Cellular Assays for High-Throughput Screening for Modulators of Trk Receptor Tyrosine Kinases

Trk receptor tyrosine kinases are required for signal transduction initiated by neurotrophins leading to cell proliferation, differentiation, survival and death. Alterations in Trk kinase activity have been linked to various diseases. To address the need for cell-based assays for screening and studying the selectivity of Trk kinase modulators, we developed high-throughput cell-based assays for Trk receptor kinases using nuclear factor of activated T-cells (NFAT) beta-lactamase reporter lines stably expressing full length human Trk kinases. These assays were functionally validated with cognate neurotrophin(s), inhibitors and TRK RNAi oligos and demonstrated for their utility in identifying potent and selective modulators of Trk receptor kinases

HTS-Compatible β-Lactamase Transcriptional Reporter Gene Assay for Interrogating the Heat Shock Response Pathway

Moderate environmental and physiological stressors are known to initiate protective heat shock response (HSR) leading to cell survival. HSR is largely mediated by the activation of heat shock factor (HSF), resulting in increased heat shock protein expression. Dysregulation of the HSR signaling has been associated with various diseases including cancer, inflammation and neurodegenerative disorders. Compounds that can modulate HSR have been pursued for the treatment of these diseases. To facilitate the discovery of HSR modulators, we developed a high-throughput amenable betalactamase transcriptional reporter gene assay for monitoring the function of HSF. HeLa cells were engineered to express the beta-lactamase reporter under the control of HSF response elements (HSE) present in the HSP70 gene promoter. The HSE-beta lactamase (HSE-bla) reporter gene assay was validated by using HSF-specific siRNAs and known small molecule modulators. Taking the advantage of fluorescence resonance energy transfer (FRET)-based cell permeable betalactamase substrate, this assay can be miniaturized into 1536-well format. Our results demonstrate that the assay is robust and can be applied to high-throughput screening (HTS) for modulators of HSR

The influence of a CYP1A2 polymorphism on the ergogenic effects of caffeine

<p>Abstract</p> <p>Background</p> <p>Although caffeine supplementation improves performance, the ergogenic effect is variable. The cause(s) of this variability are unknown. A (C/A) single nucleotide polymorphism at intron 1 of the cytochrome P450 (CYP1A2) gene influences caffeine metabolism and clinical outcomes from caffeine ingestion. The purpose of this study was to determine if this polymorphism influences the ergogenic effect of caffeine supplementation.</p> <p>Methods</p> <p>Thirty-five trained male cyclists (age = 25.0 ± 7.3 yrs, height = 178.2 ± 8.8 cm, weight = 74.3 ± 8.8 kg, VO₂max = 59.35 ± 9.72 ml·kg^-1·min^-1) participated in two computer-simulated 40-kilometer time trials on a cycle ergometer. Each test was performed one hour following ingestion of 6 mg·kg^-1of anhydrous caffeine or a placebo administered in double-blind fashion. DNA was obtained from whole blood samples and genotyped using restriction fragment length polymorphism-polymerase chain reaction. Participants were classified as AA homozygotes (N = 16) or C allele carriers (N = 19). The effects of treatment (caffeine, placebo) and the treatment × genotype interaction were assessed using Repeated Measures Analysis of Variance.</p> <p>Results</p> <p>Caffeine supplementation reduced 40 kilometer time by a greater (<it>p </it>< 0.05) magnitude in AA homozygotes (4.9%; caffeine = 72.4 ± 4.2 min, placebo = 76.1 ± 5.8 min) as compared to C allele carriers (1.8%; caffeine = 70.9 ± 4.3 min, placebo = 72.2 ± 4.2 min).</p> <p>Conclusions</p> <p>Results suggest that individuals homozygous for the A allele of this polymorphism may have a larger ergogenic effect following caffeine ingestion.</p

Evaluating insecticide resistance across African districts to aid malaria control decisions

