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Find video protocols related to scientific articles indexed in Pubmed.
Recent genetic findings in schizophrenia and their therapeutic relevance.
J. Psychopharmacol. (Oxford)
PUBLISHED: 10-16-2014
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Over 100 loci are now associated with schizophrenia risk as identified by single nucleotide polymorphisms (SNPs) in genome-wide association studies. These findings mean that 'genes for schizophrenia' have unquestionably been found. However, many questions remain unanswered, including several which affect their therapeutic significance. The SNPs individually have minor effects, and even cumulatively explain only a modest fraction of the genetic predisposition. The remainder likely results from many more loci, from rare variants, and from gene-gene and gene-environment interactions. The risk SNPs are almost all non-coding, meaning that their biological significance is unclear; probably their effects are mediated via an influence on gene regulation, and emerging evidence suggests that some key molecular events occur during early brain development. The loci include novel genes of unknown function as well as genes and pathways previously implicated in the pathophysiology of schizophrenia, e.g. NMDA receptor signalling. Genes in the latter category have the clearer therapeutic potential, although even this will be a challenging process because of the many complexities concerning the genetic architecture and mediating mechanisms. This review summarises recent schizophrenia genetic findings and some key issues they raise, particularly with regard to their implications for identifying and validating novel drug targets.
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CCR1 plays a critical role in modulating pain through hematopoietic and non-hematopoietic cells.
PLoS ONE
PUBLISHED: 08-29-2014
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Inflammation is associated with immune cells infiltrating into the inflammatory site and pain. CC chemokine receptor 1 (CCR1) mediates trafficking of leukocytes to sites of inflammation. However, the contribution of CCR1 to pain is incompletely understood. Here we report an unexpected discovery that CCR1-mediated trafficking of neutrophils and CCR1 activity on non-hematopoietic cells both modulate pain. Using a genetic approach (CCR1-/- animals) and pharmacological inhibition of CCR1 with selective inhibitors, we show significant reductions in pain responses using the acetic acid-induced writhing and complete Freund's adjuvant-induced mechanical hyperalgesia models. Reductions in writhing correlated with reduced trafficking of myeloid cells into the peritoneal cavity. We show that CCR1 is highly expressed on circulating neutrophils and their depletion decreases acetic acid-induced writhing. However, administration of neutrophils into the peritoneal cavity did not enhance acetic acid-induced writhing in wild-type (WT) or CCR1-/- mice. Additionally, selective knockout of CCR1 in either the hematopoietic or non-hematopoietic compartments also reduced writhing. Together these data suggest that CCR1 functions to significantly modulate pain by controlling neutrophil trafficking to the inflammatory site and having an unexpected role on non-hematopoietic cells. As inflammatory diseases are often accompanied with infiltrating immune cells at the inflammatory site and pain, CCR1 antagonism may provide a dual benefit by restricting leukocyte trafficking and reducing pain.
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Anthropogenic nutrients and harmful algae in coastal waters.
J. Environ. Manage.
PUBLISHED: 08-28-2014
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Harmful algal blooms (HABs) are thought to be increasing in coastal waters worldwide. Anthropogenic nutrient enrichment has been proposed as a principal causative factor of this increase through elevated inorganic and/or organic nutrient concentrations and modified nutrient ratios. We assess: 1) the level of understanding of the link between the amount, form and ratio of anthropogenic nutrients and HABs; 2) the evidence for a link between anthropogenically generated HABs and negative impacts on human health; and 3) the economic implications of anthropogenic nutrient/HAB interactions. We demonstrate that an anthropogenic nutrient-HAB link is far from universal, and where it has been demonstrated, it is most frequently associated with high biomass rather than low biomass (biotoxin producing) HABs. While organic nutrients have been shown to support the growth of a range of HAB species, insufficient evidence exists to clearly establish if these nutrients specifically promote the growth of harmful species in preference to benign ones, or if/how they influence toxicity of harmful species. We conclude that the role of anthropogenic nutrients in promoting HABs is site-specific, with hydrodynamic processes often determining whether blooms occur. We also find a lack of evidence of widespread significant adverse health impacts from anthropogenic nutrient-generated HABs, although this may be partly due to a lack of human/animal health and HAB monitoring. Detailed economic evaluation and cost/benefit analysis of the impact of anthropogenically generated HABs, or nutrient reduction schemes to alleviate them, is also frequently lacking.
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Expression of ZNF804A in Human Brain and Alterations in Schizophrenia, Bipolar Disorder, and Major Depressive Disorder: A Novel Transcript Fetally Regulated by the Psychosis Risk Variant rs1344706.
JAMA Psychiatry
PUBLISHED: 08-28-2014
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The single-nucleotide polymorphism rs1344706 in the zinc finger protein 804A gene (ZNF804A) shows genome-wide association with schizophrenia and bipolar disorder. Little is known regarding the expression of ZNF804A and the functionality of rs1344706.
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The role of group II metabotropic glutamate receptors in cognition and anxiety: Comparative studies in GRM2(-/-), GRM3(-/-) and GRM2/3(-/-) knockout mice.
Neuropharmacology
PUBLISHED: 08-23-2014
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Group II metabotropic glutamate receptors (mGlu2 and mGlu3, encoded by GRM2 and GRM3) have been implicated in both cognitive and emotional processes, although their precise role remains to be established. Studies with knockout (KO) mice provide an important approach for investigating the role of specific receptor genes in behaviour. In the present series of experiments we extended our prior characterisation of GRM2/3(-/-) double KO mice and, in complementary experiments, investigated the behavioural phenotype of single GRM2(-/-) and GRM3(-/-) mice. We found no consistent effect on anxiety in either the double or single KO mice. The lack of an anxiety phenotype in any of the lines contrasts with the clear anxiolytic effects of mGlu2/3 ligands. Motor co-ordination was impaired in GRM2/3(-/-) mice, but spared in single GRM2(-/-) and GRM3(-/-) mice. Spatial working memory (rewarded alternation) testing on the elevated T-maze revealed a deficit in GRM2(-/-) mice throughout testing, whereas GRM3(-/-) mice exhibited a biphasic effect (initially impaired, but performing better than controls by the end of training). A biphasic effect on activity levels was seen for the GRM2(-/-) mice. Overall, the phenotype in both GRM2(-/-) and GRM3(-/-) mice was less pronounced - if present at all - compared to GRM2/3(-/-) mice, across the range of task domains. This is consistent with possible redundancy of function and/or compensation in the single KO lines. Results are discussed with reference to a possible role for group II metabotropic glutamate receptors at the interface between arousal and behavioural performance, according to an inverted U-shaped function.
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CLEC-2 expression is maintained on activated platelets and on platelet microparticles.
Blood
PUBLISHED: 08-22-2014
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The C-type lectin-like receptor CLEC-2 mediates platelet activation through a hem-immunoreceptor tyrosine-based activation motif (hemITAM). CLEC-2 initiates a Src- and Syk-dependent signaling cascade that is closely related to that of the 2 platelet ITAM receptors: glycoprotein (GP)VI and Fc?RIIa. Activation of either of the ITAM receptors induces shedding of GPVI and proteolysis of the ITAM domain in Fc?RIIa. In the present study, we generated monoclonal antibodies against human CLEC-2 and used these to measure CLEC-2 expression on resting and stimulated platelets and on other hematopoietic cells. We show that CLEC-2 is restricted to platelets with an average copy number of ?2000 per cell and that activation of CLEC-2 induces proteolytic cleavage of GPVI and Fc?RIIa but not of itself. We further show that CLEC-2 and GPVI are expressed on CD41+ microparticles in megakaryocyte cultures and in platelet-rich plasma, which are predominantly derived from megakaryocytes in healthy donors, whereas microparticles derived from activated platelets only express CLEC-2. Patients with rheumatoid arthritis, an inflammatory disease associated with increased microparticle production, had raised plasma levels of microparticles that expressed CLEC-2 but not GPVI. Thus, CLEC-2, unlike platelet ITAM receptors, is not regulated by proteolysis and can be used to monitor platelet-derived microparticles.
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How Cannabis Causes Paranoia: Using the Intravenous Administration of ?9-Tetrahydrocannabinol (THC) to Identify Key Cognitive Mechanisms Leading to Paranoia.
Schizophr Bull
PUBLISHED: 07-18-2014
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Paranoia is receiving increasing attention in its own right, since it is a central experience of psychotic disorders and a marker of the health of a society. Paranoia is associated with use of the most commonly taken illicit drug, cannabis. The objective was to determine whether the principal psychoactive ingredient of cannabis-?(9)-tetrahydrocannabinol (THC)-causes paranoia and to use the drug as a probe to identify key cognitive mechanisms underlying paranoia. A randomized, placebo-controlled, between-groups test of the effects of intravenous THC was conducted. A total of 121 individuals with paranoid ideation were randomized to receive placebo, THC, or THC preceded by a cognitive awareness condition. Paranoia was assessed extensively via a real social situation, an immersive virtual reality experiment, and standard self-report and interviewer measures. Putative causal factors were assessed. Principal components analysis was used to create a composite paranoia score and composite causal variables to be tested in a mediation analysis. THC significantly increased paranoia, negative affect (anxiety, worry, depression, negative thoughts about the self), and a range of anomalous experiences, and reduced working memory capacity. The increase in negative affect and in anomalous experiences fully accounted for the increase in paranoia. Working memory changes did not lead to paranoia. Making participants aware of the effects of THC had little impact. In this largest study of intravenous THC, it was definitively demonstrated that the drug triggers paranoid thoughts in vulnerable individuals. The most likely mechanism of action causing paranoia was the generation of negative affect and anomalous experiences.
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Resting GABA and glutamate concentrations do not predict visual gamma frequency or amplitude.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 06-09-2014
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Gamma band oscillations arise in neuronal networks of interconnected GABAergic interneurons and excitatory pyramidal cells. A previous study found a correlation between visual gamma peak frequency, as measured with magnetoencephalography, and resting GABA levels, as measured with magnetic resonance spectroscopy (MRS), in 12 healthy volunteers. If true, this would allow studies in clinical populations testing modulation of this relationship, but this finding has not been replicated. We addressed this important question by measuring gamma oscillations and GABA, as well as glutamate, in 50 healthy volunteers. Visual gamma activity was evoked using an established gratings paradigm, and we applied a beamformer spatial filtering technique to extract source-reconstructed gamma peak frequency and amplitude from the occipital lobe. We determined gamma peak frequency and amplitude from the location with maximal activation and from the location of the MRS voxel to assess the relationship of GABA with gamma. Gamma peak frequency was estimated from the highest value of the raw spectra and by a Gaussian fit to the spectra. MRS data were acquired from occipital cortex. We did not replicate the previously found correlation between gamma peak frequency and GABA concentration. Calculation of a Bayes factor provided strong evidence in favor of the null hypothesis. We also did not find a correlation between gamma activity and glutamate or between gamma and the ratio of GABA/glutamate. Our results suggest that cortical gamma oscillations do not have a consistent, demonstrable relationship to excitatory/inhibitory network activity as proxied by MRS measurements of GABA and glutamate.
