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Aligned collagen architecture is a characteristic feature of the tumor extracellular matrix (ECM) and has been shown to facilitate cancer metastasis using 3D in vitro models. Additional features of the ECM, such as pore size and stiffness, have also been shown to influence cellular behavior and are implicated in cancer progression. While there are several methods to produce aligned matrices to study the effect on cell behavior in vitro, it is unclear how the alignment itself may alter these other important features of the matrix. In this study, we have generated aligned collagen matrices and characterized their pore sizes and mechanical properties at the micro- and macro-scale. Our results indicate that collagen alignment can alter pore-size of matrices depending on the polymerization temperature of the collagen. Furthermore, alignment does not affect the macro-scale stiffness but alters the micro-scale stiffness in a temperature independent manner. Overall, these results describe the manifestation of confounding variables that arise due to alignment and the importance of fully characterizing biomaterials at both micro- and macro-scales. [ABSTRACT FROM AUTHOR]

The upwelling of deep waters from the oxygen minimum zone in the Northeast Pacific from the continental slope to the shelf and into the Salish Sea during spring and summer offers a unique opportunity to study ecosystem functioning in the form of benthic fluxes along natural gradients. Using the ROV ROPOS we collected sediment cores from 10 sites in May and July 2011, and September 2013 to perform shipboard incubations and flux measurements. Specifically, we measured benthic fluxes of oxygen and nutrients to evaluate potential environmental drivers of benthic flux variation and ecosystem functioning along natural gradients of temperature and bottom water dissolved oxygen concentrations. The range of temperature and dissolved oxygen encountered across our study sites allowed us to apply a suite of multivariate analyses rarely used in flux studies to identify bottom water temperature as the primary environmental driver of benthic flux variation and organic matter remineralization. Redundancy analysis revealed that bottom water characteristics (temperature and dissolved oxygen), quality of organic matter (chl a:phaeo and C:N ratios) and sediment characteristics (mean grain size and porosity) explained 51.5% of benthic flux variation. Multivariate analyses identified significant spatial and temporal variation in benthic fluxes, demonstrating key differences between the Northeast Pacific and Salish Sea. Moreover, Northeast Pacific slope fluxes were generally lower than shelf fluxes. Spatial and temporal variation in benthic fluxes in the Salish Sea were driven primarily by differences in temperature and quality of organic matter on the seafloor following phytoplankton blooms. These results demonstrate the utility of multivariate approaches in differentiating among potential drivers of seafloor ecosystem functioning, and indicate that current and future predictive models of organic matter remineralization and ecosystem functioning of soft-muddy shelf and slope seafloor habitats should consider bottom water temperature variation. Bottom temperature has important implications for estimates of seasonal and spatial benthic flux variation, benthic–pelagic coupling, and impacts of predicted ocean warming at high latitudes. [ABSTRACT FROM AUTHOR]

Recombinant subunit vaccines should contain minimal non-pathogen motifs to reduce potential off-target reactivity. We recently developed a vaccine antigen against respiratory syncytial virus (RSV), which comprised the fusion (F) glycoprotein stabilized in its pre-fusion trimeric conformation by “DS-Cav1” mutations and by an appended C-terminal trimerization motif or “foldon” from T4-bacteriophage fibritin. Here we investigate the creation of a cysteine zipper to allow for the removal of the phage foldon, while maintaining the immunogenicity of the parent DS-Cav1+foldon antigen. Constructs without foldon yielded RSV F monomers, and enzymatic removal of the phage foldon from pre-fusion F trimers resulted in their dissociation into monomers. Because the native C terminus of the pre-fusion RSV F ectodomain encompasses a viral trimeric coiled-coil, we explored whether introduction of cysteine residues capable of forming inter-protomer disulfides might allow for stable trimers. Structural modeling indicated the introduced cysteines to form disulfide “rings”, with each ring comprising a different set of inward facing residues of the coiled-coil. Three sets of rings could be placed within the native RSV F coiled-coil, and additional rings could be added by duplicating portions of the coiled-coil. High levels of neutralizing activity in mice, equivalent to that of the parent DS-Cav1+foldon antigen, were elicited by a 4-ring stabilized RSV F trimer with no foldon. Structure-based alteration of a viral coiled-coil to create a cysteine zipper thus allows a phage trimerization motif to be removed from a candidate vaccine antigen. [ABSTRACT FROM AUTHOR]

Whole genome sequencing revealed the presence of a genomic anomaly in the region of 4.7 to 4.9 Mb of the Pseudomonas syringae pv. tomato (Pst) DC3000 genome. The average read depth coverage of Pst DC3000 whole genome sequencing results suggested that a 165 kb segment of the chromosome had doubled in copy number. Further analysis confirmed the 165 kb duplication and that the two copies were arranged as a direct tandem repeat. Examination of the corresponding locus in Pst NCPPB1106, the parent strain of Pst DC3000, suggested that the 165 kb duplication most likely formed after the two strains diverged via transposition of an ISPsy5 insertion sequence (IS) followed by unequal crossing over between ISPsy5 elements at each end of the duplicated region. Deletion of one copy of the 165 kb region demonstrated that the duplication facilitated enhanced growth in some culture conditions, but did not affect pathogenic growth in host tomato plants. These types of chromosomal structures are predicted to be unstable and we have observed resolution of the 165 kb duplication to single copy and its subsequent re-duplication. These data demonstrate the role of IS elements in recombination events that facilitate genomic reorganization in P. syringae. [ABSTRACT FROM AUTHOR]

