Search Results (29)

Cold atmospheric plasma (CAP) treatment induces cancer cell death through the generation of reactive oxygen and nitrogen species (RONS). However, the efficacy of RONS delivery into cells remains limited by membrane permeability. Here, we investigated whether combining CAP with pulsed electric fields (PEFs) could enhance cancer cell death through increased intracellular RONS uptake. HeLa cells were treated with argon atmospheric pressure plasma jet (Ar-APPJ), PEF, or their combination. The combined treatment showed significantly enhanced cell death compared to single treatments. While PEF treatment alone induced membrane permeabilization, the combination with Ar-APPJ resulted in more pronounced and sustained membrane disruption, as evidenced by increased calcein leakage. This enhanced effect was attributed to Ar-APPJ-induced lipid peroxidation interfering with membrane resealing after PEF-induced electroporation. We also demonstrated that PEF-induced membrane electroporation facilitates the intracellular uptake of CAP-generated RONS. These findings provide mechanistic insights into the synergistic effects of combined CAP and PEF treatments, suggesting enhanced cell death via multiple pathways. Full article

Background: Following injury, older adults exhibit slow recovery of muscle function. Age-related impairment of sarcolemmal membrane repair may contribute to myocyte death, increasing the need for myogenesis and prolonging recovery. Dietary fish oil (FO) is a common nutritional supplement that may alter plasma membrane composition to enhance the response to membrane injury. Methods: We assessed effects of an 8-week dietary intervention on muscle contractile recovery in aged (22 mo.) rats on control (n = 5) or FO (control + 33 g/kg FO (45% eicosapentaenoic acid; 10% docosahexaenoic acid); n = 5) diets 1-week after contusion injury, as well as adult (8 mo., n = 8) rats on the control diet. Results: Recovery was reduced in aged rats on the control diet vs. adults (63 vs. 80%; p = 0.042), while those on the FO diet recovered similarly to (78%) adults. To directly assess sarcolemma injury, C2C12 cells were cultured in media with and without FO (1, 10, and 100 μg/mL; 24 or 48 h) and injured with an infrared laser in medium containing FM4-64 dye as a marker of sarcolemmal injury. FO reduced the area under the FM4-64 fluorescence-time curve at all concentrations after both 24 and 48 h supplementation. Conclusions: These preliminary data suggest FO might aid recovery of muscle function following injury in older adults by enhancing membrane resealing and repair. Full article

Dysferlinopathies refer to a spectrum of muscular dystrophies that cause progressive muscle weakness and degeneration. They are caused by mutations in the DYSF gene, which encodes the dysferlin protein that is crucial for repairing muscle membranes. This review delves into the clinical spectra of dysferlinopathies, their molecular mechanisms, and the spectrum of emerging therapeutic strategies. We examine the phenotypic heterogeneity of dysferlinopathies, highlighting the incomplete understanding of genotype-phenotype correlations and discussing the implications of various DYSF mutations. In addition, we explore the potential of symptomatic, pharmacological, molecular, and genetic therapies in mitigating the disease’s progression. We also consider the roles of diet and metabolism in managing dysferlinopathies, as well as the impact of clinical trials on treatment paradigms. Furthermore, we examine the utility of animal models in elucidating disease mechanisms. By culminating the complexities inherent in dysferlinopathies, this write up emphasizes the need for multidisciplinary approaches, precision medicine, and extensive collaboration in research and clinical trial design to advance our understanding and treatment of these challenging disorders. Full article

Antimicrobial peptides are key components of the immune system. These peptides affect the membrane in various ways; some form nano-sized pores, while others only produce minor defects. Since these peptides are increasingly important in developing antimicrobial drugs, understanding the mechanism of their interactions with lipid bilayers is critical. Here, using atomic force microscopy (AFM), we investigated the effect of a synthetic hybrid peptide, CM15, on the membrane surface comprising E. coli polar lipid extract. Direct imaging of supported lipid bilayers exposed to various concentrations of the peptide revealed significant membrane remodeling. We found that CM15 interacts with supported lipid bilayers and forms membrane-spanning defects very quickly. It is found that CM15 is capable of remodeling both leaflets of the bilayer. For lower CM15 concentrations, punctate void-like defects were observed, some of which re-sealed themselves as a function of time. However, for CM15 concentrations higher than 5 µM, the defects on the bilayers became so widespread that they disrupted the membrane integrity completely. This work enhances the understanding of CM15 interactions with the bacterial lipid bilayer. Full article

