Histochemistry and Cell Biology最新文献

Eukaryotic genomes store information on many levels, including their linear DNA sequence, the posttranslational modifications of its constituents (epigenetic modifications), and its three-dimensional folding. Understanding how this information is stored and read requires multidisciplinary collaborations from many branches of science beyond biology, including physics, chemistry, and computer science. Concurrent recent developments in all these areas have enabled researchers to image the genome with unprecedented spatial and temporal resolution. In this review, we focus on what single-molecule imaging and tracking of individual proteins in live cells have taught us about chromatin structure and dynamics. Starting with the basics of single-molecule tracking (SMT), we describe some advantages over in situ imaging techniques and its current limitations. Next, we focus on single-nucleosome studies and what they have added to our current understanding of the relationship between chromatin dynamics and transcription. In celebration of Robert Feulgen’s ground-breaking discovery that allowed us to start seeing the genome, we discuss current models of chromatin structure and future challenges ahead.

One hundred years ago, Robert Feulgen published a landmark paper in which he described the first method to stain DNA in cells and tissues. Although a century has passed since the discovery by Feulgen and Rossenbeck, the chemical reaction still exerts an important influence in current histochemical studies. Its contribution in diverse fields, spanning from biomedicine to plant biology, has paved the way for the most significant studies that constitute our current knowledge. The possibility to specifically explore the DNA in cell nuclei while quantifying its content makes it a contemporary and timeless method. Indeed, many histocytochemical studies following the 1924 paper have led to a deep understanding of genome organization in general as well as several specific mechanisms (e.g. DNA duplication or tumour pathology) that, nowadays, constitute some of the most fundamental pillars in biological investigations. In this review, we discuss the chemistry and application of the Feulgen reaction to both light and electron microscopy.

Emerging evidence indicates the presence of vascular abnormalities and ischemia in biliary atresia (BA), although specific mechanisms remain undefined. This study examined both human and experimental BA. Structural and hemodynamic features of hepatic arteries were investigated by Doppler ultrasound, indocyanine green angiography, microscopic histology, and invasive arterial pressure measurement. Opal multiplex immunohistochemistry, western blot, and RT-PCR were applied to assess Notch3 expression and the phenotype of hepatic arterial smooth muscle cells (HASMCs). We established animal models of Notch3 inhibition, overexpression, and knockout to evaluate the differences in overall survival, hepatic artery morphology, peribiliary hypoxia, and HASMC phenotype. Hypertrophic hepatic arteriopathy was evidenced by an increased wall-to-lumen ratio and clinically manifested as hepatic arterial hypertension, decreased hepatic artery perfusion, and formation of hepatic subcapsular vascular plexuses (HSVPs). We observed a correlation between overactivation of Notch3 and phenotypic disruption of HASMCs with the exacerbation of peribiliary hypoxia. Notch3 signaling mediated the phenotype alteration of HASMCs, resulting in arterial wall thickening and impaired oxygen supply in the portal microenvironment. Inhibition of Notch3/Hey1 ameliorates portal hypoxia by restoring the balance of contractile/synthetic HASMCs, thereby preventing hypertrophic arteriopathy in BA.

The unique properties of superparamagnetic iron oxide nanoparticles (SPIONs) enable their use as magnetic biosensors, targeted drug delivery, magnetothermia, magnetic resonance imaging, etc. Today, SPIONs are the only type of metal oxide nanoparticles approved for biomedical application. In this work, we analyzed the cellular response to the previously reported luminescent silica coated SPIONs of the two cell types: M-HeLa cells and primary motor neuron culture. Both internalization pathways and intracellular fate of SPIONs have been compared for these cell lines using fluorescence and transmission electron microscopy. We also applied a pharmacological approach to analyze the endocytosis pathways of SPIONs into the investigated cell lines. The penetration of SPIONs into M-HeLa cells is already noticeable within 30 s of incubation through both caveolin-dependent endocytosis and micropinocytosis. However, incubation for a longer time (1 h at least) is required for the internalization of SPIONs into motor neuron culture cells provided by dynamin-dependent endocytosis and macropinocytosis. The intracellular colocalization assay reveals that the lysosomal internalization pathway of SPIONs is also dependent on the cell type. The lysosomal pathway is much more pronounced for M-HeLa cells compared with motor neurons. The emphasized differences in cellular responses of the two cell lines open up new opportunities in the application of SPIONs in the diagnostics and therapy of cancer cells.