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No effect of schizophrenia risk genes MIR137, TCF4, and ZNF804A on macroscopic brain structure.
Schizophr. Res.
PUBLISHED: 05-19-2014
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Single nucleotide polymorphisms (SNPs) within the MIR137, TCF4, and ZNF804A genes show genome-wide association to schizophrenia. However, the biological basis for the associations is unknown. Here, we tested the effects of these genes on brain structure in 1300 healthy adults. Using volumetry and voxel-based morphometry, neither gene-wide effects-including the combined effect of the genes-nor single SNP effects-including specific psychosis risk SNPs-were found on total brain volume, grey matter, white matter, or hippocampal volume. These results suggest that the associations between these risk genes and schizophrenia are unlikely to be mediated via effects on macroscopic brain structure.
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Computational methods for pseudogene annotation based on sequence homology.
Methods Mol. Biol.
PUBLISHED: 05-15-2014
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The number of complete genome sequences explodes more and more with each passing year. Thus, methods for genome annotation need to be honed constantly to handle the deluge of information. Annotation of pseudogenes (i.e., gene copies that appear not to make a functional protein) in genomes is a persistent problem; here, we overview pseudogene annotation methods that are based on the detection of sequence homology in genomic DNA.
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An accountability framework to promote healthy food environments.
Public Health Nutr
PUBLISHED: 02-25-2014
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To review the available literature on accountability frameworks to construct a framework that is relevant to voluntary partnerships between government and food industry stakeholders.
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Classifying prion and prion-like phenomena.
Prion
PUBLISHED: 02-20-2014
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The universe of prion and prion-like phenomena has expanded significantly in the past several years. Here, we overview the challenges in classifying this data informatically, given that terms such as "prion-like", "prion-related" or "prion-forming" do not have a stable meaning in the scientific literature. We examine the spectrum of proteins that have been described in the literature as forming prions, and discuss how "prion" can have a range of meaning, with a strict definition being for demonstration of infection with in vitro-derived recombinant prions. We suggest that although prion/prion-like phenomena can largely be apportioned into a small number of broad groups dependent on the type of transmissibility evidence for them, as new phenomena are discovered in the coming years, a detailed ontological approach might be necessary that allows for subtle definition of different "flavors" of prion / prion-like phenomena.
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Lack of marked cyto- and genotoxicity of cristobalite in devitrified (heated) alkaline earth silicate wools in short-term assays with cultured primary rat alveolar macrophages.
Inhal Toxicol
PUBLISHED: 02-06-2014
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Alkaline earth silicate (AES) wools are low-biopersistence high-temperature insulation wools. Following prolonged periods at high temperatures they may devitrify, producing crystalline silica (CS) polymorphs, including cristobalite, classified as carcinogenic to humans. Here we investigated the cytotoxic and genotoxic significance of cristobalite present in heated AES wools. Primary rat alveolar macrophages were incubated in vitro for 2?h with 200?µg/cm² unheated/heated calcium magnesium silicate wools (CMS1, CMS2, CMS3; heat-treated for 1 week at, or 4 weeks 150?°C below, their respective classification temperatures) or magnesium silicate wool (MS; heated for 24?h at 1260?°C). Types and quantities of CS formed, and fiber size distribution and shape were determined by X-ray diffraction and electron microscopy. Lactate dehydrogenase release and alkaline and hOGG1-modified comet assays were used, ± aluminum lactate (known to quench CS effects), for cytotoxicity/genotoxicity screening. Cristobalite content of wools increased with heating temperature and duration, paralleled by decreases in fiber length and changes in fiber shape. No marked cytotoxicity, and nearly no (CMS) or only slight (MS) DNA-strand break induction was observed, compared to the CS-negative control Al?O?, whereas DQ12 as CS-positive control was highly active. Some samples induced slight oxidative DNA damage, but no biological endpoint significantly correlated with free CS, quartz, or cristobalite. In conclusion, heating of AES wools mediates changes in CS content and fiber length/shape. While changes in fiber morphology can impact biological activity, cristobalite content appears minor or of no relevance to the intrinsic toxicity of heated AES wools in short-term assays with rat alveolar macrophages.
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Prospective observational cohort study of the association between thromboelastometry, coagulation and platelet parameters and bleeding in patients with haematological malignancies- the ATHENA study.
Br. J. Haematol.
PUBLISHED: 02-04-2014
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Previous studies have shown that total platelet count (TPC) inadequately predicts bleeding in thrombocytopenic patients with haematological malignancies. This prospective cohort study evaluated whether rotational thromboelastometry (ROTEM), coagulation or other platelet parameters were more strongly associated with bleeding than TPC. Adults treated at two UK haematology centres for haematological malignancy were enrolled if they had thrombocytopenia (TPC ? 50 × 10(9) /l) at beginning of, or during treatment (International Standard Randomized Controlled Trial Number 81226121). TPC and bleeding symptoms were recorded daily for up to 30 d or until platelet count recovery, hospital discharge or death. Blood samples were tested thrice weekly using ROTEM, Platelet Function Analyser (PFA)-100(®) , coagulation and platelet cytometry assays. Bleeding symptoms and TPC from 49/50 enrolled participants who completed the study were recorded on 754/760 study days. Mean platelet volume and PFA-100(®) closure times were frequently inestimatable because of thrombocytopenia. TPC, absolute immature platelet number (AIPN) and ROTEM maximum clot firmness were significantly associated with bleeding on the day after blood sampling. Only AIPN was associated with bleeding after adjustment of test results for TPC (Odds Ratio 0·52, 95% confidence interval 0·28-0·97; P = 0·038). In a predictive model, AIPN was superior to TPC for predicting bleeding. This study indicates that AIPN may be more clinically useful than TPC at predicting bleeding.
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Adaptive change inferred from genomic population analysis of the ST93 epidemic clone of community-associated methicillin-resistant Staphylococcus aureus.
Genome Biol Evol
PUBLISHED: 02-01-2014
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Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has emerged as a major public health problem around the world. In Australia, ST93-IV[2B] is the dominant CA-MRSA clone and displays significantly greater virulence than other S. aureus. Here, we have examined the evolution of ST93 via genomic analysis of 12 MSSA and 44 MRSA ST93 isolates, collected from around Australia over a 17-year period. Comparative analysis revealed a core genome of 2.6 Mb, sharing greater than 99.7% nucleotide identity. The accessory genome was 0.45 Mb and comprised additional mobile DNA elements, harboring resistance to erythromycin, trimethoprim, and tetracycline. Phylogenetic inference revealed a molecular clock and suggested that a single clone of methicillin susceptible, Panton-Valentine leukocidin (PVL) positive, ST93 S. aureus likely spread from North Western Australia in the early 1970s, acquiring methicillin resistance at least twice in the mid 1990s. We also explored associations between genotype and important MRSA phenotypes including oxacillin MIC and production of exotoxins (?-hemolysin [Hla], ?-hemolysin [Hld], PSM?3, and PVL). High-level expression of Hla is a signature feature of ST93 and reduced expression in eight isolates was readily explained by mutations in the agr locus. However, subtle but significant decreases in Hld were also noted over time that coincided with decreasing oxacillin resistance and were independent of agr mutations. The evolution of ST93 S. aureus is thus associated with a reduction in both exotoxin expression and oxacillin MIC, suggesting MRSA ST93 isolates are under pressure for adaptive change.
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Inter- and intra-individual variability in alpha peak frequency.
Neuroimage
PUBLISHED: 01-19-2014
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Converging electrophysiological evidence suggests that the alpha rhythm plays an important and active role in cognitive processing. Here, we systematically studied variability in posterior alpha peak frequency both between and within subjects. We recorded brain activity using MEG in 51 healthy human subjects under three experimental conditions - rest, passive visual stimulation and an N-back working memory paradigm, using source reconstruction methods to separate alpha activity from parietal and occipital sources. We asked how alpha peak frequency differed within subjects across cognitive conditions and regions of interest, and looked at the distribution of alpha peak frequency between subjects. In both regions we observed an increase of alpha peak frequency from resting state and passive visual stimulation conditions to the N-back paradigm, with a significantly higher alpha peak frequency in the 2-back compared to the 0-back condition. There was a trend for a greater increase in alpha peak frequency during the N-back task in the occipital vs. parietal cortex. The average alpha peak frequency across all subjects, conditions, and regions of interest was 10.3 Hz with a within-subject SD of 0.9 Hz and a between-subject SD of 2.8 Hz. We also measured beta peak frequencies, and except in the parietal cortex during rest, found no indication of a strictly harmonic relationship with alpha peak frequencies. We conclude that alpha peak frequency in posterior regions increases with increasing cognitive demands, and that the alpha rhythm operates across a wider frequency range than the 8-12 Hz band many studies tend to include in their analysis. Thus, using a fixed and limited alpha frequency band might bias results against certain subjects and conditions.
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Application of the maximum cumulative ratio (MCR) as a screening tool for the evaluation of mixtures in residential indoor air.
Sci. Total Environ.
PUBLISHED: 01-16-2014
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The maximum cumulative ratio (MCR) method allows the categorisation of mixtures according to whether the mixture is of concern for toxicity and if so whether this is driven by one substance or multiple substances. The aim of the present study was to explore, by application of the MCR approach, whether health risks due to indoor air pollution are dominated by one substance or are due to concurrent exposure to various substances. Analysis was undertaken on monitoring data of four European indoor studies (giving five datasets), involving 1800 records of indoor air or personal exposure. Application of the MCR methodology requires knowledge of the concentrations of chemicals in a mixture together with health-based reference values for those chemicals. For this evaluation, single substance health-based reference values (RVs) were selected through a structured review process. The MCR analysis found high variability in the proportion of samples of concern for mixture toxicity. The fraction of samples in these groups of concern varied from 2% (Flemish schools) to 77% (EXPOLIS, Basel, indoor), the variation being due not only to the variation in indoor air contaminant levels across the studies but also to other factors such as differences in number and type of substances monitored, analytical performance, and choice of RVs. However, in 4 out of the 5 datasets, a considerable proportion of cases were found where a chemical-by-chemical approach failed to identify the need for the investigation of combined risk assessment. Although the MCR methodology applied in the current study provides no consideration of commonality of endpoints, it provides a tool for discrimination between those mixtures requiring further combined risk assessment and those for which a single-substance assessment is sufficient.
