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Introduction: Resilience is a strengths-based construct that is useful for understanding differences in health and wellbeing among youth. There are a range of validated survey instruments available to measure resilience for Aboriginal and Torres Strait Islander (hereafter respectfully Indigenous) youth. However, standard international instruments should only be used if they have been subjected to a rigorous cross-cultural adaptation process and psychometric evaluation in the target population to ensure their validity. The aim of the study was to validate an adapted Child and Youth Resilience Measure (CYRM-28) within a sample of Indigenous Australian boarding school students. Method: The CYRM-28, augmented with an additional 11 site specific items was administered to a purposive sample of Australian Indigenous boarding school students ( n = 233) as part of the broader T4S survey instrument that captures demographic information and measures resilience, psychological distress and risk, and service usage. Confirmatory factor analysis was undertaken to verify the relationship between the observed variables with the theoretical constructs of the CYRM-28 and previous findings on the factor structure. Cronbach alpha was also calculated to assess the internal consistency of the CYRM-28 within this sample. Results: Survey data were not a good fit for any previously identified models of the CYRM-28, although the inclusion of a site-specific variable improved the overall fit statistics. Two separate scales were confirmed that capture the sources and expressions of resilience for Indigenous Australian boarding school students. This structure is different to previous findings in relation to the CYRM-28, but consistent with conceptualizations of resilience as a dynamic process. Conclusions: The findings are useful in guiding the future use of the CYRM-28 instrument, explorations of Indigenous youth resilience, and for services working with Indigenous youth in out of home care situations. They highlight contextual differences in the measurement of resilience and the importance of validating standard instruments that have been subjected to rigorous cross-cultural adaptation processes. The two scales offer practical guidance to human services working with Indigenous youth on strategies to build and monitor resilience in Indigenous Australian youth and contribute to the emergent understanding of their resilience.

Serine integrases catalyze the site-specific insertion of viral DNA into a host's genome. The minimal requirements and irreversible nature of this integration reaction have led to the use of serine integrases in applications ranging from bacterial memory storage devices to gene therapy. Our understanding of how the integrase proteins recognize the viral (attP) and host (attB) attachment sites is limited, with structural data available for only a Listeria integrase C-terminal domain (CTD) bound to an attP half-site. Here we report quantitative binding and saturation mutagenesis analyses for the Listeria innocua prophage attP site and a new 2.8-Å crystal structure of the CTD•attP half site. We find that Int binds with high affinity to attP (6.9 nM), but the Int CTD binds to attP half-sites with only 7- to 10-fold lower affinity, supporting the idea that free energy is expended to open an Int dimer for attP binding. Despite the 50-bp Int-attP interaction surface, only 20 residues are sensitive to mutagenesis, and of these, only 6 require a specific residue for efficient Int binding and integration activity. One of the integrase DNA-binding domains, the recombinase domain, appears to be primarily non-specific. Several substitutions result in an improved attP site, indicating that higher-efficiency attachment sites can be obtained through site engineering. These findings advance our understanding of serine integrase function and provide important data for efforts towards engineering this family of enzymes for a variety of biotechnology applications., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Next-generation reduced representation sequencing (RRS) approaches show great potential for resolving the structure of wild populations. However, the population structure of species that have shown rapid demographic recovery following severe population bottlenecks may still prove difficult to resolve due to high gene flow between subpopulations. Here, we tested the effectiveness of the RRS method Genotyping-By-Sequencing (GBS) for describing the population structure of the New Zealand fur seal (NZFS, Arctocephalus forsteri ), a species that was heavily exploited by the 19th century commercial sealing industry and has since rapidly recolonized most of its former range from a few isolated colonies. Using 26,026 neutral single nucleotide polymorphisms (SNPs), we assessed genetic variation within and between NZFS colonies. We identified low levels of population differentiation across the species range (<1% of variation explained by regional differences) suggesting a state of near panmixia. Nonetheless, we observed subtle population substructure between West Coast and Southern East Coast colonies and a weak, but significant ( p  = 0.01), isolation-by-distance pattern among the eight colonies studied. Furthermore, our demographic reconstructions supported severe bottlenecks with potential 10-fold and 250-fold declines in response to Polynesian and European hunting, respectively. Finally, we were able to assign individuals treated as unknowns to their regions of origin with high confidence (96%) using our SNP data. Our results indicate that while it may be difficult to detect population structure in species that have experienced rapid recovery, next-generation markers and methods are powerful tools for resolving fine-scale structure and informing conservation and management efforts.

Sexual behavior in teleost fish is highly plastic. It can be attributed to the relatively few sex differences found in adult brain transcriptomes. Environmental and hormonal factors can influence sex-specific behavior. Androgen treatment stimulates behavioral masculinization. Sex dimorphic gene expression in developing teleost brains and the molecular basis for androgen-induced behavioral masculinization are poorly understood. In this study, juvenile zebrafish (Danio rerio) were treated with 100 ng/L of 17 alpha-methyltestosterone (MT) during sexual development from 20 days post fertilization to 40 days and 60 days post fertilization. We compared brain gene expression patterns in MT-treated zebrafish with control males and females using RNA-Seq to shed light on the dynamic changes in brain gene expression during sexual development and how androgens affect brain gene expression leading to behavior masculinization. We found modest differences in gene expression between juvenile male and female zebrafish brains. Brain aromatase (cyp19a1b), prostaglandin 3a synthase (ptges3a), and prostaglandin reductase 1 (ptgr1) were among the genes with sexually dimorphic expression patterns. MT treatment significantly altered gene expression relative to both male and female brains. Fewer differences were found among MT-treated brains and male brains compared to female brains, particularly at 60 dpf. MT treatment upregulated the expression of hydroxysteroid 11-beta dehydrogenase 2 (hsd11b2), deiodinase, iodothyronine, type II (dio2), and gonadotrophin releasing hormones (GnRH) 2 and 3 (gnrh2 and gnrh3) suggesting local synthesis of 11-ketotestosterone, triiodothyronine, and GnRHs in zebrafish brains which are influenced by androgens. Androgen, estrogen, prostaglandin, thyroid hormone, and GnRH signaling pathways likely interact to modulate teleost sexual behavior.

The physiological and cellular adaptations to extreme fasting in northern elephant seals ( Mirounga angustirostris, NES) are remarkable and may help to elucidate endocrine mechanisms that regulate lipid metabolism and energy homeostasis in mammals. Recent studies have highlighted the importance of thyroid hormones in the maintenance of a lipid-based metabolism during prolonged fasting in weaned NES pups. To identify additional molecular regulators of fasting, we used a transcriptomics approach to examine changes in global gene expression profiles before and after 6-8 wk of fasting in weaned NES pups. We produced a de novo assembly and identified 98 unique protein-coding genes that were differentially expressed between early and late fasting. Most of the downregulated genes were associated with lipid, carbohydrate, and protein metabolism. A number of downregulated genes were also associated with maintenance of the extracellular matrix, consistent with tissue remodeling during weight loss and the multifunctional nature of blubber tissue, which plays both metabolic and structural roles in marine mammals. Using this data set, we predict potential mechanisms by which NES pups sustain metabolism and regulate adipose stores throughout the fast, and provide a valuable resource for additional studies of extreme metabolic adaptations in mammals.

