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Abstract Background Recent studies demonstrated that elevated osmolarity could induce adipocyte dedifferentiation, representing an appealing procedure to generate multipotent stem cells. Here we aim to elucidate the molecular mechanisms that underlie osmotic induction of adipocyte reprogramming. Methods To induce dedifferentiation, the 3T3-L1 or SVF adipocytes were cultured under the hypertonic pressure in 2% PEG 300 medium. Adipocyte dedifferentiation was monitored by aspect ratio measurement, Oil Red staining and qPCR to examine the morphology, lipid droplets, and specific genes of adipocytes, respectively. The osteogenic and chondrogenic re-differentiation capacities of dedifferentiated adipocytes were also examined. To investigate the mechanisms of the osmotic stress-induced dedifferentiation, extracellular vesicles (EVs) were collected from the reprograming cells, followed by proteomic and functional analyses. In addition, qPCR, ELISA, and TNF-α neutralizing antibody (20 ng/ml) was applied to examine the activation and effects of the TNF-α signaling. Furthermore, we also analyzed the Wnt signaling by assessing the activation of β-catenin and applying BML-284, an agonist of β-catenin. Results Hypertonic treatment induced dedifferentiation of both 3T3-L1 and the primary stromal vascular fraction (SVF) adipocytes, characterized by morphological and functional changes. Proteomic profiling revealed that hypertonicity induced extracellular vesicles (EVs) containing mitochondrial molecules including NDUFA9 and VDAC. Functionally, the mitochondrial EVs (MEVs) stimulated TNF-α signaling that activates Wnt-β-catenin signaling and adipocyte dedifferentiation. Neutralizing TNF-α inhibited hypertonic dedifferentiation of adipocytes. In addition, direct activation of Wnt-β-catenin signaling using BML-284 could efficiently induce adipocyte dedifferentiation while circumventing the apoptotic effect of the hypertonic treatment. Conclusions Hypertonicity prompts the adipocytes to release MEVs, which in turn enhances the secretion of TNF-α as a pro-inflammatory cytokine during the stress response. Importantly, TNF-α is essential for the activation of the Wnt/β-catenin signaling that drives adipocyte dedifferentiation. A caveat of the hypertonic treatment is apoptosis, which could be circumvented by direct activation of the Wnt/β-catenin signaling using BML-284.

Background: Recent studies demonstrated that elevated osmolarity could induce adipocyte dedifferentiation, representing an appealing procedure to generate multipotent stem cells. Here we aim to elucidate the molecular mechanisms that underlie osmotic induction of adipocyte reprogramming. Methods: To induce dedifferentiation, the 3T3-L1 or SVF adipocytes were cultured under the hypertonic pressure in 2% PEG 300 medium. Adipocyte dedifferentiation was monitored by aspect ratio measurement, Oil Red staining and qPCR to examine the morphology, lipid droplets, and specific genes of adipocytes, respectively. The osteogenic and chondrogenic re-differentiation capacities of dedifferentiated adipocytes were also examined. To investigate the mechanisms of the osmotic stress-induced dedifferentiation, extracellular vesicles (EVs) were collected from the reprograming cells, followed by proteomic and functional analyses. In addition, qPCR, ELISA, and TNF-α neutralizing antibody (20 ng/ml) was applied to examine the activation and effects of the TNF-α signaling. Furthermore, we also analyzed the Wnt signaling by assessing the activation of β-catenin and applying BML-284, an agonist of β-catenin. Results: Hypertonic treatment induced dedifferentiation of both 3T3-L1 and the primary stromal vascular fraction (SVF) adipocytes, characterized by morphological and functional changes. Proteomic profiling revealed that hypertonicity induced extracellular vesicles (EVs) containing mitochondrial molecules including NDUFA9 and VDAC. Functionally, the mitochondrial EVs (MEVs) stimulated TNF-α signaling that activates Wnt-β-catenin signaling and adipocyte dedifferentiation. Neutralizing TNF-α inhibited hypertonic dedifferentiation of adipocytes. In addition, direct activation of Wnt-β-catenin signaling using BML-284 could efficiently induce adipocyte dedifferentiation while circumventing the apoptotic effect of the hypertonic treatment. Conclusions: Hypertonicity prompts the adipocytes to release MEVs, which in turn enhances the secretion of TNF-α as a pro-inflammatory cytokine during the stress response. Importantly, TNF-α is essential for the activation of the Wnt/β-catenin signaling that drives adipocyte dedifferentiation. A caveat of the hypertonic treatment is apoptosis, which could be circumvented by direct activation of the Wnt/β-catenin signaling using BML-284. [ABSTRACT FROM AUTHOR]

