The various grass-induced epichloëcyclins of the Epichloë spp. are ribosomally synthesized and post-translationally modified peptides (RiPPs), produced as small, secreted cyclopeptides from a ...single gene, gigA. Here, four clustered and coregulated genes (gigA, gigB, gigC, and kexB) with predicted roles in epichloëcyclin production in Epichloë festucae were evaluated through gene disruption. Subsequent chemical analysis indicates that GigB is a DUF3328 domain-containing protein associated with cyclization of epichloëcyclins; GigC is a methyltransferase enzyme responsible for N-methylation of desmethylepichloëcyclins; and KexB is a subtilisin-like enzyme, partly responsible for the propeptide cleavage of epichloëcyclin intermediates. Symbiotic effects on the host phenotype were not observed for gigA, gigC, or kexB mutants, although ΔgigB infection correlated with increased host tiller height and biomass, while only ΔkexB exhibited an effect on endophyte morphology. Disrupting epichloëcyclin biosynthesis showed negligible influence on the biosynthesis of E. festucae-associated alkaloids. Epichloëcyclins may perform other secondary metabolism functions in Epichloë and other fungi.
The imbalance between osteogenesis and osteoclastogenesis is a feature of bone metabolic disease. Cadmium (Cd) exposure causes human bone loss and osteoporosis (OP) through bioaccumulation of the ...food chain. However, the impact of Cd on bone tissues and the underlying molecular mechanisms are not well-characterized. In the current study, we found that the Cd concentration in bone tissues of OP patients was higher than normal subjects; meanwhile, the nuclear silent information regulator of transcription 1 (SIRT1) protein expression level was significantly decreased, which is a new star molecule to treat OP. It is further revealed that SIRT1 activation markedly reprograms bone metabolic and stress-response pathways that incline with osteoblast (OB) apoptosis. Suppressing reactive oxygen species (ROS) release with N-acetyl-l-cysteine (NAC) abolished Cd-induced reduction of SIRT1 protein, deacetylation of P53, OB apoptosis, and attenuated OP. Conversely, overexpression of SIRT1 suppressed Cd-induced ROS release. SIRT1 overexpression in vivo and in vitro dampened PGC-1α protein, acetylation of P53 at lysine 382, and caspase-dependent apoptosis. These results reveal that ROS/SIRT1 controls P53 acetylation and coordinates OB apoptosis involved in the onset of OP.
Di-(2-Ethylhexyl) phthalate (DEHP) and microplastics (MPs) have released widespread residues to the environment and possess the ability to cause damage to humans and animals. However, there are still ...gaps in the study of damage to neurons caused by DEHP and MPs in mice cerebra and whether they have combined toxic effects. To investigate the underlying mechanism of action, mice were fed 200 mg/kg DEHP and 10 mg/L MPs in vivo. In vitro, NS20Y (CBNumber: CB15474825) cells were treated with 25 μM DEHP and 775 mg/L MPs. Next, qRT-PCR and western blot analysis were performed to evaluate PI3K/AKT pathway genes, mitochondrial dynamics-related genes, apoptosis-related genes, and GSK-3β and its associated genes, mRNA, and protein expression. To determine pathological changes in the mice cerebra, hematoxylin and eosin (H&E) staining, transmission electron microscopy, and TUNEL staining were employed. To determine the levels of reactive oxygen species (ROS) and apoptosis cells in vitro, ROS staining, acridine orange/ethidium bromide (AO/EB) staining, and flow cytometry were performed. Our results demonstrated that DEHP and MPs caused changes in mitochondrial function, and GSK-3β and its associated gene expression in mice through the PI3K/AKT pathway, which eventually led to apoptosis of neurons. Moreover, our findings showed that DEHP and MPs have a combined toxic effect on mice cerebra. Our findings facilitate the understanding of the neurotoxic effects of DEHP and MPs on neurons in the cerebra of mice and help identify the important role of maintaining normal mitochondrial function in protecting cerebrum health.
