Biochemistry, Biophysics, and Structural Biology Commons^™

Natural Phaeosphaeride A Derivatives Overcome Drug Resistance Of Tumor Cells And Modulate Signaling Pathways, Victoria Abzianidze, Natalia Moiseeva, Diana Suponina, Sofya Zakharenkova, Nadezhda Rogovskaya, Lidia Laletina, Alvin A. Holder, Denis Krivorotov, Alexander Bogachenkov, Alexander Garabadzhiu, Anton Ukolov, Vyacheslav Kosorukov

Chemistry & Biochemistry Faculty Publications

n the present study, natural phaeosphaeride A (PPA) derivatives are synthesized. Anti-tumor studies are carried out on the PC3, K562, HCT-116, THP-1, MCF-7, A549, NCI-H929, Jurkat, and RPMI8226 tumor cell lines, and on the human embryonic kidney (HEK293) cell line. All the compounds synthesized turned out to have better efficacy than PPA towards the tumor cell lines listed. Among them, three compounds exhibited an ability to overcome the drug resistance of tumor cells associated with the overexpression of the P-glycoprotein by modulating the work of this transporter. Luminex xMAP technology was used to assess the effect of five synthesized compounds …

Biomechanical And Biophysical Properties Of Breast Cancer Cells Under Varying Glycemic Regimens, Diganta Dutta, Xavier-Lewis Palmer, Jose Ortega-Rodas, Vasundhara Balraj, Indrani Ghosh Dastider, Surabhi Chandra

Electrical & Computer Engineering Faculty Publications

Diabetes accelerates cancer cell proliferation and metastasis, particularly for cancers of the pancreas, liver, breast, colon, and skin. While pathways linking the 2 disease conditions have been explored extensively, there is a lack of information on whether there could be cytoarchitectural changes induced by glucose which predispose cancer cells to aggressive phenotypes. It was thus hypothesized that exposure to diabetes/high glucose alters the biomechanical and biophysical properties of cancer cells more than the normal cells, which aids in advancing the cancer. For this study, atomic force microscopy indentation was used through microscale probing of multiple human breast cancer cells (MCF-7, …

Secretion Of Proteins And Antibody Fragments From Transiently Transfected Endothelial Progenitor Cells, Loree Heller, Reynald Thinard, Melanie Chevalier, Sezgi Arpag, Yu Jing, Ruth Greferath, Richard Heller, Claude Nicolau

Bioelectrics Publications

In neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis and amyotrophic lateral sclerosis, neuroinflammation can lead to blood-brain barrier (BBB) breakdown. After intravenous or intra-arterial injection into mice, endothelial progenitor cells (EPCs) home to the damaged BBB to promote neurovascular repair. Autologous EPCs transfected to express specific therapeutic proteins offer an innovative therapeutic option. Here, we demonstrate that EPC transfection by electroporation with plasmids encoding the reporter protein GFP or an anti-beta-amyloid antibody fragment (Fab) leads to secretion of each protein. We also demonstrate the secreted anti-beta-amyloid Fab protein functions in beta-amyloid aggregate solubilization.

Protonic Capacitor: Elucidating The Biological Significance Of Mitochondrial Cristae Formation, James Weifu Lee

Chemistry & Biochemistry Faculty Publications

For decades, it was not entirely clear why mitochondria develop cristae? The work employing the transmembrane-electrostatic proton localization theory reported here has now provided a clear answer to this fundamental question. Surprisingly, the transmembrane-electrostatically localized proton concentration at a curved mitochondrial crista tip can be significantly higher than that at the relatively flat membrane plane regions where the proton-pumping respiratory supercomplexes are situated. The biological significance for mitochondrial cristae has now, for the first time, been elucidated at a protonic bioenergetics level: 1) The formation of cristae creates more mitochondrial inner membrane surface area and thus more protonic capacitance for …

Non-Contact Trapping And Stretching Of Biological Cells Using Dual-Beam Optical Stretcher On Microfluidic Platform, Aotuo Dong, Balaadithya Uppalapati, Shariful Islam, Brandon Gibbs, Ganesan Kamatchi, Sacharia Albin, Makarand Deo

Electrical & Computer Engineering Faculty Publications

Optical stretcher is a tool in which two counter-propagating, slightly diverging, and identical laser beams are used to trap and axially stretch microparticles in the path of light. In this work, we utilized the dual-beam optical stretcher setup to trap and stretch human embryonic kidney (HEK) cells and mammalian breast cancer (MBC) cells. Experiments were performed by exposing the HEK cells to counter-propagating laser beams for 30 seconds at powers ranging from 100 mW to 561 mW. It was observed that the percentage of cell deformation increased from 16.7% at 100 mW to 40.5% at 561 mW optical power. The …

