Objective To establish an efficient method for isolating migrasomes from RAW264.7 macrophages and identifying these isolated migrasomes. Methods Scanning electron microscopy was used to observe the morphological characteristics of migrasomes produced by RAW264.7 cells. A 0.45 μm filter was employed for reverse filtration and elution to isolate the migrasomes. The morphological characteristics of the migrasomes were then observed using transmission electron microscopy. Western blot analysis was performed to determine the expression of characteristic markers of the migrasomes. The RNA carried by the migrasomes was analysed by using LabChip bioanalyzer. Results Scanning electron microscopy revealed that the migrasomes, with membranous structures, were attached to the tip or bifurcation of the retraction fiber formed in the tail of RAW264.7 cells. Transmission electron microscopy showed that the isolated migrasomes had a typical oval vesicle-like structure with wrinkled membrane surfaces. Western blot analysis confirmed the expression of the characteristic markers phosphatidylinositol glycan anchor biosynthesis class K (PIGK), epidermal growth factor domain-specific O-linked N-acetylglucosamine transferase (EOGT) and tetraspanin 4 (TSPAN4) in the migrasomes, while the EV (extracellular vesicle) markers tumor susceptibility gene 101 (TSG101) and Arabidopsis homolog of apoptosis-linked gene 2-interacting protein X (ALIX) were not detected. Furthermore, the isolated migrasomes were found to be rich in small RNA, which were approximately 25-200 nt in length. Conclusion A method for the extraction of well-structured and high quality migrasomes from macrophages is established.
Extracellular Vesicles ; Microscopy, Electron, Transmission ; RNA ; Macrophages

OBJECTIVES: This study aims to investigate the effects of ionizing radiation on the secretion of the paracellular pathway in rat submandibular glands (SMGs) and reveal the changes in the tight junction (TJ) protein claudin-4. METHODS: A total of 24 Wistar rats were randomly divided into control and irradiation groups. The irradiation groups were further divided into 1, 4, and 12 weeks groups after irradiation. One-time 20 Gy irradiation was given to the SMG area on the experimental side of the irradiation group. At 1, 4, and 12 weeks after irradiation, the secretion of SMGs was measured using the Schirmer's test. The pathological changes in the gland tissues were observed under light microscopy after hematoxylin⁃eosin (HE) staining. The changes in the TJ ultrastructure were observed under transmission electron microscopy. The immunofluorescence staining and Western blot were used to detect the expression levels of muscarinic acetylcholine M3 receptor, aquaporin 5 (AQP5), and claudin-4 protein. RESULTS: At 1, 4, and 12 weeks after irradiation, the secretion of SMGs in the irradiation group was significantly decreased and lower than that in the control group ( CONCLUSIONS The changes in the TJ structure, the upregulation of the claudin-4 expression, and the damage in the paracellular pathway were involved in the hyposecretion of SMGs after irradiation.
Animals ; Microscopy, Electron, Transmission ; Radiation, Ionizing ; Rats ; Rats, Wistar ; Submandibular Gland ; Tight Junctions

Objective: To investigate the relationship of electromagnetic radiation (EMR) from 900 MHz cellphone frequency with testicular oxidative damage and its influence on the Prdx2 protein expression in the rat testis, and to explore the mechanism of Guilingji Capsules (GC) alleviating oxidative damage to the testis tissue. METHODS: Fifty healthy SD male rats were randomly divided into five groups of equal number, sham-EMR, 4-h EMR, 8-h EMR, 4-h EMR+GC and 8-h EMR+GC and exposed to 900 MHz EMR (370 μW/cm2) for 0, 4 or 8 hours daily for 15 successive days. The rats of the latter two groups were treated intragastrically with GC suspension and those of the first three groups with pure water after exposure to EMR each day. After 15 days of exposure and treatment, all the rats were sacrificed and their testis tissue collected for observation of the histomorphological and ultrastructural changes by HE staining and transmission electron microscopy, measurement of the levels of serum glutathione (GSH), superoxide dismutase (SOD) and malondialdehyde (MDA) with thiobarbiuric acid and determination of the Prdx2 protein expression by immunohistochemistry and Western blot. RESULTS: Compared with the rats in the sham-EMR group, those in the 4-h and 8-h EMR groups showed different degrees of histomorphological and ultrastructural changes in the testis tissue, significantly decreased levels of GSH (［80.62 ± 10.99］ vs ［69.58 ± 4.18］ and ［66.17 ± 8.45］ mg/L, P < 0.05) and SOD (［172.29 ± 10.98］ vs ［158.92 ± 6.46］ and ［148.91 ± 8.60］ U/ml, P < 0.05) and increased level of MDA (［7.51 ± 1.73］ vs ［9.84 ± 1.03］ and ［11.22 ± 2.13］ umol/ml, P < 0.05), even more significantly in the 8-h than in the 4-h EMR group (P < 0.05). In comparison with the sham-EMR group, the expression of the Prdx2 protein was markedly downregulated in the 4-h and 8-h EMR groups (0.56 ± 0.03 vs 0.49 ± 0.03, 0.21 ± 0.01, P < 0.05), but again upregulated in the 4-h and 8-h EMR+GC groups (0.55±0.03 and 0.37±0.04) (P < 0.05). CONCLUSIONS Electromagnetic radiation from cellphones can cause ultrastructural damage to the testis tissue of male rats, while Guilingji Capsules can alleviate it, presumably by upregulating the Prdx2 protein expression in the testis tissue and reducing testicular oxidative damage.
Animals ; Capsules ; Cell Phone ; Drugs, Chinese Herbal/therapeutic use* ; Electromagnetic Radiation ; Glutathione/blood* ; Male ; Malondialdehyde/blood* ; Microscopy, Electron, Transmission ; Oxidative Stress ; Peroxiredoxins/metabolism* ; Radiation Injuries, Experimental/drug therapy* ; Rats ; Superoxide Dismutase/blood* ; Testis/pathology* ; Thiobarbituric Acid Reactive Substances/analysis*

