Hypotonic Conditions Research Articles

Color‐Tunable Elastic Photonic Shells With High Color Saturation and Deformability

AbstractPhotonic microspheres containing colloidal arrays are useful as structural colorants in inks and cosmetics. However, low color saturation and low deformability of the microspheres restrict the uses. To overcome the limitations, elastic photonic shells with enhanced color saturation and high deformability using water‐in‐oil‐in‐water (W/O/W) double‐emulsion templates are designed. The elastic shells are produced by photocuring acrylate‐based resin containing non‐close‐packed arrays of silica particles in the middle oil layer. The presence of two concentric interfaces and water‐soluble molecules in the inner and continuous water phases at high concentrations improve the crystalline order of silica particles. In addition, the absence of a relatively disordered core reduces incoherent random scattering, thereby enhancing color saturation. Although the shells themselves are elastically deformable, the liquid core renders them significantly more deformable with small stiffness and negligible hysteresis, which is potentially beneficial for improving the fluidity of suspensions. Moreover, the structural colors of the elastic shells are post‐tunable by osmotic inflation of the semipermeable shells in hypotonic conditions. The high color saturation and high deformability as well as color tunability will provide a new opportunity for structural coloration with the elastic photonic shells as a colorant in inks and cosmetics.

Effects of salinity changes on larval survival, growth, and development of the edible sea cucumber, Holothuria leucospilota (Brandt, 1835) at two developmental stages

For large-scale artificial breeding of sea cucumber (Holothuria leucospilota), it is necessary to understand how environmental parameters affect larval rearing. In this study, the effects of salinity changes on survival, growth, and development of the mid- and late-auricularia of H. leucospilota were estimated within a 7-day culture period. Salinity shock is usually accompanied by water replacement during hatching. To replicate the occurrence of salinity changes in actual larval production, 1/2 of the larval rearing water (initial salinity ∼31.6 ppt) was replaced daily by the experimental waters at different salinity levels (18, 22, 26, 30, 34, 38, and 42 ppt). On day 7, larval survival rates varied with salinity, while the highest survival rates (44.37–46.63% for mid-auricularia; 50.71–53.98% for late-auricularia) were observed in normal salinities (30 and 34 ppt) and the lowest survival rates (15.71–23.96% for mid-auricularia; 24.11–29.57% for late-auricularia) were observed in extreme salinities (18, 22, and 42 ppt). For any sampling event, the body length of mid-auricularia in normal salinities (30 and 34 ppt) were continued to increase during the 7-day culture period. The highest length of late-auricularia was reached on day 3 (880.01 ± 84.38 and 889.06 ± 112.11 µm, respectively), then decreased to 745.31 ± 127.64 and 742.94 ± 98.43 µm on day 7 due to metamorphosis. On day 7, the proportion of mid-auricularia larvae without development potential was the lowest (36.67%) at a salinity level of 34 ppt, and the proportions were increased with both decreasing/increasing salinities. However, this proportion was much lower for the late-auricularia at the same salinity. The decrease in salinity could promote metamorphosis of the late-auricularia, and the highest proportion of doliolaria (24.32%) was observed at salinity level of 26 ppt on day 7. In extreme salinities (18, 22, and 42 ppt), the hyaline spheres (indication of larval competence) in late-auricularia were less obvious than those in other salinities. The ability in resisting salinity shock was increased with larval development, where late-auricularia had better tolerance to salinity shock than mid-auricularia, whereas larvae at both stages were more tolerant to hypertonic than hypotonic conditions. Based on our results, we propose to rear the mid- and late-auricularia at a comparatively high level of salinity (e.g., ∼31 ppt) to promote their survival and performance, while to decrease the salinity (e.g., ∼26 ppt) in the last period of late-auricularia to increase their competence to metamorphose into doliolaria.

Correlation between sphingomyelin and the membrane stability of mammalian erythrocytes.

