Transcriptomics integrated with metabolomics to characterize key pigment compounds and genes related to anthocyanin biosynthesis in Zanthoxylum bungeanum peel.

Zanthoxylum bungeanum is an important condiment with high economic value and its peel color is one of the main quality indexes. However, the key pigment compounds and related genes are still unclear affecting the quality control of the plants. In this study, the contents of four types of pigments were measured in Z. bungeanum and flavonoids were identified as the most important pigments. Based on the targeted flavonoid metabolomics of Z. bungeanum peels, 14 key pigment compounds were screened out from 152 flavonoids, among which cyanidin-3-O-rutinoside and cyanidin-3-O-glucoside were the most critical compounds for peel color. They were further verified to be present in nine varieties of Z. bungeanum by HPLC fingerprints. The 14 compounds were all associated with flavonoid and anthocyanin biosynthesis pathways and the 39 differentially expressed genes related to these pathways were annotated and screened based on transcriptomics. The genes ZbDFR, ZbANS, and ZbUFGT were identified as three key genes for anthocyanin synthesis in Z. bungeanum peels. Further qRT-PCR results confirmed the reliability of transcriptomics and the accuracy of gene screening. Subsequent protein induced expression demonstrated that ZbANS and ZbUFGT were expressed after 12 h induced by IPTG while ZbDFR was expressed after 15 h. Further transient and stable transformation analysis confirmed that both anthocyanin content and the expression of ZbDFR were significantly increased in overexpression Z. bungeanum leaves and Nicotiana benthamiana. The functional effect of stable transformation of ZbDFR was more significant than that of transient transformation with a 7.67-fold/1.49-fold difference in total anthocyanin content and a 42.37-fold/12.32-fold difference in the expression of ZbDFR. This study provides new insights into the chemical composition and the molecular mechanisms of Z. bungeanum peel color and lays an effective foundation for the color quality control, multi-purpose utilization of Z. bungeanum and the creation of new germplasm.

Clinical application of enhanced recovery after surgery concept in laparoscopic treatment of pediatric acute appendicitis.

BACKGROUND: This study assesses whether enhanced recovery after surgery (ERAS) is beneficial in treating acute appendicitis in pediatrics by laparoscopic techniques. METHOD: The children with acute appendicitis (n = 116) were divided into the ERAS group (n = 54) and the control group (n = 62). Then the preoperative data, intraoperative observation indexes, and postoperative data were analyzed. RESULTS: There was no significant difference in preoperative data and intraoperative observation indexes between the two groups. C-reactive protein (CRP) and white blood cell (WBC) in the ERAS group were significantly lower than those in the control group 3 days after the operation. Moreover, no significant difference in the visual analog score (VAS) between the two groups 3 days after the operation, but the other postoperative observation indexes in the ERAS group were significantly better than those in the control group. Nausea and vomiting in the ERAS group were significantly lower than those in the control group, with no significant difference in other complications between the two groups. CONCLUSION: ERAS could improve children's comfort, reduce some postoperative complications, reduce hospitalization expenses, and speed up recovery from acute appendicitis treated by laparoscopy. Therefore, it has clinical application value.

ZbMYB111 Expression Positively Regulates ZbUFGT-Mediated Anthocyanin Biosynthesis in Zanthoxylum bungeanum with the Involvement of ZbbHLH2.

Anthocyanin (ACN)-derived pigmentation in the red Zanthoxylum bungeanum peel is an essential commercial trait. Therefore, exploring the metabolic regulatory networks involved in peel ACN levels in this species is crucial for improving its quality. However, its underlying transcriptional regulatory mechanisms are still unknown. This transcriptomic and bioinformatics study not only discovered a new TF (ZbMYB111) as a potential regulator for ACN biosynthesis in Z. bungeanum peel, but also deciphered the underlying molecular mechanisms of ACN biosynthesis. Overexpression of ZbMYB111 and flavonoid 3-O-glucosyltransferase (ZbUFGT) induced ACN accumulation in both Z. bungeanum peels and callus along with Arabidopsis thaliana and tobacco flowers, whereas their silencing impaired ACN biosynthesis. Therefore, the dual-luciferase reporter, yeast-one-hybrid, and electrophoretic mobility shift assays showed that ZbMYB111 directly interacted with the ZbUFGT promoter to activate its expression. This diverted the secondary metabolism toward the ACN pathway, thereby promoting ACN accumulation.

Efficient preparation of hyperoside and quercitrin from Zanthoxylum bungeanum Maxim leaves using an integrated surfactant-based aqueous two-phase system, back-extraction and adsorption separation.

Zanthoxylum bungeanum leaves (ZBL) are of great medicinal value for being rich in hyperoside and quercitrin. In this study, a novel, efficient and economical continuous process was established. First, aqueous two-phase system (ATPS) composed of Triton X-100/(NH4)2SO4 was employed to enrich hyperoside and quercitrin from ZBL extracts and the recoveries reached 98.53% and 99.12%. Then back-extraction with dichloromethane-water system was adopted to separate hyperoside and quercitrin from Triton X-100 micelles which were recycled and the recoveries reached 86.58% and 85.19%. Finally, S-8 macroporous resin was used for removing the salt introduced in ATPS and the final recoveries reached 82.38% and 81.81%, much higher than the total flavonoids recovery as 69.08%. Furthermore, scale-up experiment certified that the continuous process was feasible for industrial production. Efficiently and economically, this method achieved a great breakthrough in purity and provided a novel reference for further purification and phase-forming component recycle.

