Ultrastructural characteristics and morphological relationships of cardiomyocytes and telocytes in the myocardium of the bullfrog (Rana catesbeiana).

Telocytes (TCs) are distinctive interstitial cells due to their characteristic structures and heterogeneity. They are suggested to participate in tissue repair/regeneration. TCs have been identified in many organs of various mammals. However, data on TCs in lower animals are still very limited. In this work, TCs were identified in the myocardium of the bullfrog (Rana catesbeiana) by light and transmission electron microscopy (TEM). The structural relationships between TCs and neighbouring cell types were measured using the ImageJ (FiJi) morphometric software. TCs with slender Tps (telepodes) were located around cardiomyocytes (CMC). TEM revealed TCs with long Tps in the stroma between CMC. The homocellular tight junctions were observed between the Tps. The Tps were also very close to the neighbouring CMC. The distance between Tps and CMC was 0.15 ± 0.08 µm. Notably, Tps were observed to adhere to the periphery of the satellite cells. The Tps and the satellite cells established heterocellular structural connections by tight junctions. Additionally, Tps were frequently observed in close proximity to mast cells (MCs). The distance between the Tps and the MCs was 0.19 ± 0.09 µm. These results confirmed that TCs are present in the myocardium of the bullfrog, and that TCs established structural relationships with neighbouring cell types, including satellite cells and MCs. These findings provide the anatomical evidence to support the note that TCs are involved in tissue regeneration.

Transcriptome analysis reveals a potential regulatory mechanism of the lnc-5423.6/IGFBP5 axis in the early stages of mouse thymic involution.

Age-related thymic involution is one of the significant reasons for induced immunity decline. Recent evidence has indicated that lncRNAs are widely involved in regulating organ development. However, the lncRNA expression profiles in mouse thymic involution have not been reported. In this study, we collect mouse thymus at the ages of 1 month, 3 months, and 6 months for sequencing to observe the lncRNA and gene expression profiles in the early stages of thymic involution. Through bioinformatics analysis, a triple regulatory network of lncRNA-miRNA-mRNA that contains 29 lncRNAs, 145 miRNAs and 12 mRNAs that may be related to thymic involution is identified. Among them, IGFBP5 can reduce the viability, inhibit proliferation and promote apoptosis of mouse medullary thymic epithelial cell line 1 (MTEC1) cells through the p53 signaling pathway. In addition, miR-193b-3p can alleviate MTEC1 cell apoptosis by targeting IGFBP5. Notably, lnc-5423.6 can act as a molecular sponge of miR-193b-3p to regulate the expression of IGFBP5. In summary, lnc-5423.6 enhances the expression of IGFBP5 by adsorption of miR-193b-3p, thereby promoting MTEC1 cell apoptosis.

The effect of prophylactic chemotherapy on treatment outcome of postmolar gestational trophoblastic neoplasia.

OBJECTIVE: To evaluate whether prophylactic chemotherapy (P-chem) increased the drug resistance rate of postmolar GTN and whether the first-line chemotherapy should be different from P-chem. METHODS: Postmolar GTN received P-Chem was defined as P-Chem group. Postmolar GTN without P-chem was randomly selected as control group according to the ratio of 1:3 (P-chem:control) and matched by age for low risk and high risk GTN separately. RESULTS: Totally 455 low-risk and 32 high-risk postmolar GTN patients were included. WHO risk score, chemotherapy cycles to achieve hCG normalization and resistant rate were similar between P-chem (27 cases) and control (81 cases) group. Among low-risk GTN patients, interval from hydatidiform mole to GTN was significantly longer in P-chem group than control (44 vs 69 days, P = 0.001). Total chemotherapy cycles and resistant rate were similar between low-risk GTN treated with same agent as P-chem (group A) and alternative agent (group B). But group A needed more chemotherapy cycles to achieve hCG normalization than group B. CONCLUSIONS: P-chem delayed the time to GTN diagnosis, but didn't increase risk score or lead to drug resistance of postmolar GTN. Alternative agent different from P-chem had the potential of enhancing chemotherapy response in low- risk postmolar GTN.

