Mechanism and effectiveness of enzymatically induced phosphate precipitation (EIPP) in stabilizing coexisting lead, zinc, and cadmium in tailings.

Lead-zinc (Pb-Zn) tailings ponds carry the risk of multiple heavy metals (HMs) contamination and pile destabilization. This poses requirements for in-situ applicable, low-distribution, and effective stabilization/solidification (S/S) methods. For this, the novel enzymatically induced phosphate precipitation (EIPP) method was implemented in this study. Its mechanism and performance on stabilization of composite Pb, Zn, and cadmium (Cd) in tailings were explored and evaluated under typical erosion conditions for the first time. Results show that the EIPP stabilized HMs by chemically transforming the unstable carbonate-bound HMs to stable phosphate precipitates and by physically encapsulating tailings particles with newberyite precipitates. The stabilization effect on the three HMs was ranked as Pb > Zn > Cd. Comparing magnesium resources for the EIPP reactants, the EIPP utilizing Mg(CH3COO)2 was more effective at decontamination than MgCl2 because its special pre-activation and re-precipitation function enhanced the chemical transformation function of EIPP. The EIPP stabilization was confirmed to reduce simulated acid rain-leachable and bio-extractive HMs by about 90% and 60%, respectively. Under the prolonged acid attack, treated HMs were ultimately leached through the dissolution mechanism. Zn exhibited significant instability in highly acidic conditions (pH = 2.5-3.5), where its cumulative leaching toxicity after long-term dissolution warrants attention. Overall, EIPP presents a novel and effective strategy for on-site mitigation of composite HMs pollution.

Development and characterization of hydrogel-in-oleogel (bigel) systems and their application as a butter replacer for bread making.

BACKGROUND: Butter has been widely used in bakery products and it contains high level of saturated fats. However, excessive consumption of saturated fats would increase the risk of chronic disease. This study was to fabricate water-in-oil (W/O) type bigels as butter replacers to improve the quality attributes of breads. RESULTS: A stable water-in-oil (W/O) type bigel system was fabricated based on mixed oleogelators (rice bran wax and glycerol monostearate) and sodium alginate hydrogel. The ratios of oleogel to hydrogel could significantly affect the stability, microstructure and rheological properties of bigels. All of the bigels exhibited solid-like properties, with increased oleogel fractions, and the network structure of bigel became more compact and orderly with smaller sodium alginate gel particles. Meanwhile, the viscoelastic modulus and firmness of bigel increased, contributing to a higher stability. The bigel dough exhibited lower gel strength and relatively higher extensibility compared to the butter dough. Regardless of oleogel fractions, all the bigel produced bread with a higher specific volume and softer texture than the butter bread. When the oleogel fractions was less than 80%, increasing the oleogel fractions was more beneficial for improving the specific volume, softness and fluffy structure of bread. CONCLUSION: W/O type bigel as butter replacers showed great potential in improving the appearance, structure and textural properties of bread. © 2023 Society of Chemical Industry.

Spatiotemporally controlled AuNPs@ZIF-8 based nanocarriers for synergistic photothermal/chemodynamic therapy.

High efficiency and spatio-temporal control remains a challenge for multi-modal synergistic cancer therapy. Herein, based on gold nanoparticles (AuNPs) and zeolite-like imidazole skeleton material (ZIF-8), a spatio-temporal controllable photothermal/ chemical dynamic/ chemotherapy three modal synergistic anti-tumor nano-carrier (HAZD) was developed. HAZD has a size of 128.75 ± 11.86 nm, a drug loading ratio of 21.5 ± 2.2 % and an encapsulation efficiency of 71.8 ± 1.7 %. Stability, acid responsive release character, outstanding catalytic ability to generate ROS, relatively high thermal conversion efficiency up to 62.38 % and spatio-temporal controllable abilities are also found within this nano-carrier. Furthermore, HAZD performed good antitumor ability in vivo with the comprehensive effects of photothermal/ chemical dynamic/ chemotherapy. The tumor growth inhibition value is 97.1 % within 12 days, indicating its great potential in multi-modal synergistic cancer therapy. STATEMENT OF SIGNIFICANCE: Cancer remains one of the major culprits that seriously harm human health currently. With the development of materials and nanotechnology, great improvements have been made in multimodal anti-tumor strategies. However, temporal- and spatial-controllable multi-modal synergistic nanocarriers are urgently awaited for efficient and low-toxicity tumor therapy. This article proposes a spatio-temporally controllable three-modal anti-tumor strategy and designs an anti-tumor drug delivery system based on gold nanoparticles (AuNPs) and zeolite-like imidazole skeleton material (ZIF-8), which shows acid-responsive release characteristics, catalytic ability to generate ROS, relatively high thermal conversion efficiency up to 62.38 %, as well as spatio-temporal controllable abilities. Moreover, it demonstrates outstanding anti-tumor ability, with a tumor growth inhibition value of 97.1 % within 12 days, revealing its significant potential for future personalized and precise anti-tumor treatments.

