vBD3 challenge. Furthermore, the use of IL-21 and IL-15 genes was able to increase Ab-dependent cellular cytotoxicity and complement-dependent lysis of Env-expressing target cells through augmentation of Env-specific IgG Ab levels. These data indicate that the plasmid-delivered IL-21 and IL-15 can increase the magnitude of the response to DNA vaccines.

Bolesta, Elizabeth; Kowalczyk, Aleksandra; Wierzbicki, Andrzej; Eppolito, Cheryl; Kaneko, Yutaro; Takiguchi, Masafumi; Stamatatos, Leonidas; Shrikant, Protul A.; Kozbor, Danuta

2008-01-01

210

Function of the cytoplasmic domain of a retroviral transmembrane protein: p15E-p2E cleavage activates the membrane fusion capability of the murine leukemia virus Env protein.  

PubMed Central

In the murine leukemia viruses (MuLVs), the Env complex is initially cleaved by a cellular protease into gp70SU and pre15ETM. After the virus particle is released from the cell, the C-terminal 16 residues are removed from the cytoplasmic domain of pre15E by the viral protease, yielding the mature p15ETM and p2E. We have investigated the function of this cleavage by generating a Moloney MuLV mutant, termed p2E-, in which the Env coding region terminates at the cleavage site. This mutant synthesizes only the truncated, mature form of TM rather than its extended precursor. When cells expressing this truncated Env protein are cocultivated with NIH 3T3 cells, they induce rapid cell-cell fusion. Thus, the truncated form, which is normally found in virions but not in virus-producing cells, is capable of causing membrane fusion. We conclude that the 16-residue p2E tail inhibits this activity of Env until the virus has left the cell. p2E- virions were found to be infectious, though with a lower specific infectivity than that of the wild type, showing that p2E does not play an essential role in the process of infection. Fusion was also observed with a chimeric p2E- virus in which gp70SU and nearly all of p15ETM are derived from amphotropic, rather than Moloney, MuLV. In a second mutant, an amino acid at the cleavage site was changed. The pre15E protein in this mutant is not cleaved. While the mutant Env complex is incorporated into virions, these particles have a very low specific infectivity. This result suggests that the cleavage event is essential for infectivity, in agreement with the idea that removal of p2E activates the membrane fusion capability of the Env complex. Images

Rein, A; Mirro, J; Haynes, J G; Ernst, S M; Nagashima, K

1994-01-01

211

HIV-1 Reverse Transcriptase (RT) Polymorphism 172K Suppresses the Effect of Clinically Relevant Drug Resistance Mutations to Both Nucleoside and Non-nucleoside RT Inhibitors*  

PubMed Central

Polymorphisms have poorly understood effects on drug susceptibility and may affect the outcome of HIV treatment. We have discovered that an HIV-1 reverse transcriptase (RT) polymorphism (RT172K) is present in clinical samples and in widely used laboratory strains (BH10), and it profoundly affects HIV-1 susceptibility to both nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) when combined with certain mutations. Polymorphism 172K significantly suppressed zidovudine resistance caused by excision (e.g. thymidine-associated mutations) and not by discrimination mechanism mutations (e.g. Q151M complex). Moreover, it attenuated resistance to nevirapine or efavirenz imparted by NNRTI mutations. Although 172K favored RT-DNA binding at an excisable pre-translocation conformation, it decreased excision by thymidine-associated mutation-containing RT. 172K affected DNA handling and decreased RT processivity without significantly affecting the kcat/Km values for dNTP. Surface plasmon resonance experiments revealed that RT172K decreased DNA binding by increasing the dissociation rate. Hence, the increased zidovudine susceptibility of RT172K results from its increased dissociation from the chain-terminated DNA and reduced primer unblocking. We solved a high resolution (2.15 ?) crystal structure of RT mutated at 172 and compared crystal structures of RT172R and RT172K bound to NNRTIs or DNA/dNTP. Our structural analyses highlight differences in the interactions between ?-helix E (where 172 resides) and the active site ?9-strand that involve the YMDD loop and the NNRTI binding pocket. Such changes may increase dissociation of DNA, thus suppressing excision-based NRTI resistance and also offset the effect of NNRTI resistance mutations thereby restoring NNRTI binding.

Hachiya, Atsuko; Marchand, Bruno; Kirby, Karen A.; Michailidis, Eleftherios; Tu, Xiongying; Palczewski, Krzysztof; Ong, Yee Tsuey; Li, Zhe; Griffin, Daniel T.; Schuckmann, Matthew M.; Tanuma, Junko; Oka, Shinichi; Singh, Kamalendra; Kodama, Eiichi N.; Sarafianos, Stefan G.

2012-01-01

212

Computer-facilitated collaboration: experiences building SNOMED-RT.  

PubMed Central

Collaborative development involving both individuals and groups is often less efficient than independent development because of communication overhead and integration costs. Despite the decreased development efficiency, collaborations promise more general-purpose products because of the opportunity for integration, with negotiation and reconciliation of diverse perspectives. Collaborations are also perhaps less costly when considered in contexts where there is significant duplication of effort. Computer-facilitated collaboration can reduce the communication and integration burden such that the increased effort required to manage a successful collaboration focuses primarily on the development of shared conceptual model among the developers by requiring that the work product be independently reproducible. This reproducibility requirement incorporates formal quality assurance processes into the development process. In this paper, we describe our initial experiences developing SNOMED-RT using such a computer-facilitated collaborative process. We quantify the extra costs incurred to achieve consistency in our efforts and reproducibility of our results. Images Figure 2

Levy, D. H.; Dolin, R. H.; Mattison, J. E.; Spackman, K. A.; Campbell, K. E.

1998-01-01

213

Influence of RT-qPCR Primer Position on EGFR Interference Efficacy in Lung Cancer Cells  

Microsoft Academic Search

Real-time quantitative RT-PCR (RT-qPCR) is a “gold” standard for measuring steady state mRNA levels in RNA interference assays.\\u000a The knockdown of the epidermal growth factor receptor (EGFR) gene with eight individual EGFR small interfering RNAs (siRNAs)\\u000a was estimated by RT-qPCR using three different RT-qPCR primer sets. Our results indicate that accurate measurement of siRNA\\u000a efficacy by RT-qPCR requires careful attention

Gang Chen; Peter Kronenberger; Erik Teugels; Jacques De Grève

214

Kopplung PDP-11 (RT-11) PDP-11 (RSX-11M) (Link between PDP-11 (RT-11) and PDP-11 (RSX-11M)).  

National Technical Information Service (NTIS)

This paper describes a link between two PDP11 computers running under the RSX11M respective the RT11 operating system. Every asynchronous, serial line-unit is suitable for interfacing. While in RSX the standard-terminal-driver is used, RT11 employs the ha...

G. Erdei

1980-01-01

215

Recombination between feline leukemia virus subgroup B or C and endogenous env elements alters the in vitro biological activities of the viruses.  

PubMed Central

An important question in feline leukemia virus (FeLV) pathogenesis is whether, as in murine leukemia virus infection, homologous recombination between the infecting FeLV and the noninfectious endogenous FeLV-like proviruses serves as a significant base for the generation of proximal pathogens. To begin an analysis of this issue, several recombinant FeLVs were produced by using two different approaches: (i) the regions of the viral envelope (env) gene of a cloned FeLV (subgroup B virus [FeLV-B], Gardner-Arnstein strain) and those of two different endogenous proviral loci were exchanged to create specific FeLV chimeras, and (ii) vectors containing endogenous env and molecularly cloned infectious FeLV-C (Sarma strain) DNA sequences were coexpressed by transfection in nonfeline cells to facilitate recombination. The results of these combined approaches showed that up to three-fourths of the envelope glycoprotein (gp70), beginning from the N-terminal end, could be replaced by endogenous FeLV sequences to produce biologically active chimeric FeLVs. The in vitro replication efficiency or cell tropism of the recombinants appeared to be influenced by the amount of gp70 sequences replaced by the endogenous partner as well as by the locus of origin of the endogenous sequences. Additionally, a characteristic biological effect, aggregation of feline T-lymphoma cells (3201B cell line), was found to be specifically induced by replicating FeLV-C or FeLV-C-based recombinants. Multiple crossover sites in the gp70 protein selected under the conditions used for coexpression were identified. The results of induced coexpression were also supported by rapid generation of FeLV recombinants when FeLV-C was used to infect the feline 3201B cell line that constitutively expresses high levels of endogenous FeLV-specific mRNAs. Furthermore, a large, highly conserved open reading frame in the pol gene of an endogenous FeLV provirus was identified. This observation, particularly in reference to our earlier finding of extensive mutations in the gag gene, reveals a target area for potentially productive homologous recombination upstream of the functional endogenous env gene. Images

Pandey, R; Ghosh, A K; Kumar, D V; Bachman, B A; Shibata, D; Roy-Burman, P

1991-01-01

216

Bone marrow cells carrying the env-pX transgene play a role in the severity but not prolongation of arthritis in human T-cell leukaemia virus type-I transgenic rats: a possible role of articular tissues carrying the transgene in the prolongation of arthritis  

PubMed Central

Transgenic rats carrying the env-pX gene of human T-cell leukaemia virus type-I (env-pX rats) were immunized with type II collagen (CII), and chronological alterations of arthritis were compared with findings of collagen-induced arthritis (CIA) in wildtype Wistar–King–Aptekman–Hokudai (WKAH) rats. Arthritis induced by CII in env-pX rats was more severe and persisted longer than CIA in WKAH rats. To determine whether the phenomenon is caused mainly by the transgene-carrying lymphocytes or articular tissues, we immunized lethally irradiated env-pX and WKAH rats with reciprocal bone marrow cell (BMC) transplantation. A severe but transient arthritis was induced by CII in WKAH rats reconstituted by env-pX BMC (w/tB/CII rats). On the other hand, in env-pX rats reconstituted by WKAH BMC, arthritis persisted longer than in w/tB/CII rats, although the degree was less at an early phase after CII immunization. These findings suggest that articular tissues rather than the BMCs carrying the env-pX transgene play a role in the prolongation of arthritis in env-pX rats, although BMCs carrying the transgene are associated with the severity of arthritis. When inflammatory cytokines in synovial cells isolated from env-pX rats before they developed arthritis were examined, interleukin-6 (IL-6) was detected at a higher level than in synovial cells from WKAH rats, thus suggesting the critical role of IL-6 in env-pX arthritis.

Abe, Asami; Ishizu, Akihiro; Ikeda, Hitoshi; Hayase, Hiroko; Tsuji, Takahiro; Miyatake, Yukiko; Tsuji, Muneharu; Fugo, Kazunori; Sugaya, Toshiaki; Higuchi, Masato; Matsuno, Takeo; Yoshiki, Takashi

2004-01-01

217

HIV-specific humoral and cellular immunity in rabbits vaccinated with recombinant human immunodeficiency virus-like gag-env particles  

SciTech Connect

Recombinant human immunodeficiency virus type-1 (HIV-1)-like gag-env particles produced in mammalian cells were inoculated into two New Zealand white rabbits. In parallel, two control rabbits were inoculated with the homologous HIV-1 virions inactivated by ultraviolet light (uv) and psoralen treatments. The humoral and cellular immune responses to HIV-1 were evaluated for both groups of animals. Recombinant particles elicited humoral immunity that was specific for all the viral structural proteins. The antibodies recognized both denatured and nondenatured proteins. Moreover, the sera neutralized the in vitro infectivity of the homologous virus in CEM cells. Importantly, the recombinant particles also generated a T helper response by priming with the HIV proteins. Similar results were observed with inactivated virus immunization. Therefore, the authors results suggest that the recombinant HIV-like particles elicit functional humoral immunity as well as cellular immunity and represent a novel vaccine candidate for AIDS.

Haffar, O.K.; Smithgall, M.D.; Moran, P.A.; Travis, B.M.; Zarling, J.M.; Hu, S.L. (Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute-Seattle, WA (USA))

1991-08-01

218

Differential evolutionary fate of an ancestral primate endogenous retrovirus envelope gene, the EnvV syncytin, captured for a function in placentation.  

PubMed

Syncytins are envelope genes of retroviral origin that have been co-opted for a role in placentation. They promote cell-cell fusion and are involved in the formation of a syncytium layer--the syncytiotrophoblast--at the materno-fetal interface. They were captured independently in eutherian mammals, and knockout mice demonstrated that they are absolutely required for placenta formation and embryo survival. Here we provide evidence that these "necessary" genes acquired "by chance" have a definite lifetime with diverse fates depending on the animal lineage, being both gained and lost in the course of evolution. Analysis of a retroviral envelope gene, the envV gene, present in primate genomes and belonging to the endogenous retrovirus type V (ERV-V) provirus, shows that this captured gene, which entered the primate lineage >45 million years ago, behaves as a syncytin in Old World monkeys, but lost its canonical fusogenic activity in other primate lineages, including humans. In the Old World monkeys, we show--by in situ analyses and ex vivo assays--that envV is both specifically expressed at the level of the placental syncytiotrophoblast and fusogenic, and that it further displays signs of purifying selection based on analysis of non-synonymous to synonymous substitution rates. We further show that purifying selection still operates in the primate lineages where the gene is no longer fusogenic, indicating that degeneracy of this ancestral syncytin is a slow, lineage-dependent, and multi-step process, in which the fusogenic activity would be the first canonical property of this retroviral envelope gene to be lost. PMID:23555306

Esnault, Cécile; Cornelis, Guillaume; Heidmann, Odile; Heidmann, Thierry

2013-03-28

219

Endogenous retrovirus sequences as a novel class of tumor-specific antigens: an example of HERV-H env encoding strong CTL epitopes.  

PubMed

Our genome consists to about 8% of human endogenous retroviral (HERV) sequences. These HERVs have been discussed to be linked to human diseases for decades. Recently, a detailed analysis of a HERV-H sequence located on chromosome Xp22.3 revealed a strong expression in a subset of gastrointestinal cancers whereas expression in normal tissues and in other cancer entities was low. In the present study, we used the reverse immunology approach to test the immunological potential of this HERV-H ORF on Xp22.3. A total of ten peptides displaying HLA-A2.1-binding motifs were selected from the predicted env protein sequence. Stimulation of peripheral T cells with retroviral peptides (RVPs) presented by autologous antigen-presenting cells clearly resulted in sustained proliferation of predominantly CD8(+) T cells. High numbers of IFN-?-secreting T cells were detectable after several weekly stimulations with RVP mixes. Reactivity observed in RVP-Mix-stimulated cultures was attributable to RVP03, RVP09 and to a lower extend to RVP08, suggesting those to be highly immunogenic epitopes. Besides killing of RVP-loaded target cells, up to 40% specific lysis of colorectal carcinoma cell lines endogenously expressing this HERV-H Xp22.3 ORF was achieved. These data demonstrate that human T cells can be sensitized toward HERV peptides and moreover posses a high lytic potential toward HERV-H expressing CRC cells. Additionally, these data hint toward endogenous ENV protein expression followed by proteasomal degradation and presentation in the context of HLA molecules. Finally, our data strengthen the view that HERV-encoded sequences should be considered as a new class of tumor-specific antigens. PMID:22187063

Mullins, Christina S; Linnebacher, Michael

2011-12-21

220

Differential Evolutionary Fate of an Ancestral Primate Endogenous Retrovirus Envelope Gene, the EnvV Syncytin, Captured for a Function in Placentation  

PubMed Central

Syncytins are envelope genes of retroviral origin that have been co-opted for a role in placentation. They promote cell–cell fusion and are involved in the formation of a syncytium layer—the syncytiotrophoblast—at the materno-fetal interface. They were captured independently in eutherian mammals, and knockout mice demonstrated that they are absolutely required for placenta formation and embryo survival. Here we provide evidence that these “necessary” genes acquired “by chance” have a definite lifetime with diverse fates depending on the animal lineage, being both gained and lost in the course of evolution. Analysis of a retroviral envelope gene, the envV gene, present in primate genomes and belonging to the endogenous retrovirus type V (ERV-V) provirus, shows that this captured gene, which entered the primate lineage >45 million years ago, behaves as a syncytin in Old World monkeys, but lost its canonical fusogenic activity in other primate lineages, including humans. In the Old World monkeys, we show—by in situ analyses and ex vivo assays—that envV is both specifically expressed at the level of the placental syncytiotrophoblast and fusogenic, and that it further displays signs of purifying selection based on analysis of non-synonymous to synonymous substitution rates. We further show that purifying selection still operates in the primate lineages where the gene is no longer fusogenic, indicating that degeneracy of this ancestral syncytin is a slow, lineage-dependent, and multi-step process, in which the fusogenic activity would be the first canonical property of this retroviral envelope gene to be lost.

Esnault, Cecile; Cornelis, Guillaume; Heidmann, Odile; Heidmann, Thierry

2013-01-01

221

The Streptomycin-Treated Mouse Intestine Selects Escherichia coli envZ Missense Mutants That Interact with Dense and Diverse Intestinal Microbiota  

PubMed Central

Previously, we reported that the streptomycin-treated mouse intestine selected nonmotile Escherichia coli MG1655 flhDC deletion mutants of E. coli MG1655 with improved colonizing ability that grow 15% faster in vitro in mouse cecal mucus and 15 to 30% faster on sugars present in mucus (M. P. Leatham et al., Infect. Immun. 73:8039–8049, 2005). Here, we report that the 10 to 20% remaining motile E. coli MG1655 are envZ missense mutants that are also better colonizers of the mouse intestine than E. coli MG1655. One of the flhDC mutants, E. coli MG1655 ?flhD, and one of the envZ missense mutants, E. coli MG1655 mot-1, were studied further. E. coli MG1655 mot-1 is more resistant to bile salts and colicin V than E. coli MG1655 ?flhD and grows ca. 15% slower in vitro in mouse cecal mucus and on several sugars present in mucus compared to E. coli MG1655 ?flhD but grows 30% faster on galactose. Moreover, E. coli MG1655 mot-1 and E. coli MG1655 ?flhD appear to colonize equally well in one intestinal niche, but E. coli MG1655 mot-1 appears to use galactose to colonize a second, smaller intestinal niche either not colonized or colonized poorly by E. coli MG1655 ?flhD. Evidence is also presented that E. coli MG1655 is a minority member of mixed bacterial biofilms in the mucus layer of the streptomycin-treated mouse intestine. We offer a hypothesis, which we call the “Restaurant” hypothesis, that explains how nutrient acquisition in different biofilms comprised of different anaerobes can account for our results.

Leatham-Jensen, Mary P.; Frimodt-M?ller, Jakob; Adediran, Jimmy; Mokszycki, Matthew E.; Banner, Megan E.; Caughron, Joyce E.; Krogfelt, Karen A.; Conway, Tyrrell

2012-01-01

222

Highly Effective Control of an AIDS Virus Challenge in Macaques by Using Vesicular Stomatitis Virus and Modified Vaccinia Virus Ankara Vaccine Vectors in a Single-Boost Protocol  

Microsoft Academic Search

Previous studies have shown that vaccination and boosting of rhesus macaques with attenuated vesicular stomatitis virus (VSV) vectors encoding Env and Gag proteins of simian immunodeficiency virus-human immunodeficiency virus (SHIV) hybrid viruses protect rhesus macaques from AIDS after challenge with the highly pathogenic SHIV 89.6P (23). In the present study, we compared the effectiveness of a single prime-boost protocol consisting

Elizabeth Ramsburg; Nina F. Rose; Preston A. Marx; Megan Mefford; Douglas F. Nixon; Walter J. Moretto; David Montefiori; Patricia Earl; Bernard Moss; John K. Rose

2004-01-01

223

Real-time RT-PCR for norovirus screening in shellfish  

Microsoft Academic Search

Real-time RT-PCR, combining amplification and detection of virus-specific amplicons, is a promising tool for norovirus detection in environmental or food samples such as shellfish. We developed a real-time RT-PCR assay based on one-step detection using single primer sets and probes for norovirus genogroups I and II. Seventy and seven RT-PCR units of genogroup I and II reference norovirus strains, respectively,

F. Loisy; R. L. Atmar; P. Guillon; P. Le Cann; M. Pommepuy; F. S. Le Guyader

2005-01-01

224

Effects of Contextual Similarity and Target-Repetition Proportion on Negative Priming in RT Distributional Analyses  

Microsoft Academic Search

Participants' reaction time (RT) data in a prime-probe flanker task (e.g., ABA–CAC) were analyzed in terms of the characteristics of RT distribution to examine possible mechanisms that produce negative priming. When the prime and probe were presented in the same context and the proportion of repetition-target trials (TRP) was 0.33, negative priming increased as a function of RT bins, supporting

Chi-Shing Tse; Keith A. Hutchison; Yongna Li

2011-01-01

225

Formation of High-Beta Plasma and Stable Confinement of Toroidal Electron Plasma in RT1  

Microsoft Academic Search

The Ring Trap 1 (RT-1) device is a laboratory magnetosphere generated by a levitated superconducting magnet. The goals of RT-1 are to realize stable formation of ultra high-beta plasma suitable for burning advanced fusion fuels, and confinement of toroidal non-neutral plasmas including antimatter particles. RT- 1 has produced high-beta plasma in the magnetospheric configuration. The effects of coil levitation and

Haruhiko Saitoh

2010-01-01

226

Messenger RNA-based RT-PCR detection of viable Salmonella  

Microsoft Academic Search

The objective of our study was to investigate whether certain regions in the rpoD gene of Salmonella were suited for the RT-PCR detection of viable Salmonella cells. We performed RT-PCR on RNA extracted from viable, and heat- or ethanol-killed Salmonella cells. Using RT-PCR, RNA was easily detected in viable cells. For the heat-killed cells mRNA was undetectable after 1h when

Nancy Rijpens; Geert Jannes; Lieve Herman

2002-01-01

227

ESCRIPT-RT: Reactive transport simulation in PYTHON using ESCRIPT  

NASA Astrophysics Data System (ADS)

We present ESCRIPT-RT, a new reactive transport simulation code for fully saturated porous media which is based on a finite element method (FEM) combined with three other components: (i) a Gibbs minimisation solver for equilibrium modelling of fluid–rock interactions, (ii) an equation of state for pure water to calculate fluid properties and (iii) a thermodynamically consistent material database to determine rocks' material properties. Using decoupling of most of the standard governing equations, this code solves sequentially for temperature, pressure, mass transport and chemical equilibrium. In contrast, pressure and Darcy flow velocities are solved as a coupled system. The reactive transport itself is performed using the masses of chemical elements instead of chemical species. In such way it requires less computing memory and time than the majority of other packages. The code is based on ESCRIPT, a parallelised platform which supports efficient stepwise simulation of realistic geodynamic scenarios at multiple scales. It is particularly suitable to analyse hydrothermal systems involving geometrically complex geological structures with strong permeability contrasts and subject to complex fluid–rock chemical interactions. The modular architecture of the code and its high level Python interface also provide flexibility for modellers who can easily modify or add new feedbacks between the different physical processes. In addition, the implemented abstract user interface allows geologists to run the code without knowledge of the underlying numerical implementation. As an example we show the simulation of hydrothermal gold precipitation in a granite–greenstone geological sequence, which illustrates the important coupling between thermal response and mass transfer to the localisation of gold.

Poulet, T.; Gross, L.; Georgiev, D.; Cleverley, J.

2012-08-01

228

A scanning tunneling microscopy study of distyrylbenzene on Ag/Ge(111)-(sqr rt of 3 x sqr rt of 3)R30 degrees.  

PubMed

The adsorption and self-organized monolayers of trans,trans-distyrylbenzene (tt-DSB) and cis,cis-distyrylbenzene (cc-DSB) on Ag/Ge(111)-(sqr rt of 3 x sqr rt of 3)R30 degrees (Ag/Ge(111)-sqr rt of 3) were studied by low-temperature scanning tunneling microscopy (STM) in ultrahigh vacuum. tt-DSB and cc-DSB overlayers were prepared by vapor deposition at a substrate temperature of 200 K and imaged after the samples were cooled to 100 K. High-resolution images allow identification of the internal structure of individual tt-DSB molecules with three phenyl rings and their molecular arrangements on the Ag/Ge(111)-sqr rt of 3 surface. It is found that the intermolecular distance between two terminal phenyl rings in tt-DSB is about twice the lattice constant of Ag/Ge(111)-sqr rt of 3. Such a lattice match makes Ag/Ge(111)-sqr rt of 3 an ideal substrate for tt-DSB self-organization and the formation of a (3 x 1) overlayer unit cell. The structural model and the molecule registry corresponding to STM images for the adlayers of tt-DSB on Ag/Ge(111)-sqr rt of 3 are proposed and discussed. For cc-DSB adsorption on Ag/Ge(111)-sqr rt of 3, uniform molecular overlayers with two discernible molecular images corresponding to two major types of cc-DSB conformers were observed. The coexistence of multiple conformers and the mismatch of molecular dimension of cc-DSB with the substrate unit cell length limit the growth of large cc-DSB domains. PMID:17973407

Wu, H C; Tsai, C-S; Chou, L-W; Lee, Y-R; Jiang, J C; Su, C; Lin, J-C

2007-10-31

229

Divergent Evolution in Reverse Transcriptase (RT) of HIV-1 Group O and M Lineages: Impact on Structure, Fitness, and Sensitivity to Nonnucleoside RT Inhibitors?  

PubMed Central

Natural evolution in primate lentiviral reverse transcriptase (RT) appears to have been constrained by the necessity to maintain function within an asymmetric protein composed of two identical primary amino acid sequences (66 kDa), of which one is cleaved (51 kDa). In this study, a detailed phylogenetic analysis now segregates groups O and M into clusters based on a cysteine or tyrosine residue located at position 181 of RT and linked to other signature residues. Divergent evolution of two group O (C181 or Y181) and the main (Y181 only) HIV-1 lineages did not appreciably impact RT activity or function. Group O RT structural models, based on group M subtype B RT crystal structures, revealed that most evolutionarily linked amino acids appear on a surface-exposed region of one subunit while in a noncatalytic RT pocket of the other subunit. This pocket binds nonnucleoside RT inhibitors (NNRTI); therefore, NNRTI sensitivity was used to probe enzyme differences in these group O and M lineages. In contrast to observations showing acquired drug resistance associated with fitness loss, the C181Y mutation in the C181 group O lineage resulted in a loss of intrinsic NNRTI resistance and was accompanied by fitness loss. Other mutations linked to the NNRTI-resistant C181 lineage also resulted in altered NNRTI sensitivity and a net fitness cost. Based on RT asymmetry and conservation of the intricate reverse transcription process, millions of years of divergent primate lentivirus evolution may be constrained to discrete mutations that appear primarily in the nonfunctional, solvent-accessible NNRTI binding pocket.