Tyrosine Hydroxylase-Immunopositive Cells and Melanin in the Mesencephalon of Yugan Black-Bone Fowl.

Background: The Yugan blackbone fowl (YBF) is a special poultry with hyperpigmentation in various organs, including feather. However, the mechanism of hyperpigmentation is limited, and the melanic information of other organs except skin is rare. Aims and Objectives: In this study, we attempt to get an insight of the mechanism of melanogenesis of birds. Materials and Methods: The mesencephalon of YBF was observed by light microscopy with hematoxylineosin and tyrosine hydroxylase (TH) immunohistochemistry. Results: The TH immunopositive cells were found in the mesencephalon. Moreover, the melanin was also observed in the connective tissue of the mesencephalon. Conclusion: Our results confirmed the existence of melanin andTH immunopositive cells in the mesencephalon of YBF. These results provide a reference for further study on the mechanism of melanogenesis/hyperpigmentation in birds.

Telocytes and their structural relationships with surrounding cell types in the skin of silky fowl by immunohistochemistrical, transmission electron microscopical and morphometric analysis.

Telocytes (TCs), a novel type of interstitial cells, were identified in various animals. Since TCs have not observed in avian skin, hence, we carried out immunohistochemistrical and transmission electron microscopical studies in the skin of the silky fowl to investigate the TCs. TCs appear as CD34, c-Kit, and PDGFRα immunopositive. The elongated TCs with 2 long and thin telopodes (Tps) are located in the dermis. Generally, a TC possesses a fusiform, ovoid and polygonal cell body with 2 Tps (lengths = 5.27-21.85 µm), which are uneven in thickness including thick sections - podoms (diameters = 0.40-0.47 µm) and thin sections - podomers (diameters = 0.03-0.04 µm). TCs/Tps are observed frequently in close proximity to neighboring cell types/structures, such as adipocytes, collagen fibers, and capillaries. Under a magnified field, homocellular TCs/Tps contacts are observed through gap junctions (distances = 0.01-0.05 µm), whereas some of TCs/Tps have heterocellular close contacts by point contacts with surrounding cells, including stem cells and melanocytes. The multivisicular bodies, especially exosomes (diameters = 0.09-0.23 µm) releasing from TCs/Tps are observed in close proximity to TCs/Tps. Our results illustrated that the novel type of interstitial cells - TCs are present in the dermis of the silky fowl, and they have special structural relationships with surrounding cell types. The study provides histological evidence for TCs involvement in intercellular communication, skin regeneration, and pigmentogenesis in avian skin.

Structural Relationships Between Interstitial Cells of Cajal and Smooth Muscle Cells/Nerve Fibers in the Gastric Muscularis Mucosae of Chinese Giant Salamander.

Interstitial cells of Cajal (ICC) play an essential role in the motility of the gastrointestinal tract, and they have been identified in many laboratory animals and in humans. However, the information of ICC in lower animals is still very limited. In the present study, ICC were identified in the gastric muscularis mucosae of an amphibian­the Chinese giant salamander, by c-Kit immunohistochemistry and transmission electron microscopy. ICC showed c-Kit immunoreactivity and had spindle-shaped cell bodies and 1­2 long processes. ICC were located between smooth muscle cells (SMC) in gastric muscularis mucosae. Ultrastructurally, ICC appeared as polygon-, spindle-, and awl-shaped with long cytoplasmic prolongations between SMC. ICC had distinctive characteristics, such as nuclei with peripheral electron-dense heterochromatin, caveolae, and abundant intracytoplasmatic vacuoles, mitochondria, and rough endoplasmic reticula. Moreover, lamellar bodies and two types of condensed granules were observed in the cytoplasm of ICC. Notably, ICC establish close contacts with each other. Moreover, ICC establish gap junctions with SMC. In addition, ICC were frequently observed close to nerve fibers. In summary, the present study demonstrated the presence of ICC in the gastric muscularis mucosae of the Chinese giant salamander.

Effect of Baicalin on Bacterial Secondary Infection and Inflammation Caused by H9N2 AIV Infection in Chickens.

