Molecular Biology Commons^™

A Pathway To Solving The Structure Of Cl-Par-4 Tumor Suppressor Protein: Challenges & Findings, Krishna Raut, Samjhana Pandey, Andrea M. Clark, Komala Ponniah, Steven M. Pascal

College of Sciences Posters

Prostate apoptosis response-4 (Par-4) is a pro-apoptotic tumor suppressor protein. Down-regulation of this protein has been reported in a myriad of cancers. Conversely, up-regulation of Par-4 is found to be associated with several neurodegenerative disorders. Par-4 is unique in the sense it can selectively induce apoptosis in cancer cells. For this, caspase-dependent intracellular cleavage of Par-4 is essential to produce the functionally active fragment, cl-Par-4 (caspase-cleaved Par-4). The cl-Par-4 protein inhibits the NF-κB-mediated cell survival pathway and causes selective apoptosis in various tumor cells.

Our laboratory is interested in determining the structure of cl-Par-4 and understanding it’s interaction with various …

Biophysical Characterization Of The Par-4 Tumor Suppressor: Evidence Of Structure Outside The Coiled Coil Domain And Interactions With Platinum Chemotherapeutics, Andrea Megan Clark

Chemistry & Biochemistry Theses & Dissertations

Prostate apoptosis response-4 (Par-4) is an apoptosis-inducing tumor suppressor protein. Full-length Par-4 has previously been shown to be a predominantly intrinsically disordered protein (IDP) under neutral conditions, with significant regular secondary structure evident only within the C-terminal coiled coil domain. However, IDPs can gain ordered structure through the process of induced folding, which often occurs under non-neutral conditions. Previous work has shown that the Par-4 leucine zipper, which is a subset of the C-terminal coiled coil domain, is disordered under neutral conditions, but forms a dimeric coiled coil at acidic pH. Increase in ionic strength was also shown to increase …

Electropermeabilization Does Not Correlate With Plasma Membrane Lipid Oxidation, Olga Michel, Andrei G. Pakhomov, Maura Casciola, Jolanta Saczko, Julita Kulbacka, Olga N. Pakhomova

Bioelectrics Publications

The permeabilized condition of the cell membrane after electroporation can last minutes but the underlying mechanisms remain elusive. Previous studies suggest that lipid peroxidation could be responsible for the lasting leaky state of the membrane. The present study aims to link oxidation within the plasma membrane of live cells to permeabilization by electric pulses. We have introduced a method for the detection of oxidation by ratiometric fluorescence measurements of BODIPY-C11 dye using total internal reflection fluorescence (TIRF) microscopy, limiting the signal to the cell membrane. CHO-K1 cells were cultured on glass coverslips coated with an electroconductive indium tin oxide (ITO) …

Modulation Of Biological Responses To 2 Ns Electrical Stimuli By Field Reversal, Esin B. Sözer, P. Thomas Vernier

Bioelectrics Publications

Nanosecond bipolar pulse cancellation, a recently discovered Phenomenon, is modulation of the effects of a unipolar electric pulse exposure by a second pulse of opposite polarity. This attenuation of biological response by reversal of the electric field direction has been reported with pulse durations from 60 ns to 900 ns for a wide range of endpoints, and it is not observed with conventional electroporation pulses of much longer duration (> 100 mu s) where pulses are additive regardless of polarity. The most plausible proposed mechanisms involve the field-driven migration of ions to and from the membrane interface (accelerated membrane discharge). …

Accurate Flexible Refinement Of Atomic Models Against Medium-Resolution Cryo-Em Maps Using Damped Dynamics, Julio A. Kovacs, Vitold E. Galkin, Willy Wriggers

Mechanical & Aerospace Engineering Faculty Publications

Background: Dramatic progress has recently been made in cryo-electron microscopy technologies, which now make possible the reconstruction of a growing number of biomolecular structures to near-atomic resolution. However, the need persists for fitting and refinement approaches that address those cases that require modeling assistance.