Tunicates are marine invertebrates that compose the closest phylogenetic group to the vertebrates. These chordates present a particularly diverse range of regenerative abilities and life-history strategies. Consequently, tunicates provide an extraordinary perspective into the emergence and diversity of these traits. Here we describe the genome sequencing, annotation and analysis of the Stolidobranchian Botrylloides leachii. We have produced a high-quality 159 Mb assembly, 82% of the predicted 194 Mb genome. Analysing genome size, gene number, repetitive elements, orthologs clustering and gene ontology terms show that B. leachii has a genomic architecture similar to that of most solitary tunicates, while other recently sequenced colonial ascidians have undergone genome expansion. In addition, ortholog clustering has identified groups of candidate genes for the study of colonialism and whole-body regeneration. By analysing the structure and composition of conserved gene linkages, we observed examples of cluster breaks and gene dispersions, suggesting that several lineage-specific genome rearrangements occurred during tunicate evolution. We also found lineage-specific gene gain and loss within conserved cell-signalling pathways. Such examples of genetic changes within conserved cell-signalling pathways commonly associated with regeneration and development that may underlie some of the diverse regenerative abilities observed in tunicates. Overall, these results provide a novel resource for the study of tunicates and of colonial ascidians.

Establishing the sex of individuals in wild systems can be challenging and often requires genetic testing. Genotyping-by-sequencing (GBS) and other reduced-representation DNA sequencing (RRS) protocols (e.g., RADseq, ddRAD) have enabled the analysis of genetic data on an unprecedented scale. Here, we present a novel approach for the discovery and statistical validation of sex-specific loci in GBS data sets. We used GBS to genotype 166 New Zealand fur seals (NZFS, Arctocephalus forsteri) of known sex. We retained monomorphic loci as potential sex-specific markers in the locus discovery phase. We then used (i) a sex-specific locus threshold (SSLT) to identify significantly male-specific loci within our data set; and (ii) a significant sex-assignment threshold (SSAT) to confidently assign sex in silico the presence or absence of significantly male-specific loci to individuals in our data set treated as unknowns (98.9% accuracy for females; 95.8% for males, estimated via cross-validation). Furthermore, we assigned sex to 86 individuals of true unknown sex using our SSAT and assessed the effect of SSLT adjustments on these assignments. From 90 verified sex-specific loci, we developed a panel of three sex-specific PCR primers that we used to ascertain sex independently of our GBS data, which we show amplify reliably in at least two other pinniped species. Using monomorphic loci normally discarded from large SNP data sets is an effective way to identify robust sex-linked markers for nonmodel species. Our novel pipeline can be used to identify and statistically validate monomorphic and polymorphic sex-specific markers across a range of species and RRS data sets., (© 2018 John Wiley & Sons Ltd.)

The fission yeast Schizosaccharomyces pombe has become well established as a model species for studying conserved cell-level biological processes, especially the mechanics and regulation of cell division. PomBase integrates the S. pombe genome sequence with traditional genetic, molecular, and cell biological experimental data as well as the growing body of large datasets generated by emerging high-throughput methods. This chapter provides insight into the curation philosophy and data organization at PomBase, and provides a guide to using PomBase for infrequent visitors and anyone considering exploring S. pombe in their research.

Phenotypic plasticity represents an elegant adaptive response of individuals to a change in their environment. Bluehead wrasses (Thalassoma bifasciatum) exhibit astonishing sexual plasticity, including female-to-male sex change and discrete male morphs that differ strikingly in behavior, morphology, and gonadal investment. Using RNA-seq transcriptome profiling, we examined the genes and physiological pathways underlying flexible behavioral and gonadal differences among female, dominant (bourgeois) male, and female-mimic (sneaker) male blueheads. For the first time in any organism, we find that female mimicry by sneaker males has a transcriptional signature in both the brain and the gonad. Sneaker males shared striking similarity in neural gene expression with females, supporting the idea that males with alternative reproductive phenotypes have "female-like brains." Sneaker males also overexpressed neuroplasticity genes, suggesting that their opportunistic reproductive strategy requires a heightened capacity for neuroplasticity. Bourgeois males overexpressed genes associated with socio-sexual behaviors (e.g., isotocin), but also neuroprotective genes and biomarkers of oxidative stress and aging, indicating a hitherto unexplored cost to these males of attaining the reproductively privileged position at the top of the social hierarchy. Our novel comparison of testicular transcriptomes in a fish with male sexual polymorphism associates greater gonadal investment by sneaker males with overexpression of genes involved in cell proliferation and sperm quality control. We propose that morphological female-mimicry by sneaker male teleosts entails pervasive downregulation of androgenesis genes, consistent with low androgen production in males lacking well-developed secondary sexual characters., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The New Zealand collembolan genus Holacanthella contains the largest species of springtails (Collembola) in the world. Using Illumina technology we have sequenced and assembled a draft genome and transcriptome from Holacanthella duospinosa (Salmon). We have used this annotated assembly to investigate the genetic basis of a range of traits critical to the evolution of the Hexapoda, the phylogenetic position of H. duospinosa and potential horizontal gene transfer events., Results: Our genome assembly was ~375 Mbp in size with a scaffold N50 of ~230 Kbp and sequencing coverage of ~180×. DNA elements, LTRs and simple repeats and LINEs formed the largest components and SINEs were very rare. Phylogenomics (370,877 amino acids) placed H. duospinosa within the Neanuridae. We recovered orthologs of the conserved sex determination genes thought to play a role in sex determination. Analysis of CpG content suggested the absence of DNA methylation, and consistent with this we were unable to detect orthologs of the DNA methyltransferase enzymes. The small subunit rRNA gene contained a possible retrotransposon. The Hox gene complex was broken over two scaffolds. For chemosensory ability, at least 15 and 18 ionotropic glutamate and gustatory receptors were identified, respectively. However, we were unable to identify any odorant receptors or their obligate co-receptor Orco. Twenty-three chitinase-like genes were identified from the assembly. Members of this multigene family may play roles in the digestion of fungal cell walls, a common food source for these saproxylic organisms. We also detected 59 and 96 genes that blasted to bacteria and fungi, respectively, but were located on scaffolds that otherwise contained arthropod genes., Conclusions: The genome of H. duospinosa contains some unusual features including a Hox complex broken over two scaffolds, in a different manner to other arthropod species, a lack of odorant receptor genes and an apparent lack of environmentally responsive DNA methylation, unlike many other arthropods. Our detection of candidate horizontal gene transfer candidates confirms that this phenomenon is occurring across Collembola. These findings allow us to narrow down the regions of the arthropod phylogeny where key innovations have occurred that have facilitated the evolutionary success of Hexapoda.

In a range of taxa, the relatedness between mates influences both pre- and post-mating processes of sexual selection. However, relatively little is known about the genetic loci facilitating such a bias, with the exception of the major histocompatibility complex. Here, we performed tightly controlled replicated in vitro fertilization trials to explore the impact of relatedness on two possible mechanisms of cryptic female choice (CFC) in Chinook salmon ( Oncorhynchus tshawytscha ). We tested (i) whether relatedness of mates, assessed using 682 single nucleotide polymorphisms (SNPs) on 29 SNP-linkage groups (LGs), biases a male's sperm velocity in ovarian fluid (a parameter previously shown to predict male fertilization success), and (ii) whether relatedness of mates governs fertilization success via other mechanisms, probably via sperm-egg interactions. We found that relatedness on three LGs explained the variation in sperm velocity, and relatedness on two LGs explained fertilization success, which might indicate the presence of genes important in sperm-ovarian fluid and sperm-egg interactions in these genomic regions. Mapping of the SNPs on these LGs to the rainbow trout genome revealed two genes that affect fertility in humans and represent candidate genes for further studies. Our results thereby provide a novel contribution to the understanding of the mechanism of CFC., (© 2017 The Author(s).)