H9N2 subtype avian influenza virus (H9N2 AIV) is a low pathogenic virus that is widely prevalent all over the world. H9N2 AIV causes immunosuppression in the host and often leads to high rates of mortality due to secondary infection with Escherichia. Due to the drug resistance of bacteria, many antibiotics are not effective in the treatment of secondary bacterial infection. Therefore, the purpose of this study is to find effective nonantibiotic drugs for the treatment of H9N2 AIV infection-induced secondary bacterial infection and inflammation. This study proves, for the first time, that baicalin, a Chinese herbal medicine, can regulate Lactobacillus to replace Escherichia induced by H9N2 AIV, so as to resolve the intestinal flora disorder. In addition, baicalin can effectively prevent intestinal bacterial translocation of SPF chickens' post-H9N2 AIV infection, thus inhibiting secondary bacterial infection. Furthermore, baicalin can effectively treat H9N2 AIV-induced inflammation by inhibiting intestinal structural damage, inhibiting damage to ileal mucus layer construction and tight junctions, improving antioxidant capacity, affecting blood biochemical indexes, and inhibiting the production of inflammatory cytokines. Taken together, these results provide a new theoretical basis for clinical prevention and control of H9N2 AIV infection-induced secondary bacterial infection and inflammation.

Effects of Castration on miRNA, lncRNA, and mRNA Profiles in Mice Thymus.

Thymic degeneration and regeneration are regulated by estrogen and androgen. Recent studies have found that long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are involved in organ development. In this study, RNA sequencing (RNA-seq) results showed that ovariectomy significantly affected 333 lncRNAs, 51 miRNAs, and 144 mRNAs levels (p < 0.05 and |log2fold change| > 1), and orchiectomy significantly affected 165 lncRNAs, 165 miRNAs, and 208 mRNA levels in the thymus. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that differentially expressed genes (DEGs) were closely related to cell development and immunity. Next, we constructed two lncRNA-miRNA-mRNA networks using Cytoscape based on the targeting relationship between differentially expressed miRNAs (DEMs) and DEGs and differentially expressed lncRNAs (DELs) analyzed by TargetScan and miRanda. Besides, we screened DEGs that were significantly enriched in GO and in ceRNA networks to verify their expression in thymocytes and thymic epithelial cells (TECs). In addition, we analyzed the promoter sequences of DEGs, and identified 25 causal transcription factors. Finally, we constructed transcription factor-miRNA-joint target gene networks. In conclusion, this study reveals the effects of estrogen and androgen on the expression of miRNAs, lncRNAs, and mRNAs in mice thymus, providing new insights into the regulation of thymic development by gonadal hormones and non-coding RNAs.

Ultrastructure of telocytes, a new type of interstitial cells in the myocardium of the Chinese giant salamander (Andrias davidianus).

Telocytes (TCs) are new interstitial cells, and they are involved in tissue regeneration, particularly in heart. Therefore, TCs are suggested to be a promising cell in regenerative medicine. However, the information of location structural characteristics and functions of TCs is still limited. In this study, cardiac TCs of the Chinese giant salamanders (Andrias davidianus) were identified by transmission electron microscopy. TCs were located in the interstitium between cardiomyocytes (CM). TCs possessed distinctive ultrastructural characteristics, including one to two very long and thin moniliform telopodes (Tps), emerging points from the cell body, caveolae, dichotomous branchings, labyrinthic systems, neighbouring exosomes and homo-cellular contacts between Tps. TCs/Tps were frequently observed in close proximity to cardiomyocytes. Moreover, Tps established hetero-cellular contacts with cardiomyocytes. Our results confirm the presence of TCs in the myocardium of the A. davidianus. This will help us to better understand roles of TCs in amphibian hearts.

Let-7b Regulates Myoblast Proliferation by Inhibiting IGF2BP3 Expression in Dwarf and Normal Chicken.

