Comprehensive analysis of CXXX sequence space reveals that S. cerevisiae GGTase-I mainly relies on a2X substrate determinants.

Many proteins undergo a post-translational lipid attachment, which increases their hydrophobicity, thus strengthening their membrane association properties or aiding in protein interactions. Geranylgeranyltransferase-I (GGTase-I) is an enzyme involved in a three-step post-translational modification (PTM) pathway that attaches a 20-carbon lipid group called geranylgeranyl at the carboxy-terminal cysteine of proteins ending in a canonical CaaL motif (C - cysteine, a - aliphatic, L - often leucine, but can be phenylalanine, isoleucine, methionine, or valine). Genetic approaches involving two distinct reporters were employed in this study to assess S. cerevisiae GGTase-I specificity, for which limited data exists, towards all 8000 CXXX combinations. Orthogonal biochemical analyses and structure-based alignments were also performed to better understand the features required for optimal target interaction. These approaches indicate that yeast GGTase-I best modifies the Cxa[L/F/I/M/V] sequence that resembles but is not an exact match for the canonical CaaL motif. We also observed that minor modification of non-canonical sequences is possible. A consistent feature associated with well-modified sequences was the presence of a non-polar a2 residue and a hydrophobic terminal residue, which are features recognized by mammalian GGTase-I. These results thus support that mammalian and yeast GGTase-I exhibit considerable shared specificity.

A Humanized Yeast System for Evaluating the Protein Prenylation of a Wide Range of Human and Viral CaaX Sequences.

The C-terminal CaaX sequence (cysteine-aliphatic-aliphatic-any of several amino acids) is subject to isoprenylation on the conserved cysteine and is estimated to occur in 1-2% of proteins within yeast and human proteomes. Recently, non-canonical CaaX sequences in addition to shorter and longer length CaX and CaaaX sequences have been identified that can be prenylated. Much of the characterization of prenyltransferases has relied on the yeast system because of its genetic tractability and availability of reporter proteins, such as the a-factor mating pheromone, Ras GTPase, and Ydj1 Hsp40 chaperone. To compare the properties of yeast and human prenyltransferases, including the recently expanded target specificity of yeast farnesyltransferase, we have developed yeast strains that express human farnesyltransferase or geranylgeranyltransferase-I in lieu of their yeast counterparts. The humanized yeast strains display robust prenyltransferase activity that functionally replaces yeast prenyltransferase activity in a wide array of tests, including the prenylation of a wide variety of canonical and non-canonical human CaaX sequences, virus encoded CaaX sequences, non-canonical length sequences, and heterologously expressed human proteins HRas and DNAJA2. These results reveal highly overlapping substrate specificity for yeast and human farnesyltransferase, and mostly overlapping substrate specificity for GGTase-I. This yeast system is a valuable tool for further defining the prenylome of humans and other organisms, identifying proteins for which prenylation status has not yet been determined.

A comprehensive in vivo screen of yeast farnesyltransferase activity reveals broad reactivity across a majority of CXXX sequences.

The current understanding of farnesyltransferase (FTase) specificity was pioneered through investigations of reporters like Ras and Ras-related proteins that possess a C-terminal CaaX motif that consists of 4 amino acid residues: cysteine-aliphatic1-aliphatic2-variable (X). These studies led to the finding that proteins with the CaaX motif are subject to a 3-step post-translational modification pathway involving farnesylation, proteolysis, and carboxylmethylation. Emerging evidence indicates, however, that FTase can farnesylate sequences outside the CaaX motif and that these sequences do not undergo the canonical 3-step pathway. In this work, we report a comprehensive evaluation of all possible CXXX sequences as FTase targets using the reporter Ydj1, an Hsp40 chaperone that only requires farnesylation for its activity. Our genetic and high-throughput sequencing approach reveals an unprecedented profile of sequences that yeast FTase can recognize in vivo, which effectively expands the potential target space of FTase within the yeast proteome. We also document that yeast FTase specificity is majorly influenced by restrictive amino acids at a2 and X positions as opposed to the resemblance of CaaX motif as previously regarded. This first complete evaluation of CXXX space expands the complexity of protein isoprenylation and marks a key step forward in understanding the potential scope of targets for this isoprenylation pathway.

