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Electroporation technique induced by high-voltage pulses has been successfully used to ablate tumor cells while preserving the function of normal blood vessels. Generally, the tumor blood vessels can provide a pathway to draw nutrients for tumor growth and contribute to invasion and metastasis, which is an obstacle to tumor treatment. The electroporation study of the endothelial cell, which is important in the vasculature microenvironment, is helpful to investigate the influence on both tumor and normal blood vessels. This study built a multicell-layer model of the vascular microenvironment to investigate the discriminative electroporation effect between normal and tumor blood vessels by high-frequency bipolar pulses (HFBPs) and monopolar pulses (MPs). The simulation results showed that both pore number and electroporation region in normal blood vessels are significantly lower than those in tumor blood vessels. The rich vascular smooth muscle cells existed in the normal blood vessels play a protective function for endothelial cells, compared with tumor blood vessels. However, the differences in pore number and electroporation region between normal and tumor blood vessels are gradually smaller with an increased electric field, which demonstrates that the electroporation pulse with higher intensity damages both normal and tumor blood vessels. HFBPs generate a weaker electroporation effect on both normal and tumor blood vessels than traditional MP. However, HFBPs are more suitable to electroporate tumor blood vessels, while preserving the normal blood vessels. Moreover, this study could also provide a multicell-layer model that can be used to analyze the cell electroporation effect in the vascular microenvironment. [ABSTRACT FROM AUTHOR]

Electroporation is a complex, iterative, and nonlinear phenomenon often studied through numerical simulations. In recent years, simulations of tissue electroporation have been conducted with static models. However, the results of a static model simulation are restricted to a fixed protocol signature of the pulsed electric field. In this paper, we describe a novel dynamic model of tissue electroporation that also accounts for tissue dispersion and temperature to allow time-domain simulations. We have implemented the biological dispersion of potato tubers and thermal analysis in a commercial finite-element method software. A cell electroporation model was adapted to account for the increase in tissue conductivity. The model yielded 12 parameters divided into three dynamic states of electroporation. The thermal analysis describes the dependence of tissue conductivity on temperature. The model parameters were evaluated using experiments with vegetal tissue (Solanum tuberosum) under electrochemotherapy protocols. The proposed model can accurately predict the conductivity of tissue under electroporation from 100 to 1000 V/cm. A negligible thermal effect was observed at 1000 V/cm, with a temperature increase of 0.89 ° C. We believe that the proposed model is suitable to describe the electroporation at the tissue level and provides a hint of the effects on the cell membrane. [ABSTRACT FROM AUTHOR]

This document, published in the Journal of Applied Physics, discusses the importance of multiscale modeling and analysis in the field of biophysics. The authors emphasize the need for tools and analyses that integrate across physical scales and time scales in order to advance medical science. The document includes a collection of studies on various topics such as cardiac biomechanics, tissue mechanics, cerebral fluid flow, cell mechanics, protein modeling, and molecular fluorescence microscopy. The authors hope that readers will find these studies informative and inspiring. [Extracted from the article]

From the microscopic electroporation to the irregular distribution of cell populations, the inactivation efficacy of pulsed electric fields (PEFs) from in vitro experiments has lacked a unified physical model due to its cross-scale complexity. Inspired by a coarse-grained approach from the percolation theory, the inactivation process is simulated from a simple yet robust lattice model, where the spatiotemporal heterogeneity of the collective structure and the stochastic PEF strike are portrayed as random matrices, while also accounting for the rules of single-cell electroporation and subsequent death. Beyond successfully simulating the inactivation of monolayer adherent cells and suspended cells, which are in good agreement with in vitro results, our model reveals that (1) macroscopically three-staged inactivation pattern originates from the "accelerate–uniform–decelerate" transition of inactivation velocity, and (2) the inactivation patterns obey a universal scaling law under varied field strength, which is not satisfied under varied pulsed widths. The simulation not only sheds light on the PEF inactivation of the macroscopic cell collectives but also provides a simple and generalized numerical method for predicting PEF efficacy in experiments or engineering. [ABSTRACT FROM AUTHOR]

BACKGROUND: The effect of contact force (CF) on lesion formation is not clear during pulsed field ablation (PFA). The aim of this study was to evaluate the impact of CF, PFA, and their interplay through the PFA index (PF index) formula on the ventricular lesion size in swine. METHODS: PFA was delivered through the CF-sensing OMNYPULSE catheter. Predefined PFA applications (×3, ×6, ×9, and ×12) were delivered maintaining low (5-25 g), high (26-50 g), and very high (51-80 g) CFs. First, PFA lesions were evaluated on necropsy in 11 swine to investigate the impact of CF/PFA-and their integration in the PF index equation-on lesion size (study characterization). Then, 3 different PF index thresholds-300, 450, and 600-were tested in 6 swine to appraise the PF index accuracy to predict the ventricular lesion depth (study validation). RESULTS: In the study characterization data set, 111 PFA lesions were analyzed. CF was 32±17 g. The average lesion depth and width were 3.5±1.2 and 12.0±3.5 mm, respectively. More than CF and PFA dose alone, it was their combined effect to impact lesion depth through an asymptotically increasing relationship. Likewise, not only was the PF index related to lesion depth in the study validation data set (r2=0.66; P

Methanotrophic bacteria are promising hosts for methane bioconversion to biochemicals or bioproducts. However, due to limitations associated with long genetic manipulation timelines and, lack of choice in genetic tools required for strain engineering, methanotrophs are currently not employed for bioconversion technologies. In this study, a rapid and reproducible electroporation protocol is developed for type 1 methanotroph, Methylotuvimicrobium alcaliphilum using common laboratory solutions, analyzing optimal electroshock voltages and post-shock cell recovery time. Successful reproducibility of the developed method was achieved when different replicative plasmids were assessed on lab adapted vs. wild-type M. alcaliphilum strains (DASS vs. DSM19304). Overall, a ∼ 3-fold decrease in time is reported with use of electroporation protocol developed here, compared to conjugation, which is the traditionally employed approach. Additionally, an inducible (3-methyl benzoate) and a constitutive (sucrose phosphate synthase) promoter is characterized for their strength in driving gene expression.

Chlamydomonas reinhardtii , a unicellular green alga, is a prominent model for green biotechnology and for studying organelles' function and biogenesis, such as chloroplasts and cilia. However, the stable expression of foreign genes from the nuclear genome in C. reinhardtii faces several limitations, including low expression levels and significant differences between clones due to genome position effects, epigenetic silencing, and time-consuming procedures. We developed a robust transient expression system in C. reinhardtii to overcome these limitations. We demonstrated efficient entry of in vitro-transcribed mRNA into wall-less cells and enzymatically dewalled wild-type cells via electroporation. The endogenous or exogenous elements can facilitate efficient transient expression of mRNA in C. reinhardtii , including the 5' UTR of PsaD and the well-characterized Kozak sequence derived from the Chromochloris zofingiensis. In the optimized system, mRNA expression was detectable in 120 h with a peak around 4 h after transformation. Fluorescently tagged proteins were successfully transiently expressed, enabling organelle labeling and real-time determination of protein sub-cellular localization. Remarkably, transiently expressed IFT46 compensated for the ift46–1 mutant phenotype, indicating the correct protein folding and function of IFT46 within the cells. Additionally, we demonstrated the feasibility of our system for studying protein-protein interactions in living cells using bimolecular fluorescence complementation. In summary, the established transient expression system provides a powerful tool for investigating protein localization, function, and interactions in C. reinhardtii within a relatively short timeframe, which will significantly facilitate the study of gene function, genome structure, and green biomanufacturing in C. reinhardtii and potentially in other algae. • Successful establishment of an RNA-based transient expression system in Chlamydomonas. • Function of 5' UTR on gene expression occurs at the post-transcriptional level in Chlamydomonas. • Analysis of the localization, function, and interactions of proteins can be completed within one week. [ABSTRACT FROM AUTHOR]

