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Childhood sexual abuse (CSA) is a prevalent form of violence in our society. The exact number of women sexually abused as children is not known, as the estimated rates vary from 6% to 62%. This study examined the psychological characteristics of women with and without a history of CSA. A nonclinical sample of 255 undergraduate women served as volunteer participants. The variables measured included: Adult Romantic Attachment, Depression, Anxiety, Traumatic Symptoms, Cognitive Distortions, Maladaptive Schemas, and Borderline Personality Features. Women who reported a history of abuse evidenced marked differences from those who did not across a broad spectrum of variables. A majority of CSA survivors did not seek any treatment. These results are discussed relative to prevention and early intervention efforts that are necessary to assist this underserved population. [ABSTRACT FROM AUTHOR]

The present paper compared drinking and drug use in Adult Children of Alcoholics (ACOAs), compared to non-ACOAs, among college students. Participants were 572 undergraduates. ACOAs were more likely to be current drug users than non-ACOAs. ACOAs reported initiating alcohol use earlier than non-ACOAs; however, ACOAs did not drink more often or more heavily than non-ACOAs. Among ACOAs, suspecting one's mother or both parents of alcohol abuse predicted greater likelihood of lifetime drinking; suspecting one's father of alcohol abuse was associated with more frequent drinking during the school year. The fact that ACOA status was not associated with the frequency and amount of alcohol consumed may reflect that 73.3% of the sample were drinkers, and 31.9% met criteria for binge drinking in the previous month. [ABSTRACT FROM AUTHOR]

This paper examines the construct validity and measurement invariance of the Parasocial Relationships in Social Media (PRISM) survey which was designed to provide researchers with a valid and reliable tool for measuring parasocial relationships developed in a social media context. A confirmatory factor analysis indicated the survey provides an adequate measure of parasocial relationships with online, social media celebrities, replicating the factor structure found by Boyd and colleagues when they developed PRISM and providing evidence of the construct validity of the survey. Additionally, scalar measurement invariance was achieved which supports the survey's ability to compare parasocial relationships across different social media platforms., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Boyd et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Flow condensation heat transfer coefficients and pressure drop data were collected in a small-scale vapor compression cycle. The test section consisted of seven parallel, 0.95-mm-diameter square channels which utilized a heat flux block to measure the temperature gradients of the heat leaving during the condensation process. Heat transfer coefficients and pressure drops were measured using the temperatures and pressures measured during experiments. Experimental flow condensation heat transfer coefficient and pressure drop data are tabulated for R134a, R513A, and R450A for a range of mass fluxes (i.e., 200 - 500 kg/m 2 s) and qualities (i.e., 0.2 - 0.8) at a saturation temperature of 40°C. The heat transfer coefficient uncertainties for all experiments were ± 6.3 - 21.2%, with an average uncertainty of ± 9.8%. Data include refrigerant saturation temperature, wall temperature, mass flux, quality, condensation heat transfer coefficient and its uncertainty, and pressure drop. The data tabulated are the raw data from the paper "Flow condensation heat transfer and pressure drop performance of R134a alternative refrigerants R513A and R450A in a 0.95-mm-diameter minichannel," published by the International Journal of Heat and Mass Transfer [1]., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (© 2022 The Authors.)

Objective: Patients with rheumatoid arthritis (RA) often receive opioid analgesics for pain management. We examined the association between mental health conditions and the risk of chronic opioid therapy., Methods: A retrospective cohort of veterans with RA initiating opioid use was assembled using Veterans Health Administration databases (2001-2012). Mental health conditions included anxiety (N = 1108, 12.9%), depression (N = 1912, 22.2%), bipolar disease (N = 131, 1.5%), and post-traumatic stress disorder (N = 768, 8.9%) and were identified by ICD coded diagnoses and use of specific medications. Cohort members were followed from opioid initiation through chronic opioid therapy, defined as the continuous availability of opioids for at least 90 days. Multivariable Cox proportional hazard regression models assessed the association between mental health conditions and chronic opioid therapy accounting for relevant covariates. Subgroup analyses examined whether the strength of the observed association varied by the duration of the initial opioid prescription., Results: We identified 14,767 patients with RA with 22,452 episodes of opioid use initiation. Mental health conditions were identified in 8607 (38.3%) patients. Compared with patients without mental health conditions, patients with mental health conditions have a higher risk of developing chronic opioid therapy (469.3 vs 378.1 per 1000 person-years, adjusted hazard ratio [aHR] 1.18, 95% CI 1.09, 1.29). The increased risk was highest for those with a history of opioid use disorder (aHR 1.94, 95% CI 1.09, 3.46) and also elevated for those with other substance use disorders (aHR 1.35, 95% CI 1.05, 1.73). Duration of the initial opioid prescription was independently associated with chronic opioid therapy, regardless of the estimated opioid daily dose., Conclusions: History of mental health conditions and duration of the initial opioid prescription were associated with an increased risk of chronic opioid therapy among patients with RA.Key Points• Approximately a third of patients with RA are exposed to opioid analgesics.• Patients with RA and history of mental health disease, especially substance use disorders, who initiate opioid use have an increased risk of chronic opioid therapy.• This study provides insight in an underrepresented population of mainly male patients with RA.

Purpose: Two previously validated algorithms to identify sudden cardiac death using administrative data showed high positive predictive value. We evaluated the agreement between the algorithms using data from a common source population., Methods: We conducted a cross-sectional study to assess the percent agreement between deaths identified by two sudden cardiac death algorithms using Tennessee Medicaid and death certificate data from 2007 through 2014. The source population included all deceased patients aged 18 to 64 years with Medicaid enrollment in the 6 months prior to death. To identify sudden cardiac deaths, algorithm 1 used only hospital/emergency department (ED) claims from encounters at the time of death, and algorithm 2 required death certificates and used claims data for specific exclusion criteria., Results: We identified 34 107 deaths in the source population over the study period. The two algorithms identified 4372 potential sudden cardiac deaths: Algorithm 1 identified 3117 (71.3%) and algorithm 2 identified 1715 (39.2%), with 460 (10.5%) deaths identified by both algorithms. Of the deaths identified by algorithm 1, 1943 (62.3%) had an underlying cause of death not specified in algorithm 2. Of the deaths identified by algorithm 2, 1053 (61.4%) had no record of a hospital or ED encounter at the time of death, and 202 (11.8%) had a discharge diagnosis code not specified in algorithm 1., Conclusions: We found low agreement between the two algorithms for identification of sudden cardiac deaths because of differences in sudden cardiac death definitions and data sources., (© 2019 John Wiley & Sons, Ltd.)

