Integrated Proteomics and Transcriptomics Analyses Reveals the Possible Antifungal Mechanism of an Indoloquinoline Alkaloid Neocryptolepine against Rhizoctonia solani.

Rhizoctonia solani causes serious plant diseases. Neocryptolepine presented the significant antifungal activity against R. solani, however the mode of action is unclear. In this paper, we investigated the potential mode of action of neocryptolepine against R. solani integrated the proteomics and transcriptomics. Results showed that after treatment with neocryptolepine, 1012 differentially expressed proteins and 10 920 differentially expressed genes of R. solani were found, most of them were enriched in mitochondrial respiratory chain. It affected oxidative phosphorylation led to the enrichment of ROS and the decrease of MMP, and inhibited complex III activity with the inhibition rate of 63.51% at 10 µg/mL. The mitochondrial structural and function were damaged. Cytochrome b-c1 complex subunit Rieske (UQCRFS1) with the high binding score to neocryptolepine was found as a potential target. In addition, it inhibited the sclerotia formation and presented antifungal efficacy by decreasing the diameter of a wound in potato in a concentration-dependent manner. Above results indicated that neocryptolepine inhibited the complex III activity by binding UQCRFS1 and blocked the ion transfer to cause the death of R. solani mycelia. This study laid the foundation for the future development of neocryptolepine as an alternative biofungicide.

Acaricidal activity of strophanthidin derivatives against Psoroptes cuniculi and their inhibitory effect on Na+-K+-ATPase.

In our previous studies, we found that as the active gradients of Adonis coerulea, cardenolides and cardiac glycosides presented toxicity against mites by inhibiting Na+-K+-ATPase. In this paper, after evaluating the acaricidal activity of the commercial cardiac aglycones/glycosides, serials of novel strophanthidin derivatives were designed and synthesized with an efficient and simple route under mild conditions, and their toxicity against mites, the cytotoxicity and inhibitory effect on Na+-K+-ATP enzyme in PC12 cells were investigated. Results showed among of all compounds, including 9 commercial agent and 32 synthesized strophanthidin derivatives, QXG-1 presented the strongest toxicity against mites with the LC50 value of 320.0 µg/mL. C-19 group of strophanthidin substituted with glycinemethylester would increase the toxicity against mites, and the hydroxyl group at C-5 play the vital role in terms of the toxicity. At the given concentration, QXG-1 displayed the safety against PC12 (10.0 µg/mL) in vitro and mice (3.2 mg/kg) in acute toxicity test, and strong inhibitory effect on Na+-K+-ATPase. It could be used as a promising acaricidal agent. This study lays the foundation to develop of QXG-1 as a relatively safe and alternative acaricidal agent.

High Value-Added Application of Two Renewable Sources as Healthy Food: The Nutritional Properties, Chemical Compositions, Antioxidant, and Antiinflammatory Activities of the Stalks of Rheum officinale Baill. and Rheum tanguticum Maxim. ex Regel.

Rhubarb plants (Rheum officinale and R. tanguticum) have edible stalks. In this work, we aimed to compare the nutritional properties, chemical compositions, and bioactivities of R. officinale (SRO) and R. tanguticum (SRT) stalks and to analyze the composition-function relationship. Results showed that the two stalks were good sources of fiber, as well as minerals. They contained abundant essential amino acids and essential fatty acids to regulate the immunity and prevent some chronic diseases; the contents of polyunsaturated fatty acids were 2,244.32 mg/100 g and 2,844.69 mg/100 g, respectively. The antioxidant activity were also proved. Metabolomics showed that SRO and SRT contained abundant phenolic acids. Due to the higher concentrations of flavones, SRT has better antiinflammatory activities than SRO by inhibiting NF-κB signaling pathway. Rhubarb stalks exhibited good safety in acute toxicity and cytotoxicity tests. This work indicated that the two stalks have nutritional value, safety, and bioactivities, and could be used as sources of nutritional ingredients for regulating the immunity of body in food industry.

A value-added application of eugenol as acaricidal agent: The mechanism of action and the safety evaluation.

