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The Vanderbilt University School of Medicine administers degree programs that provide students with the knowledge, skills, and attitudes they will need to practice safe, effective, ethical, evidence-based, and patient-centered health care in the 21st century, and to contribute to the knowledge base supporting it.

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3D Co-culture of hiPSC-Derived Cardiomyocytes With Cardiac Fibroblasts Improves Tissue-Like Features of Cardiac Spheroids
(Frontiers in Molecular Biosciences, 2020-02-14) Beauchamp, Philippe; Jackson, Christopher B.; Ozhathil, Lijo Cherian; Agarkova, Irina; Galindo, Cristi L.; Sawyer, Douglas B.; Suter, Thomas M.; Zuppinger, Christian
Purpose: Both cardiomyocytes and cardiac fibroblasts (CF) play essential roles in cardiac development, function, and remodeling. Properties of 3D co-cultures are incompletely understood. Hence, 3D co-culture of cardiomyocytes and CF was characterized, and selected features compared with single-type and 2D culture conditions. Methods: Human cardiomyocytes derived from induced-pluripotent stem cells (hiPSC-CMs) were obtained from Cellular Dynamics or Ncardia, and primary human cardiac fibroblasts from ScienCell. Cardiac spheroids were investigated using cryosections and whole-mount confocal microscopy, video motion analysis, scanning-, and transmission-electron microscopy (SEM, TEM), action potential recording, and quantitative PCR (qPCR). Results: Spheroids formed in hanging drops or in non-adhesive wells showed spontaneous contractions for at least 1 month with frequent media changes. SEM of mechanically opened spheroids revealed a dense inner structure and no signs of blebbing. TEM of co-culture spheroids at 1 month showed myofibrils, intercalated disc-like structures and mitochondria. Ultrastructural features were comparable to fetal human myocardium. We then assessed immunostained 2D cultures, cryosections of spheroids, and whole-mount preparations by confocal microscopy. CF in co-culture spheroids assumed a small size and shape similar to the situation in ventricular tissue. Spheroids made only of CF and cultured for 3 weeks showed no stress fibers and strongly reduced amounts of alpha smooth muscle actin compared to early spheroids and 2D cultures as shown by confocal microscopy, western blotting, and qPCR. The addition of CF to cardiac spheroids did not lead to arrhythmogenic effects as measured by sharp-electrode electrophysiology. Video motion analysis showed a faster spontaneous contraction rate in co-culture spheroids compared to pure hiPSC-CMs, but similar contraction amplitudes and kinetics. Spontaneous contraction rates were not dependent on spheroid size. Applying increasing pacing frequencies resulted in decreasing contraction amplitudes without positive staircase effect. Gene expression analysis of selected cytoskeleton and myofibrillar proteins showed more tissue-like expression patterns in co-culture spheroids than with cardiomyocytes alone or in 2D culture. Conclusion: We demonstrate that the use of 3D co-culture of hiPSC-CMs and CF is superior over 2D culture conditions for co-culture models and more closely mimicking the native state of the myocardium with relevance to drug development as well as for personalized medicine.
Akt Signaling in Macrophage Polarization, Survival, and Atherosclerosis
(International Journal of Molecular Sciences, 2019-06-01) Linton, MacRae F.; Moslehi, Javid J.; Babaev, Vladimir R.
The PI3K/Akt pathway plays a crucial role in the survival, proliferation, and migration of macrophages, which may impact the development of atherosclerosis. Changes in Akt isoforms or modulation of the Akt activity levels in macrophages significantly affect their polarization phenotype and consequently atherosclerosis in mice. Moreover, the activity levels of Akt signaling determine the viability of monocytes/macrophages and their resistance to pro-apoptotic stimuli in atherosclerotic lesions. Therefore, elimination of pro-apoptotic factors as well as factors that antagonize or suppress Akt signaling in macrophages increases cell viability, protecting them from apoptosis, and this markedly accelerates atherosclerosis in mice. In contrast, inhibition of Akt signaling by the ablation of Rictor in myeloid cells, which disrupts mTORC2 assembly, significantly decreases the viability and proliferation of blood monocytes and macrophages with the suppression of atherosclerosis. In addition, monocytes and macrophages exhibit a threshold effect for Akt protein levels in their ability to survive. Ablation of two Akt isoforms, preserving only a single Akt isoform in myeloid cells, markedly compromises monocyte and macrophage viability, inducing monocytopenia and diminishing early atherosclerosis. These recent advances in our understanding of Akt signaling in macrophages in atherosclerosis may have significant relevance in the burgeoning field of cardio-oncology, where PI3K/Akt inhibitors being tested in cancer patients can have significant cardiovascular and metabolic ramifications.
Akt Signaling in Macrophage Polarization, Survival, and Atherosclerosis
(International Journal of Moledular Sciences, 2019-06-01) Linton, MacRae F.; Moslehi, Javid J.; Babaev, Vladimir R.
