Disruption of Chromatin Dynamics by Hypotonic Stress Suppresses HR and Shifts DSB Processing to Error-Prone SSA.

The processing of DNA double-strand breaks (DSBs) depends on the dynamic characteristics of chromatin. To investigate how abrupt changes in chromatin compaction alter these dynamics and affect DSB processing and repair, we exposed irradiated cells to hypotonic stress (HypoS). Densitometric and chromosome-length analyses show that HypoS transiently decompacts chromatin without inducing histone modifications known from regulated local chromatin decondensation, or changes in Micrococcal Nuclease (MNase) sensitivity. HypoS leaves undisturbed initial stages of DNA-damage-response (DDR), such as radiation-induced ATM activation and H2AX-phosphorylation. However, detection of ATM-pS1981, γ-H2AX and 53BP1 foci is reduced in a protein, cell cycle phase and cell line dependent manner; likely secondary to chromatin decompaction that disrupts the focal organization of DDR proteins. While HypoS only exerts small effects on classical nonhomologous end-joining (c-NHEJ) and alternative end-joining (alt-EJ), it markedly suppresses homologous recombination (HR) without affecting DNA end-resection at DSBs, and clearly enhances single-strand annealing (SSA). These shifts in pathway engagement are accompanied by decreases in HR-dependent chromatid-break repair in the G2-phase, and by increases in alt-EJ and SSA-dependent chromosomal translocations. Consequently, HypoS sensitizes cells to ionizing radiation (IR)-induced killing. We conclude that HypoS-induced global chromatin decompaction compromises regulated chromatin dynamics and genomic stability by suppressing DSB-processing by HR, and allowing error-prone processing by alt-EJ and SSA.

Gelling hypotonic polymer solution for extended topical drug delivery to the eye.

Eye-drop formulations should hold as high a concentration of soluble drug in contact with ocular epithelium for as long as possible. However, eye tears and frequent blinking limit drug retention on the ocular surface, and gelling drops typically form clumps that blur vision. Here, we describe a gelling hypotonic solution containing a low concentration of a thermosensitive triblock copolymer for extended ocular drug delivery. On topical application, the hypotonic formulation forms a highly uniform and clear thin layer that conforms to the ocular surface and resists clearance from blinking, increasing the intraocular absorption of hydrophilic and hydrophobic drugs and extending the drug-ocular-epithelium contact time with respect to conventional thermosensitive gelling formulations and commercial eye drops. We also show that the conformal gel layer allows for therapeutically relevant drug delivery to the posterior segment of the eyeball in pigs. Our findings highlight the importance of formulations that conform to the ocular surface before viscosity enhancement for increased and prolonged ocular surface contact and drug absorption.

Action of Protein Tyrosine Kinase Inhibitors on the Hypotonicity-Stimulated Trafficking Kinetics of Epithelial Na+ Channels (ENaC) in Renal Epithelial Cells: Analysis Using a Mathematical Model.

BACKGROUND/AIMS: Epithelial Na+ channels (ENaCs) play crucial roles in control of blood pressure by determining the total amount of renal Na+ reabsorption, which is regulated by various factors such as aldosterone, vasopressin, insulin and osmolality. The intracellular trafficking process of ENaCs regulates the amount of the ENaC-mediated Na+ reabsorption in the collecting duct of the kidney mainly by determining the number of ENaC expressed at the apical membrane of epithelial cells. Although we previously reported protein tyrosine kinases (PTKs) contributed to the ENaC-mediated epithelial Na+ reabsorption, we have no information on the role of PTKs in the intracellular ENaC trafficking. METHODS: Using the mathematical model recently established in our laboratory, we studied the effect of PTKs inhibitors (PTKIs), AG1296 (10 µM: an inhibitor of the PDGF receptor (PDGFR)) and AG1478 (10 µM: an inhibitor of the EGF receptor (EGFR)) on the rates of the intracellular ENaC trafficking in renal epithelial A6 cells endogenously expressing ENaCs. RESULTS: We found that application of PTKIs significantly reduced the insertion rate of ENaC to the apical membrane by 56%, the recycling rate of ENaC by 83%, the cumulative time of an individual ENaC staying in the apical membrane by 27%, the whole life-time after the first insertion of ENaC by 47%, and the cumulative Na+ absorption by 61%, while the degradation rate was increased to 3.8-fold by application of PTKIs. These observations indicate that PTKs contribute to the processes of insertion, recycling and degradation of ENaC in the intracellular trafficking process under a hypotonic condition. CONCLUSION: The present study indicates that application of EGFR and PDGFR-inhibitable PTKIs reduced the insertion rate (kI), and the recycling rate (kR) of ENaCs, but increased degradation rate (kD) in renal A6 epithelial cells under a hypotonic condition. These observations indicate that hypotonicity increases the surface expression of ENaCs by increasing the insertion rate (kI) and the recycling rate (kR) of ENaCs associated with a decrease in the degradation rate but without any significant effects on the endocytotic rate (kE) in EGFR and PDGFR-related PTKs-mediated pathways.

