Hemp Growing

This hypothesis ties into a study that assessed “quality of life” based on measures of alcoholism, substance abuse, criminality and a questionnaire in adults with ADHD who had previously been treated with stimulants versus those that had not. This study found that ADHD patients treated as children had a higher quality of life than those treated in adolescence by the researcher’s measured index. This hypothesis – that onset of treatment at a younger age correlates with effectiveness of treatment – may indicate a link between age of prescription and likeliness to experience SUD. A younger child is much less likely to have access to any form of street drug, especially stimulants such as cocaine or methamphetamine. If prescribed at a younger age, amphetamine sensitization would occur during a time in the child’s life in which they would have no outlet through which to act on drug cravings. Since the average duration of treatment lasts between 33.8 to 42 months, the medication would likely stop before the child reaches adolescence. Withdrawal, a series of negative symptoms that occur in the absence of a drug after a prolonged period of abuse, would therefore occur when the individual would most likely have limited access to illegal stimulants. As a result, a young child treated with amphetamines would not necessarily have an increased risk for abuse of other stimulants by the time he or she reached adolescence – when illicit drugs are more readily found. In contrast, if the onset of treatment were to start in the early teens , drug-seeking behavior would peak just as illicit substances became more available. This hypothesis is supported by one study that found that ADHD individuals whose treatment persisted into adolescence were more likely to become dependent on cigarettes than those whose treatment ended earlier. The individuals whose treatment had stopped before adolescence went through the sensitization/withdrawal process before cigarettes became available to them,custom grow rooms either through legal or illegal means.

The effect of amphetamine is also hypothesized to be greater under both temporal and environmental cues previously associated with administration. It is likely that a child treated at a younger age would move out of an environment previously associated with amphetamine and therefore have a decreased sensitivity to amphetamine at an older age compared to an individual who started treatment in adolescence. The child treated at a younger age would therefore be less likely to abuse their prescription and eventually other illicit drugs. Thus, ADHD treatment at a younger age seems to have little or no effect on drug abuse during adolescence and adulthood, while treatment that continues into adolescence may raise the risk of non-prescription stimulant abuse. Lastly, adolescents typically experience much more stressful environments as more responsibility is given to them at both home and school. The stress of adolescence may synergize with the effects described above, and thus further increase the likelihood of stimulant abuse. On the other hand, if amphetamine prescription is initiated before adolescence, the individual will not have the same added level of stress, and thus will be less driven to abuse their medication or drugs with similar effects.Although much evidence points to an increased risk of substance abuse with amphetamine treatment, many investigators have concluded that amphetamine use does not increase a patient’s likelihood of later developing SUD, and that it may actually exert a protective effect against substance abuse later in life based on population-level studies – that is, some have concluded that stimulant-based treatment of ADHD early in life may decrease drug abuse later in life. For instance, Barkley and colleagues, the same group whose results indicated a significant increase in cocaine use amongst ADHD patients treated with stimulants, still concluded that treatment of ADHD had no effect on the likelihood of using a number of drugs. A similar study that followed 56 medicated and 19 unmedicated patients found that there was no association between treatment and drug abuse.

A study that followed 285 treated and 84 untreated ADHD patients also concluded that SUD did not develop as a result of stimulant treatment. A meta-analysis of several studies also found that for any category of drug use, stimulant treatment decreased the risk that an individual would abuse drugs in general. Review papers on the subject of SUD and its relationship with ADHD have also come to the conclusion that childhood treatment with stimulants is negatively correlated with substance abuse.Although many studies conclude that stimulant treatment is protective against the development of SUD when prescribed to ADHD patients, the validity of these studies is questionable. For instance, many of the studies that come to this conclusion are funded in full or in part by drug companies such as Pfizer or Eli Lilly, which manufacture ADHD medications. These studies have clear financial biases in terms of their conclusions. Reviews and meta-analyses are particularly dubious when a conflicting financial interest exists, because they may select papers that suggest a desired result. In addition, studies with larger sample sizes and meta analyses tend to group all types of substance abuse into one category, or simply distinguish between “drug abuse” and “alcohol/ tobacco use” categories. Large bins of categorization produce a confounding variable, because stimulant drugs are known to reinforce and prime other stimulant drugs most reliably. The fact that amphetamine treatment has been suggested to protect against or have no correlation with the use of depressants such as marijuana or alcohol makes placing all drugs of abuse into one category especially problematic. The decreased risk factor for depressant use and the increased risk factor for stimulant use interfere with each other when considered together, thus concealing any specific trends that might exist. Of two predominant studies that separated “substance abuse” into individual drugs or drug subcategories, one study found a significant increase in cocaine use, while the other found no significant increase. However, the latter study had a small sample size of 56 medicated ADHD patients and 19 non-medicated patients. It is possible that if larger sample sizes were obtained, a significant increase would have become apparent.

This conclusion seems increasingly likely since the prevalence of stimulant abuse in society is generally not as high as for other drugs such as cannabis or alcohol, especially amongst ADHD patients in general. Therefore, a much larger sample size is needed to compare stimulant-specific abuse amongst ADHD patients. Furthermore, if treatment with stimulants does in fact exert a protective effect against general drug abuse and not illicit stimulant abuse, the analysis of drug abuse in general as a single category would actually downplay the increase in stimulant abuse amongst patients. Untreated subjects would be much more likely than treated subjects to participate in non-stimulant abuse, confounding a large portion of studies. Based on the idea that those with a later onset of treatment have a higher potential for stimulant abuse, it is probable that if the age of treatment onset were compared, patients with a later onset of treatment would show a specific increase in illicit stimulant abuse in adolescence and possibly into adulthood. However, those treated at a younger age may not have a statistically higher percentage of abuse of any drug. If it is true that subjects treated earlier are less likely to abuse stimulants than those treated later in adolescence, any study that does not compare age of onset and likelihood to develop stimulant-specific abuse possesses a significant weakness. Most of the studies that come to the conclusion of a negative correlation between amphetamine treatment and substance abuse fail to accurately assess age of treatment onset when evaluating data,montel grow racks thus mixing information from individuals that may have a higher risk of drug dependence with those that may have a lower risk of drug dependence because of age of treatment onset. Finally, none of these studies take into account the differences between methylphenidate and amphetamine. Since amphetamine has been shown to have an increased potential for abuse compared to methylphenidate and other ADHD medications, these studies therefore downplay the exposure to risk of substance dependence that is put forth with amphetamine prescription.The majority of population studies that have concluded that stimulant-based treatment has no effect on the development of substance abuse later in life fail to take into account all of the factors necessary to produce accurate correlations.Current knowledge regarding the effects of amphetamines on stimulant-specific abuse in animals and general drug abuse in humans is not consistent. Studies on animal models have concluded that amphetamines specifically raise the tendency to self-administer stimulants, such as cocaine and nicotine, largely due to the sensitization of the rewarding effects of amphetamine that results in drug-seeking behavior.On the other hand, other population-level studies based on surveys and meta-analyses have concluded that stimulant prescription has no correlation with the development of substance abuse. These studies, however, all possess one or more of the following flaws: failing to distinguish between stimulants and depressants in terms of drugs abused by patients; failing to distinguish between amphetamine medication and other stimulant treatment; working with sample sizes far too small to accurately reflect the level of dependence that might develop to stimulants, specifically; and failing to consider the age of the patient at treatment onset. Taken together, evidence suggests that amphetamine treatment of ADHD causes a small increase in potential for stimulant drug abuse and possibly a decreased potential abuse of depressants. The risk for developing stimulant abuse is likely dependent on age of onset of stimulant prescription, with those treated in adolescence and young adulthood at a higher risk. However, there are no conclusive studies to verify this hypothesis. Considering that the amphetamine treatment for ADHD is on the rise, it would be prudent for an independent research group concerned with the health of ADHD patients to conduct a large scale study that accounts for the variables mentioned above, using a large population of both treated and untreated ADHD patients to test specific dependence of stimulant class drugs that arise from treatment with amphetamines.

Another potential method of study might include comparing the number of formerly treated ADHD versus untreated ADHD patients amongst a population known to have abused stimulants, adjusting for the percentage of treated versus untreated ADHD individuals amongst the ADHD population. A conclusive study on this matter would allow parents, schools, and physicians to more accurately consider the treatments available for children with ADHD.Thirty male participants were recruited, among which were 15 pathological gamblers and 15 matched healthy controls . The PET data from these participants have been reported in a previous study comparing baseline dopamine synthesis capacity between groups . PGs were recruited through advertisement and addiction treatment centres and reported not to be medicated or in treatment for their gambling at the time of the PET study. HCs were recruited through advertisement. All gamblers qualified as PGs as they met ≥ 5 DSM-IV-TR criteria for pathological gambling. Current drug use disorder was a reason for exclusion for all participants. Additionally, participants were excluded if they were currently following psychiatric treatment, drank more than four alcoholic beverages daily, used marijuana more than once per month, were using medication, had a lifetime history of schizophrenia, bipolar disorder, attention deficit hyperactivity disorder, autism, bulimia, anorexia, anxiety disorder, obsessive–compulsive disorder or had a past 6-month history of major depressive episode. Nine participants were not included due to technical problems with the electro-oculography recordings . Data loss was related to EOG amplifier saturation . Three participants were kept in the analyses despite minor data loss . One gambler was further excluded because he met the DSM-IV-TR criteria for past year marijuana dependence, which is known to influence sEBR measures . As a result, the final sample comprised 20 participants . All participants provided written informed consent to take part in the study, which was approved by the regional research ethics committee .The study took place at the Radboud University Medical Center. Upon arrival of the participants, spontaneous eye blink rate was measured using EOG recordings . Approximately 1 h before entering the PET scanner, participants received 150 mg of carbidopa and 400 mg of entacapone to reduce peripheral metabolism of [18F]DOPA and increase tracer availability in the brain while having no psychotropic side effects. The participants further performed computerized tasks not reported here.We followed standard procedures for the acquisition, preprocessing and analysis of the EOG data .

Application of GA to developing na2 tassels enhanced their feminisation, supporting this hypothesis.When considering mutants with pleiotropic phenotypes, it is difficult to ascertain whether we are observing epistasis, additivity, or synergy simply because there may be epistasis for one phenotype, but not for another. Thus it is important to take into account all metrics when evaluating the mutant interactions.The fun and na2 mutants interact both additively and synergistically in different tissues. The fun mutant acts additively with na2 to reduce the overall height of the plant by further affecting the tassel and internodes. Since the d5;na2 double mutant also showed additive effects for height31, this could point to a role for FUN in the GA pathway. On the other hand, the synergistic interaction between fun and na2 at the auricle would point to FUN’s involvement in the BR pathway. These two data need not be in contradiction – Best et al.’s work clearly showed that the BR and GA pathways impinge on one another31, and the FUN protein itself may be involved in this crosstalk. Though there is very mild feminisation in the bri1::RNAi tassel, this is not an originally described phenotype of the bri1::RNAi line, nor is it common in family AV802 . Further, the metric of branch number does not indicate feminisation in the bri1::RNAi tassel, though bri1::RNAi is slightly shorter which can be considered a feminine inflorescence trait. Lack of feminisation in the bri1::RNAi tassel is surprising since removing BR causes feminisation in the tassel in the na1 and na2 mutants. Combining the bri1::RNAi line with fun leads to more feminisation than fun alone, implying not simple epistasis by the feminised tassel of fun, but rather an enhancement of the very mild feminisation caused by bri1::RNAi. Similarly,vertical cannabis grow the reduced height phenotype is enhanced in the double mutant . Phenotype enhancement, or additivity, is also observed at the auricle.

Though auricle size was not found to be smaller in bri1::RNAi plants in family AV802, a smaller auricle in bri1::RNAi plants has been reported75. Thus we can consider the completely absent auricle phenotype observed in bri1::RNAi;fun plants in family AV802, as compared to the bri1::RNAi and fun single mutants of AV802, as an enhancement of the reduced auricle associated with fun. No leaf width phenotype has been reported for bri1::RNAi plants, and fun appears to have simple epistasis of the leaf width phenotype.According to almost all metrics measured, fun is epistatic to bin2::RNAi. The oversized auricle of bin2::RNAi is completely abolished in the double mutant. Since increased leaf angle has been linked to BR hypersensitivity in rice76 as well as appearing in this bin2::RNAi line, it is reasonable to assume that this monstrous auricle is a product of BR hypersensitivity, and by extension, auricle growth is promoted by BR. The fact that the fun mutation abolishes this auricle growth is strong support for the function of FUN in the BR pathway, downstream of bin2::RNAi. On the other hand, the retention of leaf blade margin crenulations in the double mutant does not fit into this explanation, unless FUN is simply not expressed along the margin, which would be consistent with the Wab1;fun double mutant . The strong feminisation seen in the double mutant is further support for the placement of fun downstream of bin2::RNAi in the BR signalling pathway. BR is known to accumulate in developing anthers, and hence is a hormone associated with masculinity in maize. The fact that loss of normal FUN produces feminisation in the bin2::RNAi background also supports the hypothesis that FUN is in the BR pathway, downstream of BIN2. Taking together these phenotypes and interactions, this analysis supports the hypothesis that fun functions late in BR pathway, perhaps at its intersection with GA. Additivity with na2 for height is observed as with the d5 GAknockout mutant. At the same time fun enhances the feminisation phenotype of BR deficient na2 and acts additively with the BR insensitive bri1:RNAi while abolishing the phenotypes of the hyper-responder bin2:RNAi. Figure 4-11 shows FUN’s tentative placement in the BR pathway.