Tebit, Denis M.; Lobritz, Michael; Lalonde, Matthew; Immonen, Taina; Singh, Kamlendra; Sarafianos, Stefanos; Herchenroder, Ottmar; Krausslich, Hans-Georg; Arts, Eric J.

2010-01-01

230

TRAUMATIC SUBMACULAR HEMORRHAGE TREATED WITH rt-PA AND SF6 HEMORRAGIA SUBMACULAR TRAUMÁTICA TRATADA CON rt-PA y SF6  

Microsoft Academic Search

Case report: This patient was afflicted by a trau- matic submacular hemorrhage. A posterior vitrec- tomy was performed and intravitreal rt-PA and SF6 were administered. Four weeks later, the visual acuity had increased from 0.1 to 0.8. No complica- tions due to the treatment with rt-PA were reported. Discussion: It is known that waiting for the sponta- neous blood removal

HERAS-MULERO H; GARCÍA-GÓMEZ PJ; SÁDABA-ECHARRI LM; SALINAS-ALAMÁN A; GARCÍA-LAYANA A

231

Spinal reirradiation after short-course RT for metastatic spinal cord compression  

SciTech Connect

Purpose: To investigate the feasibility and effectiveness of reirradiation (re-RT) for in-field recurrence of metastatic spinal cord compression after primary RT with 1 x 8 Gy or 5 x 4 Gy. Methods and Materials: A total of 62 patients, treated with 1 x 8 Gy (n = 34) or 5 x 4 Gy (n = 28) between January 1995 and August 2003, received re-RT for in-field recurrence of metastatic spinal cord compression. The median time to recurrence was 6 months (range, 2-40 months). Re-RT was performed with 1 x 8 Gy (after 1 x 8 Gy or 5 x 4 Gy, n = 34), 5 x 3 Gy (after 1 x 8 Gy or 5 x 4 Gy, n = 15), or 5 x 4 Gy (after 1 x 8 Gy, n = 13). The cumulative biologically effective dose (primary RT plus re-RT) was 80-100 Gy{sub 2}. The median follow-up after re-RT was 8 months (range, 2-42 months). Motor function was evaluated up to 6 months after re-RT. Results: After re-RT, 25 patients (40%) showed improvement of motor function, 28 (45%) had no change, and 9 (15%) had deterioration. Of the 16 previously nonambulatory patients, 6 (38%) regained the ability to walk. No second in-field recurrence in the same spinal region was observed after re-RT. The outcome was not significantly influenced by the radiation schedule. Radiation myelopathy was not observed. Conclusions: Spinal re-RT with 1 x 8 Gy, 5 x 3 Gy, or 5 x 4 Gy for in-field recurrence of metastatic spinal cord compression appears safe and effective. Myelopathy seems unlikely, if the cumulative biologically effective dose is {<=}100 Gy{sub 2}.

Rades, Dirk [Department of Radiation Oncology, University Hospital Hamburg-Eppendorf, Hamburg (Germany)]. E-mail: Rades.Dirk@gmx.net; Stalpers, Lukas J.A. [Department of Radiotherapy, Academic Medical Center, Amsterdam (Netherlands); Veninga, Theo [Department of Radiotherapy, Dr. Bernard Verbeeten Institute, Tilburg (Netherlands); Hoskin, Peter J. [Mount Vernon Centre for Cancer Treatment, Northwood, Middlesex (United Kingdom)

2005-11-01

232

Potential Utility of the Real-Time TMPA-RT Precipitation Estimates in Streamflow Prediction.  

National Technical Information Service (NTIS)

We investigate the potential utility of the real-time Tropical Rainfall Measuring Mission (TRMM) Multi-satellite Precipitation Analysis (TMPA-RT) data for streamflow prediction, both through direct comparisons of TMPA-RT estimates with a gridded gauge pro...

D. P. Lettenmaier F. Su G. J. Huffman H. Gao

2010-01-01

233

Effects of Contextual Similarity and Target-Repetition Proportion on Negative Priming in RT Distributional Analyses  

ERIC Educational Resources Information Center

Participants' reaction time (RT) data in a prime-probe flanker task (e.g., ABA-CAC) were analyzed in terms of the characteristics of RT distribution to examine possible mechanisms that produce negative priming. When the prime and probe were presented in the same context and the proportion of repetition-target trials (TRP) was 0.33, negative…

Tse, Chi-Shing; Hutchison, Keith A.; Li, Yongna

2011-01-01

234

Validation of the RT3 in the measurement of physical activity in children  

Microsoft Academic Search

Summary The aim of this study was to assess the validity of the RT3 accelerometer, and its inbuilt algorithm, in measuring inactivity, walking and running in children. Twenty children, aged 7—12 years, participated in the study. The RT3 was compared to physiological energy expenditure obtained via a wireless portable ergospirometric system. Data analysis was performed using Bland and Altman plots

Juliette Hussey; Kathleen Bennett; Jamie O. Dwyer; Sinead Langford; Christopher Bell; John Gormley

2007-01-01

235

SPoRT-WRF: Evaluating the Impact of NASA Datasets on Convective Forecasts.  

National Technical Information Service (NTIS)

Short-term Prediction Research and Transition (SPoRT) seeks to improve short-term, regional weather forecasts using unique NASA products and capabilities SPoRT has developed a unique, real-time configuration of the NASA Unified Weather Research and Foreca...

A. Molthan B. Zavodsky D. Kozlowski J. Case

2012-01-01

236

The SPoRT Center - Infusing NASA Technology Into NWS WFO  

NSDL National Science Digital Library

This Webcast introduces the SPoRT Center, a joint NASA and National Weather Service project to provide unique NASA datasets to several forecast offices and evaluate their usefulness and impact on forecast operations. The presentation provides a description of the SPoRT Center, examples of its collaborations with weather forecast offices, and demonstrates use of MODIS data, AMSR-E derived products and lightning flash density product applications. It also includes mention of the projects the SPoRT Center will likely undertake in the future. The information contained in this Webcast reflects the status of the SPoRT program as of the summer of 2006. Since the SPoRT program evolves to meet NASA program objectives, some of the capabilities or activities portrayed in this presentation may have changed since its original production.

Spangler, Tim

2007-02-28

237

Viral RNA Levels and env Variants in Semen and Tissues of Mature Male Rhesus Macaques Infected with SIV by Penile Inoculation  

PubMed Central

HIV is shed in semen but the anatomic site of virus entry into the genital secretions is unknown. We determined viral RNA (vRNA) levels and the envelope gene sequence in the SIVmac 251 viral populations in the genital tract and semen of 5 adult male rhesus monkeys (Macaca mulatta) that were infected after experimental penile SIV infection. Paired blood and semen samples were collected from 1–9 weeks after infection and the monkeys were necropsied eleven weeks after infection. The axillary lymph nodes, testes, epididymis, prostate, and seminal vesicles were collected and vRNA levels and single-genome analysis of the SIVmac251 env variants was performed. At the time of semen collection, blood vRNA levels were between 3.09 and 7.85 log10 vRNA copies/ml plasma. SIV RNA was found in the axillary lymph nodes of all five monkeys and in 3 of 5 monkeys, all tissues examined were vRNA positive. In these 3 monkeys, vRNA levels (log10 SIVgag copies/ug of total tissue RNA) in the axillary lymph node (6.48±0.50) were significantly higher than in the genital tract tissues: testis (3.67±2.16; p<0.05), epididymis (3.08±1.19; p<0.0001), prostate (3.36±1.30; p<0.01), and seminal vesicle (2.67±1.50; p<0.0001). Comparison of the SIVmac251 env viral populations in blood plasma, systemic lymph node, and genital tract tissues was performed in two of the macaques. Visual inspection of the Neighbor-Joining phylograms revealed that in both animals, all the sequences were generally distributed evenly among all tissue compartments. Importantly, viral populations in the genital tissues were not distinct from those in the systemic tissues. Our findings demonstrate striking similarity in the viral populations in the blood and male genital tract tissues within 3 months of penile SIV transmission.

Fieni, Francis; Stone, Mars; Ma, Zhong-Min; Dutra, Joseph; Fritts, Linda; Miller, Christopher J.

2013-01-01

238

Comparison of a nucleoprotein gene based RT-PCR with real time RT-PCR for diagnosis of avian influenza in clinical specimens.  

PubMed

A nucleoprotein (NP) gene based reverse transcription polymerase chain reaction (npRT-PCR) assay was developed in our laboratory which could detect 35.09% of the experimental samples negative for virus isolation in first passage but positive by third passage. Reducing the reaction volume to 12.5 ?l did not alter the test sensitivity and the results did not vary when duplicate samples were run in a different thermal cycler. The positive and negative agreements of this test in clinical specimens were compared with a matrix gene based real time RT-PCR with virus isolation as standard. A total of 516 clinical specimens including tissues, swabs and feces submitted from various States of India as part of active surveillance for avian influenza were tested by npRT-PCR, RRT-PCR and virus isolation in 9-11 day old embryonated specific pathogen free chicken eggs. The positive and negative agreements of npRT-PCR with virus isolation were found to be 0.909±0.022 and 0.980±0.004 respectively and that of RRT-PCR with virus isolation were 0.902±0.023 and 0.977±0.005 respectively. Since the positive and negative agreements of both npRT-PCR and RRT-PCR tests were similar, we suggest that this test can be used by peripheral veterinary laboratories that do not have real time PCR facility for active surveillance of AIV. PMID:21723575

Nagarajan, S; Murugkar, H V; Tosh, C; Behera, P; Khandia, R; Jain, R; Katare, M; Syed, Z; Tripati, S; Dubey, S C

2011-07-01

239

The Receptor Complex Associated with Human T-Cell Lymphotropic Virus Type 3 (HTLV3) Env-Mediated Binding and Entry Is Distinct from, but Overlaps with, the Receptor Complexes of HTLV1 and HTLV2  

Microsoft Academic Search

Little is known about the transmission or tropism of the newly discovered human retrovirus, human T-cell lymphotropic virus type 3 (HTLV-3). Here, we examine the entry requirements of HTLV-3 using independently expressed Env proteins. We observed that HTLV-3 surface glycoprotein (SU) binds efficiently to both activated CD4 and CD8 T cells. This contrasts with both HTLV-1 SU, which primarily binds

Kathryn S. Jones; Ying K. Huang; Sebastien A. Chevalier; Philippe V. Afonso; Cari Petrow-Sadowski; Daniel C. Bertolette; Antoine Gessain; Francis W. Ruscetti; Renaud Mahieux

2009-01-01

240

Trafficking of Porin-Deficient Salmonella typhimurium Mutants inside HeLa Cells: ompR and envZ Mutants Are Defective for the Formation of Salmonella-Induced Filaments  

Microsoft Academic Search

Outer membrane porin genes of Salmonella typhimurium, including ompC, ompF, and tppB, are regulated by the products of ompB, a two-component regulatory locus encoding OmpR and EnvZ. S. typhimurium ompR mutants are attenuated in mice, but to date no one has studied the intracellular trafficking of S. typhimurium porin-deficient mutants. In this study, isogenic transposon mutants of S. typhimurium with

SCOTT D. MILLS; SHARON R. RUSCHKOWSKI; MURRY A. STEIN; B. BRETT FINLAY

1998-01-01

241

Clinical investigation survival prediction in high-grade gliomas by MRI perfusion before and during early stage of RT  

SciTech Connect

Purpose: To determine whether cerebral blood volume (CBV) and cerebral blood flow can predict the response of high-grade gliomas to radiotherapy (RT) by taking into account spatial heterogeneity and temporal changes in perfusion. Methods and Materials: Twenty-three patients with high-grade gliomas underwent conformal RT, with magnetic resonance imaging perfusion before and at Weeks 1-2 and 3-4 during RT. Tumor perfusion was classified as high, medium, or low. The prognostic values of pre-RT perfusion and the changes during RT for early prediction of tumor response to RT were evaluated. Results: The fractional high-CBV tumor volume before RT and the fluid-attenuated inversion recovery imaging tumor volume were identified as predictors for survival (p = 0.01). Changes in tumor CBV during the early treatment course also predicted for survival. Better survival was predicted by a decrease in the fractional low-CBV tumor volume at Week 1 of RT vs. before RT, a decrease in the fractional high-CBV tumor volume at Week 3 vs. Week 1 of RT, and a smaller pre-RT fluid-attenuated inversion recovery imaging tumor volume (p = 0.01). Conclusion: Early temporal changes during RT in heterogeneous regions of high and low perfusion in gliomas might predict for different physiologic responses to RT. This might also open the opportunity to identify tumor subvolumes that are radioresistant and might benefit from intensified RT.

Cao Yue [Department of Radiation Oncology, University of Michigan, Ann Arbor, MI (United States) and Department of Radiology, University of Michigan, Ann Arbor, MI (United States)]. E-mail: yuecao@med.umich.edu; Tsien, Christina I. [Department of Radiation Oncology, University of Michigan, Ann Arbor, MI (United States); Nagesh, Vijaya [Department of Radiation Oncology, University of Michigan, Ann Arbor, MI (United States); Junck, Larry [Department of Neurology, University of Michigan, Ann Arbor, MI (United States); Haken, Randall ten [Department of Radiation Oncology, University of Michigan, Ann Arbor, MI (United States); Ross, Brian D. [Department of Radiology, University of Michigan, Ann Arbor, MI (United States); Chenevert, Thomas L. [Department of Radiology, University of Michigan, Ann Arbor, MI (United States); Lawrence, Theodore S. [Department of Radiation Oncology, University of Michigan, Ann Arbor, MI (United States)

2006-03-01

242

Detection of immunoreactive epitopes in proteins encoded by gag, env, and pol genes of human T-lymphotropic virus type I using synthetic peptides  

SciTech Connect

Reactivity of 26 synthetic peptides that comprise 12 to 26 amino acid residues corresponding to segments of the p19 (gag), gp46 (env), and pol proteins (pol) of human T-lymphotropic virus type I toward 31 positive sera was studied using enzyme-linked immunosorbent assay. Specific reactivity with high titers of antibodies (presented in reciprocal dilution values) was detected for the synthetic peptides corresponding to fragments 110-130 and 100-130 (titers up to 4050) of p19, 174-197 (up to 800), 186-201 (up to to 4050), 191-215 (up to 1350), 242-257 (up to 800), and 272-292 (up to 450) of gp46. Immunoreactivity of seven peptides, fragments of pol-proteins, was weak. New linear epitopes in the regions 145-158, 272-277, and 292-300 of gp46 were detected. In addition, location of the known linear epitopes in p19 and gp46 was refined on the basis of comparative study of overlapping peptides from these proteins. 25 refs., 4 tabs.

Yaroslavtseva, N.G.; Kornilaeva, G.V.; Pashkova, T.A. [Ivanovskii Inst. of Virology, Moscow (Russian Federation)] [and others

1995-10-01

243

Prevalence of fowl glioma-inducing virus in chickens of zoological gardens in Japan and nucleotide variation in the env gene.  

PubMed

Fowl glioma-inducing virus (FGV), which belongs to subgroup A of avian leukosis virus (ALV), is tumorigenic in the nervous system. In a zoological garden in Japan, approximately 40% of chickens, including Japanese fowls, were infected with FGV. Because this zoological garden plays a role as a major supplier of Japanese fowl for other zoological gardens, FGV infection is suspected to have spread among ornamental chickens. In this study, the prevalence of the disease was examined in a total of 129 chickens in three other zoological gardens by nested polymerase chain reaction (PCR), reverse transcription nested PCR and enzyme-linked immunosorbent assay. Twenty-six to 56 percent of the fowls in each of the examined gardens were positive by nested PCR. The phylogenetic analysis revealed that the 3' untranslated region, including the specific sequence of FGV, of the 14 isolated ALVs showed high sequence identity and a close relationship with FGV. In addition, the env gene of the isolates frequently showed mutations and deletions of nucleotides. These results suggest that FGV is prevalent among ornamental chickens kept in zoological gardens in Japan. PMID:18525168

Hatai, Hitoshi; Ochiai, Kenji; Murakami, Mariko; Imanishi, Syunsuke; Tomioka, Yukiko; Toyoda, Takeshi; Ohashi, Kazuhiko; Umemura, Takashi

2008-05-01

244

Human immunodeficiency virus type 1 Vif does not influence expression or virion incorporation of gag-, pol-, and env-encoded proteins.  

PubMed Central

The Vif protein of human immunodeficiency virus type 1 is required for productive replication in peripheral blood lymphocytes and a limited number of immortalized T-lymphoid lines (nonpermissive cells). In contrast, Vif is fully dispensable for virus replication in other T-cell lines (permissive cells). Because the infection phenotype of released virions is determined by producer cells and by the presence of Vif in those cells, we have analyzed the protein contents of purified viral particles in an attempt to define compositional differences that could explain the infection phenotype. Surprisingly, we were unable to discern any Vif- or cell-type-dependent quantitative or qualitative difference in the Gag, Pol, and Env proteins of virions or virus-producing cells that correlates with virus infectivity. We were, however, able to demonstrate that Vif itself is present in virions and, using semiquantitative Western blotting (immunoblotting), that there is an average of 30 to 80 molecules of Vif incorporated into each virion. Importantly, parallel analyses of total lysates of the producer cells revealed that the cell-associated expression levels of Vif are close to those of the Gag proteins. Given the dramatically higher abundance of Vif in cells than in virions, we speculate that Vif exerts its principal activity during the processes of virus assembly and budding and that this function could be of a structural-conformational nature.

Fouchier, R A; Simon, J H; Jaffe, A B; Malim, M H

1996-01-01

245

Induction of disease by a molecularly cloned highly pathogenic simian immunodeficiency virus/human immunodeficiency virus chimera is multigenic.  

PubMed

One of three full-length infectious molecular clones of SHIV(DH12R), designated SHIV(DH12R-CL-7) and obtained from productively infected rhesus monkey peripheral blood mononuclear cells, directed rapid and irreversible loss of CD4+ T cells within 3 weeks of its inoculation into Indian rhesus monkeys. Induction of complete CD4+ T-cell depletion by SHIV(DH12R-CL-7) was found to be dependent on inoculum size. The acquisition of this pathogenic phenotype was accompanied by the introduction of 42 amino acid substitutions into multiple genes of parental nonpathogenic SHIV(DH12). Transfer of the entire SHIV(DH12R-CL-7) env gene into the genetic background of nonpathogenic SHIV(DH12) failed to confer the rapid CD4+ T-lymphocyte-depleting syndrome; similarly, the substitution of gag plus pol sequences from SIV(smE543) for analogous SIV(mac239) genes in SHIV(DH12R-CL-7) attenuated the pathogenic phenotype. Amino acid changes affecting multiple viral genes are necessary, but insufficient by themselves, to confer the prototypically rapid and irreversible CD4+ T-cell-depleting phenotype exhibited by molecularly cloned SHIV(DH12R-CL-7). PMID:15113931

Sadjadpour, Reza; Theodore, Theodore S; Igarashi, Tatsuhiko; Donau, Olivia K; Plishka, Ronald J; Buckler-White, Alicia; Martin, Malcolm A

2004-05-01

246

A novel detection system for the genetically modified canola (Brassica rapa) line RT73.  

PubMed

The herbicide-tolerant genetically modified Roundup Ready canola (Brassica napus) line RT73 has been approved worldwide for use in animal feed and human food. However, RT73 Brassica rapa lines derived from interspecific crosses with RT73 B. napus have not been approved in Japan. Here, we report on a novel system using individual kernel analyses for the qualitative detection of RT73 B. rapa in canola grain samples. We developed a duplex real-time polymerase chain reaction (PCR) method to discriminate B. napus and B. rapa DNA using scatter plots of the end-point analyses; this method was able to discriminate a group comprising B. rapa and Brassica juncea from a group comprising B. napus, Brassica carinata, and Brassica oleracea. We also developed a duplex real-time PCR method for the simultaneous detection of an RT73-specific sequence and an endogenous FatA gene. Additionally, a DNA-extraction method using 96-well silica-membrane plates was developed and optimized for use with individual canola kernels. Our detection system could identify RT73 B. rapa kernels in canola grain samples enabling the accurate and reliable monitoring of RT73 B. rapa contamination in canola, thus playing a role in its governmental regulation in Japan. PMID:21049930

Akiyama, Hiroshi; Makiyama, Daiki; Nakamura, Kosuke; Sasaki, Nobuhiro; Minegishi, Yasutaka; Mano, Junichi; Kitta, Kazumi; Ozeki, Yoshihiro; Teshima, Reiko

2010-11-04

247

The Receptor Complex Associated with Human T-Cell Lymphotropic Virus Type 3 (HTLV-3) Env-Mediated Binding and Entry Is Distinct from, but Overlaps with, the Receptor Complexes of HTLV-1 and HTLV-2?  

PubMed Central

Little is known about the transmission or tropism of the newly discovered human retrovirus, human T-cell lymphotropic virus type 3 (HTLV-3). Here, we examine the entry requirements of HTLV-3 using independently expressed Env proteins. We observed that HTLV-3 surface glycoprotein (SU) binds efficiently to both activated CD4+ and CD8+ T cells. This contrasts with both HTLV-1 SU, which primarily binds to activated CD4+ T cells, and HTLV-2 SU, which primarily binds to activated CD8+ T cells. Binding studies with heparan sulfate proteoglycans (HSPGs) and neuropilin-1 (NRP-1), two molecules important for HTLV-1 entry, revealed that these molecules also enhance HTLV-3 SU binding. However, unlike HTLV-1 SU, HTLV-3 SU can bind efficiently in the absence of both HSPGs and NRP-1. Studies of entry performed with HTLV-3 Env-pseudotyped viruses together with SU binding studies revealed that, for HTLV-1, glucose transporter 1 (GLUT-1) functions at a postbinding step during HTLV-3 Env-mediated entry. Further studies revealed that HTLV-3 SU binds efficiently to naïve CD4+ T cells, which do not bind either HTLV-1 or HTLV-2 SU and do not express detectable levels of HSPGs, NRP-1, and GLUT-1. These results indicate that the complex of receptor molecules used by HTLV-3 to bind to primary T lymphocytes differs from that of both HTLV-1 and HTLV-2.

Jones, Kathryn S.; Huang, Ying K.; Chevalier, Sebastien A.; Afonso, Philippe V.; Petrow-Sadowski, Cari; Bertolette, Daniel C.; Gessain, Antoine; Ruscetti, Francis W.; Mahieux, Renaud

2009-01-01

248

HPRS-103 (exogenous avian leukosis virus, subgroup J) has an env gene related to those of endogenous elements EAV-0 and E51 and an E element found previously only in sarcoma viruses.  

PubMed

The avian leukosis and sarcoma virus (ALSV) group comprises eight subgroups based on envelope properties. HPRS-103, an exogenous retrovirus recently isolated from meat-type chicken lines, is similar to the viruses of these subgroups in group antigen but differs from them in envelope properties and has been assigned to a new subgroup, J. HPRS-103 has a wide host range in birds, and unlike other nontransforming ALSVs which cause late-onset B-cell lymphomas, HPRS-103 causes late-onset myelocytomas. Analysis of the sequence of an infectious clone of the complete proviral genome indicates that HPRS-103 is a multiple recombinant of at least five ALSV sequences and one EAV (endogenous avian retroviral) sequence. The HPRS-103 env is most closely related to the env gene of the defective EAV-E51 but divergent from those of other ALSV subgroups. Probing of restriction digests of line 0 chicken genomic DNA has identified a novel group of endogenous sequences (EAV-HP) homologous to that of the HPRS-103 env gene but different from sequences homologous to EAV and E51. Unlike other replication-competent nontransforming ALSVs, HPRS-103 has an E element in its 3' noncoding region, as found in many transforming ALSVs. A deletion found in the HPRS-103 U3 EFII enhancer factor-binding site is also found in all replication-defective transforming ALSVs (including MC29, which causes rapid-onset myelocytomas). PMID:7815543

Bai, J; Payne, L N; Skinner, M A

1995-02-01

249

HPRS-103 (exogenous avian leukosis virus, subgroup J) has an env gene related to those of endogenous elements EAV-0 and E51 and an E element found previously only in sarcoma viruses.  

PubMed Central

The avian leukosis and sarcoma virus (ALSV) group comprises eight subgroups based on envelope properties. HPRS-103, an exogenous retrovirus recently isolated from meat-type chicken lines, is similar to the viruses of these subgroups in group antigen but differs from them in envelope properties and has been assigned to a new subgroup, J. HPRS-103 has a wide host range in birds, and unlike other nontransforming ALSVs which cause late-onset B-cell lymphomas, HPRS-103 causes late-onset myelocytomas. Analysis of the sequence of an infectious clone of the complete proviral genome indicates that HPRS-103 is a multiple recombinant of at least five ALSV sequences and one EAV (endogenous avian retroviral) sequence. The HPRS-103 env is most closely related to the env gene of the defective EAV-E51 but divergent from those of other ALSV subgroups. Probing of restriction digests of line 0 chicken genomic DNA has identified a novel group of endogenous sequences (EAV-HP) homologous to that of the HPRS-103 env gene but different from sequences homologous to EAV and E51. Unlike other replication-competent nontransforming ALSVs, HPRS-103 has an E element in its 3' noncoding region, as found in many transforming ALSVs. A deletion found in the HPRS-103 U3 EFII enhancer factor-binding site is also found in all replication-defective transforming ALSVs (including MC29, which causes rapid-onset myelocytomas).

