Kir6.2-K ATP channels alter glycolytic flux to modulate cortical activity, arousal, and sleep-wake homeostasis.

Metabolism plays an important role in the maintenance of vigilance states (e.g. wake, NREM, and REM). Brain lactate fluctuations are a biomarker of sleep. Increased interstitial fluid (ISF) lactate levels are necessary for arousal and wake-associated behaviors, while decreased ISF lactate is required for sleep. ATP-sensitive potassium (K ATP ) channels couple glucose-lactate metabolism with neuronal excitability. Therefore, we explored how deletion of neuronal K ATP channel activity (Kir6.2-/- mice) affected the relationship between glycolytic flux, neuronal activity, and sleep/wake homeostasis. Kir6.2-/- mice shunt glucose towards glycolysis, reduce neurotransmitter synthesis, dampen cortical EEG activity, and decrease arousal. Kir6.2-/- mice spent more time awake at the onset of the light period due to altered ISF lactate dynamics. Together, we show that Kir6.2-K ATP channels act as metabolic sensors to gate arousal by maintaining the metabolic stability of each vigilance state and providing the metabolic flexibility to transition between states. Highlights: Glycolytic flux is necessary for neurotransmitter synthesis. In its absence, neuronal activity is compromised causing changes in arousal and vigilance states despite sufficient energy availability. With Kir6.2-K ATP channel deficiency, the ability to both maintain and shift between different vigilance states is compromised due to changes in glucose utilization. Kir6.2-K ATP channels are metabolic sensors under circadian control that gate arousal and sleep/wake transitions.

Development of a systematic social observation tool for monitoring use of harm reduction supplies.

BACKGROUND: Harm reduction services such as safer injection supply distribution are essential to reducing morbidity and mortality among people who use drugs (PWUD); however, local use of harm reduction supplies (e.g., tourniquets, saline solution) is difficult to routinely and systematically monitor. The purpose of this study was to develop and validate a systematic social observation tool designed to assess use of harm reduction supplies at the street block level. METHODS: Data collection took place on a random sample of 150 blocks located throughout the Kensington neighborhood of North Philadelphia from November 2021 to January 2022. We measured inter-rater reliability by two-way mixed-effects intra-class correlation coefficients (ICC) with the consistency agreement definition and internal consistency reliability using Cronbach's alpha and McDonald's omega. Exploratory factor analysis with principal component extraction and promax rotation assessed internal consistency. We validated scales against locations of public syringe disposal boxes, a proxy measure for areas of concentrated drug use, using logistic regression. RESULTS: Naloxone canisters, syringe caps, saline and sterile water solution bottles showed the highest reliability (ICC≥0.7). Items also showed high internal consistency (alpha, omega>0.7). Exploratory factor analysis identified one, three-item scale with high internal consistency: syringe caps, vials, and baggies (alpha = 0.85; omega = 0.85)-all supplies used concurrently with drug injection but not discarded in syringe disposal boxes. Drug use (OR = 1.78, 95 % CI = (1.48, 2.23)), harm reduction (OR = 3.53, 95 % CI = (2.20, 6.12)), and EFA scales (OR = 1.85, 95 %CI = (1.51, 2.34)) were significantly and positively associated with being within walking distance (≤0.25 miles or 0.4 km) of a syringe disposal box. CONCLUSION: This study provides an efficient tool with high reliability and validity metrics to assess community uptake of harm reduction supplies designed for use by community organizations, policy makers, or other groups providing resources to PWUD.

Protein salvage and repurposing in evolution: Phospholipase D toxins are stabilized by a remodeled scrap of a membrane association domain.