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Increased burst-firing of ventral tegmental area dopaminergic neurons in d-amino acid oxidase knockout mice in vivo.
Eur. J. Neurosci.
PUBLISHED: 01-14-2014
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d-Amino acid oxidase (DAO) degrades the N-methyl-d-aspartate (NMDA) receptor co-agonist d-serine, and is implicated in schizophrenia as a risk gene and therapeutic target. In schizophrenia, the critical neurochemical abnormality affects dopamine, but to date there is little evidence that DAO impacts on the dopamine system. To address this issue, we measured the electrophysiological properties of dopaminergic (DA) and non-DA neurons in the ventral tegmental area (VTA) of anaesthetised DAO knockout (DAO(-/-) ) and DAO heterozygote (DAO(+/-) ) mice as compared with their wild-type (DAO(+/+) ) littermates. Genotype was confirmed at the protein level by western blotting and immunohistochemistry. One hundred and thirty-nine VTA neurons were recorded in total, and juxtacellular labelling of a subset revealed that neurons immunopositive for tyrosine hydroxylase had DA-like electrophysiological properties that were distinct from those of neurons that were tyrosine hydroxylase-immunonegative. In DAO(-/-) mice, approximately twice as many DA-like neurons fired in a bursting pattern than in DAO(+/-) or DAO(+/+) mice, but other electrophysiological properties did not differ between genotypes. In contrast, non-DA-like neurons had a lower firing rate in DAO(-/-) mice than in DAO(+/-) or DAO(+/+) mice. These data provide the first direct evidence that DAO modulates VTA DA neuron activity, which is of interest for understanding both the glutamatergic regulation of dopamine function and the therapeutic potential of DAO inhibitors. The increased DA neuron burst-firing probably reflects increased availability of d-serine at VTA NMDA receptors, but the site, mechanism and mediation of the effect requires further investigation, and may include both direct and indirect processes.
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Acclimation and toxicity of high ammonium concentrations to unicellular algae.
Mar. Pollut. Bull.
PUBLISHED: 01-03-2014
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A literature review on the effects of high ammonium concentrations on the growth of 6 classes of microalgae suggests the following rankings. Mean optimal ammonium concentrations were 7600, 2500, 1400, 340, 260, 100 ?M for Chlorophyceae, Cyanophyceae, Prymnesiophyceae, Diatomophyceae, Raphidophyceae, and Dinophyceae respectively and their tolerance to high toxic ammonium levels was 39,000, 13,000, 2300, 3600, 2500, 1200 ?M respectively. Field ammonium concentrations <100 ?M would not likely reduce the growth rate of most microalgae. Chlorophytes were significantly more tolerant to high ammonium than diatoms, prymnesiophytes, dinoflagellates, and raphidophytes. Cyanophytes were significantly more tolerant than dinoflagellates which were the least tolerant. A smaller but more complete data set was used to estimate ammonium EC?? values, and the ranking was: Chlorophyceae>Cyanophyceae, Dinophyceae, Diatomophyceae, and Raphidophyceae. Ammonia toxicity is mainly attributed to NH? at pHs >9 and at pHs <8, toxicity is likely associated with the ammonium ion rather than ammonia.
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Procoagulant and platelet-derived microvesicle absolute counts determined by flow cytometry correlates with a measurement of their functional capacity.
J Extracell Vesicles
PUBLISHED: 01-01-2014
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Flow cytometry is the most commonly used technology to measure microvesicles (MVs). Despite reported limitations of this technique, MV levels obtained using conventional flow cytometry have yielded many clinically relevant findings, such as associations with disease severity and ability to predict clinical outcomes. This study aims to determine if MV enumeration by flow cytometry correlates with a measurement of their functional capacity, as this may explain how flow cytometry generates clinically relevant results.
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Response of bacterial metabolic activity to riverine dissolved organic carbon and exogenous viruses in estuarine and coastal waters: implications for CO2 emission.
PLoS ONE
PUBLISHED: 01-01-2014
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A cross-transplant experiment between estuarine water and seawater was conducted to examine the response of bacterial metabolic activity to riverine dissolved organic carbon (DOC) input under virus-rich and virus-free conditions, as well as to exogenous viruses. Riverine DOC input increased bacterial production significantly, but not bacterial respiration (BR) because of its high lability. The bioavailable riverine DOC influenced bulk bacterial respiration in two contrasting ways; it enhanced the bulk BR by stimulating bacterial growth, but simultaneously reduced the cell-specific BR due to its high lability. As a result, there was little stimulation of the bulk BR by riverine DOC. This might be partly responsible for lower CO2 degassing fluxes in estuaries receiving high sewage-DOC that is highly labile. Viruses restricted microbial decomposition of riverine DOC dramatically by repressing the growth of metabolically active bacteria. Bacterial carbon demand in the presence of viruses only accounted for 7-12% of that in the absence of viruses. Consequently, a large fraction of riverine DOC was likely transported offshore to the shelf. In addition, marine bacteria and estuarine bacteria responded distinctly to exogenous viruses. Marine viruses were able to infect estuarine bacteria, but not as efficiently as estuarine viruses, while estuarine viruses infected marine bacteria as efficiently as marine viruses. We speculate that the rapid changes in the viral community due to freshwater input destroyed the existing bacteria-virus relationship, which would change the bacterial community composition and affect the bacterial metabolic activity and carbon cycling in this estuary.
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Interaction networks of prion, prionogenic and prion-like proteins in budding yeast, and their role in gene regulation.
PLoS ONE
PUBLISHED: 01-01-2014
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Prions are transmissible, propagating alternative states of proteins. Prions in budding yeast propagate heritable phenotypes and can function in large-scale gene regulation, or in some cases occur as diseases of yeast. Other 'prionogenic' proteins are likely prions that have been determined experimentally to form amyloid in vivo, and to have prion-like domains that are able to propagate heritable states. Furthermore, there are over 300 additional 'prion-like' yeast proteins that have similar amino-acid composition to prions (primarily a bias for asparagines and glutamines). Here, we examine the protein functional and interaction networks that involve prion, prionogenic and prion-like proteins. Set against a marked overall preference for N/Q-rich prion-like proteins not to interact with each other, we observe a significant tendency of prion/prionogenic proteins to interact with other, N/Q-rich prion-like proteins. This tendency is mostly due to a small number of networks involving the proteins NUP100p, LSM4p and PUB1p. In general, different data analyses of functional and interaction networks converge to indicate a strong linkage of prionogenic and prion-like proteins, to stress-granule assembly and related biological processes. These results further elucidate how prions may impact gene regulation, and reveal a broader horizon for the functional relevance of N/Q-rich prion-like domains.
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D-amino acid oxidase is expressed in the ventral tegmental area and modulates cortical dopamine.
Front Synaptic Neurosci
PUBLISHED: 01-01-2014
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D-amino acid oxidase (DAO, DAAO) degrades the NMDA receptor co-agonist D-serine, modulating D-serine levels and thence NMDA receptor function. DAO inhibitors are under development as a therapy for schizophrenia, a disorder involving both NMDA receptor and dopaminergic dysfunction. However, a direct role for DAO in dopamine regulation has not been demonstrated. Here, we address this question in two ways. First, using in situ hybridization and immunohistochemistry, we show that DAO mRNA and immunoreactivity are present in the ventral tegmental area (VTA) of the rat, in tyrosine hydroxylase (TH)-positive and -negative neurons, and in glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes. Second, we show that injection into the VTA of sodium benzoate, a DAO inhibitor, increases frontal cortex extracellular dopamine, as measured by in vivo microdialysis and high performance liquid chromatography. Combining sodium benzoate and D-serine did not enhance this effect, and injection of D-serine alone affected dopamine metabolites but not dopamine. These data show that DAO is expressed in the VTA, and suggest that it impacts on the mesocortical dopamine system. The mechanism by which the observed effects occur, and the implications of these findings for schizophrenia therapy, require further study.
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The use of high-throughput DNA sequencing in the investigation of antigenic variation: application to Neisseria species.
PLoS ONE
PUBLISHED: 01-01-2014
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Antigenic variation occurs in a broad range of species. This process resembles gene conversion in that variant DNA is unidirectionally transferred from partial gene copies (or silent loci) into an expression locus. Previous studies of antigenic variation have involved the amplification and sequencing of individual genes from hundreds of colonies. Using the pilE gene from Neisseria gonorrhoeae we have demonstrated that it is possible to use PCR amplification, followed by high-throughput DNA sequencing and a novel assembly process, to detect individual antigenic variation events. The ability to detect these events was much greater than has previously been possible. In N. gonorrhoeae most silent loci contain multiple partial gene copies. Here we show that there is a bias towards using the copy at the 3' end of the silent loci (copy 1) as the donor sequence. The pilE gene of N. gonorrhoeae and some strains of Neisseria meningitidis encode class I pilin, but strains of N. meningitidis from clonal complexes 8 and 11 encode a class II pilin. We have confirmed that the class II pili of meningococcal strain FAM18 (clonal complex 11) are non-variable, and this is also true for the class II pili of strain NMB from clonal complex 8. In addition when a gene encoding class I pilin was moved into the meningococcal strain NMB background there was no evidence of antigenic variation. Finally we investigated several members of the opa gene family of N. gonorrhoeae, where it has been suggested that limited variation occurs. Variation was detected in the opaK gene that is located close to pilE, but not at the opaJ gene located elsewhere on the genome. The approach described here promises to dramatically improve studies of the extent and nature of antigenic variation systems in a variety of species.
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Assessing and controlling risks from the emission of organic chemicals from construction products into indoor environments.
