CRISPR/Cas9-Mediated Knockout of the PxJHBP Gene Resulted in Increased Susceptibility to Bt Cry1Ac Protoxin and Reduced Lifespan and Spawning Rates in Plutella xylostella.

Juvenile hormone binding protein (JHBP) is a key regulator of JH signaling, and crosstalk between JH and 20-hydroxyecdysone (20E) can activate and fine-tune the mitogen-activated protein kinase cascade, leading to resistance to insecticidal proteins from Bacillis thuringiensis (Bt). However, the involvement of JHBP in the Bt Cry1Ac resistance of Plutella xylostella remains unclear. Here, we cloned a full-length cDNA encoding JHBP, and quantitative real-time PCR (qPCR) analysis showed that the expression of the PxJHBP gene in the midgut of the Cry1Ac-susceptible strain was significantly higher than that of the Cry1Ac-resistant strain. Furthermore, CRISPR/Cas9-mediated knockout of the PxJHBP gene significantly increased Cry1Ac susceptibility, resulting in a significantly shorter lifespan and reduced fertility. These results demonstrate that PxJHBP plays a critical role in the resistance to Cry1Ac protoxin and in the regulation of physiological metabolic processes associated with reproduction in adult females, providing valuable insights to improve management strategies of P. xylostella.

Enhanced resistance to Bacillus thuringiensis Cry1Ac toxin mediated by the activation of prophenoloxidase in a cosmopolitan pest.

Plutella xylostella has evolved resistance to Bacillus thuringiensis Cry1Ac toxin over a long evolutionary period. Enhanced immune response is an important factor in insect resistance to a variety of insecticides, and whether phenoloxidase (PO), an immune protein, is involved in resistance to Cry1Ac toxin in P. xylostella remains unclear. Here, spatial and temporal expression patterns showed that prophenoloxidase (PxPPO1 and PxPPO2) in the Cry1S1000-resistant strain was more highly expressed in eggs, 4th instar, head, and hemolymph than those in G88-susceptible strain. The results of PO activity analysis showed that after treatment with Cry1Ac toxin PO activity was about 3 times higher than that before treatment. Furthermore, knockout of PxPPO1 and PxPPO2 significantly increased the susceptibility to Cry1Ac toxin. These findings were further supported by the knockdown of Clip-SPH2, a negative regulator of PO, which resulted in increased PxPPO1 and PxPPO2 expression and Cry1Ac susceptibility in the Cry1S1000-resistant strain. Finally, the synergistic effect of quercetin showed that larval survival decreased from 100 % to <20 % compared to the control group. This study will provide a theoretical basis for the analysis of immune-related genes (PO) genes involved in the resistance mechanism and pest control of P. xylostella.

Interaction behaviors of sulfamethoxazole and microplastics in marine condition: Focusing on the synergistic effects of salinity and temperature.

Microplastics and antibiotics are two common pollutants in the ocean. However, due to changes of salinity and temperature in the ocean, their interaction are significantly different from that of fresh water, and the mechanism remains unclear. Here, the interactions of sulfamethoxazole (SMZ) and microplastics were studied at different temperatures and salinities. The saturation adsorption capacity of SMZ in polypropylene (PP), polyethylene (PE), styrene (PS), polyvinyl chloride (PVC), and synthetic resins (ABS) were highest at the temperature of 20 °C, with 0.118 ± 0.002 mg·g-1, 0.106 ± 0.004 mg·g-1, 0.083 ± 0.002 mg·g-1, 0.062 ± 0.007 mg·g-1 and 0.056 ± 0.003 mg·g-1, respectively. The effect of temperature reduction is more significant than temperature rise. The intraparticle diffusion model is appropriate to PP, when film diffusion model suited for PS. The salinity has a more significant effect than temperature on different microplastics, due to the electrostatic adsorption and iron exchange. With the increase in salinity from 0.05% to 3.5%, the adsorption capacity of microplastics on SMZ fell by 53.3 ± 5%, and there was no discernible difference of various microplastics. The hydrogen bond and π-π conjugation of microplastics play an important role in the adsorption of SMZ. These findings further deepen the understanding of the interaction between microplastics and antibiotics in the marine environment.

Analysis of the Effect of Plutella xylostella Polycalin and ABCC2 Transporter on Cry1Ac Susceptibility by CRISPR/Cas9-Mediated Knockout.

