eScholarship@UMMS

    NMR and MD studies combined to elucidate inhibitor and water interactions of HIV-1 protease and their modulations with resistance mutations

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    Over the last two decades, both the sensitivity of NMR and the time scale of molecular dynamics (MD) simulation have increased tremendously and have advanced the field of protein dynamics. HIV-1 protease has been extensively studied using these two methods, and has presented a framework for cross-evaluation of structural ensembles and internal dynamics by integrating the two methods. Here, we review studies from our laboratories over the last several years, to understand the mechanistic basis of protease drug-resistance mutations and inhibitor responses, using NMR and crystal structure-based parallel MD simulations. Our studies demonstrate that NMR relaxation experiments, together with crystal structures and MD simulations, significantly contributed to the current understanding of structural/dynamic changes due to HIV-1 protease drug resistance mutations

    Neuronal vulnerability and multilineage diversity in multiple sclerosis

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    Multiple sclerosis (MS) is a neuroinflammatory disease with a relapsing-remitting disease course at early stages, distinct lesion characteristics in cortical grey versus subcortical white matter and neurodegeneration at chronic stages. Here we used single-nucleus RNA sequencing to assess changes in expression in multiple cell lineages in MS lesions and validated the results using multiplex in situ hybridization. We found selective vulnerability and loss of excitatory CUX2-expressing projection neurons in upper-cortical layers underlying meningeal inflammation; such MS neuron populations exhibited upregulation of stress pathway genes and long non-coding RNAs. Signatures of stressed oligodendrocytes, reactive astrocytes and activated microglia mapped most strongly to the rim of MS plaques. Notably, single-nucleus RNA sequencing identified phagocytosing microglia and/or macrophages by their ingestion and perinuclear import of myelin transcripts, confirmed by functional mouse and human culture assays. Our findings indicate lineage- and region-specific transcriptomic changes associated with selective cortical neuron damage and glial activation contributing to progression of MS lesions

    The Possible Role of the Angiotensin System in the Pathophysiology of Schizophrenia: Implications for Pharmacotherapy

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    A growing body of literature has elucidated the involvement of the central renin-angiotensin system (RAS) in various neuropsychiatric diseases. While consensus on the exact mechanism of the central RAS in schizophrenia pathophysiology does not currently exist, increasing evidence reveals promise in harnessing the therapeutic potential of RAS modulation in the treatment of schizophrenia. In this review, we examine how the central RAS affects inflammation, glutamate, dopamine, gamma-aminobutyric acid (GABA), and peroxisome proliferator-activated receptor (PPAR)-gamma, all of which are associated with schizophrenia etiology. In addition, a recent study has demonstrated the therapeutic potential of RAS modulators, especially angiotensin II type 1 receptor blockers (ARBs), as adjunctive therapy to the currently available antipsychotic medications for schizophrenia treatment. With a greater understanding of how RAS inhibition directly modulates neurotransmitter balance in the brain, it is possible that compounds with RAS-inhibiting properties could be used to optimize physiological levels of glutamate, dopamine, and GABA, and the balance among the three neurotransmitters, analogously to how antipsychotic medications mediate the dopaminergic pathways. It can be hoped that a novel approach based on this concept, such as adjunctive telmisartan therapy, may offer practical interventional strategies to address currently unmet therapeutic needs in patients with schizophrenia, especially those with treatment-resistant schizophrenia

    Orbital Atherectomy for Calcific Coronary Artery Disease in Patients With Severe Aortic Stenosis: A Safety and Feasibility Study

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    BACKGROUND: Percutaneous revascularization followed by transcatheter aortic valve replacement (TAVR) has been increasingly utilized as an alternative to surgery in patients with severe aortic stenosis (AS) and coronary artery disease (CAD). In many of these patients, the coronary arteries are severely calcified and may best be treated with atherectomy; however, atherectomy is not routinely performed in severe AS patients due to safety concerns. There is a paucity of data on the safety of orbital atherectomy (OA) in patients with severe AS and concurrent calcific CAD. METHODS: We retrospectively analyzed the medical records of all patients with severe AS who underwent OA-facilitated percutaneous coronary intervention (PCI) at our center between September 1, 2015 and November 1, 2018. RESULTS: Twenty-four patients (mean age, 82.5 +/- 7.6 years) were identified. Mean aortic valve area was 0.68 +/- 0.26 cm and mean aortic valve gradient was 43 +/- 17.7 mm Hg. All PCIs were successful (mean diameter stenosis, 80.8 +/- 11%; mean number of passes, 5.3 +/- 3.3). Two patients had planned hemodynamic support, with left ventricular assist device and intra-aortic balloon pump; none of the patients required vasopressors during PCI. There was a slight reduction in heart rate during OA (71.6 bpm vs 63.3 bpm; P=.02), with no major procedure-related clinical events. Only 1 patient (4.2%) with pre-existing conduction system disease required transient pacing from his permanent pacemaker during OA. All procedures were completed successfully and there were no periprocedural deaths or clinical myocardial infarctions. CONCLUSION: OA-facilitated PCI can be safely performed in patients with severe AS and severely calcified CAD with low risk of complications. There was no significant change in blood pressure and heart rate during OA, with minimal need for temporary pacing