Residue behaviors, degradation, processing factors, and risk assessment of pesticides in citrus from field to product processing.

Pesticide residues in citrus may cause health risks in related juice products, and bring much uncertainty during the processing procedures. In this study, based on the dispersive solid-phase extraction (d-SPE) and UPLC-MS/MS, the residual levels of ten analytes in citrus and its processed products were monitored. The results showed that dissipation of the pesticides followed the first-order kinetics and the half-lives in citrus varied greatly, ranging from 6.36 to 63.0 days. The terminal residues of the five pesticides at harvest time were <0.01-0.302 and <0.01-0.124 mg/kg in raw citrus and citrus flesh, respectively, all of which were lower than the corresponding maximum residue limits (MRLs) of 0.5-1 mg/kg. In the processing experiments, the residues of ten analytes in sterilized juice, concentrated juice, and citrus essential oil were in the range of <0.01 to 0.442 mg/kg, <0.01 to 1.16 mg/kg, and <0.01 to 44.0 mg/kg, respectively, and the corresponding processing factors (PFs) were 0.127-1.00, 0.023-3.06, and 0.006-39.2. Particularly, in citrus essential oil, the PFs of etoxazole, fluazinam, lufenuron and spirotetramat-keto-hydroxy were 1.68-39.2, exhibiting obvious enrichment effects. By integrating the residue data of the field trials and the PFs, the acute and chronic dietary risks of the target pesticides in citrus juice were 0.031-1.83 % and 0.002-2.51 %, respectively, which were far lower than 100 %, demonstrating no unacceptable risk to human health. This work provides basic data for the establishment of the MRLs and dietary exposure risk assessment for processed citrus products.

Design and Optimization of an Index Finger Exoskeleton With Semi-Wrapped Fixtures and Series Elastic Actuators.

This paper proposes a novel index finger exoskeleton with semi-wrapped fixtures and elastomer-based clutched series elastic actuators. The semi-wrapped fixture is similar to a clip, which improves the convenience of donning/doffing and connection stability. The elastomer-based clutched series elastic actuator can limit the maximum transmission torque and improve passive safety. Second, the kinematic compatibility of the exoskeleton mechanism for the proximal interphalangeal joint is analyzed, and its kineto-statics model is built. To avoid the damage caused by the force along the phalanx, considering the individual difference in the size of the finger segment, a two-level optimization method is proposed to minimize the force along the phalanx. Finally, the performance of the proposed index finger exoskeleton is tested. Statistical results indicate that the donning/doffing time of the semi-wrapped fixture is significantly less than that of the Velcro. Compared with the Velcro, the average value of the maximum relative displacement between the fixture and the phalanx is reduced by 59.7%. Compared with the exoskeleton before optimization, the maximum force along the phalanx generated by the exoskeleton after optimization is reduced by 23.65%. The experimental results show that the proposed index finger exoskeleton can improve the convenience of donning/doffing, connection stability, comfort, and passive safety.

Multivariate response surface methodology assisted modified QuEChERS method for the rapid determination of 39 pesticides and metabolites in medlar.