Bai, J; Payne, L N; Skinner, M A

1995-01-01

250

Interleukin12 (IL12) Enhancement of the Cellular Immune Response against Human Immunodeficiency Virus Type 1 Env Antigen in a DNA Prime\\/Vaccinia Virus Boost Vaccine Regimen Is Time and Dose Dependent: Suppressive Effects of IL12 Boost Are Mediated by Nitric Oxide  

Microsoft Academic Search

We previously demonstrated that codelivery of interleukin-12 (IL-12) with the human immunodeficiency virus type 1 (HIV-1) Env antigen from a recombinant vaccinia virus (rVV) can enhance the specific anti-Env cell-mediated immune (CMI) response. In the present study, we have investigated the effects of IL-12 in mice when it is expressed in a DNA prime\\/VV boost vaccine regimen. The delivery of

M. MAGDALENA GHERARDI; JUAN C. RAMIREZ; MARIANO ESTEBAN

2000-01-01

251

The RT-18: a new screening tool to assess young adult risk-taking behavior  

PubMed Central

Risk-taking behavior is a major determinant of health and plays a central role in various diseases. Therefore, a brief questionnaire was developed to assess risk taking among young adults with known different levels of risk-taking behavior (social drinkers and recreational drug users). In Study 1, N = 522 university students completed the RT-18 risk taking questionnaire. N = 100 students were retested after 2 to 4 weeks and performed the Cambridge Gambling Task (CGT). Mean RT-18 score was 7.69 and Cronbach’s alpha was 0.886. The test-retest reliability was r = 0.94. Significant correlation was found between the RT-18 score and CGT scores of risk taking, bet proportion, and risk adjustment. In Study 2, N = 7834 young adult social drinkers, and recreational drug users, mean RT-18 score was 9.34 and Cronbach’s alpha was 0.80. Factor analysis showed that the RT-18 comprises two factors assessing level of risk-taking behavior and risk assessment. Men scored significantly higher than women on the RT-18. Recreational drug users had significantly higher scores when compared to social drinkers. In Study 3 of N = 1000 students, construct validity was confirmed by showing that the RT-18 outcome correlates significantly with scores on the Stimulating-Instrumental Risk Inventory. In conclusion, the RT-18 is a valid and reliable screening tool to differentiate levels of risk-taking behavior. This short scale is quick and practical to administer, imposing minimal demands on participants. The RT-18 is able to differentiate risk taking and risk assessment which can help target appropriate intervention strategies.

de Haan, Lydia; Kuipers, Esther; Kuerten, Yvanca; van Laar, Margriet; Olivier, Berend; Verster, Joris Cornelis

2011-01-01

252

Character of HBV (hepatitis B virus) polymerase gene rtM204V/I and rtL180M mutation in patients with lamivudine resistance  

PubMed Central

Objectives: To investigate the relationship between HBV (hepatitis B virus) polymerase gene 180 and 204 sites mutation and lamivudine resistance. Methods: One hundred forty-one patients with lamivudine resistance after lamivudine treatment and 60 chronic hepatitis B patients without lamivudine treatment were enrolled in this study. The serum HBV DNA mutation was analyzed by sequence detection via polymerase chain reaction (PCR). The sequences of the same patient were analyzed before and after lamivudine treatment. Results: One hundred and nine lamivudine resistance patients had HBV YMDD (tyrosine-methionine-aspartate-aspartate) mutation. Among them, 45 patients had rtL180M/M204V mutation (41.28%), 28 patients had rtL180M/M204I mutation (25.70%) and 36 patients had rtM204I mutation (33.02%). There were 6 patients with rtL180M mutation in 32 lamivudine resistance patients. Sixty chronic hepatitis patients without lamivudine treatment had no mutations. Conclusions: HBV mutations, which play an important role in lamivudine resistance usually locate at polymerase gene 204 site; 180 site mutation was also observed in these patients. Evaluation of the anti-virus therapy by surveillance of the two sites mutations is of importance.

Li, Min-wei; Hou, Wei; Wo, Jian-er; Liu, Ke-zhou

2005-01-01

253

The mutation T477A in HIV1 reverse transcriptase (RT) restores normal proteolytic processing of RT in virus with Gag-Pol mutated in the p51-RNH cleavage site  

Microsoft Academic Search

BACKGROUND: The p51 subunit of the HIV-1 reverse transcriptase (RT) p66\\/p51 heterodimer arises from proteolytic cleavage of the RT p66 subunit C-terminal ribonuclease H (RNH) domain during virus maturation. Our previous work showed that mutations in the RT p51?RNH cleavage site resulted in virus with defects in proteolytic processing of RT and significantly attenuated infectivity. In some cases, virus fitness

Michael E Abram; Stefan G Sarafianos; Michael A Parniak

2010-01-01

254

CYCLIC VARIATIONS OF ORBITAL PERIOD AND LONG-TERM LUMINOSITY IN CLOSE BINARY RT ANDROMEDAE  

SciTech Connect

Solutions of standard VR light curves for the eclipsing binary RT And were obtained using the PHOEBE program (ver. 0.3a). Absolute parameters of the stellar components were then determined, enabling them to be positioned on the mass-luminosity diagram. Times of minima data ({sup O} - C curve) were analyzed using the method of Kalimeris et al. A cyclic variation in the orbital period and brightness, with timescales of about 11.89 and 12.50 yr were found, respectively. This is associated with a magnetic activity cycle modulating the orbital period of RT And via the Applegate mechanism. To check the consistency of the Applegate model, we have estimated some related parameters of the RT And system. The calculated parameters were in accordance with those estimated by Applegate for other similar systems, except B, the subsurface magnetic field of which shows a rather high value for RT And.

Manzoori, Davood [Department of Physics, University of Mohaghegh Ardabili, P.O. Box 179, Ardabil (Iran, Islamic Republic of)], E-mail: d.manzoori@uma.ac.ir

2009-12-15

255

Detecting Newcastle disease virus in combination of RT-PCR with red blood cell absorption  

PubMed Central

Reverse transcription-polymerase chain reaction (RT-PCR) has limited sensitivity when treating complicated samples, such as feces, waste-water in farms, and nucleic acids, protein rich tissue samples, all the factors may interfere with the sensitivity of PCR test or generate false results. In this study, we developed a sensitive RT-PCR, combination of red blood cell adsorption, for detecting Newcastle disease virus (NDV). One pair of primers which was highly homologous to three NDV pathotypes was designed according to the consensus nucleocapsid protein (NP) gene sequence. To eliminate the interfere of microbes and toxic substances, we concentrated and purified NDV from varied samples utilizing the ability of NDV binding red blood cells (RBCs). The RT-PCR coupled with red blood cell adsorption was much more sensitive in comparison with regular RT-PCR. The approach could also be used to detect other viruses with the property of hemagglutination, such as influenza viruses.

2011-01-01

256

Aerobic and Resistance Training Effects on Energy Intake: The STRRIDE AT/RT Study  

PubMed Central

Purpose Our study characterizes food and energy intake responses to long-term aerobic (AT) and resistance training (RT) during a controlled 8-month trial. Methods In the STRRIDE AT/RT trial, overweight/obese sedentary dyslipidemic men and women were randomized to AT (n = 39), RT (n = 38), or a combined treatment (AT/RT; n = 40) without any advice to change their food intakes. Quantitative food intake assessments (QDI) and food frequency questionnaires (FFQ) were collected at baseline (BEF) and after 8 mo. training (END); body mass (BM) and fat free mass (FFM) were also assessed. Results In AT and AT/RT, respectively, meaningful decreases in reported energy intake (REI) (?217 and ?202 kcal; p < 0.001) and in intakes of fat (?14.9 and ?14.9 g; p < 0.001, p = 0.004), protein (?8.3 and ?10.7 g; p = 0.002, p < 0.001), and carbohydrate (?28.1 and ?14.7 g; p = 0.001, p = 0.030) were found by FFQ. REI relative to FFM decreased (p < 0.001 and p=0.002) as did intakes of fat (?0.2 and ?0.3 g; p = 0.003 and p = 0.014) and protein (?0.1 and ?0.2 g; p = 0.005 and p < 0.001) in AT and AT/RT and carbohydrate (?0.5 g; p<0.003) in AT only. For RT, REI by QDI decreased (?3.0 kcal/kg FFM; p=0.046), as did fat intake (?0.2 g; p = 0.033). BM decreased in AT (?1.3 kg, p=0.006) and AT/RT (?1.5 kg, p = 0.001) but was unchanged (0.6 kg, p = 0.176) in RT. Conclusions Previously sedentary subjects completing 8 months of AT or AT/RT reduced their intakes of kcal and macronutrients and BM. In RT, fat intakes and REI (when expressed per FFM) decreased, BM was unchanged, and FFM increased.

Bales, Connie W.; Hawk, Victoria H.; Granville, Esther O.; Rose, Sarah B.; Shields, Tamlyn; Bateman, Lori; Willis, Leslie; Piner, Lucy; Slentz, Cris A.; Houmard, Joseph A.; Gallup, Dianne; Samsa, Greg P.; Kraus, William E.

2012-01-01

257

Duplex nested RT-PCR for detection of Nipah virus RNA from urine specimens of bats  

Microsoft Academic Search

A method for duplex nested RT-PCR (nRT-PCR) with internal control (IC) for the detection of Nipah virus RNA is described. Incorporation of IC RNA distinguished false and true negative results. The extrinsic RNA was added directly to the PCR master mix and co-amplified with virus specific RNA in a duplex reaction to determine the presence of PCR inhibitor. Limit of

Supaporn Wacharapluesadee; Thiravat Hemachudha

2007-01-01

258

RT-IPC: An IPC Extension for Real-Time Mach  

Microsoft Academic Search

Interprocess communication (IPC) provides the fundamental mechanism for the Machmicrokernel to be extensible and flexible. Mach IPC provides efficient communicationmechanisms for many applications. However, it does not provide sufficient functionalityfor real-time applications which have rigid timing constraints among threads. In RealTimeMach (RT-Mach), we have extended Mach IPC to be priority inversion free forreal-time applications.This paper describes the Real-Time IPC (RT-IPC)...

Takuro Kitayama; Hideyuki Tokuda; Tatsuo Nakajima

1993-01-01

259

Search for new non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-1 RT).  

PubMed

The derivatives of dibenzoxazocinone, dibenzoxadiazocine, dibenzoxadiazonine, and benzodiazepine systems were synthesized as potential lead compounds for inhibitors of HIV-1 reverse transcriptase. The suggested structures were derived from analysis of the described spatial and physicochemical requirements for HIV-1 RT inhibitors. All of the evaluated compounds (apart from the oxadiazocine derivatives) showed a week inhibitory activity on recombinant HIV-1 RT from Escherichia coli. PMID:12602800

Brzezi?ska, Elzbieta; Glinka, Ryszard

260

Moving from Batch to Field Using the RT3D Reactive Transport Modeling System  

Microsoft Academic Search

The public domain reactive transport code RT3D (Clement, 1997) is a general-purpose numerical code for solving coupled, multi-species reactive transport in saturated groundwater systems. The code uses MODFLOW to simulate flow and several modules of MT3DMS to simulate the advection and dispersion processes. RT3D employs the operator-split strategy which allows the code solve the coupled reactive transport problem in a

T. P. Clement; T. R. Gautam

2002-01-01

261

RT-PCR, nucleotide, amino acid and phylogenetic analyses of enterovirus type 71 strains from Asia  

Microsoft Academic Search

A specific and sensitive method based on RT-PCR was developed to detect enterovirus 71 (EV71) from patients with hand, foot and mouth disease, myocarditis, aseptic meningitis and acute flaccid paralysis. RT-PCR primers from conserved parts of the VP1 capsid gene were designed on the basis of good correlation with sequences of EV71 strains. These primers successfully amplified 44 strains of

Sunita Singh; Vincent T. K. Chow; K. P. Chan; A. E. Ling; C. L. Poh

2000-01-01

262

Improvement of HRV Quantification Using cRNA-Based Standards for Real Time RT-PCR  

Microsoft Academic Search

Real Time RT-PCR developed in recent years represents an useful tool in the diagnosis of RNA viruses. In order to accurately\\u000a quantify and normalize a RNA target, efficiency of reverse-transcription must be considered. In this study, a cRNA-standard-based\\u000a quantitative Real Time RT-PCR have been developed for HRV quantification on bronchoalveolar lavage (BAL) specimens. Results\\u000a has been compared to a quantitative

Maria Elena Terlizzi; Massimiliano Bergallo; Sara Astegiano; Francesca Sidoti; Stefano Gambarino; Paolo Solidoro; Cristina Costa; Rossana Cavallo

2011-01-01

263

Method study for RT-Flex HPCR low-speed diesel engine injecting law measuring  

Microsoft Academic Search

For researching the injecting law of low-speed HPCR diesel engine, a novel measuring method is invited. This method provide a certain reference to solve the problem that RT-Flex HPCR low-speed diesel engine fuel injection law can not be measured directly under the present conditions. Under different load working conditions on the RT-Flex60C low-speed diesel engine HPCR (high pressure common-rail) HIL(Hardware

Wang Zheng; Yang Jian-guo

2010-01-01

264

Quantitation of RT-PCR amplified cytokine mRNA by aequorin-based bioluminescence immunoassay  

Microsoft Academic Search

We described here a bioluminescence-based immunoassay for the quantitation of RT-PCR amplified cytokine mRNA. This technique uses a standard RT-PCR procedure, with the following modifications. The forward primer in the PCR reaction is labeled with a 5? biotin molecule. Following PCR, a digoxigenin-conjugated oligonucleotide probe is hybridized to the target biotin-labeled DNA template. The hybridized duplex is captured onto a

Lihua Xiao; Chunfu Yang; Cecilia O. Nelson; Brian P. Holloway; Venkatachalam Udhayakumar; Altaf A. Lal

1996-01-01

265

Direct and rapid detection of porcine epidemic diarrhea virus by RT-PCR  

Microsoft Academic Search

To establish a practical method for detecting porcine epidemic diarrhea virus (PEDV), the use of primers derived from sequences that amplify the M protein genes of PEDV in a RT-PCR detection system was investigated. Primers were designed to amplify a 854-bp fragment by RT-PCR. This reaction was specific to the PEDV RNA but not to that of other viral genera

Kiyoyasu Ishikawa; Hideto Sekiguchi; Tomoe Ogino; Shoko Suzuki

1997-01-01

266

Natural and Enhanced Attenuation of Chlorinated Solvents Using RT3D  

Microsoft Academic Search

RT3D (Reactive Transport in 3-Dimensions) is a reactive transport code that can be applied to model solute fate and transport for many different purposes. This document specifically addresses application of RT3D for modeling related to evaluation and implementation of Monitored Natural Attenuation (MNA). Selection of MNA as a remedy requires an evaluation process to demonstrate that MNA will meet the

Christian D. Johnson; Michael J. Truex; T. P. Clement

2006-01-01

267

Quantitative RT-PCR methods for mature microRNA expression analysis.  

PubMed

This chapter describes two methods to measure expression of mature miRNA levels using qRT-PCR. The first method uses stem-loop RT primers to produce cDNA for specific miRNAs, a technique that our laboratory has modified to increase the number of miRNAs being reverse transcribed within a single RT reaction from one (as suggested by the manufacturer) to five. The second method uses a modified oligo(dT) technique to reverse transcribe all transcripts within an RNA sample; therefore, target miRNA and normalizing mRNA can be analyzed from the same RT reaction. We examined the level of miRNA-132, a miRNA known to be upregulated in granulosa cells following hCG treatment, using both of these methods. Data were normalized to GAPDH or snU6 and evaluated by DeltaDeltaCt and standard curve analysis. There was no significant difference (P > 0.05) in miRNA-132 expression between the stem-loop and modified oligo(dT) RT methods indicating that both are statistically equivalent. However, from a technical point of view, the modified oligo(dT) method was less time consuming and required only a single RT reaction to reverse transcribe both miRNA and mRNA. PMID:20300990

Fiedler, Stephanie D; Carletti, Martha Z; Christenson, Lane K

2010-01-01

268

Does inhibiting Sur1 complement rt-PA in cerebral ischemia?  

PubMed Central

Hemorrhagic transformation (HT) associated with recombinant tissue plasminogen activator (rt-PA) complicates and limits its use in stroke. Here, we provide a focused review on the involvement of matrix metalloproteinase 9 (MMP-9) in rt-PA–associated HT in cerebral ischemia, and we review emerging evidence that the selective inhibitor of the sulfonylurea receptor 1 (Sur1), glibenclamide (U.S. adopted name, glyburide), may provide protection against rt-PA–associated HT in cerebral ischemia. Glyburide inhibits activation of MMP-9, ameliorates edema formation, swelling, and symptomatic hemorrhagic transformation, and improves preclinical outcomes in several clinically relevant models of stroke, both without and with rt-PA treatment. A retrospective clinical study comparing outcomes in diabetic patients with stroke treated with rt-PA showed that those who were previously on and were maintained on a sulfonylurea fared significantly better than those whose diabetes was managed without sulfonylureas. Inhibition of Sur1 with injectable glyburide holds promise for ameliorating rt-PA–associated HT in stroke.

Simard, J. Marc; Geng, Zhihua; Silver, Frank L.; Sheth, Kevin N.; Kimberly, W. Taylor; Stern, Barney J.; Colucci, Mario; Gerzanich, Volodymyr

2012-01-01

269

Detection of canine distemper virus (CDV) through one step RT-PCR combined with nested PCR.  

PubMed

A one step reverse transcription PCR (RT-PCR) combined nested PCR was set up to increase efficiency in the diagnosis of canine distemper virus (CDV) infection after developement of nested PCR. Two PCR primer sets were designed based on the sequence of nucleocapsid gene of CDV Onderstepoort strain. One-step RT-PCR with the outer primer pair was revealed to detect 10(2) PFU/ml. The sensitivity was increased hundredfold using the one-step RT-PCR combined with the nested PCR. Specificity of the PCR was also confirmed using other related canine virus and peripheral blood mononuclear cells (PBMC) and body secretes of healthy dogs. Of the 51 blood samples from dogs clinically suspected of CD, 45 samples were revealed as positive by one-step RT-PCR combined with nested PCR. However, only 15 samples were identified as positive with a single one step RT-PCR. Therefore approximately 60% increase in the efficiency of the diagnosis was observed by the combined method. These results suggested that one step RT-PCR combined with nested PCR could be a sensitive, specific, and practical method for diagnosis of CDV infection. PMID:14614296

Kim, Y H; Cho, K W; Youn, H Y; Yoo, H S; Han, H R

2001-04-01

270

An efficient virus concentration method and RT-nested PCR for detection of rotaviruses in environmental water samples  

Microsoft Academic Search

Water samples were concentrated by the modified adsorption–elution technique followed by speedVac reconcentration of the filter eluates. Reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) was used to detect rotavirus RNA in concentrates of the water. The detection limit of the rotavirus determined by RT-nested PCR alone was about 1.67 plaque forming units (PFU) per RT-PCR assay and that by RT-nested

Leera Kittigul; Som Ekchaloemkiet; Fuangfa Utrarachkij; Kanokrat Siripanichgon; Dusit Sujirarat; Supornvit Pungchitton; Augsana Boonthum

2005-01-01

271

Relationships Between Educator Beliefs, Perceptions of Educational Practices and Skills, PS\\/RtI Implementation, and Educational Outcomes  

Microsoft Academic Search

This study examined the relationships between pilot school status and Problem-Solving\\/Response to Intervention (PS\\/RtI) implementation, educator variables and PS\\/RtI implementation, and PS\\/RtI implementation and student and systemic outcomes following the final year of a 3-year PS\\/RtI implementation Project. School-Based Leadership Team (SBLT) members from 34 pilot schools in seven demonstration districts received training, as well as ongoing technical assistance and

Kevin Stockslager

2011-01-01

272

A novel method for the normalization of microRNA RT-PCR data  

PubMed Central

Background MicroRNAs (miRNAs) are short non-coding RNA molecules that regulate mRNA transcript levels and translation. Deregulation of microRNAs is indicated in a number of diseases and microRNAs are seen as a promising target for biomarker identification and drug development. miRNA expression is commonly measured by microarray or real-time polymerase chain reaction (RT-PCR). The findings of RT-PCR data are highly dependent on the normalization techniques used during preprocessing of the Cycle Threshold readings from RT-PCR. Some of the commonly used endogenous controls themselves have been discovered to be differentially expressed in various conditions such as cancer, making them inappropriate internal controls. Methods We demonstrate that RT-PCR data contains a systematic bias resulting in large variations in the Cycle Threshold (CT) values of the low-abundant miRNA samples. We propose a new data normalization method that considers all available microRNAs as endogenous controls. A weighted normalization approach is utilized to allow contribution from all microRNAs, weighted by their empirical stability. Results The systematic bias in RT-PCR data is illustrated on a microRNA dataset obtained from primary cutaneous melanocytic neoplasms. We show that through a single control parameter, this method is able to emulate other commonly used normalization methods and thus provides a more general approach. We explore the consistency of RT-PCR expression data with microarray expression by utilizing a dataset where both RT-PCR and microarray profiling data is available for the same miRNA samples. Conclusions A weighted normalization method allows the contribution of all of the miRNAs, whether they are highly abundant or have low expression levels. Our findings further suggest that the normalization of a particular miRNA should rely on only miRNAs that have comparable expression levels.

2013-01-01

273

Factors Influencing Real-Time RT-PCR Results: Application of Real-Time RT-PCR for the Detection of Leukemia Translocations  

Microsoft Academic Search

A variety of factors that influence real-time reverse transcriptase (RT)-PCR results were examined. Real- time reaction volumes can be decreased from the manufacturers suggested volumes (50µl) to as little as 5µl. Errors introduced through volume handlings can be significantly mitigated through the use of electronic pipettes. Random hexamers are compared against the use of a specific reverse transcription primer. Negative

John D. Curry; Cliona McHale; Martyn T. Smith

274

Serum stimulates sodium uptake by rat papillary collecting duct cells (RtPC) in culture  

SciTech Connect

RtPC play an important role in the regulation of sodium excretion by the kidney. RtPC were obtained from Sprague-Dawley rats and cultured on filter-bottom cups in a serum-free medium for 5 days. For the next 24 hr the cells were grown either in S- or in Dulbecco's/F12 supplemented with 10% serum (S+). In 12 primary cultures, the transepithelial resistance (Rt) was significantly higher in S+ (118 +/- 12 ohm cm/sup 2/, n = 45) than in S- (74 +/- 13, n = 41). Short-circuit current was not different between the groups. Na uptake was measured from the apical solution after 60 sec exposure to isotope in a low Na (27 mM) Ringer (same cells as Rt). Despite the similar current, uptake was significantly higher in S+ than S-; 0.61 +/- 0.05 vs 0.23 +/- 0.04 nmol/cm/sup 2/. /sup 5/H-thymidine uptake was similar in S+ and S- indicating that differences in cell number did not account for the increased Na uptake. Supplementation of S- during the final 24 hr with indomethacin, aldosterone, vasopressin, adenosine, or hexamethylene bisacetamide did not significantly increase Na uptake. Thus, an unidentified serum factor increases Rt and Na uptake. The lack of correlation between current and Na uptake suggests that uptake occurs via an electroneutral mechanism.

Husted, R.F.; Stokes, J.B.

1986-03-01

275

Association Between RT-Induced Changes in Lung Tissue Density and Global Lung Function  

SciTech Connect

Purpose: To assess the association between radiotherapy (RT)-induced changes in computed tomography (CT)-defined lung tissue density and pulmonary function tests (PFTs). Methods and Materials: Patients undergoing incidental partial lung RT were prospectively assessed for global (PFTs) and regional (CT and single photon emission CT [SPECT]) lung function before and, serially, after RT. The percent reductions in the PFT and the average changes in lung density were compared (Pearson correlations) in the overall group and subgroups stratified according to various clinical factors. Comparisons were also made between the CT- and SPECT-based computations using the Mann-Whitney U test. Results: Between 1991 and 2004, 343 patients were enrolled in this study. Of these, 111 patients had a total of 203 concurrent post-RT evaluations of changes in lung density and PFTs available for the analyses, and 81 patients had a total of 141 concurrent post-RT SPECT images. The average increases in lung density were related to the percent reductions in the PFTs, albeit with modest correlation coefficients (range, 0.20-0.43). The analyses also indicated that the association between lung density and PFT changes is essentially equivalent to the corresponding association with SPECT-defined lung perfusion. Conclusion: We found a weak quantitative association between the degree of increase in lung density as defined by CT and the percent reduction in the PFTs.

Ma Jinli [Department of Radiation Oncology, Duke University Medical Center, Durham, NC (United States); Department of Radiation Oncology, Fudan University Cancer Hospital, Shanghai (China); Zhang Junan; Zhou Sumin [Department of Radiation Oncology, Duke University Medical Center, Durham, NC (United States); Hubbs, Jessica L. [Department of Radiation Oncology, University of North Carolina School of Medicine, Chapel Hill, NC (United States); Foltz, Rodney J. [Department of Pulmonary Medicine, Duke University Medical Center, Durham, NC (United States); Hollis, Donna R. [Department of Biostatistics, Duke University Medical Center, Durham, NC (United States); Light, Kim L. [Department of Radiation Oncology, Duke University Medical Center, Durham, NC (United States); Wong, Terence Z. [Department of Radiology, Duke University Medical Center, Durham, NC (United States); Kelsey, Christopher R. [Department of Radiation Oncology, Duke University Medical Center, Durham, NC (United States); Marks, Lawrence B. [Department of Radiation Oncology, University of North Carolina School of Medicine, Chapel Hill, NC (United States)], E-mail: marks@med.unc.edu

2009-07-01

276

Simultaneous Detection of three Arbovirus using a Triplex RT-PCR Enzyme Hybridization Assay*  

PubMed Central

Arboviruses represent a serious problem to public health and agriculture worldwide. Fast, accurate identification of the viral agents of arbovirus-associated disease is essential for epidemiological surveillance and laboratory investigation. We developed a cost-effective, rapid, and highly sensitive one-step “triplex RT-PCR enzyme hybridization” assay for simultaneous detections of Japanese Encephallitis virus (JEV, Flaviviridae), Getah virus (GETV, Togaviridae), and Tahyna virus (TAHV, Bunyaviridae) using three pairs of primers to amplify three target sequences in one RT-PCR reaction. The analytical sensitivity of this assay was 1 PFU/mL for JEV, 10 PFU/mL for GETV, and 10 PFU/mL for TAHV. This assay is significantly more rapid and less expensive than the traditional serological detection and single RT-PCR reaction methods. When “triplex RT-PCR enzyme hybridization” was applied to 29 cerebrospinal fluid (CSF) samples that were JEV-positive by normal RT-PCR assay, all samples were strongly positive for JEV, but negative for GETV and TAHV, demonstrating a good sensitivity, specificity, and performance at CSF specimen detection.

