The amount of dilution required to achieve odorless air delivers a crude point of departure but should acknowledge the large error and be presented with only one significant figure in the final results. Thresholds for odorants typically vary by several orders of magnitude, especially the ODTC50 . Further, reliable methods may not have been used, and results for odor detection and odor recognition are sometimes mixed up. Within a controlled setting, two approaches to sensory testing of dilutions by panelists are used. One is the Odor Profile Method , which uses sugar solutions for calibration, and the other uses odor disappearance upon sufficient dilution. Both rely on dilution equipment, typically a dynamic dilution olfactometer that delivers sampled air diluted with odorless air to a nose port where the dilution is smelled by the panelist. Concentrations are presented in ascending order to avoid desensitization and anticipation bias. Statistics are then used on the panelists’ results to determine the ODTC50 for the odorant or mixture tested. For a single odorant, OPM is used to assign an odor note and intensity to each dilution. The Weber-Fechner law is applied, meaning that the logarithm of the concentration is taken and then the intensity results are fit to a line through linear regression. Extrapolation to intensity score 1 yields the ODTC50 value. Each odorant can have a vastly different slope. For example, a 200-fold change in concentrations of 1-propanol and n-amyl buterate caused a 15-fold and 0.5- fold change in odor intensity, respectively . For mixtures , the ODTC50 can be determined by forced choice, typically “triangle,” methods using the same dynamic dilution equipment . While inhaling at the nose port,commercial racks the panelist rotates between three choices and then selects which is the diluted sample. A point estimate is generated rather than a curve, and no odor description is gathered.

ASTM Method E679-04 has been used to determine the ODTC50 values for a range of odorants . Such methods have been used in the drinking water industry to set ODTC50 values for methyl tertiary butyl ether and improvements to ASTM Method E679-04 have been suggested for drinking water . In Europe, under EN 13725, the final dataset typically only includes the data for the four or more panelists whose results are the most consistent with the overall panel’s geometric mean value. Also, panelists may be presented with 2 samples instead of 3. The dilution result is called “European odour unit” , which is defined as “the amount of odorants that, when evaporated into 1 m3 of neutral gas at standard conditions, elicits a physiological response from a panel equivalent to that elicited by 1 European reference odour mass [123 μg n-butanol] evaporated in 1 m3 of neutral gas at standard conditions” . Thus, the European approach accounts for the variation in detection thresholds of the panelists. Despite these strictures, proficiency tests in 2007 and 2008 found two thirds of the European laboratories claiming to work in accordance to the EN 13725 standard failed to demonstrate compliance with the required performance criteria . Dilution equipment can also be used to evaluate the odor hedonic tone, as is required in the Netherlands . Panelists express the degree of pleasantness using a 9-point scale ranging from H -4 to H +4 . The logarithm of the odor concentration is plotted versus the H-value, which approximates a straight line. Using linear regression, the level of dilution required to achieve Dutch regulatory criteria can be estimated. OPM used to determine the ODTC50 goes against the guidance upon which it was built, yet still delivers useful results . Both the FPA and APHA Method 2170 advise against conducting statistical analysis on the intensity scores. The scores are categorical rankings and not an interval scale. In other words, an intensity score of 8 is not necessarily twice as intense as a score of 4. The intensity scale was not designed for 1 to be the ODTC50 value, and historically used the symbol. Surprisingly, such “against the guidance” extrapolation was found to be not off-the-mark from ODTC50 values from other studies , which may be confirmation of the enormous range of such values .

Day-to-day variation by panels is typically within an order of magnitude . Faint environmental odors are sensed but cannot be measured by dynamic dilution olfactometry . Typically, dynamic dilution values are never given for levels lower than 10 OUE/m3 , and generally the lower values start in a range of 50–100 OUE/m3 . The use of human subjects as panelists is strictly controlled . International codes of conduct apply as do reviews by a human subjects review boards to protect participants.Exposure limits are intended to protect the health of populations and arrive at quite different results for workers versus the general public. Workers are considered healthier and less diverse in susceptibility than the general population. They also only spend a portion of their day and week on-the-job. Political pressures, too, may influence worker limits to prevent onerous restrictions to industry . The morbid or lethal outcomes of occupation exposures likely overshadowed concerns about impact on olfactory function. The end result is that worker exposure limits are usually many times higher than those for residential exposures. A survey by National Geographic revealed that factory workers reported poorer senses of smell , although sensory loss is typically gradual and may be confused with aging . Sensory-impaired workers can miss out on warning signals that impact their nutrition and quality of life. Monitoring of workplace air for hazardous chemicals cannot be replaced by sensory cues, due to a portion of the workforce having little or no sense of smell , odors can create a workplace nuisance for most employees. Workers, too, are subject to the psychological strain of odor exposure, and setting and occupational exposure limit above the odor threshold may lead to perceived risk and well as physical response . For these reasons, occupational exposure limits consider odor. OELs have long considered irritation . In the United States and Europe, about 40% of the OELs are set to avoid sensory irritation . Sensitization is especially a concern, and OELs should be set low enough to avoid such initial, triggering exposures .

Only three chemicals are regulated by OSHA based on “obnoxious odor” and worker complaints: isopropyl ether, phenyl ether, and vinyl toluene . Critical reviews suggest setting OELs by taking into account both irritation and odor thresholds . For residential exposures, sensory effects are considered in emergency situations but rarely for ongoing, long-term exposures . The latter protect from cancer and non-cancer effects by focusing on system organ toxicity. Whether odor is a health-protective signal varies from odorant to odorant. Carcinogens have especially low exposure limits, so the ODTC50 tends to be well above the residential exposure limit. Benzene and formaldehyde are prime examples of such carcinogens . Because ODTC50 values range over several orders of magnitude, they commonly overlap with exposure limits. Re-visiting the work by Rosenfeld et al. and adding the ODTC50 ranges from AIHA , this overlap became apparent for hydrogen sulfide and ammonia .Exposure assessment involves the measurement of odorant concentrations in the hope the subset measured is responsible for the odor. It also involves the use of human panels to identify odor characteristics . Such categorical measures cannot be used in a quantitative risk assessment; however, they do provide qualitative information. The measurement of the number of dilutions required to remove an odor is plagued by inconsistent results and driven by the final remaining odorant in the dilution . When exposure measurements have been determined for a subset of odorants, the readings can be compared to the exposure limits presented in Section 4.4. This is the risk characterization. For residential exposure, the comparison is with the ODTC50 by a ratio known as the “Odor Activity Value” . Such a comparison, however, carries with it the inherent weakness in the numerator and denominator values in the ratio, undermining some of its usefulness. Specifically, ODTC50 values usually span odors of magnitude, and measured concentrations are typically a snapshot in time. Some studies find the inputs to OAVs too variable and are now pursuing other methods instead . In addition, for ongoing rather than intermittent odor exposures, comparisons with health based exposure limits for residents, such as the USEPA Reference Concentration or California EPA Reference Exposure Level ,greenhouse rolling benches can be performed for non-cancer effects. For carcinogens, the cancer risk estimate involves the inhalation USEPA Unit Risk Factor or equivalent. Note that exposure assessment usually has less uncertainty than other portions of risk assessment . Mixtures are challenging for risk assessment. Combined effects of sensory irritants can be considered additive as a first approximation . The interplay of odorants, however, is often unknown . Hydrogen-sulfide-equivalents are a proposed approach , similar to the dioxin equivalents for the 17 dioxin-like congeners and the carbon dioxide equivalents for various greenhouse gases.As a literature review, where no exposure data were generated, the case-study approach is used to illustrate how odorant concentration data can be used in conjunction with odor threshold information to characterize the health risks.

Although the most significant risks are due to chemical exposures in the workplace , the focus of this paper is on residential exposures to environmental odors, so worker exposure studies were excluded .Not only have OAVs been evaluated for on-site worker exposures , they also have been evaluated for odorants from sewers across Australia over a period of 3.5 years . Both efforts were to prioritize the subset of odorants monitored within the mixture to identify “high priority” odorants for further analysis. For the sewer study, the upper bound concentrations were compared to the ODTC50 values from Nagata and Takeuchi . The ranking by OAV reduced the number of compounds from 31 to 8 “high-priority” odorants. Hydrogen sulfide and methyl mercaptan dominated across all sites , and other volatile sulfur compounds also ranked high for most sites. Diethyl sulfide, limonene, toluene and m,p-xylenes each ranked high for fewer sites. The limitations of such ranking are substantial. The choice of ODTC50 value is paramount and typically ranges over several orders of magnitude across studies, complicating OAV calculations and their utility as a ranking scheme. Although Sivret et al. acknowledged the enormous ranges of ODTC50 values available in the compilation by van Gemert in several graphs, the final ranking was performed using the single values found in Nagata and Takeuchi for simplicity. Presenting the final OAVs as ranges would have muddied the results. Finally, those compounds without ODTC50 values available in the literature were dropped from the ranking, essentially rewarding lack of data for potentially odorous compounds. As with any study of mixtures, the analyzed odorants are only a subset and may not reflect the overall, total odor experienced by residents. Risk assessment is often predictive rather than retrospective. The impacts of a proposed oil well in Hermosa Beach, California, were forecast . Except for upset conditions, the anticipated negative health outcomes were largely nuisance-related . The evaluation concluded that the oil well would have no substantial effect on community health, which was based in part on a risk assessment of hydrogen sulfide as the odorant of primary concern. The results of the modeling indicated that fugitive emissions from normal operations could produce concentrations greater than the odor threshold without mitigation, which would reach nearby residences. Concentrations could be as high as 6 times the odor threshold, primarily driven by hydrogen sulfide. The acute REL for hydrogen sulfide would only be exceeded, according to the model, during accidental or unplanned release. Odor impacts from normal operations were, therefore, considered potentially significant without mitigation, so an Odor Minimization Plan was required.Over years, the residents of communities north of Denver have complained of intermittent, unpredictable “tar” and “asphalt” odors. The symptoms in Globeville, Colorado, included burning eyes and throat, headaches, skin irritation and sleep problems. A USEPA funded environmental justice study was conducted there in 2012 . Air samples were collected from locations near potential sources and within the community over a period of 7 months. Out of a list of 23 analytes, the most prevalent were hexane, heptane, benzene, toluene, m,p-xylenes and naphthalene. The maximum concentrations were below odor and toxicity thresholds , except for the carcinogens , which were considered within normal ranges for urban air.

The inclusion of an intercept, however, fundamentally changes the slopes of the lines and does fundamentally alter the relationship. The original Weber-Fechner law included no intercept . Researchers added the 0.5 intercept to account for “the definition of the odour threshold concentration which states that 50% of the panellists perceive weak odour while the others perceive no odour” and proceeded to use an intensity scale that ranged from 0 to 5 . Other researchers also used the equation with the 0.5 intercept, but the intensity scale ranged from 0 to 6 . The effect of the 0.5 intercept in both of these studies was to assign an intensity score of 0.5 to the ODTC50 concentration, which had nothing to do with the percent of the panelists perceiving or not perceiving and odor. Another researcher allowed the intercept to float uniquely for each odorant and used a 0 to 12 intensity scale , which scale been used in flavor and drinking water profiling . The results of these three approaches are dramatically different because the fixed intercept forces all lines to the same point while the floating intercept allows for very different linear fits to the data. Further research is needed into the equation that best fits the relationship between concentration and intensity scales.To use the above equations, an intensity scale needs to be used that offers interval scaling, i.e., a doubling of the score means a doubling in perceived intensity. Initial work by Arthur D. Little avoided such scaling and applied symbols and words to relative intensity bins . Researchers turned this into a mathematical scale and chose at one point to multiply the scores by a factor of 4 . After this arbitrary expansion, the score 1 was then deemed to be the odor detection threshold concentration for 50% of panelists , although such an approach is advised against in the guidance . This approach to odor intensity is included in the Odor Profile Method,vertical grow rack which is presented in Section 4.2.The predicted ODTC50 using the 0-12 scale for several chemicals was within the range of the values from more traditional methods .

Although the intensity scale is ordinal , it is handled as a metric scale , presumably because it simply works. As with analytical instruments, the zero intensity score is not included in the linear regressions. Note that the above equations use the base-10 logarithm rather than the natural logarithm, which is used in most science, perhaps because decibels use the base-10 logarithm. Another method of finding the intensity of an odorant is through repeated dilutions of the sample . These dilutions are presented to panelists from high concentration to low concentration using continuous airflow to a nose port by a dynamic dilution instrument . Two odorless blanks and the sample are presented, and the panelist chooses which one is odorous . In Asia, the standard method is 3 bags instead of continuous flow. Points along the intensity curve can be observed, but it is typically the ODTC50 that is sought. Both methods, using an intensity scale or dilutions, are used to analyze mixtures as well. Mixtures are more complex and do not necessarily follow the above associations. Specifically, the number of dilutions required to reach the odor-detection threshold for a mixture does not properly reflect the actual sensory intensity , as demonstrated by a study of fecal odorants . In other words, odor intensities increase and decrease with concentration at different rates for different odorants, not to mention the antagonistic and synergistic effects that also occur. A comparison of two intensity methods commonly used to evaluate single odorants were compared . One method used various vials of n-butanol as reference points to provide a sniffed intensity scale for panelists , while the other used three tasted solutions of sugar at different concentrations . The latter approach was applied to odor research based on the cross-modal premise that our senses are linked, so taste can be used to inform the sense of smell . Although the two anchors were not necessarily parallel at the high end of their scales , they compared favorably . Therefore, the result of this study cannot be used to say the two scales are similar beyond this one study’s approach.

