Injecting drug use predicts active tuberculosis in a national cohort of people living with HIV from 2000 to 2014

OBJECTIVES: Tuberculosis (TB) is common in people living with HIV (PLHIV), leading to worse clinical outcomes including increased mortality. We investigated risk factors for developing TB following HIV diagnosis. DESIGN: Adults aged ≥15 years first presenting to health services for HIV care in England, Wales or Northern Ireland from 2000-2014 were identified from national HIV surveillance data and linked to TB surveillance data. METHODS: We calculated incidence rates for TB occurring >91 days after HIV diagnosis and investigated risk factors using multivariable Poisson regression. RESULTS: 95,003 adults diagnosed with HIV were followed for 635,591 person-years (PY); overall incidence of TB was 344/100,000PY (95% confidence interval 330-359). TB incidence was high for people who acquired HIV through injecting drugs (PWID; men 876 [696-1,104], women 605 [528-593]) and black Africans born in high TB incidence countries (644 [612-677]). The adjusted incidence rate ratio (IRR) for TB amongst PWID was 4.79 [3.35-6.85] for men and 6.18 [3.49-10.93] for women, compared to men who have sex with men. The adjusted IRR for TB in black Africans from high-TB countries was 4 27 (3 42-5 33), compared to white UK-born individuals. Lower time-updated CD4 count was associated with increased rates of TB. CONCLUSIONS: PWID had the greatest risk of TB; incidence rates were comparable to those in black Africans from high TB incidence countries. Most TB cases in PWID were UK-born, and likely acquired TB through transmission within the UK. Earlier HIV diagnosis and quicker initiation of ART should reduce TB incidence in these populations

Antiretroviral Therapy Outcomes in HIV-Infected Children after Adjusting Protease Inhibitor Dosing during Tuberculosis Treatment

Modification of ritonavir-boosted lopinavir (LPV/r)-based antiretroviral therapy is required for HIV-infected children co-treated for tuberculosis (TB). We aimed to determine virologic and toxicity outcomes among TB/HIV co-treated children with the following modifications to their antiretroviral therapy (ART): (1) super-boosted LPV/r, (2) double-dose LPV/r or (3) ritonavir.A medical record review was conducted at two clinical sites in Johannesburg, South Africa. The records of children 6-24 months of age initiating LPV/r-based therapy were reviewed. Children co-treated for TB were categorized based on the modifications made to their ART regimen and were compared to children of the same age at each site not treated for TB. Included are 526 children, 294 (56%) co-treated for TB. All co-treated children had more severe HIV disease, including lower CD4 percents and worse growth indicators, than comparisons. Children in the super-boosted group (n = 156) were as likely to be virally suppressed (<400 copies/ml) at 6 months as comparisons (69.2% vs. 74.8%, p = 0.36). Children in the double-dose (n = 47) and ritonavir groups (n = 91) were significantly less likely to be virally suppressed at 6 months (53.1% and 49.3%) than comparisons (74.8% and 82.1%; p = 0.02 and p<0.0001, respectively). At 12 months only children in the ritonavir group still had lower rates of virological suppression relative to comparisons (63.9% vs 83.3% p<0.05). Grade 1 or greater ALT elevations were more common in the super-boosted (75%) than double-dose (54.6%) or ritonavir (33.9%) groups (p = 0.09 and p<0.0001) but grade 3/4 elevations were observed in 3 (13.6%) of the super-boosted, 7 (15.9%) of the double-dose and 5 (8.9%) of the ritonavir group (p = 0.81 and p = 0.29).Good short-term virologic outcomes were achieved in children co-treated for TB and HIV who received super-boosted LPV/r. Treatment limiting toxicity was rare. Strategies for increased dosing of LPV/r with TB treatment warrant further investigation