Background: Sex hormones play important roles in teleost ovarian and testicular development. In zebrafish, ovarian differentiation appears to be dictated by an oocyte-derived signal via Cyp19a1a aromatase-mediated estrogen production. Androgens and aromatase inhibitors can induce female-to-male sex reversal, however, the mechanisms underlying gonadal masculinisation are poorly understood. We used histological analyses together with RNA sequencing to characterise zebrafish gonadal transcriptomes and investigate the effects of 17α-methyltestosterone on gonadal differentiation., Results: At a morphological level, 17α-methyltestosterone (MT) masculinised gonads and accelerated spermatogenesis, and these changes were paralleled in masculinisation and de-feminisation of gonadal transcriptomes. MT treatment upregulated expression of genes involved in male sex determination and differentiation (amh, dmrt1, gsdf and wt1a) and those involved in 11-oxygenated androgen production (cyp11c1 and hsd11b2). It also repressed expression of ovarian development and folliculogenesis genes (bmp15, gdf9, figla, zp2.1 and zp3b). Furthermore, MT treatment altered epigenetic modification of histones in zebrafish gonads. Contrary to expectations, higher levels of cyp19a1a or foxl2 expression in control ovaries compared to MT-treated testes and control testes were not statistically significant during early gonad development (40 dpf)., Conclusion: Our study suggests that both androgen production and aromatase inhibition are important for androgen-induced gonadal masculinisation and natural testicular differentiation in zebrafish.

Background: Acute coronary syndrome, including acute myocardial infarction (AMI), is one of the leading causes for hospitalization, with AMI 30-day readmission rates around 20%. Supporting patient information needs and increasing adherence to recommended self-management behaviors during transition from hospital to home has the potential to improve patient outcomes. Text messages have been effective in other interventions and may be suitable to provide support to patients during this transition period., Objective: The goal of this study is to pilot test a text messaging intervention program (Txt2Prevent) that supports acute coronary syndrome patients for 60 days postdischarge. The primary objective is to compare self-management, as measured by the Health Education Impact Questionnaire, between patients receiving only usual care versus those who receive usual care plus the Txt2Prevent intervention. The secondary objectives are to compare medication adherence, health-related quality of life, self-efficacy, health care resource use (and associated costs), all-cause and cardiovascular disease (CVD) readmission, and all-cause and CVD mortality rates between the 2 groups. The third objective is to assess acceptability of the text messaging intervention and feasibility of the study protocol., Methods: This is a randomized controlled trial with blinding of outcome assessors. The Txt2Prevent program includes automated text messages to patients about standard follow-up care, general self-management, and healthy living. The content of the text messages was informed by and developed based on interviews with patients, discharge materials, theoretical domains of behavior, and a clinical advisory group composed of patients, clinicians, and researchers. We will recruit 76 consecutive cardiac in-patients with acute coronary syndrome who are treated with either medical management or percutaneous coronary intervention from a hospital in Vancouver, Canada., Results: Assessments at baseline will include measures for demographic information, self-management, health-related quality of life, and self-efficacy. Assessments at follow-up will include medication adherence, readmissions, health care resource use, and mortality in addition to the reassessment of baseline measures. Baseline assessments are done in-person while follow-up assessments are completed through a combination of mailed packages and phone calls. Semistructured interviews with participants will also be performed to better understand participant experiences managing their condition and with the text messages., Conclusions: This study will determine preliminary efficacy, feasibility, and acceptability of the Txt2Prevent program to support acute coronary syndrome patients in the transition to home following hospital discharge. The results of this study will be used to inform a larger trial., Trial Registration: ClinicalTrials.gov NCT02336919; https://clinicaltrials.gov/ct2/show/NCT02336919 (Archived by WebCite at http://www.webcitation.org/6qMjEqo6O)., (©Emily S Ross, Brodie M Sakakibara, Martha H Mackay, David GT Whitehurst, Joel Singer, Mustafa Toma, Kitty K Corbett, Harriette GC Van Spall, Kimberly Rutherford, Bobby Gheorghiu, Jillianne Code, Scott A Lear. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 23.05.2017.)

The pathogen-host interactions database (PHI-base) is available at www.phi-base.org PHI-base contains expertly curated molecular and biological information on genes proven to affect the outcome of pathogen-host interactions reported in peer reviewed research articles. In addition, literature that indicates specific gene alterations that did not affect the disease interaction phenotype are curated to provide complete datasets for comparative purposes. Viruses are not included. Here we describe a revised PHI-base Version 4 data platform with improved search, filtering and extended data display functions. A PHIB-BLAST search function is provided and a link to PHI-Canto, a tool for authors to directly curate their own published data into PHI-base. The new release of PHI-base Version 4.2 (October 2016) has an increased data content containing information from 2219 manually curated references. The data provide information on 4460 genes from 264 pathogens tested on 176 hosts in 8046 interactions. Prokaryotic and eukaryotic pathogens are represented in almost equal numbers. Host species belong ∼70% to plants and 30% to other species of medical and/or environmental importance. Additional data types included into PHI-base 4 are the direct targets of pathogen effector proteins in experimental and natural host organisms. The curation problems encountered and the future directions of the PHI-base project are briefly discussed., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objectives: We aimed to calculate trends in incidence and prevalence rates of long-term opioid use for non-cancer pain, as well as to describe the characteristics of long-term opioid users and their patterns of opioid use., Methods: We used population-based linked health care and socio-demographic administrative data for British Columbia (BC) between 2005 and 2012. We included individuals who had at least one episode of long-term opioid use during the study period and who were not cancer or palliative care patients., Results: Long-term users comprised only 10% of all individuals prescribed opioids for non-cancer pain, but accounted for 64% of all opioid prescriptions and 87% of all morphine equivalents dispensed in BC during this period. While the incidence rate did not significantly change, the prevalence rate increased by 27% for men and 22% for women. In 2012, there were 3.80 (3.72-3.88) new long-term opioid users per 1,000 men and 4.42 (4.34-4.51) new users per 1,000 women. At the same time, there were 18.3 (95% CI 18.1-18.5) existing long-term users per 1,000 men and 21.7 users (95% CI 21.5-21.9) per 1,000 women. Overall, 2.4% of BC residents were long-term users of prescription opioids in 2012. Most long-term users had one continuous episode of use spanning multiple years. Almost two thirds took opioids every other day or more frequently., Conclusion: There is a growing population of long-term opioid users for non-cancer pain in BC, with higher incidence and prevalence rates observed among women than among men.