Environ Sci Process Impacts
PUBLISHED: 11-05-2013
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Construction products can be a significant source of indoor pollutants, including volatile organic compounds that may be a risk to the health and well-being of building occupants. There are currently a number of schemes for the labelling of products according to their potential to emit organic compounds. Assessment of the complex mixtures of compounds that may be released has mandated the development of test methods that allow the determination of the concentrations of the chemicals released from products in controlled test chamber environments. In response to concerns about the financial burden faced by manufacturers required to test products according to the various different labelling schemes currently in existence, the European Commission has investigated the scope for greater harmonisation. This initiative has sought to harmonise the process for the assessment of emissions data, complementing work led by the European standards organisation focussed on harmonising the test chamber procedures. The current labelling schemes have a range of requirements with respect to the number of chemicals to be quantified. A comparison of 13 schemes worldwide has identified 15 lists of target compounds, with a total of 611 chemicals occurring on at least one of the target lists. While harmonisation may clarify and perhaps simplify these requirements, at least in Europe, it can be expected that future changes to product formulations, the introduction of new products and our increasing knowledge about the potential risks to health, will require continued development of new and improved measurement techniques. There is, therefore, a particular challenge for analytical chemists to ensure the efficient provision of high quality emissions data and thereby ultimately enable effective control of risks to human health through the prevention or reduction of indoor air pollution.
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Swept-frequency feedback interferometry using terahertz frequency QCLs: a method for imaging and materials analysis.
Opt Express
PUBLISHED: 10-10-2013
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The terahertz (THz) frequency quantum cascade laser (QCL) is a compact source of high-power radiation with a narrow intrinsic linewidth. As such, THz QCLs are extremely promising sources for applications including high-resolution spectroscopy, heterodyne detection, and coherent imaging. We exploit the remarkable phase-stability of THz QCLs to create a coherent swept-frequency delayed self-homodyning method for both imaging and materials analysis, using laser feedback interferometry. Using our scheme we obtain amplitude-like and phase-like images with minimal signal processing. We determine the physical relationship between the operating parameters of the laser under feedback and the complex refractive index of the target and demonstrate that this coherent detection method enables extraction of complex refractive indices with high accuracy. This establishes an ultimately compact and easy-to-implement THz imaging and materials analysis system, in which the local oscillator, mixer, and detector are all combined into a single laser.
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Innate immune pathways in afferent lymph following vaccination with poly(I:C)-containing liposomes.
Innate Immun
PUBLISHED: 09-17-2013
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Many modern vaccines use defined adjuvants to stimulate the innate immune system and shape the adaptive immune response. The exact nature of these innate signals and whether immune differentiation can originate within the periphery is not known. Here we used an ovine lymphatic cannulation model to characterise the cellular and transcriptomic profile of the afferent lymph following injection of a liposomal vaccine formulation incorporating diphtheria toxoid and the innate stimulator poly(I:C) over a 78-h period. The response to this vaccine featured an early activation of broad pro-inflammatory pathways (e.g. TLR signalling and inflammasome pathways) and the transient recruitment of granulocytes into the lymph. At 24?h a more monocytic cellular profile arose coinciding with a transition to a specific antiviral response characterised by the up-regulation of genes associated with the receptors typical for the viral mimic, poly(I:C) (e.g. TLR3, RIG-I and MDA5). At the latest time points the up-regulation of IL-17A and IL-17F suggested that Th17 cells may participate in the earliest adaptive response to this vaccine. These data provide the most comprehensive picture of the cellular and molecular mechanisms that link the periphery to the draining lymph node following vaccination, and indicate that the immune response is capable of specialising within the periphery.
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Do Neuronal Autoantibodies Cause Psychosis? A Neuroimmunological Perspective.
Biol. Psychiatry
PUBLISHED: 06-27-2013
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In the last decade, autoantibodies targeting proteins on the neuronal surface and that are believed to be directly pathogenic have been described in patients with autoimmune encephalitis. Since then, new antigenic targets have been discovered, and new clinical phenotypes have been recognized. The psychotic disorders are one example of this expanding spectrum. Here, we consider the defining criteria of antibody-mediated central nervous system disease and the extent to which the psychiatric data currently satisfy those criteria. We discuss the implications these findings have for our understanding, nosology, and treatment of psychiatric disorders.
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Crystalline silica in heated man-made vitreous fibres: A review.
Regul. Toxicol. Pharmacol.
PUBLISHED: 06-26-2013
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Refractory ceramic fibres (RCF) and alkaline earth silicate (AES) wools are types of man-made vitreous fibre (MMVF) that are used in demanding high-temperature industrial applications, generally above 900°C and up to 1400°C. When exposed to prolonged high temperatures, MMVF can devitrify with the formation of cristobalite and other crystalline silica species, which is of potential concern because crystalline silica (CS) is classified as carcinogenic. This article reviews the chemico-physical processes and morphological consequences of fibre devitrification, the forms and micro-location of CS produced, and the toxicity of devitrified fibres and the CS species formed in this way. It also examines scenarios for worker exposure to the products of fibre devitrification in industries using RCF and/or AES wools. We identify gaps in knowledge and make recommendations for future research.
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Platelet counting with the BD Accuri(TM) C6 flow cytometer.
Platelets
PUBLISHED: 06-17-2013
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Abstract The Accuri™ C6 is a compact flow cytometer that uses a peristaltic pump with a laminar flow fluidic system and can measure absolute cell counts. In this study we have evaluated this method with the International Reference Method (IRM) simultaneously measured on both the Accuri™ C6 and a reference flow cytometer. After optimisation of sample labelling conditions, final dilutions and flow cytometer settings, a comparison of the absolute fluorescent platelet count with the RBC/platelet ratio on the C6 and the IRM was then performed in 144 patient samples with a full range of platelet counts (range 2-650?×?10(9)/l). The platelet/RBC ratio method determined on the Accuri™ agreed well with the IRM (R(2?)=?0.99, bias?=?2.3 (Bland Altman) and R(2?)=?0.96, bias?=?1.02 at counts <50?×?10(9)/l). The absolute platelet count also agreed well with the IRM (R(2?)=?0.97, bias?=?-0.16 and R(2?)=?0.91, bias?=?3.7 at <50?×?10(9)/l). The C6 absolute platelet count and RBC/platelet ratio methods also agreed well (R(2?)=?0.99, bias?=?-2.5 and R(2?)=?0.95, bias?=?2.71 at counts <50?×?10(9)/l). Reproducibility studies on the C6 gave CVs of <5% for the RB/platelet ratio and <12% for the absolute cell counts. The C6 also demonstrated excellent linearity on diluted samples with both volume and ratio methods (R(2?)=?0.99). As one might expect, the absolute platelet count is therefore slightly more inaccurate than the RBC/platelet ratio particularly at platelet counts <50?×?10(9)/l as it is likely to be more sensitive to pipetting error. The Accuri™ C6 provides a simple, rapid and reliable method for measuring platelet counts by either the RBC/platelet or direct volume methods. The direct volume method can also be used to determine platelet counts within purified platelet preparations or concentrates in the absence of RBC.
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An atlas of over 90,000 conserved noncoding sequences provides insight into crucifer regulatory regions.
Nat. Genet.
PUBLISHED: 06-04-2013
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Despite the central importance of noncoding DNA to gene regulation and evolution, understanding of the extent of selection on plant noncoding DNA remains limited compared to that of other organisms. Here we report sequencing of genomes from three Brassicaceae species (Leavenworthia alabamica, Sisymbrium irio and Aethionema arabicum) and their joint analysis with six previously sequenced crucifer genomes. Conservation across orthologous bases suggests that at least 17% of the Arabidopsis thaliana genome is under selection, with nearly one-quarter of the sequence under selection lying outside of coding regions. Much of this sequence can be localized to approximately 90,000 conserved noncoding sequences (CNSs) that show evidence of transcriptional and post-transcriptional regulation. Population genomics analyses of two crucifer species, A. thaliana and Capsella grandiflora, confirm that most of the identified CNSs are evolving under medium to strong purifying selection. Overall, these CNSs highlight both similarities and several key differences between the regulatory DNA of plants and other species.
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Analysis of the small RNA transcriptional response in multidrug-resistant Staphylococcus aureus after antimicrobial exposure.
Antimicrob. Agents Chemother.
PUBLISHED: 06-03-2013
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The critical role of noncoding small RNAs (sRNAs) in the bacterial response to changing conditions is increasingly recognized. However, a specific role for sRNAs during antibiotic exposure has not been investigated in Staphylococcus aureus. Here, we used Illumina RNA-Seq to examine the sRNA response of multiresistant sequence type 239 (ST239) S. aureus after exposure to four antibiotics (vancomycin, linezolid, ceftobiprole, and tigecycline) representing the major classes of antimicrobials used to treat methicillin-resistant S. aureus (MRSA) infections. We identified 409 potential sRNAs and then compared global sRNA and mRNA expression profiles at 2 and 6 h, without antibiotic exposure and after exposure to each antibiotic, for a vancomycin-susceptible strain (JKD6009) and a vancomycin-intermediate strain (JKD6008). Exploration of this data set by multivariate analysis using a novel implementation of nonnegative matrix factorization (NMF) revealed very different responses for mRNA and sRNA. Where mRNA responses clustered with strain or growth phase conditions, the sRNA responses were predominantly linked to antibiotic exposure, including sRNA responses that were specific for particular antibiotics. A remarkable feature of the antimicrobial response was the prominence of antisense sRNAs to genes encoding proteins involved in protein synthesis and ribosomal function. This study has defined a large sRNA repertoire in epidemic ST239 MRSA and shown for the first time that a subset of sRNAs are part of a coordinated transcriptional response to specific antimicrobial exposures in S. aureus. These data provide a framework for interrogating the role of staphylococcal sRNAs in antimicrobial resistance and exploring new avenues for sRNA-based antimicrobial therapies.
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Testing platelet function.
Hematol. Oncol. Clin. North Am.
PUBLISHED: 05-30-2013
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Platelet function tests have been traditionally used to aid in the diagnosis and management of patients with bleeding problems. Given the role of platelets in atherothrombosis, several dedicated platelet function instruments are now available that are simple to use and can be used as point-of-care assays. These can provide rapid assessment of platelet function within whole blood without the requirement of sample processing. Some tests can be used to monitor antiplatelet therapy and assess risk of bleeding and thrombosis, although current guidelines advise against this. This article discusses the potential utility of tests/instruments that are available.
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Decreased striatal dopamine in group II metabotropic glutamate receptor (mGlu2/mGlu3) double knockout mice.
