The Dysferlinopathies Conundrum: Clinical Spectra, Disease Mechanism and Genetic Approaches for Treatments.

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.

Monitoring optoporated process on mammalian cells by real-time measurement of membrane resealing time.

Significance: Resealing time based loading efficiency of optoporation is the key parameter for drug or gene delivery. This work describes a comparatively simple optical approach to directly measure the cell membrane resealing time of the gold nanoparticle mediated photoporation. Aim: To establish a membrane potential detection optical system, which can provide a direct measurement of resealing time of the optoporated cells. Approach: Voltage sensitive dye has been used to label the gold nanoparticle covered cell before laser activation and the resealing time was estimated from the voltage change due to the fluorescence light intensity change before and after laser activation. The approach has been validated by the simulated data based on diffusion model and Monte Carlo simulation and the experimental data obtained from a flow cytometry analysis. Results: The measured resealing time after perforation varied from 28.6 to 163.8 s on Hela cells when the irradiation fluence was increased, with a correlation coefficient (R2) of 0.9938. This result is in agreement with the resealing time (1-2 min) of photothermal porated Hela cells measured by electrical impedance method. The intracellular delivery efficiency of extracellular macromolecular under the same irradiation fluence depends mainly on diffusion velocity rather than pore size. Conclusion: The method described here can be used to directly measure resealing time of optoporated cells for accurately estimating the loading efficiency on discovering the mechanism of optoporation.

Early Endosomes Act as Local Exocytosis Hubs to Repair Endothelial Membrane Damage.

The plasma membrane of a cell is subject to stresses causing ruptures that must be repaired immediately to preserve membrane integrity and ensure cell survival. Yet, the spatio-temporal membrane dynamics at the wound site and the source of the membrane required for wound repair are poorly understood. Here, it is shown that early endosomes, previously only known to function in the uptake of extracellular material and its endocytic transport, are involved in plasma membrane repair in human endothelial cells. Using live-cell imaging and correlative light and electron microscopy, it is demonstrated that membrane injury triggers a previously unknown exocytosis of early endosomes that is induced by Ca2+ entering through the wound. This exocytosis is restricted to the vicinity of the wound site and mediated by the endosomal soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) VAMP2, which is crucial for efficient membrane repair. Thus, the newly identified Ca2+ -evoked and localized exocytosis of early endosomes supplies the membrane material required for rapid resealing of a damaged plasma membrane, thereby providing the first line of defense against damage in mechanically challenged endothelial cells.

Landscape of Cellular Bioeffects Triggered by Ultrasound-Induced Sonoporation.

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.

Faster calcium recovery and membrane resealing in repeated sonoporation for delivery improvement.

In sonoporation-based macromolecular delivery, repetitive microbubble cavitation in the bloodstream results in repeated sonoporation of cells or sonoporation of non-sonoporated neighboring cells (i.e., adjacent to the sonoporated host cells). The resealing and recovery capabilities of these two types of sonoporated cells affect the efficiency and biosafety of sonoporation-based delivery. Therefore, an improved understanding of the preservation of viability in these sonoporated cells is necessary. Using a customized platform for single-pulse ultrasound exposure (pulse length 13.33 µs, peak negative pressure 0.40 MPa, frequency 1.5 MHz) and real-time recording of membrane perforation and intracellular calcium fluctuations (using propidium iodide and Fluo-4 fluorescent probes, respectively), spatiotemporally controlled sonoporation was performed to administer first and second single-site sonoporations of a single cell or single-site sonoporation of a neighboring cell. Two distinct intracellular calcium changes, reversible and irreversible calcium fluctuations, were identified in cells undergoing repeat reversible sonoporation and in neighboring cells undergoing reversible sonoporation. In addition to an increased proportion of reversible calcium fluctuations that occurred with repeated sonoporation compared with that in the initial sonoporation, repeated sonoporation resulted in significantly shorter calcium fluctuation durations and faster membrane resealing than that produced by initial sonoporation. Similarly, compared with those in sonoporated host cells, the intracellular calcium fluctuation recovery and membrane perforation resealing times were significantly shorter in sonoporated neighboring cells. These results demonstrated that the function recovery and membrane resealing capabilities after a second sonoporation or sonoporation of neighboring cells were potentiated in the short term. This could aid in sustaining the long-term viability of sonoporated cells, therefore improving delivery efficiency and biosafety. This investigation provides new insight into the resealing and recovery capabilities in re-sonoporation of sonoporated cells and sonoporation of neighboring cells and can help develop safe and efficient strategies for sonoporation-based drug delivery.

