Histochemistry and Cell Biology

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Online ISSN: 1432-119X
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  • Qi YaoQi Yao
  • Huaiyuan ZhangHuaiyuan Zhang
  • Collin StandishCollin Standish
  • [...]
  • Jialing XiangJialing Xiang
Protocol for microphotograph of regions of interest in infarct scar samples. After sample visualization with light microscopy (H&E and Masson’s trichrome), regions of interest are defined, i.e., in the central area of the infarct scar. For each technique, 21 histological samples and 105 microphotographs (five per sample) were analyzed. Using histopathological references, the same regions are manually co-localized in each sample and microphotographs are taken using the described protocol. Finally, Masson’s trichrome microphotographs were optimized with digital removal of non-collagenous structures. Note that the Picrosirius red image (center left) depicts a macro photograph without polarized light microscopy. This macro caption as well as the Masson’s trichrome (upper) one have been edited for brightness and contrast to increase understandability. Bar (macro images) = 1 mm; bar (microphotographs) = 100 µm. H&E Hematoxylin–Eosin
Protocol for CBO index measurement by Fourier analysis. a Microphotograph of a pig myocardial scar sample stained with H&E and visualized with confocal microscopy. A Best Fit filter has been applied. b Conversion to grey scale 16. c FFT has been applied to the image and the 2D power plot has been obtained. d The spectrum gain has been adjusted to obtain a more defined power plot. e Manual segmentation to select the central frequencies in the FFT power plot. f Automatic major and minor axis measurement after implementing an area filter range and Smoothing, Pre-Filter, and Convex Hull filters. bar = 100 µm. For each technique, 21 histological samples and 105 microphotographs (five per sample) were analyzed. CBO collagen bundle orientation, FFT fast Fourier transform. H&E Hematoxylin–Eosin
Bland–Altman plots and limits of agreement for CBO index measurement by Fourier analysis. In each plot, the average collagen orientation index as measured by the two different methods being compared is presented in the x-axis, and differences in collagen orientation index measurements between these two methods are plotted in the y-axis. Limits of agreement are graphically represented (as dashed lines and shadowed area) and calculated as mean ± 2 SDs of the mean difference between the measurements. For each technique, 21 histological samples and 105 microphotographs (five per sample) were analyzed. a–d Plots for interrater reliability (reproducibility) of measurements in each technique. e–j Plots for consistency between staining and microscopy techniques. CBO  collagen bundle orientation, SDs standard deviations
Examples of CBO index in infarct scarring with each staining technique. Samples were stained with Masson’s trichrome (visualized with light microscopy, a and e), optimized Masson’s trichrome with digital removal of non-collagenous structures (b and f), Picrosirius red (visualized with polarized light microscopy, c and g) and H&E (visualized with confocal microscopy, d and h). In the first row (a–d), a case of parallel-oriented collagen bundles is shown. Collagen orientation index for each image is 0.67, 0.52, 0.41, and 0.35. In the second row (e–h), a case of randomly oriented collagen bundles is depicted. Collagen orientation index for each image is 0.88, 0.8, 0.82, and 0.82. bar = 100 µm. For each technique, 21 histological samples and 105 microphotographs (five per sample) were analyzed. CBO collagen bundle orientation, H&E Hematoxylin–Eosin
Histogram of CBO index measurements in the different techniques. a Histogram in Masson’s trichrome and examples of a parallel (a1) and random (a2) CBO index pattern in myocardial scar. Bin width: 0.05. Bin centers: 0.61, 0.67, 0.74, 0.79, 0.83, 0.88, 0.92 and 0.97. b Histogram in optimized Masson’s trichrome with digital removal of non-collagenous structures and examples of a parallel (b1) and random (b2) CBO index pattern in myocardial scar. Bin width: 0.075. Bin centers: 0.47, 0.49, 0.6, 0.66, 0.74, 0.82, 0.89 and 0.95. c Histogram in Picrosirius red and examples of a parallel (c1) and random (c2) CBO index pattern in myocardial scar. Bin width: 0.07. Bin centers: 0.4, 0.48, 0.55, 0.61, 0.69, 0.76, 0.82, 0.89 and 0.95. d Histogram in H&E + confocal and examples of a parallel (d1) and random (d2) CBO index pattern in myocardial scar. Bin width: 0.08. Bin centers: 0.28, 0.33, 0.4, 0.49, 0.57, 0.63, 0.73, 0.8, 0.87 and 0.93. For each technique, 21 histological samples and 105 microphotographs (five per sample) were analyzed. CBO collagen bundle orientation, H&E Hematoxylin and Eosin
  • Víctor Marcos-GarcésVíctor Marcos-Garcés
  • Cesar Rios-NavarroCesar Rios-Navarro
  • Fabián Gómez-TorresFabián Gómez-Torres
  • [...]
  • Amparo Ruiz-SauriAmparo Ruiz-Sauri
Collagen bundle orientation (CBO) in myocardial infarct scars plays a major role in scar mechanics and complications after infarction. We aim to compare four histopathological methods for CBO measurement in myocardial scarring. Myocardial infarction was induced in 21 pigs by balloon coronary occlusion. Scar samples were obtained at 4 weeks, stained with Masson’s trichrome, Picrosirius red, and Hematoxylin–Eosin (H&E), and photographed using light, polarized light microscopy, and confocal microscopy, respectively. Masson’s trichrome images were also optimized to remove non-collagenous structures. Two observers measured CBO by means of a semi-automated, Fourier analysis protocol. Interrater reliability and comparability between techniques were studied by the intraclass correlation coefficient (ICC) and Bland–Altman (B&A) plots and limits of agreement. Fourier analysis showed an almost perfect interrater reliability for each technique (ICC ≥ 0.95, p < 0.001 in all cases). CBO showed more randomly oriented values in Masson’s trichrome and worse comparability with other techniques (ICC vs. Picrosirius red: 0.79 [0.47–0.91], p = 0.001; vs. H&E-confocal: 0.70 [0.26–0.88], p = 0.005). However, optimized Masson’s trichrome showed almost perfect agreement with Picrosirius red (ICC 0.84 [0.6–0.94], p < 0.001) and H&E-confocal (ICC 0.81 [0.54–0.92], p < 0.001), as well as these latter techniques between each other (ICC 0.84 [0.60–0.93], p < 0.001). In summary, a semi-automated, Fourier-based method can provide highly reproducible CBO measurements in four different histopathological techniques. Masson’s trichrome tends to provide more randomly oriented CBO index values, probably due to non-specific visualization of non-collagenous structures. However, optimization of Masson’s trichrome microphotographs to remove non-collagenous components provides an almost perfect comparability between this technique, Picrosirius red and H&E-confocal.
  • Jobran M. MoshiJobran M. Moshi
  • Monique UmmelenMonique Ummelen
  • Jos L. V. BroersJos L. V. Broers
  • [...]
  • Anton H. N. HopmanAnton H. N. Hopman
SOX2 expression in high-grade cervical intraepithelial neoplasia (CIN3) and cervical squamous cell carcinoma is increased compared to that in the normal cervical epithelium. However, data on the expression and histological distribution of SOX2 in squamous epithelium during progression of CIN are largely lacking. We studied SOX2 expression throughout the epithelium in 53 cases of CIN1, 2, and 3. In general, SOX2 expression increased and expanded from basal/parabasal to the intermediate/superficial compartment during early stages of progression of CIN. An unexpected, specific expression pattern was found in areas classified as CIN2 and CIN3. This pattern was characterized by the absence or low expression of SOX2 in the basal/parabasal compartment and variable levels in the intermediate and superficial compartments. It was significantly associated with CIN3 ( p = 0.009), not found in CIN1 and only seen in part of the CIN2 lesions. When the different patterns were correlated with the genetic make-up and presence of HPV, the CIN3-related pattern contained HPV-positive cells in the basal/parabasal cell compartment that were disomic. This is in contrast to the areas exhibiting the CIN1 and CIN2 related patterns, which frequently exhibited aneusomic cells. Based on their SOX2 localisation pattern, CIN1 and CIN2 could be delineated from CIN3. These data shed new light on the pathogenesis and dynamics of progression in premalignant cervical lesions, as well as on the target cells in the epithelium for HPV infection.
Effects of IPZ treatment on the maturation and cell cycle progression of porcine oocytes. A Representative images of the first polar body (PB1) extrusion in the control and IPZ-treated groups. Arrow: PB1. Scale bar = 100 μm. B The PB1 extrusion rate of the control and IPZ-treated groups. n = 105. C The proportion of oocytes arrested at different stages after IPZ treatment. n = 105. The letter “n” indicates the total number of oocytes in each group of three independent replicates. a,b,c Values with different superscript letters above columns in the same stage indicate significant differences (P < 0.05)
Effects of IPZ treatment on cytoskeletal dynamics of porcine oocytes during GV-to-MI stage. A Representative images of spindle morphology and chromosome alignment in the control and IPZ-treated groups. n = 105. Green: α-tubulin, blue: chromosome. Scale bar = 5 μm. After 28 h of culture, most oocytes assembled multipolar, malformed spindles and mislocated, condensed chromosomes in the IPZ-treated group. B Percentages of oocytes with aberrant spindles in the control and IPZ-treated groups. n = 105. C, D The expression of p-MAPK in control and IPZ-treated oocytes after 28 h of culture. E Representative images of actin filaments in the control and IPZ-treated groups. Red: actin. Scale bar = 20 μm. F The percentage of oocytes with actin filaments mislocalized to the cytoplasm in the control and IPZ-treated groups after 28 h of culture. n = 105. G Representative images of TPX2 subcellular localization in control and IPZ-treated oocytes. Blue: chromosome, green: α-tubulin, red: TPX2. Scale bar = 5 μm. After 28 h of culture, TPX2 was scattered around the aberrant microtubules, showing an irregular distribution in the IPZ-treated group. H–K The protein expression of TPX2, AURKA and TACC3 in control and IPZ-treated oocytes after 28 h of culture
Effects of IPZ treatment on the maturation and cell cycle progression of porcine oocytes during MI-to-MII stage. A Representative images of PB1 extrusion in the control and IPZ-treated groups after a total of 44 h of culture (25 μM IPZ was added for the latter 16 h of culture). Arrow: PB1. Scale bar = 100 μm. B The PB1 extrusion rate of the control and IPZ-treated groups after IPZ treatment. n = 105. C The proportion of oocytes arrested at different stages after IPZ treatment. n = 105
Effects of IPZ treatment on cytoskeletal dynamics of porcine oocytes during the MI-to-MII stage. A Representative images of spindle morphology and chromosome alignment in control and IPZ-treated cells after a total of 36 h of culture (25 μM IPZ was added for the latter 8 h of culture). Scale bar = 5 μm. Blue: chromosome, green: α-tubulin. B Percentages of oocytes with aberrant spindles in the control and IPZ-treated groups after 36 h of culture. n = 105. C Representative images of spindle morphology and chromosome alignment in control and IPZ-treated oocytes after 44 h of culture (25 μM IPZ was added for the latter 16 h of culture). Scale bar = 5 μm. Blue: chromosome, green: α-tubulin. D Percentages of oocytes with aberrant spindles in the control and IPZ-treated groups after 44 h of culture. n = 105. E, F The expression of p-MAPK in control and IPZ-treated oocytes after 44 h of culture. G Representative images of actin filaments in the control and IPZ-treated groups after 44 h of culture. Red: actin. Scale bar = 20 μm. H The percentage of oocytes with actin filaments mislocalized to the cytoplasm in the control and IPZ-treated groups after 44 h of culture. n = 105. I Representative images of TPX2 subcellular localization in control and IPZ-treated oocytes after 44 h of culture (25 μM IPZ was added for the latter 16 h of culture). Blue: chromosome, green: α-tubulin, red: TPX2. Scale bar = 5 μm. In the IPZ-treated group, TPX2 was irregularly scattered around the aberrant microtubules without obvious enrichment. J–M The protein expression of TPX2, AURKA, and TACC3 in control and IPZ-treated oocytes after 44 h of culture