Further, the n-butanol method advises against translating the results into perceived odor intensities . A comparison with the other anchors would be informative .Note the similarities between odor-detection thresholds and method detection limits for analytical chemical analyses. Both are statistical measures that become less precise and highly variable when near these limits, with sensory thresholds showing greater variability, often of two orders of magnitude . Both the physical setting and panelist’s unique situation can lead to such large variability, even from one day to the next. Therefore, presenting an ODTC50 as a range rather than a single value is recommended . If data are only generated suprathreshold, then a Weber-Fechner plot can be used to crudely estimate an ODTC50. Quality controls should include method blanks and spiked samples, and analyzing samples in triplicate allows for the calculation of standard deviations .For risk assessment of conventional air pollutants, frequency and duration are temporal aspects used in the calculation of exposure and indicate the appropriate hazard benchmark . For odor assessment, frequency and duration also are part of the sensory experience because they alter the perception of odor . An individual can become “used” to an odor and no longer able to detect it. Because odors are sensed within a few seconds or less, any averaging times applied to measurements may miss the peaks that trigger the complaints. Noting the frequency and duration of odor events can help inspectors and facility operators identify odor sources based on operation schedules and weather patterns.English speakers typically refer to a smell by its source . When forced to avoid using source terms, the descriptors “stinky,” “fragrant” and “musty” are used most often. Subsistence hunter-gatherers in the Malay Peninsula, by comparison, have a rich olfactory language, naming smells as easily as colors .

Dutch participants in a study had the same facial expressions as the hunter-gatherers when smelling odorants; however, the words selected to describe the odors were vastly different, the Dutch words tending be vaguer . Overall, smell tends to be poorly coded in languages as compared to other senses, yet this seems to be based in cultural norms rather than any neurological reason .As a starting point, the previous reviews of odor-measurement methods conducted by doctoral students were consulted . These reviews built upon a substantial review , an effort sponsored by groups in Australia and Spain. To update the prior work, a literature search was conducted post-2010 to gather the most recent methods and critiques. The search was conducted online and at the UCLA and CARB physical libraries. When relevant articles or books were found, the “cited by” function was used to discover even more up-to-date information. Reviews of the latest approaches to odor exposure measurement and risk assessment were sought. Finally, relevant websites and posted materials that are not typically available in scientific journals were searched. The starting point for risk assessment methodology was the foundational work by the National Research Council , now updated for the 21st century . The programs that grew out of the original work have issued their own guidance, which was also consulted. Such programs include pesticides , site remediation , exposure factors and California-specific work . International efforts, such as the review of the chemicals in commerce in the European Union , were also consulted. The 1,556-page tome edited by Dennis Paustenbach titled “Human and Ecological Risk Assessment: Theory and Practice” was an additional starting point. Risk assessment principles and terminology will be used to organize and structure the field of odor exposure assessment. The risk assessment framework will not be applied per se to environmental odor cases but, rather,hydroponic shelf system offer well-established concepts, conventions and terminology that can be applied to odors. As one example, the challenge of evaluating real-world chemical mixtures rather than a single chemical at a time applies to both fields. At the core of the evaluation will be the scientific merits of the various approaches to ensure any recommendations are evidence-based. The strengths and shortcomings of popular sensory and instrument methods will be reviewed.Sensory methods use the human nose as the detector. Because odor complaints arise from this same detector, it is the “gold standard.” As discussed in Section 2.3, our sense of smell can detect odor notes, although our vocabulary may struggle to supply the right words. The odor hedonic tone is more easily assigned. The odor intensity may be assigned categorical words or scores or placed on a scale. The scaling of intensity has led to the techniques discussed in this section. As with all else pertaining to odors, evaluating individual odorants is an oversimplification of their contribution to the total odor of a mixture.While chemical analyses can identify a subset of odorants and their concentrations in ambient air, chemical analyses often do not directly relate to human sensory experiences .

Odor samples usually contain multiple odorants that can have synergistic and/or antagonistic effects on each other, altering the overall perception. Odor is not simply additive, unlike concentration. Further, the human nose is usually more sensitive than analytical techniques. Therefore, the most accurate way to evaluate an odor it to judge its properties “as is.” This direct approach is called “odor profiling” and can be performed at the location where the odor is observed or by capturing a sample and transporting it to where a trained observer is located . A specific version of this approach, the Odor Profile Method , has been developed based largely on flavor profiling for the food and drinking water industries, specifically Method 2710 “Flavor Profile Analysis” by the APHA . OPM includes two parts: first, identifying one or more odor notes in the sample and, second, determining the odor intensity for each odor note. Duration of the odor at the site fenceline can also be included as a third factor . OPM can be part of a diagnostic investigation or an ongoing monitoring program. Rather than have panelists use their own natural, naïve language to describe the odor notes, a standardized vocabulary has been developed. Note, however, that only up to 2 odorants per mixture can be recognized by trained panelists . The standardized vocabulary has been tabulated for several industries and displayed graphically as wheels. Appendix A includes a collection of odor wheels, and Table 3.4 is a side-by-side presentation of their content. Over the years, such wheels have contributed a standardized way to classify, communicate, and identify odor notes, and sometimes the underlying odorants, in emissions . Odor wheels consist of three rings: an inner ring of general odor notes, a middle ring of more specific odor notes within each segment and an outer ring of potential odorants associated with each odor note. Their development has been described . The OPM intensity scale, described and critiqued in Section 2.3 , is used for each odor note. This scale is anchored on three sugar solutions tasted by mouth with cross-modal sensory translation to smell. The 7-point OPM intensity scale is: threshold , slight , weak , medium , medium strong , strong , and very strong . Before an individual becomes a panelist, their sense of smell is verified. The OPM uses the University of Pennsylvania Smell Identification Test , which is the best-known test of smell that uses micro-encapsulated odorants . As such, it is highly portable and often used in field studies. It has been well-standardized against age, gender and correlates well with the results of quantitative odor-detection tests . A passing score is 70% of the 40 questions. Further guidance on the selection of panelists is provided by ASTM Method E1440 . A minimum of four panelists is required by OPM. They may not have a cold, mustache, wear perfume, eat food or drink during the session. They are trained using the applicable odor wheel as well as the intensity scale .

Sociostructural factors that contribute to substance use-related risk factors, such as incarceration, community context, and social networks, are gaining increasing research focus. Social networks contribute to the context and uptake of drug use via modeling and the sharing of information on how to use substances, drug procurement, and how to sustain usage. Previous studies show that Black men report learning about cannabis from peers and family members and use cannabis to cope with racial discrimination, financial challenges, pain, and other stressors; but the perceptions and attitudes toward cannabis use and other “harder” drugs are not well described. Whether consumed for self-medicative or recreational purposes, many participants in prior studies endorse daily usage as being a non-problematic, normalized, and a beneficial part of their lives. Many providers utilize the Diagnostic and Statistical Manual of Mental Disorders fifth edition to determine problematic cannabis use, and assessments are supported by varied metrics and assessment tools. The DSM-5 determines a person may be diagnosed with cannabis use disorder if they meet two of their designated nine criteria that primarily evaluate ability to cease/decrease cannabis use, frequency and amount consumed, and impairment of ability to achieve daily tasks. A person’s social network can influence that person to use substances in ways that are problematic, but these networks have also been found to mediate problematic substance use and recidivism by supporting persistent recovery. By characterizing social networks and contexts to inform the development of effective interventions, researchers and policymakers can leverage social structures for behavioral change. To generate further understanding of this topic as well as support potential interventions,plant growing rack this paper explores how one’s social network contributes to cannabis perception and usage patterns among BMSM who have been involved with the criminal legal system.These data were collected via a supplement to a NIDA funded three-site study designed to develop agent-based models to examine the impact of various interventions on HIV in BMSM who experience incarceration.

The parent study involved three of the four largest jail systems in the country . Interviews from Chicago and Houston were included in this analysis because they were collected first using similar interviewing approaches. Los Angeles data collection was incomplete at the time of this paper and used more in depth approaches, building on what was learned from the Chicago and Houston interviews.We used these data to qualitatively examine perceptions of cannabis use in BMSM in Chicago, IL, and Houston, TX. Eligibility criteria were: age 18–34 years old; Black or African American identified; cisgender male; had sex with a man in the 12 months prior to their interview; incarcerated for at least 24 h; and misuse of opioids or use of illegal drugs other than opioids or marijuana in their lifetime. Researchers conducted 25 interviews that lasted approximately 60 min each using a semi-structured interview guide to explore participants’ experiences with substances, social networks, and incarceration. Participants received a $50 incentive. University of Chicago institutional review board provided oversight to all study procedures.Interviews were audio-recorded and transcribed, then coded and analyzed. Minority Stress Teory and the Social Ecological Teory were used to understand how contextual factors produce a social environment that encourages sustained or exacerbated substance misuse. By using a modified version of Braun and Clarke’s six-phase guide to a deductive-inductive thematic analysis, we systematically identify and offer insights into themes observed across the sample. Dedoose was used for data coding and management.This paper expands on how social networks and socioenvironments can influence drug use; importantly, we emphasize how a majority of participants reported that their peers and family both positively influenced their desire to consume cannabis as well as negatively influenced their desire to consume “heavier” drugs . Participant narratives of their cannabis usage further emphasized their drug-use behavior as being agentic; rather than passively absorbing influential factors, interviewees endorsed actively considering social contexts and potential consequences when engaging in cannabis related decision-making and risk-assessment.

While most participants endorsed a generally positive relationship with cannabis and did not find their usage disruptive to their daily lives, a small number felt their reliance on cannabis to manage physical and emotional needs was problematic. Social networks as an influencing force for cannabis use is of interest as these same networks could also be leveraged as an intervention for cannabis use disorder. For example, there are several network interventions that could be utilized to promote treatments or health care seeking for cannabis use disorder. A community opinion leader has been successfully utilized for other HIV prevention interventions, and is currently being tested to promote COVID-19 prevention behaviors. Criteria for diagnosing cannabis use disorder vary and effective and durable cannabis use disorder treatments are elusive and impact the likelihood that individuals’ will seek treatment or stop using, particularly when these individuals are embedded in environments where use is pervasive and accepted. Future research and practice examining the role of mediating factors for cannabis usage, such as anxiety and depression management, has the potential to improve treatments. Our findings add to a growing body of research elucidating how social relationships, including peer and family relationships, as well as negative life events influence cannabis usage patterns. Shifting statutes around the legality of cannabis and efforts to reduce recidivism highlight the importance of research on the interplay of incarceration, substance use and cessation, and recidivism. Our findings did not reveal significant differences in how the legality and illegality of cannabis in Chicago and Houston respectively influenced the cannabis use contexts and perceptions of our participants; this may be because all already had experienced incarceration and used substances as a study entry criterion. Analysis of how cannabis consumption behavior differs between legislative jurisdictions should be pursued in future studies, as the landscape of cannabis acceptability and availability is rapidly yet inconsistently evolving across cities.ceptions and consumption in the life course of BMSM who had been incarnated. As such, cannabis perceptions and experiences were not explored in-depth during interviews; however, our analysis is strengthened by not having used leading questions during participant interviews. Additionally, all participants resided in two large metropolitan areas and had a history of incarceration, which may affect generalizability to broader BMSM populations.

