Hemp Growing

Cresco Labs Inc reported assisting more than 1000 expungement seekers. Green Thumb Industries Inc contributed opening day proceeds from 4 store launches toward the Last Prisoner Project and Florida Rights Restoration Coalition, and Cresco Labs Inc collected donations at 1 store for the Last Prisoner Project. Cannabis companies developed initiatives nominally promoting diversity, equity, and inclusion. These initiatives involved diversifying cannabis industry employment and promoting special populations .Both activities expanded industry involvement in communities through hiring, goodwill, and creating local retailers. Five companies reported internal diversity, equity, and inclusion efforts. Canopy Growth Corporation planned to create diversity benchmarks and report its progress; we were unable to verify whether they implemented those plans. Curaleaf Holdings Inc created internal employee resource groups for minority, LGBTQ+ , parent, and female employees and established a diversity, equity, and inclusion task force. Green Thumb Industries Inc stated it would cultivate a diverse culture and workforce through equitable employment, salary, and promotional efforts; we did not find implemented plans. Innovative Industrial Properties established a diversity, equality, and inclusion policy but reported no activities. Trulieve established a diversity, equity, and inclusion task force to build its workforce, partnerships, and events and diversify product suppliers. Trulieve planned involvement in work fairs and internships to create a more diverse workforce and to train its employees in diversity, equity, and inclusion. Cresco Labs Inc hosted a business workshop with Michigan’s Marijuana Regulatory Agency as part of the agency’s social equity program. Green Thumb Industries Inc and Cresco Labs Inc used business incubators and licensing programs to help underrepresented groups establish businesses.

Cresco Labs Inc held 13 incubator events in Illinois during 2019 and 2020 that served 255 people and 50 businesses,indoor plant table contributing 2062 staff hours and spending more than $775 000 on licensing fees and incubator events. Curaleaf Holdings Inc partnered with 1 minority-owned business and Women Grow, an organization supporting women in the cannabis industry, as part of its 420 × 25 supplier diversity initiative seeking to secure 420 product suppliers from underrepresented demographic groups in the cannabis industry by 2025. Columbia Care107 planned to partner with businesses owned by members of minority groups and women to support social equity initiatives in Illinois and helped 2 social equity license applicants there develop applications with diversity and community engagement plans after purchasing minority stakes in their enterprises. Trulieve and Cresco Labs Inc132 sponsored the Historically Black Colleges and Universities Cannabis Equity Initiative seeking to increase Black employment in the cannabis industry. Trulieve provided $15 000 for internships and $20 000 for scholarships administered by the Thurgood Marshall College Fund, the largest Black college organization serving 47 member institutions, to prepare students for cannabis industry careers. Trulieve and Cresco Labs Inc claimed involvement with National Cannabis Roundtable efforts emphasizing improving industry equity. Four companies promoted to special populations by partnering with nonprofits, participating in events, and fundraising using special products. Canopy Growth Corporation and Trulieve sponsored or participated in LGBTQ celebrations. Trulieve sold special edition products, including limited edition Pride products and TruSwag, to raise funds for LGBTQ organizations. Curaleaf Holdings Inc contributed to social justice, equity, and women’s organizations, and Canopy Growth Corporation and Green Thumb Industries Inc participated in campaigns addressing systemic racism, usually with business coalitions.

Seven companies made charitable contributions at national, state, and local levels . Canopy Growth Corporation’s national efforts focused on fundraising for education, breast cancer,mental health, and veterans. Curaleaf Holdings Inc targeted breast cancer, selling pink “Pre-rolls With a Purpose” and pink vaporizer pens with plans to dedicate some proceeds to breast cancer nonprofit organizations in 9 states. Curaleaf Holdings Inc joined the Ice Bucket Challenge to raise funds for research and treatment of amyotrophic lateral sclerosis, sold limited edition Rhythm for a Cause vaporizer pens through its dispensaries, and partnered with organizations in 3 states to promote breast cancer awareness. Green Thumb Industries Inc indicated it would donate store opening proceeds to the National Giving Alliance, dedicated to helping low-income and homeless persons. Green Thumb Industries Inc donated more than $34 000 from Dogwalker product sales to 5 animal shelters and partnered with American Corporate Partners, an organization helping veterans transition to civilian life. Grow Generation Corp announced a partnership with Whole Food’s Whole Cities Foundation to donate hydroponics to local community gardens in the US. As part of Cresco Lab Inc’s Make a Difference initiative, employees participated in 75 activities that the company claimed benefitted more than 50 communities across 8 states. Five companies assisted COVID-19 pandemic relief efforts during 2020. Canopy Growth Corporation gave $20 000 to Wounded Warriors Canada’s trauma therapy programs and its mental health assistance related to COVID-19. Canopy Growth Corporation also donated personal protective equipment, converted facilities to produce hand sanitizer, and gave Ontario, Canada, 40 000 surgical masks and 25 000 N95 masks. Canopy Growth Corporation’s subsidiary BioSteel Sports Nutrition Inc contributed $2 million in hydration mix to US and Canadian emergency workers.176-178 Curaleaf Holdings Inc gave frontline workers food. Trulieve donated more than 150 computers to assist distance learning in racial and ethnic minority communities. 

Cresco Labs Inc pledged to hire 250 COVID-19–affected workers, and stated it would pay employees extra during the pandemic. Grow Generation Corp pledged as much as $500 000 in equipment to communities and customers affected by COVID-19. Canopy Growth Corporation, Curaleaf Holdings Inc, Green Thumb Industries Inc, Innovative Industrial Properties, and Trulieve announced or participated in local philanthropic initiatives, including job training, community causes, food banks, homeless shelters, women’s services, a campaign to raise funds for Ronald McDonald charities in Canada , animal shelters, veteran organization, a reforestation organization, housing, and social equity and antipoverty nonprofits. Curaleaf Holdings Inc and Green Thumb Industries Inc planned or claimed to donate proceeds from retail store openings to local charities. Two companies incorporated their CSR programs, with Curaleaf Holdings Inc announcing it would contribute to Black Owned Maine’s family relief program as part of its Rooted in Good platform and store opening, and to food charities as part of its Feed the Block initiative. Trulieve employees locally volunteered for its Make a Difference initiative. Two companies promoted cannabis’ medical utility . Canopy Growth Corporation funded research investigating whether medical cannabis treated sleep disorders or mental health conditions. Canopy Growth Corporation and Columbia Care funded studies on whether cannabis could serve as therapy for opioid misuse or an alternative pain treatment.Canopy Growth Corporation offered cannabis education programs for physicians from at least 2016 to 2019 and partnered with the Canadian AIDS Society, an organization representing local Canadian AIDS and HIV groups, to develop medical cannabis treatment protocols for chronic pain. Canopy Growth Corporation partnered with the Beckley Foundation, a drug reform and psychedelic research think tank, to form Beckley Canopy Therapeutics to research cannabis based medicines. Three companies sought expanded medical access. Curaleaf Holdings Inc planned to fund the Veterans Cannabis Project, a nonprofit organization increasing veteran access to medical cannabis, by dedicating proceeds from branded prerolls. The nonprofit identified Curaleaf Holdings Inc as 1 of 3 partners. Canopy Growth Corporation funded a national patient survey conducted in July and August 2020 in partnership with Medical Cannabis Canada, a volunteer-run medical cannabis access nonprofit. Canopy Growth Corporation sought the survey to lower medical use barriers and gain access to academic institution, hydroponics flood table nonprofit organizations, and government officials. Three companies used CSR activities that they claimed combatted youth use or drugged driving . Cronos Group Inc and Cresco Labs Inc developed marketing regulations that they claimed reduced advertising exposure and appeal to youths. Canopy Growth Corporation developed youth prevention materials with Canadian Students for Sensible Drug Policy and Parent Action on Drugs. Canadian Students for Sensible Drug Policy was a former chapter of international grassroots drug policy reform organization Students for Sensible Drug Policy. Parent Action on Drugs, which was created in 1983 to prevent use of drugs and alcohol by youths, disbanded in 2019 due to insufficient funding. Canopy Growth Corporation framed its youth prevention programming as helping youths and “young adults make healthy, responsible decisions on the use of cannabis.” Canopy Growth Corporation also partnered with Mothers Against Drunk Driving Canada to sponsor advertisements discouraging cannabis-influenced driving. Six companies claimed they were mitigating their environmental impacts by reducing product waste and pollution. Curaleaf Holdings Inc formed a sustainability committee. Trulieve and GrowGeneration Corp were among 17 cofounders of the Sustainable Cannabis Coalition trade association. In 2019, Canopy Growth Corporation partnered with recycling company TerraCycle to pilot a recycling program for cannabis packaging in Canada, collecting more than 1 million pieces of waste. In 2020, Canopy Growth Corporation and Cronos Group Inc piloted a program with the Cannabis Council of Canada to collect cannabis vaping devices for recycling.Curaleaf Holdings Inc and Green Thumb Industries Inc listed sustainability as CSR program pillars. Innovative Industrial Properties, which sells real estate to medical cannabis companies,claimed it was sustainable because it reused existing properties over construction. 

Our results suggest CSR activities of cannabis companies are similar to those of the tobacco industry, which enabled the latter to recruit customers and allies, encourage consumption, expand markets, legitimize its product, and deter regulation. Cannabis company donations to and partnerships with advocacy organizations could generate goodwill and consumption among minority and LGBTQ+ communities, a tactic tobacco companies have used to market to those groups. Collaboration with and funding of advocacy organizations have also been used historically by tobacco companies to form partnerships that allowed them to build policy coalitions supporting their agenda . Cannabis companies also created business partnerships that could expand their reach. Although social equity programs developed by state governments allocate licenses to communities and individuals impacted by criminalization, they often impose regulatory and financial barriers. Proof currently exists that some cannabis companies assisted social equity license applicants in exchange for control of proposed businesses, providing the businesses with increased market access. Cannabis companies have publicized CSR activities similar to those of the tobacco industry, which has previously used such concerns to generate reasons for policy engagement with government officials. Industry research on cannabis as an opioid substitute and to treat mental health disorders and insomnia mirrored tobacco industry pharmaceuticalization, the strategy of selling nicotine replacement therapy to legitimize tobacco company products as therapeutic. Pharmaceuticalization may normalize and promote cannabis as a health or medical treatment akin to the tobacco industry’s sale of nicotine replacement therapy, possibly providing cannabis companies additional markets to increase consumption and profits. Cannabis company CSR activities regarding youth prevention, cannabis-influenced driving, and sustainability shared commonalities with CSR activities of the tobacco industry, portraying companies as addressing harmful business effects while sidestepping concerns. The tobacco industry has used youth prevention, marketing, and environmental CSR programs to displace effective educational programs and regulation. Cannabis companies stated that youth prevention programming helped prevent preteen cannabis use without messaging against consumption and promoted recycling while avoiding occupational risks arising from workplace exposure to toxins and secondhand smoke exposure where cannabis is consumed. Prevention programming directed at youths by the tobacco industry was historically less effective than government prevention programming and promoted youth consumption via forbidden fruit messaging. In the US, youths had a 12.8% rate of current cannabis use compared with 15.4% rate of electronic cigarette use and a 3.3% rate of combustible cigarette use in January through June 2021. These higher prevalence rates warrant public health messaging that discourages use and avoids normalizing consumption. Cannabis industry promotion of recycling and sustainability programs may divert attention from adoption of regulations preventing harmful environmental pollution. A comparable case is tobacco industry campaigns encouraging individuals to pick up cigarette butts, the largest source of litter globally, rather than accepting responsibility for manufacturing products that are not environmentally sustainable and modifying production practices.This study has some limitations. We included cannabis companies with the largest market capitalizations, which are not necessarily reflective of the entire industry. The publicly available materials included in our analysis are likely incomplete. We excluded video broadcasts and social media posts, potentially affecting our results because social media platforms are used by companies to advertise as well as promote their CSR activities. Despite these limitations, our findings provide detail on CSR strategies pursued by cannabis companies, including similarities with tobacco companies. Further study is warranted regarding cannabis companies’ use of CSR to influence regulation, improve public image, and secure market access.

With respect to international surveillance systems, the UNODC administers two separate surveillance systems that collect data from all participating UN member states: the Annual Reports Questionnaire surveillance system that collects price and purity/ potency data, and the Drug Seizures Database that collects seizure data. Finally, the European Monitoring Centre for Drugs and Drug Addiction administers the Reitox drug surveillance system network, which aggregates data from several country-level surveillance systems in Europe, as described below. Table 1 presents surveillance systems that matched search criteria. First, purity and/or potency of illegal drugs generally remained stable or increased overall during the study period. Second, the price of illegal drugs, with few exceptions, generally decreased. Third, seizures of cannabis, cocaine and opiates generally increased in major drug production regions and major domestic markets. Figure 1 presents data from the US Drug Enforcement Administration’s System To Retrieve Information from Drug Evidence . As can be seen, between 1990 and 2007 , the purity of heroin and cocaine, and the potency of cannabis herb in the US increased, while the inflation-adjusted and purity-adjusted retail street prices of these three drugs declined. Specifically, heroin purity increased by 60% , cocaine purity increased by 11% and cannabis herb potency increased by 161% during this time. During the same period, the prices of heroin,rolling flood tables cocaine and cannabis decreased 81% , 80% and 86% , respectively. Figure 2 presents data collected by the UNODC on the street price of cocaine and opiates in participating European countries .

