Transcription factors containing both C2H2 and homeobox domains play different roles in Verticillium dahliae.

Verticillium dahliae causes Verticillium wilt in more than 200 plant species worldwide. As a soilborne fungus, it forms melanized microsclerotia and colonizes the xylem of host plants. Our previous study revealed a subfamily of C2H2-homeobox transcription factors in V. dahliae, but their biological roles remain unknown. In this study, we systematically characterized the functions of seven C2H2-homeobox transcription factors in V. dahliae. Deletion of VdChtf3 and VdChtf6 significantly decreased the production of melanized microsclerotia, and knockout of VdChtf1 and VdChtf4 enhanced virulence. Loss of VdChtf2 and VdChtf6 increased conidium production, whereas loss of VdChtf5 and VdChtf7 did not affect growth, conidiation, microsclerotial formation, or virulence. Further research showed that VdChtf3 activated the expression of genes encoding pectic enzymes to participate in microsclerotial formation. In addition, VdChtf4 reduced the expression of VdSOD1 to disturb the scavenging of superoxide radicals but induced the expression of genes related to cell wall synthesis to maintain cell wall integrity. These findings highlight the diverse roles of different members of the C2H2-homeobox gene family in V. dahliae. IMPORTANCE: Verticillium dahliae is a soilborne fungus that causes plant wilt and can infect a variety of economic crops and woody trees. The molecular basis of microsclerotial formation and infection by this fungus remains to be further studied. In this study, we analyzed the functions of seven C2H2-homobox transcription factors. Notably, VdChtf3 and VdChtf4 exhibited the most severe defects, affecting phenotypes associated with critical developmental stages in the V. dahliae disease cycle. Our results indicate that VdChtf3 is a potential specific regulator of microsclerotial formation, modulating the expression of pectinase-encoding genes. This finding could contribute to a better understanding of microsclerotial development in V. dahliae. Moreover, VdChtf4 was associated with cell wall integrity, reactive oxygen species (ROS) stress resistance, and increased virulence. These discoveries shed light on the biological significance of C2H2-homeobox transcription factors in V. dahliae's adaptation to the environment and infection of host plants.

Graphene Oxide Nanosheets Toxicity in Mice Is Dependent on Protein Corona Composition and Host Immunity.

Two-dimension graphene oxide (GO) nanosheets with high and low serum protein binding profiles (high/low hard-bound protein corona/HChigh/low) are used in this study as model materials and screening tools to investigate the underlying roles of the protein corona on nanomaterial toxicities in vivo. We proposed that the in vivo biocompatibility/nanotoxicity of GO is protein corona-dependent and host immunity-dependent. The hypothesis was tested by injecting HChigh/low GO nanosheets in immunocompetent ICR/CD1 and immunodeficient NOD-scid II2rγnull mice and performed histopathological and hematological evaluation studies on days 1 and 14 post-injection. HClow GO induced more severe acute lung injury compared to HChigh GO in both immunocompetent and immunodeficient mice, with the effect being particularly pronounced in immunocompetent animals. Additionally, HClow GO caused more significant liver injury in both types of mice, with immunodeficient mice being more susceptible to its hepatotoxic effects. Moreover, administration of HClow GO resulted in increased hematological toxicity and elevated levels of serum pro-inflammatory cytokines in immunocompromised and immunocompetent mice, respectively. Correlation studies were conducted to explore the impact of distinct protein corona compositions on resulting toxicities in both immunocompetent and immunodeficient mice. This facilitated the identification of consistent patterns, aligning with those observed in vitro, thus indicating a robust in vitro-in vivo correlation. This research will advance our comprehension of how hard corona proteins interact with immune cells, leading to toxicity, and will facilitate the development of improved immune-modulating nanomaterials for therapeutic purposes.

Crafting Core-Shell Heterostructures with Enriched Active Centers for High-Energy-Density Symmetric Lithium-Ion Batteries.