Lipid compositions of mammalian erythrocyte membranes are different among species. Therefore, the information on hemolysis from mammalian erythrocytes is useful to understand membrane properties of human erythrocytes. In this work, pressure-induced hemolysis and hypotonic one were examined using erythrocytes of human, sheep, cow, cat, dog, pig, horse, rat, and mouse. Pressure-induced hemolysis was suppressed by membrane sphingomyelin, whereas hypotonic hemolysis decreased upon increment of cell diameter. Mass spectra of erythrocyte membrane lipids demonstrated that sphingomyelin with an acyl chain 24:1 was associated with the suppression of pressure-induced hemolysis. In cow erythrocytes, pressure-induced hemolysis was greatly suppressed and the detachment of cytoskeletal proteins from the membrane under hypotonic conditions was also inhibited. Taken together, these results suggest that sphingomyelin with 24:1 fatty acid plays an important role in the stability of the erythrocyte membrane, perhaps via cholesterol.

Transcriptomic changes in single yeast cells under various stress conditions

BackgroundThe stress response of Saccharomyces cerevisiae has been extensively studied in the past decade. However, with the advent of recent technology in single-cell transcriptome profiling, there is a new opportunity to expand and further understanding of the yeast stress response with greater resolution on a system level. To understand transcriptomic changes in baker’s yeast S. cerevisiae cells under stress conditions, we sequenced 117 yeast cells under three stress treatments (hypotonic condition, glucose starvation and amino acid starvation) using a full-length single-cell RNA-Seq method.ResultsWe found that though single cells from the same treatment showed varying degrees of uniformity, technical noise and batch effects can confound results significantly. However, upon careful selection of samples to reduce technical artifacts and account for batch-effects, we were able to capture distinct transcriptomic signatures for different stress conditions as well as putative regulatory relationships between transcription factors and target genes.ConclusionOur results show that a full-length single-cell based transcriptomic analysis of the yeast may help paint a clearer picture of how the model organism responds to stress than do bulk cell population-based methods.

Hypo-osmolarity induces apoptosis resistance via TRPV2-mediated AKT-Bcl-2 pathway.

In cirrhosis, several molecular alterations such as resistance to apoptosis could accelerate carcinogenesis. Recently, mechanotransduction has been attracting attention as one of the causes of these disturbances. In patients with cirrhosis, the serum sodium levels progressively decrease in the later stage of cirrhosis, and hyponatremia leads to serum hypo-osmolality. Since serum sodium levels in patients with cirrhosis with liver cancer are inversely related to cancer's number, size, stage, and cumulative survival, we hypothesized that hypo-osmolality-induced mechanotransduction under cirrhotic conditions might contribute to oncogenesis and/or progression of hepatocellular carcinoma (HCC). In this study, we adjusted osmosis of culture medium by changing the sodium chloride concentration and investigated the influence of hypotonic conditions on the apoptosis resistance of an HCC cell line, HepG2, using a serum-deprivation-induced apoptosis model. By culturing the cells in a serum-free medium, the levels of an antiapoptotic protein Bcl-2 were downregulated. In contrast, the hypotonic conditions caused apoptosis resistance by upregulation of Bcl-2. Next, we examined which pathway was involved in the apoptosis resistance. Hypotonic conditions enhanced AKT signaling, and constitutive activation of AKT in HepG2 cells led to upregulation of Bcl-2. Moreover, we revealed that the enhancement of AKT signaling was caused by intracellular calcium influx via a mechanosensor, TRPV2. Our findings suggested that hyponatremia-induced serum hypotonic in patients with cirrhosis promoted the progression of hepatocellular carcinoma.NEW & NOTEWORTHY Our study first revealed that hypo-osmolarity-induced mechanotransduction enhanced calcium-mediated AKT signaling via TRPV2 activation, resulting in contributing to apoptosis resistance. The finding indicates a possible view that liver cirrhosis-induced hyponatremia promotes hepatocellular carcinogenesis.

