Validamycin A Inhibited FB1 Biosynthesis by the Target FvNth in Fusarium verticillioides.

Validamycin A (VMA) is an antifungal antibiotic derived from Streptomyces hygroscopicus commonly used in plant disease management. Surprisingly, VMA was discovered to impede the production of fumonisin B1 (FB1) in agricultural settings. However, the specific target of VMA in Fusarium verticillioides remained unclear. To unravel the molecular mechanism of VMA, ultrastructural observations unveiled damage to mitochondrial membranes. Trehalase (FvNth) was pinpointed as the target of VMA by utilizing a 3D-printed surface plasmon resonance sensor. Molecular docking identified Trp285, Arg447, Asp452, and Phe665 as the binding sites between VMA and FvNth. A ΔFvnth mutant lacking amino acids 250-670 was engineered through homologous recombination. Transcriptome analysis indicated that samples treated with VMA and ΔFvnth displayed similar expression patterns, particularly in the suppression of the FUM gene cluster. VMA treatment resulted in reduced trehalase and ATPase activity as well as diminished production of glucose, pyruvic acid, and acetyl-CoA. Conversely, these effects were absent in samples treated with ΔFvnth. This research proposes that VMA hinders acetyl-CoA synthesis by trehalase, thereby suppressing the FB1 biosynthesis. These findings present a novel target for the development of mycotoxin control agents.

A theoretical study on the activity and selectivity of IDO/TDO inhibitors.

Indoleamine 2,3-dioxygenase 1 (IDO) is a tryptophan (Trp) metabolic enzyme along the kynurenine (NFK) pathway. Under pathological conditions, IDO overexpressed by tumor cells causes depletion of tryptophan and the accumulation of metabolic products, which inhibit the local immune response and form immune escape. Therefore, the suppression of IDO activity is one of the strategies for tumor immunotherapy, and drug design for this target has been the focus of research for more than two decades. Apart from IDO, tryptophan dioxygenase (TDO) of the same family can also catalyze the same biochemical reaction in the human body, but it has different tissue distribution and substrate selectivity from IDO. Based on the principle of drug design with high potency and low cross-reactivity to specific targets, in this subject, the activity and selectivity of IDO and TDO toward small molecular inhibitors were studied from the perspective of thermodynamics and kinetics. The aim was to elucidate the structural requirements for achieving favorable biological activity and selectivity of IDO and TDO inhibitors. Specifically, the interactions of inhibitors from eight families with IDO and TDO were initially investigated through molecular docking and molecular dynamics simulations, and the thermodynamic data for binding of inhibitors were predicted by the molecular mechanics/generalized Born surface area (MM/GBSA) method. Secondly, we explored the free energy landscape of JKloops, the kinetic control element of IDO/TDO, using temperature replica exchange molecular dynamics (T-REMD) simulations and elucidated the connection between the rules of IDO/TDO conformational changes and the inhibitor selectivity mechanism. Furthermore, the binding and dissociation processes of the C1 inhibitor (NLG919) were simulated by the adaptive steering molecular dynamics (ASMD) method, which not only addressed the possible stable, metastable, and transition states for C1 inhibitor-IDO/TDO interactions, but also accurately predicted kinetic data for C1 inhibitor binding and dissociation. In conclusion, we have constructed a complete process from enzyme (IDO/TDO) conformational activation to inhibitor binding/dissociation and used the thermodynamic and kinetic data of each link as clues to verify the control mechanism of IDO/TDO on inhibitor selectivity. This is of great significance for us to understand the design principles of tumor immunotherapy drugs and to avoid drug resistance of immunotherapy drugs.

Efficacy of a novel affitoxin targeting MOMP against Chlamydia trachomatis in vitro and in vivo.

Targeted therapy is an attractive approach for treating infectious diseases. Affibody molecules have similar capability to antibodies that facilitate molecular recognition in both diagnostic and therapeutic applications. Targeting major outer membrane protein (MOMP) for treating infection of Chlamydia trachomatis, one of the most common sexually transmitted pathogens, is a promising therapeutic approach. Previously, we have reported a MOMP-specific affibody (ZMOMP:461) from phage display library. Here, we first fused it with modified Pseudomonas Exotoxin (PE38KDEL) and a cell-penetrating peptide (CPP) to develop an affitoxin, Z461X-CPP. We then verified the addition of both toxin and CPPs that did not affect the affinitive capability of ZMOMP:461 to MOMP. Upon uptake by C.trachomatis-infected cells, Z461X-CPP induced cell apoptosis in vitro. In animal model, Z461X significantly shortened the duration of C. trachomatis infection and prevented pathological damage in mouse reproductive system. These findings provide compelling evidence that the MOMP-specific affitoxin has great potential for targeting therapy of C. trachomatis infection.