Decellularization of Large Tendon Specimens: Combination of Manually Performed Freeze-Thaw Cycles and Detergent Treatment.

Reliable decellularization techniques applicable to tendon tissue play a critical role in the field of current tissue engineering. Particularly, an application as three-dimensional culture model for in vitro research and translational approaches to establish graft-based tendon repair as a routine clinical tool represent two main application fields for decellularized tendon scaffolds. Considering methodological issues of tendon decellularization, one of the major challenges lies in the preservation of the tendon-specific extracellular matrix (ECM) architecture to reflect natural tissue characteristic as best as possible. Concurrently, further requirements for high-quality decellularized biological tendon scaffolds include not only the reduction of resident cells, but also an ensured cytocompatibility.To date, a large number and a wide variety of decellularization protocols for natural tendon tissue have already been investigated and usually, physical as well as chemical and/or enzyme-based treatments are used for the purpose of decellularization. However, to the best of our knowledge, there is a lack of evidence-based protocols for the processing of full-thickness large tendon samples, such as the equine flexor tendons.Therefore, the here presented protocol describes a reliable procedure to decellularize equine superficial digital flexor tendons by using a combined treatment of physical decellularization in the form of repetitive freeze-thaw cycles, and of chemical decellularization with the non-ionic detergent Triton X-100. The decellularization effectiveness evaluated by reduction of cell and DNA content, the influence of decellularization on the morphology of the tendon extracellular matrix (ECM) as well as the cytocompatibility of the decellularized tendon scaffolds obtained have been investigated previously. Based on this previous study, the here present protocol is an effective procedure, particularly applicable for large tendon specimens.

Pulsatile Lipid Vesicles under Osmotic Stress.

The response of lipid bilayers to osmotic stress is an important part of cellular function. Recent experimental studies showed that when cell-sized giant unilamellar vesicles (GUVs) are exposed to hypotonic media, they respond to the osmotic assault by undergoing a cyclical sequence of swelling and bursting events, coupled to the membrane's compositional degrees of freedom. Here, we establish a fundamental and quantitative understanding of the essential pulsatile behavior of GUVs under hypotonic conditions by advancing a comprehensive theoretical model of vesicle dynamics. The model quantitatively captures the experimentally measured swell-burst parameters for single-component GUVs, and reveals that thermal fluctuations enable rate-dependent pore nucleation, driving the dynamics of the swell-burst cycles. We further extract constitutional scaling relationships between the pulsatile dynamics and GUV properties over multiple timescales. Our findings provide a fundamental framework that has the potential to guide future investigations on the nonequilibrium dynamics of vesicles under osmotic stress.

Low osmolality and shear stress during liposuction impair cell viability in autologous fat grafting.

BACKGROUND: Liposuction and subsequent autologous fat grafting have become essential techniques for fat augmentation in plastic surgery. However, standard harvesting techniques that ensure the survival of adipocytes and stromal vascular fraction (SVF) cells and thus preserve the transplanted fat volume are lacking. In particular, the effect of different parameters of the tumescent solution has not been studied in this context. We hypothesized that the osmolality of the tumescent solution could have a significant effect on the survival of adipocytes and SVF cells. METHODS: We developed two distinct in vitro models based on freshly harvested excision fat from patients undergoing surgical treatment. First, we investigated the effect of osmolality by incubating excision fat in different tumescent solutions and analyzed the total cell survival and the differentiation potential of SVF cells. Vital whole-mount staining, isolation yield of SVF cells, clonogenicity, and osteogenic and adipogenic differentiation capacities were analyzed. Second, we addressed the additional effect of mechanical stress by simulating a liposuction on pieces of excision fat after incubation with the tumescent solutions. RESULTS: Osmolality of the tumescent solution by itself did not have a significant effect on adipocyte and SVF viability or SVF differentiation. However, when osmolality was combined with liposuction, a significant trend toward lower viability and more lipid droplets with lower osmolality was observed. Especially, SVF viability was significantly lower after liposuction with a hypotonic (150 mOsm/kg) solution. CONCLUSION: This study demonstrates the considerable effect of osmolality during liposuction and may lead to the development of "cell-protective" tumescent solutions.

