Isotonic Conditions Research Articles

Impact of surface-area-to-volume ratio, internal viscosity and membrane viscoelasticity on red blood cell deformability measured in isotonic condition

Osmotic gradient ektacytometry is the gold standard to assess red blood cell (RBC) deformability. It has been proposed that, when measured in isotonic condition, RBC deformability at low shear stress would depend on membrane elasticity while it would be influenced by internal viscosity when measured at high shear stress, but this hypothesis needs to be further addressed. Healthy RBCs were rigidified by treatment with lysolecithine (LPC), diamide or nystatine associated with hyperosmolar solutions (OSMO), which reduces membrane surface area, decreases membrane elasticity or promotes cell dehydration, respectively. Diamide treatment resulted in a decrease in isotonic RBC deformability at all shear stresses tested (i.e. from 0.3 to 30 Pa). LPC and OSMO treatments caused a decrease in isotonic RBC deformability above 3 Pa only. Isotonic RBC deformability from patients with hereditary spherocytosis or sickle cell disease was mainly decreased above 1.69 Pa. Our findings indicate that decreased isotonic RBC deformability at shear stresses above 3 Pa would be related to a reduction in the surface-area-to-volume ratio and/or to a loss of membrane elasticity and/or to an increase in internal viscosity while a decrease of RBC deformability below 3 Pa would reflect a loss of membrane elasticity.

Role of NFAT5 in the Immune System and Pathogenesis of Autoimmune Diseases.

The nuclear factor of activated T cells (NFAT5), also known as a tonicity-responsive enhancer-binding protein, was originally identified as a key transcription factor involved in maintaining cellular homeostasis against hypertonic and hyperosmotic environments. Although NFAT5 has been expressed and studied in various types of hyperosmolar tissues, evidence has emerged that NFAT5 plays a role in the development and activation of immune cells, especially T cells and macrophages. The immune-regulatory function of NFAT5 is achieved by inducing different target genes and different signaling pathways in both tonicity-dependent and -independent manners. Particularly in response to hyperosmotic stress, NFAT5 induces the generation of pathogenic TH17 cells and pro-inflammatory macrophages, contributing to autoimmune and inflammatory diseases. Meanwhile, with tonicity-independent stimuli, including activation of the Toll-like receptors and inflammatory cytokines, NFAT5 also can be activated and promotes immune cell survival, proliferation, migration, and angiogenesis. Moreover, under isotonic conditions, NFAT5 has been implicated in the pathogenesis of a variety of inflammatory and autoimmune diseases including rheumatoid arthritis. This review describes the current knowledge of NFAT5, focusing on its immune-regulatory functions, and it highlights the importance of NFAT5 as a novel therapeutic target for chronic inflammatory diseases.

Syringeable Self-Organizing Gels that Trigger Gold Nanoparticle Formation for Localized Thermal Ablation.

Block copolymer dispersions that form gels at body temperature and that additionally are able to reduce a gold salt to nanoparticles (AuNPs) directly in the final formulation under mild conditions were designed as hybrid depots for photothermal therapy. The in situ gelling systems may retain AuNPs in the application zone for a long time so that localized elevations of temperature can be achieved each time the zone is irradiated. To carry out the work, dispersions were prepared covering a wide range of poloxamine Tetronic 1307:gold salt molar ratios in NaCl media (also varying from pure water to hypertonic solution). Even at copolymer concentrations well above the critical micelle concentration, the reducing power of the copolymer was maintained, and AuNPs were formed in few hours without extra additives. Varying the copolymer and NaCl concentrations allowed a fine tuning of nanoparticles’ shape from spherical to triangular nanoplates, which determined that the surface plasmon resonance showed a maximum intensity at 540 nm or at 1000 nm, respectively. The information gathered on the effects of (i) the poloxamine concentration on AuNPs’ size and shape under isotonic conditions, (ii) the AuNPs on the temperature-induced gelling transition, and (iii) the gel properties on the photothermal responsiveness of the AuNPs during successive irradiation cycles may help the rational design of one-pot gels with built-in temperature and light responsiveness.