Current research strives to create sustainable and ecofriendly organic electrode materials (OEMs) due to the rising concerns about traditional inorganic electrode materials that call for substantial resource consumption in battery manufacturing. However, OEMs often exhibit unbalanced performance, with high capacity conflicting with a long lifespan. Herein, a 2D fully conjugated covalent organic framework featuring abundant C═O and C═N groups (HTPT-COF) was strategically synthesized by coupling 2,3,7,8-tetraamino-1,4,6,9-tetraketone with hexaketocyclohexane octahydrate. It stabilizes the enriched active centers by an extended π-conjugated skeleton, thereby affording a high theoretical capacity in conjunction with potential structure stability. To further unlock the barriers of fast charge, the HTPT-COF was interwoven around highly conductive carbon nanotubes, creating a robust core-sheath heterostructure (HTPT-COF@CNT). Consequently, the crafted HTPT-COF@CNT achieves large reversible capacities of 507.7 mA h g-1, high-rate performance (247.8 mA h g-1 at 20.0 A g-1), and long-term durability (1000 cycles). Aiming to streamline the process and cut the cost of battery manufacturing, all-organic symmetric batteries were well fabricated using HTPT-COF@CNT as both cathode and anode, demonstrating high energy/power density (up to 191.7 W h kg-1 and 3800.3 W kg-1, respectively) and long-term stability over 1000 cycles. Such HTPT-COF@CNT represents a promising sustainable electrode that effectively addresses irreconcilable contradictions encountered by OEMs, boosting the development of advanced organic batteries with high capacity and cycling stability.

[Material basis and mechanism of Bletillae Rhizoma for melasma, gastrointestinal hemorrhage, lung cancer and bronchoplumonary inflammation as "homotherapy for heteropathy" based on UHPLC-Q-Exactive Orbitrap HRMS coupled with network pharmacology and molecular docking].

Based on UHPLC-Q-Exactive Orbitrap HRMS coupled with the network pharmacology and molecular docking, the common material basis and molecular mechanisms of Bletillae Rhizoma for melasma, gastrointestinal hemorrhage, lung cancer and bronchoplumonary inflammation as "homotherapy for heteropathy" were explored. The fingerprint of 17 batches of Bletillae Rhizoma from different areas was established using HPLC, and the similarity analysis was carried out. The common chemical components of the 17 batches of Bletillae Rhizoma were identified using UHPLC-Q-Exactive Orbitrap HRMS. Depending on the bioavailability and drug-like properties of the common components, the active chemical components were screened, and then their protein targets were collected using the Traditional Chinese Medicine Database and Analysis Platform(TCMSP) and SwissTargetPrediction database. The protein targets related to diseases were retrieved from the databases DrugBank, TTD and GeneCards to produce a Venn diagram. The shared targets were obtained between drugs and diseases as "homotherapy for heteropathy" targets. The protein-protein interaction(PPI) was analyzed with the STRING database, and KEGG and GO analyses of the "homotherapy for heteropathy" targets were performed using the Bioconductor database. Cytoscape 3.7.2 software was employed to construct the "chemical components of Bletillae Rhizoma-homotherapy for heteropathy targets" network and PPI network, and topological analysis was conducted to screen out the key active chemical components and core targets. Finally, the affinity between the active components and core targets was evaluated using the molecular docking by AutoDock Vina 4.2.6, which verified the interaction between them. Thirteen common peaks were identified by fingerprint chromatography, and the similarity between different batches was 0.941-0.998. Fifty-three chemical components were identified by mass spectrometry in Bletillae Rhizoma, and 18 common chemical constituents were obtained in the 17 batches of Bletillae Rhizoma. Network pharmacologic screening showed that the pharmacodynamic substances of Bletillae Rhizoma for melasma, gastrointestinal hemo-rrhage, lung cancer and bronchoplumonary inflammation with "homotherapy for heteropathy" were 11 compounds, such as polysaccharides, biphenanthrenes, dihydrophenanthrenes and bibenzyls. There were 42 common targets identified for the treatment of different diseases. These targets were involved in biological processes such as cell response to chemical stress, reactive oxygen species and positive regulation of protein kinase B signal transduction. They were also involved in 121 signaling pathways, encompassing vital pathways such as PI3K-Akt, ErbB, Rap1, FoxO, MAPK and estrogen. Molecular docking results showed a strong affinity between the key active components and the core targets. This study provides a preliminary explanation of how Bletillae Rhizoma exerts its therapeutic effect on chloasma, gastrointestinal hemorrhage, lung cancer, and bronchopneumonic lesions as "homotherapy for heteropathy" through a combined action involving multiple components, targets, and pathways. These findings offer a certain theoretical basis for the further deve-lopment and application of Bletillae Rhizoma.

Rhizobacterial Bacillus enrichment in soil enhances smoke tree resistance to Verticillium wilt.