    Systems approach reveals photosensitivity and PER2 level as determinants of clock-modulator efficacy

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    In mammals, the master circadian clock synchronizes daily rhythms of physiology and behavior with the day-night cycle. Failure of synchrony, which increases the risk for numerous chronic diseases, can be treated by phase adjustment of the circadian clock pharmacologically, for example, with melatonin, or a CK1delta/epsilon inhibitor. Here, using in silico experiments with a systems pharmacology model describing molecular interactions, and pharmacokinetic and behavioral experiments in cynomolgus monkeys, we find that the circadian phase delay caused by CK1delta/epsilon inhibition is more strongly attenuated by light in diurnal monkeys and humans than in nocturnal mice, which are common preclinical models. Furthermore, the effect of CK1delta/epsilon inhibition strongly depends on endogenous PER2 protein levels, which differs depending on both the molecular cause of the circadian disruption and the patient\u27s lighting environment. To circumvent such large interindividual variations, we developed an adaptive chronotherapeutics to identify precise dosing regimens that could restore normal circadian phase under different conditions. Our results reveal the importance of photosensitivity in the clinical efficacy of clock-modulating drugs, and enable precision medicine for circadian disruption

    Inactivating mutations and X-ray crystal structure of the tumor suppressor OPCML reveal cancer-associated functions

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    OPCML, a tumor suppressor gene, is frequently silenced epigenetically in ovarian and other cancers. Here we report, by analysis of databases of tumor sequences, the observation of OPCML somatic missense mutations from various tumor types and the impact of these mutations on OPCML function, by solving the X-ray crystal structure of this glycoprotein to 2.65 A resolution. OPCML consists of an extended arrangement of three immunoglobulin-like domains and homodimerizes via a network of contacts between membrane-distal domains. We report the generation of a panel of OPCML variants with representative clinical mutations and demonstrate clear phenotypic effects in vitro and in vivo including changes to anchorage-independent growth, interaction with activated cognate receptor tyrosine kinases, cellular migration, invasion in vitro and tumor growth in vivo. Our results suggest that clinically occurring somatic missense mutations in OPCML have the potential to contribute to tumorigenesis in a variety of cancers

    Effective mismatch repair depends on timely control of PCNA retention on DNA by the Elg1 complex

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    Proliferating cell nuclear antigen (PCNA) is a sliding clamp that acts as a central co-ordinator for mismatch repair (MMR) as well as DNA replication. Loss of Elg1, the major subunit of the PCNA unloader complex, causes over-accumulation of PCNA on DNA and also increases mutation rate, but it has been unclear if the two effects are linked. Here we show that timely removal of PCNA from DNA by the Elg1 complex is important to prevent mutations. Although premature unloading of PCNA generally increases mutation rate, the mutator phenotype of elg1Delta is attenuated by PCNA mutants PCNA-R14E and PCNA-D150E that spontaneously fall off DNA. In contrast, the elg1Delta mutator phenotype is exacerbated by PCNA mutants that accumulate on DNA due to enhanced electrostatic PCNA-DNA interactions. Epistasis analysis suggests that PCNA over-accumulation on DNA interferes with both MMR and MMR-independent process(es). In elg1Delta, over-retained PCNA hyper-recruits the Msh2-Msh6 mismatch recognition complex through its PCNA-interacting peptide motif, causing accumulation of MMR intermediates. Our results suggest that PCNA retention controlled by the Elg1 complex is critical for efficient MMR: PCNA needs to be on DNA long enough to enable MMR, but if it is retained too long it interferes with downstream repair steps

    Intersectional effects of racial and gender discrimination on cardiovascular health vary among black and white women and men in the CARDIA study