The resealing factor S100A11 interacts with annexins and extended synaptotagmin-1 in the course of plasma membrane wound repair.

After damage, cells repair their plasma membrane in an active process that is driven by Ca2+ entering through the wound. This triggers a range of Ca2+-regulated events such as the translocation of different Ca2+-binding proteins to the wound site which likely function in the repair process. The translocated proteins include Ca2+/phospholipid binding proteins of the annexin (ANX) family and S100A11, an EF hand-type Ca2+-binding protein which can interact with ANX. The molecular mechanism by which S100A11 mediates PM wound repair remains poorly understood although it likely involves interactions with ANX. Here, using S100A11 knockout endothelial cells and expression of S100A11 mutants, we show that endothelial S100A11 is essential for efficient plasma membrane wound repair and engages in Ca2+-dependent interactions with ANXA1 and ANXA2 through its C-terminal extension (residues 93-105). ANXA2 but not ANXA1 translocation to the wound is substantially inhibited in the absence of S100A11; however, the repair defect in S100A11 knockout cells is rescued by ectopic expression of an ANX interaction-defective S100A11 mutant, suggesting an ANX-independent role of S100A11 in membrane wound repair. In search for other interaction partners that could mediate this action of S100A11 we identify extended synaptotagmin 1 (E-Syt1), a protein tether that regulates endoplasmic reticulum-plasma membrane contact sites. E-Syt1 binds to S100A11 in the presence of Ca2+ and depletion of E-Syt1 interferes with wound site recruitment of S100A11 and proper membrane resealing. Thus, the role of S100A11 in membrane wound repair does not exclusively dependent on ANX interactions and a Ca2+-regulated S100A11-E-Syt1 complex acts as a yet unrecognized component of the membrane resealing machinery.

Calcium supplementation of bioinks reduces shear stress-induced cell damage during bioprinting.

During bioprinting, cells are suspended in a viscous bioink and extruded under pressure through small diameter printing needles. The combination of high pressure and small needle diameter exposes cells to considerable shear stress, which can lead to cell damage and death. Approaches to monitor and control shear stress-induced cell damage are currently not well established. To visualize the effects of printing-induced shear stress on plasma membrane integrity, we add FM 1-43 to the bioink, a styryl dye that becomes fluorescent when bound to lipid membranes, such as the cellular plasma membrane. Upon plasma membrane disruption, the dye enters the cell and also stains intracellular membranes. Extrusion of alginate-suspended NIH/3T3 cells through a 200µm printing needle led to an increased FM 1-43 incorporation at high pressure, demonstrating that typical shear stresses during bioprinting can transiently damage the plasma membrane. Cell imaging in a microfluidic channel confirmed that FM 1-43 incorporation is caused by cell strain. Notably, high printing pressure also impaired cell survival in bioprinting experiments. Using cell types of different stiffnesses, we find that shear stress-induced cell strain, FM 1-43 incorporation and cell death were reduced in stiffer compared to softer cell types and demonstrate that cell damage and death correlate with shear stress-induced cell deformation. Importantly, supplementation of the suspension medium with physiological concentrations of CaCl2greatly reduced shear stress-induced cell damage and death but not cell deformation. As the sudden influx of calcium ions is known to induce rapid cellular vesicle exocytosis and subsequent actin polymerization in the cell cortex, we hypothesize that calcium supplementation facilitates the rapid resealing of plasma membrane damage sites. We recommend that bioinks should be routinely supplemented with physiological concentrations of calcium ions to reduce shear stress-induced cell damage and death during extrusion bioprinting.