The sex-linked dwarf chicken is caused by the mutation of growth hormone receptor (GHR) gene and characterized by shorter shanks, lower body weight, smaller muscle fiber diameter and fewer muscle fiber number. However, the precise regulatory pathways that lead to the inhibition of skeletal muscle growth in dwarf chickens still remain unclear. Here we found a let-7b mediated pathway might play important role in the regulation of dwarf chicken skeletal muscle growth. Let-7b has higher expression in the skeletal muscle of dwarf chicken than in normal chicken, and the expression of insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which is a translational activator of IGF2, showed opposite expression trend to let-7b. In vitro cellular assays validated that let-7b directly inhibits IGF2BP3 expression through binding to its 3'UTR region, and the protein level but not mRNA level of IGF2 would be reduced in let-7b overexpressed chicken myoblast. Let-7b can inhibit cell proliferation and induce cell cycle arrest in chicken myoblast through let-7b-IGF2BP3-IGF2 signaling pathway. Additionally, let-7b can also regulate skeletal muscle growth through let-7b-GHR-GHR downstream genes pathway, but this pathway is non-existent in dwarf chicken because of the deletion mutation of GHR 3'UTR. Notably, as the loss binding site of GHR for let-7b, let-7b has enhanced its binding and inhibition on IGF2BP3 in dwarf myoblast, suggesting that the miRNA can balance its inhibiting effect through dynamic regulate its binding to target genes. Collectively, these results not only indicate that let-7b can inhibit skeletal muscle growth through let-7b-IGF2BP3-IGF2 signaling pathway, but also show that let-7b regulates myoblast proliferation by inhibiting IGF2BP3 expression in dwarf and normal chickens.

Age and sex differences in microRNAs expression during the process of thymus aging.

The gender-biased thymus involution and the importance of microRNAs (miRNAs, miRs) expression in modulating the thymus development have been reported in many studies. However, how males and females differ in so many ways in thymus involution remains unclear. To address this question, we investigated the miRNA expression profiles in both untreated 3- and 12-month-old female and male mice thymuses. The results showed that 7 and 18 miRNAs were defined as the sex- and age-specific miRNAs, respectively. The expression of miR-181c-5p, miR-20b-5p, miR-98b-5p, miR-329-3p, miR-341-5p, and miR-2137 showed significant age-difference in mice thymus by quantitative polymerase chain reaction. High expression levels of miR-2137 were detected in mice thymic epithelial cells and gradually increased during the process of thymus aging. MiR-27b-3p and miR-378a-3p of the female-biased miRNAs were confirmed as the sex- and estrogen-responsive miRNAs in mice thymus in vivo. Their potential target genes and the pathway were identified by the online software. Possible regulation roles of sex- and age-specific miRNA expression during the process of thymus aging were discussed. Our results suggested that these miRNAs may be potential biomarkers for the study of sex- and age-specific thymus aging and involution.

MiR-181a-5p regulates 3T3-L1 cell adipogenesis by targeting Smad7 and Tcf7l2.

MicroRNAs are highly conserved non-coding small RNAs participating in almost all kinds of biological activities. MiR-181a has been reported to be involved in the differentiation of porcine primary preadipocytes, but the profound effect of miR-181a-5p on 3T3-L1 adipocyte differentiation and proliferation is still unclear. In this study, we found that supplementation of miR-181a-5p in 3T3-L1 cells significantly promoted the adipogenesis and inhibited cell proliferation with increased expression of adipogenic marker genes including peroxisome proliferator-activated receptor gamma (Pparγ), CCAAT/enhancer-binding protein alpha (C/ebpα), fatty acid-binding protein 4 (Fabp4), and Adiponectin, accompanied by an accumulation of lipid droplet, an increase of triglyceride content, and a decrease of cell proliferation. Furthermore, by using the luciferase assay, Smad7 and Tcf7l2, two important members of transforming growth factor-ß (TGFß) and Wnt signaling pathway, were proven to be the direct target genes of miR-181a-5p. Moreover, supplementation of miR-181a-5p in 3T3-L1 cells altered the expressions of proteins involved in the TGFß signaling pathway, such as TGFBR1, p-SMAD3, SMAD4, c-MYC, and p15. Taken together, these results indicate that miR-181a-5p promotes 3T3-L1 preadipocyte differentiation and adipogenesis through regulating TGFß/Smad and Wnt signaling pathway by directly targeting Smad7 and Tcf7l2.