First report of Botrytis cinerea causing gray mold on Alternanthera philoxeroides in China.

Alternanthera philoxeroides is a perennial herbaceous plant used as a forage crop (Wang et al. 2005) and is known to have medicinal properties. One of notable active components is flavonoids, which have been found to exhibit anti-Hepatitis B Virus activity (Li et al. 2016). In 2021, a leaf spot on A. philoxeroides was observed in the science and education experimental park of Hebei Agricultural University (38°49'38″ N, 115°26'39″ E). Initial symptoms included leaf tissue water loss, chloro-sis and elliptical lesions scattered across the leaf margin with further development leading to ellipse-shaped disease spots and leaf wilting (Fig. 1A). In the field, 50 plants of A. philoxeroides were randomly selected to investigate and quantify dis-ease. Incidence of leaf disease was approximately 25%, and the infected leaves ex-hibited an average affected area of about 20%. In order to identify the pathogen, three diseased plants were randomly selected from different areas. Stems and leaves of diseased plants were cut into pieces (2 to 3 mm × 5 mm) and disinfested with 1% sodium hypochlorite for 1 minute. After rinsing with sterile water three times, each lesion sample was isolated and purified on PDA at 25°C. Eventually, all samples pro-duced morphologically consistent colonies of pure strains. From the 9 isolates ob-tained, ZLQ-1 was selected as a representative isolate for further study. Colonies were initially white, turning gray from the centre, then gray-brown with cottony aerial hyphae, and finally growing black, stiff, round or irregular sclerotia (0.6 to 4.0 mm × 1.1 to 4.2 mm, n=50) (Fig1. B, C). ZLQ-1 exhibited branched conidia with en-larged apical cells. The conidia of this isolate were unicellular, ovoid or ellipsoid in shape, with dimensions ranging from 5.8 to 16.9 µm × 6.3 to 11.2 µm (n=50) (Fig. 1D). These morphological characteristics were consistent with Botrytis cinerea (Ellis, 1971). The genes of internal transcribed spacer (ITS), heat shock protein (HSP60), DNA-dependent RNA polymerase subunit II (RPB2), and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were amplified with specific primers ITS1/ITS4, HSP60-F/HSP60-R, RPB2-F/RPB2-R and G3PDH-F/G3PDH-R (Aktaruzzaman et al., 2022). Sequences were deposited into GenBank with accession numbers ON479490 for ITS, ON572246 for G3PDH, ON572248 for HSP60, ON572247 for RPB2. BLASTn analysis showed that the ITS sequence shared 99.62% similarity to B. cinerea (CP009808), and the sequences of the other three nuclear protein-encoding genes (G3PDH, HSP60, and RPB2) showed at least 99.9% identity with the genome of B. ci-nerea (B05.10) (Staats et al. 2005). We have inoculated 10 healthy A. philoxeroides leaves with a suspension of 1x105 spores/mL, and used sterile water treatment as control (Aktaruzzaman et al., 2022). Each leaf was inoculated with 10 µL spore sus-pension. After 7 days in a controlled incubation environment (25℃, 40%RH), the plants inoculated with conidial suspensions displayed lesions covered in a gray-white mycelial layer, resembling those observed in the field (Fig. 1E-G). In con-trast, the plants inoculated with sterile water remained unaffected. Morphological and PCR analysis confirmed that the pathogen responsible for the observed symp-toms was B. cinerea. Koch's postulates were fulfilled as the same pathogen was con-sistently re-isolated from the inoculated leaves and confirmed to be B. cinerea through morphological and molecular methods. This is the first reported case of B. cinerea causing gray mold on A. philoxeroides in China. It is important to monitor and prevent B. cinerea infection during cultivation to ensure the production of healthy Chinese medicine and feed.

Novel Bifunctional Affibody Molecules with Specific Binding to Both EBV LMP1 and LMP2 for Targeted Therapy of Nasopharyngeal Carcinoma.