BMC Neurosci
PUBLISHED: 04-30-2013
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Group II metabotropic glutamate receptors (mGlu2 and mGlu3, encoded by Grm2 and Grm3) have been the focus of attention as treatment targets for a number of psychiatric conditions. Double knockout mice lacking mGlu2 and mGlu3 (mGlu2/3-/-) show a subtle behavioural phenotype, being hypoactive under basal conditions and in response to amphetamine, and with a spatial memory deficit that depends on the arousal properties of the task. The neurochemical correlates of this profile are unknown. Here, we measured tissue levels of dopamine, 5-HT, noradrenaline and their metabolites in the striatum and frontal cortex of mGlu2/3-/- double knockout mice, using high performance liquid chromatography. We also measured the same parameters in mGlu2-/- and mGlu3-/- single knockout mice.
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Cell cultured chondrocyte implantation and scaffold techniques for osteochondral talar lesions.
Foot Ankle Clin
PUBLISHED: 03-08-2013
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Cell cultured techniques have gained interest and popularity in osteochondral defects because, unlike bone marrow stimulation methods, where fibrocartilage fills the defect, they allow for the regeneration of "hyaline-like cartilage" with better stiffness, resilience, and wear characteristics. Osteochondral defects in the ankle are a rare but challenging problem to treat in young active patients. If left alone, they can cause pain and reduced function and risk progressive degenerative changes in the joint. Clinical results of cell cultured and scaffold technology in the ankle, although still limited by small studies and midterm follow-up, are certainly encouraging.
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Effect of seawater-sewage cross-transplants on bacterial metabolism and diversity.
Microb. Ecol.
PUBLISHED: 02-28-2013
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Bioassays experiments were conducted to determine the metabolic and community composition response of bacteria to transplants between relatively pristine coastal seawater and sewage-impacted seawater. There were four treatments: (1) pristine seawater bacteria?+?pristine seawater (Pb?+?Pw), (2) sewage-impacted bacteria?+?sewage-impacted water (Sb?+?Sw), (3) pristine seawater bacteria?+?sewage-impacted water (Pb?+?Sw), and (4) sewage-impacted bacteria?+?pristine seawater (Sb?+?Pw). Sewage-derived DOC was more labile and readily utilized by bacteria, which favored the growth of high nucleic acid (HNA) bacteria, resulting in high bacterial production (BP, 113?±?4.92 to 130?±?15.8 ?g C l(-1)?day(-1)) and low respiration rate (BR, <67?±?11.3 ?g C l(-1)?day(-1)), as well as high bacterial growth efficiency (BGE, 0.68?±?0.09 to 0.71?±?0.05). In contrast, at the relatively pristine site, bacteria utilized natural marine-derived dissolved organic matter (DOM) at the expense of lowering their growth efficiency (BGE, <0.32?±?0.02) with low BP (<62?±?6.3 ?g C l(-1)?day(-1)) and high BR 133?±?14.2 ?g C l(-1)?day(-1)). Sewage DOM input appeared to alter the partitioning of carbon between respiration and production of bacteria, resulting in a shift toward higher BGE, which would not enhance oxygen consumption. Taxonomic classification based on 454 pyrosequencing reads of the 16S rRNA gene amplicons revealed that changes in bacterial community structure occurred when seawater bacteria were transferred to the eutrophic sewage-impacted water. Sewage DOM fueled the growth of Gammma-proteobacteria and Epsilson-proteobacteria and reduced the bacterial richness, but the changes in the community were not apparent when sewage-impacted bacteria were transferred to pristine seawater.
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Biosynthesis of ebelactone A: isotopic tracer, advanced precursor and genetic studies reveal a thioesterase-independent cyclization to give a polyketide ?-lactone.
J. Antibiot.
PUBLISHED: 02-22-2013
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Macrocyclization of polyketides generates arrays of molecular architectures that are directly linked to biological activities. The four-membered ring in oxetanones (?-lactones) is found in a variety of bioactive polyketides (for example, lipstatin, hymeglusin and ebelactone), yet details of its molecular assembly have not been extensively elucidated. Using ebelactone as a model system, and its producer Streptomyces aburaviensis ATCC 31860, labeling with sodium [1-(13)C,(18)O2]propionate afforded ebelactone A that contains (18)O at all oxygen sites. The pattern of (13)C-(18)O bond retention defines the steps for ebelactone biosynthesis, and demonstrates that ?-lactone ring formation occurs by attack of a ?-hydroxy group onto the carbonyl moiety of an acyclic precursor. Reaction of ebelactone A with N-acetylcysteamine (NAC) gives the ?-hydroxyacyl thioester, which cyclizes quantitatively to give ebelactone A in aqueous ethanol. The putative gene cluster encoding the polyketide synthase (PKS) for biosynthesis of 1 was also identified; notably the ebelactone PKS lacks a terminal thioesterase (TE) domain and no stand alone TE was found. Thus the formation of ebelactone is not TE dependent, supporting the hypothesis that cyclization occurs on the PKS surface in a process that is modeled by the chemical cyclization of the NAC thioester.
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The current and potential impact of genetics and genomics on neuropsychopharmacology.
Eur Neuropsychopharmacol
PUBLISHED: 01-30-2013
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One justification for the major scientific and financial investments in genetic and genomic studies in medicine is their therapeutic potential, both for revealing novel targets for drugs which treat the disease process, as well as allowing for more effective and safe use of existing medications. This review considers the extent to which this promise has yet been realised within psychopharmacology, how things are likely to develop in the foreseeable future, and the key issues involved. It draws primarily on examples from schizophrenia and its treatments. One observation is that there is evidence for a range of genetic influences on different aspects of psychopharmacology in terms of discovery science, but far less evidence that meets the standards required before such discoveries impact upon clinical practice. One reason is that results reveal complex genetic influences that are hard to replicate and usually of very small effect. Similarly, the slow progress being made in revealing the genes that underlie the major psychiatric syndromes hampers attempts to apply the findings to identify novel drug targets. Nevertheless, there are some intriguing positive findings of various kinds, and clear potential for genetics and genomics to play an increasing and major role in psychiatric drug discovery.
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Pramanicin analog induces apoptosis in human colon cancer cells: critical roles for Bcl-2, Bim, and p38 MAPK signaling.
PLoS ONE
PUBLISHED: 01-08-2013
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Pramanicin (PMC) is an antifungal agent that was previously demonstrated to exhibit antiangiogenic and anticancer properties in a few in vitro studies. We initially screened a number of PMC analogs for their cytotoxic effects on HCT116 human colon cancer cells. PMC-A, the analog with the most potent antiproliferative effect was chosen to further interrogate the underlying mechanism of action. PMC-A led to apoptosis through activation of caspase-9 and -3. The apoptotic nature of cell death was confirmed by abrogation of cell death with pretreatment with specific caspase inhibitors. Stress-related MAPKs JNK and p38 were both activated concomittantly with the intrinsic apoptotic pathway. Moreover, pharmacological inhibition of p38 proved to attenuate the cell death induction while pretreatment with JNK inhibitor did not exhibit a protective effect. Resistance of Bax -/- cells and the protective nature of caspase-9 inhibition indicate that mitochondria play a central role in PMC-A induced apoptosis. Early post-exposure elevation of cellular Bim and Bax was followed by a marginal Bcl-2 depletion and Bid cleavage. Further analysis revealed that Bcl-2 downregulation occurs at the mRNA level and is critical to mediate PMC-A induced apoptosis, as ectopic Bcl-2 expression substantially spared the cells from death. Conversely, forced expression of Bim proved to significantly increase cell death. In addition, analyses of p53-/- cells demonstrated that Bcl-2/Bim/Bax modulation and MAPK activations take place independently of p53 expression. Taken together, p53-independent transcriptional Bcl-2 downregulation and p38 signaling appear to be the key modulatory events in PMC-A induced apoptosis.
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Regulation of sialidase production in clostridium perfringens by the orphan sensor histidine kinase ReeS.
PLoS ONE
PUBLISHED: 01-01-2013
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Clostridium perfringens is ubiquitous in nature and is often found as a commensal of the human and animal gastrointestinal tract. It is the primary etiological agent of clostridial myonecrosis, or gas gangrene, a serious infection that results in extensive tissue necrosis due to the action of one or more potent extracellular toxins. ?-toxin and perfringolysin O are the major extracellular toxins involved in the pathogenesis of gas gangrene, but histotoxic strains of C. perfringens, such as strain 13, also produce many degradative enzymes such as collagenases, hyaluronidases, sialidases and the cysteine protease, ?-clostripain. The production of many of these toxins is regulated either directly or indirectly by the global VirSR two-component signal transduction system. By isolating a chromosomal mutant and carrying out microarray analysis we have identified an orphan sensor histidine kinase, which we have named ReeS (regulator of extracellular enzymes sensor). Expression of the sialidase genes nanI and nanJ was down-regulated in a reeS mutant. Since complementation with the wild-type reeS gene restored nanI and nanJ expression to wild-type levels, as shown by quantitative reverse transcription-PCR and sialidase assays we concluded that ReeS positively regulates the expression of these sialidase genes. However, mutation of the reeS gene had no significant effect on virulence in the mouse myonecrosis model. Sialidase production in C. perfringens has been previously shown to be regulated by both the VirSR system and RevR. In this report, we have analyzed a previously unknown sensor histidine kinase, ReeS, and have shown that it also is involved in controlling the expression of sialidase genes, adding further complexity to the regulatory network that controls sialidase production in C. perfringens.
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Sexually dimorphic effects of catechol-O-methyltransferase (COMT) inhibition on dopamine metabolism in multiple brain regions.
PLoS ONE
PUBLISHED: 01-01-2013
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The catechol-O-methyltransferase (COMT) enzyme metabolises catecholamines. COMT inhibitors are licensed for the adjunctive treatment of Parkinsons disease and are attractive therapeutic candidates for other neuropsychiatric conditions. COMT regulates dopamine levels in the prefrontal cortex (PFC) but plays a lesser role in the striatum. However, its significance in other brain regions is largely unknown, despite its links with a broad range of behavioural phenotypes hinting at more widespread effects. Here, we investigated the effect of acute systemic administration of the brain-penetrant COMT inhibitor tolcapone on tissue levels of dopamine, noradrenaline, and the dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). We examined PFC, striatum, hippocampus and cerebellum in the rat. We studied both males and females, given sexual dimorphisms in several aspects of COMTs function. Compared with vehicle, tolcapone significantly increased dopamine levels in the ventral hippocampus, but did not affect dopamine in other regions, nor noradrenaline in any region investigated. Tolcapone increased DOPAC and/or decreased HVA in all brain regions studied. Notably, several of the changes in DOPAC and HVA, particularly those in PFC, were more prominent in females than males. These data demonstrate that COMT alters ventral hippocampal dopamine levels, as well as regulating dopamine metabolism in all brain regions studied. They demonstrate that COMT is of significance beyond the PFC, consistent with its links with a broad range of behavioural phenotypes. Furthermore, they suggest that the impact of tolcapone may be greater in females than males, a finding which may be of clinical significance in terms of the efficacy and dosing of COMT inhibitors.
