Development of a Rapid Insulin Assay by Homogenous Time-Resolved Fluorescence

Direct measurement of insulin is critical for basic and clinical studies of insulin secretion. However, current methods are expensive and time-consuming. We developed an insulin assay based on homogenous time-resolved fluorescence that is significantly more rapid and cost-effective than current commonly used approaches. This assay was applied effectively to an insulin secreting cell line, INS-1E cells, as well as pancreatic islets, allowing us to validate the assay by elucidating mechanisms by which dopamine regulates insulin release. We found that dopamine functioned as a significant negative modulator of glucose-stimulated insulin secretion. Further, we showed that bromocriptine, a known dopamine D2/D3 receptor agonist and newly approved drug used for treatment of type II diabetes mellitus, also decreased glucose-stimulated insulin secretion in islets to levels comparable to those caused by dopamine treatment

An XMM-Newton Observation of the Local Bubble Using a Shadowing Filament in the Southern Galactic Hemisphere

We present an analysis of the X-ray spectrum of the Local Bubble, obtained by simultaneously analyzing spectra from two XMM-Newton pointings on and off an absorbing filament in the Southern galactic hemisphere (b ~ -45 deg). We use the difference in the Galactic column density in these two directions to deduce the contributions of the unabsorbed foreground emission due to the Local Bubble, and the absorbed emission from the Galactic halo and the extragalactic background. We find the Local Bubble emission is consistent with emission from a plasma in collisional ionization equilibrium with a temperature $\log T_{LB} = 6.06^{+0.02}_{-0.04}$ and an emission measure of 0.018 cm^{-6} pc. Our measured temperature is in good agreement with values obtained from ROSAT All-Sky Survey data, but is lower than that measured by other recent XMM-Newton observations of the Local Bubble, which find $\log T_{LB} \approx 6.2$ (although for some of these observations it is possible that the foreground emission is contaminated by non-Local Bubble emission from Loop I). The higher temperature observed towards other directions is inconsistent with our data, when combined with a FUSE measurement of the Galactic halo O VI intensity. This therefore suggests that the Local Bubble is thermally anisotropic. Our data are unable to rule out a non-equilibrium model in which the plasma is underionized. However, an overionized recombining plasma model, while observationally acceptable for certain densities and temperatures, generally gives an implausibly young age for the Local Bubble (\la 6 \times 10^5 yr).Comment: Accepted for publication in the Astrophysical Journal. 16 pages, 9 figure

Limits of applicability of the advection-dispersion model in aquifers containing connected high-conductivity channels

This is the published version. Copyright American Geophysical Union[1] The macrodispersion model from stochastic transport theory is demonstrated to be of limited utility when applied to heterogeneous aquifer systems containing narrow connected pathways. This is so even when contrasts in hydraulic conductivity (K) are small and variance in ln K is less than 0.10. We evaluated how well an advection-dispersion model (ADM) could be used to represent solute plumes transported through mildly heterogeneous three-dimensional (3-D) systems characterized by a well-connected dendritic network of 10 cm wide high-K channels. Each high-K channel network was generated using an invasion percolation algorithm and consisted of ∼10% by volume high-K regions. Contrasts in K between the channels and matrix were varied systematically from 2:1 to 30:1, corresponding to ln K values ranging from 0.04 to 1.05. Simulations involved numerical models with 3-D decimeter discretization, and each model contained 2–4 million active cells. Transport through each channel network considered only the processes of advection and molecular diffusion. In every case, the temporal change in the second spatial moment of concentrations was linear, with R2 values ranging from 0.97 to 0.99. The third spatial moment, or alternatively, the skewness coefficient values, indicated significant tailing downstream of the plume center. For each case, a corresponding ADM was used to simulate transport through the system. The corresponding ADM employed the effective mean hydraulic conductivity that reproduced the total discharge through the channel network system under an identical ambient gradient. Dispersivity values used in the ADM were obtained from the temporal change in the second spatial moments of concentrations for the plumes in the channel network systems and ranged from 0.014 m to 0.85 m. The results indicate that as the conductivity contrast between the channels and matrix increased, the simulated plumes in the channel network system became more and more asymmetric, with little solute dispersed upstream of the plume center and extensive downstream spreading of low concentrations. Distinctly different spreading was found upstream versus downstream of the plume center. The ADM failed to capture this asymmetry. Comparison of each plume in the channel network system with the corresponding plume produced using the corresponding ADM showed a maximum correlation of only 0.64 and a minimum fractional error of 0.29 for cases in which the log K variance was ∼0.20 (ln K variance was ∼1.0). At early times the correlations were as low as 0.40. The greatest correlation occurred at late times and for cases in which a wide source was considered

