Permeabilized Cells Research Articles

Temporal and Spatial Characterization of CUL3KLHL20-driven Targeted Degradation of BET family, BRD Proteins by the Macrocycle-based Degrader BTR2004.

Targeted protein degradation (TPD) is a promising new therapeutic modality that leverages the endogenous cellular protein degradation machinery of the ubiquitin-proteasome system (UPS) to degrade selected proteins. Recently, we developed a synthetic macrocycle ligand to recruit CUL3KLHL20 E3 ligase for TPD. Using this KLHL20 ligand, we constructed the PROTAC BTR2004, which demonstrated potent degradation of BET family proteins BRD 2, 3, and 4. As the TPD field expands, it is important to understand the cellular and biochemical properties of all utilized E3 ligases. Herein we report the temporal and spatial processes of BTR2004-facilitated BET family protein degradation by KLHL20: The target protein degradation kinetics, BTR2004 intracellular activity half-life, and the onset of BTR2004 cell permeabilization. Employing proximity ligation and confocal microscopy techniques, we also illustrate the subcellular location of the ternary complex assembly upon BTR2004 treatment. These characterizations provide further insight into the processes that govern TPD and features that could be incorporated when designing future PROTAC molecules.

Flow Cytometry Analysis of Tissue Factor Expression in Human Platelets.

A subset of circulating human platelets stores Tissue Factor (TF) intracellularly, the key activator of the blood coagulation cascade and thrombus formation. Upon platelet activation, TF is exposed on the cell membrane, where it binds to FVII, ultimately leading to thrombin generation. Considering that (1) levels of TF-positive platelets increase in various clinical settings, contributing to the patient's prothrombotic phenotype, and (2) different drugs can modulate platelet-associated TF expression, a standardized method for assessing TF-positive platelets is valuable, as its evaluation has been controversial in the past. Here, we outline a protocol for measuring the percentage of TF-positive platelets using flow cytometry in whole blood and platelet-rich plasma (PRP)/washed platelets. This protocol aims to provide detailed instructions for quantifying the percentage of TF-positive platelets by assessing the protein (1) intracellularly in resting conditions, and (2) on the cell surface, in both resting and activated conditions. The first section provides essential information for correctly performing blood withdrawal to ensure that pre-analytical procedures do not affect the results. Next, the protocol focuses on sample preparation and labeling procedures for flow cytometry analysis. Detailed steps for cell stimulation, labeling, fixation, and permeabilization -- where necessary -- are outlined. Finally, instructions for flow cytometry settings to correctly identify the platelet population and analyze TF-positive events are described. Lastly, the method includes the procedure for preparing PRP if TF-positive platelets are to be measured in isolated platelets. Since only a subset of platelets contains TF, it is important to ensure that these platelets are not lost during the centrifugation steps required to obtain PRP.

Integrated neural network and PSO hybrid approach for production of citrulline using immobilized permeabilized Pseudomonas furukawaii.

In the present study, nutraceutical citrulline was produced using immobilization of permeabilized whole cells of Pseudomonas furukawaii, an efficient producer of ADI. Since ADI is intracellularly localized, various additives such as SDS, Triton X-100, and EDTA were used to permeabilize the cell to improve substrate accessibility and ADI activity. The maximum ADI activity was observed with 0.25mg mL-1 biomass concentration treated with 0.5mmol L-1 EDTA for 15min using OFAT approach. Optimization of permeabilization conditions of P. furukawaii cells using novel neural networks and particle swarm optimization (PSO-NN) led to maximum ADI activity with 0.10mmol L-1 EDTA and 0.30mg mL-1 biomass. Further, the morphological characterization of immobilized cells was assessed by FESEM and FTIR. An optimal citrulline production of 1.19mmol L-1 was achieved at 2.5% sodium alginate with 20mmol L-1 arginine at 38°C, and 180min of incubation. The immobilized cells retained 90.3% productivity after seven reuse cycles. Thus, the formulated immobilized whole-cell biocatalyst, with higher stability offers cost-effective methods of citrulline production.

