Knowledge of how DNA interacts with other biomolecules is not only essential for understanding the biological processes but also crucial for the development of sensors. With these applications in mind, recent advancements of single-molecule techniques such as fluorescence microscopy and optical tweezers have enabled real-time measurement of biomechanics, binding interactions, and conformational dynamics of protein-DNA complexes at unprecedented details. Inspired by nature, we have created novel molecular platforms by harnessing the power of DNA dynamics (which we call “DNA dance”) that are suitable for mechanistic studies of protein-DNA interactions and detection of disease biomarkers. In this presentation, I will highlight our recent work that exploits the power of DNA dance for biomedical applications. For references, please visit: https://blogs.vcu.edu/sndhakal/index.php/publications/

The concentration of ?-crystallin in the eye lens cytoplasm decreases with a corresponding increase in membrane-bound ?-crystallin during cataract formation. The eye lens membrane consists of exceedingly high cholesterol (Chol) concentration, forming cholesterol bilayer domains (CBDs) within the membrane. The role of high Chol content in the eye lens membrane is unclear. In this study, we probed the role of Chol and CBDs in ?-crystallin binding to membranes made of four major phospholipids (PLs) of the eye lens membrane, i.e., phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylserine (PS), and phosphatidylethanolamine (PE), using the electron paramagnetic resonance (EPR) spin-labeling method. For the Chol/PC, Chol/SM*, and Chol/PS membranes, the maximum membrane surface occupied (MMSO) by ?-crystallin and binding affinity (Ka) of ?-crystallin binding to membranes decreased with an increase in Chol concentration. The MMSO and Ka became zero for the Chol/PC and Chol/SM* membranes at 50 and 60 mol% Chol, respectively, representing that complete inhibition of ?-crystallin binding was observed before the formation of CBDs within the Chol/PC membrane and after the formation of CBDs within the Chol/SM* membrane. The MMSO and Ka did not reach zero even at 60 mol% Chol for the Chol/PS membrane, representing that CBDs at 60 mol% Chol were insufficient for the complete inhibition of ?-crystallin binding to the Chol/PS membrane. The MMSO and Ka were zero at 0, 9, 33 mol% Chol in the Chol/PE* membrane, representing no binding of ?-crystallin to the Chol/PE* membrane with and without Chol. The mobility parameter of the Chol/PC, Chol/SM*, and Chol/PS membranes decreased with an increase in ?-crystallin concentration, representing these membranes became less mobile near the headgroup regions with an increase in ?-crystallin binding. However, with an increase in Chol concentration, the decrease in the mobility parameter was less pronounced, representing that Chol antagonized the ability of ?-crystallin to decrease the mobility of the membrane near the headgroup regions. The maximum splitting of the membranes remained the same with an increase in ?-crystallin concentration, representing that the order of the membranes near the headgroup regions remained the same with ?-crystallin concentration. However, the maximum splitting of the membranes increased with an increase in Chol concentration in the membranes, representing these membranes become more ordered with an increase in Chol concentration. Our data represent that Chol and CBDs decrease hydrophobicity near the headgroup region of the Chol/PL membranes, decreasing the hydrophobic binding of ?-crystallin to the hydrophobic core of the membrane. Our results give a molecular-level understanding of the positive physiological role of high Chol content in the eye lens membrane in preventing ?-crystallin binding to the membrane and possibly protecting against cataract formation and progression.