The Ran-GTP/importin β pathway has been implicated in a diverse array of mitotic functions in somatic mitosis; however, the possible meiotic roles of Ran-GTP/importin β in mammalian oocyte meiosis are still not fully understood. In the present study, importazole (IPZ), a small molecule inhibitor of the interaction between Ran and importin β was used to explore the potential meiotic roles of Ran-GTP/importin β in porcine oocytes undergoing meiosis. After IPZ treatment, the extrusion rate of the first polar body (PB1) was significantly decreased, and a higher proportion of the oocytes were arrested at the germinal vesicle breakdown (GVBD) stage. Moreover, IPZ treatment led to severe defects in metaphase I (MI) spindle assembly and chromosome alignment during the germinal vesicle (GV)-to-MI stage, as well as failure of metaphase II (MII) spindle reassembly and homologous chromosome segregation during the MI-to-MII stage. Notably, IPZ treatment decreased TPX2 expression and abnormal subcellular localization. Furthermore, the expression levels of aurora kinase A (AURKA) and transforming acidic coiled-coil 3 (TACC3) were significantly reduced after IPZ treatment. Collectively, these data indicate that the interaction of Ran-GTP and importin β is essential for proper spindle assembly and successful chromosome segregation during two consecutive meiotic divisions in porcine oocytes, and regulation of this complex might be related to its effect on the TPX2 signaling cascades. Graphical abstract
Vessel segment undergoing neutrophil extravasation during cutaneous inflammation. a Representative time-lapse still image of an inflamed vessel from intravital fluorescence microscopy showing the endothelial cell layer (red), transmigrating neutrophils (green), and platelets (white). Original movie in Supplementary Movie 1. b, c Electron micrographs of inflamed mouse skin sample, conventionally fixated and processed with CLEM techniques, exhibit poor preservation of the ultrastructure (asterisk marking bulging membranes of platelets) and distortion of the sections because of weak epon infiltration (arrowhead). # vessel lumen, end endothelial cell, neu neutrophil, pl platelet, *bulging membranes. Scale bar in a = 10 µm, scale bar in b, c = 5 µm
Introduction of landmarks for CLEM orientation. Illustrated workflow describing all steps introducing landmarks, which are essential to reposition ROI at the electron microscope exactly at the light microscopical location recorded by IVM in a dorsal skin chamber (a). The printed stamp on the skin supports to locate the laser point, which illuminates the last position of the IVM settings (ROI) b. After fixation, punching out, and resizing the skin to a minimum, the sample is glued to a carbon grid and marked on one corner c. The endogenous, visible vessels in the tissue serve as the next landmarks while repositioning the sample on the confocal stage f, where the fluorescently labeled vessel network in the fixed tissue serve as landmarks to relocate the ROI at the confocal microscope g, h. With the help of the carbon-gridded cover slip and its imprinted letters, the position of the ROI can be documented with respect to these very letters d, e. Together with the distance in the z-direction between the ROI and the coverslip, this information about the x–y location defines the starting point for the microtome sectioning j further illustrated in Fig. 3. The measured z height in the sample thus guides this approach to successfully find the correlative view between light and electron microscope (CLEM) k
Target trimming and sectioning. a Outline of the CLEM challenge: retrieve a specific area from skin tissue, recorded by IVM (left panel), repositioned several times through the entire sample preparation (middle panel) and reimaging the area exactly at the ROI (*) in the electron microscope (right panel). The trimming and sectioning of the embedded sample block needs to be done in a controlled manner as further illustrated in b. After detachment from the coverslip, the block surface is covered with traces of the carbon grid (left). The resin material is trimmed away from the ROI, identified by the letter of the carbon imprint, resulting in a small square block (second image). One corner is cut off, defining a new reference point. Next, the ROI is approached by careful sectioning with µm/nm step size, until the z-height of the vessel of interest is reached. The approach is documented with 200-nm-thick sections, stained with toluidine blue, which are continuously compared with the pattern of the vessel network as imaged in the confocal microscope on the prefixed sample. The overlay of all corresponding layers (fluorescence image, carbon grid, and semithin and thin section) prove that the vessel has been retrieved in a correlative mode and allows further investigation at higher resolution in the electron microscope
Analyzing the ROI by thin sectioning, thick sectioning, or serial block-face sectioning. Electron micrographs of successfully recovered CLEM vessels of inflamed mouse skin. Throughout the vessel, several cellular interactions can be observed and studied in more detail with respect to the surrounding tissue components such as the endothelial cell (end) and transmigrating neutrophil (neu) leaving the lumen of the vessel (#). To provide more “volume” information, several techniques can be applied. Serial consecutive sections (60 nm thin or 200 nm thick) can be used to follow structures in 3D on a normal transmission electron microscope a, b. Physical distortions occur regularly, marked by arrowheads. An alternative approach is automated sectioning by SBF-SEM. The sample block is continuously cut via an ultramicrotome, accommodated within the instrument. The scanning detector scans the surface after every section, resulting in a large 3D volume of the selected ROI that can be recorded and analyzed as single sections c. Here, the contour of a transmigrating neutrophil is highlighted in green, and additionally annotated in Supplementary Movie 3. Scale bar = 5 µm
The nanometer spatial resolution of electron microscopy imaging remains an advantage over light microscopy, but the restricted field of view that can be inspected and the inability to visualize dynamic cellular events are definitely drawbacks of standard transmission electron microscopy (TEM). Several methods have been developed to overcome these limitations, mainly by correlating the light microscopical image to the electron microscope with correlative light and electron microscopy (CLEM) techniques. Since there is more than one method to obtain the region of interest (ROI), the workflow must be adjusted according to the research question and biological material addressed. Here, we describe in detail the development of a three-dimensional CLEM workflow for mouse skin tissue exposed to an inflammation stimulus and imaged by intravital microscopy (IVM) before fixation. Our aim is to relocate a distinct vessel in the electron microscope, addressing a complex biological question: how do cells interact with each other and the surrounding environment at the ultrastructural level? Retracing the area over several preparation steps did not involve any specific automated instruments but was entirely led by anatomical and artificially introduced landmarks, including blood vessel architecture and carbon-coated grids. Successful retrieval of the ROI by electron microscopy depended on particularly high precision during sample manipulation and extensive documentation. Further modification of the TEM sample preparation protocol for mouse skin tissue even rendered the specimen suitable for serial block-face scanning electron microscopy (SBF-SEM).
Histological slides are an important tool in the diagnosis of tumors as well as of other diseases that affect cell shapes and distributions. Until now, the research concerning an optimal staining time has been mainly done empirically. In experimental investigations, it is often not possible to stain an already-stained slide with another stain to receive further information. To overcome these challenges, in the present paper a continuum-based model was developed for conducting a virtual (re-)staining of a scanned histological slide. This model is capable of simulating the staining of cell nuclei with the dye hematoxylin (C.I. 75,290). The transport and binding of the dye are modeled (i) along with the resulting RGB intensities (ii). For (i), a coupled diffusion–reaction equation is used and for (ii) Beer–Lambert’s law. For the spatial discretization an approach based on the finite element method (FEM) is used and for the time discretization a finite difference method (FDM). For the validation of the proposed model, frozen sections from human liver biopsies stained with hemalum were used. The staining times were varied so that the development of the staining intensity could be observed over time. The results show that the model is capable of predicting the staining process. The model can therefore be used to perform a virtual (re-)staining of a histological sample. This allows a change of the staining parameters without the need of acquiring an additional sample. The virtual standardization of the staining is the first step towards universal cross-site comparability of histological slides.
Effects of anesthesia on behavioral rhythm of rats. a Schedule of the behavioral rhythm analysis. White and black bars indicate light and dark conditions, respectively. Shaded bars correspond to the light period of the normal light–dark (LD) cycle in constant dark (DD) condition. Measurement of locomotor activity started on day 1 and changed to the DD condition on day 3, while anesthesia treatment was performed on day 5. Panels b, c, and d show representative double-plotted actograms of rats in the sevoflurane, propofol, and dexmedetomidine groups with corresponding controls, respectively. Activity was represented by double-plotted actograms, in which 48 h of activity is plotted in one row, with the last 24 h of each row overlapping with the first 24 h of the next row. Note that anesthesia administration (red arrow) is shown twice in the double plot but is actually a single administration. Light-blue vertical line in the figure indicates the point at which the rats were transferred to constant darkness (DD) conditions. The green line indicates the trace of the onset of the active phase of the rats
Effect of 4-h anesthetic administration from 8:00 to 12:00 on Period2 (Per2) expression in rat suprachiasmatic nucleus (SCN). Anesthesia was administered from 8:00 to 12:00 (inactive period of rats), and Per2 expression in the SCN was assessed by in situ hybridization (ISH). a Schedule of anesthetic treatment and brain sampling. b Groups with brain sampled at 12:00 immediately after cessation of anesthesia. Photomicrographs in the upper panel shows representative ISH images of brain containing the SCN area, while the lower panel shows the quantification of Per2. c Groups with brain sampled at 16:00 on the same day. d Groups with brain sampled at 12:00 on the next day. Values expressed as mean ± standard error on the mean (SEM) (n = 4) (*p < 0.05 (Student’s t test), scale bars: 200 µm)
Effects of 4-h anesthetic administration from 20:00 to 24:00 on Per2 expression in rat SCN. Anesthesia was administered from 20:00 to 24:00 (active period of rats), and Per2 expression in the SCN was assessed by ISH. a Schedule of anesthetic treatment and brain sampling. b Representative ISH images of brain sections including the SCN region (upper panel) and Per2 quantification (lower panel). Scale bars: 200 µm, values expressed as mean ± SEM (n = 6)
The suprachiasmatic nucleus (SCN) of the hypothalamus is a nucleus that regulates circadian rhythms through the cyclic expression of clock genes. It has been suggested that circadian-rhythm-related, adverse postoperative events, including sleep disturbances and delirium, are partly caused by anesthesia-induced disruption of clock-gene expression. We examined the effects of multiple general anesthetics on the expression cycle of Period2 (Per2), one of the clock genes that regulate circadian rhythms in the SCN, and on the behavioral rhythms of animals. Rats were treated with sevoflurane, propofol, and dexmedetomidine for 4 h. The expression of Per2 in SCN was analyzed using in situ hybridization, and the behavioral rhythm before and after anesthesia was analyzed. Per2 expression in the SCN decreased significantly immediately after anesthesia in all groups compared with corresponding control groups. However, Per2 returned to normal levels within 24 h, and there was no phase change in the gene expression cycle or behavioral rhythm. This study suggests that acute suppression of Per2 expression may be a general phenomenon induced by general anesthesia, but that the molecular mechanism of the body clock is resilient to disturbances to some extent.