Verticillium wilt, caused by the soilborne fungus Verticillium dahliae, poses a serious threat to the health of more than 200 plant species worldwide. Although plant rhizosphere-associated microbiota can influence plant resistance to V. dahliae, empirical evidence underlying Verticillium wilt resistance of perennial trees is scarce. In this study, we systemically investigated the effect of the soil microbiota on the resistance of smoke trees (Cotinus coggygria) to Verticillium wilt using field, greenhouse and laboratory experiments. Comparative analysis of the soil microbiota in the two stands of smoke trees suggested that Bacillus represented the most abundant and key microbial genus related to potential disease suppression. Smoke tree seedlings were inoculated with isolated Bacillus strains, which exhibited disease suppressiveness and plant growth-promoting properties. Furthermore, repletion of Bacillus agents to disease conducive soil significantly resulted in reduced incidence of smoke tree wilt and increased resistance of the soil microbiota to V. dahliae. Finally, we explored a more effective combination of Bacillus agents with the fungicide propiconazole to combat Verticillium wilt. The results establish a foundation for the development of an effective control for this disease. Overall, this work provides a direct link between Bacillus enrichment and disease resistance of smoke trees, facilitating the development of green control strategies and measurements of soil-borne diseases.

Enhanced efficacy of brucine dissolving-microneedles as a targeted delivery system in rheumatoid arthritis treatment: a comprehensive pharmacokinetic-pharmacodynamic analysis.

Our previous studies have shown the therapeutic efficacy of brucine dissolving-microneedles (Bru-DMNs) in treating rheumatoid arthritis (RA). Bru delivered via the DMNs can bypass some of the issues related to oral and systemic delivery, including extensive enzymatic activity, liver metabolism and in the case of systemic delivery via hypodermic needles, pain resulting from injections and needle stick injury. However, the underlying mechanism of Bru-DMNs against RA has not been investigated in depth at the pharmacokinetic-pharmacodynamic (PK-PD) level. In this study, a microdialysis-based method combined with ultra-performance liquid chromatography-tandem mass spectrometry was developed for the simultaneous and continuous sampling and quantitative analysis of blood and joint cavities in fully awake RA rats. The acquired data were analyzed by the PK-PD analysis method. Bru delivered via microneedles showed enhanced distribution and prolonged retention in the joint cavity compared to its administration in blood. The correlation between the effect of Bru and its concentration at the action site was indirect. In this study, we explored the mechanism of Bru-DMNs against RA and established a visualization method to express the PK-PD relationship of Bru-DMNs against RA. This study provides insights into the mechanism of action of drugs with potential side effects administered transdermally for RA treatment.

Preparation and evaluation of the PD0721­DOX antibody­drug conjugate targeting EGFRvIII to inhibit glioblastoma.

Epidermal growth factor receptor variant III (EGFRvIII) is prominently expressed in various epithelial tumors. PD0721, a single-chain antibody (scFv), has been developed to specifically target EGFRvIII. Although doxorubicin (DOX) is an essential treatment approach for glioblastoma (GBM), its toxic effects and limited targeting capabilities are a challenge. To overcome the above limitations, antibody-drug conjugates (ADCs) have been developed to exploit the specificity of monoclonal antibodies in directing potent cytotoxic drugs to tumor cells expressing the target antigens. The present study aimed to conjugate DOX with PD0721 scFv to construct a PD0721-DOX ADC targeting EGFRvIII and examine its targeting effect and in vitro anti-GBM activity. PD0721-DOX ADC was generated by combining PD0721 scFv with DOX, using dextran T-10 as a linker. The drug-to-antibody ratio (DAR) was measured by ultraviolet and visible spectrophotometry (UV-Vis). A series of techniques, including cytotoxicity assays, immunofluorescence, cell internalization and flow cytometry assays were employed to evaluate the targeting efficacy and anti-GBM activity of the PD0721-DOX ADC. Following the conjugation of PD0721 scFv with DOX, the UV-Vis results showed a noticeable red shift in the maximum absorbance. The DAR of PD0721 scFv and DOX was 9.23:1. Cytotoxicity assays demonstrated that DK-MG cells treatment with PD0721-DOX ADC at 10 and 20 µg/ml significantly increased cytotoxicity compared with U-87MG ATCC cells (all P<0.01). Confocal microscopy revealed distinct green and red fluorescence in EGFRvIII-expressing DK-MG cells, while no fluorescence was observed in EGFRvIII negative U-87MG ATCC cells. Furthermore, compared with U-87MG ATCC cells, DK-MG cells showed effective internalization of the PD0721-DOX ADC (P<0.001). Finally, flow cytometric analyses indicated that the PD0721-DOX ADC significantly promoted the apoptosis of DK-MG cells compared with U-87MG ATCC cells (P<0.01). In summary, the current study suggested that the PD0721-DOX ADC could exhibit a notable targeting efficacy and potent anti-GBM activity.

[Chemical constituents from Alangium chinense subsp. pauciflorum].

