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Sample records for apobec3 includes retroviral

  1. APOBEC3G enhances lymphoma cell radioresistance by promoting cytidine deaminase-dependent DNA repair.

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    Nowarski, Roni; Wilner, Ofer I; Cheshin, Ori; Shahar, Or D; Kenig, Edan; Baraz, Leah; Britan-Rosich, Elena; Nagler, Arnon; Harris, Reuben S; Goldberg, Michal; Willner, Itamar; Kotler, Moshe

    2012-07-12

    APOBEC3 proteins catalyze deamination of cytidines in single-stranded DNA (ssDNA), providing innate protection against retroviral replication by inducing deleterious dC > dU hypermutation of replication intermediates. APOBEC3G expression is induced in mitogen-activated lymphocytes; however, no physiologic role related to lymphoid cell proliferation has yet to be determined. Moreover, whether APOBEC3G cytidine deaminase activity transcends to processing cellular genomic DNA is unknown. Here we show that lymphoma cells expressing high APOBEC3G levels display efficient repair of genomic DNA double-strand breaks (DSBs) induced by ionizing radiation and enhanced survival of irradiated cells. APOBEC3G transiently accumulated in the nucleus in response to ionizing radiation and was recruited to DSB repair foci. Consistent with a direct role in DSB repair, inhibition of APOBEC3G expression or deaminase activity resulted in deficient DSB repair, whereas reconstitution of APOBEC3G expression in leukemia cells enhanced DSB repair. APOBEC3G activity involved processing of DNA flanking a DSB in an integrated reporter cassette. Atomic force microscopy indicated that APOBEC3G multimers associate with ssDNA termini, triggering multimer disassembly to multiple catalytic units. These results identify APOBEC3G as a prosurvival factor in lymphoma cells, marking APOBEC3G as a potential target for sensitizing lymphoma to radiation therapy.

  2. Evidence for restriction of ancient primate gammaretroviruses by APOBEC3 but not TRIM5alpha proteins.

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    David Perez-Caballero

    2008-10-01

    Full Text Available Because of evolutionary pressures imposed through episodic colonization by retroviruses, many mammals express factors, such as TRIM5alpha and APOBEC3 proteins, that directly restrict retroviral replication. TRIM5 and APOBEC restriction factors are most often studied in the context of modern primate lentiviruses, but it is likely that ancient retroviruses imposed the selective pressure that is evident in primate TRIM5 and APOBEC3 genes. Moreover, these antiretroviral factors have been shown to act against a variety of retroviruses, including gammaretroviruses. Endogenous retroviruses can provide a 'fossil record' of extinct retroviruses and perhaps evidence of ancient TRIM5 and APOBEC3 antiviral activity. Here, we investigate whether TRIM5 and APOBEC3 proteins restricted the replication of two groups of gammaretroviruses that were endogenized in the past few million years. These endogenous retroviruses appear quite widespread in the genomes of old world primates but failed to colonize the human germline. Our analyses suggest that TRIM5alpha proteins did not pose a major barrier to the cross-species transmission of these two families of gammaretroviruses, and did not contribute to their extinction. However, we uncovered extensive evidence for inactivation of ancient gammaretroviruses through the action of APOBEC3 cytidine deaminases. Interestingly, the identities of the cytidine deaminases responsible for inactivation appear to have varied in both a virus and host species-dependent manner. Overall, sequence analyses and reconstitution of ancient retroviruses from remnants that have been preserved in the genomes of modern organisms offer the opportunity to probe and potentially explain the evolutionary history of host defenses against retroviruses.

  3. Host Restriction of Lentiviruses and Viral Countermeasures: APOBEC3 and Vif

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    Valgerdur Andrésdóttir

    2013-07-01

    Full Text Available It is becoming increasingly clear that organisms have developed a variety of mechanisms to fight against viral infection. The viruses have developed means of counteracting these defences in various ways. The APOBEC3 proteins are a mammalian-specific family of nucleic acid cytidine deaminases that block retroviral infection. These inhibitors are counteracted by the Vif proteins encoded by most lentiviruses. In this paper, we will review the interaction of the lentiviral Vif proteins with the APOBEC3 proteins, with an emphasis on sheep APOBEC3 and maedi-visna virus (MVV Vif.

  4. HIV restriction by APOBEC3 in humanized mice.

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    John F Krisko

    2013-03-01

    Full Text Available Innate immune restriction factors represent important specialized barriers to zoonotic transmission of viruses. Significant consideration has been given to their possible use for therapeutic benefit. The apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 (APOBEC3 family of cytidine deaminases are potent immune defense molecules capable of efficiently restricting endogenous retroelements as well as a broad range of viruses including Human Immunodeficiency virus (HIV, Hepatitis B virus (HBV, Human Papilloma virus (HPV, and Human T Cell Leukemia virus (HTLV. The best characterized members of this family are APOBEC3G (A3G and APOBEC3F (A3F and their restriction of HIV. HIV has evolved to counteract these powerful restriction factors by encoding an accessory gene designated viral infectivity factor (vif. Here we demonstrate that APOBEC3 efficiently restricts CCR5-tropic HIV in the absence of Vif. However, our results also show that CXCR4-tropic HIV can escape from APOBEC3 restriction and replicate in vivo independent of Vif. Molecular analysis identified thymocytes as cells with reduced A3G and A3F expression. Direct injection of vif-defective HIV into the thymus resulted in viral replication and dissemination detected by plasma viral load analysis; however, vif-defective viruses remained sensitive to APOBEC3 restriction as extensive G to A mutation was observed in proviral DNA recovered from other organs. Remarkably, HIV replication persisted despite the inability of HIV to develop resistance to APOBEC3 in the absence of Vif. Our results provide novel insight into a highly specific subset of cells that potentially circumvent the action of APOBEC3; however our results also demonstrate the massive inactivation of CCR5-tropic HIV in the absence of Vif.

  5. Expression of APOBEC3B mRNA in Primary Breast Cancer of Japanese Women

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    Tokunaga, Eriko; Yamashita, Nami; Tanaka, Kimihiro; Inoue, Yuka; Akiyoshi, Sayuri; Saeki, Hiroshi; Oki, Eiji; Kitao, Hiroyuki; Maehara, Yoshihiko

    2016-01-01

    Recent studies have identified the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3B (APOBEC3B) as a source of mutations in various malignancies. APOBEC3B is overexpressed in several human cancer types, including breast cancer. In this study, we analyzed APOBEC3B mRNA expression in 305 primary breast cancers of Japanese women using quantitative reverse transcription-PCR, and investigated the relationships between the APOBEC3B mRNA expression and clinicopathological characteristics, prognosis, and TP53 mutations. The expression of APOBEC3B mRNA was detected in 277 tumors and not detected in 28 tumors. High APOBEC3B mRNA expression was significantly correlated with ER- and PR-negativity, high grade and high Ki67 index. The APOBEC3B mRNA expression was highest in the triple-negative and lowest in the hormone receptor-positive/HER2-negative subtypes. The TP53 gene was more frequently mutated in the tumors with high APOBEC3B mRNA expression. High APOBEC3B mRNA expression was significantly associated with poor recurrence-free survival in all cases and the ER-positive cases. These findings were almost consistent with the previous reports from the Western countries. In conclusion, high APOBEC3B mRNA expression was related to the aggressive phenotypes of breast cancer, high frequency of TP53 mutation and poor prognosis, especially in ER-positive tumors. PMID:27977754

  6. Noninfectious retrovirus particles drive the APOBEC3/Rfv3 dependent neutralizing antibody response.

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    Diana S Smith

    2011-10-01

    Full Text Available Members of the APOBEC3 family of deoxycytidine deaminases counteract a broad range of retroviruses in vitro through an indirect mechanism that requires virion incorporation and inhibition of reverse transcription and/or hypermutation of minus strand transcripts in the next target cell. The selective advantage to the host of this indirect restriction mechanism remains unclear, but valuable insights may be gained by studying APOBEC3 function in vivo. Apobec3 was previously shown to encode Rfv3, a classical resistance gene that controls the recovery of mice from pathogenic Friend retrovirus (FV infection by promoting a more potent neutralizing antibody (NAb response. The underlying mechanism does not involve a direct effect of Apobec3 on B cell function. Here we show that while Apobec3 decreased titers of infectious virus during acute FV infection, plasma viral RNA loads were maintained, indicating substantial release of noninfectious particles in vivo. The lack of plasma virion infectivity was associated with a significant post-entry block during early reverse transcription rather than G-to-A hypermutation. The Apobec3-dependent NAb response correlated with IgG binding titers against native, but not detergent-lysed virions. These findings indicate that innate Apobec3 restriction promotes NAb responses by maintaining high concentrations of virions with native B cell epitopes, but in the context of low virion infectivity. Finally, Apobec3 restriction was found to be saturable in vivo, since increasing FV inoculum doses resulted in decreased Apobec3 inhibition. By analogy, maximizing the release of noninfectious particles by modulating APOBEC3 expression may improve humoral immunity against pathogenic human retroviral infections.

  7. The Role of the Antiviral APOBEC3 Gene Family in Protecting Chimpanzees against Lentiviruses from Monkeys.

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    Lucie Etienne

    2015-09-01

    Full Text Available Cross-species transmissions of viruses from animals to humans are at the origin of major human pathogenic viruses. While the role of ecological and epidemiological factors in the emergence of new pathogens is well documented, the importance of host factors is often unknown. Chimpanzees are the closest relatives of humans and the animal reservoir at the origin of the human AIDS pandemic. However, despite being regularly exposed to monkey lentiviruses through hunting, chimpanzees are naturally infected by only a single simian immunodeficiency virus, SIVcpz. Here, we asked why chimpanzees appear to be protected against the successful emergence of other SIVs. In particular, we investigated the role of the chimpanzee APOBEC3 genes in providing a barrier to infection by most monkey lentiviruses. We found that most SIV Vifs, including Vif from SIVwrc infecting western-red colobus, the chimpanzee's main monkey prey in West Africa, could not antagonize chimpanzee APOBEC3G. Moreover, chimpanzee APOBEC3D, as well as APOBEC3F and APOBEC3H, provided additional protection against SIV Vif antagonism. Consequently, lentiviral replication in primary chimpanzee CD4(+ T cells was dependent on the presence of a lentiviral vif gene that could antagonize chimpanzee APOBEC3s. Finally, by identifying and functionally characterizing several APOBEC3 gene polymorphisms in both common chimpanzees and bonobos, we found that these ape populations encode APOBEC3 proteins that are uniformly resistant to antagonism by monkey lentiviruses.

  8. Impact of functional germline variants and a deletion polymorphism in APOBEC3A and APOBEC3B on breast cancer risk and survival in a Swedish study population.

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    Göhler, Stella; Da Silva Filho, Miguel Inacio; Johansson, Robert; Enquist-Olsson, Kerstin; Henriksson, Roger; Hemminki, Kari; Lenner, Per; Försti, Asta

    2016-01-01

    The C → T mutation signature caused by APOBEC family members contributes to the development of breast cancer (BC). Also overexpression of APOBEC3B and a ~29.5-kb deletion polymorphism between APOBEC3A and APOBEC3B have been associated with increased BC risk. We investigated in a population-based study, with 782 Swedish BC cases and 1559 controls, associations between potentially functional germline variants in APOBEC3A or APOBEC3B gene and BC risk and survival. Additionally, we identified deletion polymorphism carriers and explored possible associations with BC. No evidence of association between any germline variant, including the deletion polymorphism, and BC risk or survival was observed. Only APOBEC3A promoter polymorphism rs5757402 was associated with low stage (OR = 0.69, 95 % CI 0.50-0.96, dominant model). The reported association between the deletion polymorphism and BC risk was not confirmed in the Swedish population, nor did any genotyped germline variant show any association with BC risk or survival.

  9. Feline Immunodeficiency Virus Evolutionarily Acquires Two Proteins, Vif and Protease, Capable of Antagonizing Feline APOBEC3.

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    Yoshikawa, Rokusuke; Takeuchi, Junko S; Yamada, Eri; Nakano, Yusuke; Misawa, Naoko; Kimura, Yuichi; Ren, Fengrong; Miyazawa, Takayuki; Koyanagi, Yoshio; Sato, Kei

    2017-06-01

    The interplay between viral and host proteins has been well studied to elucidate virus-host interactions and their relevance to virulence. Mammalian genes encode apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins, which act as intrinsic restriction factors against lentiviruses. To overcome APOBEC3-mediated antiviral actions, lentiviruses have evolutionarily acquired an accessory protein, viral infectivity factor (Vif), and Vif degrades host APOBEC3 proteins via a ubiquitin/proteasome-dependent pathway. Although the Vif-APOBEC3 interaction and its evolutionary significance, particularly those of primate lentiviruses (including HIV) and primates (including humans), have been well investigated, those of nonprimate lentiviruses and nonprimates are poorly understood. Moreover, the factors that determine lentiviral pathogenicity remain unclear. Here, we focus on feline immunodeficiency virus (FIV), a pathogenic lentivirus in domestic cats, and the interaction between FIV Vif and feline APOBEC3 in terms of viral virulence and evolution. We reveal the significantly reduced diversity of FIV subtype B compared to that of other subtypes, which may associate with the low pathogenicity of this subtype. We also demonstrate that FIV subtype B Vif is less active with regard to feline APOBEC3 degradation. More intriguingly, we further reveal that FIV protease cleaves feline APOBEC3 in released virions. Taken together, our findings provide evidence that a lentivirus encodes two types of anti-APOBEC3 factors, Vif and viral protease. IMPORTANCE During the history of mammalian evolution, mammals coevolved with retroviruses, including lentiviruses. All pathogenic lentiviruses, excluding equine infectious anemia virus, have acquired the vif gene via evolution to combat APOBEC3 proteins, which are intrinsic restriction factors against exogenous lentiviruses. Here we demonstrate that FIV, a pathogenic lentivirus in domestic cats, antagonizes feline APOBEC3

  10. APOBEC3D and APOBEC3F potently promote HIV-1 diversification and evolution in humanized mouse model.

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    Kei Sato

    2014-10-01

    Full Text Available Several APOBEC3 proteins, particularly APOBEC3D, APOBEC3F, and APOBEC3G, induce G-to-A hypermutations in HIV-1 genome, and abrogate viral replication in experimental systems, but their relative contributions to controlling viral replication and viral genetic variation in vivo have not been elucidated. On the other hand, an HIV-1-encoded protein, Vif, can degrade these APOBEC3 proteins via a ubiquitin/proteasome pathway. Although APOBEC3 proteins have been widely considered as potent restriction factors against HIV-1, it remains unclear which endogenous APOBEC3 protein(s affect HIV-1 propagation in vivo. Here we use a humanized mouse model and HIV-1 with mutations in Vif motifs that are responsible for specific APOBEC3 interactions, DRMR/AAAA (4A or YRHHY/AAAAA (5A, and demonstrate that endogenous APOBEC3D/F and APOBEC3G exert strong anti-HIV-1 activity in vivo. We also show that the growth kinetics of 4A HIV-1 negatively correlated with the expression level of APOBEC3F. Moreover, single genome sequencing analyses of viral RNA in plasma of infected mice reveal that 4A HIV-1 is specifically and significantly diversified. Furthermore, a mutated virus that is capable of using both CCR5 and CXCR4 as entry coreceptor is specifically detected in 4A HIV-1-infected mice. Taken together, our results demonstrate that APOBEC3D/F and APOBEC3G fundamentally work as restriction factors against HIV-1 in vivo, but at the same time, that APOBEC3D and APOBEC3F are capable of promoting viral diversification and evolution in vivo.

  11. APOBEC3 deletion polymorphism is associated with breast cancer risk among women of European ancestry.

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    Xuan, Dennis; Li, Guoliang; Cai, Qiuyin; Deming-Halverson, Sandra; Shrubsole, Martha J; Shu, Xiao-Ou; Kelley, Mark C; Zheng, Wei; Long, Jirong

    2013-10-01

    Copy number variations occur frequently in the genome and are a significant source of human genetic variation accounting for disease. Recently, we discovered a common deletion located in the APOBEC3A and APOBEC3B genes significantly associated with breast cancer in Chinese women. Investigating this locus in other populations would be an expedient way to evaluate the generalizability of the novel finding. We analyzed the APOBEC3 deletion in a large study of 3273 European-ancestry women (including 1671 breast cancer cases and 1602 controls) from the population-based Nashville Breast Health Study. All participants were genotyped using real-time qualitative PCR. Logistic regression was used to derive odds ratios (ORs) and 95% confidence intervals (CIs) for the associations between the deletion polymorphism and breast cancer risk. The APOBEC3 deletion was observed in 12.4% of cases and 10.4% of controls. The deletion was significantly associated with breast cancer risk, with ORs and 95% CIs of 1.21 (1.02-1.43) associated with one-copy deletion and 2.29 (1.04-5.06) associated with two-copy deletion compared with women with no deletion (P for trend = 0.005). The positive association of the APOBEC3 deletion with breast cancer risk was similar for estrogen receptor-positive and estrogen receptor-negative breast cancer and was not modified by known breast cancer risk factors. Results from this study confirmed the association of the APOBEC3 deletion with breast cancer risk among women of European ancestry.

  12. Insights into the motif preference of APOBEC3 enzymes.

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    Diako Ebrahimi

    Full Text Available We used a multivariate data analysis approach to identify motifs associated with HIV hypermutation by different APOBEC3 enzymes. The analysis showed that APOBEC3G targets G mainly within GG, TG, TGG, GGG, TGGG and also GGGT. The G nucleotides flanked by a C at the 3' end (in +1 and +2 positions were indicated as disfavoured targets by APOBEC3G. The G nucleotides within GGGG were found to be targeted at a frequency much less than what is expected. We found that the infrequent G-to-A mutation within GGGG is not limited to the inaccessibility, to APOBEC3, of poly Gs in the central and 3'polypurine tracts (PPTs which remain double stranded during the HIV reverse transcription. GGGG motifs outside the PPTs were also disfavoured. The motifs GGAG and GAGG were also found to be disfavoured targets for APOBEC3. The motif-dependent mutation of G within the HIV genome by members of the APOBEC3 family other than APOBEC3G was limited to GA→AA changes. The results did not show evidence of other types of context dependent G-to-A changes in the HIV genome.

  13. APOBEC3 Interference during Replication of Viral Genomes

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    Luc Willems

    2015-06-01

    Full Text Available Co-evolution of viruses and their hosts has reached a fragile and dynamic equilibrium that allows viral persistence, replication and transmission. In response, infected hosts have developed strategies of defense that counteract the deleterious effects of viral infections. In particular, single-strand DNA editing by Apolipoprotein B Editing Catalytic subunits proteins 3 (APOBEC3s is a well-conserved mechanism of mammalian innate immunity that mutates and inactivates viral genomes. In this review, we describe the mechanisms of APOBEC3 editing during viral replication, the viral strategies that prevent APOBEC3 activity and the consequences of APOBEC3 modulation on viral fitness and host genome integrity. Understanding the mechanisms involved reveals new prospects for therapeutic intervention.

  14. A novel role for APOBEC3: Susceptibility to sexual transmission of murine acquired immunodeficiency virus (mAIDS is aggravated in APOBEC3 deficient mice

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    Jones Philip H

    2012-06-01

    Full Text Available Abstract Background APOBEC3 proteins are host factors that restrict infection by retroviruses like HIV, MMTV, and MLV and are variably expressed in hematopoietic and non-hematopoietic cells, such as macrophages, lymphocytes, dendritic, and epithelia cells. Previously, we showed that APOBEC3 expressed in mammary epithelia cells function to limit milk-borne transmission of the beta-retrovirus, mouse mammary tumor virus. In this present study, we used APOBEC3 knockout mice and their wild type counterpart to query the role of APOBEC3 in sexual transmission of LP-BM5 MLV – the etiological agent of murine AIDs (mAIDs. Results We show that mouse APOBEC3 is expressed in murine genital tract tissues and gametes and that genital tract tissue of APOBEC3-deficient mice are more susceptible to infection by LP-BM5 virus. APOBEC3 expressed in genital tract tissues most likely plays a role in decreasing virus transmission via the sexual route, since mice deficient in APOBEC3 gene have higher genitalia and seminal plasma virus load and sexually transmit the virus more efficiently to their partners compared to APOBEC3+ mice. Moreover, we show that female mice sexually infected with LP-BM5 virus transmit the virus to their off-spring in APOBEC3-dependent manner. Conclusion Our data indicate that genital tissue intrinsic APOBEC3 restricts genital tract infection and limits sexual transmission of LP-BM5 virus.

  15. Hypermutation by intersegmental transfer of APOBEC3G cytidine deaminase.

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    Nowarski, Roni; Britan-Rosich, Elena; Shiloach, Tamar; Kotler, Moshe

    2008-10-01

    Deamination of cytidine residues in single-stranded DNA (ssDNA) is an important mechanism by which apolipoprotein B mRNA-editing, catalytic polypeptide-like (APOBEC) enzymes restrict endogenous and exogenous viruses. The dynamic process underlying APOBEC-induced hypermutation is not fully understood. Here we show that enzymatically active APOBEC3G can be detected in wild-type Vif(+) HIV-1 virions, albeit at low levels. In vitro studies showed that single enzyme-DNA encounters result in distributive deamination of adjacent cytidines. Nonlinear translocation of APOBEC3G, however, directed scattered deamination of numerous targets along the DNA. Increased ssDNA concentrations abolished enzyme processivity in the case of short, but not long, DNA substrates, emphasizing the key role of rapid intersegmental transfer in targeting the deaminase. Our data support a model by which APOBEC3G intersegmental transfer via monomeric binding to two ssDNA segments results in dispersed hypermutation of viral genomes.

  16. Suppression of APOBEC3-mediated restriction of HIV-1 by Vif

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    Feng, Yuqing; Baig, Tayyba T.; Love, Robin P.; Chelico, Linda

    2014-01-01

    The APOBEC3 restriction factors are a family of deoxycytidine deaminases that are able to suppress replication of viruses with a single-stranded DNA intermediate by inducing mutagenesis and functional inactivation of the virus. Of the seven human APOBEC3 enzymes, only APOBEC3-D, -F, -G, and -H appear relevant to restriction of HIV-1 in CD4+ T cells and will be the focus of this review. The restriction of HIV-1 occurs most potently in the absence of HIV-1 Vif that induces polyubiquitination and degradation of APOBEC3 enzymes through the proteasome pathway. To restrict HIV-1, APOBEC3 enzymes must be encapsidated into budding virions. Upon infection of the target cell during reverse transcription of the HIV-1 RNA into (-)DNA, APOBEC3 enzymes deaminate cytosines to form uracils in single-stranded (-)DNA regions. Upon replication of the (-)DNA to (+)DNA, the HIV-1 reverse transcriptase incorporates adenines opposite to the uracils thereby inducing C/G to T/A mutations that can functionally inactivate HIV-1. APOBEC3G is the most studied APOBEC3 enzyme and it is known that Vif attempts to thwart APOBEC3 function not only by inducing its proteasomal degradation but also by several degradation-independent mechanisms, such as inhibiting APOBEC3G virion encapsidation, mRNA translation, and for those APOBEC3G molecules that still become virion encapsidated, Vif can inhibit APOBEC3G mutagenic activity. Although most Vif variants can induce efficient degradation of APOBEC3-D, -F, and -G, there appears to be differential sensitivity to Vif-mediated degradation for APOBEC3H. This review examines APOBEC3-mediated HIV restriction mechanisms, how Vif acts as a substrate receptor for a Cullin5 ubiquitin ligase complex to induce degradation of APOBEC3s, and the determinants and functional consequences of the APOBEC3 and Vif interaction from a biological and biochemical perspective. PMID:25206352

  17. Analysis of functional germline variants in APOBEC3 and driver genes on breast cancer risk in Moroccan study population

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    Marouf, Chaymaa; Göhler, Stella; Filho, Miguel Inacio Da Silva; Hajji, Omar; Hemminki, Kari; Nadifi, Sellama; Försti, Asta

    2016-01-01

    Breast cancer (BC) is the most prevalent cancer in women and a major public health problem in Morocco. Several Moroccan studies have focused on studying this disease, but more are needed, especially at the genetic and molecular levels. Therefore, we investigated the potential association of several functional germline variants in the genes commonly mutated in sporadic breast cancer. In this case–control study, we examined 36 single nucleotide polymorphisms (SNPs) in 13 genes (APOBEC3A, APOBEC3B, ARID1B, ATR, MAP3K1, MLL2, MLL3, NCOR1, RUNX1, SF3B1, SMAD4, TBX3, TTN), which were located in the core promoter, 5’-and 3’UTR or which were nonsynonymous SNPs to assess their potential association with inherited predisposition to breast cancer development. Additionally, we identified a ~29.5-kb deletion polymorphism between APOBEC3A and APOBEC3B and explored possible associations with BC. A total of 226 Moroccan breast cancer cases and 200 matched healthy controls were included in this study. The analysis showed that12 SNPs in 8 driver genes, 4 SNPs in APOBEC3B gene and 1 SNP in APOBEC3A gene were associated with BC risk and/or clinical outcome at P ≤ 0.05 level. RUNX1-rs8130963 (odds ratio (OR) = 2.25; 95 % CI 1.42-3.56; P = 0.0005; dominant model), TBX3-rs8853 (OR = 2.04; 95 % CI 1.38-3.01; P = 0.0003; dominant model), TBX3-rs1061651 (OR = 2.14; 95 % CI1.43-3.18; P = 0.0002; dominant model), TTN-rs12465459 (OR = 2.02; 95 % confidence interval 1.33-3.07; P = 0.0009; dominant model), were the most significantly associated SNPs with BC risk. A strong association with clinical outcome were detected for the genes SMAD4 -rs3819122 with tumor size (OR = 0.45; 95 % CI 0.25-0.82; P = 0.009) and TTN-rs2244492 with estrogen receptor (OR = 0.45; 95 % CI 0.25-0.82; P = 0.009). Our results suggest that genetic variations in driver and APOBEC3 genes were associated with the risk of BC and may have impact on clinical outcome. However, the reported association between the

  18. Analysis of single-nucleotide polymorphisms in the APOBEC3H gene of domestic cats (Felis catus) and their association with the susceptibility to feline immunodeficiency virus and feline leukemia virus infections.

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    de Castro, Fernanda Luz; Junqueira, Dennis Maletich; de Medeiros, Rúbia Marília; da Silva, Tailene Rabello; Costenaro, Jamile Girardi; Knak, Marcus Braga; de Matos Almeida, Sabrina Esteves; Campos, Fabrício Souza; Roehe, Paulo Michel; Franco, Ana Cláudia

    2014-10-01

    Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are widely distributed retroviruses that infect domestic cats (Felis catus). Restriction factors are proteins that have the ability to hamper retroviruses' replication and are part of the conserved mechanisms of anti-viral immunity of mammals. The APOBEC3 protein family is the most studied class of restriction factors; they are cytidine deaminases that generate hypermutations in provirus DNA during reverse transcription, thus causing hypermutations in the viral genome, hindering virus replication. One of the feline APOBEC3 genes, named APOBEC3H, encodes two proteins (APOBEC3H and APOBEC3CH). In other mammals, APOBEC3H single-nucleotide polymorphisms (SNPs) can alter the stability and cellular localization of the encoded protein, thus influencing its subcellular localization and reducing its anti-viral effect. In cats, the association of APOBEC3H SNPs with susceptibility to retroviral infections was not yet demonstrated. Therefore, this study aimed the investigation on the variability of APOBEC3H and the possible association with FIV/FeLV infections. DNA obtained from whole blood of fifty FIV- and/or FeLV-infected cats and fifty-nine FIV- and/or FeLV-uninfected cats were used as templates to amplify two different regions of the APOBEC3H, with subsequent sequencing and analysis. The first region was highly conserved among all samples, while in the second, six single-nucleotide variation points were identified. One of the SNPs, A65S (A65I), was significantly correlated with the susceptibility to FIV and/or FeLV infections. On the other hand, the haplotype analysis showed that the combination "GGGGCC" was positively correlated with the lack of FIV and/or FeLV infections. Our results indicate that, as previously shown in other mammals, variability of restriction factors may contribute to susceptibility of domestic cats to retroviral infections; however, these results should be confirmed by more

  19. Molecular cloning and characterization of APOBEC3 family in tree shrew.

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    Luo, Meng-Ting; Fan, Yu; Mu, Dan; Yao, Yong-Gang; Zheng, Yong-Tang

    2018-03-10

    The APOBEC3 family is a series antiviral factors that inhibit the replication of many viruses, such as HIV-1 and HBV. Tree shrews (Tupaia belangeri) possess great potential as an animal model for human diseases and therapeutic responses. However, the APOBEC3 family is unknown in tree shrews. Recent work has showed the presence of the APOBEC3 family in tree shrews. In this work, the cDNA sequences of five APOBEC3 members were identified in tree shrews, namely, tsAPOBEC3A, -3C, -3F, -3G and -3H. The results showed that their sequences encoded a zinc (Z)-coordinating-domain as a characteristic of APOBEC3 proteins. Phylogenetic analysis revealed that the tree shrew APOBEC3 (tsAPOBEC3) genes have occurred independently and that they are clustered with other mammalian APOBEC3 members. Transcript expression analysis indicated that tsAPOBEC3 genes are constitutively expressed, and high in immune-related tissues. tsAPOBEC3 gene expression was up-regulated in hepatocytes and PBMCs by IFN-α stimulation. Finally, tsAPOBEC3 proteins could edit both sides of DNA by inserting G→A and C→T hypermutations. Overall, the results suggest that the tsAPOBEC3 family could play a key role in defense immunity through distinct editing mechanisms. Our results provided insights into the genetic basis for the development of a tree shrew model for studying viral infection. Future studies will focus on deepening our understanding on the antiviral functions of these editing enzymes in tree shrew. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. D316 is critical for the enzymatic activity and HIV-1 restriction potential of human and rhesus APOBEC3B

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    McDougle, Rebecca M.; Hultquist, Judd F.; Stabell, Alex C.; Sawyer, Sara L.; Harris, Reuben S.

    2013-01-01

    APOBEC3B is one of seven human APOBEC3 DNA cytosine deaminases that function to inhibit the replication and persistence of retroelements and retroviruses. Human APOBEC3B restricts the replication of HIV-1 in HEK293 cells, while our laboratory clone of rhesus macaque APOBEC3B did not. We mapped the restriction determinant to a single amino acid difference that alters enzymatic activity. Human APOBEC3B D316 is catalytically active and capable of restricting HIV-1 while rhesus APOBEC3B N316 is n...

  1. The restriction of zoonotic PERV transmission by human APOBEC3G.

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    Stefán R Jónsson

    Full Text Available The human APOBEC3G protein is an innate anti-viral factor that can dominantly inhibit the replication of some endogenous and exogenous retroviruses. The prospects of purposefully harnessing such an anti-viral defense are under investigation. Here, long-term co-culture experiments were used to show that porcine endogenous retrovirus (PERV transmission from pig to human cells is reduced to nearly undetectable levels by expressing human APOBEC3G in virus-producing pig kidney cells. Inhibition occurred by a deamination-independent mechanism, likely after particle production but before the virus could immortalize by integration into human genomic DNA. PERV inhibition did not require the DNA cytosine deaminase activity of APOBEC3G and, correspondingly, APOBEC3G-attributable hypermutations were not detected. In contrast, over-expression of the sole endogenous APOBEC3 protein of pigs failed to interfere significantly with PERV transmission. Together, these data constitute the first proof-of-principle demonstration that APOBEC3 proteins can be used to fortify the innate anti-viral defenses of cells to prevent the zoonotic transmission of an endogenous retrovirus. These studies suggest that human APOBEC3G-transgenic pigs will provide safer, PERV-less xenotransplantation resources and that analogous cross-species APOBEC3-dependent restriction strategies may be useful for thwarting other endogenous as well as exogenous retrovirus infections.

  2. Human APOBEC3 induced mutation of human immunodeficiency virus type-1 contributes to adaptation and evolution in natural infection.

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    Eun-Young Kim

    2014-07-01

    Full Text Available Human APOBEC3 proteins are cytidine deaminases that contribute broadly to innate immunity through the control of exogenous retrovirus replication and endogenous retroelement retrotransposition. As an intrinsic antiretroviral defense mechanism, APOBEC3 proteins induce extensive guanosine-to-adenosine (G-to-A mutagenesis and inhibit synthesis of nascent human immunodeficiency virus-type 1 (HIV-1 cDNA. Human APOBEC3 proteins have additionally been proposed to induce infrequent, potentially non-lethal G-to-A mutations that make subtle contributions to sequence diversification of the viral genome and adaptation though acquisition of beneficial mutations. Using single-cycle HIV-1 infections in culture and highly parallel DNA sequencing, we defined trinucleotide contexts of the edited sites for APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H. We then compared these APOBEC3 editing contexts with the patterns of G-to-A mutations in HIV-1 DNA in cells obtained sequentially from ten patients with primary HIV-1 infection. Viral substitutions were highest in the preferred trinucleotide contexts of the edited sites for the APOBEC3 deaminases. Consistent with the effects of immune selection, amino acid changes accumulated at the APOBEC3 editing contexts located within human leukocyte antigen (HLA-appropriate epitopes that are known or predicted to enable peptide binding. Thus, APOBEC3 activity may induce mutations that influence the genetic diversity and adaptation of the HIV-1 population in natural infection.

  3. APOBEC3G and HIV-1: strike and counterstrike.

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    Soros, Vanessa B; Greene, Warner C

    2007-02-01

    APOBEC3G (A3G), a deoxycytidine deaminase, is a powerful host antiretroviral factor that can restrict HIV-1 infection. This restriction is counteracted by the HIV-1 virion infectivity factor (Vif) protein, whose activity culminates in depletion of A3G from infected cells. In the absence of Vif, viruses encapsidate A3G, which acts in part to mutate viral DNA formed during reverse transcription upon subsequent infection of a new cell. Cellular A3G also functions as a post-entry restriction factor for HIV in resting CD4 T cells, where it resides in a low molecular mass form. Unfortunately, this barrier is forfeited when CD4 T cells are activated because A3G is recruited into inactive high molecular mass ribonucleoprotein complexes. In addition to restricting HIV, A3G and related deaminases may counter other retroviruses and protect the cell from endogenous mobile retroelements. Understanding A3G complex assembly and its interplay with HIV Vif may make possible future development of a new class of HIV therapeutic agents.

  4. APOBEC3G inhibits elongation of HIV-1 reverse transcripts.

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    Kate N Bishop

    2008-12-01

    Full Text Available APOBEC3G (A3G is a host cytidine deaminase that, in the absence of Vif, restricts HIV-1 replication and reduces the amount of viral DNA that accumulates in cells. Initial studies determined that A3G induces extensive mutation of nascent HIV-1 cDNA during reverse transcription. It has been proposed that this triggers the degradation of the viral DNA, but there is now mounting evidence that this mechanism may not be correct. Here, we use a natural endogenous reverse transcriptase assay to show that, in cell-free virus particles, A3G is able to inhibit HIV-1 cDNA accumulation not only in the absence of hypermutation but also without the apparent need for any target cell factors. We find that although reverse transcription initiates in the presence of A3G, elongation of the cDNA product is impeded. These data support the model that A3G reduces HIV-1 cDNA levels by inhibiting synthesis rather than by inducing degradation.

  5. Distinct determinants in HIV-1 Vif and human APOBEC3 proteins are required for the suppression of diverse host anti-viral proteins.

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    Wenyan Zhang

    Full Text Available APOBEC3G (A3G and related cytidine deaminases of the APOBEC3 family of proteins are potent inhibitors of many retroviruses, including HIV-1. Formation of infectious HIV-1 requires the suppression of multiple cytidine deaminases by Vif. HIV-1 Vif suppresses various APOBEC3 proteins through the common mechanism of recruiting the Cullin5-ElonginB-ElonginC E3 ubiquitin ligase to induce target protein polyubiquitination and proteasome-mediated degradation. The domains in Vif and various APOBEC3 proteins required for APOBEC3 recognition and degradation have not been fully characterized.In the present study, we have demonstrated that the regions of APOBEC3F (A3F that are required for its HIV-1-mediated binding and degradation are distinct from those reported for A3G. We found that the C-terminal cytidine deaminase domain (C-CDD of A3F alone is sufficient for its interaction with HIV-1 Vif and its Vif-mediated degradation. We also observed that the domains of HIV-1 Vif that are uniquely required for its functional interaction with full-length A3F are also required for the degradation of the C-CDD of A3F; in contrast, those Vif domains that are uniquely required for functional interaction with A3G are not required for the degradation of the C-CDD of A3F. Interestingly, the HIV-1 Vif domains required for the degradation of A3F are also required for the degradation of A3C and A3DE. On the other hand, the Vif domains uniquely required for the degradation of A3G are dispensable for the degradation of cytidine deaminases A3C and A3DE.Our data suggest that distinct regions of A3F and A3G are targeted by HIV-1 Vif molecules. However, HIV-1 Vif suppresses A3F, A3C, and A3DE through similar recognition determinants, which are conserved among Vif molecules from diverse HIV-1 strains. Mapping these determinants may be useful for the design of novel anti-HIV inhibitors.

  6. Human Papillomavirus 16 E6 Upregulates APOBEC3B via the TEAD Transcription Factor.

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    Mori, Seiichiro; Takeuchi, Takamasa; Ishii, Yoshiyuki; Yugawa, Takashi; Kiyono, Tohru; Nishina, Hiroshi; Kukimoto, Iwao

    2017-03-15

    The cytidine deaminase APOBEC3B (A3B) underlies the genetic heterogeneity of several human cancers, including cervical cancer, which is caused by human papillomavirus (HPV) infection. We previously identified a region within the A3B promoter that is activated by the viral protein HPV16 E6 in human keratinocytes. Here, we discovered three sites recognized by the TEAD family of transcription factors within this region of the A3B promoter. Reporter assays in HEK293 cells showed that exogenously expressed TEAD4 induced A3B promoter activation through binding to these sites. Normal immortalized human keratinocytes expressing E6 (NIKS-E6) displayed increased levels of TEAD1/4 protein compared to parental NIKS. A series of E6 mutants revealed that E6-mediated degradation of p53 was important for increasing TEAD4 levels. Knockdown of TEADs in NIKS-E6 significantly reduced A3B mRNA levels, whereas ectopic expression of TEAD4 in NIKS increased A3B mRNA levels. Finally, chromatin immunoprecipitation assays demonstrated increased levels of TEAD4 binding to the A3B promoter in NIKS-E6 compared to NIKS. Collectively, these results indicate that E6 induces upregulation of A3B through increased levels of TEADs, highlighting the importance of the TEAD-A3B axis in carcinogenesis. IMPORTANCE The expression of APOBEC3B (A3B), a cellular DNA cytidine deaminase, is upregulated in various human cancers and leaves characteristic, signature mutations in cancer genomes, suggesting that it plays a prominent role in carcinogenesis. Viral oncoproteins encoded by human papillomavirus (HPV) and polyomavirus have been reported to induce A3B expression, implying the involvement of A3B upregulation in virus-associated carcinogenesis. However, the molecular mechanisms causing A3B upregulation remain unclear. Here, we demonstrate that exogenous expression of the cellular transcription factor TEAD activates the A3B promoter. Further, the HPV oncoprotein E6 increases the levels of endogenous TEAD1

  7. Inhibition of hepatitis B virus replication by APOBEC3G in vitro and in vivo.

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    Lei, Yan-Chang; Hao, You-Hua; Zhang, Zheng-Mao; Tian, Yong-Jun; Wang, Bao-Ju; Yang, Yan; Zhao, Xi-Ping; Lu, Meng-Ji; Gong, Fei-Li; Yang, Dong-Liang

    2006-07-28

    To investigate the effect of APOBEC3G mediated antiviral activity against hepatitis B virus (HBV) in cell cultures and replication competent HBV vector-based mouse model. The mammalian hepatoma cells Huh7 and HepG2 were cotransfected with various amounts of CMV-driven expression vector encoding APOBEC3G and replication competent 1.3 fold over-length HBV. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA. The expression of HBcAg in transfected cells was detected by western blot. HBV DNA and RNA from intracellular core particles were examined by Northern and Southern blot analyses. To assess activity of the APOBEC3G in vivo, an HBV vector-based model was used in which APOBEC3G and the HBV vector were co-delivered via high-volume tail vein injection. Levels of HBsAg and HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by ELISA and quantitative PCR analysis respectively. There was a dose dependent decrease in the levels of intracellular core-associated HBV DNA and extracellular production of HBsAg and HBeAg. The levels of intracellular core-associated viral RNA also decreased, but the expression of HBcAg in transfected cells showed almost no change. Consistent with in vitro results, levels of HBsAg in the sera of mice were dramatically decreased. More than 1.5 log10 decrease in levels of serum HBV DNA and liver HBV RNA were observed in the APOBEC3G-treated groups compared with the control groups. These findings indicate that APOBEC3G could suppress HBV replication and antigen expression both in vivo and in vitro, promising an advance in treatment of HBV infection.

  8. Copy Number Variation of HLA-DQA1 and APOBEC3A/3B Contribute to the Susceptibility of Systemic Sclerosis in the Chinese Han Population.

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    Guo, Shicheng; Li, Yuan; Wang, Yi; Chu, Haiyan; Chen, Yulin; Liu, Qingmei; Guo, Gang; Tu, Wenzhen; Wu, Wenyu; Zou, Hejian; Yang, Li; Xiao, Rong; Ma, Yanyun; Zhang, Feng; Xiong, Momiao; Jin, Li; Zhou, Xiaodong; Wang, Jiucun

    2016-05-01

    Systemic sclerosis (SSc) is a systemic connective tissue disease caused by a genetic aberrant. The involvement of the copy number variations (CNV) in the pathogenesis of SSc is unclear. We tried to identify some CNV that are involved with the susceptibility to SSc. A genome-wide CNV screening was performed in 20 patients with SSc. Five SSc-associated common CNV that included HLA-DRB5, HLA-DQA1, IRGM, CDC42EP3, and APOBEC3A/3B were identified from the screening and were then validated in 365 patients with SSc and 369 matched healthy controls. Three hundred forty-four CNV (140 gains and 204 losses) and 2 CNV hotspots (6q21.3 and 22q11.2) were found in the SSc genomes (covering 24.2 megabases), suggesting that CNV were ubiquitous in the SSc genome and played important roles in the pathogenesis of SSc. The high copy number of HLA-DQA1 was a significantly protective factor for SSc (OR 0.07, p = 2.99 × 10(-17)), while the high copy number of APOBEC3A/B was a significant risk factor (OR 3.45, p = 6.4 × 10(-18)), adjusted with sex and age. The risk prediction model based on genetic factors in logistic regression showed moderate prediction ability, with area under the curve = 0.80 (95% CI 0.77-0.83), which demonstrated that APOBEC3A/B and HLA-DQA1 were powerful biomarkers for SSc risk evaluation and contributed to the susceptibility to SSc. CNV of HLA-DQA1 and APOBEC3A/B contribute to the susceptibility to SSc in a Chinese Han population.

  9. First-In-Class Small Molecule Inhibitors of the Single-Strand DNA Cytosine Deaminase APOBEC3G

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ming; Shandilya, Shivender M.D.; Carpenter, Michael A.; Rathore, Anurag; Brown, William L.; Perkins, Angela L.; Harki, Daniel A.; Solberg, Jonathan; Hook, Derek J.; Pandey, Krishan K.; Parniak, Michael A.; Johnson, Jeffrey R.; Krogan, Nevan J.; Somasundaran, Mohan; Ali, Akbar; Schiffer, Celia A.; Harris, Reuben S. (Pitt); (UMASS, MED); (SLUHSC); (UCSF); (UMM)

    2012-04-04

    APOBEC3G is a single-stranded DNA cytosine deaminase that comprises part of the innate immune response to viruses and transposons. Although APOBEC3G is the prototype for understanding the larger mammalian polynucleotide deaminase family, no specific chemical inhibitors exist to modulate its activity. High-throughput screening identified 34 compounds that inhibit APOBEC3G catalytic activity. Twenty of 34 small molecules contained catechol moieties, which are known to be sulfhydryl reactive following oxidation to the orthoquinone. Located proximal to the active site, C321 was identified as the binding site for the inhibitors by a combination of mutational screening, structural analysis, and mass spectrometry. Bulkier substitutions C321-to-L, F, Y, or W mimicked chemical inhibition. A strong specificity for APOBEC3G was evident, as most compounds failed to inhibit the related APOBEC3A enzyme or the unrelated enzymes E. coli uracil DNA glycosylase, HIV-1 RNase H, or HIV-1 integrase. Partial, but not complete, sensitivity could be conferred to APOBEC3A by introducing the entire C321 loop from APOBEC3G. Thus, a structural model is presented in which the mechanism of inhibition is both specific and competitive, by binding a pocket adjacent to the APOBEC3G active site, reacting with C321, and blocking access to substrate DNA cytosines.

  10. Lineage-Specific Viral Hijacking of Non-canonical E3 Ubiquitin Ligase Cofactors in the Evolution of Vif Anti-APOBEC3 Activity

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    Joshua R. Kane

    2015-05-01

    Full Text Available HIV-1 encodes the accessory protein Vif, which hijacks a host Cullin-RING ubiquitin ligase (CRL complex as well as the non-canonical cofactor CBFβ, to antagonize APOBEC3 antiviral proteins. Non-canonical cofactor recruitment to CRL complexes by viral factors, to date, has only been attributed to HIV-1 Vif. To further study this phenomenon, we employed a comparative approach combining proteomic, biochemical, structural, and virological techniques to investigate Vif complexes across the lentivirus genus, including primate (HIV-1 and simian immunodeficiency virus macaque [SIVmac] and non-primate (FIV, BIV, and MVV viruses. We find that CBFβ is completely dispensable for the activity of non-primate lentiviral Vif proteins. Furthermore, we find that BIV Vif requires no cofactor and that MVV Vif requires a novel cofactor, cyclophilin A (CYPA, for stable CRL complex formation and anti-APOBEC3 activity. We propose modular conservation of Vif complexes allows for potential exaptation of functions through the acquisition of non-CRL-associated host cofactors while preserving anti-APOBEC3 activity.

  11. Structural determinants of APOBEC3B non-catalytic domain for molecular assembly and catalytic regulation

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    Xiao, Xiao; Yang, Hanjing; Arutiunian, Vagan; Fang, Yao; Besse, Guillaume; Morimoto, Cherie; Zirkle, Brett; Chen, Xiaojiang S. (USC)

    2017-05-30

    The catalytic activity of human cytidine deaminase APOBEC3B (A3B) has been correlated with kataegic mutational patterns within multiple cancer types. The molecular basis of how the N-terminal non-catalytic CD1 regulates the catalytic activity and consequently, biological function of A3B remains relatively unknown. Here, we report the crystal structure of a soluble human A3B-CD1 variant and delineate several structural elements of CD1 involved in molecular assembly, nucleic acid interactions and catalytic regulation of A3B. We show that (i) A3B expressed in human cells exists in hypoactive high-molecular-weight (HMW) complexes, which can be activated without apparent dissociation into low-molecular-weight (LMW) species after RNase A treatment. (ii) Multiple surface hydrophobic residues of CD1 mediate the HMW complex assembly and affect the catalytic activity, including one tryptophan residue W127 that likely acts through regulating nucleic acid binding. (iii) One of the highly positively charged surfaces on CD1 is involved in RNA-dependent attenuation of A3B catalysis. (iv) Surface hydrophobic residues of CD1 are involved in heterogeneous nuclear ribonucleoproteins (hnRNPs) binding to A3B. The structural and biochemical insights described here suggest that unique structural features on CD1 regulate the molecular assembly and catalytic activity of A3B through distinct mechanisms.

  12. The 29.5 kb APOBEC3B Deletion Polymorphism Is Not Associated with Clinical Outcome of Breast Cancer.

    Science.gov (United States)

    Liu, Jingjing; Sieuwerts, Anieta M; Look, Maxime P; van der Vlugt-Daane, Michelle; Meijer-van Gelder, Marion E; Foekens, John A; Hollestelle, Antoinette; Martens, John W M

    2016-01-01

    Increased APOBEC3B mRNA levels are associated with a hypermutator phenotype and poor prognosis in ER-positive breast cancer patients. In addition, a 29.5 kb deletion polymorphism of APOBEC3B, resulting in an APOBEC3A-B hybrid transcript, has been associated with an increased breast cancer risk and the hypermutator phenotype. Here we evaluated whether the APOBEC3B deletion polymorphism also associates with clinical outcome of breast cancer. Copy number analysis was performed by quantitative PCR (qPCR) in primary tumors of 1,756 Dutch breast cancer patients. The APOBEC3B deletion was found in 187 patients of whom 16 carried a two-copy deletion and 171 carried a one-copy deletion. The prognostic value of the APOBEC3B deletion for the natural course of the disease was evaluated among 1,076 lymph-node negative (LNN) patients who did not receive adjuvant systemic treatment. No association was found between APOBEC3B copy number values and the length of metastasis-free survival (MFS; hazard ratio (HR) = 1.00, 95% confidence interval (CI) = 0.90-1.11, P = 0.96). Subgroup analysis by ER status also did not reveal an association between APOBEC3B copy number values and the length of MFS. The predictive value of the APOBEC3B deletion was assessed among 329 ER-positive breast cancer patients who received tamoxifen as the first-line therapy for recurrent disease and 226 breast cancer patients who received first-line chemotherapy for recurrent disease. No association between APOBEC3B copy number values and the overall response rate (ORR) to either tamoxifen (odds ratio (OR) = 0.88, 95% CI = 0.69-1.13, P = 0.31) or chemotherapy (OR = 0.97, 95% CI = 0.71-1.33, P = 0.87) was found. Thus, in contrast to APOBEC3B mRNA levels, the APOBEC3B deletion polymorphism has neither a prognostic nor a predictive value for breast cancer patients. Although a correlation exists between APOBEC3B copy number and mRNA expression, it is relatively weak. This suggests that other mechanisms exist that may

  13. APOBEC3A is an oral cancer prognostic biomarker in Taiwanese carriers of an APOBEC deletion polymorphism.

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    Chen, Ting-Wen; Lee, Chi-Ching; Liu, Hsuan; Wu, Chi-Sheng; Pickering, Curtis R; Huang, Po-Jung; Wang, Jing; Chang, Ian Yi-Feng; Yeh, Yuan-Ming; Chen, Chih-De; Li, Hsin-Pai; Luo, Ji-Dung; Tan, Bertrand Chin-Ming; Chan, Timothy En Haw; Hsueh, Chuen; Chu, Lichieh Julie; Chen, Yi-Ting; Zhang, Bing; Yang, Chia-Yu; Wu, Chih-Ching; Hsu, Chia-Wei; See, Lai-Chu; Tang, Petrus; Yu, Jau-Song; Liao, Wei-Chao; Chiang, Wei-Fan; Rodriguez, Henry; Myers, Jeffrey N; Chang, Kai-Ping; Chang, Yu-Sun

    2017-09-06

    Oral squamous cell carcinoma is a prominent cancer worldwide, particularly in Taiwan. By integrating omics analyses in 50 matched samples, we uncover in Taiwanese patients a predominant mutation signature associated with cytidine deaminase APOBEC, which correlates with the upregulation of APOBEC3A expression in the APOBEC3 gene cluster at 22q13. APOBEC3A expression is significantly higher in tumors carrying APOBEC3B-deletion allele(s). High-level APOBEC3A expression is associated with better overall survival, especially among patients carrying APOBEC3B-deletion alleles, as examined in a second cohort (n = 188; p = 0.004). The frequency of APOBEC3B-deletion alleles is ~50% in 143 genotyped oral squamous cell carcinoma -Taiwan samples (27A3B -/- :89A3B +/- :27A3B +/+ ), compared to the 5.8% found in 314 OSCC-TCGA samples. We thus report a frequent APOBEC mutational profile, which relates to a APOBEC3B-deletion germline polymorphism in Taiwanese oral squamous cell carcinoma that impacts expression of APOBEC3A, and is shown to be of clinical prognostic relevance. Our finding might be recapitulated by genomic studies in other cancer types.Oral squamous cell carcinoma is a prevalent malignancy in Taiwan. Here, the authors show that OSCC in Taiwanese show a frequent deletion polymorphism in the cytidine deaminases gene cluster APOBEC3 resulting in increased expression of A3A, which is shown to be of clinical prognostic relevance.

  14. Crystal Structure of the DNA Deaminase APOBEC3B Catalytic Domain.

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    Shi, Ke; Carpenter, Michael A; Kurahashi, Kayo; Harris, Reuben S; Aihara, Hideki

    2015-11-20

    Functional and deep sequencing studies have combined to demonstrate the involvement of APOBEC3B in cancer mutagenesis. APOBEC3B is a single-stranded DNA cytosine deaminase that functions normally as a nuclear-localized restriction factor of DNA-based pathogens. However, it is overexpressed in cancer cells and elicits an intrinsic preference for 5'-TC motifs in single-stranded DNA, which is the most frequently mutated dinucleotide in breast, head/neck, lung, bladder, cervical, and several other tumor types. In many cases, APOBEC3B mutagenesis accounts for the majority of both dispersed and clustered (kataegis) cytosine mutations. Here, we report the first structures of the APOBEC3B catalytic domain in multiple crystal forms. These structures reveal a tightly closed active site conformation and suggest that substrate accessibility is regulated by adjacent flexible loops. Residues important for catalysis are identified by mutation analyses, and the results provide insights into the mechanism of target site selection. We also report a nucleotide (dCMP)-bound crystal structure that informs a multistep model for binding single-stranded DNA. Overall, these high resolution crystal structures provide a framework for further mechanistic studies and the development of novel anti-cancer drugs to inhibit this enzyme, dampen tumor evolution, and minimize adverse outcomes such as drug resistance and metastasis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. D316 is critical for the enzymatic activity and HIV-1 restriction potential of human and rhesus APOBEC3B.

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    McDougle, Rebecca M; Hultquist, Judd F; Stabell, Alex C; Sawyer, Sara L; Harris, Reuben S

    2013-06-20

    APOBEC3B is one of seven human APOBEC3 DNA cytosine deaminases that function to inhibit the replication and persistence of retroelements and retroviruses. Human APOBEC3B restricts the replication of HIV-1 in HEK293 cells, while our laboratory clone of rhesus macaque APOBEC3B did not. We mapped the restriction determinant to a single amino acid difference that alters enzymatic activity. Human APOBEC3B D316 is catalytically active and capable of restricting HIV-1 while rhesus APOBEC3B N316 is not; swapping these residues alters the activity and restriction phenotypes respectively. Genotyping of primate center rhesus macaques revealed uniform homozygosity for aspartate at position 316. Considering the C-to-T nature of the underlying mutation, we suspect that our rhesus APOBEC3B cDNA was inactivated by its own gene product during subcloning in Escherichia coli. This region has been previously characterized for its role in substrate specificity, but these data indicate it also has a fundamental role in deaminase activity. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Viral subversion of APOBEC3s: Lessons for anti-tumor immunity and tumor immunotherapy.

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    Borzooee, Faezeh; Asgharpour, Mahdi; Quinlan, Emma; Grant, Michael D; Larijani, Mani

    2017-12-06

    APOBEC3s (A3) are endogenous DNA-editing enzymes that are expressed in immune cells including T lymphocytes. A3s target and mutate the genomes of retroviruses that infect immune tissues such as the human immunodeficiency virus (HIV). Therefore, A3s were classically defined as host anti-viral innate immune factors. In contrast, we and others showed that A3s can also benefit the virus by mediating escape from adaptive immune recognition and drugs. Crucially, whether A3-mediated mutations help or hinder HIV, is not up to chance. Rather, the virus has evolved multiple mechanisms to actively and maximally subvert A3 activity. More recently, extensive A3 mutational footprints in tumor genomes have been observed in many different cancers. This suggests a role for A3s in cancer initiation and progression. On the other hand, multiple anti-tumor activities of A3s have also come to light, including impact on immune checkpoint molecules and possible generation of tumor neo-antigens. Here, we review the studies that reshaped the view of A3s from anti-viral innate immune agents to host factors exploited by HIV to escape from immune recognition. Viruses and tumors share many attributes, including rapid evolution and adeptness at exploiting mutations. Given this parallel, we then discuss the pro- and anti-tumor roles of A3s, and suggest that lessons learned from studying A3s in the context of anti-viral immunity can be applied to tumor immunotherapy.

  17. Free Energy Profile of APOBEC3G Protein Calculated by a Molecular Dynamics Simulation

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    Fukunishi, Yoshifumi; Hongo, Saki; Lintuluoto, Masami; Matsuo, Hiroshi

    2012-01-01

    The human APOBEC3G protein (A3G) is a single-stranded DNA deaminase that inhibits the replication of retrotransposons and retroviruses, including HIV-1. Atomic details of A3G’s catalytic mechanism have started to emerge, as the structure of its catalytic domain (A3Gctd) has been revealed by NMR and X-ray crystallography. The NMR and crystal structures are similar overall; however, differences are apparent for β2 strand (β2) and loops close to the catalytic site. To add some insight into these differences and to better characterize A3Gctd dynamics, we calculated its free energy profile by using the Generalized-Born surface area (GBSA) method accompanied with a molecular dynamics simulation. The GBSA method yielded an enthalpy term for A3Gctd’s free energy, and we developed a new method that takes into account the distribution of the protein’s dihedral angles to calculate its entropy term. The structure solved by NMR was found to have a lower energy than that of the crystal structure, suggesting that this conformation is dominant in solution. In addition, β2-loop-β2’ configuration was stable throughout a 20-ns molecular dynamics (MD) simulation. This finding suggests that in solution A3Gctd is not likely to adopt the continuous β2 strand configuration present in the APOBEC2 crystal structure. In the NMR structure, the solvent water accessibility of the catalytic Zn2+ was limited throughout the 20-ns MD simulation. This result explains previous observations in which A3G did not bind or catalyze single cytosine nucleotide, even when at excessive concentrations. PMID:24832225

  18. An ancient history of gene duplications, fusions and losses in the evolution of APOBEC3 mutators in mammals

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    Münk Carsten

    2012-05-01

    Full Text Available Abstract Background The APOBEC3 (A3 genes play a key role in innate antiviral defense in mammals by introducing directed mutations in the DNA. The human genome encodes for seven A3 genes, with multiple splice alternatives. Different A3 proteins display different substrate specificity, but the very basic question on how discerning self from non-self still remains unresolved. Further, the expression of A3 activity/ies shapes the way both viral and host genomes evolve. Results We present here a detailed temporal analysis of the origin and expansion of the A3 repertoire in mammals. Our data support an evolutionary scenario where the genome of the mammalian ancestor encoded for at least one ancestral A3 gene, and where the genome of the ancestor of placental mammals (and possibly of the ancestor of all mammals already encoded for an A3Z1-A3Z2-A3Z3 arrangement. Duplication events of the A3 genes have occurred independently in different lineages: humans, cats and horses. In all of them, gene duplication has resulted in changes in enzyme activity and/or substrate specificity, in a paradigmatic example of convergent adaptive evolution at the genomic level. Finally, our results show that evolutionary rates for the three A3Z1, A3Z2 and A3Z3 motifs have significantly decreased in the last 100 Mya. The analysis constitutes a textbook example of the evolution of a gene locus by duplication and sub/neofunctionalization in the context of virus-host arms race. Conclusions Our results provide a time framework for identifying ancestral and derived genomic arrangements in the APOBEC loci, and to date the expansion of this gene family for different lineages through time, as a response to changes in viral/retroviral/retrotransposon pressure.

  19. Random mutagenesis MAPPIT analysis identifies binding sites for Vif and Gag in both cytidine deaminase domains of Apobec3G.

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    Isabel Uyttendaele

    Full Text Available The mammalian two-hybrid system MAPPIT allows the detection of protein-protein interactions in intact human cells. We developed a random mutagenesis screening strategy based on MAPPIT to detect mutations that disrupt the interaction of one protein with multiple protein interactors simultaneously. The strategy was used to detect residues of the human cytidine deaminase Apobec3G that are important for its homodimerization and its interaction with the HIV-1 Gag and Vif proteins. The strategy is able to identify the previously described head-to-head homodimerization interface in the N-terminal domain of Apobec3G. Our analysis further detects two new potential interaction surfaces in the N-and C-terminal domain of Apobec3G for interaction with Vif and Gag or for Apobec3G dimerization.

  20. APOBEC3 cytidine deaminases in double-strand DNA break repair and cancer promotion.

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    Nowarski, Roni; Kotler, Moshe

    2013-06-15

    High frequency of cytidine to thymidine conversions was identified in the genome of several types of cancer cells. In breast cancer cells, these mutations are clustered in long DNA regions associated with single-strand DNA (ssDNA), double-strand DNA breaks (DSB), and genomic rearrangements. The observed mutational pattern resembles the deamination signature of cytidine to uridine carried out by members of the APOBEC3 family of cellular deaminases. Consistently, APOBEC3B (A3B) was recently identified as the mutational source in breast cancer cells. A3G is another member of the cytidine deaminases family predominantly expressed in lymphoma cells, where it is involved in mutational DSB repair following ionizing radiation treatments. This activity provides us with a new paradigm for cancer cell survival and tumor promotion and a mechanistic link between ssDNA, DSBs, and clustered mutations. Cancer Res; 73(12); 3494-8. ©2013 AACR. ©2013 AACR.

  1. APOBEC3G Variants and Protection against HIV-1 Infection in Burkina Faso.

    Science.gov (United States)

    Compaore, Tegwinde Rebeca; Soubeiga, Serge Theophile; Ouattara, Abdoul Karim; Obiri-Yeboah, Dorcas; Tchelougou, Damehan; Maiga, Mamoudou; Assih, Maleki; Bisseye, Cyrille; Bakouan, Didier; Compaore, Issaka Pierre; Dembele, Augustine; Martinson, Jeremy; Simpore, Jacques

    2016-01-01

    Studies on host factors, particularly the APOBEC3G gene, have previously found an association with AIDS progression in some populations and against some HIV-1 strains but not others. Our study had two main objectives: firstly, to screen a population from Burkina Faso for three variants of APOBEC3G previously described, and secondly to analyze the effect of these three variants and their haplotypes on HIV-1 infection with Circulating Recombinant Forms (CRFs) present in Burkina Faso. This case control study involved 708 seropositive and seronegative individuals. Genotyping was done by the TaqMan allelic discrimination method. Minor allele frequencies of rs6001417 (p<0.05), rs8177832 (P<0.05), and rs35228531 (P<0.001) were higher in seronegative subjects. The rs6001417 and rs8177832 SNPs were associated with HIV-1 infection in an additive model (P<0.01). Furthermore the SNP rs35228531 was also associated with HIV-1 infection in a dominant model (P<0.001). Odds ratio analysis of genotypes and alleles of the different APOBEC3G variants showed that there is a strong association between the minor genetic variants, genotype of the three SNPs, and HIV-1 status. Haplotype analysis demonstrated that rs6001417, rs8177832, and rs35228531 are in linkage disequilibrium. The haplotype GGT from the rs6001417, rs8177832 and rs35228531 respectively has a protective effect OR = 0.54 [0.43-0.68] with P<0.001. There was also associations between the haplotypes GGC OR = 1.6 [1.1;-2.3] P<0.05, and CGC OR = 5.21 [2.4-11.3] P<0.001, which increase the risk of infection by HIV-1 from almost two (2) to five (5) fold. This study demonstrates an association of rs6001417, rs8177832, and rs35228531 of APOBEC3G with HIV-1 infection in a population from Burkina Faso.

  2. APOBEC3G Variants and Protection against HIV-1 Infection in Burkina Faso.

    Directory of Open Access Journals (Sweden)

    Tegwinde Rebeca Compaore

    Full Text Available Studies on host factors, particularly the APOBEC3G gene, have previously found an association with AIDS progression in some populations and against some HIV-1 strains but not others. Our study had two main objectives: firstly, to screen a population from Burkina Faso for three variants of APOBEC3G previously described, and secondly to analyze the effect of these three variants and their haplotypes on HIV-1 infection with Circulating Recombinant Forms (CRFs present in Burkina Faso. This case control study involved 708 seropositive and seronegative individuals. Genotyping was done by the TaqMan allelic discrimination method. Minor allele frequencies of rs6001417 (p<0.05, rs8177832 (P<0.05, and rs35228531 (P<0.001 were higher in seronegative subjects. The rs6001417 and rs8177832 SNPs were associated with HIV-1 infection in an additive model (P<0.01. Furthermore the SNP rs35228531 was also associated with HIV-1 infection in a dominant model (P<0.001. Odds ratio analysis of genotypes and alleles of the different APOBEC3G variants showed that there is a strong association between the minor genetic variants, genotype of the three SNPs, and HIV-1 status. Haplotype analysis demonstrated that rs6001417, rs8177832, and rs35228531 are in linkage disequilibrium. The haplotype GGT from the rs6001417, rs8177832 and rs35228531 respectively has a protective effect OR = 0.54 [0.43-0.68] with P<0.001. There was also associations between the haplotypes GGC OR = 1.6 [1.1;-2.3] P<0.05, and CGC OR = 5.21 [2.4-11.3] P<0.001, which increase the risk of infection by HIV-1 from almost two (2 to five (5 fold. This study demonstrates an association of rs6001417, rs8177832, and rs35228531 of APOBEC3G with HIV-1 infection in a population from Burkina Faso.

  3. Self-cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage.

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    Suspène, Rodolphe; Mussil, Bianka; Laude, Hélène; Caval, Vincent; Berry, Noémie; Bouzidi, Mohamed S; Thiers, Valérie; Wain-Hobson, Simon; Vartanian, Jean-Pierre

    2017-04-07

    Foreign and self-cytoplasmic DNA are recognized by numerous DNA sensor molecules leading to the production of type I interferons. Such DNA agonists should be degraded otherwise cells would be chronically stressed. Most human APOBEC3 cytidine deaminases can initiate catabolism of cytoplasmic mitochondrial DNA. Using the human myeloid cell line THP-1 with an interferon inducible APOBEC3A gene, we show that cytoplasmic DNA triggers interferon α and β production through the RNA polymerase III transcription/RIG-I pathway leading to massive upregulation of APOBEC3A. By catalyzing C→U editing in single stranded DNA fragments, the enzyme prevents them from re-annealing so attenuating the danger signal. The price to pay is chromosomal DNA damage in the form of CG→TA mutations and double stranded DNA breaks which, in the context of chronic inflammation, could drive cells down the path toward cancer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. The Antiviral and Cancer Genomic DNA Deaminase APOBEC3H Is Regulated by an RNA-Mediated Dimerization Mechanism.

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    Shaban, Nadine M; Shi, Ke; Lauer, Kate V; Carpenter, Michael A; Richards, Christopher M; Salamango, Daniel; Wang, Jiayi; Lopresti, Michael W; Banerjee, Surajit; Levin-Klein, Rena; Brown, William L; Aihara, Hideki; Harris, Reuben S

    2018-01-04

    Human APOBEC3H and homologous single-stranded DNA cytosine deaminases are unique to mammals. These DNA-editing enzymes function in innate immunity by restricting the replication of viruses and transposons. APOBEC3H also contributes to cancer mutagenesis. Here, we address the fundamental nature of RNA in regulating human APOBEC3H activities. APOBEC3H co-purifies with RNA as an inactive protein, and RNase A treatment enables strong DNA deaminase activity. RNA-binding-defective mutants demonstrate clear separation of function by becoming DNA hypermutators. Biochemical and crystallographic data demonstrate a mechanism in which double-stranded RNA mediates enzyme dimerization. Additionally, APOBEC3H separation-of-function mutants show that RNA binding is required for cytoplasmic localization, packaging into HIV-1 particles, and antiviral activity. Overall, these results support a model in which structured RNA negatively regulates the potentially harmful DNA deamination activity of APOBEC3H while, at the same time, positively regulating its antiviral activity. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Feline Immunodeficiency Virus Vif N-Terminal Residues Selectively Counteract Feline APOBEC3s.

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    Gu, Qinyong; Zhang, Zeli; Cano Ortiz, Lucía; Franco, Ana Cláudia; Häussinger, Dieter; Münk, Carsten

    2016-12-01

    Feline immunodeficiency virus (FIV) Vif protein counteracts feline APOBEC3s (FcaA3s) restriction factors by inducing their proteasomal degradation. The functional domains in FIV Vif for interaction with FcaA3s are poorly understood. Here, we have identified several motifs in FIV Vif that are important for selective degradation of different FcaA3s. Cats (Felis catus) express three types of A3s: single-domain A3Z2, single-domain A3Z3, and double-domain A3Z2Z3. We proposed that FIV Vif would selectively interact with the Z2 and the Z3 A3s. Indeed, we identified two N-terminal Vif motifs (12LF13 and 18GG19) that specifically interacted with the FcaA3Z2 protein but not with A3Z3. In contrast, the exclusive degradation of FcaA3Z3 was regulated by a region of three residues (M24, L25, and I27). Only a FIV Vif carrying a combination of mutations from both interaction sites lost the capacity to degrade and counteract FcaA3Z2Z3. However, alterations in the specific A3s interaction sites did not affect the cellular localization of the FIV Vif protein and binding to feline A3s. Pulldown experiments demonstrated that the A3 binding region localized to FIV Vif residues 50 to 80, outside the specific A3 interaction domain. Finally, we found that the Vif sites specific to individual A3s are conserved in several FIV lineages of domestic cat and nondomestic cats, while being absent in the FIV Vif of pumas. Our data support a complex model of multiple Vif-A3 interactions in which the specific region for selective A3 counteraction is discrete from a general A3 binding domain. Both human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV) Vif proteins counteract their host's APOBEC3 restriction factors. However, these two Vif proteins have limited sequence homology. The molecular interaction between FIV Vif and feline APOBEC3s are not well understood. Here, we identified N-terminal FIV Vif sites that regulate the selective interaction of Vif with either feline APOBEC3Z

  6. A Naturally Occurring Domestic Cat APOBEC3 Variant Confers Resistance to Feline Immunodeficiency Virus Infection.

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    Yoshikawa, Rokusuke; Izumi, Taisuke; Yamada, Eri; Nakano, Yusuke; Misawa, Naoko; Ren, Fengrong; Carpenter, Michael A; Ikeda, Terumasa; Münk, Carsten; Harris, Reuben S; Miyazawa, Takayuki; Koyanagi, Yoshio; Sato, Kei

    2016-01-01

    Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; A3) DNA cytosine deaminases can be incorporated into progeny virions and inhibit lentiviral replication. On the other hand, viral infectivity factor (Vif) of lentiviruses antagonizes A3-mediated antiviral activities by degrading A3 proteins. It is known that domestic cat (Felis catus) APOBEC3Z3 (A3Z3), the ortholog of human APOBEC3H, potently suppresses the infectivity of vif-defective feline immunodeficiency virus (FIV). Although a recent report has shown that domestic cat encodes 7 haplotypes (hap I to hap VII) of A3Z3, the relevance of A3Z3 polymorphism in domestic cats with FIV Vif has not yet been addressed. In this study, we demonstrated that these feline A3Z3 variants suppress vif-defective FIV infectivity. We also revealed that codon 65 of feline A3Z3 is a positively selected site and that A3Z3 hap V is subject to positive selection during evolution. It is particularly noteworthy that feline A3Z3 hap V is resistant to FIV Vif-mediated degradation and still inhibits vif-proficient viral infection. Moreover, the side chain size, but not the hydrophobicity, of the amino acid at position 65 determines the resistance to FIV Vif-mediated degradation. Furthermore, phylogenetic analyses have led to the inference that feline A3Z3 hap V emerged approximately 60,000 years ago. Taken together, these findings suggest that feline A3Z3 hap V may have been selected for escape from an ancestral FIV. This is the first evidence for an evolutionary "arms race" between the domestic cat and its cognate lentivirus. Gene diversity and selective pressure are intriguing topics in the field of evolutionary biology. A direct interaction between a cellular protein and a viral protein can precipitate an evolutionary arms race between host and virus. One example is primate APOBEC3G, which potently restricts the replication of primate lentiviruses (e.g., human immunodeficiency virus type 1 [HIV-1] and simian

  7. New World feline APOBEC3 potently controls inter-genus lentiviral transmission.

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    Konno, Yoriyuki; Nagaoka, Shumpei; Kimura, Izumi; Yamamoto, Keisuke; Kagawa, Yumiko; Kumata, Ryuichi; Aso, Hirofumi; Ueda, Mahoko Takahashi; Nakagawa, So; Kobayashi, Tomoko; Koyanagi, Yoshio; Sato, Kei

    2018-04-10

    The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; A3) gene family appears only in mammalian genomes. Some A3 proteins can be incorporated into progeny virions and inhibit lentiviral replication. In turn, the lentiviral viral infectivity factor (Vif) counteracts the A3-mediated antiviral effect by degrading A3 proteins. Recent investigations have suggested that lentiviral vif genes evolved to combat mammalian APOBEC3 proteins, and have further proposed that the Vif-A3 interaction may help determine the co-evolutionary history of cross-species lentiviral transmission in mammals. Here we address the co-evolutionary relationship between two New World felids, the puma (Puma concolor) and the bobcat (Lynx rufus), and their lentiviruses, which are designated puma lentiviruses (PLVs). We demonstrate that PLV-A Vif counteracts the antiviral action of APOBEC3Z3 (A3Z3) of both puma and bobcat, whereas PLV-B Vif counteracts only puma A3Z3. The species specificity of PLV-B Vif is irrespective of the phylogenic relationships of feline species in the genera Puma, Lynx and Acinonyx. We reveal that the amino acid at position 178 in the puma and bobcat A3Z3 is exposed on the protein surface and determines the sensitivity to PLV-B Vif-mediated degradation. Moreover, although both the puma and bobcat A3Z3 genes are polymorphic, their sensitivity/resistance to PLV Vif-mediated degradation is conserved. To the best of our knowledge, this is the first study suggesting that the host A3 protein potently controls inter-genus lentiviral transmission. Our findings provide the first evidence suggesting that the co-evolutionary arms race between lentiviruses and mammals has occurred in the New World.

