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1

A MIV-150/Zinc Acetate Gel Inhibits SHIV-RT Infection in Macaque Vaginal Explants  

PubMed Central

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

2

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

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

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

5

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

6

Enhanced antiretroviral therapy in rhesus macaques improves RT-SHIV viral decay kinetics.  

PubMed

Using an established nonhuman primate model, rhesus macaques were infected intravenously with a chimeric simian immunodeficiency virus (SIV) consisting of SIVmac239 with the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase from clone HXBc2 (RT-SHIV). The impacts of two enhanced (four- and five-drug) highly active antiretroviral therapies (HAART) on early viral decay and rebound were determined. The four-drug combination consisted of an integrase inhibitor, L-870-812 (L-812), together with a three-drug regimen comprising emtricitabine [(-)-FTC], tenofovir (TFV), and efavirenz (EFV). The five-drug combination consisted of one analog for each of the four DNA precursors {using TFV, (-)-FTC, (-)-?-D-(2R,4R)-1,3-dioxolane-2,6-diaminopurine (amdoxovir [DAPD]), and zidovudine (AZT)}, together with EFV. A cohort treated with a three-drug combination of (-)-FTC, TFV, and EFV served as treated controls. Daily administration of a three-, four-, or five-drug combination of antiretroviral agents was initiated at week 6 or 8 after inoculation and continued up to week 50, followed by a rebound period. Plasma samples were collected routinely, and drug levels were monitored using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Viral loads were monitored with a standard TaqMan quantitative reverse transcriptase PCR (qRT-PCR) assay. Comprehensive analyses of replication dynamics were performed. RT-SHIV infection in rhesus macaques produced typical viral infection kinetics, with untreated controls establishing persistent viral loads of >10(4) copies of RNA/ml. RT-SHIV loads at the start of treatment (V0) were similar in all treated cohorts (P > 0.5). All antiretroviral drug levels were measureable in plasma. The four-drug and five-drug combination regimens (enhanced HAART) improved suppression of the viral load (within 1 week; P < 0.01) and had overall greater potency (P < 0.02) than the three-drug regimen (HAART). Moreover, rebound viremia occurred rapidly following cessation of any treatment. The enhanced HAART (four- or five-drug combination) showed significant improvement in viral suppression compared to the three-drug combination, but no combination was sufficient to eliminate viral reservoirs. PMID:24777106

North, Thomas W; Villalobos, Andradi; Hurwitz, Selwyn J; Deere, Jesse D; Higgins, Joanne; Chatterjee, Payel; Tao, Sijia; Kauffman, Robert C; Luciw, Paul A; Kohler, James J; Schinazi, Raymond F

2014-07-01

7

Enhanced Antiretroviral Therapy in Rhesus Macaques Improves RT-SHIV Viral Decay Kinetics  

PubMed Central

Using an established nonhuman primate model, rhesus macaques were infected intravenously with a chimeric simian immunodeficiency virus (SIV) consisting of SIVmac239 with the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase from clone HXBc2 (RT-SHIV). The impacts of two enhanced (four- and five-drug) highly active antiretroviral therapies (HAART) on early viral decay and rebound were determined. The four-drug combination consisted of an integrase inhibitor, L-870-812 (L-812), together with a three-drug regimen comprising emtricitabine [(?)-FTC], tenofovir (TFV), and efavirenz (EFV). The five-drug combination consisted of one analog for each of the four DNA precursors {using TFV, (?)-FTC, (?)-?-d-(2R,4R)-1,3-dioxolane-2,6-diaminopurine (amdoxovir [DAPD]), and zidovudine (AZT)}, together with EFV. A cohort treated with a three-drug combination of (?)-FTC, TFV, and EFV served as treated controls. Daily administration of a three-, four-, or five-drug combination of antiretroviral agents was initiated at week 6 or 8 after inoculation and continued up to week 50, followed by a rebound period. Plasma samples were collected routinely, and drug levels were monitored using liquid chromatography-tandem mass spectrometry (LC–MS-MS). Viral loads were monitored with a standard TaqMan quantitative reverse transcriptase PCR (qRT-PCR) assay. Comprehensive analyses of replication dynamics were performed. RT-SHIV infection in rhesus macaques produced typical viral infection kinetics, with untreated controls establishing persistent viral loads of >104 copies of RNA/ml. RT-SHIV loads at the start of treatment (V0) were similar in all treated cohorts (P > 0.5). All antiretroviral drug levels were measureable in plasma. The four-drug and five-drug combination regimens (enhanced HAART) improved suppression of the viral load (within 1 week; P < 0.01) and had overall greater potency (P < 0.02) than the three-drug regimen (HAART). Moreover, rebound viremia occurred rapidly following cessation of any treatment. The enhanced HAART (four- or five-drug combination) showed significant improvement in viral suppression compared to the three-drug combination, but no combination was sufficient to eliminate viral reservoirs. PMID:24777106

North, Thomas W.; Villalobos, Andradi; Hurwitz, Selwyn J.; Deere, Jesse D.; Higgins, Joanne; Chatterjee, Payel; Tao, Sijia; Kauffman, Robert C.; Luciw, Paul A.; Kohler, James J.

2014-01-01

8

Fatal Immunopathogenesis by SIV\\/HIV1 (SHIV) Containing a Variant Form of the HIV1 sf 33 env Gene in Juvenile and Newborn Rhesus Macaques  

Microsoft Academic Search

SIV\\/HIV-1 (SHIV) chimeric clones, constructed by substituting portions of the pathogenic molecular clone SIVmac239 with counterpart portions from HIV-1 clones, provide a means to analyze functions of selected HIV-1 genes in vivo in nonhuman primates. Our studies focused on SHIVsf33, which contains the vpu, tat, rev, and env genes of the cytopathic, T-cell line tropic clone HIV-1sf33 (subtype-B); this clone

Paul A. Luciw; Carol P. Mandell; Sunee Himathongkham; Jinling Li; Tesi A. Low; Kim A. Schmidt; Karen E. S. Shaw; Cecilia Cheng-Mayer

1999-01-01

9

Selection of unadapted, pathogenic SHIVs encoding newly transmitted HIV-1 envelope proteins.  

PubMed

Infection of macaques with chimeric viruses based on SIVMAC but expressing the HIV-1 envelope (Env) glycoproteins (SHIVs) remains the most powerful model for evaluating prevention and therapeutic strategies against AIDS. Unfortunately, only a few SHIVs are currently available. Furthermore, their generation has required extensive adaptation of the HIV-1 Env sequences in macaques so they may not accurately represent HIV-1 Env proteins circulating in humans, potentially limiting their translational utility. We developed a strategy for generating large numbers of SHIV constructs expressing Env proteins from newly transmitted HIV-1 strains. By inoculating macaques with cocktails of multiple SHIV variants, we selected SHIVs that can replicate and cause AIDS-like disease in immunologically intact rhesus macaques without requiring animal-to-animal passage. One of these SHIVs could be transmitted mucosally. We demonstrate the utility of the SHIVs generated by this method for evaluating neutralizing antibody administration as a protection against mucosal SHIV challenge. PMID:25211081

Del Prete, Gregory Q; Ailers, Braiden; Moldt, Brian; Keele, Brandon F; Estes, Jacob D; Rodriguez, Anthony; Sampias, Marissa; Oswald, Kelli; Fast, Randy; Trubey, Charles M; Chertova, Elena; Smedley, Jeremy; LaBranche, Celia C; Montefiori, David C; Burton, Dennis R; Shaw, George M; Markowitz, Marty; Piatak, Michael; KewalRamani, Vineet N; Bieniasz, Paul D; Lifson, Jeffrey D; Hatziioannou, Theodora

2014-09-10

10

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

PubMed Central

The increasing prevalence of human immunodeficiency virus type 1 (HIV-1) subtype C infection worldwide calls for efforts to develop a relevant animal model for evaluating AIDS candidate vaccines. In China, the prevalent HIV strains comprise a circulating recombinant form, BC (CRF07_BC), in which the envelope belongs to subtype C. To evaluate potential AIDS vaccines targeting Chinese viral strains in nonhuman primate models, we constructed a simian-human immunodeficiency virus (SHIV) carrying most of the envelope sequence of a primary HIV-1 clade C strain isolated from an HIV-positive intravenous drug user from YunNan province in China. Infection of six Chinese rhesus macaques with SHIV-CN97001 resulted in a low level of viremia and no significant alteration in CD4+ T-lymphocyte counts. To determine whether in vivo adaptation would enhance the infectivity of SHIV-CN97001, the parental infectious strain was serially passaged through eight Chinese rhesus macaques. The hallmarks of SHIV infectivity developed gradually, as shown by the increasingly elevated peak viremia with each passage. These findings establish that the R5-tropic SHIV-CN97001/Chinese rhesus macaque model should be very useful for evaluation of HIV-1 subtype C vaccines in China. PMID:21895680

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

2012-01-01

11

SHIV antigen immunization alters patterns of immune responses to SHIV/malaria coinfection and protects against life-threatening SHIV-related malaria.  

PubMed

Whether vaccination against a virus can protect against more virulent coinfection with the virus and additional pathogen(s) remains poorly characterized. Overlapping endemicity of human immunodeficiency virus (HIV) and malaria suggests that HIV/malaria coinfection frequently complicates acute and chronic HIV infection. Here we showed that vaccination of macaques with recombinant Listeria ?actA prfA* expressing simian/human immunodeficiency virus (SHIV) gag and env elicited Gag- and Env-specific T-cell responses, and protected against life-threatening SHIV-related malaria after SHIV/Plasmodium fragile coinfection. SHIV antigen immunization reduced peak viremia, resisted SHIV/malaria-induced lymphoid destruction, and blunted coinfection-accelerated decline of CD4(+) T-cell counts after SHIV/malaria coinfection. SHIV antigen immunization also weakened coinfection-driven overreactive proinflammatory interferon-? (IFN?) responses and led to developing T helper cell 17/22 (Th17/Th22) responses after SHIV/malaria coinfection. The findings suggest that vaccination against AIDS virus can alter patterns of immune responses to the SHIV/malaria coinfection and protect against life-threatening SHIV-related malaria. PMID:23568175

Frencher, James T; Ryan-Pasyeur, Bridgett K; Huang, Dan; Wang, Ri Cheng; McMullen, Phillip D; Letvin, Norman L; Collins, William E; Freitag, Nancy E; Malkovsky, Miroslav; Chen, Crystal Y; Shen, Ling; Chen, Zheng W

2013-07-15

12

Partial protection against multiple RT-SHIV162P3 vaginal challenge of rhesus macaques by a silicone elastomer vaginal ring releasing the NNRTI MC1220  

PubMed Central

Objectives The non-nucleoside reverse transcriptase inhibitor MC1220 has potent in vitro activity against HIV type 1 (HIV-1). A liposome gel formulation of MC1220 has previously been reported to partially protect rhesus macaques against vaginal challenge with a simian HIV (SHIV). Here, we describe the pre-clinical development of an MC1220-releasing silicone elastomer vaginal ring (SEVR), including pharmacokinetic (PK) and efficacy studies in macaques. Methods In vitro release studies were conducted on SEVRs loaded with 400 mg of MC1220, using simulated vaginal fluid (SVF, n?=?4) and 1?:?1 isopropanol/water (IPA/H2O, n?=?4) as release media. For PK evaluation, SEVRs were inserted into adult female macaques (n?=?6) for 30 days. Following a 1week washout period, fresh rings were placed in the same animals, which were then challenged vaginally with RT-SHIV162P3 once weekly for 4 weeks. Results SEVRs released 1.66 and 101 mg of MC1220 into SVF and IPA/H2O, respectively, over 30 days, the differential reflecting the low aqueous solubility of the drug. In macaque PK studies, MC1220 was consistently detected in vaginal fluid (peak 845 ng/mL) and plasma (peak 0.91 ng/mL). Kaplan–Meier analysis over 9weeks showed significantly lower infection rates for animals given MC1220-containing SEVRs than placebo rings (hazard ratio 0.20, P?=?0.0037). Conclusions An MC1220-releasing SEVR partially protected macaques from vaginal challenge. Such ring devices are a practical method for providing sustained, coitally independent protection against vaginal exposure to HIV-1. PMID:23109186

Fetherston, Susan M.; Geer, Leslie; Veazey, Ronald S.; Goldman, Laurie; Murphy, Diarmaid J.; Ketas, Thomas J.; Klasse, Per Johan; Blois, Sylvain; La Colla, Paolo; Moore, John P.; Malcolm, R. Karl

2013-01-01

13

A combination HIV vaccine based on Tat and Env proteins was immunogenic and protected macaques from mucosal SHIV challenge in a pilot study  

Microsoft Academic Search

HIV native Tat and V2 loop-deleted Env (Env?V2) proteins already proved safe and immunogenic in phase I clinical testing as single vaccine components. Further, a phase II vaccine trial with Tat showed intensification of the therapeutic effects of HAART in successfully treated HIV-infected individuals. Here a pilot study assessed the immunogenicity and protective efficacy of an HIV\\/AIDS vaccine based on

Flavia Ferrantelli; Maria Teresa Maggiorella; Ilaria Schiavoni; Leonardo Sernicola; Erika Olivieri; Stefania Farcomeni; Maria Rosaria Pavone-Cossut; Sonia Moretti; Roberto Belli; Barbara Collacchi; Indresh K. Srivastava; Fausto Titti; Aurelio Cafaro; Susan W. Barnett; Barbara Ensoli

2011-01-01

14

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

15

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

PubMed Central

We recently reported that rhesus macaques inoculated with CD4-binding-competent and CD4-binding-defective soluble YU2-derived HIV-1 envelope glycoprotein (Env) trimers in adjuvant generate comparable levels of Env-specific binding antibodies (Abs) and T cell responses. We also showed that Abs directed against the Env coreceptor binding site (CoRbs) were elicited only in animals immunized with CD4-binding-competent trimers and not in animals immunized with CD4-binding-defective trimers, indicating that a direct interaction between Env and CD4 occurs in vivo. To investigate both the overall consequences of in vivo Env-CD4 interactions and the elicitation of CoRbs-directed Abs for protection against heterologous simian-human immunodeficiency virus (SHIV) challenge, we exposed rhesus macaques immunized with CD4-binding-competent and CD4-binding-defective trimers to the CCR5-tropic SHIV-SF162P4 challenge virus. Compared to unvaccinated controls, all vaccinated animals displayed improved control of plasma viremia, independent of the presence or absence of CoRbs-directed Abs prior to challenge. Immunization resulted in plasma responses that neutralized the heterologous SHIV challenge stock in vitro, with similar neutralizing Ab titers elicited by the CD4-binding-competent and CD4-binding-defective trimers. The neutralizing responses against both the SHIV-SF162P4 stock and a recombinant virus pseudotyped with a cloned SHIV-SF162P4-derived Env were significantly boosted by the SHIV challenge. Collectively, these results suggest that the capacity of soluble Env trimers to interact with primate CD4 in vivo and to stimulate the production of moderate titers of CoRbs-directed Abs did not influence the magnitude of the neutralizing Ab recall response after viral challenge or the subsequent control of viremia in this heterologous SHIV challenge model. PMID:20610729

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

2010-01-01

16

Passive immunization of macaques with polyclonal anti-SHIV IgG against a heterologous tier 2 SHIV: outcome depends on IgG dose  

PubMed Central

Background A key goal for HIV-1 envelope immunogen design is the induction of cross-reactive neutralizing antibodies (nAbs). As AIDS vaccine recipients will not be exposed to strains exactly matching any immunogens due to multiple HIV-1 quasispecies circulating in the human population worldwide, heterologous SHIV challenges are essential for realistic vaccine efficacy testing in primates. We assessed whether polyclonal IgG, isolated from rhesus monkeys (RMs) with high-titer nAbs (termed SHIVIG), could protect RMs against the R5-tropic tier-2 SHIV-2873Nip, which was heterologous to the viruses or HIV-1 envelopes that had elicited SHIVIG. Results SHIVIG demonstrated binding to HIV Gag, Tat, and Env of different clades and competed with the broadly neutralizing antibodies b12, VRC01, 4E10, and 17b. SHIVIG neutralized tier 1 and tier 2 viruses, including SHIV-2873Nip. NK-cell depletion decreased the neutralizing activity of SHIVIG 20-fold in PBMC assays. Although SHIVIG neutralized SHIV-2873Nip in vitro, this polyclonal IgG preparation failed to prevent acquisition after repeated intrarectal low-dose virus challenges, but at a dose of 400 mg/kg, it significantly lowered peak viremia (P?=?0.001). Unexpectedly, single-genome analysis revealed a higher number of transmitted variants at the low dose of 25 mg/kg, implying increased acquisition at low SHIVIG levels. In vitro, SHIVIG demonstrated complement-mediated Ab-dependent enhancement of infection (C’-ADE) at concentrations similar to those observed in plasmas of RMs treated with 25 mg/kg of SHIVIG. Conclusion Our primate model data suggest a dual role for polyclonal anti-HIV-1 Abs depending on plasma levels upon virus encounter. PMID:24444350

2014-01-01

17

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

PubMed Central

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

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

2015-01-01

18

Protective efficacy of a global HIV-1 mosaic vaccine against heterologous SHIV challenges in rhesus monkeys.  

PubMed

The global diversity of HIV-1 represents a critical challenge facing HIV-1 vaccine development. HIV-1 mosaic antigens are bioinformatically optimized immunogens designed for improved coverage of HIV-1 diversity. However, the protective efficacy of such global HIV-1 vaccine antigens has not previously been evaluated. Here, we demonstrate the capacity of bivalent HIV-1 mosaic antigens to protect rhesus monkeys against acquisition of infection following heterologous challenges with the difficult-to-neutralize simian-human immunodeficiency virus SHIV-SF162P3. Adenovirus/poxvirus and adenovirus/adenovirus vector-based vaccines expressing HIV-1 mosaic Env, Gag, and Pol afforded a significant reduction in the per-exposure acquisition risk following repetitive, intrarectal SHIV-SF162P3 challenges. Protection against acquisition of infection correlated with vaccine-elicited binding, neutralizing, and functional nonneutralizing antibodies, suggesting that the coordinated activity of multiple antibody functions may contribute to protection against difficult-to-neutralize viruses. These data demonstrate the protective efficacy of HIV-1 mosaic antigens and suggest a potential strategy for the development of a global HIV-1 vaccine. PAPERCLIP: PMID:24243013

Barouch, Dan H; Stephenson, Kathryn E; Borducchi, Erica N; Smith, Kaitlin; Stanley, Kelly; McNally, Anna G; Liu, Jinyan; Abbink, Peter; Maxfield, Lori F; Seaman, Michael S; Dugast, Anne-Sophie; Alter, Galit; Ferguson, Melissa; Li, Wenjun; Earl, Patricia L; Moss, Bernard; Giorgi, Elena E; Szinger, James J; Eller, Leigh Anne; Billings, Erik A; Rao, Mangala; Tovanabutra, Sodsai; Sanders-Buell, Eric; Weijtens, Mo; Pau, Maria G; Schuitemaker, Hanneke; Robb, Merlin L; Kim, Jerome H; Korber, Bette T; Michael, Nelson L

2013-10-24

19

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

PubMed Central

The current study assessed the immunogenicity and protective efficacy of various prime-boost vaccine regimens in rhesus macaques using combinations of recombinant DNA (rDNA), recombinant MVA (rMVA), and subunit gp140 protein. The rDNA and rMVA vectors were constructed to express Env from HIV-1 subtype CRF01_AE and Gag-Pol from CRF01_AE or SIVmac 239. One of the rMVAs, MVA/CMDR, has been recently tested in humans. Immunizations were administered at months 0 and 1 (prime) and months 3 and 6 (boost). After priming, HIV env-specific serum IgG was detected in monkeys receiving gp140 alone or rMVA but not in those receiving rDNA. Titers were enhanced in these groups after boosting either with gp140 alone or with rMVA plus gp140. The groups that received the rDNA prime developed env-specific IgG after boosting with rMVA with or without gp140. HIV Env-specific serum IgG binding antibodies were elicited more frequently and of higher titer, and breadth of neutralizing antibodies was increased with the inclusion of the subunit Env boost. T cell responses were measured by tetramer binding to Gag p11c in Mamu-A*01 macaques, and by IFN-gamma ELISPOT assay to SIV-Gag. T cell responses were induced after vaccination with the highest responses seen in macaques immunized with rDNA and rMVA. Macaques were challenged intravenously with a novel SHIV-E virus (SIVmac239 Gag-Pol with an HIV-1 subtype E-Env CAR402). Post challenge with SHIV-E, antibody titers were boosted in all groups and peaked at 4 weeks. Robust T cell responses were seen in all groups post challenge and in macaques immunized with rDNA and rMVA a clear boosting of responses was seen. A greater than 2 log drop in RNA copies/ml at peak viremia and earlier set point was achieved in macaques primed with rDNA, and boosted with rMVA/SHIV-AE plus gp140. Post challenge viremia in macaques immunized with other regimens was not significantly different to that of controls. These results demonstrate that a gp140 subunit and inclusion of SIV Gag-Pol may be critical for control of SHIV post challenge. PMID:22234262

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

2012-01-01

20

AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges.  

PubMed

Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs). However, even the best bNAbs neutralize 10-50% of HIV-1 isolates inefficiently (80% inhibitory concentration (IC80) > 5 ?g ml(-1)), suggesting that high concentrations of these antibodies would be necessary to achieve general protection. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean half-maximum inhibitory concentration (IC50) < 0.05 ?g ml(-1)). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2 and simian immunodeficiency virus isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46 and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17-77 ?g ml(-1) of fully functional rhesus eCD4-Ig for more than 40 weeks, and these macaques were protected from several infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well-characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine. PMID:25707797

Gardner, Matthew R; Kattenhorn, Lisa M; Kondur, Hema R; von Schaewen, Markus; Dorfman, Tatyana; Chiang, Jessica J; Haworth, Kevin G; Decker, Julie M; Alpert, Michael D; Bailey, Charles C; Neale, Ernest S; Fellinger, Christoph H; Joshi, Vinita R; Fuchs, Sebastian P; Martinez-Navio, Jose M; Quinlan, Brian D; Yao, Annie Y; Mouquet, Hugo; Gorman, Jason; Zhang, Baoshan; Poignard, Pascal; Nussenzweig, Michel C; Burton, Dennis R; Kwong, Peter D; Piatak, Michael; Lifson, Jeffrey D; Gao, Guangping; Desrosiers, Ronald C; Evans, David T; Hahn, Beatrice H; Ploss, Alexander; Cannon, Paula M; Seaman, Michael S; Farzan, Michael

2015-03-01

21

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

22

Anti-HIV IgA Isotypes: Differential Virion Capture and Inhibition of Transcytosis are Linked to Prevention of Mucosal R5 SHIV Transmission  

PubMed Central

Objective Although passive immunization with anti-HIV-1 Env IgG1 neutralizing monoclonal Abs (nmAbs) prevented simian-human immunodeficiency virus (SHIV) infection in rhesus monkeys (RMs), IgA nmAbs have not been tested. Here, we sought to determine whether human anti-HIV-1 dimeric (d)IgA1, dIgA2, and IgG1 differ in their ability to prevent mucosal R5 SHIV acquisition in RMs. Design DIgA1, dIgA2, and IgG1 versions of nmAb HGN194 were applied intrarectally (i.r.) in three RM groups 30 min before i.r. SHIV challenge. Methods After a control pharmacokinetic study showed that nmAb concentrations in rectal fluids over time were similar for all HGN194 isotypes, control and nmAb-treated animals were challenged i.r. with an R5 SHIV, and viral loads were monitored. Results Unexpectedly, dIgA1 provided the best protection in vivo – although all nmAbs showed similar neutralizing activity in vitro. Five out of the six dIgA1-treated RMs remained virus-free compared to only one out of six animals given dIgA2 (P=0.045 by log rank test) and two out of six RMs treated with IgG1 forms of the nmAb (P=0.12). Protection correlated significantly with virion capture activity by a given nmAb form, as well as inhibition of transcytosis of cell-free virus across an epithelial cell layer in vitro. Conclusions Our data imply that dIgA1-mediated capturing of virions in mucosal secretions and inhibition of transcytosis can provide significant prevention of lentiviral acquisition – over and above direct virus neutralization. Vaccine strategies that induce mucosal IgA, especially IgA1, should be developed as first-line of defense against HIV-1, a virus predominantly transmitted mucosally. PMID:23775002

Watkins, Jennifer D.; Sholukh, Anton M.; Mukhtar, Muhammad M.; Siddappa, Nagadenahalli B.; Lakhashe, Samir K.; Kim, Mikyung; Reinherz, Ellis L.; Gupta, Sandeep; Forthal, Donald N.; Sattentau, Quentin; Villinger, Francois; Corti, Davide; Ruprecht, Ruth M.

2014-01-01

23

Tat protein vaccination of cynomolgus macaques influences SHIV89.6P cy243 epitope variability  

Microsoft Academic Search

In a previous study we showed that vaccination with the native Tat protein controlled virus replication in five out of seven\\u000a monkeys against challenge with the simian human immunodeficiency virus (SHIV)-89.6Pcy243 and that this protection correlated with T helper (Th)-1 response and cytotoxic T lymphocyte (CTL) activity. To address the\\u000a evolution of the SHIV-89.6Pcy243 both in control and vaccinated infected

Barbara Ridolfi; Domenico Genovese; Claudio Argentini; Maria Teresa Maggiorella; Leonardo Sernicola; Stefano Buttò; Fausto Titti; Alessandra Borsetti; Barbara Ensoli

2008-01-01

24

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

SciTech Connect

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

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

2007-05-10

25

Primate immune responses to HIV-1 Env formulated in the saponin-based adjuvant AbISCO-100 in the presence or absence of TLR9 co-stimulation  

PubMed Central

Protein-based vaccines require adjuvants to achieve optimal responses. Toll-like receptor (TLR) 9 agonists were previously shown to improve responses to protein-based vaccines, such as the Hepatitis B virus vaccine formulated in alum. Here, we used CpG-C together with the clinically relevant saponin-based adjuvant AbISCO-100/Matrix-M (AbISCO), to assess if TLR9 co-stimulation would quantitatively or qualitatively modulate HIV-1 envelope glycoprotein (Env)-specific B and T cell responses in rhesus macaques. The macaques were inoculated with soluble Env trimers in AbISCO, with or without the addition of CpG-C, using an interval similar to the Hepatitis B virus vaccine. Following a comprehensive evaluation of antigen-specific responses in multiple immune compartments, we show that the Env-specific circulating IgG, memory B cells and plasma cells displayed similar kinetics and magnitude in the presence or absence of CpG-C and that there was no apparent difference between the two groups in the elicited HIV-1 neutralizing antibody titers or antigen-specific CD4+ T cell responses. Importantly, the control of SHIV viremia was significantly improved in animals from both Env-immunized groups relative to adjuvant alone controls, demonstrating the potential of AbISCO to act as a stand-alone adjuvant for Env-based vaccines. PMID:25762407

Martinez, Paola; Sundling, Christopher; O'Dell, Sijy; Mascola, John R.; Wyatt, Richard T.; Karlsson Hedestam, Gunilla B.

2015-01-01

26

Primate immune responses to HIV-1 Env formulated in the saponin-based adjuvant AbISCO-100 in the presence or absence of TLR9 co-stimulation.  

PubMed

Protein-based vaccines require adjuvants to achieve optimal responses. Toll-like receptor (TLR) 9 agonists were previously shown to improve responses to protein-based vaccines, such as the Hepatitis B virus vaccine formulated in alum. Here, we used CpG-C together with the clinically relevant saponin-based adjuvant AbISCO-100/Matrix-M (AbISCO), to assess if TLR9 co-stimulation would quantitatively or qualitatively modulate HIV-1 envelope glycoprotein (Env)-specific B and T cell responses in rhesus macaques. The macaques were inoculated with soluble Env trimers in AbISCO, with or without the addition of CpG-C, using an interval similar to the Hepatitis B virus vaccine. Following a comprehensive evaluation of antigen-specific responses in multiple immune compartments, we show that the Env-specific circulating IgG, memory B cells and plasma cells displayed similar kinetics and magnitude in the presence or absence of CpG-C and that there was no apparent difference between the two groups in the elicited HIV-1 neutralizing antibody titers or antigen-specific CD4+ T cell responses. Importantly, the control of SHIV viremia was significantly improved in animals from both Env-immunized groups relative to adjuvant alone controls, demonstrating the potential of AbISCO to act as a stand-alone adjuvant for Env-based vaccines. PMID:25762407

Martinez, Paola; Sundling, Christopher; O'Dell, Sijy; Mascola, John R; Wyatt, Richard T; Karlsson Hedestam, Gunilla B

2015-01-01

27

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

28

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

29

Envelope Glycoprotein Determinants of Increased Fusogenicity in a Pathogenic Simian-Human Immunodeficiency Virus (SHIV-KB9) Passaged In Vivo  

Microsoft Academic Search

Changes in the envelope glycoprotein ectodomains of a nonpathogenic simian-human immunodeficiency virus (SHIV-89.6) that was serially passaged in vivo have been shown to be responsible for the increased pathogenicity of the resulting virus, SHIV-KB9 (G. B. Karlsson, et al., J. Exp. Med. 188:1159-1171, 1998). The 12 amino acid changes in the envelope glycoprotein ectodomains resulted in increased chemokine receptor- binding

BIJAN ETEMAD-MOGHADAM; YING SUN; EMMA K. NICHOLSON; MARK FERNANDES; KWA LIOU; RAUL GOMILA; JULIETTE LEE; JOSEPH SODROSKI

2000-01-01

30

Floating JMaRT  

E-print Network

We define a new partially solvable system of equations that parametrises solutions to six-dimensional N=(1,0) ungauged supergravity coupled to tensor multiplets. We obtain this system by applying a series of dualities on the known floating brane system, imposing that it allows for the JMaRT solution. We construct an explicit multi-centre solution generalising the JMaRT solution, with an arbitrary number of additional BPS centres on a line. We describe explicitly the embedding of the JMaRT solution in this system in five dimensions.

Guillaume Bossard; Stefanos Katmadas

2014-12-16

31

Floating JMaRT  

E-print Network

We define a new partially solvable system of equations that parametrises solutions to six-dimensional N=(1,0) ungauged supergravity coupled to tensor multiplets. We obtain this system by applying a series of dualities on the known floating brane system, imposing that it allows for the JMaRT solution. We construct an explicit multi-centre solution generalising the JMaRT solution, with an arbitrary number of additional BPS centres on a line. We describe explicitly the embedding of the JMaRT solution in this system in five dimensions.

Bossard, Guillaume

2014-01-01

32

Aaron Shepard's RT Page  

NSDL National Science Digital Library

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

Shepard, Aaron.

1996-01-01

33

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

34

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.

35

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

36

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

37

Passive transfer of modest titers of potent and broadly neutralizing anti-HIV monoclonal antibodies block SHIV infection in macaques  

PubMed Central

It is widely appreciated that effective human vaccines directed against viral pathogens elicit neutralizing antibodies (NAbs). The passive transfer of anti–HIV-1 NAbs conferring sterilizing immunity to macaques has been used to determine the plasma neutralization titers, which must be present at the time of exposure, to prevent acquisition of SIV/HIV chimeric virus (SHIV) infections. We administered five recently isolated potent and broadly acting anti-HIV neutralizing monoclonal antibodies (mAbs) to rhesus macaques and challenged them intrarectally 24 h later with either of two different R5-tropic SHIVs. By combining the results obtained from 60 challenged animals, we determined that the protective neutralization titer in plasma preventing virus infection in 50% of the exposed monkeys was relatively modest (?1:100) and potentially achievable by vaccination. PMID:25155019

Shingai, Masashi; Donau, Olivia K.; Plishka, Ronald J.; Buckler-White, Alicia; Mascola, John R.; Nabel, Gary J.; Nason, Martha C.; Montefiori, David; Moldt, Brian; Poignard, Pascal; Diskin, Ron; Bjorkman, Pamela J.; Eckhaus, Michael A.; Klein, Florian; Mouquet, Hugo; Cetrulo Lorenzi, Julio Cesar; Gazumyan, Anna; Burton, Dennis R.; Nussenzweig, Michel C.

2014-01-01

38

Passive transfer of modest titers of potent and broadly neutralizing anti-HIV monoclonal antibodies block SHIV infection in macaques.  

PubMed

It is widely appreciated that effective human vaccines directed against viral pathogens elicit neutralizing antibodies (NAbs). The passive transfer of anti-HIV-1 NAbs conferring sterilizing immunity to macaques has been used to determine the plasma neutralization titers, which must be present at the time of exposure, to prevent acquisition of SIV/HIV chimeric virus (SHIV) infections. We administered five recently isolated potent and broadly acting anti-HIV neutralizing monoclonal antibodies (mAbs) to rhesus macaques and challenged them intrarectally 24 h later with either of two different R5-tropic SHIVs. By combining the results obtained from 60 challenged animals, we determined that the protective neutralization titer in plasma preventing virus infection in 50% of the exposed monkeys was relatively modest (?1:100) and potentially achievable by vaccination. PMID:25155019

Shingai, Masashi; Donau, Olivia K; Plishka, Ronald J; Buckler-White, Alicia; Mascola, John R; Nabel, Gary J; Nason, Martha C; Montefiori, David; Moldt, Brian; Poignard, Pascal; Diskin, Ron; Bjorkman, Pamela J; Eckhaus, Michael A; Klein, Florian; Mouquet, Hugo; Cetrulo Lorenzi, Julio Cesar; Gazumyan, Anna; Burton, Dennis R; Nussenzweig, Michel C; Martin, Malcolm A; Nishimura, Yoshiaki

2014-09-22

39

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

40

The recombinant maize ribosome-inactivating protein transiently reduces viral load in SHIV89.6 infected Chinese Rhesus Macaques.  

PubMed

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

41

In Vitro Neutralization of Low Dose Inocula at Physiological Concentrations of a Monoclonal Antibody Which Protects Macaques against SHIV Challenge  

PubMed Central

Background Passive transfer of antibodies can be protective in the simian human immunodeficiency virus (SHIV) – rhesus macaque challenge model. The human monoclonal antibody IgG1 b12 neutralizes human immunodeficiency type 1 (HIV-1) in vitro and protects against challenge by SHIV. Our hypothesis is that neutralizing antibodies can only completely inactivate a relatively small number of infectious virus. Methods And Findings We have used GHOST cell assays to quantify individual infectious events with HIV-1SF162 and its SHIV derivatives: the relatively neutralization sensitive SHIVSF162P4 isolate and the more resistant SHIVSF162P3. A plot of the number of fluorescent GHOST cells with increasing HIV-1SF162 dose is not linear. It is likely that with high-dose inocula, infection with multiple virus produces additive fluorescence in individual cells. In studies of the neutralization kinetics of IgG1 b12 against these isolates, events during the absorption phase of the assay, as well as the incubation phase, determine the level of neutralization. It is possible that complete inactivation of a virus is limited to the time it is exposed on the cell surface. Assays can be modified so that neutralization of these very low doses of virus can be quantified. A higher concentration of antibody is required to neutralize the same dose of resistant SHIVSF162P3 than the sensitive SHIVSF162P4. In the absence of selection during passage, the density of the CCR5 co-receptor on the GHOST cell surface is reduced. Changes in the CD4 : CCR5 density ratio influence neutralization. Conclusions Low concentrations of IgG1 b12 completely inactivate small doses of the neutralization resistant SHIV SF162P3. Assays need to be modified to quantify this effect. Results from modified assays may predict protection following repeated low-dose shiv challenges in rhesus macaques. It should be possible to induce this level of antibody by vaccination so that modified assays could predict the outcome of human trials. PMID:23977339

Davis, David; Koornstra, Wim; Fagrouch, Zahra; Verschoor, Ernst J.; Heeney, Jonathan L.; Bogers, Willy M. J. M.

2013-01-01

42

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

43

Trimethoprim-Sulfamethoxazole Treatment Does Not Reverse Obstructive Pulmonary Changes in Pneumocystis-Colonized Non-Human Primates with SHIV Infection  

PubMed Central

Background Despite antiretroviral therapy and trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis, Pneumocystis pneumonia (PCP) remains an important serious opportunistic infection in HIV-infected persons. Pneumocystis (Pc) colonization in HIV-infected individuals and in HIV-uninfected smokers is associated with chronic obstructive pulmonary disease (COPD). We previously developed a non-human primate (NHP) model of HIV infection and Pc colonization and demonstrated that Pc colonization correlated with COPD development. In the present study, we examined kinetics of COPD development in NHP and tested the effect of Pc burden reduction on pulmonary function by TMP-SMX treatment. Methods Cynomolgus macaques (n=16) were infected with simian/human immunodeficiency virus (SHIV89.6P) and natural Pc colonization was examined by nested polymerase chain reaction (PCR) of serial bronchoalveolar lavage (BAL) fluid and anti-Pc serology. Results Eleven of 16 monkeys became Pc-colonized by 16 weeks post-SHIV infection. Pc colonization of SHIV-infected monkeys led to progressive declines in pulmonary function as early as 4 weeks following Pc detection. SHIV-infected, Pc-negative monkeys maintained normal lung function. At 25 weeks post SHIV-infection, TMP-SMX treatment was initiated in 7 Pc-positive (Pc+) (20mg/kg TMP, 100mg/kg SMX, daily for 48 weeks) and 5 Pc-negative (Pc-) monkeys. Four SHIV+/Pc+ remained untreated for the duration of the experiment. Detection frequency of Pc in BAL fluid (p<0.001), as well as plasma Pc antibody titers (p=0.02), were significantly reduced in TMP-SMX-treated macaques compared to untreated. Conclusion Reduction of Pc colonization by TMP-SMX treatment did not improve pulmonary function, supporting the concept that Pc-colonization results in early, permanent obstructive changes in the lungs of immunosuppressed macaques. PMID:24121760

Kling, Heather M.; Shipley, Timothy W.; Guyach, Siobhan; Tarantelli, Rebecca; Morris, Alison; Norris, Karen A.

2014-01-01

44

AIDS and optic neuritis in a rhesus monkey infected with the R5 clade C SHIV-1157ipd3N4  

PubMed Central

A Chinese rhesus macaque infected with the pathogenic CCR5-tropic clade C simian-human immunodeficiency virus, SHIV-1157ipd3N4, had persistent viremia, depletion of CD4+ T cells to <200 cells/?l, opportunistic infections, coagulopathy and gradual development of bilateral blindness. MRI revealed marked thickening of both optic nerves. Histopathological evaluation showed diffuse cellular infiltration at necropsy, and a focus of infected cells. This is the first report of CNS pathology following chronic infection with an obligate R5 SHIV. PMID:20412378

Garcia, Ana Patricia; Siddappa, Nagadenahalli B.; Li, Qingsheng; Haase, Ashley T.; Paul, Katherine; Stroud, Fawn; Zhang, Xiaodong; Fountain, Jack A.; Villinger, Francois; Novembre, Francis J.; Else, James G; Secor, W. Evan; Ruprecht, Ruth M.

2011-01-01

45

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

46

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.

