Isotonic Conditions Research Articles

Bacterial-host adhesion dominated by collagen subtypes remodelled by osmotic pressure.

Environmental osmolarity plays a crucial role in regulating the functions and behaviors of both host cells and pathogens. However, it remains unclear whether and how environmental osmotic stimuli modulate bacterial‒host interfacial adhesion. Using single-cell force spectroscopy, we revealed that the interfacial adhesion force depended nonlinearly on the osmotic prestimulation of host cells but not bacteria. Quantitatively, the adhesion force increased dramatically from 25.98 nN under isotonic conditions to 112.45 or 93.10 nN after the host cells were treated with the hypotonic or hypertonic solution. There was a strong correlation between the adhesion force and the number of host cells harboring adherent/internalized bacteria. We further revealed that enhanced overexpression levels of collagen XV and II were responsible for the increases in interfacial adhesion under hypotonic and hypertonic conditions, respectively. This work provides new opportunities for developing host-directed antibacterial strategies related to interfacial adhesion from a mechanobiological perspective.

Donor age contributes more to the rheological properties of stored red blood cells than donor sex and biological age distribution

Variation in the quality of stored red cell concentrates (RCCs) has been linked to the biological age distribution of red blood cell (RBCs) subpopulations within blood products. Teen male RCCs contain higher proportions of biologically old RBC subpopulations, with poorer quality. This study sought to assess the contribution of donor sex and age on the deformability characteristics of different ages of RBC subpopulations in stored RCCs. On days 5, 14, 21, and 42 of hypothermic storage, RCCs from healthy teen male, senior male, teen female and senior female donors were biologically age profiled. The deformability of the resulting young (Y-RBCs) and old (O-RBCs) RBC subpopulations in addition to unseparated RBCs was assessed using ektacytometry. Over storage, donor age influenced the rheological profile of RBC subpopulations, with teen male RCCs having the largest reduction in Ohyper (the osmolality in the hypertonic region corresponding to 50% of the EImax). The strongest correlations between Ohyper and MCHC (R2 > 0.5) were witnessed with O-RBCs from senior donors, and to a lesser extent with teen males. O-RBCs from teens, particularly males, had higher elongation indices both under isotonic conditions and in the presence of an increasing osmotic gradient. Consistent across RBCs regardless of their biological age, teen samples were discovered to be more rigid (higher KEI). The donor-to-donor variation in age distribution of RBC subpopulations and its downstream effect on deformability serves as further evidence that factors beyond chronological aging could potentially impact the quality of red cell concentrates and variations in transfusion outcomes.

Quantification of hemolysis rates from pulsed field ablation

Abstract Introduction Pulsed electric fields induce hemolysis of red blood cells as a dose-dependent effect of the electric field. Voltage pulsations induce a transmembrane potential across the cell membrane and either open up or create pores in the red cells. In isotonic conditions, the pores allow passage of potassium and sodium ions but not sucrose or hemoglobin, and leakage of ions leads to an osmotic imbalance which in turn causes a colloidal hemolysis of the red cells. Objective To quantify hemolysis rates during pulsed field ablation (PFA). Methods We created a computational model of PFA to determine the total volume of blood hemolyzed from each pulse during catheter ablation using pulsed field energy. The percentage hemolysis was calculated as a function of electric field intensity utilizing existing published experimental data. A single electrode delivering a single pulse of 100 microseconds duration was modeled at voltages of 1 kV, 1.5 kV, and 2 kV. Results The total volume of hemolyzed blood per pulse increased with increasing applied voltage (Figure). For 2 electrodes in direct tissue contact, the electrode surface exposed to the blood pool generated 19 microliters of hemolyzed red blood cells with a single pulse of a 2 kV field strength. In the case of a single pulse train consisting of 50 pulses, using a catheter configuration with 20 electrodes, an estimate of total volume per pulse train is approximately 9.5 mL of hemolyzed blood. In the case of electrodes not in direct contact with endocardial tissue, and exposed to the blood pool, up to double this volume could be anticipated per pulse train. Hemolyzed volume can be reduced with better tissue contact (reducing exposure to blood), or potentially with the use of irrigation to dilute the local red blood cell concentration. Increased tonicity of the irrigant fluid may also reduce the osmotic pressure increases that result in hemolysis. Conclusions Typical voltages utilized in pulsed field ablation can cause significant hemolysis, which is exacerbated by inadequate tissue contact. A greater number of electrodes and number of pulses delivered increases the risk, whereas better tissue contact, and the use of irrigation, may reduce it.

