Hemp Growing

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Each of the three genes was cloned into separateepisomal vectors, transformed into A. nidulans, and the resulting transformants were grown on CD-ST agar plates . Following 5 days of growth in CD-ST, the sample media were extracted and analyzed by liquid chromatography-mass spectrometry . To obtain compounds for structural determination, we first optimized the culturing conditions to get high titers from shake flask culture. By examining the organic extracts from cells and media separately, we determined that most of the compounds were secreted into the media. We also observed that a spore inoculum size of 104 spores/mL led to the highest titers of the four compounds whereas inoculum sizes of 108 spores/mL and higher gave low production of target compounds . Notably, when the molecules were produced at high titers, A. nidulans adopted a morphology of globular pellets. As the titer drops upon increased inoculum size, A. nidulans grew as dispersed filaments . The unnatural pairing can compromise the performance of overall biosynthesis by a “ratelimiting” component such as Ti_OvaBC. Aside from the molecular recognition of the acyl chain by the SAT domain active site, a successful acyl chain transfer also requires complementary protein-protein interactions between HRPKS ACP domain and NRPKS SAT domain.Previous studies have shown that although for some non-cognate HRPKS-NRPKS pairs, acyl chain could occur and new products emerged without any protein engineering efforts,4×4 flood tray for others replacement of SAT domain is necessary to compensate for the otherwise undermined inter-domain communication.While the sequence identity between ACPs of Ti_OvaA and Ma_OvaA is 62%, the identities between NRPKS SAT of Ti_OvaB and Ma_OvaB is lowered to 48%.

Such moderate sequence identity between the SAT domains implies that the recognition sites between ACP from noncognate HRPKS and SAT can be weakened or even abolished. Therefore, to generate a combination that exclusively produces 3 at a higher titer, protein engineering endeavors that improve the compatibility between unnatural HRPKS and NRPKS enzymes are necessary. Alternatively, further genome mining of related clusters may lead to one that can produce olivetolic acid robustly in a heterologous host. In summary, we have discovered a novel platform to produce OA and its analogs from filamentous fungi. The platform consists of an HRPKS and an NRPKS, known to produce resorcylic acid moieties in tandem, and a separate TE enzyme. This platform represents a new strategy to produce these cannabinoid precursors in microbes without relying on the OAS and OAC found in Cannabis sativa.Small quantities of olivetolic acid are produced in Cannabis sativa and chemical synthesis of the compound has proven to be difficult. Therefore, we hypothesized utilizing a Design of Experiments approach on our novel platform can further increase titer and thereby effectively solve the issue of low production. With regards to increasing production of fungal secondary metabolites and enzyme expression, DOE has proven to be an effective tool. DOE has been used for increased secondary metabolite production in bacteria, increased lipase production in fungi, increased xylanase production in fungi, and increased lignocellulolytic production in fungi, amongst other uses. We therefore sought to utilize a DOE approach to optimize the olivetolic acid and olivetolic acid analogs’ titer produced by our novel platform. DOE has proven to be more effective than one-factor at a time analysis because it greatly reduces the number of experiments needed and considers the interactive effects that factors can have with each other. The DOE approach begins at screening and ends at optimization and predictive modeling. First, screening is done to identify the factors that are most significant in the parameter response.

We decided to focus our DOE approach on the media that our A. nidulans strain producing olivetolic acid and its analogs is cultured in. Since our DOE approach is focused on media, we tested the effects that nine different components have on the production of sphaerophorolcarboxylic acid, olivetolic acid, and the analogs. Utilizing JMP statistical software, we performed an initial screening run of 24 experiments in order to identify the 3-5 most significant facts. To perform the screening experiments, we had to decide between a variety of screening platforms provided by JMP. JMP offers two types of screening designs: classical screening designs which include fractional, factorial, Plackett-Burman, regular fractional factorial, Cotter, and mixed-level designs and main effects screening designs: screening designs focused on measuring main effects with negligible interactions between the factors. We opted for a classical screen design because we wanted to include the interactions between factors and considered the different types from there.Two-level full fractional factorial designs account for all the combination of the factor levels. Two-level refers to a high value and a low value for the factor tested. The total number of runs for this design is the product of the factor levels. For example, for a two level full fractional design, the total number of runs would be 2n where n is the number of factors tested. The design also estimates that all of the effects are uncorrelated and that there are no interactions between the factors, i.e., the factors are orthogonal to each other.From a screening point of view, we determined the full fractional factorial design to be inefficient and cumbersome to do since it would require us to generate 29 types of media to test. The next classical screening design to consider was two-level regular fractional factorial.Two-level regular fractional factorial designs are similar to full fraction factorial designs; however, instead of the number of runs being equal to 2n where n is the number of factors tested, regular fractional factorial design runs are equal to 2n-k where k<n. In other words, a two-level regular fractional factorial is just a fraction of the full two-level fractional factorial design and therefore like the full factorial design, considers all the factors to be orthogonal to each other.

Since we could determine the fraction we would use, this was seen as an adequate design to choose; however, we wanted to consider some interactions between factors we determined to not follow through with this design and next looked at Cotter designs. Cotter designs are useful due to the ability to test a large number of factors in a small number of runs and are also useful if one is interested in the interaction between factors. Cotter designs are upheld by what is known as the principle of effect sparsity. These designs operate under the assumption that if one of the components of the sum of factors has an active effect , the sum of the factors will display the response.However, this can be potentially misleading and lead to false negatives if for example, one factor has a positive effect on the response, while the other factor has a negative effect, therefore totaling the sum of the factors to be zero/near zero, and therefore failing to show an effect. We determined not to go through with this design due to the false negative risks. We proceeded then briefly to mixed level designs. Mixed level designs are typically used when screening categorical or discrete factors containing varied factor levels.For example, one can be screening for the effect that light and a four-level media component have on the titer of a metabolite. Since we desired to keep the screen simple with just two levels for our factors since and we did not have any qualitative factors,hydroponic tray we proceeded to our last design, the Plackett-Burman design. Plackett-Burman designs are somewhat like regular fractional factorial designs except the total number of runs are a multiple of four rather than a power of two. Additionally, interactive effects between factors in a Plackett-Burman design are only partially confounded by the main effects which differs from regular fractional factorial where the interactive effects are completely confounded by the main effects and are therefore indistinguishable from each other.Plackett Burman designs are typically utilized when testing for the main effects among a variety of factors and so with this in mind, we chose to utilize the Plackett-Burman design as our screening design. We implemented the two-level Plackett-Burman design and generated 24 runs to test, with each run being a different media composition. For the two levels , we determined these values to input: temperature , pH , starch , NaCl , dextrose , yeast extract , casein acidic digest , trace elements , and 20x nitrate salts . We cultured the strain in these different medias, assaying the titer in media sets of 4 with CD-ST as the control media in each run. We inputted the data into the JMP software and performed analysis of variance and generated a Pareto chart from ANOVA and noted the results. ANOVA is a widely used tool to determine if there are statistical differences between means of different groups.