Last, we did not have other information to corroborate whether participants’ cannabis consumption was problematic, neutral, or effective as self-treatment.use as being influenced by social and familial networks, the desire to satisfy physical and emotional needs, and preconceptions of the risks and benefits involved in “heavy” drug and cannabis usage. Furthermore, we expand on previous findings with the novel observation that participants behaviors related to “heavy” drug and cannabis usage were the result of their own risk assessments versus passive reactions to impersonal socio-environmental factors. Given the expanded availability of cannabis, with expanding legalized recreational use, and marketing that is often directly speaks to the rationales expressed by our participants, further qualitative research will facilitate understanding of the nuanced of risk-assessments people make related to cannabis use. This is particularly important for individuals who were previously incarcerated for cannabis-related charges. Study findings strengthen and expand our understanding of drug consumption-related decision-making, revealing opportunities for future targeted behavioral interventions.Cannabis sativa has a long history of cultivation for a variety of uses including food, fibre, medicine,indoor vertical garden system and recreational drugs . Cannabis produces many different secondary compounds such as cannabinoids, flavonoids, stilbenoids, alkaloids, lignanamides, and phenolic amides . D9 -Tetrahydrocannabinolic acid , a product of the cannabinoid class, is the primary psychoactive agent. This compound is produced as an acid in the glandular trichomes of in- florescence bracts and undergoes decarboxylation with age or heating to D9 -tetrahydrocannabinol . Cannabis cultivars differ substantially in economic traits that range from marijuana, arguably the most widespread illicit drug, to hemp fibre derived from the stems of the plant. Marijuana consists of the dried female inflorescences in which the quantity of THC exceeds that of cannabidiol, and potency varies among cultivars by several orders of magnitude . Marijuana cultivars are known to have THC levels exceeding 2–24% of inflorescence dry weight whereas hemp cultivars produce substantially less THC but rather high levels of CBD . THCA and CBDA share the same biosynthetic pathway except for the last step in which THCA synthase and CBDA synthase produce THCA or CBDA, respectively . Recent evidence suggests that the genes encoding the two synthases are allelic . CBD and THC are enatiomers, but only THC elicits psychotropic effects, whereas CBD may mediate anti-psychotropic effects , a difference highlighting the stereo-selectivity of receptors in the human body that bind these compounds. Although classified as a drug without therapeutic value in the United States, ingestion of THC is widely regarded as having effects including pain relief and appetite stimulation, that may, among other things, increase the tolerance of cancer patients to chemotherapy . Dronabinol, a synthetic analogue of THC, is approved for use as an appetite stimulant in the United States as a Schedule III drug . Cesamet , another synthetic analogue, is used as an anti-emetic for patients undergoing cancer therapy. The natural product Sativex is approved for use in the UK and is derived from Cannabis cultivars containing both THC and CBD, and is used to treat pain symptoms associated with multiple sclerosis. Compounds from Cannabis sativa are of undeniable medical interest, and subtle differences in the chemical nature of these compounds can greatly influence their pharmacological properties. For these reasons, a better understanding of the secondary metabolic pathways that lead to the synthesis of bio-active natural products in Cannabis is needed .

Knowledge of genetics underlying cannabinoid biosynthesis is also needed to engineer drug-free and distinctive Cannabis varieties capable of supplying hemp fibre and oil seed. In this report, RNA from mature glands isolated from the bracts of female inflorescences was converted into cDNA and cloned to produce a cDNA library. DNA from over 2000 clones has been sequenced and characterized. Candidate genes for almost all of the enzymes required to convert primary metabolites into THCA have been identified. Expression levels of many of the candidate genes for the pathways were compared between isolated glands and intact inflorescence leaves.Seeds from the marijuana cultivar Skunk no. 1 were provided by HortaPharm BV and imported under a US Drug Enforcement Administration permit to a registered controlled substance research facility. Plants were grown under hydroponic conditions in a secure growth chamber yielding cannabinoid levels in mature plants as reported in Datwyler and Weiblen . Approximately 5 g of tissue was harvested from mature female inflorescences 8 weeks after the onset of flowering. Tissue was equally distributed into four 50 ml tubes containing 20 ml phosphate buffered saline as described by Sambrook et al. , but made with all potassium salts and mixed at maximum speed with a Vortex 2 Genie for four repetitions of 30 s mixing followed by 30 s rest on ice, for a total of 2 min of mixing. Material was sieved through four layers of 131 mm plastic mesh and the flow-through was split into two 50 ml tubes and spun in a centrifuge for 30 s at 500 rpm. Supernatants were decanted and pellets were resuspended in PBS. The suspensions were combined into one tube and pelleted as before. The resulting pellet was diluted into 100 ll of PBS. Five ll were used for cell counting with a haemocytometer, and the total suspension was estimated to contain 70 000 intact glands. Plant residue was incinerated by a DEA-registered reverse distributor .Full-length cDNAs from CAN24, 383, and 1069 contigs encoding putative polyketide synthases were used as templates for PCR reactions. Sequences identical to CAN24 and 1069 are available in GenBank with accession numbers AB164375 and AAL92879, respectively. The GenBank accession for CAN383 is GQ222379. The PCR reactions were designed to add 5# NcoI and 3# BamHI restriction enzyme sites to the ends of each sequence. After digestion with NcoI and BamHI, the inserts were cloned into the corresponding sites of pHIS8 , which adds eight histidine residues to the N-terminus of the encodedprotein. Clones corresponding to each PKS-related gene were sequenced. The Lasergene MegAlign program using the Clustal W algorithm was used to generate the alignment of PKS genes.

Cannabis farmers and the cannabis industry in general have to navigate the same pitfalls that we see [elsewhere] in the California regulatory landscape. But when you add in [the cannabis industry’s] lack of access to banking, and [its] inability to transfer product across state lines — it makes it even more difficult for folks involved in this industry.An overarching issue is the tendency of localities to move toward indoor cultivation. We only have about 17 localities in the state — counties or cities — that allow sun grown cultivation [cultivation without supplemental light]. We’re not doing enough to educate localities and regulators about the energy impacts of high-intensity lighting, or [the drawbacks] of setting up systems where the only way you can cultivate cannabis commercially is through very energy-intensive methods — which go very much against California’s goals [for reducing] carbon emissions. I think the California Department of Food and Agriculture could talk more about sustainable cultivation — about implementing [incentive programs similar to those developed for] other industries — so that, from the get go, we’re establishing sustainable systems, rather than going back 10 or 15 years later to do a greening of the cannabis industry.If I had to rank the type of cultivator that licensed cultivators are okay seeing law enforcement go after, number one would be people that are doing environmental degradation on public land. Folks in the forest or the national park who are harming the environment — I would say that almost no one would ever disagree [with enforcement against them]. The attitude changes when we talk about people on private land. Because even though nobody wants folks to be diverting from streams,mobile vertical grow racks there is a sense that “It’s their land,” and maybe they’re trying to do better.

That’s part of the culture up here [in the Emerald Triangle]. A lot of folks came up here to buy big pieces of land [partly because] they wanted privacy, and to be themselves on their land. No one wants to see environmental degradation, but when it comes to private land, they may say “Is there a way you can go in and try to help [noncompliant growers] before law enforcement comes in? Can you go and give them a warning?” In terms of people on private land who are not harming the environment, there is a strong belief that law enforcement should not be involved. Maybe these individuals want to become compliant but can’t afford to become compliant. So instead of law enforcement prioritizing them, we should instead offer support and say, “What can we do to support you in transitioning to the regulated market?” It’s not an all-or-nothing thing. There are definitely people cultivating without a license who are way more egregious than others.Over the next decade, I do. I think that there are prob ably two main components that have to happen before we can start thinking about cannabis like other industries — one, of course, being banking. We cannot be treated like any other industry when we cannot bank. Until banking is allowed and we can get small business loans, we will not be like any other industry. The second thing is being able to ship across state lines. You can’t ship wine to every state, but you can ship it to most states, and the ability of states like California and Oregon, or California and Washington, to enter into an agreement so cannabis can flow across the borders — that’s another way that we will be able to be treated like every other industry. Until then, you have to cap production [at the level] your state can consume. Do we say, “Florida, you can only grow so many oranges because [your oranges] all have to stay in Florida?” That doesn’t make sense. I’m hoping that both [banking and interstate shipments] will happen in the next 10 years. I think banking will happen this year — the SAFE Banking Act [a cannabis banking bill] was introduced in Congress this year with over 100 sponsors from both sides of the aisle.Public complaints about odors dominate the complaints received by air pollution agencies in the United States and Europe .

As populations urbanize and move closer to industry, the volume of complaints has risen. For example, in Europe between 13 and 20% of the population is affected by odor annoyance, while in highly urban areas the fraction has risen to 25% . The inherent variability in the perception of odors makes the regulation of odors a challenge. This means that odors cannot be regulated directly like other air pollutants by setting concentration limits. Instead, human perceptions of odor have to be considered within the regulations. Some neighborhoods have highly sensitive residents, while other communities have become accustomed to an odor and rely on its source for their jobs.Fugitive emissions from such industries can lead to odor complaints due to the extremely low sensory thresholds. Further, upsets and their associated larger releases are especially problematic at oil refineries , where it can take a long time to fix the problem. Historically, odors have been regulated under public nuisance law or when permitting a new or modified facility by conducting dispersion modeling within a theoretical odor impact assessment. The latter approach is outside the scope of this paper because it has been reviewed elsewhere . The use of dispersion modeling within a theoretical odor impact assessment has only achieved partial success as evidenced by the ongoing volume of odor complaints. Odor as regulated under public nuisance law for existing facilities, rather than proposed new or modified facilities, is the focus of this paper. Public nuisance law originated in British Common Law. Such law distinguishes between “private nuisance” and “public nuisance” . Almost always, nuisance odor complaints fall under the latter category rather than individual rights . To address nuisance odor complaints, regulators may set subjective or objective standards. Based on population-based health studies of toxic air pollutants, objective standards have been set for pollutants. Odors, however, are multidimensional and require difficult-to-enforce standards. Unsurprisingly, the setting of odor standards varies greatly throughout the world and depends heavily on the industrial sector, type of activity generating the odor and level of public outcry.

Applying public nuisance law to odors has been criticized : “A direct measure of annoyance is typically made by an inspector or authorized officer of the state,indoor farming systems and if there are no complaints there is by definition, no problem. The inherent subjectivity built into the approach is a problem, as is its susceptibility to political influence or community pressure. It lacks continuity of regulation for both the community and the industry concerned and does not offer a ‘target’ for the design and management of odor control systems.” As an alternative, odor intensity limits have been set in some jurisdictions. Such limits remain sensory-based, bringing with them the inherent variability in human responses to odors, and typically are set at a certain number of volumes of odorless air required to dilute one volume of sample air until the odor is no longer detected by 50% of panelists . Dynamic dilution olfactometry, which requires a dilution instrument and evaluation by a trained panel, is typically used to set this limit.In air quality guidelines for Europe, the World Health Organization recommended guideline concentration values for six odorants , which are presented along with their sensory thresholds in Table 2.3. Five of the odorants have their WHO guidelines near or below their odor detection thresholds. The exception, hydrogen sulfide, has a WHO guideline slightly above the odor recognition threshold. Developing a guideline value for each of the thousands of odorants would be daunting. Further, odorants are typically present as mixtures, which alters the overall perceived odor. Therefore, the WHO recommended that future work for sensory annoyance should probably focus on odors as perceived by individuals rather than on the component substances .Based on the information gathered from around the world, a targeted literature search and phone interviews of odor experts and air inspectors were conducted to provide current perspectives on odor investigations. Literature searches were conducted online and at the University of California, Los Angeles, and California Air Resources Board physical libraries. The focus was post-2010, and when relevant articles or books were found the “cited by” function was used to find even more up-to-date information. A California-specific literature search was conducted as well. Reviews of the latest approaches to odor investigation were sought. Finally, relevant websites and posted materials not typically available in scientific journals were searched. To collect the latest information on nuisance odor investigations from California’s air districts and international experts, phone interviews were conducted. The interviewees’ insights were gathered and any available guidance documents and relevant case studies were requested.California regulates odors under California Health and Safety Code Section 41700, which states “A person shall not discharge from any source whatsoever such quantities of air contaminants or other material which cause injury, detriment, nuisance, or annoyance to any considerable number of people.” California primarily regulates its air quality through 35 local air districts, although the California Air Resources Board addresses odor complaints from mobile sources. In addition, CalRecycle offers odor guidance to composting operations.

CARB has a long history of addressing nuisance odor complaints. In the 1970s, questionnaires were used to collect information from residents near pulp mills on exposure to the odor and on health and annoyance reactions . In general, the frequency with which odor was noticed and the frequency and intensity with which respondents were bothered by the odor were correlated with perceived odor intensity and frequency as measured by dynamic dilution olfactometry within each community. More recently, an agreement was formed between CARB and the local air districts regarding the handling of odor complaints . It stated that CARB handles mobile source odor complaints and, for stationary sources, the local air district is phoned as soon as possible, but at least within one business day. It then gives local air districts 15 working days after receipt of the CARB letter to provide a written complaint resolution or summary progress report. Finally, CARB agreed to subscribe to an over-the-phone verbal translation service that it would make available to the local air districts. Several of the local air districts were contacted to gather information on how they currently investigate nuisance odor complaints. The information gathered is summarized next. One observation during the interviews was that CARB trains the air district staff on smokestack opacity measurements using their own eyes . Training regarding environmental odor evaluation could be developed as well. In 2008, SCAQMD Rule 410 took effect to address odors from trash transfer stations and material recovery facilities. Sufficiently large facilities were required to have an approved odor management plan with extra requirements for even larger facilities. In 2009, a panel of 10 air inspectors used odor profiling to evaluate a trash transfer station . Based on the observed odors, specific odorants were suggested and then confirmed analytically. In 2017, SCAQMD adopted Rule 415 to address odors from rendering facilities . The rule requires building enclosures with ventilation to odor control systems for odorous operations and best management practices, such as covers for trucks and trailers and time limits for moving materials during the receiving and rendering process. Also in 2017, Rule 1430 was adopted to reduce odors from grinding operations at metal forging facilities . Four confirmed odor complaints over six months trigger additional odor management controls, such as enclosures.Air inspectors use their own noses to confirm the complaint, which does not necessarily need to match the complainant’s description of the odor. Each person’s ability to describe odors varies too greatly to require consistent descriptions. Rather, sensing that “something” is there is sufficient. The next step is to identify the source and move upwind to verify that the odor is not coming from elsewhere. Air inspectors are assigned to specific areas and know the sources within their area well. They are trained on how to respond by pairing junior staff with more senior mentors. For more complex odors, monitoring and meteorological data are used. Hydrogen sulfide monitors have been used for direct measurements and SUMMA evacuated canisters and Tedlar™ bags have been used to collected air samples. Sometimes a SUMMA canister is given to a complainant to collect a sample over time.

