Incarvine C and its analogues inhibit the formation of cell cytoskeleton by targeting Rac1.

Ras-related C3 botulinum toxin substrate 1 (Rac1) has emerged as a key regulator in the treatment of cancer metastasis because of its involvement in the formation of cell plate pseudopods and effects on cell migration. In this study, we found that incarvine C, a natural product isolated from Incarvillea sinensis, and its seven analogues exhibited antitumour activity by inhibiting cell cytoskeleton formation, with moderate cytotoxicity. Accordingly, these compounds inhibited the cytoskeleton-mediated migration and invasion of MDA-MB-231 cells, with inhibition rates ranging from 37.30 % to 69.72 % and 51.27 % to 70.90 % in vitro, respectively. Moreover, they induced G2/M phase cell cycle arrest in MDA-MB-231 cells. A pull-down assay revealed that the interaction between Rac1 and its downstream effector protein PAK1 was inhibited by these compounds and that the compound Ano-6 exhibited substantial activity, with an inhibition rate of more than 90 %. Molecular docking showed that incarvine C and its analogues could bind to the nucleotide-binding pocket of Rac1, maintaining high levels of inactivated Rac1. As Ano-6 exhibited significant activity in vitro, its anti-cancer activity was tested in vivo. Four weeks of oral treatment with Ano-6 was well-tolerated in mice, and it induced a potential anti-tumour response in xenografts of MDA-MB-231 cells. Further studies demonstrated that Ano-6 was enriched in tumour tissues after 2 h of administration and induced an increase in the number of dead tumour cells. In summary, these findings not only reveal the mechanism of incarvine C but also provide a new molecular template for Rac1 inhibitors and identify a promising candidate for breast cancer treatment.

Treatment of Coking Wastewater Using Hydrodynamic Cavitation Coupled with Fenton Oxidation Process.

Effective and economical processes for the advanced treatment of coking wastewater were urgently needed to reduce the persistent organic pollutants of external drainage. In the present work, we investigated the degradation of organic pollutants in coking wastewater through IHC/FO (imping stream hydrodynamic cavitation (IHC) coupled with the Fenton oxidation (FO) process) and IHC alone for their feasibility in the advanced treatment of coking wastewater. To select the optimum parameters, attention was paid to the effects of main operation conditions including inlet fluid pressure, medium temperature, initial pH, reaction time, and initial Fe(II) and initial H2O2 concentrations. The results showed that the effects of conditions that need energy to be maintained (such as initial pH and inlet pressure) on the organic pollutant removal efficiency through IHC/FO were less pronounced than those through IHC alone. Moreover, the application of IHC/FO could remove more organic pollutants from coking wastewater than IHC even at an energy-efficient condition. For example, the highest COD removal efficiency of 12.5% was achieved in the IHC treatment at 0.4 MPa, pH 3, and 60 min for the reaction time. In the case of IHC/FO, the maximum COD removal of 33.2% was obtained at pH 7, 0.1 MPa, 12 mmol/L H2O2, and 3 mmol/L Fe2+ after reacting for 15 min. The ultraviolet and visible spectrophotometry (UV-Vis) absorption spectra and gas chromatography and mass spectrometry (GC-MS) analysis further revealed that the kinds and amounts of pollutants (especially those that had benzenes) remaining in water treated through IHC/FO were much fewer and smaller than in water treated through IHC alone. The better performances of IHC/FO than IHC alone were likely related to the more hydroxyl radicals produced through IHC/FO. Taken together, our findings indicate that IHC/FO has great application potential in the advanced treatment of coking wastewater.

A New Monoterpene Alkaloid from Incarvillea sinensis with Migration Inhibitory Activity on Cancer Cell.

A novel monoterpene alkaloid, named incarvine G, was isolated from the Incarvillea sinensis Lam. Its chemical structure was elucidated using comprehensive spectroscopic methods. Incarvine G is an ester compound comprised of a monoterpene alkaloid and glucose. This compound showed evident inhibition on cell migration, invasion, and cytoskeleton formation of human MDA-MB-231 with low cytotoxicity.

Citric acid inhibits Cd uptake by improving the preferential transport of Mn and triggering the defense response of amino acids in grains.