It is a collection of different statistical models and is used to determine whether the variance of a specific effect or factor interaction is statistically significant.The Pareto chart puts the ANOVA data in a simple to understand form, displaying whether the factor or factor interaction has exceeded the t- value limit and Bonferroni limit. The t-value refers to the value of the difference relative to the variation of the data tested. It is a value that represents the ratio of the difference between the estimated value of factor and its hypothesized value to its standard error.126 The Bonferroni limit is the value from the Bonferroni method that answers which factors means are significantly different from each other. Factors and factor interactions above the Bonferroni limit indicate that they are statistically significant and have a great effect on the parameter response, factors and interactions between the t-value limit and Bonferroni limit are indicated as potentially significant, and factors and interactions below the t-value limit are noted as insignificant.Based on the Pareto chart, we noted that increased temperature, addition of dextrose, addition of yeast extract, and addition of NaCl all had values above the Bonferroni limit indicating that these factors significantly affected the titer. However, three of values were all labeled blue indicating a negative result. Increased nitrate salts which had a negative effect on the media based on its blue distinction had a t-value greater than the t-limit but lower than the Bonferroni limit whereas increased casein acidic digest and increased starch which were also labeled blue had t-values lower than the t-limit indicating that these factors are not statistically significant. For factors having a positive effect on the titer labeled orange, NaCl salt had a tvalue greater than the Bonferroni limit indicating its statistically significance and increased nitrate salts had a t-value lower than the t-limit indicating it is not significant. From the screening data, we proceeded with two factors for our optimization experiments: NaC1 and nitrate salts. Although it was tabulated as not significant, we chose to include nitrate salts mainly because we did not want to optimize just one factor and since nitrate salts were already included in the media. We also included addition of MgSO4 since that was noted in the literature to increase metabolite production.Once we obtained the three factors, we utilized the response surface methodology optimization approach for optimization of the media, seeking to add these factors to our CD-ST media containing starch, trace elements, and casein acidic digest as the base since these factors had no statistical significance towards the titer. RSM is a widely used method for modeling/predictive modeling.RSM optimizes the factors correlating to a response with the inclusion of the effects interactions between the factors have. RSM has proven to be just as effective in modeling as a 3-level full factorial design, but its advantage is that RSM greatly reduces the number of experiments needed to form an accurate model. As an example, in a 3- level factorial design utilizing 3 factors, one would need to do 27 experiments to get an accurate model as opposed to the 15 experiments one would need for a central composite design RSM approach. As the number of factors increase, the difference between the number of experiments needed for a 3-level full factorial design vs a central composite design RSM approach greatly increases, which is why RSM is the advantageous approach. Reports of the effectiveness of increasing secondary metabolite production in fungi with the RSM approach have been recorded. Talukdar et al. observed a 7-fold increase in antibiotic production of Penicillium verruculosum MKH7 utilizing an RSM approach on the media used.

Under these conditions the integrated system was able to produce cold and dehumified air that is in the safe range for server racks. In this case the return air from server is considered for first stage cooling air which has lower relative humidity and higher potential for evaporative cooling.However, the system is able to provide only 97CFM of air flow under these conditions, which is less than the maximum air flow demand for each server rack. This suggests that the current integrated SOFC-LDD system cannot continuously provide all the power and cooling required for the server rack if it is operated continuously at 12kW. However, both the server demand dynamics and day-night weather variations, could lead to dynamic conditions that with some storage of the concentrated solution could be well-matched to the server demands for power and for cooling and dehumidification. Next step will consider these dynamics for several real-world weather and server power. For the second weather condition with 35˚C and 45%RH the system can produce 96CFM cold and dry air for each server rack. In this case the outside air is considered for the first stage cooling air. Please note that the SOFC system is running based upon design operating conditions for the system. Further investigation is needed to evaluate the SOFC system available heat under different operating conditions that could produce higher quality exhaust heat likely at the expense of electrical efficiency. The results are presented for two scenarios; in first scenario, liquid desiccant provides dehumidified air for one single server rack for the entire year,indoor weed growing accessories the fuel cell runs at full load, and the dehumidifier is sized based on server rack, and the regenerator is sized based on FC exhaust.

In second scenario, liquid desiccant provides dehumidified air for two rows , Fuel cell runs at 70% load, dehumidifier is sized based on cooling required for two rows, and regenerator is sized based on equivalent of two rows fuel cell exhaust. The reason for row level fuel cells running at 70% and in rack level running at 100% is that when fuel cell provides power for only one rack, it is more likely that the single server rack runs at full load, however, when several fuel cells provide power for 20 racks, the load of serves will be balanced to the data center overall utilization .Figure 64 to Figure 69 show dehumidifier results for Illinois. In dehumidifier, two air streams are send to the system, one is the supply air which is a mixture of outside air and return air and the second stream is the cooling air that is send to the dehumidifier to keep the temperature constant by evaporative cooling. Figure 64 and Figure 65 show the supply air and cooling air inlet and outlet temperature. The temperature of the streams stays almost the same even though the dehumidification is an exothermic process, which is due to the indirect evaporation of water to the second air stream. Figure 66 shows the amount of return air. For Illinois during May to September dehumidification is needed. Other time of the year the humidity of supply air is within the acceptable range. As the outside air humidity is higher the use of return air increases due to its lower humidity compared to outdoor air. However, the return air percentages depend both on temperature and humidity of outdoor air and return air to keep the supply air humidity within the acceptable range for servers. Figure 67 shows the humidity ratio of inlet and outlet supply air. The SOFC systems installed at NFCRC are BlueGEN cogeneration systems. BlueGEN is a commercially available CHP unit, built and sold by SOLIDpower. Figure 87 shows the eight BlueGEN units as installed in the NFCRC laboratory.