Many viral infections, including the COVID-19 infection, are associated with the hindrance of blood oxygenation due to the accumulation of fluid, inflammatory cells, and cell debris in the lung alveoli. This condition is similar to Acute Respiratory Distress Syndrome (ARDS). Mechanical positive-pressure ventilation is often used to treat this condition, even though it might collapse pulmonary capillaries, trapping red blood cells and lowering the lung’s functional capillary density. We posit that the hyperosmotic-hyperoncotic infusion should be explored as a supportive treatment for ARDS. As a first step in verifying the feasibility of this ARDS treatment, we model the dynamics of alveolar fluid extraction by osmotic effects. These are induced by increasing blood plasma osmotic pressure in response to the increase of blood NaCl concentration. Our analysis of fluid drainage from a plasma-filled pulmonary alveolus, in response to the intravenous infusion of 100 ml of 1.28 molar NaCl solution, shows that alveoli empty of fluid in approximately 15 min. These modeling results are in accordance with available experimental and clinical data; no new data were collected. They are used to calculate the temporal change of blood oxygenation, as oxygen diffusion hindrance decreases upon absorption of the alveolar fluid into the pulmonary circulation. Our study suggests the extraordinary speed with which beneficial effects of the proposed ARDS treatment are obtained and highlight its practicality, cost-efficiency, and avoidance of side effects of mechanical origin.

Many viral infections, including the COVID-19 infection, are associated with the hindrance of blood oxygenation due to the accumulation of fluid, inflammatory cells, and cell debris in the lung alveoli. This condition is similar to Acute Respiratory Distress Syndrome (ARDS). Mechanical positive-pressure ventilation is often used to treat this condition, even though it might collapse pulmonary capillaries, trapping red blood cells and lowering the lung's functional capillary density. We posit that the hyperosmotic-hyperoncotic infusion should be explored as a supportive treatment for ARDS. As a first step in verifying the feasibility of this ARDS treatment, we model the dynamics of alveolar fluid extraction by osmotic effects. These are induced by increasing blood plasma osmotic pressure in response to the increase of blood NaCl concentration. Our analysis of fluid drainage from a plasma-filled pulmonary alveolus, in response to the intravenous infusion of 100 ml of 1.28 molar NaCl solution, shows that alveoli empty of fluid in approximately 15 min. These modeling results are in accordance with available experimental and clinical data; no new data were collected. They are used to calculate the temporal change of blood oxygenation, as oxygen diffusion hindrance decreases upon absorption of the alveolar fluid into the pulmonary circulation. Our study suggests the extraordinary speed with which beneficial effects of the proposed ARDS treatment are obtained and highlight its practicality, cost-efficiency, and avoidance of side effects of mechanical origin., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li, Martini, Intaglietta and Tartakovsky.)

Nuclear Ca2+ is important for the regulation of several nuclear processes such as gene expression. Localized Ca2+ signals (LCSs) in skeletal muscle fibers of mice have been mainly studied as Ca2+ release events from the sarcoplasmic reticulum. Their location with regard to cell nuclei has not been investigated.Our study is based on the hypothesis that LCSs associated with nuclei are present in skeletal muscle fibers of adult mice. Therefore we carried out experiments addressing this question and we found novel Ca2+ signals associated with nuclei of skeletal muscle fibers (with possibly attached satellite cells). We measured localized nuclear and perinuclear Ca2+ signals (NLCSs and PLCSs) alongside cytosolic localized Ca2+ signals (CLCSs) during a hypertonic treatment. We also observed NLCSs under isotonic conditions. The NLCSs and PLCSs are Ca2+ signals in the range of micrometer (FWHM (full width at half maximum): 2.75 µm±0.27 µm (NLCSs) and 2.55 µm±0.17 µm (PLCSs), S.E.M.). Additionally, global nuclear Ca2+ signals (NGCSs) were observed.To investigate which type of Ca2+ channels contribute to the Ca2+ signals associated with nuclei in skeletal muscle fibers, we performed measurements with the RyR blocker dantrolene, the DHPR blocker nifedipine or the IP3R blocker Xestospongin C. We observed Ca2+ signals associated with nuclei in the presence of each blocker. Nifedipine and dantrolene had an inhibitory effect on the fraction of fibers with PLCSs. The situation for the fraction of fibers with NLCSs is more complex indicating that RyR is less important for the generation of NLCSs compared to the generation of PLCSs. The fraction of fibers with NLCSs and PLCSs is not reduced in the presence of Xestospongin C.The localized perinuclear and intranuclear Ca2+ signals may be a powerful tool for the cell to regulate adaptive processes as gene expression. The intranuclear Ca2+ signals may be particularly interesting in this respect.