2-O-α-D-glucosyl glycerol production by whole-cell biocatalyst of lactobacilli encapsulating sucrose phosphorylase with improved glycerol affinity and conversion rate

Background2-O-α-D-glucosyl glycerol (2-αGG) is a valuable ingredient in cosmetics, health-care and food fields. Sucrose phosphorylase (SPase) is a favorable choice for biosynthesis of 2-αGG, while its glucosyl-acceptor affinity and thermodynamic feature remain largely unknown, limiting 2-αGG manufacturing.ResultsHere, three SPases were obtained from lactobacilli and bifidobacteria, and the one encoded by Lb. reuteri SDMCC050455 (LrSP) had the best transglucosylation ability, with 2-αGG accounting for 86.01% in the total product. However, the LrSP exhibited an initial forward reaction rate of 11.83/s and reached equilibrium of 56.90% at 110 h, indicating low glycerol affinity and conversion rate. To improve catalytic efficiency, the LrSP was overexpressed in Lb. paracasei BL-SP, of which the intracellular SPase activity increased by 6.67-fold compared with Lb. reuteri SDMCC050455. After chemically permeabilized with Triton X-100, the whole-cell biocatalysis of Lb. paracasei BL-SP was prepared and showed the highest activity, with the initial forward reaction rate improved to 50.17/s and conversion rate risen to 80.79% within 17 h. Using the whole-cell biocatalyst, the final yield of 2-αGG was 203.21 g/L from 1 M sucrose and 1 M glycerol.ConclusionThe food grade strain Lb. paracasei was used for the first time as cell factory to recombinantly express the LrSP and construct a whole-cell biocatalyst for the production of 2-αGG. After condition optimization and cell permeabilization, the whole-cell biocatalyst exhibited 23.89% higher equilibrium conversion and 9.10-fold of productivity compared with the pure enzyme catalytic system. This work would provide a reference for large-scale bioprocess of 2-αGG.

DDEL-18. OPTIMIZED METHOD TO QUANTIFY TTFIELDS-INDUCED PERMEABILIZATION OF CELL MEMBRANES

Abstract Tumor Treating Fields (TTFields) therapy is FDA-approved for glioblastoma. Using plate-based readouts of FITC-dextran fluorescent probes in U87 human glioblastoma cells, we previously reported increased uptake of FITC-dextrans (4-20kDa) post-TTFields exposure. However, this approach is unable to distinguish FITC-dextran uptake in live-vs-dead cells post-TTFields exposure. We demonstrate flow cytometry (FC) as a more robust and economical method to quantify the amount of FITC-dextrans entering the cells. We first identified the optimal [FITC-dextran] for the FC-based approach (titration 0-6.5mg/mL). Cells were then seeded onto coverslips and exposed to 200kHz TTFields, 1-3V/cm peak-to-peak (inovitro systemTM, Novocure), or no-TTFields (control), for 24-72h. Cells were harvested and stained for 1h using 4kDa or 20kDa FITC-dextrans. Cells were washed and analyzed by FC for mean±SD fluorescence intensity, and compared by unpaired t-test. Dead cells were excluded using propidium iodide staining. In the control vs. TTFields group, 4kDa FITC-dextrans had significantly increased uptake at 24h (4.9±0.3 vs. 8.0±1.7, p=0.01), 48h (6.5±0.2 vs. 8.3±1.0, p=0.01), and 72h (5.7±0.3 vs. 8.8±1.0, p=0.001). Likewise, in the TTFields group, the 20kDa FITC-dextrans had significantly increased uptake at 24h (5.6±0.3 vs. 7.5±0.9, p=0.006), 48h (4.8±0.3 vs. 6.6±0.8, p=0.006), and 72h (5.3±0.2 vs. 11.3±2.6, p=0.004). In the TTFields group, the 20kDa FITC-dextrans had significantly increased uptake between the 24h and 72h timepoints (p=0.03). The FC-based method utilized 0.36mg of FITC-dextrans/sample, much less compared to the 1-3mg recommended for plate-based methods. Another advantage of the FC-based method was exclusion of the dead cell population after TTFields exposure. We validated a FC-based-method that is economical and can reproducibly quantify the membrane permeabilization induced by TTFields. Compared to control conditions, TTFields increase membrane permeabilization of cells that remain alive after TTFields exposure, in a time-dependent manner for the 20kDa probe.

