An efficient visual screening of gene knockout mutants in the insect pathogenic fungus Beauveria bassiana.

Beauveria bassiana is an entomopathognic fungus, which is widely employed in the biological control of pests. Gene disruption is a common method for studying the functions of genes involved in fungal development or its interactions with hosts. However, generating gene deletion mutants was a time-consuming work. The transcriptional factor OpS3 has been identified as a positive regulator of a red secondary metabolite oosporein in B. bassiana. In this study, we have designed a new screening system by integrating a constitutive OpS3 expression cassette outside one of the homologous arms of target gene. Ectopic transformants predominantly exhibit a red colour with oosporein production, while knockout mutants appear as white colonies due to the loss of the OpS3 expression cassette caused by recombinant events. This screening strategy was used to obtain the deletion mutants of both tenS and NRPS genes. Correct mutants were obtained by screening fewer than 10 mutants with a positive efficiency ranging from 50% to 75%. This system significantly reduces the workload associated with DNA extraction and PCR amplification, thereby enhancing the efficiency of obtaining correct transformants in B. bassiana.

BpAFP, a Broussonetia papyrifera latex chitinase, exhibits a dual role in resisting to both Verticillium wilt disease and lepidopterous pests, Plutella xylostella and Prodenia litura.

Paper mulberry (Broussonetia papyrifera) is a fast-growing tree known for its tolerance to diverse biotic and abiotic stresses. To explore genes combating Verticillium wilt, a devasting and formidable disease damage to cotton and many economically significant crops, we purified an antifungal protein, named BpAFP, from the latex of paper mulberry. Based on peptide fingerprint, we cloned the full cDNA sequence of BpAFP and revealed that BpAFP belongs to Class I chitinases, sharing 74 % identity with B. papyrifera leaf chitinase, PMAPII. We further introduced BpAFP into Arabidopsis, tobacco, and cotton. Transgenic plants exhibited significant resistance to Verticillium wilt. Importantly, BpAFP also demonstrated insecticidal activity against herbivorous pests, Plutella xylostella, and Prodenia litura, when feeding the larvae with transgenic leaves. Our finding unveils a dual role of BpAFP in conferring resistance to both plant diseases and lepidopterous pests.

Expression of a viral ecdysteroid UDP-glucosyltransferase enhanced the insecticidal activity of the insect pathogenic fungus Beauveria bassiana.

BACKGROUND: Entomopathogenic fungi, such as Beauveria bassiana, hold promise as biological control agents against insect pests. However, the efficacy of these fungi can be hindered by insect immune responses. One strategy to enhance fungal virulence is to manipulate host immune by targeting key regulatory molecules like 20-hydroxyecdysone (20E). RESULTS: In this study, we engineered B. bassiana strains to constitutively express the enzyme ecdysteroid UDP-glucosyltransferase (EGT), which inactivates 20E, a crucial insect molting hormone. The engineered strain Bb::EGT-1 exhibited robust expression of EGT, leading to a significant reduction in insect 20E levels upon infection. Moreover, infection with Bb::EGT-1 resulted in accelerated larval mortality. Immune responses analysis revealed repression of insect immune response genes and decreased phenoloxidase (PO) activity in larvae infected with Bb::EGT-1. Microbiome analysis indicated alterations in bacterial composition within infected insects, with increased abundance observed during infection with Bb::EGT-1. Additionally, the presence of bacteria hindered hyphal emergence from insect cadavers, suggesting a role for microbial competition in fungal dissemination. CONCLUSIONS: Constitutive expression of EGT in B. bassiana enhances fungal virulence by reducing insect 20E levels, suppressing immune responses, and altering the insect microbiome. These findings highlighted the potential of engineered fungi as effective biocontrol agents against insect pests and provide insights into the complex interactions between entomopathogenic fungi, their hosts, and associated microbes. © 2024 Society of Chemical Industry.

Synthesis and antitumor activities of novel 3-(6-aminopyridin-3-yl)benzamide derivatives: Inducing cell cycle arrest and apoptosis via AURKB transcription inhibition.

Here, a series of 3-(6-aminopyridin-3-yl) benzamide derivatives were designed and synthesized. Cell viability assay indicated that most compounds exhibited potent antiproliferative activity against all the tested cancer cells. Among them, compound 7l displayed the best antiproliferative activity particularly in A549 cells, with an IC50 value of 0.04 ± 0.01 µM. RNA-seq analysis was employed to explore the potential pathways related to the antiproliferative activity of compound 7l. The data revealed that 7l exerted antiproliferative activity mainly by regulating cell cycle, DNA replication and p53 signaling pathway. Indeed, compound 7l induced G2/M phase arrest by AURKB transcription inhibition and resulted in cell apoptosis via p53 signaling pathway. Most importantly, compound 7l demonstrated potent antitumor activity in A549 xenograft tumor model. Collectively, 7l might be a promising lead compound for the development of new therapeutic agents for AURKB overexpressed or mutated cancers.