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    Testing hypotheses from the emerging Identity Pathology (IP) framework, we assessed race-gender differences in the effects of reporting experiences of racial and gender discrimination simultaneously compared with racial or gender discrimination alone, or no discrimination, on future cardiovascular health (CVH). Data were from a sample of 3758 black or white adults in CARDIA, a community-based cohort recruited in Birmingham, AL; Chicago, IL; Minneapolis, MN, and Oakland, CA in 1985-6 (year 0). Racial and gender discrimination were assessed using the Experiences of Discrimination scale. CVH was evaluated using a 12-point composite outcome modified from the Life\u27s Simple 7, with higher scores indicating better health. Multivariable linear regressions were used to evaluate the associations between different perceptions of discrimination and CVH scores two decades later by race and gender simultaneously. Reporting racial and gender discrimination in \u3e /=2 settings were 48% of black women, 42% of black men, 10% of white women, and 5% of white men. Year 30 CVH scores (mean, SD) were 7.9(1.4), 8.1(1.6), 8.8(1.6), and 8.7(1.3), respectively. Compared with those of their race-gender groups reporting no discrimination, white women reporting only gender-based discrimination saw an adjusted score difference of +0.3 (95% CI: 0.0,0.6), whereas white men reporting only racial discrimination had on average a 0.4 (95% CI: 0.1,0.8) higher score, and scores among white men reporting both racial and gender discrimination were on average 0.6 (95% CI: 1.1,-0.1) lower than those of their group reporting no discrimination. Consistent with predictions of the IP model, the associations of reported racial and gender discrimination with future CVH were different for different racially-defined gender groups. More research is needed to understand why reported racial and gender discrimination might better predict deterioration in CVH for whites than blacks, and what additional factors associated with gender and race contribute variability to CVH among these groups

    Scholarly Productivity and Impact: Developing a Quantifiable, Norm-based Benchmarking Methodology for Academic Emergency Medicine

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    BACKGROUND: Quantifying and benchmarking scholarly productivity of emergency medicine faculty is challenging. While performance indicators including publication and citation counts are available, use of indicators to create normative references has lagged. The authors developed methodology to benchmark emergency medicine academician scholarly productivity (e.g., publications over time) and impact (e.g., citations per publication over time) against an appropriate reference group. METHODS: The methodology includes: 1) define time frame and scholarly metrics; 2) identify representative population; 3) reconcile alternative author names; 4) use analytic tool to identify scholarly output; 5) build database containing metrics; and 6) create benchmarking statistics, including subsamples. This study included emergency medicine faculty from 2011 to 2015, with total peer-reviewed publications and citations per publication as scholarly metrics. RESULTS: In the United States at the time of the search (2016) there were 200 academic emergency departments, 186 with public faculty listings, which yielded 6,727 academicians. For each academician, the authors calculated statistics about peer-reviewed publications and average citations per publication from 2011 to 2015 and created benchmarking rulers using percentile ranks. Productivity by year of graduation with terminal degree was compared within each subsample, finding that newly graduated faculty demonstrated higher productivity than their within-rank peers who graduated earlier. Finally, benchmarking tables were created that allow comparison of peer-reviewed publication counts and citations per publication for individual academicians against the norm. CONCLUSIONS: This benchmarking method can serve as a model for norm-based scaling of scholarly productivity for emergency medicine. This has important implications for performance review, promotion and hiring, and evaluating group productivity

    Still Coming Out of the Dark: Enduring Effects of Simulation-Based Communication Skills Training for Radiology Residents-Four-Year Follow-Up

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    PURPOSE: To evaluate the long-term efficacy of simulation-based communication skills training for radiology residents. METHOD AND MATERIALS: The simulation-based communication skills training curriculum was developed in 2014. The curriculum included a teaching module based on the essential elements of communication. Two sets of 6 communication scenarios encountered by radiologist were created. First and fourth year radiology residents reviewed the teaching module and completed the 6 simulated scenarios. They then underwent debriefing sessions, received faculty and staff evaluations. Four years later, the former first year residents (now fourth years) reviewed the teaching module again and repeated the simulation. They again underwent debriefing sessions after the simulation. This time the residents\u27 communication skills were evaluated by faculty and staff. RESULTS: A total of 5 residents participated in this simulation-based skills training. The resident performance 4 years after initial training show not only that residents maintained their improved scores, but also that their scores improved further as compared to after the initial training. The average overall score for all but 1 resident increased at the 4 year follow-up simulation. From 2014 to 2018, the average score of all the residents increased from 72.4% to 81.4%. Comparison of the average scores of each student across 6 stations from 2014 to 2018 showed a statistically significant difference between the scores after 4 years (P= 0.014). CONCLUSIONS: Simulation-based communication skills training is effective and long lasting
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