Ginkgolides with anti-PAF activity from Ginkgo biloba L.

Four undescribed ginkgolides, including two rare sesquiterpene ginkgolides (compounds 1 and 2) and two diterpenoid ginkgolides (compounds 3 and 4), were isolated from Ginkgo biloba L. The structures of these four ginkgolides were identified based on extensive spectroscopic analysis, DP4+ probability analysis and X-ray diffraction. Compounds 1 and 2 exhibited excellent antiplatelet aggregation activities with IC50 values of 1.20 ± 0.25 and 4.11 ± 0.34 µM, respectively.

Metabolomic analysis revealed the edible and extended-application potential of specific Polygonum multiflorum tissues.

The diverse applications of various tissues of Polygonum Multiflorum (PM) encompass the use of its leaf and bud as tea and vegetables, as well as the utilization of its expanded root tubers and caulis as medicinal substances. However, previous studies in the field of metabolomics have primarily focused on the medicinal properties of PM. In order to investigate the potential for broader applications of other tissues within PM, a metabolomic analysis was conducted for the first time using UPLC-Q-TOF-MS/MS on 15 fresh PM tissues. A total of 231 compounds, including newly discovered compounds such as torosachrysone and dihydro-trihydroxystilbene acid derivatives, were identified within PM. Through clustering analysis, the PM tissues were categorized into edible and medicinal parts, with edible tissues exhibiting higher levels of phenolic acids, organic acids, and flavonoids, while the accumulation of quinones, dianthrones, stilbenes, and xanthones was observed in medicinal tissues. Comparative analysis demonstrated the potential application of discarded tissues, such as unexpanded root tuber (an industrial alternative to expanded root tuber) and young caulis (with edible potential). Moreover, the quantification of representative metabolites indicated that flowers and buds contained significant amounts of flavonoids or phenolic acids, suggesting their potential as functional food. Additionally, the edible portion of PM exhibited a high content of quercitrin, ranging from 0.59 to 10.37 mg/g. These findings serve as a valuable point of reference for the expanded utilization of PM tissues, thereby mitigating resource waste in this plant.

Basic leucine zipper gene VvbZIP61 is expressed at a quantitative trait locus for high monoterpene content in grape berries.

The widely appreciated muscat flavor of grapes and wine is mainly attributable to the monoterpenes that accumulate in ripe grape berries. To identify quantitative trait loci (QTL) for grape berry monoterpene content, an F1 mapping population was constructed by a cross between two grapevine genotypes, one with neutral aroma berries (cv. 'Beifeng') and the other with a pronounced muscat aroma (elite Vitis vinifera line '3-34'). A high-density genetic linkage map spanning 1563.7 cM was constructed using 3332 SNP markers that were assigned to 19 linkage groups. Monoterpenes were extracted from the berry of the F1 progeny, then identified and quantified by gas chromatography-mass spectrometry. Twelve stable QTLs associated with the amounts of 11 monoterpenes in berries were thus identified. In parallel, the levels of RNA in berries from 34 diverse cultivars were estimated by RNA sequencing and compared to the monoterpene content of the berries. The expression of five genes mapping to stable QTLs correlated well with the monoterpene content of berries. These genes, including the basic leucine zipper VvbZIP61 gene on chromosome 12, are therefore considered as potentially being involved in monoterpene metabolism. Overexpression of VvbZIP61 in Vitis amurensis callus through Agrobacterium-mediated transformation significantly increased the accumulation of several monoterpenes in the callus, including nerol, linalool, geranial, geraniol, ß-myrcene, and D-limonene. It is hypothesized that VvbZIP61 expression acts to increase muscat flavor in grapes. These results advance our understanding of the genetic control of monoterpene biosynthesis in grapes and provide important information for the marker-assisted selection of aroma compounds in grape breeding.

Qualitative and quantitative analysis of triterpenoids in different tissues of Pulsatilla chinensis.