In Taxus, both taxadiene synthase and geranylgeranyl diphosphate synthase are localized to the plastids. This is important to note when introducing TXS in yeast, as localization sequence removal is key to improving performance, with truncation of the first 60aa yielding the largest taxadiene titers . Taxadiene-5α-hydroxylase then converts taxadiene to taxadiene-5α-ol , this is the primary modification of terpene scaffold. T5αol is then acetylated at the 5C position replacing the hydroxide. Taxadien-5α-acetate then undergoes numerous oxidations, an epoxidation, dehydration and benzylation to produce 10-deacetylbaccatin III. The final bioconversion of 10-deacetylbaccatin III to Taxol requires five additional enzymes: phenylalanine aminomutase, β-phenylalanoyl-CoA ligase, [baccatin III 3-amino, 13-phenylpropanyltransferase], taxane-2α-hydroxylase, and a N-benzoyl transferase . Currently, many of the enzymes required for Taxol biosynthesis have yet to be experimentally validated, though multiple research teams are working to characterize the pathway . The daunting task of engineering Taxol production into yeast is made even more challenging due to the spectrum of byproducts generated in the first two steps of the pathway, which drastically diminishes metabolic flux to downstream reactions. The taxadiene to T5αol conversion by T5αH is especially problematic in yeast, producing more than ten dead-end alternative products, with T5αol as a minor product. Prior to this work, the structure of most of these molecules were yet to be experimentally verified which complicates optimization of T5αOH activity and T5αol production. Tobacco, specifically Nicotiana benthamiana,indoor grow trays can serve as an excellent platform for investigating the biosynthetic characteristics of plant derived enzymes with Agrobacteriumbased transient expression.

Efforts to engineer plant natural product biosynthetic pathways in yeast can be greatly enhanced when coupled with these tobacco-based investigations. Especially for high-throughput gene discovery, as plant derived enzymes frequently require modification/truncation for proper functionality in yeast while remaining active in tobacco without modification. We sought to bolster our investigations into T5αH activity with parallel experiments in yeast and tobacco, with the goal of deciphering the T5aH product spectrum and confirming T5aol biosynthesis. Characterizing the array of oxidized products produced by the T5αH reaction with taxadiene is made more perplexing when compared to the GCMS analysis of Taxus cells grown in tissue culture. We found that the spectrum of oxidations generated by T5αH is a phenomenon only observed in our transient production systems , with these additional byproducts absent in Taxus tissue . This could suggest a secondary level of regulation in Taxus that restricts the “unproductive” oxidation of taxadiene at the 5C position, potentially via allosteric regulation of T5αH, the presence of peripheral proteins that modify reaction kinetics, or substrate shuttling via metabolon formation. Interestingly, while the product profile is fairly consistent in both the yeast and tobacco transient systems, the relative and absolute abundance of each molecule are quite diverse. Taxane production in yeast is carried out in a modified line engineered to overproduce GGPP , the primary substrate of the taxadiene diterpene scaffold. TXS was further modified to improve taxadiene production with the removal of the 60aa localization sequence, an N-term maltose binding protein fusion, and a C-Term ERG20 fusion . Two copies of the augmented TXS were integrated into the yeast genome with a galactose inducible promoter to allow for pathway activation after reaching desired cell culture density. After confirmation of taxadiene production, T5αH and a CPR from Taxus were integrated into the 2x TXS strain with a galactose inducible promoter for T5αol production. This strain was then used for metabolite extraction and GCMS analysis for comparison to the T5αH product profile generated in the tobacco system, as well as molecular structure confirmation.

More than eleven oxidized products, both mono-oxidized taxadiene and di-oxidized taxadiene , are produced in both the yeast and tobacco systems . This demonstrates that not only does the T5αH reaction result in varying oxidations of taxadiene, but also has the capacity for sequential oxidations of taxadiene, further complicating optimization of T5αol production specifically. Unfortunately, the ablation of non-specific T5αH activity will be crucial for the successful integration of the Taxol biosynthetic pathway in yeast. We have currently identified the structure of eight MOTDs and DOTDs produced by T5αH via product purification and NMR analysis. Four of these are MOTDs, one being T5αol and the other three forming epoxides from the hydroxide added at the 5C position . The four DOTDs identified in this work have a myriad of secondary oxidations, with one undergoing a rearrangement of the primary taxadiene scaffold . These data, in conjunction with findings that these alternative products are not found in Taxus cells, suggest that the T5αH reaction with taxadiene is not specific in these transient hosts, with the capacity to form numerous transition states during the reaction. This could potentially be attributed to a sub optimal redox coupling between T5αH and the CPR257 . Even though both enzymes were mined from Taxus, the interaction between them may be altered when outside their native context or orientation. This also strengthens the argument that additional, unknown/unidentified structural proteins or redox intermediaries are present in Taxus that modulate the reaction. One difference observed when comparing the GCMS data from tobacco and yeast is the relative abundance ofOCT compared to T5αol . In yeast, the OCT/T5αol ratio is ~2/1, while in tobacco OCT and T5αol levels are relatively equal . Overall, the differences observed are most likely attributed to altered enzyme behavior in yeast and tobacco, as interactions with endogenous proteins and substrates would be quite varied in the plant and fungal host. Comparisons between the absolute abundance and production titers for these products in both systems are not currently viable, as there was no standardization done to normalize between samples. Another important aspect of P450 activity that needs to be explored in future experiments is the balancing of CPR and P450 levels. Over expression of both the CPR and P450 can lead to a suboptimal redox coupling if the stoichiometry of CPR and P450 are not properly balanced. Experiments that alter the level of CPR expression with constitutive promoters of varying strength could inform decisions for optimizing the concentration of redox partners, which in turn should improve T5αol production in yeast.

A major challenge when engineering functional P450s from plants into yeast is maintaining robust expression and proper ER localization. P450s, along with their general redox partners CPR and Cytochrome b5 reductase, require ER anchoring for proper assembly and functionality of the electron transport chain coupled to the oxidative reaction carried out by the P450. Confocal microscopy is a great tool for the examination of enzyme expression and localization dynamics when coupled with fluorescent reporter protein fusions* . To better understand how enzymes from plants behave in yeast, we generated a suite of fusion proteins consisting of an enzyme of interest and a fluorescent protein for visualization of expression/localization dynamics . In addition to our investigation of specific enzyme behavior in yeast, we sought to better understand how the expression vector used in each experiment influences protein production across a population. A common method for enzyme characterization in yeast utilizes high-copy plasmids with an auxotrophic selection marker for expression*. Unlike most antibiotic-based selection methods, kanamycin selection in E. coli for example, auxotrophic selection has a degree of inefficiency. It is possible for “cheaters” not carrying the plasmid containing the selectable marker to persist by scavenging the deficient amino acid from dead cells in culture. When comparing the expression patterns of an integrated fluorescent protein and various fusion proteins carried on an auxotrophic plasmid we observed a drastic effect on expression across the population . Using either of the plasmid-based vectors for fusion protein expression resulted in a mixed population when visualized, specifically when analyzing the binary pattern of expression vs. no expression in each cell . While other factors likely contribute to this phenomenon,vertical grow racks for sale there is a consistent pattern observed when comparing the expression of stably integrated and plasmid-based expression vectors. Even though genetic integration of an expression vector yields homogenous expression in a population, and thus consistency in production titers and peripheral observations, plasmid-based expression has many utilities when engineering biosynthetic pathways in yeast. While not advised for strains engineered for large-scale production, plasmid-based expression is excellent for initial experiments aimed to characterize unknown biosynthetic enzymes. This is due to the high throughput manner in which enzymes can be tested and optimized prior to genetic integration, providing a primary system for investigating novel biosynthetic pathways in yeast while building a final production strain. A key observation can be made when comparing the localization pattern of a full-length dioxygenase with that of the same enzyme with the localization sequence truncated. Expression of the full length sequence in yeast results in a unfunctional enzyme, which is corroborated by the abnormal localization pattern. Truncation of the native localization sequence restores biosynthetic functionality as well as the proper cytosolic localization in yeast . These data demonstrate the utility of confocal microscopy for the visualization of enzyme behavior, which can inform the interpretation of production titers when rebuilding biosynthetic pathways in yeast. Another aspect of enzyme expression and localization we wanted to explore was stability over time. Taxadiene-5α-acetyltransferase , a soluble and cytosolic enzyme from the Taxol pathway that acetylates T5αol, was fused to eGFP and visualized over time post-induction . At 8hr post-induction TAT:eGFP is seen properly localized to the cytosol with minor puncta forming in a few cells. Though, when visualized at 24hr PI the majority has been shunted into numerous puncta in each cell, with limited fluorescence remaining in the cytosol. Complete amalgamation of TAT:eGFP into these puncta can be seen at 48hr PI. This could be an explanation for the lack of biosynthetic activity for TAT when introduced into the T5αH production strain, though minimal levels of T5αol could also restrict activity. While the mechanism responsible for this shift from dispersed cytosolic localization to compartmentalized bodies has not been identified, it is hypothesized that the enzyme is being trafficked into proteosomes or peroxisomes for turnover.

Especially when compared to a peroxisome localized fusion protein, which shows a similar pattern seen at 48hr PI . Observing this localization pattern over time has highlighted additional aspects for optimization when engineering biosynthetic pathways in yeast, persistence of localization and stability/half-life. Ensuring that the enzymes required for target molecule biosynthesis have robust expression, along with persistence in both localization and half-life, is crucial when optimizing production strains for large scale synthesis schemes. An additional characteristic of enzyme behavior in yeast was observed when comparing localization dynamics over time of an ER and a cytosolically localized enzyme. Ketoreductase 11 and 23 involved in saponin biosynthesis were selected for visual comparison. KR11 was tagged at the C-term with eYFP and KR23 was tagged at the C-term with CFP to analysis localization simultaneously. KR11:eYFP and KR23:CFP were cloned into the same plasmid-based expression vector with Gal1 and Gal10 driving their expression. When visualizing the localization of both KR11:eYFP and KR23:CFP at 8hr PI, KR11:eYFP is seen properly localized to the cytosol while KR23:CFP is properly localized to the ER membrane . After confirming both enzymes were expressed and properly localized at 8hr PI, we visualized the same culture at 24hr PI. As previously observed with other cytosolically localized enzymes, KR11:eYFP had been shunted into small bodies with very little remaining in the cytosol. KR23:CFP on the other hand retains proper ER localization in most cells, while showing limited loss in overall abundance . This phenomenon demonstrates an incongruity in behavior between ER-anchored and cytosolically localized enzymes from plants when expressed in yeast. The ER localized enzymes are largely occluded from accumulation to the “unknown bodies” that capture the cytosolic enzyme. While the mechanism of action responsible for these observations has yet to be characterized, these findings highlight aspects of enzyme behavior that should be explored when engineering biosynthetic pathways from plants into yeast. Unfortunately, the P450s required for Taxol biosynthesis fail to properly localize to the ER membrane when expressed in yeast. This issue presents a major challenge for engineering a functional Taxol biosynthesis pathway due to the required redox coupling with ER bound enzymes. One option for reconstituting proper ER localization is to exchange the native ER anchor region of the P450 with an anchor sequence known to properly bind the ER membrane in yeast. This chimeric protein would hopefully have restored ER localization while retaining the native biosynthetic characteristics.