Abstract P296: High Salt (NaCl) Induces Metabolic Modifications and Affects Classic and Alternative Macrophage Activation

High intake of dietary sodium is a risk factor for cardiovascular disease. Previously, we have shown that an increase in extracellular hypertonic sodium (+40 mM Na + ; HS) inhibits alternatively-activated M2 macrophage (M(IL4+IL13)) gene signature and function, while boosting classic M1 macrophage polarization (M(LPS)) and bacterial killing capacity. M1 macrophages predominantly rely on glycolysis, whereas M2 macrophages use oxidative phosphorylation, fueled by the oxidation of fatty acids, at a late phase of activation (24 h). The aim of this study was to elucidate the role of HS on the metabolic signature of M1 and M2 macrophages. Therefore, we quantified 13 C incorporation into intermediates of central carbon metabolism via GC-MS based pulsed stable isotope resolved metabolomics (pSIRM), and performed Seahorse analyses at varying time points during M1/M2-activation. After 24 h, LPS-induced increase in basal ECAR (extracellular acidification as surrogate parameter for glycolysis) and lactate production were further boosted under HS compared to isotonic salt conditions (NS), whereas mitochondrial respiration (OCR) was not affected. On the other hand, IL4+IL13-induced increase in OCR was decreased under HS compared to NS, while ECAR was not affected at this stage. Regarding TCA-cycle, we have found an early (1 h and 3 h post-stimulation) increase in citrate levels in M1 and M2 macrophages independent of HS, which further increased after 24 h in M1 and M1+HS. Interestingly, both in early and late phase of activation, glucose-derived 13 C incorporation into citrate was lower in HS-treated compared to NS-treated M0, M1 and M2 macrophages, suggesting an alternative replenishing source. Consistently, other TCA-cycle intermediates increased after 24h (especially in M1 and even further in M1+HS), whereas glucose-derived 13 C incorporation was minimal. Our data suggests that under hypertonic sodium macrophages re-direct TCA-cycle intermediates into the production of pro-inflammatory mediators (as described for late-phase M1 macrophages), whereas TCA-cycle itself is replenished by other alternative metabolic substrates. These HS-induced metabolic modifications might play an important role for macrophage activation and function.

Role of the Occluding Septate Junction Protein Mesh in Epithelial Integrity and Ion Transport in the Drosophila Renal Tubules

Background/objectiveEpithelial occluding junctions are specialized cell‐cell junctions that establish and maintain cell polarity and control paracellular ion, solute and water transport. Tight junctions are the occluding junctions in the epithelia of vertebrates, whereas the epithelia of invertebrates possess septate junctions (SJs). Molecular and genetic analysis of Drosophila SJs have identified a number of transmembrane as well as cytoplasmic scaffolding proteins expressed in a tissue‐specific manner. However, many questions about the role of these proteins in SJ assembly, maintenance and function remain open. Recently, an integral protein, Mesh, has been identified within the SJs of Drosophila midgut and renal tubule epithelia, and shown to be required for the barrier function of the midgut. Here, we report, for the first time, examination of the role of Mesh in adult fly renal tubules, whose function in the maintenance of internal homeostasis relies on regulated transepithelial ion and water secretion/absorption.Methodsmesh knockdown in the fly tubules was achieved using the GAL4‐UAS system. Expression and localization of Mesh and a scaffolding SJ protein, Discs large (Dlg), in control and mutant tubules were determined using confocal microscopy and available antibodies. Transepithelial fluid and K+ transport in the main segment of the control and mesh knockdown tubules were measured using the Ramsay assay and a K+‐specific electrode.ResultsAdult fly renal tubules consist of four major morphologically and functionally distinct segments and have two cell types, the larger principal cells and smaller intercalated stellate cells. We found that developmental mesh knockdown in the tubule principal cells was associated with enlarged initial and main segments of the adult fly tubule and reduced septate junctional localization of a scaffolding protein, Dlg, especially in the enlarged initial segment. In addition, the main segment transepithelial fluid and K+ transport in isotonic condition was abolished in mesh knockdown tubules compared to the control groups.Conclusions and significanceThese data suggest that the SJ protein Mesh is required for SJ assembly, epithelial integrity and ion transport in the fly renal tubule epithelium. Since the Mesh mammalian homolog, Susd2, is also highly expressed in the mouse renal epithelium with unknown function, these studies pave the way for further characterization of the Mesh/Susd2 family of proteins in both invertebrates and vertebrates.Support or Funding InformationThis project was supported by NIH/NIDDK to ARRThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