Tracing Actin Filament Bundles In Three-Dimensional Electron Tomography Density Maps Of Hair Cell Stereocilia, Salim Sazzed, Junha Song, Julio Kovacs, Willi Wriggers, Manfred Auer, Jing He

Computer Science Faculty Publications

Cryo-electron tomography (cryo-ET) is a powerful method of visualizing the three-dimensional organization of supramolecular complexes, such as the cytoskeleton, in their native cell and tissue contexts. Due to its minimal electron dose and reconstruction artifacts arising from the missing wedge during data collection, cryo-ET typically results in noisy density maps that display anisotropic XY versus Z resolution. Molecular crowding further exacerbates the challenge of automatically detecting supramolecular complexes, such as the actin bundle in hair cell stereocilia. Stereocilia are pivotal to the mechanoelectrical transduction process in inner ear sensory epithelial hair cells. Given the complexity and dense arrangement of actin …

Inactivation Of Myeloma Cancer Cells By Helium And Argon Plasma Jets: The Effect Comparison And The Key Reactive Species, Zeyu Chen, Qingjie Cui, Chen Chen, Dehui Xu, Dingxin Liu, H. L. Chen, Michael G. Kong

Bioelectrics Publications

In plasma cancer therapy, the inactivation of cancer cells under plasma treatment is closely related to the reactive oxygen and nitrogen species (RONS) induced by plasmas. Quantitative study on the plasma-induced RONS that related to cancer cells apoptosis is critical for advancing the research of plasma cancer therapy. In this paper, the effects of several reactive species on the inactivation of LP-1 myeloma cancer cells are comparatively studied with variable working gas composition, surrounding gas composition, and discharge power. The results show that helium plasma jet has a higher cell inactivation efficiency than argon plasma jet under the same discharge …

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 …

Wild-Type P53 Enhances Endothelial Barrier Function By Mediating Rac1 Signalling And Rhoa Inhibition, Nektarios Barabutis, Christiana Dimitropoulou, Betsy Gregory, John D. Catravas

Bioelectrics Publications

Inflammation is the major cause of endothelial barrier hyper-permeability, associated with acute lung injury and acute respiratory distress syndrome. This study reports that p53 "orchestrates" the defence of vascular endothelium against LPS, by mediating the opposing actions of Rac1 and RhoA in pulmonary tissues. Human lung microvascular endothelial cells treated with HSP90 inhibitors activated both Rac1- and P21-activated kinase, which is an essential element of vascular barrier function. 17AAG increased the phosphorylation of both LIMK and cofilin, in contrast to LPS which counteracted those effects. Mouse lung microvascular endothelial cells exposed to LPS exhibited decreased expression of phospho-cofilin. 17AAG treatment …

Cold Atmospheric Plasma As A Potential Tool For Multiple Myeloma Treatment, Dehui Xu, Yujing Xu, Qingjie Cui, Dingxin Liu, Zhijie Liu, Xiaohua Wang, Yanjie Yang, Niaojuan Feng, Rong Liang, Hailan Chen, Kai Ye, Michael G. Kong

Bioelectrics Publications

Multiple myeloma (MM) is a fatal and incurable hematological malignancy thus new therapy need to be developed. Cold atmospheric plasma, a new technology that could generate various active species, could efficiently induce various tumor cells apoptosis. More details about the interaction of plasma and tumor cells need to be addressed before the application of gas plasma in clinical cancer treatment. In this study, we demonstrate that He+O₂ plasma could efficiently induce myeloma cell apoptosis through the activation of CD95 and downstream caspase cascades. Extracellular and intracellular reactive oxygen species (ROS) accumulation is essential for CD95-mediated cell apoptosis in response …

Asymmetric Patterns Of Small Molecule Transport After Nanosecond And Microsecond Electropermeabilization, Esin B. Sözer, C. Florencia Pocetti, P. Thomas Vernier

Bioelectrics Publications

Imaging of fluorescent small molecule transport into electropermeabilized cells reveals polarized patterns of entry, which must reflect in some way the mechanisms of the migration of these molecules across the compromised membrane barrier. In some reports, transport occurs primarily across the areas of the membrane nearest the positive electrode (anode), but in others cathode-facing entry dominates. Here we compare YO-PRO-1, propidium, and calcein uptake into U-937 cells after nanosecond (6 ns) and microsecond (220 µs) electric pulse exposures. Each of the three dyes exhibits a different pattern. Calcein shows no preference for anode- or cathode-facing entry that is detectable with …

