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.

It is already understood that by preheating the air before it enters the cathode results in better SOFC performance because the temperature difference from inlet to outlet in the stack is much less and therefore can respond faster to load fluctuations. As is already understood, SOFC systems best serve stationary power generation applications. SolidPower recommends using its Engen-2500 system for multi-family houses, hotels, restaurants, public buildings, schools, or small companies. It can be installed in different configurations including as a stand-alone mCHP unit with water storage where it can be combined with heat and power systems that use renewable energy source or as a series of multiple modules, integrated with the heating system of the building. The particular application of the Engen-2500, discussed throughout this thesis, is for providing power to individual server racks, which typically range from a handful of servers to dozens depending on the class of server. Fuel cells are generally suitable for constant power generation applications. They perform best when fuel and oxygen supplies are steady and the load demand remains constant. Therefore, Microsoft’s vision of disconnecting large data center facilities from the electric grid and relying solely on SOFCs for providing power is definitely possible. Data center power consumption has both short and long-term variations due to workload fluctuations and servers turning on and off. The variations in load can be represented by three categories: instantaneous load changes, short term load changes, and long-term load changes. For the first case, the load of a server can change almost instantaneously reacting to a workload. This may be caused by a change in CPU utilization from 0% to 100% and can occur within milliseconds. Some of these types of rapid fluctuations in load can potentially be absorbed by the server’s power supply with its internal capacitors, however, larger instantaneous changes must be handled using an external energy storage system in cooperation with the SOFC system. Short-term load changes occur over several seconds or minutes and can be handled by the SOFC system as it ramps up or down accordingly. Yet, in extreme situations where the server has to be cold rebooted,curing cannabis the SOFC lags behind the sudden spikes in power consumption. For these extreme situations, it is always necessary to include a battery in order to keep up with the drastic load changes.

If the load changes are predictable , the SOFC system can increase its power production ahead of time to follow the load demand. Doing so may lead to reduced end-to-end efficiency of energy usage, therefore this practice is not recommended if trying to maximize production efficiency. The ideal case for applying SOFC systems in data centers is when there are long-term load changes that occur over days and weeks. These changes are typically much slower than all fuel cell’s ramp rates and therefore are no issue for the SOFC’s ability to keep up with the load demand. Under load fluctuations, it would be helpful to understand how much over provisioning of the fuel cell is necessary. The only case where SOFCs struggle is when the servers cause large load changes due to startup and shutdown processes. By staggering server power on and off events over time instead of all at once, a single server sized battery can be shared by multiple servers in a rack. Therefore, the SOFC would not need to be aggressively over provisioned. After receiving the Engen-2500 system from SolidPower and connecting it to the load bank, natural gas, and water pipelines in the testing bay facility of the NFCRC, the initial round of testing began. It was agreed upon with members of the fuel cell team at UCI, Microsoft, and SolidPower that the testing framework would begin with less severe transients in order to avoid causing damage to the stack or system components early on. The first step to characterizing and understanding the performance of any fuel cell system is to subject it to steady-state analysis. A testing matrix was established to neatly organize and document the user-constrained parameters and the corresponding values for other important parameters. For the steady-state analyses, the cathode outlet temperature and fuel utilization were the two parameters that were defined as user-constrained with the two outlet temperature values selected to be 760°C and 770°C and the three fuel utilization values selected to be 0.70, 0.72, and 0.75 as shown in Table 5. In an ideal world, a fuel cell would be able to maintain a constant voltage output for any amount of current demanded. This would appear as a flat line on the current-voltage plot at the peak voltage level, which refers to the open-circuit voltage – defined as the difference of electrical potential between the two electrodes when disconnected from a circuit . However, due to real-world physical limitations, the actual voltage output of a real fuel cell is less than the ideal thermodynamically predicted voltage due to unavoidable losses that is discussed in detail in Section 6 of this thesis.