History‐Dependence of the Force‐Velocity Relationship in mdm Mouse Muscles

The force generated by a muscle is inversely related to the velocity of the shortening. Maximum shortening velocity (Vmax) has been predicted from highly controlled, isotonic conditions which are rarely encountered in vivo. During natural movements, muscles undergo stretch‐shortening cycles, which alter power output and predict higher Vmax than traditional isotonic experiments. We investigated the mechanism underlying the differences in the FV relationship. We hypothesized that for a given force, Vmax is greater during work loop experiments because during stretch a muscle can store elastic energy that can be recovered upon shortening. It is possible that the giant elastic protein, titin, functions as a spring in active muscle and therefore contributes to the storage and recovery of elastic energy during work loops but not isotonic contractions. To test these hypotheses, we compared the FV relationships of soleus muscles from wild type and mdm mice with a deletion in the N2A region of titin. Results showed that Vmax is lower under isotonic conditions compared to work loops in both genotypes but the difference was more than 20% greater in wild type muscles. Thus, titin, cross‐bridge kinetics or some combination of both may explain the observed differences. This work contributes to a growing body of evidence of titin's potential role as a spring in active muscle and sheds light on the underlying mechanism of active muscle stiffness.Support or Funding InformationNSF IOS‐1456150 awarded to J.Monroy, APS UGSRFThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

WNK3 and WNK4 Exhibit Opposite Sensibility for Cell Volume and Intracellular Chloride Concentration

BackgroundThe intracellular chloride concentration [Cl−]i and cell volume modulates the activity of the electroneutral cation‐coupled chloride cotransporters (CCCs) such as the NKCCs and the KCCs. Cell shrinkage or reduction of [Cl−]i induce phosphorylation of CCCs, which activates NKCCs and inhibits KCCs, promoting ion influx, while cell swelling or increase of [Cl−]i induces dephosphorylation, that inhibits NKCCs and activates KCCs, stimulating ion efflux. We have shown that the effect of [Cl−]i towards CCCs is traduced by the WNK1 or WNK4 kinases. However, cell volume and [Cl−]i moves in opposite directions. Cell shrinkage increases [Cl−]i and cell swelling decreases [Cl−]i. Thus, the effect of cell volume towards CCCs must be traduced by a different kinase. Because WNK3 bypasses the tonicity requirements for regulation of the CCCs (de Los Heros, PNAS 2006), we tested the hypothesis that WNK3 is sensitive to cell volume, rather than to the [Cl−]i.MethodsXenopus oocytes were microinjected with the Na‐Cl cotransporter (NCC) or the K‐Cl cotransporter (KCC4) cRNA alone or together with WNK3 or WNK4 wild type, or mutants in which catalytic activity and/or the chloride‐binding site has been eliminated (WNK3‐DA; WNK3‐L295/297F; WNK3‐DA‐LLFF; WNK4‐DA; WNK4‐L322F) or with chimeric constructs obtained by swapping the amino, kinase or carboxyl terminal domains between WNK3 and WNK4. The effect of [Cl−]i or extracellular tonicity on WNKs effects on NCC or KCC4 was assessed by tracer 22Na+ or 86Rb+ uptake, respectively. The effect on extracellular tonicity on WNKs activity was assessed as WNK phosphorylation at the activating serine of the T‐loop with specific antibodies by Western blot, as a surrogate of activity.ResultsDepletion of [Cl−]i increases the activity of WNK4 and thus increases the NCC activity. Elimination of WNK4 chloride‐binding site turned it into a constitutively active kinase toward NCC. In contrast the degree of activation of NCC by WNK3 was not affected by reduction of [Cl−]i and elimination of the chloride‐binding site in WNK3 had no effect on the activity of the kinase. Compared to isotonic condition, WNK3 phosphorylation significantly diminished or increased by 50% in hypotonic or hypertonic conditions, respectively. The phosphorylation of WNK4 was not affected by similar changes in tonicity. Chimeric proteins between WNK3 and WNK4 revealed that the carboxyl terminal domain modulates chloride affinity in WNKs. That is, swapping the WNK4 carboxyl terminal domain into WNK3 amino and kinase domain rendered this construct sensitive to changes in the [Cl−]i.DiscussionWe previously demonstrated that WNK4 is sensitive to [Cl−]i (Bazúa‐Valenti, JASN 2015). Here we show that WNK3 is not sensitive to changes of the [Cl−]i, but is modulated by changes in cell volume. In addition, WNK3, but not WNK4, phosphorylation was sensitive to changes in cell volume. Hypotonicity reduces WNK3 phosphorylation, while hypertonicity increased it. Thus, we propose that while WNK4 is a chloride sensitive kinase, WNK3 is a volume sensitive kinase, modulating the effect of cell volume changes towards CCCs activity.Support or Funding InformationFronteras de la Ciencia Grant No. 23 from Conacyt to GGThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hypertonicity‐induced cation channels in HepG2 cells: architecture and role in proliferation vs. apoptosis