During the pelagic larval phase, fish dispersal may be influenced passively by surface currents or actively determined by swimming behaviour. In situ observations of larval swimming are few given the constraints of field sampling. Active behaviour is therefore often inferred from spatial patterns in the field, laboratory studies, or hydrodynamic theory, but rarely are these approaches considered in concert. Ichthyoplankton survey data collected during 2004 and 2006 from coastal Newfoundland show that changes in spatial heterogeneity for multiple species do not conform to predictions based on passive transport. We evaluated the interaction of individual larvae with their environment by calculating Reynolds number as a function of ontogeny. Typically, larvae hatch into a viscous environment in which swimming is inefficient, and later grow into more efficient intermediate and inertial swimming environments. Swimming is therefore closely related to length, not only because of swimming capacity but also in how larvae experience viscosity. Six of eight species sampled demonstrated consistent changes in spatial patchiness and concomitant increases in spatial heterogeneity as they transitioned into more favourable hydrodynamic swimming environments, suggesting an active behavioural element to dispersal. We propose the tandem assessment of spatial heterogeneity and hydrodynamic environment as a potential approach to understand and predict the onset of ecologically significant swimming behaviour of larval fishes in the field. [ABSTRACT FROM AUTHOR]

Physical activity reduces the incidence and severity of psychiatric disorders such as anxiety and depression. Similarly, voluntary wheel running produces anxiolytic- and antidepressant-like effects in rodent models. The specific neurobiological mechanisms underlying the beneficial properties of exercise, however, remain unclear. One relevant pharmacological target in the treatment of psychiatric disorders is the 5-HT2C receptor (5-HT2CR). Consistent with data demonstrating the anxiogenic consequences of 5-HT2CR activation in humans and rodents, we have previously reported that site-specific administration of the selective 5-HT2CR agonist CP-809101 in the lateral/basolateral amygdala (BLA) increases shock-elicited fear while administration of CP-809101 in the dorsal striatum (DS) interferes with shuttle box escape learning. These findings suggest that activation of 5-HT2CR in discrete brain regions contributes to specific anxiety- and depression-like behaviors and may indicate potential brain sites involved in the anxiolytic and antidepressant effects of exercise. The current studies tested the hypothesis that voluntary wheel running reduces the behavioral consequences of 5-HT2CR activation in the BLA and DS, specifically enhanced shock-elicited fear and interference with shuttle box escape learning. After 6 weeks of voluntary wheel running or sedentary conditions, the selective 5-HT2CR agonist CP-809101 was microinjected into either the BLA or the DS of adult Fischer 344 rats, and shock-elicited fear and shuttle box escape learning was assessed. Additionally, in-situ hybridization was used to determine if 6 weeks of voluntary exercise changed levels of 5-HT2CR mRNA. We found that voluntary wheel running reduced the behavioral effects of CP-809101 and reduced levels of 5-HT2CR mRNA in both the BLA and the DS. The current data indicate that expression of 5-HT2CR mRNA in discrete brain sites is sensitive to physical activity status of the organism, and implicates the 5-HT2CR as a target for the beneficial effects of physical activity on mental health. [ABSTRACT FROM AUTHOR]

RNA-Seq has provided valuable insights into global gene expression in a wide variety of organisms. Using a modified RNASeq approach and Illumina's high-throughput sequencing technology, we globally identified 5'-ends of transcripts for the plant pathogen Pseudomonas syringae pv. tomato str. DC3000. A substantial fraction of 5'-ends obtained by this method were consistent with results obtained using global RNA-Seq and 59RACE. As expected, many 5'-ends were positioned a short distance upstream of annotated genes. We also captured 5'-ends within intergenic regions, providing evidence for the expression of un-annotated genes and non-coding RNAs, and detected numerous examples of antisense transcription, suggesting additional levels of complexity in gene regulation in DC3000. Importantly, targeted searches for sequence patterns in the vicinity of 5'-ends revealed over 1200 putative promoters and other regulatory motifs, establishing a broad foundation for future investigations of regulation at the genomic and single gene levels. [ABSTRACT FROM AUTHOR]

Aims: Long term survivors of haematopoietic stem cell transplantation (HSCT) for β-thalassemia major are designated "ex-thalassaemics". Whether ex-thalassaemics continue to harbour residual myocardial dysfunction and thereby stand the risk of heart failure-related morbidity and mortality is unknown. The aim of this study was to assess the prevalence and predictors of subclinical left ventricular (LV) dysfunction in an apparently normal ex-thalassaemic population. Methods: We conducted a single centre cross-sectional study among 62 ex-thalassaemic patients, who had undergone HSCT for β-thalassaemia major at our centre. The primary outcome variable was LV systolic dysfunction, as assessed by 1) LV global longitudinal strain (GLS) derived by 2D speckle tracking echocardiography and 2) LV ejection fraction (EF) derived by 2D Simpsons Biplane method. Results: Among the 62 patients included in the study, 7 [11.3%] were found to have LV systolic dysfunction, all of which were subclinical. Of these, 4 [6.5%] had abnormal GLS and LVEF, 2 [3.2%] had abnormal GLS with normal LVEF, and 1 [1.6%] had abnormal LVEF with low normal mean GLS. There were no statistically significant predictors of LV dysfunction in this cohort. Conclusion: There was a high prevalence of subclinical myocardial dysfunction in the ex-thalassaemic population reiterating the need for close follow up of these patients. 2D Speckle tracking echocardiography-derived LV global longitudinal strain is an effective tool in detecting subclinical myocardial dysfunction in this cohort. [ABSTRACT FROM AUTHOR]