Dong, Dan; Fu, Shi-hong; Wang, Li-hua; Lv, Zhi; Li, Tai-yuan; Liang, Guo-dong

2013-01-01

277

Real-time quantification of microRNAs by stem-loop RT-PCR.  

PubMed

A novel microRNA (miRNA) quantification method has been developed using stem-loop RT followed by TaqMan PCR analysis. Stem-loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30,000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem-loop RT-PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem-loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency. PMID:16314309

Chen, Caifu; Ridzon, Dana A; Broomer, Adam J; Zhou, Zhaohui; Lee, Danny H; Nguyen, Julie T; Barbisin, Maura; Xu, Nan Lan; Mahuvakar, Vikram R; Andersen, Mark R; Lao, Kai Qin; Livak, Kenneth J; Guegler, Karl J

2005-11-27

278

Comparative evaluation of 'TaqMan' RT-PCR and RT-PCR ELISA for immunological monitoring of renal transplant recipients.  

PubMed

By sequentially monitoring cytokine gene expression (using RT-PCR ELISA technology) in peripheral blood cells of renal transplant recipients in the early post-operatively period we have shown that expression patterns correlate with clinical events, namely acute allograft rejection. This strategy may have the potential of predicting acute rejection prior to clinical detection. Unfortunately, the technique used was time consuming and only semi-quantitative and, therefore, not suitable for clinical application. In this study, we have sought to confirm the results of the early work using a real time quantitative RT-PCR technique ('TaqMan'), which may be applicable in the clinical laboratory. 'TaqMan' primers and probes were designed for Interleukin (IL)-4 and IL-10 using Primer Express software. Cytokine gene expression for both cytokines was re-measured in stored cDNA samples from 27 non-rejectors and 14 patients experiencing an episode of biopsy proven acute rejection. Compared to pre-transplant levels, IL-4 gene expression fell significantly on post-operative days 2 and 7 before returning to baseline values by day 14 in the non-rejectors. In the rejectors, the initial significant fall was again seen, but with an earlier return to pre-transplant levels at the time of rejection diagnosis. This was followed by a further significant fall in levels 48 h after the initiation of anti-rejection therapy. These different patterns for rejectors and non-rejectors were seen using both techniques. For IL-10, gene expression increased significantly following transplantation throughout the study period when compared to baseline values. This pattern was seen using both techniques. In the rejectors, there were different patterns seen depending on the technique used. When using RT-PCR ELISA, the initial rise was again seen followed by a return to baseline values at the time of rejection diagnosis followed by a further significant rise in gene expression after the start of anti-rejection treatment. The pattern resembled those of the non-rejectors when expression was measured using 'TaqMan'. This study has confirmed that sequential monitoring of cytokine gene expression, measured in peripheral blood mononuclear cells, detects significant changes that correlate with clinical events in renal transplant recipients, including acute rejection, although not all changes detected with RT-PCR ELISA were confirmed. Therefore, real time quantitative RT-PCR technology may be useful in monitoring the immunological status of these patients in the early post-operative period. PMID:12727477

Gibbs, Paul J; Tan, Lam Chin; Sadek, Sami A; Howell, W Martin

279

High-Throughput RT-PCR for small-molecule screening assays  

PubMed Central

Quantitative measurement of the levels of mRNA expression using real-time reverse transcription polymerase chain reaction (RT-PCR) has long been used for analyzing expression differences in tissue or cell lines of interest. This method has been used somewhat less frequently to measure the changes in gene expression due to perturbagens such as small molecules or siRNA. The availability of new instrumentation for liquid handling and real-time PCR analysis as well as the commercial availability of start-to-finish kits for RT-PCR has enabled the use of this method for high-throughput small-molecule screening on a scale comparable to traditional high-throughput screening (HTS) assays. This protocol focuses on the special considerations necessary for using quantitative RT-PCR as a primary small-molecule screening assay, including the different methods available for mRNA isolation and analysis.

Bittker, Joshua A.

2012-01-01

280

Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples  

PubMed Central

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a valuable tool for measuring gene expression in biological samples. However, unique challenges are encountered when studies are performed on cells microdissected from tissues derived from animal models or the clinic, including specimen related issues, variability of RNA template quality and quantity, and normalization. qRT-PCR using small amounts of mRNA derived from dissected cell populations requires adaptation of standard methods to allow meaningful comparisons across sample sets. The protocol described here presents the rationale, technical steps, normalization strategy, and data analysis necessary to generate reliable gene expression measurements of transcripts from dissected samples. The entire protocol from tissue microdissection through qRT-PCR analysis requires approximately 16 hours.

Erickson, Heidi S.; Albert, Paul S.; Gillespie, John W.; Rodriguez-Canales, Jaime; Linehan, W. Marston; Pinto, Peter A.; Chuaqui, Rodrigo F.; Emmert-Buck, Michael R.

2009-01-01

281

Reliability of RT3 accelerometer for measuring mobility in people with multiple sclerosis: pilot study.  

PubMed

This pilot study investigated the test-retest reliability of an RT3 accelerometer (RT3) for measuring motion in people with multiple sclerosis (MS). Ten people with MS (mean age 49 yr; Extended Disability Status Scale mean +/- standard deviation = 3.4 +/- 1.3) and ten nondisabled people (mean age 40 yr) wore the RT3 while they performed three discrete mobility tasks on two occasions separated by 1 week. The intraclass correlation coefficients (ICCs) calculated from the RT3 motion data for the group with MS were 0.64 for the 5-minute walk test (p = 0.01), 0.50 for the timed up and go test (p = 0.05), and 0.76 for the stair-climbing task (p = 0.002). For the control group, these values were 0.65 (p = 0.01), -0.04 (p = 0.54), and 0.39 (p = 0.11), respectively. We found that the RT3 can potentially provide stable data when measuring walking, but a more robust, yet participant-friendly, method of attaching the RT3 is required. Both participant groups demonstrated inconsistencies in motor-task performance, highlighting a potential source of measurement error that would need to be addressed when future studies are designed. Based on the results of the 5-minute walk test in this study, a sample of 53 participants would be required to obtain an ICC value with a 95% confidence interval of width 0.2 using two repeat measurements. PMID:18247259

Hale, Leigh; Williams, Kimberly; Ashton, Craig; Connole, Tim; McDowell, Hayley; Taylor, Colleen

2007-01-01

282

Quantitative RT-PCR for titration of replication-defective recombinant Semliki Forest virus.  

PubMed

Virus titration may constitute a drawback in the development and use of replication-defective viral vectors like Semliki Forest virus (SFV). The standardization and validation of a reverse transcription quantitative PCR (qRT-PCR) method for SFV titration is presented here. The qRT-PCR target is located within the nsp1 gene of the non-structural polyprotein SFV region (SFV RNA), which allows the strategy to be used for several different recombinant SFV constructs. Titer determinations were carried out by performing virus titration and infection assays with SFVs containing an RNA coding region for the rabies virus glycoprotein (RVGP) or green fluorescent protein (GFP). Results showed that the standardized qRT-PCR is applicable for different SFV constructs, and showed good reproducibility. To evaluate the correlation between the amount of functional SFV RNA in a virus lot and its infectivity in BHK-21 cell cultures, a temperature mediated titer decrease was performed and successfully quantitated by qRT-PCR. When used for cell infection at the same multiplicity of infection (MOI), the temperature treated SFV-RVGP samples induced the same levels of RVGP expression. Similarly, when different SFV-GFP lots with different virus titers, as accessed by qRT-PCR, were used for cell infection at the same MOI, the cultures showed comparable amounts of fluorescent cells. The data demonstrate a good correlation between the amount of virus used for infection, as measured by its SFV RNA, and the protein synthesis in the cells. In conclusion, the qRT-PCR method developed here is accurate and enables the titration of replication-defective SFV vectors, an essential aid for viral vector development as well as for establishment of production bioprocesses. PMID:23933080

Puglia, Ana L P; Rezende, Alexandre G; Jorge, Soraia A C; Wagner, Renaud; Pereira, Carlos A; Astray, Renato M

2013-08-06

283

Optical and ultraviolet activity in RT Lacertae during 1985 and 1986  

SciTech Connect

Continued monitoring of optical spectra of RT Lac, a peculiar RS CVn system, showed a large range of activity in the H-alpha line. At times, there was no excess emission or absorption, and at others, much. During active times, the excess emission varied greatly with phase and seemed to come from an extended region. Two UV observations, taken contemporaneously with optical spectra, also showed strong variations in the emission-line strengths. The data are consistent with intermittent mass transfer and imply that RT Lac is similar to other objects which have transient disks. 22 references.

Huenemoerder, D.P.

1988-05-01

284

Simulating Rayleigh-Taylor (RT) instability using PPM hydrodynamics @scale on Roadrunner (u)  

SciTech Connect

The effect of initial conditions on the self-similar growth of the RT instability is investigated using a hydrodynamics code based on the piecewise-parabolic-method (PPM). The PPM code was converted to the hybrid architecture of Roadrunner in order to perform the simulations at extremely high speed and spatial resolution. This paper describes the code conversion to the Cell processor, the scaling studies to 12 CU's on Roadrunner and results on the dependence of the RT growth rate on initial conditions. The relevance of the Roadrunner implementation of this PPM code to other existing and anticipated computer architectures is also discussed.

Woodward, Paul R [Los Alamos National Laboratory; Dimonte, Guy [Los Alamos National Laboratory; Rockefeller, Gabriel M [Los Alamos National Laboratory; Fryer, Christopher L [Los Alamos National Laboratory; Dimonte, Guy [Los Alamos National Laboratory; Dai, W [Los Alamos National Laboratory; Kares, R. J. [Los Alamos National Laboratory

2011-01-05

285

Cell-type-specific responses of RT4 neural cell lines to dibutyryl-cAMP: branch determination versus maturation.  

PubMed

This report describes the induction of cell-type-specific maturation, by dibutyryl-cAMP and testololactone, of neuronal and glial properties in a family of cell lines derived from a rat peripheral neurotumor, RT4. This maturation allows further understanding of the process of determination because of the close lineage relationship between the cell types of the RT4 family. The RT4 family is characterized by the spontaneous conversion of one of the cell types, RT4-AC (stem-cell type), to any of three derivative cell types, RT4-B, RT4-D, or RT4-E, with a frequency of about 10(-5). The RT4-AC cells express some properties characteristic of both neuronal and glial cells. Of these neural properties expressed by RT4-AC cells, only the neuronal properties are expressed by the RT4-B and RT4-E cells, and only the glial properties are expressed by the RT4-D cells. This in vitro cell-type conversion of RT4-AC to three derivative cell types is a branch point for the coordinate regulation of several properties and seems to resemble determination in vivo. In our standard culture conditions, several other neuronal and glial properties are not expressed by these cell types. However, addition of dibutyryl-cAMP induces expression of additional properties, in a cell-type-specific manner: formation of long cellular processes in the RT4-B8 and RT4-E5 cell lines and expression of high-affinity uptake of gamma-aminobutyric acid, by a glial-cell-specific mechanism, in the RT4-D6-2 cell line. These new properties are maximally expressed 2-3 days after addition of dibutyryl-cAMP. This indicates that conversion of RT4-AC to the derivative cell types is also a branch point for the regulation of cell-type-specific properties whose expression is responsive to cAMP. Thus, the potential for maturation in response to increased cAMP is a property that segregates in a cell-type-specific manner and is activated at the determinational level in this system. PMID:3029777

Droms, K; Sueoka, N

1987-03-01

286

Serum thyroid hormone levels in patients with fulminant hepatitis: Usefulness of rT3 and the rT3\\/T3 ratio as prognostic indices  

Microsoft Academic Search

Summary  To evaluate thyroid function in 19 patients with fulminant hepatitis (FH), we have measured total and free 3,5,3?-triiodothyronine\\u000a (T3) and thyroxine (T4), 3,3?,5?-triiodothyronine (reverse T3, rT3), thyroidstimulating hormone (TSH) and thyroxin-binding\\u000a globulin (TBG) in patients with FH, compared with those of 80 patients with other various liver diseases and of 10 healthy\\u000a controls. Patients with FH showed the lowest values

Takashi Kano; Takao Kojima; Takeshi Takahashi; Yasutoshi Muto

1987-01-01

287

Specific detection of Nipah virus using real-time RT-PCR (TaqMan)  

Microsoft Academic Search

Nipah and Hendra viruses belong to the novel Henipavirus genus of the Paramyxoviridae family. Its zoonotic circulation in bats and recent emergence in Malaysia with fatal consequences for humans that were in close contact with infected pigs, has made the reinforcement of epidemiological and clinical surveillance systems a priority. In this study, TaqMan™ RT-PCR of the Nipah nucleoprotein has been

Vanessa Guillaume; Annabelle Lefeuvre; Caroline Faure; Philippe Marianneau; Robin Buckland; Sai Kit Lam; T. Fabian Wild; Vincent Deubel

2004-01-01

288

Detection of Rift Valley fever virus in mosquitoes by RT-PCR  

Microsoft Academic Search

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect Rift Valley fever (RVF) virus RNA in experimentally infected mosquitoes was developed. The specificity of the assay was evaluated with three other phleboviruses; sandfly fever Sicilian (Sabin), sandfly fever Naples (Sabin) and Punta Toro (MSP 3) viruses. The relative sensitivity of the assay, determined by using RVF virus RNA extracted from

M. S. Ibrahim; M. J. Turell; F. K. Knauert; R. S. Lofts

1997-01-01

289

Liquid phase fluorescence in situ RT-PCR analysis for gene expression analysis in woody stems.  

PubMed

We explore a rapid in situ RT-PCR protocol for gene expression studies in woody stem tissues. In situ RT-PCR was performed using fluorescent dye-conjugated nucleic acid and the fluorescence signals derived from target RNAs were detected using confocal laser scanning microscopy. The signal to background ratio was greatly enhanced by performing two rounds of PCR reactions, first without the fluorescent dye and second with the dye. Using this protocol, we obtained strong gene-specific signals in secondary stem tissues. The signals were PCR-dependent, as shown by the lack of cytoplasmic signals in the tissue sections in which either DNA polymerase or primers were omitted from PCR reactions, and were RNA-dependent, as shown by great reduction of cytoplasmic signals when sections were treated with RNase before RT reactions. To verify our protocol, transcript localization of the rbcS gene was examined in secondary stems of hybrid aspen ( Populus tremula L. x tremuloides Michx.) and compared to the chlorophyll autofluorescence signal. The in situ RT-PCR signals form the rbcS gene and chlorophyll autofluorescence co-localized in the same cell types. The signal was also confirmed by Northern blot analysis of isolated RNA from the cambium and developing xylem, thus confirming the validity of the protocol. Some difficulties of in situ transcript localization and the interpretation of the signal distribution in the secondary tissues are discussed. PMID:15095134

Gray-Mitsumune, M; Abe, H; Takahashi, J; Sundberg, B; Mellerowicz, E J

290

Extracción Simple de Ácidos Nucléicos para la Detección de Viroides de Cítricos Mediante RT-PCR  

Microsoft Academic Search

A simple nucleic acids extraction method is described for the detection of citrus viroids by reverse transcription and polymerase chain reaction (RT-PCR). The method involves the use of minimal amounts (250 mg) of citrus tissue and the adding of 1% polyvinylpyrrolidone to the extraction buffer. The complete protocol can be accomplished in 8 h with a least 18 samples simultaneously.

Isidro Humberto Almeyda-León; María Magdalena Iracheta-Cárdenas; Fermín Orona-Castro; Craig J. Kahlke; Mario Alberto Rocha-Peña; Nuevo León

2003-01-01

291

Simplified procedure for detection of enteric pathogenic viruses in shellfish by RT-PCR  

Microsoft Academic Search

Epidemiological evidence linking the transmission of enteric viral disease to shellfish has been known for a long time. A variety of methods have been described for the detection of viral contaminants in shellfish using RT-PCR. However, these methods generally include numerous, often fastidious and time consuming steps for virus release from shellfish tissues and viral RNA isolation. A simplified procedure

O. Legeay; Y. Caudrelier; C. Cordevant; L. Rigottier-Gois; M. Lange

2000-01-01

292

Prediction of lung cancer histological types by RT-qPCR gene expression in FFPE specimens.  

PubMed

Lung cancer histologic diagnosis is clinically relevant because there are histology-specific treatment indications and contraindications. Histologic diagnosis can be challenging owing to tumor characteristics, and it has been shown to have less-than-ideal agreement among pathologists reviewing the same specimens. Microarray profiling studies using frozen specimens have shown that histologies exhibit different gene expression trends; however, frozen specimens are not amenable to routine clinical application. Herein, we developed a gene expression-based predictor of lung cancer histology for FFPE specimens, which are routinely available in clinical settings. Genes predictive of lung cancer histologies were derived from published cohorts that had been profiled by microarrays. Expression of these genes was measured by quantitative RT-PCR (RT-qPCR) in a cohort of patients with FFPE lung cancer. A histology expression predictor (HEP) was developed using RT-qPCR expression data for adenocarcinoma, carcinoid, small cell carcinoma, and squamous cell carcinoma. In cross-validation, the HEP exhibited mean accuracy of 84% and ? = 0.77. In separate independent validation sets, the HEP was compared with pathologist diagnoses on the same tumor block specimens, and the HEP yielded similar accuracy and precision as the pathologists. The HEP also exhibited good performance in specimens with low tumor cellularity. Therefore, RT-qPCR gene expression from FFPE specimens can be effectively used to predict lung cancer histology. PMID:23701907

Wilkerson, Matthew D; Schallheim, Jason M; Hayes, D Neil; Roberts, Patrick J; Bastien, Roy R L; Mullins, Michael; Yin, Xiaoying; Miller, C Ryan; Thorne, Leigh B; Geiersbach, Katherine B; Muldrew, Kenneth L; Funkhouser, William K; Fan, Cheng; Hayward, Michele C; Bayer, Steven; Perou, Charles M; Bernard, Philip S

2013-05-20

293

Policy Implications at the State and District Level with RtI for Gifted students  

ERIC Educational Resources Information Center

As a field, gifted education does not endorse any one approach to serving students because of the range of student abilities and resulting concomitant diverse needs. Therefore, service delivery in gifted education is still heavily teacher dependent. Yet, many of the components of Response to Intervention (RtI) are employed in gifted education,…

Brown, Elissa F.; Abernethy, Sherry H.

2009-01-01

294

CONVERSION FROM RT-11 TO MICRO-RSX FOR REAL-TIME DATA COLLECTION AND ANALYSIS  

EPA Science Inventory

Many scientists with DEC microcomputers use the RT-11 operating system for the acquisition of real-time data in the laboratory. For these researchers, the work required to learn a new operating system and the time needed to reprogram software prevents them from upgrading their la...

295

Hardware-in-the-loop testing of PV control systems using RT-Lab simulator  

Microsoft Academic Search

This paper presents a testing system for power converters control units. A hardware-in-the-loop test bench is designed for assessing control unit performances. A photovoltaic generator, coupled with a boost circuit, is software-simulated within a real-time environment, RT-Lab and coupled in closed loop with the physical control unit to be tested.

O. Cra?ciun; A. Florescu; S. Bacha; I. Munteanu; A. I. Bratcu

2010-01-01

296

Selecting control genes for RT-QPCR using public microarray data  

Microsoft Academic Search

BACKGROUND: Gene expression analysis has emerged as a major biological research area, with real-time quantitative reverse transcription PCR (RT-QPCR) being one of the most accurate and widely used techniques for expression profiling of selected genes. In order to obtain results that are comparable across assays, a stable normalization strategy is required. In general, the normalization of PCR measurements between different

Vlad Popovici; Darlene R. Goldstein; Janine Antonov; Rolf Jaggi; Mauro Delorenzi; Pratyaksha Wirapati

2009-01-01

297

RT-PCR for confirmation of echovirus 30 isolated in Belém, Brazil  

Microsoft Academic Search

Echovirus (Echo) 30 or human enterovirus B is the most frequent enterovirus associated with meningitis cases. Epidemics and outbreaks of this disease caused by Echo 30 have occurred in several countries. In Brazil, Echo 30 has been isolated from sporadic cases and outbreaks that occurred mainly in the south and southeast regions. We used RT-PCR to examine Echo 30 isolates

Maria de Lourdes C. Gomes; Lauze Lee A. Ferreira; Rodrigo Henryque Gomes; Letícia M. Lamarão; Edna da Silveira; Lílian da Silva Rodrigues; Cláudio Ferreira da Silva; Eduardo Ribeiro de Almeida

2007-01-01

298

Chapter 6: Response to Intervention (RtI) and Students with Emotional and Behavioral Disorders  

ERIC Educational Resources Information Center

|We review the concept of response to intervention (RtI) as it is being applied to emotional and behavioral disorders (EDB) in the early part of the 21st century, examining how it differs from and incorporates features of other approaches to addressing those problems, including pre-referral interventions, applied behavior analysis, functional…

Kauffman, James M.; Bruce, Andrew; Lloyd, John Wills

2012-01-01

299

Quantitation of human brain GABA A receptor ? isoforms by competitive RT–PCR  

Microsoft Academic Search

We have developed a competitive RT–PCR assay, adapted from Lewohl et al. [Brain Res. Brain Res. Protoc. 1 (1997) 347], for the quantitation of GABAA receptor ? isoforms in human brain using an internal standard that shares high sequence homology to the targets. The internal standard is identical to the ?1 sequence except for a 61 bp deletion and the

S. Tracey Buckley; Peter R. Dodd

2003-01-01

300

A Proposed Service Aid for the Receiver-Transmitter, Radio Rt-859/Apx-72.  

National Technical Information Service (NTIS)

The design and use of a proposed service aid for the Receiver-transmitter, Radio RT-859/APX-72 is described. The proposed service aid would follow the design used with the service aid constructed for the APX-72 Service Test Model. The service aid was desi...

E. C. Bean

1968-01-01

301

R7912 Transient Digitizer FORTRAN Compatible Subroutines for the PDP-11 (RT-11).  

National Technical Information Service (NTIS)

Assembly-language software subroutines were developed for the Tektronix R7912 waveform digitizer which are RT-11 FORTRAN compatible and run on the PDP-11. Data collected from the digitizer are stored on a file in both raw and normalized form, and the leve...

L. M. Watkins

1978-01-01

302

Recent Upgrades to NASA SPoRT Initialization Datasets for the Environmental Modeling System.  

National Technical Information Service (NTIS)

The NASA Short-term Prediction Research and Transition (SPoRT) Center has developed several products for its NOAA/National Weather Service (NWS) partners that can initialize specific fields for local model runs within the NOAA/NWS Science and Training Res...

A. L. Molthan B. T. Zavodsky F. J. Lafontaine J. L. Case R. A. Rozumalski

2012-01-01

303

Human mammaglobin RT-PCR assay for detection of occult breast cancer cells in hematopoietic products  

Microsoft Academic Search

Materials and methods: A nested RT-PCR assay for mammaglobin was developed. Sensitivity was determined by serial dilution assays with breast cancer cell lines, human breast cancers and normal breast tissue. Specificity was evaluated in hematopoietic samples from healthy volunteers and patients with hematological malignancies or solid tumors other than breast cancer. Results: The mammaglobin transcript was detected in all 15

A. L. Silva; M. J. Tomé; A. E. Correia; J. L. Passos-Coelho

2002-01-01

304

Type-A influenza virus detection and quantitation by real-time RT-PCR  

Technology Transfer Automated Retrieval System (TEKTRAN)

Real-time RT-PCR (RRT-PCR) is a relatively new technology which has been used for AIV detection since the early 2000’s for routine surveillance, during outbreaks and for research. Some of the advantages of RRT-PCR are: quantitative nature, scalability, cost, high sensitivity, high specificity, and ...

305

Clinical usefulness of RT-PCR detection of hematogenous prostate cancer spread  

Microsoft Academic Search

Understaging is commonly associated with therapeutic failure of surgical intervention in apparently localized prostate cancers. Methods that specifically detect prostate cancer cells in the circulation may be able to identify metastatic cancers and thus aid in the selection of the most adequate therapy. The high sensitivity and specificity of the reverse transcriptase-polymerase chain reaction (RT-PCR) encouraged various groups to investigate

N. S. Verkaik; F. H. Schröder; J. C. Romijn

1997-01-01

306

Multiplex nested RT-PCR for the detection of porcine enteric viruses  

Microsoft Academic Search

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine group A rotavirus (PRV-A) are major viruses causing enteric diseases of piglets. A multiplex nested reverse transcription polymerase chain reaction (multiplex nested RT-PCR) was developed for the detection of these viruses in field samples from piglets with diarrhea. A mixture of (1) three external pairs of primers, yielding in

Abid Nabil Ben Salem; A. Chupin Sergei; P. Bjadovskaya Olga; G. Andreeva Olga; Aouni Mahjoub; B. Prokhvatilova Larissa

2010-01-01

307

Constrained use of CCR5 on CD4+ lymphocytes by R5X4 HIV-1: Efficiency of Env-CCR5 interactions and low CCR5 expression determine a range of restricted CCR5-mediated entry  

PubMed Central

R5X4 HIV-1 have impaired utilization of CCR5 on primary CD4+ lymphocytes but the mechanisms responsible are not well defined. Using a panel of diverse R5X4 Envs we identified a spectrum of CCR5 use on CD4+ lymphocytes. Greater lymphocyte CCR5 use correlated with relative resistance to CCR5 mAbs and small molecule antagonists. Increasing CCR5 expression on lymphocytes increased the proportion of entry mediated by CCR5 for all R5X4 isolates except 89.6. In cell lines with regulated CCR5 expression, strains with greater lymphocyte CCR5 use better exploited limiting levels of CCR5. Introduction of an R306S mutation in the 89.6 V3 domain enhanced its utilization of CCR5 at low levels and switched its preference to CCR5 for lymphocyte entry. Thus, the degree to which R5X4 HIV-1 use primary lymphocyte CCR5 is determined by low CCR5 expression coupled with variations in the efficiency of Env-CCR5 interactions, which is in part governed by V3 sequences.

Loftin, Lamorris M.; Kienzle, Martha F.; Yi, Yanjie; Lee, Benhur; Lee, Fang-Hua; Gray, Lachlan; Gorry, Paul R.; Collman, Ronald G.