The glycerophosphodiester phosphodiesterase (GDPD)-like SMaseD/PLD domain family, which includes phospholipase D (PLD) toxins in recluse spiders and actinobacteria, evolved anciently in bacteria from the GDPD. The PLD enzymes retained the core (ß/α)8 barrel fold of GDPD, while gaining a signature C-terminal expansion motif and losing a small insertion domain. Using sequence alignments and phylogenetic analysis, we infer that the C-terminal motif derives from a segment of an ancient bacterial PLAT domain. Formally, part of a protein containing a PLAT domain repeat underwent fusion to the C terminus of a GDPD barrel, leading to attachment of a segment of a PLAT domain, followed by a second complete PLAT domain. The complete domain was retained only in some basal homologs, but the PLAT segment was conserved and repurposed as the expansion motif. The PLAT segment corresponds to strands ß7-ß8 of a ß-sandwich, while the expansion motif as represented in spider PLD toxins has been remodeled as an α-helix, a ß-strand, and an ordered loop. The GDPD-PLAT fusion led to two acquisitions in founding the GDPD-like SMaseD/PLD family: (1) a PLAT domain that presumably supported early lipase activity by mediating membrane association, and (2) an expansion motif that putatively stabilized the catalytic domain, possibly compensating for, or permitting, loss of the insertion domain. Of wider significance, messy domain shuffling events can leave behind scraps of domains that can be salvaged, remodeled, and repurposed.

APOE modulates microglial immunometabolism in response to age, amyloid pathology, and inflammatory challenge.

The E4 allele of Apolipoprotein E (APOE) is associated with both metabolic dysfunction and a heightened pro-inflammatory response: two findings that may be intrinsically linked through the concept of immunometabolism. Here, we combined bulk, single-cell, and spatial transcriptomics with cell-specific and spatially resolved metabolic analyses in mice expressing human APOE to systematically address the role of APOE across age, neuroinflammation, and AD pathology. RNA sequencing (RNA-seq) highlighted immunometabolic changes across the APOE4 glial transcriptome, specifically in subsets of metabolically distinct microglia enriched in the E4 brain during aging or following an inflammatory challenge. E4 microglia display increased Hif1α expression and a disrupted tricarboxylic acid (TCA) cycle and are inherently pro-glycolytic, while spatial transcriptomics and mass spectrometry imaging highlight an E4-specific response to amyloid that is characterized by widespread alterations in lipid metabolism. Taken together, our findings emphasize a central role for APOE in regulating microglial immunometabolism and provide valuable, interactive resources for discovery and validation research.

APOE4 drives transcriptional heterogeneity and maladaptive immunometabolic responses of astrocytes.

Apolipoprotein E4 (APOE4) is the strongest risk allele associated with the development of late onset Alzheimer's disease (AD). Across the CNS, astrocytes are the predominant expressor of APOE while also being critical mediators of neuroinflammation and cerebral metabolism. APOE4 has been consistently linked with dysfunctional inflammation and metabolic processes, yet insights into the molecular constituents driving these responses remain unclear. Utilizing complementary approaches across humanized APOE mice and isogenic human iPSC astrocytes, we demonstrate that ApoE4 alters the astrocyte immunometabolic response to pro-inflammatory stimuli. Our findings show that ApoE4-expressing astrocytes acquire distinct transcriptional repertoires at single-cell and spatially-resolved domains, which are driven in-part by preferential utilization of the cRel transcription factor. Further, inhibiting cRel translocation in ApoE4 astrocytes abrogates inflammatory-induced glycolytic shifts and in tandem mitigates production of multiple pro-inflammatory cytokines. Altogether, our findings elucidate novel cellular underpinnings by which ApoE4 drives maladaptive immunometabolic responses of astrocytes.

Establishing the Relative Accuracy of Using City Directories as Proxies to Define and Reconstruct Historical Alcohol Environments.

OBJECTIVE: Research on alcohol environments has established that poorer and minoritized communities are frequently overburdened by off-premise outlets (e.g., liquor stores). These outlets have more associated harms, including increased alcohol consumption and crime rates. Little, if any, research has shown how these socio-spatial disparities in exposure have grown or shifted over time, and no studies have established a method for re-creating historical alcohol environments. METHOD: Our results suggest that in our study city of Flint, MI, disparities in the alcohol environment have narrowed since 1950. Although liquor stores are still more likely to be located in poorer and more heavily African American neighborhoods, the pattern has become insignificant over time. Furthermore, the number of alcohol outlets per capita has declined. Thus, although the city remains more overburdened with alcohol outlets than its suburbs, the disparity has shrunk. CONCLUSIONS: This work has implications for those working in alcohol prevention and policy, as well as in urban planning. Practitioners and researchers can use this method to model alcohol availability over time in their own communities, which helps better inform the discussion on disparities experienced in poor and minoritized neighborhoods.

Peer Recovery Coaches Perceptions of Their Work and Their Implications for Training, Support and Personal Recovery.