The PI3K/Akt pathway plays a crucial role in the survival, proliferation, and migration of macrophages, which may impact the development of atherosclerosis. Changes in Akt isoforms or modulation of the Akt activity levels in macrophages significantly affect their polarization phenotype and consequently atherosclerosis in mice. Moreover, the activity levels of Akt signaling determine the viability of monocytes/macrophages and their resistance to pro-apoptotic stimuli in atherosclerotic lesions. Therefore, elimination of pro-apoptotic factors as well as factors that antagonize or suppress Akt signaling in macrophages increases cell viability, protecting them from apoptosis, and this markedly accelerates atherosclerosis in mice. In contrast, inhibition of Akt signaling by the ablation of Rictor in myeloid cells, which disrupts mTORC2 assembly, significantly decreases the viability and proliferation of blood monocytes and macrophages with the suppression of atherosclerosis. In addition, monocytes and macrophages exhibit a threshold effect for Akt protein levels in their ability to survive. Ablation of two Akt isoforms, preserving only a single Akt isoform in myeloid cells, markedly compromises monocyte and macrophage viability, inducing monocytopenia and diminishing early atherosclerosis. These recent advances in our understanding of Akt signaling in macrophages in atherosclerosis may have significant relevance in the burgeoning field of cardio-oncology, where PI3K/Akt inhibitors being tested in cancer patients can have significant cardiovascular and metabolic ramifications.
Analysis of apoB Concentrations Across Early Adulthood and Predictors for Rates of Change Using CARDIA Study Data
(Journal of Lipid Research, 2022-12) Wilkins, John T.; Ning, Hongyan; Sniderman, Allan; Stone, Neil; Otvos, James; Jacobs, David R.; Shah, Ravi; Murthy, Venkatesh L.; Rana, Jamal; Allen, Norrina; Lloyd-Jones, Donald M.
The cumulative exposure to apolipoprotein B (apoB)-containing lipoproteins in the blood during early adult life is a central determinant of atherosclerotic cardiovascular disease risk. To date, the patterns and rates of change in apoB through early adult life have not been described. Here, we used NMR to measure apoB concentrations in up to 3055 Coronary Artery Risk Development in Young Adults (CARDIA) Study participants who attended the years 2 (Y2), 7 (Y7), 15 (Y15), 20 (Y20), and 30 (Y30) exams. We examined individual-level spaghetti plots of apoB change, and we calculated average annualized rate of apoB concentration change during follow-up. We used multivariable linear regression models to assess the associations between CARDIA participant characteristics and annualized rates of apoB change. Male sex, higher measures of adiposity, lower HDL-C, lower Healthy Eating Index, and higher blood pressures were observed more commonly in individuals with higher apoB level at Y2 and Y20. Inter- and intra-individual variation in apoB concentration over time was substantial-while the mean (SD) rate of change was 0.52 (1.0) mg/dl/year, the range of annualized rates of change was -6.26 to +9.21 mg/dl/year. At baseline, lower first apoB measurement, female sex, White race, lower BMI, and current tobacco use were associated with apoB increase. We conclude that the significant variance in apoB level over time and the modest association between baseline measures and rates of apoB change suggest that the ability to predict an individual's future apoB serum concentrations, and thus their cumulative apoB exposure, after a one-time assessment in young adulthood is low.
The anti-parasitic agent suramin and several of its analogues are inhibitors of the DNA binding protein Mcm10
(Open Biology, 2019-08) Paulson, Carolyn N.; John, Kristen; Baxley, Ryan M.; Kurniawan, Fredy; Orellana, Kayo; Francis, Rawle; Sobeck, Alexandra; Eichman, Brandt F.; Chazin, Walter J.; Aihara, Hideki; Georg, Gunda I.; Hawkinson, Jon E.; Bielinsky, Anja-Katrin
Minichromosome maintenance protein 10 (Mcm10) is essential for DNA unwinding by the replisome during S phase. It is emerging as a promising anti-cancer target as MCM10 expression correlates with tumour progression and poor clinical outcomes. Here we used a competition-based fluorescence polarization (FP) high-throughput screening (HTS) strategy to identify compounds that inhibit Mcm10 from binding to DNA. Of the five active compounds identified, only the anti-parasitic agent suramin exhibited a dose-dependent decrease in replication products in an in vitro replication assay. Structure-activity relationship evaluation identified several suramin analogues that inhibited ssDNA binding by the human Mcm10 internal domain and full-length Xenopus Mcm10, including analogues that are selective for Mcm10 over human RPA. Binding of suramin analogues to Mcm10 was confirmed by surface plasmon resonance (SPR). SPR and FP affinity determinations were highly correlated, with a similar rank between affinity and potency for killing colon cancer cells. Suramin analogue NF157 had the highest human Mcm10 binding affinity (FP K-i 170 nM, SPR K-D 460 nM) and cell activity (IC50 38 mu M). Suramin and its analogues are the first identified inhibitors of Mcm10 and probably block DNA binding by mimicking the DNA sugar phosphate backbone due to their extended, polysulfated anionic structures.
