These are representative sample records from Science.gov related to your search topic.
For comprehensive and current results, perform a real-time search at Science.gov.
1

RT-SHIV subpopulation dynamics in infected macaques during anti-HIV therapy  

Microsoft Academic Search

BACKGROUND: To study the dynamics of wild-type and drug-resistant HIV-1 RT variants, we developed a methodology that follows the fates of individual genomes over time within the viral quasispecies. Single genome sequences were obtained from 3 pigtail macaques infected with a recombinant simian immunodeficiency virus containing the RT coding region from HIV-1 (RT-SHIV) and treated with short-course efavirenz monotherapy 13

Wei Shao; Mary Kearney; Frank Maldarelli; John W Mellors; Robert M Stephens; Jeffrey D Lifson; Vineet N KewalRamani; Zandrea Ambrose; John M Coffin; Sarah E Palmer

2009-01-01

2

A MIV-150/zinc acetate gel inhibits SHIV-RT infection in macaque vaginal explants.  

PubMed

To extend our observations that single or repeated application of a gel containing the NNRTI MIV-150 (M) and zinc acetate dihydrate (ZA) in carrageenan (CG) (MZC) inhibits vaginal transmission of simian/human immunodeficiency virus (SHIV)-RT in macaques, we evaluated safety and anti-SHIV-RT activity of MZC and related gel formulations ex vivo in macaque mucosal explants. In addition, safety was further evaluated in human ectocervical explants. The gels did not induce mucosal toxicity. A single ex vivo exposure to diluted MZC (1?30, 1?100) and MC (1?30, the only dilution tested), but not to ZC gel, up to 4 days prior to viral challenge, significantly inhibited SHIV-RT infection in macaque vaginal mucosa. MZC's activity was not affected by seminal plasma. The antiviral activity of unformulated MIV-150 was not enhanced in the presence of ZA, suggesting that the antiviral activity of MZC was mediated predominantly by MIV-150. In vivo administration of MZC and CG significantly inhibited ex vivo SHIV-RT infection (51-62% inhibition relative to baselines) of vaginal (but not cervical) mucosa collected 24 h post last gel exposure, indicating barrier effect of CG. Although the inhibitory effect of MZC (65-74%) did not significantly differ from CG (32-45%), it was within the range of protection (?75%) against vaginal SHIV-RT challenge 24 h after gel dosing. Overall, the data suggest that evaluation of candidate microbicides in macaque explants can inform macaque efficacy and clinical studies design. The data support advancing MZC gel for clinical evaluation. PMID:25259616

Barnable, Patrick; Calenda, Giulia; Ouattara, Louise; Gettie, Agegnehu; Blanchard, James; Jean-Pierre, Ninochka; Kizima, Larisa; Rodríguez, Aixa; Abraham, Ciby; Menon, Radhika; Seidor, Samantha; Cooney, Michael L; Roberts, Kevin D; Sperling, Rhoda; Piatak, Michael; Lifson, Jeffrey D; Fernandez-Romero, Jose A; Zydowsky, Thomas M; Robbiani, Melissa; Teleshova, Natalia

2014-01-01

3

A Potent Combination Microbicide that Targets SHIV-RT, HSV-2 and HPV  

PubMed Central

Prevalent infection with human herpes simplex 2 (HSV-2) or human papillomavirus (HPV) is associated with increased human immunodeficiency virus (HIV) acquisition. Microbicides that target HIV as well as these sexually transmitted infections (STIs) may more effectively limit HIV incidence. Previously, we showed that a microbicide gel (MZC) containing MIV-150, zinc acetate (ZA) and carrageenan (CG) protected macaques against simian-human immunodeficiency virus (SHIV-RT) infection and that a ZC gel protected mice against HSV-2 infection. Here we evaluated a modified MZC gel (containing different buffers, co-solvents, and preservatives suitable for clinical testing) against both vaginal and rectal challenge of animals with SHIV-RT, HSV-2 or HPV. MZC was stable and safe in vitro (cell viability and monolayer integrity) and in vivo (histology). MZC protected macaques against vaginal (p<0.0001) SHIV-RT infection when applied up to 8 hours (h) prior to challenge. When used close to the time of challenge, MZC prevented rectal SHIV-RT infection of macaques similar to the CG control. MZC significantly reduced vaginal (p<0.0001) and anorectal (p?=?0.0187) infection of mice when 106 pfu HSV-2 were applied immediately after vaginal challenge and also when 5×103 pfu were applied between 8 h before and 4 h after vaginal challenge (p<0.0248). Protection of mice against 8×106 HPV16 pseudovirus particles (HPV16 PsV) was significant for MZC applied up to 24 h before and 2 h after vaginal challenge (p<0.0001) and also if applied 2 h before or after anorectal challenge (p<0.0006). MZC provides a durable window of protection against vaginal infection with these three viruses and, against HSV-2 and HPV making it an excellent candidate microbicide for clinical use. PMID:24740100

Kizima, Larisa; Rodríguez, Aixa; Kenney, Jessica; Derby, Nina; Mizenina, Olga; Menon, Radhika; Seidor, Samantha; Zhang, Shimin; Levendosky, Keith; Jean-Pierre, Ninochka; Pugach, Pavel; Villegas, Guillermo; Ford, Brian E.; Gettie, Agegnehu; Blanchard, James; Piatak, Michael; Lifson, Jeffrey D.; Paglini, Gabriela; Teleshova, Natalia; Zydowsky, Thomas M.; Robbiani, Melissa; Fernández-Romero, José A.

2014-01-01

4

A potent combination microbicide that targets SHIV-RT, HSV-2 and HPV.  

PubMed

Prevalent infection with human herpes simplex 2 (HSV-2) or human papillomavirus (HPV) is associated with increased human immunodeficiency virus (HIV) acquisition. Microbicides that target HIV as well as these sexually transmitted infections (STIs) may more effectively limit HIV incidence. Previously, we showed that a microbicide gel (MZC) containing MIV-150, zinc acetate (ZA) and carrageenan (CG) protected macaques against simian-human immunodeficiency virus (SHIV-RT) infection and that a ZC gel protected mice against HSV-2 infection. Here we evaluated a modified MZC gel (containing different buffers, co-solvents, and preservatives suitable for clinical testing) against both vaginal and rectal challenge of animals with SHIV-RT, HSV-2 or HPV. MZC was stable and safe in vitro (cell viability and monolayer integrity) and in vivo (histology). MZC protected macaques against vaginal (p<0.0001) SHIV-RT infection when applied up to 8 hours (h) prior to challenge. When used close to the time of challenge, MZC prevented rectal SHIV-RT infection of macaques similar to the CG control. MZC significantly reduced vaginal (p<0.0001) and anorectal (p?=?0.0187) infection of mice when 10(6) pfu HSV-2 were applied immediately after vaginal challenge and also when 5×10(3) pfu were applied between 8 h before and 4 h after vaginal challenge (p<0.0248). Protection of mice against 8×10(6) HPV16 pseudovirus particles (HPV16 PsV) was significant for MZC applied up to 24 h before and 2 h after vaginal challenge (p<0.0001) and also if applied 2 h before or after anorectal challenge (p<0.0006). MZC provides a durable window of protection against vaginal infection with these three viruses and, against HSV-2 and HPV making it an excellent candidate microbicide for clinical use. PMID:24740100

Kizima, Larisa; Rodríguez, Aixa; Kenney, Jessica; Derby, Nina; Mizenina, Olga; Menon, Radhika; Seidor, Samantha; Zhang, Shimin; Levendosky, Keith; Jean-Pierre, Ninochka; Pugach, Pavel; Villegas, Guillermo; Ford, Brian E; Gettie, Agegnehu; Blanchard, James; Piatak, Michael; Lifson, Jeffrey D; Paglini, Gabriela; Teleshova, Natalia; Zydowsky, Thomas M; Robbiani, Melissa; Fernández-Romero, José A

2014-01-01

5

Variation of human immunodeficiency virus type-1 reverse transcriptase within the simian immunodeficiency virus genome of RT-SHIV.  

PubMed

RT-SHIV is a chimera of simian immunodeficiency virus (SIV) containing the reverse transcriptase (RT)-encoding region of human immunodeficiency virus type 1 (HIV-1) within the backbone of SIVmac239. It has been used in a non-human primate model for studies of non-nucleoside RT inhibitors (NNRTI) and highly active antiretroviral therapy (HAART). We and others have identified several mutations that arise in the "foreign" HIV-1 RT of RT-SHIV during in vivo replication. In this study we catalogued amino acid substitutions in the HIV-1 RT and in regions of the SIV backbone with which RT interacts that emerged 30 weeks post-infection from seven RT-SHIV-infected rhesus macaques. The virus set points varied from relatively high virus load, moderate virus load, to undetectable virus load. The G196R substitution in RT was detected from 6 of 7 animals at week 4 post-infection and remained in virus from 4 of 6 animals at week 30. Virus from four high virus load animals showed several common mutations within RT, including L74V or V75L, G196R, L214F, and K275R. The foreign RT from high virus load isolates exhibited as much variation as that of the highly variable envelope surface glycoprotein, and 10-fold higher than that of the native RT of SIVmac239. Isolates from moderate virus load animals showed much less variation in the foreign RT than the high virus load isolates. No variation was found in SIVmac239 genes known to interact with RT. Our results demonstrate substantial adaptation of the foreign HIV-1 RT in RT-SHIV-infected macaques, which most likely reflects selective pressure upon the foreign RT to attain optimal activity within the context of the chimeric RT-SHIV and the rhesus macaque host. PMID:24498008

Wadford, Debra A; Kauffman, Robert C; Deere, Jesse D; Aoki, Scott T; Stanton, Richard A; Higgins, Joanne; Van Rompay, Koen K A; Villalobos, Andradi; Nettles, James H; Schinazi, Raymond F; Pedersen, Niels C; North, Thomas W

2014-01-01

6

Variation of Human Immunodeficiency Virus Type-1 Reverse Transcriptase within the Simian Immunodeficiency Virus Genome of RT-SHIV  

PubMed Central

RT-SHIV is a chimera of simian immunodeficiency virus (SIV) containing the reverse transcriptase (RT)-encoding region of human immunodeficiency virus type 1 (HIV-1) within the backbone of SIVmac239. It has been used in a non-human primate model for studies of non-nucleoside RT inhibitors (NNRTI) and highly active antiretroviral therapy (HAART). We and others have identified several mutations that arise in the "foreign" HIV-1 RT of RT-SHIV during in vivo replication. In this study we catalogued amino acid substitutions in the HIV-1 RT and in regions of the SIV backbone with which RT interacts that emerged 30 weeks post-infection from seven RT-SHIV-infected rhesus macaques. The virus set points varied from relatively high virus load, moderate virus load, to undetectable virus load. The G196R substitution in RT was detected from 6 of 7 animals at week 4 post-infection and remained in virus from 4 of 6 animals at week 30. Virus from four high virus load animals showed several common mutations within RT, including L74V or V75L, G196R, L214F, and K275R. The foreign RT from high virus load isolates exhibited as much variation as that of the highly variable envelope surface glycoprotein, and 10-fold higher than that of the native RT of SIVmac239. Isolates from moderate virus load animals showed much less variation in the foreign RT than the high virus load isolates. No variation was found in SIVmac239 genes known to interact with RT. Our results demonstrate substantial adaptation of the foreign HIV-1 RT in RT-SHIV-infected macaques, which most likely reflects selective pressure upon the foreign RT to attain optimal activity within the context of the chimeric RT-SHIV and the rhesus macaque host. PMID:24498008

Wadford, Debra A.; Kauffman, Robert C.; Deere, Jesse D.; Aoki, Scott T.; Stanton, Richard A.; Higgins, Joanne; Van Rompay, Koen K. A.; Villalobos, Andradi; Nettles, James H.; Schinazi, Raymond F.; Pedersen, Niels C.; North, Thomas W.

2014-01-01

7

MIV-150-Containing Intravaginal Rings Protect Macaque Vaginal Explants against SHIV-RT Infection  

PubMed Central

Recent studies demonstrated that intravaginal rings (IVRs) containing 100 mg of the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 significantly protect macaques against a chimeric simian-human immunodeficiency virus that expresses the HIV-1 HxB2 reverse transcriptase (SHIV-RT) when present before and after vaginal challenge. The objectives of this study were to (i) evaluate the pharmacodynamics (PD) of MIV-150 in vaginal fluids (VF) and in ectocervical and vaginal tissues following 100-mg MIV-150 IVR exposure and to (ii) gain more insight whether pharmacokinetics (PK) of MIV-150 can predict PD. MIV-150 in VF collected at 1 day and 14 days post-MIV-150 IVR insertion inhibited ex vivo SHIV-RT infection in vaginal biopsy specimens from untreated animals (not carrying IVRs) in a dose-dependent manner. Previous PK studies demonstrated a significant increase of ectocervical and vaginal tissue MIV-150 concentrations 14 days versus 1 day post-IVR insertion, with the highest increase in vaginal tissue. Therefore, we tested PD of MIV-150 in tissues 14 days post-MIV-150 IVR insertion. Ex vivo SHIV-RT infection of vaginal, but not ectocervical, tissues collected 14 days post-MIV-150 IVR insertion was significantly inhibited compared to infection at the baseline (prior to MIV-150 IVR exposure). No changes in vaginal and ectocervical tissue infection were observed after placebo IVR exposure. Overall, these data underscore the use of the ex vivo macaque explant challenge models to evaluate tissue and VF PK/PD of candidate microbicides before in vivo animal efficacy studies. The data support further development of MIV-150-containing IVRs. PMID:24614384

Ouattara, Louise A.; Barnable, Patrick; Mawson, Paul; Seidor, Samantha; Zydowsky, Thomas M.; Kizima, Larisa; Rodriguez, Aixa; Fernández-Romero, José A.; Cooney, Michael L.; Roberts, Kevin D.; Gettie, Agegnehu; Blanchard, James; Robbiani, Melissa

2014-01-01

8

MIV-150-containing intravaginal rings protect macaque vaginal explants against SHIV-RT infection.  

PubMed

Recent studies demonstrated that intravaginal rings (IVRs) containing 100 mg of the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 significantly protect macaques against a chimeric simian-human immunodeficiency virus that expresses the HIV-1 HxB2 reverse transcriptase (SHIV-RT) when present before and after vaginal challenge. The objectives of this study were to (i) evaluate the pharmacodynamics (PD) of MIV-150 in vaginal fluids (VF) and in ectocervical and vaginal tissues following 100-mg MIV-150 IVR exposure and to (ii) gain more insight whether pharmacokinetics (PK) of MIV-150 can predict PD. MIV-150 in VF collected at 1 day and 14 days post-MIV-150 IVR insertion inhibited ex vivo SHIV-RT infection in vaginal biopsy specimens from untreated animals (not carrying IVRs) in a dose-dependent manner. Previous PK studies demonstrated a significant increase of ectocervical and vaginal tissue MIV-150 concentrations 14 days versus 1 day post-IVR insertion, with the highest increase in vaginal tissue. Therefore, we tested PD of MIV-150 in tissues 14 days post-MIV-150 IVR insertion. Ex vivo SHIV-RT infection of vaginal, but not ectocervical, tissues collected 14 days post-MIV-150 IVR insertion was significantly inhibited compared to infection at the baseline (prior to MIV-150 IVR exposure). No changes in vaginal and ectocervical tissue infection were observed after placebo IVR exposure. Overall, these data underscore the use of the ex vivo macaque explant challenge models to evaluate tissue and VF PK/PD of candidate microbicides before in vivo animal efficacy studies. The data support further development of MIV-150-containing IVRs. PMID:24614384

Ouattara, Louise A; Barnable, Patrick; Mawson, Paul; Seidor, Samantha; Zydowsky, Thomas M; Kizima, Larisa; Rodriguez, Aixa; Fernández-Romero, José A; Cooney, Michael L; Roberts, Kevin D; Gettie, Agegnehu; Blanchard, James; Robbiani, Melissa; Teleshova, Natalia

2014-05-01

9

RT-SHIV, an infectious CCR5-tropic chimeric virus suitable for evaluating HIV reverse transcriptase inhibitors in macaque models  

PubMed Central

Background Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are an important category of drugs for both chemotherapy and prevention of human immunodeficiency virus type 1 (HIV-1) infection. However, current non-human primate (NHP) models utilizing simian immunodeficiency virus (SIV) or commonly used chimeric SHIV (SIV expressing HIV-1 envelope) are inadequate due to the insensitivity to NNRTIs. To develop a NHP model for evaluation of NNRTI compounds, we characterized a RT-SHIV virus that was assembled by replacing the SIVmac239 reverse transcriptase (RT) with that of HIV-1HXB2. Since RT-SHIV exhibited in vitro characteristics of high infectivity, CCR5-usage, and sensitivity to HIV-1 specific NNRTIs, this virus was thought to be suitable for mucosal transmission and then was used to carry out a vaginal transmission study in pigtail macaques (Macaca nemestrina). Results RT-SHIV exhibited in vitro characteristics of an infectious CCR5-tropic chimeric virus. This virus was not only highly sensitive to HIV-1 RT specific NNRTIs; its replication was also inhibited by a variety of NRTIs and protease inhibitors. For in vivo vaginal transmission studies, macaques were either pretreated with a single dose of DMPA (depot medroxyprogesterone acetate) or left untreated before intravaginal inoculation with 500 or 1,000 TCID50 of RT-SHIV. All macaques became systemically infected by 2 or 3 weeks post-inoculation exhibiting persistent high viremia, marked CD4+T cell depletion, and antiviral antibody response. DMPA-pretreated macaques showed a higher mean plasma viral load after the acute infection stage, highly variable antiviral antibody response, and a higher incidence of AIDS-like disease as compared with macaques without DMPA pretreatment. Conclusion This chimeric RT-SHIV has exhibited productive replication in both macaque and human PBMCs, predominantly CCR5-coreceptor usage for viral entry, and sensitivity to NNRTIs as well as other anti-HIV compounds. This study demonstrates rapid systemic infection in macaques following intravaginal exposure to RT-SHIV. This RT-SHIV/macaque model could be useful for evaluation of NNRTI-based therapies, microbicides, or other preventive strategies. PMID:19891783

2009-01-01

10

Analysis of Multiply Spliced Transcripts in Lymphoid Tissue Reservoirs of Rhesus Macaques Infected with RT-SHIV during HAART  

PubMed Central

Highly active antiretroviral therapy (HAART) can reduce levels of human immunodeficiency virus type 1 (HIV-1) to undetectable levels in infected individuals, but the virus is not eradicated. The mechanisms of viral persistence during HAART are poorly defined, but some reservoirs have been identified, such as latently infected resting memory CD4+ T cells. During latency, in addition to blocks at the initiation and elongation steps of viral transcription, there is a block in the export of viral RNA (vRNA), leading to the accumulation of multiply-spliced transcripts in the nucleus. Two of the genes encoded by the multiply-spliced transcripts are Tat and Rev, which are essential early in the viral replication cycle and might indicate the state of infection in a given population of cells. Here, the levels of multiply-spliced transcripts were compared to the levels of gag-containing RNA in tissue samples from RT-SHIV-infected rhesus macaques treated with HAART. Splice site sequence variation was identified during development of a TaqMan PCR assay. Multiply-spliced transcripts were detected in gastrointestinal and lymphatic tissues, but not the thymus. Levels of multiply-spliced transcripts were lower than levels of gag RNA, and both correlated with plasma virus loads. The ratio of multiply-spliced to gag RNA was greatest in the gastrointestinal samples from macaques with plasma virus loads <50 vRNA copies per mL at necropsy. Levels of gag RNA and multiply-spliced mRNA in tissues from RT-SHIV-infected macaques correlate with plasma virus load. PMID:24505331

Deere, Jesse D.; Kauffman, Robert C.; Cannavo, Elda; Higgins, Joanne; Villalobos, Andradi; Adamson, Lourdes; Schinazi, Raymond F.; Luciw, Paul A.; North, Thomas W.

2014-01-01

11

Exposure to MIV-150 from a High-Dose Intravaginal Ring Results in Limited Emergence of Drug Resistance Mutations in SHIV-RT Infected Rhesus Macaques  

PubMed Central

When microbicides used for HIV prevention contain antiretroviral drugs, there is concern for the potential emergence of drug-resistant HIV following use in infected individuals who are either unaware of their HIV infection status or who are aware but still choose to use the microbicide. Resistant virus could ultimately impact their responsiveness to treatment and/or result in subsequent transmission of drug-resistant virus. We tested whether drug resistance mutations (DRMs) would emerge in macaques infected with simian immunodeficiency virus expressing HIV reverse transcriptase (SHIV-RT) after sustained exposure to the potent non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 delivered via an intravaginal ring (IVR). We first treated 4 SHIV-RT-infected animals with daily intramuscular injections of MIV-150 over two 21 day (d) intervals separated by a 7 d drug hiatus. In all 4 animals, NNRTI DRMs (single and combinations) were detected within 14 d and expanded in proportion and diversity with time. Knowing that we could detect in vivo emergence of NNRTI DRMs in response to MIV-150, we then tested whether a high-dose MIV-150 IVR (loaded with >10 times the amount being used in a combination microbicide IVR in development) would select for resistance in 6 infected animals, modeling use of this prevention method by an HIV-infected woman. We previously demonstrated that this MIV-150 IVR provides significant protection against vaginal SHIV-RT challenge. Wearing the MIV-150 IVR for 56 d led to only 2 single DRMs in 2 of 6 animals (430 RT sequences analyzed total, 0.46%) from plasma and lymph nodes despite MIV-150 persisting in the plasma, vaginal fluids, and genital tissues. Only wild type virus sequences were detected in the genital tissues. These findings indicate a low probability for the emergence of DRMs after topical MIV-150 exposure and support the advancement of MIV-150-containing microbicides. PMID:24586674

Hsu, Mayla; Keele, Brandon F.; Aravantinou, Meropi; Krawczyk, Noa; Seidor, Samantha; Abraham, Ciby J.; Zhang, Shimin; Rodriguez, Aixa; Kizima, Larisa; Derby, Nina; Jean-Pierre, Ninochka; Mizenina, Olga; Gettie, Agegnehu; Grasperge, Brooke; Blanchard, James; Piatak, Michael J.; Lifson, Jeffrey D.; Fernández-Romero, José A.; Zydowsky, Thomas M.; Robbiani, Melissa

2014-01-01

12

Exposure to MIV-150 from a high-dose intravaginal ring results in limited emergence of drug resistance mutations in SHIV-RT infected rhesus macaques.  

PubMed

When microbicides used for HIV prevention contain antiretroviral drugs, there is concern for the potential emergence of drug-resistant HIV following use in infected individuals who are either unaware of their HIV infection status or who are aware but still choose to use the microbicide. Resistant virus could ultimately impact their responsiveness to treatment and/or result in subsequent transmission of drug-resistant virus. We tested whether drug resistance mutations (DRMs) would emerge in macaques infected with simian immunodeficiency virus expressing HIV reverse transcriptase (SHIV-RT) after sustained exposure to the potent non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 delivered via an intravaginal ring (IVR). We first treated 4 SHIV-RT-infected animals with daily intramuscular injections of MIV-150 over two 21 day (d) intervals separated by a 7 d drug hiatus. In all 4 animals, NNRTI DRMs (single and combinations) were detected within 14 d and expanded in proportion and diversity with time. Knowing that we could detect in vivo emergence of NNRTI DRMs in response to MIV-150, we then tested whether a high-dose MIV-150 IVR (loaded with >10 times the amount being used in a combination microbicide IVR in development) would select for resistance in 6 infected animals, modeling use of this prevention method by an HIV-infected woman. We previously demonstrated that this MIV-150 IVR provides significant protection against vaginal SHIV-RT challenge. Wearing the MIV-150 IVR for 56 d led to only 2 single DRMs in 2 of 6 animals (430 RT sequences analyzed total, 0.46%) from plasma and lymph nodes despite MIV-150 persisting in the plasma, vaginal fluids, and genital tissues. Only wild type virus sequences were detected in the genital tissues. These findings indicate a low probability for the emergence of DRMs after topical MIV-150 exposure and support the advancement of MIV-150-containing microbicides. PMID:24586674

Hsu, Mayla; Keele, Brandon F; Aravantinou, Meropi; Krawczyk, Noa; Seidor, Samantha; Abraham, Ciby J; Zhang, Shimin; Rodriguez, Aixa; Kizima, Larisa; Derby, Nina; Jean-Pierre, Ninochka; Mizenina, Olga; Gettie, Agegnehu; Grasperge, Brooke; Blanchard, James; Piatak, Michael J; Lifson, Jeffrey D; Fernández-Romero, José A; Zydowsky, Thomas M; Robbiani, Melissa

2014-01-01

13

RT-SHIV, an infectious CCR5-tropic chimeric virus suitable for evaluating HIV reverse transcriptase inhibitors in macaque models  

Microsoft Academic Search

BACKGROUND: Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are an important category of drugs for both chemotherapy and prevention of human immunodeficiency virus type 1 (HIV-1) infection. However, current non-human primate (NHP) models utilizing simian immunodeficiency virus (SIV) or commonly used chimeric SHIV (SIV expressing HIV-1 envelope) are inadequate due to the insensitivity to NNRTIs. To develop a NHP model for evaluation

Yonghou Jiang; Baoping Tian; Mohammed Saifuddin; Michael B Agy; Peter Emau; J Scott Cairns; Che-Chung Tsai

2009-01-01

14

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

15

Infection of macaques with chimeric simian and human immunodeficiency viruses containing Env from subtype F  

Microsoft Academic Search

Summary.  ?Chimeric simian and human immunodeficiency viruses (SHIVs) are useful for investigating the pathogenicity of human immunodeficiency\\u000a virus (HIV-1) and to develop an anti-HIV-1 vaccine. We attempted to construct SHIVs containing Env from various subtypes,\\u000a because almost all SHIVs which have been reported so far have Env from HIV-1 that belongs to subtype B. Two infectious SHIVs\\u000a containing Env from two

T. Kuwata; T. Takemura; J. Takehisa; T. Miura; M. Hayami

2002-01-01

16

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  

PubMed Central

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 either mock treated, or treated for 4 weeks starting at 8 or 24 h postinfection (p.i.) with GW420867. While such therapy led to undetectable plasma viremia in three of six monkeys, a transient plasma viremia was noted in the other three treated animals at 2 to 4 weeks following cessation of therapy. Following this transient viremia all drug-treated animals showed low or undetectable levels of plasma viremia up to the last sample examined at 90 weeks p.i. Despite low and/or undetectable viremia, virus-specific cytotoxic T lymphocyte and viral Env-specific proliferative responses were seen in the peripheral blood mononuclear cells of both mock- and drug-treated animals as early as 3 weeks p.i. Such virus-specific cellular responses, however, were better maintained in the drug-treated animals than the mock-treated animals. In contrast to the virus-specific cellular response, the magnitude and kinetics of virus specific humoral responses appeared to correlate with the detection of viremia. These data support the view that a short-term PEP with GW420867 permits the generation and maintenance of long-lasting virus-specific cell-mediated immune responses while markedly reducing viral loads to undetectable levels for a prolonged period of time (90 weeks) and leads to long-term disease protection. This model provides a unique means to define mechanisms and correlates of disease protection. PMID:10846052

Mori, Kazuyasu; Yasutomi, Yasuhiro; Sawada, Shuzo; Villinger, Francois; Sugama, Kazushige; Rosenwith, Brigitte; Heeney, Jonathan L.; Überla, Klaus; Yamazaki, Shudo; Ansari, Aftab A.; Rübsamen-Waigmann, Helga

2000-01-01

17

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.  

PubMed

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 either mock treated, or treated for 4 weeks starting at 8 or 24 h postinfection (p.i.) with GW420867. While such therapy led to undetectable plasma viremia in three of six monkeys, a transient plasma viremia was noted in the other three treated animals at 2 to 4 weeks following cessation of therapy. Following this transient viremia all drug-treated animals showed low or undetectable levels of plasma viremia up to the last sample examined at 90 weeks p.i. Despite low and/or undetectable viremia, virus-specific cytotoxic T lymphocyte and viral Env-specific proliferative responses were seen in the peripheral blood mononuclear cells of both mock- and drug-treated animals as early as 3 weeks p.i. Such virus-specific cellular responses, however, were better maintained in the drug-treated animals than the mock-treated animals. In contrast to the virus-specific cellular response, the magnitude and kinetics of virus specific humoral responses appeared to correlate with the detection of viremia. These data support the view that a short-term PEP with GW420867 permits the generation and maintenance of long-lasting virus-specific cell-mediated immune responses while markedly reducing viral loads to undetectable levels for a prolonged period of time (90 weeks) and leads to long-term disease protection. This model provides a unique means to define mechanisms and correlates of disease protection. PMID:10846052

Mori, K; Yasutomi, Y; Sawada, S; Villinger, F; Sugama, K; Rosenwith, B; Heeney, J L; Uberla, K; Yamazaki, S; Ansari, A A; Rübsamen-Waigmann, H

2000-07-01

18

Robust suppression of env-SHIV viremia in M. nemestrina by 3-drug ART is independent of timing of initiation during chronic infection  

PubMed Central

Background Nonhuman primates (NHPs) are an important model organism for studies of HIV pathogenesis and pre-clinical evaluation of anti-HIV therapies. The successful translation of NHP-derived data to clinically relevant anti-HIV studies will require better understanding of the viral strains and NHP species used, and their responses to existing antiretroviral therapies (ART). Methods Five pigtailed macaques (M. nemestrina) were productively infected with the SIV/HIV chimeric virus SHIV-1157ipd3N4 following intravenous challenge. After 8 or 27 weeks, ART (PMPA, FTC, Raltegravir) was initiated. Viral load, T-Cell counts, and production of SHIV-specific antibodies were monitored throughout the course of infection and ART. Results ART led to a rapid and sustained decrease in plasma viral load. Suppression of plasma viremia by ART was independent of the timing of initiation during chronic infection. Conclusions We present a new NHP model of HIV infection on antiretroviral therapy, which should prove applicable to multiple clinically relevant anti-HIV approaches. PMID:24025078

Peterson, Christopher W; Younan, Patrick; Polacino, Patricia S; Maurice, Nicholas J; Miller, Hannah W; Prlic, Martin; Jerome, Keith R; Woolfrey, Ann E; Hu, Shiu-Lok; Kiem, Hans-Peter

2013-01-01

19

Sequential emergence and clinical implications of viral mutants with K70E and K65R mutation in reverse transcriptase during prolonged tenofovir monotherapy in rhesus macaques with chronic RT-SHIV infection  

PubMed Central

Background We reported previously on the emergence and clinical implications of simian immunodeficiency virus (SIVmac251) mutants with a K65R mutation in reverse transcriptase (RT), and the role of CD8+ cell-mediated immune responses in suppressing viremia during tenofovir therapy. Because of significant sequence differences between SIV and HIV-1 RT that affect drug susceptibilities and mutational patterns, it is unclear to what extent findings with SIV can be extrapolated to HIV-1 RT. Accordingly, to model HIV-1 RT responses, 12 macaques were inoculated with RT-SHIV, a chimeric SIV containing HIV-1 RT, and started on prolonged tenofovir therapy 5 months later. Results The early virologic response to tenofovir correlated with baseline viral RNA levels and expression of the MHC class I allele Mamu-A*01. For all animals, sensitive real-time PCR assays detected the transient emergence of K70E RT mutants within 4 weeks of therapy, which were then replaced by K65R mutants within 12 weeks of therapy. For most animals, the occurrence of these mutations preceded a partial rebound of plasma viremia to levels that remained on average 10-fold below baseline values. One animal eventually suppressed K65R viremia to undetectable levels for more than 4 years; sequential experiments using CD8+ cell depletion and tenofovir interruption demonstrated that both CD8+ cells and continued tenofovir therapy were required for sustained suppression of viremia. Conclusion This is the first evidence that tenofovir therapy can select directly for K70E viral mutants in vivo. The observations on the clinical implications of the K65R RT-SHIV mutants were consistent with those of SIVmac251, and suggest that for persons infected with K65R HIV-1 both immune-mediated and drug-dependent antiviral activities play a role in controlling viremia. These findings suggest also that even in the presence of K65R virus, continuation of tenofovir treatment as part of HAART may be beneficial, particularly when assisted by antiviral immune responses. PMID:17417971

Van Rompay, Koen KA; Johnson, Jeffrey A; Blackwood, Emily J; Singh, Raman P; Lipscomb, Jonathan; Matthews, Timothy B; Marthas, Marta L; Pedersen, Niels C; Bischofberger, Norbert; Heneine, Walid; North, Thomas W

2007-01-01

20

Residual Viremia in an RT-SHIV Rhesus Macaque HAART Model Marked by the Presence of a Predominant Plasma Clone and a Lack of Viral Evolution  

PubMed Central

Highly active antiretroviral therapy (HAART) significantly reduces HIV-1 replication and prevents progression to AIDS. However, residual low-level viremia (LLV) persists and long-lived viral reservoirs are maintained in anatomical sites. These reservoirs permit a recrudescence of viremia upon cessation of therapy and thus HAART must be maintained indefinitely. HIV-1 reservoirs include latently infected resting memory CD4+ T-cells and macrophages which may contribute to residual viremia. It has not been conclusively determined if a component of LLV may also be due to residual replication in cells with sub-therapeutic drug levels and/or long-lived chronically infected cells. In this study, RT-SHIVmac239 diversity was characterized in five rhesus macaques that received a five-drug HAART regimen [tenofovir, emtricitabine, zidovudine, amdoxovir, (A, C, T, G nucleoside analogs) and the non-nucleoside reverse transcriptase (RT) inhibitor efavirenz]. Before maximal viral load suppression, longitudinal plasma viral RNA RT diversity was analyzed using a 454 sequencer. After suppression, LLV RT diversity (amino acids 65-210) was also assessed. LLV samples had viral levels less than our standard detection limit (50 viral RNA copies/mL) and few transient blips <200 RNA copies/mL. HAART was discontinued in three macaques after 42 weeks of therapy resulting in viral rebound. The level of viral divergence and the prevalence of specific alleles in LLV was similar to pre-suppression viremia. While some LLV sequences contained mutations not observed in the pre-suppression profile, LLV was not characterized by temporal viral evolution or apparent selection of drug resistance mutations. Similarly, resistance mutations were not detected in the viral rebound population. Interestingly, one macaque maintained a putative LLV predominant plasma clone sequence. Together, these results suggest that residual replication did not markedly contribute to LLV and that this model mimics the prevalence and phylogenetic characteristics of LLV during human HAART. Therefore, this model may be ideal for testing HIV-1 eradication strategies. PMID:24505452

Kauffman, Robert C.; Villalobos, Andradi; Bowen, Joanne H.; Adamson, Lourdes; Schinazi, Raymond F.

2014-01-01

21

Characterization of vaginal transmission of a simian human immunodeficiency virus (SHIV) encoding the reverse transcriptase gene from HIV1 in Chinese rhesus macaques  

Microsoft Academic Search

Replication competent recombinant simian-human immunodeficiency virus encoding the reverse transcriptase gene (RT SHIV) from HIV-1 was characterized for vaginal transmission in rhesus macaques. RT SHIV was shown to transmit efficiently via the vaginal route in macaques with detectable plasma viremia persisting for a year in some animals. Analyses of virus load in tissues of infected animals revealed accumulation of viral

Ranajit Pal; Jeremy Nuttall; Lindsey Galmin; Deborah Weiss; Hye-Kyung Chung; Joseph Romano

2009-01-01

22

Protection against Mucosal SHIV Challenge by Peptide and Helper-Dependent Adenovirus Vaccines  

PubMed Central

Groups of rhesus macaques that had previously been immunized with HIV-1 envelope (env) peptides and first generation adenovirus serotype 5 (FG-Ad5) vaccines expressing the same peptides were immunized intramuscularly three times with helper-dependent adenovirus (HD-Ad) vaccines expressing only the HIV-1 envelope from JRFL. No gag, pol, or other SHIV genes were used for vaccination. One group of the FG-Ad5-immune animals was immunized three times with HD-Ad5 expressing env. One group was immunized by serotype-switching with HD-Ad6, HD-Ad1, and HD-Ad2 expressing env. Previous work demonstrated that serum antibody levels against env were significantly higher in the serotype-switched group than in the HD-Ad5 group. In this study, neutralizing antibody and T cell responses were compared between the groups before and after rectal challenge with CCR5-tropic SHIV-SF162P3. When serum samples were assayed for neutralizing antibodies, only weak activity was observed. T cell responses against env epitopes were higher in the serotype-switched group. When these animals were challenged rectally with SHIV-SF162P3, both the Ad5 and serotype-switch groups significantly reduced peak viral loads 2 to 10-fold 2 weeks after infection. Peak viral loads were significantly lower for the serotype-switched group as compared to the HD-Ad5-immunized group. Viral loads declined over 18 weeks after infection with some animals viremia reducing nearly 4 logs from the peak. These data demonstrate significant mucosal vaccine effects after immunization with only env antigens. These data also demonstrate HD-Ad vectors are a robust platform for vaccination. PMID:20107521

Weaver, Eric A.; Nehete, Pramod N.; Nehete, Bharti P.; Buchl, Stephanie J.; Palmer, Donna; Montefiori, David C.; Ng, Philip; Sastry, K. Jagannadha; Barry, Michael A.

2009-01-01

23

A simian-human immunodeficiency virus carrying the rt gene from Chinese CRF01_AE strain of HIV is sensitive to nucleoside reverse transcriptase inhibitors and has a highly genetic stability in vivo.  

PubMed

Human immunodeficiency virus (HIV)-1 subtype CRF01_AE is one of the major HIV-1 subtypes that dominate the global epidemic. However, its drug resistance, associated mutations, and viral fitness have not been systemically studied, because available chimeric simian-HIVs (SHIVs) usually express the HIV-1 reverse transcriptase (rt) gene of subtype B HIV-1, which is different from subtype CRF01_AE HIV-1. In this study, a recombinant plasmid, pRT-SHIV/AE, was constructed to generate a chimeric RT-SHIV/AE by replacing the rt gene of simian immunodeficiency virus (SIVmac239) with the counterpart of Chinese HIV-1 subtype CRF01_AE. The infectivity, replication capacity, co-receptor tropism, drug sensitivity, and genetic stability of RT-SHIV/AE were characterized. The new chimeric RT-SHIV/AE effectively infected and replicated in human T cell line and rhesus peripheral blood mononuclear cells (rhPBMC). The rt gene of RT-SHIV/AE lacked the common mutation (T215I) associated with drug resistance. RT-SHIV-AE retained infectivity and immunogenicity, similar to that of its counterpart RT-SHIV/TC virus following intravenous inoculation in Chinese rhesus macaque. RT-SHIV-AE was more sensitive to nucleoside reverse transcriptase inhibitors (NRTIs) than the RT-SHIV/TC. RT-SHIV/AE was genetically stable in Chinese rhesus macaque. The new chimeric RT-SHIV/AE may be a valuable tool for evaluating the efficacy of the rt-based antiviral drugs against the subtype CRF01_AE HIV-1. PMID:24709063

Wang, Wei; Yao, Nan; Ju, Bin; Dong, Zhihui; Cong, Zhe; Jiang, Hong; Qin, Chuan; Wei, Qiang

2014-06-01

24

Sequential emergence and clinical implications of viral mutants with K70E and K65R mutation in reverse transcriptase during prolonged tenofovir monotherapy in rhesus macaques with chronic RT-SHIV infection  

Microsoft Academic Search

BACKGROUND: We reported previously on the emergence and clinical implications of simian immunodeficiency virus (SIVmac251) mutants with a K65R mutation in reverse transcriptase (RT), and the role of CD8+ cell-mediated immune responses in suppressing viremia during tenofovir therapy. Because of significant sequence differences between SIV and HIV-1 RT that affect drug susceptibilities and mutational patterns, it is unclear to what

Koen KA Van Rompay; Jeffrey A Johnson; Emily J Blackwood; Raman P Singh; Jonathan Lipscomb; Timothy B Matthews; Marta L Marthas; Niels C Pedersen; Norbert Bischofberger; Walid Heneine; Thomas W North

2007-01-01

25

Rhesus macaques vaccinated with consensus envelopes elicit partially protective immune responses against SHIV SF162p4 challenge.  

PubMed

The development of a preventative HIV/AIDS vaccine is challenging due to the diversity of viral genome sequences, especially in the viral envelope (Env???). Since it is not possible to directly match the vaccine strain to the vast number of circulating HIV-1 strains, it is necessary to develop an HIV-1 vaccine that can protect against a heterologous viral challenge. Previous studies from our group demonstrated that a mixture of wild type clade B Env(gp160s) were able to protect against a heterologous clade B challenge more effectively than a consensus clade B Envg(p160) vaccine. In order to broaden the immune response to other clades of HIV, in this study rhesus macaques were vaccinated with a polyvalent mixture of purified HIV-1 trimerized consensus Envg(p140) proteins representing clades A, B, C, and E. The elicited immune responses were compared to a single consensus Env(gp140) representing all isolates in group M (Con M). Both vaccines elicited anti- Env(gp140) IgG antibodies that bound an equal number of HIV-1 Env(gp160) proteins representing clades A, B and C. In addition, both vaccines elicited antibodies that neutralized the HIV-1(SF162) isolate. However, the vaccinated monkeys were not protected against SHIV(SF162p4) challenge. These results indicate that consensus Env(gp160) vaccines, administered as purified Env(gp140) trimers, elicit antibodies that bind to Env(gp160s) from strains representing multiple clades of HIV-1, but these vaccines did not protect against heterologous SHIV challenge. PMID:23548077

Eugene, Hermancia S; Pierce-Paul, Brooke R; Cragio, Jodi K; Ross, Ted M

2013-01-01

26

Virus replication and disease progression inversely correlate with SIV tat evolution in morphine-dependent and SIV\\/SHIV-infected Indian rhesus macaques  

Microsoft Academic Search

We analyzed the association between evolution of the 5? exon of tat and disease progression in an SIV\\/SHIV macaque model of opiate dependence and AIDS. Cloned tat sequences were obtained by RT-PCR amplification of 3 plasma viruses (recovered at different times) from 6 morphine-dependent and 2 control Indian rhesus macaques inoculated with SHIVKU-1B SHIV89.6P and SIV\\/17E-Fr. Approximately ten clones were

Richard J. Noel; Anil Kumar

2006-01-01

27

In vitro infection of primate PBMC with simian/human immunodeficiency virus, SHIV(SF33A): correlation to in vivo outcome.  

PubMed

The macaque/SIV animal system is an important model for studying AIDS pathogenesis and for evaluating the efficacy of vaccines and anti-viral therapeutics. However, differences between HIV-1 and SIV envelope proteins exist that render the SIV/macaque model of limited value when examining envelope determinants of retroviral pathogenesis. To overcome this problem, we utilized a chimeric virus, SHIV(SF33), containing the env gene from HIV-1SF33 in the context of the molecular clone SIVmac239, in the macaque animal model. In this study SHIV(SF33A), a pathogenic virus that evolved in vivo from a rhesus macaque infected intravenously with the molecular clone SHIV(SF33) was used in both in vitro and in vivo studies. By using a cell culture system, we examined the biological properties of our parental and animal-adapted chimeric viruses and compared in vitro susceptibility to in vivo studies. PMID:9747947

Harouse, J M; Tan, R C; Gettie, A; Dailey, P; Marx, P A; Luciw, P A; Cheng-Mayer, C

1998-01-01

28

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

29

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. PMID:23844034

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

30

Comparison of systemic and mucosal immunization with helper-dependent adenoviruses for vaccination against mucosal challenge with SHIV.  

PubMed

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. PMID:23844034

Weaver, Eric A; Nehete, Pramod N; 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

31

In vivo adaptation of SHIV(SF162): chimeric virus expressing a NSI, CCR5-specific envelope protein.  

PubMed

The chemokine receptor CCR5 is known to be a critical determinant of human immunodeficiency virus (HIV) transmission and pathogenesis in the human host. Towards the development of a macaque model to evaluate the efficacy of vaccines and therapeutics against infection with CCR5-specific viruses, and to delineate the pathogenic properties of such viruses, we constructed a chimeric simian human immunodeficiency virus, SHIV(SF162), containing the env, tat, rev, and vpu genes from HIV-1(SF162) (R5, MT/NSI) in the context of the molecular clone simian immunodeficiency virus, SIV(mac239). Virus generated from this molecular clone was used to intravenously infect two juvenile macaques, followed by three consecutive serial blood/bone marrow transfusions. Animals infected with parental SHIV(SF162) (P1) had detectable levels of viral replication (as determined by p27(gag) production) within days of infection; however, viral set-points fell below detection by Week 3. Late passage animals (P3 and P4) had a two-log increase in the level of plasma p27(gag) antigen. These results demonstrate that in vivo serial passage of the R5-specific SHIV(SF162) enhanced its replicative capacity. PMID:10593481

Tan, R C; Harouse, J M; Gettie, A; Cheng-Mayer, C

1999-01-01

32

Multimodality vaccination against clade C SHIV: Partial protection against mucosal challenges with a heterologous tier 2 virus.  

PubMed

We sought to test whether vaccine-induced immune responses could protect rhesus macaques (RMs) against upfront heterologous challenges with an R5 simian-human immunodeficiency virus, SHIV-2873Nip. This SHIV strain exhibits many properties of transmitted HIV-1, such as tier 2 phenotype (relatively difficult to neutralize), exclusive CCR5 tropism, and gradual disease progression in infected RMs. Since no human AIDS vaccine recipient is likely to encounter an HIV-1 strain that exactly matches the immunogens, we immunized the RMs with recombinant Env proteins heterologous to the challenge virus. For induction of immune responses against Gag, Tat, and Nef, we explored a strategy of immunization with overlapping synthetic peptides (OSP). The immune responses against Gag and Tat were finally boosted with recombinant proteins. The vaccinees and a group of ten control animals were given five low-dose intrarectal (i.r.) challenges with SHIV-2873Nip. All controls and seven out of eight vaccinees became systemically infected; there was no significant difference in viremia levels of vaccinees vs. controls. Prevention of viremia was observed in one vaccinee which showed strong boosting of virus-specific cellular immunity during virus exposures. The protected animal showed no challenge virus-specific neutralizing antibodies in the TZM-bl or A3R5 cell-based assays and had low-level ADCC activity after the virus exposures. Microarray data strongly supported a role for cellular immunity in the protected animal. Our study represents a case of protection against heterologous tier 2 SHIV-C by vaccine-induced, virus-specific cellular immune responses. PMID:25245933

Lakhashe, Samir K; Byrareddy, Siddappa N; Zhou, Mingkui; Bachler, Barbara C; Hemashettar, Girish; Hu, Shiu-Lok; Villinger, Francois; Else, James G; Stock, Shannon; Lee, Sandra J; Vargas-Inchaustegui, Diego A; Cofano, Egidio Brocca; Robert-Guroff, Marjorie; Johnson, Welkin E; Polonis, Victoria R; Forthal, Donald N; Loret, Erwann P; Rasmussen, Robert A; Ruprecht, Ruth M

2014-11-12

33

Soluble HIV-1 Env trimers in adjuvant elicit potent and diverse functional B cell responses in primates  

PubMed Central

Broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoproteins (Envs) have proven difficult to elicit by immunization. Therefore, to identify effective Env neutralization targets, efforts are underway to define the specificities of bNAbs in chronically infected individuals. For a prophylactic vaccine, it is equally important to define the immunogenic properties of the heavily glycosylated Env in healthy primates devoid of confounding HIV-induced pathogenic factors. We used rhesus macaques to investigate the magnitude and kinetics of B cell responses stimulated by Env trimers in adjuvant. Robust Env-specific memory B cell responses and high titers of circulating antibodies developed after trimer inoculation. Subsequent immunizations resulted in significant expansion of Env-specific IgG-producing plasma cell populations and circulating Abs that displayed increasing avidity and neutralization capacity. The neutralizing activity elicited with the regimen used was, in most aspects, superior to that elicited by a regimen based on monomeric Env immunization in humans. Despite the potency and breadth of the trimer-elicited response, protection against heterologous rectal simian-HIV (SHIV) challenge was modest, illustrating the challenge of eliciting sufficient titers of cross-reactive protective NAbs in mucosal sites. These data provide important information for the design and evaluation of vaccines aimed at stimulating protective HIV-1 immune responses in humans. PMID:20679401

Sundling, Christopher; Forsell, Mattias N.E.; O'Dell, Sijy; Feng, Yu; Chakrabarti, Bimal; Rao, Srinivas S.; Loré, Karin; Mascola, John R.; Wyatt, Richard T.; Douagi, Iyadh

2010-01-01

34

Sequence variation in the gp 120 region of SHIV-CN97001 during in vivo passage  

Microsoft Academic Search

SHIV-CN97001 played an important role in assessing the immune effect and strategy of the AIDS vaccine which included genes\\u000a of the predominant prevalent HIV-1 strain in China. In this study, SHIV-CN97001 was in vivo passaged serially to construct pathogenic SHIV-CN97001\\/rhesus macaques model. To identify variation in the gp120 region of SHIV-CN97001 during passage, the fragments of gp120 gene were amplified

Qiang Liu; Gui-bo Yang; Yue Ma; Chen-li Qiu; Jie-jie Dai; Hui Xing; Yi-ming Shao

2008-01-01

35

SMART LIGHTING ENGINEERING RESEARCH CENTER Dr. Shiv Kalyanaraman  

E-print Network

SMART LIGHTING ENGINEERING RESEARCH CENTER Presents Dr. Shiv Kalyanaraman Chief Scientist, IBM exciting opportunities for deploying Smart Lighting, sensor networks and controls systems in advanced transportation management systems. The Smart Lighting ERC is pleased to host Rensselaer Alum and former ECSE

Linhardt, Robert J.