Electron-transferring flavoprotein and its dehydrogenase contributed to growth development and virulence in Beauveria bassiana.

Electron-transferring flavoprotein (Etf) and its dehydrogenase (Etfdh) are integral components of the electron transport chain in mitochondria. In this study, we characterize two putative etf genes (Bbetfa and Bbetfb) and their dehydrogenase gene Bbetfdh in the entomopathogenic fungus Beauveria bassiana. Individual deletion of these genes caused a significant reduction in vegetative growth, conidiation, and delayed conidial germination. Lack of these genes also led to abnormal metabolism of fatty acid and increasing lipid body accumulation. Furthermore, the virulence of Bbetfs and Bbetfdh deletion mutants was severely impaired due to decreasing infection structure formation. Additionally, all deletion strains showed reduced ATP synthesis compared to the wild-type strain. Taken together, Bbetfa and Bbetfb, along with Bbetfdh, play principal roles in fungal vegetative growth, conidiation, conidial germination, and pathogenicity of B. bassiana due to their essential functions in fatty acid metabolism.

An Oxidoreductase-like Protein is Required for Verticillium dahliae Infection and Participates in the Metabolism of Host Plant Defensive Compounds.

Verticillium dahliae, a notorious phytopathogenic fungus, is responsible for vascular wilt diseases in numerous crops. Uncovering the molecular mechanisms underlying pathogenicity is crucial for controlling V. dahliae. Herein, we characterized a putative oxidoreductase-like protein (VdOrlp) from V. dahliae that contains a functional signal peptide. While the expression of VdOrlp was low in artificial media, it significantly increased during host infection. Deletion of VdOrlp had minimal effects on the growth and development of V. dahliae but severely impaired its pathogenicity. Metabolomic analysis revealed significant changes in organic heterocyclic compounds and phenylpropane compounds in cotton plants infected with ΔVdOrlp and V991. Furthermore, VdOrlp expression was induced by lignin, and its deletion affected the metabolism of host lignin and phenolic acids. In conclusion, our results demonstrated that VdOrlp plays an important role in the metabolism of plant phenylpropyl lignin and organic heterocyclic compounds and is required for fungal pathogenicity in V. dahliae.

Three-Dimensional Polyoxometalate Organic Frameworks with One-Dimensional Channels Constructed by Multiple Helical Chains Based on 22-Core Ln/Mn/Mo Clusters for Proton Conduction.

Two unique 22-core sandwich {[Mn6Mo6O37]Ln3[MnMo6O24]} (Ln = La or Pr) units have been assembled, featuring an undisclosed {Mn6Mo6} cluster. This assembly is subsequently integrated into two three-dimensional polyoxometalate organic frameworks, which exhibit one-dimensional hydrophilic hexagonal channels formed by six intertwined 63 helical chains, leading to effective proton conduction primarily facilitated by an abundance of water molecules within the channels.

Bbhox2 is a key regulator for conidiation and virulence in Beauveria bassiana.

Beauveria bassiana, a well-known filamentous biocontrol fungus, is the main pathogen of numerous field and forest pests. To explore the potential factors involved in the fungal pathogenicity, Bbhox2, an important and conserved functional transcription factor containing homeodomain was carried out by functional analysis. Homologous recombination was used to disrupt the Bbhox2 gene in B.bassiana. The conidia yield of the deletant fungal strain was significantly reduced. The conidial germination was faster, and stress tolerance to Congo red and high osmotic agents were decreased compared with that in the wildtype. Additionally, ΔBbhox2 showed a dramatic reduction in virulence no matter in topical inoculations or in intra-hemolymph injections against Galleria mellonella larvae, which is likely due to the failure of appressorium formation and the defect in producing hyphal body. These results indicate that the Bbhox2 gene markedly contributes to conidiation and pathogenicity in B. bassiana.

Zeaamine, a new amine from roots of Zea mays and its cytotoxic activity against CT26 and SW480 cell lines.