Lipophilic but not hydrophilic statin functionally inhibit volume-activated chloride channels by inhibiting NADPH oxidase in monocytes.

Volume-activated Cl- channels (VACCs) can be activated by hypotonic solutions and have been identified in many cell types. Here, we investigated the effects of different statins on VACCs in monocytes. Whole-cell patch clamp recordings demonstrated that a hypotonic solution induced 5-nitro-2- (3-phenylpropylamino) benzoic acid (NPPB)- and 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS)-sensitive VACC currents in human peripheral monocytes and RAW 264.7 cells. The VACC currents were inhibited by the lipophilic statin (simvastatin) but not by the hydrophilic simvastatin acid and pravastatin. A low-molecular-weight superoxide anion scavenger (tiron, 1 mM) and inhibitor of NADPH oxidase (DPI 10 µM) was able to abolish the VACC currents. A hypotonic solution increased the reactive oxygen species (ROS) detected by the fluorescence of dichlorodihydrofluorescein (DCF), which was abolished by tiron and DPI. NPPB, DIDS, and simvastatin but not pravastatin decreased the fluorescence of DCF. Simvastatin could not further decrease VACC currents when pretreated with tiron or DPI, whereas exogenous H2O2 (100 µM), increased the VACC currents and overcame the blockade of VACC currents by simvastatin. Functionally, hypotonic solution increased the TNF-α mRNA expression, which could be decreased by tiron, DPI, NPPB, DIDS and simvastatin but not pravastatin. However, simvastatin could not decrease the TNF-α expression further when pretreatment with tiron, DPI, NPPB or DIDS. We conclude that lipophilic (simvastatin) rather than hydrophilic statin inhibit VACCs and decrease hyposmolality induced inflammation in monocytes by inhibiting NADPH oxidase.

Optimizing Irreversible Electroporation Ablation with a Bipolar Electrode.

PURPOSE: To optimize single-insertion bipolar irreversible electroporation (IRE) by characterizing effects of electric parameters and controlling tissue electric properties in a porcine model. MATERIALS AND METHODS: Single-insertion electrode bipolar IRE was performed in 28 in vivo pig livers (78 ablations). First, effects of voltage (2,700-3,000 V), number of pulses, repeated cycles (1-6 cycles), and pulse width (70-100 µs) were studied. Next, electric conductivity was altered by instillation of hypertonic and hypotonic fluids. Finally, effects of thermal stabilization were assessed using internal electrode cooling. Treatment effect was evaluated 2-3 hours after IRE. Dimensions were compared and subjected to statistical analysis. RESULTS: Delivering 3,000 V at 70 µs for a single 90-pulse cycle yielded 3.8 cm ± 0.4 × 2.0 cm ± 0.3 of ablation. Applying 6 cycles of energy increased ablation to 4.5 cm ± 0.4 × 2.6 cm ± 0.3 (P < .001). Further increasing pulse lengths to 100 µs (6 cycles) increased ablation to 5.0 cm ± 0.4 × 2.9 cm ± 0.3 (P < .001) but resulted in electric spikes and system crashes in 40%-50% of cases. Increasing tissue electric conductivity via hypertonic solution instillation in surrounding tissues increased frequency of generator crashes, whereas continuous instillation of distilled water eliminated this arcing phenomenon but reduced ablation to 2.3 cm ± 0.1. Controlled instillation of distilled water when electric arcing was suspected from audible popping produced ablations of 5.3 cm ± 0.6 × 3.1 cm ±0.3 without crashes. Finally, 3.1 cm ± 0.1 short-axis ablation was achieved without system crashes with internal electrode perfusion at 37°C versus 2.3 cm ± 0.1 with 4°C-10°C perfusion (P < .001). CONCLUSIONS: Bipolar IRE ablation zones can be increased with repetitive high voltage and greater pulse widths accompanied by either judicious instillation of hypotonic fluids or internal electrode perfusion to minimize unwanted electric arcing.