  8. Human and murine APOBEC3s restrict replication of koala retrovirus by different mechanisms.

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    Nitta, Takayuki; Ha, Dat; Galvez, Felipe; Miyazawa, Takayuki; Fan, Hung

    2015-08-08

    Koala retrovirus (KoRV) is an endogenous and exogenous retrovirus of koalas that may cause lymphoma. As for many other gammaretroviruses, the KoRV genome can potentially encode an alternate form of Gag protein, glyco-gag. In this study, a convenient assay for assessing KoRV infectivity in vitro was employed: the use of DERSE cells (initially developed to search for infectious xenotropic murine leukemia-like viruses). Using infection of DERSE and other human cell lines (HEK293T), no evidence for expression of glyco-gag by KoRV was found, either in expression of glyco-gag protein or changes in infectivity when the putative glyco-gag reading frame was mutated. Since glyco-gag mediates resistance of Moloney murine leukemia virus to the restriction factor APOBEC3, the sensitivity of KoRV (wt or putatively mutant for glyco-gag) to restriction by murine (mA3) or human APOBEC3s was investigated. Both mA3 and hA3G potently inhibited KoRV infectivity. Interestingly, hA3G restriction was accompanied by extensive G → A hypermutation during reverse transcription while mA3 restriction was not. Glyco-gag status did not affect the results. These results indicate that the mechanisms of APOBEC3 restriction of KoRV by hA3G and mA3 differ (deamination dependent vs. independent) and glyco-gag does not play a role in the restriction.

  9. Inhibition of APOBEC3G activity impedes double-stranded DNA repair.

    Science.gov (United States)

    Prabhu, Ponnandy; Shandilya, Shivender M D; Britan-Rosich, Elena; Nagler, Adi; Schiffer, Celia A; Kotler, Moshe

    2016-01-01

    The cellular cytidine deaminase APOBEC3G (A3G) was first described as an anti-HIV-1 restriction factor, acting by directly deaminating reverse transcripts of the viral genome. HIV-1 Vif neutralizes the activity of A3G, primarily by mediating degradation of A3G to establish effective infection in host target cells. Lymphoma cells, which express high amounts of A3G, can restrict Vif-deficient HIV-1. Interestingly, these cells are more stable in the face of treatments that result in double-stranded DNA damage, such as ionizing radiation and chemotherapies. Previously, we showed that the Vif-derived peptide (Vif25-39) efficiently inhibits A3G deamination, and increases the sensitivity of lymphoma cells to ionizing radiation. In the current study, we show that additional peptides derived from Vif, A3G, and APOBEC3F, which contain the LYYF motif, inhibit deamination activity. Each residue in the Vif25-39 sequence moderately contributes to the inhibitory effect, whereas replacing a single residue in the LYYF motif completely abrogates inhibition of deamination. Treatment of A3G-expressing lymphoma cells exposed to ionizing radiation with the new inhibitory peptides reduces double-strand break repair after irradiation. Incubation of cultured irradiated lymphoma cells with peptides that inhibit double-strand break repair halts their propagation. These results suggest that A3G may be a potential therapeutic target that is amenable to peptide and peptidomimetic inhibition. © 2015 FEBS.

  10. Estimating the fraction of progeny virions that must incorporate APOBEC3G for suppression of productive HIV-1 infection

    International Nuclear Information System (INIS)

    Thangavelu, Pulari U.; Gupta, Vipul; Dixit, Narendra M.

    2014-01-01

    The contest between the host factor APOBEC3G (A3G) and the HIV-1 protein Vif presents an attractive target of intervention. The extent to which the A3G–Vif interaction must be suppressed to tilt the balance in favor of A3G remains unknown. We employed stochastic simulations and mathematical modeling of the within-host dynamics and evolution of HIV-1 to estimate the fraction of progeny virions that must incorporate A3G to render productive infection unsustainable. Using three different approaches, we found consistently that a transition from sustained infection to suppression of productive infection occurred when the latter fraction exceeded ∼0.8. The transition was triggered by A3G-induced hypermutations that led to premature stop codons compromising viral production and was consistent with driving the basic reproductive number, R 0 , below unity. The fraction identified may serve as a quantitative guideline for strategies targeting the A3G–Vif axis. - Highlights: • We perform simulations and mathematical modeling of the role of APOBEC3G in suppressing HIV-1 infection. • In three distinct ways, we estimate that when over 80% of progeny virions carry APOBEC3G, productive HIV-1 infection would be suppressed. • Our estimate of this critical fraction presents quantitative guidelines for strategies targeting the APOBEC3G–Vif axis

  11. APOBEC3G-induced hypermutation of human immunodeficiency virus type-1 is typically a discrete "all or nothing" phenomenon

    DEFF Research Database (Denmark)

    Armitage, Andrew E; Deforche, Koen; Chang, Chih-Hao

    2012-01-01

    The rapid evolution of Human Immunodeficiency Virus (HIV-1) allows studies of ongoing host-pathogen interactions. One key selective host factor is APOBEC3G (hA3G) that can cause extensive and inactivating Guanosine-to-Adenosine (G-to-A) mutation on HIV plus-strand DNA (termed hypermutation). HIV ...

  12. APOBEC3B-Mediated Cytidine Deamination Is Required for Estrogen Receptor Action in Breast Cancer

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    Manikandan Periyasamy

    2015-10-01

    Full Text Available Estrogen receptor α (ERα is the key transcriptional driver in a large proportion of breast cancers. We report that APOBEC3B (A3B is required for regulation of gene expression by ER and acts by causing C-to-U deamination at ER binding regions. We show that these C-to-U changes lead to the generation of DNA strand breaks through activation of base excision repair (BER and to repair by non-homologous end-joining (NHEJ pathways. We provide evidence that transient cytidine deamination by A3B aids chromatin modification and remodelling at the regulatory regions of ER target genes that promotes their expression. A3B expression is associated with poor patient survival in ER+ breast cancer, reinforcing the physiological significance of A3B for ER action.

  13. E138K and M184I mutations in HIV-1 reverse transcriptase coemerge as a result of APOBEC3 editing in the absence of drug exposure.

    Science.gov (United States)

    Fourati, Slim; Malet, Isabelle; Lambert, Sidonie; Soulie, Cathia; Wirden, Marc; Flandre, Philippe; Fofana, Djeneba B; Sayon, Sophie; Simon, Anne; Katlama, Christine; Calvez, Vincent; Marcelin, Anne-Geneviève

    2012-08-24

    Recent clinical trials with rilpivirine combined with emtricitabine and tenofovir revealed that patients failing treatment, frequently, harbored viruses encoding resistance-associated mutations in the HIV-1 reverse transcriptase at position E138K and M184I. We show here that APOBEC3 proteins play a role in the emergence of these drug resistance mutations. We used a Vif mutant that has suboptimal activity against APOBEC3 to assess the in-vitro frequency of APOBEC3-induced resistance mutations in reverse transcriptase. To assess the degree of in-vivo G-to-A viral hypermutation, a large amount of data of HIV-1 RT proviral sequences from peripheral blood mononuclear cells (PBMCs) recovered from infected patients under HAART was analyzed. In-vitro replication experiments in cell lines with and without APOBEC3 expression suggest that APOBEC3-driven mutagenesis contributes to the generation of both M184I and E138K within HIV proviral repository in the absence of drug exposure. Additionally, analysis of 601 patients PBMCs sequences revealed that the copresence of mutations E138K and M184I were never detected in nonhypermutated sequences, whereas these mutations were found at a high frequency (24%) in the context of APOBEC3 editing and in the absence of exposure to etravirine-rilpivirine. We demonstrate using in-vitro experiments and analyzing patients PBMCs sequences that M184I and E138K resistance-associated mutations may pre-exist in proviral reservoir at a high frequency prior to drug exposure, as a result of APOBEC3 editing. Thus, incomplete neutralization of one or more APOBEC3 proteins may favor viral escape to rilpivirine-emtricitabine. © 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins

  14. Human APOBEC3G drives HIV-1 evolution and the development of drug resistance

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharya, Tamoy [Los Alamos National Laboratory; Kim, Eun - Young [FEINBERG SCHOOL OF MEDS; Koning, Fransje [KING' S COLLEGE LONDON; Malim, Michael [KING' S COLLEGE LONDON; Wolinsky, Steven M [FEINBERG SCHOOL OF MEDS

    2008-01-01

    Human APOBEC3G (hA3G) is an innate virus restriction factor that induces deamination of specific cytidine residues in single-stranded human immunodeficiency virus type 1 (HIV-1) DNA. Whereas destructive hA3G editing leads to a profound loss of HIV-1 infectivity, more limited editing could be a source of adaptation and diversification. Here we show that the presence of hA3G in T-cells can drive the development of diversity in HIV-1 populations and that under selection pressure imposed by the nucleotide analog reverse transcriptase inhibitor 3TC ((-)2',3'-dideoxy-3'-thiacytidine), a single point mutation that confers 3TC resistance, methionine 184 to isoleucine (M1841), emerges rapidly and reaches fixation. These results provide strong evidence that mutation by hA3G is an important source of genetic variation on which natural selection acts to shape the structure of the viral population and drive the tempo of HIV-1 evolution.

  15. Retroelements versus APOBEC3 family members: No great escape from the magnificent seven

    Directory of Open Access Journals (Sweden)

    Juan F Arias

    2012-08-01

    Full Text Available Retroelements comprise a large and successful family of transposable genetic elements that, through intensive infiltration, have shaped the genomes of humans and other mammals over millions of years. In fact, retrotransposons now account for approximately 45% of the human genome. Based on their genomic mobility, some retroelements are known to be the cause of genetic diseases, not only in germ and embryonic stem cells but also in somatic cells, posing a threat to genomic stability throughout all cellular populations. In response, mammals have developed intrinsic immunity mechanisms that provide resistance against the threat of the deleterious effects of retrotransposition. Among these, seven members of the APOBEC3 (A3 family of cytidine deaminases serve as highly active, intrinsic, antiretroviral host factors. Certain A3 proteins effectively counteract infections of retroviruses such as HIV-1, as well as those of other virus families, while also blocking the transposition of retroelements. Based on their preferential expression in the germ cells, in which retrotransposons may be active, it is likely that A3 proteins were acquired through mammalian evolution primarily to inhibit retrotransposition and thereby maintain genomic stability in these cells. This review summarizes the recent advances in our understanding of the interplay between the retroelements currently active in the human genome and the anti-retroelement A3 proteins.

  16. Inhibition of a NEDD8 Cascade Restores Restriction of HIV by APOBEC3G.

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    David J Stanley

    2012-12-01

    Full Text Available Cellular restriction factors help to defend humans against human immunodeficiency virus (HIV. HIV accessory proteins hijack at least three different Cullin-RING ubiquitin ligases, which must be activated by the small ubiquitin-like protein NEDD8, in order to counteract host cellular restriction factors. We found that conjugation of NEDD8 to Cullin-5 by the NEDD8-conjugating enzyme UBE2F is required for HIV Vif-mediated degradation of the host restriction factor APOBEC3G (A3G. Pharmacological inhibition of the NEDD8 E1 by MLN4924 or knockdown of either UBE2F or its RING-protein binding partner RBX2 bypasses the effect of Vif, restoring the restriction of HIV by A3G. NMR mapping and mutational analyses define specificity determinants of the UBE2F NEDD8 cascade. These studies demonstrate that disrupting host NEDD8 cascades presents a novel antiretroviral therapeutic approach enhancing the ability of the immune system to combat HIV.

  17. Inhibition of APOBEC3G Activity Impedes Double-Strand DNA Repair

    Science.gov (United States)

    Prabhu, Ponnandy; Shandilya, Shivender; Britan-Rosich, Elena; Nagler, Adi; Schiffer, Celia A.; Kotler, Moshe

    2015-01-01

    The cellular cytidine deaminase APOBEC3G (A3G) was first described as an anti-HIV-1 restriction factor by directly deaminating reverse transcripts of the viral genome. HIV-1 Vif neutralizes the activity of A3G, primarily by mediating degradation of A3G to establish effective infection in host target cells. Lymphoma cells, which express high amounts of A3G, can restrict Vif-deficient HIV-1. Interestingly, these cells are more stable in the face of treatments that result in dsDNA damage, such as ionizing irradiation (IR) and chemotherapies. Previously, we showed that the Vif-derived peptide (Vif25-39) efficiently inhibits A3G deamination, and increases sensitivity of lymphoma cells to IR. In the current study, we show that additional peptides derived from Vif, A3G and A3F, which contain the LYYF motif, inhibit deamination activity. Each residue in the Vif25-39 sequence moderately contributes to the inhibitory effect, while, replacing a single amino acid in the LYYF motif completely abrogate inhibition of deamination. Treatment of A3G-expressing lymphoma cells exposed to ionizing radiation with the new inhibitory peptides reduces double-strand break (DSB) repair after radiation. Incubation of cultured irradiated lymphoma cells with peptides that inhibit DSB repair halts their propagation. These results suggest that A3G may be a potential therapeutic target amenable to peptide and peptidomimetic inhibition. PMID:26460502

  18. Basal transcription of APOBEC3G is regulated by USF1 gene in hepatocyte

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Yanli [Department of Infectious Diseases, Zhengzhou University People' s Hospital (Henan Provincial People' s Hospital), Zhengzhou, 450003 (China); Li, Hui [The Central Hospital of Wuhan, Tongji Medical College Huazhong University of Science Technology, Wuhan, 430000 (China); Zhang, Xiaoju [Department of Respiratory Medicine, Zhengzhou University People' s Hospital (Henan Provincial People' s Hospital), Zhengzhou, 450003 (China); Shang, Jia [Department of Infectious Diseases, Zhengzhou University People' s Hospital (Henan Provincial People' s Hospital), Zhengzhou, 450003 (China); Kang, Yi, E-mail: kykangyi@163.com [Department of Infectious Diseases, Zhengzhou University People' s Hospital (Henan Provincial People' s Hospital), Zhengzhou, 450003 (China)

    2016-01-29

    Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G, A3G) exert antiviral defense as an important factor of innate immunity. A variety of cytokines such as IFN-γ,IL2,IL15,IL7 could induce the transcription of A3G. However, the regulation of other nuclear factor on the transcription of A3G have not been reported at the present. To gain new insights into the transcriptional regulation of this restriction factor, we cloned and characterized the promoter region of A3G and investigate the modulation of USF1 gene on the transcription of A3G. We identified a 232 bp region that was sufficient to regulate the activity of full promoter. Transcriptional start sites (TSS) were identified by the luciferase reporter assays of plasmids containing full or shorter fragments of the A3G promoter. The results demonstrated that the core promoter of A3G is located within the region -159/-84 relative to the TSS. Transcriptional activity of A3G core promoter regulated by USF1 was dependent on an E-box (located at position -91/-86 relative to the major TSS) and was abolished after mutation of this DNA element. USF1 gene can take part in basal transcription regulation of the human A3G gene in hepatocyte, and the identified E-box represented a binding site for the USF1. - Highlights: • The core promoter of A3G is located within the region −159/−84 relative to the TSS. • Transcriptional activity of A3G core promoter regulated by USF1 was dependent on an E-box (located at position −91/−86 relative to the major TSS). • USF1 gene can take part in basal transcription regulation of the human A3G gene in hepatocyte.

  19. Targeting APOBEC3A to the viral nucleoprotein complex confers antiviral activity

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    Strebel Klaus

    2007-08-01

    Full Text Available Abstract Background APOBEC3 (A3 proteins constitute a family of cytidine deaminases that provide intracellular resistance to retrovirus replication and to transposition of endogenous retroelements. A3A has significant homology to the C-terminus of A3G but has only a single cytidine deaminase active site (CDA, unlike A3G, which has a second N-terminal CDA previously found to be important for Vif sensitivity and virus encapsidation. A3A is packaged into HIV-1 virions but, unlike A3G, does not have antiviral properties. Here, we investigated the reason for the lack of A3A antiviral activity. Results Sequence alignment of A3G and A3A revealed significant homology of A3A to the C-terminal region of A3G. However, while A3G co-purified with detergent-resistant viral nucleoprotein complexes (NPC, virus-associated A3A was highly detergent-sensitive leading us to speculate that the ability to assemble into NPC may be a property conveyed by the A3G N-terminus. To test this model, we constructed an A3G-3A chimeric protein, in which the N-terminal half of A3G was fused to A3A. Interestingly, the A3G-3A chimera was packaged into HIV-1 particles and, unlike A3A, associated with the viral NPC. Furthermore, the A3G-3A chimera displayed strong antiviral activity against HIV-1 and was sensitive to inhibition by HIV-1 Vif. Conclusion Our results suggest that the A3G N-terminal domain carries determinants important for targeting the protein to viral NPCs. Transfer of this domain to A3A results in A3A targeting to viral NPCs and confers antiviral activity.

  20. Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.

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    Monica K Akre

    Full Text Available Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80-90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.

  1. Analysis of APOBEC3A/3B germline deletion polymorphism in breast, cervical and oral cancers from South India and its impact on miRNA regulation.

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    Revathidevi, Sundaramoorthy; Manikandan, Mayakannan; Rao, Arunagiri Kuha Deva Magendhra; Vinothkumar, Vilvanathan; Arunkumar, Ganesan; Rajkumar, Kottayasamy Seenivasagam; Ramani, Rajendran; Rajaraman, Ramamurthy; Ajay, Chandrasekar; Munirajan, Arasambattu Kannan

    2016-09-01

    Breast cancer and cervical cancer are the leading causes of death in women worldwide as well as in India, whilst oral cancer is the top most common cancer among Asian especially in Indian men in terms of both incidence and mortality rate. Genetic factors determining the predisposition to cancer are being explored to identify the signature genetic variations associated with these cancers. Recently, a germline deletion polymorphism in APOBEC3 gene cluster which completely deletes APOBEC3B coding region has been studied for its association with cancer risk. We screened the germline deletion polymorphism in 409 cancer patients (224 breast cancer, 88 cervical cancer and 97 oral cancer samples), 478 controls and 239 cervical cancer tissue DNAs of South Indian origin. The results suggest that the APOBEC3A/3B deletion polymorphism is not significantly associated with cancer risk in our study population (OR 0.739, 95 % CI, p value 0.91457). Considering the viral restriction property of APOBEC3s, we also screened cervical cancer tissue DNAs for the human papilloma virus infection. We observed a gradual increase in the frequency of HPV16 infection from AA/BB cases (66.86 %) to AA/-- cases (71.43) which signifies the impact of this deletion polymorphism in HPV infection. In addition, we performed in silico analysis to understand the effect of this polymorphism on miRNA regulation of the APOBEC3A/3B fusion transcript. Only 8 APOBEC3B targeting miRNAs were observed to regulate the fusion transcript of which miR-34b-3p and miR-138-5p were found to be frequently downregulated in cancers suggesting miRNA-mediated deregulation of APOBEC3A expression in cancer patients harbouring this particular deletion polymorphism.

  2. Contribution of APOBEC3G/F activity to the development of low-abundance drug-resistant human immunodeficiency virus type 1 variants.

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    Noguera-Julian, M; Cozzi-Lepri, A; Di Giallonardo, F; Schuurman, R; Däumer, M; Aitken, S; Ceccherini-Silberstein, F; D'Arminio Monforte, A; Geretti, A M; Booth, C L; Kaiser, R; Michalik, C; Jansen, K; Masquelier, B; Bellecave, P; Kouyos, R D; Castro, E; Furrer, H; Schultze, A; Günthard, H F; Brun-Vezinet, F; Metzner, K J; Paredes, R

    2016-02-01

    Plasma drug-resistant minority human immunodeficiency virus type 1 variants (DRMVs) increase the risk of virological failure to first-line non-nucleoside reverse transcriptase inhibitor antiretroviral therapy (ART). The origin of DRMVs in ART-naive patients, however, remains unclear. In a large pan-European case-control study investigating the clinical relevance of pre-existing DRMVs using 454 pyrosequencing, the six most prevalent plasma DRMVs detected corresponded to G-to-A nucleotide mutations (V90I, V106I, V108I, E138K, M184I and M230I). Here, we evaluated if such DRMVs could have emerged from apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3G/F (APOBEC3G/F) activity. Out of 236 ART-naive subjects evaluated, APOBEC3G/F hypermutation signatures were detected in plasma viruses of 14 (5.9%) individuals. Samples with minority E138K, M184I, and M230I mutations, but not those with V90I, V106I or V108I, were significantly associated with APOBEC3G/F activity (Fisher's P 0.5% of sample sequences with an APOBEC3G/F signature. Mutations E138K, M184I and M230I co-occurred in the same sequence as APOBEC3G/F signatures in 3/9 (33%), 5/11 (45%) and 4/8 (50%) of samples, respectively; such linkage was not found for V90I, V106I or V108I. In-frame STOP codons were observed in 1.5% of all clonal sequences; 14.8% of them co-occurred with APOBEC3G/F signatures. APOBEC3G/F-associated E138K, M184I and M230I appeared within clonal sequences containing in-frame STOP codons in 2/3 (66%), 5/5 (100%) and 4/4 (100%) of the samples. In a re-analysis of the parent case control study, the presence of APOBEC3G/F signatures was not associated with virological failure. In conclusion, the contribution of APOBEC3G/F editing to the development of DRMVs is very limited and does not affect the efficacy of non-nucleoside reverse transcriptase inhibitor ART. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  3. HBV/HIV co-infection and APOBEC3G polymorphisms in a population from Burkina Faso.

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    Compaore, Tegwinde Rebeca; Diarra, Birama; Assih, Maleki; Obiri-Yeboah, Dorcas; Soubeiga, Serge Theophile; Ouattara, Abdoul Karim; Tchelougou, Damehan; Bisseye, Cyrille; Bakouan, Didier Romuald; Compaore, Issaka Pierre; Dembele, Augustine; Djigma, Wendkuuni Florencia; Simpore, Jacques

    2016-07-22

    Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G (APOBEC3G) is a potent host defense factor, which interferes with HIV-1 and HBV. Our study had three objectives, to screen a population of HIV-1 infected and uninfected patients in Burkina Faso for HBV, to screen the population for APOBEC3G variants rs6001417, rs8177832, and rs35228531 previously described, and to analyze the effect of these three variants and their haplotypes on HIV-1/HBV co-infection in Burkina Faso. HBV detection was performed on samples from HIV-1 infected and uninfected subjects using rapid detection tests and real-time PCR. APOBEC3 genotyping was done by the TaqMan allelic discrimination method. Fisher Exact test, Odds ratio (OR), confidence intervals (CI) at 95 %, Linkage disequilibrium (LD) summary statistics and haplotype frequencies were calculated. The prevalence of HBV was 56.7 % among HIV-1 positive patients of our study while it was about 12.8 % among HIV-1 seronegative subjects. Genotype E was the genotype of HBV present in our hepatitis B positive samples. Minor allele frequencies of rs6001417, rs8177832, and rs35228531 were higher in seronegative subjects. The T minor allele of variant rs35228531 was protective against HIV-1/HBV co-infection with OR = 0.61, 95 % CI (0.42-0.90), p = 0.013. There was also an association between the GGT haplotype and protection against HIV-1/HBV co-infection, OR = 0.57, 95 % CI (0.33-0.99), p = 0.050. The other haplotypes present in the population were not statistically significant. There minor allele T of the rs35228531 was protective against HIV mono-infection OR = 0.53, 95 % CI (0.3-0.93), P = 0.030. But there was no effect of protection against HBV mono-infection. APOBEC3G through its variants rs6001417, rs8177832, and rs35228531, in this study interferes with HIV-1/HBV co-infection could be due the HIV-1 mono-infection in a population from Burkina Faso.

  4. Restriction of equine infectious anemia virus by equine APOBEC3 cytidine deaminases.

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    Zielonka, Jörg; Bravo, Ignacio G; Marino, Daniela; Conrad, Elea; Perković, Mario; Battenberg, Marion; Cichutek, Klaus; Münk, Carsten

    2009-08-01

    The mammalian APOBEC3 (A3) proteins comprise a multigene family of cytidine deaminases that act as potent inhibitors of retroviruses and retrotransposons. The A3 locus on the chromosome 28 of the horse genome contains multiple A3 genes: two copies of A3Z1, five copies of A3Z2, and a single copy of A3Z3, indicating a complex evolution of multiple gene duplications. We have cloned and analyzed for expression the different equine A3 genes and examined as well the subcellular distribution of the corresponding proteins. Additionally, we have tested the functional antiretroviral activity of the equine and of several of the human and nonprimate A3 proteins against the Equine infectious anemia virus (EIAV), the Simian immunodeficiency virus (SIV), and the Adeno-associated virus type 2 (AAV-2). Hematopoietic cells of horses express at least five different A3s: A3Z1b, A3Z2a-Z2b, A3Z2c-Z2d, A3Z2e, and A3Z3, whereas circulating macrophages, the natural target of EIAV, express only part of the A3 repertoire. The five A3Z2 tandem copies arose after three consecutive, recent duplication events in the horse lineage, after the split between Equidae and Carnivora. The duplicated genes show different antiviral activities against different viruses: equine A3Z3 and A3Z2c-Z2d are potent inhibitors of EIAV while equine A3Z1b, A3Z2a-Z2b, A3Z2e showed only weak anti-EIAV activity. Equine A3Z1b and A3Z3 restricted AAV and all equine A3s, except A3Z1b, inhibited SIV. We hypothesize that the horse A3 genes are undergoing a process of subfunctionalization in their respective viral specificities, which might provide the evolutionary advantage for keeping five copies of the original gene.

  5. A novel Bayesian method for detection of APOBEC3-mediated hypermutation and its application to zoonotic transmission of simian foamy viruses.

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    Frederick A Matsen

    2014-02-01

    Full Text Available Simian Foamy Virus (SFV can be transmitted from non-human primates (NHP to humans. However, there are no documented cases of human to human transmission, and significant differences exist between infection in NHP and human hosts. The mechanism for these between-host differences is not completely understood. In this paper we develop a new Bayesian approach to the detection of APOBEC3-mediated hypermutation, and use it to compare SFV sequences from human and NHP hosts living in close proximity in Bangladesh. We find that human APOBEC3G can induce genetic changes that may prevent SFV replication in infected humans in vivo.

  6. Determinants of FIV and HIV Vif sensitivity of feline APOBEC3 restriction factors.

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    Zhang, Zeli; Gu, Qinyong; Jaguva Vasudevan, Ananda Ayyappan; Hain, Anika; Kloke, Björn-Philipp; Hasheminasab, Sascha; Mulnaes, Daniel; Sato, Kei; Cichutek, Klaus; Häussinger, Dieter; Bravo, Ignacio G; Smits, Sander H J; Gohlke, Holger; Münk, Carsten

    2016-07-01

    Feline immunodeficiency virus (FIV) is a global pathogen of Felidae species and a model system for Human immunodeficiency virus (HIV)-induced AIDS. In felids such as the domestic cat (Felis catus), APOBEC3 (A3) genes encode for single-domain A3Z2s, A3Z3 and double-domain A3Z2Z3 anti-viral cytidine deaminases. The feline A3Z2Z3 is expressed following read-through transcription and alternative splicing, introducing a previously untranslated exon in frame, encoding a domain insertion called linker. Only A3Z3 and A3Z2Z3 inhibit Vif-deficient FIV. Feline A3s also are restriction factors for HIV and Simian immunodeficiency viruses (SIV). Surprisingly, HIV-2/SIV Vifs can counteract feline A3Z2Z3. To identify residues in feline A3s that Vifs need for interaction and degradation, chimeric human-feline A3s were tested. Here we describe the molecular direct interaction of feline A3s with Vif proteins from cat FIV and present the first structural A3 model locating these interaction regions. In the Z3 domain we have identified residues involved in binding of FIV Vif, and their mutation blocked Vif-induced A3Z3 degradation. We further identified additional essential residues for FIV Vif interaction in the A3Z2 domain, allowing the generation of FIV Vif resistant A3Z2Z3. Mutated feline A3s also showed resistance to the Vif of a lion-specific FIV, indicating an evolutionary conserved Vif-A3 binding. Comparative modelling of feline A3Z2Z3 suggests that the residues interacting with FIV Vif have, unlike Vif-interacting residues in human A3s, a unique location at the domain interface of Z2 and Z3 and that the linker forms a homeobox-like domain protruding of the Z2Z3 core. HIV-2/SIV Vifs efficiently degrade feline A3Z2Z3 by possible targeting the linker stretch connecting both Z-domains. Here we identified in feline A3s residues important for binding of FIV Vif and a unique protein domain insertion (linker). To understand Vif evolution, a structural model of the feline A3 was

  7. Heat-stable molecule derived from Streptococcus cristatus induces APOBEC3 expression and inhibits HIV-1 replication.

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    Ziqing Wang

    Full Text Available Although most human immunodeficiency virus type 1 (HIV-1 cases worldwide are transmitted through mucosal surfaces, transmission through the oral mucosal surface is a rare event. More than 700 bacterial species have been detected in the oral cavity. Despite great efforts to discover oral inhibitors of HIV, little information is available concerning the anti-HIV activity of oral bacterial components. Here we show that a molecule from an oral commensal bacterium, Streptococcus cristatus CC5A can induce expression of APOBEC3G (A3G and APOBEC3F (A3F and inhibit HIV-1 replication in THP-1 cells. We show by qRT-PCR that expression levels of A3G and A3F increase in a dose-dependent manner in the presence of a CC5A extract, as does A3G protein levels by Western blot assay. In addition, when the human monocytic cell line THP-1 was treated with CC5A extract, the replication of HIV-1 IIIB was significantly suppressed compared with IIIB replication in untreated THP-1 cells. Knock down of A3G expression in THP-1 cells compromised the ability of CC5A to inhibit HIV-1 IIIB infectivity. Furthermore, SupT1 cells infected with virus produced from CC5A extract-treated THP-1 cells replicated virus with a higher G to A hypermutation rate (a known consequence of A3G activity than virus used from untreated THP-1 cells. This suggests that S. cristatus CC5A contains a molecule that induces A3G/F expression and thereby inhibits HIV replication. These findings might lead to the discovery of a novel anti-HIV/AIDS therapeutic.

  8. Molecular cloning and anti-HIV-1 activities of APOBEC3s from northern pig-tailed macaques (Macaca leonina).

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    Zhang, Xiao-Liang; Song, Jia-Hao; Pang, Wei; Zheng, Yong-Tang

    2016-07-18

    Northern pig-tailed macaques (NPMs, Macaca leonina) are susceptible to HIV-1 infection largely due to the loss of HIV-1-restricting factor TRIM5α. However, great impediments still exist in the persistent replication of HIV-1 in vivo, suggesting some viral restriction factors are reserved in this host. The APOBEC3 proteins have demonstrated a capacity to restrict HIV-1 replication, but their inhibitory effects in NPMs remain elusive. In this study, we cloned the NPM A3A-A3H genes, and determined by BLAST searching that their coding sequences (CDSs) showed 99% identity to the corresponding counterparts from rhesus and southern pig-tailed macaques. We further analyzed the anti-HIV-1 activities of the A3A-A3H genes, and found that A3G and A3F had the greatest anti-HIV-1 activity compared with that of other members. The results of this study indicate that A3G and A3F might play critical roles in limiting HIV-1 replication in NPMs in vivo. Furthermore, this research provides valuable information for the optimization of monkey models of HIV-1 infection.

  9. Fab-based inhibitors reveal ubiquitin independent functions for HIV Vif neutralization of APOBEC3 restriction factors.

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    Jennifer M Binning

    2018-01-01

    Full Text Available The lentiviral protein Viral Infectivity Factor (Vif counteracts the antiviral effects of host APOBEC3 (A3 proteins and contributes to persistent HIV infection. Vif targets A3 restriction factors for ubiquitination and proteasomal degradation by recruiting them to a multi-protein ubiquitin E3 ligase complex. Here, we describe a degradation-independent mechanism of Vif-mediated antagonism that was revealed through detailed structure-function studies of antibody antigen-binding fragments (Fabs to the Vif complex. Two Fabs were found to inhibit Vif-mediated A3 neutralization through distinct mechanisms: shielding A3 from ubiquitin transfer and blocking Vif E3 assembly. Combined biochemical, cell biological and structural studies reveal that disruption of Vif E3 assembly inhibited A3 ubiquitination but was not sufficient to restore its packaging into viral particles and antiviral activity. These observations establish that Vif can neutralize A3 family members in a degradation-independent manner. Additionally, this work highlights the potential of Fabs as functional probes, and illuminates how Vif uses a multi-pronged approach involving both degradation dependent and independent mechanisms to suppress A3 innate immunity.

  10. APOBEC3G inhibits HIV-1 RNA elongation by inactivating the viral trans-activation response element.

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    Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

    2014-07-29

    Deamination of cytidine residues in viral DNA is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient human immunodeficiency virus type 1 (HIV-1) replication. dC-to-dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here, we demonstrate that A3G provides an additional layer of defense against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. We further show that free single-stranded DNA (ssDNA) termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3'+5' ends is sufficient for A3G deamination. These results identify A3G as an efficient mutator and that deamination of (-)SSDNA results in an early block of HIV-1 transcription. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Incomplete APOBEC3G/F Neutralization by HIV-1 Vif Mutants Facilitates the Genetic Evolution from CCR5 to CXCR4 Usage.

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    Alteri, Claudia; Surdo, Matteo; Bellocchi, Maria Concetta; Saccomandi, Patrizia; Continenza, Fabio; Armenia, Daniele; Parrotta, Lucia; Carioti, Luca; Costa, Giosuè; Fourati, Slim; Di Santo, Fabiola; Scutari, Rossana; Barbaliscia, Silvia; Fedele, Valentina; Carta, Stefania; Balestra, Emanuela; Alcaro, Stefano; Marcelin, Anne Genevieve; Calvez, Vincent; Ceccherini-Silberstein, Francesca; Artese, Anna; Perno, Carlo Federico; Svicher, Valentina

    2015-08-01

    Incomplete APOBEC3G/F neutralization by a defective HIV-1Vif protein can promote genetic diversification by inducing G-to-A mutations in the HIV-1 genome. The HIV-1 Env V3 loop, critical for coreceptor usage, contains several putative APOBEC3G/F target sites. Here, we determined if APOBEC3G/F, in the presence of Vif-defective HIV-1 virus, can induce G-to-A mutations at V3 positions critical to modulation of CXCR4 usage. Peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM) from 2 HIV-1-negative donors were infected with CCR5-using 81.A-VifWT virus (i.e., with wild-type [WT] Vif protein), 81.A-VifE45G, or 81.A-VifK22E (known to incompletely/partially neutralize APOBEC3G/F). The rate of G-toA mutations was zero or extremely low in 81.A-VifWT- and 81.A-VifE45G-infected PBMC from both donors. Conversely, G-to-A enrichment was detected in 81.A-VifK22E-infected PBMC (prevalence ranging from 2.18% at 7 days postinfection [dpi] to 3.07% at 21 dpi in donor 1 and from 10.49% at 7 dpi to 8.69% at 21 dpi in donor 2). A similar scenario was found in MDM. G-to-A mutations occurred at 8 V3 positions, resulting in nonsynonymous amino acid substitutions. Of them, G24E and E25K strongly correlated with phenotypically/genotypically defined CXCR4-using viruses (P = 0.04 and 5.5e-7, respectively) and increased the CXCR4 N-terminal binding affinity for V3 (WT, -40.1 kcal/mol; G24E, -510 kcal/mol; E25K, -522 kcal/mol). The analysis of paired V3 and Vif DNA sequences from 84 HIV-1-infected patients showed that the presence of a Vif-defective virus correlated with CXCR4 usage in proviral DNA (P = 0.04). In conclusion, incomplete APOBEC3G/F neutralization by a single Vif amino acid substitution seeds a CXCR4-using proviral reservoir. This can have implications for the success of CCR5 antagonist-based therapy, as well as for the risk of disease progression. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Non-Retroviral Fossils in Vertebrate Genomes

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    Masayuki Horie

    2011-10-01

    Full Text Available Although no physical fossils of viruses have been found, retroviruses are known to leave their molecular fossils in the genomes of their hosts, the so-called endogenous retroviral elements. These have provided us with important information about retroviruses in the past and their co-evolution with their hosts. On the other hand, because non‑retroviral viruses were considered not to leave such fossils, even the existence of prehistoric non-retroviral viruses has been enigmatic. Recently, we discovered that elements derived from ancient bornaviruses, non-segmented, negative strand RNA viruses, are found in the genomes of several mammalian species, including humans. In addition, at approximately the same time, several endogenous elements of RNA viruses, DNA viruses and reverse-transcribing DNA viruses have been independently reported, which revealed that non-retroviral viruses have played significant roles in the evolution of their hosts and provided novel insights into virology and cell biology. Here we review non-retroviral virus-like elements in vertebrate genomes, non-retroviral integration and the knowledge obtained from these endogenous non-retroviral virus-like elements.

  13. RNA binding to APOBEC3G induces the disassembly of functional deaminase complexes by displacing single-stranded DNA substrates

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    Polevoda, Bogdan; McDougall, William M.; Tun, Bradley N.; Cheung, Michael; Salter, Jason D.; Friedman, Alan E.; Smith, Harold C.

    2015-01-01

    APOBEC3G (A3G) DNA deaminase activity requires a holoenzyme complex whose assembly on nascent viral reverse transcripts initiates with A3G dimers binding to ssDNA followed by formation of higher-order A3G homo oligomers. Catalytic activity is inhibited when A3G binds to RNA. Our prior studies suggested that RNA inhibited A3G binding to ssDNA. In this report, near equilibrium binding and gel shift analyses showed that A3G assembly and disassembly on ssDNA was an ordered process involving A3G dimers and multimers thereof. Although, fluorescence anisotropy showed that A3G had similar nanomolar affinity for RNA and ssDNA, RNA stochastically dissociated A3G dimers and higher-order oligomers from ssDNA, suggesting a different modality for RNA binding. Mass spectrometry mapping of A3G peptides cross-linked to nucleic acid suggested ssDNA only bound to three peptides, amino acids (aa) 181–194 in the N-terminus and aa 314–320 and 345–374 in the C-terminus that were part of a continuous exposed surface. RNA bound to these peptides and uniquely associated with three additional peptides in the N- terminus, aa 15–29, 41–52 and 83–99, that formed a continuous surface area adjacent to the ssDNA binding surface. The data predict a mechanistic model of RNA inhibition of ssDNA binding to A3G in which competitive and allosteric interactions determine RNA-bound versus ssDNA-bound conformational states. PMID:26424853

  14. Vif of feline immunodeficiency virus from domestic cats protects against APOBEC3 restriction factors from many felids.

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    Zielonka, Jörg; Marino, Daniela; Hofmann, Henning; Yuhki, Naoya; Löchelt, Martin; Münk, Carsten

    2010-07-01

    To get more insight into the role of APOBEC3 (A3) cytidine deaminases in the species-specific restriction of feline immunodeficiency virus (FIV) of the domestic cat, we tested the A3 proteins present in big cats (puma, lion, tiger, and lynx). These A3 proteins were analyzed for expression and sensitivity to the Vif protein of FIV. While A3Z3s and A3Z2-Z3s inhibited Deltavif FIV, felid A3Z2s did not show any antiviral activity against Deltavif FIV or wild-type (wt) FIV. All felid A3Z3s and A3Z2-Z3s were sensitive to Vif of the domestic cat FIV. Vif also induced depletion of felid A3Z2s. Tiger A3s showed a moderate degree of resistance against the Vif-mediated counter defense. These findings may imply that the A3 restriction system does not play a major role to prevent domestic cat FIV transmission to other Felidae. In contrast to the sensitive felid A3s, many nonfelid A3s actively restricted wt FIV replication. To test whether Vif(FIV) can protect also the distantly related human immunodeficiency virus type 1 (HIV-1), a chimeric HIV-1.Vif(FIV) was constructed. This HIV-1.Vif(FIV) was replication competent in nonpermissive feline cells expressing human CD4/CCR5 that did not support the replication of wt HIV-1. We conclude that the replication of HIV-1 in some feline cells is inhibited only by feline A3 restriction factors and the absence of the appropriate receptor or coreceptor.

  15. Vif of Feline Immunodeficiency Virus from Domestic Cats Protects against APOBEC3 Restriction Factors from Many Felids▿

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    Zielonka, Jörg; Marino, Daniela; Hofmann, Henning; Yuhki, Naoya; Löchelt, Martin; Münk, Carsten

    2010-01-01

    To get more insight into the role of APOBEC3 (A3) cytidine deaminases in the species-specific restriction of feline immunodeficiency virus (FIV) of the domestic cat, we tested the A3 proteins present in big cats (puma, lion, tiger, and lynx). These A3 proteins were analyzed for expression and sensitivity to the Vif protein of FIV. While A3Z3s and A3Z2-Z3s inhibited Δvif FIV, felid A3Z2s did not show any antiviral activity against Δvif FIV or wild-type (wt) FIV. All felid A3Z3s and A3Z2-Z3s were sensitive to Vif of the domestic cat FIV. Vif also induced depletion of felid A3Z2s. Tiger A3s showed a moderate degree of resistance against the Vif-mediated counter defense. These findings may imply that the A3 restriction system does not play a major role to prevent domestic cat FIV transmission to other Felidae. In contrast to the sensitive felid A3s, many nonfelid A3s actively restricted wt FIV replication. To test whether VifFIV can protect also the distantly related human immunodeficiency virus type 1 (HIV-1), a chimeric HIV-1.VifFIV was constructed. This HIV-1.VifFIV was replication competent in nonpermissive feline cells expressing human CD4/CCR5 that did not support the replication of wt HIV-1. We conclude that the replication of HIV-1 in some feline cells is inhibited only by feline A3 restriction factors and the absence of the appropriate receptor or coreceptor. PMID:20444897

  16. Retroviral superinfection resistance

    NARCIS (Netherlands)

    Nethe, Micha; Berkhout, Ben; van der Kuyl, Antoinette C.

    2005-01-01

    The retroviral phenomenon of superinfection resistance (SIR) defines an interference mechanism that is established after primary infection, preventing the infected cell from being superinfected by a similar type of virus. This review describes our present understanding of the underlying mechanisms

  17. Retroviral DNA Integration

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    2016-01-01

    The integration of a DNA copy of the viral RNA genome into host chromatin is the defining step of retroviral replication. This enzymatic process is catalyzed by the virus-encoded integrase protein, which is conserved among retroviruses and LTR-retrotransposons. Retroviral integration proceeds via two integrase activities: 3′-processing of the viral DNA ends, followed by the strand transfer of the processed ends into host cell chromosomal DNA. Herein we review the molecular mechanism of retroviral DNA integration, with an emphasis on reaction chemistries and architectures of the nucleoprotein complexes involved. We additionally discuss the latest advances on anti-integrase drug development for the treatment of AIDS and the utility of integrating retroviral vectors in gene therapy applications. PMID:27198982

  18. Catalytic analysis of APOBEC3G involving real-time NMR spectroscopy reveals nucleic acid determinants for deamination.

    Directory of Open Access Journals (Sweden)

    Keisuke Kamba

    Full Text Available APOBEC3G (A3G is a single-stranded DNA-specific cytidine deaminase that preferentially converts cytidine to uridine at the third position of triplet cytosine (CCC hotspots. A3G restricts the infectivity of viruses, such as HIV-1, by targeting CCC hotspots scattered through minus DNA strands, reverse-transcribed from genomic RNA. Previously, we developed a real-time NMR method and elucidated the origin of the 3'→5' polarity of deamination of DNA by the C-terminal domain of A3G (CD2, which is a phenomenon by which a hotspot located closer to the 5'-end is deaminated more effectively than one less close to the 5'-end, through quantitative analysis involving nonspecific binding to and sliding along DNA. In the present study we applied the real-time NMR method to analyze the catalytic activity of CD2 toward DNA oligonucleotides containing a nucleotide analog at a single or multiple positions. Analyses revealed the importance of the sugar and base moieties throughout the consecutive 5 nucleotides, the CCC hotspot being positioned at the center. It was also shown that the sugar or base moieties of the nucleotides outside this 5 nucleotide recognition sequence are also relevant as to CD2's activity. Analyses involving DNA oligonucleotides having two CCC hotspots linked by a long sequence of either deoxyribonucleotides, ribonucleotides or abasic deoxyribonucleotides suggested that the phosphate backbone is required for CD2 to slide along the DNA strand and to exert the 3'→5' polarity. Examination of the effects of different salt concentrations on the 3'→5' polarity indicated that the higher the salt concentration, the less prominent the 3'→5' polarity. This is most likely the result of alleviation of sliding due to a decrease in the affinity of CD2 with the phosphate backbone at high salt concentrations. We also investigated the reactivity of substrates containing 5-methylcytidine (5mC or 5-hydroxymethylcytidine, and found that A3G exhibited low

  19. Molecular modeling of human APOBEC3G to predict the binding modes of the inhibitor compounds IMB26 and IMB35

    Directory of Open Access Journals (Sweden)

    Zhixin Zhang

    2013-07-01

    Full Text Available APOBEC3G(A3G is a host cytidine deaminase that incorporates into HIV-1 virions and efficiently inhibits viral replication. The virally encoded protein Vif binds to A3G and induces its degradation, thereby counteracting the antiviral activity of A3G. Vif-mediated A3G degradation clearly represents a potential target for anti-HIV drug development. Currently, there is an urgent need for understanding the three dimensional structure of full-length A3G. In this work, we use a homology modeling approach to propose a structure for A3G based on the crystal structure of APOBEC2 (APO2 and the catalytic domain structure of A3G. Two compounds, IMB26 and IMB35, which have been shown to bind to A3G and block degradation by Vif, were docked into the A3G model and the binding modes were generated for further analysis. The results may be used to design or optimize molecules targeting Vif–A3G interaction, and lead to the development of novel anti-HIV drugs.

  20. Identification of a Cullin5-ElonginB-ElonginC E3 complex in degradation of feline immunodeficiency virus Vif-mediated feline APOBEC3 proteins.

    Science.gov (United States)

    Wang, Jiawen; Zhang, Wenyan; Lv, Mingyu; Zuo, Tao; Kong, Wei; Yu, Xianghui

    2011-12-01

    Various feline APOBEC3 (fA3) proteins exhibit broad antiviral activities against a wide range of viruses, such as feline immunodeficiency virus (FIV), feline foamy virus (FFV), and feline leukemia virus (FeLV), as well as those of other species. This activity can be counteracted by the FIV Vif protein, but the mechanism by which FIV Vif suppresses fA3s is unknown. In the present study, we demonstrated that FIV Vif could act via a proteasome-dependent pathway to overcome fA3s. FIV Vif interacted with feline cellular proteins Cullin5 (Cul5), ElonginB, and ElonginC to form an E3 complex to induce degradation of fA3s. Both the dominant-negative Cul5 mutant and a C-terminal hydrophilic replacement ElonginC mutant potently disrupted the FIV Vif activity against fA3s. Furthermore, we identified a BC-box motif in FIV Vif that was essential for the recruitment of E3 ubiquitin ligase and also required for FIV Vif-mediated degradation of fA3s. Moreover, despite the lack of either a Cul5-box or a HCCH zinc-binding motif, FIV Vif specifically selected Cul5. Therefore, FIV Vif may interact with Cul5 via a novel mechanism. These finding imply that SOCS proteins may possess distinct mechanisms to bind Cul5 during formation of the Elongin-Cullin-SOCS box complex.

  1. Our retroviral heritage.

    Science.gov (United States)

    Patience, C; Wilkinson, D A; Weiss, R A

    1997-03-01

    Darwin could not have foretold that we are descended from viruses as well as from apes. While there is clear evidence that viral diseases, such as polio and rabies, affected ancient civilizations, viruses were not defined until the early years of this century, shortly after the rediscovery of mendelian genetics. That retroviral genomes can oscillate between infectious and genetic modes of transmission seemed preposterous before the discovery of reverse transcription in 1970. Those of us who had earlier provided mendelian evidence for germ-line transmission of retroviruses were subject of friendly ridicule. Today, the shunting of genetic elements between chromosomes and RNA, and the generation of processed pseudogenes, seems commonplace. It is timely, however, to revisit the topic of human endogenous retroviruses-the subject of this article.

  2. Retroviral integration: Site matters

    Science.gov (United States)

    Demeulemeester, Jonas; De Rijck, Jan

    2015-01-01

    Here, we review genomic target site selection during retroviral integration as a multistep process in which specific biases are introduced at each level. The first asymmetries are introduced when the virus takes a specific route into the nucleus. Next, by co‐opting distinct host cofactors, the integration machinery is guided to particular chromatin contexts. As the viral integrase captures a local target nucleosome, specific contacts introduce fine‐grained biases in the integration site distribution. In vivo, the established population of proviruses is subject to both positive and negative selection, thereby continuously reshaping the integration site distribution. By affecting stochastic proviral expression as well as the mutagenic potential of the virus, integration site choice may be an inherent part of the evolutionary strategies used by different retroviruses to maximise reproductive success. PMID:26293289

  3. Identifying Cancer Driver Genes Using Replication-Incompetent Retroviral Vectors

    Directory of Open Access Journals (Sweden)

    Victor M. Bii

    2016-10-01

    Full Text Available Identifying novel genes that drive tumor metastasis and drug resistance has significant potential to improve patient outcomes. High-throughput sequencing approaches have identified cancer genes, but distinguishing driver genes from passengers remains challenging. Insertional mutagenesis screens using replication-incompetent retroviral vectors have emerged as a powerful tool to identify cancer genes. Unlike replicating retroviruses and transposons, replication-incompetent retroviral vectors lack additional mutagenesis events that can complicate the identification of driver mutations from passenger mutations. They can also be used for almost any human cancer due to the broad tropism of the vectors. Replication-incompetent retroviral vectors have the ability to dysregulate nearby cancer genes via several mechanisms including enhancer-mediated activation of gene promoters. The integrated provirus acts as a unique molecular tag for nearby candidate driver genes which can be rapidly identified using well established methods that utilize next generation sequencing and bioinformatics programs. Recently, retroviral vector screens have been used to efficiently identify candidate driver genes in prostate, breast, liver and pancreatic cancers. Validated driver genes can be potential therapeutic targets and biomarkers. In this review, we describe the emergence of retroviral insertional mutagenesis screens using replication-incompetent retroviral vectors as a novel tool to identify cancer driver genes in different cancer types.

  4. Vpr14-88-Apobec3G fusion protein is efficiently incorporated into Vif-positive HIV-1 particles and inhibits viral infection.

    Science.gov (United States)

    Ao, Zhujun; Yu, Zhe; Wang, Lina; Zheng, Yingfeng; Yao, Xiaojian

    2008-04-16

    APOBEC3G (A3G), a deoxycytidine deaminase, is a potent host antiviral factor that can restrict HIV-1 infection. During Vif-negative HIV-1 replication, A3G is incorporated into HIV-1 particles, induces mutations in reverse transcribed viral DNA and inhibits reverse transcription. However, HIV-1 Vif counteracts A3G's activities by inducing its degradation and by blocking its incorporation into HIV-1 particles. Thus, it is interesting to elucidate a mechanism that would allow A3G to escape the effects of Vif in order to rescue its potent antiviral activity and to provide a possible novel therapeutic strategy for treating HIV-1 infection. In this study, we generated an R88-A3G fusion protein by fusing A3G to a virion-targeting polypeptide (R14-88) derived from HIV-1 Vpr protein and compared its antiviral effects relative to those of HA-tagged native A3G (HA-A3G). Our study showed that transient expression of the R88-A3G fusion protein in both Vif(-) and Vif(+) HIV-1 producing cells drastically inhibited viral infection in HeLa-CD4-CCR5-cells, CD4(+) C8166 T cells and human primary PBMCs. Moreover, we established CD4(+) C8166 T cell lines that stably express either R88-A3G or HA-A3G by transduction with VSV-G-pseudotyped lentiviral vector that harbor expression cassettes for R88-A3G or HA-A3G, respectively, and tested their susceptibility to Vif(+) HIV-1 infection. Our results clearly reveal that expression of R88-A3G in transduced CD4(+) C8166 cells significantly blocked Vif(+) HIV-1 infection. In an attempt to understand the mechanism underlying the antiviral activity of R88-A3G, we demonstrated that R88-A3G was efficiently incorporated into viral particles in the presence of Vif. Moreover, PCR analysis revealed that R88-A3G significantly inhibited viral cDNA synthesis during the early stage of Vif(+) virus infection. Our results clearly indicate that R88 delivers A3G into Vif(+) HIV-1 particles and inhibits infectivity and spread of the virions among CD4(+) T cells

  5. Retroviral superinfection resistance

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    van der Kuyl Antoinette C

    2005-08-01

    Full Text Available Abstract The retroviral phenomenon of superinfection resistance (SIR defines an interference mechanism that is established after primary infection, preventing the infected cell from being superinfected by a similar type of virus. This review describes our present understanding of the underlying mechanisms of SIR established by three characteristic retroviruses: Murine Leukaemia Virus (MuLV, Foamy Virus (FV, and Human Immunodeficiency Virus (HIV. In addition, SIR is discussed with respect to HIV superinfection of humans. MuLV resistant mice exhibit two genetic resistance traits related to SIR. The cellular Fv4 gene expresses an Env related protein that establishes resistance against MuLV infection. Another mouse gene (Fv1 mediates MuLV resistance by expression of a sequence that is distantly related to Gag and that blocks the viral infection after the reverse transcription step. FVs induce two distinct mechanisms of superinfection resistance. First, expression of the Env protein results in SIR, probably by occupancy of the cellular receptors for FV entry. Second, an increase in the concentration of the viral Bet (Between-env-and-LTR-1-and-2 protein reduces proviral FV gene expression by inhibition of the transcriptional activator protein Tas (Transactivator of spumaviruses. In contrast to SIR in FV and MuLV infection, the underlying mechanism of SIR in HIV-infected cells is poorly understood. CD4 receptor down-modulation, a major characteristic of HIV-infected cells, has been proposed to be the main mechanism of SIR against HIV, but data have been contradictory. Several recent studies report the occurrence of HIV superinfection in humans; an event associated with the generation of recombinant HIV strains and possibly with increased disease progression. The role of SIR in protecting patients from HIV superinfection has not been studied so far. The phenomenon of SIR may also be important in the protection of primates that are vaccinated with live

  6. Transcriptional Silencing of Retroviral Vectors

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Duch, M.; Pedersen, F.S.

    1996-01-01

    Although retroviral vector systems have been found to efficiently transduce a variety of cell types in vitro, the use of vectors based on murine leukemia virus in preclinical models of somatic gene therapy has led to the identification of transcriptional silencing in vivo as an important problem....... Extinction of long-term vector expression has been observed after implantation of transduced hematopoietic cells as well as fibroblasts, myoblasts and hepatocytes. Here we review the influence of vector structure, integration site and cell type on transcriptional silencing. While down-regulation of proviral...... transcription is known from a number of cellular and animal models, major insight has been gained from studies in the germ line and embryonal cells of the mouse. Key elements for the transfer and expression of retroviral vectors, such as the viral transcriptional enhancer and the binding site for the t...

  7. Non-Retroviral Fossils in Vertebrate Genomes

    OpenAIRE

    Masayuki Horie; Keizo Tomonaga

    2011-01-01

    Although no physical fossils of viruses have been found, retroviruses are known to leave their molecular fossils in the genomes of their hosts, the so-called endogenous retroviral elements. These have provided us with important information about retroviruses in the past and their co-evolution with their hosts. On the other hand, because non-retroviral viruses were considered not to leave such fossils, even the existence of prehistoric non-retroviral viruses has been enigmatic. Recently, we di...

  8. Enhanced gene expression from retroviral vectors

    Directory of Open Access Journals (Sweden)

    Micklem David R

    2008-02-01

    Full Text Available Abstract Background Retroviruses are widely used to transfer genes to mammalian cells efficiently and stably. However, genetic elements required for high-level gene expression are incompatible with standard systems. The retroviral RNA genome is produced by cellular transcription and post-transcriptional processing within packaging cells: Introns present in the retroviral genomic transcript are removed by splicing, while polyadenylation signals lead to the production of ineffective truncated genomes. Furthermore strong enhancer/promoters within the retroviral payload lead to detrimental competition with the retroviral enhancer/promoter. Results By exploiting a new method of producing the retroviral genome in vitro it is possible to produce infectious retroviral particles carrying a high-level expression cassette that completely prohibits production of infectious retroviral particles by conventional methods. We produced an expression cassette comprising a strong enhancer/promoter, an optimised intron, the GFP open reading frame and a strong polyadenylation signal. This cassette was cloned into both a conventional MMLV retroviral vector and a vector designed to allow in vitro transcription of the retroviral genome by T7 RNA polymerase. When the conventional retroviral vector was transfected into packaging cells, the expression cassette drove strong GFP expression, but no infectious retrovirus was produced. Introduction of the in vitro produced uncapped retroviral genomic transcript into the packaging cells did not lead to any detectable GFP expression. However, infectious retrovirus was easily recovered, and when used to infect target primary human cells led to very high GFP expression – up to 3.5 times greater than conventional retroviral LTR-driven expression. Conclusion Retroviral vectors carrying an optimized high-level expression cassette do not produce infectious virions when introduced into packaging cells by transfection of DNA

  9. Membrane interaction of retroviral Gag proteins

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    Robert Alfred Dick

    2014-04-01

    Full Text Available Assembly of an infectious retroviral particle relies on multimerization of the Gag polyprotein at the inner leaflet of the plasma membrane. The three domains of Gag common to all retroviruses-- MA, CA, and NC-- provide the signals for membrane binding, assembly, and viral RNA packaging, respectively. These signals do not function independently of one another. For example, Gag multimerization enhances membrane binding and is more efficient when NC is interacting with RNA. MA binding to the plasma membrane is governed by several principles, including electrostatics, recognition of specific lipid head groups, hydrophobic interactions, and membrane order. HIV-1 uses many of these principles while Rous sarcoma virus (RSV appears to use fewer. This review describes the principles that govern Gag interactions with membranes, focusing on RSV and HIV-1 Gag. The review also defines lipid and membrane behavior, and discusses the complexities in determining how lipid and membrane behavior impact Gag membrane binding.

  10. Patterns of evolution of host proteins involved in retroviral pathogenesis

    Directory of Open Access Journals (Sweden)

    Kaessmann Henrik

    2006-02-01

    Full Text Available Abstract Background Evolutionary analysis may serve as a useful approach to identify and characterize host defense and viral proteins involved in genetic conflicts. We analyzed patterns of coding sequence evolution of genes with known (TRIM5α and APOBEC3G or suspected (TRIM19/PML roles in virus restriction, or in viral pathogenesis (PPIA, encoding Cyclophilin A, in the same set of human and non-human primate species. Results and conclusion This analysis revealed previously unidentified clusters of positively selected sites in APOBEC3G and TRIM5α that may delineate new virus-interaction domains. In contrast, our evolutionary analyses suggest that PPIA is not under diversifying selection in primates, consistent with the interaction of Cyclophilin A being limited to the HIV-1M/SIVcpz lineage. The strong sequence conservation of the TRIM19/PML sequences among primates suggests that this gene does not play a role in antiretroviral defense.

  11. HIV-1 genome is often defective in PBMCs and rectal tissues after long-term HAART as a result of APOBEC3 editing and correlates with the size of reservoirs.

    Science.gov (United States)

    Fourati, Slim; Lambert-Niclot, Sidonie; Soulie, Cathia; Malet, Isabelle; Valantin, Marc Antoine; Descours, Benjamin; Ait-Arkoub, Zaina; Mory, Benoit; Carcelain, Guislaine; Katlama, Christine; Calvez, Vincent; Marcelin, Anne Geneviève

    2012-10-01

    Precise characterization of viruses present in reservoirs in long-term pretreated patients will be a major issue to consider in the context of viral eradication. We assessed the frequency of defective viruses present in cellular reservoirs. Peripheral blood mononuclear cells (PBMCs) and rectal biopsy samples were compared between five patients on successful long-term highly active antiretroviral therapy (HAART) (>7 years without blips) and five untreated patients. Molecular cloning and sequencing of the reverse transcriptase region were used to detect the presence of and quantify in-frame stop codons in HIV quasi-species. The relationship between the size of the reservoir and the frequency of defective genomes was assessed. Defective genomes were systematically detected in all patients on long-term HAART in both compartments (PBMCs and rectal tissues), with a higher level of defective genomes per sample compared with PBMCs of untreated patients. A high level of defective genomes was correlated with a small size of HIV proviral DNA. Regarding the nucleotide context, guanine (G) to adenine (A) substitution at tryptophan positions was responsible for the appearance of 89% of all in-frame stop codons in the context of G-to-A hypermutation, likely reflecting APOBEC3 footprints on the viral genome. We propose a scenario whereby defective genomes accumulate during HAART treatment, eventually reaching a viral extinction threshold. In the context of viral eradication, measurement of the relative amounts of defective and non-defective viruses (by molecular cloning and ultradeep sequencing) should be used as a new criterion for eradicating HIV.

  12. New windows into retroviral RNA structures.

    Science.gov (United States)

    Jayaraman, Dhivya; Kenyon, Julia Claire

    2018-01-25

    The multiple roles of both viral and cellular RNAs have become increasingly apparent in recent years, and techniques to model them have become significantly more powerful, enabling faster and more accurate visualization of RNA structures. Techniques such as SHAPE (selective 2'OH acylation analysed by primer extension) have revolutionized the field, and have been used to examine RNAs belonging to many and diverse retroviruses. Secondary structure probing reagents such as these have been aided by the development of faster methods of analysis either via capillary or next-generation sequencing, allowing the analysis of entire genomes, and of retroviral RNA structures within virions. Techniques to model the three-dimensional structures of these large RNAs have also recently developed. The flexibility of retroviral RNAs, both structural and functional, is clear from the results of these new experimental techniques. Retroviral RNA structures and structural changes control many stages of the lifecycle, and both the RNA structures themselves and their interactions with ligands are potential new drug targets. In addition, our growing understanding of retroviral RNA structures is aiding our knowledge of cellular RNA form and function.

  13. The Host RNAs in Retroviral Particles

    Directory of Open Access Journals (Sweden)

    Alice Telesnitsky

    2016-08-01

    Full Text Available As they assemble, retroviruses encapsidate both their genomic RNAs and several types of host RNA. Whereas limited amounts of messenger RNA (mRNA are detectable within virion populations, the predominant classes of encapsidated host RNAs do not encode proteins, but instead include endogenous retroelements and several classes of non-coding RNA (ncRNA, some of which are packaged in significant molar excess to the viral genome. Surprisingly, although the most abundant host RNAs in retroviruses are also abundant in cells, unusual forms of these RNAs are packaged preferentially, suggesting that these RNAs are recruited early in their biogenesis: before associating with their cognate protein partners, and/or from transient or rare RNA populations. These RNAs’ packaging determinants differ from the viral genome’s, and several of the abundantly packaged host ncRNAs serve cells as the scaffolds of ribonucleoprotein particles. Because virion assembly is equally efficient whether or not genomic RNA is available, yet RNA appears critical to the structural integrity of retroviral particles, it seems possible that the selectively encapsidated host ncRNAs might play roles in assembly. Indeed, some host ncRNAs appear to act during replication, as some transfer RNA (tRNA species may contribute to nuclear import of human immunodeficiency virus 1 (HIV-1 reverse transcription complexes, and other tRNA interactions with the viral Gag protein aid correct trafficking to plasma membrane assembly sites. However, despite high conservation of packaging for certain host RNAs, replication roles for most of these selectively encapsidated RNAs—if any—have remained elusive.

  14. Application of Live-Cell RNA Imaging Techniques to the Study of Retroviral RNA Trafficking

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    Darrin V. Bann

    2012-06-01

    Full Text Available Retroviruses produce full-length RNA that serves both as a genomic RNA (gRNA, which is encapsidated into virus particles, and as an mRNA, which directs the synthesis of viral structural proteins. However, we are only beginning to understand the cellular and viral factors that influence trafficking of retroviral RNA and the selection of the RNA for encapsidation or translation. Live cell imaging studies of retroviral RNA trafficking have provided important insight into many aspects of the retrovirus life cycle including transcription dynamics, nuclear export of viral RNA, translational regulation, membrane targeting, and condensation of the gRNA during virion assembly. Here, we review cutting-edge techniques to visualize single RNA molecules in live cells and discuss the application of these systems to studying retroviral RNA trafficking.

  15. Two genetic determinants acquired late in Mus evolution regulate the inclusion of exon 5, which alters mouse APOBEC3 translation efficiency.

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    Jun Li

    2012-01-01

    Full Text Available Mouse apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like editing complex 3 (mA3, an intracellular antiviral factor, has 2 allelic variations that are linked with different susceptibilities to beta- and gammaretrovirus infections among various mouse strains. In virus-resistant C57BL/6 (B6 mice, mA3 transcripts are more abundant than those in susceptible BALB/c mice both in the spleen and bone marrow. These strains of mice also express mA3 transcripts with different splicing patterns: B6 mice preferentially express exon 5-deficient (Δ5 mA3 mRNA, while BALB/c mice produce exon 5-containing full-length mA3 mRNA as the major transcript. Although the protein product of the Δ5 mRNA exerts stronger antiretroviral activities than the full-length protein, how exon 5 affects mA3 antiviral activity, as well as the genetic mechanisms regulating exon 5 inclusion into the mA3 transcripts, remains largely uncharacterized. Here we show that mA3 exon 5 is indeed a functional element that influences protein synthesis at a post-transcriptional level. We further employed in vitro splicing assays using genomic DNA clones to identify two critical polymorphisms affecting the inclusion of exon 5 into mA3 transcripts: the number of TCCT repeats upstream of exon 5 and the single nucleotide polymorphism within exon 5 located 12 bases upstream of the exon 5/intron 5 boundary. Distribution of the above polymorphisms among different Mus species indicates that the inclusion of exon 5 into mA3 mRNA is a relatively recent event in the evolution of mice. The widespread geographic distribution of this exon 5-including genetic variant suggests that in some Mus populations the cost of maintaining an effective but mutagenic enzyme may outweigh its antiviral function.

  16. The design of artificial retroviral restriction factors

    International Nuclear Information System (INIS)

    Yap, Melvyn W.; Mortuza, Gulnahar B.; Taylor, Ian A.; Stoye, Jonathan P.

    2007-01-01

    In addition to the ability to bind the retroviral capsid protein, the retroviral restriction factors Fv1, Trim5α and Trim5-CypA share the common property of containing sequences that promote self-association. Otherwise Fv1 and Trim5α appear unrelated. Mutational analyses showed that restriction was invariably lost when changes designed to disrupt the sequences responsible for multimerization were introduced. A novel restriction protein could be obtained by substituting sequences from the self-associating domain of Fv1 for the Trim5 sequences in Trim5-CypA. Similarly, a fusion protein containing cyclophilin A joined to arfaptin2, a protein known to form extended dimers, was also shown to restrict HIV-1. Hence, multimerization of a capsid-binding domain could be the common minimum design feature for capsid-dependent retroviral restriction factors. However, not all domains that promote multimerization can substitute for the N-terminal domains of Fv1 and Trim5α. Moreover, only CypA can provide a capsid-binding site with different N-terminal domains. It is suggested that the spatial relationship between the multiple target binding sites may be important for restriction

  17. Improved Coinfection with Amphotropic Pseudotyped Retroviral Vectors

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    Yuehong Wu

    2009-01-01

    Full Text Available Amphotropic pseudotyped retroviral vectors have typically been used to infect target cells without prior concentration. Although this can yield high rates of infection, higher rates may be needed where highly efficient coinfection of two or more vectors is needed. In this investigation we used amphotropic retroviral vectors produced by the Plat-A cell line and studied coinfection rates using green and red fluorescent proteins (EGFP and dsRed2. Target cells were primary human fibroblasts (PHF and 3T3 cells. Unconcentrated vector preparations produced a coinfection rate of ∼4% (defined as cells that are both red and green as a percentage of all cells infected. Optimized spinoculation, comprising centrifugation at 1200 g for 2 hours at 15∘C, increased the coinfection rate to ∼10%. Concentration by centrifugation at 10,000 g or by flocculation using Polybrene increased the coinfection rate to ∼25%. Combining the two processes, concentration by Polybrene flocculation and optimized spinoculation, increased the coinfection rate to 35% (3T3 or >50% (PHF. Improved coinfection should be valuable in protocols that require high transduction by combinations of two or more retroviral vectors.

  18. Identification of endogenous retroviral reading frames in the human genome

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    Wiuf Carsten

    2004-10-01

    Full Text Available Abstract Background Human endogenous retroviruses (HERVs comprise a large class of repetitive retroelements. Most HERVs are ancient and invaded our genome at least 25 million years ago, except for the evolutionary young HERV-K group. The far majority of the encoded genes are degenerate due to mutational decay and only a few non-HERV-K loci are known to retain intact reading frames. Additional intact HERV genes may exist, since retroviral reading frames have not been systematically annotated on a genome-wide scale. Results By clustering of hits from multiple BLAST searches using known retroviral sequences we have mapped 1.1% of the human genome as retrovirus related. The coding potential of all identified HERV regions were analyzed by annotating viral open reading frames (vORFs and we report 7836 loci as verified by protein homology criteria. Among 59 intact or almost-intact viral polyproteins scattered around the human genome we have found 29 envelope genes including two novel gammaretroviral types. One encodes a protein similar to a recently discovered zebrafish retrovirus (ZFERV while another shows partial, C-terminal, homology to Syncytin (HERV-W/FRD. Conclusions This compilation of HERV sequences and their coding potential provide a useful tool for pursuing functional analysis such as RNA expression profiling and effects of viral proteins, which may, in turn, reveal a role for HERVs in human health and disease. All data are publicly available through a database at http://www.retrosearch.dk.

  19. Amplification and chromosomal dispersion of human endogenous retroviral sequences

    Energy Technology Data Exchange (ETDEWEB)

    Steele, P.E.; Martin, M.A.; Rabson, A.B.; Bryan, T.; O' Brien, S.J.

    1986-09-01

    Endogenous retroviral sequences have undergone amplification events involving both viral and flanking cellular sequences. The authors cloned members of an amplified family of full-length endogenous retroviral sequences. Genomic blotting, employing a flanking cellular DNA probe derived from a member of this family, revealed a similar array of reactive bands in both humans and chimpanzees, indicating that an amplification event involving retroviral and associated cellular DNA sequences occurred before the evolutionary separation of these two primates. Southern analyses of restricted somatic cell hybrid DNA preparations suggested that endogenous retroviral segments are widely dispersed in the human genome and that amplification and dispersion events may be linked.

  20. Amplification and chromosomal dispersion of human endogenous retroviral sequences

    International Nuclear Information System (INIS)

    Steele, P.E.; Martin, M.A.; Rabson, A.B.; Bryan, T.; O'Brien, S.J.

