Serum IgE is higher in smokers than in nonsmokers and is possibly higher in ETS exposed subjects

Evidence for a healthy worker effect is that in many of the studies, workers had higher baseline FEV1 values compared with those of control groups or with advancing tenure . There is other evidence in the occupational literature on diagnosed occupational asthma in bakers, and on allergic sensitization to platinum salts and to TDI, that risk is greatest in the initial 1- to 2-year period of employment . Except for the case report of “diesel asthma” , none of the occupational studies reviewed above performed standard spirometric tests to diagnose asthma, and none followed workers prospectively from the start of employment.One common indoor air pollutant high in PAHs is environmental tobacco smoke . ETS also contains other toxic air pollutants, including 29 air toxics of 49 major components , making it difficult to ascribe effects to any one pollutant.This suggests an acute enhancement of IgE responses is possible, but whether the initial expression of allergic sensitization is enhanced by ETS is in dispute. A quantitative meta-analysis of studies up to April 1997 showed no association between parental smoking during pregnancy or infancy and atopic sensitization by skin prick tests in children without asthma or wheezing disorders . There was considerable inconsistency across studies . Other more-recent reviews have concluded that the relationship between ETS exposurein school-age children and the development of both asthma and allergy is poorly understood . A recent study of 5,762 school-age children had sufficient power to find a significant association between in utero exposure to maternal smoking without subsequent ETS exposure and history of physician diagnosed asthma, current asthma, and asthma requiring medication .

The same study showed that although current or past ETS exposure occurring only after birth was associated with reports of wheezing,grow racking it was not associated with asthma prevalence. Furthermore, combined in utero plus postnatal exposures did not increase risk of asthma beyond in utero exposures alone. The finding that maternal smoking during pregnancy has a stronger relationship to asthma onset than later ETS exposures was supported by several other studies that separated maternal in utero exposures from postnatal exposures . It is conceivable that in utero exposures to ETS shifts the immune response toward a TH2-type pattern as a result of the adjuvant action of PAH components interacting with in utero allergen exposures, which are now believed to lead to atopic sensitization before birth . It is plausible that postnatal coexposures would do the same, but the epidemiologic data are inconsistent for the relationship between ETS exposure and childhood asthma incidence. On the other hand, there is a preponderance of evidence linking ETS to acute exacerbations of asthma in asthmatic children. A recent meta-analysis concluded that studies showed an excess incidence of wheezing in smoking households, particularly in nonatopic children, suggesting a “wheezy bronchitis” pattern; however, in children with diagnosed asthma, parental smoking was associated with greater severity rather than incidence . A quantitative meta-analysis of studies up to April 1997 for 25 studies of asthma prevalence showed a pooled odds ratio for asthma of 1.21 if either parent smoked . Well-conducted panel studies are still needed to evaluate acute exposure–response relationships using repeated measures methods. A recent daily panel study over 3 months in 74 asthmatic children showed that acute asthma symptom severity, PEF, and bronchodilator use was associated with ETS exposure . There is less information about adultonset asthma. A cohort study of 451 nonsmoking asthmatic adults found that acute asthma severity, asthma-specific quality of life, and health status were associated with self-reported ETS exposure . Cohort studies have also shown increased risk of developing adult asthma from ETS , including occupational exposures . Among 3,914 nonsmoking adults followed 10 years, the relative risk for asthma onset from 10 years of working with a smoker was 1.45 .

A large survey of 4,197 never-smoking adults showed an elevated risk of physician-diagnosed asthma from any ETS exposure [OR 1.39 ] but no increased risk of allergic rhinitis . Reviews that have included other epidemiologic studies have concluded that although ETS is consistently associated with adult asthma onset, the number of studies is limited and the magnitude of effects are small, with limited dose–response information . One question that remains to be answered is what are the chemical determinants of associations between asthma and ETS, which is a complex mixture of particle and gas-phase components? Do PAHs play a major role in these associations?The urban exposure most relevant to the potential importance of PAHs to asthma is exposure to automobile and truck traffic. An earlier descriptive study spurred interest in potential adjuvant effects of DEP on IgEmediated respiratory allergic responses . This was a cross-sectional study of 3,133 Japanese persons that showed the prevalence of cedar pollen allergy was higher near busy highways despite equivalent local exposure to cedar pollen in less-busy areas. No epidemiologic studies have used quantitative exposure estimates of either DEP or ambient PAHs. However, European research has had access to black smoke measurements. A panel study of 61 children in the summer showed stronger associations for black smoke than for PM10 in relation to PEF, respiratory symptoms, and bronchodilator use . The authors hypothesized that black smoke may be a better surrogate for fine particles emitted by diesel engines or for other chemicals that may be the causal components in DE. Ambient NO2 could additionally serve as a marker for traffic exposure. Studnicka et al. explicitly used outdoor NO2 as a surrogate to show “traffic-related pollution” was associated with asthma prevalence among 843 children living in areas of lower Austria without local industrial emissions of air pollution. Numerous epidemiologic studies have shown associations between traffic density and asthma prevalence or morbidity. All but one were conducted in Europe and Asia . Fifteen of these have been in children , four in adults , and one study in both children and adults . All but seven have been purely cross-sectional studies. Krämer et al. conducted a cross-sectional study of atopic sensitization and asthma diagnosis but had a prospective outcome assessment of atopic symptoms for 1 year along with seasonal NO2 measurements. Other designs include three case–control studies of hospital admissions , and one case–control study of California Medicaid claims for asthma .

Another study was a mixture of cross-sectional, survey nested case–control, and historical cohort . One study of adult Japanese women was cross-sectional for symptom prevalence and also tested longitudinal models for 10 seasonal repeated measures for lung function in a sub-sample . Eleven looked at traffic density, but no air pollution measurements were used in effect estimates or as confirmation of exposure gradients ; four had traffic density, black smoke and/or NO2 ; and five used combustion-related air pollution measurements near the home as modeled surrogates for traffic exposures . Hirsch et al. briefly mentioned results for truck traffic, focusing instead on predicted home exposures from one hundred eighty-two 1-km2 grid measurements of CO, benzene, NO2, SO2, and O3. Pershagen et al. used predicted NO2 from models involving traffic data near the home and background ambient NO2 data,grow racks indoor with home residence time as a weighting factor. Oosterlee et al. investigated respiratory symptom prevalence and asthma in relation to busy and quiet streets predicted with model calculations of NO2 concentrations using the Dutch CAR model . Only four studies have separately assessed exposures from truck versus automobile traffic , two of which examined the same children in South Holland using actual 1-year measurements of traffic density in relation to lung function and symptoms . Another study in Germany had only self-reported truck traffic density in relation to symptoms . Except for one study , all of the above studies examining truck traffic showed increased risks in respiratory symptoms including wheeze from higher truck traffic density near the home . The Holland studies showed greater increased risks in respiratory symptoms including wheeze and lung function deficits from higher truck traffic than from automobile density near the home. Both Holland studies confirmed the possible relevance of DE by finding that black smoke measurements at the children’s schools were also associated with increased symptoms and lung function deficits . Astudy in Italy also found increased prevalence of asthma and symptoms from truck and bus traffic but not overall traffic . Only the study by Wyler et al. failed to show any difference between truck and car traffic in strengths of association; positive associations were limited to atopic sensitization. Although most of the traffic studies did not report associations by gender, four did find adverse effects of traffic-related exposures in children to be stronger in girls than in boys , while two other showed null results for both genders . In the study by Wyler et al. in adults, associations between pollen sensitization and home traffic density were larger for women than men. These gender differences are unexplained. Although differences in the perception of symptoms or reporting bias are possible, this does not explain the considerably larger lung function deficits in girls reported by Brunekreef et al. .

Negative results in the studies of traffic related exposures may be due to weaknesses that lead to exposure and outcome misclassification, which generally, but not always, lead to bias toward the null hypothesis if the misclassification is independent of systematic errors . This bias was possible in studies that used are awide exposure estimates without assessments of micro-environmental exposures or traffic near the home and school , or that relied entirely or partly on self-reported exposures . Nevertheless, most of these studies still showed positive associations between traffic and respiratory outcomes. Except for pulmonary function tests and tests for atopic sensitization , respiratory outcomes, including physician-diagnosed illnesses, were either abstracted from administrative databases or self-reported for the remaining studies. All but a sub-sample of two studies were subject to cross sectional or case–control biases. One of these biases stems from the use of current exposure. Current exposure may not be a good surrogate for exposure during past times that are more temporally relevant to current disease status. This is because outcomes may have an onset in the past, or because outcomes were previous illnesses or exacerbations of disease recalled in survey questionnaires. An important assumption is that current residence near traffic is a proxy for past exposures, and some, but not all, of the studies screened for residence times . One resultant systematic bias that could lead to null results is differential migration away from busy streets by symptomatic subjects. This is supported by the finding of Oosterlee et al. that parents with children having respiratory symptoms live an average of 2.6 years shorter at the present address than those of asymptomatic children. A positive bias, on the other hand, could have occurred from socioeconomic status , which was not always controlled for. This is important because people living on busy streets may be poorer. Clearly, well-designed prospective cohort studies and repeated measures panel studies are needed to assess the question of whether exposure to primary pollutants from traffic, which include air toxics, are risk factors for the onset or exacerbation of asthma and other respiratory allergic illnesses in children and adults. One epidemiologic approach that may prove useful to define source-specific air pollutant exposures such as traffic-related exposures is the use of principal component factor analysis with varimax rotation using available criteria pollutant data. One large survey study used this approach in Taiwan . They recruited 331,686 middle school children who were nonsmokers and were enrolled in schools within 2 km of 1 of 55 monitoring stations. They compared asthma prevalence rates with air pollution concentrations and found positive associations with asthma prevalence for NOx and CO. These gases had factor loadings over 0.91, along with inverse loadings for O3 of –0.92, likely from scavenging of O3 by NOx . For an interquartile increase in CO and in NOx , the prevalence of either physician diagnosed or questionnaire-based asthma increased around 1% for both boys and girls. Asthma was not associated with PM10 or SO2, except for an unexpected inverse association in boys for PM10. The association of acute asthma with CO is supported in a Seattle panel study of 133 asthmatic children and is likely explained by more causal components of vehicle exhaust and other combustion byproducts .

An additional limitation emerges from uncertainty in interpreting data from the initial impactor stage

While pathways for exposure to occupant-borne bacteria have mainly focused on touching fomites and on person-to-person direct contact, our results reinforce an understanding that humans may also be exposed to bioaerosol-borne microorganisms originating from other building occupants. These emissions might be produced by direct shedding into air or they might result from shedding followed by deposition and then resuspension. While the relative proportions of these mechanisms are unknown, there is evidence that both mechanisms may be significant. The increase in total particulate matter mass in larger sizes demonstrates a potentially significant role for resuspension. For direct emissions, previous literature indicates that the shedding of desquamated skin is a significant contributor to indoor aerosols . The emission rate of bacteria associated with skin shedding has been reported to range up to 6.8 · 105 culturable colonies per person-hour , which is  12% of the value for total human micro-biota emissions reported here. We note that previous studies that focus on the bacteria shed from skin desquamation have demonstrated that the majority of skin bacteria are associated with particles larger than the 3–5 lm peak observed in our study . We note that the high enrichment of human microflora in larger-size bins of the indoor bioaerosol during occupancy indicates an increase in human bacterial emissions relative to total bacterial emissions in the larger size ranges . The results provided here represent an early inquiry into the impacts of human occupancy on the type and character of biological aerosols present in indoor air. Linking occupant characteristics such as health status, clothing type, and individual activity is an important area of future research to produce directly applicable data on how specific characteristics of human occupancy impact exposure to etiological agents. Environmental parameters, such as season, building materials and furnishings,cannabis grow rack and geographic location also should be considered. Limitations of this study included the restriction to one environment and that biological measurements were made from two indoor and two outdoor impactor samplers.

Future investigations should extend this approach to multiple environments to determine the extent to which results such as these are broadly observed.One aspect is the undefined upper size limit for particles collected on this stage. For analysis in this paper, we have assumed a sharp cut at 20 lm. Irrespective of this assumption, the mass and microbial genome concentration measurements for this upper size section are robust. However, the graphical presentation in Figure 1 would shift with a different upper size limit, with height changing so as to maintain a constant area for that section. More significantly, converting from measured concentrations to emission factors depends on knowing the section-averaged particle deposition rate, k, which in turn depends on particle size. Overall, the literature lacks good empirical data on indoor deposition rates for particles larger than 10 lm. Furthermore, size-resolved deposition rates for bacteria and fungi have not been reported. The lack of microbe-specific deposition data may be a source of additional error owing to differences in shape and density from the olive-oil droplets studied by Thatcher et al. . We based our analysis on empirical measurements reported by Thatcher et al. and extrapolated to cover the largest size section. If we had used gravitational settling as the basis for determining the loss-rate coefficient, then choosing 15 lm as the upper size limit would have reduced the loss-rate coefficient by 29%, whereas choosing 25 lm as the upper size limit would have increased it by 33%. For this range of aerodynamic diameters applied as the upper limit on the largest impactor size stage, there would be a ±20% corresponding uncertainty in emission rates. Errors in estimating the emission rates for other particle size sections are much smaller because of three factors: well-defined upper size limits, stronger basis for the loss-rate coefficient, and less importance of the smaller loss-rate coefficients as compared to ventilation as a particle removal mechanism.Relationship to place is powerful and fundamental to human experience. Each of us was born somewhere and has an ancestral lineage that connects us to that place, or to distant locales, known and imagined.

