In Vitro HIV-1 Evolution in Response to Triple Reverse Transcriptase Inhibitors & In Silico Phenotypic Analysis

Background

Effectiveness of ART regimens strongly depends upon complex interactions between the selective pressure of drugs and the evolution of mutations that allow or restrict drug resistance.

Methods

Four clinical isolates from NRTI-exposed, NNRTI-naive subjects were passaged in increasing concentrations of NVP in combination with 1 µM 3 TC and 2 µM ADV to assess selective pressures of multi-drug treatment. A novel parameter inference procedure, based on a stochastic viral growth model, was used to estimate phenotypic resistance and fitness from in vitro combination passage experiments.

Results

Newly developed mathematical methods estimated key phenotypic parameters of mutations arising through selective pressure exerted by 3 TC and NVP. Concentrations of 1 µM 3 TC maintained the M184V mutation, which was associated with intrinsic fitness deficits. Increasing NVP concentrations selected major NNRTI resistance mutations. The evolutionary pathway of NVP resistance was highly dependent on the viral genetic background, epistasis as well as stochasticity. Parameter estimation indicated that the previously unrecognized mutation L228Q was associated with NVP resistance in some isolates.

Conclusion

Serial passage of viruses in the presence of multiple drugs may resemble the selection of mutations observed among treated individuals and populations in vivo and indicate evolutionary preferences and restrictions. Phenotypic resistance estimated here “in silico” from in vitro passage experiments agreed well with previous knowledge, suggesting that the unique combination of “wet-” and “dry-lab” experimentation may improve our understanding of HIV-1 resistance evolution in the future.     

Solar Simulated Ultraviolet Radiation Induces Global Histone Hypoacetylation in Human Keratinocytes

Ultraviolet radiation (UVR) from sunlight is the primary effector of skin DNA damage. Chromatin remodeling and histone post-translational modification (PTM) are critical factors in repairing DNA damage and maintaining genomic integrity, however, the dynamic changes of histone marks in response to solar UVR are not well characterized. Here we report global changes in histone PTMs induced by solar simulated UVR (ssUVR). A decrease in lysine acetylation of histones H3 and H4, particularly at positions of H3 lysine 9, lysine 56, H4 lysine 5, and lysine 16, was found in human keratinocytes exposed to ssUVR. These acetylation changes were highly associated with ssUVR in a dose-dependent and time-specific manner. Interestingly, H4K16ac, a mark that is crucial for higher order chromatin structure, exhibited a persistent reduction by ssUVR that was transmitted through multiple cell divisions. In addition, the enzymatic activities of histone acetyltransferases were significantly reduced in irradiated cells, which may account for decreased global acetylation. Moreover, depletion of histone deacetylase SIRT1 in keratinocytes rescued ssUVR-induced H4K16 hypoacetylation. These results indicate that ssUVR affects both HDAC and HAT activities, leading to reduced histone acetylation.     

On the Evolutionary History of Uleiella chilensis,a Smut Fungus Parasite of Araucaria araucana in South America: Uleiellales ord. nov. in Ustilaginomycetes

The evolutionary history, divergence times and phylogenetic relationships of Uleiella chilensis (Ustilaginomycotina, smut fungi) associated with Araucaria araucana were analysed. DNA sequences from multiple gene regions and morphology were analysed and compared to other members of the Basidiomycota to determine the phylogenetic placement of smut fungi on gymnosperms. Divergence time estimates indicate that the majority of smut fungal orders diversified during the Triassic–Jurassic period. However, the origin and relationships of several orders remain uncertain. The most recent common ancestor between Uleiella chilensis and Violaceomyces palustris has been dated to the Lower Cretaceous. Comparisons of divergence time estimates between smut fungi and host plants lead to the hypothesis that the early Ustilaginomycotina had a saprobic lifestyle. As there are only two extant species of Araucaria in South America, each hosting a unique Uleiella species, we suggest that either coevolution or a host shift followed by allopatric speciation are the most likely explanations for the current geographic restriction of Uleiella and its low diversity. Phylogenetic and age estimation analyses, ecology, the unusual life-cycle and the peculiar combination of septal and haustorial characteristics support Uleiella chilensis as a distinct lineage among the Ustilaginomycotina. Here, we describe a new ustilaginomycetous order, the Uleiellales to accommodate Uleiella. Within the Ustilaginomycetes, Uleiellales are sister taxon to the Violaceomycetales.     

The BCL2 -938C>A Promoter Polymorphism Is Associated with Risk for and Time to Aseptic Loosening of Total Hip Arthroplasty

Aseptic loosening is a major cause of revision surgery of total hip arthroplasty (THA). Only few host factors affecting aseptic loosening have been identified until now, although they are urgently needed to identify and possibly treat those patients at higher risk for aseptic loosening. To determine whether the functional single nucleotide polymorphism (SNP) c.-938C>A (rs2279115), located in the promoter region of the BCL2 gene has an impact on aseptic loosening of THA we genotyped and analyzed 234 patients suffering from aseptic loosening and 231 patients after primary THA. The polymorphism is associated with risk for aseptic loosening with the CC genotype at highest risk for aseptic loosening, Odds Ratio CC vs. AA 1.93, 95%CI 1.15–3.25, p = 0.013. In contrast, low risk AA genotype carriers that still developed aseptic loosening showed a significantly shorter time to aseptic loosening than patients carrying the C allele (p = 0.004). These results indicate that the BCL2 -938C>A polymorphism influences the occurrence and course of aseptic loosening and suggests this polymorphism as an interesting candidate for prospective studies and analyses in THA registers.     