Efficient preconcentration is critical for sensitive and selective electrochemical detection of metal ions, but rapid specific enrichment with depressed absorption of interfering ions at the ...electrode is challenging. Here, we proposed an electric field-induced specific preconcentration to boost the analytical performance of DNA-based electrochemical sensors for Hg2+ detection. As for such preconcentration, a positive external electric field was first used to enrich Hg2+ at an electrode assembled with T-rich DNA, thus boosting T–Hg2+–T recognitions. The following applied inverse electric field strips the nonspecifically absorbed Hg2+ and other interfering ions, thus depressing matrix interferences via self-cleaning. Based on this principle, we designed a portable device to realize programmable control of electric fields; a T–Hg2+–T recognition-based electrochemical sensor was thus fabricated as a model platform to assess the feasibility of electric field-induced preconcentration. The experimental results revealed that such a strategy decreased the time of T–Hg2+–T-based recognition from 60 to 20 min and led to detection with better reproducibility by depressing the influence of free Hg2+ as well as interfering ions. This strategy offered Hg2+ detection limits of 0.01 pMthree-fold better than that without preconcentrationwithin 22 min. The proposed preconcentration strategy offers a new way to enhance the analytical performance of sensing at the solid–liquid interface.
Succinate dehydrogenase inhibitor (SDHI) fungicides have a wide spectrum of fungicidal effects on a variety of fungi causing plant diseases, including Sclerotinia stem rot caused by Sclerotinia ...sclerotiorum. However, the consistent use of site-specific SDHI fungicides can result in the development of resistant isolates with mutations in the SDHB, SDHC, or SDHD subunit thereby leading to a rapid decline of fungicide performance. In this study, we found that SDHC was genetically evolved into two isotypes SDHC1 and SDHC2 in S. sclerotiorum but not involved in the sensitivity to SDHI fungicides. In addition, we demonstrated that the A11V substitution in SDHB was not involved in the resistance of S. sclerotiorum to boscalid, and this substitution widely emerged in the field populations. Meanwhile, the P226L substitution in SDHB was demonstrated to confer boscalid resistance in S. sclerotiorum. The result of cross-resistance showed that the SDHB-P226L substitution exhibited a positive cross-resistance between boscalid and carboxin, fluopyram, pydiflumetofen, flubeneteram, pyraziflumid, fluindapyr, or penthiopyrad. Taken together, our results indicated that the P226L substitution in SDHB resulted in the resistance of S. sclerotiorum to SDHI fungicides but suffered from fitness penalty, especially the homozygous mutants conferring the P226L substitution in SDHB.
The slow catalytic rate of the carboxylation enzyme d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a major barrier to increasing the rate of carbon assimilation from the atmosphere ...into the biosphere. It is of great importance to establish a method to improve the carboxylation efficiency of Rubisco. Inspired by the assembly of Rubisco in carboxysomes, herein, we presented a rational protein engineering approach for the construction of one-dimensional (1D) protein arrays of type III Rubisco through designed π–π stacking interactions by using crystal structural information as a guide. In aqueous solutions, the dimensions of these 1D protein arrays collectively span nearly the entire nano- and micrometer scale (200 nm to 5.0 μm) by adjusting protein and NaCl concentrations. As a result, the stacked Rubisco assemblies increase by 40% in the carboxylase activity, while their turnover number (k cat) is around twofold larger than that of wild-type III Rubisco. Notably, upon heat treatment at temperature up to 75 °C for 30 min, most of the assembled nanostructures and the enzyme activity are retained. More importantly, the initial relative activity of stacked assemblies retained 91% after 10 cycles of reuse. This work provides a simple, effective solution for the improvement of the CO2 carboxylation efficiency of Rubisco.
Monitoring agrochemical distribution within plant tissues delivers significant insights into the adsorption, distribution, metabolism, and elimination of agrochemicals. Detection and imaging of the ...safener cyprosulfamide (CSA) and the herbicide thiencarbazone-methyl (TCM) after micro-droplet application on the surface of maize leaves (Zea mays L.) have been achieved using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). The agrochemicals were deposited onto the adaxial surface of maize leaves on growing plants, and their uptake, distribution, and metabolism were investigated at four timepoints (3 h, 24 h, 4 days, and 7 days) to assess the influence of CSA treatment on TCM metabolism. MALDI MSI visualized significant changes for the metabolism of TCM after 24 h. Although TCM metabolism was detected neither in the control without the safener nor in the approach with CSA on the second leaf, the co-application on the same leaf showed significant metabolism of the herbicide by detecting the metabolite N-demethylated TCM. These findings suggest that safener protection against herbicide injury is a rapid process in which CSA and TCM need to be present in the same tissues. This study showcases the use of MALDI MSI to visualize and analyze indirect interactions of two substances in planta.
