The single-cell opioid responses in the context of HIV (SCORCH) consortium.

Substance use disorders (SUD) and drug addiction are major threats to public health, impacting not only the millions of individuals struggling with SUD, but also surrounding families and communities. One of the seminal challenges in treating and studying addiction in human populations is the high prevalence of co-morbid conditions, including an increased risk of contracting a human immunodeficiency virus (HIV) infection. Of the ~15 million people who inject drugs globally, 17% are persons with HIV. Conversely, HIV is a risk factor for SUD because chronic pain syndromes, often encountered in persons with HIV, can lead to an increased use of opioid pain medications that in turn can increase the risk for opioid addiction. We hypothesize that SUD and HIV exert shared effects on brain cell types, including adaptations related to neuroplasticity, neurodegeneration, and neuroinflammation. Basic research is needed to refine our understanding of these affected cell types and adaptations. Studying the effects of SUD in the context of HIV at the single-cell level represents a compelling strategy to understand the reciprocal interactions among both conditions, made feasible by the availability of large, extensively-phenotyped human brain tissue collections that have been amassed by the Neuro-HIV research community. In addition, sophisticated animal models that have been developed for both conditions provide a means to precisely evaluate specific exposures and stages of disease. We propose that single-cell genomics is a uniquely powerful technology to characterize the effects of SUD and HIV in the brain, integrating data from human cohorts and animal models. We have formed the Single-Cell Opioid Responses in the Context of HIV (SCORCH) consortium to carry out this strategy.

Moderator effects of working memory on the stability of ADHD symptoms by dopamine receptor gene polymorphisms during development.

We tested the hypothesis that dopamine D1 and D2 receptor gene (DRD1 and DRD2, respectively) polymorphisms and the development of working memory skills can interact to influence symptom change over 10 years in children with attention-deficit/hyperactivity disorder (ADHD). Specifically, we examined whether improvements in working memory maintenance and manipulation from childhood to early adulthood predicted the reduction of ADHD symptoms as a function of allelic variation in DRD1 and DRD2. Participants were 76 7-11-year-old children with ADHD who were genotyped and prospectively followed for almost 10 years. ADHD symptoms were rated using the Attention Problems scale on the Child Behavior Checklist, and verbal working memory maintenance and manipulation, measured by Digit Span forward and backward, respectively, were assessed at baseline and follow-up. After correction for multiple testing, improvements in working memory manipulation, not maintenance, predicted reduction of symptomatology over development and was moderated by major allele homozygosity in two DRD1 polymorphisms (rs4532 and rs265978) previously linked with variation in D1 receptor expression. Depending on genetic background, developmental factors including age-dependent variation in DRD1 penetrance may facilitate the link between improvements in higher-order working memory and the remission of symptoms in individuals with childhood-diagnosed ADHD. Furthermore, the current findings suggest that DRD1 might contribute minimally to the emergence of symptoms and cognitive difficulties associated with ADHD in childhood, but may act as a modifier gene of these clinical features and outcome during later development for those with ADHD.

Mismatch Rate of Empirical Antimicrobial Treatment in Fracture-Related Infections.