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Mitigation of off-target adrenergic binding and effects on cardiovascular function in the discovery of novel ribosomal S6 kinase 2 inhibitors.
J. Pharmacol. Exp. Ther.
PUBLISHED: 11-29-2011
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We previously reported the discovery of a novel ribosomal S6 kinase 2 (RSK2) inhibitor, (R)-5-Methyl-1-oxo-2,3,4,5-tetrahydro-1H-[1,4]diazepino[1,2-a] indole-8-carboxylic acid [1-(3-dimethylamino-propyl)-1H-benzoimidazol-2-yl]-amide (BIX 02565), with high potency (IC(50) = 1.1 nM) targeted for the treatment of heart failure. In the present study, we report that despite nanomolar potency at the target, BIX 02565 elicits off-target binding at multiple adrenergic receptor subtypes that are important in the control of vascular tone and cardiac function. To elucidate in vivo the functional consequence of receptor binding, we characterized the cardiovascular (CV) profile of the compound in an anesthetized rat CV screen and telemetry-instrumented conscious rats. Infusion of BIX 02565 (1, 3, and 10 mg/kg) in the rat CV screen resulted in a precipitous decrease in both mean arterial pressure (MAP; to -65 ± 6 mm Hg below baseline) and heart rate (-93 ± 13 beats/min). In telemetry-instrumented rats, BIX 02565 (30, 100, and 300 mg/kg p.o. QD for 4 days) elicited concentration-dependent decreases in MAP after each dose (to -39 ± 4 mm Hg on day 4 at T(max)); analysis by Demming regression demonstrated strong correlation independent of route of administration and influence of anesthesia. Because of pronounced off-target effects of BIX 02565 on cardiovascular function, a high-throughput selectivity screen at adrenergic ?(1A) and ?(2A) was performed for 30 additional RSK2 inhibitors in a novel chemical series; a wide range of adrenergic binding was achieved (0-92% inhibition), allowing for differentiation within the series. Eleven lead compounds with differential binding were advanced to the rat CV screen for in vivo profiling. This led to the identification of potent RSK2 inhibitors (cellular IC(50) <0.14 nM) without relevant ?(1A) and ?(2A) inhibition and no adverse cardiovascular effects in vivo.
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Colistin-resistant, lipopolysaccharide-deficient Acinetobacter baumannii responds to lipopolysaccharide loss through increased expression of genes involved in the synthesis and transport of lipoproteins, phospholipids, and poly-?-1,6-N-acetylglucosamine.
Antimicrob. Agents Chemother.
PUBLISHED: 10-24-2011
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We recently demonstrated that colistin resistance in Acinetobacter baumannii can result from mutational inactivation of genes essential for lipid A biosynthesis (Moffatt JH, et al., Antimicrob. Agents Chemother. 54:4971-4977). Consequently, strains harboring these mutations are unable to produce the major Gram-negative bacterial surface component, lipopolysaccharide (LPS). To understand how A. baumannii compensates for the lack of LPS, we compared the transcriptional profile of the A. baumannii type strain ATCC 19606 to that of an isogenic, LPS-deficient, lpxA mutant strain. The analysis of the expression profiles indicated that the LPS-deficient strain showed increased expression of many genes involved in cell envelope and membrane biogenesis. In particular, upregulated genes included those involved in the Lol lipoprotein transport system and the Mla-retrograde phospholipid transport system. In addition, genes involved in the synthesis and transport of poly-?-1,6-N-acetylglucosamine (PNAG) also were upregulated, and a corresponding increase in PNAG production was observed. The LPS-deficient strain also exhibited the reduced expression of genes predicted to encode the fimbrial subunit FimA and a type VI secretion system (T6SS). The reduced expression of genes involved in T6SS correlated with the detection of the T6SS-effector protein AssC in culture supernatants of the A. baumannii wild-type strain but not in the LPS-deficient strain. Taken together, these data show that, in response to total LPS loss, A. baumannii alters the expression of critical transport and biosynthesis systems associated with modulating the composition and structure of the bacterial surface.
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Balancing the benefits and risks of public-private partnerships to address the global double burden of malnutrition.
Public Health Nutr
PUBLISHED: 10-13-2011
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Transnational food, beverage and restaurant companies, and their corporate foundations, may be potential collaborators to help address complex public health nutrition challenges. While UN system guidelines are available for private-sector engagement, non-governmental organizations (NGO) have limited guidelines to navigate diverse opportunities and challenges presented by partnering with these companies through public-private partnerships (PPP) to address the global double burden of malnutrition.
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Evidence for retrogene origins of the prion gene family.
PLoS ONE
PUBLISHED: 09-12-2011
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The evolutionary origin of prion genes, only known to exist in the vertebrate lineage, had remained elusive until recently. Following a lead from interactome investigations of the murine prion protein, our previous bioinformatic analyses revealed the evolutionary descent of prion genes from an ancestral ZIP metal ion transporter. However, the molecular mechanism of evolution remained unexplored. Here we present a computational investigation of this question based on sequence, intron-exon, synteny and pseudogene analyses. Our data suggest that during the emergence of metazoa, a cysteine-flanked core domain was modularly inserted, or arose de novo, in a preexisting ZIP ancestor gene to generate a prion-like ectodomain in a subbranch of ZIP genes. Approximately a half-billion years later, a genomic insertion of a spliced transcript coding for such a prion-like ZIP ectodomain may have created the prion founder gene. We document that similar genomic insertions involving ZIP transcripts, and probably relying on retropositional elements, have indeed occurred more than once throughout evolution.
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Transgenic overexpression of the type I isoform of neuregulin 1 affects working memory and hippocampal oscillations but not long-term potentiation.
Cereb. Cortex
PUBLISHED: 08-30-2011
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Neuregulin 1 (NRG1) is a growth factor involved in neurodevelopment and plasticity. It is a schizophrenia candidate gene, and hippocampal expression of the NRG1 type I isoform is increased in the disorder. We have studied transgenic mice overexpressing NRG1 type I (NRG1(tg-type I)) and their wild-type littermates and measured hippocampal electrophysiological and behavioral phenotypes. Young NRG1(tg-type I) mice showed normal memory performance, but in older NRG1(tg-type I) mice, hippocampus-dependent spatial working memory was selectively impaired. Hippocampal slice preparations from NRG1(tg-type I) mice exhibited a reduced frequency of carbachol-induced gamma oscillations and an increased tendency to epileptiform activity. Long-term potentiation in NRG1(tg-type I) mice was normal. The results provide evidence that NRG1 type I impacts on hippocampal function and circuitry. The effects are likely mediated via inhibitory interneurons and may be relevant to the involvement of NRG1 in schizophrenia. However, the findings, in concert with those from other genetic and pharmacological manipulations of NRG1, emphasize the complex and pleiotropic nature of the gene, even with regard to a single isoform.
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Evaluation of the INNOVANCE PFA P2Y test cartridge: sensitivity to P2Y(12) blockade and influence of anticoagulant.
Platelets
PUBLISHED: 08-17-2011
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Monitoring of platelet ADP receptor P2Y(12) inhibition may be performed by a variety of platelet function assays. Given the lack of sensitivity of the existing PFA-100® cartridge formulations to detect P2Y(12) inhibition, a new cartridge for the PFA-100 (INNOVANCE® PFA P2Y) has recently been developed. The performance of the new PFA-100 test cartridge was compared with standard collagen/ADP (CADP) and collagen/epinephrine (CEPI) cartridges, light transmission aggregometry, vasodilator-stimulated phosphoprotein, the VerifyNow® P2Y(12) assay and multiple electrode aggregometry. In this study, 20 normal blood samples anticoagulated with either citrate or hirudin were spiked with two different clinically relevant concentrations (1 and 10?µM final concentration) of the prasugrel active metabolite (R-138727, Lilly/Daiichi Sankyo) for 30?min at 37°C. Comparison of the platelet function tests demonstrated that all tests (except CADP and CEPI) were substantially inhibited by 10?µM R-138727. Intermediate results were typically obtained with 1?µM R-138727 in citrated blood. However, both MEA ADP and ADPHS tests were highly sensitive to 1?µM R-138727 in hirudin anticoagulated blood. Further comparison of citrate or hirudin blood samples (N?=?5) revealed that all platelet tests (except CEPI) became more sensitive to 1?µM R-138727 in hirudinized blood. The INNOVANCE PFA P2Y cartridge proved to be sensitive to P2Y(12) inhibition and was comparable to other currently available platelet function tests. The sensitivity of all platelet function tests for detecting in vitro inhibition of P2Y(12) is markedly different depending on the anticoagulant used.
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Fractionation of spatial memory in GRM2/3 (mGlu2/mGlu3) double knockout mice reveals a role for group II metabotropic glutamate receptors at the interface between arousal and cognition.
Neuropsychopharmacology
PUBLISHED: 08-10-2011
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Group II metabotropic glutamate receptors (mGluR2 and mGluR3, encoded by GRM2 and GRM3) are implicated in hippocampal function and cognition, and in the pathophysiology and treatment of schizophrenia and other psychiatric disorders. However, pharmacological and behavioral studies with group II mGluR agonists and antagonists have produced complex results. Here, we studied hippocampus-dependent memory in GRM2/3 double knockout (GRM2/3(-/-)) mice in an iterative sequence of experiments. We found that they were impaired on appetitively motivated spatial reference and working memory tasks, and on a spatial novelty preference task that relies on animals exploratory drive, but were unimpaired on aversively motivated spatial memory paradigms. GRM2/3(-/-) mice also performed normally on an appetitively motivated, non-spatial, visual discrimination task. These results likely reflect an interaction between GRM2/3 genotype and the arousal-inducing properties of the experimental paradigm. The deficit seen on appetitive and exploratory spatial memory tasks may be absent in aversive tasks because the latter induce higher levels of arousal, which rescue spatial learning. Consistent with an altered arousal-cognition relationship in GRM2/3(-/-) mice, injection stress worsened appetitively motivated, spatial working memory in wild-types, but enhanced performance in GRM2/3(-/-) mice. GRM2/3(-/-) mice were also hypoactive in response to amphetamine. This fractionation of hippocampus-dependent memory depending on the appetitive-aversive context is to our knowledge unique, and suggests a role for group II mGluRs at the interface of arousal and cognition. These arousal-dependent effects may explain apparently conflicting data from previous studies, and have translational relevance for the involvement of these receptors in schizophrenia and other disorders.