Upregulation of LRRC8A by m5C modification-mediated mRNA stability suppresses apoptosis and facilitates tumorigenesis in cervical cancer.

Cervical cancer (CC) is one of the most common gynecological malignancies with poor prognosis for advanced CC patients. LRRC8A is a volume-regulated anion channel protein involved in cellular homeostasis, but its role in CC remains largely unknown. In this study, we found that LRRC8A is elevated in CC and associated with poor prognosis. LRRC8A maintains cell survivals under the hypotonic condition, and promotes tumorigenesis through apoptosis suppression in vitro and in vivo. Notably, LRRC8A is upregulated by NSUN2-mediated m5C modification. m5C modified-LRRC8A mRNA is bound by the RNA binding protein YBX1 followed by the increased RNA stability. Moreover, loss of NSUN2 suppresses the proliferation and metastasis of CC cells, and NSUN2 expression is positively correlated with LRRC8A expression in CC. Altogether, our study demonstrates that the NSUN2-m5C-LRRC8A axis is crucial and would be a potential therapeutic target for CC.

The rpoS gene confers resistance to low osmolarity conditions in Salmonella enterica serovar Typhi.

Salmonella enterica serovars Typhimurium and Typhi are enteropathogens that differ in host range and the diseases that they cause. We found that exposure to a combination of hypotonicity and the detergent Triton X-100 significantly reduced the viability of the S. Typhi strain Ty2 but had no effect on the S. Typhimurium strain SL1344. Further analysis revealed that hypotonicity was the critical factor: incubation in distilled water alone was sufficient to kill Ty2, while the addition of sodium chloride inhibited killing in a dose-dependent manner. Ty2's loss of viability in water was modified by culture conditions: bacteria grown in well-aerated shaking cultures were more susceptible than bacteria grown under less aerated static conditions. Ty2, like many S. Typhi clinical isolates, has an inactivating mutation in the rpoS gene, a transcriptional regulator of stress responses, whereas most S. Typhimurium strains, including SL1344, have the wild-type gene. Transformation of Ty2 with a plasmid expressing wild-type rpoS, but not the empty vector, significantly increased survival in distilled water. Moreover, an S. Typhi strain with wild-type rpoS had unimpaired survival in water. Inactivation of the wild-type gene in this strain significantly reduced survival, while replacement with an arabinose-inducible allele of rpoS restored viability in water under inducing conditions. Our observations on rpoS-dependent differences in susceptibility to hypotonic conditions may be relevant to the ability of S. Typhi and S. Typhimurium to tolerate the various environments they encounter during the infectious cycle. They also have implications for the handling of these organisms during experimental manipulations.

Achog1 is required for the asexual sporulation, stress responses and pigmentation of Aspergillus cristatus.

Aspergillus cristatus is the dominant fungus during the fermentation of Fuzhuan brick tea; hypotonic conditions only induce its sexual development to produce ascospores, while hypertonic conditions only induce its asexual development to produce conidia, indicating that osmotic stress can regulate spore production in A. cristatus. However, the underlying regulatory mechanism is unclear. In this study, the role of Achog1, which is homologous to hog1 from Saccharomyces cerevisiae, in sporulation, different kinds of stress responses and pigment production was investigated. Deletion mutants of Achog1 were obtained by homologous recombination. Phenotypic observations showed that the time required to produce conidia was delayed, and the number of conidia produced was significantly reduced in the deletion mutants of Achog1 in hypertonic media, indicating that Achog1 plays a positive role in asexual development. Stress sensitivity tests showed that ΔAchog1 strains were sensitive to hyperosmolarity, and the order of the sensitivity of ΔAchog1 to different osmotic regulators was 3 M sucrose >3 M NaCl >3 M sorbitol. Moreover, the deletion mutants were sensitive to high oxidative stress. pH sensitivity tests indicated that Achog1 inhibited the growth of A. cristatus under alkaline stress. Additionally, pigmentation was decreased in the Achog1 deletion mutants compared with the WT. All the above developmental defects were reversed by the reintroduction of the Achog1 gene in ΔAchog1. Pull-down and LC-MS/MS analysis showed that the expression levels of proteins interacting with Achog1 were significantly different under low and high osmotic stress, and proteins related to conidial development were present only in the cultures treated with hyperosmotic stress. Transcription profiling data showed that Achog1 suppressed the expression of several genes related to asexual development, osmotic and oxidative stress resistance. On the basis of gene knockout, pull-down mass spectrometry and RNA-seq analyses, a regulatory pathway for Achog1 was roughly identified in A. cristatus.

