Standing genetic variation and the evolution of drug resistance in HIV

Drug resistance remains a major problem for the treatment of HIV. Resistance can occur due to mutations that were present before treatment starts or due to mutations that occur during treatment. The relative importance of these two sources is unknown. We study three different situations in which HIV drug resistance may evolve: starting triple-drug therapy, treatment with a single dose of nevirapine and interruption of treatment. For each of these three cases good data are available from literature, which allows us to estimate the probability that resistance evolves from standing genetic variation. Depending on the treatment we find probabilities of the evolution of drug resistance due to standing genetic variation between 0 and 39%. For patients who start triple-drug combination therapy, we find that drug resistance evolves from standing genetic variation in approximately 6% of the patients. We use a population-dynamic and population-genetic model to understand the observations and to estimate important evolutionary parameters. We find that both, the effective population size of the virus before treatment, and the fitness of the resistant mutant during treatment, are key-parameters that determine the probability that resistance evolves from standing genetic variation. Importantly, clinical data indicate that both of these parameters can be manipulated by the kind of treatment that is used.Comment: 33 pages 6 figure

Legalised non-consensual sterilisation - eugenics put into practice before 1945, and the aftermath. Part 1: USA, Japan, Canada and Mexico.

In the late 19th century, eugenics, a pseudo-scientific doctrine based on an erroneous interpretation of the laws of heredity, swept across the industrialised world. Academics and other influential figures who promoted it convinced political stakeholders to enact laws authorising the sterilisation of people seen as ‘social misfits’. The earliest sterilisation Act was enforced in Indiana, in 1907; most States in the USA followed suit and so did several countries, with dissimilar political regimes. The end of the Second World War saw the suspension of Nazi legislation in Germany, including that regulating coerced sterilisation. The year 1945 should have been the endpoint of these inhuman practices but, in the early post-war period, the existing sterilisation Acts were suspended solely in Germany and Austria. Only much later did certain countries concerned – not Japan so far - officially acknowledge the human rights violations committed, issue apologies, and develop reparation schemes for the victims’ benefit

Mechanism of Disruption of the Amt-GlnK Complex by PII-Mediated Sensing of 2-Oxoglutarate

GlnK proteins regulate the active uptake of ammonium by Amt transport proteins by inserting their regulatory T-loops into the transport channels of the Amt trimer and physically blocking substrate passage. They sense the cellular nitrogen status through 2-oxoglutarate, and the energy level of the cell by binding both ATP and ADP with different affinities. The hyperthermophilic euryarchaeon Archaeoglobus fulgidus possesses three Amt proteins, each encoded in an operon with a GlnK ortholog. One of these proteins, GlnK2 was recently found to be incapable of binding 2-OG, and in order to understand the implications of this finding we conducted a detailed structural and functional analysis of a second GlnK protein from A. fulgidus, GlnK3. Contrary to Af-GlnK2 this protein was able to bind both ATP/2-OG and ADP to yield inactive and functional states, respectively. Due to the thermostable nature of the protein we could observe the exact positioning of the notoriously flexible T-loops and explain the binding behavior of GlnK proteins to their interaction partner, the Amt proteins. A thermodynamic analysis of these binding events using microcalorimetry evaluated by microstate modeling revealed significant differences in binding cooperativity compared to other characterized PII proteins, underlining the diversity and adaptability of this class of regulatory signaling proteins

Functional and Transcriptional Induction of Aquaporin-1 Gene by Hypoxia; Analysis of Promoter and Role of Hif-1α

Aquaporin-1 (AQP1) is a water channel that is highly expressed in tissues with rapid O2 transport. It has been reported that this protein contributes to gas permeation (CO2, NO and O2) through the plasma membrane. We show that hypoxia increases Aqp1 mRNA and protein levels in tissues, namely mouse brain and lung, and in cultured cells, the 9L glioma cell line. Stopped-flow light-scattering experiments confirmed an increase in the water permeability of 9L cells exposed to hypoxia, supporting the view that hypoxic Aqp1 up-regulation has a functional role. To investigate the molecular mechanisms underlying this regulatory process, transcriptional regulation was studied by transient transfections of mouse endothelial cells with a 1297 bp 5′ proximal Aqp1 promoter-luciferase construct. Incubation in hypoxia produced a dose- and time-dependent induction of luciferase activity that was also obtained after treatments with hypoxia mimetics (DMOG and CoCl2) and by overexpressing stabilized mutated forms of HIF-1α. Single mutations or full deletions of the three putative HIF binding domains present in the Aqp1 promoter partially reduced its responsiveness to hypoxia, and transfection with Hif-1α siRNA decreased the in vitro hypoxia induction of Aqp1 mRNA and protein levels. Our results indicate that HIF-1α participates in the hypoxic induction of AQP1. However, we also demonstrate that the activation of Aqp1 promoter by hypoxia is complex and multifactorial and suggest that besides HIF-1α other transcription factors might contribute to this regulatory process. These data provide a conceptual framework to support future research on the involvement of AQP1 in a range of pathophysiological conditions, including edema, tumor growth, and respiratory diseases