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Origins and evolution of the HET-s prion-forming protein: searching for other amyloid-forming solenoids.
PLoS ONE
PUBLISHED: 07-29-2011
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The HET-s prion-forming domain from the filamentous fungus Podospora anserina is gaining considerable interest since it yielded the first well-defined atomic structure of a functional amyloid fibril. This structure has been identified as a left-handed beta solenoid with a triangular hydrophobic core. To delineate the origins of the HET-s prion-forming protein and to discover other amyloid-forming proteins, we searched for all homologs of the HET-s protein in a database of protein domains and fungal genomes, using a combined application of HMM, psi-blast and pGenThreader techniques, and performed a comparative evolutionary analysis of the N-terminal alpha-helical domain and the C-terminal prion-forming domain of HET-s. By assessing the tandem evolution of both domains, we observed that the prion-forming domain is restricted to Sordariomycetes, with a marginal additional sequence homolog in Arthroderma otae as a likely case of horizontal transfer. This suggests innovation and rapid evolution of the solenoid fold in the Sordariomycetes clade. In contrast, the N-terminal domain evolves at a slower rate (in Sordariomycetes) and spans many diverse clades of fungi. We performed a full three-dimensional protein threading analysis on all identified HET-s homologs against the HET-s solenoid fold, and present detailed structural annotations for identified structural homologs to the prion-forming domain. An analysis of the physicochemical characteristics in our set of structural models indicates that the HET-s solenoid shape can be readily adopted in these homologs, but that they are all less optimized for fibril formation than the P. anserina HET-s sequence itself, due chiefly to the presence of fewer asparagine ladders and salt bridges. Our combined structural and evolutionary analysis suggests that the HET-s shape has "limited scope" for amyloidosis across the wider protein universe, compared to the generic left-handed beta helix. We discuss the implications of our findings on future identification of amyloid-forming proteins sharing the solenoid fold.
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Guidelines for the laboratory investigation of heritable disorders of platelet function.
Br. J. Haematol.
PUBLISHED: 07-26-2011
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The guideline writing group was selected to be representative of UK-based medical experts. MEDLINE was systematically searched for publications in English up to the Summer of 2010 using key words platelet, platelet function testing and platelet aggregometry. Relevant references generated from initial papers and published guidelines/reviews were also examined. Meeting abstracts were not included. The writing group produced the draft guideline, which was subsequently revised and agreed by consensus. Further comment was made by members of the Haemostasis and Thrombosis Task Force of the British Committee for Standards in Haematology. The guideline was then reviewed by a sounding board of approximately 40 UK haematologists, the British Committee for Standards in Haematology (BCSH) and the British Society for Haematology Committee and comments incorporated where appropriate. Criteria used to quote levels and grades of evidence are as outlined in appendix 7 of the Procedure for Guidelines Commissioned by the BCSH [http://www.bcshguidelines.com/BCSH_PROCESS/EVIDENCE_LEVELS_AND_GRADES_OF_RECOMMENDATION/43_GRADE.html]. The objective of this guideline is to provide healthcare professionals with clear guidance on platelet function testing in patients with suspected bleeding disorders. The guidance may not be appropriate to patients receiving antiplatelet therapy and in all cases individual patient circumstances may dictate an alternative approach.
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A flow cytometric method for platelet counting in platelet concentrates.
Transfusion
PUBLISHED: 07-25-2011
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The platelets (PLTs) in PLT concentrates are counted with hematology analyzers, but varying results among different hematology analyzers are observed, making comparisons very difficult. Due to the absence of red blood cells in PLT concentrates, the International Council for Standardization in Hematology (ICSH) reference method was modified to be used for PLT concentrates and validated in an international comparative study.
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A morphometric, immunohistochemical, and in situ hybridization study of the dorsal raphe nucleus in major depression, bipolar disorder, schizophrenia, and suicide.
J Affect Disord
PUBLISHED: 07-21-2011
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Several lines of evidence implicate 5-hydroxytryptamine (5-HT, serotonin) in the pathophysiology of mood disorders and suicide. However, it is unclear whether these conditions include morphological involvement of the dorsal raphe nucleus (DRN), the origin of most forebrain 5-HT innervation.
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Importance of the COMT gene for sex differences in brain function and predisposition to psychiatric disorders.
Curr Top Behav Neurosci
PUBLISHED: 07-20-2011
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As outlined elsewhere in this volume, sex differences can affect brain function and its dysfunction in psychiatric disorders. It is known that genetic factors contribute to these sex dimorphisms, but the individual genes have rarely been identified. The catechol-O-methyltransferase (COMT) gene, which encodes an enzyme that metabolises catechol compounds, including dopamine, is a leading candidate in this regard. COMTs enzyme activity, and the neurochemistry and behaviour of COMT knockout mice are both markedly sexually dimorphic. Furthermore, genetic associations between COMT and psychiatric phenotypes frequently show differences between men and women. Although many of these differences are unconfirmed or minor, some appear to be of reasonable robustness and magnitude and are reviewed in this chapter. Sexually dimorphic effects of COMT are usually attributed to transcriptional regulation by oestrogens; however, a careful examination of the literature suggests that additional mechanisms are likely to be at least as important. Here, we review the evidence for a sexually dimorphic influence of COMT upon psychiatric phenotypes and brain function, and discuss potential mechanisms by which this may occur. We conclude that despite the evidence being incomplete, there are accumulating and in places compelling data showing that COMT has markedly sexually dimorphic effects on brain function and its dysfunction in psychiatric disorders. Although oestrogenic regulation of COMT is probably partially responsible for these sex differences, other mechanisms are likely also involved. Since sex differences in the genetic architecture of brain function and psychiatric disorders are the rule not the exception, we anticipate that additional evidence will emerge for sexual dimorphisms, not only in COMT but also in many other autosomal genes.
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Genetic mouse models relevant to schizophrenia: taking stock and looking forward.
Neuropharmacology
PUBLISHED: 07-19-2011
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Genetic mouse models relevant to schizophrenia complement, and have to a large extent supplanted, pharmacological and lesion-based rat models. The main attraction is that they potentially have greater construct validity; however, they share the fundamental limitations of all animal models of psychiatric disorder, and must also be viewed in the context of the uncertain and complex genetic architecture of psychosis. Some of the key issues, including the choice of gene to target, the manner of its manipulation, gene-gene and gene-environment interactions, and phenotypic characterization, are briefly considered in this commentary, illustrated by the relevant papers reported in this special issue.
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Syncytiotrophoblast microvesicles released from pre-eclampsia placentae exhibit increased tissue factor activity.
PLoS ONE
PUBLISHED: 07-12-2011
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Pre-eclampsia is a complication of pregnancy associated with activation of coagulation. It is caused by the placenta, which sheds increased amounts of syncytiotrophoblast microvesicles (STBM) into the maternal circulation. We hypothesized that STBM could contribute to the haemostatic activation observed in pre-eclampsia.
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The PrP-like proteins Shadoo and Doppel.
Top Curr Chem
PUBLISHED: 07-06-2011
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An almost unique place within protein databases, twenty-five years of study has underscored the enigmatically subtle role of PrP(C) in normal cell biology. It seems that PrP has evolved (and survived) to perform a function that does not have a precedent amongst transmembrane cell-surface proteins, perhaps representing a new type of plasma membrane ecosystem. In a context where we await a clarifying insight to unify a panoply of PrP(C) data into logical molecular framework, the GPI-anchored N-glycosylated Doppel and Sho proteins are tantalizing in that they correspond roughly to the front and back halves of PrP(C) itself. These molecules may be simpler - and more "understandable" - entities that can be pursued in parallel to PrP(C), and could open up the biology of mammalian prion proteins from fresh directions. Dpl has a profound role in successful gametogenesis that warrants close scrutiny and a case for deeper study can be made for Sho, a recently discovered CNS-expressed protein with many parallels to established facets of PrP biochemistry. In an aerial view of biomedical research, Sho and Dpl can be considered as adjacent islands in a prion protein archipelago. As such, the coming years of molecular exploration should be extremely interesting.
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Terahertz imaging through self-mixing in a quantum cascade laser.
Opt Lett
PUBLISHED: 07-05-2011
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We demonstrate terahertz (THz) frequency imaging using a single quantum cascade laser (QCL) device for both generation and sensing of THz radiation. Detection is achieved by utilizing the effect of self-mixing in the THz QCL, and, specifically, by monitoring perturbations to the voltage across the QCL, induced by light reflected from an external object back into the laser cavity. Self-mixing imaging offers high sensitivity, a potentially fast response, and a simple, compact optical design, and we show that it can be used to obtain high-resolution reflection images of exemplar structures.
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The dominant Australian community-acquired methicillin-resistant Staphylococcus aureus clone ST93-IV [2B] is highly virulent and genetically distinct.
PLoS ONE
PUBLISHED: 06-28-2011
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Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 has spread rapidly across North America, and CA-MRSA is also increasing in Australia. However, the dominant Australian CA-MRSA strain, ST93-IV [2B] appears distantly related to USA300 despite strikingly similar clinical and epidemiological profiles. Here, we compared the virulence of a recent Australian ST93 isolate (JKD6159) to other MRSA, including USA300, and found that JKD6159 was the most virulent in a mouse skin infection model. We fully sequenced the genome of JKD6159 and confirmed that JKD6159 is a distinct clone with 7616 single nucleotide polymorphisms (SNPs) distinguishing this strain from all other S. aureus genomes. Despite its high virulence there were surprisingly few virulence determinants. However, genes encoding ?-hemolysin, Panton-Valentine leukocidin (PVL) and ?-type phenol soluble modulins were present. Genome comparisons revealed 32 additional CDS in JKD6159 but none appeared to encode new virulence factors, suggesting that this clones enhanced pathogenicity could lie within subtler genome changes, such as SNPs within regulatory genes. To investigate the role of accessory genome elements in CA-MRSA epidemiology, we next sequenced three additional Australian non-ST93 CA-MRSA strains and compared them with JKD6159, 19 completed S. aureus genomes and 59 additional S. aureus genomes for which unassembled genome sequence data was publicly available (82 genomes in total). These comparisons showed that despite its distinctive genotype, JKD6159 and other CA-MRSA clones (including USA300) share a conserved repertoire of three notable accessory elements (SSCmecIV, PVL prophage, and pMW2). This study demonstrates that the genetically distinct ST93 CA-MRSA from Australia is highly virulent. Our comparisons of geographically and genetically diverse CA-MRSA genomes suggest that apparent convergent evolution in CA-MRSA may be better explained by the rapid dissemination of a highly conserved accessory genome from a common source.