Classical mutants tasselseed1and ts2 were first described by Emerson in 1920 following the “freak” class exhibition of the Annual Corn Show in Lincoln, Nebraska 1913-1487. Together with the classical mutant silkless1, described by Jones in 192588, these mutants have elucidated the role of JA in sex determination in Zea mays. The ts mutants bear tassels that are heavy with seeds, so that they bend over from their own weight at maturity. All or most flowers will be female, but the tassel retains a normal degree of branching that is not seen in the ear or fun mutant tassels87. Upon cloning, ts1 was found to be a lipoxygenase that catalyses a step in the JA biosynthetic pathway35. While the function of the alcohol dehydrogenase encoded by ts236 has not been definitively shown, the similarity of the ts1 and ts2 phenotype, the lack of any interaction in the ts1, ts2 double mutant, and rescue of the ts2 phenotype by JA application35 supports the hypothesis that it is also in the JA pathway. The sk1 mutant is described as developing normal cobs but failing to produce any silks such that even stripping back the husk leaves and pollinating directly onto the ear failed to produce any kernels. No differences in the tassel nor the vegetative parts of the plant were originally noted but it was later observed that sk1 has less tassel branches than normal siblings. Combining sk1 with the mutants ts1 and ts2 put sk1 in the same pathway. Though in the first generations of the double mutant sk1 and ts2, partial epistasis was observed, two further rounds of self pollinations of these double mutants revealed complete epistasis by ts2. That is, both the tassel and the ear bear silks. From this it was concluded that sk1 in some way inhibits the silk killing product of ts292, supposedly, JA35,36. This has been supported by the cloning of SK1 as a uridine diphosphate -glycosyltransferase, the overexpression of which resulted in very low JA accumulation in developing tassels and a feminised tassel phenotype37. In summary: SK1 breaks down JA that would otherwise lead to pistil abortion. In a normal tassel, JA accumulates in the pistils and they abort; in a normal ear, SK1 degrades the JA and prevents silk abortion.Since addition of JA was unable to fully rescue the feminised tassel of fun plants, FUN is unlikely to be deficient in JA.

Though failure to correct the feminised phenotype was more obvious in the first application of JA family AV920 still showed a failure to rescue by JA application when branch number is considered. AV920 was not ideal for this experiment because the fun plants in this family were not heavily feminised. This could have been due to the fact that they were grown in the winter greenhouse – greenhouse grown fun plants have been observed to be less feminised than those grown in the field, and the winter greenhouse is particularly sub-optimal for growing corn. Further these plants were the fun-2 allele, which has not been adequately characterised, but may have lower feminisation severity than the original fun-1 allele. Finally, the genealogy of these plants contains a recent cross to Mo17, which may have reduced the severity of the phenotype ,vertical farming system as well as the fact that some of the fun plants were heterozygous for ts1 . Nevertheless, JA application to fun and ts1 plants in family AV920 did not refute the original experiment showing that JA is unable to fully rescue the feminised tassel phenotype of fun plants. ts1 and fun can be considered additive in their effects on feminisation of the tassel. Double mutant tassels were more heavily feminised than either double mutant, supporting the hypothesis that ts1 and fun are in different pathways. The lack of lower floret abortion in the tassel of ts1 and ts1;fun plants compared to successful lower floret abortion in fun plants further supports the hypothesis that fun is not deficient in JA since JA is required for lower floret abortion in the ear. The loss of silks in the sk1;fun further refutes the hypothesis that FUN is involved in the production of JA. A JA biosynthetic mutant would be expected to retain silks in combination with sk1, as previously shown with ts1 and ts2. The retention of feminised traits in the form of glabrous, thickened glumes in the double mutant implies an additive interaction – the fun mutation is still causing aspects of feminisation in the tassel, though the lack of functional SK1 cannot protect the silk from abortion by the action of JA. Branch loss in both sk1 and fun, and the additivity in the double is a complex set of observations. Though branch loss is a feminine trait since the ear is branchless and tassels are branched, the sk1 mutant also has branch loss. sk1 is presumably high in JA due to the loss of the SK1 gene that is responsible for JA degradation. This high JA allows silk abortion, and so would seem to be a masculine characteristic. Paradoxically, this loss of functional SK1 is also associated with the feminine trait of less branching in the tassel, implying a role for JA in branch inhibition during tassel development. Since there is an additive interaction between the sk1 and fun mutations, and addition of JA to developing fun tassels lead to tassels that resembled the sk1;fun double mutant tassels, the hypothesis of FUN being outside of the JA pathway is supported.If we consider JA and BR as masculinising hormones in Zea mays, GA can be thought of as a feminising hormone. In the 50s, Nickerson showed that application of GA directly to the whorl during tassel development was sufficient to induce feminisation of the terminal inflorescence. In the early 80s it was shown that developing ears have GA levels two orders of magnitude higher than in developing tassels.

The d1 mutant in maize was shown to block steps in the GA biosynthesis pathway and when the gene was cloned it was found to be a GA3 oxidase that catalyses the final step in bioactive GA synthesis. While the most striking phenotype of d1 is its tiny stature, more pertinent to this discussion is its ear phenotype, dubbed “anther ear”. Without the presence of GA, the anthers of the ear do not arrest and instead grow out to produce bisexual flowers in the ear while the flowers of the tassel are normal63. As such it is perhaps erroneous to call GA a simple feminising hormone – rather it is a male killer hormone. The mRNA coding for D1 protein was shown by in situ to accumulate in stamen primordia of the developing ear which undergo cell cycle arrest and ceases growth early in its development. d1 mutant plants do not undergo this stamen primordia arrest in the ear. Since the fun mutant was feminised in the tassel, we made crosses to GA mutants and applied paclobutrazol which is an antagonist of the GA pathway and has applications as a plant growth retardant and fungicide95. PBZ blocks the entkaurene oxidation step of GA biosynthesis, and thus plants treated with this compound are unable to produce GA. PBZ has been used to investigate the role of GA in maize, and to elucidate the nature of dwarf mutants in the GA pathway. The dominant D9 mutant was obtained from the maize stock centre and is similar to the D8 maize mutant, which both bear similarities to the biosynthetic d1 mutant. D8 and D9 are both dwarf, have increased tillering, and display varying degrees of anther ear. All the D8 alleles described have anther ear, while D9 displays full anther ear in some backgrounds, but in others it develops anthers up to the point that they are visible with a magnifying glass. The D8 locus on chromosome 1 encodes a DELLA protein, and D9 has been shown to encode a duplicate of this protein located on chromosome 5. DELLA proteins are part of the Gibberellic Acid signalling pathway, and act to repress the GA response.

By binding to a different, non-competitive allosteric site on the GABAB receptor, PAMs allow endogenous GABA, binding at its original orthosteric site, to retain its potency and efficacy, reducing the risk of tolerance development and side effects. A number of PAMs have been studied as potential pharmacotherapies for AUD, and have demonstrated reductions in alcohol-associated behaviors and ethanol self-administration in preclinical models. When directly contrasted against baclofen, a GABAB PAM had a better profile, with dose-dependent reduction of relapse-like alcohol drinking and with no signs of sedation. One such novel GABAB PAM, ASP8062, appears particularly promising as it has been shown to significantly increase the affinity and efficacy of endogenous GABA binding in human and rat GABAB receptors in vitro and with oral administration in an in vivo rodent model of fbromyalgia, demonstrating the ability of oral formulated ASP8062 to cross the blood-brain-barrier. ASP8062 has recently progressed to clinical development. Two Phase I clinical trials with a combined total of 112 participants evaluated single ascending doses and multiple ascending doses of ASP8062, respectively. These studies found that ASP8062 was well tolerated in humans, with no evidence of drug effects on safety, cognition, drug withdrawal, or suicidal ideation. One additional clinical trial, assessing the safety and efficacy of ASP8062 for alcohol use disorder , is currently underway. Overall, ASP8062, and GABAB PAMs in general, appear to be well tolerated in humans and decrease alcohol self-administration in animals. These agents present a potential pathway to better utilize the GABAergic system and reduce the side effects seen with GABAB agonists.Ghrelin,4×8 tray grow a peptide produced by endocrine cells primarily in the stomach, is thought to regulate growth hormone secretion, food intake, and glucose homeostasis. Ghrelin is also thought to play a role in AUD.

Ghrelin signaling is required for stimulation of the reward system by alcohol, and higher ghrelin levels are associated with higher self-reported measures of alcohol craving . In human laboratory studies, intravenous ghrelin administration has been shown to increase the urge to drink, increase alcohol self-administration, and modulate brain activity in regions involved in reward processing and stress regulation. Preclinical studies with ghrelin receptor antagonists have shown reductions in alcohol conditioned place preference, alcohol intake and preference, and alcohol-elicited nucleus accumbens dopamine release in rodents. PF-5190457 is a ghrelin receptor inverse agonist that inhibits GHS-R1a constitutive activity as well as blocking its activation by ghrelin. In a preliminary clinical study in 12 heavy-drinking individuals, PF-5190457, compared to placebo, reduced alcohol craving and cue-reactivity to alcohol. Additionally, when administered in combination with alcohol, PF-5190457 was safe and well-tolerated with no drug-alcohol interactions. This was the first clinical study of a GHS-R1a inverse agonist in a sample of heavy alcohol drinkers. PF-5190457 may increase somnolence, heart rate, and lower blood glucose concentrations, although clinical results indicate that these side effects were not exacerbated by alcohol co-administration and in general PF-5190457 is well tolerated. In summary, preclinical and early clinical evidence support additional research toward investigating PF-5190457 as a pharmacological approach to treat AUD.Cannabidiol , one of the main compounds found in Cannabis sativa, has shown promise as a novel therapeutic to treat AUD. CBD is nonintoxicating and has diverse pharmacological effects throughout the central nervous system, including functioning as a negative allosteric modulator of CB1 and CB2 receptors, and blocking anandamide update and inhibiting enzymatic hydrolysis. CBD may also interact with non-endocannabinoid signaling systems, including the serotonergic system and the opioidergic system, among others. Preclinical studies have shown that CBD reduces alcohol administration, decreases motivation for alcohol, reduces relapse-like behavior, and improves withdrawal symptoms in animals exposed to chronic alcohol.

Evidence in healthy individuals demonstrates that CBD is well tolerated, does not interact with the subjective effects of alcohol, and has no abuse liability. Two recent studies investigated signals for potential efficacy of CBD as a treatment for heroin use disorder and cannabis use disorder. Regarding heroin use disorder, acute CBD reduced cueinduced craving for heroin and reduced anxiety in a sample of 42 abstinent individuals, which was maintained one-week following the last CBD exposure. The cannabis use disorder study found that CBD was more efficacious at reducing cannabis use than placebo in a sample of 48 subjects. In both clinical samples, CBD was well tolerated and not associated with serious adverse events. CBD is currently being evaluated as a potential treatment for AUD, AUD with comorbid PTSD, and alcohol withdrawal in AUD in three clinical trials . In brief, preclinical evidence and clinical evidence in other substance use disorders indicate the promise of CBD as a novel therapeutic for AUD.This qualitative literature review discusses the efficacy, mechanism of action, and tolerability of approved, repurposed, and novel pharmacotherapies for the treatment of AUD. This information is summarized in Table 1. As of 2018, the APA recommends acamprosate and naltrexone for the treatment of AUD and suggests gabapentin and topiramate for patients with the goal of reducing alcohol consumption or achieving abstinence, while disulfram is suggested for achieving and maintaining abstinence only. Similarly, while not included in the APA’s recommendations, aripiprazole and mifepristone are associated with drinking reduction, while baclofen shows association with abstinence and mixed results with drinking reduction. Additional repurposed medications show clinical effectiveness for the treatment of AUD. Some of these appear to have particular promise in specific cases, such as varenicline’s use for nicotine and alcohol co-users, baclofen for individuals with liver disease, and aripiprazole for more impulsive individuals. Novel agents such as GET73 and ASP8062 have also reduced alcohol intake in preclinical studies.

In summary, while currently approved medications are somewhat effective, there remains a crucial need to develop new and improved pharmacotherapies for AUD. Novel and repurposed agents show significant promise as treatments that may improve upon currently approved pharmacotherapies.While considerable progress has been made in this field, there are a number of areas which require our attention to realize the benefit of AUD pharmacotherapy. First, despite the prevalence of AUD, the rate of seeking treatment for AUD remains very low. In order for anyone to benefit from the advances in medication development reviewed herein, the treatment gap must be closed. This will require engagement at multiple levels, from prevention to public education about AUD and the available treatments. Researchers and clinicians can help in these efforts by reducing stigma surrounding AUD and other substance use disorders by choosing appropriate language to describe these disorders and the people who are affected by them. A related issue is the need to improve access to FDA approved pharmacotherapies for AUD. A recent analysis of the 2019 National Survey on Drug Use and Health found that only 1.6% of people with a past-year AUD received an evidence-based medication to treat their AUD. Medication use was associated with living in a large metropolitan area,horticulture solutions use of the emergency department, and receiving mental health care, indicating that these services and residing in an urban environment appear to increase access to evidence based medications. There is also a great need to improve the education of physicians and clinicians on the availability of evidence-based treatments for AUD. Ongoing efforts in this area are underway by the American Society of Addiction Medicine and the American Academy of Addiction Psychiatry, as well as by the National Institute on Alcohol Abuse and Alcoholism. To further improve access to treatments and increase treatment-seeking, there is a need to increase the menu of approved pharmacological treatments towards AUD, especially those that have shown promise internationally. Currently, the FDA only accepts two primary outcomes for Phase 3 trials: abstinence and no heavy-drinking days. These outcomes do not always mirror the goals of patients with AUD for their recovery, which may be better reflected by a harm reduction endpoint.