Another very noticeable fact about the system performance is the slight voltage differences for the same current load demand. From Figures 24 and 25 the slight voltage differences observed between the two stacks in the system are thought to be caused by the slight temperature differences of the two stacks and the manufacturing variation amongst the cells. The benefits of understanding the steady-state performance by looking at the polarization curve is to provide additional information for the overall data center design such as the DC bus, power supply specifications, and DC/DC converters. For all energy conversion devices, it is always of great importance to consider and compare the operational efficiency. The fundamentals of efficiency can be broken down and represented by two important concepts, ideal and real efficiency. Since fuel cell devices produce electric and not mechanical work, thermodynamic theory suggests that the electric work available is limited by the change in Gibbs free energy, therefore, the ideal efficiency of a fuel cell is limited by. The ideal fuel cell efficiency is defined as the amount of useful energy that can be extracted from the process relative to the total energy evolved by that process. For a fuel cell, the maximum amount of energy available to do work is given by the ratio of Gibbs free energy to enthalpy. These losses are illustrated in the polarization plots and the voltage efficiency can be characterized by the ratio of the real operating voltage to the thermodynamically reversible voltage. Note that the operating voltage depends upon the current drawn, therefore the higher current demand, the lower the voltage efficiency. The fuel utilization efficiency accounts for the fact that not all of the provided fuel is used by the fuel cells. Some of the fuel may undergo side reactions that do not produce electric power or the fuel may simply flow through the fuel cell without ever reacting. The fuel utilization efficiency is therefore a ratio of the fuel used by the cells to generate electric current to the total fuel provided. With this understanding of real fuel cell efficiency and considering the initial round of steady-state tests, the steady-state efficiencies of Engen-2500 stack and system were determined. Considering the case for fuel utilization of 75% and a cathode outlet temperature of 770°C, the corresponding stack and system efficiencies are shown in Figure 26. As is evident, the electrical efficiency of just the stack alone is well above 52%, which is a remarkable electrical efficiency for a 2.5 kW electric generator. The additional stack efficiencies for all other cathode outlet temperatures and fuel utilizations listed in Table 5 are displayed in Appendix D at the end of this thesis. From previous fuel cell modeling efforts performed at the NFCRC, a spatially and temporally resolved fuel cell model was developed using the Matlab and Simulink interface environments for experimental verification. The Matlab environment was particularly chosen for modelling development efforts because of its versatility and widespread adoption in the engineering community. Simulink is a graphical block diagram environment for multi-domain simulation and model based design,cannabis dryer which is extremely useful for modeling, controlling and simulating dynamic systems. The SOFC dynamic model developed at the NFCRC and adapted for the purposes of experimental verification must inherently be accurate and sophisticated to achieve reasonable and verifiable results. The NFCRC fuel cell system model incorporates all the necessary components to analyze and assess the dynamic performance for both the SOFC and MCFC fuel cell types. Additional changes can be made to restructure the model to study other fuel cell types like PEMFCs; however, for the purposes of this thesis, the SOFC system model was utilized and adapted to accurately model the Engen-2500 experimental system.

Fabian Mueller, a previous NFCRC graduate researcher, was one of the first students to work on and develop the NFCRC dynamic fuel cell modeling tools. He incorporated modeling strategies from Rivera , Xue et al. , Roberts and Gemmen , Smugeresky, Roberts et al. , and Lim et al. , using control volumes to spatially discretize system components and apply dynamic conservation equations. The preliminary modeling strategies developed by these scholars were shown to accurately capture the dynamics of fuel cell systems. However, additional details were necessary to improve the precision of the system model. Mueller outlines a host of modelling assumptions that are important to consider for a simplified analysis and incorporated them into the NFCRC fuel cell model.An essential feature of the SOFC system model is the capability of spatially resolving each component for varying degrees of resolution, dependent on the desired precision. This capability provides the user with greater accuracy and precision than relying on bulk or equivalent circuit models, especially for scaling analyses. Keep in mind that increasing the model resolution increases the precision of the localized analysis at the expense of significant computing resources and additional time to complete the simulation. Therefore, the user must be aware of the computational power of his or her computer and select a discretization resolution that maximizes the precision of the analysis while minimizing the time to reach completion. Spatial resolution yields descriptive localized analyses of the internal temperature profile and heat transfer across an individual cell and each component of the SOFC system for a given moment in time during the dynamic analysis. For the fuel cell stack, spatial resolution is achieved by taking a single cell and dividing it into a grid of smaller elements, which are referred to as nodes. Each node is broken down further into five distinct segments that comprise a single cell: the oxidant separator plate, cathode gas stream, electrolyte , anode gas stream, and fuel separator plate. Figure 27 indicates the typical structure of one SOFC cell that includes the five segments mentioned.Spatially resolving each segment of a single cell permits the localized dynamic analysis of the conservation of mass, energy, and momentum equations while also locally evaluating the temperature, species mole fractions, pressure, and other required characteristics. The dynamic analysis of one cell is then scaled to the number of cells in the stack, ultimately representing the dynamic characteristics of the entire stack component. The same discretization method just described is applied to all components of the Engen-2500 SOFC system. The transport phenomena and electrochemical reactions evaluated at each locally resolved temperature, species mole fraction, and pressure, determine the performance of each component in the SOFC system. Constructing the system model requires integration of the multiple individual components, which have been resolved following the particular component physics, chemistry and electrochemistry. The complex interactions of the integrated components are captured through simultaneous solutions from the dynamic system analysis. For higher-temperature fuel cells used in combined heat and power systems, the fuel cell stack often appears to be quite a small and insignificant part of the whole system. The extra components required depend greatly on the type of fuel cell and the fuel used. In SOFC systems, fuel and air enter the SOFC stack and electricity, exhaust gas, and hot water or steam exit the system. The difference between an SOFC stack and an SOFC system is generally referred to as the “balance-of-plant” . BOP equipment may differ for each application depending on the size of the system, the operating pressure, and the fuel used. If an SOFC stack is to be tested for commercial applications, the test is usually performed in a complete system with balance-of-plant components included and the stack integrated into the system.