Over half of all modern medical drugs target membrane proteins. Despite their significance in modern medicine, relatively few studies investigate membrane proteins due to the difficulties in studying membrane proteins in vitro. Therefore, it is required to have well established methods for incorporating the protein into native like membrane environments when studying the structural and dynamic properties of membrane proteins using biophysical methods. We are investigating the voltage gated potassium channel accessory protein, KCNE3. KCNE3 modulates the function and trafficking of several voltage gated potassium channels, including KCNQ1. Mutations in KCNE3 can lead to several diseases such as long QT syndrome (LQTS), cystic fibrosis, secretory diarrhea, periodic paralysis, tinnitus, and M�ni�re�s disease. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopic techniques have been utilized to assess the viability of various membrane mimic environments. EPR is a well-established method for studying the structure of membrane protein in vitro. The CW-EPR spectral lineshape analysis will be conducted on inside spin label probes (T71C and R81C) and outside probe (S101C and M102C) in various membrane environments such as DPC detergent micelles, POPC/POPG liposomes, and POPC/POPG lipodisq nanoparticles in order to understand their effect on dynamic properties of KCNE3. This study will provide guidance for selecting a better membrane mimetic environment for membrane protein EPR spectroscopic studies.

Krishna Neupane, Sneha Munshi, Meng Zhao, Sandaru M Ileperuma, Aaron Lyons, Dustin B. Ritchie, Noel Q. Hoffer, Abhishek Narayan and Michael T. Woodside

Department of Physics, University of Alberta, Edmonton AB T6G 2E1, Canada

The COVID-19 pandemic is caused by SARS coronavirus 2 (SARS-CoV-2). As new variants have been challenging vaccines, antiviral therapeutics targeting crucial viral processes will become an essential tool to ending the pandemic. SARS-CoV-2 uses ?1 programmed ribosomal frameshifting (?1 PRF) to control expression of key viral proteins. ?1 PRF involves a shift in the reading frame of the ribosome at a specific location in the RNA message, stimulated by a pseudoknot structure in the mRNA located 5�7 nt downstream of the �slippery� sequence where the reading-frame shift occurs. Altering ?1 PRF activity impairs virus replication, suggesting that this activity may be therapeutically targeted. We found that a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit ?1 PRF was similarly effective against SARS-CoV-2. Additionally, we found that most mutations from a pane of six mutations in the pseudoknot structure did not change ?1 PRF levels, even when base-pairing was disrupted, suggesting SARS-CoV-2 may be less sensitive to ?1 PRF modulation; the small-molecule ?1 PRF inhibitor showed a similar effect on all of the 6 mutants, indicating that anti-frameshifting activity can be resistant to natural pseudoknot mutations. Moreover, towards understanding the mechanism of the small molecule inhibitor, we studied how this pseudoknot responded to the mechanical tension applied by ribosomes during translation. We probed its structural dynamics under force using optical tweezers. Unfolding curves revealed that the frameshift signal formed multiple different structures: at least two pseudoknotted conformers with distinct unfolding forces and pathways, as well as alternative stem-loop structures. Remarkably, we found that the two pseudoknotted conformers were consistent with models having different fold topologies, one forming a ring-knot�not seen in any other frameshifting element�and the other without threading. These results solve the folding mechanism of the frameshift signal in SARS-CoV2 and highlight the conformational heterogeneity of this RNA, with important implications for structure-based drug-discovery efforts.

Laser-tissue interaction is of great interest both in physics and medical science due to the complex interaction of light with tissue and the potential application of therapeutic use. While there are many interesting phenomena that light exhibits with tissue such as scattering and fluorescence, the transportation of light energy into tissue is of major interest in many medical applications. If a biological tissue is illuminated either by continuous or pulsed laser, three phenomena are possible, namely, photochemical, photothermal, and photomechanical. These effects depend on the peak power and wavelength of the laser as well as the thermal properties of biological tissues. Here, photochemical, photothermal, and photomechanical phenomena of light-tissue interaction and their therapeutic applications are discussed.