Cross-sections of fetal corneas 10–11 wg (a, e, i) and 20 wg (b, f, j) as well as normal adult corneas (c, g, k) and ARK (d, h, l) labeled with antibodies (green) against Notch1 (a–d), Dlk1 (e–h), and Numb (i–l). The corneal epithelium is shown at the top and the stroma below, in all photographs of all figures. Cell nuclei are labeled blue with DAPI (a–l). Immunolabeling against Notch1 (a–d) was detected as streaks in the stroma and strongly around the basal layers of the epithelium in all fetal and adult corneas (a–c), whereas in the ARK corneas, Notch1 was absent in the epithelium and only very scarce in the stroma (d). In the 20 wg fetal corneas, the labeling of the epithelium against Notch1 was slightly surpassed by the strong DAPI labeling of the epithelial cell nuclei (b). Dlk1 (e–h) labeled the epithelial cells and streaks in the stroma of all fetal corneas, more abundantly in the anterior region (e, f, asterisk), whereas in the adult corneas labeling was only present in the epithelium (g). In the ARK corneas, Dlk1 immunolabeling was present both in the epithelium and in streaks, more pronounced in the anterior pannus (h). In all fetal corneas, Abs against Numb (i–l), another inhibitor of Notch1, labeled the epithelial cells and streaks in the stroma (i, j, asterisk), but stromal labeling was more abundant in the 20 wg (j) than in the 10–11 wg fetal corneas (i). Numb labeling in adult corneas was present in the epithelial cells and in sporadic streaks in the stroma (k). In ARK, Numb immunolabeling was detected in the epithelium and anterior pannus (l) in a pattern similar to that of Dlk1. Bars, 100 μm
Cross-sections of fetal corneas 10–11 wg (a, e, i), 20 wg (b, f, j), normal adult corneas (c, g, k), and ARK (d, h, l) labeled with antibodies (green) against Wnt5a (a–d), Wnt7a (e–h), and β-catenin (i–l). Cell nuclei are labeled blue with DAPI (a–l). The Abs against Wnt5a abundantly labeled both the epithelial cells and streaks in the stroma of the fetal corneas (a, b). The stromal labeling was more abundant in the 10–11 wg (a) than in the 20 wg fetal corneas (b), in which the streaks were more profuse in the anterior region (b). In the adult corneas, only the epithelium was labeled (c), whereas in ARK corneas both the epithelium and the anterior pannus were labeled (d). Immunolabeling against Wnt7a was found in the epithelial cells and as stromal streaks in all fetal samples (e, f). In contrast, in the adult corneas, labeling was present in the epithelium but only in extremely sparse steaks in the stroma (g). In ARK, the epithelium and anterior pannus as well as the rest of the stroma were labeled by Wnt7a (h). β-Catenin immunolabeling was present abundantly in the contours of epithelial cells but only discretely in stromal streaks, in a similar pattern in the 10–11 wg (i) and 20 wg fetal corneas (j). In the adult corneas, labeling was present in the epithelial cells more intensively in the basal region but absent in the stroma (k). The staining pattern in the ARK corneas was similar to that of the fetal corneas, with β-catenin immunolabeling delineating the contours of epithelial cells and present in streaks in the anterior pannus (l). Bars, 100 μm
Cross-sections of fetal corneas 10–11 wg (a, e, i, m) and 20 wg (b, f, j, n), adult normal corneas (c, g, k, o), and ARK (d, h, l, p) labeled with antibodies (green) against Hes1 (a–d), Gli1 (e–h), mTOR (i–l), and p-rpS6 (m–p). Tissue preservation of the fetal tissue was variable. Cell nuclei are labeled blue with DAPI (a–p). Abs against Hes1 strongly immunolabeled the epithelial cells of the fetal corneas (a, b). These Abs labeled streaks in the stroma more profusely in the 10–11 wg (a) than in the 20 wg fetal corneas (b) and more intensively in the anterior stroma (b). In contrast, in the adult corneas, immunolabeling was not detected (c), whereas Hes1 was present in the epithelium and in the anterior stroma of the ARK corneas (d). Immunolabeling with Abs against Gli1 was present in the epithelial cells in all fetal corneas (e, f). Labeling in the stroma was present in streaks in all fetal corneas (e, f). Immunolabeling for these Abs was not observed in adult corneas (g). In the ARK corneas, the Abs against Gli1 labeled the epithelium and the anterior pannus (h). The inserts in a–h show the epithelium at higher magnification. The Ab against mTOR abundantly labeled the epithelial cells of all fetal corneas (i, j) but marked the epithelium in the 20 wg fetal corneas more intensively (j). The stroma in all fetal corneas was labeled in streaks (i, j) but was more abundantly marked in the 10–11 wg fetal corneas (i). This Ab did not immunolabel the adult corneas (k), but the epithelium and anterior pannus were labeled by this Ab in the ARK corneas (l). The Ab against p-rpS6 labeled the epithelial cells and abundant streaks in the stroma of all fetal corneas in a likewise pattern in both 10–11 wg (m) and 20 wg fetal corneas (n). The stroma in the adult corneas was not labeled and the surface of the epithelium was only scarcely labeled, suggesting sticky adherence to the epithelial surface (o). In the ARK corneas, immunostaining against p-rpS6 was present in the epithelium and anterior pannus (p). Bars, 100 μm
Gene expression of Notch1 (NOTCH1), Dlk1 (DLK1), Numb (NUMB) (a), Wnt5A (WNT5A), Wnt7A (WNT7A), β-catenin (CTNNB1) (b), and Hes1 (HES1), mTOR (mTOR), and rps6 (RPS6) (c) in 9–12 wg fetal corneas as compared with adult cornea. There was a 3.89-fold increase in Notch1 gene expression, 1540-fold increase in Dlk1 gene expression, and 0.64-fold decrease in Numb gene expression in 9–12 wg fetal corneas. (a). Expression of Wnt5A, Wnt7A, and β-catenin genes was increased by 1.85-, 3.57-, and 2.05-fold, respectively. (b). Gene expression of Hes1, mTOR, and rps6 was increased by 2.73-, 1.89-, and 1.78-fold, respectively (c). Values are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
We aimed to study aniridia-related keratopathy (ARK) relevant cell signaling pathways [Notch1, Wnt/β-catenin, Sonic hedgehog (SHH) and mTOR] in normal human fetal corneas compared with normal human adult corneas and ARK corneas. We found that fetal corneas at 20 weeks of gestation (wg) and normal adult corneas showed similar staining patterns for Notch1; however 10–11 wg fetal corneas showed increased presence of Notch1. Numb and Dlk1 had an enhanced presence in the fetal corneas compared with the adult corneas. Fetal corneas showed stronger immunolabeling with antibodies against β-catenin, Wnt5a, Wnt7a, Gli1, Hes1, p-rpS6, and mTOR when compared with the adult corneas. Gene expression of Notch1, Wnt5A, Wnt7A, β-catenin, Hes1, mTOR, and rps6 was higher in the 9–12 wg fetal corneas compared with adult corneas. The cell signaling pathway differences found between human fetal and adult corneas were similar to those previously found in ARK corneas with the exception of Notch1. Analogous profiles of cell signaling pathway activation between human fetal corneas and ARK corneas suggests that there is a less differentiated host milieu in ARK.
Monoclonal immunoglobulin-G (IgG) antibodies are now emerging as therapeutic tools to tackle various disorders, including those affecting the brain. However, little is known about how these IgG molecules behave in the brain. To better understand the potential behavior of IgG molecules in the brain, here we established a specific protocol to immunolocalize rat IgG injected into mouse striatum with an anti-rat IgG antibody. Using double immunolabeling, IgG-like immunoreactivity (IR) was mainly found in neurons but scarcely observed in glia 1 h after intrastriatal injection of IgG, whereas some surrounding glia contained IgG-like IR 24 h after injection. However, preabsorption with a large excess of rat IgG to confirm the authenticity of this labeling failed to eliminate this neuronal IgG-like IR but rather exhibited nuclear staining in glial cells. Because this unexpected nuclear staining escalated with increasing amount of absorbing IgG, we postulated that this nuclear staining is due to formation of immune complex IgG–anti-IgG, which can be removed by centrifugal filtration. As expected, this nuclear staining in glial cells was eliminated after centrifugal filtration of the IgG/anti-IgG mixture, and authentic IgG-like IR was chiefly detected in the cytoplasm of neurons around the injection channel. This study is the first demonstration of neuronal redistribution of injected IgG in the mouse brain. Neuronal internalization of exogenous IgG may be advantageous especially when the therapeutic targets of monoclonal IgG are intraneuronal such as neurofibrillary tangles or Lewy bodies.
Adipogenic differentiation of wild-type and RB1 mutant OAMSCs: phase-contrast images of the adipogenic differentiation—days 0, 7 14, and 21 of wild-type and RB1 mutant OAMSCs (a). Adipocyte foci (black arrows) appear on day 14 in wild type and on day 7 in RB1 mutants. Adipocyte foci were counted in four fields with total area (field of view) of 16.72 mm². The number of adipocytic foci showed statistically significant increase (p < 0.001; ANOVA) in the RB1 mutant compared with wild type on days 7, 14, and 21 (b). Representative phase-contrast images of the adipocyte foci on day 21 of differentiation showing globular, refractile, intracellular lipid vesicles in wild type and RB1 mutants (c). Scale bars: 300 µm (panel a), 30 µm (panel c)
BODIPY staining of differentiated adipocytes: wild-type and RB1 mutant OAMSCs differentiated for 21 days using StemPro adipogenic differentiation kit and stained with BODIPY for 20 min at 37 °C and imaged using fluorescence microscopy (a). BODIPY-positive cells in wild type (n = 31) and RB1 mutant (n = 86) were analyzed for fluorescence intensity. RB1 mutant adipocytes showed statistically significant increase in the fluorescence intensity per cell (p < 0.0001) compared with the wild type (b). Scale bar, 50 µm (panel a)
Comparative gene expression profiles of adipogenic markers during adipogenesis of wild-type and RB1 mutant OAMSCs: temporal gene expression levels of PPARG and CEBPA (transcription factors); FABP4, GLUT4, and LPL (adipocyte markers); LEP (white adipocyte marker); and LHX8, PGC1A, and UCP1 (brown adipocyte markers) during the adipogenesis of RB1 mutants compared with wild type. Significant difference in the expression of the adipogenic markers in the RB1 mutant OAMSCs during the days of differentiation (0, 7, 14, and 21) compared with the wild type is represented by * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, ns no significance. The p value below the graph represents the overall significance between the wild type and RB1 mutant
Immunocytochemical staining of UCP1 on differentiated adipocytes: wild-type and RB1 mutant OAMSCs differentiated for 21 days using StemPro adipogenic differentiation kit were subjected to immunocytochemical staining for UCP1 (a). DAPI-positive cells in wild type (n = 558) and RB1 mutant (n = 632) were counted for UCP1 positivity. RB1 mutant adipocytes showed statistically significant increase in UCP1 expression compared with wild type (p = 0.0013) (b). Scale bar, 50 µm (panel a)
Cell cycle analysis of the differentiated adipocytes by flow cytometry and immunocytochemistry: cell cycle analysis of the wild-type and RB1 mutant adipocytes was carried out using BD FACSLyric flow cytometer (a). RB1 mutant adipocytes showed statistically significant increase in the number of cells in G2/M phase (p = 0.0325), with no significant difference in G0/G1 phase (p = 0.6479) or S phase (p = 0.1306) of cell cycle (b). Wild-type and RB1 mutant OAMSCs differentiated for 21 days using StemPro adipogenic differentiation kit were subjected to immunocytochemical staining for mitotic phase marker PH3 (d). DAPI-positive cells in wild type (n = 831) and RB1 mutant (n = 994) were counted for PH3 positivity. RB1 mutant showed statistically significant increase in PH3 expression compared with wild type (p < 0.0001) (c). Scale bars, 50 µm (panel d)
Retinoblastoma (RB1) protein is a multifunctional protein that plays an important role in cell cycle regulation and cell differentiation, including adipogenesis. A detailed literature search to understand the role of RB1 in adipogenesis revealed that the nature of the RB1 inactivation (in vivo/in vitro) led to differences in adipogenesis. The majority of these studies were animal-based, and the only study in humans employed an in vitro mode of RB1 inactivation. To overcome these differences and lack of human studies, we sought to explore the role of RB1 in adipogenesis using orbital adipose mesenchymal stem cells (OAMSCs) from patients with retinoblastoma that innately carry a heterozygous RB1 mutation. We hypothesized that these patient-derived RB1 mutant OAMSCs can model in vivo RB1 inactivation in humans. Our study revealed increased adipogenesis with a bias toward brown adipogenesis in the RB1 mutant in addition to an increased number of adipocytes in the mitotic phase.
HLA-G immunoexpression according to the tested clones. A 4H84-negative case (original magnification 600×); B 4H84-positive case showing a cytoplasmic expression with membrane reinforcement (original magnification 600×); C MEM-G/2-negative case (original magnification 600×); D MEM-G/2-positive case showing cytoplasmic expression but lacking membrane reinforcement (original magnification 600×). All scale bars: 50 microns
Survival analysis. a Disease-free survival differences according to stage (stage II vs. stage III). b Disease-free survival differences comparing HLA-G positive (stratified according to antibody clone) and negative cases
Identifying innovative molecules involved in the tumor immune escape process could help refine the survival stratification of colorectal cancer (CRC) patients. HLA-G, a non-classical HLA molecule, physiologically involved in tolerogenic mechanisms, has recently emerged as a relevant prognostic marker in other tumor types, but ambiguous data are reported in the CRC setting. This study aims to evaluate the HLA-G expression and prognostic potential in a series of stage II/III CRCs. HLA-G expression was evaluated in 100 pT3 CRC cases by means of immunohistochemistry using the 4H84 and MEM-G/2 monoclonal antibodies. We observed heterogeneous expression of HLA-G showing different ranges: 4H84 expression ranged from > 1 to 40%—median 7%; MEM-G/2 expression ranged from 20 to 90%—median 50%. HLA-G positivity (any intensity > 1%) varied according to the antibody employed, identifying: 8 4H84 positive, 34 MEM-G/2 positive, 6 double-positive and 52 negative cases. Correlation with clinico-pathologic data showed a significant association with a poor tumor differentiation in stage III right-sided CRC subgroup (p = 0.043), while no other pathologic variable was significantly associated. Survival analysis revealed a reduced disease-free survival rate (HR 4.304613; p = 0.031) in the subgroup of CRC-related death cases, while no correlations were observed considering the whole series and the overall survival. In conclusion, HLA-G is a promising CRC prognostic marker however much work is still required regarding technical aspects and evaluation of expression.
CTP biosynthesis is carried out by two pathways: salvage and de novo. CTPsyn catalyzes the latter. The study of CTPsyn activity in mammalian cells began in the 1970s, and various fascinating discoveries were made regarding the role of CTPsyn in cancer and development. However, its ability to fit into a cellular serpent-like structure, termed ‘cytoophidia,’ was only discovered a decade ago by three independent groups of scientists. Although the self-assembly of CTPsyn into a filamentous structure is evolutionarily conserved, the enzyme activity upon this self-assembly varies in different species. CTPsyn is required for cellular development and homeostasis. Changes in the expression of CTPsyn cause developmental changes in Drosophila melanogaster. A high level of CTPsyn activity and formation of cytoophidia are often observed in rapidly proliferating cells such as in stem and cancer cells. Meanwhile, the deficiency of CTPsyn causes severe immunodeficiency leading to immunocompromised diseases caused by bacteria, viruses, and parasites, making CTPsyn an attractive therapeutic target. Here, we provide an overview of the role of CTPsyn in cellular and disease perspectives along with its potential as a drug target.
Recent evidence indicates that targeting IL-6 provides broad therapeutic approaches to several diseases. In patients with cancer, autoimmune diseases, severe respiratory infections [e.g. coronavirus disease 2019 (COVID-19)] and wound healing, IL-6 plays a critical role in modulating the systemic and local microenvironment. Elevated serum levels of IL-6 interfere with the systemic immune response and are associated with disease progression and prognosis. As already noted, monoclonal antibodies blocking either IL-6 or binding of IL-6 to receptors have been used/tested successfully in the treatment of rheumatoid arthritis, many cancer types, and COVID-19. Therefore, in the present review, we compare the impact of IL-6 and anti-IL-6 therapy to demonstrate common (pathological) features of the studied diseases such as formation of granulation tissue with the presence of myofibroblasts and deposition of new extracellular matrix. We also discuss abnormal activation of other wound-healing-related pathways that have been implicated in autoimmune disorders, cancer or COVID-19.