Methods: In this paper, we describe algorithms to optimize the performance of such medium-resolution refinement methods. These algorithms aim to automatically optimize the parameters that define the density shape of the flexibly fitted model, as well as the time-dependent damper cutoff distance. Atomic distance constraints can be prescribed for cases where extra containment of parts of …

Dielectric Properties Of Isolated Adrenal Chromaffin Cells Determined By Microfluidic Impedance Spectroscopy, A. C. Sabuncu, M. Stacey, G. L. Graviso, N. Semenova, P. T. Vernier, N. Leblanc, I. Chatterjee, J. Zaklit

Bioelectrics Publications

Knowledge of the dielectric properties of biological cells plays an important role in numerical models aimed at understanding how high intensity ultrashort nanosecond electric pulses affect the plasma membrane and the membranes of intracellular organelles. To this end, using electrical impedance spectroscopy, the dielectric properties of isolated, neuroendocrine adrenal chromaffin cells were obtained. Measured impedance data of the cell suspension, acquired between 1 kHz and 20 MHz, were fit into a combination of constant phase element and Cole-Cole models from which the effect of electrode polarization was extracted. The dielectric spectrum of each cell suspension was fit into a Maxwell-Wagner …

Esope-Equivalent Pulsing Protocols For Calcium Electroporation: An In Vitro Optimization Study On 2 Cancer Cell Models, Stefania Romeo, Anna Sannino, Maria Rosaria Scarfi, P. Thomas Vernier, Ruggero Cadossi, Julie Gehl, Olga Zeni

Bioelectrics Publications

Reversible electroporation is used to increase the uptake of chemotherapeutic drugs in local tumor treatment (electrochemotherapy) by applying the pulsing protocol (8 rectangular pulses, 1000 V/cm, 100 µs) standardized in the framework of the European Standard Operating Procedure on Electrochemotherapy multicenter trial. Currently, new electrochemotherapy strategies are under development to extend its applicability to tumors with different histology. Electrical parameters and drug type are critical factors. A possible approach is to test pulse parameters different from European Standard Operating Procedure on Electrochemotherapy but with comparable electroporation yield (European Standard Operating Procedure on Electrochemotherapy-equivalent protocols). Moreover, the use of non-toxic drugs …

Coordination Of Different Ligands To Copper(Ii) And Cobalt(Iii) Metal Centers Enhances Zika Virus And Dengue Virus Loads In Both Arthropod Cells And Human Keratinocytes, Shovan Dutta, Michael J. Celestine, Supreet Khanal, Alexis Huddleston, Colin Simms, Jessa Faye Arca, Amlam Mitra, Loree Heller, Piotr Kraj, Michael Ledizet, John F. Anderson, Girish Neelakanta, Alvin A. Holder, Hameeda Sultana

Biological Sciences Faculty Publications

Trace elements such as copper and cobalt have been associated with virus-host interactions. However, studies to show the effect of conjugation of copper(II) or cobalt(III) metal centers to thiosemicarbazone ligand(s) derived from either food additives or mosquito repellent such as 2-acetylethiazole or citral, respectively, on Zika virus (ZIKV) or dengue virus (serotype 2; DENV2) infections have not been explored. In this study, we show that four compounds comprising of thiosemicarbazone ligand derived from 2-acetylethiazole viz., (E)-N-ethyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide (acetylethTSC) (compound 1), a copper(II) complex with acetylethTSC as a ligand (compound 2), a thiosemicarbazone ligand-derived from citral (compound 3) and a cobalt(III) complex …

Activation Of The Phospholipid Scramblase Tmem16f By Nanosecond Pulsed Electric Field (Nspef) Facilitates Its Diverse Cytophysiological Effects, Claudia Muratori, Andrei G. Pakhomov, Elena Gianulis, Jade Meads, Maura Casciola, Peter A. Mollica, Olga N. Pakhomova

