The high-quality genome of Grona styracifolia uncovers the genomic mechanism of high levels of schaftoside, a promising drug candidate for treatment of COVID-19.

Recent study has evidenced that traditional Chinese medicinal (TCM) plant-derived schaftoside shows promise as a potential drug candidate for COVID-19 treatment. However, the biosynthetic pathway of schaftoside in TCM plants remains unknown. In this study, the genome of the TCM herb Grona styracifolia (Osbeck) H.Ohashi & K.Ohashi (GSO), which is rich in schaftoside, was sequenced, and a high-quality assembly of GSO genome was obtained. Our findings revealed that GSO did not undergo recent whole genome duplication (WGD) but shared an ancestral papilionoid polyploidy event, leading to the gene expansion of chalcone synthase (CHS) and isoflavone 2'-hydroxylase (HIDH). Furthermore, GSO-specific tandem gene duplication resulted in the gene expansion of C-glucosyltransferase (CGT). Integrative analysis of the metabolome and transcriptome identified 13 CGTs and eight HIDHs involved in the biosynthetic pathway of schaftoside. Functional studies indicated that CGTs and HIDHs identified here are bona fide responsible for the biosynthesis of schaftoside in GSO, as confirmed through hairy root transgenic system and in vitro enzyme activity assay. Taken together, the ancestral papilionoid polyploidy event expanding CHSs and HIDHs, along with the GSO-specific tandem duplication of CGT, contributes, partially if not completely, to the robust biosynthesis of schaftoside in GSO. These findings provide insights into the genomic mechanisms underlying the abundant biosynthesis of schaftoside in GSO, highlighting the potential of GSO as a source of bioactive compounds for pharmaceutical development.

Selection and validation of reference genes for RT-qPCR analysis in Desmodium styracifolium Merr.

Gene expression valuated by reverse transcription-quantitative PCR (RT-qPCR) are often applied to study the gene function. To obtain accurate and reliable results, the usage of stable reference genes is essential for RT-qPCR analysis. The traditional southern Chinese medicinal herb, Desmodium styracifolium Merr is well known for its remarkable effect on the treatment of urination disturbance, urolithiasis, edema and jaundice. However, there are no ready-made reference genes identified for D. styracifolium. In this study, 13 novel genes retrieved from transcriptome datasets of four different tissues were reported according to the coefficient of variation (CV) and maximum fold change (MFC) of gene expression. The expression stability of currently used Leguminosae ACT6 was compared to the 13 candidate reference genes in different tissues and 7-day-old seedlings under different experimental conditions, which was evaluated by five statistical algorithms (geNorm/NormFinder/BestKeeper/ΔCT/RefFinder). Our results indicated that the reference gene combinations of PP + UFM1, CCRP4 + BRM and NFD6 + NCLN1 were the most stable reference genes in leaf, stem and root tissues, respectively. The most stable reference gene combination for all tissues was CCRP4 + CUL1. In addition, the most stable reference genes for different experimental conditions were distinct, for instance SMUP1 for MeJA treatment, ERDJ2A + SMUP1 for SA treatment, NCLN1 + ERDJ2A for ABA treatment and SF3B + VAMP721d for salt stress, respectively. Our results lay a foundation for achieving accurate and reliable RT-qPCR results so as to correctly understand the function of genes in D. styracifolium. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02954-x.

Cep63 knockout inhibits the malignant phenotypes of papillary thyroid cancer cell line TPC­1.

The present study was designed to observe the expression of the centrosomal protein 63 in papillary thyroid cancer (PTC) tissues and cells and to explore the clinical significance of Cep63 expression in PTC. Primary PTC tissues and matched normal thyroid tissues were collected, and the Cep63 expression level was determined by reverse transcription­quantitative PCR and western blotting. A stable Cep63­knockout cell line was constructed to assess the proliferation, invasion, migration and apoptosis abilities in vitro. A subcutaneous tumorigenesis model was established in nude mice to evaluate the effect of Cep63 on tumor growth and proliferation in vivo. Western blotting was used to explore the relevant signaling pathways. The results revealed that the expression level of Cep63 in PTC tissues was significantly increased. The proliferation, invasion and migration abilities of TPC­1 cells were decreased after Cep63 knockout, and silencing of Cep63 resulted in TPC­1 cell cycle arrest in the S phase. Mechanistically, Cep63 knockout inhibited the activation of the Janus kinase/signal transducer and activator of transcription 3 signaling pathway. In conclusion, Cep63 knockout significantly inhibited biological functions of TPC­1 cells in vitro and in vivo, indicating that Cep63 may be an important oncogene of PTC.