Introduction: Valid algorithms for identification of cardiovascular (CV) deaths allow researchers to reliably assess the CV safety of medications, which is of importance to regulatory science, patient safety, and public health., Objective: The aim was to conduct a systematic review of algorithms to identify CV death in administrative health plan claims databases., Methods: We searched MEDLINE, EMBASE, and Cochrane Library for English-language studies published between January 1, 2012 and October 17, 2017. We examined references in systematic reviews to identify earlier studies. Selection included any observational study using electronic health care data to evaluate the sensitivity, specificity, positive predictive value (PPV), or negative predictive value (NPV) of algorithms for CV death (sudden cardiac death [SCD], myocardial infarction [MI]-related death, or stroke-related death) among adults aged ≥ 18 years in the United States. Data were extracted by two independent reviewers, with disagreements resolved through further discussion and consensus. The Quality Assessment of Diagnostic Accuracy Studies-2 instrument was used to assess the risk of bias., Results: Five studies (n = 4 on SCD, n = 1 on MI- and stroke-related death) were included after a review of 2053 citations. All studies reported algorithm PPVs, with incomplete reporting on other accuracy parameters. One study was at low risk of bias, three studies were at moderate risk of bias, and one study was at unclear risk of bias. Two studies identified community-occurring SCD: one identified events using International Classification of Disease, Ninth Revision (ICD-9) codes on death certificates and other criteria from medical claims (PPV = 86.8%) and the other identified events resulting in hospital presentation using first-listed ICD-9 codes on emergency department or inpatient medical claims (PPV = 92.3%). Two studies used death certificates alone to identify SCD (PPV = 27% and 32%, respectively). One study used medical claims to identify CV death (PPV = 36.4%), coronary heart disease mortality (PPV = 28.3%), and stroke mortality (PPV = 34.5%)., Conclusion: Two existing algorithms based on medical claims diagnoses with or without death certificates can accurately identify SCD to support pharmacoepidemiologic studies. Developing valid algorithms identifying MI- and stroke-related death should be a research priority. PROSPERO 2017 CRD42017078745.

Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein which contains a kinase domain and GTPase domain among other regions. Individuals possessing gain of function mutations in the kinase domain such as the most prevalent G2019S mutation have been associated with an increased risk for the development of Parkinson's disease (PD). Given this genetic validation for inhibition of LRRK2 kinase activity as a potential means of affecting disease progression, our team set out to develop LRRK2 inhibitors to test this hypothesis. A high throughput screen of our compound collection afforded a number of promising indazole leads which were truncated in order to identify a minimum pharmacophore. Further optimization of these indazoles led to the development of MLi-2 (1): a potent, highly selective, orally available, brain-penetrant inhibitor of LRRK2.

Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two- to threefold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of familial and sporadic Parkinson's disease (PD). That the most prevalent mutation, G2019S, leads to increased kinase activity has led to a concerted effort to identify LRRK2 kinase inhibitors as a potential disease-modifying therapy for PD. An internal medicinal chemistry effort identified several potent and highly selective compounds with favorable drug-like properties. Here, we characterize the pharmacological properties of cis-2,6-dimethyl-4-(6-(5-(1-methylcyclopropoxy)-1H-indazol-3-yl)pyrimidin-4-yl)morpholine (MLi-2), a structurally novel, highly potent, and selective LRRK2 kinase inhibitor with central nervous system activity. MLi-2 exhibits exceptional potency in a purified LRRK2 kinase assay in vitro (IC50 = 0.76 nM), a cellular assay monitoring dephosphorylation of LRRK2 pSer935 LRRK2 (IC50 = 1.4 nM), and a radioligand competition binding assay (IC50 = 3.4 nM). MLi-2 has greater than 295-fold selectivity for over 300 kinases in addition to a diverse panel of receptors and ion channels. Acute oral and subchronic dosing in MLi-2 mice resulted in dose-dependent central and peripheral target inhibition over a 24-hour period as measured by dephosphorylation of pSer935 LRRK2. Treatment of MitoPark mice with MLi-2 was well tolerated over a 15-week period at brain and plasma exposures >100× the in vivo plasma IC50 for LRRK2 kinase inhibition as measured by pSer935 dephosphorylation. Morphologic changes in the lung, consistent with enlarged type II pneumocytes, were observed in MLi-2-treated MitoPark mice. These data demonstrate the suitability of MLi-2 as a compound to explore LRRK2 biology in cellular and animal models., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

The purpose of this article is to describe the development and implementation of a grading software system, accessible from any platform, that engages today's generation of students and replaces paper grading. Set up at one U.S. dental school in an all-access, anytime (24/7) web-based program accessed through tablets, the software allows for a comparison between students' self-grading and instructor grading. This comparison facilitates student-faculty interaction, promoting discussion and student learning. The software can also be used for practical examination grading in which blinded grading between instructors is possible. The data gathered can produce descriptive reports students can draw upon to encourage self-learning and guided learning, propelling students to a better understanding of critical principles as they progress through multiple psychomotor skill sets. Other reports generated by the software allow for instructor calibration, exporting of grades directly into the university grading report system, and visual analysis of trends within each class. In a post-course survey, students (56 percent response rate) and faculty (79 percent response rate) agreed that the electronic grading was more efficient and allowed more time for faculty-student interaction than the previous grading system, thus creating an environment more conducive to learning. Overall, the software has improved students' perception of enhanced kinetic skills, while facilitating administration of preclinical projects and practical examinations.