Plasma membrane repair is an essential cellular mechanism that reseals membrane disruptions after a variety of insults, and compromised repair capacity can contribute to the progression of many diseases. Neurodegenerative diseases are marked by membrane damage from many sources, reduced membrane integrity, elevated intracellular calcium concentrations, enhanced reactive oxygen species production, mitochondrial dysfunction, and widespread neuronal death. While the toxic intracellular effects of these changes in cellular physiology have been defined, the specific mechanism of neuronal death in certain neurodegenerative diseases remains unclear. An abundance of recent evidence indicates that neuronal membrane damage and pore formation in the membrane are key contributors to neurodegenerative disease pathogenesis. In this review, we have outlined evidence supporting the hypothesis that membrane damage is a contributor to neurodegenerative diseases and that therapeutically enhancing membrane repair can potentially combat neuronal death. Full article

Pancreatic cancer has no symptoms until the disease has advanced and is aggressive cancer with early metastasis. Up to now, the only curative treatment is surgical resection, which is possible in the early stages of the disease. Irreversible electroporation treatment offers new hope for patients with unresectable tumors. Irreversible electroporation (IRE) is a type of ablation therapy that has been explored as a potential treatment for pancreatic cancer. Ablation therapies involve the use of energy to destroy or damage cancer cells. IRE involves using high-voltage, low-energy electrical pulses to create resealing in the cell membrane, causing the cell to die. This review summarizes experiential and clinical findings in terms of the IRE applications. As was described, IRE can be a non-pharmacological approach (electroporation) or combined with anticancer drugs or standard treatment methods. The efficacy of irreversible electroporation (IRE) in eliminating pancreatic cancer cells has been demonstrated through both in vitro and in vivo studies, and it has been shown to induce an immune response. Nevertheless, further investigation is required to assess its effectiveness in human subjects and to comprehensively understand IRE’s potential as a treatment option for pancreatic cancer. Full article

The plasma membrane (sarcolemma) of skeletal muscle myofibers is susceptible to injury caused by physical and chemical stresses during normal daily movement and/or under disease conditions. These acute plasma membrane disruptions are normally compensated by an intrinsic membrane resealing process involving interactions of multiple intracellular proteins including dysferlin, annexin, caveolin, and Mitsugumin 53 (MG53)/TRIM72. There is new evidence for compromised muscle sarcolemma repair mechanisms in Amyotrophic Lateral Sclerosis (ALS). Mitochondrial dysfunction in proximity to neuromuscular junctions (NMJs) increases oxidative stress, triggering MG53 aggregation and loss of its function. Compromised membrane repair further worsens sarcolemma fragility and amplifies oxidative stress in a vicious cycle. This article is to review existing literature supporting the concept that ALS is a disease of oxidative-stress induced disruption of muscle membrane repair that compromise the integrity of the NMJs and hence augmenting muscle membrane repair mechanisms could represent a viable therapeutic strategy for ALS. Full article

Sonoporation is the process of transient pore formation in the cell membrane triggered by ultrasound (US). Numerous studies have provided us with firm evidence that sonoporation may assist cancer treatment through effective drug and gene delivery. However, there is a massive gap in the body of literature on the issue of understanding the complexity of biophysical and biochemical sonoporation-induced cellular effects. This study provides a detailed explanation of the US-triggered bioeffects, in particular, cell compartments and the internal environment of the cell, as well as the further consequences on cell reproduction and growth. Moreover, a detailed biophysical insight into US-provoked pore formation is presented. This study is expected to review the knowledge of cellular effects initiated by US-induced sonoporation and summarize the attempts at clinical implementation. Full article

