Interactions between polycyclic musks and human lactoferrin: Multi-spectroscopic methods and docking simulation.

Galaxolide (1,3,4,6,7,8-hexahydro-4,6,6,7,8-hexamethylcyclopenta-γ-2-benzopyrane; HHCB) and Tonalide (7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene; AHTN) are "pseudo-persistent" pollutants that can cause DNA damage, endocrine disruption, organ toxicity, and reproductive toxicity in humans. HHCB and AHTN are readily enriched in breast milk, so exposure of infants to HHCB and AHTN is of concern. Here, the molecular mechanisms through which HHCB and AHTN interact with human lactoferrin (HLF) are investigated using computational simulations and spectroscopic methods to identify indirectly how HHCB and AHTN may harm infants. Molecular docking and kinetic simulation studies indicated that HHCB and AHTN can interact with and alter the secondary HLF structure. The fluorescence quenching of HLF by HHCB, AHTN was static with the forming of HLF-HHCB, HLF-AHTN complex, and accompanied by non-radiative energy transfer and that 1:1 complexes form through interaction forces. Time-resolved fluorescence spectroscopy indicated that binding to small molecules does not markedly change the HLF fluorescence lifetime. Three-dimensional fluorescence spectroscopy indicated that HHCB and AHTN alter the peptide chain backbone structure of HLF. Ultraviolet-visible absorption spectroscopy, simultaneous fluorescence spectroscopy, Fourier-transform infrared spectroscopy, and circular dichroism spectroscopy indicated that HHCB and AHTN change the secondary HLF conformation. Antimicrobial activity experiments indicated that polycyclic musks decrease lactoferrin activity and interact with HLF. These results improve our understanding of the mechanisms involved in the toxicities of polycyclic musks bound to HLF at the molecular level and provide theoretical support for mother-and-child health risk assessments.

Interaction between laccase and diethylstilbestrol based on multispectral and chromatography analyses.

Diethylstilbestrol (DES) is a synthetic form of oestrogen that does not easily degrade in the environment and can be harmful to human health. Herein, the mechanism of the interaction between laccase and DES was investigated by various spectroscopic means and high-performance liquid chromatography (HPLC). The results of fluorescence experiments showed that the quenching of intrinsic fluorescence of laccase by DES was due to a static quenching, forming a binding site. According to the Förster non-radiative energy transfer theory (FRET), the action distance R0 between DES and laccase was 4.708 nm, r was 5.81 nm, and the energy transfer efficiency E was 22.08%, respectively. Both UV-Vis absorption spectra and FT-IR spectra indicated changes in the conformation and surroundings of the enzyme and changed in the secondary structure of laccase. Multispectral synthesis showed that the interaction of laccase with DES caused a change in the secondary structure of laccase. The degradation experiments showed that laccase could degrade DES, and the DES content decreased with time. This study provides a new theoretical basis and experimental method for further research on the reaction mechanism of the laccase degradation of DES. It may also provide a reference basis for human biological and environmental safety evaluations.

Synthesis of 4-dimethylaminobenzyl chrysin ester-Zn fluorescent chemical sensor for the determination of Cr(VI) in water.

Cr(VI) is a type of dangerous effluent that has caused great harm to human health and the environment. Recognition and perception of Cr(VI) by artificial receptors has attracted extensive attention. A novel fluorescent chemical sensor based on the 5,7-dihydroxyflavone skeleton was designed and synthesized for the selective recognition of Cr(VI). As confirmed by fluorescence technology, the fluorescent probe 4-dimethylaminobenzyl chrysin ester-Zn (DBC-Zn) showed high sensitivity and selectivity for dichromate and a fast response (less than 30 sec) recognition. The fluorescence intensity of DBC-Zn varies linearly with the concentration of Cr(VI) in the range 0.1-1 µM. The detection limit of Cr2 O7 2- by DBC-Zn is 2.3 nM, which is far lower than the national safe drinking water standard stipulated by the US Environmental Protection Agency (1.9 µM). The quenching mechanism of the probe can be attributed to the interaction of the dynamic quenching effect and the fluorescence internal filtration effect. In addition, the probe has good stability in both neutral and alkaline environments, and the accuracy of quantitative analysis of Cr2 O7 2- in lake water or tap water is more than 80%. The test paper based on DBC-Zn can effectively detect Cr2 O7 2- at the concentration of 100 ppb. This shows that the probe has a certain practical application value.

Small GTPase Rab7-mediated FgAtg9 trafficking is essential for autophagy-dependent development and pathogenicity in Fusarium graminearum.

Fusarium graminearum is a fungal pathogen that causes Fusarium head blight (FHB) in wheat and barley. Autophagy is a highly conserved vacuolar degradation pathway essential for cellular homeostasis in which Atg9 serves as a multispanning membrane protein important for generating membranes for the formation of phagophore assembly site. However, the mechanism of autophagy or autophagosome formation in phytopathogens awaits further clarifications. In this study, we identified and characterized the Atg9 homolog (FgAtg9) in F. graminearum by live cell imaging, biochemical and genetic analyses. We find that GFP-FgAtg9 localizes to late endosomes and trans-Golgi network under both nutrient-rich and nitrogen starvation conditions and also show its dynamic actin-dependent trafficking in the cell. Further targeted gene deletion of FgATG9 demonstrates that it is important for growth, aerial hyphae development, and pathogenicity in F. graminearum. Furthermore, the deletion mutant (ΔFgatg9) shows severe defects in autophagy and lipid metabolism in response to carbon starvation. Interestingly, small GTPase FgRab7 is found to be required for the dynamic trafficking of FgAtg9, and co-immunoprecipitation (Co-IP) assays show that FgAtg9 associates with FgRab7 in vivo. Finally, heterologous complementation assay shows that Atg9 is functionally conserved in F. graminearum and Magnaporthe oryzae. Taken together, we conclude that FgAtg9 is essential for autophagy-dependent development and pathogenicity of F. graminearum, which may be regulated by the small GTPase FgRab7.