Characterization of inflammation in chronic rhinosinusitis with (CRSwNP) and without nasal polyps (CRSsNP) is an ongoing research process. To overcome limitations of current cytologic techniques, we investigated whether immunofluorescence multiplex image cytometry could quantify intact neutrophils, eosinophils, and other immune cells in solid upper airway mucosa. We used a four-channel immunofluorescence-microscopy technique for the simultaneous detection of the leukocyte marker CD45, the neutrophil marker myeloperoxidase, two eosinophil markers, i.e., major basic protein and eosinophil peroxidase, and DAPI (4′,6-diamidin-2-phenylindole), in formalin-fixed paraffin-embedded upper airway tissue samples of patients with CRSwNP and CRSsNP, as well as of patients free of CRS with inferior turbinate hypertrophy (controls). Image acquisition and analysis were performed with TissueFAXS and StrataQuest (TissueGnostics, Vienna, Austria), respectively. Positive and negative immunostaining were differentiated with a specific fluorescence signal/background signal ratio. Isotype controls were used as negative controls. In six controls, nine patients with CRSsNP, and 11 patients with CRSwNP, the median area scanned and median cell count per patient were 14.2 mm² and 34,356, respectively. In CRSwNP, the number of eosinophils was three times higher (23%) than that of neutrophils (7%). Three times more immune cells were encountered in CRSwNP (33%) compared to CRSsNP (11%). In controls, inflammation was balanced between the epithelial layer and lamina propria, in contrast to CRS (three times more pronounced inflammation in the lamina propria). The quantification of intact neutrophils, eosinophils, and other immune cells in solid tissue with undisrupted architecture seems feasible with immunofluorescence multiplex image cytometry.

Abstract

Proteins can be successfully localized in post-mortem (PM) brain tissue sections if the time until PM tissue sampling is not too long. In this study, we show that this also applies to the localization of RNA and in particular to the RNA of microglia-specific receptor proteins using the probes and the RNAscope^™ Multiplex Fluorescent Detection Kit v2 from Advanced Cell Diagnostics. Brains were removed from killed mice after different PM delays and processed into paraffin sections. In sections of brains from animals whose cadavers had been kept at room temperature (21 °C) before tissue removal, ubiquitously expressed RNAs of genes with low to high expression levels (Polr2a, PPIB, and UBC) were reliably detected in the brain sections even if tissue removal was delayed by up to 48 h. In addition, microglia-specific G protein-coupled receptor RNA (Gpr34, P2ry12) could be reliably assigned to microglia by simultaneous labeling of the microglia with microglia-specific antibodies (Iba1 or P2ry12). Only after a delay of 48 h until tissue removal were the receptor RNA signals significantly lower. The reduction in receptor RNA signals could be delayed if the animal cadavers were stored at 4 °C until the brains were removed. Tissue sections of PM brain samples allow the spatial and cellular localization of specific RNA, at least if the sampling takes place within the first 24 h of PM.

c-Jun NH₂-terminal protein kinase (JNK) and p38 are stress-activated mitogen-activated protein kinases (MAPK) that are phosphorylated by various stimuli. It has been reported that the loss of desmoglein (DSG) 3, a desmosomal transmembrane core molecule, in keratinocytes impairs cell-cell adhesion accompanied by p38 MAPK activation. To understand the biological role of DSG3 in desmosomes and its relationship with stress-activated MAPKs, we established DSG3 knockout keratinocytes (KO cells). Wild-type cells showed a linear localization of DSG1 to cell-cell contacts, whereas KO cells showed a remarkable reduction despite the increased protein levels of DSG1. Cell-cell adhesion in KO cells was impaired over time, as demonstrated by dispase-based dissociation assays. The linear localization of DSG1 to cell-cell contacts and the strength of cell-cell adhesion were promoted by the pharmacological inhibition of JNK. Conversely, pharmacological activation of JNK, but not p38 MAPK, in wild-type cells reduced the linear localization of DSG1 in cell-cell contacts. Our data indicate that DSG1 and DSG2 in KO cells cannot compensate for the attenuation of cell-cell adhesion strength caused by DSG3 deficiency and that JNK inhibition restores the strength of cell-cell adhesion by increasing the linear localization of DSG1 in cell-cell contacts in KO cells. Inhibition of JNK signaling may improve cell-cell adhesion in diseases in which DSG3 expression is impaired.