Bioelectrics Publications

Nanosecond pulsed electric fields (nsPEF) are emerging as a novel modality for cell stimulation and tissue ablation. However, the downstream protein effectors responsible for nsPEF bioeffects remain to be established. Here we demonstrate that nsPEF activate TMEM16F (or Anoctamin 6), a protein functioning as a Ca²⁺-dependent phospholipid scramblase and Ca²⁺-activated chloride channel. Using confocal microscopy and patch clamp recordings, we investigated the relevance of TMEM16F activation for several bioeffects triggered by nsPEF, including phosphatidylserine (PS) externalization, nanopore-conducted currents, membrane blebbing, and cell death. In HEK 293 cells treated with a single 300-ns pulse of 25.5 kV/cm, …

A Balanced Approach To Adaptive Probability Density Estimation, Julio Kovacs, Cailee Helmick, Willy Wriggers

Mechanical & Aerospace Engineering Faculty Publications

Our development of a Fast (Mutual) Information Matching (FIM) of molecular dynamics time series data led us to the general problem of how to accurately estimate the probability density function of a random variable, especially in cases of very uneven samples. Here, we propose a novel Balanced Adaptive Density Estimation (BADE) method that effectively optimizes the amount of smoothing at each point. To do this, BADE relies on an efficient nearest-neighbor search which results in good scaling for large data sizes. Our tests on simulated data show that BADE exhibits equal or better accuracy than existing methods, and visual tests …

Computing Spatiotemporal Heat Maps Of Lipid Electropore Formation: A Statistical Approach, Willy Wriggers, Frederica Castellani, Julio A. Kovacs, P. Thomas Vernier

Mechanical & Aerospace Engineering Faculty Publications

We extend the multiscale spatiotemporal heat map strategies originally developed for interpreting molecular dynamics simulations of well-structured proteins to liquids such as lipid bilayers and solvents. Our analysis informs the experimental and theoretical investigation of electroporation, that is, the externally imposed breaching of the cell membrane under the influence of an electric field of sufficient magnitude. To understand the nanoscale architecture of electroporation, we transform time domain data of the coarse-grained interaction networks of lipids and solvents into spatial heat maps of the most relevant constituent molecules. The application takes advantage of our earlier graph-based activity functions by accounting for …

Quantitative Limits On Small Molecule Transport Via The Electropermeome - Measuring And Modeling Single Nanosecond Perturbations, Esin B. Sözer, Zachary A. Levine, P. Thomas Vernier

Bioelectrics Publications

The detailed molecular mechanisms underlying the permeabilization of cell membranes by pulsed electric fields (electroporation) remain obscure despite decades of investigative effort. To advance beyond descriptive schematics to the development of robust, predictive models, empirical parameters in existing models must be replaced with physics- and biology-based terms anchored in experimental observations. We report here absolute values for the uptake of YO-PRO-1, a small-molecule fluorescent indicator of membrane integrity, into cells after a single electric pulse lasting only 6 ns. We correlate these measured values, based on fluorescence microphotometry of hundreds of individual cells, with a diffusion-based geometric analysis of pore-mediated …

Characterization And Analysis Of Real-Time Capillary Convective Pcr Toward Commercialization, Xianbo Qiu, Shiyin Zhang, Lanju Mei, Di Wu, Ke Li, Shengxiang Ge, Xiangzhong Ye, Ningshao Xia, Michael G. Mauk

Mechanical & Aerospace Engineering Faculty Publications

Almost all the reported capillary convective polymerase chain reaction (CCPCR) systems to date are still limited to research use stemming from unresolved issues related to repeatability, reliability, convenience, and sensitivity. To move CCPCR technology forward toward commercialization, a couple of critical strategies and innovations are discussed here. First, single- and dual-end heating strategies are analyzed and compared between each other. Especially, different solutions for dual-end heating are proposed and discussed, and the heat transfer and fluid flow inside the capillary tube with an optimized dual-end heating strategy are analyzed and modeled. Second, real-time CCPCR is implemented with light-emitting diode and …

Effects Of High Voltage Nanosecond Electric Pulses On Eukaryotic Cells (In Vitro): A Systematic Review, Tina Batista Napotink, Matej Reberšek, P. Thomas Vernier, Babara Mali, Damijan Miklavčič