Na+ conducting hypertonicity-induced cation channels (HICCs) are key players in the volume restoration of osmotically shrunken cells and, under isotonic conditions, considered as mediators of proliferation - thereby opposing apoptosis. In an siRNA screen of ion channels and transporters in HepG2 cells, with the regulatory volume increase (RVI) as read-out, δENaC, TRPM2 and TRPM5 were identified as HICCs. Subsequently, all permutations of these channels were tested in RVI and patch-clamp recordings and, at first sight, HICCs were found to operate in an independent mode. However, there was synergy in the siRNA perturbations of HICC currents. Accordingly, proximity ligation assays showed that δENaC was located in proximity to TRPM2 and TRPM5 suggesting a physical interaction. Furthermore, δENaC, TRPM2 and TRPM5 were identified as mediators of HepG2 proliferation- their silencing enhanced apoptosis. Our study defines the architecture of HICCs in human hepatocytes as well as their molecular functions. Hypertonicity-induced cation channels (HICCs) are a substantial element in the regulatory volume increase (RVI) of osmotically shrunken cells. Under isotonic conditions, they are key effectors in the volume gain preceding proliferation; HICC repression, in turn, significantly increases apoptosis rates. Despite these fundamental roles of HICCs in cell physiology, very little is known concerning the actual molecular architecture of these channels. Here, an siRNA screening of putative ion channels and transporters was performed, in HepG2 cells, with the velocity of RVI as the read-out; in this first run, δENaC, TRPM2 and TRPM5 could be identified as HICCs. In the second run, all permutations of these channels were tested in RVI and patch-clamp recordings, with special emphasis on the non-additivity and additivity of siRNAs - which would indicate molecular interactions or independent ways of channel functioning. At first sight, the HICCs in HepG2 cells appeared to operate rather independently. However, a proximity ligation assay revealed that δENaC was located in proximity to both TRPM2 and TRPM5. Furthermore, a clear synergy of HICC current knock-downs (KDs) was observed. δENaC, TRPM2 and TRPM5 were defined as mediators of HepG2 cell proliferation and their silencing increased the rates of apoptosis. This study provides a molecular characterization of the HICCs in human hepatocytes and of their role in RVI, cell proliferation and apoptosis.

