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Sample records for rt env shiv

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

    PubMed Central

    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

    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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    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

    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

  4. Shiv Grewal, Ph.D. - Publications

    Cancer.gov

    Supplementary Information - Shiv Grewal's Laboratory The revised Supplementary Table S3 from the paper “Mtr4-like protein coordinates nuclear RNA processing for heterochromatin assembly and for telomere maintenance” Cell 155:1061-1074 can be downloaded he

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

    PubMed Central

    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

    2015-01-01

    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)1,2. However even the best bNAbs neutralize 10–50% of HIV-1 isolates inefficiently (IC80 > 5 ?g/ml), suggesting that high concentrations of these antibodies would be necessary to achieve general protection3–6. 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 IC50 < 0.05 ?g/ml). 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 SIV 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 to 77 ?g/ml of fully functional rhesus eCD4-Ig for 40 weeks, and these macaques were protected from multiple 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

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

    PubMed

    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

    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

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

    PubMed Central

    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.

    2015-01-01

    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)1,2, 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)3 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

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

    PubMed Central

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

    2012-01-01

    Background.The impact of pre-exposure prophylaxis (PrEP) with antiretrovirals on breakthrough HIV or SHIV infection is not fully documented. We addressed the hypothesis that SHIVSF162P3 infection despite active PrEP results in altered early immune parameters, compared with untreated infection. Methods.Eleven rhesus macaques were infected during repeated, rectal, low-dose SHIVSF162P3 exposures while receiving concurrent oral PrEP (Truvada [n = 2] or GS7340 [n = 4]) or as untreated controls (n = 5). We measured SHIV RNA, inflammatory cytokines, CD4 cells, and SHIV-specific and memory T cells until 20 weeks after peak viremia. Results.SHIV infection during PrEP resulted in 100-fold lower peak viremia and lower IL-15, IL-18, and IL-1Ra levels, compared with controls (P < .05; Wilcoxon rank-sum test). Unlike controls, PrEP-treated macaques showed no significant CD4 cell count reduction during acute infection and developed more SHIV-specific central memory T cells, relative to controls. After in vivo CD8 cell depletion, viral load increased to similar levels, indicating that CD8 cells were critical for viral control in both groups. Conclusions.PrEP with antiretrovirals has beneficial effects on early SHIV infection even when infection is not prevented. Although long-term immune control could not be examined in this SHIV infection model, our results suggest that PrEP results in improved early disease parameters in breakthrough infections. PMID:22740713

  9. Prevention of Vaginal SHIV Transmission in Macaques by a Coitally-Dependent Truvada Regimen

    PubMed Central

    Radzio, Jessica; Aung, Wutyi; Holder, Angela; Martin, Amy; Sweeney, Elizabeth; Mitchell, James; Bachman, Shanon; Pau, Chou-Pong; Heneine, Walid; García-Lerma, J. Gerardo

    2012-01-01

    Background Daily pre-exposure prophylaxis (PrEP) with Truvada (a combination of emtricitabine (FTC) and tenofovir (TFV) disoproxil fumarate (TDF)) is a novel HIV prevention strategy recently found to prevent HIV transmission in men who have sex with men and heterosexual couples. We previously showed that a coitally-dependent Truvada regimen protected macaques against rectal SHIV transmission. Here we examined FTC and tenofovir TFV exposure in vaginal tissues after oral dosing and assessed if peri-coital Truvada also protects macaques against vaginal SHIV infection. Methods The pharmacokinetic profile of emtricitabine (FTC) and tenofovir (TFV) was evaluated at first dose. FTC and TFV levels were measured in blood plasma, rectal, and vaginal secretions. Intracellular concentrations of FTC-triphosphate (FTC-TP) and TFV-diphosphate (TFV-DP) were measured in PBMCs, rectal tissues, and vaginal tissues. Efficacy of Truvada in preventing vaginal SHIV infection was assessed using a repeat-exposure vaginal SHIV transmission model consisting of weekly exposures to low doses of SHIV162p3. Six pigtail macaques with normal menstrual cycles received Truvada 24 h before and 2 h after each weekly virus exposure and six received placebo. Infection was monitored by serology and PCR amplification of SHIV RNA and DNA. Results As in humans, the concentration of FTC was higher than the concentration of TFV in vaginal secretions. Also as in humans, TFV levels in vaginal secretions were lower than in rectal secretions. Intracellular TFV-DP concentrations were also lower in vaginal tissues than in rectal tissues. Despite the low vaginal TFV exposure, all six treated macaques were protected from infection after 18 exposures or 4 full menstrual cycles. In contrast, all 6 control animals were infected. Conclusions We modeled a peri-coital regimen with two doses of Truvada and showed that it fully protected macaques from repeated SHIV exposures. Our results open the possibility for simplified PrEP regimens to prevent vaginal HIV transmission in women. PMID:23226529

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

    SciTech Connect

    Devitt, Gerard; Emerson, Vanessa; Holtkotte, Denise; Pfeiffer, Tanya; Pisch, Thorsten; Bosch, Valerie . E-mail: v.bosch@dkfz.de

    2007-05-10

    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.

  11. Human Non-neutralizing HIV-1 Envelope Monoclonal Antibodies Limit the Number of Founder Viruses during SHIV Mucosal Infection in Rhesus Macaques

    PubMed Central

    Liao, Hua-Xin; Pollara, Justin; Liu, Pinghuang; Alam, S. Munir; Zhang, Ruijun; Cocklin, Sarah L.; Shen, Xiaoying; Duffy, Ryan; Xia, Shi-Mao; Schutte, Robert J.; Pemble IV, Charles W.; Dennison, S. Moses; Li, Hui; Chao, Andrew; Vidnovic, Kora; Evans, Abbey; Klein, Katja; Kumar, Amit; Robinson, James; Landucci, Gary; Forthal, Donald N.; Montefiori, David C.; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Rerks-Ngarm, Supachai; Robb, Merlin L.; Michael, Nelson L.; Kim, Jerome H.; Soderberg, Kelly A.; Giorgi, Elena E.; Blair, Lily; Korber, Bette T.; Moog, Christiane; Shattock, Robin J.; Schmitz, Joern E.; Moody, M. A.; Gao, Feng; Ferrari, Guido; Shaw, George M.; Haynes, Barton F.

    2015-01-01

    HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4+ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses. PMID:26237403

  12. Human non-neutralizing HIV-1 envelope monoclonal antibodies limit the number of founder viruses during SHIV mucosal infection in rhesus macaques

    DOE PAGESBeta

    Santra, Sampa; Tomaras, Georgia D.; Warrier, Ranjit; Nicely, Nathan I.; Liao, Hua -Xin; Pollara, Justin; Liu, Pinghuang; Alam, S. Munir; Zhang, Ruijun; Cocklin, Sarah L.; et al

    2015-08-03

    HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4? T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant regionmore »of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.« less

  13. Human non-neutralizing HIV-1 envelope monoclonal antibodies limit the number of founder viruses during SHIV mucosal infection in rhesus macaques

    SciTech Connect

    Santra, Sampa; Tomaras, Georgia D.; Warrier, Ranjit; Nicely, Nathan I.; Liao, Hua -Xin; Pollara, Justin; Liu, Pinghuang; Alam, S. Munir; Zhang, Ruijun; Cocklin, Sarah L.; Shen, Xiaoying; Duffy, Ryan; Xia, Shi -Mao; Schutte, Robert J.; Pemble IV, Charles W.; Dennison, S. Moses; Li, Hui; Chao, Andrew; Vidnovic, Kora; Evans, Abbey; Klein, Katja; Kumar, Amit; Robinson, James; Landucci, Gary; Forthal, Donald N.; Montefiori, David C.; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Rerks-Ngarm, Supachai; Robb, Merlin L.; Michael, Nelson L.; Kim, Jerome H.; Soderberg, Kelly A.; Giorgi, Elena E.; Blair, Lily; Korber, Bette T.; Moog, Christiane; Shattock, Robin J.; Letvin, Norman L.; Schmitz, Joern E.; Moody, M. A.; Gao, Feng; Ferrari, Guido; Shaw, George M.; Haynes, Barton F.; Douek, Daniel C.

    2015-08-03

    HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4? T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.

  14. NRM 2061/ENV 2005 Critical Thinking Papers

    E-print Network

    Brown, Gregory G.

    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

  15. ENVE 417 HYDRAULIC DESIGN TOPIC SYLLABUS

    E-print Network

    Clark, Shirley E.

    . Water Supply and Pollution Control, 7th Edition. Prentice-Hall. 2004. Supplemental Textbooks: PublicENVE 417 HYDRAULIC DESIGN TOPIC SYLLABUS Current Catalog Data: Design of water and waste water conveyance systems and storage facilities. Design of Storm Sewers, Inlets, Detention Basins, Culverts

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

    PubMed Central

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

    2009-01-01

    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 MSRV and its precise relation to HERV-W were hitherto unknown. Results By mapping HERV-W env cDNA sequences (n = 332) from peripheral blood mononuclear cells of patients with MS and healthy controls onto individual genomic HERV-W env elements, we identified seven transcribed HERV-W env loci in these cells, including ERVWE1. Transcriptional activity of individual HERV-W env elements did not significantly differ between patients with MS and controls. Remarkably, almost 30% of HERV-W env cDNAs were recombined sequences that most likely arose in vitro between transcripts from different HERV-W env elements. Re-analysis of published MSRV env sequences revealed that all of them can be explained as originating from genomic HERV-W env loci or recombinations among them. In particular, a MSRV env clone previously used for the generation of monoclonal antibody 6A2B2, detecting an antigen in MS brain lesions, appears to be derived from a HERV-W env locus on chromosome Xq22.3. This locus harbors a long open reading frame for an N-terminally truncated HERV-W Env protein. Conclusion Our data clarify the origin of MSRV env sequences, have important implications for the status of MSRV, and open the possibility that a protein encoded by a HERV-W env element on chromosome Xq22.3 may be expressed in MS brain lesions. PMID:19368703

  17. Lentivirus-mediated Gene Transfer in Hematopoietic Stem Cells Is Impaired in SHIV-infected, ART-treated Nonhuman Primates.

    PubMed

    Younan, Patrick M; Peterson, Christopher W; Polacino, Patricia; Kowalski, John P; Obenza, Willimark; Miller, Hannah W; Milless, Brian P; Gafken, Phil; DeRosa, Stephen C; Hu, Shiu-Lok; Kiem, Hans-Peter

    2015-05-01

    Recent studies have demonstrated that genetically modified hematopoietic stem cells (HSCs) can reduce HIV viremia. We have developed an HIV/AIDS-patient model in Simian/human immunodeficiency virus (SHIV)-infected pigtailed macaques that are stably suppressed on antiretroviral therapy (ART: raltegravir, emtricitabine and tenofovir). Following SHIV infection and ART, animals undergo autologous HSC transplantation (HSCT) with lentivirally transduced cluster of differentiation (CD)34(+) cells expressing the mC46 anti-HIV fusion protein. We show that SHIV(+), ART-treated animals had very low gene marking levels after HSCT. Pretransduction CD34(+) cells contained detectable levels of all three ART drugs, likely contributing to the low gene transfer efficiency. Following HSCT recovery and the cessation of ART, plasma viremia rebounded, indicating that myeloablative total body irradiation cannot completely eliminate viral reservoirs after autologous HSCT. The kinetics of recovery following autologous HSCT in SHIV(+), ART-treated macaques paralleled those observed following transplantation of control animals. However, T-cell subset analyses demonstrated a high percentage of C-C chemokine receptor 5 (CCR5)-expressing CD4(+) T-cells after HSCT. These data suggest that an extended ART interruption time may be required for more efficient lentiviral transduction. To avoid complications associated with ART interruption in the context of high percentages of CD4(+)CCR5(+)T-cells after HSCT, the use of vector systems not impaired by the presence of residual ART may also be beneficial. PMID:25648264

  18. [Research on construction of sheep lung adenomas virus pEGFP-C1/exJSRV-env and induction of malignant transformation in NIH3T3].

    PubMed

    Zhang, Yu-Fei; Liu, Yue; Wang, Zhuan-Jia; Sun, Xiao-Lin; Liu, Shu-Ying

    2014-05-01

    This study aims to construct a eukaryotic expression system for envelope gene of Jaagsiekte sheep retrovirus, observes its localization in 293T cells, and investigates the potential in inducing malignant transformation of NIH3T3 cells. By RT-PCR, the full-length cDNA of envelope gene of Jaagsiekte sheep retrovirus (exJSRV-env) was amplified from the extract of naturally infected sheep lung. The clone of target gene was sub-cloned into eukaryotic expression system pEGFP-C1, and validated by PCR, restriction endonuclease, and sequencing. Bioinformatic analysis concerning biological function and cellular localiza tion of exJSRV-env was also performed. The recombinant clone of exJSRV-env was transfected into 293T cells and NIH3T3 cells by Lipofectamine LTX. The expression and celluar localization in 293T cells were validated by confocal microscopy. Soft agar colony formation assay was employed to test the anchorage-independent growth of NIH3T3. DNA sequencing and restriction enzyme digestion with Kpn I and Hind III indicated the correct construction of the recombinant plasmid, which was named pEGFP-C1/exJSRV-env. Amino acid sequence alignment of exJSRV-env with reference sequences found 85%-100% homogeneity. A YRNM motif was discovered at the cytoplasmic tail of envelope gene, which is exclusively found in exogenous viruses. Phylogenetic tree analysis showed that our clone of exJSRV-env clustered closely with pathogenic exogenous Jaagsiekte sheep retroviruses. Fluorescence microscopy indicated typical membrane localization of exJSRV-env protein. NIH3T3 cells transfected with exJSRV-env lost contact inhibition, and acquired colony forming ability in soft agar. This study indicated that envelope protein of Jaagsiekte sheep retrovirus can induce malignant transformation of mouse fibroblast cell NIH3T3. Discoveries of this study provide a basis for further structural and functional research on Jaagsiekte sheep retrovirus envelope protein. PMID:25118382

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

    NASA Astrophysics Data System (ADS)

    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

    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.

  20. ENV 6105 Page 1 of 6 Fall 2011 Air Pollution

    E-print Network

    Stuart, Amy L.

    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

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

    PubMed Central

    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

    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

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

    SciTech Connect

    Yankee, Thomas M. Sheffer, Darlene; Liu Zhengian; Dhillon, Sukhbir; Jia Fenglan; Chebloune, Yahia; Stephens, Edward B.; Narayan, Opendra

    2009-01-05

    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.

  3. Estimating the impact of vaccination in acute SHIV-SIV infection

    SciTech Connect

    Ribeiro, Ruy

    2008-01-01

    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.

  4. parssppt.h cc_env.h (ubiquitous)

    E-print Network

    Necula, George

    main.cc parssppt.h cc_env.h (ubiquitous) cc_lang.h (ubiquitous) cc.gr.gen.h cc_elaborate.h integrity.h parssppt.cc lexer.h cc_env.cc cc_scope.h cc_err.h builtinops.h mflags.h cc_lang.cc cc.gr.gen.cc cc_tokens.h ccparse.h kandr.h cc_elaborate.cc integrity.cc astvisit.h cc_tcheck.cc cc_ast.h

  5. Mannose-specific lectins that inhibit HIV infection bind nonspecifically to HIV Env-expressing cells.

    PubMed

    Chau, Deborah; Yee, Michael; Gebremedhin, Senait; Cheung, Jennifer; Chino, Takahiro; Düzgüne?, Nejat

    2015-02-01

    An approach to curing HIV/AIDS is to specifically kill all infected cells. Because the lectins, Hippeastrum hybrid agglutinin (HHA) and Galanthus nivalis agglutinin (GNA), are potent inhibitors of HIV infection and bind the oligomannans on the HIV Env protein, we hypothesized that they would bind specifically to cells expressing the HIV Env protein on their plasma membrane. Flow cytometry experiments indicated, however, that these lectins bind equivalently to both Env-expressing and control cells without Env. PMID:25868224

  6. ENVIRONMENTAL ENGINEERING (ENVE) CLASS OF 2019 Name: Last, First, ID Minimum 127 SH

    E-print Network

    Papautsky, Ian

    .. 3 GEOG 6071C Geog Informa Syst 3 ENVE 4011 Air Pollution Cont.... 3 PD 4001 Prof. Development... 1FS CVE 3002C Soil Mech & Lab..... 4 ENVE 4093L Flu Mech & Hyd Sy.. 2 18SS ENVE 3040 C & E System Anal

  7. ENVIRONMENTAL ENGINEERING (ENVE) CLASS OF 2018 Name: Last, First, ID Minimum 127 SH

    E-print Network

    Papautsky, Ian

    .. 3 GEOG 6071C Geog Informa Syst 3 ENVE 4011 Air Pollution Cont.... 3 PD 4001 Prof. Development... 1FS CVE 3002C Soil Mech & Lab..... 4 ENVE 4093L Flu Mech & Hyd Sy.. 2 17SS ENVE 3040 C & E System Anal

  8. ENVIRONMENTAL ENGINEERING (ENVE) CLASS OF 2017 Name: Last, First, ID Minimum 127 SH

    E-print Network

    Papautsky, Ian

    .. 3 GEOG 6071C Geog Informa Syst 3 ENVE 4011 Air Pollution Cont.... 3 PD 4001 Prof. Development... 1FS CVE 3002C Soil Mech & Lab..... 4 ENVE 4093L Flu Mech & Hyd Sy.. 2 16SS ENVE 3040 C & E System Anal

  9. ENVIRONMENTAL ENGINEERING (ENVE) CLASS OF 2020 Name: Last, First, ID Minimum 127 SH

    E-print Network

    Papautsky, Ian

    .. 3 GEOG 6071C Geog Informa Syst 3 ENVE 4011 Air Pollution Cont.... 3 PD 4001 Prof. Development... 1FS CVE 3002C Soil Mech & Lab..... 4 ENVE 4093L Flu Mech & Hyd Sy.. 2 19SS ENVE 3040 C & E System Anal

  10. 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

    Hout, David R.; Gomez, Lisa M.; Pacyniak, Erik; Miller, Jean-Marie; Hill, M. Sarah; Stephens, Edward B. . E-mail: estephen@kumc.edu

    2006-05-10

    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.

  11. Appreciating HIV-1 diversity: subtypic differences in ENV

    SciTech Connect

    Gnanakaran, S; Shen, Tongye; Lynch, Rebecca M; Derdeyn, Cynthia A

    2008-01-01

    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.

  12. Trimeric HIV Env provides epitope occlusion mediated by hypervariable loops

    PubMed Central

    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

    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

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

    SciTech Connect

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

    2007-10-10

    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.

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

    SciTech Connect

    Vargas, Amandine Thiery, Maxime Lafond, Julie Barbeau, Benoit

    2012-03-30

    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.

  15. SimEnvVis: A Climate Data Visualization Wizard

    NASA Astrophysics Data System (ADS)

    Heitzler, Magnus; Nocke, Thomas

    2013-04-01

    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.

  16. A method to determine the duration of the eclipse phase for in vitro infection with a highly pathogenic SHIV strain

    PubMed Central

    Kakizoe, Yusuke; Nakaoka, Shinji; Beauchemin, Catherine A. A.; Morita, Satoru; Mori, Hiromi; Igarashi, Tatsuhiko; Aihara, Kazuyuki; Miura, Tomoyuki; Iwami, Shingo

    2015-01-01

    The time elapsed between successful cell infection and the start of virus production is called the eclipse phase. Its duration is specific to each virus strain and, along with an effective virus production rate, plays a key role in infection kinetics. How the eclipse phase varies amongst cells infected with the same virus strain and therefore how best to mathematically represent its duration is not clear. Most mathematical models either neglect this phase or assume it is exponentially distributed, such that at least some if not all cells can produce virus immediately upon infection. Biologically, this is unrealistic (one must allow for the translation, transcription, export, etc. to take place), but could be appropriate if the duration of the eclipse phase is negligible on the time-scale of the infection. If it is not, however, ignoring this delay affects the accuracy of the mathematical model, its parameter estimates, and predictions. Here, we introduce a new approach, consisting in a carefully designed experiment and simple analytical expressions, to determine the duration and distribution of the eclipse phase in vitro. We find that the eclipse phase of SHIV-KS661 lasts on average one day and is consistent with an Erlang distribution. PMID:25996439

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

    PubMed Central

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

    2013-01-01

    Human immunodeficiency virus type 1 (HIV-1) is completely dependent upon the Env protein to enter cells. The virus typically replicates in activated CD4+ T cells due to viral entry requirements for the CCR5 coreceptor and for high surface levels of the CD4 receptor. This is the case for the transmitted virus and for most of the virus sampled in the blood. Over the course of infection, the env gene can evolve to encode a protein with altered receptor and coreceptor usage allowing the virus to enter alternative host cells. In about 50% of HIV-1 infections, the viral population undergoes coreceptor switching, usually late in disease, allowing the virus to use CXCR4 to enter a different subset of CD4+ T cells. Neurocognitive disorders occur in about 10% of infections, also usually late in disease, but caused (ultimately) by viral replication in the brain either in CD4+ T cells or macrophage and/or microglia. Expanded host range is significantly intertwined with pathogenesis. Identification and characterization of such HIV-1 variants may be useful for early detection which would allow intervention to reduce viral pathogenesis in these alternative cell types. PMID:22237899

  18. NMobTec-EnvEdu: M-Learning System for Environmental Education

    ERIC Educational Resources Information Center

    Cavus, Nadire

    2008-01-01

    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…

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

    PubMed Central

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

    2003-01-01

    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

  20. Cytoplasmic sensing by the inner membrane histidine kinase EnvZ.

    PubMed

    Foo, Yong Hwee; Gao, Yunfeng; Zhang, Hongfang; Kenney, Linda J

    2015-09-01

    Two-component regulatory systems drive signal transduction in bacteria. The simplest of these employs a membrane sensor kinase and a cytoplasmic response regulator. Environmental sensing is typically coupled to gene regulation. The histidine kinase EnvZ and its cognate response regulator OmpR regulate expression of outer membrane proteins (porins) in response to osmotic stress. We used hydrogen:deuterium exchange mass spectrometry to identify conformational changes in the cytoplasmic domain of EnvZ (EnvZc) that were associated with osmosensing. The osmosensor localized to a seventeen amino acid region of the four-helix bundle of the cytoplasmic domain and flanked the His(243) autophosphorylation site. High osmolality increased autophosphorylation of His(243), suggesting that these two events were linked. The transmembrane domains were not required for osmosensing, but mutants in the transmembrane domains altered EnvZ activity. A photoactivatable fusion protein composed of EnvZc fused to the fluorophore mEos2 (EnvZc-mEos2) was as capable as EnvZc in supporting OmpR-dependent ompF and ompC transcription. Over-expression of EnvZc reduced activity, indicating that the EnvZ/OmpR system is not robust. Our results support a model in which osmolytes stabilize helix one in the four-helix bundle of EnvZ by increased hydrogen bonding of the peptide backbone, increasing autophosphorylation and downstream signaling. The likelihood that additional histidine kinases use similar cytoplasmic sensing mechanisms is discussed. PMID:25937465

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

    PubMed Central

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

    2015-01-01

    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

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

    PubMed

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

    2015-04-01

    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

  3. Phylogenetics of HIV-1 subtype G env: Greater complexity and older origins than previously reported.

    PubMed

    Tongo, Marcel; Essomba, René G; Nindo, Frederick; Abrahams, Fatima; Nanfack, Aubin Joseph; Fokam, Joseph; Takou, Desire; Torimiro, Judith N; Mpoudi-Ngole, Eitel; Burgers, Wendy A; Martin, Darren P; Dorfman, Jeffrey R

    2015-10-01

    HIV-1 subtype G has played an early and central role in the emergent complexity of the HIV-1 group M (HIV-1M) epidemic in central/west Africa. Here, we analysed new subtype G env sequences sampled from 8 individuals in Yaoundé, Cameroon during 2007-2010, together with all publically available subtype G-attributed full-length env sequences with known sampling dates and locations. We inferred that the most recent common ancestor (MRCA) of the analysed subtype G env sequences most likely occurred in ?1953 (95% Highest Posterior Density interval [HPD] 1939-1963): about 15years earlier than previous estimates. We found that the subtype G env phylogeny has a complex structure including seven distinct lineages, each likely dating back to the late 1960s or early 1970s. Sequences from Angola, Gabon and the Democratic Republic of Congo failed to group consistently in these lineages, possibly because they are related to more ancient sequences that are poorly sampled. The circulating recombinant form (CRF), CRF06_cpx env sequences but not CRF25_cpx env sequences are phylogenetically nested within the subtype G clade. This confirms that the CRF06_cpx env plausibly was derived through recombination from a subtype G parent, and suggests that the CRF25_cpx env was likely derived from an HIV-1M lineage related to the MRCA of subtype G that has remained undiscovered and may be extinct. Overall, this fills important gaps in our knowledge of the early events in the spread of HIV-1M. PMID:26190450

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

    PubMed Central

    2014-01-01

    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

  5. Mucosal Immunization with Newcastle Disease Virus Vector Coexpressing HIV-1 Env and Gag Proteins Elicits Potent Serum, Mucosal, and Cellular Immune Responses That Protect against Vaccinia Virus Env and Gag Challenges

    PubMed Central

    Khattar, Sunil K.; Manoharan, Vinoth; Bhattarai, Bikash; LaBranche, Celia C.; Montefiori, David C.

    2015-01-01

    ABSTRACT Newcastle disease virus (NDV) avirulent strain LaSota was used to coexpress gp160 Env and p55 Gag from a single vector to enhance both Env-specific and Gag-specific immune responses. The optimal transcription position for both Env and Gag genes in the NDV genome was determined by generating recombinant NDV (rNDV)-Env-Gag (gp160 located between the P and M genes and Gag between the HN and L genes), rNDV-Gag-Env (Gag located between the P and M genes and gp160 between the HN and L genes), rNDV-Env/Gag (gp160 followed by Gag located between the P and M genes), and rNDV-Gag/Env (Gag followed by gp160 located between the P and M genes). All the recombinant viruses replicated at levels similar to those seen with parental NDV in embryonated chicken eggs and in chicken fibroblast cells. Both gp160 and Gag proteins were expressed at high levels in cell culture, with gp160 found to be incorporated into the envelope of NDV. The Gag and Env proteins expressed by all the recombinants except rNDV-Env-Gag self-assembled into human immunodeficiency virus type 1 (HIV-1) virus-like particles (VLPs). Immunization of guinea pigs by the intranasal route with these rNDVs produced long-lasting Env- and Gag-specific humoral immune responses. The Env-specific humoral and mucosal immune responses and Gag-specific humoral immune responses were higher in rNDV-Gag/Env and rNDV-Env/Gag than in the other recombinants. rNDV-Gag/Env and rNDV-Env/Gag were also more efficient in inducing cellular as well as protective immune responses to challenge with vaccinia viruses expressing HIV-1 Env and Gag in mice. These results suggest that vaccination with a single rNDV coexpressing Env and Gag represents a promising strategy to enhance immunogenicity and protective efficacy against HIV. PMID:26199332

  6. Implementing RtI with Gifted Students

    ERIC Educational Resources Information Center

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

    2012-01-01

    "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…

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

    PubMed Central

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

    2014-01-01

    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

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

    USGS Publications Warehouse

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

    2014-01-01

    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.

  9. Inside the Envelope: Endogenous Retrovirus-K Env as a Biomarker and Therapeutic Target

    PubMed Central

    Nadeau, Marie-Josée; Manghera, Mamneet; Douville, Renée N.

    2015-01-01

    Due to multiple ancestral human retroviral germ cell infections, the modern human genome is strewn with relics of these infections, termed endogenous retroviruses (ERVs). ERV expression has been silenced due to negative selective pressures and genetic phenomena such as mutations and epigenetic silencing. Nonetheless, select ERVs have retained the capacity to be damaging to their host when reawakened. Much of the current research on the ERVK Env protein strongly suggests a causal or contributive role in the pathogenesis of various cancers, autoimmune and infectious diseases. Additionally, there is a small body of research suggesting that ERVK Env has been domesticated for use in placental development, akin to the ERVW syncytin. Though much is left to ascertain, the innate immune response to ERVK Env expression has been partially characterized and appears to be due to a region located in the transmembrane domain of the Env protein. In this review, we aim to highlight ERVK Env as a biomarker for inflammatory conditions and explore its use as a future therapeutic target for cancers, HIV infection and neurological disease. PMID:26617584

  10. Cleavage-independent HIV-1 Env trimers engineered as soluble native spike mimetics for vaccine design.

    PubMed

    Sharma, Shailendra Kumar; de Val, Natalia; Bale, Shridhar; Guenaga, Javier; Tran, Karen; Feng, Yu; Dubrovskaya, Viktoriya; Ward, Andrew B; Wyatt, Richard T

    2015-04-28

    Viral glycoproteins mediate entry by pH-activated or receptor-engaged activation and exist in metastable pre-fusogenic states that may be stabilized by directed rational design. As recently reported, the conformationally fixed HIV-1 envelope glycoprotein (Env) trimers in the pre-fusion state (SOSIP) display molecular homogeneity and structural integrity at relatively high levels of resolution. However, the SOSIPs necessitate full Env precursor cleavage, which requires endogenous furin overexpression. Here, we developed an alternative strategy using flexible peptide covalent linkage of Env subdomains to produce soluble, homogeneous, and cleavage-independent Env mimics, called native flexibly linked (NFL) trimers, as vaccine candidates. This simplified design avoids the need for furin co-expression and, in one case, antibody affinity purification to accelerate trimer scale-up for preclinical and clinical applications. We have successfully translated the NFL design to multiple HIV-1 subtypes, establishing the potential to become a general method of producing native-like, well-ordered Env trimers for HIV-1 or other viruses. PMID:25892233

  11. ? Light Chain Bias Associated With Enhanced Binding and Function of Anti-HIV Env Glycoprotein Antibodies.

    PubMed

    Sajadi, Mohammad M; Farshidpour, Maham; Brown, Eric P; Ouyang, Xin; Seaman, Michael S; Pazgier, Marzena; Ackerman, Margaret E; Robinson, Harriet; Tomaras, Georgia; Parsons, Matthew S; Charurat, Manhattan; DeVico, Anthony L; Redfield, Robert R; Lewis, George K

    2016-01-01

    The humoral response to human immunodeficiency virus (HIV) remains incompletely understood. In this report, we describe biased ? light chain use during the HIV Env glycoprotein (Env) response in HIV infection and vaccination. We examined HIV Env binding (and neutralization) in the context of light chain use in subjects with acute HIV infection, chronic HIV infection, and among HIV vaccinees. In all populations tested, there was a ? chain bias for HIV Env binding antibodies, compared with other HIV antigens (such as p24) or tetanus toxoid. In subjects with chronic HIV infection, a ? bias was noted for neutralization, with ? antibodies accounting for up to 90% of all neutralization activity observed. This is the first report of antibody function in a human infection being tied to light chain use. In HIV infection, antibodies expressing ? light chains tended to have longer CDRL3s, increased light chain contact with HIV Env, and less hypermutation in the heavy chain, compared with antibodies using the ? light chain. These data also support an evolutionary model for the understanding the various ? to ? light chain ratios observed across species and suggest that the ? light chain bias against HIV provides the host an advantage in developing a more efficient humoral response. PMID:26347575

  12. Parenteral Administration of Capsule Depolymerase EnvD Prevents Lethal Inhalation Anthrax Infection.

    PubMed

    Negus, David; Vipond, Julia; Hatch, Graham J; Rayner, Emma L; Taylor, Peter W

    2015-12-01

    Left untreated, inhalation anthrax is usually fatal. Vegetative forms of Bacillus anthracis survive in blood and tissues during infection due to elaboration of a protective poly-?-d-glutamic acid (PDGA) capsule that permits uncontrolled bacterial growth in vivo, eventually leading to overwhelming bacillosis and death. As a measure to counter threats from multidrug-resistant strains, we are evaluating the prophylactic and therapeutic potential of the PDGA depolymerase EnvD, a stable and potent enzyme which rapidly and selectively removes the capsule from the surface of vegetative cells. Repeated intravenous administration of 10 mg/kg recombinant EnvD (rEnvD) to mice infected with lethal doses of B. anthracis Ames spores by inhalation prevented the emergence of symptoms of anthrax and death; all animals survived the 5-day treatment period, and 70% survived to the end of the 14-day observation period. In contrast to results in sham-treated animals, the lungs and spleen of rEnvD-dosed animals were free of gross pathological changes. We conclude that rEnvD has potential as an agent to prevent the emergence of inhalation anthrax in infected animals and is likely to be effective against drug-resistant forms of the pathogen. PMID:26438506

  13. 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

    Hout, David R.; Gomez, Melissa L.; Pacyniak, Erik; Gomez, Lisa M.; Fegley, Barbara; Mulcahy, Ellyn R.; Hill, M. Sarah; Culley, Nathan; Pinson, David M.; Nothnick, Warren; Powers, Michael F.; Wong, Scott W.; Stephens, Edward B. . E-mail: estephen@kumc.edu

    2006-01-20

    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.

  14. 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...

  15. Interinstrument Reliability of the RT3 Accelerometer

    ERIC Educational Resources Information Center

    Reneman, Michiel

    2010-01-01

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

  16. Using CoRT Thinking in Schools.

    ERIC Educational Resources Information Center

    Melchior, Timothy M.; And Others

    1988-01-01

    Describes the use of Edward de Bono's CoRT (Cognitive Research Trust) program in English classes during the past five years at Memorial Junior High School in Valley Stream, New York. CoRT tools were used to analyze literary characters and plot development and to generate and organize ideas for writing assignments. (TE)

  17. HSV-2-driven increase in the expression of ?4?7 correlates with increased susceptibility to vaginal SHIV(SF162P3) infection.

    PubMed

    Goode, Diana; Truong, Rosaline; Villegas, Guillermo; Calenda, Giulia; Guerra-Perez, Natalia; Piatak, Michael; Lifson, Jeffrey D; Blanchard, James; Gettie, Agegnehu; Robbiani, Melissa; Martinelli, Elena

    2014-12-01

    The availability of highly susceptible HIV target cells that can rapidly reach the mucosal lymphoid tissues may increase the chances of an otherwise rare transmission event to occur. Expression of ?4?7 is required for trafficking of immune cells to gut inductive sites where HIV can expand and it is expressed at high level on cells particularly susceptible to HIV infection. We hypothesized that HSV-2 modulates the expression of ?4?7 and other homing receptors in the vaginal tissue and that this correlates with the increased risk of HIV acquisition in HSV-2 positive individuals. To test this hypothesis we used an in vivo rhesus macaque (RM) model of HSV-2 vaginal infection and a new ex vivo model of macaque vaginal explants. In vivo we found that HSV-2 latently infected RMs appeared to be more susceptible to vaginal SHIVSF162P3 infection, had higher frequency of ?4?7high CD4+ T cells in the vaginal tissue and higher expression of ?4?7 and CD11c on vaginal DCs. Similarly, ex vivo HSV-2 infection increased the susceptibility of the vaginal tissue to SHIVSF162P3. HSV-2 infection increased the frequencies of ?4?7high CD4+ T cells and this directly correlated with HSV-2 replication. A higher amount of inflammatory cytokines in vaginal fluids of the HSV-2 infected animals was similar to those found in the supernatants of the infected explants. Remarkably, the HSV-2-driven increase in the frequency of ?4?7high CD4+ T cells directly correlated with SHIV replication in the HSV-2 infected tissues. Our results suggest that the HSV-2-driven increase in availability of CD4+ T cells and DCs that express high levels of ?4?7 is associated with the increase in susceptibility to SHIV due to HSV-2. This may persists in absence of HSV-2 shedding. Hence, higher availability of ?4?7 positive HIV target cells in the vaginal tissue may constitute a risk factor for HIV transmission. PMID:25521298

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

    PubMed Central

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

    2012-01-01

    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

  19. NRM 2061/ENV 2005 Environmental Documentaries: Knowledge to Action Environmental Studies Department and

    E-print Network

    Brown, Gregory G.

    systems to environmental problems and their solutions; 5. Describe a wide spectrum of environmental advocacy methods; 6. Communicate the wide range of environmental problems by the local and global communityNRM 2061/ENV 2005 Environmental Documentaries: Knowledge to Action Environmental Studies Department

  20. ENVS 4000, Spring (Jan-Apr) 2007 Impacts of Climate Change

    E-print Network

    Johnson, Dan L.

    quantitative methods of analysis, attend and participate in class, contribute current events news, write shortENVS 4000, Spring (Jan-Apr) 2007 Impacts of Climate Change This capstone Environmental Science: science and technology - paleoclimate - climate change theory and modeling; uncertainty and risk - global

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

    E-print Network

    Kececioglu, John

    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

  2. MAXIMUM LIKELIHOOD ANALYSIS OF ADAPTIVE EVOLUTION IN HIV-1 GP120 ENV GENE

    E-print Network

    Yang, Ziheng

    MAXIMUM LIKELIHOOD ANALYSIS OF ADAPTIVE EVOLUTION IN HIV-1 GP120 ENV GENE ZIHENG YANG Galton structure and function of the protein and thus under purifying selection. Some proteins also have variable. These amino acids are also important to the structure and function of the protein, although in a different way

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

    SciTech Connect

    Ye Ling; Sun Yuliang; Lin Jianguo; Bu Zhigao; Wu Qingyang; Jiang, Shibo; Steinhauer, David A.; Compans, Richard W.; Yang Chinglai . E-mail: chyang@emory.edu

    2006-08-15

    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.

  4. A Multiple siRNA-Based Anti-HIV/SHIV Microbicide Shows Protection in Both In Vitro and In Vivo Models

    PubMed Central

    Raulji, Payal; Mohapatra, Subhra; Mohapatra, Shyam S

    2015-01-01

    Human immunodeficiency virus (HIV) types 1 and 2 (HIV-1 and HIV-2) are the etiologic agents of AIDS. Most HIV-1 infected individuals worldwide are women, who acquire HIV infections during sexual contact. Blocking HIV mucosal transmission and local spread in the female lower genital tract is important in preventing infection and ultimately eliminating the pandemic. Microbicides work by destroying the microbes or preventing them from establishing an infection. Thus, a number of different types of microbicides are under investigation, however, the lack of their solubility and bioavailability, and toxicity has been major hurdles. Herein, we report the development of multifunctional chitosan-lipid nanocomplexes that can effectively deliver plasmids encoding siRNA(s) as microbicides without adverse effects and provide significant protection against HIV in both in vitro and in vivo models. Chitosan or chitosan-lipid (chlipid) was complexed with a cocktail of plasmids encoding HIV-1-specific siRNAs (psiRNAs) and evaluated for their efficacy in HEK-293 cells, PBMCs derived from nonhuman primates, 3-dimensional human vaginal ectocervical tissue (3D-VEC) model and also in non-human primate model. Moreover, prophylactic administration of the chlipid to deliver a psiRNA cocktail intravaginally with a cream formulation in a non-human primate model showed substantial reduction of SHIV (simian/human immunodeficiency virus SF162) viral titers. Taken together, these studies demonstrate the potential of chlipid-siRNA nanocomplexes as a potential genetic microbicide against HIV infections. PMID:26407080

  5. Foamy Combinatorial Anti-HIV Vectors with MGMTP140K Potently Inhibit HIV-1 and SHIV Replication and Mediate Selection In Vivo

    PubMed Central

    Kiem, Hans-Peter; Wu, Robert A.; Sun, Guihua; von Laer, Dorothee; Rossi, John J.; Trobridge, Grant D.

    2011-01-01

    Highly active antiretroviral therapy (HAART) has greatly reduced the morbidity and mortality from HIV infection, but AIDS continues to be a serious health problem worldwide. Despite enormous efforts to develop a vaccine, there is still no cure, and alternative approaches including gene therapy should be explored. Here we developed and compared combinatorial foamy virus (FV) anti-HIV vectors that also express a mutant methylguanine methyltransferase (MGMTP140K) transgene to increase the percentage of gene modified cells after transplantation. These FV vectors inhibit replication of HIV-1 and also the simian immunodeficiency virus/HIV-1 (SHIV) chimera that can be used in monkey AIDS gene therapy studies. We identified a combinatorial FV vector that expresses 3 anti-HIV transgenes and inhibits viral replication by over 4 logs in a viral challenge assay. This FV anti-HIV vector expresses an HIV fusion inhibitor and two shRNAs targeted to HIV-1 tat and rev, and can be produced at high titer (3.8×107 transducing units/ml) using improved helper plasmids suitable for clinical use. Using a competitive repopulation assay, we show that human CD34+ cells transduced with this combinatorial FV vector efficiently engraft in a mouse xenotransplantation model, and that the percentage of transduced repopulating cells can be increased after transplantation. PMID:19741733

  6. Modulation of Env Content in Virions of Simian Immunodeficiency Virus: Correlation with Cell Surface Expression and Virion Infectivity

    PubMed Central

    Yuste, Eloísa; Reeves, Jacqueline D.; Doms, Robert W.; Desrosiers, Ronald C.

    2004-01-01

    Specific mutations were created in the cytoplasmic domain of the gp41 transmembrane protein of simian immunodeficiency virus strain 239 (SIV239). The resultant strains included a mutant in which Env residue 767 was changed to a stop codon, a double mutant in which positions 738 and 739 were changed to stop codons, another mutant in which a prominent endocytosis motif was changed from YRPV to GRPV by the substitution of tyrosine 721, and a final combination mutant bearing Q738stop, Q739stop, and Y721G mutations. The effects of these mutations on cell surface expression, on Env incorporation into virions, and on viral infectivity were examined. The molar ratio of Gag to gp120 of 54:1 that we report here for SIV239 virions agrees very well with the ratio of 60:1 reported previously by Chertova et al. (E. Chertova, J. W. Bess, Jr., B. J. Crise, R. C. Sowder II, T. M. Schaden, J. M. Hilburn, J. A. Hoxie, R. E. Benveniste, J. D. Lifson, L. E. Henderson, and L. O. Arthur, J. Virol. 76:5315-5325, 2002), although they were determined by very different methodologies. Assuming 1,200 to 2,500 Gag molecules per virion, this corresponds to 7 to 16 Env trimers per SIV239 virion particle. Although all of the mutations increased Env levels in virions, E767stop had the most dramatic effect, increasing the Env content per virion 25- to 50-fold. Increased levels of Env content in virions correlated strictly with higher levels of Env expression on the cell surface. The increased Env content with the E767stop mutation also correlated with an increased infectivity, but the degree of change was not proportional: the 25- to 50-fold increase in Env content only increased infectivity 2- to 3-fold. All of the mutants replicated efficiently in the CEMx174 and Rh221-89 cell lines. Although some of these findings have been reported previously, our findings show that the effects of the cytoplasmic domain of gp41 on the Env content in virions can be dramatic, that the Env content in virions correlates strictly with the levels of cell surface expression, and that the Env content in virions can determine infectivity; furthermore, our results define a particular change with the most dramatic effects. PMID:15194752

  7. Analysis of the human env-specific cytotoxic T-lymphocyte (CTL) response in natural human immunodeficiency virus type 1 infection: low prevalence of broadly cross-reactive env-specific CTL.

    PubMed Central

    Carmichael, A; Jin, X; Sissons, P

    1996-01-01

    Major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) are part of the cellular immune response to persistent virus infections. Candidate vaccines against human immunodeficiency virus type 1 (HIV-1) should elicit broad cross-reactive immunity to confer protection against different strains of HIV-1. As it is likely that candidate vaccines will include the envelope gene product Env, we determined the proportion of CTL clones which recognized variable and conserved determinants in three env variants during natural infection. Limiting dilution analysis was used to characterize numerous short-term CTL clones derived from peripheral blood of HIV-1-infected subjects, using split-well analysis to assay cytotoxicity against target cells expressing gp160env of HIV-1 strains IIIB, MN, and RF. In 9 of 12 HIV-1-infected subjects, at the clonal level most env-specific CTL recognized determinant(s) within one env variant but not in the other variants. In some subjects, CTL recognized multiple nonconserved determinants in different variants. The pattern of recognition of different env variants was relatively stable over time. In most of the patients studied, the proportion of CTL which showed cross-recognition of conserved determinants shared among the three strains was low. Two novel CTL epitopes within gp41 were identified by using 15-mer peptides of the HIV-SF2 sequence. When specific peptide was used to stimulate CTL precursors in vitro, the frequency of peptide-specific CTL precursors was very high, but the CTL elicited by this stimulation were highly strain specific. We conclude that the use of a single HIV env variant to detect CTL activity can underestimate the magnitude and complexity of the env-specific CTL response. The low prevalence of CTL clones which show cross-recognition of conserved determinants may have implications for immunization strategies based solely on env; to elicit broadly cross-reactive CTL other, more conserved viral antigens are likely to be needed in addition to env. Because of its capacity to distinguish CTL responses against different virus strains, limiting dilution analysis is particularly appropriate to quantitate the immune responses generated by candidate env-based vaccines. PMID:8970969

  8. EnvMine: A text-mining system for the automatic extraction of contextual information

    PubMed Central

    2010-01-01

    Background For ecological studies, it is crucial to count on adequate descriptions of the environments and samples being studied. Such a description must be done in terms of their physicochemical characteristics, allowing a direct comparison between different environments that would be difficult to do otherwise. Also the characterization must include the precise geographical location, to make possible the study of geographical distributions and biogeographical patterns. Currently, there is no schema for annotating these environmental features, and these data have to be extracted from textual sources (published articles). So far, this had to be performed by manual inspection of the corresponding documents. To facilitate this task, we have developed EnvMine, a set of text-mining tools devoted to retrieve contextual information (physicochemical variables and geographical locations) from textual sources of any kind. Results EnvMine is capable of retrieving the physicochemical variables cited in the text, by means of the accurate identification of their associated units of measurement. In this task, the system achieves a recall (percentage of items retrieved) of 92% with less than 1% error. Also a Bayesian classifier was tested for distinguishing parts of the text describing environmental characteristics from others dealing with, for instance, experimental settings. Regarding the identification of geographical locations, the system takes advantage of existing databases such as GeoNames to achieve 86% recall with 92% precision. The identification of a location includes also the determination of its exact coordinates (latitude and longitude), thus allowing the calculation of distance between the individual locations. Conclusion EnvMine is a very efficient method for extracting contextual information from different text sources, like published articles or web pages. This tool can help in determining the precise location and physicochemical variables of sampling sites, thus facilitating the performance of ecological analyses. EnvMine can also help in the development of standards for the annotation of environmental features. PMID:20515448

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

    PubMed Central

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

    2015-01-01

    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

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

    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.

  11. Crystal structure, conformational fixation and entry-related interactions of mature ligand-free HIV-1 Env.

    PubMed

    Kwon, Young Do; Pancera, Marie; Acharya, Priyamvada; Georgiev, Ivelin S; Crooks, Emma T; Gorman, Jason; Joyce, M Gordon; Guttman, Miklos; Ma, Xiaochu; Narpala, Sandeep; Soto, Cinque; Terry, Daniel S; Yang, Yongping; Zhou, Tongqing; Ahlsen, Goran; Bailer, Robert T; Chambers, Michael; Chuang, Gwo-Yu; Doria-Rose, Nicole A; Druz, Aliaksandr; Hallen, Mark A; Harned, Adam; Kirys, Tatsiana; Louder, Mark K; O'Dell, Sijy; Ofek, Gilad; Osawa, Keiko; Prabhakaran, Madhu; Sastry, Mallika; Stewart-Jones, Guillaume B E; Stuckey, Jonathan; Thomas, Paul V; Tittley, Tishina; Williams, Constance; Zhang, Baoshan; Zhao, Hong; Zhou, Zhou; Donald, Bruce R; Lee, Lawrence K; Zolla-Pazner, Susan; Baxa, Ulrich; Schön, Arne; Freire, Ernesto; Shapiro, Lawrence; Lee, Kelly K; Arthos, James; Munro, James B; Blanchard, Scott C; Mothes, Walther; Binley, James M; McDermott, Adrian B; Mascola, John R; Kwong, Peter D

    2015-07-01

    As the sole viral antigen on the HIV-1-virion surface, trimeric Env is a focus of vaccine efforts. Here we present the structure of the ligand-free HIV-1-Env trimer, fix its conformation and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies but not poorly neutralizing ones. We coupled these compatibility considerations with binding antigenicity to engineer conformationally fixed Envs, including a 201C 433C (DS) variant specifically recognized by broadly neutralizing antibodies. DS-Env retained nanomolar affinity for the CD4 receptor, with which it formed an asymmetric intermediate: a closed trimer bound by a single CD4 without the typical antigenic hallmarks of CD4 induction. Antigenicity-guided structural design can thus be used both to delineate mechanism and to fix conformation, with DS-Env trimers in virus-like-particle and soluble formats providing a new generation of vaccine antigens. PMID:26098315

  12. Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike

    PubMed Central

    Lee, Jeong Hyun; Leaman, Daniel P.; Kim, Arthur S.; Torrents de la Peña, Alba; Sliepen, Kwinten; Yasmeen, Anila; Derking, Ronald; Ramos, Alejandra; de Taeye, Steven W.; Ozorowski, Gabriel; Klein, Florian; Burton, Dennis R.; Nussenzweig, Michel C.; Poignard, Pascal; Moore, John P.; Klasse, Per Johan; Sanders, Rogier W.; Zwick, Michael B.; Wilson, Ian A.; Ward, Andrew B.

    2015-01-01

    The recent identification of three broadly neutralizing antibodies (bnAbs) against gp120–gp41 interface epitopes has expanded the targetable surface on the HIV-1 envelope glycoprotein (Env) trimer. By using biochemical, biophysical and computational methods, we map the previously unknown trimer epitopes of two related antibodies, 3BC315 and 3BC176. A cryo-EM reconstruction of a soluble Env trimer bound to 3BC315 Fab at 9.3?Å resolution reveals that the antibody binds between two gp41 protomers, and neutralizes the virus by accelerating trimer decay. In contrast, bnAb 35O22 binding to a partially overlapping quaternary epitope at the gp120–gp41 interface does not induce decay. A conserved gp41-proximal glycan at N88 was also shown to play a role in the binding kinetics of 3BC176 and 3BC315. Finally, our data suggest that the dynamic structure of the Env trimer influences exposure of bnAb epitopes. PMID:26404402

  13. Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike

    NASA Astrophysics Data System (ADS)

    Lee, Jeong Hyun; Leaman, Daniel P.; Kim, Arthur S.; Torrents de La Peña, Alba; Sliepen, Kwinten; Yasmeen, Anila; Derking, Ronald; Ramos, Alejandra; de Taeye, Steven W.; Ozorowski, Gabriel; Klein, Florian; Burton, Dennis R.; Nussenzweig, Michel C.; Poignard, Pascal; Moore, John P.; Klasse, Per Johan; Sanders, Rogier W.; Zwick, Michael B.; Wilson, Ian A.; Ward, Andrew B.

    2015-09-01

    The recent identification of three broadly neutralizing antibodies (bnAbs) against gp120-gp41 interface epitopes has expanded the targetable surface on the HIV-1 envelope glycoprotein (Env) trimer. By using biochemical, biophysical and computational methods, we map the previously unknown trimer epitopes of two related antibodies, 3BC315 and 3BC176. A cryo-EM reconstruction of a soluble Env trimer bound to 3BC315 Fab at 9.3 Å resolution reveals that the antibody binds between two gp41 protomers, and neutralizes the virus by accelerating trimer decay. In contrast, bnAb 35O22 binding to a partially overlapping quaternary epitope at the gp120-gp41 interface does not induce decay. A conserved gp41-proximal glycan at N88 was also shown to play a role in the binding kinetics of 3BC176 and 3BC315. Finally, our data suggest that the dynamic structure of the Env trimer influences exposure of bnAb epitopes.

  14. 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

    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

    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

  15. Barmish WorkshopBarmish Workshop RT 200RT 20099 Uncertainty and Randomization

    E-print Network

    Tempo, Roberto

    the hyperlink structure When arriving at a page with several outgoing links, one is chosen at random and spiders which move continuously along the web #12;IEIIT-CNR Barmish WorkshopBarmish Workshop ©©RT 200RT 20099 For each node we count the number of outgoing links and normalize them to 1 Hyperlink matrix

  16. A Native-Like SOSIP.664 Trimer Based on an HIV-1 Subtype B env Gene

    PubMed Central

    Pugach, Pavel; Ozorowski, Gabriel; Cupo, Albert; Ringe, Rajesh; Yasmeen, Anila; de Val, Natalia; Derking, Ronald; Kim, Helen J.; Korzun, Jacob; Golabek, Michael; de los Reyes, Kevin; Ketas, Thomas J.; Julien, Jean-Philippe; Burton, Dennis R.; Wilson, Ian A.; Sanders, Rogier W.; Klasse, P. J.

    2015-01-01

    ABSTRACT Recombinant trimeric mimics of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) spike should expose as many epitopes as possible for broadly neutralizing antibodies (bNAbs) but few, if any, for nonneutralizing antibodies (non-NAbs). Soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A strain BG505 approach this ideal and are therefore plausible vaccine candidates. Here, we report on the production and in vitro properties of a new SOSIP.664 trimer derived from a subtype B env gene, B41, including how to make this protein in low-serum media without proteolytic damage (clipping) to the V3 region. We also show that nonclipped trimers can be purified successfully via a positive-selection affinity column using the bNAb PGT145, which recognizes a quaternary structure-dependent epitope at the trimer apex. Negative-stain electron microscopy imaging shows that the purified, nonclipped, native-like B41 SOSIP.664 trimers contain two subpopulations, which we propose represent an equilibrium between the fully closed and a more open conformation. The latter is different from the fully open, CD4 receptor-bound conformation and may represent an intermediate state of the trimer. This new subtype B trimer adds to the repertoire of native-like Env proteins that are suitable for immunogenicity and structural studies. IMPORTANCE The cleaved, trimeric envelope protein complex is the only neutralizing antibody target on the HIV-1 surface. Many vaccine strategies are based on inducing neutralizing antibodies. For HIV-1, one approach involves using recombinant, soluble protein mimics of the native trimer. At present, the only reliable way to make native-like, soluble trimers in practical amounts is via the introduction of specific sequence changes that confer stability on the cleaved form of Env. The resulting proteins are known as SOSIP.664 gp140 trimers, and the current paradigm is based on the BG505 subtype A env gene. Here, we describe the production and characterization of a SOSIP.664 protein derived from a subtype B gene (B41), together with a simple, one-step method to purify native-like trimers by affinity chromatography with a trimer-specific bNAb, PGT145. The resulting trimers will be useful for structural and immunogenicity experiments aimed at devising ways to make an effective HIV-1 vaccine. PMID:25589637

  17. Table of Contents Inside the RT Dashboard...................................................................................................................................2

    E-print Network

    Wu, Chien H.

    Table of Contents Inside the RT Dashboard.................................................................................................................................................8 Customizing Your Dashboard

  18. 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

  19. WP AFB,WP AFB, DaytonDayton RT 2006RT 2006 1 Robust and Randomized Control Design

    E-print Network

    Tempo, Roberto

    /s (14 m/s) #12;IEIIT-CNR WP AFB,WP AFB, DaytonDayton ©© RT 2006RT 2006 6 Flight Envelope (Limits) Wing, DaytonDayton ©© RT 2006RT 2006 11 Aircraft Dynamics - 1 Aircraft math model implementation within an off-line flight simulator AIRCRAFT AERODYNAMICS CONTROL SURFACES STICK INPUT COMPENSATOR POWER PLANT THROTTLE

  20. MicroRT - Small animal conformal irradiator

    SciTech Connect

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

    2007-12-15

    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.

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

    PubMed Central

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

    2003-01-01

    Mounting evidence points to a role for CD4+ T-helper (Th) cell activities in controlling human immunodeficiency virus type 1 (HIV-1) infection. To determine the induction and evolution of Th responses following acute infection, we prospectively analyzed Env- and Gag-specific Th responses longitudinally for 92 patients with acute (n = 28) or early (n = 64) HIV-1 infection (median, 55 days postinfection [DPI]). The probability of detecting HIV-1-specific lymphoproliferative responses was remarkably low, and when present, the responses were more likely to be Gag specific than Env specific (16 versus 5%). Env-specific responses were significantly more common in patients presenting at <30 DPI than in those presenting at 30 to 365 DPI (21 versus 0.5%, P = 0.001). By contrast, Gag-specific responses occurred with similar frequencies among subjects presenting at <30 DPI and 30 to 365 DPI (13 versus 17%, P = 0.6). After treatment, and regardless of the duration of infection before therapy, Gag-specific Th responses predominated. Furthermore, some acutely infected subjects lost detectable Env-specific Th proliferative responses, which failed to reemerge upon treatment. Detailed analysis for one such subject revealed Env-specific lymphoproliferation at 11 DPI but no detectable Env-specific lymphoproliferation or ex vivo gamma interferon (IFN-?) secretion at multiple subsequent time points. Env-specific CD4+ T-cell clones from 11 DPI recognized six epitopes in both conserved and variable regions within gp120 and gp41, exhibited major histocompatibility complex-restricted cytotoxicity, and secreted high levels of antiviral cytokines. T-cell receptor clonal transcript analyses and autologous virus sequencing revealed that Th cells induced during acute infection were maintained and there were no Th escape mutations. Subsequent analysis for this subject and six of seven others revealed detectable IFN-?-secreting cells, but only following in vitro gp160 stimulation. In summary, we conclude that Env-specific Th responses are elicited very early in acute infection and may precede Gag-specific responses. The inability to detect Env-specific Th responses over time and despite antiretroviral therapy may reflect low frequencies and impaired proliferative capacity, and viral escape is not necessary for this to occur. PMID:12552005

  2. Structural insights into key sites of vulnerability on HIV-1 Env and influenza HA.

    PubMed

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

    2012-11-01

    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

  3. 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

    Nehete, Pramod N.; Nehete, Bharti P.; Hill, Lori; Manuri, Pallavi R.; Baladandayuthapani, Veerabhadran; Feng Lei; Simmons, Johnny; Sastry, K. Jagannadha

    2008-01-05

    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.

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

    E-print Network

    Nowak, Robert S.

    Assignment due Topic Instructor Jan 20 Introduction to course, Tragedy of the Commons Nowak & Miller 22 27 29 · Principles of resource management Nowak 5 Tragedy of the Commons, Nevada Env. Commission 10 BN #2 due Global the plus/minus system of grading. Required text: The Drama of the Commons, edited by E. Ostrom, T. Dietz, N

  5. Intelligence and RT: A Modified Hick Paradigm and a New RT Paradigm.

    ERIC Educational Resources Information Center

    Neubauer, Aljoscha C.