Hypertonicity and/or acidosis induce marked rheological changes under hypoxic conditions in sickle trait red blood cells.

Deformability and sickling of red blood cells (RBCs) from individuals with sickle cell trait (SCT) was evaluated under harsh biophysical conditions that mimic certain vascular beds invivo. RBC deformability in osmotic-gradient ektacytometry was decreased in HbAS (SCT) compared to HbAA (wild-type) RBCs at supraphysiological osmolalities. RBC deformability was also measured by oxygen-gradient ektacytometry. Whereas RBC sickling was not observed under isotonic and neutral pH conditions, hypertonicity and acidosis alone or in combination induced reversible sickling of SCT RBC. These data suggest that hyperosmolality and/or acidosis enhance hypoxia-induced sickling of SCT RBC.

Osmotic injury and cytotoxicity for hMSCs in contact with Me2SO: The effect of cell size distribution

The paper discusses the impact of cell size on cytotoxicity and expansion lysis during the osmotic excursions resulting from the contact of hMSCs from UCB with Me2SO. It builds upon the mathematical model recently presented by the authors, which pertains to a population of cells with uniform size. The objective is to enhance the model's relevance by incorporating the more realistic scenario of cell size distribution, utilizing a Population Balance Equations approach. The study compares the capability of the multiple-sized model to the single-sized one to describe system behavior experimentally measured through cytofluorimetry and Coulter counter when, first, suspending hMSCs in hypertonic solutions of Me2SO (at varying osmolality, system temperature, and contact times), and then (at room temperature) pelleting by centrifugation before suspending the cells back to isotonic conditions. Simulations demonstrate that expansion lysis and cytotoxic effect are not affected by cell size for the specific system hMSCs/Me2SO, thus confirming what was found so far by the authors through a single-size model. On the other hand, simulations show that, when varying the adjustable parameters of the model that are expected to change from cell to cell lineages, expansion lysis is sensitive to cell size, while cytotoxicity is not, being mainly influenced by external CPA concentration and contact duration. More specifically, it is found that smaller cells suffer expansion lysis more than larger ones. The findings suggest that different cells from hMSCs may require a multiple-sized model to assess cell damage during osmotic excursions in cryopreservation.

TRPV4 and chloride channels mediate volume sensing in trabecular meshwork cells.

Aqueous humor drainage from the anterior eye determines intraocular pressure (IOP) under homeostatic and pathological conditions. Swelling of the trabecular meshwork (TM) alters its flow resistance but the mechanisms that sense and transduce osmotic gradients remain poorly understood. We investigated TM osmotransduction and its role in calcium and chloride homeostasis using molecular analyses, optical imaging, and electrophysiology. Anisosmotic conditions elicited proportional changes in TM cell volume, with swelling, but not shrinking, evoking elevations in intracellular calcium concentration [Ca2+]TM. Hypotonicity-evoked calcium signals were sensitive to HC067047, a selective blocker of TRPV4 channels, whereas the agonist GSK1016790A promoted swelling under isotonic conditions. TRPV4 inhibition partially suppressed hypotonicity-induced volume increases and reduced the magnitude of the swelling-induced membrane current, with a substantial fraction of the swelling-evoked current abrogated by Cl- channel antagonists 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS) and niflumic acid. The transcriptome of volume-sensing chloride channel candidates in primary human was dominated by ANO6 transcripts, with moderate expression of ANO3, ANO7, and ANO10 transcripts and low expression of LTTRC genes that encode constituents of the volume-activated anion channel. Imposition of 190 mosM but not 285 mosM hypotonic gradients increased conventional outflow in mouse eyes. TRPV4-mediated cation influx thus works with Cl- efflux to sense and respond to osmotic stress, potentially contributing to pathological swelling, calcium overload, and intracellular signaling that could exacerbate functional disturbances in inflammatory disease and glaucoma.NEW & NOTEWORTHY Intraocular pressure is dynamically regulated by the flow of aqueous humor through paracellular passages within the trabecular meshwork (TM). This study shows hypotonic gradients that expand the TM cell volume and reduce the outflow facility in mouse eyes. The swelling-induced current consists of TRPV4 and chloride components, with TRPV4 as a driver of swelling-induced calcium signaling. TRPV4 inhibition reduced swelling, suggesting a novel treatment for trabeculitis and glaucoma.