Pulsatilla chinensis (P.chinensis) is a traditional Chinese medicine used for the treatment of intestinal amebiasis diseases, vaginal trichomoniasis and bacterial infections. Tritepenoid saponins were important components of P.chinensis. Therefore, we asssessmented expression profiling of triterpenoids in different fresh tissues of P.chinensis by ultra high performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) and ultra high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QQQ-MS). Firstly, we identified 132 triterpenoids, including 119 triterpenoid saponins, 13 triterpenoid acids and forty seven of them were first determined in Pulsatilla genus, including new aglycones and new ways of rhamnose linking to the aglycone. Secondly, we established the analytical method to analysis triterpenoids content of P.chinensis and comprehensively verified the analytical method by linearity, precision, repeatability, stability and recovery. At last, we quantified 119 triterpenoids simultaneously based on UHPLC-QQQ-MS. The results show that the types and contents of triterpenoids had obvious tissue distribution. New components like rhamnose directly linked to the aglycone mainely distributed in aboveground tissues. Additionally, We identified 15 chemical ingredients as differential components between the aboveground and underground tissues of P.chinensis. This study provides an efficient analysis strategy for the qualitative and quantitative analysis of triterpenoids in P.chinensis even in other traditional Chinese medicines. At the same time, it provides important informations to explain the biosynthetic pathway of triterpenoid saponins in P.chinensis.

The triterpenoid saponin content difference is associated with the two type oxidosqualene cyclase gene copy numbers of Pulsatilla chinensis and Pulsatilla cernua.

Pulsatilla chinensis is an important medicinal herb, its dried radix is used to treat the inflammation since ancient China. Triterpenoid saponins are proved to be the main active compounds of Pulsatilla genus. The triterpenoid saponin contents vary widely in different Pulsatilla species. But no enzyme involved in the triterpenoid saponin biosynthetic pathway was identified in Pulsitilla genus. This seriously limits the explanation of the triterpene content difference of Pulsatilla species. In this article, we obtained two oxidosqualene cyclase (OSC) genes from P. chinensis and P. cernua by touchdown PCR and anchored PCR. These two OSCs converted 2,3-oxidosqualene into different triterpenoids. The OSC from P. cernua is a monofunctional enzyme for ß-amyrin synthesis, while the OSC from P. chinensis is a multifunctional enzyme for lupeol and ß-amyrin synthesis, and the lupeol is the main product. Then we identified the 260th amino acid residue was the key site for the product difference by gene fusion and site-directed mutant technology. When the 260th amino acid residue was tryptophan (W260) and phenylalanine (F260), the main catalysate was ß-amyrin and lupeol, respectively. Then we found that the expression of these two genes was strongly correlated with the lupeol-type and ß-amyrin-type triterpenoid contents in P. cernua and P. chinensis. Finally, we found the gene copy number difference of these two genotypes leaded to the triterpenoid diversity in P. cernua and P. chinensis. This study provides useful information for the molecular breeding and quality improvement of P. chinensis and a molecular marker to identify the P. chinensis decoction pieces.

Functional characterization and key residues engineering of a regiopromiscuity O-methyltransferase involved in benzylisoquinoline alkaloid biosynthesis in Nelumbo nucifera.

Lotus (Nelumbo nucifera), an ancient aquatic plant, possesses a unique pharmacological activity that is primarily contributed by benzylisoquinoline alkaloids (BIAs). However, only few genes and enzymes involved in BIA biosynthesis in N. nucifera have been isolated and characterized. In the present study we identified the regiopromiscuity of an O-methyltransferase, designated NnOMT6, isolated from N. nucifera; NnOMT6 was found to catalyze the methylation of monobenzylisoquinoline 6-O/7-O, aporphine skeleton 6-O, phenylpropanoid 3-O, and protoberberine 2-O. We further probed the key residues affecting NnOMT6 activity via molecular docking and molecular dynamics simulation. Verification using site-directed mutagenesis revealed that residues D316, N130, L135, N176A, D269, and E328 were critical for BIA O-methyltransferase activities; furthermore, N323A, a mutant of NnOMT6, demonstrated a substantial increase in catalytic efficiency for BIAs and a broader acceptor scope compared with wild-type NnOMT6. To the best of our knowledge, this is the first study to report the O-methyltransferase activity of an aporphine skeleton without benzyl moiety substitutions in N. nucifera. The study findings provide biocatalysts for the semisynthesis of related medical compounds and give insights into protein engineering to strengthen O-methyltransferase activity in plants.

miR­151a­5p promotes the proliferation and metastasis of colorectal carcinoma cells by targeting AGMAT.