    1991-01-01

    The relationship between speed of information processing (SIP) and psychometric intelligence was investigated by giving 60 college students (22 males and 38 females) 2 choice reaction time (RT) tests (modified Hick paradigm) and Raven's Advanced Progressive Matrices. Results support an association between intelligence and SIP. (SLD)

  6. First-in-Human Evaluation of a Hexon Chimeric Adenovirus Vector Expressing HIV-1 Env (IPCAVD 002)

    PubMed Central

    Baden, Lindsey R.; Walsh, Stephen R.; Seaman, Michael S.; Johnson, Jennifer A.; Tucker, Robert P.; Kleinjan, Jane A.; Gothing, Jon A.; Engelson, Brian A.; Carey, Brittany R.; Oza, Avinash; Bajimaya, Shringkhala; Peter, Lauren; Bleckwehl, Chelsea; Abbink, Peter; Pau, Maria G.; Weijtens, Mo; Kunchai, Meghan; Swann, Edith M.; Wolff, Mark; Dolin, Raphael; Barouch, Dan H.

    2014-01-01

    Background.?We report the first-in-human safety and immunogenicity assessment of a prototype hexon chimeric adenovirus (Ad) serotype 5 (Ad5) vector containing the hexon hypervariable regions of Ad serotype 48 (Ad48) and expressing human immunodeficiency virus (HIV) type 1 EnvA. Methods.?Forty-eight Ad5 and Ad48 seronegative, HIV-uninfected subjects were enrolled in a randomized, double-blind, placebo-controlled, dose escalation phase 1 study. Four groups of 12 subjects received 109 to 1011 viral particles (vp) of the Ad5HVR48.EnvA.01 vaccine (n = 10 per group) or placebo (n = 2 per group) at week 0 or weeks 0, 4, and 24. Safety and immunogenicity were assessed. Results.?Self-limited reactogenicity was observed after the initial immunization in the highest (1011 vp) dose group. Responses in vaccinees included Ad48 neutralizing antibody (nAb) titers higher than Ad5 nAb titers, EnvA-specific enzyme-linked immunosorbent assay titers, and EnvA-specific enzyme-linked immunospot assay responses, and these responses generally persisted at week 52. At week 28 in the 109, 1010, and 1011 vp 3-dose groups, geometric mean EnvA enzyme-linked immunosorbent assay titers were 5721, 10 929, and 3420, respectively, and Ad48 nAb titers were a median of 1.7-fold higher than for Ad5. Conclusions.?Ad5HVR48.ENVA.01 was safe, well tolerated, and immunogenic at all doses tested. Vector-elicited nAb responses were greater for Ad48 than Ad5, confirming that Ad-specific nAbs in humans are primarily, but not exclusively, directed against the hexon hypervariable regions. Clinical Trials Registration.?NCT00695877. PMID:24719474

  7. Design of Lipid Nanocapsule Delivery Vehicles for Multivalent Display of Recombinant Env Trimers in HIV Vaccination

    PubMed Central

    2015-01-01

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

  8. 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

    Thomas, Elaine R.; Dunfee, Rebecca L.; Stanton, Jennifer; Bogdan, Derek; Taylor, Joann; Kunstman, Kevin; Bell, Jeanne E.; Wolinsky, Steven M.; Gabuzda, Dana . E-mail: dana_gabuzda@dfci.harvard.edu

    2007-03-30

    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.

  9. 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

    Kraus, Matthias H.; Parrish, Nicholas F.; Shaw, Katharina S.; Decker, Julie M.; Keele, Brandon F.; Salazar-Gonzalez, Jesus F.; Grayson, Truman; McPherson, David T.; Ping, Li-Hua; Anderson, Jeffrey A.; Swanstrom, Ronald; Williamson, Carolyn; Shaw, George M.; Hahn, Beatrice H.

    2010-02-20

    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.

  10. Exclusive Decoration of Simian Immunodeficiency Virus Env with High-Mannose Type N-Glycans Is Not Compatible with Mucosal Transmission in Rhesus Macaques

    PubMed Central

    Karsten, Christina B.; Buettner, Falk F. R.; Cajic, Samanta; Nehlmeier, Inga; Neumann, Berit; Klippert, Antonina; Sauermann, Ulrike; Reichl, Udo; Gerardy-Schahn, Rita; Rapp, Erdmann; Stahl-Hennig, Christiane

    2015-01-01

    Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelope (Env) proteins are extensively decorated with N-glycans, predominantly of the high-mannose type. However, it is unclear how high-mannose N-glycans on Env impact viral spread. We show that exclusive modification of SIV Env with these N-glycans reduces viral infectivity and abrogates mucosal transmission, despite increasing viral capture by immune cell lectins. Thus, high-mannose N-glycans have opposed effects on SIV infectivity and lectin reactivity, and a balance might be required for efficient mucosal transmission. PMID:26355090

  11. Experiment vs simulation RT WFNDEC 2014 benchmark: CIVA results

    NASA Astrophysics Data System (ADS)

    Tisseur, D.; Costin, M.; Rattoni, B.; Vienne, C.; Vabre, A.; Cattiaux, G.; Sollier, T.

    2015-03-01

    The French Atomic Energy Commission and Alternative Energies (CEA) has developed for years the CIVA software dedicated to simulation of NDE techniques such as Radiographic Testing (RT). RT modelling is achieved in CIVA using combination of a determinist approach based on ray tracing for transmission beam simulation and a Monte Carlo model for the scattered beam computation. Furthermore, CIVA includes various detectors models, in particular common x-ray films and a photostimulable phosphor plates. This communication presents the results obtained with the configurations proposed in the World Federation of NDEC 2014 RT modelling benchmark with the RT models implemented in the CIVA software.

  12. s.s. PACIFIC EXPLORER ~RT III -BELOW DECK ARRANGEMENTS

    E-print Network

    s.s. PACIFIC EXPLORER ~RT III - BELOW DECK ARRANGEMENTS AND REFRIGERATION EQUI PMEN"r .. I ..···...············.· ··· ~............. 17 Refrigeration System........................... 22 Power Plant Operational Problems......... .............. .... 31 Personnel

  13. Experiment vs simulation RT WFNDEC 2014 benchmark: CIVA results

    SciTech Connect

    Tisseur, D. Costin, M. Rattoni, B. Vienne, C. Vabre, A. Cattiaux, G.; Sollier, T.

    2015-03-31

    The French Atomic Energy Commission and Alternative Energies (CEA) has developed for years the CIVA software dedicated to simulation of NDE techniques such as Radiographic Testing (RT). RT modelling is achieved in CIVA using combination of a determinist approach based on ray tracing for transmission beam simulation and a Monte Carlo model for the scattered beam computation. Furthermore, CIVA includes various detectors models, in particular common x-ray films and a photostimulable phosphor plates. This communication presents the results obtained with the configurations proposed in the World Federation of NDEC 2014 RT modelling benchmark with the RT models implemented in the CIVA software.

  14. Human Ubc9 is involved in intracellular HIV-1 Env stability after trafficking out of the trans-Golgi network in a Gag dependent manner.

    PubMed

    Bohl, Christopher R; Abrahamyan, Levon G; Wood, Charles

    2013-01-01

    The cellular E2 Sumo conjugase, Ubc9 interacts with HIV-1 Gag, and is important for the assembly of infectious HIV-1 virions. In the previous study we demonstrated that in the absence of Ubc9, a defect in virion assembly was associated with decreased levels of mature intracellular Envelope (Env) that affected Env incorporation into virions and virion infectivity. We have further characterized the effect of Ubc9 knockdown on HIV Env processing and assembly. We found that gp160 stability in the endoplasmic reticulum (ER) and its trafficking to the trans-Golgi network (TGN) were unaffected, indicating that the decreased intracellular mature Env levels in Ubc9-depleted cells were due to a selective degradation of mature Env gp120 after cleavage from gp160 and trafficked out of the TGN. Decreased levels of Gag and mature Env were found to be associated with the plasma membrane and lipid rafts, which suggest that these viral proteins were not trafficked correctly to the assembly site. Intracellular gp120 were partially rescued when treated with a combination of lysosome inhibitors. Taken together our results suggest that in the absence of Ubc9, gp120 is preferentially degraded in the lysosomes likely before trafficking to assembly sites leading to the production of defective virions. This study provides further insight in the processing and packaging of the HIV-1 gp120 into mature HIV-1 virions. PMID:23861967

  15. The conserved dileucine- and tyrosine-based motifs in MLV and MPMV envelope glycoproteins are both important to regulate a common Env intracellular trafficking

    PubMed Central

    Blot, Vincent; Lopez-Vergès, Sandra; Breton, Marie; Pique, Claudine; Berlioz-Torrent, Clarisse; Grange, Marie-Pierre

    2006-01-01

    Background Retrovirus particles emerge from the assembly of two structural protein components, Gag that is translated as a soluble protein in the cytoplasm of the host cells, and Env, a type I transmembrane protein. Because both components are translated in different intracellular compartments, elucidating the mechanisms of retrovirus assembly thus requires the study of their intracellular trafficking. Results We used a CD25 (Tac) chimera-based approach to study the trafficking of Moloney murine leukemia virus and Mason-Pfizer monkey virus Env proteins. We found that the cytoplasmic tails (CTs) of both Env conserved two major signals that control a complex intracellular trafficking. A dileucine-based motif controls the sorting of the chimeras from the trans-Golgi network (TGN) toward endosomal compartments. Env proteins then follow a retrograde transport to the TGN due to the action of a tyrosine-based motif. Mutation of either motif induces the mis-localization of the chimeric proteins and both motifs are found to mediate interactions of the viral CTs with clathrin adaptors. Conclusion This data reveals the unexpected complexity of the intracellular trafficking of retrovirus Env proteins that cycle between the TGN and endosomes. Given that Gag proteins hijack endosomal host proteins, our work suggests that the endosomal pathway may be used by retroviruses to ensure proper encountering of viral structural Gag and Env proteins in cells, an essential step of virus assembly. PMID:16978406

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

    PubMed Central

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

    2013-01-01

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

  17. Structures of HIV-1 Env V1V2 with broadly neutralizing antibodies reveal commonalities that enable vaccine design.

    PubMed

    Gorman, Jason; Soto, Cinque; Yang, Max M; Davenport, Thaddeus M; Guttman, Miklos; Bailer, Robert T; Chambers, Michael; Chuang, Gwo-Yu; DeKosky, Brandon J; Doria-Rose, Nicole A; Druz, Aliaksandr; Ernandes, Michael J; Georgiev, Ivelin S; Jarosinski, Marissa C; Joyce, M Gordon; Lemmin, Thomas M; Leung, Sherman; Louder, Mark K; McDaniel, Jonathan R; Narpala, Sandeep; Pancera, Marie; Stuckey, Jonathan; Wu, Xueling; Yang, Yongping; Zhang, Baoshan; Zhou, Tongqing; Program, Nisc Comparative Sequencing; Mullikin, James C; Baxa, Ulrich; Georgiou, George; McDermott, Adrian B; Bonsignori, Mattia; Haynes, Barton F; Moore, Penny L; Morris, Lynn; Lee, Kelly K; Shapiro, Lawrence; Mascola, John R; Kwong, Peter D

    2016-01-01

    Broadly neutralizing antibodies (bNAbs) against HIV-1 Env V1V2 arise in multiple donors. However, atomic-level interactions had previously been determined only with antibodies from a single donor, thus making commonalities in recognition uncertain. Here we report the cocrystal structure of V1V2 with antibody CH03 from a second donor and model Env interactions of antibody CAP256-VRC26 from a third donor. These V1V2-directed bNAbs used strand-strand interactions between a protruding antibody loop and a V1V2 strand but differed in their N-glycan recognition. Ontogeny analysis indicated that protruding loops develop early, and glycan interactions mature over time. Altogether, the multidonor information suggested that V1V2-directed bNAbs form an 'extended class', for which we engineered ontogeny-specific antigens: Env trimers with chimeric V1V2s that interacted with inferred ancestor and intermediate antibodies. The ontogeny-based design of vaccine antigens described here may provide a general means for eliciting antibodies of a desired class. PMID:26689967

  18. Estimating Energy Expenditure with the RT3 Triaxial Accelerometer

    ERIC Educational Resources Information Center

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

    2009-01-01

    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…

  19. An Inverse Scattering Construction of the JMaRT Fuzzball

    E-print Network

    Despoina Katsimpouri; Axel Kleinschmidt; Amitabh Virmani

    2014-12-17

    We present an inverse scattering construction in STU supergravity of the two-charge 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.

  20. se-rt www.se-rt.de - 2008-2009 Software-Engineering Rat&Tat -TTI GmbH Version 1.10 Grundlagen des Testens

    E-print Network

    Wagner, Stefan

    1 se-rt www.se-rt.de - © 2008-2009 Software-Engineering Rat&Tat - TTI GmbH Version 1.10 Grundlagen Folie 2se-rt www.se-rt.de - © 2008-2009 Software-Engineering Rat&Tat - TTI GmbH Version 1) Kapitel 7: Test-Standards (3h) 2 se-rt www.se-rt.de - © 2008-2009 Software-Engineering Rat&Tat - TTI Gmb

  1. se-rt www.se-rt.de - 2009 Software-Engineering Rat&Tat -TTI GmbH C# 1.01 Einfhrung in die

    E-print Network

    Wagner, Stefan

    1 se-rt www.se-rt.de - © 2009 Software-Engineering Rat&Tat - TTI GmbH C# 1.01 Einführung in die Programmiersprache C# Rainer Schmidberger Rainer.Schmidberger@informatik.uni-stuttgart.de se-rt www.se-rt.de - © 2009 Architektur 2 Folie 3se-rt www.se-rt.de - © 2009 Software-Engineering Rat&Tat - TTI GmbH C# 1.01 C# und .NET

  2. Construction and tropism characterisation of recombinant viruses exhibiting HIV-1 env gene from seminal strains.

    PubMed

    Lawrence, Philip; Berlier, Willy; Delezay, Olivier; Palle, Sabine; Olivier, Thomas; Saoudin, Henia; Mottin, Stéphane; Lucht, Frédéric; Pozzetto, Bruno; Bourlet, Thomas

    2009-04-10

    Genetic differences between blood and mucosal-derived HIV-1 strains have been widely reported. As amplification of HIV-1 strains from mucosal samples including semen or saliva by co-culture has low sensitivity, we developed the construction of chimeric viruses expressing wild-type seminal HIV-1 envelope protein. Chimeric viruses were produced by co-transfection of a V1-V3 deleted pNL 43 vector and PCR fragments spanning the deleted region, amplified from HIV-1 RNA positive seminal plasma samples. After an initial testing of co-receptor usage by a tropism recombinant test, replication capacity and amplification of these recombinant viruses were assessed using PBMC. Four chimeric replicative strains, all using CXCR4 as coreceptor, were produced. The interaction between cell-free viral particles and reporter cell lines was assessed by confocal microscopy. These replicative chimeras exhibiting HIV-1 env from seminal strains represent useful tools for the in vitro study of the heterosexual transmission of HIV-1 and testing of microbicide activity. PMID:19232661

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

    NASA Technical Reports Server (NTRS)

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

    1991-01-01

    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.

  4. Potential Of VIRAC* RT-32 And RT-16 Antennas To Serve As Satellite Ground Station

    NASA Astrophysics Data System (ADS)

    Bleiders, M.; Trokss, J.; Elerts, M.

    2015-02-01

    The basic application of RT-32 and RT-16 parabolic antennas is radio astronomy observations, both the radio-telescopes have been upgraded with state-of-the art cryogenic receivers, and now a large-scale modernization of the infrastructure is underway. Since the radio-astronomical observations are not full-time activities, a research work has been done to clear up whether these antennas, besides the mentioned activities, can be used as a satellite ground station. The main goal of this added functionality is to make possible the use of the extremely high reception systems' figure-of-merit thus raising the satellite downlink data rates without increasing the on-board power consumption, which would be particularly important for developers of small satellites. In this paper, the progress in the research project is reported, which includes successful S-band satellite signal reception experiments and possible options as to integration of the related equipment into the system so that both functionalities could successfully coexist. Performance of the existing and the upgraded antenna positioning systems is estimated to determine if the latter are usable even for servicing low-Earth orbiting satellites. In addition, possible options are considered as to upgrading the system with automatic beam tracking capability, which would increase the antenna pointing accuracy even further.

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

    SciTech Connect

    Wyatt, Linda S. Belyakov, Igor M.; Earl, Patricia L.; Berzofsky, Jay A.; Moss, Bernard

    2008-03-15

    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.

  6. Predicting Gene Structures from Multiple RT-PCR Tests

    NASA Astrophysics Data System (ADS)

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

    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.

  7. Disulfide Sensitivity in the Env Protein Underlies Lytic Inactivation of HIV-1 by Peptide Triazole Thiols.

    PubMed

    Bailey, Lauren D; Kalyana Sundaram, Ramalingam Venkat; Li, Huiyuan; Duffy, Caitlin; Aneja, Rachna; Rosemary Bastian, Arangassery; Holmes, Andrew P; Kamanna, Kantharaju; Rashad, Adel A; Chaiken, Irwin

    2015-12-18

    We investigated the mode of action underlying lytic inactivation of HIV-1 virions by peptide triazole thiol (PTT), in particular the relationship between gp120 disulfides and the C-terminal cysteine-SH required for virolysis. Obligate PTT dimer obtained by PTT SH cross-linking and PTTs with serially truncated linkers between pharmacophore isoleucine-ferrocenyltriazole-proline-tryptophan and cysteine-SH were synthesized. PTT variants showed loss of lytic activity but not binding and infection inhibition upon SH blockade. A disproportionate loss of lysis activity vs binding and infection inhibition was observed upon linker truncation. Molecular docking of PTT onto gp120 argued that, with sufficient linker length, the peptide SH could approach and disrupt several alternative gp120 disulfides. Inhibition of lysis by gp120 mAb 2G12, which binds at the base of the V3 loop, as well as disulfide mutational effects, argued that PTT-induced disruption of the gp120 disulfide cluster at the base of the V3 loop is an important step in lytic inactivation of HIV-1. Further, PTT-induced lysis was enhanced after treating virus with reducing agents dithiothreitol and tris (2-carboxyethyl)phosphine. Overall, the results are consistent with the view that the binding of PTT positions the peptide SH group to interfere with conserved disulfides clustered proximal to the CD4 binding site in gp120, leading to disulfide exchange in gp120 and possibly gp41, rearrangement of the Env spike, and ultimately disruption of the viral membrane. The dependence of lysis activity on thiol-disulfide interaction may be related to intrinsic disulfide exchange susceptibility in gp120 that has been reported previously to play a role in HIV-1 cell infection. PMID:26458166

  8. Tiered categorization of a diverse panel of HIV-1 Env pseudoviruses for assessment of neutralizing antibodies.

    PubMed

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

    2010-02-01

    The restricted neutralization breadth of vaccine-elicited antibodies is a major limitation of current human immunodeficiency virus-1 (HIV-1) candidate vaccines. In order to permit the efficient identification of vaccines with enhanced capacity for eliciting cross-reactive neutralizing antibodies (NAbs) and to assess the overall breadth and potency of vaccine-elicited NAb reactivity, we assembled a panel of 109 molecularly cloned HIV-1 Env pseudoviruses representing a broad range of genetic and geographic diversity. Viral isolates from all major circulating genetic subtypes were included, as were viruses derived shortly after transmission and during the early and chronic stages of infection. We assembled a panel of genetically diverse HIV-1-positive (HIV-1(+)) plasma pools to assess the neutralization sensitivities of the entire virus panel. When the viruses were rank ordered according to the average sensitivity to neutralization by the HIV-1(+) plasmas, a continuum of average sensitivity was observed. Clustering analysis of the patterns of sensitivity defined four subgroups of viruses: those having very high (tier 1A), above-average (tier 1B), moderate (tier 2), or low (tier 3) sensitivity to antibody-mediated neutralization. We also investigated potential associations between characteristics of the viral isolates (clade, stage of infection, and source of virus) and sensitivity to NAb. In particular, higher levels of NAb activity were observed when the virus and plasma pool were matched in clade. These data provide the first systematic assessment of the overall neutralization sensitivities of a genetically and geographically diverse panel of circulating HIV-1 strains. These reference viruses can facilitate the systematic characterization of NAb responses elicited by candidate vaccine immunogens. PMID:19939925

  9. Measurement of RT amplitudes and wavelengths of laser driven plates

    SciTech Connect

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

    1997-10-16

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

  10. Co-expression of miRNA targeting the expression of PERK, but not PKR, enhances cellular immunity from an HIV-1 Env DNA vaccine.

    PubMed

    Wheatley, Adam K; Kramski, Marit; Alexander, Marina R; Toe, Jesse G; Center, Rob J; Purcell, Damian F J

    2011-01-01

    Small non-coding micro-RNAs (miRNA) are important post-transcriptional regulators of mammalian gene expression that can be used to direct the knockdown of expression from targeted genes. We examined whether DNA vaccine vectors co-expressing miRNA with HIV-1 envelope (Env) antigens could influence the magnitude or quality of the immune responses to Env in mice. Human miR-155 and flanking regions from the non-protein encoding gene mirhg155 were introduced into an artificial intron within an expression vector for HIV-1 Env gp140. Using the miR-155-expressing intron as a scaffold, we developed novel vectors for miRNA-mediated targeting of the cellular antiviral proteins PKR and PERK, which significantly down-modulated target gene expression and led to increased Env expression in vitro. Finally, vaccinating BALB/c mice with a DNA vaccine vector delivering miRNA targeting PERK, but not PKR, was able to augment the generation of Env-specific T-cell immunity. This study provides proof-of-concept evidence that miRNA effectors incorporated into vaccine constructs can positively influence vaccine immunogenicity. Further testing of vaccine-encoded miRNA will determine if such strategies can enhance protective efficacy from vaccines against HIV-1 for eventual human use. PMID:21464971

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

    SciTech Connect

    Li Kejun; Zhang, Shujing; Kronqvist, Malin; Ekstroem, Maria; Wallin, Michael; Garoff, Henrik . E-mail: henrik.garoff@cbt.ki.se

    2007-04-25

    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.

  12. Hyperthermic Fibrinolysis with rt-PA: In Vitro Results

    SciTech Connect

    Schwarzenberg, Helmut; Mueller-Huelsbeck, Stefan; Brossman, Joachim; Christian Glueer, Claus; Bruhn, Hans Dieter; Heller, Martin

    1998-03-15

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

  13. Enhancing Transport of Hydrogenophaga flava ENV735 for Bioaugmentation of Aquifers Contaminated with Methyl tert-Butyl Ether

    PubMed Central

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

    2002-01-01

    The gasoline oxygenate methyl tert-butyl ether (MTBE) has become a widespread contaminant in groundwater throughout the United States. Bioaugmentation of aquifers with MTBE-degrading cultures may be necessary to enhance degradation of the oxygenate in some locations. However, poor cell transport has sometimes limited bioaugmentation efforts in the past. The objective of this study was to evaluate the transport characteristics of Hydrogenophaga flava ENV735, a pure culture capable of growth on MTBE, and to improve movement of the strain through aquifer solids. The wild-type culture moved only a few centimeters in columns of aquifer sediment. An adhesion-deficient variant (H. flava ENV735:24) of the wild-type strain that moved more readily through sediments was obtained by sequential passage of cells through columns of sterile sediment. Hydrophobic and electrostatic interaction chromatography revealed that the wild-type strain is much more hydrophobic than the adhesion-deficient variant. Electrophoretic mobility assays and transmission electron microscopy showed that the wild-type bacterium contains two distinct subpopulations, whereas the adhesion-deficient strain has only a single, homogeneous population. Both the wild-type strain and adhesion-deficient variant degraded MTBE, and both were identified by 16S rRNA analysis as pure cultures of H. flava. The effectiveness of surfactants for enhancing transport of the wild-type strain was also evaluated. Many of the surfactants tested were toxic to ENV735; however, one nonionic surfactant, Tween 20, enhanced cell transport in sand columns. Improving microbial transport may lead to a more effective bioaugmentation strategy for MTBE-contaminated sites where indigenous oxygenate degraders are absent. PMID:12406751

  14. A limited number of simian immunodeficiency virus (SIV) env variants are transmitted to rhesus macaques vaginally inoculated with SIVmac251.

    PubMed

    Stone, Mars; Keele, Brandon F; Ma, Zhong-Min; Bailes, Elizabeth; Dutra, Joseph; Hahn, Beatrice H; Shaw, George M; Miller, Christopher J

    2010-07-01

    Single-genome amplification (SGA) and sequencing of HIV-1 RNA in plasma of acutely infected humans allows the identification and enumeration of transmitted/founder viruses responsible for productive systemic infection. Use of this strategy as a means for identifying transmitted viruses suggested that intrarectal simian immunodeficiency virus (SIV) inoculation of macaques recapitulates key features of human rectal infection. However, no studies have used the SGA strategy to identify vaginally transmitted virus(es) in macaques or to determine how early SIV diversification in vaginally infected animals compares with HIV-1 in humans. We used SGA to amplify 227 partial env sequences from a SIVmac251 challenge stock and from seven rhesus macaques at the earliest plasma viral RNA-positive time point after low- and high-dose intravaginal inoculation. Sequences were analyzed phylogenetically to determine the relationship of transmitted/founder viruses within and between each animal and the challenge stock. In each animal, discrete low-diversity env sequence lineages were evident, and these coalesced phylogenetically to identical or near-identical env sequences in the challenge stock, thus confirming the validity of the SGA sequencing and modeling strategy for identifying vaginally transmitted SIV. Between 1 and 10 viruses were responsible for systemic infection, similar to humans infected by sexual contact, and the set of viruses transmitted to the seven animals studied represented the full genetic constellation of the challenge stock. These findings recapitulate many of the features of sexual HIV-1 transmission in women. Furthermore, the SIV rhesus macaque model can be used to understand the factors that influence the transmission of single versus multiple SIV variants. PMID:20463069

  15. NASA SPoRT GOES-R Proving Ground Activities

    NASA Technical Reports Server (NTRS)

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

    2010-01-01

    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.

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

    PubMed Central

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

    2015-01-01

    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 modeled 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. PMID:25410397

  17. Model Building and Refinement of a Natively Glycosylated HIV-1 Env Protein by High-Resolution Cryoelectron Microscopy.

    PubMed

    Lee, Jeong Hyun; de Val, Natalia; Lyumkis, Dmitry; Ward, Andrew B

    2015-10-01

    Secretory and membrane proteins from mammalian cells undergo post-translational modifications, including N-linked glycosylation, which can result in a large number of possible glycoforms. This sample heterogeneity can be problematic for structural studies, particularly X-ray crystallography. Thus, crystal structures of heavily glycosylated proteins such as the HIV-1 Env viral spike protein have been determined by removing the majority of glycans. This step is most frequently carried out using Endoglycosidase H (EndoH) and requires that all expressed glycans be in the high-mannose form, which is often not the native glycoform. With significantly improved technologies in single-particle cryoelectron microscopy, we demonstrate that it is now possible to refine and build natively glycosylated HIV-1 Env structures in solution to 4.36 Å resolution. At this resolution we can now analyze the complete epitope of a broadly neutralizing antibody (bnAb), PGT128, in the context of the trimer expressed with native glycans. PMID:26388028

  18. Position-specific automated processing of V3 env ultra-deep pyrosequencing data for predicting HIV-1 tropism.

    PubMed

    Jeanne, Nicolas; Saliou, Adrien; Carcenac, Romain; Lefebvre, Caroline; Dubois, Martine; Cazabat, Michelle; Nicot, Florence; Loiseau, Claire; Raymond, Stéphanie; Izopet, Jacques; Delobel, Pierre

    2015-01-01

    HIV-1 coreceptor usage must be accurately determined before starting CCR5 antagonist-based treatment as the presence of undetected minor CXCR4-using variants can cause subsequent virological failure. Ultra-deep pyrosequencing of HIV-1 V3 env allows to detect low levels of CXCR4-using variants that current genotypic approaches miss. However, the computation of the mass of sequence data and the need to identify true minor variants while excluding artifactual sequences generated during amplification and ultra-deep pyrosequencing is rate-limiting. Arbitrary fixed cut-offs below which minor variants are discarded are currently used but the errors generated during ultra-deep pyrosequencing are sequence-dependant rather than random. We have developed an automated processing of HIV-1 V3 env ultra-deep pyrosequencing data that uses biological filters to discard artifactual or non-functional V3 sequences followed by statistical filters to determine position-specific sensitivity thresholds, rather than arbitrary fixed cut-offs. It allows to retain authentic sequences with point mutations at V3 positions of interest and discard artifactual ones with accurate sensitivity thresholds. PMID:26585833

  19. Position-specific automated processing of V3 env ultra-deep pyrosequencing data for predicting HIV-1 tropism

    PubMed Central

    Jeanne, Nicolas; Saliou, Adrien; Carcenac, Romain; Lefebvre, Caroline; Dubois, Martine; Cazabat, Michelle; Nicot, Florence; Loiseau, Claire; Raymond, Stéphanie; Izopet, Jacques; Delobel, Pierre

    2015-01-01

    HIV-1 coreceptor usage must be accurately determined before starting CCR5 antagonist-based treatment as the presence of undetected minor CXCR4-using variants can cause subsequent virological failure. Ultra-deep pyrosequencing of HIV-1 V3 env allows to detect low levels of CXCR4-using variants that current genotypic approaches miss. However, the computation of the mass of sequence data and the need to identify true minor variants while excluding artifactual sequences generated during amplification and ultra-deep pyrosequencing is rate-limiting. Arbitrary fixed cut-offs below which minor variants are discarded are currently used but the errors generated during ultra-deep pyrosequencing are sequence-dependant rather than random. We have developed an automated processing of HIV-1 V3 env ultra-deep pyrosequencing data that uses biological filters to discard artifactual or non-functional V3 sequences followed by statistical filters to determine position-specific sensitivity thresholds, rather than arbitrary fixed cut-offs. It allows to retain authentic sequences with point mutations at V3 positions of interest and discard artifactual ones with accurate sensitivity thresholds. PMID:26585833

  20. Quantitative RT-PCR for measuring gene expression.

    PubMed

    Riedy, M C; Timm, E A; Stewart, C C

    1995-01-01

    Classical Northern blot analysis for measuring mRNA requires too many cells to be practical for cell sorting. Yet, measurement of gene expression in small subsets within a heterogeneous population of cells is often desired. The PCR in combination with prior reverse transcription (RT-PCR) of the mRNA of interest provides a means for measuring gene expression using as few as one cell. When RT-PCR is performed, the reliability of the data can be highly subjective due to the efficiency of both RT and PCR steps. This subjectivity can be eliminated by a technique for quantitating specific RNA molecules using an internal RNA competitive reference standard (RNA-CRS), which is identical to the sequence of interest except for a deletion of 80 bases. Here we illustrate a strategy for quantitative PCR using a RNA-CRS, synthesized solely using nonplasmid-based PCR techniques. The competitive reaction consists of a constant quantity of wild-type mRNA (from 100-1000 cells) added individually to tubes containing a serially decreasing amount of RNA-CRS. The RT-PCR is performed on these samples, then the resulting product is analyzed by gel electrophoresis and densitometry. The procedure for preparing the RNA-CRS and subsequent RT-PCR steps are described in detail. PMID:7702857

  1. AWIPS II Application Development, a SPoRT Perspective

    NASA Technical Reports Server (NTRS)

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

    2014-01-01

    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.

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

    PubMed

    Yu, Xuelian; Ghildyal, Reena

    2015-01-01

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

  3. HIV-1 envelope glycoprotein (Env) fuses viral and cell mem-branes, allowing entry of the virus into host cells to initiate

    E-print Network

    Napp, Nils

    Reports HIV-1 envelope glycoprotein (Env) fuses viral and cell mem- branes, allowing entry? We have previously screened many HIV-1 primary iso- lates and identified two (clade A 92UG037 of the cytoplasmic domain on antigenic characteristics of HIV-1 envelope glycoprotein Jia Chen,1,2 James M. Kovacs,1

  4. 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

  5. Predicting Gene Structures from Multiple RT-PCR Tests

    E-print Network

    Vinar, Tomas

    splicing variants. Keywords: gene finding, RT-PCR, NP-completeness, dynamic program- ming, splicing graph 1 in the presence of ubiquitous alternative splicing (Guigo et al., 2006). Nonetheless, prediction accuracy of gene. As an input to our algorithm, we use a set of potential transcripts of one gene represented as a splicing

  6. Linear-Convex Control and Duality R.T. Rockafellar

    E-print Network

    Goebel, Rafal

    . For simplicity of presentation, but also with control engineering applications in mind, we specialize the keyLinear-Convex Control and Duality R.T. Rockafellar and Rafal Goebel April 2, 2007 Abstract An optimal control problem with linear dynamics and convex but not necessarily quadratic and possibly

  7. 6RT59775RA700681H Science, Service, Stewardship

    E-print Network

    6RT59775RA700681H NOAA FISHERIES SERVICE Science, Service, Stewardship Wild Bottlenose Dolphin Conservation Bottlenose dolphins are distributed throughout the coastal waters of the Southeastern U viewing bottlenose dolphins in the wild, but it also puts the animals at greater risk to human

  8. Single-Chain Soluble BG505.SOSIP gp140 Trimers as Structural and Antigenic Mimics of Mature Closed HIV-1 Env

    PubMed Central

    Georgiev, Ivelin S.; Joyce, M. Gordon; Yang, Yongping; Sastry, Mallika; Zhang, Baoshan; Baxa, Ulrich; Chen, Rita E.; Druz, Aliaksandr; Lees, Christopher R.; Narpala, Sandeep; Schön, Arne; Van Galen, Joseph; Chuang, Gwo-Yu; Gorman, Jason; Harned, Adam; Pancera, Marie; Stewart-Jones, Guillaume B. E.; Cheng, Cheng; Freire, Ernesto; McDermott, Adrian B.; Mascola, John R.

    2015-01-01

    ABSTRACT Similar to other type I fusion machines, the HIV-1 envelope glycoprotein (Env) requires proteolytic activation; specifically, cleavage of a gp160 precursor into gp120 and gp41 subunits creates an N-terminal gp41 fusion peptide and permits folding from an immature uncleaved state to a mature closed state. While the atomic-level consequences of cleavage for HIV-1 Env are still being determined, the uncleaved state is antigenically distinct from the mature closed state, and cleavage has been reported to be essential for mimicry of the mature viral spike by soluble versions of Env. Here we report the redesign of a current state-of-the-art soluble Env mimic, BG505.SOSIP, to make it cleavage independent. Specifically, we replaced the furin cleavage site between gp120 and gp41 with Gly-Ser linkers of various lengths. The resultant linked gp120-gp41 constructs, termed single-chain gp140 (sc-gp140), exhibited different levels of structural and antigenic mimicry of the parent cleaved BG505.SOSIP. When constructs were subjected to negative selection to remove subspecies recognized by poorly neutralizing antibodies, trimers of high antigenic mimicry of BG505.SOSIP could be obtained; negative-stain electron microscopy indicated these to resemble the mature closed state. Higher proportions of BG505.SOSIP-trimer mimicry were observed in sc-gp140s with linkers of 6 or more residues, with a linker length of 15 residues exhibiting especially promising traits. Overall, flexible linkages between gp120 and gp41 in BG505.SOSIP can thus substitute for cleavage, and sc-gp140s that closely mimicked the vaccine-preferred mature closed state of Env could be obtained. IMPORTANCE The trimeric HIV-1 envelope glycoprotein (Env) is the sole target of virus-directed neutralizing antibody responses and a primary focus of vaccine design. Soluble mimics of Env have proven challenging to obtain and have been thought to require proteolytic cleavage into two-component subunits, gp120 and gp41, to achieve structural and antigenic mimicry of mature Env spikes on virions. Here we show that replacement of the cleavage site between gp120 and gp41 in a lead soluble gp140 construct, BG505.SOSIP, with flexible linkers can result in molecules that do not require cleavage to fold efficiently into the mature closed state. Our results provide insights into the impact of cleavage on HIV-1 Env folding. In some contexts such as genetic immunization, optimized cleavage-independent soluble gp140 constructs may have utility over the parental BG505.SOSIP, as they would not require furin cleavage to achieve mimicry of mature Env spikes on virions. PMID:25740988

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

    PubMed Central

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

    2014-01-01

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

  10. Inhibition of HIV-1 Env-Mediated Cell-Cell Fusion by Lectins, Peptide T-20, and Neutralizing Antibodies

    PubMed Central

    Yee, Michael; Konopka, Krystyna; Balzarini, Jan; Düzgüne?, Nejat

    2011-01-01

    Background: Broadly cross-reactive, neutralizing human monoclonal antibodies, including 2F5, 2G12, 4E10 and IgG1 b12, can inhibit HIV-1 infection in vitro at very low concentrations. We examined the ability of these antibodies to inhibit cell-cell fusion between Clone69TRevEnv cells induced to express the viral envelope proteins, gp120/gp41 (Env), and highly CD4-positive SupT1 cells. The cells were loaded with green and red-orange cytoplasmic fluorophores, and fusion was monitored by fluorescence microscopy. Results: Cell-cell fusion was inhibited completely by the carbohydrate binding proteins (CBPs), Hippeastrum hybrid (Amaryllis) agglutinin (HHA), and Galanthus nivalis (Snowdrop) agglutinin (GNA), and by the peptide, T-20, at relatively low concentrations. Anti-gp120 and anti-gp41 antibodies, at concentrations much higher than those required for neutralization, were not particularly effective in inhibiting fusion. Monoclonal antibodies b12, m14 IgG and 2G12 had moderate inhibitory activity; the IC50 of 2G12 was about 80 µg/ml. Antibodies 4E10 and 2F5 had no inhibitory activity at the concentrations tested. Conclusions: These observations raise concerns about the ability of neutralizing antibodies to inhibit the spread of viral genetic material from infected cells to uninfected cells via cell-cell fusion. The interaction of gp120/gp41 with cell membrane CD4 may be different in cell-cell and virus-cell membrane fusion reactions, and may explain the differential effects of antibodies in these two systems. The fluorescence assay described here may be useful in high throughput screening of potential HIV fusion inhibitors. PMID:21660189

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

    PubMed

    Balasuriya, Udeni B R

    2014-01-01

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

  12. Identification of Rauscher murine leukemia virus-specific mRNAs for the synthesis of gag- and env-gene products.

    PubMed Central

    Zaane, D V; Gielkens, A L; Hesselink, W G; Bloemers, H P

    1977-01-01

    Polyadenylylated mRNA isolated from cells infected with Rauscher murine leukemia virus was fractionated by centrifugation in in a denaturing sucrose gradient into different sizes. Each RNA fraction was injected into oocytes of Xenopus laevis and the virus-specific products were analyzed by immunoprecipitation with polyvalent and monospecific antisera against polypeptides of Rauscher murine leukemia virus, and then by gel electrophoresis and scintillation autoradiography. It was shown that a 35S mRNA species directs the synthesis of a precursor of the internal or group-specific antigens of the virion (the gag-gene products). A 22S mRNA species directs the synthesis of two viral envelope polypeptides and their precursor polypeptide (env-gene products). The results indicate that the gag- and env-related polypeptides of Rauscher murine leukemia virus are synthesized uncoordinately and provide evidence for open and closed cistrons on the virus-specific mRNAs. Images PMID:266707

  13. Comparison of the conventional multiplex RT-PCR, real time RT-PCR and Luminex xTAG(®) RVP fast assay for the detection of respiratory viruses.

    PubMed

    Choudhary, Manohar L; Anand, Siddharth P; Tikhe, Shamal A; Walimbe, Atul M; Potdar, Varsha A; Chadha, Mandeep S; Mishra, Akhilesh C

    2016-01-01

    Detection of respiratory viruses using polymerase chain reaction (PCR) is sensitive, specific and cost effective, having huge potential for patient management. In this study, the performance of an in-house developed conventional multiplex RT-PCR (mRT-PCR), real time RT-PCR (rtRT-PCR) and Luminex xTAG(®) RVP fast assay (Luminex Diagnostics, Toronto, Canada) for the detection of respiratory viruses was compared. A total 310 respiratory clinical specimens predominantly from pediatric patients, referred for diagnosis of influenza A/H1N1pdm09 from August 2009 to March 2011 were tested to determine performance characteristic of the three methods. A total 193 (62.2%) samples were detected positive for one or more viruses by mRT-PCR, 175 (56.4%) samples by real time monoplex RT-PCR, and 138 (44.5%) samples by xTAG(®) RVP fast assay. The overall sensitivity of mRT-PCR was 96.9% (95% CI: 93.5, 98.8), rtRT-PCR 87.9% (95% CI: 82.5, 92.1) and xTAG(®) RVP fast was 68.3% (95% CI: 61.4, 74.6). Rhinovirus was detected most commonly followed by respiratory syncytial virus group B and influenza A/H1N1pdm09. The monoplex real time RT-PCR and in-house developed mRT-PCR are more sensitive, specific and cost effective than the xTAG(®) RVP fast assay. J. Med. Virol. 88:51-57, 2016. © 2015 Wiley Periodicals, Inc. PMID:26100490

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

    PubMed

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

    2015-01-01

    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

  15. Propidium monoazide reverse transcriptase PCR and RT-qPCR for detecting infectious enterovirus and norovirus.

    PubMed

    Karim, Mohammad R; Fout, G Shay; Johnson, Clifford H; White, Karen M; Parshionikar, Sandhya U

    2015-07-01

    Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the public health significance of positive findings are limited. In this study, PMA RT-PCR and RT-qPCR assays were evaluated for selective detection of infectious poliovirus, murine norovirus (MNV-1), and Norwalk virus. Viruses were inactivated using heat, chlorine, and ultraviolet light (UV). Infectious and non-infectious viruses were treated with PMA before RT-PCR and RT-qPCR. PMA RT-PCR was able to differentiate selectively between infectious and heat and chlorine inactivated poliovirus. PMA RT-PCR was able to differentiate selectively between infectious and noninfectious murine norovirus only when inactivated by chlorine. However, PMA RT-PCR could not differentiate infectious Norwalk virus from virus particles rendered non-infectious by any treatment. PMA RT-PCR assay was not able to differentiate between infectious and UV inactivated viruses suggesting that viral capsid damage may be necessary for PMA to enter and bind to the viral genome. PMA RT-PCR on naked MNV-1 and Norwalk virus RNA suggest that PMA RT-PCR can be used to detect intact, potentially infectious MNV-1 and Norwalk viruses and can be used to exclude the detection of free viral RNA by PCR assay. PMID:25796356

  16. Applications of LANCE Data at SPoRT

    NASA Technical Reports Server (NTRS)

    Molthan, Andrew

    2014-01-01

    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

  17. Phorbol diester-inducible, cyclosporine-suppressible transcription from a novel promoter within the mouse mammary tumor virus env gene

    SciTech Connect

    Elliott, J.F.; Pohajdak, B.; Talbot, D.J.; Shaw, J.; Paetkau, V. )

    1988-04-01

    The mouse T-cell lymphoma cell line EL4.E1 constitutively synthesizes mouse mammary tumor virus (MMTV) transcripts encoding either the entire proviral genome or segments of it. In addition to these conventional mRNAs, however, an mRNA of about 1 kilobase accumulates after induction of these cells with phorbol myristate acetate (PMA). The accumulation of this transcript is strongly inhibited by the immunosuppressive agent cyclosporin A. Its pattern of induction by PMA and suppression by cyclosporin A is thus the same as seen for several lymphokine mRNAs in these cells, including interleukin-2 and granulocyte-macrophage colony-stimulating factor. The short MMTV transcript is the most abundant PMA-induced transcript in EL4.E1 cells, but was not found in a series of other leukocyte tumor cell lines. It is initiated from a novel promoter within the env gene, and a segment of 1,161 nucleotides is then spliced out. The major part of the transcript is a copy of the long terminal repeat (LTR) of MMTV. The MMTV proviral genomes in these cells, and the short transcript, contain a 491-nucleotide deletion in the LTR compared with the normal MMTV provirus. The resulting open reading frame could encode a protein of molecular weight 22,800, which is a likely candidate for an LTR-related protein with a similar molecular weight recently described in this system.

  18. Timing of use of cod liver oil, a vitamin D source, and multiple sclerosis risk: The EnvIMS study

    PubMed Central

    Cortese, Marianna; Riise, Trond; Bjørnevik, Kjetil; Holmøy, Trygve; Kampman, Margitta T; Magalhaes, Sandra; Pugliatti, Maura; Wolfson, Christina; Myhr, Kjell-Morten

    2015-01-01

    Background: Low vitamin D levels have been associated with an increased risk of multiple sclerosis (MS), although it remains unknown whether this relationship varies by age. Objective: The objective of this paper is to investigate the association between vitamin D3 supplementation through cod liver oil at different postnatal ages and MS risk. Methods: In the Norwegian component of the multinational case-control study Environmental Factors In Multiple Sclerosis (EnvIMS), a total of 953 MS patients with maximum disease duration of 10 years and 1717 controls reported their cod liver oil use from childhood to adulthood. Results: Self-reported supplement use at ages 13–18 was associated with a reduced risk of MS (OR 0.67, 95% CI 0.52–0.86), whereas supplementation during childhood was not found to alter MS risk (OR 1.01, 95% CI 0.81–1.26), each compared to non-use during the respective period. An inverse association was found between MS risk and the dose of cod liver oil during adolescence, suggesting a dose-response relationship (p trend = 0.001) with the strongest effect for an estimated vitamin D3 intake of 600–800 IU/d (OR 0.46, 95% CI 0.31–0.70). Conclusions: These findings not only support the hypothesis relating to low vitamin D as a risk factor for MS, but further point to adolescence as an important susceptibility period for adult-onset MS. PMID:25948625

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

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

    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.

  20. The 17 nucleotides downstream from the env gene stop codon are important for murine leukemia virus packaging.

    PubMed

    Yu, S S; Kim, J M; Kim, S

    2000-09-01

    We have identified a previously unknown nucleotide sequence important for the packaging of murine leukemia virus. This nucleotide sequence is located downstream from the stop codon of the env gene but does not overlap the polypurine tract. Deletion of 17 bp from this region resulted in a more than 10-fold decrease in viral titer. Consistent with this result, the deletion mutant showed a 20- to 30-fold drop in the amount of virion RNA in the culture supernatant. The total amount of virion protein in the culture supernatant was comparable for the deletion mutant and the parental virus, suggesting that the mutant construct could release the empty viral particles. These results suggested that the packaging signal sequence might be present at the two extreme sites of the viral genome, one in the region around the splice donor sequence downstream from the 5' long terminal repeat (LTR) and the other immediately upstream from the 3' LTR. Implications for gene therapy, especially in regard to construction of retroviral vectors and packaging constructs, are discussed. PMID:10954583

  1. [Considerations for normalisation of RT-qPCR in oncology].

    PubMed

    Ho-Pun-Cheung, A; Cellier, D; Lopez-Crapez, E

    2008-01-01

    Gene expression analysis has many applications in the management of cancer, including diagnosis, prognosis, and therapeutic care. In this context, the reverse transcription quantitative polymerase chain reaction (RT-qPCR) has become the "gold standard" for mRNA quantification. However, this technique involves several critical steps such as RNA extraction, cDNA synthesis, quantitative PCR, and analysis, which all can be source of variation. To obtain biologically meaningful results, data normalisation is required to correct sample-to-sample variations that may be introduced during this multistage process. Normalisation can be carried out against a housekeeping gene, total RNA mass, or cell number. Careful choice of the normalization method is crucial, as any variation in the reference will introduce errors in the quantification of mRNA transcripts. By reviewing the different methods available and their related problems, the aim of this article is to provide recommendations for the set up of an appropriate normalisation strategy for RT-qPCR data in oncology. PMID:18390422

  2. Towards magnetic resonance imaging guided radiation therapy (MRIgRT)

    NASA Astrophysics Data System (ADS)

    Stanescu, Teodor Marius

    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.

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

    E-print Network

    Edinburgh, University of

    Bin Jia, Khe Chai Sim et al: CU-HTK RT03 Mandarin CTS system CU-HTK RT03 Mandarin CTS System Bin Jia, Khe Chai Sim, Mark Gales, Thomas Hain, Andrew Liu, Phil Woodland, Kai Yu & the HTK STT Team May 19th 2003 Cambridge University Engineering Department Rich Transcription Workshop 2003 60 #12;Bin Jia

  4. Env-2dCD4(S60C) complexes act as super immunogens and elicit potent, broadly neutralizing antibodies against clinically relevant human immunodeficiency virus type 1 (HIV-1).

    PubMed

    Killick, Mark A; Grant, Michelle L; Cerutti, Nichole M; Capovilla, Alexio; Papathanasopoulos, Maria A

    2015-11-17

    The ability to induce a broadly neutralizing antibody (bNAb) response following vaccination is regarded as a crucial aspect in developing an effective vaccine against human immunodeficiency virus type 1 (HIV-1). The bNAbs target the HIV-1 envelope glycoprotein (Env) which is exposed on the virus surface, thereby preventing cell entry. To date, conventional vaccine approaches such as the use of Env-based immunogens have been unsuccessful. We expressed, purified, characterized and evaluated the immunogenicity of several unique HIV-1 subtype C Env immunogens in small animals. Here we report that vaccine immunogens based on Env liganded to a two domain CD4 variant, 2dCD4(S60C) are capable of consistently eliciting potent, broadly neutralizing antibody responses in New Zealand white rabbits against a panel of clinically relevant HIV-1 pseudoviruses. This was irrespective of the Env protein subtype and context. Importantly, depletion of the anti-CD4 antibodies appeared to abrogate the neutralization activity in the rabbit sera. Taken together, this data suggests that the Env-2dCD4(S60C) complexes described here are "super" immunogens, and potentially immunofocus antibody responses to a unique epitope spanning the 2dCD4(60C). Recent data from the two available anti-CD4 monoclonal antibodies, Ibalizumab and CD4-Ig (and bispecific variants thereof) have highlighted that the use of these broad and potent entry inhibitors could circumvent the need for a conventional vaccine targeting HIV-1. Overall, the ability of the unique Env-2dCD4(S60C) complexes to elicit potent bNAb responses has not been described previously, reinforcing that further investigation for their utility in preventing and controlling HIV-1/SIV infection is warranted. PMID:26432912

  5. Mutations of the Rous sarcoma virus env gene that affect the transport and subcellular location of the glycoprotein products

    PubMed Central

    1984-01-01

    The envelope glycoproteins of Rous sarcoma virus (RSV), gp85 and gp37, are anchored in the membrane by a 27-amino acid, hydrophobic domain that lies adjacent to a 22-amino acid, cytoplasmic domain at the carboxy terminus of gp37. We have altered these cytoplasmic and transmembrane domains by introducing deletion mutations into the molecularly cloned sequences of a proviral env gene. The effects of the mutations on the transport and subcellular localization of the Rous sarcoma virus glycoproteins were examined in monkey (CV-1) cells using an SV40 expression vector. We found, on the one hand, that replacement of the nonconserved region of the cytoplasmic domain with a longer, unrelated sequence of amino acids (mutant C1) did not alter the rate of transport to the Golgi apparatus nor the appearance of the glycoprotein on the cell surface. Larger deletions, extending into the conserved region of the cytoplasmic domain (mutant C2), resulted in a slower rate of transport to the Golgi apparatus, but did not prevent transport to the cell surface. On the other hand, removal of the entire cytoplasmic and transmembrane domains (mutant C3) did block transport and therefore did not result in secretion of the truncated protein. Our results demonstrate that the C3 polypeptide was not transported to the Golgi apparatus, although it apparently remained in a soluble, nonanchored form in the lumen of the rough endoplasmic reticulum; therefore, it appears that this mutant protein lacks a functional sorting signal. Surprisingly, subcellular localization by internal immunofluorescence revealed that the C3 protein (unlike the wild type) did not accumulate on the nuclear membrane but rather in vesicles distributed throughout the cytoplasm. This observation suggests that the wild-type glycoproteins (and perhaps other membrane-bound or secreted proteins) are specifically transported to the nuclear membrane after their biosynthesis elsewhere in the rough endoplasmic reticulum. PMID:6094591

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

    NASA Astrophysics Data System (ADS)

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

    2008-10-01

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

  7. 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

    Swanson, Erika L.; Indelicato, Daniel J.; Louis, Debbie; Flampouri, Stella; Li, Zuofeng; Morris, Christopher G.; Paryani, Nitesh; Slopsema, Roelf

    2012-08-01

    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.

  8. EarthEnv-DEM90: A nearly-global, void-free, multi-scale smoothed, 90m digital elevation model from fused ASTER and SRTM data

    NASA Astrophysics Data System (ADS)

    Robinson, Natalie; Regetz, James; Guralnick, Robert P.

    2014-01-01

    A variety of DEM products are available to the public at no cost, though all are characterized by trade-offs in spatial coverage, data resolution, and quality. The absence of a high-resolution, high-quality, well-described and vetted, free, global consensus product was the impetus for the creation of a new DEM product described here, 'EarthEnv-DEM90'. This new DEM is a compilation dataset constructed via rigorous techniques by which ASTER GDEM2 and CGIAR-CSI v4.1 products were fused into a quality-enhanced, consistent grid of elevation estimates that spans ?91% of the globe. EarthEnv-DEM90 was assembled using methods for seamlessly merging input datasets, thoroughly filling voids, and smoothing data irregularities (e.g. those caused by DEM noise) from the approximated surface. The result is a DEM product in which elevational artifacts are strongly mitigated from the input data fusion zone, substantial voids are filled in the northern-most regions of the globe, and the entire DEM exhibits reduced terrain noise. As important as the final product is a well defined methodology, along with new processing techniques and careful attention to final outputs, that extends the value and usability of the work beyond just this single product. Finally, we outline EarthEnv-DEM90 acquisition instructions and metadata availability, so that researchers can obtain this high-resolution, high-quality, nearly-global new DEM product for the study of wide-ranging global phenomena.

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

    PubMed Central

    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

    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

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

    NASA Technical Reports Server (NTRS)

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

    1986-01-01

    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.

  11. Rapid detection of equine influenza virus H3N8 subtype by insulated isothermal RT-PCR (iiRT-PCR) assay using the POCKIT™ Nucleic Acid Analyzer.

    PubMed

    Balasuriya, Udeni B R; Lee, Pei-Yu Alison; Tiwari, Ashish; Skillman, Ashley; Nam, Bora; Chambers, Thomas M; Tsai, Yun-Long; Ma, Li-Juan; Yang, Pai-Chun; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

    2014-10-01

    Equine influenza (EI) is an acute, highly contagious viral respiratory disease of equids. Currently, equine influenza virus (EIV) subtype H3N8 continues to be the most important respiratory pathogen of horses in many countries around the world. The need to achieve a rapid diagnosis and to implement effective quarantine and movement restrictions is critical in controlling the spread of EIV. In this study, a novel, inexpensive and user-friendly assay based on an insulated isothermal RT-PCR (iiRT-PCR) method on the POCKIT™, a field-deployable device, was described and validated for point-of-need detection of EIV-H3N8 in clinical samples. The newly established iiRT-PCR assay targeting the EIV HA3 gene was evaluated for its sensitivity using in vitro transcribed (IVT) RNA, as well as ten-fold serial dilutions of RNA extracted from the prototype H3N8 strain A/equine/Miami/1/63. Inclusivity and exclusivity panels were tested for specificity evaluation. Published real-time RT-PCR (rRT-PCR) assays targeting the NP and HA3 genes were used as the reference standards for comparison of RNA extracted from field strains and from nasal swab samples collected from experimentally infected horses, respectively. Limit of detection with a 95% probability (LoD95%) was estimated to be 11copies of IVT RNA. Clinical sensitivity analysis using RNA prepared from serial dilutions of a prototype EIV (Miami 1/63/H3N8) showed that the iiRT-PCR assay was about 100-fold more sensitive than the rRT-PCR assay targeting the NP gene of EIV subtype H3N8. The iiRT-PCR assay identified accurately fifteen EIV H3N8 strains and two canine influenza virus (CIV) H3N8 strains, and did not cross-react with H6N2, H7N7, H1N1 subtypes or any other equine respiratory viral pathogens. Finally, 100% agreement was found between the iiRT-PCR assay and the universal influenza virus type A rRT-PCR assay in detecting the EIV A/equine/Kentucky/7/07 strain in 56 nasal swab samples collected from experimentally inoculated horses. Therefore, the EIV H3N8 subtype specific iiRT-PCR assay along with the portable POCKIT™ Nucleic Acid Analyzer provides a highly reliable, sensitive and specific on-site detection system of both equine and canine influenza viruses. PMID:24992669

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

    PubMed Central

    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

    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

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

    SciTech Connect

    Rades, Dirk . E-mail: Rades.Dirk@gmx.net; Stalpers, Lukas J.A.; Veninga, Theo; Hoskin, Peter J.

    2005-11-01

    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}.

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

    PubMed Central

    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

    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

  15. 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 Central

    Mikell, Iliyana; Stamatatos, Leonidas

    2012-01-01

    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

  16. Peptide Triazole Inactivators of HIV-1 Utilize a Conserved Two-Cavity Binding Site at the Junction of the Inner and Outer Domains of Env gp120

    PubMed Central

    Aneja, Rachna; Rashad, Adel A.; Li, Huiyuan; Sundaram, Ramalingam Venkat Kalyana; Duffy, Caitlin; Bailey, Lauren D.; Chaiken, Irwin

    2015-01-01

    We used coordinated mutagenesis, synthetic design, and flexible docking to investigate the structural mechanism of Env gp120 encounter by peptide triazole (PT) inactivators of HIV-1. Prior results demonstrated that the PT class of inhibitors suppresses binding at both CD4 and coreceptor sites on Env and triggers gp120 shedding, leading to cell-independent irreversible virus inactivation. Despite these enticing anti-HIV-1 phenotypes, structural understanding of the PT–gp120 binding mechanism has been incomplete. Here we found that PT engages two inhibitor ring moieties at the junction between the inner and outer domains of the gp120 protein. The results demonstrate how combined occupancy of two gp120 cavities can coordinately suppress both receptor and coreceptor binding and conformationally entrap the protein in a destabilized state. The two-cavity model has common features with small molecule gp120 inhibitor binding sites and provides a guide for further design of peptidomimetic HIV-1 inactivators based on the PT pharmacophore. PMID:25860784

  17. CAMELEON-RT: a Software Architecture Reference Model for Distributed, Migratable, and Plastic User

    E-print Network

    CAMELEON-RT: a Software Architecture Reference Model for Distributed, Migratable, and Plastic User the problem space of distributed, migratable and plastic user interfaces, and presents CAMELEON-RT1 for distributed, migratable, and plastic user inter- faces. We have developed an early implementation of a run

  18. Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR

    E-print Network

    Quake, Stephen R.

    Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR Luigi by a microfluidic chip-based ``digital RT-PCR'' assay that can count template molecules in cDNA samples prepared revealed significant differences in the level of the housekeeping transcript GAPDH across the surveyed

  19. Evaluation of reference genes in Vibrio parahaemolyticus for gene expression analysis using quantitative RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize the viru...

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

    E-print Network

    de Souza, Romualdo T.

    R.T. deSouza, Indiana University Careers in Academia seminar UIUC Acknowledgments ·Students who Results V. Challenges associated with supporting and spreading CALM V. Summary VI Career advice #12;R.T. deSouza, Indiana University Careers in Academia seminar UIUC Only 26% scored high

  1. Propidium monoazide reverse transcription PCR and RT-qPCR for detecting infectious enterovirus and norovirus

    EPA Science Inventory

    Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the publ...

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

    E-print Network

    Schultz, Tanja

    recognition, lectures, distant speech, CHIL, RT-06S. 1. Introduction In this paper, we present the ISL's most described our improvements over the CHIL [4] eval- uation system of January 2005 [2]. The main improvements segmentation algorithm compared to the one used in the RT-04S evaluation system [8]. CMU ICSI NIST TED CHIL Hub

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

    NASA Technical Reports Server (NTRS)

    Su, Fengge; Gao, Huilin; Huffman, George J.; Lettenmaier, Dennis P.

    2010-01-01

    We investigate the potential utility of the real-time Tropical Rainfall Measuring Mission (TRMM) Multi-satellite Precipitation Analysis (TMPA-RT) data for streamflow prediction, both through direct comparisons of TMPA-RT estimates with a gridded gauge product, and through evaluation of streamflow simulations over four tributaries of La Plata Basin (LPB) in South America using the two precipitation products. Our assessments indicate that the relative accuracy and the hydrologic performance of TMPA-RT-based streamflow simulations generally improved after February 2005. The improvements in TMPA-RT since 2005 are closely related to upgrades in the TMPA-RT algorithm in early February, 2005 which include use of additional microwave sensors (AMSR-E and AMSU-B) and implementation of different calibration schemes. Our work suggests considerable potential for hydrologic prediction using purely satellite-derived precipitation estimates (no adjustments by in situ gauges) in parts of the globe where in situ observations are sparse.

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

    PubMed Central

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

    1994-01-01

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

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

    PubMed

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

    2013-01-01

    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

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

    PubMed Central

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

    2013-01-01

    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

  7. Development of RT-components for the M-3 Strawberry Harvesting Robot

    NASA Astrophysics Data System (ADS)

    Yamashita, Tomoki; Tanaka, Motomasa; Yamamoto, Satoshi; Hayashi, Shigehiko; Saito, Sadafumi; Sugano, Shigeki

    We are now developing the strawberry harvest robot called “M-3” prototype robot system under the 4th urgent project of MAFF. In order to develop the control software of the M-3 robot more efficiently, we innovated the RT-middleware “OpenRTM-aist” software platform. In this system, we developed 9 kind of RT-Components (RTC): Robot task sequence player RTC, Proxy RTC for image processing software, DC motor controller RTC, Arm kinematics RTC, and so on. In this paper, we discuss advantages of RT-middleware developing system and problems about operating the RTC-configured robotic system by end-users.

  8. 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

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

    2012-01-01

    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

  9. 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 ...

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

    SciTech Connect

    Haffar, O.K.; Smithgall, M.D.; Moran, P.A.; Travis, B.M.; Zarling, J.M.; Hu, S.L. )

    1991-08-01

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

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

    SciTech Connect

    Manzoori, Davood

    2009-12-15

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

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

    NASA Technical Reports Server (NTRS)

    Fuell, Kevin K.; Molthan, Andrew L.

    2012-01-01

    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.

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

    NASA Technical Reports Server (NTRS)

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

    2012-01-01

    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.

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

    E-print Network

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

    2014-11-24

    FFPE (formalin-fixed paraffin- embedded) diagnostic biopsies. In this study, we investigated the possibility of COO-classification using FFPE RNA samples by massive parallel quantitative reverse transcription PCR (qRT-PCR). We established a protocol...

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

    NASA Technical Reports Server (NTRS)

    Zavodsky, Bradley

    2012-01-01

    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.

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

    PubMed

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

    2007-06-01

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

  17. Assessing the effects of physical and perceived luminance contrast on RT and TMS-induced percepts.

    PubMed

    Knight, Ramisha; Mazzi, Chiara; Savazzi, Silvia

    2015-12-01

    Simple reaction times (RTs) are inversely related to the luminance of a visual region, with RT increasing as luminance decreases, and decreasing as luminance increases. A potential discrepancy in the link between luminance and RT, however, stems from the perception of luminance itself. Here, we tested whether RT is modulated by a measureable amount of light (luminance) or perceptual amount of light (brightness), as two test regions having the same luminance can be perceived as having different brightness. The current study investigates the effects of brightness using probes and artificial percepts, i.e., transcranial magnetic stimulation (TMS)-induced light and dark percepts. In Experiment 1, participants performed a RT task to light and dark probes presented over two backgrounds, one exhibiting a physical luminance and the other exhibiting perceptual brightness. Experiment 2 tested whether perceptual brightness could influence RT and frequency of artificial percepts. We found that while brightness contrast modulated RT to the dark probes, the frequency of artificial percepts was susceptible to both physical and perceived changes in luminance. These data suggest that some behavioral responses can be influenced by an illusion of brightness, wherein there is no actual change in luminance, as well as the perception of TMS-induced percepts. PMID:26314754

  18. Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme

    PubMed Central

    Moser, Michael J.; DiFrancesco, Robert A.; Gowda, Krishne; Klingele, Audrey J.; Sugar, Darby R.; Stocki, Stacy; Mead, David A.; Schoenfeld, Thomas W.

    2012-01-01

    Viral metagenomic libraries are a promising but previously untapped source of new reagent enzymes. Deep sequencing and functional screening of viral metagenomic DNA from a near-boiling thermal pool identified clones expressing thermostable DNA polymerase (Pol) activity. Among these, 3173 Pol demonstrated both high thermostability and innate reverse transcriptase (RT) activity. We describe the biochemistry of 3173 Pol and report its use in single-enzyme reverse transcription PCR (RT-PCR). Wild-type 3173 Pol contains a proofreading 3?-5? exonuclease domain that confers high fidelity in PCR. An easier-to-use exonuclease-deficient derivative was incorporated into a PyroScript RT-PCR master mix and compared to one-enzyme (Tth) and two-enzyme (MMLV RT/Taq) RT-PCR systems for quantitative detection of MS2 RNA, influenza A RNA, and mRNA targets. Specificity and sensitivity of 3173 Pol-based RT-PCR were higher than Tth Pol and comparable to three common two-enzyme systems. The performance and simplified set-up make this enzyme a potential alternative for research and molecular diagnostics. PMID:22675552

  19. Natural and Enhanced Attenuation of Chlorinated Solvents Using RT3D

    SciTech Connect

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

    2006-07-25

    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.

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

    NASA Technical Reports Server (NTRS)

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

    2012-01-01

    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

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

    PubMed Central

    Humes, Daryl; Overbaugh, Julie

    2011-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2012-01-01

    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.

  3. Equine Viperin Restricts Equine Infectious Anemia Virus Replication by Inhibiting the Production and/or Release of Viral Gag, Env, and Receptor via Distortion of the Endoplasmic Reticulum

    PubMed Central

    Tang, Yan-Dong; Na, Lei; Zhu, Chun-Hui; Shen, Nan; Yang, Fei; Fu, Xian-Qiu; Wang, Yu-Hong; Fu, Li-Hua; Wang, Jia-Yi; Lin, Yue-Zhi; Wang, Xue-Feng; Wang, Xiaojun

    2014-01-01

    ABSTRACT Viperin is an endoplasmic reticulum (ER)-associated multifunctional protein that regulates virus replication and possesses broad antiviral activity. In many cases, viperin interferes with the trafficking and budding of viral structural proteins by distorting the membrane transportation system. The lentivirus equine infectious anemia virus (EIAV) has been studied extensively. In this study, we examined the restrictive effect of equine viperin (eViperin) on EIAV replication and investigated the possible molecular basis of this restriction to obtain insights into the effect of this cellular factor on retroviruses. We demonstrated that EIAV infection of primary equine monocyte-derived macrophages (eMDMs) upregulated the expression of eViperin. The overexpression of eViperin significantly inhibited the replication of EIAV in eMDMs, and knockdown of eViperin transcription enhanced the replication of EIAV in eMDMs by approximately 45.8%. Further experiments indicated that eViperin restricts EIAV at multiple steps of viral replication. The overexpression of eViperin inhibited EIAV Gag release. Both the ?-helix domain and radical S-adenosylmethionine (SAM) domain were required for this activity. However, the essential motifs in SAM were different from those reported for the inhibition of HIV-1 Gag by human viperin. Furthermore, eViperin disrupted the synthesis of both EIAV Env and receptor, which consequently inhibited viral production and entry, respectively, and this disruption was dependent on the eViperin ?-helix domain. Using immunofluorescence assays and electron microscopy, we demonstrated that the ?-helix domain is responsible for the distortion of the endoplasmic reticulum (ER). Finally, EIAV did not exhibit counteracting eViperin at the protein level. IMPORTANCE In previous studies, viperin was indicated as restricting virus replications primarily by the inhibition of virus budding. Here, we show that viperin may have multiple antiviral mechanisms, including the reduction of EIAV Gag budding and Env expression, and these activities are dependent on different viperin domains. We especially demonstrate that the overexpression of viperin inhibits EIAV entry by decreasing the level of virus receptor. Therefore, viperin restriction of viruses is determined largely by the dependence of virus on the cellular membrane transportation system. PMID:25122784

  4. Circulation of subtype A and gagA/envB recombinant HIV type 1 strains among injecting drug users in St. Petersburg, Russia, correlates with geographical origin of infections.

    PubMed

    Lukashov, V V; Huismans, R; Rakhmanova, A G; Lisitsina, Z N; Akhtyrskaya, N A; Vlasov, N N; Melnick, O B; Goudsmit, J

    1999-11-20

    Countries of the former Soviet Union are experiencing an emerging HIV-1 epidemic due to a rapid expansion of HIV-1 among injecting drug users (IDUs). To study the molecular epidemiology of HIV-1 among IDUs in St. Petersburg, Russia, virus sequences were obtained from 22 individuals. Phylogenetic analysis of the env and gag regions revealed circulation of two major HIV-1 populations, one belonging to HIV-1 subtype A, and another being a recombinant of subtype A and B viruses (gagA/envB). Both virus populations were highly homogeneous, with a mean pairwise genetic distance of <2%, and similar to viruses obtained earlier from IDUs in other regions of the former Soviet Union. Distribution of the two major HIV-1 genotypes in St. Petersburg correlated with geographical origin of infections. In one individual, a virus type previously unseen among IDUs was found, which demonstrates the possibility that new viruses are entering this risk group. PMID:10580409

  5. Absolute and Relative Contraindications to IV rt-PA for Acute Ischemic Stroke

    PubMed Central

    Rabinstein, Alejandro A.

    2015-01-01

    Most of the contraindications to the administration of intravenous (IV) recombinant tissue plasminogen activator (rtPA) originated as exclusion criteria in major stroke trials. These were derived from expert consensus for the National Institute of Neurological Disorders and Stroke (NINDS) trial. Despite the fact that the safety and efficacy of IV rtPA has been repeatedly confirmed in large international observational studies over the past 20 years, most patients with acute ischemic stroke disappointingly still do not receive thrombolytic treatment. Some of the original exclusion criteria have proven to be unnecessarily restrictive in real-world clinical practice. It has been suggested that application of relaxed exclusion criteria might increase the IV thrombolysis rate up to 20% with comparable outcomes to thrombolysis with more conventional criteria. We review the absolute and relative contraindications to IV rtPA for acute ischemic stroke, discussing the underlying rationale and evidence supporting these exclusion criteria. PMID:26288669

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

    PubMed

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

    2007-10-01

    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

  7. Eight-drug/radiation therapy program (MOPP/ABDV/RT) for advanced Hodgkin's disease

    SciTech Connect

    Straus, D.J.; Myers, J.; Passe, S.

    1980-07-15

    Eighty-four evaluable patients with advanced Hodgkin's disease (Stages IIB, IIIA age > 35 or mixed cellularity or lymphocyte depletion histology, IIIB, IVA, and IVB) were treated with alternating monthly MOPP and Adriamycin, bleomycin, dacarbazine, and vinblastine (ABDV). Radiation therapy (RT), 2000 rads in two weeks, was given to areas of initial bulky disease in untreated patients. Complete remission (CR) rates were 80% for previously untreated, 65% for prior RT or minimal chemotherapy treated, and 50% for heavily pretreated patients. Among 49 previously untreated patients there were no primary treatment failures. The estimated two-year relapse rate for the CR group was 9%. The therapeutic effectiveness of this program may have been due to either or both of the following elements: (1) two non-cross-resistant drug combinations; (2) low dose adjuvant RT to initial sites of bulky disease. These early results are among the best reported for the treatment of advanced Hodgkin's disease.

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

    PubMed Central

    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

    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

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

    NASA Technical Reports Server (NTRS)

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

    1995-01-01

    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.

  10. Complete epitopes for vaccine design derived from a crystal structure of the broadly neutralizing antibodies PGT128 and 8ANC195 in complex with an HIV-1 Env trimer.

    PubMed

    Kong, Leopold; Torrents de la Peña, Alba; Deller, Marc C; Garces, Fernando; Sliepen, Kwinten; Hua, Yuanzi; Stanfield, Robyn L; Sanders, Rogier W; Wilson, Ian A

    2015-10-01

    The HIV-1 envelope gp160 glycoprotein (Env) is a trimer of gp120 and gp41 heterodimers that mediates cell entry and is the primary target of the humoral immune response. Broadly neutralizing antibodies (bNAbs) to HIV-1 have revealed multiple epitopes or sites of vulnerability, but mapping of most of these sites is incomplete owing to a paucity of structural information on the full epitope in the context of the Env trimer. Here, a crystal structure of the soluble BG505 SOSIP gp140 trimer at 4.6?Å resolution with the bNAbs 8ANC195 and PGT128 reveals additional interactions in comparison to previous antibody-gp120 structures. For 8ANC195, in addition to previously documented interactions with gp120, a substantial interface with gp41 is now elucidated that includes extensive interactions with the N637 glycan. Surprisingly, removal of the N637 glycan did not impact 8ANC195 affinity, suggesting that the antibody has evolved to accommodate this glycan without loss of binding energy. PGT128 indirectly affects the N262 glycan by a domino effect, in which PGT128 binds to the N301 glycan, which in turn interacts with and repositions the N262 glycan, thereby illustrating the important role of neighboring glycans on epitope conformation and stability. Comparisons with other Env trimer and gp120 structures support an induced conformation for glycan N262, suggesting that the glycan shield is allosterically modified upon PGT128 binding. These complete epitopes of two broadly neutralizing antibodies on the Env trimer can now be exploited for HIV-1 vaccine design. PMID:26457433

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

    PubMed

    Wang, Dapeng; Tian, Peng

    2014-02-17

    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

  12. [An electronic medical record information system of DICOM-RT module-based in radiation therapy].

    PubMed

    Xia, Deguo; Zhou, Linghong; Lei, Li

    2012-06-01

    Electronic medical records (EMR) is the clinical diagnosis, guiding intervention and digital medical service record of outpatient, hospital patients (or care object) in medical institution. And it is the complete, detailed clinical information resource which has produced and recorded in all previous medical treatments. Radiotherapy electronic medical records contain texts, images and graphics, therefore the information is more complicated. This paper proposes an EMR information system based on DICOM-RT standard, through the use of seven objects of DICOM-RT to achieve the information exchange and sharing between different systems, equipments, convenient radiotherapy treatment data management, improve the efficiency of radiation treatment. PMID:22826932

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

    SciTech Connect

    Woodward, Paul R; Dimonte, Guy; Rockefeller, Gabriel M; Fryer, Christopher L; Dimonte, Guy; Dai, W; Kares, R. J.

    2011-01-05

    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.

  14. Detection of hepatoma cells in peripheral blood of HCC patients by nested RT-PCR

    PubMed Central

    Liu, Yu-Hui; Zhou, Rou-Li; Rui, Jing-An

    1998-01-01

    AIM: To offer a more simple method with a high sensitivity and specificity for detection of hepatoma cells in peripheral blood of the patients with HCC. METHODS: Improved nested RT-PCR method was used to detect the expression of AFP mRNA in nuclear cells separated from peripheral venous blood. RESULTS: AFP mRNA contained in ten hepatoma cells was detected from 2 mL peripheral blood. CONCLUSION: The improved nested RT-PCR assay for AFP mRNA expressed in cancer cells in peripheral blood might be a valuable method for clinical diagnosis of HCC. PMID:11819249

  15. nature biotechnology VOLUME 29 NUMBER 8 AUGUST 2011 735 A rt i c l e s

    E-print Network

    Quake, Stephen R.

    nature biotechnology VOLUME 29 NUMBER 8 AUGUST 2011 735 A rt i c l e s Recombinant therapeutic because of their advantages in producing complex therapeutics and manufacturing adaptability. CHO cells transfection, gene amplification and clone selection in CHO cells are well characterized and widely used

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  17. Real time RT-PCR for the H5 HA subtype

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real-time RT-PCR (RRT-PCR) for type A influenza detection is widely used with subsequent tests for subtype identification. Due to the importance of the H5 HA subtype, rapid and sensitive detection of the H5 HA subtype is particularly useful due to the importance of the recent Asian H5N1 HPAI viruse...

  18. Quantifying a bystander response following microbeam irradiation using single-cell RT-PCR analyses

    E-print Network

    that are irradiated also demon- strate several responses seen in hit cellsdthe so-called bystander effect of this bystander effect, since it has the ability to place known numbers of charged particles (pro- tons or aQuantifying a bystander response following microbeam irradiation using single-cell RT-PCR analyses

  19. nature immunology VOLUME 13 NUMBER 12 DECEMBER 2012 1155 A rt i c l e s

    E-print Network

    Zhijie, Liu

    nature immunology VOLUME 13 NUMBER 12 DECEMBER 2012 1155 A rt i c l e s The host innate immuneDepartment of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA. 2Baylor Institute for Immunology Research, Baylor Research Institute, Dallas

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

    ERIC Educational Resources Information Center

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

    2013-01-01

    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…

  1. Climate Change: A Catastrophe in Slow Motion R.T. Pierrehumbert*

    E-print Network

    of the impact life has on global climate, the imminent global warming caused by humans does not stand out of geological time, human- induced climate change, known more familiarly as "global warming," is a catastrophe1 Climate Change: A Catastrophe in Slow Motion R.T. Pierrehumbert* I. INTRODUCTION The word

  2. 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...

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

    ERIC Educational Resources Information Center

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

    2010-01-01

    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…

  4. DEVELOPMENT OF MULTIPLEX REAL-TIME RT-PCR AS A DIAGNOSTIC TOOL FOR AVIAN INFLUENZA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiplex real-time RT-PCR (RRT-PCR) assay for the simultaneous detection of the H5 and H7 hemagglutinin (HA) subtypes was developed with hydrolysis type probes labeled with the FAM (H5 probe) and ROX (H7 probe) dyes. The sensitivity of the H5-H7 subtyping assay was determined, using in vitro tran...

  5. 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...

  6. AnnuAl RepoRt 2009-2010 Identity, Privacy and Security Institute

    E-print Network

    Prodiæ, Aleksandar

    Century Health Promotion in the Age of Social Networks: Youth Perspectives on New Media for Health Beyond, 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, PrIvacy

  7. RT-DEFORM: Interactive Ray Tracing of Dynamic Scenes using BVHs Christian Lauterbach

    E-print Network

    North Carolina at Chapel Hill, University of

    RT-DEFORM: Interactive Ray Tracing of Dynamic Scenes using BVHs Christian Lauterbach University for interactive ray tracing of de- formable or animated models. Unlike many of the recent ap- proaches for ray. In practice, our system can ray trace these models at 3-13 frames a second on a desktop PC including secondary

  8. arXiv:math.RT/0502035v228Jul2005 REFLECTION FUNCTORS AND SYMPLECTIC REFLECTION

    E-print Network

    Gan, Wee Liang

    arXiv:math.RT/0502035v228Jul2005 REFLECTION FUNCTORS AND SYMPLECTIC REFLECTION ALGEBRAS FOR WREATH PRODUCTS WEE LIANG GAN Abstract. We construct reflection functors on categories of modules over deformed applications to the representation theory of symplectic reflection algebras of wreath product groups. 1

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

    E-print Network

    Kay, Mark A.

    of aged muscle tissues, such as signaling via the Wnt, Notch, transform- ing growth factor- and fibroblastnature medicine VOLUME 20 | NUMBER 3 | MARCH 2014 255 a rt i c l e s During aging, skeletal muscle strength progressively declines (sarcopenia), leading to reduced mobility, muscle function and quality

  10. nature biotechnology VOLUME 32 NUMBER 5 MAY 2014 473 A rt i c l e s

    E-print Network

    nature biotechnology VOLUME 32 NUMBER 5 MAY 2014 473 A rt i c l e s Metabolic engineering has made engineering (IME) emerged as an approach to identify such distal genetic factors. IME uses combinatorial, enzymatic steps closely associated with the product- forming pathway have been engineered to prune side

  11. 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...

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

    E-print Network

    Pedram, Massoud

    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

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

    E-print Network

    Alvarez, Pedro J.

    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

  14. 240 VOLUME 31 NUMBER 3 MARCH 2013 nature biotechnology A rt i c l e s

    E-print Network

    Welch, Greg

    240 VOLUME 31 NUMBER 3 MARCH 2013 nature biotechnology A rt i c l e s The staple crop chickpea nitrogen fixation, chickpea seeds are a primary source of human dietary protein1. Chickpea is a member this subfamily, chickpea is most closely related to crops such as alfalfa (Medicago sativa), clover (Trifolium

  15. The Practical Teaching of Thinking Using the CoRT Method.

    ERIC Educational Resources Information Center

    De Bono, Edward

    1986-01-01

    The CoRT (Cognitive Research Trust) is a five-step program in direct instruction of thinking skills which increase the number and diversity of ideas as well as help the individual establish goals, set priorities, improve interactions with others, and incorporate feeling into thinking. (DB)

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

    NASA Technical Reports Server (NTRS)

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

    2009-01-01

    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.

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

    E-print Network

    Meeden, Lisa A.

    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

  18. 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

    Wang Meihua; Cairncross, Gregory; Shaw, Edward

    2010-07-01

    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.

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

    PubMed Central

    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

    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

  20. The reproduction number R(t) in structured and nonstructured populations.

    PubMed

    Burr, Tom; Chowell, Gerardo

    2009-04-01

    Using daily counts of newly infected individuals, Wallinga and Teunis (WT) introduced a conceptually simple method to estimate the number of secondary cases per primary case (R(t)) for a given day. The method requires an estimate of the generation interval probability density function (pdf), which specifies the probabilities for the times between symptom onset in a primary case and symptom onset in a corresponding secondary case. Other methods to estimate R(t) are based on explicit models such as the SIR model; therefore, one might expect the WT method to be more robust to departures from SIR- type behavior. This paper uses simulated data to compare the quality of daily R(t) estimates based on a SIR model to those using the WT method for both structured (classical SIR assumptions are violated) and nonstructured (classical SIR assumptions hold) populations. By using detailed simulations that record the infection day of each new infection and the donor-recipient identities, the true R(t) and the generation interval pdf is known with negligible error. We find that the generation interval pdf is time dependent in all cases, which agrees with recent results reported elsewhere. We also find that the WT method performs essentially the same in the structured populations (except for a spatial network) as it does in the nonstructured population. And, the WT method does as well or better than a SIR-model based method in three of the four structured populations. Therefore, even if the contact patterns are heterogeneous as in the structured populations evaluated here, the WT method provides reasonable estimates of R(t), as does the SIR method. PMID:19364151

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

    PubMed

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

    2013-01-01

    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

  2. SPoRT - An End-to-End R2O Activity

    NASA Technical Reports Server (NTRS)

    Jedlovec, Gary J.

    2009-01-01

    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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  4. 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....

  5. GreenRT: A Framework for the Design of Power-Aware Soft Real-Time Applications

    E-print Network

    Mori, Greg

    GreenRT: A Framework for the Design of Power-Aware Soft Real-Time Applications Bo Chen, William Pak, AMDs PowerNow, and Intels Enhanced SpeedStep. With DVFS, soft real-time applications lend themselvesRT, a frame- work for incorporating power-awareness into soft real-time applications. The framework consists

  6. A SYBR GREEN REAL-TIME RT-PCR METHOD TO DETECT AND QUANTITATE NORWALK VIRUS IN STOOLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was performed with three objectives: a) to develop a real-time reverse transcription-PCR (rt RT-PCR) procedure for a commonly used laboratory strain of Norwalk virus (NV), b) to evaluate the potential of sample dilution and heat release of viral RNA as an alternative to more complex proce...

  7. Abstract--The RT3S E-learning environment enables experts in vascular medicine to prepare educational training

    E-print Network

    Petrakis, Euripides G.M.

    Abstract--The RT3S E-learning environment enables experts in vascular medicine to prepare environment. The RT3S authoring environment is easy to use and customized (i.e., it can be adapted year in the UK. The symptoms are more common to those who smoke, the diabetics and to patients

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

    NASA Technical Reports Server (NTRS)

    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

    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.

  9. Genetic Analyses of HIV-1 env Sequences Demonstrate Limited Compartmentalization in Breast Milk and Suggest Viral Replication within the Breast That Increases with Mastitis ?

    PubMed Central

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

    2010-01-01

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

  10. A DICOM-RT based ePR radiation therapy information system for managing brain tumor patients

    NASA Astrophysics Data System (ADS)

    Liu, Brent J.; Law, Maria; Huang, H. K.; Zee, C. S.; Chan, Lawrence

    2005-04-01

    The need for comprehensive clinical image data and relevant information in image-guided Radiation Therapy (RT) is becoming steadily apparent. Multiple standalone systems utilizing the most technological advancements in imaging, therapeutic radiation, and computerized treatment planning systems acquire key data during the RT treatment course of a patient. One example are patients treated for brain tumors of greater sizes and irregular shapes that utilize state-of-the-art RT technology to deliver pinpoint accurate radiation doses. One such system, the Cyberknife, is a radiation treatment system that utilizes image-guided information to control a multi-jointed, six degrees of freedom, robotic arm to deliver precise and required radiation dose to the tumor site of a cancer patient. The image-guided system is capable of tracking the lesion orientations with respect to the patient"s position throughout the treatment process. This is done by correlating live radiographic images with pre-operative, CT and MR imaging information to determine relative patient and tumor position repeatedly over the course of the treatment. The disparate and complex data generated by the Cyberknife system along with related data is scattered throughout the RT department compromising an efficient clinical workflow since the data crucial for a clinical decision may be time-consuming to retrieve, temporarily missing, or even lost. To address these shortcomings, the ACR-NEMA Standards Committee extended its DICOM (Digital Imaging & Communications in Medicine) Standard from Radiology to RT by ratifying seven DICOM RT objects starting in 1997. However, they are rarely used by the RT community in daily clinical operations. In the past, the research focus of an RT department has primarily been developing new protocols and devices to improve treatment process and outcomes of cancer patients with minimal effort dedicated to integration of imaging and information systems. Our research, tightly-coupling radiology and RT information systems, represents a new frontier for medical informatics research that has never been previously considered. By combining our past experience in medical imaging informatics, DICOM-RT expertise, and system integration, we propose to test our hypothesis using a brain tumor case model that a DICOM-RT electronic patient record (ePR) system can improve clinical workflow efficiency for treatment and management of patients. This RT ePR system integrated with clinical images and RT data can impact the RT department in a similar fashion as PACS has already successfully done for Radiology. As a first step, the specific treatment case of patients with brain tumors specifically patients treated with the Cyberknife system will be the initial proof of concept for the research design, implementation, evaluation, and clinical relevance.

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

    NASA Astrophysics Data System (ADS)

    Jedlovec, G.; Fuell, K.; Smith, M. R.; Stano, G. T.; Molthan, A.

    2011-12-01

    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.

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

    NASA Technical Reports Server (NTRS)

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

    2011-01-01

    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.

  13. Emulating a crowded intracellular environment in vitro dramatically improves RT-PCR performance

    SciTech Connect

    Lareu, Ricky R.; Harve, Karthik S.; Raghunath, Michael

    2007-11-09

    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.

  14. Feasibility of small animal cranial irradiation with the microRT system

    SciTech Connect

    Kiehl, Erich L.; Stojadinovic, Strahinja; Malinowski, Kathleen T.; Limbrick, David; Jost, Sarah C.; Garbow, Joel R.; Rubin, Joshua B.; Deasy, Joseph O.; Khullar, Divya; Izaguirre, Enrique W.; Parikh, Parag J.; Low, Daniel A.; Hope, Andrew J.

    2008-10-15

    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.

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

    NASA Technical Reports Server (NTRS)

    Zavodsky, Bradley

    2011-01-01

    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.

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

    PubMed

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

    2014-04-01

    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

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

    NASA Technical Reports Server (NTRS)

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

    2012-01-01

    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.

  18. Bacterial reference genes for gene expression studies by RT-qPCR: survey and analysis.

    PubMed

    Rocha, Danilo J P; Santos, Carolina S; Pacheco, Luis G C

    2015-09-01

    The appropriate choice of reference genes is essential for accurate normalization of gene expression data obtained by the method of reverse transcription quantitative real-time PCR (RT-qPCR). In 2009, a guideline called the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) highlighted the importance of the selection and validation of more than one suitable reference gene for obtaining reliable RT-qPCR results. Herein, we searched the recent literature in order to identify the bacterial reference genes that have been most commonly validated in gene expression studies by RT-qPCR (in the first 5 years following publication of the MIQE guidelines). Through a combination of different search parameters with the text mining tool MedlineRanker, we identified 145 unique bacterial genes that were recently tested as candidate reference genes. Of these, 45 genes were experimentally validated and, in most of the cases, their expression stabilities were verified using the software tools geNorm and NormFinder. It is noteworthy that only 10 of these reference genes had been validated in two or more of the studies evaluated. An enrichment analysis using Gene Ontology classifications demonstrated that genes belonging to the functional categories of DNA Replication (GO: 0006260) and Transcription (GO: 0006351) rendered a proportionally higher number of validated reference genes. Three genes in the former functional class were also among the top five most stable genes identified through an analysis of gene expression data obtained from the Pathosystems Resource Integration Center. These results may provide a guideline for the initial selection of candidate reference genes for RT-qPCR studies in several different bacterial species. PMID:26149127

  19. Detection of enteroviruses and hepatitis a virus in water by consensus primer multiplex RT-PCR

    PubMed Central

    Li, Jun-Wen; Wang, Xin-Wei; Yuan, Chang-Qing; Zheng, Jin-Lai; Jin, Min; Song, Nong; Shi, Xiu-Quan; Chao, Fu-Huan

    2002-01-01

    AIM: To develop a rapid detection method of enteroviruses and Hepatitis A virus (HAV). METHODS: A one-step, single-tube consensus primers multiplex RT-PCR was developed to simultaneously detect Poliovirus, Coxsackie virus, Echovirus and HAV. A general upstream primer and a HAV primer and four different sets of primers (5 primers) specific for Poliovirus, Coxsacki evirus, Echovirus and HAV cDNA were mixed in the PCR mixture to reverse transcript and amplify the target DNA. Four distinct amplified DNA segments representing Poliovirus, Coxsackie virus, Echovirus and HAV were identified by gel electrophoresis as 589-, 671-, 1084-, and 1128 bp sequences, respectively. Semi-nested PCR was used to confirm the amplified products for each enterovirus and HAV. RESULTS: All four kinds of viral genome RNA were detected, and producing four bands which could be differentiated by the band size on the gel. To confirm the specificity of the multiplex PCR products, semi-nested PCR was performed. For all the four strains tested gave positive results. The detection sensitivity of multiplex PCR was similar to that of monoplex RT-PCR which was 24 PFU for Poliovrus, 21 PFU for Coxsackie virus, 60 PFU for Echovirus and 105 TCID50 for HAV. The minimum amount of enteric viral RNA detected by semi-nested PCR was equivalent to 2.4 PFU for Poliovrus, 2.1 PFU for Coxsackie virus, 6.0 PFU for Echovirus and 10.5 TCID50 for HAV. CONCLUSION: The consensus primers multiplex RT-PCR has more advantages over monoplex RT-PCR for enteric viruses detection, namely, the rapid turnaround time and cost effectiveness. PMID:12174381

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

    PubMed

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

    2013-04-01

    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. PMID:25806219

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

    NASA Astrophysics Data System (ADS)

    Saitoh, Haruhiko

    2010-11-01

    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].

  2. NASA SPoRT Initialization Datasets for Local Model Runs in the Environmental Modeling System

    NASA Technical Reports Server (NTRS)

    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

    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.

  3. Validation of reference genes for real-time quantitative RT-PCR studies in Talaromyces marneffei.

    PubMed

    Dankai, Wiyada; Pongpom, Monsicha; Vanittanakom, Nongnuch

    2015-11-01

    Talaromyces marneffei (or Penicillium marneffei) is an opportunistic pathogen that can cause disseminated disease in human immunodeficiency virus (HIV)-infected patients, especially in Southeast Asia. T. marneffei is a thermally dimorphic fungus. Typically, T. marneffei has an adaptive morphology. It grows in a filamentous form (mould) at 25°C and can differentiate to produce asexual spores (conidia). In contrast, at 37°C, it grows as yeast cells that divide by fission. This study aimed to validate a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) for gene expression analysis in T. marneffei. Analysis of relative gene expression by qRT-PCR requires normalization of data using a proper reference gene. However, suitable reference genes have not been identified in gene expression studies across different cell types or under different experimental conditions in T. marneffei. In this study, four housekeeping genes were selected for analysis: ?-actin (act); glyceraldehyde-3-phosphate dehydrogenase (gapdh); ?-tubulin (benA) and 18S rRNA. Two analysis programs; BestKeeper and geNorm software tools were used to validate the expression of the candidate normalized genes. The results indicated that the actin gene was the one which was most stably expressed and was recommended for use as the endogenous control for gene expression analysis of all growth forms in T. marneffei by qRT-PCR under normal and stress conditions. PMID:26327538

  4. Investigation of Reference Genes in Vibrio parahaemolyticus for Gene Expression Analysis Using Quantitative RT-PCR

    PubMed Central

    Ma, Yue-jiao; Sun, Xiao-hong; Xu, Xiao-yan; Zhao, Yong; Pan, Ying-jie; Hwang, Cheng-An; Wu, Vivian C. H.

    2015-01-01

    Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize its virulence factors and understand the effect of environmental conditions on its pathogenicity. However, there is not a stable gene in V. parahaemolyticus that has been identified for use as a reference gene for qRT-PCR. This study evaluated the stability of 6 reference genes (16S rRNA, recA, rpoS, pvsA, pvuA, and gapdh) in 5 V. parahaemolyticus strains (O3:K6-clinical strain-tdh+, ATCC33846-tdh+, ATCC33847-tdh+, ATCC17802-trh+, and F13-environmental strain-tdh+) cultured at 4 different temperatures (15, 25, 37 and 42°C). Stability values were calculated using GeNorm, NormFinder, BestKeeper, and Delta CT algorithms. The results indicated that recA was the most stably expressed gene in the V. parahaemolyticus strains cultured at different temperatures. This study examined multiple V. parahaemolyticus strains and growth temperatures, hence the finding provided stronger evidence that recA can be used as a reference gene for gene expression studies in V. parahaemolyticus. PMID:26659406

  5. Development of a versatile and stable internal control system for RT-qPCR assays.

    PubMed

    Felder, Eva; Wölfel, Roman

    2014-11-01

    RT-qPCR, an established method for the detection of RNA viruses, requires internal RNA controls for the correct interpretation of PCR results. Robust and versatile RT-PCR controls can be achieved for example by packaging RNA into a virus-derived protein shell. In this study a MS2-based internal control system was developed, that allows stable and universal packing of different RNAs into non-infectious, non-lytic MS2-based viral like particles (VLPs). Two competitive internal controls for a hantavirus assay and a Crimean-Congo Hemorrhagic Fever Virus (CCHFV) assay were cloned for the expression of VLPs. The expression of VLPs containing the RNA of interest could be induced with arabinose in Escherichia coli. The VLPs proved to be temperature resistant and could be frozen and thawed several times without degradation. Distinction of IC RNA from the target RNA was facilitated by a clear shift in the melting temperature or by specific hybridization signals. Furthermore, target and IC PCR amplification could be easily distinguished by their size in gel-electrophoretic analyses. Limits of detection were determined, demonstrating that the application of the IC did not reduce the sensitivity of the target RT-qPCR reactions. The system can be adapted to nearly any required sequence, resulting in a highly flexible method with broad range applications. PMID:25072380

  6. A Tool Set for the Genome-Wide Analysis of Neurospora crassa by RT-PCR

    PubMed Central

    Hurley, Jennifer M.; Dasgupta, Arko; Andrews, Peter; Crowell, Alexander M.; Ringelberg, Carol; Loros, Jennifer J.; Dunlap, Jay C.

    2015-01-01

    Neurospora crassa is an important model organism for filamentous fungi as well as for circadian biology and photobiology. Although the community-accumulated tool set for the molecular analysis of Neurospora is extensive, two components are missing: (1) dependable reference genes whose level of expression are relatively constant across light/dark cycles and as a function of time of day and (2) a catalog of primers specifically designed for real-time PCR (RT-PCR). To address the first of these we have identified genes that are optimal for use as reference genes in RT-PCR across a wide range of expression levels; the mRNA/transcripts from these genes have potential for use as reference noncycling transcripts outside of Neurospora. In addition, we have generated a genome-wide set of RT-PCR primers, thereby streamlining the analysis of gene expression. In validation studies these primers successfully identified target mRNAs arising from 70% (34 of 49) of all tested genes and from all (28) of the moderately to highly expressed tested genes. PMID:26248984

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

    PubMed Central

    McMillan, Mary; Pereg, Lily

    2014-01-01

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

  8. Two RT-PCR based assays to detect human metapneumovirus in nasopharyngeal aspirates.

    PubMed

    López-Huertas, María Rosa; Casas, Inmaculada; Acosta-Herrera, Belsy; García, María Luz; Coiras, María Teresa; Pérez-Breña, Pilar

    2005-10-01

    Two sensitive and specific RT-PCR assays were standardised for testing the presence of human metapneumovirus. A total of 300 nasopharyngeal aspirates collected from infants suffering from bronchiolitis since October 2000 to June 2003 and shown previously as negative to common respiratory viruses were examined. Matrix and polymerase viral genes, which show a low rate of variation, were chosen to design amplification assays to ensure that any genotype of the human metapneumovirus could be detected. A RT-PCR followed by a reverse line blotting hybridisation was developed for viral polymerase gene. For the matrix gene, after the RT-PCR assay, a subsequent nested PCR was carried out. Both assays had similar sensitivity, equivalent to 0.1 TCID50 of human metapneumovirus strain NL/1/99 which was used as the positive control. The human metapneumovirus was present in 16.6% of the specimens studied. The approaches described below are not only a robust method for rapid diagnosis of the human metapneumovirus, but also to establish an etiological surveillance tool for epidemiological studies. Based on the results obtained, human metapneumovirus infections in Madrid followed a seasonal pattern, with most of the infections occurring between February and April. PMID:15961167

  9. SPoRT Participation in the GOES-R and JPSS Proving Grounds

    NASA Technical Reports Server (NTRS)

    Jedlovec, Gary; Fuell, Kevin; Smith, Matthew

    2013-01-01

    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.

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

    PubMed

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

    2015-01-01

    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

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

    PubMed Central

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

    2015-01-01

    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

  12. Head-to-head comparison of poxvirus NYVAC and ALVAC vectors expressing 1 identical HIV-1 clade C immunogens in prime/boost combination with Env protein 2 in non-human primates

    E-print Network

    García-Arriaza, Juan; Perdiguero, Beatriz; Heeney, Jonathan; Seaman, Michael; Montefiori, David C.; Labranche, Celia; Yates, Nicole L.; Shen, Xiaoying; Tomaras, Georgia D.; Ferrari, Guido; Foulds, Kathryn E.; McDermott, Adrian; Kao, Shing-Fen; Roederer, Mario; Hawkins, Natalie; Self, Steve; Yao, Jiansheng; Farrell, Patrick; Phogat, Sanjay; Tartaglia, Jim; Barnett, Susan W.; Burke, Brian; Cristillo, Anthony; Weiss, Deborah; Lee, Carter; Kibler, Karen; Jacobs, Bert; Asbach, Benedikt; Wagner, Ralf; Ding, Song; Pantaleo, Giuseppe; Esteban, Mariano

    2015-06-03

    isolates 479 ? (strain MW965.26), whereas immunization with A2AP2 (B/E, AIDSVAX) elicited a 480 ? better neutralization against HIV-1 clade B virus isolates (MN-3). Moreover, the 481 ? kinetics of the neutralization response showed that the higher levels... -to-head comparison of NYVAC and ALVAC vectors expressing Gag-Pol-Env 596 ? from SIV has been undertaken, but in SIVmac251-infected rhesus macaques treated 597 ? with antiretroviral therapy (ART). These findings demonstrated that both vectors were 598 ? immunogenic...

  13. Biomarker discovery for colon cancer using a 761 gene RT-PCR assay

    PubMed Central

    Clark-Langone, Kim M; Wu, Jenny Y; Sangli, Chithra; Chen, Angela; Snable, James L; Nguyen, Anhthu; Hackett, James R; Baker, Joffre; Yothers, Greg; Kim, Chungyeul; Cronin, Maureen T

    2007-01-01

    Background Reverse transcription PCR (RT-PCR) is widely recognized to be the gold standard method for quantifying gene expression. Studies using RT-PCR technology as a discovery tool have historically been limited to relatively small gene sets compared to other gene expression platforms such as microarrays. We have recently shown that TaqMan® RT-PCR can be scaled up to profile expression for 192 genes in fixed paraffin-embedded (FPE) clinical study tumor specimens. This technology has also been used to develop and commercialize a widely used clinical test for breast cancer prognosis and prediction, the Onco typeDX™ assay. A similar need exists in colon cancer for a test that provides information on the likelihood of disease recurrence in colon cancer (prognosis) and the likelihood of tumor response to standard chemotherapy regimens (prediction). We have now scaled our RT-PCR assay to efficiently screen 761 biomarkers across hundreds of patient samples and applied this process to biomarker discovery in colon cancer. This screening strategy remains attractive due to the inherent advantages of maintaining platform consistency from discovery through clinical application. Results RNA was extracted from formalin fixed paraffin embedded (FPE) tissue, as old as 28 years, from 354 patients enrolled in NSABP C-01 and C-02 colon cancer studies. Multiplexed reverse transcription reactions were performed using a gene specific primer pool containing 761 unique primers. PCR was performed as independent TaqMan® reactions for each candidate gene. Hierarchal clustering demonstrates that genes expected to co-express form obvious, distinct and in certain cases very tightly correlated clusters, validating the reliability of this technical approach to biomarker discovery. Conclusion We have developed a high throughput, quantitatively precise multi-analyte gene expression platform for biomarker discovery that approaches low density DNA arrays in numbers of genes analyzed while maintaining the high specificity, sensitivity and reproducibility that are characteristics of RT-PCR. Biomarkers discovered using this approach can be transferred to a clinical reference laboratory setting without having to re-validate the assay on a second technology platform. PMID:17697383

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

    PubMed Central

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

    2014-01-01

    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

  15. RT-qPCR-based microneutralization assay for human cytomegalovirus using fibroblasts and epithelial cells.

    PubMed

    Wang, Xiao; Peden, Keith; Murata, Haruhiko

    2015-12-16

    Human cytomegalovirus (HCMV) is a leading cause of congenital infection that can result in serious disabilities in affected children. To facilitate HCMV vaccine development, a microscale neutralization assay based on reverse transcription quantitative PCR (RT-qPCR) was developed to quantify HCMV-neutralizing antibodies. Our approach relies on the generation of crude lysates from virus-infected cells that are amenable to direct analysis by RT-qPCR, thereby circumventing rate-limiting procedures associated with sample RNA extraction and purification. By serial passaging of the laboratory HCMV strain AD169 in epithelial cells (ARPE-19), a revertant virus with restored epithelial cell tropism, designated AD169(wt131), was obtained. AD169 and AD169(wt131) were evaluated in both epithelial cells (ARPE-19) and fibroblasts (MRC-5) by one-step RT-qPCR targeting the immediate-early gene IE1 transcript of HCMV. Expression kinetics indicated that RT-qPCR assessment could be conducted as early as 6h post-infection. Human serum samples (n=30) from healthy donors were tested for HCMV-specific IgG using a commercially available ELISA and for HCMV-neutralizing activity using our RT-qPCR-based neutralization assay. In agreement with the ELISA results, higher neutralizing activity was observed in the HCMV IgG seropositive group when compared with the HCMV IgG seronegative group. In addition, HCMV IgG seropositive human sera exhibited higher neutralizing titers using epithelial cells compared with using fibroblasts (geometric mean titers of 344 and 8 in ARPE-19 cells and MRC-5 cells, respectively). Our assay was robust to variation in input virus dose. In addition, a simple lysis buffer containing a non-ionic detergent was successfully demonstrated to be a less costly alternative to commercial reagents for cell-lysate preparation. Thus, our rapid HCMV neutralization assay may be a straightforward and flexible high-throughput tool for measuring antibody responses induced by vaccination and natural infection. PMID:26552003

  16. A DICOM-RT radiation oncology ePR with decision support utilizing a quantified knowledge base from historical data

    NASA Astrophysics Data System (ADS)

    Documet, Jorge R.; Liu, Brent; Le, Anh; Law, Maria

    2008-03-01

    During the last 2 years we have been working on developing a DICOM-RT (Radiation Therapy) ePR (Electronic Patient Record) with decision support that will allow physicists and radiation oncologists during their decision-making process. This ePR allows offline treatment dose calculations and plan evaluation, while at the same time it compares and quantifies treatment planning algorithms using DICOM-RT objects. The ePR framework permits the addition of visualization, processing, and analysis tools, which combined with the core functionality of reporting, importing and exporting of medical studies, creates a very powerful application that can improve the efficiency while planning cancer treatments. Usually a Radiation Oncology department will have disparate and complex data generated by the RT modalities as well as data scattered in RT Information/Management systems, Record & Verify systems, and Treatment Planning Systems (TPS) which can compromise the efficiency of the clinical workflow since the data crucial for a clinical decision may be time-consuming to retrieve, temporarily missing, or even lost. To address these shortcomings, the ACR-NEMA Standards Committee extended its DICOM (Digital Imaging & Communications in Medicine) standard from Radiology to RT by ratifying seven DICOM RT objects starting in 1997 [1,2]. However, they are not broadly used yet by the RT community in daily clinical operations. In the past, the research focus of an RT department has primarily been developing new protocols and devices to improve treatment process and outcomes of cancer patients with minimal effort dedicated to integration of imaging and information systems. Our attempt is to show a proof-of-concept that a DICOM-RT ePR system can be developed as a foundation to perform medical imaging informatics research in developing decision-support tools and knowledge base for future data mining applications.

  17. The Combined Approach to Lysis Utilizing Eptifibatide and rt-PA in Acute Ischemic Stroke

    PubMed Central

    Pancioli, Arthur M.; Broderick, Joseph; Brott, Thomas; Tomsick, Thomas; Khoury, Jane; Bean, Judy; del Zoppo, Gregory; Kleindorfer, Dawn; Woo, Daniel; Khatri, Pooja; Castaldo, John; Frey, James; Gebel, James; Kasner, Scott; Kidwell, Chelsea; Kwiatkowski, Thomas; Libman, Richard; Mackenzie, Richard; Scott, Phillip; Starkman, Sidney; Thurman, R. Jason

    2008-01-01

    Background and Purpose Multiple approaches are being studied to enhance the rate of thrombolysis for acute ischemic stroke. Treatment of myocardial infarction with a combination of a reduced-dose fibrinolytic agent and a glycoprotein (GP) IIb/IIIa receptor antagonist has been shown to improve the rate of recanalization versus fibrinolysis alone. The combined approach to lysis utilizing eptifibatide and recombinant tissue-type plasminogen activator (rt-PA) (CLEAR) stroke trial assessed the safety of treating acute ischemic stroke patients within 3 hours of symptom onset with this combination. Methods The CLEAR trial was a National Institutes of Health/National Institute of Neurological Disorders and Stroke–funded multicenter, double-blind, randomized, dose-escalation and safety study. Patients were randomized 3:1 to either low-dose rt-PA (tier 1=0.3 mg/kg, tier 2=0.45 mg/kg) plus eptifibatide (75 ?g/kg bolus followed by 0.75 ?g/kg per min infusion for 2 hours) or standard-dose rt-PA (0.9 mg/kg). The primary safety end point was the incidence of symptomatic intracerebral hemorrhage within 36 hours. Secondary analyses were performed regarding clinical efficacy. Results Ninety-four patients (40 in tier 1 and 54 in tier 2) were enrolled. The combination group of the 2 dose tiers (n=69) had a median age of 71 years and a median baseline National Institutes of Health Stroke Scale (NIHSS) score of 14, and the standard-dose rt-PA group (n=25) had a median age of 61 years and a median baseline NIHSS score of 10 (P=0.01 for NIHSS score). Fifty-two (75%) of the combination treatment group and 24 (96%) of the standard treatment group had a baseline modified Rankin scale score of 0 (P=0.04). There was 1 (1.4%; 95% CI, 0% to 4.3%) symptomatic intracranial hemorrhage in the combination group and 2 (8.0%; 95% CI, 0% to 19.2%) in the rt-PA–only arm (P=0.17). During randomization in tier 2, a review by the independent data safety monitoring board demonstrated that the safety profile of combination therapy at the tier 2 doses was such that further enrollment was statistically unlikely to indicate inadequate safety for the combination treatment group, the ultimate out-come of the study. Thus, the study was halted. There was a trend toward increased clinical efficacy of standard-dose rt-PA compared with the combination treatment group. Conclusions The safety of the combination of reduced-dose rt-PA plus eptifibatide justifies further dose-ranging trials in acute ischemic stroke. PMID:18772447

  18. A DICOM-RT Based ePR radiation therapy information system for decision-support of brain tumor patients

    NASA Astrophysics Data System (ADS)

    Liu, B. J.; Law, M.; Huang, H. K.; Zee, C. S.; Chan, L.

    2006-03-01

    The need for comprehensive clinical image data and relevant information in image-guided Radiation Therapy (RT) is becoming steadily apparent. Multiple standalone systems utilizing the most technological advancements in imaging, therapeutic radiation, and computerized treatment planning systems acquire key data during the RT treatment course of a patient. One example are patients treated for brain tumors of greater sizes and irregular shapes that utilize state-of-the-art RT technology to deliver pinpoint accurate radiation doses. Various treatment options are available to the patient from Radiation Therapy to Stereotactic Radiosurgery and utilize different RT modalities. The disparate and complex data generated by the RT modalities along with related data scattered throughout the RT department in RT Information/Management systems, Record & Verify systems, and Treatment Planning Systems (TPS) compromise an efficient clinical workflow since the data crucial for a clinical decision may be time-consuming to retrieve, temporarily missing, or even lost. To address these shortcomings, the ACR-NEMA Standards Committee extended its DICOM (Digital Imaging & Communications in Medicine) Standard from Radiology to RT by ratifying seven DICOM RT objects starting in 1997. However, they are rarely used by the RT community in daily clinical operations. In the past, the research focus of an RT department has primarily been developing new protocols and devices to improve treatment process and outcomes of cancer patients with minimal effort dedicated to integration of imaging and information systems. By combining our past experience in medical imaging informatics research, DICOM-RT expertise, and system integration, our research involves using a brain tumor case model to show proof-of-concept that a DICOM-Standard electronic patient record (ePR) system can be developed as a foundation to perform medical imaging informatics research in developing decision-support tools and knowledge base for future data mining applications. As an initial first step, we will develop a methodology to perform medical imaging informatics research on a clinical scenario where brain tumor patients undergo treatment planning for either radiosurgery or radiation therapy. Specifically, we will research the "inverse treatment planning" process that is used for those types of treatments and integrate decision-support knowledge and tools designed to assist in the decision-making process, thus introducing an improved "knowledge-enhanced treatment planning" approach.

  19. Simplified strategy for detection of recombinant human immunodeficiency virus type 1 group M isolates by gag/env heteroduplex mobility assay. Study Group on Heterogeneity of HIV Epidemics in African Cities.

    PubMed

    Heyndrickx, L; Janssens, W; Zekeng, L; Musonda, R; Anagonou, S; Van der Auwera, G; Coppens, S; Vereecken, K; De Witte, K; Van Rampelbergh, R; Kahindo, M; Morison, L; McCutchan, F E; Carr, J K; Albert, J; Essex, M; Goudsmit, J; Asjö, B; Salminen, M; Buvé, A; van Der Groen, G

    2000-01-01

    We developed a heteroduplex mobility assay in the gag gene (gag HMA) for the identification of group M subtypes A to H. The assay covers the region coding for amino acid 132 of p24 to amino acid 20 of p7 (according to human immunodeficiency virus type 1 [HIV-1] ELI, 460 bp). The gag HMA was compared with sequencing and phylogenetic analysis of an evaluation panel of 79 HIV-1 group M isolates isolated from infected individuals from different geographic regions. Application of gag HMA in combination with env HMA on 252 HIV-1- positive plasma samples from Bénin, Cameroon, Kenya, and Zambia revealed a high prevalence of a variety of intersubtype recombinants in Yaoundé, Cameroon (53.8%); Kisumu, Kenya (26.8%); and Cotonou, Bénin (41%); no recombinants were identified among the samples from Ndola, Zambia. The AG(IbNG) circulating recombinant form, as determined by gag HMA, was found to be the most common intersubtype recombinant in Yaoundé (39.4%) and Cotonou (38.5%). Using a one-tube reverse transcriptase PCR protocol, this gag HMA in combination with env HMA is a useful tool for rapidly monitoring the prevalence of the various genetic subtypes as well as of recombinants of HIV-1. Moreover, this technology can easily be applied in laboratories in developing countries. PMID:10590125

  20. Ra p po Rt d 'act i v i te 2 0 1 0 Musum national d'Histoire naturelle

    E-print Network

    Rbosa a., BURESCH K. C.,SOGIN E., SCHwARTz J., CHUBB C. & HANLON R.T. - 2010. Cuttlefish dynamic camouflage: responses to substrate choice and integration of multiple visual cues. Proceedings of the Royal

  1. Analytical approach for selecting normalizing genes from a cDNA microarray platform to be used in q-RT-PCR assays: A cnidarian case study

    E-print Network

    discussed. © 2007 Published by Elsevier B.V. Keywords: Cnidarians; Coral; Housekeeping genes; q-RT-PCRAnalytical approach for selecting normalizing genes from a cDNA microarray platform to be used in q-RT-PCR; accepted 27 August 2007 Abstract Research in gene function using Quantitative Reverse Transcription PCR (q-RT-PCR

  2. HIV RT Binding to DNA Primer-Template Tethered to a Quantum Dot DJ Wright, Jon Kawulok, Angela Rudolph, James A. Brozik

    E-print Network

    Collins, Gary S.

    HIV RT Binding to DNA Primer-Template Tethered to a Quantum Dot DJ Wright, Jon Kawulok, Angela-template to the catalyzing enzyme HIV reverse transcriptase (HIV RT) molecules tethered to a biotinylated layer of bovine' prime end of the DNA, a biotinylated HIV RT molecule was linked to an SA molecule at 5E-6 mg/ml which

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

    NASA Technical Reports Server (NTRS)

    Molthan, Andrew L.

    2010-01-01

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

  4. Genotyping and Classification of Tunisian Strains of Avian Reovirus using RT-PCR and RFLP Analysis.

    PubMed

    Kort, Ymene Hellal; Bourogâa, Hager; Gribaa, Latifa; Hassen, Jihene; Ghram, Abdelgelil

    2015-03-01

    Since 1998, avian reovirus (ARV) infection has been detected in broiler and breeding chicken flocks in Tunisia. The genotype of avian reoviruses was established using simple and rapid approaches. Reverse transcription PCR (RT-PCR) on both sigma C (?C) and sigma B (?B)-encoding genes followed by restriction fragment length polymorphism (RFLP) analyses were used to better characterize Tunisian isolated strains. The RT-PCR amplified fragments of 738 and 540 bp for ?C- and ?B-encoding genes, respectively, of 15 ARV Tunisian strains. DNA fragments amplified from S 1133 vaccine and isolated strains were digested with different restrictions enzymes. RFLP on the ?C gene indicated that the field isolates and the S 1133 vaccine strain have identical profiles when separately digested with TaqI, PstI, DdeI, and HincII. Considering the ?B gene, RFLP profiles were identical with RsaI, BclI, DpnII, and NciI restriction enzymes for all the strains. However, using MseI and AciI enzymes, it was shown that all tested isolates could be clearly distinguished from the vaccine strain. ARV strains could be classified in groups with strong relatedness. Strain-typing based on cleavage site results are in agreement with ARV clustering based on nucleotide sequences of both the ?C and ?B genes. RT-PCR-RFLP provides a simple and a rapid approach for genotyping ARV isolates, especially when a large number of isolates are being studied. Additionally, this approach may also determine whether a new variant strain has been introduced into a flock or if a given virus strain is being spread from one flock to another. PMID:26292528

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

    SciTech Connect

    Nanstad, R.K.; McCabe, D.E.; Swain, R.L.; Miller, M.K. )

    1992-12-01

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

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

    PubMed

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

    2014-05-01

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

  7. Regional nodal recurrence in breast cancer patients treated with conservative surgery and radiation therapy (BCS+RT)

    SciTech Connect

    Pejavar, Sunanda; Wilson, Lynn D.; Haffty, Bruce G. . E-mail: hafftybg@umdnj.edu

    2006-12-01

    Purpose: To review regional nodal (RN) management and identify predictors of RN relapse in patients treated with breast conserving surgery and radiation therapy (BCS+RT). Methods and Materials: Patients with Stage I and II breast cancer (N = 1920) underwent BCS+RT from 1973 to 2003. Patients undergoing RN were treated with a median dose of 46 Gy. Patients undergoing axillary dissection (AXD, N = 1330) were treated to the breast alone if node-negative (N = 984), and to the breast and supraclavicular fossa if node-positive (N = 346). Patients who did not undergo AXD (N = 590) were treated with RT to the supraclavicular fossa and axilla. Sentinel node biopsy (SNB) was performed on 126 patients. SN-negative patients (N = 110) were treated with tangents only. There were 16 SN-positive patients who did not undergo complete AXD and were treated with RT. Results: As of September 2005, there have been 36 RN relapses for an actuarial nodal control rate (NCR) of 98% at 10 years. There was no difference in NCR between those undergoing AXD (NCR = 97.4%) and those receiving RT without AXD (NCR = 97.9%). In multivariate analysis, young age, non-Caucasian race, and pathologic nodal status correlated with increased risk of nodal relapse. Of the 126 patients undergoing SNB, there was only 1 nodal recurrence. None of the 16 SN-positive patients treated with RT without AXD had nodal failure. Conclusions: In patients undergoing BCS+RT, both regional nodal irradiation and AXD (including SNB) resulted in equally high rates of regional nodal control. Nodal RT may also be an effective treatment for SN-positive patients.