Bioelectrics Publications

For this systematic review, 203 published reports on effects of electroporation using nanosecond high-voltage electric pulses (nsEP) on eukaryotic cells (human, animal, plant) in vitro were analyzed. A field synopsis summarizes current published data in the field with respect to publication year, cell types, exposure configuration, and pulse duration. Published data were analyzed for effects observed in eight main target areas (plasma membrane, intracellular, apoptosis, calcium level and distribution, survival, nucleus, mitochondria, stress) and an additional 107 detailed outcomes. We statistically analyzed effects of nsEP with respect to three pulse duration groups: A: 1–10 ns, B: 11–100 ns and C: …

Diffuse, Non-Polar Electropermeabilization And Reduced Propidium Uptake Distinguish The Effect Of Nanosecond Electric Pulses, Iurii Semenov, Christian W. Zemlin, Olga N. Pakhomova, Shu Xiao, Andrei G. Pakhomov

Bioelectrics Publications

Ca²⁺ activation and membrane electroporation by 10-ns and 4-ms electric pulses (nsEP and msEP) were compared in rat embryonic cardiomyocytes. The lowest electric field which triggered Ca²⁺ transients was expectedly higher for nsEP (36 kV/cm)than forms EP (0.09 kV/cm) but the respective doses were similar (190 and460 mJ/g). At higher intensities, both stimuli triggered prolonged firing in quiescent cells. An increase of basal Ca²⁺ level by N10 nM in cells with blocked voltage-gated Ca²⁺ channels and depleted Ca²⁺ depot occurred at 63 kV/cm (nsEP) or 0.14 kV/cm (msEP) and was regarded as electroporation threshold. These …

Cellular Regulation Of Extension And Retraction Of Pseudopod-Like Blebs Produced By Nanosecond Pulsed Electric Field, Mikhail A. Rassokhin, Andrei G. Pakhomov

Bioelectrics Publications

Recently we described a new phenomenon of anodotropic pseudopod-like blebbing in U937 cells exposed to nanosecond pulsed electric field (nsPEF). In Ca²⁺ -free buffer such exposure initiates formation of pseudopod-like blebs (PLBs), protrusive cylindrical cell extensions that are distinct from apoptotic and necrotic blebs. PLBs nucleate predominantly on anode-facing cell pole and extend toward anode during nsPEF exposure. Bleb extension depends on actin polymerization and availability of actin monomers. Inhibition of intracellular Ca²⁺ , cell contractility, and RhoA produced no effect on PLB initiation. Meanwhile, inhibition of WASP by wiskostatin causes dose-dependent suppression of PLB growth. Soon after …

Tracing Beta Strands Using Strandtwister From Cryo-Em Density Maps At Medium Resolutions, Dong Si, Jing He

Computer Science Faculty Publications

Major secondary structure elements such as α helices and β sheets can be computationally detected from cryoelectron microscopy (cryo-EM) density maps with medium resolutions of 5–10 A˚ . However, a critical piece of information for modeling atomic structures is missing, because there are no tools to detect β strands from cryo-EM maps at medium resolutions. We propose a method, StrandTwister, to detect the traces of β strands through the analysis of twist, an intrinsic nature of a β sheet. StrandTwister has been tested using 100 β sheets simulated at 10 A˚ resolution and 39 β sheets computationally detected from cryo-EM …

Calcium-Mediated Pore Expansion And Cell Death Following Nanoelectroporation, Olga N. Pakhomova, Betsy Gregory, Iurii Semenov, Andrei G. Pakhomov

Bioelectrics Publications

Opening of long-lived pores in the cell membrane is the principal primary effect of intense, nanosecond pulsed electric field (nsPEF). Here we demonstrate that the evolution of pores, cell survival, the time and the mode of cell death (necrotic or apoptotic) are determined by the level of external Ca²⁺ after nsPEF. We also introduce a novel, minimally disruptive technique for nsEP exposure of adherent cells on indium tin oxide (ITO)-coated glass coverslips, which does not require cell detachment and enables fast exchanges of bath media. Increasing the Ca²⁺ level from the nominal 2–5 μM to 2 mM for …

