Repurposing Acitretin as an Antipseudomonal Agent Targeting the Pseudomonas aeruginosa Iron-Regulated Heme Oxygenase.

Iron is an essential micronutrient for the survival and virulence of the bacterial pathogen Pseudomonas aeruginosa. To overcome iron withholding and successfully colonize a host, P. aeruginosa uses a variety of mechanisms to acquire iron, including the secretion of high-affinity iron chelators (siderophores) or the uptake and utilization of heme. P. aeruginosa heme oxygenase (HemO) plays pivotal roles in heme sensing, uptake, and utilization and has emerged as a therapeutic target for the development of antipseudomonal agents. Using a high-throughput fluorescence quenching assay combined with minimum inhibitory concentration measurements, we screened the Selleck Bioactive collection of 2100 compounds and identified acitretin, a Food and Drug Administration-approved oral retinoid, as a potent and selective inhibitor of HemO. Acitretin binds to HemO with a KD value of 0.10 ± 0.02 µM and inhibits the growth of P. aeruginosa PAO1 with an IC50 of 70 ± 18 µg/mL. In addition, acitretin showed good selectivity for HemO, which uniquely generates BVIXß/δ, over human heme oxygenase (hHO1) and other BVIXα-producing homologues such as the heme oxygenases from Neisseria meningitidis (nmHO) and Acinetobacter baumannii (abHO). The binding of acitretin within the HemO active site was confirmed by 1H-15N heteronuclear single-quantum coherence nuclear magnetic resonance, and molecular modeling provided further insight into potential interactions of acitretin with residues specific for orienting heme in the ß/δ selective HemO. Moreover, at 20 µM, acitretin inhibited the enzymatic activity of HemO in P. aeruginosa cells by >60% and effectively blocked the ability of P. aeruginosa to sense and acquire heme as demonstrated in the ß-galactosidase transcriptional reporter assay.

A longitudinal study of drinking and depression as predictors of insomnia in alcohol-dependent individuals.

Insomnia and depressive symptoms are common symptoms among alcohol-dependent (AD) patients. AD individuals (N = 364) were assessed during 2004-2009 in the Midwestern United States at baseline and 6-month intervals with the Sleep Problems Questionnaire, Time-Line Follow-Back interview, and the depression subscale of the Brief Symptom Inventory. Hierarchical Linear Modeling was used to analyze the data in this longitudinal study. When modeled separately, both quantity of drinking (p < .01) and depression (p < .001) predicted insomnia severity, controlling for time, age, and gender. Drinking also predicted depressive symptoms (p < .001), and its effect on insomnia was mediated by depression severity (p < .001).

Multiple dimensions of spirituality in recovery: a lagged mediational analysis of Alcoholics Anonymous' principal theoretical mechanism of behavior change.

Alcoholics Anonymous (AA) states that recovery is possible through spiritual experiences and spiritual awakenings. Research examining spirituality as a mediator of AA's effect on drinking has been mixed. It is unknown whether such findings are due to variations in the operationalization of key constructs, such as AA and spirituality. To answer these questions, the authors used a longitudinal model to test 2 dimensions of AA as focal predictors and 6 dimensions of spirituality as possible mediators of AA's association with drinking. Data from the first 18 months of a 3-year longitudinal study of 364 alcohol-dependent individuals were analyzed. Structural equation modeling was used to replicate the analyses of Kelly et al. (Alcohol Clin Exp Res. 2011;35:454-463) and to compare AA attendance and AA involvement as focal predictors. Multiple regression analyses were used to determine which spirituality dimensions changed as the result of AA participation. A trimmed, data-driven model was employed to test multiple mediation paths simultaneously. The findings of the Kelly et al. study were replicated. AA involvement was a stronger predictor of drinking outcomes than AA attendance. AA involvement predicted increases in private religious practices, daily spiritual experiences, and forgiveness of others. However, only private religious practices mediated the relationship between AA and drinking.

Persistent insomnia, abstinence, and moderate drinking in alcohol-dependent individuals.

