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Background: Microbial dysbiosis has been reported to contribute to development of neurodegenerative diseases, however, there is a need to identify causative/prognostic indicators., Objectives: To comparatively analyze gut microbiome composition in symptomatic LBD (dementia/mild cognitive impairment), iRBD, and cohabiting controls without LBD or iRBD., Methods: 16S rRNA amplicon sequencing was performed in 38 cases (27 LBD, 11 iRBD) and 39 cohabitant controls. 19 non-cohabitant healthy controls (HCs) were also included to contrast differences between cohabitant cases and controls., Results: Microbiome composition of cohabitant controls and LBD and iRBD cases were strikingly similar. No differences were observed between LBD, and iRBD only showed reduced Bacteroides, compared with cohabitant controls. There were several taxonomic differences in gut microbiome composition between non-cohabitant HCs and cases., Conclusions: Minimal microbiome differences were observed between iRBD or LBD cases and cohabitant controls. These findings underscore the importance of using cohabiting controls in future gut microbiome studies., Competing Interests: Declaration of competing interest 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., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Extreme global warming can produce hydroclimate changes that remain poorly understood for sub-tropical latitudes. Late Palaeocene-early Eocene (LPEE; ~58-52 Ma) proto-Mediterranean zones of the western Tethys offer opportunities to assess hydroclimate responses to massive carbon cycle perturbations. Here, we reconstruct LPEE hydroclimate conditions of these regions and find that carbon cycle perturbations exerted controls on orbitally forced hydroclimate variability. Long-term (~6 Myr) carbon cycle changes induced a gradual precipitation/moisture reduction, which was exacerbated by some short-lived (<200 kyr) carbon cycle perturbations that caused rapid warming and exceptionally dry conditions in western Tethyan continental areas. Hydroclimate recovery following the greatest short-lived global warming events took ~24-27 kyr. These observations support the notion that anthropogenically driven warming can cause widespread aridification with impacts that may last tens of thousands of years., (© 2024. The Author(s).)

Introduction: Rates of cigarette use remain elevated among those living in rural areas. Depressive symptoms, risky alcohol use, and weight concerns frequently accompany cigarette smoking and may adversely affect quitting. Whether treatment for tobacco use that simultaneously addresses these issues affects cessation outcomes is uncertain., Methods: The study was a multicenter, two-group, randomized controlled trial involving mostly rural veterans who smoke (N = 358) receiving treatment at one of five Veterans Affairs Medical Centers. The study randomly assigned participants to a tailored telephone counseling intervention or referral to their state tobacco quitline. Both groups received guideline-recommended smoking cessation pharmacotherapy, selected using a shared decision-making approach. The primary outcome was self-reported seven-day point prevalence abstinence (PPA) at three and six months. The study used salivary cotinine to verify self-reported quitting at six months., Results: Self-reported PPA was significantly greater in participants assigned to Tailored Counseling at three (OR = 1.66; 95 % CI: 1.07-2.58) but not six (OR = 1.35; 95 % CI: 0.85-2.15) months. Post hoc subgroup analyses examining treatment group differences based on whether participants had a positive screen for elevated depressive symptoms, risky alcohol use, and/or concerns about weight gain indicated that the cessation benefit of Tailored Counseling at three months was limited to those with ≥1 accompanying concern (OR = 2.02, 95 % CI: 1.20-3.42). Biochemical verification suggested low rates of misreporting., Conclusions: A tailored smoking cessation intervention addressing concomitant risk factors enhanced short-term abstinence but did not significantly improve long-term quitting. Extending the duration of treatment may be necessary to sustain treatment effects., Competing Interests: Declaration of competing interest None of the authors has a conflict of interest to disclose related to this work., (Published by Elsevier Inc.)

Out-of-equilibrium, strong correlation in a many-body system can trigger emergent properties that act to constrain the natural dissipation of energy and matter. Signs of such self-organization appear in the avalanche, bifurcation, and quench of a state-selected Rydberg gas of nitric oxide to form an ultracold, strongly correlated ultracold plasma. Work reported here focuses on the initial stages of avalanche and quench and uses the mm-wave spectroscopy of an embedded quantum probe to characterize the intermolecular interaction dynamics associated with the evolution to plasma. Double-resonance excitation prepares a Rydberg gas of nitric oxide composed of a single selected state of principal quantum number, n 0 . Penning ionization, followed by an avalanche of electron-Rydberg collisions, forms a plasma of NO + ions and weakly bound electrons, in which a residual population of n 0 Rydberg molecules evolves to a state of high orbital angular momentum, ℓ. Predissociation depletes the plasma of low-ℓ molecules. Relaxation ceases and n 0 ℓ(2) molecules with ℓ ≥ 4 persist for very long times. At short times, varying excitation spectra of mm-wave Rydberg-Rydberg transitions mark the rate of electron-collisional ℓ-mixing. Deep depletion resonances that persist for long times signal energy redistribution in the basis of central-field Rydberg states. The widths and asymmetries of Fano line shapes witness the degree to which coupling in the arrested bath (i) broadens the allowed transition and (ii) mixes the local network of levels in the ensemble.