Background: Mosquitoes in the Culex pipiens complex are among the most medically important vectors for human disease worldwide and include major vectors for lymphatic filariasis and West Nile virus transmission. However, detailed genetic studies in the complex are limited by the number of genetic markers available. Here, we describe methods for the rapid and efficient identification and development of single locus, highly polymorphic microsatellite markers for Cx. pipiens complex mosquitoes via in silico screening of the Cx. quinquefasciatus genome sequence. Methodology/Principal Findings: Six lab colonies representing four Cx. pipiens and two Cx. quinquefasciatus populations were utilized for preliminary assessment of 38 putative loci identified within 16 Cx. quinquefasciatus supercontig assemblies (CpipJ1) containing previously mapped genetic marker sequences. We identified and validated 12 new microsatellite markers distributed across all three linkage groups that amplify consistently among strains representing the complex. We also developed four groups of 3-5 microsatellite loci each for multiplex-ready PCR. Field collections from three cities in Indiana were used to assess the multiplex groups for their application to natural populations. All were highly polymorphic (Mean = 13.0 alleles) per locus and reflected high polymorphism information content (PIC) (Mean = 0.701). Pairwise FST indicated population structuring between Terre Haute and Fort Wayne and between Terre Haute and Indianapolis, but not between Fort Wayne and Indianapolis. In addition, we performed whole genome comparisons of microsatellite motifs and abundance between Cx. quinquefasciatus and the primary vectors for dengue virus and malaria parasites, Aedes aegypti and Anopheles gambiae, respectively. Conclusions/Significance: We demonstrate a systematic approach for isolation and validation of microsatellites for the Cx. pipiens complex by direct screen of the Cx. quinquefasciatus genome supercontig assemblies. The genome density of microsatellites is greater in Cx. quinquefasciatus (0.26%) than in Ae. aegypti (0.14%), but considerably lower than in An. gambiae (0.77%). [ABSTRACT FROM AUTHOR]

Evaluating and understanding biodiversity in marine ecosystems are both necessary and challenging for conservation. This paper compiles and summarizes current knowledge of the diversity of marine taxa in Canada's three oceans while recognizing that this compilation is incomplete and will change in the future. That Canada has the longest coastline in the world and incorporates distinctly different biogeographic provinces and ecoregions (e.g., temperate through ice-covered areas) constrains this analysis. The taxonomic groups presented here include microbes, phytoplankton, macroalgae, zooplankton, benthic infauna, fishes, and marine mammals. The minimum number of species or taxa compiled here is 15,988 for the three Canadian oceans. However, this number clearly underestimates in several ways the total number of taxa present. First, there are significant gaps in the published literature. Second, the diversity of many habitats has not been compiled for all taxonomic groups (e.g., intertidal rocky shores, deep sea), and data compilations are based on short-term, directed research programs or longer-term monitoring activities with limited spatial resolution. Third, the biodiversity of large organisms is well known, but this is not true of smaller organisms. Finally, the greatest constraint on this summary is the willingness and capacity of those who collected the data to make it available to those interested in biodiversity meta-analyses. Confirmation of identities and intercomparison of studies are also constrained by the disturbing rate of decline in the number of taxonomists and systematists specializing on marine taxa in Canada. This decline is mostly the result of retirements of current specialists and to a lack of training and employment opportunities for new ones. Considering the difficulties encountered in compiling an overview of biogeographic data and the diversity of species or taxa in Canada's three oceans, this synthesis is intended to serve as a biodiversity baseline for a new program on marine biodiversity, the Canadian Healthy Ocean Network. A major effort needs to be undertaken to establish a complete baseline of Canadian marine biodiversity of all taxonomic groups, especially if we are to understand and conserve this part of Canada's natural heritage. [ABSTRACT FROM AUTHOR]

In vitro studies have shown that the phosphoprotein osteopontin (OPN) inhibits the nucleation and growth of hydroxyapatite (HA) and other biominerals. In vivo, OPN is believed to prevent the calcification of soft tissues. However, the nature of the interaction between OPN and HA is not understood. In the computational part of the present study, we used molecular dynamics simulations to predict the adsorption of 19 peptides, each 16 amino acids long and collectively covering the entire sequence of OPN, to the {100} face of HA. This analysis showed that there is an inverse relationship between predicted strength of adsorption and peptide isoelectric point (P<0.0001). Analysis of the OPN sequence by PONDR (Predictor of Naturally Disordered Regions) indicated that OPN sequences predicted to adsorb well to HA are highly disordered. In the experimental part of the study, we synthesized phosphorylated and non-phosphorylated peptides corresponding to OPN sequences 65-80 (pSHDHMDDDDDDDDDGD) and 220-235 (pSHEpSTEQSDAIDpSAEK). In agreement with the PONDR analysis, these were shown by circular dichroism spectroscopy to be largely disordered. A constant-composition/ seeded growth assay was used to assess the HA-inhibiting potencies of the synthetic peptides. The phosphorylated versions of OPN65-80 (IC50 = 1.93 μg/ml) and OPN220-235 (IC50 = 1.48 μg/ml) are potent inhibitors of HA growth, as is the nonphosphorylated version of OPN65-80 (IC50 = 2.97 μg/ml); the nonphosphorylated version of OPN220-235 has no measurable inhibitory activity. These findings suggest that the adsorption of acidic proteins to Ca2+-rich crystal faces of biominerals is governed by electrostatics and is facilitated by conformational flexibility of the polypeptide chain. [ABSTRACT FROM AUTHOR]

Despite substantial research and policy interest in pixel level cropland allocation data, few sources are available that span a large geographic area. The data used for much of this research are derived from complex modeling techniques that may include model simulation and other data processing. We develop a transparent econometric framework that uses pixel level biophysical measurements and aggregate cropland statistics to develop pixel level cropland allocation predictions. Such pixel level land use data can be used to investigate the impact of human activities on the environment. Validation exercises show that our approach is effective at downscaling cropland allocation to multiple levels of resolution. [ABSTRACT FROM AUTHOR]