  8. NASA SPoRT Modeling and Data Assimilation Research and Transition Activities Using WRF, LIS and GSI

    NASA Technical Reports Server (NTRS)

    Case, Jonathan L.; Blankenship, Clay B.; Zavodsky, Bradley T.; Srikishen, Jayanthi; Berndt, Emily B.

    2014-01-01

    weather research and forecasting ===== The NASA Short-term Prediction Research and Transition (SPoRT) program has numerous modeling and data assimilation (DA) activities in which the WRF model is a key component. SPoRT generates realtime, research satellite products from the MODIS and VIIRS instruments, making the data available to NOAA/NWS partners running the WRF/EMS, including: (1) 2-km northwestern-hemispheric SST composite, (2) daily, MODIS green vegetation fraction (GVF) over CONUS, and (3) NASA Land Information System (LIS) runs of the Noah LSM over the southeastern CONUS. Each of these datasets have been utilized by specific SPoRT partners in local EMS model runs, with select offices evaluating the impacts using a set of automated scripts developed by SPoRT that manage data acquisition and run the NCAR Model Evaluation Tools verification package. SPoRT is engaged in DA research with the Gridpoint Statistical Interpolation (GSI) and Ensemble Kalman Filter in LIS for soil moisture DA. Ongoing DA projects using GSI include comparing the impacts of assimilating Atmospheric Infrared Sounder (AIRS) radiances versus retrieved profiles, and an analysis of extra-tropical cyclones with intense non-convective winds. As part of its Early Adopter activities for the NASA Soil Moisture Active Passive (SMAP) mission, SPoRT is conducting bias correction and soil moisture DA within LIS to improve simulations using the NASA Unified-WRF (NU-WRF) for both the European Space Agency's Soil Moisture Ocean Salinity and upcoming SMAP mission data. SPoRT has also incorporated real-time global GVF data into LIS and WRF from the VIIRS product being developed by NOAA/NESDIS. This poster will highlight the research and transition activities SPoRT conducts using WRF, NU-WRF, EMS, LIS, and GSI.

  9. Gene expression profile of circulating tumor cells in breast cancer by RT-qPCR

    PubMed Central

    2011-01-01

    Background Circulating tumor cells (CTCs) have been associated with prognosis especially in breast cancer and have been proposed as a liquid biopsy for repeated follow up examinations. Molecular characterization of CTCs is difficult to address since they are very rare and the amount of available sample is very limited. Methods We quantified by RT-qPCR CK-19, MAGE-A3, HER-2, TWIST1, hTERT ?+?+, and mammaglobin gene transcripts in immunomagnetically positively selected CTCs from 92 breast cancer patients, and 28 healthy individuals. We also compared our results with the CellSearch system in 33 of these patients with early breast cancer. Results RT-qPCR is highly sensitive and specific and can detect the expression of each individual gene at the one cell level. None of the genes tested was detected in the group of healthy donors. In 66 operable breast cancer patients, CK-19 was detected in 42.4%, HER-2 in 13.6%, MAGE-A3 in 21.2%, hMAM in 13.6%, TWIST-1 in 42.4%, and hTERT ?+?+ in 10.2%. In 26 patients with verified metastasis, CK-19 was detected in 53.8%, HER-2 in 19.2%, MAGE-A3 in 15.4%, hMAM in 30.8%, TWIST-1 in 38.5% and hTERT ?+?+in 19.2%. Our preliminary data on the comparison between RT-qPCR and CellSearch in 33 early breast cancer patients showed that RT-qPCR gives more positive results in respect to CellSearch. Conclusions Molecular characterization of CTCs has revealed a remarkable heterogeneity of gene expression between breast cancer patients. In a small percentage of patients, CTCs were positive for all six genes tested, while in some patients only one of these genes was expressed. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome for these patients is known. PMID:21967632

  10. VizieR Online Data Catalog: RT Aur and SZ tau UBV light curves (Evans+, 2015)

    NASA Astrophysics Data System (ADS)

    Evans N.R., Szabo R., Derekas A., Szabados L., Cameron C., Matthews J.M., Sasselov D., Kuschnig R., Rowe J.F., Guenther D.B., Moffat A.F.J., Rucinski S.M., Weiss W.W.

    2015-07-01

    The MOST satellite is a photometric satellite fully described in Walker et al. (2003PASP..115.1023W), with the first science presented in Matthews et al. (2004Natur.430...51M). For SZ Tau, observations were obtained in 2012 November (JD 2456238 - JD 2456257) resulting in a continuous data set covering 19d with a cadence of 1-min. For RT Aur, the observations were carried out in 2012 December 2012 (JD 2456278 - JD 2456300). For this target, the 22d-long observations were interleaved with another target, resulting in gaps in the light curve. (4 data files).

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

    NASA Technical Reports Server (NTRS)

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

    2012-01-01

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

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

  13. Using the SPoRT POES/GOES Hybrid Product in OCONUS Forecasting

    NASA Technical Reports Server (NTRS)

    Smith, Matt; Fuell, Kevin; Nelson, Jim

    2014-01-01

    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.

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

    NASA Technical Reports Server (NTRS)

    Jedlovec, Gary J.

    2013-01-01

    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.

  15. Challenges in Transitioning Research Data to Operations: The SPoRT Paradigm

    NASA Technical Reports Server (NTRS)

    Jedloved, Gary J.; Smith, Matt; McGrath, Kevin

    2010-01-01

    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 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.

  16. In Vitro Examination of the Thrombolytic Efficacy of Desmoteplase and Therapeutic Ultrasound Compared with rt-PA.

    PubMed

    Roessler, Florian C; Wang, Zhihua; Schumacher, Sabrina; Ohlrich, Marcus; Kaps, Manfred; Menciassi, Arianna; Eggers, Jürgen

    2015-12-01

    The aim of the study described here was to evaluate the thrombolytic efficacy of combined treatment with the fibrin-selective plasminogen activator desmoteplase (DSPA) and therapeutic ultrasound (sonothrombolysis [STL]) compared with conventional rt-PA (recombinant tissue plasminogen activator) treatment in vitro. Lysis rates were determined by the weight loss of platelet-rich plasma (PRP) clots treated with rt-PA (60 kU/mL) or DSPA (2 ?g/mL) combined with pulsed wave ultrasound (2 MHz, 0.179 W/cm(2)). To reveal the individual effects of medication and ultrasound, lysis rates were also determined for DSPA monotherapy and for combined treatment with rt-PA and ultrasound. Clots solely placed in plasma served as the control group. Lysis increased significantly with rt-PA (26.5 ± 7.8%) and DSPA (30.5 ± 6%) compared with the control group (18.2 ± 5.9%) (each p < 0.001). DSPA lysis was more effective than rt-PA lysis (without STL: p = 0.015, with STL: p = 0.01). Combined treatment with DSPA and 2-MHz STL significantly exceeded rt-PA lysis (32.8% vs. 26.5%, p < 0.001). PMID:26349583

  17. Effects of the protonation state in the interaction of an HIV-1 reverse transcriptase (RT) amino acid, Lys101, and a non nucleoside RT inhibitor, GW420867X.

    PubMed

    Galembeck, Sérgio E; Bickelhaupt, F Matthias; Fonseca Guerra, Célia; Galembeck, Eduardo

    2014-07-01

    Interactions between an inhibitor and amino acids from a binding pocket could help not only to understand the nature of these interactions, but also to support the design of new inhibitors. In this paper, we explore the key interaction between a second generation non-nucleoside reverse transcriptase inhibitor (NNRTI), GW420867X, and HIV-1 RT amino acid Lys101 (K101), by quantum mechanical methods. The neutral, protonated, and zwitterionic complexes of GW420867X-K101 were studied. The interaction energies were determined by SCS-MP2/def2-cc-pVQZ, and the electron density was analyzed by natural bond orbital (NBO), atoms in molecules (AIM) and reduced gradient analysis. A large increase in the interaction was observed with the tautomerization of neutral or neutral protonated species. The monomers interact by two medium-strength hydrogen bonds, one partially covalent and another noncovalent. There are some van der Waals intramolecular interactions that are topologically unstable. The nature of the intermolecular interactions was also analyzed using quantitative molecular orbital (MO) theory in combination with an energy decomposition analysis (EDA) based on dispersion-corrected density functional theory (DFT) at BLYP-D/TZ2P. PMID:24965933

  18. RT real-time PCR-based quantification of Uromyces fabae in planta.

    PubMed

    Voegele, Ralf T; Schmid, Annette

    2011-09-01

    Quantification of obligate biotrophic parasites has been a long-standing problem in plant pathology. Many attempts have been made to determine how much of a pathogen is present in infected plant tissue. Methods of quantification included scoring disease symptoms, microscopic evaluation, determination of specific compounds like Ergosterol, and lately nucleic acid-based technologies. All of these methods have their drawbacks, and even real-time PCR may not be quantitative if for example the organism of interest has specific and differing numbers of nuclei in different infection structures. We applied reverse transcription (RT) real-time PCR to quantify Uromyces fabae within its host plant Vicia faba. We used three different genes, which have been shown to be constitutively expressed. Our analyses show an exponential increase of fungal material between 4 and 9 days post inoculation and thereafter reaching a steady state of around 45% of total RNA. We also used haustorium-specific genes to determine the amount of haustoria present at each time point. These analyses parallel the development of the whole fungus with the exception of the steady-state level, which is only around 5% of the total RNA. This indicates that RT real-time PCR is a suitable method for quantification of obligate biotrophic parasites, and also for the differentiation of developmental stages. PMID:21707731

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

    PubMed

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

    2007-04-01

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

  20. ASePCR: alternative splicing electronic RT–PCR in multiple tissues and organs

    PubMed Central

    Kim, Namshin; Lim, Dajeong; Lee, Sanghyuk; Kim, Heebal

    2005-01-01

    RT–PCR is one of the most powerful and direct methods to detect transcript variants due to alternative splicing (AS) that increase transcript diversity significantly in vertebrates. ASePCR is an efficient web-based application that emulates RT–PCR in various tissues. It estimates the amplicon size for a given primer pair based on the transcript models identified by the reverse e-PCR program of the NCBI. The tissue specificity of each PCR band is deduced from the tissue information of expressed sequence tag (EST) sequences compatible with each transcript structure. The output page shows PCR bands like a gel electrophoresis in various tissues. Each band in the output picture represents a putative isoform that could happen in a tissue-specific manner. It also shows the EST alignment and tissue information in the genome browser. Furthermore, the user can compare the AS patterns of orthologous genes in other species. The ASePCR, available at , supports the transcriptome models of the RefSeq, Ensembl, ECgene and AceView for human, mouse, rat and chicken genomes. It will be a valuable web resource to explore the transcriptome diversity associated with different tissues and organs in multiple species. PMID:15980562

  1. Quantification of nascent transcription by bromouridine immunocapture nuclear run-on RT-qPCR.

    PubMed

    Roberts, Thomas C; Hart, Jonathan R; Kaikkonen, Minna U; Weinberg, Marc S; Vogt, Peter K; Morris, Kevin V

    2015-08-01

    Nuclear run-on (NRO) is a method that measures transcriptional activity via the quantification of biochemically labeled nascent RNA molecules derived from nuclear isolates. Widespread use of this technique has been limited because of its technical difficulty relative to steady-state total mRNA analyses. Here we describe a detailed protocol for the quantification of transcriptional activity in human cell cultures. Nuclei are first isolated and NRO transcription is performed in the presence of bromouridine. Labeled nascent transcripts are purified by immunoprecipitation, and transcript levels are determined by reverse-transcription quantitative PCR (RT-qPCR). Data are then analyzed using standard techniques described elsewhere. This method is rapid (the protocol can be completed in 2 d) and cost-effective, exhibits negligible detection of background noise from unlabeled transcripts, requires no radioactive materials and can be performed from as few as 500,000 nuclei. It also takes advantage of the high sensitivity, specificity and dynamic range of RT-qPCR. PMID:26182239

  2. Pregnancies and menstrual function before and after combined radiation (RT) and chemotherapy (TVPP) for Hodgkin's disease

    SciTech Connect

    Lacher, M.J.; Toner, K.

    1986-01-01

    The menstrual cycle, pregnancies, and offspring were evaluated before and after initial combined radiation (RT) and chemotherapy with thiotepa, vinblastine, vincristine, procarbazine, and prednisone (TVPP), in 34 women between the ages of 18 and 44 (median 26.5 years) treated for Stage II and Stage III Hodgkin's disease. The median range of follow-up is 83.1 months (range 40.5-140). After therapy 94.1% (32/34) continued to menstruate. Two of the four patients over the age of 35 ceased to menstruate. All patients under the age of 35 continued to menstruate (30/30). Age at the time of diagnosis was the only factor affecting change in menses with a significant probability (p = .001) that women greater than 30 years of age will experience some change in menstrual pattern. Seventeen pregnancies occurred in 12 women after therapy; 2 had 4 elective abortions; 10 delivered 12 children with normal physical development; 1 will deliver six months from now. Twelve of thirteen patients who wanted to become pregnant have conceived. The ability to become pregnant and deliver normal children after intensive treatment with combined radiation and chemotherapy (RT/TVPP) was comparable to the patients' pretreatment record.

  3. Transcripts of developmentally regulated Plasmodium falciparum genes quantified by real-time RT-PCR.

    PubMed

    Blair, Peter L; Witney, Adam; Haynes, J David; Moch, J Kathleen; Carucci, Daniel J; Adams, John H

    2002-05-15

    Plasmodium falciparum intraerythrocytic development is a complex process. Development proceeds rapidly from the trophozoite phase of nutrient acquisition and growth through to the synthetic and reproductive schizont phase, which ends with production of new invasive merozoites. During this process, the malaria parasite must express a series of different gene products, depending on its metabolic and synthetic needs. We are particularly interested in the development of the merozoite's organelles in the apical complex, which form during the later schizont stages. We have used quantitative real-time RT-PCR fluorogenic 5' nuclease assays (TaqMan) for the first time on malaria parasites for analysis of erythrocytic stage-specific gene expression. We analyzed transcripts of the P.falciparum eba-175 and other erythrocyte binding-like (ebl) family genes in temperature-synchronized parasites and found ebl genes have tightly controlled, stage-specific transcription. As expected, eba-175 transcripts were abundant only at the end of schizont development in a pattern most common among ebl, including baebl, pebl and jesebl. The maebl transcript pattern was unique, peaking at mid-late trophozoite stage, but absent in late-stage schizonts. ebl-1 demonstrated another pattern of expression, which peaked during mid-schizont stage and then significantly diminished in late-stage schizonts. Our analysis demonstrates that using real-time RT-PCR fluorogenic 5' nuclease assays is a sensitive, quantitative method for analysis of Plasmodium transcripts. PMID:12000842

  4. Transcripts of developmentally regulated Plasmodium falciparum genes quantified by real-time RT–PCR

    PubMed Central

    Blair, Peter L.; Witney, Adam; Haynes, J. David; Moch, J. Kathleen; Carucci, Daniel J.; Adams, John H.

    2002-01-01

    Plasmodium falciparum intraerythrocytic development is a complex process. Development proceeds rapidly from the trophozoite phase of nutrient acquisition and growth through to the synthetic and reproductive schizont phase, which ends with production of new invasive merozoites. During this process, the malaria parasite must express a series of different gene products, depending on its metabolic and synthetic needs. We are particularly interested in the development of the merozoite’s organelles in the apical complex, which form during the later schizont stages. We have used quantitative real-time RT–PCR fluorogenic 5? nuclease assays (TaqMan®) for the first time on malaria parasites for analysis of erythrocytic stage-specific gene expression. We analyzed transcripts of the P.falciparum eba-175 and other erythrocyte binding-like (eb l) family genes in temperature-synchronized parasites and found ebl genes have tightly controlled, stage-specific transcription. As expected, eba-175 transcripts were abundant only at the end of schizont development in a pattern most common among ebl, including baebl, pebl and jesebl. The maebl transcript pattern was unique, peaking at mid-late trophozoite stage, but absent in late-stage schizonts. ebl-1 demonstrated another pattern of expression, which peaked during mid-schizont stage and then significantly diminished in late-stage schizonts. Our analysis demonstrates that using real-time RT–PCR fluorogenic 5? nuclease assays is a sensitive, quantitative method for analysis of Plasmodium transcripts. PMID:12000842

  5. An improved RT-IPCR for detection of pyrene and related polycyclic aromatic hydrocarbons.

    PubMed

    Meng, X Y; Li, Y S; Zhou, Y; Sun, Y; Qiao, B; Si, C C; Hu, P; Lu, S Y; Ren, H L; Liu, Z S; Qiu, H J; Liu, J Q

    2016-04-15

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous homogeneous chemicals which are well known by carcinogens, mutagens and endocrine disorder. Here, an improved real-time immuno-PCR (RT-IPCR) was developed for detection of pyrene and its homologs in water samples. The PAHs in sample compete with pyrene-modified DNA to bind with monoclonal antibody (McAb) coated on PCR plate. The reporter DNA was exponentially amplified by real-time PCR instrument using Fast Start universal SYBR Green Master (ROX) kit. Only two reaction steps were needed to accomplish the detection. The assay had a good linear range from 5pmolL(-1) to 5nmolL(-1) with a detection limit of 3.5pmolL(-1). For application assay, the average recoveries from tap water, lake water and mineral water were 98.4%, 98.2% and 99.7%, respectively which showed a good correlation (R(2)=0.9906) with those from GC-MS. The results indicated that the improved RT-IPCR seems to be a potential method for simple and ultrasensitive detection of pyrene and some homologues in environment water samples. PMID:26609944

  6. Helical Tomotherapy-Based STAT RT: Dosimetric Evaluation for Clinical Implementation of a Rapid Radiation Palliation Program

    SciTech Connect

    McIntosh, Alyson; Dunlap, Neal; Sheng, Ke; Geezey, Constance; Turner, Benton; Blackhall, Leslie; Weiss, Geoffrey; Lappinen, Eric; Larner, James M.; Read, Paul W.

    2010-01-01

    Helical tomotherapy-based STAT radiation therapy (RT) uses an efficient software algorithm for rapid intensity-modulated treatment planning, enabling conformal radiation treatment plans to be generated on megavoltage computed tomography (MVCT) scans for CT simulation, treatment planning, and treatment delivery in one session. We compared helical tomotherapy-based STAT RT dosimetry with standard linac-based 3D conformal plans and standard helical tomotherapy-based intensity-modulated radiation therapy (IMRT) dosimetry for palliative treatments of whole brain, a central obstructive lung mass, multilevel spine disease, and a hip metastasis. Specifically, we compared the conformality, homogeneity, and dose with regional organs at risk (OARs) for each plan as an initial step in the clinical implementation of a STAT RT rapid radiation palliation program. Hypothetical planning target volumes (PTVs) were contoured on an anthropomorphic phantom in the lung, spine, brain, and hip. Treatment plans were created using three planning techniques: 3D conformal on Pinnacle{sup 3}, helical tomotherapy, and helical tomotherapy-based STAT RT. Plan homogeneity, conformality, and dose to OARs were analyzed and compared. STAT RT and tomotherapy improved conformality indices for spine and lung plans (CI spine = 1.21, 1.17; CI lung = 1.20, 1.07, respectively) in comparison with standard palliative anteroposterior/posteroanterior (AP/PA) treatment plans (CI spine = 7.01, CI lung = 7.30), with better sparing of heart, esophagus, and spinal cord. For palliative whole-brain radiotherapy, STAT RT and tomotherapy reduced maximum and mean doses to the orbits and lens (maximum/mean lens dose: STAT RT = 2.94/2.65 Gy, tomotherapy = 3.13/2.80 Gy, Lateral opposed fields = 7.02/3.65 Gy), with an increased dose to the scalp (mean scalp dose: STAT RT = 16.19 Gy, tomotherapy = 15.61 Gy, lateral opposed fields = 14.01 Gy). For bony metastatic hip lesions, conformality with both tomotherapy techniques (CI = 1.01 each) is superior to AP/PA treatments (CI = 1.21), as expected. Helical tomotherapy-based STAT RT treatment planning provides clinically acceptable dosimetry, with conformality and homogeneity that is superior to standard linac-based 3D conformal planning and is only slightly inferior to standard helical tomotherapy IMRT dosimetry. STAT RT facilitates rapid treatment planning and delivery for palliative radiation of patients with metastatic disease, with relative sparing of adjacent OARs compared with standard 3D conformal plans.

  7. SU-E-J-42: Customized Deformable Image Registration Using Open-Source Software SlicerRT

    SciTech Connect

    Gaitan, J Cifuentes; Chin, L; Pignol, J; Kirby, N; Pouliot, J; Lasso, A; Pinter, C; Fichtinger, G

    2014-06-01

    Purpose: SlicerRT is a flexible platform that allows the user to incorporate the necessary images registration and processing tools to improve clinical workflow. This work validates the accuracy and the versatility of the deformable image registration algorithm of the free open-source software SlicerRT using a deformable physical pelvic phantom versus available commercial image fusion algorithms. Methods: Optical camera images of nonradiopaque markers implanted in an anatomical pelvic phantom were used to measure the ground-truth deformation and evaluate the theoretical deformations for several DIR algorithms. To perform the registration, full and empty bladder computed tomography (CT) images of the phantom were obtained and used as fixed and moving images, respectively. The DIR module, found in SlicerRT, used a B-spline deformable image registration with multiple optimization parameters that allowed customization of the registration including a regularization term that controlled the amount of local voxel displacement. The virtual deformation field at the center of the phantom was obtained and compared to the experimental ground-truth values. The parameters of SlicerRT were then varied to improve spatial accuracy. To quantify image similarity, the mean absolute difference (MAD) parameter using Hounsfield units was calculated. In addition, the Dice coefficient of the contoured rectum was evaluated to validate the strength of the algorithm to transfer anatomical contours. Results: Overall, SlicerRT achieved one of the lowest MAD values across the algorithm spectrum, but slightly smaller mean spatial errors in comparison to MIM software (MIM). On the other hand, SlicerRT created higher mean spatial errors than Velocity Medical Solutions (VEL), although obtaining an improvement on the DICE to 0.91. The large spatial errors were attributed to the poor contrast in the prostate bladder interface of the phantom. Conclusion: Based phantom validation, SlicerRT is capable of achieving comparable DIR accuracy to commercial programs such as MIM and VEL.

  8. Detection of dermcidin for sweat identification by real-time RT-PCR and ELISA.

    PubMed

    Sakurada, Koichi; Akutsu, Tomoko; Fukushima, Hisayo; Watanabe, Ken; Yoshino, Mineo

    2010-01-30

    We evaluated the performance of real-time RT-PCR and ELISA assays for detection of dermcidin (DCD) in sweat and body-fluid stains. DCD, a small antibiotic peptide secreted into human sweat, was detected by real-time RT-PCR in 7-day-old stains containing as small as 10 microL of sweat, and the assay showed high specificity when testing 7-day-old stains containing 30 microL of other body-fluid. ELISA using anti-human dermcidin mouse monoclonal antibody detected DCD sweat diluted up to approximately 10,000-fold and could specifically detect DCD in 10 microL of body-fluid stains. The performance of the two assays was tested during winter on samples that simulated forensic case samples: an undershirt and a sock worn for 20 h, a handkerchief used to wipe the brow several times within 12h, a cap and a cotton glove worn for 4h, and a white robe worn at intervals for 2 years. The result showed that the former assay detected DCD in all sites of the undershirt examined (armpit, back, and breast), and the latter gave a relatively high OD value in the armpit among the three sites. For the socks, although the latter assay gave very high OD values in both the center and toe of the foot sole, the former could not detect DCD in both of them. These results indicate that highly damp conditions, such as inside a shoe, might promote the degradation of mRNA in samples such as socks. In the other case samples, sweat was adequately detected by both assays. This study is the first demonstration of the use of real-time RT-PCR to sensitively identify sweat among body-fluid stains, and it confirmed that dermcidin was an excellent marker for sweat identification. In addition, the usefulness of ELISA was also verified. Positive sweat identification using these assays is expected to assist forensic practice. PMID:19914015

  9. Transitioning NPOESS Data to Weather Offices: The SPoRT Paradigm with EOS Data

    NASA Technical Reports Server (NTRS)

    Jedlovec, Gary

    2009-01-01

    Real-time satellite information provides one of many data sources used by NWS weather forecast offices (WFOs) to diagnose current weather conditions and to assist in short-term forecast preparation. While GOES satellite data provides relatively coarse spatial resolution coverage of the continental U.S. on a 10-15 minute repeat cycle, polar orbiting imagery has the potential to provide snapshots of weather conditions at high-resolution in many spectral channels. Additionally, polar orbiting sounding data can provide additional information on the thermodynamic structure of the atmosphere in data sparse regions of at asynoptic observation times. The NASA Short-term Prediction Research and Transition (SPoRT) project has demonstrated the utility of polar orbiting MODIS and AIRS data on the Terra and Aqua satellites to improve weather diagnostics and short-term forecasting on the regional and local scales. SPoRT scientists work directly forecasters at selected WFOS in the Southern Region (SR) to help them ingest these unique data streams into their AWIPS system, understand how to use the data (through on-site and distance learn techniques), and demonstrate the utility of these products to address significant forecast problems. This process also prepares forecasters for the use of similar observational capabilities from NPOESS operational sensors. NPOESS environmental data records (EDRs) from the Visible 1 Infrared Imager I Radiometer Suite (VIIRS), the Cross-track Infrared Sounder (CrlS) and Advanced Technology Microwave Sounder (ATMS) instruments and additional value-added products produced by NESDIS will be available in near real-time and made available to WFOs to extend their use of NASA EOS data into the NPOESS era. These new data streams will be integrated into the NWs's new AWIPS II decision support tools. The AWIPS I1 system to be unveiled in WFOs in 2009 will be a JAVA-based decision support system which preserves the functionality of the existing systems and offers unique development opportunities for new data sources and applications in the Service Orientated Architecture ISOA) environment. This paper will highlight some of the SPoRT activities leading to the integration of VllRS and CrIS/ATMS data into the display capabilities of these new systems to support short-term forecasting problems at WFOs.

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

    NASA Technical Reports Server (NTRS)

    Bell, Jordan R.; Case, Jonathan L.; Molthan, Andrew L.

    2011-01-01

    The NASA Short-term Prediction Research and Transition (SPoRT) Center develops new products and techniques that can be used in operational meteorology. The majority of these products are derived from NASA polar-orbiting satellite imagery from the Earth Observing System (EOS) platforms. One such product is a Greenness Vegetation Fraction (GVF) dataset, which is produced from Moderate Resolution Imaging Spectroradiometer (MODIS) data aboard the NASA EOS Aqua and Terra satellites. NASA SPoRT began generating daily real-time GVF composites at 1-km resolution over the Continental United States (CONUS) on 1 June 2010. The purpose of this study is to compare the National Centers for Environmental Prediction (NCEP) climatology GVF product (currently used in operational weather models) to the SPoRT-MODIS GVF during June to October 2010. The NASA Land Information System (LIS) was employed to study the impacts of the new SPoRT-MODIS GVF dataset on land surface models apart from a full numerical weather prediction (NWP) model. For the 2010 warm season, the SPoRT GVF in the western portion of the CONUS was generally higher than the NCEP climatology. The eastern CONUS GVF had variations both above and below the climatology during the period of study. These variations in GVF led to direct impacts on the rates of heating and evaporation from the land surface. The second phase of the project is to examine the impacts of the SPoRT GVF dataset on NWP using the Weather Research and Forecasting (WRF) model. Two separate WRF model simulations were made for individual severe weather case days using the NCEP GVF (control) and SPoRT GVF (experimental), with all other model parameters remaining the same. Based on the sensitivity results in these case studies, regions with higher GVF in the SPoRT model runs had higher evapotranspiration and lower direct surface heating, which typically resulted in lower (higher) predicted 2-m temperatures (2-m dewpoint temperatures). The opposite was true for areas with lower GVF in the SPoRT model runs. These differences in the heating and evaporation rates produced subtle yet quantifiable differences in the simulated convective precipitation systems for the selected severe weather case examined.

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

    NASA Technical Reports Server (NTRS)

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

    2012-01-01

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

  12. Selection of reference genes for quantitative real time RT-PCR during dimorphism in the zygomycete Mucor circinelloides.

    PubMed

    Valle-Maldonado, Marco I; Jácome-Galarza, Irvin E; Gutiérrez-Corona, Félix; Ramírez-Díaz, Martha I; Campos-García, Jesús; Meza-Carmen, Víctor

    2015-03-01

    Mucor circinelloides is a dimorphic fungal model for studying several biological processes including cell differentiation (yeast-mold transitions) as well as biodiesel and carotene production. The recent release of the first draft sequence of the M. circinelloides genome, combined with the availability of analytical methods to determine patterns of gene expression, such as quantitative Reverse transcription-Polymerase chain reaction (qRT-PCR), and the development of molecular genetic tools for the manipulation of the fungus, may help identify M. circinelloides gene products and analyze their relevance in different biological processes. However, no information is available on M. circinelloides genes of stable expression that could serve as internal references in qRT-PCR analyses. One approach to solve this problem consists in the use of housekeeping genes as internal references. However, validation of the usability of these reference genes is a fundamental step prior to initiating qRT-PCR assays. This work evaluates expression of several constitutive genes by qRT-PCR throughout the morphological differentiation stages of M. circinelloides; our results indicate that tfc-1 and ef-1 are the most stable genes for qRT-PCR assays during differentiation studies and they are proposed as reference genes to carry out gene expression studies in this fungus. PMID:25391770

  13. Real-time RT-PCR assays for the rapid and differential detection of dolphin and porpoise morbilliviruses.

    PubMed

    Grant, Rebecca J; Banyard, Ashley C; Barrett, Tom; Saliki, Jeremiah T; Romero, Carlos H

    2009-03-01

    Real-time RT-PCR (rtRT-PCR) assays for identifying and differentiating infections caused by dolphin morbillivirus (DMV) and porpoise morbillivirus (PMV) were developed by targeting the hypervariable C-terminal domain of the nucleocapsid (N) gene. Total DMV and PMV RNA extracted from infected Vero cells expressing the canine signaling lymphocyte-activation molecule (SLAM) produced positive cycle threshold (C(T)) values after the 17th and 25th cycles, respectively. The assays were then validated using infected cetacean tissue RNA. The assays were specific for either DMV or PMV and did not cross-react with canine distemper virus (CDV), phocid distemper virus (PDV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV) and measles virus (MV). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was targeted as control for RNA quality, and a consensus GAPDH probe that reacted with 11 different marine mammal species, generating positive C(T) values ranging from the 21st to the 37th cycle was used. The rtRT-PCR assays have advantages over conventional assays in that they are rapid, easier to scale up, and are less prone to cross-contamination and have improved the limit of detection and specificity. PMID:19084557

  14. Diagnosis of Cetacean morbillivirus: A sensitive one step real time RT fast-PCR method based on SYBR(®) Green.

    PubMed

    Sacristán, Carlos; Carballo, Matilde; Muñoz, María Jesús; Bellière, Edwige Nina; Neves, Elena; Nogal, Verónica; Esperón, Fernando

    2015-12-15

    Cetacean morbillivirus (CeMV) (family Paramyxoviridae, genus Morbillivirus) is considered the most pathogenic virus of cetaceans. It was first implicated in the bottlenose dolphin (Tursiops truncatus) mass stranding episode along the Northwestern Atlantic coast in the late 1980s, and in several more recent worldwide epizootics in different Odontoceti species. This study describes a new one step real-time reverse transcription fast polymerase chain reaction (real-time RT-fast PCR) method based on SYBR(®) Green to detect a fragment of the CeMV fusion protein gene. This primer set also works for conventional RT-PCR diagnosis. This method detected and identified all three well-characterized strains of CeMV: porpoise morbillivirus (PMV), dolphin morbillivirus (DMV) and pilot whale morbillivirus (PWMV). Relative sensitivity was measured by comparing the results obtained from 10-fold dilution series of PMV and DMV positive controls and a PWMV field sample, to those obtained by the previously described conventional phosphoprotein gene based RT-PCR method. Both the conventional and real-time RT-PCR methods involving the fusion protein gene were 100- to 1000-fold more sensitive than the previously described conventional RT-PCR method. PMID:26454114

  15. RT3: A Windows program for the Renner Teller analysis of ?2 states of triatomic molecules

    NASA Astrophysics Data System (ADS)

    He, Sheng-Gui; Clouthier, Dennis J.

    2008-05-01

    In this work we present Windows and Fortran programs which can be used for the Renner-Teller analysis of the vibronic levels of ?2 states of linear, triatomic molecules. The programs can do least squares fitting of term values relative to the lowest energy level within a single ?2 state, of combination differences between vibronic levels within a single ?2 state or of transitions between the levels of two different ?2 states. The algorithm allows for the inclusion of Renner-Teller, spin-orbit, vibrational anharmonicity, Fermi resonance and Sears resonance terms in the Hamiltonian matrices. The Windows program RT3, written in Visual Basic 6.0 with a Fortran DLL engine for the numerically intensive computations, facilitates the construction and editing of input files and comparisons of input and output. For very large calculations, the Fortran code may be run as a stand-alone program on mainframe or other computers, but the Windows version offers significant advantages for common usage. Program summaryTitle of program: RT3 Catalogue identifier: AEAL_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEAL_v1_0.html Program obtainable from: CPC Program Library, Queen's University of Belfast, N. Ireland Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 37 477 No. of bytes in distributed program, including test data, etc.: 3 665 414 Distribution format: tar.gz Programming language: Visual Basic 6.0, Fortran 90 Operating systems: Windows 98, Windows XP, Windows Vista, UNIX Classification: 16.2 Nature of problem: The vibronic levels of triatomic molecules in ?2 states are complicated by spin-orbit and vibrational-orbital angular momentum coupling (Renner-Teller) effects that make them difficult to assign and analyze. The RT3 program, available in Windows and Fortran versions, can be used to calculate the vibronic energy levels of such systems and fit experimental data to a Hamiltonian that includes Renner-Teller, spin-orbit, vibrational anharmonicity, Fermi resonance and Sears resonance effects. Solution method: The RT3 program solves for the Renner-Teller levels of a ?2 state by diagonalizing large truncated Hamiltonian matrices whose elements are generated in a harmonic oscillator basis set. Given an initial input set of constants, the resulting energy levels can be used to least squares fit or predict vibronic term values (relative to the lowest energy level), combination differences between vibronic levels within a electronic state, and transitions between the vibronic levels of two different ?2 states. Running time: 6.0 S to run the Transitions.ir3 example file on an Intel Pentium 4 2 GHz CPU with the Windows XP operating system.

  16. Observation of Classical RM Instability Feeding Ablative RT Growth in Planar Plastic Targets

    NASA Astrophysics Data System (ADS)

    Aglitskiy, Y.; Metzler, N.; Karasik, M.; Serlin, V.; Obenschain, S. P.; Schmitt, A. J.; Velikovich, A. L.; Gardner, J. H.; Varadarajan, J.; Walsh, T.

    2004-11-01

    We observed a classical Richtmyer-Meshkov growth at the shocked embedded foam/plastic interface evolving into an ablative Rayleigh-Taylor growth. Our advanced two-slab targets consisted of a RF foam layer separated by a 2-D sinusoidal interface from a solid plastic layer. The targets were irradiated from the foam side by 4 ns Nike KrF laser pulses at 50 TW/cm2, with and without a 3.5 ns long, 3% foot preceding the main pulse. An observed linear growth of mass modulation amplitude in the positive direction characteristic of the light-to-heavy classical RM instability continued until the amplitude reached a peak value, then its variation changed direction and gradually evolved into the ablative RT growth with a reversed phase. Both the observed timing and amplitude of the peak scaled as predicted when the laser pulse shape was modified by adding a foot.

  17. SU-E-T-194: From Dicom-RT to Radiobiological Dose Metrics in 5 Minutes

    SciTech Connect

    Whelan, B; Holloway, L

    2014-06-01

    Purpose: To develop a flexible and standalone framework for batch calculation of radiobiological dose metrics from Dicom-RT. Methods: Software has been developed which allows (1) The calculation of DVH data from DICOM dose and structure files (DVHgenerator), (2) Calculation of a wide range of radiobiological metrics from this data (CompPlanGui). Both these tools are run via graphical user interface (GUI), making them fast and simple. Part 1 is a new tool which has not previously been published, whilst part 2 is a GUI overlay for the previously published software ‘Comp-Plan’ (Holloway et. al., Medical Dosimetry, 2012), previously reliant on command line interface. The time taken for an experienced user to evaluate a test case of 6 plans with and without CompPlanGUI was quantified. Results: The DVH-generator has been found to be faster, more robust and require far less physical memory then using alternative software solutions for the same purpose. The Comp Plan GUI significantly reduces the amount of time required to set up a base directory, eliminates code crashes arising from typographical errors, and renders the code far more accessible to non-expert users. It took an experienced user of the code around 3 minutes to set up a base directory of 6 plans compared around 8 minutes without, indicating that using CompPlanGUI reduced setup time by over 50%. Conclusion: A standalone GUI based framework has developed which allows for the batch calculation of radiobiological dose metrics directly from Dicom-RT files. As with the original code, this work will be made freely available on request, as well as via matlab file exchange.

  18. Reference Gene Validation for RT-qPCR, a Note on Different Available Software Packages

    PubMed Central

    De Spiegelaere, Ward; Dern-Wieloch, Jutta; Weigel, Roswitha; Schumacher, Valérie; Schorle, Hubert; Nettersheim, Daniel; Bergmann, Martin; Brehm, Ralph; Kliesch, Sabine; Vandekerckhove, Linos; Fink, Cornelia

    2015-01-01

    Background An appropriate normalization strategy is crucial for data analysis from real time reverse transcription polymerase chain reactions (RT-qPCR). It is widely supported to identify and validate stable reference genes, since no single biological gene is stably expressed between cell types or within cells under different conditions. Different algorithms exist to validate optimal reference genes for normalization. Applying human cells, we here compare the three main methods to the online available RefFinder tool that integrates these algorithms along with R-based software packages which include the NormFinder and GeNorm algorithms. Results 14 candidate reference genes were assessed by RT-qPCR in two sample sets, i.e. a set of samples of human testicular tissue containing carcinoma in situ (CIS), and a set of samples from the human adult Sertoli cell line (FS1) either cultured alone or in co-culture with the seminoma like cell line (TCam-2) or with equine bone marrow derived mesenchymal stem cells (eBM-MSC). Expression stabilities of the reference genes were evaluated using geNorm, NormFinder, and BestKeeper. Similar results were obtained by the three approaches for the most and least stably expressed genes. The R-based packages NormqPCR, SLqPCR and the NormFinder for R script gave identical gene rankings. Interestingly, different outputs were obtained between the original software packages and the RefFinder tool, which is based on raw Cq values for input. When the raw data were reanalysed assuming 100% efficiency for all genes, then the outputs of the original software packages were similar to the RefFinder software, indicating that RefFinder outputs may be biased because PCR efficiencies are not taken into account. Conclusions This report shows that assay efficiency is an important parameter for reference gene validation. New software tools that incorporate these algorithms should be carefully validated prior to use. PMID:25825906

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

    NASA Astrophysics Data System (ADS)

    Goto, Takuya; Yatsuka, Eiichi; Morikawa, Junji; Ogawa, Yuichi

    2006-06-01

    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 the case in which the internal coil was mechanically supported, plasma densities up to ne=7× 1016 m-3 were produced with a filling gas pressure of pn=4× 10-2 Pa, and plasma could not be available at a gas pressure less than 10-2 Pa due to the loss of high energy electrons by the supporting structure. It has been clearly demonstrated that the levitation of the internal coil, which was followed by the removal of the supporting structure from the plasma confinement region, improves plasma parameters markedly. The levitation of the internal coil enables plasma production at a lower filling pressure ( pn=1.5× 10-3 Pa). It also enables over-dense plasma production (ne=1.5× 1017 m-3, while cutoff density is 7.6× 1016 m-3 for 2.45 GHz microwave). Since the calculation shows that the efficiency of mode conversion from fast X-mode wave to electron Bernstein wave (FX-SX-B conversion) is sufficiently high (˜87%) in the Mini-RT device, the over-dense plasma production is thought to be caused by heating with electron Bernstein wave (EBW).

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

    NASA Technical Reports Server (NTRS)

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

    2012-01-01

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

  1. A sensitive and reliable RT-nested PCR assay for detection of Citrus tristeza virus from naturally infected citrus plants.

    PubMed

    Adkar-Purushothama, Charith Raj; Maheshwar, P K; Sano, Teruo; Janardhana, G R

    2011-05-01

    A specific and sensitive reverse transcriptase-nested polymerase chain reaction assay (RT-nPCR) was developed for the detection of Citrus tristeza virus (CTV) from naturally infected citrus samples. Two sets of primer pairs were designed by alignment of nucleotide sequences available in GenBank database for different genotypes of CTV. RT-nPCR reaction components and thermal cycling parameters were optimized and reaction conditions were standardized. Sequencing of the PCR products from direct and nested-PCR reactions confirmed the specificity of both primer pairs. Presence of CTV specific amplicons in asymptomatic samples which were collected from diseased orchards indicated the sensitivity of the test. As RT-nPCR technique, developed in the present study, is specific and efficient in detecting CTV, this could be envisioned for diagnostic applications and surveillance. PMID:21298268

  2. Substitution at rt269 in Hepatitis B Virus Polymerase Is a Compensatory Mutation Associated with Multi-Drug Resistance

    PubMed Central

    Kim, Beom Kyung; Park, Yong Kwang; Park, Eun-Sook; Ahn, Sang Hoon; Shin, Gu-Choul; Park, Soree; Kang, Hong Seok; Rhee, Jin-Kyu; Yang, Sung-Il; Chong, Youhoon; Kim, Kyun-Hwan

    2015-01-01

    The emergence of compensatory mutations in the polymerase gene of drug resistant hepatitis B virus (HBV) is associated with treatment failure. We previously identified a multi-drug resistant HBV mutant, which displayed resistance towards lamivudine (LMV), clevudine (CLV), and entecavir (ETV), along with a strong replication capacity. The aim of this study was to identify the previously unknown compensatory mutations, and to determine the clinical relevance of this mutation during antiviral therapy. In vitro mutagenesis, drug susceptibility assay, and molecular modeling studies were performed. The rtL269I substitution conferred 2- to 7-fold higher replication capacity in the wild-type (WT) or YMDD mutation backbone, regardless of drug treatment. The rtL269I substitution alone did not confer resistance to LMV, ETV, adefovir (ADV), or tenofovir (TDF). However, upon combination with YMDD mutation, the replication capacity under LMV or ETV treatment was enhanced by several folds. Molecular modeling studies suggested that the rtL269I substitution affects template binding, which may eventually lead to the enhanced activity of rtI269-HBV polymerase in both WT virus and YMDD mutant. The clinical relevance of the rtL269I substitution was validated by its emergence in association with YMDD mutation in chronic hepatitis B (CHB) patients with sub-optimal response or treatment failure to LMV or CLV. Our study suggests that substitution at rt269 in HBV polymerase is associated with multi-drug resistance, which may serve as a novel compensatory mutation for replication-defective multi-drug resistant HBV. PMID:26322642

  3. Genome-Wide Identification and Validation of Reference Genes in Infected Tomato Leaves for Quantitative RT-PCR Analyses

    PubMed Central

    Müller, Oliver A.; Grau, Jan; Thieme, Sabine; Prochaska, Heike; Adlung, Norman; Sorgatz, Anika; Bonas, Ulla

    2015-01-01

    The Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv) causes bacterial spot disease of pepper and tomato by direct translocation of type III effector proteins into the plant cell cytosol. Once in the plant cell the effectors interfere with host cell processes and manipulate the plant transcriptome. Quantitative RT-PCR (qRT-PCR) is usually the method of choice to analyze transcriptional changes of selected plant genes. Reliable results depend, however, on measuring stably expressed reference genes that serve as internal normalization controls. We identified the most stably expressed tomato genes based on microarray analyses of Xcv-infected tomato leaves and evaluated the reliability of 11 genes for qRT-PCR studies in comparison to four traditionally employed reference genes. Three different statistical algorithms, geNorm, NormFinder and BestKeeper, concordantly determined the superiority of the newly identified reference genes. The most suitable reference genes encode proteins with homology to PHD finger family proteins and the U6 snRNA-associated protein LSm7. In addition, we identified pepper orthologs and validated several genes as reliable normalization controls for qRT-PCR analysis of Xcv-infected pepper plants. The newly identified reference genes will be beneficial for future qRT-PCR studies of the Xcv-tomato and Xcv-pepper pathosystems, as well as for the identification of suitable normalization controls for qRT-PCR studies of other plant-pathogen interactions, especially, if related plant species are used in combination with bacterial pathogens. PMID:26313760

  4. Genome-Wide Identification and Validation of Reference Genes in Infected Tomato Leaves for Quantitative RT-PCR Analyses.

    PubMed

    Müller, Oliver A; Grau, Jan; Thieme, Sabine; Prochaska, Heike; Adlung, Norman; Sorgatz, Anika; Bonas, Ulla

    2015-01-01

    The Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv) causes bacterial spot disease of pepper and tomato by direct translocation of type III effector proteins into the plant cell cytosol. Once in the plant cell the effectors interfere with host cell processes and manipulate the plant transcriptome. Quantitative RT-PCR (qRT-PCR) is usually the method of choice to analyze transcriptional changes of selected plant genes. Reliable results depend, however, on measuring stably expressed reference genes that serve as internal normalization controls. We identified the most stably expressed tomato genes based on microarray analyses of Xcv-infected tomato leaves and evaluated the reliability of 11 genes for qRT-PCR studies in comparison to four traditionally employed reference genes. Three different statistical algorithms, geNorm, NormFinder and BestKeeper, concordantly determined the superiority of the newly identified reference genes. The most suitable reference genes encode proteins with homology to PHD finger family proteins and the U6 snRNA-associated protein LSm7. In addition, we identified pepper orthologs and validated several genes as reliable normalization controls for qRT-PCR analysis of Xcv-infected pepper plants. The newly identified reference genes will be beneficial for future qRT-PCR studies of the Xcv-tomato and Xcv-pepper pathosystems, as well as for the identification of suitable normalization controls for qRT-PCR studies of other plant-pathogen interactions, especially, if related plant species are used in combination with bacterial pathogens. PMID:26313760

  5. Development and evaluation of a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of type II porcine reproductive and respiratory syndrome virus.

    PubMed

    Gao, Ming; Cui, Jin; Ren, Yudong; Suo, Siqingaowa; Li, Guangxing; Sun, Xuejiao; Su, Dingding; Opriessnig, Tanja; Ren, Xiaofeng

    2012-10-01

    The objective of this study was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of type II porcine reproductive and respiratory syndrome virus (PRRSV). Based on sequence alignment, four primers were designed amplifying the M gene of type II PRRSV and were subsequently utilized in an RT-LAMP assay. The RT-LAMP product had a ladder-like pattern of bands and the optimal reaction condition for this assay was determined to be 40 min at 63°C. Comparative analysis indicated that the RT-LAMP method was more sensitive than a conventional RT-PCR assay and comparable to a real-time PCR assay. In addition, the RT-LAMP assay was capable of detecting type II PRRSV in field samples and differentiating type II PRRSV from seven other porcine viruses which are all associated frequently with similar clinical symptoms. PMID:22659065

  6. Predictive Models for Regional Hepatic Function Based upon 99mTc-IDA SPECT and Local Radiation Dose for Physiological Adaptive RT

    PubMed Central

    Wang, Hesheng; Feng, Mary; Frey, Kirk A.; Ten Haken, Randall K.; Lawrence, Theodore S.; Cao, Yue

    2013-01-01

    Purpose High dose radiation therapy (RT) for intrahepatic cancer is limited by the development of liver injury. This study investigated whether regional hepatic function assessed prior to and during the course of RT using 99mTc-labeled immindodiacetic acid (IDA) SPECT could predict regional liver function reserve after RT. Methods and Materials Fourteen patients treated with RT for intrahepatic cancers underwent dynamic 99mTc-IDA SPECT scans prior to RT, during, and one month after completion of RT. Indocyanine green (ICG) tests (a measure of overall liver function) were performed within 1 day of each scan. 3D volumetric hepatic extraction fraction (HEF) images of the liver were estimated by deconvolution analysis. After co-registration of the CT/SPECT and the treatment planning CT, HEF dose-response functions during and post-RT were generated. The volumetric mean of the HEFs in the whole liver was correlated with ICG clearance time. Three models, Dose, Priori and Adaptive models, were developed using multivariate linear regression to assess whether the regional HEFs measured before and during RT helped predict regional hepatic function post-RT. Results The mean of the volumetric liver HEFs was significantly correlated with ICG clearance half-life time (r = ?0.80, p<0.0001), for all time points. Linear correlations between local doses and regional HEFs one month post-RT were significant in 12 patients. In the priori model, regional HEF post-RT was predicted by the planned dose and regional HEF assessed prior to RT (R=0.71, p<0.0001). In the adaptive model, regional HEF post-RT was predicted by regional HEF re-assessed during RT and the remaining planned local dose (R=0.83, p<0.0001). Conclusions 99mTc-IDA SPECT obtained during RT could be used to assess regional hepatic function and helped predict post-RT regional liver function reserve. This could support individualized adaptive radiation treatment strategies to maximize tumor control and minimize the risk of liver damage. PMID:23688813

  7. Applications of NASA and NOAA Satellite Observations by NASA's Short-term Prediction Research and Transition (SPoRT) Center in Response to Natural Disasters

    NASA Technical Reports Server (NTRS)

    Molthan, Andrew L.; Burks, Jason E.; McGrath, Kevin M.; Jedlovec, Gary J.

    2012-01-01

    NASA s Short-term Prediction Research and Transition (SPoRT) Center supports the transition of unique NASA and NOAA research activities to the operational weather forecasting community. SPoRT emphasizes real-time analysis and prediction out to 48 hours. SPoRT partners with NOAA s National Weather Service (NWS) Weather Forecast Offices (WFOs) and National Centers to improve current products, demonstrate future satellite capabilities and explore new data assimilation techniques. Recently, the SPoRT Center has been involved in several activities related to disaster response, in collaboration with NOAA s National Weather Service, NASA s Applied Sciences Disasters Program, and other partners.

  8. The impact of flattening-filter-free beam technology on 3D conformal RT

    PubMed Central

    2013-01-01

    Background The removal of the flattening filter (FF) leads to non-uniform fluence distribution with a considerable increase in dose rate. It is possible to adapt FFF beams (flattening-filter-free) in 3D conformal radiation therapy (3D CRT) by using field in field techniques (FiF). The aim of this retrospective study is to clarify whether the quality of 3D CRT plans is influenced by the use of FFF beams. Method This study includes a total of 52 CT studies of RT locations that occur frequently in clinical practice. Dose volume targets were provided for the PTV of breast (n=13), neurocranium (n=11), lung (n=7), bone metastasis (n=10) and prostate (n=11) in line with ICRU report 50/62. 3D CRT planning was carried out using FiF methods. Two clinically utilized photon energies are used for a Siemens ARTISTE linear accelerator in FFF mode at 7MVFFF and 11MVFFF as well as in FF mode at 6MVFF and 10MVFF. The plan quality in relation to the PTV coverage, OAR (organs at risk) and low dose burden as well as the 2D dosimetric verification is compared with FF plans. Results No significant differences were found between FFF and FF plans in the mean dose for the PTV of breast, lung, spine metastasis and prostate. The low dose parameters V5Gy and V10Gy display significant differences for FFF and FF plans in some subgroups. The DVH analysis of the OAR revealed some significant differences. Significantly more fields (1.9 – 4.5) were necessary in the use of FFF beams for each location (p<0.0001) in order to achieve PTV coverage. All the tested groups displayed significant increases (1.3 – 2.2 times) in the average number of necessary MU with the use of FFF beams (p<0.001). Conclusions This study has shown that the exclusive use of a linear accelerator in FFF mode is feasible in 3D CRT. It was possible to realize RT plans in comparable quality in typical cases of clinical radiotherapy. The 2D dosimetric validation of the modulated fields verified the dose calculation and thus the correct reproduction of the characteristic FFF parameters in the planning system that was used. PMID:23725479

  9. Implementation and commissioning of an integrated micro-CT/RT system with computerized independent jaw collimation

    SciTech Connect

    Jensen, Michael D.; Hrinivich, W. Thomas; Jung, Jongho A.; Holdsworth, David W.; Department of Medical Biophysics, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 3K7; Department of Surgery, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 3K7 ; Drangova, Maria; Chen, Jeff; Department of Medical Biophysics, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 3K7; Department of Oncology, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 3K7 ; Wong, Eugene; Department of Medical Biophysics, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 3K7; Department of Oncology, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 3K7; Department of Physics and Engineering, London Regional Cancer Program, London Health Sciences Centre, 800 Commissioners Road East, London, Ontario N6A 5W9

    2013-08-15

    Purpose: To design, construct, and commission a set of computer-controlled motorized jaws for a micro-CT/RT system to perform conformal image-guided small animal radiotherapy.Methods: The authors designed and evaluated a system of custom-built motorized orthogonal jaws, which allows the delivery of off-axis rectangular fields on a GE eXplore CT 120 preclinical imaging system. The jaws in the x direction are independently driven, while the y-direction jaws are symmetric. All motors have backup encoders, verifying jaw positions. Mechanical performance of the jaws was characterized. Square beam profiles ranging from 2 × 2 to 60 × 60 mm{sup 2} were measured using EBT2 film in the center of a 70 × 70 × 22 mm{sup 3} solid water block. Similarly, absolute depth dose was measured in a solid water and EBT2 film stack 50 × 50 × 50 mm{sup 3}. A calibrated Farmer ion chamber in a 70 × 70 × 20 mm{sup 3} solid water block was used to measure the output of three field sizes: 50 × 50, 40 × 40, and 30 × 30 mm{sup 2}. Elliptical target plans were delivered to films to assess overall system performance. Respiratory-gated treatment was implemented on the system and initially proved using a simple sinusoidal motion phantom. All films were scanned on a flatbed scanner (Epson 1000XL) and converted to dose using a fitted calibration curve. A Monte Carlo beam model of the micro-CT with the jaws has been created using BEAMnrc for comparison with the measurements. An example image-guided partial lung irradiation in a rat is demonstrated.Results: The averaged random error of positioning each jaw is less than 0.1 mm. Relative output factors measured with the ion chamber agree with Monte Carlo simulations within 2%. Beam profiles and absolute depth dose curves measured from the films agree with simulations within measurement uncertainty. Respiratory-gated treatments applied to a phantom moving with a peak-to-peak amplitude of 5 mm showed improved beam penumbra (80%–20%) from 3.9 to 0.8 mm.Conclusions: A set of computer-controlled motorized jaws for a micro-CT/RT system were constructed with position reliably better than a tenth of a millimeter. The hardware system is ready for image-guided conformal radiotherapy for small animals with capability of respiratory-gated delivery.

  10. Removal of real-time reverse transcription polymerase chain reaction (RT-PCR) inhibitors associated with cloacal swab samples and tissues for improved diagnosis of Avian influenza virus by RT-PCR.

    PubMed

    Das, Amaresh; Spackman, Erica; Pantin-Jackwood, Mary J; Suarez, David L

    2009-11-01

    Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) is routinely used for the rapid detection of Avian influenza virus (AIV) in clinical samples, but inhibitory substances present in some clinical specimens can reduce or block PCR amplification. Most commercial RNA extraction kits have limited capacity to remove inhibitors from clinical samples, but using a modified commercial protocol (Ambion MagMAX, Applied Biosystems, Foster City, CA) with an added high-salt wash of 2 M NaCl and 2 mM ethylenediamine tetra-acetic acid was shown to improve the ability of the kit to remove inhibitors from cloacal swabs and some tissues. Real-time RT-PCR was carried out in the presence of an internal positive control to detect inhibitors present in the purified RNA. Cloacal swabs from wild birds were analyzed by real-time RT-PCR comparing RNA extracted with the standard (MagMAX-S) and modified (MagMAX-M) protocols. Using the standard protocol on 2,668 samples, 18.4% of the samples had evidence of inhibitor(s) in the samples, but the modified protocol removed inhibitors from all but 21 (4.8%) of the problem samples. The modified protocol was also tested for RNA extraction from tissues using a TRIzol-MagMAX-M hybrid protocol. Tissues from chickens and ducks experimentally infected with high-pathogenicity Asian H5N1 AIV were analyzed by real-time RT-PCR, and the limit of detection of the virus was improved by 0.5-3.0 threshold cycle units with the RNA extracted by the MagMAX-M protocol. The MagMAX-M protocol reported in the present study can be useful in extracting high-quality RNA for accurate detection of AIV from cloacal swabs and tissues by real-time RT-PCR. PMID:19901277

  11. Absolute mRNA quantification of Pseudomonas fluorescens Pf-5 by qRT-PCR using universal RNA controls

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real time quantitative RT-PCR is considered as standard for gene expression and mRNA estimate. As a calibration standard, a conserved control gene such as housekeeping genes is commonly used for data normalization and analysis. A significant problem has been observed with increased applications; h...

