Efficient Unbiased Sampling of Protein Dynamics using Reinforcement ...

Wednesday, February 21, 2018 with a resolution down to 100 nm possible. Therefore, we are able to construct 3D artificial networks for guided neurite growth: A Nanoscribe Photonic Professional GT is used to write a pillar structure resembling a medieval castle with total dimensions of 600
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100 mm. Pillars of different height with cavities on top, which act as attachment sites for the somata of the neurons, are connected to each other through channels to guide the neurites from one pillar to the next in a precise predefined way. The inner diameter of the channels, 3.6 mm, is tailored to fit the axon-diameter of 2 - 3 mm. To specifically enhance cell attachment, poly-D-lysin is printed directly into the cavities of the pillars. The substrate is cultured with granule cells from murine cerebella. Cell activity after at least 5 days in vitro is shown through basic patch-clamp measurements. The work presented here is a prove of principle for an approach to build complex, tailor-made 3D neural circuits. 3333-Pos Board B541 Focused Ultrasound Evoked Responses in Dorsal Root Ganglion Neurons (DRG) and HEK293 Cells Danny M. Florez-Paz1, Chi-Kun Tong1, Benjamin U. Hoffman1, Stephen A. Lee2, Elisa E. Konofagou2, Ellen A. Lumpkin1. 1 Physiology & Cellular Biophysics, Columbia University, New York, NY, USA, 2Biomedical Engineering, Columbia University, New York, NY, USA. Focused ultrasound (US) is a noninvasive technique to modulate peripheral activity, and holds promise for the treatment of sensory disorders and inflammatory diseases. However, the mechanisms underlying US-evoked responses in sensory neurons are still unclear. Here, we explored the neuromodulatory capabilities of US in sensory neurons and HEK293 cells. First, we developed an in vitro system integrating an US transducer (3.1 MHz) and a 3D-printed recording chamber, combined with calcium imaging and electrophysiological techniques to study US-evoked cellular activation. To establish parameters for activation, we analyzed US in DRG neurons using calcium imaging. We found robust, repeatable and reversible calcium responses, that increased with US pressure, over a wide range of sonication pressures. US activated 40% of neurons with both small and large diameter cell bodies (N=152). We next recorded US-evoked currents using whole-cell patch-clamp technique. In current-clamp, US evoked action potentials in 33% of sensory neurons (N=21). Using holding potentials from 90 mV to þ50 mV (N=10), currentvoltage relation curves did not reverse, suggesting Ca2þ or Naþ conductances, however in HEK293 cells, same experiments showed curves reversal close to 0 mV (N=8). To implicate specific channels involved in US-evoked currents, we tested US in HEK293 cells, followed by perfusion with ruthenium red (RR; 10mM), a non-specific cation channel blocker (N=5). Three cells showed reduced currents, whereas two cells showed enhanced currents in the presence of RR, with thresholds of 1.6-1.74 MPa and 1.25-1.37 MPa respectively. These results suggest there might be two populations of HEK293 cells and/or channels that have different US activation thresholds, and showing different RR sensitivity. Together, these results suggest multiple ion-channel mechanisms underlying US-evoke responses in different cells. 3334-Pos Board B542 Optical Recordings of Action Potentials in E18 Rat Hippocampal Neurons Exposed to 10-NS Electric Pulses Iurii Semenov, Shu Xiao, Andrei Pakhomov. Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA. It is currently unclear if nanosecond pulses can be used to trigger action potentials (APs) in excitable cells with no accompanied plasma membrane (PM) electropermeabilization. Present study shows that APs can be triggered in neurons by 10-ns electric pulses (EPs) with no signs of sustained PM damage related to electropermeabilization. The experiments were performed on coverslips. Cells were loaded with FluoVolt. Dye emission was observed with CCD Camera. Fast imaging at 3000 frames/s was utilized. Pair of 100-mm rod electrodes was used for EPs delivery. To find threshold of AP generation, neurons were exposed to a single 10 ns pulse in the range from 4 to 93 kV/cm. PM responses were evaluated before and after blockage of voltage-gated Naþ-channels with tetrodotoxin (TTX). Before TTX was added, 4% cells fired APs upon exposure to 4.5 kV/cm and this amount grew to 15%, and 60% with field intensity increased to 5.8 kV/ cm and 8.8 kV/cm, respectively. All APs were 1) reproducible with repeated exposures and; 2) indistinguishable from the APs triggered by conventional stimulation in the same neurons. In the presence of TTX 6% neurons responded with moderate PM depolarization to 10.1 kV/cm EPs and 60% of the cells were depolarized when field intensity reached 13.4 kV/cm. PM depolarization at these conditions was directly proportional to applied field intensity. Repeated ns-EPs applications led to the diminished PM responses. To study phenomenon of APs stimulation by ns-EPs further, we stimulated cells with multiple 10-ns

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EPs using field intensity twofold lower than the threshold level established. We found that stimulation with 100 EPs at 10 kHz results in steadily triggered APs while 1 kHz failed to produce APs and resulted in sustained TTX insensitive PM depolarization.