The role of Acpbs2 in the asexual sporulation, stress responseand carbon metabolism of Aspergillus cristatus.

Aspergillus cristatus is the dominant fungus during the fermentation of Fuzhuan brick tea, hypotonic conditions only induced its sexual development to produce ascospores, while hypertonic conditions only induced its asexual development to produce conidia, indicating that osmotic stress can regulate spore production in A. cristatus. However, the underlying regulatory mechanism is unclear. In this study, the roles of Acpbs2, which is homologous to pbs2 from Saccharomyces cerevisiae, in sporulation, stress responses, the color of colonies, and carbon metabolism were explored in A. cristatus. Deletion mutants of Acpbs2 were obtained by homologous recombination. The time required to produce conidia was delayed, and the number of conidia produced was significantly reduced in hypertonic media in ΔAcpbs2 by phenotypic observations, indicating that Acpbs2 plays a positive role in asexual development. Stress sensitivity tests showed that the order of the sensitivity of ΔAcpbs2 to different osmotic regulators was 3 M NaCl > 3 M sucrose > 3 M sorbitol. Moreover, the deletion mutants were sensitive to high oxidative stress. The growth of the Acpbs2 deletion mutant was inhibited under alkaline-pH stress, indicating that Acpbs2 is involved in high pH stress tolerance. Additionally, compared with the wild type, the colony color of the Acpbs2 deletion mutant became lighter. All the above developmental defects were reversed by the reintroduction of the Acpbs2 gene in ΔAcpbs2. Transcriptome data showed that Acpbs2 regulated the expression of several genes related to conidial development, osmotic stress, oxidative stress, and carbon metabolism. More importantly, the interaction between Acpbs2 and its downstream gene Achog1 was verified by yeast two-hybrid assays. We speculated that this interaction might regulate the osmotic stress response, the oxidative stress response, and asexual sporulation in A. cristatus, which will be one of the focuses of our future research.

Hypotonic induction of aquaporin5 expression in rat astrocytes through p38 MAPK pathway.

Brain oedema is a common pathological phenomenon following many diseases and may lead to severe secondary damage. Astrocytes are the most numerous cells in the brain. Five aquaporins (AQPs) have been found in mature astrocytes, which play crucial roles in water transportation. However, most studies have focused on AQP4 or AQP9 and whether another aquaporin such as AQP5 involved in brain oedema is unclear. Here, we addressed the issue that the expression pattern of AQP5 in rat astrocytes in vitro was altered in the hypotonic condition through some mitogen-activated protein kinases (MAPK) pathways. Primary astrocytes were randomly divided into the control group and the hypotonic group. Cell viability was evaluated by MTT test. Immunofluorescence, Western blotting and real-time PCR were used to detect the expression of AQP5. Western blotting was used to detect the variation of MAPK pathway. The present study demonstrated that incubation of astrocytes in the hypotonic medium produced an increase inAQP5 expression, and AQP5 peaked at 6-12 h after hypotension solution exposure. In addition, MAPK pathways were set in motion under hypotension, but not all branches. Only the p38 inhibitor can inhibit AQP5 expression in cultured astrocytes. AQP5 is directly related to the extracellular hypotonic stimuli in astrocytes, which could be regulated through the p38 MAPK pathway.