We crave the experience of travel, and we insert visual notations of our journeys into our everyday lives with bits of natural detritus, mementos and photographs. Each object, relic or image, imbued with the distilled essence of its place of origin, is reframed by and reframes its new surroundings. Although we may appreciate a saved pebble for its essential qualities as a stone, it also holds its own history and has the potential to evoke deeply personal, experiential memories. Artist’s expressions of their relationships to place have long served as important indicators of the shifting formal innovations and philosophical tenor of their times. The Impressionists’ aspiration to capture the transient light and color in a landscape with fleeting brushwork and a subjective palette was central to their desire to represent modernity in the nineteenth century. For the emerging middle class at that time, the rapidly changing urban world had stimulated a desire for scenes of rural landscape and life. Such scenes reassured them there were still places where timeless values could be found. The duality of this situation opened the possibility for a technically and conceptually innovative avantgarde, and as artists expanded the paradigm for modern subject matter throughout the twentieth century, some began to merge formal radicalism with social commentary about the changing landscape. The importance of the relationship between nature and culture intensified during the late 1960s, as artists began to eradicate the boundaries between form, process and meaning, shifting their relationship to the location and presentation of their work. Two major trajectories eventually emerged. Land- or Earthworks artists asserted their relationship to nature with performance-based works or large scale sculpture in remote locations. Minimalists responded to this institutional shift and telluricurge by transporting earth and other natural materials into the white cube of the gallery. As artists embraced this new aesthetic, their work also evolved to incorporate formal eclecticism, performative practice, and the primacy of conceptual expression. The legacy of this period has been a generation of artists who have adapted these methodologies to establish ideational, emotional and aesthetic bridges between the personal and public. Their confrontation of the not-sodelicate balance between nature and culture has compelled many of them to create works that commingle elements of the natural world with personal narrative to showcase issues that might otherwise be overlooked.

Parker’s deep artistic and philosophical connections to the materials of nature is shared by a growing number of contemporary artists involved with liberating natural materials from their utilitarian identities and presenting them for aesthetic, symbolic and scientific contemplation. Among them is Wolfgang Laib,cannabis grower racks whose art is largely concerned with gathering pollen, which he exhibits as material and art object. Laib’s sensibility is similar to Parker’s in that he allows the pollen to retain its own character and meaning. His process of gathering it also mimics the bee’s rites of collection. Laib’s holistic attitude toward his primary material, the creatures that cultivate it, and his reference to nature as “his monastery” have poetic resonance with Parker’s empathy toward the land, its output, and place.2 But Parker also says her “personal hunger to have connection with the land is a form of spirituality.” She speaks of her paintings, which reduce the landscape to geometrically patterned fields of color, as “more about the spirit of the land than a direct replication of it.” The material and pieces that speak most deeply of Parker’s connection to the land are those that are comprised of earth or that tell stories about it. For several years she has been collecting soil samples from various farms, which she uses as subject and medium. “The soil is really the palette, and you can’t make anything if the soil isn’t of quality.” During her exhibition’s opening, Parker glamorized the experience of soil quality by setting small portions of damp soil in wine glasses for people to test its aroma. For “Palette,” she displayed small samples in see-through containers in a box, creating an earth palette of assorted colors and textures. One particularly meaningful soil sample was gathered from her partner’s family farm in France, which “had not been disked or turned over for a hundred years. There was something poignant to me about these people living on their land, who don’t turn some of it over; who don’t impact it in any major way. I loved that soil and the idea that there’s a place where people don’t mess with the earth.” Parker’s personal identification with the soil is distilled in “Clod,” a chunk of earth which fl oats serenely atop a transparent Plexiglass pedestal, as her emblematic alter ego.Numerous observational epidemiologic studies have demonstrated that physical activity is inversely related to cardiovascular morbidity and mortality. Physical activity may contribute up to 20% – 30% reduced risk of coronary heart disease. However, studies have shown that different types of physical activities may have different effects on the risk of cardiovascular disease and may interact together. For example, some leisure time activities such as walking, stair climbing, and cycling provide protection against CVD, whereas others, such as intensive domestic physical activity, may not offer protection against CVD. There are also interactive effects between lack of exercise and sitting at work and between demanding household work and sitting at work on the association with increased risk of acute myocardial infarction. Therefore, if we use a single summary measurement to reflect physical activity, such as METS, the association between physical activity and risk of CVD might be biased because subjects who have the same measured value may have a distinct combination of physical activities. Furthermore, studying different types of physical activity in isolation may not adequately consider any joint and interactive associations on the risk of CVD. Previous models that incorporate one type of physical activity of interest and other types of physical activity for exploring the effects of each type of physical activity on CVD may be problematic because of the concomitant change in total physical activity.

As one type of physical activity increases, total physical activity increases as well, given that the other physical activities are fixed. Hence, the effect estimate of one type of physical activity does not present its pure effect, but includes the effects of total physical activity. In order to overcome these challenges in the analysis of physical activity data, we used the method of principal component analysis to identify physical activity patterns that take into account combinations of physical activities. We used both parametric and semi-parametric regression models to examine the association between derived physical activity patterns and risk of acute myocardial infarction . Data from a population-based, casecontrol study in Costa Rica were utilized for purposes of this investigation.In Costa Rica, CVD has been the country’s leading cause of death since 1970 and the mortality rate for CVD has been declining since 2002 according to 2007 Health in the Americas, a report from World Health Organization. The participants in this study are cases and controls from a case-control study of non-fatal myocardial infarction conducted in the Central Valley in Costa Rica from 1994 to 2004. The study design and population have been described previously. In brief, eligible cases were men and women who were diagnosed as survivors of a first AMI by two independent cardiologists at any of the six recruiting hospitals in the Central Valley of Costa Rica during the period 1994-2004. All cases met the World Health Organization criteria for AMI. Enrollment was carried out while cases were in the hospital’s step-down-unit. One free-living control subject for each case, matched for age , sex, and area of residence , was randomly selected using information available at the National Census and Statistics Bureau of Costa Rica. Participation rates were 98% for cases and 88% for controls. Cases and controls provided informed consent on documents approved by the Human Subjects Committee of the Harvard School of Public Health and the University of Costa Rica.Trained interviewers visited all study participants at their homes for purposes of collecting sociodemographic characteristics, physical activity, lifestyle, medical history, smoking, and dietary data by use of a standardized questionnaire. They visited cases, on average, within 3 weeks of hospital discharge and when possible, by the same interviewer. Identical questionnaires and data collection procedures were used for cases and controls.

A few users also mentioned lack of shade and crowding as a concern

Blogs also have a relatively low volume of conversations . However, Blogs display the second highest reach score , and they are tied with Forums for the highest Impact score . Blogs are a type of online media that straddles the line between news outlets and social media. Like Twitter, blog content is produced by a small minority of online users who make up 9.6% of the US population, but blog posts are consumed daily by 77% of internet users . Additionally, blogs generate considerable user engagement through user comments . The high impact and reach scores show that blogs are an effective medium for communication about our topic. As confirmed by other studies, blogs are important in a strategy for science communication and disseminating research and marketing . Outreach and engagement strategies should however also consider the possibility that blogs may decline in relevance in the future due to lower readership from younger demographics , this is reflected in our findings that show a slight decrease in content from blogs in the second year of our data Another potential drawback is that effective blogs require a considerable investment of time to produce—with success greatly depending on higher length, quality, and frequent publication of new posts . Beginning farmers.org is a blog site that generated 15 hits from our query, but we otherwise did not identify any other blogs that emerged as a leader in producing content around pastured poultry and integrated production, indicating both a possible gap and opportunity for communication and outreach. Our data shows that online forums generate not only a high volume , but also high impact and reach . Results displayed on Table 1 show that including Reddit, six of the top ten websites with the highest volume of relevant posts are online forums. Thus,seed growing rack we are able to identify these specific platforms as hot spots of conversations around our research topic, which present an opportunity for targeted extension engagement in the future.

Backyard chickens.com, a popular hobbyist poultry keeper forum, ranks #1 in sites with the highest volume of posts from a single site , highest impact score and second highest impact score of any website. Other popular online forum sites are not poultry or agriculture specific. They include Reddit.com, one of the leading content sharing online community platforms in the digital world ; 4channel.com, a similar discussion board platform commonly distinguished by its controversial counterculture communities and political movements such as Anonymous and the Alt-right; uber people.net, an online community for Uber drivers; and finally, survivalist boards.com, a community of people who practice outdoor survival skills as a hobby. Finally, although YouTube trails behind other content types in terms of volume , it still ranks 4 th in terms of sites that produced relevant posts . Due to the high volume of non-functional URLs and links, missing post body text, and/or irrelevant posts generated by the web scraper, we were unable to include posts from YouTube.com in the manual text analysis. Reach and impact data were also not available for us to give a more in-depth assessment of this platform. It is entirely possible that some relevant posts were not picked up since the Brand watch web scraper captures text content rather than video content. Although in the 2nd year of our study YouTube declined by over 80%, its relevance cannot be completely disregarded since overall usage of this platform has increased steadily in recent years to include 82% of US adults in 2022. Whereas social media platforms are sometimes known to skew towards promulgating negative opinions , this dynamic was not observed for the topic of pastured poultry and integrated production. Across all platforms, conversations tended to focus much more on the beneficial attributes rather than barriers related to the key attributes we identified. As shown in figure 4, soil health and animal welfare, autonomy topped the charts with over 150 mentions. Other attributes like autonomy, ease of use, weed control, and pest control were also frequently mentioned. Overwhelmingly, the potential for pastured poultry to contribute to soil fertility and soil health is stated as a benefit or motivator for producers.

The term “soil”, and related phrases such as “soil health” and “organic matter” topped the most mentioned charts and therefore unsurprisingly emerged as the most discussed attribute in manual review of social media posts. Animal welfare follows closely behind soil as the second most mentioned attribute. In most cases, online users state a belief that chickens are happy and healthy when allowed to forage and graze on pasture, and report their usage of mobile chicken coops as a way to allow pasture grazing while protecting flocks from predators. There is also a frequent mention of using integrated poultry-vegetable production to control weeds. In the 91 posts we identified that brought up this attribute, many online users described employing poultry to clear unwanted vegetation either at the end of a cropping season, or to start a new plot of land for cultivation. Online users also frequently mentioned chickens’ tendency to consume insects , although sometimes they frame access to insects on pasture as a benefit to chicken nutritional needs, while other times it is framed as an ecosystem service that chickens provide for removing pests from crops. Our research also revealed some value-based drivers for poultry in integrated production systems. The benefit of autonomy was the 3rd most mentioned attribute : in many posts, people who discussed pastured poultry and/or poultry-crop integrations related their motivations to their desire to increase self-reliance in the face of supply chain disruptions, high food costs, and mistrust towards the food system. We were able to identify several of the platforms or accounts as belonging to the “homesteading”, “prepper”, or “survivalist” communities. This finding is somewhat unexpected as this aspect of pastured poultry and integrated cropping was mentioned only once in the literature we reviewed by Sossidou et al., who wrote “there are many social benefits of raising pastured poultry, such as family work ethics, community involvement and improving lifestyles. This alternative enterprise fits with the farmers desire to live on the farm and to be self-sufficient and self-directed” . Others expressed a value for integrated systems because they are perceived to be more natural or sustainable. At a small extent, some social media posts also expressed counter-industrial values, contrasting integrated production favorably in contrast to large-scale commercial production; and overall intrinsic value towards practicing such types of sustainable production from which they derive a sense of happiness, well-being, and satisfaction . Overall, positive or neutral sentiment far outnumbered negative sentiments by a ratio of around 9:1. Of the negative sentiments expressed, ease of use, animal welfare, and economic viability of pastured poultry and integrated production were most frequently mentioned. Negative sentiments related to ease of use predominantly occurred in online forums, which tells us that many people are trialing the systems and turning to discussion boards for troubleshooting.

Users commonly discussed challenges around moving their poultry flocks around for rotational grazing. This often involved the heavy weight of the mobile chicken coops, which varied depending on the building design and material. Other complaints revolved around undesired labor, time or logistics involved in executing this type of production system. Negative sentiments on the animal welfare topic often revolved around challenges related to predation, with users either having lost birds due to predation or expressing doubt that chickens can be adequately protected in an outdoor mobile chicken coop. Another concern was around exposure of chickens to the elements,indoor grow rack with some users expressing that mobile coops were not sufficient to house chicken in the winter.Doubts and concerns around the economic viability of pastured poultry and integrated production were captured solely from online forum posts, with some users arguing that such farming systems cannot be profitable as a source of income. A much smaller share of concerns was expressed around the expenses related to implementing the system. This may indicate that the economic challenges are perceived by many to lie in access to market or marketing rather than cost of entry. It was unclear in many cases whether the users were speaking from firsthand experience or merely for the sake of theoretical debate. It is possible to speculate that negative sentiments were absent from platforms like Twitter and Reddit because they are being used to promote pasture and integrated systems rather than to debate its efficacy. Finally, although based on keyword identification through natural language processing, mentions of terms related to egg production and broiler production are roughly even, manual review of social networking content indicates that the number of posts discussing layer production is almost twice as numerous as posts mentioning broiler production. This indicates that online users may be more interested in egg production, but that both are important since broiler production was also mentioned at a relatively high volume. Figure 5 allows us to compare the proportional breakdowns of the attributes mentioned in each source type. We found that across all three source types, attributes related to soil health rank amongst the top 3 most discussed topics, whereas the ratios of prevalent conversation around other topics differed across source types. For Reddit users, the larger share of conversations mentioned autonomy compared to other content types. Forums revolved at a relatively greater percentage around ease of use and animal welfare. Twitter users are mentioning climate change 3x more than users on the other sites.

