The Fujian oyster ( Crassostrea angulate ) is an important marine bivalve mollusk with high economic value. Gene function research and gene editing techniques have broad application prospects in oyster. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely used for genome engineering in many species. CRISPR-mediated gene editing has also been used successfully in the Pacific oyster through direct delivery of the CRISPR/Cas9 components into oyster embryos by microinjection. However, the low throughput and operational difficulties associated with microinjection is one of the factors limiting the widespread application of CRISPR/Cas9 in oysters. In this study, we attempted to deliver the CRISPR/Cas9-system into the embryos of C. angulate by electroporation. An all-in-one CRISPR/Cas9 vector plasmid was used as CRISPR/Cas9 system in this study. Electroporation was carried out using both eggs and blastula larvae. A large number of larvae became malformed or die after electroporation. A single base substitution mutation was detected in the D-larvae developed from electroporated eggs. Our results demonstrate that the CRISPR/Cas9 system can be delivered into embryos of C. angulate for gene editing by electroporation, which provides a reference and will further contribute to the future application of electroporation in mollusks.
Retinoic acid (RA) signaling pathways mediated by RA receptors (RARs) are essential for many physiological processes such as organ development, regeneration, and differentiation in animals. Recent studies reveal that RARs identified in several mollusks, including Pacific oyster Crassostrea gigas , have a different function mechanism compared with that in chordates. In this report, we identified the molecular characteristics of CgRAR to further explore the mechanism of RAR in mollusks. RT-qPCR analysis shows that CgRAR has a higher expression level in the hemocytes and gonads, indicating that CgRAR may play roles in the processes of development and metabolism. The mRNA expression level of both CgRAR and CgRXR was analyzed by RT-qPCR after injection with RA. The elevated expression of CgRAR and CgRXR was detected upon all- trans -RA (ATRA) exposure. Finally, according to the results of Yeast Two-Hybrid assay and co-immunoprecipitation analysis, CgRAR and CgRXR can interact with each other through the C-terminal region. Taken together, our results suggest that CgRAR shows a higher expression level in gonads and hemocytes. ATRA exposure up-regulates the expression of CgRAR and CgRXR. Besides, CgRAR can interact with CgRXR to form a heterodimer complex.
A new gene, SG1, was identified in a slow-greening mutant (sg1) isolated from an ethylmethanesulphonate-mutagenized population of Arabidopsis thaliana. The newly formed leaves of sg1 were initially albino, but gradually became pale green. After 3 weeks, the leaves of the mutant were as green as those of the wild-type plants. Transmission electron microscopic observations revealed that the mutant displayed delayed proplastid to chloroplast transition. The results of map-based cloning showed that SG1 encodes a chloroplast-localized tetratricopeptide repeat-containing protein. Quantitative real-time reverse transcription–PCR data demonstrated the presence of SG1 gene expression in all tissues, particularly young green tissues. The sg1 mutation disrupted the expression levels of several genes associated with chloroplast development, photosynthesis, and chlorophyll biosynthesis. The results of genetic analysis indicated that gun1 and gun4 partially restored the expression patterns of the previously detected chloroplast-associated genes, thereby ameliorating the slow-greening phenotype of sg1. Taken together, the results suggest that the newly identified protein, SG1, is required for chloroplast development in Arabidopsis.
Microbial dysbiosis has an increasingly appreciated impact on carcinogenesis and cervicovaginal microbiome plays a critical role in microenvironmental inflammation. Here we investigated the involvement of the female genital tract microbiome in a gynaecological cancer. We transferred Peptostreptococcus species into the uteri of BALB/c nude mice and observed indoleacrylic acid (IAA) production. Intratumoral injection of conditioned medium from bacterial cultures into tumour-xenografted mice for 2 months promoted tumour growth. IAA upregulated IL-10 expression by M2 macrophages, which increased IFN-γ expression by CD8+ T cells. IFN-γ induced indoleamine-2,3-dioxygenase 1 (IDO1) expression in endometrial cancer (EC) cell line. Co-culture of IDO1-expressing EC cells with peripheral blood mononuclear cells upregulated the proportion of regulatory T cells and decreased the M1/M2 ratio. Importantly, P. anaerobius was more abundant amongst the uterine microbiota of clinical EC patient samples than control samples. IAA, IDO1, and the kynurenine/tryptophan ratio were all higher in EC tissue than in healthy tissue, and the M1/M2 ratio was lower. Thus, we conclude that the overabundance of uterine commensal Peptostreptococcus species in EC induces IDO1 expression by producing IAA. Our study sheds light on the link between IDO1 induction and uterine Peptostreptococcus dysbiosis.