Our second analysis focuses on behavior of jobs to quantify the probability that a job will have to span more racks to find resources compared to the minimum number of racks the job can oc cupy based on its requested number of nodes. In addition, this way we capture the correlation of different resource types that are assigned to the same job. In particular, we sample 210 randomly chosen timestamps from our dataset. For each timestamp, we record which jobs are executing, their resource utilization, and their size in nodes. Because KNL jobs lack memory bandwidth measure ments, we focus only on Haswell jobs. For each job’s resource utilization metric, such as memory occupancy, we measure the maximum utilization among all nodes reserved to the job through out the job’s execution. We use a job’s maximum node utilization to account for the worst-case scenario. Then, we execute 16 of the following random experiments for each randomly chosen timestamp and report the maximumprobability across experiments. In each random experiment, we look at all jobs running at the chosen timestamp and assign them to nodes. Though job placement that prioritizes resource disaggregation would likely yield better results, we leave this as future work, since such job placement policies are still emerging. Therefore, in each experiment , we allocate racks of Haswell nodes and assign them to jobs in a random fashion. For each job, the number of nodes is the same as when it was running on Cori. For each randomly chosen rack, we reserve resources to cover the job’s utilization at the randomly chosen timestamp. If the job still has resource requirements remaining, then we continue by allocating another random rack. At the end of each experiment,cannabis growing equipment we record the percentage of jobs that had to allocate resources from more racks than the minimum number of racks they could be placed in based on their size in nodes.

These are the jobs that had to span more racks because of a lack of resources. This analysis is performed for each resource type separately. Results are shown in Figure 14 . Without reducing resources, there is still some worst-case probability for a job to span more racks than the minimum to allocate memory capacity because of unfavorable random job placement. However, the average across our random experiments re mains near zero . With a 20% reduction, the same worst-case probability becomes 11%, with a 50% reduction the probability becomes 22.3%, and with 80% reduction the probability becomes 56%. For NIC and memory bandwidth, for up to a reduction of 85% the probability is near zero. For a reduction of 95%, the probability for NIC bandwidth is 12.2% and for memory bandwidth 15.5%. While these results are sensitive to our assumptions and random choices of our algorithm, they indicate that intra-rack disaggregation suffices the majority of the time except when reducing resources aggressively.To illustrate potential benefits and thus further motivate intra-rack resource disaggregation, we use per-node statistics to calculate an average resource utilization of each rack. We do this for the node-to-rack mapping in Cori but also for a large set of randomized mappings of nodes to racks, because Cori’s scheduler is agnostic to re source disaggregation. This increases the computational complexity of this analysis, so we only perform it across four days of our data set. For each mapping, we use node statistics to calcu late per-rack utilization for each resource, by average across the four-day sampling period. We then take the maximum utilization for each resource among all the node-to-rack mappings and all racks within each mapping, to capture the worst-case rack utilization for each metric. We use that maximum to derive how much we could reduce in-rack resources and still satisfy the worst-case average rack utilization in our chosen four-day period. This does not indicate that no job will have to cross a rack boundary to find resources, or that application performance will be unaffected. Instead, this analysis focuses on resource utilization statistics. Memory band width reduction is based on the per-node theoretical maximum of 136 GB/s from the eight memory modules.

The percentage we can reduce each resource and still satisfy the worst-case average rack utilization is shown in Table 2. As shown, except for memory capacity in KNL racks, the other resources can be reduced substantially. Resource disaggregation-aware job scheduling may further improve these findings.Based on Section 4, there are also opportunities for rack-level disaggregation in ML workloads and GPU-accelerated systems. We observe a strong variability of resource requirements among different neural networks and therefore application domains but also among inference and training . Inference usually requires higher CPU-to-GPU ratios than training and uses less GPU memory. In contrast, training leads to high GPU utilization in terms of computation and memory but lower CPU utilization. However, this also varies with the workload. Job schedulers can use a disaggregated system by allocating more CPUs and less GPU memory for inference and give the remaining resources to a training job, which requires lots of memory and generally less CPU time. While we observe that GPU utilization is generally high, CPU resources and network bandwidth are underutilized most of the time. Although we cannot determine peak bandwidth demands with our methodology, we see that the average bandwidth utilization over longer periods is low. As we strong-scale training to multiple nodes, the bandwidth demands increase but other resources become less utilized, such as GPU memory . A disaggregated system allows us to provision unused GPU memory for other jobs, and since bandwidth depends on the scale, we can give more bandwidth to large-scale jobs and less bandwidth to small-scale jobs.Sampling a production system has significant value, because it demonstrates what users actually execute and how resources are used in practice. At the same time, sampling a production system inevitably has practical limitations. For instance, it requires privileged access and sampling infrastructure in place. In addition, even though Cori is a top 20 system that executes a wide array of open-science HPC applications, observations are affected by the set of applications and hardware configuration of the system. Therefore, HPC systems substantially different than Cori can use our analysis as a framework to repeat a similar study. In addition, sampling typically does not capture application executable files or input data sets.