Isotonic versus Hypotonic Parenteral Maintenance Fluids in Very Severe Pneumonia.

OBJECTIVE: To compare the risk of hyponatremia between hypotonic and isotonic parenteral maintenance solutions (PMS) administered to children with very severe pneumonia, admitted in the general pediatric ward. METHODS: A randomized controlled open label trial was conducted in the pediatrics department of a tertiary care medical college hospital including euvolemic children 2 mo to 5 y of age, fulfilling the WHO clinical definition of very severe pneumonia and requiring PMS. They were randomized to receive either isotonic PMS (0.9% saline in 5% dextrose and potassium chloride 20 meq/L) or hypotonic PMS (0.18% saline in 5% dextrose and potassium chloride 20 meq/L) at standard rates for next 24 h. RESULTS: A total of 119 children were randomized (59: Isotonic; 60: Hypototonic PMS). Nine (15%) children in the isotonic PMS group and 29 (48%) in the hypotonic PMS group developed hyponatremia during the study period, (p <0.001) with a relative risk being 3.16 (95% CI 1.64 to 6.09). Mean serum sodium was significantly lower in the hypotonic group compared to the isotonic group (p < 0.001 each at 6, 12 and 24 h). The difference in mean change in serum sodium from baseline was also significant at 12 and 24 h (5.4 and 5.8 meq/L respectively; p < 0.001 each). CONCLUSIONS: This study demonstrates the rationality of the use of isotonic PMS in children with respiratory infections, a condition regularly encountered by most pediatricians.

Osmolarity of prevalent eye drops, side effects, and therapeutic approaches.

PURPOSE: Little is known about how the osmolarity of ophthalmic formulations affects the ocular surface. Because hyperosmolar eye drops could be therapeutic for treating corneal edema, this article presents an ex vivo model of corneal edema for testing ophthalmic drugs based on their osmolarity. The respective osmolarity of common eye drops found in the German market is also analyzed here. METHODS: For modeling corneal edema, an Ex Vivo Eye Irritation Test was used to simulate an ocular anterior chamber with a physiological corneal barrier. To induce corneal edema, the anterior chamber was supplied with a hypoosmolar medium (148 mOsm/L) for 24 hours. Preserved and preservative-free 5% sodium chloride (hyperosmolar Omnisorb and Ocusalin 5% UD) were used for 1 hour, on 5 corneas each, to test their efficiency to reduce corneal edema in this model. Corneal thickness was determined by optical coherence tomography. Osmolarity of 87 common eye drops was measured by freezing point osmometry. RESULTS: Ex vivo, the tested hypoosmolar condition induced corneal edema from 450 µm (±50 µm) at baseline to 851 µm (±94 µm, P < 0.0001). Omnisorb and Ocusalin 5% UD significantly reduced the corneal thickness by 279 µm (±28 µm, P < 0.001) for Omnisorb and 258 µm (±29 µm, P < 0.001) for Ocusalin 5% UD. Forty-three (49%) of the tested products had an osmolarity below and 44 (51%) above the physiological tear osmolarity of 289 mOsm/L. Osmolarity values of less than 200 mOsm/L were found in lubricant drops. The highest osmolarity was detected in Omnisorb (1955 mOsm/L). CONCLUSIONS: The Ex Vivo Eye Irritation Test has proven to be a reliable novel model of corneal edema for evaluating osmotic eye drops. Osmolarity measurements revealed a wide range from hypotonic to hypertonic formulations for commonly marketed ophthalmic drugs.

Effect of a new remineralizing biomaterial on the color of dental enamel.