A 'principles and practice' tutorial-style review of the application of solution-phase NMR in the analysis of the mechanisms of homogeneous organic and organometallic reactions and processes. This review of 345 references summarises why solution-phase NMR spectroscopy is uniquely effective in such studies, allowing non-destructive, quantitative analysis of a wide range of nuclei common to organic and organometallic reactions, providing exquisite structural detail, and using instrumentation that is routinely available in most chemistry research facilities. The review is in two parts. The first comprises an introduction to general techniques and equipment, and guidelines for their selection and application. Topics include practical aspects of the reaction itself, reaction monitoring techniques, NMR data acquisition and processing, analysis of temporal concentration data, NMR titrations, DOSY, and the use of isotopes. The second part comprises a series of 15 Case Studies, each selected to illustrate specific techniques and approaches discussed in the first part, including in situ NMR ( 1/2 H, 10/11 B, 13 C, 15 N, 19 F, 29 Si, 31 P), kinetic and equilibrium isotope effects, isotope entrainment, isotope shifts, isotopes at natural abundance, scalar coupling, kinetic analysis (VTNA, RPKA, simulation, steady-state), stopped-flow NMR, flow NMR, rapid injection NMR, pure shift NMR, dynamic nuclear polarisation, 1 H/ 19 F DOSY NMR, and in situ illumination NMR., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Aims: To test the effectiveness of an intervention, Al-Anon Intensive Referral (AIR), to facilitate participation in Al-Anon Family Groups (Al-Anon)., Design, Setting and Participants: Multi-site, randomized controlled trial of AIR versus usual care (UC), with follow-up assessments at 3, 6 and 12 months. The 12-month follow-up rate was 74%. Residential alcohol use disorder (AUD) treatment programs in three US locations. Concerned others (COs) of patients in treatment for AUD. COs were mainly women (77%) who were patients' spouses (33%) or parents (25%)., Intervention and Comparator: AIR (n = 128) consisted of four sessions over 3 months with an Al-Anon coach. UC (n = 151) was the treatment program's offer of educational sessions for COs., Measurements: Primary outcome: COs' self-reports of any Al-Anon attendance (yes or no) at 3 months., Secondary Outcomes: number of Al-Anon meetings and the CO-patient relationship (stressors, resources). Potential predictors of outcomes examined in generalized linear mixed models were their baseline value, time, CO-patient relationship type (marital or non-marital), treatment program and condition., Findings: There was no effect of condition for the primary outcome (28% in AIR, 21% in UC; Bayes factor = 1.86). Relationship stressors at follow-ups were more severe for COs in a marital relationship with the patient than for COs in a non-marital relationship [β = 2.19, 95% confidence interval (CI) = 1.07, 3.32]. For CO-patient relationship resources at follow-ups, the main effect for condition was significant (β = 1.33, 95% CI = 0.04, 2.61). COs assigned to the AIR condition had more resources than COs who were in the UC condition., Conclusions: Relative to usual care, Al-Anon Intensive Referral was not associated with increases in participation of concerned others in Al-Anon, but was associated with more resources in the concerned other-patient relationship., (© 2021 Society for the Study of Addiction. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Introduction: The Al-Anon mutual-help program helps concerned others (COs; e.g., families, friends) of persons with excessive alcohol use. Despite widespread availability of Al-Anon meetings, participation is limited and little is known about how to best facilitate engagement. Al-Anon Intensive Referral (AIR) was developed to facilitate COs' engagement in Al-Anon and is being tested in a randomized controlled trial (RCT). Toward the end of the recruitment for the RCT, a qualitative formative evaluation was conducted to learn about facilitators, barriers, and recommendations for AIR implementation in substance use disorder (SUD) treatment clinics., Methods: Thirty-one directors and staff at ten VA and community SUD clinics were interviewed. Semi-structured interviews were based on the Consolidated Framework for Implementation Research and were thematically analyzed to identify facilitators, barriers, and recommendations for AIR implementation., Results: Perceived facilitators of AIR implementation included AIR's face validity, adaptability, and alignment with staff values and skills, requiring only minimal training. Several interviewees in community settings thought AIR would fit with their current practices (e.g., family groups), and some clinics reported having sufficient staff available for delivering AIR. Perceived barriers included limited staff time, and VA clinics having limited resources for providing services to COs. Furthermore, many clients have no COs, or COs who are unwilling or unable to engage with them. Recommendations included fitting AIR within existing workflows and focusing on COs with highest readiness to receive support. Interviewees also thought AIR could be adapted to a website format or smartphone app, which may expand its reach while decreasing staff burden and cost; however, it may not be as effective and appealing to some demographic groups (e.g., older COs)., Conclusions: AIR has strong potential for implementation in SUD treatment settings, but clinics vary on implementation capacity. Most clinics could implement AIR partially (e.g., case-by-case basis) while clinics with sufficient capacity (e.g., staff time) could implement it more fully. These findings can also inform implementation of other interventions for concerned others., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Posttraumatic stress disorder (PTSD) dramatically increases the risk of both substance use disorder (SUD) and suicide in veterans. Military-related trauma, however, may not be the only or most significant trauma experienced by veterans. Trauma exposure is high among those joining the military. This study sought to identify the prevalence of five types of childhood trauma (emotional, physical, and sexual abuse and emotional and physical neglect) and three adult trauma symptom clusters (intrusive thoughts, avoidance, and hyperarousal) among veterans seeking SUD treatment and to clarify the associations between types of trauma and specific symptom clusters. Veterans at three Veterans Affairs (VA) SUD treatment facilities in the Midwest completed surveys at treatment entry ( n 1 = 195) and at 6-month follow-up ( n 2 = 138). Measures included the Childhood Trauma Questionnaire-Short Form and the PTSD Checklist, either a military or a civilian version, depending on whether the most traumatic event occurred in or out of the military. The prevalence of childhood trauma was high, ranging from 40.5% experiencing physical abuse down to 22.8% experiencing sexual abuse. At baseline, 60.2% of the military trauma group met criteria for PTSD, compared with 33.9% of the civilian trauma group, a significant difference, χ 2 (1, N = 195) = 14.46, p < .01. Childhood emotional and physical abuse were moderately associated with intrusion and hyperarousal in the military trauma group, but in the civilian trauma group a broader spectrum of childhood traumas were associated with a broader array of symptom clusters, including avoidance. At follow-up, symptoms improved and were less associated with childhood trauma. These findings illuminate the persistence of effects of childhood trauma and recommend more targeted PTSD treatments.

Background: This study of dyads composed of patients in treatment for alcohol use disorders (AUDs) and their Concerned Others (COs) examined (1) COs' attendance at three types of help (educational sessions, treatment sessions with the patient, and treatment sessions without the patient) during a 3-month period after patients' admission, and (2) whether COs' use of help was associated with better treatment outcomes reported by COs about patients, and patients about themselves., Methods: Dyads (n = 277) were assessed when patients entered AUD treatment and 3 months later (80 %). COs rated their patient's AUD, and patients rated their own AUD, on the Brief Addiction Monitor. At 3-month follow-up, COs answered three items about help (education, treatment with patient, treatment without patient) from their patient's treatment program. Main analyses were analyses of covariance to examine associations between COs' help use and patients' outcomes., Results: At 3-month follow-up, COs who had obtained one type of help were significantly more likely to have also obtained the other two types of help. However, relatively few COs (39 %) obtained any type of help. COs who obtained help rated their patient as having more protection against future substance use. And, when COs had obtained help, patients rated themselves as having less risk of future substance use, and had attended a greater number of AA meetings, than when COs had not obtained help., Conclusions: Findings suggest that COs' use of help can benefit patients in AUD treatment. Clinicians and researchers should increase the availability and use of help among COs., (Published by Elsevier B.V.)