Citric acid (CA) can regulate the balance of anions and cations in plants, and improve their resistance to heavy metals. It is not clear if foliar application with CA has any effect on migration of Cd and Mn in rice plant. In this work, a low-Cd-accumulating indica rice line (P7) and a high-Cd-accumulating line (HZ) were used to investigate the influence of CA on the transport of Cd and Mn as well as amino acid metabolism in grains. Content of Cd in grains and other organs increased with the increase of Cd content (0.1-2.4 mg kg-1) in soil, while decreased with the foliar application with CA. With the increase of Cd content in rice grains, the content of most amino acids in HZ, P7, HZ+CA and P7 + CA showed an obvious decline trend. Foliar application with CA efficiently raised the Mn:Cd ratio in grains and nodes of both HZ and P7. Meanwhile, the expression levels of OsNramp2, 3 and 5 in panicles were efficiently enhanced by CA application when plants grew in soil with Cd content of 0.6-2.4 mg kg-1. The increasing effect of CA on the content of 4 amino acids (i.e., Glu, Phe, Thr and Ala) in grains was related to varieties and Cd pollution. These results indicate that foliar application with CA can regulate the transport of Cd and Mn in the opposite directions in tissues and inhibit Cd accumulation in grains by enhancing expression of OsNRAMP 2, 3 or 5 and triggering the defense response of some amino acids in Cd-contaminated environment.

Factors affecting user satisfaction with new energy vehicles: A field survey in Shanghai and Nanjing.

Based on the diffusion of innovation theory (DOI), this paper aims to explore the potential factors that influence user satisfaction with new energy vehicles (NEVs). Furthermore, we introduce the term "personal environmental awareness" (PEA) and attempt to examine its contingent effects. A moderated hierarchical linear regression analysis (MHLRA) method is conducted to analyze the data collected from a field survey of 335 early-drivers of NEVs. Empirical results show that experiences related to usefulness, experience of ease-of-use, total cost, driving range, and infrastructure readiness are important factors that influence user satisfaction with NEVs. In the case of moderating effects, we found that PEA might strengthen the positive effects of experienced usefulness, experienced ease-of-use, driving range, and infrastructure readiness in terms of user satisfaction with NEVs. However, the moderating role of PEA in the relationship between total cost and user satisfaction is not statistically significant. Our research findings might provide some useful insights for market managers, academic researchers and policy makers.

Gadolinium inhibits cadmium transport by blocking non-selective cation channels in rice seedlings.

Non-selective cation channels (NSCCs) play important roles in uptake of heavy metals in plants. However, little information is available concerning the contribution of NSCCs to cadmium (Cd) transport in rice seedlings. Results from the hydroponic experiment showed that the inhibition of 2.7 µM Cd on the development of rice roots was alleviated by adding 0.1 mM gadolinium (Gd) in nutrient solution, companied by reduction of Cd content by 55.3% in roots and by 45.0% in shoots. Inhibition of Gd on Cd accumulation in cytoplasm fraction (F3) was much greater than that in cell walls (F1) and organelles (F2) in roots. After increasing concentrations of Mn and Zn in nutrient solution, adding 0.1 mM Gd resulted in reductions in Cd content by 89.1%, in micronutrients by 54.9% and in macronutrients by 5.4% in roots, respectively. Cd stress resulted in significant increase of PC2∼4 and free amino acids, but decrease of V-ATPase activity by 32.3% in roots. These results indicate that NSCCs make a great contribution to uptake of Cd in rice seedlings and opportunities for Cd to be transported by NSCCs can be efficiently reduced by blocking NSCCs and/or increasing essential microelements like Mn and Zn.

Rice vegetative organs alleviate cadmium toxicity by altering the chemical forms of cadmium and increasing the ratio of calcium to manganese.

