Extraction and identification of exosomes from three different sources of human ovarian granulosa cells and analysis of their differential miRNA expression profiles.

OBJECTIVE: As important functional cells in the ovary, ovarian granulosa cells are involved in the regulation of oocyte growth and development and play an important role in the study of female fertility preservation. Based on the importance of granulosa cell functionalism, in this study, we analyzed the exosome secretion capacity of human ovarian granulosa cells (SVOG/KGN-cell line, PGC-primary cells) and the differences in their miRNA expression. METHODS: Cells were identified by hematoxylin-eosin staining (HE) and FSHR immunofluorescence staining; CCK8 and colony-forming assay were performed to compare cell proliferation capacity; exosomes were extracted and identified by ultra-high speed centrifugation, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot analysis (WB), and the expression profile of each cellular exosomal miRNA was analyzed by miRNA high-throughput sequencing. RESULTS: The proliferative abilities of the three granulosa cells differed, but all had the ability to secrete exosomes. In the exosomes of SVOG, KGN, and PGC cells, 218, 327, and 471 miRNAs were detected, respectively. When compared to the exosomal miRNAs of PGC cells, 111 miRNAs were significantly different in SVOG, and 70 miRNAs were washed two significantly different in KGN cells. These differential miRNA functions were mainly enriched in the cell cycle, cell division/differentiation, multicellular biogenesis, and protein binding. CONCLUSION: Human ovarian granulosa cells of different origins are capable of secreting exosomes, but there are still some differences in their exosomes and exosomal miRNAs, and experimental subjects should be selected rationally according to the actual situation.

Transcriptome analysis of Procambarus clarkii infected with infectious hypodermal and haematopoietic necrosis virus.

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is a major viral pathogen in cultured penaeid shrimp. IHHNV has many hosts, mainly including crustaceans. It has recently been reported that Procambarus clarkii can be infected by IHHNV. In the present study, we studied the hepatopancreas of P. clarkii by transcriptome high-throughput sequencing to analyze the response of P. clarkii to IHHNV infection. After de novo assembly, there were 400,340,760 clean reads. A total of 237 differentially expressed genes (DEGs) were obtained, including 77 significantly up-regulated unigenes and 160 significantly down-regulated ones. The expression levels of 12 immune-related DEGs were validated by qRT-PCR, substantiating the reliability of RNA-Seq results. The enrichment analysis of DEGs showed that the immune-related pathways were closely related to apoptosis and phagocytosis. Moreover, a large number of pathways related to metabolic function were down-regulated, suggesting that IHHNV infection might affect the growth of P. clarkii.

A novel real-time PCR approach for detection of infectious hypodermal and haematopoietic necrosis virus (IHHNV) in the freshwater crayfish Procambarus clarkii.

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) infects many crustacean hosts, including cultured penaeid shrimp. In the present study, we aimed to develop a novel sensitive SYBR Green-based real-time PCR method to specifically amplify DNA fragments of IHHNV. Our newly developed real-time PCR method with a 195-bp amplicon specifically detected IHHNV and showed no cross reaction with white spot syndrome virus (WSSV), hepatopancreatic parvovirus (HPV), Enterocytozoon hepatopenaei (EHP), infectious myonecrosis virus (IMNV) and yellow-head virus (YHV). This method could detect as low as one single copy of IHHNV plasmid DNA, more sensitive than other SYBR Green-based real-time PCR methods and less expensive and more convenient than the TaqMan probe-based real-time PCR. Moreover, our data using the newly designed method showed that 80% of IHHNV-fed Procambarus clarkii samples were IHHNV positive. Our findings further confirmed that P. clarkii can be infected by IHHNV.

The involvement of osteopontin and matrix metalloproteinase- 9 in the migration of endometrial epithelial cells in patients with endometriosis.

