Mass spectrometry-based thermal shift assay for protein-ligand binding analysis.

Described here is a mass spectrometry-based screening assay for the detection of protein-ligand binding interactions in multicomponent protein mixtures. The assay utilizes an oxidation labeling protocol that involves using hydrogen peroxide to selectively oxidize methionine residues in proteins in order to probe the solvent accessibility of these residues as a function of temperature. The extent to which methionine residues in a protein are oxidized after specified reaction times at a range of temperatures is determined in a MALDI analysis of the intact proteins and/or an LC-MS analysis of tryptic peptide fragments generated after the oxidation reaction is quenched. Ultimately, the mass spectral data is used to construct thermal denaturation curves for the detected proteins. In this proof-of-principle work, the protocol is applied to a four-protein model mixture comprised of ubiquitin, ribonuclease A (RNaseA), cyclophilin A (CypA), and bovine carbonic anhydrase II (BCAII). The new protocol's ability to detect protein-ligand binding interactions by comparing thermal denaturation data obtained in the absence and in the presence of ligand is demonstrated using cyclosporin A (CsA) as a test ligand. The known binding interaction between CsA and CypA was detected using both the MALDI- and LC-MS-based readouts described here.

Thermodynamic analysis of a molecular chaperone binding to unfolded protein substrates.

Molecular chaperones are a highly diverse group of proteins that recognize and bind unfolded proteins to facilitate protein folding and prevent nonspecific protein aggregation. The mechanisms by which chaperones bind their protein substrates have been studied for decades. However, there are few reports about the affinity of molecular chaperones for their unfolded protein substrates. Thus, little is known about the relative binding affinities of different chaperones and about the relative binding affinities of chaperones for different unfolded protein substrates. Here we describe the application of SUPREX (stability of unpurified proteins from rates of H-D exchange), an H-D exchange and MALDI-based technique, in studying the binding interaction between the molecular chaperone Hsp33 and four different unfolded protein substrates, including citrate synthase, lactate dehydrogenase, malate dehydrogenase, and aldolase. The results of our studies suggest that the cooperativity of the Hsp33 folding-unfolding reaction increases upon binding with denatured protein substrates. This is consistent with the burial of significant hydrophobic surface area in Hsp33 when it interacts with its substrate proteins. The SUPREX-derived K(d) values for Hsp33 complexes with four different substrates were all found to be within the range of 3-300 nM.

Quantitative model of the phase behavior of recombinant pH-responsive elastin-like polypeptides.

Quantitative models are required to engineer biomaterials with environmentally responsive properties. With this goal in mind, we developed a model that describes the pH-dependent phase behavior of a class of stimulus responsive elastin-like polypeptides (ELPs) that undergo reversible phase separation in response to their solution environment. Under isothermal conditions, charged ELPs can undergo phase separation when their charge is neutralized. Optimization of this behavior has been challenging because the pH at which they phase separate, pHt, depends on their composition, molecular weight, concentration, and temperature. To address this problem, we developed a quantitative model to describe the phase behavior of charged ELPs that uses the Henderson-Hasselbalch relationship to describe the effect of side-chain ionization on the phase-transition temperature of an ELP. The model was validated with pH-responsive ELPs that contained either acidic (Glu) or basic (His) residues. The phase separation of both ELPs fit this model across a range of pH. These results have important implications for applications of pH-responsive ELPs because they provide a quantitative model for the rational design of pH-responsive polypeptides whose transition can be triggered at a specified pH.

Dental topography and molar wear in Alouatta palliata from Costa Rica.

Paleoprimatologists depend on relationships between form and function of teeth to reconstruct the diets of fossil species. Most of this work has been limited to studies of unworn teeth. A new approach, dental topographic analysis, allows the characterization and comparison of worn primate teeth. Variably worn museum specimens have been used to construct species-specific wear sequences so that measurements can be compared by wear stage among taxa with known differences in diet. This assumes that individuals in a species tend to wear their molar teeth in similar ways, a supposition that has yet to be tested. Here we evaluate this assumption with a longitudinal study of changes in tooth form over time in primates. Fourteen individual mantled howling monkeys (Alouatta palliata) were captured and then recaptured after 2, 4, and 7 years when possible at Hacienda La Pacifica in Costa Rica between 1989-1999. Dental impressions were taken each time, and molar casts were produced and analyzed using dental topographic analysis. Results showed consistent decreases in crown slope and occlusal relief. In contrast, crown angularity, a measure of surface jaggedness, remained fairly constant except with extreme wear. There were no evident differences between specimens collected in different microhabitats. These results suggest that different individual mantled howling monkeys wear their teeth down in similar ways, evidently following a species-specific wear sequence. Dental topographic analysis may therefore be used to compare morphology among similarly worn individuals from different species.

Archaeological Excavations at 18AP44: 193 Main Street, Annapolis, Maryland, 1985-1987

193 Main Street (18AP44) is located between Main Street and Duke of Gloucester Street. The property was used ass a yard related to residential and commercial buildings during the 18th and 19th centuries. In the 1930's a movie theatre and parking lot were built on the property. That structure was torn down in the 1980's and a three-story commercial building was constructed. Archaeological excavations were conducted on the property from 1985-1987. A preliminary report was written in 1986 by Paul A. Shackel. This report is the final report on the archaeological investigations at 193 Main Street.

