Leslie Tung

Johns Hopkins University, Baltimore, Maryland, United States

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Publications (122)430.63 Total impact

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    ABSTRACT: -The mechanisms of the electrocardiographic changes and arrhythmias in Brugada syndrome (BrS) remain controversial. Mutations in the sodium channel gene, SCN5A and regulatory proteins that reduce or eliminate sodium current (INa) have been linked to BrS. We studied the properties of a BS-associated SCN5A mutation in a protein kinase A (PKA) consensus phosphorylation site, R526H. -In-vitro PKA phosphorylation was detected in the I-II linker peptide of wild type (WT) channels but not R526H or S528A (phosphorylation site) mutants. Cell surface expression of R526H and S528A channels were reduced compared with WT. Whole-cell INa through all channel variants revealed no significant differences in the steady-state activation, inactivation and recovery from inactivation. Peak current densities of the mutants were significantly reduced compared to WT. Infection of 2D cultures of neonatal rat ventricular myocytes with WT and mutant channels increased conduction velocity (CV) compared with non-infected cells. PKA stimulation significantly increased peak INa and CV of WT but not mutant channels. Oxidant stress inhibits cardiac Na currents; WT and mutant INa decreases with the intracellular application of NADH, an effect that is reversed by PKA stimulation in WT but not in R526H or S528A channels. -We identified a family with BrS and an SCN5A mutation in a PKA consensus phosphorylation site. The BrS mutation R526H is associated with a reduction in the basal level of INa and a failure of PKA stimulation to augment the current which may contribute to the predisposition to arrhythmias in patients with BrS, independent of the precipitants.
    Circulation Cardiovascular Genetics 05/2014; · 6.73 Impact Factor
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    ABSTRACT: The electrophysiological consequences of cardiomyocyte and myofibroblast interactions remain unclear, and the contribution of mechanical coupling between these two cell types is still poorly understood. In this study, we examined the time course and mechanisms by which addition of myofibroblasts activated by transforming growth factor-beta (TGF-β) influence the conduction velocity (CV) of neonatal rat ventricular cell monolayers. We observed that myofibroblasts affected CV within 30 minutes of contact and that these effects were temporally correlated with membrane deformation of cardiomyocytes by the myofibroblasts. Expression of dominant negative RhoA in the myofibroblasts impaired both myofibroblast contraction and myofibroblast-induced slowing of cardiac conduction, whereas overexpression of constitutive RhoA had little effect. To determine the importance of mechanical coupling between these cell types, we examined the expression of the two primary cadherins in the heart (N- and OB-cadherin) at cell-cell contacts formed between myofibroblasts and cardiomyocytes. Although OB-cadherin was frequently found at myofibroblast-myofibroblast contacts, very little expression was observed at myofibroblast-cardiomyocyte contacts. The myofibroblast-induced slowing of cardiac conduction was not prevented by silencing of OB-cadherin in the myofibroblasts, and could be reversed by inhibitors of mechanosensitive channels (gadolinium or streptomycin) and cellular contraction (blebbistatin). In contrast, N-cadherin expression was commonly observed at myofibroblast-cardiomyocyte contacts, and silencing of N-cadherin in myofibroblasts prevented the myofibroblast-dependent slowing of cardiac conduction. We propose that myofibroblasts can impair the electrophysiological function of cardiac tissue through the application of contractile force to the cardiomyocyte membrane via N-cadherin junctions.
    Journal of Molecular and Cellular Cardiology 01/2014; · 5.15 Impact Factor
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    ABSTRACT: Light-mediated silencing and stimulation of cardiac excitability, an important complement to electrical stimulation, promises important discoveries and therapies. To date, cardiac optogenetics has been studied with patch-clamp, multielectrode arrays, video microscopy, and an all-optical system measuring calcium transients. The future lies in achieving simultaneous optical acquisition of excitability signals and optogenetic control, both with high spatio-temporal resolution. Here, we make progress by combining optical mapping of action potentials with concurrent activation of channelrhodopsin-2 (ChR2) or halorhodopsin (eNpHR3.0), via an all-optical system applied to monolayers of neonatal rat ventricular myocytes (NRVM). Additionally, we explore the capability of ChR2 and eNpHR3.0 to shape action-potential waveforms, potentially aiding the study of short/long QT syndromes that result from abnormal changes in action potential duration (APD). These results show the promise of an all-optical system to acquire action potentials with precise temporal optogenetics control, achieving a long-sought flexibility beyond the means of conventional electrical stimulation.
    Scientific reports. 01/2014; 4:6125.