The Aspergillus fumigatus Mismatch Repair MSH2 Homolog Is Important for Virulence and Azole Resistance
(mSphere, 2019-07) dos Reis, Thaila Fernanda; Silva, Lilian Pereira; de Castro, Patricia Alves; do Carmo, Rafaela Andrade; Marini, Marjorie Mendes; da Silveira, Jose Franco; Ferreira, Beatriz Henriques; Rodrigues, Fernando; Lind, Abigail Lee; Rokas, Antonis; Goldman, Gustavo H.
The genetic stability of every living organism depends on accurate DNA replication and repair systems. Here, we investigated the Aspergillus fumigatus MSH2 mismatch repair (MMR) gene MshA and how it impacts virulence and the evolution of azole resistance. We examined mshA gene variation in 62 environmental and clinical A. fumigatus strains. We have observed 12 strains with variants (18.2%), and 8 strains among them showed missense variants. We demonstrated that A. fumigatus mshA null mutants are haploid and have conserved karyotypes with discrete gross chromosomal rearrangements. The Delta mshA strains are not sensitive to several DNA-damaging agents. The lack of mshA caused a significant reduction of virulence of A. fumigatus in a neutropenic murine model of invasive pulmonary aspergillosis and in the invertebrate alternative model Galleria mellonella. Wild-type and Delta mshA populations did not show any significant changes in drug resistance acquisition after they were transferred 10 times in minimal medium in the absence of any stress. However, these populations rapidly acquired virulence in the Delta mshA background and high levels of resistance to posaconazole in the presence of this drug (at least 200-fold-higher levels of resistance than those derived from the wild-type strain). Taken together, these results suggest that genetic instability caused by Delta mshA mutations can confer an adaptive advantage, mainly increasing posaconazole resistance and virulence acquisition. IMPORTANCE Invasive aspergillosis (IA) has emerged as one of the most common life-threatening fungal diseases in immunocompromised patients, with mortality rates as high as 90%. Systemic fungal infections such as IA are usually treated with triazoles; however, epidemiological research has shown that the prevalence of azole-resistant Aspergillus fumigatus isolates has increased significantly over the last decade. There is very little information about the importance of genomic stability for A. fumigatus population structure, azole resistance, and virulence. Here, we decided to investigate whether the mismatch repair system could influence A. fumigatus azole resistance and virulence, focusing on one of the components of this system, MSH2. Although the mutation frequency of mshA (the A. fumigatus MSH2 homologue) is low in environmental and clinical isolates, our results indicate that loss of mshA function can provide increased azole resistance and virulence when selected for. These results demonstrate the importance of genetic instability in A. fumigatus as a possible mechanism of evolving azole resistance and establishing fitness in the host.
Association of Autologous Tumor Lysate-Loaded Dendritic Cell Vaccination With Extension of Survival Among Patients With Newly Diagnosed and Recurrent Glioblastoma A Phase 3 Prospective Externally Controlled Cohort Trial
(JAMA Oncology, 2022-11-17) Thompson, Reid C.
IMPORTANCE Glioblastoma is the most lethal primary brain cancer. Clinical outcomes for glioblastoma remain poor, and new treatments are needed. OBJECTIVE To investigate whether adding autologous tumor lysate-loaded dendritic cell vaccine (DCVax L) to standard of care (SOC) extends survival among patients with glioblastoma. DESIGN, SETTING, AND PARTICIPANTS This phase 3, prospective, externally controlled nonrandomized trial compared overall survival (OS) in patients with newly diagnosed glioblastoma (nGBM) and recurrent glioblastoma (rGBM) treated with DCVax-L plus SOC vs contemporaneous matched external control patients treated with SOC. This international, multicenter trial was conducted at 94 sites in 4 countries from August 2007 to November 2015. Data analysis was conducted from October 2020 to September 2021. INTERVENTIONS The active treatment was DCVax-L plus SOC temozolomide. The nGBM external control patients received SOC temozolomide and placebo; the rGBM external controls received approved rGBM therapies. MAIN OUTCOMES AND MEASURES The primary and secondary end points compared overall survival (OS) in nGBM and rGBM, respectively, with contemporaneous matched external control populations from the control groups of other formal randomized clinical trials. RESULTS A total of 331 patients were enrolled in the trial, with 232 randomized to the DCVax-L group and 99 to the placebo group. Median OS (mOS) for the 232 patients with nGBM receiving DCVax-L was 19.3 (95% CI, 17.5-21.3) months from randomization (22.4 months from surgery) vs 16.5 (95% CI, 16.0-17.5) months from randomization in control patients (HR = 0.80; 98% CI, 0.00-0.94; P = .002). Survival at 48 months from randomization was 15.7% vs 9.9%, and at 60 months, it was 13.0% vs 5.7%. For 64 patients with rGBM receiving DCVax-L, mOS was 13.2 (95% CI, 9.7-16.8) months from relapse vs 7.8 (95% CI, 7.2-8.2) months among control patients (HR, 0.58; 98% CI, 0.00-0.76; P < .001). Survival at 24 and 30 months after recurrence was 20.7% vs 9.6% and 11.1% vs 5.1%, respectively. Survival was improved in patients with nGBM with methylated MGMT receiving DCVax-L compared with external control patients (HR, 0.74; 98% CI, 0.55-1.00; P = .03). CONCLUSIONS AND RELEVANCE In this study, adding DCVax-L to SOC resulted in clinically meaningful and statistically significant extension of survival for patients with both nGBM and rGBM compared with contemporaneous, matched external controls who received SOC alone.