Abstract: Epilepsy is one of the most common neurological diseases affecting over 2.5 million people in the United States. Laser ablation and responsive neural stimulation are among a few methods used in the treatment of focal-onset seizures. However, there are no universally established criteria to identify seizure onset zone or the propagation pathways. Quantitative analysis of neurophysiology and neuroimaging of brain functional connectivity can help examine the abnormal network in epileptic patients. Our previous findings have shown that high frequency (>50 Hz) from pre-ictal intra-cortical electroencephalography (iEEG) could be used to predict seizure onset zone long before the visual onset. However, the relationship between high-frequency activity and infra-slow frequency (<0.1 Hz) in relation to epilepsy in inter-ictal iEEG and pre-ictal iEEG has not been well understood. In this research, we propose using novel spectral interdependency methods like spectral coherence and Granger Causality (GC) to look into neural synchrony and information flow from non-stationary iEEG recordings during the course of epileptic seizures. Our results on three epileptic patients show overlap between infra-slow (<0.1 Hz) neural information flow patterns and the high-frequency (>50 Hz) neural information flow patterns from pre-ictal and inter-ictal iEEG recordings. The results are consistent for two specific Granger Causal measures – GC sink (neural information flow from all electrodes to the seizure onset electrodes) and total flow of information to the seizure onset electrode. These findings suggest we might be able to use infra-slow pre-ictal and inter-ictal periods to localize seizure onset zones for the surgical evaluation of prospective patients.

DNA double-strand breaks (DSBs) are inevitable due to endogenous and exogenous DNA-damaging factors such as reactive oxygen species (ROS) and ionizing radiation. If DSBs are left unrepaired, it will undermine genomic integrity and cause the accumulation of chromosomal abnormalities. For this reason, nature has evolved a well-conserved mechanism called homologous recombination (HR) to repair DSBs. During HR, strand exchange between two homologous chromosomes occurs, leading to the formation of a critical intermediate structure known as Holliday junction (HJ). HJs, free in solution, are highly dynamic molecules as they can spontaneously undergo two types of conformational dynamics: flip-flop motion and branch migration. The dynamic behavior of the junction is critical to maintain genomic integrity. Herein, we designed a mobile HJ analog and explored its branch migration dynamics/kinetics at the single-molecule level using Fluorescence Resonance Energy Transfer (FRET). We have used dimethyl sulfoxide (DMSO) and polyethylene glycol (PEG6000) to reflect intracellular solvent properties and molecular crowding, respectively. Specifically, we observed that the buffer additives significantly enhance branch migration, perhaps by destabilizing dsDNA. Further, the single-molecule DNA construct containing a mobile HJ that we have developed may serve as a suitable platform for studying mechanochemical properties of many other HJ-binding proteins.

Video games have become a prominent part of the lives of children and young adults in today�s culture with thousands of games releasing each year. With this trend emerging towards producing more games each year and the increasing number of those who play them, finding how they affect their perceptual decision-making abilities of those who play them continuously and what changes in the brain to allow for these behavioral performance changes is required to be understood. Previous studies have shown that video game playing can improve information processing and working memory capabilities. In this rapid sampling fMRI study, we used a modified version of the moving dots perceptual decision-making task and examined in 52 participants how video game playing changed the behavior and the brain. We found out that the decision Video Game players were able to outperform non-Gamer players overall in Accuracy and in every difficulty and speed setting in Response Time. The functional image analyst revealed that performance differences correlated with different adaptive signal changes from base activity in regions inside the decision-making network as well as functional connectivity differences between these regions. These results help improve our understanding of how the brain�s abilities to integrate sparse sensory information and to make moment to moment decisions can change over time with intense and engaging perceptual motor activities like video game playing.

An error-free detection of DNA/RNA biomarkers is highly desired to enable early diagnosis of diseases. Because DNA biomarkers are circular in some specific cancer cells, there must be a robust method to detect and distinguish between linear and circular targets. We have recently developed a fluorescence resonance energy transfer (FRET)-based sensor to detect linear DNA targets, in which the sensor exhibits a static mid-FRET state in the absence of target but it switches between a low and high-FRET state in the presence of target, allowing an error-free detection. The limit of detection (LOD) of this sensor was determined to be ~50 femtomolar (fM). We are currently implementing this sensor for a high-confidence detection of circular DNA targets. In this regard, using C-circles as models, our preliminary results show that the sensor exhibits a FRET state that is different from the one observed without the target. Once optimized, this sensing platform has a high potential to be used in early diagnosis of certain cancers.

