Nicotinic acid induces apoptosis of glioma cells <i>via</i> the calcium-dependent endoplasmic reticulum stress pathway

Malignant glioma is one of the most common and deadly tumors in the central nervous system while developing effective treatments for this devastating disease remains a challenge. Previously, we demonstrated that the vitamin nicotinic acid (NA) inhibits glioma invasion. Here, we show that high-dose NA induces apoptosis of malignant glioma cells in vitro and in vivo. In cultured U251 glioma cells treated with NA, we detected ER stress that was likely caused by elevated intracellular calcium levels. The elevated calcium can be attributed to the activation of TRPV1, a cation channel that has been implicated in cutaneous flushing caused by NA administration. Our data further suggested that NA-induced apoptosis is mediated by the calcium-dependent proteases called calpains, whose activities are drastically upregulated by NA. NA-induced apoptosis of U251 cells can be attenuated by blocking calpain activity or knocking down TRPV1. These results reveal a novel function of NA in regulating glioma cell apoptosis via the calcium-dependent ER stress pathway and imply a potential application of NA for the treatment of malignant glioma.     

Geometric quantification of the plant endoplasmic reticulum

One of the most difficult obstacles to make biological sciences more quantitative is the lack of understanding the interplay of form and function. Each cell is full of complex‐shaped objects, which moreover change their form over time. To tackle this problem, we suggest the use of geometric invariants that are able to produce precise reference points to compare a cell's different functional elements such as organelles under fixed and varying physiological conditions. In this paper, we look at the topology of an almost static sample of the plant cortical endoplasmic reticulum (ER) under close‐to‐normal physiological conditions using a multi‐disciplinary approach combining confocal microscopy, image processing techniques, visualization, computational geometry and graph theory. Data collected from a series of optical sections taken at short, regular intervals along the optical axis are used to reconstruct the ER in three dimensions. A graph structure of the ER network is obtained after thinning the ER geometry to its essential features. The graph is the final and most abstract quantification of the ER and serves very well as a geometrical invariant, even and very importantly, in cases in which the ER sample is moving or slightly changing shape during image acquisition. Moreover, graph theoretic features, such as the number of nodes and their degrees and the number of edges and their lengths, are very robust against different kinds of small perturbations that should not change the ER function. We will also attach surface areas and volumes estimated for the plant ER network as weights to the graph, allowing an even more precise quantitative characterization of this organelle. In total, we have compared 28 different samples under similar experimental conditions. The methods used in this paper should also be applicable to the quantification of other organelles in which geometric abstraction is possible to analyse function. Finally, by the use of confocal microscopy, our techniques will be transferable to situations in which protein markers can move inside the organelle's lumen and/or on the membrane surface to test further aspects of protein distribution.     

Tanshinone IIA protects intestinal epithelial cells from ferroptosis through the upregulation of <i>GPX4</i> and <i>SLC7A11</i>

Background: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. The destruction of the intestinal epithelial barrier is one of the major pathological processes in IBD pathology. Growing evidence indicated that epithelial cell ferroptosis is linked to IBD and is considered a target process. Methods: RAS-selective lethal 3 (RSL3) was used to induce ferroptosis in intestinal epithelial cell line No. 6 (IEC-6) cells, and cell ferroptosis and the effects of tanshinone IIA (Tan IIA) were determined by cell counting kit-8 (CCK-8), reactive oxygen species (ROS) staining, Giemsa staining and transmission electron microscope (TEM). The cell viability of natural product library compounds was determined by CCK-8. The expression of ferroptosis-related genes were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Results: Treatment of IEC-6 cells results in the accumulation of ROS and typical morphological characteristics of ferroptosis. RSL3 treatment caused rapid cellular cytotoxicity which could be reversed by ferrostatin-1 (Fer-1) in IEC-6 cells. Natural product library screening revealed that Tan IIA is a potent inhibitor of IEC-6 cell ferroptosis. Tan IIA could significantly protect the RSL3-induced ferroptosis of IEC-6 cells. Furthermore, the ferroptosis suppressors, glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), and miR-17-92 were found to be early response genes in RSL3-treated cells. Treatment of IEC-6 cells with Tan IIA resulted in upregulation of GPX4, SLC7A11, and miR-17-92. Conclusion: Our study demonstrated that Tan IIA protects IEC-6 cells from ferroptosis through the upregulation of GPX4, SLC7A11, and miR-17-92. The findings might provide a theoretical grounding for the future application of Tan IIA to treat or prevent IBD.     