Chemical constituents of 70% ethanol extract of Alangium chinense subsp. pauciflorum were investigated. The 70% ethanol extract of A. chinense subsp. pauciflorum was isolated and purified by D-101 macroporous resins, silica gel, Sephadex LH-20 and other methods. As a result, nineteen compounds were isolated and identified as 4-cyclohexene-1α,2α,3α-triol-1-O-ß-D-glucoside(1), 1ß,4α,6α,13-tetrahydroxy-eudesm-11(12)-ene(2), sucrose(3), 1'-O-benzyl-α-L-rhamnopyranosyl-(1″→6')-ß-D-glucopyranoside(4), bis(2-ethylhexyl)benzene-1,2-dicarboxylate(5),(Z)-10-heneicosenoic acid(6), di-O-methylcrenati(7), methyl-α-D-fructofuranoside(8), ß-daucosterol(9), syringic acid(10), vanillicacid(11), octacosanol(12), isoarborinol(13), 2,7-dihydroxy-6-methyl-4-(1-methylethyl)-1-naphthalenecarboxylate(14),vanillin(15), coniferyl aldehyde(16), 9(11)-dehydroergosterolperoxide(17), 5α,8α-epidioxy-(22E,24R)-ergosta-6,22-dien-3ß-ol(18), ß-sitosterol(19), respectively. Compounds 1 and 2 were new compounds, compounds 5-11, 13, 15-18 were isolated from Alangium for the first time.The anti-inflammatory activity of compourd 1 was determinded by the LPS-induced RAW264.7 macrophage inflammation model. The results showed that the new compound 1 has a certain inhibitory effect on LPS-induced NO production of RAW264.7 cells, and the inhibitory rate was 54.57%.

Linker Mediated Electronic-State Manipulation of Conjugated Organic Polymers Enabling Highly Efficient Oxygen Reduction.

Conjugated polymers with tailorable composition and microarchitecture are propitious for modulating catalytic properties and deciphering inherent structure-performance relationships. Herein, we report a facile linker engineering strategy to manipulate the electronic states of metallophthalocyanine conjugated polymers and uncover the vital role of organic linkers in facilitating electrocatalytic oxygen reduction reaction (ORR). Specifically, a set of cobalt phthalocyanine conjugated polymers (CoPc-CPs) wrapped onto carbon nanotubes (denoted CNTs@CoPc-CPs) are judiciously crafted via in situ assembling square-planar cobalt tetraaminophthalocyanine (CoPc(NH2)4) with different linear aromatic dialdehyde-based organic linkers in the presence of CNTs. Intriguingly, upon varying the electronic characteristic of organic linkers from terephthalaldehyde (TA) to 2,5-thiophenedicarboxaldehyde (TDA) and then to thieno/thiophene-2,5-dicarboxaldehyde (bTDA), their corresponding CNTs@CoPc-CPs exhibit gradually improved electrocatalytic ORR performance. More importantly, theoretical calculations reveal that the charge transfer from CoPc units to electron-withdrawing linkers (i.e., TDA and bTDA) drives the delocalization of Co d-orbital electrons, thereby downshifting the Co d-band energy level. Accordingly, the active Co centers with more positive valence state exhibit optimized binding energy toward ORR-relevant intermediates and thus a balanced adsorption/desorption pathway that endows significant enhancement in electrocatalytic ORR. This work demonstrates a molecular-level engineering route for rationally designing efficient polymer catalysts and gaining insightful understanding of electrocatalytic mechanisms.

An enhanced cardio-protective effect of nanoparticles loaded with active components from Polygonum orientale L. against isoproterenol-induced myocardial ischemia in rats.

In this study, nanoparticles loaded with active components from Polygonum orientale L. (PO), a traditional Chinese herb known for its anti-myocardial ischemic properties, were investigated for cardio-protective properties. Specifically, OVQ-Nanoparticles (OVQ-NPs) with Orientin (Ori), Vitexin (Vit), and Quercetin (Que) was obtained by double emulsion-solvent evaporation method. The OVQ-NPs exhibited a spherical shape, with a uniform size distribution of 136.77 ± 3.88 nm and a stable ζ-potential of -13.40 ± 2.24 mV. Notably, these nanoparticles exhibited a favorable sustained-release characteristic, resulting in an extended circulation time within the living organism. Consequently, the administration of these nanoparticles resulted in significant improvements in electrocardiograms and heart mass index of myocardial ischemic rats induced by isoproterenol, as well as decreased serum levels of CK, LDH, and AST. Furthermore, the results of histopathological examination, such as H&E staining and TUNEL staining, confirmed a reduced level of cardiac tissue pathology and apoptosis. Moreover, the quantification of biochemical indicators (SOD, MDA, GSH, NO, TNF-α, and IL-6) demonstrated that OVQ-NPs effectively mitigated myocardial ischemia by regulating oxidative stress and inflammatory pathways. In conclusion, OVQ-NPs demonstrate promising therapeutic potential as an intervention for myocardial ischemia, providing a new perspective on traditional Chinese medicine treatment in this area.

