Modulation of mechanosensitive genes during embryonic aortic arch development.

BACKGROUND: Early embryonic aortic arches (AA) are a dynamic vascular structures that are in the process of shaping into the great arteries of cardiovascular system. Previously, a time-lapsed mechanosensitive gene expression map was established for AA subject to altered mechanical loads in the avian embryo. To validate this map, we investigated effects on vascular microstructure and material properties following the perturbation of key genes using an in-house microvascular gene knockdown system. RESULTS: All siRNA vectors show a decrease in the expression intensity of desired genes with no significant differences between vectors. In TGFß3 knockdowns, we found a reduction in expression intensities of TGFß3 (≤76%) and its downstream targets such as ELN (≤99.6%), Fbn1 (≤60%), COL1 (≤52%) and COL3 (≤86%) and an increase of diameter in the left AA (23%). MMP2 knockdown also reduced expression levels in MMP2 (≤30%) and a 6-fold increase in its downstream target COL3 with a decrease in stiffness of the AA wall and an increase in the diameter of the AA (55%). These in vivo measurements were confirmed using immunohistochemistry, western blotting and a computational growth model of the vascular extracellular matrix (ECM). CONCLUSIONS: Localized spatial genetic modification of the aortic arch region governs the vascular phenotype and ECM composition of the embryo and can be integrated with mechanically-induced congenital heart disease models.

Treating scars after burns with pulsed electric fields in the rat model.

Reducing scar size after severe burn injuries is an important and challenging medical, technology and social problem. We have developed a battery-powered pulsed electric field (PEF) device and surface needle electrode applicator to deliver pulsed electric fields to the healing dorsal burn wound in rats. PEF was used to treat residual burn wounds caused by metal contact in rats starting 10 days after the injury for 4 months every 11 or 22 days for 4 months using varying time applied voltages at 250-350V range, 400mA current, 40 pulses, 70 µs duration each, delivered at pulse repetition frequency 10 Hz at 5 locations inside the wound. We found 40-45% reduction in the scar size in comparison with untreated controls in both upper and lower dorsal locations on rats' backs two months after the last PEF application. We have not detected significant histopathological differences in the center of the scars besides the thickness of the newly generated epidermis, which was thicker in the PEF treated group.We showed that minimally invasively applied pulsed electric fields through needle electrodes are effective method and device for treating residual burn wounds in the rat model, reducing the size of the resulting scars, without any adverse reaction.

Tuning the Locally Enhanced Electric Field Treatment (LEEFT) between Electrophysical and Electrochemical Mechanisms for Bacteria Inactivation.

Efficient drinking water disinfection methods are critical for public health. Locally enhanced electric field treatment (LEEFT) is an antimicrobial method that uses sharp structures, like metallic nanowires, to enhance the electric field at tips and cause bacteria inactivation. Electroporation is the originally designed mechanism of LEEFT. Although oxidation is typically undesired due to byproduct generation and electrode corrosion, it can enhance the overall disinfection efficiency. In this work, we conduct an operando investigation of LEEFT, in which we change the electrical parameters to tune the mechanisms between electrophysical electroporation and electrochemical oxidation. Pure electroporation (i.e., without detectable oxidation) could be achieved under a duty cycle of ≤0.1% and a pulse width of ≤2 µs. Applying 2 µs pulses at 7-8 kV/cm and 0.1% duty cycle results in 80-100% bacteria inactivation with pure electroporation. A higher chance of oxidation is found with a higher duty cycle and a longer pulse width, where the antimicrobial efficiency could also be enhanced. For water with a higher conductivity, a higher antimicrobial efficiency can be achieved under the same treatment conditions, and electrochemical reactions could be induced more easily. The findings shown in this work improve the fundamental understanding of LEEFT and help optimize the performance of LEEFT in real applications.

Well Plate-Based Localized Electroporation Workflow for Rapid Optimization of Intracellular Delivery.