Tissue Barrier-on-Chip: A Technology for Reproducible Practice in Drug Testing.

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.

The role of intraamniotic inflammation in threatened midtrimester miscarriage.

BACKGROUND: The assessment and management of patients with threatened midtrimester miscarriage is a clinical challenge because the etiology of this condition is poorly understood. OBJECTIVE: This study aimed to examine the frequency of intraamniotic infection or inflammation and the effect of antibiotics in patients presenting with regular uterine contractions and intact membranes before 20 weeks of gestation. STUDY DESIGN: This retrospective study comprised patients who met the following criteria: (1) singleton gestation, (2) gestational age before 20 weeks, (3) the presence of regular uterine contractions confirmed by a tocodynamometer (8 or more contractions in 60 minutes), (4) intact amniotic membranes, and (5) transabdominal amniocentesis performed for the evaluation of the microbiologic and inflammatory status of the amniotic cavity. Samples of amniotic fluid were cultured for aerobic and anaerobic bacteria and genital mycoplasmas, and polymerase chain reaction was performed to detect Ureaplasma species. Amniotic fluid was tested for white blood cell counts and matrix metalloproteinase-8 concentrations to diagnose intraamniotic inflammation. Patients with intraamniotic inflammation, or intraamniotic infection, were treated with antibiotics (a combination of ceftriaxone, clarithromycin, and metronidazole). Treatment success was defined as the resolution of intraamniotic infection/inflammation at the follow-up amniocentesis or delivery after 34 weeks of gestation. RESULTS: 1) Intraamniotic inflammation was present in 88% (15/17) of patients, whereas infection was detectable in only 2 cases; 2) objective evidence of resolution of intraamniotic inflammation after antibiotic treatment was demonstrated in 100% (4/4) of patients who underwent a follow-up amniocentesis; 3) 30% (5/15) of women receiving antibiotics delivered after 34 weeks of gestation (3 of the 5 patients had a negative follow-up amniocentesis, and 2 of the women were without a follow-up amniocentesis); 4) the overall treatment success of antibiotics was 40% (6/15; 4 cases of objective evidence of resolution of intra-amniotic inflammation and 5 cases of delivery after 34 weeks of gestation). CONCLUSION: The prevalence of intraamniotic inflammation in patients who presented with a threatened midtrimester miscarriage was 88% (15/17), and, in most cases, microorganisms could not be detected. Antibiotic treatment, administered to patients with intraamniotic inflammation, was associated with either objective resolution of intraamniotic inflammation or delivery after 34 weeks of gestation in 40% (6/15) of the cases.

Nature of bilayer lipids affects membranes deformation and pore resealing during nanoparticle penetration.

Interactions of nanoparticles (NPs) with lipid membranes have enormous biological implications especially for gene delivery applications. In this work, using all-atom steered- and molecular dynamics simulations, we investigated deformation of lipid membranes and pore closure during a NP penetration process. Three membrane bilayer models built from 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), dipalmitoylphosphatidylcholine (DPPC) and dilauroylphosphatidylcholine (DLPC), and a NP formed by 2 short interfering RNA (siRNA) and 6 polyethylenimine (PEI) molecules were used. Our results showed that different membrane lipids could lead to differences in pore formation (symmetric vs. asymmetric), and could undergo different levels of pore-mediated flip-flops during the closure. DLPC showed the largest number of flip-flops among the three lipid membranes. In addition, introduction of hydrophobic linoleic acid (LA) substitution onto the PEIs was found to facilitate pore formation, since the long LA tails could insert themselves into the hydrophobic region of the membrane where the lipid tails were less aligned. Compared with DPPC, POPC and DLPC membranes had less alignment of lipid tails in the bilayer, which promoted the insertion of LA tails and hence NP entry into the cell. Our observations provide valuable insight into the membrane deformations and pore dynamics during NP penetration and will be important for the design of NP carriers for effective gene delivery.

Spatiotemporal Dynamics and Mechanisms of Actin Cytoskeletal Re-modeling in Cells Perforated by Ultrasound-Driven Microbubbles.