Altering Cd chemical form is one of the mechanisms to alleviate Cd toxicity in rice plant. Field experiments were carried out in this study to investigate the potential of rice vegetative organs in altering Cd into insoluble chemical forms in the natural environment. Experimental results showed that more than 80% of Cd in rice roots existed in the insoluble forms. Uppermost nodes altered Cd into insoluble form preferentially and generally had higher content of insoluble Cd than other organs. Rachises displayed a slow increasing trend in soluble Cd when total Cd in roots was less than 1.8 mg kg-1. However, when Cd content in roots exceeded 2.8 mg kg-1, the ratio of insoluble to soluble Cd remained stable at 85:15 in rachises and roots, and at 75:25 in uppermost nodes and flag leaves. Cd concentration in grains was greatly lower than that in vegetative organs, and closely correlated with the content of soluble Cd in rachises (r = 0.991**) as well as in uppermost nodes. Soluble Cd in the uppermost nodes displayed a much lower mobility than that in other organs. Accumulation of soluble Cd was always companied by decrease of Ca and increase of Mn in roots, uppermost nodes and rachises. A small increase of soluble Cd from 0.05 to 0.1 mg kg-1 caused a sharp decline of Ca:Mn ratio in roots and rachises. Roots and nodes had much higher Ca:Mn ratio than rachises when soluble Cd was less than 0.5 mg kg-1 in them. These results indicate that vegetative organs have a great potential to alter more than 75% Cd into insoluble forms and increasing Ca:Mn ratio may be another way to alleviate Cd toxicity by establishing new ionic homeostasis in rice plants.

Complete genome sequence of soil actinobacteria Streptomyces cavourensis TJ430.

A new actinobacteria Streptomyces cavourensis TJ430 was isolated from the mountain soil collected from the southwest of China. In previous study, TJ430 showed striking bactericidal activities and strong ability of antibiotic production. Here, we report complete genome of this bacterium, consisting of 7.6 Mb linear chromosome and 0.2 Mb plasmids. It was predicted 6450 genes in chromosome and 225 genes in plasmids, as well as 12 gene islands in chromosome. Abundant genes have predicted functions in antibiotic metabolism and stress resistance. A whole-genome comparison of S. cavourensis TJ430, S. coelicolor A3(2), and S. lividans 66 indicates that TJ430 has a relatively high degree of strain specificity. The 16S rRNA phylogenetic tree shows the high identities (99.79%) of TJ430 with S. cavourensis DSM40300. TJ430 is a new and rare Streptomyces species, and analysis of its genome helps us to better understand primary metabolism mechanism of this isolate, as well as the evolutionary biology.

[Isolation, identification of TJ430 strain and characterization of its antifungal metabolite].

OBJECTIVE: To screen new agro-antibiotics, rare actinomycetes were isolated by improved separation methods from soil samples and the chemical structure of the antifungal active product was elucidated. METHODS: Dry heating method was used for soil samples pretreatment and the improved HV separation medium for rare actinomycetes separation; agar block rapid screening was used for the rapid evaluation of rare actinomycetes biological activity. For the identification of a strain numbered TJ430, morphology observation, cell chemical composition analysis, physiological and biochemical analysis, enzymology characteristics analysis, 16 S rDNA sequence analysis, and DNA hybridization method were used. Bioactive crude extract from fermentation was purified by column chromatography and preparative chromatography; infrared spectroscopy and high resolution mass spectrometry was used for structure elucidation of bioactive ingredient. RESULTS: A total of 570 rare actinomycetes strains were isolated. Antibacterial activity of rapid screen showed that the numbed TJ430 strain showed excellent anti oomycetes and broad-spectrum antifungal activity. Strain identification results show that the strain is a S. cavourensis. The molecular formulas of the effective ingredient is C40H66N3O11, molecular weight is 765. Amino, methyl, methylene, carbonyl, covalent bond, isopropyl and other chemical groups should contained in the molecular. CONCLUSION: The characterized antibacterial active ingredient has good development prospect.

Effects of elevation and geomorphology on cadmium, lead and chromium enrichment in paddy soil and rice: A case study in the Xiangtan basin of China.