Background and objective: The mechanism of dyspnoea associated with pleural effusion is uncertain. A cohort of patients requiring thoracoscopy for unilateral exudative effusion were investigated for associations between dyspnoea and suggested predictors: impaired ipsilateral diaphragm movement, effusion volume and restricted lung inflation. Methods: Baseline Dyspnoea Index, respiratory function, and ultrasound assessment of ipsilateral diaphragm movement were assessed prior to thoracoscopy, when effusion volume was measured. Transitional Dyspnoea Index (change from baseline) was assessed 4 and 8 weeks after thoracoscopy. Pearson product moment assessed bivariate correlations and a general linear model examined how well total lung capacity (measuring restricted lung inflation), effusion volume and impaired diaphragm movement predicted Baseline Dyspnoea Index. Un-paired t tests compared the groups with normal and impaired diaphragm movement. Results: 19 patients were studied (14 malignant etiology). Total lung capacity was associated with Baseline Dyspnoea Index (r = 0.68, P = 0.003). Effusion volume (r = -0.138, P = 0.60) and diaphragm movement (P = 0.09) were not associated with Baseline Dyspnoea Index. Effusion volume was larger with impaired diaphragm movement compared to normal diaphragm movement (2.16 ±SD 0.95 vs.1.16 ±0.92 L, P = 0.009). Total lung capacity was lower with impaired diaphragm movement compared to normal diaphragm movement (65.4 ±10.3 vs 78.2 ±8.6% predicted, P = 0.011). The optimal general linear model to predict Baseline Dyspnoea Index used total lung capacity alone (adjusted R2 = 0.42, P = 0.003). In nine participants with controlled effusion, baseline effusion volume (r = 0.775, P = 0.014) and total lung capacity (r = -0.690, P = 0.040) were associated with Transitional Dyspnoea Index. Conclusions: Restricted lung inflation was the principal predictor of increased dyspnoea prior to thoracoscopic drainage of effusion, with no independent additional association with either effusion volume or impaired ipsilateral diaphragm movement. Restricted lung inflation may be an important determinant of the dyspnoea associated with pleural effusion. [ABSTRACT FROM AUTHOR]

To understand coastal dispersal dynamics of Atlantic cod (Gadus morhua), we examined spatiotemporal egg and larval abundance patterns in coastal Newfoundland. In recent decades, Smith Sound, Trinity Bay has supported the largest known overwintering spawning aggregation of Atlantic cod in the region. We estimated spawning and dispersal characteristics for the Smith Sound-Trinity Bay system by fitting ichthyoplankton abundance data to environmentally-driven, simplified box models. Results show protracted spawning, with sharply increased egg production in early July, and limited dispersal from the Sound. The model for the entire spawning season indicates egg export from Smith Sound is 13%•day−1 with a net mortality of 27%•day–1. Eggs and larvae are consistently found in western Trinity Bay with little advection from the system. These patterns mirror particle tracking models that suggest residence times of 10–20 days, and circulation models indicating local gyres in Trinity Bay that act in concert with upwelling dynamics to retain eggs and larvae. Our results are among the first quantitative dispersal estimates from Smith Sound, linking this spawning stock to the adjacent coastal waters. These results illustrate the biophysical interplay regulating dispersal and connectivity originating from inshore spawning of coastal northwest Atlantic. [ABSTRACT FROM AUTHOR]

The formation of spatial genetic structure (SGS) may originate from different patterns of seed deposition in the landscape, and is mostly determined by seed dispersal limitation. After dispersal, mechanisms such as filtering by environmental factors or attack by herbivores/pathogens throughout plant development stages, and potentially either disrupt or intensify SGS patterns. We investigated how the genotype of Protium subserratum (Burseraceae), a common tree species in the Ducke Reserve, Brazil, is distributed across the landscape. We used seven microsatellite markers to assess the SGS among plants at different life stages and in different environments. By quantifying the patterns of relatedness among plants of different sizes, we inferred the ontogenetic stage in which SGS changes occurred, and compared these effects across soil types. Relatedness among seedlings decreased when distance between seedlings increased, especially for the youngest seedlings. However, this trend was not continued by older plants, as relatedness values were higher among neighboring individuals of the juvenile and adult size class. Contrasting relatedness patterns between seedlings and larger individuals suggests a trade-off between the negative effects of being near closely-related adults (e.g. due to herbivore and pathogen attack) and the advantage of being in a site favorable to establishment. We also found that soil texture strongly influenced density-dependence patterns, as young seedlings in clay soils were more related to each other than were seedlings in bottomland sandy soils, suggesting that the mechanisms that create and maintain patterns of SGS within a population may interact with environmental heterogeneity. [ABSTRACT FROM AUTHOR]

Cyclodextrins are sugar compounds that are increasingly finding medicinal uses due to their ability to complex with hydrophobic molecules. One cyclodextrin in particular, 2-hydroxypropyl-b-cyclodextrin (HPβCD), is used as a carrier to solubilize lipophilic drugs and is itself being considered as a therapeutic agent for treatment of Niemann-Pick Type C disease, due to its ability to mobilize cholesterol. Results from toxicological studies suggest that HPbCD is generally safe, but a recent study has found that it causes hearing loss in cats. Whether the hearing loss occurred via death of cochlear hair cells, rendering it permanent, was unexplored. In the present study, we examined peripheral auditory function and cochlear histology in mice after subcutaneous injection of HPbCD to test for hearing loss and correlate any observed auditory deficits with histological findings. On average, auditory brainstem response thresholds were elevated at 4, 16, and 32 kHz in mice one week after treatment with 8,000 mg/kg. In severely affected mice all outer hair cells were missing in the basal half of the cochlea. In many cases, surviving hair cells in the cochlear apex exhibited abnormal punctate distribution of the motor protein prestin, suggesting long term changes to membrane composition and integrity. Mice given a lower dose of 4,000 mg/kg exhibited hearing loss only after repeated doses, but these threshold shifts were temporary. Therefore, cyclodextrin-induced hearing loss was complex, involving cell death and other more subtle influences on cochlear physiology. [ABSTRACT FROM AUTHOR]