The aim of this research was to describe the effects of altering the composition of a modified remineralizing agent (MRA) and the osmotic pressure on tooth color by using spectrophotometric analysis. One hundred and four (104) human premolars and molars were randomly divided into 2 groups of 52 specimens each. Group 1 was treated with the remineralizing agent MRA 55, (remineralizing agent 1), a 50% - 50% by weight mixture of coarse-grain and fine-grain generating minerals, and group 2 was treated with the remineralizing agent MRA 91 (remineralizing agent 2), containing the same minerals in a 90% - 10% proportion. Each group was divided into 2 subgroups with 26 specimens each, which were stored as follows: subgroups A were stored in synthetic saliva with isotonic osmotic pressure (IP), and subgroups B in hypotonic osmotic pressure saliva (HP). The initial and final readings were taken with a Vita Easy Shade spectrophotometer. Color parameters (L*, a*, b*) and whiteness indices (WIC, WIO, W) were calculated from the readings. The color changes (deltaL, deltaA, deltaB, and deltaE) and whiteness indices were compared and analyzed with descriptive analyses. The variables deltaL, deltaA, deltaB, deltaE, and the whiteness index W were analyzed with an analysis of variance (ANOVA), and the indices WIC and WIO were analyzed with a nonparametric Kruskal-Wallis ANOVA. The results indicate that combination A2 (MRA 91 and IP) affected variables deltaB and deltaE, while combination B1 (MRA 55 and HP) affected variables deltaA, deltaB and the whiteness index WIO. Only MRA 91 affected the variable deltaL. The osmotic pressure of saliva and the remineralizing agent used affect the color of dental enamel.

Effect of a new remineralizing biomaterial on the color of dental enamel

El objetivo de este trabajo fue describir el efecto de la composición de una sustancia remineralizante (SRM) y de la presión osmótica sobre el color dental mediante espectrofotometría. Se tomaron 104 premolares y molares humanos repartidos aleatoriamente en 2 grupos, cada uno de 52 especímenes. El grupo 1 se trató con la sustancia remineralizante SRM 55 (agente remineralizante 1) mezcla de 50 por ciento - 50 por ciento de mineral de grano fino y otro mineral de grano grueso y el grupo 2 se trató con la sustancia remineralizante SRM 91(agente remineralizante 2) contienen los mismos minerales en proporción 90 por ciento - 10 por ciento. A su vez cada grupo se dividió en 2 subgrupos, cada uno de 26 especímenes que se almacenaron así: Un subgrupo en saliva sintética con presión osmótica isotónica (PI) y el otro con presión osmótica hipotónica (PH). Se tomaron registros iniciales y finales con el espectrofotómetro Vita Easy Shade®. Con las lecturas se calcularon losparámetros de color (L*; a*; b*) y los índices de blanqueamiento (WIC; WIO; W). Los cambios de color (ΔL; ΔA; ΔB; yΔE) y los índices de blanqueamiento se compararon y se trataron todos mediante un análisis descriptivo. Las variables ΔA, ΔL, ΔB, ΔE e índice de blanqueamiento W se trataron con ANOVA y los índices WIC y WIO con un análisis de varianza no paramétrico Kruskal Wallis. Los resultados indican que la combinación A2 (SRM 91 y PI) afectó las variables ΔB y ΔE. La combinación B1 (SRM 55 Y PH) afectó las variables ΔA, ΔB y el índice de blanqueamiento WIO. Solamente SRM 91afectó la variable ΔL. La presión osmótica de la saliva y la sustancia remineralizante afectan el color del esmalte dental...

Effect of a new remineralizing biomaterial on the color of dental enamel. / Effect of a new remineralizing biomaterial on the color of dental enamel.

The aim of this research was to describe the effects of altering the composition of a modified remineralizing agent (MRA) and the osmotic pressure on tooth color by using spectrophotometric analysis. One hundred and four (104) human premolars and molars were randomly divided into 2 groups of 52 specimens each. Group 1 was treated with the remineralizing agent MRA 55, (remineralizing agent 1), a 50

- 50

by weight mixture of coarse-grain and fine-grain generating minerals, and group 2 was treated with the remineralizing agent MRA 91 (remineralizing agent 2), containing the same minerals in a 90