Evaluations Of A Mechanistic Hypothesis For The Influence Of Extracellular Ions On Electroporation Due To High-Intensity, Nanosecond Pulsing, V. Sridhara, R. P. Joshi

Electrical & Computer Engineering Faculty Publications

The effect of ions present in the extracellular medium on electroporation by high-intensity, short-duration pulsing is studied through molecular dynamic simulations. Our simulation results indicate that mobile ions in the medium might play a role in creating stronger local electric fields across membranes that then reinforce and strengthen electroporation. Much faster pore formation is predicted in higher conductivity media. However, the impact of extracellular conductivity on cellular inflows, which depend on transport processes such as electrophoresis, could be different as discussed here. Our simulation results also show that interactions between cations (Na+ in this case) and the carbonyl oxygen of …

Numerical Study Of Lipid Translocation Driven By Nanoporation Due To Multiple High-Intensity, Ultrashort Electrical Pulses, Viswanadham Sridhara, Ravindra P. Joshi

Electrical & Computer Engineering Faculty Publications

The dynamical translocation of lipids from one leaflet to another due to membrane permeabilization driven by nanosecond, high-intensity (>100 kV/cm) electrical pulses has been probed. Our simulations show that lipid molecules can translocate by diffusion through water-filled nanopores which form following high voltage application. Our focus is on multiple pulsing, and such simulations are relevant to gauge the time duration over which nanopores might remain open, and facilitate continued lipid translocations and membrane transport. Our results are indicative of a N^1/2 scaling with pulse number for the pore radius. These results bode well for the use of pulse …

Primary Pathways Of Intracellular Ca2+ Mobilization By Nanosecond Pulsed Electric Field, Iurii Semenov, Shu Xiao, Andrei G. Pakhomov

Bioelectrics Publications

Permeabilization of cell membranous structures by nanosecond pulsed electric field (nsPEF) triggers transient rise of cytosolic Ca²⁺ concentration ([Ca²⁺]_i), which determines multifarious downstream effects. By using fast ratiometric Ca²⁺ imaging with Fura-2, we quantified the external Ca²⁺ uptake, compared it with Ca²⁺ release from the endoplasmic reticulum (ER), and analyzed the interplay of these processes. We utilized CHO cells which lack voltage-gated Ca²⁺ channels, so that the nsPEF-induced [Ca²⁺]_i changes could be attributed primarily to electroporation. We found that a single 60-ns pulse caused fast [Ca2+]_i increase …

Microfluidic Impedance Spectroscopy As A Tool For Quantitative Biology And Biotechnology, Ahmet C. Sabuncu, Jie Zhuang, Juergen F. Kolb, Ali Beskok

Mechanical & Aerospace Engineering Faculty Publications

A microfluidic device that is able to perform dielectric spectroscopy is developed. The device consists of a measurement chamber that is 250 μm thick and 750 μm radius. Around 1000 cells fit inside the chamber assuming average quantities for cell radius and volume fraction. This number is about 1000 folds lower than the capacity of conventional fixtures. A T-cell leukemia cell line Jurkat is tested using the microfluidic device. Measurements of deionized water and salt solutions are utilized to determine parasitic effects and geometric capacitance of the device. Physical models, including Maxwell-Wagner mixture and double shell models, are used to …

Protein Folding By 'Levels Of Separation': A Hypothesis, Lesley H. Greene, Terri M. Grant

Chemistry & Biochemistry Faculty Publications

The protein folding process has been studied both computationally and experimentally for over 30 years. To date there is no detailed mechanism to explain the formation of long-range interactions between the transition and native states. Long-range interactions are the principle determinants of the tertiary structure. We present a theoretical model which proposes a mechanism for the acquisition of these interactions as they form in a modified version of ‘degrees of separation’, that we term ‘levels of separation’. It is based on the integration of network science and biochemistry. (C) 2012 Federation of European Biochemical Societies.