2010-01-01

308

Identification of Reference Genes for qRT-PCR Analysis in Yesso Scallop Patinopecten yessoensis  

PubMed Central

Background Bivalves comprise around 30,000 extant species and have received much attention for their importance in ecosystems, aquaculture and evolutionary studies. Despite the increasing application of real-time quantitative reverse transcription PCR (qRT-PCR) in gene expression studies on bivalve species, little research has been conducted on reference gene selection which is critical for reliable and accurate qRT-PCR analysis. For scallops, systematic evaluation of reference genes that can be used among tissues or embryo/larva stages is lacking, and ?-actin (ACT) is most frequently used as qRT-PCR reference gene without validation. Results In this study, 12 commonly used candidate reference genes were selected from the transcriptome data of Yesso scallop (Patinopecten yessoensis) for suitable qRT-PCR reference genes identification. The expression of these genes in 36 tissue samples and 15 embryo/larva samples under normal physiological conditions was examined by qRT-PCR, and their expression stabilities were evaluated using three statistic algorithms, geNorm, NormFinder, and comparative ?Ct method. Similar results were obtained by the three approaches for the most and the least stably expressed genes. Final comprehensive ranking for the 12 genes combing the results from the three programs showed that, for different tissues, DEAD-box RNA helicase (HELI), ubiquitin (UBQ), and 60S ribosomal protein L16 (RPL16) were the optimal reference genes combination, while for different embryo/larva stages, gene set containing Cytochrome B (CB), Cytochrome C (CC), Histone H3.3 (His3.3), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recommended for qRT-PCR normalization. ACT was among the least stable genes for both adult tissues and embryos/larvae. Conclusions This work constitutes the first systematic analysis on reference genes selection for qRT-PCR normalization in scallop under normal conditions. The suitable reference genes we recommended will be useful for the identification of genes related to biological processes in Yesso scallop, and also in the reference gene selection for other scallop or bivalve species.

Feng, Liying; Yu, Qian; Li, Xue; Ning, Xianhui; Wang, Jing; Zou, Jiajun; Zhang, Lingling; Wang, Shi; Hu, Jingjie; Hu, Xiaoli; Bao, Zhenmin

2013-01-01

309

Effect of pooling and multiplexing on the detection of bluetongue virus RNA by real-time RT-PCR  

Microsoft Academic Search

Real-time RT-PCR (RT-qPCR) was used routinely for laboratory diagnosis during the 2006\\/2007 bluetongue virus (BTV) serotype 8 epidemic. In the present study the impact of pooling and multiplexing strategies on RT-qPCR are assessed. To avoid any bias in the pooling experiments, 121 BTV-8 positive blood samples with a low to high viral load were selected and pooled individually with nine

F. Vandenbussche; T. Vanbinst; E. Vandemeulebroucke; N. Goris; C. Sailleau; S. Zientara; K. De Clercq

2008-01-01

310

Quantitation of canine coronavirus RNA in the faeces of dogs by TaqMan RT-PCR  

Microsoft Academic Search

A TaqMan® fluorogenic reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed for the detection and quantitation of canine coronavirus (CCoV) RNA in the faeces of naturally or experimentally infected dogs. The CCoV fluorogenic RT-PCR assay, which targeted the ORF5 (M gene), was more sensitive than a conventional RT-PCR assay targeting the same gene, showing a detection limit of 10 copies

Nicola Decaro; Annamaria Pratelli; Marco Campolo; Gabriella Elia; Vito Martella; Maria Tempesta; Canio Buonavoglia

2004-01-01

311

Modeling Variably Saturated Multispecies Reactive Groundwater Solute Transport with MODFLOW-UZF and RT3D.  

PubMed

A numerical model was developed that is capable of simulating multispecies reactive solute transport in variably saturated porous media. This model consists of a modified version of the reactive transport model RT3D (Reactive Transport in 3 Dimensions) that is linked to the Unsaturated-Zone Flow (UZF1) package and MODFLOW. Referred to as UZF-RT3D, the model is tested against published analytical benchmarks as well as other published contaminant transport models, including HYDRUS-1D, VS2DT, and SUTRA, and the coupled flow and transport modeling system of CATHY and TRAN3D. Comparisons in one-dimensional, two-dimensional, and three-dimensional variably saturated systems are explored. While several test cases are included to verify the correct implementation of variably saturated transport in UZF-RT3D, other cases are included to demonstrate the usefulness of the code in terms of model run-time and handling the reaction kinetics of multiple interacting species in variably saturated subsurface systems. As UZF1 relies on a kinematic-wave approximation for unsaturated flow that neglects the diffusive terms in Richards equation, UZF-RT3D can be used for large-scale aquifer systems for which the UZF1 formulation is reasonable, that is, capillary-pressure gradients can be neglected and soil parameters can be treated as homogeneous. Decreased model run-time and the ability to include site-specific chemical species and chemical reactions make UZF-RT3D an attractive model for efficient simulation of multispecies reactive transport in variably saturated large-scale subsurface systems. PMID:23131109

Bailey, Ryan T; Morway, Eric D; Niswonger, Richard G; Gates, Timothy K

2012-11-06

312

Suppression of Virus Load by Highly Active Antiretroviral Therapy in Rhesus Macaques Infected with a Recombinant Simian Immunodeficiency Virus Containing Reverse Transcriptase from Human Immunodeficiency Virus Type 1  

Microsoft Academic Search

We have modeled highly active antiretroviral therapy (HAART) for AIDS in rhesus macaques infected with a chimera (RT-SHIV) of simian immunodeficiency virus containing reverse transcriptase from human immu- nodeficiency virus type-1 (HIV-1). Seven RT-SHIV-infected macaques were treated with a combination of efavirenz (200 mg orally once daily), lamivudine (8 mg\\/kg subcutaneously once daily), and tenofovir (30 mg\\/kg subcutaneously once daily).

Thomas W. North; Koen K. A. Van Rompay; Joanne Higgins; Timothy B. Matthews; Debra A. Wadford; Niels C. Pedersen; Raymond F. Schinazi

2005-01-01

313

Detection and quantitation of two cucurbit criniviruses in mixed infection by real-time RT-PCR.  

PubMed

Cucurbit chlorotic yellows virus (CCYV) and Cucurbit yellow stunting disorder virus (CYSDV) are whitefly-transmitted criniviruses infecting cucurbit crops inducing similar symptoms. Single and multiplex RT-PCR protocols were developed and evaluated on cucurbit samples collected from commercial greenhouses. Primers and probes were designed from the highly conserved heat shock protein 70 homolog (Hsp70h) gene. Conventional RT-PCR and multiplex RT-PCR assays showed high specificity and suitability for routine screening. TaqMan-based quantitative real-time RT-PCR (RT-qPCR) protocols were also developed for the detection and quantitation of both viruses occurring in single or mixed infection. The assays proved to be highly specific with no cross amplification. RT-qPCR assays showed a 100-1000 times improved sensitivity over conventional RT-PCR. Virus titers in mixed infections were compared to singly infected plants by RT-qPCR. CYSDV and CCYV titers decreased in double infected plants. This paper reports highly specific conventional RT-PCR and quantitative real-time PCR assays for detection, quantitation and differentiation between two closely related cucurbit-infecting criniviruses. PMID:23810855

Abrahamian, Peter E; Seblani, Rewa; Sobh, Hana; Abou-Jawdah, Yusuf

2013-06-26

314

RT-PCR Analysis of Pain Genes: Use of Gel-Based RT-PCR for Studying Induced and Tissue-Enriched Gene Expression  

PubMed Central

Frequently, it is important to ascertain whether a molecule that is involved in one model of pain is also involved in other models of pain. Similarly, it may be important to determine whether a molecule involved in nociception in one tissue is also expressed in other tissues and to ascertain the degree of enrichment. Additionally, before initiating a complex set of experiments or purchasing an expensive immunoassay kit, it may be useful to obtain initial supporting evidence to justify the time and money. Is the transcript for the target receptor, protein, or peptide precursor present in, for example, the dorsal root ganglion? And, if present, how abundant is it? Here is where the power of PCR can be applied to obtain a quick but informative answer. In this chapter, we mainly detail the use of gel-based RT-PCR and also provide suggestions on tissue dissection and interpretation of results. The use of gel-based RT-PCR can address many of the questions of abundance or tissue specificity with a minimum of expense and time.

Mitchell, Kendall; Iadarola, Michael J.

2012-01-01

315

Human Immunodeficiency Virus 1 (HIV1)Specific Reverse Transcriptase (RT) Inhibitors may Suppress the Replication of Specific Drug-Resistant (E138K)RT HIV1 Mutants or Select for Highly Resistant (Y181C --> C181I)RT HIV1 Mutants  

Microsoft Academic Search

Mutant HIV-1 that expresses a Glu138--> Lys substitution in its RT [(E138K)RT] is resistant to the HIV-1-specific RT inhibitor 2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5''-(4''-amino-1'',2''- oxathiole-2'',2''-dioxide)pyrimidine (TSAO). However, cell cultures infected with this mutant were completely protected against virus-mediated destruction by micromolar concentrations of the HIV-1-specific RT inhibitors tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione (TIBO), nevirapine, and bis(heteroaryl)piperazine (BHAP). In contrast, cells infected with a virus mutant that

Jan Balzarini; Anna Karlsson; Vinod V. Sardana; Emilio A. Emini; Maria-Jose Camarasa; Erik de Clercq

1994-01-01

316

SPoRT's Participation in the GOES-R Proving Ground Activity  

NASA Astrophysics Data System (ADS)

The next generation geostationary satellite, GOES-R, will carry two new instruments with unique atmospheric and surface observing capabilities, the Advanced Baseline Imager (ABI) and the Geostationary Lightning Mapper (GLM), to study short-term weather processes. The ABI will bring enhanced multispectral observing capabilities with frequent refresh rates for regional and full disk coverage to geostationary orbit to address many existing and new forecast challenges. The GLM will, for the first time, provide the continuous monitoring of total lightning flashes over a hemispherical region from space. NOAA established the GOES-R Proving Ground activity several years ago to demonstrate the new capabilities of these instruments and to prepare forecasters for their day one use. Proving Ground partners work closely with algorithm developers and the end user community to develop and transition proxy data sets representing GOES-R observing capabilities. This close collaboration helps to maximize refine algorithms leading to the delivery of a product that effectively address a forecast challenge. The NASA Short-term Prediction Research and Transition (SPoRT) program has been a participant in the NOAA GOES-R Proving Ground activity by developing and disseminating selected GOES-R proxy products to collaborating WFOs and National Centers. Established in 2002 to demonstrate the weather and forecasting application of real-time EOS measurements, the SPoRT program has grown to be an end-to-end research to operations activity focused on the use of advanced NASA modeling and data assimilation approaches, nowcasting techniques, and unique high-resolution multispectral data from EOS satellites to improve short-term weather forecasts on a regional and local scale. Participation in the Proving Ground activities extends SPoRT's activities and taps its experience and expertise in diagnostic weather analysis, short-term weather forecasting, and the transition of research and experimental data to operational decision support systems like NAWIPS, AWIPS, AWIPS2, and Google Earth. Recent SPoRT Proving Ground activities supporting the development and use of a pseudo GLM total lightning product and the transition of the AWG's Convective Initiation (CI) product, both of which were available in AWIPS and AWIPS II environments, by forecasters during the Hazardous Weather Testbed (HWT) Spring Experiment. SPoRT is also providing a suite of SEVIRI and MODIS RGB image products, and a high resolution composite SST product to several National Centers for use in there ongoing demonstration activities. Additionally, SPoRT has involved numerous WFOs in the evaluation of a GOES-MODIS hybrid product which brings ABI-like data sets in front of the forecaster for everyday use. An overview of this activity will be presented at the conference.

Jedlovec, G.; Fuell, K.; Smith, M. R.; Stano, G. T.; Molthan, A.

2011-12-01

317

Detection of infectious salmon anaemia virus by real-time RT-PCR.  

PubMed

A one-tube real-time reverse transcription-polymerase chain reaction (RT-PCR) using LightCycler technology and SYBR Green chemistry that quantitatively detects infectious salmon anaemia virus (ISAV) in biological samples is described. The assay utilized primers targeting ISAV RNA segment 8, with ISAV isolate U5575-1 as template. The entire optimized assay, including one cycle of reverse transcription, 50 cycles of complementary DNA amplification, and data acquisition and analysis took only 80min. The melting curve and gel electrophoresis analyses of real-time RT-PCR showed harmony with each other as a virus-specific single melting peak and a product of the expected size of 211 bp were obtained. A regression line between the mean threshold cycle (Ct) values and viral template concentrations over a 1:10(5) dilution range with an r(2) value of 0.962 and a slope of -3.771 indicated that the assay was highly reproducible. This assay was 100 times more sensitive than the conventional one-tube RT-PCR assay when compared on the same sample. Analysis of different tissues from fish that survived an ISAV experimental infection further confirmed that real-time RT-PCR was more sensitive than regular RT-PCR for detection of ISAV nucleic acids. Temporal analysis of ISAV-infected TO cell cultures showed that the amount of the specific viral RNA increased more than 100-fold within 32 h post-inoculation (p.i.) and over 1200-fold by 144 h p.i. The melting curve analysis throughout the duration of the infection sampled had a single melting peak suggesting that the virus population was uniform in the targeted region. Quantitative analysis of CHSE-214 cell cultures infected with different ISAV isolates indicated that ISAV isolates, based on their ability to replicate and cause cytopathic effects in CHSE-214 cells, may be differentiated into three CHSE phenotypes: replicating cytopathic, replicating non-cytopathic, and non-replicating. Thus, the SYBR Green real-time RT-PCR is a sensitive, rapid, and highly reproducible assay that can be used to quantitate ISAV in biological samples. PMID:15019258

Munir, Khalid; Kibenge, Frederick S B

2004-04-01

318

Data mining DICOM RT objects for quality control in radiation oncology  

NASA Astrophysics Data System (ADS)

Our goal in this paper is to data mine the wealth of information contained in the dose-volume objects used in external beam radiotherapy treatment planning. In addition, by performing computational pattern recognition on these mined objects, the results may help identify predictors for unsafe dose delivery. This will ultimately enhance current clinical registries by the inclusion of detailed dose-volume data employed in treatments. The most efficient way of including dose-volume information in a registry is through DICOM RT objects. With this in mind, we have built a DICOM RT specific infrastructure, capable of integrating with larger, more general clinical registries, and we will present the results of data mining these sets.

Deshpande, Ruchi R.; DeMarco, John; Low, Daniel; Le, Anh H.; Liu, Brent J.

2012-02-01

319

Quantitative RT-PCR Specific for Precursor and Mature miRNAs.  

PubMed

Quantification of microRNAs (miRNAs) in cells or primary tissues is one of the most important steps in elucidating their biological functions. However, miRNAs are challenging molecules for PCR amplification due to the stable hairpin of the precursor form and the small size of the mature miRNA, which is roughly the same length as the primers used in standard PCR. To date, different assays were introduced for the specific and sensitive quantification of the mature form of miRNAs. In this chapter we describe the extraction of RNA from microdissected tissue and the quantification of miRNAs using two different methods (stem-loop qRT-PCR and polyT adaptor qRT-PCR). PMID:24166308

Zöllner, Hannah; Hahn, Stephan A; Maghnouj, Abdelouahid

2014-01-01

320

Emulating a crowded intracellular environment in vitro dramatically improves RT-PCR performance  

SciTech Connect

The polymerase chain reaction's (PCR) phenomenal success in advancing fields as diverse as Medicine, Agriculture, Conservation, or Paleontology is based on the ability of using isolated prokaryotic thermostable DNA polymerases in vitro to copy DNA irrespective of origin. This process occurs intracellularly and has evolved to function efficiently under crowded conditions, namely in an environment packed with macromolecules. However, current in vitro practice ignores this important biophysical parameter of life. In order to more closely emulate conditions of intracellular biochemistry in vitro we added inert macromolecules into reverse transcription (RT) and PCR. We show dramatic improvements in all parameters of RT-PCR including 8- to 10-fold greater sensitivity, enhanced polymerase processivity, higher specific amplicon yield, greater primer annealing and specificity, and enhanced DNA polymerase thermal stability. The faster and more efficient reaction kinetics was a consequence of the cumulative molecular and thermodynamic effects of the excluded volume effect created by macromolecular crowding.

Lareu, Ricky R. [Tissue Modulation Laboratory, Division of Bioengineering, Faculty of Engineering, National University of Singapore, Division Office Block E3A 04-15, 7 Engineering Drive 1, Singapore 117574 (Singapore); NUS Tissue Engineering Program and Department of Orthopedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore (Singapore); Harve, Karthik S. [Tissue Modulation Laboratory, Division of Bioengineering, Faculty of Engineering, National University of Singapore, Division Office Block E3A 04-15, 7 Engineering Drive 1, Singapore 117574 (Singapore); Raghunath, Michael [Tissue Modulation Laboratory, Division of Bioengineering, Faculty of Engineering, National University of Singapore, Division Office Block E3A 04-15, 7 Engineering Drive 1, Singapore 117574 (Singapore); Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (Singapore)], E-mail: bierm@nus.edu.sg

2007-11-09

321

Feasibility of small animal cranial irradiation with the microRT system  

SciTech Connect

Purpose: To develop and validate methods for small-animal CNS radiotherapy using the microRT system. Materials and Methods: A custom head immobilizer was designed and built to integrate with a pre-existing microRT animal couch. The Delrin couch-immobilizer assembly, compatible with multiple imaging modalities (CT, microCT, microMR, microPET, microSPECT, optical), was first imaged via CT in order to verify the safety and reproducibility of the immobilization method. Once verified, the subject animals were CT-scanned while positioned within the couch-immobilizer assembly for treatment planning purposes. The resultant images were then imported into CERR, an in-house-developed research treatment planning system, and registered to the microRTP treatment planning space using rigid registration. The targeted brain was then contoured and conformal radiotherapy plans were constructed for two separate studies: (1) a whole-brain irradiation comprised of two lateral beams at the 90 degree sign and 270 degree sign microRT treatment positions and (2) a hemispheric (left-brain) irradiation comprised of a single A-P vertex beam at the 0 degree sign microRT treatment position. During treatment, subject animals (n=48) were positioned to the CERR-generated treatment coordinates using the three-axis microRT motor positioning system and were irradiated using a clinical Ir-192 high-dose-rate remote after-loading system. The radiation treatment course consisted of 5 Gy fractions, 3 days per week. 90% of the subjects received a total dose of 30 Gy and 10% received a dose of 60 Gy. Results: Image analysis verified the safety and reproducibility of the immobilizer. CT scans generated from repeated reloading and repositioning of the same subject animal in the couch-immobilizer assembly were fused to a baseline CT. The resultant analysis revealed a 0.09 mm average, center-of-mass translocation and negligible volumetric error in the contoured, murine brain. The experimental use of the head immobilizer added {+-}0.1 mm to microRT spatial uncertainty along each axis. Overall, the total spatial uncertainty for the prescribed treatments was {+-}0.3 mm in all three axes, a 0.2 mm functional improvement over the original version of microRT. Subject tolerance was good, with minimal observed side effects and a low procedure-induced mortality rate. Throughput was high, with average treatment times of 7.72 and 3.13 min/animal for the whole-brain and hemispheric plans, respectively (dependent on source strength). Conclusions: The method described exhibits conformality more in line with the size differential between human and animal patients than provided by previous prevalent approaches. Using pretreatment imaging and microRT-specific treatment planning, our method can deliver an accurate, conformal dose distribution to the targeted murine brain (or a subregion of the brain) while minimizing excess dose to the surrounding tissue. Thus, preclinical animal studies assessing the radiotherapeutic response of both normal and malignant CNS tissue to complex dose distributions, which closer resemble human-type radiotherapy, are better enabled. The procedural and mechanistic framework for this method logically provides for future adaptation into other murine target organs or regions.

Kiehl, Erich L.; Stojadinovic, Strahinja; Malinowski, Kathleen T.; Limbrick, David; Jost, Sarah C.; Garbow, Joel R.; Rubin, Joshua B.; Deasy, Joseph O.; Khullar, Divya; Izaguirre, Enrique W.; Parikh, Parag J.; Low, Daniel A.; Hope, Andrew J. [Washington University School of Medicine, St. Louis, Missouri 63110 (United States); Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298 (United States); Washington University School of Medicine, St. Louis, Missouri 63110 (United States); Princess Margaret Hospital, Toronto, Ontario M5G 2M9 (Canada)

2008-10-15

322

Free cancer cell detection in peritoneal cavity in gastric cancer patients by RT-PCR for CEA.  

National Technical Information Service (NTIS)

Authors applied RT-PCR assay to detecting CEA expressing free cancer cells in peritoneal cavity of 114 gastric cancer patients to find an indication for prophylactic treatment to prevent peritoneal recurrence. Sixty-three of 114 cases were positive for RT...

J. I. Lee N. M. Moon N. S. Paik D. W. Choi H. Y. Bang

1997-01-01

323

A DICOM-RT based ePR radiation therapy information system for managing brain tumor patients  

Microsoft Academic Search

The need for comprehensive clinical image data and relevant information in image-guided Radiation Therapy (RT) is becoming steadily apparent. Multiple standalone systems utilizing the most technological advancements in imaging, therapeutic radiation, and computerized treatment planning systems acquire key data during the RT treatment course of a patient. One example are patients treated for brain tumors of greater sizes and irregular

Brent J. Liu; Maria Law; H. K. Huang; C. S. Zee; Lawrence Chan

2005-01-01

324

A DICOM-RT Based ePR radiation therapy information system for decision-support of brain tumor patients  

Microsoft Academic Search

The need for comprehensive clinical image data and relevant information in image-guided Radiation Therapy (RT) is becoming steadily apparent. Multiple standalone systems utilizing the most technological advancements in imaging, therapeutic radiation, and computerized treatment planning systems acquire key data during the RT treatment course of a patient. One example are patients treated for brain tumors of greater sizes and irregular

B. J. Liu; M. Law; H. K. Huang; C. S. Zee; L. Chan

2006-01-01

325

HIV-1 Tropism Determination Using a Phenotypic Env Recombinant Viral Assay Highlights Overestimation of CXCR4-Usage by Genotypic Prediction Algorithms for CRRF01_AE and CRF02_AG  

PubMed Central

Background Human Immunodeficiency virus type-1 (HIV) entry into target cells involves binding of the viral envelope (Env) to CD4 and a coreceptor, mainly CCR5 or CXCR4. The only currently licensed HIV entry inhibitor, maraviroc, targets CCR5, and the presence of CXCX4-using strains must be excluded prior to treatment. Co-receptor usage can be assessed by phenotypic assays or through genotypic prediction. Here we compared the performance of a phenotypic Env-Recombinant Viral Assay (RVA) to the two most widely used genotypic prediction algorithms, Geno2Pheno[coreceptor] and webPSSM. Methods Co-receptor tropism of samples from 73 subtype B and 219 non-B infections was measured phenotypically using a luciferase-tagged, NL4-3-based, RVA targeting Env. In parallel, tropism was inferred genotypically from the corresponding V3-loop sequences using Geno2Pheno[coreceptor] (5–20% FPR) and webPSSM-R5X4. For discordant samples, phenotypic outcome was retested using co-receptor antagonists or the validated Trofile® Enhanced-Sensitivity-Tropism-Assay. Results The lower detection limit of the RVA was 2.5% and 5% for X4 and R5 minority variants respectively. A phenotype/genotype result was obtained for 210 samples. Overall, concordance of phenotypic results with Geno2Pheno[coreceptor] was 85.2% and concordance with webPSSM was 79.5%. For subtype B, concordance with Geno2pheno[coreceptor] was 94.4% and concordance with webPSSM was 79.6%. High concordance of genotypic tools with phenotypic outcome was seen for subtype C (90% for both tools). Main discordances involved CRF01_AE and CRF02_AG for both algorithms (CRF01_AE: 35.9% discordances with Geno2Pheno[coreceptor] and 28.2% with webPSSM; CRF02_AG: 20.7% for both algorithms). Genotypic prediction overestimated CXCR4-usage for both CRFs. For webPSSM, 40% discordance was observed for subtype A. Conclusions Phenotypic assays remain the most accurate for most non-B subtypes and new subtype-specific rules should be developed for non-B subtypes, as research studies more and more draw conclusions from genotypically-inferred tropism, and to avoid unnecessarily precluding patients with limited treatment options from receiving maraviroc or other entry inhibitors.

Mulinge, Martin; Lemaire, Morgane; Servais, Jean-Yves; Rybicki, Arkadiusz; Struck, Daniel; da Silva, Eveline Santos; Verhofstede, Chris; Lie, Yolanda; Seguin-Devaux, Carole; Schmit, Jean-Claude; Bercoff, Danielle Perez

2013-01-01

326

A combination anti-HIV-1 gene therapy approach using a single transcription unit that expresses antisense, decoy, and sense RNAs, and trans-dominant negative mutant Gag and Env proteins.  