Molecule and Microstructure Modulations of Cyano-Containing Electrodes for High-Performance Fully Organic Batteries.

Cyano-containing electrodes usually promise high theoretical potentials while suffering from uncontrollable self-dissolution and sluggish reaction kinetics. Herein, to remedy their limitations, an unprecedented core-shell heterostructured electrode of carbon nanotubes encapsulated in poly(1,4-dicyanoperfluorobenzene sulfide) (CNT@PFDCB) is rationally crafted via molecule and microstructure modulations. Specifically, the linkage of sulfide bridges of PFDCB prevents the active cyano groups from dissolving, resulting in a robust structure. The fluorinations modulate the electronic configurations in frontier orbitals, allowing higher electrical conductivity and elevated output voltage. Combined with the core-shell architecture to unlock the sluggish diffusion kinetics for both electrons and guest ions, the CNT@PFDCB exhibits an impressive capacity (203.5 mAh g-1), remarkable rate ability (127.6 mAh g-1 at 3.0 A g-1), and exceptional cycling stability (retaining 81.1 % capacity after 3000 cycles at 1.0 A g-1). Additionally, the Li-storage mechanisms regarding PFDCB are thoroughly revealed by in situ attenuated total reflection infrared spectroscopy, in situ Raman spectroscopy, and theoretical simulations, which involve the coordination interaction between Li ions and cyano groups and the electron delocalization along the conjugated skeleton. More importantly, a practical fully organic cell based on the CNT@PFDCB is well-validated that demonstrates a tremendous potential of cyanopolymer as the cathode to replace its inorganic counterparts.

Integration of metabolomics and transcriptomics to reveal the mechanism of Gerberae piloselloidis herba in alleviating bronchial asthma.

ETHNOPHARMACOLOGICAL RELEVANCE: Gerberae Piloselloides Herba (GPH) is derived from Gerbera piloselloides (Linn.) Cass. It is a commonly used traditional medicine in China, featured by its special bioactivities as antitussive, expectorant, anti-asthma, anti-bacterial and anti-tumor. It is often used as an effective treatment for cough and sore throat as well as bronchial asthma (BA) in China. It was demonstrated in our previous studies that GPH exerted significant effects on the treatment of BA, but its underlying mechanism remains unclear. AIM OF THE STUDY: This study was aimed at revealing the mechanism through which GPH protects against BA. MATERIALS AND METHODS: The protective effect of GPH against BA was evaluated in a mouse model of BA induced by ovalbumin. Through integrated metabolomics and transcriptomics analysis, the most critical pathways were discovered. The effects of GPH in regulating these pathways was verified through molecular biology experiments and molecular docking. RESULTS: GPH have anti-BA effects. In plasma and lung tissue, 5 and 17 differentially expressed metabolites (DEMs), respectively, showed a reversed tendency in the GPH group compared with the model group; apart from gamma-aminobutyric acid and butyrylcarnitine, these DEMs might aid in BA diagnosis. The DEMs were involved primarily in the regulation of lipid metabolism, followed by glucose metabolism and amino acid metabolism. Transcriptomic analysis indicated that GPH modulated 268 differentially expressed genes (DEGs). Integration analysis of metabolomics and transcriptomics revealed that GPH might regulate the PPAR signaling pathway, thus affecting the expression of key gene targets such as Cyp4a12a, Cyp4a12b, Adh7, Acaa1b and Gpat2; controlling fatty acid degradation, unsaturated fatty acid biosynthesis, glycerophospholipid metabolism and other lipid metabolic pathways; and ameliorating BA. This possibility was confirmed through reverse-transcription quantitative polymerase chain reaction, western blotting, immunofluorescence and molecular docking. CONCLUSION: GPH was found to activate the PPAR signaling pathway, decrease the levels of Cyp4a12a and Cyp4a12b, and increase the levels of Adh7, Acaa1b and Gpat2, thereby regulating lipid metabolism disorder, decreasing the generation of inflammatory mediators and limiting lung injury.

Kss1 of Verticillium dahliae regulates virulence, microsclerotia formation, and nitrogen metabolism.