47

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

48

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

49

ANTIBODY MEDIATED IMMUNOTHERAPY OF MACAQUES CHRONICALLY INFECTED WITH SHIV SUPPRESSES VIREMIA  

PubMed Central

Neutralizing antibodies (NAbs) can confer immunity to primate lentiviruses by blocking infection in macaque models of AIDS1–4. However, earlier studies of anti-HIV 1 NAbs administered to infected individuals or humanized mice, reported poor control of virus replication and the rapid emergence of resistant variants 5–7. A new generation of anti-HIV 1 monoclonal antibodies (mAbs), possessing extraordinary potency and breadth of neutralizing activity, has recently been isolated from infected individuals 8. These NAbs target different regions of the HIV 1 envelope glycoprotein including the CD4 binding site (bs), glycans located in the V1/V2, V3, and V4 regions, and the membrane proximal external region of gp419–14. We have examined two of the new antibodies, directed to the CD4 bs and the V3 region (3BNC117 and 10-1074 respectively) for their ability to block infection and suppress viremia in macaques infected with the R5 tropic SHIVAD8 virus, which emulates many of the pathogenic and immunogenic properties of HIV 1 during infections of rhesus macaques15,16. Either antibody alone can potently block virus acquisition. When administered individually to recently infected monkeys, the 10-1074 antibody caused a rapid decline in virus loads to undetectable levels for 4 to 7 days, followed by virus rebound during which neutralization resistant variants became detectable. When administered together, a single treatment rapidly suppressed plasma viremia for 3 to 5 weeks in some long-term chronically SHIV infected animals with low CD4+ T cell levels. A second cycle of anti-HIV 1 mAb therapy, administered to two previously treated animals, successfully controlled virus rebound. These results suggest 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. PMID:24172896

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

2014-01-01

50

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

51

Presence of env-like sequences in Quercus suber retrotransposons.  

PubMed

The main difference between LTR retrotransposons and retroviruses is the presence of the envelope (env) gene in the latter, downstream of the pol gene. The env gene is involved in their infectious capacity. Here we report the presence of env-like sequences in the genome of Quercus suber (cork oak), one of the most economically important Portuguese species. These gene sequences were isolated through DNA amplification between RNaseH conserved motifs and 3' LTR, based on the structure of copia retrotransposons. Phylogenetic analysis revealed that almost all the clones isolated are clustered with Cyclops-2, a Ty3-gypsy element identified in Pisum sativum, except one clustered with gypsy and copia retroelements found in different species. This suggests the existence of a potential ancestral sequence of the env gene, prior to the separation of Ty3-gypsy and Ty1-copia retrotransposons. Additionally, the isolated env-like sequences showed 26-39% of homology with env-like sequences characterized in viruses. The origin of env-like sequences in retrotransposons from host plant taxa is discussed. PMID:21063063

Carvalho, M; Ribeiro, T; Viegas, W; Morais-Cecilio, L; Rocheta, M

2010-01-01

52

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

53

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

Microsoft Academic Search

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

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

2009-01-01

54

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

SciTech Connect

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

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

2007-10-10

55

Systemic Dendritic Cell Mobilization Associated with Administration of FLT3 Ligand to SIV- and SHIV-Infected Macaques  

PubMed Central

Abstract Reports indicate that myeloid and plasmacytoid dendritic cells (mDCs and pDCs), which are key effector cells in host innate immune responses, can be infected with HIV-1 and are reduced in number and function during the chronic phase of HIV disease. Furthermore, it was recently demonstrated that a sustained loss of mDCs and pDCs occurs in SIV-infected macaques. Since loss of functional DC populations might impair innate immune responses to opportunistic microorganisms and neoplastic cells, we explored whether inoculation of naive and SIV- or SHIV-infected pigtailed macaques with the hematopoietic cytokine FLT3-ligand (FLT3-L) would expand the number of mDCs and pDCs in vivo. After the macaques received supraphysiologic doses of FLT3-L, mDCs, pDCs, and monocytes increased up to 45-fold in blood, lymph nodes, and bone marrow (BM), with DC expansion in the BM preceding mobilization in blood and lymphoid tissues. FLT3-L also increased serum levels of IL-12, at least transiently, and elicited higher surface expression of HLA-DR and the activation markers CD25 and CD69 on NK and T cells. During and after treatment of infected animals, APCs increased in number and were activated; however, CD4+ T cell numbers, virion RNA, and anti-SIV/SHIV antibody titers remained relatively stable, suggesting that FLT3-L might be a safe modality to expand DC populations and provide therapeutic benefit during chronic lentivirus infections. PMID:20001520

Reeves, R. Keith; Wei, Qing; Stallworth, Jackie

2009-01-01

56

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

57

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

58

Trimeric HIV Env provides epitope occlusion mediated by hypervariable loops  

PubMed Central

Hypervariable loops of HIV-1 Env protein gp120 are speculated to play roles in the conformational transition of Env to the receptor binding-induced metastable state. Structural analysis of full-length Env-based immunogens, containing the entire V2 loop, displayed tighter association between gp120 subunits, resulting in a smaller trimeric diameter than constructs lacking V2. A prominent basal quaternary location of V2 and V3? that challenges previous reports would facilitate gp41-independent gp120-gp120 interactions and suggests a quaternary mechanism of epitope occlusion facilitated by hypervariable loops. Deletion of V2 resulted in dramatic exposure of basal, membrane-proximal gp41 epitopes, consistent with its predicted basal location. The structural features of HIV-1 Env characterized here provide grounds for a paradigm shift in loop exposure and epitope occlusion, while providing substantive rationale for epitope display required for elicitation of broadly neutralizing antibodies, as well as substantiating previous pertinent literature disregarded in recent reports. PMID:25395053

Moscoso, Carlos G.; Xing, Li; Hui, Jinwen; Hu, Jeffrey; Kalkhoran, Mohammad Baikoghli; Yenigun, Onur M.; Sun, Yide; Paavolainen, Lassi; Martin, Loïc; Vahlne, Anders; Zambonelli, Carlo; Barnett, Susan W.; Srivastava, Indresh K.; Cheng, R. Holland

2014-01-01

59

ENVS 607: Graduate Seminar in POLITICAL Spring 2014  

E-print Network

in shaping human relationships with the environment. This year's seminar examines the political ecologyENVS 607: Graduate Seminar in POLITICAL ECOLOGY Spring 2014 Tu/Th 10:00-11:50 am, 249 Columbia Prof. Peter Walker, Dept. of Geography, 100 Condon Hall, 6-4541, pwalker@uoregon.edu Political ecology

60

RFID-Env: methods and software simulation for RFID environments  

Microsoft Academic Search

Purpose – The purpose of this paper is to present the results of an analytical and experimental research for the development of an innovative product designated RFID environment (RFID-Env). This software is designed for the use of professionals in computer systems and plant engineering who are engaged in research and development (R&D) of ultra high frequency (UHF) passive radio frequency

Marcelo Cunha de Azambuja; Carlos Fernando Jung; Carla Schwengber ten Caten; Fabiano Passuelo Hessel

2010-01-01

61

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.

62

COURSE OUTLINE ENV 399 Special Topic: ENVIRONMENTAL LAW  

E-print Network

to environmental issues (e.g., constitutional law, criminal law, international law, municipal law, Aboriginal, executive, and judicial branches of government; types of law (e.g., civil law, common law, criminal lawCOURSE OUTLINE ENV 399 Special Topic: ENVIRONMENTAL LAW Instructor: Dr. David R. Boyd Prerequisites

63

ENVS 4000, Spring (Jan-Apr) 2008 Environmental Impacts  

E-print Network

in assignments and resources: envs4000@gmail.com This capstone Environmental Science course includes lectures, readings, presentations and discussions related to environmental impacts of natural phenomena, which in some cases are in part a result of human activity. Topics include environmental impacts of development

Johnson, Dan L.

64

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

65

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

66

European Commission DG ENV Soil biodiversity: functions, threats and  

E-print Network

for these ecosystem services depend on soil, and soil biodiversity is the driving force behind their regulation the state of knowledge of soil biodiversity, its functions, its contribution to ecosystem services and itsEuropean Commission DG ENV Soil biodiversity: functions, threats and tools for policy makers

Paris-Sud XI, Université de

67

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

68

UV-inactivated vaccinia virus (VV) in a multi-envelope DNA-VV-protein (DVP) HIV-1 vaccine protects macaques from lethal challenge with heterologous SHIV  

PubMed Central

The pandemic of HIV-1 has continued for decades, yet there remains no licensed vaccine. Previous research has demonstrated the effectiveness of a multi-envelope, multi-vectored HIV-1 vaccine in a macaque-SHIV model, illustrating a potential means of combating HIV-1. Specifically, recombinant DNA, vaccinia virus (VV) and purified protein (DVP) delivery systems were used to vaccinate animals with dozens of antigenically-distinct HIV-1 envelopes for induction of immune breadth. The vaccinated animals controlled disease following challenge with a heterologous SHIV. This demonstration suggested that the antigenic cocktail vaccine strategy, which has succeeded in several other vaccine fields (e.g. pneumococcus), might also succeed against HIV-1. The strategy remains untested in an advanced clinical study, in part due to safety concerns associated with the use of replication-competent VV. To address this concern, we designed a macaque study in which psoralen/ultraviolet light-inactivated VV (UV VV) was substituted for replication-competent VV in the multi-envelope DVP protocol. Control animals received a vaccine encompassing no VV, or no vaccine. All VV vaccinated animals generated an immune response toward VV, and all vaccinated animals generated an immune response toward HIV-1 envelope. After challenge with heterologous SHIV 89.6P, animals that received replication-competent VV or UV VV experienced similar outcomes. They exhibited reduced peak viral loads, maintenance of CD4+ T cell counts and improved survival compared to control animals that received no VV or no vaccine; there were 0/15 deaths among all animals that received VV and 5/9 deaths among controls. Results define a practical means of improving VV safety, and encourage advancement of a promising multi-envelope DVP HIV-1 vaccine candidate. PMID:22425790

Jones, Bart G; Sealy, Robert E; Zhan, Xiaoyan; Freiden, Pamela J; Surman, Sherri L; Blanchard, James L.; Hurwitz, Julia L

2012-01-01

69

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

70

RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release.  

PubMed

The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag protein expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. PMID:24971705

López, Claudia S; Sloan, Rachel; Cylinder, Isabel; Kozak, Susan L; Kabat, David; Barklis, Eric

2014-08-01

71

The Secretion of IL-22 from Mucosal NKp44+ NK Cells Is Associated with Microbial Translocation and Virus Infection in SIV/SHIV-Infected Chinese Macaques  

PubMed Central

Microbial translocation (MT) causes systemic immune activation in chronic human immunodeficiency virus (HIV) infection. The role of a novel subtype of innate lymphoid cells, the NKp44+ NK cells, in HIV/simian immunodeficiency virus- (SIV-) induced MT remains unknown. In this study, 12 simian-human immunodeficiency virus- (SHIV-) infected macaques were chosen and split into two groups based on the MT level. Blood and Peripheral lymphoid tissue were sampled for flow cytometric analysis, viral load detection, and interleukin testing. Then, six naive Chinese macaques were used to determine the dynamics of cytokine secretion from mucosal NKp44+ NK cells in different phases of SIV infection. As a result, the degranulation capacity and IL-22 production of mucosal NKp44+ NK cells were associated with the MT level in the SHIV-infected macaques. And the number of mucosal NKp44+ NK cells and IL-22 secretion by these cells were lower in the chronic phase than in the early acute phase of SIV infection. The number of mucosal NKp44+ NK cells and interleukin-22 (IL-22) secretion by these cells increased before MT occurred. Therefore, we conclude that a decline in IL-22 production from mucosal NKp44+ NK cells induced by virus infection may be one of the causes of microbial translocation in HIV/SIV infection.

Wu, Fangxin; Cong, Zhe; Liu, Kejian; Qin, Chuan

2014-01-01

72

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

73

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

74

P20-08. Glycosylation: an important factor in Env diversity  

E-print Network

) deficient and fusion (F) region deleted oligomers were expressed either as recombinant vaccinia virus produced proteins or as recombinant pro- teins following transfection of 293T epithelial cells. Env gp140CF proteins were denatured, reduced, and alkylated.... To characterize the glycan antigenicity and heter- ogeneity of expressed recombinant envelopes, we have analyzed glycosylation profiles on clade B and clade C Env gp140 oligomers, and characterized Env glycosylation diversity. Methods Six gp140 cleavage (C...

Desaire, Heather; Haynes, Barton F.; Go, Eden P.; Liao, Hua-Xin; Sutherland, Laura L.; Chang, Qing; Zhang, Ying; Irungu, Janet W.; Alam, S. Munir

2009-10-22

75

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

76

An Efficiently Cleaved HIV-1 Clade C Env Selectively Binds to Neutralizing Antibodies  

PubMed Central

An ideal HIV-1 Env immunogen is expected to mimic the native trimeric conformation for inducing broadly neutralizing antibody responses. The native conformation is dependent on efficient cleavage of HIV-1 Env. The clade B isolate, JRFL Env is efficiently cleaved when expressed on the cell surface. Here, for the first time, we report the identification of a native clade C Env, 4-2.J41 that is naturally and efficiently cleaved on the cell surface as confirmed by its biochemical and antigenic characteristics. In addition to binding to several conformation-dependent neutralizing antibodies, 4-2.J41 Env binds efficiently to the cleavage-dependent antibody PGT151; thus validating its native cleaved conformation. In contrast, 4-2.J41 Env occludes non-neutralizing epitopes. The cytoplasmic-tail of 4-2.J41 Env plays an important role in maintaining its conformation. Furthermore, codon optimization of 4-2.J41 Env sequence significantly increases its expression while retaining its native conformation. Since clade C of HIV-1 is the prevalent subtype, identification and characterization of this efficiently cleaved Env would provide a platform for rational immunogen design. PMID:25822521

Bansal, Manish; Shukla, Brihaspati N.; Patil, Shilpa; Shrivastava, Tripti; Samal, Sweety; Goswami, Sandeep; King, C. Richter; Bhattacharya, Jayanta; Chakrabarti, Bimal K.

2015-01-01

77

Expression of Human Endogenous Retrovirus env Genes in the Blood of Breast Cancer Patients  

PubMed Central

Human endogenous retroviruses (HERV) env proteins have been recently reported to be significantly up-regulated in certain cancers. Specifically, mRNA and protein levels of HERV-K (HML-2) are up-regulated in the blood plasma or serum of breast cancer patients. Here, we collected blood samples of 49 breast cancer patients and analyzed mRNA expressions of various HERVs env genes including HERV-R, HERV-H, HERV-K, and HERV-P by real-time PCR. The expression of env genes were significantly increased in the blood of primary breast cancer patients but were decreased in patients undergoing chemotherapy to a similar level with benign patients. When we compared the group currently undergoing chemotherapy and those patients undergoing chemotherapy simultaneously with radiotherapy, HERVs env genes were reduced more in the chemotherapy only group, suggesting that chemotherapy is more effective in reducing HERV env gene expression than is radiotherapy. Among chemotherapy groups, HERV env gene expression was the lowest in the taxotere- or taxol-treated group, suggesting that taxotere and taxol can reduce HERVs env expression. These data suggest the potential to use HERVs env genes as a diagnosis marker for primary breast cancer, and further studies are needed to identify the mechanism and physiological significance of the reduction of HERV env gene expression during chemotherapy. PMID:24964007

Rhyu, Dong-Won; Kang, Yun-Jeong; Ock, Mee-Sun; Eo, Jung-Woo; Choi, Yung-Hyun; Kim, Wun-Jae; Leem, Sun-Hee; Yi, Joo-Mi; Kim, Heui-Soo; Cha, Hee-Jae

2014-01-01

78

Expression of human endogenous retrovirus env genes in the blood of breast cancer patients.  

PubMed

Human endogenous retroviruses (HERV) env proteins have been recently reported to be significantly up-regulated in certain cancers. Specifically, mRNA and protein levels of HERV-K (HML-2) are up-regulated in the blood plasma or serum of breast cancer patients. Here, we collected blood samples of 49 breast cancer patients and analyzed mRNA expressions of various HERVs env genes including HERV-R, HERV-H, HERV-K, and HERV-P by real-time PCR. The expression of env genes were significantly increased in the blood of primary breast cancer patients but were decreased in patients undergoing chemotherapy to a similar level with benign patients. When we compared the group currently undergoing chemotherapy and those patients undergoing chemotherapy simultaneously with radiotherapy, HERVs env genes were reduced more in the chemotherapy only group, suggesting that chemotherapy is more effective in reducing HERV env gene expression than is radiotherapy. Among chemotherapy groups, HERV env gene expression was the lowest in the taxotere- or taxol-treated group, suggesting that taxotere and taxol can reduce HERVs env expression. These data suggest the potential to use HERVs env genes as a diagnosis marker for primary breast cancer, and further studies are needed to identify the mechanism and physiological significance of the reduction of HERV env gene expression during chemotherapy. PMID:24964007

Rhyu, Dong-Won; Kang, Yun-Jeong; Ock, Mee-Sun; Eo, Jung-Woo; Choi, Yung-Hyun; Kim, Wun-Jae; Leem, Sun-Hee; Yi, Joo-Mi; Kim, Heui-Soo; Cha, Hee-Jae

2014-01-01

79

PALATAL DYSMORPHOGENESIS: QUANTITATIVE RT-PCR  

EPA Science Inventory

ABSTRACT Palatal Dysmorphogenesis : Quantitative RT-PCR Gary A. Held and Barbara D. Abbott Reverse transcription PCR (RT-PCR) is a very sensitive method for detecting mRNA in tissue samples. However, as it is usually performed it is does not yield quantitativ...

80

Rayon (en Rt) Masse (en Mt)  

E-print Network

Nom Jour Année Rayon (en Rt) Masse (en Mt) Distance au Soleil (en UA) Distance au Soleil (en av) Échelle : 1mm représente 60 000 km, soit environ dix rayons terrestres av = année-voiture = distance parcourue en un an par une voiture roulant à 120 km/h #12;Soleil Rotation 25-36j Rayon (en Rt) 109 Masse (en

Liège, Université de

81

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.hoffman@math.tamu.edu Professor Texas A&M University Department of Mathematics SMaRT Camp 2013 SUMMER MATHEMATICS RESEARCH

Boas, Harold P.

82

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: National Science Foundation TAMU Department of Mathematics Camp Date: June 10 ­ June 23, 2012 Application

83

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

84

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

85

Role of the Cytoplasmic Tail of Ecotropic Moloney Murine Leukemia Virus Env Protein in Fusion Pore Formation  

Microsoft Academic Search

Fusion between cells expressing envelope protein (Env) of Moloney murine leukemia virus and target cells were studied by use of video fluorescence microscopy and electrical capacitance measurements. When the full-length 632-amino-acid residue Env was expressed, fusion did not occur at all for 3T3 cells as target and only somewhat for XC6 cells. Expression of Env 616*—a construct of Env with

GRIGORY B. MELIKYAN; RUBEN M. MARKOSYAN; SOFYA A. BRENER; YANINA ROZENBERG; FREDRIC S. COHEN

2000-01-01

86

Clustering and Mobility of HIV-1 Env at Viral Assembly Sites Predict Its Propensity To Induce Cell-Cell Fusion  

PubMed Central

HIV-1 Env mediates virus attachment to and fusion with target cell membranes, and yet, while Env is still situated at the plasma membrane of the producer cell and before its incorporation into newly formed particles, Env already interacts with the viral receptor CD4 on target cells, thus enabling the formation of transient cell contacts that facilitate the transmission of viral particles. During this first encounter with the receptor, Env must not induce membrane fusion, as this would prevent the producer cell and the target cell from separating upon virus transmission, but how Env's fusion activity is controlled remains unclear. To gain a better understanding of the Env regulation that precedes viral transmission, we examined the nanoscale organization of Env at the surface of producer cells. Utilizing superresolution microscopy (stochastic optical reconstruction microscopy [STORM]) and fluorescence recovery after photobleaching (FRAP), we quantitatively assessed the clustering and dynamics of Env upon its arrival at the plasma membrane. We found that Gag assembly induced the aggregation of small Env clusters into larger domains and that these domains were completely immobile. Truncation of the cytoplasmic tail (CT) of Env abrogated Gag's ability to induce Env clustering and restored Env mobility at assembly sites, both of which correlated with increased Env-induced fusion of infected and uninfected cells. Hence, while Env trapping by Gag secures Env incorporation into viral particles, Env clustering and its sequestration at assembly sites likely also leads to the repression of its fusion function, and thus, by preventing the formation of syncytia, Gag helps to secure efficient transfer of viral particles to target cells. PMID:23637402

Roy, Nathan H.; Chan, Jany; Lambelé, Marie

2013-01-01

87

Expression of the fusogenic HERV-FRD Env glycoprotein (syncytin 2) in human placenta is restricted to villous cytotrophoblastic cells.  

E-print Network

1 Expression of the fusogenic HERV-FRD Env glycoprotein (syncytin 2) in human placenta in the human placenta [4-6]. The products of these two genes are glycoproteins named HERV-W Env glycoprotein (syncytin 1) and HERV-FRD Env glycoprotein (syncytin 2). Syncytin 1 induces the formation of syncytia

Paris-Sud XI, Université de

88

Inhibition of avian leukosis virus subgroup J replication by miRNA targeted against env.  

PubMed

No effective vaccine has been developed against the subgroup J avian leukosis virus (ALV-J). The genetic diversity of ALV-J might be related to the env gene, therefore, we selected conserved sequences of the env gene and designed interference sequence. In this study, microRNAs (miRNAs) were designed and synthesized, corresponding to conserved regions of the env gene. These miRNAs were cloned into the linearized eukaryotic expression vector. The recombinant plasmids were transfected into DF-1 cells. After transfection, the cells were inoculated with ALV-J. In reporter assays, the transfection efficiency is 80% by indirect immunofluorescence (IFA). Expression of the virus envelope glycoprotein was measured by IFA and western blotting assays. The relative expression of env gene was determined using quantitative PCR. Our results show that the mi-env 231 and mi-env 1384 could effectively suppress the replication of ALV-J with an efficiency of 68.7-75.2%. These data suggest that the miRNAs targeting the env can inhibit replication of ALV-J efficiently. This finding provides evidence that miRNAs could be used as a potential tool against ALV infection. PMID:23546824

Wang, Wei; Zhang, Zai-Ping; Tian, Jin; Xiao, Zhi-Guang; Meng, Qing-Wen

2013-08-01

89

Rapid evolution of the env gene leader sequence in cats naturally infected with feline immunodeficiency virus  

PubMed Central

Analysing the evolution of feline immunodeficiency virus (FIV) at the intra-host level is important in order to address whether the diversity and composition of viral quasispecies affect disease progression. We examined the intra-host diversity and the evolutionary rates of the entire env and structural fragments of the env sequences obtained from sequential blood samples in 43 naturally infected domestic cats that displayed different clinical outcomes. We observed in the majority of cats that FIV env showed very low levels of intra-host diversity. We estimated that env evolved at a rate of 1.16×10?3 substitutions per site per year and demonstrated that recombinant sequences evolved faster than non-recombinant sequences. It was evident that the V3–V5 fragment of FIV env displayed higher evolutionary rates in healthy cats than in those with terminal illness. Our study provided the first evidence that the leader sequence of env, rather than the V3–V5 sequence, had the highest intra-host diversity and the highest evolutionary rate of all env fragments, consistent with this region being under a strong selective pressure for genetic variation. Overall, FIV env displayed relatively low intra-host diversity and evolved slowly in naturally infected cats. The maximum evolutionary rate was observed in the leader sequence of env. Although genetic stability is not necessarily a prerequisite for clinical stability, the higher genetic stability of FIV compared with human immunodeficiency virus might explain why many naturally infected cats do not progress rapidly to AIDS. PMID:25535323

Hughes, Joseph; Biek, Roman; Litster, Annette; Willett, Brian J.; Hosie, Margaret J.

2015-01-01

90

Rapid evolution of the env gene leader sequence in cats naturally infected with feline immunodeficiency virus.  

PubMed

Analysing the evolution of feline immunodeficiency virus (FIV) at the intra-host level is important in order to address whether the diversity and composition of viral quasispecies affect disease progression. We examined the intra-host diversity and the evolutionary rates of the entire env and structural fragments of the env sequences obtained from sequential blood samples in 43 naturally infected domestic cats that displayed different clinical outcomes. We observed in the majority of cats that FIV env showed very low levels of intra-host diversity. We estimated that env evolved at a rate of 1.16×10(-3) substitutions per site per year and demonstrated that recombinant sequences evolved faster than non-recombinant sequences. It was evident that the V3-V5 fragment of FIV env displayed higher evolutionary rates in healthy cats than in those with terminal illness. Our study provided the first evidence that the leader sequence of env, rather than the V3-V5 sequence, had the highest intra-host diversity and the highest evolutionary rate of all env fragments, consistent with this region being under a strong selective pressure for genetic variation. Overall, FIV env displayed relatively low intra-host diversity and evolved slowly in naturally infected cats. The maximum evolutionary rate was observed in the leader sequence of env. Although genetic stability is not necessarily a prerequisite for clinical stability, the higher genetic stability of FIV compared with human immunodeficiency virus might explain why many naturally infected cats do not progress rapidly to AIDS. PMID:25535323

B?czkowski, Pawe? M; Hughes, Joseph; Biek, Roman; Litster, Annette; Willett, Brian J; Hosie, Margaret J

2015-04-01

91

ALV-J GP37 Molecular Analysis Reveals Novel Virus-Adapted Sites and Three Tyrosine-Based Env Species  

PubMed Central

Compared to other avian leukosis viruses (ALV), ALV-J primarily induces myeloid leukemia and hemangioma and causes significant economic loss for the poultry industry. The ALV-J Env protein is hypothesized to be related to its unique pathogenesis. However, the molecular determinants of Env for ALV-J pathogenesis are unclear. In this study, we compared and analyzed GP37 of ALV-J Env and the EAV-HP sequence, which has high homology to that of ALV-J Env. Phylogenetic analysis revealed five groups of ALV-J GP37 and two novel ALV-J Envs with endemic GP85 and EAV-HP-like GP37. Furthermore, at least 15 virus-adapted mutations were detected in GP37 compared to the EAV-HP sequence. Further analysis demonstrated that three tyrosine-based motifs (YxxM, ITIM (immune tyrosine-based inhibitory motif) and ITAM-like (immune tyrosine-based active motif like)) associated with immune disease and oncogenesis were found in the cytoplasmic tail of GP37. Based on the potential function and distribution of these motifs in GP37, ALV-J Env was grouped into three species, inhibitory Env, bifunctional Env and active Env. Accordingly, 36.91%, 61.74% and 1.34% of ALV-J Env sequences from GenBank are classified as inhibitory, bifunctional and active Env, respectively. Additionally, the Env of the ALV-J prototype strain, HPRS-103, and 17 of 18 EAV-HP sequences belong to the inhibitory Env. And models for signal transduction of the three ALV-J Env species were predicted. Our findings and models provide novel insights for identifying the roles and molecular mechanism of ALV-J Env in the unique pathogenesis of ALV-J. PMID:25849207

Shang, Jianjun; Tian, Xiaoyan; Yang, Jialiang; Chen, Hongjun; Shao, Hongxia; Qin, Aijian

2015-01-01

92

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

93

Protease gene structure and env gene variability of the AIDS virus.  

PubMed

The protease gene structure and the env gene variability have been precisely compared between the AIDS virus and members of the HTLV/BLV family. The conserved amino acid sequence (LVDT) which is repeated in the proteases of the HTLV/BLV family is not repeated in AIDS virus. Comparative analysis of the env gene sequences reveals the striking fact that the env gene of AIDS virus is 8-12-times more variable than those of the HTLV/BLV family. Within the AIDS virus env gene, the surface glycoprotein region is more liable to vary than is the transmembrane region; unexpectedly, however, this liability is not a characteristic feature of the AIDS virus because it is more prominent in other retroviruses including members of the HTLV/BLV family. PMID:3009215

Yasunaga, T; Sagata, N; Ikawa, Y

1986-04-21

94

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

95

Use of RT-Defective HIV Virions: New Tool to Evaluate Specific Response in Chronic Asymptomatic HIV-Infected Individuals  

PubMed Central

Background Generation of new reagents that can be used to screen or monitor HIV-1-specific responses constituted an interesting field in the development of HIV vaccines to improve their efficacy. Methods We have evaluated the specific T cell response against different types of NL4-3 virions (including NL4-3 aldrithiol-2 treated, NL4-3/?RT and R5 envelopes: NL4-3/?RT/?Env[AC10] and NL4-3/?RT/?Env[Bal]) and against pools of overlapping peptides (15 mer) encompassing the HIV-1 Gag and Nef regions. Cryopreserved PBMC from a subset of 69 chronic asymptomatic HIV positive individuals have been employed using different techniques including IFN-? ELISPOT assay, surface activation markers and intracellular cytokine staining (ICS) by flow cytometry. Results The differential response obtained against NL4-3 aldrithiol-2 treated and NL4-3/?RT virions (25% vs 55%, respectively) allow us to divide the population in three groups: “full-responders” (positive response against both viral particles), “partial-responders” (positive response only against NL4-3/?RT virions) and “non-responders” (negative responses). There was no difference between X4 and R5 envelopes. The magnitude of the total responses was higher against NL4-3/?RT and was positively correlated with gender and inverse correlated with viral load. On the contrary CD4+ T cell count was not associated with this response. In any case responses to the viruses tended to be lower in magnitude than those detected by the overlapping peptides tested. Finally we have found an increased frequency of HLA-B27 allele (23% vs 9%) and a significant reduction in some activation markers (CD69 and CD38) on T cells surface in responders vs non-responders individuals. Conclusions In summary these virions could be considered as alternative and useful reagents for screening HIV-1-specific T cell responses in HIV exposed uninfected people, HIV infected patients and to assess immunogenicity of new prototypes both in vitro and in vaccine trials, by a feasible, simply, effective and low cost assay. PMID:23516578

Arberas, Hodei; García-Pérez, Javier; García, Felipe; Bargalló, Manuel Enric; Maleno, María José; Gatell, José María; Mothe, Beatriz; Alcami, José; Sánchez-Palomino, Sonsoles; Plana, Montserrat

2013-01-01

96

Purification of MBP-EnvZ fusion proteins using an automated system.  

PubMed

Bacteria use two-component signal transduction systems to detect and respond to environmental changes. These systems have been studied systematically in Escherichia coli as a model organism. Most of the signal transduction systems present in E. coli are conserved in related pathogenic bacteria; however, differences in regulation by these systems have been reported from one bacterial species to another [Oropeza, R., and Calva, E. (2009). The cysteine 354 and 277 residues of Salmonella enterica serovar Typhi EnvZ are determinants of autophosphorylation and OmpR phosphorylation. FEMS Microbiol. Lett.292, 282-290]. Our laboratory has been interested in studying the OmpR/EnvZ two-component system in S. enterica. In S. enterica serovar Typhi (Typhi), it regulates the expression of the porin genes, namely ompC, ompF, ompS1, and ompS2. OmpR proteins are identical between E. coli and Typhi, but several differences exist between the EnvZ proteins. To define whether some differences in porin regulation are due to changes on EnvZ, we decided to overexpress and purify E. coli, Typhi, and S. enterica serovar Typhimurium (Typhimurium) EnvZ proteins fused to the maltose-binding protein (MBP) as a purification tag. Differences in the autophosphorylation level of these proteins were evidenced. Hence, considering the differences at the amino acid level between E. coli and Typhi EnvZ proteins, several mutations were introduced in the Typhi EnvZ protein in order to try to find the amino acids affecting the enzymatic activity of the protein. We found that Cys354 plays an important role in defining the enzymatic activity of this histidine kinase. Here, we report the automated purification of a collection of MBP-EnvZ fusions using a mini-chromatography commercial system, but adapting an amylose affinity column packed by ourselves. PMID:20946843

Oropeza, Ricardo; Calva, Edmundo

2010-01-01

97

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

98

Testing Operating Systems with RT-Tester  

E-print Network

for operating systems RT-Tester test automation system Example: ARINC 653 operating system test in Linux environment can exercise the most general behaviour at the SUT-OS API which is possible for "real" applications may possess pre-programmed scenarios for robustness testing and time-critical API call sequences 5

Peleska, Jan - Fachbereich 3

99

Science Fiction Atmospheres R.T. Pierrehumbert  

E-print Network

Science Fiction Atmospheres R.T. Pierrehumbert The University of Chicago October 11, 2005 One of the first science fiction books I can remember, apart from early encounters with Mr. Bass and his mushroom ­ the obligatory part of the monthly shipment from a sci- ence fiction book club ­ and swiftly made its way up

Pierrehumbert, Raymond

100

RESORT - REMOTE SERVICE PROVISION FOR RT SYSTEMS  

Microsoft Academic Search

This paper describes the ongoing RESORT project which is developing a state of the art prototype system for remote service provision for RT (rehabilitation technology) products. RESORT stands for Remote Service of Rehabilitation Technology and is an EU funded R&D project in the TAP sector Disabled and Elderly (DE-4208). 1. Background

Paul Panek; Wolfgang L. Zagler; Christian Beck; Gottfried Seisenbacher; Theodor Isporidi; Andreas Hochgatterer; Alexander Seebacher; Nick Hine; Paul Sergeant; Ruud de Vries; Jos van Well

2000-01-01

101

Protection of rhesus macaques against disease progression from pathogenic SHIV-89.6PD by vaccination with phage-displayed HIV-1 epitopes.  

PubMed

The antigenic polymorphism of HIV-1 is a major obstacle in developing an effective vaccine. Accordingly, we screened random peptide libraries (RPLs) displayed on phage with antibodies from HIV-infected individuals and identified an array of HIV-specific epitopes that behave as antigenic mimics of conformational epitopes of gp120 and gp41 proteins. We report that the selected epitopes are shared by a collection of HIV-1 isolates of clades A-F. The phage-borne epitopes are immunogenic in rhesus macaques, where they elicit envelope-specific antibody responses. Upon intravenous challenge with 60 MID50 of pathogenic SHIV-89.6PD, all monkeys became infected; however, in contrast to the naive and mock-immunized monkeys, four of five mimotope-immunized monkeys experienced lower levels of peak viremia, followed by viral set points of undetectable or transient levels of viremia and a mild decline of CD4+ T cells, and were protected from progression to AIDS-like illness. These results provide a new approach to the design of broadly protective HIV-1 vaccines. PMID:11689887

Chen, X; Scala, G; Quinto, I; Liu, W; Chun, T W; Justement, J S; Cohen, O J; vanCott, T C; Iwanicki, M; Lewis, M G; Greenhouse, J; Barry, T; Venzon, D; Fauci, A S

2001-11-01

102

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

103

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

104

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

PubMed Central

The extensive carbohydrate coat, the variability of protein structural features on HIV-1 envelope glycoproteins (Env), and the steric constraints of the virus-cell interface during infection, present challenges to the elicitation of effective full-length (?150 kDa), neutralizing antibodies against HIV. These hurdles have motivated the engineering of smaller antibody derivatives that can bind Env and neutralize the virus. To further understand the mechanisms by which these proteins neutralize HIV-1, we carried out cryoelectron tomography of native HIV-1 BaL virions complexed separately to two small (?15 kDa) HIV-neutralizing proteins: A12, which binds the CD4-binding site on Env, and m36, whose binding to Env is enhanced by CD4 binding. We show that despite their small size, the presence of these proteins and their effects on the quaternary conformation of trimeric Env can be visualized in molecular structures derived by cryoelectron tomography combined with subvolume averaging. Binding of Env to A12 results in a conformational change that is comparable to changes observed upon its binding to the CD4-binding site antibody, b12. In contrast, binding of Env to m36 results in an “open” quaternary conformation similar to that seen with binding of soluble CD4 or the CD4i antibody, 17b. Because these small neutralizing proteins are less sterically hindered than full-length antibodies at zones of virus-cell contact, the finding that their binding has the same structural consequences as that of other broadly neutralizing antibodies highlights their potential for use in therapeutic applications. PMID:23267106

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

2013-01-01

105

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

106

Maturation cleavage of the murine leukemia virus Env precursor separates the transmembrane subunits to prime it for receptor triggering  

PubMed Central

The Env protein of murine leukemia virus matures by two cleavage events. First, cellular furin separates the receptor binding surface (SU) subunit from the fusion-active transmembrane (TM) subunit and then, in the newly assembled particle, the viral protease removes a 16-residue peptide, the R-peptide from the endodomain of the TM. Both cleavage events are required to prime the Env for receptor-triggered activation. Cryoelectron microscopy (cryo-EM) analyses have shown that the mature Env forms an open cage-like structure composed of three SU–TM complexes, where the TM subunits formed separated Env legs. Here we have studied the structure of the R-peptide precursor Env by cryo-EM. TM cleavage in Moloney murine leukemia virus was inhibited by amprenavir, and the Envs were solubilized in Triton X-100 and isolated by sedimentation in a sucrose gradient. We found that the legs of the R-peptide Env were held together by trimeric interactions at the very bottom of the Env. This suggested that the R-peptide ties the TM legs together and that this prevents the activation of the TM for fusion. The model was supported by further cryo-EM studies using an R-peptide Env mutant that was fusion-competent despite an uncleaved R-peptide. The Env legs of this mutant were found to be separated, like in the mature Env. This shows that it is the TM leg separation, normally caused by R-peptide cleavage, that primes the Env for receptor triggering. PMID:22547812

Löving, Robin; Wu, Shang-Rung; Sjöberg, Mathilda; Lindqvist, Birgitta; Garoff, Henrik

2012-01-01

107

Intracellular trafficking of Gag and Env proteins and their interactions modulate pseudotyping of retroviruses.  

PubMed

Glycoproteins derived from most retroviruses and from several families of enveloped viruses can form infectious pseudotypes with murine leukemia virus (MLV) and lentiviral core particles, like the MLV envelope glycoproteins (Env) that are incorporated on either virus type. However, coexpression of a given glycoprotein with heterologous core proteins does not always give rise to highly infectious viral particles, and restrictions on pseudotype formation have been reported. To understand the mechanisms that control the recruitment of viral surface glycoproteins on lentiviral and retroviral cores, we exploited the fact that the feline endogenous retrovirus RD114 glycoprotein does not efficiently pseudotype lentiviral cores derived from simian immunodeficiency virus, whereas it is readily incorporated onto MLV particles. Our results indicate that recruitment of glycoproteins by the MLV and lentiviral core proteins occurs in intracellular compartments and not at the cell surface. We found that Env and core protein colocalization in intracytoplasmic vesicles is required for pseudotype formation. By investigating MLV/RD114 Env chimeras, we show that signals in the cytoplasmic tail of either glycoprotein differentially influenced their intracellular localization; that of MLV allows endosomal localization and hence recruitment by both lentiviral and MLV cores. Furthermore, we found that upon membrane binding, MLV core proteins could relocalize Env glycoproteins in late endosomes and allow their incorporation on viral particles. Thus, intracellular colocalization, as well as interactions between Env and core proteins, may influence the recruitment of the glycoprotein onto viral particles and generate infectious pseudotyped viruses. PMID:15194792

Sandrin, Virginie; Muriaux, Delphine; Darlix, Jean-Luc; Cosset, François-Loïc

2004-07-01

108

Robot Vision for RT-Middleware Framework  

Microsoft Academic Search

In conventional robot system development the different robot parts (sensors, processing elements, actuators) are combined together in a compact, self contained system. The need for faster development, system reconfigurability and flexibility required the introduction of middleware frameworks for robot systems. One promising middleware framework is the RT-middleware technology (and its implementation named OpenRTM-aist), which is getting more and more popular

G. Sziebig; A. Gaudia; P. Korondi; N. Ando; B. Solvang

2007-01-01

109

Human foamy virus polypeptides: identification of env and bel gene products.  