Association of Psychosocial Factors on COVID-19 Testing among YWCA Service Recipients
(International Journal of Environmental Research and Public Health, 2023-01-11) Blasingame, Miaya; Mallett, Veronica; Cook, Mekeila; Im, Wansoo; Wilus, Derek; Kimbrough, Robin; Ikwuezunma, Gini; Orok, Ekemini; Reed, Breia; Akanbi, Victoria; Amoo-Asante, Aurdie
The purpose of this study was to examine how psychosocial factors affect receipt of COVID-19 testing among Black and Hispanic women. In this cross-sectional study of Black and Hispanic women who received services from the YWCAs in Atlanta, El Paso, Nashville, and Tucson between 2019 and 2021 (n = 662), we used Patient-Reported Outcomes Measurement Information Systems (PROMIS) item bank 1.0 short forms to examine the impact of psychosocial factors (i.e., depression, anxiety, social isolation, instrumental support, emotional support, and companionship) on COVID-19 testing. Multivariable logistic regression models were used to estimate odds ratios and 95% confidence intervals for receipt of a COVID-19 test associated with psychosocial factors while adjusting for confounders. There was little effect of moderate/severe depressions or anxiety on receipt of COVID-19 testing. Black (odds ratio [OR] 0.58, 95% confidence interval [CI] 0.26-1.29) and Hispanic (OR 0.61, 95% CI 0.38-0.96) women with high levels of emotional support were less likely to receive the COVID-19 test. While high levels of instrumental support was associated with less likely receipt of the COVID-19 test among Black women (OR 0.75, 95% CI 0.34-1.66), it was associated with more likely receipt among Hispanic women (OR 1.19, 95% CI 0.74-1.92). Our findings suggest that certain psychosocial factors influence one's decision to get a COVID-19 test which can be useful in encouraging preventive healthcare such as screening and vaccination.
Automated cell boundary and 3D nuclear segmentation of cells in suspension
(Scientific Reports, 2019-07-15) Kesler, Benjamin; Li, Guoliang; Thiemicke, Alexander; Venkat, Rohit; Neuert, Gregor
To characterize cell types, cellular functions and intracellular processes, an understanding of the differences between individual cells is required. Although microscopy approaches have made tremendous progress in imaging cells in different contexts, the analysis of these imaging data sets is a long-standing, unsolved problem. The few robust cell segmentation approaches that exist often rely on multiple cellular markers and complex time-consuming image analysis. Recently developed deep learning approaches can address some of these challenges, but they require tremendous amounts of data and well-curated reference data sets for algorithm training. We propose an alternative experimental and computational approach, called CellDissect, in which we first optimize specimen preparation and data acquisition prior to image processing to generate high quality images that are easier to analyze computationally. By focusing on fixed suspension and dissociated adherent cells, CellDissect relies only on widefield images to identify cell boundaries and nuclear staining to automatically segment cells in two dimensions and nuclei in three dimensions. This segmentation can be performed on a desktop computer or a computing cluster for higher throughput. We compare and evaluate the accuracy of different nuclear segmentation approaches against manual expert cell segmentation for different cell lines acquired with different imaging modalities.
Benign Neurofibroma/Schwannoma Hybrid Peripheral Nerve Sheath Tumor of the Ulnar Nerve Harboring a Metastatic Papillary Thyroid Carcinoma Deposit: A Case Report of Tumor-to-Tumor Metastasis
(Case Reports In Pathology, 2022-12-14) Colazo, Juan M.; Perez, Alexander N.; Judice, Anthony D.; Quirion, Julia; Prieto-Granada, Carlos N.; Holt, Ginger E.
A 74-year-old man with a medical history significant for papillary thyroid cancer (PTC) presented with a rapidly enlarging grape-sized mass in his right medial arm with paresthesia in the ulnar nerve distribution. Imaging was suspicious for a peripheral nerve sheath tumor (PNST), but an ultrasound-guided biopsy was equivocal. The mass was excised with final histopathology demonstrating a benign neurofibroma/schwannoma hybrid nerve sheath tumor (N/S HNST) harboring a metastatic PTC deposit, ultimately mimicking the rare glandular schwannoma subtype. Next-generation sequencing (NGS) of the lesion demonstrated somatic variants in BRAF and TERT (common in PTC) and NF2 (common in PNSTs). After excision, the patient's nerve symptoms improved. A postsurgical PET/CT scan also showed progression in the lungs/mediastinum. Due to the metastatic nature of his PTC, he was treated with 14 mg of Lenvima (lenvatinib) daily, and his PET/CT surveillance was performed at more frequent intervals. Tumor-to-tumor metastasis (TTM) is a rare occurrence. To our knowledge, this is the first case reported on PTC metastasizing into a benign (hybrid) PNST, which mimicked glandular schwannoma. Symptomatology, imaging characteristics, NGS, and histopathological characteristics that can decipher between different benign PNST subtypes (schwannoma, neurofibroma, glandular, hybrid, etc.), malignant PNSTs (MPNSTs), and TTM are described.
