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Harvesting Happiness: The Joy of Growing Your Own Cannabis

One debate in the HIV literature is the extent to which HIV disease is associated with accelerated aging as opposed to other comorbid conditions or other lifestyle factors associated with HIV. However, it is difficult to differentiate the effect of HIV itself versus the downstream effects of HIV or lifestyle factors associated with risk of contracting HIV . For example, many studies have documented higher risk of vascular risk factors including hyperlipidemia, type II diabetes, hypertension, and abdominal obesity in PWH likely due to the cardiometabolic side effects of ART, chronic immune activation, comorbid conditions that are more common in PWH , and increased risk of chronic stressors . Cysique & Brew propose that vascular cognitive impairment is implicated in the pathogenesis of neurocognitive impairment in PWH, particularly older PWH, given that cardiovascular and cerebrovascular conditions can cause alterations in the blood-brain barrier, altered vascular reactivity, and brain changes, particularly in white matter. A recent meta analysis by McIntosh et al., found that cardiovascular disease, particularly type II diabetes, hyperlipidemia, and current smoking, are associated with an increased risk of cognitive impairment in PWH. CVD has been associated with brain changes in PWH, but the majority find an association with abnormal white matter , which was not examined in the current study. Several vascular risk factors were examined as covariates and were not found to be significantly associated with cognitive outcomes; although it is important to note that this group is limited in that participants with more significant vascular comorbidities such as stroke or myocardial infarction were excluded for these analyses. Nevertheless,vertical grow equipment further exploration of vascular risk factors and how they are associated with cognition and brain aging in this cohort and PWH more broadly is of course warranted to further understand the effects of HIV versus the effects of comorbid conditions associated with HIV.

Comparing the results of the current study to the middle-aging literature is difficult. First, while brain changes due to AD pathology can begin in mid-life, it is still several years from midlife to when one would develop late-onset aMCI/AD; thus decades-long studies are needed to better understand brain changes in mid-life and how they relate to late-life AD. Therefore, the literature is sparse and generally relies on AD risk to examine memory and neuroimaging in mid-life. Second, many studies with an aging focus examine a memory composite and thus it is difficult to discern the association between delayed recall versus recognition and brain integrity from these studies. Even older-adult studies often do not specifically examine recognition memory as again they either examine aMCI diagnosis, which in the older adult literature does not necessarily imply recognition impairment, or a memory domain. Third, given the AD focus of middle-aging studies, many middle-aging studies focus on the medial temporal lobe and do not explore other regions such as the basal ganglia or the prefrontal cortex. From the sparse middle-aging research that examines both memory and neuroimaging, there is some indication that memory is associated with several neuroimaging correlates, most notably the medial temporal lobe. For example, the Wisconsin Registry for Alzheimer’s Prevention study, which focuses on adults aged 40-65 and is enriched with a family history of AD, has reported memory and neuroimaging associations. For example, in a study of 261 WRAP participants, those with subjective memory complaints had significant cortical thinning in the entorhinal, fusiform, posterior cingulate, and inferior parietal cortices and reduced amygdala volume compared to participants without subjective memory complaints. Subjective memory complaints were also associated with worse verbal memory . In 109 participants in the WRAP study, participants that were Aβ+, determined via PET imaging, exhibited significantly thinner entorhinal cortex, accelerated age-associated thinning of the parahippocampal gyrus, and performed worse across cognitive measures, although not significantly worse, compared with the Aβ− group . Approximately 65% of WRAP participants were female and approximately 95% of participants were non-Hispanic white. In a study of 210 adults aged 40-59 by Ritchie et al. , worse spatial recall and visual recognition as well as greater dementia risk were associated with lower brain and hippo campal volume. Overall, the middle-aging literature is quite limited, so it is difficult to discern if episodic memory, regardless of the type of memory , reliably associates with the medial temporal lobe in middle age.