Insomnia is common, persistent, and increases the risk for relapse in alcohol-dependent (AD) patients. Abstinence has long been considered the best strategy for allowing sleep to normalize, although how many and which patients respond to abstinence is unknown. The aims of this study were to investigate the prevalence and correlates of both baseline and persistent insomnia in AD patients. The course of sleep problems in response to abstinence, moderate drinking, or relapse following treatment was also examined. A naturalistic longitudinal outcomes study interviewed 267 patients (69% male; mean age of 44 years) with DSM-IV alcohol dependence at baseline and 6 months later (84% follow-up rate) . The Sleep Problems Questionnaire, Time-Line Follow-Back Interview, and Brief Symptom Inventory measured insomnia, drinking, and psychiatric symptoms, respectively. Simple correlations, logistic regression, and repeated measures analysis of variance were used to analyze the data. At baseline, 47% of patients were classified with insomnia, which was independently predicted by female gender and psychiatric severity. Both abstinence and moderate drinking outcomes significantly predicted a reduction of insomnia symptoms after controlling for gender and psychiatric severity. Among patients with baseline insomnia, however, insomnia persisted in over 60% of cases, which was predicted by baseline insomnia severity. Moreover, insomnia persisted in one-quarter of patients despite abstinence. Treatment aimed at preventing relapse to heavy drinking provides good first-line therapy for insomnia in AD patients, but some may require insomnia-specific evaluation and treatment in addition to substance-focused treatment and psychiatric care.

Heat maps: a technique for classifying and analyzing drinking behavior.

Heat maps are presented here as an innovative technique for evaluating longitudinal drinking outcomes. The Life Transitions Study followed alcohol dependent individuals for 2.5 years during 2004-2009 in a Midwestern city (N = 364). The TimeLine Follow-Back obtained drinking information. Heat map results were compared with those obtained using growth mixture modeling. Heat map classes differed significantly on baseline clinical and demographic indicators. Data were gathered with support from NIAAA R01AA014442.

Predictors of membership in Alcoholics Anonymous in a sample of Successfully remitted alcoholics.

This study identifies factors associated with Alcoholics Anonymous (AA) membership in a sample of 81 persons who have achieved at least one year of total abstinence from alcohol and other drugs. Forty-four were AA members, 37 were not. Logistic regression was used to test the cross-sectional associations of baseline demographic, substance-related, spiritual and religious, and personality variables with AA membership. Significant variables from the bivariate analyses were included in a multivariate model controlling for previous AA involvement. Having more positive views of God and more negative consequences of drinking were significantly associated with AA membership. This information can be used by clinicians to identify clients for whom AA might be a good fit, and can help others overcome obstacles to AA or explore alternative forms of abstinence support.

Recombinant Production of Biliverdin IXß and δ Isomers in the T7 Promoter Compatible Escherichia coli Nissle.

The ability to obtain purified biliverdin IX (BVIX) isomers other than the commercially available BVIXα is limited due to the low yields obtained by the chemical coupled oxidation of heme. Chemical oxidation requires toxic chemicals, has very poor BVIX yields (<0.05%), and is not conducive to scalable production. Alternative approaches utilizing recombinant E. coli BL21 expressing a cyanobacterial heme oxygenase have been employed for the production BVIXα, but yields are limited by the rate of endogenous heme biosynthesis. Furthermore, the emerging roles of BVIXß and BVIXδ in biology and their lack of commercial availability has led to a need for an efficient and scalable method with the flexibility to produce all three physiologically relevant BVIX isomers. Herein, we have taken advantage of an optimized non-pathogenic E. coli Nissle (EcN(T7)) strain that encodes an endogenous heme transporter and an integrated T7 polymerase gene. Protein production of the Pseudomonas aeruginosa BVIXß and BVIXδ selective heme oxygenase (HemO) or its BVIXα producing mutant (HemOα) in the EcN(T7) strain provides a scalable method to obtain all three isomers, that is not limited by the rate of endogenous heme biosynthesis, due to the natural ability of EcN(T7) to transport extracellular heme. Additionally, we have optimized our previous LC-MS/MS protocol for semi-preparative separation and validation of the BVIX isomers. Utilizing this new methodology for scalable production and separation we have increased the yields of the BVIXß and -δ isomers >300-fold when compared to the chemical oxidation of heme.

Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies.

Misfolded α-synuclein is a major component of Lewy bodies, which are a hallmark of Parkinson's disease (PD). A large body of evidence shows that α-synuclein can aggregate into amyloid fibrils, but the relationship between α-synuclein self-assembly and Lewy body formation remains unclear. Here, we show, both in vitro and in a Caenorhabditis elegans model of PD, that α-synuclein undergoes liquid‒liquid phase separation by forming a liquid droplet state, which converts into an amyloid-rich hydrogel with Lewy-body-like properties. This maturation process towards the amyloid state is delayed in the presence of model synaptic vesicles in vitro. Taken together, these results suggest that the formation of Lewy bodies may be linked to the arrested maturation of α-synuclein condensates in the presence of lipids and other cellular components.

Decreased Drinking and Alcoholics Anonymous are Associated with Different Dimensions of Spirituality.

Alcoholics Anonymous (AA) is a spiritual program and involvement in it has been associated with increases in spirituality. Some who pursue recovery outside AA also use spirituality for support. Decreasing drinking without AA involvement might result in spiritual change, but this has not been explored in previous research. This study investigates drinking and AA behavior to determine their association with seven dimensions of subsequent spirituality. METHODS: A 30-month panel study recruited 364 individuals with alcohol dependence. Multilevel models examined drinking and AA at six months as predictors of both the levels and trajectories of seven dimensions of spirituality assessed five times over 6 - 30 months. RESULTS: Controlling for AA involvement, less drinking was associated with higher levels of purpose in life, self-forgiveness, and spiritual/religious practices. Controlling for drinking, greater AA involvement was associated with higher levels of positive religious coping, daily spiritual experiences, forgiveness of others, and spiritual/religious practices. Neither AA nor drinking predicted trajectories of spirituality. Data visualizations identified a pattern of elevated purpose in life and self-forgiveness among individuals who were abstinent and among individuals who drank less intensely. CONCLUSIONS: Reduced drinking influenced aspects of spirituality that have been shown to respond to experience and maturation. AA was associated with aspects of spirituality embedded in the 12 steps which have been shown to be responsive to learning and modeling. This knowledge has the potential to inform decisions about recovery options, and contributes to theoretical understandings of the nature of spiritual change over the course of addiction recovery.

Forgiveness and alcohol problems among people entering substance abuse treatment.

Forgiveness is argued to be highly relevant to problematic substance use, yet supportive empirical evidence is lacking. Findings are presented from a longitudinal study exploring the relationship between religiousness and spirituality (RS) variables and alcohol use disorders. We examined forgiveness of self (ForSelf), of others (ForOthers), and by God (ByGod), hypothesizing positive relationships with RS and negative relationships with alcohol use and consequences, at both baseline (N = 157) and six-month follow-up (N = 126). ForSelf scores were significantly lower than ForOthers and ByGod scores, and ForOthers scores increased modestly over time. ByGod was most consistently associated with other RS variables. ForSelf and ForOthers were associated with alcohol-related variables at both time points, and baseline ForSelf and ForOthers were associated with fewer drinking consequences at follow-up, but not after controlling for other pertinent variables. ForSelf may be most difficult to achieve and thus most important to recovery, thereby preventing full recovery and fostering relapses.

Trajectories of alcohol use over time among adults with alcohol dependence.

This study sought to identify trajectories of drinking behavior change over time in a sample of adults with current alcohol use disorder (AUD). We conducted secondary analyses of seven waves of data from a prospective longitudinal study of 364 adults (mean age=44.0 years, SD=12.8 years) who met criteria for DSM-IV alcohol dependence (AD), 74.4% of whom were entering alcohol treatment. Participants were followed for 2 1/2 to 3 years with in-person interviews every 6 months. Results from latent class growth analyses of drinks per drinking day over 3 years indicated five trajectory classes: 1) Moderate Baseline→Slow Decline; 2) Heavy Baseline→Stable Abstinent; 3) Heavy Baseline→Slow Decline; 4) Heaviest Baseline→Steep Decline, and 5) Heaviest Baseline→Stable Heavy. Findings are consistent with previous research and suggest that these trajectory classes might represent longitudinal phenotypes of alcohol involvement across diverse samples. Treatment modality, Alcoholics Anonymous involvement, and purpose in life were associated with diverse trajectories of drinking behavior among adults with AD. AA involvement was associated with higher odds of membership in trajectory classes that showed declines in drinking from baseline, and having higher purpose in life predicted lower odds of membership in the Heaviest Baseline→Stable Heavy class. AA involvement predicts different pathways of recovery characterized by stable abstinence, steep declines, and/or slower declines in drinking over time. Higher purpose in life may protect against chronic heavy drinking by strengthening motivations to pursue goals that are unrelated to substance use.