Operating on natural gas, each unit can produce power modulated from 500We to 2kWe ; however, it achieves its highest net electrical efficiency of 62% at a 1.5kWe output. The BlueGEN systems are typically operated and controlled remotely by SOLIDpower. However, through an online human machine interface, the power output profile and as a result fuel utilization are controllable by the user. To be able to install the BlueGEN we need to provide six connections: fuel , electrical, flue gas, internet, water, and drain connections. BlueGEN requires an uninterrupted supply of natural gas, and it only operates on the second family of gases . Gas supply pressure to the BlueGEN appliance must be 9–20mbar so an Elster Jeavons gas pressure regulator is used to avoid fluctuations due to variations in the main gas supply pressure.The BlueGEN systems require an electrical connection to successfully operate during startup and to export the electricity produced during normal operation. For electrical connection the grid availability is 208VAC 3 phase, 60 hertz. Each fuel cell needs 120VAC 1 phase, 60 hertz. 3 transformers are used where two of them provide electrical connection needed for six BlueGENs and one of them runs two BlueGENs. The main water supply pressure must be at least 1bar and no more than 10bar . During operation, the appliance can consume up to 30litres of water per day, depending on heat and condensate recovery. The BlueGEN appliance has an internal water storage tank for the process water and this tank will fill from the main water supply intermittently. The water consumption rate may be up to 1 liter per minute when this tank is filling. Operation of water needs to be uninterrupted. Wastewater is ejected from the BlueGEN appliance under pressure . The main water supply connection, drain, and overflow rejection connection configuration can be seen in Figure 88. Water is distributed in a manifold to each fuel cell line and each fuel cell has its own valve for water shut down.

For water rejection line, overflow and wastewater will be collected in a tank through a sloped pipe and then will be pumped to the drain. Figure 89 and Figure 90 show the pump used for fuel cell water rejection connection, manifold, and valves for fuel cell supply water, respectively. Looking at SOFC dynamic data, it is observed that BlueGEN electrical efficiency is the highest, roughly 64%, at full load. BlueGEN efficiency is the lowest at 30% at 300W of output . The BlueGEN system is recommended to run between 1500W and 500W. At the lowest recommended power, the efficiency is roughly 43%. As expected, at loads lower than the nominal power current density drops, and because of lower polarization, voltage increases. Experimental results show that BlueGEN control system is designed to keep air flow constant and that it changes the fuel utilization and fuel flow proportional to the output power. BlueGEN stack temperature difference is designed to stay at 50℃ at full load. The experimental results show that by reducing the load, the temperature difference decreases. The exit temperature drops below the inlet temperature at 700W. At low current density the endothermicity of reformation reaction is higher that exothermicity of electrochemical reaction which causes temperature drop along the cell. Note that while electrochemical efficiency increases at part-load, fuel utilization is lowered to maintain the overall thermal balance so that the overall system efficiency drops at part-load conditions. At these part-load conditions the fuel cell stack operates at overall endothermic conditions, which must be matched by heat from the thermal oxidizer that converts the unspent fuel at the lower fuel utilization conditions.In another dynamic test,rolling benches the BlueGEN system was tested through the following profile shown in Figure 95 in 24h for 42 days. During this cycle, the power decreases between 1500W to 500W with 100W increments. In each power step the system is given 1 hour to reach to steady state and then it has 15 minutes to ramp down to the next power level. At this writing the BlueGEN system has been running for more than 6000hr from the time of installation at the NFCRC. Figure 104 shows the power profile of BlueGEN system over this 6000h operating period. After 4000 hours of steady-state operation the system runs the first dynamic load Figure 93, then after a couple of days of steady operation it goes through the 24h dynamic load cycles for 1000hr followed by the one-week dynamic cycle. Figure 105 shows the voltage change over the 600hr operation. The system operation is divided to three region and Table 22 shows the degradation results for each of the regions of operation. During the first 1000hr of operation as the system is new the degradation rate is twice that of the regular steady-state operation. Also, the system degrades twice as fast as steady-state operation while operating under highly dynamic operating conditions.

As the system degrades, the fuel consumption increases and as a result the overall system efficiency drops. For cell level tests, experiments were carried out using a commercial anode supported solid oxide cell. The cell anode side consists of two layers, a thick Ni-YSZ anode support acting as the mechanical support, current collector and gas diffusion layer, and the other is the functional layer with a more dense structure near the electrode-electrolyte interface . The electrolyte consists of 8% mol Y2O3-ZrO2 on which Gadolinium Doped Ceria oxide barrier layer is deposited to prevent the formation of insulating SrZrO3. The cathode active layer was made up oflanthanum strontium cobalt ferric oxide in contact with the cathode current collector of similar thermal expansion. The cell had an active area of 12.6cm2 . The experimental setup of the single cell used for each of the three 1000hr tests is shown in Figure 107. The furnace keeps the temperature at constant. The JV scan and EIS measurements were performed with a Zahner Zennium impedence analyzer. Zanher positive terminal connects to the cathode and the negative terminal to anode. The voltage response is also measured via the same system. During EIS measurements constant loads of 0.1, 0.5, and 0.9A/cm2 were applied to the cell. For applying constant loas of 0.5A/cm2 during the 1000hr polarization a load box is connected to the setup. The fuel cell air and fuels are supplied by Voegtlin Red-y smart series mass flow controllers. The purity level of hydrogen, nitrogen and ammonia used in the experiments are 99.999%, 99.999% and 99.99% respectively. Nitrogen and hydrogen are stored as compressed gases while ammonia is stored in liquid form in commercial cylinders.The single-cells were tested with three different fuel composition as shown in Each test runs on a pristine single cell SOFC. Cell 1 on pure H2, cell 2 on H2-N2 case, and cell 3 on NH3 directly, which includes the internal decomposition reaction . A nominal operating temperature of 750°C is chosen for the tests. After mounting each cell, each SOFC cell is reduced in H2 and N2 atmosphere. Each cell runs for 1000hr at 0.5A/cm2 to compare long term effect of different fuel on SOFC degradation. Dynamic current density – Voltage scans were performed every 100hr from Open Circuit Voltage to 700mV at 5mV/s with 1mV resolution. The JV scans are followed by three EIS measurements at 0.1, 0.5, and 0.9A/cm2 and 200mA perturbation signal for a frequency range of 1KHz to 200KHz. The quality of EIS spectra was verified using the Kramer Kronig’s test. The overall test duration of test for each cell was 47 days. Table 23 to compare the degradation effects between in-situ and ex-situ ammonia reforming with that of pure hydrogen for a duration of 1000h. Each test runs on a pristine single cell SOFC. Cell 1 on pure H2, cell 2 on H2-N2 case, and cell 3 on NH3 directly, which includes the internal decomposition reaction . A nominal operating temperature of 750°C is chosen for the tests. After mounting each cell, each SOFC cell is reduced in H2 and N2 atmosphere. Each cell runs for 1000hr at 0.5A/cm2 to compare long term effect of different fuel on SOFC degradation. Dynamic current density – Voltage scans were performed every 100hr from Open Circuit Voltage to 700mV at 5mV/s with 1mV resolution. The JV scans are followed by three EIS measurements at 0.1, 0.5, and 0.9A/cm2 and 200mA perturbation signal for a frequency range of 1KHz to 200KHz. The quality of EIS spectra was verified using the Kramer Kronig’s test.