A new amine, zeaamine (1), along with nine known compounds (2-10), were isolated from the roots of Zea mays. Among these, compound 2 was first isolated from this plant, and compound 3 was first isolated from the roots. In the current investigation, the cytotoxicity against CT26 and SW480 cells of the compounds were evaluated. Zeaamine (1) exhibited moderately affected CT26 and SW480 cells with IC50 values of 17.91 and 10.21 µM.

Roots of Zea mays L.: As a Potential Source to Treat Sodium Oxalate-Induced Renal Cell Injury.

Zea mays L. is an annual grass of the Gramineae family and is known as one of the cereal crops. Its by-products exhibited significant medicinal properties. In some regions of China, water extracts of Z. mays roots (RM) are utilized to treat kidney stones, but no research has been reported. In our present study, a bioassay-guided isolation method was used to yield five new lignans (1-5) as well as 15 known components, among which 8-15 and 17-20 were first identified from the genus. The fractions and all components were evaluated for their abilities to inhibit sodium oxalate-induced injury to human proximal tubular HK-2 cells. Fraction 50W and compounds 3, 4, and 11 exhibited the most potent activities. Further investigation indicated that these potential agents inhibited the LDH release, decreased the MDA and H2O2 concentrations, and increased the level of SOD2 in HK-2 cells. These results indicated that RM is a promising and valuable crop waste for further development and utilization in nephrolithiasis pharmaceutical research.

Dual-Ligand Synergistic Targeting Anti-Tumor Nanoplatforms with Cascade-Responsive Drug Release.

Dual-ligand targeting drug delivery nanoplatforms are considered a promising tool for enhancing the specificity of chemotherapy. However, serious off-target delivery has been observed in current dual-ligand targeting nanoplatforms, as each ligand can independently recognize receptors on the cell membrane surface and guide drug nanocarriers to different cells. To overcome this barrier, a dual-ligand synergistic targeting (DLST) nanoplatform is developed, which can guide chemotherapy treatment specifically to cancer cells simultaneously overexpressing two receptors. This nanoplatform consists of a singlet oxygen (1O2) photosensitizer-loaded nanocarrier and a drug-loaded nanocarrier with 1O2 responsiveness, which were, respectively, decorated with a pair of complementary DNA sequences and two different ligands. For cancer cells overexpressing both receptors, two nanocarriers can be internalized in larger quantities to cause DNA hybridization-induced nanocarrier aggregation, which further activates 1O2-triggered drug release under light irradiation. For cells overexpressing a single receptor, only one type of nanocarrier can be internalized in a large quantity, leading to blocked drug release due to the ultrashort action radius of 1O2. In vivo evaluation showed this DLST nanoplatform displayed highly specific tumor treatment with minimized long-term toxicity. This is a highly efficient drug delivery system for DLST chemotherapy, holding great potential for clinical applications.

Cell detoxification of secondary metabolites by P4-ATPase-mediated vesicle transport.

Mechanisms for cellular detoxification of drug compounds are of significant interest in human health. Cyclosporine A (CsA) and tacrolimus (FK506) are widely known antifungal and immunosuppressive microbial natural products. However, both compounds can result in significant side effects when used as immunosuppressants. The insect pathogenic fungus Beauveria bassiana shows resistance to CsA and FK506. However, the mechanisms underlying the resistance have remained unknown. Here, we identify a P4-ATPase gene, BbCRPA, from the fungus, which confers resistance via a unique vesicle mediated transport pathway that targets the compounds into detoxifying vacuoles. Interestingly, the expression of BbCRPA in plants promotes resistance to the phytopathogenic fungus Verticillium dahliae via detoxification of the mycotoxin cinnamyl acetate using a similar pathway. Our data reveal a new function for a subclass of P4-ATPases in cell detoxification. The P4-ATPases conferred cross-species resistance can be exploited for plant disease control and human health protection.

Three New Constituents with Anti-Inflammatory and Antimicrobial Activities in Vitro from the Roots of Capsicum annuum L.

Three new compounds, including two new sesquiterpenes (1-2), named Annuumine E-F, and one new natural product, 3-hydroxy-2,6-dimethylbenzenemethanol (3), together with seventeen known compounds (4-20) were isolated from the ethanol extract of the roots of Capsicum annuum L. Among them, five compounds (4, 5, 9, 10 and 20) were isolated from this plant for the first time. The structures of new compounds (1-3) were determined via detailed analysis of the IR, HR-ESI-MS and 1D and 2D NMR spectra. The anti-inflammatory activities of the isolated compounds were evaluated by their ability to reduce NO release by LPS-induced RAW 264.7 cells. Notably, compound 11 exhibited moderate anti-inflammatory activity (IC50 =21.11 µM). Moreover, the antibacterial activities of the isolated compounds were also evaluated.