  12. Smithsonian Institution 2008 AnnuAl RePoRT CHARTinG CouRSe From the Secretary

    E-print Network

    Miller, Scott

    Smithsonian Institution 2008 AnnuAl RePoRT CHARTinG CouRSe » #12;From the Secretary It is my great pleasure to introduce the Smithsonian's achievements in 2008, the year that I became the 12th Secretary of the Smithsonian. I am privileged to arrive at the Institution at a moment of significant opportunity, one in which

  13. Using a Response to Intervention (RtI) Framework with 1st Grade Students: A Model for Occupational Therapy Practitioners

    ERIC Educational Resources Information Center

    McGuire, Beatriz

    2012-01-01

    The most recent reauthorization of the Individual's with Disabilities Education Improvement Act (IDEA, 2004) allows for the expansion of occupational therapy's role in school-based practice to include students who have not been identified for special education through early intervening services such as response to intervention (RtI).…

  14. Methods for detection and differentiation of existing and new crinivirus species through multiplex and degenerate primer RT-PCR.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A method was developed for rapid identification and differentiation of both known and novel crinivirus species involving both multiplex and degenerate reverse transcription-polymerase chain reaction (RT-PCR). The multiplex method can discriminate among known criniviruses infecting vegetable and sma...

  15. Supporting Valid Decision Making: Uses and Misuses of Assessment Data within the Context of RtI

    ERIC Educational Resources Information Center

    Ball, Carrie R.; Christ, Theodore J.

    2012-01-01

    Within an RtI problem-solving context, assessment and decision making generally center around the tasks of problem identification, problem analysis, progress monitoring, and program evaluation. We use this framework to discuss the current state of the literature regarding curriculum based measurement, its technical properties, and its utility for…

  16. Response to Intervention (RtI) in the Social, Emotional, and Behavioral Domains: Current Challenges and Emerging Possibilities

    ERIC Educational Resources Information Center

    Saeki, Elina; Jimerson, Shane R.; Earhart, James; Hart, Shelley R.; Renshaw, Tyler; Singh, Renee D.; Stewart, Kaitlyn

    2011-01-01

    As many schools move toward a three-tier model that incorporates a Response to Intervention (RtI) service delivery model in the social, emotional, and behavioral domains, school psychologists may provide leadership. The decision-making process for filtering students through multiple tiers of support and intervention and examining change is an area…

  17. Application of qRT-PCR and RNA-Seq analysis for the identification of housekeeping genes useful

    E-print Network

    Yoder, John I.

    Application of qRT-PCR and RNA-Seq analysis for the identification of housekeeping genes useful genes that have stable expression across tissues and life stages. Since no S. hermonthica housekeeping genes have been established for this purpose, we evaluated the suitability of six candidate housekeeping

  18. USING PORTABLE REAL-TIME POLYMERASE CHAIN REACTION (RT-PCR) ASSAYS TO DETECT SALMONELLA IN RAW MILK

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The purpose of this study was to determine the efficacy of a portable RT-PCR system for detecting Salmonella spp. in raw milk. The bulk milk samples (200) chosen for this study were a subset of a larger study; the subset contained 24 samples that were culture-positive for Salmonella and 176 that we...

  19. Evaluation of Reference Genes for RT-qPCR Studies in the Seagrass Zostera muelleri Exposed to Light Limitation.

    PubMed

    Schliep, M; Pernice, M; Sinutok, S; Bryant, C V; York, P H; Rasheed, M A; Ralph, P J

    2015-01-01

    Seagrass meadows are threatened by coastal development and global change. In the face of these pressures, molecular techniques such as reverse transcription quantitative real-time PCR (RT-qPCR) have great potential to improve management of these ecosystems by allowing early detection of chronic stress. In RT-qPCR, the expression levels of target genes are estimated on the basis of reference genes, in order to control for RNA variations. Although determination of suitable reference genes is critical for RT-qPCR studies, reports on the evaluation of reference genes are still absent for the major Australian species Zostera muelleri subsp. capricorni (Z. muelleri). Here, we used three different software (geNorm, NormFinder and Bestkeeper) to evaluate ten widely used reference genes according to their expression stability in Z. muelleri exposed to light limitation. We then combined results from different software and used a consensus rank of four best reference genes to validate regulation in Photosystem I reaction center subunit IV B and Heat Stress Transcription factor A- gene expression in Z. muelleri under light limitation. This study provides the first comprehensive list of reference genes in Z. muelleri and demonstrates RT-qPCR as an effective tool to identify early responses to light limitation in seagrass. PMID:26592440

  20. The Use of Collagenase to Improve the Detection of Plant Viruses in Vector Nematodes by RT/PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tomato ringspot virus (ToRSV) Tobacco ringspot virus (TRSV) and Tobacco rattle virus (TRV) are transmitted to healthy plants by viruliferous nematodes in the soil. We developed a method for extraction of genomic viral RNA from virus particles carried within nematodes and a sensitive nested RT/PCR ...

  1. arXiv:math.RT/0702855v128Feb2007 IMAGINARY HIGHEST-WEIGHT REPRESENTATION THEORY AND

    E-print Network

    Sydney, University of

    }. Equivalent partitions define similar highest-weight theories. All partitions of the root system of a finitearXiv:math.RT/0702855v128Feb2007 IMAGINARY HIGHEST-WEIGHT REPRESENTATION THEORY AND SYMMETRIC FUNCTIONS BENJAMIN J. WILSON Abstract. Affine Lie algebras admit non-classical highest-weight theories

  2. Characterization of cytokine expression induced by avian influenza virus infection with real-time RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Knowledge of how birds react to infection from avian influenza virus is critical to understanding disease pathogenesis and host response. The use of real-time (R), reverse-transcriptase (RT), PCR to measure innate immunity, including cytokine and interferon gene expression, has become a standard tec...

  3. A collaborative framework for contributing DICOM RT PHI (Protected Health Information) to augment data mining in clinical decision support

    NASA Astrophysics Data System (ADS)

    Deshpande, Ruchi; Thuptimdang, Wanwara; DeMarco, John; Liu, Brent J.

    2014-03-01

    We have built a decision support system that provides recommendations for customizing radiation therapy treatment plans, based on patient models generated from a database of retrospective planning data. This database consists of relevant metadata and information derived from the following DICOM objects - CT images, RT Structure Set, RT Dose and RT Plan. The usefulness and accuracy of such patient models partly depends on the sample size of the learning data set. Our current goal is to increase this sample size by expanding our decision support system into a collaborative framework to include contributions from multiple collaborators. Potential collaborators are often reluctant to upload even anonymized patient files to repositories outside their local organizational network in order to avoid any conflicts with HIPAA Privacy and Security Rules. We have circumvented this problem by developing a tool that can parse DICOM files on the client's side and extract de-identified numeric and text data from DICOM RT headers for uploading to a centralized system. As a result, the DICOM files containing PHI remain local to the client side. This is a novel workflow that results in adding only relevant yet valuable data from DICOM files to the centralized decision support knowledge base in such a way that the DICOM files never leave the contributor's local workstation in a cloud-based environment. Such a workflow serves to encourage clinicians to contribute data for research endeavors by ensuring protection of electronic patient data.

  4. Sequence analysis of proviral HIV RT amplified directly by a semi-quantitative technique from AZT treated patients.

    PubMed

    Stein, C A; Levantis, P; Oxford, J S

    1994-10-01

    To minimise possible arbitrary selective effects of culturing HIV, proviral RT DNA was isolated directly from PBMCs of four patients treated for 6-14 months with AZT. RT DNA was amplified by PCR and sequenced directly without further in vitro manipulation. Eighteen changes additional to those 4 or 5 changes previously shown by genetic reconstruction experiments [Kellam et al.: Proceedings of the National Academy of Sciences of the United States of America 89:1934-1938, 1992] were found in the 14 different sequences analysed. Substitutions clustered in two defined areas of the RT, from amino acids 60 to 70 and from 180 to 220. Mutations were observed at each of the two areas independently or at both sites simultaneously. Amino acid changes in RT from patients harbouring resistant strains of HIV-1 were found in positions 60 (Val), 62 (Ala), 93 (Gly), 100 (Phe), 161 (Pro), 173 (Asn), 177 (Glu), 180 (Ile), 181 (Tyr), 182 (Leu), 186 (Asp), 194 (Gln), 196 (Glu), 200 (Ile), 209 (Val), 210 (Trp), 211 (Lys), and 214 (Phe) in addition to those described previously. It was anticipated that multiple proviral DNAs would be present in a single clinical sample. Therefore end point dilution PCR methodology was used, which allowed sequence analysis of separate proviral DNA molecules from the patients' proviral DNA. Even in patients who had received AZT for more than 10 months wild-type "AZT-sensitive" RTs co-existed with mutated "AZT-resistant" RTs in the same patient sample. PMID:7531752

  5. A Robust Plant RNA Isolation Method for Affymetrix Genechip® Analysis and Quantitative Real-Time RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microarray analysis and quantitative real-time RT-PCR are the major high-throughput techniques that are used to study transcript profiles. One of the major limitations in these technologies is the isolation maximum yield of highly-pure RNA from plant tissues rich in complex polysaccharides, polyphen...

  6. A faster method to detect norovirus in oysters using probe hybridization to isolate target RNA before RT-PCR.

    PubMed

    Ye, Xunyan; Ellender, Rudolph D; Wang, Shiao Y

    2013-04-01

    Human Noroviruses (HuNoVs) are the most frequent cause of outbreaks of acute gastroenteritis following the ingestion of raw or improperly cooked oysters. Although highly sensitive methods to detect HuNoV in oysters using reverse transcriptase-polymerase chain reaction (RT-PCR) are available, rapid methods to process samples for RT-PCR are still needed. The conventional approach is to concentrate the virus first before RNA purification to maximize assay sensitivity, but the procedures used are cumbersome. We developed a new hybridization method that is much faster and more effective compared to existing technology. The procedure includes an initial extraction of total RNA from the digestive diverticula of oysters using TRI Reagent, followed by HuNoV RNA purification using a capture probe and then HuNoV detection by real-time RT-PCR. The detection limit is approximately 100 PCR detection units of HuNoV per sample. Compared to published methods that require an initial virus concentration step before RNA extraction, the new method is much faster to complete. Approximately 3?h are needed to purify HuNoV RNA using the new method compared to at least 8?h using conventional methods. Coupled with real-time RT-PCR, the new method can detect HuNoV in contaminated oysters within 8?h. The effectiveness of the method was demonstrated using live artificially contaminated oysters and wild oysters. PMID:23510496

  7. Nature GeNetics VOLUME 47 | NUMBER 7 | JULY 2015 717 A rt i c l e s

    E-print Network

    Kaski, Samuel

    Nature GeNetics VOLUME 47 | NUMBER 7 | JULY 2015 717 A rt i c l e s The mainstream application influencing success of clinical genome sequencing across a broad spectrum of disorders Jenny C Taylor1, Katherine Bull2,10, Ondrej Cais11, Holger Cario12, Helen Chapel13, Richard R Copley1,2, Richard Cornall10

  8. A rt i c l e s 566 VOLUME 17 | NUMBER 5 | MAY 2011 nAture medicine

    E-print Network

    Jeong, Jaeseung

    A rt i c l e s 566 VOLUME 17 | NUMBER 5 | MAY 2011 nAture medicine ADHD is a common psychiatric in ADHD has gained considerable attention2. However, many ADHD susceptibility loci identified by genome with the phenotypic heterogeneity and strong heritability of ADHD, predicts the existence of diverse ADHD

  9. Evaluation of Reference Genes for RT-qPCR Studies in the Seagrass Zostera muelleri Exposed to Light Limitation

    PubMed Central

    Schliep, M.; Pernice, M.; Sinutok, S.; Bryant, C. V.; York, P. H.; Rasheed, M. A.; Ralph, P. J.

    2015-01-01

    Seagrass meadows are threatened by coastal development and global change. In the face of these pressures, molecular techniques such as reverse transcription quantitative real-time PCR (RT-qPCR) have great potential to improve management of these ecosystems by allowing early detection of chronic stress. In RT-qPCR, the expression levels of target genes are estimated on the basis of reference genes, in order to control for RNA variations. Although determination of suitable reference genes is critical for RT-qPCR studies, reports on the evaluation of reference genes are still absent for the major Australian species Zostera muelleri subsp. capricorni (Z. muelleri). Here, we used three different software (geNorm, NormFinder and Bestkeeper) to evaluate ten widely used reference genes according to their expression stability in Z. muelleri exposed to light limitation. We then combined results from different software and used a consensus rank of four best reference genes to validate regulation in Photosystem I reaction center subunit IV B and Heat Stress Transcription factor A- gene expression in Z. muelleri under light limitation. This study provides the first comprehensive list of reference genes in Z. muelleri and demonstrates RT-qPCR as an effective tool to identify early responses to light limitation in seagrass. PMID:26592440

  10. THE EFFECT OF VARIOUS DISINFECTANTS ON DETECTION OF AVIAN INFLUENZA VIRUS BY REAL TIME RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An avian influenza real time RT-PCR (RRT-PCR) test was previously shown to be a rapid and sensitive method to identify avian influenza virus-infected birds in live bird markets (LBMs). The test can also be used to identify avian influenza virus (AIV) from environmental samples. Consequently, the u...

  11. Standardized RT-PCR conditions for detection and identification of eleven viruses of potato and Potato spindle tuber viroid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Standardized RT-PCR procedures were developed and validated for detection of Alfalfa mosaic virus (AMV), Impatiens necrotic spot virus (INSV), Tobacco rattle virus (TRV), Tomato spotted wilt virus (TSWV), Potato leaf roll virus (PLRV), Potato mop top virus (PMTV), Potato virus A (PVA), Potato viru...

  12. Evaluation of an updated real-time RT-PCR test for the identification of the H7 subtype

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rapid detection of avian influenza (AI) virus and identification of the H5 and H7 subtypes is critical for wild bird monitoring programs. A real-time RT-PCR test for identification of the H7 subtype in North America was first reported in 2002. With the recent surveillance in wild birds it was disc...

  13. Nature GeNetics volume 42 | number 2 | february 2010 105 A rt i c l e s

    E-print Network

    Abecasis, Goncalo

    of continuous gly- cemic phenotypes in nondiabetic individuals can complement the genetic analyses of diabetesNature GeNetics volume 42 | number 2 | february 2010 105 A rt i c l e s Impaired beta-cell function and insulin resistance are key determinants of type 2 diabetes (T2D). Hyperglycemia in the fasting state

  14. Simultaneous detection of Cymbidium mosaic virus and Odontoglossum ringspot virus in orchids using multiplex RT-PCR.

    PubMed

    Kim, Su Min; Choi, Sun Hee

    2015-12-01

    A system for simultaneous detection of two orchid-infecting viruses was developed and applied to several orchid species. The detection system involved multiplex reverse transcription-polymerase chain reaction (RT-PCR) and could simultaneously identify Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) from the orchid species studied. Multiplex RT-PCR was conducted using two virus-specific primer pairs and an internal control pair of primers to amplify the CymMV and ORSV coat protein regions, and orchid 18S rDNA, respectively. For optimization of multiplex RT-PCR conditions, serial dilutions of total RNA and cDNA were performed and the detection limit of the system was evaluated. The optimized multiplex detection system for CymMV and ORSV was applied to various orchid species, including several cultivars of Doritaenopsis, Cymbidium, Dendrobium, and Phalaenopsis to test the efficacy of this method. Our results indicate that the multiplex RT-PCR detection system will be a rapid, simple, and precise diagnosis tool in a range of orchid species. PMID:26542829

  15. 656 VOLUME 45 | NUMBER 6 | JUNE 2013 Nature GeNetics A rt i c l e s

    E-print Network

    Kaski, Samuel

    656 VOLUME 45 | NUMBER 6 | JUNE 2013 Nature GeNetics A rt i c l e s S. pneumoniae is a human nasopharyngeal commensal and respiratory pathogen that represents a major cause of pneumonia, bacteremia and meningitis. The bacterium's best-characterized virulence factor is its polysaccharide capsule, of which

  16. Antibody-mediated protection against mucosal simian-human immunodeficiency virus challenge of macaques immunized with alphavirus replicon particles and boosted with trimeric envelope glycoprotein in MF59 adjuvant.

    PubMed

    Barnett, Susan W; Burke, Brian; Sun, Yide; Kan, Elaine; Legg, Harold; Lian, Ying; Bost, Kristen; Zhou, Fengmin; Goodsell, Amanda; Zur Megede, Jan; Polo, John; Donnelly, John; Ulmer, Jeffrey; Otten, Gillis R; Miller, Christopher J; Vajdy, Michael; Srivastava, Indresh K

    2010-06-01

    We have previously shown that rhesus macaques were partially protected against high-dose intravenous challenge with simian-human immunodeficiency virus SHIV(SF162P4) following sequential immunization with alphavirus replicon particles (VRP) of a chimeric recombinant VEE/SIN alphavirus (derived from Venezuelan equine encephalitis virus [VEE] and the Sindbis virus [SIN]) encoding human immunodeficiency virus type 1 HIV-1(SF162) gp140DeltaV2 envelope (Env) and trimeric Env protein in MF59 adjuvant (R. Xu, I. K. Srivastava, C. E. Greer, I. Zarkikh, Z. Kraft, L. Kuller, J. M. Polo, S. W. Barnett, and L. Stamatatos, AIDS Res. Hum. Retroviruses 22:1022-1030, 2006). The protection did not require T-cell immune responses directed toward simian immunodeficiency virus (SIV) Gag. We extend those findings here to demonstrate antibody-mediated protection against mucosal challenge in macaques using prime-boost regimens incorporating both intramuscular and mucosal routes of delivery. The macaques in the vaccination groups were primed with VRP and then boosted with Env protein in MF59 adjuvant, or they were given VRP intramuscular immunizations alone and then challenged with SHIV(SF162P4) (intrarectal challenge). The results demonstrated that these vaccines were able to effectively protect the macaques to different degrees against subsequent mucosal SHIV challenge, but most noteworthy, all macaques that received the intramuscular VRP prime plus Env protein boost were completely protected. A statistically significant association was observed between the titer of virus neutralizing and binding antibodies as well as the avidity of anti-Env antibodies measured prechallenge and protection from infection. These results highlight the merit of the alphavirus replicon vector prime plus Env protein boost vaccine approach for the induction of protective antibody responses and are of particular relevance to advancing our understanding of the potential correlates of immune protection against HIV infection at a relevant mucosal portal of entry. PMID:20392857

  17. Q-RT-PCR for assessing archaea, bacteria, and fungi during leaf decomposition in a stream.

    PubMed

    Manerkar, Mayura A; Seena, S; Bärlocher, Felix

    2008-10-01

    Leaf disks of Tilia cordata were exposed for up to 5 weeks in a first-order stream in Nova Scotia, Canada. The exponential decay rate k was 0.008 day(-1). Ergosterol levels increased linearly to a maximum of 134 microg g(-1) dry leaf mass. Release of conidia peaked at 700 day(-1) mg(-1) on leaves that had been exposed for 3 weeks; after 5 weeks, it declined to 15 mg(-1). In total, 23 taxa of aquatic hyphomycetes were distinguished. Anguillospora filiformis contributed over 76% of the conidia during weeks 1, 2, and 3, and 16.5% in week 5. Three sets of primers specific for Bacteria, Archaea, and Fungi were applied in quantitative real-time polymerase chain reaction (Q-RT-PCR) to estimate relative DNA amounts. Archaeal DNA was consistently present at low levels. Bacterial and fungal DNA peaked between weeks 2 and 3, and declined in week 5. With the exception of week 1, fungal DNA exceeded bacterial DNA by between 12 and 110%. PMID:18264658

  18. Genotyping of GII.4 and GIIb norovirus RT-PCR amplicons by RFLP analysis.

    PubMed

    Ramirez, Stefania; Giammanco, Giovanni M; De Grazia, Simona; Colomba, Claudia; Martella, Vito; Arista, Serenella

    2008-02-01

    GII.4 and GIIb/Hilversum norovirus (NoV) strains appear to have a prominent epidemiological role in outbreaks or sporadic cases of human gastroenteritis. Sequence analysis, although laborious, is the reference method used for characterization of noroviruses. In this study a screening test is proposed to characterize GIIb and GII.4 NoVs based on restriction fragment length polymorphism (RFLP) analysis of amplicons obtained from the RNA-dependent RNA polymerase (RdRp) region. Virtual analysis of 793 RdRp sequences of GGI and GGII NoVs, retrieved from GenBank, and representative of global geographical origins on a long-time period, permitted the selection of four restriction enzymes, XmnI, AhdI, BstXI, and AcuI, suitable for correct identification of GIIb and GII.4 NoV genotypes. Experimental analysis by the RT-PCR RFLP analysis of 41 NoV strains detected in Palermo during the years 2002-2005 allowed to recognize all the Italian strains as belonging to GIIb/Hilversum or GII.4, and sequence analysis confirmed these results. The PCR-RFLP protocol developed in this study proved to be a simple and reliable proxy for sequence-based classification of the GIIb/Hilversum and GII.4 NoV variants displaying high specificity (100%) and sensitivity (94%). PMID:17953996

  19. Microhard MHX 2420 Orbital Performance Evaluation Using RT Logic T400CS

    NASA Technical Reports Server (NTRS)

    Kearney, Stuart; Lombardi, Mark; Attai, Watson; Oyadomari, Ken; Al Rumhi, Ahmed Saleh Nasser; Rakotonarivo, Sebastien; Chardon, Loic; Gazulla, Oriol Tintore; Wolfe, Jasper; Salas, AlbertoGuillen; DeWald, Jon; Alena, Richard

    2012-01-01

    A major upfront cost of building low cost Nanosatellites is the communications sub-system. Most radios built for space missions cost over $4,000 per unit. This exceeds many budgets. One possible cost effective solution is the Microhard MHX2420, a commercial off-the-shelf transceiver with a unit cost under $1000. This paper aims to support the Nanosatellite community seeking an inexpensive radio by characterizing Microhard's performance envelope. Though not intended for space operations, the ability to test edge cases and increase average data transfer speeds through optimization positions this radio as a solution for Nanosatellite communications by expanding usage to include more missions. The second objective of this paper is to test and verify the optimal radio settings for the most common cases to improve downlinking. All tests were conducted with the aid of the RT Logic T400CS, a hardware-in-the-loop channel simulator designed to emulate real-world radio frequency (RF) link effects. This study provides recommended settings to optimize the downlink speed as well as the environmental parameters that cause the link to fail.

  20. Detection of human enteric viruses in stream water with RT-PCR and cell culture.

    USGS Publications Warehouse

    Denis-Mize, K.; Fout, G.S.; Dahling, D.R.; Francy, D.S.

    2004-01-01

    A multiplex RT-PCR method was used to measure virus occurrence at five stream water sites that span a range of hydroclimatic, water-quality, and land-use characteristics. The performance of the molecular method was evaluated in comparison with traditional cell culture and Escherichia coli membrane filtration assays. The study incorporated multiple quality controls and included a control for virus recovery during the sampling procedure as well as controls to detect potentially false-negative and false-positive data. Poliovirus recovery ranged from 16 to 65% and was variable, even in samples collected within the same stream. All five sites were positive for viruses by both molecular and cell culture-based virus assays. Enteroviruses, reoviruses, rotaviruses, and hepatitis A viruses were detected, but the use of the quality controls proved critical for interpretation of the molecular data. All sites showed evidence of faecal contamination, and culturable viruses were detected in four samples that would have met the US Environmental Protection Agency's recommended E. coli guideline for safe recreational water.

  1. Real Time RT-PCR Assays for Detection and Typing of African Horse Sickness Virus

    PubMed Central

    Bachanek-Bankowska, Katarzyna; Maan, Sushila; Castillo-Olivares, Javier; Manning, Nicola M.; Maan, Narender Singh; Potgieter, Abraham C.; Di Nardo, Antonello; Sutton, Geoff; Batten, Carrie; Mertens, Peter P. C.

    2014-01-01

    Although African horse sickness (AHS) can cause up to 95% mortality in horses, naïve animals can be protected by vaccination against the homologous AHSV serotype. Genome segment 2 (Seg-2) encodes outer capsid protein VP2, the most variable of the AHSV proteins. VP2 is also a primary target for AHSV specific neutralising antibodies, and consequently determines the identity of the nine AHSV serotypes. In contrast VP1 (the viral polymerase) and VP3 (the sub-core shell protein), encoded by Seg-1 and Seg-3 respectively, are highly conserved, representing virus species/orbivirus-serogroup-specific antigens. We report development and evaluation of real-time RT-PCR assays targeting AHSV Seg-1 or Seg-3, that can detect any AHSV type (virus species/serogroup-specific assays), as well as type-specific assays targeting Seg-2 of the nine AHSV serotypes. These assays were evaluated using isolates of different AHSV serotypes and other closely related orbiviruses, from the ‘Orbivirus Reference Collection’ (ORC) at The Pirbright Institute. The assays were shown to be AHSV virus-species-specific, or type-specific (as designed) and can be used for rapid, sensitive and reliable detection and identification (typing) of AHSV RNA in infected blood, tissue samples, homogenised Culicoides, or tissue culture supernatant. None of the assays amplified cDNAs from closely related heterologous orbiviruses, or from uninfected host animals or cell cultures. PMID:24721971

  2. Stable Reference Gene Selection for RT-qPCR Analysis in Nonviruliferous and Viruliferous Frankliniella occidentalis

    PubMed Central

    Pan, Huipeng; Ma, Yabin; Zhang, Deyong; Liu, Yong; Zhang, Zhanhong; Zheng, Changying; Chu, Dong

    2015-01-01

    Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for measuring and evaluating gene expression during variable biological processes. To facilitate gene expression studies, normalization of genes of interest relative to stable reference genes is crucial. The western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), the main vector of tomato spotted wilt virus (TSWV), is a destructive invasive species. In this study, the expression profiles of 11 candidate reference genes from nonviruliferous and viruliferous F. occidentalis were investigated. Five distinct algorithms, geNorm, NormFinder, BestKeeper, the ?Ct method, and RefFinder, were used to determine the performance of these genes. geNorm, NormFinder, BestKeeper, and RefFinder identified heat shock protein 70 (HSP70), heat shock protein 60 (HSP60), elongation factor 1 ?, and ribosomal protein l32 (RPL32) as the most stable reference genes, and the ?Ct method identified HSP60, HSP70, RPL32, and heat shock protein 90 as the most stable reference genes. Additionally, two reference genes were sufficient for reliable normalization in nonviruliferous and viruliferous F. occidentalis. This work provides a foundation for investigating the molecular mechanisms of TSWV and F. occidentalis interactions. PMID:26244556

  3. RT-PCR analysis of dystrophin mRNA in DND/BMD patients

    SciTech Connect

    Ciafaloni, E.; Silva, H.A.R. de; Roses, A.D.

    1994-09-01

    Duchenne and Becker muscular dystrophies (DMD, BMD) are X-linked recessive disorders caused by mutations in the dystrophin (dys) gene. The majority of these mutations are intragenic deletions of duplications routinely detected by Southern biots and multiplex PCR. The remainder are very likely, smaller mutations, mostly point-mutations. Detection of these mutations is very difficult due to the size and complexity of the dys gene. We applied RT-PCR to analyse the entire dys mRNA of three DMD patients with no detectable genomic defect. In two unrelated patients, a duplication of the 62 bp exon 2 was identified. This causes a frameshift sufficient to explain the DMD phenotype. In the third patient, who had congenital DMD and severe mental retardation, a complex pattern of aberrant splicing at the 3-prime exons 67-79 was observed. Sural nerve biopsy in this patient showed the complete absence of Dp116. PCR-SSCP studies are presently in progress to identify the mutations responsible for the aberrant splicing patterns.

  4. A multiplex RT-PCR assay for the detection of fish picornaviruses.

    PubMed

    Mor, Sunil K; Phelps, Nicholas B D; Barbknecht, Marisa; Hoffman, Michael A; Goyal, Sagar M

    2015-09-01

    With the emergence of high profile fish diseases in the Great Lakes region, surveillance and regulatory inspections of fish populations have increased. This has resulted in a better understanding of known pathogens and isolation of many new pathogens of fish. In this study, a multiplex RT-PCR assay was developed for the detection of three newly discovered fish picornaviruses: bluegill picornavirus-1 (BGPV-1), fathead minnow picornavirus (FHMPV), and eel picornavirus-1 (EPV-1). This assay was found to be very sensitive with a detection limit of 81.9pg/?l of extracted RNA from a pool of FHMPV and BGPV-1 and was able to detect 501 and 224 gene copies/?l of BGPV-1 and FHMPV, respectively. The assay was highly reproducible and did not cross react with other closely related pathogens. We believe that this new assay provides a rapid and cost effective tool for confirming cell culture isolates and conducting prevalence studies of these newly detected fish picornaviruses. PMID:25962537

  5. Detection of Fasciola hepatica in infected intermediate hosts using RT-PCR.

    PubMed

    Rognlie, M C; Dimke, K L; Knapp, S E

    1994-10-01

    Fasciola hepatica, the common bile duct fluke, is an economically important parasite of domestic livestock. Current research interest is directed toward an understanding of the parasite's biology at the intermediate host level. To permit study of seasonal transmission patterns and parasite/intermediate host interactions, a fasciolid-specific assay has been developed to detect infected snail vectors. This assay uses the reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify specifically a region of F. hepatica small-subunit rRNA, followed by hybridization to an F. hepatica-specific probe. The assay does not cross-react with 2 trematodes outside of the Fasciolidae but does detect Fascioloides magna rRNA. Sequence alignment with additional small-subunit rRNAs shows Fasciolopsis buski would also cross-react with the assay. The detection limit of the assay is 10 fg of fluke total RNA with 5 micrograms of snail RNA added as background. Additionally, the assay detects individual infected snails immediately after miracidial exposure and throughout the parasite's development period. PMID:7523650

  6. Survey and RT-PCR Based Detection of Cardamom mosaic virus Affecting Small Cardamom in India.

    PubMed

    Biju, C N; Siljo, A; Bhat, A I

    2010-10-01

    Mosaic or marble or katte disease caused by Cardamom mosaic virus (CdMV) is an important production constraint in all cardamom growing regions of the world. In the present study, 84 cardamom plantations in 44 locations of Karnataka and Kerala were surveyed. The incidence of the disease ranged from 0 to 85%. The incidence was highest in Madikeri (Karnataka) while no incidence was recorded in Peermade (Kerala). In general, incidence and severity of the disease was higher in cardamom plantations of Karnataka. A procedure for total RNA isolation from cardamom and detection of CdMV through reverse transcription-polymerase chain reaction (RT-PCR) using primers targeting the conserved region of coat protein was standardized and subsequently validated by testing more than 50 field cardamom samples originating from Karnataka and Kerala states. The method can be used for indexing the planting material and identifying resistant lines/cultivars before either they are further multiplied in large scale or incorporated in breeding. PMID:23637495

  7. Permeable Reactive Treatment (PeRT) Wall for Rads and Metals. Innovative Technology Summary Report

    SciTech Connect

    2000-09-01

    Organic and inorganic contamination of groundwater is widespread at Department of Energy (DOE), Department of Defense (DOD), other federal, and industrial sites. Contamination at a majority of these sites is present in shallow, unconfined aquifers, which may impact human health and the environment. Although there are many treatment methods, for organic contamination, relatively few technologies are effective in treating inorganic contamination, such as metals and radionuclide, in situ. Because metals are commonly adsorbed to clays and organic matter in an aquifer, groundwater pump and treat technology can be expensive and ineffective. Desorption of these metals into the aquifer is a long-term issue, difficult to address. A permeable reactive treatment (PeRT) wall, also referred to as a permeable reactive barrier, is a zone of reactive material that is placed in a contaminated aquifer so that the concentrations of dissolved inorganic contaminants are reduced as the groundwater passes through the material. The reactive material can be emplaced directly in the path of groundwater flow via trenching or injection or as a reactive liner in a landfill. This document contains information on the above-mentioned technology, including description, applicability, cost, and performance data.

  8. Saving resources: avian influenza surveillance using pooled swab samples and reduced reaction volumes in real-time RT-PCR.

    PubMed

    Fereidouni, Sasan R; Harder, Timm C; Gaidet, Nicolas; Ziller, Mario; Hoffmann, Bernd; Hammoumi, Saliha; Globig, Anja; Starick, Elke

    2012-12-01

    The occurrence of highly pathogenic (HP) avian influenza (AI) H5N1 in Asia and its spread to Africa and Europe prompted costly monitoring programs of wild birds and domestic poultry. AI virus excretion is tested by examining avian swab samples by real-time reverse transcription PCR (RT-qPCR). In this study, pools of swab samples and a reagents volume reduction per RT-qPCR were evaluated as measures of economization. Viral transport medium and faecal matrices were spiked with different low pathogenic AI virus strains and tested for loss of target RNA during all processing steps as individual rayon swabs or in sample pools of 5, 10 and 15 swabs. Fresh faeces from Mallard ducks and other aquatic bird species as sample matrix resulted in loss of AIV RNA of about 90% compared to transport medium. Due to sample RNA dilution in pools the likelihood of detection of single positive samples is decreasing with increasing size of sample pools. However, pools of five samples containing only one positive sample consistently gave positive results. Similarly, no differences in detection rates were obtained when analyzing 1030 wild bird swab samples either individually or in pools of five. Reducing the reaction volume of influenza A virus generic as well as of subtype-specific RT-qPCRs to 12.5 ?l (2.5 ?l template) instead of 25 ?l did not adversely affect the limit of detection of these RT-qPCRs. A significant economic benefit without impeding detection efficacy can be achieved when sample pools of five samples are analyzed by RT-qPCR using a reduction of the reaction mix to the half of the original volume. PMID:22925717

  9. Comparison of Plasmin with rt-PA in Lysis of Cerebral Thromboemboli Retrieved from Patients with Acute Ischemic Stroke

    PubMed Central

    Marder, Victor J.; Blinc, Ales; Gruber, Theresa; Tratar, Gregor; Sabovic, Miso; Starkman, Sidney; Jahan, Reza; Duckwiler, Gary; Vinuela, Fernando; Tateshima, Satoshi; Liebeskind, David; Ovbiagele, Bruce; Ali, Letisha; Kim, Doojin; Gonzalez, Nestor; Vespa, Paul M.; Saver, Jeffrey L.

    2011-01-01

    Background and purpose Plasmin is a direct-acting thrombolytic with a better safety profile than recombinant tissue-type plasminogen activator (rt-PA) in animal models. With the application of retrieval devices for managing acute ischemic stroke, extracted thromboemboli are available for ex vivo examination. We ask whether such thrombi are amenable to plasmin thrombolysis and whether such activity is different with rt-PA. Methods Thromboembolic fragments (total 29) were retrieved from the intracranial carotid artery system of 15 patients with acute ischemic stroke and randomly assigned to ex vivo thrombolysis with plasmin or rt-PA. After an initial 2-hour exposure, residual material was exposed to the other agent for an additional 2 hours. Thrombolysis was quantified by change in thrombus area and released D-dimer. Results Plasmin induced significant ex vivo thrombolysis of cerebral arterial thromboemboli, decreasing area by 45.9 +/? 29.4% and 69.2 +/? 52.5% and inducing median D-dimer release of 108,180 ?g/L (range 16,780 - 668,050 ?g/L) and 16,905 ?g/L (range 240 - 403,085 ?g/L) during the first and second 2-hour incubation periods, respectively. These changes were not different from those obtained with rt-PA, which decreased area by 34.7 +/? 57.8% (P=0.63) and by 68.4 +/? 26.9% (P=0.97), and induced median D-dimer release of 151,990 ?g/L (9,870 - 338,350 ?g/L) (P=0.51) and 34,520 ?g/L (range 3,794 - 325,400 ?g/L) (P=0.19), during the first and second 2-hour incubations. Conclusions Retrieved human cerebral thromboemboli were amenable to ex vivo lysis by plasmin, the rate and degree of which was not different than that achieved with rt-PA. PMID:21700944

  10. Receiving And Data Acquisition Systems Of Rt-32 For Vlbi Observations / Rt-32 Uztveršanas Un Datu Re?istr?cijas Sist?mas Vlbi Nov?rojumiem

    NASA Astrophysics Data System (ADS)

    Bezrukovs, Vl.; Shmeld, I.; Nechaeva, M.; Trokss, J.; Bezrukovs, D.; Klapers, M.; Berzins, A.; Lesins, A.; Dugin, N.

    2012-12-01

    Radiotelescope RT-32 is a fully steerable 32-m parabolic antenna located at Irbene and belonging to Ventspils International Radio Astronomy Centre (VIRAC). Currently, the work on upgrading and repair of its receiving hardware and data acquisition systems is of high priority for the VIRAC. One of the main scientific objectives for the VIRAC Radioastronomical observatory is VLBI (very long baseline interferometry) observations in centimetre wavelengths in collaboration with world VLBI networks, such as European VLBI network (EVN), Low Frequency VLBI network (LFVN), and others. During the last years the room in the secondary focus of telescope was reconstructed, and several new receivers were installed. Currently, RT-32 observations are carried out in four different bands: 92 cm, 18 cm, 6 cm, and 2.5 cm. First three of them are already successfully employed in diversified VLBI experiments. The receiver on 2.5 cm band has only one linear polarized chain and is used mainly for the methanol maser single dish observations. The apparatus system of RT-32 is equipped with two independent VLBI data acquisition systems: TN-16, and DBBC in combination with MK5b. Both systems are employed in interferometric observations depending on the purpose of experiment and the enabled radiotelescopes. The current status of RT-32, the availability of its receiving and data acquisition units for VLBI observations and the previous VLBI sessions are discussed. Radioteleskops RT-32 ir Ventspils Starptautiskajam Radioastronomijas Centram (VSRC) piederoša pilnas piedzi?as 32 m diametra parabolisk? antena. Pašreiz visaktu?l?kie VSRC veicamie darbi ir saist?ti ar RT-32 uztveroš?s aparat?ras un datu re?istr?šanas sist?mas labošanu un moderniz?ciju. Viens no radioastronomisk?s observatorijas galvenajiem zin?tniskajiem uzdevumiem ir seviš?i lielas b?zes interferometriskie (VLBI) nov?rojumi centimetru vi??u garumu diapazon? sadarb?b? ar pasaules VLBI t?kla partneriem, t?diem k? Eiropas VLBI t?kls, Zemo frekven?u VLBI t?kls (LFVN) un citiem. P?d?jos gados rekonstru?ta teleskopa sekund?raj? fokus? izvietot? uztv?r?ju telpa un taj? uzst?d?ti vair?ki jauni uztv?r?ji. Pašreiz radioteleskops ?auj veikt nov?rojumus ?etros vi??u garumu diapazonos: 92 cm, 18 cm, 6 cm un 2.5 cm. No min?tajiem pirmie 3 jau tiek veiksm?gi izmantoti daž?dos VLBI eksperimentos. 2.5 cm uztv?r?jam ir tikai viens line?r?s polariz?cijas kan?ls, kuru izmanto galvenok?rt metanola m?zeru nov?rojumiem viena teleskopa rež?m?. RT-32 aparat?ru veido divas neatkar?gas VLBI datu re?istr?cijas sist?mas: TN-16 un DBBC kop? ar Mark5b. Abas sist?mas izmanto interferometriskajos nov?rojumos atkar?b? no eksperimentu m?r?a un radioteleskopa iesp?j?m. Apl?kots Irbenes RT-32 radioteleskopa pašreiz?jais statuss, t? VLBI nov?rojumiem piem?rot?s uztveršanas un datu re?istr?cijas iek?rtas, k? ar? notikušaj?s VLBI sesij?s uzkr?t? pieredze.

  11. Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth disease virus and look-alike disease viruses

    SciTech Connect

    Hindson, B J; Reid, S M; Baker, B R; Ebert, K; Ferris, N P; Bentley Tammero, L F; Lenhoff, R J; Naraghi-Arani, P; Vitalis, E A; Slezak, T R; Hullinger, P J; King, D P

    2007-07-26

    A high-throughput multiplexed assay was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.

  12. Detection of a broad range of class I and II Newcastle disease viruses using a multiplex real time RT-PCR assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prompt detection of virulent strains of Newcastle disease virus (vNDV) using real time RT-PCR (rRT-PCR) is challenging due to the broad genetic variability across two clades comprising 18 recognized genotypes. A large proportion of class I low virulence Newcastle disease viruses (loNDV) recently id...

  13. Selection of reference genes for RT-qPCR analysis in the monarch butterfly, Danaus plexippus (L.), a migrating bio-indicator

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring and evaluating changes in gene expression. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. In this study, expres...

  14. VARIATION OF THE EXPRESSION OF ENDOGENOUS "HOUSEKEEPING" GENES IN B[A]P TREATED MOUSE LUNGS MEASURED BY qRT-PCR

    EPA Science Inventory

    Quantitative RT-PCR is frequently used to analyze gene expression in different experimental systems. In this assay, housekeeping genes are frequently used to normalize for the variability between samples (relative quantification). We have examined the utility of using qRT-PCR and...

  15. Controls Field Exam Consider a satellite orbiting the earth. The satellite's distance from earth is denoted by r(t). The satellite

    E-print Network

    de Weck, Olivier L.

    Controls Field Exam Question 1 Consider a satellite orbiting the earth. The satellite's distance from earth is denoted by r(t). The satellite is put on orbit such that its nominal distance to earth on the satellite. The dynamics of the satellite can be described the following differential equation: ¨r(t) = 2 r

  16. Reference gene validation for quantitative RT-PCR during biotic and abiotic stresses in Vitis vinifera.

    PubMed

    Borges, Alexandre Filipe; Fonseca, Catarina; Ferreira, Ricardo Boavida; Lourenço, Ana Maria; Monteiro, Sara

    2014-01-01

    Grapevine is one of the most cultivated fruit crop worldwide with Vitis vinifera being the species with the highest economical importance. Being highly susceptible to fungal pathogens and increasingly affected by environmental factors, it has become an important agricultural research area, where gene expression analysis plays a fundamental role. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is currently amongst the most powerful techniques to perform gene expression studies. Nevertheless, accurate gene expression quantification strongly relies on appropriate reference gene selection for sample normalization. Concerning V. vinifera, limited information still exists as for which genes are the most suitable to be used as reference under particular experimental conditions. In this work, seven candidate genes were investigated for their stability in grapevine samples referring to four distinct stresses (Erysiphe necator, wounding and UV-C irradiation in leaves and Phaeomoniella chlamydospora colonization in wood). The expression stability was evaluated using geNorm, NormFinder and BestKeeper. In all cases, full agreement was not observed for the three methods. To provide comprehensive rankings integrating the three different programs, for each treatment, a consensus ranking was created using a non-weighted unsupervised rank aggregation method. According to the last, the three most suitable reference genes to be used in grapevine leaves, regardless of the stress, are UBC, VAG and PEP. For the P. chlamydospora treatment, EF1, CYP and UBC were the best scoring genes. Acquaintance of the most suitable reference genes to be used in grapevine samples can contribute for accurate gene expression quantification in forthcoming studies. PMID:25340748

  17. RT-PCR analysis of Deformed wing virus in honeybees (Apis mellifera) and mites (Varroa destructor).

    PubMed

    Yue, Constanze; Genersch, Elke

    2005-12-01

    Deformed wing virus (DWV) is a honeybee viral pathogen either persisting as an inapparent infection or resulting in wing deformity. The occurrence of deformity is associated with the transmission of DWV through Varroa destructor during pupal stages. Such infections with DWV add to the pathology of V. destructor and play a major role in colony collapse in the course of varroosis. Using a recently developed RT-PCR protocol for the detection of DWV, individual bees and mites originating from hives differing in Varroa infestation levels and the occurrence of crippled bees were analysed. It was found that 100 % of both crippled and asymptomatic bees were positive for DWV. However, a significant difference in the spatial distribution of DWV between asymptomatic and crippled bees could be demonstrated: when analysing head, thorax and abdomen of crippled bees, all body parts were always strongly positive for viral sequences. In contrast, for asymptomatic bees viral sequences could be detected in RNA extracted from the thorax and/or abdomen but never in RNA extracted from the head. DWV replication was demonstrated in almost all DWV-positive body parts of infected bees. Analysing individual mites for the presence of DWV revealed that the percentage of DWV-positive mites differed between mite populations. In addition, it was demonstrated that DWV was able to replicate in some but not all mites. Interestingly, virus replication in mites was correlated with wing deformity. DWV was also detected in the larval food, implicating that in addition to transmission by V. destructor DWV is also transmitted by feeding. PMID:16298989

  18. Mitochondrial genome sequencing of chondrocytes in osteoarthritis by human mitochondria RT2 Profiler™ PCR array.

    PubMed

    Li, Zhifu; Shen, Jun; Chen, Yuxian; Pan, Janying; Zeng, Hua; Fang, Hang; Ye, Zhiqiang; Zeng, Chun; Zhang, Rongkai; Cai, Daozhang

    2012-07-01

    Mitochondria are not only the main energy generators of the cell, but also mediate several critical biochemical processes such as apoptosis, proliferation and redox homeostasis. As such, mitochondrial dysfunctions can lead to a wide variety of human diseases, including cancer and osteoarthritis (OA). In OA, mitochondrial-associated signaling has been implicated in the molecular events leading to cartilage degradation, including oxidative stress, defective chondrocyte biosynthesis and growth responses, increased cytokine-induced chondrocyte inflammation and matrix catabolism, cartilage matrix calcification and increased chondrocyte apoptosis. Thus, the mitochondrial genome represents an attractive target for molecular therapy and OA research has focused on determining its role in chondrocyte metabolism and subsequent cartilage degradation. In this study, we analyzed the mitochondrial gene expression changes that characterize chondrocytes in OA using the Human Mitochondria RT² Profiler™ PCR Array. Twenty-six differentially expressed genes were identified that discriminated chondrocytes in OA from those in normal cartilage, including 17 upregulated and 9 downregulated genes. These genes represent diverse functional categories, including mitochondrial membrane polarization and potential, mitochondrial transport, small molecule transport, targeting proteins to the mitochondria, mitochondrial protein import, outer and inner membrane translocation, mitochondrial fission and fusion, mitochondrial localization and apoptosis. Western blot analysis confirmed that the p53 upregulated modulator of apoptosis (PUMA; encoded by the BB3 gene) was significantly upregulated in OA cartilage. In conclusion, our study generates a differential mitochondrial gene expression profile for chondrocytes in OA and demonstrates that mitochondrial genome dysregulation occurs in cartilage cells during OA. Finally, our results indicate that PUMA may be a new diagnostic and therapeutic target for OA. PMID:22504941

  19. effect of embryonic genotype on reference gene selection for RT-qPCR normalization.

    PubMed

    Llobat, L; Marco-Jiménez, F; Peñaranda, D S; Saenz-de-Juano, M D; Vicente, J S

    2012-08-01

    To obtain more reliable results with biological significance, it requires data normalization using an appropriate internal control gene. In rabbits, there are classic stable reference genes that have been identified for normalization in oocytes and pre-implantation stage embryos. However, effects of embryonic genotype on reference gene selection have not been elucidated. The aim of this study was to test (i) the stability of mRNA transcription level for histone (H2afz) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes in rabbit blastocysts from two lines selected by different criteria (litter size and post-weaning daily weight gain) and (ii) its influence on biological significance examined by means of a set of embryonic transcripts, such as POU5F1 (Oct-4), epidermal growth factor receptor (erbB3), transforming growth factor-beta2, vascular endothelial growth factor and gamma interferon (Ifn-gamma). The geNorm, NormFinder and BestKeeper programs showed similar results, pointing out that H2afz and GAPDH were the most stable reference genes in rabbits selected on litter size at weaning. Moreover, our study revealed that embryonic genotype affected target gene expression when a single reference gene was used to analyse mRNA expression in blastocysts. Results showed that GAPDH gene is better than H2afz for gene expression studies of both embryo genotypes. A normalization factor derived from H2afz and GAPDH is likely to be appropriate when RT-qPCR was performed in rabbit embryos with different genotypes. PMID:22044783

  20. Reference Gene Validation for Quantitative RT-PCR during Biotic and Abiotic Stresses in Vitis vinifera

    PubMed Central

    Borges, Alexandre Filipe; Fonseca, Catarina; Ferreira, Ricardo Boavida; Lourenço, Ana Maria; Monteiro, Sara

    2014-01-01

    Grapevine is one of the most cultivated fruit crop worldwide with Vitis vinifera being the species with the highest economical importance. Being highly susceptible to fungal pathogens and increasingly affected by environmental factors, it has become an important agricultural research area, where gene expression analysis plays a fundamental role. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is currently amongst the most powerful techniques to perform gene expression studies. Nevertheless, accurate gene expression quantification strongly relies on appropriate reference gene selection for sample normalization. Concerning V. vinifera, limited information still exists as for which genes are the most suitable to be used as reference under particular experimental conditions. In this work, seven candidate genes were investigated for their stability in grapevine samples referring to four distinct stresses (Erysiphe necator, wounding and UV-C irradiation in leaves and Phaeomoniella chlamydospora colonization in wood). The expression stability was evaluated using geNorm, NormFinder and BestKeeper. In all cases, full agreement was not observed for the three methods. To provide comprehensive rankings integrating the three different programs, for each treatment, a consensus ranking was created using a non-weighted unsupervised rank aggregation method. According to the last, the three most suitable reference genes to be used in grapevine leaves, regardless of the stress, are UBC, VAG and PEP. For the P. chlamydospora treatment, EF1, CYP and UBC were the best scoring genes. Acquaintance of the most suitable reference genes to be used in grapevine samples can contribute for accurate gene expression quantification in forthcoming studies. PMID:25340748

  1. Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth and look-alike disease viruses

    SciTech Connect

    Hindson, B J; Baker, B R; Bentley Tammero, L F; Lenhoff, R J; Naraghi-Arani, P; Vitalis, E A; Slezak, T R; Hullinger, P J; Reid, S M; Ebert, K; Ferris, N P; King, D P

    2007-09-18

    A high-throughput multiplexed assay (Multiplex Version 1.0) was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRTPCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.

  2. Detection of HER2 status in breast cancer: comparison of current methods with MLPA and real-time RT-PCR.

    PubMed

    Pazhoomand, Reza; Keyhani, Elahe; Banan, Mehdi; Najmabadi, Hossein; Khodadadi, Faranak; Iraniparast, Alireza; Feiz, Farnaz; Majidzadeh, Keivan; Bahman, Ideh; Moghadam, Fatemeh Aghakhani; Sobhani, Atoosa Madadkar; Muhammadnejad, Ahad; Abedini, Seyedeh Sedigheh; Behjati, Farkhondeh

    2013-01-01

    Human epidermal growth factor receptor (HER) status is an important prognostic factor in breast cancer. There is no globally accepted method for determining its status, and which method is most precise is still a matter of debate. We here analyzed HER2 mRNA expression by quantitative reverse transcription-PCR (qRT-PCR) and HER2 DNA amplification using multiplex ligation-dependent probe amplification (MLPA). In parallel, we performed a routine evaluation of HER2 protein by immunohistochemistry (IHC). To assess the accuracy of the RT-PCR and MLPA techniques, a combination of IHC and fluorescence in situ hybridization (FISH) was used, substituting FISH when the results of IHC were ambiguous (2+) and for those IHC results that disagreed with MLPA and qRT-PCR, this approach being termed IHC-FISH. The IHC results for four samples were not compatible with the MLPA and qRT-PCR results; the MLPA and qRT-PCR results for these samples were confirmed by FISH. The correlations between IHC-FISH and qRT-PCR or MLPA were 0.945 and 0.973, respectively. The ASCO/CAP guideline IHC/FISH correlation with MLPA was (0.827) and with RT-PCR was (0.854). The correlations between the IHC results (0, 1+ as negative, and 3+ as positive) and qRT-PCR and MLPA techniques were 0.743 and 0.831, respectively. Given the shortcomings of IHC analysis and greater correlations between MLPA, qRT-PCR, and FISH methods than IHC analysis alone with each of these three methods, we propose that MLPA and real-time PCR are good alternatives to IHC. However a suitable cut-off point for qRT- PCR is a prerequisite for determining the exact status of HER2. PMID:24460343

  3. Duplex Real-Time RT-PCR Assays for the Detection and Typing of Epizootic Haemorrhagic Disease Virus

    PubMed Central

    Viarouge, Cyril; Breard, Emmanuel; Zientara, Stephan; Vitour, Damien; Sailleau, Corinne

    2015-01-01

    Epizootic haemorrhagic disease virus (EHDV) may cause severe clinical episodes in some species of deer and sometimes in cattle. Laboratory diagnosis provides a basis for the design and timely implementation of disease control measures. There are seven distinct EHDV serotypes, VP2 coding segment 2 being the target for serotype specificity. This paper reports the development and validation of eight duplex real-time RT-PCR assays to simultaneously amplify the EHDV target (S9 for the pan-EHDV real-time RT-PCR assay and S2 for the serotyping assays) and endogenous control gene Beta-actin. Analytical and diagnostic sensitivity and specificity, inter- and intra-assay variation and efficiency were evaluated for each assay. All were shown to be highly specific and sensitive. PMID:26161784

  4. Reference gene selection for real-time RT-PCR normalization in rice field eel (Monopterus albus) during gonad development.

    PubMed

    Hu, Qing; Guo, Wei; Gao, Yu; Tang, Rong; Li, Dapeng

    2014-12-01

    Real-time reverse transcriptase (RT) polymerase chain reaction (PCR) requires data normalization using an appropriate reference gene in order to obtain more reliable results with biological significance. We cloned a partial sequence of elongation factor-1-? (EF1?) and ribosomal protein L17 (RPL17) from Monopterus albus. We investigated the suitability of five commonly used reference genes [18S ribosomal RNA (18S), cytoskeletal protein (?-actin), glyceraldehyde phosphate dehydrogenase (GAPDH), EF1? and RPL17] as potential quantitative reference genes for normalizing real-time RT-PCR data generated in gonads of different developmental stages and in other tissues of M. albus. Analysis of the data indicated that 18S, ?-actin and GAPDH are not suitable as reference genes because of their levels of variations of expression. EF1? and RPL17 might be suitable as reference genes in the gonads of different developmental stages as well as in other tissues of M. albus. PMID:25079246

  5. The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids.

    PubMed

    Fleming, Rachel I; Harbison, SallyAnn

    2010-07-01

    With current methodology, DNA profiling can identify an individual from a sample of biological material but it does not reveal what body fluid or tissue source the DNA profile originated from. We have developed a multiplex PCR system using messenger RNA (mRNA) that can identify blood, saliva, semen and menstrual blood in individual stains or in mixtures of body fluids. Messenger RNA transcripts specific to each type of body fluid have been identified and a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) system developed to identify these body fluids along with three housekeeping genes. This multiplex can detect semen and seminal fluid (semen without spermatozoa present). Furthermore, we have targeted the co-isolation of RNA and DNA from the same sample and, with the RT-PCR multiplex, we can determine the type of body fluid present as well as generate a DNA profile(s) from the same stain. PMID:20457026

  6. Multiplex titration RT-PCR: rapid determination of gene expression patterns for a large number of genes

    NASA Technical Reports Server (NTRS)

    Nebenfuhr, A.; Lomax, T. L.

    1998-01-01

    We have developed an improved method for determination of gene expression levels with RT-PCR. The procedure is rapid and does not require extensive optimization or densitometric analysis. Since the detection of individual transcripts is PCR-based, small amounts of tissue samples are sufficient for the analysis of expression patterns in large gene families. Using this method, we were able to rapidly screen nine members of the Aux/IAA family of auxin-responsive genes and identify those genes which vary in message abundance in a tissue- and light-specific manner. While not offering the accuracy of conventional semi-quantitative or competitive RT-PCR, our method allows quick screening of large numbers of genes in a wide range of RNA samples with just a thermal cycler and standard gel analysis equipment.

  7. RT3D Reaction Modules for Natural and Enhanced Attenuation of Chloroethanes, Chloroethenes, Chloromethanes, and Daughter Products

    SciTech Connect

    Johnson, Christian D.; Truex, Michael J.

    2006-07-25

    This document describes a suite of MNA/EA reaction modules that were developed for addressing complex chlorinated solvent reactions using RT3D. As an introduction, an overview of these MNA/EA reaction modules is presented, including discussions of similarities between reaction modules, the purpose of key reaction parameters, and important considerations for using the reaction modules. Subsequent sections provide the details of the reaction kinetics (conceptual model and equations), data input requirements, and example (batch reactor) results for each reaction module. This document does not discuss reaction module implementation or validation; such information will accompany the software in the form of release notes or a supplement to the RT3D manual.

  8. [Development of SYBR Green I real-time RT-PCR for the detection of Ebola virus].

    PubMed

    Liu, Yang; Shi, Zi-Xue; Ma, Yu-Kun; Wang, Hao-Ting; Wang, Zong-Yao; Shao, Dong-Hua; Wei, Jian-Chao; Wang, Shao-Hui; Li, Bei-Bei; Wang, Shui-Ming; Liu, Xue-Hui; Ma, Zhi-Yong

    2012-09-01

    In order to establish a rapid and accurate method for the detection of Ebola virus (EBOV), the primers used in SYBR Green I real-time RT-PCR were designed based on the EBOV NP gene sequences published in GenBank. The SYBR Green I real-time RT-PCR was established and optimized for the detection of EBOV. The EBOV RNA that was transcribed in vitro was used as a template. The sensitivity of this method was found to reach 1.0 x 10(2) copies/microL and the detection range was 10(2) - 10(10). No cross reaction with RNA samples from Marburg virus, Dengue virus, Xinjiang hemorrhagic fever virus, Japanese encephalitis virus, Influenza virus (H1N1 and H3N2) and Porcine reproductive and respiratory syndrome virus E genomic RNA was found. The method would be useful for the detection and monitoring of EBOV in China. PMID:23233935

  9. Assessment of RT-qPCR normalization strategies for accurate quantification of extracellular microRNAs in murine serum.

    PubMed

    Roberts, Thomas C; Coenen-Stass, Anna M L; Wood, Matthew J A

    2014-01-01

    Extracellular microRNAs (miRNAs) are under investigation as minimally-invasive biomarkers for a wide range of disease conditions. We have recently shown in a mouse model of the progressive muscle-wasting condition Duchenne muscular dystrophy (DMD) that a set of highly elevated serum miRNAs reflects the regenerative status of muscle. These miRNAs are promising biomarkers for monitoring DMD disease progression and the response to experimental therapies. The gold standard miRNA detection methodology is Reverse Transcriptase-quantitative Polymerase Chain Reaction (RT-qPCR), which typically exhibits high sensitivity and wide dynamic range. Accurate determination of miRNA levels is affected by RT-qPCR normalization method and therefore selection of the optimal strategy is of critical importance. Serum miRNA abundance was measured by RT-qPCR array in 14 week old mice, and by individual RT-qPCR assays in a time course experiment spanning 48 weeks. Here we utilize these two datasets to assess the validity of three miRNA normalization strategies (a) normalization to the average of all Cq values from array experiments, (b) normalization to a stably expressed endogenous reference miRNA, and (c) normalization to an external spike-in synthetic oligonucleotide. Normalization approaches based on endogenous control miRNAs result in an under-estimation of miRNA levels by a factor of ?2. An increase in total RNA and total miRNA was observed in dystrophic serum which may account for this systematic bias. We conclude that the optimal strategy for this model system is to normalize to a synthetic spike-in control oligonucleotide. PMID:24586621

  10. qRT9, a quantitative trait locus controlling root thickness and root length in upland rice.