The Bacillus anthracis S-layer is an exoskeleton-like structure that imparts mechanical and osmotic stabilization to the cell wall.

Surface layers (S-layers) are 2D paracrystalline protein monolayers covering the cell envelope of many prokaryotes and archaea. Proposed functions include a role in cell support, as scaffolding structure, as molecular sieve, or as virulence factor. Bacillus anthracis holds two S-layers, composed of Sap or EA1, which interchange in early and late exponential growth phase. We previously found that acute disruption of B. anthracis Sap S-layer integrity, by means of nanobodies, results in severe morphological cell surface defects and cell collapse. Remarkably, this loss of function is due to the destruction of the Sap lattice structure rather than detachment of monomers from the cell surface. Here, we combine force nanoscopy and light microscopy observations to probe the contribution of the S-layer to the mechanical, structural, and functional properties of the cell envelope, which have been so far elusive. Our experiments reveal that cells with a compromised S-layer lattice show a decreased compressive stiffness and elastic modulus. Furthermore, we find that S-layer integrity is required to resist cell turgor under hypotonic conditions. These results present compelling experimental evidence indicating that the S-layers can serve as prokaryotic exoskeletons that support the cell wall in conferring rigidity and mechanical stability to bacterial cells.

Novel treatment for hypotonic airway obstruction and severe obstructive sleep apnea using a nasopharyngeal airway device with 3D printing innovation.

Pediatric obstructive sleep apnea impacts child and familial well-being. Airway management in patients with hypotonic pharyngeal conditions is complex. Some patients may benefit from continuous positive airway pressure or bilevel positive airway pressure, others may require further invasive measures for treatment. There is critical need for treatment alternatives for patients with pharyngeal hypotonia. This is a retrospective case series. Collaboratively with patients, families, biomedical engineers, and medical professionals, a long-term nasopharyngeal airway (NPA) was created to bypass upper airway obstruction. Two patients used a safety pin and tape attachment, and two patients used a novel 3D-printed, self-supporting nasal securement. All 4 patients had polysomnography before and during NPA use. Paired 1-tailed t-tests were conducted to compare apnea-hypopnea index, hypopnea index, obstructive index, and oxygen nadir. Compared to baseline polysomnography, repeat polysomnography with the NPA in place demonstrated statistically significant improvement for apnea-hypopnea index (75.8 ± 36.6 events/h to 8.9 ± 2.9 events/h, P = .03), hypopnea index (45.4 ± 25.8 events/h to 7.7 ± 3.2 events/h, P = .04), and oxygen saturation nadir (60.3 ± 13.0% to 79.3 ± 8.7%, P = .01). The NPA had been used for over 1 year in 3 of the 4 children. Those using the safety pin and tape did report skin irritation due to adhesive required to keep device in place. Current management of severe upper airway obstruction and obstructive sleep apnea in hypotonic pharyngeal conditions requires a team-based approach to care. A long-term NPA device may be an alternative or temporizing option to continuous positive airway pressure, upper airway surgery, or tracheostomy in children with pharyngeal hypotonia and severe obstructive sleep apnea. Larger studies of this approach are underway to assess efficacy in a range of obstructive sleep apnea severity in this population. Powell AR, Srinivasan S, Helman JL, etal. Novel treatment for hypotonic airway obstruction and severe obstructive sleep apnea using a nasopharyngeal airway device with 3D printing innovation. J Clin Sleep Med. 2022;18(10):2497-2502.