Overexpressed TP73 induces apoptosis in medulloblastoma

Abstract Background Medulloblastoma is the most common malignant brain tumor of childhood. Children who relapse usually die of their disease, which reflects resistance to radiation and/or chemotherapy. Improvements in outcome require a better understanding of the molecular basis of medulloblastoma growth and treatment response. TP73 is a member of the TP53 tumor suppressor gene family that has been found to be overexpressed in a variety of tumors and mediates apoptotic responses to genotoxic stress. In this study, we assessed expression of TP73 RNA species in patient tumor specimens and in medulloblastoma cell lines, and manipulated expression of full-length TAp73 and amino-terminal truncated ΔNp73 to assess their effects on growth. Methods We analyzed medulloblastoma samples from thirty-four pediatric patients and the established medulloblastoma cell lines, Daoy and D283MED, for expression of TP73 RNA including the full-length transcript and the 5'-terminal variants that encode the ΔNp73 isoform, as well as TP53 RNA using quantitative real time-RTPCR. Protein expression of TAp73 and ΔNp73 was quantitated with immunoblotting methods. Clinical outcome was analyzed based on TP73 RNA and p53 protein expression. To determine effects of overexpression or knock-down of TAp73 and ΔNp73 on cell cycle and apoptosis, we analyzed transiently transfected medulloblastoma cell lines with flow cytometric and TUNEL methods. Results Patient medulloblastoma samples and cell lines expressed full-length and 5'-terminal variant TP73 RNA species in 100-fold excess compared to non-neoplastic brain controls. Western immunoblot analysis confirmed their elevated levels of TAp73 and amino-terminal truncated ΔNp73 proteins. Kaplan-Meier analysis revealed trends toward favorable overall and progression-free survival of patients whose tumors display TAp73 RNA overexpression. Overexpression of TAp73 or ΔNp73 induced apoptosis under basal growth conditions in vitro and sensitized them to cell death in response to chemotherapeutic agents. Conclusion These results indicate that primary medulloblastomas express significant levels of TP73 isoforms, and suggest that they can modulate the survival and genotoxic responsiveness of medulloblastomas cells

AtriplaR/anti-TB combination in TB/HIV patients. Drug in focus

Co-administration of anti-tuberculosis and antiretroviral therapy is often inevitable in high-burden countries where tuberculosis is the most common opportunistic infection associated with HIV/AIDS. Concurrent use of rifampicin and several antiretroviral drugs is complicated by pharmacokinetic drug-drug interaction. Pubmed and Google search following the key words tuberculosis, HIV, emtricitabine, tenofovir efavirenz, interaction were used to find relevant information on each drug of the fixed dose combination AtriplaR RESULTS: Information on generic name, trade name, pharmacokinetic parameter, metabolism and the pharmacokinetic interaction with Anti-TB drugs of emtricitabine, tenofovir, and efavirenz was obtained. Fixed dose combination of emtricitabine/tenofovir/efavirenz (ATRIPLAR) which has been approved by Food and Drug Administration shows promising results as far as safety and efficacy is concerned in TB/HIV co-infection patients, hence can be considered effective and safe antiretroviral drug in TB/HIV management for adult and children above 3 years of age

Allelic Variation, Alternative Splicing and Expression Analysis of Psy1 Gene in Hordeum chilense Roem. et Schult

Background: The wild barley Hordeum chilense Roem. et Schult. is a valuable source of genes for increasing carotenoid content in wheat. Tritordeums, the amphiploids derived from durum or common wheat and H. chilense, systematically show higher values of yellow pigment colour and carotenoid content than durum wheat. Phytoene synthase 1 gene (Psy1) is considered a key step limiting the carotenoid biosynthesis, and the correlation of Psy1 transcripts accumulation and endosperm carotenoid content has been demonstrated in the main grass species. Methodology/Principal findings: We analyze the variability of Psy1 alleles in three lines of H. chilense (H1, H7 and H16) representing the three ecotypes described in this species. Moreover, we analyze Psy1 expression in leaves and in two seed developing stages of H1 and H7, showing mRNA accumulation patterns similar to those of wheat. Finally, we identify thirtysix different transcripts forms originated by alternative splicing of the 59 UTR and/or exons 1 to 5 of Psy1 gene. Transcripts function is tested in a heterologous complementation assay, revealing that from the sixteen different predicted proteins only four types (those of 432, 370, 364 and 271 amino acids), are functional in the bacterial system. Conclusions/Significance: The large number of transcripts originated by alternative splicing of Psy1, and the coexistence of functional and non functional forms, suggest a fine regulation of PSY activity in H. chilense. This work is the first analysis of H. chilense Psy1 gene and the results reported here are the bases for its potential use in carotenoid enhancement in duru