Verticillium dahliae causes destructive vascular wilt diseases on more than 200 plant species, including economically important crops and ornamental trees worldwide. The melanized microsclerotia (MS) enable V. dahliae to survive for years in soil, thus the fungus is especially difficult to control once it has become established. Previously, we found that the mitogen activated protein kinase VdSte11 (MAPKKK) plays key roles in MS formation, penetration, and virulence in V. dahliae. In this study, two MAPK homologs of the yeast Ste7p and Kss1p were identified and characterized in V. dahliae. Deletion of VdSte7 or VdKss1 reuslted in severe defects in melaninized MS formation and virulence. Furthermore, phosphorylation assays demonstrated that VdSte11 and VdSte7 can phosphorylate VdKss1 in V. dahliae. Proteomic analysis revealed a significant change in sterol biosynthesis with a fold change of ≥ 1.2 after the deletion of VdKss1. In addition, phosphoproteomic analysis showed that VdKss1 was involved in the regulation of nitrogen metabolism. Finally, we identified VdRlm1 as a potentially downstream target of VdKss1, which is involved in regulating ammonium nitrogen utilization. This study sheds light on the network of regulatory proteins in V. dahliae that affect MS formation and nitrogen metabolism.

CRISPR-based platforms for the specific and dual detection of defoliating/nondefoliating strains of Verticillium dahliae.

BACKGROUND: Verticillium dahliae is a soil-borne pathogenic fungus that causes Verticillium wilt disease on more than 400 plant species worldwide. Because of its broad host range and its ability to survive long term in the soil, there are few effective control measures for V. dahliae once it has become established. Accurate, sensitive, and rapid detection of V. dahliae is crucial for limiting pathogen entry into new regional environments and early management of Verticillium wilt. RESULTS: In this study, we developed a method to detect V. dahliae based on recombinase polymerase amplification (RPA) and CRISPR/Cas technology and used fluorescence and lateral flow test strips to monitor the outcomes. Through the establishment and optimization of RPA-CRISPR/Cas13a detection, the sensitivity of the fluorescence method was 1 am for genomic DNA (gDNA) within 20 min, whereas the sensitivity of the lateral flow strip method was 100 am for gDNA in 30 min. The field applicability of RPA-CRISPR/Cas13a was also validated by the detection of V. dahliae on smoke trees (Cotinus coggygria) in Xiangshan Park, Beijing, China. Finally, diplex detection for defoliating and nondefoliating pathotypes of V. dahliae was established by combining CRISPR-Cas12a/Cas13a with specific target genes. CONCLUSION: Taken together, this study achieved rapid, sensitive, and accurate detection of V. dahliae and the differentiation of defoliating and nondefoliating pathotypes and provides potential for field-deployable diagnostic tools for rapid and ultrasensitive detection. © 2023 Society of Chemical Industry.

Metabolites-Based Network Pharmacology to Preliminarily Verify In Vitro Anti-Inflammatory Effect of Ardisiacrispin B.

Ardisiae Crenatae Radix is an ethnic medicinal herb with good anti-inflammatory activity. Ardisiacrispin B is one of the main components in Ardisiae Crenatae Radix extract, with a content of up to 16.27%, and it may be one of the pharmacological components through which Ardisiae Crenatae Radix exerts anti-inflammatory activity. At present, reports on ardisiacrispin B mainly focus on anti-tumor effects, and there have been no reports on anti-inflammatory activities. As a triterpenoid saponin, due to its large molecular weight and complex structure, the composition of substances that function in the body may include other forms after metabolism, in addition to compounds with original structures. Exploring the anti-inflammatory effects on the prototypes and metabolites of the compound may provide a more comprehensive response to the characteristics of ardisiacrispin B's anti-inflammatory action. In this study, ardisiacrispin B was analyzed for metabolites to explore its metabolic processes in vivo. Subsequently, the anti-inflammatory effects of the prototypes and metabolites were further analyzed through network pharmacology, with the expectation of discovering the signaling metabolic pathways through which they may act. Finally, the anti-inflammatory effects of ardisiacrispin B in vitro and the effects on key signaling pathways at the protein level were explored. The results of this study showed that the isolated compounds were confirmed to be ardisiacrispin B. After the metabolite analysis, a total of 26 metabolites were analyzed, and the metabolism process in rats mainly involves oxidation, dehydration, glucuronide conjugation, and others. Speculation as to the anti-inflammatory molecular mechanisms of the prototypes and metabolites of ardisiacrispin B revealed that it may exert its anti-inflammatory effects mainly by affecting the PI3K-AKT pathway. Further anti-inflammatory mechanisms demonstrated that ardisiacrispin B had a good anti-inflammatory effect on LPS-induced RAW264.7 cells and a strong inhibitory effect on NO, TNF-α, and IL-1ß release in cells. Furthermore, it had significant inhibitory effects on the expression of PI3K, P-PI3K, AKT, and P-AKT. This study supplements the gaps in the knowledge on the in vivo metabolic process of ardisiacrispin B and explores its anti-inflammatory mechanism, providing an experimental basis for the development and utilization of pentacyclic triterpenoids.