Background: Human cadavers are crucial to numerous aspects of health care, including initial and continuing training of medical doctors and advancement of medical research. Concerns have periodically been raised about the limited number of whole body donations. Little is known, however, about a unique form of donation, namely co-donations or instances when married individuals decide to register at the same time as their spouse as whole body donors. Our study aims to determine the extent of whole body co-donation and individual factors that might influence co-donation. Methods and Findings: We reviewed all records of registrants to the University of Hawaii Medical School's whole body donation program from 1967 through 2006 to identify married registrants. We then examined the 806 married individuals' characteristics to understand their decision to register alone or with their spouse. We found that married individuals who registered at the same time as their spouse accounted for 38.2 percent of married registrants. Sex differences provided an initial lens to understand co- donation. Wives were more likely to co-donate than to register alone (p = 0.002). Moreover, registrants' main occupational background had a significant effect on co-donations (p = 0.001). Married registrants (regardless of sex) in female-gendered occupations were more likely to co-donate than to donate alone (p = 0.014). Female-gendered occupations were defined as ones in which women represented more than 55 percent of the workforce (e.g., preschool teachers). Thus, variations in donors' occupational backgrounds explained co-donation above and beyond sex differences. Conclusions: Efforts to secure whole body donations have historically focused on individual donations regardless of donors' marital status. More attention needs to be paid, however, to co-donations since they represent a non-trivial number of total donations. Also, targeted outreach efforts to male and female members of female-gendered occupations might prove a successful way to increase donations through co-donations. [ABSTRACT FROM AUTHOR]

The influence of membrane cholesterol content on a variety of ion channel conductances in numerous cell models has been shown, but studies exploring its role in auditory hair cell physiology are scarce. Recent evidence shows that cholesterol depletion affects outer hair cell electromotility and the voltage-gated potassium currents underlying tall hair cell development, but the effects of cholesterol on the major ionic currents governing auditory hair cell excitabilityare unknown. We investigated the effects of a cholesterol-depleting agent (methyl beta cyclodextrin, MβCD) on ion channels necessary for the early stages of sound processing. Large-conductance BK-type potassium channels underlie temporal processing and open in a voltage- and calcium-dependent manner. Voltage-gated calcium channels (VGCCs) are responsible for calcium-dependent exocytosis and synaptic transmission to the auditory nerve. Our results demonstrate that cholesterol depletion reduced peak steady-state calcium-sensitive (BK-type) potassiumcurrent by 50% in chick cochlear hair cells. In contrast, MβCD treatment increased peak inward calcium current (∼30%), ruling out loss of calcium channel expression or function as a cause of reduced calcium-sensitive outward current. Changes in maximal conductance indicated a direct impact of cholesterol on channel number or unitary conductance. Immunoblotting following sucrose-gradient ultracentrifugation revealed BK expression in cholesterol-enriched microdomains. Both direct impacts of cholesterol on channel biophysics, as well as channel localization in the membrane, may contribute to the influence of cholesterol on hair cell physiology. Our results reveal a new role for cholesterol in the regulation of auditory calcium and calcium-activated potassium channels and add to the growing evidence that cholesterol is a key determinant in auditory physiology. [ABSTRACT FROM AUTHOR]

The malaria mosquito, Anopheles stephensi, and other mosquitoes modulate their biology to match the time-of-day. In the present work, we used a non-hypothesis driven approach (untargeted proteomics) to identify proteins in mosquito tissue, and then quantified the relative abundance of the identified proteins from An. stephensi bodies. Using these quantified protein levels, we then analyzed the data for proteins that were only detectable at certain times-of-the day, highlighting the need to consider time-of-day in experimental design. Further, we extended our time-of-day analysis to look for proteins which cycle in a rhythmic 24-hour (“circadian”) manner, identifying 31 rhythmic proteins. Finally, to maximize the utility of our data, we performed a proteogenomic analysis to improve the genome annotation of An. stephensi. We compare peptides that were detected using mass spectrometry but are ‘missing’ from the An. stephensi predicted proteome, to reference proteomes from 38 other primarily human disease vector species. We found 239 such peptide matches and reveal that genome annotation can be improved using proteogenomic analysis from taxonomically diverse reference proteomes. Examination of ‘missing’ peptides revealed reading frame errors, errors in gene-calling, overlapping gene models, and suspected gaps in the genome assembly. [ABSTRACT FROM AUTHOR]