MicroRNA-181a-5p enhances cell proliferation in medullary thymic epithelial cells via regulating TGF-ß signaling.

The expression profiles of miRNAs in thymus tissues from mice of different age have been demonstrated in our previous study. After an integrated analysis of the miRNA expression profiles, we demonstrated that the expression of miR-181a-5p was significantly decreased in thymic epithelial cells (TECs) from 10- to 19-month-old mice when compared with that in TECs from 1-month-old mice by quantitative reverse transcriptase polymerase chain reaction. We hypothesized that miR-181a-5p in TECs might be associated with the age-related thymus involution through regulating some genes or signaling pathway. To test this hypothesis, the mouse medullary thymic epithelial cells (MTEC1) were used. Transfection with miR-181a-5p mimic promoted the proliferation of MTEC1 cells, but did not affect apoptosis. The effect was reversed when the expression of miR-181a-5p was suppressed in MTEC1 cells. Furthermore, the transforming growth factor beta receptor I (Tgfbr1) was confirmed as a direct target of miR-181a-5p by luciferase assay. Moreover, it was found that overexpression of miR-181a-5p down-regulated the phosphorylation of Smad3 and blocked the activation of the transforming growth factor beta signaling. Nevertheless, an inversely correlation was observed between the expression of Tgfbr1 and miR-181a-5p in TECs derived from mice of different age. Collectively, we provide evidence that miR-181a-5p may be an important endogenous regulator in the proliferation of TECs, and the expression levels of miR-181a-5p in TECs may be associated with the age-related thymus involution.

MicroRNA-195a-5p inhibits mouse medullary thymic epithelial cells proliferation by directly targeting Smad7.

MiR-195 has been implicated in inhibiting cell proliferation in different types of tumors. Whether it contributes to the process of thymic epithelial cells (TECs) proliferation remains unclear. In this study, we found that miR-195a-5p was highly up-regulated in the TECs isolated from the aging mice. Further experiments showed that miR-195a-5p mimic transfection inhibited the proliferation of mouse medullary thymic epithelial cell line 1 (MTEC1), whereas the transfection of miR-195a-5p inhibitor in MTEC1 had the opposite effect. In addition, miR-195a-5p had no obvious effect on MTEC1 apoptosis. Furthermore, Smad7, a negative regulator of transforming growth factor ß pathway, was confirmed as a direct target of miR-195a-5p by luciferase assays. Taken together, our results indicate that miR-195a-5p inhibits MTEC1 proliferation, at least in part, via down-regulation of Smad7.

MicroRNA-125b-5p inhibits proliferation and promotes adipogenic differentiation in 3T3-L1 preadipocytes.

Previous evidence has indicated that the microRNA-125b (miR-125b) family plays important roles in the regulation of cancer cell growth, development, differentiation, and apoptosis. However, whether they contribute to the process of adipocyte differentiation remains unclear. In the present study, we revealed that the expression level of miR-125b-5p, a member of miR-125b family, was dramatically up-regulated during differentiation of 3T3-L1 preadipocyte into mature adipocyte. Supplement of miR-125b-5p into 3T3-L1 cells promoted adipogenic differentiation as evidenced by increased lipid droplets and mRNA levels of adipocyte-specific molecular markers, including peroxisome proliferators-activated receptor γ, CCAAT/enhancer-binding protein α, fatty acid-binding protein 4, and lipoprotein lipase, and by triglyceride accumulation. CCK-8 assay showed that miR-125b-5p supplementation significantly inhibited cell proliferation. Flow cytometry analysis showed that miR-125b-5p impaired G1/S phase transition as well as the mRNA and protein expression of G1/S-related genes, such as Cyclin D2, Cyclin D3, and CDK4. Nevertheless, it had no effect on apoptosis. Additionally, by target gene prediction, we demonstrated that smad4 may be a potential target of miR-125b-5p in mouse 3T3-L1 preadipocytes, accounting for some of miR-125b-5p's functions. Taken together, these data indicated that miR-125b-5p may serve as an important positive regulator in adipocyte differentiation, at least partially through down-regulating smad4.

