Cyclophilin effector Al106 of mirid bug Apolygus lucorum inhibits plant immunity and promotes insect feeding by targeting PUB33.

Apolygus lucorum (Meyer-Dur; Heteroptera: Miridae) is a major agricultural pest infesting crops, vegetables, and fruit trees. During feeding, A. lucorum secretes a plethora of effectors into its hosts to promote infestation. However, the molecular mechanisms of these effectors manipulating plant immunity are largely unknown. Here, we investigated the molecular mechanism underlying the effector Al106 manipulation of plant-insect interaction by RNA interference, electrical penetration graph, insect and pathogen bioassays, protein-protein interaction studies, and protein ubiquitination experiment. Expression of Al106 in Nicotiana benthamiana inhibits pathogen-associated molecular pattern-induced cell death and reactive oxygen species burst, and promotes insect feeding and plant pathogen infection. In addition, peptidyl-prolyl cis-trans isomerase (PPIase) activity of Al106 is required for its function to inhibit PTI.Al106 interacts with a plant U-box (PUB) protein, PUB33, from N. benthamiana and Arabidopsis thaliana. We also demonstrated that PUB33 is a positive regulator of plant immunity. Furthermore, an in vivo assay revealed that Al106 inhibits ubiquitination of NbPUB33 depending on PPIase activity. Our findings revealed that a novel cyclophilin effector may interact with plant PUB33 to suppress plant immunity and facilitate insect feeding in a PPIase activity-dependent manner.

Apolygus lucorum effector Al6 promotes insect feeding performance on soybean plants: RNAi analysis and feeding behaviour study with electrical penetration graph.

The mirid bug Apolygus lucorum, a dominant mirid species in northern China, is a notorious polyphagous pest with more than 200 hosts, including several major crops such as cotton and soybean, resulting in massive economic loss. Studies of insect salivary effectors may provide a novel control strategy for A. lucorum. An A. lucorum effector, that is, Al6, that inhibits plant immunity by using glutathione peroxidase to repress reactive oxidase accumulation was previously identified. In this study, we further explored the molecular functions of Al6 associated with feeding behaviour and insect survival on soybean, a major host of A. lucorum, using RNA interference and electrical penetration graph (EPG) techniques. We initially observed the injury symptom of this mirid bug and characterized feeding behaviour on soybean leaves using EPG. Our results revealed that A. lucorum preferred to feed on young plant organs such as tender leaves, shoots and buds. This mirid bug used cell rupture as a feeding strategy to ingest cell contents from plant tissues. Subsequently, we silenced the Al6 gene using RNAi and investigated the feeding behaviour, honeydew excretion, body weight, and survival rates of A. lucorum on soybean after Al6 knockdown. Our results demonstrated that silencing of Al6 significantly reduced feeding duration, amount of honeydew secretion, body weight, and survival rates of A. lucorum. Thus, our findings provide a novel molecular target of plant-mediated RNAi for the control of A. lucorum.

Metastasis patterns and prognosis of octogenarians with metastatic breast cancer: A large-cohort retrospective study.

BACKGROUND: Breast cancer may differ biologically in patients aged over 80 years. The objective of the current study was to analyze the metastasis patterns and prognosis of elderly patients with metastatic breast cancer (MBC) and compare it to patients of other ages. METHODS: The Surveillance, Epidemiology, and End Results (SEER) database was utilized to select MBC patients from 2010 to 2015. Chi-squared test was used to compare clinicopathological characteristics among different aged groups. The Kaplan-Meier method and multivariate Cox model were utilized for survival analysis. RESULTS: A total of 10479 MBC patients were included, among which 1036 (9.9%) patients were aged over 80 years. Compared with other aged group, the elderly patients tended to have a higher proportion of HR+/Her2- subtype, white race, lower tumor differentiation, and receive less treatment, including surgery, chemotherapy and radiotherapy (P< 0.001). MBC patients with different age presented with distinctive metastatic patterns. The older patients were more likely to have lung metastasis, but less likely to have bone, brain, liver and multiple sites metastasis than the younger group (P <0.001). The proportion of TNBC subtype increased substantially in the older patients with brain metastasis, compared to the younger and middle-aged group. The old age was demonstrated to significantly associate with worse prognosis of MBC patients. Additionally, our findings also showed that older MBC patients could achieve dramatical overall survival benefit from surgery (HR = 0.58; P <0.001) and chemotherapy (HR = 0.59; P <0.001), but not the radiotherapy (HR = 0.96; P = 0.097). CONCLUSION: The elderly MBC patients presented with distinctive metastatic patterns, clinical characteristics, and prognostic outcomes compared with younger patients. Our findings could assist clinicians in making appropriate therapeutic decision.