Background: Urinary 5-hydroxyindoleacetic acid (5-HIAA) is a first-line investigation for gastrointestinal neuroendocrine tumours that secrete serotonin. It also has clinical utility for monitoring disease progression and therapeutic response., Aim: To develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for urinary 5-hydroxyindoleacetic acid that incorporates a supported liquid extraction and 13 C-labelled internal standard., Methods: Samples were diluted in ammonium acetate containing a 13 C-labelled internal standard (5-hydroxyindole-3a,4,5,6,7,7a- 13 C 6 -3-acetic acid). Supported liquid extraction was performed followed by chromatographic separation using the 2.1 × 30 mm CORTECS® UPLC® T3 column. Mass spectrometry detection (Waters Xevo TQ-XS) was performed in electrospray positive mode using the transitions 192.3 > 146.4 m/z (quantifier) and 192.3 > 118.4 m/z (qualifier) for 5-hydroxyindoleacetic acid and 198.2 > 152.4 m/z for 13 C-5-HIAA., Results: A well-defined 5-hydroxyindoleacetic acid peak was observed at 0.8 min with a run time of 2.4 min. The assay was linear (r 2  > 0.99) to 382  µ mol/L, with a lower limit of quantification of 5.3  µ mol/L (CV <15%). Analysis of 29 external quality assurance samples showed good agreement between our method and the UKNEQAS method mean (4.7% positive bias). The intra- and inter-assay precision was within acceptable limits, and the assay was stable up to 96 h postextraction with minimal carryover., Conclusion: We have developed a robust LC-MS/MS method with semi-automated extraction that offers an improved run time and performance over the existing, labour-intensive, HPLC method. The method was quick, precise, showed good agreement with UKNEQAS external quality assurance material and is in routine service for clinical samples.

Objective: Growing up with an adult with an alcohol use disorder (AUD) is common and negatively affects adult functioning. This study examined two questions concerning the lived experience of growing up in a home with AUD., Method and Results: The first question asked how adults entering AUD treatment (n = 402) who had this lived experience (58%) compared to those who did not (42%) on indicators of alcohol use severity. Patients with lived experience reported alcohol use at a younger age, more times having been arrested and charged, and greater risk for future substance use. The second question examined concordance between patients and their concerned others on this lived experience (n = 277 dyads) and patients' treatment outcomes 3 months later. The associations between patients' lived experience and better treatment outcomes were stronger when patients' concerned others had a concordant lived experience. When patient-concerned other dyads reported concordant lived experiences at baseline, patients had lower substance use and risk scores at the 3-month follow-up than when the dyads reported discordant lived experiences with regard to growing up in a home with AUD; effect sizes were small., Conclusions: Concordance and discordance on this lived experience could be considered in treatment planning for patients with AUD and their concerned others. Providers could ask about each member's childhood and aim interventions at helping dyads discuss their childhoods in ways that validate each other's needs and provide emotional support, without stigmatization. Delivery may consider relationship type (spousal or other) and be in educational or treatment sessions that include the dyad or one member.

The mechanism of CF 2 transfer from TMSCF 3 ( 1 ), mediated by TBAT (2-12 mol %) or by NaI (5-20 mol %), has been investigated by in situ/stopped-flow 19 F NMR spectroscopic analysis of the kinetics of alkene difluorocyclopropanation and competing TFE/ c- C 3 F 6 /homologous perfluoroanion generation, 13 C/ 2 H KIEs, LFERs, CF 2 transfer efficiency and selectivity, the effect of inhibitors, and density functional theory (DFT) calculations. The reactions evolve with profoundly different kinetics, undergoing autoinhibition (TBAT) or quasi-stochastic autoacceleration (NaI) and cogenerating perfluoroalkene side products. An overarching mechanism involving direct and indirect fluoride transfer from a CF 3 anionoid to TMSCF 3 ( 1 ) has been elucidated. It allows rationalization of why the NaI-mediated process is more effective for less-reactive alkenes and alkynes, why a large excess of TMSCF 3 ( 1 ) is required in all cases, and why slow-addition protocols can be of benefit. Issues relating to exothermicity, toxicity, and scale-up are also noted.

Migrant farmwork is often characterized by harsh working conditions that carry significant physical and mental health consequences. Using a learned helplessness framework, the current study examined the extent to which discrimination, immigration legal status difficulties, and adverse childhood experiences moderated the effects of harsh working conditions on depression and anxiety. The study also examined the extent to which harsh working conditions mediated the effects of discrimination, immigration legal status difficulties, and adverse childhood experiences on depression and anxiety. Participants were 241 migrant farmworkers recruited in the Midwest. Participants completed interviews consisting of the Migrant Farmworker Stress Index (MFWSI), Adverse Childhood Events scale (ACEs), Everyday Discrimination Scale, the Centers for Epidemiology Scale for Depression (CES-D), and the seven item Generalized Anxiety Disorder scale (GAD-7). Tests of indirect effects suggested, working conditions mediated the effects of ACEs, immigration legal status fears, and discrimination on CES-D and GAD-7 scores ( p -values < .05). Higher ACEs and discrimination also appeared to be associated with larger effects of harsh working conditions on depression and anxiety ( p -values < .05), while legal status fears did not significantly moderate the effect of harsh working conditions on either outcome ( p -values > .05). Likely through different mechanisms, adverse childhood experiences, discrimination and immigration legal status are associated with higher risk of harsh working conditions and subsequently these conditions account for much of the relations between these three stressors with depression and anxiety. Additionally, discrimination and adverse childhood experiences appear to then enhance the effects of working conditions.

Objective: This study examined patients in treatment for alcohol use disorders ("Patients") and their "concerned others" (COs-family and friends): (1) Did Patients' functioning differ according to COs' study participation? Among Patients with participating COs, (2) did Patients and COs agree on Patients' functioning, and (3) was Patients' functioning associated with COs' functioning and quality of CO-Patient relationships?, Method: Four-hundred and two Patients (mean age = 44, majority white men) and 277 COs (mean age = 52, majority white women) completed validated assessments., Results: (1) Unexpectedly, Patients who did not identify a CO for potential study participation had more protective factors against future substance use and more readiness to participate in Alcoholics Anonymous (AA) than patients who did identify a CO. (2) Patients had higher scores than COs did when rating the Patient's protective factors, viewed the Patient-CO relationship as having more resources and fewer stressors than COs did, and reported fewer incidents of violence toward the CO than the CO did. (3) Patients had higher risk factors scores when their COs binge drank, and the Patient-CO relationship had more stressors and violence. Patients had higher protective factors scores when COs had greater readiness for Al-Anon participation, and Patients had attended more AA meetings, reported more resources in their relationship with their CO, and used more negotiation tactics when in conflict with their CO., Conclusions: Findings suggest that interventions to improve Patients' functioning should be broadened beyond COs who are spouses or partners, decrease COs' binge drinking, facilitate 12-step participation, decrease relationship stressors and conflict, and increase relationship resources., (Published by Elsevier Inc.)