Herbicide resistance-endowing ACCase gene mutations in hexaploid wild oat (Avena fatua): insights into resistance evolution in a hexaploid species

Many herbicide-resistant weed species are polyploids, but far too little about the evolution of resistance mutations in polyploids is understood. Hexaploid wild oat (Avena fatua) is a global crop weed and many populations have evolved herbicide resistance. We studied plastidic acetyl-coenzyme A carboxylase (ACCase)-inhibiting herbicide resistance in hexaploid wild oat and revealed that resistant individuals can express one, two or three different plastidic ACCase gene resistance mutations (Ile-1781-Leu, Asp-2078-Gly and Cys-2088-Arg). Using ACCase resistance mutations as molecular markers, combined with genetic, molecular and biochemical approaches, we found in individual resistant wild-oat plants that (1) up to three unlinked ACCase gene loci assort independently following Mendelian laws for disomic inheritance, (2) all three of these homoeologous ACCase genes were transcribed, with each able to carry its own mutation and (3) in a hexaploid background, each individual ACCase resistance mutation confers relatively low-level herbicide resistance, in contrast to high-level resistance conferred by the same mutations in unrelated diploid weed species of the Poaceae (grass) family. Low resistance conferred by individual ACCase resistance mutations is likely due to a dilution effect by susceptible ACCase expressed by homoeologs in hexaploid wild oat and/or differential expression of homoeologous ACCase gene copies. Thus, polyploidy in hexaploid wild oat may slow resistance evolution. Evidence of coexisting non-target-site resistance mechanisms among wild-oat populations was also revealed. In all, these results demonstrate that herbicide resistance and its evolution can be more complex in hexaploid wild oat than in unrelated diploid grass weeds. Our data provide a starting point for the daunting task of understanding resistance evolution in polyploids

Cbf gene regulation in wheat in response to varying cold acclimation induction temperatures