Genome editing is recognized as a powerful tool in agriculture and research, enhancing our understanding of genetic function, diseases, and productivity. However, its progress in buffaloes has lagged behind other mammals due to several challenges, including long gestational periods, single pregnancies, and high raising costs. In this study, we aimed to generate MSTN-edited buffaloes, known for their distinctive double-muscling phenotype, as a proof of concept. To meet our goal, we used somatic cell nuclear transfer (SCNT) and zygotic electroporation (CRISPR-EP) technique. For this, we firstly identified the best transfection method for introduction of RNP complex into fibroblast which was further used for SCNT. For this, we compared the transfection, cleavage efficiency and cell viability of nucleofection and lipofection in adult fibroblasts. The cleavage, transfection efficiency and cell viability of nucleofection group was found to be significantly (P ≤ 0.05) higher than lipofection group. Four MSTN edited colony were generated using nucleofection, out of which three colonies was found to be biallelic and one was monoallelic. Further, we compared the efficacy, embryonic developmental potential and subsequent pregnancy outcome of SCNT and zygotic electroporation. The blastocyst rate of electroporated group was found to be significantly (P ≤ 0.05) higher than SCNT group. However, the zygotic electroporation group resulted into two pregnancies which were confirmed to be MSTN edited. Since, the zygotic electroporation does not require complex micromanipulation techniques associated with SCNT, it has potential for facilitating the genetic modification in large livestock such as buffaloes. The present study lays the basis for inducing genetic alternation with practical or biological significance. • Genome editing is a well-known method for introducing targeted genetic alterations into livestock genomes, however its progress in buffaloes has lagged behind. • In the present study, we aimed to generate MSTN-edited buffaloes using SCNT and Zygotic electroporation. • The best transfection method was identified for introduction of RNP complex into buffalo fibroblast which was further used for single cell isolation and generation of MSTN edited embryos via SCNT. • The embryonic developmental potential and subsequent pregnancy outcome of SCNT and zygotic electroporation were compared. [ABSTRACT FROM AUTHOR]

Most simulations of electric field driven bioeffects have considered spherical cellular geometries or probed symmetrical structures for simplicity. This work assesses cellular transmembrane potential build-up and electroporation in a Jurkat cell that includes the endoplasmic reticulum (ER) and mitochondria, both of which have complex shapes, in response to external nanosecond electric pulses. The simulations are based on a time-domain nodal analysis that incorporates membrane poration utilizing the Smoluchowski model with angular-dependent changes in membrane conductivity. Consistent with prior experimental reports, the simulations show that the ER requires the largest electric field for electroporation, while the inner mitochondrial membrane (IMM) is the easiest membrane to porate. Our results suggest that the experimentally observed increase in intracellular calcium could be due to a calcium induced calcium release (CICR) process that is initiated by outer cell membrane breakdown. Repeated pulsing and/or using multiple electrodes are shown to create a stronger poration. The role of mutual coupling, screening, and proximity effects in bringing about electric field modifications is also probed. Finally, while including greater geometric details might refine predictions, the qualitative trends are expected to remain. [ABSTRACT FROM AUTHOR]

The reactive species-independent nature of cold plasma's electric field is pivotal in biomedical applications. This work proposes to connect the plasma fluid model and the asymptotic Smoluchowski model for electroporation, providing a unified framework to investigate the evolution of the electric field in the biological substrate and the multi-stage electroporation response of the human cell. Two common substrates with distinct dielectric properties, namely, the cultivation medium and epidermis, are selected to report three stages of ionization wave (IW)–substrate interaction. The three-stage streamer discharge dynamics (restrike, axial-radial transition, and radial expansion of IW) induce three-stage cell electroporation dynamics (slow charging, fast charging, and electroporation), though the two processes are asynchronous. Specifically, the inner membrane covered the cell nucleus with ultra-short charging time that undergoes only the first two discharge stages in both substrates. Whether the cell membrane is exposed to the third stage of discharge depends on the permittivity of the substrate. The asynchrony can be attributed to the difference in the charging time of the cell membranes and substrates affected by the substrate permittivity. The presented model can provide quantitative insights into the cell electroporation induced by the IW–substrate interaction and theoretical guidance for plasma biomedical applications. [ABSTRACT FROM AUTHOR]

Cellular heterogeneity plays a significant role in understanding biological processes, such as cell cycle and disease progression. Microfluidics has emerged as a versatile tool for manipulating single cells and analyzing their heterogeneity with the merits of precise fluid control, small sample consumption, easy integration, and high throughput. Specifically, integrating microfluidics with electrical techniques provides a rapid, label-free, and non-invasive way to investigate cellular heterogeneity at the single-cell level. Here, we review the recent development of microfluidic-based electrical strategies for single-cell manipulation and analysis, including dielectrophoresis- and electroporation-based single-cell manipulation, impedance- and AC electrokinetic-based methods, and electrochemical-based single-cell detection methods. Finally, the challenges and future perspectives of the microfluidic-based electrical techniques for single-cell analysis are proposed. [ABSTRACT FROM AUTHOR]

Introduction: Pulsed‐field ablation (PFA) is a novel nonthermal energy that shows unique features that can be of use beyond pulmonary vein ablation, like tissue selectivity or proximity rather than contact dependency. Methods and Results: We report three cases of right focal atrial tachycardias arising from the superior cavoatrial junction and the crista terminalis, in close relationship with the phrenic nerve, effectively ablated using a commercially available PFA catheter designed for pulmonary vein isolation without collateral damage. Conclusion: PFA can be useful for treating right atrial tachycardias involving sites near the phrenic nerve, avoiding the need for complex nerve‐sparing strategies. [ABSTRACT FROM AUTHOR]

Introduction Methods Results Conclusion Pulsed field ablation (PFA) has emerged as an innovative therapy for cardiac arrhythmias. Drawing parallels with PFA's application in solid tumors, calcium chloride (CaCl2) as an adjuvant therapy, known as calcium electroporation, may amplify PFA's apoptotic effects. We propose that PFA in the atrium could enhance calcium uptake through PFA‐created pores, thereby increasing ablation efficacy even at reduced power levels by exploiting PFA's permeabilization effects.We conducted in vivo ablations on the atria of seven pigs using low PFA power (250 V, 20 μs for 50 pulses at 200 ms intervals). Post‐PFA, we randomly administered an infusion of either 200 mg/2 ml CaCl2 (calcium group) or saline (control) directly to the ablation site via the catheter tip. We evaluated reduction in electrogram voltage amplitude, electrocardiography (ECG) parameters, ablation lesion parameters, and histology after PFA.Nineteen lesions from control and calcium groups were examined. Control lesions showed no voltage decrease post‐PFA, whereas calcium‐treated lesions exhibited a significant voltage reduction. Gross pathology indicated marked differences in maximum lesion surface diameter, depth, and volume between the lesion groups. Histologically, calcium group lesions were characterized by a more severe acute PFA response with contraction band necrosis, myocytolysis and nuclear pyknosis in adjacent myocardium, in addition to microhemorrhages.Infusing calcium chloride locally after PFA markedly improves the immediate efficacy of electroporation in porcine atria. This study suggests that calcium electroporation could bolster PFA outcomes without higher energy levels, potentially diminishing associated risks. These preliminary findings warrant further research into the long‐term efficacy and potential clinical application of calcium electroporation in PFA. [ABSTRACT FROM AUTHOR]