Adolescent substance users have also been found to exhibit abnormalities in brain function, structure, and volume. However, given the nature of human studies, it is difficult to establish a causal link between early life exposure and the development of these conditions, especially as drug co-use is not often considered and may partially explain inconsistent findings noted in prior studies. Nicotine acts in the brain via the neuronal nicotinic acetylcholine receptors, which are ligand-gated ion channels expressed on both presynaptic and postsynaptic membranes. Rodent models have shown that adolescent nicotine exposure alone may lead to behavioral alterations during adulthood. For instance, in male and female rats, adolescent nicotine enhances nicotine reward and intake during adulthood. Nicotine during adolescence has also been shown to increase depression-associated behaviors, decrease exploratory activity, and induce deficits in context conditioning to shock-associated cues in adult rats. However, in these studies, differences were not found with anxiety associated behaviors, extinction of contextual conditioning, or cued fear responses. In mice, sex dependent effects have been noted, with adolescent nicotine consumption leading to decreased anxiety-associated behaviors in adult females, but not males. With regard to cannabinoids, Δ9-tetrahydrocannabinol has been classified as the main psychoactive component in cannabis and exerts its actions on cannabinoid 1 and cannabinoid 2 receptors in the brain and periphery. Differential patterns of expression for these receptors are found across adolescent development and between males and females,drying room and notably CB1 receptors exhibit the highest level of expression during the developmental period of mid-adolescence.

Following THC administration in adolescence, adult female, but not male, rats exhibit depression-associated behaviors, but no changes in anxiety-associated or general locomotor behaviors were observed. Interestingly, the depression-associated behavioral effects found in females were paralleled by significantly reduced CB1 receptor expression and activity in the amygdala, ventral tegmental area and nucleus accumbens, whereas similar changes were not found in the ventral tegmental area and nucleus accumbens of males. Further, administration of WIN 55,212–2, a CB1 and CB2 specific agonist, during adolescence has similarly been shown to increase depressive-like behaviors, as well as palatable food intake, during adulthood in male rats. Together, these prior findings demonstrate that early life exposure to either nicotine or cannabinoid agonists alone can alter later affective and cognitive function, which introduces the possibility of potential synergistic or opposing effects under co-use conditions. In the current studies, we sought to examine whether nicotine and cannabinoid coexposure during mid-adolescence would result in altered affective and reward-seeking behavior during adulthood. While prior studies have examined each drug and/or behavioral measure independently, the current investigations represent the first study of a co-exposure condition, which is commonly found in human subjects, and the resulting effects on multiple cognitive and affective measures. To this end, adolescent mice were exposed to the cannabinoid receptor agonist, WIN55,212–2, and/or nicotine and then assessed for cognitive, anxiety-related and depression-related behaviors during adulthood.

Drug exposure occurred during postnatal day 38–49, which corresponds to mid-adolescence in rodents or ~13–17 years of age in humans. Based on prior evidence of differential responses for males and females with drug-related effects and baseline receptor expression, male and female mice were examined in a within-sex manner. Further, given that significant differences were found in behavioral measures at the moderate dose of the cannabinoid agonist, a second study was then conducted to examine whether these effects would be maintained with a lower dose of the cannabinoid agonist. Together, these studies provide evidence that adolescent drug exposure alters affective and reward-related behaviors during adulthood in a sex- and drug-dependent manner.Male and female wildtype C57BL/6J mice were derived from breeders in our laboratory animal facilities. Mice were maintained in an environmentally controlled vivarium on a 12 h reversed light/dark cycle. Food and water were provided ad libitum until behavioral training commenced. During food training, subjects were mildly food restricted to 85–90% of their free-feeding bodyweight, and water was provided ad libitum. Following food training and the lever reversal task, food and water were again provided ad libitum for at least 5 days prior to subsequent behavioral assessments. All experiments were conducted in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at the University of California, Irvine.The cannabinoid receptor agonist WIN55,212–2 mesylate was dissolved in vehicle containing 1% DMSO, 1% Tween-80, and 98% saline . The doses of WIN55,212–2 administered were 2 or 0.2mg/kg intraperitoneally .