Therefore, reconstructing our analysis in a system simulator is impossible but also impractical due to the vast slowdown of a simulator for large-scale simulations compared to a real system. Similarly, sampling a production system has no method to differentiate when application demands exceed available resources as well as the resulting slow down. For this reason and our 1s sampling period, our study focuses on sustained behavior and cannot make claims for the impact of resource disaggregation to application performance.While it is hard to speculate how important HPC applications will evolve over the next decade, we have witnessed little change in HPC fundamental algorithms during the previous decade. It is those fundamentals that currently cause imbalance that motivates resource disaggregation. Another consideration is application resource demands relative to available resources in future systems. For instance, if future applications require significantly more memory than the memory available per CPU today, then this may motivate full system disaggregation of memory, especially if there is significant variability across applications. Similarly, if a subset of future applications request non-volatile memory , then this may also motivate full system disaggregation of NVM, similar to how file system storage is disaggregated today.However,cannabis drying trays future systems may have larger racks or nodes with more resources, strengthening the case for intra-rack resource disaggregation. When it comes to specialized fixed-function accel erators, a key question is how much data transfer they require and how many applications can use them. This can help determine which fixed-function accelerators should be disaggregated within racks, hierarchically, or across the system. Different resources can be disaggregated at different ranges. Ultimately, the choice should be made for each resource type for a given mix of applications, following an analysis similar to our study.Future work should explore the performance and cost trade off when allocating resources to applications whose utilization is dynamic. For instance, providing enough memory bandwidth to satisfy only the application’s average demand is more likely to degrade the application’s performance, but increases average resource utilization. Future work should also consider the impact of resource dis aggregation to application performance, which should also consider the underlying hardware to implement resource disaggregation and the software stack. Job scheduling for heterogeneous HPC systems should be aware of job resource usage and disaggregation hardware limitations. For instance, scheduling all nodes of an application in the same rack benefits locality but also increases the probability that all nodes will stress the same resource, thus hurting resource disaggregation.

By the end of the 20th century, the most pervasive world-changing technology was the internet because of how it revolutionized the daily productivity of modern society. Mark Schueler, a Ph.D. student from Southampton University illustrates the explosive, “Growth of the Internet,” from its inception until more recent years in Figure 1, showing just how rapidly new technology can pique the public’s interest when it positively influences the majority. The World Economic Forum estimates about 2.5 billion people are connected to the internet today; a third of the world’s population. It is projected that 4 billion users will be connected by 2020, more than half the global population. With so much of the world’s population currently having little or no internet connectivity, this poses the question: Can the infrastructure that society counts on to carry all this digital traffic keep up with the accelerating demand? There is a growing need to produce the most computing power per square foot at the lowest possible cost of energy and resources. More recently, the electricity used by data centers has garnered the most intense interest, partly because of the importance of these facilities to the broader economy and because the power used by the individual data centers rivals some large industrial facilities. In a report to Congress from the United States Environmental Protection Agency discussing data center electricity use leading up to 2006 , it is quickly apparent how the electricity consumption nearly tripled from year 2000 to year 2006. Had people chosen to disregard improving energy efficiency and technology within U.S. data centers, then the domestic energy conversion would have nearly doubled by 2011. Fortunately, the data center industry has remained aware of the vast energy required to operate their infrastructure and has experimented with improving data center operation as recent data would suggest. In 2008, data center electricity demand was approximately 69 billion kWh or 1.8% of the total 2008 U.S. electricity sales. It comes as no surprise with how connected everyone is to the internet that nowadays data centers are likely to be increasingly large, powerful, energy-intensive, always running and out-of-sight. Because of the sheer size and numbers of servers involved, data centers are loaded with energy inefficiencies. Considering a traditional data center connected to the electric grid, less than 35% of the energy from the fuel source that is supplied to the power plant is delivered to the data center. The most significant inefficiencies result from power plant generation losses and transmission and distribution losses. Figure 3 outlines the process of power loss through the transportation and distribution network of the electric grid starting from the fuel source and ending with the power supplied to the consumer. It is evident that the largest inefficiency stems from the power generated at the power plant level with additional losses associated with the transmission and distribution to the data center, where the data center receives roughly 30% of the total energy that could have been supplied ideally from the fuel source. Considering the operations within a data center, there are further losses associated with the infrastructure required for daily reliable operation systems. The additional power consumed by the cooling, lighting, and energy storage, means approximately less than 17.5% of the energy supplied to the power plant is ultimately delivered to the servers.Recognizing the significant energy losses at the power plant level, Microsoft made a commitment in May 2012 to make their operations carbon neutral: to achieve net zero emissions for their data centers, software development labs, offices, and employee business air travel.