The human brain consists of highly interconnected neurons, the organization of which into local-area and large-scale networks with activity flow on the networks underlie remarkably efficient computations in perception, cognition and action. Breakdowns or changes in activity flow dynamics are commonly associated with brain dysfunctions. Although modern brain activity recordings with high-resolution neuroimaging and multi-electrode electrophysiology provide unique opportunities to study brain activities, there are no direct experimental methods to study activity flow and we have to resort to statistical and modeling methods of the data. In this talk, the speaker plans to describe Granger causality methods for computing network activity flow from brain time series data and present some key findings on network flow dynamics in human decision-making and epileptic seizures.

Eye lens�s fiber cell plasma membrane consists of an extremely high cholesterol (Chol) level with Chol content greater than 50 mol%, unlike most cellular membranes where Chol content is less than 50 mol%. At this high concentration, Chol induces the formation of pure cholesterol bilayer domains (CBDs), which coexist with the surrounding phospholipid-cholesterol domain (PCD). In this study, we employ atomic force microscopy (AFM) to study the properties of high Chol-containing membrane relevant to eye lens membrane, where we varied the Chol content from 0 to 75 mol % in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) membrane. The surface roughness of the membrane decreased initially and increased as the Chol content in the membrane exceeds 60 mol%. We propose this increase in surface roughness of membrane with higher Chol content results due to the formation of CBDs. Force spectroscopy on the membrane with Chol content of 50 mol% or lesser exhibited single breakthrough events, whereas two distinct puncture events were observed for membranes with the Chol content greater than 50 mol%. We propose that the first puncture force corresponds to the membranes containing coexisting PCD and CBDs. In contrast, the second puncture force corresponds to the ‘CBD water pocket’ formed due to coexisting CBDs and PCD. Membrane thickness increased substantially above 50 mol% Chol due to the increase in water layer thickness above the mica surface resulting from the significantly higher polarity of CBD than the surrounding PCDs. Membrane area compressibility modulus (KA) increases with an increase in Chol content until it reaches 60 mol%, and with further increment in Chol content, CBDs are formed, and KA starts to decrease. Our results report the increase in membrane roughness and decrease KA at very high Chol content (>60 mol%) relevant to the eye lens membrane.

Since more than four decades, muon spin rotation and relaxation (uSR) technique in water and ice has been reported by several groups. Most of the previous studies were focused on muonium (Mu = u+e-) detection, its relaxation, reaction and frequencies in water and ice. The behavior of muon in ice and water is not clearly understood yet.

Since the water is indispensable component in biological sample, the detailed study of muon and Mu in ice and water is necessary to understand/apply the muon technique to life sciences and medical/clinical fields. In order to apply the muon method for detection of oxygen in hypoxia in tumor, electron transfer in protein, function of DNA, photosynthesis, etc., we have started the systematic experimental and theoretical study in water and constituents of protein towards whole targeted system. In the program, I will discuss both experimental results and theoretical interpretation (DFT calculations and Quantum simulation) of the temperature dependent behavior of muon in water and ice.

O6 alkylguanine-DNA alkyltransferase (AGT) is a methyl damaged repair protein. It repairs methyl lesion at O6 point of guanine base of DNA. We have performed steered molecular dynamics (SMD) simulation to examine the decoupling mechanism of AGT from methyl damaged DNA. This mechanism in-turn gives the binding strength of DNA-AGT complex. Outcomes of the study are also compared with the binding mechanism of these molecules after the methyl repaired in DNA. The atomic level investigations show that the binding affinity during the methylated condition of DNA is greater than after the methyl transfer. This result is also supported by the estimation of contact surface area between DNA and AGT during the decoupling of the complexes in these condition.