The distributions of heavy metals in paddy fields and rice along river valleys were studied to explore the key factors affecting the accumulation of heavy metals in the upstream terraces and downstream plains. Results from 975 sampling sites showed that elevation, growing season and soil organic matter (OM) had significant effects on the content of Cd and Pb in topsoil and rice. The content of Cd (0.47-0.66 mg kg-1) and Pb (49.9-68.6 mg kg-1) in paddy fields with low elevation (30-60 m) in the downstream plains was significantly higher than the content of Cd (0.29-0.38 mg kg-1) and Pb (43.9-56.3 mg kg-1) in the upstream terraces with high altitude (60-90 m). In the double-rice production area, late rice generally produced grains with higher Cd and Pb content than early rice. Soil Cd was positively increased with the content of OM, especially in the downstream plains. When elevation was used for principal component analysis, plains with low elevation were grouped together with high content of total and soluble Cd, OM and Pb in soil, as well as high content of Cd and Pb in late rice. Altitude is one of the key factors affecting Cd content in rice. Although content of Cr (93.7-138.0 mg kg-1) was significantly higher than that of Cd and Pb in soil, content of Cr was lower than that of Cd in rice. These results indicate that paddy fields with elevation of 30-60 m in the downstream plains had high risk to produce late rice with Cd and Pb content exceeding the food safety standard 0.2 mg kg-1, which may be resulted from the driving force of runoff on soil soluble Cd and Pb from terraces to alluvial plains in river valleys.

Interactions between rice starch and flavor components and their impact on flavor.

Flavor is considered one of the most significant factors affecting food quality. However, it is often susceptible to environmental factors, so encapsulation is highly necessary to facilitate proper handling and processing. In this study, the structural changes in starch encapsulation and their effects on flavor retention were investigated using indica starch (RS) as a matrix to encapsulate three flavoring compounds, namely nonanoic acid, 1-octanol, and 2-pentylfuran. The rheological and textural results suggested that the inclusion of flavor compounds improved the intermolecular interactions between starch molecules, resulting in a significant increase in the physicochemical properties of starch gels in the order: nonanoic acid > 1-octanol > 2-pentylfuran. The XRD results confirmed the successful preparation of v-starch. Additionally, the inclusion complexes (ICs) were characterized using FT-IR, SEM, and DSC techniques. The results showed that v-starch formed complexes with Flavor molecules. The higher enthalpy of the complexes suggested that the addition of alcohols and acids could improve the intermolecular complexation between starch molecules. The retention rates of three flavor compounds in starch were determined using HS-GC, with the values of 51.7 %, 32.37 %, and 35.62 %. Overall, this study provides insights into novel approaches to enhance the quality and flavor retention, improve the storability and stability, reduce losses during processing and storage, and extend the shelf life of starchy products.

Insight into the Effects of Hydrodynamic Cavitation at Different Ionic Strengths on Physicochemical and Gel Properties of Myofibrillar Protein from Tilapia (Oreochromis niloticus).

Effects of different ionic strengths (0.2, 0.4, and 0.6 mol/L) and different hydrodynamic cavitation (HC) treatment times (0, 1, 2, 3, and 4 min) on the conformation and gel properties of tilapia myofibrillar proteins (TMP) were investigated. The results showed that the solubility of TMP was significantly enhanced (p < 0.05) with the increase in NaCl concentration, and the gel characteristics were significantly improved. After HC treatment of TMP, the average particle size was significantly reduced (p < 0.05) and solubility was significantly enhanced (p < 0.05) with the increase in treatment time, the internal hydrophobic groups and reactive sulfhydryl groups were exposed. The intrinsic fluorescence spectra showed the unfolding of the spatial tertiary structure of proteins, and the circular dichroism spectroscopy showed the significant reduction in the content of α-helix in the secondary structure of the proteins (p < 0.05). In addition, the WHC and gel strength of the TMP heat-induced gels were enhanced, which improved the microstructure of the gels, and scanning electron microscopy showed that the gel network of the TMP gels became denser and more homogeneous. Dynamic rheology results showed that HC treatment resulted in a significant increase in the final G' and G" values of TMP. In conclusion, HC treatment was able to improve the physicochemical structure and gel properties of TMP at different ionic strengths. This study presents a novel processing technique for the quality maintenance aspect of salt-reduced surimi gel products.

Enhanced fabrication of size-controllable chitosan-genipin nanoparticles using orifice-induced hydrodynamic cavitation: Process optimization and performance evaluation.