SQSTM1/p62 Promotes Cell Growth and Triggers Autophagy in Papillary Thyroid Cancer by Regulating the AKT/AMPK/mTOR Signaling Pathway.

BACKGROUND: Thyroid cancer is one of the most common endocrine malignancies worldwide, and papillary thyroid cancer (PTC) is the most common pathologic type of thyroid cancer. SQSTM1/p62 activity mediates different biological functions. This study aimed to investigate the effect of SQSTM1/p62, a multifunctional receptor, on biological function and autophagy characteristics in the human PTC cell line TPC-1. METHODS: A total of 105 primary PTC samples and matched adjacent normal thyroid tissue samples were obtained to evaluate the expression of p62 in clinical patients. A similar p62 expression pattern was found in PTC cell lines and normal human thyroid follicular epithelial cells. To evaluate the effect of SQSTM1/p62 on TPC-1 cells, we constructed the p62 knockout cell line p62-KO-TPC-1. Cell proliferation, cell cycle, and cell apoptosis were analyzed by colony formation tests, Cell Counting Kit-8 (CCK-8) assays and flow cytometry in vitro. TPC-1 and p62-KO-TPC-1 human PTC cell lines in the logarithmic growth phase were subcutaneously implanted into BALB/c nude mice to verify their proliferation effect in vivo. Furthermore, western blotting and immunohistochemistry (IHC) were used to detect the expression of AKT/AMPK/mTOR signaling pathway-related proteins. RESULTS: Overall, p62 expression was higher in tumor tissues than in normal tissues in 73 of 105 PTC patients (69.5%). The expression level of p62 in the PTC cell line was higher than that in the normal thyroid cell line. Our data indicated that in vitro, p62 deficiency could decrease the number of colonies, inhibit cell growth and the cell cycle, and induce apoptosis. Tumor xenograft experiments in BALB/c nude mice corroborated these findings. Moreover, the molecular mechanism was explored by western blotting, and we found that the AMPK/AKT/mTOR pathway was involved. CONCLUSIONS: The results indicate that p62 might mediate cell autophagy and apoptosis in TPC-1 cells via the AMPK/AKT/mTOR pathway and could be used as a potential therapeutic approach for PTC.

The Antitumour Effect of Prunella vulgaris Extract on Thyroid Cancer Cells In Vitro and In Vivo.

Prunella vulgaris, a traditional Chinese medicine, has been used to treat various benign and malignant tumours for centuries in China. In our previous studies, Prunella vulgaris extract (PVE) was shown to promote apoptosis in differentiated thyroid cancer (DTC) cells. However, whether other mechanisms are involved in the antitumour effect of PVE in thyroid cancer (TC) cells remains unclear. The present study aimed to investigate the antiproliferative and antimigratory effects of PVE on TC cell lines both in vitro and in vivo. First, the TPC-1 and SW579 human TC cell lines were screened by MTT assay for their high level of sensitivity to PVE. Then, the results of cell growth curve and colony formation assay and cell cycle analyses, wound healing, and migration assays demonstrated that PVE inhibited the proliferation and migration of TPC-1 and SW579 cells. Moreover, the antitumour effect of PVE was verified in a subcutaneous xenotransplanted tumour model. Next, MKI67, PCNA, CTNNB1, and CDH1 were screened by qRT-PCR for their significantly differential expression levels in xenograft tissue with and without PVE treatment, and expression of MKI67, PCNA, and CDH1 was verified by Western blot. Finally, an integrated bioinformatics analysis containing protein-protein interaction network, KEGG pathway, and GO analysis was conducted to explore more potential antitumour mechanisms of PVE. In summary, PVE could inhibit the proliferation and migration of TC cells both in vitro and in vivo, which may have been achieved by modulation of the expression of MKI67, PCNA, and CDH1. These data suggest that PVE has the potential to be developed into a new anticancer drug for the treatment of TC.

Generation and identification of a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes.