PubMed Central

Human foamy virus (HFV) proteins were identified in human cells cultured in vitro by immunoprecipitation and immunoblotting with specific antisera. Among several viral polypeptides, four glycoproteins of approximately 160, 130, 70, and 48 kDa were identified in HFV-infected cells. gp130 was shown to represent the intracellular env precursor, and gp70 and gp48 were shown to represent the external and transmembrane env proteins, respectively. The nature of gp160, which shares sequences with the env, bel1, and bel2 proteins, is not yet resolved. In addition, a p62 identified with bel1- and bel2-specific antisera likely corresponds to the bet gene product. Images PMID:8388512

Giron, M L; Rozain, F; Debons-Guillemin, M C; Canivet, M; Peries, J; Emanoil-Ravier, R

1993-01-01

110

Cryo-EM structure of a fully glycosylated soluble cleaved HIV-1 Env trimer  

PubMed Central

The HIV-1 envelope glycoprotein (Env) trimer contains the receptor binding sites and membrane fusion machinery that introduce the viral genome into the host cell. As the only target for broadly neutralizing antibodies (bnAbs), Env is a focus for rational vaccine design. We present a cryo-electron microscopy reconstruction and structural model of a cleaved, soluble SOSIP gp140 trimer in complex with a CD4 binding site (CD4bs) bnAb, PGV04, at 5.8 Å resolution. The structure reveals the spatial arrangement of Env components, including the V1/V2, V3, HR1 and HR2 domains, and shielding glycans. The structure also provides insights into trimer assembly, gp120-gp41 interactions, and the CD4bs epitope cluster for bnAbs, which covers a more extensive area and defines a more complex site of vulnerability than previously described. PMID:24179160

Lyumkis, Dmitry; Julien, Jean-Philippe; de Val, Natalia; Cupo, Albert; Potter, Clinton S.; Klasse, Per Johan; Burton, Dennis R.; Sanders, Rogier W.; Moore, John P.; Carragher, Bridget; Wilson, Ian A.; Ward, Andrew B.

2014-01-01

111

Long-term central and effector SHIV-specific memory T cell responses elicited after a single immunization with a novel lentivector DNA vaccine.  

PubMed

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

112

Rectal Application of a Highly Osmolar Personal Lubricant in a Macaque Model Induces Acute Cytotoxicity but Does Not Increase Risk of SHIV Infection  

PubMed Central

Background Personal lubricant use is common during anal intercourse. Some water-based products with high osmolality and low pH can damage genital and rectal tissues, and the polymer polyquaternium 15 (PQ15) can enhance HIV replication in vitro. This has raised concerns that lubricants with such properties may increase STD/HIV infection risk, although in vivo evidence is scarce. We use a macaque model to evaluate rectal cytotoxicity and SHIV infection risk after use of a highly osmolar (>8,000 mOsm/kg) water-based lubricant with pH of 4.4, and containing PQ15. Methods Cytotoxicity was documented by measuring inflammatory cytokines and epithelial tissue sloughing during six weeks of repeated, non-traumatic lubricant or control buffer applications to rectum and anus. We measured susceptibility to SHIVSF162P3 infection by comparing virus doses needed for rectal infection in twenty-one macaques treated with lubricant or control buffer 30 minutes prior to virus exposure. Results Lubricant increased pro-inflammatory cytokines and tissue sloughing while control buffer (phosphate buffered saline; PBS) did not. However, the estimated AID50 (50% animal infectious dose) was not different in lubricant- and control buffer-treated macaques (p = 0.4467; logistic regression models). Conclusions Although the test lubricant caused acute cytotoxicity in rectal tissues, it did not increase susceptibility to infection in this macaque model. Thus neither the lubricant-induced type/extent of inflammation nor the presence of PQ15 affected infection risk. This study constitutes a first step in the in vivo evaluation of lubricants with regards to HIV transmission. PMID:25853710

Vishwanathan, Sundaram A.; Morris, Monica R.; Wolitski, Richard J.; Luo, Wei; Rose, Charles E.; Blau, Dianna M.; Tsegaye, Theodros; Zaki, Sherif R.; Garber, David A.; Jenkins, Leecresia T.; Henning, Tara C.; Patton, Dorothy L.; Hendry, R. Michael; McNicholl, Janet M.; Kersh, Ellen N.

2015-01-01

113

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

114

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

115

Virion instability of human immunodeficiency virus type 1 reverse transcriptase (RT) mutated in the protease cleavage site between RT p51 and the RT RNase H domain.  

PubMed

Each of the human immunodeficiency virus type 1 (HIV-1) pol-encoded enzymes, protease (PR), reverse transcriptase (RT), and integrase (IN), is active only as a dimer (or higher-order oligomer in the case of IN), but only RT comprises subunits of different masses. RT is a heterodimer of 66-kDa and 51-kDa subunits. The latter is formed by HIV PR-catalyzed cleavage of p66 during virion maturation, resulting in the removal of the RNase H (RNH) domain of a p66 subunit. In order to study the apparent need for RT heterodimers in the context of the virion, we introduced a variety of mutations in the RT p51-RNH protease cleavage site of an infectious HIV-1 molecular clone. Surprisingly, rather than leading to virions with increased RT p66 content, most of the mutations resulted in significantly attenuated virus that contained greatly decreased levels of RT that in many cases was primarily p51 RT. IN levels were also reduced in several mutants. However, most mutants showed normal levels of the Pr160(gag-pol) precursor polyprotein, suggesting that reduced virion RT arose from proteolytic instability rather than decreased incorporation. Mutant virion p24 Gag levels were equivalent to wild type, indicating that Gag incorporation and processing were not affected. Repeated passage of MT-2 cells exposed to mutant viruses led to the appearance of virus with improved replication capacity; these virions contained normally processed RT at near-wild-type levels. These results imply that additional proteolytic processing of RT to the p66/p51 heterodimer is essential to provide proteolytic stability of RT during HIV-1 maturation. PMID:16140771

Abram, Michael E; Parniak, Michael A

2005-09-01

116

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

117

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 and understanding of various issues in conservation biology. Our objectives during laboratories will be develop

118

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

119

Env sequence determinants in CXCR4-using human immunodeficiency virus type-1 subtype C.  

PubMed

HIV-1 subtype C (HIV-1C) CXCR4-using virus is isolated infrequently and is poorly characterized. Understanding HIV-1C env characteristics has implications for the clinical use of antiretrovirals that target viral entry. A total of 209 env clones derived from 10 samples with mixed CCR5-(R5), CXCR4-using (X4) or dual-tropic HIV-1C were phenotyped for coreceptor usage. Intra-patient X4 and R5 variants generally formed distinct monophyletic phylogenetic clusters. X4 compared to R5 envs had significantly greater amino acid variability and insertions, higher net positive charge, fewer glycosylation sites and increased basic amino acid substitutions in the GPGQ crown. Basic amino acid substitution and/or insertion prior to the crown are highly sensitive characteristics for predicting X4 viruses. Chimeric env functional studies suggest that the V3 loop is necessary but often not sufficient to impart CXCR4 utilization. Our studies provide insights into the unique genotypic characteristics of X4 variants in HIV-1C. PMID:22954962

Lin, Nina H; Becerril, Carlos; Giguel, Francoise; Novitsky, Vladimir; Moyo, Sikhulile; Makhema, Joseph; Essex, Myron; Lockman, Shahin; Kuritzkes, Daniel R; Sagar, Manish

2012-11-25

120

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.

121

ENVS 474 -Planning Studio 2013 Urban Transition Studio (UTS) Planning Series  

E-print Network

Topic: State Street Corridor Urban Design Huxley College | Urban Planning and Sustainable Development 31 ENVS 474 - Planning Studio 2013 Urban Transition Studio (UTS) Planning Series 2013 UTS Studio, and national and international planning goals and/or principles. Design alternatives emphasize sustainability

Zaferatos, Nicholas C.

122

HIV1 encodes a sequence overlapping env gp41 with highly significant similarity to  

E-print Network

HIV­1 encodes a sequence overlapping env gp41 with highly significant similarity to selenium, paralleling the loss of CD4+ T cells, has been widely documented in HIV­1 infections. As recently reviewed (1 HIV patients, and children as well as adults [2, 3, 4, 5, 6, 7]. The observations of a number

Kececioglu, John

123

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 for landscape-scale renewable resource management in the United States. Environmental Science & Policy 4

Nowak, Robert S.

124

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

125

Furin cleavage of the Moloney murine leukemia virus Env precursor reorganizes the spike structure  

PubMed Central

The trimeric Moloney murine leukemia virus Env protein matures by two proteolytic cleavages. First, furin cleaves the Env precursor into the surface (SU) and transmembrane (TM) subunits in the cell and then the viral protease cleaves the R-peptide from TM in new virus. Here we analyzed the structure of the furin precursor, by cryoelectron microscopy. We transfected 293T cells with a furin cleavage site provirus mutant, R466G/K468G, and produced the virus in the presence of amprenavir to also inhibit the R-peptide cleavage. Although Env incorporation into particles was inhibited, enough precursor could be isolated and analyzed by cryoelectron microscopy to yield a 3D structure at 22 Å resolution. This showed an open cage-like structure like that of the R-peptide precursor and the mature Env described before. However, the middle protrusion of the protomeric unit, so prominently pointing out from the side of the more mature forms of the Env, was absent. Instead, there was extra density in the top protrusion. This suggested that the C-terminal SU domain was associated alongside the receptor binding N-terminal SU domain in the furin precursor. This was supported by mapping with a SU C-terminal domain-specific antigen binding fragment. We concluded that furin cleavage not only separates the subunits and liberates the fusion peptide at the end of TM but also allows the C-terminal domain to relocate into a peripheral position. This conformational change might explain how the C-terminal domain of SU gains the potential to undergo disulfide isomerization, an event that facilitates membrane fusion. PMID:24711391

Sjöberg, Mathilda; Wu, Shang-Rung; Löving, Robin; Rantalainen, Kimmo; Lindqvist, Birgitta; Garoff, Henrik

2014-01-01

126

Furin cleavage of the Moloney murine leukemia virus Env precursor reorganizes the spike structure.  

PubMed

The trimeric Moloney murine leukemia virus Env protein matures by two proteolytic cleavages. First, furin cleaves the Env precursor into the surface (SU) and transmembrane (TM) subunits in the cell and then the viral protease cleaves the R-peptide from TM in new virus. Here we analyzed the structure of the furin precursor, by cryoelectron microscopy. We transfected 293T cells with a furin cleavage site provirus mutant, R466G/K468G, and produced the virus in the presence of amprenavir to also inhibit the R-peptide cleavage. Although Env incorporation into particles was inhibited, enough precursor could be isolated and analyzed by cryoelectron microscopy to yield a 3D structure at 22 Å resolution. This showed an open cage-like structure like that of the R-peptide precursor and the mature Env described before. However, the middle protrusion of the protomeric unit, so prominently pointing out from the side of the more mature forms of the Env, was absent. Instead, there was extra density in the top protrusion. This suggested that the C-terminal SU domain was associated alongside the receptor binding N-terminal SU domain in the furin precursor. This was supported by mapping with a SU C-terminal domain-specific antigen binding fragment. We concluded that furin cleavage not only separates the subunits and liberates the fusion peptide at the end of TM but also allows the C-terminal domain to relocate into a peripheral position. This conformational change might explain how the C-terminal domain of SU gains the potential to undergo disulfide isomerization, an event that facilitates membrane fusion. PMID:24711391

Sjöberg, Mathilda; Wu, Shang-Rung; Löving, Robin; Rantalainen, Kimmo; Lindqvist, Birgitta; Garoff, Henrik

2014-04-22

127

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. Public organizations, health institutions, small, medium and large businesses, schools and CEGEPs that this project is completed in its entirety. #12;6 RectoR's RepoRt 2010-2011 Philosophy, Oncology, Music, Music

Charette, André

128

From RT Inhibitor to RT / IN Dual Inhibitor: A Rational Design Zhengqiang Wang and Robert Vince  

E-print Network

intervention Structural and binding information well established Assay Table 1. Anti-RT, anti-IN and anti-HIV Center for Drug Design Academic Health Center University of Minnesota Minneapolis MN 55455Center for Drug at least 3 drugs and 2 mechanisms of action Suppress viral replication Improve life quality Major Drawbacks

Thomas, David D.

129

ENVS 2xx: Introduction to Environmental Studies Introduction to Environmental Studies would be team-taught, with teaching responsibility rotating among  

E-print Network

ENVS 2xx: Introduction to Environmental Studies Introduction to Environmental Studies would be team a different team of instructors might use to teach the course. ' Template for ENVS 2xx The following 9 #12;ENVS 2xx Introduction to Environmental Studies (Murphy and Kelrick) Nature is only

Gering, Jon C.

130

The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm  

PubMed Central

Two-component systems mediate bacterial signal transduction, employing a membrane sensor kinase and a cytoplasmic response regulator (RR). Environmental sensing is typically coupled to gene regulation. Understanding how input stimuli activate kinase autophosphorylation remains obscure. The EnvZ/OmpR system regulates expression of outer membrane proteins in response to osmotic stress. To identify EnvZ conformational changes associated with osmosensing, we used HDXMS to probe the effects of osmolytes (NaCl, sucrose) on the cytoplasmic domain of EnvZ (EnvZc). Increasing osmolality decreased deuterium exchange localized to the four-helix bundle containing the autophosphorylation site (His243). EnvZc exists as an ensemble of multiple conformations and osmolytes favoured increased helicity. High osmolality increased autophosphorylation of His243, suggesting that these two events are linked. In-vivo analysis showed that the cytoplasmic domain of EnvZ was sufficient for osmosensing, transmembrane domains were not required. Our results challenge existing claims of robustness in EnvZ/OmpR and support a model where osmolytes promote intrahelical H-bonding enhancing helix stabilization, increasing autophosphorylation and downstream signalling. The model provides a conserved mechanism for signalling proteins that respond to diverse physical and mechanical stimuli. PMID:22543870

Wang, Loo Chien; Morgan, Leslie K; Godakumbura, Pahan; Kenney, Linda J; Anand, Ganesh S

2012-01-01

131

Turning of the receptor-binding domains opens up the murine leukaemia virus Env for membrane fusion.  

PubMed

The activity of the membrane fusion protein Env of Moloney mouse leukaemia virus is controlled by isomerization of the disulphide that couples its transmembrane (TM) and surface (SU) subunits. We have arrested Env activation at a stage prior to isomerization by alkylating the active thiol in SU and compared the structure of isomerization-arrested Env with that of native Env. Env trimers of respective form were isolated from solubilized particles by sedimentation and their structures were reconstructed from electron microscopic images of both vitrified and negatively stained samples. We found that the protomeric unit of both trimers formed three protrusions, a top, middle and a lower one. The atomic structure of the receptor-binding domain of SU fitted into the upper protrusion. This was formed similar to a bent finger. Significantly, in native Env the tips of the fingers were directed against each other enclosing a cavity below, whereas they had turned outward in isomerization-arrested Env transforming the cavity into an open well. This might subsequently guide the fusion peptides in extended TM subunits into the target membrane. PMID:18800055

Wu, Shang-Rung; Sjöberg, Mathilda; Wallin, Michael; Lindqvist, Birgitta; Ekström, Maria; Hebert, Hans; Koeck, Philip J B; Garoff, Henrik

2008-10-22

132

Cytotoxic T cells and neutralizing antibodies induced in rhesus monkeys by virus-like particle HIV vaccines in the absence of protection from SHIV infection.  

PubMed

HIV Pr55gag has in the absence of other viral components the capacity to self assemble in budding noninfectious virus-like particles (VLP). The immunological spectrum of the HIV-1IIIB gag-derived VLP was expanded either by stable anchoring of chimeric modified gp 120 on the surface of the VLP (type 1) or by replacing sequences of the Pr55gag precursor by the V3 loop and a linear portion of the CD4 binding domain (type 2). This noninfectious antigen delivery system was evaluated for immunogenicity and efficacy in rhesus macaques without adjuvants. Intramuscular immunization with both types of VLP induced high titers of gag-specific antibodies ranging from 1/8000 to 1/510,000 for type 1 VLP and from 1/4000 to 1/16,000 for type 2 VLP. Only animals immunized with type 1 VLP developed substantial endpoint titers of env-specific antibodies (1/2000-1/32,000) with a neutralizing capacity at serum dilutions of 1/32-1/128. Gag- and env-specific cytotoxic T lymphocyte (CTL) activity was induced by both types of VLP at similar levels. Four weeks after the last immunization animals were challenged intravenously with 20 MID50 of the cell free homologous envelope simian/HIV-1IIIB chimeric challenge stock Despite HIV-1-specific neutralizing and CTL responses, all vaccinated animals became infected. PMID:9614868

Wagner, R; Teeuwsen, V J; Deml, L; Notka, F; Haaksma, A G; Jhagjhoorsingh, S S; Niphuis, H; Wolf, H; Heeney, J L

1998-05-25

133

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

PubMed Central

Background Ten to 30% of HIV-1 infected subjects develop broadly neutralizing antibodies (bNAbs) during chronic infection. We hypothesized that immunizing rabbits with viral envelope glycoproteins (Envs) from these patients may induce bNAbs, when formulated as a trimeric protein and in the presence of an adjuvant. Methods Based on in vitro neutralizing activity in serum, patients with bNAbs were selected for cloning of their HIV-1 Env. Seven stable soluble trimeric gp140 proteins were generated from sequences derived from four adults and two children infected with either clade A or B HIV-1. From one of the clade A Envs both the monomeric and trimeric Env were produced for comparison. Rabbits were immunized with soluble gp120 or trimeric gp140 proteins in combination with the adjuvant dimethyl dioctadecyl ammonium/trehalose dibehenate (CAF01). Env binding in rabbit immune serum was determined using ELISAs based on gp120-IIIB protein. Neutralizing activity of IgG purified from rabbit immune sera was measured with the pseudovirus-TZMbl assay and a PBMC-based neutralization assay for selected experiments. Results It was initially established that gp140 trimers induce better antibody responses over gp120 monomers and that the adjuvant CAF01 was necessary for such strong responses. Gp140 trimers, based on HIV-1 variants from patients with bNAbs, were able to elicit both gp120IIIB specific IgG and NAbs to Tier 1 viruses of different subtypes. Potency of NAbs closely correlated with titers, and an gp120-binding IgG titer above a threshold of 100,000 was predictive of neutralization capability. Finally, peptide inhibition experiments showed that a large fraction of the neutralizing IgG was directed against the gp120 V3 region. Conclusions Our results indicate that the strategy of reverse immunology based on selected Env sequences is promising when immunogens are delivered as stabilized trimers in CAF01 adjuvant and that the rabbit is a valuable model for HIV vaccine studies. PMID:24023951

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

2013-01-01

134

Defense-in-depth by mucosally administered anti-HIV dimeric IgA2 and systemic IgG1 mAbs: Complete protection of rhesus monkeys from mucosal SHIV challenge.  

PubMed

Although IgA is the most abundantly produced immunoglobulin in humans, its role in preventing HIV-1 acquisition, which occurs mostly via mucosal routes, remains unclear. In our passive mucosal immunizations of rhesus macaques (RMs), the anti-HIV-1 neutralizing monoclonal antibody (nmAb) HGN194, given either as dimeric IgA1 (dIgA1) or dIgA2 intrarectally (i.r.), protected 83% or 17% of the RMs against i.r. simian-human immunodeficiency virus (SHIV) challenge, respectively. Data from the RV144 trial implied that vaccine-induced plasma IgA counteracted the protective effector mechanisms of IgG1 with the same epitope specificity. We thus hypothesized that mucosal dIgA2 might diminish the protection provided by IgG1 mAbs targeting the same epitope. To test our hypothesis, we administered HGN194 IgG1 intravenously (i.v.) either alone or combined with i.r. HGN194 dIgA2. We enrolled SHIV-exposed, persistently aviremic RMs protected by previously administered nmAbs; RM anti-human IgG responses were undetectable. However, low-level SIV Gag-specific proliferative T-cell responses were found. These animals resemble HIV-exposed, uninfected humans, in which local and systemic cellular immune responses have been observed. HGN194 IgG1 and dIgA2 used alone and the combination of the two neutralized the challenge virus equally well in vitro. All RMs given only i.v. HGN194 IgG1 became infected. In contrast, all RMs given HGN194 IgG1+dIgA2 were completely protected against high-dose i.r. SHIV-1157ipEL-p challenge. These data imply that combining suboptimal defenses at the mucosal and systemic levels can completely prevent virus acquisition. Consequently, active vaccination should focus on defense-in-depth, a strategy that seeks to build up defensive fall-back positions well behind the fortified frontline. PMID:25769884

Sholukh, Anton M; Watkins, Jennifer D; Vyas, Hemant K; Gupta, Sandeep; Lakhashe, Samir K; Thorat, Swati; Zhou, Mingkui; Hemashettar, Girish; Bachler, Barbara C; Forthal, Donald N; Villinger, Francois; Sattentau, Quentin J; Weiss, Robin A; Agatic, Gloria; Corti, Davide; Lanzavecchia, Antonio; Heeney, Jonathan L; Ruprecht, Ruth M

2015-04-21

135

Human Immunodeficiency Virus Env-Independent Infection of Human CD4? Cells  

PubMed Central

CD4? epithelial cells covering mucosal surfaces serve as the primary barrier to prevent human immunodeficiency virus type 1 (HIV-1) infection. We used HIV-1 vectors carrying the enhanced green fluorescent protein gene as a reporter gene to demonstrate that HIV-1 can infect some CD4? human epithelial cell lines with low but significant efficiencies. Importantly, HIV-1 infection of these cell lines is independent of HIV-1 envelope proteins. The Env-independent infection of CD4? cells by HIV-1 suggests an alternative pathway for HIV-1 transmission. Even on virions bearing Env, a neutralizing antibody directed against gp120 is incapable of neutralizing the infection of these cells, thus raising potential implications for HIV-1 vaccine development. PMID:11069994

Pang, Shen; Yu, Duan; An, Dong Sung; Baldwin, Gayle C.; Xie, Yiming; Poon, Betty; Chow, Yen-Hung; Park, No-Hee; Chen, Irvin S. Y.

2000-01-01

136

Disease progression and evolution of the HIV1 env gene in 24 infected infants  

Microsoft Academic Search

We have studied the relationship between disease progression and HIV-1 evolution in 24 infants classified as rapid or non-rapid progressors, during nearly the entire disease progression cycle from infection to AIDS. Specifically, we examined the temporal relationship between clinical status and changes in genetic diversity, divergence, selection and recombination at the C2V3C3 region of the env gene during a period

Antonio Carvajal-Rodríguez; David Posada; Marcos Pérez-Losada; Emily Keller; Elaine J. Abrams; Raphael P. Viscidi; Keith A. Crandall

2008-01-01

137

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

138

Protection against SHIV-KB9 Infection by Combining rDNA and rFPV Vaccines Based on HIV Multiepitope and p24 Protein in Chinese Rhesus Macaques  

PubMed Central

Developing an effective vaccine against HIV infection remains an urgent goal. We used a DNA prime/fowlpox virus boost regimen to immunize Chinese rhesus macaques. The animals were challenged intramuscularly with pathogenic molecularly cloned SHIV-KB9. Immunogenicity and protective efficacy of vaccines were investigated by measuring IFN-? levels, monitoring HIV-specific binding antibodies, examining viral load, and analyzing CD4/CD8 ratio. Results show that, upon challenge, the vaccine group can induce a strong immune response in the body, represented by increased expression of IFN-?, slow and steady elevated antibody production, reduced peak value of acute viral load, and increase in the average CD4/CD8 ratio. The current research suggests that rapid reaction speed, appropriate response strength, and long-lasting immune response time may be key protection factors for AIDS vaccine. The present study contributes significantly to AIDS vaccine and preclinical research. PMID:22474488

Li, Chang; Shen, Zhenwei; Li, Xiao; Bai, Jieying; Zeng, Lin; Tian, Mingyao; Song, Ying Jin; Ye, Ming; Du, Shouwen; Ren, Dayong; Liu, Cunxia; Zhu, Na; Sun, Dandan; Li, Yi; Jin, Ningyi

2012-01-01

139

AMS CHEN CBEN CHGN CVL EBGN ELEC ENV PHGN GEGN GPGN MECH MTGN MNGN PEGN Direct Methods  

E-print Network

X X X X X X X X X X Program Code Applied Math and AMS Chemical Engineering CHEN CBEN Chemistry and CHGN Civil Engineering CVL Economics and EBGN Electrical Engineering ELEC Environmental ENV Engineering

140

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

141

Construction and characterization of the recombinant Moloney murine leukemia viruses bearing the mouse Fv-4 env gene.  

PubMed Central

A nucleotide sequence of the mouse Fv-4 env gene was completed. Structural comparison revealed a close relationship of Fv-4 to the ecotropic Cas-Br-E murine leukemia virus isolated from a wild mouse in southern California. Various portions of the env gene of Moloney murine leukemia virus were replaced by the corresponding Fv-4 env sequence to construct recombinant murine leukemia virus clones. Infectivity of these recombinants was checked by the S+L- cell focus induction assay and the XC cell syncytium formation assay. Recombinants bearing the following Fv-4 env sequence retained ecotropic infectivity; the AccI-BamHI and BamHI-BalI regions coding for the N- and C-terminal halves of Fv-4 gp70SU, respectively; and the BalI-NcoI region encoding the cleavage site between gp70SU and p15(E)TM of the Fv-4 env. However, when the Fv-4 sequence was substituted for the p15(E)TM-coding NcoI-EcoRV region or the AccI-EcoRV region covering almost the entire env gene, infectivity was undetectable in our assays. The recombinant clone containing the Fv-4 AccI-EcoRV region, i.e., almost the entire Fv-4 env sequence, was introduced with pSV2neo into NIH 3T3 cells, and a G418r cell line named NIH(Fv4)-2 was isolated. The NIH(Fv4)-2 cell released viral particles that contained reverse transcriptase, Fv-4 env molecules as well as the other viral proteins, and viral genomic RNA. However, proviral DNA synthesis was not detected upon inoculation of this virus in NIH 3T3 cells. The loss of infectivity of the recombinant virus bearing the Fv-4 AccI-EcoRV region appeared to be caused by failure in an early step of replication. Images PMID:2304138

Masuda, M; Yoshikura, H

1990-01-01

142

Antibody potency relates to the ability to recognize the closed, pre-fusion form of HIV Env.  

PubMed

HIV's envelope glycoprotein (Env) is the sole target for neutralizing antibodies. The structures of many broadly neutralizing antibodies (bNAbs) in complex with truncated Env subunits or components have been reported. However, their interaction with the intact Env trimer, and the structural determinants that underlie neutralization resistance in this more native context are less well understood. Here we use hydrogen/deuterium exchange to examine the interactions between a panel of bNAbs and native-like Env trimers (SOSIP.664 trimers). Highly potent bNAbs cause only localized effects at their binding interface, while the binding of less potent antibodies is associated with elaborate changes throughout the trimer. In conjunction with binding kinetics, our results suggest that poorly neutralizing antibodies can only bind when the trimer transiently samples an open state. We propose that the kinetics of such opening motions varies among isolates, with Env from neutralization-sensitive viruses opening more frequently than Env from resistant viruses. PMID:25652336

Guttman, Miklos; Cupo, Albert; Julien, Jean-Philippe; Sanders, Rogier W; Wilson, Ian A; Moore, John P; Lee, Kelly K

2015-01-01

143

HIV-1 Nef responsiveness is determined by Env variable regions involved in trimer association and correlates with neutralization sensitivity.  

PubMed

HIV-1 Nef and the unrelated murine leukemia virus glycoGag similarly enhance the infectivity of HIV-1 virions. We now show that the effects of Nef and glycoGag are similarly determined by variable regions of HIV-1 gp120 that control Env trimer association and neutralization sensitivity. Whereas neutralization-sensitive X4-tropic Env proteins conferred high responsiveness to Nef and glycoGag, particles bearing neutralization-resistant R5-tropic Envs were considerably less affected. The profoundly different Nef/glycoGag responsiveness of a neutralization-resistant and a neutralization-sensitive R5-tropic Env could be switched by exchanging their gp120 V1/V2 regions, which also switches their neutralization sensitivity. Within V1/V2, the same determinants governed Nef/glycoGag responsiveness and neutralization sensitivity, indicating that these phenotypes are mechanistically linked. The V1/V2 and V3 regions, which form an apical trimer-association domain, together determined the Nef and glycoGag responsiveness of an X4-tropic Env. Our results suggest that Nef and glycoGag counteract the inactivation of Env spikes with relatively unstable apical trimer-association domains. PMID:24209751

Usami, Yoshiko; Göttlinger, Heinrich

2013-11-14

144

Antibody potency relates to the ability to recognize the closed, pre-fusion form of HIV Env  

NASA Astrophysics Data System (ADS)

HIV’s envelope glycoprotein (Env) is the sole target for neutralizing antibodies. The structures of many broadly neutralizing antibodies (bNAbs) in complex with truncated Env subunits or components have been reported. However, their interaction with the intact Env trimer, and the structural determinants that underlie neutralization resistance in this more native context are less well understood. Here we use hydrogen/deuterium exchange to examine the interactions between a panel of bNAbs and native-like Env trimers (SOSIP.664 trimers). Highly potent bNAbs cause only localized effects at their binding interface, while the binding of less potent antibodies is associated with elaborate changes throughout the trimer. In conjunction with binding kinetics, our results suggest that poorly neutralizing antibodies can only bind when the trimer transiently samples an open state. We propose that the kinetics of such opening motions varies among isolates, with Env from neutralization-sensitive viruses opening more frequently than Env from resistant viruses.

Guttman, Miklos; Cupo, Albert; Julien, Jean-Philippe; Sanders, Rogier W.; Wilson, Ian A.; Moore, John P.; Lee, Kelly K.

2015-02-01

145

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

146

An inverse scattering construction of the JMaRT fuzzball  

NASA Astrophysics Data System (ADS)

We present an inverse scattering construction in STU supergravity of the twocharge single-rotation JMaRT fuzzball. The key element in our construction is the fact that with appropriate changes in the parameters, the JMaRT fuzzball can be smoothly connected to the Myers-Perry instanton.

Katsimpouri, Despoina; Kleinschmidt, Axel; Virmani, Amitabh

2014-12-01

147

RT systems overview JS sep 2006 simons@astron.nl  

E-print Network

Basic Detection Techniques Radio Telescope SystemsRadio Telescope Systems anan overviewoverview #12;RT systems overview JS sep 2006 OverviewOverview Radio frequency radiation Basic telescope system Radio imaging Antenna properties Noise characterisation of a radio telescope #12;RT systems overview JS sep 2006

Peletier, Reynier

148

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

PubMed Central

Use of Env in HIV vaccine development has been disappointing. Here we show that, in the presence of a biologically active Tat subunit vaccine, a trimeric Env protein prevents in monkeys virus spread from the portal of entry to regional lymph nodes. This appears to be due to specific interactions between Tat and Env spikes that form a novel virus entry complex favoring R5 or X4 virus entry and productive infection of dendritic cells (DCs) via an integrin-mediated pathway. These Tat effects do not require Tat-transactivation activity and are blocked by anti-integrin antibodies (Abs). Productive DC infection promoted by Tat is associated with a highly efficient virus transmission to T cells. In the Tat/Env complex the cysteine-rich region of Tat engages the Env V3 loop, whereas the Tat RGD sequence remains free and directs the virus to integrins present on DCs. V2 loop deletion, which unshields the CCR5 binding region of Env, increases Tat/Env complex stability. Of note, binding of Tat to Env abolishes neutralization of Env entry or infection of DCs by anti-HIV sera lacking anti-Tat Abs, which are seldom present in natural infection. This is reversed, and neutralization further enhanced, by HIV sera containing anti-Tat Abs such as those from asymptomatic or Tat-vaccinated patients, or by sera from the Tat/Env vaccinated monkeys. Thus, both anti-Tat and anti-Env Abs are required for efficient HIV neutralization. These data suggest that the Tat/Env interaction increases HIV acquisition and spreading, as a mechanism evolved by the virus to escape anti-Env neutralizing Abs. This may explain the low effectiveness of Env-based vaccines, which are also unlikely to elicit Abs against new Env epitopes exposed by the Tat/Env interaction. As Tat also binds Envs from different clades, new vaccine strategies should exploit the Tat/Env interaction for both preventative and therapeutic interventions. PMID:23152803

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

2012-01-01

149

The cysteine 354 and 277 residues of Salmonella enterica serovar Typhi EnvZ are determinants of autophosphorylation and OmpR phosphorylation.  

PubMed

An initial biochemical characterization of the Salmonella enterica serovar Typhi (S. Typhi) EnvZ sensor protein and several mutant derivatives was performed. Autophosphorylation levels were higher for Escherichia coli EnvZ, intermediate for S. enterica serovar Typhimurium EnvZ and very low for S. Typhi EnvZ, in spite of their high amino acid sequence identity. Consequently, OmpR phosphorylation was related to EnvZ autophosphorylation. Among the mutant derivatives, a C354G mutation in S. Typhi EnvZ resulted in a substantial increase in autophosphorylation, while mutation of its other cysteine residue at position 277 to L or S decreased the EnvZ autophosphorylation level. Upon heterodimerization, the S. Typhi C354G mutant complemented the wild type in vitro, increasing the EnvZ-P yield of both monomers, in accordance with the model where EnvZ autophosphorylation occurs in trans, indicating that dimer formation is a dynamic process. Hence, the C354 and the C277 residues are fundamental in determining the particular intrinsic biochemical characteristics of EnvZ. PMID:19187206

Oropeza, Ricardo; Calva, Edmundo

2009-03-01

150

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

151

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

PubMed

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

Chigor, Vincent Nnamdigadi; Okoh, Anthony Ifeanyi

2012-11-01

152

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

153

Feline immunodeficiency virus Gag- and Env-specific immune responses after vaginal versus intravenous infection.  

PubMed

To better understand the correlation of mucosal and systemic immune responses with lentiviral containment, we contrasted the early mucosal and systemic immune responses induced by vaginal versus intravenous exposure of cats to feline immunodeficiency virus (FIV) isolates of differing pathogenicity and clade (i.e., FIV-B-2542 and FIV-A-PPR). We found that despite divergence in viral genotype, the mucosal and systemic immune responses induced differed more with route of exposure than virus isolate. In intravenously exposed cats, Gag-specific antibody (both IgG and IgA isotype) predominated in the serum, saliva, and vaginal wash fluid irrespective of infecting virus isolate. While Env-specific responses were more variable, they were more often detected in vaginally infected cats. Both IgG and IgA directed against Gag and Env were consistently present in vaginal wash fluids independent of route of infection or virus isolate. FIV Gag- and Env-specific cytotoxic lymphocytes (CTLs) were detected in blood and tissue lymphocytes of cats infected with either virus strain but were greatest in intravenously infected animals. Likewise, FIV-specific CTLs were detected in CD8(+) vaginal lymphocytes of animals infected by either route but were also more frequent in intravenously inoculated animals. In summary, we found qualitative differences in the immune responses following vaginal infection but no evidence (1) that mucosal immune responses were enhanced in vaginally exposed cats, (2) that local mucosal infection led to measurably greater immune responses in either compartment; or (3) that more prominent immune responses correlated with lower viral burden. PMID:11788028

Burkhard, M J; Mathiason, C K; Bowdre, T; Hoover, E A

2001-12-10

154

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

155

Drosophila germline invasion by the endogenous retrovirus gypsy: involvement of the viral env gene.  

PubMed

The endogenous retrovirus gypsy is expressed at high levels in mutant flamenco female flies. Gypsy viral particles extracted from such flies can infect naive flamenco individuals raised in the presence of these extracts mixed into their food. This results in the integration of new proviruses into the germline genome. These proviruses can then increase their copy number by (1) expression in the flamenco female somatic cells, (2) transfer into the oocyte and (3) integration into the genome of the progeny. Surprisingly, unlike the infection observed in the feeding experiments, this strategy of endogenous proviral multiplication does not seem to involve the expression of the viral env gene. PMID:12225916

Pelisson, A; Mejlumian, L; Robert, V; Terzian, C; Bucheton, A

2002-10-01

156

Nucleotide sequence of the env-specific segment of NFS-Th-1 xenotropic murine leukemia virus.  

PubMed Central

The sequence of 863 contiguous nucleotides encompassing portions of the pol and env genes of NFS-Th-1 xenotropic proviral DNA was determined. This region of the xenotropic murine leukemia virus genome contains and env-specific segment that hybridizes exclusively to xenotropic and mink cell focus-forming but not to ecotropic proviral DNAs (C. E. Buckler et al., J. Virol. 41:228-236, 1982). The unique xenotropic env segment contained several characteristic deletions and insertions relative to the analogous region in AKR and Moloney ecotropic murine leukemia viruses. Portions of an endogenous env segment cloned from a BALB/c mouse embryo gene library that had a restriction map and hybridization properties typical of xenotropic viruses (A. S. Khan et al., J. Virol. 44:625-636, 1982) were also sequenced. The sequence of the endogenous env gene was very similar to the comparable region of the NFS-Th-1 xenotropic virus containing the characteristic deletions and insertions previously observed and could represent a segment of an endogenous xenotropic provirus. PMID:6298457

Repaske, R; O'Neill, R R; Khan, A S; Martin, M A

1983-01-01

157

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

158

Structural insights into key sites of vulnerability on HIV-1 Env and Influenza HA  

PubMed Central

Summary Human immunodeficiency virus-1 (HIV-1) envelope protein (Env) and influenza hemagglutinin (HA) are the surface glycoproteins responsible for viral entry into host cells, the first step in the virus life cycle necessary to initiate infection. These glycoproteins exhibit a high degree of sequence variability and glycosylation, which are used as strategies to escape host immune responses. Nonetheless, antibodies with broadly neutralizing activity against these viruses have been isolated that have managed to overcome these barriers. Here, we review recent advances in the structural characterization of these antibodies with their viral antigens that defines a few sites of vulnerability on these viral spikes. These broadly neutralizing antibodies tend to focus their recognition on the sites of similar function between the two viruses: the receptor binding site and membrane fusion machinery. However, some sites of recognition are unique to the virus neutralized, such as the dense shield of oligomannose carbohydrates on HIV-1 Env. These observations are discussed in the context of structure-based design strategies to aid in vaccine design or development of antivirals. PMID:23046130

Julien, Jean-Philippe; Lee, Peter S.; Wilson, Ian A.