The peer recovery workforce, including individuals in sustained recovery from substance use, has grown rapidly in the previous decades. Peer recovery coaches represent a scalable, resource-efficient, and acceptable approach to increasing service delivery, specifically among individuals receiving substance use services in low-resource communities. Despite the potential to improve access to care in traditionally underserved settings, there are a number of barriers to successfully integrating peer recovery coaches in existing recovery services. The current study presents results from two focus groups composed of peer recovery coaches. Findings suggest that peer recovery coaches report discordance between their perceived role and their daily responsibilities and experience both inter- and intrapersonal challenges that impact their own recovery processes. These results point to several promising policy and structural changes that may support and enhance this growing workforce.

Are Perfectionistic Standards Associated with Burnout? Multidimensional Perfectionism and Compassion Experiences Among Professional MFTs.

The current study addressed the role that perfectionism plays among professionals in this field of marriage and family therapy (MFT). Specifically, this study provides information about PS (personal standards) perfectionism and EC (evaluative concerns) and their relationship with both compassion satisfaction and compassion fatigue. The sample included 247 marriage and family therapists who answered demographic questions along with completing the professional quality of life scale (ProQOL; as reported by Stamm, B. H. (2009). Professional Quality of Life: Compassion Satisfaction and Fatigue Version 5 (ProQOL). Retrieved from http://www.proqol.org/ProQol_Test.html; Stamm in The concise ProQOL manual, ProQOL.org, 2010;) and the multidimensional perfectionism scale (MPS; Hewitt and Flett, Journal of Personality and Social Psychology 60:456-470, 1991). Results indicated three significant findings: (1) higher levels of both self-oriented and socially oriented perfectionism are correlated with higher levels of both burnout and secondary traumatic stress; (2) as years of work as an MFT increase, level of burnout decreases; and (3) women demonstrated statistically significantly higher scores in PS perfectionism than men. Limitations, implications, and future directions are discussed.

Organizational Leaders Perceptions of Barriers to Accessing Behavioral Health Services in a Low-Resource Community.

Little is known about how to effectively implement behavioral health programs in low-resource communities. Leaders from 20 community-serving behavioral health organizations in Flint, MI, were asked about their organizations and the barriers that they, and the populations they serve, face in providing and accessing behavioral health services. Barriers are reported using a mixed-methods analysis, reporting the number and percentage of organizations that experienced the barrier along with example quotations from the organization leaders. The most frequently reported barrier to providing services was finding adequate funding (50%) while the most frequently reported barrier for accessing services was finding adequate and reliable transportation (30%). Comparisons of these findings with barriers reported by providers in different settings and those seeking services are discussed. These comparisons may provide an important next step in identifying areas where providers perceptions and the needs of the population are misaligned and for systemic improvements more broadly.

Using trajectory modeling of spatio-temporal trends to illustrate disparities in COVID-19 death in flint and Genesee County, Michigan.

COVID-19's rapid onset left many public health entities scrambling. But establishing community-academic partnerships to digest data and create advocacy steps offers an opportunity to link research to action. Here we document disparities in COVID-19 death uncovered during a collaboration between a health department and university research center. We geocoded COVID-19 deaths in Genesee County, Michigan, to model clusters during two waves in spring and fall 2020. We then aggregated these deaths to census block groups, where group-based trajectory modeling identified latent patterns of change and continuity. Linking with socioeconomic data, we identified the most affected communities. We discovered a geographic and racial gap in COVID-19 deaths during the first wave, largely eliminated during the second. Our partnership generated added and immediate value for community partners, including around prevention, testing, treatment, and vaccination. Our identification of the aforementioned racial disparity helped our community nearly eliminate disparities during the second wave.

Human APOE ɛ3 and APOE ɛ4 Alleles Have Differential Effects on Mouse Olfactory Epithelium.

