A Microfluidic Device for Kinetic Optimization of Protein Crystallization and In Situ Structure Determination

The unprecedented economies of scale and unique mass transport properties of microfluidic devices made them viable nano-volume protein crystallization screening platforms. However, realizing the full potential of microfluidic crystallization requires complementary technologies for crystal optimization and harvesting. In this paper, we report a microfluidic device which provides a link between chip-based nanoliter volume crystallization screening and structure analysis through “kinetic optimization” of crystallization reactions and in situ structure determination. Kinetic optimization through systematic variation of reactor geometry and actuation of micromechanical valves is used to screen a large ensemble of kinetic trajectories that are not practical with conventional techniques. Using this device, we demonstrate control over crystal quality, reliable scale-up from nanoliter volume reactions, facile harvesting and cryoprotectant screening, and protein structure determination at atomic resolution from data collected in-chip

Implementation and performance of SIBYLS: a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline at the Advanced Light Source.

The SIBYLS beamline (12.3.1) of the Advanced Light Source at Lawrence Berkeley National Laboratory, supported by the US Department of Energy and the National Institutes of Health, is optimized for both small-angle X-ray scattering (SAXS) and macromolecular crystallography (MX), making it unique among the world's mostly SAXS or MX dedicated beamlines. Since SIBYLS was commissioned, assessments of the limitations and advantages of a combined SAXS and MX beamline have suggested new strategies for integration and optimal data collection methods and have led to additional hardware and software enhancements. Features described include a dual mode monochromator [containing both Si(111) crystals and Mo/B(4)C multilayer elements], rapid beamline optics conversion between SAXS and MX modes, active beam stabilization, sample-loading robotics, and mail-in and remote data collection. These features allow users to gain valuable insights from both dynamic solution scattering and high-resolution atomic diffraction experiments performed at a single synchrotron beamline. Key practical issues considered for data collection and analysis include radiation damage, structural ensembles, alternative conformers and flexibility. SIBYLS develops and applies efficient combined MX and SAXS methods that deliver high-impact results by providing robust cost-effective routes to connect structures to biology and by performing experiments that aid beamline designs for next generation light sources

Software for the high-throughput collection of SAXS data using an enhanced Blu-Ice/DCS control system

The Blu-Ice GUI and Distributed Control System (DCS) developed in the Macromolecular Crystallography Group at the Stanford Synchrotron Radiation Laboratory has been optimized, extended and enhanced to suit the specific needs of the SAXS endstation at the SIBYLS beamline at the Advanced Light Source. The customizations reported here provide one potential route for other SAXS beamlines in need of robust and efficient beamline control software

Nbs1 Flexibly Tethers Ctp1 and Mre11-Rad50 to Coordinate DNA Double-Strand Break Processing and Repair

SummaryThe Nijmegen breakage syndrome 1 (Nbs1) subunit of the Mre11-Rad50-Nbs1 (MRN) complex protects genome integrity by coordinating double-strand break (DSB) repair and checkpoint signaling through undefined interactions with ATM, MDC1, and Sae2/Ctp1/CtIP. Here, fission yeast and human Nbs1 structures defined by X-ray crystallography and small angle X-ray scattering (SAXS) reveal Nbs1 cardinal features: fused, extended, FHA-BRCT1-BRCT2 domains flexibly linked to C-terminal Mre11- and ATM-binding motifs. Genetic, biochemical, and structural analyses of an Nbs1-Ctp1 complex show Nbs1 recruits phosphorylated Ctp1 to DSBs via binding of the Nbs1 FHA domain to a Ctp1 pThr-Asp motif. Nbs1 structures further identify an extensive FHA-BRCT interface, a bipartite MDC1-binding scaffold, an extended conformational switch, and the molecular consequences associated with cancer predisposing Nijmegen breakage syndrome mutations. Tethering of Ctp1 to a flexible Nbs1 arm suggests a mechanism for restricting DNA end processing and homologous recombination activities of Sae2/Ctp1/CtIP to the immediate vicinity of DSBs