Efficient and nontoxic delivery of foreign cargo into cells is a critical step in many biological studies and cell engineering workflows with applications in areas such as biomanufacturing and cell-based therapeutics. However, effective molecular delivery into cells involves optimizing several experimental parameters. In the case of electroporation-based intracellular delivery, there is a need to optimize parameters like pulse voltage, duration, buffer type, and cargo concentration for each unique application. Here, we present the protocol for fabricating and utilizing a high-throughput multi-well localized electroporation device (LEPD) assisted by deep learning-based image analysis to enable rapid optimization of experimental parameters for efficient and nontoxic molecular delivery into cells. The LEPD and the optimization workflow presented herein are relevant to both adherent and suspended cell types and different molecular cargo (DNA, RNA, and proteins). The workflow enables multiplexed combinatorial experiments and can be adapted to cell engineering applications requiring in vitro delivery. Key features • A high-throughput multi-well localized electroporation device (LEPD) that can be optimized for both adherent and suspended cell types. • Allows for multiplexed experiments combined with tailored pulse voltage, duration, buffer type, and cargo concentration. • Compatible with various molecular cargoes, including DNA, RNA, and proteins, enhancing its versatility for cell engineering applications. • Integration with deep learning-based image analysis enables rapid optimization of experimental parameters.

A simple method for gene expression in endo- and ectodermal cells in mouse embryos before neural tube closure.

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.

A high-titer scalable Chinese hamster ovary transient expression platform for production of biotherapeutics.

Transient gene expression (TGE) in Chinese hamster ovary (CHO) cells offers a route to accelerate biologics development by delivering material weeks to months earlier than what is possible with conventional cell line development. However, low productivity, inconsistent product quality profiles, and scalability challenges have prevented its broader adoption. In this study, we develop a scalable CHO-based TGE system achieving 1.9 g/L of monoclonal antibody in an unmodified host. We integrated continuous flow-electroporation and alternate tangential flow (ATF) perfusion to enable an end-to-end closed system from N-1 perfusion to fed-batch 50-L bioreactor production. Optimization of both the ATF operation for three-in-one application-cell growth, buffer exchange, and cell mass concentration-and the flow-electroporation process, led to a platform for producing biotherapeutics using transiently transfected cells. We demonstrate scalability up to 50-L bioreactor, maintaining a titer over 1 g/L. We also show comparable quality between both transiently and stably produced material, and consistency across batches. The results confirm that purity, charge variants and N-glycan profiles are similar. Our study demonstrates the potential of CHO-based TGE platforms to accelerate biologics process development timelines and contributes evidence supporting its feasibility for manufacturing early clinical material, aiming to strengthen endorsement for TGE's wider implementation.

Irreversible electroporation of localised prostate cancer downregulates immune suppression and induces systemic anti-tumour T-cell activation - IRE-IMMUNO study.

OBJECTIVES: To prospectively compare systemic anti-tumour immune responses induced by irreversible electroporation (IRE) and robot-assisted radical prostatectomy (RARP) in patients with localised intermediate-risk prostate cancer (PCa). PATIENTS AND METHODS: Between February 2021 and June 2022, before and after treatment (at 5, 14 and 30 days) peripheral blood samples of 30 patients with localised PCa were prospectively collected. Patient inclusion criteria were: International Society of Urological Pathologists Grade 2-3, clinical cancer stage ≤T2c, prostate-specific antigen level <20 ng/mL). Patients were treated with IRE (n = 20) or RARP (n = 10). Frequency and activation status of lymphocytic and myeloid immune cell subsets were determined using flow cytometry. PCa-specific T-cell responses to prostatic acid phosphatase (PSAP) and cancer testis antigen (New York oesophageal squamous cell carcinoma 1 [NY-ESO-1]) were determined by interferon-γ enzyme-linked immunospot assay (ELISpot). Repeated-measures analysis of variance and two-sided Student's t-tests were used to compare immune responses over time and between treatment cohorts. RESULTS: Patient and tumour characteristics were similar between the cohorts except for age (median 68 years [IRE] and 62 years [RARP], P = 0.01). IRE induced depletion of systemic regulatory T cells (P = 0.0001) and a simultaneous increase in activated cytotoxic T-lymphocyte antigen 4 (CTLA-4)+ cluster of differentiation (CD)4+ (P < 0.001) and CD8+ (P = 0.032) T cells, consistent with reduction of systemic immune suppression allowing for effector T-cell activation, peaking 14 days after IRE. Effects were positively correlated with tumour volume/ablation size. Accordingly, IRE induced expansion of PSAP and/or NY-ESO-1 specific T-cell responses in four of the eight immune competent patients. Temporarily increased activated myeloid derived suppressor cell frequencies (P = 0.047) were consistent with transient immunosuppression after RARP. CONCLUSIONS: Irreversible electroporation induces a PCa-specific systemic immune response in patients with localised PCa, aiding conversion of the tumour microenvironment into a more immune permissive state. Therapeutic efficacy might be further enhanced by combination with CTLA-4 checkpoint inhibition, potentially opening up a new synergistic treatment paradigm for high-risk localised or (oligo)metastatic disease.