BACKGROUND: Alzheimer's disease (AD) is a progressive age-dependent disorder whose risk is affected by genetic factors. Better models for investigating early effects of risk factors such as apolipoprotein E (APOE) genotype are needed. OBJECTIVE: To determine whether APOE genotype produces neuropathologies in an AD-susceptible neural system, we compared effects of human APOE ɛ3 (E3) and APOE ɛ4 (E4) alleles on the mouse olfactory epithelium. METHODS: RNA-Seq using the STAR aligner and DESeq2, immunohistochemistry for activated caspase-3 and phosphorylated histone H3, glucose uptake after oral gavage of 2-[1,2-3H (N)]-deoxy-D-glucose, and Seahorse Mito Stress tests on dissociated olfactory mucosal cells. RESULTS: E3 and E4 olfactory mucosae show 121 differentially abundant mRNAs at age 6 months. These do not indicate differences in cell type proportions, but effects on 17 odorant receptor mRNAs suggest small differences in tissue development. Ten oxidoreductases mRNAs important for cellular metabolism and mitochondria are less abundant in E4 olfactory mucosae but this does not translate into differences in cellular respiration. E4 olfactory mucosae show lower glucose uptake, characteristic of AD susceptibility and consistent with greater expression of the glucose-sensitive gene, Asns. Olfactory sensory neuron apoptosis is unaffected at age 6 months but is greater in E4 mice at 10 months. CONCLUSION: Effects of human APOE alleles on mouse olfactory epithelium phenotype are apparent in early adulthood, and neuronal loss begins to increase by middle age (10 months). The olfactory epithelium is an appropriate model for the ability of human APOE alleles to modulate age-dependent effects associated with the progression of AD.

Symptoms of Depression and Anxiety Among Black Medical Students: the Role of Peer Connectedness and Perceived Discrimination.

This study examined the association between discrimination, peer connectedness, and mental health symptoms among Black medical students. Data were collected from a convenience sample of Black medical students via an anonymous electronic questionnaire (n = 733) in year 2020. The Patient-Reported Outcomes Measurement Information System Depression and Anxiety forms were used to measure depression and anxiety symptoms. Structural equation modeling was used to examine the association between discrimination, peer connectedness, and mental health symptoms (Mplus 7.3). The majority of the participants were female (80%), approximately 40% were third or fourth year medical school students, and 13% had a clinical diagnosis of depression/anxiety before medical school. About half of the students reported being watched more closely than their classmates, and 66% reported feeling the need to work twice as hard as others to get the same treatment or evaluation. The majority of students reported that their peers were supportive of their academic success (60.7%), and 53% reported that students often or always invited them to social outings. The mean T-score for depressive symptoms was 53.6 (SD = 7.8), and the mean T-score for anxiety symptoms was 58.6 (SD = 8.4). Overall, findings indicated a high prevalence of anxiety and depression symptoms among Black medical students, and increased discrimination was associated with more mental health symptoms among males. Additionally, increased peer connectedness was associated with fewer symptoms of anxiety among males and females and fewer depressive symptoms among females. Addressing discrimination among medical students may improve mental health among Black medical students.

Exploring the Obesity Paradox in A Murine Model of Sepsis: Improved Survival Despite Increased Organ Injury in Obese Mice.

ABSTRACT: Despite the known deleterious effects of obesity, clinical data indicate that overweight or obese patients experience higher rates of sepsis survival compared to normal and underweight patients; a phenomenon called the obesity paradox. Results from preclinical sepsis studies have not been able to replicate these findings. The objective of this study was to test the existence of the obesity paradox in a murine model of cecal slurry (CS)-induced sepsis with insulin-resistant diet-induced obese mice. Male C57BL/6 mice were provided high-fat (HFD) or low-fat (LFD) diets for 20 weeks. HFD-fed mice experienced higher rates of survival compared to LFD-fed mice after septic challenge induced by CS injection (66% vs. 25%, P = 0.01, survival assessed for 14 days). Despite the survival advantage, HFD-fed mice had higher rates of positive bacterial cultures and increased markers of kidney injury. Circulating levels of IL-6, IL-1ß, TNFα, and IL-23 were equivalent 24 h after CS-injection; however, IL-17A was uniquely increased in HFD-fed mice. While LFD-fed mice maintained euglycemia, HFD-fed mice were hyperglycemic 6 and 12 h after CS-injection. Stable isotope resolved metabolomics analysis of liver tissue showed diverging pathways of glucose utilization during sepsis, with LFD-fed mice significantly upregulating glycolytic activity and HFD-fed mice decreasing glucose entry into the TCA cycle. This murine study corroborates clinical data that obesity confers a survival benefit in sepsis, albeit at the expense of more significant organ injury. The mechanisms promoting survival in the obese remain unknown; however, this model appears to be well-poised to begin answering this question. Differences in glucose utilization are a novel target to investigate this paradox.