Biophysical Tools To Study Cellular Mechanotransduction, Ismeel Muhamed, Farhan Chowdhury, Venkat Maruthamuthu

Mechanical & Aerospace Engineering Faculty Publications

The cell membrane is the interface that volumetrically isolates cellular components from the cell's environment. Proteins embedded within and on the membrane have varied biological functions: reception of external biochemical signals, as membrane channels, amplification and regulation of chemical signals through secondary messenger molecules, controlled exocytosis, endocytosis, phagocytosis, organized recruitment and sequestration of cytosolic complex proteins, cell division processes, organization of the cytoskeleton and more. The membrane's bioelectrical role is enabled by the physiologically controlled release and accumulation of electrochemical potential modulating molecules across the membrane through specialized ion channels (e.g., Na^⁺, Ca²⁺, K^⁺ channels). …

Introduction To Fifth Special Issue On Electroporation-Based Technologies And Treatments, Damijan Miklavčič, Lluis M. Mir, P. Thomas Vernier

Bioelectrics Publications

This special issue of the Journal of Membrane Biology contains reports on recent developments in the field of electroporation by participants in the International Workshop and Postgraduate Course on Electroporation-Based Technologies and Treatments held in November 2014 in Ljubljana. This was the eighth session of what is now an annual event, first organized in 2003.

Altered Connexin 43 Expression Underlies Age-Dependent Decrease Of Regulatory T Cell Suppressor Function In Nonobese Diabetic Mice, Michel Kuczma, Cong-Yi Wang, Leszek Ignatowicz, Robert Gourdi, Piotr Kraj

Biological Sciences Faculty Publications

Type 1 diabetes is one of the most extensively studied autoimmune diseases, but the cellular and molecular mechanisms leading to T cell–mediated destruction of insulin-producing β cells are still not well understood. In this study, we show that regulatory T cells (T_regs) in NOD mice undergo age-dependent loss of suppressor functions exacerbated by the decreased ability of activated effector T cells to upregulate Foxp3 and generate T_regs in the peripheral organs. This age-dependent loss is associated with reduced intercellular communication mediated by gap junctions, which is caused by impaired upregulation and decreased expression of connexin 43. Regulatory …

Numerical Geometry Of Map And Model Assessment, Willy Wriggers, Jing He

Mechanical & Aerospace Engineering Faculty Publications

We are describing best practices and assessment strategies for the atomic interpretation of cryo-electron microscopy (cryo-EM) maps. Multiscale numerical geometry strategies in the Situs package and in secondary structure detection software are currently evolving due to the recent increases in cryo-EM resolution. Criteria that aim to predict the accuracy of fitted atomic models at low (worse than 8 angstrom) and medium (4-8 angstrom) resolutions remain challenging. However, a high level of confidence in atomic models can be achieved by combining such criteria. The observed errors are due to map-model discrepancies and due to the effect of imperfect global docking strategies. …

Picosecond To Terahertz Perturbation Of Interfacial Water And Electropermeabilization Of Biological Membranes, P. Thomas Vernier, Zachary A. Levine, Ming-Chak Ho, Shu Xiao, Iurii Semenov, Andrei G. Pakhomov

Bioelectrics Publications

Non-thermal probing and stimulation with subnanosecond electric pulses and terahertz electromagnetic radiation may lead to new, minimally invasive diagnostic and therapeutic procedures and to methods for remote monitoring and analysis of biological systems, including plants, animals, and humans. To effectively engineer these still-emerging tools, we need an understanding of the biophysical mechanisms underlying the responses that have been reported to these novel stimuli. We show here that subnanosecond (≤500 ps) electric pulses induce action potentials in neurons and cause calcium transients in neuroblastoma-glioma hybrid cells, and we report complementary molecular dynamics simulations of phospholipid bilayers in electric fields in which …

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 …

Dielectric Characterization Of Coastal Cartilage Chondrocytes, Michael W. Stacey, Ahmet C. Sabuncu, Ali Beskok

Bioelectrics Publications

BACKGROUND: Chondrocytes respond to biomechanical and bioelectrochemical stimuli by secreting appropriate extracellular matrix proteins that enable the tissue to withstand the large forces it experiences. Although biomechanical aspects of cartilage are well described, little is known of the bioelectrochemical responses. The focus of this study is to identify bioelectrical characteristics of human costal cartilage cells using dielectric spectroscopy.