A difference-in-difference approach to examining the impact of cannabis legalization on disparities in the use of cigarettes and cannabis in the United States, 2004-17.

AIMS: To estimate the impact of recreational and medical cannabis laws (RCL, MCL) on the use of cannabis and cigarettes in the United States. DESIGN: A difference-in-difference approach was applied to data from the 2004-17 National Survey on Drug Use and Health (NSDUH). SETTING: United States. PARTICIPANTS: Nationally representative cross-sectional survey of Americans aged 12 years and older (combined analytical sample for 2004-17, n = 783 663). MEASUREMENTS: Data on past-month use of (1) cigarettes and (2) cannabis were used to classify respondents into four groups: cigarette and cannabis co-use, cigarette-only use, cannabis-only use or no cigarette or cannabis use. State of residence was measured by self-report. MCL/RCL status came from state government websites. FINDINGS: Difference-in-difference analyses suggest that MCL was associated with an increase in cigarette-cannabis co-use overall [adjusted odds ratio (aOR) = 1.09; 95% confidence interval (CI) = 1.02-1.16], with the greatest increases among those aged 50 years and above (aOR = 1.60; CI = 1.39-1.84), married (aOR = 1.19; CI = 1.07-1.31), non-Hispanic (NH) black (aOR = 1.14; CI = 1.02-1.07) and with a college degree or above (aOR = 1.15; CI = 1.06-1.24). MCL was associated with increases in cigarette-only use among those aged 50 years and above (aOR = 1.07; CI = 1.01-1.14) and NH black (aOR = 1.16; CI = 1.06-1.27) and increases in cannabis-only use among those aged 50 years and above (aOR = 1.24; CI = 1.07-1.44) and widowed/divorced/separated (aOR = 1.18; CI = 1.01-1.37). RCL was associated with an increase in cannabis-only use overall (aOR = 1.21; 95% CI = 1.09-1.34), a decline in cigarette-only use overall (aOR = 0.89; 95% CI = 0.81-0.97) and increases in co-use among those who were married (aOR = 1.24; CI = 1.02-1.50) and aged 50 years and above (aOR = 1.37; CI = 1.03-1.84). CONCLUSIONS: Recreational and medical cannabis legalization have had a varying impact on the use, and co-use, of cannabis and cigarettes in the United States.

Association between fatal opioid overdose and state medical cannabis laws in US national survey data, 2000-2011.

AIMS: Most information on the relationship between medical cannabis laws (MCL) and the risk for opioid overdose fatality has been based on studies with ecological designs. To contribute additional information, we used a novel case-control design and individual-level data from national surveys to assess whether state medical cannabis laws were associated with reduced risk of fatal opioid overdose between 2000-2011. METHODS: Data from participants surveyed in the National Health Interview Survey (NHIS) between 1986-2011 were included. For those sampled between 1986-2009, detailed mortality follow-up data were available from the National Death Index up to 12/31/2011. Opioid overdose decedents (n = 791) were classified as cases. Between 2000-2011, all cases arising in a given year were matched to adult controls who were surveyed the same year and eligible for mortality follow-up (n = 723,920). The distribution of exposure to state MCL was contrasted between cases and controls, providing an approximation of the rate ratio of fatal opioid overdose associated with MCLs. Due to a NHIS sample redesign, we stratified analysis using timeframes before and after 2005. RESULTS: Overall, compared to controls, cases were more likely to be male, middle-aged, non-Hispanic White, separated/divorced; less educated, and have a family income below the poverty threshold. No overall association between state MCLs and the rate of opioid overdose was observed between 2000-2005 (aOR = 1.22, 95% CI: 0.83-1.79) or between 2006-2011 (aOR = 0.87, 95% CI: 0.60-1.25). No significant difference between sampling timeframes was observed (ratio of aOR's = 0.71, 95% CI: 0.49-1.01). CONCLUSIONS: We found no overall protective relationship between state MCLs and opioid overdose. Future research with more recent mortality data and more refined cannabis policy classifications would be useful. The importance of the study is two-fold. First, the findings provide an additional source of information countering claims of a protective effect of MCLs on opioid overdoses, suggesting that other solutions to the opioid overdose crisis are needed. Second, the study offers a potentially useful design to answer important population-level public health questions.