In these countries, between 1990 and 2009, the aggregate average retail street price of cocaine decreased by 51%, from US$198/g to US $98/g . Similarly, the aggregate average price of opiates in Europe decreased 74%, from a high of US $295/g in 1990 to US$77/g in 2009 .With respect to opiate seizures, the Golden Triangle includes parts of Thailand, Laos, Vietnam and Myanmar, and according to the UNODC, this region is the second largest supplier of heroin globally, although production has declined throughout the last decade, with opium production decreasing by approximately 60% and 90%in Myanmar and Laos, respectively. In this region, trends in seizures of opium have fluctuated; 3198 kg of opium were seized in 1990, with a high of 12 462 kg seized in 2007 before a steep decline to 1225 kg in 2010 . Similarly, seizures of heroin fluctuated, with a decrease of more than half, from 1337 kg in 1990, to 627 kg in 2010 , and a peak of 1565 kg seized in 2009. In Afghanistan, which is believed to supply more than 90% of the world’s opium,30 seizures of raw and prepared opium increased by more than 12 000%, from 453 kg in 1990 to 57 023 kg in 2010, and seizures of heroin increased by more than 600%, from 1256 kg in 1990 to 9036 kg in 2010 . With respect to cocaine seizures, according to the UNODC, Latin America’s Andean region, which includes Peru, Bolivia and Colombia, is the primary global supplier of this drug, as coca leaf is grown exclusively in this region. While seizures of cocaine in the Andean region decreased 81%, from 97 437 kg in 1990 to 17 835 kg in 2007 , seizures of coca leaf increased 188% from 601 038 kg in 1990 to 1.73 million kilograms in 2007 . During the same period, the area of cocacultivation in this region declined slightly, from approximately 210 000 to 180 000 ha . Finally, according to the UNODC, major areas of cannabis cultivation exist in North Africa, Afghanistan and North America.

These areas are net exporters of cannabis, although most cannabis-producing countries also produce the drug for internal consumption. In North Africa , seizures of cannabis herb increased by 208% from 67 930 kg in 1990 to 209 445 kg in 2007 . In North America , seizures of cannabis herb increased by 288% from 782 607 kg in 1990 to 3.05 million kilograms in 2007 . In Afghanistan, while data on cannabis herb seizures are not available, seizures of cannabis resin increased 630% from 5068 kg in 1990 to 36 972 kg in 2006 .Longitudinal data from government surveillance systems demonstrate that during the past two decades there has been a general pattern of increased illegal drug supply as defined through lower price and higher purity of heroin, cocaine and cannabis. During the same period, patterns of drug seizures either increased or remained stable, although the trends detected in some of these indicators did not reach statistical significance. As such, we conclude, consistent with previous studies,19 that the global supply of illicit drugs has likely not been reduced in the previous two decades. In particular, the data presented in this study suggest that the supply of opiates and cannabis, in particular, have increased, given the increasing potency and decreasing prices of these illegal commodities. These results have implications for the development of evidence based drug policies, particularly given the interest in novel drug policy approaches in a number of settings in Latin America, North America and Europe. As noted elsewhere, there are limitations of ecological analyses based on international surveillance systems. First, some states collect little or no data on indicators of illegal drug supply, whereas other states spend significant energy on monitoring drug availability. Second, even in states that closely track indicators of supply, the degree to which seized samples of illegal drugs reflect purity of retail drugs sold on the street is subject to variation, though where possible we presented purity-adjusted prices to address this limitation. 

Nevertheless, the long-term trends in increasing purity and decreasing price presented here likely reflect the overall trends in many regions, though it should be noted that in some regions , indicators of price and purity may have been strongly influenced by a few countries such as the UK and Spain. In addition, some exceptions in the trends were observed. Australia for instance, while experiencing a significant decrease in the prices of heroin as well as cannabis, did not experience a significant decrease in the price of cocaine, which may reflect the geographic isolation of the region or other market factors. It is also of note that Australia’s ‘heroin drought’, which saw a sudden drop in measures of the supply and availability of heroin, appears to have had a limited long-term impact on supply, though some experts suggest that it may have resulted in higher levels of poly substance use among Australian heroininjectors. Third, limitations in longitudinal data collection precluded our ability to include amphetamine-type stimulants and other emerging synthetic substances, as this data is limited to certain countries and the focus of this study was on regional trends. It is noteworthy in this regard that the production of synthetic substances—as well as indoor cannabis cultivation—present particular challenges for supply reduction strategies, given that these drugs can be mass produced in clandestine locations regardless of climate or other factors that limit traditional drug production. Finally, while this review focused on patterns of price and purity of selected illegal drugs, these measures are only a marker of drug supply and do not measure other factors determining availability and concomitant rates of drug use. These limitations in assessing global drug supply using classic proxy measures such as price, purity and, to a lesser extent, seizures, flood and drain tray suggest that there may be a need to expand the range of measures systematically collected by governments and international bodies such as the UNODC and the European Monitoring Centre for Drugs and Drug Addiction. In particular, meaningfully incorporating measures derived from street-level questionnaires of people who use drugs may provide a more reliable metric of supply and availability. Indeed, some bodies, such as Australia’s IDRS, collect such data, and this methodological approach should be considered by those coordinating surveillance of illegal drugs. Other bodies have also prioritised emphasising measures of community health including reduced HIV infections, reduced drug-related violence and reductions in number of individuals incarcerated. In summary, longitudinal illegal drug surveillance systems demonstrate a general global pattern of falling drug prices and increasing drug purity and potency, alongside a relatively consistent pattern of increasing seizures of illegal drugs. Although source data have limitations and there are some exceptions to these trends, these findings should be useful given the current debates and drug policy experimentation under way in Latin America, North America and Europe. It is hoped that this study highlights the need to re-examine the effectiveness of national and international drug strategies that place a disproportionate emphasis on supply reduction at the expense of evidence-based prevention and treatment of problematic illegal drug use.Death by suicide among Native American peoples is an alarming, racialized health disparity .

According to the Office of Minority Health, Native Americans young people ages 15 to 24 years old die by suicide at a disproportionately higher rate compared to white youth of the same age . Within California, suicide-related behavioral health outcomes for Native American youth are also worse than white youth. From 2017-2019, on average 20.0% of Native American high school students reported experiencing suicidal ideation compared to 17.6% of white youth . Attempting to address this crisis, the US Department of Health and Human Services Substance Abuse and Mental Health Services Administration spent approximately $20,000,000.00 per year on tribal-specific suicide prevention efforts from 2018 to 2023 . Despite these efforts, suicide rates have continued to rise over the past decade perpetuating suicide as the leading cause of non-accidental death for Native American young people ages 15 to 24 years old . This shortcoming has urged some scholars to rethink Native American youth suicide by urging a examination of key theoretical constructs to better direct suicide prevention efforts in tribal communities . These critics of mainstream suicidology argue that the domination of a psycho-centric conceptualization of suicide-related behavior overemphasizes the risk factors related to psychopathologies, maladaptive cognitive schema, and other mentally disordered characteristics . As such, the invitation to rethink Native American youth suicide emphasizes aneed to examine suicide and suicide-related behavior beyond this narrow psycho-centric focus to be more engaged with social, structural, and cultural dimensions of tribal health. This paper uses an Indigenous-based theoretical approach to guide a examination of the multi-level, wholistic factors that may influence suicide-related behavior among Native American young people in California. Indigenous Wholistic Theory constructs health as multifaceted, amalgamating spiritual, emotional, mental, and physical domains . Aligned with tribal constructions of health and wellness, positive health outcomes occur when these domains align with responsive historical, social, political, and economic contexts . Subsequently, maladaptive health outcomes—like the exhibition of suicidal ideation— can arise from dysfunction influenced by determinants across any of these domains and related contexts. In this paper, Indigenous Wholistic Theory provides a framework to introduce and situate a broader array of factors that may influence suicide-related behavior in a manner culturally relevant to the lived experienced of Native American youth. A computational, algorithmic approach is employed to understand how a more wholistic array of factors from various levels can predict the individualized presence of suicidal ideation among Native American youth in California . This approach diverges from traditional regression methods that inquire about the statistical associations between risk factors and health outcomes at the population level. This paper incorporates prediction modeling techniques for three purposes relevant to Native American youth in California: 1) identify a model comprising a selection of factors that combine to best predict the individualized presence of suicidal ideation; 2) evaluate how the selected model can discriminate between the presence and absence of suicidal ideation; and 3) assess the agreement between the model’s predicted outcomes with the actual outcomes of the input dataset . Additionally, this paper leverages a substantiation of the Indigenous Computational Approach to guide its inquiry . As such, this paper asks the following research question: which combination of factors can predict suicidal ideation among Native American high schoolers in California? The Indigenous Computational Approach is a response to the inherent limitations of Western research methodologies that employ computational methods within the domain of social science research. By establishing a framework rooted in Indigenous ways of knowing and being, the Indigenous Computational Approach underscores the imperative of recognizing and valuing Indigenous knowledge systems that center tribal voices and perspectives.

The recent discoveries of a variety of medical cannabis with different preparations to treat and or cure a number of serious disorders and its newly discovered industrial applications have given momentum to the quest for exploring, exploiting, and protecting natural resources with commercial potential. The quantity and composition of cannabinoids, in particular, THC and CBD, have been targeted by extensive research by breeders, the scientific community, and legislative authorities for almost half a century. Here, we considered that concentration and the ratio of THC/CBD could be the crucial key points for the initiation of a breeding program towards different end products in the huge cannabis/hemp market. Although cannabis has a long history of cultural use in Iran, there is little detailed information about the Iranian cannabis market, including the chemical characteristics of the locally available cannabis land races, which are stored in the CGRC gene bank. To complete the puzzle, the current study was conducted as the primary report of screening THC and CBD contents of 20 selected native cannabis populations collected from different locations in Iran towards initiating anew breeding program for different industrial and medical purposes. The populations under study differed in morphological features including total height , thin or squat growth, leaf shape, phyllotaxy, number of nodes, number of lateral inflorescences, internode length, compact or non-compact inflorescence, flowering time , seeding time , seed features, etc., and although not reported here,dry rack cannabis this is in line with earlier research reported that chemical phenotypes can be characterized by different morphological features. Danziger and colleagues reported architecture of cannabis plant may considerably affect the cannabinoids profile, which has significant pharmaceutical and economic importance.

Additionally, phenotype markers that can facilitate preliminary identification and selection as a supplement to chemical and genetic analysis developed in 2021. They showed significant morphological differences in terms of leaf color, leaflet shape, large and compact inflorescences, and dense and resinous trichomes, which were identified between 21 cultivars covering three chemical phenotypes . They also reported that modern cannabis cultivars are morphologically distinguished by a morphological feature by users and breeders. Among the studied populations, three dwarf populations including Saq-01, Pir-01, and Naq-01 distributed in the north-west along with 10 other populations were located in the group of Type I . These three unique populations were assumed to be marijuana, as they have similar features to those reported recently with thinner stems, more branches, and a higher density of floral tissues than industrial hemp plants. In addition, the plants of population of Ard-01 were morphologically and chemically distinct.Cannabis is an economically important species and is predicted to become a significant commercial crop with unprecedented market growth potential. Recent publication showed among individual cannabis plant parts including roots, leaves, stem bark and inflorescence, cannabis inflorescence was characterized by the highest concentration of cannabinoids in three chemovars. They stated that the comprehensive profile of bioactive metabolites can rediscover therapeutic potential for each part of cannabis from their traditional use. Chemical screening of natural populations can help identify chemical diversity, which is a primary step for improving breeding programs in this plant. Taken together, the cannabis definitions are different based on scientific and political assignations. The significant difference in cannabinoid content of cannabis is supported by numerous studies showing that the most important classification of cannabis types that vary widely among political jurisdictions is that of the drug type and the fiber type .

THC is the major cannabinoid in marijuana types, while CBD predominates in fiber-type hemps. The cultivation of cannabis varieties containing up to 0.2% ∆-9-THC with no indication of permitted percentage of the other compounds, first of all being non-addictive psychoactive cannabidiol , have been recently allowed by two European regulations and Italian law. These varieties have been used for food, oils, fiber, powder, and bioengineering. Recent studies on chemical composition and quantification of hemp industrial varieties indicated ∆9-THC content was lower compared to other cannabinoids. Additionally, ∆8-THC was detected only in one hemp oil sample at too low a concentration. Additionally, a report in 2019 indicated that, among several cannabinoids, only CBDA was determined to show a different concentration in hemp inflorescences samples. Additionally, according to their study, THCV was not found in the hemp inflorescence samples analyzed, and ∆-9-THC and ∆-8-THC were detected at low concentrations, below the legal limit. Therefore, their results confirmed the classification of the studied samples as fiber . In order to grow consumer interest in hemp oilseed supplements, four main cannabinoids of CBD, CBDA, CBN, and ∆-9-THC in an oil matrix of seven commercial hemp oil supplements have been determined. They reported that the cannabinoid composition is required to be monitored in such supplements, as in some cases, the cannabinoids concentration in analyzed samples differed significantly from those declared by the manufacturers. In addition, cannabis chemotypes have been reported using the biochemical composition, in particular, the THC/CBD ratio in many publications.

