Design of a novel quantitative PCR (QPCR)-based protocol for genotyping mice carrying the neuroprotective Wallerian degeneration slow (Wlds) gene

<p>Abstract</p> <p>Background</p> <p>Mice carrying the spontaneous genetic mutation known as Wallerian degeneration slow (<it>Wld</it>^<it>s</it>) have a unique neuroprotective phenotype, where axonal and synaptic compartments of neurons are protected from degeneration following a wide variety of physical, toxic and inherited disease-inducing stimuli. This remarkable phenotype has been shown to delay onset and progression in several mouse models of neurodegenerative disease, suggesting that <it>Wld</it>^<it>s</it>-mediated neuroprotection may assist in the identification of novel therapeutic targets. As a result, cross-breeding of <it>Wld</it>^{<it>s </it>}mice with mouse models of neurodegenerative diseases is used increasingly to understand the roles of axon and synapse degeneration in disease. However, the phenotype shows strong gene-dose dependence so it is important to distinguish offspring that are homozygous or heterozygous for the mutation. Since the <it>Wld</it>^{<it>s </it>}mutation comprises a triplication of a region already present in the mouse genome, the most stringent way to quantify the number of mutant <it>Wld</it>^{<it>s </it>}alleles is using copy number. Current approaches to genotype <it>Wld</it>^{<it>s </it>}mice are based on either Southern blots or pulsed field gel electrophoresis, neither of which are as rapid or efficient as quantitative PCR (QPCR).</p> <p>Results</p> <p>We have developed a rapid, robust and efficient genotyping method for <it>Wld</it>^{<it>s </it>}using QPCR. This approach differentiates, based on copy number, homozygous and heterozygous <it>Wld</it>^{<it>s </it>}mice from wild-type mice and each other. We show that this approach can be used to genotype mice carrying the spontaneous <it>Wld</it>^{<it>s </it>}mutation as well as animals expressing the <it>Wld</it>^{<it>s </it>}transgene.</p> <p>Conclusion</p> <p>We have developed a QPCR genotyping method that permits rapid and effective genotyping of <it>Wld</it>^{<it>s </it>}copy number. This technique will be of particular benefit in studies where <it>Wld</it>^{<it>s </it>}mice are cross-bred with other mouse models of neurodegenerative disease in order to understand the neuroprotective processes conferred by the <it>Wld</it>^{<it>s </it>}mutation.</p

Imaging Atherosclerosis.

Advances in atherosclerosis imaging technology and research have provided a range of diagnostic tools to characterize high-risk plaque in vivo; however, these important vascular imaging methods additionally promise great scientific and translational applications beyond this quest. When combined with conventional anatomic- and hemodynamic-based assessments of disease severity, cross-sectional multimodal imaging incorporating molecular probes and other novel noninvasive techniques can add detailed interrogation of plaque composition, activity, and overall disease burden. In the catheterization laboratory, intravascular imaging provides unparalleled access to the world beneath the plaque surface, allowing tissue characterization and measurement of cap thickness with micrometer spatial resolution. Atherosclerosis imaging captures key data that reveal snapshots into underlying biology, which can test our understanding of fundamental research questions and shape our approach toward patient management. Imaging can also be used to quantify response to therapeutic interventions and ultimately help predict cardiovascular risk. Although there are undeniable barriers to clinical translation, many of these hold-ups might soon be surpassed by rapidly evolving innovations to improve image acquisition, coregistration, motion correction, and reduce radiation exposure. This article provides a comprehensive review of current and experimental atherosclerosis imaging methods and their uses in research and potential for translation to the clinic.J.M.T. is supported by a Wellcome Trust research training fellowship (104492/Z/14/Z). M.D is supported by the British Heart Foundation (FS/14/78/31020). N.R.E. is supported by a research training fellowship from the Dunhill Medical Trust (RTF44/0114). A.J.B. is supported by the British Heart Foundation. J.H.F.R. is part-supported by the HEFCE, the NIHR Cambridge Biomedical Research Centre, the British Heart Foundation, and the Wellcome Trust.This is the final version of the article. It first appeared from the American Heart Association via http://dx.doi.org/10.1161/CIRCRESAHA.115.30624

A multidrug ABC transporter with a taste for salt.