36

2005 Nature Publishing Group Protection of macaques from vaginal SHIV  

E-print Network

-378806, CMPD167 and C52L potently inhibit SHIV-162P3 infection of macaque and human peripheral blood suggested partial protection8 . We adjusted the gel pH to 6.0, which allowed us to now dissolve CMPD167 at 5 mM. At pH 6.0, HMC was neither inhibitory nor inflammatory. It was used in all the following studies

Cai, Long

37

Protective effects of nef-deleted SHIV or that having IFN? against disease induced with a pathogenic virus early after vaccination  

Microsoft Academic Search

Summary. To clarify the involvement of primitive non-specific immune responses in the protective effects of a live, attenuated virus, each two rhesus macaques were intravenously immunized with an attenuated chimeric simian and human immunodeficiency virus (SHIV) in which the nef gene was deleted (SHIV-NI) or a SHIV having human IFN-? inserted into the deleted nef region (SHIV IFN-?). These immunized

Y. Enose; M. Kita; T. Yamamoto; H. Suzuki; A. Miyake; R. Horiuchi; K. Ibuki; K. Kaneyasu; T. Kuwata; E. Takahashi; K. Shinohara; T. Miura; M. Hayami

2004-01-01

38

Enhanced neonatal Fc receptor function improves protection against primate SHIV infection.  

PubMed

To protect against human immunodeficiency virus (HIV-1) infection, broadly neutralizing antibodies (bnAbs) must be active at the portals of viral entry in the gastrointestinal or cervicovaginal tracts. The localization and persistence of antibodies at these sites is influenced by the neonatal Fc receptor (FcRn), whose role in protecting against infection in vivo has not been defined. Here, we show that a bnAb with enhanced FcRn binding has increased gut mucosal tissue localization, which improves protection against lentiviral infection in non-human primates. A bnAb directed to the CD4-binding site of the HIV-1 envelope (Env) protein (denoted VRC01) was modified by site-directed mutagenesis to increase its binding affinity for FcRn. This enhanced FcRn-binding mutant bnAb, denoted VRC01-LS, displayed increased transcytosis across human FcRn-expressing cellular monolayers in vitro while retaining Fc?RIIIa binding and function, including antibody-dependent cell-mediated cytotoxicity (ADCC) activity, at levels similar to VRC01 (the wild type). VRC01-LS had a threefold longer serum half-life than VRC01 in non-human primates and persisted in the rectal mucosa even when it was no longer detectable in the serum. Notably, VRC01-LS mediated protection superior to that afforded by VRC01 against intrarectal infection with simian-human immunodeficiency virus (SHIV). These findings suggest that modification of FcRn binding provides a mechanism not only to increase serum half-life but also to enhance mucosal localization that confers immune protection. Mutations that enhance FcRn function could therefore increase the potency and durability of passive immunization strategies to prevent HIV-1 infection. PMID:25119033

Ko, Sung-Youl; Pegu, Amarendra; Rudicell, Rebecca S; Yang, Zhi-yong; Joyce, M Gordon; Chen, Xuejun; Wang, Keyun; Bao, Saran; Kraemer, Thomas D; Rath, Timo; Zeng, Ming; Schmidt, Stephen D; Todd, John-Paul; Penzak, Scott R; Saunders, Kevin O; Nason, Martha C; Haase, Ashley T; Rao, Srinivas S; Blumberg, Richard S; Mascola, John R; Nabel, Gary J

2014-10-30

39

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. PMID:19925400

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

2009-01-01

40

A pilot study comparing the development of EIAV Env-specific antibodies induced by DNA/recombinant vaccinia-vectored vaccines and an attenuated Chinese EIAV vaccine.  

PubMed

Data from successful attenuated lentiviral vaccine studies indicate that fully mature Env-specific antibodies characterized by high titer, high avidity, and the predominant recognition of conformational epitopes are associated with protective efficacy. Although vaccination with a DNA prime/recombinant vaccinia-vectored vaccine boost strategy has been found to be effective in some trials with non-human primate/simian/human immunodeficiency virus (SHIV) models, it remains unclear whether this vaccination strategy could elicit mature equine infectious anemia virus (EIAV) Env-specific antibodies, thus protecting vaccinated horses against EIAV infection. Therefore, in this pilot study we vaccinated horses using a strategy based on DNA prime/recombinant Tiantan vaccinia (rTTV)-vectored vaccines encoding EIAV env and gag genes, and observed the development of Env-specific antibodies, neutralizing antibodies, and p26-specific antibodies. Vaccination with DNA induced low titer, low avidity, and the predominant recognition of linear epitopes by Env-specific antibodies, which was enhanced by boosting vaccinations with rTTV vaccines. However, the maturation levels of Env-specific antibodies induced by the DNA/rTTV vaccines were significantly lower than those induced by the attenuated vaccine EIAV(FDDV). Additionally, DNA/rTTV vaccines did not elicit broadly neutralizing antibodies. After challenge with a virulent EIAV strain, all of the vaccinees and control horses died from EIAV disease. These data indicate that the regimen of DNA prime/rTTV vaccine boost did not induce mature Env-specific antibodies, which might have contributed to immune protection failure. PMID:23171359

Meng, Qinglai; Lin, Yuezhi; Ma, Jian; Ma, Yan; Zhao, Liping; Li, Shenwei; Yang, Kai; Zhou, Jianhua; Shen, Rongxian; Zhang, Xiaoyan; Shao, Yiming

2012-12-01

41

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. PMID:21246052

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; Fernández-Romero, Jose A.; Zydowsky, Thomas M.; Robbiani, Melissa

2011-01-01

42

NRM 2061/ENV 2005 Critical Thinking Papers  

E-print Network

NRM 2061/ENV 2005 Critical Thinking Papers Nuts and Bolts: Papers should be short (3 pages or 750 no more than 250 words on your summary). Critical thinking and reflection. Critical thinking is the use of cognitive skills or strategies that are purposeful, reasoned, and goal directed. Critical thinking

Brown, Gregory G.

43

Persistence of virus reservoirs in ART-treated SHIV-infected rhesus macaques after autologous hematopoietic stem cell transplant.  

PubMed

Despite many advances in AIDS research, a cure for HIV infection remains elusive. Here, we performed autologous hematopoietic stem cell transplantation (HSCT) in three Simian/Human Immunodeficiency Virus (SHIV)-infected, antiretroviral therapy (ART)-treated rhesus macaques (RMs) using HSCs collected prior to infection and compared them to three SHIV-infected, ART-treated, untransplanted control animals to assess the effect of conditioning and autologous HSCT on viral persistence. As expected, ART drastically reduced virus replication, below 100 SHIV-RNA copies per ml of plasma in all animals. After several weeks on ART, experimental RMs received myeloablative total body irradiation (1080 cGy), which resulted in the depletion of 94-99% of circulating CD4+ T-cells, and low to undetectable SHIV-DNA levels in peripheral blood mononuclear cells. Following HSC infusion and successful engraftment, ART was interrupted (40-75 days post-transplant). Despite the observed dramatic reduction of the peripheral blood viral reservoir, rapid rebound of plasma viremia was observed in two out of three transplanted RMs. In the third transplanted animal, plasma SHIV-RNA and SHIV DNA in bulk PBMCs remained undetectable at week two post-ART interruption. No further time-points could be assessed as this animal was euthanized for clinical reasons; however, SHIV-DNA could be detected in this animal at necropsy in sorted circulating CD4+ T-cells, spleen and lymph nodes but not in the gastro-intestinal tract or tonsils. Furthermore, SIV DNA levels post-ART interruption were equivalent in several tissues in transplanted and control animals. While persistence of virus reservoir was observed despite myeloablation and HSCT in the setting of short term ART, this experiment demonstrates that autologous HSCT can be successfully performed in SIV-infected ART-treated RMs offering a new experimental in vivo platform to test innovative interventions aimed at curing HIV infection in humans. PMID:25254512

Mavigner, Maud; Watkins, Benjamin; Lawson, Benton; Lee, S Thera; Chahroudi, Ann; Kean, Leslie; Silvestri, Guido

2014-09-01

44

Persistence of Virus Reservoirs in ART-Treated SHIV-Infected Rhesus Macaques after Autologous Hematopoietic Stem Cell Transplant  

PubMed Central

Despite many advances in AIDS research, a cure for HIV infection remains elusive. Here, we performed autologous hematopoietic stem cell transplantation (HSCT) in three Simian/Human Immunodeficiency Virus (SHIV)-infected, antiretroviral therapy (ART)-treated rhesus macaques (RMs) using HSCs collected prior to infection and compared them to three SHIV-infected, ART-treated, untransplanted control animals to assess the effect of conditioning and autologous HSCT on viral persistence. As expected, ART drastically reduced virus replication, below 100 SHIV-RNA copies per ml of plasma in all animals. After several weeks on ART, experimental RMs received myeloablative total body irradiation (1080 cGy), which resulted in the depletion of 94–99% of circulating CD4+ T-cells, and low to undetectable SHIV-DNA levels in peripheral blood mononuclear cells. Following HSC infusion and successful engraftment, ART was interrupted (40–75 days post-transplant). Despite the observed dramatic reduction of the peripheral blood viral reservoir, rapid rebound of plasma viremia was observed in two out of three transplanted RMs. In the third transplanted animal, plasma SHIV-RNA and SHIV DNA in bulk PBMCs remained undetectable at week two post-ART interruption. No further time-points could be assessed as this animal was euthanized for clinical reasons; however, SHIV-DNA could be detected in this animal at necropsy in sorted circulating CD4+ T-cells, spleen and lymph nodes but not in the gastro-intestinal tract or tonsils. Furthermore, SIV DNA levels post-ART interruption were equivalent in several tissues in transplanted and control animals. While persistence of virus reservoir was observed despite myeloablation and HSCT in the setting of short term ART, this experiment demonstrates that autologous HSCT can be successfully performed in SIV-infected ART-treated RMs offering a new experimental in vivo platform to test innovative interventions aimed at curing HIV infection in humans. PMID:25254512

Lawson, Benton; Lee, S. Thera; Chahroudi, Ann; Kean, Leslie; Silvestri, Guido

2014-01-01

45

The long-acting integrase inhibitor GSK744 protects macaques from repeated intravaginal SHIV challenge.  

PubMed

Daily preexposure prophylaxis (PrEP) with Truvada is a proven HIV prevention strategy; however, its effectiveness is limited by low adherence. Antiretroviral drug formulations that require infrequent dosing may increase adherence and thus PrEP effectiveness. We investigated whether monthly injections of a long-acting formulation of the HIV integrase inhibitor GSK1265744 (GSK744 LA) prevented simian/human immunodeficiency virus (SHIV) infection by vaginal challenge in macaques. Female pigtail macaques (n = 12) were exposed to intravaginal inoculations of SHIV twice a week for up to 11 weeks. Half of the animals received a GSK744 LA injection every 4 weeks, and half received placebo. GSK744 LA, at plasma concentrations achievable with quarterly injections in humans, protected all six macaques from infection. Placebo controls were all infected after a median of 4 (range, 2 to 20) vaginal challenges with SHIV. Efficacy was related to high and sustained vaginal and plasma drug concentrations that remained above the protein-adjusted 90% inhibitory concentration during the dosing cycles. These data support advancement of GSK744 LA as a potential PrEP candidate for women. PMID:25589631

Radzio, Jessica; Spreen, William; Yueh, Yun Lan; Mitchell, James; Jenkins, Leecresia; García-Lerma, J Gerardo; Heneine, Walid

2015-01-14

46

Progestin-based contraceptive suppresses cellular immune responses in SHIV-infected rhesus macaques.  

PubMed

Nine rhesus macaques in groups of three received a single dose of the injectable progestin-based contraceptive Depo-Provera 5 weeks prior to challenge intravaginally with varying doses of a mixture of the pathogenic CXCR4 (X4)-SHIV(SF33A) and CCR5 (R5)-SHIV(SF162P3) isolates. As controls, seven Depo-naive animals were inoculated once with a high-dose of the mixed inoculum. Irrespective of inoculum dose, acute viremia was higher in the Depo-treated than in the Depo-naive animals. Further, genetic complexity of the replicating virus was greater and replication of the X4 virus was favored in dually infected animals treated with Depo-Provera. Analysis of cellular immune responses revealed slower response rates in virus-specific IFN-gamma production to SIV Gag in the Depo-treated macaques. The immunosuppressive effect of Depo-Provera on mounting an antiviral cellular immune response may account for the increase viral burden and diversity, and the predominance of X4 virus replication in SHIV infected macaques that were administered the progestin-based contraceptive. PMID:16730772

Trunova, Nataliya; Tsai, Lily; Tung, Stephanie; Schneider, Eric; Harouse, Janet; Gettie, Agegnehu; Simon, Viviana; Blanchard, James; Cheng-Mayer, Cecilia

2006-08-15

47

BIOCHEMISTRY: RT Slides Homeâ?¦  

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)

2008-11-14

48

R5 Clade C SHIV Strains with Tier 1 or 2 Neutralization Sensitivity: Tools to Dissect Env Evolution and to Develop AIDS Vaccines in Primate Models  

Microsoft Academic Search

BackgroundHIV-1 clade C (HIV-C) predominates worldwide, and anti-HIV-C vaccines are urgently needed. Neutralizing antibody (nAb) responses are considered important but have proved difficult to elicit. Although some current immunogens elicit antibodies that neutralize highly neutralization-sensitive (tier 1) HIV strains, most circulating HIVs exhibiting a less sensitive (tier 2) phenotype are not neutralized. Thus, both tier 1 and 2 viruses are

Nagadenahalli B. Siddappa; Jennifer D. Watkins; Klemens J. Wassermann; Ruijiang Song; Wendy Wang; Victor G. Kramer; Samir Lakhashe; Michael Santosuosso; Mark C. Poznansky; Francis J. Novembre; François Villinger; James G. Else; David C. Montefiori; Robert A. Rasmussen; Ruth M. Ruprecht; Peter Sommer

2010-01-01

49

ENVS 340: Topics in Pollution: Gulf Oil Spill Spring 2011  

E-print Network

ENVS 340: Topics in Pollution: Gulf Oil Spill Spring 2011 ENVS 340 Topics in Pollution: Gulf Oil Oil Spill based on scientific research. Our report is due ~May 8. Our first goal is to determine Spill BIOL 378 Environmental Toxicology Instructor: Dr. Susan Allen-Gil Office: CNS 253 Phone: 274

50

ENV 6105 Page 1 of 6 Fall 2011 Air Pollution  

E-print Network

ENV 6105 Page 1 of 6 Fall 2011 Syllabus Air Pollution ENV 6105.901 (ref# 90504) Fall 2011 Course Description: A study of air pollution. Emphasis is given to principles underlying our understanding of ambient air pollution, its sources, its effects, and mechanisms for its management. Credit Hours and Work

Stuart, Amy L.

51

A long-acting integrase inhibitor protects female macaques from repeated high-dose intravaginal SHIV challenge.  

PubMed

Long-acting GSK1265744 (GSK744 LA) is a strand transfer inhibitor of the HIV/SIV (simian immunodeficiency virus) integrase and was shown to be an effective preexposure prophylaxis (PrEP) agent in a low-dose intrarectal SHIV (simian-human immunodeficiency virus) rhesus macaque challenge model. We examined the pharmacokinetics and efficacy of GSK744 LA as PrEP against repeat high-dose intravaginal SHIV challenge in female rhesus macaques treated with Depo-Provera (depot medroxyprogesterone acetate), which promotes viral transmission vaginally. When Depo-Provera-treated female rhesus macaques were dosed with GSK744 LA (50 mg/kg) monthly, systemic and tissue drug concentrations were lower than previously observed in male rhesus macaques. GSK744 concentrations were fivefold lower on average in cervical tissues than in rectal tissues. Eight female rhesus macaques were treated with GSK744 LA at week 0, and four female rhesus macaques served as controls. All animals received a high-dose challenge of SHIV162P3 at week 1. No infection was detected in GSK744 LA-treated rhesus macaques, whereas viremia was detected 1 to 2 weeks after SHIV challenge in all control animals. The GSK744 LA-treated rhesus macaques were given a second administration of drug at week 4 and further challenged at weeks 5 and 7. GSK744 LA treatment protected six of eight female rhesus macaques against three high-dose SHIV challenges, whereas all control animals became infected after the first challenge (P = 0.0003, log-rank test). These results support further clinical development of GSK744 LA for PrEP. PMID:25589630

Andrews, Chasity D; Yueh, Yun Lan; Spreen, William R; St Bernard, Leslie; Boente-Carrera, Mar; Rodriguez, Kristina; Gettie, Agegnehu; Russell-Lodrigue, Kasi; Blanchard, James; Ford, Susan; Mohri, Hiroshi; Cheng-Mayer, Cecilia; Hong, Zhi; Ho, David D; Markowitz, Martin

2015-01-14

52

The impact of highly active antiretroviral therapy by the oral route on the CD8 subset in monkeys infected chronically with SHIV 89.6P  

Microsoft Academic Search

The objective of this study was to assess the impact of highly active antiretroviral therapy (HAART) by an oral route on the peripheral blood CD8 subset in the monkeys infected persistently with a pathogenic strain, SHIV89.6P. Two rhesus macaques were inoculated intravenously with SHIV89.6P, then treated with the combination of AZT, 3TC and Lopinavir\\/Ritonavir (LPV\\/RTV) as recommended in humans by

Kazuhisa Yoshimura; Eiji Ido; Hisashi Akiyama; Tetsuya Kimura; Manabu Aoki; Hajime Suzuki; Hiroaki Mitsuya; Masanori Hayami; Shuzo Matsushita

2003-01-01

53

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

54

Antibody-mediated immunotherapy of macaques chronically infected with SHIV suppresses viraemia  

NASA Astrophysics Data System (ADS)

Neutralizing antibodies can confer immunity to primate lentiviruses by blocking infection in macaque models of AIDS. However, earlier studies of anti-human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies administered to infected individuals or humanized mice reported poor control of virus replication and the rapid emergence of resistant variants. A new generation of anti-HIV-1 monoclonal antibodies, possessing extraordinary potency and breadth of neutralizing activity, has recently been isolated from infected individuals. These neutralizing antibodies target different regions of the HIV-1 envelope glycoprotein including the CD4-binding site, glycans located in the V1/V2, V3 and V4 regions, and the membrane proximal external region of gp41 (refs 9, 10, 11, 12, 13, 14). Here we have examined two of the new antibodies, directed to the CD4-binding site and the V3 region (3BNC117 and 10-1074, respectively), for their ability to block infection and suppress viraemia in macaques infected with the R5 tropic simian-human immunodeficiency virus (SHIV)-AD8, which emulates many of the pathogenic and immunogenic properties of HIV-1 during infections of rhesus macaques. Either antibody alone can potently block virus acquisition. When administered individually to recently infected macaques, the 10-1074 antibody caused a rapid decline in virus load to undetectable levels for 4-7days, followed by virus rebound during which neutralization-resistant variants became detectable. When administered together, a single treatment rapidly suppressed plasma viraemia for 3-5weeks in some long-term chronically SHIV-infected animals with low CD4+ T-cell levels. A second cycle of anti-HIV-1 monoclonal antibody therapy, administered to two previously treated animals, successfully controlled virus rebound. These results indicate that immunotherapy or a combination of immunotherapy plus conventional antiretroviral drugs might be useful as a treatment for chronically HIV-1-infected individuals experiencing immune dysfunction.

Shingai, Masashi; Nishimura, Yoshiaki; Klein, Florian; Mouquet, Hugo; Donau, Olivia K.; Plishka, Ronald; Buckler-White, Alicia; Seaman, Michael; Piatak, Michael; Lifson, Jeffrey D.; Dimitrov, Dimiter; Nussenzweig, Michel C.; Martin, Malcolm A.

2013-11-01

55

The Recombinant Maize Ribosome-Inactivating Protein Transiently Reduces Viral Load in SHIV89.6 Infected Chinese Rhesus Macaques  

PubMed Central

Ribosome inactivating proteins (RIPs) inhibit protein synthesis by depurinating the large ribosomal RNA and some are found to possess anti-human immunodeficiency virus (HIV) activity. Maize ribosome inactivating protein (RIP) has an internal inactivation loop which is proteolytically removed for full catalytic activity. Here, we showed that the recombinant active maize RIP protected chimeric simian-human immunodeficiency virus (SHIV) 89.6-infected macaque peripheral blood mononuclear cells from lysis ex vivo and transiently reduced plasma viral load in SHIV89.6-infected rhesus macaque model. No evidence of immune dysregulation and other obvious side-effects was found in the treated macaques. Our work demonstrates the potential development of maize RIP as an anti-HIV agent without impeding systemic immune functions. PMID:25606813

Wang, Rui-Rui; Au, Ka-Yee; Zheng, Hong-Yi; Gao, Liang-Min; Zhang, Xuan; Luo, Rong-Hua; Law, Sue Ka-Yee; Mak, Amanda Nga-Sze; Wong, Kam-Bo; Zhang, Ming-Xu; Pang, Wei; Zhang, Gao-Hong; Shaw, Pang-Chui; Zheng, Yong-Tang

2015-01-01

56

Planning for RtI  

ERIC Educational Resources Information Center

In 2004 the Individuals with Disabilities Education Act authorized funding for Response to Intervention (RtI) instruction in the United States. By 2011, 71 percent of school districts had adopted RtI (Institute of Education Sciences 2011). The goal of RtI is to provide personalized, just-in-time intervention in reading and math for students who…

Robins, Jennifer; Antrim, Patricia

2013-01-01

57

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

Microsoft Academic Search

BackgroundThe 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

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

2011-01-01

58

Longitudinal study to assess the safety and efficacy of a live-attenuated SHIV vaccine in long term immunized rhesus macaques  

SciTech Connect

Live-attenuated viruses derived from SIV and SHIV have provided the most consistent protection against challenge with pathogenic viruses, but concerns regarding their long-term safety and efficacy have hampered their clinical usefulness. We report a longitudinal study in which we evaluated the long-term safety and efficacy of {delta}vpuSHIV{sub PPC}, a live virus vaccine derived from SHIV{sub PPC}. Macaques were administered two inoculations of {delta}vpuSHIV{sub PPC}, three years apart, and followed for eight years. None of the five vaccinated macaques developed an AIDS-like disease from the vaccine. At eight years, macaques were challenged with pathogenic SIV and SHIV. None of the four macaques with detectable cellular-mediated immunity prior to challenge had detectable viral RNA in the plasma. This study demonstrates that multiple inoculations of a live vaccine virus can be used safely and can significantly extend the efficacy of the vaccine, as compared to a single inoculation, which is efficacious for approximately three years.

Yankee, Thomas M. [Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, 3025 WHW - MS 3029, 3901 Rainbow Boulevard, Kansas City, KS 66160 (United States)], E-mail: tyankee@kumc.edu; Sheffer, Darlene; Liu Zhengian; Dhillon, Sukhbir; Jia Fenglan; Chebloune, Yahia [Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, 3025 WHW - MS 3029, 3901 Rainbow Boulevard, Kansas City, KS 66160 (United States); Stephens, Edward B. [Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, 3025 WHW - MS 3029, 3901 Rainbow Boulevard, Kansas City, KS 66160 (United States); Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Narayan, Opendra [Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, 3025 WHW - MS 3029, 3901 Rainbow Boulevard, Kansas City, KS 66160 (United States)

2009-01-05

59

EFFECTIVE, LOW TITER, ANTIBODY PROTECTION AGAINST LOW-DOSE REPEATED MUCOSAL SHIV CHALLENGE IN MACAQUES  

PubMed Central

Neutralizing antibodies are thought crucial to HIV vaccine protection but a major hurdle is the high antibody concentrations likely required as suggested by studies in animal models1. However, these studies typically apply a large virus inoculum to ensure infection in control animals in single challenge experiments. In contrast, most human infection via sexual encounter probably involves repeated exposures to much lower doses of virus2–4. Therefore, animal studies may have overestimated protective antibody levels in humans. To investigate the impact of virus challenge dose on antibody protection, we repeatedly exposed macaques intravaginally to low doses of a CCR5 coreceptor-using SHIV (an HIV/SIV chimera) in the presence of antibody at plasma concentrations leading to relatively modest neutralization titers of the order of 1:5 IC90 values in a PBMC assay. An effector function deficient variant of the neutralizing antibody was also included. The results show that a significantly greater number of challenges are required to infect animals treated with neutralizing antibody than control antibody-treated animals, and the notion that effector function may contribute to antibody protection is supported. Overall, the results imply that lower levels of antibody than considered hereto may provide benefit in the context of typical human exposure to HIV-1. PMID:19525965

Hessell, Ann J.; Poignard, Pascal; Hunter, Meredith; Hangartner, Lars; Tehrani, David M.; Bleeker, Wim K.; Parren, Paul W.H.I.; Marx, Preston A.; Burton, Dennis R.

2015-01-01

60

Development of a reliable dual-gene amplification RT-PCR assay for the detection of Turkey Meningoencephalitis virus in Turkey brain tissues.  

PubMed

The Turkey Meningoencephalitis virus (TMEV) causes neuroparalytic signs, paresis, in-coordination, morbidity and mortality in turkeys. In parallel to the increased worldwide scientific interest in veterinary avian flaviviruses, including the Bagaza, Tembusu and Tembusu-related BYD virus, TMEV-caused disease also reemergence in commercial turkeys during late summer of 2010. While initially TMEV was detected by NS5-gene RT-PCR, subsequently, the env-gene RT-PCR was employed. As lately several inconsistencies were observed between the clinical, serological and molecular detection of the TMEV env gene, this study evaluated whether genetic changes occurred in the recently isolated viruses, and sought to optimize and improve the direct TMEV amplification from brain tissues of affected turkeys. The main findings indicated that no changes occurred during the years in the TMEV genome, but the PCR detection sensitivities of the env and NS5 genes differed. The RT-PCR and RNA purification were optimized for direct amplification from brain tissues without pre-replication of clinical samples in tissue cultures or in embryonated eggs. The amplification sensitivity of the NS5-gene was 10-100 times more than the env-gene when separate. The new dual-gene amplification RT-PCR was similar to that of the NS5 gene, therefore the assay can be considered as a reliable diagnostic assay. Cases where one of the two amplicons would be RT-PCR negative would alert and warn on the virus identity, and possible genetic changes. In addition, the biochemical environment of the dual-gene amplification reaction seemed to contribute in deleting non-specific byproducts that occasionally appeared in the singular RT-PCR assays on RNA purified from brain tissues. PMID:22705084

Davidson, Irit; Raibstein, Israel; Al-Tori, Amira; Khinich, Yevgeny; Simanov, Michael; Yuval, Chanoch; Perk, Shimon; Lublin, Avishai

2012-11-01

61

Evaluation of -2 RANTES vaginal microbicide formulations in a nonhuman primate simian/human immunodeficiency virus (SHIV) challenge model.  

PubMed

A potential strategy to combat the worldwide AIDS epidemic is to develop a vaginal microbicide that prevents the sexual transmission of HIV-1. One approach for preventing vaginal HIV transmission is to block the viral coreceptor CCR5 with naturally occurring chemokine ligands. In this study, we used a cynomolgus macaque model to evaluate whether a variant of the CCR5 ligand RANTES (-2 RANTES), tested alone or in a nonphospholipid liposome carrier (Novasomes 7474), blocks vaginal challenge with a CCR5-tropic simian/human immunodeficiency virus (SHIV(162P3)). When tested in vitro, the synthetic chemokine potently inhibited SHIV(162P3) infection of cynomolgus macaque peripheral blood mononuclear cells (PBMC). Colposcopic examinations of treated animals and histological examination of cervicovaginal biopsies showed minimal signs of tissue inflammation following vaginal application of Novasomes 7474, -2 RANTES formulated in Novasomes 7474, or -2 RANTES alone. Following vaginal challenge with SHIV(162P3), complete protection was observed in four of six animals treated vaginally with -2 RANTES (0.13 mM) formulated in Novasomes 7474. However, the same proportion of animals was protected by treatment with Novasomes 7474 carrier alone. Two of five animals treated with 0.5 mM -2 RANTES in PBS were protected from infection. Further, all animals were infected when treated with lower chemokine concentrations. These findings indicate that natural CCR5 ligands may have limited efficacy in stringent nonhuman primate models for vaginal infection. In comparison, liposomal agents such as Novasomes 7474 provide comparatively robust protection against vaginal transmission. PMID:17263630

Kish-Catalone, Tina; Pal, Ranajit; Parrish, John; Rose, Nicholas; Hocker, Lindsey; Hudacik, Lauren; Reitz, Marvin; Gallo, Robert; Devico, Anthony

2007-01-01

62

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

63

Tropical Conservation and Ecology in Costa Rica ENV SCI 499  

E-print Network

Tropical Conservation and Ecology in Costa Rica ENV SCI 499 Assistant Professor Mathew Dornbush National Park. Located on Costa Rica's Pacific Coast, Carara is characterized by high biological diversity with park staff enhances group discussions by providing a Costa Rican perspective to Costa Rica's natural

Dornbush, Mathew E.

64

ENVS 404: Internship Syllabus Contact Information: Internship Coordinator  

E-print Network

1 ENVS 404: Internship Syllabus Contact Information: Internship Coordinator Peg Boulay Environmental Leadership Program Co-Director, Internship Coordinator and Academic Adviser 242 Columbia Hall 541-346-5945 boulay@uoregon.edu Note: The Internship Coordinator position may also be filled by a Graduate Teaching

65

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

66

The quantity and diversity of infectious viruses in various tissues of SHIV-infected monkeys at the early and AIDS stages  

Microsoft Academic Search

Summary. To detect the major sites of viral replication in immunodeficiency virus-infected individuals, we quantified proviral DNA and infectious viruses using quantitative PCR and a plaque assay, respectively, in various tissues of SHIV KU-2-infected monkeys in the early and AIDS stages of infection. Compared the quantity of infectious virus among PBMC and the lymphoid tissues, the mesenteric lymph node had

A. Miyake; Y. Enose; S. Ohkura; H. Suzuki; T. Kuwata; T. Shimada; S. Kato; O. Narayan; M. Hayami

2004-01-01

67

Protective properties of non-nucleoside reverse transcriptase inhibitor (MC1220) incorporated into liposome against intravaginal challenge of Rhesus Macaques with RT-SHIV  

Microsoft Academic Search

In the absence of an effective vaccine against HIV, it is urgent to develop an effective alternative such as a microbicide. Single and repeated applications of MC1220 microbicide were evaluated in macaques. First, animals were given a single application of 0.5% or 1.5% MC1220-containing liposomal gel. A second group were treated with 0.5% MC1220 once a day for 4days. The

Mélanie Caron; Guillaume Besson; Sonia Lekana-Douki Etenna; Armel Mintsa-Ndong; Spyridon Mourtas; Antonia Radaelli; Carlo De Giuli Morghen; Roberta Loddo; Paolo La Colla; Sophia G. Antimisiaris; Mirdad Kazanji

2010-01-01

68

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

69

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.

70

Direct Involvement of HERV-W Env Glycoprotein in Human Trophoblast Cell Fusion and Differentiation  

Microsoft Academic Search

We recently demonstrated that the product of the HERV-W env gene, a retroviral envelope protein also dubbed syncytin, is a highly fusogenic membrane glycoprotein inducing the formation of syncytia on interac- tion with the type D mammalian retrovirus receptor. In addition, the detection of HERV-W Env protein (Env-W) expression in placental tissue sections led us to propose a role for

Jean-Louis Frendo; Delphine Olivier; Valerie Cheynet; Jean-Luc Blond; Olivier Bouton; Michel Vidaud; Michele Rabreau; Daniele Evain-Brion; Francois Mallet

2003-01-01

71

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

2012-11-01

72

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. 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

2012-01-01

73

Induction of potent local cellular immunity with low dose X4 SHIV(SF33A) vaginal exposure.  

PubMed

Intravaginal inoculation of rhesus macaques with varying doses of the CXCR4 (X4)-tropic SHIV(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(50)) resulted in high viremia and precipitous CD4+ T cell loss, occult infection was observed in seven of eight macaques exposed to 500 TCID(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(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. PMID:17574643

Tasca, Silvana; Tsai, Lily; Trunova, Nataliya; Gettie, Agegnehu; Saifuddin, Mohammed; Bohm, Rudolf; Chakrabarti, Lisa; Cheng-Mayer, Cecilia

2007-10-10

74

Transcriptional and functional studies of Human Endogenous Retrovirus envelope EnvP(b) and EnvV genes in human trophoblasts  

SciTech Connect

HERV (Human Endogenous Retrovirus)-encoded envelope proteins are implicated in the development of the placenta. Indeed, Syncytin-1 and -2 play a crucial role in the fusion of human trophoblasts, a key step in placentation. Other studies have identified two other HERV env proteins, namely EnvP(b) and EnvV, both expressed in the placenta. In this study, we have fully characterized both env transcripts and their expression pattern and have assessed their implication in trophoblast fusion. Through RACE analyses, standard spliced transcripts were detected, while EnvV transcripts demonstrated alternative splicing at its 3 Prime end. Promoter activity and expression of both genes were induced in forskolin-stimulated BeWo cells and in primary trophoblasts. Although we have confirmed the fusogenic activity of EnvP(b), overexpression or silencing experiments revealed no impact of this protein on trophoblast fusion. Our results demonstrate that both env genes are expressed in human trophoblasts but are not required for syncytialization.

Vargas, Amandine, E-mail: amandine.vargas@voila.fr; Thiery, Maxime, E-mail: thiery.maxime@courrier.uqam.ca; Lafond, Julie, E-mail: lafond.julie@uqam.ca; Barbeau, Benoit, E-mail: barbeau.benoit@uqam.ca

2012-03-30

75

The emergence and characterization of macrophage-tropic SIV\\/HIV chimeric viruses (SHIVs) present in CD4+ T cell-depleted rhesus monkeys  

Microsoft Academic Search

Highly pathogenic simian immunodefi- ciency virus\\/human immunodeficiency virus type 1 chimeric viruses (SHIVs) induce an extremely rapid, systemic, and irreversible depletion of CD4 T lymphocytes following their inoculation into rhesus macaques. Confocal fluorescence mi- croscopy was used to demonstrate that high levels of viremia in infected animals were sustained by virus-producing tissue macrophage (m) following the irreversible elimination of CD4

Tatsuhiko Igarashi; Hiromi Imamichi; Charles R. Brown; Vanessa M. Hirsch; Malcolm A. Martin

2003-01-01

76

Functional Impairment of Central Memory CD4 T Cells Is a Potential Early Prognostic Marker for Changing Viral Load in SHIV-Infected Rhesus Macaques  

Microsoft Academic Search

In HIV infection there is a paucity of literature about the degree of immune dysfunction to potentially correlate and\\/or predict disease progression relative to CD4+ T cells count or viral load. We assessed functional characteristics of memory T cells subsets as potential prognostic markers for changing viral loads and\\/or disease progression using the SHIV-infected rhesus macaque model. Relative to long-term

Hong He; Pramod N. Nehete; Bharti Nehete; Eric Wieder; Guojun Yang; Stephanie Buchl; K. Jagannadha Sastry

2011-01-01

77

SimEnvVis: A Climate Data Visualization Wizard  

NASA Astrophysics Data System (ADS)

To efficiently make sense of complex climate data, climate scientists need to choose and utilize appropriate analysis tools in respect to specific sets of tasks. Among these, visual analysis tools, like those originating from the field of visual analytics, efficiently support to communicate such information by directly addressing human visual perception. However, climate scientists often are not aware of or not familiar with the large variety of available visual analysis tools or are underestimating their potential benefit for common research tasks and thus reducing the probability to use most suitable ones and therefore impairing the knowledge discovery process. To address this problem, SimEnvVis was developed as an easy-to-use wizard-based software system guiding the user step-by-step in choosing most appropriate visualization and visual analytics tools from a large and easily extendable repository consisting of script-based and interactive tools with different application foci (spatial, temporal or abstract data) and supported techniques (e.g. glyphs, isocontours, stream visualization). Considering the analysis context (e.g. data characteristics, user preferences and analysis tasks) SimEnvVis automatically evaluates the attached tools using a combination of a vector-based and a rule-based mechanism. Based on the users decision, the selected visual analysis tool is launched using a template which is dynamically parameterized by taking into account the analysis context. By displaying the session history in different modes as well as providing the possibility to start SimEnvVis in first-time-user mode to reduce GUI complexity and hide tools which are under development the wizard is in particular useful for novice users. This way, SimEnvVis increases the probability for the usage of appropriate visual analysis tools, lowers the obstacles of familiarization with them and therefore accelerates the knowledge discovery process as well as positively contributes to the quality of its results. With this contribution, we provide a description of the wizard as well as visual analysis use cases to illustrate its application.

Heitzler, Magnus; Nocke, Thomas

2013-04-01

78

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

PubMed Central

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

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

2013-01-01

79

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

80

Regulation of human immunodeficiency virus env expression by the rev gene product.  

PubMed Central

A single simian virus 40 late replacement vector which expresses both the rev and envelope (env) genes of human immunodeficiency virus was used to examine the mechanism underlying the dependence of env gene expression on the rev protein. When rev was deleted from the vector, no envelope protein expression could be detected in transfected cells, and the levels of cytoplasmic env mRNA were dramatically reduced. In contrast to this, the levels of env RNA in total cellular RNA preparations were similar with or without rev coexpression, and analysis of nuclear RNA showed that the levels of nuclear env RNA were increased in the absence of rev. These results suggest that rev functions to regulate nuclear export of env mRNA. It was possible to restore env expression from the vector lacking rev by supplying rev in trans, provided that a cis-acting sequence was also present. This sequence was mapped to a 854-base-pair region within the env open reading frame, and it was shown that the sequence could be moved but that it worked only in its original orientation. Images PMID:2704072

Hammarskjöld, M L; Heimer, J; Hammarskjöld, B; Sangwan, I; Albert, L; Rekosh, D

1989-01-01

81

Direct Involvement of HERV-W Env Glycoprotein in Human Trophoblast Cell Fusion and Differentiation  

PubMed Central

We recently demonstrated that the product of the HERV-W env gene, a retroviral envelope protein also dubbed syncytin, is a highly fusogenic membrane glycoprotein inducing the formation of syncytia on interaction with the type D mammalian retrovirus receptor. In addition, the detection of HERV-W Env protein (Env-W) expression in placental tissue sections led us to propose a role for this fusogenic glycoprotein in placenta formation. To evaluate this hypothesis, we analyzed the involvement of Env-W in the differentiation of primary cultures of human villous cytotrophoblasts that spontaneously differentiate by cell fusion into syncytiotrophoblasts in vitro. First, we observed that HERV-W env mRNA and glycoprotein expression are colinear with primary cytotrophoblast differentiation and with expression of human chorionic gonadotropin (hCG), a marker of syncytiotrophoblast formation. Second, we observed that in vitro stimulation of trophoblast cell fusion and differentiation by cyclic AMP is also associated with a concomitant increase in HERV-W env and hCG mRNA and protein expression. Finally, by using specific antisense oligonucleotides, we demonstrated that inhibition of Env-W protein expression leads to a decrease of trophoblast fusion and differentiation, with the secretion of hCG in culture medium of antisense oligonucleotide-treated cells being decreased by fivefold. Taken together, these results strongly support a direct role for Env-W in human trophoblast cell fusion and differentiation. PMID:12724415

Frendo, Jean-Louis; Olivier, Delphine; Cheynet, Valérie; Blond, Jean-Luc; Bouton, Olivier; Vidaud, Michel; Rabreau, Michèle; Evain-Brion, Danièle; Mallet, François

2003-01-01

82

Implementing RtI with Gifted Students  

ERIC Educational Resources Information Center

"Implementing RtI With Gifted Students" shares how RtI can fit within the framework of gifted education programming models. This edited book will serve as a reference guide for those interested in learning more about RtI and how it might be effectively implemented to meet the needs of all gifted students. Chapters contributed by top gifted…

Coleman, Mary Ruth, Ed.; Johnsen, Susan K., Ed.

2012-01-01

83

The Majority of CD4+ T-Cell Depletion during Acute Simian-Human Immunodeficiency Virus SHIV89.6P Infection Occurs in Uninfected Cells  

PubMed Central

ABSTRACT Untreated human immunodeficiency virus (HIV) infection is characterized by depletion of CD4+ T cells, ultimately leading to the impairment of host immune defenses and death. HIV-infected CD4+ T cells die from direct virus-induced apoptosis and CD8 T-cell-mediated elimination, but a broader and more profound depletion occurs in uninfected CD4+ T cells via multiple indirect effects of infection. We fit mathematical models to data from experiments that tested an HIV eradication strategy in which five macaques with a proportion of CD4+ T cells resistant to simian-human immunodeficiency virus (SHIV) entry were challenged with SHIV89.6P, a highly pathogenic dual-tropic chimeric SIV-HIV viral strain that results in rapid loss of both SHIV-susceptible and SHIV-resistant CD4+ T cells. Our results suggest that uninfected (bystander) cell death accounts for the majority of CD4+ T-lymphocyte loss, with at least 60% and 99% of CD4+ T cell death occurring in uninfected cells during acute and established infection, respectively. Mechanisms to limit the profound indirect killing effects associated with HIV infection may be associated with immune preservation and improved long-term survival. IMPORTANCE HIV infection induces a massive depletion of CD4+ T cells, leading to profound immunodeficiency, opportunistic infections, and eventually death. While HIV induces apoptosis (programmed cell death) by directly entering and replicating in CD4+ T cells, uninfected CD4+ T cells also undergo apoptosis due to ongoing toxic inflammation in the region of infection. In this paper, we use mathematical models in conjunction with data from simian-human immunodeficiency virus SHIV89.6P infection in macaques (a model of HIV infection in humans) to estimate the percentage of cell death that occurs in uninfected cells during the initial period of infection. We reveal that the vast majority of cell death occurs in these cells, which are not infected. The “bystander effects” that lead to enormous reductions in the number of uninfected CD4+ T cells may be a target for future interventions that aim to limit the extent of damage caused by HIV. PMID:24390339

Matrajt, Laura; Younan, Patrick M.; Kiem, Hans-Peter

2014-01-01

84

SMaRT CAMP 2013 The SMaRT (Summer Mathematics Research Training) Camp,  

E-print Network

SMaRT CAMP 2013 The SMaRT (Summer Mathematics Research Training) Camp, is a two-week program (979) 845-7554 Fax (979) 845-6028 www.math.tamu.edu SMaRT High School Summer Camp 2013 Sponsored by is filled. Support: Room and board will be covered for accepted participants. Information about SMaRT, visit

Boas, Harold P.

85

SMaRT CAMP 2012 The SMaRT (Summer Mathematics Research Training) Camp,  

E-print Network

SMaRT CAMP 2012 The SMaRT (Summer Mathematics Research Training) Camp, is a two-week program at (979) 845-7554 Fax (979) 845-6028 www.math.tamu.edu SMaRT High School Summer Camp 2012 Sponsored by is filled. Support: Room and board will be covered for accepted participants. Information about SMaRT, visit

86

High throughput functional analysis of HIV-1 env genes without cloning  

PubMed Central

Functional human immunodeficiency virus type -1 env clones have been widely used for vaccine design, neutralization assays, and pathogenesis studies. However, obtaining bona fide functional env clones is a time consuming and labor intensive process. A new high throughput method has been developed to characterize HIV-1 env genes. Multiple rev/env gene cassettes were obtained from each of seven HIV-1 strains using single genome amplification (SGA) PCR. The CMV promoter was amplified separately by PCR. A promoter PCR (pPCR) method was developed to link both PCR products using an overlapping PCR method. Pseudovirions were generated by cotransfection of pPCR products and pSG3?env backbone into 293T cells. After infecting TZM-bl cells, 75 out of 87 (86%) of the rev/env gene cassettes were functional. Pseudoviruses generated with pPCR products or corresponding plasmid DNA showed similar sensitivity to six HIV-1 positive sera and three monoclonal antibodies, suggesting neutralization properties are not altered in pPCR pseudovirions. Furthermore, sufficient amounts of pseudovirions can be obtained for a large number of neutralization assays. The new pPCR method eliminates cloning, transformation, and plasmid DNA preparation steps in the generation of HIV-1 pseudovirions, this allows for quick analysis of multiple env genes from HIV-1 infected individuals. PMID:17416428

Kirchherr, Jennifer L; Lu, Xiaozhi; Kasongo, Webster; Chalwe, Victor; Mwananyanda, Lawrence; Musonda, Rosemary M; Xia, Shi-Mao; Scearce, Richard M; Liao, Hua-Xin; Montefiori, David C; Haynes, Barton F; Gao, Feng

2007-01-01

87

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. PMID:22514633

Wang, Yufei; Whittall, Trevor; Rahman, Durdana; Bunnik, Evelien M.; Vaughan, Robert; Schøller, Jørgen; Bergmeier, Lesley A.; Montefiori, David; Singh, Mahavir; Schuitemaker, Hanneke; Lehner, Thomas

2012-01-01

88

Differential binding of neutralizing and non-neutralizing antibodies to native-like soluble HIV-1 Env trimers, uncleaved Env proteins, and monomeric subunits  

PubMed Central

Background The trimeric envelope glycoproteins (Env) on the surface of HIV-1 virions are the targets for neutralizing antibodies (NAbs). No candidate HIV-1 immunogen has yet induced potent, broadly active NAbs (bNAbs). Part of the explanation may be that previously tested Env proteins inadequately mimic the functional, native Env complex. Trimerization and the proteolytic processing of Env precursors into gp120 and gp41 profoundly alter antigenicity, but soluble cleaved trimers are too unstable to serve as immunogens. By introducing stabilizing mutations (SOSIP), we constructed soluble, cleaved Env trimers derived from the HIV-1 subtype A isolate BG505 that resemble native Env spikes on virions both structurally and antigenically. Results We used surface plasmon resonance (SPR) to quantify antibody binding to different forms of BG505 Env: the proteolytically cleaved SOSIP.664 trimers, cleaved gp120-gp41ECTO protomers, and gp120 monomers. Non-NAbs to the CD4-binding site bound only marginally to the trimers but equally well to gp120-gp41ECTO protomers and gp120 monomers, whereas the bNAb VRC01, directed to the CD4bs, bound to all three forms. In contrast, bNAbs to V1V2 glycan-dependent epitopes bound preferentially (PG9 and PG16) or exclusively (PGT145) to trimers. We also explored the antigenic consequences of three different features of SOSIP.664 gp140 trimers: the engineered inter-subunit disulfide bond, the trimer-stabilizing I559P change in gp41ECTO, and proteolytic cleavage at the gp120-gp41ECTO junction. Each of these three features incrementally promoted native-like trimer antigenicity. We compared Fab and IgG versions of bNAbs and validated a bivalent model of IgG binding. The NAbs showed widely divergent binding kinetics and degrees of binding to native-like BG505 SOSIP.664. High off-rate constants and low stoichiometric estimates of NAb binding were associated with large amounts of residual infectivity after NAb neutralization of the corresponding BG505.T332N pseudovirus. Conclusions The antigenicity and structural integrity of cleaved BG505 SOSIP.664 trimers render these proteins good mimics of functional Env spikes on virions. In contrast, uncleaved gp140s antigenically resemble individual gp120-gp41ECTO protomers and gp120 monomers, but not native trimers. Although NAb binding to functional trimers may thus be both necessary and sufficient for neutralization, the kinetics and stoichiometry of the interaction influence the neutralizing efficacy of individual NAbs. PMID:24884783

2014-01-01

89

Construction and characterization of recombinant VLPs and Semliki-Forest virus live vectors for comparative evaluation in the SHIV monkey model.  

PubMed

For testing of recombinant virus-like particles (VLPs) in the SHIV monkey model, SIVmac239 Pr56gag precursor-based pseudovirions were modified by HIV-1 gp160 derived peptides. First, well-characterized epitopes from the HIV-1 envelope glycoprotein were inserted into the Pr56gag precursor by replacing defined regions that were shown to be dispensable for virus particle formation. Expression of these chimeric proteins in a baculovirus expression system resulted in efficient assembly and release of non-infectious, hybrid VLPs. In a second approach the HIV-1IIIB external glycoprotein gp120 was covalently linked to an Epstein-Barr virus derived transmembrane domain. Coexpression of the hybrid envelope derivative with the Pr56gag precursor yielded recombinant SIV derived Pr56gag particles with the HIV-1 gp120 firmly anchored on the VLP surface. Immunization of rhesus monkeys with either naked VLPs or VLPs adsorbed to alum induced substantial serum antibody titers and promoted both T helper cell and cytotoxic T lymphocyte responses. Furthermore, priming macaques with the corresponding set of recombinant Semliki-Forest viruses tended to enhance the immunological outcome. Challenge of the immunized monkeys with chimeric SHIV resulted in a clearly accelerated reduction of the plasma viremia as compared to control animals. PMID:10223337

Notka, F; Stahl-Hennig, C; Dittmer, U; Wolf, H; Wagner, R

1999-03-01

90

CD4+ T Cells Modified by the Endoribonuclease MazF Are Safe and Can Persist in SHIV-infected Rhesus Macaques  

PubMed Central

MazF, an endoribonuclease encoded by Escherichia coli, specifically cleaves the ACA (adenine–cytosine–adenine) sequence of single-stranded RNAs. Conditional expression of MazF under the control of the HIV-1 LTR promoter rendered CD4+ T cells resistant to HIV-1 replication without affecting cell growth. To investigate the safety, persistence and efficacy of MazF-modified CD4+ T cells in a nonhuman primate model in vivo, rhesus macaques were infected with a pathogenic simian/human immunodeficiency virus (SHIV) and transplanted with autologous MazF-modified CD4+ T cells. MazF-modified CD4+ T cells were clearly detected throughout the experimental period of more than 6 months. The CD4+ T cell count values increased in all four rhesus macaques. Moreover, the transplantation of the MazF-modified CD4+ T cells was not immunogenic, and did not elicit cellular or humoral immune responses. These data suggest that the autologous transplantation of MazF-modified CD4+ T cells in the presence of SHIV is effective, safe and not immunogenic, indicating that this is an attractive strategy for HIV-1 gene therapy. PMID:24914931

Saito, Naoki; Chono, Hideto; Shibata, Hiroaki; Ageyama, Naohide; Yasutomi, Yasuhiro; Mineno, Junichi

2014-01-01

91

Budding and secretion of HIV Gag–Env virus-like particles from recombinant human adenovirus infected cells  

Microsoft Academic Search

We have characterized the assembly, budding and extra-cellular release of human immunodeficiency virus (HIV) Gag–Env virus-like particles (VLPs) from human embryonic kidney cells (293 cells expressing the E1a protein of adenovirus) infected with recombinant replication-defective human adenovirus type 5. Recombinant human adenovirus vectors expressing the chimeric Gag–Env protein were constructed by inserting the gag–env fusion gene into the E1a region

Lizhong Luo; Yan Li; C Yong Kang

2003-01-01

92

Molecular identification of erythrocytic necrosis virus (ENV) from the blood of Pacific herring (Clupea pallasii).  

