[Identification of genes involved in biosynthesis of paeoniflorin in Paeonia lactiflora based on transcriptome analysis].

Paeoniflorin, a representative pinane monoterpene glycoside, is the main active component and quality index of Paeoniae Radix Alba and Paeoniae Radix Rubra.The possible biosynthesis of paeoniflorin is as follows: GPP is derived from mevalonate(MVA) and/or 2-C-methyl-D-erythritol 4-phosphate(MEP) pathway(s) followed by the catalysis with terpene synthase, cytochrome P450(CYP450), UDP-glucuronosyltransferase(UGT), and acyltransferase(AT), respectively.This study aims to explore the genes rela-ted to the biosynthesis of paeoniflorin.To be specific, the cDNA libraries for flowers, leaves, and roots of Paeonia lactiflora were established and sequenced.A total of 30 609 open reading frames(ORFs) were yielded.Through functional annotation and expression analysis of all CYP450 genes in the transcriptome, 11 CYP450 genes belonging to CYP71 A and CYP71 D subfamilies and showing expression trend consistent with monoterpene synthase PlPIN that may be involved in paeoniflorin biosynthesis were screened out.Subsequently, 7 UGT genes and 9 AT genes demonstrating the expression trend consistent with PlPIN which were possibly involved in paeoniflorin biosynthesis were further screened by functional annotation analysis, full-length sequence analysis, expression analysis, and phylogeny analysis.This study provided a systematic screening method with smaller number of candidate genes, thus reducing the workload of functional gene verification.The result laid a foundation for analyzing the biosynthesis pathway of paeoniflorin and the formation mechanism.

Coinfection pulmonary mucormycosis and aspergillosis with disseminated mucormycosis involving gastrointestinalin in an acute B-lymphoblastic leukemia patient.

Pulmonary mucormycosis and aspergillosis with disseminated mucormycosis involving gastrointestinalin is a very rare but lethal infection leading to extreme mortality. Herein, we present a unique case of pulmonary coinfection with Cunninghamella bertholletiae and Aspergillus flavus, with disseminated mucormycosis involving the jejunum caused by C. bertholletiae in an acute B-lymphocytic leukemia (B-ALL) patient with familial diabetes. Early administration of active antifungal agents at optimal doses and complete resection of all infected tissues led to improved therapeutic outcomes.

Clinical application of combined detection of SARS-CoV-2-specific antibody and nucleic acid.

BACKGROUND: The global outbreak of human severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection represents an urgent need for readily available, accurate and rapid diagnostic tests. Nucleic acid testing of respiratory tract specimens for SARS-CoV-2 is the current gold standard for diagnosis of coronavirus disease 2019 (COVID-19). However, the diagnostic accuracy of reverse transcription polymerase chain reaction (RT-PCR) tests for detecting SARS-CoV-2 nucleic acid may be lower than optimal. The detection of SARS-CoV-2-specific antibodies should be used as a serological non-invasive tool for the diagnosis and management of SARS-CoV-2 infection. AIM: To investigate the diagnostic value of SARS-CoV-2 IgM/IgG and nucleic acid detection in COVID-19. METHODS: We retrospectively analyzed 652 suspected COVID-19 patients, and 206 non-COVID-19 patients in Wuhan Integrated TCM and Western Medicine Hospital. Data on SARS-CoV-2 nucleic acid tests and serum antibody tests were collected to investigate the diagnostic value of nucleic acid RT-PCR test kits and immunoglobulin (Ig)M/IgG antibody test kits. The χ2 test was used to compare differences between categorical variables. A 95% confidence interval (CI) was provided by the Wilson score method. All analyses were performed with IBM SPSS Statistics version 22.0 (IBM Corp., Armonk, NY, United States). RESULTS: Of the 652 suspected COVID-19 patients, 237 (36.3%) had positive nucleic acid tests, 311 (47.7%) were positive for IgM, and 592 (90.8%) were positive for IgG. There was a significant difference in the positive detection rate between the IgM and IgG test groups (P < 0.001). Using the RT-PCR results as a reference, the specificity, sensitivity, and accuracy of IgM/IgG combined tests for SARS-CoV-2 infection were 98.5%, 95.8%, and 97.1%, respectively. Of the 415 suspected COVID-19 patients with negative nucleic acid test results, 366 had positive IgM/IgG tests with a positive detection rate of 88.2%. CONCLUSION: Our data indicate that serological IgM/IgG antibody combined test had high sensitivity and specificity for the diagnosis of SARS-CoV-2 infection, and can be used in combination with RT-PCR for the diagnosis of SARS-CoV-2 infection.

Diversity of O-Glycosyltransferases Contributes to the Biosynthesis of Flavonoid and Triterpenoid Glycosides in Glycyrrhiza uralensis.