Overcoming fixation and permeabilization challenges in flow cytometry by optical barcoding and multi-pass acquisition.

The fixation and permeabilization of cells are essential for labeling intracellular biomarkers in flow cytometry. However, these chemical treatments often alter fragile targets, such as cell surface and fluorescent proteins (FPs), and can destroy chemically-sensitive fluorescent labels. This reduces measurement accuracy and introduces compromises into sample workflows, leading to losses in data quality. Here, we demonstrate a novel multi-pass flow cytometry approach to address this long-standing problem. Our technique utilizes individual cell barcoding with laser particles, enabling sequential analysis of the same cells with single-cell resolution maintained. Chemically-fragile protein markers and their fluorochrome conjugates are measured prior to destructive sample processing and adjoined to subsequent measurements of intracellular markers after fixation and permeabilization. We demonstrate the effectiveness of our technique in accurately measuring intracellular FPs and methanol-sensitive antigens and fluorophores, along with various surface and intracellular markers. This approach significantly enhances assay flexibility, enabling accurate and comprehensive cellular analysis without the constraints of conventional one-time measurement flow cytometry. This innovation paves new avenues in flow cytometry for a wide range of applications in immuno-oncology, stem cell research, and cell biology.

Monitoring ER Ca2+ by Luminescence with Low Affinity GFP-Aequorin Protein (GAP).

The endoplasmic reticulum (ER) is the main cellular reservoir of Ca2+, able to accumulate high amounts of calcium close to the millimolar range and to release it upon cell activation. Monitoring of Ca2+ dynamics within the ER lumen is best achieved using genetically encoded and targeted reporters. Luminescent probes based on the photoprotein aequorin have provided significant insight to measure subcellular Ca2+. Here we describe a robust and quantitative method based on the Ca2+ indicator of the GFP-Aequorin Protein (GAP) family, targeted to the ER lumen. A low Ca2+ affinity version of GAP, GAP1, carrying mutations in two EF-hands of aequorin, reconstituted with coelenterazine n has a reduced affinity for Ca2+ such that it conforms with the [Ca2+] values found in the ER and it slows the consumption of the probe by Ca2+. This feature is advantageous because it avoids fast aequorin consumption allowing long-term (longer than 1h) ER Ca2+ measurements. GAP1 targeted to the ER allows monitoring of resting [Ca2+]ER and Ca2+ dynamics in intact cells stimulated with IP3-produced agonists. In addition, GAP1 can record Ca2+ mobilization in permeabilized cells challenged with IP3. We also provide a detailed calibration procedure which allows to accurately convert the luminescence signal into [Ca2+]ER.

Lysosome Targeted Nanoparticle Aggregation Reverses Immunosuppressive Tumor Microenvironment for Cancer Immunotherapy.

Nanotechnology has proven its enormous application value in clinical practice. However, current research on nanomedicines mainly focuses on developing nanoparticles as delivery carriers to maximize the bioavailability of therapeutic agents, with little attention on exploring their potential to directly regulate physiological processes. In this study, inspired by the lysosomal swelling caused by excessive accumulation of undegraded substances, this work presents a lysosomal-targeting aggregated nanoparticle (LTANP) for cancer treatment. By rationally engineering surface composition, properties, and interparticle interactions, LTANP achieves efficient tumor accumulation and selective targeted aggregation in lysosomes of cancer cells, leading to unrelievable lysosomal swelling, and ultimately inducing lysosomal membrane permeabilization (LMP) of cancer cells. Further analysis shows that nanoparticle aggregation-mediated LMP can effectively trigger immunogenic cell death (ICD) by impairing autophagy-lysosome pathway, evoking robust antitumor immune responses and reversing tumor immunogenicity from "cold" to "hot" in a melanoma model. Additionally, LTANP can combine with clinically approved programmed death ligand-1 (PD-L1) antibodies to further unleash T cell-mediated antitumor immunity, significantly enhancing antitumor performance, inhibiting tumor recurrence and metastasis. This work demonstrates the potential of rationally engineered nanostructures in directly combating cancer and provides novel insights for the development of advanced nanoparticle-based cancer treatment.

