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.     

Detection of Proton Movement Directly across Viral Membranes To Identify Novel Influenza Virus M2 Inhibitors

The influenza virus M2 protein is a well-validated yet underexploited proton-selective ion channel essential for influenza virus infectivity. Because M2 is a toxic viral ion channel, existing M2 inhibitors have been discovered through live virus inhibition or medicinal chemistry rather than M2-targeted high-throughput screening (HTS), and direct measurement of its activity has been limited to live cells or reconstituted lipid bilayers. Here, we describe a cell-free ion channel assay in which M2 ion channels are incorporated into virus-like particles (VLPs) and proton conductance is measured directly across the viral lipid bilayer, detecting changes in membrane potential, ion permeability, and ion channel function. Using this approach in high-throughput screening of over 100,000 compounds, we identified 19 M2-specific inhibitors, including two novel chemical scaffolds that inhibit both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of viral bilayer ion permeability also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane. In addition to its application to M2 and potentially other ion channels, this technology enables direct measurement of the electrochemical and biophysical characteristics of viral membranes.     

AMP-activated protein kinase and ATP-citrate lyase are two distinct molecular targets for ETC-1002, a novel small molecule regulator of lipid and carbohydrate metabolism

ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid β-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca²⁺/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease.     

Mosaic Evolution of Silk Genes in Aliatypus Trapdoor Spiders (Mygalomorphae, Antrodiaetidae)

Spider silk genes are composed mostly of repetitive sequence that is flanked by non-repetitive terminal regions. Inferences about the evolutionary processes that influenced silk genes have largely been made from analyses using distantly related taxa and ancient silk gene duplicates. These studies have relied on comparisons across the conserved non-repetitive terminal regions to determine orthologous and paralogous relationships, as well as the influence of selection on silk genes. While the repetitive region heavily influences silk fiber mechanical properties, few molecular evolutionary analyses have been conducted on this region due to difficulty in determining homology. Here, we sample internal repetitive and carboxy terminal regions from all extant species of the trapdoor spider genus, Aliatypus. Aliatypus spiders are highly dispersal limited and rely on their silk lined burrow for protection. We determine positional homology across species for the carboxy terminal regions and relative positional homology for the internal repetitive regions. Gene trees based on each of these regions are in good agreement with the Aliatypus species tree, which indicates we sampled single spidroin orthologs in each species. In addition, we find that purifying selection and concerted evolution have acted to conserve Aliatypus spidroin internal repetitive regions. In contrast, selection testing identifies evidence of sites that evolved under positive selection and amino acid replacements that result in radical physicochemical changes in the carboxy terminal region. These findings indicate that comparison of spidroin orthologs across a comprehensive sample of congenerics reveal molecular evolutionary patterns obscured from studies using higher-level sampling of silk encoding genes.     

Post-translational membrane insertion of an endogenous YidC substrate

Membrane protein insertion is controlled by proteinaceous factors embedded in the lipid bilayer. Bacterial inner membrane proteins utilise the Sec translocon as the major facilitator of insertion; however some proteins are Sec independent and instead require only YidC. A common feature of YidC substrates is the exposure of a signal anchor sequence when translation is close to completion; this allows minimal time for targeting and favours a post-translational insertion mechanism. Despite this there is little evidence of YidC's post-translational activity. Here we develop an experimental system that uncouples translation and insertion of the endogenous YidC substrate F₀c (subunit c of the F₀F₁ ATP synthase). In this process we (i) develop a novel one step purification method for YidC, including an on column membrane reconstitution, (ii) isolate a soluble form of F₀c and (iii) show that incubation of F₀c with YidC proteoliposomes results in a high level of membrane integration. Conformational analyses of inserted F₀c through Blue Native PAGE and fluorescence quenching reveal a native, oligomerised structure. These data show that YidC can act as a post-translational insertase, a finding which could explain the absence of a ribosome binding domain on YidC. This correlates with the post-translational activity of other YidC family members lacking the ribosome binding domain.     

Sexual differentiation of the adolescent rodent brain: Hormonal influences and developmental mechanisms

This article is part of a Special Issue “Puberty and Adolescence”.     

Pyrido[2,3-d]pyrimidines: Discovery and preliminary SAR of a novel series of DYRK1B and DYRK1A inhibitors

DYRK1B is a kinase over-expressed in certain cancer cells (including colon, ovarian, pancreatic, etc.). Recent publications have demonstrated inhibition of DYRK1B could be an attractive target for cancer therapy. From a data-mining effort, the team has discovered analogues of pyrido[2,3-d]pyrimidines as potent enantio-selective inhibitors of DYRK1B. Cells treated with a tool compound from this series showed the same cellular effects as down regulation of DYRK1B with siRNA. Such effects are consistent with the proposed mechanism of action. Progress of the SAR study is presented.     

Vertical Transmission of Respiratory Syncytial Virus Modulates Pre- and Postnatal Innervation and Reactivity of Rat Airways

Background

Environmental exposure to respiratory syncytial virus (RSV) is a leading cause of respiratory infections in infants, but it remains unknown whether this infection is transmitted transplacentally from the lungs of infected mothers to the offspring. We sought to test the hypothesis that RSV travels from the respiratory tract during pregnancy, crosses the placenta to the fetus, persists in the lung tissues of the offspring, and modulates pre- and postnatal expression of growth factors, thereby predisposing to airway hyperreactivity.

Methodology

Pregnant rats were inoculated intratracheally at midterm using recombinant RSV expressing red fluorescent protein (RFP). Viral RNA was amplified by RT-PCR and confirmed by sequencing. RFP expression was analyzed by flow cytometry and viral culture. Developmental and pathophysiologic implications of prenatal infection were determined by analyzing the expression of genes encoding critical growth factors, particularly neurotrophic factors and receptors. We also measured the expression of key neurotransmitters and postnatal bronchial reactivity in vertically infected lungs, and assessed their dependence on neurotrophic signaling using selective biological or chemical inhibition.

Principal Findings

RSV genome was found in 30% of fetuses, as well as in the lungs of 40% of newborns and 25% of adults. RFP expression was also shown by flow cytometry and replicating virus was cultured from exposed fetuses. Nerve growth factor and its TrkA receptor were upregulated in RSV- infected fetal lungs and co-localized with increased cholinergic innervation. Acetylcholine expression and smooth muscle response to cholinergic stimulation increased in lungs exposed to RSV in utero and reinfected after birth, and blocking TrkA signaling inhibited both effects.

Conclusions/Significance

Our data show transplacental transmission of RSV from mother to offspring and persistence of vertically transmitted virus in lungs after birth. Exposure to RSV in utero is followed by dysregulation of neurotrophic pathways predisposing to postnatal airway hyperreactivity upon reinfection with the virus.