BACKGROUND: Endometriosis, which shares certain characteristics with cancers, may cause abnormal expression of proteins involved in cell migration. Endometrial epithelial cells (EECs) are believed to play an important role in endometriotic migration. The aim of this study was to investigate the relationship between the expression of osteopontin (OPN) and matrix metalloproteinase-9 (MMP-9) in endometriotic migration. METHODS: We performed primary culture of EECs and investigated the expression of OPN and MMP-9 in EECs regulated by 17beta-estradiol (E2). OPN-specific siRNA interference was used to down-regulate OPN and to explore the corresponding change in MMP-9 expression. Real-time RT-PCR, western blot analysis and flow cytometry were used to determine the expression levels of OPN and MMP-9. Gelatin zymography was performed to observe the enzymatic activity of MMP-9 in conditioned media. Transwell and wound scratch assays were performed to investigate the migration ability of EECs. RESULTS: The expression levels of OPN and MMP-9 in normal EECs (NEECs) were inferior to those in EECs from patients with endometriosis (EEECs). The expression levels of OPN and MMP-9 from stage III/IV EEECs and secretory-phase EECs were higher than those of stage I/II EEECs or proliferative-phase EECs. The expression levels of OPN and MMP-9 in EEECs were increased by E2 treatment and remarkably decreased by siRNA interference. Active MMP-9 expression increased with E2 treatment and decreased with siRNA treatment in EEECs compared with the same treatments in NEECs. The migratory abilities of EEECs were enhanced after cells were treated with E2; in contrast, these abilities were reduced by siRNA interference. In NEECs, active MMP-9 and cellular migration abilities were only minimally influenced by E2 and siRNA treatment. CONCLUSIONS: The present study suggests that the up-regulation of MMP-9 via activation of OPN induced by estrogen may correlate with the migration of endometrial epithelial cells in patients with endometriosis.

Quantification of derivatized phenylalanine and tyrosine in dried blood spots using liquid chromatography with tandem spectrometry for newborn screening of phenylketonuria.

Phenylketonuria (PKU) is an autosomal genetic disorder caused by a deficiency of the phenylalanine hydroxylase (PAH) enzyme. The lack of PAH results in the inability of phenylalanine (PHE) to transform into tyrosine (TYR). Consequently, this leads to the accumulation of PHE in the blood samples of newborns causing metabolic diseases such as irreversible neurological problems. An analysis was required for determining the values of PHE and TYR in blood samples from newborn babies. In this study, therefore, we developed a derivatized method to monitor PHE and TYR in plasma samples using liquid phase chromatography linked with quadrupole mass spectrometry. Accessible formaldehyde isotopes and cyanoborohydride were used to react with PHE and TYR amino groups to generate h2-formaldehyde-modified PHE and TYR (as standards) and d2-formaldehyde-modified PHE and TYR (as internal standards). We used tandem mass spectrometry for multiple reaction monitoring. We demonstrated a derivatized method suitable for the PKU screening of newborns. The recoveries for PHE and TYR were 85% and 90%, respectively. Furthermore, we compared the values of PHE and TYR in different human plasma sample storage methods, including direct plasma and dried blood spots, and the results showed no significant difference.

Circ_0075960 targets the miR-202-5p/CTNND1 axis to promote the growth and migration of endometrial carcinoma cells via regulating Wnt/ß-catenin signaling activity.

BACKGROUND: Endometrial carcinoma (EC) is one of the most common malignant tumors of the female reproductive tract, involving multiple molecular alterations. Circular RNA (circRNA) dysregulation is frequently observed in EC tissues, suggesting the involvement of circRNA in EC development. We aimed to investigate the role of circ_0075960 in EC. METHODS: Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot assays were applied for expression analysis. CCK-8, EdU, colony formation, flow cytometry and wound healing assays were employed for functional analysis. The predicted binding relationship between miR-202-5p and circ_0075960 or CTNND1 was validated by dual-luciferase reporter experiment. In vivo animal models were constructed in nude mice to verify the role of circ_0075960 in tumor growth. RESULTS: Circ_0075960 and CTNND1 were upregulated, while miR-202-5p was downregulated in EC. Knockdown of circ_0075960 induced EC cell apoptosis, suppressed cell proliferation and migration, and repressed tumor growth in animal models. MiR-202-5p was targeted by circ_0075960 and it directly bound to CTNND1 3'UTR. The inhibition of circ_0075960 knockdown or miR-202-5p enrichment on EC cell proliferation and migration was reversed by miR-202-5p depletion or CTNND1 overexpression, respectively. Circ_0075960 targeted miR-202-5p to positively regulate CTNND1 expression. Moreover, circ_0075960 knockdown weakened the activity of Wnt/ß-catenin signaling via targeting the miR-202-5p/CTNND1 axis. CONCLUSION: Circ_0075960 targets the miR-202-5p/CTNND1 axis to modulate Wnt/ß-catenin signaling activity, thus contributing to the malignant development of EC.