To limit tail biting incidence, most pig producers in Europe tail dock their piglets. This is despite EU Council Directive 2008/120/EC banning routine tail docking and allowing it only as a last resort. The paper aims to understand what it takes to fulfil the intentions of the Directive by examining economic results of four management and housing scenarios, and by discussing their consequences for animal welfare in the light of legal and ethical considerations. The four scenarios compared are: 'Standard Docked', a conventional housing scenario with tail docking meeting the recommendations for Danish production (0.7 m2/pig); 'Standard Undocked', which is the same as 'Standard Docked' but with no tail docking, 'Efficient Undocked' and 'Enhanced Undocked', which have increased solid floor area (0.9 and 1.0 m2/pig, respectively) provision of loose manipulable materials (100 and 200 g/straw per pig per day) and no tail docking. A decision tree model based on data from Danish and Finnish pig production suggests that Standard Docked provides the highest economic gross margin with the least tail biting. Given our assumptions, Enhanced Undocked is the least economic, although Efficient Undocked is better economically and both result in a lower incidence of tail biting than Standard Undocked but higher than Standard Docked. For a pig, being bitten is worse for welfare (repeated pain, risk of infections) than being docked, but to compare welfare consequences at a farm level means considering the number of affected pigs. Because of the high levels of biting in Standard Undocked, it has on average inferior welfare to Standard Docked, whereas the comparison of Standard Docked and Enhanced (or Efficient) Undocked is more difficult. In Enhanced (or Efficient) Undocked, more pigs than in Standard Docked suffer from being tail bitten, whereas all the pigs avoid the acute pain of docking endured by the pigs in Standard Docked. We illustrate and discuss this ethical balance using numbers derived from the above-mentioned data. We discuss our results in the light of the EU Directive and its adoption and enforcement by Member States. Widespread use of tail docking seems to be accepted, mainly because the alternative steps that producers are required to take before resorting to it are not specified in detail. By tail docking, producers are acting in their own best interests. We suggest that for the practice of tail docking to be terminated in a way that benefits animal welfare, changes in the way pigs are housed and managed may first be required.

Background: Regenerative capacity differs greatly between animals. In vertebrates regenerative abilities are highly limited and tissue or organ specific. However the closest related chordate to the vertebrate clade, Botrylloides leachi, can undergo whole body regeneration (WBR). Therefore, research on WBR in B. leachi has focused on pathways known to be important for regeneration in vertebrates. To obtain a comprehensive vision of this unique process we have carried out the first de novo transcriptome sequencing for multiple stages of WBR occurring in B. leachi. The identified changes in gene expression during B. leachi WBR offer novel insights into this remarkable ability to regenerate., Results: The transcriptome of B. leachi tissue undergoing WBR were analysed using differential gene expression, gene ontology and pathway analyses. We observed up-regulation in the expression of genes involved in wound healing and known developmental pathways including WNT, TGF-β and Notch, during the earliest stages of WBR. Later in WBR, the expression patterns in several pathways required for protein synthesis, biogenesis and the organisation of cellular components were up-regulated., Conclusions: While the genes expressed early on are characteristic of a necessary wound healing response to an otherwise lethal injury, the subsequent vast increase in protein synthesis conceivably sustains the reestablishment of the tissue complexity and body axis polarity within the regenerating zooid. We have, for the first time, provided a global overview of the genes and their corresponding pathways that are modulated during WBR in B. leachi.

Background: The proposed study was developed in response to increased suicide risk identified in Aboriginal and Torres Strait Islander students who are compelled to attend boarding schools across Queensland when there is no secondary schooling provision in their remote home communities. It will investigate the impact of a multicomponent mentoring intervention to increase levels of psychosocial resilience. We aim to test the null hypothesis that students' resilience is not positively influenced by the intervention. The 5-year project was funded by the Australian National Health and Medical Research Council from December 2014., Methods/design: An integrated mixed methods approach will be adopted; each component iteratively informing the other. Using an interrupted time series design, the primary research methods are quantitative: 1) assessment of change in students' resilience, educational outcomes and suicide risk; and 2) calculation of costs of the intervention. Secondary methods are qualitative: 3) a grounded theoretical model of the process of enhancing students' psychosocial resilience to protect against suicide. Additionally, there is a tertiary focus on capacity development: more experienced researchers in the team will provide research mentorship to less experienced researchers through regular meetings; while Indigenous team members provide cultural mentorship in research practices to non-Indigenous members., Discussion: Australia's suicide prevention policy is progressive but a strong service delivery model is lacking, particularly for Indigenous peoples. The proposed research will potentially improve students' levels of resilience to mitigate against suicide risk. Additionally, it could reduce the economic and social costs of Indigenous youth suicide by obtaining agreement on what is good suicide prevention practice for remote Indigenous students who transition to boarding schools for education, and identifying the benefits-costs of an evidence-based multi-component mentoring intervention to improve resilience.

Background: Teleost fishes exhibit remarkably diverse and plastic sexual developmental patterns. One of the most astonishing is the rapid socially controlled female-to-male (protogynous) sex change observed in bluehead wrasses (Thalassoma bifasciatum). Such functional sex change is widespread in marine fishes, including species of commercial importance, yet its underlying molecular basis remains poorly explored., Methods: RNA sequencing was performed to characterize the transcriptomic profiles and identify genes exhibiting sex-biased expression in the brain (forebrain and midbrain) and gonads of bluehead wrasses. Functional annotation and enrichment analysis were carried out for the sex-biased genes in the gonad to detect global differences in gene products and genetic pathways between males and females., Results: Here we report the first transcriptomic analysis for a protogynous fish. Expression comparison between males and females reveals a large set of genes with sex-biased expression in the gonad, but relatively few such sex-biased genes in the brain. Functional annotation and enrichment analysis suggested that ovaries are mainly enriched for metabolic processes and testes for signal transduction, particularly receptors of neurotransmitters and steroid hormones. When compared to other species, many genes previously implicated in male sex determination and differentiation pathways showed conservation in their gonadal expression patterns in bluehead wrasses. However, some critical female-pathway genes (e.g., rspo1 and wnt4b) exhibited unanticipated expression patterns. In the brain, gene expression patterns suggest that local neurosteroid production and signaling likely contribute to the sex differences observed., Conclusions: Expression patterns of key sex-related genes suggest that sex-changing fish predominantly use an evolutionarily conserved genetic toolkit, but that subtle variability in the standard sex-determination regulatory network likely contributes to sexual plasticity in these fish. This study not only provides the first molecular data on a system ideally suited to explore the molecular basis of sexual plasticity and tissue re-engineering, but also sheds some light on the evolution of diverse sex determination and differentiation systems.

Objectives: We quantify patterns in prescription opioid dispensations to individuals who suffered a prescription opioid-related death. In addition, we examine the relationship between opioid dispensations and prescription opioid-related deaths in geographic regions of British Columbia (BC)., Methods: We used population-based administrative data on prescription drug dispensations to identify patterns in prescription opioid dispensations to individuals who suffered a prescription opioid-related death. We also computed the quantity of prescription opioids dispensed (morphine equivalents) in small geographic regions in BC from 2004 to 2013. We identified prescription opioid-related deaths in these small geographic areas using mortality data from BC Vital Statistics and investigated the relationship between rates of prescription opioid dispensing and rates of prescription opioid death in small geographic areas in BC by sex. We examined differences in our results when limiting opioid dispensations to strong opioids and weak opioids., Results: Many individuals who suffered a prescription opioid-related death did not have an active opioid prescription in the 60 days before death (46% of women and 71% of men). Rates of prescription opioid dispensing and opioid-related deaths vary substantially across geographic regions in BC. The area-level relationship between rate of prescription opioid dispensing and rate of unintentional prescription opioid-related death is positive and statistically significant for both men and women (P<0.001). This relationship holds when opioid prescribing is limited to strong opioids., Conclusion: Targeted efforts to reduce high levels of opioid prescribing in BC, particularly dispensations of strong opioids and codeine, may substantially reduce opioid-related harms.