Trends in cannabis use among adults with children in the home in the United States, 2004-2017: impact of state-level legalization for recreational and medical use.

BACKGROUND AND AIMS: Cannabis use among parents may be increasing with legalization, but perception of associated risk has declined. The study investigated the association between cannabis legalization and cannabis use among adults with children in the home over time in the United States (US). DESIGN: A difference-in-difference approach was applied to public and restricted-use data from the 2004-2017 National Survey on Drug Use and Health (NSDUH), an annual cross-sectional survey. SETTING: A representative sample of the United States. PARTICIPANTS/CASES: Respondents ages 18+ with children living in the home drawn from the NSDUH (n = 287,624), which is administered to non-institutionalized civilians in the 50 states and District of Columbia. MEASUREMENTS: Exposures were year and state-level cannabis policy in state of residence annually. Outcomes were past-30-day cannabis use and daily cannabis use. Sociodemographic variables included age, gender, marital status, annual family income, race/ethnicity, educational attainment, and strength of state-level tobacco control. FINDINGS: In 2017, past-month cannabis use (11.9%, 9.3%, and 6.1%) and daily cannabis use (4.2%, 3.2%, and 2.3%) were more common in states with recreational marijuana laws (RML), followed by states with medical marijuana laws (MML) and without legal cannabis use, respectively. RML and MML were associated with significantly higher prevalence of past-month cannabis use (adjusted odds ratio [AOR] = 1.28, 95% confidence interval [CI] = 1.12-1.46; AOR = 1.12, 95% CI = 1.03-1.22) and daily cannabis use (AOR = 1.25, 95% CI = 1.03-1.51; AOR = 1.16, 95% CI = 1.02-1.32), respectively. The impact of MML was particularly salient among adults ages 50+ and the highest income and education subgroups. CONCLUSIONS: Among adults with children living in the home, cannabis use appears to be more common in US states with legalized cannabis use compared with states with no legal cannabis use. Recreational legalization appears to increase use among adults with children in the home broadly across nearly all sociodemographic groups, whereas the effect of legalization for medical use is heterogeneous by age and socioeconomic status.

Impact of state-level cannabis legalization on poly use of alcohol and cannabis in the United States, 2004-2017.

BACKGROUND: Cannabis policy has shifted toward legalization in many parts of the United States (US). While attention has been focused on whether legalization will lead to changes in cannabis use, it is conceivable that legalization will also affect use of substances that individuals frequently use with cannabis. This study assessed whether cannabis legalization impacted the prevalence of poly use of cannabis and alcohol from 2004 to 2017 and estimated the prevalence of cannabis and alcohol poly use in 2017. METHODS: Public and restricted-use data from the US 2004-2017 National Survey on Drug Use and Health were analyzed. Data on past-month cannabis and alcohol use were assessed each year. Cannabis legalization was determined by the presence or absence of medical marijuana laws (MML) and recreational marijuana laws (RML) in each state. Difference-in-difference approach was used to estimate the association of MML and RML on cannabis and alcohol use overall and by sociodemographic subgroups (e.g., age, income, education). RESULTS: Between 2004 and 2017, poly use of cannabis and alcohol increased while alcohol-only use decreased. MML were associated with increases in poly use only among adults over age 50 and among those with higher annual incomes and higher education, while RML were associated with increases in poly use broadly among adults across sociodemographic groups. CONCLUSIONS: Legalization of cannabis was associated with increases in cannabis-alcohol poly use in the US. RML were associated with increases across demographics, while the impact of MML was more limited to certain sociodemographic groups.

Trends in anxiety among adults in the United States, 2008-2018: Rapid increases among young adults.