Dielectrophoretic Choking Phenomenon In A Converging-Diverging Microchannel, Ye Ai, Shizhi Qian, Sheng Liu, Sang W. Joo

Mechanical & Aerospace Engineering Faculty Publications

Experiments show that particles smaller than the throat size of converging-diverging microchannels can sometimes be trapped near the throat. This critical phenomenon is associated with the negative dc dielectrophoresis arising from nonuniform electric fields in the microchannels. A finite-element model, accounting for the particle-fluid-electric field interactions, is employed to investigate the conditions for this dielectrophoretic (DEP) choking in a converging-diverging microchannel for the first time. It is shown quantitatively that the DEP choking occurs for high nonuniformity of electric fields, high ratio of particle size to throat size, and high ratio of particle's zeta potential to that of microchannel. © …

Regulation Of Intracellular Calcium Concentration By Nanosecond Pulsed Electric Fields, Shaka S. Scarlett, Jody A. White, Peter F. Blackmore, Karl H. Schoenbach, Juergen Kolb

Bioelectrics Publications

Changes in [Ca²⁺]_i response of individual Jurkat cells to nanosecond pulsed electric fields (nsPEFs) of 60 ns and field strengths of 25, 50, and 100 kV/cm were investigated. The magnitude of the nsPEF-induced rise in [Ca²⁺]_i was dependent on the electric field strength. With 25 and 50 kV/cm, the [Ca²⁺]_i response was due to the release of Ca²⁺ from intracellular stores and occurred in less than 18 ms. With 100 kV/cm, the increase in [Ca²⁺]_i was due to both internal release and to influx across the plasma …

Pressure-Driven Transport Of Particles Through A Converging-Diverging Microchannel, Ye Ai, Sang W. Joo, Xiangchun Xuan, Shizhi Qian

Mechanical & Aerospace Engineering Faculty Publications

Pressure-driven transport of particles through a symmetric converging-diverging microchannel is studied by solving a coupled nonlinear system, which is composed of the Navier-Stokes and continuity equations using the arbitrary Lagrangian-Eulerian finite-element technique. The predicted particle translation is in good agreement with existing experimental observations. The effects of pressure gradient, particle size, channel geometry, and a particle's initial location on the particle transport are investigated. The pressure gradient has no effect on the ratio of the translational velocity of particles through a converging-diverging channel to that in the upstream straight channel. Particles are generally accelerated in the converging region and then …

In Vivo Murine Melanoma Tumor Responses To Nanosecond Pulsed Electric Field Treatment, Xinhua Chen

Theses and Dissertations in Biomedical Sciences

High intensity nanosecond pulsed electric fields (nsPEF) were applied to melanoma tumors to observe functional and structural biological changes and to investigate the possible molecular mechanisms responsible. An animal model was set up by injecting B16F10 mouse melanoma cells into SKH-1 mice. A treatment (Tx) of 100 pulses: 300 nanosecond duration; 40 kV/cm field strength; at 0.5 Hz rate were delivered to melanoma tumors in 120 mice. The nsPEF Txcaused tumor self-destruction with sharply decreased cell volumes and shrunken nuclei. The apoptotic biochemical tests confirmed nsPEF Tx induced apoptosis in a time-dependent manner. Examination of gross vessel and micro-vessel density …

The Cellular And Molecular Dynamics Of The Queuosine Modification In Transfer Rna: Definition, Modulation, Deficiencies And Effect Of The Queuosine Modification System, Rana C. Morris

Theses and Dissertations in Biomedical Sciences

The presence of the queuosine modification in the wobble position of tRNA^asn, tRN^asp, tRNA^his, and tRNA^tyr is associated with a decrease in cellular growth rate, an increase in the ability to withstand environmental stress, and differentiation of pleuripotent cells into mature phenotypes. The loss of this normal modification is strongly correlated with neoplastic transformation and tumor progression of a wide variety of cancers.

The "normal" system for formation of the queuosine modification in tRNA was studied in human fibroblast cell cultures and in mouse, rat and human liver tissues. The queuosine modification system …