Introduction: Eugenol is a major component of essential oils of several plants, it exhibits significant antiparasitic and acaricidal activities, yet its molecular targets remain unknown. Objectives: We aimed to systematically investigate the mechanism of action and the potential targets of eugenol against P. cuniculi, and evaluate the safety for laying the theoretical foundation for clinical application as an acaricide. Methods: Using RNA-Seq analysis, surface plasmon resonance analysis and RNA interference assay, the mode of action of eugenol against Psoroptes cuniculi was investigated. The effect on the mitochondrial membrane potential and complex I of PC12 cells and C6/36 cells was assayed to investigate the species specificity of eugenol in insects and mammals. Finally, a safety evaluation of eugenol in vivo was performed. Results: Eugenol inhibited complex I activity of the mitochondrial respiratory chain in the oxidative phosphorylation pathway by binding to NADH dehydrogenase chain 2 and resulted in the death of mites. The inhibition rates were 37.89% for 50 µg/mL and 60.26% for 100 µg/mL, respectively. Further experiments indicated that the difference in the complex I sequence between insects and mammals led to the different affinity of eugenol to specific peptide, resulting in species specificity. Eugenol exhibited significant inhibitory effects against the mitochondrial membrane potential and complex I in Aedes albopictus C6/36 cells but was not active in rat PC12 cells. Insect cells were particularly sensitive to eugenol. In contrast to the known inhibitor rotenone, eugenol had better safety and did not result in Parkinson's disease or other diseases in rats. Conclusion: This is the first report on acaricidal eugenol targeting complex I of the mitochondrial respiratory chain. This work lays the foundation for the development of eugenol as an environmentally alternative acaricidal agent.

The acaricidal mechanism and active compounds against Psoroptes cuniculi of the methanol extract of Adonis coerulea Maxim II: Integrated proteomics and SPR analysis.

Adonis coerulea Maxim. as a folk medicine, presented acaricidal acitvity. However, the mode of action and active compounds were unclear. In this study, using proteomics and surface plasmon resonance (SPR) technology the mode of action and active compounds of A. coerulea were investigated, as well as a sensitive and environmentally friendly analytical method developed. Proteomics analysis results showed that after treatment of mites with A. coerulea methanol extract (MEAC), 135 proteins were differentially expressed, most of them enriched in the myocardium pathway and participated in the function of the inflated cystic organ. Na+-K+-ATPase may be a potential target. Then, it was used as a target to capture the compounds from the extract using a SPR test. Twelve compounds were found, five compounds, namely ellagic acid, ouabain, convallatoxin, strophanthidin and cymarin presented the higher affinity with Na+-K+-ATPase in molecular docking test. Further study showed that the latter four compounds presented the stronger cytotoxicity and the inhibitory effect on Na+-K+-ATPase with IC50 values ranging with 2.38-0.56 µg/mL, and also showed toxicity against Psoroptes cuniculi. These results indicated that MEAC presented toxicity against mites by inhibiting Na+-K+-ATPase, and cardiac glycosides may be active compounds of this plant in terms of its acaricidal activity. Only 10 g of plant was used to investigate its active compounds. This study lays the foundation for developing sensitive methods for active compound detection.

Acaricidal activity and enzyme inhibitory activity of active compounds of essential oils against Psoroptes cuniculi.

Plant essential oils and its chemical compositions are commonly applied in medicinal and other industries due to their broad advanced pharmacological activities. In the present study, we systematically evaluated the acaricidal activities of twelve compounds of essential oils against Psoroptes cuniculi in vitro and in vivo. In addition, to support the clinic uses, their toxicities against immortalized human keratinocytes (HaCaT) and human liver cells (HL-7702) and skin irritation were studied for evaluating the liver and skin safety. The possible mechanism of action of certain chemical were investigated by determining the inhibitory activities against cytochrome P450 (P450) acetylcholinesterase (AChE) and glutathione-S-transferase (GST). Among all tested compounds, eugenol exhibited the best acaricidal activity with LC50 value of 56.61 µg/ml in vitro. Meanwhile, after the treatment of eugenol for five times within 10 days, the P. cuniculi were eliminated in the naturally infested rabbits, no skin irritation was found in rabbits treated by eugenol. Moreover, eugenol presented no or weak cytotoxicity against HaCaT cells and HL-7702 cells with IC50 values of greater than 100 µg/ml. Furthermore, the moderate inhibitory activities of eugenol against mites P450 and AChE were demonstrated. Above results indicated that eugenol presented the promising acaricidal activity against P. cuniculi in vitro and in vivo, is safe for both humans and animals at the given doses. This work lays the foundation for the development of eugenol as an environmentally friendly acaricide agent.

[The role of occlusal factors in the occurrence of vertical root fracture].