A Microfluidic Device for Kinetic Optimization of Protein Crystallization and In Situ Structure Determination

The unprecedented economies of scale and unique mass transport properties of microfluidic devices made them viable nano-volume protein crystallization screening platforms. However, realizing the full potential of microfluidic crystallization requires complementary technologies for crystal optimization and harvesting. In this paper, we report a microfluidic device which provides a link between chip-based nanoliter volume crystallization screening and structure analysis through “kinetic optimization” of crystallization reactions and in situ structure determination. Kinetic optimization through systematic variation of reactor geometry and actuation of micromechanical valves is used to screen a large ensemble of kinetic trajectories that are not practical with conventional techniques. Using this device, we demonstrate control over crystal quality, reliable scale-up from nanoliter volume reactions, facile harvesting and cryoprotectant screening, and protein structure determination at atomic resolution from data collected in-chip

Adopting an external focus of attention alters intracortical inhibition within the primary motor cortex

Although it is well established that an external (EF) compared to an internal (IF) or neutral focus of attention enhances motor performance, little is known about the underlying neural mechanisms. This study aimed to clarify whether the focus of attention influences not only motor performance but also activity of the primary motor cortex (M1) when executing identical fatiguing tasks of the right index finger (first dorsal interosseous). Transcranial magnetic stimulation (TMS) at intensities below motor threshold was applied over M1 to assess and compare the excitability of intracortical inhibitory circuits.Methods: In session 1, 14 subjects performed an isometric finger abduction at 30% of their maximal force to measure the time to task failure (TTF) with either an IF or EF. In session 2, the same task was performed with the other focus. In sessions 3 and 4, subthreshold TMS (subTMS) and paired-pulse TMS were applied to the contralateral M1 to compare the activity of cortical inhibitory circuits within M1 during EF and IF.Results: With an EF, TTF was significantly prolonged (P = 0.01), subTMS-induced electromyographical suppression enhanced (P = 0.001) and short-interval intracortical inhibition (SICI) increased (P = 0.004).Conclusion: The level of intracortical inhibition was previously shown to influence motor performance. Our data shed new light on the ability to instantly modulate the activity of inhibitory circuits within M1 by changing the type of attentional focus. The increased inhibition with EF might contribute to the better movement efficiency, which is generally associated with focusing externally

Changing use of surgical antibiotic prophylaxis in Thika Hospital, Kenya: a quality improvement intervention with an interrupted time series design.

INTRODUCTION: In low-income countries, Surgical Site Infection (SSI) is a common form of hospital-acquired infection. Antibiotic prophylaxis is an effective method of preventing these infections, if given immediately before the start of surgery. Although several studies in Africa have compared pre-operative versus post-operative prophylaxis, there are no studies describing the implementation of policies to improve prescribing of surgical antibiotic prophylaxis in African hospitals. METHODS: We conducted SSI surveillance at a typical Government hospital in Kenya over a 16 month period between August 2010 and December 2011, using standard definitions of SSI and the extent of contamination of surgical wounds. As an intervention, we developed a hospital policy that advised pre-operative antibiotic prophylaxis and discouraged extended post-operative antibiotics use. We measured process, outcome and balancing effects of this intervention in using an interrupted time series design. RESULTS: From a starting point of near-exclusive post-operative antibiotic use, after policy introduction in February 2011 there was rapid adoption of the use of pre-operative antibiotic prophylaxis (60% of operations at 1 week; 98% at 6 weeks) and a substantial decrease in the use of post-operative antibiotics (40% of operations at 1 week; 10% at 6 weeks) in Clean and Clean-Contaminated surgery. There was no immediate step-change in risk of SSI, but overall, there appeared to be a moderate reduction in the risk of superficial SSI across all levels of wound contamination. There were marked reductions in the costs associated with antibiotic use, the number of intravenous injections performed and nursing time spent administering these. CONCLUSION: Implementation of a locally developed policy regarding surgical antibiotic prophylaxis is an achievable quality improvement target for hospitals in low-income countries, and can lead to substantial benefits for individual patients and the institution