Reduced NMDA receptor functioning is hypothesized to underlie the cognitive and negative symptoms associated with schizophrenia. However, because direct activation of the NMDA receptor is accompanied by neurotoxicity, mechanisms that activate the glycine co-agonist site on the NMDA receptor could carry greater therapeutic potential. In the current study, the effects of two glycine transporter 1 (GlyT1) inhibitors, RG1678 and ORG25935, were characterized in the object-retrieval detour (ORD) task in scopolamine-impaired rhesus monkeys and, using positron emission tomography (PET), the GlyT1 occupancy to efficacy relationship of each compound was established. Scopolamine exerted a significant decrease in accuracy in the ORD task. Lower doses of RG1678 (0.3 and 1.0 mg/kg, p.o.) significantly attenuated the impact of scopolamine, whereas the highest dose tested (1.8 mg/kg) did not. The predicted GlyT1 occupancies of RG1678 at the effective doses were ~10 and 30 %. ORG25935 (0.1, 0.3, and 1 mg/kg, p.o.) also significantly attenuated the impact of scopolamine on the ORD task, whereas 3 mg/kg did not. The predicted GlyT1 occupancies of ORG25935 at the effective doses ranged from 16 to 80 %. These data suggest that GlyT1 inhibitors have the potential to improve performance on prefrontal cortex-dependent tests such as the ORD task, but that efficacy is lost when higher occupancies are achieved. Importantly, recent Ph2B data published by Roche suggests that low but not high doses of RG1678 improved negative symptoms in patients with schizophrenia, highlighting the potential translational nature of the current preclinical findings.

Purpose: To quantify the prevalence of prescribed opioid analgesics among pregnant women enrolled in Tennessee Medicaid from 1995 to 2009., Methods: Retrospective cohort study of 277,555 pregnancies identified from birth and fetal death certificates, and linked to previously validated, computerized pharmacy records. Poisson regression was used to estimate trends over time, rate ratios, and 95% confidence intervals (CI)., Results: During the study period, 29% of pregnant women filled a prescription for an opioid analgesic. From 1995 to 2009, any pregnancy-related use increased 1.90-fold (95% CI, 1.83-1.98), first trimester use increased 2.27-fold (95% CI, 2.14-2.41), and second or third trimester use increased 2.02-fold (95% CI, 1.93-2.12), after adjusting for maternal characteristics. Any pregnancy-related, first trimester, and second or third trimester use were each more likely among mothers who were at least 21 years old, white, non-Hispanic, prima gravid, resided in nonurban areas, enrolled in Medicaid owing to disability, and who had less than a high school education., Conclusions: Opioid analgesic use by Tennessee Medicaid-insured pregnant women increased nearly 2-fold from 1995 to 2009. Additional study is warranted to understand the implications of this increased use., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Purpose: To quantify maternal use of atypical antipsychotics, typical antipsychotics, anticonvulsants, and lithium during pregnancy., Methods: Tennessee birth and death records were linked to Tennessee Medicaid data to conduct a retrospective cohort study of 296,817 women enrolled in Tennessee Medicaid throughout pregnancy who had a live birth or fetal death from 1985 to 2005., Results: During the study time period, the adjusted rate of use of any study medication during pregnancy increased from nearly 14 to 31 per 1000 pregnancies (β = 0.08, 95% CI = 0.07, 0.09). Significant increases were reported in use of anticonvulsants alone among mothers with pain and other psychiatric disorders, atypical antipsychotics alone among mothers with bipolar disorders, schizophrenia, unipolar depressive disorders, and other psychiatric disorders, and more than one studied medication for mothers with epilepsy, pain disorders, bipolar disorders, unipolar depressive disorders, and other psychiatric disorders. Significant decreases were reported in use of lithium alone and typical antipsychotics alone for all clinically meaningful diagnosis groups., Conclusions: There was a substantial increase in use of atypical antipsychotics alone, anticonvulsants alone, and medications from multiple studied categories among Tennessee Medicaid-insured pregnant women during the study period. Further examination of the maternal and fetal consequences of exposure to these medications during pregnancy is warranted., (Copyright © 2012 John Wiley & Sons, Ltd.)

Background: Little is known about the extent of antiepileptic drug (AED) use in pregnancy, particularly for newer agents. Our objective was to assess whether AED use has increased among pregnant women in the US, 2001-2007., Methods:   We analysed data from the Medication Exposure in Pregnancy Risk Evaluation Program (MEPREP) database, 1 January 2001 to 31 December 2007. We identified liveborn deliveries among women, aged 15-45 years on delivery date, who were members of MEPREP health plans (n=585615 deliveries). Pregnancy exposure to AEDs, determined through outpatient pharmacy dispensing files. Older AEDs were available for clinical use before 1993; other agents were considered newer AEDs. Information on sociodemographic and medical/reproductive factors was obtained from linked birth certificate files. Maternal diagnoses were identified based on ICD-9 codes., Results:   Prevalence of AED use during pregnancy increased between 2001 (15.7 per 1000 deliveries) and 2007 (21.9 per 1000 deliveries), driven primarily by a fivefold increase in the use of newer AEDs. Thirteen per cent of AED-exposed deliveries involved a combination of two or more AEDs. Psychiatric disorders were the most prevalent diagnoses, followed by epileptic and pain disorders, among AED users regardless of AED type, year of conception or gestational period., Conclusions:   AED use during pregnancy increased between 2001 and 2007, driven by a fivefold increase in the use of newer AEDs. Nearly one in eight AED-exposed deliveries involved the concomitant use of more than one AED. Additional investigations of the reproductive safety of newer AEDs may be needed., (© 2012 Blackwell Publishing Ltd.)