Fourth-generation bike sharing models may also incentivize user based redistribution by employing demand-based pricing where users receive a price reduction or credit for docking bicycles at empty docking locations. A third feature of fourth-generation systems is the seamless integration of bike sharing with public transportation and other alternative modes, such as taxis and car sharing via smart cards, which support numerous transportation modes on a single card. In 2009, the Yélo bike sharing system was launched in La Rochelle, France. This system includes a smart card, which is fully integrated with the public transportation system. This facilitates multi-modal transportation linkages and user convenience, which could lead to greater auto ownership and usage reductions, as more daily trips are supported by alternative modes. However, creating a program that coordinates various forms of transportation on a single card is challenging, as this can be costly and often requires multi-agency involvement. Another area for improvement is bicycle security, which can be supported by ongoing technological advancement, such as the design and integration of GPS units into more robust bicycle frames that further enhance existing locking mechanisms, deter bike theft, and facilitate bike recovery. However, adding GPS units is costly and can potentially increase financial losses, if bikes with built-in GPS are vandalized or stolen. Finally, to target a larger scope of bike sharing users, fourth-generation systems may be more likely to incorporate electric bicycles, which enable longer-distance trips; encourage cycling on steeper hills and slopes; and lessen physical exertion requirements, particularly when users are commuting or making work trips in business attire. Over the past century, California has built an extraordinarily complex water management system with hundreds of dams and a vast distribution network that spans the state. This system generates electricity, provides flood protection, delivers reliable water supplies to 40 million people and sup ports one of the most productive agricultural regions in the world. Yet development of the state’s water manage ment system has come at a price.

Damming waterways,microgreens grow rack diverting water from rivers and streams and altering natural flow patterns have transformed the state’s freshwater ecosystems, leading to habitat degradation, declines of freshwater species and loss of services that river ecosystems provide, including high-quality drinking water, fishing and recreational opportunities, and cultural and aesthetic values. The state aims to accommodate human water needs while maintaining sufficient stream flow for the environment. To support this mission, scientists from the U.S. Geological Survey , The Nature Conservancy and UC have developed new techniques and tools that are advancing sustainable water management in California. At the center of these new advances is the need to understand the natural ebbs and flows in the state’s rivers and streams. Natural patterns in stream flow are characterized by seasonal and annual variation in timing , magnitude , duration and frequency . California’s native freshwater species are highly adapted to these seasonally dynamic changes in stream flows. For example, salmon migration is triggered by pulses of stream flow that follow winter’s first storms, reproduction of foothill yellow-legged frogs is synchronized with the predictable spring snow melt in the Sierra Nevada, and many native fish breed on seasonally inundated flood plains, where juveniles take advantage of productive, slow-moving waters to feed and grow. When rivers are modified by dams, diversions and other activities, flows no longer behave in ways that support native species, contributing to population declines and ultimate extinction. Thus, understanding natural stream flow patterns and the role they play in supporting ecosystem health is an essential first step for developing management strategies that balance human and ecosystem needs. Unfortunately, our ability to assess alteration of natural stream flow patterns, and the ecosystem consequences, is hindered by the absence of stream flow data. California’s stream flow gauging network offers only a limited perspective on how much water is moving through our state’s rivers. In fact, it’s been estimated that 86% of California’s significant rivers and streams are poorly gauged and nearly half of the state’s historic gauges have been taken offline due to lack of funding . Of those gauges that are still in operation, most are located on rivers that are highly modified by human activities and gauge records prior to impacts are limited. These limitations can be partially overcome with modeling approaches to predict the attributes of natural stream flow expected in the absence of human influence.

The predictions can then be compared to measured stream flow at gauging locations, or they can be used to estimate natural flow conditions in ungauged streams. In 2010, Carlisle et al. developed a modeling technique to predict natural attributes of stream flow and assessed stream flow alteration at gauges throughout the United States . Soon after, UC and TNC scientists began using the approach to expand and further refine the technique for applications in California . The models have evolved over time, but all rely on stream flow monitoring data from USGS gauges located on streams with minimal influence from upstream human activities. These are referred to as reference gauges. Some reference gauge data come from historical measurements made before significant modification of flows occurred, such as the years prior to the building of a dam. The remaining data are from reference gauges located in California watersheds that remain minimally altered by human influence. Once reference gauges were identified and flow records obtained from the USGS web-based retrieval system, we used geographic information systems to characterize the watersheds above each reference gauge based on their physical attributes, such as topography, geology and soils . We also assembled monthly precipitation and temperature climate data for the past 65 years for each watershed. The watershed variables and climate data were then compiled and statistically evaluated in relation to observed flow conditions at the reference sites using a machine-learning approach that uses the power of modern computers to search for predictive relationships in large data sets. An advantage of machine-learning techniques is the ability to make predictions from multiple model iterations , which tends to increase accuracy. Once we had developed and evaluated models using observed stream flow data from reference gauges, we could predict stream flow attributes for any portion of a stream or river in California for which the climate and watershed characteristics were known . Additional technical details of the modeling approach are provided in Carlisle et al. 2016 and Zimmerman et al. 2018.In a study led by Zimmerman et al. , we applied the machine-learning technique to assess patterns of stream flow modification in California. We did this by predicting natural monthly flows at 540 streams throughout California with long-term USGS gauging stations and comparing those predictions with ob served conditions. We then assessed how observed flow conditions at the gauges deviated from predictions and recorded the frequency and degree to which flows were either higher or lower than natural expected levels, while considering the uncertainty of model predictions. We found evidence of widespread stream flow modification in California . The vast majority of sites experienced at least 1 month of modified flows over the past 20 years and many sites were modified most of the time .