Chemical phenotypes can be used to define cannabis varieties with different chemical variants and different morphological features to classify C. sativa into three principal classes differ in their THC/CBD ratios: chemotype I ; chemotype II ; and chemotype III. Additionally, cannabis varieties were classified into three groups: chemotype I , intermediate type , which has an intermediate ratio close to 1.0, and chemotype III, which exhibit a low total THC/total CBD ratio ≤ 1.0. On the other hand, three main classes of cannabis have been suggested based on THC/CBD ratio: THC-type plants with THC/CBD ≥ 10, intermediate-type plants with THC/CBD ≈ 1, and CBD-type plants with THC/CBD ≤ 0.1. Marchei and colleagues stated that, while the THC content in light cannabis has to be within 0.2%, CBD content is highly variable, ranging from 2 to 40%. Additionally, serum THC/CBD concentration ratio was used as a useful biomarker to identify use of light cannabis , illegal THC cannabis , and medical cannabis. Another study on chemotaxonomic discrimination indicated significant chemical differences in three chemotypes, so that CBD dominant varieties had higher amounts of total CBD, while THC dominant varieties had higher total THC, and intermediate varieties were generally equal to or in between those in CBD-dominant and THC-dominant varieties. They finally showed that chemotype markers could be used as chemical fingerprints for quality standardization or variety identification for clinical studies and cannabis product manufacturing. THC and CBD variations among populations of this Iranian collection enabled us to define studied populations as three different groups: Type I , Type II , and Type III with a prevalence of THC, both THC and CBD in an approximately equal proportions, and CBD, respectively . Genetic diversity in Iranian cannabis germplasm has been assessed by merging the data with the marijuana and hemp data prepared by to elucidate the relationship of Iranian cannabis with marijuana and fiber type accessions. Finally, they categorized Iranian cannabis populations into marijuana and hemp clusters and reported that natural populations of cannabis in Iran in general more closely fit the profile of marijuana than hemp. Additionally, in this study, we have used the same sources to fingerprint THCand CBD, and these populations were defined as Type I, Type II, and Type III, thus revealing, on the basis of chemistry, these three distinct types. In this research, most plants of population Ard-01, which were also morphologically distinct with a THC/CBD ratio around one due to equivalent THC and CBD concentrations, were assigned to class Type II,roll bench defined as an intermediate THC/CBD ratio. This supports the findings of a genetic diversity study reported earlier, indicating Ard-01 was failed to group with either the marijuana or hemp clusters. This is an interesting finding and may have immediate significance for commercialization, promising for therapeutic purposes in the production of medications with formula requirement of THC/CBD ratio around the unity such as Sativex®. This is an important medication for the suppression of spasticity and pain associated with multiple sclerosis. It is worth noting that sequencing the whole genome of this morphologically, chemically, and genetically distinct population would be of value, as this should provide further insight into the genetic basis of the three chemotypes described here. Furthermore, a concentration of THC ≤ 0.3% and higher amount of CBD in the plants from the location Sam-01 that was located in class Type III is another significant finding in this study, worthy of further investigation, and may reflect a tight control of cannabinoid type and content for cultivation and could provide pre-breeding germplasm resources for future development of hemp crops, particularly in nations where there is a strict regulatory environment around the production of high THC crops.

Although most of the focus has been on identifying plants with higher concentrations of THC for the recreational drug industry, and those with higher concentrations of CBD for medicinal, fiber, and grain purposes, there is also evidence reporting therapeutic benefits for CBD with anti-convulsive, anti-epileptic, antimicrobial, and anti-parkinsonian properties, which are more important recently due to the lack of psychotropic effects associated with CBD consumption, as well as FDA-approved CBD drugs such as Epidiolex® . Berman and colleagues found that, despite the similarity in CBD contents, not all equally high-CBD cannabis extracts produced the same effects. They stated that, as cannabinoids profiling of diverse medical cannabis plants are different, analyzing the effects of specific cannabis compositions for pharmacological-based research is critical. Therefore, it seems likely in the future that both THC and CBD content may be of wide relevance for further development within the pharmaceutical industry. This is supported by a previous study reported that the combination of the psychoactive cannabis ∆9-THC with other non-psychotropic cannabinoids such as CBD demonstrated a higher activity than THC alone. Additionally, previous findings showed that all three major products—food, fiber, and medicine—were extracted from the same crop of the accessions from Darchula district in the northwest of Nepal. In addition, some Iranian cannabis populations were evaluated using wood and fiber anatomy and stem biometry characteristics. They suggested that both populations of Bsh-01 and Zah-01 are significant candidates in terms of fiber anatomy, fiber length, and stem biometry and can be considered for textile and paper industries, while in this study, the aforesaid populations were defined as Type I and, despite the prevalence of THC, also have eligible fiber anatomy characteristics. Our research showed that populations of Nhv-01, Ban-01, Sir-01, Rmhz-01, Ark-01, and Sam-01 are defined as Type III. However, in the earlier study, some populations are expected to be putative high-potential fiber populations, indicating that both populations Ban-01 and Nhv-01 have strong fiber characteristics such as a higher average of bast and woody cores and, alongside populations of Rmhz-01 and Ark-01, can be considered an option for breeding programs towards producing fiber. The results of this study contribute important pre-breeding information for cannabis breeders to improve breeding programs utilizing this collection. Although the uses of these populations cannot be predicted with certainty, as accessions high in THC or high in CBD are Type I and III, respectively, they are not necessarily “hemp” or “medicinal marijuana” in the classic sense; however, according to previous study using genomic data, Iranian populations were located in two distinct marijuana and hemp clusters. Therefore,populations of this collection assumed to offer a range of measurable health benefits in the pharmaceutical, dietary supplement industries, dual-proposal , and even renewable and sustainable feed stock for the production of bio-fuels. Additionally, fiber anatomy is required to assess for fiber production purposes.In general, correlation analysis and PCA results revealed a positive correlation between temperature variables and the two target chemical metabolite contents, and a negative correlation between latitude as well as elevation and metabolites content . It is clear that higher temperatures promote cannabinoid biosynthesis, as found in this research. We also found a negative correlation between THC and CBD concentrations, but it was not significant. The THC and CBD biosynthesis pathways have been elucidated and show that cannabigerolic acid is a prerequisite for both CBD and THC biosynthesis and then follows two pathways to synthesize carboxylic acids , and these acidic forms of cannabinoids, upon heating or smoking, decarboxylate to their neutral forms. THCA synthase and CBDA synthase enzymes that catalyze the reaction of THCA to THC and CBDA to CBD, respectively, compete with each other for CBGA and expedite neutral cannabinoids creation and their levels.

ZEV infrastructure credits are capped at 5% of the prior quarter’s deficit generation – 2.5% for hydrogen fueling and 2.5% for DC fast charging equipment. Applications for ZEV infrastructure credits are open through 2025 and are valid for 15 years in the case of hydrogen infrastructure, and 5 years in the case of DC fast charging infrastructure. On one level, the addition of infrastructure credits represents a major departure from the original design of the LCFS as it does not directly subsidize the consumption of a low carbon .fuel. Rather, the credits subsidize a fixed cost of providing network infrastructure that may encourage adoption of EVs, the technology which may in turn use a low carbon fuel. In the same way, however, the infrastructure credit can reduce the very effect that LCFS critics have focused on as the central flaw in the regulations design: the encouragement of low, but still nonzero carbon fuel. While infrastructure credits may spur vehicle adoption, their effect on expanding driving miles would be second order. At the same time, if the amount of infrastructure credits awarded through the program were significant enough to ease compliance, these credits can have the effect of lowering the overall LCFS credit price, and therefore reduce even the diluted carbon price effect on end-use fuel prices. The magnitude of any price-suppression effect would depend upon both the quantity of infrastructure credits and the slope of the LCFS compliance cost curve.Initially, there were no formal limits on how high LCFS credit prices could rise, although legal challenges to the regulation effectively delayed implementation,cannabis grow equipment freezing the standard from 2013 through 2015, and effectively limited demand for credits and their pass-through to fuel prices.

However, as the lawsuits were resolved in favor of continued implementation of the LCFS and the standard declined steadily in the last several years , credit prices have risen steadily and raised increasing concerns about the cost of the regulation.32 In its 2015 re-adoption rule, the ARB introduced the credit clearance market, which is a cost-containment mechanism that would in theory limit price increases under some scenarios.Entities in need of LCFS credits for purposes of immediate compliance can purchase credits in the credit clearance market at a price no higher than the prescribed maximum of $200 per ton in 2016 and adjusted for inflation thereafter . If these entities are unable to purchase sufficient credits in this market to reach compliance, then they may carry over their deficits to future periods. Carryover deficits grow by 5% per year, meaning that firms pay an ‘interest’ penalty for deferring compliance. However, firms that hold credits are not required to sell in the credit clearance market, and they would not do so if they believed that they be able to sell their credits at a higher price in the future. Thus, the credit clearance market provides only a soft cap. However, ARB is currently proposing to impose a hard price cap of $200 per ton in 2016 dollars for LCFS credit transactions. To help facilitate compliance under this cap, it proposes a mechanism to ‘borrow credits’ from future residential electric vehicle charging. Under this mechanism, obligated entities could use credits expected to be generated in 2026- 2030 to meet unmet annual deficit obligations in 2020 – 2025. These cost-containment mechanisms are suited for dealing with a transient disruption in clean fuel supply or some other cause of a short-term supply-demand imbalance of LCFS credits. Because of the requirement that borrowed credits be restored with interest, it will not be effective at containing costs in an environment of chronic, long-term credit supply demand imbalance.

The future prospects of the regulation are therefore linked to the potential supply and demand balance through the next 11 years of the program. A circumstance where compliance is only feasible through high cost fuels or sharp reductions in fuel consumption would push credit prices above the maximum credit price for the credit clearance market. One objective of this paper is to assess the potential likelihood of such an outcome. In 2019, ARB is proposing amendments that would backstop this cost containment mechanism, enforcing additional borrowing of future credit generation from residential electricity charging for electric vehicles at the maximum credit price, with a rolling payback schedule enforced on utilities that will borrow the credits, up to a cumulative total of 10 million borrowed credits.This section outlines data and methods used to project business-as-usual for LCFS credit and deficit generation to 2030. In this paper we use the term business-as-usual frequently, and take it to mean, regarding LCFS credit demand, the continuation of historical trends through the compliance period. For LCFS credit supply, BAU refers to a continuation of current alternative fuel mix trends to 2030. Therefore, the uncertainty in the projections stems from the estimation of BAU demand, which against an assumed steady state of supply, yields a distribution of net deficits accumulate over the period 2019 to 2030, on which we base subsequent analysis.We are interested forecasting demand for fuel and vehicle miles under BAU economic conditions. Demand for fuel and vehicle miles are highly dependent on other economic variables. Demand for both fuel and vehicle miles will be influenced by general economic activity and oil prices. In a booming economy, consumers travel more and purchase more fuel.

Our aim is to fit an econometric model that characterizes past trends in key credit demand variables such as fuel consumption and key input prices for the gasoline and diesel fuel “pools,” namely oil price and soybean prices, vehicle miles traveled, and an indicator of the state economy.The estimates from that model are then used to simulate relationships moving forward to project potential credit demand.We use data available from 1987 to 2018 for the six dependent variables to fit the VEC model. Because our data are measured at the quarterly level, we have a total of 124 observations for each variable.California GSP was collected from the Bureau of Economic Analysis .Theoil prices used in our model are Europe Brent spot prices FOB collected from the Energy Information Administration at the monthly level and aggregated to quarterly averages.We chose to use Brent oil prices rather than West Texas Intermediate prices because Brent prices are more relevant to California markets. Historical vehicle miles traveled on California highways are reported by the California Department of Transportation, CalTrans, at the monthly level.On-highway VMT data are reported in the aggregate, and not divided into gasoline and diesel vehicles.Our model also requires soybean prices, which we collect from the Agricultural Marketing Service at the United States Department of Agriculture .We aggregate monthly spot prices in Central Illinois to quarterly averages to be used in the model. The main variables of interest in our model are gasoline and diesel consumption and VMT in California as we need to forecast BAU fuel demand in order to construct a distribution of LCFS deficits. We collect monthly prime supplier sales volumes for California reformulated gasoline from the EIA.This measure captures all finished gasoline that is consumed in California, including imports to the state. We assume all gasoline is consumed in the transportation sector. Measuring diesel fuel consumption is more nuanced. The EIA reports monthly sales volumes for refiners at each step in the supply chain. We aggregate wholesale and retail sales volumes for No.2 distillate to construct a measure of consumption of No.2 distillate. According to data from the EIA, 99 percent of No.2 distillate is used for diesel fuel in California. Therefore we calculate sales volumes of CARB diesel, which is ultra-low sulfur diesel sold in California, as 99 percent of No.2 distillate sales. The diesel pool, however, comprises biomass based diesel ,vertical grow rack which includes bio-diesel and renewable diesel, as well as petroleum diesel. BBD demand was negligible prior to 2011, but has been increasing in the years since. Therefore, we construct the measure for diesel fuel consumption as the sum of BBD and ULSD. The EIA does not report sales of BBD, so we use volumes reported by CARB in the LCFS quarterly summary, since the years of substantial BBD demand occur in that time period. We aggregate monthly CARB diesel sales from the EIA to quarterly totals and add quarterly volumes of BBD from CARB. The LCFS regulates fuel used in the California transportation sector. Therefore, to accurately estimate the number of deficits generated from CARB diesel using our data, we need to measure the amount of diesel fuel consumed in California that is allocated to the transportation sector. Since 1992, approximately 70% of distillate consumed in California has been used on highway in the transportation sector.We therefore assume, in accordance with our definition of BAU, that 70% of all CARB diesel will be consumed in the transportation sector in each year over the 2019-2030 compliance period.

