Locomotor hyperactivity in 14-3-3Zeta KO mice is associated with dopamine transporter dysfunction

Dopamine (DA) neurotransmission requires a complex series of enzymatic reactions that are tightly linked to catecholamine exocytosis and receptor interactions on pre- and postsynaptic neurons. Regulation of dopaminergic signalling is primarily achieved through reuptake of extracellular DA by the DA transporter (DAT) on presynaptic neurons. Aberrant regulation of DA signalling, and in particular hyperactivation, has been proposed as a key insult in the presentation of schizophrenia and related neuropsychiatric disorders. We recently identified 14-3-3ζ as an essential component of neurodevelopment and a central risk factor in the schizophrenia protein interaction network. Our analysis of 14-3-3ζ-deficient mice now shows that baseline hyperactivity of knockout (KO) mice is rescued by the antipsychotic drug clozapine. 14-3-3ζ KO mice displayed enhanced locomotor hyperactivity induced by the DA releaser amphetamine. Consistent with 14-3-3ζ having a role in DA signalling, we found increased levels of DA in the striatum of 14-3-3ζ KO mice. Although 14-3-3ζ is proposed to modulate activity of the rate-limiting DA biosynthesis enzyme, tyrosine hydroxylase (TH), we were unable to identify any differences in total TH levels, TH localization or TH activation in 14-3-3ζ KO mice. Rather, our analysis identified significantly reduced levels of DAT in the absence of notable differences in RNA or protein levels of DA receptors D1–D5. Providing insight into the mechanisms by which 14-3-3ζ controls DAT stability, we found a physical association between 14-3-3ζ and DAT by co-immunoprecipitation. Taken together, our results identify a novel role for 14-3-3ζ in DA neurotransmission and provide support to the hyperdopaminergic basis of pathologies associated with schizophrenia and related disorders.H Ramshaw, X Xu, EJ Jaehne, P McCarthy, Z Greenberg, E Saleh, B McClure, J Woodcock, S Kabbara, S Wiszniak, Ting-Yi Wang, C Parish, M van den Buuse, BT Baune, A Lopez and Q Schwar

Genetic diversity of Brazilian isolates of feline immunodeficiency virus

We isolated Feline immunodeficiency virus (FIV) from three adult domestic cats, originating from two open shelters in Brazil. Viruses were isolated from PBMC following co-cultivation with the feline T-lymphoblastoid cell line MYA-1. All amplified env gene products were cloned directly into pGL8MYA. The nucleic acid sequences of seven clones were determined and then compared with those of previously described isolates. The sequences of all of the Brazilian virus clones were distinct and phylogenetic analysis revealed that all belong to subtype B. Three variants isolated from one cat and two variants were isolated from each of the two other cats, indicating that intrahost diversity has the potential to pose problems for the treatment and diagnosis of FIV infection

Decay of interspecific avian flock networks along a disturbance gradient in Amazonia

Our understanding of how anthropogenic habitat change shapes species interactions is in its infancy. This is in large part because analytical approaches such as network theory have only recently been applied to characterize complex community dynamics. Network models are a powerful tool for quantifying how ecological interactions are affected by habitat modification because they provide metrics that quantify community structure and function. Here, we examine how large-scale habitat alteration has affected ecological interactions among mixed-species flocking birds in Amazonian rainforest. These flocks provide a model system for investigating how habitat heterogeneity influences non-trophic interactions and the subsequent social structure of forest-dependent mixed-species bird flocks. We analyse 21 flock interaction networks throughout a mosaic of primary forest, fragments of varying sizes and secondary forest (SF) at the Biological Dynamics of Forest Fragments Project in central Amazonian Brazil. Habitat type had a strong effect on network structure at the levels of both species and flock. Frequency of associations among species, as summarized by weighted degree, declined with increasing levels of forest fragmentation and SF. At the flock level, clustering coefficients and overall attendance positively correlated with mean vegetation height, indicating a strong effect of habitat structure on flock cohesion and stability. Prior research has shown that trophic interactions are often resilient to large-scale changes in habitat structure because species are ecologically redundant. By contrast, our results suggest that behavioural interactions and the structure of non-trophic networks are highly sensitive to environmental change. Thus, a more nuanced, system-by-system approach may be needed when thinking about the resiliency of ecological networks. © 2013 The Author(s) Published by the Royal Society. All rights reserved

Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

Background: Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. Principal Findings: We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L−1 of culture from E. coli with specific activities of 1000 U (U = 1 µmol hydrogen evolved mg−1 min−1). Significance: The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.United States. Dept. of Energy. (contract DE-AC36-08-GO28308

Heterologous Expression and Maturation of an NADP-Dependent [NiFe]-Hydrogenase: A Key Enzyme in Biofuel Production

Hydrogen gas is a major biofuel and is metabolized by a wide range of microorganisms. Microbial hydrogen production is catalyzed by hydrogenase, an extremely complex, air-sensitive enzyme that utilizes a binuclear nickel-iron [NiFe] catalytic site. Production and engineering of recombinant [NiFe]-hydrogenases in a genetically-tractable organism, as with metalloprotein complexes in general, has met with limited success due to the elaborate maturation process that is required, primarily in the absence of oxygen, to assemble the catalytic center and functional enzyme. We report here the successful production in Escherichia coli of the recombinant form of a cytoplasmic, NADP-dependent hydrogenase from Pyrococcus furiosus, an anaerobic hyperthermophile. This was achieved using novel expression vectors for the co-expression of thirteen P. furiosus genes (four structural genes encoding the hydrogenase and nine encoding maturation proteins). Remarkably, the native E. coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from P. furiosus. Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2). The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins

Correlations of EGFR mutations and increases in EGFR and HER2 copy number to gefitinib response in a retrospective analysis of lung cancer patients

<p>Abstract</p> <p>Background</p> <p>Gefitinib, a small molecule tyrosine kinase inhibitor of the Epidermal Growth Factor Receptor (<it>EGFR</it>), has shown limited efficacy in the treatment of lung cancer. Recognized clinical predictors of response to this drug, specifically female, non-smoker, Asian descent, and adenocarcinoma, together suggest a genetic basis for drug response. Recent studies have addressed the relationship between response and either sequence mutations or increased copy number of specific receptor tyrosine kinases. We set out to examine the relationship between response and the molecular status of two such kinases, <it>EGFR </it>and <it>HER2</it>, in 39 patients treated with gefitinib at the BC Cancer Agency.</p> <p>Methods</p> <p>Archival patient material was reviewed by a pathologist and malignant cells were selectively isolated by laser microdissection or manual recovery of cells from microscope slides. Genomic DNA was extracted from 37 such patient samples and exons 18–24, coding for the tyrosine kinase domain of <it>EGFR</it>, were amplified by PCR and sequenced. <it>EGFR </it>and <it>HER2 </it>copy number status were also assessed using FISH in 26 samples. Correlations between molecular features and drug response were assessed using the two-sided Fisher's exact test.</p> <p>Results</p> <p>Mutations previously correlated with response were detected in five tumours, four with exon 19 deletions and one with an exon 21 missense L858R point mutation. Increased gene copy number was observed in thirteen tumours, seven with <it>EGFR </it>amplification, three with <it>HER2 </it>amplification, and three with amplification of both genes. In our study cohort, a correlation was not observed between response and <it>EGFR </it>mutations (exon 19 deletion p = 0.0889, we observed a single exon 21 mutation in a non-responder) or increases in <it>EGFR </it>or <it>HER2 </it>copy number (p = 0.552 and 0.437, respectively).</p> <p>Conclusion</p> <p>Neither mutation of <it>EGFR </it>nor increased copy number of <it>EGFR </it>or <it>HER2 </it>was diagnostic of response to gefitinib in this cohort. However, validation of these features in a larger sample set is appropriate. Identification of additional predictive biomarkers beyond <it>EGFR </it>status may be necessary to accurately predict treatment outcome.</p

Clinical practice guidelines within the Southern African development community: a descriptive study of the quality of guideline development and concordance with best evidence for five priority diseases

