Hemp Growing

The stability in adolescents’ e-cigarette preferred type or brand also has not been examined. E-cigarette brands that are popular today among adolescents can deliver nicotine from a single compact pod that equals that of a pack of cigarettes, in attractive flavors, and with easy concealment for use in settings where cigarettes may be forbidden . These characteristics may facilitate the progression from intermittent to frequent use and nicotine dependence. Alternatively, low nicotine content and/or low device appeal may result in adolescents losing interest in e-cigarettes over time, with diminishing frequency and dependence risk. Among adults, research indicates that evolving from a simpler e-cigarette device to a more complex modifiable device is a common pattern and is associated with greater dependence on e-cigarettes. Despite rapid growth in the e-cigarette market in recent years, research has not yet examined whether or how adolescents’ preferred devices change over time, particularly with regard to nicotine delivery and exposure. Finally, minimal research has examined changes in adolescents’ reasons for initiating, continuing, and/or quitting e-cigarette use over time. In cross-sectional survey studies, adolescents’ top reasons for experimenting with e-cigarettes include curiosity, appealing flavors, friends’ use, and perceived benefits compared with cigarettes. However, reasons may shift over time, as adolescents move from experimentation to sustained use. The literature on youth initiation and transition to regular use of combustible cigarettes shows that media/marketing and social influences motivate initiation,hydroponic table whereas the drive for nicotine due to addiction motivates regular use. These nicotine product use patterns observed with combustible cigarettes warrant investigation with e-cigarettes.

With e-cigarettes, adolescents who begin experimenting because of curiosity or appealing flavors may subsequently use to alleviate withdrawal symptoms. The present study followed a cohort of adolescent e-cigarette users over 12 months’ time to examine patterns of e-cigarette use frequency, nicotine exposure, and dependence, product use and flavor preference, and motivators to use and cease use. The primary objectives were to determine persistence in e-cigarette and dual use and the stability in frequency and dependence measures of e-cigarette use. We also examined changes in device and e-liquid preferences and reasons for using e-cigarettes. This longitudinal study adds to the literature by providing an understanding of shifts in tobacco and nicotine product use over time among adolescents based on self-report and biomarkers of exposure.Adolescents from the San Francisco Bay Area who reported having used an e-cigarette at least once in the past 30 days and at least 10 times in their lives were recruited for a longitudinal study on teen vaping between May 2015 and April 2017. Advertisements were posted on social media and in the community around the Bay Area. Interested individuals were directed to the study Web site, where they could submit their information to be contacted by study staff to complete eligibility screening. Eligible participants who provided informed consent were scheduled for a baseline session where they completed self-report measures and provided a saliva sample for cotinine testing. Participants returned for follow-up measures and cotinine testing 6 and 12 months after baseline. Study incentives were $30 for the baseline, $35 for the 6-month, and $40 for the 12-month follow-up visits. Parental consent was waived under California law 6929. Cessation information and local treatment options were provided. The research design and study procedures were approved by the University of California, San Francisco Institutional Review Board.In this longitudinal study of adolescent e-cigarette use with self-reported and biomarker data, 80.3% of the sample continued to use e-cigarettes at 12 months, with significantly greater ecigarette use frequency, dependence, and nicotine exposure. The percentage of daily e-cigarette users doubled from 14.5% at baseline to 29.8% at 12-month follow-up.

The patterns of ecigarette use observed over time indicate substantial persistence and the use of greater amounts of nicotine over time . These findings lend support to concerns regarding the addictiveness of e-cigarettes for adolescents. In the United States, prevalence of past-month e-cigarette use increased dramatically among adolescents in 2018, whereas cigarette use declined and cannabis use remained constant. Results of this study suggest that increased prevalence of recent e-cigarette use may lead to frequent use, dependence, and greater nicotine exposure. Dependence scores at baseline were low on average, with most participants meeting a classification of “not dependent,” and 13.3% meeting a classification of moderate to heavy dependence. By 12 months, the percentage classified with moderate to heavy dependence increased to 23.3%. These findings would suggest that factors other than dependence are driving early use of e-cigarettes, and that over the course of just 1 year, more teens become daily users and more heavily dependent. Along with the self-reported increase in frequency of e-cigarette use and dependence, cotinine levels increased over time, reflecting increased exposure to nicotine. The increase in cotinine levels may be both the result of increased dependence and a catalyst for the development of dependence. Adolescents who become increasingly dependent on e-cigarettes may increase their nicotine use, thereby worsening dependence. Notably, devices with higher nicotine yield became increasingly popular over the course of the 12-month trial, consistent with the reports of greater nicotine dependence and higher cotinine levels. Transitions from single to dual and dual to single nicotine product use were observed in approximately one in three users over the study period. None of the baseline dual users abstained from both products at either follow-up, which may be partially due to their higher dependence on e-cigarettes at baseline, as well as the normalization of smoking behavior and associations between smoking cues that can perpetuate use of both products. Consistent with prior research, adolescent participants offered a wide range of reasons for e-cigarette use. The top three reasons for initiating and continuing use were socializing, enjoyment, and flavors. The top three reasons for quitting were a desire for self-improvement, difficulty maintaining an e-cigarette device, and getting in trouble for vaping at home or school. The top flavors were fruit, menthol/mint, and candy.

Taken together with experimental research demonstrating the influence of flavors on adolescents’ product choices, these findings suggest that efforts to reduce adolescent e-cigarette use ought to include regulatory action that addresses kid-friendly flavors. Little research has examined adolescents’ reasons for quitting e-cigarette use, and our findings preliminarily suggest that adolescents perceive parental controls and appropriate disciplinary consequences to be impactful.In 2018 in the United States, over 650 bicyclists died, and there were almost 158,000 bicycle-automobile collision–related injuries.1 Current bicycle injury prevention measures that have been proven include bicycle helmet programs and bicycle helmet laws.Promising prevention measures include active lighting, increased rider visibility, and roadway modifications.Trauma registries can be used to identify modifiable injury risk factors for trauma prevention efforts, including bicyclist collisions with automobiles. However, these may miss factors useful for prevention of bicycle-automobile collisions, such as vehicle speeds, driver intoxication, and patient group characteristics, such as financial stress, educational level, and languages spoken. Geographic information systems use software that can relate seemingly unrelated data to provide better understanding of spatial patterns and relationships. The GIS studies in the trauma literature include optimizing trauma center location,identifying under serviced areas for quality trauma care,hot spot and cluster analysis for traffic collisions,and helicopter basing and efficacy.Trauma registries typically already contain some geospatial data, such as home and injury location addresses. Traffic records databases contain details about bicyclist automobile collisions not typically found in trauma registries, such as vehicle speeds, driver intoxication,grow rack street and lighting conditions, and driver or cyclist fault.These records also include accurate exact collision locations, allowing GIS mapping. The GIS analysis also allows the use of census tract demographic data for both the collision location and patient’s home for analysis.We hypothesize that GIS analysis of trauma registry data matched with a traffic records database could identify additional risk factors for bicycle-automobile injury helmet use or intoxication. We also hypothesize that the addition of GIS analysis to the trauma registry will better inform injury prevention efforts.The trauma registry of the UC San Diego Level I trauma center was used retrospectively to identify bicycle-motor vehicle collision admissions from January 1, 2010, to December 31, 2018. Data collected included demographics, home and injury location addresses, injury severity scores, blood alcohol, toxicology, helmet use, hospital length of stay and mortality. Matching of the registry cases with the California Statewide Integrated Traffic Records System was done to provide collision, bicyclist, driver, and geospatial information for bicycle-automobile collisions within the County of San Diego for the same period. Statewide Integrated Traffic Records System is administered by the California Highway Patrol, and includes all traffic collisions in California with a law enforcement report. Matching was deterministic and was done by bicyclist age, bicyclist sex, zip code, date and time ±1 hour between the SWITRS collision time and the trauma registry admission time transfers from outside the County of San Diego were excluded. Outcomes of interest included toxicology—available as “Alcohol Involved” in SWITRS collision data and “Party Sobriety” under SWITRS party data or from blood alcohol and urine toxicology in the trauma registry. Helmet use was found as reported in SWITRS or the trauma registry. Missing variables in either database were managed by excluding such cases from analysis using that variable. Geocoding, mapping, and geospatial analysis of matched case SWITRS collision locations was done using ArcGIS Pro 2.6 .

Registry home addresses of cases were also geocoded and used with US Census Bureau census tract data to provide the below poverty level percentage for home census tracts. The educational attainment level for cases was done by selecting the predominant education level within the patient’s home address census tract from the US Census Bureau’s American Community Survey 2014 to 2018 5-year estimates, Table B15002. The language spoken at home was selected by home address census tract in the U.S. Census Bureau’s American Community Survey 2014 to 2018 5-year estimates, Table B16007. Locations of bike lanes in San Diego County for analysis of their associations with collisions were obtained from SanGIS for Bike Paths , which are physically separated from traffic; bike lanes , which are defined by pavement striping and signage on streets; and Bike Routes , which are shared use with motor vehicle traffic in the same travel lane and designated by signs only. Locations of alcoholic beverage control licenses in San Diego County for analysis of association with intoxicated bicyclist collisions was obtained from the California Department of Alcoholic Beverage Control via SanGIS and esri. The spatial clustering of bicycle-automobile collisions and hot spots were assessed using spatial auto correlation via the Getis-Ord Gi* statistic in ArcGIS Pro.Analyses of descriptive and tabular data were performed with IBM SPSS Statistics 27. χ2 Analysis was done for categorical data, means were compared via ANOVA and distributions of educational level, LOS, and head-neck Abbreviated Injury Scale were compared with the Mann-Whitney U test. Two-sided p values less than 0.05 were considered significant. Binary logistic regression was used for the outcome variables of helmet use and toxicology, selecting factors with a p value less than 0.01 in the univariate analysis. The SWITRS data were used under the terms of the data use agreement provided by the California Highway Patrol. The study was exempted from further review by the UC San Diego Human Research Protections Program.We have shown that GIS analysis of trauma registry data matched with a traffic accident records database can identify additional risk factors for bicycle-automobile injuries. We have also shown that our injury prevention efforts will be better informed by the hotspot analysis which clearly demonstrates clusters in specific geographic areas of the catchment area. The ability to add census tract data to registry data shows associations of education level and poverty level on bicycle helmet use. Census tract data also provides useful information for injury prevention efforts such as the predominant language spoken at home in target areas. Trauma registries, whether trauma center-based or nationally collected, should be constructed to allow geospatial analysis. Helmet use by bicyclists has been shown to reduce the risk of serious injury.Despite this, we saw relatively low use of helmets in admitted bicyclists.This may be reflected in our results showing adverse census tract poverty level and educational levels having an association with lack of helmet use. Unhelmeted cyclists in this study were also less likely to be discharged to rehabilitation or long-term care facilities, this is likely due to their being relatively underinsured compared with helmeted cyclists.

Adjacent research has found that sleep quality is malleable and thus can be improved even in clinical populations.These findings offer promising evidence for advancement in clinical staging models and future sleep-related therapies for both CHR and overt psychosis, such as an upcoming trial by Waite et al.Another budding direction of research involves computational advances in causal discovery analysis, offering innovative approaches to addressing causality in the context of observational data.Such methods require careful consideration of assumptions and properties underlying the data at hand,but could be utilized in future analyses of these and other prospective longitudinal datasets involving CHR and other clinical populations. Limitations of this study include the use of a self-report to assess sleep. The PSQI and the RU-SATED have high validity and reliability and subjective sleep problems have been the primary focus of sleep treatments. Such assessments, however, are fundamentally different from electrophysiological sleep characteristics that may be differentially associated with conversion and symptom levels. Therefore, the use of both self-report and electrophysiological sleep measures over time may inform clinical risk models and constitute targets for intervention. Other notable limitations include the significant dropout rates, the absence of ongoing sleep assessments succeeding the 8-month follow-up, and the relatively small sample size of our converting group . Furthermore, we acknowledge that pre-registering our study hypotheses would have strengthened our findings.The COVID-19 pandemic has produced major disruptions in daily life. A recent review concluded that the world population is experiencing increased stress, anxiety, and depression due to the pandemic and associated mitigation measures . In late June 2020, 40% of U.S. adults reported experiencing mental health symptoms,hydroponic table a stress disorder, or increasing substance use to cope with pandemic-related stress . Individuals with problematic substance use are particularly vulnerable to COVID-19 illness and psychosocial effects of the pandemic, such as stress and substance use treatment disruptions .

Disruptions in treatment increase risk of overdose, a serious complication of substance use disorders . Moreover, depression and anxiety commonly co-occur with substance use disorders . Evidence suggests that in the general population, increases in alcohol consumption during COVID-19 may be driven by stress, depression, and anxiety . Effects may be even more pronounced among individuals with problematic substance use. Periods of intense stress can give light to pre-existing fissures and vulnerabilities in people’s daily lives. A better understanding of substance use problems, depressive symptoms, and anxiety during the COVID-19 pandemic among people with problematic substance use can inform intervention efforts needed now and post-pandemic. Various pandemic-related situational factors may differentially affect substance use problems . According to behavioral economics, the COVID- 19 pandemic and associated mitigation measures may decrease the negative consequences of substance use . Many individuals are now working from home and have a more flexible schedule, making some of the negative consequences of substance use less relevant than before the pandemic. Simultaneously, the pandemic has limited the availability of alternative rewarding activities that are incompatible with substance use . Substance use and associated problems may increase if individuals are home more often and unable to engage in their usual activities, especially in the evenings . Moreover, other responsibilities at home may have increased during the pandemic. New consequences associated with substance use may emerge as lifestyles, schedules, and responsibilities shift. In sum, relationships between pandemic-related lifestyle factors, substance use, and substance use problems are likely complex and multifaceted. To better understand how individuals with problematic substance use are experiencing the pandemic, this exploratory, cross-sectional study examined associations between substance use problems, mental health symptoms, and pandemicrelated increased family responsibilities and stressors. Participants were recruited June 25 to August 18, 2020 for a randomized controlled trial of Woebot for Substance Use Disorders, a novel digital therapeutic for reducing problematic substance use.