Light Touch Contact Improves Pain-Evoked Postural Instability During Quiet Standing

To investigate if attention to additional sensory information from the fingertip can improve postural stability during pain, which is known to impair balance. In 16 healthy volunteers, experimental pain was induced by intramuscular injection of hypertonic saline in the right vastus medialis muscle (isotonic saline used as nonpainful control, intramuscular injection in the same location). Pain intensity was assessed on an 11-point numeric rating scale (NRS; 0 representing "no pain" and 10 "maximum pain"). Subjects were asked to stand as still as possible on a force plate for 40 seconds with their eyes closed. Their postural stability was quantified by the area and velocity of center of pressure (CoP) displacement. The CoP was recorded with and without pain during two different conditions: 1) no touch and 2) the subjects were asked to lightly touch a curtain with their right index finger and focus their attention on keeping it as still as possible. Hypertonic injections induced higher NRS scores compared with control injections (P < 0.05). During the hypertonic injection condition, the CoP area and velocity in both directions increased during no touch compared with the light touch condition (P < 0.05). No differences were found during light touch between the hypertonic and isotonic injection conditions. Although experimental knee-related pain impaired postural stability, lightly touching a curtain with the fingertip decreased postural sway during painful conditions. Providing additional sensory information while pain patients are performing balance exercises may improve postural stability and increase the quality of exercise, consequent rehabilitation protocols, and clinical outcomes.

Photobiomodulation improves motor response in patients with spinal cord injury submitted to electromyographic evaluation: randomized clinical trial.

NCT 03031223.

Hypotonic Challenge of Endothelial Cells Increases Membrane Stiffness with No Effect on Tether Force

Regulation of cell volume is a fundamental property of all mammalian cells. Multiple signaling pathways are known to be activated by cell swelling and to contribute to cell volume homeostasis. Although cell mechanics and membrane tension have been proposed to couple cell swelling to signaling pathways, the impact of swelling on cellular biomechanics and membrane tension have yet to be fully elucidated. In this study, we use atomic force microscopy under isotonic and hypotonic conditions to measure mechanical properties of endothelial membranes including membrane stiffness, which reflects the stiffness of the submembrane cytoskeleton complex, and the force required for membrane tether formation, reflecting membrane tension and membrane-cytoskeleton attachment. We find that hypotonic swelling results in significant stiffening of the endothelial membrane without a change in membrane tension/membrane-cytoskeleton attachment. Furthermore, depolymerization of F-actin, which, as expected, results in a dramatic decrease in the cellular elastic modulus of both the membrane and the deeper cytoskeleton, indicating a collapse of the cytoskeleton scaffold, does not abrogate swelling-induced stiffening of the membrane. Instead, this swelling-induced stiffening of the membrane is enhanced. We propose that the membrane stiffening should be attributed to an increase in hydrostatic pressure that results from an influx of solutes and water into the cells. Most importantly, our results suggest that increased hydrostatic pressure, rather than changes in membrane tension, could be responsible for activating volume-sensitive mechanisms in hypotonically swollen cells.

Observation of Dynamic Surfactant Adsorption Facilitated by Divalent Cation Bridging.

Dynamic evidence of the mechanism for surfactant adsorption to surfaces of like charge has been observed. Additionally, removal and retention of surfactant molecules on the surface were observed as a function of time. A decrease in surface charge is observed when metal counterions are introduced and is dependent on charge density as well as valency of the metal ion. When surfactant species are also present with the metals, a dramatic increase in surface charge arises. We observed that the rate and quantity of surfactant adsorption can be controlled by the presence of divalent Ca2+. Under isotonic conditions the introduction of Ca2+ is also easily distinguishable from that of monovalent Na+ and provides dynamic evidence of the divalent "cation bridging" phenomenon. Dynamic changes to surface charge are experimentally determined by utilizing current monitoring to quantify the zeta potential in a microfluidic device.