HepG2 cells undergo regulatory volume decrease by mechanically induced efflux of water and solutes.

This study challenges the conventional belief that animal cell membranes lack a significant hydrostatic gradient, particularly under anisotonic conditions, as demonstrated in the human hepatoma cell line HepG2. The Boyle van't Hoff (BvH) relation describes volumetric equilibration to anisotonic conditions for many cells. However, the BvH relation is simple and does not include many cellular components such as the cytoskeleton and actin cortex, mechanosensitive channels, and ion pumps. Here we present alternative models that account for mechanical resistance to volumetric expansion, solute leakage, and active ion pumping. We found the BvH relation works well to describe hypertonic volume equilibration but not hypotonic volume equilibration. After anisotonic exposure and return isotonic conditions cell volumes were smaller than their initial isotonic volume, indicating solutes had leaked out of the cell during swelling. Finally, we observed HepG2 cells undergo regulatory volume decrease at both 20°C and 4°C, indicating regulatory volume decrease to be a relatively passive phenomenon and not driven by ion pumps. We determined the turgor-leak model, which accounts for mechanical resistance and solute leakage, best fits the observations found in the suite of experiments performed, while other models were rejected.

The contribution of different sperm parameters to better explain ram semen cryoresistance

In order to determine an effective procedure for explaining ram sperm cryoresistance and develop a new model for breeders classification, a retrospective study was conducted using sperm analysis data obtained over two consecutive years from a total of 82 sessions of ram semen cryopreservation. In each session, fresh ejaculates from eight males were collected via artificial vagina, pooled and frozen in liquid nitrogen vapors. After thawing, a total of 19,084 sperm tracks and 11,319 morphometric measurements were analysed. Clustering analyses were applied to establish motile and morphometric sperm subpopulations. Additionally, plasma and acrosome membrane integrity, as well mitochondrial activity using flow cytometry immediately after sperm thawing and following hypoosmotic shock test (HOST) was assessed. To develop a Ram Sperm Cryoresistance Index, Principal Component Analyses (PCA) using 22 variables were conducted. In the first PCA, the parameters that best explain cryoresistance include total motility (TM), motile subpopulation 2 (motSP2, which groups slow, very linear spermatozoa with low lateral head displacement), morphometric subpopulation 1 (morphSP1, grouping spermatozoa with the smallest head size and lowest shape values), sperm plasma membrane integrity immediately after thawing and following hypoosmotic shock test. These parameters collectively account for 77.34 % of the accumulated variance. To emphasize their importance, a second PCA was performed, revealing significant higher weighting coefficients for the quantity (TM) and quality (motSP2) of sperm movement after thawing, compared to the head size and shape of the thawed sperm (morphSP1). Furthermore, HOST Viability played a more decisive role than what was observed under isotonic conditions.

Differential effects of stimulation frequency on isometric and concentric isotonic contractile function in human quadriceps.