Participants were recruited through Qualtrics Research Services, Stanford University listservs, a Facebook advertising campaign, and word-of-mouth. Eligibility criteria were U.S. residence, age 18-65, English literate, owning a smart phone, with past-year use of a substance, and problematic substance use . Exclusion criteria were history of severe alcohol or drug withdrawal, liver conditions, opioid overdose, or psychotic symptoms; past-year suicide attempt; past-year medical problems from drug or alcohol use; opioid use without concurrent medication-assisted treatment; past Woebot use; and pregnancy. The study protocol, approved by Stanford IRB, randomized consented participants to the Woebot for Substance Use Disorders intervention for 8 weeks or to a waitlist control condition. Only baseline measures, completed prior to randomization, were analyzed in the present study. Participants received a $25 Amazon gift card for completing the baseline survey.Correlations between substance use problems, pandemic impacts and behaviors, and mental health symptoms are presented in Table 1. Significant correlations indicated that participants who, during COVID-19, struggled with responsibilities at home, had greater mental health impacts, greater personal growth, frequented bars or large gatherings, and reported more depression and anxiety symptoms had higher SIP-AD scores. Participants who, during COVID- 19, struggled with responsibilities at home, had difficulty getting necessities, had greater mental health impacts, and worried more about their children had higher GAD-7 and PHQ-8 scores. Anxiety and depression scores were highly correlated. Participants who lost a job or income during the pandemic had higher PHQ-8 scores. Struggling with home responsibilities was associated with job or income loss, greater difficulty getting necessities, greater pandemic mental health impacts, less personal growth during the pandemic, avoiding large gatherings, and avoiding school or work. Difficulty getting necessities was associated with greater mental health impacts. Personal growth was associated with lower likelihood of avoiding large gatherings, and worrying about one’s children was associated with greater likelihood of avoiding non-essential travel. The COVID-19 precautions were significantly associated with each other. Multi–variable analyses are presented in Table 2. Participants with greater pandemic related mental health effects had higher SIP-AD, PHQ-8, and GAD-7 scores.

Younger participants had higher SIP-AD scores, and participants without a college degree had higher GAD-7 scores . Age was not associated with GAD-7 or PHQ-8 scores, and education was not associated with SIP-AD or PHQ-8 scores. Sex, race/ethnicity, marital status, and other pandemic-related variables were not associated with SIPAD, GAD-7, or PHQ-8 scores. Because only participants with children completed the “worries about children” measure, we repeated the GAD-7 and PHQ-8 models excluding this variable. In the full sample, females had higher GAD-7 scores than males . Otherwise,grow rack only pandemic-related mental health effects remained associated with GAD-7 and PHQ-8 scores. During the COVID-19 pandemic, 40.6% of surveyed adults with substance use problems reported struggling with responsibilities at home. Struggling with responsibilities at home during the pandemic was associated with more substance use problems and greater depression and anxiety symptoms. Obtaining, using, and recovering from substances can impair one’s ability to fulfill obligations. New responsibilities resulting from the pandemic, such as full-time childcare due to school closures, may be difficult to fulfill while struggling with substance use. Results are consistent with extant literature showing that unpaid caregiving during COVID-19 was associated with increased substance use . Individuals struggling with substance use, anxiety, and depression may need support in meeting their own needs as well as their family’s needs during the pandemic. Additionally, frequenting bars or large gatherings, experiencing negative mental health effects, and perceiving more positive personal growth from the pandemic were associated with more substance use problems. Difficulty getting necessities, experiencing more negative mental health effects, and greater worry about one’s children’s well-being was associated with greater depression and anxiety symptoms. In multi-variable models, controlling for demographic characteristics, negative mental health effects of the pandemic were the strongest correlate of substance use problems, depressive symptoms, and anxiety symptoms. Participants with high scores on this measure reported frequently thinking about COVID-19, worrying about their health and/or the health of their family and friends, experiencing stress due to changes in social contacts and their lifestyles, worsening of their mental/emotional health, and sleep disruptions. Findings suggest that the COVID-19 pandemic is producing major concerns that may contribute to mental health symptoms, including problematic substance use. Many individuals are using substances to cope with stress and uncertainty around the pandemic . A nationally representative sample of U.S. adults conducted early in the pandemic identified increased risk of depressive symptoms among people with lower income, fewer savings, and more stressors . People with problematic substance use may also be at elevated risk for depressive symptoms. While some evidence suggests that pandemic-induced psychological distress is lessening in the United States , people with problematic substance use are vulnerable to the negative effects of the pandemic . In this study, participants with more substance use problems were less likely to avoid bars and large gatherings, corroborating concerns that substance use may increase risk of contracting COVID-19 . Participants struggling to control their substance use may have found it difficult to avoid settings in which they use. Paradoxically, individuals with greater substance use problems also perceived greater personal growth from the pandemic in the forms of strengthened relationships, new possibilities, awareness of personal strength, spiritual change, and increased appreciation of life. People with problematic substance use often experience intense emotions .

Experiencing intense emotions may have led individuals with substance use problems to be deeply affected by both positive and negative pandemic-related changes. Additionally, perceiving greater personal growth was associated with lower likelihood of struggling with responsibilities at home and lower likelihood of avoiding large gatherings. Participants who perceived personal growth may be a subset whose daily lives were less strongly affected by the pandemic. Study data are cross-sectional, and causal pathways cannot be determined. There may be bidirectional relationships between substance use problems and pandemic-related mental health symptoms and stressors. While pandemic-related stress may have worsened mental health symptoms and substance use, it is also plausible that individuals with preexisting mental health symptoms and more substance use problems were negatively impacted by the pandemic than those with milder symptoms. Longitudinal research is needed to fully understand how substance use and pandemic-related circumstances may impact one another. Results are also subject to recall bias, as all measures were self-reported. Participants may have had difficulty accurately reporting their substance use and mental health symptoms from the past two weeks. Data were not collected on general life stressors unrelated to the pandemic. Individuals with high levels of stress may have experienced more pandemic-related stressors, mental health symptoms, and substance use problems. Lastly, the sample was predominantly non-Hispanic white. People of color are at increased risk of contracting and experiencing complications from COVID-19 . Moreover, Hispanic and Black individuals were more likely to report increased substance use than non-Hispanic white or Asian adults, potentially due to increased stress . Different vulnerabilities may interact to influence experiences of the pandemic. All participants were enrolled in a clinical trial, were not experiencing severe medical problems from their substance use, owned smartphones, and were proficient in English. Hence, findings may not generalize to more impoverished, medically complicated, or diverse groups. Future research into pandemic-related stressors and substance use should aim to recruit a more diverse sample. Smoking and alcohol misuse often co-occur. In the United States, the prevalence of nicotine dependence among individuals with alcohol dependence is 45.4%, while the prevalence of any alcohol use disorder among adults with nicotine dependence is 22.8% . These co-dependent individuals have more difficulty quitting smoking . An outstanding problem among those with substance use disorders is their disproportionate valuation of the drug and their disproportionate allocation of resources to obtaining the drug compared to participating in other daily activities . This imbalance between drug-related vs. regular activities reflects reinforced drug consumption patterns , and the differences in how drugs and non-drug reinforcers exhibit differential reinforcement strengths can be operationalized using a concept known as Relative Reinforcing Efficacy . One validated laboratory approach to measuring the RRE of drugs is hypothetical purchasing tasks, which assess changes in drug purchase and consumption as a function of increasing drug price . The consumption pattern can yield the demand curve modeled by Q = Q0∗10k , an exponentiated version of the classic equation by Hursh and Silberberg . Q represents consumption at price C; Q0 represents consumption at or near price zero, α represents the rate of change in demand elasticity, and k is the span of consumption values in log units. Other demand indices derived from the demand curve include: break point , Omax , and Pmax .

To bypass the thiolase in the pathway, we chose a new source for 3-ketovaleryl-CoA, namely betaoxidation of valeric acid. Like fatty acid oxidation, the new pathway first activates valeric acid, a biomass-derived chemical, into valeryl-CoA by a CoA ligase. An acyl-CoA dehydrogenase then oxidizes valeryl-CoA into 2-pentenyl-CoA, which is then transformed by an enoyl-CoA hydratase and a 3- hydroxyacyl-CoA dehydrogenase into 3-ketovaleryl-CoA . We believed this pathway would be more efficient than the PhaA-dependent pathway for bypassing the highly reversible and promiscuous thiolase-catalyzed reaction. We then tested the proposed beta-oxidation LMVA pathway, which accepts valeric acid into C6-isoprenol. Two plasmids were constructed to express the pathway : 1) pJL01, modified from pJL15, containing genes encoding NudB, Micrococcus luteus acyl-CoA dehydrogenase, and E. coli FadB, a bifunctional enzyme that catalyzes the hydration and dehydrogenation reactions ; 2) pJL02, modified from pJL10, containing the genes that encode the enzymes in the LMVA pathway and a Cannabis sativa acyl activating enzyme, CsAAE1, for valeryl-CoA production . E. coli BL21 transformed with pJL01 and pJL02 was grown in the presence of valeric acid and induced to express the pathway genes. GC-FID and GC-MS confirmed the production of C6-isoprenol at 27.3 mg/L . In the production broth, we also detected isoprenol at 5.8 mg/L . Compared to the thiolase LMVA pathway, C6- isoprenol production increased fourfold, and the ratio of C6-isoprenol with isoprenol increased to 4.7 from 1.3. Next, we screened CoA ligase and acyl-CoA dehydrogenase homologs, catalyzing the beta-oxidation pathway’s first two steps. We chose CoA ligase and acyl-CoA dehydrogenase candidates that have been overexpressed in E. coli and assayed in vitro in previous studies. Moreover, most of these candidates have known kinetic data for medium chain fatty acid substrates .

For the CoA ligase,plant bench indoor we selected the phenylacetate-CoA ligase from Streptomyces coelicolor A3 , ORF26 from Streptomyces aizumensis , the medium-chain fatty acyl-CoA ligase from Streptomyces sp. SN-593 , and the CoA ligase from Penicillium chrysogenum , in addition to CsAAE1 from Cannabis sativa. For the acyl-CoA dehydrogenase, we selected the short-chain acyl-CoA dehydrogenase from Pseudomonas putida KT2440 and the butyryl-CoA dehydrogenase from Megasphaera elsdenii , in addition to the acyl-CoA oxidase from Micrococcus luteus. The gene sequences were codon-optimized for E. coli and cloned into the pJL02 and pJL01 plasmid series . With the constructs in hand, we tested C6-isoprenol production in E. coli. First, we screened the CoA ligases, catalyzing the first step in beta-oxidation, with the well-characterized MeD as the acyl-CoA dehydrogenase. After production, we used GC-FID to quantify C6-isoprenol in the broths. The C6-isoprenol production varied substantially with different CoA ligases, suggesting this is a key step in the whole pathway. Among the genes we tested, PcCL gave rise to the highest production at 58.6 mg/L , a result consistent with the reported kinetics data . Also, we noticed that the combination of CsCL with MeD had a decreased production of 6.9 mg/L, down from the 27.3 mg/L produced by CsCL/MlD, suggesting that MlD is a better homolog for the second step . This hypothesis was supported by the screening results of the second step catalyzed by acyl-CoA dehydrogenase. The PcCL and MlD pair gave the highest C6-isoprenol production at 110.3 mg/L, so we used this pair of genes for the first two steps of the beta-oxidation pathway in the following experiments. Endpoint optical density analysis was performed to evaluate the impact of the expression of different beta-oxidation genes on cell growth. The results indicated the expression of the CoA ligases and acyl-CoA dehydrogenase barely impacts the growth. The slight growth inhibition effect in the high C6- isoprenol production, e.g., the PcCL/MlD, may result from the toxicity of C6-isoprenol.

After optimizing the pathway genes to increase the production efficiency, we turned to the host genes that may degrade the key intermediate, 3-ketovaleryl-CoA. As mentioned before, this intermediate can be degraded by a thiolase into acetyl-CoA and propionyl-CoA. Therefore we focused on two E. coli chromosomal thiolase genes, atoB and yqeF, which have substrate preference for short-chain betaketoacyl-CoA and were proposed to degrade 3-ketovalerylCoA. We conducted in-frame single-gene knockouts to delete these two genes in E. coli BL21 in sequence, and PCR confirmed the genotypes of the knockout mutants . The intermediate strain E. coli BL21 ΔatoBand the final double knockout strain E. coli BL21 ΔatoB ΔyqeF were then used for C6-isoprenol production using the plasmid combination of pJL01-MlD/pJL02-PcCL. The production of C6-isoprenol and the consumption of valeric acid were quantified. The results showed that while the knockout of atoB did not impact the C6-isoprenol titer, the double knockout strain, 6C02, increased the C6-isoprenol production to 390 mg/L, and the C6- isoprenol yield from valeric acid doubled to 44 mol% over the wild-type strain . We noticed even after knocking out the thiolase genes, only around half of the valeric acid is transformed into C6-isoprenol. The loss of valeric acid may come from the evaporation and the flux into side directions in the metabolic network, such as the degradation of 3-ketovaleryl-CoA by other thiolases in E. coli .In the C6-isoprenol runs, we noticed that the levels of isoprenol were generally negatively correlated to the levels of C6-isoprenol. For the sources of isoprenol, we reason that in addition to the E. coli native MEP pathway, the LMVA pathway may contribute the major portion via acetoacetylCoA, instead of 3-ketovaleryl-CoA, to C6-isoprenol. Through the LMVA pathway, the productions of C6-isoprenol and isoprenol use the same precursor, acetyl-CoA, resulting in the negatively correlated levels of C6-isoprenol and isoprenol. Also, the knockout of the short-chain acyl-CoA thiolase increased the supply of acetoacetyl-CoA and the production of IPP, resulting in an increased isoprenol titer of 14.4 mg/L compared to 2.1 mg/L for the wild-type strain. 