Malaria vector control may be compromised by resistance to insecticides in vector populations. Actions to mitigate against resistance rely on surveillance using standard susceptibility tests, but there are large gaps in the monitoring data across Africa. Using a published geostatistical ensemble model, we have generated maps that bridge these gaps and consider the likelihood that resistance exceeds recommended thresholds. Our results show that this model provides more accurate next-year predictions than two simpler approaches. We have used the model to generate district-level maps for the probability that pyrethroid resistance in Anopheles gambiae s.l. exceeds the World Health Organization thresholds for susceptibility and confirmed resistance. In addition, we have mapped the three criteria for the deployment of piperonyl butoxide-treated nets that mitigate against the effects of metabolic resistance to pyrethroids. This includes a critical review of the evidence for presence of cytochrome P450-mediated metabolic resistance mechanisms across Africa. The maps for pyrethroid resistance are available on the IR Mapper website, where they can be viewed alongside the latest survey data

Nanoceria Distribution and Effects Are Mouse-Strain Dependent

Prior studies showed nanoparticle clearance was different in C57BL/6 versus BALB/c mice, strains prone to Th1 and Th2 immune responses, respectively. Objective: Assess nanoceria (cerium oxide, CeO2 nanoparticle) uptake time course and organ distribution, cellular and oxidative stress, and bioprocessing as a function of mouse strain. Methods: C57BL/6 and BALB/c female mice were i.p. injected with 10 mg/kg nanoceria or vehicle and terminated 0.5 to 24 h later. Organs were collected for cerium analysis; light and electron microscopy with elemental mapping; and protein carbonyl, IL-1β, and caspase-1 determination. Results: Peripheral organ cerium significantly increased, generally more in C57BL/6 mice. Caspase-1 was significantly elevated in the liver at 6 h, to a greater extent in BALB/c mice, suggesting inflammasome pathway activation. Light microscopy revealed greater liver vacuolation in C57BL/6 mice and a nanoceria-induced decrease in BALB/c but not C57BL/6 mice vacuolation. Nanoceria increased spleen lymphoid white pulp cell density in BALB/c but not C57BL/6 mice. Electron microscopy showed intracellular nanoceria particles bioprocessed to form crystalline cerium phosphate nanoneedles. Ferritin accumulation was greatly increased proximal to the nanoceria, forming core-shell-like structures in C57BL/6 but even distribution in BALB/c mice. Conclusions: BALB/c mice were more responsive to nanoceria-induced effects, e.g. liver caspase-1 activation, reduced liver vacuolation, and increased spleen cell density. Nanoceria uptake, initiation of bioprocessing, and crystalline cerium phosphate nanoneedle formation were rapid. Ferritin greatly increased with a macrophage phenotype-dependent distribution. Further study will be needed to understand the mechanisms underlying the observed differences

GAWMerge expands GWAS sample size and diversity by combining array-based genotyping and whole-genome sequencing

Genome-wide association studies (GWAS) have made impactful discoveries for complex diseases, often by amassing very large sample sizes. Yet, GWAS of many diseases remain underpowered, especially for non-European ancestries. One cost-effective approach to increase sample size is to combine existing cohorts, which may have limited sample size or be case-only, with public controls, but this approach is limited by the need for a large overlap in variants across genotyping arrays and the scarcity of non-European controls. We developed and validated a protocol, Genotyping Array-WGS Merge (GAWMerge), for combining genotypes from arrays and whole-genome sequencing, ensuring complete variant overlap, and allowing for diverse samples like Trans-Omics for Precision Medicine to be used. Our protocol involves phasing, imputation, and filtering. We illustrated its ability to control technology driven artifacts and type-I error, as well as recover known disease-associated signals across technologies, independent datasets, and ancestries in smoking-related cohorts. GAWMerge enables genetic studies to leverage existing cohorts to validly increase sample size and enhance discovery for understudied traits and ancestries

Ground and In-Flight Calibration of the OSIRIS-REx Camera Suite

The OSIRIS-REx Camera Suite (OCAMS) onboard the OSIRIS-REx spacecraft is used to study the shape and surface of the mission’s target, asteroid (101955) Bennu, in support of the selection of a sampling site. We present calibration methods and results for the three OCAMS cameras—MapCam, PolyCam, and SamCam—using data from pre-flight and in-flight calibration campaigns. Pre-flight calibrations established a baseline for a variety of camera properties, including bias and dark behavior, flat fields, stray light, and radiometric calibration. In-flight activities updated these calibrations where possible, allowing us to confidently measure Bennu’s surface. Accurate calibration is critical not only for establishing a global understanding of Bennu, but also for enabling analyses of potential sampling locations and for providing scientific context for the returned sample