OBJECTIVES: To evaluate the current standard of care regarding empirical antimicrobial therapy in fracture-related infections (FRIs). DESIGN: Retrospective cohort study. SETTING: Level I Trauma Center. PATIENT SELECTION CRITERIA: Adult patients treated for FRI with surgical debridement and empirical antibiotics between September 1, 2014, and August 31, 2022. Patients were excluded if less than 5 tissue samples for culture were taken, culture results were negative, or there was an antibiotic-free window of less than 3 days before debridement. OUTCOME MEASURES AND COMPARISONS: FRI microbial etiology, antimicrobial resistance patterns (standardized antimicrobial panels were tested for each pathogen), the mismatch rate between empirical antimicrobial therapy and antibiotic resistance of causative microorganism(s), and mismatching risk factors. RESULTS: In total, 75 patients were included [79% (59/75) men, mean age 51 years]. The most prevalent microorganisms were Staphylococcus aureus (52%, 39/75) and Staphylococcus epidermidis (41%, 31/75). The most frequently used empirical antibiotic was clindamycin (59%, 44/75), followed by combinations of gram-positive and gram-negative covering antibiotics (15%, 11/75). The overall mismatch rate was 51% (38/75) [95% confidence interval (CI), 0.39-0.62] and did not differ between extremities [upper: 31% (4/13) (95% CI, 0.09-0.61), lower: 55% (33/60) (95% CI, 0.42-0.68, P = 0.11)]. Mismatching empirical therapy occurred mostly in infections caused by S. epidermidis and gram-negative bacteria. Combination therapy of vancomycin with ceftazidime produced the lowest theoretical mismatch rate (8%, 6/71). Polymicrobial infections were an independent risk factor for mismatching (OR: 8.38, 95% CI, 2.53-27.75, P < 0.001). CONCLUSIONS: In patients with FRI, a mismatching of empirical antibiotic therapy occurred in half of patients, mainly due to lack of coverage for S. epidermidis , gram-negative bacteria, and polymicrobial infections. Empirical therapy with vancomycin and ceftazidime produced the lowest theoretical mismatch rates. This study showed the need for the consideration of gram-negative coverage in addition to standard broad gram-positive coverage. Future studies should investigate the effect of the proposed empirical therapy on long-term outcomes. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

HIV-related cognitive impairment shows bi-directional association with dopamine receptor DRD1 and DRD2 polymorphisms in substance-dependent and substance-independent populations.

It has been postulated that drugs of abuse act synergistically with HIV, leading to increased neurotoxicity and neurocognitive impairment. The CNS impacts of HIV and drug use converge on the mesocorticolimbic dopamine (DA) system, which contains two main receptor subtypes: dopamine receptors 1 (DRD1) and 2 (DRD2). DRD1 and DRD2 have been linked to substance dependence; whether they predict HIV-associated neurocognitive disorder (HAND) is unclear. Using an advanced-stage HIV+ population, we sought to determine if drug dependence impacts the contribution of DA receptor polymorphisms on neurocognition. We observed that both DRD1 and DRD2 polymorphisms were associated with opiate and cocaine dependence (P < 0.05) in Caucasian subjects, but not African-American individuals. Using linear regression analysis, we examined the polymorphisms for associations with neuropsychological performance in global and cognitive domain T-scores (Motor, Processing Speed, Verbal Fluency, Learning, Memory, Executive Functioning, Working Memory) while controlling for opiate and cocaine dependency. In the Motor domain, we observed an association for two DRD2 polymorphisms (P < 0.05) in Caucasian subjects. The effects differed for substance dependence groups as the direction of the correlations with DRD2 were opposite to what was seen in subjects without these dependencies. In African-American subjects, associations were observed in nearly every domain, and again, the direction of the correlation differed between substance-dependent and substance-independent groups. We conclude that studies to examine genetic risk for HAND must carefully account for substance dependence patterns when assaying dopaminergic systems, as the neurobiological substrates of cognition in HIV populations may vary with tonic alterations secondary to chronic substance exposures.

An innovative assessment tool for evaluating narrative feedback quality among Medicine and Biomedical Sciences students.