Many insects, including the Plutella xylostella (L.), have developed varying degrees of resistance to many insecticides, including Bacillus thuringiensis (Bt) toxins, the bioinsecticides derived from Bt. The polycalin protein is one of the potential receptors for Bt toxins, and previous studies have confirmed that the Cry1Ac toxin can bind to the polycalin protein of P. xylostella, but whether polycalin is associated with the resistance of Bt toxins remains controversial. In this study, we compared the midgut of larvae from Cry1Ac-susceptible and -resistant strains, and found that the expression of the Pxpolycalin gene was largely reduced in the midgut of the resistant strains. Moreover, the spatial and temporal expression patterns of Pxpolycalin showed that it was mainly expressed in the larval stage and midgut tissue. However, genetic linkage experiments showed that the Pxpolycalin gene and its transcript level were not linked to Cry1Ac resistance, whereas both the PxABCC2 gene and its transcript levels were linked to Cry1Ac resistance. The larvae fed on a diet containing the Cry1Ac toxin showed no significant change in the expression of the Pxpolycalin gene in a short term. Furthermore, the knockout of polycalin and ATP-binding cassette transporter subfamily C2 (ABCC2) genes separately by CRISPR/Cas9 technology resulted in resistance to decreased susceptibility to Cry1Ac toxin. Our results provide new insights into the potential role of polycalin and ABCC2 proteins in Cry1Ac resistance and the mechanism underlying the resistance of insects to Bt toxins.

Optimization and Application of CRISPR/Cas9 Genome Editing in a Cosmopolitan Pest, Diamondback Moth.

The CRISPR/Cas9 system is an efficient tool for reverse genetics validation, and the application of this system in the cell lines provides a new perspective on target gene analysis for the development of biotechnology tools. However, in the cell lines of diamondback moth, Plutella xylostella, the integrity of the CRISPR/Cas9 system and the utilization of this cell lines still need to be improved to ensure the application of the system. Here, we stabilize the transfection efficiency of the P. xylostella cell lines at different passages at about 60% by trying different transfection reagents and adjusting the transfection method. For Cas9 expression in the CRIPSPR/Cas9 system, we identified a strong endogenous promoter: the 217-2 promoter. The dual-luciferase and EGFP reporter assay demonstrated that it has a driving efficiency close to that of the IE1 promoter. We constructed pB-Cas9-Neo plasmid and pU6-sgRNA plasmid for CRISPR/Cas9 system and subsequent cell screening. The feasibility of the CRISPR/Cas9 system in P. xylostella cell lines was verified by knocking out endogenous and exogenous genes. Finally, we generated a transgenic Cas9 cell line of P. xylostella that would benefit future exploitation, such as knock-in and multi-threaded editing. Our works provides the validity of the CRISPR/Cas9 system in the P. xylostella cell lines and lays the foundation for further genetic and molecular studies on insects, particularly favoring gene function analysis.

Two-stage hybrid microalgal electroactive wetland-coupled anaerobic digestion for swine wastewater treatment in South China: Full-scale verification.

Constructed wetlands have been widely used for organic wastewater treatment owing to low operating costs and simple maintenance. However, there are some disadvantages such as unstable efficiency in winter. In this study, a microalgal electroactive biofilm-constructed wetland was coupled with anaerobic digestion for full-scale treatment of swine wastewater. In a 12-month outdoor trial, the overall removal efficiencies of chemical oxygen demand, ammonium, nitrate, total nitrogen, total phosphorus, and nitrite reached 98.26%/95.14%, 97.96%/92.07%, 85.45%/66.04%, 95.07%/91.48%, 91.44%/91.52%, and 85.45%/84.67% in summer/winter, respectively. Hydrolytic bacteria were dominant in the anaerobic digestion part, and Cyanobium, Shewanella, and Azoarcus were enriched in the microalgal electroactive biofilm. The operating cost of the entire system was approximately 0.118 $/m3 of wastewater. These results confirm that the microalgal electroactive biofilm significantly enhances the efficiency and stability of constructed wetlands. In conclusion, the anaerobic digestion-microalgal electroactive biofilm-constructed wetland is technically and economically feasible for the treatment of swine wastewater.