In December 2019, a new coronavirus disease emerged characterized as a viral infection with a high level of transmission in Wuhan, China which ranks among the 10 deadliest plagues in history was declared a global health emergency in 2020 by world health organization. Here, we analyzed the daily basis data for South Asian Association for Regional Cooperation (SAARC) countries: India, Pakistan, Bangladesh, Nepal, Srilanka, Afghanistan, Maldives and Bhutan by plotting curves for cummulative confirmed cases and death cases from 23 January to 31 August, 2020. The COVID-19 infections and deaths have largely been uneven within and between countries. In terms of both infected individuals and deaths, India has the highest number of COVID cases whereas Bhutan has lowest. Using logistic equation we analyze the COVID-19 data in Nepal between 23 January to 30 September, 2020. The logistic curve shows the dependence of number of

infected individuals on time with the coefficient of determination to 0.99. The maximum number of infected individuals is predicted to be 492576 with 95% confidence interval (509382, 475770). Thus study describes transmission dynamics and forecast the future trend of the disease trend of COVID-19 in Nepal if the current scenario continues.

Keywords: COVID-19, Logistic Model, Coefficient of determination, Confidence interval, Growth rate.

Free energy of solvation in aqueous environment has a vital role to understand many phenomenon. In this work, we have performed molecular dynamics simulations of a system of protonated lysine in water. OPLS-AA force field parameters and four different water models: TIP3P, SPC, SPC/E and TIP4P were used during the simulations. Free energy of solvation of protonated lysine in aqueous medium has been reported at 310 K temperature using Thermodynamic integration (TI) and Bennett acceptance ratio (BAR) methods. We have also analyzed the individual contributions of van der Waals and coulomb interactions on the free energy of solvation. The estimated values are compared with previously reported value.

Key words: Free energy of solvation, Molecular Dynamics Simulations, Thermodynamic Integration, Bennett acceptance ratio

COVID-19, which is caused due to SARS-CoV 2, is a newly identified highly infectious disease. This disease was reported to appear first in Wuhan, China on 1 December, 2019. It has affected almost every country including Nepal and had caused pandemic situation. SARS-CoV 2 is a new virus, most of its properties are not known and still under intense investigation. Particular treatment for this disease has not been found yet, though a number of vaccine are used recently. In this case, mathematical modelling study is important to understand control strategies for the spread of newly found coronavirus. Here, we present a mathematical model, commonly known as logistic model for the modelling and prediction of COVID-19 infection in Nepal. This model is particularly based on differential equation for population growth developed by Verhulst. We collected and analyzed daily basis data provided by Ministry of Health and Population, Government of Nepal in the period (23 January – 18 September) 2020. For the modelling we used Desmos mathematical graphing software. The results show a good fit between the observed data and the data predicted by logistic model as indicated by coefficient of determination 0.98 for confirmed cases and 0.99 for death cases. From our calculation, the maximum number of infected individuals at the end was found to be 275014 with 95% confidence interval limit (284833, 265195). Similarly the estimated size for death was found to be 1732 with 95% confidence interval limit (1800, 1663). This model study is expected to predicting the upcoming trend of COVID-19 in Nepal based on current mode thereby making basis for medical and governmental decision making.

Keywords : COVID-19, SARS-CoV 2, Modelling, Logistic Model, Pandemic

Influenza A is a highly contagious and a pathogenic virus that causes serious respiratory illness, the complications from which can be fatal even to young and healthy adults. On average, approximately 250000 to 500000 people die each year from the complications due to Influenza A worldwide. Frequently occurring mutations can evade the vaccine-developed immune response, so the vaccines need to updated continuously. Drug resistance to some of the existing drugs have already been seen in this virus. Hence new antiviral therapeutics need to be explored in order to prevent further morbidity and mortality. To develop such a novel antiviral therapeutic, knowledge of the viral lifecycle and interaction between the viral proteins and host cell lipids and proteins is crucial, as influenza viruses hijack these cellular components during infection.