Hence, we proposed that acetoacetyl-CoA is readily accepted by the LMVA pathway, and it’s betaoxidation precursor, butyric acid, might be a substate of the beta-oxidation LMVA pathway. To test this hypothesis, we fed 1 g/L butyric acid instead of valeric acid to strain 6C02 containing pJL01-MlD and pJL02-PcCL. After production, GC-FID and GC-MS confirmed the isoprenol production, quantified at 301.8 mg/L . This result validates butyric acid is a good substrate for the beta-oxidation LMVA pathway for IPP/isoprenol production. The successful transformation of butyric acid into C5 alcohols by C4A suggests that the beta-oxidation LMVA pathway has substrate promiscuity. To explore the substrate spaces of this pathway, we tested other fatty acids as substrates. Without supplementing hexanoic acid, E. coli 6C02 with the beta-oxidation LMVA pathway also produces a small amount of C7-isoprenol, confirmed by the synthetic standard using GC-FID and GC-MS . Supplementing hexanoic acid increased the production of C7-isoprenol significantly . Therefore, the betaoxidation LMVA pathway can activate hexanoic acid and transform it to C7-isoprenol, albeit with low efficiency. C7-isoprenol production without hexanoic acid supplementation is likely from endogenous hexanoyl-CoA in E. coli. We also tried fatty acid analogs with functional group substitutes, including 5- chloro-valeric acid,greenhouse rolling racks 4-pentenoic acid, 4-amino-butyric acid, 5,5,5-trifluorovaleric acid, and 4- bromobutyric acid. We conducted comparative GC-FID/GC-MS analysis and fragment search in GC-MS for lack of standards of the expected alcohol products. However, none of the expected substituted alcohols were detected, suggesting these fatty acid analogs are poor substrates for the beta-oxidation LMVA pathway . The substrate promiscuity assays suggest the beta-oxidation LMVA pathway can produce IPP and C7-IPP, expanding the product chemical space of the homoterpene biosynthesis platform. The isopentenols, including isoprenol and prenol, are drop-in alcohol biofuels and have versatile potential fuel applications: prenol is one of the top 10 Department of Energy Co-Optimization of Fuels & Engines gasoline blendstocks and has synergistic blending effects for research octane number , and isoprenol is the precursor of 1,4- dimethylcyclooctane , a high-performance jet fuel blend stock . Previous studies have shown that for alcohol biofuels, molecules with longer chain lengths have a better blend stability with conventional fuels, and are less hygroscopic than their shorter chain congeners . Our pathway to C6-isoprenol and C7-isoprenol from biomass-derived fatty acids makes it possible to produce these chain-extended isoprenol analogs sustainably. With the synthesized C6-isoprenol and C7-isoprenol, we were able to test some important fuel properties of these novel isoprenol analogs. We first estimated their water solubility based on their logP values. High water solubility contributes to the high hygroscopic nature of alcohol fuels, increasing the possibility of phase separation when blended with conventional hydrocarbon fuel. Also, for microbial bio-fuel production process, molecules with high logP and low water solubility will partition into the organic phase in a two-phase extractive fermentation, resulting in low product toxicity to the producing microbes . Increasing carbon chain length leads to decreasing polarity of alcohols, resulting in lower water solubilities. The logP of isoprenol analogs were determined using an HPLC method, with C4-C8 strain chain alcohols as references. The result revealed that the one-carbon increase of the chain length of isoprenol decreases the water solubility to 22.12 g/L from 65.36 g/L. Moreover, the addition of two carbons to isoprenol further decreases the water solubility to 6.6 g/L .

The decreasing trend of the water solubility was expected, and these data reflect the trend quantitatively. The energy density of the isoprenol analogs was determined by testing their gross heats of combustion using a standard method . The energy density tests revealed C6-isoprenol has a higher heating value of 35.524 MJ/kg, while C7-isoprenol had an HHV of 39.468 MJ/kg . These numbers are similar or higher to the HHVs of isopentenols tested in the same batch. We also determined the RONs of the 10% alcohol RBOB gasoline blends using the recently published AFIDA method . The results indicated that C6- and C7-isoprenols have comparable RON boosting effects to isopentenols, making these two chemicals potential blend stocks for gasoline blends. We previously constructed the homoterpene biosynthesis platform as a proof of concept that introduces terpene structural diversity at the precursor stage. Here we further optimized this platform towards practical application. The most significant change is the upstream pathway to the key intermediate, 3- ketovaleryl-CoA. Like the natural LMVA pathways, our previous pathway starts from propionyl-CoA, condensed by a thiolase into 3-ketovaleryl-CoA.First, thermodynamic analysis indicated the condensation reaction catalyzed by thiolase is endergonic with a positive Gibbs free energy change , suggesting the thiolase catalyzed condensation reaction is thermodynamically unfavorable. This calculation is consistent with the finding that PhaA homologs catalyze the degradation reaction better than the condensation reaction . Second, to our knowledge, almost all the reported thiolases that convert propionyl-CoA and acetyl-CoA into 3-ketovaleryl-CoA also convert two molecules of acetyl-CoA into acetoacetyl-CoA. Our later experiment using butyric acid as substrate in the beta-oxidation LMVA pathway suggests the LMVA pathway readily accepts acetoacetyl-CoA into IPP . Considering these two points and the relatively high concentration of acetyl-CoA in E. coli , we reasoned that it would be difficult for the thiolase LMVA pathway to make HIPP over IPP, complicating C16, C17 and C18 terpene biosynthesis. Instead, the beta-oxidation pathway is a more specific way to produce isopentenyl pyrophosphate analogs. Although background IPP production remains, either via the native MEP pathway or from endogenous acetoacetyl-CoA transformed by the LMVA pathway, the ratio of HIPP/IPP production is significantly improved, as the molar ratio of C6-isoprenol:isoprenol is over 60 in the production run using the E. coli BL21 ΔatoB host with 1 g/L valeric acid feeding. The high production of HIPP and its dominant content in the isopentenyl pyrophosphate analog pool will benefit future homoterpene biosynthesis efforts. Using isoprenol analogs as the final product, we successfully optimized the flux to HIPP in the homoterpene biosynthesis platform. The enzymes after the LMVA pathway leading to complex terpenes are more challenging to optimize because of their elusive enzymology and unusual substrates. Following HIPP production, an IDI is supposed to isomerize HIPP to HDMAPP. We could not detect the corresponding alcohol of HDMAPP, C6-prenol, in the E. coli production run with the expression of NudB and the thiolase LMVA pathway containing the IDI from Bombyx mori. While this IDI was confirmed in vitro to transform HIPP to HDMAPP specifically , the absence of C6-prenol in the production run suggests that this IDI does not work well in E. coli, or the hydrolyzed product of HDMAPP, HDMAP, is not well accepted by the E. coli endogenous phosphatases. Incorporating this IDI into the optimized beta-oxidation LMVA pathway may increase HDMAPP production by increasing substrate supply. Other Lepidopteran IDIs are also candidates for enzyme screening of this step.

All scenes include background music and audio effects consistent with the scene and the participants’interaction. The NTP Cue VR paradigm begins with 3 “test scenes,” which are approximately 3 minutes in duration, depending on participant comfort and abilities with the VR hardware. The first scene is the Practice Room. This is a square room with cubes systematically placed around corners of the room. The participants are asked to gaze at each of the boxes to confirm that the eye-tracking is functioning as intended. Then, the participants are asked to practice using the controllers to teleport to 4 different locations in the room. The second scene is the Practice Slider room, which instructs the participants how to answer the survey questions and provides the opportunity to practice adjusting the slider to answer the scales. The third test scene is the Blink Calibration room. In this scene, the participants are asked to blink 5 times after being prompted by an audio signal. The purpose of this room is to collect pupil diameter data when the participants actively blink to assist with increasing the accuracy of blink detection algorithms. Following the completion of the initial test scenes, the 2 mood surveys are presented, and the 6 scenes are pseudorandomized within scene type such that the general scene order is maintained . The participants are then placed in each scene for 5 minutes. The entire paradigm is approximately 30 minutes in duration.There are 2 types of data recorded within each scene, regular time series and event-based data that is recorded at event onset. Regular time series data are collected at every 10-millisecond interval , independent of the frame time. The following data are recorded periodically: timestamp, raw gaze intersection point,vertical agriculture position and forward direction of the participants’ headset, and pupil diameter and eye openness .

The following events and corresponding timestamps are recorded when they occur: blinks, including number of blinks and the object of gaze at the time of the blink; button presses on the controller, including time, button pressed, and object of interaction ; and object of gaze when eye gaze switches to a new object.However, this raycasting method did not perform well in our experiments, especially for very small objects, owing to the limited precision and accuracy of the eye tracker, microsaccades, etc. Therefore, for small objects of interest, we utilized the G2OM algorithm provided by the Tobii XR SDK, which is a machine learning–based object selection algorithm that aims to improve small object– and fast-moving object–tracking. Based on our testing, this algorithm improved object selection over the naïve method but still lacked selection quality. Thus, to further improve object selection, we introduced an additional mechanism to “lock” the object selection when an object is manipulated such that whenever a participant actively picks up a virtual object, the object selection algorithm will always select the picked object until the participant releases the object. If the participant is not interacting with an object, the G2OM algorithm is employed, or if no small objects are within the field, naïve raycasting is employed. To calculate eye-gaze statistics toward active and neutral cue objects, 4 dictionaries corresponding to 4 different types of objects are initialized prior to the start of participant involvement in the paradigm. These dictionaries are then used to store the cumulating gaze fixation or dwell time durations as values for individual objects belonging to each object and type. When a participant gazes at an object, the object is searched in the dictionary on the basis of its name and type. If the object was encountered before, the current fixation time is added to its cumulative fixation time. If the object had not been encountered before, a new entry is created for the object.

The fixation time is then calculated as the difference between the timestamp of current entry and that of the next line of entry. Following the completion of the paradigm, total fixation time indices are produced, which reflect the sum of values within each dictionary . The mean fixation time indices are also created, which reflect the total fixation time divided by the number of objects gazed at by the participant. Initially, we tested a measurement of eye openness, as calculated by the HTC SRanipal SDK, as an indicator for blink detection. However, given the lack of established thresholds of eye openness for blink detection, we instead chose to rely on estimates of pupil diameter. Consistent with previous studies, an eyeblink is herein defined as complete eyelid closure with the pupil covered for 50-500 milliseconds. For any given time point, we consider a missing pupil diameter reading as a possible complete eyelid closure where the pupil is completely covered by the eyelid. These eye closure durations are blink candidates. If either pupil is covered for less than 50 milliseconds, the candidate is discarded as it is more likely owing to noise or an eye tracker limitation. If either pupil is covered for more than 500 milliseconds, the candidate is also discarded as this is more consistent with a micro-sleep. Using this blink detection definition, the blink count for the majority of the current participants fell within 12-40 blinks per minute, which appears to align with the consensus of spontaneous blink rates in the literature.Following consent procedures, participants undergo an extensive clinical interview and complete several self-report questionnaires covering demographic, psychological health , and substance use domains. The TLFB has high test-retest reliability for intervals ranging from 30 to 360 days prior to the interview date, with an intraclass correlation coefficient=0.92 for “Total number of cigarettes smoked per interval”. Thus, past 90-day NTP use episode count from the TLFB was used in the quantitative analyses presented below.