Recent work has found that harm reduction endpoints, including reductions in WHO based drinking levels, are associated with improvements in physical health and quality of life and can be used as efficacy outcomes in clinical trials. The acceptance of these outcomes as clinical trial endpoints could have a substantial impact on the medication development field and ultimately result in a larger pharmacotherapy toolbox for clinicians.Another area of development is the move towards personalized treatment, also referred to as precision medication. AUD is a highly heterogenous disorder, and it unlikely that any medication will work for all individuals with an AUD. As such, there have been efforts to use precision medicine approaches to tailor pharmacotherapies to individuals with different presentations of AUD. Studies have taken several approaches towards personalized treatments, such as pharmacogenetics, sex differences, family history, severity of alcohol withdrawal, drinking phenotypes, and biobehavioral markers. However, even these efforts may be overly simplistic given the complex nature of AUD. It is likely that personalized treatment approaches will need to account for multiple factors to truly tailor treatments to individual patients. Conversely, the public health significance of the improved efficacy of AUD pharmacotherapy with clinically accessible phenotypes argue for wider dissemination and implementation of precision treatment recommendations identified to date. A final issue to consider is the need to develop treatments for individuals with AUD and comorbid psychiatric disorders and for individuals with AUD and AALD. AUD often co-occurs with other psychiatric disorders, including other substance use disorders, personality disorders, major depressive disorder, anxiety disorders, and PTSD. The development of integrated treatments, including combined behavioral and pharmacological interventions, which simultaneously address AUD and other cooccurring disorders, is difficult due to the complexity of treating multiple disorders and the limited understanding of the underlying mechanisms. However, this is a necessary area of research given the high rates of comorbidity in the AUD population. Treatment options for individuals with AALD are limited; of the FDA-approved medications, only acamprosate can be used without concerns of hepatotoxicity. Of the non-FDA medications that may prove useful in this population, only baclofen has been evaluated in an RCT. There is a clear need to develop additional treatments for this population.Unisexual flowers are the exception in angiosperms; the vast majority of flowering plants bear hermaphroditic flowers that have both pollen producing stamens and ovule-containing ovaries . Nevertheless, a substantial number of angiosperms produce nonhermaphroditic flowers. In a recent data analysis, 5-6% of plant species were found to be dioecious, but 43% of plant families were shown to contain dioecious members. The rarity of dioecy at the species level, coupled with its widespread occurrence across the angiosperm lineage, shows that dioecy must have evolved multiple times. In this chapter, we will first discuss the possible benefits and costs to producing imperfect flowers, before considering the likely routes evolution has taken to produce these floral types, and the molecular mechanisms that underlie the development of imperfect flowers. Through a sequence of case studies, this chapter will detail a likely route that an ancestor bearing perfect flowers could take to arrive in the derived states of dioecy and monoecy, through the intermediary steps of protogyny and protandry; andro- and gynomonoecy; and andro- and gynodioecy .A historical theory for why imperfect flowers might be beneficial was that it could promote outcrossing. Clearly a dioecious species is an obligate outcrosser, since the male and female flowers exist on separate plants. However, it is not so clear that this would be true for monoecious plants. It was hypothesised that if monoecy were to promote outcrossing inherently, monoecious plants would not have to rely on other costly mechanisms to promote outcrossing such as molecular self-incompatibility . Studies have not shown this to be the case, indeed monoecy is just as common in self-compatible species as it is it in SI species5 , though of course this does not rule out the possibility that avoiding self-fertilisation could still be a factor in the evolution of imperfect flowers, especially for dioecious plants. Instead of making a theory and trying to find data to support it, it is more fruitful to look at the natural world and make a theory to explain it. For this, we might ask: “what kinds of plants are likely to bear imperfect flowers?”. Analysis of large datasets has shown that species with imperfect flowers are likely to be viniferous or woody, wind-pollinated plants with small green flowers, and for dioecious plants, the bearing of fleshy fruits . One theory proposed by Bertin to explain enrichment in wind-pollinated plants is the relatively low cost of their flowers allows for monoecy to develop.

Emerging data indicate that e-cig use may adversely affect the pulmonary and cardiovascular systems. Also, limited evidence exists on the potential of e-cigs to cause carcinogenic effects. To date, clinical studies have primarily investigated the acute effects of vaping. By design, epidemiologic studies have explored the “association” of e-cig use with disease outcomes. So, the “causal” relationship between vaping and disease pathology remains largely unknown. Currently, determining the long-term health consequences of vaping is a top research priority. Of utmost importance is the long-term effects of vaping, especially among adolescents whose developing brains are more susceptible to influences on learning, memory, and attention. Future well-defined controlled experimental studies are needed to establish the mechanisms by which chronic vaping may lead to adverse health consequences in humans. These investigations are expected to identify the constituent of e-cig vapor, which are of most relevance to disease development. Though it is generally accepted that e-cig use exposes the users to fewer and lower levels of toxicants and carcinogens as compared to smoking, the net public health effect of vaping continues to be debated. The scientific community, regulatory authorities, and the general public are faced with competing views on the health risks or benefits of vaping. On the one hand, proponents of e-cigs advocate for: “harm reduction” potential of vaping, especially for smokers who are unable/unwilling to quit; and utility of vaping for smoking cessation. On the other hand, opponents argue against the adoption of an alternative tobacco product whose long-term health effects are yet to be determined. To reach a consensus, more conclusive scientific evidence will be needed; the available data favor the stance that vaping is likely to be less harmful than smoking, with the caveat that it may still pose a health risk on users,dry racks for plants which would otherwise be eliminated if neither product were used.

On June 22, 2009, with broad bipartisan support from Congress, President Obama signed into law the Family Smoking Prevention and Tobacco Control Act , also known as “Tobacco Control Act”. The FSPTCA granted FDA immediate authority and unprecedented power to regulate cigarettes,cigarette tobacco, roll-your-own tobacco, smokeless tobacco, and any other tobacco products that the agency, by regulation, deems to be subject to the law . On August 8, 2016, when FDA’s “Deeming Rule” took effect, many of the regulatory and statutory requirements that had been in place for manufacturers of the originally regulated tobacco products since passage of the FSPTCA in 2009, became applicable to the deemed products, including e-cigs and all other electronic nicotine delivery systems , cigars, pipe tobacco, nicotine gels, hookah tobacco, and any future products meeting the statutory definition of “tobacco product.” The applicable statutory provisions include the requirement that deemed products that meet the definition of a new tobacco product must receive premarket authorization from the FDA to be legally marketed. The premarket authorization requires a Premarket Tobacco Product Application for any new tobacco product seeking an FDA marketing order. In the deeming rule and subsequent guidance documents, FDA stated that it intended to defer enforcing the premarket review requirements, for a period of time, with respect to “deemed” new tobacco products that were on the market as of the effective date of the deeming rule. This policy did not extend to deemed new tobacco products that entered the market after the rule’s effective date . Under a federal court order, manufacturers of deemed new tobacco products that were on the market as of the deeming rule’s effective date, were required to submit premarket review applications by September 9, 2020. Following the court order, FDA accelerated its planning and preparation to receive a large number of applications by the premarket application deadline. Accordingly, the agency received thousands of submissions, representing more than 6.5 million products by the set deadline.

Per the court order, products for which applications were submitted by the deadline of September 9, 2020, could generally remain on the market for up to a year from the date of the application—or until September 9, 2021, at the latest—pending FDA review, although FDA retains enforcement discretion. Over the last year, FDA has worked to review thousands of PMTAs, representing products from roughly 500 companies.The vast majority of the PMTAs are for ENDS products. Under the PMTA pathway, manufacturers or importers must demonstrate to the FDA that permitting the marketing of the new tobacco product would be “appropriate for the protection of the public health,” among other things. That statutory standard requires the FDA to consider the risks and benefits to the population as a whole, including users and non-users of tobacco products. The FDA’s review of PMTAs includes assessment of a tobacco product’s components, ingredients, additives, toxicological profile, and health effects, as well as its manufacturing, packaging, and labeling processes, and data from consumer perception research , and the applicant’s description of marketing plans for the product. When reviewing PMTAs for ENDS products, FDA’s task is to follow the direction Congress provided in the law by taking into account the increased or decreased likelihood that existing users of tobacco products will stop using such products, as well as the increased or decreased likelihood that those who do not use tobacco products will start using such products. In making this determination, the impact of such products on youth initiation and use is a critical consideration. So far, the FDA has issued ruling on over 96% of the PMTAs submitted on or before the September 9, 2020 deadline, including 295 marketing denial orders for more than 1,089,000 flavored ENDS products. Products subject to a MDO for a premarket application may not be introduced or delivered for introduction into interstate commerce. If the product is already on the market, the product must be removed from the market or risk enforcement. On October 12, 2021, the FDA granted permission to R.J. Reynolds to market its Vuse Solo closed ENDS device and two accompanying tobacco-flavored e-liquid pods with a nicotine strength of 4.8%, which approximates the nicotine content of a pack of cigarettes.

The FDA simultaneously issued MDOs for ten other flavors submitted under Vuse Solo but declined to state which ones. RJR is the second-largest tobacco company in the United States , and a wholly owned subsidiary of Reynolds American Inc., after merging with the U.S. operations of British American Tobacco in 2004 . In a statement announcing the decision, the FDA said that “The authorized products’ aerosols are significantly less toxic than combusted cigarettes based on available data.” The statement concluded that “For these products, the FDA determined that the potential benefit to smokers who switch completely or significantly reduce their cigarette use, would outweigh the risk to youth,4 x 8 grow tray provided the applicant follows postmarketing requirements aimed at reducing youth exposure and access to the products”. The FDA’s decision came on the heels of the 2021 NYTS report showing an estimated 2.06 million U.S. middle and high school students as current users of e-cigs, with Puff Bar, Vuse, and Juul among the most popular brands. In its decision, the FDA stated that it was aware that 10% of high school students who used e-cigs, named Vuse as their favorite brand in the 2021 NYTS. Vuse has become the fastest-growing e-cig brand, accounting for over 26% market share in the top five markets, being the market leader in Canada, the UK, France, and Germany . In the United States, Vuse continues to outperform other brands, becoming the second largest and fastest growing player in the market in 2021. Currently, Vuse owns 33% of the market share in the United States whereas Juul owns 40%. The FDA’s decision to grant marketing orders for Vuse Solo and accompanying e-liquid pods marks a milestone in vaping regulations as this is the first time ever that a set of vaping products gets clearance from the agency. The cleared products can now be mass marketed and sold legally in the United States. As expected, the watershed decision by the FDA to green light Vuse Solo vaping products has been praised by the industry as well as proponents of e-cigs for harm reduction. Simultaneously, there have been swift and harsh criticisms of the decision by many experts in public health and medicine and numerous healthcare advocacy groups. As the FDA continues its premarket reviewing of the remaining PMTAs for ENDS products, including major brands such as Juul, and while teens’ favorite products, specifically Puff Bars , are being ordered off the market , Vuse is expected to continue its growth momentum owing to its “first-mover” advantage and industry leading FEELM ceramic coil. Alcohol use disorder is a highly prevalent, chronic, relapsing condition characterized by an impaired ability to stop or control alcohol use despite clinically significant impairment, distress, or other adverse consequences. It is the most common substance use disorder: in 2016, 100.4 million people globally were estimated to have an AUD. This disorder represents a significant public health concern. The WHO estimates that alcohol consumption is responsible for 5.9% of all deaths and 5.1% of global disease burden. Alcohol use and misuse is thought to contribute to over 200 related diseases and health conditions globally, including cardiovascular disease, cancer, liver cirrhosis, and injuries. AUD is also often comorbid with other substance use disorders, major depressive disorder, bipolar disorder, and other psychiatric disorders. In the USA alone, AUD is estimated to contribute to about 88,000 deaths each year. An estimated 44.6 million adults per year in the USA suffer from AUD, and 93.4 million adults in the USA will meet or have met AUD criteria at some point in their life.

Furthermore, the economic burden of AUD is estimated to be approximately $250 billion across the USA. Although AUD is an important public health concern, the disorder remains severely under treated with only 7% of adults with AUD in the USA and less than 10% in Europe receiving pharmacotherapy and/or psychotherapy treatment. Furthermore, the lag between the age at which AUD onsets and the age of first AUD treatment has been estimated to be eight years on average. Pharmacotherapies have seen limited use in the treatment of AUD, partially due to lack of addiction treatment training for medical professionals, lack of awareness regarding medication options , reluctance to prescribe and take medications, perceived low efficacy of medication, and stigma surrounding treatment. Indeed, only three pharmacotherapies are approved by the US Food and Drug Administration for use in AUD treatment. These medications are disulfram , acamprosate, and naltrexone. The European Medicines Agency similarly recognizes only these same three medications, as well as nalmefene, as established pharmacotherapies for AUD. These medications are only modestly effective and are under-utilized in treatment. A study conducted in 2019 found that only 1.6% of adults in the USA with past-year AUD received evidence-based AUD pharmacotherapies. Treatment outcomes for AUD differ widely across patients and medications. While abstinence may be desirable, it is infrequently obtained, such that only 16% of individuals in treatment for AUD achieve abstinence. Furthermore, evidence does not support abstinence as the only approach in the treatment of AUD. Not all individuals with AUD consider abstinence to be a goal of their recovery; only 2–6% of goals set in patient-driven treatment center on attaining alcohol abstinence. Non-abstinent recovery, including reductions in drinking and heavy drinking in particular , has been recognized to have health and societal value and has gained traction as a treatment target. Indeed, non-abstinent AUD recovery has been shown to be sustainable for up to 10 years following treatment. Despite growing recognition of the benefits of harm reduction, however, the most commonly prescribed pharmacotherapy to treat AUD remains disulfram, a medication advised strictly for abstinence. Furthermore, the heterogeneity of AUD suggests that it will be unlikely that one single medication will be effective for all individuals with an AUD. Therefore, there is a pressing need for the development of novel, diverse, and effective pharmacological treatment options for AUD with the hopes of increasing utilization and improving care.Specifically, this review provides a brief overview of currently approved medications and identifies new and repurposed agents “on the horizon” for which evidence indicates a potentially effective application toward AUD treatment.The following sections examine pharmacotherapies approved or in-development for AUD. Medications were selected for this qualitative review by considering gaps in existing review articles and the expertise of all authors; information was gathered via qualitative PubMed literature searches.