BACKGROUND: We previously demonstrated that continuous exposure to nitrous acid gas (HONO) for 4 weeks, at a concentration of 3.6 parts per million (ppm), induced pulmonary emphysema-like alterations in guinea pigs. In addition, we found that HONO affected asthma symptoms, based on the measurement of respiratory function in rats exposed to 5.8 ppm HONO. This study aimed to investigate the dose-response effects of HONO exposure on the histopathological alterations in the respiratory tract of guinea pigs to determine the lowest observed adverse effect level (LOAEL) of HONO. METHODS: We continuously exposed male Hartley guinea pigs (n = 5) to four different concentrations of HONO (0.0, 0.1, 0.4, and 1.7 ppm) for 4 weeks (24 h/day). We performed histopathological analysis by observing lung tissue samples. We examined samples from three guinea pigs in each group under a light microscope and measured the alveolar mean linear intercept (Lm) and the thickness of the bronchial smooth muscle layer. We further examined samples from two guinea pigs in each group under a scanning electron microscope (SEM) and a transmission electron microscope (TEM). RESULTS: We observed the following dose-dependent changes: pulmonary emphysema-like alterations in the centriacinar regions of alveolar ducts, significant increase in Lm in the 1.7 ppm HONO-exposure group, tendency for hyperplasia and pseudostratification of bronchial epithelial cells, and extension of the bronchial epithelial cells and smooth muscle cells in the alveolar duct regions. CONCLUSIONS These histopathological findings suggest that the LOAEL of HONO is < 0.1 ppm.
Alveolar Epithelial Cells ; drug effects ; Animals ; Bronchi ; drug effects ; Dose-Response Relationship, Drug ; Emphysema ; chemically induced ; Epithelial Cells ; drug effects ; Guinea Pigs ; Hyperplasia ; chemically induced ; Inhalation Exposure ; adverse effects ; Lung ; drug effects ; pathology ; ultrastructure ; Male ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Myocytes, Smooth Muscle ; drug effects ; Nitrous Acid ; toxicity

Novel coronavirus (SARS-CoV-2) is found to cause a large outbreak started from Wuhan since December 2019 in China and SARS-CoV-2 infections have been reported with epidemiological linkage to China in 25 countries until now. We isolated SARS-CoV-2 from the oropharyngeal sample obtained from the patient with the first laboratory-confirmed SARS-CoV-2 infection in Korea. Cytopathic effects of SARS-CoV-2 in the Vero cell cultures were confluent 3 days after the first blind passage of the sample. Coronavirus was confirmed with spherical particle having a fringe reminiscent of crown on transmission electron microscopy. Phylogenetic analyses of whole genome sequences showed that it clustered with other SARS-CoV-2 reported from Wuhan.
China ; Coronavirus ; Crowns ; Genome ; Humans ; Korea ; Microscopy, Electron ; Microscopy, Electron, Transmission ; Phylogeny ; Vero Cells