Electrically evoked contractions are used to assess the relationship between frequency input and contractile output to characterize inherent muscle function, and these have been done mostly with isometric contractions (i.e., no joint rotation). The purpose was to compare the electrically stimulated frequency and contractile function relationship during isometric (i.e., torque) with isotonic (i.e., concentric torque, angular velocity, and mechanical power) contractions. The knee extensors of 16 (5 female) young recreationally active participants were stimulated (∼1-2.5 s) at 14 frequencies from 1 to 100 Hz. This was done during four conditions, which were isometric and isotonic at loads of 0 (unloaded), 7.5%, and 15% isometric maximal voluntary contraction (MVC), and repeated on separate days. Comparisons across contractile parameters were made as a % of 100 Hz. Independent of the load, the mechanical power-frequency relationship was rightward shifted compared with isometric torque-frequency, concentric torque-frequency, and velocity-frequency relationships (all P ≤ 0.04). With increasing load (0%-15% MVC), the isotonic concentric torque-frequency relationship was shifted leftward systematically from 15 to 30 Hz (all P ≤ 0.04). Conversely, the same changes in load caused a rightward shift in the velocity-frequency relationship from 1 to 40 Hz (all P ≤ 0.03). Velocity was leftward shifted of concentric torque in the unloaded isotonic condition from 10 to 25 Hz (all P ≤ 0.03), but concentric torque was leftward shifted of velocity at 15% MVC isotonic condition from 10 to 50 Hz (all P ≤ 0.03). Therefore, isometric torque is not a surrogate to evaluate dynamic contractile function. Interpretations of evoked contractile function differ depending on contraction type, load, and frequency, which should be considered relative to the specific task.NEW & NOTEWORTHY In whole human muscle, we showed that the electrically stimulated power-frequency relationship was rightward shifted of the stimulated isometric torque-frequency relationship independent of isotonic load, indicating that higher stimulation frequencies are needed to achieve tetanus. Therefore, interpretations of evoked contractile function differ depending on contraction type (isometric vs. dynamic), load, and frequency. And thus, isometric measures may not be appropriate as a surrogate assessment when evaluating dynamic isotonic contractile function.

Visible light photocatalytic response of Fe doped TiO2: Inactivation of Escherichia coli

Heterogeneous photocatalysis has been widely investigated for the removal of pollutants and pathogenic microorganisms. Various modified photocatalysts were developed for the beneficial use of solar light. E. coli inactivation by Fe doping of TiO2 (P-25) and sol–gel prepared (SynTiO2) was achieved upon solar irradiation in both isotonic (IsoT) and water matrix (WM) conditions. First order kinetic rate constants were comparatively higher for bare SynTiO2 both in IsoT (k = 0.254 min−1) and WM (k = 0.180 min−1) solutions. WM exerted an overall retardation effect for all photocatalysts except for 0.50 % Fe-TiO2 and 0.25 % Fe-SynTiO2. Release of intracellular components was followed by spectroscopic parameters as well as dissolved organic carbon (DOC) contents. E. coli cell destruction was related to the released protein, carbohydrate, and K+ ion contents. Solar photocatalytic inactivation of E. coli using novel sol–gel prepared TiO2 and Fe doped sol–gel TiO2 was successfully demonstrated as an alternative and promising method for disinfection purposes.

The Effect of Hypertonic Osmotic Stress and Its Duration on the Level and Activity of Executioner Caspase Enzymes in Blood Anuclear Cells (Platelets)

Background: Caspases are protease enzymes critical for apoptosis. Caspase-3 and caspase-7, known as executioner caspases, are present in anuclear cells such as platelets. Platelet mitochondria can engage in the intrinsic pathway of apoptosis during stress and cellular damage. Hypertonic osmotic stress influences not only cell shape but also induces chemical changes within the cell. This study examines the function of executioner caspases in platelets under hypertonic osmotic stress due to the absence of a caspase-independent apoptosis pathway in these nucleus-free cells. Methods: Platelets were exposed to osmotic stress in a hypertonic environment (1.7% sodium chloride), with an isotonic environment (0.9% sodium chloride) serving as the control. Both environments were maintained at 37°C for durations of 15 minutes and 24 hours. Following this, platelets were lysed using ultrasound waves, and cellular membranes were removed by centrifugation. The supernatants were then used to measure caspase-3 level (ng/mL) using an ELISA method and caspase-3/7 activity (nmol/min/L) using a colorimetric method. Results: The study confirmed the presence of executioner caspases in platelets under non-stressed isotonic conditions. Following 15 minutes and 24 hours of hypertonic osmotic stress, caspase-3 level increased as determined by ELISA. However, caspase-3/7 activity initially decreased. With extended stress duration from 15 minutes to 24 hours, both the level and activity of these enzymes increased significantly (P-value < 0.05). Conclusions: Exposure to a hypertonic osmotic environment containing 1.7% sodium chloride increases caspase-3 level in anuclear cells such as platelets, while initially reducing caspase-3/7 activity. Prolonging the duration of stress to 24 hours enhances both the level and activity of these enzymes.