Nuclear Ca2+ is important for the regulation of several nuclear processes such as gene expression. Localized Ca2+ signals (LCSs) in skeletal muscle fibers of mice have been mainly studied as Ca2+ release events from the sarcoplasmic reticulum. Their location with regard to cell nuclei has not been investigated. Our study is based on the hypothesis that LCSs associated with nuclei are present in skeletal muscle fibers of adult mice. Therefore, we carried out experiments addressing this question and we found novel Ca2+ signals associated with nuclei of skeletal muscle fibers (with possibly attached satellite cells). We measured localized nuclear and perinuclear Ca2+ signals (NLCSs and PLCSs) alongside cytosolic localized Ca2+ signals (CLCSs) during a hypertonic treatment. We also observed NLCSs under isotonic conditions. The NLCSs and PLCSs are Ca2+ signals in the range of micrometer [FWHM (full width at half maximum): 2.75 ± 0.27µm (NLCSs) and 2.55 ± 0.17µm (PLCSs), S.E.M.]. Additionally, global nuclear Ca2+ signals (NGCSs) were observed. To investigate which type of Ca2+ channels contribute to the Ca2+ signals associated with nuclei in skeletal muscle fibers, we performed measurements with the RyR blocker dantrolene, the DHPR blocker nifedipine or the IP3R blocker Xestospongin C. We observed Ca2+ signals associated with nuclei in the presence of each blocker. Nifedipine and dantrolene had an inhibitory effect on the fraction of fibers with PLCSs. The situation for the fraction of fibers with NLCSs is more complex indicating that RyR is less important for the generation of NLCSs compared to the generation of PLCSs. The fraction of fibers with NLCSs and PLCSs is not reduced in the presence of Xestospongin C. The localized perinuclear and intranuclear Ca2+ signals may be a powerful tool for the cell to regulate adaptive processes as gene expression. The intranuclear Ca2+ signals may be particularly interesting in this respect. [ABSTRACT FROM AUTHOR]

We investigated the mechanism underlying the perception of extracellular changes in osmotic pressure in Vallisneria gigantea Graebner and transgenic Arabidopsis thaliana (L.) Heynh. expressing cytoplasmic aequorin. Hypertonic and hypotonic treatments of A. thaliana leaves each rapidly induced a Ca2+ transient. Both responses were essentially dependent on the presence of extracellular Ca2+ and were sensitive to Gd3+, a potential blocker of stretch-activated Ca2+ channels. Immediately after plasmolysis caused by hypertonic treatment and subsequent deplasmolysis caused by hypotonic treatment, the cells did not respond to a second hypertonic treatment and exhibited an impaired adhesion of the plasma membrane (PM) to the cell wall (CW). Recovery of the responsiveness required about 6 h. By contrast, no refractory phenomenon was observed in response to hypotonic treatment. Pretreatment with cellulase completely inhibited the Ca2+ transient induced by hypertonic treatment, but it did not affect the response to hypotonic treatment. V. gigantea mesophyll cells pretreated with cellulase exhibited an impaired adhesion of the PM to the CW. The leaf cells of multicellular plants can respond to both hypertonic and hypotonic treatments through the stretch-activated Ca2+ channels, whereas cellulase-sensitive adhesion of the PM to the CW is involved only in the response to hypertonic treatment. [ABSTRACT FROM AUTHOR]

