Linkage Between SecA Dimerization and Ligand Binding

Abstract. Introduction and Objectives:. There have, yet, been only few attempts to phonetically characterize the vocalizations of pain, although there is wide agreement that moaning, groaning, or other nonverbal utterance can be indicative of pain. We studied the production of vowels “u,” “a,” “i”, and “schwa” (central vowel, sounding like a darker “e” as in hesitations like “ehm”)—as experimental approximations to natural vocalizations. Methods:. In 50 students vowel production and self-report ratings were assessed during painful and nonpainful heat stimulation (hot water immersion) as well as during baseline (no-stimulation). The phonetic parameters extracted were pitch (mean F0), phonatory fluctuations (range F0) and loudness (acoustic energy level). Results:. Only for the vowels “u” and “schwa,” which might be considered best approximations to moaning and groaning, did pitch and loudness increase during pain. Furthermore, changes from nonpainful to painful stimulations in these parameters also significantly predicted concurrent changes in pain ratings. Conclusion:. Vocalization characteristics of pain seem to be best described by an increase in pitch and in loudness. Future studies using more specific and comprehensive phonetic analyses will surely help to provide an even more precise characterization of vocalizations because of pain

Computationally-predicted CB1 cannabinoid receptor mutants show distinct patterns of salt-bridges that correlate with their level of constitutive activity reflected in G protein coupling levels, thermal stability, and ligand binding

The cannabinoid receptor 1 (CB1), a member of the class A G-protein-coupled receptor (GPCR) family, possesses an observable level of constitutive activity. Its activation mechanism, however, has yet to be elucidated. Previously we discovered dramatic changes in CB1 activity due to single mutations; T3.46A, which made the receptor inactive, and T3.46I and L3.43A, which made it essentially fully constitutively active. Our subsequent prediction of the structures of these mutant receptors indicated that these changes in activity are explained in terms of the pattern of salt-bridges in the receptor region involving transmembrane domains 2, 3, 5, and 6. Here we identified key salt-bridges, R2.37 + D6.30 and D2.63 + K3.28, critical for CB1 inactive and active states, respectively, and generated new mutant receptors that we predicted would change CB1 activity by either precluding or promoting these interactions. We find that breaking the R2.37 + D6.30 salt-bridge resulted in substantial increase in G-protein coupling activity and reduced thermal stability relative to the wild-type reflecting the changes in constitutive activity from inactive to active. In contrast, breaking the D2.63 + K3.28 salt-bridge produced the opposite profile suggesting this interaction is critical for the receptor activation. Thus, we demonstrate an excellent correlation with the predicted pattern of key salt-bridges and experimental levels of activity and conformational flexibility. These results are also consistent with the extended ternary complex model with respect to shifts in agonist and inverse agonist affinity and provide a powerful framework for understanding the molecular basis for the multiple stages of CB1 activation and that of other GPCRs in general

Computational analysis of the CB1 carboxyl-terminus in the receptor-G protein complex: Computational Analysis of the CB1 Carboxyl-Terminus

Despite the important role of the carboxyl-terminus (Ct) of the activated brain cannabinoid receptor one (CB1) in the regulation of G protein signaling, a structural understanding of interactions with G proteins is lacking. This is largely due to the highly flexible nature of the CB1 Ct that dynamically adapts its conformation to the presence of G proteins. In the present study, we explored how the CB1 Ct can interact with the G protein by building on our prior modeling of the CB1-Gi complex (Shim J-Y, Ahn KH, Kendall DA. The Journal of Biological Chemistry 2013;288:32449-32465) to incorporate a complete CB1 Ct (Glu416Ct–Leu472Ct). Based upon the structural constraints from NMR studies, we employed ROSETTA to predict tertiary folds, ZDOCK to predict docking orientation, and molecular dynamics (MD) simulations to obtain two distinct plausible models of CB1 Ct in the CB1-Gi complex. The resulting models were consistent with the NMR-determined helical structure (H9) in the middle region of the CB1 Ct. The CB1 Ct directly interacted with both Gα and Gβ and stabilized the receptor at the Gi interface. The results of site-directed mutagenesis studies of Glu416Ct, Asp423Ct, Asp428Ct, and Arg444Ct of CB1 Ct suggested that the CB1 Ct can influence receptor-G protein coupling by stabilizing the receptor at the Gi interface. This research provided, for the first time, models of the CB1 Ct in contact with the G protein

Computational Prediction and Biochemical Analyses of New Inverse Agonists for the CB1 Receptor

