A 3D structural model and dynamics of hepatitis C virus NS3/4A protease (genotype 4a,strain ED43) suggest conformational instability of the catalytic triad: implications in catalysis and drug resistivity

Egypt has the highest prevalence of hepatitis C virus (HCV) infection worldwide with a frequency of 15%. More than 90% of these infections are due to genotype 4, and the subtype 4a (HCV-4a) predominates. Moreover, due to the increased mobility of people, HCV-4a has recently spread to several European countries. The protease domain of the HCV nonstructural protein 3 (NS3) has been targeted for inhibition by several drugs. This approach has had marked success in inhibiting genotype 1 (HCV-1), the predominant genotype in the USA, Europe, and Japan. However, HCV-4a was found to resist inhibition by a number of these drugs, and little progress has been made to understand the structural basis of its drug resistivity. As a step forward, we sequenced the NS3 HCV-4a protease gene (strain ED43) and subsequently built a 3D structural model threaded through a template crystal structure of HCV-1b NS3 protease. The model protease, HCV-4a, shares 83% sequence identity with the template protease, HCV-1b, and has nearly identical rigid structural features. Molecular dynamics simulations predict similar overall dynamics of the two proteases. However, local dynamics and 4D analysis of the interactions between the catalytic triad residues (His57, Asp81, and Ser139) indicate conformational instability of the catalytic site in HCV-4a NS3 protease. These results suggest that the divergent dynamics behavior, more than the rigid structure, could be related to the altered catalytic activity and drug resistivity seen in HCV-4a.     

A Prospective Study of Seasonal Variation in Shift‐Work Tolerance

Seasonal effects on shift‐work tolerance were assessed using the Standardized Shiftwork Index and the 21‐item Hamilton Depression Scale. Participants (N=88) mainly worked a two‐day, two‐night, four‐off rotation with 12 h shifts changing at 06∶00 and 18∶00 h in Vancouver, Canada. At this latitude (～49° N), daylength varies seasonally from ～16 to ～8 h, and both daily commutes occur in the dark in mid‐winter and in sunlight in mid‐summer. Questionnaires were completed twice, near the summer and winter solstices (order counterbalanced). Outcome variables were mood, general psychological health, sleep quality, chronic fatigue, physical health, job satisfaction, and social and domestic disruption. Of these, general psychological health and mood were significantly worse in winter, while sleep was more disturbed in summer. In winter, 31% exceeded the cutoff for psychological distress, and >70% scored in the higher than normal range for depressive symptoms. In summer, the proportions dropped to 19% and 53%, respectively. Measures of physical health and psychosocial well‐being showed no seasonal effects. Relationships among explanatory and outcome variables, assessed by linear regression and canonical correlations, were also stable across season. Neuroticism was the strongest predictor of tolerance to shift work. Age was predictive only of sleep disturbance in both summer and winter. These results indicate that time of year can affect important outcome measures in shift‐work assessment and intervention studies. The high average scores on measures of psychological distress and depression in winter suggest that at northern latitudes, some shift schedules may increase the risk of seasonal‐type depression.     

Wastewater Treatment Effluent Reduces the Abundance and Diversity of Benthic Bacterial Communities in Urban and Suburban Rivers

In highly urbanized areas, wastewater treatment plant (WWTP) effluent can represent a significant component of freshwater ecosystems. As it is impossible for the composition of WWTP effluent to match the composition of the receiving system, the potential exists for effluent to significantly impact the chemical and biological characteristics of the receiving ecosystem. We assessed the impacts of WWTP effluent on the size, activity, and composition of benthic microbial communities by comparing two distinct field sites in the Chicago metropolitan region: a highly urbanized river receiving effluent from a large WWTP and a suburban river receiving effluent from a much smaller WWTP. At sites upstream of effluent input, the urban and suburban rivers differed significantly in chemical characteristics and in the composition of their sediment bacterial communities. Although effluent resulted in significant increases in inorganic nutrients in both rivers, surprisingly, it also resulted in significant decreases in the population size and diversity of sediment bacterial communities. Tag pyrosequencing of bacterial 16S rRNA genes revealed significant effects of effluent on sediment bacterial community composition in both rivers, including decreases in abundances of Deltaproteobacteria, Desulfococcus, Dechloromonas, and Chloroflexi sequences and increases in abundances of Nitrospirae and Sphingobacteriales sequences. The overall effect of the WWTP inputs was that the two rivers, which were distinct in chemical and biological properties upstream of the WWTPs, were almost indistinguishable downstream. These results suggest that WWTP effluent has the potential to reduce the natural variability that exists among river ecosystems and indicate that WWTP effluent may contribute to biotic homogenization.     

Lymphatic and Angiogenic Candidate Genes Predict the Development of Secondary Lymphedema following Breast Cancer Surgery

The purposes of this study were to evaluate for differences in phenotypic and genotypic characteristics in women who did and did not develop lymphedema (LE) following breast cancer treatment. Breast cancer patients completed a number of self-report questionnaires. LE was evaluated using bioimpedance spectroscopy. Genotyping was done using a custom genotyping array. No differences were found between patients with (n = 155) and without LE (n = 387) for the majority of the demographic and clinical characteristics. Patients with LE had a significantly higher body mass index, more advanced disease and a higher number of lymph nodes removed. Genetic associations were identified for four genes (i.e., lymphocyte cytosolic protein 2 (rs315721), neuropilin-2 (rs849530), protein tyrosine kinase (rs158689), vascular cell adhesion molecule 1 (rs3176861)) and three haplotypes (i.e., Forkhead box protein C2 (haplotype A03), neuropilin-2 (haplotype F03), vascular endothelial growth factor-C (haplotype B03)) involved in lymphangiogensis and angiogenesis. These genetic associations suggest a role for a number of lymphatic and angiogenic genes in the development of LE following breast cancer treatment.     