PubMed

Viral erythrocytic necrosis (VEN) is a condition affecting the red blood cells of more than 20 species of marine and anadromous fishes in the North Atlantic and North Pacific Oceans. Among populations of Pacific herring (Clupea pallasii) on the west coast of North America the disease causes anemia and elevated mortality in periodic epizootics. Presently, VEN is diagnosed by observation of typical cytoplasmic inclusion bodies in stained blood smears from infected fish. The causative agent, erythrocytic necrosis virus (ENV), is unculturable and a presumed iridovirus by electron microscopy. In vivo amplification of the virus in pathogen-free laboratory stocks of Pacific herring with subsequent virus concentration, purification, DNA extraction, and high-throughput sequencing were used to obtain genomic ENV sequences. Fragments with the highest sequence identity to the family Iridoviridae were used to design four sets of ENV-specific polymerase chain reaction (PCR) primers. Testing of blood and tissue samples from experimentally and wild infected Pacific herring as well as DNA extracted from other amphibian and piscine iridoviruses verified the assays were specific to ENV with a limit of detection of 0.0003 ng. Preliminary phylogenetic analyses of a 1448 bp fragment of the putative DNA polymerase gene supported inclusion of ENV in a proposed sixth genus of the family Iridoviridae that contains other erythrocytic viruses from ectothermic hosts. This study provides the first molecular evidence of ENV's inclusion within the Iridoviridae family and offers conventional PCR assays as a means of rapidly surveying the ENV-status of wild and propagated Pacific herring stocks. PMID:25263493

Emmenegger, Eveline J; Glenn, Jolene A; Winton, James R; Batts, William N; Gregg, Jacob L; Hershberger, Paul K

2014-11-01

93

Antibody to gp41 MPER Alters Functional Properties of HIV-1 Env without Complete Neutralization  

PubMed Central

Human antibody 10E8 targets the conserved membrane proximal external region (MPER) of envelope glycoprotein (Env) subunit gp41 and neutralizes HIV-1 with exceptional potency. Remarkably, HIV-1 containing mutations that reportedly knockout 10E8 binding to linear MPER peptides are partially neutralized by 10E8, producing a local plateau in the dose response curve. Here, we found that virus partially neutralized by 10E8 becomes significantly less neutralization sensitive to various MPER antibodies and to soluble CD4 while becoming significantly more sensitive to antibodies and fusion inhibitors against the heptad repeats of gp41. Thus, 10E8 modulates sensitivity of Env to ligands both pre- and post-receptor engagement without complete neutralization. Partial neutralization by 10E8 was influenced at least in part by perturbing Env glycosylation. With unliganded Env, 10E8 bound with lower apparent affinity and lower subunit occupancy to MPER mutant compared to wild type trimers. However, 10E8 decreased functional stability of wild type Env while it had an opposite, stabilizing effect on MPER mutant Envs. Clade C isolates with natural MPER polymorphisms also showed partial neutralization by 10E8 with altered sensitivity to various gp41-targeted ligands. Our findings suggest a novel mechanism of virus neutralization by demonstrating how antibody binding to the base of a trimeric spike cross talks with adjacent subunits to modulate Env structure and function. The ability of an antibody to stabilize, destabilize, partially neutralize as well as alter neutralization sensitivity of a virion spike pre- and post-receptor engagement may have implications for immunotherapy and vaccine design. PMID:25058619

Kim, Arthur S.; Leaman, Daniel P.; Zwick, Michael B.

2014-01-01

94

Molecular identification of erythrocytic necrosis virus (ENV) from the blood of Pacific herring (Clupea pallasii)  

USGS Publications Warehouse

Viral erythrocytic necrosis (VEN) is a condition affecting the red blood cells of more than 20 species of marine and anadromous fishes in the North Atlantic and North Pacific Oceans. Among populations of Pacific herring (Clupea pallasii) on the west coast of North America the disease causes anemia and elevated mortality in periodic epizootics. Presently, VEN is diagnosed by observation of typical cytoplasmic inclusion bodies in stained blood smears from infected fish. The causative agent, erythrocytic necrosis virus (ENV), is unculturable and a presumed iridovirus by electron microscopy. In vivo amplification of the virus in pathogen-free laboratory stocks of Pacific herring with subsequent virus concentration, purification, DNA extraction, and high-throughput sequencing were used to obtain genomic ENV sequences. Fragments with the highest sequence identity to the family Iridoviridae were used to design four sets of ENV-specific polymerase chain reaction (PCR) primers. Testing of blood and tissue samples from experimentally and wild infected Pacific herring as well as DNA extracted from other amphibian and piscine iridoviruses verified the assays were specific to ENV with a limit of detection of 0.0003 ng. Preliminary phylogenetic analyses of a 1448 bp fragment of the putative DNA polymerase gene supported inclusion of ENV in a proposed sixth genus of the family Iridoviridae that contains other erythrocytic viruses from ectothermic hosts. This study provides the first molecular evidence of ENV's inclusion within the Iridoviridae family and offers conventional PCR assays as a means of rapidly surveying the ENV-status of wild and propagated Pacific herring stocks.

Emmenegger, Eveline J.; Glenn, Jolene A.; Winton, James R.; Batts, William N.; Gregg, Jacob L.; Hershberger, Paul K.

2014-01-01

95

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. PMID:17873075

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

2007-01-01

96

Direct evidence for intracellular anterograde co-transport of M-PMV Gag and Env on microtubules  

PubMed Central

The intracellular transport of Mason-Pfizer monkey virus (M-PMV) assembled capsids from the pericentriolar region to the plasma membrane (PM) requires trafficking of envelope glycoprotein (Env) to the assembly site via the recycling endosome. However, it is unclear if Env-containing vesicles play a direct role in trafficking capsids to the PM. Using live cell microscopy, we demonstrate, for the first time, anterograde co-transport of Gag and Env. Nocodazole disruption of microtubules had differential effects on Gag and Env trafficking, with pulse-chase assays showing a delayed release of Env-deficient virions. Particle tracking demonstrated an initial loss of linear movement of GFP-tagged capsids and mCherry-tagged Env, followed by renewed movement of Gag but not Env at 4 h post-treatment. Thus, while delayed capsid trafficking can occur in the absence of microtubules, efficient anterograde transport of capsids appears to be mediated by microtubule-associated Env-containing vesicles. PMID:24418544

Pereira, Lara E.; Clark, Jasmine; Grznarova, Petra; Wen, Xiaoyun; LaCasse, Rachel; Ruml, Tomas; Spearman, Paul

2014-01-01

97

Disease progression and evolution of the HIV-1 env gene in 24 infected infants  

E-print Network

Disease progression and evolution of the HIV-1 env gene in 24 infected infants§ Antonio Carvajal Available online 1 November 2007 Abstract We have studied the relationship between disease progression disease progression cycle from infection to AIDS. Specifically, we examined the temporal relationship

Posada, David

98

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

99

Fall 2013 ENVS Additions Area 3A Upper Division Natural Science  

E-print Network

and Environment (CRN 17996) [WEB] Area 3B Humanities Elective LA 421 (Godfrey) Landscape Photography (CRN 18172) Policy Elective EC 410 (Mastromonaco) Env Economics (CRN 16880) Sustainable Design and Practice Elective Sustainable Design and Practice ELECTIVE Area. This is due to the change of the former 3B Area "Design" which

100

Conservation Biology-BIOL 21200/ENVS 21200 Course Information and Policies  

E-print Network

Conservation Biology- BIOL 21200/ENVS 21200 Course Information and Policies General Info Time: Tu will periodically collaborate with members of the Conservation Psychology course to share perspectives in conservation biology, including status and trends, case studies, and theories in a multidisciplinary setting

101

ENV/NRES 467 / 667 Regional and Global Issues Spring 2005 Resource and Land Management  

E-print Network

ENV/NRES 467 / 667 Regional and Global Issues Spring 2005 Resource and Land Management Readings of Natural Resources Policy and Management. Yale University Press, New Haven, 372 p. GE170 .F68 2000 (on reserve in Life & Health Sciences Library) *Mullner SA, Hubert WA, Wesche TA (2001) Evolving paradigms

Nowak, Robert S.

102

Service Learning Travel Course ENV SCI 499: Tropical Conservation and Ecology in Costa Rica  

E-print Network

Service Learning Travel Course ENV SCI 499: Tropical Conservation and Ecology in Costa Rica National Park, Costa Rica for the first two weeks in January. Direct student involvement with park staff challenges. Located on Costa Rica's Pacific Coast, Carara National Park manages 4,700 hectares (11,600 acres

Dornbush, Mathew E.

103

LAW AND THE ENVIRONMENT ENVS 411/511 Summer 2014: July 21-August 13  

E-print Network

LAW AND THE ENVIRONMENT ENVS 411/511 Summer 2014: July 21-August 13 Mon./Tues./Wed./Thurs. 10:00-11:50 Columbia 142 Overview: This course provides students with an understanding of laws that regulate the environment as well as the skills to analyze and apply these laws to current issues. By the end of this course

104

Dean's RepoRt New Appointments  

E-print Network

Dean's RepoRt FY09 / 39 Aa Aa P New Appointments phYsiCian­huManitaRian is new gLoBaL heaLth Chai than Paul," said Dean Jeffrey Flier. "His scholarship and international work have made him one of investigation. Szostak's prize-winning work focused on the stability of chromosomes in yeast cells

Goodrich, Lisa V.

105

Tennessee Business and economic RepoRT  

E-print Network

Tennessee Business and economic RepoRT The sTaTe's economic ouTlook spRing 2011 #12;Matthew N By thE Center for Business and Economic Research College of Business Administration the University of tennessee Knoxville, tennessee Tennessee Business and economic ouTlook The sTaTe's economic ouTlook spring 2011 #12;ii

Tennessee, University of

106

INHIBITORY AND ENHANCING EFFECTS OF IFN-? AND IL4 ON SHIV KUREPLICATION IN RHESUS MACAQUE MACROPHAGES: CORRELATION BETWEEN TH 2CYTOKINES AND PRODUCTIVE INFECTION IN TISSUE MACROPHAGES DURING LATE-STAGE INFECTION  

Microsoft Academic Search

HIV-1 is dual-tropic for CD4+T lymphocytes and macrophages, but virus production in the macrophages becomes manifest only during late-stage infection, after CD4+T cell functions are lost, and when opportunistic pathogens begin to flourish. In this study, the SHIV\\/macaque model of HIV pathogenesis was used to assess the role of cytokines in regulating virus replication in the two cell types. We

Shilpa Buch; David Pinson; Christopher L. King; Ravi Raghavan; Yueping Hou; Zhuang Li; Istvan Adany; Andre Hicks; Francois Villinger; Anil Kumar; Opendra Narayan

2001-01-01

107

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

108

Highly complex neutralization determinants on a monophyletic lineage of newly transmitted subtype C HIV-1 Env clones from India  

PubMed Central

Little is known about the neutralization properties of HIV-1 in India to optimally design and test vaccines. For this reason, a functional Env clone was obtained from each of ten newly acquired, heterosexually transmitted HIV-1 infections in Pune, Maharashtra. These clones formed a phylogenetically distinct genetic lineage within subtype C. As Env-pseudotyped viruses the clones were mostly resistant to IgG1b12, 2G12 and 2F5 but all were sensitive to 4E10. When compared to a large multi-subtype panel of Env-pseudotyped viruses (subtypes B, C and CRF02_AG) in neutralization assays with a multi-subtype panel of HIV-1-positive plasma samples, the Indian Envs were remarkably complex antigenically. With the exception of the Indian Envs, results of a hierarchical clustering analysis showed a strong subtype association with the patterns of neutralization susceptibility. From these patterns we were able to identify 19 neutralization cluster-associated amino acid signatures in gp120 and 14 signatures in the ectodomain and cytoplasmic tail of gp41. We conclude that newly transmitted Indian Envs are antigenically complex in spite of close genetic similarity. Delineation of neutralization-associated amino acid signatures provides a deeper understanding of the antigenic structure of HIV-1 Env. PMID:19167740

Kulkarni, Smita S.; Lapedes, Alan; Tang, Haili; Gnanakaran, S.; Daniels, Marcus G.; Zhang, Ming; Bhattacharya, Tanmoy; Li, Ming; Polonis, Victoria R.; McCutchan, Francine E.; Morris, Lynn; Ellenberger, Dennis; Butera, Salvatore T.; Bollinger, Robert C.; Korber, Bette T.; Paranjape, Ramesh S.; Montefiori, David C.

2009-01-01

109

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

110

Substitution of the transmembrane domain of Vpu in simian-human immunodeficiency virus (SHIV{sub KU1bMC33}) 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  

SciTech Connect

The Vpu protein of human immunodeficiency virus type 1 has been shown to shunt the CD4 receptor molecule to the proteasome for degradation and to enhance virus release from infected cells. The exact mechanism by which the Vpu protein enhances virus release is currently unknown but some investigators have shown that this function is associated with the transmembrane domain and potential ion channel properties. In this study, we determined if the transmembrane domain of Vpu could be functionally substituted with that of the prototypical viroporin, the M2 protein of influenza A virus. We constructed chimeric vpu gene in which the transmembrane domain of Vpu was replaced with that of the M2 protein of influenza. This chimeric vpu gene was substituted for the vpu gene in the genome of a pathogenic simian human immunodeficiency virus, SHIV{sub KU-1bMC33}. The resulting virus, SHIV{sub M2}, synthesized a Vpu protein that had a slightly different M{sub r} compared to the parental SHIV{sub KU-1bMC33}, reflecting the different sizes of the two Vpu proteins. The SHIV{sub M2} was shown to replicate with slightly reduced kinetics when compared to the parental SHIV{sub KU-1bMC33} but electron microscopy revealed that the site of maturation was similar to the parental virus SHIV{sub KU1bMC33}. We show that the replication and spread of SHIV{sub M2} could be blocked with the antiviral drug rimantadine, which is known to target the M2 ion channel. Our results indicate a dose dependent inhibition of SHIV{sub M2} with 100 {mu}M rimantadine resulting in a >95% decrease in p27 released into the culture medium. Rimantadine did not affect the replication of the parental SHIV{sub KU-1bMC33}. Examination of SHIV{sub M2}-infected cells treated with 50 {mu}M rimantadine revealed numerous viral particles associated with the cell plasma membrane and within intracytoplasmic vesicles, which is similar to HIV-1 mutants lacking a functional vpu. To determine if SHIV{sub M2} was as pathogenic as the parental SHIV{sub KU-1bMC33} virus, two pig-tailed macaques were inoculated and followed for up to 8 months. Both pig-tailed macaques developed severe CD4{sup +} T cell loss within 1 month of inoculation, high viral loads, and histological lesions consistent with lymphoid depletion similar to the parental SHIV{sub KU-1bMC33}. Taken together, these results indicate for the first time that the TM domain of the Vpu protein can be functionally substituted with the TM of M2 of influenza A virus, and shows that compounds that target the TM domain of Vpu protein of HIV-1 could serve as novel 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, Melissa L. [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); Gomez, Lisa M. [Department of Anatomy and Cell Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160 (United States); Fegley, Barbara [Department of Anatomy and Cell Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160 (United States); Mulcahy, Ellyn R. [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); Culley, Nathan [Laboratory Animal Resources, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160 (United States); Pinson, David M. [Department of Laboratory Medicine and Pathology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160 (United States); Nothnick, Warren [Department of Obstetrics and Gynecology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160 (United States); Powers, Michael F.; Wong, Scott W. [Vaccine and Gene Therapy Institute, Oregon National Primate Research Center, Oregon University for the Health Sciences, Beaverton, OR 97003 (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

2006-01-20

111

A Gag-Pol\\/Env-Rev SIV239 DNA vaccine improves CD4 counts, and reduce viral loads after pathogenic intrarectal SIV mac251 challenge in Rhesus Macaques  

Microsoft Academic Search

DNA vaccines are an important vaccine approach for many infectious diseases including human immunodeficiency virus (HIV). Recently, there have been exciting results reported for plasmid vaccination in pathogenic SHIV model systems. In these studies, plasmid vaccines supplemented by IL-2 Ig cytokine gene adjuvants or boosted by recombinant MVA vectors expressing relevant SIV and HIV antigens prevented CD4+ T-cell loss and

Karuppiah Muthumani; Mark Bagarazzi; Dan Conway; Daniel S. Hwang; Kelledy Manson; Richard Ciccarelli; Zimmra Israel; David C. Montefiori; Kenneth Ugen; Nancy Miller; Jong Kim; Jean Boyer; David B. Weiner

2003-01-01

112

Saccharomyces cerevisiae Env7 Is a Novel Serine/Threonine Kinase 16-Related Protein Kinase and Negatively Regulates Organelle Fusion at the Lysosomal Vacuole  

PubMed Central

Membrane fusion depends on conserved components and is responsible for organelle biogenesis and vesicular trafficking. Yeast vacuoles are dynamic structures analogous to mammalian lysosomes. We report here that yeast Env7 is a novel palmitoylated protein kinase ortholog that negatively regulates vacuolar membrane fusion. Microscopic and biochemical studies confirmed the localization of tagged Env7 at the vacuolar membrane and implicated membrane association via the palmitoylation of its N-terminal Cys13 to -15. In vitro kinase assays established Env7 as a protein kinase. Site-directed mutagenesis of the Env7 alanine-proline-glutamic acid (APE) motif Glu269 to alanine results in an unstable kinase-dead allele that is stabilized and redistributed to the detergent-resistant fraction by interruption of the proteasome system in vivo. Palmitoylation-deficient Env7C13-15S is also kinase dead and mislocalizes to the cytoplasm. Microscopy studies established that env7? is defective in maintaining fragmented vacuoles during hyperosmotic response and in buds. ENV7 function is not redundant with a similar role of vacuolar membrane kinase Yck3, as the two do not share a substrate, and ENV7 is not a suppressor of yck3?. Bayesian phylogenetic analyses strongly support ENV7 as an ortholog of the gene encoding human STK16, a Golgi apparatus protein kinase with undefined function. We propose that Env7 function in fusion/fission dynamics may be conserved within the endomembrane system. PMID:23166297

Manandhar, Surya P.; Ricarte, Florante; Cocca, Stephanie M.

2013-01-01

113

HIV Sequence Variation Associated With env Antisense Adoptive T-cell Therapy in the hNSG Mouse Model  

Microsoft Academic Search

The first use of lentiviral vectors in humans involved transduction of mature T-cells with an human immunodeficiency virus (HIV)–derived env antisense (envAS) vector to protect cells from HIV infection. In that study, only a minority of the patient T-cell population could be gene-modified, raising the question of whether the altered cells could affect replicating HIV populations. We investigated this using

Rithun Mukherjee; Gabriela Plesa; Scott Sherrill-Mix; Max W Richardson; James L Riley; Frederic D Bushman

2010-01-01

114

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 PMID:7143566

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

1982-01-01

115

Influence of Random Genetic Drift on Human Immunodeficiency Virus Type 1 env Evolution During Chronic Infection  

Microsoft Academic Search

Human immunodeficiency virus type 1 (HIV-1) has high replication and mutation rates that generate large census populations and high levels of genetic variation. We examined the roles of natural selection, population growth, random genetic drift, and recombination in shaping the variation in 1509 C2-V5 env sequences derived from nine men with chronic HIV-1 infection. These sequences were obtained from clinical

Daniel Shriner; Raj Shankarappa; Mark A. Jensen; David C. Nickle; John E. Mittler; Joseph B. Margolick; James I. Mullins

2004-01-01

116

Aerobic Biodegradation of N-Nitrosodimethylamine by the Propanotroph Rhodococcus ruber ENV425?  

PubMed Central

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 [14C]NDMA to 14CO2, 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.

2009-01-01

117

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

2009-08-01

118

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. PMID:23202829

Chigor, Vincent Nnamdigadi; Okoh, Anthony Ifeanyi

2012-01-01

119

RectoR's RepoRt 4 RectoR's RepoRt 2010-2011  

E-print Network

for our social, health and education networks, whilst acting as a fertile business and innovation have the great value of appeal- ing to all players involved in the university mission. In addition that this project is completed in its entirety. #12;6 RectoR's RepoRt 2010-2011 Philosophy, Oncology, Music, Music

Charette, André

120

Infection of monkeys by simian-human immunodeficiency viruses with transmitted/founder clade C HIV-1 envelopes.  

PubMed

Simian-human immunodeficiency viruses (SHIVs) that mirror natural transmitted/founder (T/F) viruses in man are needed for evaluation of HIV-1 vaccine candidates in nonhuman primates. Currently available SHIVs contain HIV-1 env genes from chronically-infected individuals and do not reflect the characteristics of biologically relevant HIV-1 strains that mediate human transmission. We chose to develop clade C SHIVs, as clade C is the major infecting subtype of HIV-1 in the world. We constructed 10 clade C SHIVs expressing Env proteins from T/F viruses. Three of these ten clade C SHIVs (SHIV KB9 C3, SHIV KB9 C4 and SHIV KB9 C5) replicated in naïve rhesus monkeys. These three SHIVs are mucosally transmissible and are neutralized by sCD4 and several HIV-1 broadly neutralizing antibodies. However, like natural T/F viruses, they exhibit low Env reactivity and a Tier 2 neutralization sensitivity. Of note, none of the clade C T/F SHIVs elicited detectable autologous neutralizing antibodies in the infected monkeys, even though antibodies that neutralized a heterologous Tier 1 HIV-1 were generated. Challenge with these three new clade C SHIVs will provide biologically relevant tests for vaccine protection in rhesus macaques. PMID:25462344

Asmal, Mohammed; Luedemann, Corinne; Lavine, Christy L; Mach, Linh V; Balachandran, Harikrishnan; Brinkley, Christie; Denny, Thomas N; Lewis, Mark G; Anderson, Hanne; Pal, Ranajit; Sok, Devin; Le, Khoa; Pauthner, Matthias; Hahn, Beatrice H; Shaw, George M; Seaman, Michael S; Letvin, Norman L; Burton, Dennis R; Sodroski, Joseph G; Haynes, Barton F; Santra, Sampa

2015-01-15

121

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. PMID:23593376

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

2013-01-01

122

Long-Term Central and Effector SHIV-Specific Memory T Cell Responses Elicited after a Single Immunization with a Novel Lentivector DNA Vaccine  

PubMed Central

Prevention of HIV acquisition and replication requires long lasting and effective immunity. Given the state of HIV vaccine development, innovative vectors and immunization strategies are urgently needed to generate safe and efficacious HIV vaccines. Here, we developed a novel lentivirus-based DNA vector that does not integrate in the host genome and undergoes a single-cycle of replication. Viral proteins are constitutively expressed under the control of Tat-independent LTR promoter from goat lentivirus. We immunized six macaques once only with CAL-SHIV-IN? DNA using combined intramuscular and intradermal injections plus electroporation. Antigen-specific T cell responses were monitored for 47 weeks post-immunization (PI). PBMCs were assessed directly ex vivo or after 6 and 12 days of in vitro culture using antigenic and/or homeostatic proliferation. IFN-? ELISPOT was used to measure immediate cytokine secretion from antigen specific effector cells and from memory precursors with high proliferative capacity (PHPC). The memory phenotype and functions (proliferation, cytokine expression, lytic content) of specific T cells were tested using multiparametric FACS-based assays. All immunized macaques developed lasting peripheral CD8+ and CD4+ T cell responses mainly against Gag and Nef antigens. During the primary expansion phase, immediate effector cells as well as increasing numbers of proliferating cells with limited effector functions were detected which expressed markers of effector (EM) and central (CM) memory phenotypes. These responses contracted but then reemerged later in absence of antigen boost. Strong PHPC responses comprising vaccine-specific CM and EM T cells that readily expanded and acquired immediate effector functions were detected at 40/47 weeks PI. Altogether, our study demonstrated that a single immunization with a replication-limited DNA vaccine elicited persistent vaccine-specific CM and EM CD8+ and CD4+ T cells with immediate and readily inducible effector functions, in the absence of ongoing antigen expression. PMID:25337803

Arrode-Brusés, Géraldine; Moussa, Maha; Baccard-Longere, Monique; Villinger, François; Chebloune, Yahia

2014-01-01

123

Instruments of RT-2 experiment onboard CORONAS-PHOTON and their test and evaluation I: ground calibration of RT-2/S and RT-2/G  

NASA Astrophysics Data System (ADS)

Phoswich detectors (RT-2/S & RT-2/G) are major scientific payloads of the RT-2 Experiment onboard the CORONAS-PHOTON mission, which was launched into a polar Low Earth Orbit of around 550 km on 2009 January 30. These RT-2 instruments are designed and developed to observe solar flares in hard X-rays and to understand the energy transport processes associated with these flares. Apart from this, these instruments are capable of observing Gamma Ray Bursts (GRBs) and Cosmic diffuse X-ray background (CDXRB). Both detectors consist of identical NaI(Tl) and CsI(Na) scintillation crystals in a Phoswich combination, having the same diameter (116 mm) but different thicknesses. The normal working energy range is from 15 keV to 150 keV, but may be extendable up to ~1 MeV. In this paper, we present the RT-2/S and RT-2/G instruments and discuss their testing and calibration results. We used different radio-active sources to calibrate both detectors. The radio-active source 57Co (122 keV) is used for onboard calibration of both instruments. During its lifetime (˜3-5 years), RT-2 is expected to cover the peak of the 24th solar cycle.

Debnath, Dipak; Nandi, Anuj; Rao, A. R.; Malkar, J. P.; Hingar, M. K.; Kotoch, T. B.; Sreekumar, S.; Madhav, V. P.; Chakrabarti, Sandip K.

2011-02-01

124

Involvement of EnvZ-OmpR two-component system in virulence control of Escherichia coli in Drosophila melanogaster.  

PubMed

Bacteria adapt to environmental changes by altering gene expression patterns with the aid of signal transduction machinery called the two-component regulatory system (TCS), which consists of two protein components, a sensor kinase and response regulator. We examined the role of the TCS in bacterial adaptation to host environments using genetically tractable organisms, Escherichia coli as a pathogen and Drosophila melanogaster as a host. To determine the strength of the transcription promoters of TCS-encoding genes in Drosophila, adult flies were infected with a series of E. coli strains that expressed GFP driven by the promoters of genes coding for 27 sensor kinases and 32 response regulators of E. coli TCS followed by the measurement of fluorescence intensities. We further analyzed EnvZ-OmpR among the TCS encoded by genes having stronger promoters. A mutant E. coli strain lacking EnvZ-OmpR had a higher pathogenic effect on fly survival than that of the parental strain, and the forced expression of envZ and ompR in the mutant strain lowered its pathogenicity. The lack of EnvZ-OmpR did not affect the growth of E. coli in a culture medium as well as the level of colony-formable E. coli in flies. An increase in E. coli virulence with the loss of EnvZ-OmpR was observed in flies defective in an Imd-mediated humoral response, and both the mutant and parental strains were equally engulfed by hemocytes in vitro. These results suggest that EnvZ-OmpR mitigated the virulence of E. coli in Drosophila by a mechanism not accompanied by a change of bacterial burden. This behavior of E. coli is most likely a bacterial strategy to achieve persistent infection. PMID:23886953

Pukklay, Pattraporn; Nakanishi, Yoshinobu; Nitta, Mao; Yamamoto, Kaneyoshi; Ishihama, Akira; Shiratsuchi, Akiko

2013-08-23

125

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 Central

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

2011-01-01

126

The ENVS works by using the principle of stereo disparity. Just as our eyes capture two slightly different images and our brain combines  

E-print Network

of the environment in real time effectively enables the user to navigate the environment without use of the eyesThe ENVS works by using the principle of stereo disparity. Just as our eyes capture two slightly in the ENVS captures two images and the processor computes a depth map by estimating the disparity between

Ward, Koren

127

Antigenic and 3D structural characterization of soluble X4 and hybrid X4-R5 HIV-1 Env trimers  

PubMed Central

Background HIV-1 is decorated with trimeric glycoprotein spikes that enable infection by engaging CD4 and a chemokine coreceptor, either CCR5 or CXCR4. The variable loop 3 (V3) of the HIV-1 envelope protein (Env) is the main determinant for coreceptor usage. The predominant CCR5 using (R5) HIV-1 Env has been intensively studied in function and structure, whereas the trimeric architecture of the less frequent, but more cytopathic CXCR4 using (X4) HIV-1 Env is largely unknown, as are the consequences of sequence changes in and near V3 on antigenicity and trimeric Env structure. Results Soluble trimeric gp140 Env constructs were used as immunogenic mimics of the native spikes to analyze their antigenic properties in the context of their overall 3D structure. We generated soluble, uncleaved, gp140 trimers from a prototypic T-cell line-adapted (TCLA) X4 HIV-1 strain (NL4-3) and a hybrid (NL4-3/ADA), in which the V3 spanning region was substituted with that from the primary R5 isolate ADA. Compared to an ADA (R5) gp140, the NL4-3 (X4) construct revealed an overall higher antibody accessibility, which was most pronounced for the CD4 binding site (CD4bs), but also observed for mAbs against CD4 induced (CD4i) epitopes and gp41 mAbs. V3 mAbs showed significant binding differences to the three constructs, which were refined by SPR analysis. Of interest, the NL4-3/ADA construct with the hybrid NL4-3/ADA CD4bs showed impaired CD4 and CD4bs mAb reactivity despite the presence of the essential elements of the CD4bs epitope. We obtained 3D reconstructions of the NL4-3 and the NL4-3/ADA gp140 trimers via electron microscopy and single particle analysis, which indicates that both constructs inherit a propeller-like architecture. The first 3D reconstruction of an Env construct from an X4 TCLA HIV-1 strain reveals an open conformation, in contrast to recently published more closed structures from R5 Env. Exchanging the X4 V3 spanning region for that of R5 ADA did not alter the open Env architecture as deduced from its very similar 3D reconstruction. Conclusions 3D EM analysis showed an apparent open trimer configuration of X4 NL4-3 gp140 that is not modified by exchanging the V3 spanning region for R5 ADA. PMID:24884925

2014-01-01

128

Paleovirology of ‘syncytins’, retroviral env genes exapted for a role in placentation  

PubMed Central

The development of the emerging field of ‘paleovirology’ allows biologists to reconstruct the evolutionary history of fossil endogenous retroviral sequences integrated within the genome of living organisms and has led to the retrieval of conserved, ancient retroviral genes ‘exapted’ by ancestral hosts to fulfil essential physiological roles, syncytin genes being undoubtedly among the most remarkable examples of such a phenomenon. Indeed, syncytins are ‘new’ genes encoding proteins derived from the envelope protein of endogenous retroviral elements that have been captured and domesticated on multiple occasions and independently in diverse mammalian species, through a process of convergent evolution. Knockout of syncytin genes in mice provided evidence for their absolute requirement for placenta development and embryo survival, via formation by cell–cell fusion of syncytial cell layers at the fetal–maternal interface. These genes of exogenous origin, acquired ‘by chance’ and yet still ‘necessary’ to carry out a basic function in placental mammals, may have been pivotal in the emergence of mammalian ancestors with a placenta from egg-laying animals via the capture of a founding retroviral env gene, subsequently replaced in the diverse mammalian lineages by new env-derived syncytin genes, each providing its host with a positive selective advantage. PMID:23938756

Lavialle, Christian; Cornelis, Guillaume; Dupressoir, Anne; Esnault, Cécile; Heidmann, Odile; Vernochet, Cécile; Heidmann, Thierry

2013-01-01

129

RT systems overview JS sep 2006 simons@astron.nl  

E-print Network

Antenna properties Noise characterisation of a radio telescope #12;RT systems overview JS sep 2006 overview JS sep 2006 EExamples of radio telescopesxamples of radio telescopes Yagi, horn receiver Single telescopesxamples radio telescopes -- many small antennasmany small antennas #12;RT systems overview JS sep 2006

Peletier, Reynier

130

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

131

Phylogenetic and Genetic Analysis of Feline Immunodeficiency Virus gag, pol, and env Genes from Domestic Cats Undergoing Nucleoside Reverse Transcriptase Inhibitor Treatment or Treatment-Naïve Cats in Rio de Janeiro, Brazil? †  

PubMed Central

Feline immunodeficiency virus (FIV) is the Lentivirus responsible for an immunodeficiency-like disease in domestic cats (Felis catus). FIV is divided into five phylogenetic subtypes (A, B, C, D, and E), based on genetic diversity. Knowledge of the geographical distribution of subtypes is relevant for understanding different disease progressions and for vaccine development. In this study, viral sequences of 26 infected cats from Rio de Janeiro, 8 undergoing treatment with zidovudine (AZT) for at least 5 years, were successfully amplified from blood specimens. gag capsid (CA), pol reverse transcriptase (RT), and env gp120 (V3-V4) regions were analyzed to determine subtypes and to evaluate potential mutations related to antiretroviral drug resistance among treated cats. Subtyping based on phylogenetic analysis was performed by the neighbor-joining and maximum likelihood methods. All of the sequences clustered with subtype B in the three regions, exhibiting low genetic variability. Additionally, we found evidence that the same virus is circulating in animals in close contact. The analysis of FIV RT sequences identified two new putative mutations related to drug resistance located in the RT “finger” domain, which has 60% identity to human immunodeficiency virus (HIV) sequence. Amino acid change K?R at codons 64 and 69 was found in 25% and 37.5% of the treated animals, respectively. These signatures were comparable to K65R and K70R thymidine-associated mutations found in the HIV-1 HXB2 counterpart. This finding strongly suggests a position correlation between the mutations found in FIV and the K65R and K70R substitutions from drug-resistant HIV-1 strains. PMID:18550661

Martins, Angelica N.; Medeiros, Sheila O.; Simonetti, Jose P.; Schatzmayr, Hermann G.; Tanuri, Amílcar; Brindeiro, Rodrigo M.

2008-01-01

132

ENV/ERS 467/667: Regional and Global Issues in Environmental and Natural Resource Sciences Spring 2004  

E-print Network

pollution 19 Metals 24 Guest lecturer 26 Quiz Nevada Env. Commission, Miller Mar 2 4 9 11 BN #3 due Invasive examples of case studies as well as individual and group investigations to: 1. identify critical scientific%). Content includes items such as careful consideration of data and results, critical examination of problems

Nowak, Robert S.

133

Specific Interaction of a Novel Foamy Virus Env Leader Protein with the N-Terminal Gag Domain  

PubMed Central

Cryoelectron micrographs of purified human foamy virus (HFV) and feline foamy virus (FFV) particles revealed distinct radial arrangements of Gag proteins. The capsids were surrounded by an internal Gag layer that in turn was surrounded by, and separated from, the viral membrane. The width of this layer was about 8 nm for HFV and 3.8 nm for FFV. This difference in width is assumed to reflect the different sizes of the HFV and FFV MA domains: the HFV MA domain is about 130 residues longer than that of FFV. The distances between the MA layer and the edge of the capsid were identical in different particle classes. In contrast, only particles with a distended envelope displayed an invariant, close spacing between the MA layer and the Env membrane which was absent in the majority of particles. This indicates a specific interaction between MA and Env at an unknown step of morphogenesis. This observation was supported by surface plasmon resonance studies. The purified N-terminal domain of FFV Gag specifically interacted with synthetic peptides and a defined protein domain derived from the N-terminal Env leader protein. The specificity of this interaction was demonstrated by using peptides varying in the conserved Trp residues that are known to be required for HFV budding. The interaction with Gag required residues within the novel virion-associated FFV Env leader protein of about 16.5 kDa. PMID:11483744

Wilk, Thomas; Geiselhart, Verena; Frech, Matthias; Fuller, Stephen D.; Flügel, Rolf M.; Löchelt, Martin

2001-01-01

134

Relationship of the env genes and the endonuclease domain of the pol genes of simian foamy virus type 1 and human foamy virus.  

PubMed Central

We have molecularly cloned and sequenced a portion of the simian foamy virus type 1 (SFV-1); open reading frames representing the endonuclease domain of the polymerase (pol) and the envelope (env) genes were identified by comparison with the human foamy virus (HFV). Unlike the HFV genomic organization, the SFV-1 pol gene overlaps the env gene; thus, the open reading frames reported for HFV between pol and env is not present in SFV-1. Comparisons of predicted amino acid sequences of HFV and SFV-1 reveal that the endonuclease domains of the pol genes are about 84% related. The region predicted to encode the SFV-1 extracellular env domain is 569 codons; SFV-1 and HFV have 64% amino acid similarity in this env domain. The predicted hydrophobic transmembrane env proteins of both HFV and SFV-1 show about 73% similarity. A total of 16 potential glycosylation sites are found in SFV-1 env, and 15 are found in HFV; 11 are shared. SFV-1 has 25 cysteine residues, and HFV has 23 residues; all 23 cysteine residues of HFV are conserved in SFV-1. This sequence analysis reveals that the human and simian foamy viruses are highly related. PMID:2152825

Mergia, A; Shaw, K E; Lackner, J E; Luciw, P A

1990-01-01

135

Macrophage entry mediated by HIV Envs from brain and lymphoid tissues is determined by the capacity to use low CD4 levels and overall efficiency of fusion  

SciTech Connect

HIV infects macrophages and microglia in the central nervous system (CNS), which express lower levels of CD4 than CD4+ T cells in peripheral blood. To investigate mechanisms of HIV neurotropism, full-length env genes were cloned from autopsy brain and lymphoid tissues from 4 AIDS patients with HIV-associated dementia (HAD). Characterization of 55 functional Env clones demonstrated that Envs with reduced dependence on CD4 for fusion and viral entry are more frequent in brain compared to lymphoid tissue. Envs that mediated efficient entry into macrophages were frequent in brain but were also present in lymphoid tissue. For most Envs, entry into macrophages correlated with overall fusion activity at all levels of CD4 and CCR5. gp160 nucleotide sequences were compartmentalized in brain versus lymphoid tissue within each patient. Proline at position 308 in the V3 loop of gp120 was associated with brain compartmentalization in 3 patients, but mutagenesis studies suggested that P308 alone does not contribute to reduced CD4 dependence or macrophage-tropism. These results suggest that HIV adaptation to replicate in the CNS selects for Envs with reduced CD4 dependence and increased fusion activity. Macrophage-tropic Envs are frequent in brain but are also present in lymphoid tissues of AIDS patients with HAD, and entry into macrophages in the CNS and other tissues is dependent on the ability to use low receptor levels and overall efficiency of fusion.

Thomas, Elaine R. [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA (United States); Dunfee, Rebecca L. [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA (United States); Stanton, Jennifer [Northwestern University Medical School, Chicago, IL (United States); Bogdan, Derek [Northwestern University Medical School, Chicago, IL (United States); Taylor, Joann [Northwestern University Medical School, Chicago, IL (United States); Kunstman, Kevin [Northwestern University Medical School, Chicago, IL (United States); Bell, Jeanne E. [Department of Pathology, Western General Hospital, University of Edinburgh, Edinburgh (United Kingdom); Wolinsky, Steven M. [Northwestern University Medical School, Chicago, IL (United States); Gabuzda, Dana [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA (United States) and Department of Neurology, Harvard Medical School, Boston, MA (United States)]. E-mail: dana_gabuzda@dfci.harvard.edu

2007-03-30

136

HIV-1 Env-Specific Memory and Germinal Center B Cells in C57BL/6 Mice  

PubMed Central

Continued efforts to define the immunogenic properties of the HIV-1 envelope glycoproteins (Env) are needed to elicit effective antibody (Ab) responses by vaccination. HIV-1 is a highly neutralization-resistant virus due to conformational and glycan shielding of conserved Ab determinants on the virus spike. Elicitation of broadly neutralizing Abs that bind poorly accessible epitope regions on Env is therefore extremely challenging and will likely require selective targeting of specific sub-determinants. To evaluate such approaches there is a pressing need for in vivo studies in both large and small animals, including mice. Currently, most mouse immunization studies are performed in the BALB/c strain; however, the C57BL/6 strain offers improved possibilities for mechanistic studies due to the availability of numerous knock-out strains on this genetic background. Here, we compared Env immunogenicity in BALB/c and C57BL/6 mice and found that the magnitude of the antigen-specific response was somewhat lower in C57BL/6 than in BALB/c mice by ELISA but not significantly different by B cell ELISpot measurements. We then established protocols for the isolation of single Env-specific memory B cells and germinal center (GC) B cells from immunized C57BL/6 mice to facilitate future studies of the elicited response at the monoclonal Ab level. We propose that these protocols can be used to gain an improved understanding of the early recruitment of Env-specific B cells to the GC as well as the archiving of such responses in the memory B cell pool following immunization. PMID:25198199

Soldemo, Martina; Pedersen, Gabriel K.; Hedestam, Gunilla B. Karlsson

2014-01-01

137

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

138

Unique N-Linked Glycosylation of CasBrE Env Influences Its Stability, Processing, and Viral Infectivity but Not Its Neurotoxicity  

PubMed Central

The envelope protein (Env) from the CasBrE murine leukemia virus (MLV) can cause acute spongiform neurodegeneration analogous to that induced by prions. Upon central nervous system (CNS) infection, Env is expressed as multiple isoforms owing to differential asparagine (N)-linked glycosylation. Because N-glycosylation can affect protein folding, stability, and quality control, we explored whether unique CasBrE Env glycosylation features could influence neurovirulence. CasBrE Env possesses 6/8 consensus MLV glycosylation sites (gs) but is missing gs3 and gs5 and contains a putative site (gs*). Twenty-nine mutants were generated by modifying these three sites, individually or in combination, to mimic the amino acid sequence in the nonneurovirulent Friend 57 MLV. Three basic viral phenotypes were observed: replication defective (dead; titer < 1 focus-forming unit [FFU]/ml), replication compromised (RC) (titer = 102 to 105 FFU/ml); and wild-type-like (WTL) (titer > 105 FFU/ml). Env protein was undetectable in dead mutants, while RC and WTL mutants showed variations in Env expression, processing, virus incorporation, virus entry, and virus spread. The newly introduced gs3 and gs5 sites were glycosylated, whereas gs* was not. Six WTL mutants tested in mice showed no clear attenuation in disease onset or severity versus controls. Furthermore, three RC viruses tested by neural stem cell (NSC)-mediated brainstem dissemination also induced acute spongiosis. Thus, while unique N-glycosylation affected structural features of Env involved in protein stability, proteolytic processing, and virus assembly and entry, these changes had minimal impact on CasBrE Env neurotoxicity. These findings suggest that the Env protein domains responsible for spongiogenesis represent highly stable elements upon which the more variable viral functional domains have evolved. PMID:23698308

Renszel, Krystal M.; Traister, Russell S.

2013-01-01

139

Predicting Gene Structures from Multiple RT-PCR Tests  

NASA Astrophysics Data System (ADS)

It has been demonstrated that the use of additional information such as ESTs and protein homology can significantly improve accuracy of gene prediction. However, many sources of external information are still being omitted from consideration. Here, we investigate the use of product lengths from RT-PCR experiments in gene finding. We present hardness results and practical algorithms for several variants of the problem and apply our methods to a real RT-PCR data set in the Drosophila genome. We conclude that the use of RT-PCR data can improve the sensitivity of gene prediction and locate novel splicing variants.

Ková?, Jakub; Vina?, Tomáš; Brejová, Bro?a

140

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. PMID:2824857

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

1988-01-01

141

Design of lipid nanocapsule delivery vehicles for multivalent display of recombinant Env trimers in HIV vaccination.  

PubMed

Immunization strategies that elicit antibodies capable of neutralizing diverse virus strains will likely be an important part of a successful vaccine against HIV. However, strategies to promote robust humoral responses against the native intact HIV envelope trimer structure are lacking. We recently developed chemically cross-linked lipid nanocapsules as carriers of molecular adjuvants and encapsulated or surface-displayed antigens, which promoted follicular helper T-cell responses and elicited high-avidity, durable antibody responses to a candidate malaria antigen. To apply this system to the delivery of HIV antigens, Env gp140 trimers with terminal his-tags (gp140T-his) were anchored to the surface of lipid nanocapsules via Ni-NTA-functionalized lipids. Initial experiments revealed that the large (409 kDa), heavily glycosylated trimers were capable of extracting fluid phase lipids from the membranes of nanocapsules. Thus, liquid-ordered and/or gel-phase lipid compositions were required to stably anchor trimers to the particle membranes. Trimer-loaded nanocapsules combined with the clinically relevant adjuvant monophosphoryl lipid A primed high-titer antibody responses in mice at antigen doses ranging from 5 ?g to as low as 100 ng, whereas titers dropped more than 50-fold over the same dose range when soluble trimer was mixed with a strong oil-in-water adjuvant comparator. Nanocapsule immunization also broadened the number of distinct epitopes on the HIV trimer recognized by the antibody response. These results suggest that nanocapsules displaying HIV trimers in an oriented, multivalent presentation can promote key aspects of the humoral response against Env immunogens. PMID:25020048

Pejawar-Gaddy, Sharmila; Kovacs, James M; Barouch, Dan H; Chen, Bing; Irvine, Darrell J

2014-08-20

142

C tr ti N R rt Construction News Report  

E-print Network

Cunz Hall C tr ti N R rt Renovation Construction News Report August 2010 Bid Package 301 Site Work. · Started primary underground feed. · Completed lay out and excavation of primary / telecom duct b k Goin

143

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

144

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

145

[Thrombolysis of acute arterial occlusion with rt PA].  

PubMed

The use of thrombolytic agents to treat peripheral arterial occlusions is a new method. There have been clinical trials with Streptokinase, Urokinase and rt-PA (recombinant tissue plasminogen activator). Despite its advantages, information about complications caused by the use of rt-PA and about its place in treatment is still not complete. And there are not enough studies that are made to form a safe protocol for the use of rt-PA in the treatment of acute peripheral arterial occlusions. The aim of this study was to establish a dose range for rt-PA and to follow the patients with a protocol during and after thrombolysis. Between May 1999 to January 2000, 14 patients with symptoms of pain, poikilothermia, cyanosis and loss of function came to Istanbul Medical Faculty Emergency Surgery Unit. Bolus injection of 5 mgr of rt-PA was followed by 15 minutes of interval. The extent of thrombolysis was checked by angiography and then bolus injection of 5 mgr of rt-PA was repeated. After angiographic control, patients having insufficient thrombolysis, received 0.05 mgr/kg/hour of infusion for 12 hours. At the end of 12 hours, thrombolytic treatment ended with a control angiography. A thromboembolectomy operation was made to patients still having an occlusion after thrombolysis. On the other hand, to avoid re-occlusions, all of the patients received 1.5 mgr/kg/day low molecular weight heparin (enoxyparine). PMID:11705216

Kurto?lu, M; Granit, V; Necefli, A; Kurto?lu, M; Gülo?lu, R

2001-07-01

146

Global Panel of HIV-1 Env Reference Strains for Standardized Assessments of Vaccine-Elicited Neutralizing Antibodies  

PubMed Central

ABSTRACT Standardized assessments of HIV-1 vaccine-elicited neutralizing antibody responses are complicated by the genetic and antigenic variability of the viral envelope glycoproteins (Envs). To address these issues, suitable reference strains are needed that are representative of the global epidemic. Several panels have been recommended previously, but no clear answers have been available on how many and which strains are best suited for this purpose. We used a statistical model selection method to identify a global panel of reference Env clones from among 219 Env-pseudotyped viruses assayed in TZM-bl cells with sera from 205 HIV-1-infected individuals. The Envs and sera were sampled globally from diverse geographic locations and represented all major genetic subtypes and circulating recombinant forms of the virus. Assays with a panel size of only nine viruses adequately represented the spectrum of HIV-1 serum neutralizing activity seen with the larger panel of 219 viruses. An optimal panel of nine viruses was selected and augmented with three additional viruses for greater genetic and antigenic coverage. The spectrum of HIV-1 serum neutralizing activity seen with the final 12-virus panel closely approximated the activity seen with subtype-matched viruses. Moreover, the final panel was highly sensitive for detection of many of the known broadly neutralizing antibodies. For broader assay applications, all 12 Env clones were converted to infectious molecular clones using a proviral backbone carrying a Renilla luciferase reporter gene (Env.IMC.LucR viruses). This global panel should facilitate highly standardized assessments of vaccine-elicited neutralizing antibodies across multiple HIV-1 vaccine platforms in different parts of the world. IMPORTANCE An effective HIV-1 vaccine will need to overcome the extraordinary genetic variability of the virus, where most variation occurs in the viral envelope glycoproteins that are the sole targets for neutralizing antibodies. Efforts to elicit broadly cross-reactive neutralizing antibodies that will protect against infection by most circulating strains of the virus are guided in part by in vitro assays that determine the ability of vaccine-elicited antibodies to neutralize genetically diverse HIV-1 variants. Until now, little information was available on how many and which strains of the virus are best suited for this purpose. We applied robust statistical methods to evaluate a large neutralization data set and identified a small panel of viruses that are a good representation of the global epidemic. The neutralization properties of this new panel of reference strains should facilitate the development of an effective HIV-1 vaccine. PMID:24352443

deCamp, Allan; Hraber, Peter; Bailer, Robert T.; Seaman, Michael S.; Ochsenbauer, Christina; Kappes, John; Gottardo, Raphael; Edlefsen, Paul; Self, Steve; Tang, Haili; Greene, Kelli; Gao, Hongmei; Daniell, Xiaoju; Sarzotti-Kelsoe, Marcella; Gorny, Miroslaw K.; Zolla-Pazner, Susan; LaBranche, Celia C.; Mascola, John R.; Korber, Bette T.