    1986-01-01

    Endogenous retroviral sequences have undergone amplification events involving both viral and flanking cellular sequences. The authors cloned members of an amplified family of full-length endogenous retroviral sequences. Genomic blotting, employing a flanking cellular DNA probe derived from a member of this family, revealed a similar array of reactive bands in both humans and chimpanzees, indicating that an amplification event involving retroviral and associated cellular DNA sequences occurred before the evolutionary separation of these two primates. Southern analyses of restricted somatic cell hybrid DNA preparations suggested that endogenous retroviral segments are widely dispersed in the human genome and that amplification and dispersion events may be linked

  1. High-resolution structure of a retroviral protease folded as a monomer

    International Nuclear Information System (INIS)

    Gilski, Miroslaw; Kazmierczyk, Maciej; Krzywda, Szymon; Zábranská, Helena; Cooper, Seth; Popović, Zoran; Khatib, Firas; DiMaio, Frank; Thompson, James; Baker, David; Pichová, Iva; Jaskolski, Mariusz

    2011-01-01

    The crystal structure of Mason–Pfizer monkey virus protease folded as a monomer has been solved by molecular replacement using a model generated by players of the online game Foldit. The structure shows at high resolution the details of a retroviral protease folded as a monomer which can guide rational design of protease dimerization inhibitors as retroviral drugs. Mason–Pfizer monkey virus (M-PMV), a D-type retrovirus assembling in the cytoplasm, causes simian acquired immunodeficiency syndrome (SAIDS) in rhesus monkeys. Its pepsin-like aspartic protease (retropepsin) is an integral part of the expressed retroviral polyproteins. As in all retroviral life cycles, release and dimerization of the protease (PR) is strictly required for polyprotein processing and virion maturation. Biophysical and NMR studies have indicated that in the absence of substrates or inhibitors M-PMV PR should fold into a stable monomer, but the crystal structure of this protein could not be solved by molecular replacement despite countless attempts. Ultimately, a solution was obtained in mr-rosetta using a model constructed by players of the online protein-folding game Foldit. The structure indeed shows a monomeric protein, with the N- and C-termini completely disordered. On the other hand, the flap loop, which normally gates access to the active site of homodimeric retropepsins, is clearly traceable in the electron density. The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins. The overall fold of the protein follows the retropepsin canon, but the C α deviations are large and the active-site ‘DTG’ loop (here NTG) deviates up to 2.7 Å from the standard conformation. This structure of a monomeric retropepsin determined at high resolution (1.6 Å) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral infections

  2. Structural dynamics of retroviral genome and the packaging.

    Science.gov (United States)

    Miyazaki, Yasuyuki; Miyake, Ariko; Nomaguchi, Masako; Adachi, Akio

    2011-01-01

    Retroviruses can cause diseases such as AIDS, leukemia, and tumors, but are also used as vectors for human gene therapy. All retroviruses, except foamy viruses, package two copies of unspliced genomic RNA into their progeny viruses. Understanding the molecular mechanisms of retroviral genome packaging will aid the design of new anti-retroviral drugs targeting the packaging process and improve the efficacy of retroviral vectors. Retroviral genomes have to be specifically recognized by the cognate nucleocapsid domain of the Gag polyprotein from among an excess of cellular and spliced viral mRNA. Extensive virological and structural studies have revealed how retroviral genomic RNA is selectively packaged into the viral particles. The genomic area responsible for the packaging is generally located in the 5' untranslated region (5' UTR), and contains dimerization site(s). Recent studies have shown that retroviral genome packaging is modulated by structural changes of RNA at the 5' UTR accompanied by the dimerization. In this review, we focus on three representative retroviruses, Moloney murine leukemia virus, human immunodeficiency virus type 1 and 2, and describe the molecular mechanism of retroviral genome packaging.

  3. Structural dynamics of retroviral genome and the packaging

    Directory of Open Access Journals (Sweden)

    Yasuyuki eMiyazaki

    2011-12-01

    Full Text Available Retroviruses can cause diseases such as AIDS, leukemia and tumors, but are also used as vectors for human gene therapy. All retroviruses, except foamy viruses, package two copies of unspliced genomic RNA into their progeny viruses. Understanding the molecular mechanisms of retroviral genome packaging will aid the design of new anti-retroviral drugs targeting the packaging process and improve the efficacy of retroviral vectors. Retroviral genomes have to be specifically recognized by the cognate nucleocapsid (NC domain of the Gag polyprotein from among an excess of cellular and spliced viral mRNA. Extensive virological and structural studies have revealed how retroviral genomic RNA is selectively packaged into the viral particles. The genomic area responsible for the packaging is generally located in the 5’ untranslated region (5’ UTR, and contains dimerization site(s. Recent studies have shown that retroviral genome packaging is modulated by structural changes of RNA at the 5’ UTR accompanied by the dimerization. In this review, we focus on three representative retroviruses, Moloney murine leukemia virus (MoMLV, human immunodeficiency virus type 1 (HIV-1 and 2 (HIV-2, and describe the molecular mechanism of retroviral genome packaging.

  4. Stavudine, an anti‑retroviral drug induces reactive astrocytes in ...

    African Journals Online (AJOL)

    Stavudine, an anti‑retroviral drug induces reactive astrocytes in motor cortex of albino mice. Agnes A. Nwakanma, Theresa B. Ekanem, Moses B. Ekong, Mokutima A. Eluwa, Eme E. Osim, Terkula Kpela ...

  5. Retroviral RNA Dimerization: From Structure to Functions

    Directory of Open Access Journals (Sweden)

    Noé Dubois

    2018-03-01

    Full Text Available The genome of the retroviruses is a dimer composed by two homologous copies of genomic RNA (gRNA molecules of positive polarity. The dimerization process allows two gRNA molecules to be non-covalently linked together through intermolecular base-pairing. This step is critical for the viral life cycle and is highly conserved among retroviruses with the exception of spumaretroviruses. Furthermore, packaging of two gRNA copies into viral particles presents an important evolutionary advantage for immune system evasion and drug resistance. Recent studies reported RNA switches models regulating not only gRNA dimerization, but also translation and packaging, and a spatio-temporal characterization of viral gRNA dimerization within cells are now at hand. This review summarizes our current understanding on the structural features of the dimerization signals for a variety of retroviruses (HIVs, MLV, RSV, BLV, MMTV, MPMV…, the mechanisms of RNA dimer formation and functional implications in the retroviral cycle.

  6. Retroviral insertional mutagenesis identifies genes that collaborate with NUP98-HOXD13 during leukemic transformation.

    Science.gov (United States)

    Slape, Christopher; Hartung, Helge; Lin, Ying-Wei; Bies, Juraj; Wolff, Linda; Aplan, Peter D

    2007-06-01

    The t(2;11)(q31;p15) chromosomal translocation results in a fusion between the NUP98 and HOXD13 genes and has been observed in patients with myelodysplastic syndrome (MDS) or acute myelogenous leukemia. We previously showed that expression of the NUP98-HOXD13 (NHD13) fusion gene in transgenic mice results in an invariably fatal MDS; approximately one third of mice die due to complications of severe pancytopenia, and about two thirds progress to a fatal acute leukemia. In the present study, we used retroviral insertional mutagenesis to identify genes that might collaborate with NHD13 as the MDS transformed to an acute leukemia. Newborn NHD13 transgenic mice and littermate controls were infected with the MOL4070LTR retrovirus. The onset of leukemia was accelerated, suggesting a synergistic effect between the NHD13 transgene and the genes neighboring retroviral insertion events. We identified numerous common insertion sites located near protein-coding genes and confirmed dysregulation of a subset of these by expression analyses. Among these genes were Meis1, a known collaborator of HOX and NUP98-HOX fusion genes, and Mn1, a transcriptional coactivator involved in human leukemia through fusion with the TEL gene. Other putative collaborators included Gata2, Erg, and Epor. Of note, we identified a common insertion site that was >100 kb from the nearest coding gene, but within 20 kb of the miR29a/miR29b1 microRNA locus. Both of these miRNA were up-regulated, demonstrating that retroviral insertional mutagenesis can target miRNA loci as well as protein-coding loci. Our data provide new insights into NHD13-mediated leukemogenesis as well as retroviral insertional mutagenesis mechanisms.

  7. A look inside HIV resistance through retroviral protease interaction maps.

    Directory of Open Access Journals (Sweden)

    Aleksejs Kontijevskis

    2007-03-01

    Full Text Available Retroviruses affect a large number of species, from fish and birds to mammals and humans, with global socioeconomic negative impacts. Here the authors report and experimentally validate a novel approach for the analysis of the molecular networks that are involved in the recognition of substrates by retroviral proteases. Using multivariate analysis of the sequence-based physiochemical descriptions of 61 retroviral proteases comprising wild-type proteases, natural mutants, and drug-resistant forms of proteases from nine different viral species in relation to their ability to cleave 299 substrates, the authors mapped the physicochemical properties and cross-dependencies of the amino acids of the proteases and their substrates, which revealed a complex molecular interaction network of substrate recognition and cleavage. The approach allowed a detailed analysis of the molecular-chemical mechanisms involved in substrate cleavage by retroviral proteases.

  8. Retroviral integration profiles: their determinants and implications for gene therapy

    Directory of Open Access Journals (Sweden)

    Kwang-il Lim

    2012-04-01

    Full Text Available Retroviruses have often been used for gene therapy because oftheir capacity for the long-term expression of transgenes via stableintegration into the host genome. However, retroviral integrationcan also result in the transformation of normal cells into cancercells, as demonstrated by the incidence of leukemia in a recentretroviral gene therapy trial in Europe. This unfortunate outcomehas led to the rapid initiation of studies examining variousbiological and pathological aspects of retroviral integration. Thisreview summarizes recent findings from these studies, includingthe global integration patterns of various types of retroviruses,viral and cellular determinants of integration, implications ofintegration for gene therapy and retrovirus-mediated infectiousdiseases, and strategies to shift integration to safe host genomicloci. A more comprehensive and mechanistic understanding ofretroviral integration processes will eventually make it possible togenerate safer retroviral vector platforms in the near future. [BMBreports 2012; 45(4: 207-212

  9. Adherence to anti-retroviral drugs in pregnant and lactating HIV ...

    African Journals Online (AJOL)

    Background: Anti-retroviral drugs reduce morbidity and mortality due to HIV and prevent transmission from mother to child. But compliance on anti-retroviral treatment is an essential element for the success of therapeutic goals. Objective: To assess the level of compliance of anti-retroviral treatment in pregnant and lactating ...

  10. From retroviral vector production to gene transfer: spontaneous inactivation is caused by loss of reverse transcription capacity.

    Science.gov (United States)

    Carmo, M; Panet, A; Carrondo, M J T; Alves, P M; Cruz, P E

    2008-04-01

    The loss of gene transfer capacity in retroviral vectors constitutes a major disadvantage in the development of retroviral vectors for gene therapy applications. In the present work the loss of a vector's capacity to perform reverse transcription was studied as a possible explanation for the low stability of retroviral vectors from the production stage to the target cell gene transfer event. Inactivation studies were performed with murine leukemia virus vectors at 37 degrees C and several residual activities were tested, including viral infectivity, reverse transcription capacity, reverse transcriptase (RT) activities and viral RNA stability. The results indicate a high correlation between loss of infectivity and the capacity of the virus to perform the initial steps of reverse transcription. To further understand the thermosensitivity of the reverse transcription process, the two enzyme activities of RT were investigated. The results indicate that, although the inactivation rate of the DNA polymerase is faster than that of RNase H, the decline of these two enzyme activities is significantly slower than that of reverse transcription. Also, viral RNA stability is not implicated in the loss of the virus capacity to perform reverse transcription as the rate of viral RNA degradation was very slow. Furthermore, it was observed that the amount of viral RNA that entered the cells decreased slowly due to viral inactivation at 37 degrees C. The reverse transcription process is thermolabile and this sensitivity determines the rate of retroviral inactivation. Strategies targeting stabilization of the reverse transcription complex should be pursued to improve the applicability of retroviral vectors in gene therapy studies. (c) 2008 John Wiley & Sons, Ltd.

  11. Psychological distress and adherence to highly active anti-retroviral ...

    African Journals Online (AJOL)

    Background: Mental health related risk factors for non-adherence to highly active anti-retroviral therapy (HAART) have not been investigated in Uganda and yet adherence is critical to the success of the current scale up in the provision of HAART to HIV positive individuals in rural areas of Uganda. Objective: To determine ...

  12. CD4 + CELL RESPONSE TO ANTI-RETROVIRAL THERAPY (ARTs ...

    African Journals Online (AJOL)

    East African Medical Journal Vol. 90 No. 12 (Supplement) December 2013. CD4 + CELL RESPONSE TO ANTI-RETROVIRAL THERAPY (ARTs) IN ROUTINE CLINICAL CARE OVER ONE YEAR. PERIOD IN A COHORT OF HAART NAIVE, HIV POSITIVE KENYAN PATIENTS. C. F. Otieno, MBChB, MMed (Int. Med), ...

  13. Patients' perceptions of a rural decentralised anti-retroviral therapy ...

    African Journals Online (AJOL)

    Background: Geographical and financial barriers hamper accessibility to HIV services for rural communities. The government has introduced the nurse initiated management of anti-retroviral therapy at primary health care level, in an effort to improve patient access and reduce patient loads on facilities further up the system.

  14. THE EVOLUTION OF ANTI-RETROVIRAL THERAPY IN NIGERIA ...

    African Journals Online (AJOL)

    results of a PUBMED literature search, the evolution of HIV therapy in Nigeria is .... Better understanding of HIV pathogenesis: rapid viral turnover and mechanisms of HIV resistance to anti- retroviral drugs. ... Approval of a new NNRTI (etravirine), and further evolution of 'salvage' therapy for patients with multiple failures.

  15. Deciphering the code for retroviral integration target site selection.

    Directory of Open Access Journals (Sweden)

    Federico Andrea Santoni

    2010-11-01

    Full Text Available Upon cell invasion, retroviruses generate a DNA copy of their RNA genome and integrate retroviral cDNA within host chromosomal DNA. Integration occurs throughout the host cell genome, but target site selection is not random. Each subgroup of retrovirus is distinguished from the others by attraction to particular features on chromosomes. Despite extensive efforts to identify host factors that interact with retrovirion components or chromosome features predictive of integration, little is known about how integration sites are selected. We attempted to identify markers predictive of retroviral integration by exploiting Precision-Recall methods for extracting information from highly skewed datasets to derive robust and discriminating measures of association. ChIPSeq datasets for more than 60 factors were compared with 14 retroviral integration datasets. When compared with MLV, PERV or XMRV integration sites, strong association was observed with STAT1, acetylation of H3 and H4 at several positions, and methylation of H2AZ, H3K4, and K9. By combining peaks from ChIPSeq datasets, a supermarker was identified that localized within 2 kB of 75% of MLV proviruses and detected differences in integration preferences among different cell types. The supermarker predicted the likelihood of integration within specific chromosomal regions in a cell-type specific manner, yielding probabilities for integration into proto-oncogene LMO2 identical to experimentally determined values. The supermarker thus identifies chromosomal features highly favored for retroviral integration, provides clues to the mechanism by which retrovirus integration sites are selected, and offers a tool for predicting cell-type specific proto-oncogene activation by retroviruses.

  16. Evolution of the retroviral restriction gene Fv1: inhibition of non-MLV retroviruses.

    Directory of Open Access Journals (Sweden)

    Melvyn W Yap

    2014-03-01

    Full Text Available Fv1 is the prototypic restriction factor that protects against infection by the murine leukemia virus (MLV. It was first identified in cells that were derived from laboratory mice and was found to be homologous to the gag gene of an endogenous retrovirus (ERV. To understand the evolution of the host restriction gene from its retroviral origins, Fv1s from wild mice were isolated and characterized. Most of these possess intact open reading frames but not all restricted N-, B-, NR-or NB-tropic MLVs, suggesting that other viruses could have played a role in the selection of the gene. The Fv1s from Mus spretus and Mus caroli were found to restrict equine infectious anemia virus (EIAV and feline foamy virus (FFV respectively, indicating that Fv1 could have a broader target range than previously thought, including activity against lentiviruses and spumaviruses. Analyses of the Fv1 sequences revealed a number of residues in the C-terminal region that had evolved under positive selection. Four of these selected residues were found to be involved in the novel restriction by mapping studies. These results strengthen the similarities between the two capsid binding restriction factors, Fv1 and TRIM5α, which support the hypothesis that Fv1 defended mice against waves of retroviral infection possibly including non-MLVs as well as MLVs.

  17. Retroviral Vectors: Post Entry Events and Genomic Alterations

    Directory of Open Access Journals (Sweden)

    Christof von Kalle

    2011-04-01

    Full Text Available The curative potential of retroviral vectors for somatic gene therapy has been demonstrated impressively in several clinical trials leading to sustained long-term correction of the underlying genetic defect. Preclinical studies and clinical monitoring of gene modified hematopoietic stem and progenitor cells in patients have shown that biologically relevant vector induced side effects, ranging from in vitro immortalization to clonal dominance and oncogenesis in vivo, accompany therapeutic efficiency of integrating retroviral gene transfer systems. Most importantly, it has been demonstrated that the genotoxic potential is not identical among all retroviral vector systems designed for clinical application. Large scale viral integration site determination has uncovered significant differences in the target site selection of retrovirus subfamilies influencing the propensity for inducing genetic alterations in the host genome. In this review we will summarize recent insights gained on the mechanisms of insertional mutagenesis based on intrinsic target site selection of different retrovirus families. We will also discuss examples of side effects occurring in ongoing human gene therapy trials and future prospectives in the field.

  18. The surprising activities of APOBEC3B and 5-fluorouracil

    Directory of Open Access Journals (Sweden)

    Emma L Walton

    2015-04-01

    Full Text Available In this mini-special issue on cancer, we learn how DNA editing enzymes can accelerate the development of cancer and we discover some remarkable effects of the chemotherapeutic agent, 5-fluorouracil, on the immune system. We also discuss a study revealing the continuing problem of vitamin B deficiencies in children in developing countries, and we determine how to distinguish two near-identical forms of necrotizing fasciitis.

  19. Retroviral transcriptional regulation and embryonic stem cells: war and peace.

    Science.gov (United States)

    Schlesinger, Sharon; Goff, Stephen P

    2015-03-01

    Retroviruses have evolved complex transcriptional enhancers and promoters that allow their replication in a wide range of tissue and cell types. Embryonic stem (ES) cells, however, characteristically suppress transcription of proviruses formed after infection by exogenous retroviruses and also of most members of the vast array of endogenous retroviruses in the genome. These cells have unusual profiles of transcribed genes and are poised to make rapid changes in those profiles upon induction of differentiation. Many of the transcription factors in ES cells control both host and retroviral genes coordinately, such that retroviral expression patterns can serve as markers of ES cell pluripotency. This overlap is not coincidental; retrovirus-derived regulatory sequences are often used to control cellular genes important for pluripotency. These sequences specify the temporal control and perhaps "noisy" control of cellular genes that direct proper cell gene expression in primitive cells and their differentiating progeny. The evidence suggests that the viral elements have been domesticated for host needs, reflecting the wide-ranging exploitation of any and all available DNA sequences in assembling regulatory networks. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  20. Analyzing the Genotoxicity of Retroviral Vectors in Hematopoietic Cell Gene Therapy

    Directory of Open Access Journals (Sweden)

    Luca Biasco

    2018-03-01

    Full Text Available Retroviral vectors, including those derived from gammaretroviruses and lentiviruses, have found their way into the clinical arena and demonstrated remarkable efficacy for the treatment of immunodeficiencies, leukodystrophies, and globinopathies. Despite these successes, gene therapy unfortunately also has had to face severe adverse events in the form of leukemias and myelodysplastic syndromes, related to the semi-random vector integration into the host cell genome that caused deregulation of neighboring proto-oncogenes. Although improvements in vector design clearly lowered the risk of this insertional mutagenesis, analysis of potential genotoxicity and the consequences of vector integration remain important parameters for basic and translational research and most importantly for the clinic. Here, we review current assays to analyze biodistribution and genotoxicity in the pre-clinical setting and describe tools to monitor vector integration sites in vector-treated patients as a biosafety readout.

  1. Mechanisms and factors that influence high frequency retroviral recombination

    DEFF Research Database (Denmark)

    Delviks-Frankenberry, Krista; Galli, Andrea; Nikolaitchik, Olga

    2011-01-01

    With constantly changing environmental selection pressures, retroviruses rely upon recombination to reassort polymorphisms in their genomes and increase genetic diversity, which improves the chances for the survival of their population. Recombination occurs during DNA synthesis, whereby reverse...... transcriptase undergoes template switching events between the two copackaged RNAs, resulting in a viral recombinant with portions of the genetic information from each parental RNA. This review summarizes our current understanding of the factors and mechanisms influencing retroviral recombination, fidelity...... of the recombination process, and evaluates the subsequent viral diversity and fitness of the progeny recombinant. Specifically, the high mutation rates and high recombination frequencies of HIV-1 will be analyzed for their roles in influencing HIV-1 global diversity, as well as HIV-1 diagnosis, drug treatment...

  2. Alteration of blood-brain barrier integrity by retroviral infection.

    Directory of Open Access Journals (Sweden)

    Philippe V Afonso

    2008-11-01

    Full Text Available The blood-brain barrier (BBB, which forms the interface between the blood and the cerebral parenchyma, has been shown to be disrupted during retroviral-associated neuromyelopathies. Human T Lymphotropic Virus (HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP is a slowly progressive neurodegenerative disease associated with BBB breakdown. The BBB is composed of three cell types: endothelial cells, pericytes and astrocytes. Although astrocytes have been shown to be infected by HTLV-1, until now, little was known about the susceptibility of BBB endothelial cells to HTLV-1 infection and the impact of such an infection on BBB function. We first demonstrated that human cerebral endothelial cells express the receptors for HTLV-1 (GLUT-1, Neuropilin-1 and heparan sulfate proteoglycans, both in vitro, in a human cerebral endothelial cell line, and ex vivo, on spinal cord autopsy sections from HAM/TSP and non-infected control cases. In situ hybridization revealed HTLV-1 transcripts associated with the vasculature in HAM/TSP. We were able to confirm that the endothelial cells could be productively infected in vitro by HTLV-1 and that blocking of either HSPGs, Neuropilin 1 or Glut1 inhibits this process. The expression of the tight-junction proteins within the HTLV-1 infected endothelial cells was altered. These cells were no longer able to form a functional barrier, since BBB permeability and lymphocyte passage through the monolayer of endothelial cells were increased. This work constitutes the first report of susceptibility of human cerebral endothelial cells to HTLV-1 infection, with implications for HTLV-1 passage through the BBB and subsequent deregulation of the central nervous system homeostasis. We propose that the susceptibility of cerebral endothelial cells to retroviral infection and subsequent BBB dysfunction is an important aspect of HAM/TSP pathogenesis and should be considered in the design of future therapeutics strategies.

  3. Sex-specific aspects of endogenous retroviral insertion and deletion.

    Science.gov (United States)

    Gemmell, Patrick; Hein, Jotun; Katzourakis, Aris

    2013-11-07

    We wish to understand how sex and recombination affect endogenous retroviral insertion and deletion. While theory suggests that the risk of ectopic recombination will limit the accumulation of repetitive DNA in areas of high meiotic recombination, the experimental evidence so far has been inconsistent. Under the assumption of neutrality, we examine the genomes of eighteen species of animal in order to compute the ratio of solo-LTRs that derive from insertions occurring down the male germ line as opposed to the female one (male bias). We also extend the simple idea of comparing autosome to allosome in order to predict the ratio of full-length proviruses we would expect to see under conditions of recombination linked deletion or otherwise. Using our model, we predict the ratio of allosomal to autosomal full-length proviruses to lie between32 and 23 under increasing male bias in mammals and between 1 and 2 under increasing male bias in birds. In contrast to our expectations, we find that a pattern of male bias is not universal across species and that there is a frequent overabundance of full-length proviruses on the allosome beyond the ratios predicted by our model. We use our data as a whole to argue that full-length proviruses should be treated as deleterious mutations or as effectively neutral mutations whose persistence in a full-length state is linked to the rate of meiotic recombination and whose origin is not universally male biased. These conclusions suggest that retroviral insertions on the allosome may be more prolific and that it might be possible to identify mechanisms of replication that are enhanced in the female sex.

  4. Pregnancy outcome of HIV-infected women on anti-retroviral therapy ...

    African Journals Online (AJOL)

    infected pregnant women who received anti-retroviral treatment at the University of Port Harcourt Teaching Hospital between January 2010 and December 2013. Data was analyzed using Epi Info Version 7 Statistical Package. Proportions, measures ...

  5. Gaps in the Implementation of Anti-Retroviral Treatment: A Case for ...

    African Journals Online (AJOL)

    Gaps in the Implementation of Anti-Retroviral Treatment: A Case for Addressing Gender and Mental Health Consequences of HIV Positive Individuals. JA Menon, MPS Ngoma, T Nkumbula, R Paul, S Sichimba ...

  6. Three retroviral sequences in amphibians are distinct from those in mammals and birds.

    Science.gov (United States)

    Tristem, M; Herniou, E; Summers, K; Cook, J

    1996-07-01

    We isolated and characterized three endogenous retroviral fragments from the dart-poison frog Dendrobates ventrimaculatus. These are the first retroviral sequences to be identified in amphibians, and consequently retroviruses have now been found in each of the five major vertebrate classes. Comparison of the amphibian retroviral fragments, termed DevI, DevII, and DevIII, with mammalian and avian isolates revealed significant differences between their nucleotide sequences. This suggested that they were only distantly related to the seven currently recognized retroviral genera. Additional analysis by phylogeny reconstruction showed that the amphibian retroviral fragments were approximately equally related to the Moloney leukemia-related viruses, the spumaviruses, and walleye dermal sarcoma virus. Hybridization experiments revealed that viruses closely related to DevI, DevII, and DevIII do not appear to be widespread in other vertebrates and that DevI, DevII, and DevIII are all present at high copy numbers within their amphibian hosts, typically at over 250 copies per genome. The viruses described here, along with two others which have recently been found in a fish and a reptile, indicate that there may be some major differences in the retroviruses harbored by different vertebrate classes. This suggests that further characterization of retroviruses of fish, reptiles, and amphibians will help in understanding the evolution of the whole retroviral family and may well lead to the discovery of retroviruses with novel biological properties.

  7. A revised nomenclature for transcribed human endogenous retroviral loci

    Science.gov (United States)

    2011-01-01

    Background Endogenous retroviruses (ERVs) and ERV-like sequences comprise 8% of the human genome. A hitherto unknown proportion of ERV loci are transcribed and thus contribute to the human transcriptome. A small proportion of these loci encode functional proteins. As the role of ERVs in normal and diseased biological processes is not yet established, transcribed ERV loci are of particular interest. As more transcribed ERV loci are likely to be identified in the near future, the development of a systematic nomenclature is important to ensure that all information on each locus can be easily retrieved. Results Here we present a revised nomenclature of transcribed human endogenous retroviral loci that sorts loci into groups based on Repbase classifications. Each symbol is of the format ERV + group symbol + unique number. Group symbols are based on a mixture of Repbase designations and well-supported symbols used in the literature. The presented guidelines will allow newly identified loci to be easily incorporated into the scheme. Conclusions The naming system will be employed by the HUGO Gene Nomenclature Committee for naming transcribed human ERV loci. We hope that the system will contribute to clarifying a certain aspect of a sometimes confusing nomenclature for human endogenous retroviruses. The presented system may also be employed for naming transcribed loci of human non-ERV repeat loci. PMID:21542922

  8. Retroviral Vectors for Analysis of Viral Mutagenesis and Recombination

    Directory of Open Access Journals (Sweden)

    Jonathan M.O. Rawson

    2014-09-01

    Full Text Available Retrovirus population diversity within infected hosts is commonly high due in part to elevated rates of replication, mutation, and recombination. This high genetic diversity often complicates the development of effective diagnostics, vaccines, and antiviral drugs. This review highlights the diverse vectors and approaches that have been used to examine mutation and recombination in retroviruses. Retroviral vectors for these purposes can broadly be divided into two categories: those that utilize reporter genes as mutation or recombination targets and those that utilize viral genes as targets of mutation or recombination. Reporter gene vectors greatly facilitate the detection, quantification, and characterization of mutants and/or recombinants, but may not fully recapitulate the patterns of mutagenesis or recombination observed in native viral gene sequences. In contrast, the detection of mutations or recombination events directly in viral genes is more biologically relevant but also typically more challenging and inefficient. We will highlight the advantages and disadvantages of the various vectors and approaches used as well as propose ways in which they could be improved.

  9. Retroviral insertional mutagenesis identifies Zeb2 activation as a novel leukemogenic collaborating event in CALM-AF10 transgenic mice.

    Science.gov (United States)

    Caudell, David; Harper, David P; Novak, Rachel L; Pierce, Rachel M; Slape, Christopher; Wolff, Linda; Aplan, Peter D

    2010-02-11

    The t(10;11) translocation results in a CALM-AF10 fusion gene in a subset of leukemia patients. Expression of a CALM-AF10 transgene results in leukemia, with prolonged latency and incomplete penetrance, suggesting that additional events are necessary for leukemic transformation. CALM-AF10 mice infected with the MOL4070LTR retrovirus developed acute leukemia, and ligation-mediated polymerase chain reaction was used to identify retroviral insertions at 19 common insertion sites, including Zeb2, Nf1, Mn1, Evi1, Ift57, Mpl, Plag1, Kras, Erg, Vav1, and Gata1. A total of 26% (11 of 42) of the mice had retroviral integrations near Zeb2, a transcriptional corepressor leading to overexpression of the Zeb2-transcript. A total of 91% (10 of 11) of mice with Zeb2 insertions developed B-lineage acute lymphoblastic leukemia, suggesting that Zeb2 activation promotes the transformation of CALM-AF10 hematopoietic precursors toward B-lineage leukemias. More than half of the mice with Zeb2 integrations also had Nf1 integrations, suggesting cooperativity among CALM-AF10, Zeb2, and Ras pathway mutations. We searched for Nras, Kras, and Ptpn11 point mutations in the CALM-AF10 leukemic mice. Three mutations were identified, all of which occurred in mice with Zeb2 integrations, consistent with the hypothesis that Zeb2 and Ras pathway activation promotes B-lineage leukemic transformation in concert with CALM-AF10.

  10. Murine leukemia virus-derived retroviral vector has differential integration patterns in human cell lines used to produce recombinant factor VIII

    Directory of Open Access Journals (Sweden)

    Marcela Cristina Correa de Freitas

    2014-06-01

    Full Text Available OBJECTIVE: Nowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO and baby hamster kidney cells (BHK. Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K.METHODS: This study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones.RESULTS: The retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293.CONCLUSION: The results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins.

  11. A rapid and efficient polyethylenimine-based transfection method to prepare lentiviral or retroviral vectors: useful for making iPS cells and transduction of primary cells.

    Science.gov (United States)

    Yang, Shaozhe; Shi, Haijun; Chu, Xinran; Zhou, Xiaoling; Sun, Pingnan

    2016-09-01

    To improve the efficiency, reproducibility and consistency of the PEI-based transfection method that is often used in preparation of recombinant lentiviral or retroviral vectors. The contributions to transfection efficiency of multi-factors including concentration of PEI or DNA, dilution buffer for PEI/DNA, manner to prepare PEI/DNA complexes, influence of serum, incubation time for PEI/DNA complexes, and transfection time were studied. Gentle mixing during the preparation of PEI/DNA transfection complexes is critical for a high transfection efficiency. PEI could be stored at room temperature or 4 °C, and most importantly, multigelation should be avoided. The transfection efficiency of the PEI-based new method in different types of cells, such as 293T, Cos-7, HeLa, HepG2, Hep3B, Huh7 and L02, was also higher than that of the previous method. After optimization, the titer of our lentiviral system or retroviral system produced by PEI-based new method was about 10- or 3-times greater than that produced by PEI-based previous method, respectively. We provide a rapid and efficient PEI-based method for preparation of recombinant lentiviral or retroviral vectors which is useful for making iPS cells as well as transduction of primary cell cultures.

  12. Generation of retroviral particles for the spleen necrosis virus (SNV)-based vector system and their use in transduction of various cell types.

    Science.gov (United States)

    Parveen, Zahida; Mukhtar, Muhammad; Pomerantz, Roger J

    2010-06-01

    Genetically engineered retroviruses are widely used for gene delivery into human cells. A number of investigators have studied spleen necrosis virus (SNV) as a vehicle for gene delivery. Vectors developed from SNV and its closely associated avian reticuloendotheliosis virus strain A (REV-A) can be used for gene transfer into a variety of cells, including primary hematopoietic cells and human brain and post-mitotic neuronal cells that are difficult to transduce with other vector systems. SNV-based vector systems have the advantage of being quite safe, because wild-type SNV is unable to infect human cells and has less preference for integration into transcriptionally active sites or genes. However, the generation of retroviral vectors requires cotransfection of more than one plasmid into a packaging cell line, which is a tedious process. The development of stable packaging cell lines expressing envelope (Env) proteins and the structural proteins Gag-Pol will enhance mass production of retroviral vectors for future gene therapy experiments both in vitro and in vivo. This protocol describes the generation of retroviral particles for the SNV-based vector system. These particles can then be used for transduction of various cell types; as an example, a technique for transduction of post-mitotic neurons is also presented.

  13. Determinants of non-adherence to subsidized anti-retroviral treatment in southeast Nigeria.

    Science.gov (United States)

    Uzochukwu, B S C; Onwujekwe, O E; Onoka, A C; Okoli, C; Uguru, N P; Chukwuogo, O I

    2009-05-01

    The anti-retroviral (ARV) treatment programme in Nigeria is delivered through selected teaching and mission hospitals at a free/subsidized rate. The government aims to scale up ARV treatment in the country. However, non-adherence to ARV medication can lead to viral resistance, treatment failure, toxicities and waste of financial resources. This study examined the factors responsible for non-adherence to free/subsidized ARV treatment in south-east Nigeria. The study was cross-sectional and descriptive. Information was collected from 174 patients selected by simple random sampling from the register of all patients who had been on anti-retroviral therapy (ART) for at least 12 months at the beginning of the study period. Patients were identified during their clinic visits. Information on their socio-demographic profile, ARV treatment and determinants of non-adherence to ARV treatment was obtained from those who gave consent, using pre-tested interviewer-administered questionnaires. All patients clearly understood the need to take ARV drugs throughout their lives, and what the costs entailed. They understood the need for periodic testing, the probability that complications would develop, cost of transportation to treatment site and the daily treatment regimen. Seventy-five per cent of respondents were not adhering fully to their drug regimen; the mean number of days that respondents had been off drugs was 3.57 days the preceding month. Reasons for non-adherence included: physical discomfort (side effects); non-availability of drugs at treatment site; forgetting to carry drugs during the day; fear of social rejection; treatment being a reminder of HIV status; and selling of own drugs to those unable to enrol in the projects. Being female, under 35 years, single, and having higher educational status were significantly associated with non-adherence. It is important that policy makers and programme managers address the factors responsible for non-adherence when scaling up

  14. Integration Site and Clonal Expansion in Human Chronic Retroviral Infection and Gene Therapy

    Science.gov (United States)

    Niederer, Heather A.; Bangham, Charles R. M.

    2014-01-01

    Retroviral vectors have been successfully used therapeutically to restore expression of genes in a range of single-gene diseases, including several primary immunodeficiency disorders. Although clinical trials have shown remarkable results, there have also been a number of severe adverse events involving malignant outgrowth of a transformed clonal population. This clonal expansion is influenced by the integration site profile of the viral integrase, the transgene expressed, and the effect of the viral promoters on the neighbouring host genome. Infection with the pathogenic human retrovirus HTLV-1 also causes clonal expansion of cells containing an integrated HTLV-1 provirus. Although the majority of HTLV-1-infected people remain asymptomatic, up to 5% develop an aggressive T cell malignancy. In this review we discuss recent findings on the role of the genomic integration site in determining the clonality and the potential for malignant transformation of cells carrying integrated HTLV-1 or gene therapy vectors, and how these results have contributed to the understanding of HTLV-1 pathogenesis and to improvements in gene therapy vector safety. PMID:25365582

  15. Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins

    Science.gov (United States)

    Williams, Mark

    2010-03-01

    Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins' nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone.

  16. Follistatin allows efficient retroviral-mediated gene transfer into rat liver

    International Nuclear Information System (INIS)

    Borgnon, Josephine; Djamouri, Fatima; Lorand, Isabelle; Rico, Virginie Di; Loux, Nathalie; Pages, Jean-Christophe; Franco, Dominique; Capron, Frederique; Weber, Anne

    2005-01-01

    Retroviral vectors are widely used tools for gene therapy. However, in vivo gene transfer is only effective in dividing cells, which, in liver, requires a regenerative stimulus. Follistatin is effective in promoting liver regeneration after 90% and 70% hepatectomy in rats. We studied its efficacy on liver regeneration and retroviral-mediated gene delivery in 50% hepatectomized rats. When human recombinant follistatin was infused into the portal vein immediately after 50% hepatectomy, hepatocyte proliferation was significantly higher than in control 50% hepatectomized rats. A single injection of virus particles administered 23 h after follistatin infusion resulted in more than 20% gene transduction efficiency in hepatocytes compared to 3% in control rats. It is concluded that a single injection of follistatin induces onset of proliferation in 50% hepatectomized rats and allows efficient retroviral-mediated gene transfer to the liver

  17. Chromosomal locations of members of a family of novel endogenous human retroviral genomes

    International Nuclear Information System (INIS)

    Horn, T.M.; Huebner, K.; Croce, C.; Callahan, R.

    1986-01-01

    Human cellular DNA contains two distinguishable families of retroviral related sequences. One family shares extensive nucleotide sequence homology with infectious mammalian type C retroviral genomes. The other family contains major regions of homology with the pol genes of infectious type A and B and avian type C and D retroviral genomes. Analysis of the human recombinant clone HLM-2 has shown that the pol gene in the latter family is located within an endogenous proviral genome. The authors show that the proviral genome in HLM-2 and the related recombinant clone HLM-25 are located, respectively, on human chromosomes 1 and 5. Other related proviral genomes are located on chromosomes 7, 8, 11, 14, and 17

  18. Functional cloning using pFB retroviral cDNA expression libraries.

    Science.gov (United States)

    Felts, Katherine A; Chen, Keith; Zaharee, Kim; Sundar, Latha; Limjoco, Jamie; Miller, Anna; Vaillancourt, Peter

    2002-09-01

    Retroviral cDNA expression libraries allow the efficient introduction of complex cDNA libraries into virtually any mitotic cell type for screening based on gene function. The cDNA copy number per cell can be easily controlled by adjusting the multiplicity of infection, thus cell populations may be generated in which >90% of infected cells contain one to three cDNAs. We describe the isolation of two known oncogenes and one cell-surface receptor from a human Burkitt's lymphoma (Daudi) cDNA library inserted into the high-titer retroviral vector pFB.

  19. TRIM5α selectively binds a restriction-sensitive retroviral capsid

    Directory of Open Access Journals (Sweden)

    Luban Jeremy

    2005-06-01

    Full Text Available Abstract TRIM5 is a potent retrovirus inhibitor that targets viruses bearing particular capsid (CA residues. In most primate species, retroviral restriction requires the C-terminal SPRY domain unique to the α-isoform of TRIM5, but the mechanism by which susceptible viruses are recognized and targeted for restriction is unknown. Here we show that TRIM5α binds retroviral CA from detergent-stripped virions in a SPRY-dependent manner with sufficient discrimination to account for the exquisite specificity of restriction.

  20. The use of first line Highly Active Anti-Retroviral Therapy (HAART) is ...

    African Journals Online (AJOL)

    The use of first line Highly Active Anti-Retroviral Therapy (HAART) is not associated with QTC prolongation in HIV patients. ... Mean QTc was significantly longer among patients with CD4 count 200 cells/mm3 0.445 + 0.03secs vs 0.421 + 0.03secs (P<0.001). QTc prolongation was ...

  1. Providing anti-retroviral therapy in the context of self-perceived ...

    African Journals Online (AJOL)

    Adherence to anti-retroviral treatment (ART) has been a significant step towards improving quality of life among people living with HIV. However, stigma has been described to influence adherence to ART. A cross-sectional mixed methods study was conducted to explore factors related to stigma and perceived influence of ...

  2. Reasons for Change of Anti-Retroviral Therapy (ART) Drugs: Local ...

    African Journals Online (AJOL)

    Background: Highly active anti-retroviral therapy (HAART) reduces morbidity and mortality in HIV/AIDS infected patients. HAART is used indefinitely and the regimens are changed over the course of treatment due to resistance, adverse drug reactions or access to drugs. Few studies have been done in resource constrained ...

  3. Development of retrovirally marked human T progenitor cells into mature thymocytes

    NARCIS (Netherlands)

    Staal, F. J.; Res, P. C.; Weijer, K.; Spits, H.

    1995-01-01

    Retroviral vectors have been used in most human gene therapy trials that have been undertaken. Many of these therapies have focused on the introduction of genes into hematopoietic stem cells with the goal of obtaining expression in the mature T lymphocytic progeny. It has proven difficult to achieve

  4. Kinetics of the dimerization of retroviral proteases: The "fireman's grip" and dimerization

    Czech Academy of Sciences Publication Activity Database

    Ingr, Marek; Kondrová, Taťána; Stříšovský, Kvido; Majerová, E.; Konvalinka, Jan

    2003-01-01

    Roč. 12, - (2003), s. 2173-2182 ISSN 0961-8368 R&D Projects: GA MZd NI6339 Institutional research plan: CEZ:AV0Z4055905 Keywords : retroviral protease * dimerization * HIV protease Subject RIV: CE - Biochemistry Impact factor: 3.787, year: 2003

  5. Anti-Retroviral Therapy Related Liver Injury (ARLI): A Series of 11 ...

    African Journals Online (AJOL)

    Objective: To describe anti-retroviral-related liver injury (ARLI) in HIV positive patients, their CD4+ cell counts, biochemical and viral markers and liver ultrasound. Design: Prospective, descriptive, consecutive entry study. Setting: Kisumu District Hospital liver clinic/medical outpatient clinic, Nairobi Rheumatology Clinic and ...

  6. Cancellers - Exploring the Possibility of Receptor Decoy Traps As a Superior Anti-Retroviral Strategy.

    Science.gov (United States)

    Jeremiah, Sundararaj Stanley; Ohba, Kenji; Yamamoto, Naoki

    2016-01-01

    The global Human Immunodeficiency Virus (HIV) pandemic is still spreading due to the lack of ideal anti-retroviral measures and their availability. Till date, all attempts to produce an efficient vaccine have ended with unsatisfactory results. The highly active anti-retroviral therapy (HAART) is the only effective weapon currently available and is widely being used for curtailing the HIV pandemic. However, the HAART is also expected to fail in the near future due to the emergence and dissemination of antiviral resistance. This review sheds light on the reasons for the failure of the conventional anti-viral measures against HIV and the novel anti-retroviral strategies currently being developed. The various principles to be considered for the success of a novel anti-retroviral strategy are elaborately emphasized and an innovative concept is proposed on these lines. The proposed concept intends to use receptor decoy traps (RDT) called cancellers which are erythrocytes expressing the HIV entry receptors on their surface. If successfully developed, the cancellers would be capable of active targeting of the free HIV particles leading to the trapping of the viruses within the canceller, resulting in the neutralization of infectivity of the trapped virus. The possible ways of translating this concept into reality and the probable hurdles that can be encountered in the process are subsequently discussed. Also, the scope of cancellers in therapeutic and/or preventive strategies against HIV infection is envisaged upon their successful development.

  7. Foxp3-dependent transformation of human primary CD4+ T lymphocytes by the retroviral protein tax.

    Science.gov (United States)

    Chen, Li; Liu, Dan; Zhang, Yang; Zhang, Huan; Cheng, Hua

    2015-10-23

    The retroviral Tax proteins of human T cell leukemia virus type 1 and 2 (HTLV-1 and -2) are highly homologous viral transactivators. Both viral proteins can immortalize human primary CD4+ memory T cells, but when expressed alone they rarely transform T cells. In the present study, we found that the Tax proteins displayed a differential ability to immortalize human CD4+Foxp3+ T cells with characteristic expression of CTLA-4 and GITR. Because epidermal growth factor receptor (EGFR) was reportedly expressed and activated in a subset of CD4+Foxp3+ T cells, we introduced an activated EGFR into Tax-immortalized CD4+Foxp3+ T cells. We observed that these modified cells were grown independently of exogenous IL-2, correlating with a T cell transformation phenotype. In Tax-immortalized CD4+Foxp3- T cells, ectopic expression of Foxp3 was a prerequisite for Tax transformation of T cells. Accordingly, treatment of the transformed T cells with erlotinib, a selective inhibitor of EGFR, induced degradation of EGFR in lysosome, consequently causing T cell growth inhibition. Further, we identified autophagy as a crucial cellular survival pathway for the transformed T cells. Silencing key autophagy molecules including Beclin1, Atg5 and PI3 kinase class III (PI3KC3) resulted in drastic impairment of T cell growth. Our data, therefore, unveiled a previously unidentified role of Foxp3 in T cell transformation, providing a molecular basis for HTLV-1 transformation of CD4+Foxp3+ T cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Use of retroviral-mediated gene transfer to deliver and test function of chimeric antigen receptors in human T-cells

    Directory of Open Access Journals (Sweden)

    Ana C. Parente-Pereira

    2014-07-01

    Full Text Available Chimeric antigen receptors (CARs are genetically delivered fusion molecules that elicit T-cell activation upon binding of a native cell surface molecule. These molecules can be used to generate a large number of memory and effector T-cells that are capable of recognizing and attacking tumor cells. Most commonly, stable CAR expression is achieved in T-cells using retroviral vectors. In the method described here, retroviral vectors are packaged in a two-step procedure. First, H29D human retroviral packaging cells (a derivative of 293 cells are transfected with the vector of interest, which is packaged transiently in vesicular stomatitis virus (VSV G pseudotyped particles. These particles are used to deliver the vector to PG13 cells, which achieve stable packaging of gibbon ape leukaemia virus (GALV-pseudotyped particles that are suitable for infection of human T-cells. The key advantage of the method reported here is that it robustly generates polyclonal PG13 cells that are 100% positive for the vector of interest. This means that efficient gene transfer may be repeatedly achieved without the need to clone individual PG13 cells for experimental pre-clinical testing. To achieve T-cell transduction, cells must first be activated using a non-specific mitogen. Phytohemagglutinin (PHA provides an economic and robust stimulus to achieve this. After 48-72 h, activated T-cells and virus-conditioned medium are mixed in RetroNectin-coated plasticware, which enhances transduction efficiency. Transduced cells are analyzed for gene transfer efficiency by flow cytometry 48 h following transduction and may then be tested in several assays to evaluate CAR function, including target-dependent cytotoxicity, cytokine production and proliferation.

  9. Novel Principles of Gamma-Retroviral Insertional Transcription Activation in Murine Leukemia Virus-induced End-stage Tumors

    DEFF Research Database (Denmark)

    Sokol, Martin; Wabl, Matthias; Rius Ruiz, Irene

    2014-01-01

    Background Insertional mutagenesis screens of retrovirus-induced mouse tumors have proven valuable in human cancer research and for understanding adverse effects of retroviral-based gene therapies. In previous studies, the assignment of mouse genes to individual retroviral integration sites has...... on opposite DNA strands; tandem-type activation of distal genes that are positioned adjacently on the same DNA strand; activation of genes that are not the direct integration targets; combination-type insertional mutagenesis, in which enhancer activation, alternative chimeric splicing and retroviral promoter...... transcription activation patterns that reach beyond a single and nearest annotated gene target. Awareness of this previously undescribed layer of complexity may prove important for elucidation of adverse effects in retroviral-based gene therapies. We also show that wild-type gamma-retroviruses are frequently...

  10. Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Shuohao [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Kawabe, Yoshinori; Ito, Akira [Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Kamihira, Masamichi, E-mail: kamihira@chem-eng.kyushu-u.ac.jp [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Adeno-associated virus (AAV) is capable of targeted integration in human cells. Black-Right-Pointing-Pointer Integrase-defective retroviral vector (IDRV) enables a circular DNA delivery. Black-Right-Pointing-Pointer A targeted integration system of IDRV DNA using the AAV integration mechanism. Black-Right-Pointing-Pointer Targeted IDRV integration ameliorates the safety concerns for retroviral vectors. -- Abstract: Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.

  11. Hypoxia- and radiation-inducible, breast cell-specific targeting of retroviral vectors

    International Nuclear Information System (INIS)

    Lipnik, Karoline; Greco, Olga; Scott, Simon; Knapp, Elzbieta; Mayrhofer, Elisabeth; Rosenfellner, Doris; Guenzburg, Walter H.; Salmons, Brian; Hohenadl, Christine

    2006-01-01

    To facilitate a more efficient radiation and chemotherapy of mammary tumours, synthetic enhancer elements responsive to hypoxia and ionizing radiation were coupled to the mammary-specific minimal promoter of the murine whey acidic protein (WAP) encoding gene. The modified WAP promoter was introduced into a retroviral promoter conversion (ProCon) vector. Expression of a transduced reporter gene in response to hypoxia and radiation was analysed in stably infected mammary cancer cell lines and an up to 9-fold increase in gene expression demonstrated in comparison to the respective basic vector. Expression analyses in vitro, moreover, demonstrated a widely preserved mammary cell-specific promoter activity. For in vivo analyses, xenograft tumours consisting of infected human mammary adenocarcinoma cells were established in SCID/beige mice. Immunohistochemical analyses demonstrated a hypoxia-specific, markedly increased WAP promoter-driven expression in these tumours. Thus, this retroviral vector will facilitate a targeted gene therapeutic approach exploiting the unique environmental condition in solid tumours

  12. Expression of cDNAs in human Natural Killer cell lines by retroviral transduction.

    Science.gov (United States)

    Miah, S M Shahjahan; Campbell, Kerry S

    2010-01-01

    Human NK-like cell lines are difficult to transfect using standard mammalian expression vectors and conventional transfection protocols, but they are susceptible to retroviral transduction as a means to introduce cDNAs. Our laboratory has exploited this technique to study a number of receptors in human NK cell lines. The method utilizes a bicistronic retroviral vector that co-expresses either drug resistance or enhanced green fluorescent protein (EGFP) in parallel with the gene of interest. After a single infection with recombinant retrovirus, transduced NK cells can be sorted for expression of EGFP or the transduced cell surface marker. Alternatively, cells expressing the transduced cDNAs can be selected for by treatment with neomycin, puromycin, or hygromycin. Using this method, the sorted/selected cells uniformly express the gene of interest and the expression is stable for many weeks of culture.

  13. Queratinocitos humanos modificados genéticamente por medio de un vector retroviral

    Directory of Open Access Journals (Sweden)

    C. Chamorro

    2001-07-01

    Full Text Available Los queratinocitos poseen características ideales para la terapia génica: accesibles, modifi-cables por vectores retrovirales, conservan in vitro sus propiedades de proliferación y diferen-ciación, fácil remoción por efectos adversos. Nuestro objetivo fue evaluar estas células comoblanco de transferencia de genes empleando el vector retroviral Foch-29 NeoR.

  14. PML/TRIM19-Dependent Inhibition of Retroviral Reverse-Transcription by Daxx.

    Science.gov (United States)

    Dutrieux, Jacques; Maarifi, Ghizlane; Portilho, Débora M; Arhel, Nathalie J; Chelbi-Alix, Mounira K; Nisole, Sébastien

    2015-11-01

    PML (Promyelocytic Leukemia protein), also known as TRIM19, belongs to the family of tripartite motif (TRIM) proteins. PML is mainly expressed in the nucleus, where it forms dynamic structures known as PML nuclear bodies that recruit many other proteins, such as Sp100 and Daxx. While the role of PML/TRIM19 in antiviral defense is well documented, its effect on HIV-1 infection remains unclear. Here we show that infection by HIV-1 and other retroviruses triggers the formation of PML cytoplasmic bodies, as early as 30 minutes post-infection. Quantification of the number and size of PML cytoplasmic bodies revealed that they last approximately 8 h, with a peak at 2 h post-infection. PML re-localization is blocked by reverse-transcription inhibitors and is not observed following infection with unrelated viruses, suggesting it is specifically triggered by retroviral reverse-transcription. Furthermore, we show that PML interferes with an early step of retroviral infection since PML knockdown dramatically increases reverse-transcription efficiency. We demonstrate that PML does not inhibit directly retroviral infection but acts through the stabilization of one of its well-characterized partners, Daxx. In the presence of PML, cytoplasmic Daxx is found in the vicinity of incoming HIV-1 capsids and inhibits reverse-transcription. Interestingly, Daxx not only interferes with exogenous retroviral infections but can also inhibit retrotransposition of endogenous retroviruses, thus identifying Daxx as a broad cellular inhibitor of reverse-transcription. Altogether, these findings unravel a novel antiviral function for PML and PML nuclear body-associated protein Daxx.

  15. Efficient and specific internal cleavage of a retroviral palindromic DNA sequence by tetrameric HIV-1 integrase.

    Directory of Open Access Journals (Sweden)

    Olivier Delelis

    Full Text Available BACKGROUND: HIV-1 integrase (IN catalyses the retroviral integration process, removing two nucleotides from each long terminal repeat and inserting the processed viral DNA into the target DNA. It is widely assumed that the strand transfer step has no sequence specificity. However, recently, it has been reported by several groups that integration sites display a preference for palindromic sequences, suggesting that a symmetry in the target DNA may stabilise the tetrameric organisation of IN in the synaptic complex. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the ability of several palindrome-containing sequences to organise tetrameric IN and investigated the ability of IN to catalyse DNA cleavage at internal positions. Only one palindromic sequence was successfully cleaved by IN. Interestingly, this symmetrical sequence corresponded to the 2-LTR junction of retroviral DNA circles-a palindrome similar but not identical to the consensus sequence found at integration sites. This reaction depended strictly on the cognate retroviral sequence of IN and required a full-length wild-type IN. Furthermore, the oligomeric state of IN responsible for this cleavage differed from that involved in the 3'-processing reaction. Palindromic cleavage strictly required the tetrameric form, whereas 3'-processing was efficiently catalysed by a dimer. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that the restriction-like cleavage of palindromic sequences may be a general physiological activity of retroviral INs and that IN tetramerisation is strongly favoured by DNA symmetry, either at the target site for the concerted integration or when the DNA contains the 2-LTR junction in the case of the palindromic internal cleavage.

  16. Murine leukemias with retroviral insertions at Lmo2 are predictive of the leukemias induced in SCID-X1 patients following retroviral gene therapy.

    Directory of Open Access Journals (Sweden)

    Utpal P Davé

    2009-05-01

    Full Text Available Five X-linked severe combined immunodeficiency patients (SCID-X1 successfully treated with autologous bone marrow stem cells infected ex vivo with an IL2RG-containing retrovirus subsequently developed T-cell leukemia and four contained insertional mutations at LMO2. Genetic evidence also suggests a role for IL2RG in tumor formation, although this remains controversial. Here, we show that the genes and signaling pathways deregulated in murine leukemias with retroviral insertions at Lmo2 are similar to those deregulated in human leukemias with high LMO2 expression and are highly predictive of the leukemias induced in SCID-X1 patients. We also provide additional evidence supporting the notion that IL2RG and LMO2 cooperate in leukemia induction but are not sufficient and require additional cooperating mutations. The highly concordant nature of the genetic events giving rise to mouse and human leukemias with mutations at Lmo2 are an encouraging sign to those wanting to use mice to model human cancer and may help in designing safer methods for retroviral gene therapy.

  17. Recent updates for designing CCR5 antagonists as anti-retroviral agents.

    Science.gov (United States)

    Shah, Harshil R; Savjani, Jignasa Ketan

    2018-03-10

    The healthcare system faces various challenges in human immunodeficiency virus (HIV) therapy due to resistance to Anti-Retroviral Therapy (ART) as a consequence of the evolutionary process. Despite the success of antiretroviral drugs like Zidovudine, Zalcitabine, Raltegravir WHO ranks HIV as one of the deadliest diseases with a mortality of one million lives in 2016. Thus, there emerges an urgency of developing a novel anti-retroviral agent that combat resistant HIV strains. The clinical development of ART from a single drug regimen to current triple drug combination is very slow. The progression in the structural biology of the viral envelope prompted the discovery of novel targets, which can be demonstrated a proficient approach for drug design of anti-retroviral agents. The current review enlightens the recent updates in the structural biology of the viral envelope and focuses on CCR5 as a validated target as well as ways to overcome CCR5 resistance. The article also throws light on the SAR studies and most prevalent mutations in the receptor for designing CCR5 antagonists that can combat HIV-1 infection. To conclude, the paper lists diversified scaffolds that are in pipeline by various pharmaceutical companies that could provide an aid for developing novel CCR5 antagonists. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  18. Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Mandy Y M Lo

    Full Text Available Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.

  19. Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.

    Science.gov (United States)

    Lo, Mandy Y M; Rival-Gervier, Sylvie; Pasceri, Peter; Ellis, James

    2012-01-01

    Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES) cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.

  20. Cross- and Co-Packaging of Retroviral RNAs and Their Consequences

    Directory of Open Access Journals (Sweden)

    Lizna M. Ali

    2016-10-01

    Full Text Available Retroviruses belong to the family Retroviridae and are ribonucleoprotein (RNP particles that contain a dimeric RNA genome. Retroviral particle assembly is a complex process, and how the virus is able to recognize and specifically capture the genomic RNA (gRNA among millions of other cellular and spliced retroviral RNAs has been the subject of extensive investigation over the last two decades. The specificity towards RNA packaging requires higher order interactions of the retroviral gRNA with the structural Gag proteins. Moreover, several retroviruses have been shown to have the ability to cross-/co-package gRNA from other retroviruses, despite little sequence homology. This review will compare the determinants of gRNA encapsidation among different retroviruses, followed by an examination of our current understanding of the interaction between diverse viral genomes and heterologous proteins, leading to their cross-/co-packaging. Retroviruses are well-known serious animal and human pathogens, and such a cross-/co-packaging phenomenon could result in the generation of novel viral variants with unknown pathogenic potential. At the same time, however, an enhanced understanding of the molecular mechanisms involved in these specific interactions makes retroviruses an attractive target for anti-viral drugs, vaccines, and vectors for human gene therapy.

  1. Human immunodeficiency virus infection and autoimmune hepatitis during highly active anti-retroviral treatment: a case report and review of the literature

    Directory of Open Access Journals (Sweden)

    Higgins Martha

    2011-06-01

    Full Text Available Abstract Introduction The emergence of hepatic injury in patients with human immunodeficiency virus infection during highly active therapy presents a diagnostic dilemma. It may represent treatment side effects or autoimmune disorders, such as autoimmune hepatitis, emerging during immune restoration. Case presentation We present the case of a 42-year-old African-American woman with human immunodeficiency virus infection who presented to our emergency department with severe abdominal pain and was found to have autoimmune hepatitis. A review of the literature revealed 12 reported cases of autoimmune hepatitis in adults with human immunodeficiency virus infection, only three of whom were diagnosed after highly active anti-retroviral treatment was initiated. All four cases (including our patient were women, and one had a history of other autoimmune disorders. In our patient (the one patient case we are reporting, a liver biopsy revealed interface hepatitis, necrosis with lymphocytes and plasma cell infiltrates and variable degrees of fibrosis. All four cases required treatment with corticosteroids and/or other immune modulating agents and responded well. Conclusion Our review suggests that autoimmune hepatitis is a rare disorder which usually develops in women about six to eight months after commencing highly active anti-retroviral treatment during the recovery of CD4 lymphocytes. It represents either re-emergence of a pre-existing condition that was unrecognized or a de novo manifestation during immune reconstitution.

  2. Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia

    International Nuclear Information System (INIS)

    Shimada, Hidenori; Hashimoto, Yoshiya; Nakada, Akira; Shigeno, Keiji; Nakamura, Tatsuo

    2012-01-01

    Highlights: ► Very rapid generation of human iPS cells under optimized conditions. ► Five chemical inhibitors under hypoxia boosted reprogramming. ► We performed genome-wide DNA methylation analysis. -- Abstract: Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large amounts of disease-specific cells for biomedical research. Despite their great potential, the long reprogramming process (up to 1 month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study, we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells, using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Under optimized conditions, we observed human embryonic stem (ES)-like cells as early as 6 days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate, morphology, pluripotency-associated markers, global gene expression patterns, genome-wide DNA methylation states, and the ability to differentiate into all three of the germ layers, both in vitro and in vivo. Our method, when combined with chemical inhibitors under conditions of physiological hypoxia, offers a powerful tool for rapidly generating bona fide human iPS cells and facilitates the application of i

  3. Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Shimada, Hidenori [Department of Bioartificial Organs, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan); Hashimoto, Yoshiya [Department of Biomaterials, Osaka Dental University, 8-1, Hanazonocho, Kuzuha, Hirakatashi, Osaka 573-1121 (Japan); Nakada, Akira; Shigeno, Keiji [Department of Bioartificial Organs, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan); Nakamura, Tatsuo, E-mail: nakamura@frontier.kyoto-u.ac.jp [Department of Bioartificial Organs, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan)

    2012-01-13

    Highlights: Black-Right-Pointing-Pointer Very rapid generation of human iPS cells under optimized conditions. Black-Right-Pointing-Pointer Five chemical inhibitors under hypoxia boosted reprogramming. Black-Right-Pointing-Pointer We performed genome-wide DNA methylation analysis. -- Abstract: Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large amounts of disease-specific cells for biomedical research. Despite their great potential, the long reprogramming process (up to 1 month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study, we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells, using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Under optimized conditions, we observed human embryonic stem (ES)-like cells as early as 6 days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate, morphology, pluripotency-associated markers, global gene expression patterns, genome-wide DNA methylation states, and the ability to differentiate into all three of the germ layers, both in vitro and in vivo. Our method, when combined with chemical inhibitors under conditions of physiological hypoxia, offers a powerful tool for rapidly

  4. Anti-inflammatory and vasoprotective activity of a retroviral-derived peptide, homologous to human endogenous retroviruses: endothelial cell effects.

    Directory of Open Access Journals (Sweden)

    George J Cianciolo

    Full Text Available Malignant and inflammatory tissues sometimes express endogenous retroviruses or their proteins. A highly-conserved sequence from retroviral transmembrane (TM proteins, termed the "immunosuppressive domain (ID", is associated with inhibition of immune and inflammatory functions. An octadecapeptide (MN10021 from the ID of retroviral TM protein p15E inhibits in vitro release of pro-inflammatory cytokines and increases synthesis of anti-inflammatory IL-10. We sought to determine if MN10021 has significant in vivo effects. MN10021, prepared by solid-phase synthesis, was dimerized through a naturally-occurring, carboxy-terminal cysteine. In vivo anti-inflammatory activity was determined using a murine model of sodium periodate (NaIO(4-induced peritonitis. In vivo vasoprotective effects were determined using: (1 a carrageenan-induced model of disseminated intravascular coagulation (DIC in mice; (2 a reverse passive Arthus model in guinea pigs; and (3 vasoregulatory effects in spontaneously hypertensive rats (SHR. In vitro studies included: (1 binding/uptake of MN10021 using human monocytes, cultured fibroblasts, and vascular endothelial cells (VEC; (2 gene expression by RT-PCR of MN10021-treated VEC; and (3 apoptosis of MN10021-treated VEC exposed to staurosporine or TNF-α. One-tenth nmol MN10021 inhibits 50 percent of the inflammatory response in the mouse peritonitis model. Furthermore, 73 nmol MN10021 completely protects mice in a lethal model of carrageenan-induced DIC and inhibits vascular leak in both the mouse DIC model and a guinea pig reverse passive Arthus reaction. MN10021 binds to and is taken up in a specific manner by both human monocytes and VEC but not by cultured human fibroblasts. Surprisingly, orally-administered MN10021 lowers blood pressure in SHR rats by 10-15% within 1 h suggesting a direct or indirect effect on the vascular endothelium. MN10021 and derived octapeptides induce iNOS (inducible nitric oxide synthase mRNA in VEC

  5. Lineage-specific expansions of retroviral insertions within the genomes of African great apes but not humans and orangutans.

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    Chris T Yohn

    2005-04-01

    Full Text Available Retroviral infections of the germline have the potential to episodically alter gene function and genome structure during the course of evolution. Horizontal transmissions between species have been proposed, but little evidence exists for such events in the human/great ape lineage of evolution. Based on analysis of finished BAC chimpanzee genome sequence, we characterize a retroviral element (Pan troglodytes endogenous retrovirus 1 [PTERV1] that has become integrated in the germline of African great ape and Old World monkey species but is absent from humans and Asian ape genomes. We unambiguously map 287 retroviral integration sites and determine that approximately 95.8% of the insertions occur at non-orthologous regions between closely related species. Phylogenetic analysis of the endogenous retrovirus reveals that the gorilla and chimpanzee elements share a monophyletic origin with a subset of the Old World monkey retroviral elements, but that the average sequence divergence exceeds neutral expectation for a strictly nuclear inherited DNA molecule. Within the chimpanzee, there is a significant integration bias against genes, with only 14 of these insertions mapping within intronic regions. Six out of ten of these genes, for which there are expression data, show significant differences in transcript expression between human and chimpanzee. Our data are consistent with a retroviral infection that bombarded the genomes of chimpanzees and gorillas independently and concurrently, 3-4 million years ago. We speculate on the potential impact of such recent events on the evolution of humans and great apes.

  6. [Screening of drug resistent gene by cyclical packaging rescue of hepatocellular carcinoma retroviral cDNA libraries].

    Science.gov (United States)

    Dai, Wenyan; Zhu, Ruiyu; Jin, Jian

    2016-02-01

    Multidrug resistant genes are highly expressed in hepatocellular carcinoma that seriousty affects the effect of chemotherapy. Screening of resistant genes from HCC cells and studying its mechanism of drug resistance will be helpful to improve the effecacy of chemotherapy for hepatocellular carcinoma. Here we described an alternative method called cyclical packaging rescue (CPR). First we constructed a retrovirus cDNA library of hepatoma cells and used it to infect fibroblasts. Then we added drugs to screen survival cells. The survival cells, stably integrated helper-free retroviral libraries, were recovered rapidly after transfection with plasmids expressing retroviral gag-pol and env genes. Through this method, retroviral RNAs were directly repackaged into new infectious virions. Recovered retroviral supernatant was then used to reinfect fresh target cells. When performed in concert with selection using functional assays, cDNAs regulating functional responses could be identified by enrichment through multiple rounds of retroviral library recovery and retransmission. Using CPR, we obtained several cDNAs. After a preliminary detection, we found Ribosomal protein S11 (RPS11), Ribosomal protein L6 (RPL6), Ribosomal protein L11 (RPL11), Ribosomal protein L24 (RPL24) possibly had drug resistant function.

  7. The endogenous retroviral insertion in the human complement C4 gene modulates the expression of homologous genes by antisense inhibition.

    Science.gov (United States)

    Schneider, P M; Witzel-Schlömp, K; Rittner, C; Zhang, L

    2001-02-01

    Intron 9 contains the complete endogenous retrovirus HERV-K(C4) as a 6.4-kb insertion in 60% of human C4 genes. The retroviral insertion is in reverse orientation to the C4 coding sequence. Therefore, expression of C4 could lead to the transcription of an antisense RNA, which might protect against exogenous retroviral infections. To test this hypothesis, open reading frames from the HERV sequence were subcloned in sense orientiation into a vector allowing expression of a beta-galactosidase fusion protein. Mouse L cells which had been stably transfected with either the human C4A or C4B gene both carrying the HERV insertion (LC4 cells), and L(Tk-) cells without the C4 gene were transiently transfected either with a retroviral construct or with the wild-type vector. Expression was monitored using an enzymatic assay. We demonstrated that (1) HERV-K(C4) antisense mRNA transcripts are present in cells constitutively expressing C4, (2) expression of retroviral-like constructs is significantly downregulated in cells expressing C4, and (3) this downregulation is further modulated in a dose-dependent fashion following interferon-gamma stimulation of C4 expression. These results support the hypothesis of a genomic antisense strategy mediated by the HERV-K(C4) insertion as a possible defense mechanism against exogenous retroviral infections.

  8. Seroprevalance of Helicobacter pylori amongst anti retroviral naive ...

    African Journals Online (AJOL)

    Background and objectives: HIV Infection at any stage alters patients' immunity. The pattern of several diseases including incidence and prevalence has changed due to the HIV pandemic. Infections, infestations and malignancies present more frequently and often in an unusual pattern. Helicobacter pylori is the main cause ...

  9. Retroviral transduction of murine and human hematopoietic progenitors and stem cells.

    Science.gov (United States)

    Ciuculescu, Marioara F; Brendel, Christian; Harris, Chad E; Williams, David A

    2014-01-01

    Genetic modification of cells using retroviral vectors is the method of choice when the cell population is difficult to transfect and/or requires persistent transgene expression in progeny cells. There are innumerable potential applications for these procedures in laboratory research and clinical therapeutic interventions. One paradigmatic example is the genetic modification of hematopoietic stem and progenitor cells (HSPCs). These are rare nucleated cells which reside in a specialized microenvironment within the bone marrow, and have the potential to self-renew and/or differentiate into all hematopoietic lineages. Due to their enormous regenerative capacity in steady state or under stress conditions these cells are routinely used in allogeneic bone marrow transplantation to reconstitute the hematopoietic system in patients with metabolic, inflammatory, malignant, and other hematologic disorders. For patients lacking a matched bone marrow donor, gene therapy of autologous hematopoietic stem cells has proven to be an alternative as highlighted recently by several successful gene therapy trials. Genetic modification of HSPCs using retrovirus vectors requires ex vivo manipulation to efficiently introduce the new genetic material into cells (transduction). Optimal culture conditions are essential to facilitate this process while preserving the stemness of the cells. The most frequently used retroviral vector systems for the genetic modifications of HSPCs are derived either from Moloney murine leukemia-virus (Mo-MLV) or the human immunodeficiency virus-1 (HIV-1) and are generally termed according to their genus gamma-retroviral (γ-RV) or lentiviral vectors (LV), respectively. This chapter describes in a step-by-step fashion some techniques used to produce research grade vector supernatants and to obtain purified murine or human hematopoietic stem cells for transduction, as well as follow-up methods for analysis of transduced cell populations.

  10. Cross-packaging of genetically distinct mouse and primate retroviral RNAs

    Directory of Open Access Journals (Sweden)

    Jaballah Soumeya

    2009-07-01

    Full Text Available Abstract Background The mouse mammary tumor virus (MMTV is unique from other retroviruses in having multiple viral promoters, which can be regulated by hormones in a tissue specific manner. This unique property has lead to increased interest in studying MMTV replication with the hope of developing MMTV based vectors for human gene therapy. However, it has recently been reported that related as well as unrelated retroviruses can cross-package each other's genome raising safety concerns towards the use of candidate retroviral vectors for human gene therapy. Therefore, using a trans complementation assay, we looked at the ability of MMTV RNA to be cross-packaged and propagated by an unrelated primate Mason-Pfizer monkey virus (MPMV that has intracellular assembly process similar to that of MMTV. Results Our results revealed that MMTV and MPMV RNAs could be cross-packaged by the heterologous virus particles reciprocally suggesting that pseudotyping between two genetically distinct retroviruses can take place at the RNA level. However, the cross-packaged RNAs could not be propagated further indicating a block at post-packaging events in the retroviral life cycle. To further confirm that the specificity of cross-packaging was conferred by the packaging sequences (ψ, we cloned the packaging sequences of these viruses on expression plasmids that generated non-viral RNAs. Test of these non-viral RNAs confirmed that the reciprocal cross-packaging was primarily due to the recognition of ψ by the heterologous virus proteins. Conclusion The results presented in this study strongly argue that MPMV and MMTV are promiscuous in their ability to cross-package each other's genome suggesting potential RNA-protein interactions among divergent retroviral RNAs proposing that these interactions are more complicated than originally thought. Furthermore, these observations raise the possibility that MMTV and MPMV genomes could also co-package providing substrates for

  11. Large-scale Clinical-grade Retroviral Vector Production in a Fixed-Bed Bioreactor

    Science.gov (United States)

    Wang, Xiuyan; Olszewska, Malgorzata; Qu, Jinrong; Wasielewska, Teresa; Bartido, Shirley; Hermetet, Gregory; Sadelain, Michel

    2015-01-01

    The successful genetic engineering of patient T cells with γ-retroviral vectors expressing chimeric antigen receptors or T-cell receptors for phase II clinical trials and beyond requires the large-scale manufacture of high-titer vector stocks. The production of retroviral vectors from stable packaging cell lines using roller bottles or 10- to 40-layer cell factories is limited by a narrow harvest window, labor intensity, open-system operations, and the requirement for significant incubator space. To circumvent these shortcomings, we optimized the production of vector stocks in a disposable fixed-bed bioreactor using good manufacturing practice–grade packaging cell lines. High-titer vector stocks were harvested over 10 days, representing a much broader harvest window than the 3-day harvest afforded by cell factories. For PG13 and 293Vec packaging cells, the average vector titer and the vector stocks’ yield in the bioreactor were higher by 3.2- to 7.3-fold, and 5.6- to 13.1-fold, respectively, than those obtained in cell factories. The vector production was 10.4 and 18.6 times more efficient than in cell factories for PG13 and 293Vec cells, respectively. Furthermore, the vectors produced from the fixed-bed bioreactors passed the release test assays for clinical applications. Therefore, a single vector lot derived from 293Vec is suitable to transduce up to 500 patients cell doses in the context of large clinical trials using chimeric antigen receptors or T-cell receptors. These findings demonstrate for the first time that a robust fixed-bed bioreactor process can be used to produce γ-retroviral vector stocks scalable up to the commercialization phase. PMID:25751502

  12. Maintaining therapeutic activity in the operating room: compatibility of a gamma-retroviral replicating vector with clinical materials and biofluids

    Directory of Open Access Journals (Sweden)

    Ryan Burnett

    2014-01-01

    Full Text Available Toca 511 is a novel retroviral replicating vector, encoding a modified yeast cytosine deaminase, administered to recurrent high grade glioma patients in Phase 1 trials by stereotactic, transcranial injection into the tumor or into the walls of the resection cavity. A key issue, with little published data, is vector biocompatibility with agents likely to be encountered in a neurosurgical setting. We tested biocompatibility of Toca 511 with: delivery devices; MRI contrast agents, including ProHance supporting coinjection for real time MRI-guided intratumoral delivery; hemostatic agents; biofluids (blood and cerebrospinal fluid; potential adjuvants; and a needleless vial adapter that reduces risk of accidental needle sticks. Toca 511 is stable upon thawing at ambient temperature for at least 6 hours, allowing sufficient time for administration, and its viability is not reduced in the presence of: stainless steel and silica-based delivery devices; the potential MRI contrast agent, Feraheme; ProHance at several concentrations; the hemostatic agent SURGIFOAM; blood; cerebrospinal fluid; and the needleless vial adapter. Toca 511 is not compatible with the hemostatic agent SURGICEL or with extended exposures to titanium-based biopsy needles.