Chitosan nanoparticles (NPs) possess great potential in biomedical fields. Orifice-induced hydrodynamic cavitation (HC) has been used for the enhancement of fabrication of size-controllable genipin-crosslinked chitosan (chitosan-genipin) NPs based on the emulsion cross-linking (ECLK). Experiments have been performed using various plate geometries, chitosan molecular weight and under different operational parameters such as inlet pressure (1-3.5 bar), outlet pressure (0-1.5 bar) and cross-linking temperature (40-70 °C). Orifice plate geometry was a crucial factor affecting the properties of NPs, and the optimized geometry of orifice plate was with single hole of 3.0 mm diameter. The size of NPs with polydispersity index of 0.359 was 312.6 nm at an optimized inlet pressure of 3.0 bar, and the maximum production yield reached 84.82 %. Chitosan with too high or too low initial molecular weight (e.g., chitosan oligosaccharide) was not applicable for producing ultra-fine and narrow-distributed NPs. There existed a non-linear monotonically-increasing relationship between cavitation number (Cv) and chitosan NP size. Scanning electron microscopy (SEM) test indicated that the prepared NPs were discrete with spherical shape. The study demonstrated the superiority of HC in reducing particle size and size distribution of NPs, and the energy efficiency of orifice type HC-processed ECLK was two orders of magnitude than that of ultrasonic horn or high shear homogenization-processed ECLK. In vitro drug-release studies showed that the fabricated NPs had great potential as a drug delivery system. The observations of this study can offer strong support for HC to enhance the fabrication of size-controllable chitosan-genipin NPs.

Malic acid inhibits accumulation of cadmium, lead, nickel and chromium by down-regulation of OsCESA and up-regulation of OsGLR3 in rice plant.

Malic acid (MA) plays an important role in plant tolerance to toxic metals, but its effect in restricting the transport of harmful metals remains unclear. In this study, japonica rice NPB and its fragile-culm mutant fc8 with low cellulose and thin cell wall were used to investigate the influence of MA on the accumulation of 4 toxic elements (Cd, Pb, Ni, and Cr) and 8 essential elements (K, Mg, Ca, Fe, Mn, Zn, Cu and Mo) in rice. The results showed that fc8 accumulated less toxic elements but more Ca and glutamate in grains and vegetative organs than NPB. After foliar application with MA at rice anthesis stage, the content of Cd, Pb, Ni significantly decreased by 27.9-41.0%, while those of Ca and glutamate significantly increased in both NPB and fc8. Therefore, the ratios between Cd and Ca in grains of NPB (3.4‰) and fc8 (1.5‰) were greatly higher than that in grains of NPB + MA (1.1‰) and fc8+MA (0.8‰) treatments. Meanwhile, the expression of OsCEAS4,7,8,9 for the cellulose synthesis in secondary cell walls were down-regulated and cellulose content in vegetative organs of NPB and fc8 decreased by 16.7-21.1%. However, MA application significantly up-regulated the expression of GLR genes (OsGLR3.1-3.5) and raised the activity of glutamic-oxalacetic transaminease for glutamate synthesis in NPB and fc8. These results indicate that hazard risks of toxic elements in foods can be efficiently reduced through regulating cellulose biosynthesis and GLR channels in plant by combining genetic modification in vivo and malic acid application in vitro.

[Two-stage Inhibition Effects of Burkholderia sp. Y4 Application on Cadmium Uptake and Transport in Wheat].