They are overwhelmingly positive and focus on the potential for using poultry to remove unwanted vegetation, and the improved sustainability and higher market value of integrated farming outputs. The findings also allow us to elucidate gaps in the discourse around certain topics related to pastured poultry and integrated cropping that we have identified as subjects of interest to producers. We found that discussions around relevant scientific research trials and findings, food safety, processing, and certification of goods produced through integrated pastured poultry production noticeably showed the lowest presence from social media discourse in our data set. This lack of awareness or interest may be due to bias among the social media user demographics towards people who are relatively new to this type of production system and may not be knowledgeable about certain challenges that they have yet to face. A low extent of practical experience in pastured poultry and integrated farming may also explain the low volume of perceived challenges and negative sentiment found in our data, in which case negative sentiment might be expected to increase in the future as producers confront more challenges over time.We may speculate that this demographic is unlikely to be trialing the production at such a scale as to be very concerned about certification or processing, or perhaps they simply see these topics as part of general considerations for poultry and crop production that are not uniquely associated with pastured poultry and integrated cropping. Finally, the absence of relevant scientific findings and studies mentioned in the social media posts indicates an opportunity for researchers and extensionists to engage and promote the relevance of scientific findings for the benefit of producers. According to the United Nations, the population of the world is expected to grow in the next century, which in turn encourages the development of innovative techniques to ensure agricultural sustainability. Agriculture on productive land is threatened not only by high levels of urbanization, uneven water distribution, and inclement weather, but also is threats to biodiversity that have unfavorable environmental impacts. Due to the anticipated drastic population growth and constraints on resources in the upcoming decades, only 10% of the demand for food is estimated to be met by expansion of productive lands, with the remainder relying on new techniques that can achieve higher yields. Therefore, developing novel methods to augment the ratio of crop production over used land is a vital issue. In recent years, the indoor vertical farming systems with artificial light are found to be a viable solution to resolve the in-creasing demands of future agricultural products. The IVFS are promising alternatives to open field or greenhouse agriculture because they have precisely monitoring environmental parameters and are insensitive to outdoor climates, which can boost annual sales volume per unit area up to 100 times compared to that of open lands.

Details of the RESPIRE and CRECER cohorts have been published elsewhere

Approximately 2.4 billion people worldwide rely on burning biomass fuels, including wood, crop residue, coal, and animal dung for household cooking and heating. These fuels are often burned indoors in simple stoves in often poorly ventilated rooms, emitting substantial amounts of pollutants including particulate matter, carbon monoxide, and polycyclic aromatic hydrocarbons, all components of household air pollution . It is estimated that HAP from biomass fuel is responsible for more than 1.8 million premature deaths and 60.9 million disability-adjusted life-years annually, with the greatest burden experienced in low- and middle-income countries. Women and children have the highest levels of exposure to HAP since they traditionally tend to daily household chores, including cooking and spending time in kitchens. Exposure to biomass smoke during childhood has been shown to impact lung function and increase risk of acute lower respiratory infections. However, less is known about the rates of allergic sensitization and subsequent risk of atopy, which is the tendency to develop allergic diseases such as allergic rhinitis, eczema, and allergic asthma, among children who are exposed to biomass smoke early in childhood. Prior studies focusing on outdoor air pollution suggest that exposure to traffic-related air pollution early in life may enhance the risk of aeroallergen sensitization and increase the risk of developing allergic diseases at older ages. Diesel exhaust, in particular, has been found to enhance risk of aeroallergen sensitization and promote development of allergic asthma. Environmental tobacco smoke exposure early in life has also been associated with the development of allergic sensitization in children. Studies that investigated cooking-related biomass smoke exposure in children have also found it to be associated with elevated risks of asthma, rhinitis,grow drying rack and other respiratory illnesses.

There are, however, limited data regarding the relationship between HAP, aeroallergen sensitization, and atopy in pediatric populations. A cross-sectional study conducted in rural Bavaria in southern Germany in a cohort of school-aged children from 9 to 11 years old found that children living in households using coal or wood for heating were less atopic and less likely to be sensitized towards aeroallergens than children living in households with other forms of heating. In a large study of Finnish children, there was also a negative association between woodstove heating and allergic rhinoconjunctivitis; however, this association disappeared after adjusting for other factors in the residential environment. In another population of children in Spain, the use of a biomass energy source for cooking and indoor heating was not associated with atopic dermatitis. Contrastingly, for children in rural Guatemala, a previous study suggested that the use of an open fire for cooking may be an important risk factor for asthma symptoms and severity later in life. The primary objective of this study is to examine the relationship between early biomass smoke exposure and atopy among a cohort of children enrolled in a HAP-reducing chimney stove intervention trial among a population living in the western highlands of Guatemala. In the study community, women often carry their youngest child on their back during cooking, until the child is approximately 18 months old, exposing the newborn children to high levels of HAP. We hypothesize that the availability of a vented chimney stove would reduce the children’s HAP exposure compared to those who use open fires for cooking and would be associated with risks of allergic sensitization. Participating households and children were recruited from the Randomized Exposure Study of Pollution Indoors and Respiratory Effects cohort and its follow-up study, the Chronic Respiratory Effects of Early Childhood Exposure to Respirable PM study. Briefly, 518 rural Guatemalan women with newborn children who cooked exclusively over an open fire were recruited for the RESPIRE study between October 2002 and December 2004. Households were randomized to either receive a chimney stove , which improves combustion and uses a chimney to vent emissions outdoors, or to continue to cook with their typical open fires until the end of the trial, when they also received the intervention plancha stove. CRECER, the follow up study, took place from 2006 to 2009 and revisited RESPIRE households and recruited 169 new households that were from the same geographical region, exclusively used open fires, had one child in the same age range as the RESPIRE study children and one infant less than 6 months old .

For equity purposes, these new households received a chimney stove at the end of the CRECER study when all exposure and outcome information had already been collected. All households in the study thus received a chimney stove at different time periods: RESPIRE intervention households received the stove when the index children were less than 6 months old; RESPIRE control households received the stove when the index children were approximately 18 months old; new CRECER households received the stove when the index children were approximately 5 years old, and their proxy infant siblings were 18–24 months old. The grouping and study timeline are illustrated in Figure 1. The plancha stoves provided in this study reduced the children’s biomass smoke exposure by improving combustion and venting cooking smoke outdoors. Although this may result in an increase in outdoor exposure, we would expect participants in households with plancha stoves to have lower overall biomass smoke exposure, because the total amount of smoke produced would not increase, and outdoor biomass smoke would affect children for shorter duration and lower intensity. We also hypothesized that group 1 index study children would have the lowest cumulative biomass smoke exposure because they were provided the plancha stoves earliest, followed by groups 2 and 3, respectively. To test these hypotheses, we measured carbon monoxide exposure for the study children. Personal CO exposure was used as a proxy for personal biomass smoke exposure: CO has been shown to correlate well with fine particulate matter exposure in this population, in homes using open fires or chimney stoves. Study participants wore small, passive CO diffusion tubes for 48 h every 3 months during RESPIRE and every 6 months during CRECER. Since group 3 index study children did not have CO measurements obtained when they were <18 months of age, the personal CO exposures of their younger infant siblings were used as a proxy for their early life exposures. Details on exposure assessment methodology, validation, and quality control and assurance have been extensively described elsewhere. We combined data from different measurements, including the aforementioned 48-h samples, to estimate cumulative CO exposure. We used RESPIRE CO tube measurements to estimate cumulative exposure during the first 600 days of life for groups 1 and 2 and used CRECER infant sibling CO tube measurements to estimate cumulative exposure during the first 600 days of life for group 3. Cumulative CO exposure from 600 days old to first allergy questionnaire was estimated based on CRECER CO tube measurements for the study children. We conducted a sensitivity analysis using three alternative calculations for cumulative CO exposure, two of which did not use the 600 days cut point. Details of these calculations can be found in the Table A1.Five rounds of skin prick tests were performed on 539 participants to determine allergic sensitization to six common indoor and outdoor aeroallergens . During each round of SPT, a positive control and a negative control were also performed on each child.

A positive SPT result was indicated by a wheal measurement 3 mm or greater. SPT results were considered invalid if the histamine control was negative or the saline control was positive. Results from children who reported taking antihistamine or cold medications prior to testing were also excluded. Atopic symptoms were assessed via quarterly respiratory questionnaires completed by the study children’s mothers. The QRQs were conducted three times during CRECER to ascertain the occurrence of symptoms associated with asthma,grow glide racks allergic rhinitis, and eczema. The questions were developed based on the International Study of Asthma and Allergies in Childhood questionnaire. All QRQs were conducted by field workers fluent in the participating mothers’ primary language . Details of the questionnaire development and translation processes have been published elsewhere. All questions were close ended and began with “in the last three months”. A child’s final allergic outcome was recorded as positive if the mother reported him/her having positive symptoms in any of the three QRQ rounds. Logistic regression models were used to analyze the relationship between biomass smoke exposure and the risks of developing allergic outcomes. Primary statistical analysis used study group as a categorical exposure variable based on the length of having a chimney stove in the household. Odds ratios and 95% confidence intervals were reported. In this analysis, group 1 is the baseline level, groups 2 and 3 represent intermediate and highest levels of biomass smoke exposure, respectively. Secondary statistical analysis used the estimated cumulative CO exposure as a linear continuous exposure variable. Age, sex, second-hand smoke exposure, the number of children in the family, kitchen structure , child’s average weekly temazcaluse in minutes, number and species of pets and farm animals at home, maternal history of allergic outcomes, parental education, and socioeconomic status were collected from the CRECER baseline questionnaire, and were adjusted for in the logistic regression models. We did not adjust for race because the study population was homogenous, self-identifying as Mam indigenous. Among the 557 households participating in CRECER, 20 lacking valid SPT or QRQ results were excluded. For the two households with twins that both participated in the study, only the first child recorded was kept in the analysis to ensure independence among observations. Respiratory outcomes were available for 537 children, 188 from group 1, 192 from group 2 and 157 from group 3. Valid SPT results were available for 526 children, 184 from group 1, 187 from group 2 and 155 from group 3 . The quality and precision of the five rounds of SPTs during CRECER improved with additional training and more experience of the staff, indicated by the significantly lower number of invalid tests in the later rounds. As such, the results of the last valid round of SPT for each child was recorded as the child’s final allergic sensitization outcome. Among the 526 children with valid SPT results, results for 496 participants were taken from the 5th round of SPT tests. The household-level and child-level demographic characteristics were similar among the three study groups , especially between groups 1 and 2 that were randomized in the RESPIRE study.

Study children were on average 3.6 years old at the first QRQ.Compared with groups 1 and 2, group 3 had higher proportions of maternal history of asthma, rhinitis and eczema, and slightly lower SES. This prospective cohort study followed more than 500 children in rural Guatemala over 7 years and examined associations between cooking-related biomass smoke exposure and childhood atopy outcomes. Children from households that received chimney stoves when the children were approximately 5 years old had higher risks of maternal-reported allergic asthma and rhinitis symptoms compared to children from households that received a chimney stove intervention within the first 6 months of life. A 1 ppm-year higher cumulative CO exposure and its related cumulative biomass smoke exposure was associated with 6–12% higher odds of maternal reported allergic rhinitis and conjunctivitis symptoms. No significant association was found between biomass smoke exposure and eczema or skin prick test outcomes. Notably, the overall prevalence of sensitization to cockroach in this population of rural Guatemalan children was high , which is similar to that reported for inner city children in the U.S. A summary of main results from studies that looked at household biomass smoke exposure and allergic or respiratory outcomes is presented in Table A2. Our finding that higher biomass smoke exposure was associated with maternal-reported respiratory symptoms such as wheezing, sneezing, nasal congestion and rhinorrhea in their children is consistent with other studies that looked at exposure to biomass stove use and self-reported or clinically diagnosed respiratory diseases among children. Prior studies that looked at exposure to cooking and heating-related HAP and atopy outcomes in children residing in high-income countries did not find significant associations after adjusting for lifestyle and socioeconomic factors. In this study, we did not find a significant association between biomass smoke exposure and maternal-reported eczema symptoms. Additionally, there was no significant association between biomass smoke exposure and allergic sensitization as measured by SPT.