PomBase (http://www.pombase.org) is the model organism database for the fission yeast Schizosaccharomyces pombe. PomBase provides a central hub for the fission yeast community, supporting both exploratory and hypothesis-driven research. It provides users easy access to data ranging from the sequence level, to molecular and phenotypic annotations, through to the display of genome-wide high-throughput studies. Recent improvements to the site extend annotation specificity, improve usability and allow for monthly data updates. Both in-house curators and community researchers provide manually curated data to PomBase. The genome browser provides access to published high-throughput data sets and the genomes of three additional Schizosaccharomyces species (Schizosaccharomyces cryophilus, Schizosaccharomyces japonicus and Schizosaccharomyces octosporus)., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Almost three-quarters of family practice residents in British Columbia (BC) meet criteria for burnout. We sought to understand how burnout is perceived and experienced by family medicine residents, and to identify both contributory and protective factors for resident burnout., Method: Two semi-structured focus groups were conducted with ten family practice residents from five distinct University of British Columbia training sites. Participants completed the Maslach Burnout Inventory (MBI). The data were analyzed using a thematic analysis approach., Results: Seventy percent of the focus group participants met criteria for burnout using the MBI. The experience of burnout was described as physical and emotional exhaustion, loss of motivation, isolation from loved ones, and disillusionment with the medical profession. Contributory factors included high workload, burned-out colleagues, perceived undervaluing of family medicine, lack of autonomy, and inability to achieve work-life balance. Protective factors included strong role models in medicine, feeling that one's work is valued and rotations in family medicine., Conclusions: The high level of burnout in family medicine residents in BC is a multifactorial and complex phenomenon. Training programs and faculty should be aware of burnout risk factors and strive to implement changes to reduce burnout, including allowing residents increased control over scheduling, access to counseling services and training for resident mentors.

The current build (galGal4) of the genome of the ancestor of the modern chicken, the Red Jungle Fowl, contains a single endogenous avian leukosis viral element (ALVE) on chromosome 1 (designated RSV-LTR; family ERVK). The assembly shows the ALVE provirus juxtaposed with a member of a second family of avian endogenous retroviruses (designated GGERV20; family ERVL); however, the status of the 3' end of the ALVE element as well as its flanking region remain unclear due to a gap in the reference genome sequence. In this study, we filled the gap in the assembly using a combination of long-range PCR (LR-PCR) and a short contig present in the unassembled portion of the reference genome database. Our results demonstrate that the ALVE element (ALVE-JFevB) is inserted into the putative envelope region of a GGERV20 element, roughly 1 kbp from its 3' end, and that ALVE-JFevB is complete, and depending on its expression status, potentially capable of directing the production of virus. Moreover, the unassembled portion of the genome database contains junction fragments for a second, previously characterized endogenous proviral element, ALVE-6., (©2014 Poultry Science Association Inc.)

In reptiles, sex-determining mechanisms have evolved repeatedly and reversibly between genotypic and temperature-dependent sex determination. The gene Dmrt1 directs male determination in chicken (and presumably other birds), and regulates sex differentiation in animals as distantly related as fruit flies, nematodes and humans. Here, we show a consistent molecular difference in Dmrt1 between reptiles with genotypic and temperature-dependent sex determination. Among 34 non-avian reptiles, a convergently evolved pair of amino acids encoded by sequence within exon 2 near the DM-binding domain of Dmrt1 distinguishes species with either type of sex determination. We suggest that this amino acid shift accompanied the evolution of genotypic sex determination from an ancestral condition of temperature-dependent sex determination at least three times among reptiles, as evident in turtles, birds and squamates. This novel hypothesis describes the evolution of sex-determining mechanisms as turnover events accompanied by one or two small mutations., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

Piglets reared in swine production in the USA undergo painful procedures that include castration, tail docking, teeth clipping, and identification with ear notching or tagging. These procedures are usually performed without pain mitigation. The objective of this project was to develop recommendations for pain mitigation in 1- to 28-day-old piglets undergoing these procedures. The National Pork Board funded project to develop recommendations for pain mitigation in piglets. Recommendation development followed a defined multi-step process that included an evidence summary and estimates of the efficacies of interventions. The results of a systematic review of the interventions were reported in a companion paper. This manuscript describes the recommendation development process and the final recommendations. Recommendations were developed for three interventions (CO2/O2 general anesthesia, non-steroidal anti-inflammatory drugs (NSAIDs), and lidocaine) for use during castration. The ability to make strong recommendations was limited by low-quality evidence and strong certainty about variation in stakeholder values and preferences. The panel strongly recommended against the use of a CO2/O2 general anesthesia mixture, weakly recommended for the use of NSAIDs and weakly recommended against the use of lidocaine for pain mitigation during castration of 1- to 28-day-old piglets.

Pigs are mixed into new social groups at various stages of production, and this study investigated the response of gilts to mixing with older unfamiliar sows. Four groups of 6 gilts and 1 group of 5 gilts were housed in stable groups until the most recently bred female reached d 39 of gestation. Then, stable groups were split such that 15 gilts remained in their home pens (CON) and 14 gilts were mixed (MIX) with 3 multiparous sows for 1 wk (M1), returned to the home pen for 1 wk, and then mixed with 3 different sows for 1 wk (M2). Continuous behavioral observations of gilt location in the pen, posture, aggressive, and other social interactions were made from the start of M1 and M2 for 5 h. Five-minute scans of location, posture, and behavior as well as salivary cortisol measurements were taken before, during, and after each mix week, whereas skin lesions were counted before and after each mix. During the mixed period, MIX gilts spent more (P ≤ 0.009) time in the individual feeding stalls, ventral lying, sitting, involved in aggression, and idle and less (P < 0.001) time in the straw-bedded area, lateral lying, and performing exploratory behavior (chewing, nosing, and rooting) than CON gilts. Additionally, MIX gilts had greater (P < 0.001) salivary cortisol concentrations and accumulated more (P < 0.001) skin lesions during mixing sessions compared to CON gilts. Mixed gilts in M2 spent more (P ≤ 0.013) time in the feeding stalls and ventral lying and less (P ≤ 0.029) time standing and in the dunging passageway compared to M1. Compared to M1, gilts had lower (P < 0.001) salivary cortisol concentrations and fewer (P ≤ 0.027) lesions in front and middle regions of the body during M2. During M2, salivary cortisol concentration was positively correlated with time spent in the dunging passageway (Spearman's rank correlation [rs] = 0.650, P = 0.022) and negatively correlated with time spent in the feeding stalls (rs = -0.762, P = 0.004). Results indicated that gilts adapted their behavior during M2 by using the feeding stalls more, and they became less active, especially when interacting with the sows. Despite this adaptation in M2, lesions and salivary cortisol in M IX gilts were much greater than in CON gilts, highlighting the severity of mixing gilts with multiparous sows.