Objective: To evaluate US veterans' preferences for smoking cessation counseling and pharmacotherapy., Methods: A discrete choice experiment (DCE) was conducted in 123 Veterans Health Administration primary care outpatients who planned to quit smoking within 6 months. Key attributes of tobacco cessation treatment were based on literature review and expert opinion. We used a hierarchical Bayesian approach with a logit model to estimate the part-worth utility of each attribute level and used latent class logit models to explore preference heterogeneity., Results: In the aggregate, participants valued counseling options with the following attributes: higher quit rate at 1 year, emphasis on autonomy, familiarity of the counselor, counselor's communication skills, and inclusion of printed materials on smoking cessation. Participants valued pharmacotherapy options with the following attributes: higher quit rate at 1 year, lower risk of physical side effects, zero copayment, monthly check-in calls, and less weight gain. Latent class analysis revealed distinct clusters of patients with a unique preference "phenotype.", Conclusions: Veterans have distinct preferences for attributes of cessation counseling and pharmacotherapy., Practice Implications: Identifying patients' preferences provides an opportunity for clinicians to offer tailored treatment options that better engage veterans in their own care and boost adherence to guideline-recommended counseling and pharmacotherapy., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to report., (Published by Elsevier B.V.)

The last interglacial (LIG; ~130 to ~118 thousand years ago, ka) was the last time global sea level rose well above the present level. Greenland Ice Sheet (GrIS) contributions were insufficient to explain the highstand, so that substantial Antarctic Ice Sheet (AIS) reduction is implied. However, the nature and drivers of GrIS and AIS reductions remain enigmatic, even though they may be critical for understanding future sea-level rise. Here we complement existing records with new data, and reveal that the LIG contained an AIS-derived highstand from ~129.5 to ~125 ka, a lowstand centred on 125-124 ka, and joint AIS + GrIS contributions from ~123.5 to ~118 ka. Moreover, a dual substructure within the first highstand suggests temporal variability in the AIS contributions. Implied rates of sea-level rise are high (up to several meters per century; m c -1 ), and lend credibility to high rates inferred by ice modelling under certain ice-shelf instability parameterisations.

Background: Poor functioning among Concerned Others (COs; family and friends of an individual with an alcohol or other substance use disorder) is a significant public health problem. The purpose of this study was to inform interventions for COs by examining potential correlates of 3 aspects of functioning: quality of life, mental health, and knowledge of how to handle problems related to their drinker., Methods: This study collected data from 277 COs when their friend or family member (their "drinker") was entering treatment for an alcohol use disorder. Potential correlates were the drinker's substance use severity, the CO-drinker relationship and communication, and COs' coping and perceived stigma related to involvement with their drinker., Results: In a summary analysis, only stressors in the CO-drinker relationship (the drinker's criticism, disagreement, anger, and demands) were consistently associated with poorer functioning as indicated by COs' poorer quality of life and mental health. In contrast, only COs' use of approach coping was associated with COs' knowing how to handle problems related to the drinker., Conclusions: Because reducing both relationship stressors and the link between stressors and poor functioning can be achieved through CO and drinker education and intervention, these findings inform how to effectively support COs' goals for better functioning., (Published 2019. This article is a U.S. Government work and is in the public domain in the USA.)

Background: Providers often prescribe counseling and/or medications for tobacco cessation without considering patients' treatment preferences., Objective: The primary aims of this study are to describe (1) the development of a discrete choice experiment (DCE) questionnaire designed to identify the attributes and levels of tobacco treatment that are most important to veterans; and (2) the decision-making process in choosing between hypothetical tobacco treatments., Methods: We recruited current smokers who were already scheduled for a primary care appointment within a single Veterans Affairs (VA) healthcare system. Subjects were asked to rate the importance of selected treatment attributes and were interviewed during two rounds of pilot testing of initial DCE instruments. Key attributes and levels of the initial instruments were identified by targeted literature review; the instruments were iteratively revised after each round of pilot testing. Using a 'think aloud' approach, subjects were interviewed while completing DCE choice tasks. Constant comparison techniques were used to characterize the issues raised by subjects. Findings from the cognitive interviews were used to revise the initial DCE instruments., Results: Most subjects completed the DCE questionnaire without difficulty and considered two or more attributes in choosing between treatments. Two common patterns of decision-making emerged during the cognitive interviews: (1) counting 'pros' and 'cons' of each treatment alternative; and (2) using a 'rule-out' strategy to eliminate a given treatment choice if it included an undesirable attribute. Subjects routinely discounted the importance of certain attributes and, in a few cases, focused primarily on a single 'must-have' attribute., Conclusion: Cognitive interviews provide valuable insights into the comprehension and interpretation of DCE attributes, the decision processes used by veterans during completion of choice tasks, and underlying reasons for non'-compensatory decision-making.

Community-based support group participation protects against substance use disorder (SUD) relapse, but referrals during treatment are inconsistently delivered and may not acknowledge barriers facing rural patients. This formative evaluation of a rural intensive referral intervention (RAIR) to community-based support groups for veterans seeking SUD treatment surveyed patients (N = 145) and surveyed and interviewed treatment staff (N = 28). Patients and staff did not differ significantly on quantitative ratings of the helpfulness of, or satisfaction with, seven RAIR components, but staff did not deliver the intervention consistently or as designed, citing two themes: lack of commitment and lack of resources.

Objective: A multisite field trial testing whether improved outcomes associated with intensive referral to mutual help groups (MHGs) could be maintained after the intervention was adapted for the circumstances and needs of rural veterans in treatment for substance use disorder (SUD)., Methods: In three Veterans Affairs treatment programs in the Midwest, patients (N=195) received standard referral (SR) or rural-adapted intensive referral (RAIR) and were measured at baseline and 6-month follow-up., Results: Both groups reported significant improvement at 6-months, but no significant differences between SR and RAIR groups in MHG participation, substance use, addiction severity, and posttraumatic stress symptoms. Inconsistent delivery of the intervention resulted in only one-third of the RAIR group receiving the full three sessions, but this group reported significantly greater 6-month abstinence from alcohol than those receiving no sessions., Conclusion: Further research should explore implementation problems and determine whether consistent delivery of the intervention enhances 12-step facilitation., Practice Implications: The addition of rural-specific elements to the original intensive referral intervention has not been shown to increase its effectiveness among rural veterans., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The variability of seawater temperature through time is a critical measure of climate change, yet its reconstruction remains problematic in many regions. Mg/Ca and oxygen isotope (δ 18 O C ) measurements in foraminiferal carbonate shells can be combined to reconstruct seawater temperature and δ 18 O (δ 18 O SW ). The latter is a measure of changes in local hydrology (e.g., precipitation/evaporation, freshwater inputs) and global ice volume. But diagenetic processes may affect foraminiferal Mg/Ca. This restricts its potential in many places, including the Mediterranean Sea, a strategic region for deciphering global climate and sea-level changes. High alkalinity/salinity conditions especially bias Mg/Ca temperatures in the eastern Mediterranean (eMed). Here we advance the understanding of both western Mediterranean (wMed) and eMed hydrographic variability through the penultimate glacial termination (TII) and last interglacial, by applying the clumped isotope (Δ 47 ) paleothermometer to planktic foraminifera with a novel data-processing approach. Results suggest that North Atlantic cooling during Heinrich stadial 11 (HS11) affected surface-water temperatures much more in the wMed (during winter/spring) than in the eMed (during summer). The method's paired Δ 47 and δ 18 O C data also portray δ 18 O SW . These records reveal a clear HS11 freshwater signal, which attenuated toward the eMed, and also that last interglacial surface warming in the eMed was strongly amplified by water-column stratification during the deposition of the organic-rich (sapropel) interval known as S5.