2014-01-01

147

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

148

Construction and in vitro characterization of a chimeric simian and human immunodeficiency virus with the RANTES gene  

Microsoft Academic Search

Chimeric simian–human immunodeficiency virus (SHIV) containing the env gene of HIV-1 infects macaque monkeys and provides basic information that is useful for the development of HIV-1 vaccines. Regulated-on-activation-normal-T-cell-expressed-and-secreted (RANTES), a CC-chemokine, enhances antigen-specific T helper type-1 responses against HIV-1. With the final goal of testing the adjuvant effects of RANTES in SHIV-macaque models, we constructed a SHIV having the RANTES

Yuya Shimizu; Masashi Okoba; Nanase Yamazaki; Yoshitaka Goto; Tomoyuki Miura; Masanori Hayami; Hiroo Hoshino; Takeshi Haga

2006-01-01

149

Forum in immunology Vaccines based on the native HIV Tat protein and on the combination of Tat and the structural HIV protein variant DV2 Env  

Microsoft Academic Search

The promising results obtained with the HIV-1 Tat-based vaccines in mice, monkeys and humans, a better understanding of Tat immuno- modulatory functions, as well as evidence that vaccination with trimeric V2 loop-deleted HIV-1 Env induces cross-clade neutralizing anti- bodies led to the rational design of a novel vaccine based on the combination of Tat and V2-deleted Env. © 2005 Elsevier

Barbara Ensoli; Aurelio Cafaro; Antonella Caputo; Valeria Fiorelli; Fabrizio Ensoli; Riccardo Gavioli; Flavia Ferrantelli; Andrea Cara; Fausto Titti; Mauro Magnani

150

Life+ EnvEurope DEIMS - improving access to long-term ecosystem monitoring data in Europe  

NASA Astrophysics Data System (ADS)

Long-term ecological (LTER) studies aim at detecting environmental changes and analysing its related drivers. In this respect LTER Europe provides a network of about 450 sites and platforms. However, data on various types of ecosystems and at a broad geographical scale is still not easily available. Managing data resulting from long-term observations is therefore one of the important tasks not only for an LTER site itself but also on the network level. Exchanging and sharing the information within a wider community is a crucial objective in the upcoming years. Due to the fragmented nature of long-term ecological research and monitoring (LTER) in Europe - and also on the global scale - information management has to face several challenges: distributed data sources, heterogeneous data models, heterogeneous data management solutions and the complex domain of ecosystem monitoring with regard to the resulting data. The Life+ EnvEurope project (2010-2013) provides a case study for a workflow using data from the distributed network of LTER-Europe sites. In order to enhance discovery, evaluation and access to data, the EnvEurope Drupal Ecological Information Management System (DEIMS) has been developed. This is based on the first official release of the Drupal metadata editor developed by US LTER. EnvEurope DEIMS consists of three main components: 1) Metadata editor: a web-based client interface to manage metadata of three information resource types - datasets, persons and research sites. A metadata model describing datasets based on Ecological Metadata Language (EML) was developed within the initial phase of the project. A crosswalk to the INSPIRE metadata model was implemented to convey to the currently on-going European activities. Person and research site metadata models defined within the LTER Europe were adapted for the project needs. The three metadata models are interconnected within the system in order to provide easy way to navigate the user among the related resources. 2) Discovery client: provides several search profiles for datasets, persons, research sites and external resources commonly used in the domain, e.g. Catalogue of Life , based on several search patterns ranging from simple full text search, glossary browsing to categorized faceted search. 3) Geo-Viewer: a map client that portrays boundaries and centroids of the research sites as Web Map Service (WMS) layers. Each layer provides a link to both Metadata editor and Discovery client in order to create or discover metadata describing the data collected within the individual research site. Sharing of the dataset metadata with DEIMS is ensured in two ways: XML export of individual metadata records according to the EML schema for inclusion in the international DataOne network, and periodic harvesting of metadata into GeoNetwork catalogue, thus providing catalogue service for web (CSW), which can be invoked by remote clients. The final version of DEIMS will be a pilot implementation for the information system of LTER-Europe, which should establish a common information management framework within the European ecosystem research domain and provide valuable environmental information to other European information infrastructures as SEIS, Copernicus and INSPIRE.

Kliment, Tomas; Peterseil, Johannes; Oggioni, Alessandro; Pugnetti, Alessandra; Blankman, David

2013-04-01

151

Localization of human immunodeficiency virus type 1 Gag and Env at the plasma membrane by confocal imaging.  

PubMed

Budding of lentiviruses occurs at the plasma membrane, but the preceding steps involved in particle assembly are poorly understood. Since the Gag polyprotein mediates virion assembly and budding, studies on the localization of Gag within the cell should provide insight into the mechanism of particle assembly. Here, we utilize biochemical fractionation techniques as well as high-resolution confocal imaging of live cells to demonstrate that Gag is localized at the plasma membrane in a striking punctate pattern. Mutation of the N-terminal myristoylation site results in the formation of large cytosolic complexes, whereas mutation of the N-terminal basic residue cluster in the matrix domain redirects the Gag protein to a region partially overlapping the Golgi apparatus. In addition, we show that Gag and Env colocalize at the plasma membrane and that mistargeting of a mutant Gag to the Golgi apparatus alters the pattern of surface expression of Env. PMID:10954568

Hermida-Matsumoto, L; Resh, M D

2000-09-01

152

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

153

A Novel Rabbit Monoclonal Antibody Platform To Dissect the Diverse Repertoire of Antibody Epitopes for HIV-1 Env Immunogen Design  

PubMed Central

The majority of available monoclonal antibodies (MAbs) in the current HIV vaccine field are generated from HIV-1-infected people. In contrast, preclinical immunogenicity studies have mainly focused on polyclonal antibody responses in experimental animals. Although rabbits have been widely used for antibody studies, there has been no report of using rabbit MAbs to dissect the specificity of antibody responses for AIDS vaccine development. Here we report on the production of a panel of 12 MAbs from a New Zealand White (NZW) rabbit that was immunized with an HIV-1 JR-FL gp120 DNA prime and protein boost vaccination regimen. These rabbit MAbs recognized a diverse repertoire of envelope (Env) epitopes ranging from the highly immunogenic V3 region to several previously underappreciated epitopes in the C1, C4, and C5 regions. Nine MAbs showed cross-reactivity to gp120s of clades other than clade B. Increased somatic mutation and extended CDR3 were observed with Ig genes of several molecularly cloned rabbit MAbs. Phylogenic tree analysis showed that the heavy chains of MAbs recognizing the same region on gp120 tend to segregate into an independent subtree. At least three rabbit MAbs showed neutralizing activities with various degrees of breadth and potency. The establishment of this rabbit MAb platform will significantly enhance our ability to test optimal designs of Env immunogens to gain a better understanding of the structural specificity and evolution process of Env-specific antibody responses elicited by candidate AIDS vaccines. PMID:23864612

Chen, Yuxin; Vaine, Michael; Wallace, Aaron; Han, Dong; Wan, Shengqin; Seaman, Michael S.; Montefiori, David; Wang, Shixia

2013-01-01

154

Comparison of intradermal and intramuscular delivery followed by in vivo electroporation of SIV Env DNA in macaques.  

PubMed

A panel of SIVmac251 transmitted Env sequences were tested for expression, function and immunogenicity in mice and macaques. The immunogenicity of a DNA vaccine cocktail expressing SIVmac239 and three transmitted SIVmac251 Env sequences was evaluated upon intradermal or intramuscular injection followed by in vivo electroporation in macaques using sequential vaccination of gp160, gp120 and gp140 expressing DNAs. Both intradermal and intramuscular vaccination regimens using the gp160 expression plasmids induced robust humoral immune responses, which further improved using the gp120 expressing DNAs. The responses showed durability of binding and neutralizing antibody titers and high avidity for>1 y. The intradermal DNA delivery regimen induced higher cross-reactive responses able to neutralize the heterologous tier 1B-like SIVsmE660_CG7V. Analysis of cellular immune responses showed induction of Env-specific memory responses and cytotoxic granzyme B(+) T cells in both vaccine groups, although the magnitude of the responses were ~10x higher in the intramuscular/electroporation group. The cellular responses induced by both regimens were long lasting and could be detected ~1 y after the last vaccination. These data show that both DNA delivery methods are able to induce robust and durable immune responses in macaques. PMID:23811579

Kulkarni, Viraj; Rosati, Margherita; Bear, Jenifer; Pilkington, Guy R; Jalah, Rashmi; Bergamaschi, Cristina; Singh, Ashish K; Alicea, Candido; Chowdhury, Bhabadeb; Zhang, Gen-Mu; Kim, Eun-Young; Wolinsky, Steven M; Huang, Wensheng; Guan, Yongjun; LaBranche, Celia; Montefiori, David C; Broderick, Kate E; Sardesai, Niranjan Y; Valentin, Antonio; Felber, Barbara K; Pavlakis, George N

2013-10-01

155

Genetic and neutralization sensitivity of diverse HIV-1 env clones from chronically infected patients in China.  

PubMed

As HIV-1 continues to spread in China from traditional high risk populations to the general public, its genetic makeup has become increasingly complex. However, the impact of these genetic changes on the biological and neutralization sensitivity of the virus is unknown. The current study aims to characterize the genetic, biological, and neutralization sensitivity of HIV-1 identified in China between 2004 and 2007. Based on a total of 107 full-length envelope genes obtained directly from the infected patients, we found that those viruses fell into three major genetic groups: CRF01_AE, subtype B', and subtype C/CRF07_BC/CRF08_BC/B'C. Pseudotyped viruses built upon the viable env genes have demonstrated their substantial variability in mediating viral entry and in sensitivity to neutralization by subtype-specific plasma pools and broadly neutralizing monoclonal antibodies (bnmAb). Many viruses are resistant to one or more bnmAb, including those known to have high potency against diverse viruses from outside China. Sequence and structural analysis has revealed several mechanisms by which these resistant viruses escape recognition from bnmAb. We believe that these results will help us to better understand the impact of genetic diversity on the neutralizing sensitivity of the viruses and to facilitate the design of immunogens capable of eliciting antibodies with potency and breadth similar to those of bnmAb. PMID:21325278

Shang, Hong; Han, Xiaoxu; Shi, Xuanling; Zuo, Teng; Goldin, Mark; Chen, Dan; Han, Bing; Sun, Wei; Wu, Hao; Wang, Xinquan; Zhang, Linqi

2011-04-22

156

Effects of murine leukemia virus env gene proteins on macrophage-mediated cytotoxicity in vitro  

NASA Technical Reports Server (NTRS)

F5b Tumor cells were incubated with concentrated culture supernatants taken from cells resistant (F5m) or sensitive (F5b) to contact-dependent macrophage cytotoxicity. Macrophage cell line B6MP102 and murine peritoneal macrophages killed targets incubated with supernatants taken from sensitive cells but poorly killed cells incubated in supernatants isolated from resistant cells. Membranes from cells resistant to macrophage killing, F5m, were fused into F5b cells. The fused F5b cells were killed significantly less than F5b cells fused with F5b cell membranes or untreated F5b cells. The decreased killing of F5b cells corresponded to increased concentrations of gp70(a) molecules on F5b cells. Affinity purified gp70(a) was added to cytotoxicity assays but failed to inhibit macrophage cytotoxicity. P15E molecules were detectable on both F5b and F5m cells. In addition, a synthetic peptide found to exhibit the inhibitory properties of p15E was added to cytotoxicity assays. P15E synthetic peptide also did not inhibit macrophage cytotoxicity. Therefore, env gene proteins of murine leukemia virus do not appear responsible for inducing tumor cell resistance to activated macrophage contact-dependent cytotoxicity.

Chapes, S. K.; Takemoto, L. J.; Spooner, B. S. (Principal Investigator)

1991-01-01

157

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. PMID:22843534

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

2012-01-01

158

Nested-RT-PCR and multiplex RT-PCR for diagnosis of rhinovirus infection in clinical samples.  

PubMed

Human rhinoviruses (HRVs) are positive-stranded RNA viruses belonging to the Enterovirus genus in the family of Picornaviridae. Identification of the specific strain in HRV disease has been difficult because the traditional serological method is insensitive, labor intensive, and cumbersome. With the fast progress in molecular biological technique, more sensitive and faster molecular methods have been developed, such as polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR, and real-time RT-PCR. To improve the technique for defining the links between illnesses and specific strains of HRV, we developed RT-PCR specific for HRV as routine base. A multiplex RT-PCR that simultaneously identifies 12 respiratory viruses including HRV is also routinely used in our lab. Here we have described the specific steps of methods for identification of HRV from clinical samples, such as sample preparation, isolation of total RNA, nested-RT-PCR for HRV, Seeplex(®) RV15 ACE Detection method, gel electrophoresis, how to use the QIAxcel(®) capillary electrophoresis system, and results interpretation. PMID:25261303

Yu, Xuelian; Ghildyal, Reena

2015-01-01

159

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

160

Characterization of simian and human immunodeficiency chimeric viruses re-isolated from vaccinated macaque monkeys after challenge infection  

Microsoft Academic Search

Summary.  ?Monkeys that have been vaccinated with nef-deleted SHIVs were either fully or partially protected against challenge with acute pathogenic SHIV-89.6?P. Viruses isolated\\u000a from these vaccinated monkeys were all found to be the 89.6?P challenge virus using PCR amplification and restriction enzyme\\u000a analysis of the env region of the viruses. Analysis of the 3?-end of the env region and 5?-half of

T. B. Kwofie; T. Miura; K. Ibuki; Y. Enose; H. Suzuki; M. Ui; T. Kuwata; M. Hayami

2002-01-01

161

Yeast-Elicited Cross-Reactive Antibodies to HIV Env Glycans Efficiently Neutralize Virions Expressing Exclusively High-Mannose N-Linked Glycans?  

PubMed Central

The HIV envelope (Env) protein uses a dense coat of glycans to mask conserved domains and evade host humoral immune responses. The broadly neutralizing antibody 2G12, which binds a specific cluster of high-mannose glycans on HIV Env, shows that the glycan shield can also serve as a target for neutralizing antibodies. We have described a triple mutant Saccharomyces cerevisiae strain that expresses high-mannose glycoproteins that bind to 2G12. When used to immunize rabbits, this yeast elicits antibodies that bind to gp120-associated glycans but fail to neutralize virus. Here we sought to determine the reason for these discordant results. Affinity purification of sera over columns conjugated with three 2G12-reactive yeast glycoproteins showed that these proteins could adsorb 80% of the antibodies that bind to gp120 glycans. Despite binding to monomeric gp120, these mannose-specific antibodies failed to bind cell surface-expressed trimeric Env. However, when Env was expressed in the presence of the mannosidase inhibitor kifunensine to force retention of high-mannose glycans at all sites, the purified antibodies gained the abilities to bind trimeric Env and to strongly and broadly neutralize viruses produced under these conditions. Combined, these data show that the triple mutant yeast strain elicits antibodies that bind to high-mannose glycans presented on the HIV envelope, but only when they are displayed in a manner not found on native Env trimers. This implies that the underlying structure of the protein scaffold used to present the high-mannose glycans may be critical to allow elicitation of antibodies that recognize trimeric Env and neutralize virus. PMID:20962094

Agrawal-Gamse, Caroline; Luallen, Robert J.; Liu, Bingfen; Fu, Hu; Lee, Fang-Hua; Geng, Yu; Doms, Robert W.

2011-01-01

162

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

163

Parallel Picoliter RT-PCR Assays Using Microfluidics  

E-print Network

Parallel Picoliter RT-PCR Assays Using Microfluidics Joshua S. Marcus,, W. French Anderson The development of microfluidic tools for high-throughput nucleic acid analysis has become a burgeoning area of research in the post-genome era. Here, we have developed a microfluidic chip to perform 72 parallel 450-p

Quake, Stephen R.

164

Remains of abutments for Bridge No. 1575 at MD Rt. ...  

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

Remains of abutments for Bridge No. 1575 at MD Rt. 51 in Spring Gap, Maryland, looking northeast. (Compare with HAER MD-115 photos taken 1988). - Western Maryland Railway, Cumberland Extension, Pearre to North Branch, from WM milepost 125 to 160, Pearre, Washington County, MD

165

RtI and Comprehensive Assessment: Are They Opposed?  

ERIC Educational Resources Information Center

Response to Intervention (RtI) promotes a well-integrated system connecting general, compensatory, gifted, and special education in providing high quality, standards-based instruction and intervention that are matched to students' academic, social-emotional, and behavioral needs. There are three levels to this framework. Tier 1 (or Universal) is…

Franklin-Rohr, Cheryl

2011-01-01

166

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

167

Diverse Recombinant HIV-1 Envs Fail To Activate B Cells Expressing the Germline B Cell Receptors of the Broadly Neutralizing Anti-HIV-1 Antibodies PG9 and 447-52D  

PubMed Central

ABSTRACT Broadly neutralizing antibodies (bNAbs) against HIV-1 are generated during HIV-1-infection but have not yet been elicited by immunization with recombinant forms of the viral envelope glycoprotein (Env; the target of anti-HIV-1 neutralizing antibodies). A particular type of bNAb targets the CD4-binding site (CD4-BS) region of Env. These antibodies are derived from a limited number of VH/VL genes and can bind to and neutralize diverse HIV-1 strains. Recent reports have demonstrated the limited potential of Env to activate B cells expressing the germline B cell receptor (BCR) forms of anti-CD4-BS bNAbs. A potential reason for the lack of elicitation of anti-CD4-BS bNAbs by Env immunogens is the absence of stimulation of naive B cells expressing the germline BCRs of such antibodies. Several bNAbs have been isolated from HIV-1-infected subjects that target other structurally conserved regions of Env. How frequently Env immunogens stimulate the germline BCRs that give rise to bNAbs that target Env regions other than the CD4-BS is not well understood. Here, we investigated the interactions between diverse Envs and the BCRs of known bNAbs targeting not only the CD4-BS but also conserved elements of the second and third variable Env regions. Our results indicate that Env is generally ineffective in engaging germline BCRs of bNAbs irrespective of their epitope target. Potentially, this is the result of viral evolutionary mechanisms adopted to escape broadly neutralizing antibody responses. Our results also suggest that a single Env capable of activating germline BCRs that target distinct Env epitopes will be very difficult to identify or to design. IMPORTANCE Broadly neutralizing antibodies against HIV-1 are thought to be an important component of the immune responses that a successful vaccine should elicit. Broadly neutralizing antibodies are generated by a subset of those infected by HIV-1, but so far, they have not been generated by immunization with recombinant Envelope (Env, the target of anti-HIV-1 neutralizing antibodies). Here, we provide evidence that the inability of Env to elicit the production of broadly neutralizing antibodies is due to the inability of diverse Envs to engage the germline B cell receptor forms of known broadly neutralizing antibodies. PMID:24352455

McGuire, Andrew T.; Glenn, Jolene A.; Lippy, Adriana

2014-01-01

168

Enhancing transport of hydrogenophaga flava ENV735 for bioaugmentation of aquifers contaminated with methyl tert-butyl ether.  

PubMed

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. PMID:12406751

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

2002-11-01

169

Enhancing Transport of Hydrogenophaga flava 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. PMID:12406751

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

2002-01-01

170

Detection of the human endogenous retrovirus ERV3-encoded Env-protein in human tissues using antibody-based proteomics  

PubMed Central

Objectives There is growing evidence to suggest that human endogenous retroviruses (HERVs) have contributed to human evolution, being expressed in development, normal physiology and disease. A key difficulty in the scientific evaluation of this potential viral contribution is the accurate demonstration of virally expressed protein in specific human cells and tissues. In this study, we have adopted the endogenous retrovirus, ERV3, as our test model in developing a reliable high-capacity methodology for the expression of such endogenous retrovirus-coded protein. Design Two affinity-purified polyclonal antibodies to ERV3 Env-encoded protein were generated to detect the corresponding protein expression pattern in specific human cells, tissues and organs. Participants Sampling included normal tissues from 144 individuals ranging from childhood to old age. This included more than forty different tissues and organs and some 216 different cancer tissues representing the twenty commonest forms of human cancer. Setting The Rudbeck Laboratory, Uppsala University and Uppsala University Hospital, Uppsala, Sweden. Main Outcome Measures The potential expression at likely physiological level of the ERV3Env encoded protein in a wide range of human cells, tissues and organs. Results We found that ERV3 encoded Env protein is expressed at substantive levels in placenta, testis, adrenal gland, corpus luteum, Fallopian tubes, sebaceous glands, astrocytes, bronchial epithelium and the ducts of the salivary glands. Substantive expression was also seen in a variety of epithelial cells as well as cells known to undergo fusion in inflammation and in normal physiology, including fused macrophages, myocardium and striated muscle. This contrasted strongly with the low levels expressed in other tissues types. These findings suggest that this virus plays a significant role in human physiology and may also play a possible role in disease. Conclusion This technique can now be extended to the study of other HERV genomes within the human chromosomes that may have contributed to human evolution, physiology and disease. PMID:24262892

Fei, Chen; Atterby, Christina; Edqvist, Per-Henrik; Pontén, Fredrik; Zhang, Wei Wei; Larsson, Erik; Ryan, Frank P

2014-01-01

171

HIV-1 Vaccine-Induced C1 and V2 Env-Specific Antibodies Synergize for Increased Antiviral Activities  

PubMed Central

ABSTRACT The RV144 ALVAC/AIDSVax HIV-1 vaccine clinical trial showed an estimated vaccine efficacy of 31.2%. Viral genetic analysis identified a vaccine-induced site of immune pressure in the HIV-1 envelope (Env) variable region 2 (V2) focused on residue 169, which is included in the epitope recognized by vaccinee-derived V2 monoclonal antibodies. The ALVAC/AIDSVax vaccine induced antibody-dependent cellular cytotoxicity (ADCC) against the Env V2 and constant 1 (C1) regions. In the presence of low IgA Env antibody levels, plasma levels of ADCC activity correlated with lower risk of infection. In this study, we demonstrate that C1 and V2 monoclonal antibodies isolated from RV144 vaccinees synergized for neutralization, infectious virus capture, and ADCC. Importantly, synergy increased the HIV-1 ADCC activity of V2 monoclonal antibody CH58 at concentrations similar to that observed in plasma of RV144 vaccinees. These findings raise the hypothesis that synergy among vaccine-induced antibodies with different epitope specificities contributes to HIV-1 antiviral antibody responses and is important to induce for reduction in the risk of HIV-1 transmission. IMPORTANCE The Thai RV144 ALVAC/AIDSVax prime-boost vaccine efficacy trial represents the only example of HIV-1 vaccine efficacy in humans to date. Studies aimed at identifying immune correlates involved in the modest vaccine-mediated protection identified HIV-1 envelope (Env) variable region 2-binding antibodies as inversely correlated with infection risk, and genetic analysis identified a site of immune pressure within the region recognized by these antibodies. Despite this evidence, the antiviral mechanisms by which variable region 2-specific antibodies may have contributed to lower rates of infection remain unclear. In this study, we demonstrate that vaccine-induced HIV-1 envelope variable region 2 and constant region 1 antibodies synergize for recognition of virus-infected cells, infectious virion capture, virus neutralization, and antibody-dependent cellular cytotoxicity. This is a major step in understanding how these types of antibodies may have cooperatively contributed to reducing infection risk and should be considered in the context of prospective vaccine design. PMID:24807721

Pollara, Justin; Bonsignori, Mattia; Moody, M. Anthony; Liu, Pinghuang; Alam, S. Munir; Hwang, Kwan-Ki; Gurley, Thaddeus C.; Kozink, Daniel M.; Armand, Lawrence C.; Marshall, Dawn J.; Whitesides, John F.; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Rerks-Ngarm, Supachai; Robb, Merlin L.; O'Connell, Robert J.; Kim, Jerome H.; Michael, Nelson L.; Montefiori, David C.; Tomaras, Georgia D.; Liao, Hua-Xin; Haynes, Barton F.

2014-01-01

172

AnnuAl RepoRt univeRsities must  

E-print Network

AnnuAl RepoRt 2008 2009 #12;univeRsities must contRibute to the betteRment of society. they h, endeavours that our community and our country are looking to for solutions to challenges our society faces of people Who WoRk heRe. dR. AlAn WildemAn President and Vice-Chancellor #12;9 medical education offered

173

Optics technology base R/T program overview  

NASA Technical Reports Server (NTRS)

NASA is studying a number of advanced optical systems concepts to achieve a variety of science mission goals. Most of these concepts require significant advancements in optics technology. An overview of the Optics Technology base R&T program is presented in outline form. The program structure contains six major program elements: optical materials and coatings, optics modeling, advanced optics fabrication, optical testing, wavefront sensing and control, and sensor optics technology.

Coulter, Daniel R.

1991-01-01

174

Priming B cell-mediated anti-HIV envelope responses by vaccination allows for the long-term control of infection in macaques exposed to a R5-tropic SHIV.  

PubMed

The potential of vaccine-elicited anti-HIV envelope antibodies to control HIV-infection was evaluated by immunizing macaques with the HIV envelope protein and transiently depleting them of their CD8+ cells before intravenous challenge with the pathogenic CCR5-tropic SIV/HIV chimeric virus, SHIV(SF162P4). Although sterilizing immunity was not achieved, all vaccinated animals effectively controlled infection and remained free of disease for the duration of observation (over 3 years). In contrast, during the same period, the control animals progressed to disease. Both the vaccinees and the controls developed robust cell-mediated antiviral and neutralizing antibody responses following infection. A comparative analysis of these responses suggests that the more effective long-term control of infection by the vaccinated animals is due to the more rapid development of anti-HIV envelope antibodies. These studies suggest that priming by vaccination of B cell anti-HIV envelope responses maybe crucial for the long-term control of HIV infection. PMID:15003872

Buckner, Clarisa; Gines, Leoned G; Saunders, Cheryl J; Vojtech, Lucia; Srivastava, Indresh; Gettie, Agegnehu; Bohm, Rudolph; Blanchard, James; Barnett, Susan W; Safrit, Jeffrey T; Stamatatos, Leonidas

2004-03-01

175

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. Finally, by assuming that the coupling among the gravitational and matter sectors is described solely through RT, basically by setting ?2=0, the exponent of the action (21) is constrained to n=-2/?-2. Then, as above, Eqs. (22) become a system of algebraic equations for the constants {m,?} in terms of {?,?}, which describes a set of infinite solutions, but confirms the suitability of the action with f(P)=Pn=()n for reproducing power-law expansion. As above the number of variables of the equations can be reduced by imposing ?=-3m. Nevertheless, the reconstruction of the exact expression for the gravitational action is not possible in general, and numerical calculations are required, as shown below. In addition, note that the evolution of the energy density behaves differently to the one given in GR. This may not be important for the illustration of the reconstruction procedure, as for instance, the solutions considered above, but it may seriously affect the observational constraints when a realistic evolution is studied. In this sense, the FLRW equations (19) are two differential equations for the functions ?(z) and f(RT)=f(z), expressed in terms of the redshift. They do not ensure that the solution for the energy density behaves in the proper way as observations suggest, namely ?m?(1 for dust matter, apart from the trivial solution f(RT)=-2? that recovers GR with a cosmological constant, and the usual continuity equation is satisfied again. Hence, more general actions should be considered in order to provide a realistic evolution.

Odintsov, Sergei D.; Sáez-Gómez, Diego

2013-10-01

176

Type A influenza virus detection from horses by real-time RT-PCR and insulated isothermal RT-PCR.  

PubMed

Equine influenza (EI) is a highly contagious disease of horses caused by the equine influenza virus (EIV) H3N8 subtype. EI is the most important respiratory virus infection of horses and can disrupt major equestrian events and cause significant economic losses to the equine industry worldwide. Influenza H3N8 virus spreads rapidly in susceptible horses and can result in very high morbidity within 24-48 h after exposure to the virus. Therefore, rapid and accurate diagnosis of EI is critical for implementation of prevention and control measures to avoid the spread of EIV and to reduce the economic impact of the disease. The probe-based real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assays targeting various EIV genes are reported to be highly sensitive and specific compared to the Directigen Flu A(®) test and virus isolation in embryonated hens' eggs. Recently, a TaqMan(®) probe-based insulated isothermal RT-PCR (iiRT-PCR) assay for the detection of EIV H3N8 subtype has been described. These molecular based diagnostic assays provide a fast and reliable means of EIV detection and disease surveillance. PMID:24899448

Balasuriya, Udeni B R

2014-01-01

177

The Phenylmethylthiazolylthiourea Nonnucleoside Reverse Transcriptase (RT) Inhibitor MSK-076 Selects for a Resistance Mutation in the Active Site of Human Immunodeficiency Virus Type 2 RT  

Microsoft Academic Search

The phenylmethylthiazolylthiourea (PETT) derivative MSK-076 shows, besides high potency against human immunodeficiency virus type 1 (HIV-1), marked activity against HIV-2 (50% effective concentration, 0.63 M) in cell culture. Time-of-addition experiments pointed to HIV-2 reverse transcriptase (RT) as the target of action of MSK-076. Recombinant HIV-2 RT was inhibited by MSK-076 at 23 M. As was also found for HIV-1 RT,

Joeri Auwerx; Miguel Stevens; An R. Van Rompay; Louise E. Bird; Jingshan Ren; Erik De Clercq; Bo Oberg; David K. Stammers; Anna Karlsson; Jan Balzarini

2004-01-01

178

June 4-8, 2001 Valencia, Spain Mircea Bogdan, RT2001 1  

E-print Network

June 4-8, 2001 Valencia, Spain Mircea Bogdan, RT2001 1 CDF Drift Chamber Signal Analyzer Board Bill, Spain Mircea Bogdan, RT2001 2 CDF Drift Chamber Signal Analyzer Board Drift Chamber Signals ­ Fe55 markers. #12;June 4-8, 2001 Valencia, Spain Mircea Bogdan, RT2001 3 CDF Drift Chamber Signal Analyzer

179

CERTIFICATE OF FIELD VERIFICATION AND DIAGNOSTIC TESTING CF-4R-ENV-22 Quality Insulation Installation (QII) -Insulation Stage Checklist (Page 1 of 3)  

E-print Network

CERTIFICATE OF FIELD VERIFICATION AND DIAGNOSTIC TESTING CF-4R-ENV-22 Quality Insulation Installation (QII) - Insulation Stage Checklist (Page 1 of 3) Site Address: Enforcement Agency: Permit Number: ____________ 2008 Residential Compliance Forms May 2012 All structural framing areas shall be insulated in a manner

180

CERTIFICATE OF FIELD VERIFICATION AND DIAGNOSTIC TESTING CF-4R-ENV-21 Quality Insulation Installation (QII) -Framing Stage Checklist (Page 1 of 2)  

E-print Network

CERTIFICATE OF FIELD VERIFICATION AND DIAGNOSTIC TESTING CF-4R-ENV-21 Quality Insulation: ____________ 2008 Residential Compliance Forms May 2012 Quality Insulation Installation (QII) Framing Stage Checklist Air barrier installation and preparation for insulation must be done at framing stage before

181

Yersinia pestis requires the 2-component regulatory system OmpR-EnvZ to resist innate immunity during the early and late stages of plague.  

PubMed

Plague is transmitted by fleas or contaminated aerosols. To successfully produce disease, the causal agent (Yersinia pestis) must rapidly sense and respond to rapid variations in its environment. Here, we investigated the role of 2-component regulatory systems (2CSs) in plague because the latter are known to be key players in bacterial adaptation to environmental change. Along with the previously studied PhoP-PhoQ system, OmpR-EnvZ was the only one of Y. pestis' 23 other 2CSs required for production of bubonic, septicemic, and pneumonic plague. In vitro, OmpR-EnvZ was needed to counter serum complement and leukocytes but was not required for the secretion of antiphagocyte exotoxins. In vivo, Y. pestis lacking OmpR-EnvZ did not induce an early immune response in the skin and was fully virulent in neutropenic mice. We conclude that, throughout the course of Y. pestis infection, OmpR-EnvZ is required to counter toxic effectors secreted by polymorphonuclear leukocytes in the tissues. PMID:24813471

Reboul, Angéline; Lemaître, Nadine; Titecat, Marie; Merchez, Maud; Deloison, Gaspard; Ricard, Isabelle; Pradel, Elizabeth; Marceau, Michaël; Sebbane, Florent

2014-11-01

182

Biochem. J. (2005) 385, 255264 (Printed in Great Britain) 255 Structural characterization of Escherichia coli sensor histidine kinase EnvZ  

E-print Network

that plays a pivotal role in cell adaptation to changes in extracellular osmolarity. Although the cytoplasmic-osmo- larity phenotype. Key words: bacterial signal transduction, membrane receptor, NMR, osmolarity, stress proteins, OmpF and OmpC, in response to changes in the medium osmolarity [4]. The EnvZ/OmpR system is among

Ikura, Mitsuhiko

183

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

184

HIV-1 Receptor Binding Site-Directed Antibodies Using a VH1-2 Gene Segment Orthologue Are Activated by Env Trimer Immunization  

PubMed Central

Broadly neutralizing antibodies (bNAbs) isolated from chronically HIV-1 infected individuals reveal important information regarding how antibodies target conserved determinants of the envelope glycoprotein (Env) spike such as the primary receptor CD4 binding site (CD4bs). Many CD4bs-directed bNAbs use the same heavy (H) chain variable (V) gene segment, VH1-2*02, suggesting that activation of B cells expressing this allele is linked to the generation of this type of Ab. Here, we identify the rhesus macaque VH1.23 gene segment to be the closest macaque orthologue to the human VH1-2 gene segment, with 92% homology to VH1-2*02. Of the three amino acids in the VH1-2*02 gene segment that define a motif for VRC01-like antibodies (W50, N58, flanking the HCDR2 region, and R71), the two identified macaque VH1.23 alleles described here encode two. We demonstrate that immunization with soluble Env trimers induced CD4bs-specific VH1.23-using Abs with restricted neutralization breadth. Through alanine scanning and structural studies of one such monoclonal Ab (MAb), GE356, we demonstrate that all three HCDRs are involved in neutralization. This contrasts to the highly potent CD4bs-directed VRC01 class of bNAb, which bind Env predominantly through the HCDR2. Also unlike VRC01, GE356 was minimally modified by somatic hypermutation, its light (L) chain CDRs were of average lengths and it displayed a binding footprint proximal to the trimer axis. These results illustrate that the Env trimer immunogen used here activates B cells encoding a VH1-2 gene segment orthologue, but that the resulting Abs interact distinctly differently with the HIV-1 Env spike compared to VRC01. PMID:25166308

Bale, Shridhar; Phad, Ganesh E.; Guenaga, Javier; Wilson, Richard; Soldemo, Martina; McKee, Krisha; Sundling, Christopher; Mascola, John; Li, Yuxing; Wyatt, Richard T.; Karlsson Hedestam, Gunilla B.

2014-01-01

185

The sequence complementarity between HIV-1 5' splice site SD4 and U1 snRNA determines the steady-state level of an unstable env pre-mRNA.  

PubMed Central

HIV-1 env expression from certain subgenomic vectors requires the viral regulatory protein Rev, its target sequence RRE, and a 5' splice site upstream of the env open reading frame. To determine the role of this splice site in the 5'-splice-site-dependent Rev-mediated env gene expression, we have subjected the HIV-1 5' splice site, SD4, to a mutational analysis and have analyzed the effect of those mutations on env expression. The results demonstrate that the overall strength of hydrogen bonding between the 5' splice site, SD4, and the free 5' end of the U1 snRNA correlates with env expression efficiency, as long as env expression is suboptimal, and that a continuous stretch of 14 hydrogen bonds can lead to full env expression, as a result of stabilizing the pre-mRNA. The U1 snRNA-mediated stabilization is independent of functional splicing, as a mismatch in position +1 of the 5' splice site that led to loss of detectable amounts of spliced transcripts did not preclude stabilization and expression of the unspliced env mRNA, provided that Rev enables its nuclear export. The nucleotides capable of participating in U1 snRNA:pre-mRNA interaction include positions -3 to +8 of the 5' splice site and all 11 nt constituting the single-stranded 5' end of U1 snRNA. Moreover, env gene expression is significantly decreased upon the introduction of point mutations in several upstream GAR nucleotide motifs, which are mediating SF2/ASF responsiveness in an in vitro splicing assay. This suggests that the GAR sequences may play a role in stabilizing the pre-mRNA by sequestering U1 snRNP to SD4. PMID:11333022

Kammler, S; Leurs, C; Freund, M; Krummheuer, J; Seidel, K; Tange, T O; Lund, M K; Kjems, J; Scheid, A; Schaal, H

2001-01-01

186

Comparative analysis of ELISA, one step RT-PCR and IC-RT-PCR for the detection of bean yellow Mosaic virus in gladiolus.  

PubMed

To overcome the problematic detection of Bean yellow mosaic virus (BYMV) concentration in gladiolus corms or cormlets, we tested suitability of different diagnostic methods such as ELISA, one step RT-PCR and IC-RT-PCR. Among these methods, DAS-ELISA and one step RT-PCR method was able to detect virus in leaves and it failed to detect in corms or cormlets whereas IC-RT-PCR method was very sensitive and able to detect virus in both leaves and corms or cormlets of gladiolus plants this is due to during an RNA isolation step and viral particles are enriched by antibody or plastic affinity capture whilst PCR inhibitors are eliminated from the sample during the immunocpature stage of the test. Therefore, IC-RT-PCR was the most reliable method of diagnosing BYMV in gladiolus corms or cormlets. PMID:20222572

Ganesh Selvaraj, Duraisamy; Pokorný, Radován; Holková, Ludmila

2009-01-01

187

Design and development of the RT-2/CZT payload  

NASA Astrophysics Data System (ADS)

The RT-2/CZT spectrometer is one of the satellite payloads to be flown onboard the Coronas-Photon satellite in 2008. It is a collaborative experiment between TIFR, CSP, ISRO (India) and MEPhI (Russia). It is designed to study the solar hard X-ray flare phenomena in the energy range 20 keV - 120 keV using pixilated (2.5 mm times 2.5 mm) cadmium zinc telluride detector array. These detectors along with an image coding device like coded aperture mask (CAM) or fresnel zone plate (FZP) helps in to snap images (in medium X-ray energy ranges) of the solar flares. The design characteristics of this payload are discussed.

Malkar, J. P.; Tawde, Amit; Sreekumar, S.; Hingar, M. K.; Chakrabarti, S. K.; Nandi, Anuj

188

Development of RT-LAMP and real-time RT-PCR assays for the rapid detection of the new duck Tembusu-like BYD virus.  

PubMed

A new duck Tembusu virus (TMUV), also known as BYD virus, has been identified as the causative agent for the emerging duck egg-drop syndrome in mainland China. The rapid spread and wide distribution of the new TMUV in mainland China result in heavy loss to the poultry industry and pose great threats to public health. Rapid and sensitive detection methods are critical for prevention and control of TMUV infections. In this study, a reverse-transcription loop-mediated isothermal amplification assay (RT-LAMP) and an SYBR Green-I-based real-time RT-PCR assay specific for the duck TMUV were developed and validated with laboratory and field samples, respectively. The detection limits were 1 × 10(-4) and 1 × 10(-3) PFU per reaction for the RT-LAMP assay and real-time RT-PCR assay, respectively. The specificities were analyzed with other related members of the genus Flavivirus, and no cross-reaction was observed. Furthermore, both assays were evaluated with field samples, and they exhibited high sensitivity and specificity. In addition, the real-time RT-PCR assay worked well in viral load analysis, which revealed that the spleen may be the primary target for the replication of new duck TMUV in ducks. The TMUV-specific RT-LAMP and real-time RT-PCR assays will provide useful tools for the diagnosis and epidemiological surveillance of TMUV infection. PMID:22865206

Jiang, Tao; Liu, Juan; Deng, Yong-Qiang; Su, Jing-Liang; Xu, Li-Juan; Liu, Zhi-Hui; Li, Xiao-Feng; Yu, Xue-Dong; Zhu, Shun-Ya; Gao, George Fu; Qin, E-De; Qin, Cheng-Feng

2012-12-01

189

Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus  

NASA Astrophysics Data System (ADS)

Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

Huang, Su-Hua; Yang, Tsuey-Ching; Tsai, Ming-Hong; Tsai, I.-Shou; Lu, Huang-Chih; Chuang, Pei-Hsin; Wan, Lei; Lin, Ying-Ju; Lai, Chih-Ho; Lin, Cheng-Wen

2008-10-01

190

Comparison of Three-Dimensional (3D) Conformal Proton Radiotherapy (RT), 3D Conformal Photon RT, and Intensity-Modulated RT for Retroperitoneal and Intra-Abdominal Sarcomas  

SciTech Connect

Purpose: To compare three-dimensional conformal proton radiotherapy (3DCPT), intensity-modulated photon radiotherapy (IMRT), and 3D conformal photon radiotherapy (3DCRT) to predict the optimal RT technique for retroperitoneal sarcomas. Methods and Materials: 3DCRT, IMRT, and 3DCPT plans were created for treating eight patients with retroperitoneal or intra-abdominal sarcomas. The clinical target volume (CTV) included the gross tumor plus a 2-cm margin, limited by bone and intact fascial planes. For photon plans, the planning target volume (PTV) included a uniform expansion of 5 mm. For the proton plans, the PTV was nonuniform and beam-specific. The prescription dose was 50.4 Gy/Cobalt gray equivalent CGE. Plans were normalized so that >95% of the CTV received 100% of the dose. Results: The CTV was covered adequately by all techniques. The median conformity index was 0.69 for 3DCPT, 0.75 for IMRT, and 0.51 for 3DCRT. The median inhomogeneity coefficient was 0.062 for 3DCPT, 0.066 for IMRT, and 0.073 for 3DCRT. The bowel median volume receiving 15 Gy (V15) was 16.4% for 3DCPT, 52.2% for IMRT, and 66.1% for 3DCRT. The bowel median V45 was 6.3% for 3DCPT, 4.7% for IMRT, and 15.6% for 3DCRT. The median ipsilateral mean kidney dose was 22.5 CGE for 3DCPT, 34.1 Gy for IMRT, and 37.8 Gy for 3DCRT. The median contralateral mean kidney dose was 0 CGE for 3DCPT, 6.4 Gy for IMRT, and 11 Gy for 3DCRT. The median contralateral kidney V5 was 0% for 3DCPT, 49.9% for IMRT, and 99.7% for 3DCRT. Regardless of technique, the median mean liver dose was <30 Gy, and the median cord V50 was 0%. The median integral dose was 126 J for 3DCPT, 400 J for IMRT, and 432 J for 3DCRT. Conclusions: IMRT and 3DCPT result in plans that are more conformal and homogenous than 3DCRT. Based on Quantitative Analysis of Normal Tissue Effects in Clinic benchmarks, the dosimetric advantage of proton therapy may be less gastrointestinal and genitourinary toxicity.

Swanson, Erika L. [Department of Radiation Oncology, University of Florida, Gainesville, Florida (United States)] [Department of Radiation Oncology, University of Florida, Gainesville, Florida (United States); Indelicato, Daniel J., E-mail: dindelicato@floridaproton.org [Department of Radiation Oncology, University of Florida, Gainesville, Florida (United States); University of Florida Proton Therapy Institute, Jacksonville, Florida (United States); Louis, Debbie; Flampouri, Stella; Li, Zuofeng [University of Florida Proton Therapy Institute, Jacksonville, Florida (United States)] [University of Florida Proton Therapy Institute, Jacksonville, Florida (United States); Morris, Christopher G.; Paryani, Nitesh [Department of Radiation Oncology, University of Florida, Gainesville, Florida (United States)] [Department of Radiation Oncology, University of Florida, Gainesville, Florida (United States); Slopsema, Roelf [University of Florida Proton Therapy Institute, Jacksonville, Florida (United States)] [University of Florida Proton Therapy Institute, Jacksonville, Florida (United States)

2012-08-01

191

Diagnosis of Kyasanur forest disease by nested RT-PCR, real-time RT-PCR and IgM capture ELISA.  

PubMed

Kyasanur forest disease (KFD) is a zoonotic viral disease caused by infection by a Flavivirus, a member of the family Flaviviridae. KFD is a public health concern in the Karnataka State in southern India. Available conventional diagnostic tests such as virus isolation and serological tests, such as haemagglutination inhibition and complement fixation tests are time consuming. This study reports the development of a nested RT-PCR [nRT-PCR] and a TaqMan-based real-time RT-PCR and IgM antibodies capture ELISA [MAC-ELISA] for rapid and accurate diagnosis of suspected KFD cases. The nRT-PCR and the TaqMan-based real-time RT-PCR assays were developed using gene sequences of the NS-5/non-coding region. Both the assays detected KFD viral RNA in acute phase human serum samples and can provide early diagnosis of infection. Real-time RT-PCR was found to be more sensitive than nRT-PCR, which could detect 38 copies of KFDV RNA. MAC-ELISA was developed for the detection of recent infections. Although real-time RT-PCR and nRT-PCR require expensive reagents, expensive equipment and trained personnel, the developed MAC-ELISA can be used easily in the affected areas. These tests add to the existing diagnosis arsenal against haemorrhagic viruses that are prevalent in India. These assays will also help to extend our knowledge of the pathology of KFD virus and its associated clinical features, by measuring the viral titre during infection and at the time of seroconversion. Information, which is not available currently because of the lack of appropriate diagnostic methods. In addition, early laboratory diagnosis of KFDV infection will help in the application of appropriate control measures and management of KFD cases. PMID:22874757

Mourya, Devendra T; Yadav, Pragya D; Mehla, Rajeev; Barde, Pradip V; Yergolkar, Prasanna N; Kumar, Sandeep R P; Thakare, Jyotsna P; Mishra, Akhilesh C

2012-12-01

192

Characterization of novel non-nucleoside reverse transcriptase (RT) inhibitor resistance mutations at residues 132 and 135 in the 51 kDa subunit of HIV-1 RT  

PubMed Central

Several rare and novel NNRTI [non-nucleoside reverse transcriptase (RT) inhibitor] resistance mutations were recently detected at codons 132 and 135 in RTs from clinical isolates using the yeast-based chimaeric TyHRT (Ty1/HIV-1 RT) phenotypic assay. Ile132 and Ile135 form part of the ?7–?8 loop of HIV-1 RT (residues 132–140). To elucidate the contribution of these residues in RT structure–function and drug resistance, we constructed twelve recombinant enzymes harbouring mutations at codons 132 and 135–140. Several of the mutant enzymes exhibited reduced DNA polymerase activities. Using the yeast two-hybrid assay for HIV-1 RT dimerization we show that in some instances this decrease in enzyme activity could be attributed to the mutations, in the context of the 51 kDa subunit of HIV-1 RT, disrupting the subunit–subunit interactions of the enzyme. Drug resistance analyses using purified RT, the TyHRT assay and antiviral assays demonstrated that the I132M mutation conferred high-level resistance (>10-fold) to nevirapine and delavirdine and low-level resistance (?2–3-fold) to efavirenz. The I135A and I135M mutations also conferred low level NNRTI resistance (?2-fold). Subunit selective mutagenesis studies again demonstrated that resistance was conferred via the p51 subunit of HIV-1 RT. Taken together, our results highlight a specific role of residues 132 and 135 in NNRTI resistance and a general role for residues in the ?7–?8 loop in the stability of HIV-1 RT. PMID:17286555

Nissley, Dwight V.; Radzio, Jessica; Ambrose, Zandrea; Sheen, Chih-Wei; Hamamouch, Noureddine; Moore, Katie L.; Tachedjian, Gilda; Sluis-Cremer, Nicolas

2007-01-01

193

Evidence for Extensive Polymorphism of Class I Genes in the Rat Major Histocompatibility Complex (RT1)  

Microsoft Academic Search

The major histocompatibility complex of the rat (RT1) has been poorly characterized with respect to the number, linkage, and polymorphism of class I genes. To estimate the number of class I RT1 genes and the relative extent of their polymorphism, we performed Southern blot analysis with liver DNA from rat strains expressing eight RT1 haplotypes. After digestion with EcoR1 and

Melanie Palmer; Peter J. Wettstein; Jeffrey A. Frelinger

1983-01-01

194

Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses  

Microsoft Academic Search

Three multiplex hemi-nested RT-PCR assays were developed to detect simultaneously 12 RNA respiratory viruses: influenza viruses A, B and C, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), parainfluenza virus types 1–4 (PIV-1, -2, -3 and -4), human coronavirus OC43 and 229E (HCoV) and rhinovirus (hRV). An internal amplification control was included in one of the RT-PCR assays. The RT-PCR

S. Bellau-Pujol; A. Vabret; L. Legrand; J. Dina; S. Gouarin; J. Petitjean-Lecherbonnier; B. Pozzetto; C. Ginevra; F. Freymuth

2005-01-01

195

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

Microsoft Academic Search

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 effici- ency and specificity. TaqMan miRNA assays are spe- cific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleot- ide. Furthermore, they are not

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

2005-01-01

196

The RT6 (Art2) family of ADP-ribosyltransferases in rat and mouse  

Microsoft Academic Search

Recent evidence suggests that a new member of the mono-ADP-ribosyltransferase\\/NAD glycohydrolase family, RT6, may be important in immune regulation. RT6 is expressed in two allelic forms and is present on post-thymic T cells in the rat. RT6-expressing T cells in the rat may have a regulatory role, a conclusion based on their ability to prevent autoimmune diabetes in the BB

Rita Bortell; Toshihiro Kanaitsuka; Linda A. Stevens; Joel Moss; John P. Mordes; Aldo A. Rossini; Dale L. Greiner

1999-01-01

197

A wide spectrum of collagen vascular and autoimmune diseases in transgenic rats carrying the env-pX gene of human T lymphocyte virus type I.  