Autobiographical memory for stressful events: the role of autobiographical memory in posttraumatic stress disorder.

To provide the three-way comparisons needed to test existing theories, we compared (1) most-stressful memories to other memories and (2) involuntary to voluntary memories (3) in 75 community dwelling adults with and 42 without a current diagnosis of posttraumatic stress disorder (PTSD). Each rated their three most-stressful, three most-positive, seven most-important and 15 word-cued autobiographical memories, and completed tests of personality and mood. Involuntary memories were then recorded and rated as they occurred for 2 weeks. Standard mechanisms of cognition and affect applied to extreme events accounted for the properties of stressful memories. Involuntary memories had greater emotional intensity than voluntary memories, but were not more frequently related to traumatic events. The emotional intensity, rehearsal, and centrality to the life story of both voluntary and involuntary memories, rather than incoherence of voluntary traumatic memories and enhanced availability of involuntary traumatic memories, were the properties of autobiographical memories associated with PTSD.

Reminders, Refugees, and Ramadan: Characterizing Missed Immunization Appointments among Palestinian Refugees in Jordan

There are currently over two million Palestinian refugees residing in Jordan, 370,000 of whom reside in refugee camps. Due to conflict-affiliated disease outbreaks among children in the region, the UN Relief and Works Agency for Palestine Refugees (UNRWA) has identified incomplete vaccination as a critical public health issue and has invested in the development and implementation of a text message reminder service for preventing loss-to-follow-up. Childhood immunization rates in UNRWA catchment regions are generally high, yet little is known about risk factors for missed appointments, which impose a substantial administrative burden due to the need to contact patients for rescheduling. Stronger user characterization is necessary for improved targeting and minimized cost as we develop a more robust SMS system capable of scaling across all health facilities.

This mixed-methods study prospectively recorded 6 months of immunization history among a cohort of children born in June 2014 at Taybeh Health Center in Amman. Demographic information was collected at the time of birth, and caregivers of cohort members were invited to participate in interviews that assessed immunization knowledge, preferences, decision-making, and experience with the SMS reminder system. Patients were more likely to significantly delay appointments during the Ramadan holiday and for doses further from the child date of birth. Future policies that might bridge these gaps include targeting pre-appointment SMS reminders to high-risk patients, implementing holiday shifts in clinic hours, and regularly updating patient contact information.

Improving mental health among people living with HIV: a review of intervention trials in low- and middle-income countries.

People living with HIV (PLWH) experience greater psychological distress than the general population. Evidence from high-incomes countries suggests that psychological interventions for PLWH can improve mental health symptoms, quality of life, and HIV care engagement. However, little is known about the effectiveness of mental health interventions for PLWH in low and middle-income countries (LMICs), where the large majority of PLWH reside. This systematized review aims to synthesize findings from mental health intervention trials with PLWH in LMICs to inform the delivery of mental health services in these settings. A systematic search strategy was undertaken to identify peer-reviewed published papers of intervention trials addressing negative psychological states or disorders (e.g., depression, anxiety) among PLWH in LMIC settings. Search results were assessed against pre-established inclusion and exclusion criteria. Data from papers meeting criteria were extracted for synthesis. Twenty-six papers, published between 2000 and 2014, describing 22 unique interventions were identified. Trials were implemented in sub-Saharan Africa (n=13), Asia (n=7), and the Middle East (n=2), and addressed mental health using a variety of approaches, including cognitive-behavioral (n=18), family-level (n=2), and pharmacological (n=2) treatments. Four randomized controlled trials reported significant intervention effects in mental health outcomes, and eleven preliminary studies demonstrated promising findings. Among the limited mental health intervention trials with PLWH in LMICs, few demonstrated efficacy. Mental health interventions for PLWH in LMICs must be further developed and adapted for resource-limited settings to improve effectiveness.

ENIGMA and the individual: Predicting factors that affect the brain in 35 countries worldwide.

In this review, we discuss recent work by the ENIGMA Consortium (http://enigma.ini.usc.edu) - a global alliance of over 500 scientists spread across 200 institutions in 35 countries collectively analyzing brain imaging, clinical, and genetic data. Initially formed to detect genetic influences on brain measures, ENIGMA has grown to over 30 working groups studying 12 major brain diseases by pooling and comparing brain data. In some of the largest neuroimaging studies to date - of schizophrenia and major depression - ENIGMA has found replicable disease effects on the brain that are consistent worldwide, as well as factors that modulate disease effects. In partnership with other consortia including ADNI, CHARGE, IMAGEN and others(1), ENIGMA's genomic screens - now numbering over 30,000 MRI scans - have revealed at least 8 genetic loci that affect brain volumes. Downstream of gene findings, ENIGMA has revealed how these individual variants - and genetic variants in general - may affect both the brain and risk for a range of diseases. The ENIGMA consortium is discovering factors that consistently affect brain structure and function that will serve as future predictors linking individual brain scans and genomic data. It is generating vast pools of normative data on brain measures - from tens of thousands of people - that may help detect deviations from normal development or aging in specific groups of subjects. We discuss challenges and opportunities in applying these predictors to individual subjects and new cohorts, as well as lessons we have learned in ENIGMA's efforts so far.

