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For the past twenty years research has centered on perceptions held by consumers about the quality of good produced in competing countries of origin. This paper recognizes that tradition but suggests market segments exist for each country that could be profiled and reached with marketing effort. The characteristics of potential purchasers of apparel from several Latin American countries are investigated. [ABSTRACT FROM PUBLISHER]

The opportunistic pathogen Pseudomonas aeruginosa infects the airways of people with cystic fibrosis (CF) and produces a virulence factor Cif that is associated with worse outcomes. Cif is an epoxide hydrolase that reduces cell-surface abundance of the cystic fibrosis transmembrane conductance regulator (CFTR) and sabotages pro-resolving signals. Its expression is regulated by a divergently transcribed TetR family transcriptional repressor. CifR represents the first reported epoxide-sensing bacterial transcriptional regulator, but neither its interaction with cognate operator sequences nor the mechanism of activation has been investigated. Using biochemical and structural approaches, we uncovered the molecular mechanisms controlling this complex virulence operon. We present here the first molecular structures of CifR alone and in complex with operator DNA, resolved in a single crystal lattice. Significant conformational changes between these two structures suggest how CifR regulates the expression of the virulence gene cif. Interactions between the N-terminal extension of CifR with the DNA minor groove of the operator play a significant role in the operator recognition of CifR. We also determined that cysteine residue Cys107 is critical for epoxide sensing and DNA release. These results offer new insights into the stereochemical regulation of an epoxide-based virulence circuit in a critically important clinical pathogen., (Published by Oxford University Press on behalf of Nucleic Acids Research 2024.)

Protein-protein interactions that involve recognition of short peptides are critical in cellular processes. Protein-peptide interaction surface areas are relatively small and shallow, and there are often overlapping specificities in families of peptide-binding domains. Therefore, dissecting selectivity determinants can be challenging. PDZ domains are a family of peptide-binding domains located in several intracellular signaling and trafficking pathways. These domains are also directly targeted by pathogens, and a hallmark of many oncogenic viral proteins is a PDZ-binding motif. However, amidst sequences that target PDZ domains, there is a wide spectrum in relative promiscuity. For example, the viral HPV16 E6 oncoprotein recognizes over double the number of PDZ domain-containing proteins as the cystic fibrosis transmembrane conductance regulator (CFTR) in the cell, despite similar PDZ targeting-sequences and identical motif residues. Here, we determine binding affinities for PDZ domains known to bind either HPV16 E6 alone or both CFTR and HPV16 E6, using peptides matching WT and hybrid sequences. We also use energy minimization to model PDZ-peptide complexes and use sequence analyses to investigate this difference. We find that while the majority of single mutations had marginal effects on overall affinity, the additive effect on the free energy of binding accurately describes the selectivity observed. Taken together, our results describe how complex and differing PDZ interactomes can be programmed in the cell., (© 2023 The Protein Society.)

Rationale: Specific cannabis products may differentially increase risk of initiating non-cannabis illicit drug use during adolescence., Objective: To determine whether ever- and poly-use of smoked, vaporized, edible, concentrate, or blunt cannabis products are associated with subsequent initiation of non-cannabis illicit drug use., Methods: High school students from Los Angeles completed in-classroom surveys. The analytic sample (N = 2163; 53.9% female; 43.5% Hispanic/Latino; baseline M age = 17.1 years) included students who reported never using illicit drugs at baseline (spring, 11th grade) and provided data at follow-up (fall and spring, 12th grade). Logistic regression models assessed associations between use of smoked, vaporized, edible, concentrate, and blunt cannabis at baseline (yes/no for each product) and any non-cannabis illicit drug use initiation-including cocaine, methamphetamine, psychedelics, ecstasy, heroin, prescription opioids, or benzodiazepines-at follow-up., Results: Among those who never used non-cannabis illicit drugs at baseline, ever cannabis use varied by cannabis product (smoked = 25.8%, edible = 17.5%, vaporized = 8.4%, concentrates = 3.9%, and blunts = 18.2%) and patterns of use (single product use = 8.2% and poly-product use = 21.8%). After adjustment for baseline covariates, odds of illicit drug use at follow-up were largest for baseline ever users of concentrates (aOR [95% CI] = 5.74[3.16-10.43]), followed by vaporized (aOR [95% CI] = 3.11 [2.41-4.01]), edibles (aOR [95% CI] = 3.43 [2.32-5.08]), blunts (aOR [95% CI] = 2.66[1.60-4.41]), and smoked (aOR [95% CI] = 2.57 [1.64-4.02]) cannabis. Ever use of a single product (aOR [95% CI] = 2.34 [1.26-4.34]) or 2 + products (aOR [95% CI] = 3.82 [2.73-5.35]) were also associated with greater odds of illicit drug initiation., Conclusions: For each of five different cannabis products, cannabis use was associated with greater odds of subsequent illicit drug use initiation, especially for cannabis concentrate and poly-product use., (© 2023. The Author(s).)