Objectives: The aim of the IRECAP study was to evaluate the rate of locally advanced pancreas cancer patients (LAPC) who could undergo R0 or R1 surgery after irreversible electroporation (IRE). Materials and methods: IRECAP study is a phase II, single-center, open-label, prospective, non-randomized trial registered at clinicaltrials.gov (NCT03105921). Patients with LAPC were first treated by 3-month neo-adjuvant chemotherapy in order to avoid inclusion of either patients with LAPC having become resectable after chemotherapy or patients with rapid disease progression. In cases of stable disease, IRE was performed percutaneously under CT guidance. Surgery was planned between 28 and 90 days after IRE. Tumor specimens were studied to evaluate the resection margins (R0/R1/R2). Results: Six men and 11 women were included (median age 61 years, range 37–77 years). No IRE-related death was observed. Ten patients (58%, 10/17) experienced 25 serious adverse events related to IRE. Four patients progressed between IRE and surgery and were excluded from surgery. Thirteen patients were finally operated, six withheld for pancreas resection, three for diffuse peritoneal carcinosis, two for massive vascular entrapment, and one for hepato-cellular carcinoma not diagnosed before surgery. Rate of R1-R0 was 35% (n = 6/17). Median overall survival was 31 months (95% CI; 4–undefined) for the six patients with R0/R1 resection and 21 months (95% CI; 4–25) for the 11 patients without resection or R2 resection (logrank p = 0.044). Conclusion: After neoadjuvant chemotherapy, IRE could provide R0 or R1 resection in 35% of LAPC, which seems to be associated with higher OS. Clinical relevance statement: After induction chemotherapy, stable locally advanced pancreatic cancers can be treated by irreversible electroporation, which could lead to a secondary 35% rate of R0 or R1 surgical resection which may be associated with a significantly higher overall survival. Key Points: • In cases of unresectable LAPC (locally advanced pancreatic cancer), percutaneous irreversible electroporation (pIRE) is feasible (100% success rate of the procedure), but is associated with a 58% rate of grade 3–4 adverse events. • In patients with unresectable LAPC, pIRE could lead 35% of patients to R0-R1 surgical resection. • From IRE, median overall survival was 31 months (95% CI; 4–undefined) for the patients with R0/R1 resection and 21 months (95% CI; 4–25) for the patients without resection or R2 resection (logrank p = 0.044). [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF), the most common arrhythmia, is associated with increased morbidity, mortality, and healthcare costs. Evidence indicates that rhythm control offers superior cardiovascular outcomes compared to rate control, especially when initiated early after the diagnosis of AF. Catheter ablation remains the single best therapy for AF; however, it is not free from severe complications and only a small percentage of AF patients in the Western world ultimately receive ablation. Ensuring that AF ablation is safe, effective, and efficient is essential to make it accessible to all patients. With the limitations of traditional thermal ablative energies, pulsed field ablation (PFA) has emerged as a novel non-thermal energy source. PFA targets irreversible electroporation of cardiomyocytes to achieve cell death without damaging adjacent structures. Through its capability to create rapid, selective lesions in myocytes, PFA presents a promising alternative, offering enhanced safety, reduced procedural times, and comparable, if not superior, efficacy to thermal energies. The surge of new evidence makes it challenging to stay updated and understand the possibilities and challenges of PFA. This review aims to summarize the most significant advantages of PFA and how this has translated to the clinical arena, where four different catheters have received CE-market approval for AF ablation. Further research is needed to explore whether adding new ablation targets, previously avoided due to risks associated with thermal energies, to pulmonary vein isolation can improve the efficacy of AF ablation. It also remains to see whether a class effect exists or if different PFA technologies can yield distinct clinical outcomes given that the optimization of PFA parameters has largely been empirical. [ABSTRACT FROM AUTHOR]

Purpose: Electrochemotherapy, clinically established for treating (sub)cutaneous tumors, has been standardized in the framework of the European Standard Operating Procedure on Electrochemotherapy (ESOPE). Due to common side effects of chemotherapeutic drugs, recent advances focus on non-cytotoxic agents, like calcium, to induce cell death (calcium electroporation). Therefore, this study aims to determine the efficacy of electrochemotherapy with bleomycin or cisplatin, or calcium electroporation on human hepatocellular carcinoma cells (HepG2) in vitro using the ESOPE protocol. Methods: HepG2 cell viability was measured with a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay after electrochemotherapy with the chemotherapeutic drugs bleomycin or cisplatin (0–20 µM), or after calcium electroporation (0–20 mM), to determine its efficacy on HepG2 cells in vitro using the ESOPE protocol (8 rectangular pulses, 1000 V/cm, 100 µs) compared to non-electroporated drug treatment. Results: Cell viability was significantly lower in electroporated samples, compared to their non-electroporated controls (27–75% difference). Electrochemotherapy with bleomycin and calcium electroporation, reached (almost) complete cell death (− 1 ± 3% and 2.5 ± 2%), in the lowest concentration of 2.5 µM and 2.5 mM, respectively. Electrochemotherapy with 2.5 µM cisplatin, significantly decreased cell viability to only 68% (± 7%). Conclusion: Electrochemotherapy with bleomycin or cisplatin, or calcium electroporation were more effective in reducing the HepG2 cell viability in vitro using the ESOPE protocol compared to the non-electroporated drug treatments alone. When comparing electrochemotherapy, HepG2 cells are more sensitive to bleomycin than cisplatin, in similar concentrations. Calcium electroporation has the same effectiveness as electrochemotherapy with bleomycin, but calcium potentially has a better safety profile and several treatment advantages. [ABSTRACT FROM AUTHOR]

Copyright of Przegląd Elektrotechniczny is the property of Przeglad Elektrotechniczny and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Focal therapy, a minimally invasive strategy for localized prostate cancer, has been widely employed in the targeted treatment of localized prostate cancer in recent years. We analyzed 1312 relevant papers from the last decade using Web of Science Core Collection data. Our analysis covered countries, institutions, journals, authors, keywords, and references to offer a multifaceted perspective on the development of this field. The U.S. led in publications, contributing over half of the top 10 institutions. Emberton, M from University College London was the most published and cited author. "EUROPEAN UROLOGY" was the top journal by impact factor in 2022. Analysis of references and keywords suggests the prevalence of brachytherapy-related research, while high-intensity focused ultrasound (HIFU), cryotherapy, and irreversible electroporation (IRE) are emerging as new research focuses. Consequently, more high-quality evidence is necessary to evaluate the long-term effectiveness and safety of these novel therapeutic methods. [ABSTRACT FROM AUTHOR]

Background: The non-viral production of CAR-T cells through electroporation of transposon DNA plasmids is an alternative approach to lentiviral/retroviral methods. This method is particularly suitable for early-phase clinical trials involving novel types of CAR-T cells. The primary disadvantage of non-viral methods is the lower production efficiency compared to viral-based methods, which becomes a limiting factor for CAR-T production, especially in chemotherapy-pretreated lymphopenic patients. Methods: We describe a good manufacturing practice (GMP)-compliant protocol for producing CD19 and CD123-specific CAR-T cells based on the electroporation of transposon vectors. The lymphocytes were purified from the blood of patients undergoing chemotherapy for B-NHL or AML and were electroporated with piggyBac transposon encoding CAR19 or CAR123, respectively. Electroporated cells were then polyclonally activated by anti-CD3/CD28 antibodies and a combination of cytokines (IL-4, IL-7, IL-21). The expansion was carried out in the presence of irradiated allogeneic blood-derived mononuclear cells (i.e., the feeder) for up to 21 days. Results: Expansion in the presence of the feeder enhanced CAR-T production yield (4.5-fold in CAR19 and 9.3-fold in CAR123). Detailed flow-cytometric analysis revealed the persistence of early-memory CAR-T cells and a low vector-copy number after production in the presence of the feeder, with no negative impact on the cytotoxicity of feeder-produced CAR19 and CAR123 T cells. Furthermore, large-scale manufacturing of CAR19 carried out under GMP condi tions using PBMCs obtained from B-NHL patients (starting number=200x10e6 cells) enabled the production of >50x10e6 CAR19 in 7 out of 8 cases in the presence of the feeder while only in 2 out of 8 cases without the feeder. Conclusions: The described approach enables GMP-compatible production of sufficient numbers of CAR19 and CAR123 T cells for clinical application and provides the basis for non-viral manufacturing of novel experimental CAR-T cells that can be tested in early-phase clinical trials. This manufacturing approach can complement and advance novel experimental immunotherapeutic strategies against human hematologic malignancies. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPR)-based editing tools have transformed the landscape of genome editing. However, the absence of a robust and safe CRISPR delivery method continues to limit its potential for therapeutic applications. Despite the emergence of various methodologies aimed at addressing this challenge, issues regarding efficiency and editing operations persist. We introduce a microfluidic gene delivery system, called droplet cell pincher (DCP), designed for highly efficient and safe genome editing. This approach combines droplet microfluidics with cell mechanoporation, enabling encapsulation and controlled passage of cells and CRISPR systems through a microscale constriction. Discontinuities created in cell and nuclear membranes upon passage facilitate the rapid CRISPR-system internalization into the nucleus. We demonstrate the successful delivery of various macromolecules, including mRNAs (~98%) and plasmid DNAs (~91%), using this platform, underscoring the versatility of the DCP and leveraging it to achieve successful genome engineering through CRISPR–Cas9 delivery. Our platform outperforms electroporation, the current state-of-the-art method, in three key areas: single knockouts (~6.5-fold), double knockouts (~3.8-fold), and knock-ins (~3.8-fold). These results highlight the potential of our platform as a next-generation tool for CRISPR engineering, with implications for clinical and biological cell-based research. Current approaches for the delivery of CRISPR systems into cells can be hampered by several limitations, including low delivery potential and inefficient genome editing. Here, the authors present Droplet Cell Pincher (DCP), a gene delivery system that combines droplet microfluidics with cell mechanoporation, which attains efficient genome engineering. [ABSTRACT FROM AUTHOR]