When stream flows were modified, the magnitude of modification tended to be high. On average, inflated stream flows were 10 times higher than natural expected levels, whereas depleted stream flows were 20% of natural expected levels. Overall, stream flow modification in California reflects a loss of natural seasonal variability by shifting water from the wet season to the dry season and from wet areas of the state to the drier south. Stream flow inflation was most common in dry summer months and for annual minimum flows. Conversely, flow depletion was most common in winter and spring months and for annual maximum flows. Unaltered sites tended to occur in places with relatively low population density and water management infrastructure, such as the North Coast,ebb and flow flood table whereas greater magnitude and frequency of alteration was seen in rivers that feed the massive water infrastructure in the Central Valley and the poulated Central Coast and South Coast regions. A key water management goal in California is to manage river flows to support native freshwater biodiversity. By estimating natural river flows and the degree to which they are modified, our work provides a foundation for assessing “ecological flow” needs, or the river flows necessary to sustain ecological functions, species and habitats. Assessments of ecological flow needs are generally performed at stream reach to regional scales , but rarely for an area as large and geo graphically complex as California. In 2017, a technical team that includes scientists from UC, TNC, USGS, California Trout, Southern California Coastal Water Research Project and Utah State University began developing a statewide approach for assessing ecological flows. The team has identified a set of ecologically relevant stream flow attributes for California streams that reflect knowledge of specific flow requirements for key freshwater species and habitats . Our modeling technique is now being extended to predict natural expectations for these new stream flow attributes. Model predictions of the natural range of variability for these ecologically relevant stream flow attributes will provide the basis for setting initial ecological flow criteria for all streams and rivers in California by the State Water Resources Control Board and other natural resource agencies.

These ecological flow criteria will be based on unimpaired hydro logic conditions, but they can be refined in locations where management and ecological objectives require a more detailed approach. For example, refined approaches would likely be required in rivers that must be managed for species listed under the Endangered Species Act or in rivers where substantial flow and physical habitat alteration makes reference hydrology less relevant for setting ecological flow criteria, such as in the Central Valley or in populated watersheds of coastal California. Our technical team also was involved in establishing the California Environmental Flows Work group of the California Water Quality Monitoring Council . The mission of the Work group is to advance the science of ecological flows assessment and to provide guidance to natural resource management agencies charged with balancing environmental water needs with consumptive uses. The Work group is comprised of representatives from state and federal agencies, tribes, and nongovernmental organizations involved in the management of ecological flows. It serves as a forum to facilitate communication between science and policy development and to provide a common vision for the use of tools and science-based information to support decision-making in the evaluation of ecological flow needs and allocation of water for the environment. The modeling technique described above has also been used to evaluate statewide water allocations. Grantham and Viers analyzed California’s water rights database to evaluate where and to what extent water has been allocated to human uses relative to natural supplies. They calculated the maximum annual volume of water that could be legally diverted according to the face value of all appropriative water rights in the SWRCB’s water rights database. Water rights were distributed according to their location of diversion, and the permitted diversion volumes were aggregated at the watershed scale to estimate a maximum water demand for each of the state’s watersheds. These permitted water diversion volumes were compared with modeled predictions of average annual supplies to estimate the degree of appropriation of surface water resources throughout the state . The study found that appropriative water rights exceed average supplies in more than half of the state’s large river basins, including most of the major watersheds draining to the Central Valley, such as the Sacramento, Feather, Yuba, American, Mokelumne, Tuolumne, Merced and Kern rivers. In the San Joaquin River, appropriative water rights were eight times the volume of estimated natural water supplies . The volume of water rights allocations would be much higher if pre-1914 and riparian water rights had been included, but these data were not available at the time.For example, comparisons of allocations with water use suggest that in most of California only a fraction of claimed water is being used. In a well-functioning water rights system where allocations are closely tracked and verified, an excess of water rights relative to supplies is not necessarily a problem. During water shortages, holders of junior appropriative rights would be required to curtail their water use. When water is abundant, most water rights holders should be able to fully exercise their claims.

