Human immunodeficiency virus (HIV)-specific gamma interferon secretion directed against all expressed HIV genes: relationship to rate of CD4 decline

Immune responses to human immunodeficiency virus (HIV) are detected at all stages of infection and are believed to be responsible for controlling viremia. This study seeks to determine whether gamma interferon (IFN-gamma)-secreting HIV-specific T-cell responses influence disease progression as defined by the rate of CD4 decline. The study population consisted of 31 subjects naive to antiretroviral therapy. All were monitored clinically for a median of 24 months after the time they were tested for HIV-specific responses. The rate of CD4+-T-cell loss was calculated for all participants from monthly CD4 counts. Within this population, 17 subjects were classified as typical progressors, 6 subjects were classified as fast progressors, and 8 subjects were classified as slow progressors. Peripheral blood mononuclear cells were screened for HIV-specific IFN-gamma responses to all expressed HIV genes. Among the detected immune responses, 48% of the recognized peptides were encoded by Gag and 19% were encoded by Nef gene products. Neither the breadth nor the magnitude of HIV-specific responses correlated with the viral load or rate of CD4 decline. The breadth and magnitude of HIV-specific responses did not differ significantly among typical, fast, and slow progressors. These results support the conclusion that although diverse HIV-specific IFN-gamma-secreting responses are mounted during the asymptomatic phase, these responses do not seem to modulate disease progression rates.     

Multiple mutations in heterogeneous miltefosine-resistant Leishmania major population as determined by whole genome sequencing

Background

Miltefosine (MF) is the first oral compound used in the chemotherapy against leishmaniasis. Since the mechanism of action of this drug and the targets of MF in Leishmania are unclear, we generated in a step-by-step manner Leishmania major promastigote mutants highly resistant to MF. Two of the mutants were submitted to a short-read whole genome sequencing for identifying potential genes associated with MF resistance.

Methods/Principal Findings

Analysis of the genome assemblies revealed several independent point mutations in a P-type ATPase involved in phospholipid translocation. Mutations in two other proteins—pyridoxal kinase and α-adaptin like protein—were also observed in independent mutants. The role of these proteins in the MF resistance was evaluated by gene transfection and gene disruption and both the P-type ATPase and pyridoxal kinase were implicated in MF susceptibility. The study also highlighted that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes.

Conclusions/Significance

Whole genome sequencing was used to pinpoint known and new resistance markers associated with MF resistance in the protozoan parasite Leishmania. The study also demonstrated the polyclonal nature of a resistant population with individual cells with varying susceptibilities and genotypes.     

Ray Meta: scalable de novo metagenome assembly and profiling

Voluminous parallel sequencing datasets, especially metagenomic experiments, require distributed computing for de novo assembly and taxonomic profiling. Ray Meta is a massively distributed metagenome assembler that is coupled with Ray Communities, which profiles microbiomes based on uniquely-colored k-mers. It can accurately assemble and profile a three billion read metagenomic experiment representing 1,000 bacterial genomes of uneven proportions in 15 hours with 1,024 processor cores, using only 1.5 GB per core. The software will facilitate the processing of large and complex datasets, and will help in generating biological insights for specific environments. Ray Meta is open source and available at http://denovoassembler.sf.net.     

Factors Affecting Residual Dosages of Two Formulations of Bacillus thuringiensis subsp. israelensis Tested in the Same Stream During a 3-Year Experiment

Although many field trials have been conducted using Bacillus thuringiensis subsp. israelensis (Bti)-based formulations, most have been in rivers with different biotic and abiotic conditions thus rendering the evaluation of their performance very difficult. Recently, results of a threeyear experiment using a new field procedure brought new insight into the behavior and the performance (carry) of two liquid formulations of Bti, Teknar HP-D and Vectobac 1200L, tested in the same lotic environment and under similar abiotic and biotic conditions. Factors such as discharge, water temperature and the hyporheic zone were identified as elements affecting the downstream loss of activity of both products. However, to better understand the reduction of black fly mortality along a stream (measured by using gutters), data of residual dosages of both products (measured by laboratory assay with mosquito larvae) were compared with reduction of black fly mortality. Bti toxic activity was monitored from water samples taken at different distances downstream from an application point, and from probes driven into the hyporheic zone, to study the effects of abiotic factors on the loss of the toxic crystals. Results showed that the loss of dosage was exponential for both products but more crystals were recovered from Vectobac 1200L along the stream than from Teknar HP-D. However, the latter was more efficient, i.e. less toxins were needed to kill 50% of black fly larvae both in temperate (16°C) and warmer (19.5-22°C) water. Also, a rise in water temperature had a greater effect in the kill induced by Vectobac 1200L compared to Teknar HP-D. For the same residual dosages present at the stations, longer carries of toxin activity (higher mortalities) were obtained in warmer water. Finally, the hyporheic zone was identified as a major source of loss of activity of Bti products. Large stream discharges decreased the effect of the hyporheic zone and that was reflected in longer carry of the products.     