Tese de doutoramento em Biociências, apresentada à Faculdade de Ciências e Tecnologia da Universidade de CoimbraO nemátode-das-galhas-radiculares (NGR), Meloidogyne hispanica (Mhi), tem sido detetado em todos os continentes associado a uma grande diversidade de plantas hospedeiras. Os objetivos principais deste estudo foram determinar as funções de genes efetores de M. hispanica na interação entre a planta hospedeira e o nemátode e contribuir para o desenvolvimento de novas estratégias de controlo. Em virtude de não existirem dados genómicos disponíveis para M. hispanica, alguns genes ortólogos foram selecionados, a partir de genes modelo do genoma de M. incognita e M. hapla, no "National Center for Biotechnology". Foram desenhados primers para as regiões conservadas dos efetores selecionados de M. incognita e M. hapla e usados para amplificar estes genes em M. hispanica. Este processo levou à amplificação dos genes efetores de M. hispanica: anexina-2 (nex-2); β-1,4- endoglucanase-1 e 2 (eng-1 e eng-2); cisteína protease L-catepsina (cpl-1); calreticulina (crt-1); proteína de ligação de ácidos gordos e retinol (far-1); transferase glutationa-S (gsts-1); dismutase superóxido de manganésio (mnsod); liase pectase 3 (pel-3); poligalacturonase (gp-1); proteína semelhante à alérgica do veneno 1 e 2 (vap-1 e vap-2) e 14-3-3a (14-3-3a). Os genes Mhi-cpl-1, Mhi-crt-1, Mhieng- 1, Mhi-far-1, Mhi-mnsod e Mhi-vap-1 foram diferencialmente expressos durante o desenvolvimento de M. hispanica. O cDNA foi amplificado a partir do mRNA de ovos, jovens do segundo estádio (J2) e fêmeas. Os genes Mhi-cpl-1, Mhi-crt-1, Mhi-far- 1 e Mhi-vap-1 foram detetados em todas as fases do desenvolvimento, sugerindo terem um papel em vários aspectos do ciclo de vida. No entanto, o nível de expressão mais elevado de Mhi-vap-1 foi nos J2. O gene Mhi-eng-1 foi expresso nos ovos e J2 e pode ter uma função nas primeiras fases da infecção. A expressão de Mhi-mnsod nos ovos indica que este gene poderá ter uma função na embriogénese e eclosão dos J2. Os genes cpl-1, crt-1, far-1, eng-1, mnsod e vap-1 foram sequenciados em M. arenaria, M. hapla, M. hispanica, M. incognita e M. javanica e os resultados da análise filogenética mostraram que M. hapla é a espécie mais divergente. A análise filogenética das sequências previstas para as proteínas FAR-1 e VAP-1 entre M. hispanica, outras espécies de NGR e outros nemátodes fitoparasitas revelou existir um elevado grau de conservação entre M. hispanica e as outras espécies. Os padrões de expressão temporal e espacial dos genes efetores Mhi-vap-1 e Mhi-far-1 foram analisados nos J2 através de hibridização in situ. Estes xviii transcritos foram localizados nas glândulas esofágicas subventrais, o que sugere que estas proteínas são produzidas pelos J2 e que poderão ter um papel importante nas primeiras fases do processo de infeção. Para avaliar a função do gene Mhi-vap-1 no parasitismo, foi utilizada a metodologia associada ao RNA de interferência (RNAi). O nível dos transcritos do gene vap-1 diminuiu às 48 h, indicando ser um gene susceptível ao RNAi. Na atração e penetração de J2 nas raízes de tomateiro, após tratamento com dsRNA para o silenciamento do gene Mhi-vap-1, foi possível observar, pela primeira vez, uma redução significativa do número de J2, confirmando a função deste gene no processo de infeção. Também foi detetado, pela primeira vez, que os exsudatos de raízes de tomateiro induzem alterações na expressão de alguns genes. A expressão dos genes Mhi-cpl-1, Mhi-crt-1, Mhi-far-1 e Mhi-vap-1 aumentou nos J2 após a exposição aos exsudatos de tomateiro. Estes genes, provavelmente, têm uma função estratégica nas fases iniciais do processo de infeção e sua sobreexpressão antes da infecção poderá contribuir para o sucesso do seu parasitismo. A infeção altera diferencialmente a expressão génica de numerosos genes de plantas e este estudo mostrou a expressão diferencial de PR-1 e WRKY1 no tomateiro suscetível cv. Easypeel e no pimentão resistente cv.Solero após inoculação de J2 de M. hispanica. O gene PR-1 no tomateiro pode também contribuir para o processo de regulação das primeiras vias metabólicas de defesa da planta hospedeira e nos primeiros eventos da interação nemátode-planta. A sua expressão diferencial durante o parasitismo pode estar correlacionada com os diferentes estágios de desenvolvimento dos nemátodes. Para além da identificação de vários genes efetores em M. hispanica, procedeu-se à sua caracterização molecular durante