Colorectal carcinoma (CRC) is one of the most common types of digestive cancer. It has been reported that the ectopic expression of microRNAs (miRs) plays a critical role in the occurrence and progression of CRC. In addition, it has also been suggested that miR­151a­5p may serve as a useful biomarker for the early detection and treatment of different types of cancer and particularly CRC. However, the specific effects and underlying mechanisms of miR­151a­5p in CRC remain elusive. The results of the current study demonstrated that miR­151a­5p was upregulated in CRC cell lines and clinical tissues derived from patients with CRC. Functionally, the results showed that miR­151a­5p significantly promoted CRC cell proliferation, migration and invasion. Additionally, dual luciferase reporter assays verified that agmatinase (AGMAT) was a direct target of miR­151a­5p and it was positively associated with miR­151a­5p expression. Mechanistically, miR­151a­5p could enhance the epithelial­mesenchymal transition of CRC cells. Taken together, the results of the current study revealed a novel molecular mechanism indicating that the miR­151a­5p/AGMAT axis could serve a crucial role in the regulation of CRC and could therefore be considered as a potential therapeutic strategy for CRC.

Hormone biosynthesis and metabolism members of 2OGD superfamily are involved in berry development and respond to MeJA and ABA treatment of Vitis vinifera L.

BACKGROUND: Hormones play an indispensable role during fruit ripening, nine clades in 2-oxoglutarate-dependent dioxygenase (2OGD) superfamily are responsible for the hormone biosynthesis and metabolism, but less information is known about them. RESULTS: A total of 163 Vv2OGD superfamily members were identified from grape genome, which were mainly expanded by local (tandem and proximal) duplication. Phylogenetic analysis of 2OGD members in grape and Arabidopsis indicates 37 members in Vv2OGD superfamily are related to hormone biosynthesis and metabolism process (Vv2OGD-H), which could be divided into 9 clades, gibberellin (GA) 3-oxidase (GA3ox), GA 20-oxidase (GA20ox), carbon-19 GA 2-oxidase (C19-GA2ox), carbon-20 GA 2-oxidase (C20-GA2ox), 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), dioxygenase for auxin oxidation (DAO), lateral branching oxidoreductas (LBO), downy mildew resistant 6 and DMR6-like oxygenase (DMR6/DLO) and jasmonate-induced oxygenase (JOX). Sixteen of these 37 Vv2OGD-Hs are expressed in grape berry, in which the expression patterns of VvGA2oxs, VvDAOs and VvJOXs shows a correlation with the change patterns of GAs, indole-3-acetic acid (IAA) and jasmonates (JAs), indicating the involvement of these genes in grape berry development by regulating corresponding hormones. Twelve Vv2OGD-Hs respond to methyl JA (MeJA) treatment, of which eight may lead to the inhibition of the ripening process by the crosstalk of JAs-salicylic acids (SAs), JAs-GAs and JAs-JAs, while seven Vv2OGD-Hs respond to ABA treatment may be responsible for the promotion of ripening process by the interplay of abscisic acid (ABA)-strigolactones (SLs), ABA-SAs, ABA-GAs, ABA-JAs. Especially, VvLBO1 reach an expression peak near véraison and up-regulate about four times after ABA treatment, which implies SLs and ABA-SLs crosstalk may be related to the onset of berry ripening in grape. CONCLUSIONS: This study provides valuable clues and new insights for the mechanism research of Vv2OGD-Hs in hormones regulation during the grape berry development.

Agmatinase facilitates the tumorigenesis of pancreatic adenocarcinoma through the TGFß/Smad pathway.