  13. Mesenchymal stromal cells retrovirally transduced with prodrug-converting genes are suitable vehicles for cancer gene therapy.

    Science.gov (United States)

    Ďuriniková, E; Kučerová, L; Matúšková, M

    2014-01-01

    Mesenchymal stem/stromal cells (MSC) possess a set of several fairly unique properties which make them ideally suitable both for cellular therapies and regenerative medicine. These include: relative ease of isolation, the ability to differentiate along mesenchymal and non-mesenchymal lineages in vitro and the ability to be extensively expanded in culture without a loss of differentiative capacity. MSC are not only hypoimmunogenic, but they mediate immunosuppression upon transplantation, and possess pronounced anti-inflammatory properties. They are able to home to damaged tissues, tumors, and metastases following systemic administration. The ability of homing holds big promise for tumor-targeted delivery of therapeutic agents. Viruses are naturally evolved vehicles efficiently transferring their genes into host cells. This ability made them suitable for engineering vector systems for the delivery of genes of interest. MSC can be retrovirally transduced with genes encoding prodrug-converting genes (suicide genes), which are not toxic per se, but catalyze the formation of highly toxic metabolites following the application of a nontoxic prodrug. The homing ability of MSC holds advantages compared to virus vehicles which display many shortcomings in effective delivery of the therapeutic agents. Gene therapies mediated by viruses are limited by their restricted ability to track cancer cells infiltrating into the surrounding tissue, and by their low migratory capacity towards tumor. Thus combination of cellular therapy and gene delivery is an attractive option - it protects the vector from immune surveillance, and supports targeted delivery of a therapeutic gene/protein to the tumor site.

  14. Serious treatment related adverse drug reactions amongst anti-retroviral naïve MDR-TB patients.

    Directory of Open Access Journals (Sweden)

    Martha Van der Walt

    Full Text Available BACKGROUND: Globally treatment outcomes for multidrug-resistant Mycobacterium tuberculosis (MDR-TB remain poor and this is compounded by high drug toxicity. Little is known about the influence of adverse drug reactions (ADRs on treatment outcomes in South Africa. METHODS: We evaluated the impact of severe ADRs among a prospective cohort of MDR-TB patients in South Africa (2000-2004. The HIV-infected study participants were anti-retroviral naïve. RESULTS: Of 2,079 patients enrolled, 1,390 (66.8% were included in this analysis based on known HIV test results (39.1% HIV-infected. At least one severe ADR was reported in 83 (6.9% patients with ototoxicity being the most frequent ADR experienced (38.9%. CONCLUSIONS: We found that being HIV-infected but antiretroviral naïve did not increase occurrence of SADRs in patients on second-line anti-tuberculosis drugs. Early screening and proactive management of ADRs in this patient population is essential, especially given the rollout of decentralized care and the potential for overlapping toxicity of concomitant MDR-TB and HIV treatment.

  15. Electrolyte imbalance and sleep problems during anti-retroviral therapy: an under-recognized problem

    Directory of Open Access Journals (Sweden)

    Md Dilshad Manzar

    Full Text Available Human immunodeficiency virus (HIV infection, and the anti-retroviral therapy (ART associated complications necessitate that the medical care system keeps evolving for proper management of this group of patients. Electrolyte imbalance and sleep problems are common in patients on ART. Both of these conditions are associated with increased morbidity (such as acute kidney injury, chronic kidney disease, low CD4 count, non-adherence and depression and mortality. Therefore, screening for both sleep problems and electrolytes imbalance may help to decrease the risk of complications in patients on ART.

  16. Electrolyte imbalance and sleep problems during anti-retroviral therapy: an under-recognized problem.

    Science.gov (United States)

    Manzar, Md Dilshad; Sony, Peter; Salahuddin, Mohammed; Kumalo, Abera; Geneto, Mathewos; Pandi-Perumal, Seithikurippu R; Moscovitch, Adam; BaHammam, Ahmed S

    2017-01-01

    Human immunodeficiency virus (HIV) infection, and the anti-retroviral therapy (ART) associated complications necessitate that the medical care system keeps evolving for proper management of this group of patients. Electrolyte imbalance and sleep problems are common in patients on ART. Both of these conditions are associated with increased morbidity (such as acute kidney injury, chronic kidney disease, low CD4 count, non-adherence and depression) and mortality. Therefore, screening for both sleep problems and electrolytes imbalance may help to decrease the risk of complications in patients on ART.

  17. Spontaneous silencing of humanized green fluorescent protein (hGFP) gene expression from a retroviral vector by DNA methylation

    DEFF Research Database (Denmark)

    Gram, G J; Nielsen, S D; Hansen, J E

    1998-01-01

    packaging cells returned to untreated control levels within 2 weeks. Using flow cytometric analysis, hGFP expression was detected in up to 15% of transduced MT4 cells (a CD4+ lymphocytic cell line) after coculturing with packaging cells for 4 days. A 3-day postcoculture treatment with 5-azacytidine......We have constructed a functional murine leukemia virus (MLV)-derived retroviral vector transducing two genes encoding the autofluorescent humanized green fluorescent protein (hGFP) and neomycin phosphotransferase (Neo). This was done to determine whether hGFP could function as a marker gene...... in a retroviral vector and to investigate the expression of genes in a retroviral vector. Surprisingly, clonal vector packaging cell lines showed variable levels of hGFP expression, and expression was detected in as few as 49% of the cells in a clonally derived culture. This indicated that hGFP expression...

  18. Natural history of the ERVWE1 endogenous retroviral locus

    Directory of Open Access Journals (Sweden)

    Duret Laurent

    2005-09-01

    Full Text Available Abstract Background The human HERV-W multicopy family includes a unique proviral locus, termed ERVWE1, whose full-length envelope ORF was preserved through evolution by the action of a selective pressure. The encoded Env protein (Syncytin is involved in hominoid placental physiology. Results In order to infer the natural history of this domestication process, a comparative genomic analysis of the human 7q21.2 syntenic regions in eutherians was performed. In primates, this region was progressively colonized by LTR-elements, leading to two different evolutionary pathways in Cercopithecidae and Hominidae, a genetic drift versus a domestication, respectively. Conclusion The preservation in Hominoids of a genomic structure consisting in the juxtaposition of a retrotransposon-derived MaLR LTR and the ERVWE1 provirus suggests a functional link between both elements.

  19. Representações sociais da Aids e da Terapia Anti-retroviral para pessoas vivendo com HIV

    OpenAIRE

    Torres, Tatiana de Lucena; Camargo, Brígido Vizeu

    2009-01-01

    Este estudo objetiva caracterizar a representação social (RS) da aids e da terapia anti-retroviral (TARV) para pacientes soropositivos com adesão ao tratamento, usuários dos serviços de saúde pública em Florianópolis, Santa Catarina. Todos os participantes (N = 60) apresentaram altos escores na escala de expectativa de auto-eficácia para seguir prescrição anti-retroviral (z = 4,60; dp = 0,95), indicando adesão ao tratamento. Para análise de dados, os participantes foram divididos por sexo e c...

  20. Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences.

    Directory of Open Access Journals (Sweden)

    Rick S Mitchell

    2004-08-01

    Full Text Available The completion of the human genome sequence has made possible genome-wide studies of retroviral DNA integration. Here we report an analysis of 3,127 integration site sequences from human cells. We compared retroviral vectors derived from human immunodeficiency virus (HIV, avian sarcoma-leukosis virus (ASLV, and murine leukemia virus (MLV. Effects of gene activity on integration targeting were assessed by transcriptional profiling of infected cells. Integration by HIV vectors, analyzed in two primary cell types and several cell lines, strongly favored active genes. An analysis of the effects of tissue-specific transcription showed that it resulted in tissue-specific integration targeting by HIV, though the effect was quantitatively modest. Chromosomal regions rich in expressed genes were favored for HIV integration, but these regions were found to be interleaved with unfavorable regions at CpG islands. MLV vectors showed a strong bias in favor of integration near transcription start sites, as reported previously. ASLV vectors showed only a weak preference for active genes and no preference for transcription start regions. Thus, each of the three retroviruses studied showed unique integration site preferences, suggesting that virus-specific binding of integration complexes to chromatin features likely guides site selection.

  1. Toll-like receptor 7 controls the anti-retroviral germinal center response.

    Directory of Open Access Journals (Sweden)

    Edward P Browne

    2011-10-01

    Full Text Available The development of vaccines that can enhance immunity to viral pathogens is an important goal. However, the innate molecular pathways that regulate the strength and quality of the immune response remain largely uncharacterized. To define the role of Toll-like receptor (TLR signaling in control of a model retroviral pathogen, Friend virus (FV, I generated mice in which the TLR signaling adapter Myd88 was selectively deleted in dendritic cell (DC or in B cell lineages. Deletion of Myd88 in DCs had little effect on immune control of FV, while B cell specific deletion of Myd88 caused a dramatic increase in viral infectious centers and a significantly reduced antibody response, indicating that B cell-intrinsic TLR signaling plays a crucial role, while TLR signaling in DCs is less important. I then identified the single-stranded RNA sensing protein TLR7 as being required for antibody-mediated control of FV by analyzing mice deficient in TLR7. Remarkably, B cells in infected TLR7-deficient mice upregulated CD69 and CD86 early in infection, but failed to develop into germinal center B cells. CD4 T cell responses were also attenuated in the absence of TLR7, but CD8 responses were TLR7 independent, suggesting the existence of additional pathways for detection of retroviral particles. Together these results demonstrate that the vertebrate immune system detects retroviruses in vivo via TLR7 and that this pathway regulates a key checkpoint controlling development of germinal center B cells.

  2. Evolution of endogenous non-retroviral genes integrated into plant genomes

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    Hyosub Chu

    2014-08-01

    Full Text Available Numerous comparative genome analyses have revealed the wide extent of horizontal gene transfer (HGT in living organisms, which contributes to their evolution and genetic diversity. Viruses play important roles in HGT. Endogenous viral elements (EVEs are defined as viral DNA sequences present within the genomes of non-viral organisms. In eukaryotic cells, the majority of EVEs are derived from RNA viruses using reverse transcription. In contrast, endogenous non-retroviral elements (ENREs are poorly studied. However, the increasing availability of genomic data and the rapid development of bioinformatics tools have enabled the identification of several ENREs in various eukaryotic organisms. To date, a small number of ENREs integrated into plant genomes have been identified. Of the known non-retroviruses, most identified ENREs are derived from double-strand (ds RNA viruses, followed by single-strand (ss DNA and ssRNA viruses. At least eight virus families have been identified. Of these, viruses in the family Partitiviridae are dominant, followed by viruses of the families Chrysoviridae and Geminiviridae. The identified ENREs have been primarily identified in eudicots, followed by monocots. In this review, we briefly discuss the current view on non-retroviral sequences integrated into plant genomes that are associated with plant-virus evolution and their possible roles in antiviral resistance.

  3. Passive Immunotherapy for Retroviral Disease: Influence of Major Histocompatibility Complex Type and T-Cell Responsiveness

    Science.gov (United States)

    Hasenkrug, Kim J.; Brooks, Diane M.; Chesebro, Bruce

    1995-11-01

    Administration of virus-specific antibodies is known to be an effective early treatment for some viral infections. Such immunotherapy probably acts by antibody-mediated neutralization of viral infectivity and is often thought to function independently of T-cell-mediated immune responses. In the present experiments, we studied passive antibody therapy using Friend murine leukemia virus complex as a model for an immunosuppressive retroviral disease in adult mice. The results showed that antibody therapy could induce recovery from a well-established retroviral infection. However, the success of therapy was dependent on the presence of both CD4^+ and CD8^+ T lymphocytes. Thus, cell-mediated responses were required for recovery from infection even in the presence of therapeutic levels of antibody. The major histocompatibility type of the mice was also an important factor determining the relative success of antibody therapy in this system, but it was less critical for low-dose than for high-dose infections. Our results imply that limited T-cell responsiveness as dictated by major histocompatibility genes and/or stage of disease may have contributed to previous immunotherapy failures in AIDS patients. Possible strategies to improve the efficacy of future therapies are discussed.

  4. Identification and characterization of enhancer-blocking insulators to reduce retroviral vector genotoxicity.

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    Amy C Groth

    Full Text Available The chromatin insulator cHS4 can reduce silencing chromosomal position effects and genotoxicity associated with integrating viral vectors. However, the fully active version of this element can also reduce vector titers and is only partially effective. In order to identify alternatives to cHS4, we developed a functional lentiviral vector-based reporter screen for enhancer-blocking insulators. Using this system, we screened candidate sequences that were initially identified by chromatin profiling for binding by CTCF and for DNase hypersensitivity. All 12 analyzed candidates blocked enhancer-promoter activity. The enhancer-blocking activity of the top two candidates was confirmed in two complementary plasmid-based assays. Studies in a gammaretroviral reporter vector indicated these two candidates have little to no effect on vector titers, and do not diminish vector expression in primary mouse bone marrow cultures. Subsequent assessment in a mouse in vivo tumor formation model demonstrated that both candidates reduced the rate of gammaretroviral vector-mediated genotoxicity as effectively as the cHS4 insulator. In summary, we have developed a novel lentiviral vector-based method of screening candidate elements for insulator activity, and have used this method to identify two new insulator elements capable of improving the safety of retroviral vectors without diminishing vector titers or expression. These findings expand the limited arsenal of insulators functionally validated to reduce the rate of retroviral vector-mediated genotoxicity.

  5. Association of murine lupus and thymic full-length endogenous retroviral expression maps to a bone marrow stem cell

    International Nuclear Information System (INIS)

    Krieg, A.M.; Gourley, M.F.; Steinberg, A.D.

    1991-01-01

    Recent studies of thymic gene expression in murine lupus have demonstrated 8.4-kb (full-length size) modified polytropic (Mpmv) endogenous retroviral RNA. In contrast, normal control mouse strains do not produce detectable amounts of such RNA in their thymuses. Prior studies have attributed a defect in experimental tolerance in murine lupus to a bone marrow stem cell rather than to the thymic epithelium; in contrast, infectious retroviral expression has been associated with the thymic epithelium, rather than with the bone marrow stem cell. The present study was designed to determine whether the abnormal Mpmv expression associated with murine lupus mapped to thymic epithelium or to a marrow precursor. Lethally irradiated control and lupus-prone mice were reconstituted with T cell depleted bone marrow; one month later their thymuses were studied for endogenous retroviral RNA and protein expression. Recipients of bone marrow from nonautoimmune donors expressed neither 8.4-kb Mpmv RNA nor surface MCF gp70 in their thymuses. In contrast, recipients of bone marrow from autoimmune NZB or BXSB donors expressed thymic 8.4-kb Mpmv RNA and mink cell focus-forming gp70. These studies demonstrate that lupus-associated 8.4-kb Mpmv endogenous retroviral expression is determined by bone marrow stem cells

  6. Spontaneous silencing of humanized green fluorescent protein (hGFP) gene expression from a retroviral vector by DNA methylation

    DEFF Research Database (Denmark)

    Gram, G J; Nielsen, S D; Hansen, J E

    1998-01-01

    We have constructed a functional murine leukemia virus (MLV)-derived retroviral vector transducing two genes encoding the autofluorescent humanized green fluorescent protein (hGFP) and neomycin phosphotransferase (Neo). This was done to determine whether hGFP could function as a marker gene...

  7. Green fluorescent protein as a selectable marker of fibronectin-facilitated retroviral gene transfer in primary human T lymphocytes

    NARCIS (Netherlands)

    Dardalhon, V.; Noraz, N.; Pollok, K.; Rebouissou, C.; Boyer, M.; Bakker, A. Q.; Spits, H.; Taylor, N.

    1999-01-01

    The success of gene therapy strategies for congenital and acquired blood disorders requires high levels of gene transfer into hematopoietic cells. Retroviral vectors have been extensively used to deliver foreign genes to mammalian cells and improvement of transduction protocols remains dependent on

  8. High-definition mapping of retroviral integration sites defines the fate of allogeneic T cells after donor lymphocyte infusion.

    Directory of Open Access Journals (Sweden)

    Claudia Cattoglio

    2010-12-01

    Full Text Available The infusion of donor lymphocytes transduced with a retroviral vector expressing the HSV-TK suicide gene in patients undergoing hematopoietic stem cell transplantation for leukemia/lymphoma promotes immune reconstitution and prevents infections and graft-versus-host disease. Analysis of the clonal dynamics of genetically modified lymphocytes in vivo is of crucial importance to understand the potential genotoxic risk of this therapeutic approach. We used linear amplification-mediated PCR and pyrosequencing to build a genome-wide, high-definition map of retroviral integration sites in the genome of peripheral blood T cells from two different donors and used gene expression profiling and bioinformatics to associate integration clusters to transcriptional activity and to genetic and epigenetic features of the T cell genome. Comparison with matched random controls and with integrations obtained from CD34(+ hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion. Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing. The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction.

  9. Immune responses to transgene and retroviral vector in patients treated with ex vivo-engineered T cells

    NARCIS (Netherlands)

    Lamers, C.H.; Willemsen, R.; Elzakker, P. van; Steenbergen-Langeveld, S. van; Broertjes, M.; Oosterwijk-Wakka, J.C.; Oosterwijk, E.; Sleijfer, S.; Debets, R.; Gratama, J.W.

    2011-01-01

    Adoptive transfer of immune effector cells that are gene modified by retroviral transduction to express tumor-specific receptors constitutes an attractive approach to treat cancer. In patients with metastatic renal cell carcinoma, we performed a study with autologous T cells genetically retargeted

  10. The effects of decentralizing anti-retroviral services in Nigeria on costs and service utilization: two case studies.

    Science.gov (United States)

    Johns, Benjamin; Baruwa, Elaine

    2016-03-01

    Nigeria launched a 'hub and spoke' decentralization pilot in March 2010 for the provision of anti-retroviral therapy (ART). In this programme, stable ART patients at hospitals (hubs) were referred to primary health care centres (spokes) for the continued provision of ART. The objectives of this study are to compare the cost of ART care provided through the two levels of care. We also assess if decentralization was associated with changes in patients' service utilization. Data were collected from facilities and patient records from Kaduna and Cross Rivers States. Costs were collected from the provider perspective. In Cross River, 398 patients and 528 from Kaduna were included in the retrospective cohort. The analysis utilizes separate fixed effect regressions for each state to assess differences in costs and service utilization among patients that decentralized. Uptake of decentralized services was ∼3% in Cross Rivers and ∼9% in Kaduna among active ART patients in April 2011. Patients electing to decentralize had 40% (95% CI: 13% to 67%) higher costs in Cross Rivers and 29% (-44% to -14%) lower costs in Kaduna as compared with patients that did not decentralize. Lower costs in Kaduna appear to result from shifting care to less expensive cadres of health workers (task shifting) rather than decentralization. Decentralization of health services is a complicated process and broad generalizations across settings and processes, concerning whether or not it reduces unit costs, are likely over-simplifications. Similarly, decentralization of ART services does not automatically increase access to ART care, and may limit access to ART laboratory services. This study is limited by not including costs incurred above the facility level, such as training, or costs borne by patients. © The Author 2015. Published by Oxford University Press in association with The London School of Hygiene and Tropical Medicine.

  11. Creation and validation of a ligation-independent cloning (LIC retroviral vector for stable gene transduction in mammalian cells

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    Patel Asmita

    2012-01-01

    Full Text Available Abstract Background Cloning vectors capable of retroviral transduction have enabled stable gene overexpression in numerous mitotic cell lines. However, the relatively small number of feasible restriction enzyme sequences in their cloning sites can hinder successful generation of overexpression constructs if these sequences are also present in the target cDNA insert. Results Utilizing ligation-independent cloning (LIC technology, we have modified the highly efficient retroviral transduction vector, pBABE, to eliminate reliance on restriction enzymes for cloning. Instead, the modified plasmid, pBLIC, utilizes random 12/13-base overhangs generated by T4 DNA polymerase 3' exonuclease activity. PCR-based introduction of the complementary sequence into any cDNA of interest enables universal cloning into pBLIC. Here we describe creation of the pBLIC plasmid, and demonstrate successful cloning and protein overexpression from three different cDNAs, Bax, catalase, and p53 through transduction into the human prostate cancer cell line, LNCaP or the human lung cancer line, H358. Conclusions Our results show that pBLIC vector retains the high transduction efficiency of the original pBABE while eliminating the requirement for checking individual cDNA inserts for internal restriction sites. Thus it comprises an effective retroviral cloning system for laboratory-scale stable gene overexpression or for high-throughput applications such as creation of retroviral cDNA libraries. To our knowledge, pBLIC is the first LIC vector for retroviral transduction-mediated stable gene expression in mammalian cells.

  12. Involvement of human endogenous retroviral syncytin-1 in human osteoclast fusion

    DEFF Research Database (Denmark)

    Søe, Kent; Andersen, Thomas Lykke; Hobolt-Pedersen, Anne-Sofie

    2011-01-01

    Generation of osteoclasts through fusion of mono-nucleated precursors is a key event of bone physiology and bone resorption is inefficient without osteoclast fusion. Several factors playing a critical role in the fusion process have already been recognized, but the factors involved in the actual...... fusion of the lipid bilayers of their cell membranes are still unknown. Syncytin-1 is a protein encoded by a human endogenous retroviral gene which was stably integrated into the human ancestor genome more than 24 million years ago. Upon activation, syncytin-1 is able to destabilize the lipid bilayer....... This was documented through Q-PCR, Western blot and immunofluorescence analyses. These in vitro findings were confirmed by immunohistochemical stainings in human iliac crest biopsies. A syncytin-1 inhibitory peptide reduced the number of nuclei per osteoclast by 30%, as well as TRACP activity. From a mechanistic...

  13. An audit on virological efficacy of anti-retroviral therapy in a specialist infectious disease clinic.

    LENUS (Irish Health Repository)

    Reyad, A

    2009-06-01

    We have assessed the efficacy of anti retroviral therapy (ART) using undetectable viral load (VL) (<50 RNA copies\\/ml) as a marker of virological success, in patients who have Human Immunodeficiency Virus (HIV) attending the Department of Infectious Disease. A cross-sectional review of patients\\' case notes was used to obtain their demographics and treatment details. 79% (253) of the hospital case notes of clinic population was available for analysis, which represents 90% of those receiving ART in the clinic. 166\\/253 of the cohort were receiving treatment at the time of this study and 95% (157\\/166) of these were on treatment for greater than 6 months. The total virological success rate is 93%, which is comparable to other centres and are as good as those from published clinical trials. 56% of those on therapy who have virological failure were Intravenous Drug Users (IVDUs). Case by case investigation for those with treatment failure is warranted.

  14. Apoptosis Gene Hunting Using Retroviral Expression Cloning: Identification of Vacuolar ATPase Subunit E

    Directory of Open Access Journals (Sweden)

    Claire L. Anderson

    2003-01-01

    Full Text Available Over the past 10-15 years there has been an explosion of interest in apoptosis. The delayed realisation that cell death is an essential part of life for any multicellular organism has meant that, despite the recent and rapid developments of the last decade, the precise biochemical pathways involved in apoptosis remain incomplete and potentially novel genes may, as yet, remain undiscovered. The hunt is therefore on to bridge the remaining gaps in our knowledge. Our contribution to this research effort utilises a functional cloning approach to isolate important regulatory genes involved in apoptosis. This mini-review focuses on the use and advantages of a retroviral expression cloning strategy and describes the isolation and identification of one such potential apoptosis regulatory gene, namely that encoding vacuolar ATPase subunit E.

  15. Multidrug resistance and retroviral transduction potential in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Theilade, M D; Gram, G J; Jensen, P B

    1999-01-01

    Multidrug resistance (MDR) remains a major problem in the successful treatment of small cell lung cancer (SCLC). New treatment strategies are needed, such as gene therapy specifically targeting the MDR cells in the tumor. Retroviral LacZ gene-containing vectors that were either pseudotyped...... for the gibbon ape leukemia virus (GALV-1) receptor or had specificity for the amphotropic murine leukemia virus (MLV-A) receptor were used for transduction of five SCLC cell lines differing by a range of MDR mechanisms. Transduction efficiencies in these cell lines were compared by calculating the percentage...... of blue colonies after X-Gal staining of the cells grown in soft agar. All examined SCLC cell lines were transducible with either vector. Transduction efficiencies varied from 5.7% to 33.5% independent of the presence of MDR. These results indicate that MDR does not severely impair transduction of SCLC...

  16. Interleukin-Encoding Adenoviral Vectors as Genetic Adjuvant for Vaccination against Retroviral Infection

    Science.gov (United States)

    Ohs, Inga; Windmann, Sonja; Wildner, Oliver; Dittmer, Ulf; Bayer, Wibke

    2013-01-01

    Interleukins (IL) are cytokines with stimulatory and modulatory functions in the immune system. In this study, we have chosen interleukins which are involved in the enhancement of TH2 responses and B cell functions to analyze their potential to improve a prophylactic adenovirus-based anti-retroviral vaccine with regard to antibody and virus-specific CD4+ T cell responses. Mice were vaccinated with an adenoviral vector which encodes and displays the Friend Virus (FV) surface envelope protein gp70 (Ad.pIXgp70) in combination with adenoviral vectors encoding the interleukins IL4, IL5, IL6, IL7 or IL23. Co-application of Ad.pIXgp70 with Ad.IL5, Ad.IL6 or Ad.IL23 resulted in improved protection with high control over FV-induced splenomegaly and reduced viral loads. Mice co-immunized with adenoviral vectors encoding IL5 or IL23 showed increased neutralizing antibody responses while mice co-immunized with Ad.IL6 or Ad.IL23 showed improved FV-specific CD4+ T cell responses compared to mice immunized with Ad.pIXgp70 alone. We show that the co-application of adenoviral vectors encoding specific interleukins is suitable to improve the vaccination efficacy of an anti-retroviral vaccine. Improved protection correlated with improved CD4+ T cell responses and especially with higher neutralizing antibody titers. The co-application of selected interleukin-encoding adenoviral vectors is a valuable tool for vaccination with regard to enhancement of antibody mediated immunity. PMID:24349306

  17. Retroviral-mediated gene therapy for the differentiation of primary cells into a mineralizing osteoblastic phenotype.

    Science.gov (United States)

    Phillips, Jennifer E; García, Andrés J

    2008-01-01

    Bone tissue engineering has emerged as a promising strategy for the repair of critical-sized skeletal fractures. However, the clinical application of this approach has been limited by the availability of a robust mineralizing cell source. Non-osteogenic cells, such as skin fibroblasts, are an attractive cell-source alternative because they are easy to harvest from autologous donor skin biopsies and display a high capacity for in vitro expansion. We have recently demonstrated that retroviral gene delivery of the osteoblastic transcription factor Runx2/Cbfa1 promotes osteogenic differentiation in primary dermal fibroblasts cultured in monolayer. Notably, sustained expression of Runx2 was not sufficient to promote functional osteogenesis in these cells, and co-treatment with the steroid hormone dexamethasone was required to induce deposition of biologically-equivalent matrix mineralization. On the basis of these results, we then investigated the osteogenic capacity of these genetically engineered fibroblasts when seeded on polymeric scaffolds in vitro and in vivo. These experiments demonstrated that Runx2-expressing fibroblasts seeded on collagen scaffolds produce significant levels of matrix mineralization after 28 days in vivo implantation in a subcutaneous, heterotopic site. Overall, these results offer evidence that transcription factor-based gene therapy may be a powerful strategy for the conversion of a non-osteogenic cellular phenotype into a mineralizing cell source for bone repair applications. This concept may also be applied to control functional differentiation in a broad range of cell types and tissue engineering applications. The chapter below outlines detailed methods for the isolation and ex vivo genetic modification of primary dermal fibroblasts using retroviral-mediated delivery of the Runx2 transgene in both monolayer culture and three-dimensional scaffolds.

  18. Defects in cellular sorting and retroviral assembly induced by GGA overexpression

    Directory of Open Access Journals (Sweden)

    Nagashima Kunio

    2009-09-01

    Full Text Available Abstract Background We previously demonstrated that overexpression of Golgi-localized, γ-ear containing, Arf-binding (GGA proteins inhibits retrovirus assembly and release by disrupting the function of endogenous ADP ribosylation factors (Arfs. GGA overexpression led to the formation of large, swollen vacuolar compartments, which in the case of GGA1 sequestered HIV-1 Gag. Results In the current study, we extend our previous findings to characterize in depth the GGA-induced compartments and the determinants for retroviral Gag sequestration in these structures. We find that GGA-induced structures are derived from the Golgi and contain aggresome markers. GGA overexpression leads to defects in trafficking of transferrin receptor and recycling of cation-dependent mannose 6-phosphate receptor. Additionally, we find that compartments induced by GGA overexpression sequester Tsg101, poly-ubiquitin, and, in the case of GGA3, Hrs. Interestingly, brefeldin A treatment, which leads to the dissociation of endogenous GGAs from membranes, does not dissociate the GGA-induced compartments. GGA mutants that are defective in Arf binding and hence association with membranes also induce the formation of GGA-induced structures. Overexpression of ubiquitin reverses the formation of GGA-induced structures and partially rescues HIV-1 particle production. We found that in addition to HIV-1 Gag, equine infectious anemia virus Gag is also sequestered in GGA1-induced structures. The determinants in Gag responsible for sequestration map to the matrix domain, and recruitment to these structures is dependent on Gag membrane binding. Conclusion These data provide insights into the composition of structures induced by GGA overexpression and their ability to disrupt endosomal sorting and retroviral particle production.

  19. Feline mediastinal lymphoma: a retrospective study of signalment, retroviral status, response to chemotherapy and prognostic indicators.

    Science.gov (United States)

    Fabrizio, Francesca; Calam, Amy E; Dobson, Jane M; Middleton, Stephanie A; Murphy, Sue; Taylor, Samantha S; Schwartz, Anita; Stell, Anneliese J

    2014-08-01

    Historically, feline mediastinal lymphoma has been associated with young age, positive feline leukaemia virus (FeLV) status, Siamese breed and short survival times. Recent studies following widespread FeLV vaccination in the UK are lacking. The aim of this retrospective multi-institutional study was to re-evaluate the signalment, retroviral status, response to chemotherapy, survival and prognostic indicators in feline mediastinal lymphoma cases in the post-vaccination era. Records of cats with clinical signs associated with a mediastinal mass and cytologically/histologically confirmed lymphoma were reviewed from five UK referral centres (1998-2010). Treatment response, survival and prognostic indicators were assessed in treated cats with follow-up data. Fifty-five cases were reviewed. The median age was 3 years (range, 0.5-12 years); 12 cats (21.8%) were Siamese; and the male to female ratio was 3.2:1.0. Five cats were FeLV-positive and two were feline immunodeficiency-positive. Chemotherapy response and survival was evaluated in 38 cats. Overall response was 94.7%; complete (CR) and partial response (PR) rates did not differ significantly between protocols: COP (cyclophosphamide, vincristine, prednisone) (n = 26, CR 61.5%, PR 34.0%); Madison-Wisconsin (MW) (n = 12, CR 66.7%, PR 25.0%). Overall median survival was 373 days (range, 20-2015 days) (COP 484 days [range, 20-980 days]; MW 211 days [range, 24-2015 days] [P = 0.892]). Cats achieving CR survived longer (980 days vs 42 days for PR; P = 0.032). Age, breed, sex, location (mediastinal vs mediastinal plus other sites), retroviral status and glucocorticoid pretreatment did not affect response or survival. Feline mediastinal lymphoma cases frequently responded to chemotherapy with durable survival times, particularly in cats achieving CR. The prevalence of FeLV-antigenaemic cats was low; males and young Siamese cats appeared to be over-represented. © ISFM and AAFP 2013.

  20. RAPID COMMUNICATION: Expression of an endogenous retroviral element, during early gestation in beef heifers.

    Science.gov (United States)

    McLean, K J; Crouse, M S; Crosswhite, M R; Black, D N; Dahlen, C R; Borowicz, P P; Reynolds, L P; Ward, A K; Neville, B W; Caton, J S

    2016-10-01

    Endogenous retroviral gene elements have been implicated in development and formation of the feto-maternal interface. A variant of the syncytin endogenous retroviral envelope gene family, , was recently found in ruminants. We hypothesized that mRNA would be differentially expressed in utero-placental tissues and would fluctuate during key time points of early gestation in beef heifers. Commercial Angus crossbred heifers ( = 46; ∼15 mo of age; BW = 362.3 ± 34.7kg) housed in 6-animal pens were fed daily with native grass hay and supplemented with cracked corn to gain 0.3 kg/d. The heifers were estrus synchronized, artificially inseminated, (d of breeding= d 0) and ovariohysterectomized on d 16, 22, 28, 34, 40, and 50 ( = 9, 6, 6, 7, 6, and 5, respectively) of gestation and at d 16 of the estrous cycle for non-bred, non-pregnant controls (NP; = 7). Harvested tissues were separated into maternal caruncle (CAR), intercarunclar endometrium (ICAR), and fetal membranes, (FM; chorioallantois, d 22 and later). All tissues were obtained from the ipsilateral uterine horn to the CL. Statistical analyses were conducted via the GLM procedure of SAS. Maternal CAR expression of was greater ( = 0.003) on d 50 by 81.5-fold compared to NP controls. At d 50 expression of in CAR was 190.3-fold greater than ( gestation compared to maternal ICAR (d 16 not analyzed). Expression of in FM was greater ( gestation. Therefore, we conclude that is differentially expressed in utero-placental tissues and may be involved in the establishment of pregnancy. The expression of in maternal tissues is completely novel and indicates unique functions of syncytin in ruminant pregnancy.

  1. An amphotropic retroviral vector expressing a mutant gsp oncogene: effects on human thyroid cells in vitro.

    Science.gov (United States)

    Ivan, M; Ludgate, M; Gire, V; Bond, J A; Wynford-Thomas, D

    1997-08-01

    Point mutations of the gsp protooncogene (encoding the alpha-subunit of the Gs protein) that constitutively activate the cAMP signaling pathway are a common feature of and a plausible causative mechanism for thyroid hyperfunctioning adenomas (hot nodules). To investigate the extent to which mutant gsp acting alone can induce proliferation of thyroid follicular cells, we generated an amphotropic retroviral vector (based on the pBABE-neo plasmid and psi-CRIP packaging line) to permit stable introduction of a hemagglutinin-tagged Gln227-->Leu mutant gsp gene into normal human thyrocytes in vitro. The biological activity of the vector was confirmed by detection of HA-tagged Gsp protein expression and induction of cAMP synthesis in selected target cells. Normal human thyroid follicular cells in primary monolayer culture were infected with the gsp retroviral vector or with corresponding vectors expressing mutant H-ras or neo only as positive and negative controls, respectively. Although, as before, mutant ras generated 10-20 well differentiated epithelial colonies/dish of 10(5) infected cells, with an average lifespan of 15-20 population doublings, only small groups of no more than 15-50 differentiated thyrocytes were observed with the gsp vector. In addition to standard conditions (10% FCS), infections were performed in reduced serum (1% FCS, TSH, and insulin), in the presence of isobutylylmethylxanthine, or in the presence of agents capable of closing gap junctions, with no significant difference in outcome. Although little or no proliferative response was observed regardless of the conditions, there was clear evidence of morphological response (rearrangement of the actin cytoskeleton and increased cell size). The results suggest that gsp mutation may not be a sufficient proliferogenic stimulus by itself to account for hot nodule formation.

  2. Size distribution of retrovirally marked lineages matches prediction from population measurements of cell cycle behavior

    Science.gov (United States)

    Cai, Li; Hayes, Nancy L.; Takahashi, Takao; Caviness, Verne S Jr; Nowakowski, Richard S.

    2002-01-01

    Mechanisms that regulate neuron production in the developing mouse neocortex were examined by using a retroviral lineage marking method to determine the sizes of the lineages remaining in the proliferating population of the ventricular zone during the period of neuron production. The distribution of clade sizes obtained experimentally in four different injection-survival paradigms (E11-E13, E11-E14, E11-E15, and E12-E15) from a total of over 500 labeled lineages was compared with that obtained from three models in which the average behavior of the proliferating population [i.e., the proportion of cells remaining in the proliferative population (P) vs. that exiting the proliferative population (Q)] was quantitatively related to lineage size distribution. In model 1, different proportions of asymmetric, symmetric terminal, and symmetric nonterminal cell divisions coexisted during the entire developmental period. In model 2, the developmental period was divided into two epochs: During the first, asymmetric and symmetric nonterminal cell divisions occurred, but, during the second, asymmetric and symmetric terminal cell divisions occurred. In model 3, the shifts in P and Q are accounted for by changes in the proportions of the two types of symmetric cell divisions without the inclusion of any asymmetric cell divisions. The results obtained from the retroviral experiments were well accounted for by model 1 but not by model 2 or 3. These findings demonstrate that: 1) asymmetric and both types of symmetric cell divisions coexist during the entire period of neurogenesis in the mouse, 2) neuron production is regulated in the proliferative population by the independent decisions of the two daughter cells to reenter S phase, and 3) neurons are produced by both asymmetric and symmetric terminal cell divisions. In addition, the findings mean that cell death and/or tangential movements of cells in the proliferative population occur at only a low rate and that there are no

  3. Effect of Micronutrient and Probiotic Fortified Yogurt on Immune-Function of Anti-Retroviral Therapy Naive HIV Patients  

    Directory of Open Access Journals (Sweden)

    J. Dik F. Habbema

    2011-10-01

    Full Text Available Background: Micronutrient supplementation has been shown to reduce the progression of HIV but does not have an effect on the intestinal barrier or the intestinal microbiota of HIV patients. Studies have suggested that probiotics could potentially complement micronutrients in preserving the immune-function of HIV patients. Objective: Assess the impact of micronutrient supplemented probiotic yogurt on the immune function of HIV patients. Design: We performed a randomized, double blind, controlled trial with CD4 count as primary outcome among HIV patients naïve to anti-retroviral treatment. Secondary outcomes included hematological parameters, incidence of diarrhea and clinical symptoms. A total of 112 HIV patients were randomized to receive a micronutrient fortified yogurt with (n = 55 or without additional probiotic Lactobacillus rhamnosus GR-1 (n = 57 for four weeks. Results: An average decline in CD4 count of −70 cells/μL (95% CI: −154 to −15 was observed in the micronutrient, probiotic group versus a decrease of −63 cells/μL (95% CI: −157 to −30 in the micronutrient control group (p = 0.9. Additional probiotic supplementation was well tolerated and not associated with adverse events. No difference between groups was detected in incidence of diarrhea or clinical symptoms. An improvement of hemoglobin levels was observed for all subjects, based upon a mean difference from baseline of 1.4 g/L (SD = 6 (p = 0.02. Conclusion: The addition of probiotics to a micronutrient fortified yogurt was well tolerated by HIV patients but was not associated with a further increase in CD4 count after one month.

  4. A novel, Q-PCR based approach to measuring endogenous retroviral clearance by capture protein A chromatography.

    Science.gov (United States)

    Zhang, Min; Lute, Scott; Norling, Lenore; Hong, Connie; Safta, Aurelia; O'Connor, Deborah; Bernstein, Lisa J; Wang, Hua; Blank, Greg; Brorson, Kurt; Chen, Qi

    2009-04-01

    Quantification of virus removal by the purification process during production is required for clinical use of biopharmaceuticals. The current validation approach for virus removal by chromatography steps typically involves time-consuming spiking experiments with expensive model viruses at bench scale. Here we propose a novel, alternative approach that can be applied in at least one instance: evaluating retroviral clearance by protein A chromatography. Our strategy uses a quantitative PCR (Q-PCR) assay that quantifies the endogenous type C retrovirus-like particle genomes directly in production Chinese Hamster Ovary (CHO) cell culture harvests and protein A pools. This eliminates the need to perform spiking with model viruses, and measures the real virus from the process. Using this new approach, clearance values were obtained that was comparable to those from the old model-virus spike/removal approach. We tested the concept of design space for CHO retrovirus removal using samples from a protein A characterization study, where a wide range of chromatographic operating conditions were challenged, including load density, flow rate, wash, pooling, temperature, and resin life cycles. Little impact of these variables on CHO retrovirus clearance was found, arguing for implementation of the design space approach for viral clearance to support operational ranges and manufacturing excursions. The viral clearance results from Q-PCR were confirmed by an orthogonal quantitative product-enhanced reverse transcriptase (Q-PERT) assay that quantifies CHO retrovirus by their reverse transcriptase (RT) enzyme activity. Overall, our results demonstrate that protein A chromatography is a robust retrovirus removal step and CHO retrovirus removal can be directly measured at large scale using Q-PCR assays.

  5. Characterization of Ovine A3Z1 Restriction Properties against Small Ruminant Lentiviruses (SRLVs

    Directory of Open Access Journals (Sweden)

    Lorena de Pablo-Maiso

    2017-11-01

    Full Text Available Intrinsic factors of the innate immune system include the apolipoprotein B editing enzyme catalytic polypeptide-like 3 (APOBEC3 protein family. APOBEC3 inhibits replication of different virus families by cytosine deamination of viral DNA and a not fully characterized cytosine deamination-independent mechanism. Sheep are susceptible to small ruminant lentivirus (SRLVs infection and contain three APOBEC3 genes encoding four proteins (A3Z1, Z2, Z3 and Z2-Z3 with yet not deeply described antiviral properties. Using sheep blood monocytes and in vitro-derived macrophages, we found that A3Z1 expression is associated with lower viral replication in this cellular type. A3Z1 transcripts may also contain spliced variants (A3Z1Tr lacking the cytidine deaminase motif. A3Z1 exogenous expression in fully permissive fibroblast-like cells restricted SRLVs infection while A3Z1Tr allowed infection. A3Z1Tr was induced after SRLVs infection or stimulation of blood-derived macrophages with interferon gamma (IFN-γ. Interaction between truncated isoform and native A3Z1 protein was detected as well as incorporation of both proteins into virions. A3Z1 and A3Z1Tr interacted with SRLVs Vif, but this interaction was not associated with degradative properties. Similar A3Z1 truncated isoforms were also present in human and monkey cells suggesting a conserved alternative splicing regulation in primates. A3Z1-mediated retroviral restriction could be constrained by different means, including gene expression and specific alternative splicing regulation, leading to truncated protein isoforms lacking a cytidine-deaminase motif.

  6. Mutation of Breast Cancer Cell Genomic DNA by APOBEC3B

    Science.gov (United States)

    2013-09-01

    salivary gland RNA for head and neck cancer and stomach RNA... Gland n.a. n.a. n.a. normal Hs578Bst Immortalized Mammary Gland - n.a. n.a. normal 184B5 Immortalized Mammary Gland n.a. n.a. n.a. normal HCC38 Cancer ...Carcinoma + + - normal DU4475 Cancer Mammary Gland n.a. n.a. n.a. normal BT-549 Cancer Papillary, Invasive Ductal Tumor n.a. n.a. n.a. mutant BT-483 Cancer

  7. Multifaceted counter-APOBEC3G mechanisms employed by HIV-1 Vif.

    Science.gov (United States)

    Britan-Rosich, Elena; Nowarski, Roni; Kotler, Moshe

    2011-07-29

    In the absence of human immunodeficiency virus type 1 (HIV-1) Vif protein, the host antiviral deaminase apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (A3G) restricts the production of infectious HIV-1 by deamination of dC residues in the negative single-stranded DNA produced by reverse transcription. The Vif protein averts the lethal threat of deamination by precluding the packaging of A3G into assembling virions by mediating proteasomal degradation of A3G. In spite of this robust Vif activity, residual A3G molecules that escape degradation and incorporate into newly assembled virions are potentially deleterious to the virus. We hypothesized that virion-associated Vif inhibits A3G enzymatic activity and therefore prevents lethal mutagenesis of the newly synthesized viral DNA. Here, we show that (i) Vif-proficient HIV-1 particles released from H9 cells contain A3G with lower specific activity compared with Δvif-virus-associated A3G, (ii) encapsidated HIV-1 Vif inhibits the deamination activity of recombinant A3G, and (iii) purified HIV-1 Vif protein and the Vif-derived peptide Vif25-39 inhibit A3G activity in vitro at nanomolar concentrations in an uncompetitive manner. Our results manifest the potentiality of Vif to control the deamination threat in virions or in the pre-integration complexes following entry to target cells. Hence, virion-associated Vif could serve as a last line of defense, protecting the virus against A3G antiviral activity. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. Targeted transgene insertion into the CHO cell genome using Cre recombinase-incorporating integrase-defective retroviral vectors.

    Science.gov (United States)

    Kawabe, Yoshinori; Shimomura, Takuya; Huang, Shuohao; Imanishi, Suguru; Ito, Akira; Kamihira, Masamichi

    2016-07-01

    Retroviral vectors have served as efficient gene delivery tools in various biotechnology fields. However, viral DNA is randomly inserted into the genome, which can cause problems, such as insertional mutagenesis and gene silencing. Previously, we reported a site-specific gene integration system, in which a transgene is integrated into a predetermined chromosomal locus of Chinese hamster ovary (CHO) cells using integrase-defective retroviral vectors (IDRVs) and Cre recombinase. In this system, a Cre expression plasmid is transfected into founder cells before retroviral transduction. In practical applications of site-specific gene modification such as for hard-to-transfect cells or for in vivo gene delivery, both the transgene and the Cre protein into retroviral virions should be encapsulate. Here, we generated novel hybrid IDRVs in which viral genome and enzymatically active Cre can be delivered (Cre-IDRVs). Cre-IDRVs encoding marker genes, neomycin resistance and enhanced green fluorescent protein (EGFP), flanked by wild-type and mutated loxP sites were produced using an expression plasmid for a chimeric protein of Cre and retroviral gag-pol. After analyzing the incorporation of the Cre protein into retroviral virions by Western blotting, the Cre-IDRV was infected into founder CHO cells, in which marker genes (hygromycin resistance and red fluorescent protein) flanked with corresponding loxP sites are introduced into the genome. G418-resistant colonies expressing GFP appeared and the site-specific integration of the transgene into the expected chromosomal site was confirmed by PCR and sequencing of amplicons. Moreover, when Cre-IDRV carried a gene expression unit for a recombinant antibody, the recombinant cells in which the antibody expression cassette was integrated in a site-specific manner were generated and the cells produced the recombinant antibody. This method may provide a promising tool to perform site-specific gene modification according to Cre

  9. Manifestações otoneurológicas associadas à terapia anti-retroviral Otoneurological manifestations associated with antiretroviral therapy

    Directory of Open Access Journals (Sweden)

    Andrêza Batista Cheloni Vieira

    2008-02-01

    Full Text Available Ototoxicidade e terapia anti-retroviral parecem estar associadas. O objetivo desse estudo foi avaliar essa possível correlação. Foram avaliados 779 prontuários médicos de pacientes infectados pelo HIV e regularmente acompanhados, sendo 162 tratados com terapia anti-retroviral e 122 não tratados (controle. Pacientes em tratamento eram mais velhos (média 42 anos, com maior tempo de confirmação sorológica (80 meses e com menor carga viral (p=0,00. CD4+ foi semelhante entre os grupos (P=0,60. No grupo tratado, três (1,8% casos de perda auditiva idiopática e dois (1,3% de perda auditiva relacionada a otosclerose foram observadas e ambas iniciadas após terapia anti-retroviral. Nenhuma diferença estatística relacionada à perda auditiva idiopática foi encontrada entre os grupos. Enquanto estudos descritivos consideram possível ototoxidade associada à terapia anti-retroviral, esse possível efeito adverso não foi relacionado à terapia anti-retroviral neste estudo. Contrariamente, otosclerose poderia estar correlacionada à terapia anti-retroviral. Este assunto merece ser estudado.Ototoxicity and antiretroviral therapy seem to be associated. The aim of this study was to evaluate this possible correlation. Evaluations were carried out on 779 medical records from HIV-infected patients who were being regularly followed up, of whom 162 were being treated with antiretroviral therapy and 122 were untreated (controls. The patients undergoing treatment were older (mean: 42 years, had had serological confirmation for longer times (80 months and had smaller viral loads (P = 0.00. CD4+ was similar between the groups (P = 0.60. In the treated group, three cases (1.8% of idiopathic hearing loss and two (1.3% of otosclerosis-related hearing loss were observed, which both started after antiretroviral therapy. No statistical difference relating to idiopathic hearing loss was found between the groups. While descriptive studies consider possible

  10. Complementation of a primer binding site-impaired murine leukemia virus-derived retroviral vector by a genetically engineered tRNA-like primer

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Duch, M; Lovmand, J

    1997-01-01

    , but not with a noncomplementary tRNA-like molecule. The engineered primer was shown to be involved in both the initiation of first-strand synthesis and second-strand transfer. These results provide an in vivo demonstration that the retroviral replication machinery may recognize sequence complementarity rather than actual primer...... binding site and 3' primer sequences. Use of mutated primer binding site vectors replicating via engineered primers may add additional control features to retroviral gene transfer technology....

  11. Type A-like retroviral particles in a metastatic intestinal adenocarcinoma in an emerald tree boa (Corallus caninus).

    Science.gov (United States)

    Orós, J; Lorenzo, H; Andrada, M; Recuero, J

    2004-09-01

    A metastatic intestinal papillary adenocarcinoma was diagnosed histologically in an emerald tree boa (Corallus caninus). Metastasis was detected in the liver, both kidneys, lung, and coelomic wall. Ultrastructural examination of the metastatic intestinal epithelial cells in the liver revealed the presence of a moderate number of viral particles that most closely resembled type A retroviral particles and were mainly associated with granular endoplasmic reticulum membranes. This case is the first description of type A-like retroviral particles in a neoplasm of a snake. The role of the virions in the etiology of the intestinal adenocarcinoma is uncertain. In addition, this is the first confirmed report of a metastatic intestinal adenocarcinoma in a snake.

  12. Spectrum of imaging appearances of intracranial cryptococcal infection in HIV/AIDS patients in the anti-retroviral therapy era

    International Nuclear Information System (INIS)

    Offiah, Curtis E.; Naseer, Aisha

    2016-01-01

    Cryptococcus neoformans infection is the most common fungal infection of the central nervous system (CNS) in advanced human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) patients, but remains a relatively uncommon CNS infection in both the immunocompromised and immunocompetent patient population, rendering it a somewhat elusive and frequently overlooked diagnosis. The morbidity and mortality associated with CNS cryptococcal infection can be significantly reduced by early recognition of the imaging appearances by the radiologist in order to focus and expedite clinical management and treatment. The emergence and evolution of anti-retroviral therapy have also impacted significantly on the imaging appearances, morbidity, and mortality of this neuro-infection. The constellation of varied imaging appearances associated with cryptococcal CNS infection in the HIV and AIDS population in the era of highly active anti-retroviral therapy (HAART) will be presented in this review.

  13. TRIM5α SPRY/coiled-coil interactions optimize avid retroviral capsid recognition.

    Directory of Open Access Journals (Sweden)

    Marcin D Roganowicz

    2017-10-01

    Full Text Available Restriction factors are important components of intrinsic cellular defense mechanisms against viral pathogens. TRIM5α is a restriction factor that intercepts the incoming capsid cores of retroviruses such as HIV and provides an effective species-specific barrier to retroviral infection. The TRIM5α SPRY domain directly binds the capsid with only very weak, millimolar-level affinity, and productive capsid recognition therefore requires both TRIM5α dimerization and assembly of the dimers into a multivalent hexagonal lattice to promote avid binding. Here, we explore the important unresolved question of whether the SPRY domains are flexibly linked to the TRIM lattice or more precisely positioned to maximize avidity. Biochemical and biophysical experiments indicate that the linker segment connecting the SPRY domain to the coiled-coil domain adopts an α-helical fold, and that this helical portion mediates interactions between the two domains. Targeted mutations were generated to disrupt the putative packing interface without affecting dimerization or higher-order assembly, and we identified mutant proteins that were nevertheless deficient in capsid binding in vitro and restriction activity in cells. Our studies therefore support a model wherein substantial avidity gains during assembly-mediated capsid recognition by TRIM5α come in part from tailored spacing of tethered recognition domains.

  14. Retroviral vector design studies toward hematopoietic stem cell gene therapy for mucopolysaccharidosis type I.

    Science.gov (United States)

    Pan, D; Aronovich, E; McIvor, R S; Whitley, C B

    2000-11-01

    To optimize a gene transfer system for hematopoietic stem cell gene therapy of patients with mucopolysaccharidosis (MPS) type I, 10 retroviral vectors were constructed to express the human alpha-L-iduronidase (IDUA) cDNA. These vectors were designed to evaluate the potential effects of specific promoters, the addition of selectable markers, and the use of multiple promoters versus an internal ribosome entry site for expression of IDUA and selectable maker genes. The effect of vector design was investigated in primary patient fibroblasts (F(MPS)) or murine fibroblast cell lines; while overall comparison of transgene expression was determined in patients' peripheral blood lymphocytes (PBL(MPS)) and CD34+ progenitors (PBPC(MPS)). We observed that the human PGK promoter introduced the highest IDUA activity per 1% relative transgene frequency in F(MPS). Use of the same promoter to separately regulate both the therapeutic gene and a drug-resistance gene resulted in decreased expression of the unselected gene. Co-selection using bicistronic vectors not only increased the number of transductants, but also elevated transgene expression under selective pressure in transgene-positive progenitors. Bicistronic vector LP1CD overcame down-regulation and practically introduced the highest IDUA level in unselected PBL(MPS) and an intermediate level in PBPC(MPS). These studies provide a better understanding of factors contributing to efficient gene expression in hematopoietic cells.

  15. Probing Retroviral and Retrotransposon Genome Structures: The “SHAPE” of Things to Come

    Directory of Open Access Journals (Sweden)

    Joanna Sztuba-Solinska

    2012-01-01

    Full Text Available Understanding the nuances of RNA structure as they pertain to biological function remains a formidable challenge for retrovirus research and development of RNA-based therapeutics, an area of particular importance with respect to combating HIV infection. Although a variety of chemical and enzymatic RNA probing techniques have been successfully employed for more than 30 years, they primarily interrogate small (100–500 nt RNAs that have been removed from their biological context, potentially eliminating long-range tertiary interactions (such as kissing loops and pseudoknots that may play a critical regulatory role. Selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE, pioneered recently by Merino and colleagues, represents a facile, user-friendly technology capable of interrogating RNA structure with a single reagent and, combined with automated capillary electrophoresis, can analyze an entire 10,000-nucleotide RNA genome in a matter of weeks. Despite these obvious advantages, SHAPE essentially provides a nucleotide “connectivity map,” conversion of which into a 3-D structure requires a variety of complementary approaches. This paper summarizes contributions from SHAPE towards our understanding of the structure of retroviral genomes, modifications to which technology that have been developed to address some of its limitations, and future challenges.

  16. Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Peng, H.; Armentano, D.; Mackenzie-Graham, L.; Shen, R.F.; Darlington, G.; Ledley, F.D.; Woo, S.L.C. (Baylor College of Medicine, Houston, TX (USA))

    1988-11-01

    Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. The authors report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human {alpha}{sub 1}-antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating from the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the {alpha}{sub 1} antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes.

  17. Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes

    International Nuclear Information System (INIS)

    Peng, H.; Armentano, D.; Mackenzie-Graham, L.; Shen, R.F.; Darlington, G.; Ledley, F.D.; Woo, S.L.C.

    1988-01-01

    Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. The authors report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human α 1 -antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating from the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the α 1 antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes

  18. Isolation, characterization, and genetic complementation of a cellular mutant resistant to retroviral infection

    Science.gov (United States)

    Agarwal, Sumit; Harada, Josephine; Schreifels, Jeffrey; Lech, Patrycja; Nikolai, Bryan; Yamaguchi, Tomoyuki; Chanda, Sumit K.; Somia, Nikunj V.

    2006-01-01

    By using a genetic screen, we have isolated a mammalian cell line that is resistant to infection by retroviruses that are derived from the murine leukemia virus, human immunodeficiency virus type 1, and feline immunodeficiency virus. We demonstrate that the cell line is genetically recessive for the resistance, and hence it is lacking a factor enabling infection by retroviruses. The block to infection is early in the life cycle, at the poorly understood uncoating stage. We implicate the proteasome at uncoating by completely rescuing the resistant phenotype with the proteasomal inhibitor MG-132. We further report on the complementation cloning of a gene (MRI, modulator of retrovirus infection) that can also act to reverse the inhibition of infection in the mutant cell line. These data implicate a role for the proteasome during uncoating, and they suggest that MRI is a regulator of this activity. Finally, we reconcile our findings and other published data to suggest a model for the involvement of the proteasome in the early phase of the retroviral life cycle. PMID:17043244

  19. Thyroid function among HIV/AIDS patients on highly active anti-retroviral therapy.

    Science.gov (United States)

    Thaimuta, Z L; Sekadde-Kigondu, C; Makawiti, D W

    2010-12-01

    To assess the thyroid function among Human Immunodeficiency Virus (HIV)/Acquired Immunodeficiency Syndrome (AIDS) patients on anti-retroviral drugs: stavudine, lamivudine and nevirapine and to establish the prevalence of non-thyroid illness. Laboratory based comparative cross-sectional study. Comprehensive care clinics at KNH and Mbagathi District Hospital. Eighty four HIV-infected patients on treatment with ARVs (ARV +ve) and an ARV naive (ARV naive) group of 26 HIV-infected patients. Thyroid stimulating hormone levels were not altered following treatment whereas the levels of FT4 decreased. The frequency of those with low FT4 were increasing with continued ARV use. The prevalence of non-thyroidal illness state defined by TSH within reference ranges and low FT4 was comparable among the ARV +ve and ARV naive groups (44 and 46% respectively). Progressive use of HAART causes decline in FT4 hormone levels. It is debatable whether interventions for low FT4 is necessary in ARV treatment but a longitudinal study would explain the progressive trend of thyroid hormones and implications with HAART treatment. The prevalence of NTI is comparable to both HAART users and non-users. Low levels of thyroid hormone (FT 4) may be an adaptive response by thyroid gland to minimize calorie utilisation as in chronic diseases.

  20. Fluticasone furoate induced iatrogenic Cushing syndrome in a pediatric patient receiving anti-retroviral therapy.

    Science.gov (United States)

    van den Berg, S A A; van 't Veer, N E; Emmen, J M A; van Beek, R H T

    2017-01-01

    We present a case of iatrogenic Cushing's syndrome, induced by treatment with fluticasone furoate (1-2 dd, 27.5 µg in each nostril) in a pediatric patient treated for congenital HIV. The pediatric patient described in this case report is a young girl of African descent, treated for congenital HIV with a combination therapy of Lopinavir/Ritonavir (1 dd 320/80 mg), Lamivudine (1 dd 160 mg) and Abacavir (1 dd 320 mg). Our pediatric patient presented with typical Cushingoid features (i.e. striae of the upper legs, full moon face, increased body and facial hair) within weeks after starting fluticasone furoate therapy, which was exacerbated after increasing the dose to 2 dd because of complaints of unresolved rhinitis. Biochemical analysis fitted iatrogenic Cushing's syndrome, with a repeatedly low cortisol (iatrogenic Cushing's syndrome in patients treated for HIV due to the strong inhibition of CYP3 enzymes by Ritonavir. Upon discontinuation of fluticasone treatment, the pediatric patient improved both clinically and biochemically with normalisation of cortisol and ACTH within a couple of weeks. Fluticasone therapy may induce iatrogenic Cushing's syndrome in a patient treated with anti-retroviral therapy.Pharmacogenetic analysis, in particular CYP3A genotyping, provides useful information in patients treated for HIV with respect to possible future steroid treatment.Fluticasone furoate is not detected in the Siemens Immulite cortisol binding assay.

  1. The host cell sulfonation pathway contributes to retroviral infection at a step coincident with provirus establishment.

    Science.gov (United States)

    Bruce, James W; Ahlquist, Paul; Young, John A T

    2008-11-01

    The early steps of retrovirus replication leading up to provirus establishment are highly dependent on cellular processes and represent a time when the virus is particularly vulnerable to antivirals and host defense mechanisms. However, the roles played by cellular factors are only partially understood. To identify cellular processes that participate in these critical steps, we employed a high volume screening of insertionally mutagenized somatic cells using a murine leukemia virus (MLV) vector. This approach identified a role for 3'-phosphoadenosine 5'-phosphosulfate synthase 1 (PAPSS1), one of two enzymes that synthesize PAPS, the high energy sulfate donor used in all sulfonation reactions catalyzed by cellular sulfotransferases. The role of the cellular sulfonation pathway was confirmed using chemical inhibitors of PAPS synthases and cellular sulfotransferases. The requirement for sulfonation was mapped to a stage during or shortly after MLV provirus establishment and influenced subsequent gene expression from the viral long terminal repeat (LTR) promoter. Infection of cells by an HIV vector was also shown to be highly dependent on the cellular sulfonation pathway. These studies have uncovered a heretofore unknown regulatory step of retroviral replication, have defined a new biological function for sulfonation in nuclear gene expression, and provide a potentially valuable new target for HIV/AIDS therapy.

  2. Economic evaluation of task-shifting approaches to the dispensing of anti-retroviral therapy

    Directory of Open Access Journals (Sweden)

    Foster Nicola

    2012-09-01

    Full Text Available Abstract Background A scarcity of human resources for health has been identified as one of the primary constraints to the scale-up of the provision of Anti-Retroviral Treatment (ART. In South Africa there is a particularly severe lack of pharmacists. The study aims to compare two task-shifting approaches to the dispensing of ART: Indirectly Supervised Pharmacist’s Assistants (ISPA and Nurse-based pharmaceutical care models against the standard of care which involves a pharmacist dispensing ART. Methods A cross-sectional mixed methods study design was used. Patient exit interviews, time and motion studies, expert interviews and staff costs were used to conduct a costing from the societal perspective. Six facilities were sampled in the Western Cape province of South Africa, and 230 patient interviews conducted. Results The ISPA model was found to be the least costly task-shifting pharmaceutical model. However, patients preferred receiving medication from the nurse. This related to a fear of stigma and being identified by virtue of receiving ART at the pharmacy. Conclusions While these models are not mutually exclusive, and a variety of pharmaceutical care models will be necessary for scale up, it is useful to consider the impact of implementing these models on the provider, patient access to treatment and difficulties in implementation.

  3. The host cell sulfonation pathway contributes to retroviral infection at a step coincident with provirus establishment.

    Directory of Open Access Journals (Sweden)

    James W Bruce

    2008-11-01

    Full Text Available The early steps of retrovirus replication leading up to provirus establishment are highly dependent on cellular processes and represent a time when the virus is particularly vulnerable to antivirals and host defense mechanisms. However, the roles played by cellular factors are only partially understood. To identify cellular processes that participate in these critical steps, we employed a high volume screening of insertionally mutagenized somatic cells using a murine leukemia virus (MLV vector. This approach identified a role for 3'-phosphoadenosine 5'-phosphosulfate synthase 1 (PAPSS1, one of two enzymes that synthesize PAPS, the high energy sulfate donor used in all sulfonation reactions catalyzed by cellular sulfotransferases. The role of the cellular sulfonation pathway was confirmed using chemical inhibitors of PAPS synthases and cellular sulfotransferases. The requirement for sulfonation was mapped to a stage during or shortly after MLV provirus establishment and influenced subsequent gene expression from the viral long terminal repeat (LTR promoter. Infection of cells by an HIV vector was also shown to be highly dependent on the cellular sulfonation pathway. These studies have uncovered a heretofore unknown regulatory step of retroviral replication, have defined a new biological function for sulfonation in nuclear gene expression, and provide a potentially valuable new target for HIV/AIDS therapy.

  4. CD4 lymphocyte response following anti-retroviral therapy in HIV/AIDS patients - A study in Osmania General Hospital

    Directory of Open Access Journals (Sweden)

    Srinivasa Rao Nanyam

    2016-07-01

    Full Text Available The present study aims serial four year assessment of CD4 cell response after initiation of anti-retroviral therapy (ART in patients with HIV/AIDS attending Osmania General Hospital, Hyderabad. It was a retrospective hospital based observational study. We included 110 HIV/AIDS who were on ART. Data was collected over a period of 04 years from 2005 to 2008 in the ART Centre, Upgraded Department of General Medicine, Osmania General Hospital. Data regarding CD4 cell count over 4 years was assessed for mean CD4 cell count, trends, age and sex wise distribution. All patients were on ART as per National Aids Control Organisation (NACO guidelines. Complete blood picture, serum creatinine, blood urea, serum electrolytes, liver function tests, sputum for acid fast bacilli, chest radiography, CD4 cell count in all patients, fine needle aspiration and biopsy (if necessary, magnetic resonance imaging (if necessary, computerized tomography (if necessary, colonoscopy (if necessary. The present study showed mean CD4 count improvement of 128.78 cells/mm3 after 6 months of initiation of ART, 24.77cells /mm3 after 1 year, additional 67.53 cells/mm3 after 2nd year and after 3rd year 5.59cells/mm3 from base line CD4 cell count. Improvement in CD4 count was almost equal in both male and female and in age group <25 years and above 40 years age group also. Mean CD4 cell count improvement of 240.31 cells/mm3 in females and 220.54 cells/mm3 in males to the baseline after 3 years of treatment with ART. Present study clearly shows definite improvement in CD4 cell count after ART was more than 100% irrespective of age and sex. Regular intake of drugs will improve immunologic response. Therefore strict adherence to ART /regular counseling sessions at ART centres should be stressed upon. [J Med Allied Sci 2016; 6(2.000: 68-71

  5. Screening for Cryptococcal Antigenemia in Anti-Retroviral Naïve AIDS Patients in Benin City, Nigeria

    Directory of Open Access Journals (Sweden)

    Favour Osazuwa

    2012-05-01

    Full Text Available Objectives: Cryptococcus neoformans is the most incriminated fungal pathogen causing meningitis in acquired immune deficiency syndrome (AIDS patients, and is known to constitute a major cause of deaths in AIDS patients. This study thus aimed to determine the baseline sero-prevalence of Cryptococcus neoformans infection in anti-retroviral naïve (ART-naïve AIDS patients using the serum Cryptococcal antigen (crag detection method. Baseline effect of variation in CD4 counts, as well as sex and age with sero-positivity for crag were also determined.Methods: This descriptive cross-sectional study included 150 (61 males and 89 females ART-naïve AIDS patients attending the Human Immunodeficiency Virus clinic (HIV at the University of Benin Teaching hospital, Benin City, Nigeria, within the period from February 2011- July 2011. Forty (18 males and 22 females HIV positive outpatients with CD4 counts >200 cells/µl who were ART-naive were recruited and used as controls. The sero-prevalence of crag in the patients and the control group was measured using the cryptococcal antigen latex agglutination system (CALAS (Meridian Bioscience, Europe and CD4 counts were measured using flow cytometry (Partec flow cytometer, Germany.Results: Of the 150 ART-naïve AIDS patients with CD4 counts £200 cells/µL; 19 (12.7% were positive for serum Cryptococcal antigen. ART-naïve AIDS patients with CD4 count ≤50 cells/µl had the highest prevalence of serum crag. Lower CD4 counts were significantly associated with positivity for serum crag (p<0.001. Age and sex had no significant effect on the sero-positivity for serum crag. One (2.5% of the controls was sero-positive for crag. Thus, serum crag was significantly associated with AIDS but not with HIV (p<0.001.Conclusion: This study uncovers a high prevalence of crag in ART- naïve AIDS patients in Benin City. The prevalence of crag was higher in ART-naïve AIDS patients with lower CD4 counts. There is an urgent need to

  6. Metabolic changes associated with antiretroviral therapy in HIV-positive patients Alteraciones metabólicas asociadas a la terapia anti-retroviral en pacientes HIV-positivos Alterações metabólicas associadas à terapia anti-retroviral em pacientes HIV-positivos

    Directory of Open Access Journals (Sweden)

    Sabrina Esteves de Matos Almeida

    2009-04-01

    Full Text Available OBJECTIVE: To evaluate metabolic changes associated with highly active antiretroviral therapy (HAART in HIV-positive patients, and to identify risk factors associated. METHODS: Retrospective study that included 110 HIV-positive patients who where on HAART in the city of Porto Alegre (Southern Brazil between January 2003 and March 2004. Data on demographic variables, cigarette smoking, diabetes mellitus, cholesterol and triglyceride levels, stage of HIV infection, antiretroviral therapy and HCV coinfection were collected. General linear models procedure for repeated measures was used to test the interaction between HAART and HCV coinfection or protease inhibitor treatment. RESULTS: Total cholesterol, triglycerides, and glucose levels significantly increased after receiving HAART (pOBJETIVO: Evaluar las alteraciones metabólicas asociadas a la terapia anti-retroviral potente en pacientes HIV-positivos e identificar factores de riesgo asociados. MÉTODOS: Estudio retrospectivo con 110 pacientes HIV-positivos que estaban en terapia anti-retroviral potente (HAART en la ciudad de Porto Alegre (Sur de Brasil, entre enero de 2003 y marzo de 2004. Los datos colectados incluyen variables demográficas, tabaquismo, diabetes mellitas, niveles de colesterol y triglicéridos, fase de la infección viral, terapia anti-retroviral y co-infección con hepatitis C. El análisis multivariado para medidas repetidas (General Linear Model procedure for Repeated Measures fue utilizada para analizar la interacción entre el efecto de uso de HAART y el uso de inhibidores de proteasa o co-infección por hepatitis C. RESULTADOS: Fueron observados aumentos significativos en los niveles de colesterol total, triglicéridos y glucosa posterior al tratamiento con HAART (pOBJETIVO: Avaliar as alterações metabólicas associadas à terapia anti-retroviral potente em pacientes HIV-positivos e identificar fatores de risco associados. MÉTODOS: Estudo retrospectivo com 110

  7. Perinatal genotoxicity and carcinogenicity of anti-retroviral nucleoside analog drugs

    International Nuclear Information System (INIS)

    Poirier, Miriam C.; Olivero, Ofelia A.; Walker, Dale M.; Walker, Vernon E.