Objectives: To develop a reliable instrument to objectively assess feedback quality, to use it for assessment of the quality of students' narrative feedback and to be used as a self-assessment instrument for students in their learning process. Methods: In a retrospective cohort study, 635 feedback narratives, provided by small groups of Medicine and Biomedical Sciences undergraduate students, have been extracted from available quarterly curriculum evaluation surveys. A rubric was developed based on literature and contents of our feedback education. It consists of seven subitems and has a maximum score of 20 points (sufficient score: >10 points). Rubric reliability was evaluated using intra-class correlation. The rubric was tested by analysing the feedback narratives. To test progression, we compared rubric scores between study years with a Kruskal-Wallis analysis and Dunn's post-hoc testing with Bonferroni correction. Results: The rubric has an intra-class correlation of 0.894. First year students had a mean rubric score of 11.5 points (SD 3.6), second year students 12.4 (SD 3.4) and third year students 13.1 (SD 3.6). Kruskal-Wallis testing showed significant differences in feedback quality between study years (χ2(2, N=635) = 17.53, p<0.001). Dunn's post-hoc test revealed significant differences between study years one and two (p=0.012) and one and three (p<0.001). Conclusions: The developed rubric is a reliable instrument to assess narrative feedback quality. Students were able to provide feedback of sufficient quality and quality improved across study years. The instrument will allow students to assess themselves and learn where there is still room for improvement.

HIV disease duration, but not active brain infection, predicts cortical amyloid beta deposition.

OBJECTIVE: Abnormal deposition of the antimicrobial peptide amyloid beta (Aß) is a characteristic of Alzheimer's disease. The objective of this study was to elucidate risk factors for brain Aß in a cohort enriched for HIV and other neurotropic pathogens. DESIGN: Cross-sectional cohort study. METHODS: We examined autopsy brains of 257 donors with a mean age of 52.8 years; 62% were men; and 194 were HIV+ and 63 HIV-. Hyperphosphorylated tau (p-tau) and Aß were identified in frontal and temporal regions by immunohistochemistry. APOE genotyping was performed. Clinical and neuropathological predictors for Aß were identified in univariate analyses, and then tested in multivariate regressions. RESULTS: Cortical Aß was identified in 32% of the sample, and active brain infection in 27%. Increased odds of Aß were seen with increasing age and having an APOE ε4 allele; for the overall sample, HIV+ status was protective and brain infection was not a predictor. Within the HIV+ population, predictors for Aß were duration of HIV disease and APOE alleles, but not age. When HIV disease duration and other HIV parameters were introduced into models for the entire sample, HIV disease duration was equivalent to age as a predictor of Aß. CONCLUSION: We hypothesize that dual aspects of immune suppression and stimulation in HIV, and beneficial survivor effects in older HIV+ individuals, account for HIV+ status decreasing, and HIV duration increasing, odds of Aß. Importantly, with HIV, disease duration replaces age as an independent risk for Aß, suggesting HIV-associated accelerated brain senescence.

Mice with altered serotonin 2C receptor RNA editing display characteristics of Prader-Willi syndrome.

RNA transcripts encoding the 2C-subtype of serotonin (5HT(2C)) receptor undergo up to five adenosine-to-inosine editing events to encode twenty-four protein isoforms. To examine the effects of altered 5HT(2C) editing in vivo, we generated mutant mice solely expressing the fully-edited (VGV) isoform of the receptor. Mutant animals present phenotypic characteristics of Prader-Willi syndrome (PWS) including a failure to thrive, decreased somatic growth, neonatal muscular hypotonia, and reduced food consumption followed by post-weaning hyperphagia. Though previous studies have identified alterations in both 5HT(2C) receptor expression and 5HT(2C)-mediated behaviors in both PWS patients and mouse models of this disorder, to our knowledge the 5HT(2C) gene is the first locus outside the PWS imprinted region in which mutations can phenocopy numerous aspects of this syndrome. These results not only strengthen the link between the molecular etiology of PWS and altered 5HT(2C) expression, but also demonstrate the importance of normal patterns of 5HT(2C) RNA editing in vivo.

Developmental modulation of GABA(A) receptor function by RNA editing.