    PubMed

    Li, Junzhou; Han, Yingchun; Liu, Lei; Chen, Yipeng; Du, Yanxiu; Zhang, Jing; Sun, Hongzheng; Zhao, Quanzhi

    2015-05-01

    Breeding for strong root systems is an important strategy for improving drought avoidance in rice. To clone genes responsible for strong root traits, an upland rice introgression line IL392 with thicker and longer roots than the background parent lowland rice Yuefu was selected. A quantitative trait locus (QTL), qRT9, controlling root thickness and root length was detected under hydroponic culture using 203 F(2:3) populations derived from a cross between Yuefu and IL392. The qRT9 locus explained 32.5% and 28.1% of the variance for root thickness and root length, respectively. Using 3185 F2 plants, qRT9 was ultimately narrowed down to an 11.5 kb region by substitution mapping. One putative basic helix-loop-helix (bHLH) transcription factor gene, LOC_Os09g28210 (named OsbHLH120), is annotated in this region. Sequences of OsbHLH120 in 11 upland rice and 13 lowland rice indicated that a single nucleotide polymorphism (SNP) at position 82 and an insertion/deletion (Indel) at position 628-642 cause amino acid changes and are conserved between upland rice and lowland rice. Phenotypic analysis indicated that the two polymorphisms were significantly associated with root thickness and root length under hydroponic culture. Quantitative real-time PCR showed that OsbHLH120 was strongly induced by polyethylene glycol (PEG), salt, and abscisic acid, but higher expression was present in IL392 roots than in Yuefu under PEG and salt stress. The successfully isolated locus, qRT9, enriches our knowledge of the genetic basis for drought avoidance and provides an opportunity for breeding drought avoidance varieties by utilizing valuable genes in the upland rice germplasm. PMID:25769309

  11. Real Time RT-PCR and flow cytometry to investigate wheat kernel hardness: role of puroindoline genes and proteins.

    PubMed

    Amoroso, M G; Longobardo, L; Capparelli, R

    2004-11-01

    Developing seeds from Triticum aestivum (wheat) cultivars were collected after flowering and analysed for puroindoline a and b gene expression by Real Time RT-PCR. Mature seeds were investigated for the presence and the amount of starch-associated puroindoline a and b proteins by flow cytometry. Puroindoline a gene and protein were found to have a predominant role in controlling wheat kernel hardness. PMID:15604827

  12. '~5 os~~r~t~ SCo.."""'-~r-Design and Implementation or the SUDNetwork Files:ystem

    E-print Network

    Han, Richard Y.

    ~ '~5 os~~r~t~ SCo.."""'-~r- Design and Implementation or the SUDNetwork Files:ystem Russel SandbC'" David Gold"" StnC'XlC'iman Dan Walsh Bob Lyon SunMicrosystems. Inc. 2550 Garcia Ave. Mountain View, CA. In orderto build the NFS into the UNIX 4.2 kernel in a usertransparentway, we decided to add a new interface

  13. Simultaneous detection of West Nile and Japanese encephalitis virus RNA by duplex TaqMan RT-PCR.

    PubMed

    Barros, Silvia C; Ramos, Fernanda; Zé-Zé, Líbia; Alves, Maria J; Fagulha, Teresa; Duarte, Margarida; Henriques, Margarida; Luís, Tiago; Fevereiro, Miguel

    2013-11-01

    West Nile virus (WNV) and Japanese encephalitis virus (JEV) are important mosquito-borne viruses of the Flaviviridae family, associated with encephalitis, mainly in humans and horses. WNV is also pathogen for many bird species. The incidence of human and animal WNV infections in Europe has risen, mostly in recent years, and JEV was detected in 2011 in mosquitoes collected in Italy and may emerge in Europe in the same way as other flaviviruses had emerged recently (USUTU and Bagaza virus) and should be regarded as a potential threat to public health. Prompt identification and discrimination between WNV and JEV provides critical epidemiological data for prevalence studies and public and animal health management policies. Here we describe a quantitative one-step duplex TaqMan RT-PCR, targeting non-structural protein 2A gene (NS2A-qRT-PCR), based on only one primer pair and two probes for differential diagnosis of WNV and JEV. Also this assay enables the detection of both WNV lineages (WNV-1 and WNV-2). To access the specificity of NS2A-qRT-PCR a panel of different arboviruses were used. The assay was shown to be specific for both WNV lineages (WNV-1 and WNV-2), WNV related Kunjin virus and JEV, since no cross-reactions were observed with other tested arboviruses. Sensitivity of the assay was determined using serial dilutions of in vitro-transcribed RNA from WNV and JEV. The duplex NS2A-qRT-PCR assay was shown to be very sensitive, being able to detect 10 copies of WNV and JEV RNA. This assay is a suitable tool for the diagnosis of WNV and JEV, and provides a valuable addition to the methods currently available for routine diagnosis of these zoonoses and for surveillance studies. PMID:23892127

  14. Increased sensitivity of RT-PCR for Potato virus Y detection using RNA isolated by a procedure with differential centrifugation.

    PubMed

    Zhang, Jianhua; Nie, Xianzhou; Boquel, Sébastien; Al-Daoud, Fadi; Pelletier, Yvan

    2015-12-01

    The sensitivity of reverse transcription-polymerase chain reaction (RT-PCR) for virus detection is influenced by many factors such as specificity of primers and quality of templates. These factors become extremely important for successful detection when virus concentration is low. Total RNA isolated from Potato virus Y (PVY)-infected potato plants using the sodium sulfite RNA isolation method or RNeasy plant mini kit contains a high proportion of host RNA and may also contain trace amount of phenolic and polysaccharide residues, which may inhibit RT-PCR. The goal of this study was to enhance the sensitivity of PVY detection by reducing host RNA in the extract by differential centrifugation followed by extraction using an RNeasy mini kit (DCR method). One-step RT-PCR had relatively low amplification efficiency for PVY RNA when a high proportion of plant RNA was present. SYBR Green-based real time RT-PCR showed that the RNA isolated by the DCR method had a higher cycle threshold value (Ct) for the elongation factor 1-? mRNA (Ef1?) of potato than the Ct value of the RNA extracted using the RNeasy plant mini kit, indicating that the DCR method significantly reduced the proportion of potato RNA in the extract. The detectable amount of RNA extracted using the DCR method was <0.001ng when plant sap from 10 PVY-infected and PVY-free potato leaflets in a 1.5:100 fresh weight ratio was extracted, compared with 0.01 and 0.02ng of RNA using the RNeasy plant mini kit and sodium sulfite RNA isolation methods, respectively. PMID:26210699

  15. Development of an in situ magnetic beads based RT-PCR method for electrochemiluminescent detection of rotavirus

    NASA Astrophysics Data System (ADS)

    Zhan, Fangfang; Zhou, Xiaoming

    2012-12-01

    Rotaviruses are double-stranded RNA viruses belonging to the family of enteric pathogens. It is a major cause of diarrhoeal disease in infants and young children worldwide. Consequently, rapid and accurate detection of rotaviruses is of great importance in controlling and preventing food- and waterborne diseases and outbreaks. Reverse transcription-polymerase chain reaction (RT-PCR) is a reliable method that possesses high specificity and sensitivity. It has been widely used to detection of viruses. Electrochemiluminescence (ECL) can be considered as an important and powerful tool in analytical and clinical application with high sensitivity, excellent specificity, and low cost. Here we have developed a method for the detection of rotavirus by combining in situ magnetic beads (MBs) based RT-PCR with ECL. RT of rotavirus RNA was carried out in a traditional way and the resulting cDNA was directly amplified on MBs. Forward primers were covalently bounded to MBs and reverse primers were labeled with tris-(2, 2'-bipyridyl) ruthenium (TBR). During the PCR cycling, the TBR labeled products were directly loaded and enriched on the surface of MBs. Then the MBs-TBR complexes could be analyzed by a magnetic ECL platform without any post-modification or post-incubation which avoid some laborious manual operations and achieve rapid yet sensitive detection. In this study, rotavirus from fecal specimens was successfully detected within 2 h, and the limit of detection was estimated to be 104copies/?L. This novel in situ MBs based RT-PCR with ECL detection method can be used for pathogen detection in food safety field and clinical diagnosis.

  16. GOES-R Proving Ground Activities at the NASA Short-Term Prediction Research and Transition (SPoRT) Center

    NASA Technical Reports Server (NTRS)

    Molthan, Andrew

    2011-01-01

    SPoRT is actively involved in GOES-R Proving Ground activities in a number of ways: (1) Applying the paradigm of product development, user training, and interaction to foster interaction with end users at NOAA forecast offices national centers. (2) Providing unique capabilities in collaboration with other GOES-R Proving Ground partners (a) Hybrid GOES-MODIS imagery (b) Pseudo-GLM via regional lightning mapping arrays (c) Developing new RGB imagery from EUMETSAT guidelines

  17. Immunomagnetic separation combined with RT-qPCR for determining the efficacy of disinfectants against human noroviruses.

    PubMed

    Liu, Pengbo; Kim, Myung; Schlesinger, David; Kranz, Christine; Ha, Sangdo; Ha, Jeehyoung; Slauch, James; Baek, Seungbum; Moe, Christine

    2015-01-01

    Little is known about the effectiveness of disinfectants against human noroviruses (NoV) partially because human NoV cannot be routinely cultured in laboratory. The objective of this study was to develop a NoV monoclonal antibody-conjugated immunomagnetic separation (IMS) procedure combined with real-time reverse transcription polymerase chain reaction (RT-qPCR) assays to study the in vitro efficacy of disinfectants against human NoV. Monoclonal antibodies against Norwalk virus (NV, GI.1) and NoV GII.4 were produced using unique NoV capsid proteins, and the antibodies were conjugated to magnetic Dynalbeads. The immunomagnetic beads were used to simultaneously capture intact NoV in samples and effectively remove PCR inhibitors. We examined the efficacy of ethanol, sodium hypochlorite, nine commercially available disinfectants, and one prototype disinfectant using the IMS/RT-qPCR. The sensitivity of this procedure was approximately 100 virus particles for both the NV and GII.4 viruses. The average log reductions in in vitro activities varied between disinfectants. The prototype disinfectant produced an average 3.19-log reduction in NV and a 1.38-log reduction in GII.4. The prototype disinfectant is promising of inactivating NoV. This method can be used to evaluate in vitro activity of disinfectants against human NoV. The IMS/RT-qPCR method is promising as an effective method to remove PCR inhibitors in disinfectants and enable the evaluation of the efficacy of disinfectants. PMID:25270388

  18. Detection of Langat virus by TaqMan real-time one-step qRT-PCR method

    PubMed Central

    Muhd Radzi, Siti Fatimah; Rückert, Claudia; Sam, Sing-Sin; Teoh, Boon-Teong; Jee, Pui-Fong; Phoon, Wai-Hong; Abubakar, Sazaly; Zandi, Keivan

    2015-01-01

    Langat virus (LGTV), one of the members of the tick-borne encephalitis virus (TBEV) complex, was firstly isolated from Ixodes granulatus ticks in Malaysia. However, the prevalence of LGTV in ticks in the region remains unknown. Surveillance for LGTV is therefore important and thus a tool for specific detection of LGTV is needed. In the present study, we developed a real-time quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) for rapid detection of LGTV. Our findings showed that the developed qRT-PCR could detect LGTV at a titre as low as 0.1 FFU/ml. The detection limit of the qRT-PCR assay at 95% probability was 0.28 FFU/ml as determined by probit analysis (p???0.05). Besides, the designed primers and probe did not amplify ORF of the E genes for some closely related and more pathogenic viruses including TBEV, Louping ill virus, Omsk hemorrhagic fever virus (OHFV), Alkhurma virus (ALKV), Kyasanur Forest Disease virus (KFDV) and Powassan virus (POWV) which showed the acceptable specificity of the developed assay. The sensitivity of the developed method also has been confirmed by determining the LGTV in infected tick cell line as well as LGTV- spiked tick tissues. PMID:26360297

  19. The use of collagenase to improve the detection of plant viruses in vector nematodes by RT-PCR.

    PubMed

    Martin, Robert R; Pinkerton, Jack N; Kraus, Jennifer

    2009-01-01

    Tomato ringspot virus (ToRSV), Tobacco ringspot virus (TRSV) and Tobacco rattle virus (TRV) are transmitted to healthy plants by viruliferous nematodes in the soil. A method was developed for extraction of genomic viral RNA from virus particles carried within nematodes and a sensitive nested RT-PCR detection assay. The procedure has been adapted to microscale for handling multiple samples. This assay is effective for detection of ToRSV or TRSV in Xiphinema americanum or TRV in Paratrichodorus allius. With this method, viruses can be detected in nematodes fed on infected plants or from field-collected nematodes where the percentage of viruliferous nematodes is unknown. Soil samples from four red raspberry fields infected with ToRSV were collected in 2003 and 2004. Nematodes isolated from these samples were assayed for ToRSV by RT-PCR and compared to cucumber baiting bioassay for virus transmission from the same soil samples. ToRSV was detected in nematodes throughout the season with similar frequencies by the RT-PCR assay and the transmission bioassay. PMID:18992280

  20. A novel approach to quantitating leukemia fusion transcripts by qRT-PCR without the need for standard curves.

    PubMed

    Schumacher, Jonathan A; Scott Reading, N; Szankasi, Philippe; Matynia, Anna P; Kelley, Todd W

    2015-08-01

    Acute myeloid leukemia patients with recurrent cytogenetic abnormalities including inv(16);CBFB-MYH11 and t(15;17);PML-RARA may be assessed by monitoring the levels of the corresponding abnormal fusion transcripts by quantitative reverse transcription-PCR (qRT-PCR). Such testing is important for evaluating the response to therapy and for the detection of early relapse. Existing qRT-PCR methods are well established and in widespread use in clinical laboratories but they are laborious and require the generation of standard curves. Here, we describe a new method to quantitate fusion transcripts in acute myeloid leukemia by qRT-PCR without the need for standard curves. Our approach uses a plasmid calibrator containing both a fusion transcript sequence and a reference gene sequence, representing a perfect normalized copy number (fusion transcript copy number/reference gene transcript copy number; NCN) of 1.0. The NCN of patient specimens can be calculated relative to that of the single plasmid calibrator using experimentally derived PCR efficiency values. We compared the data obtained using the plasmid calibrator method to commercially available assays using standard curves and found that the results obtained by both methods are comparable over a broad range of values with similar sensitivities. Our method has the advantage of simplicity and is therefore lower in cost and may be less subject to errors that may be introduced during the generation of standard curves. PMID:26079545

  1. Development and evaluation of a fluorogenic real-time RT-PCR for the detection of dengue 3 virus.

    PubMed

    Tan, Irene L; Dimamay, Mark Pierre S; Buerano, Corazon C; Alfon, Jhoe Anthony R; Tanig, Carol Z; Matias, Ronald R; Natividad, Filipinas F

    2010-12-01

    A dengue-3-specific real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was developed using the novel Light Upon eXtension (LUX™) fluorogenic technology. A labeled forward primer and a standard reverse primer that target a conserved region within the non-structural 1 (NS1) gene of dengue 3 strains were designed. The dengue-3-specific assay did not recognize other dengue serotypes and related flaviviruses. Using a tenfold serial dilution of plasmid DNA containing the dengue 3 NS1 gene as standards, the range of dengue virus detection was determined to be from 10(3) to 10(9) copies/ml or from 80 to 8 × 10(7) copies/reaction with an average correlation coefficient of ? 0.99. The mean intra-assay coefficient of variation (CV) at 2.01% and the mean inter-assay CV at 2.68% suggest the repeatability of the procedure. Moreover, the fluorogenic assay was evaluated by using clinical specimens and comparing test results with historical data obtained from conventional RT-PCR, which served as the criterion standard. Using patient sera as test samples, the assay demonstrated 95.45% sensitivity and 100% specificity, respectively. These results reveal that the real-time RT-PCR assay may be utilized as a rapid, convenient, and sensitive tool for the detection of dengue 3 in clinical and laboratory specimens. PMID:20981793

  2. A simple and reliable protocol for the detection of apple stem grooving virus by RT-PCR and in a multiplex PCR assay.

    PubMed

    James, D

    1999-12-01

    Primers were identified which amplify specifically a 499 bp fragment in the coat protein coding region of apple stem grooving virus (ASGV) genome. These primers were used in various RT-polymerase chain reaction (PCR) analyses for the detection of ASGV in Chenopodium quinoa, Nicotiana occidentalis, and in species of Malus and Pyrus. Isolates of ASGV in Malus and Pyrus from locations in Canada, China, Israel, Japan, Nepal, Pakistan, South Africa, and the U.S.A. were reliably detected in leaf and bark (budwood) tissue. Storage of the tissues at -80 degrees C for more than 4 months did not affect the reliability of detection by immunocapture (IC) RT-PCR. Triton-X was not necessary for the detection of ASGV by IC/RT-PCR, and it was also possible to combine the antibody incubation and virus sap incubation into a single step without any obvious loss in sensitivity. A Tube Capture (TC) RT-PCR procedure was developed that eliminated the need for antibody binding of the virus. Phosphate buffered saline with 2% PVP was identified as the most effective sample-grinding buffer for the detection of ASGV by IC/RT-PCR and TC/RT-PCR. TC/RT-PCR facilitated the simultaneous detection (multiplex PCR) of ASGV and cherry mottle leaf virus. PMID:10598077

  3. Validation and comparison of different end point and real time RT-PCR assays for detection and genotyping of porcine reproductive and respiratory syndrome virus.

    PubMed

    Drigo, Michele; Franzo, Giovanni; Belfanti, Ilaria; Martini, Marco; Mondin, Alessandra; Ceglie, Letizia

    2014-06-01

    The accuracy and rapid diagnosis of PRRSV infection is a major prerequisite for every control and/or eradication strategy. In this study two real time RT-PCR based on different chemistry analysis (TaqMan Probes and SYBR Green) have been developed and validated before comparison to an end point two-step RT-PCR validated previously. All assays were aimed at discrimination between PRRSV genotypes. Furthermore, an exogenous internal control (IC) system had also been implemented in qRT-PCR. A rigorous analytical validation, executed on infected cell cultures and serum, demonstrated good sensitivity, specificity and repeatability. In particular RT-PCR was exceptionally sensitive and could detect a viral titre in the order of a magnitude of 1 copies/?L, 10-fold lower than other qRT-PCR described in this study. Optimal diagnostic performances have been demonstrated analyzing samples retrieved from an experimental infection, with RT-PCR again outperforming real time RT-PCR assays. All tests, showing substantial agreement between them, were able to detect early stages of viraemia (1 DPI) and some animals were classified as positive until the end of the study (76 DPI). Therefore, this supports the assays usefulness in animals with different clinical conditions and in a broad range of epidemiological scenarios. The benefits and disadvantages of different assays were also considered and discussed. PMID:24613148

  4. Simultaneous diagnosis of Cetacean morbillivirus infection in dolphins stranded in the Spanish Mediterranean sea in 2011 using a novel Universal Probe Library (UPL) RT-PCR assay.

    PubMed

    Rubio-Guerri, Consuelo; Melero, Mar; Rivera-Arroyo, Belén; Bellière, Edwige Nina; Crespo, Jose Luis; García-Párraga, Daniel; Esperón, Fernando; Sánchez-Vizcaíno, Jose Manuel

    2013-07-26

    A highly sensitive and specific real-time (rt) RT-PCR assay has been developed for rapid, simultaneous detection of three strains of cetacean morbillivirus (CeMV). In this assay, two PCR primers and a hydrolysis probe from a commercially available Universal Probe Library (UPL) are used to amplify a highly conserved region within the fusion protein gene. RT-PCR is carried out on the same sample using two primer sets in parallel: one set detects the more virulent strains, dolphin morbillivirus (DMV) and porpoise morbillivirus (PMV), and the other set detects the least virulent and least common strain, pilot whale morbillivirus (PWMV). Sensitivity analysis using dilute samples containing purified DMV, PMV and PWMV showed that viral RNA detection limits in this UPL RT-PCR assay were lower than in a conventional RT-PCR assay. Our method gave no amplification signal with field samples positive for viruses related and unrelated to CeMV, such as phocine distemper virus (PDV). The reliability and robustness of the UPL RT-PCR assay were verified using tissue samples previously analyzed by conventional methods, as well as a panel of clinical samples suspected of containing CeMV. Using the UPL RT-PCR assay, we were able to associate DMV with a mass stranding of striped dolphins in the Spanish Mediterranean in 2011 with greater reliability than was possible with a conventional RT-PCR method. These results suggest that this UPL RT-PCR method is more sensitive and specific than the conventional approach, and that it may be an affordable and rapid test for routine diagnosis of three CeMV strains. PMID:23380457

  5. Enhanced control of dorsolateral prefrontal cortex neurophysiology with real-time functional magnetic resonance imaging (rt-fMRI) neurofeedback training and working memory practice.

    PubMed

    Sherwood, Matthew S; Kane, Jessica H; Weisend, Michael P; Parker, Jason G

    2016-01-01

    Real-time functional magnetic resonance imaging (rt-fMRI) neurofeedback can be used to train localized, conscious regulation of blood oxygen level-dependent (BOLD) signals. As a therapeutic technique, rt-fMRI neurofeedback reduces the symptoms of a variety of neurologic disorders. To date, few studies have investigated the use of self-regulation training using rt-fMRI neurofeedback to enhance cognitive performance. This work investigates the utility of rt-fMRI neurofeedback as a tool to enhance human cognition by training healthy individuals to consciously control activity in the left dorsolateral prefrontal cortex (DLPFC). A cohort of 18 healthy participants in the experimental group underwent rt-fMRI neurofeedback from the left DLPFC in five training sessions across two weeks while 7 participants in the control group underwent similar training outside the MRI and without rt-fMRI neurofeedback. Working memory (WM) performance was evaluated on two testing days separated by the five rt-fMRI neurofeedback sessions using two computerized tests. We investigated the ability to control the BOLD signal across training sessions and WM performance across the two testing days. The group with rt-fMRI neurofeedback demonstrated a significant increase in the ability to self-regulate the BOLD signal in the left DLPFC across sessions. WM performance showed differential improvement between testing days one and two across the groups with the highest increases observed in the rt-fMRI neurofeedback group. These results provide evidence that individuals can quickly gain the ability to consciously control the left DLPFC, and this training results in improvements of WM performance beyond that of training alone. PMID:26348555

  6. Secondary structure in the 3' UTR of EGF and the choice of reverse transcriptases affect the detection of message diversity by RT-PCR.

    PubMed

    Brooks, E M; Sheflin, L G; Spaulding, S W

    1995-11-01

    The secondary structure in mRNA is essential for many processes, but it can present a technical problem in making full-length cDNA with reverse transcriptases. Furthermore, different reverse transcriptases have differing abilities to transcribe through regions with secondary structure, which can alter the products obtained by reverse-transcribing RNA and then PCR-amplifying the product (RT-PCR). We have been interested in studying the posttranscriptional regulation of epidermal growth factor by RT-PCR and have tested the ability of several reverse transcriptases to reverse transcribe the 3'-untranslated region (3'UTR), a region that contains substantial secondary structure. When low levels of either total RNA or poly(A)+ mRNA were used, we found avian myeloblastosis virus reverse transcriptase (AMV-RT) to be the most robust of all the enzymes tested. Furthermore, contrary to reports that AMV-RT is inhibited by tRNA--which should make it less effective than Moloney murine leukemia virus reverse transcriptase (MMLV-RT) at reverse-transcribing total RNA--adding tRNA to poly(A)+ RNA actually increased the amount of specific RT-PCR product obtained with AMV-RT while it decreased the amount of product and enhanced mispriming with MMLV-RT. We found that pre-incubation of the oligo(dT) primer with total RNA at elevated temperature prior to reverse transcription improved the efficiency of both native and modified MMLV-RTs. These findings support the concept that secondary structures in RNA differentially affect the abilities of different reverse transcriptases to detect transcript diversity and raise the possibility that such structures could affect quantitation using RT-PCR with internal mRNA standards. PMID:8588921

  7. Evaluation of NASA SPoRT's Pseudo-Geostationary Lightning Mapper Products in the 2011 Spring Program

    NASA Technical Reports Server (NTRS)

    Stano, Geoffrey T.; Carcione, Brian; Siewert, Christopher; Kuhlman, Kristin M.

    2012-01-01

    NASA's Short-term Prediction Research and Transition (SPoRT) program is a contributing partner with the GOES-R Proving Ground (PG) preparing forecasters to understand and utilize the unique products that will be available in the GOES-R era. This presentation emphasizes SPoRT s actions to prepare the end user community for the Geostationary Lightning Mapper (GLM). This preparation is a collaborative effort with SPoRT's National Weather Service partners, the National Severe Storms Laboratory (NSSL), and the Hazardous Weather Testbed s Spring Program. SPoRT continues to use its effective paradigm of matching capabilities to forecast problems through collaborations with our end users and working with the developers at NSSL to create effective evaluations and visualizations. Furthermore, SPoRT continues to develop software plug-ins so that these products will be available to forecasters in their own decision support system, AWIPS and eventually AWIPS II. In 2009, the SPoRT program developed the original pseudo geostationary lightning mapper (PGLM) flash extent product to demonstrate what forecasters may see with GLM. The PGLM replaced the previous GLM product and serves as a stepping-stone until the AWG s official GLM proxy is ready. The PGLM algorithm is simple and can be applied to any ground-based total lightning network. For 2011, the PGLM used observations from four ground-based networks (North Alabama, Kennedy Space Center, Oklahoma, and Washington D.C.). While the PGLM is not a true proxy product, it is intended as a tool to train forecasters about total lightning as well as foster discussions on product visualizations and incorporating GLM-resolution data into forecast operations. The PGLM has been used in 2010 and 2011 and is likely to remain the primary lightning training tool for the GOES-R program for the near future. This presentation will emphasize the feedback received during the 2011 Spring Program. This will discuss several topics. Based on feedback from the 2010 Spring Program, SPoRT created two variant PGLM products, which NSSL produced locally and provided in real-time within AWIPS for 2011. The first is the flash initiation density (FID) product, which creates a gridded display showing the number of flashes that originated in each 8 8 km grid box. The second product is the maximum flash density (MFD). This shows the highest PGLM value for each grid point over a specific period of time, ranging from 30 to 120 minutes. In addition to the evaluation of these two new products, the evaluation of the PGLM itself will be covered. The presentation will conclude with forecaster feedback for additional improvements requested for future evaluations, such as within the 2012 Spring Program.

  8. Reduced Toxicity With Intensity Modulated Radiation Therapy (IMRT) for Desmoplastic Small Round Cell Tumor (DSRCT): An Update on the Whole Abdominopelvic Radiation Therapy (WAP-RT) Experience

    SciTech Connect

    Desai, Neil B.; Stein, Nicholas F.; LaQuaglia, Michael P.; Alektiar, Kaled M.; Kushner, Brian H.; Modak, Shakeel; Magnan, Heather M.; Goodman, Karyn; Wolden, Suzanne L.

    2013-01-01

    Purpose: Desmoplastic small round cell tumor (DSRCT) is a rare malignancy typically involving the peritoneum in young men. Whole abdominopelvic radiation therapy (WAP-RT) using conventional 2-dimensional (2D) radiation therapy (RT) is used to address local recurrence but has been limited by toxicity. Our objectives were to assess the benefit of intensity modulated radiation therapy (IMRT) on toxicity and to update the largest series on radiation for DSRCT. Methods and Materials: The records of 31 patients with DSRCT treated with WAP-RT (22 with 2D-RT and 9 with IMRT) between 1992 and 2011 were retrospectively reviewed. All received multi-agent chemotherapy and maximal surgical debulking followed by 30 Gy of WAP-RT. A further focal boost of 12 to 24 Gy was used in 12 cases. Boost RT and autologous stem cell transplantation were nearly exclusive to patients treated with 2D-RT. Toxicities were assessed with the Common Terminology Criteria for Adverse Events. Dosimetric analysis compared IMRT and simulated 2D-RT dose distributions. Results: Of 31 patients, 30 completed WAP-RT, with a median follow-up after RT of 19 months. Acute toxicity was reduced with IMRT versus 2D-RT: P=.04 for gastrointestinal toxicity of grade 2 or higher (33% vs 77%); P=.02 for grade 4 hematologic toxicity (33% vs 86%); P=.01 for rates of granulocyte colony-stimulating factor; and P=.04 for rates of platelet transfusion. Post treatment red blood cell and platelet transfusion rates were also reduced (P=.01). IMRT improved target homogeneity ([D05-D95]/D05 of 21% vs 46%) and resulted in a 21% mean bone dose reduction. Small bowel obstruction was the most common late toxicity (23% overall). Updated 3-year overall survival and progression-free survival rates were 50% and 24%, respectively. Overall survival was associated with distant metastasis at diagnosis on multivariate analysis. Most failures remained intraperitoneal (88%). Conclusions: IMRT for consolidative WAP-RT in DSRCT improves hematologic toxicity in particular. Although the long-term efficacy of current treatment options remains disappointing, the improved therapeutic index of IMRT may aid in generalizing its use and allowing the addition of novel approaches such as intraperitoneal immunotherapy.

  9. Comparison of the RT3 Research Tracker and Tritrac R3D accelerometers during a backpacking expedition by a single subject.

    PubMed

    DeVoe, Dale

    2004-10-01

    This study compared the RT3 Research Tracker accelerometer and the Tritrac R3D accelerometer in a field setting. A six-day backpacking expedition (122.4 km in length) was completed by a single subject in the Grand Canyon National Park, Arizona. The overall correlation between the counts of vector magnitude activity for the RT3 and R3D was moderate (r =.75, p<.001), with the overall calculated bias [mean difference (RT3 minus R3D) and standard deviation of the differences] across all six days estimated at 235+/-436 vector magnitude activity counts. However, agreement between the instruments is problematic; the RT3 might be 201 activity counts below or 671 activity counts above the R3D in assessing physical activity during backpacking. PMID:15560342

  10. Ten polymorphic DNA loci, including five in the rat MHC (RT1) region, form a single linkage group on rat chromosome 20

    SciTech Connect

    Remmers, E.F.; Du, Y.; Zha, H.; Goldmuntz, E.A.; Wilder, R.L.

    1995-03-01

    We have described ten markers for polymorphic loci on rat chromosome 20, including five in the rat MHC (RT1) region. These markers formed a single linkage group spanning a recombination distance of 0.40. The markers identified five expressed gene loci - RT1.N1 (thymus leukemia antigen 1), Tnfa (tumor necrosis factor {alpha}), Hspa1 (heat shock protein 70), Ggt1 ({gamma} glutamyl-transferase 1), and Prkacn2 (protein kinase C catalytic subunit binding inhibitor 2), two loci with sequences that are related to expressed genes - RT1.Aw2 (sequence related to a non-RT1A class I {alpha} chain) and Mt21 (sequence related to metallothionein 2), and three anonymous loci - D20Arb548, D20Arb234, and D20Arb249. These polymorphic markers should facilitate mapping studies and genetic monitoring of inbred rat strains. 18 refs., 2 figs., 3 tabs.

  11. Hepatitis C Virus RNA Real-Time Quantitative RT-PCR Method Based on a New Primer Design Strategy.

    PubMed

    Chen, Lida; Li, Wenli; Zhang, Kuo; Zhang, Rui; Lu, Tian; Hao, Mingju; Jia, Tingting; Sun, Yu; Lin, Guigao; Wang, Lunan; Li, Jinming

    2016-01-01

    Viral nucleic acids are unstable when improperly collected, handled, and stored, resulting in decreased sensitivity of currently available commercial quantitative nucleic acid testing kits. Using known unstable hepatitis C virus RNA, we developed a quantitative RT-PCR method based on a new primer design strategy to reduce the impact of nucleic acid instability on nucleic acid testing. The performance of the method was evaluated for linearity, limit of detection, precision, specificity, and agreement with commercial hepatitis C virus assays. Its clinical application was compared to that of two commercial kits-Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) and Kehua. The quantitative RT-PCR method delivered a good performance, with a linearity of R(2) = 0.99, a total limit of detection (genotypes 1 to 6) of 42.6 IU/mL (95% CI, 32.84 to 67.76 IU/mL), a CV of 1.06% to 3.34%, a specificity of 100%, and a high concordance with the CAP/CTM assay (R(2) = 0.97), with a means ± SD value of -0.06 ± 1.96 log IU/mL (range, -0.38 to 0.25 log IU/mL). The method was superior to commercial assays in detecting unstable hepatitis C virus RNA (P < 0.05). This quantitative RT-PCR method can effectively eliminate the influence of RNA instability on nucleic acid testing. The principle of primer design strategy may be applied to the detection of other RNA or DNA viruses. PMID:26612712

  12. Random Tagging Genotyping by Sequencing (rtGBS), an Unbiased Approach to Locate Restriction Enzyme Sites across the Target Genome

    PubMed Central

    Hilario, Elena; Barron, Lorna; Deng, Cecilia H.; Datson, Paul M.; Davy, Marcus W.; Storey, Roy D.

    2015-01-01

    Genotyping by sequencing (GBS) is a restriction enzyme based targeted approach developed to reduce the genome complexity and discover genetic markers when a priori sequence information is unavailable. Sufficient coverage at each locus is essential to distinguish heterozygous from homozygous sites accurately. The number of GBS samples able to be pooled in one sequencing lane is limited by the number of restriction sites present in the genome and the read depth required at each site per sample for accurate calling of single-nucleotide polymorphisms. Loci bias was observed using a slight modification of the Elshire et al. method: some restriction enzyme sites were represented in higher proportions while others were poorly represented or absent. This bias could be due to the quality of genomic DNA, the endonuclease and ligase reaction efficiency, the distance between restriction sites, the preferential amplification of small library restriction fragments, or bias towards cluster formation of small amplicons during the sequencing process. To overcome these issues, we have developed a GBS method based on randomly tagging genomic DNA (rtGBS). By randomly landing on the genome, we can, with less bias, find restriction sites that are far apart, and undetected by the standard GBS (stdGBS) method. The study comprises two types of biological replicates: six different kiwifruit plants and two independent DNA extractions per plant; and three types of technical replicates: four samples of each DNA extraction, stdGBS vs. rtGBS methods, and two independent library amplifications, each sequenced in separate lanes. A statistically significant unbiased distribution of restriction fragment size by rtGBS showed that this method targeted 49% (39,145) of BamH I sites shared with the reference genome, compared to only 14% (11,513) by stdGBS. PMID:26633193

  13. Identification of suitable reference genes for gene expression normalization in qRT-PCR analysis in watermelon.

    PubMed

    Kong, Qiusheng; Yuan, Jingxian; Gao, Lingyun; Zhao, Shuang; Jiang, Wei; Huang, Yuan; Bie, Zhilong

    2014-01-01

    Watermelon is one of the major Cucurbitaceae crops and the recent availability of genome sequence greatly facilitates the fundamental researches on it. Quantitative real-time reverse transcriptase PCR (qRT-PCR) is the preferred method for gene expression analyses, and using validated reference genes for normalization is crucial to ensure the accuracy of this method. However, a systematic validation of reference genes has not been conducted on watermelon. In this study, transcripts of 15 candidate reference genes were quantified in watermelon using qRT-PCR, and the stability of these genes was compared using geNorm and NormFinder. geNorm identified ClTUA and ClACT, ClEF1? and ClACT, and ClCAC and ClTUA as the best pairs of reference genes in watermelon organs and tissues under normal growth conditions, abiotic stress, and biotic stress, respectively. NormFinder identified ClYLS8, ClUBCP, and ClCAC as the best single reference genes under the above experimental conditions, respectively. ClYLS8 and ClPP2A were identified as the best reference genes across all samples. Two to nine reference genes were required for more reliable normalization depending on the experimental conditions. The widely used watermelon reference gene 18SrRNA was less stable than the other reference genes under the experimental conditions. Catalase family genes were identified in watermelon genome, and used to validate the reliability of the identified reference genes. ClCAT1and ClCAT2 were induced and upregulated in the first 24 h, whereas ClCAT3 was downregulated in the leaves under low temperature stress. However, the expression levels of these genes were significantly overestimated and misinterpreted when 18SrRNA was used as a reference gene. These results provide a good starting point for reference gene selection in qRT-PCR analyses involving watermelon. PMID:24587403

  14. ALK-rearranged lung cancer in Chinese: a comprehensive assessment of clinicopathology, IHC, FISH and RT-PCR.

    PubMed

    Li, Yuan; Pan, Yunjian; Wang, Rui; Sun, Yihua; Hu, Haichuan; Shen, Xuxia; Lu, Yongming; Shen, Lei; Zhu, Xiongzeng; Chen, Haiquan

    2013-01-01

    Approximately 3-7% of non-small cell lung cancers harbor an anaplastic lymphoma kinase (ALK) gene fusion, constituting a new molecular subtype of lung cancer that responds to crizotinib, an ALK inhibitor. Although previous studies have evaluated ALK-rearranged lung cancers, the comprehensive analysis of lung cancer in Chinese has not well assessed. Herein, we identified 44 cases of ALK-rearranged samples by fluorescent in-situ hybridization (FISH), immunohistochemistry (IHC), and reverse transcription polymerase chain reaction (RT-PCR) in a large number of surgically resected lung cancers. All 44 ALK-rearranged lung cancers were adenocarcinomas, with 2 cases having additional focal squamous components. The goal was to analyse the clinicopathological features of ALK-rearranged lung adenocarcinomas. Our data showed that a cribriform structure, prominent extracellular mucus and any type of mucous cell pattern may be either sensitive or specific to predict an ALK rearrangement. We used FISH as the standard detection method. We compared the ALK rearrangement accuracy of FISH, RT-PCR and IHC. RT-PCR could define both the ALK fusion partner and the fusion variant, but seemed unable to detect all translocations involving the ALK gene. It is noteworthy that IHC using the D5F3 antibody (Cell Signaling Technology) showed higher sensitivity and specificity than the ALK1 antibody (Dako). Therefore, we conclude that IHC remains a cost-effective and efficient technique for diagnosing ALK rearrangements and that D5F3 can be the optimal screening antibody in clinical practice. PMID:23922677

  15. Norovirus and FRNA bacteriophage determined by RT-qPCR and infectious FRNA bacteriophage in wastewater and oysters.

    PubMed

    Flannery, John; Keaveney, Sinéad; Rajko-Nenow, Paulina; O'Flaherty, Vincent; Doré, William

    2013-09-15

    Norovirus (NoV), the leading cause of adult non-bacterial gastroenteritis can be commonly detected in wastewater but the extent of NoV removal provided by wastewater treatment plants (WWTPs) is unclear. We monitored a newly commissioned WWTP with UV disinfection on a weekly basis over a six month period for NoV using RT-qPCR and for FRNA bacteriophage GA using both RT-qPCR (total concentration) and a plaque assay (infectious concentration). Mean concentrations of NoV GI and GII in influent wastewater were reduced by 0.25 and 0.41 log10 genome copies 100 ml(-1), respectively by the WWTP. The mean concentration of total FRNA bacteriophage GA was reduced by 0.35 log genome copies 100 ml(-1) compared to a reduction of infectious FRNA bacteriophage GA of 2.13 log PFU 100 ml(-1). A significant difference between concentrations of infectious and total FRNA bacteriophage GA was observed in treated, but not in untreated wastewaters. We conclude that RT-qPCR in isolation underestimates the reduction of infectious virus during wastewater treatment. We further compared the concentrations of infectious virus in combined sewer overflow (CSO) and UV treated effluents using FRNA bacteriophage GA. A greater percentage (98%) of infectious virus is released in CSO discharges than UV treated effluent (44%). Following a CSO discharge, concentrations of NoV GII and infectious FRNA bacteriophage GA in oysters from less than the limit of detection to 3150 genome copies 100 g(-1) and 1050 PFU 100 g(-1) respectively. PMID:23850211

  16. MATH 317 SUPPLEMENTARY PROBLEM SET I 1) Find the velocity, speed and acceleration at time t of the particle whose position is r(t). Describe the path

    E-print Network

    Carrell, Jim

    MATH 317 SUPPLEMENTARY PROBLEM SET I 1) Find the velocity, speed and acceleration at time t of the particle whose position is r(t). Describe the path of the particle. a) r(t) = a cos t^i + a sin t ^ + ct ^k is in the air, it experiences a downward (vertical) gravitational acceleration of 9.8 m/s2 and an eastward

  17. File: LecRlRtF Version dated April 8, 2014 2:52 pm Prof. W. Kahan Notes for Math. 128 A & B page 1/26

    E-print Network

    California at Berkeley, University of

    File: LecRlRtF Version dated April 8, 2014 2:52 pm Prof. W. Kahan Notes for Math. 128 A & B page 1/26 A & B page 3/26 Given a real equation " f(z) = 0 ", we solve it for its real root z , a zero of function;File: LecRlRtF Version dated April 8, 2014 2:52 pm Prof. W. Kahan Notes for Math. 128 A & B page 4/26

  18. Projected Applications of a "Weather in a Box" Computing System at the NASA Short-Term Prediction Research and Transition (SPoRT) Center

    NASA Technical Reports Server (NTRS)

    Jedlovec, Gary J.; Molthan, Andrew; Zavodsky, Bradley T.; Case, Jonathan L.; LaFontaine, Frank J.; Srikishen, Jayanthi

    2010-01-01

    The NASA Short-term Prediction Research and Transition Center (SPoRT)'s new "Weather in a Box" resources will provide weather research and forecast modeling capabilities for real-time application. Model output will provide additional forecast guidance and research into the impacts of new NASA satellite data sets and software capabilities. By combining several research tools and satellite products, SPoRT can generate model guidance that is strongly influenced by unique NASA contributions.

  19. RT-2 DETECTION OF QUASI-PERIODIC PULSATIONS IN THE 2009 JULY 5 SOLAR HARD X-RAY FLARE

    SciTech Connect

    Rao, A. R.; Malkar, J. P.; Hingar, M. K.; Agrawal, V. K.; Chakrabarti, S. K.; Nandi, A.; Debnath, D.; Kotoch, T. B.; Chidambaram, T. R.; Vinod, P.; Sreekumar, S.; Kotov, Y. D.; Buslov, A. S.; Yurov, V. N.; Tyshkevich, V. G.; Arkhangelskij, A. I.; Zyatkov, R. A.; Begum, S. Shaheda; Manoharan, P. K.

    2010-05-10

    We present the results of an analysis of hard X-ray observations of the C2.7 solar flare detected by the RT-2 experiment on board the Coronas-Photon satellite. We detect hard X-ray pulsations at periods of {approx}12 s and {approx}15 s. We find a marginal evidence for a decrease in period with time. We have augmented these results using the publicly available data from the RHESSI satellite. We present a spectral analysis and measure the spectral parameters.

  20. A database paradigm for the management of DICOM-RT structure sets using a geographic information system

    NASA Astrophysics Data System (ADS)

    Shao, Weber; Kupelian, Patrick A.; Wang, Jason; Low, Daniel A.; Ruan, Dan

    2014-03-01

    We devise a paradigm for representing the DICOM-RT structure sets in a database management system, in such way that secondary calculations of geometric information can be performed quickly from the existing contour definitions. The implementation of this paradigm is achieved using the PostgreSQL database system and the PostGIS extension, a geographic information system commonly used for encoding geographical map data. The proposed paradigm eliminates the overhead of retrieving large data records from the database, as well as the need to implement various numerical and data parsing routines, when additional information related to the geometry of the anatomy is desired.

  1. MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation

    PubMed Central

    Zubakov, Dmitry; Boersma, Anton W. M.; Choi, Ying; van Kuijk, Patricia F.; Wiemer, Erik A. C.

    2010-01-01

    MicroRNAs (miRNAs) are non-protein coding molecules with important regulatory functions; many have tissue-specific expression patterns. Their very small size in principle makes them less prone to degradation processes, unlike messenger RNAs (mRNAs), which were previously proposed as molecular tools for forensic body fluid identification. To identify suitable miRNA markers for forensic body fluid identification, we first screened total RNA samples derived from saliva, semen, vaginal secretion, and venous and menstrual blood for the expression of 718 human miRNAs using a microarray platform. All body fluids could be easily distinguished from each other on the basis of complete array-based miRNA expression profiles. Results from quantitative reverse transcription PCR (RT-PCR; TaqMan) assays for microarray candidate markers confirmed strong over-expression in the targeting body fluid of several miRNAs for venous blood and several others for semen. However, no candidate markers from array experiments for other body fluids such as saliva, vaginal secretion, or menstrual blood could be confirmed by RT-PCR. Time-wise degradation of venous blood and semen stains for at least 1 year under lab conditions did not significantly affect the detection sensitivity of the identified miRNA markers. The detection limit of the TaqMan assays tested for selected venous blood and semen miRNA markers required only subpicogram amounts of total RNA per single RT-PCR test, which is considerably less than usually needed for reliable mRNA RT-PCR detection. We therefore propose the application of several stable miRNA markers for the forensic identification of blood stains and several others for semen stain identification, using commercially available TaqMan assays. Additional work remains necessary in search for suitable miRNA markers for other forensically relevant body fluids. Electronic supplementary material The online version of this article (doi:10.1007/s00414-009-0402-3) contains supplementary material, which is available to authorized users. PMID:20145944

  2. Insights into the Mechanism of Severe Mitral Regurgitation: RT-3D TEE Guided Management with Pathological Correlation

    PubMed Central

    Anand, Senthil; Hamoud, Naktal; Thompson, Jess; Janardhanan, Rajesh

    2015-01-01

    Mitral valve perforation is an uncommon but important complication of infective endocarditis. We report a case of a 65-year-old man who was diagnosed to have infective endocarditis of his mitral valve. Through the course of his admission he had a rapid development of hemodynamic instability and pulmonary edema secondary to acutely worsening mitral regurgitation. While the TEE demonstrated an increase in the size of his bacterial vegetation, Real Time 3D TEE was ultimately the imaging modality through which the valve perforation was identified. Through this case report we discuss the advantages that RT-3D TEE has over traditional 2D TEE in the management of valve perforation.

  3. Specific detection of all members of the Venezuelan Equine Encephalitis complex: development of a RT-Nested PCR.

    PubMed

    Pisano, María Belén; Seco, María Paz Sánchez; Ré, Viviana Elizabeth; Farías, Adrián A; Contigiani, Marta Silvia; Tenorio, Antonio

    2012-12-01

    Venezuelan Equine Encephalitis (VEE) complex belongs to alphavirus genus in the family Togaviridae. Several species of this complex are pathogenic to humans. VEE infections can produce severe or mild disease, and many cases remain undiagnosed. A specific and sensitive reverse transcriptase nested polymerase chain reaction (RT-Nested PCR) method was developed for the detection of all VEE subtypes, including Rio Negro Virus (RNV) (subtype VI), which circulates only in Argentina. Degenerated primers were designed and thermal cycling parameters were standardized. This technique is suitable for rapid and specific detection of these viruses, and may be useful for diagnosis and surveillance. PMID:22609888

  4. The NASA Short-term Prediction and Research Transition (SPoRT) Center: A Research to Operations Test Bed

    NASA Technical Reports Server (NTRS)

    Jedlovec, Gary J.

    2005-01-01

    Over the last three years, NASA/MSFC scientists have embarked on an effort to transition unique NASA EOS data/products and research technology to selected NWSEOs in the southeast U.S. This activity, called the Short-term Prediction and - Research Transition (SPoRT) program, supports the NASA Science Mission Directorate and its Earth-Sun System Mission to develop a scientific understanding of the Earth System and its response to natural or human-induced changes that will enable improved prediction capability for climate, weather, and natural hazards. The overarching question related to weather prediction is "How well can weather forecasting duration and reliability be improved by new space-based observations, data assimilation, and modeling?" The transition activity has included the real-time delivery of MODIS data and products to several NWS Forecast Offices. Local NWS FOs have used the MODIS data to complement the coarse resolution GOES data for a number of applications. Specialized products have also been developed and made available to local and remote offices for their weather applications. Data from &e Lightning Mapping Array (LMA) network has been used in severe storm forecasts at several offices in the region. At the regional scale and forecast horizons from 0-1 day, the next generation of high-resolution mesoscale forecast and data assimilation models have been used to provide local offices with unique weather forecasts not otherwise available. The continued use of near red-time infusion of NASA science products into high-resolution mesoscale forecast and decision-making models can be expected to improve the model initialization as well as short-term forecasts. A current focus of SPoRT is to expand collaborations to include contributions from the assimilation of AMSR-E data in the ADASIARPS forecast system (OU), inclusion of MODIS SSTs and AIRS thermodynamic profiles in the WRF, and to extend the distribution of real-time MODIS and AMSR-E data and products to the Florida coastal WFOs. A SPoRT Test bed, together with input from other interagency and university partners, will provide a means and a process to effectively transition ESE observations and technology to NWS operations and decision makers at both the globdnational and regional scales. The transition of emerging experimental products into operations through the SPoRT infrastructure will allow NASA to foster and accelerate the progress of this Science Mission Directorate research strategy over the coming years.

  5. Short communication: a repeated simian human immunodeficiency virus reverse transcriptase/herpes simplex virus type 2 cochallenge macaque model for the evaluation of microbicides.

    PubMed

    Kenney, Jessica; Derby, Nina; Aravantinou, Meropi; Kleinbeck, Kyle; Frank, Ines; Gettie, Agegnehu; Grasperge, Brooke; Blanchard, James; Piatak, Michael; Lifson, Jeffrey D; Zydowsky, Thomas M; Robbiani, Melissa

    2014-11-01

    Epidemiological studies suggest that prevalent herpes simplex virus type 2 (HSV-2) infection increases the risk of HIV acquisition, underscoring the need to develop coinfection models to evaluate promising prevention strategies. We previously established a single high-dose vaginal coinfection model of simian human immunodeficiency virus (SHIV)/HSV-2 in Depo-Provera (DP)-treated macaques. However, this model does not appropriately mimic women's exposure. Repeated limiting dose SHIV challenge models are now used routinely to test prevention strategies, yet, at present, there are no reports of a repeated limiting dose cochallenge model in which to evaluate products targeting HIV and HSV-2. Herein, we show that 20 weekly cochallenges with 2-50 TCID50 simian human immunodeficiency virus reverse transcriptase (SHIV-RT) and 10(7) pfu HSV-2 results in infection with both viruses (4/6 SHIV-RT, 6/6 HSV-2). The frequency and level of vaginal HSV-2 shedding were significantly greater in the repeated exposure model compared to the single high-dose model (p<0.0001). We used this new model to test the Council's on-demand microbicide gel, MZC, which is active against SHIV-RT in DP-treated macaques and HSV-2 and human papillomavirus (HPV) in mice. While MZC reduced SHIV and HSV-2 infections in our repeated limiting dose model when cochallenging 8?h after each gel application, a barrier effect of carrageenan (CG) that was not seen in DP-treated animals precluded evaluation of the significance of the antiviral activity of MZC. Both MZC and CG significantly (p<0.0001) reduced the frequency and level of vaginal HSV-2 shedding compared to no gel treatment. This validates the use of this repeated limiting dose cochallenge model for testing products targeting HIV and HSV-2. PMID:25354024

  6. Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro

    PubMed Central

    Zhang, Haili; Zhu, Guan

    2015-01-01

    Our laboratory has previously developed a qRT-PCR assay to assess drug efficacy on the growth of Cryptosporidium parvum in vitro by detecting the levels of parasite 18S rRNA. This approach displayed up to four orders of magnitude of linear dynamic range and was much less labor-intensive than the traditional microscopic methods. However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures. Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent). Using these reagents, we are able to adapt the qRT-PCR assay into high-throughput screening of drugs in vitro (i.e., 96-well and 384-well formats for the cultivation of parasites and qRT-PCR detection, respectively). This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites. The new assay is also validated by the NIH-recommended intra-plate, inter-plate, and inter-day uniformity tests. The robustness and effectiveness of the assay are also confirmed by evaluating the anti-cryptosporidial efficacy of paromomycin and by a small scale screening of compounds. PMID:26441920

  7. Assignment of the human RT6 gene to 11q13 by PCR screening of somatic cell hybrids and in situ hybridization

    SciTech Connect

    Koch-Nolte, F.; Haag, F.; Kuehl, M.; Thiele, H.G.; Singh, S. ); Van Heyningen, V. ); Hoovers, J. ); Grzeschik, K.H. )

    1993-11-01

    RT6 is a T cell membrane protein that has attracted interest because a defect in RT6 expression is associated with susceptibility to autoimmune type I diabetes in DP-BB rats and NOD mice. Using PCR screening of human/rodent somatic cell hybrids and fluorescence in situ hybridization, the authors have determined that the gene for the human RT6 homologue is located at 11q13, centromeric to the gene for tyrosinase (TYR, albino locus) and telomeric to that for fibroblast growth factor 4 (FGF4). The data suggest that the human RT6 gene constitutes a new linkage group with TYR and the gene for olfactory marker protein (OMP) on 11q, which has a counterpart in mouse chromosome 7. Thus, in the human, the RT6 locus is dissociated from the hemoglobin [beta] chain locus (HBB) and its neighboring conserved linkage group at 11q15, in contrast to the mouse, in which RT6 shows a tighter linkage to Hbb than to Tyr. The results support the conclusion that there has been considerable intrachromosomal reshuffling of linked genes since the divergence of primates and rodents. 9 refs., 1 fig.

  8. A One-Step Real-Time RT-PCR Assay for the Detection and Quantitation of Sugarcane Streak Mosaic Virus

    PubMed Central

    Fu, Wei-Lin; Sun, Sheng-Ren; Fu, Hua-Ying; Chen, Ru-Kai; Su, Jin-Wei; Gao, San-Ji

    2015-01-01

    Sugarcane mosaic disease is caused by the Sugarcane streak mosaic virus (SCSMV; genus Poacevirus, family Potyviridae) which is common in some Asian countries. Here, we established a protocol of a one-step real-time quantitative reverse transcription PCR (real-time qRT-PCR) using the TaqMan probe for the detection of SCSMV in sugarcane. Primers and probes were designed within the conserved region of the SCSMV coat protein (CP) gene sequences. Standard single-stranded RNA (ssRNA) generated by PCR-based gene transcripts of recombinant pGEM-CP plasmid in vitro and total RNA extracted from SCSMV-infected sugarcane were used as templates of qRT-PCR. We further performed a sensitivity assay to show that the detection limit of the assay was 100 copies of ssRNA and 2?pg of total RNA with good reproducibility. The values obtained were approximately 100-fold more sensitive than those of the conventional RT-PCR. A higher incidence (68.6%) of SCSMV infection was detected by qRT-PCR than that (48.6%) with conventional RT-PCR in samples showing mosaic symptoms. SCSMV-free samples were verified by infection with Sugarcane mosaic virus (SCMV) or Sorghum mosaic virus (SrMV) or a combination of both. The developed qRT-PCR assay may become an alternative molecular tool for an economical, rapid, and efficient detection and quantification of SCSMV. PMID:26185758

  9. An immunomagnetic separation-reverse transcription polymerase chain reaction (IMS-RT-PCR) test for sensitive and rapid detection of viable waterborne Cryptosporidium parvum.

    PubMed

    Hallier-Soulier, Sylvie; Guillot, Emmanuelle

    2003-07-01

    The public health problem posed by the waterborne parasite Cryptosporidium parvum incited the water supply industry to develop very accurate analytical tools able to assess the presence of viable oocysts in drinking water. In this study, we report the development of a viability assay for C. parvum oocysts based on immunomagnetic separation and reverse transcription polymerase chain reaction (IMS-RT-PCR). The detection limit of the IMS-RT-PCR assay, which targets the hsp70 heat shock-induced mRNA, was in the range of ten viable oocysts per 100-l tap water samples. Purified Cryptosporidium parvum oocysts were exposed to heating, freezing and three chemical disinfection treatments namely, chlorination, chlorine dioxide treatment and ozonation under conventional doses used in water treatment plants, then detected by IMS-PCR and IMS-RT-PCR. The results obtained by IMS-PCR showed that none of the treatments had an effect on oocyst detection. The inactivation of oocysts by boiling resulted in no RT-PCR signal. Chlorine as well as chlorine dioxide did not influence oocyst viability as determined by IMS-RT-PCR. Ozone more effectively inactivated oocysts. The IMS-RT-PCR assay in conjunction with IMS-PCR marks the development of a combined detection and viability test which can be used for drinking water quality control as well as for reliable evaluation of treatment efficiency. PMID:12823191

  10. Selection of reference genes for RT-qPCR analysis in a predatory biological control agent, Coleomegilla maculata (Coleoptera: Coccinellidae).

    PubMed

    Yang, Chunxiao; Pan, Huipeng; Noland, Jeffrey Edward; Zhang, Deyong; Zhang, Zhanhong; Liu, Yong; Zhou, Xuguo

    2015-01-01

    Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for quantifying gene expression across various biological processes, of which requires a set of suited reference genes to normalize the expression data. Coleomegilla maculata (Coleoptera: Coccinellidae), is one of the most extensively used biological control agents in the field to manage arthropod pest species. In this study, expression profiles of 16 housekeeping genes selected from C. maculata were cloned and investigated. The performance of these candidates as endogenous controls under specific experimental conditions was evaluated by dedicated algorithms, including geNorm, Normfinder, BestKeeper, and ?Ct method. In addition, RefFinder, a comprehensive platform integrating all the above-mentioned algorithms, ranked the overall stability of these candidate genes. As a result, various sets of suitable reference genes were recommended specifically for experiments involving different tissues, developmental stages, sex, and C. maculate larvae treated with dietary double stranded RNA. This study represents the critical first step to establish a standardized RT-qPCR protocol for the functional genomics research in a ladybeetle C. maculate. Furthermore, it lays the foundation for conducting ecological risk assessment of RNAi-based gene silencing biotechnologies on non-target organisms; in this case, a key predatory biological control agent. PMID:26656102

  11. Single cell analysis of cancer cells using an improved RT-MLPA method has potential for cancer diagnosis and monitoring

    PubMed Central

    Kvastad, L.; Werne Solnestam, B.; Johansson, E.; Nygren, A. O.; Laddach, N.; Sahlén, P.; Vickovic, S.; Bendigtsen, Schirmer C.; Aaserud, M.; Floer, L.; Borgen, E.; Schwind, C.; Himmelreich, R.; Latta, D.; Lundeberg, J.