METHODS: Dielectric spectroscopy allows non-invasive probing of biological cells. An in house computer program is developed to extract dielectric properties of human costal cartilage cells from raw cell suspension impedance data measured by a microfluidic device. The dielectric properties of chondrocytes are …

Basic Features Of A Cell Electroporation Model: Illustrative Behavior For Two Very Different Pulses, Reuben S. Son, Kyle C. Smith, Thiruvallur R. Gowrishankar, P. Thomas Vernier, James C. Weaver

Bioelectrics Publications

Science increasingly involves complex modeling. Here we describe a model for cell electroporation in which membrane properties are dynamically modified by poration. Spatial scales range from cell membrane thickness (5 nm) to a typical mammalian cell radius (10 μm), and can be used with idealized and experimental pulse waveforms. The model consists of traditional passive components and additional active components representing nonequilibrium processes. Model responses include measurable quantities: transmembrane voltage, membrane electrical conductance, and solute transport rates and amounts for the representative "long" and "short" pulses. The long pulse-1.5 kV/cm, 100 μs-evolves two pore subpopulations with a valley at ~5 …

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 …

Introduction To Fourth Special Issue On Electroporation-Based Technologies And Treatments, Damijan Miklavčič, Lluis M. Mir, P. Thomas Vernier

Bioelectrics Publications

This fourth special electroporation-based technologies and treatments issue of the Journal of Membrane Biology contains reports on recent developments in the field of electroporation by participants in the 7th International Workshop and Postgraduate Course on electroporation based technologies and treatments (EBTT 2013) held in Ljubljana, November 17–23, 2013. The 65 participants included faculty members, invited lecturers, special guests, and young scientists, and students from 16 countries. In addition to lectures on the fundamentals, this year’s sessions included talks on microbial inactivation by pulsed electric fields, modeling of intracellular electroporation, electroporation in food processing, and electrotransfer-facilitated DNA vaccination.

Recruitment Of The Intracellular Ca2+ By Ultrashort Electric Stimuli: The Impact Of Pulse Duration, Iurii Semenov, Shu Xiao, Olga N. Pakhomova, Andrei G. Pakhomov

Bioelectrics Publications

Nanosecond-duration electric stimuli are distinguished by the ability to permeabilize intracellular membranes and recruit Ca²⁺ from intracellular stores. We quantified this effect in non-excitable cells (CHO) using ratiometric Ca²⁺ imaging with Fura-2. In a Ca²⁺-free medium, 10-, 60-, and 300-ns stimuli evoked Ca2⁺ transients by mobilization of Ca²⁺ from the endoplasmic reticulum. With 2 mM external Ca²⁺, the transients included both extra- and intracellular components. The recruitment of intracellular Ca²⁺ increased as the stimulus duration decreased. At the threshold of 200–300 nM, the transients were amplified by calcium-induced calcium release. We …

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.

Electric Field Exposure Triggers And Guides Formation Of Pseudopod-Like Blebs In U937 Monocytes, Mikhail A. Rassokhin, Andrei G. Pakhomov

Bioelectrics Publications

We describe a new phenomenon of anodotropic pseudopod-like blebbing in U937 cells stimulated by nanosecond pulsed electric field (nsPEF). In contrast to "regular," round-shaped blebs, which are often seen in response to cell damage, pseudopod-like blebs (PLBs) formed as longitudinal membrane protrusions toward anode. PLB length could exceed the cell diameter in 2 min of exposure to 60-ns, 10-kV/cm pulses delivered at 10-20 Hz. Both PLBs and round-shaped nsPEF-induced blebs could be efficiently inhibited by partial isosmotic replacement of bath NaCl for a larger solute (sucrose), thereby pointing to the colloid-osmotic water uptake as the principal driving force for bleb …

Oxidative Effects Of Nanosecond Pulsed Electric Field Exposure In Cells And Cell-Free Media, Olga N. Pakhomova, Vera A. Khorokhorina, Angela M. Bowman, Raminta Rodaitė-Riševičienė, Gintautas Saulis, Shu Xiao, Andrei G. Pakhomov