<p>Abstract</p> <p>Background</p> <p>Reducing the burden of disease relies on availability of evidence-based clinical practice guidelines (CPGs). There is limited data on availability, quality and content of guidelines within the Southern African Development Community (SADC). This evaluation aims to address this gap in knowledge and provide recommendations for regional guideline development.</p> <p>Methods</p> <p>We prioritised five diseases: HIV in adults, malaria in children and adults, pre-eclampsia, diarrhoea in children and hypertension in primary care. A comprehensive electronic search to locate guidelines was conducted between June and October 2010 and augmented with email contact with SADC Ministries of Health. Independent reviewers used the AGREE II tool to score six quality domains reporting the guideline development process. Alignment of the evidence-base of the guidelines was evaluated by comparing their content with key recommendations from accepted reference guidelines, identified with a content expert, and percentage scores were calculated.</p> <p>Findings</p> <p>We identified 30 guidelines from 13 countries, publication dates ranging from 2003-2010. Overall the '<it>scope and purpose' </it>and '<it>clarity and presentation' </it>domains of the AGREE II instrument scored highest, median 58%(range 19-92) and 83%(range 17-100) respectively. '<it>Stakeholder involvement' </it>followed with median 39%(range 6-75). '<it>Applicability'</it>, '<it>rigour of development' </it>and '<it>editorial independence' </it>scored poorly, all below 25%. Alignment with evidence was variable across member states, the lowest scores occurring in older guidelines or where the guideline being evaluated was part of broader primary healthcare CPG rather than a disease-specific guideline.</p> <p>Conclusion</p> <p>This review identified quality gaps and variable alignment with best evidence in available guidelines within SADC for five priority diseases. Future guideline development processes within SADC should better adhere to global reporting norms requiring broader consultation of stakeholders and transparency of process. A regional guideline support committee could harness local capacity to support context appropriate guideline development.</p

Effect of extended morning fasting upon ad libitum lunch intake and associated metabolic and hormonal responses in obese adults

Background/Objectives: Breakfast omission is positively associated with obesity and increased risk of disease. However, little is known about the acute effects of extended morning fasting upon subsequent energy intake and associated metabolic/regulatory factors in obese adults. Subjects/Methods: In a randomised cross-over design, 24 obese men (n=8) and women (n=16) extended their overnight fast by omitting breakfast consumption or ingesting a typical carbohydrate-rich breakfast of 2183±393 kJ (521±94 kcal), before an ad libitum pasta lunch 3 h later. Blood samples were obtained throughout the day until 3 h post lunch and analysed for hormones implicated in appetite regulation, along with metabolic outcomes and subjective appetite measures. Results: Lunch intake was unaffected by extended morning fasting (difference=218 kJ, 95% confidence interval −54 kJ, 490 kJ; P=0.1) resulting in lower total intake in the fasting trial (difference=−1964 kJ, 95% confidence interval −1645 kJ, −2281 kJ; P<0.01). Systemic concentrations of peptide tyrosine–tyrosine and leptin were lower during the afternoon following morning fasting (Pless than or equal to0.06). Plasma-acylated ghrelin concentrations were also lower following the ad libitum lunch in the fasting trial (P<0.05) but this effect was not apparent for total ghrelin (Pgreater than or equal to0.1). Serum insulin concentrations were greater throughout the afternoon in the fasting trial (P=0.05), with plasma glucose also greater 1 h after lunch (P<0.01). Extended morning fasting did not result in greater appetite ratings after lunch, with some tendency for lower appetite 3 h post lunch (P=0.09). Conclusions: We demonstrate for the first time that, in obese adults, extended morning fasting does not cause compensatory intake during an ad libitum lunch nor does it increase appetite during the afternoon. Morning fasting reduced satiety hormone responses to a subsequent lunch meal but counterintuitively also reduced concentrations of the appetite-stimulating hormone-acylated ghrelin during the afternoon relative to lunch consumed after breakfast

Sooty Mangabey Genome Sequence Provides Insight into AIDS Resistance in a Natural SIV Host

In contrast to infections with human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in macaques, SIV infection of a natural host, sooty mangabeys (Cercocebus atys), is non-pathogenic despite high viraemia. Here we sequenced and assembled the genome of a captive sooty mangabey. We conducted genome-wide comparative analyses of transcript assemblies from C. atys and AIDS-susceptible species, such as humans and macaques, to identify candidates for host genetic factors that influence susceptibility. We identified several immune-related genes in the genome of C. atys that show substantial sequence divergence from macaques or humans. One of these sequence divergences, a C-terminal frameshift in the toll-like receptor-4 (TLR4) gene of C. atys, is associated with a blunted in vitro response to TLR-4 ligands. In addition, we found a major structural change in exons 3-4 of the immune-regulatory protein intercellular adhesion molecule 2 (ICAM-2); expression of this variant leads to reduced cell surface expression of ICAM-2. These data provide a resource for comparative genomic studies of HIV and/or SIV pathogenesis and may help to elucidate the mechanisms by which SIV-infected sooty mangabeys avoid AIDS