Tumor progression is profoundly affected by crosstalk between cancer cells and their stroma. In the past decades, the development of bioinformatics and the establishment of organoid model systems have allowed extensive investigation of the relationship between tumor cells and the tumor microenvironment (TME). However, the interaction between tumor cells and the extracellular matrix (ECM) in odontogenic epithelial neoplasms and the ECM remodeling mechanism remain unclear. In the present study, transcriptomic comparison and histopathologic analysis revealed that TME-related genes were upregulated in ameloblastoma compared to in odontogenic keratocysts. Tumoroid analysis indicated that type I collagen is required for ameloblastoma progression. Furthermore, ameloblastoma shows the capacity to remodel the ECM independently of cancer-associated fibroblasts. In conclusion, ameloblastoma-mediated ECM remodeling contributes to the formation of an invasive collagen architecture during tumor progression.
We previously reported that the membrane skeletal protein 4.1G in the peripheral nervous system transports membrane palmitoylated protein 6 (MPP6), which interacts with the synaptic scaffolding protein Lin7 and cell adhesion molecule 4 (CADM4) in Schwann cells that form myelin. In the present study, we investigated the localization of and proteins related to MPP2, a highly homologous family protein of MPP6, in the cerebellum of the mouse central nervous system, in which neurons are well organized. Immunostaining for MPP2 was observed at cerebellar glomeruli (CG) in the granular layer after postnatal day 14. Using the high-resolution Airyscan mode of a confocal laser-scanning microscope, MPP2 was detected as a dot pattern and colocalized with CADM1 and Lin7, recognized as small ring/line patterns, as well as with calcium/calmodulin-dependent serine protein kinase (CASK), NMDA glutamate receptor 1 (GluN1), and M-cadherin, recognized as dot patterns, indicating the localization of MPP2 in the excitatory postsynaptic region and adherens junctions of granule cells. An immunoprecipitation analysis revealed that MPP2 formed a molecular complex with CADM1, CASK, M-cadherin, and Lin7. Furthermore, the Lin7 staining pattern showed small rings surrounding mossy fibers in wild-type CG, while it changed to the dot/spot pattern inside small rings detected with CADM1 staining in MPP2-deficient CG. These results indicate that MPP2 influences the distribution of Lin7 to synaptic cell membranes at postsynaptic regions in granule cells at CG, at which electric signals enter the cerebellum.
Hippocampal CA1 stratum radiatum of WT mice labeled with GFAP (a) or GLAST (b) antibodies in combination with an immunogold method for electron microscopy. a GFAP labeling is concentrated in the cytoplasm of the soma and the main branches of astrocytes. b GLAST labeling is localized in the plasma membrane of astrocytic cell bodies, main branches as well as small and thin projections. c GFAP and GLAST labeling distribution within astrocytes (cytoplasm or membrane). Astrocyte: orange. Scale bars: 1 μm
Hippocampal CA1 stratum radiatum of WT mice showing GFAP (a) and GLAST (b) staining with an immunoperoxidase method for electron microscopy. GFAP labeling is restricted to the cell body and main projections of an astrocyte. GLAST is distributed in numerous thin astroglial processes throughout the neuropil. c, d Statistics of labeled astroglia with each marker. Astroglial area (μm²) and membrane (μm) labeled with GFAP or GLAST per 100 μm². Data were analyzed by parametric tests (unpaired t-test, ****p < 0.0001). Astrocyte: orange. Scale bars: 2 μm
Hippocampal CA1 stratum radiatum of WT mice labeled for GFAP-CB1 (a) or GLAST-CB1 (b, c). CB1 receptor labeling (arrows) is found in membranes of excitatory terminals (ter, green), astrocytes (as, orange) and mitochondria (m, purple), but is concentrated in inhibitory terminals (ter, red). d CB1 receptor labeling is absent in CB1-KO tissue processed for GLAST-CB1 immunohistochemistry. e Percentage of astroglial CB1 receptors in GFAP-CB1 (4.98 ± 1.10%) and GLAST-CB1 (11.92 ± 0.64%). Dendrite, den: blue; spine, sp: blue. Scale bars: 500 nm
The cannabinoid CB1 receptor-mediated functions in astrocytes are highly dependent on the CB1 receptor distribution in these glial cells relative to neuronal sites, particularly at the nearby synapses under normal or pathological conditions. However, the portrait of the CB1 receptor distribution in astroglial compartments remains uncompleted because of the scarce CB1 receptor expression in these cells and the limited identification of astrocytes. The glial fibrillary acidic protein (GFAP) is commonly used as astroglial marker. However, because GFAP is a cytoskeleton protein mostly restricted to the astroglial cell bodies and their main branches, it seems not ideal for the localization of CB1 receptor distribution in astrocytes. Therefore, alternative markers to decipher the actual astroglial CB1 receptors are required. In this work, we have compared the glutamate aspartate transporter (GLAST) versus GFAP for the CB1 receptor localization in astrocytes. We found by immunoelectron microscopy that GLAST reveals almost three-fold astroglial area and four-fold astroglial membranes compared to GFAP. In addition, this better visualization of astrocytes was associated with the detection of 12% of the total CB1 receptor labeling in GLAST-positive astrocytes.
Zinc homeostasis is vital to immune and other organ system functions, yet over a quarter of the world’s population is zinc deficient. Abnormal zinc transport or storage protein expression has been linked to diseases, such as cancer and chronic obstructive pulmonary disorder. Although recent studies indicate a role for zinc regulation in vascular functions and diseases, detailed knowledge of the mechanisms involved remains unknown. This study aimed to assess protein expression and localization of zinc transporters of the SLC39A/ZIP family (ZIPs) and metallothioneins (MTs) in human subcutaneous microvessels and to relate them to morphological features and expression of function-related molecules in the microvasculature. Microvessels in paraffin biopsies of subcutaneous adipose tissues from 14 patients undergoing hernia reconstruction surgery were analysed for 9 ZIPs and 3 MT proteins by MQCM (multifluorescence quantitative confocal microscopy). Zinc regulation proteins detected in human microvasculature included ZIP1, ZIP2, ZIP8, ZIP10, ZIP12, ZIP14 and MT1-3, which showed differential localization among endothelial and smooth muscle cells. ZIP1, ZIP2, ZIP12 and MT3 showed significantly ( p < 0.05) increased immunoreactivities, in association with increased microvascular muscularization, and upregulated ET-1, α-SMA and the active form of p38 MAPK (Thr180/Tyr182 phosphorylated, p38 MAPK-P). These findings support roles of the zinc regulation system in microvascular physiology and diseases.
Congestive hepatopathy (CH) is a chronic liver disease (CLD) caused by impaired hepatic venous blood outflow, most frequently resulting from congestive heart failure. Although it is known that heart failure and CLDs contribute to increased risk for age-related fractures, an assessment of CH-induced skeletal alterations has not been made to date. The aim of our study was to characterize changes in bone quality in adult male cadavers with pathohistologically confirmed CH compared with controls without liver disease. The anterior mid-transverse part of the fifth lumbar vertebral body was collected from 33 adult male cadavers (age range 43–89 years), divided into the CH group (n = 15) and the control group (n = 18). We evaluated trabecular and cortical micro-architecture and bone mineral content (using micro-computed tomography), bone mechanical competence (using Vickers micro-hardness tester), vertebral cellular indices (osteocyte lacunar network and bone marrow adiposity), and osteocytic sclerostin and connexin 43 expression levels (using immunohistochemistry staining and analysis). Deterioration in trabecular micro-architecture, reduced trabecular and cortical mineral content, and decreased Vickers microhardness were noted in the CH group (p < 0.05). Reduced total number of osteocytes and declined connexin 43 expression levels (p < 0.05) implied that harmed mechanotransduction throughout the osteocyte network might be present in CH. Moreover, elevated expression levels of sclerostin by osteocytes could indicate the role of sclerostin in mediating low bone formation in individuals with CH. Taken together, these micro-scale bone alterations suggest that vertebral strength could be compromised in men with CH, implying that vertebral fracture risk assessment and subsequent therapy may need to be considered in these patients. However, further research is required to confirm the clinical relevance of our findings.
A high fructose diet is a major cause of diabetes and various metabolic disorders, including fatty liver. In this study, we investigated the effects of resveratrol and vitamin D (VitD) treatments on endoplasmic reticulum (ER) stress, oxidative stress, inflammation, apoptosis, and liver regeneration in a rat model of type 2 diabetes mellitus, namely, T2DM Sprague–Dawley rats. This T2DM rat model was created through a combination treatment of a 10% fructose diet and 40 mg/kg streptozotocin (STZ). Resveratrol (1 mg/kg/day) and VitD (170/IU/week) were administered alone and in combination to both the diabetic and control groups. Immunohistochemical staining was performed to evaluate PCNA, NF-κB, TNF-α, IL-6, IL-1β, GRP78, and active caspase-3 in liver tissue. The TUNEL method and Sirius red staining were used to determine apoptosis and fibro- sis, respectively. G6PD, 6-PGD, GR, and GST activities were measured to determine oxidative stress status. We found that the expressions of cytokines (TNF-α, IL-6, and IL-1β) correlated with NF-κB activation and were significantly increased in the T2DM rats. Increased GRP78 expression, indicating ER stress, increased in apoptotic cells, enhanced caspase-3 activa- tion, and collagen accumulation surrounding the central vein were observed in the T2DM group compared with the other groups. The combination VitD + resveratrol treatment improved antioxidant defense via increasing G6PD, 6-PGD, GR, and GST activities compared to the diabetic groups. We concluded that the combined administration of resveratrol with VitD ameliorates the adverse effects of T2DM by regulating blood glucose levels, increasing antioxidant defense mechanisms, controlling ER stress, enhancing tissue regeneration, improving inflammation, and reducing apoptosis in liver cells. In conclusion, this study indicates that the combination treatment of resveratrol + VitD can be a beneficial option for preventing liver damage in fructose-induced T2DM.
Megapinosomes are endocytic organelles found in human macrophage colony-stimulating factor (M-CSF) monocyte-derived M macrophages. They are large (several microns) and have a complex internal structure that is connected with the cytosol and consists of interconnected knots and concave bridges with sizes in the range of 100 nm. We called this structure trabecular meshwork. The luminal part of the megapinosome can be connected with luminal tubules and cisterns that form the megapinosome complex. The structures are especially well visible in scanning electron tomography when macrophages are prepared by high-pressure freezing and freeze substitution. Our research received a new impulse after studying the literature on hematopoietic cells, where very similar, most likely homologous, structures have been published in peritoneal macrophages as well as in megakaryocytes and blood platelets. In platelets, they serve as membrane storage that is used for structural changes of platelets during activation.
Fetal testis growth involves cell influx and extensive remodeling. Immediately after sex determination in mouse, macrophages enable normal cord formation and removal of inappropriately positioned cells. This study provides new information about macrophages and other immune cells after cord formation in fetal testes, including their density, distribution, and close cellular contacts. C57BL6J mouse testes from embryonic day (E) 13.5 to birth (post-natal day 0; PND0), were examined using immunofluorescence, immunohistochemistry, and RT-qPCR to identify macrophages (F4/80, CD206, MHCII), T cells (CD3), granulocytes/neutrophils (Ly6G), and germ cells (DDX4). F4/80 ⁺ cells were the most abundant, comprising 90% of CD45 ⁺ cells at E13.5 and declining to 65% at PND0. Changes in size, shape, and markers (CD206 and MHCII) documented during this interval align with the understanding that F4/80 ⁺ cells have different origins during embryonic life. CD3 ⁺ cells and F4/80 ⁻ /MHCII ⁺ were absent to rare until PND0. Ly6G ⁺ cells were scarce at E13.5 but increased robustly by PND0 to represent half of the CD45 ⁺ cells. These immunofluorescence data were in accord with transcript analysis, which showed that immune marker mRNAs increased with testis age. F4/80 ⁺ and Ly6G ⁺ cells were frequently inside cords adjacent to germ cells at E13.5 and E15.5. F4/80 ⁺ cells were often in clusters next to other immune cells. Macrophages inside cords at E13.5 and E15.5 (F4/80 Hi /CD206 ⁺ ) were different from macrophages at PND0 (F4/80 Dim /CD206 ⁻ ), indicating that they have distinct origins. This histological quantification coupled with transcript information identifies new cellular interactions for immune cells in fetal testis morphogenesis, and highlights new avenues for studies of their functional significance.
Overview of the workflow of each group
A major aim in structural cell biology is to analyze intact cells in three dimensions, visualize subcellular structures, and even localize proteins at the best possible resolution in three dimensions. Though recently developed electron microscopy tools such as electron tomography, or three-dimensional (3D) scanning electron microscopy, offer great resolution in three dimensions, the challenge is that, the better the resolution, usually the smaller the volume under investigation. Several different approaches to overcome this challenge were presented at the Microscopy Conference in Vienna in 2021. These tools include array tomography, batch tomography, or scanning transmission electron tomography, all of which can nowadays be extended toward correlative light and electron tomography, with greatly increased 3D information. Here, we review these tools, describe the underlying procedures, and discuss their advantages and limits.