Pancreatic adenocarcinoma (PAAD) is one of the most lethal malignancies. Due to the lack of typical symptoms and difficulties in early diagnosis, PAAD has a high mortality rate. Therefore, it is essential to identify novel specific biomarkers for the application of targeted therapies. A previous study suggested that agmatinase (AGMAT) may fulfill important roles in tumor progression; however, these roles and the underlying mechanisms of AGMAT involvement in PAAD have yet to be thoroughly investigated. To address this shortcoming, in the present study the expression and prognostic significance of AGMAT were analyzed via several bioinformatics databases. Gain- and loss-of-function experiments were subsequently performed to observe the impact of AGMAT on the proliferation and metastasis of PAAD cells via Cell Counting Kit 8 (CCK-8) assay, colony formation assay, and cell migration and invasion assays in vitro. In order to probe the mechanisms involved, western blot assays were performed. AGMAT was found to be overexpressed in PAAD, and it was positively associated with a poor prognosis. Stable overexpression of AGMAT was found to lead to a marked increase in cell proliferation and metastasis through activation of the transforming growth factor-ß (TGFß)/Smad pathway, and via enhancing epithelial-mesenchymal transition (EMT). In conclusion, the results of the present study suggest that AGMAT may be an oncogene, and a pivotal mechanism has been uncovered in which AGMAT facilitates the progression of PAAD tumorigenesis through the TGFß/Smad pathway.

Genome-wide identification of B-box proteins and VvBBX44 involved in light-induced anthocyanin biosynthesis in grape (Vitis vinifera L.).

MAIN CONCLUSION: Genome-wide identification, analysis and functional characterization of an unreported VvBBX gene showed a response to light and positive correlation with anthocyanin content, but also inhibition of light-induced anthocyanin synthesis. B-box (BBX) proteins are a class of zinc (Zn) finger transcription factors or regulators characterized by the presence of one or two BBX domains and play important roles in plant growth and development. However, the BBX genes' potential functions are insufficiently characterized in grape, a globally popular berry with high economic value. Here, 25 BBX family genes including a novel member (assigned VvBBX44) were identified genome widely in grape. The expression level of these VvBBXs were analyzed in 'Cabernet Sauvignon' (V. vinifera) stem, flower, leaf, tendril, petiole, and developing berries. The expression of VvBBX44 increased in developing 'Cabernet Sauvignon' berries. Its expression was inhibited in 'Jingxiu' and 'Muscat Hamburg' berry skin without sunlight. Furthermore, overexpression of VvBBX44 decreased the expression of LONG HYPOCOTYL 5 (VvHY5) and UDP-glucose flavonoid 3-O-glucosyltransferase (VvUFGT), and reduced the anthocyanin content in grape calli. Our results suggest that VvBBX44 may play an important role in grape berry coloring by directly repressing VvHY5 expression. This study provides new insights into the potential role of VvBBXs in berry development and light response and contributes to the understanding on the regulation mechanism of VvBBX44 in anthocyanin biosynthesis.

Author Correction: Whole-genome resequencing of 472 Vitis accessions for grapevine diversity and demographic history analyses.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Whole-genome resequencing of 472 Vitis accessions for grapevine diversity and demographic history analyses.

Understanding the Vitis species at the genomic level is important for cultivar improvement of grapevine. Here we report whole-genome genetic variation at single-base resolution of 472 Vitis accessions, which cover 48 out of 60 extant Vitis species from a wide geographic distribution. The variation helps to identify a recent dramatic expansion and contraction of effective population size in the domesticated grapevines and that cultivars from the pan-Black Sea region have a unique demographic history in comparison to the other domesticated cultivars. We also find selective sweeps for berry edibility and stress resistance improvement. Furthermore, we find associations between candidate genes and important agronomic traits, such as berry shape and aromatic compounds. These results demonstrate resource value of the resequencing data for illuminating the evolutionary biology of Vitis species and providing targets for grapevine genetic improvement.

VvWRKY8 represses stilbene synthase genes through direct interaction with VvMYB14 to control resveratrol biosynthesis in grapevine.