A modified QuEChERS method coupled with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was established for residue analysis of 39 pollutants (34 commonly used multi-class pesticides and 5 metabolites) in medlar matrices (fresh, dried, and medlar juice). Samples were extracted using water with 0.1 % formic acid: acetonitrile (5: 10, v/v). The phase-out salts and five different cleanup sorbents (including N-propyl ethylenediamine (PSA), octadecyl silane bonded silica gel (C18), graphitized carbon black (GCB), Carbon nanofiber (C-Fiber) and MWCNTs) were investigated to improve the purification efficiency. The Box-Behnken Design (BBD) study was employed for an optimal solution of the volume of extraction solvent, phase-out salt, and the purification sorbents for the analytical method. The average recoveries of the target analytes in the three medlar matrices ranged from 70 % to 119 % with relative standard deviations (RSDs) of 1.0 %-19.9 %. Screening of market samples (fresh and dried medlars) collected from the major producing regions in China showed that 15 pesticides and metabolites were detected in the samples at concentrations of 0.01-2.22 mg/kg, and none of which exceeded the maximum residue limits (MRLs) set in China. The results showed that the risk of food safety by consumption of medlar products caused by the use of pesticides was low. The validated method could be used for rapid and accurate screening of multi-class multi-pesticide residues in Medlar for food safety.

Paeoniflorin protects 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease mice by inhibiting oxidative stress and neuronal apoptosis through activating the Nrf2/HO-1 signaling pathway.

OBJECTIVES: This study aimed to explore the neuroprotective effects of paeoniflorin on oxidative stress and apoptosis in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) mice. METHODS: The effects of paeoniflorin on motor function in mice were evaluated by behavioral test. Then substantia nigra of mice were collected and neuronal damage was assessed using Nissl staining. Positive expression of tyrosine hydroxylase (TH) was detected by immunohistochemistry. Levels of malondialdehyde, superoxide dismutase (SOD) and glutathione were measured by biochemical method. terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay was used to detect apoptosis of dopaminergic neurons. Western blotting and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expressions of Nrf2, heme oxygenase-1 (HO-1), B-cell lymphoma-2(Bcl-2), Bax and cleaved caspase-3. RESULTS: Paeoniflorin treatment significantly ameliorated the motor performance impairment in MPTP-induced PD mice. Moreover, it notably increased the positive expression rate of TH and reduced the damage and apoptosis of dopaminergic neurons in the substantia nigra. Furthermore, paeoniflorin increased the levels of SOD and glutathione and decreased the malondialdehyde content. It also promoted Nrf2 nuclear translocation, increased the protein and mRNA expressions of HO-1 and Bcl-2 and reduced the protein and mRNA expressions of BCL2-Associated X2 (Bax) and cleaved caspase-3. Treatment with the Nrf2 inhibitor, ML385, notably reduced the effects of paeoniflorin in MPTP-induced PD mice. CONCLUSIONS: Neuroprotective effects of paeoniflorin in MPTP-induced PD mice may be mediated via inhibition of oxidative stress and apoptosis of dopaminergic neurons in substantia nigra through activation of the Nrf2/HO-1 signaling pathway.

DaDL-SChlo: protein subchloroplast localization prediction based on generative adversarial networks and pre-trained protein language model.

Chloroplast is a crucial site for photosynthesis in plants. Determining the location and distribution of proteins in subchloroplasts is significant for studying the energy conversion of chloroplasts and regulating the utilization of light energy in crop production. However, the prediction accuracy of the currently developed protein subcellular site predictors is still limited due to the complex protein sequence features and the scarcity of labeled samples. We propose DaDL-SChlo, a multi-location protein subchloroplast localization predictor, which addresses the above problems by fusing pre-trained protein language model deep learning features with traditional handcrafted features and using generative adversarial networks for data augmentation. The experimental results of cross-validation and independent testing show that DaDL-SChlo has greatly improved the prediction performance of protein subchloroplast compared with the state-of-the-art predictors. Specifically, the overall actual accuracy outperforms the state-of-the-art predictors by 10.7% on 10-fold cross-validation and 12.6% on independent testing. DaDL-SChlo is a promising and efficient predictor for protein subchloroplast localization. The datasets and codes of DaDL-SChlo are available at https://github.com/xwanggroup/DaDL-SChlo.