Methylmalonate-semialdehyde dehydrogenase mediated metabolite homeostasis essentially regulate conidiation, polarized germination and pathogenesis in Magnaporthe oryzae.

Plants generate multitude of aldehydes under abiotic and biotic stress conditions. Ample demonstrations have shown that rice-derived aldehydes enhance the resistance of rice against the rice-blast fungus Magnaporthe oryzae. However, how the fungal pathogen nullifies the inhibitory effects of host aldehydes to establish compatible interaction remains unknown. Here we identified and evaluated the in vivo transcriptional activities of M. oryzae aldehyde dehydrogenase (ALDH) genes. Transcriptional analysis of M. oryzae ALDH genes revealed that the acetylating enzyme Methylmalonate-Semialdehyde Dehydrogenase (MoMsdh/MoMmsdh) elevated activities during host invasion and colonization of the fungus. We further examined the pathophysiological importance of MoMSDH by deploying integrated functional genetics, and biochemical approaches. MoMSDH deletion mutant ΔMomsdh exhibited germination defect, hyper-branching of germ tube and failed to form appressoria on hydrophobic and hydrophilic surface. The MoMSDH disruption caused accumulation of small branch-chain amino acids, pyridoxine and AMP/cAMP in the ΔMomsdh mutant and altered Spitzenkörper organization in the conidia. We concluded that MoMSDH contribute significantly to the pathogenesis of M. oryzae by regulating the mobilization of Spitzenkörper during germ tube morphogenesis, appressoria formation by acting as metabolic switch regulating small branch-chain amino acids, inositol, pyridoxine and AMP/cAMP homeostasis.

Retrograde trafficking from the endosome to the trans-Golgi network mediated by the retromer is required for fungal development and pathogenicity in Fusarium graminearum.

In eukaryotes, the retromer is an endosome-localized complex involved in protein retrograde transport. However, the role of such intracellular trafficking events in pathogenic fungal development and pathogenicity remains unclear. The role of the retromer complex in Fusarium graminearum was investigated using cell biological and genetic methods. We observed the retromer core component FgVps35 (Vacuolar Protein Sorting 35) in the cytoplasm as fast-moving puncta. FgVps35-GFP co-localized with both early and late endosomes, and associated with the trans-Golgi network (TGN), suggesting that FgVps35 functions at the donor endosome membrane to mediate TGN trafficking. Disruption of microtubules with nocodazole significantly restricted the transportation of FgVps35-GFP and resulted in severe germination and growth defects. Mutation of FgVPS35 not only mimicked growth defects induced by pharmacological treatment, but also affected conidiation, ascospore formation and pathogenicity. Using yeast two-hybrid assays, we determined the interactions among FgVps35, FgVps26, FgVps29, FgVps17 and FgVps5 which are analogous to the yeast retromer complex components. Deletion of any one of these genes resulted in similar phenotypic defects to those of the ΔFgvps35 mutant and disrupted the stability of the complex. Overall, our results provide the first clear evidence of linkage between the retrograde transport mediated by the retromer complex and virulence in F. graminearum.

OsCRLK2, a Receptor-Like Kinase Identified by QTL Analysis, is Involved in the Regulation of Rice Quality.

The quality of rice (Oryza sativa L) is determined by a combination of appearance, flavor, aroma, texture, storage characteristics, and nutritional composition. Rice quality directly influences acceptance by consumers and commercial value. The genetic mechanism underlying rice quality is highly complex, and is influenced by genotype, environment, and chemical factors such as starch type, protein content, and amino acid composition. Minor variations in these chemical components may lead to substantial differences in rice quality. Among these components, starch is the most crucial and influential factor in determining rice quality. In this study, quantitative trait loci (QTLs) associated with eight physicochemical properties related to the rapid viscosity analysis (RVA) profile were identified using a high-density sequence map constructed using recombinant inbred lines (RILs). Fifty-nine QTLs were identified across three environments, among which qGT6.4 was a novel locus co-located across all three environments. By integrating RNA-seq data, we identified the differentially expressed candidate gene OsCRLK2 within the qGT6.4 interval. osclrk2 mutants exhibited decreased gelatinization temperature (GT), apparent amylose content (AAC) and viscosity, and increased chalkiness. Furthermore, osclrk2 mutants exhibited downregulated expression of the majority of starch biosynthesis-related genes compared to wild type (WT) plants. In summary, OsCRLK2, which encodes a receptor-like protein kinase, appears to consistently influence rice quality across different environments. This discovery provides a new genetic resource for use in the molecular breeding of rice cultivars with improved quality.