Structural basis for Mep2 ammonium transceptor activation by phosphorylation

Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation

Risk factors for virological failure and subtherapeutic antiretroviral drug concentrations in HIV-positive adults treated in rural northwestern Uganda

ABSTRACT: BACKGROUND: Little is known about immunovirological treatment outcomes and adherence in HIV/AIDS patients on antiretroviral therapy (ART) treated using a simplified management approach in rural areas of developing countries, or about the main factors influencing those outcomes in clinical practice. METHODS: Cross-sectional immunovirological, pharmacological, and adherence outcomes were evaluated in all patients alive and on fixed-dose ART combinations for 24 months, and in a random sample of those treated for 12 months. Risk factors for virological failure (>1,000 copies/mL) and subtherapeutic antiretroviral (ARV) concentrations were investigated with multiple logistic regression. RESULTS: At 12 and 24 months of ART, 72% (n=701) and 70% (n=369) of patients, respectively, were alive and in care. About 8% and 38% of patients, respectively, were diagnosed with immunological failure; and 75% and 72% of patients, respectively, had undetectable HIV RNA (<400 copies/mL). Risk factors for virological failure (>1,000 copies/mL) were poor adherence, tuberculosis diagnosed after ART initiation, subtherapeutic NNRTI concentrations, general clinical symptoms, and lower weight than at baseline. About 14% of patients had low ARV plasma concentrations. Digestive symptoms and poor adherence to ART were risk factors for low ARV plasma concentrations. CONCLUSIONS: Efforts to improve both access to care and patient management to achieve better immunological and virological outcomes on ART are necessary to maximize the duration of first-line therapy

Immunovirological response to combined antiretroviral therapy and drug resistance patterns in children: 1- and 2-year outcomes in rural Uganda

Children living with HIV continue to be in urgent need of combined antiretroviral therapy (ART). Strategies to scale up and improve pediatric HIV care in resource-poor regions, especially in sub-Saharan Africa, require further research from these settings. We describe treatment outcomes in children treated in rural Uganda after 1 and 2 years of ART start

Fast T2 gradient-spin-echo (T2-GraSE) mapping for myocardial edema quantification: first in vivo validation in a porcine model of ischemia/reperfusion

BACKGROUND: Several T2-mapping sequences have been recently proposed to quantify myocardial edema by providing T2 relaxation time values. However, no T2-mapping sequence has ever been validated against actual myocardial water content for edema detection. In addition, these T2-mapping sequences are either time-consuming or require specialized software for data acquisition and/or post-processing, factors impeding their routine clinical use. Our objective was to obtain in vivo validation of a sequence for fast and accurate myocardial T2-mapping (T2 gradient-spin-echo [GraSE]) that can be easily integrated in routine protocols. METHODS: The study population comprised 25 pigs. Closed-chest 40 min ischemia/reperfusion was performed in 20 pigs. Pigs were sacrificed at 120 min (n = 5), 24 h (n = 5), 4 days (n = 5) and 7 days (n = 5) after reperfusion, and heart tissue extracted for quantification of myocardial water content. For the evaluation of T2 relaxation time, cardiovascular magnetic resonance (CMR) scans, including T2 turbo-spin-echo (T2-TSE, reference standard) mapping and T2-GraSE mapping, were performed at baseline and at every follow-up until sacrifice. Five additional pigs were sacrificed after baseline CMR study and served as controls. RESULTS: Acquisition of T2-GraSE mapping was significantly (3-fold) faster than conventional T2-TSE mapping. Myocardial T2 relaxation measurements performed by T2-TSE and T2-GraSE mapping demonstrated an almost perfect correlation (R(2) = 0.99) and agreement with no systematic error between techniques. The two T2-mapping sequences showed similarly good correlations with myocardial water content: R(2) = 0.75 and R(2) = 0.73 for T2-TSE and T2-GraSE mapping, respectively. CONCLUSIONS: We present the first in vivo validation of T2-mapping to assess myocardial edema. Given its shorter acquisition time and no requirement for specific software for data acquisition or post-processing, fast T2-GraSE mapping of the myocardium offers an attractive alternative to current CMR sequences for T2 quantification