An asparagine-to-serine substitution at residue 370 (N370S) in glucocerebrosidase (GCase) is the most prevalent mutation leading to Gaucher's disease, the most common lysosomal storage disorder. Two types of hydrogen/deuterium exchange experiment coupled with proteolysis and liquid chromatography-mass spectrometry (HDX-MS) were used to investigate the dynamic properties and unfolding stability of wt, R495H, and N370S GCases in the presence and absence of ligands. R495H GCase is used for enzyme replacement therapy and is considered to be a wt surrogate, whereas N370S is the most prevalent mutation leading to Gaucher's disease. Time-course HDX experiments of the GCases were performed under near-physiological conditions to detect the protein's local unfolding motions at a submolecular level. In guanidine-titration experiments, HDX reactions were performed with various concentrations of a chemical denaturant to provide the global stability of the proteins. The two types of experiment showed that all three purified GCases, wt, R495H, and N370S, have virtually identical local unfolding motions and global stabilities in solution. Combined with previous X-ray crystallographic studies, which showed indistinguishable backbone conformations for N370S and R495H GCase mutants and very similar melting temperatures for the wt, R495H, and N370S mutants, all three GCases are likely to have virtually identical structural and dynamic properties in solution. The guanidine-titration experiments revealed that the pharmacological chaperone, isofagomine (IFG), interacts more weakly with the N370S mutant than with the R495H GCase; this is consistent with the higher IC(50) value of IFG against N370S than against R495H. The time-course experiments showed that IFG restricts the local unfolding motions of N370S in the same way as those of R495H when the ligand saturates the proteins., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Understanding antigen-antibody interactions at the sub-molecular level is of particular interest for scientific, regulatory, and intellectual property reasons, especially with increasing demand for monoclonal antibody therapeutic agents. Although various techniques are available for the determination of an epitope, there is no widely applicable, high-resolution, and reliable method available. Here, a combination approach using amide hydrogen/deuterium exchange coupled with proteolysis and mass spectrometry (HDX-MS) and computational docking was applied to investigate antigen-antibody interactions. HDX-MS is a widely applicable, medium-resolution, medium-throughput technology that can be applied to epitope identification. First, the epitopes of cytochrome c-E8, IL-13-CNTO607, and IL-17A-CAT-2200 interactions identified using the HDX-MS method were compared with those identified by X-ray co-crystal structures. The identified epitopes are in good agreement with those identified using high-resolution X-ray crystallography. Second, the HDX-MS data were used as constraints for computational docking. More specifically, the non-epitope residues of an antigen identified using HDX-MS were designated as binding ineligible during computational docking. This approach, termed HDX-DOCK, gave more tightly clustered docking poses than stand-alone docking for all antigen-antibody interactions examined and improved docking results significantly for the cytochrome c-E8 interaction., (Copyright © 2012 John Wiley & Sons, Ltd.)

Starting from compound 1, we utilized biostructural data to successfully evolve an existing series into a new chemotype with a promising overall profile, exemplified by 19., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Backbone amide hydrogen exchange rates can be used to describe the dynamic properties of a protein. Amide hydrogen exchange rates in a native protein may vary from milliseconds (ms) to several years. Ideally, the rates of all amide hydrogens of the analyte protein can be determined individually. To achieve this goal, monitoring of a wider time window is critical, in addition to high sequence coverage and high sequence resolution. Significant improvements have been made to hydrogen/deuterium exchange mass spectrometry methods in the past decade for better sequence coverage and higher sequence resolution. On the other hand, little effort has been made to expand the experimental time window to accurately determine exchange rates of amide hydrogens. Many fast exchanging amide hydrogens are completely exchanged before completion of a typical short exchange time point (10-30 s) and many slow exchanging amide hydrogens do not start exchanging before a typical long exchanging time point (1-3 h). Here various experimental conditions, as well as a quenched-flow apparatus, are utilized to monitor cytochrome c amide hydrogen exchange behaviors over more than eight orders of magnitude (0.0044-1 000 000 s), when converted into the standard exchange condition (pH 7 and 23°C).

Starting from lead compound 1, we demonstrate how X-ray structural data can be used to understand SAR and expediently optimize bioavailability in a novel series of AMPA receptor modulators, furnishing 5 with improved bioavailability and robust in vivo activity., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Starting from an HTS derived hit 1, application of biostructural data facilitated rapid optimization to lead 22, a novel AMPA receptor modulator. This is the first demonstration of how structure based drug design can be exploited in an optimization program for a glutamate receptor., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Recent evidence suggests that antiglucocorticoids, like conventional antidepressants, may recover depressive symptoms by boosting hippocampal neurogenesis. Here, we explore several possible antiglucocorticoid-based antidepressive therapeutic strategies. Firstly, we review specific glucocorticoid receptor/antagonist interactions. Secondly, we discuss a potential new therapeutic target, doublecortin-like kinase, which regulates glucocorticoid signaling in neuronal progenitor cells.

Statistical analysis of data from 39 proteins (13 766 amino acid residues) digested with immobilized porcine pepsin under conditions compatible with hydrogen/deuterium (H/D) exchange (<1 degrees C, <30 s) was performed to examine pepsin cleavage specificity. The cleavage of pepsin was most influenced by the amino acid residue at position P1. Phe and Leu are favored residues each with a cleavage probability greater than 40%. His, Lys, Arg, or Pro residues prohibit cleavage when found at the P1 position. Pro also cannot be at position P2 (cleavage probability <0.3%). Occupation of the P3 position by His, Lys, or Arg, or occupation of the P2' position by Pro, also leads to very little cleavage (cleavage probability <1.7%). The average cleavage probability over the entire data set was 13.6%, which is slightly lower than the value previously obtained by Powers et al. (14.8%). This is due, in part, to the larger protein sizes used in the current study. While the specificity of pepsin was similar to that previously observed, higher selectivity was observed in the present study due to less experimental variation in the conditions used to generate our database., ((c) 2008 John Wiley & Sons, Ltd.)