Molecular Identification and Antibacterial Activity Analysis of Blue Fox (Vulpes lagopus) ß-Defensins 108 and 122.

The blue fox (Vulpes lagopus), a fur-bearing animal, is an important component of the breeding industry in China. Semen quality is a key factor for the reproductive process and the breeding effectiveness of the farmed blue fox. However, bacterial contamination in semen samples utilized in artificial fertilization is very common. The ß-defensins, a class of important antimicrobial peptides in mammals, could protect the reproductive system of male animals from bacterial invasion, maintain the stability of the genital tract microenvironment and improve semen quality. In this study, molecular cloning and bioinformatics analysis were conducted to analyze the protein structure and function of blue fox ß-defensin 108 (Vulpes lagopus beta-defensin 108, vBD108) and 122 (Vulpes lagopus beta-defensin 122, vBD122). To evaluate the bacteriostatic effect of recombinant vBDs (Vulpes lagopus beta-defensins) protein, varying concentrations (0, 25, 50, 100, 200 µg/mL) were taken to evaluate the effects on Escherichia coli and Staphylococcus aureus at different times (0, 2, 4, 6, 8, 12 h). The results showed that vBD108 and vBD122 existed in different forms in protein structure and had antibacterial activity. Both proteins, at 50 µg/mL, had efficacious bacteriostatic activity. This study shows that recombinant vBD108 and vBD122 proteins have good antibacterial activity in vitro. This implies a potential role in improving semen quality and hygienic measures in the process of artificial insemination as an extender of semen dilution with antibacterial activity.

[Land use pattern change in Ejin Delta of Northwest China during 1930-2010].

The land use and landscape pattern in the lower reaches of the arid inland river basin is meaningful to water resource allocation. Based on the land use data in 1930, 1961, 1990, 2000, 2010, the purpose of this study was to quantitatively analyze the change of landscape pattern in the Ejin Delta in the lower reaches of the Heihe River Basin, a typical inland river basin in Northwest China. The results showed that the desert area accounted for 73.4% of the total research area in 2010, and the grassland 20.8%. During the past 80 years, the grassland, farmland and construction land increased. The transformation of land use types were characterized by switching to farmland and construction land. The fragmentation and. diversity of the landscape increased, while the dominance of the landscape decreased. The landscape pattern obviously lied on the water resource and had regional diversity. Land use changes tended to make the landscape well-distributed, diverse and fragmentized. At last, the driving factors and ecological environment effects of land use change were discussed. In a word, to ensure harmonious development between human and eco-hydrology, suggestions such as planning ecological resettlement, limiting farmland area, developing precision agriculture and increasing the proportion of ecological water use should be put forward.

IgG expression in trophoblasts derived from placenta and gestational trophoblastic disease and its role in regulating invasion.

Immunoglobulin G (IgG) is an important humoral immune factor, which plays a role in innate immunity of the fetus. IgG immunoreactivity was often seen in trophoblasts of placenta. Traditionally, IgG in trophoblasts was believed to be transported from the maternal blood through neonatal Fc receptor (FcRn). Here, we explored the phenomenon of IgG expression and its role in regulating invasion in trophoblasts derived from normal placenta and gestational trophoblastic disease (GTD). IgG expression was detected with an emphasis on mRNA transcripts by using reverse transcription-polymerase chain reaction and hybridization in situ, besides evaluated at the protein level with immunohistochemistry and immunofluorescence. The migration and attachment of normal trophoblast cell line (TEV-1) and choriocarcinoma cell line (JAR) were inhibited with down-regulation of IgG expression. Methotrexate promoted the differentiation of JAR cell line; however, it had little effect on the differentiation of TEV-1 cell line. IgG expression, migration, and attachment of JAR and TEV-1 cell lines were decreased in the presence of methotrexate. Furthermore, statistical analysis showed that the differences in migration and attachment were significant (P < 0.05) for JAR cell line, while no significant difference was found for TEV-1 cell line. Collectively, these results confirmed that with the progression from normal placenta to GTD, the expression of IgG was increased in trophoblasts, which might actively promote the migration and attachment of trophoblasts as an important regulating factor.