Deepening biomedical research training: Community-Building Wellness Workshops for Post-Baccalaureate Research Education Program (PREP) Trainees.

Problem: All trainees, especially those from historically minoritized backgrounds, experience stresses that may reduce their continuation in science, technology, engineering, math, and medicine (STEMM) careers. The Johns Hopkins University School of Medicine is one of ~45 institutions with a National Institutes of Health funded Postbaccalaureate Research Education Program (PREP) that provides mentoring and a year of fulltime research to prepare students from historically excluded groups for graduate school. Having experienced the conflation of stresses during the COVID-19 pandemic and related shutdown, we realized our program lacked a component that explicitly helped PREP Scholars recognize and cope with non-academic stresses (financial, familial, social, mental) that might threaten their confidence and success as scientists and future in STEMM. Intervention: We developed an early-intervention program to help Scholars develop life-long skills to become successful and resilient scientists. We developed a year-long series comprised of 9 workshops focused on community, introspection, financial fitness, emotional intelligence, mental health, and soft-skills. We recruited and compensated a cohort of PhD students and postdoctoral fellows to serve as Peer Mentors, to provide a community and the safest 'space' for Scholars to discuss personal concerns. Peer Mentors were responsible for developing and facilitating these Community-Building Wellness Workshops (CBWW). Context: CBWW were created and exectued as part of the larger PREP program. Workshops included a PowerPoint presentation by Peer Mentors that featured several case studies that prompted discussion and provided time for small-group discussions between Scholars and Peer Mentors. We also included pre- and post-work for each workshop. These touch-points helped Scholars cultivate the habit of introspection. Impact: The CBWW exceeded our goals. Both Peer Mentors and Scholars experienced strong mutual support, and Scholars developed life-long skills. Notably, several Scholars who had been experiencing financial, mental or mentor-related stress immediately brought this to the attention of program leadership, allowing early and successful intervention. At the completion of CBWW, PREP Scholars reported implementing many workshop skills into practice, were reshaping their criteria for choosing future mentors, and evaluating career decisions. Strikingly, Peer Mentors found they also benefitted from the program as well, suggesting a potential larger scope for the role of CBWW in academia. Lessons Learned: Peer Mentors were essential in creating a safe supportive environment that facilitated discussions, self-reflection, and self-care. Providing fair compensation to Peer Mentors for their professional mentoring and teaching contributions was essential and contributed meaningfully to the positive energy and impact of this program.

Spatial Heterogeneity of Brain Lipids in SIV-Infected Macaques Treated with Antiretroviral Therapy.

Human immunodeficiency virus (HIV) infection continues to promote neurocognitive impairment, mood disorders, and brain atrophy, even in the modern era of viral suppression. Brain lipids are vulnerable to HIV-associated energetic strain and may contribute to HIV-associated neurologic dysfunction due to alterations in lipid breakdown and structural lipid composition. HIV neuropathology is region dependent, yet there has not been comprehensive characterization of the spatial heterogeneity of brain lipids during infection that possibly impacts neurologic function. To address this gap, we evaluated the spatial lipid distribution using matrix laser desorption/ionization imaging mass spectrometry (MALDI-IMS) across four brain regions (parietal cortex, midbrain, thalamus, and temporal cortex), as well as the kidney for a peripheral tissue control, in a simian immunodeficiency virus (SIV)-infected rhesus macaque treated with a course of antiretroviral therapies (ARTs). We assessed lipids indicative of fat breakdown [acylcarnitines (CARs)] and critical structural lipids [phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs)] across fatty acid chain lengths and degrees of unsaturation. CARs with very long-chain, polyunsaturated fatty acids (PUFAs) were more abundant across all brain regions than shorter chain, saturated, or monounsaturated species. We observed distinct brain lipid distribution patterns for the CARs and PCs. However, no clear expression patterns emerged for PEs. Surprisingly, the kidney was nearly devoid of ions corresponding to PUFAs common in brain. PEs and PCs with PUFAs had little intensity and less density than other species, and only one CAR species was observed in kidney at high intensity. Overall, our study demonstrates the stark variation in structural phospholipids and lipid-energetic intermediates present in the virally suppressed SIV-macaque brain. These findings may be useful for identifying regional vulnerabilities to damage due to brain lipid changes in people with HIV.