Influence of calcium and amino acids on the osmium impregnation of the endoplasmic reticulum

The aim of this study was to define further the interaction between osmium and organelle content in cells prefixed with glutaraldehyde. We have studied the reaction of osmium with divalent or trivalent cations (calcium, barium, zinc, aluminum, and iron) and various amino acids in the same conditions prevalent in histological techniques, in particular with Thiéry's technique of metal impregnation. Experiments were carried out in vitro in test tubes, on cellulose acetate discs, or with an immunodiffusion apparatus. Some experiments were also carried out with tissue extracts (kidney and intestine). Our studies suggest that calcium is in general essential for the formation of osmium black, but also that lysine is reactive even in the absence of calcium and that a few amino acids–such as tryptophan, ornithine, cysteine, and aspartic acid–are only slightly reactive in the absence of calcium. Other amino acids do not seem to participate in the endoplasmic reticulum osmium impregnation even in the presence of calcium ions. Our studies also suggest that osmium reactivity reflects calcium binding sites and not only calcium content.     

Utilization of 3D printing for an intravital microscopy platform to study the intestinal microcirculation

Intravital microscopy of the intestine is a sophisticated technique that allows qualitative and quantitative in vivo observation of dynamic cellular interactions and blood flow at a high resolution. Physiological conditions of the animal and in particular of the observed organ, such as temperature and moisture are crucial for intravital imaging. Often, the microscopy stage with the animal or the organ of interest imposes limitations on how well the animal can be maintained. In addition, the access for additional oxygen supply or drug administration during the procedure is rather restricted. To address these limitations, we developed a novel intravital microscopy platform, allowing us to have improved access to the animal during the intravital microscopy procedure, as well as improved microenvironmental maintenance. The production process of this prototype platform is based on 3D printing of device parts in a single‐step process. The simplicity of production and the advantages of this versatile and customizable design are shown and discussed in this paper. Our design potentially represents a major step forward in facilitating intestinal intravital imaging using fluorescent microscopy.     

Measurements of cells in culture by scanning acoustic microscopy

A novel method allowing the determination of thickness, attenuation and impedance of both living and fixed cells in culture using a single image recorded with a scanning acoustic microscope is described. The method is based on the recording of the image data locating the surface of the cell far below the geometric focus. In this way only the normal incident acoustic wave is used for image formation. Higher values of impedance and attenuation coefficients were found in the cell periphery than in the central part of the cell. This phenomenon is suggested to be due to the different organization of cytoskeletal elements.     

Effects of high molybdenum intake on 1,2-dimethylhydrazine-induced intestinal tumors in rats

Wistar male rats, 3 months of age were given ad-libitum a nutritionally adequate diet and demineralized drinking water. The Molybdenum (Mo) and Tungsten (W) were provided in the drinking water at 200 ppm concentration. Intestinal tumors were induced by 1,2-dimethylhydrazine (DMH) given subcutaneously as 16 weekly doses at 20 mg/kg body weight. Mo in the form of (NH₄)₆ Mo₇O₂₄ 4H₂O or W in the form of (Na₂ WO₄) were provided in the drinking water two months before the first DMH treatment and were continued during 4 months more until the last DMH treatment. Three months after the last carcinogen injection, all animals were sacrificed and examined for intestinal tumors. The number, size and location of the tumors were recorded and the pathology was examined. The addition of Mo to the drinking water induced an increase of hepatic Mo content. At the end of the second month, the hepatic content of Mo was 5.61 ppm, compared with control and W groups (2.18 and 0.96 ppm, respectively). A significantly lower incidence of tumors was observed in the Mo group (47), compared with the control group given DMH alone (105) and W group (113). On the other hand, the Mo group showed a significant decrease in the numbers of multiple tumors per rat.     

Morphological characterization of the progenitor blood cells in canine and feline umbilical cord

The umbilical cord blood (UCB) is an important source of hematopoietic stem cells with great deal of interest in regenerative medicine. The UCB cells have been extensively studied as an alternative to the bone marrow transplants. The challenge is to define specific methods to purify and characterize these cells in different animal species. This study is aimed at morphological characterization of progenitor cells derived from UCB highlighting relevant differences with peripheral blood of adult in dog and cats. Therefore, blood was collected from 18 dogs and 5 cats' umbilical cords from fetus in various developmental stages. The mononuclear cells were separated using the gradient of density Histopaque-1077. Characterization of CD34+ cells was performed by flow cytometric analysis and transmission electron microscopy. Granulocytes (ancestry of the basophiles, eosinophiles, and neutrophiles) and agranulocytes (represented by immature lymphocytes) were identified. We showed for the first time the ultrastructural features of cat UCB cells.     