2012-01-01

159

A Multivalent Clade C HIV-1 Env Trimer Cocktail Elicits a Higher Magnitude of Neutralizing Antibodies than Any Individual Component  

PubMed Central

ABSTRACT The sequence diversity of human immunodeficiency virus type 1 (HIV-1) presents a formidable challenge to the generation of an HIV-1 vaccine. One strategy to address such sequence diversity and to improve the magnitude of neutralizing antibodies (NAbs) is to utilize multivalent mixtures of HIV-1 envelope (Env) immunogens. Here we report the generation and characterization of three novel, acute clade C HIV-1 Env gp140 trimers (459C, 405C, and 939C), each with unique antigenic properties. Among the single trimers tested, 459C elicited the most potent NAb responses in vaccinated guinea pigs. We evaluated the immunogenicity of various mixtures of clade C Env trimers and found that a quadrivalent cocktail of clade C trimers elicited a greater magnitude of NAbs against a panel of tier 1A and 1B viruses than any single clade C trimer alone, demonstrating that the mixture had an advantage over all individual components of the cocktail. These data suggest that vaccination with a mixture of clade C Env trimers represents a promising strategy to augment vaccine-elicited NAb responses. IMPORTANCE It is currently not known how to generate potent NAbs to the diverse circulating HIV-1 Envs by vaccination. One strategy to address this diversity is to utilize mixtures of different soluble HIV-1 envelope proteins. In this study, we generated and characterized three distinct, novel, acute clade C soluble trimers. We vaccinated guinea pigs with single trimers as well as mixtures of trimers, and we found that a mixture of four trimers elicited a greater magnitude of NAbs than any single trimer within the mixture. The results of this study suggest that further development of Env trimer cocktails is warranted. PMID:25540368

Bricault, Christine A.; Kovacs, James M.; Nkolola, Joseph P.; Yusim, Karina; Giorgi, Elena E.; Shields, Jennifer L.; Perry, James; Lavine, Christy L.; Cheung, Ann; Ellingson-Strouss, Katharine; Rademeyer, Cecelia; Gray, Glenda E.; Williamson, Carolyn; Stamatatos, Leonidas; Seaman, Michael S.; Korber, Bette T.; Chen, Bing

2014-01-01

160

Effect of Serum Lipid Levels on Stroke Outcome after rt-PA Therapy: SAMURAI rt-PA Registry  

Microsoft Academic Search

Background: The effects of lipid levels on clinical outcomes after ischemic stroke are controversial. Whether admission lipid levels and prior statin use are associated with early intracerebral hemorrhage (ICH) and long-term functional outcome after recombinant tissue plasminogen activator (rt-PA) therapy for stroke patients was investigated. Methods: Ischemic stroke patients who received intravenous rt-PA from a multicenter registry were studied. Lipid

Noriko Makihara; Yasushi Okada; Masatoshi Koga; Yoshiaki Shiokawa; Jyoji Nakagawara; Eisuke Furui; Kazumi Kimura; Hiroshi Yamagami; Yasuhiro Hasegawa; Kazuomi Kario; Satoshi Okuda; Masaki Naganuma; Kazunori Toyoda

2012-01-01

161

NASA SPoRT GOES-R Proving Ground Activities  

NASA Technical Reports Server (NTRS)

The NASA Short-term Prediction Research and Transition (SPoRT) program is a partner with the GOES-R Proving Ground (PG) helping prepare forecasters understand the unique products to come from the GOES-R instrument suite. SPoRT is working collaboratively with other members of the GOES-R PG team and Algorithm Working Group (AWG) scientists to develop and disseminate a suite of proxy products that address specific forecast problems for the WFOs, Regional and National Support Centers, and other NOAA users. These products draw on SPoRT s expertise with the transition and evaluation of products into operations from the MODIS instrument and the North Alabama Lightning Mapping Array (NALMA). The MODIS instrument serves as an excellent proxy for the Advanced Baseline Imager (ABI) that will be aboard GOES-R. SPoRT has transitioned and evaluated several multi-channel MODIS products. The true and false color products are being used in natural hazard detection by several SPoRT partners to provide better observation of land features, such as fires, smoke plumes, and snow cover. Additionally, many of SPoRT s partners are coastal offices and already benefit from the MODIS sea surface temperature composite. This, along with other surface feature observations will be developed into ABI proxy products for diagnostic use in the forecast process as well as assimilation into forecast models. In addition to the MODIS instrument, the NALMA has proven very valuable to WFOs with access to these total lightning data. These data provide situational awareness and enhanced warning decision making to improve lead times for severe thunderstorm and tornado warnings. One effort by SPoRT scientists includes a lightning threat product to create short-term model forecasts of lightning activity. Additionally, SPoRT is working with the AWG to create GLM proxy data from several of the ground based total lightning networks, such as the NALMA. The evaluation will focus on the vastly improved spatial coverage of the GLM, but with the trade-off of lower resolution compared to the NALMA. In addition to the above tasks, SPoRT will make these data available in the NWS next generation display software, AWIPS II. This has already been successfully completed for the two basic GLM proxies. SPoRT will use these products to train forecasters on the capabilities of GOES-R and foster feedback to develop additional products, visualizations, and requirements beneficial to end users needs. These developments and feedback will be made available to the GOES-R Proving Ground for the upcoming 2010 Spring Program in Norman, Oklahoma.

Stano, Geoffrey T.; Fuell, Kevin K.; Jedloec, Gary J.

2010-01-01

162

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

E-print Network

­polymerase chain reaction (RT-PCR) is much more sensitive. Ob- taining quantitative RT-PCR results, however, can­polymerase chain reaction. Relative RT-PCR with a plant-microbe interaction Dean et al.Introduction Gene expression

Hsiang, Tom

163

Impact of the HIV-1 env Genetic Context outside HR1–HR2 on Resistance to the Fusion Inhibitor Enfuvirtide and Viral Infectivity in Clinical Isolates  

PubMed Central

Resistance mutations to the HIV-1 fusion inhibitor enfuvirtide emerge mainly within the drug's target region, HR1, and compensatory mutations have been described within HR2. The surrounding envelope (env) genetic context might also contribute to resistance, although to what extent and through which determinants remains elusive. To quantify the direct role of the env context in resistance to enfuvirtide and in viral infectivity, we compared enfuvirtide susceptibility and infectivity of recombinant viral pairs harboring the HR1–HR2 region or the full Env ectodomain of longitudinal env clones from 5 heavily treated patients failing enfuvirtide therapy. Prior to enfuvirtide treatment onset, no env carried known resistance mutations and full Env viruses were on average less susceptible than HR1–HR2 recombinants. All escape clones carried at least one of G36D, V38A, N42D and/or N43D/S in HR1, and accordingly, resistance increased 11- to 2800-fold relative to baseline. Resistance of full Env recombinant viruses was similar to resistance of their HR1–HR2 counterpart, indicating that HR1 and HR2 are the main contributors to resistance. Strictly X4 viruses were more resistant than strictly R5 viruses, while dual-tropic Envs featured similar resistance levels irrespective of the coreceptor expressed by the cell line used. Full Env recombinants from all patients gained infectivity under prolonged drug pressure; for HR1–HR2 viruses, infectivity remained steady for 3/5 patients, while for 2/5 patients, gains in infectivity paralleled those of the corresponding full Env recombinants, indicating that the env genetic context accounts mainly for infectivity adjustments. Phylogenetic analyses revealed that quasispecies selection is a step-wise process where selection of enfuvirtide resistance is a dominant factor early during therapy, while increased infectivity is the prominent driver under prolonged therapy. PMID:21760896

Baatz, Franky; Nijhuis, Monique; Lemaire, Morgane; Riedijk, Martiene; Wensing, Annemarie M. J.; Servais, Jean-Yves; van Ham, Petra M.; Hoepelman, Andy I. M.; Koopmans, Peter P.; Sprenger, Herman G.; Devaux, Carole; Schmit, Jean-Claude; Perez Bercoff, Danielle

2011-01-01

164

Impact of the HIV-1 env genetic context outside HR1-HR2 on resistance to the fusion inhibitor enfuvirtide and viral infectivity in clinical isolates.  

PubMed

Resistance mutations to the HIV-1 fusion inhibitor enfuvirtide emerge mainly within the drug's target region, HR1, and compensatory mutations have been described within HR2. The surrounding envelope (env) genetic context might also contribute to resistance, although to what extent and through which determinants remains elusive. To quantify the direct role of the env context in resistance to enfuvirtide and in viral infectivity, we compared enfuvirtide susceptibility and infectivity of recombinant viral pairs harboring the HR1-HR2 region or the full Env ectodomain of longitudinal env clones from 5 heavily treated patients failing enfuvirtide therapy. Prior to enfuvirtide treatment onset, no env carried known resistance mutations and full Env viruses were on average less susceptible than HR1-HR2 recombinants. All escape clones carried at least one of G36D, V38A, N42D and/or N43D/S in HR1, and accordingly, resistance increased 11- to 2800-fold relative to baseline. Resistance of full Env recombinant viruses was similar to resistance of their HR1-HR2 counterpart, indicating that HR1 and HR2 are the main contributors to resistance. Strictly X4 viruses were more resistant than strictly R5 viruses, while dual-tropic Envs featured similar resistance levels irrespective of the coreceptor expressed by the cell line used. Full Env recombinants from all patients gained infectivity under prolonged drug pressure; for HR1-HR2 viruses, infectivity remained steady for 3/5 patients, while for 2/5 patients, gains in infectivity paralleled those of the corresponding full Env recombinants, indicating that the env genetic context accounts mainly for infectivity adjustments. Phylogenetic analyses revealed that quasispecies selection is a step-wise process where selection of enfuvirtide resistance is a dominant factor early during therapy, while increased infectivity is the prominent driver under prolonged therapy. PMID:21760896

Baatz, Franky; Nijhuis, Monique; Lemaire, Morgane; Riedijk, Martiene; Wensing, Annemarie M J; Servais, Jean-Yves; van Ham, Petra M; Hoepelman, Andy I M; Koopmans, Peter P; Sprenger, Herman G; Devaux, Carole; Schmit, Jean-Claude; Perez Bercoff, Danielle

2011-01-01

165

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

166

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

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

2008-08-01

167

Impact of Viral Dose and Major Histocompatibility Complex Class IB Haplotype on Viral Outcome in Mauritian Cynomolgus Monkeys Vaccinated with Tat upon Challenge with Simian/Human Immunodeficiency Virus SHIV89.6P ? †  

PubMed Central

The effects of the challenge dose and major histocompatibility complex (MHC) class IB alleles were analyzed in 112 Mauritian cynomolgus monkeys vaccinated (n = 67) or not vaccinated (n = 45) with Tat and challenged with simian/human immunodeficiency virus (SHIV) 89.6Pcy243. In the controls, the challenge dose (10 to 20 50% monkey infectious doses [MID50]) or MHC did not affect susceptibility to infection, peak viral load, or acute CD4 T-cell loss, whereas in the chronic phase of infection, the H1 haplotype correlated with a high viral load (P = 0.0280) and CD4 loss (P = 0.0343). Vaccination reduced the rate of infection acquisition at 10 MID50 (P < 0.0001), and contained acute CD4 loss at 15 MID50 (P = 0.0099). Haplotypes H2 and H6 were correlated with increased susceptibility (P = 0.0199) and resistance (P = 0.0087) to infection, respectively. Vaccination also contained CD4 depletion (P = 0.0391) during chronic infection, independently of the challenge dose or haplotype. PMID:20554774

Cafaro, Aurelio; Bellino, Stefania; Titti, Fausto; Maggiorella, Maria Teresa; Sernicola, Leonardo; Wiseman, Roger W.; Venzon, David; Karl, Julie A.; O'Connor, David; Monini, Paolo; Robert-Guroff, Marjorie; Ensoli, Barbara

2010-01-01

168

From sensors to semantic web: the SemsorGrid4env project  

NASA Astrophysics Data System (ADS)

Sensor networks are producing large quantities of valuable data around the planet. However advances are needed in the information management of such systems in order to make it easier to find and obtain the data. This is particularly true of software systems or models which need to automatically find appropriate data sources. The SemsorGrid4Env project is a three year European project to develop an integrated information space where new sensor network data sources can be discovered using web technologies and semantic descriptions. Rapid development of decision support systems are being developed within the context of ocean monitoring for flood and fire warnings. These are based around large quantities of sensors around the southern coast of the UK as well as a new sensor network deployment in forests in Spain. This paper will describe the design of the system, data integration issues, mashups and semantic interfaces.

Martinez, K.; Deroure, D.; Page, K.; Sadler, J.; Hutton, C.; Newman, R.; Roe, S.

2009-12-01

169

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

PubMed Central

In previous studies we have shown that a pantothenate auxotroph of Myocbacterium bovis BCG (BCG?panCD) expressing HIV-1 subtype C Gag induced Gag-specific immune responses in mice and Chacma baboons after prime-boost immunization in combination with matched rMVA and VLP vaccines respectively. In this study recombinant BCG (rBCG) expressing HIV-1 subtype C reverse transcriptase and a truncated envelope were constructed using both the wild type BCG Pasteur strain as a vector and the pantothenate auxotroph. Mice were primed with rBCG expressing Gag and RT and boosted with a recombinant MVA, expressing a polyprotein of Gag, RT, Tat and Nef (SAAVI MVA-C). Priming with rBCG?panCD expressing Gag or RT rather than the wild type rBCG expressing Gag or RT resulted in higher frequencies of total HIV-specific CD8+ T cells and increased numbers of T cells specific to the subdominant Gag and RT epitopes. Increasing the dose of rBCG from 105 cfu to 107 cfu also led to an increase in the frequency of responses to subdominant HIV epitopes. A mix of the individual rBCG?panCD vaccines expressing either Gag, RT or the truncated Env primed the immune system for a boost with SAAVI MVA-C and generated five-fold higher numbers of HIV-specific IFN-?-spot forming cells than mice primed with rBCG?panCD containing an empty vector control. Priming with the individual rBCG?panCD vaccines or the mix and boosting with SAAVI MVA-C also resulted in the generation of HIV-specific CD4+ and CD8+ T cells producing IFN-? and TNF-? and CD4+ cells producing IL-2. The rBCG vaccines tested in this study were able to prime the immune system for a boost with rMVA expressing matching antigens, inducing robust, HIV-specific T cell responses to both dominant and subdominant epitopes in the individual proteins when used as individual vaccines or in a mix. PMID:23977084

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

2013-01-01

170

Detection of Japanese yam mosaic virus by RT-LAMP.  

PubMed

Arapid and simple procedure is described to detect the genomic RNA molecule of Japanese yam mosaic potyvirus (JYMV). This method, named RT-LAMP, allows direct detection of RNA from infected plants without careful RNA extraction, rapid thermal cycling and gel electrophoresis. RT-LAMP was successfully applied to leaves, propagules and roots of Japanese yam infected with JYMV. One of the characteristics of the RT-LAMP method is its ability to synthesize an extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophospate ion, is produced yielding a white precipitate of magnesium pyrophosphate in the reaction mixture. The presence or absence of this white precipitate allows easy detection of the amplification of JYMV genomic RNA without gel electrophoresis. PMID:14505084

Fukuta, S; Iida, T; Mizukami, Y; Ishida, A; Ueda, J; Kanbe, M; Ishimoto, Y

2003-09-01

171

Pilot study on the immunogenicity of paired Env immunogens from mother-to-child transmitted HIV-1 isolates  

PubMed Central

Recent studies have reported that founder viruses play unique roles in establishing HIV-1 infection. Understanding the biological and immunological features of envelope glycoproteins (Env) from such viruses may facilitate the development of effective vaccines against HIV-1. In this report, we evaluated the immunogenicity of gp120 immunogens from two pairs of clade B and two pairs of clade C mother-to-child transmitted (MTCT) HIV-1 variants that had various levels of sensitivity to broadly neutralizing monoclonal antibodies. Individual gp120 DNA and protein vaccines were produced from each of the eight MTCT Env antigens included in the current study. Rabbits were immunized with these gp120 immunogens by the DNA prime-protein boost approach. High level Env-specific antibody responses were elicited by all MTCT gp120 immunogens. However, their abilities to elicit neutralizing antibody (NAb) responses differed and those from relatively neutralization-resistant variants tended to be more effective in eliciting broader NAb. Results of this pilot study indicated that not all MTCT Env proteins have the same potential to elicit NAb. Understanding the mechanism(s) behind such variation may provide useful information in formulating the next generation of HIV vaccines. PMID:23151449

Wang, Shixia; Kishko, Michael; Wan, Shengqin; Wang, Yan; Brewster, Frank; Gray, Glenda E.; Violari, Avye; Sullivan, John L.; Somasundaran, Mohan; Luzuriaga, Katherine; Lu, Shan

2012-01-01

172

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

PubMed Central

We have studied the antibody responses to Env and Gag antigens of human immunodeficiency virus type 1 (HIV-1) in several cohorts of HIV-1-infected individuals: long-term nonprogressors, progressors to disease, acute seroconvertors, and recipients of HIV-1 protease inhibitors. We conclude that the antibody responses to Env and Gag antigens are differentially regulated and that changes in the plasma viral load in the measurable range (500 to 10(8) RNA copies per ml) do not directly affect the antibody responses to these HIV-1 proteins. We provide quantitative estimates of HIV-1-specific immunoglobulin G concentrations in plasma, which can be in excess of 1 mg/ml for both anti-gp120 and anti-p24 once the immune response to HIV-1 has stabilized after seroconversion. We discuss the apparent paradox that the absence of anti-Gag antibodies (which have, at best, limited antiviral activity) is indicative of disease progression, while the retention of anti-Env antibodies (which do have antiviral activity) is of limited (or no) prognostic value. We show that the disappearance of anti-Gag antibodies during disease progression is highly unlikely to be due to immune complexing; instead, we believe that it reflects the loss of T-cell help that is more necessary for the anti-Gag than the anti-Env response. PMID:9060635

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

1997-01-01

173

Evidence for Positive Selection Driving the Evolution of HIV-1 env under Potent Antiviral Therapy Simon D. W. Frost,*,  

E-print Network

Evidence for Positive Selection Driving the Evolution of HIV-1 env under Potent Antiviral Therapy, and Andrew J. Leigh Brown*, *Centre for HIV Research, Institute for Cell, Animal and Population Biology with potent antiretroviral therapy, viable virus can be isolated from latently infected cells several years

Brown, Andrew Leigh

174

AWIPS II Application Development, a SPoRT Perspective  

NASA Technical Reports Server (NTRS)

The National Weather Service (NWS) is deploying its next-generation decision support system, called AWIPS II (Advanced Weather Interactive Processing System II). NASA's Short-term Prediction Research and Transition (SPoRT) Center has developed several software 'plug-ins' to extend the capabilities of AWIPS II. SPoRT aims to continue its mission of improving short-term forecasts by providing NASA and NOAA products on the decision support system used at NWS weather forecast offices (WFOs). These products are not included in the standard Satellite Broadcast Network feed provided to WFOs. SPoRT has had success in providing support to WFOs as they have transitioned to AWIPS II. Specific examples of transitioning SPoRT plug-ins to WFOs with newly deployed AWIPS II systems will be presented. Proving Ground activities (GOES-R and JPSS) will dominate SPoRT's future AWIPS II activities, including tool development as well as enhancements to existing products. In early 2012 SPoRT initiated the Experimental Product Development Team, a group of AWIPS II developers from several institutions supporting NWS forecasters with innovative products. The results of the team's spring and fall 2013 meeting will be presented. Since AWIPS II developers now include employees at WFOs, as well as many other institutions related to weather forecasting, the NWS has dealt with a multitude of software governance issues related to the difficulties of multiple remotely collaborating software developers. This presentation will provide additional examples of Research-to-Operations plugins, as well as an update on how governance issues are being handled in the AWIPS II developer community.

Burks, Jason E.; Smith, Matthew; McGrath, Kevin M.

2014-01-01

175

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

176

Stabilizing the Native Trimer of HIV-1 Env by Destabilizing the Heterodimeric Interface of the gp41 Postfusion Six-Helix Bundle  

PubMed Central

ABSTRACT The HIV-1 envelope glycoprotein (Env) is a trimer of gp120-gp41 heterodimers and is essential for viral entry. The gp41 subunit in native, prefusion trimeric Env exists in a metastable conformation and attains a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers, that drives the fusion of viral and cellular membranes. We attempted to stabilize native Env trimers by incorporation of mutations at the NHR-CHR interface that disrupt the postfusion 6-HB of gp41. The mutations V570D and I573D stabilize native Env of the HIV-1 JRFL strain and occlude nonneutralizing epitopes to a greater extent than the previously identified I559P mutation that is at the interface of the NHR trimers in the 6-HB. The mutations prevent soluble-CD4 (sCD4)-induced gp120 shedding and 6-HB formation. In the context of cell surface-expressed JRFL Env, introduction of a previously reported additional disulfide between residues A501 and T605 perturbs the native conformation, though this effect is partially alleviated by furin coexpression. The data suggest that positions 570 and 573 are surface proximal in native Env and that the NHR homotrimeric coiled coil in native Env terminates before or close to residue 573. Aspartic acid substitutions at these positions stabilize native trimers through destabilization of the postfusion 6-HB conformation. These mutations can be used to stabilize Env in a DNA vaccine format. IMPORTANCE The major protein on the surface of HIV-1 is the envelope (Env) glycoprotein. Env is a trimer of gp120-gp41 heterodimers. gp120 is involved in receptor/coreceptor binding and gp41 in the fusion of viral and cellular membranes. Like many other viral fusion proteins, the gp41 subunit in native trimeric Env exists in a metastable conformation. gp41 readily forms a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers that drives fusion of viral and cellular membranes. While it is expected that native Env is a good immunogen, its metastability results in exposure of immunodominant nonneutralizing epitopes. In the present study, we stabilize native Env trimers by incorporation of a number of different mutations at the NHR-CHR interface that disrupt the postfusion 6-HB of gp41. The stabilized constructs described here can be incorporated into DNA vaccine candidates. PMID:24920800

Kesavardhana, Sannula

2014-01-01

177

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.

178

Justin Simulator and GeRT Action Zeyn Saigol  

E-print Network

Justin Simulator and GeRT Action Learning Zeyn Saigol IR Lab, School of Computer Science University of Birmingham 24th March 2011 #12;IRLab, 24 Mar 2011 Justin and Action Learning Outline Justin Simulator Aims Strategy for Learning Action Models 2/14 #12;IRLab, 24 Mar 2011 Justin and Action Learning DLR's Justin

Yao, Xin

179

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

180

Bin Jia, Khe Chai Sim et al: CU-HTK RT03 Mandarin CTS system CU-HTK RT03 Mandarin CTS System  

E-print Network

Bin Jia, Khe Chai Sim et al: CU-HTK RT03 Mandarin CTS system CU-HTK RT03 Mandarin CTS System Bin, Khe Chai Sim et al: CU-HTK RT03 Mandarin CTS system Mandarin CTS 2003 System · Acoustic and Language Model Training Data · Mandarin Phone Sets · Tonal Decision Tree Questions · Vocal Tract Length

Edinburgh, University of

181

Bin Jia, Khe Chai Sim et al: CU-HTK RT03 Mandarin CTS system CU-HTK RT03 Mandarin CTS System  

E-print Network

Bin Jia, Khe Chai Sim et al: CU-HTK RT03 Mandarin CTS system CU-HTK RT03 Mandarin CTS System Bin, Khe Chai Sim et al: CU-HTK RT03 Mandarin CTS system Mandarin CTS 2003 System #15; Acoustic and Language Model Training Data #15; Mandarin Phone Sets #15; Tonal Decision Tree Questions #15; Vocal Tract

Hain, Thomas

182

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

183

Unique N-linked glycosylation of CasBrE Env influences its stability, processing, and viral infectivity but not its neurotoxicity.  

PubMed

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 = 10(2) to 10(5) FFU/ml); and wild-type-like (WTL) (titer > 10(5) 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; Lynch, William P

2013-08-01

184

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

185

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

186

Multiple Vaccine-Elicited Nonneutralizing Antienvelope Antibody Activities Contribute to Protective Efficacy by Reducing both Acute and Chronic Viremia following Simian/Human Immunodeficiency Virus SHIV89.6P Challenge in Rhesus Macaques?  

PubMed Central

We have shown that following priming with replicating adenovirus type 5 host range mutant (Ad5hr)-human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) recombinants, boosting with gp140 envelope protein enhances acute-phase protection against intravenous simian/human immunodeficiency virus (SHIV)89.6P challenge compared to results with priming and no boosting or boosting with an HIV polypeptide representing the CD4 binding site of gp120. We retrospectively analyzed antibodies in sera and rectal secretions from these same macaques, investigating the hypothesis that vaccine-elicited nonneutralizing antibodies contributed to the better protection. Compared to other immunized groups or controls, the gp140-boosted group exhibited significantly greater antibody activities mediating antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell-mediated viral inhibition (ADCVI) in sera and transcytosis inhibition in rectal secretions. ADCC and ADCVI activities were directly correlated with antibody avidity, suggesting the importance of antibody maturation for functionality. Both ADCVI and percent ADCC killing prechallenge were significantly correlated with reduced acute viremia. The latter, as well as postchallenge ADCVI and ADCC, was also significantly correlated with reduced chronic viremia. We have previously demonstrated induction by the prime/boost regimen of mucosal antibodies that inhibit transcytosis of SIV across an intact epithelial cell layer. Here, antibody in rectal secretions was significantly correlated with transcytosis inhibition. Importantly, the transcytosis specific activity (percent inhibition/total secretory IgA and IgG) was strongly correlated with reduced chronic viremia, suggesting that mucosal antibody may help control cell-to-cell viral spread during the course of infection. Overall, the replicating Ad5hr-HIV/SIV priming/gp140 protein boosting approach elicited strong systemic and mucosal antibodies with multiple functional activities associated with control of both acute and chronic viremia. PMID:20444898

Xiao, Peng; Zhao, Jun; Patterson, L. Jean; Brocca-Cofano, Egidio; Venzon, David; Kozlowski, Pamela A.; Hidajat, Rachmat; Demberg, Thorsten; Robert-Guroff, Marjorie

2010-01-01

187

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

188

Influence of random genetic drift on human immunodeficiency virus type 1 env evolution during chronic infection.  

PubMed Central

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 visits that reflect the first 6-13.7 years of infection. Pairwise comparisons of nonsynonymous and synonymous distances, Tajima's D test, Fu and Li's D* test, and a test of recurrent mutation revealed evidence for episodes of nonneutral evolution in a total of 22 out of 145 blood samples, representing six of the nine individuals. Using three coalescent-based maximum-likelihood estimators, we found viral effective population sizes in all nine individuals to be approximately 10(3). We also show that a previous estimate of the effective population size of approximately 10(5) based on rare haplotype frequencies decreases to approximately 10(3) upon correcting a biased sampling procedure. We conclude that the genetic variation in these data sets can be explained by a predominance of random genetic drift of neutral mutations with brief episodes of natural selection that were frequently masked by recombination. PMID:15082537

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

2004-01-01

189

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

190

Obacunone represses Salmonella pathogenicity islands 1 and 2 in an envZ-dependent fashion.  

PubMed

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; Pillai, Suresh D; Patil, Bhimanagouda S

2012-10-01

191

AdaBoost.RT: a boosting algorithm for regression problems  

Microsoft Academic Search

A boosting algorithm, AdaBoost.RT, is proposed for regression problems. The idea is to filter out examples with a relative estimation error that is higher than the pre-set threshold value, and then follow the AdaBoost procedure. Thus it requires to select the sub-optimal value of relative error threshold to demarcate predictions from the predictor as correct or incorrect. Some experimental results

D. P. Solomatine; D. L. Shrestha

2004-01-01

192

THE ISL RT04 MANDARIN BROADCAST NEWS EVALUATION SYSTEM  

Microsoft Academic Search

This paper describes our effort in developing a Mandarin Broadcast News system for the RT-04f (Rich Transcription) evaluation. Starting from a legacy system, we revisited all the issues including partitioning, acoustic modeling, lan- guage modeling, decoding and system combination strate- gies. We have achieved a sizable improvement, from 21.2% to 5.2% on the development set, from 42.7% to 22.4% mea-

Hua Yu; Yik-Cheung Tam; Thomas Schaaf; Qin Jin; Mohamed Noamany; Tanja Schultz

2004-01-01

193

A Machine Learning Approach for Identifying Amino Acid Signatures in the HIV Env Gene Predictive of Dementia  

PubMed Central

The identification of nucleotide sequence variations in viral pathogens linked to disease and clinical outcomes is important for developing vaccines and therapies. However, identifying these genetic variations in rapidly evolving pathogens adapting to selection pressures unique to each host presents several challenges. Machine learning tools provide new opportunities to address these challenges. In HIV infection, virus replicating within the brain causes HIV-associated dementia (HAD) and milder forms of neurocognitive impairment in 20–30% of patients with unsuppressed viremia. HIV neurotropism is primarily determined by the viral envelope (env) gene. To identify amino acid signatures in the HIV env gene predictive of HAD, we developed a machine learning pipeline using the PART rule-learning algorithm and C4.5 decision tree inducer to train a classifier on a meta-dataset (n?=?860 env sequences from 78 patients: 40 HAD, 38 non-HAD). To increase the flexibility and biological relevance of our analysis, we included 4 numeric factors describing amino acid hydrophobicity, polarity, bulkiness, and charge, in addition to amino acid identities. The classifier had 75% predictive accuracy in leave-one-out cross-validation, and identified 5 signatures associated with HAD diagnosis (p<0.05, Fisher’s exact test). These HAD signatures were found in the majority of brain sequences from 8 of 10 HAD patients from an independent cohort. Additionally, 2 HAD signatures were validated against env sequences from CSF of a second independent cohort. This analysis provides insight into viral genetic determinants associated with HAD, and develops novel methods for applying machine learning tools to analyze the genetics of rapidly evolving pathogens. PMID:23166702

Holman, Alexander G.; Gabuzda, Dana

2012-01-01

194

Starspot activity and period change in RT CrB  

NASA Astrophysics Data System (ADS)

The light curves of RT CrB in the B and V bands observed by ?bano?lu et al. (1985, Ap&SS, 112, 133), and in the V band and the radial velocity curves observed by Sabby and Lacy (2003, AJ, 125, 1448), are analyzed using the Wilson-Devinney code. The results show that the distortions in the light curve observed by Sabby and Lacy (2003) can be fitted by two spots, a hot spot on the primary component and a cool spot on the secondary star. The temperature ratios of the spotted region to the photosphere, Ts/Tph, are 1.181(±0.053) and 0.803(±0.057) respectively. Combining the radial velocity curves with the light curves, our analysis gives reliable, accurate estimates of the physical parameters of the system, M1 = 1.35(±0.01)M? and R1 = 2.88(±0.05)R? for the primary (hotter) component, M2 = 1.36(±0.01)M? and R2 = 2.92(±0.04)R? for the secondary (cooler) component. In addition, the orbital period variations of RT CrB are investigated based on all available times of light minima collected from literature and databases. We find that the orbital period exhibits a possible long-term period decrease with a rate of dP/dt = -3.11 × 10-7 d yr-1, suggesting that RT CrB is undergoing an angular momentum loss via magnetic braking.

Xiang, Fu-Yuan; Xiao, Ting-Yu; Yu, Yun-Xia

2015-02-01

195

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

PubMed

Gypsy is an infectious endogenous retrovirus of Drosophila melanogaster. The gypsy proviruses replicate very efficiently in the genome of the progeny of females homozygous for permissive alleles of the flamenco gene. This replicative transposition is correlated with derepression of gypsy expression, specifically in the somatic cells of the ovaries of the permissive mothers. The determinism of this amplification was studied further by making chimeric mothers containing different permissive/restrictive and somatic/germinal lineages. We show here that the derepression of active proviruses in the permissive soma is necessary and sufficient to induce proviral insertions in the progeny, even if the F1 flies derive from restrictive germ cells devoid of active proviruses. Therefore, gypsy endogenous multiplication results from the transfer of some gypsy-encoded genetic material from the soma towards the germen of the mother and its subsequent insertion into the chromosomes of the progeny. This transfer, however, is not likely to result from retroviral infection of the germline. Indeed, we also show here that the insertion of a tagged gypsy element, mutant for the env gene, occurs at high frequency, independently of the production of gypsy Env proteins by any transcomplementing helper. The possible role of the env gene for horizontal transfer to new hosts is discussed. PMID:10228177

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

1999-05-01

196

Generation of hybrid genes and proteins by vaccinia virus-mediated recombination: application to human immunodeficiency virus type 1 env.  

PubMed Central

The ability of poxviruses to undergo intramolecular recombination within tandemly arranged homologous sequences can be used to generate chimeric genes and proteins. Genes containing regions of nucleotide homology will recombine to yield a single sequence composed of portions of both original genes. A recombinant virus containing two genes with a number of conserved regions will yield a population of recombinant viruses containing a spectrum of hybrid sequences derived by recombination between the original genes. This scheme has been used to generate hybrid human immunodeficiency virus type 1 env genes. Recombinant vaccinia viruses that contain two divergent env genes in tandem array have been constructed. In the absence of selective pressure to maintain both genes, recombination between conserved homologous regions in these genes generated a wide range of progeny, each of which expressed a novel variant polypeptide encoded by the newly created hybrid env gene. Poxvirus-mediated recombination may be applied to map type-specific epitopes, to create novel pharmaceuticals such as hybrid interferons, to study receptor-binding or enzyme substrate specificities, or to mimic the antigenic diversity found in numerous pathogens. Images PMID:2243381

Gritz, L; Destree, A; Cormier, N; Day, E; Stallard, V; Caiazzo, T; Mazzara, G; Panicali, D

1990-01-01

197

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

PubMed Central

Background Infections with HIV still represent a major human health problem worldwide and a vaccine is the only long-term option to fight efficiently against this virus. Standardized assessments of HIV-specific immune responses in vaccine trials are essential for prioritizing vaccine candidates in preclinical and clinical stages of development. With respect to neutralizing antibodies, assays with HIV-1 Env-pseudotyped viruses are a high priority. To cover the increasing demands of HIV pseudoviruses, a complete cell culture and transfection automation system has been developed. Methodology/Principal Findings The automation system for HIV pseudovirus production comprises a modified Tecan-based Cellerity system. It covers an area of 5×3 meters and includes a robot platform, a cell counting machine, a CO2 incubator for cell cultivation and a media refrigerator. The processes for cell handling, transfection and pseudovirus production have been implemented according to manual standard operating procedures and are controlled and scheduled autonomously by the system. The system is housed in a biosafety level II cabinet that guarantees protection of personnel, environment and the product. HIV pseudovirus stocks in a scale from 140 ml to 1000 ml have been produced on the automated system. Parallel manual production of HIV pseudoviruses and comparisons (bridging assays) confirmed that the automated produced pseudoviruses were of equivalent quality as those produced manually. In addition, the automated method was fully validated according to Good Clinical Laboratory Practice (GCLP) guidelines, including the validation parameters accuracy, precision, robustness and specificity. Conclusions An automated HIV pseudovirus production system has been successfully established. It allows the high quality production of HIV pseudoviruses under GCLP conditions. In its present form, the installed module enables the production of 1000 ml of virus-containing cell culture supernatant per week. Thus, this novel automation facilitates standardized large-scale productions of HIV pseudoviruses for ongoing and upcoming HIV vaccine trials. PMID:23300558

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

2012-01-01

198

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.  

PubMed

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 virus Env the ability of two VV recombinants expressing HIV-1 Env B (VV WR Env and the highly attenuated modified VV Ankara [MVA] Env) to boost cellular immune responses in the spleen and in the lymph nodes draining the genital and rectal tracts. Groups of mice were primed by the intranasal route with 10(4) PFU of influenza virus Env and boosted 14 days later by the intraperitoneal or intranasal route with 10(7) PFU of MVA Env or VV WR Env, while the control group received two immunizations with influenza virus Env. We found that the combined immunization (Flu/VV) increased more than 60 times the number of gamma interferon-specific CD8(+) T cells compared to the Flu/Flu scheme. Significantly, boosting with MVA Env by the intraperitoneal route induced a response 1.25 or 2.5 times (spleen or genital lymph nodes) higher with respect to that found after the boost with VV WR Env. Mice with an enhanced CD8(+) T-cell response also had an increased Th1/Th2 ratio, evaluated by the cytokine pattern secreted following in vitro restimulation with gp160 protein and by the specific immunoglobulin G2a (IgG2a)/IgG1 ratio in serum. By the intranasal route recombinant WR Env booster gave a more efficient immune response (10 and 1.3 times in spleen and genital lymph nodes, respectively) than recombinant MVA Env. However, the scheme influenza virus Env/MVA Env increased four times the response in the spleen, giving a low but significant response in the genital lymph nodes compared with a single intranasal immunization with MVA Env. These results demonstrate that the combination Flu/MVA in prime-booster immunization regimens is an effective vaccination approach to generate cellular immune responses to HIV antigens at sites critical for protective responses. PMID:12768024

Gherardi, M Magdalena; Nájera, José Luis; Pérez-Jiménez, Eva; Guerra, Susana; García-Sastre, Adolfo; Esteban, Mariano

2003-06-01

199

A comparative study of digital RT-PCR and RT-qPCR for quantification of Hepatitis A virus and Norovirus in lettuce and water samples.  

PubMed

Sensitive and quantitative detection of foodborne enteric viruses is classically achieved by quantitative RT-PCR (RT-qPCR). Recently, digital PCR (dPCR) was described as a novel approach to genome quantification without need for a standard curve. The performance of microfluidic digital RT-PCR (RT-dPCR) was compared to RT-qPCR for detecting the main viruses responsible for foodborne outbreaks (human Noroviruses (NoV) and Hepatitis A virus (HAV)) in spiked lettuce and bottled water. Two process controls (Mengovirus and Murine Norovirus) were used and external amplification controls (EAC) were added to examine inhibition of RT-qPCR and RT-dPCR. For detecting viral RNA and cDNA, the sensitivity of the RT-dPCR assays was either comparable to that of RT-qPCR (RNA of HAV, NoV GI, Mengovirus) or slightly (around 1 log10) decreased (NoV GII and MNV-1 RNA and of HAV, NoV GI, NoV GII cDNA). The number of genomic copies determined by dPCR was always from 0.4 to 1.7 log10 lower than the expected numbers of copies calculated by using the standard qPCR curve. Viral recoveries calculated by RT-dPCR were found to be significantly higher than by RT-qPCR for NoV GI, HAV and Mengovirus in water, and for NoV GII and HAV in lettuce samples. The RT-dPCR assay proved to be more tolerant to inhibitory substances present in lettuce samples. This absolute quantitation approach may be useful to standardize quantification of enteric viruses in bottled water and lettuce samples and may be extended to quantifying other human pathogens in food samples. PMID:25725459

Coudray-Meunier, Coralie; Fraisse, Audrey; Martin-Latil, Sandra; Guillier, Laurent; Delannoy, Sabine; Fach, Patrick; Perelle, Sylvie

2015-05-18

200

Hardware-In-the-Loop Simulation of Power Drives with RT-LAB  

Microsoft Academic Search

This paper presents the RT-LAB Electrical Drive Simulator technology along with practical applications. The RT-LAB simulation software enables the parallel simulation of power drives and electric circuits on clusters of PC running QNX or RT-Linux operating systems at sample time below 10 ?s. Using standard Simulink models including SimPowerSystems models, RT-LAB build computation and communication tasks necessary to make parallel

Christian Dufour; S. Abourida; J. Belanger

2005-01-01

201

SU Detection for RT-03f at Cambridge University Marcus Tomalin, Sue Tranter, Phil Woodland  

E-print Network

Detection for RT-03f at Cambridge University The Prosodic Feature Model Five SU sub-types defined: · SU SSU Detection for RT-03f at Cambridge University Marcus Tomalin, Sue Tranter, Phil Woodland & the CU-HTK STT Team 13th November Cambridge University Engineering Department RT-03f Workshop 13th November 2003

Hain, Thomas

202

Avian influenza virus detection and quantitation by real-time RT-PCR  

Technology Transfer Automated Retrieval System (TEKTRAN)

Real-time RT-PCR (rRT-PCR) has been used for avian influenza virus (AIV) detection since the early 2000’s for routine surveillance, during outbreaks and for research. Some of the advantages of rRT-PCR are: high sensitivity, high specificity, rapid time-to-result, scalability, cost, and its inherentl...