Licorice (Glycyrrhiza uralensis) is a popular medicinal plant containing more than 70 flavonoid and triterpenoid glycosides. Thus far, only a few reports are available on the glycosylation enzymes involved in their biosynthesis. In this work, we mined the transcriptome data of G. uralensis and discovered 43 candidate genes for O-glycosyltransferase (O-GT). Among them, 17 genes could be expressed in E. coli, and functions of the enzymes were analyzed by catalyzing eight native substrates. As a result, we characterized 11 O-GTs, including isoflavone 7-O-GTs, flavonol 3-O-GTs, and promiscuous O-GTs catalyzing flavones, chalcones, and triterpenoids. They could efficiently synthesize key licorice compounds such as liquiritin, isoliquiritin, ononin, and 3-O-ß-d-glucuronosyl glycyrrhetinic acid. The diversity of O-GTs contributes to the biosynthesis of various glycosides in licorice. These enzymes could also be used as biocatalytic tools to synthesize other bioactive O-glycosides.

Molecular and Structural Characterization of a Promiscuous C-Glycosyltransferase from Trollius chinensis.

Herein, the catalytic promiscuity of TcCGT1, a new C-glycosyltransferase (CGT) from the medicinal plant Trollius chinensis is explored. TcCGT1 could efficiently and regio-specifically catalyze the 8-C-glycosylation of 36 flavones and other flavonoids and could also catalyze the O-glycosylation of diverse phenolics. The crystal structure of TcCGT1 in complex with uridine diphosphate was determined at 1.85 Šresolution. Molecular docking revealed a new model for the catalytic mechanism of TcCGT1, which is initiated by the spontaneous deprotonation of the substrate. The spacious binding pocket explains the substrate promiscuity, and the binding pose of the substrate determines C- or O-glycosylation activity. Site-directed mutagenesis at two residues (I94E and G284K) switched C- to O-glycosylation. TcCGT1 is the first plant CGT with a crystal structure and the first flavone 8-C-glycosyltransferase described. This provides a basis for designing efficient glycosylation biocatalysts.

UGT73F17, a new glycosyltransferase from Glycyrrhiza uralensis, catalyzes the regiospecific glycosylation of pentacyclic triterpenoids.

The regiospecific glycosylation of pentacyclic triterpenoids by UGT73F17, a new glycosyltransferase from Glycyrrhiza uralensis, is highlighted. UGT73F17 exhibited strict substrate specificity toward the carboxyl group at C-30/C-29 of pentacyclic triterpenoids, and showed high promiscuity to sugar donors. UGT73F17 represents the first identified triterpenoid 30/29-O-glycosyltransferase, and could be used as an effective biocatalyst to synthesize glycosyl ester saponins.

A comprehensive review on phytochemistry, pharmacology, and flavonoid biosynthesis of Scutellaria baicalensis.

CONTEXT: Scutellaria baicalensis Georgi (Lamiaceae) is a popular medicinal plant. Its roots are used as the famous traditional Chinese medicine Huang-Qin, which is recorded in Chinese Pharmacopoeia, European Pharmacopoeia, and British Pharmacopoeia. OBJECTIVE: This review comprehensively summarizes research progress in phytochemistry, pharmacology, and flavonoid biosynthesis of S. baicalensis. METHODS: English and Chinese literature from 1973 to March 2018 was collected from databases including Web of Science, SciFinder, PubMed, Elsevier, Baidu Scholar (Chinese), and CNKI (Chinese). Scutellaria baicalensis, chemical constituents, phytochemistry, biological activities, and biosynthesis were used as the key words. RESULTS: A total of 126 small molecules (1-126) and 6 polysaccharides have been isolated from S. baicalensis. The small molecules can be classified into four structural types, namely, free flavonoids, flavonoid glycosides, phenylethanoid glycosides, and other small molecules. Extracts of S. baicalensis and its major chemical constituents have been reported to possess anti-viral, anti-tumor, anti-bacterial, antioxidant, anti-inflammatory, hepatoprotective, and neuroprotective activities. Key steps in the biosynthetic pathways of Scutellaria flavonoids have also been summarized. CONCLUSIONS: This article could be helpful for researchers who are interested in the chemical constituents, bioactivities, biosynthesis, and clinical applications of S. baicalensis.

Cyclic bisbibenzyls induce growth arrest and apoptosis of human prostate cancer PC3 cells.

