Facilitation of Electroporative Drug Uptake and Cell Killing by Electrosensitization

Cell permeabilization by electric pulses (EP), or electroporation, is widely used for intracellular delivery of drugs and plasmids, as well as for tumour and tissue ablation. We found that cells pre-treated with 100-mus EP develop delayed hypersensitivity to subsequent EP applications. Sensitizing B16 and CHO cells by splitting a single train of eight 100-mus EP into two trains of four EP each (with 5-min. interval) decreased the LD(50) 1.5-2 times. Sensitization profoundly enhanced the electroporation-assisted uptake of bleomycin, a cell-impermeable cytotoxic agent accepted for killing tumours by electrochemotherapy. EP exposures that were not lethal per se caused cell death in the presence of bleomycin and proportionally to its concentration. Sensitizing cells by a split-dose EP exposure increased bleomycin-mediated lethality to the same extent as a 10-fold increase in bleomycin concentration when using a single EP dose. Likewise, sensitization by a split-dose EP exposure (without changing the overall dose, pulse number, or amplitude) enhanced the electroporative uptake of propidium up to fivefold. Enhancement of the electroporative uptake appears a key mechanism of electrosensitization and may benefit electrochemotherapy and numerous applications that employ EP for cell permeabilization

Two Modes of Cell Death Caused by Exposure to Nanosecond Pulsed Electric Field

High-amplitude electric pulses of nanosecond duration, also known as nanosecond pulsed electric field (nsPEF), are a novel modality with promising applications for cell stimulation and tissue ablation. However, key mechanisms responsible for the cytotoxicity of nsPEF have not been established. We show that the principal cause of cell death induced by 60- or 300-ns pulses in U937 cells is the loss of the plasma membrane integrity (‘‘nanoelectroporation’’), leading to water uptake, cell swelling, and eventual membrane rupture. Most of this early necrotic death occurs within 1–2 hr after nsPEF exposure. The uptake of water is driven by the presence of pore-impermeable solutes inside the cell, and can be counterbalanced by the presence of a pore-impermeable solute such as sucrose in the medium. Sucrose blocks swelling and prevents the early necrotic death; however the long-term cell survival (24 and 48 hr) does not significantly change. Cells protected with sucrose demonstrate higher incidence of the delayed death (6–24 hr post nsPEF). These cells are more often positive for the uptake of an early apoptotic marker dye YO-PRO-1 while remaining impermeable to propidium iodide. Instead of swelling, these cells often develop apoptotic fragmentation of the cytoplasm. Caspase 3/7 activity increases already in 1 hr after nsPEF and poly-ADP ribose polymerase (PARP) cleavage is detected in 2 hr. Staurosporin-treated positive control cells develop these apoptotic signs only in 3 and 4 hr, respectively. We conclude that nsPEF exposure triggers both necrotic and apoptotic pathways. The early necrotic death prevails under standard cell culture conditions, but cells rescued from the necrosis nonetheless die later on by apoptosis. The balance between the two modes of cell death can be controlled by enabling or blocking cell swelling

Diagnosis of Ovarian Cancer Using Decision Tree Classification of Mass Spectral Data

Recent reports from our laboratory and others support the SELDI ProteinChip technology as a potential clinical diagnostic tool when combined with n-dimensional analyses algorithms. The objective of this study was to determine if the commercially available classification algorithm biomarker patterns software (BPS), which is based on a classification and regression tree (CART), would be effective in discriminating ovarian cancer from benign diseases and healthy controls. Serum protein mass spectrum profiles from 139 patients with either ovarian cancer, benign pelvic diseases, or healthy women were analyzed using the BPS software. A decision tree, using five protein peaks, resulted in an accuracy of 81.5% in the cross-validation analysis and 80% in a blinded set of samples in differentiating the ovarian cancer from the control groups. The potential, advantages, and drawbacks of the BPS system as a bioinformatic tool for the analysis of the SELDI high-dimensional proteomic data are discussed