Resveratrol (Res) is a stilbenoid, a group of plant phenolic metabolites derived from stilbene that possess activities against pests, pathogens, and abiotic stresses. Only a few species, including grapevine (Vitis), synthesize and accumulate Res. Although stilbene synthases (STSs) have been isolated and characterized in several species, the gene regulatory mechanisms underlying stilbene biosynthesis are still largely unknown. Here, we characterize a grapevine WRKY transcription factor, VvWRKY8, that regulates the Res biosynthetic pathway. Transient and stable overexpression of VvWRKY8 in grapevine results in decreased expression of VvSTS15/21 and VvMYB14, as well as in a reduction of Res accumulation. VvWRKY8 does not bind to or activate the promoters of VvMYB14 and VvSTS15/21; however, it physically interacts with VvMYB14 proteins through their N-terminal domains to prevent them from binding to the VvSTS15/21 promoter. Application of exogenous Res results in the stimulation of VvWRKY8 expression and in a decrease of VvMYB14 and VvSTS15/21 expression in grapevine suspension cells, and in the activation of the VvWRKY8 promoter in tobacco leaves. These results demonstrate that VvWRKY8 represses VvSTS15/21 expression and Res biosynthesis through interaction with VvMYB14. In this context, the VvMYB14-VvSTS15/21-Res-VvWRKY8 regulatory loop may be an important mechanism for the fine-tuning of Res biosynthesis in grapevine.

CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.).

The type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has been successfully applied to edit target genes in multiple plant species. However, it remains unknown whether this system can be used for genome editing in grape. In this study, we described genome editing and targeted gene mutation in 'Chardonnay' suspension cells and plants via the CRISPR/Cas9 system. Two single guide RNAs (sgRNAs) were designed to target distinct sites of the L-idonate dehydrogenase gene (IdnDH). CEL I endonuclease assay and sequencing results revealed the expected indel mutations at the target site, and a mutation frequency of 100% was observed in the transgenic cell mass (CM) as well as corresponding regenerated plants with expression of sgRNA1/Cas9. The majority of the detected mutations in transgenic CM were 1-bp insertions, followed by 1- to 3-nucleotide deletions. Off-target activities were also evaluated by sequencing the potential off-target sites, and no obvious off-target events were detected. Our results demonstrated that the CRISPR/Cas9 system is an efficient and specific tool for precise genome editing in grape.

Identification of genomic sites for CRISPR/Cas9-based genome editing in the Vitis vinifera genome.

BACKGROUND: CRISPR/Cas9 has been recently demonstrated as an effective and popular genome editing tool for modifying genomes of humans, animals, microorganisms, and plants. Success of such genome editing is highly dependent on the availability of suitable target sites in the genomes to be edited. Many specific target sites for CRISPR/Cas9 have been computationally identified for several annual model and crop species, but such sites have not been reported for perennial, woody fruit species. In this study, we identified and characterized five types of CRISPR/Cas9 target sites in the widely cultivated grape species Vitis vinifera and developed a user-friendly database for editing grape genomes in the future. RESULTS: A total of 35,767,960 potential CRISPR/Cas9 target sites were identified from grape genomes in this study. Among them, 22,597,817 target sites were mapped to specific genomic locations and 7,269,788 were found to be highly specific. Protospacers and PAMs were found to distribute uniformly and abundantly in the grape genomes. They were present in all the structural elements of genes with the coding region having the highest abundance. Five PAM types, TGG, AGG, GGG, CGG and NGG, were observed. With the exception of the NGG type, they were abundantly present in the grape genomes. Synteny analysis of similar genes revealed that the synteny of protospacers matched the synteny of homologous genes. A user-friendly database containing protospacers and detailed information of the sites was developed and is available for public use at the Grape-CRISPR website ( http://biodb.sdau.edu.cn/gc/index.html ). CONCLUSION: Grape genomes harbour millions of potential CRISPR/Cas9 target sites. These sites are widely distributed among and within chromosomes with predominant abundance in the coding regions of genes. We developed a publicly-accessible Grape-CRISPR database for facilitating the use of the CRISPR/Cas9 system as a genome editing tool for functional studies and molecular breeding of grapes. Among other functions, the database allows users to identify and select multi-protospacers for editing similar sequences in grape genomes simultaneously.