Intrinsically disordered proteins (IDPs) often coordinate transient interactions with multiple proteins to mediate complex signals within large protein networks. Among these, the IDP hub protein G3BP1 can form complexes with cytoplasmic phosphoprotein Caprin1 and ubiquitin peptidase USP10; the resulting control of USP10 activity contributes to a pathogenic virulence system that targets endocytic recycling of the ion channel CFTR. However, while the identities of protein interactors are known for many IDP hub proteins, the relationship between pairwise affinities and the extent of protein recruitment and activity is not well understood. Here, we describe in vitro analysis of these G3BP1 affinities and show tryptophan substitutions of specific G3BP1 residues reduce its affinity for both USP10 and Caprin1. We show that these same mutations reduce the stability of complexes between the full-length proteins, suggesting that copurification can serve as a surrogate measure of interaction strength. The crystal structure of G3BP1 TripleW (F15W/F33W/F124W) mutant reveals a clear reorientation of the side chain of W33, creating a steric clash with USP10 and Caprin1. Furthermore, an amino-acid scan of USP10 and Caprin1 peptides reveals similarities and differences in the ability to substitute residues in the core motifs as well as specific substitutions with the potential to create higher affinity peptides. Taken together, these data show that small changes in component binding affinities can have significant effects on the composition of cellular interaction hubs. These specific protein mutations can be harnessed to manipulate complex protein networks, informing future investigations into roles of these networks in cellular processes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: Despite the substantial influence these acute alcohol-related problems cause globally, past research has failed historically to capture the dynamic nature of drinking events, including how multiple factors (ie, individual, group, and environmental) interact to affect event-level intoxication. Fortunately, technology (eg, transdermal alcohol monitors) and smartphone surveys have provided researchers with new avenues to measure the complex nature of alcohol consumption. This paper presents the methods of a pilot study that sought to measure event-level alcohol consumption in a natural drinking group of college students. Methods: Ten groups of friends (N=49) were followed for 2 weeks with daily diary surveys, continuous activity trackers, hourly geographic ecological momentary assessments (EMAs) on 4 separate drinking occasions, and a transdermal alcohol monitor during one group-based social event. Results: On average, participants responded to > 75% of both daily diaries and EMAs and were compliant with activity trackers on 96% of monitoring days. Over 90% of the sample had usable transdermal data and after smoothing, peak transdermal alcohol contents ranged from 0.13 to 0.395 during the observation evening. Conclusion: The lessons learned during this pilot study can provide a building block for future work in this area, especially as data collection in alcohol research rapidly advances.

Introduction Little is known about the momentary patterns and predictors of substance use among young adults who experience homelessness. To enhance understanding of substance use patterns, smartphone-based ecological momentary assessment (EMA) was utilized to examine the real-time association between affect and substance use. Methods : 251 young adults (aged 18-27) with history of homelessness were recruited from supportive housing programs and drop-in facilities in Los Angeles. Exploratory factor analysis was used to examine the latent structure of positive and negative affective states and mixed-effects logistic regression models were completed separately for both the full remaining sample ( n  = 227) and a subsample of alcohol or cannabis users ( n  = 145) to evaluate whether positive or negative affect predicted lead, recent, or lagged substance use. Results : Greater positive affect within-person was associated with greater odds of alcohol or cannabis use within the past two hours, and participants who reported feeling more negative than their peers experienced greater odds of reporting use within the past 4 h and the following two hours. Conclusion : Results suggest that individuals experience a heightened positive mood compared to their own average mood, concurrently or immediately after engaging in alcohol or cannabis use. Heightened positive mood might be an anticipatory effect of drinking or cannabis use. Future research should consider a longer study period to capture multiple drinking or drug use events over a longer period and consider more environmental exposures that may influence the frequency or intensity of substance use.

This study investigated HIV risk among homeless and formerly homeless young adults by examining risky sex behaviors (e.g., condomless sex, exchange sex, and sex with multiple persons) using 90-day and daily recall methods. Data came from a sample of young adults (aged 18-27) with current (n = 101) or past (n = 109) homelessness experience in Los Angeles, California, recruited between 2017 and 2019. Baseline surveys queried demographics and sexual history. Daily retrospective surveys queried sexual events. Multiple logistic regressions were used to test the effects of demographic characteristics including homelessness history, relationship status, substance use, and sexual history on risky sex outcomes. In this sample, 26% reported never using a condom during anal or vaginal sex in the past 90 days, 5% reported testing positive for HIV, 82% had limited to no knowledge of preexposure prophylaxis, and 8% reported having had exchange sex during a 7-day measurement period, with those experiencing homelessness more likely to report. The study suggests supportive housing can reduce the occurrence of exchange sex but that HIV prevention services are still needed in homeless and housing programs to promote safe sexual practices., (© 2021. The Author(s).)

A recent workshop titled "Developing Models to Study Polymicrobial Infections," sponsored by the Dartmouth Cystic Fibrosis Center (DartCF), explored the development of new models to study the polymicrobial infections associated with the airways of persons with cystic fibrosis (CF). The workshop gathered 35+ investigators over two virtual sessions. Here, we present the findings of this workshop, summarize some of the challenges involved with developing such models, and suggest three frameworks to tackle this complex problem. The frameworks proposed here, we believe, could be generally useful in developing new model systems for other infectious diseases. Developing and validating new approaches to study the complex polymicrobial communities in the CF airway could open windows to new therapeutics to treat these recalcitrant infections, as well as uncovering organizing principles applicable to chronic polymicrobial infections more generally.