Glucoraphanin and sulforaphane mitigate TNFα-induced Caco-2 monolayers permeabilization and inflammation

Intestinal permeabilization is central to the pathophysiology of chronic gut inflammation. This study investigated the efficacy of glucoraphanin (GR), prevalent in cruciferous vegetables, particularly broccoli, and its derivative sulforaphane (SF), in inhibiting tumor necrosis factor alpha (TNFα)-induced Caco-2 cell monolayers inflammation and permeabilization through the regulation of redox-sensitive events. TNFα binding to its receptor led to a rapid increase in oxidant production and subsequent elevation in the mRNA levels of NOX1, NOX4, and Duox2. GR and SF dose-dependently mitigated both these short- and long-term alterations in redox homeostasis. Downstream, GR and SF inhibited the activation of the redox-sensitive signaling cascades NF-κB (p65 and IKK) and MAPK ERK1/2, which contribute to inflammation and barrier permeabilization. GR (1 μM) and SF (0.5–1 μM) prevented TNFα-induced monolayer permeabilization and the associated reduction in the levels of the tight junction (TJ) proteins occludin and ZO-1. Both GR and SF also mitigated TNFα-induced increased mRNA levels of the myosin light chain kinase, which promotes TJ opening. Molecular docking suggests that although GR is mostly not absorbed, it could interact with extracellular and membrane sites in NOX1. Inhibition of NOX1 activity by GR would mitigate TNFα receptor downstream signaling and associated events. These findings support the concept that not only SF, but also GR, could exert systemic health benefits by protecting the intestinal barrier against inflammation-induced permeabilization, in part by regulating redox-sensitive pathways. GR has heretofore not been viewed as a biologically active molecule, but rather, the benign precursor of highly active SF. The consumption of GR and/or SF-rich vegetables or supplements in the diet may offer a means to mitigate the detrimental consequences of intestinal permeabilization, not only in disease states but also in conditions characterized by chronic inflammation of dietary and lifestyle origin.

A New Approach for Studying Poly(ADP-Ribose) Polymerase Inhibitors Using Permeabilized Adherent Cells.

Poly(ADP-ribose) polymerase (PARP) inhibitors have been proposed as pharmacological agents in the treatment of various diseases. Recently, factors and mechanisms responsible for regulating PARP catalytic activity have been identified, some of which can significantly influence the effectiveness of inhibitors of this enzyme. Inthis regard, it is important to develop new models and methods that would reflect the cellular context in which PARP functions. We proposed to use digitonin-permeabilized adherent cells to study poly(ADP-ribosyl)ation reaction (PARylation) in order to maintain the nuclear localization of PARP and to control the concentrations of its substrate (NAD+) and tested compounds in the cell. A specific feature of the approach is that before permeabilization, cellular PARP is converted to the DNA-bound state under conditions preventing premature initiation of the PARylation reaction. Experiments were carried out in rat H9c2 cardiomyoblasts. The activity of PARP in permeabilized cells was analyzed by measuring the immunofluorescence of the reaction product poly(ADP-ribose). The method was verified in the studies of PARP inhibition by the classic inhibitor 3-aminobenzamide and a number of new 7-methylguanine derivatives. One of them, 7,8-dimethylguanine, was found to be a stronger inhibitor compared to 7-methylguanine, due to a formation of additional hydrophobic contact with the protein. The proposed approach opens up new prospects for studying the mechanisms of PARP activity regulation in cells and can be used in high-throughput screening of PARP inhibitors.