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    ABSTRACT: Methods for obtaining cardiomyocytes from human embryonic stem cells (hESCs) are improving at a significant rate. However, the characterization of these cardiomyocytes (CMs) is evolving at a relatively slower rate. In particular, there is still uncertainty in classifying the phenotype (ventricular-like, atrial-like, nodal-like, etc.) of an hESC-derived cardiomyocyte (hESC-CM). While previous studies identified the phenotype of a CM based on electrophysiological features of its action potential, the criteria for classification were typically subjective and differed across studies. In this paper, we use techniques from signal processing and machine learning to develop an automated approach to discriminate the electrophysiological differences between hESC-CMs. Specifically, we propose a spectral grouping-based algorithm to separate a population of CMs into distinct groups based on the similarity of their action potential shapes. We applied this method to a dataset of optical maps of cardiac cell clusters dissected from human embryoid bodies. While some of the nine cell clusters in the dataset are presented with just one phenotype, the majority of the cell clusters are presented with multiple phenotypes. The proposed algorithm is generally applicable to other action potential datasets and could prove useful in investigating the purification of specific types of CMs from an electrophysiological perspective.
    IEEE Transactions on Biomedical Engineering 01/2014; 61(9):2389-2395. · 2.35 Impact Factor
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    ABSTRACT: Cultured heart cells have long been valuable for characterizing biological mechanism and disease pathogenesis. However, these preparations have limitations, relating to immaturity in key properties like excitation-contraction coupling and β-adrenergic stimulation. Progressive attenuation of the latter is intimately related to pathogenesis and therapy in heart failure. Highly valuable would be a long-term culture system that emulates the structural and functional changes that accompany disease and development, while concurrently permitting ready access to underlying molecular events. Accordingly, we here produce functional monolayers of adult guinea-pig ventricular myocytes (aGPVMs) that can be maintained in long-term culture for several weeks. At baseline, these monolayers exhibit considerable myofibrillar organization and a significant contribution of sarcoplasmic reticular (SR) Ca(2+) release to global Ca(2+) transients. In terms of electrical signaling, these monolayers support propagated electrical activity and manifest monophasic restitution of action-potential duration and conduction velocity. Intriguingly, β-adrenergic stimulation increases chronotropy but not inotropy, indicating selective maintenance of β-adrenergic signaling. It is interesting that this overall phenotypic profile is not fixed, but can be readily enhanced by chronic electrical stimulation of cultures. This simple environmental cue significantly enhances myofibrillar organization as well as β-adrenergic sensitivity. In particular, the chronotropic response increased, and an inotropic effect now emerges, mimicking a reversal of the progression seen in heart failure. Thus, these aGPVM monolayer cultures offer a valuable platform for clarifying long elusive features of β-adrenergic signaling and its plasticity.
    Journal of Molecular and Cellular Cardiology 09/2013; · 5.15 Impact Factor
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    ABSTRACT: Mechanical stresses directly affect many cellular processes, including signal transduction, growth, differentiation, and survival. Cells can themselves generate such stresses by activating myosin to contract the actin cytoskeleton, which in turn can regulate both cell-substrate and cell-cell interactions. We are studying mechanical forces at cell-cell and cell-substrate interactions using arrays of selectively patterned flexible PDMS microposts combined with the ability to apply local chemical stimulation. Micropipette ``spritzing'', a laminar flow technique, uses glass micropipettes mounted on a microscope stage to deliver drugs to controlled regions within a cellular construct while cell traction forces are recorded via the micropost array. The pipettes are controlled by micromanipulators allowing for rapid and precise movement across the array and the ability to treat multiple constructs within a sample. This technique allows for observing the propagation of a chemically induced mechanical stimulus through cell-cell and cell-substrate interactions. We have used this system to administer the acto-myosin inhibitors Blebbistatin and Y-27632 to single cells and observed the subsequent decrease in cell traction forces. Experiments using trypsin-EDTA have shown this system to be capable of single cell manipulation through removal of one cell within a pair configuration while leaving the other cell unaffected.