Mechanisms of amphetamine action illuminated through optical monitoring of dopamine synaptic vesicles in Drosophila brain

Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. Here we show in rodents that acute pharmacological inhibition of the vesicular monoamine transporter (VMAT) blocks amphetamine-induced locomotion and self-administration without impacting cocaine-induced behaviours. To study VMAT’s role in mediating amphetamine action in dopamine neurons, we have used novel genetic, pharmacological and optical approaches in Drosophila melanogaster. In an ex vivo whole-brain preparation, fluorescent reporters of vesicular cargo and of vesicular pH reveal that amphetamine redistributes vesicle contents and diminishes the vesicle pH-gradient responsible for dopamine uptake and retention. This amphetamine-induced deacidification requires VMAT function and results from net H+ antiport by VMAT out of the vesicle lumen coupled to inward amphetamine transport. Amphetamine-induced vesicle deacidification also requires functional dopamine transporter (DAT) at the plasma membrane. Thus, we find that at pharmacologically relevant concentrations, amphetamines must be actively transported by DAT and VMAT in tandem to produce psychostimulant effects

Increased localization of APP-C99 in mitochondria-associated ER membranes causes mitochondrial dysfunction in Alzheimer disease

In the amyloidogenic pathway associated with Alzheimer disease (AD), the amyloid precursor protein (APP) is cleaved by beta-secretase to generate a 99-aa C-terminal fragment (C99) that is then cleaved by c-secretase to generate the beta-amyloid (Ab) found in senile plaques. In previous reports, we and others have shown that c-secretase activity is enriched in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) and that ER-mitochondrial connectivity and MAM function are upregulated in AD. We now show that C99, in addition to its localization in endosomes, can also be found in MAM, where it is normally processed rapidly by c-secretase. In cell models of AD, however, the concentration of unprocessed C99 increases in MAM regions, resulting in elevated sphingolipid turnover and an altered lipid composition of both MAM and mitochondrial membranes. In turn, this change in mitochondrial membrane composition interferes with the proper assembly and activity of mitochondrial respiratory supercomplexes, thereby likely contributing to the bioenergetic defects characteristic of AD.We thank Drs. Orian Shirihai and Marc Liesa (UCLA) for assistance with the Seahorse measurements, Dr. Huaxi Xu (Sanford Burnham Institute) for the APP-DKO MEFs and Dr. Mark Mattson (NIH) for the PS1 knock-in mice, Drs. Arancio and Teich for the APP-KO mice tissues used in these studies, Dr. Hua Yang (Columbia University) for mouse husbandry, and Drs. Marc Tambini, Ira Tabas, and Serge Przedborski for helpful comments. This work was supported by the Fundacion Alfonso Martin Escudero (to M.P.); the Alzheimer's Drug Discovery Foundation, the Ellison Medical Foundation, the Muscular Dystrophy Association, the U.S. Department of Defense W911NF-12-1-9159 and W911F-15-1-0169), and the J. Willard and Alice S. Marriott Foundation (to E.A.S.); the U.S. National Institutes of Health (P01-HD080642 and P01-HD032062 to E.A.S.; NS071571 and HD071593 to M.F.M.; R01-NS056049 and P50-AG008702 to G.D.P.; 1S10OD016214-01A1 to G.S.P. and F.P.M, and K01-AG045335 to E.A.-G.), the Lucien Cote Early Investigator Award in Clinical Genetics from the Parkinson's Disease Foundation (PDF-CEI-1364 and PDF-CEI-1240) to C.G.-L., and National Defense Science and Engineering Graduate Fellowship (FA9550-11-C-0028) to R.R.A.S