Storage Stability of Two Liquid Formulations of Bacillus thuringiensis subsp. israelensis and Effect of Freezing Over Time

Over the last two decades, many tests have been performed in the field to investigate the behaviour (persistence, carry, loss of activity, etc.) of different Bacillus thuringiensis subsp. israelensis ( Bti ) formulations. Depending on the experimental protocols, a single container of a formulation could be used more than once over time and field samples containing Bti may have to be frozen to preserve them for bioassays to be performed later. Thus, it is necessary to know how long a formulation could keep its level of efficacy and also the effects of time on frozen samples. Our results showed that the efficacy of two commercial liquid formulations of Bti (Teknar HP-D and Vectobac 1200L) when tested against Aedes triseriatus behaved differently over time when kept at room temperature. Teknar HP-D remained stable for the first two years and its LC 50 increased by 20% the third year. For Vectobac 1200L, although its larvicidal activity was better than that of Teknar HP-D every year, there was an increase in LC 50 by 22% the second year and by another 20% the third year for a total loss of activity of 46% over the three-year study. The efficacy of suspensions made with both formulations was greatly affected by freezing and the loss of efficacy increased over time. About half of the efficacy of Teknar HP-D was lost after one week of freezing and stayed at that level for three months, while with Vectobac 1200L, no significant effect of freezing was seen after one month, when compared to fresh material. However, both products showed similar efficacy after three or six months of freezing. Overall, the LC 50 s of both products had increased by a factor of about 2.5 after six months of freezing.     

Semi-automated ninhydrin assay of Kjeldahl nitrogen

The staining and photography of nucleic acids in polyacrylamide gels is a somewhat involved and certainly time-consuming procedure. Ultraviolet scanning methods (1) may be used to record the location of uv-absorbing bands of material in polyacrylamide gels, but this is a complicated and inefficient way of storing experimental information. Recently, we have published a method (2) for the location and isolation of nucleic acids from unstained polyacrylamide gels (entitled UV Shadowing). An extension of this method is the technique of Direct-Contact Photography.     

Quantification of ketoprofen enantiomers in human plasma based on solid-phase extraction and enantioselective column chromatography

An HPLC method for the quantification of ketoprofen enantiomers in human plasma is described. Following extraction with a disposable C₁₈ solid-phase extraction column, separation of ketoprofen enantiomers and I.S. (3,4-dimethoxy benzoic acid) was achieved using a chiral column [Chirex 3005; (R)-1-naphthylglycine 3,5-dinitrobenzoic acid] with the mobile phase, 0.02 M ammonium acetate in methanol, set at a flow-rate of 1.2 ml/min. Baseline separation of ketoprofen enantiomers and I.S., free from interferences, was achieved in less than 20 min. The calibration curves (n = 14) were linear over the concentration range of 0.16 to 5.00 μg/ml per enantiomer [mean r² of 0.999 for both enantiomers, root mean square error were 0.015 for R(−) and 0.013 for S(+)]. The inter-day coefficient of variation for duplicate analysis of spiked samples was less than 7% and the accuracy was more than 93% over the concentration range of 0.2 to 4.0 μg/ml for individual enantiomer using 1 ml of plasma sample. This method has been applied to a pharmacokinetic study from healthy human volunteers following the administration of a ketoprofen extended release product (200 mg). This method is simple, fast and should find wide application in monitoring pharmacokinetic studies of ketoprofen.     

Persistence of Toxic Activity and Recycling of Bacillus thuringiensis var. israelensis in Cold Water: Field Experiments Using Diffusion Chambers in a Pond

The persistence of the larvicidal activity of Bacillus thuringiensis var. israelensis (Bti) was tested over a five-month period in a low-temperature aquatic environment. Diffusion chambers filled with a suspension of Bti (100 mg l - 1) and different experimental substrates (pond water, periphyton, sediments and vegetation), but without mosquito larvae, were placed near the bottom of a large pond and removed at various intervals to measure residual toxic activity to mosquito larvae, spore concentration and proteolytic activity. Within the pond water substrate, 50% of the initial toxicity was still present after one month in cold water, while with the periphyton substrate, 30% remained in the liquid fraction after the same period of exposure. Within the vegetation substrate (blue-joint grass, Calamagrostis canadensis), an average of 30% of the initial toxicity was still present in the liquid fraction between day 84 to day 154. Solid fractions of vegetation became toxic very early and remained toxic for five months. At the end (day 154), there was still 54% of the original toxic activity put in the chambers associated with the vegetation samples. In the absence of mosquito larvae, spore recycling was observed in the chambers especially with sediments and vegetation. But spore recycling did not appear to play a major role in the observed persistence, but rather rapid absorption onto vegetation substrates was responsible for the persistence of Bti in a cold climate.