Trajectory Modeling of Spatio-Temporal Trends in COVID-19 Incidence in Flint and Genesee County, Michigan.

PURPOSE: The establishment of community-academic partnerships to digest data and create actionable policy and advocacy steps is of continuing importance. In this paper, we document COVID-19 racial and geographic disparities uncovered via a collaboration between a local health department and university research center. METHODS: We leverage individual level data for all COVID-19 cases aggregated to the census block group level, where group-based trajectory modeling was employed to identify latent patterns of change and continuity in COVID-19 diagnoses. RESULTS: Linking with socioeconomic data from the census, we identified the types of communities most heavily affected by each of Michigan's two waves (in spring and fall of 2020). This includes a geographic and racial gap in COVID-19 cases during the first wave, which is largely eliminated during the second wave. CONCLUSIONS: Our work has been extremely valuable for community partners, informing community-level response toward testing, treatment, and vaccination. In particular, identifying and conducting advocacy on the sizeable racial disparity in COVID-19 cases during the first wave in spring 2020 helped our community nearly eliminate disparities throughout the second wave in fall 2020.

APOΕ4 lowers energy expenditure in females and impairs glucose oxidation by increasing flux through aerobic glycolysis.

BACKGROUND: Cerebral glucose hypometabolism is consistently observed in individuals with Alzheimer's disease (AD), as well as in young cognitively normal carriers of the Ε4 allele of Apolipoprotein E (APOE), the strongest genetic predictor of late-onset AD. While this clinical feature has been described for over two decades, the mechanism underlying these changes in cerebral glucose metabolism remains a critical knowledge gap in the field. METHODS: Here, we undertook a multi-omic approach by combining single-cell RNA sequencing (scRNAseq) and stable isotope resolved metabolomics (SIRM) to define a metabolic rewiring across astrocytes, brain tissue, mice, and human subjects expressing APOE4. RESULTS: Single-cell analysis of brain tissue from mice expressing human APOE revealed E4-associated decreases in genes related to oxidative phosphorylation, particularly in astrocytes. This shift was confirmed on a metabolic level with isotopic tracing of 13C-glucose in E4 mice and astrocytes, which showed decreased pyruvate entry into the TCA cycle and increased lactate synthesis. Metabolic phenotyping of E4 astrocytes showed elevated glycolytic activity, decreased oxygen consumption, blunted oxidative flexibility, and a lower rate of glucose oxidation in the presence of lactate. Together, these cellular findings suggest an E4-associated increase in aerobic glycolysis (i.e. the Warburg effect). To test whether this phenomenon translated to APOE4 humans, we analyzed the plasma metabolome of young and middle-aged human participants with and without the Ε4 allele, and used indirect calorimetry to measure whole body oxygen consumption and energy expenditure. In line with data from E4-expressing female mice, a subgroup analysis revealed that young female E4 carriers showed a striking decrease in energy expenditure compared to non-carriers. This decrease in energy expenditure was primarily driven by a lower rate of oxygen consumption, and was exaggerated following a dietary glucose challenge. Further, the stunted oxygen consumption was accompanied by markedly increased lactate in the plasma of E4 carriers, and a pathway analysis of the plasma metabolome suggested an increase in aerobic glycolysis. CONCLUSIONS: Together, these results suggest astrocyte, brain and system-level metabolic reprogramming in the presence of APOE4, a 'Warburg like' endophenotype that is observable in young females decades prior to clinically manifest AD.

Diagnostic Measures of Disease Progression in Cattle Following Natural Infection with Bovine Leukemia Virus.