The moderate dose of WIN was selected based on prior studies demonstrating altered neural function with adolescent exposure in mice and rats, and the low dose of WIN was selected since this amount of drug has been shown to sustain daily reinforcing self-administration behavior in adolescent rats. -Nicotine hydrogen tartrate salt was dissolved in 0.9% sterile saline and adjusted to pH 7.4. Nicotine was administered at a dose of 0.36 mg/kg, subcutaneous ; this dose is within the rewarding range of the dose response function that also elicits a behavioral response in adolescent C57BL/6J mice. Peripheral injections were administered at a volume of 10 mL/kg.On PND 70, subjects were mildly food restricted and trained to press a lever in an operant chamber for food pellets under a fixedratio 5, time out 20 s schedule of reinforcement. Each session was performed using 2 retractable levers . Completion of the response criteria on the active lever resulted in the delivery of a food pellet. Responses on the inactive lever were recorded but had no scheduled consequences. Once stable responding was achieved , the lever assignment was switched to examine cognitive flexibility. In the reversal task, the previous inactive lever became active, in that food pellets were earned in accordance with the established FR5TO20s schedule. In contrast, the previously active lever became inactive, in which responses were recorded but without scheduled consequence. All behavioral responses were automatically recorded by Med Associates software.The elevated plus maze was composed of 4 opaque runways 5 cm wide and 35 cm in length, which were elevated 40 cm from the floor. Two opposing closed runways had opaque walls 15 cm in height, whereas the other two opposing sides did not contain walls . Subjects were placed in the center portion of the elevated plus maze and behavior was recorded for 5 min with a video camera. Behavior was scored by two blinded experimenters with ANY-maze software.Subjects were habituated to sucrose pellet consumption for 2 days prior to sucrose testing, during which time 60 mg of sucrose pellets was provided for each subject in the home cage. On the third day, subjects were individually examined in home cage conditions, but were single housed and provided 200 mg of total sucrose pellets in a dish. All subjects were maintained under ad libitum full food conditions, and thus were not food restricted during testing. Sucrose eaten was recorded at specified intervals by experimenters blinded to the group condition. At the end of each session, experimenters examined the cage for breakage or disintegration of sucrose pellets; this occurred on only a few occasions and in these instances, the remnant amount was calculated and included in the final mg amount of sucrose remaining. Mice were required to consume at least one 20mg sucrose pellet within the first 30-min time period for inclusion in the study.Subjects were examined for their daily intake of mouse chow. Mice were restricted to daily feeding sessions of 6 hr periods. During these sessions, subjects were individually housed and provided full access to consume 6–8 grams of standard chow , and water was provided in the feed cages ad libitum. Food was weighed prior to and after each session. After 3 days of habituation to the feeding protocol, data were collected on the fourth day and analyzed across groups.A cylindrical tank was filled with room temperature water at a level of 15cm from the bottom of the tank. For testing, each subject was held by the tail, and slowly placed in the water. Mice were videotaped for the 5 min swim test duration. Analysis of distance traveled was assessed with AnyMaze software,how to trim cannabis and the quantity of immobility bouts was hand scored by two separate experimenters to ensure accurate assessments.Given that these studies sought to investigate the effects of drug exposure relative to the control condition within each sex, statistical comparisons were performed separately for males and females based on this a priori hypothesis. Data were analyzed by a t-test, one-way or two-way ANOVA with Prism 7 software , as appropriate. Data obtain across sessions was analyzed with a repeated measures two-way ANOVA. Significant main or interaction effects were followed by Bonferroni post-hoc comparison with correction for multiple comparisons.

The criterion for significance was set at α = 0.05.In an initial cohort, we assessed whether drug condition would affect change in body weight during the duration of the drug injections from postnatal day 38 to PND 49 . Change in body weight was also compared to adulthood at PND70, prior to the commencement of behavioral assessments. Groups did not differ in body weight at PND 38 following random group assignment.For both males and females, post-hoc tests revealed significant differences between the number of active and inactive lever presses for all groups from sessions 3–7, but the groups did not differ from one another when comparing responding among drug conditions on each lever. After establishing consistent responding on the active lever, cognitive flexibility was examined in the reversal task. Subjects were required to switch their lever pressing behavior, as the active and inactive lever assignments were reversed. After establishing consistent responding on the active lever, cognitive flexibility was examined in the reversal task. Subjects were required to switch their lever pressing behavior, as the active and inactive lever assignments were reversed. Given the growing incidence of nicotine and cannabis experimentation during adolescence, we sought to examine whether such exposure would lead to altered behavioral effects during adulthood. In these studies, we found that male adolescent exposure to a moderate dose of the cannabinoid receptor agonist, WIN55,212–2 , led to increased cognitive flexibility in a learning reversal task, decreased anxiety-associated behaviors, and increased natural reward consumption, but no differences in general locomotor or depression-related behavior. Interestingly, the co-exposure condition of both nicotine and the moderate dose of WIN led to similar behavioral profiles as WIN alone in these measures, suggesting that a potentiative or additive effect was not present for these behaviors. However, with regard to the number of lane crosses in the elevated plus maze, the nicotine and WIN co-exposure condition appeared to exert a counteractive effect on the WIN-induced increase in exploratory behavior at the moderate dose, suggesting an opposing effect with adolescent exposure to both drugs. With regard to females, the moderate dose of WIN induced a lower body weight during the adolescent period, but co-exposure with nicotine appeared to exert an opposing effect that resulted in no difference from the control condition. However, these effects of WIN on body weight were transitory, as the difference in females did not persist into adulthood. For the behavioral assessments, female subjects were overall more resistant to the long-term effects of adolescent drug exposure. Group differences were only found in the sucrose consumption test, in which the moderate dose WIN females exhibited decreased natural reward consumption compared to the control females. However, differences from the control were not found with the female nicotine and WIN co-exposure condition for sucrose consumption, suggesting that the presence of nicotine ameliorated the actions of WIN on reward circuitry during the adolescent period. In contrast, adolescent exposure to a low dose of WIN had no effect on physiological or behavioral measures, either alone or in the presence of nicotine, for both males and females. Taken together, these findings demonstrate that while adolescent cannabinoid agonist exposure at a moderate dose exerts variable effects on both physiological and behavioral measures in males and females, co-administration of nicotine surprisingly counteracted some of these effects by normalizing to control levels. While prior studies have examined the effects of adolescent exposure of either nicotine or WIN alone on later behaviors, the current findings represent the first examination of the effects of co-exposure during mid-adolescence and subsequent long-term effects on adult behavior. This age range was selected based on the correlation to human adolescence with higher levels of experimentation and more recurrent patterns of drug consumption than that found in younger individuals. With regard to nicotine alone, opposing effects have been found in male Sprague-Dawley rats with increased depression-associated behaviors, but no difference in anxiety-associated behaviors, during adulthood. However, these behavioral differences were only found at higher nicotine doses approximately twice that administered in the current study.

The San Francisco Medical Cannabis Act sets up a permitting system for MCDs and places certain restrictions on their location and operation. Speaking directly to the issue of crime, Section 3308 of the Act states that dispensaries must “provide and maintain adequate security on the premises, including lighting and alarms reasonably designed to ensure the safety of persons and protect the premises from theft.” The Act further requires that all MCDs comply with California state law as well as guidelines written in 2008 by then-Attorney General Jerry Brown entitled “Guidelines For The Security And Non-Diversion Of Marijuana Grown For Medical Use”. Brown’s guidelines state that “a properly organized and operated collective or cooperative that dispenses medical marijuana through a storefront may be lawful under California law” if they meet certain requirements, including: operating on a not-for-profit basis, obtaining all of the relevant permits and licenses, taking steps to verify that their members are qualified patients or caregivers under state law, acquiring and distributing only marijuana that has been cultivated legally, prohibiting sales to non-members, and providing adequate security . Regarding security, Brown states that MCDs must “provide adequate security to ensure that patients are safe and that the surrounding homes or businesses are not negatively impacted by nuisance activity such as loitering or crime.” Expanding on this theme, the San Francisco Medical Cannabis Act prohibits “any breach of peace… or any disturbance of public order or decorum by any tumultuous,cannabis grow room riotous or disorderly conduct” within permitted MCDs.MCDs are required to submit security plans as part of their permit application.This study examines whether such security protocols amount to capable guardianship, which is an effective deterrent against crime according to routine activities theory .