Endogenous retroviral elements (ERVs) are prolific components of the genomes of complex species, typically occupying more sequence space than do essential, protein-encoding genes. Much of what we know today about the structure and function, as well as the evolution and pathogenic potential, of ERVs was fleshed out over several decades during the last century using the avian leukosis virus subgroup E-related (ALVE) family of endogenous retroviruses of chickens as a model system. A critical enabling factor in the elucidation of ALVE structure and function is the ability to detect and unambiguously identify specific ALVE proviral elements and to develop accurate element profiles for individual chickens under study. Currently, the most common approach for ALVE locus detection involves element-specific PCR assays carried out using primers that target host DNA near the insertion site of the provirus (i.e., the upstream and downstream flanks of the unoccupied site). Here we describe a new approach for proviral detection that exploits restriction enzyme sites in flanking DNA to develop ALVE element profiles more rapidly than with assays currently in use. Moreover, unlike element-specific PCR tests, the "profiling" assay detects novel ALVEs for which insertion sites have not yet been identified as well as previously characterized elements.

Serine integrases catalyze the integration and excision of phage genomes into and out of bacterial chromosomes in a highly specific and directional manner, making these proteins powerful tools for genome engineering. In 2013, the first structure of a serine integrase-DNA complex was reported. This work revealed how the phage attP sequence is recognized by the integrase and provided important clues about how serine integrases bind to other attachment site sequences. The resulting structural models indicate that distinct spatial arrangements of integrase domains are present for each attachment site complex. Here we describe how serine integrases may exploit this site-dependent domain arrangement to regulate the direction of recombination. We also discuss how phage-encoded recombination directionality factors could change this directionality by altering the nature of inter-subunit interactions., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Serine integrases catalyze the integration of bacteriophage DNA into a host genome by site-specific recombination between 'attachment sites' in the phage (attP) and the host (attB). The reaction is highly directional; the reverse excision reaction between the product attL and attR sites does not occur in the absence of a phage-encoded factor, nor does recombination occur between other pairings of attachment sites. A mechanistic understanding of how these enzymes achieve site-selectivity and directionality has been limited by a lack of structural models. Here, we report the structure of the C-terminal domains of a serine integrase bound to an attP DNA half-site. The structure leads directly to models for understanding how the integrase-bound attP and attB sites differ, why these enzymes preferentially form attP × attB synaptic complexes to initiate recombination, and how attL × attR recombination is prevented. In these models, different domain organizations on attP vs. attB half-sites allow attachment-site specific interactions to form between integrase subunits via an unusual protruding coiled-coil motif. These interactions are used to preferentially synapse integrase-bound attP and attB and inhibit synapsis of integrase-bound attL and attR. The results provide a structural framework for understanding, testing and engineering serine integrase function.

Large serine recombinases (LSRs) catalyze the movement of DNA elements into and out of bacterial chromosomes using site-specific recombination between short DNA "attachment sites". The LSRs that function as bacteriophage integrases carry out integration between attachment sites in the phage (attP) and in the host (attB). This process is highly directional; the reverse excision reaction between the product attL and attR sites does not occur in the absence of a phage-encoded recombination directionality factor, nor does recombination typically occur between other pairings of attachment sites. Although the mechanics of strand exchange are reasonably well understood through studies of the closely related resolvase and invertase serine recombinases, many of the fundamental aspects of the LSR reactions have until recently remained poorly understood on a structural level. In this review, we discuss the results of several years worth of biochemical and molecular genetic studies of LSRs in light of recently described structural models of LSR-DNA complexes. The focus is understanding LSR domain structure, how LSRs bind to the attP and attB attachment sites, and the differences between attP-binding and attB-binding modes. The simplicity, site-selectivity and strong directionality of the LSRs has led to their use as important tools in a number of genetic engineering applications in a wide variety of organisms. Given the important potential role of LSR enzymes in genetic engineering and gene therapy, understanding the structure and DNA-binding properties of LSRs is of fundamental importance for those seeking to enhance or alter specificity and functionality in these systems.

This report deals with the identification of novel elements belonging to a family of endogenous retroviruses, designated endogenous avian leukosis virus-type proviral elements (ALVE), that reside in the genome of the chicken and are closely related to exogenous avian leukosis viruses. The study of ALVE elements in the chicken genome serves as a model system for understanding the interplay between endogenous viruses and their vertebrate hosts in general, including humans. In this report, we characterize the insertion sites and describe locus-specific, diagnostic polymerase chain reaction-based assays for three previously discovered, but as yet not localized, ALVE elements. In addition, we assess the proviral integrity, provide the complete element sequence and examine the genomic environs of the three broiler-derived elements.

The Gene Ontology (GO) Consortium (GOC, http://www.geneontology.org) is a community-based bioinformatics resource that classifies gene product function through the use of structured, controlled vocabularies. Over the past year, the GOC has implemented several processes to increase the quantity, quality and specificity of GO annotations. First, the number of manual, literature-based annotations has grown at an increasing rate. Second, as a result of a new 'phylogenetic annotation' process, manually reviewed, homology-based annotations are becoming available for a broad range of species. Third, the quality of GO annotations has been improved through a streamlined process for, and automated quality checks of, GO annotations deposited by different annotation groups. Fourth, the consistency and correctness of the ontology itself has increased by using automated reasoning tools. Finally, the GO has been expanded not only to cover new areas of biology through focused interaction with experts, but also to capture greater specificity in all areas of the ontology using tools for adding new combinatorial terms. The GOC works closely with other ontology developers to support integrated use of terminologies. The GOC supports its user community through the use of e-mail lists, social media and web-based resources.

Summary: InterMine is an open-source data warehouse system that facilitates the building of databases with complex data integration requirements and a need for a fast customizable query facility. Using InterMine, large biological databases can be created from a range of heterogeneous data sources, and the extensible data model allows for easy integration of new data types. The analysis tools include a flexible query builder, genomic region search and a library of 'widgets' performing various statistical analyses. The results can be exported in many commonly used formats. InterMine is a fully extensible framework where developers can add new tools and functionality. Additionally, there is a comprehensive set of web services, for which client libraries are provided in five commonly used programming languages., Availability: Freely available from http://www.intermine.org under the LGPL license., Contact: g.micklem@gen.cam.ac.uk, Supplementary Information: Supplementary data are available at Bioinformatics online.

The prenatal period is of critical importance in defining how individuals respond to their environment throughout life. Stress experienced by pregnant females has been shown to have detrimental effects on offspring biology in humans and a variety of other species. It also is becoming increasingly apparent that prenatal events can have important consequences for the behavior, health, and productivity of offspring in farmed species. Pregnant cattle may experience many potentially important stressors, for instance, relating to their social environment, housing system and physical environment, interactions with humans and husbandry procedures, and their state of health. We examined the available literature to provide a review of the implications of prenatal stress for offspring welfare in cattle. The long-term effects of dystocia on cattle offspring also are reviewed. To ensure a transparent and repeatable selection process, a systematic review approach was adopted. The research literature clearly demonstrates that prenatal stress and difficult births in beef and dairy cattle both have implications for offspring welfare and performance. Common husbandry practices, such as transport, were shown to influence offspring biology and the importance of environmental variables, including thermal stress and drought, also were highlighted. Maternal disease during pregnancy was shown to negatively impact offspring welfare. Moreover, dystocia-affected calves suffer increased mortality and morbidity, decreased transfer of passive immunity, and important physiological and behavioral changes. This review also identified considerable gaps in our knowledge and understanding of the effects of prenatal stress in cattle.