An additional issue related to product regulation of marijuana edibles is the high THC potency per package without adequate requirements that these products clearly be demarcated to explicitly communicate the actual size of an individual serving to the consumer.In Colorado and Washington, product regulations allow for each package to contain up to ten 10 mg servings of THC or 100 mg of THC. Poor product labeling in Colorado and Washington contributed to an increase in calls to poison control centers and self-reports of adult intoxication.In Colorado, marijuana-related calls to the poison control center increased from 44 in 2006 to 227 in 2015,while in Washington calls increased by 79% from 111 in 2010 to 199 in 2014.Since commercialization, calls increased by 55% from 129 in 2012 to 199 in 2014.Liquor control boards in charge of approving products prior to market release allowed fruit and candy flavored marijuana products to enter the legal markets in Washington and Colorado. Despite a rule that the Washington State Liquor and Cannabis Control Board not approve any marijuana-infused edible products “especially appealing to children” such as, but not limited to, “gummy candies, lollipops, cotton candy, or brightly colored products” did not block approval of fruit flavored sodas and candy, chocolate and peanut butter flavored cookies and brownies, and chocolate truffles,including Mirth Provisions’ Legal Sparkling Rainier Cherry Soda and Nana’s Secret Soda in Orange Cream and Peach Flavors.Colorado does not have even such nominal restrictions and similar products have entered the market, including Dixie Elixir’s Crispy Cracken and Chocolate Cherry Pretzel. Marijuana edibles may be a safer alternative for adult consumers than marijuana cigarettes because they avoid combustion. However, because edibles are being produced in a wide array of flavors and variations that often are appealing to children, it is questionable whether these products contribute to less harm. Avoiding these harms could be achieved through tight regulation,plant growing stand including low limits on potency, large warning labels, accurate labeling, standardization of dosing, and standardization of packaging to avoid accidental ingestion by children and adults.

There is concern that the high potency of these products as well as their appeal to children may result in adverse health consequences.Indeed, it is likely that such youth appealing products are a major contributor to an increase in accidental childhood ingestion since legalization in Colorado402 and Washington.Prior to legalization in Colorado and Washington there were few cases involving marijuana related accidental poisonings in children. Children admitted to the emergency room for accidental marijuana ingestion increased from 0 to 14 two years following liberalization of medical marijuana laws in Colorado.Following implementation of retail marijuana laws in Colorado in 2013, an additional 14 children were admitted to the hospital for ingestion of edibles,with the first 9 occurring in the first 6 months of legalization.Washington, which modeled its product labeling and potency rules on Colorado’s,experienced a similar increase in adverse outcomes. In 2014, 45% of calls to poison control center were related to marijuana intoxication for those under age twenty – since legalization in 2012, these calls have increased from 50 in 2012 to 90 in 2014. Significantly, the highest number of calls in 2015 were regarding children under the age of five.Of the calls reported for the first nine months of 2015, 51% were in the marijuana/cannabis category, 42% were associated with infused-products, and 7% were related to marijuana oil. Youth accounted for 43% of the statewide calls during this nine-month period in 2015. National data from the United States show similar trends for accidental childhood ingestion. Between 2005-2011 there was an annual 30% increase in marijuana exposure in medical marijuana states while non-medical marijuana states showed no increase.To address the issue of accidental consumption of marijuana edibles, Colorado and Oregon enacted legislation requiring marijuana producers to place a THC warning symbol on their products . Colorado, Washington, and Oregon developed mass media campaigns aimed at preventing youth marijuana use , not general market campaigns designed to minimize overall population use as is done for tobacco. These campaigns were targeted at youth with messages on health risks of impaired memory, developmental delays, increased risk for addiction, depression, anxiety, psychosis, or other mental illnesses.

Messages related to the consequences of marijuana use include ineligibility for receiving financial aid and how marijuana-related charges may lead to school suspensions and expulsions. State health departments public awareness messages in Colorado and Washington directed to adults only cautioned adults, particularly new users, to “be safe and sensible” when using newly legal marijuana rather than discouraging use altogether. Colorado contracted with the University of Colorado to evaluate the impact of its mass media campaign on change risk perceptions and use behaviors as well as increasing knowledge of marijuana laws, health impacts of use, safe storage practices, and prevention.Adult exposure to the 2015 “Good to Know Campaign” was associated with an increased likelihood of accurately identifying retail marijuana laws compared to adults with zero exposure, with the proportion adult acute awareness increasing from 62% to 73% at follow up. There were moderate effects on knowledge of harms associated with use and perceptions of risk related to underage use , use around children , and high risk use . The survey did not question respondents whether or not the campaign impacted use behavior or thoughts on quitting, intentions to quit, or quit attempts .Taxation can both be used to raise marijuana prices artificially in order to discourage consumption,and to prevent taxpayers from subsidizing the regulatory, public education, and prevention and control program, and the marijuana education and research program and adjusted periodically for inflation. Additional tax increases could be used as a way to raise the price to reduce marijuana initiation and promote cessation. While all four US states that had legalized recreational marijuana as of October 2016 and Uruguay tax marijuana, these tax rates were not set at levels designed to cover regulatory, public health education, and medical costs associated with marijuana legalization.In Colorado,Washington,and Oregon, state legislators were directed by the ballot initiatives voters enacted to adjust the retail sales tax to make retail marijuana competitive with black market prices. Washington and Oregon ballot initiatives also include criteria for adjusting marijuana taxes to discourage use, and an additional requirement in Oregon to maximize net revenue from the marijuana tax. In Uruguay, officials of the IRRCA have determined that marijuana will be taxed at $1 per gram to compete with black market prices, despite national legislation requiring that government officials develop and fund an enforcement system and education and prevention programs.