Effects of Exercise Intensity on Cardiovascular Variables During Concentric and Eccentric Resistive Knee Extensors Exercises in Adults under Isotonic Condition

Effects of Exercise Intensity on Cardiovascular Variables During Concentric and Eccentric Resistive Knee Extensors Exercises in Adults under Isotonic Condition

Glycine Induces Migration of Microglial BV-2 Cells via SNAT-Mediated Cell Swelling

Background/Aims: The neutral, non-essential amino acid glycine has manifold functions and effects under physiological and pathophysiological conditions. Besides its function as a neurotransmitter in the central nervous system, glycine also exerts immunomodulatory effects and as an osmolyte it participates in cell volume regulation. During phagocytosis, glycine contributes to (local) cell volume-dependent processes like lamellipodium formation. Similar to the expansion of the lamellipodium we assume that glycine also affects the migration of microglial cells in a cell volume-dependent manner. Methods: Mean cell volume (MCV) and cell migration were determined using flow cytometry and trans-well migration assays, respectively. Electrophysiological recordings of the cell membrane potential (V_mem) and swelling-dependent chloride (Cl^-) currents (ICl_swell, VSOR, VRAC) were performed using the whole-cell patch clamp technique. Results: In the murine microglial cell line BV-2, flow cytometry analysis revealed that glycine (5 mM) increases the MCV by ∼9%. The glycine-dependent increase in MCV was suppressed by the partial sodium-dependent neutral amino acid transporter (SNAT) antagonist MeAIB and augmented by the Cl^- current blocker DCPIB. Electrophysiological recordings showed that addition of glycine activates a Cl^- current under isotonic conditions resembling features of the swelling-activated Cl^- current (ICl_swell). The cell membrane potential (V_mem) displayed a distinctive time course after glycine application; initially, glycine evoked a rapid depolarization mediated by Na⁺-coupled glycine uptake via SNAT, followed by a further gradual depolarization, which was fully suppressed by DCPIB. Interestingly, glycine significantly increased migration of BV-2 cells, which was suppressed by MeAIB, suggesting that SNAT is involved in the migration process of microglial cells. Conclusion: We conclude that glycine acts as a chemoattractant for microglial cells presumably by a cell volume-dependent mechanism involving SNAT-mediated cell swelling.

Osmotic behaviour of human mesenchymal stem cells: Implications for cryopreservation.

Aimed at providing a contribution to the optimization of cryopreservation processes, the present work focuses on the osmotic behavior of human mesenchymal stem cells (hMSCs). Once isolated from the umbilical cord blood (UCB) of three different donors, hMSCs were characterized in terms of size distribution and their osmotic properties suitably evaluated through the exposure to hypertonic and isotonic aqueous solutions at three different temperatures. More specifically, inactive cell volume and cell permeability to water and di-methyl sulfoxide (DMSO) were measured, being cell size determined using impedance measurements under both equilibrium and dynamic conditions. Experimental findings indicate that positive cell volume excursions are limited by the apparent increase of inactive volume, which occurs during both the shrink-swell process following DMSO addition and the subsequent restoration of isotonic conditions in the presence of hypertonic solutions of impermeant or permeant solutes. Based on this evidence, hMSCs must be regarded as imperfect osmometers, and their osmotic behavior described within a scenario no longer compatible with the simple two-parameter model usually utilized in the literature. In this respect, the activation of mechano-sensitive ion-channels seemingly represents a reasonable hypothesis for rationalizing the observed osmotic behavior of hMSCs from UCB.