- 10

proportion. Each group was divided into 2 subgroups with 26 specimens each, which were stored as follows: subgroups A were stored in synthetic saliva with isotonic osmotic pressure (IP), and subgroups B in hypotonic osmotic pressure saliva (HP). The initial and final readings were taken with a Vita Easy Shade spectrophotometer. Color parameters (L*, a*, b*) and whiteness indices (WIC, WIO, W) were calculated from the readings. The color changes (deltaL, deltaA, deltaB, and deltaE) and whiteness indices were compared and analyzed with descriptive analyses. The variables deltaL, deltaA, deltaB, deltaE, and the whiteness index W were analyzed with an analysis of variance (ANOVA), and the indices WIC and WIO were analyzed with a nonparametric Kruskal-Wallis ANOVA. The results indicate that combination A2 (MRA 91 and IP) affected variables deltaB and deltaE, while combination B1 (MRA 55 and HP) affected variables deltaA, deltaB and the whiteness index WIO. Only MRA 91 affected the variable deltaL. The osmotic pressure of saliva and the remineralizing agent used affect the color of dental enamel.

Stabilization of erythrocytes against oxidative and hypotonic stress by tannins isolated from sumac leaves (Rhus typhina L.) and grape seeds (Vitis vinifera L.).

Erythrocytes are constantly exposed to ROS due to their function in the organism. High tension of oxygen, presence of hemoglobin iron and high concentration of polyunsaturated fatty acids in membrane make erythrocytes especially susceptible to oxidative stress. A comparison of the antioxidant activities of polyphenol-rich plant extracts containing hydrolysable tannins from sumac leaves (Rhus typhina L.) and condensed tannins from grape seeds (Vitis vinifera L.) showed that at the 5-50 µg/ml concentration range they reduced to the same extent hemolysis and glutathione, lipid and hemoglobin oxidation induced by erythrocyte treatment with 400 µM ONOO(-) or 1 mM HClO. However, extract (condensed tannins) from grape seeds in comparison with extract (hydrolysable tannins) from sumac leaves stabilized erythrocytes in hypotonic NaCl solutions weakly. Our data indicate that both hydrolysable and condensed tannins significantly decrease the fluidity of the surface of erythrocyte membranes but the effect of hydrolysable ones was more profound. In conclusion, our results indicate that extracts from sumac leaves (hydrolysable tannins) and grape seeds (condensed tannins) are very effective protectors against oxidative damage in erythrocytes.

Isolation of hemoglobin from bovine erythrocytes by controlled hemolysis in the membrane bioreactor.

In this work, we describe an optimized procedure based on gradual hemolysis for the isolation of hemoglobin derived from bovine slaughterhouse erythrocytes in a membrane bioreactor. The membrane bioreactor system that provided high yields of hemoglobin (mainly oxyhemoglobin derivate) and its separation from the empty erythrocyte membranes (ghosts) was designed at a pilot scale. Ten different concentrations of hypotonic media were assessed from the aspect of the extent of hemolysis, hematocrit values of the erythrocyte suspensions, cell swelling, and membrane deformations induced by decreased salt concentration. Effective gradual osmotic hemolysis with an extent of hemolysis of 88% was performed using 35 mM Na-phosphate/NaCl buffer of pH 7.2-7.4. Under these conditions most of the cell membranes presented the appearance of the normal ghosts under phase contrast microscope. The hemoglobin purity of >80% was confirmed by SDS-PAGE. Kinetic studies showed that maximal concentration of hemoglobin was reached after 40 min, but the process cycle at which recovery of 83% was achieved lasted for 90 min. The dynamics of both steps, (1) transport through the membrane of erythrocytes during process of hemolysis and (2) transport through the reactor filters, were evaluated.

Cryobiological properties of immature zebrafish oocytes assessed by their ability to be fertilized and develop into hatching embryos.

As a step to develop a cryopreservation method for zebrafish oocytes, we investigated the cryobiological properties of immature oocytes at stage III by examining their ability to mature and to develop into hatching embryos after fertilization. When oocytes were chilled at -5°C for 30min, the maturation rate decreased, but the rates of fertilization and hatching were not significantly different from those of controls. When oocytes were exposed to hypotonic solutions for 60min at 25°C, the rates of maturation, fertilization, and hatching decreased in a solution with 0.16Osm/kg or below. When oocytes were exposed to hypertonic solutions (containing sucrose) at 25°C for 30min, the maturation rate decreased in solution with 0.51Osm/kg, whereas the hatching rate decreased with lower osmolality (0.40Osm/kg). In an experiment on the toxicity of cryoprotectants (∼10%, at 25°C), it was found that glycerol and ethylene glycol were toxic both by the assessment of maturation and hatching. Propylene glycol, DMSO and methanol were less toxic by the assessment of maturation, but were found to be toxic by the assessment of hatching. Methanol was the least toxic, but it was less effective to make a solution vitrify than propylene glycol. Therefore, a portion of methanol was replaced with propylene glycol. The replacement increased the toxicity, but could be effective to reduce chilling injury at -5°C. These results clarified the sensitivity of immature oocytes to various cryobiological properties accurately, which will be useful for realizing cryopreservation of zebrafish oocytes.