In cities that do not regulate MCDs, it is difficult to determine when and where dispensaries operate, and for how long. It is also much more difficult, in the case of unregulated dispensaries, to infer whether MCDs implement security protocols amounting to capable guardianship against crime, from a routine activities perspective . Simply put, San Francisco is the largest California city to have enacted meaningful legislation with respect to MCDs. It has done so in a way that reasonably controls for crime, at least in theoretical terms. Thus it provides an excellent case study for analyzing the spatial relationship between crime and locally regulated MCDs. All data analyzed in this study are for the year 2010, a year in which there were 26 permitted MCDs in San Francisco. Although it is too early to say, it is possible, due to the current federal crackdown on MCDs in California and MCDs, that 2010 will end up being the last full year for which this type of analysis can be conducted. Since research for this project began in the summer of 2011, at least ten Bay Area MCDs have closed, including five in San Francisco, under pressure from Melinda Haag, the US Attorney for the Northern District of California.Not surprisingly, Haag’s justification for the crackdown is that MCD’s attract crime and endanger communities. Crime data were collected from the San Francisco Police Department in late 2011. The Crime Analysis Unit provided lists of serious crimes reported in 2010 along with the date and approximate location of each crime. Here, “serious crimes” refer to those classified as Part I offenses by the Federal Bureau of Investigation in its Uniform Crime Reports. The crime variables used in this analysis include measures of “violent crime” as well as “property crime” ; both as total counts and as rates per 1,000 residents. The lists of crimes and addresses were geocoded and aggregated into census tracts using ArcGIS software.Geocoding refers to the process by which tabular data are attributed spatial components by a geodatabase.

Geocoding resulted in a successful match for more than 98% of all crimes, which were the aggregated into census tracts using a “spatial join” analysis. The remaining 1-2% of crimes were discarded from analysis. In addition, some reported crimes were removed from analysis because their geocoding confidence ratings were below 95%. In the end 43,688 reported crimes were analyzed out of the original 44,422 for which the San Francisco Police Department provided 2010 data. For the purpose of analysis, these crime measures were aggregated together by census tract. This measure was then transformed by natural logarithm to address a right-skewed distribution . The primary independent variable under examination is the density of MCDs. Lists of MCD names and addresses were compiled using information provided by the San Francisco Department of Public Health. These MCDs were located across 16 census tracts primarily in the downtown area, as illustrated by Maps 4.1 and 4.2. The MCD addresses were geocoded to 100%. As with crime frequency, data for MCDs are presented in two forms. Descriptive statistics presented in Table 4.1 include MCD density as the number of dispensaries per square mile in a given census tract. For the regression analyses presented by Table 4.2, this variable is transformed by natural logarithm to address a right-skewed distribution.Crime rates by census tract are presented by Maps 4.1 and 4.2 . All data presented are for the year 2010. MCD locations are marked by green crosses. These addresses were obtained from the San Francisco Department of Public Health. Crime rates were calculated using data obtained from the San Francisco Police Department. Census tracts are assigned to one of five classes based on their crime rates. Because crime rates were transformed by natural logarithm to address a right-skewed distribution, units are not given. As Maps 4.1 and 4.2 illustrate, MCDs are largely concentrated in downtown San Francisco. This could confound the relationship between MCD density and crime. Downtown areas are densely populated and highly trafficked. In terms of routine activities theory , they contain larger numbers of likely offenders and suitable targets.

The high rate and volume of human activity also poses a challenge in terms of guardianship. Thus it is likely that these areas will have high rates of crime, independent of any other factor . This potentially confounding factor highlights the need to consider other variables in the forthcoming analysis, namely, the “exogenous sources of social disorganization” identified by Sampson and Groves . The story told by Maps 4.1 and 4.2 is too simple to be of use to policymakers wishing to understand the relationship between MCDs and crime. Alongside MCD density, it is also important to analyze socioeconomic disadvantage, family stability, and residential turnover. I discuss these factors in the following section.In addition to the primary variables already discussed, data were also collected for several neighborhood characteristics that could potentially confound the relationship between MCDs and crime. These neighborhood characteristics are drawn from social disorganization theory, which associates higher rates of crime with socioeconomic disadvantage, family disruption, residential instability, and population heterogeneity . From these, the present study examines the criminogenic effect of poverty, unemployment, percent of single-parent households, percent of housing units that are vacant, and percent of the population between the ages of 18 and 24. Demographic data are collected from the American Community Survey database of the United States Census Bureau via the American FactFinder website, as well as the Demographic Research Unit of the California Department of Finance. With regard to the census data, variables are constructed from the ACS 5-year estimates for the year 2010.Criminological research has found that indicators of socioeconomic disadvantage—including poverty and unemployment—have been associated with higher crime rates . In the present study economic data are collected from the ACS. The U.S. Census Bureau calculates the poverty status of individuals based on whether their total income in the past 12 months falls below the applicable poverty threshold,grow trays which is determined by age, family size, and family composition . The 2010 poverty thresholds range from $11,139 for a single individual living alone to $42,156 for a family of eight or more people living in the same household.For the present analysis, “poverty” means the number of individuals with incomes under their applicable poverty threshold in the past twelve months, divided by the total number of people for whom poverty status is calculated within a given tract.According to the United States Census Bureau, an individual is considered unemployed if he or she did not have a job and has been actively looking for work during the last four weeks and was available to start a job at the time of the survey . “Unemployment”, in the present analysis, means the unemployment rate in each tract as estimated by the ACS.