Participants were prompted to report craving and pain ratings within the W-SUDs app on a daily basis

Post treatment survey completion was 50.5% , with better retention among those with a higher CAGE-AID score at screening . Retention was lowest among those with a CAGE-AID score of 2 and higher for those scoring 3 or 4 . Retention was unrelated to participant demographic characteristics, previous use of Woebot, psychiatric diagnoses, primary problematic substance, depressive symptoms, pain, cravings, confidence, substance use occasions, AUDIT-C scores, or DAST-10 scores . Missing data on individual survey items was minimal. In a single instance, a participant’s average score values were imputed when missing 1 item on the PHQ-8.The data were aggregated so that if participants provided multiple ratings within a day, the scores were averaged. To examine changes over time, generalized estimating equationlinear models were run with week entered as a factor, setting week 1 as the reference category. W-SUDs, an automated conversational agent, was feasible to deliver, engaging, and acceptable and was associated with significant improvements pre- to post treatment in self-reported measures of substance use, confidence, craving, depression, and anxiety and in-app measures of craving. The W-SUDs app registration rate among those who completed the baseline survey was 78.9% , comparable with other successful mobile health interventions. As expected, the use of the W-SUDs app was highest early in treatment and declined over the 8 weeks. Study of engagement with digital health apps has been growing, with no consensus yet on ideal construct definitions.Simply reporting the number of messages or minutes spent on an app over time may undermine clarity and genuine understanding of the type and manifestation of app utilization related to clinical outcomes of interest. Further research in this area is warranted. The observed reductions from pre- to post treatment measures of depression and anxiety symptoms were consistent with a previous evaluation of Woebot conducted with college students self-identified as having symptoms of anxiety and depression. Furthermore, in this study,growing racks treatment-related reductions in depression and anxiety symptoms were associated with declines in problematic substance use.

Declines in depressive symptoms observed from pre- to post treatment were greater among the participants in therapy. This study also examined working alliance, proposed to mediate clinical outcomes in traditional therapeutic settings. Traditionally, working alliance has been characterized as the cooperation and collaboration in the therapeutic relationship between the patient and the therapist. The role of working alliance in relationally based systems and digital therapeutics has been previously considered; the potential of alliance to mediate outcomes in Woebot should be further validated in future studies adequately powered to examine mediators of change. Measures of physical pain did not change with the use of W-SUDs as reported in pre- and post treatment measures or within the app; however, the sample’s baseline ratings of pain intensity and pain interference were low. Although not a direct intervention target, pain was measured due to the potential for use of substances to self-treat physical pain and the possibility that pain may worsen if substance use was reduced, which was not observed here. Within-app lesson completion and content acceptability were high for the overall sample, although there was a wide range of use patterns. Most participants used all facets of the W-SUDs app: tracked their mood, cravings, and pain; completed on average over 7 psycho educational lessons; and used tools in the W-SUDs app. Only about half of the sample completed the post treatment assessment, with better retention among those screening higher on the CAGE-AID. That is, those with more severe substance use problems at the start of the study, and hence in greater need of the intervention, were more likely to complete the post treatment evaluation. None of the other measured variables distinguished those who did and did not complete the post treatment evaluation. This level of attrition is commensurate with other digital mental health solution trial attrition rates. By addressing problematic substance use, including but not limited to alcohol, the W-SUDs intervention supports and extends a growing body of literature on the use of automated conversational agents and other mobile apps to support behavioral health. A systematic review of mobile and web-based interventions targeting the reduction of problematic substance use found that most web-based interventions produced significant short-term improvements in at least one measure of problematic substance use. Mobile apps were less common than web-based interventions, with weaker evidence of efficacy and some indication of causing harm. However, mobile interventions can be efficacious.

Electronic screening and brief intervention programs, which use mobile tools to screen for excessive alcohol use and deliver personalized feedback, have been found to effectively reduce alcohol consumption and alcohol-related problems. However, rigorous evaluation trials of digital interventions targeting non-alcohol substance use are limited. Furthermore, although a systematic review concluded that conversational agents showed preliminary efficacy in reducing psychological distress among adults with mental health concerns compared with inactive control conditions, this is the first published study of a conversational agent adapted for substance use.Study strengths include study enrollment being double the initial recruitment goal, reflecting interest in W-SUDs. Most participants reported lifetime psychiatric diagnoses, and approximately half of the participants endorsed current moderate-to-severe levels of depression or anxiety. W-SUDs was used on average twice per week during the 8-week program. From pre- to post treatment with W-SUDs, participants reported significant improvements in multiple measures of substance use and mood. The delivery modality of W-SUDs offered easy, immediate, and stigma-free access to emotional support and substance use recovery information, particularly relevant during a time of global physical distancing and sheltering in place. More time spent at home, coupled with reduced access to in-person mental health care, may have increased enrollment and engagement with the app. Although further data on recruitment and enrollment are warranted, these early findings suggest that individuals with SUDs are indeed interested in obtaining support for this condition from a fully digitalized conversational agent. This study had a single-group design, and the outcomes were short term and limited to post treatment, thus limiting the strength of inferences that can be drawn. The sample was predominately female and identified as non-Hispanic White, and the majority were employed full-time. Non-Hispanic White participants reported higher program acceptability on 2 of the 4 measures compared with participants from other racial or ethnic groups. Future research on W-SUDs will use a randomized design, with longer follow-up, and focus on recruitment of a more diverse population to better inform racial or ethnic cultural programmatic tailoring, using quotas to ensure racial or ethnic diversity in sampling. Notably, although recruited from across the United States, nearly all participants were sheltering in place at the time of study enrollment due to the COVID-19 pandemic, which may have affected substance use patterns and mood as well as interest in a digital health intervention. Notably, however, alcohol sales in the United States increased during the COVID-19 pandemic.

The primary outcomes of substance use, cravings, confidence, mood, and program acceptability were standard measures with demonstrated validity and reliability. The limitations were that all were self-reported,indoor grow rack and acceptability measures were not open-ended or qualitative. Few participants were misusing opioids, likely due to study exclusion designed to mitigate risk, namely, the requirement of engagement with medication-assisted treatment and no history of opioid overdose requiring Narcan . Notably, nearly 1400 people with interest in a program for those with substance use concerns were excluded due to low severity on the CAGE-AID screener. Worth testing is the utility of digital health programs for early intervention on substance misuse that is sub-syndromal. Building upon the findings of this study, future research will evaluate W-SUDs in a randomized controlled trial with a more racially or ethnically diverse sample, balanced on sex and primary problematic substance of use; will employ greater strategies for study retention ; and will be conducted during a period with less restrictions on social contacts and physical mobility. Randomized controlled evaluations of conversational agent interventions relative to other treatment modalities are required. Self-cutting can be understood clinically as a symptomatic behavior, on the one hand, and as a bodily practice embedded in a cultural imaginary and identity on the other. Youths may identify with “Emo” or “Goth” culture which lionize depression and cultivate self-cutting as a cultural practice . Popular concern about perceived dangers of self-cutting has at times been heightened to the point that one cultural historian suggested that “Cutting has become a new moral panic about the dangers confronting today’s youth” . Anthropology has not been disposed toward addressing cutting as a problematic cultural or clinical phenomenon given the disciplinary propensity to understand body mutilation and modification in terms of rituals and cultural practices. This is perhaps because ritual meaning is not so dependent on distinguishing whether harm is inflicted by others or by oneself or on differentiating cultural practice from psychopathology.One other anthropological observation has been provided by Lester, who notes that current explanations of self-harm can be grouped into four categories: communicating emotional pain, emotional or physiological self-regulation, interpersonal strategy, and cultural trend. She notes that these categories share the idea that self-harm manifests individual pathology or dysfunction, with the cultural assumption of the individual as a rational actor. In contrast, an anthropological perspective emphasizes the “cultural actor who embodies and responds to cultural systems of meaning to internal psychological or physiological states” .

Emphasizing the powerful symbolic significance and long cross-cultural record of self-harm and blood shedding as ritual and even therapeutic practices, she suggests that contemporary cutting may be seen as privatized and decontextualized social rituals affecting transformation parallel to collective initiation rituals that operate in a cycle of self-harm and repair, especially in the case of adolescent girls struggling with the aftermath of sexual abuse and/or with contradictory gender messages . Sociocultural characteristics of a typical “self-cutter” emerged in the 1960s as Euro-American, attractive, intelligent, and possibly sexually adventurous teenage girls, that Brickman claimed was partially taken up in medical discourse in a manner that “pathologizes the female body, relying on the notion of ‘femininity as a disease’” . Gilman took exception to assumptions of pathology with the provocative claim that “self-cutting is a reasonable response to an irrational world” . From a clinical vantage point, self-cutting is often viewed as a type of injury or harm to the self. The historical backdrop to this development can be traced to Menninger’s attention to self-mutilation as distinguished from suicidality. The distinction between “delicate” and “coarse” self-cutting was made by Pao , with Weissman focusing on wrist-cutting syndrome and Pattison and Kahan proposing the existence of a deliberate self-harm syndrome. Favazza provided cases of extreme and highly unusual forms of self-mutilation in excruciating detail, with an attempt to classify types based on severity. With the provisional emergence of non-suicidal self-injury disorder criteria in the fifth version of the Statistical and Diagnostic Manual of Mental Disorders DSM-V ,1 the distinction between self-harm as within a normative or pathological range remains equivocal. This is illustrative of the manner in which conceptualizations of self-cutting continue to be embedded in a complex cultural history of changes in the incidence, popular awareness, and social conditions in which such phenomena occur.While it is possible to find clinical, psychometric, survey, and historical approaches to the phenomenon of self-cutting, we lack an ethnographic account with a substantive locus in the interactions of individuals, grounded in the specificity of bodily experience and the immediacy of struggle in the face of existential precarity . In this article, we take a step toward such an account with a discussion situated at the intersection of two anthropological concerns. First is the ethnographic understanding of experiential specificity through anthropological adaptation of phenomenological method . Drawing on this approach, we understand experience as meaningful sensory perception in temporal context and within particular cultural, social, and interpersonal settings and subjectivity as the more or less enduring structure of experience. With respect to mental illness, this approach invites anthropological recognition of struggle as a fundamental human process that comes to light in the context of lived experience . Second is the ongoing anthropological concern with adolescence as a stage in the life course at which identity is consolidated and people approach full cultural membership but which is also fraught with challenges to well-being that anthropology can contribute to understanding in a way relevant to mental health policy and practice .

Each cytokine was measured in duplicate and represented as the average value±SD

Our findings suggest that cannabinoids are capable of altering the differentiation and activation of cells involved in human cell-mediated immunity. Functional coupling of cannabinoid receptors to G-protein activity was assessed by measuring forskolin-induced cAMP levels in CHO-CB2 cells and fresh human monocytes. CHO-CB2 cells were cultured overnight at 5×105 cells/well in a 6-well plate. The next day, DMSO was added and cells cultured for an additional 18 h. THC , JWH-015 , the combination of SR144528 and THC , or diluent alone, were then added to the wells and incubated for 15 min prior to an 18 h stimulation with 50 μM forskolin . For studies with monocytes, 5× 106 PBMC were placed into each well of a 24-well plate and monocytes allowed to adhere for 2 h in X-VIVO-15 medium. Non-adherent cells were then removed, wells rinsed, and the remaining monolayer treated in a manner identical to that described for CHO-CB2 cells. Forboth cell types, supernatants were harvested at the end of the forskolin stimulation and stored at −80 °C until assayed in duplicate for cAMP using a standard competitive enzyme immunoassay kit . The final reaction product was read in a plate reader at 405 nm and the amount of cAMP determined by regression analysis. DC and THC-DC were evaluated for their capacity to activate T cells in a standard MLR assay . Allogeneic CD45RA+ T cells were isolated by negative selection with specific antibody and immunomagnetic beads, then labeled using the Vybrant CDSE/CFSE Cell Tracer Kit according to the manufacturer’s protocol. DC were cultured with 2×105 T cells at 1:50 DC:T cell ratios in X-VIVO 15 medium in 96 well round-bottom plates at 37 °C in a humidified CO2 incubator. For some experiments, DC and THC-DC were matured by culture with 20 μg/ml SAC for 18–24 h prior to co-culture with the T cells. In other experiments, the co-cultures were supplemented with 2 ng/ml of either IL-7, IL-12 or IL-15. On day 5 of co-culture,vertical grow trays the T cells were collected and analyzed by FACS for proliferation and cell surface marker expression .