A comprehensive evaluation of the dissipation of a myclobutanil plant protection product was performed in tomato and grape samples. Different temperature conditions (3 and 22 °C) were evaluated. A ...biphasic kinetic model provided a suitable adjustment (R 2 > 0.95), with persistence (residual level, RL50) lower than 24 days in all cases. Solid–liquid extraction and ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-Q-Orbitrap-HRMS) were used for metabolites’ elucidation, identifying six myclobutanil metabolites, four out of them described for the first time and one of them confirmed using 1H, 13C, (1H-1H)-COSY, (1H-13C)-HMQC, and (1H-13C)-HMBC nuclear magnetic resonance (NMR). Their degradation curves were also evaluated, increasing their concentrations when the myclobutanil concentration decreases. Additionally, coformulants present in the commercial formulation were monitored employing headspace solid-phase microextraction method (HS-SPME)-gas chromatography coupled to HRMS (GC-Q-Orbitrap-HRMS). Seven coformulants were quantified in tomato samples. Their dissipation curves were studied, and it was observed that they were almost degraded 12 days after application.
An accurate controlled-release strategy of pesticides is considered desirable in sustainable agriculture. A site-specific nanorelease system of dazomet (DZ) was proposed by employing the zeolitic ...imidazolate framework-8 composite (DZ@ZIF-8) by a one-pot method. The synthetic parameters of DZ@ZIF-8 were optimized, and the loading content of DZ was maximized. ZIF-8 endowed DZ with a pH-sensitive behavior. The collapse of the DZ@ZIF-8 structure and the site-specific release of DZ were triggered by acidic substances produced by Botrytis cinerea. In vitro and pot experiments showed that the fungicidal activity of DZ@ZIF-8 was about 36.3 and 42.7% higher than that of DZ, respectively. DZ is conventionally used before a crop is planted because of its volatility and toxicity. However, DZ@ZIF-8 could avoid phytotoxicity of DZ to plants, which made the application of DZ possible during plant growth. Moreover, the acute toxicity to zebrafish changed from high to moderate levels. This study highlights a potential strategy that improves DZ effective utilization and reduces side effects.
In order to prevent and control the effects of pesticide residues on human health and the ecological environment, the rapid, highly sensitive, and selective detection of multiple pesticide residues ...has become an urgent problem to be solved. Herein, a lab-on-a-molecule probe based on a host–guest complex (ThT@Q8 probe) has been developed to simultaneously analyze multiple aromatic pesticides under single wavelength excitation, such as fuberidazole, thiabendazole, carbendazim, thidiazuron, and tricyclazole. The fluorescence titration spectra of the ThT@Q8 probe with the five pesticides mentioned above showed that the fluorescence intensity exhibited a good linear correlation with the pesticide concentration and the limit of detection was as low as 10–7 M. Because the ThT@Q8 probe exhibits diverse fluorescence color changes to the five pesticides studied under a 365 nm ultraviolet lamp, we fabricated a single probe used to detect multiple analytes in the RGB triple channel by extracting the RGB variations. Principal component analysis and linear discriminant analysis proved that the ThT@Q8 probe can recognize and distinguish five pesticides and can be applied at different concentrations. In real samples, the ThT@Q8 probe recognized and distinguished five pesticides in tap water and Huaxi River water. The 1H NMR spectra results proved that a charge-transfer complex of ThT and pesticides in the Q8 cavity may be formed. Moreover, we selected a test strip as a carrier to detect pesticides. The results indicate it can be used to quickly and conveniently detect different pesticides due to the rapid color change. Besides, the ThT@Q8 probe has good cell permeability and can be used to detect pesticide residues in living cells. This work has laid the foundation for the qualitative and quantitative multitarget detection of pesticide residues.