Research examining crime rates in the United States during the 1990’s suggests that the job market can provide powerful explanations for criminal behavior . Poverty and unemployment are important measures in the model currently being tested, as they control for varying levels of socioeconomic disadvantage across city neighborhoods, which according to social disorganization theory affect crime rates in significant ways .In this study I use “family stability” as an inverse measure of family disruption. I calculate family stability by taking the number of individuals living in married couple family housing and dividing it by the number of people living in single-parent family households. Scholars of both routine activities theory and social disorganization theory predict that higher concentrations of married-couple families are associated with lower crime rates in urban areas, because more parents can provide more supervision and therefore more social control. From a routine activities perspective, both “family stability” and “residential stability” correspond with the notion of capable guardianship. According to social disorganization theory, residential turnover weakens a community’s social cohesion and therefore its ability to deter and prevent crime within its territory. In this study I use vacancy rates as measure of residential instability. I calculate “vacancy” by dividing the number of vacant housing units within a census tract by the total number of units within that tract. Data for this measure comes from the Census 2010 Redistricting Plan.Another indicator of residential turnover discussed in the criminological literature is the percent of the sample population that is young . The idea is that neighborhoods with a high concentration of young adults will have correspondingly fewer older adults and children, which results in a lack of social cohesion and crime-preventive capacity much in the same way as the other precursors of “social disorganization” already discussed. The variable “percent young” was constructed using ACS population estimates by dividing the total number of individuals between the ages of 18 and 29 by the total tract population.Although they are not of particular theoretical interest, the following measures are included as demographic control variables: population size, percent of the population that is male, and percent of land that is commercially zoned. Population size and gender composition are adapted from the 2010 Census. “Percent of land commercially zoned” was calculated in ArcGIS using zoning shape files provided by the San Francisco Planning Department. Tables 4.1 and 4.2 present descriptive statistics for the measures analyzed in this study. Here the crime data are provided as total counts by category, but in the regression analysis that follows the crime variables are transformed by natural logarithm to address a right-skewed distribution . All other variables are presented as described in the previous section. A total of 189 census tracts within San Francisco are analyzed using data for the year 2010. Five census tracts were removed from analysis because only partial data were available; these were low population tracks with no MCDs and therefore their loss is not analytically significant. Of the tracts analyzed, the average population size is 4,234. The average crime rate is 197.38 property crimes per year . The average violent crime rate is much lower: only 28.65 reported instances per year . According to the results of regression analyses presented in Table 4.3, the current model is better at explaining property crime than it is violent crime. The simplest explanation for this is that substantially more property crimes are committed on a yearly basis than violent crimes, as illustrated by Table 4.1 below on the following page.Descriptive statistics for the independent variables analyzed by this study are also presented in Table 4.2 These include “% Unemployed” and “% Under Poverty” as measures of socioeconomic disadvantage; “% of Housing Units Vacant” and “% of Population Ages 18-29” and measures of residential instability; and “% In Married-Couple Families” as a measure of family stability .

Potential transmission routes of SARS-CoV-2 from animals and animal products are shown in Fig. 2. While SARS-CoV-2 does not cause food borne illness, the virus has caused enormous disruptions to the global food supply chain. The risk of SARS-CoV-2 to food safety was detailed elsewhere . Animal product industries usually rely on the presence of metallic surfaces and the maintenance of low humidity and low temperature . However, these conditions can be favorable for the viability and transmissibility of SARS-CoV-2. While there was a significant reduction and exponential decay in the infectious titer, SARS-CoV-2 remained viable in aerosols for up to 3 h, on plastics for up to 72 h, and on stainless steel for up to 48 h after application . The viability of the virus on different environmental surfaces is shown in Table 1. In addition to rapid work speed and multiple long shifts, food industry workplaces tend to be very crowded with employees in close proximity to one another. Additionally, because of the loud mechanical noise, workers tend to talk louder or shout. These factors can increase the chances of the transmission of SARS-CoV-2 to the co-workers. In many industries, there is a significant number of younger employees. If the manpower in food industry mainly consists of youthful workforce, there can also be a high possibility of having an infected asymptomatic employee who could infect other employees. Infectious bronchitis is an avian disease caused by infectious bronchitis virus, a member of genus Gammacoronavirus in the family Coronaviridae, and is a major respiratory disease of poultry affecting lungs, kidney,drying cannabis and the reproductive tract and causing tremendous economic losses in the poultry industry worldwide . It causes multiple characteristic consequences such as kidney damage, decreased egg production, and deteriorated egg quality .

Another significant problem of infectious bronchitis is its rapid spread. Within 48 h, the whole flock can be infected and remains as a reservoir, even after recovery. The infection usually spreads horizontally from hen to hen, but not from hen to chick. It is still possible that the infection gets transmitted via contaminated eggshells in the hatcheries . Although chickens are not known to be infected with SARS-CoV-2 through the intranasal route, COVID-19 can still have economic negative impacts on the poultry industry . Different poultry species are susceptible to coronaviruses infections leading to the development of enteric diseases, respiratory diseases, and kidney diseases. Examples include turkey infected with Turkey coronavirus , quail infected with Quail coronavirus , guineafowl infected with Guineafowl coronavirus , and pheasant infected with Pheasant coronavirus .Porcine coronaviruses belong to four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and D . The porcine epizootic diarrhea virus and the transmissible gastroenteritis virus are alphacoronaviruses of swine which infect epithelial cells of the gastrointestinal tract. Another alphacoronavirus named the porcine respiratory coronavirus has no affinity to the gastrointestinal tract. A fourth alphacoronavirus is swine acute diarrhea syndrome coronavirus , which causes acute diarrhea syndrome in piglets. A betacoronavirus is porcine haemagglutinating encephalomyelitis virus , while a deltacoronavirus is porcine deltacoronavirus . Due to similarities in protein characteristics, swine can be potential reservoir for the transmission of SARS-CoV and SARS-CoV-2 . PDCoV is an emerging swine pathogen. Importantly, it was recently detected from three sick Haitian children and is now recognized as a zoonotic pathogen .Coronaviruses of ruminants include bovine coronavirus which infects cattle, sheep, goat, buffalo, llamas, alpacas, and can cause respiratory distress . Examples include bubaline coronavirus that infects water buffalo, alpaca coronavirus that infects alpaca, and dromedary camel coronavirus that infects camels .

In addition, MERS-CoV causes infections in respiratory tracts of camels. The seroprevalence of MERS-CoV in dromedaries was reported to be more than 90% in different countries in Africa, Asia, and the Middle East . There is not enough evidence to suggest that cattle might have play a role in the COVID-19 pandemic. In a study on six animals, only two cattle tested positive for the virus in nasal swabs and showed specific seroconversion, indicating low susceptibility of cattle to SARS-CoV-2 infection . However, close contact of infected humans with large numbers of cattle may still lead to anthropozoonotic infections in cattle . The reason for the low susceptibility to infection is the low expression of ACE2 in the respiratory tracts of these ruminants . The SARS-CoV-2 outbreak in December, 2019 has been linked to Huanan Seafood Market in Wuhan, China . A second wave in June 2020 has been traced to Xinfadi Seafood Market in Beijing, China . Frozen seafood items contaminated with SARS-CoV-2 have been reported in China . Salmon-attached SARS-CoV-2 stayed in a viable status for at least 8 days at 4 ◦C, and 2 days at 25 ◦C . It is noteworthy that at 4 ◦C is the temperature of refrigerators, cold rooms, and transport carriers for storage of fish before selling in the fish or seafood market. Thus, the import and export of frozen and refrigerated fish can be a source of transmission of fish-attached SARS-CoV-2 across countries and continents. Imported frozen cod package surfaces from China showed signs of contamination by SARS-CoV-2 . This calls for strict inspection measures for the detection of SARS-CoV-2 in imported and exported fish during the pandemic . Evidence suggests that cold-storage foods may present a risk for the transmission of SARS-CoV-2 between different countries . Ferrets and cats have been shown to be permissive to infections with SARS-CoV-2 .