The effect of drinking goals at treatment entry on longitudinal alcohol use patterns among adults with alcohol dependence.

BACKGROUND: Drinking goals at treatment entry are a promising, yet under-studied mechanism of change in alcohol use following treatment. It is not known who, upon treatment entry, is likely to desire abstinence as a drinking goal and whether desiring abstinence as a drinking goal influences alcohol use following treatment. METHODS: Data from a 2.5-year longitudinal study of alcohol-dependent adults from 3 treatment sites is examined in a secondary data analysis. At treatment entry, participants reported sociodemographic and clinical characteristics, as well as whether they desired abstinence as a drinking goal or not. At each subsequent wave, participants reported their alcohol use. RESULTS: Bivariate analyses showed that individuals from a VA outpatient treatment site, men, and racial or ethnic minorities were most likely to desire abstinence as a drinking goal at treatment entry. Multi-level mixed effects regression models indicated that individuals who at baseline desired abstinence as a drinking goal sustained higher percentage of days abstinent and higher percentage of days since last drink 2.5 years following treatment entry, compared to individuals who did not desire abstinence. CONCLUSIONS: Understanding who is most likely to desire the specific drinking goal of abstinence can assist clinicians in anticipating client response to goal setting. Furthermore, by understanding the benefits and risks associated with drinking goals, clinicians can focus attention to individuals who desire a more risk-laden goal, including goals of non-abstinence, and tailor interventions, including motivational interviewing techniques, to support effective goals.

Gender and extroversion as moderators of the association between Alcoholics Anonymous and sobriety.

OBJECTIVE: Although women make up one third of Alcoholics Anonymous (AA) membership, research on gender and AA has been limited. Findings in the literature are mixed, with few empirical investigations of factors that may moderate any gender differences found. AA is highly interpersonal, and research has found that women are more extroverted than men. The current study explores the impact of AA on sobriety, gender differences in the relationship between AA and sobriety, and whether extroversion can inform our understanding of gender differences. METHOD: A sample of 276 alcohol-dependent adults (180 men, 96 women) was recruited from four sites and followed prospectively for 2.5-3 years. Participants completed the Timeline Followback interview. AA membership was assessed by an item from the Alcoholics Anonymous Involvement scale. Multiple logistic regression analyses were conducted to determine whether gender, extroversion, AA membership, and their interaction would predict sobriety status at follow-up. RESULTS: AA membership significantly increased the odds of achieving a year of sobriety, and this relationship was stronger for women than men (odds ratio [OR] = 4.42, 95% CI [1.14, 17.18]). There were no main or interactive effects of extroversion on sobriety. CONCLUSIONS: A A was founded by men, and early in its history it was exclusively attended by men. Some have criticized AA for women because of its emphasis on "powerlessness." Despite its historical beginnings and such debate, this study joins others in finding evidence that women fare better in AA than do men. In this sample, extroversion did not moderate the association between gender and sobriety. Further research is needed on gender differences in AA and its explanatory factors.

The Role of Religiousness on Substance-Use Disorder Treatment Outcomes: A Comparison of Black and White Adolescents.