INTRODUCTION: In a time of global uncertainty, understanding the psychological health of the American public is imperative. There are no current data on anxiety trends among adults in the United States (US) over time. This study aimed to investigate prevalence of anxiety among US adults from 2008 to 2018. METHODS: Data from the National Survey on Drug Use and Health (NSDUH), which is an annual, cross-sectional survey on substance use and mental health in the US, were analyzed in 2020. Prevalence of past-month anxiety was estimated among those ages ≥18, by survey year from 2008 to 2018. Time trends were tested using logistic regression. RESULTS: Anxiety increased from 5.12% in 2008 to 6.68% in 2018 (p < 0.0001) among adult Americans. Stratification by age revealed the most notable increase from 7.97% to 14.66% among respondents 18-25 years old (p < 0.001), which was a more rapid increase than among 26-34 and 35-49 year olds (differential time trend p < 0.001). Anxiety did not significantly increase among those ages 50 and older. Anxiety increased more rapidly among those never married and with some college education, relative to their respective counterparts. Apart from age, marital status and education, anxiety increased consistently among sociodemographic groups. CONCLUSIONS: Anxiety is increasing among adults under age 50 in the US, with more rapid increase among young adults. To prepare for a healthier adulthood and given direct and indirect (via 24/7 media) exposure to anxiety-provoking world events, prophylactic measures that can bolster healthy coping responses and/or treatment seeking seem warranted on a broad scale.

Cannabis use among US adults with anxiety from 2008 to 2017: The role of state-level cannabis legalization.

BACKGROUND: Cannabis use is more common among adults with anxiety. Cannabis legalization is occurring rapidly across the United States (US) and individuals may use cannabis to cope with anxiety. This study investigated whether cannabis use across the US has changed differentially by anxiety status and by state cannabis legalization for medical (MML) and/or recreational use (RML). METHODS: Public and restricted-use data from the 2004 to 2017 National Survey on Drug Use and Health, an annual cross-sectional, nationally representative survey of US individuals, were analyzed. The prevalence of past-30-day cannabis use by anxiety status in 2017 was estimated among respondents ages ≥18 (n = 42,554) by sociodemographics and state-level cannabis law. Weighted logistic regressions with continuous year as the predictor for the linear time trend were used to examine the time trends in cannabis use by anxiety and cannabis law status from 2004 to 2017 (total combined analytic sample n = 398,967). RESULTS: Cannabis use was consistently two to three times higher among those with high anxiety compared to those with some or no anxiety and was higher in states with RML compared to MML or no MML/RML. Cannabis use has increased over time among those with and without anxiety overall, in MML states, and in states without MML/RML; with a faster increase in cannabis use among those with high anxiety compared to lower anxiety in states with MML. CONCLUSIONS: Cannabis use is increasing among American adults overall, yet is disproportionately common among Americans with anxiety especially among those residing in states where cannabis has been legalized.

Protein Isoprenylation in Yeast Targets COOH-Terminal Sequences Not Adhering to the CaaX Consensus.

Protein isoprenylation targets a subset of COOH-terminal Cxxx tetrapeptide sequences that has been operationally defined as a CaaX motif. The specificity of the farnesyl transferase toward each of the possible 8000 combinations of Cxxx sequences, however, remains largely unresolved. In part, it has been difficult to consolidate results stemming from in vitro and in silico approaches that yield a wider array of prenylatable sequences relative to those known in vivo We have investigated whether this disconnect results from the multistep complexity of post-translational modification that occurs in vivo to CaaX proteins. For example, the Ras GTPases undergo isoprenylation followed by additional proteolysis and carboxymethylation events at the COOH-terminus. By contrast, Saccharomyces cerevisiae Hsp40 Ydj1p is isoprenylated but not subject to additional modification. In fact, additional modifications are detrimental to Ydj1p activity in vivo We have taken advantage of the properties of Ydj1p and a Ydj1p-dependent growth assay to identify sequences that permit Ydj1p isoprenylation in vivo while simultaneously selecting against nonprenylatable and more extensively modified sequences. The recovered sequences are largely nonoverlapping with those previously identified using an in vivo Ras-based yeast reporter. Moreover, most of the sequences are not readily predicted as isoprenylation targets by existing prediction algorithms. Our results reveal that the yeast CaaX-type prenyltransferases can utilize a range of sequence combinations that extend beyond the traditional constraints for CaaX proteins, which implies that more proteins may be isoprenylated than previously considered.

Association Between Prescription Drug Monitoring Programs and Nonfatal and Fatal Drug Overdoses: A Systematic Review.