Testicular ectopia: A case report.

This paper mainly describes three cases of children with ectopic testis, of which two patients with transverse testicular ectopia (TTE) and one with perineal ectopic testis (PET). All patients who underwent orchidopexy at the same pediatric surgical unit in the Affiliated Hospital of Jining Medical University (Jining, China) between June 2010 and February 2021 were retrospectively evaluated (age range, 14-34 months). A total of two patients (67%) was admitted with asymptomatic unilateral inguinal masses and the contralateral testis missing; the first patient was diagnosed with TTE intraoperatively, whereas the other patient was diagnosed with TTE through physical examination and ultrasound preoperatively. The third patient (33%) was admitted with the right testis missing and a left perineal mass, which was confirmed using PET by physical and ultrasound examination before the operation. The first two patients underwent transseptal orchidopexy, whereas the third patient underwent simple orchidopexy. Postoperative complications were not observed (follow-up, 10-24 months). The low incidence and poor understanding of ectopic testis compels us to report our findings and further discuss this particular disease of testicular ectopia, including its pathogenesis, diagnostic and treatment options.

Functional validation of ZbFAD2 and ZbFAD3 in the alkylamide biosynthesis pathway from Zanthoxylum bungeanum Maxim.

The spicy taste and medicinal properties of Zanthoxylum bungeanum are imparted by several alkylamides. Although most studies have focused on their isolation and identification, few have reported their biosynthesis pathways. Among the differentially expressed genes (DEGs) reported in the numerous varieties of Z. bungeanum, some might contribute to alkylamide biosynthesis. However, they are not yet functionally validated. The present study explored the function of two genes, ZbFAD2 and ZbFAD3, in the alkylamide biosynthesis pathway, and their stable and transient expression in Arabidopsis thaliana and Nicotiana benthamiana were also analyzed. As compared with the wild-type (WT), the fatty acid content analysis indicated that ZbFAD2-A. thaliana transgenic seeds had lower oleic acid and higher linoleic acid contents, while the ZbFAD3-A. thaliana transgenic seeds showed lower linoleic acid and higher α-linolenic acid levels. Moreover, hydroxy-α-sanshool, a major alkylamide, was considerably higher in the ZbFAD2-N. benthamiana transgenic plants (0.2167 ± 0.0026 mg/g) than in the WT (0.0875 ± 0.0049 mg/g), while it was lower in the ZbFAD3-N. benthamiana transgenic plants (0.0535 ± 0.0037 mg/g). These results suggest that both ZbFAD2 and ZbFAD3 are vital alkylamide biosynthesis enzymes in Z. bungeanum. Our study not only helps to scale up the alkylamide production, but also establishes the role of the uncharacterized genes.

Lentivirus-mediated siRNA knockdown of SPHK1 inhibits proliferation and tumorigenesis of neuroblastoma.

BACKGROUND: The overexpression of sphingosine kinase 1 (SPHK1) is responsible for the progress of many cancers. However, the role of SPHK1 in the development and progression of neuroblastoma (NB) remain largely unknown. Here in this study, we explored whether silencing SPHK1 by lentivirus-mediated siRNA could be employed as a potential therapeutic target for NB. MATERIALS AND METHODS: Lentivirus was adopted to load SPHK1 siRNA. The results were obtained using RT-qPCR, Western blot, cell proliferation assay, transwell cell migration/invasion assays as well as in vivo xenograft tumor models in nude mice. RESULTS: Our results demonstrated that SPHK1 mRNA was upregulated in SH-SY5Y and SK-N-SH cells as well as in human NB tissues. SPHK1 knockdown by siRNA resulted in impaired proliferation, increased apoptosis, as well as impaired migration and invasion of SH-SY5Y and SK-N-SH cells. In addition, the in vivo study suggested that SPHK1 knockdown significantly reduced the tumorigenesis of SH-SY5Y xenograft model. Furthermore, intratumorally administered lentivirus-SPHK1 siRNA could significantly inhibit tumor growth in an SH-SY5Y xenograft mice model. Intensive investigations on mechanism revealed that these effects were achieved through the deactivation of STAT3 pathways. CONCLUSION: These data suggest that SPHK1 inhibition via downregulation of STAT3 pathways by lentivirus-mediated siRNA knockdown can significantly suppress NB progression, which could be a promising target for future gene therapy of NB.

MiR-374a promotes the proliferation of human osteosarcoma by downregulating FOXO1 expression.

MiRNAs play crucial roles in development of cancer. However, the underlying mechanisms of miRNAs in osteosarcoma (OS) are poorly understood. In the present study, we reported that the expression of miR-374a was markedly upregulated in OS tissues and OS cells compared with the matched adjacent normal tissues and human osteoclast h-FOB cell lines. Overexpression of miR-374a promoted the proliferation and anchorage-independent growth of OS cells, whereas inhibition of miR-374a showed opposite effect. Furthermore, we identified that FOXO1 is the functional target of miR-374a. MiR-374a-induced proliferation was correlated with FOXO1, upregulating of the cell cycle regulator cyclin D1 and downregulating of cyclin-dependent kinase inhibitors p27. In functional assays, FOXO1 downregulation is required for miR-374a-induced OS cell proliferation. In sum, our data provide compelling evidence that a novel mechanism of FOXO1 suppression mediated by miR-374a in OS.