These high value products split into two major classes: high value small molecules and proteins. The anti-malarial drug Artemisinin is a high value small molecule naturally produced in non-model plants. Fast-growing tobacco plants have recently been engineered to produce the drug, potentially expanding production capacity. Plants have been the primary production platform for high value small molecules for millennia – medicines, spices, and drugs have mostly been sourced from plant hosts. In modern times, the large-scale production of cannabis and opium poppies attest to the scalability and cost-efficacy of in planta small molecule production. Despite advances in microbial engineering and synthetic chemistry, plants remain the production platforms of choice for these high value small molecules, demonstrating the low cost and high scale that can be achieved with plant systems. With an appropriate ideotype compromising between fermentable sugars and high-value co-products, engineered lignocellulosic biomass crops may be economically viable. Rather than a single ideotype for all biomass crops, different crops may be more amenable hosts to particular applications. Complex metabolic pathways to produce high value small molecules have been successfully implemented in model plant species, and some biomass crops seem particularly amenable to metabolic engineering for high value small molecule production as well as overall modifications to the total carbon allocation . Ultimately, engineered feedstock crops that produce co-products may help offset costs associated with a future plant based bio-economy that will have to compete with petrochemicals.Dedicated crops have been used in first-generation food-to-ethanol production for over 100 years,cannabis vertical farming and in the United States annual production has increased 10-fold since 199022.

Ethanol accounts for over 90% of all bio-fuel produced in the United States, nearly all of which is derived from dedicated fields of corn, consuming 38% of corn production. The production of bio-fuel products from food crops causes competition between food and fuel, raising the price of staple foodstuffs. Lignocellulosic ‘second generation’ bio-fuels substantially reduce this problem by either growing on marginal land where food crops are not viable, or by production from agricultural residues rather than diverting a food crop into the bio-fuel pathway. Bio-fuels have sometimes been presented as an environmentally friendly and low-carbon alternative to fossil fuels, but current implementations have failed to deliver substantial GHG emission reductions. Bio-fuels grown from established agricultural fields generally achieve GHG emission reductions of 20–80% compared to fossil fuels95. However, land use change associated with the conversion of natural land to bio-fuel production leads to a ‘carbon debt’ that takes decades to centuries to pay back, negating any GHG savings96. Furthermore, conversion of natural land to bio-fuel production is a major driver of rainforest loss97. Growing bio-fuel feedstock on marginal lands and producing bio-fuel as one of multiple products are the two main strategies to reduce this trade-off. Here we consider re-designing bio-fuel feedstock crops to reduce cell wall recalcitrance, increase biomass per acre, and generate additional products not only add value but also improve resource use efficiency. Modern biotechnology has expanded the possibilities of crop ideotypes by allowing for plant phenotypes not attainable through classical breeding. Petrochemical fuels have been instrumental for global industrialization, and their use remains indispensable at present. However, climate considerations as well as the practical limitations inherent in using a finite resource call for the development of alternative sources of liquid fuel and materials. Plant biomass is the most viable means of production sufficiently scalable to take the place of petrochemicals in the economy of the future, and ideotype breeding serves as a useful paradigm for the design and improvement of biomass feedstock crops. No matter the goal of a plant engineering project, gene expression and the regulation of its expression is crucial for synthetic biology applications.

At its core, the regulation of when, where, and how strong a gene is expressed underpins the phenomenon of multicellular life. Intricate networks of transcriptional elements harness complex arrangements of “logic gates” to conduct the development of a multi-system organism from a single embryonic cell. The development of synthetic regulatory systems that provide control of when a gene is expressed , where it is expressed , and the level of expression without disrupting endogenous processes would allow for the engineering of complex, multilayered, synthetic gene circuits in future endeavors. In the work presented here, we sought to develop novel tools for the modulation of transgene expression in plants by developing synthetic orthogonal regulatory elements. These elements allow for the refinement of the engineering scheme by providing the means to tune the desired synthetic genetic circuit with a higher level of precision. We also developed a series of synthetic genetic circuits building upon our preliminary work with synthetic transcriptional regulators for deployment in sorghum. Agricultural biotechnology strategies often require the precise regulation of multiple genes to effectively modify complex plant traits. However, most efforts are hindered by a lack of characterized tools that allow for reliable and targeted expression of transgenes. We have successfully engineered a library of synthetic transcriptional regulators that modulate expression strength in planta. By leveraging orthogonal regulatory systems from Saccharomyces spp., we have developed a strategy for the design of synthetic activators, synthetic repressors, and synthetic promoters and have validated their utility in Nicotiana benthamiana and Arabidopsis thaliana. This characterization of contributing genetic elements that dictate gene expression represents a foundation for the rational design of refined synthetic regulators. Our findings demonstrate that these tools provide variation in transcriptional output while enabling the concerted expression of multiple genes in a tissue-specific and environmentally responsive manner, providing a basis for generating complex genetic circuits that process endogenous and environmental stimuli. Plants offer a unique platform to address many imminent challenges that face society, as future engineering efforts hold promise in promoting sustainable agriculture, renewable energy, and green technologies. However, the tools to effectively modify and engineer plants are still in their infancy.

One major hurdle has been the development of genetic parts that enable precise control of transgene expression in plants. Many genetic and metabolic engineering strategies require robust and accurate control of multiple genes to optimize synthetic pathways, regulate flux, and introduce new traits. The ability to modulate gene expression provides a direct approach to address these tasks. However, the majority of plant engineering efforts are limited to a small number of characterized constitutive promoters, which may result in unintended pleiotropic effects or toxicity issues and are limited in their range of expression strength. This work was inspired by previous studies that have successfully engineered orthogonal gene expression tools employing a reductionist and modular approach to parts design. Our unique strategy builds upon this approach by blending elements from both yeast and plants and could theoretically be applied to any TF type. Additionally, many of these previous systems were constructed using repetitive cis-element DNA sequences in the promoter design, while an important aspect of our approach is maximizing sequence diversity while tuning promoter strength. Specifically, we sought to build an expansive and diverse library of synthetic transcriptional regulators for plant engineering with components from various yeast transcription factor systems in conjunction with plant-specific regulatory DNA sequences. With this approach we developed a method that can be expanded beyond the well-characterized Gal4- based synthetic systems that have been used in the past . In general, parts design was approached in three ways: altering TF DNA-binding dynamics at the regulatory promoter by introducing DNA cis-element variation with randomly concatenated cis-elements,cannabis drying rack modulating RNA polymerase II recruitment by testing various plant minimal promoters, and directly modifying TFs from disparate families through truncation and fusion of activation or repression domains to generate new synthetic TFs, or trans-elements . A major finding of our work is that plant minimal promoter sequences can be leveraged for the design of chimeric promoters. By utilizing cis-elements from yeast in combination with a plant minimal promoter, we can generate functional synthetic promoters that interact with both the orthologous yeast TF as well as the endogenous transcriptional machinery of the plant. The minimal promoter, also known as a core promoter, lies directly upstream of the transcription start site and is where the transcription pre-initiation complex including RNA-polymerase II binds. Thus, the minimal promoter region facilitates the assembly and stabilization of the pre-initiation complex leading to variable levels of basal and activated transcription determined by this interaction. The binding of additional TFs, specifically orthogonal trans-elements in this work, at the regulatory promoter upstream of the minimal promoter can stimulate or repress this basal level of transcription providing multiple layers of regulation131. Using this strategy, we characterized a diverse set of synthetic promoters and synthetic trans-elements composed of discrete genetic parts from various TF families. Promoters were constructed with a collection of TF-binding cis-elements appended upstream of a plant minimal promoter.We tuned promoter strength by modifying basal transcription through minimal promoter variation and modulating the binding dynamics of the trans-element with concatenated cis-element variation. The initial library of synthetic promoters was developed for the yeast TF, Gal4, by appending random combinations of five well-characterized Gal4 upstream activation sequences , to varied plant minimal promoters. Through our approach, we explored how these CCEs and minimal promoters contribute to the overall strength of a synthetic promoter.

We then expanded upon this design strategy to TFs from disparate protein families and assembled a full suite of trans-elements and synthetic promoters for each. By expanding our library to contain both cis- and trans-element variation, we generated and tested more than 500 unique promoter/TF pairs resulting in a wide range of transcriptional output potential. Our library of TF binding cis-elements, plant minimal promoters, and TF fusion proteins demonstrates a novel method for the design, construction, and characterization of new tools for the controlled modulation of gene expression for various plant synthetic biology applications. As a proof of concept for our promoter design strategy, we utilized the well-characterized TF Gal4 fused to the VP16 activation domain as the activating trans-element. The heterologous nature of yeast TFs like Gal4 provides an opportunity to leverage a purely orthogonal system in plants, decoupling the transcriptional regulation of transgenes from those endogenous to the genome. To test the abundance of parts generated in this study, we used a high throughput transient expression assay in Nicotiana benthamiana. This system allows for the rapid screening of parts using a combinatorial approach, with each element cloned into individual binary vectors. A diversity of known Gal4 cis-elements were manually curated from native promoters in the yeast Gal regulon, each with a distinct nucleotide sequence and assumed to exhibit a diversity of dissociation constants with Gal4. We hypothesized that these deviations would lead to variation in the transcriptional output of each synthetic promoter, depending on the combination and position of cis-elements used in the CCE design. Minimal promoters from a collection of plant promoter sequences were appended to the CCEs generated with these Gal4 cis-elements to produce complete synthetic promoters . This strategy also presents an opportunity to investigate the effect the minimal promoter has on gene expression outside the context of its endogenous sequence. Additionally, introducing nucleotide diversity to our promoter constructs may limit the potential for transcriptional silencing often observed when identical sequences are used multiple times in gene stacking efforts. We screened these randomized combinations to measure the strength of the promoter in the presence of our synthetic trans element and the basal expression of our promoters in the absence of the TF. A measurement of Green Fluorescent Protein fluorescence was used as the proxy for transcriptional output, while the constitutive expression of a Red Fluorescent Protein was used as a normalization metric, with the ratio of GFP over RFP providing normalized values for the output of each construct. As expected, we observed a distribution of expression strengths while avoiding the usage of identical sequences, demonstrating a strategy for tuning gene expression . In many cases, it is not necessary to constitutively express all transgenes, and thus state-specific regulation of gene expression provides an additional dimension of control over a genetic circuit. For example, the constitutive over expression of a given protein may act as a sink on cellular resources resulting in overall detrimental effects. Similarly, various agricultural traits often result in fitness costs, and thus targeted expression of these genes may curtail unintended consequences. To further evaluate the efficacy of our parts in planta, we generated stable lines in A. thaliana.

As a land-grant institution, UC receives federal support; were UC to engage in work that directly supports or enhances marijuana production or profitability, it would be inviolation of federal law and risk losing federal support. As a result, UC research on California cannabis production has been limited and focused on the geography of production and its environmental impacts . These studies have documented the negative effects of production on waterways, natural habitats and wildlife. While such effects are not unique to cannabis agriculture perse, they do present a significant threat to environmental quality and sensitive species in the watersheds where cannabis is grown . Science-based best management practices to mitigate or avoid impacts have not been developed for cannabis. Because information on cannabis production practices is so limited, it is currently not possible to identify key points of intervention to address the potential negative impacts of production. As a first step toward understanding cannabis production practices, we developed a statewide survey on cultivation techniques, pest and disease management, water use, labor and regulatory compliance. The objective was to provide a starting point from which UC scientists could build research and extension programs that promote best management practices — which are allowable as long as their intended purpose is not to improve yields, quality or profitability. Survey results also establish a baseline for documenting changes in cultivation practices over time as legal cannabis production evolves in California. To characterize key aspects of cannabis production in California, we developed an anonymous online survey using Qualtrics survey software . A web-based survey that masked participants’ identity was determined to be the most suitable approach given that in-person interviews were limited by legal restrictions on UC researchers visiting cannabis farms,vertical racking system and mail or telephone surveys were constrained by the lack of any readily available mailing address or telephone contact information for most cannabis growers, who are understandably discrete with this information.

Survey questions focused on operational features , pest and water management, labor, farm revenue and grower demographics. Two draft surveys were reviewed by a subset of cannabis growers to improve the relevance of the questions and terminology. A consistent critique was that the survey was too long and asked for too much detail, taking up to 2 hours to complete, and that such a large time commitment would significantly reduce the response. We therefore made the survey more concise by eliminating or rephrasing many detailed questions across various aspects of cannabis production. The final survey included 37 questions: 12 openended and 25 structured . Structured questions presented either a list of answer choices or a text box to fill in with a number. Each list of answer choices included an “Other” option with a box for growers to enter text. Open-ended questions had a text entry box with no character limit. Condensing the survey to capture more respondents resulted in less detailed data, but the overall nature of the survey remained the same — a survey to broadly characterize multiple aspects of cannabis production in California. Data from the survey has supported and contextualized research by other scientists on specific aspects of cannabis production, such as water use , permitting , law enforcement , testing requirements , crop prices and perceptions of cannabis cultivation in the broader community . Recruitment of survey participants leveraged networks of California cannabis growers who had organized themselves for various economic and political purposes . These were a combination of county, regional and large statewide organizations, with many growers affiliating with multiple groups. We identified the organizations through online searches and social media and sent recruitment emails to their membership list-serves. The emails contained an explanation of the survey goals, a link to the survey website and a message from the grower organization that endorsed the survey and encouraged members to participate. The emails were sent in July 2018 to approximately 17,500 email addresses, although not all members of these organizations necessarily cultivated cannabis, and the organizations noted that their mailing lists somewhat overlapped the lists of other groups that we contacted.