PubMed

To investigate the pathogenesis of human T lymphocyte virus type I (HTLV-I)- related diseases, the env-pX gene of HTLV-I was introduced into the germline of inbred Wistar-King-Aptekman-Hokudai rats. A wide spectrum of collagen vascular diseases was evident in the transgenic rats, including chronic destructive arthritis similar to rheumatoid arthritis, necrotizing arteritis mimicking polyarteritis nodosa, polymyositis, myocarditis, dermatitis, and chronic sialoadenitis and dacryoadenitis resembling Sjögren's syndrome in humans. Thymic atrophy with the depletion of CD4 and CD8 double-positive thymocytes was also observed. In these animals, a number of autoantibodies, including high titers of rheumatoid factor, were present in the serum. We propose that the HTLV-I env-pX gene region may play a pathogenetic role in the development of collagen vascular and autoimmune diseases associated with autoimmune phenomenon. PMID:9040015

Yamazaki, H; Ikeda, H; Ishizu, A; Nakamaru, Y; Sugaya, T; Kikuchi, K; Yamada, S; Wakisaka, A; Kasai, N; Koike, T; Hatanaka, M; Yoshiki, T

1997-02-01

198

env Sequences of Simian Immunodeficiency Viruses from Chimpanzees in Cameroon Are Strongly Related to Those of Human Immunodeficiency Virus Group N from the Same Geographic Area  

PubMed Central

Human immunodeficiency virus type 1 (HIV-1) group N from Cameroon is phylogenetically close, in env, to the simian immunodeficiency virus (SIV) cpz-gab from Gabon and SIVcpz-US of unknown geographic origin. We screened 29 wild-born Cameroonian chimpanzees and found that three (Cam3, Cam4, and Cam5) were positive for HIV-1 by Western blotting. Mitochondrial DNA sequence analysis demonstrated that Cam3 and Cam5 belonged to Pan troglodytes troglodytes and that Cam4 belonged to P. t. vellerosus. Genetic analyses of the viruses together with serological data demonstrated that at least one of the two P. t. troglodytes chimpanzees (Cam5) was infected in the wild, and revealed a horizontal transmission between Cam3 and Cam4. These data confirm that P. t. troglodytes is a natural host for HIV-1-related viruses. Furthermore, they show that SIVcpz can be transmitted in captivity, from one chimpanzee subspecies to another. All three SIVcpz-cam viruses clustered with HIV-1 N in env. The full Cam3 SIVcpz genome sequence showed a very close phylogenetic relationship with SIVcpz-US, a virus identified in a P. t. troglodytes chimpanzee captured nearly 40 years earlier. Like SIVcpz-US, SIVcpz-cam3 was closely related to HIV-1 N in env, but not in pol, supporting the hypothesis that HIV-1 N results from a recombination event. SIVcpz from chimpanzees born in the wild in Cameroon are thus strongly related in env to HIV-1 N from Cameroon, demonstrating the geographic coincidence of these human and simian viruses and providing a further strong argument in favor of the origin of HIV-1 being in chimpanzees. PMID:10590144

Corbet, Sylvie; Müller-Trutwin, Michaela C.; Versmisse, Pierre; Delarue, Severine; Ayouba, Ahidjo; Lewis, John; Brunak, Soren; Martin, Paul; Brun-Vezinet, Françoise; Simon, François; Barre-Sinoussi, Françoise; Mauclere, Philippe

2000-01-01

199

env sequences of simian immunodeficiency viruses from chimpanzees in Cameroon are strongly related to those of human immunodeficiency virus group N from the same geographic area.  

PubMed

Human immunodeficiency virus type 1 (HIV-1) group N from Cameroon is phylogenetically close, in env, to the simian immunodeficiency virus (SIV) cpz-gab from Gabon and SIVcpz-US of unknown geographic origin. We screened 29 wild-born Cameroonian chimpanzees and found that three (Cam3, Cam4, and Cam5) were positive for HIV-1 by Western blotting. Mitochondrial DNA sequence analysis demonstrated that Cam3 and Cam5 belonged to Pan troglodytes troglodytes and that Cam4 belonged to P. t. vellerosus. Genetic analyses of the viruses together with serological data demonstrated that at least one of the two P. t. troglodytes chimpanzees (Cam5) was infected in the wild, and revealed a horizontal transmission between Cam3 and Cam4. These data confirm that P. t. troglodytes is a natural host for HIV-1-related viruses. Furthermore, they show that SIVcpz can be transmitted in captivity, from one chimpanzee subspecies to another. All three SIVcpz-cam viruses clustered with HIV-1 N in env. The full Cam3 SIVcpz genome sequence showed a very close phylogenetic relationship with SIVcpz-US, a virus identified in a P. t. troglodytes chimpanzee captured nearly 40 years earlier. Like SIVcpz-US, SIVcpz-cam3 was closely related to HIV-1 N in env, but not in pol, supporting the hypothesis that HIV-1 N results from a recombination event. SIVcpz from chimpanzees born in the wild in Cameroon are thus strongly related in env to HIV-1 N from Cameroon, demonstrating the geographic coincidence of these human and simian viruses and providing a further strong argument in favor of the origin of HIV-1 being in chimpanzees. PMID:10590144

Corbet, S; Müller-Trutwin, M C; Versmisse, P; Delarue, S; Ayouba, A; Lewis, J; Brunak, S; Martin, P; Brun-Vezinet, F; Simon, F; Barre-Sinoussi, F; Mauclere, P

2000-01-01

200

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

201

Preclinical and early clinical development of GNbAC1, a humanized IgG4 monoclonal antibody targeting endogenous retroviral MSRV-Env protein.  

PubMed

ABSTRACT Monoclonal antibodies (mAbs) play an increasing important role in the therapeutic armamentarium against multiple sclerosis (MS), an inflammatory and degenerative disorder of the central nervous system. Most of the mAbs currently developed for MS are immunomodulators blocking the inflammatory immune process. In contrast with mAbs targeting immune function, GNbAC1, a humanized IgG4 mAb, targets the multiple sclerosis associated retrovirus envelope (MSRV-Env) protein, an upstream factor in the pathophysiology of MS. MSRV-Env protein is of endogenous retroviral origin, expressed in MS brain lesions, and it is pro-inflammatory and toxic to the remyelination process, by preventing the differentiation of oligodendrocyte precursor cells. We present the preclinical and early clinical development results of GNbAC1. The specificity of GNbAC1 for its endogenous retroviral target is described. Efficacy of different mAb versions of GNbAC1 were assessed in MSRV-Env induced experimental allergic encephalitis (EAE), an animal model of MS. Because the target MSRV-Env is not expressed in animals, no relevant animal model exists for a proper in vivo toxicological program. An off-target 2-week toxicity study in mice was thus performed, and it showed an absence of safety risk. Additional in vitro analyses showed an absence of complement or antibody-dependent cytotoxicity as well as a low level of cross-reactivity to human tissues. The first-in-man clinical study in 33 healthy subjects and a long-term clinical study in 10 MS patients showed that GNbAC1 is well tolerated in humans without induction of immunogenicity and that it induces a pharmacodynamic response on MSRV biomarkers. These initial results suggest that the mAb GNbAC1 could be a safe long-term treatment for patients with MS with a unique therapeutic mechanism of action. PMID:25427053

Curtin, François; Perron, Hervé; Kromminga, Arno; Porchet, Hervé; Lang, Alois B

2014-11-26

202

env Sequences of Simian Immunodeficiency Viruses from Chimpanzees in Cameroon Are Strongly Related to Those of Human Immunodeficiency Virus Group N from the Same Geographic Area  

Microsoft Academic Search

Human immunodeficiency virus type 1 (HIV-1) group N from Cameroon is phylogenetically close, in env ,t o the simian immunodeficiency virus (SIV) cpz-gab from Gabon and SIVcpz-US of unknown geographic origin. We screened 29 wild-born Cameroonian chimpanzees and found that three (Cam3, Cam4, and Cam5) were positive for HIV-1 by Western blotting. Mitochondrial DNA sequence analysis demonstrated that Cam3 and

SYLVIE CORBET; MICHAELA C. MULLER-TRUTWIN; PIERRE VERSMISSE; SEVERINE DELARUE; AHIDJO AYOUBA; JOHN LEWIS; SOREN BRUNAK; PAUL MARTIN; FRANCOISE BRUN-VEZINET; FRANCOIS SIMON; FRANCOISE BARRE-SINOUSSI; PHILIPPE MAUCLERE

2000-01-01

203

Differential repositioning of the second transmembrane helices from E. coli Tar and EnvZ upon moving the flanking aromatic residues.  

PubMed

Aromatic tuning, i.e. repositioning aromatic residues found at the cytoplasmic end of transmembrane (TM) domains within bacterial receptors, has been previously shown to modulate signal output from the aspartate chemoreceptor (Tar) and the major osmosensor EnvZ of Escherichia coli. In the case of Tar, changes in signal output consistent with the vertical position of the native Trp-Tyr aromatic tandem within TM2 were observed. In contrast, within EnvZ, where a Trp-Leu-Phe aromatic triplet was repositioned, the surface that the triplet resided upon was the major determinant governing signal output. However, these studies failed to determine whether moving the aromatic residues was sufficient to physically reposition the TM helix within a membrane. Recent coarse-grained molecular dynamics (CG-MD) simulations predicted displacement of Tar TM2 upon moving the aromatic residues at the cytoplasmic end of the helix. Here, we demonstrate that repositioning the Trp-Tyr tandem within Tar TM2 displaces the C-terminal boundary of the helix relative to the membrane. In a similar analysis of EnvZ, an abrupt initial displacement of TM2 was observed but no subsequent movement was seen, suggesting that the vertical position of TM2 is not governed by the location of the Trp-Leu-Phe triplet. Our results also provide another set of experimental data, i.e. the resistance of EnvZ TM2 to being displaced upon aromatic tuning, which could be useful for subsequent refinement of the initial CG-MD simulations. Finally, we discuss the limitations of these methodologies, how moving flanking aromatic residues might impact steady-state signal output and the potential to employ aromatic tuning in other bacterial membrane-spanning receptors. PMID:25445668

Botelho, Salomé C; Enquist, Karl; von Heijne, Gunnar; Draheim, Roger R

2015-02-01

204

One-Step RT-PCR protocols improve the rate of dengue diagnosis compared to Two-Step RT-PCR approaches.  

PubMed

Dengue is the most important arboviral disease transmitted to humans. In our laboratory, we have been working on the standardization of the polymerase chain reaction (PCR) diagnosis of this disease. In this work, we compared five commercial kits regularly used on reverse-transcription polymerase chain reaction (RT-PCR) protocols: two Two-Step kits (SuperScript II RT/Super Mix kit and reverse transcription system/Taq DNA polymerase) and three One-Step kits (ready-to-go RT-PCR Beads kit, QIAGEN One-Step RT-PCR kit, and AcessQuick RT-PCR system). Thirty-one serum samples of patients with clinical diagnosis of dengue fever (DF) were analyzed by RT-PCR and serology. RNA extraction was done with the QIAamp Viral RNA kit, and cDNA synthesis and PCR done according to the manufacturer's protocol for the five kits. Out of the 31 serum samples collected from patients suspected of having dengue, 27 were IgM-positive, confirming the dengue diagnosis. Out of those, 24 were positive by the ready-to-go RT-PCR Beads kit, 25 were positive by AcessQuick RT-PCR system and 27 were positive by QIAGEN One-Step RT-PCR kit. On the other hand, only six samples were positive by the SuperScript II RT/Super Mix kits and 10 were positive by reverse transcription system/Taq DNA polymerase kit. The best performance observed with the One-Step kits was confirmed in spiked samples with known quantities of dengue-1 virus since they detected up 1 x 10(2) PFU/ml, while the most sensitive Two-Step kit detected up 1 x 10(4) PFU/ml. These data show that One-Step RT-PCR kits yielded a higher rate of dengue virus detection than the Two-Step kits and correlated well with the serological diagnosis. PMID:15163417

De Paula, Sérgio Oliveira; de Melo Lima, Cristiane; Torres, Maria Paula; Pereira, Márcia Rodrigues Garbin; Lopes da Fonseca, Benedito Antônio

2004-08-01

205

In vitro cell fusion between CD4(+) and HIV-1 Env(+) T cells generates a diversity of syncytia varying in total number, size and cellular content.  

PubMed

Syncytia formation in HIV infections is driven by the virus fusion-active molecules (Env) interacting with membrane components of hosts cells. HIV-syncytia are usually interpreted as pathogenic entities and although they may potentially vary in size, numbers and types of constituent cells, little is known about the extent and significance of their diversity. Here, we describe numerically the cell population dynamics and the diversity of syncytia produced in the in vitro cell-fusion between two Jurkat T cell lines, one CD4(+) and the other Env(+). Cell-fusion partners were differentially stained with the lipophilic DiI and DiO, or with the cytoplasmic CMFDA and CMTMR tracers and syncytia showing double fluorescence were counted in a flow cytometer. The total number of syncytia formed, their size, cellular complexity and ratio of CD4(+)/Env(+) cells recruited, varied significantly in relation with time of reaction and initial proportions of fusion partners. The considerable structural diversity of syncytia formed, in so limited an in vitro cell fusion reaction, suggests that a greater heterogeneity may be formed in the natural course of disease. Identification of the main determinants of syncytia diversity allows for a detailed study of the relation between the syncytia structure and function. PMID:17014923

López-Balderas, N; Huerta, L; Villarreal, C; Rivera-Toledo, E; Sandoval, G; Larralde, C; Lamoyi, E

2007-02-01

206

HIV-1 Neutralization Profile and Plant-Based Recombinant Expression of Actinohivin, an Env Glycan- Specific Lectin Devoid of T-Cell Mitogenic Activity  

E-print Network

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, and recombinant producibility. Two validated assay systems based on human peripheral blood mononuclear cell (hPBMC) infection with primary isolates and TZM-bl cell infection with Env-pseudotyped viruses were employed to characterize AH’s anti-HIV-1 activity. In hPMBCs, AH exhibited nanomolar neutralizing activity against primary viruses with diverse cellular tropisms, but did not cause mitogenicity or cytotoxicity that are often associated with other anti-HIV lectins. In the TZM-bl-based assay, AH showed broad anti-HIV-1 activity against clinically-relevant, mucosally transmitting strains of clades B and C. By contrast, clade A viruses showed strong resistance to AH. Correlation analysis suggested that HIV-19s AH susceptibility is significantly linked to the N-glycans at the Env C2 and V4 regions. For

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; Carl V. Hanson; Haruo Tanaka

207

Generation of a monkey-tropic human immunodeficiency virus type 1 carrying env from a CCR5-tropic subtype C clinical isolate.  

PubMed

Several derivatives of human immunodeficiency virus type 1 (HIV-1) that evade macaque restriction factors and establish infection in pig-tailed macaques (PtMs) have been described. These monkey-tropic HIV-1s utilize CXCR4 as a co-receptor that differs from CCR5 used by most currently circulating HIV-1 strains. We generated a new monkey-tropic HIV-1 carrying env from a CCR5-tropic subtype C HIV-1 clinical isolate. Using intracellular homologous recombination, we generated an uncloned chimeric virus consisting of at least seven types of recombination breakpoints in the region between vpr and env. The virus increased its replication capacity while maintaining CCR5 tropism after in vitro passage in PtM primary lymphocytes. PtM infection with the adapted virus exhibited high peak viremia levels in plasma while the virus was undetectable at 12-16 weeks. This virus serves as starting point for generating a pathogenic monkey-tropic HIV-1 with CCR5-tropic subtype C env, perhaps through serial passage in macaques. PMID:25010265

Otsuki, Hiroyuki; Yoneda, Mai; Igarashi, Tatsuhiko; Miura, Tomoyuki

2014-07-01

208

The Environmental-Data Automated Track Annotation (Env-DATA) System: Linking Animal Tracks with Environmental Data  

NASA Astrophysics Data System (ADS)

The movement of animals is strongly influenced by external factors in their surrounding environment such as weather, habitat types, and human land use. With the advances in positioning and sensor technologies, it is now possible to capture data of animal locations at high spatial and temporal granularities. Likewise, modern technology provides us with an increasing access to large volumes of environmental data, some of which changes on an hourly basis. Although there have been strong developments in computational methods for the analysis of movement in its environmental context, there remain challenges in efficiently linking the spatiotemporal locations of animals with the appropriate environmental conditions along their trajectories. To this end, our new Environmental-Data Automated Track Annotation (Env-DATA) system enhances Movebank, an open portal of animal tracking data, by automating access to environmental variables from global remote sensing, weather, and ecosystem products. The system automates the download and decryption of the data from open web resources of remote sensing and weather data, and provides several interpolation methods from the native grid resolution and structure to a global regular grid linked with the movement tracks in space and time. The system is open and free to any user with movement data. The aim is to facilitate new understanding and predictive capabilities of spatiotemporal patterns of animal movement in response to dynamic and changing environments from local to global scales. The system is illustrated with a series of case studies of pan-American migrations of turkey vultures, and foraging flights of Galapagos Albatross.

Bohrer, G.; Dodge, S.; Weinzierl, R.; Davidson, S. C.; Kays, R.; Douglas, D. C.; Brandes, D.; Bildstein, K.; Wikelski, M.

2013-12-01

209

Loss of antigenic epitopes as the result of env gene recombination in retrovirus-induced leukemia in immunocompetent mice.  

PubMed

The murine leukemia virus, E-55+ virus, induces a thymic lymphoma/leukemia in 100% of BALB.K mice infected as adults after a latent period of 4 months or more (Pozsgay et al., Virology 173, 330-334, 1989). Two molecular clones of virus designated E-55+ and E-55- based on their ability to encode the E-55 epitope detected by the monoclonal antibody 55 (mAb 55) were isolated from a leukemic BALB.K mouse inoculated with a biologically cloned E-55+ virus (Chesebro et al., Virology 112, 131-144, 1981). Env gene sequence analysis of E-55+ and E-55- clones showed that the E-55- virus was generated from the E-55+ virus as the result of a recombination between E-55+ virus and the endogenous ecotropic virus, emv-1, carried in the genome of the BALB.K mouse strain. The recombinant E-55- virus is replication competent. This recombination event and the consequential expression of E-55- virus consistently occur in immunocompetent BALB.K mice inoculated with the E-55+ virus and appear to play a role in the loss of epitopes recognized by virus neutralizing antibodies. The loss of these epitopes apparently allows the virus to evade the host immune response. PMID:7678475

Tumas, K M; Poszgay, J M; Avidan, N; Ksiazek, S J; Overmoyer, B; Blank, K J; Prystowsky, M B

1993-02-01

210

Advances in Lecture Recognition: The ISL RT-06S Evaluation System Christian Fugen1  

E-print Network

Advances in Lecture Recognition: The ISL RT-06S Evaluation System Christian F¨ugen1 , Matthias W lecture recognition system devel- oped at the Interactive Systems Laboratories (ISL), for individual head recognition, lectures, distant speech, CHIL, RT-06S. 1. Introduction In this paper, we present the ISL's most

Schultz, Tanja

211

Convergent Evolution of Reverse Transcriptase (RT) Genes of Human Immunodeficiency Virus Type 1 Subtypes E and B following Nucleoside Analogue RT Inhibitor Therapies  

PubMed Central

Changes in the drug susceptibility, gene lineage, and deduced amino acid sequences of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) subtype E following 3?-azido-3?-deoxythymidine (AZT) monotherapy or AZT–2?,3?-dideoxyinosine combination therapy were examined with sequential virus isolates from a single family. The changes were compared to those reported for HIV-1 subtype B, revealing striking similarities in selected phenotype and amino acids independent of differences in the RT backbone sequences that constantly distinguish the two subtypes. Particularly, identical amino acid substitutions were present simultaneously at four different positions (D67N, K70R, T215F, and K219Q) for high-level AZT resistance. These data suggest that HIV-1 subtypes E and B evolve convergently at the phenotypic and amino acid levels when the nucleoside analogue RT inhibitors act as selective forces. PMID:10799614

Sato, Hironori; Tomita, Yasuhiro; Shibamura, Kayo; Shiino, Teiichiro; Miyakuni, Tuyoshi; Takebe, Yutaka

2000-01-01

212

Molecular and biological characterization of simian-human immunodeficiency virus-like particles produced by recombinant fowlpox viruses  

Microsoft Academic Search

Virus-like particles (VLPs) mimicking the simian-human immunodeficiency virus SHIV89.6P (VLPSHIV) were produced by co-infection of Vero cells with fowlpox SIVgag\\/pol (FPgag\\/polSIV) and fowlpox HIV-1env89.6P (FPenv89.6P) recombinant viruses. As a necessary prerequisite for a more efficient vaccine approach, ultrastructural, functional and molecular characterizations of VLPSHIV were performed in the SHIV-macaque model to verify the similarity of these particles to SHIV89.6P. Here

Carlo Zanotto; Manuela Paganini; Veronica Elli; Valeria Basavecchia; Margherita Neri; Carlo De Giuli Morghen; Antonia Radaelli

2005-01-01

213

Pyruvate minimizes rtPA toxicity from in vitro oxygen-glucose deprivation and reoxygenation.  

PubMed

Clinical application of recombinant tissue plasminogen activator (rtPA) for stroke is limited by hemorrhagic transformation, which narrows rtPA's therapeutic window. In addition, mounting evidence indicates that rtPA is potentially neurotoxic if it traverses a compromised blood brain barrier. Here, we demonstrated that pyruvate protects cultured HT22 neuronal and primary microvascular endothelial cells co-cultured with primary astrocytes from oxygen glucose deprivation (OGD)/reoxygenation stress and rtPA cytotoxicity. After 3 or 6h OGD, cells were reoxygenated with 11mmol/L glucose±pyruvate (8mmol/L) and/or rtPA (10µg/ml). Measured variables included cellular viability (calcein AM and annexin-V/propidium iodide), reactive oxygen species (ROS; mitosox red and 2',7'-dichlorofluorescein diacetate), NADPH, NADP(+) and ATP contents (spectrophotometry), matrix metalloproteinase-2 (MMP2) activities (gelatin zymography), and cellular contents of MMP2, tissue inhibitor of metalloproteinase-2 (TIMP2), and phosphor-activation of anti-apoptotic p70s6 kinase, Akt and Erk (immunoblot). Pyruvate prevented the loss of HT22 cells after 3h OGD±rtPA. After 6h OGD, rtPA sharply lowered cell viability; pyruvate dampened this effect. Three hours OGD and 4h reoxygenation with rtPA increased ROS formation by about 50%. Pyruvate prevented this ROS formation and doubled cellular NADPH/NADP(+) ratio and ATP content. In endothelial cell monolayers, 3h OGD and 24h reoxygenation increased FITC-dextran leakage, indicating disruption of intercellular junctions. Although rtPA exacerbated this effect, pyruvate prevented it while sharply lowering MMP2/TIMP2 ratio and increasing phosphorylation of p70s6 kinase, Akt and Erk. Pyruvate protects neuronal cells and microvascular endothelium from hypoxia-reoxygenation and cytotoxic action of rtPA while reducing ROS and activating anti-apoptotic signaling. These results support the proposed use of pyruvate as an adjuvant to dampen the side effects of rtPA treatment, thereby extending rtPA's therapeutic window. PMID:23891792

Ryou, Myoung-Gwi; Choudhury, Gourav Roy; Winters, Ali; Xie, Luokun; Mallet, Robert T; Yang, Shao-Hua

2013-09-12

214

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. 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.

2013-01-01

215

Transition and Evaluation of RGB Imagery to WFOs and National Centers by NASA SPoRT  

NASA Technical Reports Server (NTRS)

MODIS Snow/Cloud and True Color RGB imagery has been used by SPoRT partners since 2004 to examine changes in surface features such as snow cover, vegetation, ocean color, fires, smoke plumes, and oil spills.

Fuell, Kevin K.; Molthan, Andrew L.

2012-01-01

216

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

NASA Technical Reports Server (NTRS)

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 method to more efficiently handle the increase channels/frequency of ABI. Challenges for RGB production exist. Internal vs. external production, Bit depth needed, Adding quantitative information, etc. SPoRT forming group to address these issues. SPoRT is leading efforts on the application of total lightning in operations and to educate users of this new capability. Training in many forms is used to support testbed activities and is a key part to the transition process.

Fuell, Kevin; Jedlovec, Gary; Molthan, Andrew; Stano, Geoffrey

2012-01-01

217

Development of Real Time RT-PCR Assays for Neuraminidase Subtyping of  

E-print Network

Development of Real Time RT-PCR Assays for Neuraminidase Subtyping of Avian Influenza Virus Yanyan glycoproteins, hemagglutinin (HA) and neuraminidase (NA). To date, there are total 16 HA and 9 NA subtypes

Mandoiu, Ion

218

Discrimination of infectious hepatitis A viruses by propidium monoazide real-time RT-PCR.  

PubMed

The discrimination of infectious and inactivated viruses remains a key obstacle when using quantitative RT-PCR (RT-qPCR) to quantify enteric viruses. In this study, propidium monoazide (PMA) and RNase pretreatments were evaluated for the detection and quantification of infectious hepatitis A virus (HAV). For thermally inactivated HAV, PMA treatment was more effective than RNase treatment for differentiating infectious and inactivated viruses, with HAV titers reduced by more than 2.4 log(10) units. Results showed that combining 50 ?M of PMA and RT-qPCR selectively quantify infectious HAV in media suspensions. Therefore, PMA treatment previous to RT-qPCR detection is a promising alternative to assess HAV infectivity. PMID:23412764

Sánchez, Gloria; Elizaquível, Patricia; Aznar, Rosa

2012-03-01

219

Ethidium monoazide does not inhibit RT-PCR amplification of nonviable avian influenza RNA.  

PubMed

A critical obstacle to using PCR to quantify viral titers is the distinguishment of viable and nonviable genomic material. Pretreatments of ethidium monoazide (EMA) have been effective in preventing PCR amplification of DNA from nonviable bacteria. To test whether an EMA pretreatment could be used with RT-PCR to quantify viable RNA virions, avian influenza virus (AIV) survival was measured in water through 28d using cell culture titration and real-time RT-PCR with or without EMA pretreatment. Cell culture titration yielded significantly lower titers than both RT-PCR procedures, and there was no significant difference between RT-PCR results with or without EMA. Ineffective binding of EMA to AIV RNA may have allowed nonviable AIV RNA to amplify. Furthermore, since AIV inactivation may take place by means other than membrane disruption, any pretreatment distinguishing viable and nonviable AIV virions by membrane integrity may not be practical. PMID:19941905

Graiver, David A; Saunders, Samuel E; Topliff, Christina L; Kelling, Clayton L; Bartelt-Hunt, Shannon L

2010-03-01

220

Detection of Banana mild mosaic virus and Banana virus X by polyvalent degenerate oligonucleotide RT-PCR (PDO-RT-PCR).  

PubMed

Viruses are important constraints to the movement and propagation of plant germplasm, especially for vegetatively propagated crops such as banana and plantain. Their control relies primarily on the use of virus-free plant material, whose production and certification requires sensitive and reliable detection methods. An existing polyvalent degenerate oligonucleotide RT-PCR (PDO-RT-PCR) assay was adapted to the detection of Banana mild mosaic virus (BanMMV) and Banana virus X, two Flexiviridae infecting Musa spp. PDO inosine-containing primers were found to be well suited to the detection of BanMMV, despite its high molecular diversity, but not to that of the highly conserved BVX, for which species-specific primers were designed. Sampling and sample processing steps were optimized in order to avoid nucleic acid purification prior to the reverse transcription step. A polyclonal anti-BanMMV antiserum was raised and successfully used for the immunocapture (IC) of BanMMV viral particles from leaf extracts, leading to the development of a PDO-IC-RT-nested PCR assay. Although the anti-BanMMV antiserum could to some extent recognize BVX viral particles, direct binding (DB) was shown to be a more efficient method for processing BVX-infected samples and a PDO-DB-RT-nested PCR assay was developed for the detection of BVX from leaf extracts. PMID:17280722

Teycheney, Pierre-Yves; Acina, Isabelle; Lockhart, Benham E L; Candresse, Thierry

2007-06-01

221

Single cell RT-PCR proceeds without the risk of genomic DNA amplification  

Microsoft Academic Search

We have previously described a method for detection of mRNAs expressed in single cells after patch-clamp recordings. The method, termed single cell RT-PCR, involves aspiration of the cell content, a reverse transcription (RT) step, and a polymerase chain reaction (PCR) using specific primers. Since the nucleus is frequently harvested together with the cytosol, genomic DNA may generate false positive results.

Flemming Fryd Johansen; Bertrand Lambolez; Etienne Audinat; Pascal Bochet; Jean Rossier

1995-01-01

222

A period investigation of two chromospherically active binary stars: RT Coronae Borealis and PW Herculis  

Microsoft Academic Search

Orbital period variations of two chromospherically active binary systems, RT CrB and PW Her, are presented. It is shown that the orbital period of RT CrB undergoes a cyclic oscillation with a period of 53.9years. For PW Her, an alternate change, with a period of 42.7years, is found to superimpose on a rapid secular increase (dP\\/dt=+3.53×10?6 days\\/year). If the period

S. B Qian; L. Y Zhu; J. J He; S Boonruksar

2003-01-01

223

Short-Term Prediction Research and Transition (SPoRT) Center: Transitioning Satellite Data to Operations  

NASA Technical Reports Server (NTRS)

The Short-term Prediction Research and Transition (SPoRT) Center located at NASA Marshall Space Flight Center has been conducting testbed activities aimed at transitioning satellite products to National Weather Service operational end users for the last 10 years. SPoRT is a NASA/NOAA funded project that has set the bar for transition of products to operational end users through a paradigm of understanding forecast challenges and forecaster needs, displaying products in end users decision support systems, actively assessing the operational impact of these products, and improving products based on forecaster feedback. Aiming for quality partnerships rather than a large quantity of data users, SPoRT has become a community leader in training operational forecasters on the use of up-and-coming satellite data through the use of legacy instruments and proxy data. Traditionally, SPoRT has supplied satellite imagery and products from NASA instruments such as the Moderate-resolution Imaging Spectroradiometer (MODIS) and the Atmospheric Infrared Sounder (AIRS). However, recently, SPoRT has been funded by the GOES-R and Joint Polar Satellite System (JPSS) Proving Grounds to accelerate the transition of selected imagery and products to help improve forecaster awareness of upcoming operational data from the Visible Infrared Imager Radiometer Suite (VIIRS), Cross-track Infrared Sounder (CrIS), Advanced Baseline Imager (ABI), and Geostationary Lightning Mapper (GLM). This presentation provides background on the SPoRT Center, the SPoRT paradigm, and some example products that SPoRT is excited to work with forecasters to evaluate.

Zavodsky, Bradley

2012-01-01

224

The T Cell Marker RT6 in A Rat Model of Autoimmune Diabetes  

Microsoft Academic Search

\\u000a The RT6 alloantigenic system of the rat was discovered in the 1970s. It was originally designated Pta, AgF, A.R.T.-2, and\\u000a RTLy-2 by the laboratories involved in its characterization. Exchange of reagents demonstrated that these laboratories had\\u000a identified the same system, and the official designation RT6 was assigned in 1982 (1).

Dale L. Greiner; Samir Malkani; Toshihiro Kanaitsuka; Rita Bortell; John Doukas; Mark Rigby; Barbara Whalen; Linda A. Stevens; Joel Moss; John P. Mordes; Aldo A. Rossini

225

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

NASA Technical Reports Server (NTRS)

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 Forecasting (WRF)WRF (ARW) that integrates all SPoRT modeling research data: (1) 2-km SPoRT Sea Surface Temperature (SST) Composite, (2) 3-km LIS with 1-km Greenness Vegetation Fraction (GVFs) (3) 45-km AIRS retrieved profiles. Transitioned this real-time forecast to NOAA's Hazardous Weather Testbed (HWT) as deterministic model at Experimental Forecast Program (EFP). Feedback from forecasters/participants and internal evaluation of SPoRT-WRF shows a cool, dry bias that appears to suppress convection likely related to methodology for assimilation of AIRS profiles Version 2 of the SPoRT-WRF will premier at the 2012 EFP and include NASA physics, cycling data assimilation methodology, better coverage of precipitation forcing, and new GVFs

Zavodsky, Bradley; Kozlowski, Danielle; Case, Jonathan; Molthan, Andrew

2012-01-01

226

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. PMID:16785513

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

2008-01-01

227

Identification of a new epitope for HIV-neutralizing antibodies in the gp41 membrane proximal external region by an Env-tailored phage display library.  

PubMed

HIV controllers are a valuable source for the identification of HIV-neutralizing antibodies, as chronic infection over decades allows extensive affinity maturation of antibodies for improved Ag recognition. We analyzed a small cohort of elite controllers (ECs) for HIV-neutralizing antibodies using a panel of standardized HIV-1 pseudovirions on TZM-bl cells. An HIV-1 Env-tailored phage display library was generated to select epitopes targeted by neutralizing antibodies in the EC26 plasma sample showing the broadest neutralizing activity. Selected Env fragments were mostly allocated to the membrane proximal external region of gp41. After preabsorbing the EC26 plasma with the selected phage EC26-2A4, we achieved 50% depletion of its neutralizing activity. Furthermore, antibodies affinity-purified with the EC26-2A4 epitope from EC26 plasma showed neutralizing activity, proving that the selected phage indeed contains an epitope targeted by neutralizing plasma antibodies. Epitope fine mapping of the purified plasma antibodies on peptide arrays identified a new epitope overlapping, but clearly distinct, from the prominent 2F5 epitope. Of note, the purified antibodies did not show autoreactivity with cardiolipin, whereas low reactivity with phosphatidylserine comparable to mAb 2F5 was observed. Thus, this new epitope represents a promising candidate for further analysis in view of HIV vaccine development. PMID:23180650

Zhou, Mingkui; Meyer, Torsten; Koch, Stefanie; Koch, Joachim; von Briesen, Hagen; Benito, José M; Soriano, Vincent; Haberl, Annette; Bickel, Markus; Dübel, Stefan; Hust, Michael; Dietrich, Ursula

2013-02-01

228

STEM-LOOP RT-qPCR for miRNAS  

PubMed Central

This unit presents a specific and sensitive quantitative reverse-transcription PCR (RT-qPCR) method for measuring individual microRNAs (miRNAs) in tissue or cultured cells. MiRNAs are 17 – 24 nucleotides (nt) in length. Standard and quantitative PCR methods require a template that is at least twice the length of either of the specific forward or reverse primers, each typically ? 20 nt in length. Thus, the target minimum length is ? 40 nt, making miRNAs too short for standard RT-qPCR methods. In this assay, each of the RT-qPCR nucleic acid reagents, including the RT-primer, the forward and reverse PCR primers, and the hydrolysis probe, contain design features that, together, optimize miRNA specificity and assay sensitivity. The RT-primer contains a highly stable stem-loop structure that lengthens the target cDNA. The forward PCR primer adds additional length with nucleotides that optimize its melting temperature (Tm) and enhance assay specificity. The reverse primer disrupts the stem loop. Assay specificity is further optimized by placement of the probe over much of the original miRNA sequence, and the probe Tm is optimized by addition of a minor groove binding (MGB) moiety. PMID:21732315

Kramer, Martha F.

2011-01-01

229

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

Microsoft Academic Search

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

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

230

Ontogeny and immunohistochemical localization of thymus-dependent and thymus-independent RT6+ cells in the rat.  

PubMed Central

RT6 is a cell surface alloantigen that identifies a regulatory subset of peripheral T cells in the rat. Diabetes-prone BB rats are deficient in peripheral RT6+ T cells and develop spontaneous autoimmune insulin-dependent diabetes mellitus. Diabetes-resistant BB rats have normal numbers of RT6+ T cells, and insulin-dependent diabetes mellitus can be induced in these animals by in vivo depletion of peripheral RT6+ cells. Athymic rats are also severely deficient in peripheral RT6+ T cells. Although very different with respect to the peripheral RT6+ cell compartment, normal, athymic, and diabetes-prone BB rats all generate RT6+ intestinal epithelial lymphocytes (IELs). The goal of these studies was to analyze the ontogeny of RT6+ IELs and peripheral lymphoid cells by in situ immunohistochemistry. We observed the following. 1) RT6+ IELs appear before alpha(beta) T-cell-receptor- expressing IELs in diabetes-prone BB, diabetes-resistant BB, and athymic WAG rats. 2) In vivo depletion of peripheral RT6+ T cells in diabetes-resistant BB rats using a cytotoxic monoclonal antibody is not accompanied by depletion of RT6+ IELs. 3) A population of RT6+ T-cell-receptor-negative IELs is present in normal, euthymic diabetes-resistant BB rats, constitutes a larger percentage of the euthymic but lymphopenic diabetes-prone BB rat IEL population, and is the predominant IEL phenotype in athymic WAG rats. These results suggest that RT6+ cells are composed of both thymus-dependent and thymus-independent cell subsets that have different developmental characteristics and may differ in function. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8669488

Waite, D. J.; Appel, M. C.; Handler, E. S.; Mordes, J. P.; Rossini, A. A.; Greiner, D. L.

1996-01-01

231

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. PMID:23487248

Bittker, Joshua A.

2012-01-01

232

[Angioneurotic orolingual edema associated with the use of rt-PA following a stroke].  

PubMed

Angioneurotic orolingual edema associated with the use of rt-PA (recombinant tissue plasminogen activator) for systemic thrombolysis are described in the literature, but only as isolated case reports. Strangely, the rate of anaphylactic reactions to rt-PA is higher (1.9%) when they are used in the treatment of acute stroke than when they are given to treat acute myocardial infarction (0.02%). Patients who are taking ACE inhibitors seem to be at increased risk of such a potentially life-threatening event. We now report on two patients, in each of whom asymmetric angioneurotic edema was observed following successful thrombolysis with rt-PA. Both these patients were taking ACE inhibitors. It was possible to avoid intubation and ventilation in both cases. Therapy with ranitidine, clemastine, and a C1 esterase inhibitor resulted in the resolution of symptomatic angioneurotic edema within hours. PMID:17694290

Laubinger, R; Guthke, K; Erdmann, U; Klein, U

2007-10-01

233

In-flight demonstration of a Real-Time Flush Airdata Sensing (RT-FADS) system  

NASA Technical Reports Server (NTRS)

A prototype real-time flush airdata sensing (RT-FADS) system has been developed and flight tested at the NASA Dryden Flight Research Center. This system uses a matrix of pressure orifices on the vehicle nose to estimate airdata parameters in real time using nonlinear regression. The algorithm is robust to sensor failures and noise in the measured pressures. The RT-FADS system has been calibrated using inertial trajectory measurements that were bootstrapped for atmospheric conditions using meteorological data. Mach numbers as high as 1.6 and angles of attack greater than 45 deg have been tested. The system performance has been evaluated by comparing the RT-FADS to the ship system airdata computer measurements to give a quantitative evaluation relative to an accepted measurement standard. Nominal agreements of approximately 0.003 in Mach number and 0.20 deg in angle of attack and angle of sideslip have been achieved.

Whitmore, Stephen A.; Davis, Roy J.; Fife, John Michael

1995-01-01

234

Inactivation conditions for human norovirus measured by an in situ capture-qRT-PCR method.  

PubMed

Human norovirus (HuNoV) is a leading cause of foodborne gastroenteritis. Unfortunately, the inactivation parameters for HuNoV in clinical, food and environmental samples have not been established. Due to the inability to cultivate HuNoV in vitro, quantitative real-time RT-PCR (qRT-PCR) is widely-used for detecting HuNoVs. However, qRT-PCR does not indicate viral infectivity. Our method employs histo-blood group antigens (HBGAs) as viral receptors/co-receptors and container-affixed capture agents to concentrate HuNoVs. The captured viruses are denatured and its genome is amplified in the same module by in situ capture qRT-PCR (ISC-qRT-PCR). Greater than three log10 reduction in the receptor-captured viral genomic signal (RCVGS) was observed when HuNoV was treated by heat at 72 °C for 4 min, by chlorine at a final concentration of 16 ppm in less than 1 min, and by UV irradiation at 1J/cm². Treatment of low-titer HuNoV (<10³ copies/sample) with 70% ethanol for 20 s reduced the RCVGS of HuNoV by two log10. However, ethanol had a limited effect on high-titer samples of HuNoV (>10³ copies/sample). The results demonstrate that ISC-qRT-PCR method could be used as an alternative method to measure encapsidated viral RNA and indirectly indicate the inactivation status of HuNoV caused by physical treatment such as heat, and chemical treatment such as chlorine, that damage the ability of the virus to bind to its receptor. PMID:24361836

Wang, Dapeng; Tian, Peng

2014-02-17

235

Generation and characterization of a Tet-On (rtTA-M2) transgenic rat  

PubMed Central

Background The tetracycline-inducible gene regulation system is a powerful tool that allows temporal and dose-dependent regulation of target transgene expression in vitro and in vivo. Several tetracycline-inducible transgenic mouse models have been described with ubiquitous or tissue-specific expression of tetracycline-transactivator (tTA), reverse tetracycline-transactivator (rtTA) or Tet repressor (TetR). Here we describe a Tet-On transgenic rat that ubiquitously expresses rtTA-M2 driven by the murine ROSA 26 promoter. Results The homozygous rat line (ROSA-rtTA-M2) generated by lentiviral vector injection, has a single integration site and was derived from the offspring of a genetic mosaic founder with multiple transgene integrations. The rtTA-M2 transgene integrated into an intron of a putative gene on chromosome 2 and does not appear to affect the tissue-specificity or expression of that gene. Fibroblasts from the ROSA-rtTA-M2 rats were transduced with a TetO7/CMV-EGFP lentivirus and exhibited doxycycline dose-dependent expression of the EGFP reporter transgene, in vitro. In addition, doxycycline-inducible EGFP expression was observed, in vivo, when the TetO7/CMV-EGFP lentivirus was injected into testis, kidney and muscle tissues of ROSA-rtTA-M2 rats. Conclusions This conditional expression rat model may have application for transgenic overexpression or knockdown studies of gene function in development, disease and gene therapy. PMID:20158911

2010-01-01

236

Identification of three feline immunodeficiency virus (FIV) env gene subtypes and comparison of the FIV and human immunodeficiency virus type 1 evolutionary patterns.  

PubMed Central

Feline immunodeficiency virus (FIV) is a lentivirus associated with AIDS-like illnesses in cats. As such, FIV appears to be a feline analog of human immunodeficiency virus (HIV). A hallmark of HIV infection is the large degree of viral genetic diversity that can develop within an infected individual and the even greater and continually increasing level of diversity among virus isolates from different individuals. Our goal in this study was to determine patterns of FIV genetic diversity by focusing on a 684-nucleotide region encompassing variable regions V3, V4, and V5 of the FIV env gene in order to establish parallels and distinctions between FIV and HIV type 1 (HIV-1). Our data demonstrate that, like HIV-1, FIV can be separated into distinct envelope sequence subtypes (three are described here). Similar to that found for HIV-1, the pairwise sequence divergence within an FIV subtype ranged from 2.5 to 15.0%, whereas that between subtypes ranged from 17.8 to 26.2%. However, the high number of synonymous nucleotide changes among FIV V3 to V5 env sequences may also include a significant number of back mutations and suggests that the evolutionary distances among FIV subtypes are underestimated. Although only a few subtype B viruses were available for examination, the pattern of diversity between the FIV A and B subtypes was found to be significantly distinct; subtype B sequences had proportionally fewer mutations that changed amino acids, compared with silent changes, suggesting a more advanced state of adaptation to the host. No similar distinction was evident for HIV-1 subtypes. The diversity of FIV genomes within individual infected cats was found to be as high as 3.7% yet twofold lower than that within HIV-1-infected people over a comparable region of the env gene. Despite these differences, significant parallels between patterns of FIV evolution and HIV-1 evolution exist, indicating that a wide array of potentially divergent virus challenges need to be considered in FIV vaccine and pathogenesis studies. PMID:8139008

Sodora, D L; Shpaer, E G; Kitchell, B E; Dow, S W; Hoover, E A; Mullins, J I

1994-01-01

237

Gene expression analysis in early embryos through reverse transcription quantitative PCR (RT-qPCR).  

PubMed

Real-time, reverse transcription quantitative PCR (RT-qPCR) is a highly sensitive and reproducible technology for the analysis of gene expression patterns. Its ability to detect minute quantities of nucleic acid from multifarious sources makes it an ideal technique for embryonic transcript quantification. However, complex cellular diversity and active transcriptome dynamics in early embryos necessitate particular caution to avoid erroneous results. This chapter is intended to outline basic methodology to design and execute RT-qPCR experiments in pre-implantation embryos. PMID:25287347

Peynot, Nathalie; Duranthon, Véronique; Khan, Daulat Raheem

2015-01-01

238

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

239

Class I and class II restriction pattern polymorphisms associated with independently derived RT1 haplotypes in inbred rats  

Microsoft Academic Search

This communication reports the DNA level identification of class I and class II sequences associated with 20 RT1 haplotypes which have been assigned previously to eight RT1 groups. Sixteen to 22 bands in genomic blots hybridized with the mouse pH-2III class I cDNA probe. Only the three RT1khaplotypes associated with identical class I restriction fragment patterns. Differences in restriction bands

Peter J. Wettstein; Susan Faas; David A. Buck

1985-01-01

240

Standardized quantitative RT-PCR assays for quantitation of yellow fever and chimeric yellow fever–dengue vaccines  

Microsoft Academic Search

Yellow fever–dengue chimeras (CYDs) are being developed currently as live tetravalent dengue vaccine candidates. Specific quantitative assays are needed to evaluate the viral load of each serotype in vaccine batches and biological samples. A quantitative real-time RT-PCR (qRT-PCR) system was developed comprising five one-step qRT-PCRs targeting the E\\/NS1 junction of each chimera, or the NS5 gene in the yellow fever

N. Mantel; M. Aguirre; S. Gulia; Y. Girerd-Chambaz; S. Colombani; C. Moste; V. Barban

2008-01-01

241

[A protease of the external membrane of Escherichia coli sensitive to environmental conditions. Its relations with the expression of envZ gene].  

PubMed

A mutant strain (called Cpr), devoid of the outer membrane protease that cleaves colicin A, has been isolated. The location on the genetic map of the cpr locus as well as its pleiotropic effects concerning chiefly: the protein composition of the outer membrane, sensitivity to phages and colicins, alteration in protease activity (cpr), are very similar to the location on the genetic map and to phenotypic properties observed in strains tpo, perA or envZ which are altered in the ompB locus. Conditions resulting in inhibition of the colicin A protease activity also result in transcriptional regulation of OmpF, OmpC and LamB proteins synthesis. The possibility for this protease to be an osmosensor of the cell's external environment is discussed. PMID:6820310

Cavard, D; Pagès, J M; Lazdunski, C

1982-12-20

242

Expression, refolding and preliminary X-ray crystallographic analysis of equine MHC class I molecule complexed with an EIAV-Env CTL epitope.  

PubMed

In order to clarify the structure and the peptide-presentation characteristics of the equine major histocompatibility complex (MHC) class I molecule, a complex of equine MHC class I molecule (ELA-A1 haplotype, 7-6 allele) with mouse ?(2)-microglobulin and the cytotoxic T lymphocyte (CTL) epitope Env-RW12 (RVEDVTNTAEYW) derived from equine infectious anaemia virus (EIAV) envelope protein (residues 195-206) was refolded and crystallized. The crystal, which belonged to space group P2(1), diffracted to 2.3 Å resolution and had unit-cell parameters a = 82.5, b = 71.4, c = 99.8 Å, ? = 102.9°. The crystal structure contained two molecules in the asymmetric unit. These results should help to determine the first equine MHC class I molecule structure presenting an EIAV CTL epitope. PMID:22232164

Yao, Shugang; Qi, Jianxun; Liu, Jun; Chen, Rong; Pan, Xiaocheng; Li, Xiaoying; Gao, Feng; Xia, Chun

2012-01-01

243

Evolution of human immunodeficiency virus type 1 env sequence variation in patients with diverse rates of disease progression and T-cell function.  

PubMed Central

We examined the relationship between env sequence variation and disease progression in 10 human immunodeficiency virus type 1 (HIV-1)-seropositive subjects selected from a longitudinal cohort receiving zidovudine therapy. Five subjects were chosen for stable clinical status and CD4 counts (slow progressors), and five were selected for rapid clinical deterioration and CD4 count decline (rapid progressors). The slow progressors had significantly lower plasma viral RNA loads and greater lymphoproliferative responses to mitogens than the rapid progressors. DNA sequences representing the C1 through C3 regions of env were amplified from two peripheral blood mononuclear cell DNA samples from each subject separated by an average of 2.5 years. Molecular clones of these amplicons were then sequenced, and DNA sequence and deduced amino acid sequence distances were compared. Inter-time point sequence comparison showed a higher rate of sequence evolution for the rapid progressors in three of five matched pairs of rapid progressors and slow progressors and for the slow progressors in the remaining two subject pairs. However, intra-time point sequence comparisons showed that four of five slow progressors developed a more diverse quasispecies over time and one showed no change. In contrast, four of five rapid progressors showed no change in quasispecies diversity over time and one showed a significant decrease in diversity. The overall C1 through C3 region quasispecies diversity in the slow progressors at baseline was lower than that for the rapid progressors, but this difference was not significant at the follow-up time points. These diversity relationships were obscured if sequence analyses were limited to the 300-bp C2 to V3 region. Thus, HIV-1 quasispecies diversity increased over time in subjects with more functional immune systems. PMID:9032317

McDonald, R A; Mayers, D L; Chung, R C; Wagner, K F; Ratto-Kim, S; Birx, D L; Michael, N L

1997-01-01

244

An operational semantics of real time design language RT-CDL  

Microsoft Academic Search

Any methodology for the design of a complex system needs a basis for specification and verification. This is particularly so for realtime systems since safety and reliability ate extremely important for these systems. As a first step, we provide au operational semantics for the language RT-CDL (Real Time Common Design Language) employing Plotkin’s labeled transition systems using the maximal paraheliim

Leo YuHsiang Liu; R. K. Shyamasundar

1989-01-01

245

Evaluation of reference genes for quantitative RT-PCR in Lolium temulentum under abiotic stress  

Technology Transfer Automated Retrieval System (TEKTRAN)

Lolium temulentum is a valuable model grass species for the study of stress in forage and turf grasses. Gene expression analysis by quantitative real time RT-PCR relies on the use of proper internal standards. The aim of this study was to identify and evaluate reference genes for use in real-time q...

246

Padlock probe-mediated qRT-PCR for DNA computing answer determination  

PubMed Central

Padlock probe-mediated quantitative real time PCR (PLP-qRT-PCR) was adapted to quantify the abundance of sequential 10mer DNA sequences for use in DNA computing to identify optimal answers of traveling salesman problems. The protocol involves: (i) hybridization of a linear PLP with a target DNA sequence; (ii) PLP circularization through enzymatic ligation; and (iii) qRT-PCR amplification of the circularized PLP after removal of non-circularized templates. The linear PLP was designed to consist of two 10-mer sequence-detection arms at the 5? and 3? ends separated by a core sequence composed of universal PCR primers, and a qRT-PCR reporter binding site. Circularization of each PLP molecule is dependent upon hybridization with target sequence and high-fidelity ligation. Thus, the number of PLP circularized is determined by the abundance of target in solution. The amplification efficiency of the PLP was 98.7% within a 0.2 pg–20 ng linear detection range between thermal cycle threshold (Ct value) and target content. The Ct values derived from multiplex qRT-PCR upon three targets did not differ significantly from those obtained with singleplex assays. The protocol provides a highly sensitive and efficient means for the simultaneous quantification of multiple short nucleic acid sequences that has a wide range of applications in biotechnology. PMID:21691417

Xiong, Fusheng; Frasch, Wayne D.