Objectives: There are significant health disparities according to sexual orientation and gender identity, particularly in mental health; however, very few mental health professionals specialise in caring for lesbian, gay, bisexual and transgender (LGBT) communities. The purpose of this study was to explore how providers with LGBT-focused practices have developed their capacity for working with these populations., Methods: Eight semi-structured interviews were conducted with practising mental health service providers with extensive experience serving LGBT individuals. Participants represented four professional disciplines: psychiatry (n = 2); social work (n = 3); psychotherapy (n = 2), and psychology (n = 1). The data were analysed for themes that were identified using a descriptive phenomenological approach., Results: All providers self-identified as members of LGBT communities; however, most agreed that this membership was not necessary to provide supportive, appropriate care for LGBT individuals. Providers described their self-identity as members of LGBT communities, associated lived experiences and recognition of the need for mental health services that are sensitive to the unique needs of LGBT individuals as influential factors in their career decisions. The lack of training opportunities and resources specific to the provision of LGBT-sensitive mental health services was highlighted. Provider recommendations included the introduction of mandatory LGBT health content in education curricula that addresses basic LGBT-related terminology, appropriate interview questions to facilitate the disclosure of sexual orientation and gender identity, information regarding the health impact of heterosexism and homophobia, and specific health care needs of sexual and gender identity minority people., Conclusions: Data from this study suggest there are few opportunities for medical providers to access training and gain expertise in the provision of care to LGBT people. Additional research is needed to consider whether the lack of LGBT health content in medical and psychiatric training programme curricula indirectly contributes to the health disparities experienced by these populations., (© Blackwell Publishing Ltd 2012.)

Tigger elements belong to the Tc1/Mariner family of DNA transposons which is remarkably widespread in nature with homologs present in organisms as diverse as fungi, plants and animals. In this report, we present the nucleotide sequence of a defragmented Tigger1 element from the genome of the American mink. The element is 2,274 bp long, carries 13 bp terminal inverted repeats (TIRs) and contains the vestiges of two open reading frames (ORFs), one of which is similar to the centromere associated protein CENP B. In addition, we estimate that the genome of the American mink contains approximately 1000 Tigger1 elements, but find no evidence for the transcription of extant elements in the mink., (Copyright © 2012 Elsevier B.V. All rights reserved.)

PomBase (www.pombase.org) is a new model organism database established to provide access to comprehensive, accurate, and up-to-date molecular data and biological information for the fission yeast Schizosaccharomyces pombe to effectively support both exploratory and hypothesis-driven research. PomBase encompasses annotation of genomic sequence and features, comprehensive manual literature curation and genome-wide data sets, and supports sophisticated user-defined queries. The implementation of PomBase integrates a Chado relational database that houses manually curated data with Ensembl software that supports sequence-based annotation and web access. PomBase will provide user-friendly tools to promote curation by experts within the fission yeast community. This will make a key contribution to shaping its content and ensuring its comprehensiveness and long-term relevance.

In an effort to comprehensively characterize the functional elements within the genomes of the important model organisms Drosophila melanogaster and Caenorhabditis elegans, the NHGRI model organism Encyclopaedia of DNA Elements (modENCODE) consortium has generated an enormous library of genomic data along with detailed, structured information on all aspects of the experiments. The modMine database (http://intermine.modencode.org) described here has been built by the modENCODE Data Coordination Center to allow the broader research community to (i) search for and download data sets of interest among the thousands generated by modENCODE; (ii) access the data in an integrated form together with non-modENCODE data sets; and (iii) facilitate fine-grained analysis of the above data. The sophisticated search features are possible because of the collection of extensive experimental metadata by the consortium. Interfaces are provided to allow both biologists and bioinformaticians to exploit these rich modENCODE data sets now available via modMine.

A key indicator of resource use within farming is technical efficiency, which measures the amount of physical output attainable from a given set of inputs. The social aspects, in particular the treatment of animals, have generally been ignored within these measurement schemas. In addition, animal welfare will affect the production technology under which farms operate, and some allowance for this is needed within the measurement approach. This is the first paper to apply animal welfare as a discriminating technology within a technical efficiency framework. Using results from an animal welfare monitoring study coupled with resource usage data, it presents an adjusted measure of technical efficiency applied to a sample of British dairy farms and compares differences in lameness management strategies for herds. We employ both a categorical and nondiscretionary variant of the data envelopment analysis approach to measure technical efficiencies and adjust for various degrees of lameness prevalence among these farms. This paper finds that farms with low rates of lameness (below 10% of the cattle herd) tend to have significantly higher technical efficiencies than those with lameness rates of above 10% of the herd. Farms that have levels of lameness of between 10 to 20% of the herd and higher levels of lameness (above 20% of the herd) did not differ significantly. Furthermore, low lameness farms are inefficient in terms of labor and stocking density, but this is outweighed by the gain in milk yield obtained on these farms. Consequently, we argue for a whole-farm, rather than a partial indicator, approach to assessing efficiency when noneconomic factors such as lameness are accounted for. From a policy perspective, we support programs that encourage active lameness management., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The behavior of groups of housed, lactating dairy cattle was observed over 2 winter housing periods on 20 organic farms and 20 conventional farms in the United Kingdom. Three methods were used: (1) 6 video-clips of 10 min duration were captured of cows feeding at sections of the feed-bunk face during the peak feeding period (0 to 90 min) and continuously observed for aggressive interactions among cows; (2) the proportion of cows at the feed-bunk face was scanned every 15 min for 4.5h to include the peak feeding period (0 to 255 min); and (3) all nonfeeding behaviors were scanned every 15 min for 2.5h after the peak feeding period (120 to 255 min). The latter scans were analyzed post hoc for measures of cow comfort (freestall farms only). Management and health data were collected on each farm. On farms with open-fronted feed-bunk faces, a greater number of aggressive interactions occurred at the feed-bunk face at peak feeding time on organic farms than on conventional farms (organic = 36.3 ± 4.4; conventional = 29.1 ± 3.0). Higher proportions of cows were at the feed-bunk face at peak feeding on organic farms than on conventional farms (organic=0.58 ± 0.04; conventional=0.48 ± 0.03). Housing type (freestall versus straw pen) explained most differences in postfeeding behavior (proportion of ruminating cow in alleyways: freestalls=0.16±0.06 vs. straw-pen=0.08 ± 0.03), with few differences between organic and conventional herds. On freestall farms, the proportions of cows on organic farms lying down postfeeding was smaller than in conventional herds (organic=0.38 ± 0.09 vs. conventional=0.43 ± 0.07). Differences in behavior around peak feeding time could be associated with the reduction in food "quality" on organic farms compared with the energy requirement of the cows, with cows on organic farms being highly motivated to feed. A correlation was observed between farms that had high amounts of lying and farms that had high lameness prevalence (R(2)=55.3), suggesting a complex relationship between comfort and pain. Overall, the behavior of dairy cows on organic farms was not different from that of conventionally reared cows, and the results suggest that most behavioral welfare problems relating to housing could be alleviated by management practices., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor-binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor-binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.