Broad dengue neutralization in mosquitoes expressing an engineered antibody
(PLoS Pathogens, 2020-01) Crowe, James E., Jr.
Author summary With limited success of traditional vector control methods to curb dengue infections and more than half of the world's population still at risk, there is a need for novel strategies to reduce its impact on public health. Recent advances in genetic technologies has allowed for precise modifications of mosquito genome to make them resistant to infections, thus breaking the transmission cycle. Here we generated engineered Ae. aegypti mosquitoes efficiently expressing a DENV-targeting single-chain variable fragment (scFv) derived from a previously characterized broadly neutralizing human antibody, which blocked infection and transmission in these mosquitoes. To our knowledge, this is the first example of an engineered transgene capable of rendering Ae. aegypti mosquitoes 100% refractory to all four serotypes of DENV. The engineered mosquitoes, in future, could easily be paired with a gene drive, capable of spreading the transgene throughout wild disease-transmitting mosquito populations and preventing further DENV transmission. Since a number of diverse and well-characterized antibodies exist against other arboviruses (eg chikungunya and Zika, this work also provides a proof-of-concept principle for developing similar genetic strategies for reducing the impact of these arboviruses. With dengue virus (DENV) becoming endemic in tropical and subtropical regions worldwide, there is a pressing global demand for effective strategies to control the mosquitoes that spread this disease. Recent advances in genetic engineering technologies have made it possible to create mosquitoes with reduced vector competence, limiting their ability to acquire and transmit pathogens. Here we describe the development of Aedes aegypti mosquitoes synthetically engineered to impede vector competence to DENV. These mosquitoes express a gene encoding an engineered single-chain variable fragment derived from a broadly neutralizing DENV human monoclonal antibody and have significantly reduced viral infection, dissemination, and transmission rates for all four major antigenically distinct DENV serotypes. Importantly, this is the first engineered approach that targets all DENV serotypes, which is crucial for effective disease suppression. These results provide a compelling route for developing effective genetic-based DENV control strategies, which could be extended to curtail other arboviruses.
Cardiolipin-Dependent Properties of Model Mitochondrial Membranes from Molecular Simulations
(Biophysical Journal, 2019-08) Wilson, Blake A.; Ramanathan, Arvind; Lopez, Carlos F.
Cardiolipin is an anionic lipid found in the mitochondrial membranes of eukaryotes ranging from unicellular microorganisms to metazoans. This unique lipid contributes to various mitochondrial functions, including metabolism, mitochondrial membrane fusion and/or fission dynamics, and apoptosis. However, differences in cardiolipin content between the two mitochondrial membranes, as well as dynamic fluctuations in cardiolipin content in response to stimuli and cellular signaling events, raise questions about how cardiolipin concentration affects mitochondrial membrane structure and dynamics. Although cardiolipin's structural and dynamic roles have been extensively studied in binary mixtures with other phospholipids, the biophysical properties of cardiolipin in higher number lipid mixtures are still not well resolved. Here, we used molecular dynamics simulations to investigate the cardiolipin-dependent properties of ternary lipid bilayer systems that mimic the major components of mitochondrial membranes. We found that changes to cardiolipin concentration only resulted in minor changes to bilayer structural features but that the lipid diffusion was significantly affected by those alterations. We also found that cardiolipin position along the bilayer surfaces correlated to negative curvature deflections, consistent with the induction of negative curvature stress in the membrane monolayers. This work contributes to a foundational understanding of the role of cardiolipin in altering the properties in ternary lipid mixtures composed of the major mitochondrial phospholipids, providing much-needed insights to help understand how cardiolipin concentration modulates the biophysical properties of mitochondrial membranes.