    2004-01-01

    The current worldwide spread of the human immunodeficiency virus-1 (HIV-1) to the heterosexual population has resulted in approximately 800 000 children born yearly to HIV-1-infected mothers. In the absence of anti-retroviral intervention, about 25% of the approximately 7000 children born yearly to HIV-1-infected women in the United States are HIV-1 infected. Administration of zidovudine (AZT) prophylaxis during pregnancy reduces the rate of infant HIV-1 infection to approximately 7%, and further reductions are achieved with the addition of lamivudine (3TC) in the clinical formulation Combivir. Whereas clinically this is a remarkable achievement, AZT and 3TC are DNA replication chain terminators known to induce various types of genotoxicity. Studies in rodents have demonstrated AZT-DNA incorporation, HPRT mutagenesis, telomere shortening, and tumorigenicity in organs of fetal mice exposed transplacentally to AZT. In monkeys, both AZT and 3TC become incorporated into the DNA from multiple fetal organs taken at birth after administration of human-equivalent protocols to pregnant dams during gestation, and telomere shortening has been found in monkey fetuses exposed to both drugs. In human infants, AZT-DNA and 3TC-DNA incorporation as well as HPRT and GPA mutagenesis have been documented in cord blood from infants exposed in utero to Combivir. In infants of mice, monkeys, and humans, levels of AZT-DNA incorporation were remarkably similar, and in newborn mice and humans, mutation frequencies were also very similar. Given the risk-benefit ratio, these highly successful drugs will continue to be used for prevention of vertical viral transmission, however evidence of genotoxicity in mouse and monkey models and in the infants themselves would suggest that exposed children should be followed well past adolescence for early detection of potential cancer hazard

  8. Cloning of the rat ecotropic retroviral receptor and studies of its expression in intestinal tissues

    Energy Technology Data Exchange (ETDEWEB)

    Puppi, M.; Henning, S.J. [Baylor College of Medicine, Houston, TX (United States)

    1995-05-01

    A long-term goal of our laboratory is to establish a rat model to study the feasibility of using the intestinal tract as a site for somatic gene therapy. As a step toward that goal, the current study reports the cloning of the rat ecotropic retroviral receptor (EcoR) cDNA and the study of various aspects of its expression in the intestinal cDNA library with mouse EcoR cDNA. A clone of approximately 7 kb, designated MP10, was obtained. Partial sequencing of MP10 from the 5{prime} end revealed a level of similarity of 92% compared with mouse EcoR. The presence of a 5{prime} untranslated region and a 3{prime} poly(A)tract, together with the overall size of the cDNA, suggest that is very close to being a full-length cDNA for this large transcript. Northern blots with MP10 showed an RNA of approximately 7.9 kb present along the entire length of the small intestine and somewhat less abundant in the colon. Developmental studies showed high levels of EcoR in fetal rat intestine, a decline in the early postnatal period, then a gradual rise to adulthood. Caco-2 cells were used to assess the expression of EcoR in proliferating compared with differentiated intestinal epithelial cells. EcoR mRNA was found to be very much more abundant in nondifferentiated cells and declined to low levels as the cells underwent spontaneous differentiation. These patterns of EcoR expression indicate that ecotropic retroviruses should be suitable vectors with which to attempt gene transfer into the intestinal epithelium. In addition, since the endogenous role of EcoR is as the y{sup +} cationic amino acid transporter, these data have significance for understanding patterns of amino acid transport in the intestinal epithelium. 37 refs., 4 figs.

  9. Activation and regulation of endogenous retroviral genes in the human pituitary gland and related endocrine tumours.

    Science.gov (United States)

    Buslei, Rolf; Strissel, Pamela L; Henke, Christine; Schey, Regina; Lang, Nadine; Ruebner, Matthias; Stolt, Claus C; Fabry, Ben; Buchfelder, Michael; Strick, Reiner

    2015-02-01

    Adenohypophysis (AH) hormone-producing cells represent the origin of diverse groups of pituitary adenomas (PA). Deregulation of hypothalamic hormone receptors, growth factors and cAMP signalling have been implicated in the aetiology of PA. Endogenous retroviruses (ERVs) are derived from past exogenous retroviral infections and represent more than 8% of the human genome. Some ERV genes encode open reading frames and produce functional proteins, for example, the ERVW-1 envelope gene Syncytin-1, essential for placentogenesis, but also deregulated in human tumours. Data concerning ERV expression in the AH and related endocrine tumours are missing. Syncytin-1 protein was analysed in normal AH (n = 15) and compared with five PA subtypes (n = 117) by immunohistochemistry. Absolute gene expression of 20 ERV functional envelope genes and ERVW-5 gag was measured. PA tissues were examined for Syncytin-1 and the cAMP signalling marker phospho-CREB-Ser133 using immunohistochemistry. Isolated primary human PA cells were treated with different hormones. Murine embryonic and adult pituitary gland ERV expressions were compared with human AH. Syncytin-1 protein colocalized with corticotropic cells of AH. In contrast, all PA demonstrated significant Syncytin-1 protein overexpression, supporting deregulation. All other ERV genes showed significant up-regulations in different PA subtypes. Phospho-CREB-Ser133 and Syncytin-1 colocalized in PA cells. Cultivated primary PA cells with ACTH or CRH induced their respective receptors and ERV genes. Syncytin-A/-B, murine orthologues to human Syncytin-1/-2, localized to embryonic and adult pituitary glands demonstrating functional mammalian conservation. Deregulated ERV genes may contribute to PA development via cAMP signalling. © 2014 British Neuropathological Society.

  10. Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites.

    Science.gov (United States)

    Serrao, Erik; Cherepanov, Peter; Engelman, Alan N

    2016-03-22

    Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

  11. Retroviral-mediated transfer and expression of human β-globin genes in cultured murine and human erythroid cells

    International Nuclear Information System (INIS)

    Weber-Benarous, A.; Cone, R.D.; London, I.M.; Mulligan, R.C.

    1988-01-01

    The authors cloned human β-globin DNA sequences from a genomic library prepared from DNA isolated from the human leukemia cell line K562 and have used the retroviral vector pZip-NeoSV(X)1 to introduce a 3.0-kilobase segment encompassing the globin gene into mouse erythroleukemia cells. Whereas the endogenous K562 β-globin gene is repressed in K562 cells, when introduced into mouse erythroleukemia cells by retroviral-mediated gene transfer, the β-globin gene from K562 cells was transcribed and induced 5-20-fold after treatment of the cells with dimethyl sulfoxide. The transcripts were correctly initiated, and expression and regulation of the K562 gene were identical to the expression of a normal human β-globin gene transferred into mouse erythroleukemia cells in the same way. They have also introduced the normal human β-globin gene into K562 cells using the same retrovirus vector. SP6 analysis of the RNA isolated from the transduced cells showed that the normal β-globin gene was transcribed at a moderately high level, before or after treatment with hemin. Based on these data, they suggest that the lack of expression of the endogenous β-globin gene in K562 cells does not result from an alteration in the gene itself and may not result from a lack of factor(s) necessary for β-lobin gene transcription. Retroviral-mediated transfer of the human β-globin gene may, however, uniquely influence expression of the gene K562 cells

  12. Retroviral Replicating Vector Delivery of miR-PDL1 Inhibits Immune Checkpoint PDL1 and Enhances Immune Responses In Vitro

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    Amy H. Lin

    2017-03-01

    Full Text Available Tumor cells express a number of immunosuppressive molecules that can suppress anti-tumor immune responses. Efficient delivery of small interfering RNAs to treat a wide range of diseases including cancers remains a challenge. Retroviral replicating vectors (RRV can be used to stably and selectively introduce genetic material into cancer cells. Here, we designed RRV to express shRNA (RRV-shPDL1 or microRNA30-derived shRNA (RRV-miRPDL1 using Pol II or Pol III promoters to downregulate PDL1 in human cancer cells. We also designed RRV expressing cytosine deaminase (yCD2 and miRPDL1 for potential combinatorial therapy. Among various configurations tested, we showed that RRV-miRPDL1 vectors with Pol II or Pol III promoter replicated efficiently and exhibited sustained downregulation of PDL1 protein expression by more than 75% in human cancer cell lines with high expression of PDL1. Immunologic effects of RRV-miRPDL1 were assessed by a trans-suppression lymphocyte assay. In vitro data showed downregulation of PDL1+ tumor cells restored activation of CD8+ T cells and bio-equivalency compared to anti-PDL1 antibody treatment. These results suggest RRV-miRPDL1 may be an alternative therapeutic approach to enhance anti-tumor immunity by overcoming PDL1-induced immune suppression from within cancer cells and this approach may also be applicable to other cancer targets.

  13. Retention of HIV-Infected Children in the First 12 Months of Anti-Retroviral Therapy and Predictors of Attrition in Resource Limited Settings: A Systematic Review.

    Science.gov (United States)

    Abuogi, Lisa L; Smith, Christiana; McFarland, Elizabeth J

    2016-01-01

    Current UNAIDS goals aimed to end the AIDS epidemic set out to ensure that 90% of all people living with HIV know their status, 90% initiate and continue life-long anti-retroviral therapy (ART), and 90% achieve viral load suppression. In 2014 there were an estimated 2.6 million children under 15 years of age living with HIV, of which only one-third were receiving ART. Little literature exists describing retention of HIV-infected children in the first year on ART. We conducted a systematic search for English language publications reporting on retention of children with median age at ART initiation less than ten years in resource limited settings. The proportion of children retained in care on ART and predictors of attrition were identified. Twelve studies documented retention at one year ranging from 71-95% amongst 31877 African children. Among the 5558 children not retained, 4082 (73%) were reported as lost to follow up (LFU) and 1476 (27%) were confirmed to have died. No studies confirmed the outcomes of children LFU. Predictors of attrition included younger age, shorter duration of time on ART, and severe immunosuppression. In conclusion, significant attrition occurs in children in the first 12 months after ART initiation, the majority attributed to LFU, although true outcomes of children labeled as LFU are unknown. Focused efforts to ensure retention and minimize early mortality are needed as universal ART for children is scaled up.

  14. Global HIV-1 transmitted drug resistance in the INSIGHT Strategic Timing of AntiRetroviral Treatment (START) trial

    DEFF Research Database (Denmark)

    Baxter, J D; Dunn, D; White, E

    2015-01-01

    of resistance testing in START trial participants. METHODS: In the Strategic Timing of AntiRetroviral Treatment (START) trial, baseline genotypic resistance testing results were collected at study entry and analysed centrally to determine the prevalence of TDR in the study population. Resistance was based...... on a modified 2009 World Health Organization definition to reflect newer resistance mutations. RESULTS: Baseline resistance testing was available in 1946 study participants. Higher rates of testing occurred in Europe (86.7%), the USA (81.3%) and Australia (89.9%) as compared with Asia (22.2%), South America (1...

  15. Target-cell-derived tRNA-like primers for reverse transcription support retroviral infection at low efficiency

    DEFF Research Database (Denmark)

    Schmitz, Alexander; Lund, Anders H; Hansen, Anette C

    2002-01-01

    Reverse transcription of a retroviral genome takes place in the cytoplasm of an infected cell by a process primed by a producer-cell-derived tRNA annealed to an 18-nucleotide primer-binding site (PBS). By an assay involving primer complementation of PBS-mutated vectors we analyzed whether t......RNA primers derived from the target cell can sustain reverse transcription during murine leukemia virus (MLV) infection. Transduction efficiencies were 4-5 orders of magnitude below those of comparable producer-cell complementations. However, successful usage of a target-cell-derived tRNA primer was proven...

  16. As representações sociais de pacientes em ínicio de terapia anti-retroviral envolvidas na adesão ao tratamento

    OpenAIRE

    Angela de Cassia Rover da Silva

    2006-01-01

    Neste estudo, buscou-se compreender as representações sociais de portadores do vírus HIV, ou da aids, sobre os aspectos que se relacionam à adesão à terapia anti-retroviral. Foram analisadas 30 entrevistas abertas, realizadas com pacientes em início de terapia anti-retroviral, atendidos em dois serviços de referência em Belo Horizonte, Minas Gerais. Na análise, explicitaram-se as categorias relacionadas à adesão, que compõem dois grandes grupos. O primeiro inclui os aspectos relacionados à ex...

  17. Lack of evidence for retroviral infections formerly related to chronic fatigue in Spanish fibromyalgia patients.

    Science.gov (United States)

    Oltra, Elisa; García-Escudero, María; Mena-Durán, Armando Vicente; Monsalve, Vicente; Cerdá-Olmedo, Germán

    2013-11-11

    types of retroviral infection formerly associated to chronic fatigue does not rule out the possibility that other viruses are involved in inciting or maintaining fibromyalgia and/or chronic fatigue conditions.

  18. Supporting children to adhere to anti-retroviral therapy in urban Malawi: multi method insights

    Directory of Open Access Journals (Sweden)

    Phiri Sam

    2009-07-01

    Full Text Available Abstract Background Ensuring good adherence is critical to the success of anti-retroviral treatment (ART. However, in resource-poor contexts, where paediatric HIV burden is high there has been limited progress in developing or adapting tools to support adherence for HIV-infected children on ART and their caregivers. We conducted formative research to assess children's adherence and to explore the knowledge, perceptions and attitudes of caregivers towards children's treatment. Methods All children starting ART between September 2002 and January 2004 (when ART was at cost in Malawi were observed for at least 6 months on ART. Their adherence was assessed quantitatively by asking caregivers of children about missed ART doses during the previous 3 days at monthly visits. Attendance to clinic appointments was also monitored. In June and July 2004, four focus group discussions, each with 6 to 8 caregivers, and 5 critical incident narratives were conducted to provide complementary contextual data on caregivers' experiences on the challenges to and opportunities of paediatric ART adherence. Results We followed prospectively 47 children who started ART between 8 months and 12 years of age over a median time on ART of 33 weeks (2–91 weeks. 72% (34/47 never missed a single dose according to caregivers' report and 82% (327/401 of clinic visits were either as scheduled, or before or within 1 week after the scheduled appointment. Caregivers were generally knowledgeable about ART and motivated to support children to adhere to treatment despite facing multiple challenges. Caregivers were particularly motivated by seeing children begin to get better; but faced challenges in meeting the costs of medicine and transport, waiting times in clinic, stock outs and remembering to support children to adhere in the face of multiple responsibilities. Conclusion In the era of rapid scale-up of treatment for children there is need for holistic support strategies that focus

  19. Production of avian retroviruses and tissue-specific somatic retroviral gene transfer in vivo using the RCAS/TVA system.

    Science.gov (United States)

    von Werder, Alexander; Seidler, Barbara; Schmid, Roland M; Schneider, Günter; Saur, Dieter

    2012-05-24

    Spatiotemporal retroviral gene transfer into specific somatic mammalian cells using the avian RCAS (replication-competent avian sarcoma-leukosis virus long terminal repeat with splice acceptor)/tumor virus A (TVA) system is a versatile tool for performing lineage tracing and gene function analysis in vivo. RCAS retroviruses carrying the subgroup A envelope transduce only genetically engineered mammalian cells that express the cognate avian retroviral receptor TVA. The RCAS/TVA gene delivery system has been successfully used in various different mouse TVA-expression models. This protocol contains a detailed description of the production of high-titer RCAS retroviruses in chicken fibroblasts and the transduction of proliferating TVA-positive somatic mammalian cells in vivo. By taking advantage of the combination of the RCAS/TVA with the 'universal' Cre/loxP system, the protocol can be used in nearly every proliferating cell type in vivo. The protocol takes 4 weeks from transfection of chicken fibroblasts, which act as the host cells for viral production, to the transduction of TVA-transgenic mice.

  20. Retroviral-mediated gene transfer of human phenylalanine hydroxylase into NIH 3T3 and hepatoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Ledley, F.D.; Grenett, H.E.; McGinnis-Shelnutt, M.; Woo, S.L.C.

    1986-01-01

    Phenylketonuria (PKU) is caused by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). A full-length human PAH cDNA sequence has been inserted into pzip-neoSV(X), which is a retroviral vector containing the bacterial neo gene. The recombinant has been transfected into Psi2 cells, which provide synthesis of the retroviral capsid. Recombinant virus was detected in the culture medium of the transfected Psi2 cells, which is capable of transmitting the human PAH gene into mouse NIH 3T3 cells by infection leading to stable incorporation of the recombinant provirus. Infected cells express PAH mRNA, immunoreactive PAH protein, and exhibit pterin-dependent phenylaline hydroxylase activity. The recombinant virus is also capable of infecting a mouse hepatoma cell line that does not normal synthesize PAH. PAH activity is present in the cellular extracts and the entire hydroxylation system is reconstituted in the hepatoma cells infected with the recombinant viruses. Thus, recombinant viruses containing human PAH cDNA provide a means for introducing functional PAH into mammalian cells of hepatic origin and can potentially be introduced into whole animals as a model for somatic gene therapy for PKU.

  1. Comprehensive profiling of retroviral integration sites using target enrichment methods from historical koala samples without an assembled reference genome

    Directory of Open Access Journals (Sweden)

    Pin Cui

    2016-03-01

    Full Text Available Background. Retroviral integration into the host germline results in permanent viral colonization of vertebrate genomes. The koala retrovirus (KoRV is currently invading the germline of the koala (Phascolarctos cinereus and provides a unique opportunity for studying retroviral endogenization. Previous analysis of KoRV integration patterns in modern koalas demonstrate that they share integration sites primarily if they are related, indicating that the process is currently driven by vertical transmission rather than infection. However, due to methodological challenges, KoRV integrations have not been comprehensively characterized. Results. To overcome these challenges, we applied and compared three target enrichment techniques coupled with next generation sequencing (NGS and a newly customized sequence-clustering based computational pipeline to determine the integration sites for 10 museum Queensland and New South Wales (NSW koala samples collected between the 1870s and late 1980s. A secondary aim of this study sought to identify common integration sites across modern and historical specimens by comparing our dataset to previously published studies. Several million sequences were processed, and the KoRV integration sites in each koala were characterized. Conclusions. Although the three enrichment methods each exhibited bias in integration site retrieval, a combination of two methods, Primer Extension Capture and hybridization capture is recommended for future studies on historical samples. Moreover, identification of integration sites shows that the proportion of integration sites shared between any two koalas is quite small.

  2. Queratinocitos derivados de piel humana modificados por el vector retroviral FOCH 29-NeoR

    Directory of Open Access Journals (Sweden)

    Luz Marina Restrepo

    2000-02-01

    Full Text Available

    En este protocolo se evaluará la eficiencia de la transducción mediada por el vector retroviral FOCH 29-NeoR derivado del virus de Friend; éste ha mostrado una alta eficiencia en la transducción, tanto de células madres hematopoyéticas como de otras líneas celulares. Se medirá su eficiencia de transducción en cultivos primarios de queratinocitos, derivados de biopsias de piel humana o de sobrantes de procedimientos quirúrgicos como circuncisiones, mastectomías y cirugía cosmética de pacientes que consultan el Hospital Universitario San Vicente de Paul, Hospital la María, la Clínica del Rosario y la Clínica León XIII.

    Las muestras de piel se procesarán en un lapso no superior a 12 horas, se eliminará el exceso de dermis y tejido conectivo por digestión con dispasa (0.6-2.4 U/ml a 37°C durante 1 hora. Las muestras serán lavadas con PBS, antibiótico (penicilina + estreptomicina y se cortarán en fragmentos de 1-2 mm; después de 2-3 horas de digestión con tripsina-EDTA (0.25% las células serán resuspendidas en KGM (Medio de crecimiento para queratinocitos y se sembrarán a una concentración de 105 - 3x105 células por plato de 100 mm; se incubarán a 37°C, 5% CO2 con cambios de medio 2-3 veces por semana. Se harán subcultivos con el fin de expandirlos y congelar una parte de las

  3. Electronic medication monitoring-informed counseling to improve adherence to combination anti-retroviral therapy and virologic treatment outcomes: a meta-analysis

    NARCIS (Netherlands)

    Langebeek, Nienke; Nieuwkerk, Pythia

    2015-01-01

    Adherence to combination anti-retroviral therapy for HIV infection is a primary determinant of treatment success, but is often suboptimal. Previous studies have suggested that electronic medication monitoring-informed counseling is among the most effective adherence intervention components. Our

  4. [Construction of retroviral vector carrying Twist gene and its induction of epithelial-mesenchymal transition in human mammary epithelial cells].

    Science.gov (United States)

    Yang, Jiajia; Hu, Ping; Zhou, Mingli; Huang, Jietao; Liu, Manran

    2013-09-01

    To construct a retroviral vector carrying Twist gene and investigate its effect on human mammary MCF10A epithelial cells. Myc-Twist was digested from pcDNA3/myc-Twist and subcloned into the retroviral vector pBABE-puro to construct a recombinant plasmid (pBABE-myc-Twist). The inserted Twist gene was confirmed by restriction enzyme digestion and DNA sequencing. The plasmid pBABE-myc-Twist and the packaging plasmid pAmpho were co-transfected into HEK293T cells for packaging of retrovirus. Meanwhile, the control plasmid pBABE-puro and the packaging plasmid were co-transfected into the other HEK293T cells as a control group. Human mammary MCF10A epithelial cells were infected with the retroviruses carrying Twist gene or the controls, and selected by puromycin. The expression of Twist in the MCF10A-Twist and MCF10A-Vector cells was determined by RT-PCR and Western blotting. The expressions of epithelial-mesenchymal transition (EMT) marker proteins induced by Twist in MCF10A cells were detected using immunofluorescence cytochemistry and Western blotting. Cell migration and invasion abilities were analyzed by Transwell(R); assay. The myc-tagged Twist gene was correctly inserted into the retroviral expression vector as a recombinant plasmid (pBABE-myc-Twist) as identified by restriction analysis and DNA sequencing. The Twist gene was efficiently delivered into human mammary MCF10A epithelial cells by the retrovirus, resulting in the stable expression of Twist mRNA and myc-tagged Twist protein as shown by RT-PCR and Western blotting, respectively. The expression of the epithelial biomarker E-cadherin was downregulated whereas, the mesenchymal marker vimentin upregulated in MCF10A-Twist cells as shown by immunofluorescence cytochemistry and Western blotting. Cell migration and invasion abilities were enhanced notably in MCF10A-Twist cells as compared with MCF10A-Vector control cells (PMCF10A cells and plays an important role in the promotion of cell migration and invasion.

  5. An Efficient Large-Scale Retroviral Transduction Method Involving Preloading the Vector into a RetroNectin-Coated Bag with Low-Temperature Shaking

    Science.gov (United States)

    Dodo, Katsuyuki; Chono, Hideto; Saito, Naoki; Tanaka, Yoshinori; Tahara, Kenichi; Nukaya, Ikuei; Mineno, Junichi

    2014-01-01

    In retroviral vector-mediated gene transfer, transduction efficiency can be hampered by inhibitory molecules derived from the culture fluid of virus producer cell lines. To remove these inhibitory molecules to enable better gene transduction, we had previously developed a transduction method using a fibronectin fragment-coated vessel (i.e., the RetroNectin-bound virus transduction method). In the present study, we developed a method that combined RetroNectin-bound virus transduction with low-temperature shaking and applied this method in manufacturing autologous retroviral-engineered T cells for adoptive transfer gene therapy in a large-scale closed system. Retroviral vector was preloaded into a RetroNectin-coated bag and incubated at 4°C for 16 h on a reciprocating shaker at 50 rounds per minute. After the supernatant was removed, activated T cells were added to the bag. The bag transduction method has the advantage of increasing transduction efficiency, as simply flipping over the bag during gene transduction facilitates more efficient utilization of the retroviral vector adsorbed on the top and bottom surfaces of the bag. Finally, we performed validation runs of endoribonuclease MazF-modified CD4+ T cell manufacturing for HIV-1 gene therapy and T cell receptor-modified T cell manufacturing for MAGE-A4 antigen-expressing cancer gene therapy and achieved over 200-fold (≥1010) and 100-fold (≥5×109) expansion, respectively. In conclusion, we demonstrated that the large-scale closed transduction system is highly efficient for retroviral vector-based T cell manufacturing for adoptive transfer gene therapy, and this technology is expected to be amenable to automation and improve current clinical gene therapy protocols. PMID:24454964

  6. In vivo mitochondrial function in HIV-infected persons treated with contemporary anti-retroviral therapy: a magnetic resonance spectroscopy study.

    Directory of Open Access Journals (Sweden)

    Brendan A I Payne

    Full Text Available Modern anti-retroviral therapy is highly effective at suppressing viral replication and restoring immune function in HIV-infected persons. However, such individuals show reduced physiological performance and increased frailty compared with age-matched uninfected persons. Contemporary anti-retroviral therapy is thought to be largely free from neuromuscular complications, whereas several anti-retroviral drugs previously in common usage have been associated with mitochondrial toxicity. It has recently been established that patients with prior exposure to such drugs exhibit irreversible cellular and molecular mitochondrial defects. However the functional significance of such damage remains unknown. Here we use phosphorus magnetic resonance spectroscopy ((31P-MRS to measure in vivo muscle mitochondrial oxidative function, in patients treated with contemporary anti-retroviral therapy, and compare with biopsy findings (cytochrome c oxidase (COX histochemistry. We show that dynamic oxidative function (post-exertional ATP (adenosine triphosphate resynthesis was largely maintained in the face of mild to moderate COX defects (affecting up to ∼10% of fibers: τ½ ADP (half-life of adenosine diphosphate clearance, HIV-infected 22.1±9.9 s, HIV-uninfected 18.8±4.4 s, p = 0.09. In contrast, HIV-infected patients had a significant derangement of resting state ATP metabolism compared with controls: ADP/ATP ratio, HIV-infected 1.24±0.08×10(-3, HIV-uninfected 1.16±0.05×10(-3, p = 0.001. These observations are broadly reassuring in that they suggest that in vivo mitochondrial function in patients on contemporary anti-retroviral therapy is largely maintained at the whole organ level, despite histochemical (COX defects within individual cells. Basal energy requirements may nevertheless be increased.

  7. Target-cell-derived tRNA-like primers for reverse transcription support retroviral infection at low efficiency

    DEFF Research Database (Denmark)

    Schmitz, Alexander; Lund, Anders H; Hansen, Anette C

    2002-01-01

    Reverse transcription of a retroviral genome takes place in the cytoplasm of an infected cell by a process primed by a producer-cell-derived tRNA annealed to an 18-nucleotide primer-binding site (PBS). By an assay involving primer complementation of PBS-mutated vectors we analyzed whether tRNA...... primers derived from the target cell can sustain reverse transcription during murine leukemia virus (MLV) infection. Transduction efficiencies were 4-5 orders of magnitude below those of comparable producer-cell complementations. However, successful usage of a target-cell-derived tRNA primer was proven...... by cases of correction of single mismatches between Akv-MLV vectors and complementary tRNA primers toward the primer sequence in the integrated vector. Thus, target-cell-derived tRNA-like primers are able to initiate first-strand cDNA synthesis and plus-strand transfer leading to a complete provirus...

  8. FLT3 ligand preserves the uncommitted CD34+CD38- progenitor cells during cytokine prestimulation for retroviral transduction

    DEFF Research Database (Denmark)

    Nielsen, S D; Husemoen, L L; Sørensen, T U

    2000-01-01

    Before stem cell gene therapy can be considered for clinical applications, problems regarding cytokine prestimulation remain to be solved. In this study, a retroviral vector carrying the genes for the enhanced version of green fluorescent protein (EGFP) and neomycin resistance (neo(r)) was used...... (SCF), FLT3 ligand, interleukin-3 (IL-3), IL-6, and IL-7 prior to transduction. Expression of the two genes was assessed by flow cytometry and determination of neomycin-resistant colonies in a selective colony-forming unit (CFU) assay, respectively. The neomycin resistance gene was expressed...... in a higher percentage of cells than the EGFP gene, but there seemed to be a positive correlation between expression of the two genes. The effect of cytokine prestimulation was therefore monitored using EGFP as marker for transduction. When SCF was compared to SCF in combination with more potent cytokines...

  9. Structure-function studies of nucleocytoplasmic transport of retroviral genomic RNA by mRNA export factor TAP

    Science.gov (United States)

    Teplova, Marianna; Wohlbold, Lara; Khin, Nyan W.; Izaurralde, Elisa; Patel, Dinshaw J.

    2011-01-01

    Messenger RNA export is mediated by the TAP-p15 heterodimer, which belongs to the family of NTF2-like export receptors. TAP-p15 heterodimers also bind to the constitutive transport element (CTE) present in simian type D retroviral RNAs, and mediate export of viral unspliced RNAs to the host cytoplasm. We have solved the crystal structure of the RNA recognition and leucine-rich repeat motifs of TAP bound to one symmetrical-half of CTE RNA. L-shaped conformations of protein and RNA are involved in a mutual molecular embrace on complex formation. We have monitored the impact of structure-guided mutations on binding affinities in vitro and transport assays in vivo. Our studies define the principles by which CTE RNA subverts the mRNA export receptor TAP, thereby facilitating nuclear export of viral genomic RNAs, and more generally, provide insights on cargo RNA recognition by mRNA export receptors. PMID:21822283

  10. Distinct roles of CD4+ T cell subpopulations in retroviral immunity: lessons from the Friend virus mouse model

    Directory of Open Access Journals (Sweden)

    Kassiotis George

    2011-09-01

    Full Text Available Abstract It is well established that CD4+ T cells play an important role in immunity to infections with retroviruses such as HIV. However, in recent years CD4+ T cells have been subdivided into several distinct populations that are differentially regulated and perform widely varying functions. Thus, it is important to delineate the separate roles of these subsets, which range from direct antiviral activities to potent immunosuppression. In this review, we discuss contributions from the major CD4+ T cell subpopulations to retroviral immunity. Fundamental concepts obtained from studies on numerous viral infections are presented along with a more detailed analysis of studies on murine Friend virus. The relevance of these studies to HIV immunology and immunotherapy is reviewed.

  11. An Optimized GD2-Targeting Retroviral Cassette for More Potent and Safer Cellular Therapy of Neuroblastoma and Other Cancers.

    Directory of Open Access Journals (Sweden)

    Simon Thomas

    Full Text Available Neuroblastoma is the commonest extra cranial solid cancer of childhood. Despite escalation of treatment regimens, a significant minority of patients die of their disease. Disialoganglioside (GD2 is consistently expressed at high-levels in neuroblastoma tumors, which have been targeted with some success using therapeutic monoclonal antibodies. GD2 is also expressed in a range of other cancer but with the exception of some peripheral nerves is largely absent from non-transformed tissues. Chimeric Antigen Receptors (CARs are artificial type I proteins which graft the specificity of a monoclonal antibody onto a T-cell. Clinical data with early CAR designs directed against GD2 have shown some promise in Neuroblastoma. Here, we describe a GD2-targeting CAR retroviral cassette, which has been optimized for CAR T-cell persistence, efficacy and safety.

  12. Identifying activated T cells in reconstituted RAG deficient mice using retrovirally transduced Pax5 deficient pro-B cells.

    Directory of Open Access Journals (Sweden)

    Nadesan Gajendran

    Full Text Available Various methods have been used to identify activated T cells such as binding of MHC tetramers and expression of cell surface markers in addition to cytokine-based assays. In contrast to these published methods, we here describe a strategy to identify T cells that respond to any antigen and track the fate of these activated T cells. We constructed a retroviral double-reporter construct with enhanced green fluorescence protein (EGFP and a far-red fluorescent protein from Heteractis crispa (HcRed. LTR-driven EGFP expression was used to enrich and identify transduced cells, while HcRed expression is driven by the CD40Ligand (CD40L promoter, which is inducible and enables the identification and cell fate tracing of T cells that have responded to infection/inflammation. Pax5 deficient pro-B cells that can give rise to different hematopoietic cells like T cells, were retrovirally transduced with this double-reporter cassette and were used to reconstitute the T cell pool in RAG1 deficient mice that lack T and B cells. By using flow cytometry and histology, we identified activated T cells that had developed from Pax5 deficient pro-B cells and responded to infection with the bacterial pathogen Listeria monocytogenes. Microscopic examination of organ sections allowed visual identification of HcRed-expressing cells. To further characterize the immune response to a given stimuli, this strategy can be easily adapted to identify other cells of the hematopoietic system that respond to infection/inflammation. This can be achieved by using an inducible reporter, choosing the appropriate promoter, and reconstituting mice lacking cells of interest by injecting gene-modified Pax5 deficient pro-B cells.

  13. Adherence to anti-retroviral therapy & factors associated with it: A community based cross-sectional study from West Bengal, India

    Directory of Open Access Journals (Sweden)

    Sobha Pahari

    2015-01-01

    Full Text Available Background & objectives: Failure to adhere to anti-retroviral therapy (ART can lead to a range of unfavourable consequences impacting upon people living with HIV (PLH and society. It is, therefore, paramount that ART adherence is measured in a reliable manner and factors associated with adherence are identified. Lack of such data from West Bengal necessitated undertaking the current study. Methods: Participants were included during August-October, 2011 from three Drop-In-Centres (DICs from the three districts of West Bengal, India. ART-adherence was calculated by using formula based on pill-count and records collected from ART-card in possession of each of the 128 consenting adult PLH. Information on self-reported adherence, socio-demography, and adherence influencing issues was also collected through interviewer-administered questionnaire. Results: Of the 128 PLH, 99 (77% and 93 (73% PLH had ≥90 per cent and ≥95 per cent adherence, respectively to ART. Conversely, subjective reporting captured much higher proportion of PLH as ′well adherent′; a finding having implications for ongoing ART programme. Factors, independently associated with poor adherence (<90%, were ′7 th to 12 th month period of ART intake′ (adjusted OR=9.5; 90% CI 1.9 - 47.3; p0 =0.02 and ′non-disclosure of HIV status to family members′ (adjusted OR=4; 90% CI 1.3 - 13; P=0.05. Results at 95 per cent adherence cut-off were similar. Interpretation & conclusions: Enabling environment, which would encourage people to disclose their HIV status and in turn seek adherence partners from families and beyond and ongoing adherence-counselling appear to be important issues in the programme. Relevance of these study findings in wider context is conceivable.

  14. Evaluation of oral manifestations and oral health status among pediatric human immunodeficiency virus patients-under anti-retroviral therapy: A cross-sectional study

    Directory of Open Access Journals (Sweden)

    Monika Aroquiadasse

    2016-01-01

    Full Text Available Introduction: The human immunodeficiency virus (HIV acquired immunodeficiency syndrome disease has evolved to become a social and economic catastrophe, with far-reaching implications affecting every phase of life of the diseased individual. Data on adults and children diagnosed with HIV infection are useful for determining populations needing prevention and treatment services. Oral lesions may be the presenting symptoms of HIV infection and may differ entirely from those manifested in the adult population. Aim and Objective: We aimed to evaluate the prevalence of HIV related oral lesions among pediatric HIV patients and to assess the oral health status of HIV infected children residing in a selected childcare facility in Puducherry. Materials and Methods: A cross-sectional study was conducted during September 2015 in child care facility for HIV infected children located in Puducherry U.T, India. All children <18 years, who are diagnosed with HIV infection and are put on anti-retroviral therapy (ART or pre-ART care, were included in the study. After obtaining informed consent from the care-givers and assent of the children, they were interviewed and examined by a team comprising a qualified dental surgeon and a trained physician. Results: Majority of the children were under first-line ART (73% and were on ART for more than 4 years. The CD4 count of 23 (52.3 was between 500–1000 cells/μL. The recent viral load assay in 32 (72.7 patients was <150/not detected. Tooth decay was the most common oral manifestation with 28 (63.6 being affected. Nonspecific lymphadenopathy 26 (59.1 was the most common coexisting systemic illness. Conclusion: This study proves that constant surveillance by monitoring the general health status, CD4 counts, viral load coupled with stringent ART care has improved the overall quality of life of these children and consequently resulted in lesser oral manifestations.

  15. Demographic and HIV-specific characteristics of participants enrolled in the INSIGHT Strategic Timing of AntiRetroviral Treatment (START) trial.

    Science.gov (United States)

    Sharma, S; Babiker, A G; Emery, S; Gordin, F M; Lundgren, J D; Neaton, J N; Bakowska, E; Schechter, M; Wiselka, M J; Wolff, M J

    2015-04-01

    The risks and benefits of initiating antiretroviral treatment (ART) at high CD4 cell counts have not been reliably quantified. The Strategic Timing of AntiRetroviral Treatment (START) study is a randomized international clinical trial that compares immediate with deferred initiation of ART for HIV-positive individuals with CD4 cell counts above 500 cells/μL. We describe the demographics, HIV-specific characteristics and medical history of this cohort. Data collected at baseline include demographics, HIV-specific laboratory values, prior medical diagnoses and concomitant medications. Baseline characteristics were compared by geographical region, gender and age. START enrolled 4685 HIV-positive participants from 215 sites in 35 countries. The median age is 36 years [interquartile range (IQR) 29-44 years], 27% are female, and 45% self-identify as white, 30% as black, 14% as Latino/Hispanic, 8% as Asian and 3% as other. The route of HIV acquisition is reported as men who have sex with men in 55% of participants, heterosexual sex in 38%, injecting drug use in 1% and other/unknown in 5%. Median time since HIV diagnosis is 1.0 year (IQR 0.4-3.0 years) and the median CD4 cell count and HIV RNA values at study entry are 651 cells/μL (IQR 584-765 cells/μL) and 12,754 HIV RNA copies/mL (IQR 3014-43,607 copies/mL), respectively. START has enrolled a diverse group of ART-naïve individuals with high CD4 cell counts who are comparable to the HIV-positive population from the regions in which they were enrolled. The information collected with this robust study design will provide a database with which to evaluate the risks and benefits of early ART use for many important outcomes. © 2015 British HIV Association.

  16. Lineage analysis of the late otocyst stage mouse inner ear by transuterine microinjection of a retroviral vector encoding alkaline phosphatase and an oligonucleotide library.

    Directory of Open Access Journals (Sweden)

    Han Jiang

    Full Text Available The mammalian inner ear subserves the special senses of hearing and balance. The auditory and vestibular sensory epithelia consist of mechanically sensitive hair cells and associated supporting cells. Hearing loss and balance dysfunction are most frequently caused by compromise of hair cells and/or their innervating neurons. The development of gene- and cell-based therapeutics will benefit from a thorough understanding of the molecular basis of patterning and cell fate specification in the mammalian inner ear. This includes analyses of cell lineages and cell dispersals across anatomical boundaries (such as sensory versus nonsensory territories. The goal of this study was to conduct retroviral lineage analysis of the embryonic day 11.5(E11.5 mouse otic vesicle. A replication-defective retrovirus encoding human placental alkaline phosphatase (PLAP and a variable 24-bp oligonucleotide tag was microinjected into the E11.5 mouse otocyst. PLAP-positive cells were microdissected from cryostat sections of the postnatal inner ear and subjected to nested PCR. PLAP-positive cells sharing the same sequence tag were assumed to have arisen from a common progenitor and are clonally related. Thirty five multicellular clones consisting of an average of 3.4 cells per clone were identified in the auditory and vestibular sensory epithelia, ganglia, spiral limbus, and stria vascularis. Vestibular hair cells in the posterior crista were related to one another, their supporting cells, and nonsensory epithelial cells lining the ampulla. In the organ of Corti, outer hair cells were related to a supporting cell type and were tightly clustered. By contrast, spiral ganglion neurons, interdental cells, and Claudius' cells were related to cells of the same type and could be dispersed over hundreds of microns. These data contribute new information about the developmental potential of mammalian otic precursors in vivo.

  17. Electronic medication monitoring-informed counseling to improve adherence to combination anti-retroviral therapy and virologic treatment outcomes: a meta-analysis.

    Science.gov (United States)

    Langebeek, Nienke; Nieuwkerk, Pythia

    2015-01-01

    Adherence to combination anti-retroviral therapy for HIV infection is a primary determinant of treatment success, but is often suboptimal. Previous studies have suggested that electronic medication monitoring-informed counseling is among the most effective adherence intervention components. Our objective was to review available evidence about the effectiveness of monitoring-informed counseling and to aggregate findings into quantitative estimates of the effect of such intervention on medication adherence and virologic treatment outcomes. We searched PubMed for papers reporting on randomized controlled trials comparing intervention groups receiving monitoring-informed counseling as one of the intervention components versus control groups not receiving such counseling for their effect on medication adherence and viral load concentrations. The standardized mean difference (SMD) in adherence and the odds ratio (OR) of undetectable HIV RNA in intervention versus control groups were the common effect sizes. Random-effect models with inverse variance weights were used to aggregate findings into pooled effect estimates with 95% confidence limits (CI). A total of 13 studies were included. Adherence was significantly higher in intervention groups than in control groups (SMD 0.51, 95% CI 0.31-0.71). Patients in intervention groups were significantly more likely to have undetectable HIV RNA concentrations than patients in control groups (OR 1.35, 95% CI 1.12-1.63). However, in studies in which monitoring-informed counseling was the only intervention component, the difference in adherence and virologic response between intervention and control groups was not statistically significant. Electronic monitoring-informed counseling improved adherence and virologic response compared with control groups not receiving such counseling in studies in which it was one out of multiple intervention components, but not in studies where it was the only intervention component.

  18. Construction and characterization in vitro of a bicistronic retroviral vector coding endostatin and interleukin-2 for use in gene therapy; Construcao e caracterizacao in vitro de um vetor retroviral bicistronico codificando endostatina e interleucina-2 para utilizacao em terapia genica

    Energy Technology Data Exchange (ETDEWEB)

    Calvo, Fernanda Bernardes

    2009-07-01

    Gene therapy has been used in preclinical studies and clinical trials in order to alleviate or cure a disease. Retroviral vectors are a tool for gene transfer is widely used. Bicistronic vectors are an attractive alternative for treatment of complex diseases. A variety of options exists to simultaneously express two genes in genetically modified cells. The most common approach relies on bicistronic vectors in which the genes are linked to each other by an internal ribosome entry site allowing co-translational expression of both cistrons. Endostatin, the C-terminal fragment of collagen XVIII, is a potent angiogenesis inhibitor. At present, ES has been widely used in anti-angiogenic in a variety of experimental tumor models, and clinical trials to test it as an anti-tumor agent are already under way. Immunotherapy has been used as adjuvant treatment for tumors and has been used in several preclinical studies and clinical trials. The objective of this project was to construct and characterize 'in vitro' an IRES-based bicistronic retroviral vector encoding endostatin and interleukin-2. The construction of the vector was performed in three stages, the final construction was analyzed by restriction analysis and sequencing. Packaging cells were prepared. The endostatin and interleukin-2 levels were determined by Dot blot. Monocistronic and bicistronic mRNA expression were analyzed by real time RT-PCR. Bicistronic vector showed high levels of virus trites, ranging from 4.20x10{sup 5} to 1.53x10{sup 6}UFC/ml. Secreted levels of endostatin and interleukin-2 ranged from 1.08 to 2.08{mu}g/10{sup 6}cells.24h and 0.66 - 0.89{mu}g/10{sup 6}cells.24h, respectively. The mRNA expression of ES in the NIH3T3 clone pLend-IRES-IL2SN was 2 times higher than the level presented by the NIH3T3 clone pLendSN. The endostatin promoted inhibition (40%) of endothelial cell proliferation. Interleukin-2 promoted a proliferation of 10.6% lymphocytes CD4 and 8.9% of CD8. We conclude that

  19. Protection of hematopoietic cells from O(6)-alkylation damage by O(6)-methylguanine DNA methyltransferase gene transfer: studies with different O(6)-alkylating agents and retroviral backbones.

    Science.gov (United States)

    Jansen, M; Bardenheuer, W; Sorg, U R; Seeber, S; Flasshove, M; Moritz, T

    2001-07-01

    Overexpression of O(6)-methylguanine DNA methyltransferase (MGMT) can protect hematopoietic cells from O(6)-alkylation damage. To identify possible clinical applications of this technology we compared the effect of MGMT gene transfer on the hematotoxicity induced by different O(6)-alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazine derivative temozolomide. In addition, various retroviral vectors expressing the MGMT-cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or temozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus-based retroviral vector (N2/Zip-PGK-MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony-forming units (CFU) was demonstrated for all four drugs. In comparison to mock-transduced controls, after transduction with N2/Zip-PGK-MGMT the IC50 for CFU increased on average 4.7-fold for ACNU, 2.5-fold for BCNU, 6.3-fold for CCNU and 1.5-fold for temozolomide. To study the effect of the retroviral backbone on hematoprotection various vectors expressing the human MGMT-cDNA from a murine embryonic sarcoma virus LTR (MSCV-MGMT) or a hybrid spleen focus-forming/murine embryonic sarcoma virus LTR (SF1-MGMT) were compared with the N2/Zip-PGK-MGMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1-MGMT construct was most efficient especially at high ACNU concentrations (8-12 microg/ml). Similar results were obtained for protection of murine high-proliferative-potential colony-forming cells. These data may help to optimize treatment design and retroviral constructs in future clinical studies aiming at hematoprotection by MGMT gene transfer.

  20. The RNA binding G-patch domain in retroviral protease is important for infectivity and D-type morphogenesis of Mason-Pfizer monkey virus

    Czech Academy of Sciences Publication Activity Database

    Bauerová, Helena; Štokrová, Jitka; Stříšovský, Kvido; Hunter, E.; Ruml, Tomáš; Pichová, Iva

    2005-01-01

    Roč. 280, č. 51 (2005), s. 42106-42112 ISSN 0021-9258 R&D Projects: GA MŠk(CZ) 1M0508; GA MŠk(CZ) 1M0520 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : retroviral protease * RNA binding domain * M-PMV * infectivity * assembly Subject RIV: CE - Biochemistry Impact factor: 5.854, year: 2005

  1. Construction and characterization in vitro of a bicistronic retroviral vector coding endostatin and interleukin-2 for use in gene therapy

    International Nuclear Information System (INIS)

    Calvo, Fernanda Bernardes

    2009-01-01

    Gene therapy has been used in preclinical studies and clinical trials in order to alleviate or cure a disease. Retroviral vectors are a tool for gene transfer is widely used. Bicistronic vectors are an attractive alternative for treatment of complex diseases. A variety of options exists to simultaneously express two genes in genetically modified cells. The most common approach relies on bicistronic vectors in which the genes are linked to each other by an internal ribosome entry site allowing co-translational expression of both cistrons. Endostatin, the C-terminal fragment of collagen XVIII, is a potent angiogenesis inhibitor. At present, ES has been widely used in anti-angiogenic in a variety of experimental tumor models, and clinical trials to test it as an anti-tumor agent are already under way. Immunotherapy has been used as adjuvant treatment for tumors and has been used in several preclinical studies and clinical trials. The objective of this project was to construct and characterize 'in vitro' an IRES-based bicistronic retroviral vector encoding endostatin and interleukin-2. The construction of the vector was performed in three stages, the final construction was analyzed by restriction analysis and sequencing. Packaging cells were prepared. The endostatin and interleukin-2 levels were determined by Dot blot. Monocistronic and bicistronic mRNA expression were analyzed by real time RT-PCR. Bicistronic vector showed high levels of virus trites, ranging from 4.20x10 5 to 1.53x10 6 UFC/ml. Secreted levels of endostatin and interleukin-2 ranged from 1.08 to 2.08μg/10 6 cells.24h and 0.66 - 0.89μg/10 6 cells.24h, respectively. The mRNA expression of ES in the NIH3T3 clone pLend-IRES-IL2SN was 2 times higher than the level presented by the NIH3T3 clone pLendSN. The endostatin promoted inhibition (40%) of endothelial cell proliferation. Interleukin-2 promoted a proliferation of 10.6% lymphocytes CD4 and 8.9% of CD8. We conclude that the IRES bicistronic vector

  2. Ex vivo γ-retroviral gene therapy of dogs with X-linked severe combined immunodeficiency and the development of a thymic T cell lymphoma.

    Science.gov (United States)

    Kennedy, Douglas R; Hartnett, Brian J; Kennedy, Jeffrey S; Vernau, William; Moore, Peter F; O'Malley, Thomas; Burkly, Linda C; Henthorn, Paula S; Felsburg, Peter J

    2011-07-15

    We have previously shown that in vivo γ-retroviral gene therapy of dogs with X-linked severe combined immunodeficiency (XSCID) results in sustained T cell reconstitution and sustained marking in myeloid and B cells for up to 4 years with no evidence of any serious adverse effects. The purpose of this study was to determine whether ex vivo γ-retroviral gene therapy of XSCID dogs results in a similar outcome. Eight of 12 XSCID dogs treated with an average of dose of 5.8 × 10(6) transduced CD34(+) cells/kg successfully engrafted producing normal numbers of gene-corrected CD45RA(+) (naïve) T cells. However, this was followed by a steady decrease in CD45RA(+) T cells, T cell diversity, and thymic output as measured by T cell receptor excision circles (TRECs) resulting in a T cell lymphopenia. None of the dogs survived past 11 months post treatment. At necropsy, few gene-corrected thymocytes were observed correlating with the TREC levels and one of the dogs was diagnosed with a thymic T cell lymphoma that was attributed to the gene therapy. This study highlights the outcome differences between the ex vivo and in vivo approach to γ-retroviral gene therapy and is the first to document a serious adverse event following gene therapy in a canine model of a human genetic disease. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Pump apparatus including deconsolidator

    Energy Technology Data Exchange (ETDEWEB)

    Sonwane, Chandrashekhar; Saunders, Timothy; Fitzsimmons, Mark Andrew

    2014-10-07

    A pump apparatus includes a particulate pump that defines a passage that extends from an inlet to an outlet. A duct is in flow communication with the outlet. The duct includes a deconsolidator configured to fragment particle agglomerates received from the passage.

  4. Optical modulator including grapene

    Science.gov (United States)

    Liu, Ming; Yin, Xiaobo; Zhang, Xiang

    2016-06-07

    The present invention provides for a one or more layer graphene optical modulator. In a first exemplary embodiment the optical modulator includes an optical waveguide, a nanoscale oxide spacer adjacent to a working region of the waveguide, and a monolayer graphene sheet adjacent to the spacer. In a second exemplary embodiment, the optical modulator includes at least one pair of active media, where the pair includes an oxide spacer, a first monolayer graphene sheet adjacent to a first side of the spacer, and a second monolayer graphene sheet adjacent to a second side of the spacer, and at least one optical waveguide adjacent to the pair.

  5. Retroviral reverse transcriptase inhibitory activity in Thai herbs and spices: screening with Moloney murine leukemia viral enzyme.

    Science.gov (United States)

    Suthienkul, O; Miyazaki, O; Chulasiri, M; Kositanont, U; Oishi, K

    1993-12-01

    Fifty-seven Thai herbs and spices were examined for their retroviral reverse transcriptase inhibitory activity. All herbs and spices were extracted with hot-water and methanol. Reverse transcriptase inhibitory activity of the extracts was determined by using Moloney Murine Leukemia Virus reverse transcriptase (M-MuLV-RT) reacted with 3H-dTTP and radioactivity measured with a scintillation counter. Eighty-one per cent (46/57) of hot-water extracts and 54% (31/57) of methanol extracts showed inhibitory activities. At a concentration of 125 micrograms/ml, 13% (6/46) of hot-water extracts, namely Eugenia caryophyllus Bullock et Harrison, Phyllanthus urinaria Linn., Terminalia belerica Roxb., Nelumbo nucifera Gaertn., Psidium guajava Linn. and Lawsonia inermis Linn., had a relative inhibitory ratio (IR) over 50%. They showed ratios of 100%, 91%, 75%, 74%, 61% and 60%, respectively. For methanol extracts, only 10% (3/31) had IR values over 50%. They were T. belerica, E. caryophyllus and N. nucifera which exhibited IR values of 83%, 54% and 54%, respectively.

  6. Identification of a high incidence region for retroviral vector integration near exon 1 of the LMO2 locus

    Directory of Open Access Journals (Sweden)

    Sakashita Kazuo

    2009-09-01

    Full Text Available Abstract Therapeutic retroviral vector integration near the oncogene LMO2 is thought to be a cause of leukemia in X-SCID gene therapy trials. However, no published studies have evaluated the frequency of vector integrations near exon 1 of the LMO2 locus. We identified a high incidence region (HIR of vector integration using PCR techniques in the upstream region close to the LMO2 transcription start site in the TPA-Mat T cell line. The integration frequency of the HIR was one per 4.46 × 104 cells. This HIR was also found in Jurkat T cells but was absent from HeLa cells. Furthermore, using human cord blood-derived CD34+ cells we identified a HIR in a similar region as the TPA-Mat T cell line. One of the X-linked severe combined immunodeficiency (X-SCID patients that developed leukemia after gene therapy had a vector integration site in this HIR. Therefore, the descriptions of the location and the integration frequency of the HIR presented here may help us to better understand vector-induced leukemogenesis.

  7. A review of ICT systems for HIV/AIDS and anti-retroviral treatment management in South Africa.

    Science.gov (United States)

    Sørensen, Tove; Rivett, Ulrike; Fortuin, Jill

    2008-01-01

    Telemedicine and e-health systems have been proposed as a support tool, to monitor and evaluate HIV/AIDS management strategies. The aim of the present study was to provide an overview of telemedicine and e-health systems for HIV/AIDS in South Africa as a basis for developing an e-health toolkit for anti-retroviral treatment (ART). An initial literature review and a subsequent interactive networking approach were chosen to identify telemedicine and e-health systems, projects and services for HIV/AIDS and ART facilities in low-resource settings and under-served areas. The literature review produced little useful information. In contrast, the face-to-face interviews and the focus group discussions provided useful information about projects and systems which had not been published. The meetings involved 1 - 5 people per session, about 30 people in total. The review showed that there were some plans for telemedicine and e-health implementation in South Africa. However, there was no all-inclusive ICT-based system in place for AIDS treatment there. With the exception of the major health information systems and electronic patient record systems, none of the telemedicine and e-health systems identified in the review were ready to be deployed across the country as a whole.

  8. Different impact of anti-retroviral regimen containing protease inhibitors on development of HIV-related Kaposi sarcoma.

    Science.gov (United States)

    Carleo, Maria Aurora; Di Martino, Filomena; Del Giudice, Annalisa; Gargiulo, Miriam; Parrella, Giovanni; Rosario, Pietro; Sangiovanni, Vincenzo; Viglietti, Rosaria; Esposito, Vincenzo; Chirianni, Antonio

    2015-01-01

    The incidence of Kaposi's sarcoma (KS), an AIDS-related malignancy, has dramatically decreased in the Highly Active Anti-retroviral Therapy (HAART) era. However, KS remains the second most frequent tumor in HIV-infected patients worldwide and has become the most common cancer in the sub-Saharan Africa. Experimental studies have demonstrated a direct anti-neoplastic effect of HAART, and overall of protease inhibitors (PIs), on KS. We describe five cases of KS in HIV-infected patients on HAART regimen, containing PIs as atazanavir/r (ATV/r), darunavir/r (DRV/r), lopinavir/r (LPV/r) and fosamprenavir (fAMP/r). Clinical and experimental observations support the hypothesis that PIs may play an important role in prevention and treatment of KS. In our study, the treatment with PIs of recent generation was not protective against the development of KS. Therefore, it could be necessary to re-evaluate the therapeutic effects of PIs and their role in the development and treatment of KS in HIV-infected patients. Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  9. Prevalence of Drug Resistance Associated Mutations Among the Anti Retroviral Therapy Exposed HIV-1 Infected Individuals in Manipur, Northeast India.

    Science.gov (United States)

    Lakhikumar Sharma, Adhikarimayum; Ramsing Singh, Thiyam; Ranjana Devi, Khuraijam; Shanjukumar Singh, Lisam

    2016-01-01

    Manipur is one of the highest HIV prevalence states of India because of its geographical location at the international border near the golden triangle of South-East Asia, but no study on drug resistance associated mutations (DRAMs) has been reported yet. A population-based study on DRAMs of HIV-1 among the anti-retroviral therapy (ART) exposed HIV-1 infected individuals of Manipur was conducted. 110 HIV-1 positive individuals who had initially exposed to first line anti-HIV drugs were recruited for the surveillance of DRAMs. Reverse transcriptase and protease genes of HIV-1 were amplified, sequenced and analyzed. Significant prevalence of DRAMs of HIV-1 was found among the ART exposed HIV-1 infected individuals of Manipur. The results revealed that 37%, 29% and 7% individuals harbor HIV-1 strains mutated at the target sites of nonnucleoside reverse transcriptase inhibitors, nucleoside reverse transcriptase inhibitors and protease inhibitors respectively. Predominant DRAMs at RT genes were M184V, T215Y, M41L and V108I and H221Y while at PR genes were M46I and I47V. Among the high risk groups, intravenous drug users have the highest number of DRAMs followed by heterosexual individuals. Analysis of viral subtype based on pol gene revealed 83% subtype C, 11.8% recombinant forms and 5.2% subtype B. DRAMs at the target sites of reverse transcriptase inhibitors are high and these were found to have developed resistance to the primary ART drugs that are used in Manipur. The findings of this study will help the clinicians to guide patients during the course of ART treatment regimes.

  10. Susceptibility of Human Oral Squamous Cell Carcinoma (OSCC H103 and H376 cell lines to Retroviral OSKM mediated reprogramming

    Directory of Open Access Journals (Sweden)

    Nalini Devi Verusingam

    2017-04-01

    Full Text Available Although numbers of cancer cell lines have been shown to be successfully reprogrammed into induced pluripotent stem cells (iPSCs, reprogramming Oral Squamous Cell Carcinoma (OSCC to pluripotency in relation to its cancer cell type and the expression pattern of pluripotent genes under later passage remain unexplored. In our study, we reprogrammed and characterised H103 and H376 oral squamous carcinoma cells using retroviral OSKM mediated method. Reprogrammed cells were characterized for their embryonic stem cells (ESCs like morphology, pluripotent gene expression via quantitative real-time polymerase chain reaction (RT-qPCR, immunofluorescence staining, embryoid bodies (EB formation and directed differentiation capacity. Reprogrammed H103 (Rep-H103 exhibited similar ESCs morphologies with flatten cells and clear borders on feeder layer. Reprogrammed H376 (Rep-H376 did not show ESCs morphologies but grow with a disorganized morphology. Critical pluripotency genes Oct4, Sox2 and Nanog were expressed higher in Rep-H103 against the parental counterpart from passage 5 to passage 10. As for Rep-H376, Nanog expression against its parental counterpart showed a significant decrease at passage 5 and although increased in passage 10, the level of expression was similar to the parental cells. Rep-H103 exhibited pluripotent signals (Oct4, Sox2, Nanog and Tra-1-60 and could form EB with the presence of three germ layers markers. Rep-H103 displayed differentiation capacity into adipocytes and osteocytes. The OSCC cell line H103 which was able to be reprogrammed into an iPSC like state showed high expression of Oct4, Sox2 and Nanog at late passage and may provide a potential iPSC model to study multi-stage oncogenesis in OSCC.

  11. Profiles of HIV-infected anti-retroviral therapy naïve children from Mumbai, India.

    Science.gov (United States)

    Paranjpe, Supriya Mayur; Sarkate, Purva Pankaj; Ingole, Nayana Avinash; Raut, Shweta Sadanand; Mehta, Preeti Rajeev

    2016-11-01

    This study aimed to investigate the demographic profiles of human immunodifficiency virus (HIV) infected anti-retroviral therapy (ART) naïve children in our hospital and their relations to the clinical, immunological and nutritional status. A cross-sectional study was conducted in an Integrated Counselling and Testing Center (ICTC) at a tertiary care hospital in Mumbai. ART naïve HIV positive children were enrolled in the study. The demographic profiles, clinical features, immunological (CD4%/CD4 count) and nutritional status of these children were recorded. The agreement between clinical, immunological and nutritional staging was determined using Cohen's kappa test. In 192 HIV-infected ART naive children enrolled with a median age of 9 years (range 3 months-14 years), 97.4% acquired infection through vertical transmission. The most common clinical presentation was fever (39.6 %), followed by generalized lymphadenopathy (32.3%), cough (22.4%) and diarrhoea (9.9%). Tuberculosis was seen in 22.9% of the children. The agreement was fair between clinical and immunological staging, and slight between nutritional, immunological and clinical staging. Perinatal transmission is the most common mode of acquiring HIV infection in children. The Prevention of Parent to Child Transmission (PPTCT) program should be strengthened for lowering the transmission rate by providing extended ART to mothers during pregnancy and breast-feeding. Tuberculosis remains a major concern in HIV-infected children. The poor correlation between WHO clinical and immunological staging emphasizes the importance of making CD4 facilities available in HIV prevalent areas. Malnutrition cannot be used as a surrogate marker for predicting stage or severity as it is common at all stages of HIV disease.

  12. Serum lipid profile of anti-retroviral (ARV) naïve human ...

    African Journals Online (AJOL)

    Background: Human immunodeficiency virus infection has become pandemic in Nigeria and affects the immune system. Most HIV/ AIDS patients develop multiple metabolic abnormalities including insulin resistance, lipodystrophy and dyslipidemia leading to increased risk of cardiovascular disease (CVD). Objective: To ...

  13. Divergent effects of Tenofovir and Retrovir (AZT) on TLR-mediated cytokine production

    DEFF Research Database (Denmark)

    Melchjorsen, Jesper; Tolstrup, Martin; Paludan, Søren Riis

      Pathogen-recognizing Toll-like receptors 2 (TLR2) and TLR4 are known to recognize a number of pathogens, including E.Coli, S. Pneumoniae and N. Meningitidis. We have studied whether a number of HIV therapeutics affect immediate proinflammatory cytokine responses in cell cultures. Preliminary...

  14. S2 from equine infectious anemia virus is an infectivity factor which counteracts the retroviral inhibitors SERINC5 and SERINC3.

    Science.gov (United States)

    Chande, Ajit; Cuccurullo, Emilia Cristiana; Rosa, Annachiara; Ziglio, Serena; Carpenter, Susan; Pizzato, Massimo

    2016-11-15

    The lentivirus equine infectious anemia virus (EIAV) encodes the small protein S2, a pathogenic determinant that is important for virus replication and disease progression in horses. No molecular function had been linked to this accessory protein. We report that S2 can replace the activity of Negative factor (Nef) in HIV-1 infectivity, being required to antagonize the inhibitory activity of Serine incorporator (SERINC) proteins on Nef-defective HIV-1. Like Nef, S2 excludes SERINC5 from virus particles and requires an ExxxLL motif predicted to recruit the clathrin adaptor, Adaptor protein 2 (AP2). Accordingly, functional endocytic machinery is essential for S2-mediated infectivity enhancement, and S2-mediated enhancement is impaired by inhibitors of clathrin-mediated endocytosis. In addition to retargeting SERINC5 to a late endosomal compartment, S2 promotes host factor degradation. Emphasizing the similarity with Nef, we show that S2 is myristoylated, and, as is compatible with a crucial role in posttranslational modification, its N-terminal glycine is required for anti-SERINC5 activity. EIAV-derived vectors devoid of S2 are less susceptible than HIV-1 to the inhibitory effect of both human and equine SERINC5. We then identified the envelope glycoprotein of EIAV as a determinant that also modulates retroviral susceptibility to SERINC5, indicating that EIAV has a bimodal ability to counteract the host factor. S2 shares no sequence homology with other retroviral factors known to counteract SERINC5. Like the primate lentivirus Nef and the gammaretrovirus glycoGag, the accessory protein from EIAV is an example of a retroviral virulence determinant that independently evolved SERINC5-antagonizing activity. SERINC5 therefore plays a critical role in the interaction of the host with diverse retrovirus pathogens.

  15. Parasitoses intestinais em portadores de HIV/AIDS nas eras prà e pÃs terapia anti-retroviral potente

    OpenAIRE

    Tatiana Paschoalette Rodrigues Bachur

    2007-01-01

    Desde os primeiros casos de AIDS descritos, alta prevalÃncia de diarrÃia de etiologia parasitÃria era reportada; antes da terapia anti-retroviral potente (HAART), era elevada a prevalÃncia de enteroparasitos em portadores de HIV/AIDS no Brasil. Com o advento da HAART, isto tem se modificado. O objetivo deste trabalho foi determinar a prevalÃncia de parasitos intestinais em portadores de HIV/AIDS atendidos no Hospital SÃo Josà de DoenÃas Infecciosas e no Hospital UniversitÃrio Walter CantÃdio,...

  16. Anti-retroviral Therapy and Pregnancy Outcomes in Developing Countries: A Systematic Review

    Directory of Open Access Journals (Sweden)

    Fekadu Mazengia Alemu, MPH

    2015-03-01

    Full Text Available Background: Despite significant efforts to understand adverse pregnancy outcome in women receiving Antiretroviral Therapy (ART, ART-related adverse birth outcomes are still poorly understood. We systematically review ART-related adverse birth outcomes among HIV-infected pregnant women; we also review the covariates associated with adverse birth outcomes in the aforementioned group. Methods: The main source for our systematic review was electronic bibliographic databases. Databases such as MEDLINE, PubMed, EMBASE and AIDSLINE were searched. Furthermore, search engines such as Google and Google Scholar were specifically searched for gray literature. Methodological quality of available literature was assessed using the Newcastle – Ottawa Quality Assessment Scale & M. Hewitt guideline. We examined a total of 1,124 papers and reviewed the studies using the PICOT criteria which stands for Patient (population, Intervention (or “Exposure”, Comparison, Outcome and Type of study. Finally, 32 methodologically fit studies were retained and included in our review. Results: Frequently observed adverse birth outcomes included low birth weight (LBW, Preterm Birth (PB, Small for Gestational Age (SGA, while still birth and congenital anomalies were infrequent. Type of regimen such as Protease Inhibitor (PI based regimens and timing of initiation of ART are some of the factors associated with adverse pregnancy outcomes. Covariates principally included malnutrition and other co-morbidities such as malaria and HIV. Conclusions and Public Health Implications: There is growing evidence in published literature suggesting that ART might be causing adverse birth outcomes among pregnant women in developing countries. There is a need to consider regimen types for HIV-infected pregnant women. There is need to design large cohort studies.

  17. Characterization of the fusion core in zebrafish endogenous retroviral envelope protein

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Jian [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072 (China); State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China); Zhang, Huaidong [CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China); Gong, Rui, E-mail: gongr@wh.iov.cn [CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China); Xiao, Gengfu, E-mail: xiaogf@wh.iov.cn [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072 (China); State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China)

    2015-05-08

    Zebrafish endogenous retrovirus (ZFERV) is the unique endogenous retrovirus in zebrafish, as yet, containing intact open reading frames of its envelope protein gene in zebrafish genome. Similarly, several envelope proteins of endogenous retroviruses in human and other mammalian animal genomes (such as syncytin-1 and 2 in human, syncytin-A and B in mouse) were identified and shown to be functional in induction of cell–cell fusion involved in placental development. ZFERV envelope protein (Env) gene appears to be also functional in vivo because it is expressible. After sequence alignment, we found ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR) which were crucial for membrane fusion. We expressed the regions of N + C protein in the ZFERV Env (residues 459–567, including predicted NHR and CHR) to characterize the fusion core structure. We found N + C protein could form a stable coiled-coil trimer that consists of three helical NHR regions forming a central trimeric core, and three helical CHR regions packing into the grooves on the surface of the central core. The structural characterization of the fusion core revealed the possible mechanism of fusion mediated by ZFERV Env. These results gave comprehensive explanation of how the ancient virus infects the zebrafish and integrates into the genome million years ago, and showed a rational clue for discovery of physiological significance (e.g., medicate cell–cell fusion). - Highlights: • ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes. • The fusion core of ZFERV Env forms stable coiled-coil trimer including three NHRs and three CHRs. • The structural mechanism of viral entry mediated by ZFERV Env is disclosed. • The results are helpful for further discovery of physiological function of ZFERV Env in zebrafish.

  18. Regions identity between the genome of vertebrates and non-retroviral families of insect viruses

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    Fan Gaowei

    2011-11-01

    Full Text Available Abstract Background The scope of our understanding of the evolutionary history between viruses and animals is limited. The fact that the recent availability of many complete insect virus genomes and vertebrate genomes as well as the ability to screen these sequences makes it possible to gain a new perspective insight into the evolutionary interaction between insect viruses and vertebrates. This study is to determine the possibility of existence of sequence identity between the genomes of insect viruses and vertebrates, attempt to explain this phenomenon in term of genetic mobile element, and try to investigate the evolutionary relationship between these short regions of identity among these species. Results Some of studied insect viruses contain variable numbers of short regions of sequence identity to the genomes of vertebrate with nucleotide sequence length from 28 bp to 124 bp. They are found to locate in multiple sites of the vertebrate genomes. The ontology of animal genes with identical regions involves in several processes including chromatin remodeling, regulation of apoptosis, signaling pathway, nerve system development and some enzyme-like catalysis. Phylogenetic analysis reveals that at least some short regions of sequence identity in the genomes of vertebrate are derived the ancestral of insect viruses. Conclusion Short regions of sequence identity were found in the vertebrates and insect viruses. These sequences played an important role not only in the long-term evolution of vertebrates, but also in promotion of insect virus. This typical win-win strategy may come from natural selection.

  19. Regions identity between the genome of vertebrates and non-retroviral families of insect viruses.

    Science.gov (United States)

    Fan, Gaowei; Li, Jinming

    2011-11-10

    The scope of our understanding of the evolutionary history between viruses and animals is limited. The fact that the recent availability of many complete insect virus genomes and vertebrate genomes as well as the ability to screen these sequences makes it possible to gain a new perspective insight into the evolutionary interaction between insect viruses and vertebrates. This study is to determine the possibility of existence of sequence identity between the genomes of insect viruses and vertebrates, attempt to explain this phenomenon in term of genetic mobile element, and try to investigate the evolutionary relationship between these short regions of identity among these species. Some of studied insect viruses contain variable numbers of short regions of sequence identity to the genomes of vertebrate with nucleotide sequence length from 28 bp to 124 bp. They are found to locate in multiple sites of the vertebrate genomes. The ontology of animal genes with identical regions involves in several processes including chromatin remodeling, regulation of apoptosis, signaling pathway, nerve system development and some enzyme-like catalysis. Phylogenetic analysis reveals that at least some short regions of sequence identity in the genomes of vertebrate are derived the ancestral of insect viruses. Short regions of sequence identity were found in the vertebrates and insect viruses. These sequences played an important role not only in the long-term evolution of vertebrates, but also in promotion of insect virus. This typical win-win strategy may come from natural selection.

  20. Structure of a Spumaretrovirus Gag Central Domain Reveals an Ancient Retroviral Capsid.

    Science.gov (United States)

    Ball, Neil J; Nicastro, Giuseppe; Dutta, Moumita; Pollard, Dominic J; Goldstone, David C; Sanz-Ramos, Marta; Ramos, Andres; Müllers, Erik; Stirnnagel, Kristin; Stanke, Nicole; Lindemann, Dirk; Stoye, Jonathan P; Taylor, William R; Rosenthal, Peter B; Taylor, Ian A

    2016-11-01

    The Spumaretrovirinae, or foamy viruses (FVs) are complex retroviruses that infect many species of monkey and ape. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. However, there is a paucity of structural information for FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. To probe the functional overlap of FV and orthoretroviral Gag we have determined the structure of a central region of Gag from the Prototype FV (PFV). The structure comprises two all α-helical domains NtDCEN and CtDCEN that although they have no sequence similarity, we show they share the same core fold as the N- (NtDCA) and C-terminal domains (CtDCA) of archetypal orthoretroviral capsid protein (CA). Moreover, structural comparisons with orthoretroviral CA align PFV NtDCEN and CtDCEN with NtDCA and CtDCA respectively. Further in vitro and functional virological assays reveal that residues making inter-domain NtDCEN-CtDCEN interactions are required for PFV capsid assembly and that intact capsid is required for PFV reverse transcription. These data provide the first information that relates the Gag proteins of Spuma and Orthoretrovirinae and suggests a common ancestor for both lineages containing an ancient CA fold.

  1. New bioinformatic tool for quick identification of functionally relevant endogenous retroviral inserts in human genome.

    Science.gov (United States)

    Garazha, Andrew; Ivanova, Alena; Suntsova, Maria; Malakhova, Galina; Roumiantsev, Sergey; Zhavoronkov, Alex; Buzdin, Anton

    2015-01-01

    Endogenous retroviruses (ERVs) and LTR retrotransposons (LRs) occupy ∼8% of human genome. Deep sequencing technologies provide clues to understanding of functional relevance of individual ERVs/LRs by enabling direct identification of transcription factor binding sites (TFBS) and other landmarks of functional genomic elements. Here, we performed the genome-wide identification of human ERVs/LRs containing TFBS according to the ENCODE project. We created the first interactive ERV/LRs database that groups the individual inserts according to their familial nomenclature, number of mapped TFBS and divergence from their consensus sequence. Information on any particular element can be easily extracted by the user. We also created a genome browser tool, which enables quick mapping of any ERV/LR insert according to genomic coordinates, known human genes and TFBS. These tools can be used to easily explore functionally relevant individual ERV/LRs, and for studying their impact on the regulation of human genes. Overall, we identified ∼110,000 ERV/LR genomic elements having TFBS. We propose a hypothesis of "domestication" of ERV/LR TFBS by the genome milieu including subsequent stages of initial epigenetic repression, partial functional release, and further mutation-driven reshaping of TFBS in tight coevolution with the enclosing genomic loci.

  2. Structure of a Spumaretrovirus Gag Central Domain Reveals an Ancient Retroviral Capsid.

    Directory of Open Access Journals (Sweden)

    Neil J Ball

    2016-11-01

    Full Text Available The Spumaretrovirinae, or foamy viruses (FVs are complex retroviruses that infect many species of monkey and ape. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. However, there is a paucity of structural information for FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. To probe the functional overlap of FV and orthoretroviral Gag we have determined the structure of a central region of Gag from the Prototype FV (PFV. The structure comprises two all α-helical domains NtDCEN and CtDCEN that although they have no sequence similarity, we show they share the same core fold as the N- (NtDCA and C-terminal domains (CtDCA of archetypal orthoretroviral capsid protein (CA. Moreover, structural comparisons with orthoretroviral CA align PFV NtDCEN and CtDCEN with NtDCA and CtDCA respectively. Further in vitro and functional virological assays reveal that residues making inter-domain NtDCEN-CtDCEN interactions are required for PFV capsid assembly and that intact capsid is required for PFV reverse transcription. These data provide the first information that relates the Gag proteins of Spuma and Orthoretrovirinae and suggests a common ancestor for both lineages containing an ancient CA fold.