For these reasons, the survey population was certainly less than 17,500 individual cannabis growers, but because we were not able to view mailing lists nor contact growers directly, and because there are no comprehensive surveys of the number of cannabis farms in California, we could not calculate a response rate or evaluate the representativeness of the sample. Respondents were given until Aug. 15, 2018, to complete the survey. All survey participants remained anonymous, and response data did not include any specific participant identifiers. In total, 101 surveys were either partially or fully completed. Responses to open-ended questions were coded before summary. Since incomplete surveys were included in this summary, the number of responses varied between questions. Each response was considered a unique grower and farm operation. As noted, survey response rate was difficult to quantify, and participants were self-selecting, which introduces bias. The survey data should be taken only as a starting point to guide more detailed evaluations of specific practices in the future, not as a basis for developing recommendations for production practices or policies.Survey respondents operated farms primarily in Humboldt , Mendocino and Nevada counties, but survey responses also came from Trinity , Santa Cruz , Sonoma , San Luis Obispo , Sacramento , Butte , Calaveras , Fresno , Los Angeles , San Diego , San Mateo and Siskiyou counties and Josephine County, Oregon . In line with California regulatory guidelines, small farms were defined as those of 10,000 sq ft or less, medium farms 10,001 to 22,000 sq ft and large farms 22,001 sq ft or more. Accordingly, 74% of farms were small, 16% were medium and 8% were large . For those growers who reported on their land use in 2013 , most farmed on land that was previously used entirely or in part for cannabis production . The other 22% indicated that the land was used in 2013 for agricultural crops, ranching, open space or “other” land uses.For this survey, we differentiated between outdoor , greenhouse and indoor farming . The most common ways to farm were all outdoors , combined outdoor and greenhouse and greenhouse only . This was followed by various combinations of greenhouse and indoor , greenhouse and other , outdoor and other , outdoor and indoor , all indoor and other .

When measured by total plants, farms with combined outdoor and greenhouse facilities were responsible for 41% of crop production, followed by outdoor and other , greenhouse only , outdoor only , greenhouse and other , outdoor and indoor , greenhouse and indoor and other . A majority of survey respondents grew their cannabis crop in raised beds , native soil and/or grow bags , followed by hydroponic systems and plastic pots . The average number of plants grown in outdoor farms was 166 , in greenhouses, 582 and indoors, 383 . When adjusted for total cropping area, this equates to 0.05 plant per sq ft for outdoor cultivation , 0.13 plant per sq ft for greenhouse cultivation and 0.64 plant per sq ft for indoor cultivation .Most growers reported groundwater as their primary water source for irrigation , with some growers reporting use of multiple water sources. Those using groundwater extracted 87% of annual volume between June and October. Of those storing water,indoor grow facility most stored exclusively well or spring water, though some stored municipal water or rainwater . Extraction to storage was greatest in summer but was relatively well distributed throughout the year. Many growers reported that adding storage was either cost prohibitive or limited by regulatory constraints. Half the respondents indicated that additional storage was not needed, 40% indicated that the high costs of building storage were limiting, and 5% reported there was insufficient water available and 5% that they were unable to obtain permits to store . Most growers reported using variable amounts of water across the growing season. Outdoor growers applied, on average, 5.5 gal per day per plant in August and 5.1 gal per day per plant in September. Greenhouse growers applied an average of 2.5 gal per day per plant in August and 2.8 gal per day per plant in September . When standardized by area, application rates were very similar between cultivation types . In our survey, growers reported using low maximum pumping rates : 53% indicated rates ranging 1 to 50 gal per minute, 7% did not know their pumping rate and the remaining 40%, who used groundwater or municipal water sources, indicated that this question did not apply to them. Growers reported 14 different arthropods, 13 diseases and nine vertebrates that had negative impacts on cannabis production . The most frequent arthropod pest was mites , followed by thrips , aphids and unknown larvae . The most common vertebrate pests were gophers, mice and rats , followed by deer and wild boars . Powdery mildew was by far the most commonly reported disease , followed by other fungal diseases such as molds and rots .

While these findings are in line with cannabis pests and diseases reported by others , survey data are self-reported data and grower identification of pests and diseases may not be entirely accurate. For instance, the complex of mites reported included russet mites, spider mites, broad mites and red mites. Growers were likely referring to hemp russet mite , two-spotted spider mite , broad mite and Carmine spider mite , respectively, but this remains unclear because there are many species of mite commonly referred to as russet mite, spider mite and red mite . This similarly applies to aphids, thrips, larvae, mildew, rots and molds. Accurate species identification of these pests and diseases will remain uncertain until they can be more systematically collected and identified by UC academics or other scientists. The most common approach to pest and disease control was to apply some type of solution or chemical to the crop , followed by augmentation of natural enemies and various cultural practices .Products specifically used for control of arthropod pests included azadirachtin , soap solution , pyrethrins and Bacillus thuringiensis . Many respondents indicated that certain products were effective against both pests and diseases, for instance microbial pesticides , oils and compost tea . Sulfur was the most commonly applied product specifically used for disease control. In addition, 29% of respondents claimed to use certified organic products for pest and disease management but did not name any product specifically. Finally, 2% of respondents reported that they did not spray for pests and diseases at all. Augmentation of natural enemies involved the introduction of predatory mites , lady beetles , predatory nematodes and other unnamed beneficial insects . Cultural practices included removal of infested plant material , insect trapping , intercropping , use of diatomaceous earth and selection of resistant cultivars .Our survey, although of limited sample size, is the first known survey of California cannabis growers and provided insights into common forms of cultivation, pest and disease management, water use and labor practices. Since completing this survey, we have discussed and/or presented the survey results with representatives from multiple cannabis grower organizations, and they confirmed that the data were generally in line with production trends. Evident in the survey results, however, was the need for more data on grower cultivation practices before best management practices or natural resource stewardship goals can be developed. All growers monitored crop health, and many reported using a preventative management strategy, but we have no information on treatment thresholds used or the efficacy of particular sprays on cannabis crops. Likewise, the details of species-level pest and disease identification, natural enemy augmentation and sanitation efforts remain unclear. Growers did not report using synthetic pesticides, which contrasts with findings from previous studies that documented a wide range of synthetic pesticide residues on cannabis . Product selection for cannabis is very limited due to a mixed regulatory environment that currently does not allow for the registration of any insecticide or fungicide for use specifically on cannabis , although growers are allowed to use products that are exempt from residue tolerance requirements, exempt from registration requirements or registered for a use that is broad enough to include cannabis .

Such responses to sexual harassment by labor unions have largely been shaped by the sexism rooted in the birth of the labor movement . It is the same male-centered philosophy that breeds the sexual harassment unions are intended to protect workers against. The first labor unions were over represented by male workers and as such, they discouraged women from reporting harassment by other union members, reinforcing labor unions as organizations perpetuating patriarchy . Despite the tumultuous past unions have had with sexual harassment, many are now taking a proactive approach to address the issue. UNITE HERE, in particular, has worked on addressing sexual harassment in the hospitality industry in cities across the country. Their Chicago Hands Off Pants On campaign, involved a member survey which found 58% of hotel workers and 77% of casino workers had been sexually harassed by a guest . The survey gave the union the data it needed to push for protections through both collective bargaining and eventually a city wide ordinance to protect both union and nonunion Chicago hotel workers . Moreover, in 2016, California members of the Service Employees International Union began their Ya Basta campaign and succeeded in winning statewide legislation in California requiring all janitors in the state to receive training on rape and sexual harassment prevention . Because of the growing relationship between labor unions and the cannabis industry, labor unions serve as a unique point of intervention for addressing sexual harassment in this particular workplace. Specifically,clone rack it useful to consider the social-ecological model proposed by Bronfenbrenner . His model had been referenced and adopted to fit several public interventions including violence prevention .

The individual, relationship, community and societal levels of the social ecological framework focus on personal factors, interpersonal relationships involving family, friends workplaces and neighborhoods, and broader structural factors such as health systems and polices, respectively . Each level of the model can be addressed to holistically grapple with the issue of violence, including sexual harassment in the workplace. Therefore, this particular study seeks to explore the community level as an opportunity for intervention, specifically through the growing relationship between the cannabis industry and labor unions.The study population was drawn from a source sample of cannabis employees represented by UFCW in Los Angeles County. Currently, UFCW Local 770 represents 20 dispensaries in Los Angeles County and approximately 400 workers to-date. In order to participate in the study, participants had to be at least 21 years of age, employed in a dispensary shop in Los Angeles County and had to be a current member of Local 770. As of August 2018, California is home to at least 358 retail stores with varying levels of permission including Temporary Approval or License to sell Medicinal or Adult-Use cannabis and cannabis products . Of the 358 legal stores located in the state, 188 are located in Los Angeles County , confirming Los Angeles area as an optimal location to investigate sexual harassment in the retail sector of the cannabis industry. This was a mixed methods study using a cross sectional design to analyze survey data from a convenience sample of dispensary workers represented by UFCW Local 770 in Los Angeles County. Surveys were used as part of this study to assess the demographics of the workforce, prevalence, frequency, types of sexual harassment occurring in the workplace, and workers’ knowledge on workplace policies regarding sexual harassment. In-depth interviews were conducted to better understand organizational risk factors of sexual harassment that are specific to cannabis dispensaries. Recruitment flyers with a description of the study were posted in the 20 Los Angeles dispensaries represented by Local 770.

The recruitment flyer included an online link and a quick response code to the survey for workers to easily access the survey through a mobile device. The recruitment flyer included an invitation to participate in an additional interview. The majority of recruitment occurred at shops as I explained the study to workers, and they volunteered to participate. Interview participants were recruited through snowball sampling. Each interview participant was provided a consent form, was explained the objectives of the study, and agreed to have their interview digitally recorded. Participants were assured that no identifiable information would be collected and any identifiable information that may have been mentioned throughout the interview was redacted from the transcript.Each interview took between 40 and 60 minutes and occurred at a location of the interviewee’s choosing . Each participant was compensated for their time with a 15-dollar gift card. In total, seven participants were interviewed as a part of this study. The interview protocol included providing participants with a copy of the interview questions so that they could follow along with the line of questions. In addition to interview questions, participants were provided with a formal definition of workplace violence and sexual harassment in order to establish a common understanding of the terms I would be using throughout the interview. Interviewees were first asked to explain their regular workday and their work responsibilities as well as their employment tenure in cannabis. They were then asked about sexual harassment in their workplace, if they had experienced, witnessed or heard about instances through word-of-mouth. They were also asked to explain the protocols of their workplace aimed at preventing harassment and protecting workers who seek redress. In an effort to be mindful of discussing the sensitive topic of sexual harassment, the questions focused more on factors leading up to harassment and the actions taken by workers, co-workers and their management as a result of such events. Finally, workers were also asked to describe interventions they would like to see implemented in their workplace to address harassment.

Throughout data collection, questions were added, removed, and re-written based on feedback from participants. Survey participants completed the survey on their mobile device or self-administered it in-person with a tablet. While visiting stores for recruitment, interested workers could take the survey on tablets provided by Local 770. Once the study was complete, all data collected, except for the city of employment variable, were made available to UFCW with additional information and recommendations on how they may better support members of their cannabis division. The final sample for the survey was 117 participants. All data were collected between October 2019 – February 2020.The survey instrument consisted of 37 questions designed to take no more than seven minutes to complete. The first section included questions on workplace city, duration of employment in the current workplace and total employment experience in the cannabis industry. The second section asked about sexual harassment policies in the workplace followed by questions regarding experiences with sexual harassment , perpetuators, and responses . The fourth section asked workers to select, from a list of examples, interventions they believed could be implanted to prevent or better handle instances of harassment in their workplace. The fifth and concluding section included basic demographic questions .In order to capture sexual harassment in the workplace, I adopted nine questions from Fitzgerald and colleague’s Sexual Experiences Questionnaire – Department of Defense Items . The SEQ-Dod was originally developed to measure sexual harassment of in the Armed Forces but has also frequently been adopted to capture harassment in the workplace . The original 24-item measures four dimensions of sexual harassment: sexist hostility, sexual hostility, unwanted sexual attention and sexual coercion. As defined by Fitzgerald , sexist hostility is defined as gender-based harassment, sexual hostility is also a form of gender-based harassment but involves behaviors that are more overtly sexual in nature, unwanted sexual attention is characterized by attention and behaviors that are unprompted and unreciprocated by the receiver, and lastly sexual coercion is more explicitly extortionist in nature and considered quid pro quo harassment. The tool has also since been shortened to capture the original four dimensions of sexual harassment in 16 questions . For this study,4×8 tray grow only the first three dimensions of sexual harassment were measured. The version of survey specifically adopted for this study was from the National Park Service’s Work Environment Survey as their study addressed shortcomings of the original tools including issues of recall bias and a lack of examples which constitute sexual harassment . All survey participants responded to 12 different questions of sexual harassment behaviors . Participants indicated how often they experienced the behavior in the last 12 months using a 6-point scale ranging from never , “once” , “once or less a month” , “2-3 times a month” , “once a week or more” , “to once or more times a day” . Because the 12 questions were adopted from the original instrument, a reliability analysis conducted to determine if the 12 items were interrelated produced a Cronbach’s alpha of 0.88, indicating good internal consistency. The Cronbach alphas for the three sub-scales of sexist hostility, sexual hostility, unwanted sexual attention were 0.88, 0.84, and 0.36, respectively.