HSP90 Inhibitors Modulate SARS-CoV-2 Spike Protein Subunit 1-Induced Human Pulmonary Microvascular Endothelial Activation and Barrier Dysfunction

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused more than 5 million deaths worldwide. Multiple reports indicate that the endothelium is involved during SARS-Cov-2-related disease (COVID-19). Indeed, COVID-19 patients display increased thrombophilia with arterial and venous embolism and lung microcapillary thrombotic disease as major determinants of deaths. The pathophysiology of endothelial dysfunction in COVID-19 is not completely understood. We have investigated the role of subunit 1 of the SARS-CoV-2 spike protein (S1SP) in eliciting endothelial barrier dysfunction, characterized dose and time relationships, and tested the hypothesis that heat shock protein 90 (HSP90) inhibitors would prevent and repair such injury. S1SP activated (phosphorylated) IKBα, STAT3, and AKT and reduced the expression of intercellular junctional proteins, occludin, and VE-cadherin. HSP90 inhibitors (AT13387 and AUY-922) prevented endothelial barrier dysfunction and hyperpermeability and reduced IKBα and AKT activation. These two inhibitors also blocked S1SP-mediated barrier dysfunction and loss of VE-cadherin. These data suggest that spike protein subunit 1 can elicit, by itself, direct injury to the endothelium and suggest a role of HSP90 inhibitors in preserving endothelial functionality

Hsp90 Inhibition Suppresses NF-κB Transcriptional Activation via Sirt-2 in Human Lung Microvascular Endothelial Cells

The ability of anti-heat shock protein 90 (Hsp90) drugs to attenuate NF-κB-mediated transcription is the major basis for their anti-inflammatory properties. While the molecular mechanisms underlying this effect are not clear, they appear to be distinct in human endothelial cells. We now show for the first time that type 2 sirtuin (Sirt-2) histone deacetylase binds human NF-κB target gene promoter and prevents the recruitment of NF-κB proteins and subsequent assembly of RNA polymerase II complex in human lung microvascular endothelial cells. Hsp90 inhibitors stabilize the Sirt-2/promoter interaction and impose a “transcriptional block,” which is reversed by either inhibition or downregulation of Sirt-2 protein expression. Furthermore, this process is independent of NF-κB (p65) Lysine 310 deacetylation, suggesting that it is distinct from known Sirt-2-dependent mechanisms. We demonstrate that Sirt-2 is recruited to NF-κB target gene promoter via interaction with core histones. Upon inflammatory challenge, chromatin remodeling and core histone H3 displacement from the promoter region removes Sirt-2 and allows NF-κB/coactivator recruitment essential for RNA Pol II-dependent mRNA induction. This novel mechanism may have important implications in pulmonary inflammation

Electroporation-Induced Electrosensitization

BACKGROUND: Electroporation is a method of disrupting the integrity of cell membrane by electric pulses (EPs). Electrical modeling is widely employed to explain and study electroporation, but even most advanced models show limited predictive power. No studies have accounted for the biological consequences of electroporation as a factor that alters the cell's susceptibility to forthcoming EPs. METHODOLOGY/PRINCIPAL FINDINGS: We focused first on the role of EP rate for membrane permeabilization and lethal effects in mammalian cells. The rate was varied from 0.001 to 2,000 Hz while keeping other parameters constant (2 to 3,750 pulses of 60-ns to 9-µs duration, 1.8 to 13.3 kV/cm). The efficiency of all EP treatments was minimal at high rates and started to increase gradually when the rate decreased below a certain value. Although this value ranged widely (0.1-500 Hz), it always corresponded to the overall treatment duration near 10 s. We further found that longer exposures were more efficient irrespective of the EP rate, and that splitting a high-rate EP train in two fractions with 1-5 min delay enhanced the effects severalfold. CONCLUSIONS/SIGNIFICANCE: For varied experimental conditions, EPs triggered a delayed and gradual sensitization to EPs. When a portion of a multi-pulse exposure was delivered to already sensitized cells, the overall effect markedly increased. Because of the sensitization, the lethality in EP-treated cells could be increased from 0 to 90% simply by increasing the exposure duration, or the exposure dose could be reduced twofold without reducing the effect. Many applications of electroporation can benefit from accounting for sensitization, by organizing the exposure either to maximize sensitization (e.g., for sterilization) or, for other applications, to completely or partially avoid it. In particular, harmful side effects of electroporation-based therapies (electrochemotherapy, gene therapies, tumor ablation) include convulsions, pain, heart fibrillation, and thermal damage. Sensitization can potentially be employed to reduce these side effects while preserving or increasing therapeutic efficiency