Purpose: The objective of this study is to determine whether rural residence is associated with trauma exposure or posttraumatic stress disorder symptoms among military veterans seeking treatment for substance use disorder (SUD) through the Department of Veterans Affairs (VA), Delivering mental health services to veterans in rural areas is a challenge, so identifying differences in the causes and outcomes of trauma exposure would assist in effectively targeting service delivery., Methods: Veterans (N = 196) entering SUD treatment at 3 Midwestern VA treatment centers were designated as either urban or rural, based on rural-urban commuting area (RUCA) codes. The veterans completed the Life Events Checklist, the Posttraumatic Stress Disorder Checklist, and the Addiction Severity Index's psychiatric status subscale. Hypothesized relationships between rural-urban residence and both trauma exposure and symptomology were tested using independent samples t tests, chi-square tests, and ordinary least squares regression., Findings: The range of traumatic experiences was similar between rural and urban veterans, and rural-urban residence was not significantly associated with the overall array of traumas experienced or the symptom measures' overall scores or subscores. Of 17 possible traumatic lifetime experiences, rural veterans differed from urban veterans on only 2, reporting significantly lower rates of transportation accidents and unwanted sexual experiences., Conclusions: In both the causes of trauma and the need for treatment, veterans residing in rural areas differ little from their urban counterparts., (© 2016 National Rural Health Association.)

Purpose: To examine the impact of a nurse-initiated tobacco cessation intervention focused on providing guideline-recommended care to hospitalized smokers., Design: Pre-post quasi-experimental trial., Setting: General medical units of four US Department of Veterans Affairs hospitals., Subjects: 898 adult Veteran smokers (503 and 395 were enrolled in the baseline and intervention periods, respectively)., Intervention: The intervention included academic detailing, adaptation of the computerized medical record, patient self-management support, and organizational support and feedback., Measures: The primary outcome was self-reported 7-day point prevalence abstinence at six months., Analysis: Tobacco use was compared for the pre-intervention and intervention periods with multivariable logistic regression using generalized estimating equations to account for clustering at the nurse level. Predictors of abstinence at six months were investigated with best subsets regression., Results: Seven-day point prevalence abstinence during the intervention period did not differ significantly from the pre-intervention period at either three (adjusted odds ratio (AOR) and 95% confidence interval (CI 95 )=0.78 [0.51-1.18]) or six months (AOR=0.92; CI 95 =0.62-1.37). Predictors of abstinence included baseline self-efficacy for refraining from smoking when experiencing negative affect (p=0.0004) and perceived likelihood of staying off cigarettes following discharge (p<0.0001)., Conclusions: Tobacco use interventions in the VA inpatient setting likely require more substantial changes in clinician behavior and enhanced post-discharge follow-up to improve cessation outcomes., (Published by Elsevier Inc.)

Introduction: Information continuity is critical to person-centered care when patients receive care from multiple healthcare systems. Patients can access their electronic health record data through patient portals to facilitate information exchange. This pilot was developed to improve care continuity for rural Veterans by (1) promoting the use of the Department of Veterans Affairs (VA) patient portal to share health information with non-VA providers, and (2) evaluating the impact of health information sharing at a community appointment., Materials and Methods: Veterans from nine VA healthcare systems were trained to access and share their VA Continuity of Care Document (CCD) with their non-VA providers. Patients and non-VA providers completed surveys on their experiences., Results: Participants (n = 620) were primarily older, white, and Vietnam era Veterans. After training, 78% reported the CCD would help them be more involved in their healthcare and 86% planned to share it regularly with non-VA providers. Veterans (n = 256) then attended 277 community appointments. Provider responses from these appointments (n = 133) indicated they were confident in the accuracy of the information (97%) and wanted to continue to receive the CCD (96%). Ninety percent of providers reported the CCD improved their ability to have an accurate medication list and helped them make medication treatment decisions. Fifty percent reported they did not order a laboratory test or another procedure because of information available in the CCD., Conclusions: This pilot demonstrates feasibility and value of patient access to a CCD to facilitate information sharing between VA and non-VA providers. Outreach and targeted education are needed to promote consumer-mediated health information exchange.

Background and Objectives: Little is known about gender differences in methamphetamine (METH)-dependent users. The objective of this study was to examine potential gender differences in four domains: drug use history, psychological burden, current symptomology, and coping strategy., Methods: One hundred twenty four METH-dependent individuals (men; n = 75) were enrolled from substance use treatment programs. Participants filled out detailed questionnaires in the four domains., Results: Men reported earlier first alcohol and drug use than women, but there was no difference in the age of first METH use or frequency of METH use. Women reported experiencing problems because of METH use at a younger age. Women were also more likely to have injected METH in the past year and they reported greater severity of drug problems compared to men. METH-dependent women had greater psychological burden, reported more use of an emotional-coping strategy, and had greater childhood emotional and sexual trauma., Conclusions: Overall, this study suggests that, unlike many other illicit drugs, severity of use and problems associated with use were not elevated in METH-dependent men compared to women. In fact, several factors indicated more severe patterns of use or risk factors in women.

Background: Tobacco use remains prevalent among Veterans of military service and those residing in rural areas. Smokers frequently experience tobacco-related issues including risky alcohol use, post-cessation weight gain, and depressive symptoms that may adversely impact their likelihood of quitting and maintaining abstinence. Telephone-based interventions that simultaneously address these issues may help to increase treatment access and improve outcomes., Methods: This study was a two-group randomized controlled pilot trial. Participants were randomly assigned to an individually-tailored telephone tobacco intervention combining counseling for tobacco use and related issues including depressive symptoms, risky alcohol use, and weight concerns or to treatment provided through their state tobacco quitline. Selection of pharmacotherapy was based on medical history and a shared decision interview in both groups. Participants included 63 rural Veteran smokers (mean age = 56.8 years; 87 % male; mean number of cigarettes/day = 24.7). The primary outcome was self-reported 7-day point prevalence abstinence at 12 weeks and 6 months., Results: Twelve-week quit rates based on an intention-to-treat analysis did not differ significantly by group (Tailored = 39 %; Quitline Referral = 25 %; odds ratio [OR]; 95 % confidence interval [CI] = 1.90; 0.56, 5.57). Six-month quit rates for the Tailored and Quitline Referral conditions were 29 and 28 %, respectively (OR; 95 % CI = 1.05; 0.35, 3.12). Satisfaction with the Tailored tobacco intervention was high., Conclusions: Telephone-based treatment that concomitantly addresses other health-related factors that may adversely affect quitting appears to be a promising strategy. Larger studies are needed to determine whether this approach improves cessation outcomes., Trial Registration: ClinicalTrials.gov identifier number NCT01592695 registered 11 April 2012.