Ribosome-associated vesicles: A dynamic subcompartment of the endoplasmic reticulum in secretory cells

The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo–electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic β-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species. We show that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture that interact with mitochondria via direct intermembrane contacts. These findings describe a new ER subcompartment within cells

The effectiveness, acceptability and cost-effectiveness of psychosocial interventions for maltreated children and adolescents: an evidence synthesis.

BACKGROUND: Child maltreatment is a substantial social problem that affects large numbers of children and young people in the UK, resulting in a range of significant short- and long-term psychosocial problems. OBJECTIVES: To synthesise evidence of the effectiveness, cost-effectiveness and acceptability of interventions addressing the adverse consequences of child maltreatment. STUDY DESIGN: For effectiveness, we included any controlled study. Other study designs were considered for economic decision modelling. For acceptability, we included any study that asked participants for their views. PARTICIPANTS: Children and young people up to 24 years 11 months, who had experienced maltreatment before the age of 17 years 11 months. INTERVENTIONS: Any psychosocial intervention provided in any setting aiming to address the consequences of maltreatment. MAIN OUTCOME MEASURES: Psychological distress [particularly post-traumatic stress disorder (PTSD), depression and anxiety, and self-harm], behaviour, social functioning, quality of life and acceptability. METHODS: Young Persons and Professional Advisory Groups guided the project, which was conducted in accordance with Cochrane Collaboration and NHS Centre for Reviews and Dissemination guidance. Departures from the published protocol were recorded and explained. Meta-analyses and cost-effectiveness analyses of available data were undertaken where possible. RESULTS: We identified 198 effectiveness studies (including 62 randomised trials); six economic evaluations (five using trial data and one decision-analytic model); and 73 studies investigating treatment acceptability. Pooled data on cognitive-behavioural therapy (CBT) for sexual abuse suggested post-treatment reductions in PTSD [standardised mean difference (SMD) -0.44 (95% CI -4.43 to -1.53)], depression [mean difference -2.83 (95% CI -4.53 to -1.13)] and anxiety [SMD -0.23 (95% CI -0.03 to -0.42)]. No differences were observed for post-treatment sexualised behaviour, externalising behaviour, behaviour management skills of parents, or parental support to the child. Findings from attachment-focused interventions suggested improvements in secure attachment [odds ratio 0.14 (95% CI 0.03 to 0.70)] and reductions in disorganised behaviour [SMD 0.23 (95% CI 0.13 to 0.42)], but no differences in avoidant attachment or externalising behaviour. Few studies addressed the role of caregivers, or the impact of the therapist-child relationship. Economic evaluations suffered methodological limitations and provided conflicting results. As a result, decision-analytic modelling was not possible, but cost-effectiveness analysis using effectiveness data from meta-analyses was undertaken for the most promising intervention: CBT for sexual abuse. Analyses of the cost-effectiveness of CBT were limited by the lack of cost data beyond the cost of CBT itself. CONCLUSIONS: It is not possible to draw firm conclusions about which interventions are effective for children with different maltreatment profiles, which are of no benefit or are harmful, and which factors encourage people to seek therapy, accept the offer of therapy and actively engage with therapy. Little is known about the cost-effectiveness of alternative interventions. LIMITATIONS: Studies were largely conducted outside the UK. The heterogeneity of outcomes and measures seriously impacted on the ability to conduct meta-analyses. FUTURE WORK: Studies are needed that assess the effectiveness of interventions within a UK context, which address the wider effects of maltreatment, as well as specific clinical outcomes. STUDY REGISTRATION: This study is registered as PROSPERO CRD42013003889. FUNDING: The National Institute for Health Research Health Technology Assessment programme