Sample preparation for the elemental mapping using the fresh frozen osteochondral unit. (A) Workflow for the sample preparation and synchrotron XRF examination. (B) Sample preparation using EXAKT bone saw. (C) Representative fresh frozen sample preparation methods for the osteochondral sample. (D) Representative freezing of the osteochondral sample using liquid nitrogen. (E) Representative freeze-embedding of the osteochondral sample. (F) Representative sample block of the osteochondral sample. (G) Representative sample preparation in cryostat. (H) Representative cryosectioning of the osteochondral unit. (I) Representative slides of the osteochondral sample after cryosectioning. (J) Representative sandwich structure for elemental mapping
Elemental mapping for the osteochondral unit in the normal group. (A) Representative H&E staining of the osteochondral unit (n = 9). (B) Representative elemental mapping images of Compton osteochondral unit (n = 9). (C) Representative elemental mapping merged images of osteochondral unit (n = 9; Zn, Ca, Sr, Pb). (D) Representative elemental mapping images of Zn osteochondral unit (n = 9). (E) Representative elemental mapping images of Ca osteochondral unit (n = 9). (F) Representative elemental mapping images of Sr osteochondral unit (n = 9). (G) Representative elemental mapping images of Pb osteochondral unit (n = 9). Scale bar, 100 μm
Quantification for elemental mapping in the osteochondral unit in the normal osteochondral unit. (A) Quantitative Compton elemental mapping images of normal osteochondral unit (n = 9). (B) Quantitative Zn elemental mapping images of normal osteochondral unit (n = 9). (C) Quantitative Ca elemental mapping images of normal osteochondral unit (n = 9). (D) Quantitative Sr elemental mapping images of normal osteochondral unit (n = 9). (E) Quantitative Pb elemental mapping images of normal osteochondral unit (n = 9). Scale bar, 100 μm
The anatomy of the osteochondral junction is complex because several tissue components exist as a unit, including uncalcified cartilage (with superficial, middle, and deep layers), calcified cartilage, and subchondral bone. Furthermore, it is difficult to study because this region is made up of a variety of cell types and extracellular matrix compositions. Using X-ray fluorescence microscopy, we present a protocol for simultaneous elemental detection on fresh frozen samples. We transferred the osteochondral sample using a tape-assisted system and successfully tested it in synchrotron X-ray fluorescence. This protocol elucidates the distinct distribution of elements at the human knee’s osteochondral junction, making it a useful tool for analyzing the co-distribution of various elements in both healthy and diseased states.
Diabetic retinopathy (DR) is one of the leading causes of blindness in the world. While there is a major focus on the study of juvenile/adult DR, the effects of hyperglycemia during early retinal development are less well studied. Recent studies in embryonic zebrafish models of nutritional hyperglycemia (high-glucose exposure) have revealed that hyperglycemia leads to decreased cell numbers of mature retinal cell types, which has been related to a modest increase in apoptotic cell death and altered cell differentiation. However, how embryonic hyperglycemia impacts cell proliferation in developing retinas still remains unknown. Here, we exposed zebrafish embryos to 50 mM glucose from 10 h postfertilization (hpf) to 5 days postfertilization (dpf). First, we confirmed that hyperglycemia increases apoptotic death and decreases the rod and Müller glia population in the retina of 5-dpf zebrafish. Interestingly, the increase in cell death was mainly observed in the ciliary marginal zone (CMZ), where most of the proliferating cells are located. To analyze the impact of hyperglycemia in cell proliferation, mitotic activity was first quantified using pH3 immunolabeling, which revealed a significant decrease in mitotic cells in the retina (mainly in the CMZ) at 5 dpf. A significant decrease in cell proliferation in the outer nuclear and ganglion cell layers of the central retina in hyperglycemic animals was also detected using the proliferation marker PCNA. Overall, our results show that nutritional hyperglycemia decreases cellular proliferation in the developing retina, which could significantly contribute to the decline in the number of mature retinal cells.
Aluminum, the third most plentiful metal in the Earth’s crust, has potential for human exposure and harm. Oxidative stress plays an essential role in producing male infertility by inducing defects in sperm functions. We aimed to investigate the role of endoplasmic reticulum (ER) stress and mitochondrial injury in the pathogenesis of aluminum chloride (AlCl 3 )-induced testicular and epididymal damage at the histological, biochemical, and molecular levels, and to assess the potential protective role of taurine. Forty-eight adult male albino rats were separated into four groups (12 in each): negative control, positive control, AlCl 3 , and AlCl 3 plus taurine groups. Testes and epididymis were dissected. Histological and immunohistochemical (Bax and vimentin) studies were carried out. Gene expression of vimentin , PCNA , CHOP , Bcl-2 , Bax , and XBP1 were investigated via quantitative real-time polymerase chain reaction (qRT-PCR), besides estimation of malondialdehyde (MDA) and total antioxidant capacity (TAC). Light and electron microscopic examinations of the testes and epididymis revealed pathological changes emphasizing both mitochondrial injury and ER stress in the AlCl 3 group. Taurine-treated rats showed a noticeable improvement in the testicular and epididymal ultrastructure. Moreover, they exhibited increased gene expression of vimentin , Bcl-2 , and PNCA accompanied by decreased CHOP , Bax , and XBP1 gene expression. In conclusion, male reproductive impairment is a significant hazard associated with AlCl 3 exposure. Both ER stress and mitochondrial impairment are critical mechanisms of the deterioration in the testes and epididymis induced by AlCl 3 , but taurine can amend this.
Bioinformatic analysis of lncRNA expression in mouse placenta using mouse FANTOM5 CAGE expression data and qPCR results for lncRNA 1600012P17Rik. a Overall lncRNA expression level and total number of lncRNA genes expressed in adult mouse organs including placenta at gestational day 10 and 17 (considered as E10 and E17, respectively). b Pie chart of the distribution of lncRNAs detected in E10 and E17 placentas into biotypes, expressed as numbers of genes and percentages. c Pie chart of the distribution of lncRNAs detected in E10 and E17 placentas into the expression levels of biotypes, expressed as tag counts and percentages. d Top 10 most highly expressed lncRNAs in E10 and E17 placentas. e Expression levels of 1600012P17Rik (P17Rik) and lincRNA Malat1 in E10 and E17 placentas and adult mouse organs. f Locations of P17Rik and its neighboring coding gene Pappa2 transcripts on mouse chromosome (chr) 1. g qPCR analysis of the organ distribution of P17Rik transcript expression (1600012P17Rik-201 [P17Rik-201] and 1600012P17Rik-202 [P17Rik-202]) during placental development from E7.5 to E18.5 and in adult organs. Data are normalized to Rn18s. Data presented as mean ± standard deviation of three independent experiments. Tukey test; values with different letters are significantly (p < 0.05) different for each placenta
In situ hybridization (ISH) analysis of P17Rik in mouse placenta. a E10.5 placenta hybridized with antisense probe for P17Rik. Weak Fast Red signals (red color) of P17Rik expression are present in the junctional zone (arrows). Hybridization with the sense probe for P17Rik (inset); specific signals are absent. b Hematoxylin–eosin (HE)-stained section corresponding to panel a. c E16.5 placenta hybridized with the antisense probe for P17Rik. More intense Fast Red signals indicating P17Rik are visible in the junctional zone (arrows), and the decidual area adjacent to the junctional zone is weakly positive (arrowheads). Hybridization with the sense probe for P17Rik (inset). d HE-stained section corresponding to panel c. e Bright-field (BF) image of P17Rik detected as chromogenic red signals in the E10.5 placenta. f Fluorescence (FL) image of P17Rik detected as red fluorescence signals in the section shown in panel e. g DAPI image of the section shown in panel e. h Merged image of the fluorescence signals (red) and DAPI-stained nuclei (blue) of the section shown in panel e. i Bright-field image of P17Rik in the E16.5 placenta. j Fluorescence image of P17Rik in the section shown in panel i. k DAPI image of the section shown in panel i. l Merged image showing fluorescence signals and DAPI-stained nuclei for the section shown in panel i. Decidua (dec), junctional zone (jz), labyrinth (lab), chorionic plate (cp), spongiotrophoblast cells (sp), glycogen trophoblast cells (gc), and parietal trophoblast giant cells (tgc) are visible. a–d bars = 1 mm; e, i bars = 100 µm
High-magnification image of P17Rik expression in the mouse placenta as revealed through ISH analysis. a High-magnification image of P17Rik expression in the junctional zone of the E10.5 placenta. b DAPI image of the section shown in panel a. c Merged image of the fluorescence signals (red) and DAPI-stained nuclei (blue) of the section shown in panel a. Note the dot-like P17Rik signals (arrows) in the nucleoplasm of spongiotrophoblast cells. Parietal trophoblast giant cells (tgc) are P17Rik negative. d HE-stained section of the E16.5 placenta. e PAS-stained section corresponding to panel d. f High-magnification image of the region within the box shown in panel d. g DAPI image of the section shown in panel f. h Merged image showing fluorescence signals (red) and DAPI-stained nuclei (blue) of the section shown in panel f. Note that the intracellular localization of P17Rik is mainly within the cytoplasm (arrows). Decidua (dec), junctional zone (jz), maternal blood sinusoid (mbs), spongiotrophoblast cells (sp), and glycogen trophoblast cells (gc) are visible. Bars = 50 µm
Regulation of the neighboring protein-coding gene Pappa2 by lncRNA P17Rik. a Validation of the 3′-poly(A) tail of P17Rik. Total RNA from the E16.5 mouse placenta was reverse-transcribed with oligo-dT primers (dT), random primers (random), or a mixture of both primers (mix); P17Rik (P17Rik-201 transcript) was analyzed through qPCR. Nonadenylated rRNA Rn18s and polyadenylated mRNA Gapdh were employed as positive controls. b Nuclear/cytoplasmic RNA fractionation of pBI-CMV3-P17Rik-transfected MC3T3-E1 cells and subsequent qPCR analysis of P17Rik. Equal amounts of total RNA (T), nuclear RNA (N), and cytoplasmic RNA (C) from each transfection were subjected to qPCR. LncRNA Neat1 and rRNA Rn18s served as nuclear and cytoplasmic RNA controls, respectively. Note that lncRNA P17Rik is present predominantly in the cytoplasm. c qPCR analysis of the organ distribution of Pappa2–202 transcript expression during placental development from E7.5 to E18.5 and in adult organs. Tukey test; values with different letters are significantly (p < 0.05) different for each placenta. d Effects of lncRNA P17Rik on the expression of its neighboring coding gene Pappa2. The transfection of pBI-CMV3 vectors [pBI-CMV3-P17Rik (P17Rik) and pBI-CMV3-cont (cont)] into Pappa2-expressing MC3T3-E1 cells (left). Pappa2 mRNA (upper right panel) and protein (lower right panel) in pBI-CMV3-transfected cells. Rn18s was used as an internal control in qPCR (c, d); Gapdh was used as an internal control in Western blot. Data presented as mean ± standard deviation of three independent experiments. *p < 0.05; Student’s t test
A few long noncoding RNAs (long ncRNAs, lncRNAs) exhibit trophoblast cell type-specific expression patterns and functional roles in mouse placenta. However, the cell- and stage-specific expression patterns and functions of most placenta-derived lncRNAs remain unclear. In this study, we explored mouse placenta-associated lncRNAs using a combined bioinformatic and experimental approach. We used the FANTOM5 database to survey lncRNA expression in mouse placenta and found that 1600012P17Rik (MGI: 1919275, designated P17Rik), a long intergenic ncRNA, was the most highly expressed lncRNA at gestational day 17. Polymerase chain reaction analysis confirmed that P17Rik was exclusively expressed in placenta and not in any of the adult organs examined in this study. In situ hybridization analysis revealed that it was highly expressed in spongiotrophoblast cells and to a lesser extent in glycogen trophoblast cells, including migratory glycogen trophoblast cells invading the decidua. Moreover, we found that it is a polyadenylated lncRNA localized mainly to the cytoplasm of these trophoblast cells. As these trophoblast cells also expressed the neighboring protein-coding gene, pappalysin 2 (Pappa2), we investigated the effects of P17Rik on Pappa2 expression using Pappa2-expressing MC3T3-E1 cells and found that P17Rik transfection increased the messenger RNA (mRNA) and protein levels of Pappa2. These results indicate that mouse placenta-specific lncRNA P17Rik modulates the expression of the neighboring protein-coding gene Pappa2 in spongiotrophoblast and glycogen trophoblast cells of mouse placenta during late gestation.