Cell-free supernatants were collected from the wells and assessed for cytokines by custom multiplex analysis .As an initial step in understanding the potential interaction between cannabinoids and human monocyte-derived DC, monocytes were evaluated for the expression of the CB1 and CB2 receptor subtypes by RT-PCR and flow cytometry . RT-PCR studies were carried out on monocytes that had been purified to >90% purity by either negative depletion or fluorescent cell sorting. mRNA encoding for both CB1 and CB2 were detected, although expression of CB2 predominated whether analyzed by standard RT-PCR or by an automated quantitative RT-PCR using cells from 4 different donors . Despite the presence of mRNA, standard flow cytometry failed to detect CB1 or CB2 receptor protein on the cell surface of monocytes even though antibodies were directed against their N-terminal epitopes. However, when cells were fixed and permeabilized, specific staining for both CB1 and CB2 was detected, consistent with the presence of intracellular protein . Intracellular background staining with isotype control mAb was minimal for CB1 but dimly-positive for CB2, likely reflecting the need for APC-labeled goat anti-mouse F2 as a secondary detection reagent. Due to these differences in fluorescent labels and staining protocols, the relative fluorescent intensity for CB1 and CB2 cannot be directly compared as measures of receptor concentration. The presence of functional CB2 receptor complexes was then assessed by measuring the impact of different cannabi-noids on forskolin-induced generation of cAMP . Using CHO-CB2 cells as a model, we confirmed that treatment with THC significantly inhibited the generation of cAMP, as did JWH-015 at p<0.01. Furthermore, the inhibition of cAMP by THC was blocked by pretreatment with SR144528, a selective CB2 receptor antagonist . The same assays were repeated using purified human monocytes . Again, an overall CB2 agonist treatment effect was present. Pretreatment with either THC or JWH-015 inhibited the forskolin-induced generation of cAMP and the effects of THC were blocked by SR144528 . While monocytes express both CB1 and CB2, the predominance of CB2 mRNA and the response of these cells to CB2-selective agents suggest that CB2 acts as the dominant cannabinoid signaling pathway. The differentiation of human monocytes into DC is associated with characteristic changes in cell surface proteins involved in antigen presentation .

To evaluate the effects of THC on this aspect of differentiation, adherent PBMC were cultured for 7 days with GM-CSF and IL-4 and examined for the expression of typical monocyte and DC markers by flow cytometry . Exposure to THC did not prevent the normal down-regulation of CD14, but did inhibit the upregulation of other cell surface markers characteristic of antigen presenting cells including CD11c, HLA-DR, CD40 and CD86. The effects were concentration-dependent, with 0.5 μg/ml THC inhibiting expression of all of these markers by 40–60%. Interestingly, the response profiles were not uniform for every protein. THC produced a uniform decrease in the expression of CD11c and CD40 on all of the cells but resulted in two distinct subsets with respect to the expression of HLA-DR and CD86 – one population that did not express these markers and one that expressed relatively normal levels . In the latter case, the relative proportions of these two subsets depended upon the concentration of THC, with higher levels of THC resulting in fewer marker-positive cells. Cannabinoids have been reported to promote the apoptosis of mouse bone marrow-derived DC under certain conditions . In order to assure that the phenotypic changes observed in our studies were not the result of poor viability, DC that had been differentiated in the presence of either THC or JWH-015 were stained with propidium iodide and Annexin-V-FITC. There was no significant impact of either cannabinoid on the number of recovered cells or on the frequency of apoptotic or dead cells . In addition to their high level expression of major histo-compatibility complex and costimulatory molecules, monocyte-derived DC are usually characterized by their capacity for antigen uptake, as well as their secretion of cytokines that promote cell mediated immunity. Receptor-mediated endocytosis was measured by the uptake of FITC-dextran and was dramatically suppressed in cells that had been exposed to THC . The production of IL-10 and IL-12 was also assessed by stimulating cells with SAC and measuring cytokines released into the culture media at 48 h following stimulation. Interestingly, while the production of IL-12 was significantly suppressed , the secretion of IL-10, which can bias T cell activation toward T helper type 2 and/or T regulatory phenotypes, was not altered . This differential effect on cytokine production is consistent with an immuno regulatory effect rather than a global suppression of DC function. The capacity for DC to stimulate the activation and proliferation of antigen-specific T cells represents a final integrated measure of their function. DC that had been generated in the presence or absence of THC were recovered from the 7 day culture of adherent PBMC, purified by negative depletion, and cultured with allogeneic Tcells in a MLR assay . In order to avoid direct effects on responder T cells, no further THC was added during the 5 days of DC:T cell co-culture. Proliferation was monitored by labeling cells with CFSE, which also allowed the phenotype of responder cells to be tracked with each cell division over time. While control DC were potent stimulators of both CD4+ and CD8+ respond-er T cells,vertical marijuana grow the proliferative response to THC-DC was severely blunted . THC-treated DC induced T cell proliferation that ranged from only 17 to 32% of control values . Furthermore, while control DC promoted the conversion from naïve CD45RA+ T cells to activated CD45RO+ clones expressing the high affinity IL-2 receptor , this functional transformation into effector/memory T cells was almost completely absent when T cells were stimulated with THC-DC .

There was a similar impact on the generation of effector cell cytokines as measured by the release of IFN-γ and TNF-α into the culture supernatant . A number of factors can help restore function to impaired antigen presenting cells or enhance their capacity to stimulate T cell responses. Given our findings with THC-DC, we hypothesized that a combination of DC activation and cytokine replacement might be effective for this purpose. In initial experiments, DC and THC-DC were exposed to heatkilled and fixed SAC for 18–24 h prior to co-culture with T cells. The goal was to replicate bacterial activation signals that might occur during an immune challenge in vivo. In other experiments, the co-cultures were supplemented with IL-7, IL-12 or IL-15 to replace key cytokines known to be involved in the proliferation and differentiation of effector/memory T cells. As demonstrated in Fig. 6, pre-treating control DC with SAC enhanced their capacity to stimulate T cell proliferation and maturation. In addition, exposing THC-DC to SAC restored some of their capacity to generate mature responder T cells. This effect correlated with the upregulation of HLA-DR, CD80 and CD86 on THC-DC . In addition, supplementing the co-cultures with IL-7 helped SAC-stimulated DC to further promote the expansion and phenotypic maturation of effector T cells, a synergistic effect that was not observed with either IL-12 or IL-15. When assessed in a limited number of experiments, IL-7 also increased the production of IFN-γ and TNF-α, consistent with a restoration of their effector/memory function . The human CB2 receptor was first cloned from a human myeloid cell line and has been reported as the predominant cannabinoid receptor sub-type expressed by immune cells . Consistent with this, we found that expression of CB2 mRNA predominated over CB1 when fresh human monocytes were purified and assayed by semi-quantitative RT-PCR techniques. However, neither cannabinoid receptor could be detected on the extracellular surface of monocytes when stained with mAbs known to be specific for their N-terminal sequences. We recently reported that CB2 may exist as an intracellular protein in immune cells and others have suggested that CB1 may also function as an intracellular receptor . Consistent with these observations, the addition of an initial fixation and permeabilization step resulted in positive staining by both anti-CB1 and anti-CB2 mAbs, but not by their respective isotype controls. Functional receptor protein was confirmed by assaying the capacity for cannabinoids to inhibit forskolin-induced changes in cAMP. Addition of THC, a pan-agonist with equal affinity for CB1 and CB2, blocked forskolin-induced cAMP in both transduced CHO-CB2 cells and in fresh human monocytes. In addition, this effect was recapitulated by exposure to JWH-015, a selective CB2 agonist, and the effects of THC were completely blocked by SR144528, a selective CB2 antagonist. These findings confirm reports that CB2 predominates as the functional cannabinoid receptor pathway in human monocytes and add the caveat that receptor expression occurs at an intracellular location rather than on the cell surface. Monocytes act as myeloid precursors that can differentiate along a number of functionally distinct pathways depending upon their interaction with cytokines, growth factors, infectious signals and other regulatory mediators . When driven to differentiate into monocyte-derived DC under the influence of GM-CSF and IL-4 , their function can also be modulated by a variety of factors . Concurrent exposure to IL-6 and macrophage-colony stimulating factor can divert differentiation toward macrophages instead of DC . Transforming growth factor -β and IL-23 promote the development of DC that promote Th17 biased responses . IL-10 promotes tolerogenic and Th2-promoting features , while a variety of toll-like receptor ligands and immuno stimulatory cytokines will promote DC that stimulate effector/memory T cells . In this setting, we hypothesized that exposure to THC during the process of DC differentiation would provide valuable insight regarding its immuno regulatory properties. Further, given the immuno suppressive effects that cannabinoids have on antigen-specific T cell responses in animals in vivo and on human T cell activation in vitro , we hypothesized that cannabinoids might render DC tolerogenic or otherwise skew their stimulatory activity. Only a few studies have examined the interaction of cannabinoids with DC and in most cases the focus has been on murine models or on the effects of cannabinoids on differentiated DC . Do et al. suggested that THC can impair immune responses by inducing DC apoptosis. However, they studied mouse bone marrow-derived DC and apoptosis occurred primarily when THC concentrations exceeded 5 μM.

LG2 encodes a basic-leucine zipper protein that functions cell-nonautonomously

The leaves of the mutant are on average 20% narrower than normal siblings . At a cellular level, the cells of the epidermis are 15-17% narrower than normal siblings, implying that this deffect in cell expansion results in the overall narrowing seen in the mature leaves . Having smaller cells may be contributing to the darker green phenotype discussed above. Smaller cells would mean a smaller vacuole that would place the chloroplasts closer together, this proximity could lead to a greater intensity of colour, perceived at a distance as an overall darker green colour. Neither the blade nor the sheath was found to differ in length from normal siblings . All leaf phenotypes are only observed on adult leaves – that is, leaves 5-6 and onward – with juvenile plants being indistinguishable from normal siblings. Maize bears typical grass leaves, comprised of a sheath and a blade, which are separated by the ligule and auricle. The sheath is most proximal to the stem and wraps around it, providing support to the stem and the leaf as a whole. The ligule is immediately distal to the sheath and consists of a fringe of epidermal derived cells on the adaxial side of the leaf. Since the placement of the ligule forms a structural barrier between the environment and the internal spaces of the plant, it could be part of a”structural immune system”. Distal to the ligule are the auricles. The auricles are two wedge-shaped structures that develop on either side of the midrib, expanding in the proximal-distal plane as they approach the margins. Auricles are derived from both epidermal and mesophyll tissue and their structural function is likely to allow the blade to bend outwards to maximise light capture. Together, the mature ligule and auricle represent the position of the blade/sheath boundary , but their position is defined by this boundary, not the other way round. The BSB is visible before ligule and auricle development and the anastomosis of intermediate veins, which also characterises the BSB, vertical grow equipment is visible in leaf mutants liguleless1 and lg2 that lack the ligule. As such, it is clear that the BSB is a fundamental component of the leaf upon which the ligule and auricle are elaborations.

The lg1 mutant is a classical mutant that lacks both ligule and auricle and possesses a diffuse BSB. The lg2 mutant is similar to lg1, but upper leaves have partial recovery of ligule and auricle at the margins, while lg1 does not display this partial recovery but has a slightly better defined boundary. lg1 leaves are slightly narrower than normal siblings. Tassel branch number is reduced in both lg1 and lg2 and the transition to the reproductive phase is delayed in lg2 mutants, such that they produce more leaves than normal siblings.The accumulation of LG2 mRNA precedes that of LG1 – it is observed in the meristem, while LG1 mRNA is only observed at the BSB. On the other hand, LG2 mRNA is observed in the lg1 background and LG1 mRNA is observed in the lg2 background at the leaf margins, so there does not seem to be a direct “switching on” of LG1 by LG2 nor vice versa. LG1 encodes a nuclear localised protein that contains a domain with significant similarity to SQUAMOSA PROMOTER-BINDING1 and SBP2 proteins from Antirrhinum majus. In contrast to LG2, LG1 functions cell-autonomously. Wavy auricle in Blade1 is a dominant mutation that results in ectopic expression of the WAB1 protein in the leaf. WAB1 functions in the tassel to turn on LG1, which in turn promotes branch initiation. When WAB1 is ectopically expressed in the leaf, the resulting activation of LG1 causes the distinctive over proliferation of the auricle at the ligule boundary as well as ectopic auricle and sheath in the leaf blade. At the blade sheath boundary the blade width of Wab1 is similar to normal siblings, but distal to this, the blade quickly narrows such that Wab1 has a very narrow blade further up the leaf. This narrowing is due to a deletion of the lateral domain of blade, as shown by analysis of the triple mutant ns1;ns2;Wab1, where both the marginal domain and lateral domains are lost. A reduction in the number of lateral veins in the Wab1 blade support the hypothesis that narrowing in the Wab1 blade is due to deletion of domains rather than loss of cell expansion. The disruption of the BSB in lg1 and lg2 made them obvious candidates for crossing to fun. As Wab1 displays hypertrophy of the auricle, and fun displays complete loss of the auricle, we made a double mutant population to ask if FUN is required for ectopic auricle growth in the Wab1 background.Families segregating for Wab1, lg2, lg1 and fun were made according to the genealogy shown in Figure 3-1. Leaf width measurements were taken at blade mid point and/or 5cm above the blade/sheath boundary. Sheath lengths were measured from the base of the leaf to the MLK at the midrib and/or the auricle at the margin closest to the base of the leaf.