In order to evaluate the feasibility of using Burkholderia sp. Y4 as a cadmium （Cd）-reducing bacterial agent in contaminated wheat fields， the changes in the rhizosphere soil microbial community and Cd available state， as well as the content and transport characteristics of Cd in the wheat root， basal node， internode， and grain under the treatment of strain Y4 were tested using microbial high-throughput sequencing， step-by-step extraction， subcellular distribution， and occurrence analyses. The results showed that root application of strain Y4 significantly reduced the root and grain Cd content of wheat by 7.7% and 30.3%， respectively， compared with that in the control treatment. The Cd content and Cd transfer factor results in wheat vegetative organs showed that strain Y4 reduced the Cd transfer factor from basal node to internode by 79.3%， and Cd content in the wheat internode stem also decreased by 50.9%. The study of Cd occurrence morphology showed that strain Y4 treatment increased the proportion of residual Cd in roots and basal ganglia， decreased the contents of inorganic and water-soluble Cd in roots， and increased the content of residual Cd in basal ganglia. Further examination of the subcellular distribution of Cd showed that the Cd content in root cell walls and basal ganglia cell fluid increased by 21.3% and 98.2%， respectively， indicating that the Cd fixation ability of root cell walls and basal ganglia cell fluid was improved by the strain Y4 treatment. In the rhizosphere soil， it was found that the microbial community structure was changed by strain Y4 application. Under the Y4 treatment， the relative abundance of Burkholderia increased from 9.6% to 11.5%， whereas that of Acidobacteriota decreased. Additionally， the relative abundance of Gemmatimonadales， Pseudomonadales， and Chitinophagales were also increased by strain Y4 treatment. At the same time， the application of strain Y4 increased the pH value of rhizosphere soil by 8.3%. The contents of exchangeable Cd， carbonate-bound Cd， and iron-manganese oxide-bound Cd in the soil decreased by 44.4%， 21.7%， and 15.9%， respectively， whereas the proportion of residual Cd reached 53.6%. Root application of strain Y4 increased the contents of nitrate nitrogen and ammonium nitrogen in the soil by 22.0% and 21.4%， respectively， and the contents of alkaline nitrogen also increased to a certain extent. In conclusion， the root application of strain Y4 not only improved soil nitrogen availability but also inhibited Cd transport and accumulation from contaminated soil to wheat grains in a "two-stage" manner by reducing Cd availability in rhizosphere soil and improving Cd interception and fixation capacity of wheat roots and basal nodes. Therefore， Burkholderia Y4 has application potential as a Cd-reducing and growth-promoting agent in wheat.

Inhibitory effects of Pseudomonas sp. W112 on cadmium accumulation in wheat grains: Reduced the bioavailability in soil and enhanced the interception by plant organs.

Microorganisms play an important role in heavy metal bioremediation and soil fertility. The effects of soil inoculation with Pseudomonas sp. W112 on Cd accumulation in wheat were investigated by analyzing the transport, subcellular distribution and speciation of Cd in the soil and plants. Pseudomonas sp. W112 application significantly decreased Cd content in the roots, internode and grains by 10.2%, 29.5% and 33.0%, respectively, and decreased Cd transfer from the basal nodes to internodes by 63.5%. Treatment with strain W112 decreased the inorganic and water-soluble Cd content in the roots and increased the proportion of residual Cd in both the roots and basal nodes. At the subcellular level, the Cd content in the root cell wall and basal node cytosol increased by 19.6% and 61.8%, respectively, indicating that strain W112 improved the ability of the root cell wall and basal node cytosol to fix Cd. In the rhizosphere soil, strain W112 effectively colonized and significantly decreased the exchangeable Cd, carbonate-bound Cd and iron-manganese oxide-bound Cd content by 43.5%, 27.3% and 17.6%, respectively, while it increased the proportion of residual Cd by up to 65.2%. Moreover, a 3.1% and 23.5% increase in the pH and inorganic nitrogen content in the rhizosphere soil, respectively, was recorded. Similarly, soil bacterial community sequencing revealed that inoculating with strain W112 increased the abundance of Pseudomonas, Thauera and Azoarcus, which are associated with inorganic nitrogen metabolism, and decreased that of Acidobacteria, which is indicative of soil alkalinization. Hence, root application of Pseudomonas sp. W112 improved soil nitrogen availability and inhibited Cd accumulation in the wheat grains in a two-stage process: by reducing the Cd availability in the rhizosphere soil and by improving Cd interception and fixation in the wheat roots and basal nodes. Pseudomonas sp. W112 may be a suitable bioremediation agent for restoring Cd-contaminated wheat fields.

Carbon emission reduction effect of innovative city pilot policies in China: based on the staggered difference-in-difference model.

Environmental economics theory suggests that the technological effects of increased innovation capacity can drive stable economic growth and act as a major means to mitigate regional environmental pollution levels. Therefore, science and technology innovation is the key to achieving low-carbon and green development. This study examined the influence of China's Pilot Policy for Innovative Cities on greenhouse gas emissions and its operational mechanism. By employing both quantitative and qualitative approaches, we successfully examined the impact of the policy on the nation's carbon emissions peaks. The findings indicated that adopting an urban pilot policy can effectively decelerate the increase of carbon emissions in cities. Additionally, the policy had a more pronounced impact on emission reduction in major urban areas and provinces. A mechanism test revealed that the policy could help reduce urban carbon emissions by implementing various technological innovations and spatial intensification. The results of this research offer significant theoretical support for adopting urban pilot policies and encourage the advancement of eco-friendly growth in Chinese urban areas.