Autophagy is involved in spermatogenesis by regulating germ cell maturation. This catabolic process increases with hyperthermic conditions to prevent the accumulation of damaged organelles. Cryptorchidism is associated with impairment of germ cell maturation revealed by the presence of immature forms of sperm cells in ejaculates. The aim of the present study was to evaluate the status of autophagy in sperm cells from cryptorchid patients. Semen samples of cryptorchid patients and normozoospermic controls were analyzed by immunocytochemistry and electron microscopy. Autophagy proteins, autophagy-related protein 9 (ATG9) and microtubule-associated protein, 1A/1B-light chain 3 (LC3) were localized by immunocytochemistry on the acrosome and on the equatorial segment of sperm cells. LC3 was also detected in the midpiece of cryptorchid sperm tail. Autophagy substrate p62 protein was present in the acrosome and in the postequatorial segment of sperm in control samples, but not in the cryptorchid ones. Transmission electron microscopy revealed double-membrane-limited autophagosomes in postequatorial part of spermatozoa head and midpiece in cryptorchid samples. Partly degraded mitochondria were frequently discerned in autophagic vacuoles. In conclusion, autophagy is increased in sperm cells from patients with cryptorchid history comparatively to control. Our work provides insights into the role of autophagy in the maturation and survival of human male gametes in pathological conditions. Thus, regulating autophagy could represent a potential way to improve sperm quality in cryptorchid men.
Adult ; Autophagy ; Case-Control Studies ; Cryptorchidism/pathology* ; Humans ; Male ; Microscopy, Electron, Transmission ; Spermatogenesis ; Spermatozoa/pathology* ; Teratozoospermia/pathology* ; Testis/pathology*

Ageratina adenophora is a noxious plant and it is known to cause acute asthma, diarrhea, depilation, and even death in livestock (Zhu et al., 2007; Wang et al., 2017). A. adenophora grows near roadsides and degraded land worldwide (He et al., 2015b). In the areas where it grows, A. adenophora is an invasive species that inhibits the growth of local plants and causes poisoning in animals that come in contact with it (Nie et al., 2012). In China, these plants can be found in Yunnan, Sichuan, Guizhou, Chongqing, and other southwestern areas (He et al., 2015a) and they have become a dominant species in these local regions. It threatens the native biodiversity and ecosystem in the invaded areas and causes serious economic losses (Wang et al., 2017). It has been reported that A. adenophora can grow in the northeast direction at a speed of 20 km per year in China (Guo et al., 2009). Because of the damage caused by A. adenophora, it ranks among the earliest alien invasive plant species in China (Wang et al., 2017).
Adenosine Triphosphatases/metabolism* ; Ageratina/toxicity* ; Animals ; Biodiversity ; Chemical and Drug Induced Liver Injury/pathology* ; China ; DNA, Mitochondrial/genetics* ; Ecosystem ; Introduced Species ; Liver/drug effects* ; Mice ; Microscopy, Electron, Transmission ; Mitochondria, Liver/pathology* ; Plant Extracts/toxicity*

The aim of the present study was to investigate the role of ferroptosis in acute lung injury (ALI) mouse model induced by oleic acid (OA). ALI was induced in the mice via the lateral tail vein injection of pure OA. The histopathological score of lung, lung wet-dry weight ratio and the protein content of bronchoalveolar lavage fluid (BALF) were used as the evaluation indexes of ALI. Iron concentration, glutathione (GSH) and malondialdehyde (MDA) contents in the lung tissues were measured using corresponding assay kits. The ultrastructure of pulmonary cells was observed by transmission electron microscope (TEM), and the expression level of prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA was detected by quantitative polymerase chain reaction (q-PCR). Protein expression levels of glutathione peroxidase 4 (GPX4), ferritin and transferrin receptor 1 (TfR1) in lung tissues were determined by Western blot. The results showed that histopathological scores of lung tissues, lung wet-dry weight ratio and protein in BALF in the OA group were higher than those of the control group. In the OA group, the mitochondria of pulmonary cells were shrunken, and the mitochondrial membrane was ruptured. The expression level of PTGS2 mRNA in the OA group was seven folds over that in the control group. Iron overload, GSH depletion and accumulation of MDA were observed in the OA group. Compared with the control group, the protein expression levels of GPX4 and ferritin in lung tissue were down-regulated in the OA group. These results suggest that ferroptosis plays a potential role in the pathogenesis of ALI in our mouse model, which may provide new insights for development of new drugs for ALI.
Acute Lung Injury ; chemically induced ; pathology ; Animals ; Apoptosis ; Bronchoalveolar Lavage Fluid ; chemistry ; Cyclooxygenase 2 ; metabolism ; Ferritins ; metabolism ; Glutathione ; analysis ; Glutathione Peroxidase ; metabolism ; Iron ; analysis ; Iron Overload ; physiopathology ; Lung ; cytology ; pathology ; Malondialdehyde ; analysis ; Mice ; Microscopy, Electron, Transmission ; Mitochondrial Membranes ; ultrastructure ; Oleic Acid