Shortly after legal sales went into effect, state legislators in Colorado,Washington,and Oregon reduced marijuana taxes to compete with the black market. Colorado reduced the retail sales tax from 10% in 2014 to 8% in 2015, while Washington consolidated the state’s three-tier tax system to a single ad valorem excise tax of 37% at the retail sales level to reduce the marijuana industry’s federal income tax liability because consumers would pay the tax and so would technically not be considered part of the retailers’ gross income. Oregon also modified its wholesale tax in 2015 to a price-based excise tax of 17% of the retail sale, with up to an additional 3% tax levied at the local level, to increase state revenue through increased sales stimulated by lower prices.In the three states where marijuana taxes were reduced, state legislators were more concerned with short-term gains of competing with the black market and maximizing state revenue than long-term public health impact and costs associated with reduced use through higher taxes. There are no requirements for marijuana to be taxed based on a percentage of tetrahydrocannabinol content, which may in effect provide incentive for manufacturers to increase the THC content of cannabis.Indeed, US marijuana producers have been increasing product potency over the last 20 years.Between 1995 and 2014, marijuana potency increased from 5% to 12%, with a corresponding decline in cannabinol. The result was a THC/CBD ratio increase from a factor of 14 in 1995 to a factor of 80 in 2014.In jurisdictions with legal marijuana sales, edibles and cannabis concentrates, where THC concentration can be as high as 70%,plant grow table has increased in recent years. A weight-based tax, or a tax based on the unit of THC per weight or volume could be a solution to this problem. Another policy worth considering from the alcohol control literature is implementation of minimum unit pricing .Evidence from Canada show that MUP for alcohol is associated with reduced consumption and alcohol-related harms.Longitudinal estimates from British Colombia suggest that a 10% increase in MUP for a given alcohol product would result in a 16.1% drop in consumption.As is the case with most parts of the new regulatory framework for marijuana, implementation of MUP for marijuana should be considered at the same time as legalization in order to avoid potential legal battles with a professionalized marijuana industry. In 2012, Scotland was the first country to pass national legislation requiring MUP for all alcohol products.However, implementation of MUP in Scotland has been met with fierce opposition from the drinks industry, with claims of MUP as a violation of European Union trade law.The US states took varied approaches to regulating restrictions on marijuana business locations, none of which protect those most likely to regularly use marijuana In Colorado, local governments were prohibited by state law from granting a license to a business within 1,000 feet of a school defined as “public or private preschool or a public or private elementary, middle, junior high, or high school or institution of higher education”, alcohol or drug treatment facility, principal campus of college, university or seminary, or a residential child care facility.Although Washington lawmakers prohibited marijuana businesses within 1,000 feet of K-12 schools, recreational center or facility, child care center, public park, public transit center, library, or any game arcade, it allowed local governments to pass rules to reduce the distance requirement to a minimum of 100 feet from areas where children and adolescents are likely to congregate.As of September 2016, four Washington cities reduced the buffer zone for marijuana businesses to 500 feet, and one city reduced its buffer zone to 100 feet for parks, recreational/community centers, libraries, childcare centers, game arcades, and public transit centers.Oregon lawmakers did not prohibit retail store locations within 1,000 feet of colleges or universities despite the fact that many college students are under 21. Retail stores in Alaska were prohibited under the legalization initiative within 500 feet of child-sensitive areas, defined as facilities that provide services for persons under 21, a building in which religious services are regularly conducted, or a correctional facility. Colleges and universities are not explicitly included. Retail outlet density is positively associated with youth and young adult smoking,heavy alcohol consumption,and marijuana use.Despite the fact that use is higher in areas where there are more retail outlets, marijuana regulatory regimes in the four US states have failed to implement licensing systems to control retail density in ways that would protect vulnerable populations . Similar to tobacco and alcohol outlets marijuana businesses appear to be concentrated in low-income, minority communities. By 2016, Colorado marijuana businesses were more likely to be located in census tracts with higher proportions of racial/ethnic minorities , lower proportion of young people, lower median household incomes , higher crime rates, and higher concentrations of alcohol outlets .Similar findings were observed in California neighborhoods with medical marijuana dispensaries.Research on US state implementation of retail marijuana laws has focused on potential impacts of these laws on risk perceptions,use,health harms and stakeholder participation in the regulatory process.There is only a limited literature on the impact of marijuana policies on use and associated harms from the experiences in the Netherlands,Uruguay,and the United States.However, variability in US state medical marijuana laws makes it difficult to make strong generalizations, which likely explains why there is no scientific consensus on how legalization will impact risk perceptions or use patterns.There is limited evidence on the complexities of how a policy is implemented and when it is implemented having a dramatic effect on health-related outcomes. It is important to consider perceptions of risk when assessing the public health impacts of marijuana legalization laws.