MicroRNA expression in the aging mouse thymus.

MicroRNAs (miRNAs) have been implicated in the process of aging in many model organisms, such as Caenorhabditis elegans, and in many organs, such as the mouse lung and human epididymis. However, the role of miRNAs in the thymus tissues of the aging mouse remains unclear. To address this question, we investigated the miRNA expression profiles in the thymuses of 1-, 10- and 19-month-old mice using miRNA array and qRT-PCR assays. A total of 223 mouse miRNAs were screened, and the expression levels of those miRNAs exhibited gradual increases and decreases over the course of thymus aging. Fifty miRNAs in the 10-month-old thymus and 81 miRNAs in the 19-month-old thymus were defined as differentially expressed miRNAs (p<0.05) in comparison with their levels in the 1-month-old mouse, and approximately one-third of these miRNAs were grouped within 11 miRNA clusters. Each miRNA cluster contained 2 to 5 miRNA genes, and most of the cluster members displayed similar expression patterns, being either increased or decreased. In addition, Ingenuity Pathway Analysis (IPA) software and the IPA database were used to analyze the 12 miRNAs that exhibited significant expression changes, revealing that as many as 15 pathways may be involved. Thus, our current study determined the expression profiles of miRNAs in the mouse thymus during the process of aging. The results suggested that these miRNAs could become meaningful biomarkers for studying thymus aging and that the aging-related alternations in miRNA expression may be involved in the regulation of cell proliferation, apoptosis, development and carcinogenesis/tumorigenesis.

Deoxynivalenol induces apoptosis in mouse thymic epithelial cells through mitochondria-mediated pathway.

Deoxynivalenol (DON) is a mycotoxin produced as a secondary metabolite by fungal species. In this report, we investigated the apoptotic effect of DON in mouse thymic epithelial cell line 1 (MTEC1). MTEC1 cell apoptosis induced by DON was confirmed by nuclei morphology change, TUNEL positive staining, annexin V/propidium iodide positive staining and increased protein levels of caspase-3, caspase-8, caspase-9 and poly(ADP-ribose) polymerase (PARP). The effects of DON on reactive oxygen species (ROS) levels and mitochondrial membrane potential were investigated via fluorescence microscope and flow cytometry, respectively. In addition, DON could significantly increase the protein levels of p53 and Bax/Bcl-2 ratio in MTEC1 cells. Taken together, our results suggest that DON causes the activation of p53, increased levels of ROS and the induction of mitochondrial dysfunction, which may contribute to DON-induced apoptosis in MTEC1 cells.

Analysis of differentially expressed genes and signaling pathways related to intramuscular fat deposition in skeletal muscle of sex-linked dwarf chickens.

Intramuscular fat (IMF) plays an important role in meat quality. However, the molecular mechanisms underlying IMF deposition in skeletal muscle have not been addressed for the sex-linked dwarf (SLD) chicken. In this study, potential candidate genes and signaling pathways related to IMF deposition in chicken leg muscle tissue were characterized using gene expression profiling of both 7-week-old SLD and normal chickens. A total of 173 differentially expressed genes (DEGs) were identified between the two breeds. Subsequently, 6 DEGs related to lipid metabolism or muscle development were verified in each breed based on gene ontology (GO) analysis. In addition, KEGG pathway analysis of DEGs indicated that some of them (GHR, SOCS3, and IGF2BP3) participate in adipocytokine and insulin signaling pathways. To investigate the role of the above signaling pathways in IMF deposition, the gene expression of pathway factors and other downstream genes were measured by using qRT-PCR and Western blot analyses. Collectively, the results identified potential candidate genes related to IMF deposition and suggested that IMF deposition in skeletal muscle of SLD chicken is regulated partially by pathways of adipocytokine and insulin and other downstream signaling pathways (TGF- ß /SMAD3 and Wnt/catenin- ß pathway).