Objectives: The current study examined the association among family history of substance use problems, childhood maltreatment, and age of first drug use in a sample of men and women seeking treatment for methamphetamine dependence. Various forms of childhood maltreatment were considered as mediators of the association between family history of substance use problems and age of first drug use., Methods: Participants (N = 99, 40% women, mean age 33) who were under treatment for methamphetamine dependence completed a baseline interview that obtained demographic information, past substance use by participants, history of drug/alcohol problems in their family of origin, and age at first use of any drug (excluding alcohol and tobacco). The Early Trauma Inventory Self-Report-Short Form was used to assess child maltreatment experiences before the age of 18., Results: Family history of substance use problems and childhood physical (but not emotional or sexual) trauma significantly predicted age of first drug use. Further, childhood physical trauma mediated the association between family history of substance use problems and age of first drug use., Conclusions: These findings suggest that the experience of childhood physical abuse may be an important mechanism through which family history of substance use is associated with an earlier age of first drug use.

Our current understanding of ocean-atmosphere-cryosphere interactions at ice-age terminations relies largely on assessments of the most recent (last) glacial-interglacial transition, Termination I (T-I). But the extent to which T-I is representative of previous terminations remains unclear. Testing the consistency of termination processes requires comparison of time series of critical climate parameters with detailed absolute and relative age control. However, such age control has been lacking for even the penultimate glacial termination (T-II), which culminated in a sea-level highstand during the last interglacial period that was several metres above present. Here we show that Heinrich Stadial 11 (HS11), a prominent North Atlantic cold episode, occurred between 135 ± 1 and 130 ± 2 thousand years ago and was linked with rapid sea-level rise during T-II. Our conclusions are based on new and existing data for T-II and the last interglacial that we collate onto a single, radiometrically constrained chronology. The HS11 cold episode punctuated T-II and coincided directly with a major deglacial meltwater pulse, which predominantly entered the North Atlantic Ocean and accounted for about 70 per cent of the glacial-interglacial sea-level rise. We conclude that, possibly in response to stronger insolation and CO2 forcing earlier in T-II, the relationship between climate and ice-volume changes differed fundamentally from that of T-I. In T-I, the major sea-level rise clearly post-dates Heinrich Stadial 1. We also find that HS11 coincided with sustained Antarctic warming, probably through a bipolar seesaw temperature response, and propose that this heat gain at high southern latitudes promoted Antarctic ice-sheet melting that fuelled the last interglacial sea-level peak.

Recurrent deposition of organic-rich sediment layers (sapropels) in the eastern Mediterranean Sea is caused by complex interactions between climatic and biogeochemical processes. Disentangling these influences is therefore important for Mediterranean palaeo-studies in particular, and for understanding ocean feedback processes in general. Crucially, sapropels are diagnostic of anoxic deep-water phases, which have been attributed to deep-water stagnation, enhanced biological production or both. Here we use an ocean-biogeochemical model to test the effects of commonly proposed climatic and biogeochemical causes for sapropel S1. Our results indicate that deep-water anoxia requires a long prelude of deep-water stagnation, with no particularly strong eutrophication. The model-derived time frame agrees with foraminiferal δ(13)C records that imply cessation of deep-water renewal from at least Heinrich event 1 to the early Holocene. The simulated low particulate organic carbon burial flux agrees with pre-sapropel reconstructions. Our results offer a mechanistic explanation of glacial-interglacial influence on sapropel formation.

Background: Although the US Department of Veterans Affairs (VA) has promoted adherence to smoking cessation guidelines since 1997, hospitalized smokers do not consistently receive assistance in quitting., Methods: In a pre-post guideline implementation trial on the inpatient medicine units of four VA hospitals, the effectiveness of a multimodal intervention (enhanced academic detailing, modification of the nursing admission template, patient education materials and quitline referral, practice facilitation and staff feedback) changing practice behavior was evaluated. Peridischarge interviews were conducted with 824 patients to assess receipt of nurses' and physicians' delivery of the 5A's (Ask, Advise, Assess, Assist, Arrange) in hospitalized smokers., Results: Subjects were significantly more likely to have received each of the 5A's from a nurse during the postimplementation period (except for "advise to quit"). More patients were assisted in quitting (75% versus 56%, adjusted odds ratio [OR] = 2.3, 95% confidence interval [CI] = 1.6, 3.1) and had follow-up arranged (23% versus 18%, adjusted OR = 1.5, 95% CI = 1.0, 2.2) by a nurse during the postimplementation period. However, unadjusted results showed no improvement in seven-day point prevalence abstinence at six-month follow-up (13.5% versus 13.9%). Nurses' self-efficacy in cessation counseling, as measured in a survey of 166 unit nurses, improved following guideline implementation., Discussion: A multifaceted intervention including enhanced academic detailing is an effective strategy for improving the delivery of smoking cessation services in medical inpatients. To promote long-term cessation, more intensive interventions are needed to ensure that motivated smokers receive guideline-recommended treatment (including pharmacotherapy and referral to outpatient cessation counseling).

Research on global ice-volume changes during Pleistocene glacial cycles is hindered by a lack of detailed sea-level records for time intervals older than the last interglacial. Here we present the first robustly dated, continuous and highly resolved records of Red Sea sea level and rates of sea-level change over the last 500,000 years, based on tight synchronization to an Asian monsoon record. We observe maximum 'natural' (pre-anthropogenic forcing) sea-level rise rates below 2 m per century following periods with up to twice present-day ice volumes, and substantially higher rise rates for greater ice volumes. We also find that maximum sea-level rise rates were attained within 2 kyr of the onset of deglaciations, for 85% of such events. Finally, multivariate regressions of orbital parameters, sea-level and monsoon records suggest that major meltwater pulses account for millennial-scale variability and insolation-lagged responses in Asian monsoon records.