MANCOVA revealed significant between groups differences in SOPS P, GAF, and the 15-Analyte Index. Post-Hoc analyses using Bonferroni correction revealed that CHR- CTrauma subjects with a history of childhood trauma demonstrated significantly higher SOPS positive symptom scores than CHR- CNoTrauma, and CHR- NCTrauma, CHR- NCNoTrauma subjects. Given that criteria for conversion to psychosis is based on increased scores in the SOPS Positive scale, total childhood trauma may be an important independent variable associated with increased baseline SOPS positive symptoms and help identify a category of individual who are highest risk for conversion to psychosis. Further, CHR-CTrauma demonstrated lower GAF as compared to CHRNCNoTrauma subjects, whereas CHR- CNoTrauma did not demonstrate differences in GAF scores compared to either group of CHR-NC subjects. This again may imply that history of childhood trauma may be an important independent variable associated with increased baseline global functioning and thus help identify a category of clinical high-risk subjects who demonstrate the lowest global functioning. Finally, the 15-Analyte index did not differ between CHR-CTrauma and CHR-CNoTrauma subjects but did differ between CHR-NCTrauma and CHR-NCNoTrauma groups. Given that higher scores on the 15-Analyte index are associated with higher SOPS overall symptoms, lower global functioning, and lower social/role functioning, this finding may be interpreted to mean that CHR subjects with any history of trauma may demonstrate a unique population of subjects with worse clinical outcomes who would benefit most from early intervention. There are several possible limitations of this study that may explain the non-significant association between history of childhood trauma and pro-inflammatory cytokines . While sample size was adequate to detect small to medium between subjects’ effects,indoor cannabis grow system due to employing multiple between-subjects comparisons, risk for Type I error was increased.

Future research may consider use of open public data sets or consortium efforts in order to pool resources and maximize recruitment of sufficient samples to conduct analyses that require a large sample of subjects, such as exploratory factor analyses of inflammatory analytes. Further, measurement of childhood trauma in this study prevents evaluation of the effect of chronicity and severity of trauma to be evaluated. Future studies should consider use of the non-abbreviated Childhood Trauma Questionnaire: CTQ, in order to capture severity and chronicity of each individual sub-type of trauma endorsed. A review by Schafer and Fisher determined that instruments assessing childhood trauma, such as the CTQ, that were originally developed for the general population are also appropriate for use among people with psychosis. However, the use of self-report measures of childhood trauma is additionally prone to bias, particularly when subjects are under the age of 18. Thus, in order to prevent bias in reporting, future studies should employ use of informants that may be able to verify experience of childhood trauma in order to increase reliability of reporting. Moreover, inflammatory analytes and childhood trauma were only evaluated from one time point. Due to the age range of the at-risk sample , this might mean that ongoing changes in inflammatory analytes and additional trauma experiences that occurred during the course of the study, were not captured. Ongoing sampling of childhood trauma on inflammation must be evaluated across time in order to establish more reliable measures of these dynamic processes. The use of cross-sectional data for mediation analyses is limited, as temporal precedence cannot be established thereby preventing implications to be drawn regarding the causal effect of childhood trauma or inflammatory markers on clinical outcomes. While a study by Simpson et al. demonstrated that self-report measurement of childhood trauma in a first episode psychosis sample remained stable and consistent across multiple time points, they found that severity of trauma reported did fluctuate across multiple assessments. If unable to collect multiple biological samples from different time points, implementation of a stress test design with blood marker sampling would allow for testing the impact of trauma on stress reactivity and inflammatory response during the course of one visit.

Additionally, the validation of inflammatory markers in psychosis is ongoing; therefore, selection of specific markers of inflammation to measure at various phases of illness is not well established, nor is there a clear understanding of what inflammatory analytes may be differentially impacted by environmental stress as compared to disease progression. Measuring a large panel of serum markers of inflammation is preferable, but studies must be well-powered in order to establish reliable effects. Evaluating profile networks of inflammatory analytes is needed to understand the dynamic activation and suppression of analytes and provide a clearer understanding of immune system regulation as a whole, as compared to understanding of single analytes. Measurement of single inflammatory analytes provides only a small snapshot of a much larger and more complex picture that is the immune system. Finally, this study lacked assessment of variables known to affect inflammatory analyte levels, namely body mass index . While this study controlled for the effects of antipsychotic, antidepressant, tobacco, and cannabis use, BMI is highly associated with inflammation and thus may have confounded findings in inflammatory analytes. Taken together these results confirm existing research that individuals at CHR for psychosis demonstrate higher total childhood trauma as compared to unaffected comparison subjects and that history of childhood trauma is associated with increased positive psychosis-risk symptoms and worse global functioning. However, total childhood trauma was not associated with inflammation in this sample, thus analyses of the mediating effect of inflammatory analytes in the relationships between childhood trauma and clinical outcomes was non-significant. Instead, this study suggests that total childhood trauma and inflammatory analytes independently predict positive psychosis risk symptoms and lower global functioning; thus, these independent effects are additive. These findings confirm the importance of assessing for childhood trauma and blood-based inflammation in at-risk subjects as a means to identify individuals who may be at the highest risk for poor clinical and functional outcome. Childhood trauma and inflammation may seem difficult variables to target through existing evidence-based psychotherapy interventions. However, there is a growing body of research that supports the use of complementary and alternative medicine psychosocial interventions that may effectively target these factors in psychosis. The goal of research of CAM in psychosis is to replicate results from studies conducted in the general population demonstrating that the use of mind-body interventions reduces reactivity to stress and chronic inflammation.

For example, research Breines et al. demonstrated that higher levels of “self-compassion,” defined by Neff as “the attitude of treating oneself with kindness and non-judgmental understanding,” are associated with reduced IL-6 response in reaction to stress. More importantly, it has been demonstrated that self-compassion is not a “trait,” but rather a modifiable and alterable “state.” Cognitively-Based Compassion Training is a meditation-based program derived from Tibetan Buddhist mind-training that has been demonstrated to enhance empathy and compassion for oneself and others. Research on CBCT in medically stable populations by Pace et al. reveals 6 weeks of compassion meditation training reduced stress-induced immune responses in a stress-test design. Further, Pace et al. demonstrate that strength of reduction in immune response was not mediated by time spent meditating, indicating that benefits of compassion training are not dependent on long hours of practice. This is very relevant to the application and effectiveness of such techniques in children or adolescents,equipment for growing weed particularly those currently experiencing mental health concerns, given that it would be impractical to expect children with mental health concerns to engage in lengthy meditation practice. In fact, Pace et al. demonstrated the feasibility of CBCT in not only adolescents, but those in foster care with a history of early life adversity. Moreover, foster care program adolescents with a history of childhood trauma demonstrated significant reductions in salivary CRP after just 6-weeks of compassion training. Compassion training has not yet been evaluated in CHR population, but there is evidence that adapted mindfulness-based interventions are not only safe and therapeutic for use in chronic psychosis populations, but also may help to decrease individual distress around positive psychosis symptoms, such as auditory hallucinations and delusions . A recent systematic review by Louise, Fitzpatrick, Strauss, Rossell, and Thomas on “third-wave” cognitive behavioral interventions in psychosis, reveals that acceptance-based interventions show moderate effects in reducing depressive symptoms, but no effect in reducing distress around psychosis symptoms or improving functional outcome. Randomized-controlled clinical trials utilizing mindfulness-based interventions for early-psychosis are currently lacking. Nonetheless, these techniques represent a promising category of psychosocial intervention warranting further study as they may modify reactivity to stress and immune response. Other CAM interventions that warrant further study in psychosis populations to target immune response and clinical outcomes include exercise, diet, and cannabidiol. Exercise and diet have been shown to have robust effects on reducing chronic inflammation and improving health outcomes in the adolescents . Research on aerobic exercise in psychosis groups has demonstrated very promising findings, indicating that moderately intense exercises, such as walking or bike riding, may improve positive and negative psychosis risk symptomatology, cognition, and functional outcome . Further, these effects have been replicated in CHR populations .For example, a recently review by Stogios et al. reveals that unmedicated individuals with psychosis demonstrate increased appetite and cravings for fatty foods, which contribute to weight gain and metabolic disturbances known to be associated with higher levels of inflammation. Wu, Wang, Bai, Huang, and Lee revealed that a 6-month combined diet and physical activity program in schizophrenia subjects resulted in reduced BMI, improved metabolic profiles of insulin and triglycerides, as well as improved psychotic symptoms. Cahn, Goodman, Peterson, Maturi, and Mills demonstrated a 3- month mindfulness, diet, and yoga combined intervention resulted in increased levels of BDNF and increased cortisol awakening response in a population of medically stable adults.

Research on novel therapeutics, such as cannabidiol , as a potential treatment for psychosis have demonstrated that CBD may not only have neuroprotective, antioxidant, and anti-inflammatory effects, but also improve disease trajectory of psychosis by reducing positive psychosis symptoms, anxiety, and cognitive deficits in first episode psychosis groups . To date, there are no studies evaluating the effects of diet, exercise, CBD, or combined interventions on immune response to stress in CHR psychosis populations; however, there is strong evidence to warrant further study of these interventions in CHR psychosis groups. Finally, therapeutic interventions that are known to improve clinical outcomes for individuals who have experienced childhood trauma may be particularly important in mitigating long term functional impairments in youth at clinical high risk for psychosis. Bendall, AlvarezJimenez, Nelson, and McGorry describe several recommendations to be considered for good, quality, assessment and intervention withing individuals at risk for psychosis endorsing a history of childhood trauma, including, systematic inquiry about childhood trauma for all individuals with psychosis, and development of individualized treatment plan adapted from cognitive behavioral therapy approaches for the treatment of psychosis and trauma, paying particular attention to pacing of treatment and repeated assessment. Evidence based psychotherapies for trauma that include focus on stress management and interpersonal effectiveness such as Skills Training in Affect and Interpersonal Regulation , may be particularly meaningful for CHR subjects who have a history of childhood trauma. Schafer and Fisher demonstrated the effectiveness and tolerability of STAIR for individuals at clinical high risk for psychosis with history of childhood trauma. However, there has been little research evaluating the effectiveness of evidence-based trauma-focused treatments in this complex population. Studies evaluating the effectiveness of trauma focused interventions may include individuals with psychosis as only a small sub-sample of participants included in the research, but co-occurring psychosis spectrum disorders are often included as exclusionary criteria in evaluation of trauma-focused treatments for individuals with a history of trauma . As previously discussed, compassion training, such as CBCT, may represent a unique category of psychosocial intervention that helps to improve stress reactivity in youth who have experienced early life adversity . Moreover, PoehlmannTynan et al. demonstrated when parents completed 8-10 weeks of CBCT their children demonstrated reduced cortisol, indicating that compassion training for parents may have cascading effects of cumulative stress on their children. Although difficult to achieve, prevention of the occurrence of childhood trauma would be an ultimate goal. Varese et al. maintain that if childhood trauma was removed from the population entirely, the number of individuals presenting with psychosis would be reduced by 33%. Thus, assessment of childhood trauma is an essential first step toward not only early intervention in, but also ultimately prevention of psychosis spectrum disorders.

Similarly, in a study of undergraduate men and women, researchers found that negative urgency, a component of emotion dysregulation that includes the tendency to act rashly when distressed, was significantly associated with problematic alcohol use and disordered eating . Finally, Loxton and Dawe found that adolescent girls who abused alcohol and engaged in disordered eating were more sensitive to reward than adolescent girls who did not engage in any of these behaviors. Overall, extant literature highlights the complex nature of ED-SUD presentations. Thus, traditional treatment programs have targeted EDs and SUDs sequentially. However, interest in integrated treatment approaches has grown , and research indicates that patients who do not receive integrated treatment have poorer treatment outcomes . Nevertheless, there is limited research on what such an integrated approach should optimally target, and there is no consensus in the field about the best treatment modality for the ED-SUD population. One potentially promising intervention for ED-SUD is Dialectical Behavior Therapy , which is a treatment based on an emotion regulation model . In DBT, psycho education on this model is provided, and patients are encouraged to accept and learn to tolerate their emotional experiences, while also learning alternative methods of coping with their emotions. DBT is a well-established treatment for individuals with multiple and severe psychological disorders , and has been adapted for use with EDs . Its further adaptation and testing for individuals with co-occurring SUDs and BPD support its use to target multiple problem areas in an integrated manner.Findings from this study are promising, suggesting that integrated DBT for EDSUD treatment is associated with decreased substance use severity and frequency,vertical farming units decreased emotional eating, and increased levels of confidence in ability to resist urges for substance use .