Background: Prescription drug monitoring programs (PDMPs) are a key component of the president's Prescription Drug Abuse Prevention Plan to prevent opioid overdoses in the United States. Purpose: To examine whether PDMP implementation is associated with changes in nonfatal and fatal overdoses; identify features of programs differentially associated with those outcomes; and investigate any potential unintended consequences of the programs. Data Sources: Eligible publications from MEDLINE, Current Contents Connect (Clarivate Analytics), Science Citation Index (Clarivate Analytics), Social Sciences Citation Index (Clarivate Analytics), and ProQuest Dissertations indexed through 27 December 2017 and additional studies from reference lists. Study Selection: Observational studies (published in English) from U.S. states that examined an association between PDMP implementation and nonfatal or fatal overdoses. Data Extraction: 2 investigators independently extracted data from and rated the risk of bias (ROB) of studies by using established criteria. Consensus determinations involving all investigators were used to grade strength of evidence for each intervention. Data Synthesis: Of 2661 records, 17 articles met the inclusion criteria. These articles examined PDMP implementation only (n = 8), program features only (n = 2), PDMP implementation and program features (n = 5), PDMP implementation with mandated provider review combined with pain clinic laws (n = 1), and PDMP robustness (n = 1). Evidence from 3 studies was insufficient to draw conclusions regarding an association between PDMP implementation and nonfatal overdoses. Low-strength evidence from 10 studies suggested a reduction in fatal overdoses with PDMP implementation. Program features associated with a decrease in overdose deaths included mandatory provider review, provider authorization to access PDMP data, frequency of reports, and monitoring of nonscheduled drugs. Three of 6 studies found an increase in heroin overdoses after PDMP implementation. Limitation: Few studies, high ROB, and heterogeneous analytic methods and outcome measurement. Conclusion: Evidence that PDMP implementation either increases or decreases nonfatal or fatal overdoses is largely insufficient, as is evidence regarding positive associations between specific administrative features and successful programs. Some evidence showed unintended consequences. Research is needed to identify a set of "best practices" and complementary initiatives to address these consequences. Primary Funding Source: National Institute on Drug Abuse and Bureau of Justice Assistance.

In-transition culture of experimentation with cannabis in Latin American college students: A new role within a potential drug use sequencing pattern.

INTRODUCTION AND AIMS: Given a scenario of intense discussion about the legal situation of cannabis users worldwide, this paper aims to investigate the role of cannabis within a drug use sequencing pattern. DESIGN AND METHODS: Data came from a representative sample of college students from 27 Brazilian capitals (n = 12 711). We analysed the patterns of transition from/to the first use of cannabis to/from the first use of alcohol, tobacco and seven other illegal drugs. Survival analysis methods were used to analyse age of onset data on all potential drug pairs. Drugs that were not specified as the target drug pair tested in each survival model were included as time-varying covariates in all models. RESULTS: We found significant transitions from alcohol [adjusted hazard ratio (aHR) = 1.41, 95% confidence interval (CI) 1.15-1.73, P < 0.001] and inhalants (aHR = 1.56, 95% CI 1.26-1.93, P < 0.001) to cannabis. Moreover, we found significant transitions from cannabis to alcohol (aHR = 2.40, 95% CI 1.47-3.91, P < 0.001), cocaine (aHR = 7.47, 95% CI = 4.26-13.09, P < 0.001), prescription opioids (aHR = 2.16, 95% CI 1.29-3.63, P < 0.01) and tranquilisers (aHR = 1.51, 95% CI 1.11-2.06, P < 0.01). DISCUSSION AND CONCLUSIONS: Overall, our findings point to a strategic role of cannabis within drug first use sequence pattern. We had an important and unexpected finding-the bi-directional relationship between alcohol and cannabis. In addition, the first use of cannabis still precedes the first use of cocaine and non-medical use of tranquilisers and prescription opioids. [Castaldelli-Maia JM, Nicastri S, Cerdá M, Kim JH, Oliveira LG, Andrade AG, Martins, SS. In-transition culture of experimentation with cannabis in Latin American college students: A new role within a potential drug use sequencing pattern. Drug Alcohol Rev 2017;00:000-000].

US Traffic Fatalities, 1985-2014, and Their Relationship to Medical Marijuana Laws.