One key application of organ-on-chip systems is the examination of drug transport and absorption through native cell barriers such the blood–brain barrier. To overcome previous hurdles related to the transferability of existing static cell cultivation protocols and polydimethylsiloxane (PDMS) as the construction material, a chip platform with key innovations for practical use in drug-permeation testing is presented. First, the design allows for the transfer of barrier-forming tissue into the microfluidic system after cells have been seeded on porous polymer or Si3N4 membranes. From this, we can follow highly reproducible models and cultivation protocols established for static drug testing, from coating the membrane to seeding the cells and cell analysis. Second, the perfusion system is a microscopable glass chip with two fluid compartments with transparent embedded electrodes separated by the membrane. The reversible closure in a clamping adapter requires only a very thin PDMS sealing with negligible liquid contact, thereby eliminating well-known disadvantages of PDMS, such as its limited usability in the quantitative measurements of hydrophobic drug molecule concentrations. Equipped with tissue transfer capabilities, perfusion chamber inertness and air bubble trapping, and supplemented with automated fluid control, the presented system is a promising platform for studying established in vitro models of tissue barriers under reproducible microfluidic perfusion conditions. Full article

Localized and reversible plasma membrane disruption is a promising technique employed for the targeted deposition of exogenous therapeutic compounds for the treatment of disease. Indeed, the plasma membrane represents a significant barrier to successful delivery, and various physical methods using light, sound, and electrical energy have been developed to generate cell membrane perforations to circumvent this issue. To restore homeostasis and preserve viability, localized cellular repair mechanisms are subsequently triggered to initiate a rapid restoration of plasma membrane integrity. Here, we summarize the known emergency membrane repair responses, detailing the salient membrane sealing proteins as well as the underlying cytoskeletal remodeling that follows the physical induction of a localized plasma membrane pore, and we present an overview of potential modulation strategies that may improve targeted drug delivery approaches. Full article

The plasma membrane separates the interior of the cells from the extracellular fluid and protects the cell from disruptive external factors. Therefore, the self-repairing capability of the membrane is crucial for cells to maintain homeostasis and survive in a hostile environment. Here, we found that micron-sized membrane pores induced by cylindrical atomic force microscope probe puncture resealed significantly (~1.3–1.5 times) faster in drug-resistant non-small cell lung cancer (NSCLC) cell lines than in their drug-sensitive counterparts. Interestingly, we found that such enhanced membrane repairing ability was due to the overexpression of annexin in drug-resistant NSCLC cells. In addition, a further ~50% reduction in membrane resealing time (i.e., from ~23 s to ~13 s) was observed through the epithelial-mesenchymal-transition, highlighting the superior viability and potential of highly aggressive tumor cells using membrane resealing as an indicator for assessing the drug-resistivity and pathological state of cancer. Full article

Defects in membrane repair contribute to the development of muscular dystrophies, such as Miyoshi muscular dystrophy 1, limb girdle muscular dystrophy (LGMD), type R2 or R12. Deciphering membrane repair dysfunctions in the development of muscular dystrophies requires precise and detailed knowledge of the membrane repair machinery in healthy human skeletal muscle cells. Using correlative light and electron microscopy (CLEM), we studied the trafficking of four members of the annexin (ANX) family, in myotubes damaged by laser ablation. Our data support a model in which ANXA4 and ANXA6 are recruited to the disruption site by propagating as a wave-like motion along the sarcolemma. They may act in membrane resealing by proceeding to sarcolemma remodeling. On the other hand, ANXA1 and A2 exhibit a progressive cytoplasmic recruitment, likely by interacting with intracellular vesicles, in order to form the lipid patch required for membrane resealing. Once the sarcolemma has been resealed, ANXA1 is released from the site of the membrane injury and returns to the cytosol, while ANXA2 remains accumulated close to the wounding site on the cytoplasmic side. On the other side of the repaired sarcolemma are ANXA4 and ANXA6 that face the extracellular milieu, where they are concentrated in a dense structure, the cap subdomain. The proposed model provides a basis for the identification of cellular dysregulations in the membrane repair of dystrophic human muscle cells. Full article