We are unaware of information that would lead us to believe a divergence from this long term could occur and we don’t consider altering this assumption in this study. Importantly, scaling diesel by a constant has no effect on the coefficient estimates in the VEC model that we use to generate our BAU simulations.The long-run coefficient estimates from the VEC cointegration model appear in Table 9. The three columns in Table 9 correspond to the three cointegrating equations specified in and and the rows to their long-run relationships with GSP, VMT, and the oil price.In the first two equations of Table 9, gasoline and diesel demand in California, the coefficients on the oil price capture the price responsiveness of demand for each fuel.The elasticity for gasoline, on the other hand, is positive but qualitatively small, and statistically insignificant at the 5% level. This may reflect fact that gasoline demand is very inelastic. The coefficients on GSP reflect the income effect. Gasoline and diesel fuel are normal goods and thus should be expected to be positively correlated with income in the state. The coefficient on VMT captures fuel economy improvements as more VMT per gallon implies fewer gallons. Because the VMT measure is not reported by vehicle type, implied fuel efficiency gains in each of the two fuel pools are not discernible. In the next section, we use the long-run coefficient estimates from Table 9, along with the short-run estimates located in the appendix in The estimates of the and Γ matrices from the VEC model in appear in Table A-4.We use the coefficient estimates from the VEC model to predict the distribution for each variable through the compliance period, 2019-2030. Specifically, we simulate 1000 potential values for each variable in each quarter during the compliance period. To this end, we assume that the potential shocks ?! that may occur in the compliance period have the same distribution as the shocks during our estimation sample period, 1987-2018. Using this assumption, we simulate potential future shocks by sampling randomly with replacement from the 1987-2018 shocks. For each random draw, we use the VEC model to generate a hypothetical path for the six variables. We repeat this exercise 1000 times to give us a distribution of potential paths.The hypothetical paths for blended gasoline, diesel, and VMT, simulated using , are described in Figure 17 with the median draw from each year and a 90% point wise confidence interval .In addition to those variables, we calculate the fuel economy of gasoline vehicles that is implied under BAU conditions. To do so, we multiply each VMT projection by the percent estimated in ARB’s EMFAC model to come from gasoline powered vehicles .Then we can express the average fuel economy, measured in miles per gallon , for gasoline vehicles by dividing gasoline VMT in each draw by the number of gallons of CaRFG. The implied fuel economy shown in Figure 17d highlights the range of efficiency gains considered in our simulations over the compliance period. This implied gasoline vehicle economy, derived from EMFAC percentages combined with our projections, is a fleet-wide average for gasoline powered vehicles only, and does not explicitly build in the recent California vehicle efficiency agreement with major automakers to reduce GHG emissions per mile for model years 2022 through 2026.For each variable in our VEC model, the level of future uncertainty grows as we move further into the future. In Figure 17a, 90% of the draws from our sample fall between 14 and 17 billion gallons of CaRFG being consumed in 2030 – a 12 percent increase and 13 percent decrease, respectively, from current levels.

The first contribution of this paper is estimating the extent to which the RIN tax, the LCFS diesel tax, and the CFP diesel tax have been passed through to wholesale diesel prices since 2015. To estimate pass through of the RIN tax, I take the approach used in Knittel et al. which exploits the fact that wholesale diesel and jet fuel prices only differ in their RIN obligation. Their sample covers 2013-2015, whereas mine covers 2015-2021, therefore providing a larger and more recent sample to leverage. I find that refiners, on average, fully passed through the cost of their RIN obligations to wholesale diesel prices over the last six years. The issue of RIN pass through has been of particular interest recently. RFS compliance costs reached an all-time high in 2021, strengthening the already substantial concern of potential harm on consumers and the refining industry, especially amidst a global pandemic. Senator Pat Toomey and his senate colleagues sent a letter to the Environmental Protection Agency – the administrator of the RFS – asking them to waive or reduce the 2020 volume requirements to mitigate the unduly burden placed on consumers and refiners via RFS compliance costs.EPA responded in kind, reducing the 2020 mandate retroactively in 2021. Still, some in the refining industry have argued that the RFS has made it harder to produce gasoline and diesel domestically, according to a recent article in Reuters. Refiners have indeed seen much larger RFS bills this year; one Pennsylvania refinery had a $350 million RIN obligation in the first three months of 2021, 500% higher than their total 2019 of $58 million.In 2021, refiners paid an implicit tax upwards of 20 cents per gallon of gasoline and diesel. Smaller refiners have been granted exemptions by EPA for their RIN obligations for several years. However,cannabis grow racks prior work suggests that smaller refineries have been able to pass through the cost of their RIN obligations at the same rate as large refineries .

This also comes at a time when gasoline and diesel prices have both reached their highest levels since 2014. The Biden administration has announced that the Department of Energy will release 50 million barrels of oil from the Strategic Petroleum Reserve to combat the high fuel prices. As of November 2021, the average retail price of diesel is $3.72/gallon and gasoline, $3.49/gallon. The unprecedented growth in RIN prices due to the soybean boom in 2020 and 2021 mentioned above raises speculation as to whether that period is driving any result of incomplete pass through. However, I present results where that period is dropped from the sample and find lower rates of pass through, not higher, especially in San Francisco. In fact, the striking increase in compliance costs during that period were largely passed through in most U.S. regions. Refiners in California and Oregon face an additional cost due to deficit obligations under the LCFS and CFP, respectively. In California, the additional cost is double for diesel; the implicit tax associated with their deficits were also upwards of 20 cents per gallon of diesel under the LCFS. In this paper, I find that refiners have passed through little to none of the LCFS tax to wholesale diesel prices. However, I find that the LCFS tax has been fully passed through to rack prices by blenders, which suggest that refiners have exercised their ability to trade their deficit obligations downstream. This suggests that LCFS compliance costs are borne by retailers and/or consumers. Due to data limitations, I am unable to estimate the pass through of the implicit CFP tax. The second contribution of this paper is estimating the extent to which implicit subsidies from the RFS, LCFS, and CFP are passed through to rack prices of blended diesel. Pass through of RIN subsidies to bio-diesel has been acknowledged but sidestepped in previous work due to data limitations and the unknown interactions of the RFS and the Blender’s Tax Credit .

The BTC awards blenders a $1 tax credit against their federal liability for every gallon of bio-diesel blended in the U.S. and it is realized in addition to the RIN, and the LCFS credit or CFP credit in California and Oregon, respectively. The BTC was implemented in 2005 and expired four times since, but retroactively reinstated each time. When expired, blenders and bio-diesel producers formed contracts to share the expected future credit and had potentially unknown impacts on the blenders’ margins and RIN prices. However, with straightforward assumptions about the BTC, I can identify the pass through of RIN subsidies to blended diesel prices. This subsidy pass-through analysis undertaken here is most like Pouliot et al. in the existing literature, which studies pass through of ethanol RIN subsidies to blended gasoline prices at racks across country. They find incomplete pass through in some regions and attribute it to lack of salience about how the subsidies impacted profit margins. I extend their empirical framework to estimate the pass through of RIN, LCFS, and CFP subsidies to blended bio-diesel prices at fuel terminals in eight U.S. cities. I utilize daily pricing data from the Oil Price Information Service and Bloomberg on rack prices and spot prices of diesel, bio-diesel, RINs, and LCFS and CFP credits to calculate daily profit margins for blenders. If a subsidy is fully passed through, blenders’ profit margins should move one-for-one with changes in said subsidy. There are significant heterogeneities in the pass through of bio-diesel subsidies across space, time, and policy. RIN subsidy pass-through is complete in the Midwest, incomplete on the West Coast, Gulf Coast, and East Coast. These results are somewhat consistent with Pouliot et al., ; they find incomplete pass-through only in the eastern U.S. This was due to an extreme event at the end of their sample in 2015 — the Exxon Mobil refinery explosion in California. My sample follows the explosion and accounts for the LCFS, allowing for more precise estimates of pass through on the West Coast. The finding that pass through is still incomplete on the East Coast may suggest that incomplete RIN subsidy pass through stems from a mechanism other than lack of salience.

As with pass through of the RIN tax, pass through of the RIN subsidy is less complete when dropping the time period with the shock to RIN prices. LCFS subsidies exhibited significantly less variation over my sample, making it difficult to recover precise estimates of pass through. However, I find that, on average none, of the LCFS subsidy is passed through to rack prices of blended diesel in California urban centers. In smaller California cities, complete pass through of the LCFS subsidy pass cannot be ruled out. After the LCFS subsidy grew to significant levels, pass through was lower, indicating lack of salience wouldn’t explain the results of incomplete pass through. LCFS subsidy pass through is lower for blends with higher bio-diesel content, which is consistent with market power in higher blends . CFP subsidy pass through is estimated even less precisely but the findings resemble a similar pattern as the LCFS. Taken together, the results outlined above can be summarized as follows. The RFS largely operates as intended in the diesel sector, however, findings are consistent with local market power in fuel blending on the coasts. With incomplete pass through of RIN subsidies in some markets and complete pass through of the tax, it may be that blenders are raising RFS compliance costs for consumers. California and Oregon exhibit less than complete RIN subsidy and LCFS subsidy pass through,cannabis drying racks which suggest blenders exercise local market power. On average, only 68 percent and 66 percent of the combined subsidies are passed through in California and Oregon, respectively. The remainder of the paper is structured as follows. Section 1.1 provides background information and institutional context on the RFS, LCFS, CFP, and the. Section 1.2 describes markets for diesel and biomass-based diesel. Section 1.3 describes the data used to execute the empirical strategy. Section 1.4 describes the empirical strategy for and presents results from estimating pass through of the implicit taxes from the RFS, LCFS, and CFP. Section 1.5 describes the empirical strategy for and presents results from estimating pass through of the implicit subsidies from the RFS, LCFS, and CFP. Section 1.6 concludes and discusses policy implications of the findings.The RFS was enacted in the 2005 Energy Policy Act and was revised and expanded as part of the Energy Independence and Security Act of 2007, sometimes referred to as RFS2. The RFS is administered by the U.S. Environmental Protection Agency and specifies a fraction of U.S. petroleum transportation fuel consumption that must be displaced by renewable fuels. Using projections of gasoline and diesel consumption from the Energy Information Administration , the mandate is communicated as a volume of renewable fuels for the upcoming two years. These volumetric mandates are called Renewable Volume Obligations . RVOs are set separately each for conventional , advanced , BBD , and cellulosic categories.

Refiners or fuel importers, the obligated parties under the RVO, can either produce biofuels or purchase credits, called Renewable Identification Numbers , generated from the production of renewable fuels. Each refiner must retire a certain number of each type of RIN each year for each gallon of gasoline or diesel that they sell. For example, in 2018, for every 100 gallons of gasoline or diesel sold, refineries had to retire a total of 10.67 RINs, including at least 2.37 advanced biofuel RINs, 1.74 bio-diesel RINs, and 0.16 cellulosic RINs. The remaining 8.14 RINs that must be retired can be of any category, but typically come from corn ethanol RINs and BBD RINs. Corn ethanol is the lowest-cost renewable fuel, however the E10 blendwall forces the market to use the next lowest-cost option, BBD, for much of the conventional RVO. Since refiners must purchase RINs for every gallon of gasoline and diesel they sell, RINs act as a tax that is used to subsidize renewable fuels. The magnitude of the taxes and subsidies depend on the economics of the underlying fuel markets. Since each category relies on different types of renewable fuels, each type of RIN has its own market price. Generally, the market price for each type of RIN reflects the expected cost of supplying the marginal gallon of the relevant renewable fuel needed to meet the RVO, relative to the cost of gasoline or diesel. Since advanced and cellulosic biofuels are much more expensive than conventional biofuels, the market price for their RINs are relatively expensive. Since BBD has been used to satisfy the conventional RVO, the market price for D6 RINs have converged to the market price for D4 RINs. RINs can be traded freely before being retired for annual compliance, and 20 percent of RINs generated in one year can be banked compliance in the next year. In an efficient RIN market, these features cause future expectations around fuel markets and policy to influence RIN prices. Lade et al., show that RIN prices follow a random walk and respond quickly to EPA announcements that change expectations around future compliance. The point of policy incidence is at the fuel terminal for the RFS. The fuel terminal is the midpoint in the supply chain of RINs. Renewable fuel producers generate RINs with every gallon of renewable fuel they supply. When RINs are generated, they are “attached” to the renewable fuel, meaning that whoever purchases that fuel receive the RIN certificate with the purchase. Terminals purchase the renewable fuels with the attached RINs and blends the renewable fuel with petroleum, which separates the RIN from the fuel. When the RIN is separated, it can be traded freely. When blenders separate RINs, they sell them to refiners who must retire RINs to EPA for compliance. Since blenders can sell RINs once separated, they are willing to pay a premium for renewable fuels. Figure 1 shows the movement of fuel and RINs through the supply chain in the context of diesel.RINs can be generated by domestic BBD producers, foreign BBD producers, and domestic BBD importers. The majority of bio-diesel consumed in the U.S. is produced domestically. In 2016, nearly a quarter of bio-diesel was imported, however in recent years imports fell below an eighth of total consumption – 10 percent in 2020. Almost no RINs generated from U.S. bio-diesel consumption are awarded to foreign producers.