    03/2013;
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    ABSTRACT: BACKGROUND: We recently demonstrated that high frequency alternating current (HFAC) electric fields can reversibly block propagation in the heart by inducing an oscillating, elevated transmembrane potential (V(m)) which maintains myocytes in a refractory state for the field duration, and can terminate arrhythmias, including ventricular fibrillation (VF). OBJECTIVE: In this study, we sought to quantify and characterize conduction block (CB) induced by HFAC fields and to determine whether the degree of CB can be used to predict defibrillation success. METHODS: Optical mapping was performed in adult guinea pig hearts (n = 14), and simulations were performed in an anatomically accurate rabbit ventricular model. HFAC fields (50-500 Hz) were applied to the ventricles. A novel power spectrum metric of CB - the loss of spectral power in the 1-30 Hz range, termed loss of conduction power (LCP) - was assessed during the HFAC field and compared with defibrillation success and VF vulnerability. RESULTS: LCP increased with field strength and decreased with frequency. Optical mapping experiments conducted on the epicardial surface showed that LCP and the size of CB regions were significantly correlated with VF initiation and termination. In simulations, sub-surface myocardial LCP and CB size were more closely correlated with VF termination than surface values. Multilinear regression analysis of simulation results revealed that while CB on both the surface and sub-surface myocardium were predictive, sub-surface myocardial CB was the better predictor of defibrillation success. CONCLUSION: HFAC fields induce a field-dependent state of conduction block, and defibrillation success is related to the degree and location of the conduction block.
    Heart rhythm: the official journal of the Heart Rhythm Society 01/2013; · 4.56 Impact Factor
  • Biophysical Journal 01/2013; 104(2):281-. · 3.67 Impact Factor
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    ABSTRACT: Since the first description of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), these cells have garnered tremendous interest for their potential use in patient-specific analysis and therapy. Additionally, hiPSC-CMs can be derived from donor cells from patients with specific cardiac disorders, enabling in vitro human disease models for mechanistic study and therapeutic drug assessment. However, a full understanding of their electrophysiological and contractile function is necessary before this potential can be realized. Here, we review this emerging field from a functional perspective, with particular emphasis on beating rate, action potential, ionic currents, multicellular conduction, calcium handling and contraction. We further review extant hiPSC-CM disease models that recapitulate genetic myocardial disease.
    Progress in Biophysics and Molecular Biology 08/2012; · 2.91 Impact Factor
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    ABSTRACT: Human embryonic stem cells have emerged as the prototypical source from which cardiomyocytes can be derived for use in drug discovery and cell therapy. However, such applications require that these cardiomyocytes (hESC-CMs) faithfully recapitulate the physiology of adult cells, especially in relation to their electrophysiological and contractile function. We review what is known about the electrophysiology of hESC-CMs in terms of beating rate, action potential characteristics, ionic currents, and cellular coupling as well as their contractility in terms of calcium cycling and contraction. We also discuss the heterogeneity in cellular phenotypes that arises from variability in cardiac differentiation, maturation, and culture conditions, and summarize present strategies that have been implemented to reduce this heterogeneity. Finally, we present original electrophysiological data from optical maps of hESC-CM clusters.
    Progress in Biophysics and Molecular Biology 08/2012; · 2.91 Impact Factor
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    ABSTRACT: In this study, we characterized the electrophysiological benefits of engrafting human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in a model of arrhythmogenic cardiac tissue. Using transforming growth factor-β treated monolayers of neonatal rat ventricular cells (NRVCs), which retain several key aspects of the healing infarct such as an excess of contractile myofibroblasts and slowed, heterogeneous conduction, we assessed the ability of hESC-CMs to improve conduction and prevent arrhythmias. Cells from beating embryoid bodies (hESC-CMs) can form functional monolayers which beat spontaneously and can be electrically stimulated, with mean action potential duration of 275 ± 36 ms and conduction velocity (CV) of 10.6 ± 4.2 cm/s (n = 3). These cells, or cells from non-beating embryoid bodies (hEBCs) were added to anisotropic, NRVC monolayers. Immunostaining demonstrated hESC-CM survival and engraftment, and dye transfer assays confirmed functional coupling between hESC-CMs and NRVCs. Conduction velocities significantly increased in anisotropic NRVC monolayers after engraftment of hESC-CMs (13.4 ± 0.9 cm/s, n = 35 vs. 30.1 ± 3.2 cm/s, n = 20 in the longitudinal direction and 4.3 ± 0.3 cm/s vs. 9.3 ± 0.9 cm/s in the transverse direction), but decreased to even lower values after engraftment of non-cardiac hEBCs (to 10.6 ± 1.3 cm/s and 3.1 ± 0.5 cm/s, n = 11, respectively). Furthermore, reentrant wave vulnerability in NRVC monolayers decreased by 20% after engraftment of hESC-CMs, but did not change with engraftment of hEBCs. Finally, the culture of hESC-CMs in transwell inserts, which prevents juxtacrine interactions, or engraftment with connexin43-silenced hESC-CMs provided no functional improvement to NRVC monolayers. These results demonstrate that hESC-CMs can reverse the slowing of conduction velocity, reduce the incidence of reentry, and augment impaired electrical propagation via gap junction coupling to host cardiomyocytes in this arrhythmogenic in vitro model.