Design, Synthesis, and Evaluation of the COX-2 Inhibitory Activities of New 1,3-Dihydro-2H-indolin-2-one Derivatives.

Thirty-three 1,3-dihydro-2H-indolin-2-one derivatives bearing α, ß-unsaturated ketones were designed and synthesized via the Knoevenagel condensation reaction. The cytotoxicity, in vitro anti-inflammatory ability, and in vitro COX-2 inhibitory activity of all the compounds were evaluated. Compounds 4a, 4e, 4i-4j, and 9d exhibited weak cytotoxicity and different degrees of inhibition against NO production in LPS-stimulated RAW 264.7 cells. The IC50 values of compounds 4a, 4i, and 4j were 17.81 ± 1.86 µM, 20.41 ± 1.61 µM, and 16.31 ± 0.35 µM, respectively. Compounds 4e and 9d showed better anti-inflammatory activity with IC50 values of 13.51 ± 0.48 µM and 10.03 ± 0.27 µM, respectively, which were lower than those of the positive control ammonium pyrrolidinedithiocarbamate (PDTC). Compounds 4e, 9h, and 9i showed good COX-2 inhibitory activities with IC50 values of 2.35 ± 0.04 µM, 2.422 ± 0.10 µM and 3.34 ± 0.05 µM, respectively. Moreover, the possible mechanism by which COX-2 recognized 4e, 9h, and 9i was predicted by molecular docking. The results of this research suggested that compounds 4e, 9h, and 9i might be new anti-inflammatory lead compounds for further optimization and evaluation.

Cytostatic Activity of Sanguinarine and a Cyanide Derivative in Human Erythroleukemia Cells Is Mediated by Suppression of c-MET/MAPK Signaling.

Sanguinarine (1) is a natural product with significant pharmacological effects. However, the application of sanguinarine has been limited due to its toxic side effects and a lack of clarity regarding its molecular mechanisms. To reduce the toxic side effects of sanguinarine, its cyanide derivative (1a) was first designed and synthesized in our previous research. In this study, we confirmed that 1a presents lower toxicity than sanguinarine but shows comparable anti-leukemia activity. Further biological studies using RNA-seq, lentiviral transfection, Western blotting, and flow cytometry analysis first revealed that both compounds 1 and 1a inhibited the proliferation and induced the apoptosis of leukemic cells by regulating the transcription of c-MET and then suppressing downstream pathways, including the MAPK, PI3K/AKT and JAK/STAT pathways. Collectively, the data indicate that 1a, as a potential anti-leukemia lead compound regulating c-MET transcription, exhibits better safety than 1 while maintaining cytostatic activity through the same mechanism as 1.

Design, Synthesis and Biological Evaluation of 6-(Imidazo[1,2-a]pyridin-6-yl)quinazoline Derivatives as Anticancer Agents via PI3Kα Inhibition.

Aberrant expression of the phosphatidylinositol 3-kinase (PI3K) signalling pathway is often associated with tumourigenesis, progression and poor prognosis. Hence, PI3K inhibitors have attracted significant interest for the treatment of cancer. In this study, a series of new 6-(imidazo[1,2-a]pyridin-6-yl)quinazoline derivatives were designed, synthesized and characterized by 1H NMR, 13C NMR and HRMS spectra analyses. In the in vitro anticancer assay, most of the synthetic compounds showed submicromolar inhibitory activity against various tumour cell lines, among which 13k is the most potent compound with IC50 values ranging from 0.09 µΜ to 0.43 µΜ against all the tested cell lines. Moreover, 13k induced cell cycle arrest at G2/M phase and cell apoptosis of HCC827 cells by inhibition of PI3Kα with an IC50 value of 1.94 nM. These results suggested that compound 13k might serve as a lead compound for the development of PI3Kα inhibitor.

Synthetic dual hormone-responsive promoters enable engineering of plants with broad-spectrum resistance.