Cell communication coordinates developmental processes, maintains homeostasis, and contributes to disease. Therefore, understanding the relationship between cells in a shared environment is crucial. Here we introduce Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours (PUFFFIN), a cell neighbour-labelling system based upon secretion and uptake of positively supercharged fluorescent protein s36GFP. We fused s36GFP to mNeonGreen or to a HaloTag, facilitating ultra-bright, sensitive, colour-of-choice labelling. Secretor cells transfer PUFFFIN to neighbours while retaining nuclear mCherry, making identification, isolation, and investigation of live neighbours straightforward. PUFFFIN can be delivered to cells, tissues, or embryos on a customisable single-plasmid construct composed of interchangeable components with the option to incorporate any transgene. This versatility enables the manipulation of cell properties, while simultaneously labelling surrounding cells, in cell culture or in vivo. We use PUFFFIN to ask whether pluripotent cells adjust the pace of differentiation to synchronise with their neighbours during exit from naïve pluripotency. PUFFFIN offers a simple, sensitive, customisable approach to profile non-cell-autonomous responses to natural or induced changes in cell identity or behaviour. Synopsis: While there are some options for labeling cellular neighbours, they often have low flexibility, require modification of multiple cells with different plasmids, or are otherwise technically challenging. To overcome these limitations, this study develops Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours (PUFFFIN), a method based on secretion and receptor-independent uptake of a positively charged fluorescent fusion molecule. PUFFFIN uses a universal uptake mechanism to deliver fluorescent label to neighbouring cells without the need to engineer them with label-uptake machinery. All PUFFFIN components are encoded on a single modular plasmid, amenable to delivery by transfection, electroporation, or lentivirus. PUFFFIN enables ultra-bright colour-of-choice labelling for unambiguous and rapid labelling of neighbours in cell culture or in vivo. PUFFFIN labelling does not compromise viability, enabling functional analysis of live neighbours. PUFFFIN labelling is used to demonstrate that pluripotent cells adjust the pace of differentiation to synchronise with their neighbours. Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours, or PUFFFIN, allows visualization and isolation of surrounding cells in culture, tissues, or embryos to profile non-cell-autonomous responses. [ABSTRACT FROM AUTHOR]

Copyright of Food Research & Development is the property of Food Research & Development Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Feline histiocytic diseases are uncommon and rarely reported. Feline progressive histiocytosis (FPH) is the most common histiocytic disease in cats, predominantly affecting middle-aged animals. The most common presentation is the cutaneous form with solitary or multiple cutaneous nodules. A female, mixed-breed 6-year-old cat was presented with a 9-month history of a nodule in the nasal planum and was diagnosed by histopathology with histiocytic proliferation. At the time of diagnosis, new nodules were discovered on the lower lip, digit, and two lesions in the tail region, with the largest measuring 1.5 cm. Supplementary immunohistochemistry, showed immunolabeling for Iba-1 that in combination with the clinical course of the disease, confirmed the diagnosis of FPH. No response to chemotherapy treatment with lomustine alternated with doxorubicin was achieved. Toceranib phosphate resulted in a transient response and, stable disease for a short period (6 weeks). Electrochemotherapy with bleomycin was initiated and resulted in partial remission. Later on, chlorambucil was also started. Ultimately, the combination of all three treatments led to a complete response and disappearance of all the lesions. FPH is considered a disease resistant to various treatments, and effective treatments have not been reported. In this case report, we describe a successful multimodal therapeutic approach that resulted in complete resolution of the FPH and long-term survival (460 days without external lesions at the time of death). Further studies are necessary to confirm the efficacy of this therapeutic approach. [ABSTRACT FROM AUTHOR]

As a widely used eukaryotic model organism, Neurosporacrassa offers advantages in genetic studies due to its diverse biology and rapid growth. Traditional genetic manipulation methods, such as homologous recombination, require a considerable amount of time and effort. In this study, we present an easy-to-use CRIPSR/Cas9 system for N.crassa, in which the cas9 sequence is incorporated into the fungal genome and naked guide RNA is introduced via electroporation. Our approach eliminates the need for constructing multiple vectors, speeding up the mutagenesis process. Using cyclosporin-resistant-1 (csr-1) as a selectable marker gene, we achieved 100% editing efficiency under selection conditions. Furthermore, we successfully edited the non-selectable gene N-acylethanolamineamidohydrolase-2 (naa-2), demonstrating the versatility of the system. Combining gRNAs targeting csr-1 and naa-2 simultaneously increased the probability of finding mutants carrying the non-selectable mutation. The system is not only user-friendly but also effective, providing a rapid and efficient method for generating loss-of-function mutants in N.crassa compared to traditional methods. [ABSTRACT FROM AUTHOR]

Background/aim: Griscelli Syndrome Type 2 (GS-2) is a rare, inherited immune deficiency caused by a mutation in the RAB27A gene. The current treatment consists of hematopoietic stem cell transplantation, but a lack of suitable donors warrants the development of alternative treatment strategies, including gene therapy. The development of mutation-specific clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 gene editing technology has opened the way for custom-designed gene correction of patientderived stem cells. In this study, we aimed to custom design CRISPR/Cas9 constructs and test their efficiency on homology-directed repair (HDR) on the correction of exon 3 and exon 7 mutations in the RAB27A gene of GS-2 patient-derived mesenchymal stem cells (MSCs) and induced pluripotent stem cells. Materials and methods: We assessed RAB27A gene and protein expression using qRT-PCR, Western Blot, and immune fluorescence in GS-2 patient-derived MSCs and induced pluripotent stem cells (iPSCs). Guide RNAs (gRNAs) and donor DNAs were designed based on patient mutations in exon 3 and exon 7 using the CHOPCHOP online tool and transfected into GS-2 MSCs and iPSCs by electroporation. The cells were cultured for 2 days and then used for mutation analysis using DNA sequencing. Results: MSCs and iPSCs from the GS-2 patients lacked RAB27A gene and protein expression. After gRNA and donor DNAs were designed and optimized, we found HDR efficiency with gRNA3.3 (10% efficiency) and gRNA7.3 (27% efficiency) for MSCs but lower efficiency in iPSCs (<5%). However, transfection of both MSCs and iPSCs resulted in massive cell death, loss of colony formation, and spontaneous differentiation. Conclusion: The use of CRISPR/Cas9 to genetically correct MSCs and iPSCs from GS-2 patients with different mutations through HDR is feasible but requires optimization of the procedure to reduce cell death and improve stem cell function before clinical application. [ABSTRACT FROM AUTHOR]

Objectives: Non-viral mediated plasmid DNA transfection by electroporation (EP) is an established method for gene transfection. In this study, the usefulness of direct EP at an intradermal (i.d.) site (DEP) with implanted electrodes to achieve a high protein expression level was investigated. In addition, DEP application with various intervals with a low application voltage was also evaluated to confirm its effect on protein expression. Methods: Green fluorescent protein (GFP)- and luciferase-encoding DNA were administrated, and GFP and luciferase were evaluated. Results: A higher protein expression level was observed after green fluorescent protein (GFP)- and luciferase-encoding DNA were delivered by i.d. injection followed by DEP application. When luciferase expression was observed with an in vivo imaging system, continuous expression was confirmed over 21 days after i.d. injection followed by DEP at 100 V. This approach provided increased gene expression levels compared with conventional EP methods via the stratum corneum layer. In addition, the effect of application voltage on luciferase expression was investigated; two-time applications (repeated DEP) at 20 V with 5 min intervals showed similar luciferase expression level to single DEP application with 100 V. Histological observations showed the skin became thicker after a single DEP at 100 V, whereas no apparent thickness changes were confirmed after repeated DEP at 20 V with 5 min intervals. Conclusions: This study revealed that direct i.d. voltage application achieved high protein expression levels even at low voltages. Skin is a promising administration site for DNA vaccines, so this approach may be effective for DNA vaccine delivery into skin tissue. [ABSTRACT FROM AUTHOR]