Role of Protein Phosphatase 1 (PP1) Upon KCCs Regulation by WNK3 Kinase During Cell Volume Regulation and Ion Transport

Background: The K-Cl cotransporters (KCCs) are SLC12A cation/chloride cotransporters (CCCs) that mediate epithelial transport, maintain cellular volume, and regulate GABA neurotransmission. These mechanisms are complex, and contain multiple sensors, transducers, and effectors, often operating in parallel pathways. It has been shown that KCCs activity is affected by direct phosphorylation and dephosphorylation. In particular dephosphorylation of important regulatory sites on KCCs is necessary to become fully activated, whereas phosphorylation by SPAK/OSR1 kinases will result in KCCs inhibition. Protein phosphatases (PP) inhibitors reduce KCCs function and so, it is generally accepted the presence of phosphatases on the KCCs signaling pathway. Several studies have proposed protein phosphatase 1 (PP1) as the major PP responsible for KCCs dephosphorylation. On the other hand, WNK3 kinases have been shown to directly regulate KCCs activity bypassing their osmotic regulation. That is wild type (WT) WNK3 phosphorylates KCCs under hypotonic conditions where they are normally active, whereas kinase dead WNK3 (DA) activates KCCs function under isotonic conditions where they are normally inhibited. On the present study we analyze the role of protein phosphatase 1 (PP1) upon KCCs regulation by WNK3 kinase during cell volume regulation and ion transport. Methods: HEK 293 cells and Xenopus laevis oocytes were either transfected or microinjected with KCC3 or KCC4 and one of the following PP1 plasmids: WT, H248K (inactive) or T320A (constitutive active), and/or WT WNK3 /DA WNK3. KCCs dephosphorylation in response to cell swelling, hypotonic conditions, was evaluated by Western blot analysis of KCCs regulatory phosphorylation site (pThr1048 in KCC3a). Phosphatase/kinase interaction and the possible formation of a complex was analyzed by transfecting cells or microinjecting oocytes with WNK3 PP1 putative binding sites mutants (WNK3 PP1a y PP1b). Phosphorylation of SPAK/OSR1 activating regulatory site Ser373 was analyzed in parallel under the same conditions. Results: WT or constitutive active mutant PP1 T320A induced KCCs Thr1048 dephosphorylation in HEK cells and X. laevis oocytes even under isotonic conditions, where KCCs are normally phosphorylated. WT WNK3 prevented KCCs T1048 dephosphorylation under hypotonic conditions, ablating their response to cell swelling. Nevertheless, when co-expressed with PP1 T320A, KCCs became dephosphorylated in both osmotic conditions eliminating WT WNK3 inhibitory effect. PP1 inactivating mutation H248K prevented KCCs Thr1048 dephosphorylation in response to hypotonic conditions. However, when co-expressed with DA WNK3, this KCCs regulatory site, became dephosphorylated under hypotonic conditions. SPAK Ser373 regulation was unaffected in any off the studied conditions. Conclusions: Our results show a direct role for PP1 on KCCs function. PP1WT and PP1T320A regulate KCCs activity by its dephosphorylation. In presence of WT WNK3, PP1 activity predominates over WNK3 surpassing the inhibitory phosphorylation of the regulatory Thr residue. KCCs are not dephosphorylated by PP1H248K under hypotonic conditions. Coexpression of kinase dead WNK3 overcomes this effect, supporting the hypothesis of a phosphatase/kinase complex regulating KCCs cell volume regulatory response. AM CF-2023-I-532, INCar 22-1345 PdlH Papiit IN228123. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Voluntary activation of human knee extensors during isotonic shortening contractions.