Background: Adults experiencing homelessness have a high burden of sleep disturbance, which may be reduced by accessing permanent supportive housing., Objectives: To assess sleep disturbances and their correlates, including demographics, activity level, health status, age-related health issues (eg, functionality and cognitive impairment), substance use, and homelessness history in a sample of permanent supportive housing (PSH) tenants., Research Design: Cross-sectional survey design., Subjects: A total of 237 formerly homeless adults between 45 and 80 years old., Measures: The Patient-Reported Outcomes Measurement Information System (PROMIS) Sleep Disturbance short form was used to measure sleep disturbance., Results: Twenty-eight percent of our sample had PROMIS scores indicative of a moderate or severe sleep disturbance. Functional impairment, pain, and mental health comorbidities were associated with increased sleep disturbance in multivariable linear regression analyses. The number of years a person experienced homelessness was inversely associated with sleep disturbance., Conclusions: This study supports the need to screen for sleep disturbances among PSH tenants. The findings suggest that supportive services in PSH may need to include integrated physical and behavioral health care, pain management, and interventions designed to address activities of daily livings to improve tenant sleep. They also suggest that improved sleep may help reduce PSH tenant pain, impairment, and mental health symptoms among PSH tenants., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: The estimated 3.5-million transition age youth (TAY) who experience homelessness in the United States annually are routinely exposed to inadequate sleep environments and other psychosocial risk factors for deficient sleep. Although staying in a shelter versus being unsheltered may facilitate sleep, research suggests that perceived safety wherever one sleeps may be just as important. In this study, which is the first known study to investigate sleep disturbances among TAY experiencing homelessness, we examine associations of sleep disturbances with sheltered status and perceived safety of usual sleep environment., Methods: We surveyed TAY (aged 18-25) experiencing homelessness in Los Angeles, CA about their sleep, psychosocial health, and living situations. Participants (n=103; 60% sheltered) self-reported sleep disturbances using the Patient-Reported Outcomes Measurement Information System Sleep Disturbance short form, while individual items assessed sheltered status and perceived safety where they usually slept. Regression analyses examined associations of sheltered status and perceived sleep environment safety with sleep disturbance, adjusting for age, sex, race, self-rated health, depression symptoms, serious mental illness, high-risk drinking, and severe food insecurity., Results: Twenty-six percent of participants reported moderate-severe sleep disturbances. Sleep disturbance was not associated with sheltered status, but was positively associated with feeling unsafe in one's sleep environment, depression symptoms, severe food insecurity, and decreased age., Conclusions: Our findings suggest that sleep disturbances among TAY experiencing homelessness are associated more closely with how safe one feels rather than one's sheltered status. This highlights the importance of providing safe places to live for sheltered and unsheltered TAY., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Epoxide hydrolases catalyze the conversion of epoxides to vicinal diols in a range of cellular processes such as signaling, detoxification, and virulence. These enzymes typically utilize a pair of tyrosine residues to orient the substrate epoxide ring in the active site and stabilize the hydrolysis intermediate. A new subclass of epoxide hydrolases that utilize a histidine in place of one of the tyrosines was established with the discovery of the CFTR Inhibitory Factor (Cif) from Pseudomonas aeruginosa . Although the presence of such Cif-like epoxide hydrolases was predicted in other opportunistic pathogens based on sequence analyses, only Cif and its homolog aCif from Acinetobacter nosocomialis have been characterized. Here we report the biochemical and structural characteristics of Cfl1 and Cfl2, two Cif-like epoxide hydrolases from Burkholderia cenocepacia . Cfl1 is able to hydrolyze xenobiotic as well as biological epoxides that might be encountered in the environment or during infection. In contrast, Cfl2 shows very low activity against a diverse set of epoxides. The crystal structures of the two proteins reveal quaternary structures that build on the well-known dimeric assembly of the α/β hydrolase domain, but broaden our understanding of the structural diversity encoded in novel oligomer interfaces. Analysis of the interfaces reveals both similarities and key differences in sequence conservation between the two assemblies, and between the canonical dimer and the novel oligomer interfaces of each assembly. Finally, we discuss the effects of these higher-order assemblies on the intra-monomer flexibility of Cfl1 and Cfl2 and their possible roles in regulating enzymatic activity., 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., (© 2021 The Authors.)

Importance: Alkaline free-base nicotine is bitter and a respiratory irritant. High-nicotine electronic cigarette (e-cigarette) products contain acid additives that change nicotine from a free-base to a protonated salt chemical form, which could improve the sensory experience of vaping, particularly among never smokers unaccustomed to inhaling free-base nicotine., Objective: To determine whether exposure to e-cigarettes with salt vs free-base nicotine formulations improves the appeal and sensory experience of vaping e-cigarettes and whether nicotine formulation effects differ by e-cigarette flavor and ever combustible cigarette smoking status., Design, Setting, and Participants: Single-visit double-blind within-participant randomized clinical trial was conducted in an academic medical center outpatient clinical research facility in Southern California. Participants were 119 individuals with past 30-day e-cigarette or combustible cigarette use aged 21 years or older recruited from November 2019 to March 2020., Interventions: Participants self-administered standardized puffs of each 10 differently flavored e-cigarette solutions using a pod-style device. Each flavor was administered in salt (benzoic acid added) and free-base (no benzoic acid) nicotine formulations with commensurate nicotine concentrations (mean, 23.6 mg/mL). The 20 solutions were administered in randomly assigned sequences. Immediately after puffing each solution, participants rated appeal and sensory attributes., Main Outcomes and Measures: Self-reported appeal (mean of like, dislike [reverse-scored], and willingness to use again ratings) and 4 sensory attributes (sweetness, smoothness, bitterness, and harshness; analyzed individually) on visual analog scales with not at all and extremely anchors (range, 0-100)., Results: Of the 119 participants; 39 (32.8%) were female. The mean (SD) age was 42.1 (14.4) years; 105 (88.2%) were ever combustible cigarette smokers, and 66 (55.5%) were current e-cigarette users. Salt vs free-base nicotine formulations produced higher ratings of appeal (salt vs free-base mean difference effect estimate: b = 12.0; 95% CI, 9.9-14.1; P < .001), sweetness (b = 9.3; 95% CI, 7.1-11.4; P < .001), and smoothness (b = 17.4; 95% CI, 15.2-19.6; P < .001) and lower ratings of bitterness (b = -13.3; 95% CI, -15.4 to -11.2; P < .001) and harshness (b = -21.0; 95% CI, -23.2 to -18.7; P < .001). Nicotine formulation effects largely generalized across different flavors and the smoothness-enhancing and harshness-reducing effects of nicotine salt were stronger in never vs ever cigarette smokers., Conclusions and Relevance: In this randomized clinical trial of adult current nicotine or tobacco product users, controlled exposure to e-cigarette puffs with salt vs free-base nicotine formulations appeared to increase product appeal and improve the sensory experience of vaping, particularly among never smokers. Regulatory policies limiting acid additives in e-cigarettes might reduce the appeal of high-nicotine e-cigarettes among populations deterred from vaping e-cigarettes that emit harsh aerosol., Trial Registration: ClinicalTrials.gov Identifier: NCT04399031.

Adults with serious mental illness engage in limited physical activity, which contributes to significant health disparities. This study explored the use of both ecological momentary assessments (EMAs) and activity trackers in adults with serious mental illness to examine the bidirectional relationship between activity and affect with multilevel modeling. Affective states were assessed up to seven times per day using EMA across 4 days. The participants (n = 20) were equipped with a waist-worn accelerometer to measure moderate to vigorous physical activity. The participants had a mean EMA compliance rate of 88.3%, and over 90% of completed EMAs were matched with 30-min windows of accelerometer wear. The participants who reported more positive affect than others had a higher probability of engaging in moderate to vigorous physical activity. Engaging in more moderate to vigorous physical activity than one's usual was associated with more negative affect. This study begins to address the effect of momentary mood on physical activity in a population of adults that is typically difficult to reach.