A novel isotonic-balanced electrolyte solution with 1% glucose for intraoperative fluid therapy in children: results of a prospective multicentre observational post-authorization safety study (PASS).

BACKGROUND: The recommendations for intraoperative fluid therapy in children have been adapted from hypotonic to isotonic electrolyte solutions with lower glucose concentrations (1-2.5% instead of 5%) to avoid hyponatremia and hyperglycemia. OBJECTIVE: The objective of this prospective multicentre observational post-authorization safety study was to evaluate the intraoperative use of a novel isotonic-balanced electrolyte solution with 1% glucose (BS-G1) with a particular focus on changes in acid-base status, electrolyte and glucose concentrations. METHODS: Following local ethics committee approval, pediatric patients aged up to 4 years with an ASA risk score of I-III undergoing intraoperative administration of BS-G1 were enrolled. Patient demographics, the performed procedure, adverse drug reactions, hemodynamic data, and the results of blood gas analysis before and after infusion were documented with a focus on changes in acid-base status, electrolyte and glucose concentrations. RESULTS: In 107 patients (ASA I-III; age 16.2 ± 15.4, range day of birth to 47.7 months; body weight 8.8 ± 4.8, range 1.6-18.8 kg), the mean volume infused was 20 ± 12.6 (range 3.6-83.3) ml·kg(-1) BS-G1. During the infusion, hemoglobin, hematocrit, anion gap, strong ion difference, and calcium decreased and chloride and glucose increased significantly within the physiologic range. All other measured parameters including sodium, bicarbonate, base excess, and lactate remained stable. Neither hypoglycemia (glucose <2.5 mmol·l(-1) ) nor hyperglycemia (glucose >10 mmol·l(-1) ) was documented after BS-G1 infusion. No adverse drug reactions were reported. CONCLUSION: The studied isotonic-balanced electrolyte solution with 1% glucose helps to avoid perioperative acid-base imbalance, hyponatremia, hyperglycemia, and ketoacidosis in infants and toddlers and may therefore enhance patient safety.

Cell swelling-induced insulin secretion from INS-1E cells is inhibited by extracellular Ca2+ and is tetanus toxin resistant.

UNLABELLED: Cell swelling-induced insulin secretion represents an alternative pathway of stimulation of insulin secretion. INS-1E rat tumor beta cells do not release insulin in response to cell swelling in presence of Ca(2+) despite a good response to glucose challenge and appropriate increase in cell volume. Surprisingly, perifusion with Ca(2+)-depleted medium showed distinct secretory response of INS-1E cells to hypotonicity. Objective of this study was further characterization of the role of Ca(2+) in secretory process in INS-1 and INS-1E cell lines. Ca(2+) depleted hypotonic medium with 10 muM BAPTA/AM (intracellular chelator) induced insulin secretion from both types of cells. We demonstrated expression of L-type Ca(2+) channel Ca(v)1.2 and non-L-type Ca(2+) channels Ca(v)2.1 (P/Q-type), Ca(v)2.2 (N-type), and Ca(v)3.1 (T-type) in both cell lines. Inhibition of L type channel with nifedipine and/or P/Q type with omega-agatoxin IVA enabled distinct response to hypotonic medium also in INS-1E cells. Tetanus toxin (TeTx) in medium containing Ca(2+) and a group of calcium channel blockers inhibited hypotonicity-induced insulin secretion from INS-1 cells but not from INS-1E cells. CONCLUSION: Hypotonicity-induced insulin secretion from INS-1E cells is inhibited by extracellular Ca(2+), does not require intracellular Ca(2+) and is TeTx resistant.