Characterisation of hederacoside C metabolites using ultrahigh-performance liquid chromatography quadrupole Orbitrap mass spectrometry based on automatic fragment ion search.

INTRODUCTION: Hederacoside C (HDC) is a bioactive natural triterpenoid saponins constituent originating from traditional Chinese medicines, playing an important role in the treatment of acute respiratory infections and chronic inflammatory bronchitis. Meanwhile, it is recognised by Korea as a botanical drug. OBJECTIVES: In order to develop an integrated template approach to analysing screening and identification of the metabolites of traditional Chinese medicines. This study will provide available information for further pharmaceutical studies of HDC and other triterpene saponins. METHODOLOGY: An analysis strategy based on ultrahigh-performance liquid chromatography quadrupole Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS) technique combined with automatic fragment ion search (FISh) was firstly exploited for the characterisation metabolites of HDC in vivo and in vitro. Accurate full mass scan combined with an on-line FISh annotations approach was developed to rapidly identify all the potential metabolites of HDC. Furthermore, FISh accurately located the structure of the target compound in a large number of mass spectral data. RESULTS: A total of 34 metabolites were detected and tentatively identified by analysing comprehensive biological samples. The results clearly demonstrated that HDC underwent general metabolic reactions including dealkylation, reduction, oxidation, desaturation, dehydration, cysteine conjugation, GSH conjugation, taurine conjugation, and glycine conjugation to produce 26 phase I and eight phase II metabolites. CONCLUSION: In the present study, UHPLC-Q-Exactive Orbitrap MS technique combined with FISh provided a rapid and efficient platform to characterise metabolites of HDC in vivo and in vitro. The proposed method could develop an integrated template approach to screen and identify the constituents and metabolites of traditional Chinese medicines.

Safety assessment and antioxidant evaluation of betulin by LC-MS combined with free radical assays.

Betulin, as a new type of natural food preservative, is widely used in various kinds of meat products. However, its detailed mechanism of action and metabolism have not been clarified. In this study, for further gain insight of the mechanism of betulin as a preservative, an efficient method has been applied for measuring the antioxidant capacity of betulin, based on the absorbance of the DPPH• and ABTS• radical cation. When the concentration of betulin was more than 2.0 mg/mL, the scavenging rate of ABTS and DPPH radical reached over 90%, which was equivalent to the antioxidant capacity of Trolox. It is indicated that betulin has significant DPPH and ABTS free radical scavenging ability. This should be one of the important mechanisms for betulin as a preservative. A sensitive method using UHPLC-Q-TOF-MS/MS was established to determine the metabolite profile in vivo and in vitro of betulin. 32 phase I and 2 phase II metabolites were structurally characterized. This study will provide theoretical support for the safety and effectiveness of betulin in the field of preservatives and provide theoretical basis for the further study of betulin and the other natural preservatives. This research also contributes to the development of the food industry.

A high-throughput metabolomics approach for the comprehensive differentiation of four Pulsatilla Adans herbs combined with a nontargeted bidirectional screen for rapid identification of triterpenoid saponins.

Pulsatilla Adans (PSA) herbs (Ranunculaceae) have been widely used in traditional medicine in China and other countries. However, the authentication and quality control of PSA herbs have always been a challenging task due to their similar morphological characteristics and the diversity of the multiple components that exist in the complicated matrix. Herein, a novel integrated strategy combining UHPLC/Q-Orbitrap-MS techniques with chemometrics analysis is proposed for the discrimination of PSA materials. We developed a comprehensive method integrating a nontargeted bidirectionally screened (NTBDS) MS data set and a targeted extraction peak area analysis for the characterization of triterpenoid saponins of PSA from different species. After that, partial least-squares discriminant analysis (PLS-DA) was performed on the obtained MS data set and the parameter variable importance for the projection (VIP) value and P value were employed to screen the valuable MS features to discriminate PSA from different species. In addition, the receiver operating characteristic (ROC) curve is used to verify the reliability of MS features. Finally, heatmap visualization was employed to clarify the distribution of the identified triterpenoid saponins, and four medicinal species of PSA were successfully differentiated. Additionally, 34 constituents were reported in PSAs for the first time, 81 triterpenoid saponins were identified as differential components, and 12 chemical ingredients were characterized as potential chemical markers to differentiate the four officinal PSA herbs. This is the first time that the differences in different PSA herbs have been observed systematically at the chemical level. The results suggested that using the identified characteristic components as chemical markers to identify different PSA herbs was effective and viable. This method provides promising perspectives in the analysis and identification of the ingredients of Chinese herbal medicines, and the identification of similar herbs from the same species.