To develop new strategies for improving the efficacy and biosafety of sonoporation-based macromolecule delivery, it is essential to understand the mechanisms underlying plasma membrane re-sealing and function recovery of the cells perforated by ultrasound-driven microbubbles. However, we lack a clear understanding of the spatiotemporal dynamics of the disrupted actin cytoskeleton and its role in the re-sealing of sonoporated cells. Here we used a customized experimental setup for single-pulse ultrasound (133.33-µs duration and 0.70-MPa peak negative pressure) exposure to microbubbles and for real-time recording of single-cell (human umbilical vein endothelial cell) responses by laser confocal microscopic imaging. We found that in reversibly sonoporated cells, the locally disrupted actin cytoskeleton, which was spatially correlated with the perforated plasma membrane, underwent three successive phases (expansion; contraction and re-sealing; and recovery) to re-model and that each phase of the disrupted actin cytoskeleton was approximately synchronized with that of the perforated plasma membrane. Moreover, compared with the closing time of the perforated plasma membrane, the same time was used for the re-sealing of the actin cytoskeleton in mildly sonoporated cells and a longer time was required in moderately sonoporated cells. Further, the generation, directional migration, accumulation and re-polymerization of globular actin polymers during the three phases drove the re-modeling of the actin cytoskeleton. However, in irreversibly sonoporated cells, the actin cytoskeleton, which underwent expansion and no contraction, was progressively de-polymerized and could not be re-sealed. Finally, we found that intracellular calcium transients were essential for the recruitment of globular actin and the re-modeling of the actin cytoskeleton. These results provide new insight into the role of actin cytoskeleton dynamics in the re-sealing of sonoporated cells and serve to guide the design of new strategies for sonoporation-based delivery.

Targeted protein degradation by Trim-Away using cell resealing coupled with microscopic image-based quantitative analysis.

"Trim-Away" technology enables rapid degradation of endogenous proteins without prior modification of protein-coding genes or mRNAs through delivery of antibodies that target proteins of interest. Although this approach can be readily applied to almost any cytosolic protein, strategies for cytosolic antibody delivery have been limited to microinjection or electroporation, which require skill-dependent operation or specialized equipment. Thus, the development of antibody delivery methods that are convenient, scalable, and preferably do not require detachment of adherent cells is required to extend the versatility of the Trim-Away method. Here, we developed a cell resealing technique optimized for Trim-Away degradation, which uses the pore-forming toxin streptolysin O (SLO) to permeabilize the cell membrane and delivered the antibodies of interest into HEK293T, HeLa, and HK-2 cell lines. We demonstrated the ability of Trim-Away protein degradation using IKKα and mTOR as targets, and we showed the availability of the developed system in antibody screening for the Trim-Away method. Furthermore, we effectively coupled Trim-Away with cyclic immunofluorescence and microscopic image-based analysis, which enables single-cell multiplexed imaging analysis. Taking advantage of this new analysis strategy, we were able to compensate for low signal-to-noise due to cell-to-cell variation, which occurs in the Trim-Away method because of the heterogenous contents of the introduced antibody, target protein, and TRIM21 in individual cells. Therefore, the reported cell resealing technique coupled with microscopic image analysis enables Trim-Away users to elucidate target protein function and the effects of target protein degradation on various cellular functions in a more quantitative and precise manner.

Surface-associated antigen induces permeabilization of primary mouse B-cells and lysosome exocytosis facilitating antigen uptake and presentation to T-cells.

B-cell receptor (BCR)-mediated antigen internalization and presentation are essential for humoral memory immune responses. Antigen encountered by B-cells is often tightly associated with the surface of pathogens and/or antigen-presenting cells. Internalization of such antigens requires myosin-mediated traction forces and extracellular release of lysosomal enzymes, but the mechanism triggering lysosomal exocytosis is unknown. Here, we show that BCR-mediated recognition of antigen tethered to beads, to planar lipid-bilayers or expressed on cell surfaces causes localized plasma membrane (PM) permeabilization, a process that requires BCR signaling and non-muscle myosin II activity. B-cell permeabilization triggers PM repair responses involving lysosomal exocytosis, and B-cells permeabilized by surface-associated antigen internalize more antigen than cells that remain intact. Higher affinity antigens cause more B-cell permeabilization and lysosomal exocytosis and are more efficiently presented to T-cells. Thus, PM permeabilization by surface-associated antigen triggers a lysosome-mediated B-cell resealing response, providing the extracellular hydrolases that facilitate antigen internalization and presentation.