Globular PDZ domains typically serve as protein-protein interaction modules that regulate a wide variety of cellular functions via recognition of short linear motifs (SLiMs). Often, PDZ mediated-interactions are essential components of macromolecular complexes, and disruption affects the entire scaffold. Due to their roles as linchpins in trafficking and signaling pathways, PDZ domains are attractive targets: both for controlling viral pathogens, which bind PDZ domains and hijack cellular machinery, as well as for developing therapies to combat human disease. However, successful therapeutic interventions that avoid off-target effects are a challenge, because each PDZ domain interacts with a number of cellular targets, and specific binding preferences can be difficult to decipher. Over twenty-five years of research has produced a wealth of data on the stereochemical preferences of individual PDZ proteins and their binding partners. Currently the field lacks a central repository for this information. Here, we provide this important resource and provide a manually curated, comprehensive list of the 271 human PDZ domains. We use individual domain, as well as recent genomic and proteomic, data in order to gain a holistic view of PDZ domains and interaction networks, arguing this knowledge is critical to optimize targeting selectivity and to benefit human health., 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., (© 2020 The Authors.)

The CFTR-associated ligand PDZ domain (CALP) binds to the cystic fibrosis transmembrane conductance regulator (CFTR) and mediates lysosomal degradation of mature CFTR. Inhibition of this interaction has been explored as a therapeutic avenue for cystic fibrosis. Previously, we reported the ensemble-based computational design of a novel peptide inhibitor of CALP, which resulted in the most binding-efficient inhibitor to date. This inhibitor, kCAL01, was designed using osprey and evinced significant biological activity in in vitro cell-based assays. Here, we report a crystal structure of kCAL01 bound to CALP and compare structural features against iCAL36, a previously developed inhibitor of CALP. We compute side-chain energy landscapes for each structure to not only enable approximation of binding thermodynamics but also reveal ensemble features that contribute to the comparatively efficient binding of kCAL01. Finally, we compare the previously reported design ensemble for kCAL01 vs the new crystal structure and show that, despite small differences between the design model and crystal structure, significant biophysical features that enhance inhibitor binding are captured in the design ensemble. This suggests not only that ensemble-based design captured thermodynamically significant features observed in vitro , but also that a design eschewing ensembles would miss the kCAL01 sequence entirely.

Objectives: To examine the longitudinal change in insomnia as adults transition from homelessness to permanent supportive housing (PSH) and whether additional factors may moderate this relationship., Methods: Standardized interviews were conducted with 331 homeless participants in Los Angeles prior to moving into PSH. Outcomes were measured 3, 6, and 12 months after move-in. Insomnia was assessed using the Sleep Condition Indicator, which is a 2-item validated short-form inventory that is intended to be used in clinical settings as a brief screening instrument for insomnia. Mixed-effects models were used to examine insomnia across all 4 measurement points and to test for interactions between time and covariates., Results: Participants were on average approximately 55 years old and had spent an average of 5.6 years homeless in their lifetime, with approximately 70% identifying as male. Sixty-two percent of the sample screened positive for insomnia disorder at baseline. There was a significantly reduced likelihood of insomnia at each measurement period compared to baseline, but no differences were found between 3, 6, and 12 months. Mental health symptoms, physical health comorbidities, tobacco consumption, and female gender were associated with an increased likelihood of insomnia., Conclusion: Findings indicate a significant decrease in insomnia after moving into PSH, regardless of time spent homeless., (Copyright © 2019 National Sleep Foundation. Published by Elsevier Inc. All rights reserved.)

Lead identification and optimization are essential steps in the development of a new drug. It requires cost-effective, selective and sensitive chemical tools. Here, we report a novel method using nanobodies that allows the efficient screening for potent ligands. The method is illustrated with the cystic fibrosis transmembrane conductance regulator inhibitory factor (Cif), a virulence factor secreted by the opportunistic pathogen Pseudomonas aeruginosa. 18 nanobodies selective to Cif were isolated by bio-panning from nanobody-phage library constructed from immunized llama. 8 out of 18 nanobodies were identified as potent inhibitors of Cif enzymatic activity with IC 50 s in the range of 0.3-6.4 μM. A nanobody VHH219 showed high affinity (K D  = 0.08 nM) to Cif and the highest inhibitory potency, IC 50  = 0.3 μM. A displacement sandwich ELISA (dsELISA) with VHH219 was then developed for classification of synthetic small molecule inhibitors according their inhibitory potency. The developed assay allowed identification of new inhibitor with highest potency reported so far (0.16 ± 0.02 μM). The results from dsELISA assay correlates strongly with a conventional fluorogenic assay (R = 0.9998) in predicting the inhibitory potency of the tested compounds. However, the novel dsELISA is an order of magnitude more sensitive and allows the identification and ranking of potent inhibitors missed by the classic fluorogenic assay method. These data were supported with Octet biolayer interferometry measurements. The novel method described herein relies solely on the binding properties of the specific neutralizing nanobody, and thus is applicable to any pharmacological target for which such a nanobody can be found, independent of any requirement for catalytic activity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This paper employs control-theoretic tools to provide guidelines for in-situ interventions aimed at reducing high-risk alcohol consumption at drinking events. A dynamical directed network model of a drinking event with external intervention, suitable for mathematical analysis and parameter estimation using field data is proposed, with insights from pharmacokinetics and psychology. Later, a characterization of a bound on blood alcohol content (BAC) trajectories is obtained via Lyapunov stability analysis, and structural controllability guarantees are obtained via a graph-theoretic method. We use the degree of controllability, given to be the trace of the system's controllability Gramian, as a metric to compare the viability of network nodes for intervention based on theoretic and heuristic centrality measures. Results of numerical examples of bars and parties, informed by field data, and the stability and controllability results, suggest that intervening in the environment in wet bars, while targeting influential individuals with high alcohol consumption motivations in private parties efficiently yield lower peak BAC levels in individuals at the drinking events.