Bioelectrics Publications

Nanosecond pulsed electric field (nsPEF) is a novel modality for permeabilization of membranous structures and intracellular delivery of xenobiotics. We hypothesized that oxidative effects of nsPEF could be a separate primary mechanism responsible for bioeffects. ROS production in cultured cells and media exposed to 300-ns PEF (1–13 kV/cm) was assessed by oxidation of 2′, 7′-dichlorodihydrofluoresein (H₂DCF), dihidroethidium (DHE), or Amplex Red. When a suspension of H₂DCF-loaded cells was subjected to nsPEF, the yield of fluorescent 2′,7′dichlorofluorescein (DCF) increased proportionally to the pulse number and cell density. DCF emission increased with time after exposure in nsPEF-sensitive Jurkat …

An Apoptosis Targeted Stimulus With Nanosecond Pulsed Electric Fields (Nspefs) In E4 Squamous Cell Carcinoma, Wei Ren, Stephen J. Beebe

Bioelectrics Publications

Stimuli directed towards activation of apoptosis mechanisms are an attractive approach to eliminate evasion of apoptosis, a ubiquitous cancer hallmark. In these in vitro studies, kinetics and electric field thresholds for several apoptosis characteristics are defined in E4 squamous carcinoma cells (SCC) exposed to ten 300 ns pulses with increasing electric fields. Cell death was [95% at the highest electric field and coincident with phosphatidylserine externalization, caspase and calpain activation in the presence and absence of cytochrome c release, decreases in Bid and mitochondria membrane potential (Δψm) without apparent changes reactive oxygen species levels or in Bcl2 and Bclxl levels. …

Never Let Me Clone? Countering An Ethical Argument Against The Reproductive Cloning Of Humans, Yvette Pearson

Philosophy Faculty Publications

In the March 2006 issue of EMBO reports, Christof Tannert, a bioethicist at the Max Delbrück Research Centre in Berlin, Germany, presented a moral argument against human reproductive cloning on the basis of Immanuel Kant’s categorical imperative (Tannert, 2006). In this article, I address some problems with Tannert’s views and show that our concerns about this prospective procedure should prompt us to scrutinize carefully the conventional procreative practices and attitudes. Indeed, if we set aside objections that are grounded in genetic determinism, many of the offensive features of human cloning are identical to problems with procreation by more conventional means, …

Selective Field Effects On Intracellular Vacuoles And Vesicle Membranes With Nanosecond Electric Pulses, Ephrem Tekle, Hammou Oubrahim, Sergey M. Dzekunov, Juergen F. Kolb, Karl H. Schoenbach

Bioelectrics Publications

Electric pulses across intact vesicles and cells can lead to transient increase in permeability of their membranes. We studied the integrity of these membranes in response to external electric pulses of high amplitude and submicrosecond duration with a primary aim of achieving selective permeabilization. These effects were examined in two separate model systems comprising of 1), a mixed population of 1,2-di-oleoyl-sn-glycero-3-phosphocholine phospholipid vesicles and in 2), single COS-7 cells, in which large endosomal membrane vacuoles were induced by stimulated endocytosis. It has been shown that large and rapidly varying external electric fields, with pulses shorter than the charging time of …

Theoretical Predictions Of Electromechanical Deformation Of Cells Subjected To High Voltages For Membrane Electroporation, R. P. Joshi, Q. Hu, K. H. Schoenbach, H. P. Hjalmarson

Bioelectrics Publications

An electromechanical analysis based on thin-shell theory is presented to analyze cell shape changes in response to external electric fields. This approach can be extended to include osmotic-pressure changes. Our calculations demonstrate that at large fields, the spherical cell geometry can be significantly modified, and even ellipsoidal forms would be inappropriate to account for the deformation. Values of the surface forces obtained from our calculations are in very good agreement with the 1–10 mN/m range for membrane rupture reported in the literature. The results, in keeping with reports in the literature, demonstrate that the final shape depends on membrane thickness. …

Mechanism For Membrane Electroporation Irreversibility Under High-Intensity, Ultrashort Electrical Pulse Conditions, R. P. Joshi, K. H. Schoenbach

Bioelectrics Publications

An improved electroporation model is used to address membrane irreversibility under ultrashort electric pulse conditions. It is shown that membranes can survive a strong electric pulse and recover provided the pore distribution has a relatively large spread. If, however, the population consists predominantly of larger radii pores, then irreversibility can result. Physically, such a distribution could arise if pores at adjacent sites coalesce. The requirement of close proximity among the pore sites is more easily satisfied in smaller organelles than in outer cell membranes. Model predictions are in keeping with recent observations of cell damage to intracellular organelles (e.g., mitochondria), …