Ice volume (and hence sea level) and deep-sea temperature are key measures of global climate change. Sea level has been documented using several independent methods over the past 0.5 million years (Myr). Older periods, however, lack such independent validation; all existing records are related to deep-sea oxygen isotope (δ(18)O) data that are influenced by processes unrelated to sea level. For deep-sea temperature, only one continuous high-resolution (Mg/Ca-based) record exists, with related sea-level estimates, spanning the past 1.5 Myr. Here we present a novel sea-level reconstruction, with associated estimates of deep-sea temperature, which independently validates the previous 0-1.5 Myr reconstruction and extends it back to 5.3 Myr ago. We find that deep-sea temperature and sea level generally decreased through time, but distinctly out of synchrony, which is remarkable given the importance of ice-albedo feedbacks on the radiative forcing of climate. In particular, we observe a large temporal offset during the onset of Plio-Pleistocene ice ages, between a marked cooling step at 2.73 Myr ago and the first major glaciation at 2.15 Myr ago. Last, we tentatively infer that ice sheets may have grown largest during glacials with more modest reductions in deep-sea temperature.

During ice-age cycles, continental ice volume kept pace with slow, multi-millennial scale, changes in climate forcing. Today, rapid greenhouse gas (GHG) increases have outpaced ice-volume responses, likely committing us to > 9 m of long-term sea-level rise (SLR). We portray a context of naturally precedented SLR from geological evidence, for comparison with historical observations and future projections. This context supports SLR of up to 0.9 (1.8) m by 2100 and 2.7 (5.0) m by 2200, relative to 2000, at 68% (95%) probability. Historical SLR observations and glaciological assessments track the upper 68% limit. Hence, modern change is rapid by past interglacial standards but within the range of 'normal' processes. The upper 95% limit offers a useful low probability/high risk value. Exceedance would require conditions without natural interglacial precedents, such as catastrophic ice-sheet collapse, or activation of major East Antarctic mass loss at sustained CO2 levels above 1000 ppmv.

Current global warming necessitates a detailed understanding of the relationships between climate and global ice volume. Highly resolved and continuous sea-level records are essential for quantifying ice-volume changes. However, an unbiased study of the timing of past ice-volume changes, relative to polar climate change, has so far been impossible because available sea-level records either were dated by using orbital tuning or ice-core timescales, or were discontinuous in time. Here we present an independent dating of a continuous, high-resolution sea-level record in millennial-scale detail throughout the past 150,000 years. We find that the timing of ice-volume fluctuations agrees well with that of variations in Antarctic climate and especially Greenland climate. Amplitudes of ice-volume fluctuations more closely match Antarctic (rather than Greenland) climate changes. Polar climate and ice-volume changes, and their rates of change, are found to covary within centennial response times. Finally, rates of sea-level rise reached at least 1.2 m per century during all major episodes of ice-volume reduction.

Objectives and Methods: Previous studies have shown that CRK3 protein kinase of Leishmania mexicana is a potential drug target. Therefore, the aim of this study was to provide an active protein kinase for chemical inhibitors testing. A system was developed to express and affinity-purify recombinant L. mexicana CRK3 protein from Escherichia coli., Results: Biochemical analysis has confirmed the expression of the pure kinase. The bacterial-expressed kinase was found to be inactive as a monomer. The mutated CRK3-E178 protein kinase was also found to be inactive., Conclusion: This study suggests that cyclin binding and phosphorylation status are both important for reconstituting protein kinase activity. Work presented by this paper has confirmed the usefulness of the prokaryotic system for production of pure homogenous recombinant protein kinase of Leishmania parasite, though this system is unable to produce active CRK3 protein kinase

Methamphetamine (METH) is a frequent drug of abuse in U.S. populations and commonly associated with psychosis. This may be a factor in frequent criminal justice referrals and lengthy treatment required by METH users. Persecutory delusions and auditory hallucinations are the most consistent symptoms of METH-associated psychosis (MAP). MAP has largely been studied in Asian populations and risk factors have varied across studies. Duration, frequency and amount of use as well as sexual abuse, family history, other substance use, and co-occurring personality and mood disorders are risk factors for MAP. MAP may be unique with its long duration of psychosis and recurrence without relapse to METH. Seven candidate genes have been identified that may be associated with MAP. Six of these genes are also associated with susceptibility, symptoms, or treatment of schizophrenia and most are linked to glutamatergic neurotransmission. Animal studies of pre-pulse inhibition, attenuation of social interaction, and stereotypy and alterations in locomotion are used to study MAP in rodents. Employing various models, rodent studies have identified neuroanatomical and neurochemical changes associated with METH use. Throughout this review, we identify key gaps in our understanding of MAP and suggest potential directions for future research.

Background: Leishmania species are parasitic protozoa that have a tightly controlled cell cycle, regulated by cyclin-dependent kinases (CDKs). Cdc2-related kinase 3 (CRK3), an essential CDK in Leishmania and functional orthologue of human CDK1, can form an active protein kinase complex with Leishmania cyclins CYCA and CYC6. Here we describe the identification and synthesis of specific small molecule inhibitors of bacterially expressed Leishmania CRK3:CYC6 using a high throughput screening assay and iterative chemistry. We also describe the biological activity of the molecules against Leishmania parasites., Methodology/principal Findings: In order to obtain an active Leishmania CRK3:CYC6 protein kinase complex, we developed a co-expression and co-purification system for Leishmania CRK3 and CYC6 proteins. This active enzyme was used in a high throughput screening (HTS) platform, utilising an IMAP fluorescence polarisation assay. We carried out two chemical library screens and identified specific inhibitors of CRK3:CYC6 that were inactive against the human cyclin-dependent kinase CDK2:CycA. Subsequently, the best inhibitors were tested against 11 other mammalian protein kinases. Twelve of the most potent hits had an azapurine core with structure activity relationship (SAR) analysis identifying the functional groups on the 2 and 9 positions as essential for CRK3:CYC6 inhibition and specificity against CDK2:CycA. Iterative chemistry allowed synthesis of a number of azapurine derivatives with one, compound 17, demonstrating anti-parasitic activity against both promastigote and amastigote forms of L. major. Following the second HTS, 11 compounds with a thiazole core (active towards CRK3:CYC6 and inactive against CDK2:CycA) were tested. Ten of these hits demonstrated anti-parasitic activity against promastigote L. major., Conclusions/significance: The pharmacophores identified from the high throughput screens, and the derivatives synthesised, selectively target the parasite enzyme and represent compounds for future hit-to-lead synthesis programs to develop therapeutics against Leishmania species. Challenges remain in identifying specific CDK inhibitors with both target selectivity and potency against the parasite.