Plants were identified by their distinctive phenotypes with double mutant phenotypes from previous generations informing identification of plants as more mutations were introduced into the family. In families AV814-7, genotypes of double and triple mutants were also deduced from the presence of single mutant plants in the family.Both Wab1 and fun lead to narrowing of the leaf blade. Since narrowing in the leaf blade of Wab1 is due to a deletion of the lateral domain of the leaf, and narrowing in fun is associated with narrow cells , an additive interaction in the double mutant is not surprising. The lack of narrowing in lg2 and the epistatic narrowing observed in the lg2;fun double mutants implies that lateral expansion of the blade is associated with elaboration of the auricle but not definition of the MLK or the ligule itself. The partial recovery of blade width close to the BSB in the Wab1 mutant where there is extensive auricle supports this hypothesis.Although Wab1 leaf blades are slightly shorter than normal siblings, the extreme shortening of Wab1;fun double mutant blades was surprising since fun blades are of normal length . Furthermore, though Wab1 displays almost no displacement of the MLK and marginal auricle location as compared to normal siblings, and fun displays a slight distal displacement of the MLK, the Wab1;fun double mutant MLK is extremely proximal to the stem as compared to the marginal auricle . This extensive synergistic interaction is mirrored in the Wab1;lg1 double but not the Wab1;lg2 double mutant. Since Wab1 is known to activate LG157 but not LG2 ,vertical grow factories these interactions would place FUN downstream of LG1. The additive interaction between lg2 and fun resulting in a loss of ligule at the midrib as well as loss of auricle at the margins shows that these genes function in different zones and pathways to elaborate the BSB. It seems likely that LG2 is responsible for elaborating the boundary at the midrib, while FUN elaborates the auricle at the margins. The different zones of action are underlined by the double mutants with Wab1 – loss of LG2 does not affect the elaboration of marginal auricle in the Wab1;lg2 double mutant , while the Wab1;fun double retains the MLK but loses the marginal auricle. In the Wab1;lg2;fun triple mutant both the marginal auricle is reduced, and the MLK is lost, as would be expected if LG2 and FUN work in different domains. It is possible that LG2 and FUN have a common upstream element and represent forks in a pathway defining and elaborating the BSB. This upstream element could be LG1, since the lg1;fun double mutant shows epistasis of the lg1 phenotype, the lg1;lg2 double mutant shows partial synergy, and the lg1 phenotype is more severe than either the lg2 or fun phenotypes. Figure 3-11 shows a tentative pathway for ligular region development that includes FUN. The lg2;fun tassel has no branches and the rachis is thickened, similar to the female inflorescence. This phenotype could be considered additive, with both lg2 and fun removing branches, but for different reasons . This analysis also underlines the idea that reduction in inflorescence branching is a female trait in maize inflorescences and that LG2 may function in promoting the masculine trait of branch initiation.

Two characterised mutants that are deffective in the synthesis of brass inosteroids in maize are nana1 and nana2. The maize mutant nana1 was isolated from the F2 of a plant with active Mutator transposons. PCR revealed a 497-bp insertion in a gene homologous to DE-ETIOLATED2 in A. thaliana. DET2 is a steroid 5α-reductase68 known to catalyse a step in the BR synthesis pathway in A. thaliana. The knockout det2 phenotype can be rescued by addition of active brassinolide and is characterised in A. thaliana by extreme dwarfism as well as increased male sterility, dark green leaves, and repression of hypocotyl etiolation in the dark71. In the maize na1 plant, levels of the substrate of the DET2 enzyme, -24- methylcholest-4-en-3-one, accumulated to 475% that of normal sibs, while downstream intermediates were reduced . While rescue by addition of brassinolide was not reported, addition of the BR inhibitor propiconazole to wild-type maize plants caused them to phenocopy the nana1 phenotypes of greatly shortened stature and feminised tassels. Since NA1 transcript was shown by in situ to accumulate in developing anthers, the role of BR in development of masculine flowers is supported. Five years after the publication of the causative mutation of the nana1 plant, the classical mutant nana2 was cloned31. Like na1, na2 is of greatly reduced height and has a feminised tassel. Since the na2 phenotype was so similar to na1, it was not surprising that a mutation was found in the maize orthologue of the A. thaliana DWF1 gene. AtDWF1 is a Δ4-sterol reductase and dwf1 mutants accumulate its substrate 24-methylenecholesterol and have low levels of campestrol indicating a block in the BR pathway. The dwf1 phenotype, like det2, can be rescued by application of brassinolide in A. thaliana. The maize na2 plant was also shown to have low campestrol and later intermediates implying a similar block in the BR biosynthesis pathway. The overall na2 phenotype is similar to na1: short stature, feminised tassel and suppressed tillering. NA2 transcript is expressed in many tissue types including seedlings, mature and growing leaves, and immature ears and tassels with the highest expression detected in developing leaf collars, which is the position of ligule and auricle.

Older tissues tend to have lower expression of NA2, supporting a role for BR in expansion and growth. Crosses with GA deficient mutants and application of GA to developing na2 plants has shown an interaction between these two hormone pathways in regulating growth and defining sex in Zea mays. GA and BR are thought to have independent roles in defining the absolute height of a plant, since the na2;d5 double mutant was shorter than either single mutant. Masculine inflorescence development requires GA and BR to work together – the feminised tassel of na2 is abolished in the double mutant, implying that GA is needed for BR defficiency dependent feminisation. This hierarchy is mirrored in the ear: the anther ear phenotype of d5 is not abolished in the na2;d5 double mutant. Together these results show that BR is not essential for the masculinisation of inflorescences, while GA is essential for emasculation. Application of GA to developing na2 tassels enhanced their feminisation, supporting this hypothesis. In Arabidopsis thaliana, BRASSINOSTEROID INSENSITIVE1 encodes a membrane-bound leucine-rich repeat receptor kinase that binds BR and sets up a signal cascade ultimately leading to BR response. BRI1 homologs in maize are ZmBRI1a and ZmBRI1b, along with other members of the family BRASSINOSTEROID INSENSITIVE1-LIKE RECEPTOR KINASE 75. The RNAi based transgenic knockdown based on the ZmBRI1a sequence, was shown by qRT-PCR to have reduced levels of all five of these predicted BR receptors in 8-week-old shoot apices. Reduced response to BR in the knockdown line was confirmed by analysis of expression of BR marker genes constitutive photomorphogenic dwarf and brassinosteroid dependent1 and a BR root growth inhibition assay. In addition, a cross with the BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATESUPPRESSOR1 -YFP line showed a change in localization pattern.

Both medications are used for the treatment of hypertension and benign prostatic hyperplasia

In an 8-week RCT in 20 participants with early-onset AUD, ondansetron and naltrexone significantly reduced drinks per drinking day and trended towards an increase in percentage of days abstinent. Another combination study in 90 participants after 7 days on ondansetron and naltrexone found that the combination decreased craving for alcohol and ventral striatal activation to alcohol cues. Ondansetron may also be suitable for individuals with biological predisposition to early-onset AUD. In an 11-week RCT of 271 participants, ondansetron was shown to reduce self-reported drinking such that patients with early-onset AUD who received ondansetron reported fewer drinks per day and more days of abstinence compared to placebo. Ondansetron, combined with cognitive behavioral therapy in an 8-week open-label trial comparing effects in early-onset versus late-onset AUD, found that drinks per drinking day and alcohol-related problems were significantly decreased among those with early-onset AUD compared to those with late-onset AUD. Furthermore, in an 11-week study with 253 participants, ondansetron at 4 μg/kg reduced overall craving significantly in participants with early-onset AUD, while a lower dose reduced craving in participants with late-onset AUD. These reductions in craving were also associated with reduced drinking and an increased percentage of abstinent days. Ondansetron is well tolerated with relatively mild side effects including diarrhea, constipation, and headache. Overall, these studies suggest a potential role for ondansetron as an AUD treatment,vertical cannabis growing systems especially in participants with early-onset AUD and possibly in combination with naltrexone.Mifepristone , a glucocorticoid receptor antagonist, works on the stress system by regulating the amygdala.

Preclinically, both systemic and central amygdala injections of mifepristone were shown to suppress yohimbine stress-induced reinstatement of alcohol seeking, indicating that the central amygdala plays an important role in mifepristone’s effects on ethanol-seeking. Recent preclinical research in primates has shown mixed results, such that mifepristone was shown to decrease chronic voluntary alcohol consumption in rhesus macaques at doses of 30 and 56 mg/kg per day, but had no effect on alcohol-seeking or self-administration in baboons at slightly lower doses of 10–30 mg/kg per day. Of note, in the former study, cessation of mifepristone treatment resulted in a rapid return to baseline intake levels, and mifepristone was not effective in preventing a relapse during early abstinence. In a human laboratory study with 56 alcohol-dependent participants, mifepristone was effective in reducing alcohol craving and consumption relative to placebo, improved liver-function markers, and was overall well tolerated. A two-week Phase 4 RCT examining the effects of mifepristone on cognition in AUD was recently conducted, but recruitment challenges rendered the results inconclusive. Of note, in this trial, participants who received mifepristone had higher Beck Depression Inventory scores compared to placebo at 4 weeks post-randomization despite similar scores at baseline between the two groups, indicating a greater severity of depression symptoms caused by the medication. Additional clinical research on mifepristone as a treatment for AUD and its potential side effects is warranted.Ibudilast is an inhibitor of phosphodiesterases -3, -4, -10, and -11 and macrophage migration inhibitory factor. Ibudilast has been shown to dose-dependently suppress pro-infammatory cytokines, such as interleukins IL-1β, IL-6, and tumor necrosis factor alpha , and to increase the anti-infammatory cytokine IL-10 and neurotrophic factors. As increases in infammation are seen in AUD, the effects of ibudilast in treating AUD are thought to be driven by its anti-infammatory and proneurotrophic qualities. Preclinically, ibudilast was demonstrated to reduce alcohol intake in two rat models, and decreased drinking selectively in alcohol-dependent mice in comparison to non-dependent mice. These pre-clinical findings align with prior rodent studies in which pharmacological inhibition of PDE also reduced alcohol intake. In a 7-day human laboratory crossover trial , ibudilast was well tolerated and decreased tonic craving in comparison to placebo.

Additionally, ibudilast improved mood during exposure to alcohol and stress cues, and reduced the mood-altering and stimulant effects of alcohol among participants with more severe depressive symptoms. Another recent 2-week RCT enrolling 52 participants found that ibudilast also significantly decreased the odds of heavy drinking during the trial by 45% compared to placebo and attenuated neural response to alcohol cues in the ventral striatum. Ibudilast appears to be well tolerated, with common adverse side effects including gastrointestinal symptoms , headaches, and depression. In the aforementioned 2-week RCT, no significant differences in side effects were seen between groups. In summary, early findings from clinical studies of ibudilast for AUD treatment appear promising and warrant further clinical investigation.Prazosin and doxazosin are alpha-1 adrenergic receptor antagonists with similar chemical structures that can readily cross the blood-brain barrier and block noradrenergic excitation of the mesolimbic dopaminergic system .While these medications show good safety and tolerability, doxazosin appears to have a better clinical profile, such as improved absorption profile and a longer half-life, leading to less frequent dosing. Adrenergic receptors regulate sympathetic nervous system activity through activation of the neurotransmitter norepinephrine. Stress physiology is disrupted with chronic alcohol use, particularly during early alcohol abstinence. In early abstinence, individuals with AUD experience more emotional dysregulation, stress, and alcohol cravings, all of which can increase the risk of relapse. Thus, alpha-1 blockers like prazosin and doxazosin may help to normalize these stress system changes seen in AUD]. Preclinical work found that prazosin reduced ethanolrelated operant responding in acute withdrawal for dependent, but not non-dependent rodents. In addition, prazosin treatment prevented yohimbine stress-induced reinstatement of ethanol seeking and attenuated ethanol intake during relapse in alcohol-preferring rats. Doxazosin decreased voluntary alcohol intake in alcohol-preferring rats in a dose-dependent manner, and this effect was likely not due to general motor impairment. Further, doxazosin significantly reduced voluntary ethanol intake in a rodent model of AUD and stress exposure . In humans, an early 6-week pilot study with 24 randomized participants with AUD revealed that prazosin treatment was associated with fewer drinking days per week and fewer drinks per week than placebo. More recently, 92 participants with AUD completed a 12-week double-blind study with prazosin and medication management. Results from intent-to-treat analyses showed that prazosin participants had greater reductions in heavy drinking and rates of drinking over time, although these effects were modest.