Observed peptide gas-phase fragmentation patterns are a complex function of many variables. To systematically probe this phenomenon, an array of 40 peptides was synthesized for study. The array of sequences was designed to hold certain variables (peptide length) constant and randomize or balance others (peptide amino acid distribution and position). A high-quality tandem mass spectrometry (MS/MS) data set was acquired for each peptide for all observed charge states on multiple MS instruments, quadrupole-time-of-flight and quadrupole ion trap. The data were analyzed as a function of total charge state and number of mobile protons. Previously known dissociation trends were observed, validating our approach. In addition, the general influence of basic amino acids on dissociation could be determined because, in contrast to the more widely studied tryptic peptides, the amino acids H, K, and R were positionally distributed. Interestingly, our results suggest that cleavage at all basic amino acids is suppressed when a mobile proton is available. Cleavage at H becomes favored only under conditions where a partially mobile proton is present, a caveat to the previously reported trend of enhanced cleavage at H. Finally, all acquired data were used as a benchmark to determine how well these sequences would have been identified in a database search using a common algorithm, Mascot.

Background: Patients with mildly abnormal or equivocal myocardial perfusion imaging (MPI) scans undergo diagnostic angiography or receive medical management. However, current guidelines mandate different treatment goals for patients with known coronary artery disease (CAD), and catheterization is often required. Coronary computed tomography angiography (CCTA) may be an effective alternative to catheterization for patients at intermediate risk for CAD., Objectives: The purpose of this study was to analyze the cost implications of CCTA before catheterization in patients with mildly abnormal or equivocal MPI scans., Methods: Patients (n = 206) with mildly abnormal or equivocal MPI scans underwent 64-detector CCTA instead of catheterization at the discretion of a treating physician. Studies were evaluated by a trained reader, and results were classified as "no evident CAD," "nonobstructive CAD," or "potentially obstructive CAD." Cost data were analyzed based on actual reimbursements for CT angiography and cardiac catheterization. We modeled the costs of two clinical approaches. "Selective catheterization" involved catheterization only if CCTA showed potentially obstructive CAD. "Immediate catheterization" considered catheterization for all patients in the cohort. Sensitivity analysis was performed on multiple variables., Results: Thirty-two percent of patients had potentially obstructive plaque on CTA. Selective catheterization saves $1454 per patient. Sensitivity analysis revealed cost savings to be preserved even if up to 81.5% of the patient cohort undergoes catheterization, as well as across wide ranges of procedural costs., Conclusion: A strategy that uses CCTA as a gatekeeper to catheterization is cost saving as opposed to initial catheterization for patients with equivocal or mildly abnormal myocardial perfusion scans.

The alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propioic acid (AMPA) subtype of glutamate receptors mediate fast excitatory neurotransmission throughout the mammalian nervous system and participate in the forms of synaptic plasticity that are considered to underlie learning and memory. Positive allosteric modulators of these receptors are the subject of much investigation because of their emerging therapeutic potential for a range of psychiatric and neurological disorders such as schizophrenia and Alzheimer's disease. This review focuses on the most recent developments in preclinical and clinical research on novel classes of AMPA receptor positive modulators and highlights how the application of biostructural studies has increased our understanding of the biophysical effects produced by these drugs.

A nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARgamma), is a ligand-dependent transcription factor involved in glucose homeostasis and adipocyte differentiation. PPARgamma is the molecular target of various natural and synthetic molecules, including anti-diabetic agents such as rosiglitazone. Amide hydrogen/deuterium-exchange (H/D-Ex), coupled with proteolysis and mass spectrometry, was applied to study the dynamics of the PPARgamma ligand binding domain (LBD) with or without molecules that modulate PPARgamma activity. The H/D-Ex patterns of ligand-free PPARgamma LBD show that the ligand binding pocket of LBD is significantly more dynamic than the rest of the LBD. Presumably, the binding pocket is intrinsically disordered in order to accommodate different ligands. The presence of two full agonists (rosiglitazone and GW1929), a partial agonist (nTZDpa), and a covalent antagonist (GW9662), changed the dynamics/conformation of PPARgamma LBD and slowed the H/D exchange rate in various regions of the protein. The full agonists slowed the H/D exchange more globally and to a greater extent than the partial agonist or the antagonist, indicating that the full agonist stabilizes the PPARgamma LBD more than the partial agonist or the antagonist. One interesting observation is that the two full agonists significantly stabilized helix 12 while the partial agonist and the antagonist did not perturb the H/D exchange of this region. The results showed that the change in protein dynamics induced by ligand binding may be an important factor for the activation of genes and that H/D-Ex is a useful method for analyzing the biological activity of drug leads.

It is generally accepted that protein and solvation dynamics play fundamental roles in the mechanisms of protein-protein binding; however, assessing their contribution meaningfully has not been straightforward. Here, hydrogen/deuterium exchange mass spectrometry (H/D-Ex) was employed to assess the role of dynamics for a high-affinity human growth hormone variant (hGHv) and the wild-type growth hormone (wt-hGH) each binding to the extracellular domain of their receptor (hGHbp). Comparative analysis of the transient fluctuations in the bound and unbound states revealed that helix-1 of hGHv undergoes significant transient unfolding in its unbound state, a characteristic that was not found in wt-hGH or apparent in the temperature factor data from the X-ray analysis of the unbound hGHv structure. In addition, upon hormone binding, an overall increase in stability was observed for the beta-sheet structure of hGHbp which included sites distant from the binding interface. On the basis of the stability, binding kinetics, and thermodynamic data presented, the increase in the binding free energy of hGHv is primarily generated by factors that appear to increase the energy of the unbound state relative to the free energy of the bound complex. This implies that an alternate route to engineer new interactions aiming to increase protein-protein association energies may be achieved by introducing certain mutations that destabilize one of the interacting molecules without destabilizing the resulting bound complex. Importantly, although the hGHv molecule is less stable than its wt-hGH counterpart, its resulting active ternary complex with two copies of hGHbp has comparable stability to the wt complex.