Given the limited research on DBT for ED-SUD, a better understanding of factors associated with ED-SUD compared to ED or SUD alone may be helpful in identifying potential treatment targets to address both disorders simultaneously. The impetus for the current study was to add to this limited literature by reproducing previous research findings in a treatment-seeking ED population and discussing how these empirical findings can guide treatment recommendations for ED-SUD. Consequently, the present study examined differences between patients with EDs only to patients with ED-SUD on demographics, psychiatric comorbidity, and self-reported eating disorder and related psychopathology. Given previous research findings, we hypothesized that individuals with EDSUD would be more likely than ED only to engage in binge eating/purging, and to have a bulimic-spectrum eating disorder, BPD symptoms, higher rates of psychiatric comorbidities, self-harm, and suicidality, greater difficulties with emotion regulation, and more reward sensitivity.Participants were 98 adult patients admitted to a partial hospital program for EDs between August 2016 and November 2018. Participants completed clinical interviews and survey measures within 14 days of treatment admission. Eating disorder and comorbidities were diagnosed using either the Mini Neuropsychiatric Interview or the Structured Clinical Interview for DSM-5 Disorder administered by trained, bachelor’s-level research assistants. Suicidality risk was assessed using the MINI suicidality module. Thirty-six patients were diagnosed with a SUD. Of those, 19.4% were diagnosed with an alcohol use disorder and 25.5% were diagnosed with a non-alcohol SUD . Of the 25 patients with a nonalcohol SUD, 20% had a sedative-hypnotic-anxiolytic use disorder , 52% had a cannabis use disorder , 20% had a stimulant use disorder , 8% had an opioid use disorder , and 4% had a hallucinogen use disorder .Table 1 presents demographic differences between ED and ED-SUD patients.

There were no significant differences in age, BMI, length of illness, history of previous treatment, gender, ethnicity, diagnosis, or engagement in purging behaviors between groups, and only trend-level differences in racial background and the likelihood of engaging in objective binge eating episodes. Table 2 presents differences in comorbidity and psychotropic medication use at admission between ED and ED-SUD patients. ED-SUD patients had a significantly greater number of psychiatric comorbidities and were more likely to be taking a mood stabilizer at treatment admission compared to ED patients. There was a trend towards ED-SUD patients being more likely to be diagnosed with panic disorder and posttraumatic stress disorder compared to ED patients. Table 3 presents the differences between ED and ED-SUD patients on self-reported measures of eating disorder and related psychopathology. ED-SUD patients had higher scores on multiple sub-scales of the DERS—DERS Goals, DERS Impulse, and DERS Strategies—compared to ED patients. Additionally, ED-SUD patients reported significantly greater SPSRQ-Reward scores than those without a SUD. There was a trend towards individuals with a SUD reporting greater STAI-Trait, DERS Total, and SPSRQ-Punishment scores.The present study sought to describe differences between ED patients with and without a SUD at treatment admission. Results demonstrated that ED-SUD patients reported a greater number of comorbid psychiatric diagnoses and were more likely to be prescribed mood stabilizers. They also reported greater difficulty engaging in goal-directed activity, higher impulsivity, more limited access to emotion regulation strategies, and higher reward sensitivity. There were trend-level differences suggesting that individuals with ED-SUD were more likely to engage in objective binge episodes, be diagnosed with panic disorder and post traumatic stress disorder, and to report higher trait anxiety, global emotion dysregulation, and sensitivity to punishment. Results are largely consistent with our hypotheses and previous research demonstrating higher rates of psychiatric comorbidity , emotion regulation difficulties, and reward sensitivity in ED-SUD samples. Partially consistent with previous research , our results suggested a trend towards a higher frequency of binge eating in ED-SUD, although there were no differences between ED and ED-SUD groups on purging. Furthermore, patients with bulimic syndromes were not significantly more likely to have a SUD.

While this is somewhat inconsistent with previous research , results support examining substance use across ED diagnoses. In contrast, with previous research, we did not find evidence for higher levels of self-harm or BPD symptoms in the ED-SUD group. Previous research supporting increased self harm in ED-SUD has been in adolescent samples , which may also explain this discrepancy. While previous research has found higher cluster B symptoms in ED-SUD , the lack of significant differences between ED and ED-SUD in our sample may be due to the relatively high scores on the BEST in both groups. Indeed, both groups scored similarly to patient samples with BPD .Overall, results demonstrating a greater number of comorbid diagnoses for the ED-SUD group support the need for integrated treatment, which is consistent with recent calls from experts within the field . DBT takes a behavioral approach, treating behaviors, regardless of their diagnostic association, according to a specific hierarchy. Given the complexity of ED-SUD cases and the tendency for these patients to vacillate between ED and substance use behaviors over time , an integrated, transdiagnostic approach may be useful in treating both behavioral presentations. Importantly, we did not find evidence for ED diagnostic differences between ED-SUD and ED only groups, lending further support for a transdiagnostic approach to ED-SUD treatment. DBT provides a comprehensive framework for effectively working with the multiple comorbidities observed in ED-SUD patients. In particular, the focus on the DBT hierarchy may help address vacillation between ED-SUD and other comorbid symptoms. The DBT hierarchy systematically addresses the most severe and life-threatening symptoms first, to help avoid shifting treatment targets throughout treatment. Additionally,weed drying room skills generalization may be particularly important in this population. Phone coaching, which is a part of DBT, may be useful in helping patients to generalize skills to multiple behaviors across environments. Regarding specific disorders, the non-statistically significant elevation in the likelihood of PTSD in the ED-SUD group compared to the ED alone group suggests that trauma symptoms may be a relevant treatment target for ED patients generally. Indeed, groups are working to develop protocols for the concurrent treatment of ED and PTSD , while existing trauma protocols are commonly used to treat PTSD in these populations such as the DBT/Prolonged Exposure protocol .Our study shows that ED-SUD patients report significantly greater difficulties with emotion regulation. More specifically, ED-SUD patients in our sample endorsed difficulties with regulating behavior when distressed, engaging in goal directed behavior when distressed, and accessing strategies for feeling better when distressed. Moreover, ED-SUD patients were more likely to already be prescribed a mood stabilizer; thus, despite previous treatment for emotion dys regulation they continued to have difficulty in this area. This is consistent with our hypothesis and points to emotion regulation as a critical treatment target. As previously discussed, DBT was specifically developed to provide education on emotion dys regulation and provide individuals with adaptive emotion regulation skills. Several skills were added to the DBT for SUD model to specifically address the heightened impulsivity reported by ED-SUD patients. These skills include Burning Bridges to persons, places, and things associated with substance abuse and Adaptive Denial of urges for substance use.The present findings that patients with ED-SUD report higher reward sensitivity to highlight the importance of assessing for and addressing temperament in this treatment population. Reward sensitivity may be an underlying mechanism that drives an individual’s substance use and ED behaviors. For instance, substance use and ED behaviors may be highly rewarding in the moment; hence, patients seek the short-term rewards of addictive behaviors despite their long-term, negative consequences. Furthermore, a potential obstacle to abstinence from ED behaviors and substances of abuse is the non-rewarding aspect of abstinence . Several skills taught in DBT for SUDs target these barriers. Contingency management strategies to reduce cues and access to substances and behaviors , as well as reinforcement of adaptive behavior, are essential to treatment. Specifically, Community Reinforcement , and Abstinence Sampling focus on the reinforcement of healthy behaviors. In conjunction with findings on reward sensitivity, the trend towards the significance of increased punishment sensitivity in this ED-SUD population suggests that for some patients, holding patients accountable to treatment goals and implementing consequences and rewards accordingly may be important for behavior change.

For example, using behavioral contracts and administering drug analysis screens to monitor substance use may be helpful. The DBT skill of Pros and Cons may help patients to identifying negative consequences of substance use.The present study has several strengths, including the use of structured clinical interviews to assess diagnoses and an examination of a broad range of constructs theoretically relevant to eating and substance use disorders. As such, this study adds to the limited literature investigating factors characterizing the ED-SUD population. However, there are several limitations worth noting. First, participants were drawn from a treatment-seeking sample presenting at a higher level of care. As such, results may not be representative of individuals with ED-SUD in the broader community. The modest ED-SUD sample size may have limited our ability to detect significant differences between groups. Additionally, the present study did not assess tobacco use or caffeine use disorders, which may also be relevant substances for ED groups, given their association with appetite suppression. Further, although the present sample included males and non-binary individuals, the smaller numbers in these groups limits the generalizability of the results beyond females. Importantly, we did not assess the past history of SUD, so the relative influences of active substance use versus traits underlying substance use on our findings cannot be determined. Finally, this study reviewed factors that provide a rationale for the applicability of DBT to treat EDs and co-occurring substance use in a cross-sectional study; however, future longitudinal studies and randomized controlled trials are needed to examine outcomes to determine the efficacy of DBT to treat ED-SUDs.Psychoneuroimmunology refers to the study of interactions between behavior, neural and endocrine systems, and the immune system . Alder and Cohen state that the field of psychoneuroimmunology is intended to “emphasize the functional significance” of the relationship between mind and body systems “in addition to” and “not in place of analysis of the mechanisms governing functions within a single system.” This growing field seeks to understand the associations between environmental exposures and neural, endocrine, and immune systems, as well as the consequences of inflammatory responses on human behavior, to allow for new insights into mechanistic pathways that are involved in the development of psychopathology. Thus, identifying the impact of early life adverse experiences, such as childhood trauma, on immune system regulation, and subsequent clinical outcomes, such as functioning, provides important information regarding possible therapeutic targets for early intervention and prevention of psychopathology.

Falls result from an interaction between an individual’s underlying vulnerabilities and their exposure to environmental conditions.People experiencing homelessness have a high prevalence of factors known to be associated with falls in the general population, including chronic diseases, functional impairment, alcohol and opioid use problems.Homeless older adults have high prevalence of other factors that could be associated with falls, such as substance use and heightened exposure to physical violence.People who are homeless live in a variety of environments, including homeless shelters and unsheltered spaces that expose them to environmental hazards and violence. In each of these settings, homeless individuals have limited control over their environment, especially when living in unsheltered environments. We examined the prevalence of and risk factors for falls in a longitudinal cohort of adults aged 50 and older who were homeless at study entry. We hypothesized that homeless adults would have a high prevalence of falls and high exposure to environmental hazards. We hypothesized that factors known to be associated with falls in the general population would be associated with falls in our cohort. We further hypothesized that several factors that are plausibly related, but have not been studied , would be associated. We conducted a 3-year prospective cohort study of 350 homeless adults aged 50 and older, the Health Outcomes in People Experiencing Homelessness in Older Middle agE study.We interviewed participants at baseline and every 6 months for three years; at each interview, trained research staff administered a structured interview and conducted clinical assessments. The institutional review board of the University of California, San Francisco approved this study. The datasets we analyzed during the current study are available from the corresponding author on request. Between July 2013 and June 2014,vertical grow systems we recruited 350 adults age 50 or older who were homeless at study entry.

We recruited from all local shelters open to older adults , all free and low-cost meal programs that served at least three meals a week , one recycling center, and areas where adults slept unsheltered in Oakland, California . To create a sample that best represented the target population, including the high number of people living unsheltered in Oakland, we randomly selected potential participants using sampling frames that included encampment sites, recycling centers, shelters, and meal programs.We describe our Methods in more detail elsewhere .Eligibility criteria included: homeless according to the Homeless Emergency Assistance and Rapid Transition to Housing Act definition that includes any person living unsheltered, staying in an emergency shelter, or facing eviction in the next 14 days, age 50 years or older, English-speaking, and able to provide informed consent as determined by a teach-back mechanism.20Participants received $25 for the screening and enrollment interview, $5 for monthly check-ins, and $15 for follow-up interviews. Our primary outcome was self-reported falls in the prior six months, assessed at each study interview. We defined falling as “a sudden, unintentional change in position from an upright posture coming to rest on the floor or ground.” For descriptive purposes, among participants who reported a fall, we asked how many times the participant fell and whether they sought medical treatment for their fall. We identified demographic risk factors as time-constant and other risk factors, health status, and health-related behaviors as time-varying . We assessed age, gender, and race/ethnicity.In our analyses, we dichotomized race as Black versus non-Black. Participants reported their highest educational attainment. We classified participants as having graduated from high school or earned a General Educational Development certificate versus no high school diploma/GED. Using modified questions from the National Health and Nutrition Examination Survey , we asked participants whether a healthcare provider told them they had: myocardial infarction, congestive heart failure, stroke, arthritis, diabetes, or chronic lung disease ; we included these as separate variables.If a participant reported a medical condition at any time point, we considered them to have that condition in subsequent visits. We assessed visual impairment using the Snellen test, and defined visual impairment as corrected visual acuity <20/100.We defined hearing impairment as self-reported difficulty hearing.