A 10-week RCT comprising 41 individuals with AUD showed no significant effect of doxazosin over placebo on quantity of alcohol consumption. However, further examination of clinical moderators revealed that doxazosin significantly reduced drinks per week and heavy-drinking days among individuals with higher family history of AUD and higher standing diastolic blood pressure. Similarly, moderator analyses from a 12-week RCT of prazosin found that one’s degree of alcohol withdrawal symptoms predicted clinical response,vertical cannabis grows such that participants with high levels of withdrawal symptoms benefited the most from treatment. A small meta-analysis of 6 studies with a total of 319 participants tested the effectiveness of drugs acting on adrenergic receptors for AUD and found a significant treatment effect of prazosin and doxazosin on alcohol consumption but not abstinence. In summary, prazosin and doxazosin show some early promise as a treatment for AUD and may be particularly beneficial as a harm reduction approach and for individuals with significant alcohol withdrawal symptoms or family history of AUD as well as comorbid post-traumatic stress disorder .Alcohol use has been shown to alter the glutamate system. Chronic and binge drinking inhibit glutamate levels and transmission through blockade of NMDA receptors, subsequently leading to elevated glutamate levels during alcohol withdrawal. N-Acetylcysteine is a cysteine prodrug that works to restore glutamatergic tone in reward circuitry by improving the expression and function of the cysteine-glutamate exchanger and normalizing glial glutamate transporters. Preclinically, NAC has been shown to reduce withdrawal effects, block the development of behavioral sensitization to alcohol, and attenuate biological adaptations induced by alcohol cessation. Additionally, NAC reduced ethanol-seeking and self-administration in rodents. NAC and aspirin co-administration also reduced ethanol intake and relapse binge drinking in ethanol-preferring rats. In a recent meta-analysis of seven RCTs with a total of 245 participants, NAC compared to placebo was shown to reduce craving symptoms across a number of substance use disorders. A secondary analysis of a 12-week, multisite RCT of NAC to treat cannabis use disorder in 277 participants found that NAC increased odds of between-visit abstinence, and reduced alcohol consumption by 30%. However, a recent 5-day human laboratory study found that NAC did not attenuate alcohol self-administration. Additional clinical trials will also examine the effectiveness of NAC in adolescent and adult samples of AUD . These trials include samples with comorbid psychopathology and will use neuroimaging methods to examine the neural circuitry underlying NAC’s modulation of relevant metabolites and neural reactivity to alcohol cues. Orally-administered NAC appears to be well tolerated, with the most common adverse effects being nausea and diarrhea. Of note, NAC is currently being tested in adolescent AUD, a unique prospect as no other AUD pharmacotherapies are yet approved for adolescents. In summary, NAC represents a promising potential treatment for AUD and merits further exploration.Suvorexant is a dual orexin antagonist that is used for the treatment of insomnia. The orexin/hypocretin system is well known for its role in sleep-wake regulation but has more recently been implicated in AUD. Orexins are neuropeptides that are densely localized in the lateral hypothalamus. Orexins A and B bind to the G-protein coupled orexin-1 and orexin-2 receptors. Orexin A has equal afnity for both receptors, whereas orexin B has selectivity for orexin-2 receptors. The dense orexin projections from the lateral hypothalamus to the ventral tegmental area provide neurobiological support that orexins may influence responses to rewarding stimuli, including alcohol. Animal models demonstrate that orexin-1 receptor antagonists reduce alcohol drinking in alcohol-dependent mice and alcohol-preferring rats.

Orexin-2 antagonists also reduce alcohol drinking and reinstatement/relapse in mice and rats. Similarly, dual orexin antagonists reduce alcohol consumption in alcohol-preferring rats. Given the effectiveness of orexin antagonists in reducing alcohol drinking at the preclinical level of analysis, suvorexant has garnered interest as a drug that can be repurposed to treat AUD. While no animal or human laboratory study has directly examined the efficacy of suvorexant on alcohol-related behaviors, two ongoing Phase 2 RCTs will assess suvorexant’s potential as a treatment for AUD and comorbid AUD + insomnia . The sedative effects of suvorexant are of notable concern, particularly if individuals engage in alcohol drinking during treatment. Thus, the level of patient monitoring throughout the treatment phase and finding the optimal time of dosing to negate the additive sedative effects are important factors to consider to fully evaluate its therapeutic potential.ABT‐436 is an orally active, highly selective vasopressin type 1B receptor antagonist. V1B antagonists attenuate basal hypothalamic-pituitary-adrenal axis activity and have shown favorable effects in rat models of alcohol dependence, including attenuating reinstatement of alcohol self-administration, and diminishing alcohol intake by alcohol-preferring and alcohol-dependent rats . ABT-436 has been shown to attenuate basal HPA axis activity in humans.A 12-week RCT enrolling 150 participants found ABT-436 to be associated with an increased percentage of days of abstinence compared to placebo, as well as significantly reduced cigarette use.However, no differences were found on heavy-drinking days or alcohol craving. A subgroup analysis also showed that ABT-436 appeared to have greater efficacy among participants with high baseline stress levels. This study was the first Phase 2 clinical trial that tested a V1B receptor antagonist for AUD. The finding that ABT-436 reduced both alcohol drinking and smoking indicate a potential use for V1B antagonists to treat co-use of alcohol and nicotine. ABT-436 is generally well tolerated in humans, with the most common side effect being diarrhea. Furthermore, results indicate that patients with high levels of stress may specifically benefit from medications targeting the vasopressin receptor.N-[ benzyl] 4-methoxybutyramide is a GHB analogue that has shown promising in vitro and in vivo preclinical results as a potential agent for the treatment of AUD. The addition of GET73 to cultures of rat hippocampal neurons rescued negative ethanolinduced effects, reductions in cell viability, and increases in reactive oxygen species production, providing evidence for a neuroprotective role of GET73 as an AUD treatment. In vivo, GET73 treatment at low, non-sedative doses reduced alcohol intake and suppressed relapse in alcohol-preferring rats, as well as exerting anxiolytic effects. These effects are similar to those seen with GHB administration; however, GET73 was shown not to bind to either high- or low-affinity GHB binding sites in rat cortical membranes. Recent studies indicate that GET73 may act as a negative allosteric modulator at the metabotropic glutamate sub-type 5 receptor; however, the complete mechanism of action of GET73 remains unclear.

Findings add to this literature by specifying a boundary dose for alcohol’s stimulatory effect on eating

Given that pharmacotherapies for addiction can alter other appetitive responses, we hypothesized that IBUD would reduce tonic levels of high-fat/high-sugar food craving as well as reduce high fat/high-sugar food craving following alcohol infusion and stress. Understanding generaland domain-specific effects of IBUD will further medication development. A neuroimmune modulator such as IBUD may uniquely impact high-fat/high-sugar food craving because pro-inflammatory signaling may be associated with increased or decreased motivations to eat . Indeed, contrary to our hypothesis, IBUD increased high-fat/high-sugar food craving during psychological stress. It is uncertain why this effect emerged, although one might speculate that because IBUD inhibits pro-inflammatory signaling it reduces “sickness behavior” thereby increasing motivation to eat . Indeed, IBUD effects on high-fat/high-sugar food craving only emerged during a psychological stress task and psychological stressors acutely stimulate eating of high-fat/high-sugar food and production of pro-inflammatory cytokines , which could make modulation by IBUD more salient. Nevertheless, chronic peripheral levels of pro-inflammatory cytokines are associated with depression and increased comfort eating . In accordance with this, depressive symptomatology moderated the effects of IBUD versus PLAC on high-fat/high-sugar food craving during psychological stress reactivity: for those scoring higher in depressive symptoms,mobile vertical system grow high-fat/high-sugar food craving remained higher regardless of IBUD. For those scoring lower in depressive symptoms, IBUD increased high-fat/high-sugar food craving to a level comparable to the level experienced by those scoring higher in depressive symptoms.

These findings should be interpreted while considering that the overall mean of depressive symptoms in this study sample [M = 7.68] was below the clinical depression cutoff. Thus, effects may reflect differences in sub-clinical mood dysphoria. The associations among mood dysphoria, depression, acute and chronic neuroinflammation, and comfort eating are complex and future research is needed to tease apart inflammation mechanisms that drive changes in eating. On the other hand, IBUD did not alter high-fat/high-sugar food craving following alcohol infusion. This could be because alcohol did not generally increase high-fat/high-sugar food craving and had a non-linear effect on craving across target BrAC levels. In detail, a small amount of alcohol increased highest urge for high-fat/high-sugar food , yet more alcohol decreased this urge , and even more alcohol diminished effects . Although there is a substantial literature documenting that small alcohol doses stimulate eating , this is observed less in studies where individuals drink larger doses . The present study is the first to use a within-subjects design wherein food craving was measured at target BrAC levels.Alcohol was intravenously administered; this rules out expectancy effects and suggests that the observed nonlinear effect was mediated by pharmacological or physiological mechanisms. Intravenously administering alcohol comes with the drawback of reduced real-world validity but prior research indicates that participants who drink placebos eat similarly to those in control conditions rather than those who drink alcohol , again suggesting that the stimulatory eating effect of small alcohol doses is pharmacological or physiological. It is important to note that there was some variability across the two craving items in the results for high-fat/high-sugar food craving following psychological stress and alcohol administration. The reason for the different findings between items is uncertain. It could be that asking about highest urge felt during the tasks rather than urge felt right after the task increased response variance and improved statistical precision.

Finally—and critical to the development of IBUD for addiction indications—IBUD did not alter daily urge for high-fat/high-sugar food. This effect remained across all days of each condition in the trial. Although not what we hypothesized, in terms of medication development, this suggests that the effects of IBUD are domain-specific. The trial showed that IBUD reduced tonic levels of alcohol craving . Other research on IBUD has demonstrated its safety and initial efficacy for methamphetamine use disorder and opioid use disorder . IBUD, however, did not reduce tonic levels of high-fat/high-sugar food craving. IBUD seems to be influencing drug-specific mechanisms rather than general appetitive mechanisms. This is in contrast to other addiction medications such as naltrexone and naloxone, which have been shown to have a notable effect on food cravings and eating . The results alternatively highlight that multiple pathways regulate eating including inflammation, satiety, and reward signaling . Although some of these pathways may overlap with drug use, others may not. The present study findings should be interpreted while considering limitations. The sample was moderately sized and only powered to detect large effect sizes . Also, the sample comprised individuals with AUD. This sampling is typical when studies test if pharmacotherapies for addiction alter eating , yet a trial examining IBUD effects in a sample with obesity or eating disorders may yield different results. In addition, the items measuring high-fat/high-sugar food craving may have been too vague. Providing a clear definition of high-fat/high-sugar food with examples may have improved measurement precision. The present study nonetheless had several methodological strengths. Foremost, IBUD is a novel medication for addictions with support from theoretical rationale and strong preclinical data. The randomized, placebo-control, crossover design reduced study bias and error variance because participants served as their own controls. The design additionally included observation of medication adherence and a standardized meal prior to alcohol administration.

Another strength of the design is measurement of the effect of IBUD on tonic food craving as well as food craving following stress and alcohol administration. Calls in the medical field have emphasized that studying multiple behavior intersections in treatment may increase health benefits, maximize health promotion, and reduce health care costs . Understanding intersections between eating and alcohol use—and intersections in the context of medication effects—may be important to promoting overall health in addiction treatment. Tobacco industry has continuously used product modifications, such as manipulating smoke pH with various chemical additives, to make combustible cigarettes more appealing to novice users. For decades, Philip Morris and other tobacco companies have used ammonia as a relatively innocuous additive for a variety of purposes, including augmenting certain flavors, cutting costs by expanding or “puffing up” the volume of cured tobacco leaves, preparing reconstituted tobacco sheet , denicotinizing tobacco , and even lowering/removing tobacco smoke carcinogens. In the early 1960s, while evaluating the impact of ammoniated recons used in Marlboro cigarettes, Philip Morris discovered that freebase or unbound nicotine, as opposed to nicotine bound to other molecules , is more volatile and vaporizable,movable vertical grow rack system thus being highly bio-available. This led to the development of low-yield cigarettes that still had the nicotine kick necessary to keep customers “satisfied”. The freebased low-yield version of Marlboro cigarettes became the world’s most popular cigarette; Marlboro has remained the top selling cigarette brand in the world since 1972. The commercial success of Marlboro persuaded the rest of the tobacco industry to utilize ammonia to convert nicotine to its freebase form, as part of a new process for manufacturing cigarettes. However, this was achieved only after competitors uncovered Philip Morris’ “secret” of freebasing nicotine in cigarettes by reverse engineering the chemistry of Marlboro cigarettes. More than half a century later, a relatively unknown vape company, Juul Labs Inc., recognized the utility of salt-based nicotine for a novel electronic cigarette device, called JUUL. Started in 2017, Juul Inc. is a spin off company from Pax Labs, which was a manufacturer of vaporizing devices for cannabis and loose-leaf tobacco; Pax Labs was preceded by Ploom as the original company for e-cig development. Remarkably, JUUL’s use of salt-based nicotine, which is significantly less aversive than freebase nicotine, made it very quickly popular among naïve users, particularly adolescents and youth. The high content of nicotine in JUUL, which was claimed to be equivalent to nicotine content of a pack of 20 cigarettes, also made JUUL highly appealing to adult smokers, seeking a putatively less-harmful alternative to combustible cigarettes. Shortly after its launch, JUUL became the preeminent vaping product on the market and a dominant player in the vaping industry. In December 2018, Altria, one of the world’s largest cigarette manufacturers and the parent company of Philip Morris USA, acquired a 35% stake in JUUL. This made Altria a major force in both the tobacco and vaping markets. Nearly 1 year later, the US Surgeon General declared “youth vaping” an “epidemic” in the United States, and the US Food and Drug Administration called JUUL a particular cause for concern. Not long after, in August 2019, the Centers for Disease Control and Prevention , the FDA, state and local health departments, and other clinical and public health partners reported a nation-wide outbreak of vaping related severe lung illnesses and deaths, also referred to as “ecig, or vaping, product use-associated lung injury ”. Most EVALI cases reported using e-cig products containing vitamin E acetate and tetrahydrocannabinol , the principal psychoactive component of cannabis. However, the CDC did not rule out the etiologic involvement of other substances present in non-THC containing e-cig products. The EVALI outbreak lasted for several months, but declined considerably by February 2020. Worldwide, there are around 1.1 billion current cigarette smokers aged 15 or older, of whom 942 million are males and 175 million are females.