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Evolution of multidrug resistance during Staphylococcus aureus infection involves mutation of the essential two component regulator WalKR.
PLoS Pathog.
PUBLISHED: 05-20-2011
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Antimicrobial resistance in Staphylococcus aureus is a major public health threat, compounded by emergence of strains with resistance to vancomycin and daptomycin, both last line antimicrobials. Here we have performed high throughput DNA sequencing and comparative genomics for five clinical pairs of vancomycin-susceptible (VSSA) and vancomycin-intermediate ST239 S. aureus (VISA); each pair isolated before and after vancomycin treatment failure. These comparisons revealed a frequent pattern of mutation among the VISA strains within the essential walKR two-component regulatory locus involved in control of cell wall metabolism. We then conducted bi-directional allelic exchange experiments in our clinical VSSA and VISA strains and showed that single nucleotide substitutions within either walK or walR lead to co-resistance to vancomycin and daptomycin, and caused the typical cell wall thickening observed in resistant clinical isolates. Ion Torrent genome sequencing confirmed no additional regulatory mutations had been introduced into either the walR or walK VISA mutants during the allelic exchange process. However, two potential compensatory mutations were detected within putative transport genes for the walK mutant. The minimal genetic changes in either walK or walR also attenuated virulence, reduced biofilm formation, and led to consistent transcriptional changes that suggest an important role for this regulator in control of central metabolism. This study highlights the dramatic impacts of single mutations that arise during persistent S. aureus infections and demonstrates the role played by walKR to increase drug resistance, control metabolism and alter the virulence potential of this pathogen.
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?-Lactone natural products and derivatives inactivate homoserine transacetylase, a target for antimicrobial agents.
J. Antibiot.
PUBLISHED: 04-27-2011
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Homoserine transacetylase (HTA) catalyzes the transfer of an acetyl group from acetyl-CoA to the hydroxyl group of homoserine. This is the first committed step in the biosynthesis of methionine (Met) from aspartic acid in many fungi, Gram-positive and some Gram-negative bacteria. The enzyme is absent in higher eukaryotes and is important for microorganism growth in Met-poor environments, such as blood serum, making HTA an attractive target for new antimicrobial agents. HTA catalyzes acetyl transfer via a double displacement mechanism facilitated by a classic Ser-His-Asp catalytic triad located at the bottom of a narrow actives site tunnel. We explored the inhibitory activity of several ?-lactones to block the activity of HTA. In particular, the natural product ebelactone A, a ?-lactone with a hydrophobic tail was found to be a potent inactivator of HTA from Haemophilus influenzae. Synthetic analogs of ebelactone A demonstrated improved inactivation characteristics. Covalent modification of HTA was confirmed by mass spectrometry, and peptide mapping identified Ser143 as the modified residue, consistent with the known structure and mechanism of the enzyme. These results demonstrate that ?-lactone inhibitors are excellent biochemical probes of HTA and potential leads for new antimicrobial agents.
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Genetic neuropathology of schizophrenia: new approaches to an old question and new uses for postmortem human brains.
Biol. Psychiatry
PUBLISHED: 04-20-2011
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Human postmortem brain studies are critical for elucidating the pathophysiology and etiology of schizophrenia and other major mental illnesses. The traditional approach compares patients and control subjects but is potentially confounded by a number of artifacts, including medication, substance misuse, and other secondary effects of illness. Genetic advances now make possible a novel approach that focuses on how allelic variation in risk-associated genes affects expression and function of transcripts and proteins. These questions can be addressed in normal brain, overcoming to some extent the confounding effects of studying brains from subjects with schizophrenia; equally, extension of the studies to include cases also has advantages. Conceptually, the approach may be seen as the neuropathologic counterpart of genetic neuroimaging, representing a potentially powerful intermediate phenotype. For several schizophrenia susceptibility genes, the data show that risk-associated polymorphisms do affect gene expression or the function of the encoded protein; in some instances, expression of downstream or interacting partners of the gene are also altered. A further striking finding is that the implicated transcripts often appear to be enriched in, or specific to, human brain. Some also show enhanced expression in fetal brain. These considerations give unique importance to postmortem human brain tissue in elucidating the genetic mechanisms underlying schizophrenia and probably other neurodevelopmental disorders as well. Studies of this kind can provide clues as to the biological mechanisms of genetic association, especially when carried out in conjunction with experimental studies. Moreover, the data, interpreted judiciously, can strengthen the plausibility of the association itself.
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Functionally induced changes in water transport in the proximal tubule segment of rat kidneys.
Int J Nephrol Renovasc Dis
PUBLISHED: 04-13-2011
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To eliminate freezing artifacts in the proximal tubule cells, two cryotechniques were applied to normal rat kidneys, ie, freeze substitution and special freeze drying. In addition, salt depletion and salt loading were applied to groups of rats to evaluate whether the segmental structure of the proximal tubule could be altered. In the superficial part of the renal cortex of normal kidneys, the typical first segment structure in the proximal tubule was generally present in the early postglomerular fraction of the tubule. However, in the second segment, a special cellular phenomenon was constantly present, comprising a significant intercellular space that was easily identified using a light microscope. In the third segment, in which the presence of basolateral interdigitations is minimal, the small lateral space, which was found to be present in cryopreparations between neighboring cells from the normal kidney, was found to be enlarged by heavy salt loading of short duration. It is concluded that these cryotechniques demonstrate quantitative structural variations between superficial and deep nephrons, as well as the presence of extracellular areas between the cells of the second and the third segment, representing a structural background for the essential transport of water from the proximal tubules to the peritubular capillaries.
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Discordant and chameleon sequences: their distribution and implications for amyloidogenicity.
Protein Sci.
PUBLISHED: 03-25-2011
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Identification of ambiguous encoding in protein secondary structure is paramount to develop an understanding of key protein segments underlying amyloid diseases. We investigate two types of structurally ambivalent peptides, which were hypothesized in the literature as indicators of amyloidogenic proteins: discordant ?-helices and chameleon sequences. Chameleon sequences are peptides discovered experimentally in different secondary-structure types. Discordant ?-helices are ?-helical stretches with strong ?-strand propensity or prediction. To assess the distribution of these features in known protein structures, and their potential role in amyloidogenesis, we analyzed the occurrence of discordant ?-helices and chameleon sequences in nonredundant sets of protein domains (n = 4263) and amyloidogenic proteins extracted from the literature (n = 77). Discordant ?-helices were identified if discordance was observed between known secondary structures and secondary-structure predictions from the GOR-IV and PSIPRED algorithms. Chameleon sequences were extracted by searching for identical sequence words in ?-helices and ?-strands. We defined frustrated chameleons and very frustrated chameleons based on varying degrees of total ? propensity ?? propensity. To our knowledge, this is the first study to discern statistical relationships between discordance, chameleons, and amyloidogenicity. We observed varying enrichment levels for some categories of discordant and chameleon sequences in amyloidogenic sequences. Chameleon sequences are also significantly enriched in proteins that have discordant helices, indicating a clear link between both phenomena. We identified the first set of discordant-chameleonic protein segments we predict may be involved in amyloidosis. We present a detailed analysis of discordant and chameleons segments in the family of one of the amyloidogenic proteins, the Prion Protein.
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Nonsurgical management of blunt splenic injury: is it cost effective?
Am. J. Surg.
PUBLISHED: 03-18-2011
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This study analyzed outcomes and cost of splenic embolization compared with surgery for the management of blunt splenic injury.
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Functional biomarkers of musculoskeletal syndrome (MSS) for early in vivo screening of selective MMP-13 inhibitors.
J Pharmacol Toxicol Methods
PUBLISHED: 02-15-2011
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Long-term administration of non-selective matrix metalloproteinase (MMP) inhibitors, such as marimastat, in humans elicits musculoskeletal syndrome (MSS), a syndrome characterized by joint damage including pain, stiffness, and inflammation. This pathology is a significant obstacle to the clinical development of MMP inhibitors and in pre-clinical models MSS can be verified only after terminal histopathology. Consequently, we devised a longitudinal and functional readout of MSS in conscious rats treated with marimastat that was validated against terminal histological assessment.
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Sew it up! A Western Trauma Association multi-institutional study of enteric injury management in the postinjury open abdomen.
J Trauma
PUBLISHED: 02-11-2011
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Use of damage control surgery techniques has reduced mortality in critically injured patients but at the cost of the open abdomen. With the option of delayed definitive management of enteric injuries, the question of intestinal repair/anastomosis or definitive stoma creation has been posed with no clear consensus. The purpose of this study was to determine outcomes on the basis of management of enteric injuries in patients relegated to the postinjury open abdomen.
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A morphometric study of glia and neurons in the anterior cingulate cortex in mood disorder.
J Affect Disord
PUBLISHED: 02-10-2011
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The anterior cingulate cortex (ACC) is a key region for the pathophysiology and treatment of depression. However, it remains unclear whether and how the morphology of the ACC is altered in subjects with mood disorders.
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Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis.
Nanomedicine
PUBLISHED: 02-07-2011
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Cellular microvesicles and nanovesicles (exosomes) are involved in many disease processes and have major potential as biomarkers. However, developments in this area are constrained by limitations in the technology available for their measurement. Here we report on the use of fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles. In this system vesicles are visualized by light scattering using a light microscope. A video is taken, and the NTA software tracks the brownian motion of individual vesicles and calculates their size and total concentration. Using human placental vesicles and plasma, we have demonstrated that NTA can measure cellular vesicles as small as ? 50 nm and is far more sensitive than conventional flow cytometry (lower limit ? 300 nm). By combining NTA with fluorescence measurement we have demonstrated that vesicles can be labeled with specific antibody-conjugated quantum dots, allowing their phenotype to be determined.
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Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay.
Thromb. Res.
PUBLISHED: 01-22-2011
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Circulating cell-derived microparticles (MPs) have been implicated in several disease processes and elevated levels are found in many pathological conditions. The detection and accurate measurement of MPs, although attracting widespread interest, is hampered by a lack of standardisation. The aim of this study was to establish a reliable flow cytometric assay to measure distinct subtypes of MPs in disease and to identify any significant causes of variability in MP quantification.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.