The phenomenon known as bipolar cancellation is observed when biphasic nanosecond electric field pulses are used, which results in reduced electroporation efficiency when compared to unipolar pulses of the same parameters. Basically, the negative phase of the bipolar pulse diminishes the effect of the positive phase. Our study aimed to investigate how bipolar cancellation affects Ca2+ electrochemotherapy and cellular response under varying electric field intensities and pulse durations (3–7 kV/cm, 100, 300, and 500 ns bipolar 1 MHz repetition frequency pulse bursts, n = 100). As a reference, standard microsecond range parametric protocols were used (100 µs × 8 pulses). We have shown that the cancellation effect is extremely strong when the pulses are closely spaced (1 MHz frequency), which results in a lack of cell membrane permeabilization and consequent failure of electrochemotherapy in vitro. To validate the observations, we have performed a pilot in vivo study where we compared the efficacy of monophasic (5 kV/cm × ↑500 ns × 100) and biphasic sequences (5 kV/cm × ↑500 ns + ↓500 ns × 100) delivered at 1 MHz frequency in the context of Ca2+ electrochemotherapy (B16-F10 cell line, C57BL/6 mice, n = 24). Mice treated with bipolar pulses did not exhibit prolonged survival when compared to the untreated control (tumor-bearing mice); therefore, the bipolar cancellation phenomenon was also occurrent in vivo, significantly impairing electrochemotherapy. At the same time, the efficacy of monophasic nanosecond pulses was comparable to 1.4 kV/cm × 100 µs × 8 pulses sequence, resulting in tumor reduction following the treatment and prolonged survival of the animals. [ABSTRACT FROM AUTHOR]

The utilization of electroporation for delivering CRISPR/Cas9 system components has enabled efficient gene editing in mammalian zygotes, facilitating the development of genome-edited animals. In this study, our research focused on targeting the ACTG1 and MSTN genes in sheep, revealing a threshold phenomenon in electroporation with a voltage tolerance in sheep in vitro fertilization (IVF) zygotes. Various poring voltages near 40 V and pulse durations were examined for electroporating sheep zygotes. The study concluded that stronger electric fields required shorter pulse durations to achieve the optimal conditions for high gene mutation rates and reasonable blastocyst development. This investigation also assessed the quality of Cas9/sgRNA ribonucleoprotein complexes (Cas9 RNPs) and their influence on genome editing efficiency in sheep early embryos. It was highlighted that pre-complexation of Cas9 proteins with single-guide RNA (sgRNA) before electroporation was essential for achieving a high mutation rate. The use of suitable electroporation parameters for sheep IVF zygotes led to significantly high mutation rates and heterozygote ratios. By delivering Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) to zygotes through electroporation, targeting the MSTN (Myostatin) gene, a knock-in efficiency of 26% was achieved. The successful generation of MSTN-modified lambs was demonstrated by delivering Cas9 RNPs into IVF zygotes via electroporation. [ABSTRACT FROM AUTHOR]

One of the recent advancements in the field of cardiac electrophysiology is pulsed field ablation (PFA). PFA is a novel energy modality that does not rely on thermal processes to achieve ablation which, in turn, results in limited collateral damage to surrounding structures. In this review, we discuss the mechanisms, safety, efficacy, and clinical applications of PFA for the management of atrial and ventricular arrhythmias. We also summarize the published pre-clinical and clinical studies regarding this new technology. [ABSTRACT FROM AUTHOR]

Pulsed-field ablation (PFA) is an emerging ablative technology that has been used successfully to eliminate cardiac arrhythmias. As a nonthermal technique, it has significant benefits over traditional radiofrequency ablation with improved target tissue specificity and reduced risk of adverse events during cardiac applications. We investigated whether PFA is safe for use in the stomach and whether it could modulate gastric slow waves. Female weaner pigs were fasted overnight before anesthesia was induced using tiletamine hydrochloride (50 mg·mL−1) and zolazepam hydrochloride (50 mg·mL−1) and maintained with propofol (Diprivan 2%, 0.2–0.4 mg·kg−1·min−1). Pulsed-field ablation was performed on their gastric serosa in vivo. Adjacent point lesions (n = 2–4) were used to create a linear injury using bipolar pulsed-field ablation consisting of 40 pulses (10 Hz frequency, 0.1 ms pulse width, 1,000 V amplitude). High-resolution electrical mapping defined baseline and postablation gastric slow-wave patterns. A validated five-point scale was used to evaluate tissue damage in hematoxylin and eosin-stained images. Results indicated that PFA successfully induced complete conduction blocks in all cases, with lesions through the entire thickness of the gastric muscle layers. Consistent postablation slow-wave patterns emerged immediately following ablation and persisted over the study period. Pulsed-field ablation induces rapid conduction blocks as a tool to modulate slow-wave patterns, indicating it may be suitable as an alternative to radiofrequency ablation. NEW & NOTEWORTHY: Results show that pulsed-field ablation can serve as a gastric slow-wave intervention by preventing slow-wave propagation across the lesion site. Stable conduction blocks were established immediately following energy delivery, faster than previous examples of radiofrequency gastric ablation. Pulsed-field ablation may be an alternative for gastric slow-wave intervention, and further functional and posthealing studies are now warranted. [ABSTRACT FROM AUTHOR]

Pulsed electrical field (PEF) treatment with 1 kJ/kg and 3 kJ/kg of energies was applied to yellow- and purple-fleshed potatoes before frying (180 °C for 2 min). The effects of PEF on oil content, colour and bioactive compounds were investigated. Significant oil reductions were observed for PEF-treated potatoes, ≥ 20% for yellow- and ≥ 24% for purple-fleshed potatoes, and this was also confirmed in the Fourier transform infrared (FTIR) graph. PEF-treated potatoes had significantly higher phenolic compounds and antioxidant activity than those of untreated samples. Moreover, although the frying reduced total monomeric anthocyanin (TMA) contents of the purple-fleshed potato samples compared to raw potato samples (not PEF-treated and not fried), PEF-treated samples had significantly higher TMA than the untreated (not PEF-treated but fried) samples. Raw samples and fried samples were separated according to PC1 (principal component 1) in chemometric analysis, and fried samples with and without PEF treatment were also separated according to PC2 (principal component 2). When PLS (partial least squares) regression graphs were examined, it was revealed that there was a very good correlation between the oil content and redness (a*) values calculated and estimated by FTIR spectroscopy. Our study showed that PEF treatment could be succesfully used for different varieties of potato before frying. [ABSTRACT FROM AUTHOR]

Electrochemotherapy (ECT) is a treatment modality that combines the electroporation of cell membranes with chemotherapy to facilitate the transport of non‐permeant molecules into cells. Several canine and feline studies have shown promising results, suggesting that ECT can be a valid adjuvant or alternative treatment option for some tumours. The objective of this paper is to provide a bibliographic review of the principles and applications of ECT in veterinary medicine and to compare to its use in human medicine. [ABSTRACT FROM AUTHOR]