While it is significant that these studies do find associations between memory, AD risk, and the medial temporal lobe as well as other brain structures, these studies do not report prefrontal involvement like that observed in the current study. Of note, the study participants in these two studies markedly differ from the CHARTER cohort; the CHARTER cohort was not enriched for family history of AD, is predominantly male , and this sub-sample is 50.5% African American/Black and 38.4% non-Hispanic White. However, Jak et al., , which examined men in their 50s , also found that MCI diagnosis was associated with smaller hippo campal volume, although only the hippocampus was examined in this study. One curious finding was that the post hoc analyses examining a sub-sample of participants showed that better delayed recall was associated with a thinner entorhinal cortex. The aim of these analyses were to examine and confirm that prior findings are applicable to participants that were ideally treated for HIV disease and did not have any current substance use that could confound results. Although it should be noted that full sample is already a group that somewhat differs from the general population of PWH in that this group excludes PWH with severe comorbid conditions, they have little to no current substance use, and are relatively well treated for HIV as compared to the general population . Nevertheless, this finding is opposite of what was hypothesized based on the literature. While thicker cortex has been associated with cognitive dysfunction in some settings, suggesting that it is the deviation from normal cortical thickness that is meaningful , within the HIV and AD literature this has not been observed. In HAND and AD , atrophy is consistently related to worse cognitive functioning. Therefore, it is likely that this is a spurious finding. Given that this is a relatively small sub-sample and may not be generalizable, and thus this finding should not be over interpreted. To further validate the specificity of memory and medial temporal lobe relationships and show that memory is not just related to overall brain integrity, processing speed and psychomotor skills were also examined in aim 1c. It was hypothesized that these two domains would be more associated with fronto-striatal structures implicated in HAND. In the entire sample, processing speed and psychomotor skills were not significantly associated with the medial temporal lobe as hypothesized. However, they were not significantly associated with prefrontal or basal ganglia structures either. Overall, these findings were not in line with the hypotheses,vertical grow factories and given that episodic memory was not associated with medial temporal lobes, these findings do not help to demonstrate that associations with the medial temporal lobe are specific to memory and not cognitive functioning in general.

In post hoc analyses, a thinner pars orbitalis was significantly associated with worse psychomotor functioning, which was somewhat in line with the hypotheses; however, the literature would suggest that we may expect psychomotor function to be more related to basal ganglia structures, particularly the putamen . Lastly, recognition and delayed recall and their association with the primary motor cortex were examined in aim 1d. It was hypothesized that memory would be at least less associated with the motor cortex given that the primary motor cortex is spared in AD. The sub aim was explored in order to complete double dissociation. Consistent with this hypothesis, episodic memory was not significantly associated with the prefrontal cortex. However, given that memory was not associated with the medial temporal lobe, this lack of an association is not meaningful and double dissociation was not supported. When examining covariates for this aim of the study, AIDS status and APOE e4 status were associated with worse delayed recall. Regarding AIDS status, nadir CD4 count has repeatedly been associated with risk of HAND both within the CHARTER cohort and in other cohorts around the world . While nadir CD4 count was examined as a potential covariate and was not found to be associated with delayed recall, AIDS status is of course associated with nadir CD4 given that an AIDS diagnosis is defined by either an opportunistic infection or if CD4 cell count drops below 200 cells per milliliter of blood at any point in one’s life . It is thought that greater immunosuppression is associated with CNS injury and those neurologic consequences may persist even after treatment with ART and immune recovery ; this highlights the importance of HIV identification and initiation of ART to avoid immunosuppression. Based on the estimated duration of HIV, most of these participants contracted HIV either before ART was available or in the era in which ART was not recommended to be initiated until after immunosuppression. This cohort on average is characterized by a history of immuno supression with immune recovery given that there is high rates of AIDS with evidence of immune recovery as evidenced by a median CD4 count of almost 500 and high rates of current ART use. Given that ART policies have changed and it is now recommended that ART is initiated immediately after diagnosis , continued research on aging with HIV will be needed to understand different cohort effects . Nadir CD4 has been associated with thinner cortex and smaller brain volumes throughout the brain, particularly in the parietal, temporal, and frontal lobes, and the hippocampus . Therefore, it is important to reiterate that episodic memory and prefrontal regions were significantly associated with one another even when accounting for AIDS status. Interestingly, one study found that low nadir CD4 was associated with reduced functional connectivity in the memory networks in APOE e4 carriers not but non-carriers . Regarding APOE status, several studies have examined the association between memory and APOE status in middle age, finding that APOE e4 carriers have similar memory and cognitive performance to non-carriers until the mid-to-late 50s when differences start to appear . Interestingly, the association between APOE e4 status and worse memory, specifically delayed recall but not recognition, was found within this group of PWH whose mean age was in the early 50s.