Background/Aims: Long-term follow-up studies validating the clinical benefit of sustained virological response (SVR) in people with chronic hepatitis C (CHC) infection are lacking. Our aim was to identify rates and predictors of liver fibrosis progression in a large, well characterized cohort of CHC patients in whom paired liver fibrosis assessments were performed more than 10 years apart. Methods: CHC patients who had undergone a baseline liver biopsy pre-2004 and a follow up liver fibrosis assessment more than 10 years later (biopsy or liver stiffness measurement (LSM) using transient elastography [FibroScan]) were identified. Subjects who had undergone a baseline liver biopsy but had no follow up fibrosis assessment were recalled for LSM. Fibrosis was categorised as mild-moderate (METAVIR F0-2 / LSM result of ≤ 9.5 kPa) or advanced (METAVIR F3-4/ LSM >9.5 kPa). The primary objective was to assess the association between SVR and the rate of liver fibrosis progression over at least 10 years, defined as an increase from mild-moderate fibrosis at baseline liver biopsy (METAVIR F0-2) to advanced fibrosis at follow-up liver fibrosis assessment. Results: 131 subjects were included in this analysis: 69% male, 82% Caucasian, 60% G1 HCV, 25% G3 HCV. The median age at F/U fibrosis staging was 57 (IQR 54–62) years with median estimated duration of infection 33-years (IQR 29–38). At F/U, liver fibrosis assessment was performed by LSM in 86% and liver biopsy in 14%. The median period between fibrosis assessments was 14-years (IQR 12–17). 109 (83%) participants had received interferon-based antiviral therapy. 40% attained SVR. At F/U, there was a significant increase in the proportion of subjects with advanced liver fibrosis: 27% at baseline vs. 46% at F/U (p = 0.002). The prevalence of advanced fibrosis did not change among subjects who attained SVR, 30% at B/L vs 25% at F/U (p = 0.343). However, advanced fibrosis became more common at F/U among subjects with persistent viremia: 10% at B/L vs 31% at F/U (p = 0.0001). SVR was independently associated with protection from liver fibrosis progression after adjustment for other variables including baseline ALT (p = 0.011), duration of HCV infection and mode of acquisition. Conclusion: HCV eradication is associated with lower rates of liver fibrosis progression. The data support early treatment to prevent long-term liver complications of HCV infection. [ABSTRACT FROM AUTHOR]

The study of population differentiation in the context of ecological speciation is commonly assessed using populations with obvious discreteness. Fewer studies have examined diversifying populations with occasional adaptive variation and minor reproductive isolation, so factors impeding or facilitating the progress of early stage differentiation are less understood. We detected non-random genetic structuring in lake trout (Salvelinus namaycush) inhabiting a large, pristine, postglacial lake (Mistassini Lake, Canada), with up to five discernible genetic clusters having distinctions in body shape, size, colouration and head shape. However, genetic differentiation was low (FST = 0.017) and genetic clustering was largely incongruent between several population- and individual-based clustering approaches. Genotype- and phenotype-environment associations with spatial habitat, depth and fish community structure (competitors and prey) were either inconsistent or weak. Striking morphological variation was often more continuous within than among defined genetic clusters. Low genetic differentiation was a consequence of relatively high contemporary gene flow despite large effective population sizes, not migration-drift disequilibrium. Our results suggest a highly plastic propensity for occupying multiple habitat niches in lake trout and a low cost of morphological plasticity, which may constrain the speed and extent of adaptive divergence. We discuss how factors relating to niche conservatism in this species may also influence how plasticity affects adaptive divergence, even where ample ecological opportunity apparently exists. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) is a significant cause of severe respiratory illness worldwide, particularly in infants, young children, and the elderly. Although no licensed vaccine is currently available, an engineered version of the metastable RSV fusion (F) surface glycoprotein—stabilized in the pre-fusion (pre-F) conformation by “DS-Cav1” mutations—elicits high titer RSV-neutralizing responses. Moreover, pre-F-specific antibodies, often against the neutralization-sensitive antigenic site Ø in the membrane-distal head region of trimeric F glycoprotein, comprise a substantial portion of the human response to natural RSV infection. To focus the vaccine-elicited response to antigenic site Ø, we designed a series of RSV F immunogens that comprised the membrane-distal head of the F glycoprotein in its pre-F conformation. These “head-only” immunogens formed monomers, dimers, and trimers. Antigenic analysis revealed that a majority of the 70 engineered head-only immunogens displayed reactivity to site Ø-targeting antibodies, which was similar to that of the parent RSV F DS-Cav1 trimers, often with increased thermostability. We evaluated four of these head-only immunogens in detail, probing their recognition by antibodies, their physical stability, structure, and immunogenicity. When tested in naïve mice, a head-only trimer, half the size of the parent RSV F trimer, induced RSV titers, which were statistically comparable to those induced by DS-Cav1. When used to boost DS-Cav1-primed mice, two head-only RSV F immunogens, a dimer and a trimer, boosted RSV-neutralizing titers to levels that were comparable to those boosted by DS-Cav1, although with higher site Ø-directed responses. Our results provide proof-of-concept for the ability of the smaller head-only RSV F immunogens to focus the vaccine-elicited response to antigenic site Ø. Decent primary immunogenicity, enhanced physical stability, potential ease of manufacture, and potent immunogenicity upon boosting suggest these head-only RSV F immunogens, engineered to retain the pre-fusion conformation, may have advantages as candidate RSV vaccines. [ABSTRACT FROM AUTHOR]

A large and growing body of evidence has demonstrated that airborne scanning light detection and ranging (lidar) systems can be an effective tool in measuring and monitoring above-ground forest tree biomass. However, the potential of lidar as an all-round tool for assisting in assessment of carbon (C) stocks in soil and non-tree vegetation components of the forest ecosystem has been given much less attention. Here we combine the use airborne small footprint scanning lidar with fine-scale spatial C data relating to vegetation and the soil surface to describe and contrast the size and spatial distribution of C pools within and among multilayered Norway spruce (Picea abies) stands. Predictor variables from lidar derived metrics delivered precise models of above- and below-ground tree C, which comprised the largest C pool in our study stands. We also found evidence that lidar canopy data correlated well with the variation in field layer C stock, consisting mainly of ericaceous dwarf shrubs and herbaceous plants. However, lidar metrics derived directly from understory echoes did not yield significant models. Furthermore, our results indicate that the variation in both the mosses and soil organic layer C stock plots appears less influenced by differences in stand structure properties than topographical gradients. By using topographical models from lidar ground returns we were able to establish a strong correlation between lidar data and the organic layer C stock at a stand level. Increasing the topographical resolution from plot averages (~2000 m2) towards individual grid cells (1 m2) did not yield consistent models. Our study demonstrates a connection between the size and distribution of different forest C pools and models derived from airborne lidar data, providing a foundation for future research concerning the use of lidar for assessing and monitoring boreal forest C. [ABSTRACT FROM AUTHOR]