    Journal of Molecular and Cellular Cardiology 07/2012; 53(1):15-23. · 5.15 Impact Factor
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    ABSTRACT: Early afterdepolarizations (EADs) are voltage oscillations that occur during the repolarizing phase of the cardiac action potential and cause cardiac arrhythmias in a variety of clinical settings. EADs occur in the setting of reduced repolarization reserve and increased inward-over-outward currents, which intuitively explains the repolarization delay but does not mechanistically explain the time-dependent voltage oscillations that are characteristic of EADs. In a recent theoretical study, we identified a dual Hopf-homoclinic bifurcation as a dynamical mechanism that causes voltage oscillations during EADs, depending on the amplitude and kinetics of the L-type Ca(2+) channel (LTCC) current relative to the repolarizing K(+) currents. Here we demonstrate this mechanism experimentally. We show that cardiac monolayers exposed to the LTCC agonists BayK8644 and isoproterenol produce EAD bursts that are suppressed by the LTCC blocker nitrendipine but not by the Na(+) current blocker tetrodoxin, depletion of intracellular Ca(2+) stores with thapsigargin and caffeine, or buffering of intracellular Ca(2+) with BAPTA-AM. These EAD bursts exhibited a key dynamical signature of the dual Hopf-homoclinic bifurcation mechanism, namely, a gradual slowing in the frequency of oscillations before burst termination. A detailed cardiac action potential model reproduced the experimental observations, and identified intracellular Na(+) accumulation as the likely mechanism for terminating EAD bursts. Our findings in cardiac monolayers provide direct support for the Hopf-homoclinic bifurcation mechanism of EAD-mediated triggered activity, and raise the possibility that this mechanism may also contribute to EAD formation in clinical settings such as long QT syndromes, heart failure, and increased sympathetic output.
    Biophysical Journal 06/2012; 102(12):2706-14. · 3.67 Impact Factor
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    Seth H Weinberg, Leslie Tung
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    ABSTRACT: Alternans is a beat-to-beat alternation of the cardiac action potential duration (APD) or intracellular calcium (Ca(i)) transient. In cardiac tissue, alternans can be spatially concordant or discordant, of which the latter has been shown to increase dispersion of repolarization and promote a substrate for initiation of ventricular fibrillation. Alternans has been studied almost exclusively under constant cycle length pacing conditions. However, heart rate varies greatly on a beat-by-beat basis in normal and pathological conditions. The purpose of this study was to determine if applying a repetitive but non-constant pacing pattern, specifically cycle length oscillation (CLO), promotes or suppresses a proarrhythmic substrate. We performed computational simulations and optical mapping experiments to investigate the potential consequences of CLO. In a single cell computational model, CLO induced APD and Ca(i) alternans, which became "phase-matched" with the applied oscillation. As a consequence of the phase-matching, in one-dimensional cable simulations, neonatal rat ventricular myocyte monolayers, and isolated adult guinea pig hearts CLO could transiently induce spatial and electromechanical discordant alternans followed by a steady-state of concordance. Our results demonstrated that under certain conditions, CLO can initiate ventricular fibrillation in the isolated hearts. On the other hand, CLO can also exert an antiarrhythmic effect by converting an existing state of discordant alternans to concordant alternans.
    PLoS ONE 01/2012; 7(7):e40477. · 3.73 Impact Factor
  • Susan Thompson, Leslie Tung
    Biophysical Journal 01/2012; 102(3):36-. · 3.67 Impact Factor
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    ABSTRACT: Electrical impulse propagation is an essential function in cardiac, skeletal muscle, and nervous tissue. Abnormalities in cardiac impulse propagation underlie lethal reentrant arrhythmias, including ventricular fibrillation. Temporary propagation block throughout the ventricular myocardium could possibly terminate these arrhythmias. Electrical stimulation has been applied to nervous tissue to cause reversible conduction block, but has not been explored sufficiently in cardiac tissue. We show that reversible propagation block can be achieved in cardiac tissue by holding myocardial cells in a refractory state for a designated period of time by applying a sustained sinusoidal high-frequency alternating current (HFAC); in doing so, reentrant arrhythmias are terminated. We demonstrate proof of concept using several models, including optically mapped monolayers of neonatal rat ventricular cardiomyocytes, Langendorff-perfused guinea pig and rabbit hearts, intact anesthetized adult rabbits, and computer simulations of whole-heart impulse propagation. HFAC may be an effective and potentially safer alternative to direct current application, currently used to treat ventricular fibrillation.