PubMed

Oncoretroviral vectors were engineered to allow constitutive expression of an antisense RNA and the trans-activator of transcription (Tat)-inducible expression of a mRNA containing the trans-activation response (TAR) element, the Rev response element (RRE), and the efficient packaging signal (Psi(e) of human immunodeficiency virus-1 (HIV-1) RNA. Nuclear export of this mRNA by the regulator of expression of virion proteins (Rev) would allow its translation into wild type (WT) (MoTN-Ti-GE-Ri- Ter) or trans-dominant negative mutant (TDM) (MoTN-Ti-GmEm-Ri-Ter) Gag and Env proteins. Thus, the antisense RNA produced in a constitutive manner would ensure that even if there is leaky expression, no WT/TDM Gag or Env protein would be produced in the uninfected cells. If cells become infected by HIV-1, the antisense RNA would inhibit HIV-1 replication. Failure on the part of antisense RNA to inhibit virus replication would allow GE/GmEm mRNA production. The GE/GmEm mRNA would cause partial inhibition of HIV-1 replication as it contains the TAR, RRE, and Psi(e) signal sequences. Translation of GmEm mRNA would give rise to TDM Gag and Env proteins, which would further decrease progeny virus infectivity. Tat- and Rev-inducibility was demonstrated in transfected HeLa and HeLa-Tev cells. Full-length WT/TDM Gag production was confirmed by Western blot analysis. Amphotropic vector particles were used to transduce a human CD4+ T-lymphoid cell line, and the stable transductants were challenged with HIV-1. Virus replication was better inhibited by the MoTN-Ti-GE-Ri-Ter vector than by the MoTN-Ti-GmEm-Ri-Ter vector. Inhibition of HIV-1 replication was also demonstrated in transduced CD4+ human peripheral blood T lymphocytes (PBLs). Moreover, our results suggest that cloning in the reverse transcriptional orientation must be avoided to prevent antisense RNA-mediated inhibition of transgene and endogenous gene expression. PMID:11815282

Ding, Shi-Fa; Lombardi, Rocco; Nazari, Reza; Joshi, Sadhna

2002-02-01

327

Real-time continuous glucose monitoring using Guardian ®RT: from research to clinical practice  

Microsoft Academic Search

The limited number of self-monitoring blood glucose measurements is an obstacle for good metabolic control in patients with type 1 diabetes. However, continuous glucose measurement with real-time data and alarms is a recent technology that promises to improve the efficacy of treatment in diabetic patients. Guardian®RT uses a continuous telemetry display of real-time glucose values, and automatic alerts at preset

N. Tubiana-Rufi; J.-P. Riveline; D. Dardari

2007-01-01

328

Quantification of mRNA using real-time RT-PCR  

Microsoft Academic Search

The real-time reverse transcription polymerase chain reaction (RT-qPCR) addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology and diagnostics and has become the method of choice for the quantification of mRNA. Although it is often described as a “gold” standard, it is far from being a standard assay. The significant problems caused by variability of RNA

Tania Nolan; Rebecca E Hands; Stephen A Bustin

2006-01-01

329

A novel multiplex RT-PCR system detects human endogenous retrovirus-K in breast cancer  

Microsoft Academic Search

Summary. Human endogenous retrovirus HERV-K like-sequences have been implicated in certain cancers. We developed a novel multiplex RT-PCR system for HERV-K that yielded a 533?bp product together with a smaller sized product (319?bp) of the house keeping gene, histidyl tRNA synthetase (HtRNAS). The latter spanned an intron that also served to validate target cDNA. PCR amplicons of HERV-K and HtRNAS

H. Davari Ejthadi; J. H. Martin; J. Junying; D. A. Roden; M. Lahiri; P. Warren; P. G. Murray; P. N. Nelson

2005-01-01

330

Comparison of cytokine measurements using ELISA, ELISPOT and semi-quantitative RT-PCR  

Microsoft Academic Search

We have investigated the correlation between results obtained by three different methods (semi-quantitative RT-PCR, ELISA and ELISPOT) used to measure cytokine expression by mouse leukocytes. The production of the cytokines tumour necrosis factor-alpha (TNF?), interferon-gamma (IFN?) and interleukin-4 (IL-4), was analysed with all three methods. In a simple experimental murine in vivo model of leukocyte stimulation, consisting of a single

Nicolas Favre; Gérard Bordmann; Werner Rudin

1997-01-01

331

Application of RT-PCR for the detection of avian reovirus contamination in avian viral vaccines  

Microsoft Academic Search

An efficient procedure for the detection of avian reovirus (ARV)-specific RNA sequences in veterinary immunological medicinal products using reverse transcriptase polymerase chain reaction (RT-PCR) is described. Four ARV vaccine strains (1133, 1733, 2408 and Olson WVU2937), two ATCC strains (VR826 and VR856) as well as several ARV field isolates obtained from domestic, wild and pet birds could be easily detected

Silke Bruhn; Lukas Bruckner; Hans-Peter Ottiger

2005-01-01

332

Pellet-HeaRT - Proposal of an Architecture for Ontology Systems with Rules  

Microsoft Academic Search

\\u000a The ongoing research on integration of rules and ontologies has resulted in multiple solutions, rule languages and systems.\\u000a They differ in terms of aims and scope, semantics and architecture. The paper describes a proposal of a hybrid system combining\\u000a a Description Logics reasoner with a forward-chaining rule engine. An integration of a dedicated tool for modularized rule\\u000a bases, called HeaRT,

Grzegorz J. Nalepa; Weronika T. Furmanska

2010-01-01

333

Diagnosis of Norovirus outbreaks by commercial ELISA or RT-PCR  

Microsoft Academic Search

The IDEIA Norwalk-like virus (Dakocytomation Ltd., Ely, United Kingdom) and the Ridascreen Norwalk-like virus enzyme immunoassay (R-Biopharm AG, Darmstadt, Germany), were evaluated for the diagnosis of outbreaks of acute gastroenteritis.A panel of 158 fecal samples from 23 outbreaks, including confirmed rotavirus and astrovirus outbreaks, was used to determine the sensitivity and specificity of both ELISA kits relative to an RT-PCR

Erwin de Bruin; Erwin Duizer; Harry Vennema; Marion P. G. Koopmans

2006-01-01

334

Using metallic resin and aluminum alloy molds to manufacture propellers with RP\\/RT technique  

Microsoft Academic Search

Purpose – This purpose of this study is to investigate an effective method to manufacture propellers. Design\\/methodology\\/approach – The investment casting process and injection molding process have been applied separately to the rapid prototyping\\/rapid tooling (RP\\/RT) to obtain metal (Al-Si alloy) propellers and plastic (Acrylonitrile butadiene styrene – ABS) propellers. The two different manufacturing processes were compared following the same

C. Y. Hsu; C. K. Huang; G. J. Tzou

2008-01-01

335

Software Architecture for a Humanoid Robot Teleoperation Based on RT-Linux\\/Linux\\/Windows Operating System  

Microsoft Academic Search

This paper puts forward a software system architecture for the humanoid robot teleoperation system based on RT-Linux\\/Linux\\/Windows operating system. In this new method the communication control subsystem is based on Linux operating system and exchanges data with the real-time control subsystem by using network interface instead of memolink. By this architecture the teleoperation system for the remote control of humanoid

Yuepin Lu; Qiang Huang; Lei Zhang; Jiapeng Yang; Shusheng Iv; M. Keerio

2006-01-01

336

mRNA and microRNA quality control for RT-qPCR analysis  

Microsoft Academic Search

The importance of high quality sample material, i.e. non-degraded or fragmented RNA, for classical gene expression profiling is well documented. Hence, the analysis of RNA quality is a valuable tool in the preparation of methods like RT-qPCR and microarray analysis. For verification of RNA integrity, today the use of automated capillary electrophoresis is state of the art. Following the recently

C. Becker; A. Hammerle-Fickinger; I. Riedmaier; M. W. Pfaffl

2010-01-01

337

A practical approach to RT-qPCR—Publishing data that conform to the MIQE guidelines  

Microsoft Academic Search

Given the highly dynamic nature of mRNA transcription and the potential variables introduced in sample handling and in the downstream processing steps (Garson et al. (2009) [4]), a standardized approach to each step of the RT-qPCR workflow is critical for reliable and reproducible results. The MIQE provides this approach with a checklist that contains 85 parameters to assure quality results

Sean Taylor; Michael Wakem; Greg Dijkman; Marwan Alsarraj; Marie Nguyen

2010-01-01

338

Experimental comparison of relative RT-qPCR quantification approaches for gene expression studies in poplar  

Microsoft Academic Search

BACKGROUND: RT-qPCR is a powerful tool for analysing gene expression. It depends on measuring the increase in fluorescence emitted by a DNA-specific dye during the PCR reaction. For relative quantification, where the expression of a target gene is measured in relation to one or multiple reference genes, various mathematical approaches are published. The results of relative quantification can be considerably

Nicole Regier; Beat Frey

2010-01-01

339

Pre-processing Feature Selection for Improved C&RT Models for Oral Absorption.  

PubMed

There are currently thousands of molecular descriptors that can be calculated to represent a chemical compound. Utilizing all molecular descriptors in Quantitative Structure-Activity Relationships (QSAR) modeling can result in overfitting, decreased interpretability, and thus reduced model performance. Feature selection methods can overcome some of these problems by drastically reducing the number of molecular descriptors and selecting the molecular descriptors relevant to the property being predicted. In particular, decision trees such as C&RT, although they have an embedded feature selection algorithm, can be inadequate since further down the tree there are fewer compounds available for descriptor selection, and therefore descriptors may be selected which are not optimal. In this work we compare two broad approaches for feature selection: (1) a "two-stage" feature selection procedure, where a pre-processing feature selection method selects a subset of descriptors, and then classification and regression trees (C&RT) selects descriptors from this subset to build a decision tree; (2) a "one-stage" approach where C&RT is used as the only feature selection technique. These methods were applied in order to improve prediction accuracy of QSAR models for oral absorption. Additionally, this work utilizes misclassification costs in model building to overcome the problem of the biased oral absorption data sets with more highly absorbed than poorly absorbed compounds. In most cases the two-stage feature selection with pre-processing approach had higher model accuracy compared with the one-stage approach. Using the top 20 molecular descriptors from the random forest predictor importance method gave the most accurate C&RT classification model. The molecular descriptors selected by the five filter feature selection methods have been compared in relation to oral absorption. In conclusion, the use of filter pre-processing feature selection methods and misclassification costs produce models with better interpretability and predictability for the prediction of oral absorption. PMID:24050619

Newby, Danielle; Freitas, Alex A; Ghafourian, Taravat

2013-10-09

340

Formal Specification of Real-Time Systems by Transformation of UML-RT Design Models  

Microsoft Academic Search

We are motivated to complement our methodology by integrating collaboration diagrams to facilitate the specification of capsules in UML-RT design models. An improved systematic transformation method to derive a cor- rect and complete formal system specification of real-time systems is estab- lished. This article aims at integrating temporal requirements in the design stage of the life cycle of a real-time

Kawtar Benghazi Akhlaki; Manuel I. Capel; Juan Antonio Holgado Terriza; Luis E. Mendoza Morales

2006-01-01

341

Performance evaluation of an EtherCAT master using Linux and the RT Patch  

Microsoft Academic Search

This paper has the twofold goal of investigating the real-time performance of an EtherCAT master entirely built from open-source components (using Linux and the RT Patch at the operating system level) and assess its ability to support concurrent best-effort tasks without compromising the real-time ones, depending on kernel configuration. This is especially important for the successful adoption of the proposed

Marco Cereia; Ivan Cibrario Bertolotti; Stefano Scanzio

2010-01-01

342

High-beta plasma formation and observation of peaked density profile in RT1  

Microsoft Academic Search

High-beta ECH plasma is generated and stably sustained in a magnetospheric configuration, the Ring Trap 1 (RT-1) device, generated by a levitated dipole field magnet. Geomagnetic-field compensation and optimized operation have realized drastic improvements in plasma properties. The maximum local beta value has reached 70% and the pressure profiles have a rather steep gradient near the superconducting magnet. Electrons of

H. Saitoh; Z. Yoshida; J. Morikawa; Y. Yano; T. Mizushima; Y. Ogawa; M. Furukawa; Y. Kawai; K. Harima; Y. Kawazura; Y. Kaneko; K. Tadachi; S. Emoto; M. Kobayashi; T. Sugiura; G. Vogel

2011-01-01

343

Flow cytometric detection of specific gene expression in prokaryotic cells using in situ RT-PCR  

Microsoft Academic Search

Prokaryotic in situ RT-PCR was coupled with flow cytometry to detect mRNA transcripts of the toluene dioxygenase (todC1) gene in intact cells of the bacterium Pseudomonas putida F1. Recovery efficiency of fixed cells over the course of the entire in situ detection procedure was approximately 81% for both P. putida F1 and AC10R cells. It appeared that lysozyme treatment and

Feng Chen; Brian Binder; Robert E Hodson

2000-01-01

344

Validation of Array-Based Gene Expression Profiles by Real-Time (Kinetic) RT-PCR  

Microsoft Academic Search

We evaluated real-time (kinetic) reverse transcrip- tion-polymerase chain reaction (RT-PCR) to validate differentially expressed genes identified by DNA ar- rays. Gene expression of two keratinocyte subclones differing in the physical state of human papillomavi- rus (episomal or integrated) was used as a model system. High-density filter arrays identified 444 of 588 genes as either negative or expressed with less than

Mangalathu S. Rajeevan; Suzanne D. Vernon; Naovarath Taysavang; Elizabeth R. Unger

2001-01-01

345

Evaluation of biological responses to polymeric biomaterials by RT-PCR analysis  

Microsoft Academic Search

In this study, we introduce a novel research methodology, the evaluation of mRNA expression of cells contacting with polymeric materials using reverse transcription-polymerase chain reaction (RT-PCR) analysis HL-60 was used as a model of the macrophages. The expression of interleukin-1? (IL-1?) mRNA, a cytokine secreted by macrophages, was selected to estimate the extent of inflammation. The expression of IL-1? mRNA

Akio Kishida; Shinya Kato; Kaori Ohmura; Kazuhisa Sugimura; Mitsuru Akashi

1996-01-01

346

Approaches to strategic research and technology (R&T) analysis and road mapping  

Microsoft Academic Search

Increasingly, the timely and successful incorporation of innovative technologies into new systems is a critical factor in their success or failure. This is true for both commercial and government space missions. In addition, continuing progress in methodologies that may enable the effective identification of long-term technology needs and opportunities—and the guidance of ongoing research and technology (R&T) programs to address

John C. Mankins

2002-01-01

347

Applicability of RNA standards for evaluating RT-qPCR assays and platforms.  

PubMed

The availability of diverse RT-qPCR assay formats and technologies hinder comparability of data between platforms. Reference standards to facilitate platform evaluation and comparability are needed. We have explored using universal RNA standards for comparing the performance of a novel qPCR platform (Fluidigm® BioMark™) against the widely used ABI 7900HT system. Our results show that such standards may form part of a toolkit to evaluate the key performance characteristics of platforms. PMID:21332979

Devonshire, Alison S; Elaswarapu, Ramnath; Foy, Carole A

2011-02-18

348

Improved quantitative real-time RT-PCR for expression profiling of individual cells  

Microsoft Academic Search

The real-time quantitative polymerase chain reac- tion (rtqPCR) has overcome the limitations of conventional, time-consuming quantitative PCR strategies and is maturing into a routine tool to quantify gene expression levels, following reverse transcription (RT) of mRNA into complementary DNA (cDNA). Expression profiling with single-cell resolution is highly desirable, in particular for com- plex tissues like the brain that contain a

Birgit Liss

2002-01-01

349

Early diagnosis of SARS Coronavirus infection by real time RT-PCR  

Microsoft Academic Search

Background: A novel coronavirus was recently identified as the aetiological agent of Severe Acute Respiratory Syndrome (SARS). Molecular assays currently available for detection of SARS-coronavirus (SARS-Cov) have low sensitivity during the early stage of the illness. Objective: To develop and evaluate a sensitive diagnostic test for SARS by optimizing the viral RNA extraction methods and by applying real-time quantitative RT-PCR

Leo L. M. Poon; Kwok Hung Chan; On Kei Wong; Wing Cheong Yam; Kwok Yung Yuen; Yi Guan; Y. M. Dennis Lo; Joseph S. M. Peiris

2003-01-01

350

Transcriptome profiling of host-microbe interactions by differential display RT-PCR.  

PubMed

In recent years, DNA microarray has become increasingly popular as a tool to investigate global expression patterns compared to differential display RT-PCR. Although differential display RT-PCR can be labour-intensive, it has its own merits over those of DNA microarray. While the latter usually consists of a well-defined set of species-specific genes, differential display RT-PCR allows the investigation of host-microbe interactions without bias towards any mRNA transcripts. This means that the regulated transcript expression of both host and pathogen can be analysed simultaneously. In addition, novel transcripts and alternate splicing variants pertaining to the infection can also be discovered. We have investigated the response of rhabdomyosarcoma cells to infection with a neurovirulent strain of enterovirus 71 (EV71) at different time-points during the infection process compared with uninfected cells. Using differential display RT-PCR, we identified mRNAs that were up- or down-regulated. Less than half of the clones match known genes including those involved in mediating the cytoskeleton, cell cycle, cell death, protein translational machinery and cellular transport. The rest of the clones do not match any known genes, of which several are novel genes. Noteworthy is the discovery of an alternate splicing form of TRIP7, which is down-regulated during EV71 infection. The differential display technique has potentially wide applicability to elucidate the gene expression or transcriptomic profiles of host-microbe interactions, which can provide a better understanding of microbial pathogenesis. PMID:20300989

Fook, Leong Wai; Chow, Vincent T K

2010-01-01

351

A host-based rt-PCR gene expression signature to identify acute respiratory viral infection.  

PubMed

Improved ways to diagnose acute respiratory viral infections could decrease inappropriate antibacterial use and serve as a vital triage mechanism in the event of a potential viral pandemic. Measurement of the host response to infection is an alternative to pathogen-based diagnostic testing and may improve diagnostic accuracy. We have developed a host-based assay with a reverse transcription polymerase chain reaction (RT-PCR) TaqMan low-density array (TLDA) platform for classifying respiratory viral infection. We developed the assay using two cohorts experimentally infected with influenza A H3N2/Wisconsin or influenza A H1N1/Brisbane, and validated the assay in a sample of adults presenting to the emergency department with fever (n = 102) and in healthy volunteers (n = 41). Peripheral blood RNA samples were obtained from individuals who underwent experimental viral challenge or who presented to the emergency department and had microbiologically proven viral respiratory infection or systemic bacterial infection. The selected gene set on the RT-PCR TLDA assay classified participants with experimentally induced influenza H3N2 and H1N1 infection with 100 and 87% accuracy, respectively. We validated this host gene expression signature in a cohort of 102 individuals arriving at the emergency department. The sensitivity of the RT-PCR test was 89% [95% confidence interval (CI), 72 to 98%], and the specificity was 94% (95% CI, 86 to 99%). These results show that RT-PCR-based detection of a host gene expression signature can classify individuals with respiratory viral infection and sets the stage for prospective evaluation of this diagnostic approach in a clinical setting. PMID:24048524

Zaas, Aimee K; Burke, Thomas; Chen, Minhua; McClain, Micah; Nicholson, Bradly; Veldman, Timothy; Tsalik, Ephraim L; Fowler, Vance; Rivers, Emanuel P; Otero, Ronny; Kingsmore, Stephen F; Voora, Deepak; Lucas, Joseph; Hero, Alfred O; Carin, Lawrence; Woods, Christopher W; Ginsburg, Geoffrey S

2013-09-18

352

Multiplex nested RT-PCR for the detection of porcine enteric viruses.  

PubMed

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine group A rotavirus (PRV-A) are major viruses causing enteric diseases of piglets. A multiplex nested reverse transcription polymerase chain reaction (multiplex nested RT-PCR) was developed for the detection of these viruses in field samples from piglets with diarrhea. A mixture of (1) three external pairs of primers, yielding in the amplification step two different amplicons with sizes of 950 bp and 317 bp and (2) three pairs of internal primers in a second round of PCR (nested PCR), yielding two different amplicons with sizes of 792 bp and 208 bp for TGEV and porcine PRV-A, respectively. The genome of PEDV was not detected after the amplification step but it was detected in the second round of PCR, yielding amplicon with size of 291 bp. Multiplex nested RT-PCR can detect TGEV, PRV-A, and PEDV up to concentration 10(2) TCID(50)/mL, 10(1) TCID(50)/mL, and 27.2 microg/microl of RNA, respectively. A total of 175 field samples were collected from swine with diarrhea from January 2005 until July 2007. The samples were tested for the presence of three viruses by a multiplex nested RT-PCR. Dual infections with PEDV and PRV-A were identified in seven specimens (4%) (n = 6). Twenty-one (25%) infections were caused by PEDV and thirty-four infections (41%) were caused by PRV-A. The genome of TGEV was not detected in any of these field samples, however TGEV was detected in piglets infected experimentally. The multiplex nested RT-PCR is rapid, sensitive, and a cost-effective detection method for the detection of porcine enteric viruses. PMID:20170679

Ben Salem, Abid Nabil; Chupin Sergei, A; Bjadovskaya Olga, P; Andreeva Olga, G; Mahjoub, Aouni; Prokhvatilova Larissa, B

2010-02-17

353

RT lasing via nanoscale CdSe islands in a (Zn,Mg)(S,Se) matrix  

Microsoft Academic Search

New approach for high performance lasing in semiconductors using an effect of exciton-induced waveguiding is proposed and\\u000a realized. We demonstrate RT lasing in a structure without external waveguide formed by cladding of an active region with wider\\u000a bandgap layers. Stacked arrays of nanoscale CdSe islands inserted directly in a wide bandgap matrix are used as an active\\u000a media in our

I. L. Krestnikov; S. V. Ivanov; P. S. Kop’ev; N. N. Ledentsov; M. V. Maximov; A. V. Sakharov; S. V. Sorokin; C. M. Sotomayor Torres; D. Bimberg; Zh. I. Alferov

1998-01-01

354

Miniature RT-PCR systems integrated with a sample pretreatment device for virus detection  

Microsoft Academic Search

This paper presents a new chip-based reverse transcription polymerase chain reaction (RT-PCR) system integrated with a sample pretreatment device for fast DNA amplification and diagnosis of RNA- based viruses. A new two-way serpentine-shape (S- shape) pneumatic micropump and a magnetic bio- separator were developed for purification and enrichment of viruses. The target RNA-based virus would be bound onto the antibody-conjugated

Kang-Yi Lien; Wang-Ying Lin; Chih-Hao Wang; Huan-Yao Lei; Gwo-Bin Lee

2007-01-01

355

Semi-quantitative RT-PCR analysis of photoregulated gene expression in marine diatoms  

Microsoft Academic Search

The low cell densities of diatoms and other phytoplankton in culture has precluded the use of classical RNA analysis techniques for routine studies of gene expression in large numbers of samples. This has seriously hampered studies of the basic biology of such organisms. To circumvent this problem, we have developed a high-throughput semi-quantitative RT-PCR-based protocol and used it to monitor

Catherine Leblanc; Angela Falciatore; Masakatsu Watanabe; Chris Bowler

1999-01-01

356

Optimization of an EHR Mobile Application Using the UFuRT Conceptual Framework  

PubMed Central

Drchrono is an Electronic Healthcare Record (EHR) application designed specifically for the iPad. Described as portable, efficient and accessible anywhere, drchrono has several features that might be attractive to health care providers. However, EHRs have to conform to certain Federal Healthcare Information Technology Guidelines, which are evaluated in a series of 12 test procedures, defined by the Office of the National Coordinator for Health Information Technology (ONC). In this study, we evaluated Test Procedure for §170.302 (c) Maintain up-to-date problem list, in drchrono. The methodology for our evaluation was contained within Zhang’s unified framework for usability, using UFuRT, i.e. user, functional, representation and task analyses. Based upon the analysis, using Adobe Flex, we then designed a prototype that corrected or improved on perceived weaknesses in the functionality served by the test procedure. We applied the test procedure taxonomy to a prototypic modification of drchrono, and then repeated the UFuRT usability analysis. We also used a 14-item heuristic evaluation by each member of our informatics team. Our findings support a conclusion that UFuRT is a valuable tool to evaluate EHR usability and that an “up-to-date problem” list may be customized, according to healthcare provider preference.

Amith, Muhammad; Loubser, Paul G.; Chapman, John; Zoker, Kaafoe C.; Rabelo Ferreira, Fred E. R.

2012-01-01

357

Selection of suitable reference genes for RT-qPCR analyses in cyanobacteria.  

PubMed

Cyanobacteria are a group of photosynthetic prokaryotes that have a diverse morphology, minimal nutritional requirements and metabolic plasticity that has made them attractive organisms to use in biotechnological applications. The use of these organisms as cell factories requires the knowledge of their physiology and metabolism at a systems level. For the quantification of gene transcripts real-time quantitative polymerase chain reaction (RT-qPCR) is the standard technique. However, to obtain reliable RT-qPCR results the use and validation of reference genes is mandatory. Towards this goal we have selected and analyzed twelve candidate reference genes from three morphologically distinct cyanobacteria grown under routinely used laboratory conditions. The six genes exhibiting less variation in each organism were evaluated in terms of their expression stability using geNorm, NormFinder and BestKeeper. In addition, the minimum number of reference genes required for normalization was determined. Based on the three algorithms, we provide a list of genes for cyanobacterial RT-qPCR data normalization. To our knowledge, this is the first work on the validation of reference genes for cyanobacteria constituting a valuable starting point for future works. PMID:22496882

Pinto, Filipe; Pacheco, Catarina C; Ferreira, Daniela; Moradas-Ferreira, Pedro; Tamagnini, Paula

2012-04-04

358

RT-PCR is a more accurate diagnostic tool for detection of BCR-ABL rearrangement  

SciTech Connect

Detection of the Philadelphia chromosome (Ph1) or genomic Southern hybridization for clonal gene rearrangement (GSH-R) has provided very specific identification of BCR-ABL gene rearrangement. Reverse transcriptase-polymerase chain reaction (RT-PCR) is diagnostic for patterns of BCR-ABL expression which are undetected by GSH-R and/or Ph1 and provides increased sensitivity both at diagnosis and in detection of minimal residual leukemia. Fifty-three specimens (of 150 tested from 119 consecutive leukemia patients) were RT-PCR positive for BCR-ABL gene expression confirmed by hybridization of PCR products with b{sub 3}a{sub 2}, b{sub 2}a{sub 2}, or e{sub 1}a{sub 2} junction-specific oligonucleotides. In 6 cases of CML with GSH-R{sup {minus}}at diagnosis, RT-PCR provided specific BCR-ABL identification. Deletion of BCR regions, low mitotic index, or e{sub 1}a{sub 2} expression caused failure to detect GSH-R or Ph1 translocation.

Zehnbauer, B.A.; Allen, A.P.; McGrath, S.D. [Johns Hopkins Oncology Center, Balitmore, MD (United States)] [and others

1994-09-01

359

Transition from Classical RM Instability at Embedded Material Interface to Ablative RT Growth  

NASA Astrophysics Data System (ADS)

If a laser target is designed with material interface(s) embedded into it, then the imperfections of such interface(s) can contribute to seeding the ablative Rayleigh-Taylor (RT) instability at its accelerated surface. We study the case of a single rippled interface in planar geometry. As the classical Richtmyer-Meshkov (RM) growth starts at the shocked interface, rippled transmitted and reflected shock waves carry the perturbations to the unperturbed rear surface of the target and the ablation front. After the shock breakouts, rarefaction waves from both sides of the target run back to the interface. Rapid deceleration of the interface in a rarefaction wave coming from a lower-density fluid triggers its oscillation that reverses the phase of mass modulation. As a result, the subsequent ablative RT growth starts in the negative direction, in contrast with the regular cases of ablative RT growth initiated by the ripples on either front or rear surface of the target. We present the results of the theory and numerical simulations for hydrodynamic experiments with plastic-foam targets on the Nike KrF laser at NRL.