BACKGROUND: Pulsed field ablation (PFA) is a novel technology for catheter-based atrial arrhythmia treatment. Evidence of its application for ventricular arrhythmia ablation is still limited. In this study, we describe the feasibility and efficacy of focal PFA for premature ventricular contraction (PVC) ablation. METHODS: A prospective cohort of 20 patients referred for PVC ablation at 2 centers was enrolled, regardless of the presence of structural heart disease, PVC morphology, or previous ablation attempts. All procedures were performed using the CENTAURI System in combination with contact force sensing catheters and 3-dimensional electroanatomical mapping systems. Energy output and the number of applications were left to the operator’s discretion. RESULTS: Eleven (55%) procedures were conducted under general anesthesia, 6 (30%) under deep sedation, and 3 (15%) under light sedation. Muscular contraction was observed in one case (5%). Median procedural and fluoroscopy times were 95.5 and 6.55 minutes, respectively. The median number of PFA applications was 8 with a median contact force of 10g. A statistically significant (76%) reduction was observed in mean peak-to-peak bipolar electrogram voltage before and after ablation (0.707 versus 0.098 mV; P=0.008). Ventricular irritative firing was observed in 11 (55%) patients after PFA. The median follow-up was 120 days. Acute procedural success was achieved in 17 of 20 (85% [95% CI, 0.70–1]) patients. Two of the patients with procedural failure had late success with >80% clinical PVC burden suppression during follow-up, and 2 of 17 patients with acute success had late PVC recurrence, which accounts for a total of 17 of 20 (85% [95% CI, 0.70–1]) patients with chronic success. Transient ST-segment depression occurred in 1 patient, and the right bundle branch block was induced in 2 others (permanently only in one case). CONCLUSIONS: PVC ablation using a focal PFA is feasible, effective, and safe, with promising acute and long-term results in several ventricular locations. Irritative firing is frequently observed. Coronary evaluation should be considered when targeting the outflow tract. [ABSTRACT FROM AUTHOR]

Achieving high cell transfection efficiency is essential for various cell types in numerous disease applications. However, the efficient introduction of genes into natural killer (NK) cells remains a challenge. In this study, we proposed a design strategy for delivering exogenous genes into the NK cell line, NK-92, using a modified non-viral gene transfection method. Calcium phosphate/DNA nanoparticles (pDNA-CaP NPs) were prepared using co-precipitation methods and combined with low-voltage pulse electroporation to facilitate NK-92 transfection. The results demonstrated that the developed pDNA-CaP NPs exhibited a uniform diameter of approximately 393.9 nm, a DNA entrapment efficiency of 65.8%, and a loading capacity of 15.9%. Furthermore, at three days post-transfection, both the transfection efficiency and cell viability of NK-92 were significantly improved compared to standalone plasmid DNA (pDNA) electroporation or solely relying on the endocytosis pathway of pDNA-CaP NPs. This study provides valuable insights into a novel approach that combines calcium phosphate nanoparticles with low-voltage electroporation for gene delivery into NK-92 cells, offering potential advancements in cell therapy. [ABSTRACT FROM AUTHOR]

Background: Nanosecond pulsed electric fields (nsPEF)‐based electroporation is a new therapy modality potentially synergized with radiation therapy to improve treatment outcomes. To verify its treatment accuracy intraoperatively, electroacoustic tomography (EAT) has been developed to monitor in‐vivo electric energy deposition by detecting ultrasound signals generated by nsPEFs in real‐time. However, utility of EAT is limited by image distortions due to the limited‐angle view of ultrasound transducers. Methods: This study proposed a supervised learning‐based workflow to address the ill‐conditioning in EAT reconstruction. Electroacoustic signals were detected by a linear array and initially reconstructed into EAT images, which were then fed into a deep learning model for distortion correction. In this study, 56 distinct electroacoustic data sets from nsPEFs of different intensities and geometries were collected experimentally, avoiding simulation‐to‐real‐world variations. Forty‐six data were used for model training and 10 for testing. The model was trained using supervised learning, enabled by a custom rotating platform to acquire paired full‐view and single‐view signals for the same electric field. Results: The proposed method considerably improved the image quality of linear array‐based EAT, generating pressure maps with accurate and clear structures. Quantitatively, the enhanced single‐view images achieved a low‐intensity error (RMSE: 0.018), high signal‐to‐noise ratio (PSNR: 35.15), and high structural similarity (SSIM: 0.942) compared to the reference full‐view images. Conclusions: This study represented a pioneering stride in achieving high‐quality EAT using a single linear array in an experimental environment, which improves EAT's utility in real‐time monitoring for nsPEF‐based electroporation therapy. [ABSTRACT FROM AUTHOR]

Administering gadolinium-based contrast agent before electroporation allows the contrast agent to enter the cells and enables MRI assessment of reversibly electroporated regions. The aim of this study was evaluation of contrast agent entrapment in Chinese hamster ovary (CHO) cells and comparison of these results with those determined by standard in vitro methods for assessing cell membrane permeability, cell membrane integrity and cell survival following electroporation. Cell membrane permeabilization and cell membrane integrity experiments were performed using YO-PRO-1 dye and propidium iodide, respectively. Cell survival experiments were performed by assessing metabolic activity of cells using MTS assay. The entrapment of gadolinium-based contrast agent gadobutrol inside the cells was evaluated using T1 relaxometry of cell suspensions 25 min and 24 h after electroporation and confirmed by inductively coupled plasma mass spectrometry. Contrast agent was detected 25 min and 24 h after the delivery of electric pulses in cells that were reversibly electroporated. In addition, contrast agent was present in irreversibly electroporated cells 25 min after the delivery of electric pulses but was no longer detected in irreversibly electroporated cells after 24 h. Inductively coupled plasma mass spectrometry showed a proportional decrease in gadolinium content per cell with shortening of T1 relaxation time (R2 = 0.88 and p = 0.0191). Our results demonstrate that the contrast agent is entrapped in cells exposed to reversible electroporation but exits from cells exposed to irreversible electroporation within 24 h, thus confirming the hypothesis on which detection experiments in vivo were based. [ABSTRACT FROM AUTHOR]

Electrochemotherapy (ECT) is an emerging therapeutic approach gaining growing interest for its potential immunomodulatory effects in cancer treatment. This narrative review systematically examines the current state of knowledge regarding the interplay between ECT and the immune system. Through an analysis of preclinical and clinical studies, the review highlights ECT capacity to induce immunogenic cell death, activate dendritic cells, release tumor antigens, trigger inflammatory responses, and occasionally manifest systemic effects—the abscopal phenomenon. These mechanisms collectively suggest the ECT potential to influence both local tumor control and immune responses. While implications for clinical practice appear promising, warranting the consideration of ECT as a complementary treatment to immunotherapy, the evidence remains preliminary. Consequently, further research is needed to elucidate the underlying mechanisms, optimize treatment protocols, explore potential synergies, and decipher the parameters influencing the abscopal effect. As the field advances, the integration of ECT’s potential immunomodulatory aspects into clinical practice will need careful evaluation and collaboration among clinical practitioners, researchers, and policymakers.

Abstract Immunotherapeutic drugs are promising medicines for cancer treatment. A potential candidate for immunotherapy is interleukin-12 (IL-12), a cytokine well known for its ability to mediate antitumor activity. We developed a plasmid encoding human IL-12 devoid of an antibiotic resistance gene (phIL12). For the approval of phase I clinical trials in basal cell carcinoma (BCC), the regulatory agency requires non-clinical in vivo testing of the pharmacodynamic, pharmacokinetic and toxicological properties of the plasmid. As human IL-12 is not biologically active in mice, a mouse ortholog of the plasmid phIL12 (pmIL12) was evaluated. The evaluation demonstrated the antitumor effectiveness of the protein accompanied by immune cell infiltration. The plasmid was distributed throughout the body, and the amount of plasmid diminished over time in all organs except the skin around the tumor. The therapy did not cause any detectable systemic toxicity. The results of the non-clinical evaluation demonstrated the safety and efficacy of the pmIL12/phIL12 GET, and on the basis of these results, approval was obtained for the initiation of a phase I clinical study in BCC.

Abstract Background Nanosecond pulsed electric fields (nsPEF)‐based electroporation is a new therapy modality potentially synergized with radiation therapy to improve treatment outcomes. To verify its treatment accuracy intraoperatively, electroacoustic tomography (EAT) has been developed to monitor in‐vivo electric energy deposition by detecting ultrasound signals generated by nsPEFs in real‐time. However, utility of EAT is limited by image distortions due to the limited‐angle view of ultrasound transducers. Methods This study proposed a supervised learning‐based workflow to address the ill‐conditioning in EAT reconstruction. Electroacoustic signals were detected by a linear array and initially reconstructed into EAT images, which were then fed into a deep learning model for distortion correction. In this study, 56 distinct electroacoustic data sets from nsPEFs of different intensities and geometries were collected experimentally, avoiding simulation‐to‐real‐world variations. Forty‐six data were used for model training and 10 for testing. The model was trained using supervised learning, enabled by a custom rotating platform to acquire paired full‐view and single‐view signals for the same electric field. Results The proposed method considerably improved the image quality of linear array‐based EAT, generating pressure maps with accurate and clear structures. Quantitatively, the enhanced single‐view images achieved a low‐intensity error (RMSE: 0.018), high signal‐to‐noise ratio (PSNR: 35.15), and high structural similarity (SSIM: 0.942) compared to the reference full‐view images. Conclusions This study represented a pioneering stride in achieving high‐quality EAT using a single linear array in an experimental environment, which improves EAT's utility in real‐time monitoring for nsPEF‐based electroporation therapy.