Context: Disparities in the prevalence, morbidity, and mortality of multiple mental health conditions have been described between rural and urban populations. However, there is limited information regarding differences in exposure to trauma and trauma-related mental health conditions in these populations. Given the number of veterans who are returning to rural communities after serving in Operation Enduring Freedom and Operation Iraqi Freedom, differences in trauma exposure are of particular relevance. Trauma exposure is related to a variety of mental health disorders including substance use disorders (SUD)., Purpose: The objectives of this preliminary study were to describe lifetime military and nonmilitary trauma and to compare trauma history between rural and urban veterans in SUD treatment., Methods: Sixty adults in SUD treatment were enrolled at 3 Veterans Health Administration sites in Nebraska over a 3-month period in 2008. Subjects completed an interview with study staff, which assessed SUD diagnoses and childhood, lifetime, and military trauma. Rural or urban status was determined by self-report of childhood residence. Childhood trauma, lifetime trauma, and response to military trauma were compared between rural and urban veterans., Findings: Although there were no significant differences in trauma exposure between rural and urban groups, there was an association between specific types of trauma and measures typically associated with increased substance abuse severity and poorer SUD treatment outcome., Conclusion: This is the first study, to our knowledge, which compared trauma exposure between rural and urban veterans and identified an association between childhood trauma exposure and multiple SUD treatment attempts., (No claim to original US government works.)

We are reporting here, the cloning and characterization of the first cyclin from Leishmania mexicana. We have identified a cyclin-like motif from the L. major genome sequencing project. A cyclin homologue was cloned and sequenced from L. mexicana genome and it showed 96.1% amino acid identity with the putative L. major cyclin. It has also sequence identity to mitotic cyclins from other organisms. Southern analysis showed that it is present as a single copy gene. CYCa has been over-expressed in E. coli as a histidine fusion and western blot has confirmed the immunoreactive property of the recombinant cyclin, which then used to reconstitute active recombinant L. mexicana CRK3. No phosphorylation of histone HI was detected by both wild type and mutated CRK3 on the activation assays suggesting that phosphorylation status and cyclin binding are important for reconstituting protein kinase activity. The results confirm that we have isolated a cyclin molecule from L. mexicana (LmCYCa) which may play an important role in the regulation of the parasite cell cycle.

The activity of cyclin-dependent kinases (CDKs), which are key regulators of the eukaryotic cell cycle, is regulated through post-translational mechanisms, including binding of a cyclin and phosphorylation. Previously studies have shown that Leishmania mexicana CRK3 is an essential CDK that is a functional homologue of human CDK1. In this study, recombinant histidine tagged L. mexicana CRK3 and the cyclin CYCA were combined in vitro to produce an active histone H1 kinase that was inhibited by the CDK inhibitors, flavopiridol and indirubin-3'-monoxime. Protein kinase activity was observed in the absence of phosphorylation of the T-loop residue Thr178, but increased 5-fold upon phosphorylation by the CDK activating kinase Civ1 of Saccharomyces cerevisiae. Seven recombinant L. major CRKs (1, 2, 3, 4, 6, 7 and 8) were also expressed and purified, none of which were active as monomers. Moreover, only CRK3 was phosphorylated by Civ1. HA-tagged CYCA expressed in L. major procyclic promastigotes was co-precipitated with CRK3 and exhibited histone H1 kinase activity. These data indicate that in Leishmania CYCA interacts with CRK3 to form an active protein kinase, confirm the conservation of the regulatory mechanisms that control CDK activity in other eukaryotes, but identifies biochemical differences to human CDK1., (Copyright 2010 Elsevier B.V. All rights reserved.)

Indirubins known to target mammalian cyclin-dependent kinases (CDKs) and glycogen synthase kinase (GSK-3) were tested for their antileishmanial activity. 6-Br-indirubin-3'-oxime (6-BIO), 6-Br-indirubin-3'acetoxime and 6-Br-5methylindirubin-3'oxime (5-Me-6-BIO) were the most potent inhibitors of Leishmania donovani promastigote and amastigote growth (half maximal inhibitory concentration (IC(50)) values < or =1.2 microM). Since the 6-Br substitution on the indirubin backbone greatly enhances the selectivity for mammalian GSK-3 over CDKs, we identified the leishmanial GSK-3 homologues, a short (LdGSK-3s) and a long one, focusing on LdGSK-3s which is closer to human GSK-3beta, for further studies. Kinase assays showed that 5-Me-6-BIO inhibited LdGSK-3s more potently than CRK3 (the CDK1 homologue in Leishmania), whilst 6-BIO was more selective for CRK3. Promastigotes treated with 5-Me-6-BIO accumulated in the S and G2/M cell-cycle phases and underwent apoptosis-like death. Interestingly, these phenotypes were completely reversed in parasites over-expressing LdGSK-3s. This finding strongly supports that LdGSK-3s is: (i) the intracellular target of 5-Me-6-BIO, and (ii) involved in cell-cycle control and in pathways leading to apoptosis-like death. 6-BIO treatment induced a G2/M arrest, consistent with inhibition of CRK3 and apoptosis-like death. These effects were partially reversed in parasites over-expressing LdGSK-3s suggesting that in vivo 6-BIO may also target LdGSK-3s. Molecular docking of 5-Me-6-BIO in CRK3 and 6-BIO in human GSK-3beta and LdGSK-3s active sites predict the existence of functional/structural differences that are sufficient to explain the observed difference in their affinity. In conclusion, LdGSK-3s is validated as a potential drug target in Leishmania and could be exploited for the development of selective indirubin-based leishmanicidals.

Background: A wide range of unicellular eukaryotes have now been shown to undergo a form of programmed cell death (PCD) that resembles apoptosis; exhibiting morphological and, in some cases, biochemical markers typical of metazoans. However, reports that sexual and asexual stages of malaria parasites exhibit these markers have been challenged. Here we use a rodent malaria model, Plasmodium berghei, to determine whether, and what proportion of cultured ookinetes show signs of apoptosis-like death and extend the study to examine ookinetes of Plasmodium falciparum in vivo., Results: Ookinetes displayed the following markers of PCD: loss of mitochondrial membrane potential, nuclear chromatin condensation, DNA fragmentation, translocation of phosphatidylserine to the outer surface of the cell membrane and caspase-like activity. The proportion of parasites expressing apoptosis markers rose with time, particularly when cultured in phosphate buffered saline. Some ookinetes positive for apoptosis markers also had compromised membranes, which could represent a late stage in the process. When these are included a similar proportion of ookinetes display each marker. Over 50% of P. falciparum ookinetes, removed from the mosquito midgut lumen 24 h post-infection, had nuclei containing fragmented DNA., Conclusion: We have confirmed previous reports that Plasmodium ookinetes display multiple signs that suggest they die by a mechanism resembling apoptosis. This occurs in vivo and in vitro without experimental application of triggers. Our findings support the hypothesis that non-necrotic mechanisms of cell death evolved before the advent of multicellular organisms.