MNV primarily surveillance by a recombination VP1-derived ELISA in Beijing area in China.

Murine norovirus (MNV) was first found as a surrogate for human norovirus study. However, MNV infection was mostly prevalent in laboratory mice, and its immunomodulatory properties may affect the outcome of animal experiments. MNV surveillance had been performed in Europe, North America and some other countries, but not in China. Nowadays, the complete MNV virions had been used as antigen in MNV serological detection. However, the complexity in the preparation of virions might affect the antigen stability, and the virulence recovery of virion antigen had also been detected. In this study, the caspid VP1 protein was proved to be the mostly predominant antigen in MNV virions. An ELISA method using the recombination VP1 as antigen was developed (rVP1 ELISA). The rVP1 ELISA is more sensitive and less specific than the MNV virion-derived IFA method. To address the prevalence of MNV in China, a totally 600 mouse serum samples from Beijing area were tested by rVP1 ELISA and confirmed by IFA and WB. The MNV infection rate was 11.67%, but most of the MNV-positive samples were from experimental facilities (MNV rate=30.94%), not from commercial vendors (MNV rate=0.27%). Collectively, a sensitive rVP1 ELISA was developed in the current study, and the MNV investigation by rVP1 ELISA showed that MNV infection was mostly prevalent in the laboratory mice, especially the mice from experimental facilities in Beijing area in China.

[The version and academic characteristics of Mai jue kan wu (Correcting Errors in Pulse Study in Verse)].

Mai jue kan wu (Correcting Errors in Pulse Study in Verse) was written by Dai Qizong of the Yuan Dynasty without block-printing when completed. It was reviewed and checked by Wang Ji, attached with the main discourses on pulse taking from various scholars of pulse taking and Jiao shi huo mai lun (On Pulse-taking with Corrections on Its Popular Difficult Issues) and was block-printed in 1523. This book corrects the fallacies on the descriptions of pulse shape and pulse body reflecting its underlying diseases appeared in Mai jue (Pulse Study in Verse), refutes its classification of the so-called "Seven exterior", "Eight interior" and "Nine dao (channels)". It also creates the measures for the studies of pulse-taking, including "differentiating, combining, doubling, comparing and classifying".

[Observation on therapeutic effect of warming needle moxibustion on chronic pelvic inflammation of cold-damp stagnation type].

OBJECTIVE: To observe clinical therapeutic effect of warming needle moxibustion on chronic pelvic in flammation of cold-damp stagnation type. METHODS: Eighty-five cases with chronic pelvic inflammation were randomly divided into a warming needle moxibustion group of 45 cases and a Chinese drug group of 40 cases. The war ming needle moxibustion group were treated with warming needle moxibustion at Guanyuan (CV 4), Qihai (CV 6), Zusanli (ST 36) and Shenshu (BL 23) etc. and the Chinese drug group were treated with oral administration of TCM decoction, one dose each day. One course was constituted by 10 days. After treatment of 3 courses, the therapeutic effects, changes of C-reactive protein (CRP) level and the recurrence rate of the cured cases a half year later were observed. RESULTS: The total effective rate was 95.6% in the warming needle moxibustion group and 77.5% in the Chinese drug group with a significant difference between the two groups (P < 0.05). Twenty-five cases in each group were selected to determine CRP levels and results showed that before and after treatment CRP levels were (16.47 +/- 7.11) mg/L and (5.98 +/- 2.29) mg/L in the warming needle moxibustion group, and (16.32 +/- 8.19) mg/L and (8.63 +/- 2.41) mg/L in the Chinese drug group, with significantly decreased in the two groups (P < 0.01 or P < 0.05), and with a significant difference between the two group after treatment (P < 0.05). In the warming needle moxibustion group, of 23 cases followed-up, 2 cases relapsed, while in the Chinese drug group, among 9 cases followed-up, 2 cases relapsed. CONCLUSION: Warming needle moxibustion has a definite therapeutic effect on chronic pelvic inflammation of cold-damp stagnation type and better improves CRP level of the patient.