Mechanistic study of cytochrome P450 enzyme-mediated cytotoxicity of psoralen and isopsoralen.

Psoralen and isopsoralen are the major components responsible for Psoraleae Fructus-induced hepatotoxicity. This study explored the role of metabolic activation by cytochrome P450 (CYP) enzymes in psoralen- and isopsoralen-induced cytotoxicity and its potential mechanisms. Inhibitors of CYP1A2, 2C9, 2C19, 2D6, 2E1, and 3A4 were used to screen specific CYP enzymes responsible for the metabolic activation of psoralen and isopsoralen in mouse primary hepatocytes, which was verified using the corresponding transfected cell lines. Network toxicology and transcriptome analyses were performed to explore the mechanisms underlying toxicity. Psoralen and isopsoralen decreased the viability of mouse primary hepatocytes, whereas the inhibition of CYP2C9, 2C19, 2D6, and 2E1 significantly increased their viability. Psoralen-induced cytotoxicity was significantly enhanced by the overexpression of CYP2C19 in Chinese hamster ovary cells, whereas the overexpression of the above CYP enzymes did not affect the cytotoxicity of isopsoralen. Psoralen- and isopsoralen-induced cytotoxic effects were associated with putative core targets (i.e., Fn1, Thbs1, and Tlr2) and multiple signaling pathways (e.g., PI3K-Akt, MAPK, and TNF pathways). Our results demonstrate that the metabolic activation of psoralen and isopsoralen is mediated by CYP enzymes, thereby regulating multiple core targets and signaling pathways and resulting in cytotoxicity.

miR159a modulates poplar resistance against different fungi and bacteria.

Trees are inevitably attacked by different kinds of pathogens in their life. However, little is known about the regulatory factors in poplar response to different pathogen infections. MicroRNA159 (miR159) is a highly conserved microRNA (miRNA) in plants and regulates plant development and stress responses. Here, transgenic poplar overexpressing pto-miR159a (OX-159) showed antagonistic regulation mode to poplar stem disease caused by fungi Cytospora chrysosperma and bacteria Lonsdalea populi. OX-159 lines exhibited a higher susceptibility after inoculation with bacterium L. populi, whereas enhanced disease resistance to necrotrophic fungi C. chrysosperma compared with wild-type (WT) poplars. Intriguingly, further disease assay found that OX159 line rendered the poplar susceptible to hemi-biotrophic fungi Colletotrichum gloeosporioide, exhibiting larger necrosis and lower ROS accumulation than WT lines. Transcriptome analyses revealed that more down-regulated differentially expressed genes with disease-resistant domains in OX-159 line compared with WT line. Moreover, the central mediator NPR1 of salicylic acid (SA) pathway showed a decrease in expression level, while jasmonic acid/ethylene (JA/ET) signal pathway marker genes ERF, as well as PR3, MPK3, and MPK6 genes showed an increase level in OX159-2 and OX159-5 compared with WT lines. Further spatio-temporal expression analysis revealed JA/ET signaling was involved in the dynamic response process to C. gloeosporioides in WT and OX159 lines. These results demonstrate that overexpression of pto-miR159a resulted in the crosstalk changes of the downstream hub genes, thereby controlling the disease resistance of poplars, which provides clues for understanding pto-miR159a role in coordinating poplar-pathogen interactions.

[Qualitative and quantitative study of constituents in Lysionoti Herba based on UHPLC-Q-Exactive Orbitrap HRMS and HPLC-UV].