A general, self-consistent scheme for analyzing cellular electroporation for bio-medical applications is developed to probe realistic biological shapes and different length scales ranging from nanometers to hundreds of micrometers. The COMSOL Multiphysics suite is used with suitable embellishments to incorporate the details of the electroporation (EP) process and the inherent internal physics. The results are obtained for the voltage pulse driven electroporation for a Jurkat cell with mitochondria (as an example organelle) where spatial dimensions on the order of a few nanometers become important, to hundreds of cells (with Bacillus as an example) where collective effects and mutual interactions can dominate. Thus, scalable computing to generalized geometries with the ability to include complex organelles is made available. The results obtained for mitochondrial EP in Jurkat cells compare well with available data. In addition, quantitative predictions of field attenuation and shielding in Bacillus clusters are made, which point to highly nonuniform field distributions and a strong need to engineer novel electrode designs. [ABSTRACT FROM AUTHOR]

ABSTRACTIntroduction Nucleic acids represent a promising platform for creating vaccines. One disadvantage of this approach is its relatively low immunogenicity. Electroporation (EP) is an effective way to increase the DNA vaccines immunogenicity. However, due to the different configurations of devices used for EP, EP protocols optimization is required not only to enhance immunogenicity, but also to ensure greater safety and tolerability of the EP procedure.Area covered An data analysis for recent years on the DNA vaccines delivery against viral and parasitic infections using EP was carried out. The study of various EP physical characteristics, such as frequency, pulse duration, pulse interval, should be considered along with the immunogenic construct design and the site of delivery of the vaccine, through the study of the immunogenic and protective characteristics of the latter.Expert opinion Future research should focus on regulating the humoral and cellular response required for protection against infectious agents by modifying the EP protocol. Significant efforts will be directed to establishing the possibility of redirecting the immune response toward the Th1 or Th2 response by changing the EP physical parameters. It will allow for an individual selective approach during EP, depending on the pathogen type of an infectious disease.

A priority to facilitate the application of lipofection to generate genetically modified porcine embryos and animals will be the use of zona pellucida (ZP)-intact oocytes and zygotes. Recently, our group produced genetically modified embryos by lipofection of ZP-intact oocytes during in vitro fertilization (IVF). This study investigates the effect of two commercial lipofection reagents, Lipofectamine 3000 and Lipofectamine CRISPRMAX, on embryo development and mutation efficiency in ZP-intact porcine oocytes. We compared these reagents with the electroporation method and a control group using two sgRNAs targeting the CAPN3 and CD163 genes. The detrimental effects on cleavage rates were observed in both lipofection treatments compared to the control and electroporated groups. However, blastocyst rates were higher in the Lipofectamine 3000 group than in the electroporated group for both genes. Mutation parameters varied by target gene, with Lipofectamine 3000 achieving higher mutation rates for CD163, while all groups were similar for the CAPN3 gene. Overall efficiency was similar for both lipofectamines, confirming their feasibility for use. In addition, we evaluated the effect of coincubation time (4, 8, and 24 h) on IVF outcomes, embryo development, and mutation parameters. Results indicated that an 8-h coincubation period optimized fertilization and mutation efficiency without significant toxic effects. This study demonstrates that lipofection with either Lipofectamine 3000 or CRISPRMAX during IVF is an effective method for generating genetically modified porcine embryos without the need for specialized equipment or trained personnel, with efficiencies similar to or greater than electroporation. This study also highlights the importance of optimizing reagent selection and coincubation times. There is no difference between Lipofectamine 3000 and CRISPRMAXTM in terms of embryo development and mutation efficiency, and under our experimental conditions, the optimal coincubation time with lipofectamine is 8 h. • Lipofection during IVF is an effective method to generate mutant porcine embryos. • Lipofectamine 3000 and CRISPRMAX are similar in efficiency. • Lipofection maintains high blastocyst rates. • Optimal coincubation time with Lipofectamine is 8 h. [ABSTRACT FROM AUTHOR]

Background: Tumor-reactive T cells play a crucial role in anti-tumor responses, but T cells induced by DNA vaccination are time-consuming processes and exhibit limited anti-tumor efficacy. Therefore, we evaluated the anti-tumor effectiveness of reactive T cells elicited by electroporation (EP)-mediated DNA vaccine targeting epidermal growth factor receptor variant III (pEGFRvIII plasmid), in conjunction with adoptive cell therapy (ACT), involving the transfer of lymphocytes from a pEGFRvIII EP-vaccinated healthy donor. Methods: The validation of the established pEGFRvIII plasmid and EGFRvIII-positive cell model was confirmed through immunofluorescence and western blot analysis. Flow cytometry and cytotoxicity assays were performed to evaluate the functionality of antigen-specific reactive T cells induced by EP-mediated pEGFRvIII vaccines, ACT, or their combination. The anti-tumor effectiveness of EP-mediated pEGFRvIII vaccines alone or combined with ACT was evaluated in the B16F10-EGFRvIII tumor model. Results: EP-mediated pEGFRvIII vaccines elicited serum antibodies and a robust cellular immune response in both healthy and tumor-bearing mice. However, this response only marginally inhibited early-stage tumor growth in established tumor models. EP-mediated pEGFRvIII vaccination followed by adoptive transfer of lymphocytes from vaccinated healthy donors led to notable anti-tumor efficacy, attributed to the synergistic action of antigen-specific CD4+ Th1 cells supplemented by ACT and antigen-specific CD8+ T cells elicited by the EP-mediated DNA vaccination. Conclusions: Our preclinical studies results demonstrate an enhanced anti-tumor efficacy of EP-mediated DNA vaccination boosted with adoptively transferred, vaccinated healthy donor-derived allogeneic lymphocytes. [ABSTRACT FROM AUTHOR]

The lack of a widely accessible method for expressing genes of interest in wild-type embryos is a fundamental obstacle to understanding genetic regulation during embryonic development. In particular, only a few methods are available for introducing gene expression vectors into cells prior to neural tube closure, which is a period of drastic development for many tissues. In this study, we present a simple technique for injecting vectors into the amniotic cavity and allowing them to reach the ectodermal cells and the epithelia of endodermal organs of mouse embryos at E8.0 via in utero injection, using only a widely used optical fiber with an illuminator. Using this technique, retroviruses can be introduced to facilitate the labeling of cells in various tissues, including the brain, spinal cord, epidermis, and digestive and respiratory organs. We also demonstrated in utero electroporation of plasmid DNA into E7.0 and E8.0 embryos. Taking advantage of this method, we reveal the association between Ldb1 and the activity of the Neurog2 transcription factor in the mouse neocortex. This technique can aid in analyzing the roles of genes of interest during endo- and ectodermal development prior to neural tube closure. [Display omitted] • New gene transfection method for embryos at E7 and E8 before neural tube closure. • Only optical fiber and injector needed. • Gene transfection into progenitors in ecto- and endodermal tissues. • Useful to analyze the role of lethal genes during embryogenesis, such as Ldb1. [ABSTRACT FROM AUTHOR]

Protocols surrounding electroporation have long been based on trapezoidal (or near rectangular) pulsing of biological cells. Here, we revisit cellular electroporation for biomedical applications, including tumor treatment, based on a self-consistent electro-thermal analysis with sinusoidal RF excitation. Predictions for the evolution of pores and their surface angular distribution, as well as potential heating and temperature increases, are given. Our results show an optimum frequency range from 5 to 7 MHz to achieve increased mass transport without detrimental heating in Jurkat cells. Through parametrized frequency sweeps, this work establishes potential optimized regimes that could guide experimental and clinical protocols. More significantly, a possible frequency for porating healthy B-cells is predicted to be ∼2.5 MHz, with almost no poration at 7 MHz. This opens up the exciting possibility for treating malignant tissue with a well-tuned frequency range for bioeffects while minimizing deleterious effects on healthy cells and tissues. [ABSTRACT FROM AUTHOR]