o processo de infeção e possível função durante o parasitismo. Este estudo também analisou o efeito da infeção por nemátodes em dois genes importantes de defesa das plantas e as vias de sinalização que ocorrem na rizosfera antes da penetração do nemátode. Os resultados contribuem para o conhecimento e compreensão da interação nemátode-planta e indicam algumas perspetivas para o desenvolvimento de estratégias para o controlo de M. hispanica.of the M. hispanica effectors genes: annexin-2 (nex-2); β-1,4-endoglucanase-1 and 2 (eng-1 and eng-2); cathepsin L cysteine protease (cpl-1); calreticulin (crt-1); fatty acid and retinol binding protein (far-1); glutathione-S-transferase (gsts-1); manganese superoxide dismutase (mnsod); pectase lyase 3 (pel-3); polygalacturonase (gp-1); venom allergen-like protein 1 and 2 (vap-1 and vap-2) and 14-3-3a (14-3-3a). The Mhi-cpl-1, Mhi-crt-1, Mhi-eng-1, Mhi-far-1, Mhi-mnsod and Mhi-vap-1 genes were shown to be differentially expressed during M. hispanica development. The cDNA was amplified from mRNA from eggs, second-stage juveniles (J2) and females. The Mhi-cpl-1, Mhi-crt-1, Mhi-far-1 and Mhi-vap-1 genes were present in all developmental stages, suggesting having a role in various aspects of the life cycle. However, Mhi-vap-1 showed the highest level of expression in J2. The Mhi-eng-1 gene was expressed in eggs and J2 and may have a function in the early events of infection. The expression in eggs of Mhi-mnsod indicates that this gene could have a role in the embryogenesis and hatching of J2. Meloidogyne arenaria, M. hapla, M. hispanica, M. incognita and M. javanica cpl-1, crt-1, far-1, eng-1, mnsod and vap-1 genes were sequenced and phylogenetic studies revealed that M. hapla is the most divergent species. Phylogenetic analysis of the FAR-1 and VAP-1 predicted protein sequences between M. hispanica, other RKN species and other plant-parasitic nematodes indicated towards a high degree of conservation between M. hispanica and the other species. The temporal and spatial expression patterns of far-1 and vap-1 effector genes were analyzed in J2 by in situ hybridization. These transcripts were localized within the subventral oesophageal glands which suggest that these proteins are secreted by J2 and could play a crucial role in the early stages xvi of the infection process. RNA interference (RNAi) was used to evaluate the putative role of the Mhi-vap-1 gene in the parasitism, the transcript level of vap-1 gene in J2 decreased after incubation with dsRNA for 48 h, indicating the susceptibility of this gene to RNAi. It was showed for the first time that a significant reduction in nematode attraction and penetration of tomato roots was observed when M. hispanica J2 were treated with dsRNA to silence the Mhi-vap-1 gene. Therefore, the nematode effector Mhi-vap-1 gene seems to play an important role in the infection process. It was also reported for the first time that tomato root exudates induce changes in the gene expression of some nematode candidate parasitism genes. Gene expression of Mhi-cpl-1, Mhi-crt-1, Mhi-far-1 and Mhi-vap-1 genes were up-regulated in the pre-parasitic J2 after exposure to tomato root exudates. Possibly, these candidate parasitism genes have a strategic function during the early events of infection and their up-regulation prior to root infection may contribute to their successful parasitism. Nematode infection differentially changes the gene expression of numerous plant genes and this study showed differential expression of PR-1 and WRKY1 upon nematode infection in the susceptible tomato cv. Easypeel and in the resistant pepper cv. Solero. The PR-1 gene in tomato may also contribute to the process of regulation of primary host plant defence pathways and in the early events of the compatible plant-nematode interaction. Its differential expression during parasitism might be co-related with the presence of different nematode development stages. Several M. hispanica effector genes were identified and a detailed insight of their molecular characterisation, during the infection process and function during parasitism, was provided. This study also analysed the effect of nematode infection on two important plant defence genes and the signalling occurring in the rhizosphere before nematode penetration. The results contribute to further understanding the plant-nematode interaction and offer some possible avenues for the development of novels strategies for the management of M. hispanica