203

Applications of LANCE Data at SPoRT  

NASA Technical Reports Server (NTRS)

Short term Prediction Research and Transition (SPoRT) Center: Mission: Apply NASA and NOAA measurement systems and unique Earth science research to improve the accuracy of short term weather prediction at the regional/local scale. Goals: Evaluate and assess the utility of NASA and NOAA Earth science data and products and unique research capabilities to address operational weather forecast problems; Provide an environment which enables the development and testing of new capabilities to improve short term weather forecasts on a regional scale; Help ensure successful transition of new capabilities to operational weather entities for the benefit of society

Molthan, Andrew

2014-01-01

204

Development of a multiplex one step RT-PCR that detects eighteen respiratory viruses in clinical specimens and comparison with real time RT-PCR.  

PubMed

Rapid and accurate diagnosis of viral respiratory infections is crucial for patient management. Multiplex reverse transcriptase polymerase chain reaction (mRT-PCR) is used increasingly to diagnose respiratory infections and has shown to be more sensitive than viral culture and antigen detection. Objective of the present study was to develop a one-step mRT-PCR that could detect 18 respiratory viruses in three sets. The method was compared with real time RT-PCR (rRT-PCR) for its sensitivity and specificity. Clinical specimens from 843 pediatric patients with respiratory symptoms were used in the study. 503 (59.7%) samples were detected positive by mRT-PCR. Of these 462 (54.8%) exhibited presence of a single pathogen and 41 (4.9%) had multiple pathogens. rRT-PCR detected 439 (52.1%) positive samples, where 419 (49.7%) exhibited one virus and 20 (2.4%) showed co-infections. Concordance between mRT-PCR and rRT-PCR was 91.9% and kappa correlation 0.837. Sensitivity and specificity of mRT-PCR were 99.5% and 83.7% while that of rRT-PCR was 86.9% and 99.4% respectively. Rhinovirus (17.2%) was the most frequently detected virus followed by respiratory syncytial virus B (15.4%), H1N1pdm09 (8.54%), parainfluenza virus-3 (5.8%) and metapneumovirus (5.2%). In conclusion, mRT-PCR is a rapid, cost effective, specific and highly sensitive method for detection of respiratory viruses. PMID:23313883

Choudhary, Manohar L; Anand, Siddharth P; Heydari, Mostafa; Rane, Grishma; Potdar, Varsha A; Chadha, Mandeep S; Mishra, Akhilesh C

2013-04-01

205

RAMSES-RT: radiation hydrodynamics in the cosmological context  

NASA Astrophysics Data System (ADS)

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

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

2013-12-01

206

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

207

Towards magnetic resonance imaging guided radiation therapy (MRIgRT)  

NASA Astrophysics Data System (ADS)

The goal of this work is to address key aspects of the magnetic resonance imaging guided radiation therapy (MRIgRT) process of cancer sites. MRIgRT is implemented by using a system comprised of a magnetic resonance imaging (MRI) scanner coupled with a radiation source, in our case a radiotherapy accelerator (Linac). The potential benefits of MRIgRT are the real-time tracking of the tumor and neighbouring healthy anatomy during treatment irradiation leading to on-line treatment plan optimization. Ultimately, this results in an increased accuracy and efficiency of the overall treatment process. A large research effort is conducted at Cross Cancer Institute to develop a hybrid MRI-Linac system consisting of a bi-planar 0.2 T permanent magnet coupled with a 6 MV Linac. The present work is part of this project and aims to address the following key components: (a) magnetic shielding and dosimetric effects of the MRI-Linac system, (b) measure and correction of scanner-related MR image distortions, and (c) MRI-based treatment planning procedure for intracranial lesions. The first two components are essential for the optimal construction and operation of the MRI-Linac system while the third one represents a direct application of the system. The linac passive shielding was achieved by (a) adding two 10 cm thick steel (1020) plates placed at a distance of 10 cm from the structure on opposite sides of the magnet; and (b) a box lined with a 1 mm MuMetal(TM) wall surrounding the Linac. For our proposed MRI-Linac configuration (i.e. 0.2 T field and rotating bi-planar geometry) the maximum dose difference from zero magnetic field case was found to be within 6% and 12% in a water and water-lung-water phantom, respectively. We developed an image system distortion correction method for MRI that relies on adaptive thresholding and an iterative algorithm to determine the 3D distortion field. Applying this technique the residual image distortions were reduced to within the voxel resolution of the raw imaging data. We investigated a procedure for the MRI Simulation of brain lesions which consists of (a) correction of MR images for 3D distortions, (b) automatic segmentation of head sub-structures (i.e. scalp, bone, and brain) relevant for dosimetric calculations, (c) conversion of MRI datasets into CT-like images by assigning bulk CT values to head sub-structures and MRI-based dose calculations, and (d) RT plan evaluation based on isodose distributions, dosimetric parameters, dose volume histograms, and an RT ranking tool. The proposed MRI-based treatment planning procedure performed similarly to the standard clinical technique, which relies on both CT and MR imaging modalities, and is suitable for the radiotherapy of brain cancer.

Stanescu, Teodor Marius

208

Structural variability of env and gag gene products from a highly cytopathic strain of HIV-1.  

PubMed

The glycoprotein precursor of the highly cytopathic Zairian virus HIV1-NDK synthesized in CEM leukemic cells displayed a molecular mass of 140 kDa (gp140) as compared to the 160 kDa of gp160 of HIV1-LAV prototype strain. This precursor was cleaved to produce a smaller than prototype extra-cellular envelope glycoprotein (gp100) and a transmembrane component with a usual size (gp41). Immunoprecipitates from tunicamycin-treated infected cells demonstrated the presence of a non-glycosylated precursor of 100 kDa for HIV1-LAV prototype strain and 90 kDa for HIV1-NDK. Digestion of labeled precipitates with a mixture of endoglycosidase F and glycopeptidase F reduced the size of HIV1-LAV gp160 and gp120 to 100 and 60 kDa, respectively, while HIV1-NDK gp140 and gp100, after treatment with the same enzymes, displayed an apparent molecular mass of 90 kDa and 55 kDa, respectively. From these data we conclude that HIV1-LAV gp120 and HIV1-NDK gp100 differ both in their proteic moiety (60 kDa and 55 kDa, respectively) and in their carbohydrate moiety (60 kDa and 45 kDa, respectively). These differences could not be deduced from the available gene sequences of the two viruses. A chimeric virus containing the first 124 amino acid residues of the envelope glycoprotein coded by HIV1-LAV sequence and the rest by HIV1-NDK displayed normal size envelope glycoproteins, demonstrating the involvement of this N-terminal sequence in the alteration of the molecular mass characteristic of HIV1-NDK gp140 and gp100. Finally, characterization of the gag gene products from both strains demonstrated that HIV1-NDK p18 and p15 have a slower electrophoretic mobility as compared to its HIV1-LAV counterparts. Therefore, structural properties of HIV1-NDK env and gag products, reflected by their unusual electrophoretic mobilities, may be responsible for HIV1-NDK biological properties. PMID:1642554

Yahi, N; Fantini, J; Hirsch, I; Chermann, J C

1992-01-01

209

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

210

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

211

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

212

In-line alignment and Mg2+ coordination at the cleavage site of the env22 twister ribozyme  

NASA Astrophysics Data System (ADS)

Small self-cleaving nucleolytic ribozymes contain catalytic domains that accelerate site-specific cleavage/ligation of phosphodiester backbones. We report on the 2.9-Å crystal structure of the env22 twister ribozyme, which adopts a compact tertiary fold stabilized by co-helical stacking, double-pseudoknot formation and long-range pairing interactions. The U-A cleavage site adopts a splayed-apart conformation with the modelled 2?-O of U positioned for in-line attack on the adjacent to-be-cleaved P-O5? bond. Both an invariant guanosine and a Mg2+ are directly coordinated to the non-bridging phosphate oxygens at the U-A cleavage step, with the former positioned to contribute to catalysis and the latter to structural integrity. The impact of key mutations on cleavage activity identified an invariant guanosine that contributes to catalysis. Our structure of the in-line aligned env22 twister ribozyme is compared with two recently reported twister ribozymes structures, which adopt similar global folds, but differ in conformational features around the cleavage site.

Ren, Aiming; Košuti?, Marija; Rajashankar, Kanagalaghatta R.; Frener, Marina; Santner, Tobias; Westhof, Eric; Micura, Ronald; Patel, Dinshaw J.

2014-11-01

213

Intercompartmental Recombination of HIV-1 Contributes to env Intrahost Diversity and Modulates Viral Tropism and Sensitivity to Entry Inhibitors?†‡  

PubMed Central

HIV-1 circulates within an infected host as a genetically heterogeneous viral population. Viral intrahost diversity is shaped by substitutional evolution and recombination. Although many studies have speculated that recombination could have a significant impact on viral phenotype, this has never been definitively demonstrated. We report here phylogenetic and subsequent phenotypic analyses of envelope genes obtained from HIV-1 populations present in different anatomical compartments. Assessment of env compartmentalization from immunologically discrete tissues was assessed utilizing a single genome amplification approach, minimizing in vitro-generated artifacts. Genetic compartmentalization of variants was frequently observed. In addition, multiple incidences of intercompartment recombination, presumably facilitated by low-level migration of virus or infected cells between different anatomic sites and coinfection of susceptible cells by genetically divergent strains, were identified. These analyses demonstrate that intercompartment recombination is a fundamental evolutionary mechanism that helps to shape HIV-1 env intrahost diversity in natural infection. Analysis of the phenotypic consequences of these recombination events showed that genetic compartmentalization often correlates with phenotypic compartmentalization and that intercompartment recombination results in phenotype modulation. This represents definitive proof that recombination can generate novel combinations of phenotypic traits which differ subtly from those of parental strains, an important phenomenon that may have an impact on antiviral therapy and contribute to HIV-1 persistence in vivo. PMID:21471230

Brown, Richard J. P.; Peters, Paul J.; Caron, Catherine; Gonzalez-Perez, Maria Paz; Stones, Leanne; Ankghuambom, Chiambah; Pondei, Kemebradikumo; McClure, C. Patrick; Alemnji, George; Taylor, Stephen; Sharp, Paul M.; Clapham, Paul R.; Ball, Jonathan K.

2011-01-01

214

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

215

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

216

Mucosal immunization of lactating female rhesus monkeys with a transmitted/founder HIV-1 envelope induces strong Env-specific IgA antibody responses in breast milk.  

PubMed

We previously demonstrated that vaccination of lactating rhesus monkeys with a DNA prime/vector boost strategy induces strong T-cell responses but limited envelope (Env)-specific humoral responses in breast milk. To improve vaccine-elicited antibody responses in milk, hormone-induced lactating rhesus monkeys were vaccinated with a transmitted/founder (T/F) HIV Env immunogen in a prime-boost strategy modeled after the moderately protective RV144 HIV vaccine. Lactating rhesus monkeys were intramuscularly primed with either recombinant DNA (n = 4) or modified vaccinia virus Ankara (MVA) poxvirus vector (n = 4) expressing the T/F HIV Env C.1086 and then boosted twice intramuscularly with C.1086 gp120 and the adjuvant MF59. The vaccines induced Env-binding IgG and IgA as well as neutralizing and antibody-dependent cellular cytotoxicity (ADCC) responses in plasma and milk of most vaccinated animals. Importantly, plasma neutralization titers against clade C HIV variants MW965 (P = 0.03) and CAP45 (P = 0.04) were significantly higher in MVA-primed than in DNA-primed animals. The superior systemic prime-boost regimen was then compared to a mucosal-boost regimen, in which animals were boosted twice intranasally with C.1086 gp120 and the TLR 7/8 agonist R848 following the same systemic prime. While the systemic and mucosal vaccine regimens elicited comparable levels of Env-binding IgG antibodies, mucosal immunization induced significantly stronger Env-binding IgA responses in milk (P = 0.03). However, the mucosal regimen was not as potent at inducing functional IgG responses. This study shows that systemic MVA prime followed by either intranasal or systemic protein boosts can elicit strong humoral responses in breast milk and may be a useful strategy to interrupt postnatal HIV-1 transmission. PMID:23596289

Fouda, Genevieve G A; Amos, Joshua D; Wilks, Andrew B; Pollara, Justin; Ray, Caroline A; Chand, Anjali; Kunz, Erika L; Liebl, Brooke E; Whitaker, Kaylan; Carville, Angela; Smith, Shannon; Colvin, Lisa; Pickup, David J; Staats, Herman F; Overman, Glenn; Eutsey-Lloyd, Krissey; Parks, Robert; Chen, Haiyan; Labranche, Celia; Barnett, Susan; Tomaras, Georgia D; Ferrari, Guido; Montefiori, David C; Liao, Hua-Xin; Letvin, Norman L; Haynes, Barton F; Permar, Sallie R

2013-06-01

217

Real-time RT-PCR for norovirus screening in shellfish  

Microsoft Academic Search

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

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

2005-01-01

218

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

219

Recombinant, truncated CD4 molecule (rT4) binds IgG.  

PubMed

CD4 is a cell surface glycoprotein that identifies the subset of human T lymphocytes that induces sIg+ B lymphocytes to differentiate and secrete Ig after intimate T-B cell contact. In the course of studying a recombinant, truncated form of CD4 (rT4) we noticed that goat antibodies of apparently irrelevant specificities bound to immobilized rT4. To directly study whether rT4 interacts with Ig molecules, purified human IgG was added to rT4-coated wells and a dose-dependent interaction between IgG and rT4 was observed by ELISA. Purified myeloma IgG proteins bound to immobilized rT4 with the same avidity as polyclonal IgG that suggests that rT4-IgG binding was not due to the presence of anti-rT4 antibodies in the IgG fraction. IgG from 6 sera bound to rT4 in concentration dependent manner similar to purified IgG. Immobilized rT4 specifically bound IgG, and not IgM, IgA, IgD, or beta 2-microglobulin. The specific interaction of rT4 and IgG was also observed when IgG or IgM were immobilized, demonstrating that IgG binding was not a unique property of immobilized rT4. As with low affinity receptors for IgG, rT4 bound heat aggregated IgG with increased avidity. Neither anti-CD4 mAb nor dextran sulfate inhibited rT4-IgG binding. rT4 bound Fc but not F(ab)2 fragments. Each of the purified IgG subclasses; IgG1, 2, 3, and 4 bound to rT4 with similar avidity. Taken together, these data suggest that rT4 specifically interacts with a public structure on IgG Fc. PMID:2295793

Lederman, S; Yellin, M J; Cleary, A M; Gulick, R; Chess, L

1990-01-01

220

RT_BUILD: An expert programmer for implementing and simulating Ada real-time control software  

NASA Technical Reports Server (NTRS)

The RT BUILD is an expert control system programmer that creates real-time Ada code from block-diagram descriptions of control systems. Since RT BUILD embodies substantial knowledge about the implementation of real-time control systems, it can perform many, if not most of the functions normally performed by human real-time programmers. Though much basic research was done in automatic programming, RT BUILD appears to be the first application of this research to an important problem in flight control system development. In particular, RT BUILD was designed to directly increase productivity and reliability for control implementations of large complex systems.

Lehman, Larry L.; Houtchens, Steve; Navab, Massoud; Shah, Sunil C.

1986-01-01

221

Sensitivity and reproducibility of standardized-competitive RT-PCR for transcript quantification and its comparison with real time RT-PCR  

PubMed Central

Background Probe based detection assays form the mainstay of transcript quantification. Problems with these assays include varying hybridization efficiencies of the probes used for transcript quantification and the expense involved. We examined the ability of a standardized competitive RT-PCR (StaRT PCR) assay to quantify transcripts of 4 cell cycle associated genes (RB, E2F1, CDKN2A and PCNA) in two cell lines (T24 & LD419) and compared its efficacy with the established Taqman real time quantitative RT-PCR assay. We also assessed the sensitivity, reproducibility and consistency of StaRT PCR. StaRT PCR assay is based on the incorporation of competitive templates (CT) in precisely standardized quantities along with the native template (NT) in a PCR reaction. This enables transcript quantification by comparing the NT and CT band intensities at the end of the PCR amplification. The CT serves as an ideal internal control. The transcript numbers are expressed as copies per million transcripts of a control gene such as ?-actin (ACTB). Results The NT and CT were amplified at remarkably similar rates throughout the StaRT PCR amplification cycles, and the coefficient of variation was least (<3.8%) when the NT/CT ratio was kept as close to 1:1 as possible. The variability between the rates of amplification in different tubes subjected to the same StaRT PCR reaction was very low and within the range of experimental noise. Further, StaRT PCR was sensitive enough to detect variations as low as 10% in endogenous actin transcript quantity (p < 0.01 by the paired student's t-test). StaRT PCR correlated well with Taqman real time RT-PCR assay in terms of transcript quantification efficacy (p < 0.01 for all 4 genes by the Spearman Rank correlation method) and the ability to discriminate between cell types and confluence patterns. Conclusion StaRT PCR is thus a reliable and sensitive technique that can be applied to medium-high throughput quantitative transcript measurement. Further, it correlates well with Taqman real time PCR in terms of quantitative and discriminatory ability. This label-free, inexpensive technique may provide the ability to generate prognostically important molecular signatures unique to individual tumors and may enable identification of novel therapeutic targets. PMID:14741054

Pagliarulo, Vincenzo; George, Ben; Beil, Stephen J; Groshen, Susan; Laird, Peter W; Cai, Jie; Willey, James; Cote, Richard J; Datar, Ram H

2004-01-01

222

A comparison of RT-PCR, in-situ hybridisation and in-situ RT-PCR for the detection of rhinovirus infection in paraffin sections.  

PubMed

We describe an in-situ RT-PCR method for the amplification of rhinovirus (RV) in fixed, paraffin-embedded HeLa cells employed as a model for human respiratory epithelium. HeLa cells were infected in-vitro with inocula of rhinovirus-16 ranging from 10(2) to 10(6) 50% tissue culture infective doses (TCID50), incubated for 18 h then fixed and processed into paraffin blocks. Sections of the cell preparation were subjected to standard RT-PCR, in-situ hybridisation (ISH) or in-situ RT-PCR using specific oligonucleotide primers or probes directed against the 5' non-coding region of RV RNA. RT-PCR was found to be capable of detecting RV16 RNA in one 8 microns-thick section of cells infected with the lowest virus titre. ISH using digoxigenin labelled oligonucleotide probes located RV16 signal in the majority of HeLa cells at the highest virus titre, but in few or no cells with the lowest virus titre. In contrast, in-situ RT-PCR detected RV16 in the majority of cells infected with this amount of RV16. There was a slight loss of morphology and fine localisation associated with the in-situ thermal cycling process. However, the sensitivity of in-situ RT-PCR is comparable to standard RT-PCR and greater than ISH for the detection of RV. In-situ RT-PCR has wide applications for sensitive localization of low copy viral and RNA sequences within cells to investigate the role of viruses in a variety of clinical conditions. PMID:9300380

Bates, P J; Sanderson, G; Holgate, S T; Johnston, S L

1997-09-01

223

The role of the N-terminal segment of CCR5 in HIV-1 Env-mediated membrane fusion and the mechanism of virus adaptation to CCR5 lacking this segment  

PubMed Central

Background HIV-1 envelope glycoprotein (Env) induces membrane fusion as a result of sequential binding to CD4 and chemokine receptors (CCR5 or CXCR4). The critical determinants of CCR5 coreceptor function are the N-terminal domain (Nt) and the second extracellular loop. However, mutations in gp120 adapt HIV-1 to grow on cells expressing the N-terminally truncated CCR5(?18) (Platt et al., J. Virol. 2005, 79: 4357–68). Results We have functionally characterized the adapted Env (designated Env(NYP)) using a quantitative cell-cell fusion assay. The rate of fusion with target cells expressing wild-type CCR5 and the resistance to fusion inhibitors was virtually identical for wild-type Env and Env(NYP), implying that the coreceptor affinity had not increased as a result of adaptation. In contrast, Env(NYP)-induced fusion with cells expressing CCR5(?18) occurred at a slower rate and was extremely sensitive to the CCR5 binding inhibitor, Sch-C. Resistance to Sch-C drastically increased after pre-incubation of Env(NYP)- and CCR5(?18)-expressing cells at a temperature that was not permissive to fusion. This indicates that ternary Env(NYP)-CD4-CCR5(?18) complexes accumulate at sub-threshold temperature and that low-affinity interactions with the truncated coreceptor are sufficient for triggering conformational changes in the gp41 of Env(NYP) but not in wild-type Env. We also demonstrated that the ability of CCR5(?18) to support fusion and infection mediated by wild-type Env can be partially reconstituted in the presence of a synthetic sulfated peptide corresponding to the CCR5 Nt. Pre-incubation of wild-type Env- and CCR5(?18)-expressing cells with the sulfated peptide at sub-threshold temperature markedly increased the efficiency of fusion. Conclusion We propose that, upon binding the Nt region of CCR5, wild-type Env acquires the ability to productively engage the extracellular loop(s) of CCR5 – an event that triggers gp41 refolding and membrane merger. The adaptive mutations in Env(NYP) enable it to more readily release its hold on gp41, even when it interacts weakly with a severely damaged coreceptor in the absence of the sulfopeptide. PMID:17686153

Melikyan, Gregory B; Platt, Emily J; Kabat, David

2007-01-01

224

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

SciTech Connect

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

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

2005-11-01

225

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

226

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

227

Evaluation of reference genes for quantitative RT-PCR in Lolium perenne  

Technology Transfer Automated Retrieval System (TEKTRAN)

Quantitative real-time RT-PCR provides an important tool for analyzing gene expression if proper internal standards are used. The aim of this study was to identify and evaluate reference genes for use in real-time quantitative RT-PCR in perennial ryegrass (Lolium perenne L.) during plant developmen...

228

The KARL\\/KARATE System: Automatic Test Pattern Generation Based on RT Level Descriptions  

Microsoft Academic Search

A system is described for automatic test-pattern generation (ATPG) using symbolic representations and heuristics to attack the test problem at RT level, where redesigns to increase the testability are relatively cheap. In contrast to other ATPG tools based on RT-level hardware descriptions, KARATE includes tests for primitive operators and allows the modification and redefinition of fault models. KARATE has been

Gerold Affs; Reiner W. Hartenstein; Andrea Wodtko

1988-01-01

229

Stresa, Italy, 25-27 April 2007 0-LEVEL VACUUM PACKAGING RT PROCESS FOR MEMS RESONATORS  

E-print Network

AND PACKAGING DESIGN The packaging process has been done on a MEMS resonator having MOSFET detection [1Stresa, Italy, 25-27 April 2007 0-LEVEL VACUUM PACKAGING RT PROCESS FOR MEMS RESONATORS Nicolas, France, 4 CEA-LETI MINATEC, France ABSTRACT A new Room Temperature (RT) 0-level vacuum package

Paris-Sud XI, Université de

230

LITMUS RT : A Status Report # Bj orn B. Brandenburg, Aaron D. Block, John M. Calandrino,  

E-print Network

This paper describes a real­time extension to Linux called LITMUS RT , which is being designed to support operating systems such as Linux. 1 Introduction In this paper, we report on the development of a real­ time extension of Linux called LITMUS RT (LInux Testbed for MUltiprocessor Scheduling in Real­Time systems) [9

Anderson, James

231

Positional Identification of RT1-B (HLA-DQ) as Susceptibility Locus for Autoimmune Arthritis.  

PubMed

Rheumatoid arthritis (RA) is associated with amino acid variants in multiple MHC molecules. The association to MHC class II (MHC-II) has been studied in several animal models of RA. In most cases these models depend on T cells restricted to a single immunodominant peptide of the immunizing Ag, which does not resemble the autoreactive T cells in RA. An exception is pristane-induced arthritis (PIA) in the rat where polyclonal T cells induce chronic arthritis after being primed against endogenous Ags. In this study, we used a mixed genetic and functional approach to show that RT1-Ba and RT1-Bb (RT1-B locus), the rat orthologs of HLA-DQA and HLA-DQB, determine the onset and severity of PIA. We isolated a 0.2-Mb interval within the MHC-II locus of three MHC-congenic strains, of which two were protected from severe PIA. Comparison of sequence and expression variation, as well as in vivo blocking of RT1-B and RT1-D (HLA-DR), showed that arthritis in these strains is regulated by coding polymorphisms in the RT1-B genes. Motif prediction based on MHC-II eluted peptides and structural homology modeling suggested that variants in the RT1-B P1 pocket, which likely affect the editing capacity by RT1-DM, are important for the development of PIA. PMID:25672758

Haag, Sabrina; Tuncel, Jonatan; Thordardottir, Soley; Mason, Daniel E; Yau, Anthony C Y; Dobritzsch, Doreen; Bäcklund, Johan; Peters, Eric C; Holmdahl, Rikard

2015-03-15

232

RT-isoPCR: nested, high multiplex mRNA amplification.  

PubMed

RT-isoPCR provides multiplex amplification of mRNA targets using a first-stage multiplex RT-PCR reaction with subsequent isothermal amplification for individual target loci detection. We demonstrate detection of 24 mRNA targets with high specificity and sensitivity without compromising sample variation or introducing biases between targets. PMID:23964356

Søe, Martin Jensen; Warthoe, Peter

2013-10-21

233

An improved multiplex IC-RT-PCR assay distinguishes nine strains of Potato virus Y  

Technology Transfer Automated Retrieval System (TEKTRAN)

A multiplex RT-PCR assay was previously developed to identify a group of PVY isolates with unusual recombinant structures, e.g. PVYNTN-NW and SYR-III, and to differentiate them from other PVY strains. In the present study, the efficiency of this multiplex RT-PCR assay was validated and extended cons...

234

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

ERIC Educational Resources Information Center

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

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

2011-01-01

235

RT-qPCR Normalization Genes in the Red Alga Chondrus Nathalie Kowalczyk1,2  

E-print Network

RT-qPCR Normalization Genes in the Red Alga Chondrus crispus Nathalie Kowalczyk1,2 , Sylvie it as a model for red algae, its genome has been sequenced, allowing the development of molecular tools, Colle´n J (2014) RT-qPCR Normalization Genes in the Red Alga Chondrus crispus. PLoS ONE 9(2): e86574

Paris-Sud XI, Université de

236

[Validation of nested RT-PCR method for detection of measles virus in clinical samples].  

PubMed

The aim of this study was to perform the validation of nested RT-PCR method for detection of measles virus genome in laboratory of Virology Department (NIZP-PZH) conditions. The PCR reactions were made with specific primers for nucleoprotein gene. The validation of nested RT-PCR assay includes determination of limit of detection (LOD), specificity, intra- and inter-assay precision and influence of materials from clinical samples on the PCR reaction. The detection limit of nested RT-PCR was 255-510 RNA MeV/ml. The obtained results reveal high precision and lack of substances present in clinical samples influence on the PCR reaction. The application of nested RT-PCR assay for viral genomes other than MeV gave negative results. The obtained results indicated that the nested RT-PCR fulfilled validation criteria and can be used for routine laboratory diagnostics of measles virus. PMID:19382605

Makówka, Agata; Siennicka, Joanna

2008-01-01

237

A Functional Interaction between gp41 and gp120 Is Observed for Monomeric but Not Oligomeric, Uncleaved HIV-1 Env gp140  

PubMed Central

The envelope glycoprotein (Env) is the sole antigenic feature on the surface of HIV and the target for the humoral immune system. Soluble, uncleaved gp140 Env constructs truncated at the transmembrane domain are being investigated intensively as potential vaccine immunogens by many groups, and understanding their structural properties is essential. We used hydrogen/deuterium-exchange mass spectrometry and small-angle X-ray scattering to probe structural order in a panel of commonly used gp140 constructs and matched gp120 monomers. We observed that oligomeric forms of uncleaved gp140, generally presumed to be trimeric, contain a protease-resistant form of gp41 akin to the postfusion, helical bundle conformation and appear to lack specific interactions between gp120 and gp41. In contrast, the monomeric form of gp140 shows significant stabilization of the gp120 inner domain imparted by the gp41 region, demonstrating excellent agreement with past mutagenesis studies. Moreover, the gp140 monomers respond to CD4 binding in manner that is consistent with the initial stages of Env activation: CD4 binding induces structural ordering throughout gp120 while loosening its association with gp41. The results indicate that uncleaved gp140 oligomers do not represent an authentic prefusion form of Env, whereas gp140 monomers isolated from the same glycoprotein preparations in many ways exhibit function and internal structural order that are consistent with expectations for certain aspects of native Env. gp140 monomers may thus be a useful reagent for advancing structural and functional studies. PMID:23966389

Guttman, Miklos

2013-01-01

238

HIV-1 Env C2-V4 diversification in a slow-progressor infant reveals a flat but rugged fitness landscape.  

PubMed

Human immunodeficiency virus type-1 (HIV-1) fitness has been associated with virus entry, a process mediated by the envelope glycoprotein (Env). We previously described Env genetic diversification in a Zambian, subtype C infected, slow-progressor child (1157i) in parallel with an evolving neutralizing antibody response. Because of the role the Variable-3 loop (V3) plays in transmission, cell tropism, neutralization sensitivity, and fitness, longitudinally isolated 1157i C2-V4 alleles were cloned into HIV-1NL4-3-eGFP and -DsRed2 infectious molecular clones. The fluorescent reporters allowed for dual-infection competitions between all patient-derived C2-V4 chimeras to quantify the effect of V3 diversification and selection on fitness. 'Winners' and 'losers' were readily discriminated among the C2-V4 alleles. Exceptional sensitivity for detection of subtle fitness differences was revealed through analysis of two alleles differing in a single synonymous amino acid. However, when the outcomes of N?=?33 competitions were averaged for each chimera, the aggregate analysis showed that despite increasing diversification and divergence with time, natural selection of C2-V4 sequences in this individual did not appear to be producing a 'survival of the fittest' evolutionary pattern. Rather, we detected a relatively flat fitness landscape consistent with mutational robustness. Fitness outcomes were then correlated with individual components of the entry process. Env incorporation into particles correlated best with fitness, suggesting a role for Env avidity, as opposed to receptor/coreceptor affinity, in defining fitness. Nevertheless, biochemical analyses did not identify any step in HIV-1 entry as a dominant determinant of fitness. Our results lead us to conclude that multiple aspects of entry contribute to maintaining adequate HIV-1 fitness, and there is no surrogate analysis for determining fitness. The capacity for subtle polymorphisms in Env to nevertheless significantly impact viral fitness suggests fitness is best defined by head-to-head competition. PMID:23638182

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

2013-01-01

239

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

SciTech Connect

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

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

2006-03-01

240

The two triiodothyronines (T3 and rT3). Thyroid biosynthesis of T3 and rT3 and peripheral metabolism of thyroxine (author's transl).  

PubMed

Thirty per cent of the iodine in thyroglobulin is present as iodothyronines. L-thyroxine (T4) represents 90-95% of hormonal iodine, 3,5,3'-triodo-L-thyronine (T3) contains at the most two per cent of it, 3,3'5'-triodo-L-thyronine (rT3) even less, as well as traces of 3,3'-diodo-L-thyronine. The plasma concentration of T4 is about 8 microgram per 100 ml, in the case of T3 it is 120 ng and 25 ng for rT3. The cell nucleus preferentially binds T3 and rT3 and there are also some specific mitochondrial proteins which possess a high affinity for T3. L-thyroxine is dehalogenated peripherically to T3, to take care of most of the requirements in T3. The enrichment of the plasma in rT3 has been shown to occur under various experimental and pathological conditoins. The blood level of T3 varies in inverse ratio to the level of rT3 and it shows that the peripheral formation of one is compensated for by the other. The excess of the prehormone T4 is metabolised as 3,5,3',5'-tetraiodothyroacetic acid (TetrAc); its level in the blood varies in the same way as the level of T3, in particular it decreases during starvation. PMID:900879

Roche, J; Michel, R

1977-01-01

241

ScriptingRT: A Software Library for Collecting Response Latencies in Online Studies of Cognition  

PubMed Central

ScriptingRT is a new open source tool to collect response latencies in online studies of human cognition. ScriptingRT studies run as Flash applets in enabled browsers. ScriptingRT provides the building blocks of response latency studies, which are then combined with generic Apache Flex programming. Six studies evaluate the performance of ScriptingRT empirically. Studies 1–3 use specialized hardware to measure variance of response time measurement and stimulus presentation timing. Studies 4–6 implement a Stroop paradigm and run it both online and in the laboratory, comparing ScriptingRT to other response latency software. Altogether, the studies show that Flash programs developed in ScriptingRT show a small lag and an increased variance in response latencies. However, this did not significantly influence measured effects: The Stroop effect was reliably replicated in all studies, and the found effects did not depend on the software used. We conclude that ScriptingRT can be used to test response latency effects online. PMID:23805326

Schubert, Thomas W.; Murteira, Carla; Collins, Elizabeth C.; Lopes, Diniz

2013-01-01

242

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

243

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

244

Glucose metabolic rate monitored with PET 18-F-FDG for tumor response to radiotherapy (RT) or radiochemotherapy (RT+CT) in lung cancer  

SciTech Connect

Changes in tumor glucose utilization as a result of RT or RT+CT may have prognostic implication. Therefore, the goals of this study were (1) to determine the correlation between tumor control probability (TCP) and the gradient of residual glucose metabolic rate (MRGlc) in response to RT or RT+CT and (2) to define the level of residual MRGlc that corresponds with 80% of TCP (FDG-TCP>80). Quantitative and dynamic positron emission tomography (PET) using the glucose analog, 2-(18F)-fluoro-2-deoxy-D-glucose (FDG) was performed before treatment in 28 patients with stage III non-small cell lung cancer, and this was repeated 2-3 weeks after full dose RT or RT+CT in 22 patients. Thirteen patients who had adequate follow up for the status of local tumor are the subject of this report. Fifteen, 6 mm, axial slices were acquired over a region that included the tumor. Scanning was performed continuously for two hours. From these data, decay corrected tumor and blood pool time-activity curves were obtained. The glucose metabolic rate was calculated for tissue using the Sokoloff model. The baseline value (mean) of tumor MRGlc was 0.4414 {plus_minus}0.896 {mu}mol/min/gm, and it was reduced to 0.0671 {plus_minus}0.034 {mu}mol/min/gm with treatment, p+0.011. Local tumor control was obtained in 5/5 with residual MRGlc of < 0.0412 {mu}mol/min/gm, 1/2 with MRGlc 0.0532 - 0.0542, 1/4 with 0.0763-0.0888 and 0/2 with 0.160.

Choi, N.C.; Hamberg, L.M.; Hunter, G.J. [Harvard Medical School, Boston, MA (United States)] [and others

1994-05-01

245

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

Microsoft Academic Search

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

Michael E Abram; Stefan G Sarafianos; Michael A Parniak

2010-01-01

246

DEVELOPMENT OF HOMOLOGOUS VIRAL INTERNAL CONTROLS FOR USE IN RT-PCR ASSAYS OF WATERBORNE ENTERIC VIRUSES  

EPA Science Inventory

Enteric viruses often contaminate water sources causing frequent outbreaks of gastroenteritis. Reverse transcription-polymerase chain reaction (RT-PCR) assays are commonly used for detection of human enteric viruses in environmental and drinking water samples. RT-PCR provides ...

247

Complexes of HIV-1 RT, NNRTI and RNA/DNA hybrid reveal a structure compatible with RNA degradation  

PubMed Central

Structures of type-1 human immunodeficiency virus (HIV-1) reverse transcriptase (RT) have been determined in several forms, but only one contains an RNA/DNA hybrid. Here we report three structures of HIV-1 RT complexed with a non-nucleotide RT inhibitor (NNRTI) and an RNA/DNA hybrid. In the presence of an NNRTI, the RNA/DNA structure differs from all prior nucleic acid bound to RT including the RNA/DNA hybrid. The enzyme structure also differs from all previous RT–DNA complexes. As a result, the hybrid has ready access to the RNase H active site. These observations indicate that an RT–nucleic acid complex may adopt two structural states, one competent for DNA polymerization and the other for RNA degradation. RT mutations that confer drug resistance but are distant from the inhibitor-binding sites often map to the unique RT–hybrid interface that undergoes conformational changes between two catalytic states. PMID:23314251

Miller, Jennifer T.; Le Grice, Stuart F. J.; Yang, Wei

2014-01-01

248

University of soUthern California 2007The Dean's RepoRT  

E-print Network

University of soUthern California 2007The Dean's RepoRT USC Viterbi School of Engineering Students.26% of Viterbi under graduates were underrepresented minorities (Hispanic, Native American and African American

Zhou, Chongwu

249

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

250

Time Based Linux for Real-Time NOWs and MPI\\/RT  

Microsoft Academic Search

The Real-Time Message Passing Interface (MPI\\/RT) is a communication layer middleware standard that is aimed at providing guaranteed Quality of Service for data transfers on high performance networks. It poses \\

Manoj Apte; Srigurunath Chakravarthi; Anand Pillai; Anthony Skjellum; Xin Yan Zan

1999-01-01

251

Simultaneous detection and identification of four sugarcane viruses by one-step RT-PCR.  

PubMed

Sugarcane mosaic disease (SMD) caused by the Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV) and Sugarcane streak mosaic virus (SCSMV) and sugarcane yellow leaf disease (SYLD) caused by the Sugarcane yellow leaf virus (SCYLV) are the two most prevalent and economically important viral diseases of sugarcane. In this study, a one-step quadruplex reverse transcription (RT)-PCR method that employed virus-specific primers was developed for the simultaneous detection and differentiation of SCMV, SrMV, SCSMV and SCYLV. Several sets of primers for each target virus were evaluated for their sensitivity and specificity by simplex and quadruplex RT-PCR. The optimum primer combinations and concentrations, RT temperature and time, and PCR annealing temperature and extension time were determined for the quadruplex RT-PCR. The assay was then validated using sugarcane samples affected with SMD and/or SYLD collected from sugarcane breeding fields and farmers' fields in southern China. PMID:19646484

Xie, Yujia; Wang, Mingqiang; Xu, Donglin; Li, Ruhui; Zhou, Guohui

2009-12-01

252

RT @ OSU Research Research has long been part of the curriculum to become an advanced respiratory  

E-print Network

Decompensated Heart Failure Post-Discharge." Jenny Hsieh RRT. Advisors: Rami N Khayat MD, Sarah M Varekojis Ph Abstracts AARC Times AARC Education Annual Advance for Respiratory Care RT - The Journal for RCPs Focus

253

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

254

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

255

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

PubMed Central

Purpose Our study characterizes food and energy intake responses to long-term aerobic (AT) and resistance training (RT) during a controlled 8-month trial. Methods In the STRRIDE AT/RT trial, overweight/obese sedentary dyslipidemic men and women were randomized to AT (n = 39), RT (n = 38), or a combined treatment (AT/RT; n = 40) without any advice to change their food intakes. Quantitative food intake assessments (QDI) and food frequency questionnaires (FFQ) were collected at baseline (BEF) and after 8 mo. training (END); body mass (BM) and fat free mass (FFM) were also assessed. Results In AT and AT/RT, respectively, meaningful decreases in reported energy intake (REI) (?217 and ?202 kcal; p < 0.001) and in intakes of fat (?14.9 and ?14.9 g; p < 0.001, p = 0.004), protein (?8.3 and ?10.7 g; p = 0.002, p < 0.001), and carbohydrate (?28.1 and ?14.7 g; p = 0.001, p = 0.030) were found by FFQ. REI relative to FFM decreased (p < 0.001 and p=0.002) as did intakes of fat (?0.2 and ?0.3 g; p = 0.003 and p = 0.014) and protein (?0.1 and ?0.2 g; p = 0.005 and p < 0.001) in AT and AT/RT and carbohydrate (?0.5 g; p<0.003) in AT only. For RT, REI by QDI decreased (?3.0 kcal/kg FFM; p=0.046), as did fat intake (?0.2 g; p = 0.033). BM decreased in AT (?1.3 kg, p=0.006) and AT/RT (?1.5 kg, p = 0.001) but was unchanged (0.6 kg, p = 0.176) in RT. Conclusions Previously sedentary subjects completing 8 months of AT or AT/RT reduced their intakes of kcal and macronutrients and BM. In RT, fat intakes and REI (when expressed per FFM) decreased, BM was unchanged, and FFM increased. PMID:22525775

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

2012-01-01

256

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

PubMed Central

A transmission cluster of HIV-1 M:B was identified in 11 patients with a median age of 52 (range 26–65) in North-East Germany by C2V4 region sequencing of the env gene of HIV-1, who—except of one—were not aware of any risky behaviour. The 10 male and 1 female patients deteriorated immunologically, according to their information made available, within 4 years after a putative HIV acquisition. Nucleic acid sequence analysis showed a R5 virus in all patients and in 7 of 11 a crown motif of the V3 loop, GPGSALFTT, which is found rarely. Analysis of formation of this cluster showed that there is still a huge discrepancy between awareness and behaviour regarding HIV transmission in middle-aged patients, and that a local outbreak can be detected by nucleic acid analysis of the hypervariable env region. PMID:20217125

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

2010-01-01

257

COMPARISON BETWEEN REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION (RT-PCR) AND REAL-TIME RT-PCR FOR DETECTION OF FOOT-AND-MOUTH DISEASE VIRUS (FMDV)  

Technology Transfer Automated Retrieval System (TEKTRAN)

As part of Argentina's national preparedness for the rapid detection and diagnosis of foot-and-mouth disease (FMD) outbreakes, we have developed a reverse transcription polymerase chain reaction (RT-PCR) for the detection of FMDV. This assay that targeted the viral 3D polymerase-coding region speci...