  3. Development of retroviral vectors for tissue-restricted expression in chicken embryonic gonads.

    Directory of Open Access Journals (Sweden)

    Luke S Lambeth

    Full Text Available The chicken embryo has long been a useful model organism for studying development, including sex determination and gonadal differentiation. However, manipulating gene expression specifically in the embryonic avian gonad has been difficult. The viral vector RCASBP can be readily used for embryo-wide transgene expression; however global mis-expression using this method can cause deleterious off-target effects and embryo-lethality. In an attempt to develop vectors for the over-expression of sequences in chicken embryonic urogenital tissues, the viral vector RCANBP was engineered to contain predicted promoter sequences of gonadal-expressed genes. Several promoters were analysed and it was found that although the SF1 promoter produced a tissue-restricted expression pattern that was highest in the mesonephros and liver, it was also higher in the gonads compared to the rest of the body. The location of EGFP expression from the SF1 promoter overlapped with several key gonad-expressed sex development genes; however expression was generally low-level and was not seen in all gonadal cells. To further validate this sequence the key testis determinant DMRT1 was over-expressed in female embryos, which due to insufficient levels had no effect on gonad development. The female gene aromatase was then over-expressed in male embryos, which disrupted the testis pathway as demonstrated by a reduction in AMH protein. Taken together, although these data showed that the SF1 promoter can be used for functional studies in ovo, a stronger promoter sequence would likely be required for the functional analysis of gonad genes that require high-level expression.

  4. HIV sero-discordance among married HIV patients initiating anti-retroviral therapy in northern Vietnam.

    Science.gov (United States)

    Van Tam, Vu; Cuong, Do Duy; Alfven, Tobias; Phuc, Ho Dang; Chuc, Nguyen Thi Kim; Hoa, Nguyen Phuong; Diwan, Vinod; Larsson, Mattias

    2016-01-01

    In many countries in Asia, the HIV epidemic is in a concentrated phase, with high prevalence in certain risk groups, such as men who inject drugs. There is also a rapid increase of HIV among women. The latter might be due to high levels of sero-discordant couples and increasing transmission from male to female partners over time. All adult married patients initiating antiretroviral treatment at four out-patient clinics in Quang Ninh province in north-eastern Vietnam between 2007 and 2009 were asked to participate in the study. Clinical information was extracted from patients' records, and a structured questionnaire was used to collect social, demographic and economic data. Two hundred eighty-eight married patients for whom information on the HIV status of their spouse was available were included in the study. Overall, the sero-discordance rate was 58%. The sero-discordance rate was significantly higher among married males, 71% had spouses not infected, than married females, of whom 18% had spouses not infected. Other factors associated with a high rate of sero-discordance were injection drug use (IDU) history, tuberculosis (TB) history and the availability of voluntary counselling and testing (VCT) in residential locations. High sero-concordance was associated with college/university education. The sero-discordance was significantly higher among married males than married females. Other factors also related to high sero-discordance were history of IDU, history of TB and the availability of VCT in residential locations. In contrast, college/university education and female sex were significantly related to low sero-discordance. To contain the increasing HIV prevalence among women, measures should be taken to prevent transmission among sero-discordant couples. Trial registration NCT01433601.

  5. Sustainability of a community-based anti-retroviral care delivery model - a qualitative research study in Tete, Mozambique.

    Science.gov (United States)

    Rasschaert, Freya; Decroo, Tom; Remartinez, Daniel; Telfer, Barbara; Lessitala, Faustino; Biot, Marc; Candrinho, Baltazar; Van Damme, Wim

    2014-01-01

    To overcome patients' reported barriers to accessing anti-retroviral therapy (ART), a community-based delivery model was piloted in Tete, Mozambique. Community ART Groups (CAGs) of maximum six patients stable on ART offered cost- and time-saving benefits and mutual psychosocial support, which resulted in better adherence and retention outcomes. To date, Médecins Sans Frontières has coordinated and supported these community-driven activities. To better understand the sustainability of the CAG model, we developed a conceptual framework on sustainability of community-based programmes. This was used to explore the data retrieved from 16 focus group discussions and 24 in-depth interviews with different stakeholder groups involved in the CAG model and to identify factors influencing the sustainability of the CAG model. We report the findings according to the framework's five components. (1) The CAG model was designed to overcome patients' barriers to ART and was built on a concept of self-management and patient empowerment to reach effective results. (2) Despite the progressive Ministry of Health (MoH) involvement, the daily management of the model is still strongly dependent on external resources, especially the need for a regulatory cadre to form and monitor the groups. These additional resources are in contrast to the limited MoH resources available. (3) The model is strongly embedded in the community, with patients taking a more active role in their own healthcare and that of their peers. They are considered as partners in healthcare, which implies a new healthcare approach. (4) There is a growing enabling environment with political will and general acceptance to support the CAG model. (5) However, contextual factors, such as poverty, illiteracy and the weak health system, influence the community-based model and need to be addressed. The community embeddedness of the model, together with patient empowerment, high acceptability and progressive MoH involvement

  6. Incidence of WHO stage 3 and 4 conditions following initiation of anti-retroviral therapy in resource limited settings.

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    Andrea J Curtis

    Full Text Available OBJECTIVES: To determine the incidence of WHO clinical stage 3 and 4 conditions during early anti-retroviral therapy (ART in resource limited settings (RLS. DESIGN/SETTING: A descriptive analysis of routine program data collected prospectively from 25 Médecins Sans Frontières supported HIV treatment programs in eight countries between 2002 and 2010. SUBJECTS/PARTICIPANTS: 35,349 study participants with median follow-up on ART of 1.33 years (IQR 0.51-2.41. OUTCOME MEASURES: Incidence in 100 person-years of WHO stage 3 or 4 conditions during 5 periods after ART initiation. Diagnoses of conditions were made according to WHO criteria and relied upon clinical assessments supported by basic laboratory investigations. RESULTS: The incidence of any WHO clinical stage 3 or 4 condition over 3 years was 40.02 per 100 person-years (31.77 for stage 3 and 8.25 for stage 4. The incidence of stage 3 and 4 conditions fell by over 97% between months 0-3 and months 25-36 (77.81 to 2.40 for stage 3 and 28.70 to 0.64 for stage 4. During months 0-3 pulmonary tuberculosis was the most common condition diagnosed in adults (incidence 22.24 per 100 person-years and children aged 5-14 years (25.76 and oral candidiasis was the most common in children <5 years (25.79. Overall incidences were higher in Africa compared with Asia (43.98 versus 12.97 for stage 3 and 8.98 versus 7.05 for stage 4 conditions, p<0.001. Pulmonary tuberculosis, weight loss, oral and oesophageal candidiasis, chronic diarrhoea, HIV wasting syndrome and severe bacterial infections were more common in Africa. Extra-pulmonary tuberculosis, non-tuberculous mycobacterial infection, cryptococcosis, penicilliosis and toxoplasmosis were more common in Asia. CONCLUSIONS: The incidence of WHO stage 3 and 4 conditions during the early period after ART initiation in RLS is high, but greatly reduces over time. This is likely due to both the benefits of ART and deaths of the sickest patients occurring shortly

  7. Non-adherence to anti-retroviral therapy among HIV infected adults in Mon State of Myanmar

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    Win Lei Aye

    2017-05-01

    Full Text Available Abstract Background The provision of Anti-Retroviral Therapy (ART was started in Myanmar in 2005 in collaboration with the National AIDS Program and the private sector. Successful clinical management of HIV-infected patients is subject to optimal adherence. The aim of the study was to determine the prevalence of adherence to ART and identify factors associated with non-adherence to ART among HIV infected adults registered in a private sector setting in Mon State, Myanmar. Methods This cross-sectional study was conducted with adults living with HIV receiving ART at an HIV outpatient clinic between April and May 2016. A total of three hundred People Living with HIV(PLHIV were interviewed using a pretested and structured questionnaire. The 30 days Visual Analog Scale (VAS adherence instrument was used to assess the level of adherence. Multivariable logistic regression analysis was used to determine factors associated with non-adherence to ART. Results Among 300 patients (male 37.7% and female 62.3%, with a mean age of 41.3 years, standard deviation 8.7, 84% reported ≥95% adherence to ART in the past month. Among 16% of those reporting non-adherence, major reasons for skipping the medication were being busy (23%, being away from home (17.7% and being forgetful (12.3%. In multivariable logistic rgeression, low behavioural skills on ART adherence (OR = 0.31, 95% CI: 0.10-0.94, tobacco use (OR = 3.22, 95% CI:1.28-8.12, having disclosed their HIV status (OR = 0.07, 95% CI: 0.01-0.69, having a partner who was not on ART (OR = 4.25, 95% CI: 1.70-10.64 and among men, having erectile dysfunction (OR = 15.14, 95% CI: 1.41-162.66 were significant associated with ART non-adherence. Conclusion Non-adherence to ART was associated with individual moderating factors and behavioral skills. Priority measures such as addressing risk behaviour and behavioural change communication tailored to individual patients’ lifestyles requires comprehensive

  8. Comparison of anti-retroviral therapy treatment strategies in prevention of mother-to-child transmission in a teaching hospital in Ethiopia

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    Kumela K

    2015-06-01

    Full Text Available Background: More than 90% of Human immunodeficiency virus (HIV infection in children is acquired due to mother-to-child transmission, which is spreading during pregnancy, delivery or breastfeeding. Objective: To determine the effectiveness of highly active antiretroviral and short course antiretroviral regimens in prevention of mother-to-child transmission of HIV and associated factors Jimma University Specialized Hospital (JUSH. Method: A hospital based retrospective cohort study was conducted on HIV infected pregnant mothers who gave birth and had follow up at anti-retroviral therapy (ART clinic for at least 6 months during a time period paired with their infants. The primary and secondary outcomes were rate of infant infection by HIV at 6 weeks and 6 months respectively. The Chi-square was used for the comparison of categorical data multivariate logistic regression model was used to identify the determinants of early mother-to-child transmission of HIV at 6 weeks. Cox proportional hazard model was used to analyze factors that affect the 6 month HIV free survival of infants born to HIV infected mothers. Results: A total of 180 mother infant pairs were considered for the final analysis, 90(50% mothers received single dose nevirapine (sdNVP designated as regimen-3, 67 (37.2% mothers were on different types of ARV regimens commonly AZT + 3TC + NVP (regimen-1, while the rest 23 (12.8% mothers were on short course dual regimen AZT + 3TC + sdNVP (regimen-2. Early mother-to-child transmission rate at 6 weeks for regimens 1, 2 and 3 were 5.9% (4/67, 8.6% (2/23, and 15.5% (14/90 respectively. The late cumulative mother-to-child transmission rate of HIV at 6 months regardless of regimen type was 15.5% (28/180. Postnatal transmission at 6 months was 28.5% (8/28 of infected children. Factors that were found to be associated with high risk of early mother-to-child transmission of HIV include duration of ARV regimen shorter than 2 months during pregnancy

  9. Predictors of treatment failure and time to detection and switching in HIV-infected Ethiopian children receiving first line anti-retroviral therapy

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    Bacha Tigist

    2012-08-01

    Full Text Available Abstract Background The emergence of resistance to first line antiretroviral therapy (ART regimen leads to the need for more expensive and less tolerable second line drugs. Hence, it is essential to identify and address factors associated with an increased probability of first line ART regimen failure. The objective of this article is to report on the predictors of first line ART regimen failure, the detection rate of ART regime failure, and the delay in switching to second line ART drugs. Methods A retrospective cohort study was conducted from 2005 to 2011. All HIV infected children under the age of 15 who took first line ART for at least six months at the four major hospitals of Addis Ababa, Ethiopia were included. Data were collected, entered and analyzed using Epi info/ENA version 3.5.1 and SPSS version 16. The Cox proportional-hazard model was used to assess the predictors of first line ART failure. Results Data of 1186 children were analyzed. Five hundred seventy seven (48.8% were males with a mean age of 6.22 (SD = 3.10 years. Of the 167(14.1% children who had treatment failure, 70 (5.9% had only clinical failure, 79 (6.7% had only immunologic failure, and 18 (1.5% had both clinical and immunologic failure. Patients who had height for age in the third percentile or less at initiation of ART were found to have higher probability of ART treatment failure [Adjusted Hazard Ratio (AHR, 3.25 95% CI, 1.00-10.58]. Patients who were less than three years old [AHR, 1.85 95% CI, 1.24-2.76], chronic diarrhea after initiation of antiretroviral treatment [AHR, 3.44 95% CI, 1.37-8.62], ART drug substitution [AHR, 1.70 95% CI, 1.05-2.73] and base line CD4 count below 50 cells/mm3 [AHR, 2.30 95% CI, 1.28-4.14] were also found to be at higher risk of treatment failure. Of all the 167 first line ART failure cases, only 24 (14.4% were switched to second line ART with a mean delay of 24 (SD = 11.67 months. The remaining 143 (85.6% cases were diagnosed

  10. [Effect of highly active anti-retroviral therapy on prevention of mother to child transmission of HIV and on infant growth and development].

    Science.gov (United States)

    He, Yan; Luo, Yan; Ding, Yi-ling; Zheng, Yu-huang; Li, Jing; Huang, Jian; Li, Jie-min

    2011-10-01

    To identify the effect of highly active anti-retroviral therapy (HAART) on prevention of mother to child transmission (PMTCT) of HIV and on infant growth and development. A total of 16 HIV-infected women or pregnant women selected in this study received HAART before or 18 - 24 weeks after pregnancy. The treatment included taking Zidovudine (AZT) 0.3 g each time, twice a day, Lamivudine (3TC) 0.3 g each time, once a day and Nevirapine (NVP) 0.2 g each time, twice a day or Efavirenz (EFV) 0.6 g each time, once a day, as well as labor intervention and artificial feeding. The growth index for 17 infants from HIV-infected mothers (experimental group) and 16 normal infants (control group) were observed for 18 months. Neonatal hemoglobin (Hb), liver and kidney function, serum iron and calcium were detected at neonatal period and at 12(th) month, respectively. All the pregnant women were in good conditions and had tolerance with HAART. The birth weight, length and Apgar score of the newborns in the experimental group were (3.5 ± 0.9) kg, (54.2 ± 3.8) cm and 7 - 10 scores respectively, however those in the control group were (3.6 ± 0.8) kg, (55.6 ± 3.6) cm and 8 - 10 scores (t(weight) = 1.01, t(length) = 6.98, P > 0.05). Weight and length of infants in experimental group were (9.36 ± 1.8) kg and (76.3 ± 2.7) cm at 12(th) month, while those in control group were (9.86 ± 2.5) kg and (76.8 ± 2.9) cm (t(weight) = 0.83, t(length) = 1.00, P > 0.05). The level of Hb in experimental group was (126.2 ± 16.7) g/L, and was (148.6 ± 20.5) g/L in control group (t = -5.89, P = 0.11). At 12(th) month, the levels of Hb and the total bilirubin (TB) were (125.9 ± 19.8) g/L and (11.7 ± 3.5) µmol/L in experimental group; and those in the control group were (130.1 ± 18.7) g/L and (13.2 ± 3.7) µmol/L (t(Hb) = -3.82, t(TB) = -2.14, P > 0.05). Serum iron and calcium were (25.4 ± 5.7) µmol/L and (26.4 ± 7.2) µmol/L at neonatal period and were (2.3 ± 0.6) mol/L and (2.8 ± 0

  11. Endogenous Retroviral Insertions Indicate a Secondary Introduction of Domestic Sheep Lineages to the Caucasus and Central Asia between the Bronze and Iron Age

    Science.gov (United States)

    Schroeder, Oskar; Benecke, Norbert; Frölich, Kai; Peng, Zuogang; Kaniuth, Kai; Sverchkov, Leonid; Reinhold, Sabine; Belinskiy, Andrey; Ludwig, Arne

    2017-01-01

    Sheep were one of the first livestock species domesticated by humans. After initial domestication in the Middle East they were spread across Eurasia. The modern distribution of endogenous Jaagsiekte sheep retrovirus insertions in domestic sheep breeds suggests that over the course of millennia, successive introductions of improved lineages and selection for wool quality occurred in the Mediterranean region and most of Asia. Here we present a novel ancient DNA approach using data of endogenous retroviral insertions in Bronze and Iron Age domestic sheep from the Caucasus and Pamir mountain areas. Our findings support a secondary introduction of wool sheep from the Middle East between the Late Bronze Age and Iron Age into most areas of Eurasia. PMID:28632161

  12. Endogenous Retroviral Insertions Indicate a Secondary Introduction of Domestic Sheep Lineages to the Caucasus and Central Asia between the Bronze and Iron Age

    Directory of Open Access Journals (Sweden)

    Oskar Schroeder

    2017-06-01

    Full Text Available Sheep were one of the first livestock species domesticated by humans. After initial domestication in the Middle East they were spread across Eurasia. The modern distribution of endogenous Jaagsiekte sheep retrovirus insertions in domestic sheep breeds suggests that over the course of millennia, successive introductions of improved lineages and selection for wool quality occurred in the Mediterranean region and most of Asia. Here we present a novel ancient DNA approach using data of endogenous retroviral insertions in Bronze and Iron Age domestic sheep from the Caucasus and Pamir mountain areas. Our findings support a secondary introduction of wool sheep from the Middle East between the Late Bronze Age and Iron Age into most areas of Eurasia.

  13. Demographic and HIV-specific characteristics of participants enrolled in the INSIGHT Strategic Timing of AntiRetroviral Treatment (START) trial

    DEFF Research Database (Denmark)

    Sharma, S; Babiker, A G; Emery, S

    2015-01-01

    OBJECTIVES: The risks and benefits of initiating antiretroviral treatment (ART) at high CD4 cell counts have not been reliably quantified. The Strategic Timing of AntiRetroviral Treatment (START) study is a randomized international clinical trial that compares immediate with deferred initiation......, 14% as Latino/Hispanic, 8% as Asian and 3% as other. The route of HIV acquisition is reported as men who have sex with men in 55% of participants, heterosexual sex in 38%, injecting drug use in 1% and other/unknown in 5%. Median time since HIV diagnosis is 1.0 year (IQR 0.4-3.0 years) and the median......-positive population from the regions in which they were enrolled. The information collected with this robust study design will provide a database with which to evaluate the risks and benefits of early ART use for many important outcomes....

  14. Endogenous Retroviral Insertions Indicate a Secondary Introduction of Domestic Sheep Lineages to the Caucasus and Central Asia between the Bronze and Iron Age.

    Science.gov (United States)

    Schroeder, Oskar; Benecke, Norbert; Frölich, Kai; Peng, Zuogang; Kaniuth, Kai; Sverchkov, Leonid; Reinhold, Sabine; Belinskiy, Andrey; Ludwig, Arne

    2017-06-20

    Sheep were one of the first livestock species domesticated by humans. After initial domestication in the Middle East they were spread across Eurasia. The modern distribution of endogenous Jaagsiekte sheep retrovirus insertions in domestic sheep breeds suggests that over the course of millennia, successive introductions of improved lineages and selection for wool quality occurred in the Mediterranean region and most of Asia. Here we present a novel ancient DNA approach using data of endogenous retroviral insertions in Bronze and Iron Age domestic sheep from the Caucasus and Pamir mountain areas. Our findings support a secondary introduction of wool sheep from the Middle East between the Late Bronze Age and Iron Age into most areas of Eurasia.

  15. Who is accessing public-sector anti-retroviral treatment in the Free State, South Africa? An exploratory study of the first three years of programme implementation

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    Booysen Frederik

    2010-07-01

    Full Text Available Abstract Background Although South Africa has the largest public-sector anti-retroviral treatment (ART programme in the world, anti-retroviral coverage in adults was only 40.2% in 2008. However, longitudinal studies of who is accessing the South African public-sector ART programme are scarce. This study therefore had one main research question: who is accessing public-sector ART in the Free State Province, South Africa? The study aimed to extend the current literature by investigating, in a quantitative manner and using a longitudinal study design, the participants enrolled in the public-sector ART programme in the period 2004-2006 in the Free State Province of South Africa. Methods Differences in the demographic (age, sex, population group and marital status socio-economic (education, income, neo-material indicators, geographic (travel costs, relocation for ART, and medical characteristics (CD4, viral load, time since first diagnosis, treatment status among 912 patients enrolled in the Free State public-sector ART programme between 2004 and 2006 were assessed with one-way analysis of variance, Bonferroni post-hoc analysis, and cross tabulations with the chi square test. Results The patients accessing treatment tended to be female (71.1% and unemployed (83.4%. However, although relatively poor, those most likely to access ART services were not the most impoverished patients. The proportion of female patients increased (P P P P P Conclusions Our analysis showed significant changes in the demographic, socio-economic, geographic, and medical characteristics of the patients during the first three years of the programme. Knowledge of the characteristics of these patients can assist policy makers in developing measures to retain them in care. The information reported here can also be usefully applied to target patient groups that are currently not reached in the implementation of the ART programme.

  16. Production of glycosylated physiologically normal human α1-antitrypsin by mouse fibroblasts modified by insertion of a human α1-antitrypsin cDNA using a retroviral vector

    International Nuclear Information System (INIS)

    Garver, R.I. Jr.; Chytil, A.; Karlsson, S.

    1987-01-01

    α 2 -Antitrypsin (α 1 AT) deficiency is a hereditary disorder characterized by reduced serum levels of α 1 AT, resulting in destruction of the lower respiratory tract by neutrophil elastase. As an approach to augment α 1 AT levels in this disorder with physiologically normal human α 1 AT, the authors have integrated a full-length normal human α 1 AT cDNA into the genome of mouse fibroblasts. To accomplish this, the retroviral vector N2 was modified by inserting the simian virus 40 early promoter followed by the α 1 AT cDNA. Southern analysis demonstrated that the intact cDNA was present in the genome of selected clones of the transfected murine fibroblasts psi2 and infected NIH 3T3. The clones produced three mRNA transcripts containing human α 1 AT sequences, secreted an α 1 AT molecule recognized by an anti-human α 1 AT antibody, with the same molecular mass as normal human α 1 AT and that complexed with and inhibited human neutrophil elastase. The psi2 produced α 1 AT was glycosylated, and when infused intravenously into mice, it had a serum half-life similar to normal α 1 AT purified from human plasma and markedly longer than that of nonglycosylated human α 1 AT cDNA-directed yeast-produced α 1 AT. These studies demonstrate the feasibility of using a retroviral vector to insert the normal human α 1 AT cDNA into non-α 1 AT-producing cells, resulting in the synthesis and secretion of physiologically normal α 1 AT

  17. Pessoas vivendo com HIV/AIDS: variáveis associadas à adesão ao tratamento anti-retroviral Persons living with HIV/AIDS: factors associated with adherence to antiretroviral treatment

    Directory of Open Access Journals (Sweden)

    Eliane Maria Fleury Seidl

    2007-10-01

    Full Text Available O estudo objetivou descrever o comportamento de adesão ao tratamento anti-retroviral em pessoas vivendo com HIV/AIDS e investigar preditores da adesão entre as variáveis escolaridade, presença de efeitos colaterais, interrupção anterior da terapia anti-retroviral (TARV por conta própria, auto-estima, expectativa de auto-eficácia, estratégias de enfrentamento, suporte social e satisfação com a relação profissional de saúde-usuário. Adesão foi medida pelo auto-relato da perda do número de comprimidos/cápsulas dos medicamentos anti-retrovirais na última semana e mês, sendo considerada satisfatória na ocorrência de omissão inferior a 5% do total prescrito. Participaram 101 pessoas, 60,4% homens, idades entre 20 a 71 anos (M = 37,9 anos, 73,3% sintomáticos. A coleta de dados incluiu entrevista e instrumentos auto-aplicáveis. A maioria (n = 73; 72,3% relatou adesão igual ou superior a 95%. Nos resultados da regressão logística, interrupção anterior da TARV e expectativa de auto-eficácia foram preditores significativos da adesão. Faz-se necessária a qualificação da assistência pela constituição de equipes interdisciplinares, para o desenvolvimento de abordagens adequadas às dificuldades médicas e psicossociais de adesão das pessoas vivendo com HIV/AIDS.This study aimed to describe the adherence of persons living with HIV/AIDS to antiretroviral therapy (ART and to investigate adherence predictors among the following: level of schooling, presence of side effects, current or previous interruption of ART by the persons themselves, self-esteem, self-efficacy expectation, coping strategies, social support, and satisfaction with the health professional-patient relationship. Adherence was measured by self-reported number of ART pills/capsules missed during the previous week and previous month, evaluated as satisfactory when less than 5%. 101 HIV+ adults took part in this study, 60.4% males, ranging from 20 to 71 years

  18. Adesão à terapêutica anti-retroviral por indivíduos com HIV/AIDS assistidos em uma instituição do interior paulista Adhesión a la terapéutica anti-retroviral por los individuos con VIH/SIDA de uno servicio del interior paulista Adhesion to anti-retroviral therapy by individuals with HIV/AIDS attended at an institution in the interior of São Paulo

    Directory of Open Access Journals (Sweden)

    Elucir Gir

    2005-10-01

    Full Text Available A adesão indevida à terapêutica anti-retroviral acarreta sérios agravos aos indivíduos com aids. Assim, objetivou-se identificar os determinantes (facilitadores/dificultadores da adesão aos anti-retrovirais atribuídos pelos indivíduos com aids, seguidos em um hospital universitário do interior paulista. Constituíram sujeitos 200 usuários de anti-retrovirais há pelo menos 6 meses. Os dados foram coletados mediante entrevista semi-estruturada, individual e analisados quali-quantitativamente. Dos participantes, 59% eram do sexo masculino; idade média 38,2 anos, 51% cursaram ensino fundamental incompleto; 50,5% não exerciam atividade remunerada. Usavam anti-retrovirais em média há 5 anos. A quantidade diária de comprimidos variou de 3 a 24. As principais dificuldades apontadas: sabor, tamanho, quantidade, odor dos comprimidos (40%; efeitos colaterais intensos (14,4%; fatores psicológicos (13,7%; diferentes horários de medicação (10,8%. Quanto às facilidades, destacam-se horários coincidentes dos comprimidos (26,2%; nenhuma facilidade (16,4%, ingestão condicionada a algum hábito (16%. A enfermagem deve incrementar ações de vigilância supervisionada e educacionais interventivas.La terapéutica anti-retroviral requiere perfecta adhesión para evitarse complejos efectos colaterales. En esta investigación, el objetivo fue identificar los determinantes (facilitadores/dificultadores de la adhesión a los anti-retrovirales según individuos con sida, seguidos clínicamente en un hospital universitario del interior de São Paulo. Participaron del estudio 200 pacientes diagnosticados, usuarios de anti-retrovirales desde hace por lo menos 6 meses. Se realizó entrevistas semiestructuradas individuales. Los datos fueron analizados cuali-cuantitativamente. De los participantes, el 59% era del sexo masculino; edad promedia 38,2 años, el 51% no terminó la enseñanza fundamental; el 50,5% no ejercía actividad remunerada. Usaban anti

  19. Who is accessing public-sector anti-retroviral treatment in the Free State, South Africa? An exploratory study of the first three years of programme implementation.

    Science.gov (United States)

    Wouters, Edwin; Heunis, Christo; Ponnet, Koen; Van Loon, Francis; Booysen, Frederik le Roux; van Rensburg, Dingie; Meulemans, Herman

    2010-07-01

    Although South Africa has the largest public-sector anti-retroviral treatment (ART) programme in the world, anti-retroviral coverage in adults was only 40.2% in 2008. However, longitudinal studies of who is accessing the South African public-sector ART programme are scarce. This study therefore had one main research question: who is accessing public-sector ART in the Free State Province, South Africa? The study aimed to extend the current literature by investigating, in a quantitative manner and using a longitudinal study design, the participants enrolled in the public-sector ART programme in the period 2004-2006 in the Free State Province of South Africa. Differences in the demographic (age, sex, population group and marital status) socio-economic (education, income, neo-material indicators), geographic (travel costs, relocation for ART), and medical characteristics (CD4, viral load, time since first diagnosis, treatment status) among 912 patients enrolled in the Free State public-sector ART programme between 2004 and 2006 were assessed with one-way analysis of variance, Bonferroni post-hoc analysis, and cross tabulations with the chi square test. The patients accessing treatment tended to be female (71.1%) and unemployed (83.4%). However, although relatively poor, those most likely to access ART services were not the most impoverished patients. The proportion of female patients increased (P < 0.05) and their socio-economic situation improved between 2004 and 2006 (P < 0.05). The increasing mean transport cost (P < 0.05) to visit the facility is worrying, because this cost is an important barrier to ART uptake and adherence. Encouragingly, the study results revealed that the interval between the first HIV-positive diagnosis and ART initiation decreased steadily over time (P < 0.05). This was also reflected in the increasing baseline CD4 cell count at ART initiation (P < 0.05). Our analysis showed significant changes in the demographic, socio-economic, geographic

  20. Gene transfer to pre-hematopoietic and committed hematopoietic precursors in the early mouse Yolk Sac: a comparative study between in situ electroporation and retroviral transduction

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    Lécluse Yann

    2007-07-01

    Full Text Available Abstract Background Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated. While intra-embryonic precursors harbour the features of hematopoietic stem cells (HSC, precursors formed earlier in the yolk sac (YS display limited differentiation and self-renewal potentials. The mechanisms leading to the generation of the precursors in both sites are still largely unknown, as are the molecular basis underlying their different potential. A possible approach to assess the role of candidate genes is to transfer or modulate their expression/activity in both sites. We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors. Results One transduction protocol involves transient modification of gene expression through in situ electroporation of the prospective blood islands, which allows the evolution of transfected mesodermal cells in their "normal" environment, upon organ culture. Following in situ electroporation of a GFP reporter construct into the YS cavity of embryos at post-streak (mesodermal/pre-hematopoietic precursors or early somite (hematopoietic precursors stages, high GFP expression levels as well as a good preservation of cell viability is observed in YS explants. Moreover, the erythro-myeloid progeny typical of the YS arises from GFP+ mesodermal cells or hematopoietic precursors, even if the number of targeted precursors is low. The second approach, based on retroviral transduction allows a very efficient transduction of large precursor numbers, but may only be used to target 8 dpc YS hematopoietic precursors. Again, transduced cells generate a progeny quantitatively and qualitatively similar to that of control YS. Conclusion We thus provide two protocols whose combination may allow a thorough study of both early and late events of hematopoietic development in the murine YS. In situ

  1. CCR5 and CXCR4 chemokine receptor expression and β-chemokine production during early T cell repopulation induced by highly active anti-retroviral therapy

    Science.gov (United States)

    Giovannetti, A; Ensoli, F; Mazzetta, F; De Cristofaro, M; Pierdominici, M; Muratori, D S; Fiorelli, V; Aiuti, F

    1999-01-01

    Expression of chemokine receptors and β-chemokine production by peripheral blood mononuclear cells (PBMC) were determined in HIV-1-infected individuals before and after highly active anti-retroviral therapy (HAART) and their relationship to viral load, T cell phenotype and the expression of immunological activation markers was examined. We found that the expression of CCR5 is up-regulated in HIV-1-infected individuals while CXCR4 appears down-regulated on both CD4 and CD8 T cells compared with normal controls. These alterations are associated with the high levels of viral load. In addition, a relationship was observed between the degree of immune activation and chemokine receptor expression on T cells. However, after 3 months of combined anti-retroviral regimen, expression of CXCR4 significantly increased while CCR5 decreased when compared with pretherapy determinations. This was seen in strict association with a dramatic decrease of viral load and an increase of both CD45RA+/CD62L+ (naive) and CD45RA−/CD62L+ or CD45RA+/CD62L− (memory) T cells accompanied by a significant decrease of the expression of immune activation markers such as HLA-DR and CD38. At enrolment, both spontaneous and lectin-induced RANTES, macrophage inflammatory protein-1α (MIP-1α) and MIP-1β production by PBMC were higher in HIV-1-infected individuals compared with normal controls, although differences for MIP-1β were not statistically significant. However, RANTES and MIP-1α production decreased during HAART at levels closer to that determined with normal controls, while MIP-1β production was less consistently modified. These data indicate that the expression of chemokine receptors CCR5 and CXCR4 and the production of β-chemokines are altered in HIV-infected individuals, and suggest that their early modifications during HAART reflect both the peripheral redistribution of naive/memory T cell compartments and the decrease in levels of T cell activation. Such modifications in the

  2. Associations between HIV, highly active anti-retroviral therapy, and hypertensive disorders of pregnancy among maternal deaths in South Africa 2011-2013.

    Science.gov (United States)

    Sebitloane, Hannah M; Moodley, Jagidesa; Sartorius, Benn

    2017-02-01

    To explore potential relationships between HIV and highly active anti-retroviral therapy (HAART), and hypertensive disorders of pregnancy (HDP). A retrospective secondary analysis of maternal-deaths data from the 2011-2013 Saving Mothers Report from South Africa. The incidence of HIV infection amongst individuals who died owing to HDP was determined and comparisons were made based on HIV status and the use of HAART. Among 4452 maternal deaths recorded in the Saving Mothers report, a lower risk of a maternal deaths being due to HDP was observed among women who had HIV infections compared with women who did not have HIV (relative risk [RR] 0.57, 95% confidence interval [CI] 0.51-0.64). Further, reduced odds of death being due to HDP were recorded among women with AIDS not undergoing HAART compared with women with HIV who did not require treatment (RR 0.42, 95% CI 0.3-0.58). Notably, among all women with AIDS, a greater risk of death due to HDP was demonstrated among those who received HAART compared with those who did not (RR 1.15, 95% CI 1.02-1.29). HIV and AIDS were associated with a decreased risk of HDP being the primary cause of death; the use of HAART increased this risk. © 2016 International Federation of Gynecology and Obstetrics.

  3. Mouse Mammary Tumor Virus Promoter-Containing Retroviral Promoter Conversion Vectors for Gene-Directed Enzyme Prodrug Therapy are Functional in Vitro and in Vivo

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    Reinhard Klein

    2008-01-01

    Full Text Available Gene directed-enzyme prodrug therapy (GDEPT is an approach for sensitization of tumor cells to an enzymatically activated, otherwise nontoxic, prodrug. Cytochrome P450 2B1 (CYP2B1 metabolizes the prodrugs cyclophosphamide (CPA and ifosfamide (IFA to produce the cytotoxic substances phosphoramide mustard and isophosphoramide mustard as well as the byproduct acrolein. We have constructed a retroviral promoter conversion (ProCon vector for breast cancer GDEPT. The vector allows expression of CYP2B1 from the mouse mammary tumor virus (MMTV promoter known to be active in the mammary glands of transgenic animals. It is anticipated to be used for the generation of encapsulated viral vector producing cells which, when placed inside or close to a tumor, will act as suppliers of the therapeutic CYP2B1 protein as well as of the therapeutic vector itself. The generated vector was effectively packaged by virus producing cells and allowed the production of high levels of enzymatically active CYP2B1 in infected cells which sensitized them to killing upon treatment with both IFA and CPA. Determination of the respective IC50 values demonstrated that the effective IFA dose was reduced by sixteen folds. Infection efficiencies in vivo were determined using a reporter gene-bearing vector in a mammary cancer cell-derived xenograft tumor mouse model.

  4. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

    Energy Technology Data Exchange (ETDEWEB)

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku, E-mail: nakagawa@phs.osaka-u.ac.jp; Okada, Naoki, E-mail: okada@phs.osaka-u.ac.jp

    2016-04-22

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

  5. Retroviral expression of a kinase-defective IGF-I receptor suppresses growth and causes apoptosis of CHO and U87 cells in-vivo

    International Nuclear Information System (INIS)

    Seely, B Lynn; Samimi, Goli; Webster, Nicholas JG

    2002-01-01

    Phosphatidylinositol-3,4,5-triphosphate (PtdInsP3) signaling is elevated in many tumors due to loss of the tumor suppressor PTEN, and leads to constitutive activation of Akt, a kinase involved in cell survival. Reintroduction of PTEN in cells suppresses transformation and tumorigenicity. While this approach works in-vitro, it may prove difficult to achieve in-vivo. In this study, we investigated whether inhibition of growth factor signaling would have the same effect as re-expression of PTEN. Dominant negative IGF-I receptors were expressed in CHO and U87 cells by retroviral infection. Cell proliferation, transformation and tumor formation in athymic nude mice were assessed. Inhibition of IGF-IR signaling in a CHO cell model system by expression of a kinase-defective IGF-IR impairs proliferation, transformation and tumor growth. Reduction in tumor growth is associated with an increase in apoptosis in-vivo. The dominant-negative IGF-IRs also prevented growth of U87 PTEN-negative glioblastoma cells when injected into nude mice. Injection of an IGF-IR blocking antibody αIR3 into mice harboring parental U87 tumors inhibits tumor growth and increases apoptosis. Inhibition of an upstream growth factor signal prevents tumor growth of the U87 PTEN-deficient glioma to the same extent as re-introduction of PTEN. This result suggests that growth factor receptor inhibition may be an effective alternative therapy for PTEN-deficient tumors

  6. A modular lentiviral and retroviral construction system to rapidly generate vectors for gene expression and gene knockdown in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Benjamin Geiling

    Full Text Available The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG and retroviral (pREG vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs. In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems.

  7. Evaluation of the Impact of Anti-Retroviral Therapy on the Prevalence of Oral Lesions in Human Immunodeficiency Virus Infected Patients

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    P. Davoodi

    2013-10-01

    Full Text Available Introduction & Objective: Oral lesions have important diagnostic and prognostic roles in HIV infected patients. It seems that HAART reduces the prevalence of oral lesions. The aim of the present study was to evaluate the prevalence of oral lesions in HIV infected patients on/not on HAART. Materials & Methods: In this retrospective study, 40 HIV infected patients receiving HAART and 40 who were not on HAART were evaluated in Behavioral Consultation Center in Kermanshah. The diagnosis of the oral lesions was recorded by using established presump-tive clinical criteria. Data were gathered and analyzed using SPSS version 16 by chi-square test. Results: In the current study 80 HIV infected patients with mean age of 38.86 were chosen. 72.5% and 27.5% of participants were male and female respectively. The most common le-sions in those receiving HAART were hairy leukoplakia, hairy tongue and oral pigmentation. However the prevalence of these lesions had declined in comparison to those who were not on HAART but the difference was not significant (P>0.05. Although comparing lesions in the two groups showed no significant difference, the total number of lesions significantly reduced in patients receiving HAART (P=0.046 Conclusion: According to the results of the present study using anti retroviral therapy leaded to reduction in the oral lesions in HIV infected patients. However, more research in this field seems necessary. (Sci J Hamadan Univ Med Sci 2013; 20 (3:215-222

  8. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

    International Nuclear Information System (INIS)

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku; Okada, Naoki

    2016-01-01

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8 + CAR-T cells had antigen-specific cytotoxic activity. • CD4 + CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

  9. Simultaneous RNA quantification of human and retroviral genomes reveals intact interferon signaling in HTLV-1-infected CD4+ T cell lines

    Directory of Open Access Journals (Sweden)

    Moens Britta

    2012-08-01

    Full Text Available Abstract Background IFN-α contributes extensively to host immune response upon viral infection through antiviral, pro-apoptotic, antiproliferative and immunomodulatory activities. Although extensively documented in various types of human cancers and viral infections, controversy exists in the exact mechanism of action of IFN-α in human immunodeficiency virus type 1 (HIV-1 and human T-lymphotropic virus type 1 (HTLV-1 retroviral infections. Results IFN-α displayed strong anti-HIV-1 effects in HIV-1/HTLV-1 co-infected MT-4 cells in vitro, demonstrated by the dose-dependent inhibition of the HIV-1-induced cytopathic effect (IC50 = 83.5 IU/ml, p 50 = 1.2 IU/ml, p  Conclusions Taken together, our results indicate that both the absence of in vitro antiproliferative and pro-apoptotic activity as well as the modest post-transcriptional antiviral activity of IFN-α against HTLV-1, were not due to a cell-intrinsic defect in IFN-α signalisation, but rather represents a retrovirus-specific phenomenon, considering the strong HIV-1 inhibition in co-infected cells.

  10. N-linked glycosylation and sequence changes in a critical negative control region of the ASCT1 and ASCT2 neutral amino acid transporters determine their retroviral receptor functions.

    Science.gov (United States)

    Marin, Mariana; Lavillette, Dimitri; Kelly, Sean M; Kabat, David

    2003-03-01

    A widely dispersed interference group of retroviruses that includes the feline endogenous virus (RD114), baboon endogenous virus (BaEV), human endogenous virus type W (HERV-W), and type D primate retroviruses uses the human Na(+)-dependent neutral amino acid transporter type 2 (hASCT2; gene name, SLC1A5) as a common cell surface receptor. Although hamster cells are fully resistant to these viruses and murine cells are susceptible only to BaEV and HERV-W pseudotype viruses, these rodent cells both become highly susceptible to all of the viruses after treatment with tunicamycin, an inhibitor of protein N-linked glycosylation. A partial explanation for these results was recently provided by findings that the orthologous murine transporter mASCT2 is inactive as a viral receptor, that a related (ca. 55% identity) murine paralog (mASCT1; gene name, SLC1A4) mediates infections specifically of BaEV and HERV-W, and that N-deglycosylation of mASCT1 activates it as a receptor for all viruses of this interference group. Because the only two N-linked oligosaccharides in mASCT1 occur in the carboxyl-terminal region of extracellular loop 2 (ECL2), it was inferred that this region contributes in an inhibitory manner to infections by RD114 and type D primate viruses. To directly and more thoroughly investigate the receptor active sites, we constructed and analyzed a series of hASCT2/mASCT2 chimeras and site-directed mutants. Our results suggest that a hypervariable sequence of 21 amino acids in the carboxyl-terminal portion of ECL2 plays a critical role in determining the receptor properties of ASCT2 proteins for all viruses in this interference group. In addition, we analyzed the tunicamycin-dependent viral susceptibility of hamster cells. In contrast to mASCT1, which contains two N-linked oligosaccharides that partially restrict viral infections, hamster ASCT1 contains an additional N-linked oligosaccharide clustered close to the others in the carboxyl-terminal region of ECL2

  11. Estudo da Adesão à Quimioprofilaxia Anti-retroviral para a Infecção por HIV em Mulheres Sexualmente Vitimadas Study of Adherence to Antiretroviral Chemoprophylaxis for HIV Infection in Sexually Abused Women

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    Jefferson Drezett

    1999-10-01

    Full Text Available Objetivos: embora não existam dados apropriados para estabelecer sua eficácia, alguns serviços tem utilizado, profilaticamente, a terapia anti-retroviral para o HIV nos casos de violência sexual. O objetivo deste estudo foi avaliar a aceitabilidade, tolerância e adesão a um esquema quimioprofilático para o HIV. Pacientes e método: foram avaliadas 62 mulheres vítimas de estupro e/ou atentado violento ao pudor com coito ectópico anal. Os agressores foram referidos como desconhecidos. A profilaxia foi iniciada dentro das primeiras 48 h da violência e mantida por 4 semanas, sendo administrados diariamente: zidovudina, 600 mg; indinavir, 2.400 mg e lamivudina, 300 mg. Resultados: a taxa de descontinuidade foi de 24,2%, sendo em 12 casos (80% decorrente de intolerância gástrica. Os efeitos colaterais estiveram presentes em 43 casos (69,4%, sendo as náuseas e vômitos os mais freqüentes. A complexidade posológica e o tempo de uso foram fatores possivelmente associados ao uso inadequado das drogas, ocorrendo em 10,6% dos casos. Conclusão: a taxa de descontinuidade da quimioprofilaxia foi semelhante à observada em outras indicações.Purpose: some medical institutions have been prophylactically ministrating anti-HIV therapy in cases of sexual violence, although there are no appropriate basic facts to establish its efficacy. The aim of the present study was to evaluate the acceptance, tolerance and adhesion of these women under a chemoprophylaxis plan for HIV. Methods: sixty-two women victims of rape and/or anal intercourse with unknown aggressors have been evaluated. Prophylaxis has been started within the first 48 h after violence and maintained for 4 weeks, with daily administration of zidovudine, 600 mg; indinavir, 2,400 mg and lamivudine, 300 mg. Results: the discontinuance rate was 24.2%, withe 12 cases (80% due to gastric intolerance. The side effects were present in 43 cases (69.4%, including nausea and vomitting as the most

  12. (including travel dates) Proposed itinerary

    Indian Academy of Sciences (India)

    Ashok

    31 July to 22 August 2012 (including travel dates). Proposed itinerary: Arrival in Bangalore on 1 August. 1-5 August: Bangalore, Karnataka. Suggested institutions: Indian Institute of Science, Bangalore. St Johns Medical College & Hospital, Bangalore. Jawaharlal Nehru Centre, Bangalore. 6-8 August: Chennai, TN.

  13. Malignant lymphomas (including myeloproliferative disorders)

    International Nuclear Information System (INIS)

    Todd, I.D.H.

    1985-01-01

    This chapter deals with the radiotherapy and cytotoxic chemotherapy of the malignant lymphomas. Included within this group are Hodgkin's disease, non-Hodgkin's lymphoma, mycosis fungoides, and chronic lymphatic leukaemia. A further section deals with the myeloproliferative disorders, including granulocytic leukaemia, polycythaemia vera, and primary thrombocythaemia. Excluded are myeloma and reticulum cell sarcoma of bone and acute leukaemia. With regard to Hodgkin's disease, the past 25 years have seen general recognition of the curative potential of radiotherapy, at least in the local stages, and, more recently, awareness of the ability to achieve long-term survival after combination chemotherapy in generalised or in recurrent disease. At the same time the importance of staging has become appreciated and the introduction of procedures such as lymphography, staging laparotomy, and computer tomography (CT) has enormously increased its reliability. Advances have not been so dramatic in the complex group of non-Hodgkins's lymphomas, but are still very real

  14. Rinossinusites em crianças infectadas pelo HIV sob terapia anti-retroviral Rhinosinusitis in HIV-infected children undergoing antiretroviral therapy

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    Carlos Diógenes Pinheiro Neto

    2009-02-01

    Full Text Available A associação dos inibidores de protease (IP à terapia anti-retroviral provocou mudanças importantes na morbidade e mortalidade de pacientes infectados pelo HIV. OBJETIVOS: Avaliar o impacto desta associação na prevalência de rinossinusite (RS e na contagem sérica de linfócitos CD4 em crianças infectadas pelo HIV. CASUÍSTICA E MÉTODOS: A forma de estudo foi cross-sectional com 471 crianças infectadas pelo HIV. Em 1996, inibidores de protease foram liberados para terapia anti-retroviral. Desta forma, dois grupos de crianças foram formados: as que não fizeram uso de IP e as que fizeram uso desta droga após 1996. A prevalência de RS e a contagem sérica de linfócitos CD4 foram comparadas entre estes grupos. RESULTADOS: 14,4% das crianças infectadas pelo HIV apresentaram RS. A RS crônica foi mais prevalente que a RS aguda em ambos os grupos. Crianças menores de 6 anos tratadas com a associação de IP apresentaram maior prevalência de RS aguda. A associação de IP esteve associada à maior contagem de linfócitos CD4 séricos com menor prevalência de RS crônica. CONCLUSÕES: A terapia com IP esteve associada ao aumento na contagem de linfócitos CD4. Crianças abaixo dos 6 anos em uso de IP apresentaram menor tendência à cronificação da doença.The association of protease inhibitors (PI to antiretroviral therapy has generated sensible changes in morbidity and mortality of HIV-infected patients. AIM: Aims at evaluating the impact of this association on the prevalence of rhinosinusitis (RS and CD4+ lymphocyte count in HIV-infected children. METHODS: Retrospective cross-sectional study of the medical charts of 471 HIV-infected children. In 1996, protease inhibitors were approved for use as an association drug in antiretroviral therapy. Children were divided into two groups: one which did not receive PI and another which received PI after 1996. The prevalence of RS and CD4+ lymphocyte counts were compared between these groups

  15. Device including a contact detector

    DEFF Research Database (Denmark)

    2011-01-01

    The present invention relates to a probe for determining an electrical property of an area of a surface of a test sample, the probe is intended to be in a specific orientation relative to the test sample. The probe may comprise a supporting body defining a first surface. A plurality of cantilever...... of cantilever arms (12) contacting the surface of the test sample when performing the movement....... arms (12) may extend from the supporting body in co-planar relationship with the first surface. The plurality of cantilever arms (12) may extend substantially parallel to each other and each of the plurality of cantilever arms (12) may include an electrical conductive tip for contacting the area...

  16. Predictors of fertility desire among people living with HIV attending anti-retroviral clinic in a tertiary health facility in Sokoto, Northern Nigeria

    Directory of Open Access Journals (Sweden)

    A U Kaoje

    2015-01-01

    Full Text Available Introduction: The pressure on couples and particularly women to have children is strong in developing countries where a childless woman is considered a social pariah. This study aimed to determine the predictors of fertility desire among people living with HIV. Materials and Methods : This is a descriptive cross-sectional study conducted among 400 HIV-positive clients receiving follow-up care at anti-retroviral (ART clinic in a tertiary health facility in Sokoto. A list of clients that came to the clinic was compiled and served as sampling frame. A two stage sampling method was used to select study respondents from the sampling frame. Interviewer- administered closed-ended questionnaire was used to collect the required data from the respondents. Ethical approval was granted for the conduct of the study and informed consent was obtained from the respondents. Descriptive, bivariate and multivariate analyses were carried out. Results : Majority (56.2% of the respondents were females. The overall mean age was 34.5 ± 0.4 years (male 38.4 ± 0.5 years, and (females, 31.4 ± 0.4 years. A large proportion of the respondents (67.7% reported desire to have children in future. Using logistic regression analysis, younger age (adjusted odd ratio [aOR] = 2.0, P = 0.023, marital status (aOR = 1.9, P < 0.001 and number of living children (aOR = 0.7, P < 0.001 were the key factors influencing respondents desire for children. Conclusion : Many HIV-positive clients in the clinic reported desire to have children. It is recommended that full reproductive health services be provided in the clinic to help them achieve their reproductive goal without risk to their partners and new born.

  17. Outcome of anti-retroviral treatment in HIV-infected orphans and non-orphans at an ART centre in North India.

    Science.gov (United States)

    Bhattacharya, Malobika; Saxena, Romit

    2012-01-01

    Few Indian studies have reported the long-term efficacy of anti-retroviral treatment (ART) in children and in orphaned, HIV-infected children in particular. To study differences in outcome of ART in HIV-infected orphans compared with non-orphans. A retrospective study of 87 HIV-infected children who commenced ART in the period January 2006 to August 2007. The main measures were orphan status, absolute CD4 count and weight-for-height (WHZ) and height-for-age (HAZ) Z-scores. Median follow-up was 33 months. Forty (45·9%) children were orphaned. Orphans and non-orphans had similar baseline median WHZ and HAZ (-2·48 vs -2·63, P = 0·65 and -2·78 vs -2·91, P = 0·77, respectively). The two groups were similar in terms of WHO clinical stage and frequency of severe immunosuppression at presentation (P = 0·88 and 0·25, respectively). After ART initiation, the median absolute CD4 count increased progressively in both groups. Median WHZ and HAZ increased throughout the study period in the orphans and reached -1 at 27 and 39 months of ART, respectively. In the non-orphans, WHZ remained below that of the orphan group, the difference becoming statistically significant from 18 months of ART. The increment in HAZ in the non-orphan group was at par with the orphan group until 12 months of follow-up, after which it fell between 18 and 30 months. Subsequently, HAZ rose but remained below that of the orphan group. Both WHZ and HAZ failed to reach -1 in the non-orphan group. In both groups, 85% reported 100% adherence to ART. The outcome of ART is not affected by orphan status with the extended family adequately supporting orphaned children. Growth of children whose parents are HIV-infected may be constrained despite ART if there is inadequate family support.

  18. Prevalence and clinical and laboratory characteristics of kidney disease in anti-retroviral-naive human immunodeficiency virus-infected patients in South-South Nigeria

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    U H Okafor

    2016-01-01

    Full Text Available Since the emergence of acquired immune deficiency syndrome (AIDS about three decades ago, several renal disorders have been reported as common complications of human immunodeficiency virus (HIV infection. These renal disorders result from diverse etiologies. The aim of this cross-sectional study was to determine the prevalence and clinical and laboratory characteristics of anti-retroviral-naοve HIV-infected patients with impaired kidney disorder in South-South Nigeria. This study was conducted on patients presenting at the University of Benin Teaching Hospital, Benin City in South-South Nigeria for six months. The patients′ demographic data and clinical, hematological and biochemical parameters were assessed. Their glomerular filtration rate (GFR was calculated and the protein excretion was assessed from the protein- creatinine ratio. Data were analyzed using statistical software program SPSS version 15.0. Threehundred and eighty-three patients with a mean age of 35.39 ± 8.78 years and a male: female ratio of 1:1 were studied; 53.3% had evidence of kidney disorder. The main clinical features in patients with kidney disorder were evidence of fluid retention, urinary symptoms, pallor and encephalopathy. The mean systolic and diastolic blood pressures were 115.33 ± 17.17 and 72.33 ± 14.31 mm Hg, respectively. The mean estimated GFR was 52.5 mL/min/1.73 m 2 . Patients with kidney disorder had higher proteinuria (P = 0.001, lower mean CD4 cell count and packed cell volume (P = 0.019 and 0.001, respectively. Kidney disorder is a common complication in HIV-infected patients, and they have clinical and laboratory anomalies. Screening of HIV/AIDS patients at the time of diagnosis will facilitate early diagnosis of kidney disorders in them.

  19. Critical variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency.

    Science.gov (United States)

    van der Loo, J C M; Swaney, W P; Grassman, E; Terwilliger, A; Higashimoto, T; Schambach, A; Hacein-Bey-Abina, S; Nordling, D L; Cavazzana-Calvo, M; Thrasher, A J; Williams, D A; Reeves, L; Malik, P

    2012-08-01

    Patients with X-linked severe combined immunodeficiency (SCID-X1) were successfully cured following gene therapy with a gamma-retroviral vector (gRV) expressing the common gamma chain of the interleukin-2 receptor (IL2RG). However, 5 of 20 patients developed leukemia from activation of cellular proto-oncogenes by viral enhancers in the long-terminal repeats (LTR) of the integrated vector. These events prompted the design of a gRV vector with self-inactivating (SIN) LTRs to enhance vector safety. Herein we report on the production of a clinical-grade SIN IL2RG gRV pseudotyped with the Gibbon Ape Leukemia Virus envelope for a new gene therapy trial for SCID-X1, and highlight variables that were found to be critical for transfection-based large-scale SIN gRV production. Successful clinical production required careful selection of culture medium without pre-added glutamine, reduced exposure of packaging cells to cell-dissociation enzyme, and presence of cations in wash buffer. The clinical vector was high titer; transduced 68-70% normal human CD34(+) cells, as determined by colony-forming unit assays and by xenotransplantation in immunodeficient NOD.CB17-Prkdc(scid)/J (nonobese diabetic/severe combined immunodeficiency (NOD/SCID)) and NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NOD/SCID gamma (NSG))) mice; and resulted in the production of T cells in vitro from human SCID-X1 CD34(+) cells. The vector was certified and released for the treatment of SCID-X1 in a multi-center international phase I/II trial.

  20. Transfection of a retroviral construct carrying a non producer HIV-1 variant induces HIV-1 resistance in CD4+ CEMss cells.

    Science.gov (United States)

    Federico, M; Taddeo, B; Nappi, F; Nicolini, A; Rossi, G B; Verani, P

    1993-01-01

    The phenotype of Human Immunodeficiency Virus-1 (HIV)-infected HUT-78 cell clone (F12) has been described (Federico et al, AIDS Res Hum Retrov 1989; 5: 365-96). Briefly, F12 cells are: i) CD4 down-regulated, ii) non producer and iii) fully resistant to homologous superinfection. We tested whether this phenotype was dependent upon the expression of the HIV-1 genome integrated therein. The SstI/SstI F12 provirus was cloned and inserted in the pLj retroviral vector bearing the neomycin (neo)-Geneticine resistance gene. CD4+ HIV-susceptible CEMss cells were transfected with this construct in the sense orientation. Neo-resistant clones exhibited an integrated viral DNA, low viral mRNA expression and (as in F12 cells) the presence of uncleaved gp160, no gp41 and a small amount of p55 gag precursor. Superinfection of the F12/HIV-DNA-transfected CEMss clones showed that these CD4+ cells had acquired a significant (0.7-1.5 logs) resistance towards superinfection with HIV-1. This was observed in all four transfected clones where the F12/HIV DNA was expressed, but not in the control clone that was transfected with the pLj vector alone. These results confirm those that were obtained with human CD4+ CEMss cells infected with a recombinant retrovirus bearing the same SstI/SstI F12/HIV genome (Federico et al, J Gen Virol, 1993, in press). Both sets of results indicate that the expression of this genome in bio-engineered CD4+ human cells results in their intracellular immunization against HIV-1.

  1. Determinantes da aderência à terapia anti-retroviral combinada em Brasília, Distrito Federal, Brasil, 1999-2000

    Directory of Open Access Journals (Sweden)

    Cláudio Viveiros de Carvalho

    Full Text Available A aderência ao tratamento é um dos principais problemas relacionados à terapia anti-retroviral, já que a tomada incompleta dos medicamentos pode levar à resistência viral. Efeitos colaterais podem interferir com a qualidade de vida dos pacientes. Buscou-se estimar níveis de aderência à terapia e investigar seus determinantes, através de um estudo transversal. Definiram-se dois pontos de corte como boa aderência: a tomada de pelo menos 80% ou de 95% da medicação conforme a prescrição. Realizaram-se entrevistas semi-estruturadas em uma amostra seqüencial de 150 pacientes atendidos no Hospital-Dia de Brasília. Observou-se que a média de aderência foi 85,8%. As variáveis que se mostraram significativamente associadas à baixa aderência foram: idade, escolaridade, situação de emprego, rendas pessoal e familiar, uso de substâncias ilícitas, estrutura familiar e/ou comunitária, presença de infecção oportunista no momento do diagnóstico e ocorrência de efeitos colaterais relacionados à terapia. As razões de prevalência variaram de 1,6 a 4,5. Concluiu-se que variáveis sócio-econômicas e de hábitos tiveram maior força de associação com o nível de aderência do que as relacionadas com a doença ou com o tratamento.

  2. Seroprevalence of hepatitis B and delta viruses among HIV-infected population attending anti-retroviral clinic in selected health facilities in Abuja, Nigeria.

    Science.gov (United States)

    Ifeorah, I M; Bakarey, A S; Adeniji, J A; Onyemelukwe, F N

    2017-01-01

    Triple infection with human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis D virus (HDV) is rare. There is limited data on the seroprevalence of HIV/HBV/HDV tri-infection especially in Nigeria. The aim of this study was to determine the seroprevalences of HBsAg and HDV among HIV-infected individuals attending anti-retroviral (ARV) clinics in Abuja, Nigeria. In this cohort study, blood samples were collected from 1102 (male = 450; female = 652), with age range HIV-infected population attending ARV clinics at selected health facilities in Abuja, Nigeria, between April and October 2016. A well-structured questionnaire was used to capture demographic information from the respondents. Enzyme-linked immunosorbent assay (ELISA) was used to determine the seroprevalence of hepatitis B surface antigen and anti-HDV. The result was interpreted according to manufacturer's instruction. Statistical data were analyzed using SPSS software version 21, and chi-square (χ 2 ) test was used to determine association with P HIV/HBV/HDV, respectively, were found among the study population. The infection rate (13.3%) peaked at age range of 31-40 years for HBV (P = 0.002), 50% at HIV/HBV/HDV (P = 0.202). By gender, the rate was higher in males (10.9%, 10.2%, 1.1%) than females (9.8%, 4.9%, 0.5%) for HBV, HBV/HDV, and HIV/HBV/HDV infections, respectively. However, there was no significant association between infection rate and gender. This study has established that HBV and HDV prevalence is still high in the population studied and that the rate of triple infection is low. We advocate for more robust control measures for HBV which should be extended to HDV in HIV population through screening and vaccination.

  3. Harmful alcohol drinking among HIV-positive people in Nepal: an overlooked threat to anti-retroviral therapy adherence and health-related quality of life.

    Science.gov (United States)

    Pokhrel, Khem Narayan; Gaulee Pokhrel, Kalpana; Neupane, Sanjeev Raj; Sharma, Vidya Dev

    2018-01-01

    People living with Human Immunodeficiency Virus (HIV) often suffer from alcohol-use disorders resulting in their poor health and treatment outcomes. Little is known about the association of harmful alcohol drinking with their adherence to anti-retroviral therapy (ART) and health-related quality of life (QOL) in low-resource settings. This study aimed to investigate associations between harmful alcohol drinking, adherence to ART and health-related QOL in HIV-positive people, stratified by gender, in Nepal. We conducted a cross-sectional study of 682 HIV-positive people on ART to measure their self-reported harmful alcohol drinking and non-adherence to ART in the previous month of data collection. We also measured health-related QOL using a WHOQOL-HIV BREF scale. The association between harmful alcohol drinking and non-adherence to ART was examined using multiple logistic regressions. Additionally, multiple linear regressions examined association between harmful alcohol drinking and QOL. Harmful alcohol drinking was associated with non-adherence to ART among men (AOR: 2.48, 95% CI: 1.50, 4.11, p drinking. Moreover, women were more likely to have lower scores for the physical (β = -1.01, p = 0.015), social relations (β = -0.82, p = 0.033), environmental (β = -0.88, p = 0.011), and spiritual (β = -1.30, p = 0.005) domains of QOL when they had harmful alcohol drinking. Harmful alcohol drinking had a negative association with ART adherence and QOL in both HIV-positive men and women in Nepal. Screening for alcohol-use disorders and community-based counseling services should be provided while delivering ART services to improve treatment adherence and QOL.

  4. Prevalence of lipodystrophy and metabolic syndrome among HIV positive individuals on Highly Active Anti-Retroviral treatment in Jimma, South West Ethiopia.

    Science.gov (United States)

    Berhane, Tsegay; Yami, Alemishet; Alemseged, Fessahaye; Yemane, Tilahun; Hamza, Leja; Kassim, Mehedi; Deribe, Kebede

    2012-01-01

    Use of highly active antiretroviral therapy has led to significant reductions in morbidity and mortality rates. However, these agents had also given rise to the metabolic and morphologic abnormalities which are modifiable risk factors for cardiovascular diseases. Evidences elsewhere indicate growing in prevalence of these problems but studies are lacking in Ethiopia. This study was conducted to determine the prevalence of HIV-associated lipodystrophy and metabolic syndrome in patients taking highly active antiretroviral therapy. A cross-sectional study was conducted in 2010 on a sample of 313 patients taking highly active antiretroviral therapy in Jimma University specialized hospital. Structured questionnaire was used to assess patients' sociodemographic characteristics and clinical manifestations of metabolic abnormalities. Checklists were used for reviewing charts about clinical manifestations of metabolic abnormalities and immunologic profile of patients. Data was cleaned, entered in and analyzed using SPSS for windows version 16.0. Metabolic syndrome was detected in 21.1% and HIV-lipodystrophy was detected 12.1% of patients. The factors found to be independently associated with metabolic syndrome were taking the antiretroviral therapy for more than 12 months (AOR=4.2; 95% CI=1.24-14.23) and female sex (AOR=2.30; 95% CI=1.0-5.27) and the factor found to be independently associated with HIV-lipodystrophy was taking the antiretroviral therapy (AOR=3.59; 95% CI=1.03-12.54) for more than 12 months. Metabolic abnormalities were relatively common in the study population. The problems were higher among those who took anti-retroviral treatment for longer duration. Therefore, regular screening for and taking action against the metabolic abnormalities is mandatory.

  5. Prevalence of renal disease in Nigerian children infected with the human immunodeficiency virus and on highly active anti-retroviral therapy.

    Science.gov (United States)

    Iduoriyekemwen, Nosakhare J; Sadoh, Wilson E; Sadoh, Ayebo E

    2013-01-01

    Access to highly active anti-retroviral therapy (HAART) has improved the prognosis of Nigerian children infected with the human immunodeficiency virus (HIV); thus, more children are surviving. Long-term exposure to HAART is potentially nephrotoxic. We therefore aimed at assessing the prevalence of renal disease in Nigerian children infected with HIV, who are on HAART. In this cross-sectional study, we studied children, aged ten months to 17 years, infected with HIV, attending the pediatric HIV clinics of the University of Benin Teaching Hospital. Demographic and clinical data were obtained by parental interview as well as from the medical records. Each child's urine was tested for albumin and microalbuminuria using multi test strips and mitral test strips, respectively. The serum creatinine level of each child was also estimated and used in calculating the glomerular filtration rate (GFR). Renal disease was defined as the presence of significant proteinuria of 1+ and above on dipstick or the presence of microalbuminuria of ≥20 mg and/or GFR children recruited, 60 were males and 39 were females. The mean age of the children was 6.6 ± 3.5 years. All the children were on HAART and 85% had acquired the HIV infection by vertical transmission. The overall prevalence of renal disease was 16.2%. Microalbuminuria was seen in 11 children with renal disease (11.1%); 3 of them had significant proteinuria. GFR of less than 60 mL/min/1.73 m 2 was seen in five children (5.1%) with renal disease, but none had end-stage renal disease (GFR less than 15 mL/min/1.73 m 2 ). Renal disease was found to be significantly associated with advanced stage of HIV infection (P renal disease in HAART-treated Nigerian children is high and majority of them are asymptomatic of renal disease, but in the advanced stages of HIV infection.

  6. Effects of UVA1 Phototherapy on Expression of Human Endogenous Retroviral Sequence (HERV)-K10 gag in Morphea: A Preliminary Study.

    Science.gov (United States)

    Kowalczyk, Michał Jacek; Teresiak-Mikołajczak, Ewa; Dańczak-Pazdrowska, Aleksandra; Żaba, Ryszard; Adamski, Zygmunt; Osmola-Mańkowska, Agnieszka

    2017-01-28

    BACKGROUND Morphea, also known as localized scleroderma, is a rare autoimmune connective tissue disease characterized by skin fibrosis. UVA1 phototherapy is an important asset in the reduction of clinical manifestations in morphea. There are studies claiming that UV light modulates the expression of some human endogenous retroviral sequences. The aim of this study was to determine if the expression of HERV-K10 gag element is lowered by UVA1 phototherapy in morphea, a disease in which such irradiation has a soothing effect. MATERIAL AND METHODS The expression levels of the HERV-K10 gag were assessed by real-time PCR (polymerase chain reaction) in peripheral blood mononuclear cells (PBMC) and skin-punch biopsies of healthy volunteers and 9 morphea patients before and after phototherapy. Additionally, correlations between the HERV-K10 gag expression and age, disease duration, the Localized Scleroderma Skin Severity Index (LoSSI), and antinuclear antibody (ANA) titers were assessed. RESULTS In PBMC, HERV-K10 gag mRNA was significantly elevated after UVA1 phototherapy compared to healthy controls. Most of the patients responded with an increased expression level of this sequence. However, we found no statistical evidence at this point that phototherapy indeed has an effect on the HERV-K10 gag expression (there were no statistical differences in PBMC of morphea patients before and after phototherapy). Similarly, there was no statistically relevant effect of the UVA1 on the expression of HERV-K10 gag in skin. CONCLUSIONS At this point, the effect of UVA1 phototherapy on the expression of HERV-K10 gag cannot be statistically confirmed.

  7. Restriction genes for retroviruses influence the risk of multiple sclerosis

    DEFF Research Database (Denmark)

    Nexø, Bjørn A; Hansen, Bettina; Nissen, Kari K

    2013-01-01

    We recently described that the autoimmune, central nervous system disease, multiple sclerosis (MS), is genetically associated with the human endogenous retroviral locus, HERV-Fc1, in Scandinavians. A number of dominant human genes encoding factors that restrict retrovirus replication have been kn...... and a marker in BST2, associated statistically with the risk of getting MS, while markers in or near APOBEC3s and TREXs showed little or no effect. This indicates that the two TRIMs and BST2 influence the risk of disease and thus supports the hypothesis of a viral involvement....

  8. Colonização oral por Candida spp. em pacientes com infecção pelo HIV em uso de terapia anti-retroviral : estudo epidemiologico, clinico e microbiologico

    OpenAIRE

    Ana Cecilia Nastrini Delgado

    2008-01-01

    Resumo: OBJETIVOS: Avaliar a incidência de colonização oral por Candida spp. em pacientes com HIV em uso de terapia anti-retroviral, comparando os resultados dos grupos de pacientes colonizados e não colonizados, assim como estudar os aspectos microbiológicos das cepas isoladas. PACIENTES E MÉTODOS: Foi realizado estudo transversal de pacientes assistidos no HC/Unicamp, de agosto de 2003 a abril de 2004, com coleta única por paciente de swab da cavidade oral. CHROMagar Candida® e ID32C® foram...

  9. Use of anti-retroviral therapy in tuberculosis patients on second-line anti-TB regimens: a systematic review.

    Directory of Open Access Journals (Sweden)

    Matthew Arentz

    Full Text Available Use of antiretroviral therapy (ART during treatment of drug susceptible tuberculosis (TB improves survival. However, data from HIV infected individuals with drug resistant TB are lacking. Second line TB drugs when combined with ART may increase drug interactions and lead to higher rates of toxicity and greater noncompliance. This systematic review sought to determine the benefit of ART in the setting of second line drug therapy for drug resistant TB.We included individual patient data from studies that evaluated treatment of drug-resistant tuberculosis in HIV-1 infected individuals published between January 1980 and December of 2009. We evaluated the effect of ART on treatment outcomes, time to smear and culture conversion, and adverse events.Ten observational studies, including data from 217 subjects, were analyzed. Patients using ART during TB treatment had increased likelihood of cure (hazard ratio (HR 3.4, 95% CI 1.6-7.4 and decreased likelihood of death (HR 0.4, 95% CI 0.3-0.6 during treatment for drug resistant TB. These associations remained significant in patients with a CD4 less than 200 cells/mm(3 and less than 50 cells/mm(3, and when correcting for drug resistance pattern.We identified only observational studies from which individual patient data could be drawn. Limitations in study design, and heterogeneity in a number of the outcomes of interest had the potential to introduce bias.While there are insufficient data to determine if ART use increases adverse drug interactions when used with second line TB drugs, ART use during treatment of drug resistant TB appears to improve cure rates and decrease risk of death. All individuals with HIV appear to benefit from ART use during treatment for TB.

  10. A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites

    DEFF Research Database (Denmark)

    Uren, Anthony G; Mikkers, Harald; Kool, Jaap

    2009-01-01

    Insertional mutagens such as viruses and transposons are a useful tool for performing forward genetic screens in mice to discover cancer genes. These screens are most effective when performed using hundreds of mice; however, until recently, the cost-effective isolation and sequencing of insertion...... optimized for the murine leukemia virus (MuLV), and can easily be performed in a 96-well plate format for the efficient multiplex isolation of insertion sites.......Insertional mutagens such as viruses and transposons are a useful tool for performing forward genetic screens in mice to discover cancer genes. These screens are most effective when performed using hundreds of mice; however, until recently, the cost-effective isolation and sequencing of insertion...... sites has been a major limitation to performing screens on this scale. Here we present a method for the high-throughput isolation of insertion sites using a highly efficient splinkerette-PCR method coupled with capillary or 454 sequencing. This protocol includes a description of the procedure for DNA...

  11. Clonal Dominance With Retroviral Vector Insertions Near the ANGPT1 and ANGPT2 Genes in a Human Xenotransplant Mouse Model

    Directory of Open Access Journals (Sweden)

    Reinhard Haemmerle

    2014-01-01

    Full Text Available Insertional leukemogenesis represents the major risk factor of hematopoietic stem cell (HSC based gene therapy utilizing integrating viral vectors. To develop a pre-clinical model for the evaluation of vector-related genotoxicity directly in the relevant human target cells, cord blood CD34+ HSCs were transplanted into immunodeficient NOD.SCID.IL2rg−/− (NSG mice after transduction with an LTR-driven gammaretroviral vector (GV. Furthermore, we specifically investigated the effect of prolonged in vitro culture in the presence of cytokines recently described to promote HSC expansion or maintenance. Clonality of human hematopoiesis in NSG mice was assessed by high throughput insertion site analyses and validated by insertion site-specific PCR depicting a GV typical integration profile with insertion sites resembling to 25% those of clinical studies. No overrepresentation of integrations in the vicinity of cancer-related genes was observed, however, several dominant clones were identified including two clones harboring integrations in the ANGPT1 and near the ANGPT2 genes associated with deregulated ANGPT1- and ANGPT2-mRNA levels. While these data underscore the potential value of the NSG model, our studies also identified short-comings such as overall low numbers of engrafted HSCs, limited in vivo observation time, and the challenges of in-depth insertion site analyses by low contribution of gene modified hematopoiesis.