Transcriptome and metabolome profiling of the medicinal plant Veratrum mengtzeanum reveal key components of the alkaloid biosynthesis.

Veratrum mengtzeanum is the main ingredient for Chinese folk medicine known as "Pimacao" due to its unique alkaloids. A diverse class of plant-specific metabolites having key pharmacological activities. There are limited studies on alkaloid synthesis and its metabolic pathways in plants. To elucidate the alkaloid pathway and identify novel biosynthetic enzymes and compounds in V. mengtzeanum, transcriptome and metabolome profiling has been conducted in leaves and roots. The transcriptome of V. mengtzeanum leaves and roots yielded 190,161 unigenes, of which 33,942 genes expressed differentially (DEGs) in both tissues. Three enriched regulatory pathways (isoquinoline alkaloid biosynthesis, indole alkaloid biosynthesis and tropane, piperidine and pyridine alkaloid biosynthesis) and a considerable number of genes such as AED3-like, A4U43, 21 kDa protein-like, 3-O-glycotransferase 2-like, AtDIR19, MST4, CASP-like protein 1D1 were discovered in association with the biosynthesis of alkaloids in leaves and roots. Some transcription factor families, i.e., AP2/ERF, GRAS, NAC, bHLH, MYB-related, C3H, FARI, WRKY, HB-HD-ZIP, C2H2, and bZIP were also found to have a prominent role in regulating the synthesis of alkaloids and steroidal alkaloids in the leaves and roots of V. mengtzeanum. The metabolome analysis revealed 74 significantly accumulated metabolites, with 55 differentially accumulated in leaves compared to root tissues. Out of 74 metabolites, 18 alkaloids were highly accumulated in the roots. A novel alkaloid compound viz; 3-Vanilloylygadenine was discovered in root samples. Conjoint analysis of transcriptome and metabolome studies has also highlighted potential genes involved in regulation and transport of alkaloid compounds. Here, we have presented a comprehensive metabolic and transcriptome profiling of V. mengtzeanum tissues. In earlier reports, only the roots were reported as a rich source of alkaloid biosynthesis, but the current findings revealed both leaves and roots as significant manufacturing factories for alkaloid biosynthesis.

Key metabolites and mechanistic insights in forchlorfenuron controlling kiwifruit development.

Forchlorfenuron (CPPU) is a plant growth regulator widely applied on kiwifruit to improve yield, however, there are rarely reports on its effects on the nutrients of kiwifruits. Based on UHPLC-Q-TOF-MS, the effects of CPPU on metabolism profile and nutrient substances of two kiwifruit varieties during development were investigated by non-targeted metabolomics. A total of 115 metabolites were identified, and 29 differential metabolites were confirmed and quantified using certified reference standards. Metabolic profile indicated that CPPU promoted kiwifruit development during the main expansion stages at the molecular level, and the effects varied slightly for different varieties. In the early and middle stages of kiwifruit development, the anthocyanin, flavone and flavonol biosynthesis were down-regulated in both varieties, and flavanols biosynthesis was down-regulated only in Hayward variety. Arginine biosynthesis was down-regulated at all stages till the harvest. Although the synthesis of these nutrient substances in kiwifruits was mostly down-regulated by CPPU, the negative effects became mild at harvest time, and positively, the significant increase of sucrose and decrease of organic acids at harvest time could help to improve the taste of kiwifruits.

Different photoprotective strategies for white leaves between two co-occurring Actinidia species.