Paneth cells are antimicrobial peptide-secreting epithelial cells located at the bottom of the intestinal crypts of Lieberkühn. The crypts begin to form around postnatal day 7 (P7) mice, and Paneth cells usually appear within the first 2 weeks. Paneth cell dysfunction has been reported to correlate with Crohn’s disease-like inflammation, showing narrow crypts or loss of crypt architecture in mice. The morphology of dysfunctional Paneth cells is similar to that of Paneth/goblet intermediate cells. However, it remains unclear whether the formation of the crypt is related to the maturation of Paneth cells. In this study, we investigated the histological changes including epigenetic modification in the mouse ileum postnatally and assessed the effect of the methyltransferase inhibitor on epithelium development using an organoid culture. The morphological and functional maturation of Paneth cells occurred in the first 2 weeks and was accompanied by histone H3 lysine 27 (H3K27) trimethylation, although significant differences in DNA methylation or other histone H3 trimethylation were not observed. Inhibition of H3K27 trimethylation in mouse ileal organoids suppressed crypt formation and Paneth cell maturation, until around P10. Overall, our findings show that post-transcriptional modification of histones, particularly H3K27 trimethylation, leads to the structural and functional maturation of Paneth cells during postnatal development.
Histone methylation is one of the main epigenetic mechanisms by which methyl groups are dynamically added to the lysine and arginine residues of histone tails in nucleosomes. This process is catalyzed by specific histone methyltransferase enzymes. Methylation of these residues promotes gene expression regulation through chromatin remodeling. Functional analysis and knockout studies have revealed that the histone lysine methyltransferases SETD1B, SETDB1, SETD2, and CFP1 play key roles in establishing the methylation marks required for proper oocyte maturation and follicle development. As oocyte quality and follicle numbers progressively decrease with advancing maternal age, investigating their expression patterns in the ovaries at different reproductive periods may elucidate the fertility loss occurring during ovarian aging. The aim of our study was to determine the spatiotemporal distributions and relative expression levels of the Setd1b, Setdb1, Setd2, and Cxxc1 (encoding the CFP1 protein) genes in the postnatal mouse ovaries from prepuberty to late aged periods. For this purpose, five groups based on their reproductive periods and histological structures were created: prepuberty (3 weeks old; n = 6), puberty (7 weeks old; n = 7), postpuberty (18 weeks old; n = 7), early aged (52 weeks old; n = 7), and late aged (60 weeks old; n = 7). We found that Setd1b, Setdb1, Setd2, and Cxxc1 mRNA levels showed significant changes among postnatal ovary groups (P < 0.05). Furthermore, SETD1B, SETDB1, SETD2, and CFP1 proteins exhibited different subcellular localizations in the ovarian cells, including oocytes, granulosa cells, stromal and germinal epithelial cells. In general, their levels in the follicles, oocytes, and granulosa cells as well as in the germinal epithelial and stromal cells significantly decreased in the aged groups when compared the other groups (P < 0.05). These decreases were concordant with the reduced numbers of the follicles at different stages and the luteal structures in the aged groups (P < 0.05). In conclusion, these findings suggest that altered expression of the histone methyltransferase genes in the ovarian cells may be associated with female fertility loss in advancing maternal age.
The myotendinous junction (MTJ), a specialized interface for force transmission between muscle and tendon, has a unique transcriptional activity and is highly susceptible to muscle strain injury. Eccentric exercise training is known to reduce this risk of injury, but knowledge of the influence of exercise on the MTJ at the molecular and cellular levels is limited. In this study, 30 subjects were randomized to a single bout of eccentric exercise 1 week prior to tissue sampling (exercised) or no exercise (control). Samples were collected from the semitendinosus as part of reconstruction of the anterior cruciate ligament and divided into fractions containing muscle, MTJ and tendon, respectively. The concentrations of macrophages and satellite cells were counted, and the expression of genes previously known to be active at the MTJ were analyzed by real-time–quantitative PCR. An effect of the single bout of exercise was found on the expression of nestin (NES) and osteocrin (OSTN) mRNA in the MTJ and tendon fractions. Genes earlier identified at the MTJ (COL22A1, POSTN, ADAMTS8, MNS1, NCAM1) were confirmed to be expressed at a significantly higher level in the MTJ compared to muscle and tendon but were unaffected by exercise. In the exercise group a higher concentration of macrophages, but not of satellite cells, was seen in muscle close to the MTJ. The expression of NES and OSTN was higher in human semitendinosus MTJ 1 week after a single session of heavy eccentric exercise. Based on these results, NES and OSTN could have a part in explaining how the MTJ adapts to eccentric exercise.
Myelin loss with consecutive axon degeneration and impaired remyelination are the underlying causes of progressive disease in patients with multiple sclerosis. Astrocytes are suggested to play a major role in these processes. The unmasking of distinct astrocyte identities in health and disease would help to understand the pathophysiological mechanisms in which astrocytes are involved. However, the number of specific astrocyte markers is limited. Therefore, we performed immunohistochemical studies and analyzed various markers including GFAP, vimentin, S100B, ALDH1L1, and LCN2 during de- and remyelination using the toxic murine cuprizone animal model. Applying this animal model, we were able to confirm overlapping expression of vimentin and GFAP and highlighted the potential of ALDH1L1 as a pan-astrocytic marker, in agreement with previous data. Only a small population of GFAP-positive astrocytes in the corpus callosum highly up-regulated LCN2 at the peak of demyelination and S100B expression was found in a subset of oligodendroglia as well, thus S100B turned out to have a limited use as a particular astroglial marker. Additionally, numerous GFAP-positive astrocytes in the lateral corpus callosum did not express S100B, further strengthening findings of heterogeneity in the astrocytic population. In conclusion, our results acknowledged that GFAP, vimentin, LCN2, and ALDH1L1 serve as reliable marker to identify activated astrocytes during cuprizone-induced de- and remyelination. Moreover, there were clear regional and temporal differences in protein and mRNA expression levels and patterns of the studied markers, generally between gray and white matter structures.
Fast hematoxylin staining of extracted mouse whisker hair follicles. a Schematic diagram (top) and physical objects (bottom) of the observation device. After staining in hematoxylin or AKP, follicles were mounted in an observation device to be examined under a microscope. b Front and back views of whisker follicle attained by turning the observation device over. Note the difference in sharpness (white and blue arrows) and content (red arrows) in front and back views. Scale bar, 100 μm. c General (left) and enlarged views (right) of the bulge region in whisker follicles before staining under a light microscope. The location of enlarged view was labeled in black box in the general view, as in the text below. Red color indicates blood in the follicular intrinsic muscles. d Mouse whisker follicle stained with hematoxylin for 3 min without fixation (10 min of immersion in 4% paraformaldehyde) and permeabilization (10 min of immersion in 0.3% Triton X-100 permeabilization), after fixation. e Mouse whisker follicles stained 3, 5, and 15 min in hematoxylin, permeabilized, without fixation. General view (left) and enlarged view of the black box (right) of each group. f Mouse whisker follicles stained with hematoxylin for 10 min, permeabilized, nonfixed, and subsequently stained with eosin for 10 s. Note the background became red and the image became blurred. g Mouse whisker follicles stained with hematoxylin for 3 min, permeabilized, nonfixed. Enlarged view is labeled in the black boxes in general view. DP is indicated by blue arrow in the hair bulb. Scale bar, 50 μm. DP, dermal papilla; HS, hair shaft; HFSCs, hair follicle stem cells. Scale bar, 500 μm in general view of whole follicles. Scale bar, 100 μm in enlarged view unless specifically stated
Fast hematoxylin staining of human hair follicles. a Whole human hair follicle (left, general view) and hair bulge region (right, enlarged view) before staining under a light microscope. Scale bar, 500 μm in the general view of whole follicles. Scale bar, 100 μm in the enlarged view. b Hematoxylin-stained hair bulb, hair bulge, and infundibulum of the whole human hair follicle. In the general view, the shape of the hair follicle and the density of cells inside the follicle were easily identified. In the hair bulb, dermal papilla (blue arrow) was enclosed by pigmented hair matrix and was not visible. In the HS plane, the HS, IRS, and ORS were easily identified. There were many HFSCs in the HFSCs plane. Scale bar, 500 μm in the general view; scale bar, 50 μm in the enlarged view. c HFSCs identified by HE staining, IHC staining of SOX9 in 4-μm sections, and fast staining of whole human hair follicle. Scale bar, 50 μm. Enlarged view of the HFSCs in mitotic metaphase (red arrow) in the black box. Note the alignment of chromosomes at the equatorial plane of the nucleus and the division of the nucleus. Scale bar, 10 μm. d Subcutaneous gland in human hair follicles after hematoxylin staining. The membranes of the gland cells are clearly shown. Scale bar, 50 μm. IHC, immunohistochemistry; IRS, inner root sheath; ORS, outer root sheath
Pathohistological changes in hair follicles. a Hematoxylin stained intact and incomplete human hair follicles. The lower part of the IRS and ORS of the follicle was destroyed (red arrow). Transected hair follicle (blue arrow). Scale bar, 500 μm. b Hematoxylin-stained human normal anagen hair follicle and villus hair follicle. Scale bar, 200 μm. Black box, enlarged view of villus hair follicle. Note the reduction of melanin in HS and the miniaturization of the villus hair follicle. Scale bar, 100 μm. c General view of hematoxylin-stained black and white human hair follicles. Scale bar, 500 μm. d Hematoxylin-stained hair bulb, bulge, and infundibulum of black and white human hair follicles. Note the deficiency of melanin in both the matrix and the HS in the white hair follicle. Scale bar, 50 μm. Scale bar, 15 μm in enlarged view of the black box. e Quantification of HFSCs in black and white hair follicles
AKP staining of vessels in hair follicles. a AKP staining of mouse whisker follicles penetrated with different concentrations of Triton X-100. Small vessels were not stained in groups with permeabilization with 0.05% Triton X-100 or without permeabilization. Permeabilization with either 0.1%, 0.3%, or 0.5% of Triton X-100 showed the vessels (dark-purple lines) clearly. The 0.3% concentration stained more tiny vessels (blue arrows) than the 0.1% concentration and had less background than 0.5%. The high background of the 1% concentration group reduced the quality of staining. Scale bar, 200 μm. b Different AKP staining times of mouse whisker follicles. Five minutes of staining had the least background, but vessels were incompletely stained. Staining for 10 min labeled more vessels without staining of the unintended lower part of the hair bulb. After 15 and 30 min of staining, the background was increased and the unintended staining of the lower parts of the hair bulb were increased (red arrows). Scale bar, 200 μm. c AKP-stained human hair follicles before and after hyalinization. Note the reduction in background and increased transparency. Scale bar, 200 μm. d Blood vessels identified by IHC staining of CD31, AKP, and eosin staining in a 4 μm section and AKP staining of the whole human hair follicle. Note the cross-section of the vessels (red arrows) stained with 4 μm sections and the intact vessels (blue vessels) in the staining of whole human hair follicle. Scale bar, 50 μm. e Vessels in different planes of the Z axis in the same follicle. Z1 and Z3 exhibit the vessels in double faces of the follicle, and Z2 shows the hair shaft between them. Scale bar, 100 μm. f AKP staining of whole mouse whisker follicle and enlarged views of hair bulb, bulge, and infundibulum. Note the uneven distribution of vessels in the hair follicle. Scale bar, 500 μm in the general view. Scale bar, 100 μm in the enlarged view. g AKP staining of normal (left) and miniaturized (right) human hair follicles. Scale bar, 500 μm. h AKP staining of the hair bulb, bulge, and infundibulum of normal human hair follicle. Scale bar, 100 μm. i Quantification of average area covered by vessels in each follicle
Intact and healthy hair follicles are important for hair growth after hair follicle transplantation. However, effective and practical evaluation methods for the quality of hair follicles are currently lacking. In the present study, we developed a novel fast staining method for histological examination of hair follicles. The whisker follicles from mice were used to explore the staining protocols, and the final protocol for the evaluation of human hair follicles was derived from animal experiments. After extraction, human hair follicles or mouse whisker follicles were permeabilized with 0.3% Triton X-100. Subsequently, hair follicles were processed by either hematoxylin or alkaline phosphatase staining. The integrity and growth state, including the status of hair follicle stem cells and blood vessels of the extracted hair follicles, were clearly identified under a light microscope. Unhealthy hair follicles from donors or hair follicles broken during extraction were easily revealed by this method. Importantly, it took less than half an hour to obtain images of an individual hair follicle. This method is simple and practical for evaluating the quality and status of hair follicles, providing a fast-screening procedure for hair follicle transplantation.