The glycine transporter (GlyT-1b) is a Na(+)/Cl(-)-dependent electrogenic transporter which mediates the rapid re-uptake of glycine from the synaptic cleft. Based on its tissue distribution, GlyT-1 has been suggested to co-localise with the NMDA receptor where it may modulate the concentration of glycine at its co-agonist binding site. This data has led to GlyT-1 inhibitors being proposed as targets for disorders such as schizophrenia and cognitive dysfunction. Radiolabelled uptake assays (e.g. [(3)H]glycine) have been traditionally used in compound screening to identify glycine transporter inhibitors. While such an assay format is useful for testing limited numbers of compounds, the identification of novel glycine uptake inhibitors requires a functional assay compatible with high-throughput screening (HTS) of large compound libraries. Here, the authors present the development of a novel homogenous cell-based assay using the FLIPR membrane potential blue dye (Molecular Devices) and FLEXstation. Pharmacological data for the GlyT-1 inhibitors Org 24598 and ALX 5407 obtained using this novel electrogenic assay correlated well with the conventional [(3)H]-glycine uptake assay format. Furthermore, the assay has been successfully miniaturised using FLIPR(3) and therefore has the potential to be used for high-throughput screening.

The majority of immediate-early gene (IEG) studies focus on a few key brain regions associated with the class of psychoactive compound being studied. Recently, using a meta-analysis of the c-fos literature, we demonstrated the utility of c-fos profiling to classify such compounds. The present study examined acute delivery of a range of antidepressant classes; fluoxetine, imipramine, LiCl, and mirtazapine. The dual aims were to study the IEG profiles of these varying classes of antidepressants throughout the rat brain and to compare the utility of c-fos or Egr-1 as IEGs to classify clinically efficacious antidepressants. All antidepressants increased c-fos mRNA in the central amygdala, as previously shown, while c-fos was also increased in the anterior insular cortex and significantly decreased within the septum. Although acute antidepressant administration altered c-fos expression in a number of brain regions, Egr-1 expression was only significantly altered in the central amygdala, suggesting that Egr-1 may not be as useful a marker to investigate acute antidepressant treatment. The fact that these drugs, including the previously unclassified antidepressant mirtazapine, share a number of common loci of activation, which are implicated by human and animal studies in depression, adds further support to the use of IEG mapping to classify psychoactive compounds.

Body-image dissatisfaction is not uncommon and can adversely affect individuals' psychosocial functioning and quality of life. Various oft-cited surveys and a meta-analysis implicate a worsening of body image over the past several decades, especially among women and possibly among men. The present cross-sectional study examined changes in multiple facets of body image among 3,127 college students from 1983 through 2001; the same standardized assessment was used in 22 studies conducted within the same university. Results confirmed non-Black women's increasing body-image dissatisfaction until the early or mid-1990s, after which significant improvements occurred in terms of overall body-image evaluation and overweight preoccupation among both non-Black and Black women, despite heavier body weights. A reduction over time in women's investment in their appearance was also evident. Men's body image was relatively stable during the 19-year period. Explanations, limitations, and implications of the findings are discussed., (Copyright 2004 APA.)

The amino-terminal domain of apolipoprotein (apo) E4 is less susceptible to chemical and thermal denaturation than the apoE3 and apoE2 domains. We compared the urea denaturation curves of the 22-kDa amino-terminal domains of the apoE isoforms at pH 7.4 and 4.0. At pH 7.4, apoE3 and apoE4 reflected an apparent two-state denaturation. The midpoints of denaturation were 5.2 and 4.3 m urea, respectively. At pH 4.0, a pH value known to stabilize folding intermediates, apoE4 and apoE3 displayed the same order of denaturation but with distinct plateaus, suggesting the presence of a stable folding intermediate. In contrast, apoE2 proved the most stable and lacked the distinct plateau observed with the other two isoforms and could be fitted to a two-state unfolding model. Analysis of the curves with a three-state unfolding model (native, intermediate, and unfolded) showed that the apoE4 folding intermediate reached its maximal concentration ( approximately 90% of the mixture) at 3.75 m, whereas the apoE3 intermediate was maximal at 4.75 m ( approximately 80%). These results are consistent with apoE4 being more susceptible to unfolding than apoE3 and apoE2 and more prone to form a stable folding intermediate. The structure of the apoE4 folding intermediate at pH 4.0 in 3.75 m urea was characterized using pepsin proteolysis, Fourier transform infrared spectroscopy, and dynamic light scattering. From these studies, we conclude that the apoE4 folding intermediate is a single molecule with the characteristics of a molten globule. We propose a model of the apoE4 molten globule in which the four-helix bundle of the amino-terminal domain is partially opened, generating a slightly elongated structure and exposing the hydrophobic core. Since molten globules have been implicated in both normal and abnormal physiological function, the differential abilities of the apoE isoforms to form a molten globule may contribute to the isoform-specific effects of apoE in disease.

Denaturation by guanidine-HCl, urea, or heating was performed on the common isoforms of human apolipoprotein (apo) E (apoE2, apoE3, and apoE4) and their 22-kDa and 10-kDa fragments in order to investigate the effects of the cysteine/arginine interchanges at residues 112 and 158. Previous physical characterization of apoE3 established that apoE contains two domains, the 10-kDa carboxyl-terminal and 22-kDa amino-terminal domains, which unfold independently and exhibit large differences in stability. However, the physical properties of apoE2, apoE3, and apoE4 have not been compared before. Analysis by circular dichroism showed that the different isoforms have identical alpha-helical contents and guanidine-HCl denaturation confirmed that the two domains unfold independently in all three isoforms. However, guanidine-HCl, urea, and thermal denaturation showed differences in stability among the 22-kDa amino-terminal fragments of the apoE isoforms (apoE4 < apoE3 < apoE2). Furthermore, guanidine-HCl denaturation monitored by circular dichroism and fluorescence suggested the presence of a folding intermediate in apoE, most prominently in apoE4. Thus, these studies reveal that the major isoforms of apoE, which are associated with different pathological consequences, exhibit significant differences in stability.