At the outer canopy, the white leaves of Actinidia kolomikta can turn pink but they stay white in A. polygama. We hypothesized that the different leaf colors in the two Actinidia species may represent different photoprotection strategies. To test the hypothesis, leaf optical spectra, anatomy, chlorophyll a fluorescence, superoxide (O2 ˙- ) concentration, photosystem II photo-susceptibility, and expression of anthocyanin-related genes were investigated. On the adaxial side, light reflectance was the highest for white leaves of A. kolomikta, followed by its pink leaves and white leaves of A. polygama, and the absorptance for white leaves of A. kolomikta was the lowest. Chlorophyll and carotenoid content of white and pink leaves in A. kolomikta were significantly lower than those of A. polygama, while the relative anthocyanin content of pink leaves was the highest. Chloroplasts of palisade cells of white leaves in A. kolomikta were not well developed with a lower maximum quantum efficiency of PSII than the other types of leaves (pink leaves of A. kolomikta and white leaves of A. Polygama at the inner/outer canopy). After high light treatment from the abaxial surface, Fv /Fm decreased to a larger extent for white leaves of A. kolomikta than pink leaf and white leaves of A. polygama, and its non-photochemical quenching was also the lowest. White leaves of A. kolomikta showed higher O2 ˙- concentration compared to pink leaves under the same strong irradiance. The expression levels of anthocyanin biosynthetic genes in pink leaves were higher than in white leaves. These results indicate that white leaves of A. kolomikta apply a reflection strategy for photoprotection, while pink leaves resist photoinhibition via anthocyanin accumulation.

Stabilization of Pb(II) in wastewater and tailings by commercial bacteria through microbially induced phosphate precipitation (MIPP).

Microbially induced phosphate precipitation (MIPP) is an effective and eco-friendly method for Pb(II) stabilization. The phosphate-solubilizing microorganisms (PSM) for MIPP are commonly isolated from Pb(II)-contaminated sites through a series of intricate and time-consuming enrichment and purification processes. This research used ready-made commercial bacteria to develop a simple MIPP process. Bacillus subtilis (BS, CCTCC AB 98002) was selected from two commercial PSM strains owing to more effective Pb(II) removal. Compared to the most isolated microorganisms, BS released more than twice as much inorganic phosphorus (Pi) as well as had a high-level Pb(II) tolerance. BS could remove >99% of Pb(II) from 500 mg/L Pb(II)-containing water at the optimal 0.05 M sodium glycerophosphate (SGP), pH 7-9, and ≤0.03 M MgCl2, outperforming most isolated microorganisms. In addition, BS could mitigate the contamination risk of the lead­zinc tailings, by reducing the readily leachable Pb(II) concentration from 0.81 mg/L (over the regulatory limit of 0.1 mg/L) to 0.00042 mg/L. The unstable Pb(II) in the solution and tailings was ultimately stabilized to Pb5(PO4)3Cl after the SGP phosphorlysis and phosphate precipitation processes. In conclusion, commercial BS is a superior alternative to isolated microorganisms for MIPP on Pb(II) stabilization. The simple-processed and high-effective BS-based MIPP provides the MIPP method a new insight for widespread implementation in the remediation of heavy metals-containing wastewater, soil, and waste.

Stabilization and strengthening of chromium(VI)-contaminated soil via magnesium ascorbyl phosphate (MAP) and phytase addition.

Cr(VI) contamination of soil threatens the environment and reduces soil strength. Therefore, both Cr(VI) stabilization and soil reinforcement should be considered in site remediation for future construction. This study investigated a biochemical treatment process using magnesium ascorbyl phosphate (MAP) and phytase. MAP was hydrolyzed via phytase catalysis to produce ascorbic acid (AA) and MgHPO4·3H2O precipitation. The AA reduced Cr(VI) into low-toxic Cr(III), which precipitated as Cr(OH)3 and CrPO4. More than 90% of the 500 mg/kg Cr(VI) in soil was reduced by 5% MAP (wt% of soil) and 1% phytase (vol/vol of soil water) doses at the geotechnically optimal soil moisture content of 16.8%. The MgHPO4·3H2O precipitates filled soil pores and enhanced the unconfined compression strength of treated soil by more than two times. This research reports a novel and practical enzymatically induced phosphate precipitation process for the remediation of Cr(VI)-contaminated soil.

Scalable Palladium-Catalyzed Alkoxycarbonylation of Conjugated Dienes.