Orthogonal approaches required to measure proteasome composition and activity in mammalian brain tissue.

Proteasomes are large macromolecular complexes with multiple distinct catalytic activities that are each vital to human brain health and disease. Despite their importance, standardized approaches to investigate proteasomes have not been universally adapted. Here, we describe pitfalls and define straightforward orthogonal biochemical approaches essential to measure and understand changes in proteasome composition and activity in the mammalian central nervous system. Through our experimentation in the mammalian brain, we determined an abundance of catalytically active proteasomes exist with and without a 19S cap(s), the regulatory particle essential for ubiquitin-dependent degradation. Moreover, we learned that in-cell measurements using activity-based probes (ABPs) are more sensitive in determining the available activity of the 20S proteasome without the 19S cap and in measuring individual catalytic subunit activities of each ß subunit within all neuronal proteasomes. Subsequently, applying these tools to human brain samples, we were surprised to find that post-mortem tissue retained little to no 19S-capped proteasome, regardless of age, sex, or disease state. In comparing brain tissues (parahippocampal gyrus) from patients with Alzheimer's disease (AD) and unaffected individuals, the available 20S proteasome activity was significantly elevated in severe cases of AD, an observation not previously noted. Taken together, our study establishes standardized approaches for the comprehensive investigation of proteasomes in mammalian brain tissue, and we reveal new insight into brain proteasome biology.

The role of ethanolamine phosphate phospholyase in regulation of astrocyte lipid homeostasis.

Dietary lipid composition has been shown to impact brain morphology, brain development, and neurologic function. However, how diet uniquely regulates brain lipid homeostasis compared with lipid homeostasis in peripheral tissues remains largely uncharacterized. To evaluate the lipid response to dietary changes in the brain, we assessed actively translating mRNAs in astrocytes and neurons across multiple diets. From this data, ethanolamine phosphate phospholyase (Etnppl) was identified as an astrocyte-specific fasting-induced gene. Etnppl catabolizes phosphoethanolamine (PEtN), a prominent headgroup precursor in phosphatidylethanolamine (PE) also found in other classes of neurologically relevant lipid species. Altered Etnppl expression has also previously been associated with humans with mood disorders. We evaluated the relevance of Etnppl in maintaining brain lipid homeostasis by characterizing Etnppl across development and in coregulation with PEtN-relevant genes, as well as determining the impact to the brain lipidome after Etnppl loss. We found that Etnppl expression dramatically increased during a critical window of early brain development in mice and was also induced by glucocorticoids. Using a constitutive knockout of Etnppl (EtnpplKO), we did not observe robust changes in expression of PEtN-related genes. However, loss of Etnppl altered the phospholipid profile in the brain, resulting in increased total abundance of PE and in polyunsaturated fatty acids within PE and phosphatidylcholine species in the brain. Together, these data suggest that brain phospholipids are regulated by the phospholyase action of the enzyme Etnppl, which is induced by dietary fasting in astrocytes.

Dietary intake is associated with neuropsychological impairment in women with HIV.