AIM: To investigate the cytotoxic effects of four cyclic bisbibenzyls, Riccardin C (Ric), Pakyonol (Pak), Marchantin M (Mar), and Plagiochin E (Pla) against chemoresistant prostate cancer PC3 cells. METHODS: Cell growth was assayed by MTT method, and apoptotic related protein Bcl-2 and Bax, poly(ADP-ribose) polymerase (PARP) were examined by Western blotting. Cell cycle and apoptosis of PC3 cells were evaluated with flow cytometry and morphologic examinations. RESULTS: The four compounds inhibited proliferation and elicited cell death in a dose- and time-dependent manner with IC(50) values of 3.22 micromol/L for Ric, 7.98 micromol/L for Pak, 5.45 micromol/L for Mar, and 5.99 micromol/L for Pla, respectively. Furthermore, exposed to these chemicals caused a decrease in the antiapoptotic protein Bcl-2 and an increase in proapoptotic Bax expression. PARP cleavage and caspase-3 activity were also observed. CONCLUSION: The results suggest that cyclic bisbibenzyls could be used for the development of novel therapeutic chemicals against prostate cancer.

[Preparation and bioactivity evaluation of SA-mIL15 fusing protein].

AIM: To prepare and characterize streptavidin-tagged murine interleukin-15 fusion proteins. METHODS: pET24a-SA-L-mIL15 and pET21a-mIL15-L-SA plasmids were constructed and expressed in Rosetta (DE3) host bacteria to generate SA/mIL15 fusion proteins. SA-mIL15 fusion protein was purified through the Ni-NTA affinity chromatography, and mIL15-SA fusion protein through anion exchange chromatography, followed by refolding. The efficiency of surface modification of the fusion proteins on the biotinylated RM-1 tumor cells was evaluated by a flow cytometer. MTT method was used to evaluate the proliferating effect of SA/mIL15 fusion proteins on mouse spleen lymphocytes stimulated by ConA. RESULTS: Both SA-mIL15 and mIL15-SA fusion proteins were highly expressed in Rosetta (DE3) at about 20% of total bacterial proteins. They exhibited the bi-functionality: proliferation-promoting activity of mIL15 on mouse spleen lymphocytes with the specific activity of 1x10(6); IU/mg for SA-mIL15 or 2 x 10(5); IU/mg for mIL15-SA, and SA-mediated high-affinity binding to the biotinylated surfaces of RM-1 tumor cells with about 95% surface modification efficiency. CONCLUSION: SA/mIL15 bi-functional fusion proteins were generated, which made feasible the development of mIL15-surface-modified cancer cell vaccine.

Anti-inflammation role for mesenchymal stem cells transplantation in myocardial infarction.

The aim of the present study was to investigate the role of anti-inflammation for MSCs transplantation in rat models of myocardial infarction. Rats with AMI induced by occlusion of the left coronary artery were randomized to MSCs transplantation group, MI group and sham operated group. The effects of MSCs transplantation on cardiac inflammation and left ventricular remodeling in non-infarcted zone were observed after 4 weeks of MI. We found that MSC transplantation (1) decreased protein production and gene expression of inflammation cytokines TNF-alpha, IL-1beta and IL-6, (2) inhibited deposition of type I and III collagen, as well as gene and protein expression of MMP-1 and TIMP-1, (3) attenuated LV cavitary dilation and transmural infarct thinning, thus prevent myocardial remodeling after myocardial infarction, and (4) increased EF, FS, LVESP and dp/dtmax (P < 0.01), decreased LVDd, LVEDV, LVEDP (P < 0.05). Anti-inflammation role for MSCs transplantation might partly account for the cardiac protective effect in ischemic heart disease.

[Defense mechanisms of Taeniidae against host immune response].

The defense mechanisms of Taeniidae against host immune reaction were reviewed. The parasites may defend themselves from the host's immune attack by: (1)producing specific biochemicals as barriers against the damage caused by immune reactions, (2) changing surface antigens and secreting some active substances that interfere and deconstruct host's immune system and other hazards, (3) self-disguising through synthesizing homologies to host's substances in structure or function in order to avoid the immune surveillance of the host.

[Expression of Taenia solium oncosphere 45WB2 gene in Pichia pastoris system].

OBJECTIVE: To amplify and express the gene from Taenia solium oncosphere in Pichia pastoris system. METHODS: Total RNA was extracted from hatched and activated oncospheres. A 459bp specific fragment was amplified by RT-PCR. It was subcloned into the secreted expression vector pPIC9K, and identified by sequencing and then transformed into P. pastoris SMD1168 by electroporation. PCR analysis showed that 45WB2 was integrated into the genomic DNA of yeast. The recombinants were induced by 1% methanol and the culture supernatant was collected and tested by SDS-PAGE and Western blotting. RESULTS: 45WB2 gene was expressed successfully in P. pastoris and the product was recognized by human positive serum against cysticercosis. CONCLUSION: The recombinant 45WB2 expressed in P. pastoris may be used to prevent pigs from the infection of Taenia solium.