Consistent with the scoring used in the WES report , responses to these questions were summed and averaged across items to determine a sexual harassment score per sub-scale. Sexual harassment scores ranged from one to five; the greater the sexual harassment score, the greater frequency of harassment experienced by a worker. Prevalence of specific dimensions or behaviors of harassment among respondents was calculated by the percentage of respondents who reported experiencing an example of harassment at least once. The greater the percentage, the more employees experienced either a specific dimension or behaviors of sexual harassment. Respondents who answered experiencing sexual harassment at least once in their workplace were asked to report who had sexually harassment them. Options included a fellow employee , subordinate, manager, customer and other or an unknown individual. The five possible options were coded as separate dichotomous variables and scored as one if selected and zero if left blank. Participants were also asked if they had ever experienced sexual harassment by someone of the same gender. Responses options were “yes” , “no” , and “unsure” . To gauge awareness of sexual harassment policies, a question was adopted from UFCW’s questionnaire on sexual harassment in the workplace. The survey provided by UFCW was originally designed for their grocery worker division and was intended to capture worker’s knowledge of workplace policies protecting them against sexual harassment as well as the impact of sexual harassment on employee work schedules. Respondents were asked if they were aware of their retail company’s policies regarding sexual harassment. They either responded “yes” or “no” . If they responded “yes,” they were further asked to select specific policies that applied to their workplace from a list of six examples . All examples were coded as dichotomous variables. If selected, the item was coded as a one, and if left blank, the item was coded as zero. A response score ranging from one to six was calculated by summing the responses to all six items. A larger score equates to workers having a greater awareness of policies in the workplace to protect them against sexual harassment. The Cronbach alpha for these six items was 0.83, indicating good internal consistency. As part the translational nature of the project, respondents were asked if they wanted more support in their workplace to protect workers against sexual harassment. This item as also adopted from UFCW’s questionnaire. If respondents selected “yes” , they were asked to indicate which actions, from a list of seven examples, they would like to see taken in their workplace to address the issue of sexual harassment . Respondents were asked to choose all options they felt were applicable to their workplace. All seven options were converted into a dichotomized variable and scored as one if the option was selected and zero if left unselected. An intervention score was created by the summing responses to seven options. This data will be especially beneficial to informing the union how they may better service their members.Covariates included in my analysis include the age of workers, their race/ethnicity, gender identity, sexual orientation and employment status. Age was treated as a continuous variable in analysis. Respondents provided their race and ethnicity by selecting from the six provided categories, all the options they felt best applied to their identity. A seventh option for “other” was provided if they wanted to write in their response. Responses for race/ethnicity were first categorized into seven levels: White, Black, Asian, Latina/o, Asian, Native American, Hawaiian or Pacific Islander and other.

The center’s ongoing research includes a multi faceted project to assess specific aspects of Northern California’s cannabis farms, including the number and size of non-compliant cultivation sites; the environmental effects of non-compliant sites ; and the challenges to regulatory compliance that cannabis cultivators encounter. According to a grant proposal associated with the research, the project is motivated by an urgent need to understand the environmental threats posed by non-compliant farms and the reasons that some farms successfully navigate state regulations while others fail. The researchers are combining high-resolution satellite images with local and state permitting data to identify permitted and non-permitted cultivation sites. In parallel, the researchers are combining permit specifications with water use models to estimate the effects on stream flows of non-permitted versus permit ted cultivation. Additionally, they are determining which factors associated with cannabis cultivation are most closely linked to compliance — whether parcels are large or small, old or new — and, through writ ten grower surveys and in-person interviews, they are seeking to understand what stands in the way of cultivator compliance. Ultimately, the work will yield a policy report outlining ways in which state and local governments can decrease the harm of non-compliant cannabis cultivation while increasing rates of compliance. The research is supported by a grant from the Campbell Foundation, provided through the Resource Legacy Fund. In another example of CRC research focused on cannabis and the environment, last year Butsic, Jennifer Carah and additional co-authors published the results of their work on “agricultural frontiers” . These are places where,plants rack due to increased profit potential for agricultural activity, land is newly cultivated — frequently resulting in environmental impacts such as forest fragmentation and threats to sensitive species.

Such transformations, the authors write, occur when economic circumstances are altered by some new mechanism — such as, in the case of cannabis, a new legal status. The researchers, documenting the emergence of such a frontier, studied cannabis cultivation sites in Humboldt and Mendocino counties from 2012 to 2016. Using satellite imagery to develop a database of cultivation sites, the research ers correlated site characteristics such as remoteness and erosion potential with the spread of agricultural frontiers. They report that, over the study period, cannabis cultivation sites in the study area nearly doubled in number, with total acreage under cultivation likewise nearly doubling, and that a significant portion of the new cultivation occurred in areas such as sensitive watersheds. They found, for example, that nearly 90% of the areas newly developed for cannabis cultivation had been covered in natural vegetation as late as 2006. The researchers argue that agricultural frontiers can develop “almost anywhere institutions fail to prevent” them — and note that, for 18 years after medicinal cannabis use became legal in California with the 1996 Compassionate Use Act, the state devoted no funds to regulating cannabis cultivation and production. In this issue of California Agriculture, Grantham and four co-authors from the North Coast Regional Water Quality Control Board present the results of their research into cannabis cultivators’ patterns of water use in several Northern California countries. For the research that resulted in “Watering the Emerald Triangle: Irrigation sources used by cannabis cultivators in Northern California” , Grantham and his colleagues analyzed reports submitted to the board by cannabis cultivators. The researchers determined how many cultivators sourced their water from wells, surface water diversions, spring diversions and other sources; how water sourcing behavior changed over the course of a year; and how water use patterns varied ac cording to whether growers operated within the state’s legal cannabis market.

The researchers determined that cannabis growers rely on well water to a greater degree than is generally supposed — and that their reliance on well water may increase as more growers join the legal market because of well water’s less restrictive permit ting requirements. In separate research, Michael Polson — a post doctoral researcher in UC Berkeley’s Department of Environmental Science, Policy, and Management — has investigated the environmental dimensions of cannabis from an anthropological perspective. In a paper published earlier this year, Polson shows how cannabis has been identified as an environmental problem that requires public intervention . On the basis of participant observation and more than 70 interviews with subjects across the cannabis spectrum — from park rangers to environmentalists to “criminalized people” — Polson demonstrates how cannabis production has been defined as pollution — “dovetail[ing] with [cannabis] prohibition’s history of marking people and substances as socially polluting.” Polson argues, as he highlights the legacy of cannabis prohibition in environmental debates, that policy making is at its most innovative when it includes a broad range of cultivators and when stigmas are explicitly addressed. Research into the environmental aspects of cannabis is also underway at UC Davis, where Mourad Ga briel is a research associate member in UCD’s School of Veterinary Medicine. In 2018, Gabriel and co-authors, including Robert Poppenga — a professor of molecular bio-sciences at the California Animal Health and Food Safety Lab at UC Davis — published the results of their research on the effects of rodenticides on owls in northwestern California forests . The researchers, working on privately owned timberland in Humboldt and Del Norte counties, investigated the prevalence of anticoagulant rodenticides in areas char acterized by illegal cannabis cultivation. Anticoagulant rodenticides, used by some cannabis cultivators to control pests, are known to affect non-target species in urban areas and recently have been shown to affect carnivores in California’s remote forest areas as well. Gabriel and his co authors undertook to deter mine whether the northern spotted owl, a threatened species, is exposed to anti coagulant rodenticides in the study area — and also to determine if barred owls, a common species, can be used as a surrogate to determine exposure levels in northern spotted owls.

The researchers analyzed liver samples from 84 barred owls and 10 northern spotted owls. Within the study area, 70% of northern spotted owls and 40% of barred owls tested positive for anticoagulant rodenticides. The researchers hypothesize that cannabis cultivation in the area is the main source point for the presence of dangerous rodenticides. They also determined that barred owls are a suitable surrogate for determining rodenticide levels in the threatened northern spotted owl. Gabriel, in his capacities as a UC researcher and as executive director of the Integral Ecology Research Center, a nonprofit organization based in Humboldt County, is currently carrying out reclamation projects at illegal cannabis cultivation sites in California and Oregon. In a project conducted this May in the Shasta-Trinity National Forest, a team representing 11 governmental and nongovernmental entities worked at 16 cultivation sites within eight large cultivation complexes, removing 6,000 pounds of trash, which included rodenticides and more than 5 miles of irrigation lines. Mourad estimates that removal of the irrigation lines restored more than 500,000 gallons of water — daily — into affected watersheds. Agencies including the California Department of Fish and Wildlife and the Law Enforcement and Investigations arm of the U.S. Forest Service have provided grant funding for 170 such projects, 112 of which have already been completed. In an entirely different vein, UC Davis–based cannabis research has been conducted since 2016 at the UC Agricultural Issues Center , a UC Agriculture and Natural Resources statewide program operating since 1985. The center’s broad mission is to provide research based information on the economic dimensions of emerging issues in agriculture. Cannabis, then, is right in the center’s wheelhouse. Dan Sumner, the center’s director,plant growing trays reports that AIC began pursuing cannabis-related work after the 2015 passage of a set of laws known collectively as the Medical Marijuana Regulation and Safety Act. This legislation laid the groundwork for state regulation of medicinal cannabis and ultimately of the recreational cannabis industry. The lead agency in regulating commercial cannabis licenses for distributors and retailers, among other business types, is the Bureau of Cannabis Control — for which, between 2016 and 2018, the AIC prepared a Standardized Regulatory Impact Analysis . In the process, the AIC advised the BCC on the economic dimensions of various regulatory scenarios — and the bureau used the center’s analysis to inform the final cannabis regulations that it issued on Jan. 16 of this year. According to Sumner, a principal insight that the AIC furnished to the BCC was that, since illegal cannabis continues to be attractive to retail buyers because it is cheaper than cannabis from regulated retailers, “much of the cannabis sold in California [after legalization] would remain in the illegal segment.” Moreover, regulations that generate benefits for consumers at lower costs will help sustain the legal marketplace. In this issue of California Agriculture, three AIC researchers — Pablo Valdes-Donoso, a postdoctoral scholar; Robin S. Goldstein, principal economic counselor; and Sumner — present their research on California’s rather stringent system for testing cannabis that enters the legal market . All cannabis sold legally in the state is tested for more than 100 contaminants. Of those contaminants, 66 are pesticides — and tolerance for 21 of those pesticides is set at zero. In many cases, allowable levels of cannabis contaminants are lower than those established for food sold in the state. The researchers, drawing on data provided by testing laboratories and manufacturers of testing equipment, estimated how much it costs to test a pound of cannabis under California’s regulatory regime, as well as the cost of collecting samples. They concluded that the need to destroy batches of cannabis that fail testing accounts for a large share of testing costs. The research ers argue that, though the availability of certifiably safe and legal cannabis products may prompt some customers to join the regulated market, other customers will remain in the cheaper illegal market.

They speculate that, over time, increased availability of data about cannabis testing and sales will allow for greater certainty about the effect of the testing regime on cannabis prices and demand for legal cannabis. Meanwhile, UC Davis is establishing a dedicated center for research into psychoactive cannabis and industrial hemp — the Cannabis Research Initiative. According to Cindy Kiel, executive associate vice chancellor for research administration at UC Davis, the initiative will draw on the comprehensive strengths of UC Davis faculty in areas ranging from agricultural and environmental impacts to legal, economic and policy outcomes to human and animal health. In particular, the initiative will benefit from UC Davis’s strong emphasis on agricultural issues such as soils, water, genomics and plant science and from faculty interest in two-way interactions such as those between cannabis and the environment. Funding is envisioned to flow from the UC Davis budget, from research funds established in Proposition 64 and from outside sources such as industry partners. The initiative will be headed by co-directors representing the agricultural and medical sides of cannabis research. In May, UC Davis faculty members including Chemistry Professor Mark Mascal, along with col leagues from the University of Reading in the United Kingdom, published an article demonstrating that a synthetic analogue of cannabidiol is as effective as CBD in controlling seizures in rats — and that it provides several benefits in comparison to CBD. The synthetic analogue is cheaper than herbal CBD, cannot be converted into psychoactive tetrahydro cannabinol and is not restricted by the Drug Enforcement Administration’s “scheduling” apparatus. Meanwhile, the UC Davis–affiliated Western Center for Agricultural Health and Safety is studying issues such as workplace safety for cannabis workers, who face risks that include unhealthy pesticide exposure. For students, UC Davis has offered cannabis courses including the graduate-level Cannabis sativa: The Plant and Its Impact on People — and, for undergraduates, Physiology of Cannabis.A brand-new entrant into UC cannabis research is the UC Nicotine and Cannabis Policy Center at UC Merced , established just last year to study tobacco- and cannabis-related issues in public health and public policy, especially in the San Joaquin Valley. The center, partnering with local public health departments and organizations such as the American Heart Association, aims to produce tobacco and cannabis research that places special emphasis on the San Joaquin Valley’s diverse population of teens and young adults and in forms policy decisions that affect the region. The center’s flagship research initiative is a long term, survey-based effort to understand issues surrounding cannabis, tobacco and e-cigarettes.