Conformational reorganization of the amino-terminal four-helix bundle (22-kDa fragment) of apolipoprotein E (apoE) in binding to the phospholipid dimyristoylphosphatidylcholine (DMPC) to form discoidal particles was investigated by introducing single, double, and triple interhelical disulfide bonds to restrict the opening of the bundle. Interaction of apoE with DMPC was assessed by vesicle disruption, turbidimetric clearing, and gel filtration assays. The results indicate that the formation of apoE.DMPC discoidal particles occurs in a series of steps. A triple disulfide mutant, in which all four helices were tethered, did not form complexes but could release encapsulated 5-(6)-carboxylfluorescein from DMPC vesicles, indicating that the initial interaction does not involve major reorganization of the helical bundle. Initial interaction is followed by the opening of the four-helix bundle to expose the hydrophobic faces of the amphipathic helices. In this step, helices 1 and 2 and helices 3 and 4 preferentially remain paired, since these disulfide-linked mutants bound to DMPC in a manner similar to that of the 22-kDa fragment of apoE4. In contrast, mutants in which helices 2 and 3 and/or helices 1 and 4 paired bound poorly to DMPC. However, all single and double helical pairings resulted in the formation of larger discs than were formed by the 22-kDa fragment, indicating that further reorganization of the helices occurs following the initial opening of the four-helix bundle in which the protein assumes its final lipid-bound conformation. In support of this rearrangement, reducing the disulfide bonds converted the large disulfide mutant discs to normal size.

The region of apolipoprotein E (apoE) that interacts directly with the low density lipoprotein (LDL) receptor lies in the vicinity of residues 136-150, where lysine and arginine residues are crucial for full binding activity. However, defective binding of carboxyl-terminal truncations of apoE3 has suggested that residues in the vicinity of 170-183 are also important. To characterize and define the role of this region in LDL receptor binding, we created either mutants of apoE in which this region was deleted or in which arginine residues within this region were sequentially changed to alanine. Deletion of residues 167-185 reduced binding activity (15% of apoE3), and elimination of arginines at positions 167, 172, 178, and 180 revealed that only position 172 affected binding activity (2% of apoE3). Substitution of lysine for Arg(172) reduced binding activity to 6%, indicating a specific requirement for arginine at this position. The higher binding activity of the Delta167-185 mutant relative to the Arg(172) mutant (15% versus 2%) is explained by the fact that arginine residues at positions 189 and 191 are shifted in the deletion mutant into positions equivalent to 170 and 172 in the intact protein. Mutation of these residues and modeling the region around these residues suggested that the influence of Arg(172) on receptor binding activity may be determined by its orientation at a lipid surface. Thus, the association of apoE with phospholipids allows Arg(172) to interact directly with the LDL receptor or with other residues in apoE to promote its receptor-active conformation.

1 The VPAC2 and PAC1 receptors are closely related members of the Group II G protein-coupled receptor family. At the VPAC2 receptor, VIP is equipotent to PACAP-38 in stimulating cyclic AMP production, whereas at the PAC1 receptor PACAP-38 is many fold more potent than VIP. In this study, domains which confer this selectivity were investigated by constructing four chimaeric receptors in which segments of the VPAC2 receptor were exchanged with the corresponding segment from the PAC1 receptor. 2 When expressed in COS 7 cells all the chimaeric receptors bound the common ligand [125I]PACAP-27 and produced cyclic AMP in response to agonists. 3 Relative selectivity for agonists was determined primarily by the amino terminal extracellular domain of the PAC1 receptor and the VPAC2 receptor. The interchange of other domains had little effect on the potency of PACAP-38 or PACAP-27. 4 For chimaeric constructs with a PAC1 receptor amino terminal domain, the substitution of increasing portions of the VPAC2 receptor decreased the potency of VIP yet increased that of helodermin. 5 This suggests that the interaction of VIP/helodermin but not PACAP with the PAC1 receptor may be influenced (and differentially so) by additional receptor domains.

Apolipoprotein (apo) E plays an important role in lipid metabolism, and the major isoforms of apoE (apoE2, apoE3, and apoE4) have significantly different metabolic effects. Apolipoprotein E4 is associated with a higher risk of both heart disease and Alzheimer's disease (AD). Patients homozygous for apolipoprotein E2 are predisposed to type III hyperlipoproteinemia, and apoE2 may be protective against AD. Structure/function studies have proved to be a useful tool in understanding how the different apoE isoforms result in different pathological consequences. As these studies continue, it is essential to have a reliable method to produce large quantities of apoE and mutants of apoE. We describe here a method of apoE production in Escherichia coli strain BL21(DE3). The cDNA from apoE isoforms was inserted into a pET32a vector with a T7 promoter and a fusion partner (thioredoxin). The T7 promoter results in high expression of an easily purified His-tagged fusion protein. A thrombin recognition site was positioned in the expression vector so that only two novel amino acids (Gly-Ser) are added to the amino terminus of apoE following the removal of thioredoxin. Approximately 20 mg of apoE is obtained from a 1-liter culture. The major isoforms of apoE produced with this system were extensively characterized for their ability to bind the low-density lipoprotein (LDL) receptor, for their characteristic lipid association preferences, and for their stability as measured by guanidine denaturation. The recombinant proteins behaved identically to plasma-derived apoE isoforms., (Copyright 1999 Academic Press.)

Neurotransmitter transport systems are major targets for therapeutic alterations in synaptic function. We have cloned and sequenced a cDNA encoding the human type 2 glycine transporter GlyT2 from human brain and spinal cord. An open reading frame of 2391 nucleotides encodes a 797 amino acid protein that transports glycine in a Na+/Cl--dependent manner. When stably expressed in CHO cells, human GlyT2 displays a dose-dependent uptake of glycine with an apparent Km of 108 microM. This uptake is not affected by sarcosine at concentrations up to 1 mM. Radiation hybrid analysis mapped the GlyT2 gene to D11S1308 (LOD=8.988) on human chromosome 11p15.1-15.2.