    2015-01-01

    Single cell analysis techniques have great potential in the cancer genomics field. The detection and characterization of circulating tumour cells are important for identifying metastatic disease at an early stage and monitoring it. This protocol is based on transcript profiling using Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification (RT-MLPA), which is a specific method for simultaneous detection of multiple mRNA transcripts. Because of the small amount of (circulating) tumour cells, a pre-amplification reaction is performed after reverse transcription to generate a sufficient number of target molecules for the MLPA reaction. We designed a highly sensitive method for detecting and quantifying a panel of seven genes whose expression patterns are associated with breast cancer, and optimized the method for single cell analysis. For detection we used a fluorescence-dependent semi-quantitative method involving hybridization of unique barcodes to an array. We evaluated the method using three human breast cancer cell lines and identified specific gene expression profiles for each line. Furthermore, we applied the method to single cells and confirmed the heterogeneity of a cell population. Successful gene detection from cancer cells in human blood from metastatic breast cancer patients supports the use of RT-MLPA as a diagnostic tool for cancer genomics. PMID:26558529

  12. Single cell analysis of cancer cells using an improved RT-MLPA method has potential for cancer diagnosis and monitoring.

    PubMed

    Kvastad, L; Werne Solnestam, B; Johansson, E; Nygren, A O; Laddach, N; Sahlén, P; Vickovic, S; Bendigtsen, Schirmer C; Aaserud, M; Floer, L; Borgen, E; Schwind, C; Himmelreich, R; Latta, D; Lundeberg, J

    2015-01-01

    Single cell analysis techniques have great potential in the cancer genomics field. The detection and characterization of circulating tumour cells are important for identifying metastatic disease at an early stage and monitoring it. This protocol is based on transcript profiling using Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification (RT-MLPA), which is a specific method for simultaneous detection of multiple mRNA transcripts. Because of the small amount of (circulating) tumour cells, a pre-amplification reaction is performed after reverse transcription to generate a sufficient number of target molecules for the MLPA reaction. We designed a highly sensitive method for detecting and quantifying a panel of seven genes whose expression patterns are associated with breast cancer, and optimized the method for single cell analysis. For detection we used a fluorescence-dependent semi-quantitative method involving hybridization of unique barcodes to an array. We evaluated the method using three human breast cancer cell lines and identified specific gene expression profiles for each line. Furthermore, we applied the method to single cells and confirmed the heterogeneity of a cell population. Successful gene detection from cancer cells in human blood from metastatic breast cancer patients supports the use of RT-MLPA as a diagnostic tool for cancer genomics. PMID:26558529

  13. Study of housekeeping gene expression in human keratinocytes using OLISA, a long-oligonucleotide microarray and q RT-PCR.

    PubMed

    Bonnet-Duquennoy, M; Abaibou, H; Tailhardat, M; Lazou, K; Bosset, S; Le Varlet, B; Cleuziat, P; Kurfürst, R

    2006-01-01

    In recent years, applications of microarray platforms have been extended to different areas of research including cosmetic and pharmaceutical. Although microarray technology is still improving its sensitivity and flexibility, researchers often turn toward quantitative RT-PCR for data validation. Assessment of messenger RNA quantity by these methods is based on comparison with internal standard genes, mainly housekeeping genes, so called because their synthesis occurs normally at a constant level. However, numerous studies showed that expression of these genes could vary in given situations. Here, we report results on four housekeeping genes (GAPDH, beta-2 microglobulin, S40 and S26 ribosomal sub-units) with constant expression levels established on OLISA microarray using different keratinocyte cultures. Moreover, qRT-PCR validation demonstrates that S26 ribosomal is a good housekeeping gene on keratinocytes and skin studies. Our data indicate that S26 gene can be routinely used to standardize results to investigate differentially expressed genes in a healthy human skin. PMID:16581563

  14. Expression of multidrug resistance proteins (Mrps) in astrocytes of the mouse brain: a single cell RT-PCR study.

    PubMed

    Hirrlinger, Johannes; Moeller, Heinz; Kirchhoff, Frank; Dringen, Ralf

    2005-10-01

    Multidrug resistance proteins (Mrps) are ATP-driven export pumps which mediate the export of organic anions such as glutathione conjugates and glucuronides from eukaryotic cells. Within the central nervous system astrocytes have important functions in metabolism and detoxification. In such processes Mrps play essential roles. To identify the Mrp repertoire of mouse brain and of astrocytes in particular, the expression of six mouse Mrps was investigated by reverse-transcription polymerase-chain reaction (RT-PCR). Using mouse brain mRNA as source, amplification products were obtained for Mrp1, Mrp3, Mrp4, Mrp5 and Mrp6. In contrast, mRNA of Mrp2 could not be detected in mouse brain. To investigate whether individual astrocytes express different Mrps in brain, single-cell RT-PCRs were performed from the cytosol harvested from single astrocytes in acutely isolated brain slices from cortex and cerebellum of TgN(GFAP-EGFP) mice. In these mice astrocytes can readily be identified by glial fibrillary acidic protein promoter-controlled green fluorescent protein expression. Investigation of individual cortical astrocytes and Bergmann glial cells revealed that these cells express Mrp1, Mrp4 and Mrp5 and that individual astrocytes can contain mRNA of one, two or three of these Mrps simultaneously. PMID:16341585

  15. Analysis of a Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) for yellow fever diagnostic.

    PubMed

    Nunes, Marcio R T; Vianez, João Lídio; Nunes, Keley N B; da Silva, Sandro Patroca; Lima, Clayton P S; Guzman, Hilda; Martins, Lívia C; Carvalho, Valéria L; Tesh, Robert B; Vasconcelos, Pedro F C

    2015-12-15

    Yellow Fever virus (YFV) is an important human pathogen in tropical areas of Africa and South America. Although an efficient vaccine is available and has been used since the early 1940s, sylvatic YFV transmission still occurs in forested areas where anthropogenic actions are present, such as mineral extraction, rearing livestock and agriculture, and ecological tourism. In this context, two distinct techniques based on the RT-PCR derived method have been previously developed, however both methods are expensive due to the use of thermo cyclers and labeled probes. We developed isothermal genome amplification, which is a rapid, sensitive, specific and low cost molecular approach for YFV genome detection. This assay used a set of degenerate primers designed for the NS1 gene and was able to amplify, within 30min in isothermal conditions, the YFV 17D vaccine strain derived from an African wild prototype strain (Asibi), as well as field strains from Brazil, other endemic countries from South and Central America, and the Caribbean. The generic RT-LAMP assay could be helpful for YFV surveillance in field and rapid response during outbreaks in endemic areas. PMID:26459206

  16. Four-leaf clover qRT-PCR: A convenient method for selective quantification of mature tRNA.

    PubMed

    Honda, Shozo; Shigematsu, Megumi; Morichika, Keisuke; Telonis, Aristeidis G; Kirino, Yohei

    2015-01-01

    Transfer RNAs (tRNAs) play a central role in translation and also recently appear to have a variety of other functions in biological processes beyond translation. Here we report the development of Four-Leaf clover qRT-PCR (FL-PCR), a convenient PCR-based method, which can specifically quantify individual mature tRNA species. In FL-PCR, T4 RNA ligase 2 specifically ligates a stem-loop adapter to mature tRNAs but not to precursor tRNAs or tRNA fragments. Subsequent TaqMan qRT-PCR amplifies only unmodified regions of the tRNA-adapter ligation products; therefore, FL-PCR quantification is not influenced by tRNA post-transcriptional modifications. FL-PCR has broad applicability for the quantification of various tRNAs in different cell types, and thus provides a much-needed simple method for analyzing tRNA abundance and heterogeneity. PMID:25833336

  17. Expression profiling of the lignin biosynthetic pathway in Norway spruce using EST sequencing and real-time RT-PCR.

    PubMed

    Koutaniemi, Sanna; Warinowski, Tino; Kärkönen, Anna; Alatalo, Edward; Fossdal, Carl G; Saranpää, Pekka; Laakso, Tapio; Fagerstedt, Kurt V; Simola, Liisa K; Paulin, Lars; Rudd, Stephen; Teeri, Teemu H

    2007-10-01

    Lignin biosynthesis is a major carbon sink in gymnosperms and woody angiosperms. Many of the enzymes involved are encoded for by several genes, some of which are also related to the biosynthesis of other phenylpropanoids. In this study, we aimed at the identification of those gene family members that are responsible for developmental lignification in Norway spruce (Picea abies (L.) Karst.). Gene expression across the whole lignin biosynthetic pathway was profiled using EST sequencing and quantitative real-time RT-PCR. Stress-induced lignification during bending stress and Heterobasidion annosum infection was also studied. Altogether 7,189 ESTs were sequenced from a lignin forming tissue culture and developing xylem of spruce, and clustered into 3,831 unigenes. Several paralogous genes were found for both monolignol biosynthetic and polymerisation-related enzymes. Real-time RT-PCR results highlighted the set of monolignol biosynthetic genes that are likely to be responsible for developmental lignification in Norway spruce. Potential genes for monolignol polymerisation were also identified. In compression wood, mostly the same monolignol biosynthetic gene set was expressed, but peroxidase expression differed from the vertically grown control. Pathogen infection in phloem resulted in a general up-regulation of the monolignol biosynthetic pathway, and in an induction of a few new gene family members. Based on the up-regulation under both pathogen attack and in compression wood, PaPAL2, PaPX2 and PaPX3 appeared to have a general stress-induced function. PMID:17764001

  18. Validation of reference genes for RT-qPCR analysis of CYP4T expression in crucian carp

    PubMed Central

    Mo, Fei; Zhao, Jie; Liu, Na; Cao, Li-hua; Jiang, Shan-xiang

    2014-01-01

    Reference genes are commonly used for normalization of target gene expression during RT-qPCR analysis. However, no housekeeping genes or reference genes have been identified to be stable across different tissue types or under different experimental conditions. To identify the most suitable reference genes for RT-qPCR analysis of target gene expression in the hepatopancreas of crucian carp (Carassius auratus) under various conditions (sex, age, water temperature, and drug treatments), seven reference genes, including beta actin (ACTB), beta-2 microglobulin (B2M), embryonic elongation factor-1 alpha (EEF1A), glyceraldehyde phosphate dehydrogenase (GAPDH), alpha tubulin (TUBA), ribosomal protein l8 (RPL8) and glucose-6-phosphate dehydrogenase (G6PDH), were evaluated in this study. The stability and ranking of gene expression were analyzed using three different statistical programs: GeNorm, Normfinder and Bestkeeper. The expression errors associated with selection of the genes were assessed by the relative quantity of CYP4T. The results indicated that all the seven genes exhibited variability under the experimental conditions of this research, and the combination of ACTB/TUBA/EEF1A or of ACTB/EEF1A was the best candidate that raised the accuracy of quantitative analysis of gene expression. The findings highlighted the importance of validation of housekeeping genes for research on gene expression under different conditions of experiment and species. PMID:25249772

  19. Rapid detection of rabies and rabies-related viruses by RT-PCR and enzyme-linked immunosorbent assay.

    PubMed

    Whitby, J E; Heaton, P R; Whitby, H E; O'Sullivan, E; Johnstone, P

    1997-12-01

    A rapid detection method for the six established genotypes of rabies and rabies-related viral RNA using RT-PCR-ELISA is described. The detection of digoxigenin-labelled amplified products is performed by solution hybridization to two specific, biotin-labelled, capture probes, which are complementary to the inner region of the amplification products. The capture probe and amplified product hybrid are then immobilised on a streptavidin-coated microtitre plate, bound products are detected by an anti-DIG Fab fragment conjugated to peroxidase, and colorimetric reaction automatically measured. This method was up to 100-fold more sensitive than Southern blot hybridization, detecting 0.00002 TCID50/ml of a genotype 1, classical rabies virus strain. The complete detection methodology from RT-PCR to PCR-ELISA detection could be completed within 10 h. Using this procedure, we were 100% successful in detecting 60 isolates from a representative selection of the six established genotypes from all over the world. This test is a useful additional tool for the detection of the rabies and rabies-related viruses, which is easy to perform, rapid and highly sensitive. PMID:9504752

  20. Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation

    NASA Astrophysics Data System (ADS)

    Zhao, Ye; Chen, Muyan; Wang, Tianming; Sun, Lina; Xu, Dongxue; Yang, Hongsheng

    2014-11-01

    Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues (intestine, respiratory tree, and muscle) of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the ?-tubulin (TUBB) gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 (RPS18), TUBB, and NADH dehydrogenase (NADH); for respiratory tree, it was ?-actin (ACTB), TUBB, and succinate dehydrogenase cytochrome B small subunit (SDHC); and for muscle it was ?-tubulin (TUBA) and NADH dehydrogenase [ubiquinone] 1 ? subcomplex subunit 13 (NDUFA13). These combinations of internal control genes should be considered for use in further studies of gene expression in A. japonicus during aestivation.

  1. Selection of reference genes for RT-qPCR analysis in a predatory biological control agent, Coleomegilla maculata (Coleoptera: Coccinellidae)

    PubMed Central

    Yang, Chunxiao; Pan, Huipeng; Noland, Jeffrey Edward; Zhang, Deyong; Zhang, Zhanhong; Liu, Yong; Zhou, Xuguo

    2015-01-01

    Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for quantifying gene expression across various biological processes, of which requires a set of suited reference genes to normalize the expression data. Coleomegilla maculata (Coleoptera: Coccinellidae), is one of the most extensively used biological control agents in the field to manage arthropod pest species. In this study, expression profiles of 16 housekeeping genes selected from C. maculata were cloned and investigated. The performance of these candidates as endogenous controls under specific experimental conditions was evaluated by dedicated algorithms, including geNorm, Normfinder, BestKeeper, and ?Ct method. In addition, RefFinder, a comprehensive platform integrating all the above-mentioned algorithms, ranked the overall stability of these candidate genes. As a result, various sets of suitable reference genes were recommended specifically for experiments involving different tissues, developmental stages, sex, and C. maculate larvae treated with dietary double stranded RNA. This study represents the critical first step to establish a standardized RT-qPCR protocol for the functional genomics research in a ladybeetle C. maculate. Furthermore, it lays the foundation for conducting ecological risk assessment of RNAi-based gene silencing biotechnologies on non-target organisms; in this case, a key predatory biological control agent. PMID:26656102

  2. Evaluation of Reference Genes for RT-qPCR in Tribolium castaneum (Coleoptera: Tenebrionidae) Under UVB Stress.

    PubMed

    Sang, Wen; He, Li; Wang, Xiao-Ping; Zhu-Salzman, Keyan; Lei, Chao-Liang

    2015-04-01

    Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) has become a widely used technique to quantify gene expression. It is necessary to select appropriate reference genes for normalization. In the present study, we assessed the expression stability of seven candidate genes in Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) irradiated by ultraviolet B (UVB) at different developmental stages for various irradiation time periods. The algorithms of geNorm, NormFinder, and BestKeeper were applied to determine the stability of these candidate genes. Ribosomal protein genes RpS3, RpL13A, and ?-actin gene (ActB) showed the highest stability across all UVB irradiation time points, whereas expression of other normally used reference genes, such as those encoding the ?-tubulin gene TUBB and the E-cadherin gene CAD, varied at different developmental stages. This study will potentially provide more suitable reference gene candidates for RT-qPCR analysis in T. castaneum subjected to environmental stresses, particularly UV irradiation. PMID:26313197

  3. Specific Interaction between eEF1A and HIV RT Is Critical for HIV-1 Reverse Transcription and a Potential Anti-HIV Target

    PubMed Central

    Rawle, Daniel J.; Qin, Fangyun; Wang, Rui; Soares, Dinesh C.; Jin, Hongping; Sivakumaran, Haran; Lin, Min-Hsuan; Spann, Kirsten; Abbott, Catherine M.; Harrich, David

    2015-01-01

    Reverse transcription is the central defining feature of HIV-1 replication. We previously reported that the cellular eukaryotic elongation factor 1 (eEF1) complex associates with the HIV-1 reverse transcription complex (RTC) and the association is important for late steps of reverse transcription. Here we show that association between the eEF1 and RTC complexes occurs by a strong and direct interaction between the subunit eEF1A and reverse transcriptase (RT). Using biolayer interferometry and co-immunoprecipitation (co-IP) assays, we show that association between the eEF1 and RTC complexes occurs by a strong (KD ~3–4 nM) and direct interaction between eEF1A and reverse transcriptase (RT). Biolayer interferometry analysis of cell lysates with titrated levels of eEF1A indicates it is a predominant cellular RT binding protein. Both the RT thumb and connection domains are required for interaction with eEF1A. A single amino acid mutation, W252A, within the thumb domain impaired co-IP between eEF1A and RT, and also significantly reduced the efficiency of late reverse transcription and virus replication when incorporated into infectious HIV-1. Molecular modeling analysis indicated that interaction between W252 and L303 are important for RT structure, and their mutation to alanine did not impair heterodimerisation, but negatively impacted interaction with eEF1A. Didemnin B, which specifically binds eEF1A, potently inhibited HIV-1 reverse transcription by greater than 2 logs at subnanomolar concentrations, especially affecting reverse transcription late DNA synthesis. Analysis showed reduced levels of RTCs from HIV-1-infected HEK293T treated with didemnin B compared to untreated cells. Interestingly, HIV-1 with a W252A RT mutation was resistant to didemnin B negative effects showing that didemnin B affects HIV-1 by targeting the RT-eEF1A interaction. The combined evidence indicates a direct interaction between eEF1A and RT is crucial for HIV reverse transcription and replication, and the RT-eEF1A interaction is a potential drug target. PMID:26624286

  4. Characterisation of RT1-E2, a multigenic family of highly conserved rat non-classical MHC class I molecules initially identified in cells from immunoprivileged sites

    PubMed Central

    Lau, Pierre; Amadou, Claire; Brun, Hélène; Rouillon, Virginie; McLaren, Fiona; Le Rolle, Anne-France; Graham, Margaret; Butcher, Geoffrey W; Joly, Etienne

    2003-01-01

    Background So-called "immunoprivileged sites" are tissues or organs where slow allograft rejection correlates with low levels of expression of MHC class I molecules. Whilst classical class I molecules are recognised by cytotoxic T lymphocytes (CTL), some MHC class I molecules are called "non-classical" because they exhibit low polymorphism and are not widely expressed. These last years, several studies have shown that these can play different, more specialised roles than their classical counterparts. In the course of efforts to characterise MHC class I expression in rat cells obtained from immunoprivileged sites such as the central nervous system or the placenta, a new family of non-classical MHC class I molecules, which we have named RT1-E2, has been uncovered. Results Members of the RT1-E2 family are all highly homologous to one another, and the number of RT1-E2 loci varies from one to four per MHC haplotype among the six rat strains studied so far, with some loci predicted to give rise to soluble molecules. The RT1n MHC haplotype (found in BN rats) carries a single RT1-E2 locus, which lies in the RT1-C/E region of the MHC and displays the typical exon-intron organisation and promoter features seen in other rat MHC class I genes. We present evidence that: i) RT1-E2 molecules can be detected at the surface of transfected mouse L cells and simian COS-7 cells, albeit at low levels; ii) their transport to the cell surface is dependent on a functional TAP transporter. In L cells, their transport is also hindered by protease inhibitors, brefeldin A and monensin. Conclusions These findings suggest that RT1-E2 molecules probably associate with ligands of peptidic nature. The high homology between the RT1-E2 molecules isolated from divergent rat MHC haplotypes is particularly striking at the level of their extra-cellular portions. Compared to other class I molecules, this suggests that RT1-E2 molecules may associate with well defined sets of ligands. Several characteristics point to a certain similarity to the mouse H2-Qa2 and human HLA-G molecules. PMID:12837137

  5. A systematic review of human norovirus survival reveals a greater persistence of human norovirus RT-qPCR signals compared to those of cultivable surrogate viruses.

    PubMed

    Knight, Angus; Haines, John; Stals, Ambroos; Li, Dan; Uyttendaele, Mieke; Knight, Alastair; Jaykus, Lee-Ann

    2016-01-01

    Human noroviruses (hNoV) are the single largest cause of acute gastroenteritis in the western world. The efficacy of hNoV control measures remains largely unknown, partly owing to the inability to grow the virus in vitro and partly to the large number of surrogate studies of unknown relevance. A systematic review of the persistence and survival of hNoV in foods and the environment was undertaken based upon PRISMA (preferred reporting items for systematic reviews and meta analyses) guidelines to answer the questions: (1) "What are the natural hNoV persistence characteristics in food and the environment?" and (2) "How can these properties be altered by applying physical and/or chemical treatments to foods or food contact surfaces?" Over 10,000 citations were screened using defined inclusion and exclusion criteria. One hundred and twenty-six (126) citations were identified for further evaluation and data were extracted based upon the conditions of study and treatment (e.g., treatment parameters, pH, and temperature, time, infectivity, and RT-qPCR results). Since the only markers for hNoV persistence and survival were RT-qPCR data and human challenge studies, citations for further analysis were restricted to only those that included data on hNoV behavior (using RT-qPCR) as compared directly to surrogate virus behavior (using both RT-qPCR and infectivity) in the same study, and clinical studies. Based on these criteria, a total of 12 independent studies (5 for thermal inactivation and 7 for available chlorine) and 3 human challenge studies were identified. RT-qPCR always underestimated reductions in surrogate virus titre as a function of treatment when compared to infectivity. The corresponding reductions in RT-qPCR signals for hNoV under comparable conditions were nearly always less than those observed for the surrogates. These relationships were statistically significant for heat when comparing persistence of hNoV RT-qPCR signals with surrogate MNV-1 RT-qPCR signals (P equal persistence=<0.07); and for free chlorine when comparing persistence of hNoV RT-qPCR signals to those of FCV F-9 (p=<0.01). Overall the data suggest that hNoV are frequently more resistant to typical food and environmental control measures compared with cultivable surrogate viruses, when basing data on comparative RT-qPCR results. PMID:26398283

  6. Effect of resistance training on C-reactive protein, blood glucose and lipid profile in older women with differing levels of RT experience.

    PubMed

    Ribeiro, Alex S; Tomeleri, Crisieli M; Souza, Mariana F; Pina, Fábio Luiz C; Schoenfeld, Brad J; Nascimento, Matheus A; Venturini, Danielle; Barbosa, Décio S; Cyrino, Edilson S

    2015-12-01

    The purpose of this study was to analyze the effects of a progressive resistance training (RT) program on C-reactive protein (CRP), blood glucose (GLU), and lipid profile in older women with differing levels of RT experience. Sixty-five older women (68.9 ± 6.1 years, 67.1 ± 13.1 kg) were separated according to RT experience: an advanced group composed by 35 participants who previously carried out 24 weeks of RT and a novice group composed by 30 participants without previous experience in RT (n = 30). Both groups performed a RT program comprised of eight exercises targeting all the major muscles. Training was carried out 3 days/week for 8 weeks. Serum triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), GLU, and CRP concentrations were determined pre- and post- intervention after 12 h fasting. A significant group by time interaction (P < 0.05) for the TC (novice = -1.9 % vs. advanced = 1.0 %), and CRP (novice = -22.9 % vs. advanced = -54.5 %) was observed. A main effect of time (P < 0.05) was identified for the GLU (novice = -2.6 % vs. advanced = -6.6 %), TG (novice = -12.9 % vs. advanced = -5.7 %), HDL-C (novice = +6.7 % vs. advanced = +2.6 %), and LDL-C (novice = -34.0 % vs. advanced = -25.4 %). These results suggest that RT improves the metabolic profile of older women and that training for a longer period of time seems to produce more pronounced reductions mainly on CRP. PMID:26499819

  7. Development of a two-step SYBR Green I based real time RT-PCR assay for detecting and quantifying peste des petits ruminants virus in clinical samples.

    PubMed

    Abera, Tsegalem; Thangavelu, Ardhanary

    2014-12-01

    A two-step SYBR Green I based real time RT-PCR targeting the matrix (M) gene of Peste des petits ruminants virus (PPRV) was developed. The specificity of the assay was assessed against viral nucleic acid extracted from a range of animal viruses of clinical and structural similarities to PPRV including canine distemper virus, measles virus, bluetongue virus and Newcastle disease virus. But none of the viruses and no template control showed an amplification signal. Sensitivity of the same assay was assessed based on plasmid DNA copy number and with respect to infectivity titre. The lower detection limit achieved was 2.88 plasmid DNA copies/?l with corresponding Ct value of 35.93. Based on tissue culture infectivity titre the lower detection limits were 0.0001TCID50/ml and 1TCID50/ml for the SYBR green I based real time RT-PCR and conventional RT-PCR, respectively. The calculated coefficient of variations values for intra- and inter-assay variability were low, ranging from 0.21% to 1.83% and 0.44% to 1.97%, respectively. The performance of newly developed assay was evaluated on a total of 36 clinical samples suspected of PPR and compared with conventional RT-PCR. The SYBR Green I based real time RT-PCR assay detected PPRV in 32 (88.8%) of clinical samples compared to 19 (52.7%) by conventional RT-PCR. Thus, the two-step SYBR Green I based real time RT-PCR assay targeting the M gene of PPRV reported in this study was highly sensitive, specific and reproducible for detection and quantitation of PPRV nucleic acids. PMID:25194891

  8. Identification of Candidate Reference Genes in Perennial Ryegrass for Quantitative RT-PCR under Various Abiotic Stress Conditions

    PubMed Central

    Jiang, Xiaomei; Yin, Guohua; Zhang, Xinquan; Qi, Xiao; Zhang, Yu; Yan, Yanhong; Ma, Xiao; Peng, Yan

    2014-01-01

    Background Quantitative real-time reverse-transcriptase PCR (qRT-PCR) is an important technique for analyzing differences in gene expression due to its sensitivity, accuracy and specificity. However, the stability of the expression of reference genes is necessary to ensure accurate qRT-PCR assessment of expression in genes of interest. Perennial ryegrass (Lolium perenne L.) is important forage and turf grass species in temperate regions, but the expression stability of its reference genes under various stresses has not been well-studied. Methodology/Principal Findings In this study, 11 candidate reference genes were evaluated for use as controls in qRT-PCR to quantify gene expression in perennial ryegrass under drought, high salinity, heat, waterlogging, and ABA (abscisic acid) treatments. Four approaches – Delta CT, geNorm, BestKeeper and Normfinder were used to determine the stability of expression in these reference genes. The results are consistent with the idea that the best reference genes depend on the stress treatment under investigation. Eukaryotic initiation factor 4 alpha (eIF4A), Transcription elongation factor 1 (TEF1) and Tat binding protein-1 (TBP-1) were the three most stably expressed genes under drought stress and were also the three best genes for studying salt stress. eIF4A, TBP-1, and Ubiquitin-conjugating enzyme (E2) were the most suitable reference genes to study heat stress, while eIF4A, TEF1, and E2 were the three best reference genes for studying the effects of ABA. Finally, Ubiquitin (UBQ), TEF1, and eIF4A were the three best reference genes for waterlogging treatments. Conclusions/Significance These results will be helpful in choosing the best reference genes for use in studies related to various abiotic stresses in perennial ryegrass. The stability of expression in these reference genes will enable better normalization and quantification of the transcript levels for studies of gene expression in such studies. PMID:24699822

  9. Direct sequencing of hepatitis A virus and norovirus RT-PCR products from environmentally contaminated oyster using M13-tailed primers.

    PubMed

    Williams-Woods, Jacquelina; González-Escalona, Narjol; Burkhardt, William

    2011-12-01

    Human norovirus (HuNoV) and hepatitis A (HAV) are recognized as leading causes of non-bacterial foodborne associated illnesses in the United States. DNA sequencing is generally considered the standard for accurate viral genotyping in support of epidemiological investigations. Due to the genetic diversity of noroviruses (NoV), degenerate primer sets are often used in conventional reverse transcription (RT) PCR and real-time RT-quantitative PCR (RT-qPCR) for the detection of these viruses and cDNA fragments are generally cloned prior to sequencing. HAV detection methods that are sensitive and specific for real-time RT-qPCR yields small fragments sizes of 89-150bp, which can be difficult to sequence. In order to overcome these obstacles, norovirus and HAV primers were tailed with M13 forward and reverse primers. This modification increases the sequenced product size and allows for direct sequencing of the amplicons utilizing complementary M13 primers. HuNoV and HAV cDNA products from environmentally contaminated oysters were analyzed using this method. Alignments of the sequenced samples revealed ?95% nucleotide identities. Tailing NoV and HAV primers with M13 sequence increases the cDNA product size, offers an alternative to cloning, and allows for rapid, accurate and direct sequencing of cDNA products produced by conventional or real time RT-qPCR assays. PMID:21963395

  10. Expansion of the Real-Time SPoRT-Land Information System for NOAA/National Weather Service Situational Awareness and Local Modeling Applications

    NASA Technical Reports Server (NTRS)

    Case, Jonathan L; White, Kristopher D.

    2014-01-01

    The NASA Short-term Prediction Research and Transition (SPoRT) Center in Huntsville, AL is running a real-time configuration of the Noah land surface model (LSM) within the NASA Land Information System (LIS) framework (hereafter referred to as the "SPoRT-LIS"). Output from the real-time SPoRT-LIS is used for (1) initializing land surface variables for local modeling applications, and (2) displaying in decision support systems for situational awareness and drought monitoring at select NOAA/National Weather Service (NWS) partner offices. The experimental CONUS run incorporates hourly quantitative precipitation estimation (QPE) from the National Severe Storms Laboratory Multi- Radar Multi-Sensor (MRMS) which will be transitioned into operations at the National Centers for Environmental Prediction (NCEP) in Fall 2014.This paper describes the current and experimental SPoRT-LIS configurations, and documents some of the limitations still remaining through the advent of MRMS precipitation analyses in the SPoRT-LIS land surface model (LSM) simulations.

  11. The survey of porcine teschoviruses in field samples in China with a universal rapid probe real-time RT-PCR assay.

    PubMed

    Zhang, Chaofan; Wang, Zhongtian; Hu, Feng; Liu, Yebing; Qiu, Zheng; Zhou, Shun; Cui, Shangjin; Wang, Ming

    2013-04-01

    A real-time reverse transcription polymerase chain reaction (RT-PCR) based on TaqMan was established and evaluated for quantitative detection of porcine teschoviruses (PTVs). A pair of primers and a TaqMan probe targeting on the highly conserved sequence of the 5'-untranslated region (5'-UTR) of one to 11 serotypes of PTV were designed. Standard plasmid DNA containing PCR amplification of the 5'-UTR were constructed and used to develop the real-time RT-PCR. The results indicated that the real-time RT-PCR was specific for detection of PTV with a detection limit of 10 copies/?L, but not for porcine parvovirus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, pseudorabies virus, classical swine fever virus. The coefficient of variation of inter-assay and intra-assay were less than 3 %. A total of 91 clinical samples were tested by the real-time RT-PCR and virus isolation (OIE 2008) and positive rates were 79.12 % (72/91) and 57.14 % (48/91), respectively. In conclusion, the developed real-time RT-PCR assay was an effective method for detection and quantification of PTV in fields or organs of infected pigs. PMID:23138413

  12. Bank Vole Prion Protein As an Apparently Universal Substrate for RT-QuIC-Based Detection and Discrimination of Prion Strains.

    PubMed

    Orrú, Christina D; Groveman, Bradley R; Raymond, Lynne D; Hughson, Andrew G; Nonno, Romolo; Zou, Wenquan; Ghetti, Bernardino; Gambetti, Pierluigi; Caughey, Byron

    2015-06-01

    Prions propagate as multiple strains in a wide variety of mammalian species. The detection of all such strains by a single ultrasensitive assay such as Real Time Quaking-induced Conversion (RT-QuIC) would facilitate prion disease diagnosis, surveillance and research. Previous studies have shown that bank voles, and transgenic mice expressing bank vole prion protein, are susceptible to most, if not all, types of prions. Here we show that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far--a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification. Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimination of prion strains such as classical and atypical L-type bovine spongiform encephalopathy, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt-Jakob disease in humans. Comparison of protease-resistant RT-QuIC conversion products also aided strain discrimination and suggested the existence of several distinct classes of prion templates among the many strains tested. PMID:26086786

  13. Bank Vole Prion Protein As an Apparently Universal Substrate for RT-QuIC-Based Detection and Discrimination of Prion Strains

    PubMed Central

    Raymond, Lynne D.; Hughson, Andrew G.; Nonno, Romolo; Zou, Wenquan; Ghetti, Bernardino; Gambetti, Pierluigi; Caughey, Byron

    2015-01-01

    Prions propagate as multiple strains in a wide variety of mammalian species. The detection of all such strains by a single ultrasensitive assay such as Real Time Quaking-induced Conversion (RT-QuIC) would facilitate prion disease diagnosis, surveillance and research. Previous studies have shown that bank voles, and transgenic mice expressing bank vole prion protein, are susceptible to most, if not all, types of prions. Here we show that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far – a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification. Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimination of prion strains such as classical and atypical L-type bovine spongiform encephalopathy, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt-Jakob disease in humans. Comparison of protease-resistant RT-QuIC conversion products also aided strain discrimination and suggested the existence of several distinct classes of prion templates among the many strains tested. PMID:26086786

  14. Measurement of a density profile of a hot-electron plasma in RT-1 with three-chord interferometry

    NASA Astrophysics Data System (ADS)

    Saitoh, H.; Yano, Y.; Yoshida, Z.; Nishiura, M.; Morikawa, J.; Kawazura, Y.; Nogami, T.; Yamasaki, M.

    2015-02-01

    The electron density profile of a plasma in a magnetospheric dipole field configuration was measured with a multi-chord interferometry including a relativistic correction. In order to improve the accuracy of density reconstruction, a 75 GHz interferometer was installed at a vertical chord of the Ring Trap 1 (RT-1) device in addition to previously installed ones at tangential and another vertical chords. The density profile was calculated by using the data of three-chord interferometry including relativistic effects for a plasma consisting of hot and cold electrons generated by electron cyclotron resonance heating (ECH). The results clearly showed the effects of density peaking and magnetic mirror trapping in a strongly inhomogeneous dipole magnetic field.

  15. Measurement of a density profile of a hot-electron plasma in RT-1 with three-chord interferometry

    SciTech Connect

    Saitoh, H.; Yano, Y.; Yoshida, Z.; Nishiura, M.; Morikawa, J.; Kawazura, Y.; Nogami, T.; Yamasaki, M.

    2015-02-15

    The electron density profile of a plasma in a magnetospheric dipole field configuration was measured with a multi-chord interferometry including a relativistic correction. In order to improve the accuracy of density reconstruction, a 75?GHz interferometer was installed at a vertical chord of the Ring Trap 1 (RT-1) device in addition to previously installed ones at tangential and another vertical chords. The density profile was calculated by using the data of three-chord interferometry including relativistic effects for a plasma consisting of hot and cold electrons generated by electron cyclotron resonance heating (ECH). The results clearly showed the effects of density peaking and magnetic mirror trapping in a strongly inhomogeneous dipole magnetic field.

  16. Analysis on the arcelin expression in bruchid pest resistant wild pulses using real time RT-qPCR.

    PubMed

    Sakthivelkumar, Shanmugavel; Veeramani, Velayutham; Hilda, Karuppiah; Arumugam, Munusamy; Janarthanan, Sundaram

    2014-12-01

    Arcelin, the antimetabolic protein from wild pulses is a known natural insecticidal molecule. Wild pulses with high arcelin content could serve as potential source to. increase the levels of insect resistance in cultivated pulse crops. In this study, arcelin (Arl) gene expression was screened in seven stored product insect pest resistant wild pulse varieties using real time RT-qPCR. Arcelin gene specific real time PCR primers were synthesized from arcelin mRNA sequence of the wild pulse variety, Lablab purpureus. The results revealed different levels of arcelin gene expression in the tested varieties. Canavalia virosa registered significantly high content indicating its suitability for utilization of arcelin gene in developing stored product insect pest resistance with other cultivated pulses. PMID:25651613

  17. Factor VIII (F8) inversions in severe hemophilia A: Male germ cell origin and diagnosis with RT-PCR

    SciTech Connect

    Antonarakis, S.E. |; Rossiter, J.P.; Young, M.

    1994-09-01

    The Factor VIII (F8) gene, which is defective in hemophilia A, is located in the most telomeric megabase of Xq. Inversions due to intrachromosomal homologous recombination between mispaired copies of gene A located within intron 22 of the gene and about 500 kb telomeric to it account for nearly half of the cases of severe hemophilia A. We hypothesized that pairing of Xq with its homolog inhibits the inversion process, and that therefore the event originates predominantly in male germ cells. In all 21 informative cases in which the inversion originated in a maternal grandparent, DNA polymorphism analysis using markers within or very closely linked to F8, determined that it occurred in the male germline. In addition, all but one of 56 mothers of sporadic cases due to inversions were carriers. The data indicate that the F8 gene inversions leading to severe hemophilia A occur almost exclusively in male germ cells. The mean age of maternal grandfathers at the birth of their carrier daughters was 29.9 years (13 cases), i.e. not different from the mean paternal age in the general population, supporting the hypothesis that the inversions occur in meiosis. The inversions can be diagnosed by Southern blot analysis. For more rapid diagnosis we have used RT-PCR of RNA ectopically expressed in blood. Oligonucleotides were used to PCR amplify, after the initial RT reaction of RNA samples using random hexamers, either the normal transcript (F8 exons 21 to 24;312 bp product) or the novel abnormal transcript that is generated after the inversion. Both type 1 and 2 inversions can be recognized in affecteds and carriers by the presence of the diagnostic PcR product of 248 bp. Correct diagnoses were made in samples from 6 patients and 2 carriers with type 1 inversions, 2 patients and 2 carriers with type 2 inversions and 5 normal controls.

  18. Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR.

    PubMed

    Chandna, Ruby; Augustine, Rehna; Bisht, Naveen C

    2012-01-01

    The real time quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important to gain insight into function of genes. Given the increased sensitivity, ease and reproducibility of qRT-PCR, the requirement of suitable reference genes for normalization has become important and stringent. It is now known that the expression of internal control genes in living organism vary considerably during developmental stages and under different experimental conditions. For economically important Brassica crops, only a couple of reference genes are reported till date. In this study, expression stability of 12 candidate reference genes including ACT2, ELFA, GAPDH, TUA, UBQ9 (traditional housekeeping genes), ACP, CAC, SNF, TIPS-41, TMD, TSB and ZNF (new candidate reference genes), in a diverse set of 49 tissue samples representing different developmental stages, stress and hormone treated conditions and cultivars of Brassica juncea has been validated. For the normalization of vegetative stages the ELFA, ACT2, CAC and TIPS-41 combination would be appropriate whereas TIPS-41 along with CAC would be suitable for normalization of reproductive stages. A combination of GAPDH, TUA, TIPS-41 and CAC were identified as the most suitable reference genes for total developmental stages. In various stress and hormone treated samples, UBQ9 and TIPS-41 had the most stable expression. Across five cultivars of B. juncea, the expression of CAC and TIPS-41 did not vary significantly and were identified as the most stably expressed reference genes. This study provides comprehensive information that the new reference genes selected herein performed better than the traditional housekeeping genes. The selection of most suitable reference genes depends on the experimental conditions, and is tissue and cultivar-specific. Further, to attain accuracy in the results more than one reference genes are necessary for normalization. PMID:22606308

  19. RT-PCR standardization and bone mineralization after low-level laser therapy on adult osteoblast cells

    NASA Astrophysics Data System (ADS)

    do Bomfim, Fernando R. C.; Sella, Valéria R. G.; Zanaga, Jéssica Q.; Pereira, Nayara S.; Nouailhetas, Viviane L. A.; Plapler, Hélio

    2014-03-01

    Purpose: Osteoblasts are capable to produce different compounds directly connected to bone mineralization process. This study aims to standardize the reverse transcriptase polymerase chain reaction (RT-PCR) for adult osteoblasts to observe the effect of low level laser therapy on bone mineralization. Methods: Five-millimeter long fragments obtained from the mead femoral region of male Wistar rats were assigned into group A (n=10, laser) and group B (n=10, no laser), submitted to mechanic and enzymatic digestion. After 7 days, cultures of group A were irradiated daily on a single spot with a GaInAs laser, ?=808nm, 200mW/cm2, 2J/cm2, bean diameter of 0,02mm, 5 seconds for 6 days. Group B was manipulated but received no laser irradiation. After 13 days the cells were trypsinized for 15 minute and stabilized with RNA later® for RNA extraction with Trizol®. cDNA synthesis used 10?g of RNA and M-MLV® enzyme. PCR was accomplished using the ?-actin gene as a control. Another aliquot was fixed for Hematoxylin-Eosin and Von Kossa staining to visualize bone mineralization areas. Results: Under UV light we observed clearly the amplification of ?-actin gene around 400bp. HE and Von Kossa staining showed osteoblast clusters, a higher number of bone cells and well defined mineralization areas in group A. Conclusion: The cell culture, RNA extraction and RT-PCR method for adult osteoblasts was effective, allowing to use these methods for bone mineralization studies. Laser improved bone mineralization and further studies are needed involving osteogenesis, calcium release mechanisms and calcium related channels.

  20. Cationic amphipathic peptides KT2 and RT2 are taken up into bacterial cells and kill planktonic and biofilm bacteria.

    PubMed

    Anunthawan, Thitiporn; de la Fuente-Núñez, César; Hancock, Robert E W; Klaynongsruang, Sompong

    2015-06-01

    We investigated the mechanisms of two tryptophan-rich antibacterial peptides (KT2 and RT2) obtained in a previous optimization screen for increased killing of both Gram-negative and Gram-positive bacteria pathogens. At their minimal inhibitory concentrations (MICs), these peptides completely killed cells of multidrug-resistant, enterohemorrhagic pathogen Escherichia coli O157:H7 within 1-5 min. In addition, both peptides exhibited anti-biofilm activity at sub-MIC levels. Indeed, these peptides prevented biofilm formation and triggered killing of cells in mature E. coli O157:H7 biofilms at 1 ?M. Both peptides bound to bacterial surface LPS as assessed using the dansyl-polymyxin displacement assay, and were able to interact with the lipids of liposomes as determined by observing a tryptophan blue shift. Interestingly, even though these peptides were highly antimicrobial, they did not induce pore formation or aggregates in bacterial cell membranes. Instead these peptides readily penetrated into bacterial cells as determined by confocal microscopy of labeled peptides. DNA binding assays indicated that both peptides bound to DNA with higher affinity than the positive control peptide buforin II. We propose that cationic peptides KT2 and RT2 bind to negatively-charged LPS to enable self-promoted uptake and, subsequently interact with cytoplasmic membrane phospholipids through their hydrophobic domains enabling translocation across the bacterial membrane and entry into cells within minutes and binding to DNA and other cytoplasmic membrane. Due to their dual antimicrobial and anti-biofilm activities, these peptides may find use as an alternative to (or in conjunction with) conventional antibiotics to treat acute infections caused by planktonic bacteria and chronic, biofilm-related infections. PMID:25767037

  1. Evaluation and Selection of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Withania somnifera (L.) Dunal

    PubMed Central

    Singh, Varinder; Kaul, Sunil C.; Wadhwa, Renu; Pati, Pratap Kumar

    2015-01-01

    Quantitative real-time PCR (qRT-PCR) is now globally used for accurate analysis of transcripts levels in plants. For reliable quantification of transcripts, identification of the best reference genes is a prerequisite in qRT-PCR analysis. Recently, Withania somnifera has attracted lot of attention due to its immense therapeutic potential. At present, biotechnological intervention for the improvement of this plant is being seriously pursued. In this background, it is important to have comprehensive studies on finding suitable reference genes for this high valued medicinal plant. In the present study, 11 candidate genes were evaluated for their expression stability under biotic (fungal disease), abiotic (wounding, salt, drought, heat and cold) stresses, in different plant tissues and in response to various plant growth regulators (methyl jasmonate, salicylic acid, abscisic acid). The data as analyzed by various software packages (geNorm, NormFinder, Bestkeeper and ?Ct method) suggested that cyclophilin (CYP) is a most stable gene under wounding, heat, methyl jasmonate, different tissues and all stress conditions. T-SAND was found to be a best reference gene for salt and salicylic acid (SA) treated samples, while 26S ribosomal RNA (26S), ubiquitin (UBQ) and beta-tubulin (TUB) were the most stably expressed genes under drought, biotic and cold treatment respectively. For abscisic acid (ABA) treated samples 18S-rRNA was found to stably expressed gene. Finally, the relative expression level of the three genes involved in the withanolide biosynthetic pathway was detected to validate the selection of reliable reference genes. The present work will significantly contribute to gene analysis studies in W. somnifera and facilitate in improving the quality of gene expression data in this plant as well as and other related plant species. PMID:25769035

  2. Evaluation and selection of candidate reference genes for normalization of quantitative RT-PCR in Withania somnifera (L.) Dunal.

    PubMed

    Singh, Varinder; Kaul, Sunil C; Wadhwa, Renu; Pati, Pratap Kumar

    2015-01-01

    Quantitative real-time PCR (qRT-PCR) is now globally used for accurate analysis of transcripts levels in plants. For reliable quantification of transcripts, identification of the best reference genes is a prerequisite in qRT-PCR analysis. Recently, Withania somnifera has attracted lot of attention due to its immense therapeutic potential. At present, biotechnological intervention for the improvement of this plant is being seriously pursued. In this background, it is important to have comprehensive studies on finding suitable reference genes for this high valued medicinal plant. In the present study, 11 candidate genes were evaluated for their expression stability under biotic (fungal disease), abiotic (wounding, salt, drought, heat and cold) stresses, in different plant tissues and in response to various plant growth regulators (methyl jasmonate, salicylic acid, abscisic acid). The data as analyzed by various software packages (geNorm, NormFinder, Bestkeeper and ?Ct method) suggested that cyclophilin (CYP) is a most stable gene under wounding, heat, methyl jasmonate, different tissues and all stress conditions. T-SAND was found to be a best reference gene for salt and salicylic acid (SA) treated samples, while 26S ribosomal RNA (26S), ubiquitin (UBQ) and beta-tubulin (TUB) were the most stably expressed genes under drought, biotic and cold treatment respectively. For abscisic acid (ABA) treated samples 18S-rRNA was found to stably expressed gene. Finally, the relative expression level of the three genes involved in the withanolide biosynthetic pathway was detected to validate the selection of reliable reference genes. The present work will significantly contribute to gene analysis studies in W. somnifera and facilitate in improving the quality of gene expression data in this plant as well as and other related plant species. PMID:25769035

  3. Application of F(+)RNA Coliphages as Source Tracking Enteric Viruses on Parsley and Leek Using RT-PCR.

    PubMed

    Shahrampour, Dina; Yavarmanesh, Masoud; Habibi Najafi, Mohammad Bagher; Mohebbi, Mohebbat

    2015-12-01

    The objective of this study was to identify sources of fecal contamination in leek and parsley, by using four different F(+)RNA coliphage genogroups (IV, I indicate animal fecal contamination and II, III indicate human fecal contamination). Three different concentrations (10(2), 10(4), 10(6) pfu/ml) of MS2 coliphage were inoculated on the surface of parsley and leek samples for detection of phage recovery efficiency among two methods of elution concentration (PEG-precipitation and Ultracentrifugation) by performing double agar layer (DAL) assay in three replications. Highest recovery of MS2 was observed in PEG method and in 10(6) inoculation concentration. Accordingly, the PEG method was used for washing and isolation of potentially contaminated phages of 30 collected samples (15 samples from the market and 15 samples from the farm). The final solutions of PEG method were tested for the enumeration of plaques by DAL assay. Total RNA was then extracted from recovered phages, and RT-PCR was performed by using four primer sets I, II, III, and IV. Incidence of F(+)RNA coliphages was observed in 12/15 (80 %) and 10/15 (66/6 %) of samples were obtained from farm and market, respectively, using both DAL and RT-PCR test methods. Different genotypes (I, II, and IV) of F(+)RNA coliphages were found in farm samples, while only genotype I was detected in market samples by using the primer sets. Due to the higher frequency of genotype I and IV, the absence of genotype III, and also the low frequency of genotype II, it is concluded that the contamination of vegetable (parsley and leek) in Neyshabour, Iran is most likely originated from animal sources. PMID:26264153

  4. Development of a duplex one-step RT-qPCR assay for the simultaneous detection of Apple scar skin viroid and plant RNA internal control.

    PubMed

    Khan, Subuhi; Mackay, John; Liefting, Lia; Ward, Lisa

    2015-09-01

    Apple scar skin viroid (ASSVd) is an important quarantine pathogen for international movement of pome germplasm as it can cause significant damage to pip fruit. A one-step real-time RT-PCR assay was developed for the rapid and sensitive detection of ASSVd. The assay was able to detect a wide range of ASSVd isolates and was highly specific compared to a published conventional RT-PCR. The detection limit of the new assay was estimated to be about 100 copies of the ASSVd target. The assay can be run as a duplex with the nad5 internal control primers and probe to simultaneously check the PCR competency of the samples therefore reducing the risk of false negatives. It is expected that this real-time RT-PCR assay will facilitate efficient testing for ASSVd by regulatory services, and will also have a wider use for the general detection of ASSVd in a range of pip fruit. PMID:25962536

  5. IL-1RT1 signaling antagonizes IL-11 induced STAT3 dependent cardiac and antral stomach tumor development through myeloid cell enrichment

    PubMed Central

    Buzzelli, Jon N.; Pavlic, Dan I.; Chalinor, Heather V.; O'Connor, Louise; Menheniott, Trevelyan R.; Giraud, Andrew S.; Judd, Louise M.

    2015-01-01

    IL-1 is key driver of gastric tumorigenesis and is a downstream target of IL-11 signaling. Recently, IL-1 cytokines, particularly IL-1?, have been flagged as therapeutic targets for gastric cancer treatment. Here, we assess the requirement for IL-1 signaling in gastric tumorigenesis. gp130757FF xIL-1RT1?/? mice were generated to determine the pathological consequence of ablated IL-1 signaling in the IL-11 dependent gp130757FF mouse model of gastric tumorigenesis. Gastric lesions in gp130757FF xIL-1RT1?/? mice were increased in incidence and size compared to gp130757FF mice. Proximal gastric lesions originated from the cardiac region and were associated with elevated STAT3 activation, loss of specialized gastric cells and a modulated immune response including increased expression of TNF-? and MDSC associated genes. Administration of IL-11 to IL-1RT1?/? mice showed similar changes to gp130757FF xIL-1RT1?/? mice. Spleens from IL-11 treated wildtype mice showed an enrichment of MDSC and gp130757FF xIL-1RT1?/? mice had increased MDSCs in the stomach compared to gp130757FF mice. Furthermore, crossing TNF-??/? to gp130757FF mice resulted in reduced lesion size. We conclude that IL-1 signaling antagonizes IL-11/STAT3 mediated pathology and the genetic deletion of IL-1RT1 results in increased tumor burden. We provide evidence that a likely mechanism is due to IL-11/STAT3 dependent enrichment of MDSCs. PMID:25528766

  6. Relevance of Post-Stroke Circulating BDNF Levels as a Prognostic Biomarker of Stroke Outcome. Impact of rt-PA Treatment

    PubMed Central

    Rodier, Marion; Quirié, Aurore; Prigent-Tessier, Anne; Béjot, Yannick; Jacquin, Agnès; Mossiat, Claude; Marie, Christine; Garnier, Philippe

    2015-01-01

    The recombinant form of tissue plasminogen activator (rt-PA) is the only curative treatment for ischemic stroke. Recently, t-PA has been linked to the metabolism of brain-derived neurotrophic factor (BDNF), a major neurotrophin involved in post-stroke neuroplasticity. Thus, the objective of our study was to investigate the impact of rt-PA treatment on post-stroke circulating BDNF levels in humans and in animals. Serum BDNF levels and t-PA/plasmin activity were measured at hospital admission and at up to 90 days in stroke patients receiving (n = 24) or not (n = 14) rt-PA perfusion. We investigated the relationships between serum BDNF with concurrent t-PA/plasmin activity, neurological outcomes and cardiovascular scores at admission. In parallel, serum BDNF levels and t-PA/plasmin activity were assessed before and after (1, 4 and 24h) the induction of ischemic stroke in rats. Our study revealed higher serum BDNF levels and better neurological outcome in rt-PA-treated than non-treated patients. However, serum BDNF levels did not predict stroke outcome when the whole cohort of stroke patients was analyzed. By contrast, serum BDNF levels when measured at admission and at day 90 correlated with cardiovascular scores, and those at day 1 correlated with serum t-PA/plasmin activity in the whole cohort of patients whereas no association could be found in the rt-PA-treated group. In rats devoid of cardiovascular risk, no difference in post-stroke serum BDNF levels was detected between rt-PA- and vehicle-treated animals and no correlation was found between serum BDNF levels and t-PA/plasmin activity. Overall, the data suggest that serum BDNF levels may not be useful as a prognostic biomarker of stroke outcome and that endothelial dysfunction could be a confounding factor when serum BDNF levels after stroke are used to reflect of brain BDNF levels. PMID:26469350

  7. Validation of Reference Genes for RT–qPCR Analysis in Noise–Induced Hearing Loss: A Study in Wistar Rat

    PubMed Central

    Melgar–Rojas, Pedro; Alvarado, Juan Carlos; Fuentes–Santamaría, Verónica; Gabaldón–Ull, María Cruz; Juiz, José M.

    2015-01-01

    The reverse transcriptase–quantitative polymerase chain reaction (RT–qPCR) requires adequate normalization in order to ensure accurate results. The use of reference genes is the most common method to normalize RT–qPCR assays; however, many studies have reported that the expression of frequently used reference genes is more variable than expected, depending on experimental conditions. Consequently, proper validation of the stability of reference genes is an essential step when performing new gene expression studies. Despite the fact that RT–qPCR has been widely used to elucidate molecular correlates of noise–induced hearing loss (NIHL), up to date there are no reports demonstrating validation of reference genes for the evaluation of changes in gene expression after NIHL. Therefore, in this study we evaluated the expression of some commonly used reference genes (Arbp, b–Act, b2m, CyA, Gapdh, Hprt1, Tbp, Tfrc and UbC) and examined their suitability as endogenous control genes for RT–qPCR analysis in the adult Wistar rat in response to NIHL. Four groups of rats were noise–exposed to generate permanent cochlear damage. Cochleae were collected at different time points after noise exposure and the expression level of candidate reference genes was evaluated by RT–qPCR using geNorm, NormFinder and BestKeeper software to determine expression stability. The three independent applications revealed Tbp as the most stably expressed reference gene. We also suggest a group of top–ranked reference genes that can be combined to obtain suitable reference gene pairs for the evaluation of the effects of noise on gene expression in the cochlea. These findings provide essential basis for further RT–qPCR analysis in studies of NIHL using Wistar rats as animal model. PMID:26366995

  8. Low Frequency of Drug-Resistant Variants Selected by Long-Acting Rilpivirine in Macaques Infected with Simian Immunodeficiency Virus Containing HIV-1 Reverse Transcriptase.

    PubMed

    Melody, Kevin; McBeth, Sarah; Kline, Christopher; Kashuba, Angela D M; Mellors, John W; Ambrose, Zandrea

    2015-12-01

    Preexposure prophylaxis (PrEP) using antiretroviral drugs is effective in reducing the risk of human immunodeficiency virus type 1 (HIV-1) infection, but adherence to the PrEP regimen is needed. To improve adherence, a long-acting injectable formulation of the nonnucleoside reverse transcriptase (RT) inhibitor rilpivirine (RPV LA) has been developed. However, there are concerns that PrEP may select for drug-resistant mutations during preexisting or breakthrough infections, which could promote the spread of drug resistance and limit options for antiretroviral therapy. To address this concern, we administered RPV LA to macaques infected with simian immunodeficiency virus containing HIV-1 RT (RT-SHIV). Peak plasma RPV levels were equivalent to those reported in human trials and waned over time after dosing. RPV LA resulted in a 2-log decrease in plasma viremia, and the therapeutic effect was maintained for 15 weeks, until plasma drug concentrations dropped below 25 ng/ml. RT mutations E138G and E138Q were detected in single clones from plasma virus in separate animals only at one time point, and no resistance mutations were detected in viral RNA isolated from tissues. Wild-type and E138Q RT-SHIV displayed similar RPV susceptibilities in vitro, whereas E138G conferred 2-fold resistance to RPV. Overall, selection of RPV-resistant variants was rare in an RT-SHIV macaque model despite prolonged exposure to slowly decreasing RPV concentrations following injection of RPV LA. PMID:26438501

  9. Dose optimization with first-order total-variation minimization for dense angularly sampled and sparse intensity modulated radiation therapy (DASSIM-RT)

    SciTech Connect

    Kim, Hojin; Li Ruijiang; Lee, Rena; Goldstein, Thomas; Boyd, Stephen; Candes, Emmanuel; Xing Lei

    2012-07-15

    Purpose: A new treatment scheme coined as dense angularly sampled and sparse intensity modulated radiation therapy (DASSIM-RT) has recently been proposed to bridge the gap between IMRT and VMAT. By increasing the angular sampling of radiation beams while eliminating dispensable segments of the incident fields, DASSIM-RT is capable of providing improved conformity in dose distributions while maintaining high delivery efficiency. The fact that DASSIM-RT utilizes a large number of incident beams represents a major computational challenge for the clinical applications of this powerful treatment scheme. The purpose of this work is to provide a practical solution to the DASSIM-RT inverse planning problem. Methods: The inverse planning problem is formulated as a fluence-map optimization problem with total-variation (TV) minimization. A newly released L1-solver, template for first-order conic solver (TFOCS), was adopted in this work. TFOCS achieves faster convergence with less memory usage as compared with conventional quadratic programming (QP) for the TV form through the effective use of conic forms, dual-variable updates, and optimal first-order approaches. As such, it is tailored to specifically address the computational challenges of large-scale optimization in DASSIM-RT inverse planning. Two clinical cases (a prostate and a head and neck case) are used to evaluate the effectiveness and efficiency of the proposed planning technique. DASSIM-RT plans with 15 and 30 beams are compared with conventional IMRT plans with 7 beams in terms of plan quality and delivery efficiency, which are quantified by conformation number (CN), the total number of segments and modulation index, respectively. For optimization efficiency, the QP-based approach was compared with the proposed algorithm for the DASSIM-RT plans with 15 beams for both cases. Results: Plan quality improves with an increasing number of incident beams, while the total number of segments is maintained to be about the same in both cases. For the prostate patient, the conformation number to the target was 0.7509, 0.7565, and 0.7611 with 80 segments for IMRT with 7 beams, and DASSIM-RT with 15 and 30 beams, respectively. For the head and neck (HN) patient with a complicated target shape, conformation numbers of the three treatment plans were 0.7554, 0.7758, and 0.7819 with 75 segments for all beam configurations. With respect to the dose sparing to the critical structures, the organs such as the femoral heads in the prostate case and the brainstem and spinal cord in the HN case were better protected with DASSIM-RT. For both cases, the delivery efficiency has been greatly improved as the beam angular sampling increases with the similar or better conformal dose distribution. Compared with conventional quadratic programming approaches, first-order TFOCS-based optimization achieves far faster convergence and smaller memory requirements in DASSIM-RT. Conclusions: The new optimization algorithm TFOCS provides a practical and timely solution to the DASSIM-RT or other inverse planning problem requiring large memory space. The new treatment scheme is shown to outperform conventional IMRT in terms of dose conformity to both the targetand the critical structures, while maintaining high delivery efficiency.