A novel method for non-destructive efficient green recovery of multiple high value products from microalgal cells with enzymatically enhanced permeability

A method is developed for simultaneous extraction of lipids, hydrocarbons and carotenoids from primary algal biomass without the need for cell sacrifice. Enzymatically predigested cells (from previous experiments) with enhanced permeability were used, for ‘milking’ with biocompatible solvent. The continuous non-destructive extraction of lipids and carotenoids from an improved permeabilized microalgal cell using crude enzyme concoctions and biocompatible solvents has not been reported so far. Oocystis sp. cells showed considerable viability following enzymatic pretreatment and extraction using dodecane which was verified through cell viability assays, and chlorophyll content. HPLC analysis confirmed the presence of lutein and zeaxanthin. GC-MS analysis showed the presence of omega-3 alpha linolenic acid, PUFAs, and phenolics, in enzymatically digested microalgal cells. The content of fatty acids and esters in enzyme treated samples was 57.18 %, and the enzyme with dodecane treated samples showed 31.4 % fatty acids and esters content. Omega-3 linolenic acid comprised 11.66 % in enzyme treated cells and showed a marginal increase of 12.5 % in enzyme with dodecane treated cells. The method enables recovery of multiple compounds from a single biomass. In the present study, a cultivation strategy of microalgal cells with enhanced permeability, grown in biocompatible solvent is proposed, whereby maximum yield of multiple valuable metabolites can be obtained from a single biomass. The method simplifies downstream processing operations by circumventing cell harvesting and drying. The use of green extraction solvents reduces the overall carbon footprint of the process.

A bifunctional antibody conjugate marks the location of DNA binding proteins on deproteinized DNA fibers.

ABSTRACTImmunofluorescent foci of DNA Damage Response (DDR) proteins serve as surrogates for DNA damage and are frequently interpreted as denoting specific lesions. For example, Double Strand Breaks (DSBs) are potent inducers of the DDR, whose best-known factor is the phosphorylated histone variant H2AX (γ-H2AX). The association with DSBs is so well established that the reverse interpretation that γ-H2AX invariably implies DSBs is routine. However, this conclusion is inferential and has been challenged. The resolution of this question has been hampered by the lack of methods for distinguishing the location of DDR proteins relative to DSBs caused by sequence indifferent agents. Here, we describe an approach for marking the location of DDR factors in relation to DSBs on DNA fibers. We synthesized a two-arm “Y” conjugate containing biotin and trimethylpsoralen (TMP) coupled to a secondary antibody. After exposure to a DNA breaker, permeabilized mammalian cells were incubated with a primary antibody against the DDR factor followed by binding of the secondary antibody in the conjugate to the primary antibody. Exposure to longwave UV light covalently linked the psoralen to the DNA. DNA fibers were spread, and the immunofluorescence of the biotin tag denoted the location of the target protein.Abstract FigureGraphical abstract

Chiral Fluorescent Antifungal Azole Probes Detect Resistance, Uptake Dynamics, and Subcellular Distribution in Candida Species.

Azoles are essential for fungal infection treatment, yet the increasing resistance highlights the need for innovative diagnostic tools and strategies to revitalize this class of antifungals. We developed two enantiomers of a fluorescent antifungal azole probe (1 S and 1 R ), analyzing 60 Candida strains via live-cell microscopy. A database of azole distribution images in strains of Candida albicans, Candida glabrata, and Candida parapsilosis, among the most important pathogenic Candida species, was established and analyzed. This analysis revealed distinct populations of yeast cells based on the correlation between fluorescent probe uptake and cell diameter. Varied uptake levels and subcellular distribution patterns were observed in C. albicans, C. glabrata, and C. parapsilosis, with the latter displaying increased localization to lipid droplets. Comparison of the more potent fluorescent antifungal azole probe enantiomer 1 S with the moderately potent enantiomer 1 R highlighted time-dependent differences in the uptake profiles. The former displayed a marked elevation in uptake after approximately 150 min, indicating the time required for significant cell permeabilization to occur and its association with the azole's antifungal activity potency. Divergent uptake levels between susceptible and high efflux-based azole-resistant strains were detected, offering a rapid diagnostic approach for identifying azole resistance. This study highlights unique insights achievable through fluorescent antifungal azole probes, unraveling the complexities of azole resistance, subcellular dynamics, and uptake within fungal pathogens.