Changes in rapid HIV treatment initiation after national "treat all" policy adoption in 6 sub-Saharan African countries: Regression discontinuity analysis
(PLOS MEDICINE, 2019-06) Rebeiro, Peter F.; Tymejczyk, Olga; Brazier, Ellen; Yiannoutsos, Constantin T.; Vinikoor, Michael; van Lettow, Monique
Background Most countries have formally adopted the World Health Organization's 2015 recommendation of universal HIV treatment ("treat all"). However, there are few rigorous assessments of the real-world impact of treat all policies on antiretroviral treatment (ART) uptake across different contexts. Methods and findings We used longitudinal data for 814,603 patients enrolling in HIV care between 1 January 2004 and 10 July 2018 in 6 countries participating in the global International epidemiology Databases to Evaluate AIDS (IeDEA) consortium: Burundi (N = 11,176), Kenya (N = 179,941), Malawi (N = 84,558), Rwanda (N = 17,396), Uganda (N = 96,286), and Zambia (N = 425,246). Using a quasi-experimental regression discontinuity design, we assessed the change in the proportion initiating ART within 30 days of enrollment in HIV care (rapid ART initiation) after country-level adoption of the treat all policy. A modified Poisson model was used to identify factors associated with failure to initiate ART rapidly under treat all. In each of the 6 countries, over 60% of included patients were female, and median age at enrollment ranged from 32 to 36 years. In all countries studied, national adoption of treat all was associated with large increases in rapid ART initiation. Significant increases in rapid ART initiation immediately after treat all policy adoption were observed in Rwanda, from 44.4% to 78.9% of patients (34.5 percentage points [pp], 95% CI 27.2 to 41.7; p < 0.001), Kenya (25.7 pp, 95% CI 21.8 to 29.5; p < 0.001), Burundi (17.7 pp, 95% CI 6.5 to 28.9; p = 0.002), and Malawi (12.5 pp, 95% CI 7.5 to 17.5; p < 0.001), while no immediate increase was observed in Zambia (0.4 pp, 95% CI -2.9 to 3.8; p = 0.804) and Uganda (-4.2 pp, 95% CI -9.0 to 0.7; p = 0.090). The rate of rapid ART initiation accelerated sharply following treat all policy adoption in Malawi, Uganda, and Zambia; slowed in Kenya; and did not change in Rwanda and Burundi. In post hoc analyses restricted to patients enrolling under treat all, young adults (16-24 years) and men were at increased risk of not rapidly initiating ART (compared to older patients and women, respectively). However, rapid ART initiation following enrollment increased for all groups as more time elapsed since treat all policy adoption. Study limitations include incomplete data on potential ART eligibility criteria, such as clinical status, pregnancy, and enrollment CD4 count, which precluded the assessment of rapid ART initiation specifically among patients known to be eligible for ART before treat all. Conclusions Our analysis indicates that adoption of treat all policies had a strong effect on increasing rates of rapid ART initiation, and that these increases followed different trajectories across the 6 countries. Young adults and men still require additional attention to further improve rapid ART initiation.
Changing Perspectives on the Role of DnaA-ATP in Orisome Function and Timing Regulation
(Frontiers in Microbiology, 2019-08-29) Leonard, Alan C.; Rao, Prassanna; Kadam, Rohit P.; Grimwade, Julia E.
Bacteria, like all cells, must precisely duplicate their genomes before they divide. Regulation of this critical process focuses on forming a pre-replicative nucleoprotein complex, termed the orisome. Orisomes perform two essential mechanical tasks that configure the unique chromosomal replication origin, oriC to start a new round of chromosome replication: (1) unwinding origin DNA and (2) assisting with loading of the replicative DNA helicase on exposed single strands. In Escherichia coli, a necessary orisome component is the ATP-bound form of the bacterial initiator protein, DnaA. DnaA-ATP differs from DnaA-ADP in its ability to oligomerize into helical filaments, and in its ability to access a subset of low affinity recognition sites in the E. coli replication origin. The helical filaments have been proposed to play a role in both of the key mechanical tasks, but recent studies raise new questions about whether they are mandatory for orisome activity. It was recently shown that a version of E. coli oriC (oriC(allADP)), whose multiple low affinity DnaA recognition sites bind DnaA-ATP and DnaA-ADP similarly, was fully occupied and unwound by DnaA-ADP in vitro, and in vivo suppressed the lethality of DnaA mutants defective in ATP binding and ATP-specific oligomerization. However, despite their functional equivalency, orisomes assembled on oriC(allADP) were unable to trigger chromosome replication at the correct cell cycle time and displayed a hyper-initiation phenotype. Here we present a new perspective on DnaA-ATP, and suggest that in E. coli, DnaA-ATP is not required for mechanical functions, but rather is needed for site recognition and occupation, so that initiation timing is coupled to DnaA-ATP levels. We also discuss how other bacterial types may utilize DnaA-ATP and DnaA-ADP, and whether the high diversity of replication origins in the bacterial world reflects different regulatory strategies for how DnaA-ATP is used to control orisome assembly.
Correlated evolution between repertoire size and song plasticity predicts that sexual selection on song promotes open-ended learning
(eLife, 2019-09-03) Robinson, Cristina M.; Snyder, Kate T.; Creanza, Nicole
Some oscine songbird species modify their songs throughout their lives ('adult song plasticity' or 'open-ended learning'), while others crystallize their songs around sexual maturity. It remains unknown whether the strength of sexual selection on song characteristics, such as repertoire size, affects adult song plasticity, or whether adult song plasticity affects song evolution. Here, we compiled data about song plasticity, song characteristics, and mating system and then examined evolutionary interactions between these traits. Across 67 species, we found that lineages with adult song plasticity show directional evolution toward increased syllable and song repertoires, while several other song characteristics evolved faster, but in a non-directional manner. Song plasticity appears to drive bi-directional transitions between monogamous and polygynous social mating systems. Notably, our analysis of correlated evolution suggests that extreme syllable and song repertoire sizes drive the evolution of adult song plasticity or stability, providing novel evidence that sexual selection may indirectly influence open-versus closed-ended learning.