Osmotic tolerance of feline epididymal spermatozoa

During the cryopreservation process, spermatozoa are exposed to hypertonic solutions contributed by the high concentration of cryoprotectant. During addition and removal of cryoprotectant the spermatozoa are subjected to a substantial osmotic stress. Spermatozoa of different species and different stages of maturation may have different susceptibility to osmotic stress depending on the biology of the cell membrane and this will affect their tolerance to the freezing-thawing stress. The aims of this study were to determine the osmotic tolerance limits for motility, membrane integrity and mitochondrial membrane potential of feline epididymal spermatozoa and to study the effect of osmotic stress on the feline spermatozoa of different epididymal regions. Epididymal spermatozoa from three regions (caput, corpus and cauda) were pre-exposed to various osmolalities (75, 300, 600, 900, 1200 mOsm) in a single step for 10min and returned to 300 mOsm afterward. Percentage of motile spermatozoa was measured subjectively and membrane integrity (SYBR-14 positive cells) was evaluated prior to and after exposure to different osmolalities. The mitochondrial membrane potential (JC1) of spermatozoa were evaluated using flow cytometer and compared between epididymal regions (caput, corpus and cauda). All the parameters were compared using a mixed procedure. The percentage of motile epididymal spermatozoa decreased significantly when spermatozoa were exposed to 75 mOsm and 600 mOsm. Epididymal spermatozoa showed signs of damage when pre-exposed to 900 and 1200 mOsm and returned to isotonic condition as significant reduction of membrane integrity and mitochondrial membrane potential were observed (P<0.05). The plasma membrane of spermatozoa from the cauda epididymal region showed higher susceptibility to osmotic stress than the other regions as demonstrated by a significant difference between regions after return to isotonicity from 900 mOsm (P>0.01) and a difference between caput and corpus after return from 1200 mOsm (P<0.05). The corpus and cauda epididymal spermatozoa had higher percentage of spermatozoa with high mitochondrial membrane potential than those from caput when exposed to 75, 300 and 600 mOsm (P<0.05). In conclusion, a single step exposure to hypertonic solution of greater than 600 mOsm prior to return to isotonic condition can cause severe damage to sperm membrane and mitochondrial membrane potential compared to non-returning (exposure to various osmolality but not returned to isotonic condition). Changes in osmolality impacted mostly on sperm motility. Spermatozoa from cauda epididymis were more susceptible to osmotic stress compared to those from corpus and caput indicating that the maturation changes in the sperm membrane during passage through the epididymis increase susceptibility to the osmotic changes that may occur during sperm cryopreservation.

Enhancement of polypyrrole linear actuation with poly(ethylene oxide)

In the present paper, we report the electro-chemo-mechanical deformation (ECMD) measurements of pristine polypyrrole (PPy) and polypyrrole/poly(ethylene oxide) composite films doped with dodecylbenzenesulfonate (DBS) under isotonic (constant force) conditions. The PPy/DBS and PPy-PEO/DBS films, the latter made from a polymerization solution containing 5wt% PEO, were actuated potentiodynamically in an aqueous solution containing lithium bis-trifluoromethane-sulfonimide (LiTFSI). The obtained composite films were characterized with scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy and Fourier transform infrared spectroscopy (FTIR). The results confirmed the successful incorporation of PEO within the PPy film. The composite films showed a remarkable improvement in maximum strain achieved (of 7.7%), nearly double that of the pristine PPy/DBS film, had a higher conductivity, higher ion diffusion coefficient and faster strain rate. The ECMD measurements for both the pristine as well as the PEO-composite films showed cation driven actuation involving the migration of Li+ cations in the aqueous electrolyte.

Hyperosmotic stress enhances cytotoxicity of SMAC mimetics

Inhibitors of apoptosis (IAP) proteins contribute to cell death resistance in malignancies and emerged as promising targets in cancer therapy. Currently, small molecules mimicking the IAP-antagonizing activity of endogenous second mitochondria-derived activator of caspases (SMAC) are evaluated in phase 1/2 clinical trials. In cancer cells, SMAC mimetic (SM)-mediated IAP depletion induces tumor necrosis factor (TNF) secretion and simultaneously sensitizes for TNF-induced cell death. However, tumor cells lacking SM-induced autocrine TNF release survive and thus limit therapeutic efficacy. Here, we show that hyperosmotic stress boosts SM cytotoxicity in human and murine cells through hypertonicity-induced upregulation of TNF with subsequent induction of apoptosis and/or necroptosis. Hypertonicity allowed robust TNF-dependent killing in SM-treated human acute lymphoblastic leukemia cells, which under isotonic conditions resisted SM treatment due to poor SM-induced TNF secretion. Mechanistically, hypertonicity-triggered TNF release bypassed the dependency on SM-induced TNF production to execute SM cytotoxicity, effectively reducing the role of SM to TNF-sensitizing, but not necessarily TNF-inducing agents. Perspectively, these findings could extend the clinical application of SM.