A label-free LC/MS-based enzymatic activity assay for the detection of PDE5A inhibitors.

Phosphodiesterase type 5 (PDE5), a cyclic nucleotide phosphodiesterase, controls the duration of the cyclic guanosine monophosphate (cGMP) signal by hydrolyzing cGMP to GMP. Inhibiting the activity of PDE5A has proven to be an effective strategy for treating pulmonary arterial hypertension and erectile dysfunction. Current enzymatic activity assay methods for PDE5A mainly use fluorescent or isotope-labeled substrates, which are expensive and inconvenient. Here, we developed an LC/MS-based enzymatic activity assay for PDE5A without labeling, which detects the enzymatic activity of PDE5A by quantifying the substrate cGMP and product GMP at a concentration of 100 nM. The accuracy of this method was verified by a fluorescently labeled substrate. Moreover, a new inhibitor of PDE5A was identified by this method and virtual screening. It inhibited PDE5A with an IC50 value of 870 nM. Overall, the proposed strategy provides a new method for screening PDE5A inhibitors.

Mitigating the toxicity of reactive oxygen species induced by cadmium via restoring citrate valve and improving the stability of enzyme structure in rice.

The mechanism of reactive oxygen species (ROS) burst in rice cells induced by cadmium (Cd) stress remains poorly understood. The present study shows that the burst of superoxide anions (O2·-) and hydrogen peroxide (H2O2) in roots and shoots led by Cd stress was attributed to the disturbance of citrate (CA) valve and the damage of antioxidant enzyme structure in the rice seedlings. Cd accumulation in cells altered the molecular structure of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) through attacking glutamate (Glu) and other residues, leading to the significant reduction of their activities in clearing O2·- and decomposing H2O2. Citrate supplementation obviously increased the activity of antioxidant enzymes and decreased ∼20-30% of O2·- and H2O2 contents in roots and shoots. Meanwhile, the synthesis of metabolites/ligands such as CA, α-ketoglutarate (α-KG) and Glu as well as the activities of related enzymes in CA valve were remarkably improved. The activities of antioxidant enzymes were protected by CA through forming stable hydrogen-bonds between CA and antioxidant enzymes, and forming the stable chelates between ligands and Cd. These findings indicate that exogenous CA mitigated the toxicity of ROS under Cd stress by the ways of restoring CA valve function to reduce the production of ROS, and improving the stability of enzyme structure to enhance antioxidant enzymes activity.

Cadmium pollution leads to selectivity loss of glutamate receptor channels for permeation of Ca2+/Mn2+/Fe2+/Zn2+ over Cd2+ in rice plant.

The selective permeation of glutamate receptor channels (GLRs) for essential and toxic elements in plant cells is poorly understood. The present study found that the ratios between cadmium (Cd) and 7 essential elements (i.e., K, Mg, Ca, Mn, Fe, Zn and Cu) in grains and vegetative organs increased significantly with the increase of soil Cd levels. Accumulation of Cd resulted in the significant increase of Ca, Mn, Fe and Zn content and the expression levels of Ca channel genes (OsCNGC1,2 and OsOSCA1.1,2.4), while remarkable reduction of glutamate content and expression levels of GLR3.1-3.4 in rice. When planted in the same Cd-polluted soil, mutant fc8 displayed significantly higher content of Ca, Fe, Zn and expression levels of GLR3.1-3.4 than its wild type NPB. On the contrary, the ratios between Cd and essential elements in fc8 were significantly lower than that in NPB. These results indicate that Cd pollution may damage the structural integrity of GLRs by inhibiting glutamate synthesis and expression levels of GLR3.1-3.4, which leads to the increase of ion influx but the decrease of preferential selectivity for Ca2+/ Mn2+/ Fe2+/ Zn2+ over Cd2+ through GLRs in rice cells.