The interpolated twitch technique (ITT) is often used to assess voluntary activation during isometric contractions; however, this may have limited relevance to dynamic contractions. Although the ITT has been applied to relatively slow isokinetic contractions (< 150°/s), it has received limited consideration during unconstrained velocity (i.e., isotonic) contractions, despite their relevance to natural movements. Here, we explored the ITT during isotonic knee extension contractions using a modified dynamometer. Young males (n = 6) and females (n = 4) performed isometric and isotonic knee extension contractions of sub-maximal and maximal intensities with doublet (150 Hz) muscle belly stimulations to assess voluntary activation. Following each voluntary isotonic contraction (velocity range ~ 35°/s to ~ 275°/s), resting potentiated doublets were evaluated during passive joint rotation at the same angular velocity achieved during voluntary efforts, to account for force-velocity characteristics. Correlations between voluntary activation and the proportion of maximal torque or power were evaluated for isometric and isotonic contractions, respectively. Isometric voluntary activation was strongly correlated with increasing torque output (r = 0.96, p < 0.001). Doublet torque during passive joint rotation displayed a hyperbolic relationship with increasing angular velocity (r = 0.98, p < 0.001). Isotonic voluntary activation was strongly correlated with increasing power output (r = 0.89, p < 0.001). During maximal effort contractions, no differences were observed in voluntary activation between isometric and isotonic conditions (89.4% vs. 89.2%, p = 0.904). The ITT is a valid approach to evaluate voluntary activation during an isotonic contraction using a modified dynamometer. Participants were able to achieve a similar high level of voluntary activation during isometric and isotonic contractions.

Generalizing Upper Limb Force Modeling With Transfer Learning: A Multimodal Approach Using EMG and IMU for New Users and Conditions.

In the field of EMG-based force modeling, the ability to generalize models across individuals could play a significant role in its adoption across a range of applications, including assistive devices, robotic and rehabilitation devices. However, current studies have predominately focused on intra-subject modeling, largely neglecting the burden of end-user data acquisition. In this work, we propose the use of transfer learning (TL) to generalize force modeling to a new user by first establishing a baseline model trained using other users' data, and then adapting to the end-user using a small amount of new data (only 10% , 20% , and 40% of the new user data). Using a deep multimodal convolutional neural network, consisting of two CNN models, one with high-density (HD) EMG and one with motion data recorded by an Inertial Measurement Unit (IMU), our proposed TL technique significantly improved force modeling compared to leave-one-subject-out (LOSO) and even intra-subject scenarios. The TL approach increased the average R squared values of the force modeling task by 60.81%, 190.53%, and 199.79% compared to the LOSO case, and by 13.4%, 36.88%, and 45.51% compared to the intra-subject case for isotonic, isokinetic and dynamic conditions, respectively. These results show that it is possible to adapt to a new user with minimal data while improving performance significantly compared to the intra-subject scenario. We also show that TL can be used to generalize on a new experimental condition for a new user.

Intravenous Injection of Na Ions Aggravates Ang II-Induced Hypertension-Related Vascular Endothelial Injury by Increasing Transmembrane Osmotic Pressure.