Background: Young adults who experience homelessness are exposed to environments that contribute to risk behavior. However, few studies have examined how access to housing may affect the health risk behaviors of young adults experiencing homelessness., Objective: This paper describes the Log My Life study that uses an innovative, mixed-methods approach based on geographically explicit ecological momentary assessment (EMA) through cell phone technology to understand the risk environment of young adults who have either enrolled in housing programs or are currently homeless., Methods: For the quantitative arm, study participants age 18-27 respond to momentary surveys via a smartphone app that collects geospatial information repeatedly during a 1-week period. Both EMAs (up to 8 per day) and daily diaries are prompted to explore within-day and daily variations in emotional affect, context, and health risk behavior, while also capturing infrequent risk behaviors such as sex in exchange for goods or services. For the qualitative arm, a purposive subsample of participants who indicated engaging in risky behaviors are asked to complete an in-depth qualitative interview using an interactive, personalized geospatial map rendering of EMA responses., Results: Recruitment began in June of 2017. To date, 170 participants enrolled in the study. Compliance with EMA and daily diary surveys was generally high. In-depth qualitative follow-ups have been conducted with 15 participants. We expect to recruit 50 additional participants and complete analyses by September of 2019., Conclusions: Mixing the quantitative and qualitative arms in this study will provide a more complete understanding of differences in risk environments between homeless and housed young adults. Furthermore, this approach can improve recall bias and enhance ecological validity., International Registered Report Identifier (irrid): DERR1-10.2196/12112., (©Benjamin F Henwood, Brian Redline, Eldin Dzubur, Danielle R Madden, Harmony Rhoades, Genevieve F Dunton, Eric Rice, Sara Semborski, Qu Tang, Stephen S Intille. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 10.01.2019.)

Introduction: Much of the past research on the excessive consumption of alcohol by college students has focused on the interplay of individual factors and typical drinking patterns, but this is not adequate to understand behavior as it occurs. The need to understand drinking at the event-level is critical in order to develop event-level prevention. To this end, this study examined a conceptual model of college students' drinking events in order to determine the potential mediating effect of drinking motives and protective behavioral strategies (PBS) in the relationship between alcohol expectancies and event-level alcohol use and consequences., Methods: An existing data set containing information about 2279 college student drinking events were analyzed for this study. Students completed surveys during the administration of a commercial online alcohol course during 2010 and 2011. A theoretical model was analyzed with structural equation modeling., Results: Both typical use of PBS and drinking motives mediated the relationship between expectancies and event-level alcohol use and problems. Positive expectancies were associated with greater positive motives, greater motives were associated with less use of PBS, and less PBS use was then, in turn, associated with higher event-level intoxication. Lastly, higher intoxication was associated with more serious consequences during the event., Conclusion: This is the first study to simultaneously explore the relationship between these factors and event-level drinking. There is a great need to continue to further explore the dynamic nature of drinking at the event-level to illuminate potential leverage points amendable to change., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: The opioid epidemic remains a serious issue in the United States and presents additional challenges for women of childbearing age. An increasingly common complication of opioid use is neonatal abstinence syndrome (NAS), or infant withdrawal from in utero exposure to opioids., Purpose: The objective of our qualitative study was to identify service needs and barriers to care in the NAS epidemic in Ohio, which has among the highest rates of opioid use and NAS in the nation., Methods: Drawing on interviews with 18 healthcare providers, we investigated the challenges, opportunities, and service gaps in treating NAS. Open-ended questions covered opioid misuse and drug treatment, provision of and barriers to healthcare, and suggestions to improve prevention and programming. Content analysis identified major themes., Findings: Providers were primarily women (67%) and included individuals working in healthcare administrative positions, hospital settings, clinics, and social support positions for pregnant women or new mothers. Our results suggest that rather than an acute diagnosis, NAS is better conceptualized as a "cascade of care" including (1) prevention, (2) prenatal care, including drug treatment, (3) labor and delivery, and (4) aftercare. Providers identified challenges and opportunities at each stage of the cascade that could influence NAS outcomes., Implications for Practice: Our results suggest that greater resources, coordination, and cross-disciplinary education are urgently needed across the cascade of care to effectively address NAS., Implications for Research: Framing NAS as a cascade of care allows researchers to identify points along a cascade where mothers and infants require enhanced care and access to social and health services.

Protein-protein interactions have become attractive targets for both experimental and therapeutic interventions. The PSD-95/Dlg1/ZO-1 (PDZ) domain is found in a large family of eukaryotic scaffold proteins that plays important roles in intracellular trafficking and localization of many target proteins. Here, we seek inhibitors of the PDZ protein that facilitates post-endocytic degradation of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR): the CFTR-associated ligand (CAL). We develop and validate biochemical screens and identify methyl-3,4-dephostatin (MD) and its analog ethyl-3,4-dephostatin (ED) as CAL PDZ inhibitors. Depending on conditions, MD can bind either covalently or non-covalently. Crystallographic and NMR data confirm that MD attacks a pocket at a site distinct from the canonical peptide-binding groove, and suggests an allosteric connection between target residue Cys 319 and the conserved Leu 291 in the GLGI motif. MD and ED thus appear to represent the first examples of small-molecule allosteric regulation of PDZ:peptide affinity. Their mechanism of action may exploit the known conformational plasticity of the PDZ domains and suggests that allosteric modulation may represent a strategy for targeting of this family of protein-protein binding modules., (© 2018 The Author(s).)