Exceeding physiological limits of the cell membrane potential compromises structural integrity, enabling the passage of normally impermeant solutes and disrupting cell function. Electropermeabilization has been studied extensively at the cellular scale, but not at the individual membrane lesion level. We employed fast total internal reflection fluorescence (TIRF) imaging of Ca2+ entry transients to discern individual lesions in a hyperpolarized cell membrane and characterize their focality, thresholds, electrical conductance, and the lifecycle. A diffuse and momentary membrane permeabilization without a distinct pore formation was observed already at a -100 mV threshold. Polarizing down to -200 mV created focal pores with a low 50- to 300-pS conductance, which disappeared instantly once the hyperpolarization was removed. Charging to -240 mV created high-conductance (> 1 nS) pores which persisted for seconds even at zero membrane potential. With incremental hyperpolarization steps, persistent pores often emerged at locations different from those where the short-lived, low-conductance pores or diffuse permeabilization were previously observed. Attempts to polarize membrane beyond the threshold for the formation of persistent pores increased their conductance adaptively, preventing further potential build-up and "clamping" it at a certain limit (-270 ± 6 mV in HEK cells, -284 ± 5 mV in CHO cells, and -243 ± 9 mV in neurons). The data suggest a previously unknown role of electroporative lesions as a protective mechanism against a potentially fatal membrane overcharging and cell disintegration.

Abstract Introduction Radiation Therapy and IRreversible Electroporation for Intermediate Risk Prostate Cancer (RTIRE) is a phase II clinical trial testing combination of radiation therapy and irreversible electroporation for intermediate risk prostate cancer Background PCa is the most common non-cutaneous cancer in men and the second leading cause of cancer death in men. PCa treatment is associated with long term side effects including urinary, sexual, and bowel dysfunction. Management of PCa is based on risk stratification to prevent its overtreatment and associated treatment-related toxicity. There is increasing interest in novel treatment strategies, such as focal therapy, to minimize treatment associated morbidity. Focal therapy alone has yet to be included in mainstream guidelines, given ongoing concerns with potentially higher risk of recurrence. We hypothesize combining focal therapy with whole gland, reduced dose radiotherapy will provide acceptable oncologic efficacy with minimal treatment associated morbidity. RTIRE is a phase II single institution, investigator-initiated study combining a local ablative technique though local irreversible electroporation (IRE) with MR guided RT (MRgRT) to treat the entire prostate. The goal is to provide excellent oncologic outcomes and minimize treatment related side effects through leveraging benefits of locally ablative therapy with established radiation treatment techniques. Methods A total of 42 men with intermediate risk PCa per NCCN guidelines and focal grade group (GG) 2 or 3, Gleason Score (GS) 3 + 4 or GS 4 + 3, cancer in an MRI target will be enrolled. Patients with MRI visible foci of GG2/GG3 will undergo focal therapy with IRE of this lesion. Following successful focal therapy, patients will then undergo a course of reduced dose, whole gland MRgRT with either 32.5 Gy in 5 Fractions or 22 Gy in 2 fractions. The primary objective of the study is to determine safety. Secondary outcomes include evaluation of oncologic efficacy (as measured by the proportion of patients free of clinically significant cancer as defined as > Grade Group 1 at 1-year follow-up biopsy), imaging characteristics of patients pre and post RTIRE, impact on quality of life (QoL), and PSA kinetics. Discussion Combining IRE with a reduced dose radiotherapy may offer a new treatment paradigm for PCa by both reducing treatment effects of full dose radiotherapy and minimizing the risk of recurrence observed with focal therapy. Trial Registration Clinicaltrials.gov identifier: NCT05345444. Date of registration: April 25, 2022. Protocol Version: 6.0, July 7, 2023.

Background: The non-viral production of CAR-T cells through electroporation of transposon DNA plasmids is an alternative approach to lentiviral/retroviral methods. This method is particularly suitable for early-phase clinical trials involving novel types of CAR-T cells. The primary disadvantage of non-viral methods is the lower production efficiency compared to viral-based methods, which becomes a limiting factor for CAR-T production, especially in chemotherapy-pretreated lymphopenic patients. Methods: We describe a good manufacturing practice (GMP)-compliant protocol for producing CD19 and CD123-specific CAR-T cells based on the electroporation of transposon vectors. The lymphocytes were purified from the blood of patients undergoing chemotherapy for B-NHL or AML and were electroporated with piggyBac transposon encoding CAR19 or CAR123, respectively. Electroporated cells were then polyclonally activated by anti- CD3/CD28 antibodies and a combination of cytokines (IL-4, IL-7, IL-21). The expansion was carried out in the presence of irradiated allogeneic blood-derived mononuclear cells (i.e., the feeder) for up to 21 days. Results: Expansion in the presence of the feeder enhanced CAR-T production yield (4.5-fold in CAR19 and 9.3-fold in CAR123). Detailed flow-cytometric analysis revealed the persistence of early-memory CAR-T cells and a low vector-copy number after production in the presence of the feeder, with no negative impact on the cytotoxicity of feeder-produced CAR19 and CAR123 T cells. Furthermore, large-scale manufacturing of CAR19 carried out under GMP condi tions using PBMCs obtained from B-NHL patients (starting number=200x10e6 cells) enabled the production of >50x10e6 CAR19 in 7 out of 8 cases in the presence of the feeder while only in 2 out of 8 cases without the feeder. Conclusions: The described approach enables GMP-compatible production of sufficient numbers of CAR19 and CAR123 T cells for clinical application and provides the basis for non-viral manufacturing of novel experimental CAR-T cells that can be tested in early-phase clinical trials. This manufacturing approach can complement and advance novel experimental immunotherapeutic strategies against human hematologic malignancies. [ABSTRACT FROM AUTHOR]

By increasing the permeability of the cell membrane of the treated material, pulsed electric fields (PEF) enhance the internal transport of various chemical substances. Changing the distribution of these components can modify the chemical and thermal properties of the given material. This study aimed to analyze the impact of PEF (1 kV/cm; 1 and 4 kJ/kg) applied to strawberries prior to drying by various methods (convective, infrared-convective, microwave-convective, and vacuum) on the chemical and thermal properties of the obtained dried materials (sugars content, total phenolic content, and antioxidant capacity (ABTS and DPPH assays); thermal properties (TGA and DSC); and molecular composition (FTIR)). PEF could have induced and/or enhanced sucrose inversion because, compared to untreated samples, PEF-pretreated samples were characterized by a lower share of sucrose in the total sugar content but a higher share of glucose and fructose. Reduced exposure to oxygen and decreased drying temperature during vacuum drying led to obtaining dried strawberries with the highest content of antioxidant compounds, which are sensitive to these factors. All PEF-pretreated dried strawberries exhibited a lower glass transition temperature (Tg) than the untreated samples, which confirms the increased mobility of the system after the application of an electric field. [ABSTRACT FROM AUTHOR]

Efficient intracellular delivery is crucial for biotherapeutics, such as proteins, oligonucleotides, and CRISPR/Cas9 gene-editing systems, to achieve their efficacy. Despite the great efforts of developing new intracellular delivery carriers, the lack of straightforward methods for intracellular delivery quantification limits further development in this area. Herein, we designed a simple and versatile bioorthogonal luminescent reaction (BioLure assay) to analyze intracellular delivery. Our results suggest that BioLure can be used to estimate the amount of intracellularly delivered molecules after electroporation, and the estimation by BioLure is in good correlation with the results from complementary methods. Furthermore, we used BioLure assay to correlate the intracellularly-delivered RNase A amount with its tumoricidal activity. Overall, BioLure is a versatile tool for understanding the intracellular delivery process on live cells, and establishing the link between the cytosolic concentration of intracellularly-delivered biotherapeutics and their therapeutic efficacy. Efficient intracellular delivery is crucial for biotherapeutics to achieve their efficacy, however, evaluation of delivery efficiency remains challenging. Here, the authors design a bioorthogonal luminescent reaction to analyze intracellular delivery, showing the application in electroporation-mediated protein delivery. [ABSTRACT FROM AUTHOR]