2011-01-01

247

136 VOLUME 14 NUMBER 2 FEBRUARY 2013 nature immunology A rt i c l e s  

E-print Network

136 VOLUME 14 NUMBER 2 FEBRUARY 2013 nature immunology A rt i c l e s Microbial and viral of the acute responses to the parasite 1Department of Immunology, University of Texas Southwestern Medical Center, Dallas, Texas, USA. 2Department of Microbiology & Immunology, University of California, San

Arnold, Jonathan

248

nature biotechnology VOLUME 32 NUMBER 2 FEBRUARY 2014 191 A rt i c l e s  

E-print Network

nature biotechnology VOLUME 32 NUMBER 2 FEBRUARY 2014 191 A rt i c l e s Bispecific antibodies (Bs- clonal antibodies (mAbs) into a single molecule. As such, BsAbs can elicit synergistic activities antibody geometry with its well-known stability and solu- bility properties to achieve specificity

Cai, Long

249

About the RepoRt While the notion of business fostering peace is becoming  

E-print Network

Al RepoRt 315 septembeR 2012 © 2012 by the United States Institute of Peace. All rights reserved. contents Brown at USIP. 2301 Constitution Ave., NW · Washington, DC 20037 · 202.457.1700 · fax 202.429.6063 speci potential of the business sector to foster peace must account for the size of firms, whether they are state

Vertes, Akos

250

RocK coNceRt PRomotioN ROCk CONCERT PROMOTION POLICY _____________________________________________  

E-print Network

16 RocK coNceRt PRomotioN ROCk CONCERT PROMOTION POLICY a rock music concert (as defined by the Clark County Rock Concert Promotion Ordinance) must have will determine the applicant's suitability for use of UNLV's Rock Concert Promotion License Waiver. A license

Hemmers, Oliver

251

University of Maryland Center for Environmental Science 2012 AnnuAl RepoRt  

E-print Network

University of Maryland Center for Environmental Science 2012 AnnuAl RepoRt #12;annUal rEport 2012 between the mountains and sea, our research laboratories provide scientists direct access to Maryland as part of the university System of Maryland's nationally ranked graduate program in marine

Boynton, Walter R.

252

FY 2014 AgencY FinAnciAl RepoRt www.nasa.gov  

E-print Network

FY 2014 AgencY FinAnciAl RepoRt www.nasa.gov National Aeronautics and Space Administration #12;Front Cover: Outside Front Main Image: Artist concept of planets space. (Credit: NASA) Outside Front Bottom Images (left to right): Crawler-Transporter Passes Milestone Test at NASA's Kennedy Space Center

Waliser, Duane E.

253

Miniature RT-PCR system for diagnosis of RNA-based viruses.  

PubMed

This paper presents an innovative portable chip-based RT-PCR system for amplification of specific nucleic acid and detection of RNA-based viruses. The miniature RT-PCR chip is fabricated using MEMS (Micro-electro-mechanical-system) techniques, and comprises a micro temperature control module and a PDMS (polydimethylsiloxane)-based microfluidic control module. The heating and sensing elements of temperature control module are both made of platinum and are located within the reaction chambers in order to generate a rapid and uniform thermal cycling. The microfluidic control module is capable of automating testing process with minimum human intervention. In this paper, the proposed miniature RT-PCR system is used to amplify and detect two RNA-based viruses, namely dengue virus type-2 and enterovirus 71 (EV 71). The experimental data confirm the ability of the system to perform a two-step RT-PCR process. The developed miniature system provides a crucial tool for the diagnosis of RNA-based viruses. PMID:16221971

Liao, Chia-Sheng; Lee, Gwo-Bin; Liu, Hsiao-Sheng; Hsieh, Tsung-Min; Luo, Ching-Hsing

2005-01-01

254

ISSN0249-0803ISRNINRIA/RT--0401--FR+ENG Audio, Speech, and Language Processing  

E-print Network

of phoneme variants as a parameter. As no database of infant-directed speech1 containing rich phoneticapport technique ISSN0249-0803ISRNINRIA/RT--0401--FR+ENG Audio, Speech, and Language Processing 39 63 53 30 A note on the generation of allophonic rules Luc Boruta Theme : Audio, Speech

Paris-Sud XI, Université de

255

ISSN0249-0803ISRNINRIA/RT--0401--FR+ENG Audio, Speech, and Language Processing  

E-print Network

to vary the number of phoneme variants as a parameter. As no database of infant-directed speech1apport technique ISSN0249-0803ISRNINRIA/RT--0401--FR+ENG Audio, Speech, and Language Processing on the generation of allophonic rules Luc Boruta Theme : Audio, Speech, and Language Processing Perception

Paris-Sud XI, Université de

256

Training for Generalization and Maintenance in RtI Implementation: Front-Loading for Sustainability  

ERIC Educational Resources Information Center

Response to Intervention (RtI) is being implemented as a new initiative in PK-12 schools with increasing frequency. However, the model must be sustained at the school level, which is potentially difficult due to a number of challenges brought about by systems change. This article applied the Stokes and Baer (1977) framework for programming for…

Burns, Matthew K.; Egan, Andrea M.; Kunkel, Amy K.; McComas, Jennifer; Peterson, Meredith M.; Rahn, Naomi L.; Wilson, Jennifer

2013-01-01

257

2013AnnuAl RepoRt Office of the Vice President for Research  

E-print Network

with major industry and governmental groups, including Bayer CropScience and Sandia National Laboratories. I2013AnnuAl RepoRt Office of the Vice President for Research #12;2 A Letter from the Former Interim for Research 2013 Annual Report A Letter from the Former Interim Vice President for Research Dear colleagues

Rock, Chris

258

RESEARCH Open Access Development of a SYBR green I based RT-PCR  

E-print Network

RESEARCH Open Access Development of a SYBR green I based RT-PCR assay for yellow fever virus2 , Shashi Sharma3 and Paul Reiter1* Abstract Background: Yellow Fever virus (YFV) is an important for detection and quantification of YFV than other currently used methods. Background Yellow fever (YF) is one

Paris-Sud XI, Université de

259

S E A T U RT L E S Sea Turtles  

E-print Network

S E A T U RT L E S 1 U N I T 25 Sea Turtles Unit 25 NMFS OFFICE OF PROTECTED RESOURCES Silver. The authority to protect and conserve sea turtles in the marine environment is vested in the National Ma- rine at the Federal level for protection of sea turtles on land (nesting beaches). SPECIES AND STATUS Atlantic Region

260

The ISL RT-06S Speech-to-Text System Christian Fugen1  

E-print Network

The ISL RT-06S Speech-to-Text System Christian F¨ugen1 , Shajith Ikbal1 , Florian Kraft1 , Kenichi at the Interactive Systems Labora- tories (ISL), for the individual head-mounted microphone (IHM), single distant Introduction In this paper, we present the ISL's most recent speech-to-text systems for lec- tures

Schultz, Tanja

261

Department of recreational SportS AnnuAl RepoRt  

E-print Network

Department of recreational SportS AnnuAl RepoRt 2012 - 2013 #12;#12;#12;#12;1 Table of Contents 3 5. This effort is the underpinning of our endeavor to support the wellbeing of our health-conscious campus to read further to learn about the Department of recreational Sports and its impact on the campus

Escher, Christine

262

Comparison of Relative RT-PCR and Northern Blot Analyses to Measure Expression of  

E-print Network

,3-Glucanase in Nicotiana benthamiana Infected With Colltotrichum destructivum J.D. DEAN, P.H. GOODWIN* and T,3-glucanase, a translation elongation factor 1 (EF-1) gene was selected as an internal control. Northern blot compatible, and 35 cycles of amplification gave reproducible relative RT-PCR results for -1,3-glucanase gene

Hsiang, Tom

263

nature medicine VOLUME 20 | NUMBER 3 | MARCH 2014 255 a rt i c l e s  

E-print Network

nature medicine VOLUME 20 | NUMBER 3 | MARCH 2014 255 a rt i c l e s During aging, skeletal muscle for the impaired regen- eration observed during aging13. Instead, several reports attribute loss of muscle themselves2,14­18. For example, systemic factors from young mice ameliorate muscle regeneration in aged mice

Cai, Long

264

a lookahead 2010-2012 School of Dental MeDicine annual RepoRt  

E-print Network

Association of Orthodontists American Fund for Dental Health American Heart Association Jack R. Beattie, D.S.D. DEN'93 B. Douglas Amberman, D.D.S., M.S. DEN'67 American Association of Endodontists Americanthe yearsin review a lookahead 2010-2012 School of Dental MeDicine annual RepoRt #12;our past Thank

Rollins, Andrew M.

265

The association between RT-induced changes in lung tissue density and global lung function  

PubMed Central

Purpose To assess the association between RT-induced changes in computed tomography (CT)-defined lung tissue density and pulmonary function tests (PFTs). Methods and Materials Patients receiving incidental partial lung irradiation were prospectively assessed for global (PFTs) and regional (CT and SPECT [single photon emission computed tomography] scans) lung function pre- and serially post-RT. The percent reductions in PFTs and the average changes in lung density were compared (Pearson correlations) in the overall group and subgroups based on various clinical factors. Comparisons were also made between the CT- and SPECT-based computations using U test. Results From 1991–2004, 343 patients were enrolled. Of these, 111 patients had a total of 203 concurrent post-RT evaluations of changes in lung density and PFTs available for analyses, and 81 patients had a total of 141 concurrent post-RT SPECT images as well. The average increases in lung density were related to the percent reductions in PFTs, albeit with modest correlation coefficients (r) (range, 0.20 ~ 0.43). The analyses also indicate that the association between lung density and PFT changes is essentially equivalent to the corresponding association with SPECT-defined lung perfusion. Conclusion There is a weak quantitative association between the degree of increase in lung density as defined by CT and percent reduction in PFTs. PMID:19084355

Ma, Jinli; Zhang, Junan; Zhou, Sumin; Hubbs, Jessica L.; Foltz, Rodney J.; Hollis, Donna R.; Light, Kim L.; Wong, Terence Z.; Kelsey, Christopher R.; Marks, Lawrence B.

2009-01-01

266

A RT I C L E S Dynamic evolution of the innate immune system in  

E-print Network

A RT I C L E S Dynamic evolution of the innate immune system in Drosophila Timothy B Sackton1 of the innate immune system. We have identified orthologs and paralogs of 245 Drosophila melanogaster immune that immune-system genes, and especially those encoding recognition proteins, evolve under positive darwinian

Keinan, Alon

267

reaction (RT-PCR) to detect Kashmir bee virus (KBV). This technique requires time-  

E-print Network

reaction (RT-PCR) to detect Kashmir bee virus (KBV). This technique requires time- consuming virus that circumvents these steps. KBV is one of the picorna-like viruses found in honey bees. It was first isolated from a diseased adult bee of the Asian honey bee, Apis cerana [4]. KBV has also been 1. INTRODUCTION

Paris-Sud XI, Université de

268

Ethidium monoazide does not inhibit RT-PCR amplification of nonviable avian influenza RNA  

Microsoft Academic Search

A critical obstacle to using PCR to quantify viral titers is the distinguishment of viable and nonviable genomic material. Pretreatments of ethidium monoazide (EMA) have been effective in preventing PCR amplification of DNA from nonviable bacteria. To test whether an EMA pretreatment could be used with RT-PCR to quantify viable RNA virions, avian influenza virus (AIV) survival was measured in

David A. Graiver; Samuel E. Saunders; Christina L. Topliff; Clayton L. Kelling; Shannon L. Bartelt-Hunt

2010-01-01

269

a rt i c l e s nature medicine VOLUME 19 | NUMBER 4 | APRIL 2013 465  

E-print Network

a rt i c l e s nature medicine VOLUME 19 | NUMBER 4 | APRIL 2013 465 Cancer vaccines have shown+ T cell immune responses after gp100/IFA vaccination. RESULTS IFA-based vaccination induces T cell remains why many vaccinated patients show increased numbers of circulat- ing tumor-specific T cells

270

FinAnCiAl RepoRt 2010 CertifiCAtions ..................................................................... 45  

E-print Network

of the Financial Management Act 2006 from proper accounts and records to present fairly the financial transactionsFinAnCiAl RepoRt 2010 CertifiCAtions ..................................................................... 45 stAtement of Comprehensive inCome ..................... 46 stAtement of finAnCiAl position

271

LAL/RT 06-04 ATLAS ENDCAP LIQUID ARGON CALORIMETERS  

E-print Network

� � �º¹�º � ÐÐ � #12;LAL/RT 06-04 May 2006 ATLAS ENDCAP LIQUID ARGON CALORIMETERS Description and charge detection in liquid argon. They are therefore all grouped in the same vessel which must basically support and keep in place the heavy plates and the detection electrodes and maintain liquid argon at cold

Paris-Sud XI, Université de

272

The Rt. Hon. John Denham MP Secretary of State for Innovation, Universities and Skills  

E-print Network

The Rt. Hon. John Denham MP Secretary of State for Innovation, Universities and Skills DIUS 1 Victoria Street London SW1H 0ET Dear Secretary of State, We are writing to you as a group of scientists our talents, skills and leadership to an already thriving scientific community. The policies pursued

Crowther, Paul

273

Universal external RNA controls for microbial gene expression analysis using microarray and qRT-PCR  

Technology Transfer Automated Retrieval System (TEKTRAN)

We presented a set of universal external RNA quality controls for microbial mRNA expression analysis across different platforms of DNA oligo microarray and real time qRT-PCR, including using different chemistry of SYBR Green and TaqMan. The control is a powerful tool to guard reliability and reprod...

274

2009 AnnuAl RepoRt 1. Report for 2009 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2  

E-print Network

2009 AnnuAl RepoRt 1 Contents contents 1. Report for 2009 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 4. statement by the Management on the Annual Report and Auditors' Report . . . . . . . . 11 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 4.2 statement by the Management on the Annual Report

275

Control #: SF 6432-RT Title: Standard Terms and Conditions for Acquiring the Services of Sandia Retirees  

E-print Network

the Services of Sandia Retirees Owner: Procurement Policy & Quality Dept Release Date: 03/2014 Page 2 of 16Control #: SF 6432-RT Title: Standard Terms and Conditions for Acquiring the Services of Sandia Retirees Owner: Procurement Policy & Quality Dept Release Date: 03/2014 Page 1 of 16 Printed copies

276

RT-Based Administrative Models for Community Cyber Security Information Sharing  

E-print Network

RT-Based Administrative Models for Community Cyber Security Information Sharing Ravi Sandhu, Khalid Zaman Bijon, Xin Jin, and Ram Krishnan Institute for Cyber Security & Department of Computer Science Institute for Cyber Security & Department of Electrical and Computer Engineering University of Texas at San

Sandhu, Ravi

277

RT-Based Administrative Models for Community Cyber Security Information Sharing  

E-print Network

RT-Based Administrative Models for Community Cyber Security Information Sharing Ravi Sandhu, Khalid Zaman Bijon Institute for Cyber Security World-Leading Research with Real Ravi Sandhu, Khalid Zaman Bijon Institute for Cyber Security University of Texas at San Antonio Oct. 15, 2011 International

Sandhu, Ravi

278

DETECTION OF HUMAN ENTERIC VIRUSES IN STREAM WATER WITH RT-PCR AND CELL CULTURE  

EPA Science Inventory

A multiplex RT-PCR method was used to measure virus occurrence at five stream water sites that span a range of hydroclimatic, water-quality, and land-use characteristics. The performance of the molecular method was evaluated in comparison to traditional cell culture and Escherich...

279

1116 VOLUME 15 NUMBER 12 DECEMBER 2014 NATURE IMMUNOLOGY A RT I C L E S  

E-print Network

1116 VOLUME 15 NUMBER 12 DECEMBER 2014 NATURE IMMUNOLOGY A RT I C L E S Chitinase-like proteins are members of a family that include both chitotriosidase and acidic mammalian chitinase, enzymes that cleave chitin, which is a widespread structural component of arthropods, par- asites and fungi. Consistent

280

RT-PCR and real-time RT-PCR methods for the detection of potato virus Y in potato leaves and tubers.  

PubMed

Potato virus Y (PVY) is a major threat to potato crops around the world. It is an RNA virus of the family Potyviridae, exhibiting many different strains that cause a range of symptoms in potato. ELISA detection of viral proteins has traditionally been used to quantify virus incidence in a crop or seed lot. ELISA, however, cannot reliably detect the virus directly in dormant tubers, requiring several weeks of sprouting tubers to produce detectable levels of virus. Nor can ELISA fully discriminate between the wide range of strains of the virus. Several techniques for directly detecting the viral RNA have been developed which allow rapid detection of PVY in leaf or tuber tissue, and that can be used to easily distinguish between different strains of the virus. Described in this chapter are several protocols for the extraction of RNA from leaf and tuber tissues, and three detection methods based upon reverse-transcription-PCR (RT-PCR). First described is a traditional two-step protocol with separate reverse transcription of viral RNA into cDNA, then PCR to amplify the viral cDNA fragment. Second described is a one-step RT-PCR protocol combining the cDNA production and PCR in one tube and one step, which greatly reduces material and labor costs for PVY detection. The third protocol is a real-time RT-PCR procedure which not only saves on labor but also allows for more precise quantification of PVY titre. The three protocols are described in detail, and accompanied with a discussion of their relative advantages, costs, and possibilities for cost-saving modifications. While these techniques have primarily been developed for large-scale screening of many samples for determining viral incidence in commercial fields or seed lots, they are also amenable to use in smaller-scale research applications. PMID:25287492

MacKenzie, Tyler D B; Nie, Xianzhou; Singh, Mathuresh

2015-01-01

281

Cognition and Quality of Life After Chemotherapy Plus Radiotherapy (RT) vs. RT for Pure and Mixed Anaplastic Oligodendrogliomas: Radiation Therapy Oncology Group Trial 9402  

SciTech Connect

Purpose: Radiation Therapy Oncology Group 9402 compared procarbazine, lomustine, and vincristine (PCV) chemotherapy plus radiation therapy (PCV + RT) vs. RT alone for anaplastic oligodendroglioma. Here we report longitudinal changes in cognition and quality of life, effects of patient factors and treatments on cognition, quality of life and survival, and prognostic implications of cognition and quality of life. Methods and Materials: Cognition was assessed by Mini Mental Status Examination (MMSE) and quality of life by Brain-Quality of Life (B-QOL). Scores were analyzed for survivors and within 5 years of death. Shared parameter models evaluated MMSE/B-QOL with survival. Results: For survivors, MMSE and B-QOL scores were similar longitudinally and between treatments. For those who died, MMSE scores remained stable initially, whereas B-QOL slowly declined; both declined rapidly in the last year of life and similarly between arms. In the aggregate, scores decreased over time (p = 0.0413 for MMSE; p = 0.0016 for B-QOL) and were superior with age <50 years (p < 0.001 for MMSE; p = 0.0554 for B-QOL) and Karnofsky Performance Score (KPS) 80-100 (p < 0.001). Younger age and higher KPS were associated with longer survival. After adjusting for patient factors and drop-out, survival was longer after PCV + RT (HR = 0.66, 95% CI = 0.49-0.9, p = 0.0084; HR = 0.74, 95% CI = 0.54-1.01, p = 0.0592) in models with MMSE and B-QOL. In addition, there were no differences in MMSE and B-QOL scores between arms (p = 0.4752 and p = 0.2767, respectively); higher scores predicted longer survival. Conclusion: MMSE and B-QOL scores held steady in the upper range in both arms for survivors. Younger, fitter patients had better MMSE and B-QOL and longer survival.

Wang Meihua, E-mail: mwang@phila.acr.or [American College of Radiology, Philadelphia, PA (United States); Cairncross, Gregory [University of Calgary, Calgary, AB (Canada); Shaw, Edward [Wake Forest University School of Medicine, Winston-Salem, NC (United States)

2010-07-01

282

Modeling variably saturated multispecies reactive groundwater solute transport with MODFLOW-UZF and RT3D  

USGS Publications Warehouse

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.

Bailey, Ryan T.; Morway, Eric D.; Niswonger, Richard G.; Gates, Timothy K.

2013-01-01

283

Diagnosis of Ebola haemorrhagic fever by RT-PCR in an epidemic setting.  

PubMed

This study reports the first field evaluation of a new diagnostic technique for Ebola virus disease with sensitivity and specificity. Ebola virus causes rare but fulminating outbreaks in Equatorial Africa. Rapid differentiation from other infections is critical for timely implementation of public health measures. Patients usually die before developing antibodies, necessitating rapid virus detection. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed, implemented and evaluated at Centre International de Recherches Médicales de Franceville (CIRMF) in Gabon, to detect Ebola viral RNA in peripheral blood mononuclear cells (PBMC). Twenty-six laboratory-confirmed patients during and 5 after the acute phase of Ebola haemorrhagic fever, 15 healthy controls and 20 febrile patients not infected with Ebola virus were studied. RT-PCR results were compared with ELISA antigen capture, and Ebola specific IgM and IgG antibody detection. Ebola virus RNA was amplified from 26/26 specimens from the acute phase, 3/5 during recovery, 0/20 febrile patients and 1/15 negative controls. Sensitivity of RT-PCR in identifying acute infection and early convalescence compared with antigen or IgM detection was 100% and 91% respectively, and specificity compared with antigen detection and IgM assay combined was 97%. Antigen capture detected only 83% of those identified by PCR, and IgM only 67%. Ebola virus RNA was detected in all 13 fatalities, only 5 of whom had IgM and none IgG. RT-PCR detected Ebola RNA in PBMC one to three weeks after disappearance of symptoms when antigen was undetectable. RT-PCR was the most sensitive method and able to detect virus from early acute disease throughout early recovery. PMID:10686031

Leroy, E M; Baize, S; Lu, C Y; McCormick, J B; Georges, A J; Georges-Courbot, M C; Lansoud-Soukate, J; Fisher-Hoch, S P

2000-04-01

284

Does the sex of acute stroke patients influence the effectiveness of rt-PA?  

PubMed Central

Background Women have been reported to show more frequent recanalization and better recovery after intravenous (IV) recombinant tissue plasminogen activator (rt-PA) treatment for acute stroke compared with men. To investigate this we studied a series of stroke patients receiving IV rt-PA and undergoing acute transcranial doppler (TCD) examination. Methods Acute stroke patients received IV rt-PA and had acute TCD examination within 4 hours of symptom onset at 4 major stroke centers. TCD findings were interpreted using the Thrombolysis in Brain Ischemia (TIBI) flow grading system. The recanalization rates, and poor 3-month outcomes (modified Rankin scale >2) of men and women were compared using the chi-square test. Multiple regression analysis was used to assess sex as a predictor of recanalization and poor 3-month outcome after controlling for age, baseline NIH Stroke Scale (NIHSS), time to treatment, hypertension, and blood glucose. Results 369 patients had TCD examinations before or during IV rt-PA treatment. The 199 (53.9%) men and 170 (46.1%) women had mean ages of 67?±?13 and 70?±?14 years, respectively. The sexes did not differ significantly in baseline stroke severity, time to TCD examination, or time to thrombolysis. Of the men, 68 (34.2%) had complete recanalization, 58 (29.1%) had partial recanalization, and 73 (36.6%) had no recanalization. Of the women, 53 (31.2%) had complete recanalization, 46 (27%) had partial recanalization, and 71 (41.8%) had no recanalization (p?=?0.6). Multiple regression analyses showed no difference between the sexes in recanalization rate, time to recanalization, or clinical outcome at 3 months. Conclusions In our study; sex is not a significant predictor of recanalization rate, time to recanalization or 3-month outcome in stroke patients following IV rt-PA. Trial registration Data from CLOTBUST trial Clinicaltrails.gov Identifier: NCT01240356. PMID:24669960

2014-01-01

285

Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)  

PubMed Central

Gene expression profiling of formalin-fixed and paraffin-embedded (FFPE) specimens, banked from completed clinical trials and routine clinical care, has the potential to yield valuable information implicating and linking genes with clinical parameters. In order to prepare high-quality cDNA from highly fragmented FFPE-RNA, previously precluded from high-throughput analyses, we have designed a novel strategy based on the nucleic acid restoration of incomplete cDNA sequences prior to T7 in vitro transcription (IVT) amplification. We describe this strategy as complementary-template reverse-transcription (CT-RT) because short single-stranded T7-oligo-dT24-VN-DNA sequences, obtained from FFPE-RNA, are used as primers for the RT of complementary RNA templates contained in a sense-RNA library. We validated our assay by determining the correlation between expression profiles of a matched 10-year-old frozen and FFPE breast cancer sample. We show that T7 IVT-amplification of cDNA transcripts restored by CT-RT is a specific and reliable process that allows recovery of transcriptional features undetectable by direct T7 IVT-amplification of FFPE-RNA. Furthermore, CT-RT restored 35–41% of the transcripts from archived breast and cervical specimens when compared to matched frozen tissue; and profiles included tissue-specific transcripts. Our results indicate that CT-RT allows microarray profiling of severely degraded RNA that could not be analyzed by previous methods. PMID:17636051

Loudig, Olivier; Milova, Ekaterina; Brandwein-Gensler, Margaret; Massimi, Aldo; Belbin, Thomas J.; Childs, Geoffrey; Singer, Robert H.; Rohan, Thomas; Prystowsky, Michael B.

2007-01-01

286

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

287

A combination microbicide gel protects macaques against vaginal simian human immunodeficiency virus-reverse transcriptase infection, but only partially reduces herpes simplex virus-2 infection after a single high-dose cochallenge.  

PubMed

Herpes simplex virus-2 (HSV-2) infection increases HIV susceptibility. We previously established a rhesus macaque model of vaginal HSV-2 preexposure followed by cochallenge with HSV-2 and simian/human immunodeficiency virus-reverse transcriptase (SHIV-RT). Using this model, we showed that a gel containing the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 in carrageenan (CG) reduced SHIV-RT infection. To evaluate the efficacy of new generation microbicides against both viruses, we first established dual infection after single vaginal cochallenge with SHIV-RT and HSV-2 in HSV-2-naive macaques. All animals (6/6) became HSV-2 infected, with 4/6 coinfected with SHIV-RT. In a control group cochallenged with SHIV-RT and UV-inactivated HSV-2, 2/4 became SHIV-RT infected, and none had detectable HSV-2. Low-level HSV-2-specific antibody and T cell responses were detected in some HSV-2-infected animals. To test a CG gel containing MIV-150 and zinc acetate (MZC), which provided naive animals full protection from SHIV-RT for at least 8?h, MZC (vs. CG) was applied daily for 14 days followed by cochallenge 8?h later. MZC prevented SHIV-RT infection (0/9 infected, p=0.04?vs. 3/6 in CG controls), but only reduced HSV-2 infection by 20% (6/9 infected vs. 5/6 in CG, p=0.6). In HSV-2-infected animals, none of the gel-treated animals seroconverted, and only the CG controls had measurable HSV-2-specific T cell responses. This study shows the promise of MZC to prevent immunodeficiency virus infection (even in the presence of HSV-2) and reduce HSV-2 infection after exposure to a high-dose inoculum. Additionally, it demonstrates the potential of a macaque coinfection model to evaluate broad-spectrum microbicides. PMID:24117013

Hsu, Mayla; Aravantinou, Meropi; Menon, Radhika; Seidor, Samantha; Goldman, Daniel; Kenney, Jessica; Derby, Nina; Gettie, Agegnehu; Blanchard, James; Piatak, Michael; Lifson, Jeffrey D; Fernández-Romero, Jose A; Zydowsky, Thomas M; Robbiani, Melissa

2014-02-01

288

Further improvement and validation of MagMAX-96 AI/ND viral RNA isolation for efficient removal of RT-PCR inhibitors from cloacal swabs and tissues for rapid diagnosis of avian influenza virus by RT reverse transcription PCR  

Technology Transfer Automated Retrieval System (TEKTRAN)

Real time RT-PCR (RRT-PCR) is a high throughput molecular diagnostic test used for rapid detection of avian influenza virus (AIV) in clinical samples. However the performance of RRT-PCR can be adversely affected by RT-PCR inhibitors present in the sample. The tested commercial RNA extraction kits ...

289

Covariance of Charged Amino Acids at Positions 322 and 440 of HIV-1 Env Contributes to Coreceptor Specificity of Subtype B Viruses, and Can Be Used to Improve the Performance of V3 Sequence-Based Coreceptor Usage Prediction Algorithms  

PubMed Central

The ability to determine coreceptor usage of patient-derived human immunodeficiency virus type 1 (HIV-1) strains is clinically important, particularly for the administration of the CCR5 antagonist maraviroc. The envelope glycoprotein (Env) determinants of coreceptor specificity lie primarily within the gp120 V3 loop region, although other Env determinants have been shown to influence gp120-coreceptor interactions. Here, we determined whether conserved amino acid alterations outside the V3 loop that contribute to coreceptor usage exist, and whether these alterations improve the performance of V3 sequence-based coreceptor usage prediction algorithms. We demonstrate a significant covariant association between charged amino acids at position 322 in V3 and position 440 in the C4 Env region that contributes to the specificity of HIV-1 subtype B strains for CCR5 or CXCR4. Specifically, positively charged Lys/Arg at position 322 and negatively charged Asp/Glu at position 440 occurred more frequently in CXCR4-using viruses, whereas negatively charged Asp/Glu at position 322 and positively charged Arg at position 440 occurred more frequently in R5 strains. In the context of CD4-bound gp120, structural models suggest that covariation of amino acids at Env positions 322 and 440 has the potential to alter electrostatic interactions that are formed between gp120 and charged amino acids in the CCR5 N-terminus. We further demonstrate that inclusion of a “440 rule” can improve the sensitivity of several V3 sequence-based genotypic algorithms for predicting coreceptor usage of subtype B HIV-1 strains, without compromising specificity, and significantly improves the AUROC of the geno2pheno algorithm when set to its recommended false positive rate of 5.75%. Together, our results provide further mechanistic insights into the intra-molecular interactions within Env that contribute to coreceptor specificity of subtype B HIV-1 strains, and demonstrate that incorporation of Env determinants outside V3 can improve the reliability of coreceptor usage prediction algorithms. PMID:25313689

Cashin, Kieran; Sterjovski, Jasminka; Harvey, Katherine L.; Ramsland, Paul A.; Churchill, Melissa J.; Gorry, Paul R.

2014-01-01

290

A CCR5-tropic simian-HIV molecular clone capable of inducing AIDS in rhesus macaques.  

PubMed

We previously reported the derivation of a CCR5 (R5)-tropic pathogenic strain SHIVSF162P3. Here, we show that a simian-HIV (SHIV) molecular clone expressing the entire env gp160 of SHIVSF162P3, termed SHIV P3gp160, could fully recapitulate the in vivo replicative characteristics of the parental isolate. SHIV P3gp160 is mucosally transmissible, preferentially depletes memory CD4 T cells, and induced simian AIDS in 2 of 6 infected macaques. The availability of an infectious R5 SHIV molecular clone that can be transmitted mucosally and causes disease provides an important reagent for studies of lentiviral pathogenesis and AIDS vaccine research. PMID:16280691

Hsu, Mayla; Ho, Siu-Hong; Balfe, Peter; Gettie, Agegnehu; Harouse, Janet; Blanchard, James; Cheng-Mayer, Cecilia

2005-12-01

291

Middle School Teacher Satisfaction with Response to Intervention (RtI): An Assessment between Inception and Implementation  

ERIC Educational Resources Information Center

Response to intervention (RtI) is a multi-tiered process of monitoring student responses to remediation that is designed to help struggling learners succeed within the purview of regular education. Under the RtI model, students are referred to special education only after a series of documented interventions have been attempted. This study…

Zahedi, Karynn Jensen

2010-01-01

292

Real-time RT-PCR and cDNA macroarray to study the impact of the genetic polymorphism  

E-print Network

Review Real-time RT-PCR and cDNA macroarray to study the impact of the genetic polymorphism and stearoyl-CoA desaturase) was performed using real-time RT-PCR, suggesting an absence of association between

Paris-Sud XI, Université de

293

The integration of the microfluidic Geniom Biochip and the Geniom RT Analyzer as processing platform has several advantages.  

E-print Network

The integration of the microfluidic Geniom Biochip and the Geniom RT Analyzer as processing Biochip® with the fully automated processing station Geniom RT Analyzer® from febit are tools to a Geniom Biochip, stringent washing, and elu- tion of desired library fragments (Fig. 1b). febit

Cai, Long

294

RtI's Implementation and Collaborative Systems between General and Special Education in Two Middle Schools  

ERIC Educational Resources Information Center

This study investigated Response to Intervention (RtI) implementation in two Illinois middle schools and its impact on the collaborative relationship between general and special education teachers. The intended and unintended consequences of RtI implementation for general and special educators were examined. This qualitative research study was…

Kosteck, Kathleen M.

2012-01-01

295

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

296

rtM204Q May Serve as a Novel Lamivudine-Resistance-Associated Mutation of Hepatitis B Virus  

PubMed Central

Background and Aims Lamivudine (LAM) is still widely used for anti-HBV therapy in China. The study aimed to clarify whether a newly-found rtM204Q mutation from patients was associated with the drug resistance. Methods HBV complete reverse-transcriptase region was screened by direct sequencing and verified by clonal sequencing. Replication-competent plasmids containing patient-derived 1.1mer mutant or wild-type viral genome were constructed and transfected into HepG2 cells. After cultured with or without serially-diluted antiviral drugs, intracellular HBV replicative intermediates were quantitated for calculating the 50% effective concentration of drug (EC50). Results A total of 12,000 serum samples of 9,830 patients with chronic HBV infection were screened. rtM204Q mutation was detected in seven LAM-refractory patients. By contrast, rtM204I/rtM204V mutations were detected in 2,502 patients' samples. The rtM204Q emerged either alone or in concomitance with rtM204I/rtM204V, and all were accompanied with virologic breakthrough in clinical course. Clonal sequencing verified that rtM204Q mutant was predominant in viral quasispecies of these samples. Phenotypic analysis showed that rtM204Q mutant had 89.9% of replication capacity and 76-fold increased LAM EC50 of the concomitant wild-type strain. By contrast, rtM204I mutant in the sample had lower replication capacity and higher LAM resistance (46.3% and 1396-fold increased LAM EC50 of the wild-type strain) compared to rtM204Q mutant. rtM204Q mutant was susceptible to adefovir dipivoxil (ADV) in vitro and ADV/ADV+LAM rescue therapy in clinic. Conclusion rtM204Q is suggested to be a novel LAM-resistance-associated mutation. It conferred a moderate resistance with higher competent natural replication capacity compared to rtM204I mutation. PMID:24586482

Wang, Yan; Li, Xiaodong; Liu, Liming; Chen, Li; Xin, Shaojie; Xu, Dongping

2014-01-01

297

Adaptation of Subtype A Human Immunodeficiency Virus Type 1 Envelope to Pig-Tailed Macaque Cells?  

PubMed Central

The relevance of simian/human immunodeficiency virus (SHIV) infection of macaques to HIV-1 infection in humans depends on how closely SHIVs mimic HIV-1 transmission, pathogenesis, and diversity. Circulating HIV-1 strains are predominantly subtypes C and A and overwhelmingly require CCR5 for entry, yet most SHIVs incorporate CXCR4-using subtype B envelopes (Envs). While pathogenic subtype C-based SHIVs have been constructed, the subtype A-based SHIVs (SHIV-As) constructed to date have been unable to replicate in macaque cells. To understand the barriers to SHIV-A replication in macaque cells, HIVAQ23/SIVvif was constructed by engineering a CCR5-tropic subtype A provirus to express SIV vif, which counters the macaque APOBEC3G restriction. HIVAQ23/SIVvif replicated poorly in pig-tailed macaque (Ptm) lymphocytes, but viruses were adapted to Ptm lymphocytes. Two independent mutations in gp120, G312V (V3 loop) and A204E (C2 region), were identified that increased peak virus levels by >100-fold. Introduction of G312V and A204E to multiple subtype A Envs and substitution of G312 and A204 with other residues increased entry into Ptm cells by 10- to 100-fold. G312V and A204E Env variants continued to require CCR5 for entry but were up to 50- and 200-fold more sensitive to neutralization by IgG1b12 and soluble CD4 and had a 5- to 50-fold increase in their ability to utilize Ptm CD4 compared to their wild-type counterparts. These findings identify the inefficient use of Ptm CD4 as an unappreciated restriction to subtype A HIV-1 replication in Ptm cells and reveal amino acid changes to gp120 that can overcome this barrier. PMID:21325401

Humes, Daryl; Overbaugh, Julie

2011-01-01

298

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

299

A comparison of RT-PCR-based assays for the detection of HAV from shellfish.  

PubMed

The hepatitis A virus (HAV) is the most common cause of viral infection linked to shellfish consumption. The lack of correlation between the fecal coliform indicators and the presence of enteric viruses in shellfish and their harvesting waters points to the need for molecular methods to detect viruses. We compared two RT-PCR based techniques currently available for the detection of the hepatitis A virus (HAV) in shellfish. Both approaches involve extraction of viral particles by glycine buffer and concentration of virus particles by one or two PEG precipitation steps. One procedure involves as RNA extraction method the use of oligo (dT) cellulose to select poly (A) RNA, and the other uses a system in which total RNA is bound on silica membrane. Comparison of the two RT-PCR based methods highlighted the efficiency of the first approach which is less time-consuming and technically demanding than the second. PMID:15164621

Di Pinto, A; Conversano, M C; Forte, V T; La Salandra, G; Montervino, C; Tantillo, G M

2004-04-01

300

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

NASA Technical Reports Server (NTRS)

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 Resource Center Environmental Modeling System (EMS). The suite of SPoRT products for use in the EMS consists of a Sea Surface Temperature (SST) composite that includes a Lake Surface Temperature (LST) analysis over the Great Lakes, a Great Lakes sea-ice extent within the SST composite, a real-time Green Vegetation Fraction (GVF) composite, and NASA Land Information System (LIS) gridded output. This paper and companion poster describe each dataset and provide recent upgrades made to the SST, Great Lakes LST, GVF composites, and the real-time LIS runs.

Case, Jonathan L.; Lafontaine, Frank J.; Molthan, Andrew L.; Zavodsky, Bradley T.; Rozumalski, Robert A.

2012-01-01

301

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

302

Feasibility of small animal cranial irradiation with the microRT system  

PubMed Central

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° and 270° microRT treatment positions and (2) a hemispheric (left-brain) irradiation comprised of a single A-P vertex beam at the 0° 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. PMID:18975718

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.

2008-01-01

303

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

304

[RT-PCR use for the diagnostic of chronic myeloid leukaemia].  

PubMed

The molecular analysis of chromosomal abnormalities associated with hematological malignancies allowed the identification of genes involved in theses rearrangements as well as of some recurrent mechanisms. Polymerase chain reaction (PCR) tools are now available to detect these rearrangements, allowing a better follow-up of these diseases. Chronic myeloid leukemia is a myeloproliferative disorder characterized by a reciprocal translocation t(9;22)(q34;q11) which results in a bcr-abl fusion gene. Retro-transcription polymerase chain reaction (RT-PCR) is used to detect bcr-abl to establish diagnosis and to monitor patients. We report here the results of 30 patients samples tested in the hematology laboratory at Pasteur Institute, diagnosed as chronic myeloid leukemia and monitored with RT-PCR. Our results highlight the interest of molecular tools to diagnose and monitor patients mainly when cytogenetic techniques are irrelevant such as cases with complex chromosomal rearrangements or when patients achieve Philadelphia negativity after treatment. PMID:19388595

Menif, S; El Borgi, W; Jeddi, R; Belakhal, R; Elloumi, M; Laatiri, A; Meddeb, B; Dellagi, K

2006-01-01

305

Simultaneous detection and identification of four viruses infecting pepino by multiplex RT-PCR.  

PubMed

Potato virus M (PVM), pepino mosaic virus (PepMV), tomato mosaic virus (ToMV), and potato virus S (PVS) infect pepino and cause serious crop losses. In this study, a multiplex RT-PCR method was developed for simultaneous detection and differentiation of PVS, ToMV, PepMV and PVM. The method was highly reliable and sensitive; validation was accomplished by testing pepino samples collected from different regions of China. In this survey, PVM, ToMV and PVS were detected in 37.0 %, 31.0 % and 5.5 % of samples tested, respectively, confirming the widespread occurrence of these three viruses in China. PepMV was not detected in any of the samples, which indicated that this virus may not be prevalent in China. The results suggest that the new multiplex RT-PCR method has potential to be used routinely for surveys of pepino for virus infection. PMID:23338706

Ge, Beibei; Li, Qian; Liu, Guojie; Lu, Meiguang; Li, Shifang; Wang, Hongqing

2013-06-01

306

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

307

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

308

RT-EP: A Fixed-Priority Real Time Communication Protocol over Standard Ethernet  

Microsoft Academic Search

This paper presents the design and implementation of RT-EP (Real-Time Ethernet Protocol), which is a software-based token-passing Ethernet protocol for multipoint communications in real-time applica- tions, that does not require any modification to existing Ethernet hardware. The protocol allows a fixed priority to be assigned to each message, and consequently well-known schedulability analysis tech- niques can be applied. A precise

José María Martínez; Michael González Harbour

2005-01-01

309

Improved detection of barley yellow dwarf virus in single aphids using RT-PCR  

Microsoft Academic Search

The detection of a British isolate of barley yellow dwarf virus (BYDV-G, PAV-like) from individual vector aphids, using a combined assay of reverse transcription and polymerase chain reaction (RT-PCR) is reported. The method makes use of a multiplex format, including internal control primers directed at conserved regions of insect actin. The actin primers serve as controls for each stage of

E. S. G. Canning; M. J. Penrose; I. Barker; D. Coates

1996-01-01

310

Experiments of the HTS floating coil system in the mini-RT project  

Microsoft Academic Search

A magnetically levitated superconducting coil device, Mini-RT, has been constructed using a high-temperature superconductor for the purpose of examining a new magnetic confinement scheme of high-beta plasmas. The floating coil is wound with Bi-2223 Ag-sheathed tapes, and it is operated in the temperature range 20-40 K. The excitation tests of the coil were carried out and the rated current was

Nagato Yanagi; Toshiyuki Mito; Junji Morikawa; Yuichi Ogawa; Kotaro Ohkuni; Dan Hori; Shigeo Yamakoshi; Masataka Iwakuma; Toshio Uede; Ikuo Itoh; Masahiro Fukagawa; Shigeo Fukui

2004-01-01

311

A Host-Based RT-PCR Gene Expression Signature to Identify Acute Respiratory Viral Infection  

PubMed Central

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.

2014-01-01

312

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

2013-10-28

313

DISTRIBUTION OF OLIVE TREE VIRUSES IN ITALY AS REVEALED BY ONE-STEP RT-PCR  

Microsoft Academic Search

SUMMARY We have used a one-step RT-PCR protocol to detect and identify each of the eight viruses most commonly found in olive trees namely: Arabis mosaic virus (Ar- MV), Cherry leaf roll virus (CLRV), Cucumber mosaic virus (CMV), Olive leaf yellowing associated virus (OLYaV), Olive latent ring spot virus (OLRSV), Olive latent virus-1 (OLV-1), Olive latent virus-2 (OLV-2), and Strawberry

F. Faggioli; L. Ferretti; G. Albanese; R. Sciarroni; G. Pasquini; V. Lumia; M. Barba; Piazza S. Francesco

2005-01-01

314

ISSN0249-0803ISRNINRIA/RT--365--FR+ENG Thmes COM et NUM  

E-print Network

apport technique ISSN0249-0803ISRNINRIA/RT--365--FR+ENG Thèmes COM et NUM INSTITUT NATIONAL DE Abergel N° 365 Version 2 - June 2009 inria-00387324,version2-18Jun2009 #12;inria-00387324,version2-18Jun numériques Projets Oasis and Tosca Rapport technique n° 365 Version 2 - June 2009 30 pages Abstract

Boyer, Edmond

315

Function Net Modeling with UML-RT: Experiences from an Automotive Project at BMW Group  

Microsoft Academic Search

\\u000a This paper presents the function net modeling approach that has been developed within an automotive project at BMW Group aimed at software development for electronic control\\u000a units. This modeling approach provides a graphical, quite abstract representation of a (typically large) set of functions\\u000a to be realized in software or hardware. Function net models are described in UML-RT, a dialect of

Michael Von Der Beeck

2004-01-01

316

Persistence of Human Norovirus RT-qPCR Signals in Simulated Gastric Fluid.  

PubMed

Human noroviruses (HuNoV) are a leading cause of foodborne disease and are known to be environmentally persistent. Foods usually become contaminated by contact with fecal material, both on hands and on surfaces. Emerging evidence suggests that HuNoVs are also shed and potentially aerosolized during projectile vomiting, resulting in another source of contamination. The purpose of this study was to compare the persistence of HuNoV in vomitus-like material (simulated gastric fluid, SGF, pH 2.5) to that in a pH neutral buffer (phosphate buffered saline, PBS, pH 7.4) in suspension and on surfaces. Human fecal suspensions containing two HuNoV strains (GI.1 and GII.4) were suspended in SGF and PBS. Suspension and surface samples were held at room temperature, and subsamples were collected from both samples for a period up to 42 days. Subsamples were subjected to RNA isolation, with and without inclusion of an RNase pre-treatment, followed by RT-qPCR amplification. In suspension assays, the genome copy number of HuNoV GII.4 decreased by ?1.0-1.3 log10 over 42 days, irrespective of suspension buffer. On stainless steel, there was virtually no reduction in HuNoV GII.4 RT-qPCR signal over the 42-days experimental period, regardless of suspension buffer. Overall, the GI.1 RT-qPCR signal dropped more precipitously. In most cases, there were no statistically significant differences (p > 0.05) between persistence in solution or on surfaces when comparing RT-qPCR assays with and without prior RNase treatment. This study suggests that HuNoV suspended in vomitus-like material can persist for long periods, a likely contributor to foodborne transmission. PMID:25344785

Tung-Thompson, Grace; Gentry-Shields, Jennifer; Fraser, Angela; Jaykus, Lee-Ann

2014-10-26

317

Evaluation of Reference Genes for Gene Expression Analysis Using Quantitative RT-PCR in Azospirillum brasilense  

PubMed Central

Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data. PMID:24841066

McMillan, Mary; Pereg, Lily

2014-01-01

318

Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Azospirillum brasilense.  

PubMed

Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data. PMID:24841066

McMillan, Mary; Pereg, Lily

2014-01-01

319

The rtA181S mutation of hepatitis B virus primarily confers resistance to adefovir dipivoxil.  

PubMed

The study aimed to clarify clinical significance of hepatitis B virus (HBV) rtA181S mutation in Chinese HBV-infected patients. A total of 18 419 patients with chronic HBV infection from Beijing 302 Hospital were investigated. HBV complete reverse transcriptase region of polymerase was screened by direct sequencing, and the results were verified by clonal sequencing. Replication-competent mutant and wild-type HBV genomic amplicons were constructed and transfected into the HepG2 cells and cultured in the presence or absence of serially diluted nucleos(t)ide analogues. Intracellular HBV replicative intermediates were quantitated for calculating the 50% effective concentration of the drug (EC50 ). The rtA181S was detected in 98 patients with 12 kinds of mutational patterns. Genotype C and genotype B HBV infection occupied 91.8% and 8.2% in rtA181S-positive patients, in contrast to 84.6% and 15.4% in rtA181S-negative patients (P < 0.01). All rtA181S-positive patients had received nucleos(t)ide analogues. rtA181S was detected in multiple patients with virologic breakthrough. Phenotypic analysis of patient-derived viral strains showed that rtA181S, rtA181S+N236T, rtN236T and rtA181V strains had 68.5%, 49.9%, 71.4% and 66.2% of natural replication capacity of wild-type strain, and 3.7-fold, 9.8-fold, 7.9-fold and 5.6-fold increased EC50 to adefovir dipivoxil (ADV). The rtA181S strain remained susceptible to lamivudine, entecavir and tenofovir, and ADV susceptibility was restored after the mutation was eliminated through site-directed mutagenesis. Rescue therapy with entecavir or combination therapy was effective in rtA181S-related ADV-refractory patients. The rtA181S mutation confers moderate resistance to ADV. It could be induced by either lamivudine or ADV and contribute ADV treatment failure. PMID:25132017

Liu, Y; Li, X; Xin, S; Xu, Z; Chen, R; Yang, J; Liu, L; Wong, V W-S; Yang, D; Chan, H L-Y; Xu, D

2015-03-01

320

Integration of CEN\\/TC251\\/PT5-021 “VITAL” preENV standard and of “DICOM supplement 30.0” into a telemedicine system for vital signs monitoring from home  

Microsoft Academic Search

This study investigates the applicability of two `standards' addressing the robust interchange of waveform and related medical data, namely of the CEN\\/TC251\\/PT5-021 Vital Signs Information Representation (VITAL) preENV standard and the DICOM Supplement 30.0, in the context of a telemedicine application for the vital signs monitoring from their home of patients needing after-hospitalization attendance. This paper presents an object-oriented design

A. Anagnostaki; E. Kyriacou; M. Reynolds; S. Pavlopoulos; D. Koutsouris

2000-01-01

321

Universal Single-Probe RT-PCR Assay for Diagnosis of Dengue Virus Infections  

PubMed Central

Background Dengue is a mosquito-borne viral disease that has become more prevalent in the last few decades. Most patients are viremic when they present with symptoms, and early diagnosis of dengue is important in preventing severe clinical complications associated with this disease and also represents a key factor in differential diagnosis. Here, we designed and validated a hydrolysis-probe-based one-step real-time RT-PCR assay that targets the genomes of dengue virus serotypes 1–4. Methodology/Principal Findings The primers and probe used in our RT-PCR assay were designed to target the 3? untranslated region of all complete genome sequences of dengue virus available in GenBank (n?=?3,305). Performance of the assay was evaluated using in vitro transcribed RNA, laboratory-adapted virus strains, external control panels, and clinical specimens. The linear dynamic range was found to be 104–1011 GCE/mL, and the detection limit was between 6.0×102 and 1.1×103 GCE/mL depending on target sequence. The assay did not cross-react with human RNA, nor did it produce false-positive results for other human pathogenic flaviviruses or clinically important etiological agents of febrile illnesses. We used clinical serum samples obtained from returning travelers with dengue-compatible symptomatology (n?=?163) to evaluate the diagnostic relevance of our assay, and laboratory diagnosis performed by the RT-PCR assay had 100% positive agreement with diagnosis performed by NS1 antigen detection. In a retrospective evaluation including 60 archived serum samples collected from confirmed dengue cases 1–9 days after disease onset, the RT-PCR assay detected viral RNA up to 9 days after appearance of symptoms. Conclusions/Significance The validation of the RT-PCR assay presented here indicates that this technique can be a reliable diagnostic tool, and hence we suggest that it be introduced as the method of choice during the first 5 days of dengue symptoms. PMID:25522325

Alm, Erik; Lesko, Birgitta; Lindegren, Gunnel; Ahlm, Clas; Söderholm, Sandra; Falk, Kerstin I.; Lagerqvist, Nina

2014-01-01

322

A one step real-time RT-PCR assay for the quantitation of Wheat yellow mosaic virus (WYMV)  

PubMed Central

Background Wheat yellow mosaic virus (WYMV) is an important pathogen in China and other countries. It is the member of the genus Bymovirus and transmitted primarily by Polymyxa graminis. The incidence of wheat infections in endemic areas has risen in recent years. Prompt and dependable identification of WYMV is a critical component of response to suspect cases. Methods In this study, a one step real-time RT-PCR, followed by standard curve analysis for the detection and identification of WYMV, was developed. Two reference genes, 18s RNA and ?-actin were selected in order to adjust the veracity of the real-time RT-PCR assay. Results We developed a one-step Taqman-based real-time quantitative RT-PCR (RT-qPCR) assay targeting the conserved region of the 879 bp long full-length WYMV coat protein gene. The accuracy of normalized data was analyzed along with appropriate internal control genes: ?-actin and 18s rRNA which were included in detecting of WYMV-infected wheat leaf tissues. The detectable end point sensitivity in RT-qPCR assay was reaching the minimum limit of the quantitative assay and the measurable copy numbers were about 30 at106-fold dilution of total RNA. This value was close to 104-fold more sensitive than that of indirect enzyme-linked immunosorbent assay. More positive samples were detected by RT-qPCR assay than gel-based RT-PCR when detecting the suspected samples collected from 8 regions of China. Based on presented results, RT-qPCR will provide a valuable method for the quantitative detection of WYMV. Conclusions The Taqman-based RT-qPCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of WYMV than other currently used methods. PMID:23725024

2013-01-01

323

Development of one-step SYBR Green real-time RT-PCR for quantifying bovine viral diarrhea virus type-1 and its comparison with conventional RT-PCR  

E-print Network

Background Bovine viral diarrhea virus (BVDV) is a worldwide pathogen in cattle and acts as a surrogate model for hepatitis C virus (HCV). One-step real-time fluorogenic quantitative reverse transcription polymerase chain reaction (RT-PCR) assay...