Nasopharyngeal carcinoma cell screening based on nuclear targeting Surface-Enhanced Raman Scattering (SERS) detection.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant epithelial carcinoma arising from the nasopharyngeal mucosal lining. Diagnosis of NPC at early stage can improve the outcome of patients and facilitate reduction in cancer mortality. The most significant change between cancer cells and normal cells is the variation of cell nucleus. Therefore, accurately detecting the biochemical changes in nucleus between cancer cells and normal cells has great potential to explore diagnostic molecular markers for NPC. Highly sensitive surface-enhanced Raman scattering (SERS) could reflect the biochemical changes in the process of cell cancerization at the molecular level. However, rapid nuclear targeting SERS detection remains a challenge. RESULTS: A novel and accurate nuclear-targeting SERS detection method based on electroporation was proposed. With the assistance of electric pulses, nuclear-targeting nanoprobes were rapidly introduced into different NPC cells (including CNE1, CNE2, C666 cell lines) and normal nasopharyngeal epithelial cells (NP69 cell line), respectively. Under the action of nuclear localization signaling peptides (NLS), the nanoprobes entering cells were located to the nucleus, providing high-quality nuclear SERS signals. Hematoxylin and eosin (H&E) staining and in situ cell SERS imaging confirmed the excellent nuclear targeting performance of the nanoprobes developed in this study. The comparison of SERS signals indicated that there were subtle differences in the biochemical components between NPC cells and normal nasopharyngeal cells. Furthermore, SERS spectra combined with principal component analysis (PCA) and linear discriminant analysis (LDA) were employed to diagnose and distinguish NPC cell samples, and high sensitivity, specificity, and accuracy were obtained in the screening of NPC cells from normal nasopharyngeal epithelial cells. SIGNIFICANCE: To the best of our knowledge, this is the first study that employing nuclear-targeting SERS testing to screen nasopharyngeal carcinoma cells. Based on the electroporation technology, nanoprobes can be rapidly introduced into living cells for intracellular biochemical detection. Nuclear-targeting SERS detection can analyze the biochemical changes in the nucleus of cancer cells at the molecular level, which has great potential for early cancer screening and cytotoxicity analysis of anticancer drugs.

Rapid intracellular pH measurement based on electroporation- surface-enhanced Raman scattering.

In this study, electroporation-surface-enhanced Raman scattering (SERS) was applied to rapidly measure intracellular pH. The generation of a sensitive SERS probe for measuring pH in the range of 6.0-8.0 was accomplished through the conjugation of the pH-sensitive molecule 4-mercaptobenzoic acid (4-MBA) to the surface of gold nanoparticles (Au NPs) through its thiol functional group. This bioprobe was then rapidly introduced into nasopharyngeal carcinoma CNE-1 cells by electroporation, followed by SERS scanning and the fitting of intensity ratios of each detection point's Raman peaks at 1423 cm-1 and 1072 cm-1, to create the pH distribution map of CNE-1 cells. The electroporation-SERS assay introduces pH bioprobes into a living cell in a very short time and disperses the nanoprobe throughout the cytoplasm, ultimately enabling rapid and comprehensive pH analysis of the entire cell. Our work demonstrates the potential of electroporation-SERS for the biochemical analysis of live cells.

Radiation therapy and IRreversible electroporation for intermediate risk prostate cancer (RTIRE).

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.

Enhanced Cellular Doxorubicin Uptake via Delayed Exposure Following Nanosecond Pulsed Electric Field Treatment: An In Vitro Study.

The combination of nanosecond Pulsed Electric Field (nsPEF) with pharmaceuticals is a pioneering therapeutic method capable of enhancing drug uptake efficacy in cells. Utilizing nsPEFs configured at 400 pulses, an electric field strength of 15 kV/cm, a pulse duration of 100 ns, and a repetition rate of 10 pulses per second (PPS), we combined the nsPEF with a low dose of doxorubicin (DOX) at 0.5 µM. Upon verifying that cells could continuously internalize DOX from the surrounding medium within 1 h post nsPEF exposure, we set the DOX exposure period to 10 min and contrasted the outcomes of varying sequences of DOX and nsPEF administration: pulsing followed by DOX, DOX followed by pulsing, and DOX applied 40 min after pulsing. Flow cytometry, CCK-8 assays, and transmission electron microscopy (TEM) were employed to examine intracellular DOX accumulation, cell viability, apoptosis, cell cycle, and ultrastructural transformations. Our findings demonstrate that exposing cells to DOX 40 min subsequent to nsPEF treatment can effectively elevate intracellular DOX levels, decrease cell viability, and inhibit the cell cycle. This research work presents a novel approach to enhance DOX uptake efficiency with moderate conditions of both DOX and nsPEF.

