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Osmotic shock by addition of sucrose to the medium stimulates erythrocyte sphingomyelinase with subsequent ceramide formation and triggers Ca2+ entry through stimulation of cation channels. Both ceramide and Ca2+ activate an erythrocyte scramblase, leading to breakdown of phosphatidylserine asymmetry, a typical feature of apoptosis. Because erythrocytes are regularly exposed to osmotic shock during passage of kidney medulla, the present study explored the influence of NaCl and urea on erythrocyte phosphatidylserine exposure as determined by annexin binding. The percentage of annexin-binding erythrocytes increased from <5 to 80 ± 4% (n = 4) upon addition of 650 mM sucrose, an effect paralleled by activation of the cation channel and stimulation of ceramide formation. The number of annexin-binding erythrocytes increased only to 18% after addition of 325 mM NaCl and was not increased by addition of 650 mM urea. According to whole cell patch-clamp experiments, the cation conductance was activated by replacement of extracellular Cl- with gluconate at isotonic conditions or by addition of hypertonic sucrose or urea. Although stimulating the cation conductance, urea abrogated the annexin binding and concomitant increase of ceramide levels induced by osmotic cell shrinkage. In vitro sphingomyelinase assays demonstrated a direct inhibitory effect of urea on sphingomyelinase activity. Urea did not significantly interfere with annexin binding after addition of ceramide. In conclusion, both Cl- and urea blunt erythrocyte phosphatidylserine exposure after osmotic shock. Whereas Cl- is effective through inhibition of the cation conductance, urea exerts its effect through inhibition of sphingomyelinase, thus blunting formation of ceramide. [ABSTRACT FROM AUTHOR]

Human induced pluripotent stem cell (hiPSC)-derived organoids mimicking tissues and organs in vitro have advanced medical research, as they opened up new possibilities for in-depth basic research on human organ development as well as providing a human in vitro model for personalized therapeutic approaches. hiPSC-derived retinal organoids have proven to be of great value for modeling the human retina featuring a very similar cellular composition, layering, and functionality. The technically challenging imaging of three-dimensional structures such as retinal organoids has, however, raised the need for robust whole-organoid imaging techniques. To improve imaging of retinal organoids we optimized a passive clearing technique (PACT), which enables high-resolution visualization of fragile intra-tissue structures. Using cleared retinal organoids, we could greatly enhance the antibody labeling efficiency and depth of imaging at high resolution, thereby improving the three-dimensional microscopy output. In that course, we were able to identify the spatial morphological shape and organization of, e.g., photoreceptor cells and bipolar cell layers. Moreover, we used the synaptic protein CtBP2/Ribeye to visualize the interconnection points of photoreceptor and bipolar cells forming the retinal-specific ribbon synapses. [ABSTRACT FROM AUTHOR]

Osmotic shock by addition of sucrose to the medium stimulates erythrocyte sphingomyelinase with subsequent ceramide formation and triggers Ca2+ entry through stimulation of cation channels. Both ceramide and Ca2+ activate an erythrocyte scramblase, leading to breakdown of phosphatidylserine asymmetry, a typical feature of apoptosis. Because erythrocytes are regularly exposed to osmotic shock during passage of kidney medulla, the present study explored the influence of NaCl and urea on erythrocyte phosphatidylserine exposure as determined by annexin binding. The percentage of annexin-binding erythrocytes increased from <5 to 80 ± 4% (n = 4) upon addition of 650 mM sucrose, an effect paralleled by activation of the cation channel and stimulation of ceramide formation. The number of annexin-binding erythrocytes increased only to 18% after addition of 325 mM NaCl and was not increased by addition of 650 mM urea. According to whole cell patch-clamp experiments, the cation conductance was activated by replacement of extracellular Cl- with gluconate at isotonic conditions or by addition of hypertonic sucrose or urea. Although stimulating the cation conductance, urea abrogated the annexin binding and concomitant increase of ceramide levels induced by osmotic cell shrinkage. In vitro sphingomyelinase assays demonstrated a direct inhibitory effect of urea on sphingomyelinase activity. Urea did not significantly interfere with annexin binding after addition of ceramide. In conclusion, both Cl- and urea blunt erythrocyte phosphatidylserine exposure after osmotic shock. Whereas Cl- is effective through inhibition of the cation conductance, urea exerts its effect through inhibition of sphingomyelinase, thus blunting formation of ceramide. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is the most common malignant primary brain tumor in adults. GBM displays excessive and unfunctional vascularization which may, among others, be a reason for its devastating prognosis. Pericytes have been identified as the major component of the irregular vessel structure in GBM. In vitro data suggest an epithelial-to-mesenchymal transition (EMT)-like activation of glioma-associated pericytes, stimulated by GBM-secreted TGF-β, to be involved in the formation of a chaotic and dysfunctional tumor vasculature. This study investigated whether TGF-β impacts the function of vessel associated mural cells (VAMCs) in vivo via the induction of the EMT transcription factor SLUG and whether this is associated with the development of GBM-associated vascular abnormalities. Upon preventing the TGF-β-/SLUG-mediated EMT induction in VAMCs, the number of PDGFRβ and αSMA positive cells was significantly reduced, regardless of whether TGF-β secretion by GBM cells was blocked or whether SLUG was specifically knocked out in VAMCs. The reduced amount of PDGFRβ + or αSMA + cells observed under those conditions correlated with a lower vessel density and fewer vascular abnormalities. Our data provide evidence that the SLUG-mediated modulation of VAMC activity is induced by GBM-secreted TGF-β¬ and that activated VAMCs are key contributors in neo-angiogenic processes. We suggest that a pathologically altered activation of GA-Peris in the tumor microenvironment is responsible for the unstructured tumor vasculature. There is emerging evidence that vessel normalization alleviates tumor hypoxia, reduces tumor-associated edema and improves drug delivery. Therefore, avoiding the generation of an unstructured and non-functional tumor vasculature during tumor recurrence might be a promising treatment approach for GBM and identifies pericytes as a potential novel therapeutic target., Competing Interests: The authors declare no conflicts of interest. All coauthors have reviewed and approved the contents of the manuscript and that the requirements for authorship have been met.