To evaluate cognitive impairment, we used the Modified Mini-Mental State Examination . Those who scored below the 7th percentile or were unable to complete the assessment were defined as cognitively impaired. We asked participants about their ability to complete activities of daily living . We defined an ADL impairment as reporting difficulty with bathing, transferring, toileting, dressing or eating.We assessed lower extremity function with the Short Physical Performance Battery test and classified those who scored ≤10 as having reduced physical performance.We assessed urinary incontinence in the past six months by asking participants whether they had “leaked urine, even a small amount.” To assess exposure to environmental hazards at each visit, we used a residential follow-back calendar in which we asked participants to report each place they had stayed and number of nights in each setting during the prior six months.We considered being unsheltered as indicative of the highest environmental exposure. We defined an unsheltered environment as sleeping outdoors or any place not meant for human habitation .In preliminary analyses, we evaluated nights unsheltered as a 3-level variable and as a 6-level variable . Neither alternative exhibited a dose-response effect. Therefore, we used a dichotomous measure of any nights unsheltered in our analysis. Statistical Analyses To identify risk factors for falls, we chose independent variables based on our hypotheses. We assessed bivariate associations between a priori independent variables and recent falls using generalized estimating equations . We built our multivariable model by including variables with bivariate Type 1 p-values <0.20. If a categorical variable had more than two levels, we included all levels in our multivariable model if any Type 1 p-value was p<0.20. We reduced the model using backwards elimination retaining variables with p-values <0.05 in our final multivariable model. We conducted our analysis in SAS 9.4 using complete case analysis and robust confidence intervals . In a sensitivity analysis, we assessed whether we had underestimated the probability of falls due to incomplete follow-up or mortality. We examined the prevalence of falls amongthose: 1) with complete follow-up, 2) who had died during follow-up, or 3) who had not died but had missed any study visits over the 36-month study period. We used GEE to examine whether those who had died or missed visits were more likely to have experienced a fall in the past 6 months than those with complete follow-up. We included participants with a minimum of two visits.

We used weighted linear regression with a second order polynomial and zero intercept term to plot a trend line.At baseline,curing marijuana over one-third reported one or more falls in the past six months. Of the 118 participants who reported falling at baseline, 28.0% reported 4 or more falls, 35.6% two to three falls, and 36.4% one fall. One-third of participants who fell required medical treatment due to a fall. During the 36-month study, 28 participants died. Of those who survived, 183 completed all six follow-up interviews; 72 completed 4-5, 32 completed 2-3, and 21 completed one follow-up interview. We found a higher mean number of falls at baseline among those who died during follow-up 0.50) and those who had not died but had missed visits than among those who completed follow-up . Of the 350 participants, 218 reported one or more falls in at least one study visit; 107 reported falls in at least half of the visits; and 34 reported falls at all visits. Factors Associated with Falls Those who reported falls at baseline had a higher prevalence of known risk factors for falls than those who had not fallen . People with falls were significantly more likely to have less than a high school education, a history of stroke, difficulty with ADLs, mobility impairment, use of assistive device, increased urinary incontinence, and depressive symptoms. We found that those who fell were more likely to have moderate-to-high risk opioid and marijuana use, fewer social confidants, have spent at least one night unsheltered, or experienced physical assault. In models adjusted for key covariates, individual risk factors associated with significantly higher odds of falls included older age 1.00-1.06), being a woman , having non-Black race , having a history of stroke , reporting an ADL impairment , urinary incontinence , and use of an assistive device . Moderate-to-severe marijuana use and moderate-tosevere opioid use were associated with increased odds of falling. Experiencing physical assault and spending any night unsheltered in the last six months were as well. In this longitudinal study of adults 50 and older who were homeless at study enrollment, we found a high prevalence of falls. Despite a median age of 58 years, study participants reported a prevalence of falls higher than older adults with a mean age of 78 in the general population.3 Many participants fell repeatedly throughout the three-year study period; over a third of the cohort reported falls in at least half of their study visits. We found an association between falls and several factors known to increase fall risk within the general population, including older age, gender, functional impairment, urinary incontinence, use of an assistive device, and stroke. Our findings indicate that the increased risk for falls in homeless older adults results, in part, from a high prevalence of geriatric conditions and substance use known to increase fall risk.2 Some of these risk factors may be modifiable via physical and occupational therapy, although it is more difficult to intervene while someone is experiencing homelessness. As the average age of the homeless population continues to increase, the population will have increasing prevalence of geriatric risk factors.We identified novel risk factors: using marijuana, experiencing physical assault, and spending time unsheltered that contributed to the high fall prevalence in our population. Both opioid use and marijuana use were associated with increased odds of falling. Opioid use is associated with increased fall risk among older adults in the general population.However,despite research on marijuana use and injuries in community-dwelling older adults, little is known about how marijuana use impacts falls.Marijuana—like opioids—may increase falls by affecting the sensorium, inducing dizziness, confusion, and drowsiness.We found a high prevalence of marijuana use among study participants. People born in the study’s age cohort have had high prevalence of marijuana use their whole lives, including in older adulthood.As marijuana use among older adults increases, due to changes in legal status and cohort effects, there may be increased falls associated with its use. Experiencing physical assault is common among older adults who are homeless.Physical assault can increase fall risk directly , or indirectly, by causing injuries that enhance underlying individual vulnerabilities associated with falls.Future research should evaluate the role of marijuana use and physical assault in falls among housed older adults to determine whether these risk factors are unique to older adults experiencing homelessness. People who are unsheltered have increased exposure to unsafe environments, with minimal control. They may stay in isolated locations with uneven surfaces and physical barriers, such as abandoned buildings, under bridges, or along highways. Unsheltered environments lack lighting or protection against environmental hazards. Avoiding falls requires intact executive function and physical agility to be able to process external stimuli and modify movements to remain upright.For older adults with vulnerabilities—such as those common among homeless older adults—small external triggers may precipitate falls. Housed older adults are able to modify their behaviors to avoid high-risk environmental exposures that predispose them to falls. For example, they can decrease how often they walk outside on uneven surfaces or minimize their public transit use. In contrast, adults living in unsheltered settings have less ability to avoid high-risk environmental exposures.Our finding that non-Black race was associated with increased falls is consistent with research in housed adults.Homelessness is caused by an interaction between structural factors and individual risk factors. Because Black Americans face structural racism, Black Americans with less individual vulnerability are at risk for homelessness. While we adjusted for these conditions, there may be unmeasured confounders that we were unable to account for.Our study has several limitations. We rely on six-month recall of falls. Other studies of falls in older adults use time frames that range from monthly to biennial.Participants without complete study follow-up had a higher prevalence of falls, indicating that our model may have underestimated the odds of experiencing falls.

The stability in adolescents’ e-cigarette preferred type or brand also has not been examined. E-cigarette brands that are popular today among adolescents can deliver nicotine from a single compact pod that equals that of a pack of cigarettes, in attractive flavors, and with easy concealment for use in settings where cigarettes may be forbidden . These characteristics may facilitate the progression from intermittent to frequent use and nicotine dependence. Alternatively, low nicotine content and/or low device appeal may result in adolescents losing interest in e-cigarettes over time, with diminishing frequency and dependence risk. Among adults, research indicates that evolving from a simpler e-cigarette device to a more complex modifiable device is a common pattern and is associated with greater dependence on e-cigarettes. Despite rapid growth in the e-cigarette market in recent years, research has not yet examined whether or how adolescents’ preferred devices change over time, particularly with regard to nicotine delivery and exposure. Finally, minimal research has examined changes in adolescents’ reasons for initiating, continuing, and/or quitting e-cigarette use over time. In cross-sectional survey studies, adolescents’ top reasons for experimenting with e-cigarettes include curiosity, appealing flavors, friends’ use, and perceived benefits compared with cigarettes. However, reasons may shift over time, as adolescents move from experimentation to sustained use. The literature on youth initiation and transition to regular use of combustible cigarettes shows that media/marketing and social influences motivate initiation,hydroponic table whereas the drive for nicotine due to addiction motivates regular use. These nicotine product use patterns observed with combustible cigarettes warrant investigation with e-cigarettes.

With e-cigarettes, adolescents who begin experimenting because of curiosity or appealing flavors may subsequently use to alleviate withdrawal symptoms. The present study followed a cohort of adolescent e-cigarette users over 12 months’ time to examine patterns of e-cigarette use frequency, nicotine exposure, and dependence, product use and flavor preference, and motivators to use and cease use. The primary objectives were to determine persistence in e-cigarette and dual use and the stability in frequency and dependence measures of e-cigarette use. We also examined changes in device and e-liquid preferences and reasons for using e-cigarettes. This longitudinal study adds to the literature by providing an understanding of shifts in tobacco and nicotine product use over time among adolescents based on self-report and biomarkers of exposure.Adolescents from the San Francisco Bay Area who reported having used an e-cigarette at least once in the past 30 days and at least 10 times in their lives were recruited for a longitudinal study on teen vaping between May 2015 and April 2017. Advertisements were posted on social media and in the community around the Bay Area. Interested individuals were directed to the study Web site, where they could submit their information to be contacted by study staff to complete eligibility screening. Eligible participants who provided informed consent were scheduled for a baseline session where they completed self-report measures and provided a saliva sample for cotinine testing. Participants returned for follow-up measures and cotinine testing 6 and 12 months after baseline. Study incentives were $30 for the baseline, $35 for the 6-month, and $40 for the 12-month follow-up visits. Parental consent was waived under California law 6929. Cessation information and local treatment options were provided. The research design and study procedures were approved by the University of California, San Francisco Institutional Review Board.In this longitudinal study of adolescent e-cigarette use with self-reported and biomarker data, 80.3% of the sample continued to use e-cigarettes at 12 months, with significantly greater ecigarette use frequency, dependence, and nicotine exposure. The percentage of daily e-cigarette users doubled from 14.5% at baseline to 29.8% at 12-month follow-up.

The patterns of ecigarette use observed over time indicate substantial persistence and the use of greater amounts of nicotine over time . These findings lend support to concerns regarding the addictiveness of e-cigarettes for adolescents. In the United States, prevalence of past-month e-cigarette use increased dramatically among adolescents in 2018, whereas cigarette use declined and cannabis use remained constant. Results of this study suggest that increased prevalence of recent e-cigarette use may lead to frequent use, dependence, and greater nicotine exposure. Dependence scores at baseline were low on average, with most participants meeting a classification of “not dependent,” and 13.3% meeting a classification of moderate to heavy dependence. By 12 months, the percentage classified with moderate to heavy dependence increased to 23.3%. These findings would suggest that factors other than dependence are driving early use of e-cigarettes, and that over the course of just 1 year, more teens become daily users and more heavily dependent. Along with the self-reported increase in frequency of e-cigarette use and dependence, cotinine levels increased over time, reflecting increased exposure to nicotine. The increase in cotinine levels may be both the result of increased dependence and a catalyst for the development of dependence. Adolescents who become increasingly dependent on e-cigarettes may increase their nicotine use, thereby worsening dependence. Notably, devices with higher nicotine yield became increasingly popular over the course of the 12-month trial, consistent with the reports of greater nicotine dependence and higher cotinine levels. Transitions from single to dual and dual to single nicotine product use were observed in approximately one in three users over the study period. None of the baseline dual users abstained from both products at either follow-up, which may be partially due to their higher dependence on e-cigarettes at baseline, as well as the normalization of smoking behavior and associations between smoking cues that can perpetuate use of both products. Consistent with prior research, adolescent participants offered a wide range of reasons for e-cigarette use. The top three reasons for initiating and continuing use were socializing, enjoyment, and flavors. The top three reasons for quitting were a desire for self-improvement, difficulty maintaining an e-cigarette device, and getting in trouble for vaping at home or school. The top flavors were fruit, menthol/mint, and candy.

Taken together with experimental research demonstrating the influence of flavors on adolescents’ product choices, these findings suggest that efforts to reduce adolescent e-cigarette use ought to include regulatory action that addresses kid-friendly flavors. Little research has examined adolescents’ reasons for quitting e-cigarette use, and our findings preliminarily suggest that adolescents perceive parental controls and appropriate disciplinary consequences to be impactful.In 2018 in the United States, over 650 bicyclists died, and there were almost 158,000 bicycle-automobile collision–related injuries.1 Current bicycle injury prevention measures that have been proven include bicycle helmet programs and bicycle helmet laws.Promising prevention measures include active lighting, increased rider visibility, and roadway modifications.Trauma registries can be used to identify modifiable injury risk factors for trauma prevention efforts, including bicyclist collisions with automobiles. However, these may miss factors useful for prevention of bicycle-automobile collisions, such as vehicle speeds, driver intoxication, and patient group characteristics, such as financial stress, educational level, and languages spoken. Geographic information systems use software that can relate seemingly unrelated data to provide better understanding of spatial patterns and relationships. The GIS studies in the trauma literature include optimizing trauma center location,identifying under serviced areas for quality trauma care,hot spot and cluster analysis for traffic collisions,and helicopter basing and efficacy.Trauma registries typically already contain some geospatial data, such as home and injury location addresses. Traffic records databases contain details about bicyclist automobile collisions not typically found in trauma registries, such as vehicle speeds, driver intoxication,grow rack street and lighting conditions, and driver or cyclist fault.These records also include accurate exact collision locations, allowing GIS mapping. The GIS analysis also allows the use of census tract demographic data for both the collision location and patient’s home for analysis.We hypothesize that GIS analysis of trauma registry data matched with a traffic records database could identify additional risk factors for bicycle-automobile injury helmet use or intoxication. We also hypothesize that the addition of GIS analysis to the trauma registry will better inform injury prevention efforts.The trauma registry of the UC San Diego Level I trauma center was used retrospectively to identify bicycle-motor vehicle collision admissions from January 1, 2010, to December 31, 2018. Data collected included demographics, home and injury location addresses, injury severity scores, blood alcohol, toxicology, helmet use, hospital length of stay and mortality. Matching of the registry cases with the California Statewide Integrated Traffic Records System was done to provide collision, bicyclist, driver, and geospatial information for bicycle-automobile collisions within the County of San Diego for the same period. Statewide Integrated Traffic Records System is administered by the California Highway Patrol, and includes all traffic collisions in California with a law enforcement report. Matching was deterministic and was done by bicyclist age, bicyclist sex, zip code, date and time ±1 hour between the SWITRS collision time and the trauma registry admission time transfers from outside the County of San Diego were excluded. Outcomes of interest included toxicology—available as “Alcohol Involved” in SWITRS collision data and “Party Sobriety” under SWITRS party data or from blood alcohol and urine toxicology in the trauma registry. Helmet use was found as reported in SWITRS or the trauma registry. Missing variables in either database were managed by excluding such cases from analysis using that variable. Geocoding, mapping, and geospatial analysis of matched case SWITRS collision locations was done using ArcGIS Pro 2.6 .