Influenza A Hemagglutinin (HA) is viral glycoprotein responsible for binding and entry of influenza virus. HA clusters at the plasma membrane in infected cells, and these clusters have high density in progeny viruses in order to catalyze membrane fusion for viral entry. The mechanism behind the clustering of HA is not known. However, we recently showed that HA clusters are interdependent with clusters of the lipid phosphatidylinositol (4,5) biphosphate (PIP2). Hence, modulating PIP2 clusters can in turn modulate HA clusters. This approach could provide a novel strategy to reduce the infectivity of the virus, as HA cluster density directly correlates with the infectivity of the virus. Cetylpyridinium chloride (CPC) is a quaternary ammonium compound well known for its antibacterial and antiviral properties at high (mM) concentrations. However, its effect on cellular functions at lower (micromolar) has not been well elucidated. In this study we use diffraction-limited and super resolution microscopy (FPALM) to study the effect of CPC on PIP2 binding proteins at micromolar concentrations. These results help explain the mechanism for antiviral action of CPC. We also extend our study to zebrafish model and show that CPC at non-cytotoxic micromolar concentrations improves survival of zebrafish infected with influenza A virus.

The majority (>60%) of clinically important human drug targets are either integral or peripheral membrane-bound proteins, including G protein-coupled receptors, ion channels, drug- metabolizing enzymes, and transporters. Gamma-aminobutyric acid (GABA) type B receptors (GABABRs) are therapeutically important membrane proteins in the central nervous system that mediate GABA neurotransmission. Any disorders in GABABRs function are associated with various neurological disorders such as anxiety, depression, and epilepsy. Recently, our labs reconstructed a high-resolution cryo-EM structure of GABABR in its active state along with allosteric modulators bound to the transmembrane membrane domain (TMD) sites. Published in- vitro study (Ferlenghi et al., 2020, J. Pharm. Sci) showed that ligand COR659, a 2- acylaminothiophene derivative, is a positive allosteric modulator (PAM) of GABABR. We used combined molecular docking and molecular dynamics (MD) simulations to gain structural insights into the positive allosteric modulation of GABABRs by COR659. First, the ligand docked in the heterodimeric interface of GABABR, a membrane-embedded TMD binding site, where two other ligands BHFF and GS39783, were also recently solved by cryo-EM. Then, the docked complex embedded in the membrane lipid bilayer consisting of phosphatidylcholine and cholesterol lipid bilayers was followed by ~500ns all-atom MD simulations. Results demonstrate the stable binding pose of PAM due to strong hydrogen-bonded interactions with residues N698 (GB1 subunit) and Y693 (GB2 subunit) and favorably located in an aromatic cage made of residues F300 and Y697 (GB2), and Y693 and Y672 (GB1). Interestingly, the chlorophenyl ring of PAM makes strong hydrophobic contact with the steroid ring of cholesterol. The allosteric communication between the agonist binding Venus flytrap domain and PAM binding TMD was quantified using PCA analysis. Furthermore, free energy MD simulations using Metadynamics will be used to quantify the ligand-induced modulation of the GABABR energy landscape. Finally, using the ground-truth Cryo-EM data and ManifoldEM, a manifold embedding-based technique recently introduced that provides the continuous conformational motions over the energy landscape, will validate the MD results.

The use of radiation is broad in biological systems, in different areas of research mostly in health. Radiation is used to kill cancer. In radiation therapy proper calculation is done so that a maximum dose is delivered to the cancer .In spite of this precaution, radiation effects healthy tissue. This effect is especially dangerous when the tumor is located near important organs. Thus, in radiation therapy, it is important to reduce the dose and the damage to the healthy tissues and organs. The damage on the healthy tissues due to radiation therapy in cancer could be reduced by reducing the radiation dose to get the same treatment effect or by enhancing the radiation. The enhancement of radiation effect in vitro and in vivo can be obtained by targeted drug delivery on the cancer. Also photo dynamic therapy can supplement the use of radiation therapy for the treatment of cancer by targeted drug delivery