In Vitro Anti-Inflammatory Evaluation of Sesbania Procumbens Extracts

Plants used in the traditional healing system require a systematic evaluation that may give more promising data about their medicinal value and may be useful to meet the rising demand for novel agents to combat infections and diseases. The plant Sesbania procumbens is a one used traditionally by the rural population for a varietyof medicinal purposes including anti inflammatory activity. Based on this report the plant S. procumbens was selected for our research and in this study different extracts of S. procumbens were evaluated in vitro for anti-inflammatory activity. The whole plant material was collected, authenticated, aerial parts were separated, shade dried for about two weeks and made into a coarse powder by using mechanical grinder. Extraction of powdered material was done by soxhlation using the different solvents of increasing polarity viz., petroleum ether, chloroform, ethyl acetate and methanol. Dried extracts thus obtained were subjected to in vitro anti-inflammatory evaluation by human red blood cell (HRBC) membrane stabilization in normal and different temperature (54°C and −10°C) and tonicity (hypotonic and isotonic) conditions. Six different concentrations (100, 200, 400, 600, 800, 1000μg/ml) of four extracts were subjected to evaluation with indomethacin (100μg/ml) as standard control. The data thus collected was analysed by using SPSS version 15.0. All values are expressed as mean ± SD. 0.05 was considered statistically significant From the results, a dose-dependent rise in antiinflammatory activity was found in all the tested extracts. Particularly, the methanol and ethyl acetate extracts showed significant activity in their maximum testing concentration (1000µg/ml) in all the experimental approaches. The results of this study strongly supported the claims of the anti-inflammatory property of the tested extracts. Further studies such as in vivo evaluations and phytochemical screening of the extracts may give more significant data which may be useful for the development of novel chemotherapeutic agents

Poster 105: Nanowarming by Inductive Heating of Vitrified Large Articular Cartilage Specimens, In Vivo Testing with Hanford Mini Pigs

Poster 105: Nanowarming by Inductive Heating of Vitrified Large Articular Cartilage Specimens, In Vivo Testing with Hanford Mini Pigs

Uptake of Fluorescein upon Osmotic Cell Swelling is Dependent on the Volume- Regulated Anion Channel VRAC/LRRC8

Background/Aims: The permeability of the plasma membrane for some substances increases during osmotic swelling of the cell. Transport systems for cationic dyes were identified previously, but the hypotonic uptake of anionic substances remained elusive. We aimed to test for the hypotonic uptake of the anionic dye fluorescein and to identify the transport pathway. Methods: We quantified the uptake of fluorescein in hypoosmotic medium by fluorescence microscopy in several cell lines. To investigate the uptake mechanism, we used pharmacological inhibitors for the volume-regulated anion channel VRAC and a genome-edited cell line lacking the essential LRRC8A VRAC subunit. Results: Fluorescein was specifically taken up under hypotonic conditions in HeLa, MDCK, and 3T3 cells. Both pharmacological inhibition and genetic disruption of VRAC strongly diminished hypotonicity-driven dye uptake. Conclusion: Cellular uptake of fluorescein is mediated by the volume-regulated anion channel VRAC. The measurement of fluorescein accumulation can be used as a convenient method to detect and study VRAC activation.

Pikeperch (Sander lucioperca) spermatozoa motility and volume regulation under different osmotic and ionic conditions.

Pikeperch (Sander lucioperca) is a highly profitable commercial species whose economic value has greatly increased in the last decade. As in other species, the quality of spermatozoa in this species is a principal feature inherent in fertilization success and efficient natural and artificial reproduction. The capacity of fish spermatozoa to be activated and tolerate environmental changes (in osmolality, ion composition, external pH, temperature, etc.) during the motility period contributes to fertilization success. In this study, we investigated the effects of environmental osmolality and ion composition on spermatozoa motility. To determine if the activation mechanism is affected by sperm quality parameters, we measured semen characteristics such as semen volume, spermatozoa concentration, seminal fluid osmolality and ion composition, and spermatozoa lipid composition. An additional parameter of sperm quality reflecting spermatozoa osmoresistance, the swelling rate, was measured by the nephelometry method. We detected that sperm samples with the highest content of palmitic (C16:0) and palmitoleic (C16:1) acids showed the lowest motility activation under the studied conditions, suggesting that these fatty acids are possible markers for the determination of spermatozoa quality in fish. Our results show that pikeperch spermatozoa can be activated under different osmotic conditions and that cell swelling always accompanies motility. However, spermatozoa sustain their volume under hypotonic conditions when motility is not initiated, suggesting that pikeperch spermatozoa activation is mainly controlled by ion composition rather than the osmolarity of the surrounding medium.