    Science translational medicine 09/2011; 3(102):102ra96. · 10.76 Impact Factor
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    ABSTRACT: After cardiac injury, activated cardiac myofibroblasts can influence tissue electrophysiology. Because mechanical coupling through adherens junctions provides a route for intercellular communication, we tested the hypothesis that myofibroblasts exert tonic contractile forces on the cardiomyocytes and affect electric propagation via a process of mechanoelectric feedback. The role of mechanoelectric feedback was examined in transforming growth factor-β-treated monolayers of cocultured myofibroblasts and neonatal rat ventricular cells by inhibiting myofibroblast contraction and blocking mechanosensitive channels. Untreated (control) and transforming growth factor-β-treated (fibrotic) anisotropic monolayers were optically mapped for electrophysiological comparison. Longitudinal conduction velocity, transverse conduction velocity, and normalized action potential upstroke velocity (dV/dt(max)) significantly decreased in fibrotic monolayers (14.4 ± 0.7 cm/s [mean ± SEM], 4.1 ± 0.3 cm/s [n=53], and 3.1 ± 0.2% per ms [n=14], respectively) compared with control monolayers (27.2 ± 0.8 cm/s, 8.5 ± 0.4 cm/s [n=40], and 4.9 ± 0.1% per ms [n=12], respectively). Application of the excitation-contraction uncoupler blebbistatin or the mechanosensitive channel blocker gadolinium or streptomycin dramatically increased longitudinal conduction velocity, transverse conduction velocity, and dV/dt(max) in fibrotic monolayers (35.9 ± 1.5 cm/s, 10.3 ± 0.6 cm/s [n=17], and 4.5 ± 0.1% per ms [n=14], respectively). Similar results were observed with connexin43-silenced cardiac myofibroblasts. Spiral-wave induction in fibrotic monolayers also decreased after the aforementioned treatments. Finally, traction force measurements of individual myofibroblasts showed a significant increase with transforming growth factor-β, a decrease with blebbistatin, and no change with mechanosensitive channel blockers. These observations suggest that myofibroblast-myocyte mechanical interactions develop during cardiac injury, and that cardiac conduction may be impaired as a result of increased mechanosensitive channel activation owing to tension applied to the myocyte by the myofibroblast.
    Circulation 05/2011; 123(19):2083-93. · 15.20 Impact Factor
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    Leslie Tung
    The Journal of Physiology 05/2011; 589(Pt 9):2107-8. · 4.38 Impact Factor
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    ABSTRACT: In this work, we utilize a CMOS active pixel sensor in a fluorescence imaging setup. The ability to sense small light intensity changes on top of a large baseline with spatial resolution at the subcellular scale is required in fluorescence imaging. The CMOS imager presented in (1) is perfect for this application with the ability to resolve fine features coupled with high dynamic range. By using a custom imager with a relay lens we are able to realize a dramatic decrease in device size, cost and complexity of the whole system.
    International Symposium on Circuits and Systems (ISCAS 2011), May 15-19 2011, Rio de Janeiro, Brazil; 01/2011
  • Biophysical Journal 01/2011; 100(3). · 3.67 Impact Factor
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    ABSTRACT: The production of cardiomyocytes from human induced pluripotent stem cells (hiPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC) and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC), including hiPSC generated from CD34(+) cord blood using non-viral, non-integrating methods. We systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB) with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34(+) cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5%) oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64-89%) of cardiac troponin I(+) cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs. This efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically-safe nonviral human cardiac cells for regenerative medicine.
    PLoS ONE 01/2011; 6(4):e18293. · 3.73 Impact Factor

Publication Stats

2k Citations
430.63 Total Impact Points

Institutions

  • 1988–2014
    • Johns Hopkins University
      • Department of Biomedical Engineering
      Baltimore, Maryland, United States
  • 2013
    • College of William and Mary
      • Department of Applied Science
      Williamsburg, VA, United States
  • 2009–2013
    • Johns Hopkins Medicine
      • • Department of Biomedical Engineering
      • • Division of Cardiology
      Baltimore, MD, United States
  • 2012
    • University of California, Los Angeles
      Los Angeles, California, United States
  • 2006–2007
    • Duke University
      • Department of Biomedical Engineering (BME)
      Durham, NC, United States