GAP promoter‐based fed‐batch production of highly bioactive core streptavidin by Pichia pastoris

Streptavidin is a homotetrameric protein binding the vitamin biotin and peptide analogues with an extremely high affinity, which leads to a large variety of applications. The biotin‐auxotrophic yeast Pichia pastoris has recently been identified as a suitable host for the expression of the streptavidin gene, allowing both high product concentrations and productivities. However, so far only methanol‐based expression systems have been applied, bringing about increased oxygen demand, strong heat evolution and high requirements for process safety, causing increased cost. Moreover, common methanol‐based processes lead to large proportions of biotin‐blocked binding sites of streptavidin due to biotin‐supplemented media. Targeting these problems, this paper provides strategies for the methanol‐free production of highly bioactive core streptavidin by P. pastoris under control of the constitutive GAP promoter. Complex were superior to synthetic production media regarding the proportion of biotin‐blocked streptavidin. The optimized, easily scalable fed‐batch process led to a tetrameric product concentration of up to 4.16 ± 0.11 µM of biotin‐free streptavidin and a productivity of 57.8 nM h^?1 based on constant glucose feeding and a successive shift of temperature and pH throughout the cultivation, surpassing the concentration in un‐optimized conditions by a factor of 3.4. Parameter estimation indicates that the optimized conditions caused a strongly increased accumulation of product at diminishing specific growth rates (μ ≈ D < 0.01 h^?1), supporting the strategy of feeding. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:855–864, 2016     

Behavioural Type Affects Space Use in a Wild Population of Crows (Corvus corone)

While personality‐dependent dispersal is well studied, local space use has received surprisingly little attention in this context, despite the multiple consequences on survival and fitness. Regarding the coping style of individuals, recent studies on personality‐dependent space use within a habitat indicate that ‘proactive’ individuals are wider ranging than ‘reactive’ ones. However, such studies are still scarce and cover limited taxonomic diversity, and thus, more research is needed to explore whether this pattern generalises across species. We examined the link between coping style and space use in a population of crows (Corvus corone) freely inhabiting the urban zoo of Vienna, Austria. We used a binary docility rating (struggle during handling vs. no struggle) and a tonic immobility test to quantify individual coping style. Individual space use was quantified as the number of different sites at which each crow was observed, and we controlled for different number of sightings per individual by creating a space use index. Only the binary docility rating showed repeatability over time, and significantly predicted space use. In contrast to previous studies, we found that reactive crows (no struggle during handling) showed wider ranging space use within the study site than proactive individuals (who struggled during handling). The discrepancy from previous results suggests that the relationship between behavioural type and space use may vary between species, potentially reflecting differences in socioecology.     

Real-time metabolomic analysis of lactic acid bacteria as monitored by in vitro NMR and chemometrics

Introduction

Lactic acid bacteria (LAB) play an important role in the food industry as starter cultures to manufacture fermented food, and as probiotics. In recent years, there has been an increasing interest in using LAB cultures for biopreservation of food products. It is therefore of great interest to study the detailed metabolism of these bacteria.

Objectives

This study aimed at developing an efficient analytical protocol for real-time in vitro NMR measurements of LAB fermentations, from sample preparation, over data acquisition and preprocessing, to the extraction of the kinetic metabolic profiles.

Method

The developed analytical protocol is applied to an experimental design with two LAB strains (Lactobacillus rhamnosus DSM 20021 and Lactobacillus plantarum subsp. plantarum DSM 20174), two initial pH levels (pH_i 6.5 and 5.5), two levels of glucose concentration (2.5 and 0.25 g/l), and two batch fermentation replicates.

Results

The design factors proved to be strongly significant and led to interesting biological information. The protocol allowed for detailed real-time kinetic analysis of 11 major metabolites involved in the glycolysis, pyruvate catabolism, amino acid catabolism and cell energy metabolism. New biological knowledge was obtained about the different patterns of glutamine and aspartic acid consumption by the two strains. It was observed that L. plantarum consumes more glutamine at low pH (pH 5.5) whereas the opposite applies to L. rhamnosus. Regarding aspartic acid, both of the strains consume it higher at low pH, and overall L. plantarum consumes it more. L. rhamnosus did not consume aspartic acid at pH 6.5.

Conclusion

The developed analytical protocol for real-time in vitro NMR measurements of bacterial metabolism allows a relatively easy investigation of different fermentation factors such as new strains, new substrates, cohabitations, temperature, and pH and has a great potential in biopreservation studies to discover new efficient bioprotective cultures.

     

Time-Restricted Feeding Prevents Obesity and Metabolic Syndrome in Mice Lacking a Circadian Clock

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