Xuanfei Baidu Decoction protects against macrophages induced inflammation and pulmonary fibrosis via inhibiting IL-6/STAT3 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Xuanfei Baidu Decoction (XFBD), one of the "three medicines and three prescriptions" for the clinically effective treatment of COVID-19 in China, plays an important role in the treatment of mild and/or common patients with dampness-toxin obstructing lung syndrome. AIM OF THE STUDY: The present work aims to elucidate the protective effects and the possible mechanism of XFBD against the acute inflammation and pulmonary fibrosis. METHODS: We use TGF-ß1 induced fibroblast activation model and LPS/IL-4 induced macrophage inflammation model as in vitro cell models. The mice model of lung fibrosis was induced by BLM via endotracheal drip, and then XFBD (4.6 g/kg, 9.2 g/kg) were administered orally respectively. The efficacy and molecular mechanisms in the presence or absence of XFBD were investigated. RESULTS: The results proved that XFBD can effectively inhibit fibroblast collagen deposition, down-regulate the level of α-SMA and inhibit the migration of fibroblasts. IL-4 induced macrophage polarization was also inhibited and the secretions of the inflammatory factors including IL6, iNOS were down-regulated. In vivo experiments, the results proved that XFBD improved the weight loss and survival rate of the mice. The XFBD high-dose administration group had a significant effect in inhibiting collagen deposition and the expression of α-SMA in the lungs of mice. XFBD can reduce bleomycin-induced pulmonary fibrosis by inhibiting IL-6/STAT3 activation and related macrophage infiltration. CONCLUSIONS: Xuanfei Baidu Decoction protects against macrophages induced inflammation and pulmonary fibrosis via inhibiting IL-6/STAT3 signaling pathway.

The network pharmacology integrated with pharmacokinetics to clarify the pharmacological mechanism of absorbed components from Viticis fructus extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Viticis fructus (VF) has been widely used in alleviating the swelling and pain, owning to its pharmacologically active components including agnuside, 10-O-vanilloylaucubin, luteolin and casticin. AIM OF THE STUDY: The pharmacokinetic profiles of the absorbed components from aqueous and ethanolic extracts of VF in rat plasma were performed, and explored the molecular mechanisms of absorbed components via network pharmacology. MATERIALS AND METHODS: Ultra-performance liquid chromatography-tandem mass spectroscopy (UHPLC-MS/MS) was employed to identify the absorbed components from rat plasma. Liquid-liquid extraction with ethyl acetate was used to purify the plasma samples. Plasma pharmacokinetics parameters of the components absorbed were analyzed after oral administration of both extracts. Network pharmacology was used to predict the biological functions and potential signaling pathways of VF. The anti-cancer effects of VF extract and absorbed components have been confirmed by in vitro experiments. RESULTS: The method was very sensitive with lower limit of quantification (LLOQ) of 1.0, 2.5, 0.2 and 0.5 ng/mL for agnuside, 10-O-vanilloylaucubin, luteolin and casticin, respectively. With the exception of 10-O-vanilloylaucubin which was not detected in the ethanolic extract of VF, all other components were detected in both extracts in plasma. The pharmacokinetic parameters of the four components from rat plasma were significantly different between the two extracts. According to the results of network pharmacology, the absorption components of VF are enriched in 32 key pathways, and 15 pathways are related to cancer. Ultimately, the anti-cancer effects, as well as the signaling pathways of VF ethanolic extract and absorbed components were verified by in vitro experiments. CONCLUSION: The optimized, sensitive and validated UHPLC-MS/MS method was successfully applied for the plasma pharmacokinetics comparison analysis of the two VF extracts. The combination of network pharmacology and pharmacokinetics provides a useful method to elucidate the biological effects and molecular mechanism of the absorbed components of VF.

Network pharmacology strategy for revealing the pharmacological mechanism of pharmacokinetic target components of San-Ye-Tang-Zhi-Qing formula for the treatment of type 2 diabetes mellitus.