Interplay between Electric Field Strength and Number of Short-Duration Pulses for Efficient Gene Electrotransfer.

Electroporation is a method that shows great promise as a non-viral approach for delivering genes by using high-voltage electric pulses to introduce DNA into cells to induce transient gene expression. This research aimed to evaluate the interplay between electric pulse intensity and 100 µs-duration pulse numbers as an outcome of gene electrotransfer efficacy and cell viability. Our results indicated a close relationship between pulse number and electric field strength regarding gene electrotransfer efficacy; higher electric pulse intensity resulted in fewer pulses needed to achieve the same gene electrotransfer efficacy. Subsequently, an increase in pulse number had a more negative impact on overall gene electrotransfer by significantly reducing cell viability. Based on our data, the best pulse parameters to transfect CHO cells with the pMax-GFP plasmid were using 5 HV square wave pulses of 1000 V/cm and 2 HV of 1600 V/cm, correspondingly resulting in 55 and 71% of transfected cells and maintaining 79 and 54% proliferating cells. This shows ESOPE-like 100 µs-duration pulse protocols can be used simultaneously to deliver cytotoxic drugs as well as immune response regulating genetically encoded cytokines.

Electroporation with Calcium or Bleomycin: First Application in an In Vivo Uveal Melanoma Patient-Derived Xenograft Model.

Uveal melanoma (UM) represents a rare tumor of the uveal tract and is associated with a poor prognosis due to the high risk of metastasis. Despite advances in the treatment of UM, the mortality rate remains high, dictating an urgent need for novel therapeutic strategies. The current study introduces the first in vivo analysis of the therapeutic potential of calcium electroporation (CaEP) compared with electrochemotherapy (ECT) with bleomycin in a patient-derived xenograft (PDX) model based on the chorioallantoic membrane (CAM) assay. The experiments were conducted as monotherapy with either 5 or 10 mM calcium chloride or 1 or 2.5 µg/mL bleomycin in combination with EP or EP alone. CaEP and ECT induced a similar reduction in proliferative activity, neovascularization, and melanocytic expansion. A dose-dependent effect of CaEP triggered a significant induction of necrosis, whereas ECT application of 1 µg/mL bleomycin resulted in a significantly increased apoptotic response compared with untreated tumor grafts. Our results outline the prospective use of CaEP and ECT with bleomycin as an adjuvant treatment of UM, facilitating adequate local tumor control and potentially an improvement in metastatic and overall survival rates.

Analysis of In Situ Electroporation Utilizing Induced Electric Field at a Wireless Janus Microelectrode.

In situ electroporation, a non-invasive technique for enhancing the permeability of cell membranes, has emerged as a powerful tool for intracellular delivery and manipulation. This method allows for the precise introduction of therapeutic agents, such as nucleic acids, drugs, and proteins, directly into target cells within their native tissue environment. Herein, we introduce an innovative electroporation strategy that employs a Janus particle (JP)-based microelectrode to generate a localized and controllable electric field within a microfluidic chip. The microfluidic device is engineered with an indium tin oxide (ITO)-sandwiched microchannel, where the electric field is applied, and suspended JP microelectrodes that induce a stronger localized electric field. The corresponding simulation model is developed to better understand the dynamic electroporation process. Numerical simulations for both single-cell and chain-assembled cell electroporation have been successfully conducted. The effects of various parameters, including pulse voltage, duration medium conductivity, and radius of Janus microelectrode, on cell membrane permeabilization are systematically investigated. Our findings indicate that the enhanced electric intensity near the poles of the JP microelectrode significantly contributes to the electroporation process. In addition, the distribution for both transmembrane voltage and the resultant nanopores can be altered by conveniently adjusting the relative position of the JP microelectrode, demonstrating a selective and in situ electroporation technique for spatial control over the delivery area. Moreover, the obtained differences in the distribution of electroporation between chain cells can offer insightful directives for the electroporation of tissues or cell populations, enabling the precise and targeted modulation of specific cell populations. As a proof of concept, this work can provide a robust alternative technique for the study of complex and personalized cellular processes.

Clustered synapses develop in distinct dendritic domains in visual cortex before eye opening.