Determining which demographic and ecological parameters contribute to variation in population growth rate is crucial to understanding the dynamics of declining populations. This study aimed to evaluate the magnitude and mechanisms of an apparent major decline in an Atlantic Puffin Fratercula arctica population. This was achieved using a 27-year dataset to estimate changes in population size and in two key demographic rates: adult survival and breeding success. Estimated demographic variation was then related to two ecological factors hypothesised to be key drivers of demographic change, namely the abundance of the main predator at the study site, the Great Skua Stercorarius skua, and Atlantic Puffin chick food supply, over the same 27-year period. Using a population model, we assessed whether estimated variation in adult survival and reproductive success was sufficient to explain the population change observed. Estimates of Atlantic Puffin population size decreased considerably during the study period, approximately halving, whereas Great Skua population estimates increased, approximately trebling. Estimated adult Atlantic Puffin survival remained high across all years and did not vary with Great Skua abundance; however, Atlantic Puffin breeding success and quantities of fish prey brought ashore by adults both decreased substantially through the period. A population model combining best possible demographic parameter estimates predicted rapid population growth, at odds with the long-term decrease observed. To simulate the observed decrease, population models had to incorporate low immature survival, high immature emigration, or increasingly high adult non-breeding rates. We concluded that reduced recruitment of immatures into the breeding population was the most likely cause of population decrease. This study showed that increase in the size of a predator population does not always impact on the survival of adult prey and that reduced recruitment can be a crucial determinant of seabird population size but can easily go undetected. [ABSTRACT FROM AUTHOR]

Streptococcus pneumonia (the pneumococcus) is the leading vaccine preventable cause of serious infections in infants under 5 years of age. The major correlate of protection for pneumococcal infections is serotype-specific IgG antibody. More recently, antibody-independent mechanisms of protection have also been identified. Preclinical studies have found that IL-17 secreting CD4+ Th17 cells in reducing pneumococcal colonisation. This study assessed IL-17A levels in children from Fiji with high and low pneumococcal carriage density, as measured by quantitative real-time PCR (qPCR). We studied Th17 responses in 54 children who were designated as high density carriers (N=27, >8.21x105 CFU/ml) or low density carriers (N=27, <1.67x105 CFU/ml). Blood samples were collected, and isolated peripheral blood mononuclear cells (PBMCs) were stimulated for 6 days. Supernatants were harvested for cytokine analysis by multiplex bead array and/or ELISA. Th17 cytokines assayed included IL-17A, IL-21, IL-22 as well as TNF-α, IL-10, TGF-β, IL-6, IL-23 and IFNγ. Cytokine levels were significantly lower in children with high density pneumococcal carriage compared with children with low density carriage for IL-17A (p=0.002) and IL-23 (p=0.04). There was a trend towards significance for IL-22 (p=0.057) while no difference was observed for the other cytokines. These data provide further support for the role of Th17-mediated protection in humans and suggest that these cytokines may be important in the defence against pneumococcal carriage. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease Virus (FMDV) is an economically important, highly contagious picornavirus that affects both wild and domesticated cloven hooved animals. In developing countries, the effective laboratory diagnosis of foot-and-mouth disease (FMD) is often hindered by inadequate sample preservation due to difficulties in the transportation and storage of clinical material. These factors can compromise the ability to detect and characterise FMD virus in countries where the disease is endemic. Furthermore, the high cost of sending infectious virus material and the biosecurity risk it presents emphasises the need for a thermo-stable, non-infectious mode of transporting diagnostic samples. This paper investigates the potential of using FMDV lateral-flow devices (LFDs) for dry transportation of clinical samples for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by electroporation. FMDV positive samples (epithelial suspensions and cell culture isolates) representing four FMDV serotypes were applied to antigen LFDs: after which it was possible to recover viral RNA that could be detected using real-time RT-PCR. Using this nucleic acid, it was also possible to recover VP1 sequences and also successfully utilise protocols for amplification of complete FMD virus genomes. It was not possible to recover infectious FMDV directly from the LFDs, however following electroporation into BHK-21 cells and subsequent cell passage, infectious virus could be recovered. Therefore, these results support the use of the antigen LFD for the dry, non-hazardous transportation of samples from FMD endemic countries to international reference laboratories. [ABSTRACT FROM AUTHOR]

Hybrid zones provide unprecedented opportunity for the study of the evolution of reproductive isolation, and the extent of hybridization across individuals and genomes can illuminate the degree of isolation. We examine patterns of interchromosomal linkage disequilibrium (ILD) and the presence of hybridization in Atlantic cod, Gadus morhua, in previously identified hybrid zones in the North Atlantic. Here, previously identified clinal loci were mapped to the cod genome with most (∼70%) occurring in or associated with (<5 kb) coding regions representing a diverse array of possible functions and pathways. Despite the observation that clinal loci were distributed across three linkage groups, elevated ILD was observed among all groups of clinal loci and strongest in comparisons involving a region of low recombination along linkage group 7. Evidence of ILD supports a hypothesis of divergence hitchhiking transitioning to genome hitchhiking consistent with reproductive isolation. This hypothesis is supported by Bayesian characterization of hybrid classes present and we find evidence of common F1 hybrids in several regions consistent with frequent interbreeding, yet little evidence of F2 or backcrossed individuals. This work suggests that significant barriers to hybridization and introgression exist among these co-occurring groups of cod either through strong selection against hybrid individuals, or genetic incompatibility and intrinsic barriers to hybridization. In either case, the presence of strong clinal trends, and little gene flow despite extensive hybridization supports a hypothesis of reproductive isolation and cryptic speciation in Atlantic cod. Further work is required to test the degree and nature of reproductive isolation in this species. [ABSTRACT FROM AUTHOR]