In 2019, there were an estimated 155 million smokers aged between 15 and 24 years – equivalent to 20.1% of young men and 5.0% of young women, globally. Two-thirds of all current smokers began smoking by age 20, and 89% of smokers began by age 25, highlighting a critical age window during which individuals develop nicotine addiction and transition to become established smokers. Globally, 7.4 trillion cigarette-equivalents of tobacco were consumed in 2019, amounting to 20.3 billion each day. Countries with the highest consumption per person were mostly in Europe. One in three male and one in five female smokers consumed 20 or more cigarette-equivalents per day, worldwide. The 10 countries with the largest number of tobacco smokers, which together comprised nearly two-thirds of the global tobacco smoking population in 2019, were China, India, Indonesia, the United States, Russia, Bangladesh, Japan, Turkey, Vietnam, and the Philippines—one in three current tobacco smokers lived in China. In many countries, progress in reducing the prevalence of smoking did not keep a pace with population growth, resulting in significant rises in the number of young smokers. India, Egypt, and Indonesia had the largest absolute increases in number of young male smokers. Turkey, Jordan, and Zambia had the largest increases in number of young female smokers. Over half of countries, worldwide, showed no progress in reducing smoking among 15–24 years old. Youth vaping is an evolving public health problem in the United States and around the world. Results from the 2020 National Youth Tobacco Survey and Monitoringthe Future survey showed that nearly 3.6 million American teens were current users of e-cigs, of whom 80% reported using flavored products, such as fruit, mint, menthol, and candy, desserts, or other sweet-flavored eliquids. Specifically, one in five U.S. high school students and one in ten middle school students reported current use of e-cigs in 2020. The CDC and FDA analysis of the 2021 NYTS, conducted during January 18 – May 21, 2021, showed an estimated 2.06 million U.S. middle and high school students reporting current use of e-cigs. The authors, however, cautioned that because the 2021 NYTS was fully conducted amid the COVID-19 pandemic through mostly online data collection, estimates from this year’s survey should not be compared to previous NYTS survey waves that were primarily conducted on school campuses. Following a same trend, the percentage of college-age youth who vape nicotine, has risen dramatically in recent years. Between 2017 and 2019, the 30-day prevalence of e-cig use increased from 6 to 22% among college students, and from 8 to 18% among 19 to 22 year-olds not in college. Together, these data indicate a continued and dynamic evolution of the global tobacco epidemic and youth vaping epidemic. Tobacco smoking-related diseases, including cardiovascular disease, pulmonary disease, stroke, and cancer in multiple organ sites, such as the lung, mouth , throat, nose and sinuses, esophagus, bladder, kidney, and ureter, pancreas, stomach, liver, cervix and ovary, the bowel , and white blood cells , are the leading causes of preventable death, worldwide. In 2019, smoking was associated with 1.7 million deaths from ischemic heart disease, 1.6 million deaths from chronic obstructive pulmonary disease, 1.3 million deaths from tracheal, bronchus, and lung cancer, and nearly 1 million deaths from stroke.

A pen-style e-cigarette battery was used as a model variable voltage e-cigarette for this study

It is possible that the TBI group itself differed in some way that we did not account for. A potential future prospective study could integrate more clinical information about a participant’s head injury such as the location of initial insult, Glasgow Coma Scale rating, or more precision on duration of unconsciousness. Future studies could also utilize functional magnetic resonance imaging information as another means to evaluate neuropsychological performance simultaneously with metabolic imaging. Looking at the number of head injuries that a person experienced could also have been worthwhile , as neuropsychological tests of memory, attention, and motor function have shown worse performance with repeated head injury . For example, HIV– individuals with multiple TBIs, when compared to those with a single TBI, exhibit significantly poorer memory and executive functioning . Given that these two neurocognitive domains are the same as those that showed significant deficits in our study, it would be interesting to determine whether there are cumulative effects of multiple TBI on HIV-associated neurocognitive decline. Despite some limitations, our study does indicate that TBI may increase vulnerability to brain dysfunction in HIV-infected individuals. If confirmed, the results indicate that those involved with HIV care need to take head injury into account in their neurological evaluation and clinical management of HIV patients.After the 2019–2020 outbreak of e-cigarette or vaping product use-associated lung injury in which the Centers for Disease Control and Prevention reported over 2,800 hospitalizations of patients displaying symptoms of acute respiratory distress,cannabis grow supplies serious public health concerns have been raised about the safety of e-cigarettes. In the initial investigations, evidence has supported that vaping of vitamin E acetate , a synthetic form of vitamin E that was used to “cut” or dilute black market or homemade tetrahydrocannabinol , was a major cause of the onset of EVALI symptoms.

Several different mechanisms of toxicity have been proposed since the outbreak, yet the exact causative agents and molecular mechanisms through which VEA vaping emissions resulted in lung toxicity are still not well understood. VE and VEA alone are considered safe for dermatological application in skin-care products and as well as for consumption in foods and dietary supplements. Several studies since the outbreak, however, have found that e-liquids like VEA undergo major thermal decomposition during the vaping process to form products that are often more toxic than the parent oil. VEA in particular has been found to decompose into a wide range of emission products including VE, alkenes such as 1-pristene, alcohol-containing compounds such as 3,7,11-trimethyl-1-dodecanol, durohydroquinone, and durohydroquinone monoacetate, and carbonyl-containing compounds such as ketene, 4-acetoxy-2,3,5-trimethyl-6-methylene-2,4-cyclohexadienone, and duroquinone. Still, the overall risk of exposure of each identified product to those who vaped VEA is unclear. For example, ketene gas has been hypothesized to form from the cleavage of the acetate group of VEA. However, this reaction has been calculated to only be feasible at temperatures exceeding 500˚C–temperatures that are likely to only occur under “dry puff” conditions. The operating temperature of the vape device is one of many parameters–including the model of e-cigarette used, puff duration, interval between puffs, etc.–that a user may alter to customize their vaping experience. A few studies to date have investigated the impact of increased temperature on the size and volume distribution of emitted vaping aerosols, reporting that greater coil temperatures result in larger puff volumes, but decrease the size of emitted particles. A recent study in 2021 found that the emission of volatile degradation products, including various carbonyl-containing species, was significantly enhanced when temperature was increased from 170 to 280˚C. In addition, increased coil temperature and characteristics of the vape device have also been found to influence other aspects of vaping emissions, such as the release of metals and the level of carbonyl-containing compounds or radical species.

E-cigarette atomizers and heating elements are often comprised of various transition metals including nickel, iron, and chromium which not only pose a risk of metal toxicity to vape users , but may play a role in the catalysis of thermal degradation of the e-liquid. One study by Saliba et al. found that e-cigarette filament wires had a significant impact on the production of carbonyl-containing compounds from propylene glycol vaping, lowering the temperature required to form carbonyl species by nearly 200˚C. However, the factors affecting the chemical composition of e-cigarette degradation products have yet to be fully characterized. The objective of this study was to examine the influence of variable temperature on the product distribution of e-cigarette vaping emissions, using VEA as a model e-liquid. To do so, we performed a non-targeted analysis of the aerosol-phase constituents at relevant, mid-range vaping temperatures using gas chromatography/mass spectrometry . We hypothesized that elevated temperature of the heating coil during vaping could enhance thermaldegradation of VEA, causing a shift in emission product distribution and toxicity in vapers. VEA vaping emissions were produced at coil temperatures ranging between 176 to 356˚C using a variable voltage vape pen and analyzed using GC/MS with electron ionization to assess how emission product identity and concentration changes as a function of temperature. In addition, pure pyrolysis of VEA without the influence of the device was also investigated using a tube furnace to investigate potential catalysis by the device itself. The results from this study contribute to our current understanding of the toxicity mechanisms underlying VEA vaping emissions and have significant implications for the potential health risks associated with the use of other e-liquids.This vape pen has set nominal voltages of 3.3, 3.8, 4.3, and 4.8 V. These voltages were confirmed using a multi-meter to measure the actual voltage of the battery upon activation.

The set-up of the temperature measurements can be seen in S1 Fig in S1 File. The protocol for the thermocouple measurement of the e-cigarette coil and oil temperatures was adapted from Chen et al. To measure the temperature at each voltage setting, the pen was connected to a fresh cartridge that was filled with VEA standard oil until the oil level sat just above the atomizer base. The oil level in the cartridge was kept consistent between each reading,cannabis grow facility as the amount of oil in the cartridge has been previously shown to affect the temperatures the coil may reach. Three 1 mm grounded k-type thermocouple wires were connected to a 4-channel data logger . One thermocouple was kept suspended to measure the temperature of ambient air as a device control. The second thermocouple was inserted into the air flow tube of the cartridge and allowed to rest on the surface of the ceramic coil. This position was chosen to record temperature across all voltage settings as it not only provided the most consistent measurements, but certain positioning of the probe resulted in the battery shutting off, likely to prevent overheating or burning in the event of the air flow tube being blocked during real-use scenarios. The third thermocouple was inserted into the glass casing of the cartridge to submerge the end of the probe in VEA oil in contact with the atomizer. The thermocouples allowed for simultaneous measurement of the coil and the parent oil in the cartridge when the battery was activated. Temperatures were recorded by the data logger every 1 s over a 1 min cycle. The vape pen was activated by holding the power button for 4 s to heat the coil, then allowed to rest for the remainder of the cycle. A total of 13 cycles–including 3 initial preconditioning cycles–were measured.The procedure for collection of VEA vaping emissions at each temperature setting was adapted from previous studies. Prior to each collection, a fresh cartridge was filled with VEA standard oil, weighed, and preconditioned by taking 3–5 puffs. The vaping emissions were collected using a cold trap apparatus maintained at -40˚C . The particle collection efficiency of the cold trap system at the flow rate used in this study has been reported previously. To collect aerosol emissions, one 4 s puff was taken at intervals of 1 min to maintain consistency with the temperature measurement procedure. Puffs were generated at each temperature using a 0.4 L min-1 air flow rate, which was controlled by a 0.46 L min-1 critical orifice connected a diaphragm pump . For each setting, the vape pen was operated until approximately 100 mg of VEA had been consumed; this consumption was typically achieved within 10–20 puffs. In instances where more puffs were required, the vape pen was allowed to rest at 20 puffs for 10–20 minutes to prevent overheating of the battery. Condensed emission products were dissolved in 1 mL of ACN, with 10 μL of 1, 3, 5-TCB solution added to each sample as an internal standard for chemical analysis. Emissions were analyzed immediately after collection or stored at -80˚C to prevent any aging effects.To determine the impact of the device on the degradation of e-liquids, pure pyrolysis of VEA oil was simulated using a tube furnace reactor system . The schematic of the set up for these experiments is shown in S2 Fig in S1 File. An alumina crucible containing 100 mg of VEA standard oil was weighed, and then placed into a high temperature quartz tube furnace capable of reaching temperatures as high as 1200˚C.

The tube furnace was initially set to 23˚C, then ramped to each temperature setting at a rate of 10˚C min-1, and then held at the target temperature for 75 minutes to allow for VEA oil to be evenly heated. Inert argon gas was flowed through the system at a rate of 0.18 L min-1 to carry the VEA pyrolysis products into cold trap apparatus kept at -40˚C. After 75 minutes, the tube furnace was programed to return to room temperature before the alumina crucible was removed and re-weighed to determine the amount of VEA that was consumed. Pyrolysis products condensed in the cold trap were dissolved in 1 mL of ACN and concentrated to 100 μL using a gentle N2 gas stream. Then, 10 μL of 1, 3, 5-TCB solution was added to each sample as an internal standard for chemical analysis.Fig 1 shows the temperature profiles of the e-cigarette coil and VEA oil in the cartridge operated at each voltage setting. Peak coil temperature at each voltage setting was fairly consistent between each measurement with no significant increase after consecutive use, which agrees with previous reports. Though the starting temperature after 1 min of rest increased slightly with subsequent measurements, the starting temperature never exceeded 33˚C. In contrast, the temperature of the oil in the cartridge increased with each subsequent measurement until seeming to plateau. The peak temperatures of both the coil and the oil were then taken and plotted as a function of voltage, as shown in Fig 2. Coil temperature showed a strong positive linear relationship with applied voltage , whereas oil temperature increased linearly with voltage until 41˚C , where the peak temperatures at 4.3 and 4.8 V do not significantly differ. This is likely due to the specific heat capacity of VEA; at higher voltages. Visible discoloration to the oil and wick could be seen during temperature measurements, indicating that the specific heat capacity of the oil in the cartridges may have been exceeded and part of the stored VEA may have been transformed before it is vaped .The total ion chromatographs obtained from GC/MS analysis of VEA vaping emissions produced at each temperature setting are shown in Fig 3. Overall, clear temperature dependent degradation of VEA vaping emissions can be seen as the amount and abundance of degradation products substantially increases with increasing coil temperature. Analysis of the GC/MS results revealed 19 compounds that were able to be tentatively identified based on consistent NIST MS spectral library match scores of 800 or greater. One other compound, 1-pristene, was not found in the NIST library and thus was identified based on comparison with previously reported mass spectra and a mass spectrum generated with the QCEIMS program that found signature fragments of m/z 266, 111, and 126, which are consistent with our results . A summary of the identified compounds and chemical information identified from PubChem can be found in the supporting information . Many of the products described here, such as phytol, 2,3,5-trimethyl-1,4-benzenediol and 2-hydroxy-4-methoxy-3,6-dimethyl benzaldehyde, have not been previously detected from VEA vaping to our knowledge.