Many sites have scattered carcasses of deer and bears that were poached by laborers, who shoot almost anything that comes near their site. This aggressive behavior protects the plants and provides supplemental protein in the laborers diet. However, the vast majority of these carcasses are left to rot and to be eaten by vultures because growers cannot viably consume or preserve all of the meat from large mammals before it rots. The scavengers that successfully feed on carcasses have an increased risk of developing lead poisoning because they begin eating at the point of bullet entry where inner flesh is the most exposed and easily accessible. By eating from a gunshot wound, scavengers consume bullets containing lead. Over time, lead can accumulate in their bodies and cause lead poisoning. Lead poisoning caused by hunting is cited as the number one killer of the California condor, an endangered species, and poses one of the most significant threats to wild scavengers in California.Sustained inhabitance poses a significant hazard to fire prone areas throughout California. Cultivator activity can cause wildfires and the presence of a marijuana garden obstructs firefighting efforts due to safety issues. “On the Hume Lake Ranger District of the Sequoia National Forest, a wildfire in 1999 was started by a campfire in a marijuana garden. Firefighters found the garden and had to stop fire suppression activities in the area until law enforcement secured the area. This problem occurs several times every year.” Cultivators use campfires and burn volatile gases for cooking during the dry season in areas vulnerable to fire. Under prime fire conditions, a stray spark, improperly connected tank,pipp mobile systems or overturned stove can initiate an out of control fire with drastic and widespread consequences. A crucial function of sustained inhabitance is to perform counter-surveillance efforts.

Cultivators are trained by cartel employees, often veterans of the Mexican army, and equipped with weapons ranging from shotguns to assault rifles. A station is setup at a vantage point above the grow site so a watchman can detect and alert his colleagues to approaching scouts or the presence of pedestrians. While entering sites, laborers sweep trails of any prints so they can patrol for signs of entry by others. One positive development is that the use of booby-traps has drastically declined. However, the protective behavior of inhabiting guards has become increasingly aggressive year to year as a result of escalating grower competition, law enforcement pressure, high crop value, and DTOs’ demand for harvest delivery.Men on patrol have been known to threaten anybody they encounter, and even harass law enforcement and forest service employees at their homes.Their engagement in armed standoffs with law enforcement scouts has increased in conjunction with the occurrence of gunfire. By shooting at site detection scouts, cultivators create a two to three day window during which they harvest all of the buds they can carry out, and flee the area before a larger task force can return. The rate of violence and harassment towards pedestrians has also increased. In June 2006, two individuals in a remote area north of Covelo, California, came near a marijuana plantation and were shot and killed.”Two months later, in October 2006, “a man hunting in a remote location within the Mendocino National Forest was fired upon by four individuals after he inadvertently approached the edge of a grow site.”Both instances lead to the discovery of the plantations that the gunmen were trying to protect.

Due to the illegal status and consequently hazardous state of marijuana plantations, law enforcement agencies are the primary organizations responsible for site reclamation on public lands. “In the past, site reclamation has not been considered much, if any, by the law enforcement community.”Environmental cleanup and remediation are crucial to mitigating the effects of remote cultivation. Since the objectives of law enforcement agencies have changed, leaders have recognized the significance of clean up in counter-cultivation efforts, and have emphasized site reclamation in their large scale marijuana control plan. Even so, environmental cleanup and remediation remain the most neglected tasks associated with post-raid site processing. The main barriers to reclamation efforts are the high costs and intensive labor needs. Enforcement agencies, land managers and volunteer groups are increasing post-eradication cleanups, but reclamation efforts remain inadequate when compared to the damage that cultivators create. The remoteness of Cannabis grow sites makes clean up both time consuming and arduous. Clean up crews remove marijuana plants, disassemble water diversion and drip irrigation systems, and clean up camp sites and surrounding areas. As forest technician Madison Thomson states, “[cultivators] pack everything in and nothing out. We [the conservation fund] don’t have enough money or enough manpower to go into these places and clean them up. It’s a big eye sore.” Depending on the number of people, available funding and equipment involved, the site cleanup process can take anywhere from a number of days to several months. More often than not, cleanup is not completed at all. From the perspective of law enforcement agencies, it is crucial for grow sites to be cleaned up. Undoing the work cultivators put in and removing grow infrastructure minimizes the incentive for cultivators to come back. Otherwise, cultivators readily return to a location to save countless hours preparing the grow site. Equipment and trash that is dispersed across cultivated landscapes provides visual evidence of marijuana cultivation activities. Sites that are not cleaned up are indistinguishable from active cultivation plots, and provide evidence that may result in raids on unoccupied areas. The most important piece of equipment used for optimizing the cleanup process is a helicopter. Helicopters enable cultivation equipment and trash to be removed quickly and in large quantities.

Clean up crews fill up hauling nets with marijuana plants, irrigation tubing and trash bags. The nets are connected to an extended line and flown to a designated area for transport to a disposal location. A clear area is critical for helicopters to drop lines with nets into sites for equipment removal. Heavy tree canopy and forest fires hinder the use of helicopters, and greatly reduce the efficiency of cleanup efforts. What cannot be flown out due to inaccessible site conditions, limited time allocated for helicopter use, or limited funding to pay for helicopter costs, must be hiked out on peoples backs or left to rot. Marijuana removal and destruction is a historical focus of law enforcement agencies that subsists today, despite the reality that plant removal after raids actually accomplishes little to remedy the environmental damages. Plant eradication provides a measure of operational success, prevents cultivators and civilians from attaining marijuana from already raided areas, and enables land alterations including terracing and mounds to be rectified. However, the effort put into plant eradication is needless in sites that will not be cleaned up because abandoned sinsemilla Cannabis will rot and will not reproduce to grow in subsequent years. During eradication, officers, land management employees and volunteer laborers use machetes and loppers to cut down Cannabis plants at the stems. On large plots, the work is both physically demanding and time consuming. Depending on the location of the site, different strategies are used to remove eradicated plants. Traditionally, a common method was to leave the cut plants on the ground to decompose. Today, this generally occurs only in the most remote locations to prevent plant acquisition by outside parties. Another historically prevalent practice was to burn the marijuana plants on-site. However, this process releases carbon into the atmosphere and poses a potential risk of fire spreading to surrounding vegetation. To prevent forest fires, this option requires constant monitoring and can only be done during certain times of the year. The final option is to airlift or hike plants out to a landfill or a location suitable for burning. The most important structure to remove is the water diversion system. Removing miles of irrigation tubing is one of the most intensive aspects of cleanup in terms of time, effort, and money. It is the most important infrastructure to remove because DTOs invest large quantities of money and labor into its installation. When irrigation systems remain intact, DTOs retain a major incentive to return to eradicated sites in subsequent years,industrial drying rack which perpetually prevents ecological recovery. Irrigation lines alone can fill multiple helicopter removal bundles, each weighing about five hundred pounds when full.The most environmentally harmful element of irrigation systems is the dam or cistern that is used to create a catchment from which cultivators extract water. Removal of catchments requires great care to restore original flow patterns, while minimizing sedimentation and alterations to primary and ephemeral flows. Additional clean up aims to remove equipment, supplies, piles of cleared vegetation and remains from human inhabitance: this includes bags of fertilizer, chemical containers, propane tanks, camp dwellings, food preparation areas, latrine sites, scattered trash and more. The scattered trash includes composed of plastic wrapping, notebooks, clothing, tin cans, hiking packs, beer cans, pruning saws, rope, and more. It is important to gather and remove these scattered remains so that eradicated sites can be distinguished from active ones. The latrine, however, cannot be simply picked up and taken away. Cultivators generally dig at least one hole deep into the ground to be used throughout the duration of their stay. Nothing is currently done to remove the excrement aside from covering up the hole, marking the location, and allowing the contents to filter through the soil. Holistic remediation efforts attempt to restore landscapes to pre-alteration conditions, which entails watershed maintenance, surface restoration, vegetation management, and wildlife promotion. While there are astounding regularities from one DTO site to another, remediation efforts still must be suited to specific landscapes and management goals because the individual problems of each site influence reclamation solutions.

Watershed maintenance centers on restoring water courses by removing cisterns and dams. Dams must be removed in a way that will not wash the dam components downstream. This requires careful consideration about how to release collected water in a controlled manner to its original path. However, the process of diversion may permanently alter the direction of ephemeral water flow and the way in which it accumulates in pools. Equally important in preserving watershed function is preserving landscape drainage. Restoring the integrity of watershed drainage goes hand in hand with rectifying land alterations such as reservoirs, terracing and displaced vegetation that alters surface and subsurface water flow. Remediation teams attempt to fill reservoirs, holes and sumps, re-contour slopes, and disperse vegetation.It is important to fill any holes dug by cultivators because they can trap and kill small mammals. Remediators use rakes and shovels to re-grade slopes and flatten mounds to smooth soil surfaces. While re-contouring can be significant, soil replacement is inevitably an imperfect process. The removal of logs providing terrace support can cause rapid erosion, which makes terrace remediation complicated and difficult. The original grade is never reproduced perfectly, but over time, the decomposition of organic matter and normal physical processes replace soil losses. In addition, crews perform restoration tasks such as “filling in planting holes and covering the hillsides with small branches and duff to help prevent [further] erosion.”Clean up crews use the slash piles created by cultivators to control erosion in areas with bare soil. It is important to disperse cleared vegetation, to remove large berms that obstruct site accessibility, and to redistribute piles from streams and creeks. Remote landscapes generally recover very well with time through “natural or unassisted regeneration.”The dark and rich organic soils provide abundant nutrients to new sprouts, and the more inhospitable landscapes are well suited for native plants. For example, large trees that are topped off will take tens to hundreds of years to recover if at all; but successive plants will thrive in the newly sunlit areas. When remediators do restore vegetation, they spread seeds of native plant species or plant seedlings. This can provide habitat, prevent further establishment of invasive plants and mitigate erosion. Habitat maintenance and restoration is a major concern for cultivated areas containing threatened or endangered species. In certain cases, plant restoration requires regular monitoring to gauge effectiveness. The success of plant recovery depends on each site, but the most vital factors in site recovery are the prevention of cultivator return and the passing of time. Little can be done to remove the presence of chemicals from the biomass.

Petitions to reclassify Cannabis have been proposed since the 1970s based on an ever increasing literature of clinical studies and scientific research that disputes the vague classifications of “high abuse potential, a lack of accepted safety under medical supervision and no currently accepted medical use.”8 This article of the Comprehensive Drug Abuse Prevention and Control Act was brought to the forefront of legal and political debate in 1996 when the Compassionate Use Act, or Proposition 215, passed in California, followed by 13 other states, to legalize the medical use of marijuana.9 Criteria for the legal possession of medical marijuana vary from the state to county levels, but Cannabis possession and consumption remain illegal at the federal level. In the pivotal Supreme Court decision of Gonzales v. Raich, the court ruled that the federal ban on cannabis may be enforced at all levels of jurisdiction based on the Commerce Clause of the United States Constitution. Their basis was that “incidents of the traffic which are not an integral part of the interstate or foreign flow, such as manufacture, local distribution, and possession, nonetheless have a substantial and direct effect upon interstate commerce.”10 Despite the precedent set by this case, the development of legalized medical marijuana has led to significant changes in domestic Cannabis cultivation. In California, marijuana is cultivated in various amounts ranging from a single plant grown for personal consumption to thousands of plants per plot cultivated for commercial distribution. Law enforcement and US Forest Service reports indicate that Drug Trafficking Organizations control a significant portion of Cannabis cultivation in the United States and are establishing an increasing number of both indoor and outdoor growing sites. The primary DTOs operating in California are of Mexican origin and consist of the most powerful cartels in Mexico. The outdoor cultivation sites developed by DTOs are of special concern because they mainly occur on public lands.

Remote areas used by cultivators include land holdings managed by the US Forest Service, the Bureau of Land Management,pipp grow rack the National Park Service, the Fish and Wildlife Service, the Bureau of Indian Affairs, and the Bureau of Reclamation.11 However, DTOs also cultivate marijuana plots on private lands including conservation reserves, game lands, and large private land holdings. Marijuana cultivation on public lands is entrenched in California structurally, institutionally, economically, and culturally. The issues that surround marijuana cultivation and prevention efforts are both complex and large in scale. The systems and processes employed by both cultivators and law enforcement agencies are long established and the product of a dynamic progression. However, the scale of marijuana production and the adverse effects of cultivation have reached unprecedented proportions. Black market revenues have increased with the demand for marijuana, giving rise to criminal enterprises that go to any lengths necessary to maximize profits. The result is an oligopolistic market control, continued natural resource damage and sustained infringement on public safety.Marijuana is California’s largest cash crop, estimated to be worth between 10 and 14 billion dollars annually.12This immense profit incentive is the main cause for continual marijuana cultivation on public lands, and is based on high demand and inflated market prices. In the most highly concentrated cultivation counties in California, the economies of which are based on the marijuana industry, only a handful of cultivators are arrested each year. Marijuana has a great cultural significance which is apparent in clothing, artwork, music, and many other forms. People of all ages and backgrounds consume marijuana for a variety of reasons. It is a constant and accessible commodity, even more so than alcohol to some, and has become deeply ingrained in California culture. Its accessibility makes it easy for people to use regularly and to create substance based relationships where marijuana becomes routine in certain activities or social situations.