The type III secretion system (T3SS) is required for virulence in the gram-negative plant pathogen Pseudomonas syringae pv. tomato DC3000. The alternative sigma factor HrpL directly regulates expression of T3SS genes via a promoter sequence, often designated as the “hrp promoter.” Although the HrpL regulon has been extensively investigated in DC3000, it is not known whether additional regulon members remain to be found. To systematically search for HrpL-regulated genes, we used chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) and bulk mRNA sequencing (RNA-Seq) to identify HrpL-binding sites and likely hrp promoters. The analysis recovered 73 sites of interest, including 20 sites that represent new hrp promoters. The new promoters lie upstream of a diverse set of genes encoding potential regulators, enzymes and hypothetical proteins. PSPTO_5633 is the only new HrpL regulon member that is potentially an effector and is now designated HopBM1. Deletions in several other new regulon members, including PSPTO_5633, PSPTO_0371, PSPTO_2130, PSPTO_2691, PSPTO_2696, PSPTO_3331, and PSPTO_5240, in either DC3000 or ΔhopQ1-1 backgrounds, do not affect the hypersensitive response or in planta growth of the resulting strains. Many new HrpL regulon members appear to be unrelated to the T3SS, and orthologs for some of these can be identified in numerous non-pathogenic bacteria. With the identification of 20 new hrp promoters, the list of HrpL regulon members is approaching saturation and most likely includes all DC3000 effectors. [ABSTRACT FROM AUTHOR]

Among trapping mechanisms in carnivorous plants, those termed 'active' have especially fascinated scientists since Charles Darwin's early works because trap movements are involved. Fast snap-trapping and suction of prey are two of the most spectacular examples for how these plants actively catch animals, mainly arthropods, for a substantial nutrient supply. We show that Drosera glanduligera, a sundew from southern Australia, features a sophisticated catapult mechanism: Prey animals walking near the edge of the sundew trigger a touch-sensitive snap-tentacle, which swiftly catapults them onto adjacent sticky glue-tentacles; the insects are then slowly drawn within the concave trap leaf by sticky tentacles. This is the first detailed documentation and analysis of such catapult-flypaper traps in action and highlights a unique and surprisingly complex mechanical adaptation to carnivory. [ABSTRACT FROM AUTHOR]

Background: In mid-June 2009 the State of Victoria in Australia appeared to have the highest notification rate of pandemic (H1N1) 2009 influenza in the world. We hypothesise that this was because community transmission of pandemic influenza was already well established in Victoria at the time testing for the novel virus commenced. In contrast, this was not true for the pandemic in other parts of Australia, including Western Australia (WA). Methods: We used data from detailed case follow-up of patients with confirmed infection in Victoria and WA to demonstrate the difference in the pandemic curve in two Australian states on opposite sides of the continent. We modelled the pandemic in both states, using a susceptible-infected-removed model with Bayesian inference accounting for imported cases. Results: Epidemic transmission occurred earlier in Victoria and later in WA. Only 5% of the first 100 Victorian cases were not locally acquired and three of these were brothers in one family. By contrast, 53% of the first 102 cases in WA were associated with importation from Victoria. Using plausible model input data, estimation of the effective reproductive number for the Victorian epidemic required us to invoke an earlier date for commencement of transmission to explain the observed data. This was not required in modelling the epidemic in WA. Conclusion: Strong circumstantial evidence, supported by modelling, suggests community transmission of pandemic influenza was well established in Victoria, but not in WA, at the time testing for the novel virus commenced in Australia. The virus is likely to have entered Victoria and already become established around the time it was first identified in the US and Mexico. [ABSTRACT FROM AUTHOR]

Background:Cancer spread to other organs is the main cause of death of oncological patients. Migration of cancer cells from a primary tumour is the crucial step in the complex process of metastasis, therefore blocking this process is currently the main treatment strategy. Metastasis inhibitors derived from natural products, such as, migrastatin, are very promising anticancer agents. Thus, the aim of our study was to investigate the effect of six migrastatin analogues (MGSTA-1 to 6) on migration and invasion of canine mammary adenocarcinoma cell lines isolated from primary tumours and their metastases to the lungs. Canine mammary tumours constitute a valuable tool for studying multiple aspect of human cancer. Results:Our results showed that two of six fully synthetic analogues of migrastatin: MGSTA-5 and MGSTA-6 were potent inhibitors of canine mammary cancer cells migration and invasion. These data were obtained using the wound healing test, as well as trans-well migration and invasion assays. Furthermore, the treatment of cancer cells with the most effective compound (MGSTA-6) disturbed binding between filamentous F-actin and fascin1. Confocal microscopy analyses revealed that treatment with MGSTA-6 increased the presence of unbound fascin1 and reduced co-localization of F-actin and fascin1 in canine cancer cells. Most likely, actin filaments were not cross-linked by fascin1 and did not generate the typical filopodial architecture of actin filaments in response to the activity of MGSTA-6. Thus, administration of MGSTA-6 results in decreased formation of filopodia protrusions and stress fibres in canine mammary cancer cells, causing inhibition of cancer migration and invasion. Conclusion:Two synthetic migrastatin analogues (MGSTA-5 and MGSTA-6) were shown to be promising compounds for inhibition of cancer metastasis. They may have beneficial therapeutic effects in cancer therapy in dogs, especially in combination with other anticancer drugs. However, further invivo studies are required to verify this hypothesis. [ABSTRACT FROM AUTHOR]