Modeling the Mechanics of Cell Division: Influence of Spontaneous Membrane Curvature, Surface Tension, and Osmotic Pressure.

Many cell division processes have been conserved throughout evolution and are being revealed by studies on model organisms such as bacteria, yeasts, and protozoa. Cellular membrane constriction is one of these processes, observed almost universally during cell division. It happens similarly in all organisms through a mechanical pathway synchronized with the sequence of cytokinetic events in the cell interior. Arguably, such a mechanical process is mastered by the coordinated action of a constriction machinery fueled by biochemical energy in conjunction with the passive mechanics of the cellular membrane. Independently of the details of the constriction engine, the membrane component responds against deformation by minimizing the elastic energy at every constriction state following a pathway still unknown. In this paper, we address a theoretical study of the mechanics of membrane constriction in a simplified model that describes a homogeneous membrane vesicle in the regime where mechanical work due to osmotic pressure, surface tension, and bending energy are comparable. We develop a general method to find approximate analytical expressions for the main descriptors of a symmetrically constricted vesicle. Analytical solutions are obtained by combining a perturbative expansion for small deformations with a variational approach that was previously demonstrated valid at the reference state of an initially spherical vesicle at isotonic conditions. The analytic approximate results are compared with the exact solution obtained from numerical computations, getting a good agreement for all the computed quantities (energy, area, volume, constriction force). We analyze the effects of the spontaneous curvature, the surface tension and the osmotic pressure in these quantities, focusing especially on the constriction force. The more favorable conditions for vesicle constriction are determined, obtaining that smaller constriction forces are required for positive spontaneous curvatures, low or negative membrane tension and hypertonic media. Conditions for spontaneous constriction at a given constriction force are also determined. The implications of these results for biological cell division are discussed. This work contributes to a better quantitative understanding of the mechanical pathway of cellular division, and could assist the design of artificial divisomes in vesicle-based self-actuated microsystems obtained from synthetic biology approaches.

The contractile adaption to preload depends on the amount of afterload.

AimsThe Frank–Starling mechanism (rapid response (RR)) and the secondary slow response (SR) are known to contribute to increases contractile performance. The contractility of the heart muscle is influenced by pre‐load and after‐load. Because of the effect of pre‐load vs. after‐load on these mechanisms in not completely understood, we studied the effect in isolated muscle strips.Methods and resultsProgressive stretch lead to an increase in shortening/force development under isotonic (only pre‐load) and isometric conditions (pre‐ and after‐load). Muscle length with maximal function was reached earlier under isotonic (L max‐isotonic) compared with isometric conditions (L max‐isometric) in nonfailing rabbit, in human atrial and in failing ventricular muscles. Also, SR after stretch from slack to L max‐isotonic was comparable under isotonic and isometric conditions (human: isotonic 10 ± 4%, isometric 10 ± 4%). Moreover, a switch from isotonic to isometric conditions at L max‐isometric showed no SR proving independence of after‐load. To further analyse the degree of SR on the total contractile performance at higher pre‐load muscles were stretched from slack to 98% L max‐isometric under isotonic conditions. Thereby, the SR was 60 ± 9% in rabbit and 51 ± 14% in human muscle strips.ConclusionsThis work shows that the acute contractile response largely depends on the degree and type of mechanical load. Increased filling of the heart elevates pre‐load and prolongs the isotonic part of contraction. The reduction in shortening at higher levels of pre‐load is thereby partially compensated by the pre‐load‐induced SR. After‐load shifts the contractile curve to a better ‘myofilament function’ by probably influencing thin fibers and calcium sensitivity, but has no effect on the SR.