PURPOSE: To investigate the occlusal characteristics and the condition of tooth abrasion in patients with vertical root fracture and to discuss the etiology of the vertical root fracture and the relationships between occlusal disorder, tooth abrasion and vertical root fracture of molars. METHODS: Two hundred and seventy-seven cases of root fracture patients were included in the study, with 309 involved teeth. The periodontal status, occlusal wear, occlusal contact area, occlusal interference, occlusal trauma and the X-ray film were observed and evaluated. All statistical analyses were performed using SPSS13.0 software package. Date was expressed as n(%).Differences between groups were compared using X(2) test with SPSS 13.0 software package. RESULTS: Vertical root fracture occurred in patients aging 40 to 60 years, the first molar mesial root or buccal root was mostly involved. Periodontal tissue destruction was significantly greater than roots without vertical fracture, with formation of localized periodontal pocket. Severe occlusal surface wear, high and steep non-work tip, working tip abrasion, deep occlusal surface concave and lower distal edge were seen in the affected teeth.The degree of vertical root fracture was related to the site of deep occlusal surface concave. Occlusal interference or occlusal trauma was the predisposing factors of vertical root fracture. CONCLUSIONS: Vertical root fracture is associated with malocclusion and tooth abrasion. Changes of occlusion leads to over occlusal force and abnormal direction of occlusal force, which may be important factor of vertical root fracture.

Arecoline suppresses HaCaT cell proliferation through cell cycle regulatory molecules.

Betel nut chewing is the most common cause of oral submucous fibrosis (OSF). Arecoline is the main component of the betel nut, and is associated with the occurrence and development of OSF through cytotoxicity, genotoxicity and DNA damage. Similar types of stimuli elicit differential responses in different cells. In the present study, we investigated the effects of arecoline on the HaCaT epithelial and Hel fibroblast cell lines. The data showed that arecoline affected HaCaT cell morphology. MTT assay revealed that arecoline suppressed HaCaT cell proliferation. Furthermore, we found that arecoline induced the cell cycle arrest of HaCaT cells. In comparison with the untreated control cells, following treatment with ≥75 µg/ml arecoline an increased percentage of HaCaT cells remained at the G0/G1 phase of the cell cycle, accompanied by a reduced percentage of cells in the S phase. However, arecoline treatment did not significantly alter Hel cell cycle distribution. In the HaCaT epithelial cells, arecoline downregulated expression of the G1/S phase regulatory proteins cyclin D1, CDK4, CDK2, E2F1 as determined by reverse transcription-PCR analysis and western blotting. In summary, arecoline inhibits HaCaT epithelial cell proliferation and survival, in a dose-dependent manner, and cell cycle arrest in the G1/S phase, while this is not obvious in the Hel fibroblast cells. Potentially, our findings may aid in the prevention of arecoline-associated human OSF.

Combination of Caspy2 and IP-10 gene therapy significantly improves therapeutic efficacy against murine malignant neoplasm growth and metastasis.

It has been shown that Caspy2, a zebrafish active caspase, can efficiently suppress the growth of malignant tumor. The present study was designed to test whether combined gene therapy with IP-10, a potent antitumor chemokine, and Caspy2 would improve therapy efficacy. Recombinant plasmid expressing both Caspy2 and IP-10 genes was mixed with DOTAP-cholesterol nanoparticles. Immunocompetent mice bearing CT26 colon carcinoma, B16-F10 melanoma, and 4T1 breast carcinoma were treated with the complex. We found that the combined gene therapy more efficiently inhibited tumor growth, while efficiently prolonging the survival of tumor-bearing animals, compared with monotherapy. Moreover, a significant reduction in spontaneous lung metastasis could be observed in the 4T1 breast carcinoma model. Infiltration of CD8(+) T lymphocytes was also observed. In addition, apoptotic cells were widely detected by TUNEL assay and caspase-3 immunostaining in coadministered tumor tissues. The combination treatment also successfully inhibited angiogenesis and tumor cell proliferation as assessed by CD31 and Ki-67 immunostaining, respectively. Furthermore, depletion of CD8(+) T lymphocytes could significantly abrogate the antitumor activity, whereas the depletion of CD4(+) cells or natural killer cells showed partial abrogation. Rechallenged CT26 tumors were rejected in all of the surviving mice treated by combination therapy. Our results suggest that combined therapy with Caspy2 and IP-10 can significantly enhance antitumor activity by acting as an immune response initiator, apoptosis inducer, and angiogenesis inhibitor, which may be important for further applications in clinical cancer therapy.