These habitual relationships are significant in social participation and the creation of psychological reliance. Marijuana’s unique psychoactive function distinguishes it from any other drug. Marijuana can alter one’s perception in a way to provide relief from stress, or to simply to produce a pleasurable sensory perception. The most common mechanism of ingestion, inhalation, allows the psychoactive chemicals to take effect within thirty seconds.13 Marijuana is dose dependent so people can control the intensity of their high to some extent. It is a versatile natural medicine and provides viable relief from a myriad of symptoms and diseases. While individuals have their own motivation for smoking marijuana, the cannabis plant is a deeply rooted product in American culture. Marijuana cultivation by Drug Trafficking Organizations is a relatively recent development in California history. The foremost organizations that produce commercial marijuana plantations are of Mexican descent. Other major cultivators include outfits of Canadian and South American origin as well as comparably small scale independent growers. These groups began cultivating Cannabis on public lands in order to evade border security, capitalize on lucrative domestic markets, and take advantage of the optimal growing conditions provided by California lands. Remote Cannabis cultivation requires large scale capital investments as well as a high degree of risk; however, DTOs have had a great measure of success. Traditionally, major organizations conducted drug traffic across international borders. Illegal substances were produced in foreign areas and transported across United States borders. Trafficking organizations utilized a variety of methods and a large quantity of smugglers to transport a steady flow of drugs beyond US border security with minimal losses to seizure.

Mexican cartels were notorious for supplying large amounts of low quality marijuana to the southern regions of the United States. This commercial grade marijuana was often referred to as ‘Mexican Brick Weed’; a reference to the tightly condensed ‘bricks’ of dried marijuana buds packaged for international transfer. The process of drying and condensing buds distorted bud shape, reduced THC content, and decreased overall marketability, however, high volume smuggling created a surplus of marijuana that could be sold cheaply and in large quantities to compensate for the low quality of the substance. Mexican Brick Weed contained approximately 2-3 percent THC, whereas domestically produced strains of sinsemilla, marijuana without seeds, could reach levels upwards of 10 percent THC.14 In most areas of California, Mexican marijuana could not compete with domestically produced strains in terms of quality; only in competitive price. The risk involved with Cannabis cultivation enabled local growers and distributors to charge high prices, while the methods of pollination prevention, crossbreeding, and nutrient additives created highly psychoactive buds that maintained a widespread demand in California and across the nation. Domestic marijuana cultivation traditionally occurred on a small scale by California residents. Small gardens were tended by a few people on private property, or a nearby location where plots of up to one hundred plants were grown.15 While marijuana cultivation remained illegal, this method provided a form of income and sustenance for people living in rural areas. Grow sites were generally outdoors near an accessible water source, and growers lived in local proximity to their sites. These small scale plantations were the primary producers of the marijuana sold in California up until early 1980s, when large scale organizations entered into marijuana production inside the US. These policies initiated a transition in immigration practices that allowed more Mexican laborers to legally enter the United States while deterring illegal methods of entry by tightening border security. The increased border security led to higher rates of drug seizure. These shifts in immigration were reinforced by the Immigration Act of 1990, which increased the limits on legal immigration, changed the status of aliens, and further increased border security by deploying a regular presence of National Guard troops to assist with Border Patrol.17 As the presence of law enforcement troops increased, the funding for extended patrols, searches and seizures increased as well. These measures threatened the flow of non-documented laborers and restricted the ability for Mexican organizations to regularly transport drugs across the border. As more traffickers were intercepted, DTOs responded by developing products within US borders.18 During the early 1990s the Mexican cartels began to participate in the trend that originated in California in the early 1980s. In response to increased marijuana related property seizures, California citizens began to grow marijuana on public lands because it made “ownership of marijuana… difficult to prove.” Growing Cannabis outside of personal property enabled local cultivators to limit investments on land and greatly reduced both the probability of getting caught and the liability of losing their property to the state.

Commercial growers were able to diversify and decentralize their cultivation sites in order to make them less vulnerable. This cultivation model was ideal for cartels because they did not need to acquire land and could operate undetected by discreetly encroaching on remote landscapes. Meanwhile,pipp horticulture racks the dynamics of the marijuana market in California began to change as a result of state level policy changes. In 1996, the Compassionate Use Act, Proposition 215, passed 55.6 percent to 44.4 percent to allow medical marijuana to be legally grown and consumed in California, soon followed by thirteen other states. Minimal regulation of doctor prescribed medical marijuana cards allowed cultivators to easily enter into the medical marijuana market. The new legal standing of marijuana decreased the consequences for cultivation on the whole, and often enabled traditional commercial cultivation to occur under the umbrella of the Compassionate Use Act. This proposition reduced the scale of some grow sites by setting limitations on legally cultivated amounts, but it also significantly increased the number of growers. Proposition 215 created a political climate conducive to innovative cultivator practices. Access to important infrastructure and Cannabis enhancing inputs increased because cultivation efforts no longer needed to be kept completely secret. Commercial outlets and plant nurseries could supply high technology indoor and outdoor grow systems along with all of the associated inputs. As a result, more specialized systems were designed and sold commercially. As time passed, individuals increasingly experimented with techniques in crossbreeding, transgenesis, and high-tech indoor cultivation environments. Meticulous grower experimentation and utilization of extensive inputs increased the quality of high grade marijuana by increasing THC and Cannabinoid contents.Whereas the THC content of marijuana previously peaked around 12%, new strains could reach levels upward of 20%.The change in California state marijuana policy decreased the demand for lower quality imported marijuana. California’s Mediterranean climate, abundant water, and loamy organic soils provide ideal growing conditions. By increasing cultivation operations on US lands, DTOs were able to exploit the abundant resources, and domestic research and development necessary to produce high quality psychoactive Cannabis. The transition from traditional marijuana cultivation to DTO operated cultivation has transformed the scale, methods, economic scope, and environmental impact of marijuana growing. Land encroachment practices have likely increased cartel expenditures compared to traditional trafficking methods, but they have also increased market accessibility, demand and price. The marijuana market has become big business, and is estimated to be the most profitable industry in California.However, the primary profiteers are safeguarded outside of the US. Modern DTOs are international organizations governed by tight hierarchical structures, the upper ranks of which are controlled by familial ties.The logistics of their operations are meticulously planned and highly organized. DTOs are sophisticated in their methods, technologically advanced in their systems, and resourceful in their practices. They choose unusual locations to evade detection, even planting in sites not considered conducive for Cannabis cultivation. Grower methods for entering sites are both inventive and evasive. Suppliers drop off materials at inconspicuous locations during night hours in order to remain undetected. Laborers use irregular entry points and carry the supplies on their backs, hiking long distances off established paths through the darkness. These men sometimes carry more than their body weight in supplies over extremely rough terrain, and are careful to remove evidence of their presence. While some individuals use night vision goggles to navigate in the darkness, they still cross terrain that is dangerous in full daylight without added weight. DTO-established sites are setup and operated by two to fifteen people, several of which live on location. A select number of workers with specialized technical expertise rotate between sites and aid in preparation according to their skill set .The men who live on site throughout the season are usually Mexican nationals recruited out of economic desperation or to settle a debt to a Cartel. Their payment depends on the delivery of a complete harvest of marijuana buds without detection.

Following this operant learning paradigm, we wanted to assess how adolescent drug exposure might impact nicotine reinforcement in adulthood. In our previous studies we have found that exposure to the synthetic cannabinoid WIN 55,212-2 and co-exposure to nicotine and WIN together in adolescence increases subsequent adulthood intake of a low dose of nicotine in males, but decreases intake of low and moderate nicotine doses in females. Our current experiments revealed that females exposed to the higher dose of THC in adolescence self-administered more nicotine in adulthood at a moderate nicotine dose than control subjects. Males exposed to either the lower or higher dose of THC or co-exposed to nicotine and the lower dose of THC also self-administered more nicotine at this dose. These findings differ compared to our previous studies where we did not see any differences in nicotine intake at this dose following adolescent cannabinoid drug exposure in males and reduced nicotine intake in females. But given that the method of drug exposure, oral as compared to previously injected, and the drug itself, THC compared to WIN, are changed, these differences are not too surprising. Rather, it further adds to the complexity of this story when parsing out the effects of adolescent drug exposure on later drug-taking behaviors. In humans, women with a history of cannabis use are four times more likely to become regular cigarette smokers and almost three times as likely to develop nicotine dependence which is supported by our finding of increased nicotine intake in adult female mice who were previously exposed to THC.

Additionally, men with a history of cannabis use are also more likely to become daily cigarette smokers which aligns with our current findings as well. Of note,flood and drain hydroponics it was unexpected that the groups co-exposed to nicotine and the higher dose of THC did not exhibit any differences in nicotine intake. These groups did have the initial differences in cotinine levels which we suspected would alter later drug-taking. While all of the nicotine exposed groups in both sexes had a significant level of cotinine in their blood, both males and females co-exposed to the higher dose of THC and nicotine together had lower cotinine levels than those exposed to nicotine vapor alone and those co-exposed to the lower dose of THC and nicotine vapor. This suggests that the higher dose of THC may impact the metabolism of nicotine. In support of these findings, another study confirms that in human smokers, co-users of both nicotine and THC have lower cotinine levels than tobacco only smokers. Yet although males co-exposed to nicotine and the higher dose of THC exhibited a greater drive to obtain food reward that was not reflected in their nicotine intake. Thus, taken together, findings from these experiments demonstrate that in males, THC or nicotine and lower dose THC co-exposure in adolescence, and in females exposure to higher doses of THC during adolescence have persistent developmental effects and increase the drive to consume both food and nicotine in adulthood.Finally, we wanted to assess the impact of this adolescent drug exposure on cueinduced nicotine seeking in adulthood. We were first able to reliably demonstrate the incubation of nicotine craving effect in both male and female control mice. Then we found that chronic adolescent exposure to nicotine or THC differentially alters later incubation of nicotine craving based on route of administration and dose.

Specifically, both male and female mice that were exposed to nicotine via injections and females exposed to nicotine vapor during adolescence did not have enhanced nicotine-seeking as adults following an extended withdrawal period. These findings indicates that nicotine-associated cues may not induce craving across abstinence in some subjects that have an adolescent history of nicotine exposure. Surprisingly, males that were exposed to nicotine vapor during adolescence did have the incubation of nicotine craving effect in adulthood. It is important to note that the cotinine levels for males exposed to nicotine alone regardless of route of administration did not differ, which emphasizes that the duration of the daily nicotine exposure has unique implications on this later drug seeking behavior. This contention needs to be further explored with more specific studies but the notion is supported with prior findings that duration of nicotine exposure via osmotic minipumps compared to injections at the same dose alters nicotine withdrawal. Furthermore, given the differences in nicotine intake among the male THC exposure groups, we were surprised to find that they maintained the incubation of nicotine craving effect. However, another study found similar results in which adolescent THC exposure in male mice does not alter later stress- and cue-induced reinstatement of nicotine-seeking following extinction. In females, while the lower dose THC exposure group did have an increase in nicotine-seeking on day 24 as compared to day 1 of the incubation, the higher dose THC group did not. Instead, the higher dose THC female subjects exhibited an increase in active lever presses on day 1 as compared to their lever pressing during nicotine self administration.