OBJECTIVES: To determine the association of medical marijuana laws (MMLs) with traffic fatality rates. METHODS: Using data from the 1985-2014 Fatality Analysis Reporting System, we examined the association between MMLs and traffic fatalities in multilevel regression models while controlling for contemporaneous secular trends. We examined this association separately for each state enacting MMLs. We also evaluated the association between marijuana dispensaries and traffic fatalities. RESULTS: On average, MML states had lower traffic fatality rates than non-MML states. Medical marijuana laws were associated with immediate reductions in traffic fatalities in those aged 15 to 24 and 25 to 44 years, and with additional yearly gradual reductions in those aged 25 to 44 years. However, state-specific results showed that only 7 states experienced post-MML reductions. Dispensaries were also associated with traffic fatality reductions in those aged 25 to 44 years. CONCLUSIONS: Both MMLs and dispensaries were associated with reductions in traffic fatalities, especially among those aged 25 to 44 years. State-specific analysis showed heterogeneity of the MML-traffic fatalities association, suggesting moderation by other local factors. These findings could influence policy decisions on the enactment or repealing of MMLs and how they are implemented.

Is employment status in adults over 25 years old associated with nonmedical prescription opioid and stimulant use?

PURPOSE: Nonmedical use of prescription opioid and stimulants (NMUPO and NMUPS, respectively) has declined in recent years, but remains an important public health problem. Evidence regarding their relationships with employment status remains unclear. We determined the relationship between employment status and NMUPO and NMUPS. METHODS: We analyzed a cross-sectional, nationally representative, weighted sample of 58,486 adults, ages 26 years and older, using combined 2011-2013 data from the National Survey on Drug Use and Health (NSDUH). We fit two crude and two adjusted multivariable logistic regression models to assess the relationship between our two different outcomes of interest: (1) past-year NMUPO and (2) past-year NMUPS, and our exposure of interest: employment status, categorized as (1) full time, (2) part time, (3) unemployed, and (4) not in the workforce. Our adjusted models featured the following covariates: sex, race, age, marital status, and psychological distress, and other nonmedical use. RESULTS: Prevalence of NMUPO was higher than NMUPS (3.48 vs. 0.72%). Unemployed participants had the highest odds of NMUPO [aOR 1.45, 95% CI (1.15-1.82)], while those not in the workforce had the highest odds of NMUPS [aOR 1.71, 95% CI (1.22-2.37)]. Additionally, part-time and unemployed individuals had increased odds of NMUPS [aORs, 95% CI 1.59 (1.09-2.31) and 1.67 (1.11-2.37) respectively], while those not in the workforce had decreased odds of NMUPO [aOR 0.82, 95% CI (0.68-0.99)] relative to full-time participants. CONCLUSIONS: There is a need for adult prevention and deterrence programs that target nonmedical prescription drug use, especially among those unemployed or not in the workforce.

State-level medical marijuana laws, marijuana use and perceived availability of marijuana among the general U.S. population.

BACKGROUND: Little is known on how perceived availability of marijuana is associated with medical marijuana laws. We examined the relationship between medical marijuana laws (MML) and the prevalence of past-month marijuana use, with perceived availability of marijuana. METHODS: Data were from respondents included in the National Survey of Drug Use and Health restricted use data portal 2004-2013. Multilevel logistic regression of individual-level data was used to test differences between MML and non-MML states and changes in prevalence of past-month marijuana use and perceived availability from before to after passage of MML among adolescents, young adults and older adults controlling for demographics. RESULTS: Among adults 26+, past-month prevalence of marijuana use increased from 5.87% to 7.15% after MML passage (Adjusted Odds Ratio (AOR): 1.24 [1.16-1.31]), but no change in prevalence of use was found for 12-17 or 18-25 year-olds. Perceived availability of marijuana increased after MML was enacted among those 26+ but not in younger groups. Among all age groups, prevalence of marijuana use and perception of it being easily available was higher in states that would eventually pass MML by 2013 compared to those that had not. Perceived availability was significantly associated with increased risk of past-month marijuana use in all age groups. CONCLUSION: Evidence suggests perceived availability as a driver of change in use of marijuana due to MML. To date, this has only occurred in adults 26+ and different scenarios that could explain this change need to be further explored.