Actin Cytoskeletal Dynamics in Single-Cell Wound Repair.

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.

Ca2+ roles in electroporation-induced changes of cancer cell physiology: From membrane repair to cell death.

The combination of Ca2+ ions and electroporation has gained attention as potential alternative to electrochemotherapy. Ca2+ is an important component of the cell membrane repair system and its presence directly influences the dynamics of the pore cycle after electroporation which can be exploited for cancer therapies. Here, the influence of Ca2+ concentration is investigated on small molecule electrotransfer and release of Calcein from 4T1, MX-1, B16F10, U87 cancer cells after cell exposure to microsecond electric pulses. Moreover, we investigated simultaneous molecule electrotransfer and intracellular calcium ion influx when media was supplemented with different Ca2+ concentrations. Results show that increased concentrations of calcium ions reduce the electrotransfer of small molecules to different lines of cancer cells as well as the release of Calcein. These effects are related with an enhanced membrane repair mechanism. Overall, we show that the efficiency of molecular electrotransfer can be controlled by regulating Ca2+ concentration in the electroporation medium. For the first time, the cause of cancer cell death in vitro from 1 mM CaCl2 concentrations is related to the irreversible loss of Ca2+ homeostasis after cell electroporation. Our findings provide fundamental insight on the mechanisms of Ca2+ electroporation that might lead to improved therapeutic outcomes.

Phenothiazines alter plasma membrane properties and sensitize cancer cells to injury by inhibiting annexin-mediated repair.

Repair of damaged plasma membrane in eukaryotic cells is largely dependent on the binding of annexin repair proteins to phospholipids. Changing the biophysical properties of the plasma membrane may provide means to compromise annexin-mediated repair and sensitize cells to injury. Since, cancer cells experience heightened membrane stress and are more dependent on efficient plasma membrane repair, inhibiting repair may provide approaches to sensitize cancer cells to plasma membrane damage and cell death. Here, we show that derivatives of phenothiazines, which have widespread use in the fields of psychiatry and allergy treatment, strongly sensitize cancer cells to mechanical-, chemical-, and heat-induced injury by inhibiting annexin-mediated plasma membrane repair. Using a combination of cell biology, biophysics, and computer simulations, we show that trifluoperazine acts by thinning the membrane bilayer, making it more fragile and prone to ruptures. Secondly, it decreases annexin binding by compromising the lateral diffusion of phosphatidylserine, inhibiting the ability of annexins to curve and shape membranes, which is essential for their function in plasma membrane repair. Our results reveal a novel avenue to target cancer cells by compromising plasma membrane repair in combination with noninvasive approaches that induce membrane injuries.

A Novel Electroporation System for Living Cell Staining and Membrane Dynamics Interrogation.

A novel electroporation system was developed to introduce transient membrane pores to cells in a spatially and temporally controlled manner, allowing us to achieve fast electrotransfection and live cell staining as well as to systematically interrogate the dynamics of the cell membrane. Specifically, using this platform, we showed that both reversible and irreversible electroporation could be induced in the cell population, with nano-sized membrane pores in the former case being able to self-reseal in ~10 min. In addition, green fluorescent protein(GFP)-vinculin plasmid and 543 phalloidin have been delivered successively into fibroblast cells, which enables us to monitor the distinct roles of vinculin and F-actin in cell adhesion and migration as well as their possible interplay during these processes. Compared to conventional bulk electroporation and staining methods, the new system offers advantages such as low-voltage operation, cellular level manipulation and testing, fast and adjustable transfection/staining and real-time monitoring; the new system therefore could be useful in different biophysical studies in the future.