Human cannabinoid type 1 (CB1) G-protein coupled receptor is a potential therapeutic target for obesity. The previously predicted and experimentally validated ensemble of ligand-free conformations of CB1 [Scott, C. E. et al. Protein Sci. 2013, 22, 101−113; Ahn, K. H. et al. Proteins 2013, 81, 1304–1317] are used here to predict the binding sites for known CB1-selective inverse agonists including rimonabant and its seven known derivatives. This binding pocket, which differs significantly from previously published models, is used to identify 16 novel compounds expected to be CB1 inverse agonists by exploiting potential new interactions. We show experimentally that two of these compounds exhibit inverse agonist properties including inhibition of basal and agonist-induced G-protein coupling activity, as well as an enhanced level of CB1 cell surface localization. This demonstrates the utility of using the predicted binding sites for an ensemble of CB1 receptor structures for designing new CB1 inverse agonists

The National Childrens Study: Recruitment Outcomes Using the Provider-Based Recruitment Approach

In 2009, the National Children’s Study (NCS) Vanguard Study tested the feasibility of household-based recruitment and participant enrollment using a birth-rate probability sample. In 2010, the NCS Program Office launched 3 additional recruitment approaches. We tested whether provider-based recruitment could improve recruitment outcomes compared with household-based recruitment

Pan-viral serology implicates enteroviruses in acute flaccid myelitis.

Since 2012, the United States of America has experienced a biennial spike in pediatric acute flaccid myelitis (AFM)1-6. Epidemiologic evidence suggests non-polio enteroviruses (EVs) are a potential etiology, yet EV RNA is rarely detected in cerebrospinal fluid (CSF)2. CSF from children with AFM (n = 42) and other pediatric neurologic disease controls (n = 58) were investigated for intrathecal antiviral antibodies, using a phage display library expressing 481,966 overlapping peptides derived from all known vertebrate and arboviruses (VirScan). Metagenomic next-generation sequencing (mNGS) of AFM CSF RNA (n = 20 cases) was also performed, both unbiased sequencing and with targeted enrichment for EVs. Using VirScan, the viral family significantly enriched by the CSF of AFM cases relative to controls was Picornaviridae, with the most enriched Picornaviridae peptides belonging to the genus Enterovirus (n = 29/42 cases versus 4/58 controls). EV VP1 ELISA confirmed this finding (n = 22/26 cases versus 7/50 controls). mNGS did not detect additional EV RNA. Despite rare detection of EV RNA, pan-viral serology frequently identified high levels of CSF EV-specific antibodies in AFM compared with controls, providing further evidence for a causal role of non-polio EVs in AFM

Evaluation of Xpert MTB/RIF Versus AFB Smear and Culture to Identify Pulmonary Tuberculosis in Patients With Suspected Tuberculosis From Low and Higher Prevalence Settings

Background. The Xpert MTB/RIF (Xpert) assay is a rapid nucleic acid amplification test widely used in settings of high tuberculosis prevalence to detect tuberculosis as well as rpoB mutations associated with rifampin resistance. Data are needed on the diagnostic performance of Xpert in lower-prevalence settings to inform appropriate use for both tuberculosis detection and the need for respiratory isolation. Methods. Xpert was compared to 2 sputum samples, each evaluated with acid-fast bacilli (AFB) smear and mycobacterial culture using liquid and solid culture media, from participants with suspected pulmonary tuberculosis from the United States, Brazil, and South Africa. Results. Of 992 participants enrolled with evaluable results, 22% had culture-confirmed tuberculosis. In 638 (64%) US participants, 1 Xpert result demonstrated sensitivity of 85.2% (96.7% in participants with AFB smear-positive [AFB+] sputum, 59.3% with AFB smear-negative [AFB–] sputum), specificity of 99.2%, negative predictive value (NPV) of 97.6%, and positive predictive value of 94.9%. Results did not differ between higher- and low-prevalence settings. A second Xpert assay increased overall sensitivity to 91.1% (100% if AFB+, 71.4% if AFB–), with specificity of 98.9%. In US participants, a single negative Xpert result predicted the absence of AFB+/culture-positive tuberculosis with an NPV of 99.7%; NPV of 2 Xpert assays was 100%, suggesting a role in removing patients from airborne infection isolation. Xpert detected tuberculosis DNA and mutations associated with rifampin resistance in 5 of 7 participants with rifampin-resistant, culture-positive tuberculosis. Specificity for rifampin resistance was 99.5% and NPV was 98.9%. Conclusions. In the United States, Xpert testing performed comparably to 2 higher-tuberculosis-prevalence settings. These data support the use of Xpert in the initial evaluation of tuberculosis suspects and in algorithms assessing need for respiratory isolation