Cost-Effectiveness of Multigene Pharmacogenetic Testing in Patients With Acute Coronary Syndrome After Percutaneous Coronary Intervention
(Value in Health, 2020-01) Dong, Olivia M.; Wheeler, Stephanie B.; Cruden, Gracelyn; Lee, Craig R.; Voora, Deepak; Dusetzina, Stacie B.; Wiltshire, Tim
Objective: To evaluate the cost-effectiveness of multigene testing (CYP2C19, SLCO1B1, CYP2C9, VKORC1) compared with singlegene testing (CYP2C19) and standard of care (no genotyping) in acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI) from Medicare's perspective. Methods: A hybrid decision tree/Markov model was developed to simulate patients post-PCI for ACS requiring antiplatelet therapy (CYP2C19 to guide antiplatelet selection), statin therapy (SLCO1B1 to guide statin selection), and anticoagulant therapy in those that develop atrial fibrillation (CYP2C9/VKORC1 to guide warfarin dose) over 12 months, 24 months, and lifetime. The primary outcome was cost (2016 US dollar) per quality-adjusted life years (QALYs) gained. Costs and QALYs were discounted at 3% per year. Probabilistic sensitivity analysis (PSA) varied input parameters (event probabilities, prescription costs, event costs, health-state utilities) to estimate changes in the cost per QALY gained. Results: Base-case-discounted results indicated that the cost per QALY gained was $59 876, $33 512, and $3780 at 12 months, 24 months, and lifetime, respectively, for multigene testing compared with standard of care. Single-gene testing was dominated by multigene testing at all time horizons. PSA-discounted results indicated that, at the $50 000/QALY gained willingness-to-pay threshold, multigene testing had the highest probability of cost-effectiveness in the majority of simulations at 24 months (61%) and over the lifetime (81%). Conclusions: On the basis of projected simulations, multigene testing for Medicare patients post-PCI for ACS has a higher probability of being cost-effective over 24 months and the lifetime compared with single-gene testing and standard of care and could help optimize medication prescribing to improve patient outcomes.
CpG-related SNPs in the MS4A region have a dose-dependent effect on risk of late-onset Alzheimer disease
(Aging Cell, 2019-08) Thornton-Wells, Tricia A.
CpG-related single nucleotide polymorphisms (CGS) have the potential to perturb DNA methylation; however, their effects on Alzheimer disease (AD) risk have not been evaluated systematically. We conducted a genome-wide association study using a sliding-window approach to measure the combined effects of CGSes on AD risk in a discovery sample of 24 European ancestry cohorts (12,181 cases, 12,601 controls) from the Alzheimer's Disease Genetics Consortium (ADGC) and replication sample of seven European ancestry cohorts (7,554 cases, 27,382 controls) from the International Genomics of Alzheimer's Project (IGAP). The potential functional relevance of significant associations was evaluated by analysis of methylation and expression levels in brain tissue of the Religious Orders Study and the Rush Memory and Aging Project (ROSMAP), and in whole blood of Framingham Heart Study participants (FHS). Genome-wide significant (p < 5 x 10(-8)) associations were identified with 171 1.0 kb-length windows spanning 932 kb in the APOE region (top p < 2.2 x 10(-308)), five windows at BIN1 (top p = 1.3 x 10(-13)), two windows at MS4A6A (top p = 2.7 x 10(-10)), two windows near MS4A4A (top p = 6.4 x 10(-10)), and one window at PICALM (p = 6.3 x 10(-9)). The total number of CGS-derived CpG dinucleotides in the window near MS4A4A was associated with AD risk (p = 2.67 x 10(-10)), brain DNA methylation (p = 2.15 x 10(-10)), and gene expression in brain (p = 0.03) and blood (p = 2.53 x 10(-4)). Pathway analysis of the genes responsive to changes in the methylation quantitative trait locus signal at MS4A4A (cg14750746) showed an enrichment of methyltransferase functions. We confirm the importance of CGS in AD and the potential for creating a functional CpG dosage-derived genetic score to predict AD risk.