Zhang, Ni; Liu, Zhengwen; Han, Qunying; Qiu, Jianming; Chen, Jinghong; Zhang, Guoyu; Li, Zhu; Lou, Sai; Li, Na

2011-07-29

324

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

325

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

326

Amino acid mutations in the env gp90 protein that modify N-linked glycosylation of the Chinese EIAV vaccine strain enhance resistance to neutralizing antibodies.  

PubMed

The Chinese EIAV vaccine is an attenuated live-virus vaccine obtained by serial passage of a virulent horse isolate (EIAV(L)) in donkeys (EIAV(D)), and subsequently in donkey cells in vitro. In this study, we compare the env gene of the original horse virulent virus (EIAV(L)) with attenuated strains serially passaged in donkey MDM (EIAV(DLV)), and donkey dermal cells (EIAV(FDDV)). Genetic comparisons among parental and attenuated strains found that vaccine strains contained amino acid substitutions/deletions in gp90 that resulted in a loss of three potential N-linked glycosylation sites, designated g5, g9, and g10. To investigate the biological significance of these changes, reverse-mutated viruses were constructed in the backbone of the EIAV(FDDV) infectious molecular clone (pLGFD3). The resulting virus stocks were characterized for replication efficiency in donkey dermal cells and donkey MDM, and were tested for sensitivity to neutralization using sera from two ponies experimentally infected with EIAV(FDDV). The results clearly show that these mutations generated by site-directed mutagenesis resulted in cloned viruses with enhanced resistance to serum-neutralizing antibodies that were also able to recognize parental viruses. The results of this study indicate that these mutations play an important role in the attenuation of the EIAV vaccine strains. PMID:20883167

Han, Xiue; Zou, Jianhua; Wang, Xuefeng; Guo, Wei; Huo, Guicheng; Shen, Rongxian; Xiang, Wenhua

2010-10-01

327

HIV-1 Env DNA Vaccine plus Protein Boost Delivered by EP Expands B- and T-Cell Responses and Neutralizing Phenotype In Vivo  

PubMed Central

An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs), and the elicitation of antibody-dependent cellular cytotoxicity (ADCC). Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP). However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted. PMID:24391921

Muthumani, Kar; Wise, Megan C.; Broderick, Kate E.; Hutnick, Natalie; Goodman, Jonathan; Flingai, Seleeke; Yan, Jian; Bian, Chaoran B.; Mendoza, Janess; Tingey, Colleen; Wilson, Christine; Wojtak, Krzysztof; Sardesai, Niranjan Y.; Weiner, David B.

2013-01-01

328

The rat T-cell surface protein RT6 is associated with src family tyrosine kinases and generates an activation signal  

Microsoft Academic Search

RT6 is a glycosyl-phosphatidylinositol-linked surface molecule present on most mature rat T-cells. RT6+ T-cells can prevent the expression of autoimmune diabetes in the BB rat, but the mechanism is unknown. Because cross-linking of other glycosyl-phosphatidylinositol-linked T-cell proteins is known to activate T-cells, we investigated the signaling properties of RT6. Antibody cross-linking of RT6 enhanced expression of the alpha subunit of

Mark R. Rigby; Rita Bortell; Dale L. Greiner; Michael P. Czech; Jes K. Klarlund; John P. Mordes; Aldo A. Rossini

1996-01-01

329

ff! F ff . claim. Suppose F is a truncation closed L an elementary substructure of R((t)) E and the  

E-print Network

is an L R an ­elementary submodel of R((t)) E . The above claim, the truncation results of x3 of [3 submodel of R((t)) E , y 2 R((t)) E , v(y) 62 v(F ) and F \\Lambda is the smallest L R an ­elementary submodel of R((t)) E containing F (y), then F \\Lambda is truncation closed and the value group of F \\Lambda

Marker, David

330

Chemical composition and RT[sub NDT] determinations for Midland weld WF-70  

SciTech Connect

The Heavy-Section Steal Irradiation Program Tenth Irradiation Series has the objective to investigate the affects of radiation on the fracture toughness of the low-upper-shelf submerged-arc welds (B W designation WF-70) in the reactor pressure vessel of the canceled Midland Unit 1 nuclear plant. This report discusses determination of variations in chemical composition And reference temperature (RT[sub NDT]) throughout the welds. Specimens were machined from different sections and through thickness locations in both the beltline and nozzle course welds. The nil-ductility transition temperatures ranged from [minus]40 to [minus]60[degrees]C ([minus]40 and [minus]76[degrees]F) while the RT[sub NDT]S, controlled by the Charpy behavior, varied from [minus]20 to 37[degrees]C ([minus]4 to 99[degrees]F). The upper-shelf energies varied from 77 to 108 J (57 to 80 ft-lb). The combined data revealed a mean 41-J (30-ft-lb) temperature of [minus]8[degrees]C (17[degrees]F) with a mean upper-shelf energy of 88 J (65 ft-lb). The copper contents range from 0.21 to 0.34 wt % in the beltline weld and from 0.37 to 0.46 wt % in the nozzle course weld. Atom probe field ion microscope analyses indicated substantial depletion of copper in the matrix but no evidence of copper clustering. Statistical analyses of the Charpy and chemical composition results as well as interpretation of the ASME procedures for RT[sub NDT] determination are discussed.

Nanstad, R.K.; McCabe, D.E.; Swain, R.L.; Miller, M.K. (Oak Ridge National Lab., TN (United States))

1992-12-01

331

Identification of Reference Genes for Quantitative RT-PCR in Ascending Aortic Aneurysms  

PubMed Central

Hypertension and congenital aortic valve malformations are frequent causes of ascending aortic aneurysms. The molecular mechanisms of aneurysm formation under these circumstances are not well understood. Reference genes for gene activity studies in aortic tissue that are not influenced by aortic valve morphology and its hemodynamic consequences, aortic dilatation, hypertension, or antihypertensive medication are not available so far. This study determines genes in ascending aortic tissue that are independent of these parameters. Tissue specimens from dilated and undilated ascending aortas were obtained from 60 patients (age ?70 years) with different morphologies of the aortic valve (tricuspid undilated n?=?24, dilated n?=?11; bicuspid undilated n?=?6, dilated n?=?15; unicuspid dilated n?=?4). Of the studied individuals, 36 had hypertension, and 31 received ACE inhibitors or AT1 receptor antagonists. The specimens were obtained intraoperatively from the wall of the ascending aorta. We analyzed the expression levels of 32 candidate reference genes by quantitative RT-PCR (RT-qPCR). Differential expression levels were assessed by parametric statistics. The expression analysis of these 32 genes by RT-qPCR showed that EIF2B1, ELF1, and PPIA remained constant in their expression levels in the different specimen groups, thus being insensitive to aortic valve morphology, aortic dilatation, hypertension, and medication with ACE inhibitors or AT1 receptor antagonists. Unlike many other commonly used reference genes, the genes EIF2B1, ELF1, and PPIA are neither confounded by aortic comorbidities nor by antihypertensive medication and therefore are most suitable for gene expression analysis of ascending aortic tissue. PMID:23326585

Henn, Dominic; Bandner-Risch, Doris; Perttunen, Hilja; Schmied, Wolfram; Porras, Carlos; Ceballos, Francisco; Rodriguez-Losada, Noela; Schäfers, Hans-Joachim

2013-01-01

332

Intravenous rt-PA for acute stroke: comparing its effectiveness in younger and older patients  

PubMed Central

Objective: To study the short and long term differences in outcome between patients ?80 years of age and those ?79 years of age who received intravenous recombinant tissue plasminogen activator (iv rt-PA) for acute stroke within the first 3 hours of symptom onset. Methods: We studied consecutive patients treated with iv rt-PA for acute stroke, with prospective follow up of up to 3 years. Outcome measures included National Institutes of Health Stroke Scale (NIHSS) score, Barthel Index (BI), modified Rankin score (MRS), and stroke mortality. Patients were split into two groups: younger (?79 years) and older (?80 years). Results: There were 65 patients in the younger cohort and 31 patients in the older. Older patients were more likely to present with more severe baseline stroke (p = 0.04; odds ratio (OR) 3.04; 95% confidence interval (CI) 1.03 to 8.98). Stroke mortality at 90 days was 10.8% in the younger and 32.3% in the older cohort (p = 0.01). At 90 days' follow up, patients in the older cohort with more severe stroke (NIHSS score ?11) were nearly 10 times more likely to have poor outcome compared with their younger counterparts presenting with severe stroke (p = 0.001; OR = 10.36; 95% CI 2.16 to 49.20). Baseline stroke severity and age were the only independent and equal predictors for stroke outcome. No threshold was found for age or baseline stroke severity predicting outcome. Conclusion: Older patients presenting with more severe baseline stroke are much less likely to benefit from iv rt-PA as compared with their younger counterparts. PMID:16107357

Mouradian, M; Senthilselvan, A; Jickling, G; McCombe, J; Emery, D; Dean, N; Shuaib, A

2005-01-01

333

Diffuse large B-cell lymphoma: sub-classification by massive parallel quantitative RT-PCR.  

PubMed

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous entity with remarkably variable clinical outcome. Gene expression profiling (GEP) classifies DLBCL into activated B-cell like (ABC), germinal center B-cell like (GCB), and Type-III subtypes, with ABC-DLBCL characterized by a poor prognosis and constitutive NF-?B activation. A major challenge for the application of this cell of origin (COO) classification in routine clinical practice is to establish a robust clinical assay amenable to routine formalin-fixed paraffin-embedded (FFPE) diagnostic biopsies. In this study, we investigated the possibility of COO-classification using FFPE tissue RNA samples by massive parallel quantitative reverse transcription PCR (qRT-PCR). We established a protocol for parallel qRT-PCR using FFPE RNA samples with the Fluidigm BioMark HD system, and quantified the expression of the COO classifier genes and the NF-?B targeted-genes that characterize ABC-DLBCL in 143 cases of DLBCL. We also trained and validated a series of basic machine-learning classifiers and their derived meta classifiers, and identified SimpleLogistic as the top classifier that gave excellent performance across various GEP data sets derived from fresh-frozen or FFPE tissues by different microarray platforms. Finally, we applied SimpleLogistic to our data set generated by qRT-PCR, and the ABC and GCB-DLBCL assigned showed the respective characteristics in their clinical outcome and NF-?B target gene expression. The methodology established in this study provides a robust approach for DLBCL sub-classification using routine FFPE diagnostic biopsies in a routine clinical setting. PMID:25418578

Xue, Xuemin; Zeng, Naiyan; Gao, Zifen; Du, Ming-Qing

2015-01-01

334

Characterization of a thermostable 2,4-diaminopentanoate dehydrogenase from Fervidobacterium nodosum Rt17-B1.  

PubMed

2,4-Diaminopentanoate dehydrogenase (2,4-DAPDH), which is involved in the oxidative ornithine degradation pathway, catalyzes the NAD(+)- or NADP(+)-dependent oxidative deamination of (2R,4S)-2,4-diaminopentanoate (2,4-DAP) to form 2-amino-4-oxopentanoate. A Fervidobacterium nodosum Rt17-B1 gene, Fnod_1646, which codes for a protein with sequence similarity to 2,4-DAPDH discovered in metagenomic DNA, was cloned and overexpressed in Escherichia coli, and the gene product was purified and characterized. The purified protein catalyzed the reduction of NAD(+) and NADP(+) in the presence of 2,4-DAP, indicating that the protein is a 2,4-DAPDH. The optimal pH and temperature were 9.5 and 85°C, respectively, and the half-denaturation time at 90°C was 38 min. Therefore, the 2,4-DAPDH from F. nodosum Rt17-B1 is an NAD(P)(+)-dependent thermophilic-alkaline amino acid dehydrogenase. This is the first thermophilic 2,4-DAPDH reported, and it is expected to be useful for structural and functional analyses of 2,4-DAPDH and for the enzymatic production of chiral amine compounds. Activity of 2,4-DAPDH from F. nodosum Rt17-B1 was suppressed by 2,4-DAP via uncompetitive substrate inhibition. In contrast, the enzyme showed typical Michaelis-Menten kinetics toward 2,5-diaminohexanoate. The enzyme was uncompetitively inhibited by d-ornithine with an apparent Ki value of 0.1 mM. These results suggest a regulatory role for this enzyme in the oxidative ornithine degradation pathway. PMID:24326351

Fukuyama, Sadanobu; Mihara, Hisaaki; Miyake, Ryoma; Ueda, Makoto; Esaki, Nobuyoshi; Kurihara, Tatsuo

2014-05-01

335

NASA/SPoRT's GOES-R Activities in Support of Product Development, Management, and Training  

NASA Astrophysics Data System (ADS)

The NASA Short-term Prediction Research and Transition (SPoRT) Center supports many activities within the GOES-R Proving Grounds (PG). These include the development of imagery from existing instrumentation as a proxy to future Advanced Baseline Imager (ABI) capabilities on GOES-R. The Moderate Resolution Imaging Spectroradiometer (MODIS) and the Visible/Infrared Imager/Radiometer Suite (VIIRS) instruments are used to provide a glimpse of the multi-spectral capabilities that will become the norm as the number of channels and data rate dramatically increase with GOES-R. The NOAA/NWS has plans to provide operational users with all ABI channels at the highest resolution. Data fusion of individual channels into composite red, green, and blue imagery products will assist the end user with this future wave of information. While increasing the efficiency in the operational use of ABI channels, these composites provide only qualitative information. Within the GOES-R PG, SPoRT and other partners are exploring ways to include quantitative information as part of the composite imagery. However, limitations in local hardware processing and/or data bandwidth for users of the GOES-R data stream are challenges to overcome. This presentation will discuss the creation of these composite images as well as possible solutions to address these processing challenges. In a similar manner the Geostationary Lightning Mapper (GLM) to be launched on GOES-R presents several data management challenges. The GLM is a pioneering instrument to quantify total lightning from a geostationary platform. The expected data frequency from the GLM is to be at a sub-minute interval. Users of such a data set may have little experience in handling such a rapid update of information. To assist users, SPoRT is working with the NWS to develop tools within the user's decision support system to allow tracking and analysis of total lightning from a storm-based perspective. This presentation will discuss the challenges and progress of this tool development work. With new data and products comes the need for user Training. Within the GOES-R PG SPoRT is supporting the demonstration of these future products by providing various training materials to end users. A summary of training provided to operational users will be discussed.

Fuell, K. K.; Jedlovec, G.; Molthan, A.; Stano, G. T.

2012-12-01

336

NASA/SPoRT's GOES-R Activities in Support of Product Development, Management, and Training  

NASA Technical Reports Server (NTRS)

The NASA Short-term Prediction Research and Transition (SPoRT) Center supports many activities within the GOES-R Proving Grounds (PG). These include the development of imagery from existing instrumentation as a proxy to future Advanced Baseline Imager (ABI) capabilities on GOES-R. The Moderate Resolution Imaging Spectroradiometer (MODIS) and the Visible/Infrared Imager/Radiometer Suite (VIIRS) instruments are used to provide a glimpse of the multi-spectral capabilities that will become the norm as the number of channels and data rate dramatically increase with GOES-R. The NOAA/NWS has plans to provide operational users with all ABI channels at the highest resolution. Data fusion of individual channels into composite red, green, and blue imagery products will assist the end user with this future wave of information. While increasing the efficiency in the operational use of ABI channels, these composites provide only qualitative information. Within the GOES-R PG, SPoRT and other partners are exploring ways to include quantitative information as part of the composite imagery. However, limitations in local hardware processing and/or data bandwidth for users of the GOES-R data stream are challenges to overcome. This presentation will discuss the creation of these composite images as well as possible solutions to address these processing challenges. In a similar manner the Geostationary Lightning Mapper (GLM) to be launched on GOES-R presents several data management challenges. The GLM is a pioneering instrument to quantify total lightning from a geostationary platform. The expected data frequency from the GLM is to be at a sub-minute interval. Users of such a data set may have little experience in handling such a rapid update of information. To assist users, SPoRT is working with the NWS to develop tools within the user fs decision support system to allow tracking and analysis of total lightning from a storm-based perspective. This presentation will discuss the challenges and progress of this tool development work. With new data and products comes the need for user Training. Within the GOES-R PG SPoRT is supporting the demonstration of these future products by providing various training materials to end users. A summary of training provided to operational users will be discussed.

Fuell, Kevin K.; Jedlovec, Gary; Molthan, Andrew L.; Stano, Geoffrey T.

2012-01-01

337

Accurate RT-qPCR gene expression analysis on cell culture lysates.  

PubMed

Gene expression quantification on cultured cells using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) typically involves an RNA purification step that limits sample processing throughput and precludes parallel analysis of large numbers of samples. An approach in which cDNA synthesis is carried out on crude cell lysates instead of on purified RNA samples can offer a fast and straightforward alternative. Here, we evaluate such an approach, benchmarking Ambion’s Cells-to-CT kit with the classic workflow of RNA purification and cDNA synthesis, and demonstrate its good accuracy and superior sensitivity. PMID:22355736

Van Peer, Gert; Mestdagh, Pieter; Vandesompele, Jo

2012-01-01

338

The NASA Short-Term Prediction Research and Transition (SPoRT) Center: Opportunities for Collaboration in the Great Lakes Region  

NASA Technical Reports Server (NTRS)

The presentation slides include: The SPoRT Center, History and Future of SPoRT, Great Lakes Applications, Great Lakes Forecasting Issues, Applications to the WRF-EMS, Precipitation Science, Lake Effect Precipitation, Sensitivity to Microphysics, Exploring New Schemes, Opportunities for Collaboration, and SPoRT Research and Development.

Molthan, Andrew L.

2010-01-01

339

UNIVERSAL EXTERNAL RNA QUALITY CONTROLS FOR MRNA EXPRESSION ANALYSIS USING MICROBIAL DNA OLIGO MICROARRAY AND REAL TIME QUANTITATIVE RT-PCR  

Technology Transfer Automated Retrieval System (TEKTRAN)

With rapid advances of genomic sequence and development of biotechnology, gene expression analysis using microarray and real time quantitative RT-PCR (qRT-PCR) has been widely used in a broad range of fields in biology. Quality control of microarray and qRT-PCR experiments has become an important i...

340

RT-OMT: a real-time object modeling technique for designing real-time database applications  

Microsoft Academic Search

This paper describes a methodology called RT-OMT (Real-time Object Modeling Technique) for designing real-time database applications. RT-OMT adapts the OMT (Object Modeling Technique) methodology for this purpose. OMT is one of the more popular object-oriented methodologies for designing applications for complex information systems. These include database systems, executive\\/enterprise information systems, collaborative computing systems, medical information systems, and hypermedia systems. This

Bhavani Thuraisingham; Alice Schafer

1994-01-01

341

Simultaneous quantitation of two orchid viruses by the TaqMan ® real-time RT-PCR  

Microsoft Academic Search

Simultaneous quantitation of two orchid viruses, cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV), were carried out using the TaqMan® real-time RT-PCR, a novel detection technique that combines RT-PCR with the power of fluorescent detection. Four TaqMan® probes were synthesized, targeting at the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes of both viruses. The reporter dye FAM

Alvin Jin-Cherng Eun; Mui-Leng Seoh; Sek-Man Wong

2000-01-01

342

Preoperative radiotherapy (RT) for rectal cancer: Predictive factors of tumor downstaging and residual tumor cell density (RTCD): Prognostic implications  

Microsoft Academic Search

Purpose: To determine predictive factors and prognostic value of tumor downstaging and tumor sterilization after preoperative RT for rectal cancer.Methods and Materials: Between 1977 and 1994, 167 patients with a histologically proven adenocarcinoma (70 T2, 65 T3, 29 T4, and 3 local recurrences) underwent preoperative RT. Median dose was 44 Gy (5–73 Gy). Surgery was performed in a mean time

Christine Berger; Anne de Muret; Pascal Garaud; Sophie Chapet; Pascal Bourlier; Agnès Reynaud-Bougnoux; Etienne Dorval; Loïc de Calan; Noël Huten; Olivier le Floch; Gilles Calais

1997-01-01

343

Insulin treatment prevents diabetes mellitus but not thyroiditis in RT6-depleted diabetes resistant BB\\/Wor rats  

Microsoft Academic Search

Summary  Prophylactic insulin administration is known to prevent hyperglycaemia in diabetes prone BB rats and non-obese diabetic mice. This study investigated the effect of insulin treatment on the development of overt diabetes, clinically inapparent anti-islet autoreactivity, and thyroiditis in RT6-depleted diabetes resistant BB rats. Fewer than 1% of these animals develop spontaneous diabetes, but if depleted of RT6+ T cells >50%

P. A. Gottlieb; E. S. Handler; M. C. Appel; D. L. Greiner; J. P. Mordes; A. A. Rossini

1991-01-01

344

A powerful and flexible linear mixed model framework for the analysis of relative quantification RT-PCR data  

Microsoft Academic Search

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is currently viewed as the most precise technique to quantify levels of messenger RNA. Relative quantification compares the expression of a target gene under two or more experimental conditions normalized to the measured expression of a control gene. The statistical methods and software currently available for the analysis of relative quantification of RT-PCR

Juan Pedro Steibel; Rosangela Poletto; Paul M. Coussens; Guilherme J. M. Rosa

2009-01-01

345

Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue  

Microsoft Academic Search

BACKGROUND: Studies of gene expression in post mortem human brain can contribute to understanding of the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Quantitative real-time PCR (RT qPCR) is often used to analyse gene expression. The validity of results obtained using RT qPCR is reliant on accurate data normalization. Reference

David TR Coulson; Simon Brockbank; Joseph G Quinn; Suzanne Murphy; Rivka Ravid; G Brent Irvine; Janet A Johnston

2008-01-01

346

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

PubMed

Dengue virus (DENV) transmission occurs throughout the Caribbean, though laboratory confirmation and epidemiologic surveillance are limited by the availability of serotype-specific molecular diagnostics. In this study, we show that a serotype-specific DENV multiplex, real-time reverse transcriptase-PCR (RT-PCR) detected DENV RNA in significantly more samples (82/182) than a reference hemi-nested RT-PCR (57/182; P=0.01). PMID:25533614

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

2015-02-01

347

Alternative method of RT{sub NDT} determination for some reactor vessel weld metals validated by fracture toughness data  

SciTech Connect

The fracture toughness curves used for nuclear power plant operation pressure-temperature limits and for pressurized thermal shock evaluations are dependent on the reference temperature for nil-ductility transition (RT{sub NDT}). The original method to determine the RT{sub NDT} was formulated more than 20 yr ago when Section 3 of the ASME Code was adopted. At that time, there were insufficient data to judge whether some of the weld metals used in reactor vessel fabrication were unsuitable for this procedure. Presently, this causes a compliance problem for some weld metals used in nuclear reactor vessels, whereas there is no technical problem in meeting required safety margins. The RT{sub NDT} is a parameter to index degrees of irradiation embrittlement to adjust the Code reference fracture toughness curves to represent the actual degraded fracture toughness at a given fluence of a reactor vessel beltline region. When there is a problem determining RT{sub NDT} value for unirradiated material where Charpy transition temperature is the dominating criterion, an alternative RT{sub NDT} based solely on a drop-weight test was investigated for some of the weld metals. Using a new test method for fracture toughness in the transition range (ASTM, 1993), a fracture toughness curve was directly generated from a set of compact tension test data and used for validating the nil-ductility temperature (T{sub NDT}) from drop-weight test data as the sole mean for determining initial RT{sub NDT} value.

Yoon, K.K. [B and W Nuclear Technologies, Lynchburg, VA (United States)

1995-11-01

348

RT Leonis Minoris: Does It Belong to an A- or W-Type Overcontact Binary System?  

NASA Astrophysics Data System (ADS)

New photometric observations and analyses of the W UMa-type binary system RT Leonis Minoris are presented. A period study with our four times of light minimum, including other eclipse times from the literature, suggests that the orbital period of the binary star is variable. The O-C curve can be explained as a cyclic variation with an amplitude of 0.0049d and a period of 46.7yr. Photometric solutions were derived by using the 2003 version of the Wilson-Devinney method (1971, ApJ, 166, 605), and absolute parameters of the system were determined. The results indicate that RT LMi is an ambiguous W UMa-type overcontact system for the following reasons: (i) the depths of both minima are nearly equal, which makes it very difficult to distinguish whether it belongs to an A- or W-type system; (ii) the primary minima in Hoffmann's light curves are an occultation, indicating a W-type system, while the primary minimum in our light curve is a transit, suggesting an A-type light variation; and (iii) though our light curve shows an A-type variation, the photometric solutions indicate a higher secondary temperature of T2= 6513K (T1= 6400K), making the binary system, surprisingly, of W-type. These may indicate that Binnendijk's classification based on the shape of the light curve cannot reveal the physical properties of W UMa-type binary stars of this kind.

Qian, Sheng-Bang; He, Jia-Jia; Xiang, Fu-Yuan

2008-02-01

349

Multiplex RT-PCR method for the simultaneous detection of nine grapevine viruses.  

PubMed

Viral diseases are a serious pathological problem for grapevines, and in recent years the need for increasingly specific and rapid diagnostic methods for the selection of propagation materials has grown. Arabis mosaic virus, Grapevine fanleaf virus, Grapevine virus A, Grapevine virus B, Grapevine rupestris stem pitting-associated virus, Grapevine fleck virus, and Grapevine leafroll-associated viruses 1, 2, and 3 are nine of the most widespread viruses that naturally infect grapevines. A multiplex RT-PCR was developed for simultaneous detection of these nine grapevine viruses, in combination with a plant RNA internal control used as an indicator of the effectiveness of the reaction. One to ten fragments specific for the viruses and an internal control were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel. The protocol reported is an update of previously published protocols for RNA extraction and multiplex diagnosis of viruses. After several years of use and hundreds of samples tested, and following validation in several laboratories, this multiplex RT-PCR provides a reliable and rapid method for detecting grapevine viruses from a large number of samples. PMID:25287494

Gambino, Giorgio

2015-01-01

350

Dusty shells surrounding the carbon variables S Scuti and RT Capricorni  

NASA Astrophysics Data System (ADS)

For the Mass-loss of Evolved StarS (MESS) programme, the unprecedented spatial resolution of the PACS photometer on board the Herschel Space Observatory was employed to map the dusty environments of asymptotic giant branch (AGB) and red supergiant (RSG) stars. Among the morphologically heterogeneous sample, a small fraction of targets is enclosed by spherically symmetric detached envelopes. Based on observations in the 70 ?m and 160 ?m wavelength bands, we investigated the surroundings of the two carbon semiregular variables S Sct and RT Cap, which both show evidence for a history of highly variable mass-loss. S Sct exhibits a bright, spherically symmetric detached shell, 138? in diameter and co-spatial with an already known CO structure. Moreover, weak emission is detected at the outskirts, where the morphology seems indicative of a mild shaping by interaction of the wind with the interstellar medium, which is also supported by the stellar space motion. Two shells are found around RT Cap that were not known so far in either dust emission or from molecular line observations. The inner shell with a diameter of 188? shows an almost immaculate spherical symmetry, while the outer ~5' structure is more irregularly shaped. MoD, a modification of the DUSTY radiative transfer code, was used to model the detached shells. Dust temperatures, shell dust masses, and mass-loss rates are derived for both targets. Appendices are available in electronic form at http://www.aanda.org

Me?ina, M.; Kerschbaum, F.; Groenewegen, M. A. T.; Ottensamer, R.; Blommaert, J. A. D. L.; Mayer, A.; Decin, L.; Luntzer, A.; Vandenbussche, B.; Posch, Th.; Waelkens, C.

2014-06-01

351

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. PMID:24066051

Angione, Stephanie L.; Inde, Zintis; Beck, Christina M.; Artenstein, Andrew W.; Opal, Steven M.; Tripathi, Anubhav

2013-01-01

352

[Cloning and analysis of reverse transcriptase(RT) of Ty1-copia retrotransposons in Dendrobium officinale].  

PubMed

Using universal primer Ty1-copia retrotransposon RT,43 Ty1-copia like retrotransposon RT with high heterogeneity, stop codon mutation and frameshift mutation were amplified by PCR from genomic DNA of Zhejiang Lin'an (C15) and Yunnan Guangnan (A39) of Dendrobium officinale. The length of these sequences varied from 260 to 266 bp, and was rich in AT and consistency ranged from 47.1% to 97.7%. Different c/s-acting regulatory elements induced by low temperature, heat, light, all kinds of plant growth regulating substances and the starting transcription signals, corresponding to CAAT box, TATA box conserved sequences and some other regulatory elements. When being translated into amino acids, ten sequences presented stop codon mutation, five sequences presented frameshift mutation, and thirty-seven sequences presented conserved sequence "SLYGKQ" mutation. Six categories were identified through phylogenic analysis after alignment analyses of their amino acid sequences, and with other plants (eg. Triticum aestivum, Eleocharis quinqueflora) having high homology, which indicated that horizontal transmission of retrotransposon occurred among the plants in the past. PMID:24761633

Li, Cong; Si, Jin-Ping; Gao, Yan-Hui; Zhu, Yu-Qiu

2014-01-01

353

Evaluation of the efficacy of disinfectants against Puumala hantavirus by real-time RT-PCR.  

PubMed

Puumala virus, a hantavirus belonging to the Bunyaviridae family, causes a human disease known as nephropathia epidemica, a mild form of hemorrhagic fever with renal syndrome. The implementation of effective decontamination procedures is critical in hantavirus research to minimize the risk of personnel exposure. This study investigated the efficacy of Clidox((R)), Dettol((R)), ethanol, Halamid-d((R)), peracetic acid, sodium hypochloride and Virkon((R))S for inactivating Puumala virus. A real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to quantify Puumala virus before and after treatment with these products. Inactivation of Puumala virus was effective after 10min with all products except ethanol. Inactivation with absolute ethanol was effective only after 30min. Using the qRT-PCR method, this study has shown that the commercially available products Clidox((R)), Halamid-d((R)) and Virkon((R))S in particular represent a rapid and safe way to decontaminate surfaces with possible Puumala virus contamination. These products can be used in solutions of 1-2%, with contact times greater than 10min, for inactivating effectively Puumala virus. PMID:17188760

Maes, Piet; Li, Sandra; Verbeeck, Jannick; Keyaerts, Els; Clement, Jan; Van Ranst, Marc

2007-04-01

354

Detection of human beta defensin-1 and -2 by RT-competitive multiplex PCR.  

PubMed

Although, the human epithelium is constantly challenged by a broad spectrum of microorganisms, invasive infections are rather rare. Recent findings suggest the expression of antimicrobial peptides by skin cells in order to provide an innate defensive barrier. In particular, peptides of the beta-defensin family offer antimicrobial activity against different pathogens including bacteria and fungi. Within this peptide family, hBD-1 is rather constitutively expressed while hBD-2 and hBD-3 expression depends on environmental conditions. The present paper introduces RT-competitive multiplex PCR as a precise tool to detect hBD-1 and hBD-2 expression on the transcriptional level. The method makes use of co-amplification of synthetic competitors along with referring wildtype targets. Competitor- and wildtype-derived products differ in size allowing signal discrimination using agarose gel electrophoresis. Regulation of gene transcripts is evaluated by comparison of competitor and corresponding wildtype signals. It was found that primary human keratinocytes stimulated with Escherichia coli cells for 8 h offered an upregulation of hBD-2 to about 2,000 fold, while hBD-1 was only marginally regulated. RT-competitive multiplex PCR is a simple and accurate method that enables new insights into defensin regulation under physiological and pathophysiological conditions. PMID:15821924

Kippenberger, Stefan; Loitsch, Stefan; Thaci, Diamant; Kaufmann, Roland; Bernd, August

2005-05-01

355

Genetic analyses of HIV-1 env sequences demonstrate limited compartmentalization in breast milk and suggest viral replication within the breast that increases with mastitis.  

PubMed

The concentration of human immunodeficiency virus type 1 (HIV-1) is generally lower in breast milk than in blood. Mastitis, or inflammation of the breast, is associated with increased levels of milk HIV-1 and risk of mother-to-child transmission through breastfeeding. We hypothesized that mastitis facilitates the passage of HIV-1 from blood into milk or stimulates virus production within the breast. HIV-1 env sequences were generated from single amplicons obtained from breast milk and blood samples in a cross-sectional study. Viral compartmentalization was evaluated using several statistical methods, including the Slatkin and Maddison (SM) test. Mastitis was defined as an elevated milk sodium (Na(+)) concentration. The association between milk Na(+) and the pairwise genetic distance between milk and blood viral sequences was modeled using linear regression. HIV-1 was compartmentalized within milk by SM testing in 6/17 (35%) specimens obtained from 9 women, but all phylogenetic clades included viral sequences from milk and blood samples. Monotypic sequences were more prevalent in milk samples than in blood samples (22% versus 13%; P = 0.012), which accounted for half of the compartmentalization observed. Mastitis was not associated with compartmentalization by SM testing (P = 0.621), but Na(+) was correlated with greater genetic distance between milk and blood HIV-1 populations (P = 0.041). In conclusion, local production of HIV-1 within the breast is suggested by compartmentalization of virus and a higher prevalence of monotypic viruses in milk specimens. However, phylogenetic trees demonstrate extensive mixing of viruses between milk and blood specimens. HIV-1 replication in breast milk appears to increase with inflammation, contributing to higher milk viral loads during mastitis. PMID:20660189

Gantt, Soren; Carlsson, Jacquelyn; Heath, Laura; Bull, Marta E; Shetty, Avinash K; Mutsvangwa, Junior; Musingwini, Georgina; Woelk, Godfrey; Zijenah, Lynn S; Katzenstein, David A; Mullins, James I; Frenkel, Lisa M

2010-10-01

356

Archival fixed histologic and cytologic specimens including stained and unstained materials are amenable to RT-PCR.  

PubMed

Formalin-fixed and paraffin-embedded tissues are increasingly used for analysis of gene expression. However, a large proportion of archival fixed histologic specimens including spare paraffin sections and stained slides, as well as archival cytologic materials, have not been investigated for their suitability for RNA-based analysis. The current study addressed this issue by reverse transcription and polymerase chain reaction (RT-PCR) of the glucose-6-phosphate dehydrogenase (G6PD) transcript in a series of archival histologic and cytologic specimens. The histologic specimens included freshly prepared paraffin sections, spare paraffin sections, hematoxylin and eosin-stained slides, immunostained slides, and decalcified bone marrow trephines. The cytologic specimens comprised cervical smears and various stained and unstained needle aspirates and cell sediments. The G6PD was amplified for five different fragment sizes ranging from 67 bp to 453 bp. It was found that the majority of archival materials were amenable to RT-PCR of small fragments with the overall success rates of 95% and 79% for 67 bp and 151 bp of the G6PD mRNA, respectively. Neither staining nor prolonged storage up to 15 years had major negative effects on RT-PCR, although fine-needle aspirates showed a higher rate of RT-PCR of 242-bp fragment than other types of cytologic specimens and so did Papanicolaou-stained samples than May Grounwald and Giemsa-stained samples. RT-PCR of minute cell populations microdissected from immunostained sections of tonsils and t(11;18)-positive mucosa-associated lymphoid tissue lymphomas showed that as few as 100 cells were adequate for RT-PCR of G6PD and translocation-associated fusion transcript as long as the target fragment was limited to less than 150 bp. Our results demonstrate that archival fixed histologic and cytologic specimens are valuable resources for RT-PCR-based molecular investigations. PMID:12459638

Liu, Hongxiang; Huang, Xuebiao; Zhang, Yun; Ye, Hongtao; El Hamidi, Amina; Kocjan, Gabrijela; Dogan, Ahmet; Isaacson, Peter G; Du, Ming-Qing

2002-12-01

357

Detection of Middle East respiratory syndrome coronavirus using reverse transcription loop-mediated isothermal amplification (RT-LAMP)  

PubMed Central

Background The first documented case of Middle East Respiratory Syndrome coronavirus (MERS-CoV) occurred in 2012, and outbreaks have continued ever since, mainly in Saudi Arabia. MERS-CoV is primarily diagnosed using a real-time RT-PCR assay, with at least two different genomic targets required for a positive diagnosis according to the case definition of The World Health Organization (WHO) as of 3 July 2013. Therefore, it is urgently necessary to develop as many specific genetic diagnostic methods as possible to allow stable diagnosis of MERS-CoV infections. Methods Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) is a genetic diagnostic method used widely for the detection of viral pathogens, which requires only a single temperature for amplification, and can be completed in less than 1 h. This study developed a novel RT-LAMP assay for detecting MERS-CoV using primer sets targeting a conserved nucleocapsid protein region. Results The RT-LAMP assay was capable of detecting as few as 3.4 copies of MERS-CoV RNA, and was highly specific, with no cross-reaction to other respiratory viruses. Pilot experiments to detect MERS-CoV from medium containing pharyngeal swabs inoculated with pre-titrated viruses were also performed. The RT-LAMP assay exhibited sensitivity similar to that of MERS-CoV real-time RT-PCR. Conclusions These results suggest that the RT-LAMP assay described here is a useful tool for the diagnosis and epidemiologic surveillance of human MERS-CoV infections. PMID:25103205

2014-01-01

358

Heminested reverse-transcriptase polymerase chain reaction (hnRT-PCR) as a tool for rabies virus detection in stored and decomposed samples  

PubMed Central

Background The use of methods, both sensitive and specific, for rabies diagnosis are important tools for the control and prophylaxis of the disease. Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) has been used in rabies diagnosis with good results, even in decomposed materials. Additionally, molecular techniques have been used for epidemiological studies and to gain a better knowledge of viral epidemiology. Findings The aim of this work was to evaluate the RT-PCR and hnRT-PCR for rabies virus detection in original tissues stored at -20°C for different periods considering their use for rabies virus detection in stored and decomposed samples. RT-PCR and hnRT-PCR were evaluated in 151 brain samples from different animal species, thawed and left at room temperature for 72 hours for decomposition. The RT-PCR and hnRT-PCR results were compared with previous results from Direct Fluorescent Antibody Test and Mouse Inoculation Test. From the 50 positive fresh samples, 26 (52%) were positive for RT-PCR and 45 (90%) for hnRT-PCR. From the 48 positive decomposed samples, 17 (34, 3%) were positive for RT-PCR and 36 (75%) for hnRT-PCR. No false-positives results were found in the negatives samples evaluated to the molecular techniques. Conclusion These results show that the hnRT-PCR was more sensitive than RT-PCR, and both techniques presented lower sensibility in decomposed samples. The hnRT-PCR demonstrated efficacy in rabies virus detection in stored and decomposed materials suggesting it's application for rabies virus retrospective epidemiological studies. PMID:18710536

Araújo, Danielle B; Langoni, Helio; Almeida, Marilene F; Megid, Jane

2008-01-01

359

Detection and genogrouping of noroviruses from children's stools by Taqman One-step RT-PCR.  

PubMed

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate. For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups. PMID:22898754

Apaza, Sonia; Espetia, Susan; Gilman, Robert H; Montenegro, Sonia; Pineda, Susana; Herhold, Fanny; Pomari, Romeo; Kosek, Margaret; Vu, Nancy; Saito, Mayuko

2012-01-01

360

Evaluation of automated RT-PCR to accelerate the laboratory diagnosis of foot-and-mouth disease virus.  

PubMed

Automated fluorogenic (5' nuclease probe-based) reverse transcription polymerase chain reaction (RT-PCR) procedures were evaluated for the diagnosis of foot-and-mouth disease (FMD) using suspensions of vesicular epithelium, heparinised or clotted blood, milk and oesophageal-pharyngeal fluid ('probang') samples from the United Kingdom (UK) 2001 epidemic and on sera from animals experimentally infected with the outbreak serotype O FMD virus strain. A MagNA Pure LC was initially programmed to automate the nucleic acid extraction and RT procedures with the PCR amplification carried out manually by fluorogenic assay in a GeneAmp 5700 Sequence Detection System. This allowed 32 samples to be tested by one person in a typical working day or 64 samples by two people within 10-12 h. The PCR amplification was later automated and a protocol developed for one person to complete a single test incorporating 96 RT-PCR results within 2 working days or for two people to do the same thing in around 12 h. The RT-PCR results were directly compared with those obtained by the routine diagnostic tests of ELISA and virus isolation in cell culture. The results on blood, probang and milk samples were in broad agreement between the three procedures but specific RT-PCR protocols for such material have to be fully optimised as perhaps have the positive-negative acceptance criteria. However, the automated RT-PCR achieved definitive diagnostic results (positive or negative) on supernatant fluids from first passage inoculated cell cultures and its sensitivity was greater than ELISA on suspensions of vesicular epithelium (ES) and at least equivalent to that of virus isolation in cell culture. The combined tests of ELISA, virus isolation in cell culture and RT-PCR might, therefore, only be required for confirmation of a first outbreak of FMD in a previously FMD-free country. Should a prolonged outbreak subsequently occur, then either ELISA plus RT-PCR or else RT-PCR alone could be used as the laboratory diagnostic tool(s). Either approach would eliminate the requirement for sample passage in cell culture and considerably advance the issue of laboratory diagnostic test results. PMID:12505626

Reid, Scott M; Grierson, Sylvia S; Ferris, Nigel P; Hutchings, Geoffrey H; Alexandersen, Soren

2003-02-01

361

Evaluating the Impacts of NASA/SPoRT Daily Greenness Vegetation Fraction on Land Surface Model and Numerical Weather Forecasts  

NASA Technical Reports Server (NTRS)

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 Spectroradiometer (MODIS) data aboard the NASA EOS Aqua and Terra satellites. NASA SPoRT began generating daily real-time GVF composites at 1-km resolution over the Continental United States (CONUS) on 1 June 2010. The purpose of this study is to compare the National Centers for Environmental Prediction (NCEP) climatology GVF product (currently used in operational weather models) to the SPoRT-MODIS GVF during June to October 2010. The NASA Land Information System (LIS) was employed to study the impacts of the SPoRT-MODIS GVF dataset on a land surface model (LSM) apart from a full numerical weather prediction (NWP) model. For the 2010 warm season, the SPoRT GVF in the western portion of the CONUS was generally higher than the NCEP climatology. The eastern CONUS GVF had variations both above and below the climatology during the period of study. These variations in GVF led to direct impacts on the rates of heating and evaporation from the land surface. In the West, higher latent heat fluxes prevailed, which enhanced the rates of evapotranspiration and soil moisture depletion in the LSM. By late Summer and Autumn, both the average sensible and latent heat fluxes increased in the West as a result of the more rapid soil drying and higher coverage of GVF. The impacts of the SPoRT GVF dataset on NWP was also examined for a single severe weather case study using the Weather Research and Forecasting (WRF) model. Two separate coupled LIS/WRF model simulations were made for the 17 July 2010 severe weather event in the Upper Midwest using the NCEP and SPoRT GVFs, with all other model parameters remaining the same. Based on the sensitivity results, regions with higher GVF in the SPoRT model runs had higher evapotranspiration and lower direct surface heating, which typically resulted in lower (higher) predicted 2-m temperatures (2-m dewpoint temperatures). Portions of the Northern Plains states experienced substantial increases in convective available potential energy as a result of the higher SPoRT/MODIS GVFs. These differences produced subtle yet quantifiable differences in the simulated convective precipitation systems for this event.

Bell, Jordan R.; Case, Jonathan L.; LaFontaine, Frank J.; Kumar, Sujay V.

2012-01-01

362

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

PubMed Central

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

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

2014-01-01

363

Pilot Quality Control Program for Audit RT External Beams at Mexican Hospitals  

NASA Astrophysics Data System (ADS)

A pilot quality control program for audit 18 radiotherapy RT external beams at 13 Mexican hospitals is described-for eleven 60Co beams and seven photon beams of 6, 10 and 15 MV from accelerators. This program contains five parts: a) Preparation of the TLD-100 powder: washing, drying and annealing (one hour 400 °C plus 24 hrs 80 °C). b) Sending two IAEA type capsules to the hospitals for irradiation at the hospital to a nominal DW = 2 Gy.c) Preparation at the SSDL of ten calibration curves CC in the range of 0.5 Gy to 6 Gy in terms of absorbed dose to water DW for 60Co with traceability to primary laboratory NRC (Canada), according to a window irradiation: 26/10/2007-7/12/2007. d) Reading all capsules that match their hospital time irradiation and the SSDL window irradiation. f) Evaluation of the Dw imparted by the hospitals.

Álvarez R., J. T.; Tovar M., V. M.

2008-08-01

364

Picoinjection Enables Digital Detection of RNA with Droplet RT-PCR  

PubMed Central

The ability to add reagents to drops in a sequential fashion is necessary for numerous applications of microfluidics in biology. An important method for accomplishing this is picoinjection, a technique in which reagents are injected into aqueous drops using an electric field. While picoinjection has been shown to allow the precise addition of reagents to drops, its compatibility with biological reactions is yet to be thoroughly demonstrated. Here, we investigate the compatibility of picoinjection with digital RT-PCR Taqman assays, reactions that incorporate nucleic acids, enzymes, and other common biological reagents. We find that picoinjection is compatible with this assay and enables the detection of RNA transcripts at rates comparable to workflows not incorporating picoinjection. We also find that picoinjection results in negligible transfer of material between drops and that the drops faithfully retain their compartmentalization. PMID:23658657

Abate, Adam R.

2013-01-01

365

Microhard MHX2420 Orbital Performance Evaluation Using RT Logic T400CS  

NASA Technical Reports Server (NTRS)

RT Logic allows simulation of Ground Station - satellite communications: Static tests have been successful. Dynamic tests have been performed for simple passes. Future dynamic tests are needed to simulate real orbit communications. Satellite attitude changes antenna gain. Atmospheric and rain losses need to be added. STK Plug-in will be the next step to improve the dynamic tests. There is a possibility of running longer simulations. Simulation of different losses available in the STK Plug-in. Microhard optimization: Effect of Microhard settings on the data throughput have been understood. Optimized settings improve data throughput for LEO communications. Longer hop intervals make transfer of larger packets more efficient (more time between hops in frequency). Use of FEC (Reed-Solomon) reduces the number of retransmissions for long-range or noisy communications.