Aims: Irreversible electroporation is an ablation technique being adapted for the treatment of atrial fibrillation. Currently, there are many differences reported in the in vitro and pre-clinical literature for the effective voltage threshold for ablation. The aim of this study is a direct comparison of different cell types within the cardiovascular system and identification of optimal voltage thresholds for selective cell ablation. Methods: Monophasic voltage pulses were delivered in a cuvette suspension model. Cell viability and live–dead measurements of three different neuronal lines, cardiomyocytes, and cardiac fibroblasts were assessed under different voltage conditions. The immediate effects of voltage and the evolution of cell death was measured at three different time points post ablation. Results: All neuronal and atrial cardiomyocyte lines showed cell viability of less than 20% at an electric field of 1000 V/cm when at least 30 pulses were applied with no significant difference amongst them. In contrast, cardiac fibroblasts showed an optimal threshold at 1250 V/cm with a minimum of 50 pulses. Cell death overtime showed an immediate or delayed cell death with a proportion of cell membranes re-sealing after three hours but no significant difference was observed between treatments after 24 h. Conclusions: The present data suggest that understanding the optimal threshold of irreversible electroporation is vital for achieving a safe ablation modality without any side-effect in nearby cells. Moreover, the evolution of cell death post electroporation is key to obtaining a full understanding of the effects of IRE and selection of an optimal ablation threshold. Full article

The plasma membrane protects the eukaryotic cell from its surroundings and is essential for cell viability; thus, it is crucial that membrane disruptions are repaired quickly to prevent immediate dyshomeostasis and cell death. Accordingly, cells have developed efficient repair mechanisms to rapidly reseal ruptures and reestablish membrane integrity. The cortical actin cytoskeleton plays an instrumental role in both plasma membrane resealing and restructuring in response to damage. Actin directly aids membrane repair or indirectly assists auxiliary repair mechanisms. Studies investigating single-cell wound repair have often focused on the recruitment and activation of specialized repair machinery, despite the undeniable need for rapid and dynamic cortical actin modulation; thus, the role of the cortical actin cytoskeleton during wound repair has received limited attention. This review aims to provide a comprehensive overview of membrane repair mechanisms directly or indirectly involving cortical actin cytoskeletal remodeling. Full article

In Part I, by using ³¹P-NMR spectroscopy, we have shown that isolated granum and stroma thylakoid membranes (TMs), in addition to the bilayer, display two isotropic phases and an inverted hexagonal (H_II) phase; saturation transfer experiments and selective effects of lipase and thermal treatments have shown that these phases arise from distinct, yet interconnectable structural entities. To obtain information on the functional roles and origin of the different lipid phases, here we performed spectroscopic measurements and inspected the ultrastructure of these TM fragments. Circular dichroism, 77 K fluorescence emission spectroscopy, and variable chlorophyll-a fluorescence measurements revealed only minor lipase- or thermally induced changes in the photosynthetic machinery. Electrochromic absorbance transients showed that the TM fragments were re-sealed, and the vesicles largely retained their impermeabilities after lipase treatments—in line with the low susceptibility of the bilayer against the same treatment, as reflected by our ³¹P-NMR spectroscopy. Signatures of H_II-phase could not be discerned with small-angle X-ray scattering—but traces of H_II structures, without long-range order, were found by freeze-fracture electron microscopy (FF-EM) and cryo-electron tomography (CET). EM and CET images also revealed the presence of small vesicles and fusion of membrane particles, which might account for one of the isotropic phases. Interaction of VDE (violaxanthin de-epoxidase, detected by Western blot technique in both membrane fragments) with TM lipids might account for the other isotropic phase. In general, non-bilayer lipids are proposed to play role in the self-assembly of the highly organized yet dynamic TM network in chloroplasts. Full article