Posters: Molecular Dynamics III 3335-Pos Board B543 Efficient Unbiased Sampling of Protein Dynamics using Reinforcement Learning Zahra Shamsi, Diwakar Shukla. University of Illinois at Urbana-Champaign, Champaign, IL, USA. Understanding protein dynamics is crucial for a fundamental understanding of the biological process and drug design [1]. Molecular dynamics (MD) simulation is a powerful tool to help us learn about conformational changes in proteins. However, even with the use of specialized supercomputers, it is difficult to simulate proteins with relevant biological timescales [2]. Advances in the field of genomics over the last decade, have led to uncovering the substantial gene sequencing information, which can be utilized as valuable unused information to enhance the simulation samplings. On the other hand, machine learning is another fast-growing field, which finds its place in different areas in science. Reinforcement learning (RL) constitutes a significant aspect of the machine-learning field with numerous applications ranging from finance to autonomous vehicles. In this study, we outline a novel computational method to incorporate reinforcement learning concepts along with evolutionary information to guide the exploration of the long timescale behavior of the proteins [3]. The algorithm aims to enhance the sampling by identifying reaction coordinates that are relevant for sampling the system. We demonstrate the effectiveness of the algorithms by comparing the sampling to long continuous MD simulations and least-counts adaptive sampling on multiple systems such as b2-AR GPCR and Src kinase. References: [1] Agafonov, et al. Front. Mol. Biosci., 2, 1-8, 2015; [2] Shukla, et al. Nat. Commun., 5, 3397, 2014; [3] Shamsi, et al. Sci. Rep. InPress; 3336-Pos Board B544 Rational Development of HBV Capsid Inhibitors Aided by Molecular Dynamics Anna Pavlova1, Maksym Korablyov2, Chris Chipot3, James Gumbart1. 1 Physics, Georgia Institue of Technology, Atlanta, GA, USA, 2Massachusetts Institute of Technology, Boston, MA, USA, 3University of Illinois UrbanaChampaign, Urbana, IL, USA. Chronic Hepatitis B virus (HBV) infections are a major cause of liver damage and liver cancer and are presently incurable. A novel approach to combat the virus is by targeting HBV capsid assembly. Two major classes of compounds, phenylpropenamides and heteroaryldihydropyrimidines (HAP), have been shown to prevent the formation of normal capsids by binding to the intermediates of the capsid assembly. This binding can lead to either assembly into noncapsid structures or accelerated assembly that does not incorporate the viral DNA into the capsid. Unfortunately, both classes of compounds are toxic; thus, there is significant interest in discovering novel assembly modifiers. We have used MD simulations to study a tetramer of HBV capsid proteins, an important assembly intermediate, with and without bound assembly modifiers. It was found that the tetramer was highly flexible in solution; snapshots from the simulations were selected for docking using principal component analysis. The highest scoring compounds from docking were tested experimentally. After discovering one compound that showed activity against HBV, confirmed binding to the capsid protein, and low toxicity, we proceeded to dock and test similar compounds. We have discovered several compounds that are structurally distinct from previously known assembly modifiers and that have moderate activity against HBV. The HBV tetramer was simulated with the new compounds as well with previously known assembly modifiers bound. It was found that all active compounds greatly decreased the flexibility of the protein complex. Additionally, distinct configurations were sampled for the different HAP compounds while the configurations of the tetramer were similar for our new compounds. 3337-Pos Board B545 QwikMD - Gateway for Easy Simulation with VMD and NAMD Joa˜o Vieira Ribeiro1, Rafael C. Bernardi1, Till Rudack1,2, Klaus Schulten1,3, Emad Tajkhorshid1,4. 1 Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana, IL, USA, 2Department of Biophysics, Ruhr-Universit€at Bochum, Bochum, Germany, 3Department of Physics, University of Illinois, Urbana, IL, USA, 4Department of Biochemistry, University of Illinois, Urbana, IL, USA.