This study compares 41 Black and 124 White adolescents at intake and discharge from a residential treatment program for substance-use disorders. Study data were obtained as part of a larger study (N = 195) that sought to assess the relationship of helping behavior and addiction recovery. This post-hoc analysis aims to identify cultural strengths that may be associated with recovery from substance-use disorders among Black adolescents. Using regression analyses and controlling for the severity of substance use and background variables that distinguish racial groups, religious practices and behaviors at intake were examined. Specifically, Black youth and White youth were compared on treatment outcomes, including alcohol or drug use during treatment, drug craving, 12-Step work, and 12-Step helping. The burden of health and socioeconomic disparities at intake did not disproportionately disfavor Black adolescents. Outcomes related to 12-Step measures were similar between Black and White youth. White adolescents reported higher craving scores at discharge, and Black adolescents were more likely to use drugs during treatment. High levels of religiousness at treatment intake were linked to greater 12-Step work and greater 12-Step helping at discharge. High levels of religiousness at intake were not related to drug use during treatment or to craving scores at discharge. The relationship between intake levels of religiousness and treatment-related outcomes did not differ by race.

Six-month changes in spirituality and religiousness in alcoholics predict drinking outcomes at nine months.

OBJECTIVE: Although spiritual change is hypothesized to contribute to recovery from alcohol dependence, few studies have used prospective data to investigate this hypothesis. Prior studies have also been limited to treatment-seeking and Alcoholics Anonymous (AA) samples. This study included alcohol-dependent individuals, both in treatment and not, to investigate the effect of spiritual and religious (SR) change on subsequent drinking outcomes, independent of AA involvement. METHOD: Alcoholics (N = 364) were recruited for a panel study from two abstinence-based treatment centers, a moderation drinking program, and untreated individuals from the local community. Quantitative measures of SR change between baseline and 6 months were used to predict 9-month drinking outcomes, controlling for baseline drinking and AA involvement. RESULTS: Significant 6-month changes in 8 of 12 SR measures were found, which included private SR practices, beliefs, daily spiritual experiences, three measures of forgiveness, negative religious coping, and purpose in life. Increases in private SR practices and forgiveness of self were the strongest predictors of improvements in drinking outcomes. Changes in daily spiritual experiences, purpose in life, a general measure of forgiveness, and negative religious coping also predicted favorable drinking outcomes. CONCLUSIONS: SR change predicted good drinking outcomes in alcoholics, even when controlling for AA involvement. SR variables, broadly defined, deserve attention in fostering change even among those who do not affiliate with AA or religious institutions. Last, future research should include SR variables, particularly various types of forgiveness, given the strong effects found for forgiveness of self.

Mental health, not social support, mediates the forgiveness-alcohol outcome relationship.

Religiousness and spirituality are important to most Americans, and evidence suggests that they may contribute to both addiction and recovery. Forgiveness is a specific dimension of religiousness and spirituality that may enhance recovery, but the mechanism(s) through which it operates is unknown. We hypothesized that higher levels of forgiveness would be associated with higher levels of mental health and social support, which, in turn, would be associated with improved alcohol-related outcomes. Baseline and 6-month longitudinal data from a sample of 149 individuals with alcohol use disorders seeking outpatient substance abuse treatment were analyzed through multiple-mediation statistical techniques. While previous research has shown direct associations among forgiveness, alcohol-related outcomes, mental health, and social support, this study found that the direct associations between forgiveness and alcohol-related outcomes were no longer significant when mental health and social support were analyzed as mediator variables. At baseline, for each alcohol-related outcome measured (alcohol-related problems, percent heavy drinking days, percent days abstinent, and drinks per drinking day), mental health individually played a role in the relationship with both forgiveness of self and forgiveness of others, fully mediating or operating through an indirect-only pathway. For alcohol-related problems only, mental health fully mediated the relationship with forgiveness of self at follow-up and operated through an indirect-only pathway with forgiveness of others longitudinally. Social support and feeling forgiven by God were nonsignificant variables at baseline, follow-up, and longitudinally.

How Alcoholics Anonymous (AA) and Narcotics Anonymous (NA) Work: Cross-Disciplinary Perspectives.

Evidence from multiple lines of research supports the effectiveness and practical importance of Alcoholics Anonymous and Narcotics Anonymous. Conference presenters discussed the relationship between 12-Step participation and abstinence among various populations, including adolescents, women, and urban drug users. Insight from the arts and humanities placed empirical findings in a holistic context.