Registry home addresses of cases were also geocoded and used with US Census Bureau census tract data to provide the below poverty level percentage for home census tracts. The educational attainment level for cases was done by selecting the predominant education level within the patient’s home address census tract from the US Census Bureau’s American Community Survey 2014 to 2018 5-year estimates, Table B15002. The language spoken at home was selected by home address census tract in the U.S. Census Bureau’s American Community Survey 2014 to 2018 5-year estimates, Table B16007. Locations of bike lanes in San Diego County for analysis of their associations with collisions were obtained from SanGIS for Bike Paths , which are physically separated from traffic; bike lanes , which are defined by pavement striping and signage on streets; and Bike Routes , which are shared use with motor vehicle traffic in the same travel lane and designated by signs only. Locations of alcoholic beverage control licenses in San Diego County for analysis of association with intoxicated bicyclist collisions was obtained from the California Department of Alcoholic Beverage Control via SanGIS and esri. The spatial clustering of bicycle-automobile collisions and hot spots were assessed using spatial auto correlation via the Getis-Ord Gi* statistic in ArcGIS Pro.Analyses of descriptive and tabular data were performed with IBM SPSS Statistics 27. χ2 Analysis was done for categorical data, means were compared via ANOVA and distributions of educational level, LOS, and head-neck Abbreviated Injury Scale were compared with the Mann-Whitney U test. Two-sided p values less than 0.05 were considered significant. Binary logistic regression was used for the outcome variables of helmet use and toxicology, selecting factors with a p value less than 0.01 in the univariate analysis. The SWITRS data were used under the terms of the data use agreement provided by the California Highway Patrol. The study was exempted from further review by the UC San Diego Human Research Protections Program.We have shown that GIS analysis of trauma registry data matched with a traffic accident records database can identify additional risk factors for bicycle-automobile injuries. We have also shown that our injury prevention efforts will be better informed by the hotspot analysis which clearly demonstrates clusters in specific geographic areas of the catchment area. The ability to add census tract data to registry data shows associations of education level and poverty level on bicycle helmet use. Census tract data also provides useful information for injury prevention efforts such as the predominant language spoken at home in target areas. Trauma registries, whether trauma center-based or nationally collected, should be constructed to allow geospatial analysis. Helmet use by bicyclists has been shown to reduce the risk of serious injury.Despite this, we saw relatively low use of helmets in admitted bicyclists.This may be reflected in our results showing adverse census tract poverty level and educational levels having an association with lack of helmet use. Unhelmeted cyclists in this study were also less likely to be discharged to rehabilitation or long-term care facilities, this is likely due to their being relatively underinsured compared with helmeted cyclists.

Adjacent research has found that sleep quality is malleable and thus can be improved even in clinical populations.These findings offer promising evidence for advancement in clinical staging models and future sleep-related therapies for both CHR and overt psychosis, such as an upcoming trial by Waite et al.Another budding direction of research involves computational advances in causal discovery analysis, offering innovative approaches to addressing causality in the context of observational data.Such methods require careful consideration of assumptions and properties underlying the data at hand,but could be utilized in future analyses of these and other prospective longitudinal datasets involving CHR and other clinical populations. Limitations of this study include the use of a self-report to assess sleep. The PSQI and the RU-SATED have high validity and reliability and subjective sleep problems have been the primary focus of sleep treatments. Such assessments, however, are fundamentally different from electrophysiological sleep characteristics that may be differentially associated with conversion and symptom levels. Therefore, the use of both self-report and electrophysiological sleep measures over time may inform clinical risk models and constitute targets for intervention. Other notable limitations include the significant dropout rates, the absence of ongoing sleep assessments succeeding the 8-month follow-up, and the relatively small sample size of our converting group . Furthermore, we acknowledge that pre-registering our study hypotheses would have strengthened our findings.The COVID-19 pandemic has produced major disruptions in daily life. A recent review concluded that the world population is experiencing increased stress, anxiety, and depression due to the pandemic and associated mitigation measures . In late June 2020, 40% of U.S. adults reported experiencing mental health symptoms,hydroponic table a stress disorder, or increasing substance use to cope with pandemic-related stress . Individuals with problematic substance use are particularly vulnerable to COVID-19 illness and psychosocial effects of the pandemic, such as stress and substance use treatment disruptions .

Disruptions in treatment increase risk of overdose, a serious complication of substance use disorders . Moreover, depression and anxiety commonly co-occur with substance use disorders . Evidence suggests that in the general population, increases in alcohol consumption during COVID-19 may be driven by stress, depression, and anxiety . Effects may be even more pronounced among individuals with problematic substance use. Periods of intense stress can give light to pre-existing fissures and vulnerabilities in people’s daily lives. A better understanding of substance use problems, depressive symptoms, and anxiety during the COVID-19 pandemic among people with problematic substance use can inform intervention efforts needed now and post-pandemic. Various pandemic-related situational factors may differentially affect substance use problems . According to behavioral economics, the COVID- 19 pandemic and associated mitigation measures may decrease the negative consequences of substance use . Many individuals are now working from home and have a more flexible schedule, making some of the negative consequences of substance use less relevant than before the pandemic. Simultaneously, the pandemic has limited the availability of alternative rewarding activities that are incompatible with substance use . Substance use and associated problems may increase if individuals are home more often and unable to engage in their usual activities, especially in the evenings . Moreover, other responsibilities at home may have increased during the pandemic. New consequences associated with substance use may emerge as lifestyles, schedules, and responsibilities shift. In sum, relationships between pandemic-related lifestyle factors, substance use, and substance use problems are likely complex and multifaceted. To better understand how individuals with problematic substance use are experiencing the pandemic, this exploratory, cross-sectional study examined associations between substance use problems, mental health symptoms, and pandemicrelated increased family responsibilities and stressors. Participants were recruited June 25 to August 18, 2020 for a randomized controlled trial of Woebot for Substance Use Disorders, a novel digital therapeutic for reducing problematic substance use.

Participants were recruited through Qualtrics Research Services, Stanford University listservs, a Facebook advertising campaign, and word-of-mouth. Eligibility criteria were U.S. residence, age 18-65, English literate, owning a smart phone, with past-year use of a substance, and problematic substance use . Exclusion criteria were history of severe alcohol or drug withdrawal, liver conditions, opioid overdose, or psychotic symptoms; past-year suicide attempt; past-year medical problems from drug or alcohol use; opioid use without concurrent medication-assisted treatment; past Woebot use; and pregnancy. The study protocol, approved by Stanford IRB, randomized consented participants to the Woebot for Substance Use Disorders intervention for 8 weeks or to a waitlist control condition. Only baseline measures, completed prior to randomization, were analyzed in the present study. Participants received a $25 Amazon gift card for completing the baseline survey.Correlations between substance use problems, pandemic impacts and behaviors, and mental health symptoms are presented in Table 1. Significant correlations indicated that participants who, during COVID-19, struggled with responsibilities at home, had greater mental health impacts, greater personal growth, frequented bars or large gatherings, and reported more depression and anxiety symptoms had higher SIP-AD scores. Participants who, during COVID- 19, struggled with responsibilities at home, had difficulty getting necessities, had greater mental health impacts, and worried more about their children had higher GAD-7 and PHQ-8 scores. Anxiety and depression scores were highly correlated. Participants who lost a job or income during the pandemic had higher PHQ-8 scores. Struggling with home responsibilities was associated with job or income loss, greater difficulty getting necessities, greater pandemic mental health impacts, less personal growth during the pandemic, avoiding large gatherings, and avoiding school or work. Difficulty getting necessities was associated with greater mental health impacts. Personal growth was associated with lower likelihood of avoiding large gatherings, and worrying about one’s children was associated with greater likelihood of avoiding non-essential travel. The COVID-19 precautions were significantly associated with each other. Multi–variable analyses are presented in Table 2. Participants with greater pandemic related mental health effects had higher SIP-AD, PHQ-8, and GAD-7 scores.

Younger participants had higher SIP-AD scores, and participants without a college degree had higher GAD-7 scores . Age was not associated with GAD-7 or PHQ-8 scores, and education was not associated with SIP-AD or PHQ-8 scores. Sex, race/ethnicity, marital status, and other pandemic-related variables were not associated with SIPAD, GAD-7, or PHQ-8 scores. Because only participants with children completed the “worries about children” measure, we repeated the GAD-7 and PHQ-8 models excluding this variable. In the full sample, females had higher GAD-7 scores than males . Otherwise,grow rack only pandemic-related mental health effects remained associated with GAD-7 and PHQ-8 scores. During the COVID-19 pandemic, 40.6% of surveyed adults with substance use problems reported struggling with responsibilities at home. Struggling with responsibilities at home during the pandemic was associated with more substance use problems and greater depression and anxiety symptoms. Obtaining, using, and recovering from substances can impair one’s ability to fulfill obligations. New responsibilities resulting from the pandemic, such as full-time childcare due to school closures, may be difficult to fulfill while struggling with substance use. Results are consistent with extant literature showing that unpaid caregiving during COVID-19 was associated with increased substance use . Individuals struggling with substance use, anxiety, and depression may need support in meeting their own needs as well as their family’s needs during the pandemic. Additionally, frequenting bars or large gatherings, experiencing negative mental health effects, and perceiving more positive personal growth from the pandemic were associated with more substance use problems. Difficulty getting necessities, experiencing more negative mental health effects, and greater worry about one’s children’s well-being was associated with greater depression and anxiety symptoms. In multi-variable models, controlling for demographic characteristics, negative mental health effects of the pandemic were the strongest correlate of substance use problems, depressive symptoms, and anxiety symptoms. Participants with high scores on this measure reported frequently thinking about COVID-19, worrying about their health and/or the health of their family and friends, experiencing stress due to changes in social contacts and their lifestyles, worsening of their mental/emotional health, and sleep disruptions. Findings suggest that the COVID-19 pandemic is producing major concerns that may contribute to mental health symptoms, including problematic substance use. Many individuals are using substances to cope with stress and uncertainty around the pandemic . A nationally representative sample of U.S. adults conducted early in the pandemic identified increased risk of depressive symptoms among people with lower income, fewer savings, and more stressors . People with problematic substance use may also be at elevated risk for depressive symptoms. While some evidence suggests that pandemic-induced psychological distress is lessening in the United States , people with problematic substance use are vulnerable to the negative effects of the pandemic . In this study, participants with more substance use problems were less likely to avoid bars and large gatherings, corroborating concerns that substance use may increase risk of contracting COVID-19 . Participants struggling to control their substance use may have found it difficult to avoid settings in which they use. Paradoxically, individuals with greater substance use problems also perceived greater personal growth from the pandemic in the forms of strengthened relationships, new possibilities, awareness of personal strength, spiritual change, and increased appreciation of life. People with problematic substance use often experience intense emotions .

Experiencing intense emotions may have led individuals with substance use problems to be deeply affected by both positive and negative pandemic-related changes. Additionally, perceiving greater personal growth was associated with lower likelihood of struggling with responsibilities at home and lower likelihood of avoiding large gatherings. Participants who perceived personal growth may be a subset whose daily lives were less strongly affected by the pandemic. Study data are cross-sectional, and causal pathways cannot be determined. There may be bidirectional relationships between substance use problems and pandemic-related mental health symptoms and stressors. While pandemic-related stress may have worsened mental health symptoms and substance use, it is also plausible that individuals with preexisting mental health symptoms and more substance use problems were negatively impacted by the pandemic than those with milder symptoms. Longitudinal research is needed to fully understand how substance use and pandemic-related circumstances may impact one another. Results are also subject to recall bias, as all measures were self-reported. Participants may have had difficulty accurately reporting their substance use and mental health symptoms from the past two weeks. Data were not collected on general life stressors unrelated to the pandemic. Individuals with high levels of stress may have experienced more pandemic-related stressors, mental health symptoms, and substance use problems. Lastly, the sample was predominantly non-Hispanic white. People of color are at increased risk of contracting and experiencing complications from COVID-19 . Moreover, Hispanic and Black individuals were more likely to report increased substance use than non-Hispanic white or Asian adults, potentially due to increased stress . Different vulnerabilities may interact to influence experiences of the pandemic. All participants were enrolled in a clinical trial, were not experiencing severe medical problems from their substance use, owned smartphones, and were proficient in English. Hence, findings may not generalize to more impoverished, medically complicated, or diverse groups. Future research into pandemic-related stressors and substance use should aim to recruit a more diverse sample. Smoking and alcohol misuse often co-occur. In the United States, the prevalence of nicotine dependence among individuals with alcohol dependence is 45.4%, while the prevalence of any alcohol use disorder among adults with nicotine dependence is 22.8% . These co-dependent individuals have more difficulty quitting smoking . An outstanding problem among those with substance use disorders is their disproportionate valuation of the drug and their disproportionate allocation of resources to obtaining the drug compared to participating in other daily activities . This imbalance between drug-related vs. regular activities reflects reinforced drug consumption patterns , and the differences in how drugs and non-drug reinforcers exhibit differential reinforcement strengths can be operationalized using a concept known as Relative Reinforcing Efficacy . One validated laboratory approach to measuring the RRE of drugs is hypothetical purchasing tasks, which assess changes in drug purchase and consumption as a function of increasing drug price . The consumption pattern can yield the demand curve modeled by Q = Q0∗10k , an exponentiated version of the classic equation by Hursh and Silberberg . Q represents consumption at price C; Q0 represents consumption at or near price zero, α represents the rate of change in demand elasticity, and k is the span of consumption values in log units. Other demand indices derived from the demand curve include: break point , Omax , and Pmax .