Extracellular protein nanoparticles (PNs) and ions perform synergistical functions in the control of transmembrane osmotic pressure (OP) under isotonic conditions. Intravenous injection may disrupt the ion balance and alter PN levels in blood plasma, changing transmembrane OP and damaging vascular endothelial cells. Na ions were injected into AngII-induced HUVECs to simulate cell injury in vitro, and tail vein infusion of Na ions into hypertensive rats was performed to assess vascular damage. Optical measurements using an intermediate filament (IF) tension probe were conducted to detect indicators related to transmembrane OP. Immunofluorescence, Western blotting and small interfering RNA (siRNA) transfection were employed to investigate inflammasomes and the relationship between Abl2 and inflammation. Electrolyte injections with sodium ions (but not glucose and hydroxyethyl starch) induced the production of ASC and NLRP3 inflammasomes in Ang II-induced HUVECs; this in turn resulted in the disorder of calcium signals, and changes in transmembrane OP and cell permeability. Moreover, injection of Na ions into Ang II-induced HUVECs activated the mechanosensitive protein Abl2, involved in inflammation-induced transmembrane OP changes. A drug combination was identified that could induce OP recovery and block hyperpermeability induced by cytoplasmic inflammatory corpuscles in vivo and in vitro. Changes in extracellular PNs and ions following chemical stimuli (Ang II) participate in the regulation of transmembrane OP. Furthermore, injection of Na ions causes vascular endothelial injury in Ang II-induced cells in vitro and hypertension rats in vivo, suggesting it is not safe for hypertensive patients, and we propose a new drug combination as a solution.

Cell-compatible isotonic freezing media enabled by thermo-responsive osmolyte-adsorption/exclusion polymer matrices

During the long-term storage of cells, it is necessary to inhibit ice crystal formation by adding cryoprotectants. Non-cell-permeable cryoprotectants have high osmotic pressure which dehydrates cells, indirectly suppressing intracellular ice crystal formation. However, the high osmotic pressure and dehydration often damage cells. Emerging polymer-type non-cell-permeable cryoprotectants form matrices surrounding cells. These matrices inhibit the influx of extracellular ice nuclei that trigger intracellular ice crystal formation. However, these polymer-type cryoprotectants also require high osmotic pressure to exert an effective cryoprotecting effect. In this study, we designed a poly(zwitterion) (polyZI) that forms firm matrices around cells based on their high affinity to cell membranes. The polyZI successfully cryopreserved freeze-vulnerable cells under isotonic conditions. These matrices also controlled osmotic pressure by adsorbing and desorbing NaCl depending on the temperature, which is a suitable feature for isotonic cryopreservation. Although cell proliferation was delayed by the cellular matrices, washing with a sucrose solution improved proliferation.

Quantifying turgor pressure in budding and fission yeasts based upon osmotic properties

Walled cells, such as plants, fungi, and bacteria cells, possess a high internal hydrostatic pressure, termed turgor pressure, that drives volume growth and contributes to cell shape determination. Rigorous measurement of turgor pressure, however, remains challenging, and reliable quantitative measurements, even in budding yeast are still lacking. Here, we present a simple and robust experimental approach to access turgor pressure in yeasts based upon the determination of isotonic concentration using protoplasts as osmometers. We propose three methods to identify the isotonic condition – three-dimensional cell volume, cytoplasmic fluorophore intensity, and mobility of a cytGEMs nano-rheology probe – that all yield consistent values. Our results provide turgor pressure estimates of 1.0 ± 0.1 MPa for Schizosaccharomyces pombe, 0.49 ± 0.01 MPa for Schizosaccharomyces japonicus, 0.5 ± 0.1 MPa for Saccharomyces cerevisiae W303a and 0.31 ± 0.03 MPa for Saccharomyces cerevisiae BY4741. Large differences in turgor pressure and nano-rheology measurements between the Saccharomyces cerevisiae strains demonstrate how fundamental biophysical parameters can vary even among wild-type strains of the same species. These side-by-side measurements of turgor pressure in multiple yeast species provide critical values for quantitative studies on cellular mechanics and comparative evolution.

Optimization of the Green Chemistry-like Extraction of Phenolic Compounds from Grape (Vitis labrusca L.) and Blackberry (Rubus fruticosus L.) Seeds with Concomitant Biological and Antioxidant Activity Assessments.