Effect of a C‐terminal Electrostatic Interaction Domain on KCC3 Function

BackgroundThe K:Cl cotransporters (KCCs) are key players to maintain cellular volume, mediate epithelial transport and intracellular chloride concentration, and regulate GABA neurotransmission. Under isotonic conditions, KCCs are normally inhibited due to the phosphorylation of residues located within the cytoplasmic domains. On the contrary, dephosphorylation of those residues in response to hypotonic cell swelling stimulates transport activity. Dysfunction of these proteins and the processes involved in their regulation have been associated with various human diseases by alteration of ionic homeostasis mainly in the kidney and the brain. Based on the cryo‐EM structures recently reported of human KCCs, revealing structural determinants for regulation in both N‐ and C‐terminal domains, in the present study we analyzed the effect of a structural domain located at the C‐ terminal involved in electrostatic interactions. It has been stated that the side chains of residues as Gln (Q), Arg (R) and Lys (K) tend to form hydrogen bonds promoting the stabilization of tertiary and quaternary protein structures. However, whether the phosphorylation status is modified after the electrostatic disruption is unknown.MethodsXenopus laevis oocytes and HEK 293 cells were either microinjected or transfected with KCC3a wild type (WT) or C‐terminal mutants, Q905A, Q905/K934A (2M), Q905/K934/R937A (3M) and Q905/K934/R937/K938A (4M), key residues involved in electrostatic interactions and located within the C‐terminal domain of the cotransporter. The phosphorylation status of KCC3a in response to isotonic and hypotonic conditions was evaluated by immunoblot analysis of three key regulatory phosphorylation sites (pSer96, pThr991 and pThr1048). In parallel, phosphorylation status of SPAK/OSR1 activating regulatory site pSer373/pSer325 was analyzed under the same conditions.ResultsCell swelling‐induced dephosphorylation is not observed on C‐terminal electrostatic interaction domain mutants compared to WT KCC3 in X. laevis oocytes and HEK cells. All three regulatory‐phosphorylation sites in KCC3a mutants remained phosphorylated under hypotonic conditions where dephosphorylation is required for the protein to become active. These results suggest that the disruption of the hydrogen bonds could disrupt the proper folding of the protein and thus interfere with the regulatory dephosphorylation/phosphorylation signaling pathway which is known to activate the transporter after cell swelling.ConclusionsOur results show that the four residues in the electrostatic interaction domain of the C‐termini are involved in KCC3a response to hypotonic conditions affecting the phosphorylation status of the cotransporter. The side chains of these residues seem to be important in maintaining tertiary conformation given their ability to form hydrogen bonds and thus, the disruption of their interactions affects the cotransporter ability to respond to osmotic and cell volume changes.

Combination of Hypotonic Lysis and Application of Detergent for Isolation of Polyhydroxyalkanoates from Extremophiles.

Production of polyhydroxyalkanoates (PHA), microbial biopolyesters, employing extremophilic microorganisms is a very promising concept relying on robustness of such organisms against microbial contamination, which provides numerous economic and technological benefits. In this work, we took advantage of the natural susceptibility of halophilic and thermophilic PHA producers to hypotonic lysis and we developed a simple and robust approach enabling effective isolation of PHA materials from microbial cells. The method is based on the exposition of microbial cells to hypotonic conditions induced by the diluted solution of sodium dodecyl sulfate (SDS) at elevated temperatures. Such conditions lead to disruption of the cells and release of PHA granules. Moreover, SDS, apart from its cell-disruptive function, also solubilizes hydrophobic components, which would otherwise contaminate PHA materials. The purity of obtained materials, as well as the yields of recovery, reach high values (values of purity higher than 99 wt.%, yields close to 1). Furthermore, we also focused on the removal of SDS from wastewater. The simple, inexpensive, and safe technique is based on the precipitation of SDS in the presence of KCl. The precipitate can be simply removed by decantation or centrifugation. Moreover, there is also the possibility to regenerate the SDS, which would substantially improve the economic feasibility of the process.