ETHNOPHARMACOLOGICAL RELEVANCE: San-Ye-Tang-Zhi-Qing formula (SYTZQ) is an effective prescription for the treatment of pre-diabetes disorders of glycolipid metabolism in type 2 diabetes mellitus (T2DM). It consists of five Chinese herbs including Mori Folium, Nelumbinis Folium, Crataegi Folium, Salviae Miltiorrhizae Radix et Rhizoma and Paeoniae Radix Rubra. AIM OF THE STUDY: This study was aimed to reveal the pharmacological mechanism of pharmacokinetic target components of SYTZQ for the treatment of T2DM. MATERIALS AND METHODS: A rapid, precise and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to quantify simultaneously nuciferin, vitexin-4″-O-glucoside, vitexin-2″-O-rhamnoside, paeoniflorin and rosmarinic acid in rat plasma after oral administration of SYTZQ. The network pharmacology was used to analyze the effect of the compounds absorbed into the blood of SYTZQ on T2DM. The effects of paeoniflorin, nuciferine and rosmarinic acid on adipogenic differentiation were validated in vitro experiments. RESULTS: The separation was performed on an ACQUITY UHPLC HSS T3 column (2.1 mm × 100 mm, 1.7 µm) using acetonitrile and 0.1% (v/v) formic acid in water as the mobile phase in gradient elution. The calibration curves of five analytes showed good linearity (r ≥ 0.9991) with the lower limits of quantification (LLOQ) between 0.3 and 5.0 ng/mL. The recoveries and matrix effects of five analytes ranged from 81.1% to 113%. The RSDs of inter-day and intra-day precision were all within 13.7%. The validated method was successfully applied to the pharmacokinetic study of five ingredients after oral administration of SYTZQ to rat. 39 major targets and 22 candidate pathways of five compounds absorbed into the blood of rats after administration of SYTZQ were identified and successfully constructed a compound-target-disease-pathway network. It was confirmed that paeniforin, nuciferine and rosmarinic acid could decrease the adipogenicity differentiation in vitro experiments. CONCLUSIONS: The pharmacokinetic parameters indicated that the five components (nuciferin, vitexin-4″-O-glucoside, vitexin-2″-O-rhamnoside, paeoniflorin and rosmarinic acid) were absorbed and eliminated quickly in vivo. These five absorbed components were associated with 22 pathways, including insulin resistance, regulation of lipolysis in adipocytes, PI3k/AKT-, TNF-, cAMP- and cGMP-PKG-signaling pathway. Paeoniflorin, nuciferine and rosmarinic acid have the effect of inhibiting adipocyte differentiation. This study could provide more reference for quality control, and provide a firm basis for evaluating the clinical efficiency of SYTZQ.

Thrombin-based discovery strategy of bioactive-chemical quality marker combination for pollen of Typha orientalis by metabolomics coupled with chemometrics.

BACKGROUND: It is of utmost significance to choose the bioactive components as quality markers for ensuring the effectiveness of traditional Chinese medicine (TCM). Nonetheless, some markers are able to assess effectively the quality of TCM without considering the pharmacological mechanisms and intrinsic chemical complexities. OBJECTIVE: This underscores the need to discover new and efficient markers which can assess both quality and mechanism of action. Herein, a strategy of bioactive-chemical quality marker combination was proposed to improve the level of the quality control of TCM by metabolomics coupled with chemometrics. METHODS: A four-step plan was followed. Firstly, acquisition of metabolic features and component characterization of different batches of pollen of Typha orientalis C.Presl were performed using UHPLC-Q-TOF/MS. Secondly, the direct inhibitory effects of pollen of T. orientalis on thrombin was assessed by using chromogenic substrate method together with HPLC. Thereafter, bioactive-chemical marker combination associated with anti-thrombin segregation was screened using supervised classifiers. Finally, quantitative assay and prediction-model of selected markers were established for guarantying the quality of pollen of T. orientalis. RESULTS: A total of 22 compounds were annotated based on comparison with previous work from pollen of T. orientalis by UHPLC-Q-TOF/MS. Citric acid and linolenic acid inhibited the thrombin activity with IC50 values, 0.52 ± 0.02 and 0.51 ± 0.02 mg/mL, respectively. A bioactive-chemical marker combination including citric acid, linolenic acid, typhaneoside, and isorhamnetin-3-O-neohesperidoside were discovered and selected as quality markers for evaluation of pollen of T. orientalis according to their capacity for inhibiting thrombin. CONCLUSION: The thrombin-based discovery strategy of bioactive-chemical marker combination was a powerful tool for screening the quality markers for evaluation of pollen of T. orientalis.

Simultaneous Determination of Five Alkaloids by HPLC-MS/MS Combined With Micro-SPE in Rat Plasma and Its Application to Pharmacokinetics After Oral Administration of Lotus Leaf Extract.

An environment-friendly and efficient method for simultaneous determination of five alkaloids (nunciferine, O-nornuciferin, liriodenine, armepavine, and pronuciferine) in rat plasma was established by HPLC-MS/MS associated with micro-solid phase extraction (micro-SPE). The plasma sample was pretreated by using micro-SPE columns filled with polymer materials PEP-2 and eluted by little organic solvent (400 µl acetonitrile). The five alkaloids were separated with acetonitrile and 0.1% formic acid aqueous solution on Eclipse plus C18 column. The mode of positive electrospray ionization was used to measure the analytes in multiple-reaction monitoring (MRM). The determination coefficients (R2) of the five alkaloids were greater than 0.99. The lower limit of quantification (LLOQ) of O-nornuciferin, liriodenine, and armepavine was 0.5 ng·ml-1, and that of nunciferine and pronuciferine was 1 ng·ml-1. The validated method was effectively used for the pharmacokinetics of the five orally administrated alkaloids of lotus leaf extract in rat plasma.