BACKGROUND: Diet is a modifiable risk factor that may influence cognition in people with HIV. OBJECTIVES: We examined the association between dietary intake and cognition in women with HIV (WWH) and HIV-seronegative women. METHODS: An 18-item dietary National Cancer Institute screener was completed by 729 WWH and 346 HIV-seronegative Women's Interagency HIV Study participants. Daily intake frequencies of processed meats, sweet beverages, fish, whole milk, and vegetables were calculated. Participants completed biennial neuropsychological (NP) testing. NP domains included attention/working memory, executive function, processing speed, memory, learning, fluency, and motor function. NP impairment was defined as demographically adjusted T-scores (mean = 50; SD = 10) ≤40 at ≥1 visit after completing the dietary screener. Multivariable logistic regression, stratified by HIV serostatus, examined associations between intake frequency tertile (referent = lowest intake) and NP performance. RESULTS: Dietary intake frequencies of individual food line items were similar between WWH and HIV-seronegative women, except for sweet beverages, for which HIV-seronegative women reported higher intake frequencies than WWH (P values < 0.05). In WWH, multivariable-adjusted models indicated higher odds of NP impairment with higher intake frequencies of processed meat [P = 0.006; ORupper tertile = 1.91 (95% CI: 1.23-2.95; P = 0.003); ORmiddle tertile = 1.66 (95% CI: 1.14-2.42; P = 0.01)], sweet beverages [P = 0.02; ORupper tertile = 1.75 (95% CI: 1.17-2.64; P = 0.007)], fish [P = 0.01; ORupper tertile = 1.70 (95% CI: 1.10-2.64; P = 0.02)], and whole milk [P = 0.029; ORupper tertile = 1.66 (95% CI: 1.14-2.42; P = 0.008)]. Lower odds of NP impairment [P = 0.005; ORupper tertile = 0.65 (95% CI: 0.45-0.95; P = 0.02); ORmiddle tertile = 0.42 (95% CI: 0.24-0.73; P = 0.002)] were associated with higher vegetable intakes. In HIV-seronegative women, multivariable-adjusted models did not show associations between food line items/diet quality score and NP outcomes. CONCLUSIONS: Intakes of processed meat, sweet beverages, whole milk, fish, and vegetables may be associated with NP functions among WWH. Associations among WWH are not directly comparable to those among HIV-seronegative women, because models were conducted on each group separately given controls for HIV-specific covariates in WWH. Further studies are needed using more rigorous dietary assessment methods and lengthier longitudinal follow-ups.

Determining the Bioenergetic Capacity for Fatty Acid Oxidation in the Mammalian Nervous System.

The metabolic state of the brain can greatly impact neurologic function. Evidence of this includes the therapeutic benefit of a ketogenic diet in neurologic diseases, including epilepsy. However, brain lipid bioenergetics remain largely uncharacterized. The existence, capacity, and relevance of mitochondrial fatty acid ß-oxidation (FAO) in the brain are highly controversial, with few genetic tools available to evaluate the question. We have provided evidence for the capacity of brain FAO using a pan-brain-specific conditional knockout (KO) mouse incapable of FAO due to the loss of carnitine palmitoyltransferase 2, the product of an obligate gene for FAO (CPT2B-/-). Loss of central nervous system (CNS) FAO did not result in gross neuroanatomical changes or systemic differences in metabolism. Loss of CPT2 in the brain did not result in robustly impaired behavior. We demonstrate by unbiased and targeted metabolomics that the mammalian brain oxidizes a substantial quantity of long-chain fatty acids in vitro and in vivo Loss of CNS FAO results in robust accumulation of long-chain acylcarnitines in the brain, suggesting that the mammalian brain mobilizes fatty acids for their oxidation, irrespective of diet or metabolic state. Together, these data demonstrate that the mammalian brain oxidizes fatty acids under normal circumstances with little influence from or on peripheral tissues.

Phosphoglucan-bound structure of starch phosphatase Starch Excess4 reveals the mechanism for C6 specificity.

Plants use the insoluble polyglucan starch as their primary glucose storage molecule. Reversible phosphorylation, at the C6 and C3 positions of glucose moieties, is the only known natural modification of starch and is the key regulatory mechanism controlling its diurnal breakdown in plant leaves. The glucan phosphatase Starch Excess4 (SEX4) is a position-specific starch phosphatase that is essential for reversible starch phosphorylation; its absence leads to a dramatic accumulation of starch in Arabidopsis, but the basis for its function is unknown. Here we describe the crystal structure of SEX4 bound to maltoheptaose and phosphate to a resolution of 1.65 Å. SEX4 binds maltoheptaose via a continuous binding pocket and active site that spans both the carbohydrate-binding module (CBM) and the dual-specificity phosphatase (DSP) domain. This extended interface is composed of aromatic and hydrophilic residues that form a specific glucan-interacting platform. SEX4 contains a uniquely adapted DSP active site that accommodates a glucan polymer and is responsible for positioning maltoheptaose in a C6-specific orientation. We identified two DSP domain residues that are responsible for SEX4 site-specific activity and, using these insights, we engineered a SEX4 double mutant that completely reversed specificity from the C6 to the C3 position. Our data demonstrate that the two domains act in consort, with the CBM primarily responsible for engaging glucan chains, whereas the DSP integrates them in the catalytic site for position-specific dephosphorylation. These data provide important insights into the structural basis of glucan phosphatase site-specific activity and open new avenues for their biotechnological utilization.