Velikovich, A. L.; Schmitt, A. J.; Gardner, J. H.; Metzler, N.; Aglitskiy, Y.

2004-11-01

360

Improvement of HRV quantification using cRNA-based standards for real time RT-PCR.  

PubMed

Real Time RT-PCR developed in recent years represents an useful tool in the diagnosis of RNA viruses. In order to accurately quantify and normalize a RNA target, efficiency of reverse-transcription must be considered. In this study, a cRNA-standard-based quantitative Real Time RT-PCR have been developed for HRV quantification on bronchoalveolar lavage (BAL) specimens. Results has been compared to a quantitative plasmid standard-based Real Time RT-PCR previously developed by us. Large amount of pHRV was linearized and purified. Blunt ends were generated and cRNA production was carried out. Dilutions of cRNA were generated and dynamic range, intra- and inter-test variability, sensitivity, and limit of detection were evaluated. Sixty-seven BAL, previously resulted positive to our plasmid standard-based method, were evaluated using cRNA-standard quantification. cRNA curve showed a broad dynamic range with a good intra- and inter-test variability, with an average of 3.23 threshold cycles more in comparison to plasmid standard-based curve. In terms of specimen quantification, a difference of 1.07 log was found, showing a significant underrate using plasmid standard-based quantification. The method for cRNA-standard construction seems more suitable for quantification of RNA viruses, in order to normalize the quantification in reverse-transcription. PMID:21042885

Terlizzi, Maria Elena; Bergallo, Massimiliano; Astegiano, Sara; Sidoti, Francesca; Gambarino, Stefano; Solidoro, Paolo; Costa, Cristina; Cavallo, Rossana

2011-05-01

361

Effect of shear on mixing in R-T mixing layers at low Atwood numbers  

NASA Astrophysics Data System (ADS)

Effect of shear on R-T mixing is studied at two different Atwood numbers using the gas channel facility at Texas A&M University. The channel basically consists of two streams separated by a splitter plate. Pure air flows on top of the plate where as the lower density air Helium mixture flows on bottom and R-T mixing starts right after the splitter plate. Two different techniques, high resolution digital image analysis and simultaneous 3 wire cold wire Anemometry are used to measure R-T mixing growth rates. Results obtained from both the techniques are compared. Temperature is used as a marker to identify the streams and density is calculated from the temperature measured using a cold wire. Experiments are performed at Atwood numbers 0.04 and 0.1. At these Atwood numbers, effect of shear is studied by varying the velocity of one of the streams (mainly top stream). Simultaneous 3 wire cold wire Anemometry is performed at the vertical center line at three different axial locations. Different parameters obtained from these measurements including, ? (molecular mixing parameter), &'circ;^2 and vertical turbulent mass flux &'circ; v^' and their effect on mixing growth rate are discussed.

Akula, Bhanesh; Andrews, Malcolm; Ranjan, Devesh

2010-11-01

362

Optimization of an EHR mobile application using the UFuRT conceptual framework.  

PubMed

Drchrono is an Electronic Healthcare Record (EHR) application designed specifically for the iPad. Described as portable, efficient and accessible anywhere, drchrono has several features that might be attractive to health care providers. However, EHRs have to conform to certain Federal Healthcare Information Technology Guidelines, which are evaluated in a series of 12 test procedures, defined by the Office of the National Coordinator for Health Information Technology (ONC). In this study, we evaluated Test Procedure for §170.302 (c) Maintain up-to-date problem list, in drchrono. The methodology for our evaluation was contained within Zhang's unified framework for usability, using UFuRT, i.e. user, functional, representation and task analyses. Based upon the analysis, using Adobe Flex, we then designed a prototype that corrected or improved on perceived weaknesses in the functionality served by the test procedure. We applied the test procedure taxonomy to a prototypic modification of drchrono, and then repeated the UFuRT usability analysis. We also used a 14-item heuristic evaluation by each member of our informatics team. Our findings support a conclusion that UFuRT is a valuable tool to evaluate EHR usability and that an "up-to-date problem" list may be customized, according to healthcare provider preference. PMID:23304290

Amith, Muhammad; Loubser, Paul G; Chapman, John; Zoker, Kaafoe C; Rabelo Ferreira, Fred E R

2012-11-03

363

Rapid detection of polioviruses in environmental water samples by one-step duplex RT-PCR.  

PubMed

This study describes the rapid detection of polioviruses in environmental waters by a simple reverse transcriptase-polymerase chain reaction (RT-PCR) using two primer pairs for differentiation of poliovirus from non-polio enteroviruses in a single reaction by a one-step method, combining RT and PCR in a single tube. The detection by agarose gel electrophoresis yielded 2 bands of 153-bp and 293-bp for poliovirus tested without the need for further hybridization. The detection sensitivity of this one-step duplex RT-PCR, as measured with RNA extracted by heat treatment from supernatant of infected cell extracts, was 10(-1) 50% tissue culture effective doses (TCID50). This assay was used to evaluate the ability of sample concentration by membrane filter-based adsorption and elution, and purification by a simple RNA isolation based on guanidine isothiocyanate-phenol-chloroform extraction; the system yielded a detection limit of 5 x 10(-1) TCID50 seeded in 5 liters of tap water. This protocol was applied to the poliovirus detection in environmental water collected from 2 communities in Bangkok, Thailand during February and May 1998. Of 100 samples tested, 2 water samples collected from the same open sewage pipeline at one location were positive for polioviruses and one sample collected from another sewage pipeline was positive for non-polio enterovirus while a further 97 water samples were negative for both polioviruses and non-polio enteroviruses. With poliovirus detection by cell culture technique, none of the 100 samples tested was positive for poliovirus type 1, 2 or 3. RT-PCR was more sensitive, rapid, simple and cost-effective than the cell culture technique since the two water samples which were positive for polioviruses by RT-PCR failed to be detected by cell culture. Sequence data of 293-bp amplicons from positive samples were compared with those of reference poliovirus strains in the Genbank and the EMBL databases and identity to the sequence of type 1 strain Sabin was found to be 99%. PMID:11023064

Tansuphasiri, U; Vathanophas, K; Pariyanonda, A; Kittigul, L; Utrarachkij, F; Diraphat, P; Siripanichgon, K; Punchitton, S; Chitpirom, K; Cheaochantanakij, N

2000-03-01

364

Steered Molecular Dynamics Simulation on the Binding of NNRTI to HIV-1 RT  

SciTech Connect

HIV-1 reverse transcriptase (RT) is the primary target for anti-AIDS chemotherapy. Nonnucleoside RT inhibitors (NNRTIs) are very potent and most promising anti-AIDS drugs that specifically inhibit HIV-1 RT. The binding and unbinding processes of alpha-APA, an NNRTI, have been studied using nanosecond conventional molecular dynamics and steered molecular dynamics simulations. The simulation results show that the unbinding process of alpha-APA consists of three phases based on the position of alpha-APA in relation to the entrance of the binding pocket. When alpha-APA is bound in the binding pocket, the hydrophobic interactions between HIV-1 RT and alpha-APA dominate the binding; however, the hydrophilic interactions (both direct and water-bridged hydrogen bonds) also contribute to the stabilizing forces. Whereas Tyr-181 makes significant hydrophobic interactions with alpha-APA, Tyr-188 forms a strong hydrogen bond with the acylamino group (N14) of alpha-APA. These two residues have very flexible side chains and appear to act as two ''flexible clamps'' discouraging alpha-APA to dissociate from the binding pocket. At the pocket entrance, two relatively inflexible residues, Val-179 and Leu-100, gauge the openness of the entrance and form the bottleneck of the inhibitor-unbinding pathway. Two special water molecules at the pocket entrance appear to play important roles in inhibitor recognition of binding and unbinding. These water molecules form water bridges between the polar groups of the inhibitor and the residues around the entrance, and between the polar groups of the inhibitor themselves. The water-bridged interactions not only induce the inhibitor to adopt an energetically favorable conformation so the inhibitor can pass through the pocket entrance, but also stabilize the binding of the inhibitor in the pocket to prevent the inhibitor's dissociation. The complementary steered molecular dynamics and conventional molecular dynamics simulation results strongly support the hypothesis that NNRTIs inhibit HIV-1 RT polymerization activity by enlarging the DNA-binding cleft and restricting the flexibility and mobility of the p66 thumb subdomain that are believed to be essential during DNA translocation and polymerization.

Shen, Lingling; Shen, Jianhua; Luo, Xiaomin; Cheng, Feng; Xu, Yechun; Chen, Kaixian; Arnold, Edward; Ding, Jianping; Jiang, Hualiang

2003-06-01

365

Decreased Processivity of Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) Containing Didanosine-Selected Mutation Leu74Val: a Comparative Analysis of RT Variants Leu74Val and Lamivudine-Selected Met184Val  

PubMed Central

We previously showed that a didanosine-selected mutation in pNL4-3 background conferred a replication disadvantage on human immunodeficiency virus type 1, resulting in a loss of replication fitness. This work has been extended by showing that a recombinant virus with the HXBc2 backbone and reverse transcriptase (RT) fragments from pNL4-3 containing the Leu74Val mutation produce decreasing amounts of p24 antigen over a 3-week period. The HXBc2 recombinant containing the wild-type RT from pNL4-3 replicated efficiently. When the virion-associated RT containing the Leu74Val mutation was used in an RT processivity assay with homopolymer RNA template-primer, poly(A), and oligo(dT), the RT with altered Leu74Val mutation was less processive, generating fewer cDNA products in comparison to wild-type pNL4-3 RT. The replication kinetics and RT processivity of the mutant with the Leu74Val mutation were compared to those of a lamivudine-selected mutant Met184Val. In replication kinetics assays, mutant Leu74Val replicated slower than the mutant Met184Val. In a processivity assay, the mutant RTs from both viruses show comparable decreases in processivity. These observations provide biochemical evidence of decreased processivity to support the decrease in replication fitness observed with the Leu74Val or Met184Val mutations.

Sharma, Prem L.; Crumpacker, Clyde S.

1999-01-01

366

Regulation of the aprX-lipA operon of Pseudomonas fluorescens B52: differential regulation of the proximal and distal genes, encoding protease and lipase, by ompR-envZ.  

PubMed

The production of lipase and protease from psychrotrophic strains of Pseudomonas fluorescens may result in spoilage of dairy products. The lipase (lipA) and alkaline metalloprotease (aprX) genes of P. fluorescens B52 are regulated by temperature and are located at opposite ends of an operon which contains eight genes and spans 14 kb. In this report, we show that lipase activity in the supernatant of cultures of P. fluorescens strain B52 is also regulated by the homologue of the Escherichia coli EnvZ-OmpR two-component regulatory system. Differences in the regulation of lipase and protease may be related to the proximal and distal locations of aprX and lipA within the operon. PMID:15598539

McCarthy, Conor N; Woods, Rick G; Beacham, Ifor R

2004-12-15

367

The mutation T477A in HIV-1 reverse transcriptase (RT) restores normal proteolytic processing of RT in virus with Gag-Pol mutated in the p51-RNH cleavage site  

PubMed Central

Background The p51 subunit of the HIV-1 reverse transcriptase (RT) p66/p51 heterodimer arises from proteolytic cleavage of the RT p66 subunit C-terminal ribonuclease H (RNH) domain during virus maturation. Our previous work showed that mutations in the RT p51?RNH cleavage site resulted in virus with defects in proteolytic processing of RT and significantly attenuated infectivity. In some cases, virus fitness was restored after repeated passage of mutant viruses, due to reversion of the mutated sequences to wild-type. However, in one case, the recovered virus retained the mutated p51?RNH cleavage site but also developed an additional mutation, T477A, distal to the cleavage site. In this study we have characterized in detail the impact of the T477A mutation on intravirion processing of RT. Results While the T477A mutation arose during serial passage only with the F440V mutant background, introduction of this substitution into a variety of RT p51?RNH cleavage site lethal mutant backgrounds was able to restore substantial infectivity and normal RT processing to these mutants. T477A had no phenotypic effect on wild-type HIV-1. We also evaluated the impact of T477A on the kinetics of intravirion Gag-Pol polyprotein processing of p51?RNH cleavage site mutants using the protease inhibitor ritonavir. Early processing intermediates accumulated in p51?RNH cleavage site mutant viruses, whereas introduction of T477A promoted the completion of processing and formation of the fully processed RT p66/p51 heterodimer. Conclusions This work highlights the extraordinary plasticity of HIV-1 in adapting to seemingly lethal mutations that prevent RT heterodimer formation during virion polyprotein maturation. The ability of T477A to restore RT heterodimer formation and thus intravirion stability of the enzyme may arise from increased conformation flexibility in the RT p51?RNH cleavage site region, due to loss of a hydrogen bond associated with the normal threonine residue, thereby enabling proteolytic cleavage near the normal RT p51?RNH cleavage site.

2010-01-01

368

Accelerated ST-segment reduction after thrombolytic therapy with recombinant tissue plasminogen activator (rtPA) compared to urokinase.  

PubMed

Effects of therapy with urokinase (UK) and with recombinant tissue plasminogen activator (rtPA) were compared in patients with acute myocardial infarction (AMI). To achieve homogenous therapeutic conditions the comparison was restricted to patients having their first AMI and to cases of clinically successful thrombolytic therapy (defined by non-invasive criteria, such as a 50% decrease in elevated ST-segment in the worst load of a 12 lead ECG within 300 min after onset of thrombolytic therapy, complete pain resolution during thrombolytic therapy, and later confirmed by angiography 10 days after AMI). Effects of UK and rtPA on continuous multilead ST-segment analysis and cardiac proteins (creatine kinase and its isoenzyme CK-MB, aspartate transaminase and hydroxybutyrate dehydrogenase) were analyzed during 24 hours following onset of therapy. Continuous ST analysis showed a faster resolution of the elevated ST-segments after thrombolytic therapy with rtPA than with UK(p < 0.01). Accelerated idioventricular rhythms (p < 0.05) occurred sooner following rtPA than UK treatment. The wash-out of creatine kinase was increased (p < 0.01) after rtPA. Although both drugs induced comparable, angiographically controlled reperfusion, the results suggest that the process of reperfusion was accelerated during thrombolysis with rtPA compared to UK. Thrombolytic therapy of AMI with rtPA may hence improve myocardial salvage. PMID:8632624

Derad, I; Stierle, U; Giannitsis, E; Potratz, J; Born, J; Djonlagic, H; Fehm, H L

1996-01-01

369

A DICOM-RT radiation oncology ePR with decision support utilizing a quantified knowledge base from historical data  

NASA Astrophysics Data System (ADS)

During the last 2 years we have been working on developing a DICOM-RT (Radiation Therapy) ePR (Electronic Patient Record) with decision support that will allow physicists and radiation oncologists during their decision-making process. This ePR allows offline treatment dose calculations and plan evaluation, while at the same time it compares and quantifies treatment planning algorithms using DICOM-RT objects. The ePR framework permits the addition of visualization, processing, and analysis tools, which combined with the core functionality of reporting, importing and exporting of medical studies, creates a very powerful application that can improve the efficiency while planning cancer treatments. Usually a Radiation Oncology department will have disparate and complex data generated by the RT modalities as well as data scattered in RT Information/Management systems, Record & Verify systems, and Treatment Planning Systems (TPS) which can compromise the efficiency of the clinical workflow since the data crucial for a clinical decision may be time-consuming to retrieve, temporarily missing, or even lost. To address these shortcomings, the ACR-NEMA Standards Committee extended its DICOM (Digital Imaging & Communications in Medicine) standard from Radiology to RT by ratifying seven DICOM RT objects starting in 1997 [1,2]. However, they are not broadly used yet by the RT community in daily clinical operations. In the past, the research focus of an RT department has primarily been developing new protocols and devices to improve treatment process and outcomes of cancer patients with minimal effort dedicated to integration of imaging and information systems. Our attempt is to show a proof-of-concept that a DICOM-RT ePR system can be developed as a foundation to perform medical imaging informatics research in developing decision-support tools and knowledge base for future data mining applications.

Documet, Jorge R.; Liu, Brent; Le, Anh; Law, Maria

2008-04-01

370

Magnetic properties of RT2Al20 (R = Gd, Eu and Yb, T = Ti, V and Cr)  

NASA Astrophysics Data System (ADS)

Isostructural RT2Al20 series of compounds contain less than 5 at. % of rare earth ions. Thermodynamic and transport measurements were performed on solution-grown, single crystals: both R = Gd and Eu series manifest clear local moment behavior with magnetic ordering below 10 K. These low transition temperatures are consistent with the dilute nature of the rare earth ions. Unlike the RT2Zn20 series, we have not found enhanced magnetic order or near-Stoner like behavior for any member of the RT2Al20 family of compounds. The R = Yb members, however, all manifest weak Pauli paramagnetism, consistent with a divalent state for the Yb ions.

Frederick, J.; Jia, Shuang; Bud'Ko, S. L.; Canfield, P. C.

2008-03-01

371

A DICOM-RT Based ePR radiation therapy information system for decision-support of brain tumor patients  

NASA Astrophysics Data System (ADS)

The need for comprehensive clinical image data and relevant information in image-guided Radiation Therapy (RT) is becoming steadily apparent. Multiple standalone systems utilizing the most technological advancements in imaging, therapeutic radiation, and computerized treatment planning systems acquire key data during the RT treatment course of a patient. One example are patients treated for brain tumors of greater sizes and irregular shapes that utilize state-of-the-art RT technology to deliver pinpoint accurate radiation doses. Various treatment options are available to the patient from Radiation Therapy to Stereotactic Radiosurgery and utilize different RT modalities. The disparate and complex data generated by the RT modalities along with related data scattered throughout the RT department in RT Information/Management systems, Record & Verify systems, and Treatment Planning Systems (TPS) compromise an efficient clinical workflow since the data crucial for a clinical decision may be time-consuming to retrieve, temporarily missing, or even lost. To address these shortcomings, the ACR-NEMA Standards Committee extended its DICOM (Digital Imaging & Communications in Medicine) Standard from Radiology to RT by ratifying seven DICOM RT objects starting in 1997. However, they are rarely used by the RT community in daily clinical operations. In the past, the research focus of an RT department has primarily been developing new protocols and devices to improve treatment process and outcomes of cancer patients with minimal effort dedicated to integration of imaging and information systems. By combining our past experience in medical imaging informatics research, DICOM-RT expertise, and system integration, our research involves using a brain tumor case model to show proof-of-concept that a DICOM-Standard electronic patient record (ePR) system can be developed as a foundation to perform medical imaging informatics research in developing decision-support tools and knowledge base for future data mining applications. As an initial first step, we will develop a methodology to perform medical imaging informatics research on a clinical scenario where brain tumor patients undergo treatment planning for either radiosurgery or radiation therapy. Specifically, we will research the "inverse treatment planning" process that is used for those types of treatments and integrate decision-support knowledge and tools designed to assist in the decision-making process, thus introducing an improved "knowledge-enhanced treatment planning" approach.

Liu, B. J.; Law, M.; Huang, H. K.; Zee, C. S.; Chan, L.

2006-03-01

372

[Identification of enteroviruses from central nervous system infections by RT-PCR and cell culture methods].  

PubMed

Viruses are the major causes of aseptic meningitis and encephalitis. Enteroviruses account for more than 80% of the aseptic meningitis cases for which an etiologic agent is identified. The aims of the present study were to identify agents of enteroviral meningitis by viral culture and reverse transcriptase polymerase chain reaction (RT-PCR) methods, to evaluate the appropriateness of a commercial RTPCR kit for its use in routine laboratory, and to obtain epidemiological data about enteroviral meningitis. Sixty six cerebrospinal fluid (CSF) samples from patients with suspected viral central nervous system (CNS) infection by clinical and CSF biochemical findings, sent to Ege University Faculty of Medicine, Department of Medical Microbiology were included in the study. The CSF samples were all negative for tested bacteria, mycobacteria, fungi, herpes simplex virus and cytomegalovirus. Thirty-four (51.5%) of the samples were from female and 32 (48.5%) were from male patients. Twenty-three (34.8%) patients were children (5 months-18 years) and 43 (65.2%) were adults (19-86 years). Shell vial rapid cell culture method by using Vero, HEp-2 and RD cell lines was performed for virus isolation and the results were evaluated on 48th hours after staining the cells with fluorescein labeled polyclonal antibodies (Pan-Enterovirus Blend, Light Diagnostics, USA). Enteroviral RNA in the samples was detected by a commercial RT-PCR kit (Enterovirus Consensus Kit, Argene, France). Sixty-one (92.4%) of 66 samples from patients with suspected viral CNS infection were found to be negative for enterovirus both with RT-PCR and shell vial cell culture methods. Three samples (4.5%) were positive by shell vial culture method. In one CSF sample that was culture positive, RT-PCR was also positive. However, the remaining two culture positive samples yielded negative result by RT-PCR. Intermediate results with RT-PCR were obtained in two samples (3%) that were identified as negative by cell culture. Two of the three positive samples in cell culture were identified as echovirus, however, the remaining sample could not be identified due to small sample amount. As a result, the commercial assay was found non-practical and labor intensive, giving indeterminant results in some cases and missing two culture positive samples. Since it didn't have an advantage over the cell culture method used, it was found inappropriate for routine diagnosis in our laboratory. On the other hand, it has been known that nucleic acid amplification tests (NAT) have markedly improved the diagnosis of enterovirus infections by increasing the sensitivity compared with cell culture methods. An alternative NAT method should be evaluated in parallel with cell culture method especially in CSF samples of children with suspected viral central nervous system infections. PMID:21935780

K?l?ç, Ilknur; Altu?lu, Imre; Ciçek, Candan; Pullukçu, Hüsnü; Bayram, Nuri; Sirin, Hadiye; Erensoy, Selda

2011-07-01

373

Multiparametric ISODATA analysis of embolic stroke and rt-PA intervention in rat.  

PubMed

To increase the sensitivity of MRI parameters to detect tissue damage of ischemic stroke, an unsupervised analysis method, Iterative Self-Organizing Data Analysis Technique Algorithm (ISODATA), was applied to analyze the temporal evolution of ischemic damage in a focal embolic cerebral ischemia model in rat with and without recombinant tissue plasminogen activator (rt-PA) treatment. Male Wistar rats subjected to embolic stroke were investigated using a 7-T MRI system. Rats were randomized into control (n=9) and treated (n=9) groups. The treated rats received rt-PA via a femoral vein at 4 h after onset of embolic ischemia. ISODATA analysis employed parametric maps or weighted images (T1, T2, and diffusion). ISODATA results with parametric maps are superior to ISODATA with weighted images, and both of them were highly correlated with the infarction size measured from the corresponding histological section. At 24 h after embolic stroke, the average map ISODATA lesion sizes were 37.7+/-7.0 and 39.2+/-5.6 mm2 for the treated and the control group, respectively. Average histological infarction areas were 37.9+/-7.4 mm2 for treated rats and 39.4+/-6.1 mm2 for controls. The R2 values of the linear correlation between map ISODATA and histological data were 0.98 and 0.96 for treated and control rats, respectively. Both histological and map ISODATA data suggest that there is no significant difference in infarction area between non-treated and rt-PA-treated rats when treatment was administered 4 h after the onset of embolic stroke. The ISODATA lesion size analysis was also sensitive to changes of lesion size during acute and subacute stages of stroke. Our data demonstrate that the multiparameter map ISODATA approach provides a more sensitive quantitation of the ischemic lesion at all time points than image ISODATA and single MRI parametric analysis using T1, T2 or ADCw. PMID:15337614

Ding, Guangliang; Jiang, Quan; Zhang, Li; Zhang, Zhenggang; Knight, Robert A; Soltanian-Zadeh, Hamid; Lu, Mei; Ewing, James R; Li, Qingjiang; Whitton, Polly A; Chopp, Michael

2004-08-30

374

Selecting control genes for RT-QPCR using public microarray data  

PubMed Central

Background Gene expression analysis has emerged as a major biological research area, with real-time quantitative reverse transcription PCR (RT-QPCR) being one of the most accurate and widely used techniques for expression profiling of selected genes. In order to obtain results that are comparable across assays, a stable normalization strategy is required. In general, the normalization of PCR measurements between different samples uses one to several control genes (e.g. housekeeping genes), from which a baseline reference level is constructed. Thus, the choice of the control genes is of utmost importance, yet there is not a generally accepted standard technique for screening a large number of candidates and identifying the best ones. Results We propose a novel approach for scoring and ranking candidate genes for their suitability as control genes. Our approach relies on publicly available microarray data and allows the combination of multiple data sets originating from different platforms and/or representing different pathologies. The use of microarray data allows the screening of tens of thousands of genes, producing very comprehensive lists of candidates. We also provide two lists of candidate control genes: one which is breast cancer-specific and one with more general applicability. Two genes from the breast cancer list which had not been previously used as control genes are identified and validated by RT-QPCR. Open source R functions are available at Conclusion We proposed a new method for identifying candidate control genes for RT-QPCR which was able to rank thousands of genes according to some predefined suitability criteria and we applied it to the case of breast cancer. We also empirically showed that translating the results from microarray to PCR platform was achievable.