Adenosine-to-inosine (A-to-I) editing of RNA transcripts is an increasingly recognized cellular strategy to modulate the function of proteins involved in neuronal excitability. We have characterized the editing of transcripts encoding the alpha3 subunit of heteromeric GABA(A) receptors (Gabra3), in which a genomically encoded isoleucine codon (ATA) is converted to a methionine codon (ATI) in a region encoding the predicted third transmembrane domain of this subunit. Editing at this position (I/M site) was regulated in a spatiotemporal manner with approximately 90% of the Gabra3 transcripts edited in most regions of adult mouse brain, but with lower levels of editing in the hippocampus. Editing was low in whole-mouse brain at embryonic day 15 and increased during development, reaching maximal levels by postnatal day 7. GABA-evoked current in transfected cells expressing nonedited alpha3(I)beta3gamma2L GABA(A) receptors activated more rapidly and deactivated much more slowly than edited alpha3(M)beta3gamma2L receptors. Furthermore, currents from nonedited alpha3(I)beta3gamma2L receptors were strongly outwardly rectifying (corresponding to chloride ion influx), whereas currents from edited alpha3(M)beta3gamma2L receptors had a more linear current/voltage relationship. These studies suggest that increased expression of the nonedited alpha3(I) subunit during brain development, when GABA is depolarizing, may allow the robust excitatory responses that are critical for normal synapse formation. However, the strong chloride ion influx conducted by receptors containing the nonedited alpha3(I) subunit could act as a shunt to prevent excessive excitation, providing the delicate balance necessary for normal neuronal development.

ADAR1 and ADAR2 expression and editing activity during forebrain development.

The conversion of adenosine to inosine within RNA transcripts is regulated by a family of double-stranded RNA-specific adenosine deaminases referred to as adenosine deaminases that act on RNA (ADARs). Little is known regarding the developmental expression of ADAR family members or the mechanisms responsible for the specific patterns of editing observed for ADAR substrates. We have examined the spatiotemporal expression patterns for ADAR1 and ADAR2 in mouse forebrain. ADAR1 and ADAR2 are broadly distributed in most regions of the mouse forebrain by P0, including the cerebral cortex, hippocampus, and diencephalon. High expression levels were maintained into adulthood. Colocalization studies demonstrated ADAR1 and ADAR2 expression in neurons but not astrocytes. Editing for specific ADAR mRNA targets precedes high expression of ADAR proteins, suggesting that region-specific differences in editing patterns may not be mediated solely by ADAR expression levels.

A Functional 3'UTR Polymorphism (rs2235749) of Prodynorphin Alters microRNA-365 Binding in Ventral Striatonigral Neurons to Influence Novelty Seeking and Positive Reward Traits.

Genetic factors impact behavioral traits relevant to numerous psychiatric disorders and risk-taking behaviors, and different lines of evidence have indicated that discrete neurobiological systems contribute to such individual differences. In this study, we explored the relationship of genetic variants of the prodynorphin (PDYN) gene, which is enriched in the striatonigral/striatomesencephalic pathway, a key neuronal circuit implicated in positive 'Go' behavioral choice and action. Our multidisciplinary approach revealed that the single nucleotide polymorphism (SNP) rs2235749 (in high linkage disequilibrium with rs910080) modifies striatal PDYN expression via impaired binding of miR-365, a microRNA that targets the PDYN 3'-untranslated region (3'UTR), and is significantly associated to novelty- and reward-related behavioral traits in humans and translational animal models. Carriers of the rs2235749G allele exhibited increased levels of PDYN 3'UTR in vitro and had elevated mRNA expression in the medial nucleus accumbens shell (NAcSh) and caudate nucleus in postmortem human brains. There was an association of rs2235749 with novelty-seeking trait and a strong genotype-dose association with positive reinforcement behavior in control subjects, which differed in cannabis-dependent individuals. Using lentiviral miRZip-365 constructs selectively expressed in Pdyn-neurons of the NAcSh, we demonstrated that the Pdyn-miR365 interaction in the NAcSh directly influences novelty-seeking exploratory behavior and facilitates self-administration of natural reward. Overall, this translational study suggests that genetically determined miR-365-mediated epigenetic regulation of PDYN expression in mesolimbic striatonigral/striatomesencephalic circuits possibly contributes to novelty seeking and positive reinforcement traits.