Synaptic inputs to cortical neurons are highly structured in adult sensory systems, such that neighboring synapses along dendrites are activated by similar stimuli. This organization of synaptic inputs, called synaptic clustering, is required for high-fidelity signal processing, and clustered synapses can already be observed before eye opening. However, how clustered inputs emerge during development is unknown. Here, we employed concurrent in vivo whole-cell patch-clamp and dendritic calcium imaging to map spontaneous synaptic inputs to dendrites of layer 2/3 neurons in the mouse primary visual cortex during the second postnatal week until eye opening. We found that the number of functional synapses and the frequency of transmission events increase several fold during this developmental period. At the beginning of the second postnatal week, synapses assemble specifically in confined dendritic segments, whereas other segments are devoid of synapses. By the end of the second postnatal week, just before eye opening, dendrites are almost entirely covered by domains of co-active synapses. Finally, co-activity with their neighbor synapses correlates with synaptic stabilization and potentiation. Thus, clustered synapses form in distinct functional domains presumably to equip dendrites with computational modules for high-capacity sensory processing when the eyes open.

Consensus Guidelines of Irreversible Electroporation for Pancreatic Tumors: Protocol Standardization Using the Modified Delphi Technique.

Since no uniform treatment protocol for pancreatic irreversible electroporation (IRE) exists, the heterogeneity throughout literature complicates the comparison of results. To reach agreement among experts, a consensus study was performed. Eleven experts, recruited according to predefined criteria regarding previous IRE publications, participated anonymously in three rounds of questionnaires according to a modified Delphi technique. Consensus was defined as having reached ≥80% agreement. Response rates were 100, 64, and 64% in rounds 1 to 3, respectively; consensus was reached in 93%. Pancreatic IRE should be considered for stage III pancreatic cancer and inoperable recurrent disease after previous local treatment. Absolute contraindications are ventricular arrhythmias, implantable stimulation devices, congestive heart failure NYHA class 4, and severe ascites. The inter-electrode distance should be 10 to 20 mm and the exposure length should be 15 mm. After 10 test pulses, 90 treatment pulses of 1,500 V/cm should be delivered continuously, with a 90-µs pulse length. The first postprocedural contrast-enhanced computed tomography should take place 1 month post-IRE, and then every 3 months. This article provides expert recommendations regarding patient selection, procedure, and follow-up for IRE treatment in pancreatic malignancies through a modified Delphi consensus study. Future studies should define the maximum tumor diameter, response evaluation criteria, and the optimal number of preoperative FOLFIRINOX cycles.

Next-generation atrial fibrillation ablation: clinical performance of pulsed-field ablation and very high-power short-duration radiofrequency.

INTRODUCTION: Pulsed-field energy (PFA) and very high-power short-duration radiofrequency (vHPSD-RF) are two novel ablation methods for pulmonary vein isolation (PVI). Both PFA and vHPSD-RF show promise for improving efficacy, safety, and reducing procedure durations. However, direct comparisons between these two techniques are scarce. METHODS AND RESULTS: Retrospective analysis of 82 patients with symptomatic AF. Of these, 52 patients received PFA and 30 received vHPSD-RF (90 W, 4 s) as index procedure. At the 6-month follow-up, AF recurrence occurred in 4 patients following PFA and 5 patients following vHPSD-RF (p-value = 0.138). Significant improvements in the EHRA and NYHA stages were evident in both PFA (p < 0.001 and p = 0.047, respectively) and vHPSD-RF groups (p = 0.007 and p = 0.012, respectively). The total procedure duration and the left atrial dwell time were significantly shorter in the PFA group (64 ± 19 min vs. 99 ± 32 min, p < 0.001 and 41 ± 12 min vs. 62 ± 29 min, p < 0.001, respectively). The fluoroscopy time and dose area product were significantly higher in PFA (14 ± 6 vs. 9 ± 5 min, p < 0.001 and 14 ± 9 vs. 11 ± 9 Gy cm2, p = 0.046, respectively). One patient in the vHPSD-RF group suffered a stroke, not directly linked to the procedure (0 vs. 1 major complication, p = 0.366). CONCLUSION: Based on this retrospective single-center study, PFA and vHPSD-RF were associated with similar effectiveness and safety profiles. PFA was linked to shorter procedure times and higher radiation exposure compared to vHPSD-RF.

Development of a Shuttle Vector That Transforms at High Frequency for the Emerging Human Fungal Pathogen: Candida auris.