Endothelial colony-forming cells (ECFCs) are progenitors of endothelial cells with significant proliferative and angiogenic ability. ECFCs are a promising treatment option for various diseases, such as ischemic heart disease and peripheral artery disease. However, some barriers hinder the clinical application of ECFC therapeutics. One of the current obstacles is that ECFCs are dysfunctional due to the underlying disease states. ECFCs exhibit dysfunctional phenotypes in pathologic states, which include but are not limited to the following: premature neonates and pregnancy-related diseases, diabetes mellitus, cancers, haematological system diseases, hypoxia, pulmonary arterial hypertension, coronary artery diseases, and other vascular diseases. Besides, ECFCs are heterogeneous among donors, tissue sources, and within cell subpopulations. Therefore, it is important to elucidate the underlying mechanisms of ECFC dysfunction and characterize their heterogeneity to enable clinical application. In this review, we summarize the current and potential application of transcriptomic analysis in the field of ECFC biology. Transcriptomic analysis is a powerful tool for exploring the key molecules and pathways involved in health and disease and can be used to characterize ECFC heterogeneity.
Tissue regeneration is a hot topic in the field of biomedical research in this century. Material composition, surface topology, light, ultrasonic, electric field and magnetic fields (MFs) all have important effects on the regeneration process. Among them, MFs can provide nearly non-invasive signal transmission within biological tissues, and magnetic materials can convert MFs into a series of signals related to biological processes, such as mechanical force, magnetic heat, drug release, etc. By adjusting the MFs and magnetic materials, desired cellular or molecular-level responses can be achieved to promote better tissue regeneration. This review summarizes the definition, classification and latest progress of MFs and magnetic materials in tissue engineering. It also explores the differences and potential applications of MFs in different tissue cells, aiming to connect the applications of magnetism in various subfields of tissue engineering and provide new insights for the use of magnetism in tissue regeneration.
Increasing evidence has suggested that discoidin domain receptor 2 (DDR2) plays an important role in cancer development and metastasis. However, the correlation between DDR2 expression and clinical outcome in ovarian cancer has not been investigated. In this study, DDR2 expression was examined by Real-time PCR in surgically resected ovarian cancer and normal ovary tissues. Besides, DDR2 expression was analyzed immunohistochemically in 103 ovarian cancer patients, and the correlation between DDR2 expression with clinicopathologic factors was analyzed. The result showed that DDR2 mRNA expression was upregulated in ovarian cancer tissues compared with normal ovary tissues. Statistical analysis revealed that DDR2 expression correlated with tumor stage (P = 0.008) and peritoneal metastasis (P = 0.009). Patients with high DDR2 expression showed poorer 5-year overall survival (P = 0.005), and DDR2 remained an independent prognostic marker for OS (P = 0.013) in multivariate analysis. Our results suggest that DDR2 might be closely associated with ovarian cancer progression and metastasis. Its high expression may serve as a potential prognostic biomarker in human ovarian cancer.
Ca2+‑activated Cl‑ channel A2 (CLCA2), a tumor suppressor, is associated with the development of several cancers. However, little is known about CLCA2 in human cervical cancer. Therefore, the aim of the present study was to investigate the effects of CLCA2 on cervical cancer. Reverse transcription‑quantitative (RT‑q)PCR was used to examine the mRNA expression levels of CLCA2 in eight pairs of cervical cancer tissues. Immunohistochemistry was used to investigate CLCA2 protein expression in 144 archived cervical cancer specimens. The association of the CLCA2 with clinicopathological parameters was statistically evaluated. Cell proliferation and invasion capability were examined by MTT and Transwell assays, respectively. RT‑qPCR analysis revealed that CLCA2 expression was decreased in cervical cancer compared with that in adjacent normal tissues. The expression levels of CLCA2 in patients were correlated with tumor stage (P=0.028), tumor size (P=0.009), and human papillomavirus (HPV) infection status (P=0.041). In addition, CLCA2 upregulation was associated with longer overall and recurrence‑free survival time after surgery (P=0.016 and P=0.009, respectively). Multivariate Cox regression analysis demonstrated that CLCA2 expression had a predictive value for overall survival of patients with cervical cancer (P=0.017 and P=0.025, respectively). Knockdown of CLCA2 by small interfering RNA suppressed tumor cell proliferation and migration. Mechanistically, CLCA2 was involved in Wnt/β‑catenin signaling. In conclusion, the results of the present study demonstrated that CLCA2 suppressed the proliferation, migration and invasion of cervical cancer cells, and that CLCA2 may be a potential therapeutic target of cervical cancer.
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