258

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

259

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

Microsoft Academic Search

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

Kiyoyasu Ishikawa; Hideto Sekiguchi; Tomoe Ogino; Shoko Suzuki

1997-01-01

260

Rapid single-tube immunocapture RT-PCR for the detection of two yam potyviruses  

Microsoft Academic Search

An immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay has been developed for the detection of yam-infecting potyviruses. Based upon the same format two distinct simple tests have been developed, which allow the reliable diagnosis of yam mosaic virus and the tentatively named yam mild mosaic virus. By using immunocapture and a single-buffer RT-PCR reaction, the test can be performed

R. A Mumford; S. E Seal

1997-01-01

261

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

Microsoft Academic Search

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

Supaporn Wacharapluesadee; Thiravat Hemachudha

2007-01-01

262

A quantitative real-time RT-PCR assay for European eel tyrosine hydroxylase  

Microsoft Academic Search

Dopamine (DA) plays a key inhibitory role in pubertal development of the European eel, but how DAergic neuronal activity is regulated is not known in this species. In order to investigate the regulation of DA inhibition at the molecular level, we developed a quantitative real-time RT-PCR (qrtRT-PCR) assay, using the Light Cycler system, for the expression of eel tyrosine hydroxylase

Finn-Arne Weltzien; Catherine Pasqualini; Philippe Vernier; Sylvie Dufour

2005-01-01

263

Development of an RT-PCR detection method for mud crab reovirus  

Microsoft Academic Search

Mud crab reovirus (MCRV) causes high mortality in the cultivated mud crab. To control better an outbreak of this virus, a rapid, specific and sensitive detection method based on RT-PCR was developed. The MCRV detection method was designed based on the one-step and two-step RT-PCR which resulted in the amplification of predicted products of 433 and 304bp. The method is

Zhi-Xun Guo; Shao-Ping Weng; Guang Li; Siu-Ming Chan; Jian-Guo He

2008-01-01

264

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

265

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

PubMed Central

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-1?s AH susceptibility is significantly linked to the N-glycans at the Env C2 and V4 regions. For recombinant (r)AH expression, we evaluated a tobacco mosaic virus-based system in Nicotiana benthamiana plants as a means to facilitate molecular engineering and cost-effective mass production. Biochemical analysis and an Env-mediated syncytium formation assay demonstrated high-level expression of functional rAH within six days. Taken together, our study revealed AH's cross-clade anti-HIV-1 activity, apparent lack of side effects common to lectins, and robust producibility using plant biotechnology. These findings justify further efforts to develop rAH toward a candidate HIV-1 microbicide. PMID:20559567

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

2010-01-01

266

DNA prime-protein boost using subtype consensus Env was effective in eliciting neutralizing antibody responses against subtype BC HIV-1 viruses circulating in China.  

PubMed

Previously, we have shown that DNA prime-protein boost is effective in eliciting neutralizing antibodies (NAb) against randomly selected HIV-1 isolates. Given the genetic diversity of HIV-1 viruses and the unique predominant subtypes in different geographic regions, it is critical to test the DNA prime-protein boost approach against circulating viral isolates in key HIV endemic areas. In the current study, the same DNA prime-protein boost vaccine was used as in previous studies to investigate the induction of NAb responses against HIV-1 clade BC, a major subtype circulating in China. A codon optimized gp120-BC DNA vaccine, based on the consensus envelope (Env) antigen sequence of clade BC, was constructed and a stable CHO cell line expressing the same consensus BC gp120 protein was produced. The immunogenicity of this consensus gp120-BC was examined in New Zealand White rabbits by either DNA prime-protein boost or protein alone vaccination approaches. High levels of Env-specific antibody responses were elicited by both approaches. However, DNA prime-protein boost but not the protein alone immune sera contained significant levels of NAb against pseudotyped viruses expressing HIV-1 BC Env antigens. Furthermore, high frequencies of CD4 binding site-targeted antibodies were found in the DNA prime- protein boost rabbit sera indicating that the positive NAb may be the result of antibodies against conformationally sensitive epitopes on HIV-1 Env. The findings support that DNA prime-protein boost was effective in eliciting NAb against a key HIV-1 virus subtype in China. This result may lead to the development of regional HIV vaccines through this approach. PMID:23111170

Zhang, Mingshun; Zhang, Lu; Zhang, Chunhua; Hong, Kunxue; Shao, Yiming; Huang, Zuhu; Wang, Shixia; Lu, Shan

2012-11-01

267

DNA prime-protein boost using subtype consensus Env was effective in eliciting neutralizing antibody responses against subtype BC HIV-1 viruses circulating in China  

PubMed Central

Previously, we have shown that DNA prime-protein boost is effective in eliciting neutralizing antibodies (NAb) against randomly selected HIV-1 isolates. Given the genetic diversity of HIV-1 viruses and the unique predominant subtypes in different geographic regions, it is critical to test the DNA prime-protein boost approach against circulating viral isolates in key HIV endemic areas. In the current study, the same DNA prime-protein boost vaccine was used as in previous studies to investigate the induction of NAb responses against HIV-1 clade BC, a major subtype circulating in China. A codon optimized gp120-BC DNA vaccine, based on the consensus envelope (Env) antigen sequence of clade BC, was constructed and a stable CHO cell line expressing the same consensus BC gp120 protein was produced. The immunogenicity of this consensus gp120-BC was examined in New Zealand White rabbits by either DNA prime-protein boost or protein alone vaccination approaches. High levels of Env-specific antibody responses were elicited by both approaches. However, DNA prime-protein boost but not the protein alone immune sera contained significant levels of NAb against pseudotyped viruses expressing HIV-1 BC Env antigens. Furthermore, high frequencies of CD4 binding site-targeted antibodies were found in the DNA prime- protein boost rabbit sera indicating that the positive NAb may be the result of antibodies against conformationally sensitive epitopes on HIV-1 Env. The findings support that DNA prime-protein boost was effective in eliciting NAb against a key HIV-1 virus subtype in China. This result may lead to the development of regional HIV vaccines through this approach. PMID:23111170

Zhang, Mingshun; Zhang, Lu; Zhang, Chunhua; Hong, Kunxue; Shao, Yiming; Huang, Zuhu; Wang, Shixia; Lu, Shan

2012-01-01

268

Human Immunodeficiency Virus Type 1 Env Trimer Immunization of Macaques and Impact of Priming with Viral Vector or Stabilized Core Protein? †  

PubMed Central

Currently there is limited information about the quality of immune responses elicited by candidate human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env)-based immunogens in primates. Here we describe a comprehensive analysis of neutralizing antibody and T-cell responses obtained in cynomolgus macaques by three selected immunization regimens. We used the previously described YU2-based gp140 protein trimers administered in an adjuvant, preceded by two distinct priming strategies: either alphavirus replicon particles expressing matched gp140 trimers or gp120 core proteins stabilized in the CD4-bound conformation. The rationale for priming with replicon particles was to evaluate the impact of the expression platform on trimer immunogenicity. The stable core proteins were chosen in an attempt to expand selectively lymphocytes recognizing common determinants between the core and trimers to broaden the immune response. The results presented here demonstrate that the platform by which Env trimers were delivered in the priming (either protein or replicon vector) had little impact on the overall immune response. In contrast, priming with stable core proteins followed by a trimer boost strikingly focused the T-cell response on the core sequences of HIV-1 Env. The specificity of the T-cell response was distinctly different from that of the responses obtained in animals immunized with trimers alone and was shown to be mediated by CD4+ T cells. However, this regimen showed limited or no improvement in the neutralizing antibody responses, suggesting that further immunogen design efforts are required to successfully focus the B-cell response on conserved neutralizing determinants of HIV-1 Env. PMID:19004960

Mörner, Andreas; Douagi, Iyadh; Forsell, Mattias N. E.; Sundling, Christopher; Dosenovic, Pia; O'Dell, Sijy; Dey, Barna; Kwong, Peter D.; Voss, Gerald; Thorstensson, Rigmor; Mascola, John R.; Wyatt, Richard T.; Karlsson Hedestam, Gunilla B.

2009-01-01

269

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

PubMed

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

2015-01-01

270

Profiles of Human Serum Antibody Responses Elicited by Three Leading HIV Vaccines Focusing on the Induction of Env-Specific Antibodies  

Microsoft Academic Search

In the current report, we compared the specificities of antibody responses in sera from volunteers enrolled in three US NIH-supported HIV vaccine trials using different immunization regimens. HIV-1 Env-specific binding antibody, neutralizing antibody, antibody-dependent cell-mediated cytotoxicity (ADCC), and profiles of antibody specificity were analyzed for human immune sera collected from vaccinees enrolled in the NIH HIV Vaccine Trial Network (HVTN)

Michael Vaine; Shixia Wang; Qin Liu; James Arthos; David Montefiori; Paul Goepfert; M. Juliana McElrath; Shan Lu; Cheryl A. Stoddart

2010-01-01

271

Low pH Is Required for Avian Sarcoma and Leukosis Virus Env-Induced Hemifusion and Fusion Pore Formation but Not for Pore Growth  

Microsoft Academic Search

Binding of avian sarcoma and leukosis virus (ASLV) to its cognate receptor on the cell surface causes conformational changes in its envelope protein (Env). It is currently debated whether low pH is required for ASLV infection. To elucidate the role of low pH, we studied the association between ASLV subgroup B (ASLV-B) and liposomes and fusion between effector cells expressing

G. B. Melikyan; R. J. O. Barnard; R. M. Markosyan; J. A. T. Young; F. S. Cohen

2004-01-01

272

Does inhibiting Sur1 complement rt-PA in cerebral ischemia?  

PubMed

Hemorrhagic transformation (HT) associated with recombinant tissue plasminogen activator (rt-PA) complicates and limits its use in stroke. Here, we provide a focused review on the involvement of matrix metalloproteinase 9 (MMP-9) in rt-PA-associated HT in cerebral ischemia, and we review emerging evidence that the selective inhibitor of the sulfonylurea receptor 1 (Sur1), glibenclamide (U.S. adopted name, glyburide), may provide protection against rt-PA-associated HT in cerebral ischemia. Glyburide inhibits activation of MMP-9, ameliorates edema formation, swelling, and symptomatic hemorrhagic transformation, and improves preclinical outcomes in several clinically relevant models of stroke, both without and with rt-PA treatment. A retrospective clinical study comparing outcomes in diabetic patients with stroke treated with rt-PA showed that those who were previously on and were maintained on a sulfonylurea fared significantly better than those whose diabetes was managed without sulfonylureas. Inhibition of Sur1 with injectable glyburide holds promise for ameliorating rt-PA-associated HT in stroke. PMID:22994227

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

2012-09-01

273

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

274

Natural and Enhanced Attenuation of Chlorinated Solvents Using RT3D  

SciTech Connect

RT3D (Reactive Transport in 3-Dimensions) is a reactive transport code that can be applied to model solute fate and transport for many different purposes. This document specifically addresses application of RT3D for modeling related to evaluation and implementation of Monitored Natural Attenuation (MNA). Selection of MNA as a remedy requires an evaluation process to demonstrate that MNA will meet the remediation goals. The U.S. EPA, through the Office of Solid Waste and Emergency Response (OSWER) Directive 9200.4?17P, provides the regulatory context for the evaluation and implementation of MNA. In a complementary fashion, the context for using fate and transport modeling as part of MNA evaluation is described in the EPA?s technical protocol for chlorinated solvent MNA, the Scenarios Evaluation Tool for Chlorinated Solvent MNA, and in this document. The intent of this document is to describe (1) the context for applying RT3D for chlorinated solvent MNA and (2) the attenuation processes represented in RT3D, (3) dechlorination reactions that may occur, and (4) the general approach for using RT3D reaction modules (including a summary of the RT3D reaction modules that are available) to model fate and transport of chlorinated solvents as part of MNA or for combinations of MNA and selected types of active remediation.

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

2006-07-25

275

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

NASA Technical Reports Server (NTRS)

The Short-term Prediction Research and Transition Center (SPoRT) is a collaborative partnership between NASA and operational forecasting entities, including a number of National Weather Service offices. SPoRT transitions real-time NASA products and capabilities to its partners to address specific operational forecast challenges. One challenge that forecasters face is applying convection-allowing numerical models to predict mesoscale convective weather. In order to address this specific forecast challenge, SPoRT produces real-time mesoscale model forecasts using the Weather Research and Forecasting (WRF) model that includes unique NASA products and capabilities. Currently, the SPoRT configuration of the WRF model (SPoRT-WRF) incorporates the 4-km Land Information System (LIS) land surface data, 1-km SPoRT sea surface temperature analysis and 1-km Moderate resolution Imaging Spectroradiometer (MODIS) greenness vegetation fraction (GVF) analysis, and retrieved thermodynamic profiles from the Atmospheric Infrared Sounder (AIRS). The LIS, SST, and GVF data are all integrated into the SPoRT-WRF through adjustments to the initial and boundary conditions, and the AIRS data are assimilated into a 9-hour SPoRT WRF forecast each day at 0900 UTC. This study dissects the overall impact of the NASA datasets and the individual surface and atmospheric component datasets on daily mesoscale forecasts. A case study covering the super tornado outbreak across the Ce ntral and Southeastern United States during 25-27 April 2011 is examined. Three different forecasts are analyzed including the SPoRT-WRF (NASA surface and atmospheric data), the SPoRT WRF without AIRS (NASA surface data only), and the operational National Severe Storms Laboratory (NSSL) WRF (control with no NASA data). The forecasts are compared qualitatively by examining simulated versus observed radar reflectivity. Differences between the simulated reflectivity are further investigated using convective parameters along with model soundings to determine the impacts of the various NASA datasets. Additionally, quantitative evaluation of select meteorological parameters is performed using the Meteorological Evaluation Tools model verification package to compare forecasts to in situ surface and upper air observations.

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

2012-01-01

276

Nedd4-Mediated Increase in HIV-1 Gag and Env Proteins and Immunity following DNA-Vaccination of BALB/c Mice  

PubMed Central

The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins, are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteins with intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 budding have been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function for Nedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 following SHIVSF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immune modulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4+ and CD8+ T cells following SHIVSF162P3-infection of naïve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4 demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca2+/calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfection of Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gag or Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularly with plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 but not anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFN? but had a trend of increased Gag-specific IL-6, IL-17A and TNF? that was not seen following Env stimulation. Based on our initial findings, Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants further investigation. PMID:24614057

Lewis, Brad; Whitney, Stephen; Hudacik, Lauren; Galmin, Lindsey; Huaman, Maria Cecilia; Cristillo, Anthony D.

2014-01-01

277

HIV Type 1 Env Precursor Cleavage State Affects Recognition by Both Neutralizing and Nonneutralizing gp41 Antibodies  

PubMed Central

Abstract HIV-1 is relatively resistant to antibody-mediated neutralization; however, rare antibodies to the exterior envelope glycoprotein, gp120, and the transmembrane glycoprotein, gp41, can neutralize a broad array of isolates. Two antibodies, 2F5 and 4E10, are directed against the gp41 membrane proximal external region (MPER); however, the kinetic neutralization signature of these antibodies remains unresolved. Previously, we reported that the fully cleaved, cell surface envelope glycoproteins (Env) derived from the primary isolate, JR-FL, are well recognized exclusively by gp120-directed neutralizing ligands and not by nonneutralizing gp120 antibodies. However, the gp120 nonneutralizing antibodies can recognize HIV spikes that are rendered fully cleavage defective by site-directed mutagenesis. Here, we extended such analysis to gp41 neutralizing and nonneutralizing antibodies and, relative to the rules of gp120-specific antibody recognition, we observed marked contrasts. Similar to gp120 recognition, the nonneutralizing gp41 cluster 1 or cluster 2 antibodies bound much more efficiently to cleavage-defective spikes when compared to their recognition of cleaved spikes. In contrast to gp120 neutralizing antibody recognition, the broadly neutralizing gp41 antibodies 2F5 and 4E10, like the nonneutralizing gp41 antibodies, did not efficiently recognize the predominantly cleaved, primary isolate JR-FL spikes. However, if the spikes were rendered cleavage defective, recognition by both the neutralizing and nonneutralizing ligand markedly increased. CD4 interaction with the cleaved spikes markedly increased recognition by most nonneutralizing gp41 antibodies, whereas such treatment had a minimal increase of 2F5 and 4E10 recognition. These data indicate again the profound influence that cleavage imposes on the quaternary packing of primary isolate spikes and have important implications for soluble trimer candidate immunogens. PMID:21158699

Chakrabarti, Bimal K.; Pancera, Marie; Phogat, Sanjay; O'Dell, Sijy; McKee, Krisha; Guenaga, Javier; Robinson, James; Mascola, John

2011-01-01

278

Generation and Evaluation of Clade C Simian-Human Immunodeficiency Virus Challenge Stocks  

PubMed Central

ABSTRACT The development of a panel of mucosally transmissible simian-human immunodeficiency virus (SHIV) challenge stocks from multiple virus clades would facilitate preclinical evaluation of candidate HIV-1 vaccines and therapeutics. The majority of SHIV stocks that have been generated to date have been derived from clade B HIV-1 env sequences from viruses isolated during chronic infection and typically required serial animal-to-animal adaptation for establishing mucosal transmissibility and pathogenicity. To capture essential features of mucosal transmission of clade C viruses, we produced a series of SHIVs with early clade C HIV-1 env sequences from acutely HIV-1-infected individuals from South Africa. SHIV-327c and SHIV-327cRM expressed env sequences that were 99.7 to 100% identical to the original HIV-1 isolate and did not require in vivo passaging for mucosal infectivity. These challenge stocks infected rhesus monkeys efficiently by both intrarectal and intravaginal routes, replicated to high levels during acute infection, and established chronic setpoint viremia in 13 of 17 (76%) infected animals. The SHIV-327cRM challenge stock was also titrated for both single, high-dose intrarectal challenges and repetitive, low-dose intrarectal challenges in rhesus monkeys. These SHIV challenge stocks should facilitate the preclinical evaluation of vaccines and other interventions aimed at preventing clade C HIV-1 infection. IMPORTANCE We describe the development of two related clade C SHIV challenge stocks. These challenge stocks should prove useful for preclinical testing of vaccines and other interventions aimed at preventing clade C HIV-1 infection. PMID:25473043

Chang, Hui-Wen; Tartaglia, Lawrence J.; Whitney, James B.; Lim, So-Yon; Sanisetty, Srisowmya; Lavine, Christy L.; Seaman, Michael S.; Rademeyer, Cecelia; Williamson, Carolyn; Ellingson-Strouss, Katharine; Stamatatos, Leonidas; Kublin, James

2014-01-01

279

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

SciTech Connect

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

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

2009-07-01

280

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

PubMed Central

Arboviruses represent a serious problem to public health and agriculture worldwide. Fast, accurate identification of the viral agents of arbovirus-associated disease is essential for epidemiological surveillance and laboratory investigation. We developed a cost-effective, rapid, and highly sensitive one-step “triplex RT-PCR enzyme hybridization” assay for simultaneous detections of Japanese Encephallitis virus (JEV, Flaviviridae), Getah virus (GETV, Togaviridae), and Tahyna virus (TAHV, Bunyaviridae) using three pairs of primers to amplify three target sequences in one RT-PCR reaction. The analytical sensitivity of this assay was 1 PFU/mL for JEV, 10 PFU/mL for GETV, and 10 PFU/mL for TAHV. This assay is significantly more rapid and less expensive than the traditional serological detection and single RT-PCR reaction methods. When “triplex RT-PCR enzyme hybridization” was applied to 29 cerebrospinal fluid (CSF) samples that were JEV-positive by normal RT-PCR assay, all samples were strongly positive for JEV, but negative for GETV and TAHV, demonstrating a good sensitivity, specificity, and performance at CSF specimen detection. PMID:22684472

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

2013-01-01

281

Numerical simulations of Rayleigh-Taylor (RT) turbulence with complex acceleration history  

NASA Astrophysics Data System (ADS)

Complex acceleration histories of an RT unstable interface are important in validating turbulent mix models. Of particular interest are alternating stages of acceleration and deceleration, since the the associated demixing is a discriminating test of such models. We have performed numerical simulations of a turbulent RT mixing layer subjected to two stages of acceleration separated by a stage of deceleration. The profile was chosen from earlier Linear Electric Motor experiments with which we compare our results. The acceleration phases produce classical RT unstable growth (t^2) with growth rates comparable to earlier results of turbulent RT simulations. The calculations are challenging as dominant bubbles become shredded as they reverse direction in response to the reversal in g, placing increased demands on numerical resolution. The shredding to small scales is accompanied by a peaking of the molecular mixing during the RT stable stage. In general, we find that simulations agree with experiments when initialized with broadband initial perturbations, but not for an annular shell. Other effects such as the presence of surface tension in the LEM experiments (but not in our simulations) further complicate this picture.

Ramaprabhu, Praveen; Dimonte, Guy; Andrews, Malcolm

2007-11-01

282

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

283

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

PubMed Central

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a valuable tool for measuring gene expression in biological samples. However, unique challenges are encountered when studies are performed on cells microdissected from tissues derived from animal models or the clinic, including specimen related issues, variability of RNA template quality and quantity, and normalization. qRT-PCR using small amounts of mRNA derived from dissected cell populations requires adaptation of standard methods to allow meaningful comparisons across sample sets. The protocol described here presents the rationale, technical steps, normalization strategy, and data analysis necessary to generate reliable gene expression measurements of transcripts from dissected samples. The entire protocol from tissue microdissection through qRT-PCR analysis requires approximately 16 hours. PMID:19478806

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

2009-01-01

284

[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

285

ULTRASOUND-ENHANCED rt-PA THROMBOLYSIS IN AN EX VIVO PORCINE CAROTID ARTERY MODEL  

PubMed Central

Ultrasound is known to enhance recombinant tissue plasminogen activator (rt-PA) thrombolysis. In this study, occlusive porcine whole blood clots were placed in flowing plasma within living porcine carotid arteries. Ultrasonically induced stable cavitation was investigated as an adjuvant to rt-PA thrombolysis. Aged, retracted clots were exposed to plasma alone, plasma containing rt-PA (7.1 ± 3.8 ?g/mL) or plasma with rt-PA and Definity® ultrasound contrast agent (0.79 ± 0.47 ?L/mL) with and without 120-kHz continuous wave ultrasound at a peak-to-peak pressure amplitude of 0.44 MPa. An insonation scheme was formulated to promote and maximize stable cavitation activity by incorporating ultrasound quiescent periods that allowed for the inflow of Definity®-rich plasma. Cavitation was measured with a passive acoustic detector throughout thrombolytic treatment. Thrombolytic efficacy was measured by comparing clot mass before and after treatment. Average mass loss for clots exposed to rt-PA and Definity® without ultrasound (n = 7) was 34%, and with ultrasound (n = 6) was 83%, which constituted a significant difference (p < 0.0001). Without Definity® there was no thrombolytic enhancement by ultrasound exposure alone at this pressure amplitude (n = 5, p < 0.0001). In the low-oxygen environment of the ischemic artery, significant loss of endothelium occurred but no correlation was observed between arterial tissue damage and treatment type. Acoustic stable cavitation nucleated by an infusion of Definity® enhances rt-PA thrombolysis without apparent treatment-related damage in this ex vivo porcine carotid artery model. PMID:21723448

Hitchcock, Kathryn E.; Ivancevich, Nikolas M.; Haworth, Kevin J.; Caudell Stamper, Danielle N.; Vela, Deborah C.; Sutton, Jonathan T.; Pyne-Geithman, Gail J.; Holland, Christy K.

2014-01-01

286

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

287

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

288

1999-2008 Index.hu Rt. Minden jog fenntartva. A magyar szerverek tbbsge vdtelen  

E-print Network

©1999-2008 Index.hu Rt. Minden jog fenntartva. A magyar szerverek többsége védtelen Index - ugyelet@mail.index..hu 2008. augusztus 11., hétf 11:45 A BME laborjában a magyar DNS-szerverek védettségét vizsgálták meg. Az. A BME Híradástechnikai tanszékének Crysys laboratóriumában a magyar szakemberek felmérték [1], hogy mi

Bencsáth, Boldizsár

289

Selection of a simian-human immunodeficiency virus strain resistant to a vaginal microbicide in macaques.  

PubMed

PSC-RANTES binds to CCR5, inhibits human immunodeficiency virus type 1 (HIV-1) entry, and has been shown as a vaginal microbicide to protect rhesus macaques from a simian-human immunodeficiency virus chimera (SHIV(SF162-p3)) infection in a dose-dependent manner. In this study, env gene sequences from SHIV(SF162-p3)-infected rhesus macaques treated with PSC-RANTES were analyzed for possible drug escape variants. Two specific mutations located in the V3 region of gp120 (K315R) and C-helical domain of gp41 (N640D) were identified in a macaque (m584) pretreated with a 100 microM dose of PSC-RANTES. These two env mutations were found throughout infection (through week 77) but were found at only low frequencies in the inoculating SHIV(SF162-p3) stock and in the other SHIV(SF162-p3)-infected macaques. HIV-1 env genes from macaque m584 (env(m584)) and from inoculating SHIV(SF162-p3) (env(p3)) were cloned into an HIV-1 backbone. Increases in 50% inhibitory concentrations to PSC-RANTES with env(m584) were modest (sevenfold) and most pronounced in cells expressing rhesus macaque CCR5 as compared to human CCR5. Nonetheless, virus harboring env(m584), unlike inoculating virus env(p3), could replicate even at the highest tissue culture PSC-RANTES concentrations (100 nM). Dual-virus competitions revealed a dramatic increase in fitness of chimeric virus containing env(m584) (K315R/N640D) over that containing env(p3), but again, only in rhesus CCR5-expressing cells. This study is the first to describe the immediate selection and infection of a drug-resistant SHIV variant in the face of a protective vaginal microbicide, PSC-RANTES. This rhesus CCR5-specific/PSC- RANTES resistance selection is particularly alarming given the relative homogeneity of the SHIV(SF162-p3) stock compared to the potential exposure to a heterogeneous HIV-1 population in human transmission. PMID:19279098

Dudley, Dawn M; Wentzel, Jennifer L; Lalonde, Matthew S; Veazey, Ronald S; Arts, Eric J

2009-05-01

290

Massively parallel pyrosequencing highlights minority variants in the HIV-1 env quasispecies deriving from lymphomonocyte sub-populations  

PubMed Central

Background Virus-associated cell membrane proteins acquired by HIV-1 during budding may give information on the cellular source of circulating virions. In the present study, by applying immunosorting of the virus and of the cells with antibodies targeting monocyte (CD36) and lymphocyte (CD26) markers, it was possible to directly compare HIV-1 quasispecies archived in circulating monocytes and T lymphocytes with that present in plasma virions originated from the same cell types. Five chronically HIV-1 infected patients who underwent therapy interruption after prolonged HAART were enrolled in the study. The analysis was performed by the powerful technology of ultra-deep pyrosequencing after PCR amplification of part of the env gene, coding for the viral glycoprotein (gp) 120, encompassing the tropism-related V3 loop region. V3 amino acid sequences were used to establish heterogeneity parameters, to build phylogenetic trees and to predict co-receptor usage. Results The heterogeneity of proviral and viral genomes derived from monocytes was higher than that of T-lymphocyte origin. Both monocytes and T lymphocytes might contribute to virus rebounding in the circulation after therapy interruptions, but other virus sources might also be involved. In addition, both proviral and circulating viral sequences from monocytes and T lymphocytes were predictive of a predominant R5 coreceptor usage. However, minor variants, segregating from the most frequent quasispecies variants, were present. In particular, in proviral genomes harboured by monocytes, minority variant clusters with a predicted X4 phenotype were found. Conclusion This study provided the first direct comparison between the HIV-1 quasispecies archived as provirus in circulating monocytes and T lymphocytes with that of plasma virions replicating in the same cell types. Ultra-deep pyrosequencing generated data with some order of magnitude higher than any previously obtained with conventional approaches. Next generation sequencing allowed the analysis of previously inaccessible aspects of HIV-1 quasispecies, such as co-receptor usage of minority variants present in archived proviral sequences and in actually replicating virions, which may have clinical and therapeutic relevance. PMID:19216757

Rozera, Gabriella; Abbate, Isabella; Bruselles, Alessandro; Vlassi, Crhysoula; D'Offizi, Gianpiero; Narciso, Pasquale; Chillemi, Giovanni; Prosperi, Mattia; Ippolito, Giuseppe; Capobianchi, Maria R

2009-01-01

291

Structure of 2G12 Fab2 in Complex with Soluble and Fully Glycosylated HIV-1 Env by Negative-Stain Single-Particle Electron Microscopy  

PubMed Central

ABSTRACT The neutralizing anti-HIV-1 antibody 2G12 is of particular interest due to the sterilizing protection it provides from viral challenge in animal models. 2G12 is a unique, domain-exchanged antibody that binds exclusively to conserved N-linked glycans that form the high-mannose patch on the gp120 outer domain centered on a glycan at position N332. Several glycans in and around the 2G12 epitope have been shown to interact with other potent, broadly neutralizing antibodies; therefore, this region constitutes a supersite of vulnerability on gp120. While crystal structures of 2G12 and 2G12 bound to high-mannose glycans have been solved, no structural information that describes the interaction of 2G12 with gp120 or the Env trimer is available. Here, we present a negative-stain single-particle electron microscopy reconstruction of 2G12 Fab2 in complex with a soluble, trimeric Env at ?17-? resolution that reveals the antibody's interaction with its native and fully glycosylated epitope. We also mapped relevant glycans in this epitope by fitting high-resolution crystal structures and by performing neutralization assays of glycan knockouts. In addition, a reconstruction at ?26 ? of the ternary complex formed by 2G12 Fab2, soluble CD4, and Env indicates that 2G12 may block membrane fusion by induced steric hindrance upon primary receptor binding, thereby abrogating Env's interaction with coreceptor(s). These structures provide a basis for understanding 2G12 binding and neutralization, and our low-resolution model and glycan assignments provide a basis for higher-resolution studies to determine the molecular nature of the 2G12 epitope. IMPORTANCE HIV-1 is a human virus that results in the deaths of millions of people around the world each year. While there are several effective therapeutics available to prolong life, a vaccine is the best long-term solution for curbing this global epidemic. Here, we present structural data that reveal the viral binding site of one of the first HIV-1-neutralizing antibodies isolated, 2G12, and provide a rationale for its effectiveness. These structures provide a basis for higher-resolution studies to determine the molecular nature of the 2G12 epitope, which will aid in vaccine design and antibody-based therapies. PMID:24965454

Murin, Charles D.; Julien, Jean-Philippe; Sok, Devin; Stanfield, Robyn L.; Khayat, Reza; Cupo, Albert; Moore, John P.; Burton, Dennis R.; Wilson, Ian A.

2014-01-01

292

Development of a hemi-nested RT-PCR method for the specific determination of European Bat Lyssavirus 1  

Microsoft Academic Search

A simplified hemi-nested reverse transcriptase polymerase chain reaction (hnRT-PCR) has been developed to determine specifically the European Bat Lyssavirus 1 (EBLV-1) nucleoprotein gene. The specificity of this method was determined by using the seven genotypes of lyssavirus by RT-PCR, Southern blot and sequence analysis. Compared to the rabies diagnostic methods, the hnRT-PCR showed a higher sensitivity for the detection of

E Picard-Meyer; V Bruyère; J Barrat; E Tissot; M. J Barrat; F Cliquet

2004-01-01

293

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

294

Modeling benzene plume elongation mechanisms exerted by ethanol using RT3D with a general  

E-print Network

Modeling benzene plume elongation mechanisms exerted by ethanol using RT3D with a general substrate ethanol on benzene fate and transport in fuel-contaminated groundwater and to discern the most influential benzene plume elongation mechanisms. The model, developed as a module for the Reactive Transport in 3

Alvarez, Pedro J.

295

MuSA.RT: Music on the Spiral Array . Real-Time Elaine Chew  

E-print Network

by the author/owner. MM'03, November 2­8, 2003, Berkeley, California, USA. ACM 1-58113-722-2/03/0011. Figure 1 Interfaces and Presentation]: Sound and Music Computing--Systems; I.5.5 [Pattern Recogni- tionMuSA.RT: Music on the Spiral Array . Real-Time Elaine Chew and Alexandre R.J. Franc

Southern California, University of

296

Type-A influenza virus detection and quantitation by real-time RT-PCR  

Technology Transfer Automated Retrieval System (TEKTRAN)

Real-time RT-PCR (RRT-PCR) is a relatively new technology which has been used for AIV detection since the early 2000’s for routine surveillance, during outbreaks and for research. Some of the advantages of RRT-PCR are: quantitative nature, scalability, cost, high sensitivity, high specificity, and ...

297

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

E-print Network

that limit their clinical efficacy. IFA-based vaccines are water-in-oil emulsions of antigen in mineral oila rt i c l e s nature medicine VOLUME 19 | NUMBER 4 | APRIL 2013 465 Cancer vaccines have shown remains why many vaccinated patients show increased numbers of circulat- ing tumor-specific T cells

298

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...

299

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

EPA Science Inventory

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

300

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

301

AnnuAl RepoRt 2009-2010 Identity, Privacy and Security Institute  

E-print Network

institution exploring issues of Identity, Privacy and Security. Through the administrative support, policy, and social implications of today's identity, privacy, and security issues but also considersAnnuAl RepoRt 2009-2010 Identity, Privacy and Security Institute #12;IPSI IdentIty, Pr

Prodiæ, Aleksandar

302

Regional Data Assimilation of AIRS Profiles and Radiances at the SPoRT Center  

NASA Technical Reports Server (NTRS)

This slide presentation reviews the Short Term Prediction Research and Transition (SPoRT) Center's mission to improve short-term weather prediction at the regional and local scale. It includes information on the cold bias in Weather Research and Forcasting (WRF), troposphere recordings from the Atmospheric Infrared Sounder (AIRS), and vertical resolution of analysis grid.

Zavodsky, Brad; Chou, Shih-hung; Jedlovec, Gary

2009-01-01

303

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.

304

Multiplex nested RT-PCR for the detection of porcine enteric viruses  

Microsoft Academic Search

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine group A rotavirus (PRV-A) are major viruses causing enteric diseases of piglets. A multiplex nested reverse transcription polymerase chain reaction (multiplex nested RT-PCR) was developed for the detection of these viruses in field samples from piglets with diarrhea. A mixture of (1) three external pairs of primers, yielding in

Abid Nabil Ben Salem; A. Chupin Sergei; P. Bjadovskaya Olga; G. Andreeva Olga; Aouni Mahjoub; B. Prokhvatilova Larissa

2010-01-01

305

nature biotechnology VOLUME 30 NUMBER 8 AUGUST 2012 777 A rt i c l e s  

E-print Network

for single-cell microarray studies6 has been adapted for mRNA-Seq and used to generate transcriptome datanature biotechnology VOLUME 30 NUMBER 8 AUGUST 2012 777 A rt i c l e s Analyses of transcriptomes through massively parallel sequencing of cDNAs (mRNA-Seq) generates millions of short sequence fragments

Cai, Long

306

46 VOLUME 31 NUMBER 1 JANUARY 2013 nature biotechnology A rt i c l e s  

E-print Network

sequencers such as the Illumina MiSeq. We use Cuffdiff 2 to assess the response to knockdown of HOXA146 VOLUME 31 NUMBER 1 JANUARY 2013 nature biotechnology A rt i c l e s RNA-seq is a high- ment1­4. Recent studies have shown RNA-seq to be more accurate over a larger dynamic range of gene

Cai, Long

307

Original article Construction of an internal standard used in RT nested  

E-print Network

Borna Disease (BD) is an infectious immunopathological disease of the central nervous system (CNS. The results confirmed that nervous tissue has an inhibitory effect on RT-PCR, which supports the necessity natural hosts are classically described as horses and sheep but infections in donkeys, cattle, rabbits

Boyer, Edmond

308

Power Minimization Techniques at the RT-Level and Afshin Abdollahi and Massoud Pedram  

E-print Network

1 Power Minimization Techniques at the RT-Level and Below Afshin Abdollahi and Massoud Pedram Dept. of Electrical Engineering University of Southern California Los Angeles, CA 90089 U.S.A. Abstract ­ Power consumption and power-related issues have become a first-order concern for most designs and loom

Pedram, Massoud

309

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

310

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.

311

RAPID COMMUNICATION CW DFB RT diode laser-based sensor for trace-gas detection  

E-print Network

RAPID COMMUNICATION CW DFB RT diode laser-based sensor for trace-gas detection of ethane using- moelectrically cooled (TEC), distributed feedback diode laser-based spectroscopic trace-gas sensor for ultra tunable diode laser absorption spectroscopy (TDLAS) and wavelength modulation spectroscopy

312

Cycle slip Detection in the context of rtK GPs positioning of lightweight UAVs  

E-print Network

148 Cycle slip Detection in the context of rtK GPs positioning of lightweight UAVs C. Eling1 , E for a reliable detection and repair of cycle slips. Keywords RTK GPS, UAV, direct georeferencing, cycle slip, this paper is focused on the cycle slip detection based on accelerometers in a Kalman filter. By means

Behnke, Sven

313

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...