Cats were also susceptible to airborne transmission . SARS-CoV-2 infection has also been reported in dogs, tigers, and lions . Other susceptible species include mice, golden hamsters, minks, and non-human primates . Clinical signs in ferrets involved fever and loss of appetite . Both cats and non-human primates were asymptomatic . The main features of animals experimentally infected with SARS-CoV-2 are presented in Table 2. The supply chain of animal feed raw materials has been negatively impacted due to animal movement restrictions resulting from the pandemic-triggered lock downs. Regular patterns of production, supply, and consumption were severely disrupted . Thus, farm animals have been deprived from important feed ingredients in their diets. It has been reported that more than dairy farms have faced shortages of dry feed intake in Pakistan as a result of the pandemic . International cessations in exports and imports of animals’ feeds hampered the supply of several basic raw ingredients that are important for raising and fostering livestock . These raw ingredients include mixtures of carbohydrates, proteins, fats, minerals, and vitamins. As an example, Argentina-the world leader in soybean meal exports – had to reduce its exports of soybean, a critical feed ingredient, by half into feed manufacturing factories . Similarly, Brazil and U.S. have also faced hurdles in their soy meal and corn exports . Local restrictions have been impacted animal feed ingredients, as evidenced by pastoralists in African dry lands who were unable to feed their animals, typically fed on natural plants . International and local restrictions have naturally led to increased costs of animal feed materials, which impacted animal farms in different countries. In Bangladesh, there has been a 3.7% hike in dairy feed price . In India and many regions in Africa, the prices of key animal feed ingredients have increased by 15% as a result of the pandemic . In the United Kingdom, the prices of soy meal, wheat, corns, molasses,curing cannabis and other important animal feed stuffs have increased due to COVID-19 . The COVID-19 pandemic has jolted the livestock production. Pivotal livestock farming materials have been largely unavailable. These include frozen semen aliquoted in a semen straw for artificial insemination of livestock, replacement stocks , equipment , and animal feed additives , 2020. Veterinary healthcare services and other animal health preventative services have been greatly reduced during the pandemic . This caused significant delays in diagnosis and treatment of diseases. The reductions and delays resulted in halting the progress in prevention, control, and eradication of different animal and zoonotic diseases. Since zoonotic disease can also impact humans, the pandemic-triggered disruptions jeopardized human health in addition to animal health.

Furthermore, COVID-19 has negatively impacted food safety inspections, animal health extension services, and disease surveillance efforts which were important checks to prevent the spread of zoonotic diseases and other infections that impact human and animal health . This can create ripe conditions for future outbreaks and pandemics that can be detrimental to livestock health and human health . Amid the COVID-19 situation, markets have been disrupted throughout the world which ultimately affected animal production . The closure of animal markets and restrictions on export and import operations have deprived livestock producers from precious local and global marketing opportunities . In addition, intermediaries who collect animals and animal products and then process or sell them after fattening have been hit hard, which caused farmers to lose their links to major buyers . Thus, there was a sharp decline in animal processing and slaughtering capacities which further added to the turmoil in the livestock market . This has impacted both animals and animal products such as milk, eggs, and meat. Some farmers had to cull their animals or dump their animal products such as milk, which caused them significant income losses. According to the FAO, these losses have been especially severe on women who were unable to obtain the necessary nutrients for their small ruminants and poultry . The wide scale market disruption that resulted from the pandemic has impacted the agricultural workforce and caused staff shortages and layoffs in the labor force associated with animal production . There is a significant proportion of migrant workers in the livestock industry and meat plants . Many of them had to return to their home countries due to the lock downs, closures, and other measures that were implemented during the pandemic . During the peak of the pandemic, China closed livestock and poultry trading and slaughter markets in most of the nation, which results in labor shortage in slaughterhouses . Other causes for labor shortage include childcare, quarantine, and sick leave, which led to a 30% absence rate in some slaughterhouses in France and to similar issues in other countries in Africa and Asia . Owing to these factors and to other factors, the process of bringing animals and animal products to local or global markets faced multiple hurdles. The problem of labor shortage has had significant impacts on countries in South and Southeast Asia. In India, labor shortage has led to 23% food grain production loss . Labor shortage has also led to food insecurity concerns and hunger concerns for daily wage workers in the farm and non-farm sectors in Bangladesh . Food insecurity has also emerged as a major pandemic-related concern in Nepal and other countries . The worldwide demand for meat has been increasing in recent years for many factors including the rapid growth of population in many parts of the world . However, working in slaughterhouses and meat packing plants has been considered a major risk for COVID-19 infection during the pandemic . Workers in meat plants and slaughterhouses in Germany, England, Wales, and Portugal have reportedly been infected with SARS-CoV-2. . The Portugal outbreak led to short-term closure of the poultry slaughterhouse and the implementation of strict hygienic measures including health screening of all employees, adding new bathing areas, and replacing old disinfectants with stronger ones . Previous reports confirmed the presence of rotaviruses and coronaviruses in raw milk and dairy products . MERS-CoV has been shown to survive for prolonged periods in milk . The microbial composition of milk is influenced by the hygienic conditions of the animal, the animal’s feed and water, air quality, environment, and equipment used for milking . To minimize transmission of food borne pathogens, milk and dairy products are subjected to the process of pasteurization . Although some studies suggested that SARS-CoV-2 can spread via food products as it can remain viable on inanimate surfaces for hours to days , there is no proof that SARS-CoV-2 can spread directly through food, milk, milk products, or eggs . However, it is always a good practice to understand the sources of contamination of dairy products and to try to minimize all possible routes of contamination . The COVID-19 pandemic caused global economic losses and significant negative impacts on the agri-food sector, including farming, crop production, and animal production systems .