Formation of regulatory modules by local sequence duplication

Turnover of regulatory sequence and function is an important part of molecular evolution. But what are the modes of sequence evolution leading to rapid formation and loss of regulatory sites? Here, we show that a large fraction of neighboring transcription factor binding sites in the fly genome have formed from a common sequence origin by local duplications. This mode of evolution is found to produce regulatory information: duplications can seed new sites in the neighborhood of existing sites. Duplicate seeds evolve subsequently by point mutations, often towards binding a different factor than their ancestral neighbor sites. These results are based on a statistical analysis of 346 cis-regulatory modules in the Drosophila melanogaster genome, and a comparison set of intergenic regulatory sequence in Saccharomyces cerevisiae. In fly regulatory modules, pairs of binding sites show significantly enhanced sequence similarity up to distances of about 50 bp. We analyze these data in terms of an evolutionary model with two distinct modes of site formation: (i) evolution from independent sequence origin and (ii) divergent evolution following duplication of a common ancestor sequence. Our results suggest that pervasive formation of binding sites by local sequence duplications distinguishes the complex regulatory architecture of higher eukaryotes from the simpler architecture of unicellular organisms

Quality metrics for the evaluation of Rapid Response Systems: Proceedings from the third international consensus conference on Rapid Response Systems.

BACKGROUND: Clinically significant deterioration of patients admitted to general wards is a recognized complication of hospital care. Rapid Response Systems (RRS) aim to reduce the number of avoidable adverse events. The authors aimed to develop a core quality metric for the evaluation of RRS. METHODS: We conducted an international consensus process. Participants included patients, carers, clinicians, research scientists, and members of the International Society for Rapid Response Systems with representatives from Europe, Australia, Africa, Asia and the US. Scoping reviews of the literature identified potential metrics. We used a modified Delphi methodology to arrive at a list of candidate indicators that were reviewed for feasibility and applicability across a broad range of healthcare systems including low and middle-income countries. The writing group refined recommendations and further characterized measurement tools. RESULTS: Consensus emerged that core outcomes for reporting for quality improvement should include ten metrics related to structure, process and outcome for RRS with outcomes following the domains of the quadruple aim. The conference recommended that hospitals should collect data on cardiac arrests and their potential predictability, timeliness of escalation, critical care interventions and presence of written treatment goals for patients remaining on general wards. Unit level reporting should include the presence of patient activated rapid response and metrics of organizational culture. We suggest two exploratory cost metrics to underpin urgently needed research in this area. CONCLUSION: A consensus process was used to develop ten metrics for better understanding the course and care of deteriorating ward patients. Others are proposed for further development

Large-scale genome-wide association studies and meta-analyses of longitudinal change in adult lung function.

BACKGROUND: Genome-wide association studies (GWAS) have identified numerous loci influencing cross-sectional lung function, but less is known about genes influencing longitudinal change in lung function. METHODS: We performed GWAS of the rate of change in forced expiratory volume in the first second (FEV1) in 14 longitudinal, population-based cohort studies comprising 27,249 adults of European ancestry using linear mixed effects model and combined cohort-specific results using fixed effect meta-analysis to identify novel genetic loci associated with longitudinal change in lung function. Gene expression analyses were subsequently performed for identified genetic loci. As a secondary aim, we estimated the mean rate of decline in FEV1 by smoking pattern, irrespective of genotypes, across these 14 studies using meta-analysis. RESULTS: The overall meta-analysis produced suggestive evidence for association at the novel IL16/STARD5/TMC3 locus on chromosome 15 (P  =  5.71 × 10(-7)). In addition, meta-analysis using the five cohorts with ≥3 FEV1 measurements per participant identified the novel ME3 locus on chromosome 11 (P  =  2.18 × 10(-8)) at genome-wide significance. Neither locus was associated with FEV1 decline in two additional cohort studies. We confirmed gene expression of IL16, STARD5, and ME3 in multiple lung tissues. Publicly available microarray data confirmed differential expression of all three genes in lung samples from COPD patients compared with controls. Irrespective of genotypes, the combined estimate for FEV1 decline was 26.9, 29.2 and 35.7 mL/year in never, former, and persistent smokers, respectively. CONCLUSIONS: In this large-scale GWAS, we identified two novel genetic loci in association with the rate of change in FEV1 that harbor candidate genes with biologically plausible functional links to lung function