Developing Single-Molecule TPM Experiments for Direct Observation of Successful RecA-Mediated Strand Exchange Reaction

RecA recombinases play a central role in homologous recombination. Once assembled on single-stranded (ss) DNA, RecA nucleoprotein filaments mediate the pairing of homologous DNA sequences and strand exchange processes. We have designed two experiments based on tethered particle motion (TPM) to investigate the fates of the invading and the outgoing strands during E. coli RecA-mediated pairing and strand exchange at the single-molecule level in the absence of force. TPM experiments measure the tethered bead Brownian motion indicative of the DNA tether length change resulting from RecA binding and dissociation. Experiments with beads labeled on either the invading strand or the outgoing strand showed that DNA pairing and strand exchange occurs successfully in the presence of either ATP or its non-hydrolyzable analog, ATPγS. The strand exchange rates and efficiencies are similar under both ATP and ATPγS conditions. In addition, the Brownian motion time-courses suggest that the strand exchange process progresses uni-directionally in the 5′-to-3′ fashion, using a synapse segment with a wide and continuous size distribution

Identifying the favored mutation in a positive selective sweep.

Most approaches that capture signatures of selective sweeps in population genomics data do not identify the specific mutation favored by selection. We present iSAFE (for "integrated selection of allele favored by evolution"), a method that enables researchers to accurately pinpoint the favored mutation in a large region (∼5 Mbp) by using a statistic derived solely from population genetics signals. iSAFE does not require knowledge of demography, the phenotype under selection, or functional annotations of mutations

An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump

Bacterial efflux pumps confer multidrug resistance by transporting diverse antibiotics from the cell. In Gram-negative bacteria, some of these pumps form multi-protein assemblies that span the cell envelope. Here, we report the near-atomic resolution cryoEM structures of the Escherichia coli AcrAB-TolC multidrug efflux pump in resting and drug transport states, revealing a quaternary structural switch that allosterically couples and synchronizes initial ligand binding with channel opening. Within the transport-activated state, the channel remains open even though the pump cycles through three distinct conformations. Collectively, our data provide a dynamic mechanism for the assembly and operation of the AcrAB-TolC pump.This work was supported by the American Heart Association (16RNT29720001), and Grants from National Institutes of Health (P41GM103832, R01GM079429, R01GM072804, S10OD016279), the Wellcome trust, the Human Frontiers Science Program and MRC Mitochondrial Biology Unit (Grant number: U105663141). CFH is supported by a pre-doctoral training fellowship from the Keck Center of the Gulf Coast Consortia, on the NLM Training Program in Biomedical Informatics (Grant No. T15LM007093). We acknowledge the computing resources provided by the Center for Computational and Integrative Biomedical Research of Baylor College of Medicine and the Texas Advanced Computing Center at the University of Texas at Austin funded by the National Science Foundation (NSF) through grant ACI-1134872

Psychometric precision in phenotype definition is a useful step in molecular genetic investigation of psychiatric disorders

Affective disorders are highly heritable, but few genetic risk variants have been consistently replicated in molecular genetic association studies. The common method of defining psychiatric phenotypes in molecular genetic research is either a summation of symptom scores or binary threshold score representing the risk of diagnosis. Psychometric latent variable methods can improve the precision of psychiatric phenotypes, especially when the data structure is not straightforward. Using data from the British 1946 birth cohort, we compared summary scores with psychometric modeling based on the General Health Questionnaire (GHQ-28) scale for affective symptoms in an association analysis of 27 candidate genes (249 single-nucleotide polymorphisms (SNPs)). The psychometric method utilized a bi-factor model that partitioned the phenotype variances into five orthogonal latent variable factors, in accordance with the multidimensional data structure of the GHQ-28 involving somatic, social, anxiety and depression domains. Results showed that, compared with the summation approach, the affective symptoms defined by the bi-factor psychometric model had a higher number of associated SNPs of larger effect sizes. These results suggest that psychometrically defined mental health phenotypes can reflect the dimensions of complex phenotypes better than summation scores, and therefore offer a useful approach in genetic association investigations

High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structurefunction relationship of GPCRs. © 2014 Bill et al

Weakly Aggregative Modal Logic: Characterization and Interpolation

Weakly Aggregative Modal Logic (WAML) is a collection of disguised polyadic modal logics with n-ary modalities whose arguments are all the same. WAML has some interesting applications on epistemic logic and logic of games, so we study some basic model theoretical aspects of WAML in this paper. Specifically, we give a van Benthem-Rosen characterization theorem of WAML based on an intuitive notion of bisimulation and show that each basic WAML system Kn lacks Craig Interpolation

Behavioral Inhibition and Attentional Control in Adolescents: Robust Relationships with Anxiety and Depression

Behavioral inhibition (BI) has been associated with the development of internalizing disorders in children and adolescents. It has further been shown that attentional control (AC) is negatively associated with internalizing problems. The combination of high BI and low AC may particularly lead to elevated symptomatology of internalizing behavior. This study broadens existing knowledge by investigating the additive and interacting effects of BI and AC on the various DSM-IV based internalizing dimensions. A sample of non-clinical adolescents (N = 1806, age M = 13.6 years), completed the Behavioral Inhibition System/Behavioral Activation System Scales (BIS/BAS), the attentional control subscale of the Adult Temperament Questionnaire (ATQ) and the Revised Child Anxiety and Depression Scale (RCADS). As expected, BI was positively, and AC was negatively related to internalizing dimensions, with stronger associations of BI than of AC with anxiety symptoms, and a stronger association of AC than of BI with depressive symptoms. AC moderated the association between BI and all measured internalizing dimensions (i.e., symptoms of generalized anxiety disorder, social phobia, separation anxiety disorder, panic disorder, obsessive–compulsive disorder, and major depressive disorder). Since high AC may reduce the impact of high BI on the generation of internalizing symptoms, an intervention focused on changing AC may have potential for prevention and treatment of internalizing disorders