This finding suggests that the higher dose of THC during adolescence may have led to overall increased active lever pressing, which is consistent with the higher level of responding for food training, or it may be a premature incubation effect with higher immediate and persistent drug seeking behavior. Importantly, co-exposure to nicotine vapor and THC at either dose does result in the incubation of nicotine craving effect. We expected these results as human adult co-users are twice as likely as tobacco smokers who do not use cannabis to continue smoking tobacco. This could be due to the cannabinoids enhancing the effects of nicotine-associated cues in reinstating the drug-seeking behavior after a quit attempt. Moreover, for the females, although single drug exposure to nicotine or the higher dose of THC alone does not result in the incubation effect, perhaps in these poly-drug exposure conditions the nicotine and cannabinoids interact during development to alter the responsivity of nicotine-associated cues in reinstating drug-seeking behavior. Thus, the adolescent co-exposure to both drugs could result in a heightened response to the drug-associated cues later in life. Patients’ drug histories are an important factor for treating substance use disorders. Our prior findings demonstrated that acute and chronic pre-treatment with a synthetic cannabinoid can reduce nicotine intake in male and female mice; however, if either sex had been previously exposed to nicotine or cannabinoids during adolescence, this reduction does not occur. Thus, prior drug history may be a mediating factor in the effectiveness of pharmacological cessation treatments. Furthermore, in another study, the offspring mice of parents who were exposed to nicotine do not demonstrate an incubation of nicotine craving effect. This indicates that the effects of drug exposure persist not only for the later drug seeking and cue responsive behaviors in one subject, but it could have lasting generational impacts for their offspring as well. In conclusion, adolescent exposure to nicotine and/or THC does alter operant learning, drug intake, and relapse-related behaviors in adulthood in a sex-dependent manner. Taken together, these results indicate that adolescent use of cannabinoids have a persistent effect on reward consumption, which is dependent on THC dose, nicotine coexposure, and type of reward. These studies emphasize the impact of prior drug history on later drug-associated behaviors and susceptibility to relapse as well as the importance of being cognizant of adolescent drug use in patient populations when navigating personalized approaches to treating adulthood substance abuse.Diversity, equity, and inclusion have been hot topics in recent years. In particular since the worldwide protests against police brutality and social injustices in 2020, there has been a resurgence in the push for creating more equitable and inclusive environments within the field of neuroscience and academia as a whole. However, true DEI work must go beyond just increasing the number of historically excluded scholars in the laboratories and classrooms; they must also be actively included, given a voice to push for change, and supported in their efforts. My DEI efforts as a graduate student have included serving as an Interdepartmental Neuroscience Program graduate student representative, Competitive Edge peer mentor,indoor vertical farming and department representative for the Diverse Educational Community and Doctoral Experience program. In each of these roles, I strived to create safe spaces for first-generation, Black, Indigenous, Latinx scholars and women to feel welcomed in neuroscience. I also offered reassurance that they belong in the field, advocated on their behalf, and provided needed support as they navigate academia. In doing so, I went beyond simply recruiting a more diverse cohort into the neuroscience program to help ensure their retention by cultivating a community in which they could thrive.

Perhaps my greatest achievement outside my scientific research has been my efforts to support historically marginalized people in neuroscience and founding the global nonprofit, Black In Neuro. Black In Neuro is an international organization that aspires to diversify the neurosciences by building a community that celebrates and empowers Black scholars and professionals in neuroscience-related fields. We also aim to provide professional development resources and increase the visibility of Black neuroscientists to inspire the next generation and dismantle stereotypes of what a neuroscientist can be.As a PI and mentor, your reactions about these events can dictate whether your trainees ever feel safe to speak up again. Before we are scientists, we are people. Our self advocacy requires an immense amount of bravery. It is consuming, is psychologically draining and could cause future troubles for us. So, not every underrepresented person wants to confront all instances of racism head-on. But with the perpetual anti-Black violence still occurring, I want to give a few suggestions for mentors to better support their Black trainees. Make it very clear that you are an ally. Don’t wait for us to start the conversation, because your silence speaks volumes. Let it be known if your office is a safe space. Host quarterly lab meetings that are focused on efforts the lab is making to become anti-racist and on current issues affecting diverse students. Call out your colleagues on their racism. Educate yourself. Become aware of the systemic hurdles that every Black student has had to overcome to get to this point. Black Americans hold ~2% of the national wealth, which means less access to private schooling, tutors and prep programs. Standardized tests have been shown to be biased against minority students and those belonging to a lower socioeconomic status. Almost half of Black students enrolled in a post secondary institution are first-generation college students, which means they may not know about as many scholarship or internship opportunities as their peers do. This isn’t even addressing the racial profiling, subpar medical care and over-policing that takes a physiological toll. Take these into consideration when considering graduate school applicants and hiring. Furthermore, actively encourage applicants from nationally funded diversity initiatives that uplift Black students in the biomedical sciences, such as Maximizing Access to Research Careers, and Diversity Specialized Predoctoral to Postdoctoral Advancement in Neuroscience. Speak up for your Black trainees. Advocate for us when we’re not in the room. Nominate us for awards and speak highly of our efforts. Let us know about fellowships, travel grants and other opportunities that can help advance our careers. Teach us how academia works and the ‘unspoken’ etiquette in the field. Use your position of power to be a champion for equality and racial justice. You do not have to belong to an underrepresented group to support the people in that group. Demand that your departments and schools hire diversity and inclusion experts to host implicit-bias workshops and cultural-competency trainings. When they do want to hear from the current Black student and faculty perspective, find ways to compensate them for this diversity work. But also recognize that the one Black student cannot speak for all Black people. Black experiences are not a monolith. Ensure Black leaders in your field are invited to give research talks at conferences, symposia and departmental seminar series. Encourage everyone to attend, not just the underrepresented students, because perceptions and stereotypes can be changed all around. If you do not identify as the same gender, race or background as your trainee, help them find a mentor who does. My advisor and I have a mutual understanding of sexism in science. But as a white woman, she cannot fully understand how racism is compounded in my experience. One of the best things she did was to connect me with another Black neuroscientist and professor.

Catheters consisted of a 6 cm length of silastic tubing fitted to guide cannula bent at a curved right angle and encased in dental acrylic. The catheter tubing was passed subcutaneously from the animal’s back to the right jugular vein, and a 1 cm length of the catheter tip was inserted into the vein and tied with surgical silk suture. Following the surgical procedure, animals were allowed ≥48 hours to recover from surgery, then provided access to again respond for food reward. Mice were then permitted to acquire intravenous nicotine self-administration during 1 hour daily sessions, 6–7 days per week, at the standard training dose of nicotine . Each session was performed using two retractable levers . Completion of the response criteria on the active lever resulted in the delivery of an intravenous nicotine infusion . Responses on the inactive lever were recorded but had no scheduled consequences. Catheters were flushed daily with physiological sterile saline solution containing heparin . Catheter integrity was tested with the short-acting barbiturate anesthetic Brevital . Subjects and their data were removed from the study due to death or if the catheter integrity was compromised as determined by visual leakage or Brevital assessment. Behavioral responses were automatically recorded by Med Associates software.The experimental design is outlined in Figure 2.1. Following adolescent injections, mice remained drug-free until adulthood . Thereafter, they were examined for differences in cognitive behavior as reported previously. For these investigations, to ascertain the dose–response function,flood drain tray mice were tested according to the established mouse intravenous self-administration protocol.

Following an acquisition period of at least 7 days on the training dose , the animals were presented with a different dose of nicotine for at least 5 days, and the mean intake for the last two sessions was used for statistical analyses. In between each dose, subjects were returned to the 0.1 mg/kg/infusion dose for 2 days or until intake returned to baseline levels. The dose– response function occurred over a total ~35 sessions with testing sessions occurring 6 days per week. Thereafter, mice were stabilized on the moderate 0.1 mg/kg/infusion dose across three baseline sessions after successfully passing the Brevital catheter patency test. Then, subjects were challenged with an injection of the low dose WIN or vehicle control, 20 minutes prior to the nicotine self-administration session. Injections of vehicle or low dose WIN were administered in a random, counterbalanced design both within and across groups, and subjects were permitted at least 2 baseline days in between WIN/vehicle administration to return to baseline levels of nicotine intake. After the crossover experiment with the single, acute dose of WIN, mice were chronically pretreated with the same low WIN dose prior to each session across seven consecutive sessions, and nicotine intake on the seventh session was used to determine the effects of chronic coexposure during adulthood for all groups. Since the control groups for the moderate and low dose WIN cohorts exhibited similar effects with pretreatment, data were compiled into one graph for each sex. Finally, mice were again returned to self-administer the 0.1 mg/kg/infusion dose, and after achieving baseline levels of responding, they were then transitioned to respond for saline infusions . Eleven mice were required to be excluded due to death/cannibalization by cagemates , and six were excluded due to compromised catheter integrity .

Given that these studies sought to investigate the effects of drug exposure relative to the control condition within each sex, statistical comparisons were performed separately for males and females based on this a priori hypothesis. Data were analyzed by a t test, oneway or two-way analysis of variance with Prism 7 software , as appropriate. Data obtained across sessions were analyzed with a repeated measures two-way ANOVA. Significant main or interaction effects were followed by Bonferroni post hoc comparison with correction for multiple comparisons. The criterion for significance was set at α = 0.05.Adolescent exposure groups were examined for differences in nicotine intake during adulthood across low, moderate, and high self-administration doses . This approach allows for the assessment of the dose–response function, which provides a measure of responding across nicotine doses with increasing value of reinforcement and doses inducing greater aversion and/or satiation. Specifically, the WIN and nicotine/WIN adolescent exposure groups exhibited a significantly increased number of nicotine infusions compared with the control and nicotine adolescent exposure groups . Further, the groups did not differ in their saline level of responding, indicating that these differences were not due to a general increase in lever pressing behavior. Since both the WIN and nicotine/WIN exposure conditions involved a moderate dose of the cannabinoid agonist , we next addressed the possibility that this WIN dose could have masked the effects of nicotine in an interactive effect. Thus, we examined a separate cohort of mice exposed to a low dose of WIN , either in the presence or absence of nicotine. The post hoc analysis revealed an upward shift in the dose–response function for the nicotine exposure group, as compared with both the WIN and coexposure nicotine and WIN groups.

Specifically, at the 0.03 mg/kg/infusion dose, the adolescent nicotine group exhibited a significantly greater number of nicotine infusions than the adolescent WIN and nicotine/WIN coexposure groups. At the moderate 0.1 mg/kg/infusion dose, the nicotine group also demonstrated a statistically significant increase from the WIN group and nicotine/WIN coexposure group , and the nicotine/WIN coexposure group was also significantly decreased compared with the control group . No other groups significantly differed from the control, or at the saline and high dose of nicotine . Thereafter, a second set of female mice were examined for differences with the lower dose of WIN. To examine whether further exposure in adulthood to a cannabinoid agonist subsequently alters nicotine intake, mice were pretreated with the low dose of the cannabinoid agonist or vehicle prior to a nicotine self-administration session; thereafter, the mice were then repeatedly administered the low dose of the cannabinoid agonist prior to seven consecutive nicotine self-administration sessions . Interestingly, when we examined the adolescent-exposed nicotine and WIN groups, a stark contrast in responding was evidenced.Specifically, in post hoc analyses, the vehicle condition exhibited a higher level of nicotine infusions compared with pretreatment with the cannabinoid after one session and after seven consecutive sessions . Further, chronic administration of the cannabinoid agonist significantly reduced nicotine intake to a greater extent than the acute condition .In these studies, we found that adolescent cannabinoid and/or nicotine exposure exert a lasting impact on susceptibility to drug reinforcement, which is evidenced in adulthood. However, these effects were dependent on the substance of abuse , dose of the cannabinoid, and sex. Specifically, adult males exhibited increased nicotine self-administration at the lower rewarding nicotine dose following adolescent cannabinoid agonist exposure at the moderate dose , either alone or with nicotine coadministration. In contrast, adult females demonstrated an opposing effect following adolescent cannabinoid exposure at the moderate dose, in which such exposure resulted in decreased nicotine intake compared with nicotine exposure alone. However, differences were not induced within either sex with adolescent exposure to the lower dose of the cannabinoid agonist . Furthermore, after maintaining nicotine self administration, pretreatment with the low dose of the cannabinoid agonist attenuated nicotine intake in both male and female control mice,flood and drain tray and this lowering effect was evidenced both acutely and after chronic pairings. Surprisingly, the cannabinoid agonist was ineffective in altering nicotine intake in mice previously exposed to nicotine, the cannabinoid agonist, or both during adolescence; an effect that was present at both the lower and moderate doses of the cannabinoid agonist.Nicotine self-administration produces an inverted U-shaped dose–response curve, which represents the competing positive and negative properties of nicotine. The increased responding for nicotine over the ascending limb of the curve reflects the increasing reinforcing effects of nicotine as the unit dose increases. In contrast, the decreased responding over the descending limb of the curve reflects the increasing aversive properties of nicotine or satiation. Mesolimbic dopamine neurons have been primarily implicated in modulating the rewarding and reinforcing aspects of the drug, whereas the aversive signaling of nicotine appears to involve the habenulo-interpeduncular pathway.