Methyltransferases catalyze the methylation processes essential for protein/DNA repair, transcriptional regulation, and drug metabolism in vivo. More than 500 human methyltransferase polymorphisms have been identified, many of which are linked to disease. We mapped all available coding polymorphisms of seven methyltransferases onto their structures to address their structural significance, and identified a polymorphic hotspot ∼20Å from the active site in four of the proteins. Molecular dynamics simulations of these proteins reveal a common mechanism of destabilization: the mutations alter important side-chain contacts within the polymorphic site that are propagated through the protein, thereby distorting the active site. We propose that this hotspot might have arisen to modulate enzymatic activity, with decreased activity actually conferring an advantage in three of the four methyltransferases., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Protein L-isoaspartate O-methyltransferase (PIMT) repairs isoaspartate residues in damaged proteins, and it contains a Val-Ile polymorphismin in alpha5, approximately 13 A from its active site. Val119 has lower activity and thermal stability but increased affinity for endogenous substrates. Studies suggest that heterozygosity for Val/Ile favors efficient isoaspartate repair. We have performed multiple molecular dynamics simulations of 119I and 119V PIMT. Both V119 and I119 interact with the same residues throughout all of the simulations. However, the larger Ile altered the orientations of alpha5 and beta5, both of which have co-substrate binding residues on their distal ends. I119 increases the flexibility of several residues, loosening up the S-adenosylmethionine (SAM)-binding site. These subtle changes are propagated towards the isoaspartate-docking site via residues common to both active sites. The increased mobility in 119I PIMT reorients alpha3, resulting in a salt-bridge network at the substrate-binding interface that disrupts several key side-chain interactions in the isoaspartate site. In contrast, 119V PIMT remains quite rigid with little change to the co-substrate binding site, which could hinder SAM's binding and release, accounting for the decreased activity. These results shed light on the molecular basis behind the decreased activity and increased specificity for endogenous substrates of 119V PIMT relative to the 119I variant. 119I PIMT catalyzes the methylation reaction but may have difficulties recognizing and orienting specific substrates due to its distorted substrate-binding site. Heterozygosity for both the Ile and Val alleles may provide the best of both worlds, allowing the fast and specific methylation of damaged proteins.

Human catechol O-methyltransferase (COMT) contains three common polymorphisms (A22S, A52T, and V108M), two of which (A22S and V108M) render the protein susceptible to deactivation by temperature or oxidation. We have performed multiple molecular dynamics simulations of the wild-type, A22S, A52T, and V108M COMT proteins to explore the structural consequences of these mutations. In total, we have amassed more than 1.4 micros of simulation time, representing the largest set of simulations detailing the effects of polymorphisms on a protein system to date. The A52T mutation had no significant effect on COMT structure in accord with experiment, thereby serving as a good negative control for the simulation set. Residues 22 (alpha2) and 108 (alpha5) interact with each other throughout the simulations and are located in a polymorphic hotspot approximately 20 A from the active site. Introduction of either the larger Ser (22) or Met (108) tightens this interaction, pulling alpha2 and alpha5 toward each other and away from the protein core. The V108M polymorphism rearranges active-site residues in alpha5, beta3, and alpha6, increasing the S-adenosylmethionine site solvent exposure. The A22S mutation reorients alpha2, moving critical catechol-binding residues away from the substrate-binding pocket. The A22S and V108M polymorphisms evolved independently in Northern European and Asian populations. While the decreased activities of both A22S and V108M COMT are associated with an increased risk for schizophrenia, the V108M-induced destabilization is also linked with improved cognitive function. These results suggest that polymorphisms within this hotspot may have evolved to regulate COMT activity and that heterozygosity for either mutation may be advantageous.

Extramedullary plasmacytoma is uncommon, accounting for only 3% of all plasma cell neoplasms. Most lesions occur in the head and neck, primarily in the upper aerodigestive tract; only a minority occur in the larynx. Most cases of extramedullary plasmacytoma are seen in older men; presentation in an adolescent is rare. We report the case of a 13-year-old girl who presented with a 2- to 3-month history of progressive dysphonia. Physical examination identified two lesions--a nasopharyngeal mass and a laryngeal mass. Biopsy indicated that the masses were extramedullary plasmacytomas with amyloid deposition. The patient was treated with radiation therapy, and the tumors were eradicated. We also review the literature with respect to the epidemiology and presentation of extramedullary plasmacytoma of the head and neck, specifically those that affect the larynx.

The human gene for catechol O-methyltransferase has a common single-nucleotide polymorphism that results in substitution of methionine (M) for valine (V) 108 in the soluble form of the enzyme (s-COMT). 108M s-COMT loses enzymatic activity more rapidly than 108V s-COMT at physiological temperature, and the 108M allele has been associated with increased risk of breast cancer and several neuropsychiatric disorders. We used circular dichroism (CD), dynamic light scattering, and fluorescence spectroscopy to examine how the 108V/M polymorphism affects the stability of the purified, recombinant protein to heat and guanidine hydrochloride (GuHCl). COMT contains two tryptophan residues, W143 and W38Y, which are located in loops that border the S-adenosylmethionine (SAM) and catechol binding sites. We therefore also studied the single-tryptophan mutants W38Y and W143Y in order to dissect the contributions of the individual tryptophans to the fluorescence signals. The 108V and 108M proteins differed in the stability of both the tertiary structure surrounding the active site, as probed by the fluorescence yields and emission spectra, and their global secondary structure as reflected by CD. With either probe, the midpoint of the thermal transition of 108M s-COMT was 5 to 7 degrees C lower than that of 108V s-COMT, and the free energy of unfolding at 25 degrees C was smaller by about 0.4 kcal/mol. 108M s-COMT also was more prone to aggregation or partial unfolding to a form with an increased radius of hydration at 37 degrees C. The co-substrate SAM stabilized the secondary structure of both 108V and 108M s-COMT. W143 dominates the tryptophan fluorescence of the folded protein and accounts for most of the decrease in fluorescence that accompanies unfolding by GuHCl. While replacing either tryptophan by tyrosine was mildly destabilizing, the lower stability of the 108M variant was retained in all cases.

Catechol O-methyltransferase (COMT) plays important roles in the metabolism of catecholamine neurotransmitters and catechol estrogens. The development of COMT inhibitors for use in the treatment of Parkinson's disease has been aided by crystallographic structures of the rat enzyme. However, the human and rat proteins have significantly different substrate specificities. Additionally, human COMT contains a common valine-methionine polymorphism at position 108. The methionine protein is less stable than the valine polymorph, resulting in decreased enzyme activity and protein levels in vivo. Here we describe the crystal structures of the 108V and 108M variants of the soluble form of human COMT bound with S-adenosylmethionine (SAM) and a substrate analog, 3,5-dinitrocatechol. The polymorphic residue 108 is located in the alpha5-beta3 loop, buried in a hydrophobic pocket approximately 16 A from the SAM-binding site. The 108V and 108M structures are very similar overall [RMSD of C(alpha) atoms between two structures (C(alpha) RMSD)=0.2 A], and the active-site residues are superposable, in accord with the observation that SAM stabilizes 108M COMT. However, the methionine side chain is packed more tightly within the polymorphic site and, consequently, interacts more closely with residues A22 (alpha2) and R78 (alpha4) than does valine. These interactions of the larger methionine result in a 0.7-A displacement in the backbone structure near residue 108, which propagates along alpha1 and alpha5 toward the SAM-binding site. Although the overall secondary structures of the human and rat proteins are very similar (C(alpha) RMSD=0.4 A), several nonconserved residues are present in the SAM-(I89M, I91M, C95Y) and catechol- (C173V, R201M, E202K) binding sites. The human protein also contains three additional solvent-exposed cysteine residues (C95, C173, C188) that may contribute to intermolecular disulfide bond formation and protein aggregation.