Overcoming fixation and permeabilization challenges in flow cytometry by optical barcoding and multi-pass acquisition.

The fixation and permeabilization of cells are essential for labeling intracellular biomarkers in flow cytometry. However, these chemical treatments often alter fragile targets, such as cell surface and fluorescent proteins, and can destroy chemically-sensitive fluorescent labels. This reduces measurement accuracy and introduces compromises into sample workflows, leading to losses in data quality. Here, we demonstrate a novel multi-pass flow cytometry approach to address this long-standing problem. Our technique utilizes individual cell barcoding with laser particles, enabling sequential analysis of the same cells with single-cell resolution maintained. Chemically-fragile protein markers and their fluorochrome conjugates are measured prior to destructive sample processing and adjoined to subsequent measurements of intracellular markers after fixation and permeabilization. We demonstrate the effectiveness of our technique in accurately measuring intracellular fluorescent proteins and methanol-sensitive antigens and fluorophores, along with various surface and intracellular markers. This approach significantly enhances assay flexibility, enabling accurate and comprehensive cell analysis without the constraints of conventional one-time measurement flow cytometry. This innovation paves new avenues in flow cytometry for a wide range of applications in immuno-oncology, stem cell research, and cell biology.

Role of charge in enhanced nuclear transport and retention of graphene quantum dots

The nuclear pore complexes on the nuclear membrane serve as the exclusive gateway for communication between the nucleus and the cytoplasm, regulating the transport of various molecules, including nucleic acids and proteins. The present work investigates the kinetics of the transport of negatively charged graphene quantum dots through nuclear membranes, focusing on quantifying their transport characteristics. Experiments are carried out in permeabilized HeLa cells using time-lapse confocal fluorescence microscopy. Our findings indicate that negatively charged graphene quantum dots exhibit rapid transport to the nuclei, involving two distinct transport pathways in the translocation process. Complementary experiments on the nuclear import and export of graphene quantum dots validate the bi-directionality of transport, as evidenced by comparable transport rates. The study also shows that the negatively charged graphene quantum dots possess favorable retention properties, underscoring their potential as drug carriers.

Threshold Interphase Delay for Bipolar Pulses to Prevent Cancellation Phenomenon during Electrochemotherapy.

Electroporation-based procedures employing nanosecond bipolar pulses are commonly linked to an undesirable phenomenon known as the cancelation effect. The cancellation effect arises when the second pulse partially or completely neutralizes the effects of the first pulse, simultaneously diminishing cells' plasma membrane permeabilization and the overall efficiency of the procedure. Introducing a temporal gap between the positive and negative phases of the bipolar pulses during electroporation procedures may help to overcome the cancellation phenomenon; however, the exact thresholds are not yet known. Therefore, in this work, we have tested the influence of different interphase delay values (from 0 ms to 95 ms) using symmetric bipolar nanoseconds (300 and 500 ns) on cell permeabilization using 10 Hz, 100 Hz, and 1 kHz protocols. As a model mouse hepatoma, the MH-22a cell line was employed. Additionally, we conducted in vitro electrochemotherapy with cisplatin, employing reduced interphase delay values (0 ms and 0.1 ms) at 10 Hz. Cell plasma membrane permeabilization and viability dependence on a variety of bipolar pulsed electric field protocols were characterized. It was shown that it is possible to minimize bipolar cancellation, enabling treatment efficiency comparable to monophasic pulses with identical parameters. At the same time, it was highlighted that bipolar cancellation has a significant influence on permeabilization, while the effects on the outcome of electrochemotherapy are minimal.

Dynamin-2 mutations linked to neonatal-onset centronuclear myopathy impair exocytosis and endocytosis in adrenal chromaffin cells.