Identification of a Novel Human Papillomavirus by Metagenomic Analysis of Samples from Patients with Febrile Respiratory Illness

As part of a virus discovery investigation using a metagenomic approach, a highly divergent novel Human papillomavirus type was identified in pooled convenience nasal/oropharyngeal swab samples collected from patients with febrile respiratory illness. Phylogenetic analysis of the whole genome and the L1 gene reveals that the new HPV identified in this study clusters with previously described gamma papillomaviruses, sharing only 61.1% (whole genome) and 63.1% (L1) sequence identity with its closest relative in the Papillomavirus episteme (PAVE) database. This new virus was named HPV_SD2 pending official classification. The complete genome of HPV-SD2 is 7,299 bp long (36.3% G/C) and contains 7 open reading frames (L2, L1, E6, E7, E1, E2 and E4) and a non-coding long control region (LCR) between L1 and E6. The metagenomic procedures, coupled with the bioinformatic methods described herein are well suited to detect small circular genomes such as those of human papillomaviruses.     

A Luminex Assay Detects Amyloid β Oligomers in Alzheimer’s Disease Cerebrospinal Fluid

Amyloid beta (aβ) protein assembles into larger protein aggregates during the pathogenesis of Alzheimer’s disease (AD) and there is increasing evidence that soluble aβ oligomers are a critical pathologic species. Diagnostic evaluations rely on the measurement of increased tau and decreased aβ42 in the cerebrospinal fluid (CSF) from AD patients and evidence for oligomeric aβ in patient CSF is conflicting. In this study, we have adapted a monoclonal single antibody sandwich ELISA assay to a Luminex platform and found that this assay can detect oligomerized aβ42 and sAPPα fragments. We evaluated oligomeric aβ reactivity in 20 patients with AD relative to 19 age matched controls and compared these values with a commercially available Alzbio3 kit that detects tau, phosphorylated tau and aβ42 on the same diagnostic platform. We found that CSF samples of patients with AD had elevated aβ oligomers compared to control subjects (p < 0.05) and the ratio of aβ oligomers to aβ42 was also significantly elevated (p < 0.0001). Further research to develop high sensitivity analytical platforms and rigorous methods of developing stable assay standards will be needed before the analysis of oligomeric aβ becomes a routine diagnostic assay for the evaluation of late onset AD patients.     

Resveratrol Induces Premature Senescence in Lung Cancer Cells via ROS-Mediated DNA Damage

Resveratrol (RV) is a natural component of red wine and grapes that has been shown to be a potential chemopreventive and anticancer agent. However, the molecular mechanisms underlying RV''s anticancer and chemopreventive effects are incompletely understood. Here we show that RV treatment inhibits the clonogenic growth of non-small cell lung cancer (NSCLC) cells in a dose-dependent manner. Interestingly, the tumor-suppressive effect of low dose RV was not associated with any significant changes in the expression of cleaved PARP and activated caspase-3, suggesting that low dose RV treatment may suppress tumor cell growth via an apoptosis-independent mechanism. Subsequent studies reveal that low dose RV treatment induces a significant increase in senescence-associated β–galactosidase (SA-β-gal) staining and elevated expression of p53 and p21 in NSCLC cells. Furthermore, we show that RV-induced suppression of lung cancer cell growth is associated with a decrease in the expression of EF1A. These results suggest that RV may exert its anticancer and chemopreventive effects through the induction of premature senescence. Mechanistically, RV-induced premature senescence correlates with increased DNA double strand breaks (DSBs) and reactive oxygen species (ROS) production in lung cancer cells. Inhibition of ROS production by N-acetylcysteine (NAC) attenuates RV-induced DNA DSBs and premature senescence. Furthermore, we show that RV treatment markedly induces NAPDH oxidase-5 (Nox5) expression in both A549 and H460 cells, suggesting that RV may increase ROS generation in lung cancer cells through upregulating Nox5 expression. Together, these findings demonstrate that low dose RV treatment inhibits lung cancer cell growth via a previously unappreciated mechanism, namely the induction of premature senescence through ROS-mediated DNA damage.     

Measuring the immune system of the three‐spined stickleback – investigating natural variation by quantifying immune expression in the laboratory and the wild

Current understanding of the immune system comes primarily from laboratory‐based studies. There has been substantial interest in examining how it functions in the wild, but studies have been limited by a lack of appropriate assays and study species. The three‐spined stickleback (Gasterosteus aculeatus L.) provides an ideal system in which to advance the study of wild immunology, but requires the development of suitable immune assays. We demonstrate that meaningful variation in the immune response of stickleback can be measured using real‐time PCR to quantify the expression of eight genes, representing the innate response and Th1‐, Th2‐ and Treg‐type adaptive responses. Assays are validated by comparing the immune expression profiles of wild and laboratory‐raised stickleback, and by examining variation across populations on North Uist, Scotland. We also compare the immune response potential of laboratory‐raised individuals from two Icelandic populations by stimulating cells in culture. Immune profiles of wild fish differed from laboratory‐raised fish from the same parental population, with immune expression patterns in the wild converging relative to those in the laboratory. Innate measures differed between wild populations, whilst the adaptive response was associated with variation in age, relative size of fish, reproductive status and S. solidus infection levels. Laboratory‐raised individuals from different populations showed markedly different innate immune response potential. The ability to combine studies in the laboratory and in the wild underlines the potential of this toolkit to advance our understanding of the ecological and evolutionary relevance of immune system variation in a natural setting.