Antibodies are considered highly specific therapeutic agents in cancer medicines, and numerous formats have been developed. Among them, bispecific antibodies (BsAbs) have gained a lot of attention as a next-generation strategy for cancer therapy. However, poor tumor penetration is a major challenge because of their large size and thus contributes to suboptimal responses within cancer cells. On the other hand, affibody molecules are a new class of engineered affinity proteins and have achieved several promising results with their applications in molecular imaging diagnostics and targeted tumor therapy. In this study, an alternative format for bispecific molecules was constructed and investigated, named ZLMP110-277 and ZLMP277-110, that targets Epstein-Barr virus latent membrane protein 1 (LMP1) and latent membrane protein 2 (LMP2). Surface plasmon resonance (SPR), indirect immunofluorescence assay, co-immunoprecipitation, and near-infrared (NIR) imaging clearly demonstrated that ZLMP110-277 and ZLMP277-110 have good binding affinity and specificity for both LMP1 and LMP2 in vitro and in vivo. Moreover, ZLMP110-277 and ZLMP277-110, especially ZLMP277-110, significantly reduced the cell viability of C666-1 and CNE-2Z as compared to their monospecific counterparts. ZLMP110-277 and ZLMP277-110 could inhibit phosphorylation of proteins modulated by the MEK/ERK/p90RSK signaling pathway, ultimately leading to suppression of oncogene nuclear translocations. Furthermore, ZLMP110-277 and ZLMP277-110 showed significant antitumor efficacy in nasopharyngeal carcinoma-bearing nude mice. Overall, our results demonstrated that ZLMP110-277 and ZLMP277-110, especially ZLMP277-110, are promising novel prognostic indicators for molecular imaging and targeted tumor therapy of EBV-associated nasopharyngeal carcinoma.

Natural Occurrence of Mycotoxins in Maize in North China.

Mycotoxins seriously threaten the quality of maize seriously around the world. A total of 426 samples of maize kernel from northeast and northwest China were analyzed in this study. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was performed to analyze the mycotoxin contamination of maize samples. The results showed that it was contaminated by mycotoxins in maize. The average contamination levels of fumonisins, deoxynivalenol, aflatoxins, zearalenone, ochratoxin A, T-2 and HT-2 were 937, 431, 22, 27, 2 and 12 µg/kg, respectively. Concentration of mycotoxins in some samples exceeded their limit, but most were still at safe levels. The contamination level of FBs and DON were most significative. The proportion of mycotoxins exceeding the maximum limit standard was in the following order: 8.0%, 8.0%, 7.0%, 1.6%, 1.4% and 0.0%. The contamination of mycotoxins in maize varies from region to region.

EBV LMP1-C terminal binding affibody molecule downregulates MEK/ERK/p90RSK pathway and inhibits the proliferation of nasopharyngeal carcinoma cells in mouse tumor xenograft models.

Nasopharyngeal carcinoma (NPC), is an Epstein-Barr virus (EBV) associated malignancy most common in Southern China and Southeast Asia. In southern China, it is one of the major causes of cancer-related death. Despite improvement in radiotherapy and chemotherapy techniques, locoregional recurrence and distant metastasis remains the major causes for failure of treatment in NPC patients. Therefore, finding new specific drug targets for treatment interventions are urgently needed. Here, we report three potential ZLMP1-C affibody molecules (ZLMP1-C15, ZLMP1-C114 and ZLMP1-C277) that showed specific binding interactions for recombinant and native EBV LMP1 as determined by epitope mapping, co-localization and co-immunoprecipitation assays. The ZLMP1-C affibody molecules exhibited high antitumor effects on EBV-positive NPC cell lines and displayed minimal cytotoxicity towards EBV-negative NPC cell line. Moreover, ZLMP1-C277 showed higher antitumor efficacy than ZLMP1-C15 and ZLMP1-C114 affibody molecules. The ability of ZLMP1-C277 decrease the phosphorylation levels of up-stream activator phospho-Raf-1(Ser338), phospho-MEK1/2(Ser217/Ser221), phospho-ERK1/2(Thr202/Thr204), thereby leading to downstream suppression of phospho-p90RSK(Ser380) and transcription factor c-Fos. Importantly, tumor growth was reduced in tumor-bearing mice treated with ZLMP1-C277 and caused no apparent toxicity. Taken together, our findings provide evidence that ZLMP1-C277 as a promising therapeutic agent in EBV-associated NPC.