ELK1 transcription factor linked to dysregulated striatal mu opioid receptor signaling network and OPRM1 polymorphism in human heroin abusers.

BACKGROUND: Abuse of heroin and prescription opiate medications has grown to disturbing levels. Opioids mediate their effects through mu opioid receptors (MOR), but minimal information exists regarding MOR-related striatal signaling relevant to the human condition. The striatum is a structure central to reward and habitual behavior and neurobiological changes in this region are thought to underlie the pathophysiology of addiction disorders. METHODS: We examined molecular mechanisms related to MOR in postmortem human brain striatal specimens from a homogenous European Caucasian population of heroin abusers and control subjects and in an animal model of heroin self-administration. Expression of ets-like kinase 1 (ELK1) was examined in relation to polymorphism of the MOR gene OPRM1 and drug history. RESULTS: A characteristic feature of heroin abusers was decreased expression of MOR and extracellular regulated kinase signaling networks, concomitant with dysregulation of the downstream transcription factor ELK1. Striatal ELK1 in heroin abusers associated with the polymorphism rs2075572 in OPRM1 in a genotype dose-dependent manner and correlated with documented history of heroin use, an effect reproduced in an animal model that emphasizes a direct relationship between repeated heroin exposure and ELK1 dysregulation. A central role of ELK1 was evidenced by an unbiased whole transcriptome microarray that revealed ~20% of downregulated genes in human heroin abusers are ELK1 targets. Using chromatin immune precipitation, we confirmed decreased ELK1 promoter occupancy of the target gene Use1. CONCLUSIONS: ELK1 is a potential key transcriptional regulatory factor in striatal disturbances associated with heroin abuse and relevant to genetic mutation of OPRM1.

Proenkephalin mediates the enduring effects of adolescent cannabis exposure associated with adult opiate vulnerability.

BACKGROUND: Marijuana use by teenagers often predates the use of harder drugs, but the neurobiological underpinnings of such vulnerability are unknown. Animal studies suggest enhanced heroin self-administration (SA) and dysregulation of the endogenous opioid system in the nucleus accumbens shell (NAcsh) of adults following adolescent Δ(9)-tetrahydrocannabinol (THC) exposure. However, a causal link between proenkephalin (Penk) expression and vulnerability to heroin has yet to be established. METHODS: To investigate the functional significance of NAcsh Penk tone, selective viral-mediated knockdown and overexpression of Penk was performed, followed by analysis of subsequent heroin SA behavior. To determine whether adolescent THC exposure was associated with chromatin alteration, we analyzed levels of histone H3 methylation in the NAcsh via chromatin immunoprecipitation at five sites flanking the Penk gene transcription start site. RESULTS: Here we show that regulation of the Penk opioid neuropeptide gene in NAcsh directly regulates heroin SA behavior. Selective viral-mediated knockdown of Penk in striatopallidal neurons attenuates heroin SA in adolescent THC-exposed rats, whereas Penk overexpression potentiates heroin SA in THC-naïve rats. Furthermore, we report that adolescent THC exposure mediates Penk upregulation through reduction of histone H3 lysine 9 (H3K9) methylation in the NAcsh, thereby disrupting the normal developmental pattern of H3K9 methylation. CONCLUSIONS: These data establish a direct association between THC-induced NAcsh Penk upregulation and heroin SA and indicate that epigenetic dysregulation of Penk underlies the long-term effects of THC.

Cannabis-dependence risk relates to synergism between neuroticism and proenkephalin SNPs associated with amygdala gene expression: case-control study.