To bypass the thiolase in the pathway, we chose a new source for 3-ketovaleryl-CoA, namely betaoxidation of valeric acid. Like fatty acid oxidation, the new pathway first activates valeric acid, a biomass-derived chemical, into valeryl-CoA by a CoA ligase. An acyl-CoA dehydrogenase then oxidizes valeryl-CoA into 2-pentenyl-CoA, which is then transformed by an enoyl-CoA hydratase and a 3- hydroxyacyl-CoA dehydrogenase into 3-ketovaleryl-CoA . We believed this pathway would be more efficient than the PhaA-dependent pathway for bypassing the highly reversible and promiscuous thiolase-catalyzed reaction. We then tested the proposed beta-oxidation LMVA pathway, which accepts valeric acid into C6-isoprenol. Two plasmids were constructed to express the pathway : 1) pJL01, modified from pJL15, containing genes encoding NudB, Micrococcus luteus acyl-CoA dehydrogenase, and E. coli FadB, a bifunctional enzyme that catalyzes the hydration and dehydrogenation reactions ; 2) pJL02, modified from pJL10, containing the genes that encode the enzymes in the LMVA pathway and a Cannabis sativa acyl activating enzyme, CsAAE1, for valeryl-CoA production . E. coli BL21 transformed with pJL01 and pJL02 was grown in the presence of valeric acid and induced to express the pathway genes. GC-FID and GC-MS confirmed the production of C6-isoprenol at 27.3 mg/L . In the production broth, we also detected isoprenol at 5.8 mg/L . Compared to the thiolase LMVA pathway, C6- isoprenol production increased fourfold, and the ratio of C6-isoprenol with isoprenol increased to 4.7 from 1.3. Next, we screened CoA ligase and acyl-CoA dehydrogenase homologs, catalyzing the beta-oxidation pathway’s first two steps. We chose CoA ligase and acyl-CoA dehydrogenase candidates that have been overexpressed in E. coli and assayed in vitro in previous studies. Moreover, most of these candidates have known kinetic data for medium chain fatty acid substrates .

For the CoA ligase,plant bench indoor we selected the phenylacetate-CoA ligase from Streptomyces coelicolor A3 , ORF26 from Streptomyces aizumensis , the medium-chain fatty acyl-CoA ligase from Streptomyces sp. SN-593 , and the CoA ligase from Penicillium chrysogenum , in addition to CsAAE1 from Cannabis sativa. For the acyl-CoA dehydrogenase, we selected the short-chain acyl-CoA dehydrogenase from Pseudomonas putida KT2440 and the butyryl-CoA dehydrogenase from Megasphaera elsdenii , in addition to the acyl-CoA oxidase from Micrococcus luteus. The gene sequences were codon-optimized for E. coli and cloned into the pJL02 and pJL01 plasmid series . With the constructs in hand, we tested C6-isoprenol production in E. coli. First, we screened the CoA ligases, catalyzing the first step in beta-oxidation, with the well-characterized MeD as the acyl-CoA dehydrogenase. After production, we used GC-FID to quantify C6-isoprenol in the broths. The C6-isoprenol production varied substantially with different CoA ligases, suggesting this is a key step in the whole pathway. Among the genes we tested, PcCL gave rise to the highest production at 58.6 mg/L , a result consistent with the reported kinetics data . Also, we noticed that the combination of CsCL with MeD had a decreased production of 6.9 mg/L, down from the 27.3 mg/L produced by CsCL/MlD, suggesting that MlD is a better homolog for the second step . This hypothesis was supported by the screening results of the second step catalyzed by acyl-CoA dehydrogenase. The PcCL and MlD pair gave the highest C6-isoprenol production at 110.3 mg/L, so we used this pair of genes for the first two steps of the beta-oxidation pathway in the following experiments. Endpoint optical density analysis was performed to evaluate the impact of the expression of different beta-oxidation genes on cell growth. The results indicated the expression of the CoA ligases and acyl-CoA dehydrogenase barely impacts the growth. The slight growth inhibition effect in the high C6- isoprenol production, e.g., the PcCL/MlD, may result from the toxicity of C6-isoprenol.

After optimizing the pathway genes to increase the production efficiency, we turned to the host genes that may degrade the key intermediate, 3-ketovaleryl-CoA. As mentioned before, this intermediate can be degraded by a thiolase into acetyl-CoA and propionyl-CoA. Therefore we focused on two E. coli chromosomal thiolase genes, atoB and yqeF, which have substrate preference for short-chain betaketoacyl-CoA and were proposed to degrade 3-ketovalerylCoA. We conducted in-frame single-gene knockouts to delete these two genes in E. coli BL21 in sequence, and PCR confirmed the genotypes of the knockout mutants . The intermediate strain E. coli BL21 ΔatoBand the final double knockout strain E. coli BL21 ΔatoB ΔyqeF were then used for C6-isoprenol production using the plasmid combination of pJL01-MlD/pJL02-PcCL. The production of C6-isoprenol and the consumption of valeric acid were quantified. The results showed that while the knockout of atoB did not impact the C6-isoprenol titer, the double knockout strain, 6C02, increased the C6-isoprenol production to 390 mg/L, and the C6- isoprenol yield from valeric acid doubled to 44 mol% over the wild-type strain . We noticed even after knocking out the thiolase genes, only around half of the valeric acid is transformed into C6-isoprenol. The loss of valeric acid may come from the evaporation and the flux into side directions in the metabolic network, such as the degradation of 3-ketovaleryl-CoA by other thiolases in E. coli .In the C6-isoprenol runs, we noticed that the levels of isoprenol were generally negatively correlated to the levels of C6-isoprenol. For the sources of isoprenol, we reason that in addition to the E. coli native MEP pathway, the LMVA pathway may contribute the major portion via acetoacetylCoA, instead of 3-ketovaleryl-CoA, to C6-isoprenol. Through the LMVA pathway, the productions of C6-isoprenol and isoprenol use the same precursor, acetyl-CoA, resulting in the negatively correlated levels of C6-isoprenol and isoprenol. Also, the knockout of the short-chain acyl-CoA thiolase increased the supply of acetoacetyl-CoA and the production of IPP, resulting in an increased isoprenol titer of 14.4 mg/L compared to 2.1 mg/L for the wild-type strain. 

Hence, we proposed that acetoacetyl-CoA is readily accepted by the LMVA pathway, and it’s betaoxidation precursor, butyric acid, might be a substate of the beta-oxidation LMVA pathway. To test this hypothesis, we fed 1 g/L butyric acid instead of valeric acid to strain 6C02 containing pJL01-MlD and pJL02-PcCL. After production, GC-FID and GC-MS confirmed the isoprenol production, quantified at 301.8 mg/L . This result validates butyric acid is a good substrate for the beta-oxidation LMVA pathway for IPP/isoprenol production. The successful transformation of butyric acid into C5 alcohols by C4A suggests that the beta-oxidation LMVA pathway has substrate promiscuity. To explore the substrate spaces of this pathway, we tested other fatty acids as substrates. Without supplementing hexanoic acid, E. coli 6C02 with the beta-oxidation LMVA pathway also produces a small amount of C7-isoprenol, confirmed by the synthetic standard using GC-FID and GC-MS . Supplementing hexanoic acid increased the production of C7-isoprenol significantly . Therefore, the betaoxidation LMVA pathway can activate hexanoic acid and transform it to C7-isoprenol, albeit with low efficiency. C7-isoprenol production without hexanoic acid supplementation is likely from endogenous hexanoyl-CoA in E. coli. We also tried fatty acid analogs with functional group substitutes, including 5- chloro-valeric acid,greenhouse rolling racks 4-pentenoic acid, 4-amino-butyric acid, 5,5,5-trifluorovaleric acid, and 4- bromobutyric acid. We conducted comparative GC-FID/GC-MS analysis and fragment search in GC-MS for lack of standards of the expected alcohol products. However, none of the expected substituted alcohols were detected, suggesting these fatty acid analogs are poor substrates for the beta-oxidation LMVA pathway . The substrate promiscuity assays suggest the beta-oxidation LMVA pathway can produce IPP and C7-IPP, expanding the product chemical space of the homoterpene biosynthesis platform. The isopentenols, including isoprenol and prenol, are drop-in alcohol biofuels and have versatile potential fuel applications: prenol is one of the top 10 Department of Energy Co-Optimization of Fuels & Engines gasoline blendstocks and has synergistic blending effects for research octane number , and isoprenol is the precursor of 1,4- dimethylcyclooctane , a high-performance jet fuel blend stock . Previous studies have shown that for alcohol biofuels, molecules with longer chain lengths have a better blend stability with conventional fuels, and are less hygroscopic than their shorter chain congeners . Our pathway to C6-isoprenol and C7-isoprenol from biomass-derived fatty acids makes it possible to produce these chain-extended isoprenol analogs sustainably. With the synthesized C6-isoprenol and C7-isoprenol, we were able to test some important fuel properties of these novel isoprenol analogs. We first estimated their water solubility based on their logP values. High water solubility contributes to the high hygroscopic nature of alcohol fuels, increasing the possibility of phase separation when blended with conventional hydrocarbon fuel. Also, for microbial bio-fuel production process, molecules with high logP and low water solubility will partition into the organic phase in a two-phase extractive fermentation, resulting in low product toxicity to the producing microbes . Increasing carbon chain length leads to decreasing polarity of alcohols, resulting in lower water solubilities. The logP of isoprenol analogs were determined using an HPLC method, with C4-C8 strain chain alcohols as references. The result revealed that the one-carbon increase of the chain length of isoprenol decreases the water solubility to 22.12 g/L from 65.36 g/L. Moreover, the addition of two carbons to isoprenol further decreases the water solubility to 6.6 g/L .

The decreasing trend of the water solubility was expected, and these data reflect the trend quantitatively. The energy density of the isoprenol analogs was determined by testing their gross heats of combustion using a standard method . The energy density tests revealed C6-isoprenol has a higher heating value of 35.524 MJ/kg, while C7-isoprenol had an HHV of 39.468 MJ/kg . These numbers are similar or higher to the HHVs of isopentenols tested in the same batch. We also determined the RONs of the 10% alcohol RBOB gasoline blends using the recently published AFIDA method . The results indicated that C6- and C7-isoprenols have comparable RON boosting effects to isopentenols, making these two chemicals potential blend stocks for gasoline blends. We previously constructed the homoterpene biosynthesis platform as a proof of concept that introduces terpene structural diversity at the precursor stage. Here we further optimized this platform towards practical application. The most significant change is the upstream pathway to the key intermediate, 3- ketovaleryl-CoA. Like the natural LMVA pathways, our previous pathway starts from propionyl-CoA, condensed by a thiolase into 3-ketovaleryl-CoA.First, thermodynamic analysis indicated the condensation reaction catalyzed by thiolase is endergonic with a positive Gibbs free energy change , suggesting the thiolase catalyzed condensation reaction is thermodynamically unfavorable. This calculation is consistent with the finding that PhaA homologs catalyze the degradation reaction better than the condensation reaction . Second, to our knowledge, almost all the reported thiolases that convert propionyl-CoA and acetyl-CoA into 3-ketovaleryl-CoA also convert two molecules of acetyl-CoA into acetoacetyl-CoA. Our later experiment using butyric acid as substrate in the beta-oxidation LMVA pathway suggests the LMVA pathway readily accepts acetoacetyl-CoA into IPP . Considering these two points and the relatively high concentration of acetyl-CoA in E. coli , we reasoned that it would be difficult for the thiolase LMVA pathway to make HIPP over IPP, complicating C16, C17 and C18 terpene biosynthesis. Instead, the beta-oxidation pathway is a more specific way to produce isopentenyl pyrophosphate analogs. Although background IPP production remains, either via the native MEP pathway or from endogenous acetoacetyl-CoA transformed by the LMVA pathway, the ratio of HIPP/IPP production is significantly improved, as the molar ratio of C6-isoprenol:isoprenol is over 60 in the production run using the E. coli BL21 ΔatoB host with 1 g/L valeric acid feeding. The high production of HIPP and its dominant content in the isopentenyl pyrophosphate analog pool will benefit future homoterpene biosynthesis efforts. Using isoprenol analogs as the final product, we successfully optimized the flux to HIPP in the homoterpene biosynthesis platform. The enzymes after the LMVA pathway leading to complex terpenes are more challenging to optimize because of their elusive enzymology and unusual substrates. Following HIPP production, an IDI is supposed to isomerize HIPP to HDMAPP. We could not detect the corresponding alcohol of HDMAPP, C6-prenol, in the E. coli production run with the expression of NudB and the thiolase LMVA pathway containing the IDI from Bombyx mori. While this IDI was confirmed in vitro to transform HIPP to HDMAPP specifically , the absence of C6-prenol in the production run suggests that this IDI does not work well in E. coli, or the hydrolyzed product of HDMAPP, HDMAP, is not well accepted by the E. coli endogenous phosphatases. Incorporating this IDI into the optimized beta-oxidation LMVA pathway may increase HDMAPP production by increasing substrate supply. Other Lepidopteran IDIs are also candidates for enzyme screening of this step.