TintoreGazulla, Oriol; Lombardi, Mark

2012-01-01

366

Efavirenz Therapy in Rhesus Macaques Infected with a Chimera of Simian Immunodeficiency Virus Containing Reverse Transcriptase from Human Immunodeficiency Virus Type 1  

Microsoft Academic Search

The specificity of nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) for the RT of human immunodeficiency virus type 1 (HIV-1) has prevented the use of simian immunodeficiency virus (SIV) in the study of NNRTIs and NNRTI-based highly active antiretroviral therapy. However, a SIV-HIV-1 chimera (RT-SHIV), in which the RT from SIVmac239 was replaced with the RT-encoding region from HIV-1, is susceptible

Michael J. Hofman; Joanne Higgins; Timothy B. Matthews; Niels C. Pedersen; Chalet Tan; Raymond F. Schinazi; Thomas W. North

2004-01-01

367

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

NASA Technical Reports Server (NTRS)

The NASA Short-term Prediction Research and Transition (SPoRT) Center has developed several products for its National Weather Service (NWS) partners that can initialize specific fields for local model runs within the NOAA/NWS Science and Training Resource Center (STRC) Environmental Modeling System (EMS). In last year's NWA abstract on this topic, the suite of SPoRT products supported in the STRC EMS was presented, which includes a Sea Surface Temperature (SST) composite, a Great Lakes sea-ice extent, a Green Vegetation Fraction (GVF) composite, and NASA Land Information System (LIS) gridded output. This abstract and companion presentation describes recent upgrades made to the SST and GVF composites, as well as the real-time LIS runs. The Great Lakes sea-ice product is unchanged from 2011. The SPoRT SST composite product has been expanded geographically and as a result, the resolution has been coarsened from 1 km to 2 km to accommodate the larger domain. The expanded domain covers much of the northern hemisphere from eastern Asia to western Europe (0 N to 80 N latitude and 150 E to 10 E longitude). In addition, the NESDIS POES-GOES product was added to fill in gaps caused by the Moderate Resolution Imaging Spectroradiometer (MODIS) being unable to sense in cloudy regions, replacing the recently-lost Advanced Microwave Scanning Radiometer for EOS with negligible change to product fidelity. The SST product now runs twice per day for Terra and Aqua combined data collections from 0000 to 1200 UTC and from 1200 to 0000 UTC, with valid analysis times at 0600 and 1800 UTC. The twice-daily compositing technique reduces the overall latency of the previous version while still representing the diurnal cycle characteristics. The SST composites are available at approximately four hours after the end of each collection period (i.e. 1600 UTC for the nighttime analysis and 0400 UTC for the daytime analysis). The real-time MODIS GVF composite has only received minor updates in the past year. The domain was expanded slightly to extend further west, north, and east to improve coverage over parts of southern Canada. Minor adjustments were also made to the manner in which GVF is calculated from the distribution of maximum Normalized Difference Vegetation Index from MODIS. The presentation will highlight some examples of the substantial inter-annual change in GVF that occurred from 2010 to 2011 in the U.S. Southern Plains as a result of the summer 2011 drought, and the early vegetation green up across the eastern U.S. due to the very warm conditions in March 2012. Finally, the SPoRT LIS runs the operational Noah land surface model (LSM) in real time over much of the eastern half of the CONUS. The Noah LSM is continually cycled in real time, uncoupled to any model, and driven by operational atmospheric analyses over a long-term, multi-year integration. The LIS-Noah provides the STRC EMS with high-resolution (3 km) LSM initialization data that are in equilibrium with the operational analysis forcing. The Noah LSM within the SPoRT LIS has been upgraded from version 2.7.1 to version 3.2, which has improved look-up table attributes for several land surface quantities. The surface albedo field is now being adjusted based on the input real-time MODIS GVF, thereby improving the net radiation. Also, the LIS-Noah now uses the newer MODIS-based land use classification scheme (i.e. the International Biosphere-Geosphere Programme [IGBP]) that has a better depiction of urban corridors in areas where urban sprawl has occurred. STRC EMS users interested in initializing their LSM fields with high-resolution SPoRT LIS data should set up their model domain with the MODIS-IGBP 20-class land use database and select Noah as the LSM.

Case, Jonathan L.; LaFontaine, Frank J.; Molthan, Andrew L.; Zavodsky, Bradley T.; Rozumalski, Robert A.

2012-01-01

368

Absence of RT6+ T cells in diabetes-prone biobreeding/Worcester rats is due to genetic and cell developmental defects  

SciTech Connect

Diabetes-prone BB/Wor (DP) rats lack the RT6+ peripheral T cell subset whereas diabetes-resistant BB/Wor rats have normal numbers of RT6+ T cells. Lymphocyte transfusion experiments and in vivo depletion studies have demonstrated that RT6+ T cells have an important regulatory role in the pathogenesis of insulin-dependent diabetes mellitus in BB/Wor rats. In the present study, the results of genetic complementation studies indicate that the DP rat contains an intact RT6 gene, but fails to express the RT6.1 alloantigen in the functional absence of an accessory factor (provided by RT6+ cells). At the cellular level, irradiation chimeras demonstrate that the absence of RT6+ T cells in DP rats is due to an intrinsic defect that results in abnormal development and/or differentiation of prothymocytes into RT6+ T cells. The inability of DP prothymocytes to generate RT6+ T cells is not due to serum autoantibodies, lack of accessory cells, or to the presence of inhibitory cells. Inasmuch as DP bone marrow can transfer the susceptibility for diabetes to irradiated recipients, our present results suggest that an important predisposing factor for insulin-dependent diabetes mellitus in DP rats is the inability of DP prothymocytes to generate RT6+ T cells.

Angelillo, M.; Greiner, D.L.; Mordes, J.P.; Handler, E.S.; Nakamura, N.; McKeever, U.; Rossini, A.

1988-12-15

369

Sodium sulphite enhances RNA isolation and sensitivity of Cucumber mosaic virus detection by RT-PCR in black pepper.  

PubMed

Isolation of intact high quality RNA suitable for RT-PCR from black pepper is greatly hindered by the presence of polyphenols and polysaccharides. These compounds adversely affect the sensitivity of virus detection by RT-PCR. The present study evaluated the effect of sodium sulphite in enhancing RNA yield and quality in a modified acid guanidium thiocyanate-phenol-chloroform (AGPC) protocol. The results were compared with the standard AGPC method and RNeasy Plant Mini Kit (Qiagen) for detection of Cucumber mosaic virus through RT-PCR. The addition of sodium sulphite in the extraction buffer increased the sensitivity of virus detection. Higher sensitivity of detection (than obtained from the kit) was seen when sodium sulphite was used at 0.5%. Similar levels of sensitivity were also observed for the detection of Cucumber mosaic virus from Piper longum. PMID:17275931

Siju, S; Madhubala, R; Bhat, A I

2007-04-01

370

Analytical and Clinical Performance of the CDC Real Time RT-PCR Assay for Detection and Typing of Dengue Virus  

PubMed Central

Dengue is an acute illness caused by the positive-strand RNA dengue virus (DENV). There are four genetically distinct DENVs (DENV-1–4) that cause disease in tropical and subtropical countries. Most patients are viremic when they present with symptoms; therefore, RT-PCR has been increasingly used in dengue diagnosis. The CDC DENV-1–4 RT-PCR Assay has been developed as an in-vitro diagnostic platform and was recently approved by the US Food and Drug Administration (FDA) for detection of dengue in patients with signs or symptoms of mild or severe dengue. The primers and probes of this test have been designed to detect currently circulating strains of DENV-1–4 from around the world at comparable sensitivity. In a retrospective study with 102 dengue cases confirmed by IgM anti-DENV seroconversion in the convalescent sample, the RT-PCR Assay detected DENV RNA in 98.04% of the paired acute samples. Using sequencing as a positive indicator, the RT-PCR Assay had a 97.92% positive agreement in 86 suspected dengue patients with a single acute serum sample. After extensive validations, the RT-PCR Assay performance was highly reproducible when evaluated across three independent testing sites, did not produce false positive results for etiologic agents of other febrile illnesses, and was not affected by pathological levels of potentially interfering biomolecules. These results indicate that the CDC DENV-1–4 RT-PCR Assay provides a reliable diagnostic platform capable for confirming dengue in suspected cases. PMID:23875046

Santiago, Gilberto A.; Vergne, Edgardo; Quiles, Yashira; Cosme, Joan; Vazquez, Jesus; Medina, Juan F.; Medina, Freddy; Colón, Candimar; Margolis, Harold; Muñoz-Jordán, Jorge L.

2013-01-01

371

The Diagnostic Utility of Combination of HMGA2 and IMP3 qRT-PCR Testing in Thyroid Neoplasms.  

PubMed

The diagnosis of malignant thyroid tumors in some cytologic and histologic specimens remains challenging. High-mobility group A2 (HMGA2) expression and insulin-like growth factor II mRNA-binding protein-3 (IMP3) expression were evaluated by relative quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The aim of this study was to evaluate whether the combination of HMGA2 and IMP3 qRT-PCR was diagnostically useful in differentiating benign from malignant thyroid neoplasms. Fine-needle aspiration (FNA) specimens from 120 patients including 56 benign lesions and 64 carcinomas were used. The available 80 corresponding formalin-fixed paraffin-embedded (FFPE) thyroid tissues from 66 patients were also included in this study. HMGA2 and IMP3 expression levels were detected by qRT-PCR and reported as relative fold change after normalizing with a calibrator. The diagnostic utilities of HMGA2 and IMP3 qRT-PCR tests were evaluated individually and in combination. In FNA specimens, HMGA2 and IMP3 expression was consistently higher in thyroid malignancies compared with benign lesions in all subgroups except in Hürthle cell tumors. After exclusion of Hürthle cell tumors, the sensitivity was 90.2% for HMGA2, 88.2% for IMP3, and 98% for HMGA2+IMP3; the specificity was 97.1% for HMGA2, 79.4% for IMP3, and 79.4% for HMGA+IMP3. qRT-PCR data showed similar results in FFPE tissues: the sensitivity was 84.2% for HMGA2, 85.7% for IMP3, and 94.7% for HMGA2+IMP3; the specificity was 96.9% for HMGA2, 91.2% for IMP3, and 90.6% for HMGA2+IMP3. qRT-PCR data were concordant between FNA and FFPE samples for HMGA2 (97.4%) and IMP3 (96.9%). The results indicate that HMGA2 qRT-PCR with high specificity may be a useful ancillary technique to assist in the classification of difficult thyroid specimens, excluding Hürthle cell tumors. The HMGA2 and IMP3 qRT-PCR combination model with increased sensitivity and negative predictive value (96.4%) may be useful in screening thyroid cytology specimens. PMID:25356939

Jin, Long; Lloyd, Ricardo V; Henry, Michael R; Erickson, Lori A; Sebo, Thomas J; Rumilla, Kandelaria M; Zhang, Jun

2015-01-01

372

Evaluation of endogenous reference genes for analysis of gene expression with real-time RT-PCR during planarian regeneration  

Microsoft Academic Search

It is important that endogenous reference genes for real-time RT-PCR be empirically evaluated for stability in different cell\\u000a types, developmental stages, and\\/or sample treatment. To select the most stable endogenous reference genes during planarian\\u000a regeneration, three housekeeping genes, 18S rRNA, ACTB and DjEF2, were identified and established expression levels by real-time RT-PCR. The data were analyzed by GeNorm and NormFinder

Yan-qing YuwenZi-mei; Zi-mei Dong; Qing-hua Wang; Xiao-juan Sun; Chang-ying Shi; Guang-wen Chen

373

The impact of flattening-filter-free beam technology on 3D conformal RT  

PubMed Central

Background The removal of the flattening filter (FF) leads to non-uniform fluence distribution with a considerable increase in dose rate. It is possible to adapt FFF beams (flattening-filter-free) in 3D conformal radiation therapy (3D CRT) by using field in field techniques (FiF). The aim of this retrospective study is to clarify whether the quality of 3D CRT plans is influenced by the use of FFF beams. Method This study includes a total of 52 CT studies of RT locations that occur frequently in clinical practice. Dose volume targets were provided for the PTV of breast (n=13), neurocranium (n=11), lung (n=7), bone metastasis (n=10) and prostate (n=11) in line with ICRU report 50/62. 3D CRT planning was carried out using FiF methods. Two clinically utilized photon energies are used for a Siemens ARTISTE linear accelerator in FFF mode at 7MVFFF and 11MVFFF as well as in FF mode at 6MVFF and 10MVFF. The plan quality in relation to the PTV coverage, OAR (organs at risk) and low dose burden as well as the 2D dosimetric verification is compared with FF plans. Results No significant differences were found between FFF and FF plans in the mean dose for the PTV of breast, lung, spine metastasis and prostate. The low dose parameters V5Gy and V10Gy display significant differences for FFF and FF plans in some subgroups. The DVH analysis of the OAR revealed some significant differences. Significantly more fields (1.9 – 4.5) were necessary in the use of FFF beams for each location (p<0.0001) in order to achieve PTV coverage. All the tested groups displayed significant increases (1.3 – 2.2 times) in the average number of necessary MU with the use of FFF beams (p<0.001). Conclusions This study has shown that the exclusive use of a linear accelerator in FFF mode is feasible in 3D CRT. It was possible to realize RT plans in comparable quality in typical cases of clinical radiotherapy. The 2D dosimetric validation of the modulated fields verified the dose calculation and thus the correct reproduction of the characteristic FFF parameters in the planning system that was used. PMID:23725479

2013-01-01

374

Implementation and commissioning of an integrated micro-CT/RT system with computerized independent jaw collimation  

SciTech Connect

Purpose: To design, construct, and commission a set of computer-controlled motorized jaws for a micro-CT/RT system to perform conformal image-guided small animal radiotherapy.Methods: The authors designed and evaluated a system of custom-built motorized orthogonal jaws, which allows the delivery of off-axis rectangular fields on a GE eXplore CT 120 preclinical imaging system. The jaws in the x direction are independently driven, while the y-direction jaws are symmetric. All motors have backup encoders, verifying jaw positions. Mechanical performance of the jaws was characterized. Square beam profiles ranging from 2 × 2 to 60 × 60 mm{sup 2} were measured using EBT2 film in the center of a 70 × 70 × 22 mm{sup 3} solid water block. Similarly, absolute depth dose was measured in a solid water and EBT2 film stack 50 × 50 × 50 mm{sup 3}. A calibrated Farmer ion chamber in a 70 × 70 × 20 mm{sup 3} solid water block was used to measure the output of three field sizes: 50 × 50, 40 × 40, and 30 × 30 mm{sup 2}. Elliptical target plans were delivered to films to assess overall system performance. Respiratory-gated treatment was implemented on the system and initially proved using a simple sinusoidal motion phantom. All films were scanned on a flatbed scanner (Epson 1000XL) and converted to dose using a fitted calibration curve. A Monte Carlo beam model of the micro-CT with the jaws has been created using BEAMnrc for comparison with the measurements. An example image-guided partial lung irradiation in a rat is demonstrated.Results: The averaged random error of positioning each jaw is less than 0.1 mm. Relative output factors measured with the ion chamber agree with Monte Carlo simulations within 2%. Beam profiles and absolute depth dose curves measured from the films agree with simulations within measurement uncertainty. Respiratory-gated treatments applied to a phantom moving with a peak-to-peak amplitude of 5 mm showed improved beam penumbra (80%–20%) from 3.9 to 0.8 mm.Conclusions: A set of computer-controlled motorized jaws for a micro-CT/RT system were constructed with position reliably better than a tenth of a millimeter. The hardware system is ready for image-guided conformal radiotherapy for small animals with capability of respiratory-gated delivery.

Jensen, Michael D. [Department of Medical Biophysics, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 3K7 (Canada)] [Department of Medical Biophysics, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 3K7 (Canada); Hrinivich, W. Thomas; Jung, Jongho A. [Department of Physics and Astronomy, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 3K7 (Canada)] [Department of Physics and Astronomy, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 3K7 (Canada); Holdsworth, David W. [Imaging Research Laboratories, Robarts Research Institute, 100 Perth Drive, London, Ontario N6A 5K8 (Canada) [Imaging Research Laboratories, Robarts Research Institute, 100 Perth Drive, London, Ontario N6A 5K8 (Canada); Department of Medical Biophysics, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 3K7 (Canada); Department of Surgery, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 3K7 (Canada); Drangova, Maria [Imaging Research Laboratories, Robarts Research Institute, 100 Perth Drive, London, Ontario N6A 5K8, Canada and Department of Medical Biophysics, The University of Western Ontario 1151 Richmond Street, London, Ontario N6A 3K7 (Canada)] [Imaging Research Laboratories, Robarts Research Institute, 100 Perth Drive, London, Ontario N6A 5K8, Canada and Department of Medical Biophysics, The University of Western Ontario 1151 Richmond Street, London, Ontario N6A 3K7 (Canada); Chen, Jeff [Department of Physics and Engineering, London Regional Cancer Program, London Health Sciences Centre, 800 Commissioners Road East, London, Ontario N6A 5W9 (Canada) [Department of Physics and Engineering, London Regional Cancer Program, London Health Sciences Centre, 800 Commissioners Road East, London, Ontario N6A 5W9 (Canada); Department of Medical Biophysics, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 3K7 (Canada); Department of Oncology, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 3K7 (Canada); Wong, Eugene [Department of Physics and Astronomy, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 3K7 (Canada) [Department of Physics and Astronomy, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 3K7 (Canada); Department of Medical Biophysics, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 3K7 (Canada); Department of Oncology, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 3K7 (Canada); Department of Physics and Engineering, London Regional Cancer Program, London Health Sciences Centre, 800 Commissioners Road East, London, Ontario N6A 5W9 (Canada)

2013-08-15

375

Removal of real-time reverse transcription polymerase chain reaction (RT-PCR) inhibitors associated with cloacal swab samples and tissues for improved diagnosis of Avian influenza virus by RT-PCR.  

PubMed

Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) is routinely used for the rapid detection of Avian influenza virus (AIV) in clinical samples, but inhibitory substances present in some clinical specimens can reduce or block PCR amplification. Most commercial RNA extraction kits have limited capacity to remove inhibitors from clinical samples, but using a modified commercial protocol (Ambion MagMAX, Applied Biosystems, Foster City, CA) with an added high-salt wash of 2 M NaCl and 2 mM ethylenediamine tetra-acetic acid was shown to improve the ability of the kit to remove inhibitors from cloacal swabs and some tissues. Real-time RT-PCR was carried out in the presence of an internal positive control to detect inhibitors present in the purified RNA. Cloacal swabs from wild birds were analyzed by real-time RT-PCR comparing RNA extracted with the standard (MagMAX-S) and modified (MagMAX-M) protocols. Using the standard protocol on 2,668 samples, 18.4% of the samples had evidence of inhibitor(s) in the samples, but the modified protocol removed inhibitors from all but 21 (4.8%) of the problem samples. The modified protocol was also tested for RNA extraction from tissues using a TRIzol-MagMAX-M hybrid protocol. Tissues from chickens and ducks experimentally infected with high-pathogenicity Asian H5N1 AIV were analyzed by real-time RT-PCR, and the limit of detection of the virus was improved by 0.5-3.0 threshold cycle units with the RNA extracted by the MagMAX-M protocol. The MagMAX-M protocol reported in the present study can be useful in extracting high-quality RNA for accurate detection of AIV from cloacal swabs and tissues by real-time RT-PCR. PMID:19901277

Das, Amaresh; Spackman, Erica; Pantin-Jackwood, Mary J; Suarez, David L

2009-11-01

376

Applications of NASA and NOAA Satellite Observations by NASA's Short-term Prediction Research and Transition (SPoRT) Center in Response to Natural Disasters  

NASA Technical Reports Server (NTRS)

NASA s Short-term Prediction Research and Transition (SPoRT) Center supports the transition of unique NASA and NOAA research activities to the operational weather forecasting community. SPoRT emphasizes real-time analysis and prediction out to 48 hours. SPoRT partners with NOAA s National Weather Service (NWS) Weather Forecast Offices (WFOs) and National Centers to improve current products, demonstrate future satellite capabilities and explore new data assimilation techniques. Recently, the SPoRT Center has been involved in several activities related to disaster response, in collaboration with NOAA s National Weather Service, NASA s Applied Sciences Disasters Program, and other partners.

Molthan, Andrew L.; Burks, Jason E.; McGrath, Kevin M.; Jedlovec, Gary J.

2012-01-01

377

NONINVASIVE MEASUREMENT OF MENISCUS STRAIN USING HYPERELASTIC WARPING *Sun, Q; **Burks, RT; **Greis, PE; *Phatak, NS; +*Weiss, JA  

E-print Network

NONINVASIVE MEASUREMENT OF MENISCUS STRAIN USING HYPERELASTIC WARPING *Sun, Q; **Burks, RT; **Greis, and measurement of strain can be used to assess the efficacy of surgical repair techniques. However, in vivo measurement of strain in menisci is nearly impossible and in vitro measurements are difficult due

Utah, University of

378

A rt i c l e s 566 VOLUME 17 | NUMBER 5 | MAY 2011 nAture medicine  

E-print Network

A rt i c l e s 566 VOLUME 17 | NUMBER 5 | MAY 2011 nAture medicine ADHD is a common psychiatric in ADHD has gained considerable attention2. However, many ADHD susceptibility loci identified by genome with the phenotypic heterogeneity and strong heritability of ADHD, predicts the existence of diverse ADHD

Jeong, Jaeseung

379

Simple, specific molecular typing of dengue virus isolates using one-step RT-PCR and restriction fragment length polymorphism.  

PubMed

A one-step RT-PCR and one-enzyme RFLP was used to detect and distinguish among flaviviruses, including the four serotypes of dengue and the St. Louis Encephalitis, West Nile and Yellow Fever viruses in cultured virus samples or acute-phase human serum. Using a previously described RT-PCR, but novel RFLP procedure, results are obtained in 24 h with basic PCR and electrophoresis equipment. There is 95% agreement between RT-PCR/RFLP results and those achieved by indirect immunofluorescence assays, and 100% agreement between RT-PCR/RFLP results and gene sequencing. This method is more rapid than tests of cytopathic effect based on virus isolation in tissue culture, and simpler than real-time PCR. It does not require specialized equipment, radioisotopes or computer analysis and is a method that can be applied widely in the developing world. It allows for prompt determination of whether a flavivirus is the cause of illness in a febrile patient, rapid identification of dengue serotypes in circulation, and improved patient management in cases where prior dengue exposure make dengue hemorrhagic fever or dengue shock syndrome a risk. PMID:22766181

Ortiz, Alma; Capitan, Zeuz; Mendoza, Yaxelis; Cisneros, Julio; Moreno, Brechla; Zaldivar, Yamitzel; Garcia, Mariana; Smith, Rebecca E; Motta, Jorge; Pascale, Juan Miguel

2012-10-01

380

u c l a C o l l e g e R e p o rt By Aaron Dalton  

E-print Network

u c l a C o l l e g e R e p o rt By Aaron Dalton If you met someone from Europe who was tall, blond and evolutionary biology John Novembre led a team of researchers in pub- lishing a Nature article demonstrating how is a very exciting place," said John Novembre analyzes data to understand patterns of genetic variation

Grether, Gregory

381

THE EFFECT OF VARIOUS DISINFECTANTS ON DETECTION OF AVIAN INFLUENZA VIRUS BY REAL TIME RT-PCR  

Technology Transfer Automated Retrieval System (TEKTRAN)

An avian influenza real time RT-PCR (RRT-PCR) test was previously shown to be a rapid and sensitive method to identify avian influenza virus-infected birds in live bird markets (LBMs). The test can also be used to identify avian influenza virus (AIV) from environmental samples. Consequently, the u...

382

A collaborative framework for contributing DICOM RT PHI (Protected Health Information) to augment data mining in clinical decision support  

NASA Astrophysics Data System (ADS)

We have built a decision support system that provides recommendations for customizing radiation therapy treatment plans, based on patient models generated from a database of retrospective planning data. This database consists of relevant metadata and information derived from the following DICOM objects - CT images, RT Structure Set, RT Dose and RT Plan. The usefulness and accuracy of such patient models partly depends on the sample size of the learning data set. Our current goal is to increase this sample size by expanding our decision support system into a collaborative framework to include contributions from multiple collaborators. Potential collaborators are often reluctant to upload even anonymized patient files to repositories outside their local organizational network in order to avoid any conflicts with HIPAA Privacy and Security Rules. We have circumvented this problem by developing a tool that can parse DICOM files on the client's side and extract de-identified numeric and text data from DICOM RT headers for uploading to a centralized system. As a result, the DICOM files containing PHI remain local to the client side. This is a novel workflow that results in adding only relevant yet valuable data from DICOM files to the centralized decision support knowledge base in such a way that the DICOM files never leave the contributor's local workstation in a cloud-based environment. Such a workflow serves to encourage clinicians to contribute data for research endeavors by ensuring protection of electronic patient data.

Deshpande, Ruchi; Thuptimdang, Wanwara; DeMarco, John; Liu, Brent J.

2014-03-01

383

Abstract--The RT3S E-learning environment enables experts in vascular medicine to prepare educational training  

E-print Network

Abstract--The RT3S E-learning environment enables experts in vascular medicine to prepare to the needs of the tutor and of the scenario) and enables tutors (e.g., expert surgeons) to easily prepare new disease of the peripheral arteries gives rise to high morbidity and mortality rates. Around 20

Petrakis, Euripides G.M.

384

Evidence-based RT-PCR methods for the detection of the 8 most common MLL aberrations in acute leukemias.  

PubMed

MLL aberrations are detected in around 5-10% of acute myeloid and lymphatic leukemias and an additional 5% of acute myeloid leukemias show a partial internal MLL duplication (PTD). MLL rearrangements are important for therapy stratification, assessment of minimal residual disease and for targeted therapies. However, no truly evidence-based RT-PCR methods for the detection of most of these aberrations have been published yet. Based on the large data collection of MLL genomic breakpoints in acute leukemias comprising more than 1.600 cases at the Diagnostic Center for Acute Leukemias (DCAL) in Frankfurt, Germany that provide an overview over the experimentally observed fusion transcript variants, we developed RT-PCR methods for the reliable detection of the 8 most common MLL aberrations (MLL-AF4, MLL-AF6, MLL-AF9, MLL-AF10, MLL-ENL, MLL-ELL, MLL-EPS15, MLL PTD), together accounting for around 90% of MLL-r cases. The easily implementable RT-PCRs should enable a reliable detection of these MLL fusion transcripts by RT-PCR. PMID:25510485

Burmeister, Thomas; Meyer, Claus; Gröger, Daniela; Hofmann, Julia; Marschalek, Rolf

2015-02-01

385

Ultrahigh detectivity room temperature THZ-IR photodetector based on resonant tunneling spherical centered defect quantum dot (RT-SCDQD)  

Microsoft Academic Search

In this paper, a novel structure for THZ-IR photodetector based on resonant tunneling spherical centered defect quantum dot (RT-SCDQD) operating at room temperature is proposed. The proposed structure includes a quantum dot with centered defect following a resonant tunneling double barrier. It is shown that inserting a centered defect leads to considerable enhancement in absorption coefficient at long wavelength in

H. Rasooli Saghai; N. Sadoogi; A. Rostami; H. Baghban

2009-01-01

386

Molecular detection of infectious bronchitis and Newcastle disease viruses in broiler chickens with respiratory signs using Duplex RT-PCR  

PubMed Central

Infectious bronchitis (IB) and Newcastle disease (ND) are highly contagious and the most economically important diseases of the poultry affecting respiratory tract and causing economic losses in poultry industry throughout the world. In the present study, the simultaneous detection and differentiation of causative agents of these diseases were investigated using duplex-RT-PCR. RNA was extracted from vaccinal and reference strains of infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) and then cDNA was synthesized. Using two universal primer sets for detection of IBV and NDV, the duplex-RT-PCR was developed. In order to assess the efficiency of the developed duplex RT-PCR, a number of 12 broiler farms with the symptoms of respiratory tract infection was sampled (trachea, lung and kidney were sampled from affected birds suspicious for IBV and NDV infections). After RNA extraction from tissues and cDNA synthesis, the presence of IBV and NDV genome were investigated using duplex-PCR. The results showed that three of twelve examined broiler farms were positive for IBV and two farms were positive for NDV and IBV. The results revealed that the duplex-RT-PCR is a quick and sensitive procedure for simultaneously detecting IBV and NDV in birds with respiratory infections. PMID:25610585

Saba Shirvan, Aylar; Mardani, Karim

2014-01-01

387

1274 VOLUME 46 | NUMBER 12 | DECEMBER 2014 Nature GeNetics A rt i c l e s  

E-print Network

1274 VOLUME 46 | NUMBER 12 | DECEMBER 2014 Nature GeNetics A rt i c l e s Vaccination is one seizures Bjarke Feenstra1, Björn Pasternak1, Frank Geller1, Lisbeth Carstensen1, Tongfei Wang2­4, Fen Huang

Kaski, Samuel

388

2012 RepoRt IllInoIs natuRal hIstoRy suRvey  

E-print Network

2012 RepoRt IllInoIs natuRal hIstoRy suRvey IllInoIs state aRchaeologIcal suRvey IllInoIs state supplies for growing communities, discover and catalogue archaeological sites in the path of construction State Archaeological Survey Illinois State Geological Survey Illinois State Water Survey Illinois

Bashir, Rashid

389

Collaborative Inquiry: A Strategy for Assessing Response to Instruction and Intervention (RtI2) for English Learner Students  

ERIC Educational Resources Information Center

This pilot study describes elementary teachers' use of collaborative inquiry as a strategy for assessing Response to Instruction and Intervention (RtI [superscript 2]) in reading for an English Learner student. The design of the study was based on the sociocultural theory that assessment practices shape teachers' understanding of students and of…

Vineyard, Lynn

2010-01-01

390

Nature GeNetics VOLUME 45 | NUMBER 8 | AUGUST 2013 891 A rt i c l e s  

E-print Network

Nature GeNetics VOLUME 45 | NUMBER 8 | AUGUST 2013 891 A rt i c l e s A central challenge, Mickael Leclercq3,4, Robert J Williamson1, Ewa Forczek5, Zoé Joly-Lopez5, Joshua G Steffen6, Khaled M, Thomas E Bureau5, Stephen I Wright1,16 & Mathieu Blanchette3,4 Despitethecentralimportanceofnoncoding

Stinchcombe, John

391

arXiv:math.RT/0702855v128Feb2007 IMAGINARY HIGHEST-WEIGHT REPRESENTATION THEORY AND  

E-print Network

}. Equivalent partitions define similar highest-weight theories. All partitions of the root system of a finitearXiv:math.RT/0702855v128Feb2007 IMAGINARY HIGHEST-WEIGHT REPRESENTATION THEORY AND SYMMETRIC FUNCTIONS BENJAMIN J. WILSON Abstract. Affine Lie algebras admit non-classical highest-weight theories

Sydney, University of

392

Speed of Neuron Conduction Is Not the Basis of the IQ-RT Correlation: Results from a Simple Neural Model.  

ERIC Educational Resources Information Center

Using a simple neural model comprising between two and four neurons, it is concluded that speed of neuron conduction is not the probable basis of the intelligence quotient (IQ)-reaction time (RT) correlation. This result illustrates that neural modeling can be applied to biological theories of individual differences in intelligence. (SLD)

Anderson, Britt

1994-01-01

393

Smithsonian Institution 2008 AnnuAl RePoRT CHARTinG CouRSe From the Secretary  

E-print Network

Smithsonian Institution 2008 AnnuAl RePoRT CHARTinG CouRSe » #12;From the Secretary It is my great pleasure to introduce the Smithsonian's achievements in 2008, the year that I became the 12th Secretary technologies will enable us to leverage our considerable resources and talents more effectively than ever

Mathis, Wayne N.

394

Methods for detection and differentiation of existing and new crinivirus species through multiplex and degenerate primer RT-PCR.  

Technology Transfer Automated Retrieval System (TEKTRAN)

A method was developed for rapid identification and differentiation of both known and novel crinivirus species involving both multiplex and degenerate reverse transcription-polymerase chain reaction (RT-PCR). The multiplex method can discriminate among known criniviruses infecting vegetable and sma...

395

Supporting Valid Decision Making: Uses and Misuses of Assessment Data within the Context of RtI  

ERIC Educational Resources Information Center

Within an RtI problem-solving context, assessment and decision making generally center around the tasks of problem identification, problem analysis, progress monitoring, and program evaluation. We use this framework to discuss the current state of the literature regarding curriculum based measurement, its technical properties, and its utility for…

Ball, Carrie R.; Christ, Theodore J.

2012-01-01

396

ALBANY ENGINEERED COMPOSITES, INC. / R&T Engineer -Software Developer Apply at: http://www.albint.com/careers  

E-print Network

, organizing, and executing internal Research and Technology (R&T) projects primarily directed toward. Provide engineering/technical project updates and communications as needed by internal and external environment such as Citect, or LabView is a plus. Working knowledge of the Design and analysis of composite

New Hampshire, University of

397

BLIND RT60 ESTIMATION ROBUST ACROSS ROOM SIZES AND SOURCE DISTANCES Baldwin Dumortier1,2,3  

E-print Network

of a room. In many situations, the room impulse re- sponse (RIR) is not available and the RT60 must be blindly es- timated from a speech or music signal. Current methods often implicitly assume the source has been switched off [6]. It can be calculated from a room impulse response (RIR) using Schroeder

Paris-Sud XI, Université de

398

Integrated RT-PCR/nested PCR diagnosis for differentiating between subgroups of plum pox virus.  

PubMed

An RT-PCR/nested PCR technique was developed for the simultaneous detection and typing of plum pox virus (PPV) and its major types--Dideron (D), Marcus (M), El-Amar (EA) and Cherry (C). Degenerated oligonucleotides were synthesized for the general detection of PPV, flanking the coding sequence for the N-terminal portion of the coat protein (CP), within which strain-specific differences were identified. On the basis of these characteristic differences, degenerated primer pairs were designed to differentiate between the four major subgroups of the virus in nested PCR reactions. The validity of the technique was tested on viral strains and cloned cDNAs overlapping the CP region. High specificity was observed with no detectable cross-reactions. The results of general PPV detection with the new primers and those of the PCR-based detection of the 3' non-coding region of the viral genome correlated with complete coincidence. The PCR typing results correlated well with those of the RsaI-RFLP and serological typing and revealed a surprisingly high incidence of PPV-D in Hungary. PMID:11226563

Szemes, M; Kálmán, M; Myrta, A; Boscia, D; Németh, M; Kölber, M; Dorgai, L

2001-04-01

399

RT-PCR analysis of dystrophin mRNA in DND/BMD patients  

SciTech Connect

Duchenne and Becker muscular dystrophies (DMD, BMD) are X-linked recessive disorders caused by mutations in the dystrophin (dys) gene. The majority of these mutations are intragenic deletions of duplications routinely detected by Southern biots and multiplex PCR. The remainder are very likely, smaller mutations, mostly point-mutations. Detection of these mutations is very difficult due to the size and complexity of the dys gene. We applied RT-PCR to analyse the entire dys mRNA of three DMD patients with no detectable genomic defect. In two unrelated patients, a duplication of the 62 bp exon 2 was identified. This causes a frameshift sufficient to explain the DMD phenotype. In the third patient, who had congenital DMD and severe mental retardation, a complex pattern of aberrant splicing at the 3-prime exons 67-79 was observed. Sural nerve biopsy in this patient showed the complete absence of Dp116. PCR-SSCP studies are presently in progress to identify the mutations responsible for the aberrant splicing patterns.

Ciafaloni, E.; Silva, H.A.R. de; Roses, A.D. [Duke Univ. Medical Center, Durham, NC (United States)

1994-09-01

400

A plausible third body in RS CVn-type binary: RT Coronae Borealis  

NASA Astrophysics Data System (ADS)

Cyclic period changes, which are usually explained as being caused by the magnetic activity of one or both components or the light travel time effect of a third body, are a typical phenomenon in the RS CVn-type binary. Combining the newly determined eclipsing times with others compiled from literature, we analyzed the cyclic period change in the RS CVn-type binary RT CrB. The result shows that the orbital period of the binary shows a period of 3.47 yr with an amplitude of 0 d .0084. The cyclic oscillation can be attributed to the existence of a third body with a lower limit mass of 1.71(±0.19) M ?. The third body is cycling the binary at a distance of no more than 2.30(±0.19) AU which would play an important role in the evolution of this system. No detected spectral lines of the third body may suggest that the third component may itself be a close binary of 0.86 M ? stars, or more likely a dark star such as a neutron star.

Zhang, W. X.; Ran, M. W.; Feng, Y. G.

2014-12-01

401

Myriad of correlated electron effects found in the RT2Zn20 family  

NASA Astrophysics Data System (ADS)

The dilute, rare earth bearing family of RT2Zn20 (R = rare earth, T = transition metal) intermetallic compounds can be tuned to a nearly ferromagnetic Fermi liquid that is closer to the Stoner limit than Pd (for R = Y, Lu and T = Fe). The submersion of moment bearing rare earths (Gd-Tm) into this highly polarizable matrix gives rise to exceptionally high temperature ferromagnetism (e.g. TC=86 K for GdFe2Zn20) for a compound with only one moment bearing ion out of 23 in the formula unit. In addition to this d-shell correlated electron behavior, 4f hybridization gives rise to six, new, Yb-based heavy fermion compounds: YbT2Zn20 (T = Fe, Ru, Os, Co, Rh, Ir). These half dozen compounds manifest a range of Yb ion degeneracy values ( N) for T?TK, ranging from N=4-8. Not only do these compounds offer a clear testing ground for the effects of low temperature degeneracy on the correlated electron state, they also present an exceptionally clear experimental confirmation of the generalized Kadowaki-Woods formalism.

Canfield, P. C.; Jia, S.; Mun, E. D.; Bud'ko, S. L.; Samolyuk, G. D.; Torikachvili, M. S.

2008-04-01

402

Implicating Culicoides Biting Midges as Vectors of Schmallenberg Virus Using Semi-Quantitative RT-PCR  

PubMed Central

Background The recent unprecedented emergence of arboviruses transmitted by Culicoides biting midges in northern Europe has necessitated the development of techniques to differentiate competent vector species. At present these techniques are entirely reliant upon interpretation of semi-quantitative RT-PCR (sqPCR) data in the form of Cq values used to infer the presence of viral RNA in samples. Methodology/Principal Findings This study investigates the advantages and limitations of sqPCR in this role by comparing infection and dissemination rates of Schmallenberg virus (SBV) in two colony lines of Culicoides. Through the use of these behaviorally malleable lines we provide tools for demarcating arbovirus infection and dissemination rates in Culicoides which to date have prevented clear implication of primary vector species in northern Europe. The study demonstrates biological transmission of SBV in an arthropod vector, supporting the conclusions from field-caught Culicoides and provides a general framework for future assessment of vector competence of Culicoides for arboviruses using sqPCR. Conclusions/Significance When adopting novel diagnostic technologies, correctly implicating vectors of arboviral pathogens requires a coherent laboratory framework to fully understand the implications of results produced in the field. This study illustrates these difficulties and provides a full examination of sqPCR in this role for the Culicoides-arbovirus system. PMID:23520481

Veronesi, Eva; Henstock, Mark; Gubbins, Simon; Batten, Carrie; Manley, Robyn; Barber, James; Hoffmann, Bernd; Beer, Martin; Attoui, Houssam; Mertens, Peter Paul Clement; Carpenter, Simon

2013-01-01

403

Optical and UV spectroscopy of the peculiar RS CVn system, RT Lacertae  

NASA Technical Reports Server (NTRS)

Spectra in the H-alpha and H-beta regions of the peculiar double-lined RS CVn binary, RT Lacertae, were obtained in the fall of 1984. Limited International Ultraviolet Explorer (IUE) long wavelength low and high resolution spectra were obtained concurrently. The ground based spectra have shown an asymmetry with orbital phase in the H-alpha profile. The H-beta profiles were consistent with the same effect. One hemisphere showed excess emission and the other excess absorption, with a broad Gaussian emission component superposed upon the excess H-alpha line. An improved radial velocity curve, giving a better determined mass ratio and geometry was derived. This combined with the radii implied by the rotational broadening of the spectra, showed one component to be 80 to 90% filling the equilibrium Roche surface. The two-faced nature is, therfore, very likely due to mass transfer from the contact component impacting upon its companion. Low resolution ultraviolet data showed that the supposed cooler component is bluer than its companion. High resolution ultraviolet data taken during secondary eclipse showed Mg II emission strength which decreased more slowly than the area visible. The phase behavior of the low resolution data support the former situation, indicating traditional chromospheric activity.

Huenemoerder, D. P.; Barden, S. C.

1985-01-01

404

Relative quantitation goes viral: An RT-qPCR assay for a grapevine virus.  

PubMed

Accurate detection and quantitation of viruses can be beneficial to plant-virus interaction studies. In this study, three SYBR green real-time RT-PCR assays were developed to quantitate grapevine leafroll-associated virus 3 (GLRaV-3) in infected vines. Three genomic regions (ORF1a, coat protein and 3'UTR) were targeted to quantitate GLRaV-3 relative to three stably expressed reference genes (actin, GAPDH and ?-tubulin). These assays were able to detect all known variant groups of GLRaV-3, including the divergent group VI, with equal efficiency. No link could be established between the concentration ratios of the different genomic regions and subgenomic RNA (sgRNA) expression. However, a significant lower virus concentration ratio for plants infected with variant group VI compared to variant group II was observed for the ORF1a, coat protein and the 3'UTR. Significant higher accumulation of the virus in the growth tip was also detected for both variant groups. The quantitation of viral genomic regions under different conditions can contribute to elucidating disease aetiology and enhance knowledge about virus ecology. PMID:25286180

Bester, R; Pepler, P T; Burger, J T; Maree, H J

2014-10-01

405

Detection of human enteric viruses in stream water with RT-PCR and cell culture.  

USGS Publications Warehouse

A multiplex RT-PCR method was used to measure virus occurrence at five stream water sites that span a range of hydroclimatic, water-quality, and land-use characteristics. The performance of the molecular method was evaluated in comparison with traditional cell culture and Escherichia coli membrane filtration assays. The study incorporated multiple quality controls and included a control for virus recovery during the sampling procedure as well as controls to detect potentially false-negative and false-positive data. Poliovirus recovery ranged from 16 to 65% and was variable, even in samples collected within the same stream. All five sites were positive for viruses by both molecular and cell culture-based virus assays. Enteroviruses, reoviruses, rotaviruses, and hepatitis A viruses were detected, but the use of the quality controls proved critical for interpretation of the molecular data. All sites showed evidence of faecal contamination, and culturable viruses were detected in four samples that would have met the US Environmental Protection Agency's recommended E. coli guideline for safe recreational water.

Denis-Mize, K.; Fout, G.S.; Dahling, D.R.; Francy, D.S.

2004-01-01

406

Age Predicts Functional Outcome in Acute Stroke Patients with rt-PA Treatment  

PubMed Central

The standard treatment for acute ischemic stroke is thrombolytic therapy. There is limited data on prognostic factors of acute stroke with thrombolytic therapy particularly in Asian population. Acute ischemic stroke patients who were treated with thrombolytic therapy at Srinagarind Hospital between May 2008 and July 2010 were included. Factors associated with Barthel index more than 80 were studied by multiple logistic regression analysis. There were 75 patients included in the study. The mean NIHSS scores before treatment and at 3 months were 9.16 ± 4.82 and 3.83 ± 4.00, respectively, and median Barthel index at 3 months was 86. Only significant predictor for having Barthel index more than 80 points at 3 months was age (adjusted odds ratio 0.929, 95% confidence interval 0.874, 0.988). Four patients developed intracranial hemorrhage after the treatment (5%), and two died (2.6%). In conclusion, age predicts Barthel index in acute stroke patients with rt-PA treatment. PMID:24171121

Chindaprasirt, Jarin; Chattakul, Paiboon; Limpawattana, Panita; Tiamkao, Somsak; Aountri, Patcharin; Chotmongkol, Verajit

2013-01-01

407

Microhard MHX 2420 Orbital Performance Evaluation Using RT Logic T400CS  

NASA Technical Reports Server (NTRS)

A major upfront cost of building low cost Nanosatellites is the communications sub-system. Most radios built for space missions cost over $4,000 per unit. This exceeds many budgets. One possible cost effective solution is the Microhard MHX2420, a commercial off-the-shelf transceiver with a unit cost under $1000. This paper aims to support the Nanosatellite community seeking an inexpensive radio by characterizing Microhard's performance envelope. Though not intended for space operations, the ability to test edge cases and increase average data transfer speeds through optimization positions this radio as a solution for Nanosatellite communications by expanding usage to include more missions. The second objective of this paper is to test and verify the optimal radio settings for the most common cases to improve downlinking. All tests were conducted with the aid of the RT Logic T400CS, a hardware-in-the-loop channel simulator designed to emulate real-world radio frequency (RF) link effects. This study provides recommended settings to optimize the downlink speed as well as the environmental parameters that cause the link to fail.

Kearney, Stuart; Lombardi, Mark; Attai, Watson; Oyadomari, Ken; Al Rumhi, Ahmed Saleh Nasser; Rakotonarivo, Sebastien; Chardon, Loic; Gazulla, Oriol Tintore; Wolfe, Jasper; Salas, AlbertoGuillen; DeWald, Jon; Alena, Richard

2012-01-01

408

Real Time RT-PCR Assays for Detection and Typing of African Horse Sickness Virus  

PubMed Central

Although African horse sickness (AHS) can cause up to 95% mortality in horses, naïve animals can be protected by vaccination against the homologous AHSV serotype. Genome segment 2 (Seg-2) encodes outer capsid protein VP2, the most variable of the AHSV proteins. VP2 is also a primary target for AHSV specific neutralising antibodies, and consequently determines the identity of the nine AHSV serotypes. In contrast VP1 (the viral polymerase) and VP3 (the sub-core shell protein), encoded by Seg-1 and Seg-3 respectively, are highly conserved, representing virus species/orbivirus-serogroup-specific antigens. We report development and evaluation of real-time RT-PCR assays targeting AHSV Seg-1 or Seg-3, that can detect any AHSV type (virus species/serogroup-specific assays), as well as type-specific assays targeting Seg-2 of the nine AHSV serotypes. These assays were evaluated using isolates of different AHSV serotypes and other closely related orbiviruses, from the ‘Orbivirus Reference Collection’ (ORC) at The Pirbright Institute. The assays were shown to be AHSV virus-species-specific, or type-specific (as designed) and can be used for rapid, sensitive and reliable detection and identification (typing) of AHSV RNA in infected blood, tissue samples, homogenised Culicoides, or tissue culture supernatant. None of the assays amplified cDNAs from closely related heterologous orbiviruses, or from uninfected host animals or cell cultures. PMID:24721971

Bachanek-Bankowska, Katarzyna; Maan, Sushila; Castillo-Olivares, Javier; Manning, Nicola M.; Maan, Narender Singh; Potgieter, Abraham C.; Di Nardo, Antonello; Sutton, Geoff; Batten, Carrie; Mertens, Peter P. C.

2014-01-01

409

Rapid and Sensitive RT-QuIC Detection of Human Creutzfeldt-Jakob Disease Using Cerebrospinal Fluid.  

PubMed

Fast, definitive diagnosis of Creutzfeldt-Jakob disease (CJD) is important in assessing patient care options and transmission risks. Real-time quaking-induced conversion (RT-QuIC) assays of cerebrospinal fluid (CSF) and nasal-brushing specimens are valuable in distinguishing CJD from non-CJD conditions but have required 2.5 to 5 days. Here, an improved RT-QuIC assay is described which identified positive CSF samples within 4 to 14 h with better analytical sensitivity. Moreover, analysis of 11 CJD patients demonstrated that while 7 were RT-QuIC positive using the previous conditions, 10 were positive using the new assay. In these and further analyses, a total of 46 of 48 CSF samples from sporadic CJD patients were positive, while all 39 non-CJD patients were negative, giving 95.8% diagnostic sensitivity and 100% specificity. This second-generation RT-QuIC assay markedly improved the speed and sensitivity of detecting prion seeds in CSF specimens from CJD patients. This should enhance prospects for rapid and accurate ante mortem CJD diagnosis. IMPORTANCE?: A long-standing problem in dealing with various neurodegenerative protein misfolding diseases is early and accurate diagnosis. This issue is particularly important with human prion diseases, such as CJD, because prions are deadly, transmissible, and unusually resistant to decontamination. The recently developed RT-QuIC test allows for highly sensitive and specific detection of CJD in human cerebrospinal fluid and is being broadly implemented as a key diagnostic tool. However, as currently applied, RT-QuIC takes 2.5 to 5 days and misses 11 to 23% of CJD cases. Now, we have markedly improved RT-QuIC analysis of human CSF such that CJD and non-CJD patients can be discriminated in a matter of hours rather than days with enhanced sensitivity. These improvements should allow for much faster, more accurate, and practical testing for CJD. In broader terms, our study provides a prototype for tests for misfolded protein aggregates that cause many important amyloid diseases, such as Alzheimer's, Parkinson's, and tauopathies. PMID:25604790

Orrú, Christina D; Groveman, Bradley R; Hughson, Andrew G; Zanusso, Gianluigi; Coulthart, Michael B; Caughey, Byron

2015-01-01

410

Rapid and Sensitive RT-QuIC Detection of Human Creutzfeldt-Jakob Disease Using Cerebrospinal Fluid  

PubMed Central

ABSTRACT? Fast, definitive diagnosis of Creutzfeldt-Jakob disease (CJD) is important in assessing patient care options and transmission risks. Real-time quaking-induced conversion (RT-QuIC) assays of cerebrospinal fluid (CSF) and nasal-brushing specimens are valuable in distinguishing CJD from non-CJD conditions but have required 2.5 to 5 days. Here, an improved RT-QuIC assay is described which identified positive CSF samples within 4 to 14 h with better analytical sensitivity. Moreover, analysis of 11 CJD patients demonstrated that while 7 were RT-QuIC positive using the previous conditions, 10 were positive using the new assay. In these and further analyses, a total of 46 of 48 CSF samples from sporadic CJD patients were positive, while all 39 non-CJD patients were negative, giving 95.8% diagnostic sensitivity and 100% specificity. This second-generation RT-QuIC assay markedly improved the speed and sensitivity of detecting prion seeds in CSF specimens from CJD patients. This should enhance prospects for rapid and accurate ante mortem CJD diagnosis. Importance? A long-standing problem in dealing with various neurodegenerative protein misfolding diseases is early and accurate diagnosis. This issue is particularly important with human prion diseases, such as CJD, because prions are deadly, transmissible, and unusually resistant to decontamination. The recently developed RT-QuIC test allows for highly sensitive and specific detection of CJD in human cerebrospinal fluid and is being broadly implemented as a key diagnostic tool. However, as currently applied, RT-QuIC takes 2.5 to 5 days and misses 11 to 23% of CJD cases. Now, we have markedly improved RT-QuIC analysis of human CSF such that CJD and non-CJD patients can be discriminated in a matter of hours rather than days with enhanced sensitivity. These improvements should allow for much faster, more accurate, and practical testing for CJD. In broader terms, our study provides a prototype for tests for misfolded protein aggregates that cause many important amyloid diseases, such as Alzheimer’s, Parkinson’s, and tauopathies. PMID:25604790

Orrú, Christina D.; Groveman, Bradley R.; Hughson, Andrew G.; Zanusso, Gianluigi; Coulthart, Michael B.

2015-01-01

411

Comparative detection of enterovirus RNA in cerebrospinal fluid: GeneXpert system vs. real-time RT-PCR assay.  

PubMed

Enteroviruses (EVs) constitute the most common cause of aseptic meningitis in both children and adults. Molecular techniques have now been recognized as the reference standard for the diagnosis of EV infections, and the rapidity of the molecular diagnosis of EV meningitis has been shown to be a determining factor in the management of patients. The rapid documentation of EV RNA in cerebrospinal fluid (CSF) is key to adapting patient management and the therapeutic regimen. To shorten the time needed for virological documentation, we implemented EV RNA detection in two point-of-care (POC) laboratories. Here, we present the results of the POC detection of EV RNA with the Xpert EV kit on the GeneXpert integrated system, and a comparison with the real-time RT-PCR (rtRT-PCR) assay routinely used in