BACKGROUND: LmrA is a multidrug ATP-binding cassette (ABC) transporter from Lactococcus lactis with no known physiological substrate, which can transport a wide range of chemotherapeutic agents and toxins from the cell. The protein can functionally replace the human homologue ABCB1 (also termed multidrug resistance P-glycoprotein MDR1) in lung fibroblast cells. Even though LmrA mediates ATP-dependent transport, it can use the proton-motive force to transport substrates, such as ethidium bromide, across the membrane by a reversible, H(+)-dependent, secondary-active transport reaction. The mechanism and physiological context of this reaction are not known. METHODOLOGY/PRINCIPAL FINDINGS: We examined ion transport by LmrA in electrophysiological experiments and in transport studies using radioactive ions and fluorescent ion-selective probes. Here we show that LmrA itself can transport NaCl by a similar secondary-active mechanism as observed for ethidium bromide, by mediating apparent H(+)-Na(+)-Cl(-) symport. Remarkably, LmrA activity significantly enhances survival of high-salt adapted lactococcal cells during ionic downshift. CONCLUSIONS/SIGNIFICANCE: The observations on H(+)-Na(+)-Cl(-) co-transport substantiate earlier suggestions of H(+)-coupled transport by LmrA, and indicate a novel link between the activity of LmrA and salt stress. Our findings demonstrate the relevance of investigations into the bioenergetics of substrate translocation by ABC transporters for our understanding of fundamental mechanisms in this superfamily. This study represents the first use of electrophysiological techniques to analyze substrate transport by a purified multidrug transporter

Discovery of species-wide tool use in the Hawaiian crow

Funding from the Biotechnology and Biological Sciences Research Council, UK (BBSRC; grant BB/G023913/2 to C.R., and studentship to B.C.K.), the University of St Andrews (C.R.), JASSO (S.S.), and the Royal Society of London (M.B.M.). Funding for thecaptive ‘Alala propagation programme was provided by the U.S. Fish and Wildlife Service, Hawai‘i Division of Forestry and Wildlife, Moore Family Foundation, Marisla Foundation, several anonymous donors, and San Diego Zoo Global.Only a handful of bird species are known to use foraging tools in the wild1. Amongst them, the New Caledonian crow (Corvus moneduloides) stands out with its sophisticated tool-making skills2, 3. Despite considerable speculation, the evolutionary origins of this species’ remarkable tool behaviour remain largely unknown, not least because no naturally tool-using congeners have yet been identified that would enable informative comparisons4. Here we show that another tropical corvid, the ‘Alalā (C. hawaiiensis; Hawaiian crow), is a highly dexterous tool user. Although the ‘Alalā became extinct in the wild in the early 2000s, and currently survives only in captivity5, at least two lines of evidence suggest that tool use is part of the species’ natural behavioural repertoire: juveniles develop functional tool use without training, or social input from adults; and proficient tool use is a species-wide capacity. ‘Alalā and New Caledonian crows evolved in similar environments on remote tropical islands, yet are only distantly related6, suggesting that their technical abilities arose convergently. This supports the idea that avian foraging tool use is facilitated by ecological conditions typical of islands, such as reduced competition for embedded prey and low predation risk4, 7. Our discovery creates exciting opportunities for comparative research on multiple tool-using and non-tool-using corvid species. Such work will in turn pave the way for replicated cross-taxonomic comparisons with the primate lineage, enabling valuable insights into the evolutionary origins of tool-using behaviour.PostprintPeer reviewe