Ultra-high performance liquid chromatography-quadrupole-Exactive Orbitrap high resolution mass spectrometry(UHPLC-Q-Exactive Orbitrap HRMS) was employed to systematically analyze the chemical constituents in Lysionoti Herba, and high perfor-mance liquid chromatography-ultraviolet(HPLC-UV) to determine the content of main compounds. A Synergi~(TM) Hydro-RP 100 Å colu-mn(2 mm×100 mm, 2.5 µm) was used for gradient elution with acetonitrile-0.1% aqueous formic acid as the mobile phase at a flow rate of 0.2 mL·min~(-1) and a column temperature of 40 ℃. MS and MS/MS were conducted with electrospray ionization(ESI) in both positive and negative modes. The chemical components in Lysionoti Herba were identified by comparison with the retention time and mass spectra of reference compounds and the relevant mass spectral data reported in MS databases and relevant literature. Furthermore, the content of five constituents(neochlorogenic acid, chlorogenic acid, forsythoside B, acteoside, and nevadensin) in different Lysiono-ti Herba samples was simultaneously determined by HPLC-UV at the wavelength of 330 nm. A total of 84 compounds were identified in Lysionoti Herba, including 27 flavonoids, 20 phenylethanoid glycosides, 5 amino acids, 18 organic acids, 1 alkaloid, 6 nucleosides, and 7 others. The content of neochlorogenic acid, chlorogenic acid, forsythoside B, acteoside, and nevadensin showed good linear relationship(r>0.999) with the peak area within certain concentration ranges, which were 3.22-102.90, 12.84-410.82, 31.63-1 012.01, 25.00-800.11, and 4.08-130.51 µg·mL~(-1), respectively. The instrument precision, method repeatability, and solution stability all met requirement, and the average recovery rate was 97.31%-100.2%, with RSD ranging from 0.95% to 2.4%. The content of the five components varied among different Lysionoti Herba samples collected from different regions of Guizhou, and the average content of forsythoside B was the highest. The established qualitative method can rapidly and efficiently identify the chemical components of Lysionoti Herba, and the developed HPLC-UV method can simultaneously determine the content of five components in a simple, ra-pid, and accurate manner, providing a scientific basis for the quality evaluation of Lysionoti Herba.

The Transcription Factor CcRlm1 Regulates Cell Wall Maintenance and Poplar Defense Response by Directly Targeting CcChs6 and CcGna1 in Cytospora chrysosperma.

Maintenance of cell wall integrity is important for fungal cell morphology against external stresses and even virulence. Although the transcription factor Rlm1 is known to play major regulatory roles in the maintenance of cell integrity, the underlying mechanism of how Rlm1 contributes to cell wall integrity and virulence in phytopathogenic fungi remains unclear. Here, we demonstrated that CcRlm1 plays important roles in cell wall maintenance and virulence in the poplar canker fungus Cytospora chrysosperma. Among putative downstream targets, CcChs6 (chitin synthase) and CcGna1 (glucosamine 6-phosphate N-acetyltransferase) were found to be direct targets of CcRlm1 and shown to function in chitin synthesis and virulence. Furthermore, we found stronger induction of poplar defense responses when challenged with these gene deletion mutants. Collectively, these results suggest that CcRlm1 plays a critical role in the regulation of cell wall maintenance, stress response, and virulence by directly regulating CcChs6 and CcGna1 in C. chrysosperma. IMPORTANCE Cytospora chrysosperma causes canker diseases on woody plants, and the molecular basis of its infection is not well understood. This study shows that CcRlm1 is the major regulator of chitin synthesis and virulence of the poplar canker fungus. Our research contributes to further understanding the molecular basis of the interaction between C. chrysosperma and poplar.

Intranasal administration of edaravone nanoparticles improves its stability and brain bioavailability.

The clinical application of EDV, a potent antioxidant drug approved for amyotrophic lateral sclerosis (ALS), is limited by its short biological half-life and poor water solubility necessitating hospitalization during intravenous infusion. Nanotechnology-based drug delivery constitutes a powerful tool through inferring drug stability and targeted drug delivery improving drug bioavailability at the diseased site. Nose-to-brain drug delivery offers direct access to the brain bypassing the blood brain barrier and reducing systemic biodistribution. In this study, we designed EDV-loaded poly(lactic-co-glycolic acid) (PLGA)-based polymeric nanoparticles (NP-EDV) for intranasal administration. NPs were formulated by the nanoprecipitation method. Morphology, EDV loading, physicochemical properties, shelf-life stability, in vitro release and pharmacokinetic assessment in mice were conducted. EDV was efficiently loaded into ∼90 nm NPs, stable up to 30 days of storage, at ∼3% drug loading. NP-EDV reduced H2O2-induced oxidative stress toxicity in mouse microglial cell line BV-2. Optical imaging and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) showed that intranasal delivery of NP-EDV offered higher and more sustained brain uptake of EDV compared to intravenous administration. This study is the first of its kind to develop an ALS drug in a nanoparticulate formulation for nose-to-brain delivery raising hope to ALS patients where currently treatment options are limited to two clinically approved drugs only.