  12. Why do Patients in Pre-Anti Retroviral Therapy (ART) Care Default: A Cross-Sectional Study.

    Science.gov (United States)

    Chakravarty, Jaya; Kansal, Sangeeta; Tiwary, Narendra; Sundar, Shyam

    2016-01-01

    Approximately, 40% of the patients registered in the National AIDS Control Program in India are not on antiretroviral therapy (ART), i.e., are in pre-ART care. However, there are scarce data regarding the retention of pre-ART patients under routine program conditions. The main objective of this study was to find out the reasons for default among patients in pre-ART care. Patients enrolled in the ART Centre, Banaras Hindu University (BHU) between January and December 2009 and in pre-ART care were included in the study. Defaulters were those pre-ART patients who missed their last appointment of CD4 count by more than 1 month. Defaulters were traced telephonically in 2011 and those who returned and gave their consent for the study were interviewed using a semi-structured questionnaire. Out of 620 patients in pre-ART care, 384 (68.2%) were defaulters. One hundred forty-four of the defaulters were traced and only 83 reached the ART center for interview. Among defaulters who did not reach the ART center, illiterate and unmarried were significantly more and mean duration from registration to default was also significantly less as compared to those who came back for the interview. Most defaulters gave more than one reason for defaulting that were as follows: Inconvenient clinic timings (98%), need for multiple mode of transport (92%), perceived improved health (65%), distance of center from home (61%), lack of social support (62%), and financial difficulty (59%). Active tracing of pre-ART patients through outreach and strengthening of the Link ART centers will improve the retention of patients in the program.

  13. S-phase reduction in T47D human breast cancer epithelial cells induced by an S100P antisense-retroviral construct.

    Science.gov (United States)

    Beissel, Bettina; Silva, Ismael D C G; Pesquero, João B; Russo, Jose; Schor, Nestor; Bellini, Maria Helena

    2007-03-01

    S100P is expressed in several malignant neoplasms. It was previously demonstrated that S100P is involved in the very early stages of breast carcinogenesis. In the present study we used a retrovirus-mediated transfer of antisense-S100P in order to check whether the decrease in expression of this protein could lead to alterations in the cell cycle of epithelial cells of human breast cancer. The T47D breast carcinoma cell line, a human breast epithelial cell that expresses high levels of S100P, was a tool used in this study to investigate the alteration in cell cycle induced by a retrovirus-mediated transfer of antisense-S100P. First we used the real-time PCR technique to quantify the gene expression. The results showed a reduction of 63% of expression within the T47D-S100P-A/S infected population compared with control T47D-LXSN clones. To determine the impact of the S100P antisense technique on protein expression in T47D cells, we performed immunofluorescence staining and analyzed the resulting images using a confocal microscope. The images showed much less pronounced antibody marking of the S100P protein in the T47D-S100P-A/S compared with control cells. To evaluate whether the antisense approach caused any alteration in the cell cycle, we concluded the study with flow cytometric analysis of the cell distribution. Our findings indicated that, in our model, S100P-antisense cells showed a 23% reduction of cells at the S-phase. Using transduction techniques with an S100P antisense-retroviral construct we were able to demonstrate a significant reduction in S-phase of the T47D cell cycle. To the best of our knowledge, this is the first time that an antisense approach has been used against S100P mRNA in breast cancer epithelial cells. The results showed here seem to further classify S100P as a protein that might be involved in the cell cycle imbalance observed during breast carcinogenesis.

  14. Harmful alcohol drinking among HIV-positive people in Nepal: an overlooked threat to anti-retroviral therapy adherence and health-related quality of life

    Science.gov (United States)

    Pokhrel, Khem Narayan; Gaulee Pokhrel, Kalpana; Neupane, Sanjeev Raj; Sharma, Vidya Dev

    2018-01-01

    ABSTRACT Background: People living with Human Immunodeficiency Virus (HIV) often suffer from alcohol-use disorders resulting in their poor health and treatment outcomes. Little is known about the association of harmful alcohol drinking with their adherence to anti-retroviral therapy (ART) and health-related quality of life (QOL) in low-resource settings. Objective: This study aimed to investigate associations between harmful alcohol drinking, adherence to ART and health-related QOL in HIV-positive people, stratified by gender, in Nepal. Methods: We conducted a cross-sectional study of 682 HIV-positive people on ART to measure their self-reported harmful alcohol drinking and non-adherence to ART in the previous month of data collection. We also measured health-related QOL using a WHOQOL-HIV BREF scale. The association between harmful alcohol drinking and non-adherence to ART was examined using multiple logistic regressions. Additionally, multiple linear regressions examined association between harmful alcohol drinking and QOL. Results: Harmful alcohol drinking was associated with non-adherence to ART among men (AOR: 2.48, 95% CI: 1.50, 4.11, p < 0.001) and women (AOR: 2.52, 95% CI: 1.32, 4.80, p = 0.005). Men were more likely to have lower score for the psychological (β = −0.55, p = 0.021) and level of independence (β = −0.68, p = 0.018) domains when they had harmful alcohol drinking. Moreover, women were more likely to have lower scores for the physical (β = −1.01, p = 0.015), social relations (β = −0.82, p = 0.033), environmental (β = −0.88, p = 0.011), and spiritual (β = −1.30, p = 0.005) domains of QOL when they had harmful alcohol drinking. Conclusions: Harmful alcohol drinking had a negative association with ART adherence and QOL in both HIV-positive men and women in Nepal. Screening for alcohol-use disorders and community-based counseling services should be provided while delivering ART services to improve

  15. Acceptance of anti-retroviral therapy among patients infected with HIV and tuberculosis in rural Malawi is low and associated with cost of transport.

    Directory of Open Access Journals (Sweden)

    Rony Zachariah

    Full Text Available BACKGROUND: A study was conducted among newly registered HIV-positive tuberculosis (TB patients systematically offered anti-retroviral treatment (ART in a district hospital in rural Malawi in order to a determine the acceptance of ART b conduct a geographic mapping of those placed on ART and c examine the association between "cost of transport" and ART acceptance. METHODOLOGY/PRINCIPAL FINDINGS: A retrospective cross-sectional analysis was performed on routine program data for the period of February 2003 to July 2004. Standardized registers and patient cards were used to gather data. The place of residence was used to determine road distances to the Thyolo district hospital. Cost of transport from different parts of the district was based on the known cost for public transport to the road-stop closest to the patient's residence. Of 1,290 newly registered TB patients, 1,003(78% underwent HIV-testing of whom 770 (77% were HIV-positive. 742 of these individuals (pulmonary TB = 607; extra-pulmonary TB = 135 were considered eligible for ART of whom only 101(13.6% accepted ART. Cost of transport to the hospital ART site was significantly associated with ART acceptance and there was a linear trend in association between cost and ART acceptance (chi(2 for trend = 25.4, P<0.001. Individuals who had to pay 50 Malawi Kwacha (1 United States Dollar = 100 Malawi Kwacha, MW or less for a one-way trip to the Thyolo hospital were four times more likely to accept ART than those who had to pay over 100 MW (Adjusted Odds ratio = 4.0, 95% confidence interval: 2.0-8.1, P<0.001. CONCLUSIONS/SIGNIFICANCE: ART acceptance among TB patients in a rural district in Malawi is low and associated with cost of transport to the centralized hospital based ART site. Decentralizing the ART offer from the hospital to health centers that are closer to home communities would be an essential step towards reducing the overall cost and burden of travel.

  16. Who has access to counseling and testing and anti-retroviral therapy in Malawi – an equity analysis

    Directory of Open Access Journals (Sweden)

    Banda Talumba

    2009-05-01

    Full Text Available Abstract Background The HIV and AIDS epidemic in Malawi poses multiple challenges from an equity perspective. It is estimated that 12% of Malawians are living with HIV or AIDS among the 15-49 age group. This paper synthesises available information to bring an equity lens on Counselling and Testing (CT and Antiretroviral Therapy (ART policy, practice and provision in Malawi. Methods A synthesis of a wide range of published and unpublished reports and studies using a variety of methodological approaches was undertaken. The analysis and recommendations were developed, through consultation with key stakeholders in Malawi. Findings At the policy level Malawi is unique in having an equity in access to ART policy, and equity considerations are also included in key CT documents. The number of people accessing CT has increased considerably from 149,540 in 2002 to 482,364 in 2005. There is urban bias in provision of CT and more women than men access CT. ART has been provided free since June 2004 and scale up of ART provision is gathering pace. By end December 2006, there were 85,168 patients who had ever started on ART in both the public and private health sector, 39% of the patients were male while 61% were female. The majority of patients were adults, and 7% were children, aged 14 years or below. Despite free ART services, patients, especially poor rural patients face significant barriers in access and adherence to services. There are missed opportunities in strengthening integration between CT and ART and TB, Sexually Transmitted Infections (STI and maternal health services. Conclusion To promote equitable access for CT and ART in Malawi there is need to further invest in human resources for health, and seize opportunities to integrate CT and ART services with tuberculosis, sexually transmitted infections and maternal health services. This should not only promote access to services but also ensure that resources available for CT and ART strengthen

  17. Adherence to antiretroviral therapy: a qualitative study with physicians from Rio de Janeiro, Brazil Aderência à terapia anti-retroviral: um estudo qualitativo com médicos no Rio de Janeiro, Brasil

    Directory of Open Access Journals (Sweden)

    Monica Malta

    2005-10-01

    Full Text Available Brazil provides free antiretroviral (ARV therapy to some 150,000 individuals living with HIV/ AIDS. ARV regimens require optimal adherence to achieve undetectable viral loads and to avoid viral resistance. Physicians play a key role to foster ARV adherence, but until now little is known about the communication between physicians/ people living with HIV/AIDS in this setting. In-depth interviews were conducted with 40 physicians treating people living with HIV/AIDS at six public reference centers in Rio de Janeiro, Brazil. Interview topics included: experiences in the treatment of people living with HIV/AIDS, relationship and dialogue with patients, barriers/facilitators to adherence, and effectiveness of available services. Barriers to ARV adherence were mainly related to the low quality of patient-provider relationship. Other barriers were related to "chaotic" patients' lifestyles, and inadequate knowledge and/or negative beliefs about HIV/AIDS and ARV effectiveness. It is necessary to improve networking between services, establish agile referral systems, and improve health professionals' integration. These structural changes could contribute to improved adherence, resulting in improved quality of life for people living with HIV/AIDS.O Brasil fornece gratuitamente terapia anti-retroviral (ARV para cerca de 150 mil pessoas vivendo com HIV/ AIDS. A terapia ARV requer aderência ótima, visando alcançar carga viral indetectável e evitar resistência viral. Os médicos desempenham papel central quanto à aderência à ARV, mas há escassa informação sobre a comunicação entre médicos/pessoas vivendo com HIV/ AIDS. Entrevistas em profundidade foram realizadas com 40 médicos assistentes de seis hospitais de referência do Rio de Janeiro, Brasil. Tópicos da entrevista incluíram: experiências relativas ao tratamento de pessoas vivendo com HIV/AIDS, relacionamento/diálogo com pacientes, barreiras/facilitadores para aderência aos servi

  18. Retroviral proteinases and their inhibitors

    Czech Academy of Sciences Publication Activity Database

    Sedláček, Juraj

    2000-01-01

    Roč. 3, 3,4 (2000), s. 23-24 [ Proteolytic enzymes and their inhibitors in physiology and pathogenesis. 14.09.2000, Plzen] Institutional research plan: CEZ:AV0Z5052915 Subject RIV: EB - Genetics ; Molecular Biology

  19. Increased levels of regulatory T cells (Tregs) in human immunodeficiency virus-infected patients after 5 years of highly active anti-retroviral therapy may be due to increased thymic production of naive Tregs

    DEFF Research Database (Denmark)

    Kolte, L.; Gaardbo, J.C.; Skogstrand, K.

    2009-01-01

    in HIV-infected patients compared with controls, both after 1 and 5 years of HAART (Pcounts, immune activation and cytokine patterns. Furthermore, levels of naive T(regs) were elevated significantly in HIV-infected patients (P...This study determines levels of regulatory T cells (T(regs)), naive T(regs), immune activation and cytokine patterns in 15 adult human immunodeficiency virus (HIV)-infected patients receiving prolonged highly active anti-retroviral therapy (HAART) who have known thymic output, and explores if naive...... T(regs) may represent recent thymic emigrant T(regs). HIV-infected patients treated with HAART with a median of 1 and 5 years were compared with healthy controls. Percentages of T(regs) (CD3(+)CD4(+)CD25(+)CD127(low)), naive T(regs) (CD3(+)CD4(+)CD25(+)CD45RA(+)) and activation markers (CD38(+)human...

  20. Increased levels of regulatory T cells (Tregs) in human immunodeficiency virus-infected patients after 5 years of highly active anti-retroviral therapy may be due to increased thymic production of naive Tregs

    DEFF Research Database (Denmark)

    Kolte, L.; Gaardbo, J.C.; Skogstrand, Kristin

    2008-01-01

    higher in HIV-infected patients compared with controls, both after 1 and 5 years of HAART (P counts, immune activation and cytokine patterns. Furthermore, levels of naive T(regs) were elevated significantly in HIV-infected patients......Summary This study determines levels of regulatory T cells (T(regs)), naive T(regs), immune activation and cytokine patterns in 15 adult human immunodeficiency virus (HIV)-infected patients receiving prolonged highly active anti-retroviral therapy (HAART) who have known thymic output, and explores...... if naive T(regs) may represent recent thymic emigrant T(regs). HIV-infected patients treated with HAART with a median of 1 and 5 years were compared with healthy controls. Percentages of T(regs) (CD3(+)CD4(+)CD25(+)CD127(low)), naive T(regs) (CD3(+)CD4(+)CD25(+)CD45RA(+)) and activation markers (CD38...

  1. Increased levels of regulatory T cells (T(regs)) in human immunodeficiency virus-infected patients after 5 years of highly active anti-retroviral therapy may be due to increased thymic production of naive T(regs)

    DEFF Research Database (Denmark)

    Kolte, L; Gaardbo, J C; Skogstrand, K

    2008-01-01

    higher in HIV-infected patients compared with controls, both after 1 and 5 years of HAART (P counts, immune activation and cytokine patterns. Furthermore, levels of naive T(regs) were elevated significantly in HIV-infected patients......Summary This study determines levels of regulatory T cells (T(regs)), naive T(regs), immune activation and cytokine patterns in 15 adult human immunodeficiency virus (HIV)-infected patients receiving prolonged highly active anti-retroviral therapy (HAART) who have known thymic output, and explores...... if naive T(regs) may represent recent thymic emigrant T(regs). HIV-infected patients treated with HAART with a median of 1 and 5 years were compared with healthy controls. Percentages of T(regs) (CD3(+)CD4(+)CD25(+)CD127(low)), naive T(regs) (CD3(+)CD4(+)CD25(+)CD45RA(+)) and activation markers (CD38...

  2. Static, Lightweight Includes Resolution for PHP

    NARCIS (Netherlands)

    M.A. Hills (Mark); P. Klint (Paul); J.J. Vinju (Jurgen)

    2014-01-01

    htmlabstractDynamic languages include a number of features that are challenging to model properly in static analysis tools. In PHP, one of these features is the include expression, where an arbitrary expression provides the path of the file to include at runtime. In this paper we present two

  3. Article Including Environmental Barrier Coating System

    Science.gov (United States)

    Lee, Kang N. (Inventor)

    2015-01-01

    An enhanced environmental barrier coating for a silicon containing substrate. The enhanced barrier coating may include a bond coat doped with at least one of an alkali metal oxide and an alkali earth metal oxide. The enhanced barrier coating may include a composite mullite bond coat including BSAS and another distinct second phase oxide applied over said surface.

  4. Rare thoracic cancers, including peritoneum mesothelioma

    NARCIS (Netherlands)

    Siesling, Sabine; van der Zwan, Jan Maarten; Izarzugaza, Isabel; Jaal, Jana; Treasure, Tom; Foschi, Roberto; Ricardi, Umberto; Groen, Harry; Tavilla, Andrea; Ardanaz, Eva

    Rare thoracic cancers include those of the trachea, thymus and mesothelioma (including peritoneum mesothelioma). The aim of this study was to describe the incidence, prevalence and survival of rare thoracic tumours using a large database, which includes cancer patients diagnosed from 1978 to 2002,

  5. Rare thoracic cancers, including peritoneum mesothelioma

    NARCIS (Netherlands)

    Siesling, Sabine; Zwan, J.M.V.D.; Izarzugaza, I.; Jaal, J.; Treasure, T.; Foschi, R.; Ricardi, U.; Groen, H.; Tavilla, A.; Ardanaz, E.

    2012-01-01

    Rare thoracic cancers include those of the trachea, thymus and mesothelioma (including peritoneum mesothelioma). The aim of this study was to describe the incidence, prevalence and survival of rare thoracic tumours using a large database, which includes cancer patients diagnosed from 1978 to 2002,

  6. Adesão e não-adesão à terapia anti-retroviral: as duas faces de uma mesma vivência Adhesión y no adhesión a la terapia antiretroviral: las dos caras de una misma vivencia Adhesion and non adhesion to anti-retroviral therapy: the two faces of a same experience

    Directory of Open Access Journals (Sweden)

    Ana Lúcia Cardoso Nogueira da Silva

    2009-04-01

    Full Text Available O objetivo do estudo foi compreender, a partir da perspectiva de portadores e familiares, os aspectos que influenciam na adesão à terapêutica anti-retroviral. Trata-se de um estudo descritivo, de natureza qualitativa, desenvolvido no período de junho de 2006 a julho de 2007, junto a 10 indivíduos portadores do HIV/Aids, acompanhados pelo Serviço de Atendimento Especializado em Aids de Campo Mourão - PR e seus familiares. Constituem fatores facilitadores da adesão: adoção de estratégias para lembrar horários e mascarar o gosto do medicamento, ausência de efeitos colaterais, número reduzido de medicamentos a serem ingeridos e capacidade para reconhecê-los, lembrança dos sintomas da doença e o apoio da rede social. A ausência destes fatores pode culminar na não-adesão.El objetivo del estudio fue comprender, a partir de la perspectiva de portadores y familiares, los aspectos que influyen en la adhesión a la terapéutica antiretroviral. Se trata de un estudio descriptivo, de naturaleza cualitativa, desarrollado en el período de junio de 2006 a julio de 2007, junto a 10 individuos portadores del VIH/SIDA, acompañados por el Servicio de Atención Especializado en SIDA de Campo Mourão - PR y sus familiares. Constituyen factores que facilitan la adhesión: adopción de estrategias para recordar horarios y enmascarar el sabor del medicamento, ausencia de efectos colaterales, número reducido de medicamentos a ser ingeridos y capacidad para reconocerlos, recuerdo de los síntomas de la enfermedad y el apoyo de la red social. La ausencia de estos factores puede culminar en la no-adhesión.The objective of the study was to understand, from the perspective of HIV bearers and family, the aspects that influence in the adhesion to the antiretroviral therapy. It is a descriptive study, of qualitative nature, carried out from June 2006 to July 2007, with 10 HIV/Aids positive individuals, attended at the Service of Specialized Care on Aids

  7. Composite Pressure Vessel Including Crack Arresting Barrier

    Science.gov (United States)

    DeLay, Thomas K. (Inventor)

    2013-01-01

    A pressure vessel includes a ported fitting having an annular flange formed on an end thereof and a tank that envelopes the annular flange. A crack arresting barrier is bonded to and forming a lining of the tank within the outer surface thereof. The crack arresting barrier includes a cured resin having a post-curing ductility rating of at least approximately 60% through the cured resin, and further includes randomly-oriented fibers positioned in and throughout the cured resin.

  8. Including Organizational Cultural Parameters in Work Processes

    National Research Council Canada - National Science Library

    Handley, Holly A; Heacox, Nancy J

    2004-01-01

    .... In order to represent the organizational impact on the work process, five organizational cultural parameters were identified and included in an algorithm for modeling and simulation of cultural...

  9. Haemophilus influenzae Disease (Including Hib) Symptoms

    Science.gov (United States)

    ... Links Global Hib Vaccination Hib Vaccination Meningitis Pneumonia Sepsis ... Haemophilus influenzae , including H. influenzae type b or Hib, can cause many different kinds of infections . Symptoms depend on ...

  10. Human endogenous retrovirus expression is inversely related with the up-regulation of interferon-inducible genes in the skin of patients with lichen planus.

    Science.gov (United States)

    Nogueira, Marcelle Almeida de Sousa; Gavioli, Camila Fátima Biancardi; Pereira, Nátalli Zanete; de Carvalho, Gabriel Costa; Domingues, Rosana; Aoki, Valéria; Sato, Maria Notomi

    2015-04-01

    Lichen planus (LP) is a common inflammatory skin disease of unknown etiology. Reports of a common transactivation of quiescent human endogenous retroviruses (HERVs) support the connection of viruses to the disease. HERVs are ancient retroviral sequences in the human genome and their transcription is often deregulated in cancer and autoimmune diseases. We explored the transcriptional activity of HERV sequences as well as the antiviral restriction factor and interferon-inducible genes in the skin from LP patients and healthy control (HC) donors. The study included 13 skin biopsies from patients with LP and 12 controls. Real-time PCR assay identified significant decrease in the HERV-K gag and env mRNA expression levels in LP subjects, when compared to control group. The expressions of HERV-K18 and HERV-W env were also inhibited in the skin of LP patients. We observed a strong correlation between HERV-K gag with other HERV sequences, regardless the down-modulation of transcripts levels in LP group. In contrast, a significant up-regulation of the cytidine deaminase APOBEC 3G (apolipoprotein B mRNA-editing), and the GTPase MxA (Myxovirus resistance A) mRNA expression level was identified in the LP skin specimens. Other transcript expressions, such as the master regulator of type I interferon-dependent immune responses, STING (stimulator of interferon genes) and IRF-7 (interferon regulatory factor 7), IFN-β and the inflammassome NALP3, had increased levels in LP, when compared to HC group. Our study suggests that interferon-inducible factors, in addition to their role in innate immunity against exogenous pathogens, contribute to the immune control of HERVs. Evaluation of the balance between HERV and interferon-inducible factor expression could possibly contribute to surveillance of inflammatory/malignant status of skin diseases.

  11. Mother-to-child transmission of human immunodeficiency virus (HIV) among HIV-infected pregnant women on highly active anti-retroviral therapy with premature rupture of membranes at term.

    Science.gov (United States)

    Eleje, George Uchenna; Edokwe, Emeka Stephen; Ikechebelu, Joseph Ifeanyichukwu; Onubogu, Chinyere Ukamaka; Ugochukwu, Ebele Francesca; Okam, Princeston Chukwuemeka; Ibekwe, Adaobi Maryann

    2018-01-01

    To determine mother-to-child transmission (MTCT) rate and associated risk factors of human immune-deficiency virus (HIV) among HIV-infected pregnant women with term premature rupture of membranes (PROM) in comparison with those without PROM at term. All optimally managed HIV-positive pregnant women of Nnamdi Azikiwe University Teaching Hospital, on highly active anti-retroviral therapy (HAART) who had PROM at term were enrolled. Maternal HIV-1 viral load was not assessed. Follow up was for a minimum of 18 months for evidence of HIV infection. Of the 121 women with PROM at term, 46 (38.0%) were HIV sero-positive, 22/46 (47.8%) of which had their babies followed up till 18 months. The mean latency period was 10.5 ± 5.3 h in PROM group. Apart from duration of PROM (OR = 0.01; 95%CI = 0.00-0.13; p  0.05). Of the 22 (47.8%) babies followed-up in the PROM group and 13 in non-PROM group, none tested positive to HIV, given an MTCT rate of 0%. MTCT rate was 0% following term PROM and in women without PROM. Since maternal HIV-1 viral load was not assessed, we need to be critical while interpreting the findings.

  12. The surgery of peripheral nerves (including tumors)

    DEFF Research Database (Denmark)

    Fugleholm, Kåre

    2013-01-01

    Surgical pathology of the peripheral nervous system includes traumatic injury, entrapment syndromes, and tumors. The recent significant advances in the understanding of the pathophysiology and cellular biology of peripheral nerve degeneration and regeneration has yet to be translated into improved...

  13. Including Indigenous Minorities in Decision-Making

    DEFF Research Database (Denmark)

    Pristed Nielsen, Helene

    Based on theories of public sphere participation and deliberative democracy, this book presents empirical results from a study of experiences with including Aboriginal and Maori groups in political decision-making in respectively Western Australia and New Zealand...

  14. Lung Disease Including Asthma and Adult Vaccination

    Science.gov (United States)

    ... Diseases Resources Lung Disease including Asthma and Adult Vaccination Language: English (US) Español (Spanish) Recommend on Facebook ... more about health insurance options. Learn about adult vaccination and other health conditions Asplenia Diabetes Heart Disease, ...

  15. Births and deaths including fetal deaths

    Data.gov (United States)

    U.S. Department of Health & Human Services — Access to a variety of United States birth and death files including fetal deaths: Birth Files, 1968-2009; 1995-2005; Fetal death file, 1982-2005; Mortality files,...

  16. Isolators Including Main Spring Linear Guide Systems

    Science.gov (United States)

    Goold, Ryan (Inventor); Buchele, Paul (Inventor); Hindle, Timothy (Inventor); Ruebsamen, Dale Thomas (Inventor)

    2017-01-01

    Embodiments of isolators, such as three parameter isolators, including a main spring linear guide system are provided. In one embodiment, the isolator includes first and second opposing end portions, a main spring mechanically coupled between the first and second end portions, and a linear guide system extending from the first end portion, across the main spring, and toward the second end portion. The linear guide system expands and contracts in conjunction with deflection of the main spring along the working axis, while restricting displacement and rotation of the main spring along first and second axes orthogonal to the working axis.

  17. Electrochemical cell structure including an ionomeric barrier

    Science.gov (United States)

    Lambert, Timothy N.; Hibbs, Michael

    2017-06-20

    An apparatus includes an electrochemical half-cell comprising: an electrolyte, an anode; and an ionomeric barrier positioned between the electrolyte and the anode. The anode may comprise a multi-electron vanadium phosphorous alloy, such as VP.sub.x, wherein x is 1-5. The electrochemical half-cell is configured to oxidize the vanadium and phosphorous alloy to release electrons. A method of mitigating corrosion in an electrochemical cell includes disposing an ionomeric barrier in a path of electrolyte or ion flow to an anode and mitigating anion accumulation on the surface of the anode.

  18. Electric Power Monthly, August 1990. [Glossary included

    Energy Technology Data Exchange (ETDEWEB)

    1990-11-29

    The Electric Power Monthly (EPM) presents monthly summaries of electric utility statistics at the national, Census division, and State level. The purpose of this publication is to provide energy decisionmakers with accurate and timely information that may be used in forming various perspectives on electric issues that lie ahead. Data includes generation by energy source (coal, oil, gas, hydroelectric, and nuclear); generation by region; consumption of fossil fuels for power generation; sales of electric power, cost data; and unusual occurrences. A glossary is included.

  19. Diversification of Smallholder Tobacco Systems to include ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    Tobacco is the mainstay of the economy of Malawi, accounting for over 70% of export earnings. Of the 100 000 members of the National Smallholder Farmers' Association of Malawi (NASFAM), 60% rely on tobacco for their sole source of income. Like their counterparts elsewhere, they face many difficulties, including: ...

  20. BIOLOGIC AND ECONOMIC EFFECTS OF INCLUDING DIFFERENT ...

    African Journals Online (AJOL)

    The biologic and economic effects of including three agro-industrial by-products as ingredients in turkey poult diets were investigated using 48 turkey poults in a completely randomised design experiment. Diets were formulated to contain the three by-products – wheat offal, rice husk and palm kernel meal, each at 20% level ...

  1. Extending flood damage assessment methodology to include ...

    African Journals Online (AJOL)

    Optimal and sustainable flood plain management, including flood control, can only be achieved when the impacts of flood control measures are considered for both the man-made and natural environments, and the sociological aspects are fully considered. Until now, methods/models developed to determine the influences ...

  2. Including Children Dependent on Ventilators in School.

    Science.gov (United States)

    Levine, Jack M.

    1996-01-01

    Guidelines for including ventilator-dependent children in school are offered, based on experience with six such students at a New York State school. Guidelines stress adherence to the medical management plan, the school-family partnership, roles of the social worker and psychologist, orientation, transportation, classroom issues, and steps toward…

  3. Musculoskeletal ultrasound including definitions for ultrasonographic pathology

    DEFF Research Database (Denmark)

    Wakefield, RJ; Balint, PV; Szkudlarek, Marcin

    2005-01-01

    Ultrasound (US) has great potential as an outcome in rheumatoid arthritis trials for detecting bone erosions, synovitis, tendon disease, and enthesopathy. It has a number of distinct advantages over magnetic resonance imaging, including good patient tolerability and ability to scan multiple joint...

  4. Modernizing Agrifood Markets : Including Small Producers in ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    Against this baseline data, they will endeavor to identify success stories or examples of interventions that ensure small farmers' access to modernizing agrifood markets. The research will inform a set of policy recommendations to be promoted through policy platforms in a large number of developing countries, including but ...

  5. Including Students with Visual Impairments: Softball

    Science.gov (United States)

    Brian, Ali; Haegele, Justin A.

    2014-01-01

    Research has shown that while students with visual impairments are likely to be included in general physical education programs, they may not be as active as their typically developing peers. This article provides ideas for equipment modifications and game-like progressions for one popular physical education unit, softball. The purpose of these…

  6. Numerical simulation of spark ignition including ionization

    NARCIS (Netherlands)

    Thiele, M; Selle, S; Riedel, U; Warnatz, J; Maas, U

    2000-01-01

    A detailed understanding of the processes associated Midi spark ignition, as a first step during combustion, is of great importance fur clean operation of spark ignition engines. In the past 10 years. a growing concern for environmental protection, including low emission of pollutants, has increased

  7. Photoactive devices including porphyrinoids with coordinating additives

    Science.gov (United States)

    Forrest, Stephen R; Zimmerman, Jeramy; Yu, Eric K; Thompson, Mark E; Trinh, Cong; Whited, Matthew; Diev, Vlacheslav

    2015-05-12

    Coordinating additives are included in porphyrinoid-based materials to promote intermolecular organization and improve one or more photoelectric characteristics of the materials. The coordinating additives are selected from fullerene compounds and organic compounds having free electron pairs. Combinations of different coordinating additives can be used to tailor the characteristic properties of such porphyrinoid-based materials, including porphyrin oligomers. Bidentate ligands are one type of coordinating additive that can form coordination bonds with a central metal ion of two different porphyrinoid compounds to promote porphyrinoid alignment and/or pi-stacking. The coordinating additives can shift the absorption spectrum of a photoactive material toward higher wavelengths, increase the external quantum efficiency of the material, or both.

  8. Electric power monthly, September 1990. [Glossary included

    Energy Technology Data Exchange (ETDEWEB)

    1990-12-17

    The purpose of this report is to provide energy decision makers with accurate and timely information that may be used in forming various perspectives on electric issues. The power plants considered include coal, petroleum, natural gas, hydroelectric, and nuclear power plants. Data are presented for power generation, fuel consumption, fuel receipts and cost, sales of electricity, and unusual occurrences at power plants. Data are compared at the national, Census division, and state levels. 4 figs., 52 tabs. (CK)

  9. Power generation method including membrane separation

    Science.gov (United States)

    Lokhandwala, Kaaeid A.

    2000-01-01

    A method for generating electric power, such as at, or close to, natural gas fields. The method includes conditioning natural gas containing C.sub.3+ hydrocarbons and/or acid gas by means of a membrane separation step. This step creates a leaner, sweeter, drier gas, which is then used as combustion fuel to run a turbine, which is in turn used for power generation.

  10. Should Trade Agreements Include Environmental Policy?

    OpenAIRE

    Josh Ederington

    2010-01-01

    This article examines the extent to which environmental and trade policies should be treated equally, or symmetrically, in international negotiations. It reviews the recent economics literature on trade and the environment to address two questions. First, should trade negotiations include negotiations over environmental policies and the setting of binding environmental standards? Second, if there are grounds for international environmental negotiations, should environmental agreements b...

  11. Jet-calculus approach including coherence effects

    International Nuclear Information System (INIS)

    Jones, L.M.; Migneron, R.; Narayanan, K.S.S.

    1987-01-01

    We show how integrodifferential equations typical of jet calculus can be combined with an averaging procedure to obtain jet-calculus-based results including the Mueller interference graphs. Results in longitudinal-momentum fraction x for physical quantities are higher at intermediate x and lower at large x than with the conventional ''incoherent'' jet calculus. These results resemble those of Marchesini and Webber, who used a Monte Carlo approach based on the same dynamics

  12. Revisiting Hansen Solubility Parameters by Including Thermodynamics.

    Science.gov (United States)

    Louwerse, Manuel J; Maldonado, Ana; Rousseau, Simon; Moreau-Masselon, Chloe; Roux, Bernard; Rothenberg, Gadi

    2017-11-03

    The Hansen solubility parameter approach is revisited by implementing the thermodynamics of dissolution and mixing. Hansen's pragmatic approach has earned its spurs in predicting solvents for polymer solutions, but for molecular solutes improvements are needed. By going into the details of entropy and enthalpy, several corrections are suggested that make the methodology thermodynamically sound without losing its ease of use. The most important corrections include accounting for the solvent molecules' size, the destruction of the solid's crystal structure, and the specificity of hydrogen-bonding interactions, as well as opportunities to predict the solubility at extrapolated temperatures. Testing the original and the improved methods on a large industrial dataset including solvent blends, fit qualities improved from 0.89 to 0.97 and the percentage of correct predictions rose from 54 % to 78 %. Full Matlab scripts are included in the Supporting Information, allowing readers to implement these improvements on their own datasets. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Drug delivery device including electrolytic pump

    KAUST Repository

    Foulds, Ian G.

    2016-03-31

    Systems and methods are provided for a drug delivery device and use of the device for drug delivery. In various aspects, the drug delivery device combines a “solid drug in reservoir” (SDR) system with an electrolytic pump. In various aspects an improved electrolytic pump is provided including, in particular, an improved electrolytic pump for use with a drug delivery device, for example an implantable drug delivery device. A catalytic reformer can be incorporated in a periodically pulsed electrolytic pump to provide stable pumping performance and reduced actuation cycle.

  14. Aerosol simulation including chemical and nuclear reactions

    International Nuclear Information System (INIS)

    Marwil, E.S.; Lemmon, E.C.

    1985-01-01

    The numerical simulation of aerosol transport, including the effects of chemical and nuclear reactions presents a challenging dynamic accounting problem. Particles of different sizes agglomerate and settle out due to various mechanisms, such as diffusion, diffusiophoresis, thermophoresis, gravitational settling, turbulent acceleration, and centrifugal acceleration. Particles also change size, due to the condensation and evaporation of materials on the particle. Heterogeneous chemical reactions occur at the interface between a particle and the suspending medium, or a surface and the gas in the aerosol. Homogeneous chemical reactions occur within the aersol suspending medium, within a particle, and on a surface. These reactions may include a phase change. Nuclear reactions occur in all locations. These spontaneous transmutations from one element form to another occur at greatly varying rates and may result in phase or chemical changes which complicate the accounting process. This paper presents an approach for inclusion of these effects on the transport of aerosols. The accounting system is very complex and results in a large set of stiff ordinary differential equations (ODEs). The techniques for numerical solution of these ODEs require special attention to achieve their solution in an efficient and affordable manner. 4 refs

  15. Regressão de psoríase em paciente HIV-positivo após terapia anti-retroviral Regression of psoriasis in HIV patient after antiretroviral therapy

    Directory of Open Access Journals (Sweden)

    Maria de Fátima Amorin Ruiz

    2003-12-01

    Full Text Available A síndrome da imunodeficiência adquirida foi reconhecida pela primeira vez como nova doença em 1981 devido à associação atípica de sarcoma de Kaposi e pneumonia por Pneumocystis carinii em homens. A pele é sede freqüente de doenças conseqüentes a essa infecção. A psoríase é dermatose crônica que afeta proporção que varia de 1,3 a 5% dos pacientes infectados com HIV. Portadores de psoríase que apresentem formas clínicas exacerbadas e dificuldade de resposta terapêutica devem ser investigados para possível infecção pelo HIV. É relatado caso de paciente do sexo masculino, de 44 anos, que iniciou com lesões eritêmato-escamosas no couro cabeludo, nos cotovelos, joelhos, palma das mãos, planta dos pés, além de comprometimento ungueal, após infecção pelo HIV. Confirmado o diagnóstico de psoríase e introduzida a terapia anti-retroviral, houve melhora significativa das lesões.Immunodeficiency syndrome was first described as a new disease in 1981 because an unusual association of Kaposi's Sarcoma and Pneumocystis carinii pneumonia in men. The skin is a frequent site of diseases due to this infection. Psoriasis is a chronic dermatitis that affects 1.3-5% of HIV-positive patients. The case is described of a 44-year-old man with onset of erythematous scaly lesions in scalp, elbows, knees, hands, feet and nails following HIV infection. After diagnosis of psoriasis was confirmed and antiretroviral therapy instigated, he presented improvement of the psoriasis lesions.

  16. Including gauge corrections to thermal leptogenesis

    International Nuclear Information System (INIS)

    Huetig, Janine

    2013-01-01

    This thesis provides the first approach of a systematic inclusion of gauge corrections to leading order to the ansatz of thermal leptogenesis. We have derived a complete expression for the integrated lepton number matrix including all resummations needed. For this purpose, a new class of diagram has been invented, namely the cylindrical diagram, which allows diverse investigations into the topic of leptogenesis such as the case of resonant leptogenesis. After a brief introduction of the topic of the baryon asymmetry in the universe and a discussion of its most promising solutions as well as their advantages and disadvantages, we have presented our framework of thermal leptogenesis. An effective model was described as well as the associated Feynman rules. The basis for using nonequilibrium quantum field theory has been built in chapter 3. At first, the main definitions have been presented for equilibrium thermal field theory, afterwards we have discussed the Kadanoff-Baym equations for systems out of equilibrium using the example of the Majorana neutrino. The equations have also been solved in the context of leptogenesis in chapter 4. Since gauge corrections play a crucial role throughout this thesis, we have also repeated the naive ansatz by replacing the free equilibrium propagator by propagators including thermal damping rates due to the Standard Model damping widths for lepton and Higgs fields. It is shown that this leads to a comparable result to the solutions of the Boltzmann equations for thermal leptogenesis. Thus it becomes obvious that Standard Model corrections are not negligible for thermal leptogenesis and therefore need to be included systematically from first principles. In order to achieve this we have started discussing the calculation of ladder rung diagrams for Majorana neutrinos using the HTL and the CTL approach in chapter 5. All gauge corrections are included in this framework and thus it has become the basis for the following considerations

  17. Myeloid-derived suppressor cells in murine AIDS inhibit B-cell responses in part via soluble mediators including reactive oxygen and nitrogen species, and TGF-β.

    Science.gov (United States)

    Rastad, Jessica L; Green, William R

    2016-12-01

    Monocytic myeloid-derived suppressor cells (M-MDSCs) were increased during LP-BM5 retroviral infection, and were capable of suppressing not only T-cell, but also B-cell responses. In addition to previously demonstrating iNOS- and VISTA-dependent M-MDSC mechanisms, in this paper, we detail how M-MDSCs utilized soluble mediators, including the reactive oxygen and nitrogen species superoxide, peroxynitrite, and nitric oxide, and TGF-β, to suppress B cells in a predominantly contact-independent manner. Suppression was independent of cysteine-depletion and hydrogen peroxide production. When two major mechanisms of suppression (iNOS and VISTA) were eliminated in double knockout mice, M-MDSCs from LP-BM5-infected mice were able to compensate using other, soluble mechanisms in order to maintain suppression of B cells. The IL-10 producing regulatory B-cell compartment was among the targets of M-MDSC-mediated suppression. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Grand unified models including extra Z bosons

    International Nuclear Information System (INIS)

    Li Tiezhong

    1989-01-01

    The grand unified theories (GUT) of the simple Lie groups including extra Z bosons are discussed. Under authors's hypothesis there are only SU 5+m SO 6+4n and E 6 groups. The general discussion of SU 5+m is given, then the SU 6 and SU 7 are considered. In SU 6 the 15+6 * +6 * fermion representations are used, which are not same as others in fermion content, Yukawa coupling and broken scales. A conception of clans of particles, which are not families, is suggested. These clans consist of extra Z bosons and the corresponding fermions of the scale. The all of fermions in the clans are down quarks except for the standard model which consists of Z bosons and 15 fermions, therefore, the spectrum of the hadrons which are composed of these down quarks are different from hadrons at present

  19. CLIC expands to include the Southern Hemisphere

    CERN Multimedia

    Roberto Cantoni

    2010-01-01

    Australia has recently joined the CLIC collaboration: the enlargement will bring new expertise and resources to the project, and is especially welcome in the wake of CERN budget redistributions following the recent adoption of the Medium Term Plan.   The countries involved in CLIC collaboration With the signing of a Memorandum of Understanding on 26 August 2010, the ACAS network (Australian Collaboration for Accelerator Science) became the 40th member of in the multilateral CLIC collaboration making Australia the 22nd country to join the collaboration. “The new MoU was signed by the ACAS network, which includes the Australian Synchrotron and the University of Melbourne”, explains Jean-Pierre Delahaye, CLIC Study Leader. “Thanks to their expertise, the Australian institutes will contribute greatly to the CLIC damping rings and the two-beam test modules." Institutes from any country wishing to join the CLIC collaboration are invited to assume responsibility o...

  20. Musculoskeletal ultrasound including definitions for ultrasonographic pathology

    DEFF Research Database (Denmark)

    Wakefield, RJ; Balint, PV; Szkudlarek, Marcin

    2005-01-01

    Ultrasound (US) has great potential as an outcome in rheumatoid arthritis trials for detecting bone erosions, synovitis, tendon disease, and enthesopathy. It has a number of distinct advantages over magnetic resonance imaging, including good patient tolerability and ability to scan multiple joints...... in a short period of time. However, there are scarce data regarding its validity, reproducibility, and responsiveness to change, making interpretation and comparison of studies difficult. In particular, there are limited data describing standardized scanning methodology and standardized definitions of US...... pathologies. This article presents the first report from the OMERACT ultrasound special interest group, which has compared US against the criteria of the OMERACT filter. Also proposed for the first time are consensus US definitions for common pathological lesions seen in patients with inflammatory arthritis....

  1. Education Program on Fossil Resources Including Coal

    Science.gov (United States)

    Usami, Masahiro

    Fossil fuels including coal play a key role as crucial energies in contributing to economic development in Asia. On the other hand, its limited quantity and the environmental problems causing from its usage have become a serious global issue and a countermeasure to solve such problems is very much demanded. Along with the pursuit of sustainable development, environmentally-friendly use of highly efficient fossil resources should be therefore, accompanied. Kyushu-university‧s sophisticated research through long years of accumulated experience on the fossil resources and environmental sectors together with the advanced large-scale commercial and empirical equipments will enable us to foster cooperative research and provide internship program for the future researchers. Then, this program is executed as a consignment business from the Ministry of Economy, Trade and Industry from 2007 fiscal year to 2009 fiscal year. The lecture that uses the textbooks developed by this program is scheduled to be started a course in fiscal year 2010.

  2. Should Broca's area include Brodmann area 47?

    Science.gov (United States)

    Ardila, Alfredo; Bernal, Byron; Rosselli, Monica

    2017-02-01

    Understanding brain organization of speech production has been a principal goal of neuroscience. Historically, brain speech production has been associated with so-called Broca’s area (Brodmann area –BA- 44 and 45), however, modern neuroimaging developments suggest speech production is associated with networks rather than with areas. The purpose of this paper was to analyze the connectivity of BA47 ( pars orbitalis) in relation to language . A meta-analysis was conducted to assess the language network in which BA47 is involved. The Brainmap database was used. Twenty papers corresponding to 29 experimental conditions with a total of 373 subjects were included. Our results suggest that BA47 participates in a “frontal language production system” (or extended Broca’s system). The BA47  connectivity found is also concordant with a minor role in language semantics. BA47 plays a central role in the language production system.

  3. Pulmonary disorders, including vocal cord dysfunction.

    Science.gov (United States)

    Greenberger, Paul A; Grammer, Leslie C

    2010-02-01

    The lung is a very complex immunologic organ and responds in a variety of ways to inhaled antigens, organic or inorganic materials, infectious or saprophytic agents, fumes, and irritants. There might be airways obstruction, restriction, neither, or both accompanied by inflammatory destruction of the pulmonary interstitium, alveoli, or bronchioles. This review focuses on diseases organized by their predominant immunologic responses, either innate or acquired. Pulmonary innate immune conditions include transfusion-related acute lung injury, World Trade Center cough, and acute respiratory distress syndrome. Adaptive immunity responses involve the systemic and mucosal immune systems, activated lymphocytes, cytokines, and antibodies that produce CD4(+) T(H)1 phenotypes, such as for tuberculosis or acute forms of hypersensitivity pneumonitis, and CD4(+) T(H)2 phenotypes, such as for asthma, Churg-Strauss syndrome, and allergic bronchopulmonary aspergillosis. Copyright 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  4. CERN Technical Training: LABVIEW courses include RADE

    CERN Multimedia

    HR Department

    2009-01-01

    The contents of the "LabView Basic I" and "LabView Intermediate II" courses have recently been changed to include, respectively, an introduction to and expert training in the Rapid Application Development Environment (RADE). RADE is a LabView-based application developed at CERN to integrate LabView in the accelerator and experiment control infrastructure. It is a suitable solution to developing expert tools, machine development analysis and independent test facilities. The course names have also been changed to "LabVIEW Basics I with RADE Introduction" and "LabVIEW Intermediate II with Advanced RADE Application". " LabVIEW Basics I with RADE Introduction" is designed for: Users preparing to develop applications using LabVIEW, or NI Developer Suite; users and technical managers evaluating LabVIEW or NI Developer Suite in purchasing decisions; users pursuing the Certified LabVIEW Developer certification. The course pr...

  5. CERN Technical Training: LABVIEW courses include RADE

    CERN Multimedia

    HR Department

    2009-01-01

    The contents of "LabView Basic I" and "LabView Intermediate II" trainings have been recently changed to include, respectively, an introduction and an expert training on the Rapid Application Development Environment (RADE). RADE is a LabView-based application developed at CERN to integrate LabView in the accelerator and experiment control infrastructure. It is a suitable solution to develop expert tools, machine development analysis and independent test facilities. The course names have also been changed to "LabVIEW Basics I with RADE Introduction" and "LabVIEW Intermediate II with Advanced RADE Application". " LabVIEW Basics I with RADE Introduction" is designed for: Users preparing to develop applications using LabVIEW, or NI Developer Suite; users and technical managers evaluating LabVIEW or NI Developer Suite in purchasing decisions; users pursuing the Certified LabVIEW Developer certification. The course prepare...

  6. CERN Technical Training: LABVIEW courses include RADE

    CERN Multimedia

    HR Department

    2009-01-01

    The contents of the "LabView Basic I" and "LabView Intermediate II" courses have recently been changed to include, respectively, an introduction to and expert training in the Rapid Application Development Environment (RADE). RADE is a LabView-based application developed at CERN to integrate LabView in the accelerator and experiment control infrastructure. It is a suitable solution to developing expert tools, machine development analysis and independent test facilities. The course names have also been changed to "LabVIEW Basics I with RADE Introduction" and "LabVIEW Intermediate II with Advanced RADE Application". " LabVIEW Basics I with RADE Introduction" is designed for: Users preparing to develop applications using LabVIEW, or NI Developer Suite; users and technical managers evaluating LabVIEW or NI Developer Suite in purchasing decisions; users pursuing the Certified LabVIEW Developer certification. The course prepares participants to develop test and measurement, da...

  7. AMS at the ANU including biomedical applications

    International Nuclear Information System (INIS)

    Fifield, L.K.; Allan, G.L.; Cresswell, R.G.; Ophel, T.R.; King, S.J.; Day, J.P.

    1993-01-01

    An extensive accelerator mass spectrometry program has been conducted on the 14UD accelerator at the Australian National University since 1986. In the two years since the previous conference, the research program has expanded significantly to include biomedical applications of 26 Al and studies of landform evolution using isotopes produced in situ in surface rocks by cosmic ray bombardment. The system is now used for the measurement of 10 Be, 14 C, 26 Al, 36 Cl, 59 Ni and 129 I, and research is being undertaken in hydrology, environmental geochemistry, archaeology and biomedicine. On the technical side, a new test system has permitted the successful off-line development of a high-intensity ion source. A new injection line to the 14UD has been established and the new source is now in position and providing beams to the accelerator. 4 refs

  8. AMS at the ANU including biomedical applications

    Energy Technology Data Exchange (ETDEWEB)

    Fifield, L.K.; Allan, G.L.; Cresswell, R.G.; Ophel, T.R. [Australian National Univ., Canberra, ACT (Australia); King, S.J.; Day, J.P. [Manchester Univ. (United Kingdom). Dept. of Chemistry

    1993-12-31

    An extensive accelerator mass spectrometry program has been conducted on the 14UD accelerator at the Australian National University since 1986. In the two years since the previous conference, the research program has expanded significantly to include biomedical applications of {sup 26}Al and studies of landform evolution using isotopes produced in situ in surface rocks by cosmic ray bombardment. The system is now used for the measurement of {sup 10}Be, {sup 14}C, {sup 26}Al, {sup 36}Cl, {sup 59}Ni and {sup 129}I, and research is being undertaken in hydrology, environmental geochemistry, archaeology and biomedicine. On the technical side, a new test system has permitted the successful off-line development of a high-intensity ion source. A new injection line to the 14UD has been established and the new source is now in position and providing beams to the accelerator. 4 refs.

  9. A micromanipulation cell including a tool changer

    Science.gov (United States)

    Clévy, Cédric; Hubert, Arnaud; Agnus, Joël; Chaillet, Nicolas

    2005-10-01

    This paper deals with the design, fabrication and characterization of a tool changer for micromanipulation cells. This tool changer is part of a manipulation cell including a three linear axes robot and a piezoelectric microgripper. All these parts are designed to perform micromanipulation tasks in confined spaces such as a microfactory or in the chamber of a scanning electron microscope (SEM). The tool changer principle is to fix a pair of tools (i.e. the gripper tips) either on the tips of the microgripper actuator (piezoceramic bulk) or on a tool magazine. The temperature control of a thermal glue enables one to fix or release this pair of tools. Liquefaction and solidification are generated by surface mounted device (SMD) resistances fixed on the surface of the actuator or magazine. Based on this principle, the tool changer can be adapted to other kinds of micromanipulation cells. Hundreds of automatic tool exchanges were performed with a maximum positioning error between two consecutive tool exchanges of 3.2 µm, 2.3 µm and 2.8 µm on the X, Y and Z axes respectively (Z refers to the vertical axis). Finally, temperature measurements achieved under atmospheric pressure and in a vacuum environment and pressure measurements confirm the possibility of using this device in the air as well as in a SEM.

  10. Robust Unit Commitment Including Frequency Stability Constraints

    Directory of Open Access Journals (Sweden)

    Felipe Pérez-Illanes

    2016-11-01

    Full Text Available An increased use of variable generation technologies such as wind power and photovoltaic generation can have important effects on system frequency performance during normal operation as well as contingencies. The main reasons are the operational principles and inherent characteristics of these power plants like operation at maximum power point and no inertial response during power system imbalances. This has led to new challenges for Transmission System Operators in terms of ensuring system security during contingencies. In this context, this paper proposes a Robust Unit Commitment including a set of additional frequency stability constraints. To do this, a simplified dynamic model of the initial system frequency response is used in combination with historical frequency nadir data during contingencies. The proposed approach is especially suitable for power systems with cost-based economic dispatch like those in most Latin American countries. The study is done considering the Northern Interconnected System of Chile, a 50-Hz medium size isolated power system. The results obtained were validated by means of dynamic simulations of different system contingencies.

  11. Unifying all elementary particle forces including gravity

    International Nuclear Information System (INIS)

    Terazawa, H.

    1979-01-01

    It is a final goal in physics to unify all four basic forces, strong, weak, electromagnetic and gravitational. First, the unified gauge theories of strong, weak and electromagnetic interactions are discussed. There are two standard models, the model of Pati and Salam in which leptons have the fourth color, and the model of Georgi and Glashow in which a simple group SU (5) is assumed for grand unification. Two mass relations for leptons and quarks were derived, and the extension of the Georgi-Glashow model to a grand unified model of SU (6) gauge group has been made. The quantization of the electric charge of elementary particles is one of the most satisfactory features in grand unified gauge theories. The constraint relations between the gauge couplings, the weak mixing angle and the mass scale of symmetry breaking owing to the renormalization effect are not so severe as those in the grand unified models. However, the mass scale becomes far above the Planck mass in some cases. The baryon number non-conservation is one of the most intriguing features common to grand unified gauge theories. The unified models of all elementary particle forces including gravity are discussed. The discovery of weak vector bosons and the production of subquark pairs are anticipated. (Kako, I.)

  12. SEEPAGE MODEL FOR PA INCLUDING DRIFT COLLAPSE

    International Nuclear Information System (INIS)

    C. Tsang

    2004-01-01

    The purpose of this report is to document the predictions and analyses performed using the seepage model for performance assessment (SMPA) for both the Topopah Spring middle nonlithophysal (Tptpmn) and lower lithophysal (Tptpll) lithostratigraphic units at Yucca Mountain, Nevada. Look-up tables of seepage flow rates into a drift (and their uncertainty) are generated by performing numerical simulations with the seepage model for many combinations of the three most important seepage-relevant parameters: the fracture permeability, the capillary-strength parameter 1/a, and the percolation flux. The percolation flux values chosen take into account flow focusing effects, which are evaluated based on a flow-focusing model. Moreover, multiple realizations of the underlying stochastic permeability field are conducted. Selected sensitivity studies are performed, including the effects of an alternative drift geometry representing a partially collapsed drift from an independent drift-degradation analysis (BSC 2004 [DIRS 166107]). The intended purpose of the seepage model is to provide results of drift-scale seepage rates under a series of parameters and scenarios in support of the Total System Performance Assessment for License Application (TSPA-LA). The SMPA is intended for the evaluation of drift-scale seepage rates under the full range of parameter values for three parameters found to be key (fracture permeability, the van Genuchten 1/a parameter, and percolation flux) and drift degradation shape scenarios in support of the TSPA-LA during the period of compliance for postclosure performance [Technical Work Plan for: Performance Assessment Unsaturated Zone (BSC 2002 [DIRS 160819], Section I-4-2-1)]. The flow-focusing model in the Topopah Spring welded (TSw) unit is intended to provide an estimate of flow focusing factors (FFFs) that (1) bridge the gap between the mountain-scale and drift-scale models, and (2) account for variability in local percolation flux due to

  13. [Contracts including performance and management of uncertainty].

    Science.gov (United States)

    Duru, G; Garassus, P; Auray, J-P

    2013-09-01

    Since many decades in France, the most important part of ambulatory health care expenditure is represented by drug consumption. By the fact, French patient is indeed the greatest world consumer of pharmaceuticals treatments. Therefore, the regulation authorities by successive strategies, attempt to limit or even restrict market access for new drugs in the health care sector secured by public social insurance coverage. Common objectives are to assess the reimbursement to scientific studies and to fix the price of therapeutics at an acceptable level for both industries and government. New trends try then to determine recently the drug price in a dual approach, as a component of global and effective contract, including performance and outcome. The first diffusion authorization is diffusion concerned, but this concept takes into account the eventual success of new produces in long-term survey. Signed for a fixed period as reciprocal partnership between regulation authorities and pharmaceutics industries, the contract integrates two dimensions of incertitude. The first one is represented by the strategy of new treatments development according to efficacy and adapted price, and the second one is linked to the result of diffusion and determines adapted rules if eventual non-respects of the previous engagement are registered. This paper discusses problems related to this new dimension of incertitude affected by conditional drug prices in market access strategy and the adapted follow-up of new treatment diffusion fixed by "outcome" contract between French regulation administration and pharmaceutics industries in our recent economic context. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  14. Resection of thymoma should include nodal sampling.

    Science.gov (United States)

    Weksler, Benny; Pennathur, Arjun; Sullivan, Jennifer L; Nason, Katie S

    2015-03-01

    Thymoma is best treated by surgical resection; however, no clear guidelines have been created regarding lymph node sampling at the time of resection. Additionally, the prognostic implications of nodal metastases are unclear. The aim of this study was to analyze the prognostic implications of nodal metastases in thymoma. The Surveillance, Epidemiology, and End Results database was queried for patients who underwent surgical resection of thymoma with documented pathologic examination of lymph nodes. The impact of nodal status on survival and thymoma staging was examined. We identified 442 patients who underwent thymoma resection with pathologic evaluation of 1 or more lymph nodes. A median of 2 nodes were sampled per patient. Fifty-nine patients (59 of 442, 13.3%) had ≥ 1 positive node. Patients with positive nodes were younger and had smaller tumors than node-negative patients. Median survival in the node-positive patients was 98 months, compared with 144 months in node-negative patients (P = .013). In multivariable analysis, the presence of positive nodes had a significant, independent, adverse impact on survival (hazard ratio 1.945, 95% confidence interval 1.296-2.919, P = .001). The presence of nodal metastases resulted in a change in classification to a higher stage in 80% of patients, the majority from Masaoka-Koga stage III to stage IV. Nodal status seems to be an important prognostic factor in patients with thymoma. Until the prognostic significance of nodal metastases is better understood, surgical therapy for thymoma should include sampling of regional lymph nodes. Copyright © 2015 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

  15. Suplementação de N-acetilcisteína em pacientes infectados pelo HIV submetidos ao primeiro tratamento anti-retroviral: Avaliação do efeito sobre a carga viral, TNF-α, IL-6, IL-8, β2-microglobulina, IgA, IgG e IgM, haptoglobina e α1-glicoproteína ácida N-acetylcysteine supplementation of HIV-infected patients under the first anti-retroviral treatment: Evaluation of the effect on viral load, TNF-α, IL-6, IL-8, β2-microglobulin, IgA, IgG, IgM, haptoglobin and α1-acid glycoprotein

    Directory of Open Access Journals (Sweden)

    Aricio Treitinger

    2002-03-01

    Full Text Available Indivíduos infectados pelo vírus da imunodeficiência humana (HIV- 1 apresentam aumento progressivo da carga viral, da destruição do sistema de defesa imune celular e alterações imunológicas e inflamatórias, incluindo a elevação dos níveis séricos do fator de necrose tumoral alfa (TNF-α, interleucina 8 (IL-8, β2- microglobulina, IgA, IgG e IgM, haptoglobina e α1-glicoproteína ácida.O objetivo deste estudo foi avaliar os níveis séricos destes marcadores em indivíduos submetidos ao primeiro tratamento antiretroviral, suplementados ou não com N-acetilcisteína. Participaram deste estudo, duplo cego controlado por placebo, que teve a duração de 180 dias, 24 indivíduos que iniciaram a terapia antiretroviral O Grupo Estudo foi constituído por 11 indivíduos, que receberam suplementação de 600 mg/dia de Nacetilcisteína enquanto o Grupo Controle foi constituído por 13 indivíduo que receberam placebo. Os níveis dos marcadores avaliados foram determinados no dia anterior ao início do tratamento a que foram submetidos e após 60, 120 e 180 dias. Verificou-se diminuição significativa dos níveis de TNF-α (p=0,0001, IL-6 (p>0,05, IL-8 (p=0,0001, β2-microglobulina (p=0,0005, IgA (p=0,007, IgG (p=0,001, IgM (p=0,0001, haptoglobina (p=0,0001 e α1-glicoproteína ácida (p=0.012 em decorrência do tratamento anti-retroviral. A suplementação com N-acetilcisteína, na dose utilizada neste estudo, não teve efeitos aditivos ou sinérgicos sobre as variáveis analisadas. Em conclusão, a suplementação de pacientes HIV-positivos com 600 mg/dia de N-acetilcisteína não proporcionou benefícios adicionais àqueles decorrentes do tratamento anti-retroviral.Human immunodeficiency virus infection is associated with a progressive elevation of viral load and with a continuous destruction of the immune cellular defense system which is marked by immunological and inflammatory disorders characteristic of HIV-infected individuals. These

  16. An Integrated Biochemistry Laboratory, Including Molecular Modeling

    Science.gov (United States)

    Hall, Adele J. Wolfson Mona L.; Branham, Thomas R.

    1996-11-01

    The dilemma of designing an advanced undergraduate laboratory lies in the desire to teach and reinforce basic principles and techniques while at the same time exposing students to the excitement of research. We report here on a one-semester, project-based biochemistry laboratory that combines the best features of a cookbook approach (high success rate, achievement of defined goals) with those of an investigative, discovery-based approach (student involvement in the experimental design, excitement of real research). Individual modules may be selected and combined to meet the needs of different courses and different institutions. The central theme of this lab is protein purification and design. This laboratory accompanies the first semester of biochemistry (Structure and Function of Macromolecules, a course taken mainly by junior and senior chemistry and biological chemistry majors). The protein chosen as the object of study is the enzyme lysozyme, which is utilized in all projects. It is suitable for a student lab because it is easily and inexpensively obtained from egg white and is extremely stable, and its high isoelectric point (pI = 11) allows for efficient separation from other proteins by ion-exchange chromatography. Furthermore, a literature search conducted by the resourceful student reveals a wealth of information, since lysozyme has been the subject of numerous studies. It was the first enzyme whose structure was determined by crystallography (1). Hendrickson et al. (2) have previously described an intensive one-month laboratory course centered around lysozyme, although their emphasis is on protein stability rather than purification and engineering. Lysozyme continues to be the focus of much exciting new work on protein folding and dynamics, structure and activity (3 - 5). This lab course includes the following features: (i) reinforcement of basic techniques, such as preparation of buffers, simple enzyme kinetics, and absorption spectroscopy; (ii

  17. Improving adherence to ante-retroviral treatment for people with harmful alcohol use in Kariobangi, Kenya through participatory research and action

    Directory of Open Access Journals (Sweden)

    Othieno Caleb J

    2012-08-01

    Full Text Available Abstract Background Harmful alcohol use has been linked to the spread of HIV in Kenya. It also adversely affects those on antiretroviral (ARV treatment through poor compliance. This study using participatory research and action (PRA methods sought to understand factors related to alcohol abuse and non-adherence and to formulate appropriate interventions in a sample of people living with HIV and AIDS (PLWHA who were also abusing alcohol, at Kariobangi in Nairobi, Kenya. Methods Entry into the community was gained through previous PRA work in that community and PLWHA were recruited through snowballing. Working together with the community members, the researchers explored the participants’ understanding of alcohol use problem, its effects on compliance to ARV treatment and discussed possible action areas through PRA techniques that included focus group and market place discussions; visual aids such as spider diagrams, community mapping and ranking. Follow-up meetings were held to discuss the progress. Results By the final meeting, 67 PLWHA and 19 community members had been recruited. Through discussions, misconceptions regarding alcohol use were identified. It emerged that alcohol abuse was poorly recognised among both the community and health workers. Screening for alcohol use was not routinely done and protocols for managing alcohol related disorders were not available at the local health centres providing ARVs. The study participants identified improving communication, psychoeducation and screening for alcohol use as possible action areas. Poverty was identified as a major problem but the interventions to mitigate this were not easy to implement. Conclusion We propose that PRA could be useful in improving communication between the health workers and the clients attending primary health care (PHC facilities and can be applied to strengthen involvement of support groups and community health workers in follow up and counselling. Integrating these

  18. Determinantes da adesão ao tratamento anti-retroviral em Brasília, DF: um estudo de caso-controle Determinants of antiretroviral treatment adherence in Brasília, Federal District: a case-control study

    Directory of Open Access Journals (Sweden)

    Cláudio Viveiros de Carvalho

    2007-10-01

    Full Text Available Buscou-se avaliar os fatores relacionados à adesão ao tratamento anti-retroviral no Distrito Federal. De 150 pacientes entrevistados em sete centros de referência, 35 não aderentes foram definidos como casos, sendo selecionados 70 controles aderentes, pareados por idade. Avaliaram-se variáveis sócio-demográficas, hábitos, suporte social, qualidade de vida, questões relacionadas a doença, estado clínico, tratamento e serviço. Na análise bivariada, houve associação da adesão com raça/cor, escolaridade, centros de referência em que faz acompanhamento e renda familiar. Após ajuste, cor parda, centros de referência localizado no Plano Piloto, escolaridade alta e receber apoio dos amigos quanto às necessidades permaneceram associados com adesão. Retirando raça/cor do modelo, mantiveram-se centros de referência, escolaridade, profissão, renda, apoio (contar com alguém que demonstre gostar de você e satisfação com o atendimento na farmácia de dispensação. Além dos fatores já consolidados na literatura, questões relacionadas ao apoio no âmbito micro-social e aos serviços de assistência mostraram-se associados à observância terapêutica.We sought to evaluate factors relating to adherence to antiretroviral treatment in the Federal District. Out of 150 patients interviewed at seven reference centers; 35 non-adherent subjects were considered to be cases; we selected 70 age-matched adherent individuals as controls. Variables relating to sociodemographics, habits, social support, quality of life, disease-related issues, clinical conditions, treatment and healthcare services were evaluated. Bivariate analysis showed that adherence was associated with race/color, educational level, the reference center where the patient was followed up and familial income. After adjustments, mixed race (parda, reference center location in central Brasília, high educational level and friends’ support for needs remained associated with

  19. Lesões anorretais em pacientes HIV positivos usuários de terapia anti-retroviral de alta efetividade Anorrectal lesions in HIV-positive patients using highly active antiretroviral therapy

    Directory of Open Access Journals (Sweden)

    Manuel Retamozo-Palacios

    2007-06-01

    Full Text Available As lesões anorretais são comuns nos pacientes positivos para o vírus da imunodeficiência humana. A terapia antirretroviral de alta efetividade tem pouca influência na progressão das neoplasias anais. Estudou-se a prevalência das lesões anorretais em 88 pacientes HIV positivos atendidos no serviço de doenças infecto-parasitárias do Hospital Universitário de Brasília, em uso de terapia antirretroviral de alta efetividade. Dados sócio-demográficos foram coletados usando um questionário pré-elaborado e os pacientes foram submetidos a exame proctológico. Cerca de 71% relataram coito anal e 30,7% estavam em uso de inibidor de protease. A prevalência das lesões anorretais foi 36,4%, sendo as mais freqüentes: condiloma acuminado e fissura anal. O condiloma acuminado foi a lesão anorretal mais prevalente e teve associação com o uso de lopinavir/ritonavir. Sugere-se o rastreamento das lesões anorretais causadas pelo papilomavírus humano nos pacientes HIV positivos/AIDS em uso de inibidor de protease.Anorectal lesions are common in patients with human immunodeficiency virus (HIV. Highly active anti-retroviral therapy (HAART has little influence on the progression of anal neoplasms. The prevalence of anorectal lesions in 88 HIV-positive patients attended at the infectious diseases service of the University Hospital of Brasília who were using HAART was studied. Sociodemographic data were collected using a pre-prepared questionnaire and then the patients underwent proctological examination. Around 71% of the patients said they practiced anal intercourse. 30.7% were using a protease inhibitor. The prevalence of anorectal lesions was 36.4%, and condyloma acuminata and anal fissure were the most frequent of these. Condyloma acuminata was the most prevalent anorectal lesion and was strongly associated with the use of lopinavir/ritonavir. Screening for anorectal lesions caused by human papillomavirus in HIV/AIDS patients who use protease

  20. Longitudinal comparison between plasma and seminal HIV-1 viral loads during antiretroviral treatment Comparação longitudinal entre cargas virais seminais e plasmáticas do HIV-1 durante terapia anti-retroviral

    Directory of Open Access Journals (Sweden)

    Lauro Ferreira da Silva Pinto Neto

    2003-12-01

    Full Text Available This study was designed to investigate the impact of anti-retroviral therapy on both plasma and seminal HIV-1 viral loads and the correlation between viral loads in these compartments after treatment. Viral load, CD4+ and CD8+ T-cell counts were evaluated in paired plasma and semen samples from 36 antiretroviral therapy-naïve patients at baseline and on days 45, 90, and 180 of treatment. Slopes for blood and seminal viral loads in all treated patients were similar (p = 0.21. Median HIV-1 RNA titers in plasma and semen at baseline were 4.95 log10 and 4.48 log10 copies/ml, respectively. After 180 days of therapy, the median viral load declined to 3.15 log10 copies/ml (plasma and 3.2 log10 copies/ml (semen. At this timepoint 22 patients presented HIV-1 viral load below 400 copies/ml in either plasma or semen, but only 9 had viral loads below 400 copies/ml in both compartments.Este estudo foi desenhado para investigar o impacto do tratamento com anti-retrovirais na evolução das cargas virais plasmáticas e seminais do HIV-1. A carga viral do HIV-1 e a contagem de linfócitos T CD4+ e CD8+ foi determinada em amostras pareadas de sangue e sêmen de 36 pacientes virgem de tratamento nos dias 0, 45, 90 e 180 após o início da terapia. As curvas de declínio das cargas virais plasmática e seminal foram semelhantes (p= 0.21. As medianas da carga viral plasmática e seminal no pré-tratamento (dia 0 foram 4.95 e 4.48 log10 cópias/ml, respectivamente. Seis meses após o início da terapia, a mediana da carga viral plasmática era 3.15 log10 cópias/ml e a seminal 3.2 log10 cópias/ml. Neste mesmo periodo, 22 pacientes apresentavam carga viral abaixo de 400 cópias/ml no plasma e/ou sêmen, enquanto apenas 9 pacientes apresentavam carga viral abaixo do limite de detecção nos dois compartimentos.

  1. Humanized mice recapitulate key features of HIV-1 infection: a novel concept using long-acting anti-retroviral drugs for treating HIV-1.

    Directory of Open Access Journals (Sweden)

    Marc Nischang

    Full Text Available BACKGROUND: Humanized mice generate a lymphoid system of human origin subsequent to transplantation of human CD34+ cells and thus are highly susceptible to HIV infection. Here we examined the efficacy of antiretroviral treatment (ART when added to food pellets, and of long-acting (LA antiretroviral compounds, either as monotherapy or in combination. These studies shall be inspiring for establishing a gold standard of ART, which is easy to administer and well supported by the mice, and for subsequent studies such as latency. Furthermore, they should disclose whether viral breakthrough and emergence of resistance occurs similar as in HIV-infected patients when ART is insufficient. METHODS/PRINCIPAL FINDINGS: NOD/shi-scid/γ(cnull (NOG mice were used in all experimentations. We first performed pharmacokinetic studies of the drugs used, either added to food pellets (AZT, TDF, 3TC, RTV or in a LA formulation that permitted once weekly subcutaneous administration (TMC278: non-nucleoside reverse transcriptase inhibitor, TMC181: protease inhibitor. A combination of 3TC, TDF and TMC278-LA or 3TC, TDF, TMC278-LA and TMC181-LA suppressed the viral load to undetectable levels in 15/19 (79% and 14/14 (100% mice, respectively. In successfully treated mice, subsequent monotherapy with TMC278-LA resulted in viral breakthrough; in contrast, the two LA compounds together prevented viral breakthrough. Resistance mutations matched the mutations most commonly observed in HIV patients failing therapy. Importantly, viral rebound after interruption of ART, presence of HIV DNA in successfully treated mice and in vitro reactivation of early HIV transcripts point to an existing latent HIV reservoir. CONCLUSIONS/SIGNIFICANCE: This report is a unique description of multiple aspects of HIV infection in humanized mice that comprised efficacy testing of various treatment regimens, including LA compounds, resistance mutation analysis as well as viral rebound after treatment

  2. Ionic liquids, electrolyte solutions including the ionic liquids, and energy storage devices including the ionic liquids

    Science.gov (United States)

    Gering, Kevin L.; Harrup, Mason K.; Rollins, Harry W.

    2015-12-08

    An ionic liquid including a phosphazene compound that has a plurality of phosphorus-nitrogen units and at least one pendant group bonded to each phosphorus atom of the plurality of phosphorus-nitrogen units. One pendant group of the at least one pendant group comprises a positively charged pendant group. Additional embodiments of ionic liquids are disclosed, as are electrolyte solutions and energy storage devices including the embodiments of the ionic liquid.