314

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

315

DEVELOPMENT OF THE CUHTK 2004 RT04F MANDARIN CONVERSATIONAL TELEPHONE SPEECH TRANSCRIPTION SYSTEM  

E-print Network

DEVELOPMENT OF THE CUHTK 2004 RT04F MANDARIN CONVERSATIONAL TELEPHONE SPEECH TRANSCRIPTION SYSTEM M are applied to Mandarin conversational telephone speech recognition. However, since Mandarin is a tonal the resources and data that were used for the development of the Mandarin system. This work was supported

Gales, Mark

316

A Comparison of the MPCP, DPCP, and FMLP on LITMUS RT  

E-print Network

LITMUS RT , a multiprocessor real­time extension of Linux [8, 11, 12]. Our choice of Linux as a development platform was influenced by recent e#orts to introduce real­time­ oriented features in stock Linux,anderson}@cs.unc.edu Abstract. This paper presents a performance comparison of three mul­ tiprocessor real­time locking

Anderson, James

317

Exploring MultiObjective Fitness Functions and Compositions of Different Fitness Functions in rtNEAT  

E-print Network

Exploring MultiObjective Fitness Functions and Compositions of Different Fitness Functions in rtNEAT KJ Bredder and Cole Harbeck Abstract In machine learning, the fitness function is an incredibly to identify a set of sub-goal that can be explicitly rewarded as part of the fitness function. However

Meeden, Lisa A.

318

Real-time RT-PCR Assay for Detection and Differentiation of Citrus Tristeza Virus Isolates  

Technology Transfer Automated Retrieval System (TEKTRAN)

Multiplex one step real time RT-PCR assays using TaqMan probes were developed for detection and strain differentiation of Citrus tristeza virus (CTV). For broad spectrum CTV detection, a TaqMan primer and Cy5-labeled probe were designed using CP gene sequences. An internal control was developed us...

319

Real-time RT-PCR assay for detection and differentiation of Citrus tristeza virus isolates  

Technology Transfer Automated Retrieval System (TEKTRAN)

For universal detection of Citrus tristeza virus (CTV) strains by real time RT-PCR, a protocol was developed based on a set of primers and a Cy5-labeled TaqMan probe. This test included primers and a TET-labeled TaqMan probe selected on the mitochondrial nad5 gene for the simultaneous detection of ...

320

Identification and evaluation of reference genes for qRT-PCR normalization in Ganoderma lucidum.  

PubMed

Quantitative real-time reverse transcription PCR (qRT-PCR) is a rapid, sensitive, and reliable technique for gene expression studies. The accuracy and reliability of qRT-PCR results depend on the stability of the reference genes used for gene normalization. Therefore, a systematic process of reference gene evaluation is needed. Ganoderma lucidum is a famous medicinal mushroom in East Asia. In the current study, 10 potential reference genes were selected from the G. lucidum genomic data. The sequences of these genes were manually curated, and primers were designed following strict criteria. The experiment was conducted using qRT-PCR, and the stability of each candidate gene was assessed using four commonly used statistical programs-geNorm, NormFinder, BestKeeper, and RefFinder. According to our results, PP2A was expressed at the most stable levels under different fermentation conditions, and RPL4 was the most stably expressed gene in different tissues. RPL4, PP2A, and ?-tubulin are the most commonly recommended reference genes for normalizing gene expression in the entire sample set. The current study provides a foundation for the further use of qRT-PCR in G. lucidum gene analysis. PMID:24013612

Xu, Jiang; Xu, ZhiChao; Zhu, YingJie; Luo, HongMei; Qian, Jun; Ji, AiJia; Hu, YuanLei; Sun, Wei; Wang, Bo; Song, JingYuan; Sun, Chao; Chen, ShiLin

2014-01-01

321

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

322

R.T. deSouza, Indiana University Careers in Academia seminar UIUC  

E-print Network

advice #12;R.T. deSouza, Indiana University Careers in Academia seminar UIUC Only 26% scored high enough on the science test to indicate they are likely to succeed in college biology. We of the solution is to increase the accountability of students in their out of classroom time. Ã? Homework

de Souza, Romualdo T.

323

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

324

Application of a Master Equation for Quantitative mRNA Analysis Using qRT-PCR  

Technology Transfer Automated Retrieval System (TEKTRAN)

The qRT-PCR has been widely accepted as the assay of choice for mRNA quantification. Gene expression as measured by mRNA dynamics varies in response to different conditions and environmental stimuli. For conventional practice, housekeeping genes have been applied as internal reference for data nor...

325

A Comprehensive Approach to RtI: Embedding Universal Design for Learning and Technology  

ERIC Educational Resources Information Center

Response to intervention (RtI) provides tiered levels of supports to all students and allows for increasingly more intensive and individualized instruction. Similarly, universal design for learning (UDL) addresses needs of students by proactively planning for instructional, environmental, and technology supports to allow all students to…

Basham, James D.; Israel, Maya; Graden, Janet; Poth, Rita; Winston, Markay

2010-01-01

326

USE OF A MULTIPLEX RT-PCR TO DIFFERENTIATE LENTOGENIC NDV FROM END  

Technology Transfer Automated Retrieval System (TEKTRAN)

A multiplex RT-PCR has been designed to differentiate lentogenic Newcastle disease viruses from exotic Newcastle disease (END). Using computer sequence analysis and published sequences (GenBank), we have constructed lentogenic (B1/LaSota) and END (CA02)-specific primer sets. The primers amplify re...

327

Twists in Hopf algebras and RT 1 T 2= T 2 T 1 relations  

NASA Astrophysics Data System (ADS)

Let R be a matrix unitary quasi-classical solution of the Yang-Baxter equation. Considering an associative algebra defined by the relation RT 1 T 2= T 2 T 1 we find a universal twist F such that R is the image of R= F 21 F -1 in the vector representation.

Kulish, P. P.; Stolin, A.

2002-11-01

328

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

329

Minimally Invasive Surgery plus rt-PA for Intracerebral Hemorrhage Evacuation (MISTIE) Decreases Perihematomal Edema  

PubMed Central

Background and Purpose Perihematomal edema (PHE) can worsen outcomes following ICH. Reports suggest that blood degradation products lead to PHE. We hypothesized that hematoma evacuation will reduce PHE volume and that treatment with rt-PA will not exacerbate it. Methods MISTIE II tested safety and efficacy of hematoma evacuation after ICH. We conducted a semi-automated, computerized volumetric analysis on CT to assess impact of hematoma removal on PHE and 2) effects of rt-PA on PHE. Volumetric analyses were performed on Baseline Stability (BLS) and End of Treatment (EOT) scans. Results Seventy-nine surgical and 39 medical patients from MISTIE II were analyzed. Mean hematoma volume at EOT was 19.6±14.5 cc for the surgical cohort and 40.7±13.9 cc for the medical cohort (p<0.001). Edema volume at EOT was lower for the surgical cohort: 27.7±13.3 cc than medical cohort: 41.7±14.6 cc (p<0.001). Graded effect of clot removal on PHE was observed when patients with >65%, 20-65%, and <20% ICH removed were analyzed (p<0.001). Positive correlation between PHE reduction and percent of ICH removed was identified (?=0.658; p<0.001). In the surgical cohort, 69 patients underwent surgical aspiration and rt-PA (S+rt-PA) while 10 underwent surgical aspiration only (SO). Both cohorts achieved similar clot reduction: S+rt-PA, 18.9±14.5 cc; and SO, 24.5±14.0 cc (p=0.26). Edema at EOT in S+rt-PA was 28.1±13.8 cc and 24.4±8.6 cc in SO (p=0.41). Conclusions Hematoma evacuation is associated with significant reduction in PHE. Furthermore, PHE does not appear to be exacerbated by rt-PA, making such neurotoxic effects unlikely when the drug is delivered to intracranial clot. Clinical Trial Registration Information URL: http://clinicaltrials.gov/ct2/show/NCT00224770?term=MISTIE&rank=1 Clinicaltrials.gov ID: NCT00224770 PMID:23391763

Mould, W. Andrew; Carhuapoma, J. Ricardo; Muschelli, John; Lane, Karen; Morgan, Timothy C; McBee, Nichol A; Bistran-Hall, Amanda J; Ullman, Natalie L; Vespa, Paul; Martin, Neil A; Awad, Issam; Zuccarello, Mario; Hanley, Daniel F.

2014-01-01

330

Identification of Reference Genes for qRT-PCR Analysis in Yesso Scallop Patinopecten yessoensis  

PubMed Central

Background Bivalves comprise around 30,000 extant species and have received much attention for their importance in ecosystems, aquaculture and evolutionary studies. Despite the increasing application of real-time quantitative reverse transcription PCR (qRT-PCR) in gene expression studies on bivalve species, little research has been conducted on reference gene selection which is critical for reliable and accurate qRT-PCR analysis. For scallops, systematic evaluation of reference genes that can be used among tissues or embryo/larva stages is lacking, and ?-actin (ACT) is most frequently used as qRT-PCR reference gene without validation. Results In this study, 12 commonly used candidate reference genes were selected from the transcriptome data of Yesso scallop (Patinopecten yessoensis) for suitable qRT-PCR reference genes identification. The expression of these genes in 36 tissue samples and 15 embryo/larva samples under normal physiological conditions was examined by qRT-PCR, and their expression stabilities were evaluated using three statistic algorithms, geNorm, NormFinder, and comparative ?Ct method. Similar results were obtained by the three approaches for the most and the least stably expressed genes. Final comprehensive ranking for the 12 genes combing the results from the three programs showed that, for different tissues, DEAD-box RNA helicase (HELI), ubiquitin (UBQ), and 60S ribosomal protein L16 (RPL16) were the optimal reference genes combination, while for different embryo/larva stages, gene set containing Cytochrome B (CB), Cytochrome C (CC), Histone H3.3 (His3.3), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recommended for qRT-PCR normalization. ACT was among the least stable genes for both adult tissues and embryos/larvae. Conclusions This work constitutes the first systematic analysis on reference genes selection for qRT-PCR normalization in scallop under normal conditions. The suitable reference genes we recommended will be useful for the identification of genes related to biological processes in Yesso scallop, and also in the reference gene selection for other scallop or bivalve species. PMID:24069432

Feng, Liying; Yu, Qian; Li, Xue; Ning, Xianhui; Wang, Jing; Zou, Jiajun; Zhang, Lingling; Wang, Shi; Hu, Jingjie; Hu, Xiaoli; Bao, Zhenmin

2013-01-01

331

Stability indicating studies on NMITLI 118RT+ (standardized extract of withania somnifera dunal)  

PubMed Central

Background: Withania somnifera Dunal (Ashwagandha) is an Indian medicinal plant of great medicinal value; used in many clinically proven conditions. NMITLI-118RT+ is a candidate drug under a Council of Scientific and Industrial Research (CSIR) networking project. It is a chemotype of W. somnifera's root extract, which has been used for the present study. Objectives: The present investigation aims to develop and validate a simple isocratic reverse phase-high performance liquid chromatography (RP-HPLC) system for the detection and estimation of Withanolide A (marker compound) and its analytical application for stability indicating studies on NMITLI-118RT+. Material and Methods: A validated RP-HPLC method for Withanolide A was established on a Waters HPLC system and the same was used on NMITLI-118RT+ for quantification and fingerprinting purposes, and for establishing forced degradation, isothermal stress tests, and drug-excipient testing protocols as per International Conference on Harmonization (ICH) guidelines. Results: A validated method was established, which could detect the marker at a retention time of around 6.3 minutes, with a linearity range of 2-100 ?g/mL, by varying the amounts of the said marker, which were estimated in four different batches of NMITLI-118RT+. Photostability as per ICH guidelines suggested a slight loss of the active constituent and maximum degradation was afforded with alkali followed by acid, and then peroxide, in the forced degradation studies. In the drug-excipient studies, the maximum amount of active constituent could be detected in the samples with ethyl cellulose and the least with hydroxy propyl cellulose. Conclusion: The method developed here was simple and rapid. The various stability indicating studies carried out in the present investigation would be useful for formulation development and were suggestive of deciding the recommended storage conditions for NMITLI-118RT+. PMID:25210308

Ahmad, Hafsa; Khandelwal, Kiran; Pachauri, Shakti Deep; Sanghwan, Rajender Singh; Dwivedi, Anil Kumar

2014-01-01

332

A quantitative real-time RT-PCR assay for mature C. albicans biofilms  

PubMed Central

Background Fungal biofilms are more resistant to anti-fungal drugs than organisms in planktonic form. Traditionally, susceptibility of biofilms to anti-fungal agents has been measured using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide (XTT) assay, which measures the ability of metabolically active cells to convert tetrazolium dyes into colored formazan derivatives. However, this assay has limitations when applied to high C. albicans cell densities because substrate concentration and solubility are limiting factors in the reaction. Because mature biofilms are composed of high cell density populations we sought to develop a quantitative real-time RT-PCR assay (qRT-PCR) that could accurately assess mature biofilm changes in response to a wide variety of anti-fungal agents, including host immune cells. Results The XTT and qRT-PCR assays were in good agreement when biofilm changes were measured in planktonic cultures or in early biofilms which contain lower cell densities. However, the real-time qRT-PCR assay could also accurately quantify small-medium size changes in mature biofilms caused by mechanical biomass reduction, antifungal drugs or immune effector cells, that were not accurately quantifiable with the XTT assay. Conclusions We conclude that the qRT-PCR assay is more accurate than the XTT assay when measuring small-medium size effects of anti-fungal agents against mature biofilms. This assay is also more appropriate when mature biofilm susceptibility to anti-fungal agents is tested on complex biological surfaces, such as organotypic cultures. PMID:21548962

2011-01-01

333

The Accuracy of the Guardian® RT Continuous Glucose Monitor in Children with Type 1 Diabetes  

PubMed Central

Abstract Objective The aim of this study was to determine the accuracy of the Guardian® RT system (Medtronic Minimed, Northridge, CA) in young children and adolescents with type 1 diabetes (T1D) during different scenarios of glucose levels and sensor age. Methods At five clinical centers, 30 subjects between 4 and 17 years old with T1D were recruited. All subjects had a glycosylated hemoglobin level of ?10.0% and were using an insulin pump. Subjects initially used a Guardian RT for approximately 1 week at home. Each subject was then hospitalized overnight for about 18?h in a clinical research center, during which time insulin-induced hypoglycemia occurred, along with frequently sampled glucose. Results There were 1,511 laboratory glucose measurements paired with glucose measurements from 48 Guardian RT sensors. Overall, the median absolute difference (AD) was 21?mg/dL, and the median relative AD (RAD) was 14%, with 64% of sensor values meeting International Organization for Standardization home glucose meter criteria. The median AD was 27?mg/dL for reference glucose values ?60?mg/dL and 25?mg/dL for reference glucose values ?70?mg/dL. The median RAD was 19% for reference glucose values 71–120?mg/dL, 14% for reference glucose values 121–180?mg/dL, and 10% for reference glucose values >180?mg/dL. Conclusions The Guardian RT appears to perform as well in children with T1D as it has been reported to perform in adults with diabetes. The Guardian RT has an accuracy similar to that of other available continuous glucose monitors and can give important and useful clinical information. PMID:18828242

2008-01-01

334

SPoRT - An End-to-End R2O Activity  

NASA Technical Reports Server (NTRS)

Established in 2002 to demonstrate the weather and forecasting application of real-time EOS measurements, the Short-term Prediction Research and Transition (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 observational data applications from EOS satellites to improve short-term weather forecasts on a regional and local scale. SPoRT currently partners with several universities and other government agencies for access to real-time data and products, and works collaboratively with them and operational end users at 13 WFOs to develop and test the new products and capabilities in a "test-bed" mode. The test-bed simulates key aspects of the operational environment without putting constraints on the forecaster workload. Products and capabilities which show utility in the test-bed environment are then transitioned experimentally into the operational environment for further evaluation and assessment. SPoRT focuses on a suite of data and products from MODIS, AMSR-E, and AIRS on the NASA Terra and Aqua satellites, and total lightning measurements from ground-based networks. Some of the observations are assimilated into or used with various versions of the WRF model to provide supplemental forecast guidance to operational end users. SPoRT is enhancing partnerships with NOAA / NESDIS for new product development and data access to exploit the remote sensing capabilities of instruments on the NPOESS satellites to address short term weather forecasting problems. The VIIRS and CrIS instruments on the NPP and follow-on NPOESS satellites provide similar observing capabilities to the MODIS and AIRS instruments on Terra and Aqua. SPoRT will be transitioning existing and new capabilities into the AWIIPS II environment to continue the continuity of its activities.

Jedlovec, Gary J.

2009-01-01

335

Simple multiplex RT-PCR for identifying common fusion transcripts in childhood acute leukemia.  

PubMed

Nonrandom gene rearrangements have been demonstrated in leukemic cells at diagnosis. These genetic abnormalities are associated with specific types, clinical characteristics, and prognosis of acute leukemia. Common fusion transcripts in childhood acute lymphoblastic leukemia (ALL) are TEL-AML1, E2A-PBX, MLL-AF4, and BCR-ABL (p190) and in acute nonlymphoblastic leukemia (ANLL) are AML-ETO, PML-RARA, and CBFB-MYH11. Reverse transcription-polymerase chain reaction (RT-PCR) for detection of each individual fusion transcript is impractical and time consuming. The purpose of this study was to develop simple RT-PCR methods to identify common fusion transcripts of newly diagnosed acute leukemia in children. Total RNA was extracted from bone marrow samples of children diagnosed with acute leukemia. Multiplex RT-PCR panel A (ALL) included primers for TEL-AML1, E2A-PBX, MLL-AF4, and BCR-ABL (p190) whereas panel B (ANLL) composed of primers for AML-ETO, PML-RARA, and CBFB-MYH11. Known leukemic cell lines were used to serve as positive controls. Eighty three children diagnosed with ALL (n = 63) and ANLL (n = 20) were included in this study. Fusion transcripts could be identified using multiplex RT-PCR panel A for ALL and panel B for ANLL in 26/83 (31.3%) cases. In ALL samples, we found TEL-AML1 = 16/63 (25.4%), E2A-PBX = 3/63 (4.8%), MLL-AF4 = 1/63 (1.6%), and BCR-ABL = 1/63 (1.6%). Four cases of AML1-ETO (20%) and one PML-RARA (5%) were found in ANLL samples. In conclusion, our simple multiplex RT-PCR for detection of fusion transcripts in childhood acute leukemia was found to be a rapid, accurate, and effective method. PMID:18665825

Pakakasama, S; Kajanachumpol, S; Kanjanapongkul, S; Sirachainan, N; Meekaewkunchorn, A; Ningsanond, V; Hongeng, S

2008-08-01

336

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

PubMed

A numerical model was developed that is capable of simulating multispecies reactive solute transport in variably saturated porous media. This model consists of a modified version of the reactive transport model RT3D (Reactive Transport in 3 Dimensions) that is linked to the Unsaturated-Zone Flow (UZF1) package and MODFLOW. Referred to as UZF-RT3D, the model is tested against published analytical benchmarks as well as other published contaminant transport models, including HYDRUS-1D, VS2DT, and SUTRA, and the coupled flow and transport modeling system of CATHY and TRAN3D. Comparisons in one-dimensional, two-dimensional, and three-dimensional variably saturated systems are explored. While several test cases are included to verify the correct implementation of variably saturated transport in UZF-RT3D, other cases are included to demonstrate the usefulness of the code in terms of model run-time and handling the reaction kinetics of multiple interacting species in variably saturated subsurface systems. As UZF1 relies on a kinematic-wave approximation for unsaturated flow that neglects the diffusive terms in Richards equation, UZF-RT3D can be used for large-scale aquifer systems for which the UZF1 formulation is reasonable, that is, capillary-pressure gradients can be neglected and soil parameters can be treated as homogeneous. Decreased model run-time and the ability to include site-specific chemical species and chemical reactions make UZF-RT3D an attractive model for efficient simulation of multispecies reactive transport in variably saturated large-scale subsurface systems. PMID:23131109

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

2013-01-01

337

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

338

cis-Acting inhibitory elements within the pol-env region of human T-cell leukemia virus type 1 possibly involved in viral persistence.  

PubMed Central

Human T-cell leukemia virus type 1 (HTLV-1) remains latent throughout the life of the carrier, with cells containing the provirus and viral gene expression efficiently down-regulated. On a molecular level, exactly how viruses are down-regulated in vivo remains unresolved. We described here the possibility that down-regulation results from the presence of inhibitory elements within the gag-env region of the provirus in fresh peripheral blood mononuclear cells from carriers. In vitro experiments then revealed that potent cis-acting inhibitory elements (CIEs) are indeed contained in two discrete fragments from the pol region and weaker ones in the env region. The effect of CIEs is relieved by the HTLV-1 posttranscriptional regulator Rex through binding to the Rex-responsive element (RxRE), suggesting that Rex might interfere with pre-mRNA degradation and/or activate the export of mRNA molecules harboring both of the inhibitory elements and RxRE on the same RNA molecule. Thus, we propose the hypothesis that such functions of CIEs may be involved in HTLV-1 persistence. PMID:9151840

Saiga, A; Orita, S; Minoura-Tada, N; Maeda, M; Aono, Y; Asakawa, M; Nakahara, K; Kubota, R; Osame, M; Igarashi, H

1997-01-01

339

Role of Env in Resistance of Feline Immunodeficiency Virus (FIV)-Infected Cats to Superinfection by a Second FIV Strain as Determined by Using a Chimeric Virus?  

PubMed Central

A more or less pronounced resistance to superinfection by a second strain of the infecting virus has been observed in many lentivirus-infected hosts. We used a chimeric feline immunodeficiency virus (FIV), designated FIV?, containing a large part of the env gene of a clade B virus (strain M2) and all the rest of the genome of a clade A virus (a p34TF10 molecular clone of the Petaluma strain modified to grow in lymphoid cells), to gain insights into such resistance. FIV? was infectious and moderately pathogenic for cats and in vitro exhibited the neutralization specificity of the env donor. The experiments performed were bidirectional, in that cats preinfected with either parental virus were challenged with FIV? and vice versa. The preinfected animals were partially or completely protected relative to what was observed in naïve control animals, most likely due, at least in part, to the circumstance that in all the preinfecting/challenge virus combinations examined, the first and the second virus shared significant viral components. Based on the proportions of complete protection observed, the role of a strongly matched viral envelope appeared to be modest and possibly dependent on the time interval between the first and the second infection. Furthermore, complete protection and the presence of measurable neutralizing antibodies capable of blocking the second virus in vitro were not associated. PMID:17634241

Giannecchini, Simone; Pistello, Mauro; Isola, Patrizia; Matteucci, Donatella; Mazzetti, Paola; Freer, Giulia; Bendinelli, Mauro

2007-01-01

340

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

PubMed Central

The lack of success of subunit human immunodeficiency virus (HIV) type 1 vaccines to date suggests that multiple components or a complex virion structure may be required. We hypothesized that the failure of current vaccine strategies to induce protective antibodies is linked to the inability of native envelope structures to readily elicit these types of antibodies. We have previously reported on the ability of a formaldehyde-treated, heat-inactivated vaccine to induce modest antibody responses in animal vaccine models. We investigated here whether immunization for HIV with an envelope-modified, formaldehyde-stabilized, heat-inactivated virion vaccine could produce higher-titer and/or broader neutralizing antibody responses. Thus, a clade B vaccine which contains a single point mutation in gp41 (Y706C) that results in increased incorporation of oligomeric Env into virions was constructed. This vaccine was capable of inducing high-titer antibodies that could neutralize heterologous viruses, including those of clades A and C. These results further support the development of HIV vaccines with modifications in native Env structures for the induction of neutralizing antibody responses. PMID:16051814

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

2005-01-01

341

Structure-function analysis of the LytM domain of EnvC, an activator of cell wall remodeling at the Escherichia coli division site  

PubMed Central

Proteins with LytM (Peptidase_M23) domains are broadly distributed in bacteria and have been implicated in a variety of important processes, including cell division and cell-shape determination. Most LytM-like proteins that have been structurally and/or biochemically characterized are metallo-endopeptidases that cleave crosslinks in the peptidoglycan (PG) cell wall matrix. Notable exceptions are the Escherichia coli cell division proteins EnvC and NlpD. These LytM factors are not hydrolases themselves, but instead serve as activators that stimulate PG cleavage by target enzymes called amidases to promote cell separation. Here we report the structure of the LytM domain from EnvC, the first structure of a LytM factor implicated in the regulation of PG hydrolysis. As expected, the fold is highly similar to that of other LytM proteins. However, consistent with its role as a regulator, the active site region is degenerate and lacks a catalytic metal ion. Importantly, genetic analysis indicates that residues in and around this degenerate active site are critical for amidase activation in vivo and in vitro. Thus, in the regulatory LytM factors, the apparent substrate binding pocket conserved in active metallo-endopeptidases has been adapted to control PG hydrolysis by another set of enzymes. PMID:23796240

Peters, Nick T.; Morlot, Cécile; Yang, Desirée C.; Uehara, Tsuyoshi; Vernet, Thierry; Bernhardt, Thomas G.

2013-01-01

342

Evolution of cross-neutralizing antibody specificities to the CD4-BS and the carbohydrate cloak of the HIV Env in an HIV-1-infected subject.  

PubMed

Broadly neutralizing antibodies are considered an important part of a successful HIV vaccine. A better understanding of the factors underlying their development during infection and of the epitopes they target is needed to elicit similar antibody responses by vaccination. We and others reported that, on average, it takes 2 to 3 years for cross-reactive neutralizing antibodies to become detectable in the sera of HIV-1-infected subjects and that they target a limited number of epitopes on the HIV Envelope. Here we investigated the emergence and evolution of the earliest cross-reactive neutralizing antibody specificities in one HIV-1-infected individual, AC053. We defined two distinct epitopes on Env that are targeted by the broadly neutralizing antibody responses developed by AC053. The first specificity became evident at 3 years post infection and targeted the CD4-binding site of Env. Antibodies responsible for that specificity neutralized most, but not all, viruses susceptible to neutralization by the plasma antibodies of AC053. The second specificity became apparent approximately a year later. It was due to PG9-like antibodies, which were able to neutralize those viruses not susceptible to the anti-CD4-BS antibodies in AC053. These findings improve our understanding of the co-development of broadly neutralizing antibodies that target more than one epitope during natural HIV-1-infection in selected HIV+ subjects. They support the hypothesis that developing broadly neutralizing antibody responses targeting distinct epitopes by immunization could be feasible. PMID:23152926

Mikell, Iliyana; Stamatatos, Leonidas

2012-01-01

343

RT-PCR analysis of pain genes: use of gel-based RT-PCR for studying induced and tissue-enriched gene expression.  

PubMed

Frequently, it is important to ascertain whether a molecule that is involved in one model of pain is also involved in other models of pain. Similarly, it may be important to determine whether a molecule involved in nociception in one tissue is also expressed in other tissues and to ascertain the degree of enrichment. Additionally, before initiating a complex set of experiments or purchasing an expensive immunoassay kit, it may be useful to obtain initial supporting evidence to justify the time and money. Is the transcript for the target receptor, protein, or peptide precursor present in, for example, the dorsal root ganglion? And, if present, how abundant is it? Here is where the power of PCR can be applied to obtain a quick but informative answer. In this chapter, we mainly detail the use of gel-based RT-PCR and also provide suggestions on tissue dissection and interpretation of results. The use of gel-based RT-PCR can address many of the questions of abundance or tissue specificity with a minimum of expense and time. PMID:20336429

Mitchell, Kendall; Iadarola, Michael J

2010-01-01

344

RT-PCR Analysis of Pain Genes: Use of Gel-Based RT-PCR for Studying Induced and Tissue-Enriched Gene Expression  

PubMed Central

Frequently, it is important to ascertain whether a molecule that is involved in one model of pain is also involved in other models of pain. Similarly, it may be important to determine whether a molecule involved in nociception in one tissue is also expressed in other tissues and to ascertain the degree of enrichment. Additionally, before initiating a complex set of experiments or purchasing an expensive immunoassay kit, it may be useful to obtain initial supporting evidence to justify the time and money. Is the transcript for the target receptor, protein, or peptide precursor present in, for example, the dorsal root ganglion? And, if present, how abundant is it? Here is where the power of PCR can be applied to obtain a quick but informative answer. In this chapter, we mainly detail the use of gel-based RT-PCR and also provide suggestions on tissue dissection and interpretation of results. The use of gel-based RT-PCR can address many of the questions of abundance or tissue specificity with a minimum of expense and time. PMID:20336429

Mitchell, Kendall; Iadarola, Michael J.

2012-01-01

345

Misidentification of Bordetella bronchiseptica as Bordetella pertussis using a Newly Described RT-PCR Targeting the Pertactin Gene  

Technology Transfer Automated Retrieval System (TEKTRAN)

Recently a real-time PCR (RT-PCT) assay based on sequence from the gene for pertactin was proposed for identification of Bordetella pertussis. Here we report that the B. pertussis pertactin gene sequence for the region encompassing the RT-PCR probe and primers is nearly identical to that of many B....

346

Development of Virtual Airspace Simulation Technology - Real-Time (VAST-RT) Capability 2 and Experimental Plans  

NASA Technical Reports Server (NTRS)

The Virtual Airspace Simulation Technology - Real-Time (VAST-RT) Project, an element cf NASA's Virtual Airspace Modeling and Simulation (VAMS) Project, has been developing a distributed simulation capability that supports an extensible and expandable real-time, human-in-the-loop airspace simulation environment. The VAST-RT system architecture is based on DoD High Level Architecture (HLA) and the VAST-RT HLA Toolbox, a common interface implementation that incorporates a number of novel design features. The scope of the initial VAST-RT integration activity (Capability 1) included the high-fidelity human-in-the-loop simulation facilities located at NASA/Ames Research Center and medium fidelity pseudo-piloted target generators, such as the Airspace Traffic Generator (ATG) being developed as part of VAST-RT, as well as other real-time tools. This capability has been demonstrated in a gate-to-gate simulation. VAST-RT's (Capability 2A) has been recently completed, and this paper will discuss the improved integration of the real-time assets into VAST-RT, including the development of tools to integrate data collected across the simulation environment into a single data set for the researcher. Current plans for the completion of the VAST-RT distributed simulation environment (Capability 2B) and its use to evaluate future airspace capacity enhancing concepts being developed by VAMS will be discussed. Additionally, the simulation environment's application to other airspace and airport research projects is addressed.

Lehmer, R.; Ingram, C.; Jovic, S.; Alderete, J.; Brown, D.; Carpenter, D.; LaForce, S.; Panda, R.; Walker, J.; Chaplin, P.; Ballinger, D.

2006-01-01

347

Response to Intervention (RtI) Self-Efficacy among Elementary and Middle School General Education Teachers  

ERIC Educational Resources Information Center

Response to Intervention (RtI) integrates assessment and intervention within a school-wide, multi-level prevention system to maximize student achievement. RtI requires that educators collect ongoing information about student progress and provide instruction that aligns with that progress. By providing rigorous interventions prior to students…

Shirley, Tory Swearingen

2012-01-01

348

Co-regulation of polysaccharide production, motility, and expression of type III secretion genes by EnvZ/OmpR and GrrS/GrrA systems in Erwinia amylovora.  

PubMed

The EnvZ/OmpR and GrrS/GrrA systems, two widely distributed two-component systems in gamma-Proteobacteria, negatively control amylovoran biosynthesis in Erwinia amylovora, and the two systems regulate motility in an opposing manner. In this study, we examined the interplay of EnvZ/OmpR and GrrS/GrrA systems in controlling various virulence traits in E. amylovora. Results showed that amylovoran production was significantly higher when both systems were inactivated, indicating that the two systems act as negative regulators and their combined effect on amylovoran production appears to be enhanced. In contrast, reduced motility was observed when both systems were deleted as compared to that of grrA/grrS mutants and WT strain, indicating that the two systems antagonistically regulate motility in E. amylovora. In addition, glycogen accumulation was much higher in envZ/ompR and two triple mutants than that of grrS/grrA mutants and WT strain, suggesting that EnvZ/OmpR plays a dominant role in regulating glycogen accumulation, whereas levan production was significantly lower in the grrS/grrA and two triple mutants as compared with that of WT and envZ/ompR mutants, indicating that GrrS/GrrA system dominantly controls levan production. Furthermore, both systems negatively regulated expression of three type III secretion (T3SS) genes and their combined negative effect on hrp-T3SS gene expression increased when both systems were deleted. These results demonstrated that EnvZ/OmpR and GrrS/GrrA systems co-regulate various virulence factors in E. amylovora by still unknown mechanisms or through different target genes, sRNAs, or proteins, indicating that a complex regulatory network may be involved, which needs to be further explored. PMID:24218204

Li, Wenting; Ancona, Veronica; Zhao, Youfu

2014-02-01

349

SPoRT's Participation in the GOES-R Proving Ground Activity  

NASA Technical Reports Server (NTRS)

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, Gary; Fuell, Kevin; Smith, Matthew; Stano, Geoffrey; Molthan, Andrew

2011-01-01

350

Expression of nonclassical MHC class I (RT1-U) in certain neuronal populations of the central nervous system.  

PubMed

Major histocompatibility complex (MHC) class I genes consist of classical (Ia) and nonclassical (Ib) types. Recently, a set of structurally similar MHC class Ib genes in the rat, denoted RT1-U, was described. We here demonstrate expression of RT1-U mRNA using highly stringent oligonucleotide in situ hybridization in several different neuronal populations, including different motor nuclei and the substantia nigra in the rat MHC (c) and (n) haplotypes under normal conditions. The expression pattern for beta2-microglobulin mRNA was almost identical. In contrast, neuronal expression of classical MHC class I (RT1-A) was low. Interestingly, after mechanical nerve injury, glial cells predominantely upregulated expression of RT1-A, whereas neuronal expression of RT1-U remained unchanged. Neuronal expression of nonclassical MHC class I may thus be important for immune surveillance in the nervous system. PMID:10594675

Lidman, O; Olsson, T; Piehl, F

1999-12-01

351

Human endogenous retrovirus K(HML-2) Gag- and Env-specific T-cell responses are infrequently detected in HIV-1-infected subjects using standard peptide matrix-based screening.  

PubMed

T-cell responses to human endogenous retrovirus (HERV) K(HML-2) Gag and Env were mapped in HIV-1-infected subjects using 15 mer peptides. Small peptide pools and high concentrations were used to maximize sensitivity. In the 23 subjects studied, only three bona fide HERV-K(HML-2)-specific responses were detected. At these high peptide concentrations, we detected false-positive responses, three of which were mapped to an HIV-1 Gag peptide contaminant. Thus, HERV-K(HML-2) Gag- and Env-specific T-cell responses are infrequently detected by 15 mer peptide mapping. PMID:22205657

Jones, R Brad; John, Vivek M; Hunter, Diana V; Martin, Eric; Mujib, Shariq; Mihajlovic, Vesna; Burgers, Peter C; Luider, Theo M; Gyenes, Gabor; Sheppard, Neil C; Sengupta, Devi; Tandon, Ravi; Yue, Feng-Yun; Benko, Erika; Kovacs, Colin; Nixon, Douglas F; Ostrowski, Mario A

2012-02-01

352

The carbon Cepheid RT Trianguli Australis: additional evidence of triple-? and CNO cycling  

NASA Astrophysics Data System (ADS)

We have used echelle spectra of resolving power 35000 to derive chemical abundances and the 12C/13C ratio in the 1.9-d carbon Cepheid RT TrA and the Cepheid U TrA, employed as a comparison star. We confirm that RT TrA is very metal-rich with [Fe/H]=+0.4. In addition, C and N are substantially in excess, and a small deficiency in O is present. We interpret these anomalies as resulting from the appearance on the stellar surface of material enriched in 12C by the 3-? process, followed by CNO cycling to convert 12C to 13C and 14N. In addition, some 16O has been processed to 14N. The partial processing of 16O to 14N indicates that substantial 17O may be present. Proton capture seems to have enhanced 23Na from the Ne isotopes.

Wallerstein, George; Matt, Sean; Gonzalez, Guillermo

2000-01-01

353

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

354

Evaluating the Impact of AIRS Observations on Regional Forecasts at the SPoRT Center  

NASA Technical Reports Server (NTRS)

NASA Short-term Prediction Research and Transition (SPoRT) Center collaborates with operational partners of different sizes and operational goals to improve forecasts using targeted projects and data sets. Modeling and DA activities focus on demonstrating utility of NASA data sets and capabilities within operational systems. SPoRT has successfully assimilated the Atmospheric Infrared Sounder (AIRS) radiance and profile data. A collaborative project is underway with the Joint Center for Satellite Data Assimilation (JCSDA) to use AIRS profiles to better understand the impact of AIRS radiances assimilated within Gridpoint Statistical Interpolation (GSI) in hopes of engaging the operational DA community in a reassessment of assimilation methodologies to more effectively assimilate hyperspectral radiances.

Zavodsky, Bradley

2011-01-01

355

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

356

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

357

Concordance of IHC, FISH and RT-PCR for EML4-ALK rearrangements  

PubMed Central

The echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase (EML4-ALK) has emerged as the second most important driver oncogene in lung cancer and the first targetable fusion oncokinase to be identified in 4-6% of lung adenocarcinomas. Crizotinib, along with a diagnostic test—the Vysis ALK Break Apart fluorescence in situ hybridization (FISH) Probe Kit—is approved for the treatment of ALK positive advanced non-small cell lung cancer (NSCLC). However, the success of a targeted drug is critically dependent on a sensitive and specific screening assay to detect the molecular drug target. In our experience, reverse transcription polymerase chain reaction (RT-PCR)-based detection of EML4-ALK is a more sensitive and reliable approach compared to FISH and immunohistochemistry (IHC). Although ALK FISH is clinically validated, the assay can be technically challenging and other diagnostic modalities, including IHC and RT-PCR should be further explored. PMID:25806283

Teixidó, Cristina; Karachaliou, Niki; Peg, Vicente; Gimenez-Capitan, Ana

2014-01-01

358

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

359

Using metallic resin and aluminum alloy molds to manufacture propellers with RP\\/RT technique  

Microsoft Academic Search

Purpose – This purpose of this study is to investigate an effective method to manufacture propellers. Design\\/methodology\\/approach – The investment casting process and injection molding process have been applied separately to the rapid prototyping\\/rapid tooling (RP\\/RT) to obtain metal (Al-Si alloy) propellers and plastic (Acrylonitrile butadiene styrene – ABS) propellers. The two different manufacturing processes were compared following the same

C. Y. Hsu; C. K. Huang; G. J. Tzou

2008-01-01

360

Specific detection of avian pneumovirus (APV) US isolates by RT-PCR  

Microsoft Academic Search

Summary.  ?This report details the development of an RT-PCR assay for the specific detection of US isolates of avian pneumovirus (APV).\\u000a Of the several primer pairs tested, two sets of primers derived from the matrix gene of APV were able to specifically detect\\u000a the viral RNA of APV. The nucleotide sequence comparison of the PCR products of APV isolates from Minnesota

H. J. Shin; G. Rajashekara; F. F. Jirjis; D. P. Shaw; S. M. Goyal; D. A. Halvorson; K. V. Nagaraja

2000-01-01

361

Summary of Proton Test on the Actel RT54SX16 Prototype at Indiana University  

NASA Technical Reports Server (NTRS)

A summary of tests performed at the Indiana University Cyclotron Facility, on the Actel RT54SX16 prototype circuit device is presented. The devices' performances in the test is shown in both a table and a graph and was typical for devices of this class. Another summary of tests performed at the Indiana University Cyclotron Facility, on the Chip Express QYH530 device is presented.The device's performance in the test is shown in a graph and tables.