The objective of this work was to determine the phenolic composition, chemical and cellular antioxidant activity, cytotoxicity in human cells, and peroxidative inhibition of the defatted fraction of grape (Vitis labrusca) and blackberry (Rubus fruticosus) seeds. Soxhlet extraction (Sox) was used to extract the fat and obtain the degreased material. A statistical optimization study was developed to maximize the extraction of bioactive compounds and antioxidant activity from defatted grape and blackberry seeds. Simultaneous optimization was applied with a combination of 35.9 min of extraction and a solid-to-solvent ratio of 1 g of defatted grape seed to 61.28 mL of an extracting solvent (60% ethanol) and 62.1 min of extraction and a solid-to-solvent ratio of 1 g of defatted blackberry seed to 64.1 mL of an extracting solvent (60% ethanol). In the cell viability assay, HepG2 cancer cells seemed more sensitive to grape and blackberry extracts, while Ea.hy926 hybrid cells showed more resistance to their effects. In general, the extracts presented low/no cytotoxicity, exhibited a protective effect against H2O2-induced ROS production, and demonstrated antioxidant activity and a protective effect on the erythrocytes when subjected to hypotonic and isotonic conditions not presenting hemolytic behavior (5.0 to 10.0 μg GAE/mL). Thus, the results provided a broad assessment of the bioactivity of the extracts obtained using a simple and low-cost process developed by employing non-toxic solvents and with the potential to be used in technological applications.

Stretch-activated ion channel TMEM63B associates with developmental and epileptic encephalopathies and progressive neurodegeneration

By converting physical forces into electrical signals or triggering intracellular cascades, stretch-activated ion channels allow the cell to respond to osmotic and mechanical stress. Knowledge of the pathophysiological mechanisms underlying associations of stretch-activated ion channels with human disease is limited. Here, we describe 17 unrelated individuals with severe early-onset developmental and epileptic encephalopathy (DEE), intellectual disability, and severe motor and cortical visual impairment associated with progressive neurodegenerative brain changes carrying ten distinct heterozygous variants of TMEM63B, encoding for a highly conserved stretch-activated ion channel. The variants occurred de novo in 16/17 individuals for whom parental DNA was available and either missense, including the recurrent p.Val44Met in 7/17 individuals, or in-frame, all affecting conserved residues located in transmembrane regions of the protein. In 12 individuals, hematological abnormalities co-occurred, such as macrocytosis and hemolysis, requiring blood transfusions in some. We modeled six variants (p.Val44Met, p.Arg433His, p.Thr481Asn, p.Gly580Ser, p.Arg660Thr, and p.Phe697Leu), each affecting a distinct transmembrane domain of the channel, in transfected Neuro2a cells and demonstrated inward leak cation currents across the mutated channel even in isotonic conditions, while the response to hypo-osmotic challenge was impaired, as were the Ca2+ transients generated under hypo-osmotic stimulation. Ectopic expression of the p.Val44Met and p.Gly580Cys variants in Drosophila resulted in early death. TMEM63B-associated DEE represents a recognizable clinicopathological entity in which altered cation conductivity results in a severe neurological phenotype with progressive brain damage and early-onset epilepsy associated with hematological abnormalities in most individuals.

Effect of Core Yarn on Linear Actuation of Electroactive Polymer Coated Yarn Actuators

AbstractSmart textiles combine the features of conventional textiles with promising properties of smart materials such as electromechanically active polymers, resulting in textile actuators. Textile actuators comprise of individual yarn actuators, so understanding their electro‐chemo‐mechanical behavior is of great importance. Herein, this study investigates the effect of inherent structural and mechanical properties of commercial yarns, that form the core of the yarn actuators, on the linear actuation of the conducting‐polymer‐based yarn actuators. Commercial yarns were coated with poly(3,4‐ethylenedioxythiophene)‐poly(styrenesulfonate) (PEDOT:PSS) to make them conductive. Then polypyrrole (PPy) that provides the electromechanical actuation is electropolymerized on the yarn surface under controlled conditions. The linear actuation of the yarn actuators is investigated in aqueous electrolyte under isotonic and isometric conditions. The yarn actuators generated an isotonic strain up to 0.99% and isometric force of 95 mN. The isometric strain achieved in this work is more than tenfold and threefold greater than the previously reported yarn actuators. The isometric actuation force shows an increase of nearly 11‐fold over our previous results. Finally, a qualitative mechanical model is introduced to describe the actuation behavior of yarn actuators. The strain and force created by the yarn actuators make them promising candidates for wearable actuator technologies.