Background: Justice involved youth exposed to multiple forms of victimization (i.e., poly-victimization) may be at risk for long term substance use problems and difficulty in self-regulation, placing them at higher risk of long-term problematic behaviors. This study empirically identifies victimization classifications in a sample of justice involved youth and how long-term binge drinking is related to victimization experiences. We further sought to understand how self-regulatory abilities such as impulse control and emotion regulation effect emergent profiles and binge drinking trajectories., Methods: Based on a sample of 1354 justice involved youth from 15 to 25 years old, classes of victimization were extracted. Emergent classes were examined in relationship to their binge drinking trajectories using latent growth models. Finally, self-regulation was examined as a predictor of binge drinking trajectories across emergent classes., Results: The analyses indicated three classes of victimization: poly-victimized, indirectly victimized, and lowly victimized. Latent growth models revealed that the poly-victimized class had significantly steeper growth in binge drinking as compared to the indirect and low victimized patterns. Impulse and emotional regulation both significantly decelerated binge drinking only for the indirect victimization group., Conclusions: Findings highlight the need to focus on poly-victimization in understanding binge drinking trajectories as well as the role impulse control and emotional regulation play among justice involved youth. Findings are discussed through the lens of adolescent development, coping strategies, and early traumatic experiences., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The opportunistic pathogen Pseudomonas aeruginosa colonizes the lungs of susceptible individuals by deploying virulence factors targeting host defenses. The secreted factor Cif (cystic fibrosis transmembrane conductance regulator inhibitory factor) dysregulates the endocytic recycling of CFTR and thus reduces CFTR abundance in host epithelial membranes. We have postulated that the decrease in ion secretion mediated by Cif would slow mucociliary transport and decrease bacterial clearance from the lungs. To test this hypothesis, we explored the effects of Cif in cultured epithelia and in the lungs of mice. We developed a strategy to interpret the "hurricane-like" motions observed in reconstituted cultures and identified a Cif-mediated decrease in the velocity of mucus transport in vitro. Presence of Cif also increased the number of bacteria recovered at two time points in an acute mouse model of pneumonia caused by P. aeruginosa. Furthermore, recent work has demonstrated an inverse correlation between the airway concentrations of Cif and 15-epi-lipoxin A 4, a proresolving lipid mediator important in host defense and the resolution of pathogen-initiated inflammation. Here, we observe elevated levels of 15-epi-lipoxin A 4 in the lungs of mice infected with a strain of P. aeruginosa that expresses only an inactive form of cif compared with those mice infected with wild-type P. aeruginosa. Together these data support the inclusion of Cif on the list of virulence factors that assist P. aeruginosa in colonizing and damaging the airways of compromised patients. Furthermore, this study establishes techniques that enable our groups to explore the underlying mechanisms of Cif effects during respiratory infection.

Many of the problems associated with alcohol occur after a single drinking event (e.g. drink driving, assault). These acute alcohol problems have a huge global impact and account for a large percentage of unintentional and intentional injuries in the world. Nonetheless, alcohol research and preventive interventions rarely focus on drinking at the event-level since drinking events are complex, dynamic, and methodologically challenging to observe. This exploratory study provides an example of how event-level data may be collected, analyzed, and interpreted. The drinking behavior of twenty undergraduate students enrolled at a large Midwestern public university was observed during a single bar crawl event that is organized by students annually. Alcohol use was monitored with transdermal alcohol devices coupled with ecological momentary assessments and geospatial data. "Small N, Big Data" studies have the potential to advance health behavior theory and to guide real-time interventions. However, such studies generate large amounts of within subject data that can be challenging to analyze and present. This study examined how to visually display event-level data and also explored the relationship between some basic indicators and alcohol consumption.

PDZ domains play crucial roles in cell signaling processes and are therefore attractive targets for the development of therapeutic inhibitors. In many cases, C-terminal peptides are the physiological binding partners of PDZ domains. To identify both native ligands and potential inhibitors we have screened arrays synthesized by the process of inverted peptides (PIPE), a variant of SPOT synthesis that generates peptides with free C-termini. Here, we present the development of a new functionalized cellulose membrane as solid support along with the optimized PIPE PLUS technology. Improved resolution and accuracy of the synthesis were shown with peptide arrays containing both natural and non-natural amino acids. These new screening possibilities will advance the development of active, selective and metabolically stable PDZ interactors., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Several members of the SLC26A family of anion transporters associate with CFTR, forming complexes in which CFTR and SLC26A functions are reciprocally regulated. These associations are thought to be facilitated by PDZ scaffolding interactions. CFTR has been shown to be positively regulated by NHERF-1, and negatively regulated by CAL in airway epithelia. However, it is unclear which PDZ-domain protein(s) interact with SLC26A9, a SLC26A family member found in airway epithelia. We have previously shown that primary, human bronchial epithelia (HBE) from non-CF donors exhibit constitutive anion secretion attributable to SLC26A9. However, constitutive anion secretion is absent in HBE from CF donors. We examined whether changes in SLC26A9 constitutive activity could be attributed to a loss of CFTR trafficking, and what role PDZ interactions played. HEK293 coexpressing SLC26A9 with the trafficking mutant F508del CFTR exhibited a significant reduction in constitutive current compared with cells coexpressing SLC26A9 and wt CFTR. We found that SLC26A9 exhibits complex glycosylation when coexpressed with F508del CFTR, but its expression at the plasma membrane is decreased. SLC26A9 interacted with both NHERF-1 and CAL, and its interaction with both significantly increased with coexpression of wt CFTR. However, coexpression with F508del CFTR only increased SLC26A9's interaction with CAL. Mutation of SLC26A9's PDZ motif decreased this association with CAL, and restored its constitutive activity. Correcting aberrant F508del CFTR trafficking in CF HBE with corrector VX-809 also restored SLC26A9 activity. We conclude that when SLC26A9 is coexpressed with F508del CFTR, its trafficking defect leads to a PDZ motif-sensitive intracellular retention of SLC26A9., (Copyright © 2017 the American Physiological Society.)

Pseudomonas aeruginosa secretes an epoxide hydrolase with catalytic activity that triggers degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and perturbs other host defense networks. Targets of this CFTR inhibitory factor (Cif) are largely unknown, but include an epoxy-fatty acid. In this class of signaling molecules, chirality can be an important determinant of physiological output and potency. Here we explore the active-site chemistry of this two-step α/β-hydrolase and its implications for an emerging class of virulence enzymes. In combination with hydrolysis data, crystal structures of 15 trapped hydroxyalkyl-enzyme intermediates reveal the stereochemical basis of Cif's substrate specificity, as well as its regioisomeric and enantiomeric preferences. The structures also reveal distinct sets of conformational changes that enable the active site to expand dramatically in two directions, accommodating a surprising array of potential physiological epoxide targets. These new substrates may contribute to Cif's diverse effects in vivo, and thus to the success of P. aeruginosa and other pathogens during infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Purpose: To determine whether the cif gene is present in pathogenic Pseudomonas aeruginosa isolates from patients with bacterial keratitis at Aravind Eye Hospital, a referral eye care center in southern India, and from corresponding environmental isolates., Methods: Polymerase chain reaction amplification was performed on strains of P. aeruginosa isolated from ocular infections and environmental soil samples were collected from the area surrounding Aravind Eye Hospital. DNA sequencing of 16S ribosomal DNA amplicons was performed to verify strain identity., Results: We determined that 45 of 48 patient isolates carry a genomic copy of cif. Analysis of a catalog of environmental strains previously isolated from the surrounding area revealed that only 4 of 10 P. aeruginosa strains and 1 of 14 strains of related species carry the cif gene., Conclusions: This is the first study to show that P. aeruginosa strains with ocular pathogenicity carry the cif gene and that the presence of this gene may be enriched over its prevalence in the environment. Taken together, these results suggest a potential role for Cif in acute bacterial keratitis.