Katz, Richard

1998-01-01

362

Comprehensive molecular screening: from the RT-PCR to the RNA-seq  

PubMed Central

Up to now, the analysis of the mRNA expression in tumoral and non-tumoral has been conducted via RT-PCR. It is considered to be the gold standard for measuring the number of copies of specific cDNA targets. The application of RT-PCR has demonstrated that levels of RNA transcripts stratify patients and predict outcomes in a variety of diseases, providing the basis for several important clinical tests. However, the inherent variability in the quality of any quantitative PCR data makes it difficult to replicate and the analysis is time consuming in the laboratory for the analysis of one gene. Moreover, comparing expression levels across different experiments is often difficult and can require complicated normalization methods. Many techniques have been developed over the years but without good clinical applications. A new, simple and effective way to measure transcriptome composition and to discover new exons or genes is by the RNA-seq. Some advantages of this technique are high reproducibility, the large dynamic range, requirement of less sample RNA, and the ability to detect novel transcripts, alternative splicing, even in the absence of a sequenced genome. However, this RNA-Seq technique will not likely replace current RT-PCR methods, but will be complementary depending on the needs and the resources of the clinic and the laboratory as the results of the RNA-Seq will identify those genes that need to then be examined using RT-PCR methods. The application of the two complementary technologies in the routine analysis of cancer laboratories would be useful in characterizing patients and would assist oncologists in making clinical decisions, as it allows us to identify all molecular characteristics of the tumor.

Giménez-Capitán, Ana; Karachaliou, Niki; Rosell, Rafael

2013-01-01

363

Formation of High-Beta Plasma and Stable Confinement of Toroidal Electron Plasma in RT-1  

NASA Astrophysics Data System (ADS)

The Ring Trap 1 (RT-1) device is a laboratory magnetosphere generated by a levitated superconducting magnet. The goals of RT-1 are to realize stable formation of ultra high-beta plasma suitable for burning advanced fusion fuels, and confinement of toroidal non-neutral plasmas including antimatter particles. RT- 1 has produced high-beta plasma in the magnetospheric configuration. The effects of coil levitation and geomagnetic field compensation [Y. Yano et al., Plasma Fusion Res. 4, 039] resulted drastic improvements of the plasma properties, and a maximum local beta value exceeded 70%. Because plasma is generated by electron cyclotron resonance heating (ECH) in the present experiment, the plasma pressure is mainly due to hot electrons, whose bremsstrahlung was observed with an x-ray CCD camera. The pressure profiles have rather steep gradient near the superconducting coil in the strong field region. The decay rates of magnetic probe and interferometer signals have different time constants, suggesting multiple temperature components. The energy confinement time estimated from the input RF power and stored magnetic energy is on the order of 1s, which is comparable to the decay time constant of the density of hot electron component. Pure electron plasma experiments are also conducted in RT-1. Radial profiles of electrostatic potential and electron density showed that the plasma rigidly rotates in the toroidal direction in the stable confinement phase. Long time confinement of toroidal non- neutral plasma for more than 300s and inward particle diffusion to strong field regions, caused by the activation of the diocotron (Kelvin-Helmholtz) instability, have been realized [Z. Yoshida et al., Phys. Rev. Lett. 104, 235004].

Saitoh, Haruhiko

2010-11-01

364

Diagnosis of Norovirus outbreaks by commercial ELISA or RT-PCR  

Microsoft Academic Search

The IDEIA Norwalk-like virus (Dakocytomation Ltd., Ely, United Kingdom) and the Ridascreen Norwalk-like virus enzyme immunoassay (R-Biopharm AG, Darmstadt, Germany), were evaluated for the diagnosis of outbreaks of acute gastroenteritis.A panel of 158 fecal samples from 23 outbreaks, including confirmed rotavirus and astrovirus outbreaks, was used to determine the sensitivity and specificity of both ELISA kits relative to an RT-PCR

Erwin de Bruin; Erwin Duizer; Harry Vennema; Marion P. G. Koopmans

2006-01-01

365

NASA SPoRT Initialization Datasets for Local Model Runs in 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 be used to initialize local model runs within the Weather Research and Forecasting (WRF) Environmental Modeling System (EMS). These real-time datasets consist of surface-based information updated at least once per day, and produced in a composite or gridded product that is easily incorporated into the WRF EMS. The primary goal for making these NASA datasets available to the WRF EMS community is to provide timely and high-quality information at a spatial resolution comparable to that used in the local model configurations (i.e., convection-allowing scales). The current suite of SPoRT products supported in the WRF EMS include a Sea Surface Temperature (SST) composite, a Great Lakes sea-ice extent, a Greenness Vegetation Fraction (GVF) composite, and Land Information System (LIS) gridded output. The SPoRT SST composite is a blend of primarily the Moderate Resolution Imaging Spectroradiometer (MODIS) infrared and Advanced Microwave Scanning Radiometer for Earth Observing System data for non-precipitation coverage over the oceans at 2-km resolution. The composite includes a special lake surface temperature analysis over the Great Lakes using contributions from the Remote Sensing Systems temperature data. The Great Lakes Environmental Research Laboratory Ice Percentage product is used to create a sea-ice mask in the SPoRT SST composite. The sea-ice mask is produced daily (in-season) at 1.8-km resolution and identifies ice percentage from 0 100% in 10% increments, with values above 90% flagged as ice.

Case, Jonathan L.; LaFontaine, Frank J.; Molthan, Andrew L.; Carcione, Brian; Wood, Lance; Maloney, Joseph; Estupinan, Jeral; Medlin, Jeffrey M.; Blottman, Peter; Rozumalski, Robert A.

2011-01-01

366

Plasma Production by Electron Cyclotron Heating on the Internal Coil Device Mini-RT  

Microsoft Academic Search

The internal coil device Mini-RT (Ring Trap) has been constructed in order to study the extremely high beta plasma confinement predicted in two fluid relaxation theory. Plasma is produced by electron cyclotron heating with a 2.45 GHz, 2.8 kW microwave. Plasma experiments were carried out for two conditions: with a mechanically supported coil, and with a magnetically levitated one. In

Takuya Goto; Eiichi Yatsuka; Junji Morikawa; Yuichi Ogawa

2006-01-01

367

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

2015-03-01

368

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

369

RT-WLAN: a soft real-time extension to ORiNOCO Linux device driver  

Microsoft Academic Search

The current IEEE 802.11 wireless LAN (WLAN) systems are unable to support real-time applications because the underlying contention-based MAC (medium access control) protocol causes unpredictable delays. In this paper, we present the implementation details of a new RT-WLAN device driver module, which extends the original Linux device driver for the popular Agere ORiNOCO cards to support soft real-time communications. To

Amit Jain; Daji Qiao; Kang G. Shin

2003-01-01

370

Integrated RT–PCR\\/nested PCR diagnosis for differentiating between subgroups of plum pox virus  

Microsoft Academic Search

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.

Marianna Szemes; Miklós Kálmán; Arben Myrta; Donato Boscia; Mária Németh; Mária Kölber; László Dorgai

2001-01-01

371

Multiplex-nested RT-PCR to evaluate latent and lytic Epstein Barr virus gene expression  

Microsoft Academic Search

This paper describes the development of two multiplex-nested RT-PCR devised to evaluate latent\\/immortalizing (EBNA1, EBNA2, LMP1 and LMP2) and lytic [immediate early (Zebra), early, and late (VCA), respectively] Epstein Barr virus (EBV) transcripts. Subsequently, the assays have been validated evaluating the EBV latent\\/lytic gene expression in peripheral blood mononuclear cells (PBMC) from immunocompetent subjects (children with primary EBV infection, past

Massimiliano Bergallo; Cristina Costa; Sara Baro; Tiziana Musso; Luciano Balbo; Chiara Merlino; Rossana Cavallo

2007-01-01

372

Extended-rtPS Algorithm for VoIP Services in IEEE 802.16 systems  

Microsoft Academic Search

There are several scheduling algorithms for Voice over IP (VoIP) services in IEEE 802.16 systems, such as unsolicited grant service (UGS), real-time polling service (rtPS), UGS with Activity Detection (UGS-AD), and Lee's algorithm using Grant-Me bit of the generic MAC header. However, these algorithms have some problems of a waste of uplink resources, additional access delay, and MAC overhead for

Howon Lee; Taesoo Kwon; Dong-Ho Cho

2006-01-01

373

A Dynamical Shape Prior for LV Segmentation from RT3D Echocardiography?  

PubMed Central

Real-time three-dimensional (RT3D) echocardiography is the newest generation of three-dimensional (3-D) echocardiography. Segmentation of RT3D echocardiographic images is essential for determining many important diagnostic parameters. In cardiac imaging, since the heart is a moving organ, prior knowledge regarding its shape and motion patterns becomes an important component for the segmentation task. However, most previous cardiac models are either static models (SM), which neglect the temporal coherence of a cardiac sequence or generic dynamical models (GDM), which neglect the inter-subject variability of cardiac motion. In this paper, we present a subject-specific dynamical model (SSDM) which simultaneously handles inter-subject variability and cardiac dynamics (intra-subject variability). It can progressively predict the shape and motion patterns of a new sequence at the current frame based on the shapes observed in the past frames. The incorporation of this SSDM into the segmentation process is formulated in a recursive Bayesian framework. This results in a segmentation of each frame based on the intensity information of the current frame, as well as on the prediction from the previous frames. Quantitative results on 15 RT3D echocardiographic sequences show that automatic segmentation with SSDM is superior to that of either SM or GDM, and is comparable to manual segmentation. PMID:20054422

Papademetris, Xenophon; Sinusas, Albert J.; Duncan, James S.

2009-01-01

374

SPoRT Participation in the GOES-R and JPSS Proving Grounds  

NASA Technical Reports Server (NTRS)

For the last several years, the NASA Short-term Prediction Research and Transition (SPoRT) project at has been working with the various algorithm working groups and science teams to demonstrate the utility of future operational sensors for GOES-R and the suite of instruments for the JPSS observing platforms. For GOES-R, imagery and products have been developed from polar-orbiting sensors such as MODIS and geostationary observations from SEVIRI, simulated imagery, enhanced products derived from existing GOES satellites, and data from ground-based observing systems to generate pseudo or proxy products for the ABI and GLM instruments. The suite of products include GOES-POES basic and RGB hybrid imagery, total lightning flash products, quantitative precipitation estimates, and convective initiation products. SPoRT is using imagery and products from VIIRS, CrIS, ATMS, and OMPS to show the utility of data and products from their operational counterparts on JPSS. The products include VIIRS imagery in swath form, the GOES-POES hybrid, a suite of RGB products including the air mass RGB using water vapor and ozone channels from CrIS, and several DNB products. Over a dozen SPoRT collaborative WFOs and several National Centers are involved in an intensive evaluation of the operational utility of these products.

Jedlovec, Gary; Fuell, Kevin; Smith, Matthew

2013-01-01

375

Characterization of the different BCR-ABL transcripts with a single multiplex RT-PCR.  

PubMed

The diagnosis of chronic myeloid leukemia is based on detection of the Philadelphia (Ph) chromosome or the BCR-ABL gene. The junction present in the transcript may vary according to the reciprocal translocation t(9;22)(q34;11). Identification of the transcript (p190, p210 or p230) does not reveal the type of junction but this information is very important for classification of patients in clinical trials. Most identification kits do not explore p230 transcripts and are unable to determine exotic breakpoints. We have developed a clinical molecular diagnosis assay, able to identify all of the BCR-ABL transcripts and, by single assay, to characterize all of the possible transcript junctions. This technique is based on RT-PCR and PCR-capillary electrophoresis. For each patient sample, we performed RT-PCR with three different BCR primers each coupled to a specific different fluorochrome and a unique reverse ABL primer. Depending on the transcript, only one BCR primer was used for each RT-PCR. After capillary electrophoresis and fluorescence determination, we were able to identify both the transcript and its junction at the same time. PMID:15507673

Chasseriau, Jacques; Rivet, Jérôme; Bilan, Frédéric; Chomel, Jean-Claude; Guilhot, François; Bourmeyster, Nicolas; Kitzis, Alain

2004-11-01

376

Mass scale screening of common arboviral infections by an affordable, cost effective RT-PCR method  

PubMed Central

Objective To develop a rapid, cost effective RT-PCR method for the mass scale diagnosis of such diseases at the viremia stage to find out the actual disease burden in that area. Methods For this purpose, cases with the history of only short febrile illness were considered. Thus 157 samples with the history of dengue/chikungunya like illness and only 58 samples with a history of acute encephalitis syndrome (AES) were selected. Results Out of 157 samples, 42 and 74 were detected as dengue and chikungunya, respectively and out of 58 AES cases only 23 could be detected as Japanese encephalitis by this RT-PCR method. Conclusions This cost effective RT-PCR method can detect the total positive cases that remain undetected by ELISA method. Moreover, this method is capable to detect the viral RNA from patients' sera even after the appearance of IgM antibody at one fifth costs as compared with the other commercially available kits. PMID:23569876

Taraphdar, Debjani; Sarkar, Arindam; Chatterjee, Shyamalendu

2012-01-01

377

How Many Microorganisms Are Present? Quantitative Reverse Transcription PCR (qRT-PCR)  

NASA Astrophysics Data System (ADS)

Quantitative reverse transcription PCR (qRT-PCR) is a variation of conventional quantitative or real-time PCR, whereby mRNA is first converted into the complementary DNA (cDNA) by reverse transcription, the cDNA is then subsequently quantified by qPCR. The use of mRNA as the initial template allows the quantification of gene transcripts, rather than gene copy numbers. mRNA is only produced by actively metabolising cells and is produced by its corresponding gene to provide a 'blueprint' in order for a cell to manufacture a specific protein. Conventional qPCR detects not only DNA present in actively metabolising cells but also inactive and dead cells. qRT-PCR has the advantage that only actively metabolising cells are detected, hence provides a more reliable measure of microbial activity in oilfield samples. When qRT-PCR is combined with primers and probes for specific genes, the activity of microbial processes important in the oilfield, such as sulphate reduction, methanogenesis and nitrate reduction can be monitored.

Price, Andy; Álvarez, Laura Acuña; Whitby, Corinne; Larsen, Jan

378

Selection of reference genes for quantitative RT-PCR (RT-qPCR) analysis of rat tissues under physiological and toxicological conditions  

PubMed Central

In biological research the analysis of gene expression levels in cells and tissues can be a powerful tool to gain insights into biological processes. For this, quantitative RT-PCR (RT-qPCR) is a popular method that often involve the use of constitutively expressed endogenous reference (or ‘housekeeping’) gene for normalization of data. Thus, it is essential to use reference genes that have been verified to be stably expressed within the specific experimental setting. Here, we have analysed the expression stability of 12 commonly used reference genes (Actb, B2m, Gapdh, Hprt, Pgk1, Rn18s, Rpl13a, Rps18, Rps29, Sdha, Tbp and Ubc) across several juvenile and adult rat tissues (liver, adrenal, prostate, fat pad, testis and ovaries), both under normal conditions and following exposure to various chemicals during development. Employing NormFinder and BestKeeper softwares, we found Hprt and Sdha to be amongst the most stable genes across normal and manipulated tissues, with several others also being suitable for most tissues. Tbp and B2m displayed highest variability in transcript levels between tissues and developmental stages. It was also observed that the reference genes were most unstable in liver and testis following toxicological exposure. For future studies, we propose the use of more than one verified reference gene and the continuous monitoring of their suitability under various experimental conditions, including toxicological studies, based on changes in threshold (Ct) values from cDNA samples having been reverse-transcribed from a constant input concentration of RNA. PMID:25825680

Letting, Heidi; Hadrup, Niels; Hass, Ulla; Vinggaard, Anne Marie

2015-01-01

379

Comparison of dosage schedules of rt-PA in the treatment of proximal deep vein thrombosis.  

PubMed

This study assessed the efficacy and safety of increasing durations of constant-dose intravenous recombinant tissue-type plasminogen activator (rt-PA) in the treatment of deep vein thrombosis. Patients with venogram-documented proximal lower limb (popliteal, iliofemoral) or upper limb (axillary, subclavian) thrombi were given an initial 2-hour rt-PA infusion at 4 micrograms/kg/min, followed by a maintenance infusion of 1 microgram/kg/min for an additional 4, 22, or 33 hours (mean total rt-PA dosages of 54, 127, and 185 mg). A new quantitative venogram scoring system was applied to the study, based on measurements of thrombus volume before and after completion of treatment. Whereas none of the seven patients given treatment for 6 hours and only one of four given treatment for 24 hours showed significant lysis, four of seven who received a prolonged infusion for 35 hours showed lysis of more than 40% of the original thrombus. Overall, the prolonged 35-hour infusion induced 51% lysis of original thrombus, representing a thrombus volume of 16.7 ml dissolved. Hemorrhagic complications were common in all three groups, with four of 18 patients having significant bleeding, including one massive gastrointestinal hemorrhage, two patients with a decrease in hematocrit of more than 10%, and one patient with an intracranial hemorrhage who recovered completely. Pharmacokinetics of the rt-PA showed a steady state antigen concentration of 240 ng/ml and activity of 200 IU/ml during the initial 2-hour infusion and a postinfusion half-life of 5 minutes. Plasma fibrinogen concentrations decreased to approximately 40% to 50% of initial values with all three treatment regimens, but the nadir fibrinogen concentrations did not correlate with either therapeutic efficacy or bleeding complications. One patient with systemic lupus erythematosus had an unusual allergic reaction that manifested primarily as angioedema. This study suggests that rt-PA infusion of 35 hours induces greater thrombolysis of deep vein thrombosis than does a shorter course of 6 or 24 hours, without an increase in hemorrhagic complications. PMID:1583404

Marder, V J; Brenner, B; Totterman, S; Francis, C W; Rubin, R; Rao, A K; Kessler, C M; Kwaan, H C; Sharma, G V; Fong, K L

1992-05-01

380

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

381

Rapid detection and high occurrence of porcine rotavirus A, B, and C by RT-qPCR in diagnostic samples.  

PubMed

Rotaviruses are important cause of diarrhea in animals, including humans. Currently, rotavirus species A, B, C, E, and H (RVA-RVC, RVE, and RVH) have been identified in pigs. Traditionally, RVA has been considered the primary cause of diarrhea in pigs, and RVB and RVC had been described sporadically in pigs until recently. Qualitative porcine RVA, RVB, and RVC RT-PCR (RT-qPCR) assays were designed and 7508 porcine diarrheic samples, submitted to University of Minnesota, were tested to estimate the percentage of RVA, RVB, and RVC over a period of approximately 2 years (from 2009 to 2011). The individual RVA and RVC RT-qPCR assays were multiplex into a single RT-qPCR while the RVB RT-qPCR assay remained as an individual RT-qPCR. In total, 83% of the samples were positive for RVA, RVB, or RVC. As expected, RVA was detected at the highest overall percentage (62%). However, 33% and 53% of the samples were positive for RVB and RVC, respectively, indicating that both RVB and RVC are also epidemiologically important in the swine population. RVC was most predominant in young pigs (1-20 days of age), while RVA and RVB were most predominant in ?21 day old pigs. As diagnostic tools, the developed RT-qPCR assays could successfully discriminate among infecting RV species, which could lead to better surveillance and epidemiological studies for ultimately better prevention and control strategies. PMID:25194889

Marthaler, Douglas; Homwong, Nitipong; Rossow, Kurt; Culhane, Marie; Goyal, Sagar; Collins, James; Matthijnssens, Jelle; Ciarlet, Max

2014-12-01

382

Env-glycoprotein heterogeneity as a source of apparent synergy and enhanced cooperativity in inhibition of HIV-1 infection by neutralizing antibodies and entry inhibitors  

PubMed Central

We measured the inhibition of infectivity of HIV-1 isolates and derivative clones by combinations of neutralizing antibodies (NAbs) and other entry inhibitors in a single-cycle-replication assay. Synergy was analyzed both by the current linear and a new nonlinear method. The new method reduced spurious indications of synergy and antagonism. Synergy between NAbs was overall weaker than between other entry inhibitors, and no stronger where one ligand is known to enhance the binding of another. However, synergy was stronger for a genetically heterogeneous HIV-1 R5 isolate than for its derivative clones. Enhanced cooperativity in inhibition by combinations, compared with individual inhibitors, correlated with increased synergy at higher levels of inhibition, while being less variable. Again, cooperativity enhancement was stronger for isolates than clones. We hypothesize that genetic, post-translational or conformational heterogeneity of the Env protein and of other targets for inhibitors can yield apparent synergy and increased cooperativity between inhibitors. PMID:22018634

Ketas, Thomas J.; Holuigue, Sophie; Matthews, Katie; Moore, John P.

2011-01-01

383

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

384

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

PubMed Central

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

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

2013-01-01

385

Spreading of HIV-1 subtype G and envB/gagG recombinant strains among injecting drug users in Lisbon, Portugal.  

PubMed

We have evaluated the genetic diversity of HIV-1 strains infecting injecting drug users (IDUs) in Lisbon, Portugal. Heteroduplex mobility assay and/or phylogenetic analysis revealed that env (C2V3C3 or gp41) subtype B is present in 63.7% of the 135 viral samples studied, followed by subtypes G (23.7%), A (6.7%), F (5.2%), and D (0.7%). Similar analysis of gag (p24/p7) performed on 91 of the specimens demonstrated that 49.5% of the infections were caused by subtype G viruses; other gag subtypes identified were B (39.5%), F (3.3%), A and D (1.1.% each), and the recombinant circulating form CRF02_AG (5.5%). Discordant env/gag sub-types were detected in 34.1% of the strains and may reflect the presence of dual infections and/or recombinant viruses. The presumptive B/G recombinant form was highly predominant (21 of 31). The genetic pattern of HIV-1 subtype B and G strains is suggestive of multiple introductions and recombination episodes and of a longstanding presence of both subtypes in the country. C2V3C3 amino acid sequences from IDU-derived subtype G viruses presented highly significant signatures, which distinguish the variants from this transmission group. The unusually high prevalence of subtype G sequences (34.1%), independent of the geographic origin of the infected individuals, makes this IDU HIV-1 epidemic unique. PMID:12892060

Esteves, Aida; Parreira, Ricardo; Piedade, João; Venenno, Teresa; Franco, Margarida; Germano de Sousa, José; Patrício, Luis; Brum, Paula; Costa, António; Canas-Ferreira, Wanda F

2003-06-01

386

The streptomycin-treated mouse intestine selects Escherichia coli envZ missense mutants that interact with dense and diverse intestinal microbiota.  

PubMed

Previously, we reported that the streptomycin-treated mouse intestine selected nonmotile Escherichia coli MG1655 flhDC deletion mutants of E. coli MG1655 with improved colonizing ability that grow 15% faster in vitro in mouse cecal mucus and 15 to 30% faster on sugars present in mucus (M. P. Leatham et al., Infect. Immun. 73:8039-8049, 2005). Here, we report that the 10 to 20% remaining motile E. coli MG1655 are envZ missense mutants that are also better colonizers of the mouse intestine than E. coli MG1655. One of the flhDC mutants, E. coli MG1655 ?flhD, and one of the envZ missense mutants, E. coli MG1655 mot-1, were studied further. E. coli MG1655 mot-1 is more resistant to bile salts and colicin V than E. coli MG1655 ?flhD and grows ca. 15% slower in vitro in mouse cecal mucus and on several sugars present in mucus compared to E. coli MG1655 ?flhD but grows 30% faster on galactose. Moreover, E. coli MG1655 mot-1 and E. coli MG1655 ?flhD appear to colonize equally well in one intestinal niche, but E. coli MG1655 mot-1 appears to use galactose to colonize a second, smaller intestinal niche either not colonized or colonized poorly by E. coli MG1655 ?flhD. Evidence is also presented that E. coli MG1655 is a minority member of mixed bacterial biofilms in the mucus layer of the streptomycin-treated mouse intestine. We offer a hypothesis, which we call the "Restaurant" hypothesis, that explains how nutrient acquisition in different biofilms comprised of different anaerobes can account for our results. PMID:22392928

Leatham-Jensen, Mary P; Frimodt-Møller, Jakob; Adediran, Jimmy; Mokszycki, Matthew E; Banner, Megan E; Caughron, Joyce E; Krogfelt, Karen A; Conway, Tyrrell; Cohen, Paul S

2012-05-01

387

A Arts |CS Community Services | D Disabilities | ED Education | EL Elderly | ENV Environment | F Family H Health | L Literacy | P Poverty, Housing, Hunger | S Science and Technology | Y Youth  

E-print Network

as employment opportunities. Assist with Wednesday evening mixers, art and horticulture therapy programsA Arts |CS Community Services | D Disabilities | ED Education | EL Elderly | ENV Environment | F Family H Health | L Literacy | P Poverty, Housing, Hunger | S Science and Technology | Y Youth Wittenberg

Bogaerts, Steven

388

Early detection of dominant Env-specific and subdominant Gag-specific CD8+ lymphocytes in equine infectious anemia virus-infected horses using major histocompatibility complex class I/peptide tetrameric complexes  

PubMed Central

Cytotoxic T lymphocytes (CTL) are critical for control of lentiviruses, including equine infectious anemia virus (EIAV). Measurement of equine CTL responses has relied on chromium-release assays, which do not allow accurate quantitation. Recently, the equine MHC class I molecule 7-6, associated with the ELA-A1 haplotype, was shown to present both the Gag-GW12 and Env-RW12 EIAV CTL epitopes. In this study, 7-6/Gag-GW12 and 7-6/Env-RW12 MHC class I/peptide tetrameric complexes were constructed and used to analyze Gag-GW12- and Env-RW12-specific CTL responses in two EIAV-infected horses (A2164 and A2171). Gag-GW12 and Env-RW12 tetramer-positive CD8+ cells were identified in nonstimulated peripheral blood mononuclear cells as early as 14 days post-EIAV inoculation, and frequencies of tetramer-positive cells ranged from 0.4% to 6.7% of nonstimulated peripheral blood CD8+ cells during the 127-day study period. Although both horses terminated the initial viremic peak, only horse A2171 effectively controlled viral load. Neutralizing antibody was present during the initial control of viral load in both horses, but the ability to maintain control correlated with Gag-GW12-specific CD8+ cells in A2171. Despite Env-RW12 dominance, Env-RW12 escape viral variants were identified in both horses and there was no correlation between Env-RW12-specific CD8+ cells and control of viral load. Although Gag-GW12 CTL escape did not occur, a Gag-GW12 epitope variant arose in A2164 that was recognized less efficiently than the original epitope. These data indicate that tetramers are useful for identification and quantitation of CTL responses in horses, and suggest that the observed control of EIAV replication and clinical disease was associated with sustained CTL recognition of Gag-specific epitopes. PMID:15979679

Mealey, Robert H.; Sharif, Amin; Ellis, Shirley A.; Littke, Matt H.; Leib, Steven R.; McGuire, Travis C.

2012-01-01

389

Establecimiento de la técnica de RT-PCR para el diagnóstico de la fiebre porcina clásica en México Establishment of RT-PCR for classical swine fever diagnosis in Mexico  

Microsoft Academic Search

Classical swine fever (CSF) is an endemic disease in most of Mexico and results in large financial loss to the swine industry. Diagnosis is usually done with direct immunofluorescence (IF) and virus isolation (VI), though molecular techniques such as reverse transcription-polymerase chain reaction (RT-PCR) have been used in other countries. RT-PCR followed by nucleotide sequencing has been widely used for

Guadalupe Socci Escatel; Fernando Diosdado Vargas; Elvira Carrera Salas; Martha Macías García; Camila Arriaga Díaz; Antonio Morilla González

390

Reduced Estimated Glomerular Filtration Rate Is Associated with Stroke Outcome after Intravenous rt-PA: The Stroke Acute Management with Urgent Risk-Factor Assessment and Improvement (SAMURAI) rt-PA Registry  

Microsoft Academic Search

Background: The aim of this study was to determine whether renal dysfunction affects the outcome of stroke patients treated with recombinant tissue plasminogen activator (rt-PA). Methods: A retrospective, multicenter, observational study was conducted to identify the effects of underlying risk factors on intravenous rt-PA therapy using 0.6 mg\\/kg alteplase in 10 stroke centers in Japan. Consecutive stroke patients with a

Masaki Naganuma; Masatoshi Koga; Yoshiaki Shiokawa; Jyoji Nakagawara; Eisuke Furui; Kazumi Kimura; Hiroshi Yamagami; Yasushi Okada; Yasuhiro Hasegawa; Kazuomi Kario; Satoshi Okuda; Kazutoshi Nishiyama; Kazuo Minematsu; Kazunori Toyoda

2011-01-01

391

Standardized assessment of NAb responses elicited in rhesus monkeys immunized with single- or multi-clade HIV-1 envelope immunogens.  

PubMed

The genetic diversity of HIV-1 envelope glycoproteins (Env) remains a major obstacle to the development of an antibody-based AIDS vaccine. The present studies examine the breadth and magnitude of neutralizing antibody (NAb) responses in rhesus monkeys after immunization with DNA prime-recombinant adenovirus (rAd) boost vaccines encoding either single or multiple genetically distant Env immunogens, and subsequently challenged with a pathogenic simian-human immunodeficiency virus (SHIV-89.6P). Using a standardized multi-tier panel of reference Env pseudoviruses for NAb assessment, we show that monkeys immunized with a mixture of Env immunogens (clades A, B, and C) exhibited a greater breadth of NAb activity against neutralization-sensitive Tier 1 viruses following both vaccination and challenge compared to monkeys immunized with a single Env immunogen (clade B or C). However, all groups of Env-vaccinated monkeys demonstrated only limited neutralizing activity against Tier 2 pseudoviruses, which are more characteristic of the neutralization sensitivity of circulating HIV-1. Notably, the development of a post-challenge NAb response against SHIV-89.6P was similar in monkeys receiving either clade B, clade C, or clade A+B+C Env immunogens, suggesting cross-clade priming of NAb responses. In addition, vaccines encoding Env immunogens heterologous to SHIV-89.6P primed for a rapid anamnestic NAb response following infection compared to vaccines lacking an Env component. These results show that DNA/rAd immunization with multiple diverse Env immunogens is a viable approach for enhancing the breadth of NAb responses against HIV-1, and suggest that Env immunogens can prime for anamnestic NAb responses against a heterologous challenge virus. PMID:17599382

Seaman, Michael S; Leblanc, Daniel F; Grandpre, Lauren E; Bartman, Melissa T; Montefiori, David C; Letvin, Norman L; Mascola, John R

2007-10-10

392

Multiplex RT-PCR-based detections of CEA, CK20 and EGFR in colorectal cancer patients  

PubMed Central

AIM: To develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) method detecting circulating tumor cells in the peripheral blood of colorectal cancer (CRC) patients. METHODS: Peripheral blood samples were collected from 88 CRC patients and 40 healthy individuals from the blood donors’ clinic and subsequently analyzed by multiplex RT-RCR for the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and epidermal growth factor receptor (EGFR) mRNA. The analysis involved determining the detection rates of CEA, CK20 and EGFR transcripts vs disease stage and overall survival. Median follow-up period was 19 mo (range 8-28 mo). RESULTS: Rates of CEA, CK20 and EGFR detection in CRC patients were 95.5%, 78.4% and 19.3%, respectively. CEA transcripts were detected in 3 healthy volunteer samples (7.5%), whereas all control samples were tested negative for CK20 and EGFR transcripts. The increasing number of positive detections for CEA, CK20 and EGFR transcripts in each blood sample was positively correlated with Astler-Coller disease stage (P < 0.001) and preoperative serum levels of CEA (P = 0.029) in CRC patients. Data analysis using Kaplan-Meier estimator documented significant differences in the overall survival of the different CRC patient groups as formed according to the increasing number of positivity for CEA, CK20 and EGFR transcripts. CONCLUSION: These data suggest that multiplex RT-PCR assay can provide useful information concerning disease stage and overall survival of CRC patients. PMID:21157973

Tsouma, Aikaterini; Aggeli, Chrysanthi; Lembessis, Panagiotis; Zografos, George N; Korkolis, Dimitris P; Pectasides, Dimitrios; Skondra, Maria; Pissimissis, Nikolaos; Tzonou, Anastasia; Koutsilieris, Michael

2010-01-01

393

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

394

UV inactivation of adenovirus type 41 measured by cell culture mRNA RT-PCR.  

PubMed

Adenoviruses are among the most resistant waterborne pathogens to UV disinfection, yet of the 51 serologically distinct human adenoviruses, only a few have been evaluated for their sensitivities to UV irradiation. Human enteric adenoviruses (Ad40 and Ad41) are difficult to cultivate and reliably assay for infectivity, requiring weeks to obtain cytopathogenic effects (CPE). Inoculated cell cultures often deteriorate before the appearance of distinctive CPE making it difficult to obtain reliable and reproducible data regarding UV inactivation. Adenovirus is a double-stranded DNA virus and produces messenger RNA (mRNA) during replication in host cells. The presence of viral mRNA in host cells is definitive evidence of infection. We recently developed a rapid and reliable cell culture-mRNA RT-PCR assay to detect and quantify adenovirus infectivity. Viral mRNA recovered from cell cultures 5-7 days after infection was purified on oligo-dT latex, treated with DNase, and amplified by RT-PCR using the primers specific for a conserved region of the hexon late mRNA transcript. Treatment of approximately 10(4) Ad41 with different doses of 254 nm germicidal UV radiation resulted in a dose-dependent loss of infectivity. As UV doses were increased from 75 to 200 mJ/cm2, virus survival decreased and no virus infectivity (measured by detectable mRNA) was found at a dose of 225 mJ/cm2 or higher. Our results using the cell culture mRNA RT-PCR assay indicate that Ad41 is more resistant to UV radiation than in a previous study using a conventional cell culture infectivity assay. Results were more similar to those found for Ad 40 using CPE as a measure of infectivity in another previous study. PMID:16046229

Ko, Gwangpyo; Cromeans, Theresa L; Sobsey, Mark D

2005-09-01

395

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

396

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...

397

THE ISL RT04 MANDARIN BROADCAST NEWS EVALUATION SYSTEM Hua Yu, Yik-Cheung Tam, Thomas Schaaf, Sebastian Stuker, Qin Jin, Mohamed Noamany, Tanja Scultz  

E-print Network

THE ISL RT04 MANDARIN BROADCAST NEWS EVALUATION SYSTEM Hua Yu, Yik-Cheung Tam, Thomas Schaaf}@ira.uka.de ABSTRACT This paper describes our effort in developing a Mandarin Broadcast News system for the RT-04f on the RT-04f evaluation set, over a period of three months. 1. INTRODUCTION Recognition of Mandarin

Schultz, Tanja

398

SPoRT: Transitioning NASA and NOAA Experimental Data to the Operational Weather Community  

NASA Technical Reports Server (NTRS)

Established in 2002 to demonstrate the weather and forecasting application of real-time EOS measurements, the NASA Short-term Prediction Research and Transition (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. With the ever-broadening application of real-time high resolution satellite data from current EOS, Suomi NPP, and planned JPSS and GOES-R sensors to weather forecast problems, significant challenges arise in the acquisition, delivery, and integration of the new capabilities into the decision making process of the operational weather community. For polar orbiting sensors such as MODIS, AIRS, VIIRS, and CRiS, the use of direct broadcast ground stations is key to the real-time delivery of the data and derived products in a timely fashion. With the ABI on the geostationary GOES-R satellite, the data volumes will likely increase by a factor of 5-10 from current data streams. However, the high data volume and limited bandwidth of end user facilities presents a formidable obstacle to timely access to the data. This challenge can be addressed through the use of subsetting techniques, innovative web services, and the judicious selection of data formats. Many of these approaches have been implemented by SPoRT for the delivery of real-time products to NWS forecast offices and other weather entities. Once available in decision support systems like AWIPS II, these new data and products must be integrated into existing and new displays that allow for the integration of the data with existing operational products in these systems. SPoRT is leading the way in demonstrating this enhanced capability. This paper will highlight the ways SPoRT is overcoming many of the challenges presented by the enormous data volumes of current and future satellite systems to get unique high quality research data into the operational weather environment.

Jedlovec, Gary J.

2013-01-01

399

Using the SPoRT POES/GOES Hybrid Product in OCONUS Forecasting  

NASA Technical Reports Server (NTRS)

The SPoRT (Short-term Prediction and Research Transition) Program at the NASA/Marshall Space Flight Center has been providing unique NASA and NOAA data and techniques to partner Weather Forecast Offices (WFOs) for ten years. Data are provided in the Decision Support System used by WFO forecasters: AWIPS. For the last couple of years, SPoRT has been producing the POES/GOES Hybrid. This suite of products combines the strength ofl5- minute animations of GOES imagery - providing temporal continuity, with the higher resolution, relatively random availability, of polar orbiting (POES) imagery data. The product was first introduced with only MODIS data from NASA's Terra and Aqua satellites, but recently the VIIRS instrument onboard the Suomi-NPP satellite was added, providing better high-resolution coverage. These products represent SPoRT's efforts to prepare for higher resolution, higher frequency GOES-R imagery - as well as helping to move VIIRS (JPSS) data into the mainstream of weather forecasting. SPoRT generates 5 products for this dataset: Visible, Longwave Infrared (11 micrometers), Shortwave IR (3.7 micrometers), Water Vapor (6.7 micrometers), and Fog (Difference of 11 micrometer and 3.7 micrometer channels). The Water Vapor hybrid product has a Red-Blue-Green image from MODIS inlaid, since it provides even more qualitative information than water vapor alone. Animated examples of the products will be shown in this presentation. While the resolution at nadir of GOES imagery is nominally Han (4km for IR channels), the inlaid polar orbiter imagery has a resolution of 250m (lkm for IR channels). This has tremendous application in the continental US. However, in high latitudes, since the usefulness of GOES degrades poleward rapidly, the contrast of GOES and POES data is stark. The consistent temporal nature of GOES, even though at a reduced resolution at high latitudes, provides basic situational awareness, but the introduction of polar data is very helpful in seeing the big picture with clarity - even if only briefly. This presentation will offer real situations where these products helped forecasters make better informed decisions quickly. Plans to augment the product further with the addition of data from several A VHRR instruments will be described.

Smith, Matt; Fuell, Kevin; Nelson, Jim

2014-01-01

400

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

401

Challenges in Transitioning Research Data to Operations: The SPoRT Paradigm  

NASA Technical Reports Server (NTRS)

Established in 2002 to demonstrate the weather and forecasting application of real-time EOS measurements, the NASA Short-term Prediction Research and Transition (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. With the ever-broadening application of real-time high resolution satellite data from current EOS and planned NPP, JPSS, and GOES-R sensors to weather forecast problems, significant challenges arise in the acquisition, delivery, and integration of the new capabilities into the decision making process of the operational weather community. For polar orbiting sensors such as MODIS, AIRS, VIIRS, and CRiS, the use of direct broadcast ground stations is key to the real-time delivery of the data and derived products in a timely fashion. With the ABI on the geostationary GOES-R satellite, the data volume will likely increase by a factor of 5- 10 from current data streams. However, the high data volume and limited bandwidth of end user facilities presents a formidable obstacle to timely access to the data. This challenge can be addressed through the use of subsetting techniques, innovative web services, and the judicious selection of data formats. Many of these approaches have been implemented by SPoRT for the delivery of real-time products to