An original phylogenetic approach identified mitochondrial haplogroup T1a1 as inversely associated with breast cancer risk in BRCA2 mutation carriers

Introduction: Individuals carrying pathogenic mutations in the BRCA1 and BRCA2 genes have a high lifetime risk of breast cancer. BRCA1 and BRCA2 are involved in DNA double-strand break repair, DNA alterations that can be caused by exposure to reactive oxygen species, a main source of which are mitochondria. Mitochondrial genome variations affect electron transport chain efficiency and reactive oxygen species production. Individuals with different mitochondrial haplogroups differ in their metabolism and sensitivity to oxidative stress. Variability in mitochondrial genetic background can alter reactive oxygen species production, leading to cancer risk. In the present study, we tested the hypothesis that mitochondrial haplogroups modify breast cancer risk in BRCA1/2 mutation carriers. Methods: We genotyped 22,214 (11,421 affected, 10,793 unaffected) mutation carriers belonging to the Consortium of Investigators of Modifiers of BRCA1/2 for 129 mitochondrial polymorphisms using the iCOGS array. Haplogroup inference and association detection were performed using a phylogenetic approach. ALTree was applied to explore the reference mitochondrial evolutionary tree and detect subclades enriched in affected or unaffected individuals. Results: We discovered that subclade T1a1 was depleted in affected BRCA2 mutation carriers compared with the rest of clade T (hazard ratio (HR) = 0.55; 95% confidence interval (CI), 0.34 to 0.88; P = 0.01). Compared with the most frequent haplogroup in the general population (that is, H and T clades), the T1a1 haplogroup has a HR of 0.62 (95% CI, 0.40 to 0.95; P = 0.03). We also identified three potential susceptibility loci, including G13708A/rs28359178, which has demonstrated an inverse association with familial breast cancer risk. Conclusions: This study illustrates how original approaches such as the phylogeny-based method we used can empower classical molecular epidemiological studies aimed at identifying association or risk modification effects.Peer reviewe

The National Early Warning Score and its subcomponents recorded within ±24 hours of emergency medical admission are poor predictors of hospital-acquired acute kidney injury

YesBackground: Hospital-acquired Acute Kidney Injury (H-AKI) is a common cause of avoidable morbidity and mortality. Aim: To determine if the patients’ vital signs data as defined by a National Early Warning Score (NEWS), can predict H-AKI following emergency admission to hospital. Methods: Analyses of emergency admissions to York hospital over 24-months with NEWS data. We report the area under the curve (AUC) for logistic regression models that used the index NEWS (model A0), plus age and sex (A1), plus subcomponents of NEWS (A2) and two-way interactions (A3). Likewise for maximum NEWS (models B0,B1,B2,B3). Results: 4.05% (1361/33608) of emergency admissions had H-AKI. Models using the index NEWS had the lower AUCs (0.59 to 0.68) than models using the maximum NEWS AUCs (0.75 to 0.77). The maximum NEWS model (B3) was more sensitivity than the index NEWS model (A0) (67.60% vs 19.84%) but identified twice as many cases as being at risk of H-AKI (9581 vs 4099) at a NEWS of 5. Conclusions: The index NEWS is a poor predictor of H-AKI. The maximum NEWS is a better predictor but seems unfeasible because it is only knowable in retrospect and is associated with a substantial increase in workload albeit with improved sensitivity.The Health Foundatio