All study interview and self-report data were collected and managed using REDCap electronic data capture tools hosted at the University of California, San Diego. Participants then undergo the NTP Cue VR paradigm,hydroponic stands which includes repeated assessments of subjective nicotine craving and scene relevance to the individual participant . Upon completion of the paradigm, additional assessments on VR-related outcomes such as VR presence and VR-related simulator or motion sickness are administered. The IPQ total score was calculated using a simple averaging method to obtain a single average perceived presence score ranging 0-100. Similarly, the SSQ was scored in concordance with procedures outlined to assess VR-specific sickness , which involves a simple averaging method to obtain a single average sickness score with a range of 0-100. These analyses include the first 31 participants to complete the study protocol; however, data were missing for some subjects on a subset of indices owing to technological difficulties . Owing to safety restrictions related to COVID-19, no biological verification of abstinence was conducted. Group differences are not being investigated in the present pilot analyses since the goal of this study is to describe the development and general validity of the paradigm and to maximize statistical power. Statistical analyses were conducted using a repeated measures t test or Pearson correlation framework. The threshold of significance was set at P<.05 for all analyses. SPSS Statistics for Windows software was used for all analyses. This report describes our approach to the development of a novel NTP cue VR paradigm designed to simultaneously induce and assess potential eye-based objective correlates of nicotine craving in naturalistic and translatable virtual settings. The preliminary statistical analyses support the potential of this paradigm in its ability to induce subjective craving while instilling a moderate sense of presence in the virtual world and only low levels of VR-related sickness. The preliminary results outline a potential context-specific effect of NTP-related attentional bias and pupil dilation in this pilot sample. Consistent with the literature on attentional bias and pupil dilation, we observed greater Active NTP versus Neutral control cue-related effects in 2 of the 3 Active scenes . The similarity observed in the pattern of effects between attentional bias and pupil dilation provides early evidence of a potential cross-validation of these metrics. No effects were observed for the EBR metric; however, the size of this effect, if present at all, may be smaller than we are currently able to detect with the limited sample. The observed reversal of attentional bias and pupil dilation toward neutral cues in the Driving scene warrants further investigation, given the large effect size. Potential explanations for this include the presence of especially engaging neutral cues in the Driving scene, as a 360° video of a busy city street is presented in the background, which participants report as entertaining to watch. Despite the overall bias toward neutral cues reflected in the global attentional bias metric, and within the Driving scene alone, participants with greater attentional bias toward NTP cues were found to endorse greater NTP use in the previous 90 days.

This effect appears to be driven by the higher-frequency NTP users in our sample and is consistent with the literature supporting the validity of attentional bias as a clinically important indicator of nicotine addiction. Additional analyses are planned to assess direct and indirect relationships between scene eye-related outcomes and relevance to the individual, scene-specific craving level, randomization of scenes, engagement with specific cues, and NTP use groups once more data are collected. This pilot study has several strengths and limitations. Strengths include the development of a cutting-edge VR cue-reactivity task that incorporates the latest technological advances in graphic design to increase translatability to the real-world and simultaneous assessment of multiple potential eye-related indices of cue-reactivity in a 3D virtual environment. Limitations include the absence of biological verification to confirm self-reported NTP use and the inability to investigate NTP use profiles in the analyses owing to limited power. Importantly, given the limited sample size, we caution against over interpretation of our results. It remains unknown whether the absence of significant results, particularly with respect to the correlations between objective eye-related indices and subjective craving ratings, are the result of limited power to detect these relationships or true independence of these indices. However, we believe that the general pattern of scene-related effects on attentional bias and pupil dilation are encouraging and warrant further study. The identification of reliable objective correlates of craving would allow for greater examination of the underlying neurobiological processes involved, and inform new avenues for the development of psychological and pharmacological treatments.PATTERNS OF ALCOHOL intake vary dramatically over the lifespan, with the heaviest drinking and steepest trajectory of increasing alcohol problems typically observed in the mid-teens to mid-20s . In the United States, a person’s first drink is likely to occur at about age 15, and by age 18, 70% of people have consumed alcohol, 35% were ever intoxicated, and 24% admitted to consuming 5 or more drinks on an occasion . It has been estimated that the first drink in the United Kingdom may occur closer to age 14, with 70% of students in that age range having consumed alcohol . Thus, heavy drinking during adolescence may be especially prominent in the United Kingdom, which ranks near the top among 35 European countries regarding several measures of drunkenness . One common alcohol-related adverse consequence is an alcohol-related blackout , defined as not being able to remember parts or entire periods of events that occurred while drinking and awake . Almost 50% of drinkers, including college students, have ever experienced an ARB , as have 80% of individuals with alcohol use disorders . The high rate of blackouts in people with AUDs prompted the inclusion of these phenomena in alcohol questionnaires and interviews , but their prevalence in non-AUD drinkers resulted in their omission from most AUD diagnostic criteria . Blood alcohol concentrations >0.300 g/dl are associated with a 60% rate of ARBs, especially for en bloc events, although fragmentary blackouts are observed with BACs as low as ≥0.06 g/dl . Higher drinking frequencies also relate to blackouts, perhaps reflecting their association with higher quantities . In addition, the rate of ARBs may be higher in individuals consuming other drugs that affect brain functioning . Thus, alcohol quantities and frequencies and the use of substances other than alcohol are important characteristics to consider as predictors of ARBs.

The data sources for our index of American libertarian conservatism originate from the Rand Corporation’s TL-354 state-level estimates of firearms per household, data from the Tax Foundation giving the highest marginal tax rate for each state, and data from the National Council of State Legislatures on the stringency of state laws regulating the private use of cannabis. The data for our index of American social conservatism includes CDC/Guttmacher Institute data on the number of abortion clinics by state per million residents, Public Religion Research Institute data on pre-Obergefell restrictions on samesex marriage, and the JRANK Legal Encyclopedia’s rating of U.S. state-level support for the legality of prayer in public schools. Table 1 presents summary statistics of our variables, showing differences in characteristics and COVID-safety behaviors, cases, and deaths between counties above and below a 50% county-level Trump vote in the 2020 election. On average counties with a majority Trump vote are 3.5 years older, have $5,473 lower median household income lower income, have a smaller percentage of Black, Latino, and Asian residents, have economies that are more oriented toward manufacturing and less toward services, and have somewhat poorer baseline health, ranking about 2.7 percentile points below the mean of non-Trump counties. Raw differences in mask-wearing show a 17.1 percentage point difference in means for mask-wearing between Trump and non-Trump counties,grow rack 0.50σ lower rate of sheltering at home, 12.9 percentage point lower rate of COVID vaccination, and a 1.12σ lower COVID-safety index, all significant at p < 0.001.

Raw differences in health outcomes show an additional 2,300 COVID cases and 53.4 additional deaths per 100,000 residents across counties, also both significant at p < 0.001. Figure 2 shows spacial correlations across U.S. counties by 2020 presidential voting, COVID-safety behaviors, and COVID deaths per 100,000. Figure 3 provides a series of plots by state that show self-reported mask-wearing by county-level Trump voter support. In nearly each map, the blue dots lie to the northwest of red dots, which show higher levels of county voter support for President Trump and lower rates of mask-wearing. Table 2 provides the results of our state-level fixed-effect estimations in which we regress COVID-safety behaviors on the 2020 Trump vote, controlling for county characteristics. All estimates use U.S. state-level fixed effects so that identification is off differences in the Trump vote and COVID-safety behaviors between counties within a given state. Our results examine three COVID-safety variables: county-level mask-wearing, sheltering at home, and COVID-vaccination rates. We aggregate these three variables into a COVIDsafety index using the method of Kling et al. , which is the standardized sum of each of these three individually standardized variables. The results in Column 1 of Table 2 suggest that a 10 percentage point increase in the county Trump vote is associated with a 3.9 percentage point decrease in the number of people in a county stating that they wear a mask ”all the time” in public . Column 2 shows a tightly estimated coefficient close to zero for sheltering at home. Column 3 indicates that a 10 percentage point increase in the county Trump vote is associated with a 5.1 percentage point decrease in the vaccination rate . A visualization of the significant mask-wearing and vaccination results are shown over the scatter plots provided in Figures 4A and 4B, respectively. Our estimates in Column 4 suggests that a 10 percentage point increase in the county Trump vote is associated with a 0.23σdecline in our COVID-safety index.

Estimates on control variables across columns indicate that COVID-safety behavior generally increases with county median age, median income, percent Latino, percent Asian American, percent employed in manufacturing, education and health, and baseline level of county health. In general the controlled estimates display COVID-safety behavior associations with the Trump vote that are lower than raw differences between Trump and non-Trump counties, but differences by the county Trump vote remain and are highly significant. The exception is the sheltering-at-home variable, which we do not find to be affected by the Trump vote once age, income, and other county level covariates are controlled.Table 3 shows the relationship between the county-level 2020 Trump vote and COVID cases and deaths. Vaccines began to be widely available in the U.S. roughly toward the end of February 2021, and columns 1 and 2 show the impacts on COVID cases, pre- and post-vaccine respectively. Here results indicate that a 10 percentage point increase in the county Trump vote is associated with 789 additional cases per 100,000 residents in roughly the first year of the pandemic in the U.S., and 1,394 additional cases in the pandemic’s first twenty months . Columns 3 and 4 of Table 3 show outcomes for COVID deaths pre- and post-vaccine, where a 10 percentage point increase in the county Trump vote is associated with 11.5 additional COVID deaths per 100,000 residents in the pre-vaccine first year of the pandemic and 27.6 additional COVID deaths per 100,000 country residents in the first twenty months of the pandemic . A visualization of these results is provided in Figures 5A and 5B. A comparison of the pre- and post-vaccine results in Table 3 is important because February 2021 represents both the first month after President Trump’s departure from office as well as the month during which U.S. vaccination rates most quickly accelerated among the general public. The differences between columns 1-2 and 3-4 show that the statistical relationship between Trump support and COVID infections and deaths not only failed to narrow after February 2021, but actually grew, rising from about 65.7 added cases and 0.96 deaths per month during the first twelve months of the pandemic to 76.2 cases and 2.00 deaths per month during the subsequent eight months.

This divergence may have occurred because the easing of public restrictions that accompanied the vaccination-availability phase of the pandemic may have actually elevated risks to the unvaccinated and those not wearing masks in public spaces, a phenomenon likely exacerbated by the deepening political polarization over COVID safety behaviors. How confident can we be that the higher rates of COVID infection in Trump-supporting counties resulted from reduced COVID-safety behavior? The largest and most influential controlled study to date estimating the causal effects of mask-wearing on COVID infection is the extensive randomized trail carried out by Abaluck et al. , who report results from a mask-wearing intervention implemented among 342,183 adults across 600 villages in Bangladesh. The Abaluck et al. study, referenced worldwide by governments as both motivation and guide for COVID-safety behavior, reports that the 27.9 percentage point difference between treatment and control groups, created by those induced to wear masks through the experiment, reduced symptomatic COVID infection in the treatment population by 0.91 percentage points within a two-month time-frame,greenhouse grow tables or that every single percentage point increase in mask-wearing reduced COVID cases by an average of 0.016 percentage points per month. Our results for the United States associate a 10 percentage point increase in the county-level Trump vote with a 5.11 percentage point reduction in mask-wearing and an increase in COVID cases of 1.39 percentage points over a twenty-month time-frame, or that every percentage point increase in mask-wearing reduced COVID cases by an average of 0.014 percentage points per month. Our non-controlled U.S. results are thus remarkably similar to the Abaluck et al. controlled estimates of the causal effect of mask-wearing on symptomatic COVID cases, and they suggest that higher levels of COVID infection in the high Trump-vote counties are unlikely to be due to unobserved county characteristics but rather are substantially mediated by differences in COVID-safety behaviors.In this section we ascertain to what degree the COVID-safety behaviors are specifically tied to a political identity specifically tied to Trump voter support relative to two traditional strains of conservatism in the United States: American social conservatism and American libertarian conservatism, which for many decades have formed key parts of the conservative political coalition embodied within the Republican party. Our Social Conservatism index is made up of three standardized and equally weighted state-level variables representing political issues strongly tied to social-conservative concerns in the United States. They include 1) the availability of abortion, given in terms of clinics per million residents; 2) restrictions on same-sex marriage, existing before federal legalization under the Obergefell v. Hodges, 576 U.S. 644 2015 Supreme Court ruling; and 3) the degree of support for public prayer in schools, as given from ratings by the JRANK Legal Encyclopedia.

Our Libertarian Conservatism index also consists of three standardized and equally weighted variables, representing issues of concern to libertarian conservatives, which embody a preference for minimal taxation, regulation, and interference in personal freedoms and private life by government: 1) low state taxes at the highest income brackets; 2) estimated per-capita firearms ownership; 3) the stringency of state laws regulating the private use of Cannabis. Consistent with the procedure of Kling et al. , after summing each standardized variable within the index, each index itself is then itself subsequently standardized. Table 4 shows our estimates in which we compare associations of our Libertarian Conservatism index, Social Conservatism indices, and Trump voter support with COVID safety behaviors, cases and deaths. To facilitate comparisons with these indices, we also standardize our Trump-vote variable. Overall, results show that once the Trump 2020 vote is taken into account, the Social Conservatism index and Libertarian Conservatism indices have very little systematic explanatory power over COVID-safety behaviors, cases and deaths. In these estimations, a one-standard deviation difference in Trump support decreases mask-wearing by 5.2 percentage points . While the Social Conservatism index is also statistically significant , it explains less than half the variation of Trump support.While neither standardized Trump support nor the Social Conservatism index is statistically significant in explaining sheltering at home, the Libertarian Conservatism index is actually positive and modestly significant . For vaccination rates, a one standard deviation difference in Trump support reduces the percent of adults vaccinated by 7.9 percentage points while the Social Conservatism and Libertarian Conservatism indices have the unexpected positive sign . The aggregated COVID-safety index shows that a one-standard deviation increase in Trump support decreases COVID-safety behavior by 0.45σ . Once Trump support is controlled, Libertarian Conservatism is actually positive with about half the effect but in the direction of increased COVID safety. As is the COVID-safety index, COVID cases and deaths are strongly associated with the 2020 Trump vote but are insignificantly associated with Social Conservatism and Libertarian Conservatism indices. A one-standard deviation increase in Trump voter support increases COVID cases by 2,075 per 100,000 and COVID deaths by 52.4 per 100,000 residents. These results are consistent with the fact that there are relatively weak historical ties between, for example, an anti-vaccination stance and traditional American conservatism. Indeed, some such as Berezow and Campbell trace the anti-vaccination movement to the political left rather than the political right, although there is evidence that in the last decade that anti-vaccination stances have migrated from the libertarian left toward the libertarian right . Rather than being closely tied to conservative ideology per se, numerous both pre- and post-COVID studies find the factor most strongly tied to an anti-vaccine stance to be a belief in conspiracy theories . A number of papers have linked reduced levels of COVID-safety behaviors to the Trump presidency itself . In a quantitative study of behavioral responses to social media activity, Germani and Biller-Andorno find Twitter posts by President Trump to be the strongest driver of information contradicting established scientific evidence regarding the COVID vaccines. Thus rather than originating from a particular strain of traditional American conservatism, this body of evidence taken together with our results in Table 4 strongly suggest an identity more specifically related to Trump political support to principally drive differences in COVID-safety behaviors, along with the accompanying COVID infections and deaths. that bleed across state lines. As a robustness check, we estimate our models in Tables 2, 3, and 4 with Conley standard errors that account for large spatial correlations in COVID cases and deaths. Using longitudinal and latitudinal coordinates, we employ a Bartlett linear decay of the spatial error correlation to a distance of 500 kilometers from the center of each county. In estimations without state-fixed effects, we account for spatially correlated errors 500 km from the center of each state.