Study on the metabolites of betulinic acid in vivo and in vitro by ultra high performance liquid chromatography with time-of-flight mass spectrometry.

Betulinic acid is a triterpenoid organic acid with remarkable antitumor properties and is naturally present in many fruits, condiments and traditional Chinese medicines. Currently, a strategy was developed for the identification of metabolites following the in vivo and in vitro biotransformation of Betulinic acid with rat intestinal bacteria utilizing ultra high performance liquid chromatography with time-of-flight mass spectrometry with polymeric solid-phase extraction. As a result, 46 metabolites were structurally characterized. The results demonstrated that Betulinic acid is universally metabolized in vivo and in vitro, and Betulinic acid could undergo general metabolic reactions, including oxidation, methylation, desaturation, loss of O and loss of CH2 . Additionally, the main metabolic pathways in vivo and in vitro were determined by calculating the relative content of each metabolite. This is the first study of Betulinic acid metabolism in vivo, whose results provide novel and useful data for better understanding of the safety and efficacy of Betulinic acid.

UPLC-Q-TOF-MS/MS-guided dereplication of Pulsatilla chinensis to identify triterpenoid saponins.

INTRODUCTION: Triterpenoid saponins are the major bioactive constituents of Pulsatilla chinensis, playing an important role in various biological activities such as anti-tumour, cognition-enhancing, anti-biosis, anti-inflammatory, hypoglycemic and immunological adjuvant. OBJECTIVE: To establish a systematic strategy based on ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) for the efficient characterisation and identification of triterpenoid saponins in crude extracts from Pulsatilla chinensis. METHODOLOGY: In this work, the strategy includes two aspects: (1) positive mode: by target screening, we can deduce the aglycone type and the composition of sugar moiety according to the fragment ions; untargeted screening includes four steps, find unknown, formula finder, ChemSpider search and MS/MS identification; (2) negative mode: according to the MS/MS spectra, the composition of sugar chain bonded to C-28 is inferred reasonably. The extract of Pulsatilla chinensis was separated within 60 min on a C18 column and eluted with methanol and water both containing 0.1% formic acid. RESULTS: As a result, a total of 22 triterpenoid saponins (11 pairs of isomers) with four aglycone skeletons were tentatively identified or elucidated in crude extracts from Pulsatilla chinensis based on their retention times, the mass spectrometric fragmentation patterns, and MS and MS/MS data. CONCLUSION: This study provides an efficient analysis strategy to rapidly identify the triterpenoid saponins in Pulsatilla species even in traditional Chinese medicines.

UHPLC-Q-TOF-MS/MS based screening and identification of the metabolites in vivo after oral administration of betulin.

Betulin is an active natural pentacyclic triterpene ingredient with valuable anti-cancer and anti-HIV efficacies. In the present study, an efficient approach was developed to screening and identification of metabolites and to assess the metabolic profiles of betulin in vivo using UHPLC-Q-TOF-MS/MS system based on multiple mass defect filter data acquisition (MMDF) and multiple data processing techniques. Based on the proposed method, 56 phase I and 6 phase II metabolites were detected in rats after oral administration of betulin. The main biotransformation routes of betulin were identified as demethylation, dehydroxylation, deoxidization, dehydration. Conjugation with sulfate, taurine, cysteine and N-acetylcysteine groups produced 6 phase II metabolites. This study not only provided useful information for further study of the pharmacology and mechanism of betulin in vivo, but also provided essential data for further pharmaceutical studies of other pentacyclic triterpenes.