  10. Characterisation of norovirus contamination in an Irish shellfishery using real-time RT-qPCR and sequencing analysis.

    PubMed

    Rajko-Nenow, Paulina; Keaveney, Sinéad; Flannery, John; O'Flaherty, Vincent; Doré, William

    2012-11-15

    Norovirus (NoV) is the single most important agent of foodborne viral gastroenteritis worldwide. Bivalve shellfish, such as oysters, grown in areas contaminated with human faecal waste may become contaminated with human pathogens including NoV. A study was undertaken to investigate NoV contamination in oysters (Crassostrea gigas) from a shellfishery over a 24month period from October 2007 to September 2009. Oyster samples were collected monthly from a commercial shellfish harvest area classified as category B under EU regulations, but that had had been closed for commercial harvesting due to its previous association with NoV outbreaks. Real-time reverse transcription quantitative PCR (RT-qPCR) was used to determine the concentration of human NoV genogroups I and II (GI and GII) in monthly samples. Total NoV (GI and GII) concentrations in NoV positive oysters ranged from 97 to 20,080genome copiesg(-1) of digestive tissue and displayed a strong seasonal trend with greater concentrations occurring during the winter months. While NoV GII concentrations detected in oysters during both years were similar, NoV GI concentrations were significantly greater in oysters during the winter of 2008/09 than during the winter of 2007/08. To examine the NoV genotypes present in oyster samples, sequence analysis of nested RT-PCR products was undertaken. Although NoV GII.4 is responsible for the vast majority of reports of outbreaks in the community, multiple NoV genotypes were identified in oysters during this study: GI.4, GI.3, GI.2, GII.4, GII.b, GII.2, GII.12, and GII.e. NoV GI.4 was the most frequently detected genotype throughout the study period and was detected in 88.9% of positive samples, this was followed by GII.4 (43.7%) and GII.b (37.5%). This data demonstrates the diversity of NoV genotypes that can be present in sewage contaminated shellfish and that a disproportionate number of non-NoV GII.4 genotypes can be found in environmental samples compared to the number of recorded human infections associated with non-NoV GII.4 genotypes. PMID:23177049

  11. High throughput nano-liter RT-qPCR to classify soil contamination using a soil arthropod

    PubMed Central

    2011-01-01

    Background To incorporate genomics data into environmental assessments a mechanistic perspective of interactions between chemicals and induced biological processes needs to be developed. Since chemical compounds with structural similarity often induce comparable biological responses in exposed animals, gene expression signatures can serve as a starting point for the assessment of chemicals and their toxicity, but only when relevant and stable gene panels are available. To design such a panel, we isolated differentially expressed gene fragments from the soil arthropod Folsomia candida, a species often used for ecotoxicological testing. Animals were exposed to two chemically distinct compounds, being a metal (cadmium) and a polycyclic aromatic hydrocarbon (phenanthrene). We investigated the affected molecular responses resulting from either treatment and developed and validated 44 qPCR assays for their responses using a high throughput nano-liter RT-qPCR platform for the analysis of the samples. Results Suppressive subtractive hybridization (SSH) was used to retrieve stress-related gene fragments. SSH libraries revealed pathways involved in mitochondrial dysfunction and protein degradation for cadmium and biotransformation for phenanthrene to be overrepresented. Amongst a small cluster of SSH-derived cadmium responsive markers were an inflammatory response protein and an endo-glucanase. Conversely, cytochrome P450 family 6 or 9 was specifically induced by phenanthrene. Differential expressions of these candidate biomarkers were also highly significant in the independently generated test sample set. Toxicity levels in different training samples were not reflected by any of the markers' intensity of expressions. Though, a model based on partial least squares differential analysis (PLS-DA) (with RMSEPs between 9 and 22% and R2s between 0.82 and 0.97) using gene expressions of 25 important qPCR assays correctly predicted the nature of exposures of test samples. Conclusions For the application of molecular bio-indication in environmental assessments, multivariate analyses obviously have an added value over univariate methods. Our results suggest that compound discrimination can be achieved by PLS-DA, based on a hard classification of the within-class rankings of samples from a test set. This study clearly shows that the use of high throughput RT-qPCR could be a valuable tool in ecotoxicology combining high throughput with analytical sensitivity. PMID:21362169

  12. Analytical validation of a real-time RT-PCR test for Pan-American lineage H7 subtype avian influenza viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rapid detection of avian influenza virus and identification of the H5 and H7 hemagglutinin subtypes some of which are associated with high pathogenicity in poultry is critical for clinical diagnosis and wild bird monitoring programs. A real-time RT-PCR test for identification of the H7 subtype in N...

  13. J. Electrochem. Soc., Vol. 142, No. 11, November 1995 9 The Electrochemical Society, Inc. 3815 Q = In [c~a+ ~ ~ ~ -in io -RT ~ [5

    E-print Network

    Weidner, John W.

    F Q = In [c~a+ ~ ~ ~ - in io - RT ~ [5] exp ~ ~j - 1 so that a plot of Q vs. ~q yields a straight line with the intercept of in (io).This plot isthe so-called Allen-Hickling plot. This procedure has been used extensively

  14. Complete Genome Sequences of Caldicellulosiruptor sp. Strain Rt8.B8, Caldicellulosiruptor sp. Strain Wai35.B1, and “Thermoanaerobacter cellulolyticus”

    PubMed Central

    Lee, Laura L.; Izquierdo, Javier A.; Blumer-Schuette, Sara E.; Zurawski, Jeffrey V.; Conway, Jonathan M.; Cottingham, Robert W.; Huntemann, Marcel; Copeland, Alex; Chen, I-Min A.; Kyrpides, Nikos; Markowitz, Victor; Palaniappan, Krishnaveni; Ivanova, Natalia; Mikhailova, Natalia; Ovchinnikova, Galina; Andersen, Evan; Pati, Amrita; Stamatis, Dimitrios; Reddy, T.B.K.; Shapiro, Nicole; Nordberg, Henrik P.; Cantor, Michael N.; Hua, Susan X.; Woyke, Tanja

    2015-01-01

    The genus Caldicellulosiruptor contains extremely thermophilic, cellulolytic bacteria capable of lignocellulose deconstruction. Currently, complete genome sequences for eleven Caldicellulosiruptor species are available. Here, we report genome sequences for three additional Caldicellulosiruptor species: Rt8.B8 DSM 8990 (New Zealand), Wai35.B1 DSM 8977 (New Zealand), and “Thermoanaerobacter cellulolyticus” strain NA10 DSM 8991 (Japan). PMID:25977428

  15. A RT/PCR-partial restriction enzymatic mapping (PREM) method for the molecular characterisation of the large satellite RNAs of Arabis mosaic virus isolates.

    PubMed

    Wetzel, T; Bassler, A; Amren, M A W; Krczal, G

    2006-03-01

    The satellite RNA of the grapevine isolate NW of Arabis mosaic virus (ArMV) was cloned and sequenced, and showed 75% identity at the nucleotide level to the satellite RNA of the lilac isolate of ArMV. In order to survey ArMV isolates from various geographical origins and natural hosts for the presence of large satellite RNAs and analyse their degree of variability, a RT/PCR-partial restriction enzymatic mapping (PREM) method was developed. The method is based on the incorporation of 5-methyl-dCTP in the RT/PCR reaction, and the subsequent digestion of the RT/PCR products by methyl-sensitive restriction enzymes. Satellites RNAs were detected by RT/PCR in eight isolates out of 47, six of them originating from grapevine, one from hop and one from lilac. The partial restriction digestion patterns allowed to distinguish six different types of satellites. Cloning and sequencing of the different satellites confirmed these results, the PREM proving able to discriminate sequences with 96% identity. The sizes of the different satellites varied between 1092 and 1139 nucleotides, their encoded proteins between 338 and 360 amino acids. Conserved domains were found in the amino and carboxy-termini between the sequences of the proteins encoded by the satellites of the different isolates of ArMV. PMID:16216344

  16. ReGional ColleGe Review 2010 Final RepoRt CaRlton tRail ReGional ColleGe

    E-print Network

    Saskatchewan, University of

    1 ReGional ColleGe Review 2010 Final RepoRt CaRlton tRail ReGional ColleGe Glen Kobussen CEO Board Islay Ehlert St. Peter's College Board Ralph Eliasson Watrous Town Council Mike Forman Davidson Town Council Des Grace St. Peter's College Board Rob Harasymchuk St. Peter's College Board Janine Hart Humboldt

  17. The implementation of non pharmaceutical interventions(NPIs) in smaller to large communities and its relation to RO and R(t) during HIN1 pandemic 2009

    E-print Network

    Hashmi, Sahar

    2011-01-01

    This thesis focuses on the use of non-pharmaceutical interventions (NPIs) during the time of the 2009 HINI pandemic and its possible relation to RO and R(t). RO is defined as the mean number of people that a newly infected ...

  18. arXiv:1010.1184v3[math.RT]14Mar2011 PTOLEMY DIAGRAMS AND TORSION PAIRS IN THE

    E-print Network

    Holm, Thorsten

    arXiv:1010.1184v3[math.RT]14Mar2011 PTOLEMY DIAGRAMS AND TORSION PAIRS IN THE CLUSTER CATEGORY combinatorial description of Ptolemy diagrams, an infinite version of which was introduced by Ng in [15. The Ptolemy condition This concept was introduced by Iyama and Yoshino in [11, def. 2.2]. It is a triangu

  19. Use of a custom RT-PCR array to analyze toxicity pathways at different life stages in Brown Norway Rat Brain following acute Toluene exposure.

    EPA Science Inventory

    To investigate the contribution of different life stages on response to toxicants, we utilized a custom designed RT-PCR array to examine the effects of acute exposure by oral gavage of the volatile organic solvent toluene (0.00, 0.65 or 1.0 glkg) in the brains of ma1e Brown Norwa...

  20. Frequency detection of imidacloprid resistance allele in Aphis gossypii field populations by real-time PCR amplification of specific-allele (rtPASA).

    PubMed

    Zhang, Jing; Cui, Li; Xu, Xibao; Rui, Changhui

    2015-11-01

    The Aphis gossypii Glover (Hemiptera: Aphididae) is one of the most serious pests worldwide, and imidacloprid has been widely used to control this insect pest. Just like other classes of insecticides, the resistance to imidacloprid has been found in A. gossypii. An amino acid mutation (R81T) in the nicotinic acetylcholine receptor (nAChR) beta1 subunit was detected in the imidacloprid-resistant A. gossypii collected from Langfang (LF) and Dezhou (DZ) cities. To estimate the R81T mutation frequency of A. gossypii field populations, a simple, rapid and accurate rtPASA (real-time PCR amplification of specific allele) protocol was developed. The performance of the rtPASA protocol was evaluated by comparing with the data generated by a cPASA (competitive PCR amplification of specific allele) method from 50 individual genotypes. The R81T allele frequencies of the LF population (34.7%±1.3%) and DZ population (45.2%±5.2%) estimated by the rtPASA protocol matched the frequencies (LF 38.1%, DZ 48.2%) deduced by the cPASA method in specimens. The results indicated that the rtPASA format was applicable for the detection of mutation associated with imidacloprid resistance and will allow rapid and efficient monitoring of A. gossypii resistance in field populations in a high throughput format. PMID:26615144

  1. Field detection of avian influenza virus in wild birds: evaluation of a portable rRT-PCR system and freeze-dried reagents

    USGS Publications Warehouse

    Takekawa, John Y.; Iverson, Samuel A.; Schultz, Annie K.; Hill, Nichola J.; Cardona, Carol J.; Boyce, Walter M.; Dudley, Joseph P.

    2010-01-01

    Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAIV) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds is often conducted in remote regions, but results are often delayed because of limited local analytical capabilities, difficulties with sample transportation and permitting, or problems keeping samples cold in the field. In response to these challenges, the performance of a portable real-time, reverse transcriptase-polymerase chain reaction (rRT-PCR) unit (RAPID(Registered), Idaho Technologies, Salt Lake City, UT) that employed lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies) was compared to virus isolation combined with real-time RT-PCR conducted in a laboratory. This study included both field and experimental-based sampling. Field samples were collected from migratory shorebirds captured in northern California, while experimental samples were prepared by spiking fecal material with an H6N2 AIV isolate. Results indicated that the portable rRT-PCR unit had equivalent specificity to virus isolation with no false positives, but sensitivity was compromised at low viral titers. Use of portable rRT-PCR with lyophilized reagents may expedite surveillance results, paving the way to a better understanding of wild bird involvement in HPAIV H5N1 transmission.

  2. The Influence of Tier One of RtI[superscript 2] and Instructional Coaching on Teacher Instruction and Student/ELL Learning: A Multiple Case Study

    ERIC Educational Resources Information Center

    Valadez, Frances E.

    2012-01-01

    The purpose of this case study was to demonstrate the influence of Tier 1 of Response to Intervention and Instruction (RtI[superscript 2]) and instructional coaching on teachers' instruction and on students' and English Language Learners' (ELL) learning. Research was conducted in one large urban elementary school. The unit of study…

  3. Identification of RtGST-1, a novel germ cell-specific mRNA-like transcript predominantly expressed in early previtellgenic oocytes in rainbow trout (Oncorhynchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Noncoding mRNA-like transcripts play important roles in a wide range of biological events such as cell differentiation and organogenesis. We report here the identification of a novel germ cell-specific mRNA-like transcript (RtGST-1) from a rainbow trout oocyte cDNA library. The novel transcript of 1...

  4. IDENTIFICATION OF A NOVEL GERM CELL-SPECIFIC mRNA-LIKE TRANSCRIPT (RtGST-1) PREDOMINANTLY EXPRESSED IN EARLY PREVITELLGENIC OOCYTE IN RAINBOW TROUT (ONCORHYNCHUS MYKISS)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Increasing evidences have shown that noncoding mRNA-like transcripts play important roles in a wide range of biological events such as cell differentiation and organogenesis. In this study, a novel germline-specific transcript, termed RtGST-1, was identified by bioinformatics analysis of expressed s...

  5. T e c h n i ca l R e p o RT s 1074 VOLUME 20 | NUMBER 9 | SEPTEMBER 2014 naTuRe medicine

    E-print Network

    Quake, Stephen R.

    T e c h n i ca l R e p o RT s 1074 VOLUME 20 | NUMBER 9 | SEPTEMBER 2014 naTuRe medicine Glaucoma for glaucoma diagnosis and patient monitoring. IOP has wide diurnal fluctuation and is dependent on body care of patients with glaucoma. Glaucoma affects more than 65 million people worldwide and is expected

  6. A bacterial community analysis using reverse transcription (RT) PCR which detects the bacteria with high activity in a wastewater treatment reactor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This research used reverse transcription polymerase chain reaction (RT-PCR) method to help detect active bacteria in a single-tank deammonification reactor combining partial nitritation and anammox. The single-tank aerobic deammonification reactor effectively removed the ammonia in anaerobically di...

  7. Real-time RT-PCR for detection of Raspberry bushy dwarf virus, Raspberry leaf mottle virus and characterizing synergistic interactions in mixed infections

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two TaqMan-based real-time One-Step RT-PCR assays were developed for the rapid and efficient detection of Raspberry bushy dwarf virus (RBDV) and Raspberry leaf mottle virus (RLMV), two of the most common raspberry viruses in North America and Europe. The primers and probes were designed from conser...

  8. 2010-2011 ANNUAl REPoRt O F Y E S H I V A U N I V E R S I T Y

    E-print Network

    Emmons, Scott

    einstein faces of 2010-2011 ANNUAl REPoRt O F Y E S H I V A U N I V E R S I T Y Albert Einstein Students 34 Educational Milestones 36 einstein fACES Of The College of Medicine's namesake, Albert Einstein College of Medicine EINSTEIN #12;in the Laboratory ·In fiscal year 2010, Einstein received nearly $200

  9. A robust standard for absolute mRNA quantification of Saccharomyces cerevisiae by qRT-PCR using the universal RNA controls

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently developed universal external RNA controls allow comparison of expression data generated from microarray and real time qRT-PCR, including SYBR Green and TaqMan-probe based chemistry. It provides reliable controls for data normalization and analysis. In this study, we further developed stra...

  10. Optimization of Droplet Digital PCR from RNA and DNA extracts with direct comparison to RT-qPCR: Clinical implications for quantification of Oseltamivir-resistant subpopulations.

    PubMed

    Taylor, Sean C; Carbonneau, Julie; Shelton, Dawne N; Boivin, Guy

    2015-11-01

    The recent introduction of Droplet Digital PCR (ddPCR) has provided researchers with a tool that permits direct quantification of nucleic acids from a wide range of samples with increased precision and sensitivity versus RT-qPCR. The sample interdependence of RT-qPCR stemming from the measurement of Cq and ?Cq values is eliminated with ddPCR which provides an independent measure of the absolute nucleic acid concentration for each sample without standard curves thereby reducing inter-well and inter-plate variability. Well-characterized RNA purified from H275-wild type (WT) and H275Y-point mutated (MUT) neuraminidase of influenza A (H1N1) pandemic 2009 virus was used to demonstrate a ddPCR optimization workflow to assure robust data for downstream analysis. The ddPCR reaction mix was also tested with RT-qPCR and gave excellent reaction efficiency (between 90% and 100%) with the optimized MUT/WT duplexed assay thus enabling the direct comparison of the two platforms from the same reaction mix and thermal cycling protocol. ddPCR gave a marked improvement in sensitivity (>30-fold) for mutation abundance using a mixture of purified MUT and WT RNA and increased precision (>10 fold, p<0.05 for both inter- and intra-assay variability) versus RT-qPCR from patient samples to accurately identify residual mutant viral population during recovery. PMID:26315318

  11. Change your conventional practice of qRT-PCR: A simple robust quality control standard for yeast mRNA quantification analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has been realized to be the assay of choice among available techniques for mRNA quantification analysis. For conventional practice, housekeeping genes have been applied as internal reference for data normalization and a...

  12. Environmental Regulation of Plant Gene Expression: An Rt-qPCR Laboratory Project for an Upper-Level Undergraduate Biochemistry or Molecular Biology Course

    ERIC Educational Resources Information Center

    Eickelberg, Garrett J.; Fisher, Alison J.

    2013-01-01

    We present a novel laboratory project employing "real-time" RT-qPCR to measure the effect of environment on the expression of the "FLOWERING LOCUS C" gene, a key regulator of floral timing in "Arabidopsis thaliana" plants. The project requires four 3-hr laboratory sessions and is aimed at upper-level undergraduate…

  13. Development of a One-Step Immunocapture Real-Time TaqMan RT-PCR Assay for the Broad Spectrum Detection of Pepino Mosaic Virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A Real-time reverse transcription-polymerase chain reaction (RT-PCR) was developed for efficient detection of genetically diverse PepMV isolates. The novel detection system was designed to use a duo-primer system targeting the conserved region in the triple gene block 2 (TGB2) gene with a single co...

  14. Quantitative RT-PCR assay of HER2 mRNA expression in formalin-fixed and paraffin-embedded breast cancer tissues.

    PubMed

    Park, Sangjung; Wang, Hye-Young; Kim, Sunghyun; Ahn, Sungwoo; Lee, Dongsup; Cho, Yoonjung; Park, Kwang Hwa; Jung, Dongju; Kim, Seung Il; Lee, Hyeyoung

    2014-01-01

    Detection of human epidermal growth factor receptor 2 gene (HER2, also known as erbB2) expression is a preparatory process to decide a treatment strategy for breast cancer patients. 20-30% of breast cancer patients have HER2 overexpression, and they usually show poor recovery rate. For detection of HER2 expression, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) methods are conventionally used. Although these methods are accurate and reliable, their time-consuming process and high cost need a concise method with high sensitivity and accuracy. As a complementary method to the current IHC/FISH standard techniques, PCR-based methods have been developed. Here we employed a quantitative PCR method to detect HER2 expression in one hundred ninety nine formalin-fixed and paraffin-embedded (FFPE) breast cancer tissue samples from the patients treated over two years at the Yonsei University Severance Hospital, Republic of Korea. Relative expression of HER2 mRNA in the FFPE samples was analyzed using a quantitative RT-PCR (RT-qPCR) method and the obtained HER2 expression levels were compared with those from IHC/FISH methods. Our results show that the RT-qPCR method was highly concordant with IHC/FISH methods for detecting HER2 expression. Overall sensitivity and specificity of the BrightGen HER2 RT-qDx assay kit (Syantra, Calgary, Canada), which is a kit we used for RT-qPCR analyses, were 93.0% and 89.8% (P < 0.0001), respectively. The diagnostic cut-off value of HER2 RT-qDx for the clinical samples was determined by likelihood ratio, among which the highest likelihood ratio of relative HER2 mRNA levels was over 105.5 (AUC = 0.9466) with the highest sensitivity and specificity. Our study indicates that quantification of HER2 mRNA expression with the RT-qPCR could be an alternative method of conventional IHC/FISH methods. PMID:25400755

  15. Improved beta (local beta >1) and density in electron cyclotron resonance heating on the RT-1 magnetosphere plasma

    NASA Astrophysics Data System (ADS)

    Nishiura, M.; Yoshida, Z.; Saitoh, H.; Yano, Y.; Kawazura, Y.; Nogami, T.; Yamasaki, M.; Mushiake, T.; Kashyap, A.

    2015-05-01

    This study reports the recent progress in improved plasma parameters of the RT-1 device. Increased input power and the optimized polarization of electron cyclotron resonance heating (ECRH) with an 8.2 GHz klystron produce a significant increase in electron beta, which is evaluated by an equilibrium analysis of the Grad-Shafranov equation. The peak value of the local electron beta ?e is found to exceed 1. In the high-beta and high-density regime, the density limit is observed for H, D and He plasmas. The line-averaged density is close to the cutoff density for 8.2 GHz ECRH. When the filling gas pressure is increased, the density limit still exists even in the low-beta region. This result indicates that the density limit is caused by the cutoff density rather than the beta limit. From the analysis of interferometer data, we found that inward diffusion causes a peaked density profile beyond the cutoff density.

  16. Web-based documentation system with exchange of DICOM RT for multicenter clinical studies in particle therapy

    NASA Astrophysics Data System (ADS)

    Kessel, Kerstin A.; Bougatf, Nina; Bohn, Christian; Engelmann, Uwe; Oetzel, Dieter; Bendl, Rolf; Debus, Jürgen; Combs, Stephanie E.

    2012-02-01

    Conducting clinical studies is rather difficult because of the large variety of voluminous datasets, different documentation styles, and various information systems, especially in radiation oncology. In this paper, we describe our development of a web-based documentation system with first approaches of automatic statistical analyses for transnational and multicenter clinical studies in particle therapy. It is possible to have immediate access to all patient information and exchange, store, process, and visualize text data, all types of DICOM images, especially DICOM RT, and any other multimedia data. Accessing the documentation system and submitting clinical data is possible for internal and external users (e.g. referring physicians from abroad, who are seeking the new technique of particle therapy for their patients). Thereby, security and privacy protection is ensured with the encrypted https protocol, client certificates, and an application gateway. Furthermore, all data can be pseudonymized. Integrated into the existing hospital environment, patient data is imported via various interfaces over HL7-messages and DICOM. Several further features replace manual input wherever possible and ensure data quality and entirety. With a form generator, studies can be individually designed to fit specific needs. By including all treated patients (also non-study patients), we gain the possibility for overall large-scale, retrospective analyses. Having recently begun documentation of our first six clinical studies, it has become apparent that the benefits lie in the simplification of research work, better study analyses quality and ultimately, the improvement of treatment concepts by evaluating the effectiveness of particle therapy.

  17. Vitamin D receptor alleles: Cloning and characterization of the VDR gene and RT-PCR of VDR cDNA

    SciTech Connect

    Javed, A.A.; Huang, Y.; Bombard, A.T.

    1994-09-01

    Vitamin D{sub 3} receptors (VDR) function as regulators through the action of the ligand 1{alpha}, 25-dihydroxy vitamin D{sub 3}. The receptor specifically finds its ligand and exerts it effect on the regulation of the expression of target genes. It has been shown that mutations in the VDR gene affect the function of the receptors and cause a corresponding disorder state. Recently, it has been reported that common allelic variations found normally in the Caucasian (Australian) population pose varying degrees of risk for osteoporosis. We present here the cloning of the VDR gene and RT-PCR of VDR cDNA. Studies are in progress to establish allele frequency in the Black, Hispanic and Caucasian populations to systematically study the influence of allele types and to develop a risk profile for osteoporosis. The present method for detection of various alleles is based on RFLP analysis. We are developing PCR-based methods for the rapid detection and typing of alleles.

  18. Modeling benzene plume elongation mechanisms exerted by ethanol using RT3D with a general substrate interaction module

    NASA Astrophysics Data System (ADS)

    Gomez, Diego E.; de Blanc, Phillip C.; Rixey, William G.; Bedient, Phillip B.; Alvarez, Pedro J. J.

    2008-05-01

    A mathematical model was developed to evaluate the effect of the common fuel additive 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 Dimensions (RT3D) model, includes commonly considered fate and transport processes (advection, dispersion, adsorption, biodegradation, and depletion of molecular oxygen during biodegradation) and substrate interactions previously not considered (e.g., a decrease in the specific benzene utilization rate due to metabolic flux dilution and/or catabolite repression) as well as microbial population shifts. Benzene plume elongation predictions, based on literature model parameters, were on the order of 40% for a constant source of E10 gasoline (10% vol/vol ethanol), which compares favorably to field observations. For low benzene concentrations (<1 mg/L), oxygen depletion during ethanol degradation was the principal mechanism hindering benzene natural attenuation. For higher benzene concentrations (exerting an oxygen demand higher than the available dissolved oxygen), metabolic flux dilution was the dominant plume elongation process. If oxygen were not limiting, as might be the case in zones undergoing aerobic biostimulation, model simulations showed that microbial growth on ethanol could offset negative substrate interactions and enhance benzene degradation, resulting in shorter plumes than baseline conditions without ethanol.

  19. Shoulder physical activity, functional disability and task difficulties in patients with stiff shoulders: interpretation from RT3 accelerator.

    PubMed

    Yang, Jing-Lan; Lin, Jiu-Jenq; Huang, Han-Yi; Huang, Tsun-Shun; Chao, Yu Wen

    2014-08-01

    We determined whether the degree of symptom-related functional disability was related to daily physical activity of the shoulder in subjects with stiff shoulders (SSs). Responsiveness and a clinically meaningful level of discrimination between improvement and non-improvement for shoulder physical activity (SPA) were determined. Twenty-six subjects with SSs participated. Shoulder physical activity was assessed by RT3 accelerometers fixed on the humerus during daily 14-h data collection periods twice a week for 2 weeks. A moderate correlation coefficient was found between SPA and functional disability (? = .47). Based on our cohort design and sample, we suggest that SPA (higher than 101.8 counts, hard-moderate or hard tasks) during daily activity are associated with (with at least 83% probability) non-improvement in an individual with SS. Compared to the non-improvement group, the improvement group had less duration of sedentary activity, less frequency and duration of hard tasks, and more frequency and duration of easy tasks (p < 0.01). Appropriate guidance on shoulder physical activities for subjects with SS is important to consider in rehabilitation strategies for these subjects. In our sample, a hard level of shoulder physical activity and sedentary activity should be cautious for these subjects. Further study is needed to validate therapeutic effect of physical activity on the course of patient improvement in subjects with SSs. PMID:24650638

  20. One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees.

    PubMed

    Malandraki, Ioanna; Varveri, Christina; Olmos, Antonio; Vassilakos, Nikon

    2015-03-01

    A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys. PMID:25479356

  1. Microarray screening and qRT-PCR evaluation of microRNA markers for forensic body fluid identification.

    PubMed

    Park, Jong-Lyul; Park, Seong-Min; Kwon, Oh-Hyung; Lee, Han-chul; Kim, Jin-young; Seok, Hyun Ha; Lee, Woo Sik; Lee, Seung-Hwan; Kim, Yong Sung; Woo, Kwang-Man; Kim, Seon-Young

    2014-11-01

    MicroRNAs (miRNA) are a class of small (?22 nucleotides) noncoding RNAs that regulate diverse biological processes at the post-transcriptional level. MiRNAs have great potential for forensic body fluid identification because they are expressed in a tissue specific manner and are less prone to degradation. Previous studies reported several miRNAs as body fluid specific, but there are few overlaps among them. Here, we used a genome-wide miRNA microarray containing over 1700 miRNAs to assay 20 body fluid samples and identify novel miRNAs useful for forensic body fluid identification. Based on Shannon Entropy and Q-statistics, 203 miRNAs specifically expressed in each body fluid were first selected. Eight miRNAs were then selected as novel forensically relevant miRNA markers: miR-484 and miR-182 for blood, miR-223 and miR-145 for saliva, miR-2392 and miR-3197 for semen, and miR-1260b and miR-654-5p for vaginal secretions. When the eight selected miRNAs were evaluated in 40 additional body fluid samples by qRT-PCR, they showed high sensitivity and specificity for the identification of the target body fluid. We suggest that the eight miRNAs may be candidates for developing an effective molecular assay for forensic body fluid identification. PMID:24915788

  2. A scheme for radiation pressure and photon diffusion with the M1 closure in RAMSES-RT

    NASA Astrophysics Data System (ADS)

    Rosdahl, J.; Teyssier, R.

    2015-06-01

    We describe and test an updated version of radiation-hydrodynamics in the RAMSES code, that includes three new features: (i) radiation pressure on gas, (ii) accurate treatment of radiation diffusion in an unresolved optically thick medium, and (iii) relativistic corrections that account for Doppler effects and work done by the radiation to first order in v/c. We validate the implementation in a series of tests, which include a morphological assessment of the M1 closure for the Eddington tensor in an astronomically relevant setting, dust absorption in an optically semithick medium, direct pressure on gas from ionizing radiation, convergence of our radiation diffusion scheme towards resolved optical depths, correct diffusion of a radiation flash and a constant luminosity radiation, and finally, an experiment from Davis et al. of the competition between gravity and radiation pressure in a dusty atmosphere, and the formation of radiative Rayleigh-Taylor instabilities. With the new features, RAMSES-RT can be used for state-of-the-art simulations of radiation feedback from first principles, on galactic and cosmological scales, including not only direct radiation pressure from ionizing photons, but also indirect pressure via dust from multiscattered IR photons reprocessed from higher-energy radiation, both in the optically thin and thick limits.

  3. Use of Trichoderma reesei RT-P1 crude enzyme powder for ethanol fermentation of sweet sorghum fresh stalks.

    PubMed

    Siwarasak, Pongsri; Pajantagate, Pradatrat; Prasertlertrat, Kanoktip

    2012-03-01

    Use of Trichoderma reesei RT-P1 crude enzyme powder and of this powder with 10%v/v Saccharomyces cerevisiae for ethanol fermentation of sweet sorghum fresh stalks were investigated. The optimal conditions were determined by orthogonal experiment method. With T. reesei crude enzyme powder, the optimal condition for the Keller cultivar was at 25 g with 4 g enzyme loading and for the Cowley cultivar at 30 g with 5 g enzyme loading, both with 8 days fermentation at pH 5 and 30°C. At the optimal conditions above, ethanol concentration, productivity and yield of the Cowley cultivar (35.00 g/L, 0.18 g/Lh and 0.38 g ethanol/g substrate, respectively) were higher than those of the Keller cultivar (20.46 g/L, 0.11 g/Lh and 0.28 g ethanol/g substrate). The addition of 10%v/v S. cerevisiae to fermentation at the optimal conditions showed no significant variations in ethanol concentration, productivity and yield for both cultivars. PMID:22221989

  4. Methanol Masers Observations in the 3-mm Bandwidth at the Radio Telescope RT-22 CrAO

    E-print Network

    S. Yu. Zubrin; A. V. Antyufeyev; V. V. Myshenko; V. M. Shulga

    2007-12-10

    We report the beginning of the astronomical masers investigations in the 3-mm bandwidth at the radio telescope RT-22 (CrAO, Ukraine). For this purpose the special complex for maser lines investigation in 85...115 GHz frequency band is developed. It is made on the base of the low noise cryogenic Shottky-diode receiver and the high resolution Fourier-spectrometer. The cryogenic receiver has the DSB noise temperature less than 100K. The spectral channel separation of the Fourier-spectrometer is about 4kHz and the spectrometer bandwidth is 8 MHz. Results of maser observations of 8$^{0}-7^{1} $A$^{+}$ transition of methanol (95.169 GHz) towards DR-21(OH), DR-21W and NGC7538 are in good agreement with early obtained results by other authors. On the basis of the analysis of the location of masers in the NGC7538 direction we can assume that the origin of all known class I methanol masers in this region is connected with existing molecular outflows from young stars.

  5. Development and Practical Use of RT-PCR for Seed-transmitted Prune dwarf virus in Quarantine

    PubMed Central

    Lee, Siwon; Shin, Yong-Gil

    2014-01-01

    Among imported plants, seeds are the items that have many latent pathogens and are difficult to inspect. Also, they are the import and export items whose market is expected to expand. The biggest problem with seeds is viruses. Prune dwarf virus (PDV) is the virus that is commonly inspected in Prunus cerasifera, P. persica, P. armeniaca, P. mandshurica, P. cerasus, P. avium or P. serotina seeds. In this study, two RT-PCR primer sets, which can promptly and specifically diagnose plant quarantine seed-transmitted PDV, were developed; and nested PCR primers, where products amplify 739 and 673 nucleotides (nt), and an nested PCR-product, 305 nt, can be obtained as these products are amplified again, were developed. Also, a modified-positive control plasmid was developed, where the restriction enzyme XhoI, which can identify the contamination of samples from the control, was inserted. The method developed in this study has detected PDV in 18 cases since 2007, and is expected to continuously contribute to the plant quarantine in Korea. PMID:25289000

  6. Development and Practical Use of RT-PCR for Seed-transmitted Prune dwarf virus in Quarantine.

    PubMed

    Lee, Siwon; Shin, Yong-Gil

    2014-06-01

    Among imported plants, seeds are the items that have many latent pathogens and are difficult to inspect. Also, they are the import and export items whose market is expected to expand. The biggest problem with seeds is viruses. Prune dwarf virus (PDV) is the virus that is commonly inspected in Prunus cerasifera, P. persica, P. armeniaca, P. mandshurica, P. cerasus, P. avium or P. serotina seeds. In this study, two RT-PCR primer sets, which can promptly and specifically diagnose plant quarantine seed-transmitted PDV, were developed; and nested PCR primers, where products amplify 739 and 673 nucleotides (nt), and an nested PCR-product, 305 nt, can be obtained as these products are amplified again, were developed. Also, a modified-positive control plasmid was developed, where the restriction enzyme XhoI, which can identify the contamination of samples from the control, was inserted. The method developed in this study has detected PDV in 18 cases since 2007, and is expected to continuously contribute to the plant quarantine in Korea. PMID:25289000

  7. Use of silica as a carrier to recover and prepare waterborne enteric viruses for detection by RT-PCR.

    PubMed

    Leisinger, M; Metzler, A

    1997-10-01

    A rapid, efficient and inexpensive method was developed to concentrate poliovirus type 1 (PV1), rotavirus (RV) and hepatitis A virus (HAV) from artificially spiked samples of tap and surface water. The method consists of adsorbing the viruses to silicon dioxide (SiO2) in the presence of 0.5 mM AlCl3 and adjustment of the pH to 3.5. The silica-adsorbed virus was collected by low speed centrifugation. Viral RNA was then extracted with guanidium thiocyanate (GT), and environmental nucleases and inhibitors of reverse transcriptase and Taq polymerase were further eliminated from concentrates by sequential treatment with GT, ethanol and acetone. Subsequent RT-PCR allowed the detection of as few as 1 to 10 TCID50 of PV1, RV, and HAV in seeded 1 liter samples of tap water. The same protocol was then used with effluents from two local sewage treatment plants. These samples, found to be free of HAV, were most commonly contaminated with enteroviruses and rotaviruses. Addition of 1000 TCID50 of HAV, PV1 or RV to a second 1 liter sample, taken at the same time from the corresponding surface waters allowed detection of the input virus without discernible inhibition by amplification inhibitors. The newly established method seems amenable to scaling up and promising for virus monitoring in different water types. The method is rapid and results can be obtained within 24 to 36 hours. PMID:9638882

  8. Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR

    PubMed Central

    Ling, Hui; Wu, Qibin; Guo, Jinlong; Xu, Liping; Que, Youxiong

    2014-01-01

    The increasingly used real time quantitative reverse transcription-PCR (qRT-PCR) method for gene expression analysis requires one or several reference gene(s) acting as normalization factor(s). In order to facilitate gene expression studies in sugarcane (Saccharum officinarum), a non-model plant with limited genome information, the stability of 13 candidate reference genes was evaluated. The geNorm, NormFinder and deltaCt methods were used for selecting stably expressed internal controls across different tissues and under various experimental treatments. These results revealed that, among these 13 candidate reference genes, GAPDH, eEF-1a and eIF-4? were the most stable and suitable for use as normalization factors across all various experimental samples. In addition, APRT could be a candidate for examining the relationship between gene copy number and transcript levels in sugarcane tissue samples. According to the results evaluated by geNorm, combining CUL and eEF-1? in hormone treatment experiments; CAC and CUL in abiotic stress tests; GAPDH, eEF-1a and CUL in all treatment samples plus CAC, CUL, APRT and TIPS-41 in cultivar tissues as groups for normalization would lead to more accurate and reliable expression quantification in sugarcane. This is the first systematic validation of reference genes for quantification of transcript expression profiles in sugarcane. This study should provide useful information for selecting reference genes for more accurate quantification of gene expression in sugarcane and other plant species. PMID:24823940

  9. Microarray and RT-PCR screening for white spot syndrome virus immediate-early genes in cycloheximide-treated shrimp

    SciTech Connect

    Liu Wangjing; Chang Yunshiang; Wang Chunghsiung; Kou, Guang-Hsiung; Lo Chufang . E-mail: gracelow@ntu.edu.tw

    2005-04-10

    Here, we report for the first time the successful use of cycloheximide (CHX) as an inhibitor to block de novo viral protein synthesis during WSSV (white spot syndrome virus) infection. Sixty candidate IE (immediate-early) genes were identified using a global analysis microarray technique. RT-PCR showed that the genes corresponding to ORF126, ORF242 and ORF418 in the Taiwan isolate were consistently CHX-insensitive, and these genes were designated ie1, ie2 and ie3, respectively. The sequences for these IE genes also appear in the two other WSSV isolates that have been sequenced. Three corresponding ORFs were identified in the China WSSV isolate, but only an ORF corresponding to ie1 was predicted in the Thailand isolate. In a promoter activity assay in Sf9 insect cells using EGFP (enhanced green fluorescence protein) as a reporter, ie1 showed very strong promoter activity, producing higher EGFP signals than the insect Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) ie2 promoter.

  10. Construction of RNA standards for high-resolution automatic product analysis in quantitative competitive RT-PCR.

    PubMed

    Repp, R; Borkhardt, A; Gossen, R; Kreuder, J; Hammermann, J; Lampert, F

    1995-07-01

    The exponential character of PCR amplification may compromise quantitative assays because it multiplies minor sample-to-sample variations. To overcome these problems, several authors have used recombinant standard DNA or RNA molecules to be spiked into the samples in a dilution series of known copy numbers before co-amplification by PCR. To obtain an equal efficacy of reverse transcription and PCR amplification, standard and template molecules should be highly homologous. However, the limited resolution of commonly used agarose gel electrophoresis requires rather large differences in size and nucleotide sequence to separate both molecules from each other after PCR. Due to a much higher resolution, automatic post-PCR analyzing systems based on laser-induced fluorescence may help to overcome these difficulties. For using the capabilities of these systems in quantitative competitive RT-PCR, we developed a protocol to construct recombinant RNA standard molecules that only differ from the target sequence by a small deletion of 8 nucleotides. It is based on PCR-induced mutagenesis and solid-phase in vitro transcription. This protocol was applied to quantify multidrug resistance gene (MDRI) mRNA in malignant cells, but it can easily be adapted to any gene of interest. PMID:7545409

  11. Evaluation and Validation of Reference Genes for qRT-PCR Normalization in Frankliniella occidentalis (Thysanoptera:Thripidae)

    PubMed Central

    Zheng, Yu-Tao; Li, Hong-Bo; Lu, Ming-Xing; Du, Yu-Zhou

    2014-01-01

    Quantitative real time PCR (qRT-PCR) has emerged as a reliable and reproducible technique for studying gene expression analysis. For accurate results, the normalization of data with reference genes is particularly essential. Once the transcriptome sequencing of Frankliniella occidentalis was completed, numerous unigenes were identified and annotated. Unfortunately, there are no studies on the stability of reference genes used in F. occidentalis. In this work, seven candidate reference genes, including actin, 18S rRNA, H3, tubulin, GAPDH, EF-1 and RPL32, were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs BestKeeper, geNorm, Normfinder and the comparative ?Ct method. Because the rankings of the reference genes provided by each of the four programs were different, we chose a user-friendly web-based comprehensive tool RefFinder to get the final ranking. The result demonstrated that EF-1 and RPL32 displayed the most stable expression in different developmental stages; RPL32 and GAPDH showed the most stable expression at high temperatures, while 18S and EF-1 exhibited the most stable expression at low temperatures. In this study, we validated the suitable reference genes in F. occidentalis for gene expression profiling under different experimental conditions. The choice of internal standard is very important in the normalization of the target gene expression levels, thus validating and selecting the best genes will help improve the quality of gene expression data of F. occidentalis. What is more, these validated reference genes could serve as the basis for the selection of candidate reference genes in other insects. PMID:25356721

  12. RT-PCR-based tyrosine kinase display profiling of canine melanoma: IGF-1 receptor as a potential therapeutic target.

    PubMed

    Thamm, Douglas H; Huelsmeyer, Michael K; Mitzey, Ann M; Qurollo, Barbara; Rose, Barbara J; Kurzman, Ilene D

    2010-02-01

    Canine malignant melanoma (CMM) resembles human malignant melanoma in terms of metastatic behavior, refractoriness to standard therapy, and tumor antigen expression but it is largely unknown how CMM resembles human melanoma with regard to molecular pathogenesis and cellular signaling. No attempt has been made to systematically define the repertoire of tyrosine kinases (TKs) expressed in CMM. This study used a reverse transcription-PCR display technique to evaluate the expression of multiple TKs in the 17CM98 CMM cell line. RT-PCR was performed using degenerate primers coding for highly conserved regions flanking the kinase domains of many TKs and the repertoire of TKs expressed was determined using standard molecular cloning techniques. Sequencing 46 clones yielded canine homologs of insulin-like growth factor-1 receptor (IGF-1R) (50%), JAK1 (17%), PDGFR-a (11%), FGFR1 (9%), Axl (7%), Abl (4%), and PTK2 (2%). Interestingly, IGF-1R, JAK1, and Axl were detected in human melanoma using similar techniques, supporting the cross-species validity of this assay. Given the abundance of IGF-1R clones, we determined the biological effect of rhIGF-1 in 17CM98 cells. IGF-1 stimulated cell proliferation and vascular endothelial growth factor production in 17CM98, and addition of the IGF-1R inhibitor ADW742 abrogated IGF-1-induced phenotypic changes. Expression of IGF-1R mRNA was detected in five of five additional CMM cell cultures, and IGF-1R protein was detected in five of six primary tumors evaluated, suggesting that IGF-1R expression may be common in CMM and may provide a novel target for future therapy. In conclusion, this study suggests that similar TKs are expressed in human and canine melanoma, and shows potential antitumor effects of IGF-1R inhibition in CMM. PMID:19949352

  13. Differential display RT PCR of total RNA from human foreskin fibroblasts for investigation of androgen-dependent gene expression

    SciTech Connect

    Nitsche, E.M.; Moquin, A.; Adams, P.S.

    1996-05-03

    Male sexual differentiation is a process that involves androgen action via the androgen receptor. Defects in the androgen receptor, many resulting from point mutations in the androgen receptor gene, lead to varying degrees of impaired masculinization in chromosomally male individuals. To date no specific androgen regulated morphogens involved in this process have been identified and no marker genes are known that would help to predict further virilization in infants with partial androgen insensitivity. In the present study we first show data on androgen regulated gene expression investigated by differential display reverse transcription PCR (dd RT PCR) on total RNA from human neonatal genital skin fibroblasts cultured in the presence or absence of 100 nM testosterone. Using three different primer combinations, 54 cDNAs appeared to be regulated by androgens. Most of these sequences show the characteristics of expressed mRNAs but showed no homology to sequences in the database. However 15 clones with significant homology to previously cloned sequences were identified. Seven cDNAs appear to be induced by androgen withdrawal. Of these, five are similar to ETS (expression tagged sequences) from unknown genes; the other two show significant homology to the cDNAs of ubiquitin and human guanylate binding protein 2 (GBP-2). In addition, we have identified 8 cDNA clones which show homologies to other sequences in the database and appear to be upregulated in the presence of testosterone. Three differential expressed sequences show significant homology to the cDNAs of L-plastin and one to the cDNA of testican. This latter gene codes for a proteoglycan involved in cell social behavior and therefore of special interest in this context. The results of this study are of interest in further investigation of normal and disturbed androgen-dependent gene expression. 49 refs., 2 figs., 5 tabs.

  14. Ecological comparison of cellular stress responses among populations – normalizing RT-qPCR values to investigate differential environmental adaptations

    PubMed Central

    2013-01-01

    Background Rising temperatures and other environmental factors influenced by global climate change can cause increased physiological stress for many species and lead to range shifts or regional population extinctions. To advance the understanding of species’ response to change and establish links between individual and ecosystem adaptations, physiological reactions have to be compared between populations living in different environments. Although changes in expression of stress genes are relatively easy to quantify, methods for reliable comparison of the data remain a contentious issue. Using normalization algorithms and further methodological considerations, we compare cellular stress response gene expression levels measured by RT-qPCR after air exposure experiments among different subpopulations of three species of the intertidal limpet Nacella. Results Reference gene assessment algorithms reveal that stable reference genes can differ among investigated populations and / or treatment groups. Normalized expression values point to differential defense strategies to air exposure in the investigated populations, which either employ a pronounced cellular stress response in the inducible Hsp70 forms, or exhibit a comparatively high constitutive expression of Hsps (heat shock proteins) while showing only little response in terms of Hsp induction. Conclusions This study serves as a case study to explore the methodological prerequisites of physiological stress response comparisons among ecologically and phylogenetically different organisms. To improve the reliability of gene expression data and compare the stress responses of subpopulations under potential genetic divergence, reference gene stability algorithms are valuable and necessary tools. As the Hsp70 isoforms have been shown to play different roles in the acute stress responses and increased constitutive defenses of populations in their different habitats, these comparative studies can yield insight into physiological strategies of adaptation to environmental stress and provide hints for the prudent use of the cellular stress response as a biomarker to study environmental stress and stress adaptation of populations under changing environmental conditions. PMID:23680017

  15. An ex vivo RT-qPCR-based assay for human peripheral leukocyte responsiveness to glucocorticoids in surgically induced inflammation

    PubMed Central

    Gråberg, Truls; Strömmer, Lovisa; Hedman, Erik; Uzunel, Mehmet; Ehrenborg, Ewa; Wikström, Ann-Charlotte

    2015-01-01

    Introduction An assay to determine glucocorticoid (GC) responsiveness in humans could be used to monitor GC non-responsiveness in states of GC insufficiency and could provide a tool to adapt GC treatment to individual patients. We propose an ex vivo assay to test GC responsiveness in peripheral leukocytes. The assay was evaluated in a human experimental model of surgery-induced inflammation. Patients and methods Changes in expression of the GC-regulated genes GILZ, IL1R2, FKBP5, and HLA-DR and glucocorticoid receptor alpha (GR?) were determined by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in peripheral leukocytes from surgical patients and healthy blood donors (total n=60) in response to low (1 nM) and high (1 µM) dexamethasone (DEX). The final selection of a suitable endogenous control gene was based on the studies of stability during DEX treatment and inflammation. Correlations between pre- and postoperative GC-induced gene expression, the postoperative systemic inflammatory and metabolic response (CRP, IL-6, white blood cell count, cytokines, resistin, free fatty acids, glucose, insulin, and adiponectin), and the clinical outcome were analyzed. The length of stay in the intensive care unit (ICU-LOS), the length of stay in the hospital, and postoperative complications were used to measure clinical outcome. Results When the blood donors were compared to the patients, there were no significant differences in the regulation of the genes in response to DEX, except for GR?. Preoperative, but not postoperative, gene regulation of GILZ and GR? was negatively correlated to ICU-LOS (P<0.05 and P<0.01, respectively). Preoperative GILZ and FKBP5 gene regulation was negatively correlated to postoperative systemic TNF? and MIP-1? levels. Conclusion We suggest that this assay could be used to determine GC responsiveness. An alteration in preoperative GC responsiveness may be related to a patient’s ability to recover from surgically induced inflammatory stress. PMID:26316794

  16. Study design of DIACORE (DIAbetes COhoRtE) – a cohort study of patients with diabetes mellitus type 2

    PubMed Central

    2013-01-01

    Background Diabetes mellitus type 2 (DM2) is highly associated with increased risk for chronic kidney disease (CKD), end stage renal disease (ESRD) and cardiovascular morbidity. Epidemiological and genetic studies generate hypotheses for innovative strategies in DM2 management by unravelling novel mechanisms of diabetes complications, which is essential for future intervention trials. We have thus initiated the DIAbetes COhoRtE study (DIACORE). Methods DIACORE is a prospective cohort study aiming to recruit 6000 patients of self-reported Caucasian ethnicity with prevalent DM2 for at least 10 years of follow-up. Study visits are performed in University-based recruiting clinics in Germany using standard operating procedures. All prevalent DM2 patients in outpatient clinics surrounding the recruiting centers are invited to participate. At baseline and at each 2-year follow-up examination, patients are subjected to a core phenotyping protocol. This includes a standardized online questionnaire and physical examination to determine incident micro- and macrovascular DM2 complications, malignancy and hospitalization, with a primary focus on renal events. Confirmatory outcome information is requested from patient records. Blood samples are obtained for a centrally analyzed standard laboratory panel and for biobanking of aliquots of serum, plasma, urine, mRNA and DNA for future scientific use. A subset of the cohort is subjected to extended phenotyping, e.g. sleep apnea screening, skin autofluorescence measurement, non-mydriatic retinal photography and non-invasive determination of arterial stiffness. Discussion DIACORE will enable the prospective evaluation of factors involved in DM2 complication pathogenesis using high-throughput technologies in biosamples and genetic epidemiological studies. PMID:23409726

  17. Intravenous siRNA silencing of survivin enhances activity of mitomycin C in human bladder RT4 xenografts

    PubMed Central

    Cui, Minjian; Au, Jessie L.-S.; Wientjes, M. Guillaume; O’Donnell, Michael A.; Loughlin, Kevin R.; Lu, Ze

    2015-01-01

    Purpose Survivin inhibits apoptosis and enables tumor cells to escape from therapy-induced senescence. High expression of survivin is associated with bladder cancer aggressiveness and recurrence. The present study evaluated if survivin expression is reduced by siRNA and if survivin silencing enhances the activity of mitomycin C (MMC), in human RT4 bladder transitional cell tumors in vitro and in vivo. Materials and Methods The effectiveness of siRNA therapy was evaluated using two newly developed pegylated cationic liposome carriers (PCat, PPCat). Both carries used a fusogenic lipid to destabilize the endosomal membrane. One carrier (PPCat) further contained paclitaxel to enhance the in vivo delivery and transfection of survivin siRNA (siSurvivin). In vitro antitumor activity was evaluated using short and long term cytotoxicity assays (microtetrazolium and clonogenicity). In vivo intravenous therapy was evaluated in mice bearing subcutaneous tumors. Results The nontarget-siRNA had no antitumor activity in vitro or in vivo. Treatment of cultured cells with MMC at 50% cytotoxic concentration enhanced survivin mRNA and protein levels; addition of PPCat or PCat containing siSurvivin reversed the survivin induction and enhanced the MMC activity (p<0.05). In tumor-bearing mice, single agent MMC delayed tumor growth and nearly tripled the survivin protein level in residual tumors, whereas addition of PPCat-siSurvivin, which by itself yielded a minor survivin reduction (<10%), completely reversed the MMC-induced survivin and enhanced the MMC activity (p<0.05). Conclusions The results indicate effective in vivo survivin silencing and synergism between MMC and PPCat-siSurvivin. This combination represents a potentially useful chemo-gene therapy for bladder cancer. PMID:25681288

  18. Perceptual learning for multiple features: Neural correlates of changes in RT-based measures of processing dependencies.

    PubMed

    Wenger, Michael; Rhoten, Stephanie

    2015-09-01

    We previously (VSS 2014) reported EEG and behavioral data (response frequencies) supporting the hypothesis that that perceptual learning can be obtained with multiple features and that learning involves the development of non-independence at perceptual and decisional levels. This is consistent with evidence suggesting that multiple levels of processing and representation may be involved in perceptual learning. The present study explores these notions using EEG and response times (RTs), testing the hypothesis that learning of multi-feature patterns results in processing that is non-independent and parallel, as would be expected if the stimuli were learned as integral objects. Stimuli contained contrast-defined features, extracted from the 1865 drawing of the Cheshire Cat in Alice's Adventures in Wonderland. Participants began by performing a detection task implemented as a double-factorial paradigm (DFP), in which stimuli contained 0, 1, or 2 targets, at two levels of contrast, and were instructed to give a positive response if they judged the stimulus to have either 1 or 2 targets. They then practiced with all possible stimuli for 10-15 days, using an adaptive staircase procedure to track thresholds. Finally, they again performed the DFP detection task with stimuli presented at threshold and the two initial levels of contrast. EEG data were collected during both pre- and post-practice performance of the CID. Analysis of the RT data, at both the level of the mean and the distribution, suggested that the stimuli were initially processed serially, with practice resulting in a transition to dependent parallel processing and modulations of amplitudes of early ERP features, pre- and post-stimulus spectral power, and reductions in post-stimulus g-band power, selective to frontal electrodes. These results are consistent with the idea that perceptual learning of multi-feature patterns produces shifts in fundamental characteristics of processing consistent with the learning of an integral object. Meeting abstract presented at VSS 2015. PMID:26326817

  19. One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3

    PubMed Central

    2012-01-01

    Background Detection of respiratory viruses in veterinary species has traditionally relied on virus detection by isolation or immunofluorescence and/or detection of circulating antibody using ELISA or serum neutralising antibody tests. Multiplex real time PCR is increasingly used to diagnose respiratory viruses in humans and has proved to be superior to traditional methods. Bovine respiratory disease (BRD) is one of the most common causes of morbidity and mortality in housed cattle and virus infections can play a major role. We describe here a one step multiplex reverse transcriptase quantitative polymerase chain reaction (mRT-qPCR) to detect the viruses commonly implicated in BRD. Results A mRT-qPCR assay was developed and optimised for the simultaneous detection of bovine respiratory syncytial virus (BRSV), bovine herpes virus type 1 (BoHV-1) and bovine parainfluenza virus type 3 (BPI3 i & ii) nucleic acids in clinical samples from cattle. The assay targets the highly conserved glycoprotein B gene of BoHV-1, nucleocapsid gene of BRSV and nucleoprotein gene of BPI3. This mRT-qPCR assay was assessed for sensitivity, specificity and repeatability using in vitro transcribed RNA and recent field isolates. For clinical validation, 541 samples from clinically affected animals were tested and mRT-qPCR result compared to those obtained by conventional testing using virus isolation (VI) and/or indirect fluorescent antibody test (IFAT). Conclusions The mRT-qPCR assay was rapid, highly repeatable, specific and had a sensitivity of 97% in detecting 102 copies of BRSV, BoHV-1 and BPI3 i & ii. This is the first mRT-qPCR developed to detect the three primary viral agents of BRD and the first multiplex designed using locked nucleic acid (LNA), minor groove binding (MGB) and TaqMan probes in one reaction mix. This test was more sensitive than both VI and IFAT and can replace the aforesaid methods for virus detection during outbreaks of BRD. PMID:22455597

  20. Rapid detection and differentiation of dengue virus serotypes by NS1 specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in patients presenting to a tertiary care hospital in Hyderabad, India.

    PubMed

    Neeraja, M; Lakshmi, V; Lavanya, Vanjari; Priyanka, E N; Parida, M M; Dash, P K; Sharma, Shashi; Rao, P V Lakshmana; Reddy, Gopal

    2015-01-01

    Early and rapid detection of dengue virus (DENV) infection during the acute phase of illness is crucial for proper patient management and prevention of the spread of the infection. In the present study, the standardization and validation of a one step, four tube reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) for rapid detection and serotyping of the DENV targeting NS1 gene using the Genie® II flourometer was carried out. The performance of the RT-LAMP was compared to RT-PCR, CDC 1-4 Real time PCR and the NS1 antigen ELISA, IgM and IgG anti DENV antibodies. Acute DENV infection was confirmed in 250/300 patients suspected clinically of DENV infection. RT- LAMP and CDC 1-4 Real time PCR assay was positive in 148/250 patients, while 92/250 patients were positive for anti- Dengue IgM and IgG antibodies. The RT-LAMP assay and the CDC real-time RT-PCR assay showed high concordance (k=1.0). The detection rate of acute DENV infection improved to 96% (240/250) when the results of RT-LAMP were combined with NS1 Ag, IgM and IgG ELISA. The RT-LAMP had a detection limit of 100 copies for DEN-1 and DEN-2, 10 copies for DEN-3 and DEN-4 compared to 1000 copies for DEN-1 and DEN-2, 100 copies for DEN-3 and DEN-4 by the conventional RT-PCR. The assay showed 100% specificity. The RT-LAMP assay developed in this study has potential use for early clinical diagnosis, serotyping and surveillance of DENV infection in endemic countries such as India. PMID:25455901