Biophysical and electrochemical studies of protein-nucleic acid interactions

This review is devoted to biophysical and electrochemical methods used for studying protein-nucleic acid (NA) interactions. The importance of NA structure and protein-NA recognition for essential cellular processes, such as replication or transcription, is discussed to provide background for description of a range of biophysical chemistry methods that are applied to study a wide scope of protein-DNA and protein-RNA complexes. These techniques employ different detection principles with specific advantages and limitations and are often combined as mutually complementary approaches to provide a complete description of the interactions. Electrochemical methods have proven to be of great utility in such studies because they provide sensitive measurements and can be combined with other approaches that facilitate the protein-NA interactions. Recent applications of electrochemical methods in studies of protein-NA interactions are discussed in detail

Statewide Systematic Evaluation of Sudden, Unexpected Infant Death Classification: Results from a National Pilot Project

The Centers for Disease Control and Prevention funded seven states, including Kentucky, to clarify statewide death certification practices in sudden, unexpected infant death and compare state performances with national expectations. Accurate assignment of the cause and manner of death in cases of sudden, unexpected infant death is critical for accurate vital statistics data to direct limited resources to appropriate targets, and to implement optimal and safe risk reduction strategies. The primary objectives are to (1) Compare SUID death certifications recommended by the KY medical examiners with the stated cause of death text field on the hard copy death electronic death certificates and (2) Compare KY and national SUID rates. Causes of death for SUID cases recommended by the medical examiners and those appearing on the hard copy and electronic death certificates in KY were collected retrospectively for 2004 and 2005. Medical examiner recommendations were based upon a classification scheme devised by them in 2003. Coroners hard copy death certificates and the cause of death rates in KY were compared to those occurring nationally. Eleven percent of infants dying suddenly and unexpectedly did not undergo autopsy during the study interval. The KY 2003 classification scheme for SIDS is at variance with the NICHD and San Diego SIDS definitions. Significant differences in causes of death recommended by medical examiners and those appearing on the hard copy and electronic death certificates were identified. SIDS rates increased in KY in contrast to decreasing rates nationally. Nationwide adoption of a widely used SIDS definition, such as that proposed in San Diego in 2004 as well as legislation by states to ensure autopsy in all cases of sudden unexpected infant death are recommended. Medical examiners’ recommendations for cause of death should appear on death certificates. Multidisciplinary pediatric death review teams prospectively evaluating cases before death certification is recommended. Research into other jurisdictions death certification process is encouraged

Pregnant women’s awareness, knowledge and beliefs about pelvic floor muscles: a cross-sectional survey

Introduction and hypothesis: Pregnant women benefit from completing pelvic floor muscle exercises (PFMEs). The aims of the study were to evaluate pregnant women’s levels of awareness, knowledge, and beliefs about the pelvic floor muscles (PFMs) and PFMEs. Methods: A cross-sectional survey was conducted. Respondents were pregnant women over the age of 18 years who attended antenatal clinics in Western Australia (WA). Questionnaire items measured awareness and knowledge about PFMs, confidence and beliefs about engaging in PFMEs, and attendance at antenatal education (ANE) classes. Chi-squared tests examined potential associations between questionnaire items and respondent characteristics. Results: Mean gestation of respondents (n = 633 out of 850; 74% response rate) was 28.7 (+7.8) weeks and 50% were giving birth for the first time. Although 76% of respondents knew that PFMs can prevent urinary incontinence, only 27% knew that they prevented faecal incontinence and 41% thought it was normal to leak urine when pregnant. Only n = 72 (11%) were practicing PFMEs. Respondents who had attended ANE (28%) were significantly more knowledgeable about pelvic floor function (p &lt; .001) and significantly less likely to believe that leaking urine during pregnancy was normal (p = 0.02), compared with those who had not attended ANE. Respondents who did not speak English at home (18%) were significantly less knowledgeable about PFMs and PFMEs, and significantly less likely to have attended, or planned to attend, ANE classes. Conclusion: Pregnant women require more health education regarding PFMs. Education should be provided using diverse modes, especially for women from migrant backgrounds and women who do not plan to attend formal ANE classes