Characterizing the microvasculature of the human brain is critical to advance understanding of brain vascular function. Most methods rely on tissue staining and microscopy in two-dimensions, which pose several challenges to visualize the three-dimensional structure of microvessels. In this study, we used an edge-based segmentation method to extract the 3D vasculature from synchrotron radiation phase-contrast microtomography (PC-μCT) of two unstained, paraffin-embedded midbrain region of the human brain stem. Vascular structures identified in PC-μCT were validated with histology of the same specimen. Using the Deriche-Canny edge detector that was sensitive to the boundary between tissue and vascular space, we could segment the vessels independent of signal variations in PC-μCT images. From the segmented volumetric vasculature, we calculated vessel diameter, vessel length and volume fraction of the vasculature in the superior colliculi. From high resolution images, we found the most frequent vessel diameter to be between 8.6-10.2 µm. Our findings are consistent with the known anatomy showing two types of vessels with distinctive morphology: peripheral collicular vessels and central collicular vessels. The proposed method opens up new possibilities for vascular research of the central nervous system using synchrotron radiation PC-μCT of unstained human tissue., (© 2022. The Author(s).)

Background: In vitro studies are very useful to increase the knowledge of different cell types and could be the key to understand cell metabolism and function. Fish optic nerves (ON) can recover visual functions by reestablishing its structure and reconnecting the axons of ganglion cells. This is because fish show spontaneous regeneration of the central nervous system which does not occur in mammals. In addition, several studies have indicated that glial cells of ON have different properties in comparison to the glial cells from brain or retina. Consequently, providing an in vitro tool will be highly beneficial to increase the knowledge of these cells., New Method: We developed a cell culture protocol to isolate glial cells from ON of two teleost fish species, Danio rerio and Astatotilapia burtoni., Results: The optimized protocol allowed us to obtain ON cells and brain-derived cells from adult teleost fish. These cells were characterized as glial cells and their proprieties in vitro were analyzed.Comparison with Existing Method(s): Although it is striking that ON glial cells show peculiarities, their study in vitro has been limited by the only published protocol going back to the 1990s. Our protocol makes glial cells of different fish species available for experiments and studies to increase the understanding of these glial cell types., Conclusions: This validated and effective in vitro tool increases the possibilities on studies of glial cells from fish ON which implies a reduction in animal experimentation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

DJ-1/Park7 is a redox-sensitive chaperone protein counteracting oxidation and presumably contributing to the control of oxidative stress responses and thus inflammation. DJ-1 gene deletion exacerbates the progression of Parkinson's disease presumably by augmenting oxidative stress. Formation of reactive oxygen species (ROS) is paralleled by activation of the Na + /H + exchanger 1 (NHE1). ROS formation in CD4 + T cells plays a decisive role in regulating inflammatory responses. In the present study, we explored whether DJ-1 is expressed in CD4 + T cells, and affects ROS production as well as NHE1 in those cells. To this end, DJ-1 and NHE1 transcript, and protein levels were quantified by qRT-PCR and Western blotting, respectively, intracellular pH (pH i ) utilizing bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from realkalinization after an ammonium pulse, and ROS production utilizing 2',7' -dichlorofluorescin diacetate (DCFDA) fluorescence. As a result DJ-1 was expressed in CD4 + T cells. ROS formation, NHE1 transcript levels, NHE1 protein, and NHE activity were higher in CD4 + T cells from DJ-1 deficient mice than in CD4 + T cells from wild type mice. Antioxidant N-acetyl-cysteine (NAC) and protein tyrosine kinase (PTK) inhibitor staurosporine decreased the NHE activity in DJ-1 deficient CD4 + T cells, and blunted the difference between DJ-1 -/- and DJ-1 +/+ CD4 + T cells, an observation pointing to a role of ROS in the up-regulation of NHE1 in DJ-1 -/- CD4 + T cells. In conclusion, DJ-1 is a powerful regulator of ROS production as well as NHE1 expression and activity in CD4 + T cells. J. Cell. Physiol. 232: 3050-3059, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The quantity of therapeutic gene products released from genetically engineered cells can be controlled externally at different levels. The widely used approach of controlling expression, however, generally has the disadvantage that chemical substances must be applied for stimulation. An alternative strategy aims at controlling gene products at posttranslational levels such as secretion. The secretion of a therapeutic agent can be regulated if the agent is targeted to the regulated secretory pathway and stored in the secretory granules until its release. In this article we address the question of whether the release of beta-endorphin, an opioid with a potent analgesic effect, could be induced by electrically stimulating stably transfected Neuro-2a cells. Throughout this study we used the human proopiomelanocortin (POMC) gene, which is the precursor molecule for human beta-endorphin. We analyzed its subcellular localization and found it in the regulated secretory pathway in Neuro-2a cells. Using electrical field stimulation we were able to identify a stimulation pattern that significantly increased the release of beta-endorphin-immunoreactive material, although to a limited extent. This result indicates that electrical stimulation of secretion could be used to manipulate the amount of a therapeutic agent released from transplanted cells.