Age-related modifications of macrophages influenced by “inflammageing” in graft <i>vs.</i> host disease

Most studies focus on the adaptive immune cells in the GVHD pathogenesis, while little is known about innateimmune cells in GVHD occurrence and development, especially macrophages. Meanwhile, a higher incidence of graftversus host disease (GVHD) is also found in the elderly patients. Though advances have been made in themodification of macrophages influenced by the inflamm-ageing, there is still no review on the role of macrophages inGVHD and the association between GVHD and the altered macrophages by inflamm-ageing. In this review, we focuson the potential age-related modifications of macrophage in GVHD, which contributes to the change of morbidityand mortality of GVHD. Via literature review, we found that the infiltration of macrophages is associated withGVHD and macrophages are modified in inflamm-ageing state, including the proliferation, migration, phagocytosis,antigen presentation, interaction with other immune cells, and pro-fibrosis. We suppose that altered macrophagefunctions in inflamm-ageing state contribute to GVHD in elderly patients.     

Effects of human umbilical cord mesenchymal stem cells on co‐cultured ovarian carcinoma cells

Ovarian carcinoma is mainly treated by surgery aided by chemotherapy. If supplemented by stem cells treatment, its recurrence rate and mortality rate will be decreased. This is a new therapy. In this study, ovarian cancer cells were cultured together with umbilical cord mesenchymal stem cells (UCMSCs), and the interactions between them were observed. The results showed that the survival rates of UCMSCs increased to 83.8 ± 2.2% from 56.5 ± 5.5%, and the survival rates of ovarian cancer cells decreased to 16.2 ± 2.2% from 43.5 ± 5.5% with the progression of the cultural time from 24 to 96 hr. There was a significant difference between them (p < .05). It revealed that UCMSCs could inhibit the proliferation of ovarian cancer cells.     

ER-Tracker dye and BODIPY-brefeldin A differentiate the endoplasmic reticulum and Golgi bodies from the tubular-vacuole system in living hyphae of Pisolithus tinctorius

Two fluorochromes, ER-Tracker^TM Blue-White DPX dye and the fluorescent brefeldin A (BFA) derivative, BODIPY-BFA, label the endoplasmic reticulum (ER) in hyphal tips of Pisolithus tinctorius and allow its differentiation from the tubular-vacuole system at the light microscope level in living cells. The ER-Tracker dye labels a reticulate network similar in distribution to ER as seen in electron micrographs of freeze-substituted hyphae. BODIPY-BFA stains a thicker axially aligned structure with an expanded region at the apex, which is similar to that seen when hyphae are stained with ER-Tracker dye in the presence of unconjugated BFA. This structure is considered to be ER modified by BFA, a view supported by ultrastructural observations of the effect of BFA on the fungal ER. Both fluorescent probes also stain punctate structures, which are most likely to be Golgi bodies. Neither probe labels the tubular-vacuole system.     

Technical Note : Prolonged exposure of human embryonic stem cells to heat shock induces necrotic cell death

We investigated the effects of prolonged heat shock treatment on human embryonic stem cell (hESC) viability. The hESC viability steadily declined with longer exposure to heat shock treatment (43ºC). After 4 h of exposure to heat shock at 43ºC, only 56.2 ± 1.5% of cells were viable. Viability subsequently declined to 37.0 ± 3.3% and 3.5 ± 0.7% after 8 h and 16 h, respectively of heat shock treatment at 43ºC. Transmission electron micrographs showed that the morphology of the dead/dying cells after heat shock treatment was characteristic of cellular necrosis with an uncondensed chromatin and a non-intact plasma membrane. This was further confirmed by flow cytometry analysis which showed that the DNA of the dead/ dying cells was still mostly intact, unlike the characteristic DNA fragmentation observed with apoptotic cells. In conclusion, prolonged exposure to heat shock treatment was detrimental to hESC viability. Hence, any future protocols developed for either the heat shock pre-conditioning of hESC prior to transplantation or for the temporary expression of specific genes with heat shock-responsive promoters should take these results into account; to achieve an optimal balance between the duration of heat shock exposure and the attainment of the desired effects.     