The Pd(cod)Cl2-catalyzed alkoxycarbonylation of conjugated dienes to ß,γ-unsaturated esters was approached by both intramolecular phosphinesulfonate L1 and intermolecular PPh3/PTSA in this study. However, the poor solubility of the Pd/L1 complex and the labile monodentate Pd/PPh3 structure restricts the system efficiency, especially for the scale-up application. By contrast, the stable and well-soluble bidentate Xantphos system allows for the quantitative formation of 3-pentenoate (96%) on a gram scale within 6 h in weakly alkaline N-methylpyrrolidone (NMP), which also functions as a basic site to promote the rate-limiting alcoholysis step while reducing the dosage of ligand to a theoretical value.

Design, synthesis and fungicidal evaluation of novel psoralen derivatives containing sulfonohydrazide or acylthiourea moiety.

To search a novel lead structure for antiphytopathogenic fungus agent, a series of novel psoralen derivatives possessing sulfonohydrazide or acylthiourea structure were designed and synthesized, and their fungicidal activity against seven phytopathogens was evaluated. Their structures were confirmed by melting points, 1H NMR, 13C NMR and HRMS, and the typical crystal structure was determined by X-ray diffraction for validation. Preliminary fungicidal activity showed that some of the title compounds exhibited certain-to-high fungicidal activity. Compound I-13 exhibited good fungicidal activity against Botrytis cinerea, Cercospora arachidicola and Physalospora piricola with EC50 values of 12.49, 13.22 and 12.12 µg/mL, respectively. Compounds II-9 and II-15 showed over 90% inhibition against B. cinerea at 50 µg/mL in vitro. In particular, II-9 exhibited significant higher fungicidal activity with a lower EC50 value of 9.09 µg/mL than the positive control YZK-C22 (13.41 µg/mL). Our studies found that sulfonohydrazide or acylthiourea-containing psoralen derivatives were promising fungicide leads deserve for further study.

The Protective Role of Non-Photochemical Quenching in PSII Photo-Susceptibility: A Case Study in the Field.

Non-photochemical quenching (NPQ) has been regarded as a safety valve to dissipate excess absorbed light energy not used for photochemistry. However, there exists no general consensus on the photoprotective role of NPQ. In the present study, we quantified the Photosystem II (PSII) photo-susceptibilities (mpi) in the presence of lincomycin, under red light given to five shade-acclimated tree species grown in the field. Photosynthetic energy partitioning theory was applied to investigate the relationships between mpi and each of the regulatory light-induced NPQ [Y(NPQ)], the quantum yield of the constitutive nonregulatory NPQ [Y(NO)] and the PSII photochemical yield in the light-adapted state [Y(PSII)] under different red irradiances. It was found that in the low to moderate irradiance range (50-800 µmol m-2 s-1) when the fraction of open reaction centers (qP) exceeded 0.4, mpi exhibited no association with Y(NPQ), Y(NO) and Y(PSII) across species. However, when qP < 0.4 (1,500 µmol m-2 s-1), there existed positive relationships between mpi and Y(NPQ) or Y(NO) but a negative relationship between mpi and Y(PSII). It is postulated that both Y(NPQ) and Y(NO) contain protective and damage components and that using only Y(NPQ) or Y(NO) metrics to identify the photo-susceptibility of a species is a risk. It seems that qP regulates the balance of the two components for each of Y(NPQ) and Y(NO). Under strong irradiance, when both protective Y(NPQ) and Y(NO) are saturated/depressed, the forward electron flow [i.e. Y(PSII)] acts as the last defense to resist photoinhibition.

Metal ion induced dual switchable dielectric and luminescent properties in hybrid halides.

A new multi-functional organic-inorganic hybrid compound was successfully obtained by regulating metal halides. Apart from excellent luminescence properties, in particular, the introduction of a Mn halide successfully achieved a dual-switchable dielectric property, which could lead to very interesting exploration in sensors.

Novel [1,2,4]-Triazolo[3,4-b]-[1,3,4]thiadizoles as Potent Pyruvate Kinase Inhibitors for Fungal Control.