Magnetism and its microscopic origin in iron-based high-temperature superconductors

High-temperature superconductivity in the iron-based materials emerges from, or sometimes coexists with, their metallic or insulating parent compound states. This is surprising since these undoped states display dramatically different antiferromagnetic (AF) spin arrangements and N$\rm \acute{e}$el temperatures. Although there is general consensus that magnetic interactions are important for superconductivity, much is still unknown concerning the microscopic origin of the magnetic states. In this review, progress in this area is summarized, focusing on recent experimental and theoretical results and discussing their microscopic implications. It is concluded that the parent compounds are in a state that is more complex than implied by a simple Fermi surface nesting scenario, and a dual description including both itinerant and localized degrees of freedom is needed to properly describe these fascinating materials.Comment: 14 pages, 4 figures, Review article, accepted for publication in Nature Physic

Percepção de autoeficácia, assertividade sexual e uso do preservativo em jovens colombianos

El uso correcto y consistente del condón en las relaciones sexuales es un procedimiento altamente eficaz para la prevención del VIH/SIDA, así como para otras infecciones de transmisión sexual (ITS). Algunos estudios reportan que las habilidades psicosociales y de interrelación favorecen el desarrollo de prácticas sexuales protegidas, como con el uso de preservativos. El objetivo del presente estudio fue determinar en qué medida la percepción de autoeficacia y la asertividad sexual predicen el uso del condón en las relaciones sexuales en un grupo de jóvenes. Se realizó un estudio descriptivo correlacional y predictivo con una muestra de 631 jóvenes con edades entre 19 y 26 años que reportaron haber tenido relaciones o ser activos sexualmente. Los resultados muestran que la percepción de autoeficacia y la asertividad sexual fueron fuertes predictores del uso del condón en las relaciones sexuales de las mujeres, mientras que para el caso de los hombres, solo la asertividad sexual predijo su uso.O uso correto e consistente do preservativo nas relações sexuais é um procedimento altamente eficaz para a prevenção do HIV/ Aids, bem como para outras infecções de transmissão sexual (ITS). Alguns estudos mostram que as habilidades psicossociais e de inter-relação favorecem o desenvolvimento de práticas sexuais protegidas, como com o uso do preservativo. O objetivo do presente estudo foi determinar em que medida a percepção de autoeficácia e a assertividade sexual predizem o uso do preservativo nas relações sexuais em um grupo de jovens. Realizou-se um estudo descritivo correlacional e preditivo com uma amostra de 631 jovens com idades entre 19 e 26 anos que relataram ter tido relações ou ser ativos sexualmente. Os resultados mostram que a percepção de autoeficácia e a assertividade sexual foram fortes indicadores do uso do preservativo nas relações sexuais das mulheres, enquanto para o caso dos homens, só a assertividade sexual predisse seu uso.The correct and consistent use of condoms during sexual intercourse is a highly effective procedure for the prevention of HIV / AIDS as well as other sexually transmitted infections (STIs). Some studies report that psychosocial skills and interaction promote the development of safe sexual relations by using condoms. The objective of this study was to determine to what extent the perception of self-efficacy and sexual assertiveness predicts condom use in sexual activity in a group of young people. The study was descriptive correlational and predictive.The sample consisted of 645 young people aged between 19 and 26 years who reported having had sex or being sexually active. Results showed that, unlike men, the perception of selfefficacy coupled with sexual assertiveness are strong predictors of condom use in women's sexual relations, whereas for men only sexual assertiveness predicts condom use in sexual relations

Injection of Human Bone Marrow and Mononuclear Cell Extract into Infarcted Mouse Hearts Results in Functional Improvement

Background: We have previously shown that mouse whole bone marrow cell (BMC) extract results in improvement of cardiac function and decreases scar size in a mouse model of myocardial infarction (MI), in the absence of intact cells. It is not clear if thes