  3. Molecular Mechanisms of HIV Immune Evasion of the Innate Immune Response in Myeloid Cells

    Directory of Open Access Journals (Sweden)

    Mike Mashiba

    2012-12-01

    Full Text Available The expression of intrinsic antiviral factors by myeloid cells is a recently recognized mechanism of restricting lentiviral replication. Viruses that enter these cells must develop strategies to evade cellular antiviral factors to establish a productive infection. By studying the cellular targets of virally encoded proteins that are necessary to infect myeloid cells, a better understanding of cellular intrinsic antiviral strategies has now been achieved. Recent findings have provided insight into how the lentiviral accessory proteins, Vpx, Vpr and Vif counteract antiviral factors found in myeloid cells including SAMHD1, APOBEC3G, APOBEC3A, UNG2 and uracil. Here we review our current understanding of the molecular basis of how cellular antiviral factors function and the viral countermeasures that antagonize them to promote viral transmission and spread.

  4. Molecular mechanisms of HIV immune evasion of the innate immune response in myeloid cells.

    Science.gov (United States)

    Mashiba, Mike; Collins, Kathleen L

    2012-12-21

    The expression of intrinsic antiviral factors by myeloid cells is a recently recognized mechanism of restricting lentiviral replication. Viruses that enter these cells must develop strategies to evade cellular antiviral factors to establish a productive infection. By studying the cellular targets of virally encoded proteins that are necessary to infect myeloid cells, a better understanding of cellular intrinsic antiviral strategies has now been achieved. Recent findings have provided insight into how the lentiviral accessory proteins, Vpx, Vpr and Vif counteract antiviral factors found in myeloid cells including SAMHD1, APOBEC3G, APOBEC3A, UNG2 and uracil. Here we review our current understanding of the molecular basis of how cellular antiviral factors function and the viral countermeasures that antagonize them to promote viral transmission and spread.

  5. Alternative tRNA priming of human immunodeficiency virus type 1 reverse transcription explains sequence variation in the primer-binding site that has been attributed to APOBEC3G activity

    NARCIS (Netherlands)

    Das, Atze T.; Vink, Monique; Berkhout, Ben

    2005-01-01

    It is generally assumed that human immunodeficiency virus type 1 (HIV-1) uses exclusively the cellular tRNA(3)(Lys) molecule as a primer for reverse transcription. We demonstrate that HIV-1 uses not only tRNA(3)(Lys) but also an alternative tRNA primer. This tRNA was termed tRNA(5)(Lys), and the

  6. Catalyst support structure, catalyst including the structure, reactor including a catalyst, and methods of forming same

    Science.gov (United States)

    Van Norman, Staci A.; Aston, Victoria J.; Weimer, Alan W.

    2017-05-09

    Structures, catalysts, and reactors suitable for use for a variety of applications, including gas-to-liquid and coal-to-liquid processes and methods of forming the structures, catalysts, and reactors are disclosed. The catalyst material can be deposited onto an inner wall of a microtubular reactor and/or onto porous tungsten support structures using atomic layer deposition techniques.

  7. Launch Lock Assemblies Including Axial Gap Amplification Devices and Spacecraft Isolation Systems Including the Same

    Science.gov (United States)

    Barber, Tim Daniel (Inventor); Hindle, Timothy (Inventor); Young, Ken (Inventor); Davis, Torey (Inventor)

    2014-01-01

    Embodiments of a launch lock assembly are provided, as are embodiments of a spacecraft isolation system including one or more launch lock assemblies. In one embodiment, the launch lock assembly includes first and second mount pieces, a releasable clamp device, and an axial gap amplification device. The releasable clamp device normally maintains the first and second mount pieces in clamped engagement; and, when actuated, releases the first and second mount pieces from clamped engagement to allow relative axial motion there between. The axial gap amplification device normally residing in a blocking position wherein the gap amplification device obstructs relative axial motion between the first and second mount pieces. The axial gap amplification device moves into a non-blocking position when the first and second mount pieces are released from clamped engagement to increase the range of axial motion between the first and second mount pieces.

  8. Genetic Diversity of Koala Retroviral Envelopes

    Directory of Open Access Journals (Sweden)

    Wenqin Xu

    2015-03-01

    Full Text Available Genetic diversity, attributable to the low fidelity of reverse transcription, recombination and mutation, is an important feature of infectious retroviruses. Under selective pressure, such as that imposed by superinfection interference, gammaretroviruses commonly adapt their envelope proteins to use alternative receptors to overcome this entry block. The first characterized koala retroviruses KoRV subgroup A (KoRV-A were remarkable in their absence of envelope genetic variability. Once it was determined that KoRV-A was present in all koalas in US zoos, regardless of their disease status, we sought to isolate a KoRV variant whose presence correlated with neoplastic malignancies. More than a decade after the identification of KoRV-A, we isolated a second subgroup of KoRV, KoRV-B from koalas with lymphomas. The envelope proteins of KoRV-A and KoRV-B are sufficiently divergent to confer the ability to bind and employ distinct receptors for infection. We have now obtained a number of additional KoRV envelope variants. In the present studies we report these variants, and show that they differ from KoRV-A and KoRV-B envelopes in their host range and superinfection interference properties. Thus, there appears to be considerable variation among KoRVs envelope genes suggesting genetic diversity is a factor following the KoRV-A infection process.

  9. Endogenous retroviral promoter exaptation in human cancer

    Directory of Open Access Journals (Sweden)

    Artem Babaian

    2016-12-01

    Full Text Available Abstract Cancer arises from a series of genetic and epigenetic changes, which result in abnormal expression or mutational activation of oncogenes, as well as suppression/inactivation of tumor suppressor genes. Aberrant expression of coding genes or long non-coding RNAs (lncRNAs with oncogenic properties can be caused by translocations, gene amplifications, point mutations or other less characterized mechanisms. One such mechanism is the inappropriate usage of normally dormant, tissue-restricted or cryptic enhancers or promoters that serve to drive oncogenic gene expression. Dispersed across the human genome, endogenous retroviruses (ERVs provide an enormous reservoir of autonomous gene regulatory modules, some of which have been co-opted by the host during evolution to play important roles in normal regulation of genes and gene networks. This review focuses on the “dark side” of such ERV regulatory capacity. Specifically, we discuss a growing number of examples of normally dormant or epigenetically repressed ERVs that have been harnessed to drive oncogenes in human cancer, a process we term onco-exaptation, and we propose potential mechanisms that may underlie this phenomenon.

  10. Construction of retroviral recombinant containing human tissue ...

    African Journals Online (AJOL)

    USER

    2010-03-29

    Mar 29, 2010 ... was performed in triplicates. About 2 µl of 3-(4,5-Dimethylthiazol-. 2-yl)-2,5- diphenyltetrazolium bromide, a tetrazole (MTT) was added into each well, followed by incubation for 4 h. Approximately 1 ml of supernatant was removed and 0.1 ml of lysis buffer (20% SDS,. 5%DMSDD; pH 7.4) was supplemented ...

  11. Cleavage of vimentin by different retroviral proteases

    Czech Academy of Sciences Publication Activity Database

    Snášel, Jan; Shoeman, R.; Hořejší, Magdalena; Hrušková-Heidingsfeldová, Olga; Sedláček, Juraj; Ruml, Tomáš; Pichová, Iva

    2000-01-01

    Roč. 377, č. 2 (2000), s. 241-245 ISSN 0003-9861 R&D Projects: GA ČR GA203/97/0416; GA AV ČR IAA4055904 Institutional research plan: CEZ:AV0Z4055905 Subject RIV: CE - Biochemistry Impact factor: 2.576, year: 2000

  12. Genetic diversity of koala retroviral envelopes.

    Science.gov (United States)

    Xu, Wenqin; Gorman, Kristen; Santiago, Jan Clement; Kluska, Kristen; Eiden, Maribeth V

    2015-03-17

    Genetic diversity, attributable to the low fidelity of reverse transcription, recombination and mutation, is an important feature of infectious retroviruses. Under selective pressure, such as that imposed by superinfection interference, gammaretroviruses commonly adapt their envelope proteins to use alternative receptors to overcome this entry block. The first characterized koala retroviruses KoRV subgroup A (KoRV-A) were remarkable in their absence of envelope genetic variability. Once it was determined that KoRV-A was present in all koalas in US zoos, regardless of their disease status, we sought to isolate a KoRV variant whose presence correlated with neoplastic malignancies. More than a decade after the identification of KoRV-A, we isolated a second subgroup of KoRV, KoRV-B from koalas with lymphomas. The envelope proteins of KoRV-A and KoRV-B are sufficiently divergent to confer the ability to bind and employ distinct receptors for infection. We have now obtained a number of additional KoRV envelope variants. In the present studies we report these variants, and show that they differ from KoRV-A and KoRV-B envelopes in their host range and superinfection interference properties. Thus, there appears to be considerable variation among KoRVs envelope genes suggesting genetic diversity is a factor following the KoRV-A infection process.

  13. Electrolyte solutions including a phosphoranimine compound, and energy storage devices including same

    Science.gov (United States)

    Klaehn, John R.; Dufek, Eric J.; Rollins, Harry W.; Harrup, Mason K.; Gering, Kevin L.

    2017-09-12

    An electrolyte solution comprising at least one phosphoranimine compound and a metal salt. The at least one phosphoranimine compound comprises a compound of the chemical structure ##STR00001## where X is an organosilyl group or a tert-butyl group and each of R.sup.1, R.sup.2, and R.sup.3 is independently selected from the group consisting of an alkyl group, an aryl group, an alkoxy group, or an aryloxy group. An energy storage device including the electrolyte solution is also disclosed.

  14. Adesão à terapia antiretroviral para HIV/AIDS Adhesión a la terapia anti-retroviral para el vih/sida Adherence to the antiretroviral therapy for HIV/AIDS

    Directory of Open Access Journals (Sweden)

    Maria Rosa Ceccato Colombrini

    2006-12-01

    Full Text Available A não-adesão à terapêutica antiretroviral altamente eficaz (HAART é considerada, no plano individual, como um dos mais ameaçadores perigos para a efetividade do tratamento da pessoa com HIV/aids e para a disseminação de vírus-resistência, no plano coletivo. Assim, o objetivo deste estudo foi analisar, mediante revisão de literatura, os fatores de risco para não-adesão à HAART, além de agrupá-los e relacioná-los à pessoa em tratamento, à doença, ao tratamento e ao serviço de saúde e suporte social. A literatura aponta para a necessidade da realização de estudos que avaliem aspectos socioculturais, crenças, qualidade do serviço prestado, relações do cliente com a equipe multiprofissional e outros referentes à raça e aos efeitos colaterais dos anti-retrovirais. Estes estudos visam a favorecer o estabelecimento de estratégias que melhorem a adesão dos clientes à HAART, ao mesmo tempo em e que contribuem para a construção e exercício da cidadania.La no adhesión a la terapéutica anti-retroviral altamente eficaz (HAART es considerada, en el plano individual, como uno de los más amenazadores peligros para la efectividad del tratamiento de la persona con VIH/SIDA y para la diseminación del virus-resistencia, en el plano colectivo. Así, el objetivo de este estudio fue analizar, mediante revisión de la literatura, los factores de riesgo para la no adhesión a la HAART, además de agruparlos y relacionarlos a la persona en tratamiento, a la enfermedad, al tratamiento y al servicio de salud y soporte social. La literatura apunta hacia la necesidad de realizar estudios que evalúen aspectos socioculturales, creencias, calidad del servicio prestado, relaciones del cliente con el equipo multi-profesional y otros referentes a la raza y a los efectos colaterales de los anti-retrovirales. Estos estudios visan a favorecer el establecimiento de estrategias que mejoren la adhesión de los clientes a la HAART, al mismo tiempo

  15. Enteropatógenos relacionados à diarréia em pacientes HIV que fazem uso de terapia anti-retroviral Enteropathogens relating to diarrhea in HIV patients on antiretroviral therapy

    Directory of Open Access Journals (Sweden)

    Áurea Regina Telles Pupulin

    2009-10-01

    Full Text Available A etiologia do processo diarréico na AIDS pode ser causada por vírus, bactérias, fungos, protozoários e helmintos, assim como pelo próprio HIV. Este trabalho avaliou enteropatogenos relacionados à diarréia em pacientes HIV que fazem uso de terapia anti-retroviral. Os métodos parasitológicos utilizados foram Faust, Hoffmann e Kinyoun. O isolamento e cultura dos fungos foram realizados conforme metodologia recomendada por NCCLS M27-A standard. A identificação das espécies de leveduras foi realizada através da reação em cadeia da polimerase. O isolamento de bactérias, foi feito em agar Mac Conkey e agar SS, a identificação das espécies através do Enterokit B (Probac do Brasil e métodos bioquímicos. Foram avaliados 49 pacientes, 44,9% apresentaram enteroparasitas, 48,1% Candida sp com 61,5% Candida albicans, 7,6% Candida sp e 30,7% Candida não- albicans. Foram isoladas bactérias de 72% dos pacientes, 49% Escherichia coli, 13% Salmonella parathyphi, Klebsiella sp ou Proteus e 6% Citrobacter freundii ou Yersinia sp. Houve alta prevalência de Candida sp nos pacientes HIV com diarréia e foram isoladas espécies não albicans cuja presença pode ser entendida como cúmplice ou causa da infecção.The etiology of the diarrheic process in AIDS may be caused by viruses, bacteria, fungi, protozoa or helminths, as well as HIV itself. This study evaluated enteropathogens relating to diarrhea in HIV patients who were on antiretroviral therapy. The parasitological methods used were Faust, Hoffmann and Kinyoun. Isolation and culturing of fungi were carried out in accordance with the methodology recommended by the NCCLS M27-A standard. The yeast species were identified using the polymerase chain reaction (PCR. Bacteria were isolated on MacConkey and SS agar and the species were identified using Enterokit B (Probac do Brasil and biochemical methods. Forty-nine patients were evaluated: 44.89% presented enteroparasites and 48.1% presented

  16. Uso da terapia anti-retroviral entre pacientes atendidos em dois serviços públicos de referência para o HIV/aids: impacto na qualidade de vida e ocorrência de sintomas de ansiedade e depressão

    OpenAIRE

    Lorenza Nogueira Campos

    2008-01-01

    Introdução: A introdução da terapia anti-retroviral combinada (TARV) mudou a percepção do HIV/aids de uma doença fatal para crônica e potencialmente controlável. A TARV é capaz de aumentar a sobrevida, reduzir a morbidade e mortalidade e melhorar a qualidade de vida das pessoas vivendo com o HIV/aids. Sintomas psiquiátricos, como ansiedade e depressão, são comuns entre pessoas com doenças crônicas, incluindo a infecção pelo HIV/aids. Eles têm sido apontados como importantes preditores da n...

  17. Prevalence study and risk factor analysis of selected bacterial, protozoal and viral, including vector-borne, pathogens in cats from Cyprus.

    Science.gov (United States)

    Attipa, Charalampos; Papasouliotis, Kostas; Solano-Gallego, Laia; Baneth, Gad; Nachum-Biala, Yaarit; Sarvani, Elpida; Knowles, Toby G; Mengi, Sena; Morris, David; Helps, Chris; Tasker, Séverine

    2017-03-13

    Feline infectious agent studies are lacking in Cyprus. The aims of this study were to determine the prevalence and risk factors for various feline infectious agents, including feline vector-borne pathogens (FVBP), in cats from Cyprus. A cross-sectional, descriptive, multicentre study was performed on 174 feline samples [138 owned and 36 shelter-feral, including both healthy (43) and non-healthy (131), cats] from private veterinary clinics from all six districts of Cyprus. Real-time quantitative polymerase chain reaction (qPCR) assays were used to detect Mycoplasma haemofelis (Mhf), "Candidatus Mycoplasma haemominutum" (CMhm) and "Candidatus Mycoplasma turicensis" (CMt). The population was tested for four FVBP including Bartonella henselae and Leishmania spp. using qPCR, while conventional PCR assays were used to detect Ehrlichia/Anaplasma spp. and Hepatozoon spp. Serological assays were performed to detect Leishmania infantum antibodies, feline leukaemia virus (FeLV) antigen and feline immunodeficiency virus (FIV) antibodies. Statistical analysis was performed to test associations and possible risk factors between variables and infectious agents. Ninety-six (55.2%) of the 174 cats were PCR-positive for at least one infectious agent. Forty-six cats (26.4%) were haemoplasma positive, including 13 (7.5%) for Mhf, 36 (20.7%) for CMhm and 12 (6.9%) for CMt. Sixty-six cats (37.9%) were positive for Hepatozoon spp., while 19 (10.9%) were positive for B. henselae, four (2.3%) for Leishmania spp. and one (0.6%) for Ehrlichia/Anaplasma spp. Sequencing revealed the presence of Hepatozoon felis, L. infantum and Anaplasma platys. Of the 164 cats that underwent retroviral serology, 10 (6.1%) were FeLV-positive and 31 (18.9%) were FIV-positive, while L. infantum serology was positive in 7 (4.4%) of the 160 cats tested. Multivariable logistic regression revealed significant associations for various infectious agents including L. infantum with each of Hepatozoon spp. and CMt

  18. Haemophilus influenzae Disease (Including Hib) Diagnosis and Treatment

    Science.gov (United States)

    ... Search Form Controls Cancel Submit Search The CDC Haemophilus influenzae Disease (Including Hib) Note: Javascript is disabled or ... Page Diagnosis Treatment Complications Diagnosis Doctors usually diagnose Haemophilus influenzae , including H. influenzae type b or Hib, infection ...

  19. Should Relational Aggression Be Included in DSM-V?

    Science.gov (United States)

    Keenan, Kate; Coyne, Claire; Lahey, Benjamin B.

    2008-01-01

    The study examines whether relational aggression should be included in DSM-V disruptive behavior disorders. The results conclude that some additional information is gathered from assessing relational aggression but not enough to be included in DSM-V.

  20. Partially ionized plasmas including the third symposium on uranium plasmas

    Energy Technology Data Exchange (ETDEWEB)

    Krishnan, M. [ed.

    1976-09-01

    Separate abstracts are included for 28 papers on electrically generated plasmas, fission generated plasmas, nuclear pumped lasers, gaseous fuel reactor research, and applications. Five papers have been previously abstracted and included in ERA.

  1. Dictionary of scientific units including dimensionless numbers and scales

    National Research Council Canada - National Science Library

    Jerrard, H.G; McNeill, D.B

    1992-01-01

    .... The text includes the most recently accepted values of all units. Several disciplines, which have in the past employed few scientific principles and the dictionary has been extended to include examples of these.

  2. 75 FR 16513 - B&C Corporation, JR Engineering Division, Including B&C Distribution Center, Including On-Site...

    Science.gov (United States)

    2010-04-01

    ... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-70, 975A] B&C Corporation, JR Engineering Division, Including B&C Distribution Center, Including On-Site Leased Workers From B&C Services... October 2, 2009, applicable to workers of B&C Corporation, JR Engineering Division, including on-site...

  3. 20 CFR 404.1312 - World War II service included.

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false World War II service included. 404.1312... DISABILITY INSURANCE (1950- ) Wage Credits for Veterans and Members of the Uniformed Services World War II Veterans § 404.1312 World War II service included. Your service was in the active service of the United...

  4. 24 CFR 220.822 - Claim computation; items included.

    Science.gov (United States)

    2010-04-01

    ... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Claim computation; items included. 220.822 Section 220.822 Housing and Urban Development Regulations Relating to Housing and Urban... computation; items included. (a) Assignment of loan. Upon an acceptable assignment of the note and security...

  5. Solar Energy Education. Reader, Part II. Sun story. [Includes glossary

    Energy Technology Data Exchange (ETDEWEB)

    1981-05-01

    Magazine articles which focus on the subject of solar energy are presented. The booklet prepared is the second of a four part series of the Solar Energy Reader. Excerpts from the magazines include the history of solar energy, mythology and tales, and selected poetry on the sun. A glossary of energy related terms is included. (BCS)

  6. 7 CFR 1437.303 - Aquaculture, including ornamental fish.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Aquaculture, including ornamental fish. 1437.303... ASSISTANCE PROGRAM Determining Coverage Using Value § 1437.303 Aquaculture, including ornamental fish. (a) Aquaculture is a value loss crop and is compensable only in accord with restrictions set in this section...

  7. Including Exceptional Students in Your Instrumental Music Program

    Science.gov (United States)

    Mixon, Kevin

    2005-01-01

    This article describes the method and adaptations used by the author in including students with special needs in an instrumental music program. To ensure success in the program, the author shares the method he uses to include exceptional students and enumerates some possible adaptations. There are certainly other methods and modifications that…

  8. 26 CFR 1.1013-1 - Property included in inventory.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 11 2010-04-01 2010-04-01 true Property included in inventory. 1.1013-1 Section... inventory. The basis of property required to be included in inventory is the last inventory value of such property in the hands of the taxpayer. The requirements with respect to the valuation of an inventory are...

  9. A framework for including family health spillovers in economic evaluation

    NARCIS (Netherlands)

    H. Al-Janabi (Hareth); N.J.A. van Exel (Job); W.B.F. Brouwer (Werner); J. Coast (Joanna)

    2016-01-01

    textabstractHealth care interventions may affect the health of patients' family networks. It has been suggested that these health spillovers? should be included in economic evaluation, but there is not a systematic method for doing this. In this article, we develop a framework for including health

  10. 31 CFR 103.51 - Dollars as including foreign currency.

    Science.gov (United States)

    2010-07-01

    ... RECORDKEEPING AND REPORTING OF CURRENCY AND FOREIGN TRANSACTIONS General Provisions § 103.51 Dollars as including foreign currency. Wherever in this part an amount is stated in dollars, it shall be deemed to mean... 31 Money and Finance: Treasury 1 2010-07-01 2010-07-01 false Dollars as including foreign currency...

  11. Electric drive systems including smoothing capacitor cooling devices and systems

    Energy Technology Data Exchange (ETDEWEB)

    Dede, Ercan Mehmet; Zhou, Feng

    2017-02-28

    An electric drive system includes a smoothing capacitor including at least one terminal, a bus bar electrically coupled to the at least one terminal, a thermoelectric device including a first side and a second side positioned opposite the first side, where the first side is thermally coupled to at least one of the at least one terminal and the bus bar, and a cooling element thermally coupled to the second side of the thermoelectric device, where the cooling element dissipates heat from the thermoelectric device.

  12. Initiation devices, initiation systems including initiation devices and related methods

    Energy Technology Data Exchange (ETDEWEB)

    Daniels, Michael A.; Condit, Reston A.; Rasmussen, Nikki; Wallace, Ronald S.

    2018-04-10

    Initiation devices may include at least one substrate, an initiation element positioned on a first side of the at least one substrate, and a spark gap electrically coupled to the initiation element and positioned on a second side of the at least one substrate. Initiation devices may include a plurality of substrates where at least one substrate of the plurality of substrates is electrically connected to at least one adjacent substrate of the plurality of substrates with at least one via extending through the at least one substrate. Initiation systems may include such initiation devices. Methods of igniting energetic materials include passing a current through a spark gap formed on at least one substrate of the initiation device, passing the current through at least one via formed through the at least one substrate, and passing the current through an explosive bridge wire of the initiation device.

  13. Stages of Plasma Cell Neoplasms (Including Multiple Myeloma)

    Science.gov (United States)

    ... Treatment Health Professional Plasma Cell Neoplasms Treatment Research Plasma Cell Neoplasms (Including Multiple Myeloma) Treatment (PDQ®)–Patient Version General Information About Plasma Cell Neoplasms Go to Health Professional Version Key ...

  14. Including estimates of the future in today's financial statements

    OpenAIRE

    Mary Barth

    2006-01-01

    This paper explains why the question is how, not if, today's financial statements should include estimates of the future. Including such estimates is not new, but their use is increasing. This increase results primarily because standard setters believe asset and liability measures that reflect current economic conditions and up-to-date expectations of the future will result in more useful information for making economic decisions, which is the objective of financial reporting. This is why sta...

  15. Internet addiction neuroscientific approaches and therapeutical implications including smartphone addiction

    CERN Document Server

    Reuter, Martin

    2017-01-01

    The second edition of this successful book provides further and in-depth insight into theoretical models dealing with Internet addiction, as well as includes new therapeutical approaches. The editors also broach the emerging topic of smartphone addiction. This book combines a scholarly introduction with state-of-the-art research in the characterization of Internet addiction. It is intended for a broad audience including scientists, students and practitioners. The first part of the book contains an introduction to Internet addiction and their pathogenesis. The second part of the book is dedicated to an in-depth review of neuroscientific findings which cover studies using a variety of biological techniques including brain imaging and molecular genetics. The third part of the book focuses on therapeutic interventions for Internet addiction. The fourth part of the present book is an extension to the first edition and deals with a new emerging potential disorder related to Internet addiction – smartphone addicti...

  16. Ceramic substrate including thin film multilayer surface conductor

    Energy Technology Data Exchange (ETDEWEB)

    Wolf, Joseph Ambrose; Peterson, Kenneth A.

    2017-05-09

    A ceramic substrate comprises a plurality of ceramic sheets, a plurality of inner conductive layers, a plurality of vias, and an upper conductive layer. The ceramic sheets are stacked one on top of another and include a top ceramic sheet. The inner conductive layers include electrically conductive material that forms electrically conductive features on an upper surface of each ceramic sheet excluding the top ceramic sheet. The vias are formed in each of the ceramic sheets with each via being filled with electrically conductive material. The upper conductive layer includes electrically conductive material that forms electrically conductive features on an upper surface of the top ceramic sheet. The upper conductive layer is constructed from a stack of four sublayers. A first sublayer is formed from titanium. A second sublayer is formed from copper. A third sublayer is formed from platinum. A fourth sublayer is formed from gold.

  17. Including information technology project management in the nursing informatics curriculum.

    Science.gov (United States)

    Sockolow, Paulina; Bowles, Kathryn H

    2008-01-01

    Project management is a critical skill for nurse informaticists who are in prominent roles developing and implementing clinical information systems. It should be included in the nursing informatics curriculum, as evidenced by its inclusion in informatics competencies and surveys of important skills for informaticists. The University of Pennsylvania School of Nursing includes project management in two of the four courses in the master's level informatics minor. Course content includes the phases of the project management process; the iterative unified process methodology; and related systems analysis and project management skills. During the introductory course, students learn about the project plan, requirements development, project feasibility, and executive summary documents. In the capstone course, students apply the system development life cycle and project management skills during precepted informatics projects. During this in situ experience, students learn, the preceptors benefit, and the institution better prepares its students for the real world.

  18. Classical mechanics including an introduction to the theory of elasticity

    CERN Document Server

    Hentschke, Reinhard

    2017-01-01

    This textbook teaches classical mechanics as one of the foundations of physics. It describes the mechanical stability and motion in physical systems ranging from the molecular to the galactic scale. Aside from the standard topics of mechanics in the physics curriculum, this book includes an introduction to the theory of elasticity and its use in selected modern engineering applications, e.g. dynamic mechanical analysis of viscoelastic materials. The text also covers many aspects of numerical mechanics, ranging from the solution of ordinary differential equations, including molecular dynamics simulation of many particle systems, to the finite element method. Attendant Mathematica programs or parts thereof are provided in conjunction with selected examples. Numerous links allow the reader to connect to related subjects and research topics. Among others this includes statistical mechanics (separate chapter), quantum mechanics, space flight, galactic dynamics, friction, and vibration spectroscopy. An introductory...

  19. Dynamical criteria for a unified gauge theory (including gravity)

    International Nuclear Information System (INIS)

    1979-01-01

    It is shown that local gauge invariance together with dynamical (and possibly Higgs) symmetry breaking can be taken as a basis for a unified gauge theory including gravity. The criterion for the breakdown of the linear gauge symmetry of the space-time sector turns out to be the absence of a prior geometry. The usual postulates of general relativity, such as general coordinate invariance, etc., follow from the above two criteria. Gravity actions that are natural from the point of view are discussed. The extension to superspace relevant to gauge supergroups is also given, including a large number of linear and quadratic superspace invariants

  20. Solar Energy Education. Renewable energy: a background text. [Includes glossary

    Energy Technology Data Exchange (ETDEWEB)

    1985-01-01

    Some of the most common forms of renewable energy are presented in this textbook for students. The topics include solar energy, wind power hydroelectric power, biomass ocean thermal energy, and tidal and geothermal energy. The main emphasis of the text is on the sun and the solar energy that it yields. Discussions on the sun's composition and the relationship between the earth, sun and atmosphere are provided. Insolation, active and passive solar systems, and solar collectors are the subtopics included under solar energy. (BCS)

  1. Meta-structure and tunable optical device including the same

    Energy Technology Data Exchange (ETDEWEB)

    Han, Seunghoon; Papadakis, Georgia Theano; Atwater, Harry

    2017-12-26

    A meta-structure and a tunable optical device including the same are provided. The meta-structure includes a plurality of metal layers spaced apart from one another, an active layer spaced apart from the plurality of metal layers and having a carrier concentration that is tuned according to an electric signal applied to the active layer and the plurality of metal layers, and a plurality of dielectric layers spaced apart from one another and each having one surface contacting a metal layer among the plurality of metal layers and another surface contacting the active layer.

  2. Methods of producing adsorption media including a metal oxide

    Science.gov (United States)

    Mann, Nicholas R; Tranter, Troy J

    2014-03-04

    Methods of producing a metal oxide are disclosed. The method comprises dissolving a metal salt in a reaction solvent to form a metal salt/reaction solvent solution. The metal salt is converted to a metal oxide and a caustic solution is added to the metal oxide/reaction solvent solution to adjust the pH of the metal oxide/reaction solvent solution to less than approximately 7.0. The metal oxide is precipitated and recovered. A method of producing adsorption media including the metal oxide is also disclosed, as is a precursor of an active component including particles of a metal oxide.

  3. Tunable cavity resonator including a plurality of MEMS beams

    Science.gov (United States)

    Peroulis, Dimitrios; Fruehling, Adam; Small, Joshua Azariah; Liu, Xiaoguang; Irshad, Wasim; Arif, Muhammad Shoaib

    2015-10-20

    A tunable cavity resonator includes a substrate, a cap structure, and a tuning assembly. The cap structure extends from the substrate, and at least one of the substrate and the cap structure defines a resonator cavity. The tuning assembly is positioned at least partially within the resonator cavity. The tuning assembly includes a plurality of fixed-fixed MEMS beams configured for controllable movement relative to the substrate between an activated position and a deactivated position in order to tune a resonant frequency of the tunable cavity resonator.

  4. Appraisal Of Quality Of Life Of Diabetic Patients, Including Life ...

    African Journals Online (AJOL)

    Appraisal Of Quality Of Life Of Diabetic Patients, Including Life Expectancy. ... of long-term complications, development of short-term complications, and physical symptoms and lifestyle changes resulting from the demands of the diabetic ... Key words: Type 2 Diabetes, quality of life, life expectancy, diabetic complications.

  5. Assessing the Doctoral Thesis When It Includes Published Work

    Science.gov (United States)

    Sharmini, Sharon; Spronken-Smith, Rachel; Golding, Clinton; Harland, Tony

    2015-01-01

    In this article we explore how examiners assess a thesis that includes published work. An online survey was used to gather data on approaches to assessing publication-based theses (PBTs). The respondents were 62 supervisors who had experience examining PBTs across a range of disciplines at a research-intensive university in New Zealand. Nearly…

  6. 25 CFR 20.308 - What does earned income include?

    Science.gov (United States)

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false What does earned income include? 20.308 Section 20.308 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR HUMAN SERVICES FINANCIAL ASSISTANCE AND..., the sale of farm crops, livestock, or professional artists producing art work); and (b) With regard to...

  7. 34 CFR 661.20 - What must an application include?

    Science.gov (United States)

    2010-07-01

    ... 34 Education 3 2010-07-01 2010-07-01 false What must an application include? 661.20 Section 661.20 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF POSTSECONDARY EDUCATION, DEPARTMENT OF EDUCATION BUSINESS AND INTERNATIONAL EDUCATION PROGRAM How Does One Apply for a...

  8. Simple suggestions for including vertical physics in oil spill models

    International Nuclear Information System (INIS)

    D'Asaro, Eric; University of Washington, Seatle, WA

    2001-01-01

    Current models of oil spills include no vertical physics. They neglect the effect of vertical water motions on the transport and concentration of floating oil. Some simple ways to introduce vertical physics are suggested here. The major suggestion is to routinely measure the density stratification of the upper ocean during oil spills in order to develop a database on the effect of stratification. (Author)

  9. Truck Drivers And Risk Of STDs Including HIV

    Directory of Open Access Journals (Sweden)

    Bansal R.K

    1995-01-01

    Full Text Available Research Question: Whether long distance truck drivers are at a higher risk of contracting and transmitting STDs including HIV? Objectives: i To study the degree of knowledge of HIV and AIDS among long- distance truck drivers. ii Assess their sexual behaviour including condom use. iii Explore their prevailing social influences and substance abuse patterns. iv Explore their treatment seeking bahaviour as regards STDs. v Deduce their risk of contracting and transmitting STDs including HIV. Study Design: Cross- sectional interview. Setting: Transport Nagar, Indore (M.P Participants: 210 senior drivers (First drivers and 210 junior drivers (Second drivers. Study Variables: Extra-Marital sexual intercourse, condom usage, past and present history of STDs, treatment and counseling, substance abuse, social â€" cultural milieu. Outcome Variables: Risk of contraction of STDs. Statistical Analysis: Univariate analysis. Results: 94% of the drivers were totally ignorant about AIDS. 82.9% and 43.8 % of the senior and junior drivers had a history of extra- marital sex and of these only 2 regularly used condoms. 13.8% and 3.3 % of the senior and junior drivers had a past or present history suggestive of STD infection. Alcohol and Opium were regularly used by them. Conclusion: The studied drivers are at a high risk of contracting and transmitting STDs including HIV.

  10. Towards a general framework for including noise impacts in LCA

    NARCIS (Netherlands)

    Cucurachi, Stefano; Heijungs, Reinout; Ohlau, Katrin

    Purpose Several damages have been associated with the exposure of human beings to noise. These include auditory effects, i.e., hearing impairment, but also non-auditory physiological ones such as hypertension and ischemic heart disease, or psychological ones such as annoyance, depression, sleep

  11. 25 CFR 20.403 - What do protective services include?

    Science.gov (United States)

    2010-04-01

    ....403 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR HUMAN SERVICES FINANCIAL ASSISTANCE... services, which may include, but are not limited to, the following: (i) Investigating and reporting on... for assistance in solving the social problems of individuals, families, and children. (5) Coordinating...

  12. Including Critical Thinking and Problem Solving in Physical Education

    Science.gov (United States)

    Pill, Shane; SueSee, Brendan

    2017-01-01

    Many physical education curriculum frameworks include statements about the inclusion of critical inquiry processes and the development of creativity and problem-solving skills. The learning environment created by physical education can encourage or limit the application and development of the learners' cognitive resources for critical and creative…

  13. Including Leap Year in the Canonical Birthday Problem

    Science.gov (United States)

    Nandor, M. J.

    2004-01-01

    The greatest benefit of including leap year in the calculation is not to increase precision, but to show students that a problem can be solved without such presumption. A birthday problem is analyzed showing that calculating a leap-year birthday probability is not a frivolous computation.

  14. Sexually transmitted infections, including HIV, in the Netherlands in 2006

    NARCIS (Netherlands)

    MG van Veen; FDH Koedijk; IVF van der Broek; ELM Op de Coul; IM de Boer; AI van Sighem; MAB van der Sande; soa-centra; Stichting HIV Monitoring; EPI/Cib

    2007-01-01

    The nationally covered low threshold STI centres offering STI care targeted at high risk groups, provide surveillance data to monitor national trends in STI, including HIV. In 2006, chlamydia remained the most commonly diagnosed bacterial STI in the Netherlands in the STI centres, in spite of

  15. Three-humped fission barrier transmission including vibrational damping

    International Nuclear Information System (INIS)

    Calabretta, L.; Del Zoppo, A.; Ingrao, G.

    1978-01-01

    The total penetrability through a three-humped fission barrier including vibrational damping is calculated by using an optical model for fission. The Bondorf's stationary probability current theory is used for transitions among class-1, class-2 and class-3 phases. A method to calculate the partial-transmission coefficients is developed

  16. Information for Teachers (Including Classroom Activities), Skylab Student Project.

    Science.gov (United States)

    National Aeronautics and Space Administration, Washington, DC.

    This program is intended to directly involve the educational community in space experiments, many of which can be related to existing curricula. Included in this first packet are: 1) a brief description of the Skylab Program and the National Science Teachers Association-National Aeronautics and Space Administration (NSTA-NASA) Skylab Student…

  17. Smart Antenna Skins, including Conformal Array, MMICs and Applications

    NARCIS (Netherlands)

    Bogaart, F.L.M. van den

    2000-01-01

    Low-cost technologies are presented for future space-borne and airborne SAR systems. These technologies include state-of-the art highly integrated circuits to miniaturise front-end, solutions to lower-cost interconnection technologies, new beamforming aspects and new architectures. The MMICs address

  18. Including Students with Severe Disabilities in General Education Settings.

    Science.gov (United States)

    Wisniewski, Lech; Alper, Sandra

    1994-01-01

    This paper presents five systematic phases for bringing about successful regular education inclusion of students with severe disabilities. Phases include develop networks within the community, assess school and community resources, review strategies for integration, install strategies that lead to integration, and develop a system of feedback and…

  19. Including patients’ perspectives in patient information leaflets: A polyocular approach

    DEFF Research Database (Denmark)

    Fage-Butler, Antoinette Mary

    2013-01-01

    Existing research reveals that patients’ perspectives are missing from mandatory patient information leaflets (PILs). At the same time, there is overwhelming consensus that they should be included in this genre, and a corresponding need for potential approaches to tackle this problem. This paper ...

  20. Everglades National Park Including Biscayne National Park. Activity Book.

    Science.gov (United States)

    Ruehrwein, Dick

    Intended to help elementary school children learn about the resources of the Everglades and Biscayne National Parks, this activity book includes information, puzzles, games, and quizzes. The booklet deals with concepts related to: (1) the seasons; (2) fire ecology; (3) water; (4) fish; (5) mammals; (6) mosquitos; (7) birds; (8) venomous snakes;…

  1. 10 CFR 905.11 - What must an IRP include?

    Science.gov (United States)

    2010-01-01

    ... DEPARTMENT OF ENERGY ENERGY PLANNING AND MANAGEMENT PROGRAM Integrated Resource Planning § 905.11 What must an IRP include? (a) General. Integrated resource planning is a planning process for new energy... projected durability of such savings measured over time; and must treat demand and supply resources on a...

  2. LTRACK: Beam-transport calculation including wakefield effects

    International Nuclear Information System (INIS)

    Chan, K.C.D.; Cooper, R.K.

    1988-01-01

    LTRACK is a first-order beam-transport code that includes wakefield effects up to quadrupole modes. This paper will introduce the readers to this computer code by describing the history, the method of calculations, and a brief summary of the input/output information. Future plans for the code will also be described

  3. Visual Impairments, "Including Blindness." NICHCY Disability Fact Sheet #13

    Science.gov (United States)

    National Dissemination Center for Children with Disabilities, 2012

    2012-01-01

    Vision is one of the five senses. Being able to see gives tremendous access to learning about the world around--people's faces and the subtleties of expression, what different things look like and how big they are, and the physical environments, including approaching hazards. When a child has a visual impairment, it is cause for immediate…

  4. 34 CFR 429.20 - What must an application include?

    Science.gov (United States)

    2010-07-01

    ... 34 Education 3 2010-07-01 2010-07-01 false What must an application include? 429.20 Section 429.20 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF VOCATIONAL AND ADULT EDUCATION, DEPARTMENT OF EDUCATION BILINGUAL VOCATIONAL MATERIALS, METHODS, AND TECHNIQUES PROGRAM...

  5. Interviewing Objects: Including Educational Technologies as Qualitative Research Participants

    Science.gov (United States)

    Adams, Catherine A.; Thompson, Terrie Lynn

    2011-01-01

    This article argues the importance of including significant technologies-in-use as key qualitative research participants when studying today's digitally enhanced learning environments. We gather a set of eight heuristics to assist qualitative researchers in "interviewing" technologies-in-use (or other relevant objects), drawing on concrete…

  6. Oat have multifunctional uses including animal feed, human food ...

    African Journals Online (AJOL)

    Akademia Rolnicza

    2014-07-11

    Jul 11, 2014 ... Abstract. The objective of the work was to evaluate the influence of genetic and mechanical removal of hulls from oat grains on their nutrient content. The studies included three cultivars and six lines of oat grains. In grain samples of hulled (5 samples), dehulled (5 samples) and naked (4 samples) oats, the ...

  7. Modeling Electric Double-Layers Including Chemical Reaction Effects

    DEFF Research Database (Denmark)

    Paz-Garcia, Juan Manuel; Johannesson, Björn; Ottosen, Lisbeth M.

    2014-01-01

    A physicochemical and numerical model for the transient formation of an electric double-layer between an electrolyte and a chemically-active flat surface is presented, based on a finite elements integration of the nonlinear Nernst-Planck-Poisson model including chemical reactions. The model works...

  8. Including the gifted learner: perceptions of South African teachers ...

    African Journals Online (AJOL)

    Qualitative content analysis revealed the following themes: inclusive education and the learner who is gifted; curriculum differentiation; obstacles to curriculum differentiation; and possible solutions for more effectively including the gifted learner. Despite their diversity in terms of culture, language and positioning by the ...

  9. Payment Services for Global Online Systems Including Internet.

    Science.gov (United States)

    Seebeck, Bill; And Others

    1995-01-01

    A panel of four conference presenters address issues related to paying for services provided through online systems. Discussion includes the following topics: metering devices; electronic/digital cash; working within existing banking/credit card structures; provision of payment mechanisms in countries without extensive credit card usage; and…

  10. Restructuring the Public School Curriculum To Include Parenting Education Classes.

    Science.gov (United States)

    Tyree, Carolyn L.; And Others

    Although the current educational climate stresses a back-to-basics approach, there is nonetheless overwhelming evidence of a need for an appropriately structured parenting education program in the public school curriculum. Reasons for this need include the large number of teenage pregnancies and abortions. These lead teens to miss high school…

  11. Can We Include The Third Dimension During Image Mining?

    Indian Academy of Sciences (India)

    Table of contents. Can We Include The Third Dimension During Image Mining? Retrieve An Image · Content-Based Retrieval · Problem Statement · Slide 5 · CBIR Methodology · Slide 7 · Illustration : Logo Search · Illustration: Arbitrary Query · Limitations · Illustration: Change in View · Slide 12 · Illustration: Depth Variation.

  12. Microfluidic System Simulation Including the Electro-Viscous Effect

    Science.gov (United States)

    Rojas, Eileen; Chen, C. P.; Majumdar, Alok

    2007-01-01

    This paper describes a practical approach using a general purpose lumped-parameter computer program, GFSSP (Generalized Fluid System Simulation Program) for calculating flow distribution in a network of micro-channels including electro-viscous effects due to the existence of electrical double layer (EDL). In this study, an empirical formulation for calculating an effective viscosity of ionic solutions based on dimensional analysis is described to account for surface charge and bulk fluid conductivity, which give rise to electro-viscous effect in microfluidics network. Two dimensional slit micro flow data was used to determine the model coefficients. Geometry effect is then included through a Poiseuille number correlation in GFSSP. The bi-power model was used to calculate flow distribution of isotropically etched straight channel and T-junction microflows involving ionic solutions. Performance of the proposed model is assessed against experimental test data.

  13. Suddenly included: cultural differences in experiencing re-inclusion.

    Science.gov (United States)

    Pfundmair, Michaela; Graupmann, Verena; Du, Hongfei; Frey, Dieter; Aydin, Nilüfer

    2015-03-01

    In the current research, we examined whether re-inclusion (i.e. the change from a previous state of exclusion to a new state of inclusion) was perceived differently by people with individualistic and collectivistic cultural backgrounds. Individualists (German and Austrian participants) but not collectivists (Chinese participants) experienced re-inclusion differently than continued inclusion: While collectivistic participants did not differentiate between both kinds of inclusion, individualistic participants showed reduced fulfilment of their psychological needs under re-inclusion compared to continued inclusion. The results moreover revealed that only participants from individualistic cultures expressed more feelings of exclusion when re-included than when continually included. These exclusionary feelings partially mediated the relationship between the different states of inclusion and basic need fulfilment. © 2014 International Union of Psychological Science.

  14. Progressive IRP Models for Power Resources Including EPP

    Directory of Open Access Journals (Sweden)

    Yiping Zhu

    2017-01-01

    Full Text Available In the view of optimizing regional power supply and demand, the paper makes effective planning scheduling of supply and demand side resources including energy efficiency power plant (EPP, to achieve the target of benefit, cost, and environmental constraints. In order to highlight the characteristics of different supply and demand resources in economic, environmental, and carbon constraints, three planning models with progressive constraints are constructed. Results of three models by the same example show that the best solutions to different models are different. The planning model including EPP has obvious advantages considering pollutant and carbon emission constraints, which confirms the advantages of low cost and emissions of EPP. The construction of progressive IRP models for power resources considering EPP has a certain reference value for guiding the planning and layout of EPP within other power resources and achieving cost and environmental objectives.

  15. How Do We Include Underrepresented Voices in the Sustainability Conversation?

    Directory of Open Access Journals (Sweden)

    Virajita Singh

    2018-02-01

    Full Text Available In a speech given at the Regional Sustainable Development Partnerships 20th Anniversary Statewide Event in the Cargill Building on the St. Paul Campus of the University of Minnesota on November 21, 2017, Virajita Singh, Assistant Vice Provost in the Office for Equity and Diversity, addressed the question, “How do we include underrepresented voices in the sustainability conversation?” The speech describes the work of The Partnerships as observed by the speaker, and its connection to the Design for Community Resilience program. It also introduces the concepts of Partnership and Design Thinking, and suggests a process for including underrepresented voices in the work informed by Design Thinking.  

  16. 3D integrated HYDRA simulations of hohlraums including fill tubes

    Science.gov (United States)

    Marinak, M. M.; Milovich, J.; Hammel, B. A.; Macphee, A. G.; Smalyuk, V. A.; Kerbel, G. D.; Sepke, S.; Patel, M. V.

    2017-10-01

    Measurements of fill tube perturbations from hydro growth radiography (HGR) experiments on the National Ignition Facility show spoke perturbations in the ablator radiating from the base of the tube. These correspond to the shadow of the 10 μm diameter glass fill tube cast by hot spots at early time. We present 3D integrated HYDRA simulations of these experiments which include the fill tube. Meshing techniques are described which were employed to resolve the fill tube structure and associated perturbations in the simulations. We examine the extent to which the specific illumination geometry necessary to accommodate a backlighter in the HGR experiment contributes to the spoke pattern. Simulations presented include high resolution calculations run on the Trinity machine operated by the Alliance for Computing at Extreme Scale (ACES) partnership. This work was performed under the auspices of the Lawrence Livermore National Security, LLC, (LLNS) under Contract No. DE-AC52-07NA27344.

  17. Survey of state legislative programs that include passive solar energy

    Energy Technology Data Exchange (ETDEWEB)

    Weiss, S

    1979-06-01

    This report surveys and evaluates state-level solar-incentive programs, including passive solar energy. The range of programs examined focuses on financial and legal incentives designed to speed the implementation of solar heating, cooling, and hot water systems. They have been evaluated by probing the wording of the incentive legislation and by interviewing state program administrators in each state to determine: (1) the extent, if any, of passive inclusion in solar-incentive programs, and (2) the level of success that various implementation techniques have achieved for encouraging passive solar designs as opposed to the more-commonly-understood active systems. Because no states have initiated incentive legislation designed exclusively to encourage passive solar techniques, it has been essential to determine whether legislative programs explicitly or implicitly include passive solar or if they explicitly exclude it.

  18. Energy storage device including a redox-enhanced electrolyte

    Science.gov (United States)

    Stucky, Galen; Evanko, Brian; Parker, Nicholas; Vonlanthen, David; Auston, David; Boettcher, Shannon; Chun, Sang-Eun; Ji, Xiulei; Wang, Bao; Wang, Xingfeng; Chandrabose, Raghu Subash

    2017-08-08

    An electrical double layer capacitor (EDLC) energy storage device is provided that includes at least two electrodes and a redox-enhanced electrolyte including two redox couples such that there is a different one of the redox couples for each of the electrodes. When charged, the charge is stored in Faradaic reactions with the at least two redox couples in the electrolyte and in a double-layer capacitance of a porous carbon material that comprises at least one of the electrodes, and a self-discharge of the energy storage device is mitigated by at least one of electrostatic attraction, adsorption, physisorption, and chemisorption of a redox couple onto the porous carbon material.

  19. Improving the strength of amalgams by including steel fibers

    Energy Technology Data Exchange (ETDEWEB)

    Cochran, Calvin T. [Hendrix College, Conway, AR 72032 (United States); Van Hoose, James R. [Siemens, Orlando, FL 32826 (United States); McGill, Preston B. [Marshall Space Flight Center, EM20, Huntsville, AL 35812 (United States); Grugel, Richard N., E-mail: richard.n.grugel@nasa.gov [Marshall Space Flight Center, EM30, Huntsville, AL 35812 (United States)

    2012-05-30

    Highlights: Black-Right-Pointing-Pointer A room temperature liquid Ga-In alloy was successfully substituted for mercury. Black-Right-Pointing-Pointer Physically sound amalgams with included steel fibers can be made. Black-Right-Pointing-Pointer A small volume fraction inclusion of fibers increased strength by {approx}20%. - Abstract: Mercury amalgams, due to their material properties, are widely and successfully used in dental practice. They are, however, also well recognized as having poor tensile strength. With the possibility of expanding amalgam applications it is demonstrated that tensile strength can be increased some 20% by including a small amount of steel fibers. Furthermore, it is shown that mercury can be replaced with a room temperature liquid gallium-indium alloy. Processing, microstructures, and mechanical test results of these novel amalgams are presented and discussed in view of means to further improve their properties.

  20. QCD Reggeon field theory for every day: Pomeron loops included

    International Nuclear Information System (INIS)

    Altinoluk, Tolga; Kovner, Alex; Peressutti, Javier; Lublinsky, Michael

    2009-01-01

    We derive the evolution equation for hadronic scattering amplitude at high energy. Our derivation includes the nonlinear effects of finite partonic density in the hadronic wave function as well as the effect of multiple scatterings for scattering on dense hadronic target. It thus includes Pomeron loops. It is based on the evolution of the hadronic wave function derived in /cite{foam}. The kernel of the evolution equation defines the second quantized Hamiltonian of the QCD Reggeon Field Theory, H RFT beyond the limits considered so far. The two previously known limits of the evolution: dilute target (JIMWLK limit) and dilute projectile (KLWMIJ limit) are recovered directly from our final result. The Hamiltonian H RFT is applicable for the evolution of scattering amplitude for arbitrarily dense hadronic projectiles/targets - from 'dipole-dipole' to 'nucleus-nucleus' scattering processes.

  1. Smart Farming: Including Rights Holders for Responsible Agricultural Innovation

    OpenAIRE

    Kelly Bronson

    2018-01-01

    This article draws on the literature of responsible innovation to suggest concrete processes for including rights holders in the “smart” agricultural revolution. It first draws upon historical agricultural research in Canada to highlight how productivist values drove seed innovations with particular consequences for the distribution of power in the food system. Next, the article uses document analysis to suggest that a similar value framework is motivating public investment in smart farming i...

  2. Production, control and utilization of radioisotopes including radiopharmaceuticals

    International Nuclear Information System (INIS)

    Muenze, R.

    1985-05-01

    From April 29th to May 5th, 1984 27 participants from 21 developing countries stayed within an IAEA Study Tour ('Production, Control and Utilization of Radioisotopes including Radiopharmaceuticals') in the GDR. In the CINR, Rossendorf the reactor, the cyclotron, the technological centre as well as the animal test laboratory were visited. The participants were made familiar by 10 papers with the development, production and control of radiopharmaceuticals in the CINR, Rossendorf. (author)

  3. Information to Include in Curriculum Vitae | Cancer Prevention Fellowship Program

    Science.gov (United States)

    Applicants are encouraged to use their current curriculum vitae and to add any necessary information. Please include your name and a page number on each page of the curriculum vitae. Some of the information requested below will not be applicable to all individuals. Please do not print or type your information on this page. Personal Information Name (First middle last) Gender (optional) Race (optional) Date of birth Place of birth (city,

  4. Stochastic modelling of two-phase flows including phase change

    International Nuclear Information System (INIS)

    Hurisse, O.; Minier, J.P.

    2011-01-01

    Stochastic modelling has already been developed and applied for single-phase flows and incompressible two-phase flows. In this article, we propose an extension of this modelling approach to two-phase flows including phase change (e.g. for steam-water flows). Two aspects are emphasised: a stochastic model accounting for phase transition and a modelling constraint which arises from volume conservation. To illustrate the whole approach, some remarks are eventually proposed for two-fluid models. (authors)

  5. How to include farmers in the emission trading system?

    OpenAIRE

    Svendsen, Gert Tinggaard

    2011-01-01

    The EU has committed itself to an ambitious 20% reduction of greenhouse gases (GHG) by 2020 compared to the 1990 emissions level. Moreover, the EU goal beyond 2012 is to strengthen, expand and improve climate change initiatives. Therefore, there is a strong need to consider more carefully how to integrate as many sectors as possible in these efforts. Farmers, however, do not trade GHG under the Kyoto agreement. The idea of including farmers in a national emission trading system has been launc...

  6. GNSS-Based Space Weather Systems Including COSMIC Ionospheric Measurements

    Science.gov (United States)

    Komjathy, Attila; Mandrake, Lukas; Wilson, Brian; Iijima, Byron; Pi, Xiaoqing; Hajj, George; Mannucci, Anthony J.

    2006-01-01

    The presentation outline includes University Corporation for Atmospheric Research (UCAR) and Jet Propulsion Laboratory (JPL) product comparisons, assimilating ground-based global positioning satellites (GPS) and COSMIC into JPL/University of Southern California (USC) Global Assimilative Ionospheric Model (GAIM), and JPL/USC GAIM validation. The discussion of comparisons examines Abel profiles and calibrated TEC. The JPL/USC GAIM validation uses Arecibo ISR, Jason-2 VTEC, and Abel profiles.

  7. A Framework for Including Family Health Spillovers in Economic Evaluation

    Science.gov (United States)

    Al-Janabi, Hareth; van Exel, Job; Brouwer, Werner; Coast, Joanna

    2016-01-01

    Health care interventions may affect the health of patients’ family networks. It has been suggested that these “health spillovers” should be included in economic evaluation, but there is not a systematic method for doing this. In this article, we develop a framework for including health spillovers in economic evaluation. We focus on extra-welfarist economic evaluations where the objective is to maximize health benefits from a health care budget (the “health care perspective”). Our framework involves adapting the conventional cost-effectiveness decision rule to include 2 multiplier effects to internalize the spillover effects. These multiplier effects express the ratio of total health effects (for patients and their family networks) to patient health effects. One multiplier effect is specified for health benefit generated from providing a new intervention, one for health benefit displaced by funding this intervention. We show that using multiplier effects to internalize health spillovers could change the optimal funding decisions and generate additional health benefits to society. PMID:26377370

  8. A Framework for Including Family Health Spillovers in Economic Evaluation.

    Science.gov (United States)

    Al-Janabi, Hareth; van Exel, Job; Brouwer, Werner; Coast, Joanna

    2016-02-01

    Health care interventions may affect the health of patients' family networks. It has been suggested that these "health spillovers" should be included in economic evaluation, but there is not a systematic method for doing this. In this article, we develop a framework for including health spillovers in economic evaluation. We focus on extra-welfarist economic evaluations where the objective is to maximize health benefits from a health care budget (the "health care perspective"). Our framework involves adapting the conventional cost-effectiveness decision rule to include 2 multiplier effects to internalize the spillover effects. These multiplier effects express the ratio of total health effects (for patients and their family networks) to patient health effects. One multiplier effect is specified for health benefit generated from providing a new intervention, one for health benefit displaced by funding this intervention. We show that using multiplier effects to internalize health spillovers could change the optimal funding decisions and generate additional health benefits to society. © The Author(s) 2015.

  9. Modeling heart rate variability including the effect of sleep stages

    Science.gov (United States)

    Soliński, Mateusz; Gierałtowski, Jan; Żebrowski, Jan

    2016-02-01

    We propose a model for heart rate variability (HRV) of a healthy individual during sleep with the assumption that the heart rate variability is predominantly a random process. Autonomic nervous system activity has different properties during different sleep stages, and this affects many physiological systems including the cardiovascular system. Different properties of HRV can be observed during each particular sleep stage. We believe that taking into account the sleep architecture is crucial for modeling the human nighttime HRV. The stochastic model of HRV introduced by Kantelhardt et al. was used as the initial starting point. We studied the statistical properties of sleep in healthy adults, analyzing 30 polysomnographic recordings, which provided realistic information about sleep architecture. Next, we generated synthetic hypnograms and included them in the modeling of nighttime RR interval series. The results of standard HRV linear analysis and of nonlinear analysis (Shannon entropy, Poincaré plots, and multiscale multifractal analysis) show that—in comparison with real data—the HRV signals obtained from our model have very similar properties, in particular including the multifractal characteristics at different time scales. The model described in this paper is discussed in the context of normal sleep. However, its construction is such that it should allow to model heart rate variability in sleep disorders. This possibility is briefly discussed.

  10. Electrolytes including fluorinated solvents for use in electrochemical cells

    Science.gov (United States)

    Tikhonov, Konstantin; Yip, Ka Ki; Lin, Tzu-Yuan

    2015-07-07

    Provided are electrochemical cells and electrolytes used to build such cells. The electrolytes include ion-supplying salts and fluorinated solvents capable of maintaining single phase solutions with the salts at between about -30.degree. C. to about 80.degree. C. The fluorinated solvents, such as fluorinated carbonates, fluorinated esters, and fluorinated esters, are less flammable than their non-fluorinated counterparts and increase safety characteristics of cells containing these solvents. The amount of fluorinated solvents in electrolytes may be between about 30% and 80% by weight not accounting weight of the salts. Fluorinated salts, such as fluoroalkyl-substituted LiPF.sub.6, fluoroalkyl-substituted LiBF.sub.4 salts, linear and cyclic imide salts as well as methide salts including fluorinated alkyl groups, may be used due to their solubility in the fluorinated solvents. In some embodiments, the electrolyte may also include a flame retardant, such as a phosphazene or, more specifically, a cyclic phosphazene and/or one or more ionic liquids.

  11. The COG database: an updated version includes eukaryotes

    Directory of Open Access Journals (Sweden)

    Sverdlov Alexander V

    2003-09-01

    Full Text Available Abstract Background The availability of multiple, essentially complete genome sequences of prokaryotes and eukaryotes spurred both the demand and the opportunity for the construction of an evolutionary classification of genes from these genomes. Such a classification system based on orthologous relationships between genes appears to be a natural framework for comparative genomics and should facilitate both functional annotation of genomes and large-scale evolutionary studies. Results We describe here a major update of the previously developed system for delineation of Clusters of Orthologous Groups of proteins (COGs from the sequenced genomes of prokaryotes and unicellular eukaryotes and the construction of clusters of predicted orthologs for 7 eukaryotic genomes, which we named KOGs after eukaryotic orthologous groups. The COG collection currently consists of 138,458 proteins, which form 4873 COGs and comprise 75% of the 185,505 (predicted proteins encoded in 66 genomes of unicellular organisms. The eukaryotic orthologous groups (KOGs include proteins from 7 eukaryotic genomes: three animals (the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster and Homo sapiens, one plant, Arabidopsis thaliana, two fungi (Saccharomyces cerevisiae and Schizosaccharomyces pombe, and the intracellular microsporidian parasite Encephalitozoon cuniculi. The current KOG set consists of 4852 clusters of orthologs, which include 59,838 proteins, or ~54% of the analyzed eukaryotic 110,655 gene products. Compared to the coverage of the prokaryotic genomes with COGs, a considerably smaller fraction of eukaryotic genes could be included into the KOGs; addition of new eukaryotic genomes is expected to result in substantial increase in the coverage of eukaryotic genomes with KOGs. Examination of the phyletic patterns of KOGs reveals a conserved core represented in all analyzed species and consisting of ~20% of the KOG set. This conserved portion of the

  12. Global anthropogenic emissions of particulate matter including black carbon

    Science.gov (United States)

    Klimont, Zbigniew; Kupiainen, Kaarle; Heyes, Chris; Purohit, Pallav; Cofala, Janusz; Rafaj, Peter; Borken-Kleefeld, Jens; Schöpp, Wolfgang

    2017-07-01

    This paper presents a comprehensive assessment of historical (1990-2010) global anthropogenic particulate matter (PM) emissions including the consistent and harmonized calculation of mass-based size distribution (PM1, PM2. 5, PM10), as well as primary carbonaceous aerosols including black carbon (BC) and organic carbon (OC). The estimates were developed with the integrated assessment model GAINS, where source- and region-specific technology characteristics are explicitly included. This assessment includes a number of previously unaccounted or often misallocated emission sources, i.e. kerosene lamps, gas flaring, diesel generators, refuse burning; some of them were reported in the past for selected regions or in the context of a particular pollutant or sector but not included as part of a total estimate. Spatially, emissions were calculated for 172 source regions (as well as international shipping), presented for 25 global regions, and allocated to 0.5° × 0.5° longitude-latitude grids. No independent estimates of emissions from forest fires and savannah burning are provided and neither windblown dust nor unpaved roads emissions are included. We estimate that global emissions of PM have not changed significantly between 1990 and 2010, showing a strong decoupling from the global increase in energy consumption and, consequently, CO2 emissions, but there are significantly different regional trends, with a particularly strong increase in East Asia and Africa and a strong decline in Europe, North America, and the Pacific region. This in turn resulted in important changes in the spatial pattern of PM burden, e.g. European, North American, and Pacific contributions to global emissions dropped from nearly 30 % in 1990 to well below 15 % in 2010, while Asia's contribution grew from just over 50 % to nearly two-thirds of the global total in 2010. For all PM species considered, Asian sources represented over 60 % of the global anthropogenic total, and residential combustion

  13. Global anthropogenic emissions of particulate matter including black carbon

    Directory of Open Access Journals (Sweden)

    Z. Klimont

    2017-07-01

    Full Text Available This paper presents a comprehensive assessment of historical (1990–2010 global anthropogenic particulate matter (PM emissions including the consistent and harmonized calculation of mass-based size distribution (PM1, PM2. 5, PM10, as well as primary carbonaceous aerosols including black carbon (BC and organic carbon (OC. The estimates were developed with the integrated assessment model GAINS, where source- and region-specific technology characteristics are explicitly included. This assessment includes a number of previously unaccounted or often misallocated emission sources, i.e. kerosene lamps, gas flaring, diesel generators, refuse burning; some of them were reported in the past for selected regions or in the context of a particular pollutant or sector but not included as part of a total estimate. Spatially, emissions were calculated for 172 source regions (as well as international shipping, presented for 25 global regions, and allocated to 0.5°  ×  0.5° longitude–latitude grids. No independent estimates of emissions from forest fires and savannah burning are provided and neither windblown dust nor unpaved roads emissions are included. We estimate that global emissions of PM have not changed significantly between 1990 and 2010, showing a strong decoupling from the global increase in energy consumption and, consequently, CO2 emissions, but there are significantly different regional trends, with a particularly strong increase in East Asia and Africa and a strong decline in Europe, North America, and the Pacific region. This in turn resulted in important changes in the spatial pattern of PM burden, e.g. European, North American, and Pacific contributions to global emissions dropped from nearly 30 % in 1990 to well below 15 % in 2010, while Asia's contribution grew from just over 50 % to nearly two-thirds of the global total in 2010. For all PM species considered, Asian sources represented over 60 % of the global

  14. Fatores de risco para a não adesão ao tratamento com terapia antiretroviral altamente eficaz Factores de riesgo para la no-adherencia al tratamiento con terapia anti-retroviral altamente eficiente Risk factors for non-compliance to treatment with highly effective antiretroviral therapy

    Directory of Open Access Journals (Sweden)

    Maria Rosa Ceccato Colombrini

    2008-09-01

    Full Text Available O estudo objetivou: mensurar a prevalência de não-adesão à terapia anti-retroviral altamente eficaz (HAART em pacientes com AIDS; identificar se alguns fatores relacionados na literatura estavam associados com a não-adesão; estabelecer o valor preditivo dos fatores associados à não-adesão à HAART. Foi realizado um estudo analítico de prevalência (N=60. Foram considerados os três dias anteriores à entrevista e os pacientes classificados como aderentes quando ingeriam 95% ou mais do total de comprimidos prescritos por dia. A adesão foi de 73,3%. A análise de regressão logística multivariada indicou que indivíduos da raça negra apresentaram 6,48 vezes mais risco de não-adesão; aqueles que apresentaram ausência de efeito colateral tiveram um risco 7,6 vezes maior, e a cada comprimido ingerido o risco foi de 1,12. A adesão observada foi maior que a encontrada na literatura. Os fatores sociodemográficos e culturais podem interferir na adesão à HAART.Objetivo del estudio: medir la prevalencia por falta de seguimiento al tratamiento anti-retroviral altamente eficaz (HAART en pacientes con SIDA; identificar si algunos factores relacionados en la bibliografía se encuentran asociados con la falta de seguimiento; establecer el valor preditivo de los factores asociados con la falta de seguimiento al HAART. Para lo cual fue realizado un estudio analitico de prevalencia (N=60, considerándose los tres días anteriores a la entrevista, donde aquellos pacientes con ingesta del 95% o más del total de comprimidos prescritos por día eran clasificados como seguidores del tratamiento. El seguimiento fue de 73,3%. El análisis de regresion logística multi-variable mostró que sujetos de raza negra presentaron 6,48 veces mayor riesgo de no continuar con el tratamiento; aquellos individuos sin efectos colaterales tuvieron un riesgo 7,6 veces mayor, asimismo, cada comprimido ingerido produjo un riesgo de 1,12. El seguimiento observado fue

  15. Lipodystrophy syndrome associated with antiretroviral therapy in HIV patients: considerations for psychosocial aspects Síndrome de la lipodistrofia asociado con la terapia antiretroviral en pacientes con VIH: consideraciones para los aspectos psicosociales Sindrome da lipodistrofia associada com a terapia anti-retroviral em portadores do HIV: considerações para os aspectos psicossociais

    Directory of Open Access Journals (Sweden)

    Ana Paula Morais Fernandes

    2007-10-01

    son necesarios para entender mejor este complejo síndrome, proveyendo nueva información para ser utilizada en el cuidado de enfermería para pacientes con VIH que están afectados por este problema.Diversos efeitos colaterais têm sido associados à terapia anti-retroviral em portadores da infecção pelo HIV, dentre esses, a síndrome da lipodistrofia apresentando hiperlipidemia e alterações na forma do corpo, com hipertrofia adiposa central e lipoatrofia periférica, relatada pelos pacientes como um visível marcador para a identificação de portadores da infecção pelo HIV. Este estudo consiste em análise da produção científica sobre aspectos psicossociais em portadores da infecção pelo HIV que apresentam lipodistrofia associada à utilização da terapia anti-retroviral. Os resultados mostram que alterações corporais podem ser suficientemente perturbadoras para o bem-estar psicossocial, afetando a qualidade de vida e aumentando o estigma da doença, ocasionando perturbações nas relações sociais. Esta revisão possibilita uma análise preliminar dos aspectos psicossocias da lipodistrofia; entretanto, outros estudos são necessários para o melhor entendimento desta complexa síndrome, trazendo novas informações a serem utilizadas no cuidado de enfermagem a portadores da infecção pelo HIV afetados por este problema.

  16. Generalized fluid theory including non-Maxwellian kinetic effects

    Science.gov (United States)

    Izacard, Olivier

    2017-04-01

    The results obtained by the plasma physics community for the validation and the prediction of turbulence and transport in magnetized plasmas come mainly from the use of very central processing unit (CPU)-consuming particle-in-cell or (gyro)kinetic codes which naturally include non-Maxwellian kinetic effects. To date, fluid codes are not considered to be relevant for the description of these kinetic effects. Here, after revisiting the limitations of the current fluid theory developed in the 19th century, we generalize the fluid theory including kinetic effects such as non-Maxwellian super-thermal tails with as few fluid equations as possible. The collisionless and collisional fluid closures from the nonlinear Landau Fokker-Planck collision operator are shown for an arbitrary collisionality. Indeed, the first fluid models associated with two examples of collisionless fluid closures are obtained by assuming an analytic non-Maxwellian distribution function (e.g. the INMDF (Izacard, O. 2016b Kinetic corrections from analytic non-Maxwellian distribution functions in magnetized plasmas. Phys. Plasmas 23, 082504) that stands for interpreted non-Maxwellian distribution function). One of the main differences with the literature is our analytic representation of the distribution function in the velocity phase space with as few hidden variables as possible thanks to the use of non-orthogonal basis sets. These new non-Maxwellian fluid equations could initiate the next generation of fluid codes including kinetic effects and can be expanded to other scientific disciplines such as astrophysics, condensed matter or hydrodynamics. As a validation test, we perform a numerical simulation based on a minimal reduced INMDF fluid model. The result of this test is the discovery of the origin of particle and heat diffusion. The diffusion is due to the competition between a growing INMDF on short time scales due to spatial gradients and the thermalization on longer time scales. The results

  17. Early Course in Obstetrics Increases Likelihood of Practice Including Obstetrics.

    Science.gov (United States)

    Pearson, Jennifer; Westra, Ruth

    2016-10-01

    The Department of Family Medicine and Community Health Duluth has offered the Obstetrical Longitudinal Course (OBLC) as an elective for first-year medical students since 1999. The objective of the OBLC Impact Survey was to assess the effectiveness of the course over the past 15 years. A Qualtrics survey was emailed to participants enrolled in the course from 1999-2014. Data was compiled for the respondent group as a whole as well as four cohorts based on current level of training/practice. Cross-tabulations with Fisher's exact test were applied and odds ratios calculated for factors affecting likelihood of eventual practice including obstetrics. Participation in the OBLC was successful in increasing exposure, awareness, and comfort in caring for obstetrical patients and feeling more prepared for the OB-GYN Clerkship. A total of 50.5% of course participants felt the OBLC influenced their choice of specialty. For participants who are currently physicians, 51% are practicing family medicine with obstetrics or OB-GYN. Of the cohort of family physicians, 65.2% made the decision whether to include obstetrics in practice during medical school. Odds ratios show the likelihood of practicing obstetrics is higher when participants have completed the OBLC and also are practicing in a rural community. Early exposure to obstetrics, as provided by the OBLC, appears to increase the likelihood of including obstetrics in practice, especially if eventual practice is in a rural community. This course may be a tool to help create a pipeline for future rural family physicians providing obstetrical care.

  18. How to include farmers in the emission trading system?

    DEFF Research Database (Denmark)

    Svendsen, Gert Tinggaard

    2011-01-01

    The EU has committed itself to an ambitious 20 % reduction of greenhouse gases (GHG) by 2020 compared to the 1990 emissions level. Moreover, the EU goal beyond 2012 is to strengthen, expand and improve climate change initiatives. Therefore, there is a strong need to consider more carefully how...... to integrate as many sectors as possible in these efforts. Farmers, however, do not trade GHG under the Kyoto agreement. The idea of including farmers in a national emission trading system has been launched in Australia but it has not yet been applied to the EU....

  19. Smart Farming: Including Rights Holders for Responsible Agricultural Innovation

    Directory of Open Access Journals (Sweden)

    Kelly Bronson

    2018-02-01

    Full Text Available This article draws on the literature of responsible innovation to suggest concrete processes for including rights holders in the “smart” agricultural revolution. It first draws upon historical agricultural research in Canada to highlight how productivist values drove seed innovations with particular consequences for the distribution of power in the food system. Next, the article uses document analysis to suggest that a similar value framework is motivating public investment in smart farming innovations. The article is of interest to smart farming’s decision makers (from farmers to governance actors and a broader audience – anyone interested in engendering equity through innovation-led societal transitions.

  20. Luminous variable stars with naked eye: data reduction including extinction

    Science.gov (United States)

    Sigismondi, Costantino

    2014-05-01

    The observation of variable stars from urban contexts is hampered by city lights and field of view. Some bright stars like Betelgeuse and Antares are visible from the majority of cities, and during clear nights can be accurately estimated with the naked eye. The reference stars should be bright stars, not necessarily at the same altitude, including the atmospheric extinction in the data reduction. The software Stellarium 0.12.4 calculates well the standard atmospheric extinction with stars at least 10 degrees above the horizon. The accuracy of visual estimations is better than 0.1 magnitudes.