Correlation between the loss of intracellular molecules and cell viability after cell electroporation.

Control of membrane permeability to exogenous compounds by membrane electroporation can lead to cell death, which is related to permanent membrane damage, oxidation stress, leakage of intracellular molecules. In this study, we show that the predominant cell death modality after the application of high voltage electric pulses is related with inability to reseal of initial pores (first stage irreversible electroporation, FirEP). After moderately strong electric pulses, initial pores reseal, however, some cell still die later on due to electric field induced cell stress which leads to delayed cell death (late-stage irreversible electroporation, LirEP). According to our data, the period in which the majority of cells commit to either pore resealing or complete loss of barrier function depends on the intensity of electric field treatment but did not exceed 35 min. Additionally, we show that after electroporation using electric pulse parameters that induce LirEP, some cells can be rescued by supplementing medium with compounds obtained from irreversibly electroporated cells. We determined that the intracellular molecules that contribute to the increase of cell viability are larger than 30 kDa. This serves to prove that the loss of intracellular compounds plays a significant role in the decrease of cell viability after electroporation.

Extracellular-Ca2+-Induced Decrease in Small Molecule Electrotransfer Efficiency: Comparison between Microsecond and Nanosecond Electric Pulses.

Electroporation-a transient electric-field-induced increase in cell membrane permeability-can be used to facilitate the delivery of anticancer drugs for antitumour electrochemotherapy. In recent years, Ca2+ electroporation has emerged as an alternative modality to electrochemotherapy. The antitumor effect of calcium electroporation is achieved as a result of the introduction of supraphysiological calcium doses. However, calcium is also known to play a key role in membrane resealing, potentially altering the pore dynamics and molecular delivery during electroporation. To elucidate the role of calcium for the electrotransfer of small charged molecule into cell we have performed experiments using nano- and micro-second electric pulses. The results demonstrate that extracellular calcium ions inhibit the electrotransfer of small charged molecules. Experiments revealed that this effect is related to an increased rate of membrane resealing. We also employed mathematical modelling methods in order to explain the differences between the CaCl2 effects after the application of nano- and micro-second duration electric pulses. Simulation showed that these differences occur due to the changes in transmembrane voltage generation in response to the increase in specific conductivity when CaCl2 concentration is increased.

Risk factors and maternal outcomes following preterm premature rupture of membrane in the second trimester of gestation.

PURPOSE: To characterize the population of women who underwent mid-trimester preterm premature rupture of membrane (PPROM) in a country where mid-trimester abortions are legal and available. METHODS: A retrospective cross-sectional cohort study was conducted at a tertiary referral hospital, during 2013-2016. Mid-trimester defined as gestational age 13 + 0 to 23 + 6 weeks. Rupture of membrane was defined by documentation of fluid passing through the cervix on sterile speculum examination, and a positive Nitrazine (Bristol-Myers Squibb, Princeton, NJ) or erning test. All records were evaluated for medical history, laboratory data, postnatal examination, and autopsy findings, and a database was constructed. RESULTS: A total of 61 women were hospitalized for mid-trimester PPROM during the study period. Mean maternal age was 32 ± 5.98, range 20-45 years old. The majority (50, 82%) of patients decided to terminate their pregnancy before reaching the limit of viability at 24 weeks gestation. The overall prognosis of pregnancies reaching term was better than expected, with six (9.8%) patients delivering live babies and four of them born at term (36 ± 5 to 40 ± 6 weeks gestation), all after PPROM following amniocentesis or selective fetal reduction. A total of 60% of women with hypothyroidism had unbalanced TSH levels above 4.0 mIU/L prior to their pregnancy. A notable number of women (15, 24.6%) had PPROM following a pregnancy achieved by assisted reproductive technology (ART). CONCLUSIONS: Most women with diagnosed mid-trimester PPROM opted for pregnancy termination before the limit of viability when granted the choice. Possible risk factors for early PPROM are unbalanced hypothyroidism and ART. PPROM following amniocentesis can in some cases reseal and reach term, suggesting conservative treatment is a reasonable management for those cases.