Nevertheless, several lines of research suggest that individuals with substance use disorders, including MA users , have higher densities of D3 receptor levels in striatal and extrastriatal brain regions than those who do not frequently abuse drugs . Thus, it seems probable that the lower [18F]fally pride BPND among MA users primarily reflects lower D2 receptor availability in this group compared to controls. An additional limitation includes the possibility of competition with endogenous dopamine influencing [18F]fally pride BPND . That IQ was not assessed is also a limitation, because IQ has been found to be significantly correlated with delay discounting , and a group difference in the former could therefore overshadow the true group difference in the latter. There are also some caveats that should be considered when interpreting the results of this study. First, the MCQ has limited ability to provide precise estimates of discount rates for individuals who discount very steeply. That is, the choice items only probe preference up to a maximum k-equivalence value of 0.25, and this value was assigned as a conservative estimate of discount rate to individuals whose calculated k value was predicted to exceed this value . Second, BPND values were highly correlated across all VOIs examined in both groups of participants, which limits the ability to draw conclusions regarding the relative importance of D2 /D3 receptor availability in specific brain regions to discount rate. Finally, as there is some evidence that abstinence from drugs can increase temporal discounting among addicted individuals , it is possible that abstinence from MA may have amplified the difference in discount rate between MA users and controls. The results of this study may help to explain why low striatal D2 /D3 receptor availability is associated with poor treatment response among individuals with MA dependence and cocaine dependence . This view seems reasonable given that steep temporal discounting has also been linked with poor treatment response among cocainedependent individuals , and predicts relapse among smokers .

The present results lend empirical support to a theoretical model in which Trifilieff and Martinez propose that,cannabis dry rack “low D2 receptor levels and dopamine transmission in the ventral striatum lead to impulsive behavior, including the choice for smaller, immediate rewards over larger, but delayed or more effortful, rewards, which may represent an underlying behavioral pattern in addiction.” Consistent with this model, we found evidence of a negative correlation between discount rate and D2 / D3 receptor availability in the limbic striatal subdivision . The correlation in the limbic striatum did not reach statistical significance, possibly due to the high D3 /D2 receptor ratio in this region and partial volume effects. An important question for future research is to determine whether interventions that increase D2 /D3 receptor availability can reduce temporal discounting, at least among those with low D2 /D3 receptor availability. Pharmacological interventions could prove useful to this end. For example, varenicline increases striatal D2 /D3 receptor availability in rats , and in a study of human smokers, males treated with varenicline showed lower temporal discounting than placebo-treated controls . This finding is compelling considering that dorsal striatal D2 /D3 receptor availability is lower in male smokers compared to nonsmoker controls . There also is evidence that rimonabant increases striatal D2 /D3 receptor availability , and can decrease discounting of delayed rewards in rats . Nonpharmacological approaches may be useful as well, as there is preliminary evidence that intensive exercise can increase striatal D2 /D3 receptor availability in MA-dependent individuals and patients with early-stage Parkinson’s disease . Similarly, in a mouse model of Parkinson’s disease, higher striatal D2 /D3 receptor availability and D2 receptor expression was noted among those exposed to high-intensity exercise relative to non-exercising controls . Establishing a causal link between D2 /D3 receptor availability and temporal discounting is likely to have significant clinical implications.

This is because there is evidence that interventions that reduce temporal discounting are useful for treating disorders that are associated with both steep discounting and low striatal D2 /D3 receptor availability. For instance, contingency management decreases discounting among cocaine-dependent individuals and smokers , and methylphenidate decreases discounting in children with attention-deficit hyperactivity disorder . More importantly, greater reductions in discounting predict a greater likelihood of protracted abstinence among cocaine users and smokers . Thus, if a causative link between D2 /D3 receptor availability and temporal discounting is established, it may lead to the development of novel D2 /D3 -targeted interventions which could be used to more effectively treat a variety of disorders. In conclusion, the results of this study indicate that low D2 / D3 receptor availability is associated with steep temporal discounting. This link may explain why some individuals choose to continue using drugs despite knowledge of their future negative consequences, and could help to guide strategies for treating substance abuse and other psychiatric disorders.To date, this is the largest SC case series describing MABCHMINACA toxicity. In the fall of 2014, MAB-CHMINACA was responsible for more than 125 patients seeking hospital care in Baton Rouge, LA.It is a highly potent SC, which was just recently added to the Schedule 1 Controlled Substance Act in late 2015.The clinical manifestations observed in these patients appear similar to those described with other SC toxicities.The exact mechanism of MAB-CHMINACA toxicity is unknown. Affinities of SCs and their metabolites are multi-fold higher at the CB1 and CB2 receptors than that of Δ-9 THC and are thought responsible for such severe clinical manifestations.CB1 receptors are mostly located in the brain and regulate the central nervous system effects of Δ-9 THC and other cannabinoids; they are also expressed peripherally in adipocytes and skeletal muscle.The identification of CB1 receptors presynaptically on GABA and glutamatergic terminals with increased excitatory and decreased inhibitory tone may be responsible.Other postulated mechanisms include activation of non-cannabinoid receptors and drug-drug synergistic effects.Some patients demonstrated stimulant and serotonergic agent use as detected by comprehensive urine drug LC/ MS testing that could have contributed to their clinical presentation, such as sympathomimetic or serotonergic toxidromes. However, this was not universal and would not explain the fairly consistent clinical presentation of all of these patients.

Further, the relative concentrations of MABCHMINACA do not necessarily correlate with toxicity and may demonstrate the potential lipophilic distribution of the drug to tissue at the time of blood draw. For example, patient #11 had a relatively low concentration, yet expired. Interestingly, post-mortem analysis of a patient who died from MAB-CHMINACA toxicity demonstrated a relatively low amount in the adipose tissue, atypical of other SCs. The authors hypothesized the compound may require time to distribute to that tissue, and that may have been the salient issue regarding patient #11 given the length of time from medical attention.Limitations in real-time detection by routine toxicologic immunoassay screening appear to be a factor in SC use.The reasons for the abrupt, recent increase in exposures, however, remain unclear. The celebration of “4/20” correlated with this surge and might have influenced users. Management of the SC-intoxicated patient centers on meticulous supportive care, as no specific antidote currently exists. Decontamination is of little value. Given their beneficial pharmacokinetic profile, administration of benzodiazepines titrated to clinical effect can control agitation, delirium,hyperthermia and seizures. Cooling measures without antipyretic administration can also be implemented for hyperthermic patients.Endotracheal intubation may be required for the severely intoxicated patient.Serotonin plays a crucial role in emotional processes. A considerable number of imaging studies involving pictures of emotional faces show that changes in serotonergic function are associated with changes in amygdala reactivity when viewing negative facial expressions: Acute tryptophan depletion, which reduces central serotonin synthesis,planting racks leads to higher amygdala activity when processing negative face expressions . Several studies have found that acute/subacute SSRI intervention leads to a decrease in amygdala activation . Further, when the cerebral serotonin level is pharmacologically enhanced by a three-week intervention with a selective serotonin reuptake inhibitor , the ensuing decreased cerebral [11C]SB207145- PET binding in response to pharmacologically increased brain serotonin levels is associated with lower threat-related amygdala reactivity . Whereas it is relatively clear that induction of acute and subacute changes in serotonin neurotransmission leads to changes in emotional processing, less is known about how the neural processing of emotional information is affected by chronic cerebral serotonin depletion. Ecstasy, or 3,4-methylene-dioxymethamphetamine is a widely used recreational drug that has immediate effects in terms of improved mood and feelings of empathy . In this paper, we will use the term “ecstasy” when referring to the recreational human, and “MDMA” when referring to experimental human/animal studies. MDMA exerts its primary effects on the serotonin neurotransmitter system, in particular by reversing normal serotonin transporter function and hence releasing serotonin from the storage vesicles into the synaptic cleft . Animal studies show that repeated exposure to moderate and high doses of MDMA is associated with a reduction in cerebral serotonin levels and a decreased number of SERT binding sites. In humans, prolonged recreational use of ecstasy is also associated with reductions in SERT in both cortical and sub-cortical brain areas .

Most , although not all , molecular imaging studies show that the accumulated lifetime intake of ecstasy correlates negatively with SERT binding, supporting a dose-dependent relationship between recreational ecstasy use and reductions in SERT binding. Thus, it also seems plausible that in humans, an ecstasy-associated reduction in SERT is associated with reduced cerebral serotonin levels, and that the SERT changes may even result from chronically reduced serotonin levels. Additional support for serotonin depletion in recreational ecstasy use comes from the finding of increased levels of the post-synaptic serotonin 2A receptor in most , although not all , studies where ecstasy users have had their serotonin 2A receptors measured. Importantly, lowering brain serotonin levels in preclinical models leads to low SERT combined with high serotonin 2A receptor levels, and low SERT has also been found to be associated with high serotonin 2A receptor levels in humans . The reduction in subcortical SERT binding in ecstasy users seems to be reversible, since a positive correlation with time of abstinence from ecstasy intake has been observed . Taken together, data from these preclinical and clinical studies indicate that there is a causal relationship between ecstasy intake and effects on the serotonergic system. Reduced SERT and serotonin after MDMA/ecstasy exposure in combination with the observed correlation between serotonin depletion and reduced SERT binding in animal studies makes it plausible that SERT binding can be regarded as a representation of the extent of chronic—but most likely reversible—serotonin depletion in long-term ecstasy users. With MDMA being an interesting and promising candidate as adjunct to psychotherapy for treatment of post-traumatic stress disorder and possibly other conditions as well , it is important to explore the possible long-term impact of this drug on serotonergic neurotransmission, as well as the functional consequences of this. Although the multiple and not always pure MDMA doses used recreationally differ from the only few—and pure—doses employed in therapy, recreational use of MDMA/ ecstasy can serve as a model for the long-term effect of repeated doses. In the present study, we used functional magnetic resonance imaging to investigate the functional effects of long-term recreational ecstasy use, representing a model of long-term serotonin depletion, on the neural basis of emotional responses in the amygdala. We hypothesized that amygdala engagement to aversive stimuli would show a positive correlation with accumulated lifetime ecstasy use; a negative correlation with SERT binding, as assessed with positron emission tomography imaging, in the amygdala; and a negative correlation with time since last use of ecstasy, suggesting recovery of amygdala response. The degree of serotonin depletion as reflected by cerebral SERT binding was assessed with PET and the selective SERT radioligand, 11C-DASB, as described in more detail by Erritzoe et al. . In short, PET data were acquired on a GE-Advance scanner as a dynamic 90-minute emission recording after intravenous injection of the radiotracer, 11C-DASB. There was maximum of 1.6 months between the 11C-DASB PET and the fMRI experiment ; approximately half of the group had PET before MRI, and vice versa. The vast majority was scanned within one to two weeks; only two were scanned with an interval of more than a month. For each participant, a mean SERT binding value from the amygdala was calculated.

The optimal understanding of how ARBs develop requires considering a range of characteristics, preferably in a prospective study . The complex relationships with which multiple factors relate to ARB risks may indicate opportunities for more focused and efficient prevention by identifying subgroups most likely to experience ARBs and who are most likely to gain from programs aimed at decreasing heavy drinking. The larger study from which these data were extracted and a smaller investigation at another university indicated that active education about alcohol-related risk factors are associated with less intense future drinking. In the current study, the significant active education group vs. control group main effect in Table 4 supports the conclusion that decreases in maximum drinks seen with participation in the educational videos were also associated with lower levels of ARBs over time . Thus, universities and other institutions interested in decreasing the risk for ARBs and associated problems might consider developing similar education programs and focusing their efforts on subgroups of subjects with the highest ARB risk. As is true for all research, it is important to recognize caveats regarding the current work. The data were extracted from a larger study evaluating different ways of decreasing heavy drinking among students, and consistent with a prior report focusing on heavy drinking , exposure to active intervention affected ARB rates, a factor that complicates interpretation of results. However, as shown in Table 4, the current results remained robust when prevention group assignment was used as a covariate in the mixeddesign ANOVA. The relationships among ethnicity, LR and changes in drinking over time are the focus of several other papers and, due to space constraints, are not discussed in detail here . Also regarding the larger study,greenhouse benches the subjects were from a single California university, and the generalizability of results to other settings needs to be established, including gathering data on additional ethnic minorities as our analyses were limited to EA, Hispanic and Asian individuals.