Recurrent Pseudomonas aeruginosa infections coupled with robust, damaging neutrophilic inflammation characterize the chronic lung disease cystic fibrosis (CF). The proresolving lipid mediator, 15-epi lipoxin A 4 (15-epi LXA 4 ), plays a critical role in limiting neutrophil activation and tissue inflammation, thus promoting the return to tissue homeostasis. Here, we show that a secreted P. aeruginosa epoxide hydrolase, cystic fibrosis transmembrane conductance regulator inhibitory factor (Cif), can disrupt 15-epi LXA 4 transcellular biosynthesis and function. In the airway, 15-epi LXA 4 production is stimulated by the epithelial-derived eicosanoid 14,15-epoxyeicosatrienoic acid (14,15-EET). Cif sabotages the production of 15-epi LXA 4 by rapidly hydrolyzing 14,15-EET into its cognate diol, eliminating a proresolving signal that potently suppresses IL-8-driven neutrophil transepithelial migration in vitro. Retrospective analyses of samples from patients with CF supported the translational relevance of these preclinical findings. Elevated levels of Cif in bronchoalveolar lavage fluid were correlated with lower levels of 15-epi LXA 4 , increased IL-8 concentrations, and impaired lung function. Together, these findings provide structural, biochemical, and immunological evidence that the bacterial epoxide hydrolase Cif disrupts resolution pathways during bacterial lung infections. The data also suggest that Cif contributes to sustained pulmonary inflammation and associated loss of lung function in patients with CF., Competing Interests: C.D.B., C.M., B.D.H., and D.R.M. are coinventors of patent-pending Cif inhibitor compounds. B.D.L. is an inventor on patents (resolvins) licensed by Brigham and Women’s Hospital to Resolvyx Pharmaceuticals, a company that seeks to develop Resolvin therapeutics for inflammatory diseases. B.D.L. also owns equity in the company. B.D.L.’s interests were reviewed and are managed by the Brigham and Women’s Hospital and Partners HealthCare in accordance with their conflict of interest policies.

A method for development of murine Fab fragments towards extracellular domains of a surface receptor is presented. The GluA4 ionotropic glutamate receptor is used as a model system. Recombinant GluA4 ectodomain comprising both the N-terminal domain (NTD) and the ligand-binding domain (LBD) in one molecule was used for immunization. A Fab-phage library was constructed and a parallel panning approach enabled selection of murine Fab fragments towards either intact ectodomain or the isolated LBD of the GluA4 receptor. One LBD-Fab (FabL9) showed exclusive selectivity for the GluA4 LBD, over a panel of LBDs from GluA2, GluK1, GluK2 and GluD2. Soluble FabL9 was produced in amounts suitable for characterization. Competitive ELISA and rat-brain immunoprecipitation experiments confirmed that the FabL9 epitope is conserved in the LBD and in the intact native receptor. By an alignment of GluA2 and GluA4, the likely binding epitope for FabL9 was predicted. This study demonstrates a simple approach for development of antibody fragments towards specific sub-domains of a large ligand-gated ion channel, and this method could be utilized for all multi-domain surface receptors where antibody domain-selectivity may be desirable. Furthermore, we present for the first time a GluA4 subtype-specific murine Fab fragment targeting the LBD of the receptor., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The virulence factor cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is secreted by Pseudomonas aeruginosa and is the founding member of a distinct class of epoxide hydrolases (EHs) that triggers the catalysis-dependent degradation of the CFTR. We describe here the development of a series of potent and selective Cif inhibitors by structure-based drug design. Initial screening revealed 1a (KB2115), a thyroid hormone analog, as a lead compound with low micromolar potency. Structural requirements for potency were systematically probed, and interactions between Cif and 1a were characterized by X-ray crystallography. On the basis of these data, new compounds were designed to yield additional hydrogen bonding with residues of the Cif active site. From this effort, three compounds were identified that are 10-fold more potent toward Cif than our first-generation inhibitors and have no detectable thyroid hormone-like activity. These inhibitors will be useful tools to study the pathological role of Cif and have the potential for clinical application.

The CFTR inhibitory factor (Cif) is an epoxide hydrolase (EH) virulence factor secreted by the bacterium Pseudomonas aeruginosa. Sequence alignments reveal a pattern of Cif-like substitutions that proved to be characteristic of a new subfamily of bacterial EHs. At the same time, crystallographic and mutagenetic data suggest that EH activity is required for virulence and that Cif's active site remains generally compatible with a canonical two-step EH mechanism. A hallmark of this mechanism is the formation of a covalent hydroxyalkyl-enzyme intermediate by nucleophilic attack. In several well-studied EHs, this intermediate has been captured at near stoichiometric levels, presumably reflecting rate-limiting hydrolysis. Here we show by mass spectrometry that only minimal levels of the expected intermediate can be trapped with WT Cif. In contrast, substantial amounts of intermediate are recovered from an active-site mutant (Cif-E153Q) that selectively targets the second, hydrolytic release step. Utilizing Cif-E153Q and a previously reported nucleophile mutant (Cif-D129S), we then captured Cif in the substrate-bound, hydroxyalkyl-intermediate, and product-bound states for 1,2-epoxyhexane, yielding the first crystallographic snapshots of an EH at these key stages along the reaction coordinate. Taken together, our data illuminate the proposed two-step hydrolytic mechanism of a new class of bacterial virulence factor. They also suggest that the failure of WT Cif to accumulate a covalent hydroxyalkyl-enzyme intermediate reflects an active-site chemistry in which hydrolysis is no longer the rate-limiting step, a noncanonical kinetic regime that may explain similar observations with a number of other EHs.