Proteome-wide screening for the analysis of protein targeting of <i>Chlamydia pneumoniae</i> in endoplasmic reticulum of host cells and their possible implication in lung cancer development

Available reports have confirmed a link between bacterial infection and the progression of different types of cancers, including colon, lungs, and prostate cancer. Here we report the Chlamydia pneumonia proteins targeting in endoplasmic reticulum (ER) using in-silico approaches and their possible role in lung cancer etiology. We predicted 48 proteins that target human ER, which may be associated with protein folding and protein-protein interactions during infection. The results showed C. pneumoniae proteins targeting human ER and their implications in lung cancer growth. These targeted proteins may be involved in competitive interactions between host and bacterial proteins, which may change the usual pathway functions and trigger the development of lung cancer. Moreover, C. pneumoniae unfolded protein accumulation in the human ER possibly induces ER stress, consequently activating the unfolded protein response (UPR), and providing a favorable microenvironment for cancer growth. The current study showed the C. pneumoniae protein targeting in ER of host cell and their implication in lung cancer growth. These results may help researchers better manage lung cancer and establish a molecular mechanism for C. pneumoniae lung cancer association.     

Differentiation of Leydig cells in the Mongolian gerbil

Information on postnatal Leydig cell (LC) differentiation in the Mongolian gerbil has been unavailable. Therefore, current investigation was designed to examine LC lineage differentiationin this rodent, from birth to adulthood. Gerbil testes at 1 day, 1–7 weeks (w), 2 and 3 months of age were conventionally processed by light and transmission electron microscopy. Immunocytochemistry for specific markers of steroidogenic enzymes, namely 3β‐hydroxysteroid dehydrogenase (3β‐HSD) and 11β‐hydroxysteroid steroid dehydrogenase 1 (11β‐HSD1) and also for androgen receptor (AR) was performed. The establishment of adult Leydig cell populations (ALC) during testis maturation in the gerbil follows the pattern previously described in other mammalian species, with the four progressive stages of differentiation. The LC progenitors were identified at second w by 3β‐HSD expression; the first newly formed ALC were recognized at fourth w whereas immature ALC appeared at fifth w. The latter were recognized by abundance of cytoplasmic lipid, in addition to expression of 11β‐HSD1 and intense nuclear AR immunoreaction. Mature ALC in gerbil exhibited irregular eccentric nuclei and a cytoplasmic canaliculus in the perinuclear area. Only one third of mature ALC in adult gerbils showed a high expression of 11β‐HSD1 and AR expression was highly variable among them. In conclusion, the process of differentiation of ALC population in gerbil follows the pattern previously established for other rodents. However, the resulting mature ALC are strikingly different due their singular asymmetric morphology and presence of a cytoplasmic canaliculus and as well as their functional heterogeneity. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Three months of Western diet induces small intestinal mucosa alteration in TLR KO mice

Several studies support the role of Western‐style diet (WD) in inflammatory bowel disease (IBD). Toll‐like receptors/NOD‐like receptors (TLRs/NLRs) are important to maintain a healthy epithelium as well as inducing inflammation. Given that dietary factors influence IBD development, that epithelial dysfunction is thought to be involved in initiating intestinal inflammation and that TLR‐NLR are involved in maintenance of the functionality of intestinal epithelium as well as in regulating inflammation, we decided to examine the role of TLR signals in the triggering events that lead to alteration of the small intestinal epithelium associated to consumption of WD. C57BL/6J mice deficient for TLR2, 4, 9, or NOD2 and wild‐type (WT) were fed a WD or a standard diet for 3 months. The effects of WD on small intestinal samples were evaluated by histological and immunohistochemical analysis. After 3 months, WD modifies the morphology and the organization of the small intestine in TLR9 KO mice compared with WT mice and the others TLRs. The most interesting change involved the expression of proliferative and differentiation markers of WNT signaling, Ki67 and FzD5. Mice deficient in TLR2, 4, and NOD2 have a significant reduction in the proliferative cell numbers but do not show any signs of histological alterations. Our results suggest that TLR9 is an important protective factor in intestinal epithelial homeostasis and provide new insights into an unrecognized role of TLR9 signaling in the small intestinal mucosa dysfunction associated with WD.