Particularly notable is FAAH’s ability to hydrolyse bioactive fatty amides, which do not bind to any of the known cannabinoid receptors: these include the satiety factor oleoylethanolamide and the anti inflammatory/ analgesic mediator palmitoylethanolamide. FAAHtightly controls brain concentrations of these compounds, but the functional significance of this regulation is unknown. FAAH is widely distributed in the rat brain, where it is expressed at high concentrations in cell bodies and dendrites of principal neurons. In the hippocampus, neocortex and cerebellum, FAAH-positive cell bodies are juxtaposed to axon terminals that contain CB1 receptors, indicating not only that FAAH participates in the inactivation of neurally generated anandamide, but also that this process occurs post synaptically. This idea can now be tested in FAAH-deficient mice or using selective FAAH inhibitors with long-lasting systemic actions.The pig brain contains two chromatographically distinct 2-AG-hydrolysing activities, one of which is probably due to the enzyme monoacylglycerol lipase . The rat brain isoform of this cytosolic serine hydrolase has been characterized both molecularly and morphologically. It has a broad distribution in the central nervous system , which partially overlaps with that of FAAH; however, whereas FAAH is predominantly found in postsynaptic structures, MGL might be mostly associated with nerve endings. In the hippocampal CA1 field, MGL-positive axon terminals surround cell bodies of pyramidal neurons containing FAAH. This localization could reflect a functional role of presynaptic MGL in terminating RETROGRADE SIGNALLING events mediated by 2-AG .CB1 is considered to be the most abundant G-protein coupled receptor in the mammalian brain, and its presence in the neocortex, hippocampus, basal ganglia, cerebellum and brainstem accounts for most of the behavioural actions of cannabinoid drugs. The four symptoms that are often used to define cannabinoid intoxication in the rodent __ hypothermia, rigid immobility,weed drying room analgesia and decreased motor activity __ are strikingly absent in mice in which the cb1 gene has been deleted by targeted recombination.

Aside from its unusually high concentrations in the brain, CB1 is a standard Gi/o-coupled receptor and can initiate signalling events typical of this class of transducing proteins. These include closure of Ca2+ channels, opening of K+ channels, inhibition of adenylyl cyclase activity and stimulation of kinases that phosphorylate tyrosine, serine and threonine residues in proteins. Each of these mechanisms seems to have distinct functions in translating CB1 -receptor occupation into biological responses. Cannabinoid agonists inhibit N- and P/Q-type voltage-activated Ca2+ channels. This effect, which has been suggested to result from a direct interaction of Gi/o-protein β−γ subunits with the channels, might underlie CB1 -mediated depression of transmitter release at GABA synapses in the CA1 field of the hippocampus and at glutamatergic synapses in the dorsal striatum. Importantly,however, endocannabinoid-mediated suppression of GABA release in hippocampal slices seems primarily to involve N-type Ca2+ channels. Cannabinoid regulation of voltage-gated K+ currents is also implicated in presynaptic inhibition at GABA and glutamate synapses. The latter include PARALLEL FIBRE–Purkinje cell synapses in the cerebellum, as well as synapses in the nucleus accumbens and lateral amygdala. The sensitivity of these responses to PERTUSSIS TOXIN implies that they are mediated by Gi/o proteins, but it is still unclear whether transduction is direct or indirect . Inhibition of cAMP formation does not seem to be involved. On the other hand, cAMP can contribute to the regulation of neuronal gene expression by CB1 . This process, which is necessary to produce lasting changes in synaptic strength, depends on the recruitment of complex networks of intracellular protein kinases87. Two components of these networks, extracellular signal-regulated kinase and focal adhesion kinase , become activated when hippocampal slices are treated with cannabinoid agonists. This activation is mimicked by inhibitors of cAMP-dependent kinase and is lost when the slices are exposed to cell-permeant cAMP analogues, implying that it might result from a decrease in intracellular cAMP concentrations.

The involvement of ERK and FAK in synaptic plasticity indicates that these protein kinases could participate in the changes in gene expression and the persistent neural adaptations that accompany cannabinoid administration90.In the rodent and human cortices, CB1 receptors are primarily found on axon terminals of cholecystokinin-8 -positive GABA interneurons. This expression pattern dominates the neocortex, hippocampal formation and amygdala, where nerve terminals that form excitatory synapses are ostensibly devoid of CB1 immunoreactivity. However, there is evidence that excitatory terminals in these regions do contain the receptor; for example, cannabinoid agonists reduce glutamatergic transmission in the amygdala of normalmice, but fail to do so in CB1 -deficient mutants. In addition, low concentrations of CB1 messenger RNA have been found in many neurons of the cortex that do not contain GABA. CB1 receptors are also expressed at very high levels throughout the basal ganglia. In the striatum they are localized to three distinct neuronal elements: glutamatergic terminals originating in the cortex, local-circuit GABA interneurons and axon terminals of GABA projection neurons. Medium spiny neurons project to striatal outflow nuclei, where CB1 receptors are especially abundant; for example, in the globus pallidus they outnumber dopamine D1 receptors by a factor of 45 . In the cerebellum, CB1 is present on excitatory terminals of climbing and parallel fibres as well as on GABA interneurons. Smaller numbers of CB1 receptors are also found in the thalamus , hypothalamus , midbrain , medulla and spinal cord. Last, CB1 is expressed in peripheral sensory neurons, where it is localized in cells that express N52, a protein marker of mechanosensitive Aβ fibres.A few cannabinoid effects persist in CB1 -null mice, implying that this receptormight not act alone in mediating brain cannabinoid signalling. Although cannabinoid agonists lose their ability to inhibit GABA and glutamate transmission in some brain regions of adult CB1 -knockout mice, they can still reduce excitatory transmission in the hippocampal CA1 field of these animals. This discrepancy is reinforced by the finding that GABA and glutamate synapses in CA1 respond in different ways to cannabinoid drugs. For example, cannabinoid depression of excitatory currents is blocked by CAPSAZEPINE, whereas depression of inhibitory currents is not.

These results make a persuasive case for the existence of a hippocampal cannabinoid-sensitive site that is distinct from CB1 , but other evidence appears to contradict them; for example, in newborn CB1 -null mice, cannabinoid agonists affect neither GABA nor glutamate transmission. Although this difference could be due to the developmental stage of the preparation used __ adult versus one-week-old mice __ more studies are needed to establish whether the CB3 site is molecularly distinct from CB1 . A novel cannabinoid site has also been identified in the vascular endothelium, but seems to be different from CB3 because it is not antagonized by capsazepine or activated by the CB1 /CB2 agonist Win-55212-2 .Outside the brain, the endocannabinoids are produced on demand and act on cells located near their site of synthesis. For example, they are formed by circulating leukocytes and platelets, and induce vascular relaxation by interacting with cannabinoidreceptors on the surface of neighbouring endothelial and smooth muscle cells. Similar PARACRINE actions are thought to occur in the CNS, where the endocannabinoids might mediate a localized signalling mechanism through which principal neurons modify the strength of incoming synaptic inputs.When a pyramidal neuron in the CA1 field of the hippocampus is depolarized, the inhibitory GABA inputs received by that cell are transiently suppressed. This phenomenon, called depolarization-induced suppression of inhibition , is initiated postsynaptically by voltage-dependent influx of Ca2+ into the soma and dendrites of the neuron, but is expressed presynaptically through inhibition of transmitter release from axon terminals of GABAinterneuron. This indicates that a chemical messenger generated during depolarization of the pyramidal cell must travel backwards across the synapse to induce DSI . There is evidence that this retrograde signalling process involves an endocannabinoid substance, possibly 2-AG. First, CB1 agonists mimic DSI, whereas CB1 antagonists block it. Second, DSI is absent in CB1 – deficient mice81,109. Third, the GABA interneurons that are implicated in DSI express high levels of CB1 receptors, which are localized to their axon terminals. Fourth, neural activity and Ca2+ entry stimulate the hippocampal synthesis of 2-AG, but have no effect on anandamide concentrations. Nevertheless, we still don’t know whether the endocannabinoid actually crosses back to the presynaptic nerve ending or is produced there by the action of another, unidentified retrograde signal . The fact that DSI is induced in vitro by levels of neural activity that could also be encountered in vivo indicates that this process might have a role in normal brain function. Although this idea is still questioned, various results link DSI to the regulation of hippocampal GAMMA OSCILLATIONS. These network oscillations are coordinated by CB1 -positive GABA interneurons and are influenced by cannabinoid agonists,drying rack for weed raising the possibility that an endocannabinoid substance might modulate their expression and be involved in the organization of hippocampal cell assemblies. Another function of DSI might relate to synaptic plasticity. By weakening GABA-mediated inhibition, DSI could facilitate the induction of long-term potentiation in individual CA1 pyramidal neurons; this might contribute in turn to the formation of ‘place fields’ or to other forms of hippocampus-dependent learning. Such a cognitive-enhancing action would not contradict the well-known amnesic effects of cannabinoid drugs as the latter might result from a generalized, circuit-independent activation of CB1 receptors in the hippocampus and other brain areas. Outside the hippocampus, endocannabinoid-mediated DSI has been shown to occur at interneuron– principal cell synapses of the cerebellum and probably will soon be discovered elsewhere.

CB1 -bearing interneurons are selectively localized to a subdivision of the amygdala called the basolateral complex, a key station in the neural circuitry that processes emotions and a primary site of cannabinoid analgesia. This localization, and the fact that CB1 inactivation causes anxiety-like and aggressive responses in rodents, indicate that the endocannabinoid system might influence affective states through changes in the amygdala’s efferent activity. This idea is further supported by two findings: first, presentation of anxiogenic stimuli increases anandamide and 2-AG concentrations in the mouse amygdala; second, FAAH inhibitors exhibit marked anxiolytic-like properties in rats. Locally formed endocannabinoids could modify the amygdala’s output in two complementary ways.They could depress glutamate release from axon terminals originating in the cortex and other brain regions. In addition, by reducing GABA release from basolateral interneurons, they might disinhibit GABA cells in the adjacent intercalated nuclei and consequently decrease the activity of their postsynaptic targets, the pyramidal neurons in the central nucleus of the amygdala, which constitute the structure’s primary efferent pathway. Here, local administration of cannabinoid agonists inhibits GABA release and profoundly affects motor behaviours. Membrane depolarization and dopamine D2 -receptor activation stimulate striatal anandamide formation, indicating that this endocannabinoid might contribute to the regulation of basal ganglia function. In agreement with this hypothesis, the CB1 antagonist rimonabant enhances the stimulation of movement that is induced in rats by dopamine agonists, whereas the endocannabinoid transport inhibitor AM404 attenuates this stimulation in a CB1 -dependent manner. Anandamide might act at multiple sites in the basal ganglia, including GABA projection neurons, corticostriatal glutamatergic terminals and local-circuit interneurons. Local-circuit interneurons are particularly notable because of their functional resemblance to CB1 -positive interneurons in the hippocampus, with which they share not only a GABA-containing phenotype, but also the ability to discharge high-frequency bursts of action potentials that can inhibit firing in large assemblies of projection cells. Does locally released anandamide gain access to these interneurons? Or does it primarily act on medium spiny cells and their cortical afferents? We don’t know yet. But these unanswered questions do not diminish the significance of striatal endocannabinoid signalling, which is further highlighted by the effectiveness of cannabinoid agonists in the symptomatic treatment of LEVODOPA-INDUCED DYSKINESIAS and TOURETTE’S SYNDROME, two disorders with strong striatal underpinnings.Beside their actions in the amygdala, cannabinoid agonists can influence the central processing of pain by interacting with CB1 receptors in the periaqueductal grey, rostral ventromedial medulla128 and spinal trigeminal nucleus. At each of these sites, CB1 activation depresses GABA release through a presynaptic mechanism, without causing significant changes in somatic membrane conductances. In the trigeminal nucleus, glycinergic transmission also is inhibited. Painful stimuli elicit anandamide release in the rat periaqueductal grey, and systemic administration of CB1 antagonists produces HYPERALGESIA in rats and mice. So, noxious stimuli can engage a central analgesic circuit operated by the endocannabinoids, which, working in combination with a parallel mechanism in the periphery, could underlie the analgesic properties of cannabinoid drugs.