In plant immunity, the mutually antagonistic hormones salicylic acid (SA) and jasmonic acid (JA) are implicated in resistance to biotrophic and necrotrophic pathogens, respectively. Promoters that can respond to both SA and JA signals are urgently needed to engineer plants with enhanced resistance to a broad spectrum of pathogens. However, few natural pathogen-inducible promoters are available for this purpose. To address this problem, we have developed a strategy to synthesize dual SA- and JA-responsive promoters by combining SA- and JA-responsive cis elements based on the interaction between their cognate trans-acting factors. The resulting promoters respond rapidly and strongly to both SA and Methyl Jasmonate (MeJA), as well as different types of phytopathogens. When such a synthetic promoter was used to control expression of an antimicrobial peptide, transgenic plants displayed enhanced resistance to a diverse range of biotrophic, necrotrophic, and hemi-biotrophic pathogens. A dual-inducible promoter responsive to the antagonistic signals auxin and cytokinin was generated in a similar manner, confirming that our strategy can be used for the design of other biotically or abiotically inducible systems.

Design, synthesis, and biological evaluation of 6-(imidazo[1,2-a] pyridin-6-yl) quinazolin-4(3H)-one derivatives as potent anticancer agents by dual targeting Aurora kinase and ROR1.

ROR1 and Aurora kinase were overexpressed in various cancers and essential for cell proliferation, survive and metastasis. Pharmaceutical inhibition of ROR1 and Aurora kinase abrogated the activation of downstream signaling and induced cancer cell apoptosis. Hence, ROR1 and Aurora kinase considered as attractive therapeutic targets for the development of anticancer drugs. In the present work, three series of novel 6-(imidazo[1,2-a] pyridin-6-yl)-quinazolin-4(3H)-one derivatives were designed and synthesized via bioisosterism and scaffold-hopping strategies guided by FLF-13, an Aurora kinase inhibitor we discovered earlier. Most of compounds in series 2 and series 3 showed submicromolar to nanomolar inhibitory activity against multiple cancer cell lines. More importantly, compounds 12d and 12f in series 3 showed nanomolar inhibitory activity against all test cancer cells. The most promising compound 12d exhibited potent inhibitory activity against Aurora A and Aurora B with IC50 values of 84.41 nM and 14.09 nM, respectively. Accordingly, compounds 12d induced G2/M phase cell cycle arrest at 24 h and polyploidy at 48 h. It's worth noting that 12d also displayed inhibitory activity against ROR1 and induce cell apoptosis. Furthermore, 12d could significantly inhibit the tumor growth in SH-SY5Y xenograft model with tumor growth inhibitory rate (IR) up to 46.31 % at 10 mg/kg and 52.66 % at 20 mg/kg. Overall, our data suggested that 12d might serve as a promising candidate for the development of therapeutic agents for cancers with aberrant expression of ROR1 and Aurora kinases by simultaneously targeting ROR1 and Aurora kinase.

Structure optimization, synthesis, and biological evaluation of 6-(2-amino-1H-benzo[d]imidazole-6-yl)-quinazolin-4(3H)-one derivatives as potential multi-targeted anticancer agents via Aurora A/ PI3K/BRD4 inhibition.

Aurora A (Aurora kinase A), a critical regulator of cell mitosis, is frequently overexpressed in many malignant cancers, and has been considered as a promising drug target for cancer therapy. Likewise, Phosphatidylinositol 3-kinase alpha (PI3Kα) is also regarded as one of the most important targets in cancer therapy by mediating the cell growth and angiogenesis of various human cancers. In addition, Bromodomain-containing protein 4 (BRD4) modulates oncogene expressions of Myc, Aurora kinase and various RTKs. Recently, accumulating evidences indicated that hyperactivated or abnormally expressed Aurora A, PI3Kα or BRD4 are closely associated with drug resistance and poor prognosis of non-small cell lung cancer (NSCLC). Hence, simultaneous inhibition of Aurora A, PI3Kα, and BRD4 is expected to be a new strategy for NSCLC therapy. In this study, we performed further structure optimization of 6-(2-amino-1H-benzo[d]imidazole-6-yl)-quinazolin-4(3H) -one based on previous study to obtain a series of derivatives for discovering potential Aurora A, PI3Kα and BRD4 multi-targeted inhibitors. MTT assay showed that most of the newly synthesized compounds exhibited an evident anticancer activity against the NSCLC cells. Among them, the IC50 values of the most potent compound 9a were 0.83, 0.26 and 1.02 µM against A549, HCC827 and H1975 cells, respectively. In addition, 9a markedly inhibited the Aurora A and PI3Kα kinase activities with IC50 values of 10.19 nM and 13.12 nM. Compound 9a induced G2/M phase arrests and apoptosis of HCC827 cells by simultaneous inhibition of Aurora A/PI3K/ BRD4 signaling pathways. Collectively, our studies suggested that 9a might be a potential multi-targeted inhibitor for NSCLC therapy.