Popovici, Vlad; Goldstein, Darlene R; Antonov, Janine; Jaggi, Rolf; Delorenzi, Mauro; Wirapati, Pratyaksha

2009-01-01

375

Confinement of Toroidal Non-neutral Plasma in Proto-RT  

SciTech Connect

In contrast to linear configurations for non-neutral plasmas, toroidal devices allow us to trap charged particles without the use of a plugging electric field. Thus it has a potential ability to confine high-energy particles or to simultaneously trap multiple particles with different charges. In spite of the relatively long history of the study in pure toroidal magnetic field devices, toroidal non-neutral plasmas are attracting renewed interest with the use of magnetic surface configurations. Possible applications of toroidal traps for non-neutral plasmas are formation of matter-antimatter plasmas, investigation on the fundamental properties of exotic plasmas including pair (equal mass) plasmas, and experimental test on the equilibrium and stability of flowing plasmas. As an initial test on non-neutral plasmas in the toroidal magnetic-surface geometry, formation and confinement properties of pure electron plasma have been investigated at Prototype-Ring Trap (Proto-RT) device with a dipole magnetic field. Electrons can be injected by using chaotic orbit near a magnetic null line generated by the combination of dipole and vertical magnetic fields. The confinement time of electrons is limited due to the effects of collisions with remaining neutral gas, and electrons of {approx} 1012 are trapped for {approx} 0.5 s in the typical magnetic field strength of 100 G and back pressure of 4 x 10-7 Torr in Proto-RT. Although the present experiment was carried out on the single-component plasma, the result shows that a stable confinement configuration has been realized for toroidal non-neutral plasmas by using the magnetic surface configuration. Together with the experiment on the toroidal pure electron plasma in Proto-RT, preliminary prospects for the injection and trap of anti-protons and positrons in the toroidal magnetic surface configuration, and creation of multi-component plasmas will be described.

Saitoh, H.; Yoshida, Z.; Watanabe, S. [Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8583 (Japan)

2005-10-19

376

Novel biomarkers for the detection of wound infection by wound fluid RT-PCR in rats.  

PubMed

Wound infection is a form of host damage resulting from an imbalance in pathogen virulence and the host immune response. However, at present, diagnosis is based solely on bacterial numbers or inflammatory signs and is therefore not precise. Thus, infection diagnosis requires indicators of both of these factors. We focused on wound fluid because it includes both bacteria and host cells. The purpose of this study was to establish biomarkers that reflect both bacterial and host factors using the reverse transcription-polymerase chain reaction method on the centrifugal precipitation of wound fluids (wound fluid RT-PCR). We created full thickness wounds in animal models of the three groups: control, colonization and infection, which were conditioned by administration of different concentrations of Pseudomonas aeruginosa dispersion. Messenger RNA expression in bacteria and host cells was analysed. Expression of bacterial housekeeping genes was detected in the samples in the colonization and infection groups. Expression of host housekeeping genes was detected in all samples from the three groups. Expression of toxA, encoding the virulence factor exotoxin A, was detected in 90% of samples in the infection group only. Expression of Foxp3, encoding the transcription factor forkhead box P3, was detected in 100% of samples only in the colonization group. These results revealed that wound fluid RT-PCR analysis reflected both bacterial virulence and the host immune status, and we determined the combination of novel biomarkers that can discriminate these three groups. We anticipate that wound fluid RT-PCR could be applied in the future to diagnose wound infection. PMID:22141756

Asada, Mayumi; Nakagami, Gojiro; Minematsu, Takeo; Nagase, Takashi; Akase, Tomoko; Huang, Lijuan; Yoshimura, Kotaro; Sanada, Hiromi

2011-12-06

377

Strokes with Minor Symptoms: An Exploratory Analysis of the NINDS rt-PA Trials  

PubMed Central

BACKGROUND The pivotal NINDS rt-PA trials excluded ischemic stroke patients with specific minor presentations or rapidly improving symptoms. The rt-PA product label notes that its use for minor neurological deficit or rapidly improving stroke symptoms has not been evaluated. As a result, patients with low NIHSS scores are not commonly treated in clinical practice. We sought to further characterize the minor stroke patients that were included in the NINDS trials. METHODS Minor strokes were defined as NIHSS ? 5 at baseline for this retrospective analysis, as this subgroup is most commonly excluded from treatment in clinical practice and trials. Clinical stroke syndromes were defined based on prespecified NIHSS item score clusters. Clinical outcomes were reviewed generally and within these cluster subgroups. RESULTS Only 58 cases had NIHSS scores of ?0–5 in the NINDS trials (42 rt-PA and 16 placebo), and 2971 patients were excluded from the trials due to “rapidly improving” or “minor symptoms” as the primary reason. No patients were enrolled with isolated motor symptoms, isolated facial droop, isolated ataxia, dysarthria, isolated sensory symptoms, or with only symptoms/signs not captured by the NIHSS score (i.e., NIHSS=0). There were three or fewer patients with each of the other isolated deficits enrolled in the trial. CONCLUSIONS The NINDS trials excluded a substantial number of strokes with minor presentations, those that were included were small in number, and conclusions about outcomes based on specific syndromes cannot be drawn. Further prospective, systematic study of this subgroup is needed.

Khatri, Pooja; Kleindorfer, Dawn O; Yeatts, Sharon D; Saver, Jeffrey L.; Levine, Steven R.; Lyden, Patrick; Moomaw, Charles J.; Palesch, Yuko Y; Jauch, Edward; Broderick, Joseph P

2010-01-01

378

Measurement delay associated with the Guardian(R) RT continuous glucose monitoring system  

PubMed Central

Aims Using compartment modelling, we assessed the time delay between blood glucose and sensor glucose measured by the Guardian® RT continuous glucose monitoring system in young subjects with Type 1 diabetes (T1D). Methods Twelve children and adolescents with T1D treated by continuous subcutaneous insulin infusion (male/female 7/5; age 13.1 ± 4.2 years; body mass index 21.9 ± 4.3 kg/m2; mean ± sd) were studied over 19 h in a Clinical Research Facility. Guardian® RT was calibrated every 6 h and sensor glucose measured every 5 min. Reference blood glucose was measured every 15 min using a YSI 2300 STAT Plus Analyser. A population compartment model of sensor glucose–blood glucose kinetics was adopted to estimate the time delay, the calibration scale and the calibration shift. Results The population median of the time delay was 15.8 (interquartile range 15.2, 16.5) min, which was corroborated by correlation analysis between blood glucose and 15-min delayed sensor glucose. The delay has a relatively low intersubject variability, with 95% of individuals predicted to have delays between 10.4 and 24.3 min. Population medians (interquartile range) for the scale and shift are 0.800 (0.777, 0.823) (unitless) and 1.66 (1.47, 1.84) mmol/l, respectively. Conclusions In young subjects with T1D, the total time delay associated with the Guardian® RT system was approximately 15 min. This is twice that expected on physiological grounds, suggesting a 5- to 10-min delay because of data processing. Delays above 25 min are rarely to be observed.

Wei, C; Lunn, D J; Acerini, C L; Allen, J M; Larsen, A M; Wilinska, M E; Dunger, D B; Hovorka, R

2010-01-01

379

Comparing protocols for preparation of DNA-free total yeast RNA suitable for RT-PCR  

PubMed Central

Background Preparation of RNA free from DNA is a critical step before performing RT-PCR assay. Total RNA isolated from several sources, including those obtained from Saccharomyces cerevisiae, using routine methodologies are frequently contaminated with DNA, which can give rise to amplification products that mimic the amplicons expected from the RNA target. Results We investigated the efficiency of two DNase I based protocols for eliminating DNA contaminations from RNA samples obtained from yeast cells. Both procedures are very efficient in eliminating DNA contamination from RNA samples and entail three main steps, which involve treating of RNA samples with DNase I, inhibition of the enzyme by EDTA and its subsequent inactivation at 65°C. The DNase I treated samples were further purified with phenol: chloroform followed by precipitation with ice-cold ethanol (protocol I) or, alternatively, they were directly used in RT-PCR reactions (protocol II). Transcripts from ACT1, PDA1, CNA1, CNA2, TPS1 and TPS2 analyzed after each treatment showed that all mRNAs tested can be amplified if total RNA was extracted and purified after DNase I treatment, however, only TPS1, TPS2 and ACT1 mRNAs were amplified without extraction/purification step. Conclusion Although more laborious and requiring a higher initial amount of material, the inclusion of an extraction and purification step allows to prepare RNA samples that are free from DNA and from low molecular contaminants and can be applied to amplify any Saccharomyces cerevisiae mRNA by RT-PCR.

Del Aguila, Eduardo M; Dutra, Marcio B; Silva, Joab T; Paschoalin, Vania MF

2005-01-01

380

Spectroscopic and photometric observations of the short-period RS CVn star RT And  

Microsoft Academic Search

Spectroscopic observations in the range 6500-6700 Å and BVRI photometry of the eclipsing short-period RS CVn-star RT And are presented. We determined K1=130 km s-1 and K2=175.8 km s-1 by measurement of the double profiles of the lines Halpha and FeI 6678, and obtained the mass ratio q=0.74 and masses of the star components M1=1.23 Msun and M2=0.91 Msun. It

D. P. Kjurkchieva; D. V. Marchev; W. Ogloza

2001-01-01

381

Ancient RNA? RT-PCR of 50-year-old RNA identifies peach latent mosaic viroid.  

PubMed

The preservation of macromolecules is at best haphazard. Modern techniques have improved the detection of ancient DNA and proteins, but there is little information on the preservation of RNA. Fifty-year-old dried leaf material showing symptoms of peach calico disease was used successfully in RT-PCRs to amplify peach latent mosaic viroid (PLMVd) RNA and the mRNA for the large subunit of ribulose 1,5-bisphosphate carboxylase (rubisco). These results indicate that naked RNA may be preserved, under suitable conditions, for at least 50 years. The results are discussed in the context of ancient DNA and proteins and the process of fossilization. PMID:23138153

Guy, Paul L

2012-11-09

382

Gene expression profile of circulating tumor cells in breast cancer by RT-qPCR  

PubMed Central

Background Circulating tumor cells (CTCs) have been associated with prognosis especially in breast cancer and have been proposed as a liquid biopsy for repeated follow up examinations. Molecular characterization of CTCs is difficult to address since they are very rare and the amount of available sample is very limited. Methods We quantified by RT-qPCR CK-19, MAGE-A3, HER-2, TWIST1, hTERT ?+?+, and mammaglobin gene transcripts in immunomagnetically positively selected CTCs from 92 breast cancer patients, and 28 healthy individuals. We also compared our results with the CellSearch system in 33 of these patients with early breast cancer. Results RT-qPCR is highly sensitive and specific and can detect the expression of each individual gene at the one cell level. None of the genes tested was detected in the group of healthy donors. In 66 operable breast cancer patients, CK-19 was detected in 42.4%, HER-2 in 13.6%, MAGE-A3 in 21.2%, hMAM in 13.6%, TWIST-1 in 42.4%, and hTERT ?+?+ in 10.2%. In 26 patients with verified metastasis, CK-19 was detected in 53.8%, HER-2 in 19.2%, MAGE-A3 in 15.4%, hMAM in 30.8%, TWIST-1 in 38.5% and hTERT ?+?+in 19.2%. Our preliminary data on the comparison between RT-qPCR and CellSearch in 33 early breast cancer patients showed that RT-qPCR gives more positive results in respect to CellSearch. Conclusions Molecular characterization of CTCs has revealed a remarkable heterogeneity of gene expression between breast cancer patients. In a small percentage of patients, CTCs were positive for all six genes tested, while in some patients only one of these genes was expressed. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome for these patients is known.

2011-01-01

383

Evaluating the Impacts of NASA/SPoRT Daily Greenness Vegetation Fraction on Land Surface Model and Numerical Weather Forecasts.  

National Technical Information Service (NTIS)

The NASA Short-term Prediction Research and Transition (SPoRT) Center has developed a Greenness Vegetation Fraction (GVF) dataset, which is updated daily using swaths of Normalized Difference Vegetation Index data from the Moderate Resolution Imaging Spec...

F. J. LaFontaine J. L. Case J. R. Bell S. V. Kumar

2012-01-01

384

Real time TaqMan RT-PCR assay for the detection of Cucumber green mottle mosaic virus.  

PubMed

A real time reverse-transcription polymerase chain reaction (RT-PCR) was developed for efficient detection of Cucumber green mottle mosaic virus (CGMMV). The method was designed to use a duo-primer system with a TaqMan probe targeting the conserved sequence in 3' noncoding region (NCR) of CGMMV to detect isolates of this virus collected in China. The sensitivity of the real time RT-PCR assay was 0.13 pg of total RNA or 50 molecules of RNA transcripts. This level of sensitivity indicated that the one step real time RT-PCR developed in the present study could be used for routine testing assays. The real time RT-PCR method could assist in the implementation of quarantine measures for prevention and control of the disease caused by CGMMV. PMID:18359519

Hongyun, Chen; Wenjun, Zhao; Qinsheng, Gu; Qing, Chen; Shiming, Lin; Shuifang, Zhu

2008-03-24

385

Stable confinement of electron plasma and initial results on positron injection in RT-1  

NASA Astrophysics Data System (ADS)

The Ring Trap 1 (RT-1) device is a dipole field configuration generated by a levitated superconducting magnet. It offers very interesting opportunities for research on the fundamental properties on non-neutral plasmas, such as self-organization of charged particles in the strongly positive and negative charged particles on magnetic surfaces. When strong positron sources will be available in the future, the dipole field configuration will be potentially applicable to the formation of an electron-positron plasma. We have realized stable, long trap of toroidal pure electron plasma in RT-1; Magnetic levitation of the superconducting magnet resulted in more than 300s of confinement for electron plasma of ~ 1011 m-3. Aiming for the confinement of positrons as a next step, we started a positron injection experiment. For the formation of positron plasma in the closed magnetic surfaces, one of the key issues to be solved is the efficient injection method of positron across closed magnetic surfaces. In contrast to linear configurations, toroidal configurations have the advantage that they are capable of trapping high energy positrons in the dipole field configuration and consider the possibility of direct trapping of positrons emitted from a 22Na source.

Saitoh, H.; Yoshida, Z.; Morikawa, J.; Yano, Y.; Kasaoka, N.; Sakamoto, W.; Nogami, T.

2013-03-01

386

The HYP-RT hypoxic tumour radiotherapy algorithm and accelerated repopulation dose per fraction study.  

PubMed

The HYP-RT model simulates hypoxic tumour growth for head and neck cancer as well as radiotherapy and the effects of accelerated repopulation and reoxygenation. This report outlines algorithm design, parameterisation and the impact of accelerated repopulation on the increase in dose/fraction needed to control the extra cell propagation during accelerated repopulation. Cell kill probabilities are based on Linear Quadratic theory, with oxygenation levels and proliferative capacity influencing cell death. Hypoxia is modelled through oxygen level allocation based on pO(2) histograms. Accelerated repopulation is modelled by increasing the stem cell symmetrical division probability, while the process of reoxygenation utilises randomised pO(2) increments to the cell population after each treatment fraction. Propagation of 10(8) tumour cells requires 5-30 minutes. Controlling the extra cell growth induced by accelerated repopulation requires a dose/fraction increase of 0.5-1.0 Gy, in agreement with published reports. The average reoxygenation pO(2) increment of 3 mmHg per fraction results in full tumour reoxygenation after shrinkage to approximately 1 mm. HYP-RT is a computationally efficient model simulating tumour growth and radiotherapy, incorporating accelerated repopulation and reoxygenation. It may be used to explore cell kill outcomes during radiotherapy while varying key radiobiological and tumour specific parameters, such as the degree of hypoxia. PMID:22778783

Harriss-Phillips, W M; Bezak, E; Yeoh, E

2012-06-19

387

RT-PCR and cell culture infectivity assay to detect enteroviruses during drinking water treatment processes.  

PubMed

In this study, 62 water samples were collected from two water treatment plants (WTPs) in Suez Canal cities (Port Said and Ismaillia) and one plant in Cairo (Giza WTP) in addition to the beginning of the two Nile river branches (Rosetta and Damietta). Viruses were concentrated by adsorption-elution ethod sing 142 mm-diameter nitrocellulose membrane of 0.45 microm pore size and eluted with 3% beef extract at pH 9.5. The concentrated samples were inoculated for 3 successive passages in three cell culture types (Vero, BGM and RD). Enterovirus RNAs in CPE-induced samples were extracted by guanidinium thiocyanate/ phenol/chloroform and heat shock methods and detected by RT-PCR and neutralization test. The results showed that eight samples [14.5% (8/62)] contained enteroviruses most of them were polioviruses [87.5% (7/8)] and coxsackievirus type B2 [12.5% (1/8)]. The three cell cultures were of the same sensitivity to detect the isolated viruses. Also, RT-PCR followed by neutralization assay facilitates and accelerate the results. The guanidinium thiocyanate extraction method was more sensitive than heat shock method. The results turned our attention to review our technology of water treatment and disinfection step in addition to the selection of suitable intake for the drinking water treatment plants. PMID:17219867

Ali, M A; El-Esnawy, N A; Shoaeb, A R; Ibraheim, M; El-Hawaary, S E

1999-01-01

388

Establishment of a multiplex RT-PCR assay for the rapid detection of fish cytokines.  

PubMed

To monitor the expression of cytokine genes in Japanese pufferfish, a novel platform for quantitative multiplexed analysis was developed. This custom-designed multiplex RT-PCR assay was used to analyze the expression profiles of 19 cytokine genes, including pro-inflammatory (IL-1?, IL-6, IL-17A/F3, IL-18, TNF-?, TNF-N), anti-inflammatory (IL-4/13A, IL-4/13B, IL-10), T-cell proliferation/differentiation (IL-2, IL-15, IL-21, TGF-?1), B-cell activation/differentiation (IL-7, IL-6, IL-4/13A, IL-4/13B), NK cell stimulation (IL-12p35 and IL-12p40), induction of anti-viral activity (I-IFN-1 and IFN-?), and monocyte/macrophage progenitor cell proliferation (M-CSF1b) cytokines in head kidney cells under immune stimulatory conditions. The expression profiles were dissimilar in the unstimulated control and immune-stimulated cells. Moreover, increased expression profile was observed due to different stimulations for IL-1?, IL-6, IL-10, IL-12p35, IL-12p40, IL-21, TNF-?, TNF-N, I-IFN-1 and IFN-? genes. These results suggest that cytokine genes could be used as biomarkers to know the immune status of fish. The constructed multiplex RT-PCR assay will enhance understanding on immune regulation by cytokines in fish. PMID:23237907

Kono, Tomoya; Takayama, Hiroaki; Nagamine, Ryusuke; Korenaga, Hiroki; Sakai, Masahiro

2012-11-16

389

Synthesis of kifunensine thioanalogs and their inhibitory activities against HIV-RT and ?-mannosidase.  

PubMed

An efficient and practical synthesis of kifunensine thioanalogs 1a-c was reported. The bicyclic azasugars fused thiazolidin-4-one 4a-c as key intermediates were first synthesized in good yields of 74-80% via one-pot tandem Staudinger/aza-Wittig/cyclization by using the pivotal azidosugars 3a and 3b derived from D-mannose. Followed by double Pummerer rearrangements and deprotection, the target thiokifunensine 1a and its epimers 1b and 1c were obtained in good yields. Compounds 1a-c were preliminary evaluated for their HIV-RT and ?-mannosidase (Jack bean) inhibitory activities. The results showed that such compounds exhibited significant anti-HIV-RT inhibitory activity but poor inhibitory against ?-mannosidase. To gain further insight into the inhibitory mechanism of compounds 1a-c, the analog compounds 9a-c were also prepared after deprotection from 4a-c, respectively. Activity comparison between compounds 1a-c and 9a-c suggests that the better activities of 1a-c than those of the 9a-c is possibly due to the additional carbonyl at thiazolidine-4-one ring in fused bicyclic azasugars. PMID:23159373

Chen, Hua; Li, Rui; Liu, Zhenying; Wei, Sinan; Zhang, Hongzhi; Li, Xiaoliu

2012-10-27

390

Pregnancies and menstrual function before and after combined radiation (RT) and chemotherapy (TVPP) for Hodgkin's disease  

SciTech Connect

The menstrual cycle, pregnancies, and offspring were evaluated before and after initial combined radiation (RT) and chemotherapy with thiotepa, vinblastine, vincristine, procarbazine, and prednisone (TVPP), in 34 women between the ages of 18 and 44 (median 26.5 years) treated for Stage II and Stage III Hodgkin's disease. The median range of follow-up is 83.1 months (range 40.5-140). After therapy 94.1% (32/34) continued to menstruate. Two of the four patients over the age of 35 ceased to menstruate. All patients under the age of 35 continued to menstruate (30/30). Age at the time of diagnosis was the only factor affecting change in menses with a significant probability (p = .001) that women greater than 30 years of age will experience some change in menstrual pattern. Seventeen pregnancies occurred in 12 women after therapy; 2 had 4 elective abortions; 10 delivered 12 children with normal physical development; 1 will deliver six months from now. Twelve of thirteen patients who wanted to become pregnant have conceived. The ability to become pregnant and deliver normal children after intensive treatment with combined radiation and chemotherapy (RT/TVPP) was comparable to the patients' pretreatment record.

Lacher, M.J.; Toner, K.

1986-01-01

391

A qRT-PCR RFLP Method for Monitoring Highly Conserved Transgene Expression during Gene Therapy  

PubMed Central

Evaluation of the transfer efficiency of a rat heme oxygenase-1 (HO-1) transgene into mice requires differentiation of rat and mouse HO-1. However, rat and mouse HO-1 have 94% homology; antibodies and enzyme activity cannot adequately distinguish HO-1. We designed a qRT-PCR method to monitor HO-1 transcription relative to a housekeeping gene, GAPDH. The ratio of rat and mouse HO-1 mRNA could be estimated through restriction fragment length polymorphism (RFLP) analysis of the PCR products. In vitro, murine AML12 hepatocytes were transfected with rat HO-1. After 40 h, total HO-1 mRNA was enriched 2-fold relative to control cells and rat HO-1 comprised 84% of HO-1 cDNA. In vivo, the rat HO-1 transgene was cloned into a Sleeping Beauty transposase (SB-Tn) construct and injected hydrodynamically into a mouse model of sickle cell disease (SCD). After 21 days, there was a 32% enrichment of HO-1 mRNA relative to control mice and the rat transgene comprised 88% of HO-1 cDNA. After 21 days, HO-1 protein expression in liver was increased 2.5-fold. In summary, qRT-PCR RFLP is a useful and reliable method to differentiate transgene from host gene transcription, especially when the host and transgene protein are identical or highly homologous. This method has translational applications to the design, delivery and monitoring of gene therapy vectors.

Bruzzone, Carol M; Belcher, John D; Schuld, Nathan J; Newman, Kristal A; Vineyard, Julie; Nguyen, Julia; Chen, Chunsheng; Beckman, Joan D; Steer, Clifford J; Vercellotti, Gregory M

2009-01-01

392

Microdroplet sandwich real-time rt-PCR for detection of pandemic and seasonal influenza subtypes.  

PubMed

As demonstrated by the recent 2012/2013 flu epidemic, the continual emergence of new viral strains highlights the need for accurate medical diagnostics in multiple community settings. If rapid, robust, and sensitive diagnostics for influenza subtyping were available, it would help identify epidemics, facilitate appropriate antiviral usage, decrease inappropriate antibiotic usage, and eliminate the extra cost of unnecessary laboratory testing and treatment. Here, we describe a droplet sandwich platform that can detect influenza subtypes using real-time reverse-transcription polymerase chain reaction (rtRT-PCR). Using clinical samples collected during the 2010/11 season, we effectively differentiate between H1N1p (swine pandemic), H1N1s (seasonal), and H3N2 with an overall assay sensitivity was 96%, with 100% specificity for each subtype. Additionally, we demonstrate the ability to detect viral loads as low as 10(4) copies/mL, which is two orders of magnitude lower than viral loads in typical infected patients. This platform performs diagnostics in a miniaturized format without sacrificing any sensitivity, and can thus be easily developed into devices which are ideal for small clinics and pharmacies. PMID:24066051

Angione, Stephanie L; Inde, Zintis; Beck, Christina M; Artenstein, Andrew W; Opal, Steven M; Tripathi, Anubhav

2013-09-16

393

Microdroplet Sandwich Real-Time RT-PCR for Detection of Pandemic and Seasonal Influenza Subtypes  

PubMed Central

As demonstrated by the recent 2012/2013 flu epidemic, the continual emergence of new viral strains highlights the need for accurate medical diagnostics in multiple community settings. If rapid, robust, and sensitive diagnostics for influenza subtyping were available, it would help identify epidemics, facilitate appropriate antiviral usage, decrease inappropriate antibiotic usage, and eliminate the extra cost of unnecessary laboratory testing and treatment. Here, we describe a droplet sandwich platform that can detect influenza subtypes using real-time reverse-transcription polymerase chain reaction (rtRT-PCR). Using clinical samples collected during the 2010/11 season, we effectively differentiate between H1N1p (swine pandemic), H1N1s (seasonal), and H3N2 with an overall assay sensitivity was 96%, with 100% specificity for each subtype. Additionally, we demonstrate the ability to detect viral loads as low as 104 copies/mL, which is two orders of magnitude lower than viral loads in typical infected patients. This platform performs diagnostics in a miniaturized format without sacrificing any sensitivity, and can thus be easily developed into devices which are ideal for small clinics and pharmacies.

Angione, Stephanie L.; Inde, Zintis; Beck, Christina M.; Artenstein, Andrew W.; Opal, Steven M.; Tripathi, Anubhav

2013-01-01

394

Single-step multiplex RT-PCR for simultaneous and colourimetric detection of six RNA viruses in olive trees  

Microsoft Academic Search

A single-step multiplex RT-PCR was developed for the simultaneous and colourimetric detection of six RNA viruses (Cucumber mosaic virus, Cherry leaf roll virus, strawberry latent ringspot virus, Arabis mosaic virus, Olive latent-1 virus and Olive latent-2virus) which infect olive trees. Six compatible primer set for one-step RT-PCR amplification in a single closed-tube and 3? digoxigenin labelled probes were designed in

Edson Bertolini; Antonio Olmos; M. Carmen Mart??nez; Mar??a Teresa Gorris; Mariano Cambra

2001-01-01

395

Robust RT-qPCR Data Normalization: Validation and Selection of Internal Reference Genes during Post-Experimental Data Analysis  

Microsoft Academic Search

Reverse transcription and real-time PCR (RT-qPCR) has been widely used for rapid quantification of relative gene expression. To offset technical confounding variations, stably-expressed internal reference genes are measured simultaneously along with target genes for data normalization. Statistic methods have been developed for reference validation; however normalization of RT-qPCR data still remains arbitrary due to pre-experimental determination of particular reference genes.

Daijun Ling; Paul M. Salvaterra; Baochuan Lin

2011-01-01