Opportunistic pathogens exploit diverse strategies to sabotage host defenses. Pseudomonas aeruginosa secretes the CFTR inhibitory factor Cif and thus triggers loss of CFTR, an ion channel required for airway mucociliary defense. However, the mechanism of action of Cif has remained unclear. It catalyzes epoxide hydrolysis, but there is no known role for natural epoxides in CFTR regulation. It was demonstrated that the hydrolase activity of Cif is strictly required for its effects on CFTR. A small-molecule inhibitor that protects this key component of the mucociliary defense system was also uncovered. These results provide a basis for targeting the distinctive virulence chemistry of Cif and suggest an unanticipated role of physiological epoxides in intracellular protein trafficking., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

It has been suggested that the alkaline form of cytochrome c (cyt c) regulates function of this protein as an electron carrier in oxidative phosphorylation and as a peroxidase that reacts with cardiolipin (CL) during apoptosis. In this form, Met80, the native ligand to the heme iron, is replaced by a Lys. While it has become clear that the structure of cyt c changes, the extent and sequence of conformational rearrangements associated with this ligand replacement remain a subject of debate. Herein we report a high-resolution crystal structure of a Lys73-ligated cyt c conformation that reveals intricate change in the heme environment upon this switch in the heme iron ligation. The structure is surprisingly compact, and the heme coordination loop refolds into a β-hairpin with a turn formed by the highly conserved residues Pro76 and Gly77. Repositioning of residue 78 modifies the intraprotein hydrogen-bonding network and, together with adjustments of residues 52 and 74, increases the volume of the heme pocket to allow for insertion of one of the CL acyl moieties next to Asn52. Derivatization of Cys78 with maleimide creates a solution mimic of the Lys-ligated cyt c that has enhanced peroxidase activity, adding support for a role of the Lys-ligated cyt c in the apoptotic mechanism. Experiments with the heme peptide microperoxidase-8 and engineered model proteins provide a thermodynamic rationale for the switch to Lys ligation upon perturbations in the protein scaffold.

A general strategy was developed for the intracellular delivery of linear peptidyl ligands through fusion to a cell-penetrating peptide and cyclization of the fusion peptides via a disulfide bond. The resulting cyclic peptides are cell permeable and have improved proteolytic stability. Once inside the cell, the disulfide bond is reduced to produce linear biologically active peptides. This strategy was applied to generate a cell-permeable peptide substrate for real-time detection of intracellular caspase activities during apoptosis and an inhibitor for the CFTR-associated ligand (CAL) PDZ domain as a potential treatment for cystic fibrosis., (© 2015 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

PDZ domains are protein-protein interaction modules that coordinate multiple signaling and trafficking pathways in the cell and that include active therapeutic targets for diseases such as cancer, cystic fibrosis, and addiction. Our previous work characterized a PDZ interaction that restricts the apical membrane half-life of the cystic fibrosis transmembrane conductance regulator (CFTR). Using iterative cycles of peptide-array and solution-binding analysis, we targeted the PDZ domain of the CFTR-Associated Ligand (CAL), and showed that an engineered peptide inhibitor rescues cell-surface expression of the most common CFTR disease mutation ΔF508. Here, we present a series of scaffolds containing chemically modifiable side chains at all non-motif positions along the CAL PDZ domain binding cleft. Concordant equilibrium dissociation constants were determined in parallel by fluorescence polarization, isothermal titration calorimetry, and surface plasmon resonance techniques, confirming robust affinity for each scaffold and revealing an enthalpically driven mode of inhibitor binding. Structural studies demonstrate a conserved binding mode for each peptide, opening the possibility of combinatorial modification. Finally, we diversified one of our peptide scaffolds with halogenated substituents that yielded modest increases in binding affinity. Overall, this work validates our approach and provides a stereochemical foundation for further CAL inhibitor design and screening.

The glucocorticoid dexamethasone increases cystic fibrosis transmembrane conductance regulator (CFTR) abundance in human airway epithelial cells by a mechanism that requires serum- and glucocorticoid-induced protein kinase 1 (SGK1) activity. The goal of this study was to determine whether SGK1 increases CFTR abundance by phosphorylating Shank2E, a PDZ domain protein that contains two SGK1 phosphorylation consensus sites. We found that SGK1 phosphorylates Shank2E as well as a peptide containing the first SGK1 consensus motif of Shank2E. The dexamethasone-induced increase in CFTR abundance was diminished by overexpression of a dominant-negative Shank2E in which the SGK1 phosphorylation sites had been mutated. siRNA-mediated reduction of Shank2E also reduced the dexamethasone-induced increase in CFTR abundance. Taken together, these data demonstrate that the glucocorticoid-induced increase in CFTR abundance requires phosphorylation of Shank2E at an SGK1 consensus site., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) is blocked by the CFTR inhibitory factor (Cif). Originally discovered in Pseudomonas aeruginosa, Cif is a secreted epoxide hydrolase that is transcriptionally regulated by CifR, an epoxide-sensitive repressor. In this report, we investigate a homologous protein found in strains of the emerging nosocomial pathogens Acinetobacter nosocomialis and Acinetobacter baumannii ("aCif"). Like Cif, aCif is an epoxide hydrolase that carries an N-terminal secretion signal and can be purified from culture supernatants. When applied directly to polarized airway epithelial cells, mature aCif triggers a reduction in CFTR abundance at the apical membrane. Biochemical and crystallographic studies reveal a dimeric assembly with a stereochemically conserved active site, confirming our motif-based identification of candidate Cif-like pathogenic EH sequences. Furthermore, cif expression is transcriptionally repressed by a CifR homolog ("aCifR") and is induced in the presence of epoxides. Overall, this Acinetobacter protein recapitulates the essential attributes of the Pseudomonas Cif system and thus may facilitate airway colonization in nosocomial lung infections.