Gel filtration chromatographs of lipoproteins represent a superposition, or convolution, of the intrinsic polydispersity of the solute and the dispersion due to transport phenomena. We describe a deconvolution technique for improving the resolution of gel filtration chromatographs applicable to lipoproteins and other polydisperse solutes. A matrix of spreading functions, characterizing the dispersive properties of the column, was determined by fitting chromatographic data from a series of monodisperse standards with the solution to the transport equations and interpolating between the fit parameters. A successive approximation scheme was used in which a test distribution was incrementally corrected by an amount proportional to the error between the measured chromatograph and that derived from the test distribution. A nonlinear relaxing function was used to constrain the correction term such that the solution remained physically realizable (i.e., nonnegative absorbance) as it evolved. Deconvolved chromatographs of lipoproteins provided resolution of peaks that were obscured by spreading in the original data. The distribution of particle sizes within each fraction was calculated and verified experimentally by further separating the contents of fractions by gradient gel electrophoresis. Our technique, however, provided comparable resolution of the peaks without the additional experimental procedure.

We studied the ability of the rat vasoactive intestinal polypeptide (VIP) cyclic AMP responsive element (CRE) to regulate reporter gene expression through a c-fos promoter in rat sensory neurons transfected in culture by plasmid microinjection. The CRE enhanced the synergistic response of the promoter to combined potassium-evoked depolarisation and forskolin treatment in neonatal but not adult rat neurons. This corresponds to endogenous VIP expression which is induced synergistically by the same stimuli in neonatal but not adult rat neurons. We conclude that VIP expression in sensory neurons, which is induced by axotomy in vivo, could be regulated through the CRE.

We describe the analysis of the herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) promoter using an HSV-1-based vector system. Sequences under investigation for LAT promoter activity were analysed for their ability to direct chloramphenicol acetyltransferase gene expression, either in transfection assays or following their insertion into an HSV-1 vector from which the endogenous LAT promoter sequences had been removed. The analysis mapped the main determinants of LAT promoter activity during lytic infection of tissue culture cells to a 277 bp region between -279 and -2 relative to the recognized 5' end of the primary 8.3 kb transcript. The LAT promoter constructs behaved similarly in the context of the virus genome and in the plasmid-based transfection assays. Comparison of the relative activities following infection of fibroblast and neuroblastoma cell lines indicates that sequences upstream from -279 are important for LAT promoter activity in neurons.

We have cloned and sequenced a cDNA (RPR4) encoding a new member of the secretin/calcitonin/parathyroid hormone (PTH) receptor family. RPR4 was identified by PCR of rat pituitary cDNA, and a full-length clone was isolated from a rat olfactory bulb cDNA library. When RPR4 was functionally expressed in COS 7 cells, cyclic adenosine monophosphate (cAMP) production was stimulated by vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptides (PACAP-38 and PACAP-27) and helodermin, with equal potency. Peptide histidine isoleucine (PHI) and rat growth hormone releasing hormone (rGHRH) also stimulated cAMP production at lower potency. This suggests that RPR4 encodes a novel VIP receptor which we have designated the VIP2 receptor. In situ hybridisation showed that mRNA for this receptor was present mainly in the thalamus, hippocampus and in the suprachiasmatic nucleus.

We have cloned and sequenced a novel cDNA (RPR7) encoding a receptor for pituitary adenylate cyclase activating polypeptide (PACAP). RPR7 was identified by PCR of rat pituitary cDNA, and full-length clones were isolated from a rat olfactory bulb cDNA library. When expressed in COS cells, RPR7 was functionally coupled to increases in intracellular cyclic adenosine monophosphate (cAMP) in response to stimulation by PACAP-38, PACAP-27, vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI). The order of potency of these ligands was PACAP-38-PACAP-27 > VIP > PHI, suggesting that the receptor corresponds to the pharmacologically characterised PACAP Type I receptor.

Objective: Intravenous adenosine decreases infarct size in experimental models of myocardial ischaemia/reperfusion. Ischaemia/reperfusion is associated with a significant increase in cardiac release of endothelin. The effect of cardioprotective doses of adenosine on endothelin release was explored in dogs undergoing 90 min coronary occlusion and 210 min reperfusion., Methods: Dogs were assigned to intravenous adenosine in a dose of 0.15 mg.kg-1.min-1 (n = 12) or control (n = 11) during the first 150 min reperfusion. Serial endothelin levels were obtained from the coronary sinus and aortic blood and measured by radioimmunoassay., Results: Adenosine significantly reduced infarct size expressed as a percent of the risk region (28.8 6% v 14.4 2%; p = 0.03). A similar increase in aortic and coronary sinus blood endothelin was observed in both groups during temporary occlusion. A significant transcardiac increase in endothelin levels was present in the control group 60 min after reperfusion whereas no increase occurred in the adenosine treated group [control 5.6(SEM 1.9) v adenosine -0.2(1.4) pg.ml-1; p = 0.02]. Similarly, intravenous adenosine tended to prevent the increase in myocardial endothelin production seen in control animals during the early reperfusion period [control 280(146) v adenosine -57(55) pg.min-1; p = 0.05]. Endocardial blood flow in the ischaemic zone 210 min after reperfusion was significantly higher in the adenosine group, at 0.60(0.02) v 0.38(0.02) ml.min-1.g-1; p < 0.05. A significant correlation between endothelin levels, endocardial flow and infarct size was observed in the control group 3 h after reperfusion: r = 0.73, p = 0.02; r = 0.62, p = 0.03 respectively. This relationship was absent in animals treated with adenosine., Conclusions: Intravenous adenosine suppresses the release of endothelin from the previously ischaemic myocardium during the early reperfusion period. This effect may in part contribute to the improvement by adenosine in postischaemic microcirculatory flow resulting in attenuation of the "no reflow" phenomenon.