Next, the data were gathered on-line rather than in person by research staff with whom students had no personal contact, a step that might have affected the veracity of the responses, but the level of impact or direction of effect cannot be determined. Also regarding the larger study from which these data were extracted, to maximize the number of students receiving educational videos only 13% of the subjects were controls, and differences in numbers of subjects across groups may have impacted on current results. While the time frame for the current study was 55 weeks and the proportion of subjects reporting ARBs during this interval approached 50% in females, ARBs occur over many years and longer term follow ups are needed. In addition, the short time frame of reporting for the prior month for each assessment resulted in relatively low numbers of ARBs per individual per evaluation.Additional caveats are worth noting. All information about ethnic identity and blackouts involved self-reports, which may underestimate ARBs because heavy drinking can interfere with accurate recognition of whether an ARB occurred. It is also important to recognize that while the SRE has proven to be a robust predictor of future heavy drinking and alcohol problems, the present analyses did not control for years of drinking, the type of beverage consumed or other covariates. However, prior studies demonstrated that the relationship of SRE scores to heavy drinking and related consequences remained robust even after controlling for sex, weight, marijuana use or smoking histories and operated similarly in 12- year-old subjects with recent drinking onsets and in young adults . Finally, there are important subgroups among EA, Asian and Hispanic populations, which, reflecting our sample size, could not be evaluated, and additional risk factors associated with ARBs were not included in analyses. These caveats aside, the present findings indicate that the propensity toward ARBs goes beyond the amount of alcohol consumed and is related to interrelationships among ethnicities, sex, and the sensitivity to alcohol. There are important differences among subgroups of students regarding how characteristics contribute to the ARB risk. Understanding how these interrelationships operate can be important in identifying who carries the highest risk and in creating focused and efficient prevention programs.

Adolescent use of electronic vapor products , including electronic cigarettes, vaporizers, and vape pens—with and without nicotine—is an emergent public health epidemic in the U.S.Vaping nicotine during adolescence is associated with increased risk for cigarette smoking initiation and co-use of alcohol, cannabis, and other substances.Vape products not containing nicotine may also have negative health effects, although these effects are not well understood.For example, evidence suggests that vape cartridges with THC may be related to recent outbreaks of severe lung injury in the U.S5 and flavored e-liquid found in vape products with and without THC contain health harming toxins.Since 2011, past 30-day prevalence of adolescent vaping has increased steadily, peaking in 2019 at approximately 30%.7,8 Although adolescent vaping prevalence dropped to approximately 20% in 2020, it remains high.Given the potential negative health consequences associated with vaping, adolescent vaping prevention must be a public health priority. Ideally, prevention efforts will target the most vulnerable, however, there is a limited evidence base regarding differences in vulnerability across adolescent groups. One group that may be at high risk for vaping is transgender adolescents. By transgender, we mean adolescents whose gender identity is not aligned with their sex assigned at birth, and by cisgender, we mean adolescents whose gender identity aligns with their sex assigned a birth. Although limited, research with nationally-representative and population-based surveys finds transgender adolescents are more likely to smoke combustible cigarettes and vape than their cisgender peers.The model posits that chronic exposure to multilevel gender minority-related prejudice and discrimination predisposes transgender and other gender minority people to excess stress and in turn, negative health outcomes and health disparities. Indeed, past research has found tobacco- and substance use related disparities among transgender adolescents may be related to violence and victimization, community norms favoring substance use, and targeting of LGBTQ people by tobacco and alcohol companies.

While examinations of gender identity disparities in adolescent tobacco use—vaping in particular—are uncommon,grower equipment even less is known about how these disparities vary by race/ethnicity, i.e., disparities at the intersection of gender identity and race/ethnicity. Indeed, vaping among transgender adolescents of color may differ significantly from their non-Latinx white peers given exposure to multiple and intersecting individual, interpersonal, and structural level forms of racism and cisgenderism. These intersecting, multiplicative experiences of racism and cisgenderism, and resultant stress and coping can be understood through the lens of intersectionality. A theoretical framework rooted in Black feminist thought, intersectionality examines relationships between macro-level interlocking systems of power and individual-level experiences and behaviors across multiple social positions .As a tool, intersectionality provides a lens through which researchers can elucidate and explain population health disparities across multiple axes of social positions, centering the notion that “social categories are not independent and unidimensional but rather multiple, interdependent, and mutually constitutive” .Given a dearth of evidence on vaping and vaping disparities among transgender adolescents of color, the present study examines the prevalence of adolescent vaping at the intersections of gender identity and race/ethnicity in a population-based sample of adolescents in California secondary schools. We tested the hypothesis that gender identity and race/ethnicity interact such that transgender adolescents of color would evidence greater frequency of vaping compared to cisgender white adolescents . This information may provide a starting point for advancing understanding of vaping disparities among transgender adolescents of diverse races/ethnicities and informing vaping prevention and control initiatives. Data for this study come from the California Healthy Kids Survey collected in 2017-2018 and 2018-2019. One of the largest of its kind in the U.S., the CHKS is administered via paper/pencil or electronically to adolescents in California schools on a variety of health domains, including tobacco and substance use, and sociodemographics, including gender identity, ethnicity, and race. School districts receiving subsidies from the California Department of Education are required to administer the CHKS at least biennially in 7th and 9th grades and strongly encouraged to administer it in 5th and 11th grades. Districts not receiving subsidies participate voluntarily. Parents/guardians provide active, written consent for children in 5th grade and passive consent for children in 7th grade and above to participate. Student participation is voluntary and anonymous.For years 2017-2018 and 2018-2019, approximately 75% of California school districts administered the survey at least once , 39% of which administered it twice .Our main independent variables were gender identity and race/ethnicity. Gender identity was measured with the question, “Some people describe themselves as transgender when their sex at birth does not match the way they think or feel about their gender. Are you transgender?”. We categorized participants into three gender identity categories based on their response to the question: Cisgender , Transgender , and Unsure of Gender Identity . We categorized race/ethnicity based on participant responses to two separate questions: “Are you of Hispanic or Latino origin” , and “What is your race?” . Participants who indicated a Hispanic or Latino ethnicity were categorized as “Latinx” regardless of the race they endorsed. Participants who did not indicate a Latinx identity were categorized as non-Latinx white , non-Latinx Black or African American , non-Latinx Asian , non-Latinx American Indian or Alaskan Native , non-Latinx Native Hawaiian or Pacific Islander , and non-Latinx multiracial .

Our outcome variable was number of days vaped in the past 30-days, i.e., vaping frequency, measured with the item: “During the past 30 days, on how many days did you use electronic cigarettes, e-cigarettes, or other vaping device such as juul, e-hookah, hookah pens, or vape pens? ”. Past research on adolescent vaping has tended to examine vaping as a binary outcome . To allow for greater detail in modeling frequency, we re-categorized this variable on an integer ordinal scale of 0 days , 1 day , 2-9 days , and 10 or more days vaping in the past 30-days. Based on prior research finding differential patterns in vaping or other tobacco product use among adolescents by specific sociodemographic factors,we included the following potential confounders in analyses: grade, parental education, and sexual orientation. We coded grade into four categories to capture typical groupings in the U.S. context and aid model convergence: 6th -8 th , 9th -10th , 11th -12th , and other/non-traditional. Of note, the majority of adolescents participating in the CHKS are in grades 7th, 9th, and 11th; however a small proportion of students participating in the 2017-18 or 2018-19 survey indicated that they were in 6th, 8th, 10th, or 12th grade which may mean they completed the survey during a class that is primarily open to students in grades 7th, 9th , or 11th, or that their school administered the survey to all grades. We coded parental education into five categories: did not graduate from high school, graduated from high school, attended some college, graduate college, or “don’t know”, and sexual orientation into six categories: heterosexual/straight, gay/lesbian, bisexual, not sure, other , or declined to answer. Our analytical goal was to test the hypothesis that disparities in vaping frequency would be magnified among transgender adolescents of color relative to cisgender white adolescents. We pooled data from the CHKS 2017-2018 and 2018-2019 waves to increase sample size in smaller racial/ethnic and gender identity subcategories and to ensure that the maximum number of schools across the state could be included in the analysis given most schools participate biennially as opposed to annually. We restricted analyses to data from adolescents in grades 6th and above , as the 5th grade survey does not ask about gender identity or substance use . We excluded data from 26 schools that did not collect gender identity . Compared to adolescents attending schools that collected gender identity, adolescents attending schools that did not collect gender identity were less likely to report any past 30-day vaping , and more likely to identify as white and report their parents graduated from college . We further excluded observations collected via a shortened version of the survey which lacked items on substance use . Per recommendations from the survey administrator WestEd, we then excluded observations considered implausible or impossible responses and/or endorsement of an item indicating that some or all survey items were answered dishonestly .

These new analyses used generalized psychophysiological interactions , a technique that allows for the examination of connectivity between two brain areas as a specific function of the task of interest. gPPI was developed to allow for the use of more than two task conditions in the same model while also improving model fit . The evaluations were centered around two hypotheses. Hypothesis 1 is based on observations from fMRI studies of young individuals who did not have an AUD history prior to testing where regional BOLD differences were observed during placebo conditions. This prediction states that compared with high LR individuals, those with less intense response to modest alcohol doses will demonstrate lower levels of functional connectivity between the amygdala and PFC when processing emotion-laden faces. Further, we predict negatively valenced emotions will elicit a greater decrease in connectivity relative to positively valenced emotions . Given prior studies demonstrating changes to brain functional connectivity following alcohol administration, Hypothesis 2 states that low LR individuals will have lower levels of functional connectivity following alcohol for negatively valenced emotions compared with high LR individuals.In the original study from which the current data were extracted , a survey was distributed to randomly selected 18 to 25-year-old European American and white Hispanic students at the University of California, San Diego, using methods approved by the UCSD Institutional Review Board. This questionnaire gathered information on demography, substance use, and DSM-IV psychiatric disorders using questions extracted from the Semi-Structured Assessment for the Genetics of Alcoholism interview . To be included in the fMRI protocol described below, subjects were limited to those who were: right-handed; had no history of brain trauma or epilepsy; no history of alcohol or drug dependence; no current major psychiatric disorder; were not pregnant; and had no irremovable body metal. A laboratory-based alcohol challenge was then used to establish an individual’s LR.

After confirming zero baseline breath alcohol concentrations ,container for growing weed subjects drank alcohol over a 10-min period given as a 20% by volume solution in a room-temperature carbonated beverage, a protocol that produced approximately equivalent BrACs across sexes . We used a median split on self-report scores from the Subjective High Assessment Scale during the alcohol challenge to determine low versus high LR in both the prior study and the present secondary analysis of those data. Once the LR status was confirmed through the initial alcohol challenge, two MRI sessions were scheduled on nonconsecutive days within 1 week of each other whenever possible. Alcohol and placebo sessions were carried out in an MRI scanner where participants received, in random order, the same alcohol dose as in the laboratory session or placebo in sessions that included the Hariri emotional face recognition task . Complete data required for functional connectivity analyses were available from 216 fMRI scans across placebo and alcohol challenge sessions for 108 individuals. The subjects were comprised of 54 pairs of low- and high-LR participants who were matched on sex, demography, drinking frequency and usual quantities, as well as tobacco and cannabis use. The data presented here were extracted from the Hariri emotional face recognition task, which was presented to participants in the MRI scanner 60 min post-beverage consumption during placebo and alcohol fMRI sessions. The task was measured at a time close to the peak BrAC during the average alcohol session. At this point of the alcohol session, the alcohol levels were similar in the LR groups.We used a modified version of the Hariri emotional face-processing task . Participants were presented with a target face and two probe faces for 5 s, with instructions to match the probe and emotional expression of the target by pressing a button in a block design. Each block had six consecutive trials where faces were either angry, fearful, or happy. Additionally, a sensorimotor control condition was used where vertical or horizontal ovals or circles were presented for six consecutive trials in block with instructions to match the shape of the probe to the target. Each block of emotion condition and the sensorimotor control condition were presented three times in a pseudorandomized order. The task began with an 8-s fixation period and had interspersed 12-s fixation periods between each block.

There were 18 trials for each condition yielding 512 s total task time. Figure 1 illustrates the timing of the task with examples from the first two blocks . We recorded accuracy and reaction time during the task to confirm the task was carried out correctly .The Analysis of Functional Neuro Images software package was used for preprocessing data and gPPI analyses. All T1-weighted images were skull-stripped and warped into Talairach Tournoux atlas space using a 12-parameter affine transformation. Next, functional data were visually inspected for scanner artifacts. To correct for small movements over time, image repetitions were registered to a selected base volume via AFNI’s 3dvolreg program. The time series of the motion parameters were used in the linear regression analysis of individual data to control for spin history effects . Functional data were then transformed into the participant’s anatomical space and aligned to standard space using the previously computed anatomical transformation matrix and smoothed with a 4-mm FWHM Gaussian kernel.The general linear models used reference vectors for the task conditions convolved with the hemodynamic response function. Estimated motion and linear, quadratic, and cubic trends were also included as nuisance variables. The resulting model generated scaled beta coefficient maps representing the mean percent BOLD signal change for each condition of the task relative to baseline.Functional connectivity analyses were conducted using gPPI with anatomical-defined seeds in the left and right amygdala. Seed regions were chosen using Neurosynth , a meta-analytic tool for selecting fMRI activation coordinates from a database of studies , where we examined studies using tasks of emotional faces to elicit amygdala activation. Specifically, we created a 5-mm sphere around the coordinates X = −26, Y = 6, Z = −14 and resampled to the template space of our fMRI scans. Visual inspection of these seeds ensured that they were anatomically constrained to the amygdala using the Talairach Tournoux atlas. For each participant, the average time series of the BOLD signal was extracted for each seed, and trends were removed. A one parameter gamma model was used to estimate the hemodynamic response function of the task and a deconvolution of the seed’s time series with this function was calculated using the 3dTfitter AFNI program yielding scaled coefficients. PPI regressor interaction terms for each condition of the Hariri task were computed by multiplying the mean time series of the de-convolved seed with the condition vector of interest, and then convolved with a gamma basis function using the AFNI program Waver.Separate voxel-wise GLMs were conducted for each bilateral amygdala seed. Each GLM contained the PPI regressors, the physiological regressor , the psychological regressors , and nuisance variables .