Lighting during grow-out and Salmonella in broiler flocks

<p>Abstract</p> <p>Background</p> <p>Lighting is used during conventional broiler grow-out to modify bird behaviour to reach the goals of production and improve bird welfare. The protocols for lighting intensity vary. In a field study, we evaluated if the lighting practices impact the burden of <it>Salmonella </it>in broiler flocks.</p> <p>Methods</p> <p>Conventional grow-out flocks reared in the states of Alabama, Mississippi and Texas, USA in 2003 to 2006 were sampled 1 week before harvest (<it>n </it>= 58) and upon arrival for processing (<it>n </it>= 56) by collecting feathered carcass rinsate, crop and one cecum from each of 30 birds, and during processing by collecting rinsate of 30 carcasses at pre-chilling (<it>n </it>= 56) and post-chilling points (<it>n </it>= 54). Litter samples and drag swabs of litter were collected from the grow-out houses after bird harvest (<it>n </it>= 56). Lighting practices for these flocks were obtained with a questionnaire completed by the growers. Associations between the lighting practices and the burden of <it>Salmonella </it>in the flocks were tested while accounting for variation between the grow-out farms, their production complexes and companies.</p> <p>Results</p> <p>Longer relative duration of reduced lights during the grow-out period was associated with reduced detection of <it>Salmonella </it>on the exterior of birds 1 week before harvest and on the broiler carcasses at the post-chilling point of processing. In addition, starting reduced lights for ≥18 hours per day later in the grow-out period was associated with decreased detection of <it>Salmonella </it>on the exterior of broilers arriving for processing and in the post-harvest drag swabs of litter from the grow-out house.</p> <p>Conclusions</p> <p>The results of this field study show that lighting practices implemented during broiler rearing can impact the burden of <it>Salmonella </it>in the flock. The underlying mechanisms are likely to be interactive.</p

Improved limits on dark matter annihilation in the Sun with the 79-string IceCube detector and implications for supersymmetry

We present an improved event-level likelihood formalism for including neutrino telescope data in global fits to new physics. We derive limits on spin-dependent dark matter-proton scattering by employing the new formalism in a re-analysis of data from the 79-string IceCube search for dark matter annihilation in the Sun, including explicit energy information for each event. The new analysis excludes a number of models in the weak-scale minimal supersymmetric standard model (MSSM) for the first time. This work is accompanied by the public release of the 79-string IceCube data, as well as an associated computer code for applying the new likelihood to arbitrary dark matter models.Comment: 24 pages, 8 figs, 1 table. Contact authors: Pat Scott & Matthias Danninger. Likelihood tool available at http://nulike.hepforge.org. v2: small updates to address JCAP referee repor

Interpersonal polyvictimization and mental health in males

A consistent conclusion within the extant literature is that victimization and in particular polyvictimization leads to adverse mental health outcomes. A large body of literature exists as it pertains to the association between victimisation and mental health in studies utilising samples of childhood victims, female only victims, and samples of male and female victims; less research exists as it relates to males victims of interpersonal violence. The aim of the current study was therefore to identify profiles of interpersonal victimizations in an exclusively male sample and to assess their differential impact on a number of adverse mental health outcomes. Using data from 14,477 adult males from Wave 2 of the NESARC, we identified interpersonal victimization profiles via Latent Class Analysis. Multinomial Logistic Regression was subsequently utilized to establish risk across mental health disorders. A 4-class solution was optimal. Victimisation profiles showed elevated odds ratios for the presence of mental health disorders; suggesting that multiple life-course victimisation typologies exists, and that victimization is strongly associated with psychopathology. Several additional notable findings are discussed