Proceedings of the 3rd Biennial Conference of the Society for Implementation Research Collaboration (SIRC) 2015: advancing efficient methodologies through community partnerships and team science

It is well documented that the majority of adults, children and families in need of evidence-based behavioral health interventionsi do not receive them [1, 2] and that few robust empirically supported methods for implementing evidence-based practices (EBPs) exist. The Society for Implementation Research Collaboration (SIRC) represents a burgeoning effort to advance the innovation and rigor of implementation research and is uniquely focused on bringing together researchers and stakeholders committed to evaluating the implementation of complex evidence-based behavioral health interventions. Through its diverse activities and membership, SIRC aims to foster the promise of implementation research to better serve the behavioral health needs of the population by identifying rigorous, relevant, and efficient strategies that successfully transfer scientific evidence to clinical knowledge for use in real world settings [3]. SIRC began as a National Institute of Mental Health (NIMH)-funded conference series in 2010 (previously titled the “Seattle Implementation Research Conference”; $150,000 USD for 3 conferences in 2011, 2013, and 2015) with the recognition that there were multiple researchers and stakeholdersi working in parallel on innovative implementation science projects in behavioral health, but that formal channels for communicating and collaborating with one another were relatively unavailable. There was a significant need for a forum within which implementation researchers and stakeholders could learn from one another, refine approaches to science and practice, and develop an implementation research agenda using common measures, methods, and research principles to improve both the frequency and quality with which behavioral health treatment implementation is evaluated. SIRC’s membership growth is a testament to this identified need with more than 1000 members from 2011 to the present.ii SIRC’s primary objectives are to: (1) foster communication and collaboration across diverse groups, including implementation researchers, intermediariesi, as well as community stakeholders (SIRC uses the term “EBP champions” for these groups) – and to do so across multiple career levels (e.g., students, early career faculty, established investigators); and (2) enhance and disseminate rigorous measures and methodologies for implementing EBPs and evaluating EBP implementation efforts. These objectives are well aligned with Glasgow and colleagues’ [4] five core tenets deemed critical for advancing implementation science: collaboration, efficiency and speed, rigor and relevance, improved capacity, and cumulative knowledge. SIRC advances these objectives and tenets through in-person conferences, which bring together multidisciplinary implementation researchers and those implementing evidence-based behavioral health interventions in the community to share their work and create professional connections and collaborations

Two modes of cell death caused by exposure to nanosecond pulsed electric field.

High-amplitude electric pulses of nanosecond duration, also known as nanosecond pulsed electric field (nsPEF), are a novel modality with promising applications for cell stimulation and tissue ablation. However, key mechanisms responsible for the cytotoxicity of nsPEF have not been established. We show that the principal cause of cell death induced by 60- or 300-ns pulses in U937 cells is the loss of the plasma membrane integrity ("nanoelectroporation"), leading to water uptake, cell swelling, and eventual membrane rupture. Most of this early necrotic death occurs within 1-2 hr after nsPEF exposure. The uptake of water is driven by the presence of pore-impermeable solutes inside the cell, and can be counterbalanced by the presence of a pore-impermeable solute such as sucrose in the medium. Sucrose blocks swelling and prevents the early necrotic death; however the long-term cell survival (24 and 48 hr) does not significantly change. Cells protected with sucrose demonstrate higher incidence of the delayed death (6-24 hr post nsPEF). These cells are more often positive for the uptake of an early apoptotic marker dye YO-PRO-1 while remaining impermeable to propidium iodide. Instead of swelling, these cells often develop apoptotic fragmentation of the cytoplasm. Caspase 3/7 activity increases already in 1 hr after nsPEF and poly-ADP ribose polymerase (PARP) cleavage is detected in 2 hr. Staurosporin-treated positive control cells develop these apoptotic signs only in 3 and 4 hr, respectively. We conclude that nsPEF exposure triggers both necrotic and apoptotic pathways. The early necrotic death prevails under standard cell culture conditions, but cells rescued from the necrosis nonetheless die later on by apoptosis. The balance between the two modes of cell death can be controlled by enabling or blocking cell swelling