Each seed’s connectivity with other brain areas was examined separately. Whole-brain between-group differences in functional connectivity were estimated using mixed-effects ANOVAs with the AFNI 3dANOVA3 program where the within-subject factor was alcohol/placebo condition and the between-subjects’ factor was Low/High LR. We applied a cluster correction to guard against identifying false positive areas for potential functional connectivity differences. Specifically, we estimated the noise in our fMRI volumes using 3dFWHMx in AFNI and used that information to apply a cluster-size threshold of α = 0.01 for a given voxel-wise threshold of α = 0.01 to protect our α at a 0.01 level using the 3dClustSim AFNI program. Using this approach,cannabis drying the minimum volume for a significant cluster in our analyses was set at 448 µl or seven contiguous significant voxels.Connectivity analyses were carried out examining LR groups during the alcohol and placebo conditions using left and right amygdala seeds. Clusters showing significant LR, alcohol, or LR-by-alcohol interaction effects in functional connectivity differences in response to fearful, angry, and happy faces are described below and detailed in Table 2. Specifically, Table 2 lists brain regions, associated Brodmann area , peak activation, Talairach coordinates, and volume for each significant cluster. Figure 2 illustrates the pattern of functional connectivity main effects for exemplar regions, while Figure 3 illustrates the pattern of functional connectivity interaction effects for exemplar regions. For all significant clusters we also ran ANCOVA models covarying for sex and did not find any significant effect of sex in our results.During angry faces, using the left amygdala seed, the differences between low and high responders illustrate that low LR participants had lower functional connectivity in several cortical regions compared with high LR participants in both placebo and alcohol conditions. This was observed in the left medial frontal gyrus, bilateral ventral anterior cingulate, bilateral posterior cingulate, and left supramarginal gyrus. Using the right amygdala seed, the main effects of LR during processing of angry faces were identical to left amygdala such that low LR participants had diminished functional connectivity as compared to high LR participants in both placebo and alcohol conditions in the bilateral posterior cingulate. In contrast, during happy faces, significant effects were observed only with the right amygdala seed where, opposite to the LR main effects pattern observed for angry faces, low LR participants had greater functional connectivity compared with high LR participants in the right dorsal anterior cingulate gyrus and right caudate.During angry faces, within the right precuneus, lower functional connectivity was observed with the left amygdala following alcohol compared with placebo in low LR individuals, but increased connectivity following alcohol was found in high LR individuals. Interactions during processing of happy faces were characterized as a decrease in functional connectivity with right amygdala following alcohol as compared to placebo in low LR individuals, but increased connectivity following alcohol in high LR individuals in left middle frontal gyrus regions; a pattern similar to the interaction observed during angry faces.The main goal of these analyses was to expand upon the findings of Paulus et al. by evaluating LR group differences in functional connectivity. This is the first demonstration of differences in connectivity between low and high LR individuals, results that might help explain why drinkers with low LRs might require greater cognitive effort to perform optimally on some tasks. Consistent with our hypotheses, in the current analyses the most prominent patterns found were attenuation of amygdala connectivity with cortical regions in low LR participants, both during placebo and in response to alcohol, relative to high LR participants while viewing angry faces. Additionally, comparing alcohol and placebo sessions, low LR individuals exhibited decreased functional connectivity in response to alcohol, whereas high LR individuals showed increased functional connectivity while viewing both angry and happy faces. The present findings add to the regional brain changes originally reported by Paulus et al. . While those authors found no difference in amygdala regional activation patterns across LR groups, we found several differences in functional connectivity with the amygdala that varied depending on the valence of facial affect being decoded. Thus, BOLD signal changes in the cortex in response to an emotional processing task in low versus high LR individuals might reflect aberrant functional connectivity in corticoamygdalar circuits. These functional connectivity differences were observed in relatively highly functioning individuals from a nonclinical sample, some of whom carried an enhanced risk for alcohol problems through a low LR but none of whom had yet developed an AUD. Along with a growing body of evidence that the low LR phenotype is characterized by CNS differences relative to their high LR peers , these emotional processing data suggest that low LR individuals may have an altered neurobiological process in which they recognize some negative or positive social cues through decoding facial affect. It is also possible that given the pattern of brain regions in the frontal lobe, anterior cingulate, and insular cortex that showed altered amygdala functional connectivity, as well as regional BOLD signal changes in the insula, low LR individuals may have an impaired ability to recognize intoxication, or the rewarding aspects of alcohol, in social situations. Functional connections between the amygdala and cortical regions play important roles in processing human emotions. For example, connections between the PFC and amygdala facilitate the cortex’s top-down regulation in decoding emotional stimuli , allowing appraisals based on affective information to moderate goal-directed behaviors .

We ranked the continuous traits based on their heritability and performed a genome wide association of the top six with the Efficient and Parallelizable Association Container Toolbox. This would be expected to reduce the loss of power due to multiple testing of hundreds of phenotypes. The family Carnobacteriacea was excluded from the GWAS analyses since it was highly correlated with the genus Granulicatella and the latter has a more refined taxonomic resolution. It is well established that continuous traits afford greater power in both twin studies and in GWAS. Therefore, although some categorical phenotypes showed high twin heritability , for GWAS we only studied continuous traits. The analyses were all controlled for age, sex, and sequencing run among other covariates . Analysis was done independently with individuals from the two major different ancestry groups of the unrelated sample, European and Admixture. Due to the limited size of the admixture sample, only the European sample is discussed and the admixture was only considered for the meta-GWAS discussed below. To control for population stratification a kinship matrix created from all the chromosomes and the first ten principal components from the LD-pruned SNPs were included as covariates . To control for the fact that 6 traits were tested, the genome wide significance level was lowered to 8.33e-09 . Using this threshold, we found that the genus Granulicatella was significantly associated with the SNP chr7:110,659,581 within an intron of the IMMP2L gene on chromosome 7. This gene is known to be involved in mitochondrial protein trafficking. The regional Manhattan Plots in Fig. 4b show that the peak locus includes SNPs of decreasing r2 values around the peak SNP lending greater confidence to the association. Without a replication sample this result is provisional but potentially interesting. Using PLINK 1.9, which takes categorical imputed genotypes rather than the probabilistic dosage calls produced by imputation as input,hydroponic tables canada produced results consistent with this association showing the association is independent of underlying computational method.

A comparison of the 100 SNPs with lowest P values in each of the six phenotypes examined in the European sample revealed that 7 SNPs were held in common between at least two of the phenotypes. Bray Curtis PCo2, Unweighted UniFrac PCo2, and Weighted PCo2, all β- diversity measures, were most often shared . After the initial analyses of the 6 most heritable traits, a GWAS was completed in the remaining 64 continuous traits in the European sample. We have used a relatively conservative approach to controlling for population stratification . To evaluate if this may have produced false negatives, we repeated the GWAS with EPACTS kinship only, PLINK 10 PCs, and GCTA LOCO . Each consistently identified the same SNP at chr7:110,659,581 significantly associated with the trait along with nearby SNPs in high LD associated as well . No additional significant SNPs were identified consistent with the hypothesis that stratification methodology had little effect on identifying the top SNPs and that we were not “over filtering” with rigorous kinship controls. For completeness, we then carried out a GWAS analyses for the remaining 64 continuous microbial phenotypes using the EPACTS kinship only analyses adjusting significance for the additional multiple testing and found no SNPs to be significantly associated. This is perhaps not surprising given the relatively small sample size .The size of the ADM sample made it unlikely to produce statistically significant results. To glean useful information from it we combined it with the EUR data described using a meta-analysis approach that can effectively deal with population issues inherent in mixing samples of different populations. METAL is such a meta-analysis package that takes as input individual SNP P values and the direction of their effects weighted by the sample size to arrive at composite P values. The test statistics were also corrected for population stratification . The METAL analysis identified the same suggestive significant SNP on chromosome 7 that was associated with Granulicatella abundance in the EUR GWAS . However, due to the small size of the ADM sample, this SNP did not survive quality filtering in the METAL analysis and so was not a factor in the METAL analysis outcome. Analyses of Unweighted Principal Coordinate 3 yielded a SNP on chromosome 12 that reached genome wide significance in the same direction for the combined sample, though it was not robust to multiple testing correction .

Again, the regional Manhattan Plots in Fig. 5c show the peak locus includes SNPs of decreasing r2 around the peak SNP consistent with the association. The minor allele C, was shown to be consistent with lower PCo3 z-scored values . The most promising single SNP association occurred with the phenotype defined as the abundance of the genus Granulicatella. We reanalyzed the association data with the gene-based tool Knowledge-based mining system for Genome-Wide Genetic studies involved in protein processing associated with mitochondrial import and a non-coding antisense RNA INHBA- AS1 . A SNP in INHBA-AS1 had been previously identified in a dental caries GWAS along with a loci in the INHBA gene. INHBA is thought to be important to tooth development, which could have potential interesting implications to the oral micro-biome. The meta-GWAS results on the PCo3 of Unweighted UniFrac most highly associated region was the gene LIN7A on chromosome.It was possible that tobacco or other factors influenced our observation of genetic association. For example, Streptococcus abundance, a highly heritable phenotype, has also been shown to change in smokers. In addition other substances could potentially change the oral micro-biome. Among these alcohol and marijuana, though these effects have yet to be determined. However, marijuana use is correlated with poor oral health, which is often indicative of changes in the oral microbiota. We had available the self-reported tobacco, alcohol and marijuana use in 92% of our subjects for the previous six months. We therefore repeated the analyses using the three substances as covariates . As seen in Additional file 2: Figures. S15 and S16, controlling for tobacco/alcohol/marijuana use had negligible impact on the top hit on chromosome 7 for the genus Granulicatella . For the 6 highly heritable continuous traits that were analyzed, both with and without substance use covariates, results appear to be consistent with and without substance .We have shown that microbe abundance and some aspects of the microbial population structure are influenced by heritable traits in saliva.

We have ranked the “most heritable” traits using ACE/ADE modeling and GCTA-based SNP heritability and carried out an unbiased GWAS on the 6 most heritable traits. One SNP on chromosome 7 in the gene IMMPL2 reached genome-wide significance. Another gene IINHBA-AS1 on chromosome 7 achieved genome-wide significance when analyzed by KGG4 that relies on a composite association score including all SNPs in each known gene. The significance of these associations was not influenced by “p-hacking” statistical biases common in GWAS because phenotype choice was not based on previous association tests. This approach is a model for using heritability to reduce the multiple testing problems seen in many GWAS reports and it could be the method of choice in the design of GWAS studies in which sample size may be limited. Bray-Curtis, Weighted UniFrac,microgreen rack for sale and to a lesser extent Unweighted UniFrac β-diversity demonstrate that many components of the micro-biome community are heritable . While a shared environment and behavioral habits contribute to a more similar micro-biome , such studies did not control well for the clear genetic influences in their populations. When we examined the differences among MZ and DZ cotwins and age-matched unrelated individuals that we were confident cohabitated , the genetic influences remain clear. It is significant that the genetic effects are detected using measures that include all detectable OTUs. To assess heritable influences of individual microbial components, we carried out intraclass correlation analyses that show that heritability extends across nearly all observed taxa individually . The one exception is in the fusobacteria where ICC does not distinguish MZ and DZ. Possibly these organisms, known to be “bridges” between early and late colonizers on gum and tooth surfaces, may not have interaction with host proteins and could lack human genetic influences. GWAS of complex traits on relatively small samples is problematic due to the lack of statistical power. The influence of individual genes on traits that have multiple genetic components may be small. Moreover, the micro-biome is a highly complex population with interacting networks of bacteria that all may have multiple interactions with the host. A variety of covarying network modeling approaches have demonstrated how complex these communities are. It has been shown that assuming the number of causal variants and their frequency spectra for a pair of traits are similar, more heritable traits are more likely to be detectable in GWAS. Therefore we focused on those micro-biome endophenotypes with greatest additive genetic heritability for GWAS. Both ACE/ADE modeling and GCTA SNP heritability are suited to this approach. The microbial phenotypes with greatest additive genetic influence in the ACE/ADE model on the entire twin cohort were the abundance of the OTU4483015 that corresponds to an unnamed species of Granulicatella and PCo2 of Bray Curtis . The influence of additive genetics was variable depending on the trait when comparing the full sample to heritability only among cotwins that cohabitate . The variation in estimates may reflect environmental effects or loss of power between the full sample and the cohabitating sample . This again points to the complex nature of the microbe-host interactions in primarily aerobic and anaerobic environments and how human genetic influences must also be complex. As a further test of heritability prior to GWAS, we examined SNP-based heritability in our unrelated sample with GCTA. A positive correlation was observed between the ACE/ADE and GCTA ‘heritability’ estimates for continuous traits in both the full twin sample and the EUR sample . Previous studies have demonstrated that large samples are needed to produce results reaching statistical significance using GCTA.