Changes in photosynthetic activities under hypertonic conditions were studied in a terrestrial, highly desiccation-tolerant cyanobacterium, Nostoc commune, and in some desiccation-sensitive cyanobacteria. The amounts of water sustained in the colony matrix outside the N. commune cells and the cellular solute concentration were estimated by measuring the water potential, and the solute concentration was supposed to correspond to around 0.22 M sorbitol. Incubation of the colonies in 0.8 M sorbitol solution inhibited the energy transfer from the phycobilisome (PBS) anchor to PSII core complexes. At higher sorbitol concentrations, light energy absorbed by PSI, PSII, and PBS was dissipated to heat. PSI and cyclic electron flow around PSI was also deactivated by hypertonic treatment. Fv/Fm and (Fm′–F)/Fm′ values started to decrease at 0.6 and 0.3 M sorbitol and reached zero at 1.0 and 0.8 M, respectively. Decreases in these two fluorescence parameters corresponded to the decreases in PSII fluorescence (F695) and photosynthetic CO2 fixation, respectively. The intensity of delayed light emission started to decrease at 1.0 M sorbitol and became negligible at 4.0 M. Comparing these changes in N. commune with those in desiccation-sensitive species, we found that N. commune cells actively deactivates photosynthetic systems on sensing water loss. [ABSTRACT FROM AUTHOR]

The responses of cortical microtubules to sub-zero temperatures were examined in non-acclimated (NA) and cold-acclimated (CA) rye (Secale cereale L. cv. Voima) leaf and root cells, and in protoplasts isolated enzymatically from leaves. Responses of leaf and root cells to hypertonic solutions equivalent to the dehydration response of freezing (P. L. Steponkus and D. V. Lynch 1989. J. Bioenerg. Biomembr. 21: 21-41) were also examined. At the respective growth temperatures both NA and CA leaf and root cells had typical organization and abundance of cortical microtubules as observed by indirect immunofluorescence (IIF) staining. Unchanged microtubule arrays were still present in CA leaf cells after -4°C treatment, while in leaf cells of NA plants and in the root cells of both NA and CA plants microtubules were shorter and less abundant. After -10°C treatment the cortical microtubules were almost totally depolymerized in both types of root cells and in leaf cells of NA plants while CA leaf cells still had abundant cortical microtubule arrays. Semiquantitative analyses of cortical microtubules (MTs) of protoplasts confirmed the findings with intact leaf cells. Hypertonic treatment of NA and CA leaf cells gave similar effects as exposure of cells to sub-zero temperatures. However, after the hypertonic treatment, more microtubules remained present in the CA root cells than in the NA root cells, suggesting that also in root cells cold acclimation increases the dehydration stability of MTs. In conclusion, cold acclimation induces both greater frost stability and greater osmotic tolerance in the cortical microtubules of the leaf cells, and greater osmotic tolerance in the microtubules of the root cells in winter rye. [ABSTRACT FROM AUTHOR]

Nuclear Ca(2+) is important for the regulation of several nuclear processes such as gene expression. Localized Ca(2+) signals (LCSs) in skeletal muscle fibers of mice have been mainly studied as Ca(2+) release events from the sarcoplasmic reticulum. Their location with regard to cell nuclei has not been investigated. Our study is based on the hypothesis that LCSs associated with nuclei are present in skeletal muscle fibers of adult mice. Therefore, we carried out experiments addressing this question and we found novel Ca(2+) signals associated with nuclei of skeletal muscle fibers (with possibly attached satellite cells). We measured localized nuclear and perinuclear Ca(2+) signals (NLCSs and PLCSs) alongside cytosolic localized Ca(2+) signals (CLCSs) during a hypertonic treatment. We also observed NLCSs under isotonic conditions. The NLCSs and PLCSs are Ca(2+) signals in the range of micrometer [FWHM (full width at half maximum): 2.75 ± 0.27 μm (NLCSs) and 2.55 ± 0.17 μm (PLCSs), S.E.M.]. Additionally, global nuclear Ca(2+) signals (NGCSs) were observed. To investigate which type of Ca(2+) channels contribute to the Ca(2+) signals associated with nuclei in skeletal muscle fibers, we performed measurements with the RyR blocker dantrolene, the DHPR blocker nifedipine or the IP3R blocker Xestospongin C. We observed Ca(2+) signals associated with nuclei in the presence of each blocker. Nifedipine and dantrolene had an inhibitory effect on the fraction of fibers with PLCSs. The situation for the fraction of fibers with NLCSs is more complex indicating that RyR is less important for the generation of NLCSs compared to the generation of PLCSs. The fraction of fibers with NLCSs and PLCSs is not reduced in the presence of Xestospongin C. The localized perinuclear and intranuclear Ca(2+) signals may be a powerful tool for the cell to regulate adaptive processes as gene expression. The intranuclear Ca(2+) signals may be particularly interesting in this respect.