A semi-Lagrangian finite volume method for Newtonian contraction flows

A new finite volume method for solving the incompressible Navier--Stokes equations is presented. The main features of this method are the location of the velocity components and pressure on different staggered grids and a semi-Lagrangian method for the treatment of convection. An interpolation procedure based on area-weighting is used for the convection part of the computation. The method is applied to flow through a constricted channel, and results are obtained for Reynolds numbers, based on half the flow rate, up to 1000. The behavior of the vortex in the salient corner is investigated qualitatively and quantitatively, and excellent agreement is found with the numerical results of Dennis and Smith [Proc. Roy. Soc. London A, 372 (1980), pp. 393-414] and the asymptotic theory of Smith [J. Fluid Mech., 90 (1979), pp. 725-754].

A volume of fluid numerical model for wave impacts at coastal structures

The first stages in the development of a new design tool, to be used by coastal engineers to improve the efficiency, analysis, design, management and operation of a wide range of coastal and harbour structures, are described. The tool is based on a two-dimensional numerical model, NEWMOTICS-2D, using the volume of fluid (VOF) method, which permits the rapid calculation of wave hydrodynamics at impermeable natural and man-made structures. The critical hydrodynamic flow processes and forces are identified together with the equations that describe these key processes. The different possible numerical approaches for the solution of these equations, and the types of numerical models currently available, are examined and assessed. Preliminary tests of the model, using comparisons with results from a series of hydraulic model test cases, are described. The results of these tests demonstrate that the VOF approach is particularly appropriate for the simulation of the dynamics of waves at coastal structures because of its flexibility in representing the complex free surfaces encountered during wave impact and breaking. The further programme of work, required to develop the existing model into a tool for use in routine engineering design, is outlined.

Cognitive issues in information visualisation design for interaction for the visually impaired

This paper reports on the findings of a study on improving interaction design for visually impaired students, focusing upon the cognitive criteria for information visualisation.

Influence of megasonic agitation on the electrodeposition of high aspect ratio blind vias

This paper presents preliminary studies in electroplating using megasonic agitation to avoid the formation of voids within high aspect ratio microvias that are used for the redistribution of interconnects in high density interconnection technology in printed circuit boards. Through this technique, uniform deposition of metal on the side walls of the vias is possible. High frequency acoustic streaming at megasonic frequencies enables the decrease of the Nernst diffusion layer down to the sub-micron range, allowing thereby conformal electrodeposition in deep grooves. This effect enables the normally convection free liquid near the surface to be agitated. Higher throughput and better control of the material properties of the deposits can be achieved for the manufacturing of embedded interconnections and metal-based MEMS. For optimal filling performance of the microvias, a full design of experiments (DOE) and a multi-physics numerical simulation have been conducted to analyse the influence of megasonic agitation on the plating quality of the microvias. Megasonic based deposition has been found to increase the deposition rate as well as improving the quality of the metal deposits.

Megasonic agitation for enhanced electrodeposition of copper

In this paper we propose an agitation method based on megasonic acoustic streaming to overcome the limitations in plating rate and uniformity of the metal deposits during the electroplating process. Megasonic agitation at a frequency of 1 MHz allows the reduction of the thickness of the Nernst diffusion layer to less than 600 nm. Two applications that demonstrate the benefits of megasonic acoustic streaming are presented: the formation of uniform ultra-fine pitch flip-chip bumps and the metallisation of high aspect ratio microvias. For the latter application, a multi-physics based numerical simulation is implemented to describe the hydrodynamics introduced by the acoustic waves as they travel inside the deep microvias.

The application of electrostatic dry powder deposition technology to coat drug-eluting stents

Purpose: A novel methodology has been introduced to effectively coat intravascular stents with sirolimus-loaded polymeric microparticles. Methods: Dry powders of the microparticulate formulation, consisting of non-erodible polymers, were produced by a supercritical, aerosol, solvent extraction system (ASES). A design of experiment (DOE) approach was conducted on the independent variables, such as organic/CO2 phase volume ratio, polymer weight and stirring-rate, while regression analysis was utilized to interpret the influence of all operational parameters on the dependent variable of particle size. The dry powders, so formed, entered an electric field created by corona charging and were sprayed on the earthed metal stent. Furthermore, the thermal stability of sirolimus was investigated to define the optimum conditions for fusion to the metal surfaces. Results: The electrostatic dry powder deposition technology (EDPDT) was used on the metal strut followed by fusion to produce uniform, reproducible and accurate coatings. The coated stents exhibited sustained release profiles over 25 days, similar to commercial products. EDPDT-coated stents displayed significant reduced platelet adhesion. Conclusions: EDPDT appeared to be a robust accurate and reproducible technology to coat eluting stents.