To discover novel target-based fungicidal candidates, a molecular design model was established with a three-dimensional (3D) structure of Rhizoctonia solani pyruvate kinase (RsPK) simulated with the AlphaFold 2 and YZK-C22 as a fungicidal lead. A series of novel [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives were rationally designed, synthesized, evaluated for their fungicidal performance, and validated for their mode of action. The in vitro bioassays with R. solani indicated that compounds 5g, 5o, and 5z with an EC50 value ranging from 1.01 to 1.54 µg/mL displayed higher fungicidal activity than the positive control YZK-C22 with its EC50 of 3.14 µg/mL. Especially, 5o exhibited high potency and a broad spectrum against Alternaria solani, Botrytis cinerea, Cercospora arachidicola, Physalospora piricola, R. solani, and Sclerotinia sclerotiorum with its EC50 value falling between 1.54 and 13.10 µg/mL. Like all positive controls, 5g, 5o, and 5z showed excellent in vivo growth inhibition against Pseudoperonospora cubensis at 200 µg/mL. Even though the PK enzymatic inhibition assay showed that 5o was approximately 2.6 times less active than YZK-C22 (IC50: 29.14 vs 11.15 µg/mL, respectively), the similar fluorescence quenching patterns of RsPK by 5o and YZK-C22, and the docking results of interactions between RsPK and 5o or YZK-C22 implied that they might share the similar binding site in the RsPK active pocket. Our studies suggested that 5o could be used as a potent fungicidal lead for further optimization. The results of comparative molecular field analysis (CoMFA) provided a direction for further molecular design.

Design, Synthesis, and Evaluation of Novel 4-Chloropyrazole-Based Pyridines as Potent Fungicide Candidates.

A rational molecular design approach was developed in our laboratory to guide the discovery of novel sterol biosynthesis inhibitors. Based on the application of bioactivities of heterocyclic rings and molecular docking targeting the sterol biosynthesis 14α-demethylase, a series of 4-chloropyrazole-based pyridine derivatives were rationally designed, synthesized, and characterized and their fungicidal activities were also evaluated. Bioassay results showed that 7e, 7f, and 7m exhibited commendable, diverse antifungal actions that are comparable to those of the positive controls imazalil and triadimefon. The active compounds' mode of action was further studied by microscopy observations, Q-PCR, and enzyme inhibition assay and discovered that target compounds affect fungal sterol biosynthesis via disturbing RcCYP51 enzyme system. These findings support that their fungicidal mode of action still targets the cytochrome P450-dependent 14α-demethylase as the molecular design did at first. The above results strongly suggest that our rational molecular design protocol is not only practical but also efficient.

Functional Characterization of Soybean Diacylglycerol Acyltransferase 3 in Yeast and Soybean.

Diacylglycerol acyltransferases (DGAT) function as the key rate-limiting enzymes in de novo biosynthesis of triacylglycerol (TAG) by transferring an acyl group from acyl-CoA to sn-3 of diacylglycerol (DAG) to form TAG. Here, two members of the type 3 DGAT gene family, GmDGAT3-1 and GmDGAT3-2, were identified from the soybean (Glycine max) genome. Both of them were predicted to encode soluble cytosolic proteins containing the typical thioredoxin-like ferredoxin domain. Quantitative PCR analysis revealed that GmDGAT3-2 expression was much higher than GmDGAT3-1's in various soybean tissues such as leaves, flowers, and seeds. Functional complementation assay using TAG-deficient yeast (Saccharomyces cerevisiae) mutant H1246 demonstrated that GmDGAT3-2 fully restored TAG biosynthesis in the yeast and preferentially incorporated monounsaturated fatty acids (MUFAs), especially oleic acid (C18:1) into TAGs. This substrate specificity was further verified by fatty-acid feeding assays and in vitro enzyme activity characterization. Notably, transgenic tobacco (Nicotiana benthamiana) data showed that heterogeneous expression of GmDGAT3-2 resulted in a significant increase in seed oil and C18:1 levels but little change in contents of protein and starch compared to the EV-transformed tobacco plants. Taken together, GmDGAT3-2 displayed a strong enzymatic activity to catalyze TAG assembly with high substrate specificity for MUFAs, particularly C18:1, playing an important role in the cytosolic pathway of TAG synthesis in soybean. The present findings provide a scientific reference for improving oil yield and FA composition in soybean through gene modification, further expanding our knowledge of TAG biosynthesis and its regulatory mechanism in oilseeds.