However, other early markers associated with preclinical AD, such as the association between memory and medial temporal lobe structures were not, although APOE by medial temporal lobe interactions were not explored. Previous HIV studies have shown mixed results when examining the association between APOE status and cognition within PWH. Within the larger CHARTER cohort, Morgan et al. found that APOE e4 status was not associated with a greater risk of HAND; however, this study was from an earlier time point in which participants were, on average, 44.1 years old. Moreover, in another CHARTER study by Cooley et al. in a sub-sample of CHARTER participants aged 50 and over, APOE e4 status was not associated with volumetric differences on MRI or MR spectroscopy metabolite analyses. However, these structural analyses may not have had the specificity to detect more minute differences in specific regions of the brain such as the medial temporal lobe. Nevertheless, the HIV literature is mixed as a review found that some HIV studies do find worse cognitive and brain integrity in PWH who are APOE e4 carriers whereas others do not . Several HIV studies, particularly in PWH over the age of 50, have found that APOE e4 status is associated with worse brain integrity in several regions including cerebral white matter, the thalamus, and temporal, parietal, and frontal regions. Additionally, one study comparing PWH to HIV-negative controls found APOE e4 carrier status to be beneficial in younger age, consistent with the well-documented antagonistic pleiotropy effect of APOE across the lifespan, but found that the negative effect of APOE e4 status in persons over the age of 50 was stronger in PWH compared to HIV-negative participants . Despite this one study, few studies have examined if there is a synergistic effect between APOE status and HIV status on cognition and brain integrity, although animal models do suggest possible mechanisms of a synergistic interaction between HIV and APOE status .

The fun and na2 mutants interact both additively and synergistically in different tissues

In wild-type plants, BES1 is in the nucleus in a BR inducible manner. When crossed to the bri1::RNAi line, the localization is no longer nuclear. BR insensitivity in this RNAi line was further supported by the presence of BR deficient phenotypes such as short, thickened leaves, shortened stature, and decreased auricle, phenotypes that were observed in Oryza sativa bri mutants. The bri1::RNAi line was also observed to have shortened internodes, especially those between the ear and tassel, and twisting in the leaves. The twisting was sometimes so extreme that leaves had to be removed to access the tassel since they clasped it and prevented it from growing out.Mutants in BIN2 were first identified in Arabidopsis thaliana as semi-dwarf plants that were not rescued by exogenous BR. The bin2 mutant phenocopied classical BR knockout mutants, but was found to contain a gainof-function mutation in the causative gene, rather than a loss of function like bri1 or deetiolated1. Loss of function in BIN2 gives a constitutive response to BR, which manifests in A. thaliana as long wavy petioles and narrow leaves. BIN2 is a serine /threonine kinase of the Glycogen Synthase Kinase- 3 family that phosphorylates BR transcription factors to tag them for degradation in the absence of BR. Thus, when BIN2 is knocked down, the plant is unable to degrade BR transcription factors in the absence of BR and therefore constitutively responds to BR. Maize has at least 10 GSK3 like kinases that were identified by BLAST, 8 of which are expressed in leaf tissue. All 8 of the GSK3 like kinases expressed in leaf tissue were knocked down by the RNAi line created by Dr Kir, so despite the possibility of functional redundancy among these genes,vertical grow factory the knockdown of BIN2 activity is likely to be real. While BR knockouts are usually associated with shorter plants, the constitutively BR responding bin2::RNAi line had an unexpectedly shorter stature than normal siblings.

This shortened stature was due to shortened internodes, but the internodes of the tassel were found to be longer in the bin2::RNAi plants. In concurrence with suppression of BIN2 in rice, auricles were larger in the bin2::RNAi maize line, but unlike the rice line, leaf blades were wider and longer, while the rice line had narrower, longer leaves. The maize bin2::RNAi line leaves also had distinctive crenulations along their blades.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. Taking each organ in isolation before coming to a synthesis can be a helpful approach. 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 height, 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 another, 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 line75, 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, 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 reported. 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 rice 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.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,vertical grow indoor 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 , 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 ts 2. 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 bio-active 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 normal. 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 fungicide. 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 mutant96, 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 glass96. 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.