Our findings suggest that adolescent cannabinoid exposure most likely alters the function of the mesolimbic pathway, as differences were found primarily on the ascending limb of the dose– response function. In support of this notion, adolescent cannabinoid or nicotine exposure has previously been shown to alter monoaminergic signaling.However, in our study, nicotine alone was ineffective in altering later drug-taking behaviors in males, either in combination with the cannabinoid or alone. Since studies have shown that of those adolescents age 12–17 who smoke, the majority smoke one or less than one cigarette per day , the current studies focused on a rewarding dose with once daily exposure of a rewarding dose. Thus, the current results have particular relevance to experimental patterns of drug consumption found in youth. Differential patterns of expression of the cannabinoid receptors are found across adolescent development and between males and females, and CB1Rs exhibit highest level of expression during the developmental period of mid-adolescence. Thus, the potential for exogenous cannabinoids to alter synaptic and neural circuit function may be considered greatest during this time period. Indeed, prior studies have revealed an effect of CB1R activation on adolescent brain development and indicate a correlation between adolescent exposure and later cognition and reward-related function. For instance, we found that adult males exposed during adolescence to the moderate 2 mg/kg dose of WIN exhibited increased cognitive flexibility in a learning reversal task, decreased anxiety associated behaviors, and increased natural reward consumption with the same exposure paradigm. The coexposure condition of both nicotine and the moderate dose of WIN also led to similar behavioral profiles as WIN alone in these measures, suggesting that a potentiative or additive effect was not present similar to that found in the current studies with nicotine intake. With regard to females, they were found to be overall more resistant to the long-term effects of adolescent drug exposure, in which the moderate dose WIN females exhibited decreased natural reward consumption compared with the control females. Interestingly, CB1R knockout mice are resistant to nicotine-mediated locomotion and conditioned place preference, but do not differ in nicotine self-administration, as compared with wild-type mice, which suggests that the lack of CB1Rs during adulthood may affect generalized locomotor behavior and drug-conditioned memory function, but perhaps not the motivation to consume nicotine. However, given the constitutive knockout of the gene in these mice, it is possible that compensatory mechanisms occurred during development, resulting in altered expression of other receptors, potentially including cannabinoid 2 receptors and/or nAChRs.Both single and co-use of nicotine and cannabinoid products are prevalent during adolescence and adulthood. Thus, we further examined coexposure during adulthood, under both control conditions and following adolescent drug exposure. In the control group, we found that the low dose of the cannabinoid agonist reduced nicotine intake in adulthood. This represents the first demonstration of the effects of a cannabinoid agonist on intravenous nicotine self-administration in mice. These results were surprising since the CB1 receptor antagonists rimonabant and taranabant have also been shown to reduce nicotine consumption. However, when one considers that additive effects may be induced on brain reward circuitries, such as that found with reduced brain stimulation thresholds in the presence of rewarding doses of nicotine, it is likely that the presence of the cannabinoid agonist augmented the activity of the reward circuits in the brain, leading to a reduction in nicotine intake while maintaining similar circuit activation to support drug reinforcement. However, this stipulation will need to be more directly tested in future studies. We further found that the effectiveness of the cannabinoid agonist in reducing nicotine self-administration is dependent on prior drug exposure during adolescence, as all of the adolescent nicotine and/or WIN exposure groups did not differ in nicotine intake with WIN pretreatment in adulthood. Of further note, we found that this lack of responsiveness to the dampening effects of the cannabinoid agonist on nicotine intake also occurred in adolescent groups exposed to the low dose of the cannabinoid agonist. It is important to note that this level of exposure did not induce any other detectable behavioral effects during adulthood, either in this study or in our prior analysis of cognitive, anxiety-, and depression associated behaviors.

THCAS and CBDAS have been more studied and characterized than CBCAS, although CBCAS’s high sequence similarity with THCAS suggests it shares similar biological activities.With regards to localization and expression in the Cannabis sativa plant, CBDAS and THCAS have been demonstrated to be catalytically active in the glandular trichome’s extracellular cavity and need FAD as well as oxygen in order to functionally express.In contrast, the CBCAS enzyme is not oxygen dependent and can also be inhibited by hydrogen peroxide.As mentioned, THCAS and CBCAS share a 92% sequence similarity with each other, whereas THCAS and CBDAS are 84% identical to each other and CBCAS and CBDAS are 83% identical to each other; therefore, all three cyclase enzymes have high sequence similarity with each other. Sequence analysis of variants of the cyclase enzymes indicated that CBDA is likely the ancestral enzyme from which THCAS evolved from. Further sequence analysis of THCAS showed a flavinylation consensus sequence with His114 being the likely FAD-binding site, exhibiting similarity to CBDAS in this regard.Since both CBDAS and THCAS are flavinated enzymes, it is postulated that they have the same reaction mechanism. With regards to CBCAS, although much is unknown, it has been reported that based on its kinetic data , CBCAS has higher affinity for CBGA than both THCAS and CBDAS. These oxidocyclase enzymes are only capable of enzymatically acting up CBGA, not the decarboxylated CBG product.Due to the reported potent biological activity of THCP, the heptyl analog of THC,grow racks we determined to focus on achieving function expression of THCAS in our platform since our novel platform produces the heptyl variant of olivetolic acid.

As previously described, THCP was isolated from the Cannabis plant in small quantities in 2019 with pharmacological data showing that THCP has a Ki to CB1 of 1.2 mM, and a Ki to CB2 of 6.2 mM, ~ 30 and 6 times more effective in binding than THC, respectively.102 Even the THCA molecule, although unlike its decarboxylated form THC, is not psychoactive, has been investigated for its neuroprotective, anti-neoplastic, immunomodulator, and anti-inflammatory effects further making the pursuit and optimization of in vivo functional expression of THCAS a priority. Different groups have reported of engineering strains from Saccharomyces cerevisiae and Komagataella phaffi capable of functional expression of THCAS. Zirpel et al. demonstrated through the engineering of THCAS, optimization of culturing conditions, and overexpression of helper proteins, 83% conversion of CBGA to THCA in Komagataella phaffi. Zirpel et al were first able to obtain functional expression of THCAS in both S. cerevisiae and K. phaffi by utilizing a signal peptide from the vacuolar proteinase, proteinase A. The group cleaved the 28 amino acid N-terminal plastid signal peptide from the THCAS and inserted the proteinase A signal peptide. Additionally, they knocked out the proteinase A gene in both strains, responsible for degrading genes targeted to the vacuole. With functional expression in both yeast strains achieved through this process, they sought to optimize culturing conditions.The group assayed the strains at different temperatures and times and observed that the highest intracellular activity of THCAS was achieved at 15C for 192 hours. The temperature of the expression was the key change from other groups which had expressed THCAS at 30C and 37C. The expression of THCAS at 15 C increased volumetric THCAS by 6350%. Additionally concerning culturing conditions, Zirpel et al observed that addition of casamino acids, biotin, riboflavin, and yeast nitrogen base further increased THCAS functional expression.

Lastly, the overexpression of ER chaperones, Kar2p, CNE1p, the Pd1p foldase, the unfolded protein activator , as well as the FAD synthetase increased functional expression of THACS in K. phaffi, with the expression of HAC1p, having the greatest impact, increasing functional expression 4-fold.Through implementation of these concepts, we achieved functional expression of THCAS in our Saccharomyces cerevisiae strain. We already had obtained a S. cerevisiae strain with the pep4 knockout, so with transformation of the THCAS gene with the proA signal and with feeding CBGA, we observed production of THCA in YPD media at 30C. To increase functional expression, we added 13.8g/L yeast nitrogen base and 5 g/L casamino acids into the YPD media and observed a slight increase in production. To further increase expression, we overexpressed the HAC1p with the THCAS in our S. cerevisiae strain and observed a notable increase in production. We also then tested the temperature similar to Zirpel et al. The initial THCAS functional expression assays were done in media cultured at 30C. We therefore tested the temperature in a few different ways: 1) Culturing the seed culture and growth culture at 15C and feeding CBGA at day 2 2) Culturing the seed culture and growth culture at 30C and feeding CBGA at day 2 3) Culturing the seed culture and growth culture at 30C for 5 days and feeding CBGA at day 6. 4) Culturing the seed culture and growth culture at 15C for 5 days. At the 6th day, feed CBGA and culture at 30C 5) Culturing the seed culture and growth culture at 15C for 5 days and feeding CBGA at day 6. As the data indicates, option 4 had the greatest increase in functional THCAS expression, achieving greater than 50% conversion of CBGA to THCA based on UV area under the curve, a significant increase over the 3% estimated conversion rate when we initially expressed THCAS in S. cerevisiae.

In conclusion, we have demonstrated that through genome mining of fungal bio-synthetic gene clusters, we were able to identify a cluster in Metarhizium anisopliae that produces olivetolic acid, the first key intermediate in the cannabinoid bio-synthetic pathway as well as unsaturated olivetolic acid, sphaerophorolcarboxylic acid, and unsaturated sphaerophorolcarboxylic acid at moderate to high titers. We also identified homologous clusters that produced these same compounds as well as a homologous cluster found in Talaromyces islandicus that selectively produces olivetolic acid. From there, we desired to engineer our platform in three different aspects: i) by increasing the titer ii) by increasing the diversity our product profile iii) by going downstream to achieve the final elaborated cannabinoids.To increase the titer, we utilized two different approaches. In one, we utilized JMP software to develop a design of experiments approach to increase titer by optimizing the culture media. We initially screened for nine different components in the media, testing to determine which components had statistically significant effects on the titer production. We identified three components which we then optimized for using a response surface methodology approach. Using this approach, we were able to identify a culturing media that assists us in reaching titers almost 2-fold more than our original CD-ST culturing media. In our second approach to increasing the titer of our platform, we increased the malonyl-CoA pool in our model Aspergillus nidulans host by overexpressing the acetyl-CoA carboxylase gene, the enzyme responsible for converting acetyl-CoA to malonyl-CoA. Overexpressing of acetyl-CoA carboxylase helped us achieve greater than 2.5-fold increase in titer. We also desired to increase the diversity of our product profile. To do so, we also employed two different approaches. In one, we made mutations to the ketosynthase domain of our HRPKS, Ma_OvaA. The KS domain is responsible for decarboxylative Claisen condensation, extending the carbon-carbon chain length. We made mutations in the KS domain both manually and through the use of the automated Tecan liquid handling system integrated with a ThermoFisher Momentum Laboratory Automation System. We were able to identify a couple of mutations that produced different products from our original platform including divarinic acid as well as the olivetolic acid nonyl variant,grow table undecyl variant, and ethyl variant. We also further genome mining to identify more homologous clusters to our original platform. Utilizing our in-house strain database, we were able to identify a cluster in Penicillium thomii r89 that when heterologously expressed in Aspergillus nidulans, produced a nonyl variant of olivetolic acid with two degrees of unsaturation and a heptyl variant containing one degree ofunsaturation as well as a hydroxy group. Therefore, both methods were effective in producing more olivetolic acid variants and there is still much potential in discovering more variants with regards to these methods since especially with regards to the genome mining aspect, there is still much unexplored space. Lastly, we attempted to go downstream of the pathway to achieve the final elaborated cannabinoids. To do so, we mined for different prenyltransferase enzymes capable of geranylating olivetolic acid to form cannabigerolic acid. We were able to identify one in Talaromyces islandicus capable of geranylating olivetolic acid to form cannabigerolic acid in Aspergillus nidulans, giving us a platform where through the heterologous expression of four fungal genes, we were able to produce the first cannabinoid, CBGA. Utilizing CsPT4, we were also able to achieve de novo production of the heptyl variant of CBGA, when we expressed the Ma_OvA genes with CsPT4 in Saccharomyces cerevisiae. We also were able to achieve functional expression of THCAS in S. cerevisiae and were able to greatly increase expression by optimization of media, over expression of helper proteins, and culturing at lower temperatures.

Therefore, we have developed a different method for microbial production of cannabinoids, one that does not rely heavily on the cannabis plant genes and has potential to produce rare and new to-nature cannabinoids.With its roots in international treaties signed during the League of Nations Era, the transnational legal order of cannabis prohibition represents one of the most sustained efforts to develop internationally applicable standards for governing illicit markets.The vast majority of United Nations member states are now parties to the three major international drug conventions, which require criminalizing the production, distribution, and use of cannabis. Over the past decades, the cannabis prohibition TLO has come to encompass an extensive array of legal instruments for monitoring implementation efforts,disseminating information on the activities of drug trafficking networks,and facilitating cooperation among national police forces.However, despite the extensive institutionalization of this TLO, cannabis remains the most widely used illegal drug in the world. The 2018 World Drug Report estimates that at least 192 million people aged 15–64 had used cannabis in the preceding year.With the percentage of adults reporting cannabis use in North American and European countries far exceeding the international average, cannabis use has become integrated into mainstream culture in a large number of countries.In an era that is often characterized as one of a growing isomorphism of the laws and procedures governing criminal activities in different countries,the issue area of cannabis policy undergoes processes of fragmentation and polarization.Some countries continue to criminalize all forms of medical and recreational uses of cannabis. Others have sought to “separate the market” for cannabis from that of other drugs by decriminalizing the possession of small amounts of marijuana, authorizing its use for medical purposes, and establishing administrative measures for taxing and regulating the commercial sale of the drug.These reforms have gained international momentum despite resistance from key actors in the international drug control system, including the International Narcotic Control Board and the US federal government.The proliferation of cannabis liberalization reform is frequently depicted as a historical step toward the collapse not only of this TLO but of the entire edifice of the international narcotic control system of which it forms a part.How deep is the current crisis of the cannabis prohibition TLO? What are its causes and consequences? What does this case study reveal about the conditions under which criminal justice TLOs rise and fall? In this Article, I explore these questions to demonstrate the complex ways in which the cannabis prohibition TLO has served as a battleground between competing conceptions of the role of criminal law in addressing social and medical harms. Drawing on TLO theory,the Article shows that the capacity of the cannabis prohibition TLO to regulate the practices of legal actors at the international, national, and local levels has been eroded as a result of effective contestations of the input and output legitimacy of its governance endeavors. The rapid and widespread diffusion of new models of decriminalization, depenalization, and legalization has relied on the operation of mechanisms of recursive transnational lawmaking. These mechanisms originate from the indeterminacy of drug prohibition norms, the ideological contradictions between competing interpretations of their meaning, the impact of diagnostic struggles over the social issues that the international drug control system should address, and the mismatch between the actors shaping formal prohibition norms at the international level and those implementing these norms in national and local contexts.