Routine molecular manipulation of any organism is inefficient and difficult without the existence of a plasmid. Although transformation is possible in C. auris, no plasmids are available that can serve as cloning or shuttle vectors. C. auris centromeres have been well characterized but have not been explored further as molecular tools. We tested C. auris centromeric sequences to identify which, if any, could be used to create a plasmid that was stably maintained after transformation. We cloned all seven C. auris centromeric sequences and tested them for transformation frequency and stability. Transformation frequency varied significantly; however, one was found to transform at a very high frequency. A 1.7 Kb subclone of this sequence was used to construct a shuttle vector. The vector was stable with selection and maintained at ~1 copy per cell but could be easily lost when selection was removed, which suggested that the properties of the centromeric sequence were more Autonomously Replicating Sequence (ARS)-like than centromere-like when part of a plasmid. Rescue of this plasmid from transformed C. auris cells into E. coli revealed that it remained intact after the initial C. auris transformation, even when carrying large inserts. The plasmid was found to be able to transform all four clades of C. auris, with varying frequencies. This plasmid is an important new reagent in the C. auris molecular toolbox, which will enhance the investigation of this human fungal pathogen.

Irreversible Electroporation Margin Accentuation in Pancreaticoduodenectomy: A Propensity Score Matching Analysis.

BACKGROUND: Margin accentuation using irreversible electroporation (MA-IRE) improves recurrence and overall survival (OS) in pancreatic cancer patients; however, there have been limited outcome comparisons to similarly risked patients who did not receive MA-IRE. METHODS: Patients with borderline resectable or locally advanced pancreatic adenocarcinoma who underwent a pancreaticoduodenectomy (PD) between 2017 and 2022 were included. Those who did not receive neoadjuvant chemotherapy for major vessel involvement were excluded. One-to-one propensity score matching (PSM) was used to match the MA-IRE group with the corresponding non-MA-IRE control group with similar risk factors. RESULTS: A total of 36 patients were included in this study. Seventeen (47.2%) patients who underwent MA-IRE matched with 19 control patients (52.8%) with similar risk factors who did not have MA-IRE. Before matching, OS and disease-free survival (DFS) were comparable between the MA-IRE and non-MA-IRE groups. After matching, the MA-IRE group showed improved OS (746 vs. 509 days, hazard ratio 0.313; p = 0.034) compared with the non-MA-IRE group. DFS (p = 0.768), negative margin status (p = 0.317), and 30-day complication rates (p = 1.000) remained statistically different between the groups. CONCLUSIONS: MA-IRE in PD results in longer OS but does not impact margin status, DFS, or postoperative complication rates in our cohort. These findings suggest that MA-IRE is safe and potentially promotes immune cell activation rather than upfront margin mitigation.

Description of outcome and adverse events in 21 cats with locally advanced nasal planum squamous cell carcinoma treated with electrochemotherapy.

OBJECTIVES: Squamous cell carcinoma (SCC) is the most common tumour in the nasal planum of cats. Surgery has traditionally been the treatment of choice but might not be feasible in locally advanced scenarios. Electrochemotherapy (ECT) has shown good control in superficial tumours, but there is a lack of robust information about efficacy in locally advanced cases. The aim of this study was to assess the safety and efficacy of ECT in the treatment of locally advanced stage nasal planum SCC in cats. METHODS: The clinical database of a veterinary referral hospital was searched retrospectively for cats diagnosed with a locally advanced nasal planum SCC (T3N0M0 or T4N0M0) that had received ECT. Local response, adverse events and outcome were documented. The data were evaluated by inferential statistics and correlations between response, recurrence, feline immunodeficiency virus/feline leukaemia virus status, number of treatments, voltage and severity of adverse events, with Kaplan-Meier curves and log-rank tests. Statistical significance was set at P <0.05. RESULTS: In total, 21 cats were enrolled over a 4-year period. Nineteen cats achieved a complete response (CR) and two cats a partial response (PR) for an overall response rate of 100%. Cats achieving a CR had a median disease-free interval of 182 days (range 128-327) and those with a PR had a median progression-free survival of 156.5 days (range 122-191). The median time to progression was not reached. The overall survival was 453 days for a median follow-up of 341 days (range 191-989). Of the cats, 62% had grade 3 or 4 toxicities, but no deaths due to the treatment were documented. Only voltage was correlated with longer survival (P = 0.001). CONCLUSIONS AND RELEVANCE: ECT appears to be an effective treatment for feline nasal planum SCC and could be considered a first-line therapy for locally advanced cases. Toxicities reported can be severe in the short term and these could be secondary to more invasive lesions and equipment used.