BACKGROUND AND OBJECTIVES: The release of microvesicles (MVs) from mesenchymal stem cells (MSCs) has been implicated in intercellular communication, and may contribute to beneficial paracrine effects of stem cell-based therapies. We investigated the effect of administration of MSC-MVs on the therapeutic potential of carbon tetrachloride (CCL₄) induced liver fibrosis in rats.METHODS: Our work included: isolation and further identification of bone marrow MSC-MVs by transmission electron microscopy (TEM). Liver fibrosis was induced in rats by CCl4 followed by injection of prepared MSC-MVs in injured rats. The effects of MSC-MVs were evaluated by biochemical analysis of liver functions, RNA gene expression quantitation for collagen-1α, transforming growth factor β (TGF-β), interleukin-1β (IL-1β), vascular endothelial growth factor (VEGF) by real time reverse transcription PCR (RT-PCR) techniques. Finally histopathological examination of the liver tissues was assessed for all studied groups.RESULTS: BM-MSC-MVs treated group showed significant increase in serum albumin levels, VEGF quantitative gene expression (p < 0.05), while it showed a significant decrease in serum alanine transaminase (ALT) enzyme levels, quantitative gene expression of TGF-β, collagen-1α, IL-1β compared to CCL₄ fibrotic group (p < 0.05). Additionally, the histopathological assessment of the liver tissues of BM-MSC-MVs treated group showed marked decrease in the collagen deposition & improvement of histopathological picture in comparison with CCL₄ fibrotic group.CONCLUSIONS: Our study demonstrates that BM-MSC-MVs possess anti-fibrotic, anti-inflammatory, and pro-angiogenic properties which can promote the resolution of CCL₄ induced liver fibrosis in rats.
Alanine Transaminase ; Animals ; Bone Marrow ; Carbon Tetrachloride ; Collagen ; Gene Expression ; Liver Cirrhosis ; Liver ; Mesenchymal Stromal Cells ; Microscopy, Electron, Transmission ; Polymerase Chain Reaction ; Rats ; Reverse Transcription ; RNA ; Serum Albumin ; Transforming Growth Factors ; Vascular Endothelial Growth Factor A

BACKGROUND: Nano-hydroxyapatite/polyamide 66 (nHA/PA66) is a composite used widely in the repair of bone defects. However, this material is insufficient bioactivity. In contrast, D-RADA16-RGD self-assembling peptide (D-RADA16-RGD sequence containing all D-amino acids is Ac-RADARADARADARADARGDS-CONH2) shows admirable bioactivity for both cell culture and bone regeneration. Here, we describe the fabrication of a favorable biomaterial material (nHA/PA66/D-RADA16-RGD). METHODS: Proteinase K and circular dichroism spectroscopy were employed to test the stability and secondary structural properties of peptide D-RADA16-RGD respectively. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the surface of these materials. Confocal laser scanning (CLS), cell counting kit-8 tests (CCK-8), alizarin red S staining, cell immunofluorescence analysis and Western blotting were involved in vitro. Also biosafety and bioactivity of them have been evaluated in vivo. RESULTS: Proteinase K and circular dichroism spectroscopy demonstrated that D-RADA16-RGD in nHA/PA66 was able to form stable-sheet secondary structure. SEM and TEM showed that the D-RADA16-RGD material was 7–33 nm in width and 130–600 nm in length, and the interwoven pore size ranged from 40 to 200 nm. CLS suggests that cells in nHA/PA66/D-RADA16-RGD group were linked to adjacent cells with more actin filaments. CCK-8 analysis showed that nHA/PA66/D-RADA16-RGD revealed good biocompatibility. The results of Alizarin-red S staining and Western blotting as well as vivo osteogenesis suggest nHA/PA66/D-RADA16-RGD exhibits better bioactivity. CONCLUSION: This study demonstrates that our nHA/PA66/D-RADA16-RGD composite exhibits reasonable mechanical properties, biocompatibility and bioactivity with promotion of bone formation.
Actin Cytoskeleton ; Blotting, Western ; Bone Regeneration ; Cell Count ; Cell Culture Techniques ; Circular Dichroism ; Endopeptidase K ; Fluorescent Antibody Technique ; In Vitro Techniques ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Osteogenesis ; Sincalide ; Spectrum Analysis