Dynamins are large GTPases whose primary function is not only to catalyze membrane scission during endocytosis but also to modulate other cellular processes, such as actin polymerization and vesicle trafficking. Recently, we reported that centronuclear myopathy associated dynamin-2 mutations, p.A618T, and p.S619L, impair Ca2+-induced exocytosis of the glucose transporter GLUT4 containing vesicles in immortalized human myoblasts. As exocytosis and endocytosis occur within rapid timescales, here we applied high-temporal resolution techniques, such as patch-clamp capacitance measurements and carbon-fiber amperometry to assess the effects of these mutations on these two cellular processes, using bovine chromaffin cells as a study model. We found that the expression of any of these dynamin-2 mutants inhibits a dynamin and F-actin-dependent form of fast endocytosis triggered by single action potential stimulus, as well as inhibits a slow compensatory endocytosis induced by 500 ms square depolarization. Both dynamin-2 mutants further reduced the exocytosis induced by 500 ms depolarizations, and the frequency of release events and the recruitment of neuropeptide Y (NPY)-labeled vesicles to the cell cortex after stimulation of nicotinic acetylcholine receptors with 1,1-dimethyl-4-phenyl piperazine iodide (DMPP). They also provoked a significant decrease in the Ca2+-induced formation of new actin filaments in permeabilized chromaffin cells. In summary, our results indicate that the centronuclear myopathy (CNM)-linked p.A618T and p.S619L mutations in dynamin-2 affect exocytosis and endocytosis, being the disruption of F-actin dynamics a possible explanation for these results. These impaired cellular processes might underlie the pathogenic mechanisms associated with these mutations.

Differential Expression Analysis of Cutaneous Squamous Cell Carcinoma and Basal Cell Carcinoma Proteomic Profiles Sampled with Electroporation-Based Biopsy

Clinical misdiagnosis between cutaneous squamous cell carcinoma (cSCC) and basal cell carcinoma (BCC) affects treatment plans. We report a tissue sampling approach with molecular biopsy using electroporation. This method, coined e-biopsy, enables non-destructive non-thermal permeabilization of cells in the skin for vacuum-assisted extraction of biomolecules. We used e-biopsy for ex vivo proteome extraction from 3 locations per patient in 21 cSCC, 20 BCC and 7 actinic keratosis human skin samples. Using liquid chromatography mass spectrometry (LC/MS/MS), we identified 5,966 proteins observed with non-zero intensity in at least one samples. The intra-patient Pearson correlation of 0.888 ± 0.065 for BCC patients; 0.858 ± 0.077 for SCC patients; 0.876 ± 0.116 for solar actinic keratosis (AK) patients indicates high consistency of the e-biopsy sampling. The mass spectra presented significantly different proteome profiles for cSCC, BCC and AK, with several hundreds of proteins differentially expressed. Notably, our study showed that proteomes sampled with e-biopsy from cSCC and BCC lesions are different, and that proteins of CRNN, SULT1E1 and ITPK1 genes are significantly overexpressed in BCC in comparison to cSCC. Our results provide evidence that the e-biopsy approach could potentially be used as a tool to support cutaneous lesions classification with molecular pathology.

Limiting the impact of protein leakage in single-cell proteomics.

Limiting artifacts during sample preparation can significantly increase data quality in single-cell proteomics experiments. Towards this goal, we characterize the impact of protein leakage by analyzing thousands of primary single cells that were prepared either fresh immediately after dissociation or cryopreserved and prepared at a later date. We directly identify permeabilized cells and use the data to define a signature for protein leakage. We use this signature to build a classifier for identifying damaged cells that performs accurately across cell types and species.

ComI inhibits transformation in Bacillus subtilis by selectively killing competent cells.

Natural transformation mechanisms have been studied across several bacterial systems, but few examples of inhibition exist. This work investigates the mechanism of action of a plasmid-encoded transmembrane inhibitor of natural transformation. The data reveal that the peptide can cause cell permeabilization. Permeabilization is synergistic with entry of Bacillus subtilis into the "competent" state, such that cells with the ability to be transformed are preferentially killed. These findings reveal a self-preservation mechanism coupled to the physiological state of the cells that ensures that the population can maintain an unaltered plasmid and its predicted prophage.