Osmosis-Mediated Microfluidic Production of Submillimeter-Sized Capsules with an Ultrathin Shell for Cosmetic Applications.

There is a demand for submillimeter-sized capsules with an ultrathin shell with high visibility and no tactile sensation after release for cosmetic applications. However, neither bulk emulsification nor droplet microfluidics can directly produce such capsules in a controlled manner. Herein, we report the microfluidic production of submillimeter-sized capsules with a spacious lumen and ultrathin biodegradable shell through osmotic inflation of water-in-oil-in-water (W/O/W) double-emulsion drops. Monodisperse double-emulsion drops are produced with a capillary microfluidic device to have an organic solution of poly(lactic-co-glycolic acid) (PLGA) in the middle oil layer. Hypotonic conditions inflate the drops, leading to core volume expansion and oil-layer thickness reduction. Afterward, the oil layer is consolidated to the PLGA shell through solvent evaporation. The degree of inflation is controllable with the osmotic pressure. With a strong hypotonic condition, the capsule radius increases up to 330 μm and the shell thickness decreases to 1 μm so that the ratio of the thickness to radius is as small as 0.006. The large capsules with an ultrathin shell readily release their encapsulant under an external force by shell rupture. In the mechanical test of single capsules, the threshold strain for shell rupture is reduced from 75 to 12%, and the threshold stress is decreased by two orders for highly inflated capsules in comparison with noninflated ones. During the shell rupture, the tactile sensation of capsules gradually disappears as the capsules lose volume and the residual shells are ultrathin.

Study of transmembrane ion transport under tonicity imbalance using a combination of low frequency-electrical impedance spectroscopy (LF-EIS) and improved ion transport model

Transmembrane ion transport under tonicity imbalance has been investigated using a combination of low frequency-electrical impedance spectroscopy (LF-EIS) and improved ion transport model, by considering the cell diameter d [m] and the initial intracellular ion concentration c in [mM] as a function of tonicity expressed by sucrose concentration c s [mM]. The transmembrane ion transport is influenced by extracellular tonicity conditions, leading to a facilitation/inhibition of ion passage through the cell membrane. The transmembrane transport coefficient P [m s−1], which represents the ability of transmembrane ion transport, is calculated by the extracellular ion concentrations obtained by improved ion transport model and LF-EIS measurement. P is calculated as 4.11 × 10−6 and 3.44 × 10−6 m s−1 at c s of 10 and 30 mM representing hypotonic condition, 2.44 × 10−6 m s−1 at c s of 50 mM representing isotonic condition, and 3.68 × 10−6, 5.16 × 10−6 , 9.51 × 10−6, and 14.89 × 10−6 m s−1 at c s of 75, 100, 125 and 150 mM representing hypertonic condition. The LF-EIS results indicate that the transmembrane ion transport is promoted under hypertonic and hypotonic conditions compared to isotonic condition. To verify the LF-EIS results, fluorescence intensity F [–] of extracellular potassium ions is observed to obtain the temporal distribution of average potassium ion concentration within the region of 3.6 μm from cell membrane interface c ROI [mM]. The slopes of ∆c ROI /c ROI1 to time t are 0.0003, 0.0002, and 0.0006 under hypotonic, isotonic, and hypertonic conditions, where c ROI1 denotes initial c ROI , which shows the same tendency with LF-EIS result that is verified by the potassium ion fluorescence observation.