In their original paper Yang et al. showed that while increasing the sample size does decrease the error bars of the heritability estimates, the heritability estimates themselves remain relatively stable. While the GCTA estimate was not significant upon correction for multiple testing, the positive correlation between the unrelated individuals and the twin studies provides support for the conclusion that for these continuous traits genetic variation influences microbial populations. A GWAS analysis with the six most heritable continuous traits determined from the twin modeling was carried out in the European populations . The GWAS of the abundance of the genus Granulicatella identified a genome wide significant SNP on chr7 . This SNP is located in an intron of the IMMP2L gene. The GWAS meta-analyses combining the EUR and ADM samples using METAL with the same 6 traits showed no new information about the chr7 SNP due to its low frequency in the ADM population but did produce an additional association with suggestive significance, chr12:82,166,911 for the phenotype Unweighted UniFrac PCo3, though it was not robust to correction for multiple testing. This SNP is located in the gene LIN7A that is widely expressed in endothelial cells. Markers in LD with the top SNPs were also highly associated with the phenotype, but in addition, markers of somewhat lower LD that were nearby also displayed elevated significance for both hits. This provides an argument that these loci may not be due purely to chance . To be adequately powered one must have a large sample size or the single SNP effect must be very large. However, most complex traits are polygenetic and so many loci with small effects account for the variation of the trait. Therefore, where sample size is limited, it may be difficult to observe significant SNP associations. To address this, it is possible to use biological information to inform analyses and increase statistical power. This may be done by aggregating the association of multiple SNPs known to be present within a known gene. By this approach, the possibly small effects of all SNPs in the gene are combined and then the association of the entire gene may be determined. Even if no single SNP is found to be genome-wide significant the combined SNP contributions across the gene may be. One widely used gene-based GWAS analysis method is the Knowledge-based mining system for Genome-wide Genetic Studies.

Furthermore, these cytokines promoted T cell proliferation and cytokine production even in the absence of stimulation by DC . However, the addition of IL-7 to DC:T cell co-cultures had a dramatic effect on T cell proliferation, maturation and cytokine production that was restricted in part to co-cultures containing THC-DC. These studies suggest that the immuno regulatory effects of THC might be counterbalanced by the presence of a combination of DC activating signals and the production of cytokines by other cell types present in the local immune environment. In summary, our experiments demonstrate that human monocytes express functional cannabinoid receptors, even if they are not detectable on the cell surface, and that exposure to THC alters their capacity to differentiate into immuno stimulatory DC with prominent effects on antigen uptake and presentation, expression of costimulatory molecules, and production of IL-12. The end result is the generation of DC that fail to stimulate T cell proliferation or promote maturation into functional effector/memory T cells. While the effects are relatively potent when studied in isolation in vitro, there may be a number of immuno regulatory factors that could counteract or moderate the impact of cannabinoid exposure in vivo. The functional role that marijuana smoking has on host immunity and the response to immune challenges in vivo remains to be clarified. Cannabis sativa extracts have been used for centuries both as therapeutic agents and recreational drugs, primarily due to their ability to regulate neurobehavioral processes including memory, mood and appetite. The spectra of possible therapeutic uses of marijuana and its active constituents, the cannabinoids, range from handling nausea,cannabis grow system vomiting and cachexia to treatment of chronic pain, glaucoma, epileptic seizures, Parkinsonian tremor as well as multiple sclerosis. Amongst the 60 different cannabinoids identified from Cannabis preparations, D9 – tetrahydrocannabinol , the major psychoactive Cannabis constituent, and the non-psychoactive cannabidiol are the most abundant and important.

Many of the beneficial effects of cannabinoids have been attributed to their potent immuno suppressive and anti-inflammatory properties. Additionally, cannabinoids are known to possess pro-apoptotic, neuroprotective and anti-tumor properties. To date, two cannabinoid receptors have been characterized: the CB1 and the CB2 receptors. The CB1 receptor is highly expressed in neural cells and mediates the psychoactive and addictive activities of cannabinoids, while the CB2 receptor is abundantly present in the periphery including the immune system and is involved in cannabinoid immuno modulation.THC binds to both of these receptors with similar efficiency and hasbeen reported to have effects on both the nervous and the immune systems. In the last years, attention has been turned to several other phytocannabinoids, most notably to CBD. CBD displays a diversity of actions, including anticonvulsive, sedative, hypnotic, antipsychotic, anti-inflammatory and neuroprotective properties. CBD, unlike THC, is not an efficient ligand of either CB1 or CB2 and therefore, is devoid of the unwanted psychotropic effects characteristic of marijuana or THC. Thus, the effects of CBD are probably mediated through other receptors/targets, as described below and elsewhere . Phytocannabinoids were reported to affect various populations of immune cells. Both THC and CBD have been shown to decrease cytokine production in human immune cell lines and to suppress T cell proliferation and their effector functions. In response to stimulation with the bacterial endotoxin lipopolysaccharide , both monocytes and microglial cultures treated with either THC or CBD produce lower levels of cytokines such as tumor necrosis factor a , interleukin-1a , IL-1b and IL-6. However, the molecular mechanisms involved in these cannabinoid-mediated effects are not yet fully characterized. Eljaschewitsch et al.,showed that activation of CB1 and/or CB2 receptors in the murine microglial BV-2 cell line leads to rapid induction of mitogen-activated protein kinase phosphatase-1 and that this event switches off MAPK signal transduction which was activated by LPS stimulation. Altered adenosine signaling has been reported as a potential non-cannabinoid receptor mechanism by which CBD, but less so THC, can decrease inflammation. Other studies identified the nuclear receptor peroxisome proliferator-activated receptor c as a novel intracellular target, which mediates the cannabinoid-associated immunosuppression in a manner that is independent of the known cannabinoid receptors CB1 and CB2.

Other targets including the G-protein-coupled receptors GPR55 and GPR18 as well as the transient receptor potential channels were also suggested. Microglial cells are the resident macrophage-like cells of the CNS. They are highly ramified cells and their processes are very dynamic under non-pathological conditions, actively scanning their environment. These cells have important roles in brain’s innate immunity and neuronal homeostasis as well as in neuro inflammatory pathologies. Microglia can be activated by infection, injury or by endogenously released neurotoxic factors and their activation is associated with the production and secretion of a variety of compounds such as cytokines, reactive oxygen species , reactive nitrogen species, matrix metalloproteinases and prostaglandins. Although microglial activation is considered a protective mechanism involved in the clearance of pathogen infection and in regulating tissue repair and recovery, excessive or chronic activation can lead to harmful effects. Interestingly, the mechanisms that give rise to either the protective or the damaging microglial phenotypes are not fully elucidated. Enhancing the microglial-mediated innate immunity in the CNS and/or preventing the harmful effects associated with their chronic activation may offer new therapeutic approaches for the treatment of brain injury and neurodegenerative diseases. One of the most potent stimuli for microglia activation is the bacterial endotoxin LPS, that mimics infection by Gram-negative bacteria. LPS activates intracellular signaling pathways in a complex way leading to secretion of cytokines and to over expression of several markers of the immune response. Previous studies reported that LPS stimulation induces gene expression of TNFa, IL-1b, IL-6, iNOS and COX-2 as well as the production of NO and PGE2 in primary and BV-2 microglial cell cultures. We reported that CBD reduces the activity of the NF-kB pathway and upregulates the activation of STAT3 transcription factor in LPSstimulated BV-2 cells, and that both CBD and THC decrease the activation of the LPS-induced STAT1 transcription factor, a key player in IFNb-dependent pro-inflammatory processes. Moreover, performing comparative microarray analysis of genome-wide mRNA levels in the BV-2 cells, we reported that CBD, but less so THC, shows a specific gene expression profile associated with oxidative stress and glutathione depletion involving the GCN2/eIF2a/p8/ATF4/Chop-Trib3 pathway. Furthermore, the CBD-stimulated genes were shown to be controlled by nuclear factors known to be involved in regulation of stress response and inflammation, mainly via the -Nrf2/ Atf4 system and the Nrf2/Hmox axis.

We reported that CBD, but less so THC, affects the expression of genes involved in zinc homeostasis, suggesting that the regulation of zinc levels could have an important role through which CBD may exert its antioxidant and anti-inflammatory effects [14]. Although the inhibitory functions of cannabinoids on LPS activated NF-kB and IFN-b/STAT pro-inflammatory pathways and on the secretion of selected cytokines in BV-2 microglial cells has been studied, a genome-wide search for cannabinoid molecular targets in LPS-activated BV-2 cells has not yet been performed. We have, therefore, performed gene array studies and comparative gene profiling analysis of BV-2 cells treated with LPS, CBD+LPS or THC+LPS. This approach allowed us to analyze the changes induced by CBD and THC on gene expression patterns in LPS-treated BV-2 cells and to explore the genome wide interaction network affected by these treatments. In this regard,marijuana grow system a structured network knowledge-based approach to analyze genome-wide transcriptional responses in the context of known functional interactions among proteins, small molecules and phenotypes has been established. We applied this analysis to show the interactions and signaling networks elicited by the cannabinoids in LPS-stimulated BV-2 cells. Our results show that CBD is a potent modulator of microglial activation. Identification of the CBD- and THC-regulated genes and related networks provides a molecular basis for understanding the effects of these cannabinoids on LPS-activated microglia.BV-2 microglial cells were pretreated for 2 h with either THC or CBD followed by the addition of LPS to the incubation medium for another 4 h.The choice of these time points for transcriptional profiling was guided by our previous studies as well as by other reports which investigated the general temporal pattern of microglial activation by LPS. Moreover, according to our previous results, neither THC nor CBD treatments significantly affected the viability of the BV-2 microglial cells during this 6 h period. The RNA prepared from these samples was analyzed for changes in transcriptional levels using the MouseRef- 8 v1.1 Expression BeadChip Illumina Arrays. Each of these arrays has .24,000 mouse targets based on the NCBI mouse Reference Sequence Database, including 16,287 constitutive exons/islands based on the splice variants in the mouse transcriptome and NCBI LocusLink databases. The results of the analyses of the arrays showed that 32% of the transcripts were consistently ‘‘present’’ in the BV-2 RNA samples across all arrays. Moreover, clustering based on inter-array Pearson correlation coefficient indicated no batch effects. Microarray analysis based on a threshold of p# 0.005, revealed that a total of 22% of the Illumina gene set was differentially regulated across treatments. Of these, 1319 gene probe sets were upregulated and 1829 transcripts were down regulated by the LPS treatment ; and from these numbers of genes, 400 transcripts were found to be upregulated and 145 down regulated by LPS by 2-fold or more. When the fold change was set on $3-fold , we found that 226 gene products were upregulated and 33 were down regulated by LPS . The vast majority of the LPS-affected transcripts represented genes that were exclusively responsive to LPS stimulation, and not to treatment with CBD alone or THC alone . the CBD+LPS treatment was analyzed, 1381 gene probe sets were upregulated and 1666 transcripts were down regulated by CBD+LPS . From these numbers of genes, 379 gene products were found to be upregulated and 489 down regulated exclusively as a response to the combination of CBD+LPS and not affected by LPS or CBD alone . When the fold change was set on $2-fold, 502 transcripts were upregulated and 297 gene probe sets were down regulated by the CBD+LPS treatment .When LPS was applied in the presence of THC, 1216 gene probe sets were upregulated and 1638 transcripts were down regulated; and from these, 424 transcripts were found to be upregulated and 149 down regulated by 2-fold or more . From the 1216 upregulated genes, 157 transcripts were exclusively responsive to THC+LPS treatment alone and from the 1638 down regulated transcripts, 285 gene probes were affected only when THC and LPS were together . When the fold change was set on $3-fold, we found that 230 gene products were upregulated by CBD+LPS and 236 transcripts by THC+LPS whereas 51 gene products were down regulated by CBD+LPS and 31 transcripts by THC+LPS . Our results also reveal that from the 5338 transcripts found to be differentially regulated by the various treatments , 680 gene probe sets were found to be upregulated by CBD alone and 58 gene products by THC alone. CBD had also a much larger effect compared to THC on the number of down regulated genes, 524 gene products were down regulated by CBD and 36 by THC . The groups of LPS-upregulated and down regulated genes that showed a change in expression of $2-fold in either direction were subjected to gene ontology analysis allowing functional annotation using the DAVID Bio-informatics Resources and the KEGG pathways . All major Biological Processes, Cellular Components and Molecular Functions within GO for the LPS-upregulated transcripts, were for the most part, genes associated with the immune and defense responses as well as apoptosis and cell death. KEGG database analysis included genes related to Toll-like receptor pathways and antigen processing as well as to the MAPK pathways. IPA global functional analysis of the LPS-upregulated and down regulated genes confirmed the DAVID representation of genes within IPAspecific GO categories . IPA global functional analysis of the LPS-upregulated transcripts show gene products mainly implicated in cell-to-cell signaling, cellular movements, growth and proliferation, as well as cell death, immune response and signaling, including genes involved in the NF-kB pathway and in the activation of the liver X receptor/retinoic X receptor . The largest subsets of down regulated transcripts included genes known to be involved in the regulation of macro-molecules and nucleotide metabolism, differentiation and development as well as regulation of gene expression and transcription .