State Medical Marijuana Laws and the Prevalence of Opioids Detected Among Fatally Injured Drivers.

OBJECTIVES: To assess the association between medical marijuana laws (MMLs) and the odds of a positive opioid test, an indicator for prior use. METHODS: We analyzed 1999-2013 Fatality Analysis Reporting System (FARS) data from 18 states that tested for alcohol and other drugs in at least 80% of drivers who died within 1 hour of crashing (n = 68 394). Within-state and between-state comparisons assessed opioid positivity among drivers crashing in states with an operational MML (i.e., allowances for home cultivation or active dispensaries) versus drivers crashing in states before a future MML was operational. RESULTS: State-specific estimates indicated a reduction in opioid positivity for most states after implementation of an operational MML, although none of these estimates were significant. When we combined states, we observed no significant overall association (odds ratio [OR] = 0.79; 95% confidence interval [CI] = 0.61, 1.03). However, age-stratified analyses indicated a significant reduction in opioid positivity for drivers aged 21 to 40 years (OR = 0.50; 95% CI = 0.37, 0.67; interaction P < .001). CONCLUSIONS: Operational MMLs are associated with reductions in opioid positivity among 21- to 40-year-old fatally injured drivers and may reduce opioid use and overdose.

The epidemiologic principles underlying traffic safety study designs.

This article describes the epidemiological principles underlying four observational study designs commonly used to assess traffic safety: the case-control, case-crossover, culpability and quasi-induced exposure designs. We focus in particular on the specific challenges for preventing bias using each design. Whereas recruiting controls representative of the source population poses a special challenge in case-control traffic safety studies, case-crossover designs are prone to recall bias, and culpability and quasi-induced exposure studies can be undermined by difficulties assigning crash responsibility. Using causal diagrams and worked examples, we provide a simple way to teach traffic safety designs to epidemiologists and to encourage proper application of epidemiological principles among researchers designing traffic safety studies.

A shunt pathway limits the CaaX processing of Hsp40 Ydj1p and regulates Ydj1p-dependent phenotypes.

The modifications occurring to CaaX proteins have largely been established using few reporter molecules (e.g. Ras, yeast a-factor mating pheromone). These proteins undergo three coordinated COOH-terminal events: isoprenylation of the cysteine, proteolytic removal of aaX, and COOH-terminal methylation. Here, we investigated the coupling of these modifications in the context of the yeast Ydj1p chaperone. We provide genetic, biochemical, and biophysical evidence that the Ydj1p CaaX motif is isoprenylated but not cleaved and carboxylmethylated. Moreover, we demonstrate that Ydj1p-dependent thermotolerance and Ydj1p localization are perturbed when alternative CaaX motifs are transplanted onto Ydj1p. The abnormal phenotypes revert to normal when post-isoprenylation events are genetically interrupted. Our findings indicate that proper Ydj1p function requires an isoprenylatable CaaX motif that is resistant to post-isoprenylation events. These results expand on the complexity of protein isoprenylation and highlight the impact of post-isoprenylation events in regulating the function of Ydj1p and perhaps other CaaX proteins.

Older, Less Regulated Medical Marijuana Programs Have Much Greater Enrollment Rates Than Newer 'Medicalized' Programs.

Twenty-three states and the District of Columbia have passed laws implementing medical marijuana programs. The nineteen programs that were in operation as of October 2014 collectively had over one million participants. All states (including D.C.) with medical marijuana laws require physicians directly or indirectly to authorize the use of marijuana at their discretion, yet little is known about how medical marijuana programs vary regarding adherence to basic principles of medical practice and associated rates of enrollment. To explore this, we analyzed marijuana programs according to seven components of traditional medical care and pharmaceutical regulation. We then examined enrollment rates, while controlling for potentially confounding state characteristics. We found that fourteen of the twenty-four programs were nonmedical and collectively enrolled 99.4 percent of participants nationwide, with enrollment rates twenty times greater than programs deemed to be "medicalized." Policy makers implementing or amending medical marijuana programs should consider the powerful relationship between less regulation and greater enrollment. Researchers should consider variations across programs when assessing programs' population-level effects.