Coiled-coil domain containing 67 (CCDC67) gene is a tumor suppressor gene that exhibits a significant inhibitory effect on a variety of tumors. Our previous study demonstrated that the upregulation of CCDC67 gene in TPC-1 cells inhibited cell proliferation, migration and invasion, and promoted apoptosis in vitro. However, due to the lack of a suitable cell tool, these results were not validated in vivo. In the present study, a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes was generated and identified. Firstly, cDNA clones of the CCDC67 gene were obtained by reverse transcription using a custom-designed primer. The results of subsequent electrophoresis analysis and sequencing revealed that the cDNA clones of CCDC67 gene were obtained successfully, with a length of 1,862 bp. The lentiviral vectors, containing the CCDC67, luciferase reporter and puromycin acetyltransferase genes, were co-transfected with two plasmids that encode lentiviral structural proteins and envelope proteins into 293T cells. Following ultracentrifugation, the titer of lentivirus was determined by ELISA to be 5.0×108 TU/ml. The constructed lentiviral vector was used to transfect TPC-1 thyroid cancer cells, and stabilization was achieved by puromycin screening. The expression of CCDC67 gene, luciferase activity and tumorigenic ability of the generated cell line were detected. Reverse transcription-qPCR results demonstrated that the expression levels of CCDC67 gene in TPC-1 cells following transfection were increased 194,46.782-fold compared with those in the negative control group (P<0.01). A higher fluorescence intensity was detected in the generated cell line, while no detectable fluorescence was observed in untransfected TPC-1 cells. The tumorigenic ability of TPC-1-Luc-Puromycin-CCDC67 cells was verified by bioluminescence imaging and histopathological analysis using a pulmonary metastasis model. These results demonstrated that a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes was generated successfully. The TPC-1-Luc-Puromycin-CCDC67 cell line may be a helpful tool for further research on CCDC67 in vivo.

Subcellular distribution of fluoranthene in Chlorella vulgaris with the presence of cetyltrimethylammonium chloride.

This study explored the possible mechanism of the joint toxicity of binary mixtures of cetyltrimethylammonium chloride (CTAC) and fluoranthene (Flu) to the green alga Chlorella vulgaris by examining the subcellular distribution of Flu within the alga. The joint action of CTAC (100 µg L(-1)) and Flu (0-200 µg L(-1)) on the algae changed from a synergetic effect (0-50 µg L(-1)) to an antagonistic effect (50-200 µg L(-1)) with an increase of the Flu concentration. The Flu uptake was enhanced by the presence of CTAC through the intracellular detection of Flu. Furthermore, the highest amount of Flu bound to the cytosol, whereas the least amount bound to the cellular debris when synergistic effect was observed at 2.5 µg L(-1) Flu. However, the highest amount of Flu bound to the cellular debris, whereas the least amount bound to the organelles when antagonistic effect was displayed at 200 µg L(-1) Flu. The different subcellular distribution of Flu may affect the uptake of the highly toxic CTAC by the algae in the binary mixture, and consequently lead to a different level of CTAC toxicity. The abovementioned results indicate that the subcellular distribution of chemicals can be used to elucidate possible mechanisms for the joint toxicity of their binary mixtures to aquatic organisms.

Joint action of binary mixtures of cetyltrimethyl ammonium chloride and aromatic hydrocarbons on Chlorella vulgaris.

The joint action of binary mixtures of cetyltrimethyl ammonium chloride (CTAC), a cationic surfactant, and six aromatic hydrocarbons (AHs) on green algae Chlorella vulgaris was investigated. In single systems, inhibition efficiency of CTAC on the growth of algae was much higher than that of AHs (benzene, toluene, phenol, nitrobenzene, phenanthrene and fluoranthene). In combined systems, the toxicity of CTAC was enhanced by low concentrations of AHs. 96 h EC(50) value of CTAC varied from 145±13.35-56±8.27 to 56±8.27-226±8.22 µg/L when exposed to 0-1.13 and 1.13-100.84 µg/L fluoranthene, respectively. Zeta potential of algae initially increased and then decreased with the increase of fluoranthene concentration, whereas residual CTAC concentration displayed an opposite trend in the combined system. These results of this investigation showed that fluoranthene influenced the sorption of CTAC by C. vulgaris. The above results indicated that cationic surfactants and AHs have synergetic toxic effects on aquatic biota.

[Anesthetic management for cesarean section involving placenta previa].

BACKGROUND: There are few consistent anesthetic guidelines how to manage cesarean section in the presence of placenta previa. Main problem may be hemorrhage, as occasionary unexpected massive bleeding leads to life-threatening hemorrhage. METHODS: We investigated retrospectively, covering the period between April 1, 2001 and September 30, 2005, 30 women with placenta previa who had undergone cesarean section. RESULTS: Comparing general anesthesia with regional anesthesia, there was not a significant difference between the two. Comparing totalis (T) with partial (P) in the classification of placenta previa, infusion and hemorrhage in T group were more pronounced than those in the P group. Regarding these operations performed during the weekend or at night, shortage of supportive anesthesiologist was pointed out. CONCLUSIONS: These results indicate that regional and general anesthesia did not differ in the intraoperative incidence. In all cases at least two anesthesiologists and at least two venous lines are necessary to manage cesarean section in the presence of placenta previa.