Su (var) 3-9, enhancer of seste, trithorax (SET)-domain bifurcated histone lysine methyltransferase (SETDB1) plays a crucial role in maintaining intestinal stem cell homeostasis; however, its physiological function in epithelial injury is largely unknown. In this study, we investigated the role of SETDB1 in epithelial regeneration using an intestinal ischemia/reperfusion injury (IRI) mouse model. Jejunum tissues were sampled after 75 min of ischemia followed by 3, 24, and 48 h of reperfusion. Morphological evaluations were performed using light microscopy and electron microscopy, and the involvement of SETDB1 in epithelial remodeling was investigated by immunohistochemistry. Expression of SETDB1 was increased following 24 h of reperfusion and localized in not only the crypt bottom but also in the transit amplifying zone and part of the villi. Changes in cell lineage, repression of cell adhesion molecule expression, and decreased histone H3 methylation status were detected in the crypts at the same time. Electron microscopy also revealed aberrant alignment of crypt nuclei and fusion of adjacent villi. Furthermore, increased SETDB1 expression and epithelial remodeling were confirmed with loss of stem cells, suggesting SETDB1 affects epithelial cell plasticity. In addition, crypt elongation and increased numbers of Ki-67 positive cells indicated active cell proliferation after IRI; however, the expression of PCNA was decreased compared to sham mouse jejunum. These morphological changes and the aberrant expression of proliferation markers were prevented by sinefungin, a histone methyltransferase inhibitor. In summary, SETDB1 plays a crucial role in changes in the epithelial structure after IRI-induced stem cell loss.

The third most prevalent malignancy to cause mortality is hepatocellular carcinoma (HCC). The Hedgehog (Hh) signaling pathway is activated by binding to the transmembrane receptor Patched-1 (PTCH-1), which depresses the transmembrane G protein-coupled receptor Smoothened (SMO). This study was performed to examine the preventative and therapeutic effects of cannabidiol in adult rats exposed to diethyl nitrosamine (DENA)-induced HCC.A total of 50 male rats were divided into five groups of 10 rats each. Group I was the control group. Group II received intraperitoneal (IP) injections of DENA for 14 weeks. Group III included rats that received cannabidiol (CBD) orally (3-30 mg/kg) for 2 weeks and DENA injections for 14 weeks. Group IV rats received oral CBD for 2 weeks before 14 weeks of DENA injections. Group V included rats that received CBD orally for 2 weeks after their last injection of DENA. Measurements were made for alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and alpha fetoprotein (AFP). Following total RNA extraction, Smo, Hhip, Ptch-1, and Gli-1 expressions were measured using quantitative real-time polymerase chain reaction (qRT-PCR). A histopathological analysis of liver tissues was performed.The liver enzymes, oxidant-antioxidant state, morphological, and molecular parameters of the adult male rat model of DENA-induced HCC showed a beneficial improvement after CBD administration. In conclusion, by focusing on the Hh signaling system, administration of CBD showed a beneficial improvement in the liver enzymes, oxidant-antioxidant status, morphological, and molecular parameters in the DENA-induced HCC in adult male rats.

Demonstration of glycogen in tissue holds considerable diagnostic relevance across various pathological conditions, particularly in certain tumors. The histochemical staining of glycogen using methods utilizing Schiff's reagents is subject to influences arising from the type of fixative, fixation temperature, and oxidizing agents employed. This study aimed to assess diverse fixatives, fixation temperatures, and oxidizing agents, each with variable treatment durations, in conjunction with Schiff's reagent for optimal glycogen demonstration. Paraffin blocks derived from a rabbit's liver served as the experimental substrate, encompassing 340 paraffin sections subjected to different procedures. For tissues fixed at 4 °C, good staining outcomes, as determined by the periodic acid-Schiff (PAS) stain, were observed with 10% neutral buffered formalin (NBF), 80% alcohol, and Bouin's solution. Tissues fixed at room temperature (RT) demonstrated good PAS staining results with both 10% NBF and 80% alcohol. Notably, other oxidizing agents exhibited poor outcomes across all fixatives and fixation temperature, with two exceptions, as satisfactory staining results were obtained when using 5% chromic acid. Consequently, Both 10% NBF and 80% emerge as preferred fixatives of choice for glycogen demonstration when coupled with PAS stain. It is noteworthy that Bouin's solution could also provide good outcomes when fixation occurred at 4 °C.