Normal porcine urothelium in vitro and mouse urothelium in vivo and the organization of the Golgi complex in urothelial cells. a Normal porcine UCs grown on a scaffold of human amniotic membrane and labeled with antibodies against uroplakins. The strongest labeling of differentiation-associated markers uroplakins (brown) is seen in the superficial UCs (umbrella cells). b Distribution of GRASP 55 (green) in mouse urothelium. c Co-localization of GRASP 55 (green) and giantin (red) in mouse urothelium. d Perinuclear distribution of GRASP 55 in intermediate UCs. e GRASP 55 distribution over the entire cytoplasm in the umbrella cell. Note: a Immunohistochemistry on formalin-fixed paraffin section. b, c Immunofluorescence on cryo-semithin sections (thickness 300 nm) prepared using the Tokuyasu technique. d, e Immunofluorescence on optical section of the UCs in the direction of the lumen—perpendicular to the apical surface. Scale bars, 50 µm (a, b); 10 µm (c, d, e)
Representative examples of the Golgi complexes from umbrella cells. a Low magnification of the umbrella cell. The green box indicates the Golgi complex. The red box shows the ER exit site. b Enlargement of the area inside green box in (a). c Enlargement of the area inside the red box in (a). d Immuno EM based on cryosections according to Tokuyasu. Labeling for GRASP55 is present over Golgi cisternae. Scale bars 600 nm (a); 220 nm (b); 180 nm (c); 90 nm (d)
Structure of the Golgi complex and its derivatives in umbrella cells. a The Golgi complex with the presumably COPI-dependent vesicles only at the Golgi cis–side. b Ministack with COPI vesicles at all levels. c, d ER exit sites (yellow arrows in c; green box in d). e Clathrin-coated bud on the FV. f Enlargement of the area inside green box in (d). The yellow arrow shows COPII-coated bud. Deformation of round profiles typical for other cell types. g Enlargement of the area inside the blue box in (e). h Image shows tight attachment of post-Golgi transport vesicle to the FV. i Enlargement of the area inside the red box in (h). The yellow arrow shows attachment of post-Golgi transport vesicle to the FV. Scale bars 220 nm (a, c, d); 110 nm (b); 200 nm (e, h)
The Golgi complex undergoes considerable structural remodeling during differentiation of urothelial cells in vivo and in vitro. It is known that in a healthy bladder the differentiation from the basal to the superficial cell layer leads to the formation of the tightest barrier in our body, i.e., the blood–urine barrier. In this process, urothelial cells start expressing tight junctional proteins, apical membrane lipids, surface glycans, and integral membrane proteins, the uroplakins (UPs). The latter are the most abundant membrane proteins in the apical plasma membrane of differentiated superficial urothelial cells (UCs) and, in addition to well-developed tight junctions, contribute to the permeability barrier by their structural organization and by hindering endocytosis from the apical plasma membrane. By studying the transport of UPs, we were able to demonstrate their differentiation-dependent effect on the Golgi architecture. Although fragmentation of the Golgi complex is known to be associated with mitosis and apoptosis, we found that the process of Golgi fragmentation is required for delivery of certain specific urothelial differentiation cargoes to the plasma membrane as well as for cell–cell communication. In this review, we will discuss the currently known contribution of the Golgi complex to the formation of the blood–urine barrier in normal UCs and how it may be involved in the loss of the blood–urine barrier in cancer. Some open questions related to the Golgi complex in the urothelium will be highlighted.
Human periodontal ligament mesenchymal stem cells (hPDLSCs) are a promising cell type model for regenerative medicine applications due to their anti-inflammatory, immunomodulatory and non-tumorigenic potentials. Extremely low-frequency electromagnetic fields (ELF-EMF) are reported to affect biological properties such as cell proliferation and differentiation and modulate gene expression profile. In this study, we investigated the effects of an intermittent ELF-EMF exposure (6 h/day) for the standard differentiation period (28 days) and for 10 days in hPDLSCs in the presence or not of osteogenic differentiation medium (OM). We evaluated cell proliferation, de novo calcium deposition and osteogenic differentiation marker expression in sham and ELF-EMF-exposed cells. After ELF-EMF exposure, compared with sham-exposed, an increase in cell proliferation rate (p < 0.001) and de novo calcium deposition (p < 0.001) was observed after 10 days of exposure. Real-time PCR and Western blot results showed that COL1A1 and RUNX-2 gene expression and COL1A1, RUNX-2 and OPN protein expression were upregulated respectively in the cells exposed to ELF-EMF exposure along with or without OM for 10 days. Altogether, these results suggested that the promotion of osteogenic differentiation is more efficient in ELF-EMF-exposed hPDLSCs. Moreover, our analyses indicated that there is an early induction of hPDLSC differentiation after ELF-EMF application.
Mammalian pulmonary arteries divide multiple times before reaching the vast capillary network of the alveoli. Morphological analyses of the arterial branches can be challenging because more proximal branches are likely biologically distinct from more peripheral parts. Thus, it is useful to group the arterial branches into groups of coherent biology. While the generational approach of dichotomous branching is straightforward, the grouping of arterial branches in the asymmetrically branching monopodial lung is less clear. Several established classification methods return highly dissimilar groupings when employed on the same organ. Here, we established a workflow allowing the quantification of grouping results for the monopodial lung and tested various methods to group the branches of the arterial tree into coherent groups. A mouse lung was imaged by synchrotron x-ray microcomputed tomography, and the arteries were digitally segmented. The arterial tree was divided into its individual segments, morphological properties were assessed from corresponding light microscopic scans, and different grouping methods were employed, such as (fractal) generation or (Strahler) order. The results were ranked by the morphological similarity within and dissimilarity between the resulting groups. Additionally, a method from the mathematical field of cluster analysis was employed for creating a reference classification. In conclusion, there were significant differences in method performance. The Strahler order was significantly superior to the generation system commonly used to classify human lung structure. Furthermore, a clustering approach indicated more precise ways to classify the monopodial lung vasculature exist.
Mucosal hypoxia is detected in the mucosa of ulcerative colitis (UC), however the mechanism and the cause of hypoxia is not fully understood, while a dense infiltration of plasma cells is observed in the inflamed mucosa of UC. When differentiating from a B cell to a plasma cell, the energy metabolism dramatically shifts from glycolysis to oxidative phosphorylation, which results in a large amount of oxygen consumption of the plasma cell. We hypothesized that the plasma cell infiltration into the inflamed mucosa contributes to the mucosal hypoxia in UC in part. We examined the association between mucosal hypoxia and plasma cell infiltration in UC. More IgG plasma cells (but not IgA plasma cells) were distributed, and the nuclear and cell sizes were enlarged in hypoxic mucosa compared to normoxic mucosa in UC. Oxidative phosphorylation signature genes of these IgG plasma cells were markedly upregulated compared to those of other lymphoid cells infiltrating the lamina propria of inflamed mucosa of UC. Enlarged IgG plasma cells, which increase in number in the inflamed mucosa of UC, can be related to the hypoxic state of the inflamed mucosa of UC.
The urothelium is a stratified epithelium that lines the inner surface of the components of the urinary drainage system. It is composed of a layer of basal cells, one or several layers of intermediate cells, and a layer of large luminal superficial or umbrella cells. In the mouse, only a small set of markers is available that allows easy molecular distinction of these urothelial cell types. Here, we analyzed expression of S100A1, a member of the S100 family of calcium-binding proteins, in the urothelium of the two major organs of the murine urinary tract, the ureter and the bladder. Using RNA in situ hybridization analysis, we found exclusive expression of S100a1 mRNA in luminal cells of the ureter from embryonic day (E)17.5 onwards and of the bladder from E15.5 to adulthood. Immunofluorescence analysis showed that expression of S100A1 protein is confined to terminally differentiated superficial cells of both the ureter and bladder where it localized to the nucleus and cytoplasm. We conclude that S100A1 is a suitable marker for mature superficial cells in the urothelial lining of the drainage system of the developing and mature mouse.
Overexpression of ABC transporters, such as ABCB1 and ABCG2, plays an important role in mediating multidrug resistance (MDR) in cancer. This feature is also attributed to a subpopulation of cancer stem cells (CSCs), having enhanced tumourigenic potential. ABCG2 is specifically associated with the CSC phenotype, making it a valuable target for eliminating aggressive and resistant cells. Several natural and synthetic ionophores have been discovered as CSC-selective drugs that may also have MDR-reversing ability, whereas their interaction with ABCG2 has not yet been explored. We previously reported the biological activities, including ABCB1 inhibition, of a group of adamantane-substituted diaza-18-crown-6 (DAC) compounds that possess ionophore capabilities. In this study, we investigated the mechanism of ABCG2-inhibitory activity of DAC compounds and the natural ionophores salinomycin, monensin and nigericin. We used a series of functional assays, including real-time microscopic analysis of ABCG2-mediated fluorescent substrate transport in cells, and docking studies to provide comparative aspects for the transporter–compound interactions and their role in restoring chemosensitivity. We found that natural ionophores did not inhibit ABCG2, suggesting that their CSC selectivity is likely mediated by other mechanisms. In contrast, DACs with amide linkage in the side arms demonstrated noteworthy ABCG2-inhibitory activity, with DAC-3Amide proving to be the most potent. This compound induced conformational changes of the transporter and likely binds to both Cavity 1 and the NBD–TMD interface. DAC-3Amide reversed ABCG2-mediated MDR in model cells, without affecting ABCG2 expression or localization. These results pave the way for the development of new crown ether compounds with improved ABCG2-inhibitory properties.
Histological analysis and MUC5AC, MUC6, and αGlcNAc IHC of three representative cases of 54 IMA LC cases. Upper, middle, and lower rows are cases 4, 13, and 16 in supplemental Table S1, respectively. Insets in the lower right corner of each image are high-magnification views. The left column shows HE stain, indicating proliferation of columnar cells with clear cytoplasm in cases 4 and 13, and the N/C ratio appears to be smaller in cases 4 and 13 than in case 16. Next to the HE stain column, AB-PAS stain indicates that abundant neutral mucin production in cytoplasm of LC cells in cases 4 and 13. In case 16, neutral mucin is observed only on the apical side of cancer cells. Based on IHC, MUC5AC-positive cells are present to varying extent in all three representative cases, and cases 13 and 16 also exhibit MUC6 positivity. In case 4, αGlcNAc staining corresponds to MUC6-positive cells, while decreased αGlcNAc glycosylation was observed in most MUC6-positive cells in case 13, and in case 16, αGlcNAc and MUC6 staining is absent. Scale bar = 100 μm. Inset scale bar = 10 μm
MUC6-positive expression is a favorable marker in IMA LC cases. a Cumulative bar chart of MUC5AC, MUC6, and αGlcNAc expression score in 54 IMA LC cases corresponding to Table 1. b Difference between MUC6 and αGlcNAc expression scores based on IHC evaluation in 54 IMA LC cases. The y-axis shows expression sores of MUC6 or αGlcNAc. Results are expressed as means ± SEM. αGlcNAc expression scores are significantly decreased compared to MUC6 expression scores. Difference of expression scores were calculated by using Student’s t test. ***P < 0.001. c Kaplan–Meier analysis of MUC6 expression. MUC6-positive cases appeared to show a higher overall survival (OS) rate than MUC6-negative cases, but differences were not significant (P = 0.27). d MUC6-positive cases showed a significantly higher disease-free survival (DFS) rate than MUC6-negative cases (P = 0.021). e Log-rank test of differences in DFS rate among MUC6-positive/αGlcNAc-positive cases (n = 18), MUC6-positive/αGlcNAc-negative cases (n = 20), and MUC6-negative/αGlcNAc-negative cases (n = 15). Differences in DFS rate among them were not significant (P = 0.138). Differences in DFS rate between MUC6-positive/αGlcNAc-positive cases and MUC6-negative/αGlcNAc-negative cases were also not significant (P = 0.094)
Ectopic MUC6 expression suppressed cell proliferation, motility, and invasiveness in A549 cells. a Western blotting analysis of MUC6 ectopically expressed in A549 cells versus mock-transduced control cells. β-actin serves as loading control. b Analysis of proliferation of MUC6-expressing and control A549 cells, based on MTS assay. The proliferation ratio was calculated based on the value on day 0, which was set to 1. Results are expressed as means ± SD (n = 6). Representative results from four independent experiments are shown. *P < 0.05, **P < 0.01. c Transwell migration assay. Left images show control and MUC6-expressing A549 cells migrating through a Transwell membrane. Scale bar = 50 μm. The right graph shows the number of migrated cells counted in five randomly chosen fields in triplicate wells. Results are expressed as means ± SD (n = 15). Representative results from four independent experiments are shown. **P < 0.01. d Matrigel invasion assay. Left images show control and MUC6-expressing A549 cells invading Matrigel. Scale bar = 50 μm. The right graph shows the number of invaded cells counted in five randomly chosen fields in triplicate wells. Results are expressed as means ± SD (n = 15). Representative results from four independent experiments are shown. **P < 0.01. Note that both migration and invasion are suppressed by MUC6 overexpression in A549 cells. e ECM adhesion assay. Assays were performed after pre-coating plates with indicated ECM factors (see “Materials and methods”). The adherent cells were stained with crystal violet and solubilized, and the absorbance at 595 nm was measured. Results are expressed as means ± SD (n = 4), and there were no significant differences in any case between MUC6-expressing and control cells. Representative results from four independent experiments are shown. f F-actin staining of MUC6-expressing (right panels) and control (left panels) A549 cells. The top row represents low-power view (scale bar = 10 μm), and the lower two rows show higher-power views (scale bar = 10 μm). In upper and lower panels of control cells, filopodia are evident, as indicated by white arrowheads. By contrast, in MUC6-expressing cells shown in the right column, filopodia are shortened or not evident. Representative images are shown. g Quantitative analysis of the number of filopodia. The numbers of filopodia were counted in higher-power views. Results are expressed as means ± SD (n = 11). **P < 0.01. hFSCN transcript levels in control and MUC6-expressing A549 cells. Values were normalized to GAPDH expression, and the control value was set to 1. Results are expressed as means ± SD (n = 3). Representative results from four independent experiments are shown. *P < 0.05. The lower table shows the Ct values used to calculate the relative quantity shown in the graph
Gastric gland mucin consists of core protein MUC6 with residues heavily glycosylated by unique O-glycans carrying α1,4-linked N-acetylglucosamine (αGlcNAc). αGlcNAc-glycosylated MUC6 protein is seen in normal gastric and duodenal glands. Decreased αGlcNAc glycosylation on MUC6-positive tumor cells is often observed in premalignant lesions of the stomach, pancreas, and bile duct, and decreased MUC6 expression is seen in invasive cancer of these organs. Lung cancer (LC) is the most common cause of cancer death worldwide. Recently, the adenocarcinoma subtype has become the most common histological subtype of LC, and one of its invasive forms is invasive mucinous adenocarcinoma (IMA). Currently, prognostic markers of LC IMA are unknown. Here, we analyzed MUC5AC, MUC6, and αGlcNAc expression in 54 IMA LC cases. MUC5AC was positively expressed in 50 (93%), MUC6 in 38 (70%), and αGlcNAc in 19 (35%). Each expression level was scored from 0 to 3. The αGlcNAc expression score was significantly decreased relative to MUC6 (P < 0.001). Interestingly, disease-free survival was significantly higher in MUC6-positive than MUC6-negative cases based on the log-rank test (P = 0.021). For in vitro analysis, we ectopically expressed MUC6 in A549 cells, derived from LC and harboring a KRAS mutation. MUC6-expressing A549 cells showed significantly lower proliferation, motility, and invasiveness than control cells. Finally, F-actin staining in MUC6-expressing cells revealed a decrease or loss of filopodia associated with decreased levels of FSCN transcripts, which encodes an actin-bundling protein fascin1 necessary for cell migration. We conclude that MUC6 expression is a preferable prognostic biomarker in IMA LC.