This study describes the longitudinal changes in bovine leukemia virus (BLV) ELISA antibodies, proviral load (PVL), and blood lymphocyte counts (LC) observed over a 2.5-year period in naturally infected cattle. The dataset utilized was from a BLV intervention field trial on three Midwestern dairy herds. Our analysis showed ELISA false negatives were more likely to occur in cattle with low PVL and normal LC. On average, negligible changes in LC were observed during six-month intervals. Periods of lymphocytosis, defined as >10,000 lymphocytes per uL of blood, were observed in 31.5% (68/216) of BLV test-positive cattle. In BLV test-positive cows, an average increase of 2900 to 3100 proviral copies per 100,000 cells was observed during each subsequent six-month sampling interval. The difference between the minimum and maximum PVL observed for an ELISA-positive cow with 3 or more observations ranged from 0 to 115,600 copies per 100,000 cells (median: 12,900; mean: 19,200). Therefore, following the identification of ELISA-positive cattle and the assessment of PVL and LC, subsequent semiannual tests to assess disease progression may not be needed. Further work is needed to determine how available diagnostic tests can be optimized to design cost-effective testing schemes for BLV control programs.

Evaluation of a Local Ordinance to Prevent Any Underage Purchases in Liquor Stores: The Need for Enforcement.

OBJECTIVE: In June 2012, Baltimore City, MD, enacted legislation (commonly referred to as the Mosby Bill) prohibiting all liquor stores (outlets that primarily sell alcoholic beverages) from selling "any food, goods, wares, supplies, or other merchandise to any person under the age of 18." Three years after enactment, we evaluated the impact of this legislation on non-alcohol product sales among youth. METHOD: Research assistants (RAs) ages 16-20 were trained in using a standardized observational tool to quantify and record characteristics of the outlets, including products sold. A trained pair comprising one RA age 16 to 20 and one RA exactly age 18 were sent into every liquor store (i.e., packaged goods stores and bar/taverns with packaged goods sales) in Baltimore to conduct the assessment and make a non-alcohol purchase. Since the research was not conducted in concert with the police, the 18-year-old RA made the purchase attempt while the other (age 16 to 20) RA completed the assessment. RESULTS: Purchase attempts were made at 502 liquor stores, and 352 of those attempts were successful (able to make purchase without being asked for identification or age; noncompliance rate = 68.1%). Noncompliance was highest among packaged goods stores compared with bar/taverns, and in neighborhoods with a lower median household income and a higher proportion of African American residents (p < .050). Noncompliant outlets were also located closer to public schools (p < .050). CONCLUSIONS: This evaluation demonstrates that, in the absence of enforcement, ordinances are neither likely to be honored nor to achieve the intended public health benefits.

A cohesin cancer mutation reveals a role for the hinge domain in genome organization and gene expression.

The cohesin complex spatially organizes interphase chromatin by bringing distal genomic loci into close physical proximity, looping out the intervening DNA. Mutation of cohesin complex subunits is observed in cancer and developmental disorders, but the mechanisms through which these mutations may contribute to disease remain poorly understood. Here, we investigate a recurrent missense mutation to the hinge domain of the cohesin subunit SMC1A, observed in acute myeloid leukemia. Engineering this mutation into murine embryonic stem cells caused widespread changes in gene expression, including dysregulation of the pluripotency gene expression program. This mutation reduced cohesin levels at promoters and enhancers, decreased DNA loops and interactions across short genomic distances, and weakened insulation at CTCF-mediated DNA loops. These findings provide insight into how altered cohesin function contributes to disease and identify a requirement for the cohesin hinge domain in three-dimensional chromatin structure.

Functional impact of cancer-associated cohesin variants on gene expression and cellular identity.

Cohesin is a ring-shaped protein complex that controls dynamic chromosome structure. Cohesin activity is important for a variety of biological processes, including formation of DNA loops that regulate gene expression. The precise mechanisms by which cohesin shapes local chromosome structure and gene expression are not fully understood. Recurrent mutations in cohesin complex members have been reported in various cancers, though it is not clear whether many cohesin sequence variants have phenotypes and contribute to disease. Here, we utilized CRISPR/Cas9 genome editing to introduce a variety of cohesin sequence variants into murine embryonic stem cells and investigate their molecular and cellular consequences. Some of the cohesin variants tested caused changes to transcription, including altered expression of gene encoding lineage-specifying developmental regulators. Altered gene expression was also observed at insulated neighborhoods, where cohesin-mediated DNA loops constrain potential interactions between genes and enhancers. Furthermore, some cohesin variants altered the proliferation rate and differentiation potential of murine embryonic stem cells. This study provides a functional comparison of cohesin variants found in cancer within an isogenic system, revealing the relative roles of various cohesin perturbations on gene expression and maintenance of cellular identity.