The Cross Talk between TbTim50 and PIP39, Two Aspartate-Based Protein Phosphatases, Maintains Cellular Homeostasis in Trypanosoma brucei
(mSphere, 2019-07) Tripathi, Anuj; Singha, Ujjal K.; Paromov, Victor; Hill, Salisha; Pratap, Siddharth; Rose, Kristie; Chaudhuri, Minu
Trypanosoma brucei, the infectious agent of a deadly disease known as African trypanosomiasis, undergoes various stresses during its digenetic life cycle. We previously showed that downregulation of T. brucei mitochondrial inner membrane protein translocase 50 (TbTim50), an aspartate-based protein phosphatase and a component of the translocase of the mitochondrial inner membrane (TIM), increased the tolerance of procyclic cells to oxidative stress. Using comparative proteomics analysis and further validating the proteomics results by immunoblotting, here we discovered that TbTim50 downregulation caused an approximately 5-fold increase in the levels of PIP39, which is also an aspartate-based protein phosphatase and is primarily localized in glycosomes. A moderate upregulation of a number of glycosomal enzymes was also noticed due to TbTim50 knockdown. We found that the rate of mitochondrial ATP production by oxidative phosphorylation decreased and that substrate-level phosphorylation increased due to depletion of TbTim50. These results were correlated with relative increases in the levels of trypanosome alternative oxidase and hexokinase and a reduced-growth phenotype in low-glucose medium. The levels and activity of the mitochondrial superoxide dismutase and glutaredoxin levels were increased due to TbTim50 knockdown. Furthermore, we show that TbTim50 downregulation increased the cellular AMP/ATP ratio, and as a consequence, phosphorylation of AMP-activated protein kinase (AMPK) was increased. Knocking down both TbTim50 and TbPIP39 reduced PIP39 levels as well as AMPK phosphorylation and reduced T. brucei tolerance to oxidative stress. These results suggest that TbTim50 and PIP39, two protein phosphatases in mitochondria and glycosomes, respectively, cross talk via the AMPK pathway to maintain cellular homeostasis in the procyclic form of T. brucei. IMPORTANCE Trypanosoma brucei, the infectious agent of African trypanosomiasis, must adapt to strikingly different host environments during its digenetic life cycle. Developmental regulation of mitochondrial activities is an essential part of these processes. We have shown previously that mitochondrial inner membrane protein translocase 50 in T. brucei (TbTim50) possesses a dually specific phosphatase activity and plays a role in the cellular stress response pathway. Using proteomics analysis, here we have elucidated a novel connection between TbTim50 and a protein phosphatase of the same family, PIP39, which is also a differentiation-related protein localized in glycosomes. We found that these two protein phosphatases cross talk via the AMPK pathway and modulate cellular metabolic activities under stress. Together, our results indicate the importance of a TbTim50 and PIP39 cascade for communication between mitochondria and other cellular parts in regulation of cell homeostasis in T. brucei.
Deletion of Tet proteins results in quantitative disparities during ESC differentiation partially attributable to alterations in gene expression
(BMC Developmental Biology, 2019-07-08) Reimer, Michael, Jr.; Pulakanti, Kirthi; Shi, Linzheng; Abel, Alex; Liang, Mingyu; Malarkannan, Subramaniam; Rao, Sridhar
BackgroundThe Tet protein family (Tet1, Tet2, and Tet3) regulate DNA methylation through conversion of 5-methylcytosine to 5-hydroxymethylcytosine which can ultimately result in DNA demethylation and play a critical role during early mammalian development and pluripotency. While multiple groups have generated knockouts combining loss of different Tet proteins in murine embryonic stem cells (ESCs), differences in genetic background and approaches has made it difficult to directly compare results and discern the direct mechanism by which Tet proteins regulate the transcriptome. To address this concern, we utilized genomic editing in an isogenic pluripotent background which permitted a quantitative, flow-cytometry based measurement of pluripotency in combination with genome-wide assessment of gene expression and DNA methylation changes. Our ultimate goal was to generate a resource of large-scale datasets to permit hypothesis-generating experiments.ResultsWe demonstrate a quantitative disparity in the differentiation ability among Tet protein deletions, with Tet2 single knockout exhibiting the most severe defect, while loss of Tet1 alone or combinations of Tet genes showed a quantitatively intermediate phenotype. Using a combination of transcriptomic and epigenomic approaches we demonstrate an increase in DNA hypermethylation and a divergence of transcriptional profiles in pluripotency among Tet deletions, with loss of Tet2 having the most profound effect in undifferentiated ESCs.ConclusionsWe conclude that loss of Tet2 has the most dramatic effect both on the phenotype of ESCs and the transcriptome compared to other genotypes. While loss of Tet proteins increased DNA hypermethylation, especially in gene promoters, these changes in DNA methylation did not correlate with gene expression changes. Thus, while loss of different Tet proteins alters DNA methylation, this change does not appear to be directly responsible for transcriptome changes. Thus, loss of Tet proteins likely regulates the transcriptome epigenetically both through altering 5mC but also through additional mechanisms. Nonetheless, the transcriptome changes in pluripotent Tet2(-/-) ESCs compared to wild-type implies that the disparities in differentiation can be partially attributed to baseline alterations in gene expression.
Delineating the Molecular Basis of the Calmodulin–bMunc13-2 Interaction by Cross-Linking/Mass Spectrometry—Evidence for a Novel CaM Binding Motif in bMunc13-2
(Cells, 2020-01) Piotrowski, Christine; Moretti, Rocco; Ihling, Christian H.; Haedicke, Andre; Liepold, Thomas; Lipstein, Noa; Meiler, Jens; Jahn, Olaf; Sinz, Andrea
Exploring the interactions between the Ca2+ binding protein calmodulin (CaM) and its target proteins remains a challenging task. Members of the Munc13 protein family play an essential role in short-term synaptic plasticity, modulated via the interaction with CaM at the presynaptic compartment. In this study, we focus on the bMunc13-2 isoform expressed in the brain, as strong changes in synaptic transmission were observed upon its mutagenesis or deletion. The CaM-bMunc13-2 interaction was previously characterized at the molecular level using short bMunc13-2-derived peptides only, revealing a classical 1-5-10 CaM binding motif. Using larger protein constructs, we have now identified for the first time a novel and unique CaM binding site in bMunc13-2 that contains an N-terminal extension of a classical 1-5-10 CaM binding motif. We characterize this motif using a range of biochemical and biophysical methods and highlight its importance for the CaM-bMunc13-2 interaction.

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