BACKGROUND: Many young people experiment with cannabis, yet only a subgroup progress to dependence suggesting individual differences that could relate to factors such as genetics and behavioral traits. Dopamine receptor D2 (DRD2) and proenkephalin (PENK) genes have been implicated in animal studies with cannabis exposure. Whether polymorphisms of these genes are associated with cannabis dependence and related behavioral traits is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Healthy young adults (18-27 years) with cannabis dependence and without a dependence diagnosis were studied (N = 50/group) in relation to a priori-determined single nucleotide polymorphisms (SNPs) of the DRD2 and PENK genes. Negative affect, Impulsive Risk Taking and Neuroticism-Anxiety temperamental traits, positive and negative reward-learning performance and stop-signal reaction times were examined. The findings replicated the known association between the rs6277 DRD2 SNP and decisions associated with negative reinforcement outcomes. Moreover, PENK variants (rs2576573 and rs2609997) significantly related to Neuroticism and cannabis dependence. Cigarette smoking is common in cannabis users, but it was not associated to PENK SNPs as also validated in another cohort (N = 247 smokers, N = 312 non-smokers). Neuroticism mediated (15.3%-19.5%) the genetic risk to cannabis dependence and interacted with risk SNPs, resulting in a 9-fold increase risk for cannabis dependence. Molecular characterization of the postmortem human brain in a different population revealed an association between PENK SNPs and PENK mRNA expression in the central amygdala nucleus emphasizing the functional relevance of the SNPs in a brain region strongly linked to negative affect. CONCLUSIONS/SIGNIFICANCE: Overall, the findings suggest an important role for Neuroticism as an endophenotype linking PENK polymorphisms to cannabis-dependence vulnerability synergistically amplifying the apparent genetic risk.

Hyperphagia-mediated obesity in transgenic mice misexpressing the RNA-editing enzyme ADAR2.

ADAR2 is a double-stranded RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-specific conversion of adenosine to inosine. To examine the physiologic consequences resulting from ADAR2 misexpression, we have generated mutant mice expressing either wild-type or deaminase-deficient ADAR2 transgenes under the control of the human cytomegalovirus promoter. Transgenic mice expressing either wild-type or inactive ADAR2 isoforms demonstrated adult onset obesity characterized by hyperglycemia, hyperleptinemia, and increased adiposity. Paired feeding analysis revealed that mutant mice on caloric restriction had a growth rate and body composition indistinguishable from wild-type littermates, indicating that the observed obesity predominantly results from hyperphagia rather than a metabolic derangement. The observation that expression of catalytically inactive ADAR2 also is capable of producing an obese phenotype in mutant animals suggests that ADAR2 may possess additional biological activities beyond those required for the site-selective deamination of adenosine or may interfere with the actions of other double-stranded RNA-specific binding proteins in the cell.

A linkage disequilibrium map of the 1-Mb 15q12 GABA(A) receptor subunit cluster and association to autism.

Autism is a complex genetic neuropsychiatric condition characterized by deficits in social interaction and language and patterns of repetitive or stereotyped behaviors and restricted interests. Chromosome 15q11.2-q13 is a candidate region for autism susceptibility based on observations of chromosomal duplications in a small percentage of affected individuals and findings of linkage and association. We performed linkage disequilibrium (LD) mapping across a 1-Mb interval containing a cluster of GABA(A) receptor subunit genes (GABRB3, GABRA5, and GABRG3) which are good positional and functional candidates. Intermarker LD was measured for 59 single nucleotide polymorphism (SNP) markers spanning this region, corresponding to an average marker spacing of 17.7 kb(-1). We identified haplotype blocks, and characterized these blocks for common (>5%) haplotypes present in the study population. At this marker resolution, haplotype blocks comprise <50% of the DNA in this region, consistent with a high local recombination rate. Identification of haplotype tag SNPs reduces the overall number of markers necessary to detect all common alleles by only 12%. Individual SNPs and multi-SNP haplotypes were examined for evidence of allelic association to autism, using a dataset of 123 multiplex autism families. Six markers individually, across GABRB3 and GABRA5, and several haplotypes inclusive of those markers, demonstrated nominally significant association. These results are positively correlated with the position of observed linkage. These studies support the existence of one or more autism risk alleles in the GABA(A) receptor subunit cluster on 15q12 and have implications for analysis of LD and association in regions with high local recombination.