Early-life consumption of high-fat and sugar-rich foods is recognized as a major contributor for the onset of metabolic dysfunction and its related disorders, including diabetes and nonalcoholic fatty liver disease. The lifelong impact of early unhealthy eating habits that start at younger ages remains unclear. Therefore, to better understand the effects of diet, it is essential to evaluate the structural and functional changes induced in metabolic organs and potential mechanisms underlying those changes. To investigate the long-term effects of eating habits, young male rats were exposed to high-sugar and high-energy diets. After 14 weeks, body composition was assessed, and histopathological changes were analyzed in the liver and adipose tissue. Serum biochemical parameters were also determined. Expression of inflammatory markers in the liver was evaluated by immunohistochemistry. Our results revealed that serum levels of glucose, creatinine, aspartate transaminase (AST), alanine transaminase (ALT), and lipid profile were increased in rats red high-sugar and high-energy diets. Histopathological alterations were observed, including abnormal hepatocyte organization and lipid droplet accumulation in the liver, and abnormal structure of adipocytes. In both unhealthy diet groups, hepatic expression of Toll-like receptor 4 (TLR4), cyclooxygenase 2 (COX-2), and E-selectin were increased, as well as a biomarker of oxidative stress. Together, our data demonstrated that unhealthy diets induced functional and structural changes in the metabolic organs, suggesting that proinflammatory and oxidative stress mechanisms trigger the hepatic alterations and metabolic dysfunction.
The glycerol channel AQP7 facilitates glycerol efflux from adipose tissue (AT), and AQP7 deficiency has been suggested to promote obesity. However, the release of glycerol from AT is not fully blocked in AQP7-deficient mice, which suggests that either alternative glycerol channels are present in AT or significant simple diffusion of glycerol occurs. Previous investigations of the expression of other aquaglyceroporins (AQP3, AQP9, AQP10) than AQP7 in AT are contradictory. Therefore, we here aim at determining the cellular localization of AQP3 and AQP9 in addition to AQP7 in human and mouse AT using well-characterized antibodies for immunohistochemistry (IHC) and immunoblotting as well as available single-cell transcriptomic data from human and mouse AT. We confirm that AQP7 is expressed in endothelial cells and adipocytes in human AT and find ex vivo evidence for interaction between AQP7 and perilipin-1 in adipocytes. In addition, labeling for AQP7 in human AT also includes CD68-positive cells. No labeling for AQP3 or AQP9 was identified in endothelial cells or adipocytes in human or mouse AT using IHC. Instead, in human AT, AQP3 was predominantly found in erythrocytes, whereas AQP9 expression was observed in a small number of CD15-positive cells. The transcriptomic data revealed that AQP3 mRNA was found in a low number of cells in most of the identified cell clusters, whereas AQP9 mRNA was found in myeloid cell clusters as well as in clusters likely representing mesothelial progenitor cells. No AQP10 mRNA was identified in human AT. In conclusion, the presented results do not suggest a functional overlap between AQP3/AQP9/AQP10 and AQP7 in human or mouse white AT.
We hypothesized that odontoblasts release exosomes as well as dental pulp cells and focused on the exosome membrane marker CD63. Odontoblasts are well-differentiated mesenchymal cells that produce dentin. Dental pulp, a tissue complex formed with odontoblasts, releases exosomes to epithelial cells and stimulates their differentiation to ameloblasts. However, the localization of CD63 in differentiated odontoblasts is poorly understood. Therefore, herein, we aimed to reveal the expression of CD63 in odontoblasts during tooth development. We first investigated the localization of CD63 in mouse incisors and molars using immunofluorescence. In adult mouse incisors, the anti-CD63 antibody was positive in mature odontoblasts and dental pulp cells but not in pre-odontoblasts along the ameloblasts in the apical bud. Additionally, the anti-CD63 antibody was observed as a vesicular shape in the apical area of odontoblast cytosol and inside Tomes’ fibers. The anti-CD63 antibody-positive vesicles were also observed using immunoelectron microscopy. Moreover, during mouse mandibular molar tooth morphogenesis (E16 to postnatal 6 weeks), labeling of anti-CD63 antibody was positive in the odontoblasts at E18. In contrast, the anti-CD63 antibody was positive in the dental pulp after postnatal day 10. Furthermore, anti-CD63 antibody was merged with the multivesicular body marker Rab7 in dental pulp tissues but not with the lysosome marker Lamp1. Finally, we determined the effect of a ceramide-generation inhibitor GW4869 on the mouse organ culture of tooth germ in vitro. After 28 days of GW4869 treatment, both CD63 and Rab7 were negative in Tomes’ fibers, but were positive in control odontoblasts. These results suggest that CD63-positive vesicular organelles are important for mouse tooth morphogenesis.
Polyethylene glycol (PEG) is an amphiphilic polymer that has many uses in medical as well as biological applications. Recently, PEG can be used mainly in development of drug delivery systems (DDS) based nanomaterials. PEG characterized with higher solubility, biologically inert and has the ability to escape from immune cells (stealthiness) after systemic injection. Most challenging problem towards PEGylated nanomaterials is the quick elimination from blood stream after repeated dose of systemic injection. This phenomenon scientifically called accelerated blood clearance (ABC). Therefore, in this study, the effect of the dose concentration on the ABC induction will be investigated using quantitative, histological and immunohistochemical analysis. The higher dose concentration (2 mg/kg) of PEGylated gold nanoparticles (PEG-AuNPs) reduced the ABC phenomenon when intravenously injected into the pre-injected mice with the same dose. Contradictory, lower dose concentration (˂ 1 mg/kg) significantly induced the ABC phenomenon by the rapid elimination of the second dose of PEG-AuNPs from blood stream. To explain, the relationship between the dose concentration and the induction of ABC phenomenon, the biodistribution of PEG-AuNPs in liver and spleen (reticuloendothelial systems (RES))-rich organs was investigated. The injected dose of PEG-AuNPs accumulated mainly in the hepatic kupffer cells and hepatocytes. Similarly, spleen red pulp received higher amount of the injected dose of PEG-AuNPs. But, the biodistriution profiles of PEG-AuNPs after the first and second dose (different dose concentrations) are different in RES-rich organs. Additionally, the number of B lymphocytes, which have important role to produce anti-PEG IgM, affected by the repeated dose of PEG-AuNPs in the spleen. For effective DDS-based nanomaterials development, dose optimization of PEGylated nanomaterials is important to reduce ABC phenomenon effect. Interestingly, the role of RES-rich organs, in controlling the ABC phenomenon effect of PEGylated nanomaterials, needs further investigations.
After their assembly by budding into the lumen of the intermediate compartment (IC) at the endoplasmic reticulum (ER)–Golgi interface, coronaviruses (CoVs) are released from their host cells following a pathway that remains poorly understood. The traditional view that CoV exit occurs via the constitutive secretory route has recently been questioned by studies suggesting that this process involves unconventional secretion. Here, using the avian infectious bronchitis virus (IBV) as a well-established model virus, we have applied confocal microscopy to investigate the pathway of CoV egress from epithelial Vero cells. We report a novel effect of IBV infection on cellular endomembranes, namely, the compaction of the pericentrosomal endocytic recycling compartment (ERC) defined by the GTPase Rab11, which coincides with the previously described Golgi fragmentation, as well as virus release. Despite Golgi disassembly, the IC elements containing the major IBV membrane protein (M)—which mostly associates with newly formed virus particles—maintain their close spatial connection with the Rab11-positive endocytic recycling system. Moreover, partial colocalization of the M protein with Rab11 was observed, whereas M displayed negligible overlap with LAMP-1, indicating that IBV egress does not occur via late endosomes or lysosomes. Synchronization of virus release using temperature-shift protocols was accompanied by increased colocalization of M and Rab11 in vesicular and vacuolar structures in the pericentrosomal region and at the cell periphery, most likely representing IBV-containing transport carriers. In conclusion, these results add CoVs to the growing list of viruses exploiting the endocytic recycling apparatus defined by Rab11 for their assembly and/or release.
Positive and negative controls for immunohistochemistry. a Positive control for Sox2 (red nuclei) and pRb (brown nuclei) used in every double labeling run; note cytoplasmic reddish Sox2 signals within the germinal layer. b Lack of immunosignal within the isotype-control for Sox2 using IgG mouse as primary antibody. c Slight
Parvovirus infections in dogs and cats are restricted to highly mitotically active tissues, predominantly to the epithelium of the gastrointestinal tract and, in cases of prenatal infections in cats, also to Purkinje cell neuroblasts. The evidence of parvovirus-infected mature feline neurons gave rise to reconsider the dogma of post-mitotically fixed and terminally differentiated neurons in the adult central nervous system. To elucidate the postulated capability of certain terminally differentiated feline neurons to re-enter the cell cycle, immunohistochemical double labeling using the transcription factor Sox2 and the tumor suppressor and cell cycle regulator retinoblastoma protein in its phosphorylated state (pRb) was performed. Formalin-fixed and paraffin-embedded brain tissue negative for parvovirus-antigen from 14 cats was compared to brain tissue from 13 cats with immunohistochemically confirmed cerebral parvovirus infection; the 27 cats were aged between 50 days of gestation (E50) and 5 years. Both groups revealed nuclear Sox2 and pRb immunosignals in numerous neurons, suggesting a more active state than mature neurons should have. Accordingly, parvovirus is not exclusively involved in the reactivation of the cell cycle machinery in those post-mitotic, terminally differentiated feline neurons.
The present immunohistochemical study was performed to examine the number, distribution, and chemical coding of intrinsic substance P (SP) neurons and nerve fibers within the esophagus and discuss their functional roles. Many SP neurons and nerve fibers were found in the myenteric plexus, and the SP neurons gradually decreased from the oral side toward the aboral side of the esophagus. Double-immunolabeling showed that most SP neurons were cholinergic (positive for choline acetyltransferase), and few were nitrergic (positive for nitric oxide synthase). Some cholinergic SP nerve terminals surrounded cell bodies of several myenteric neurons. In the muscularis mucosa and lower esophageal sphincter, and around blood vessels, numerous SP nerve endings were present, and many of them were cholinergic. Also, SP nerve endings were found on only a few motor endplates of the striated muscles, and most of them were calcitonin gene-related peptide (CGRP)-positive. Retrograde tracing using Fast Blue (FB) showed that numerous sensory neurons in the dorsal root ganglia (DRGs) and nodose ganglion (NG) projected to the esophagus, and most FB-labeled SP neurons were CGRP-positive. These results suggest that the intrinsic SP neurons in the rat esophagus may play roles as, at least, motor neurons, interneurons, and vasomotor neurons, which are involved in local regulation of smooth muscle motility, neuronal transmission, and blood circulation, respectively. Moreover, SP nerve endings on only a minority of motor endplates may be extrinsic, derived from DRGs or NG, and possibly detect chemical circumstances within motor endplates to modulate esophageal motility.
Top-cited authors
Ayça Aksoy
  • The Scientific & Technological Research Council of Turkey
Erdal Karaoz
  • Liv Hospital
Sebastian Malkusch
  • Goethe-Universität Frankfurt am Main
Ayla Eker Sariboyaci
  • Eskisehir Osmangazi University
Selda Ayhan