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α-Glucosidase (AG) is a bifunctional enzyme, it has a capacity to synthesize 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) from L-ascorbic acid (L-AA) and low-cost maltose under mild conditions, but it can also hydrolyze AA-2G, which leads to low synthesis efficiency of AA-2G. Main Methods and Major Results This study introduces a rational molecular design strategy to regulate enzymatic reactions based on inhibiting the formation of ground state of enzyme-substrate complex. Y215 was analyzed as the key amino acid site affecting the affinity of AG to AA-2G and L-AA. For the purpose of reducing the hydrolysis efficiency of AA-2G, the mutant Y215W was obtained by analyzing the molecular docking binding energy and hydrogen bond formation between AG and the substrates. Compared with the wild type, Isothermal Titration Calorimetry(ITC) results showed that the equilibrium dissociation constant (KD) of the mutant for AA-2G was doubled; the Michaelis constant (Km) for AA-2G was reduced by 1.15 times; and the yield of synthetic AA-2G was increased by 39%. Conclusions and Implications Our work also provides a new reference strategy for the molecular modification of multifunctional enzymes and other enzymes in cascade reactions system.

Poly(ADP-ribosyl)ation (PARylation) is a posttranslational modification reversibly catalyzed by poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolases (PARGs) and plays a key role in multiple cellular processes. The molecular mechanisms by which PARylation regulates innate immunity remain largely unknown in eukaryotes. Here we show that Arabidopsis UBC13A and UBC13B, the major drivers of lysine 63 (K63)-linked polyubiquitination, directly interact with PARPs/PARGs. Activation of pathogen-associated molecular pattern (PAMP)-triggered immunity promotes these interactions and enhances PARylation of UBC13. Both parp1 parp2 and ubc13a ubc13b mutants are compromised in immune responses with increased accumulation of total pathogenesis-related (PR) proteins but decreased accumulation of secreted PR proteins. Protein disulfide-isomerases (PDIs), essential components of endoplasmic reticulum quality control (ERQC) that ensure proper folding and maturation of proteins destined for secretion, complex with PARPs/PARGs and are PARylated upon PAMP perception. Significantly, PARylation of UBC13 regulates K63-linked ubiquitination of PDIs, which may further promote their disulfide isomerase activities for correct protein folding and subsequent secretion. Taken together, these results indicate that plant immunity is coordinately regulated by PARylation and K63-linked ubiquitination.

Poly(ADP-ribosyl)lation (PARylation) is a posttranslational modification that plays an important role in a variety of biological processes in both animals and plants. Identification of PARylated substrates is the key to elucidating the regulatory mechanism of PARylation. Several approaches have been developed to identify PARylated substrates over the past decade; however, a reliable and efficient method is needed to demonstrate PARylated proteins. Here, we report a simple and sensitive assay of PARylated proteins using a clickable 6-alkyne-NAD

Recently, cadmium (Cd) contamination in paddy soils has become a highly concerning pollution problem. Endophytic microbes in rice not only affect the plant growth but also contribute to ion absorption by the roots. Therefore, they are a promising, ecologically sound means of reducing the Cd transport from soils to shoots and grains of the plant. In this study, a Cd-resistant endophytic bacterium, named 181-22, with high Cd absorption capacity (90.8%) was isolated from the roots of rice planting in heavily Cd-contaminated paddy soils and was identified as Bacillus koreensis CGMCC 19,468. The strain significantly increased fresh weight of roots and shoots (44.4% and 42.7%) and dry weight of roots and shoots (71.3% and 39.9%) and decreased Cd content in the rice roots (12.8%), shoots (34.3%), and grains (39.1%) under Cd stress compared to uninoculated plant by colonizing rice roots via seed inoculation. Moreover, colonization of 181-22 reprogrammed rice physiology to alleviate Cd stress by increasing pigment and total protein content, regulating Cd-induced oxidative stress enzymes such as superoxide dismutase and catalase and reducing malondialdehyde. Thus, B. koreensis 181-22 has the potential to protect rice against Cd stress and can be used as a biofertilizer to bioremediate paddy soils contaminated with Cd. KEY POINTS: • Bacillus koreensis 181-22 colonized the inside of rice roots at high numbers via seed inoculation. • B. koreensis 181-22 promoted rice growth and decreased Cd accumulation in grains. • B. koreensis 181-22 regulated the physiological response to alleviated Cd stress in rice.

As a valid tumor marker, vascular endothelial growth factor 165 (VEGF165) is an effective therapeutic target for anticancer treatments. Aptamers hold great promise for the development of anti-VEGF strategies. In this study, anti-VEGF165 ssDNA aptamers were screened using a semirational design and a multilevel screening strategy. Recombinant human VEGF165 protein was used as a target for the construction of an ssDNA virtual aptamer library with ssDNA that had one sole secondary structure. After silicon-assisted prescreening, circular dichroism and isothermal titration calorimetry were used to further screen for candidates. Three aptamers (nos. 524, 529, and 64) with one sole secondary and tertiary structure, showing a high affinity for VEGF165, were identified. TheiK/isubD/subvalues obtained using surface plasmon resonance analysis were 36.3, 288, and 79.3 nM for aptamers 524, 529, and 64, respectively. Cytological tests revealed that the three aptamers inhibit rhVEGF165-induced proliferation of HUVECs. Specifically, aptamer 529 had the strongest inhibitory effect (nearly 100% inhibition). The screening strategy used in our study showed improved screening efficiency relative to other methods and resulted in aptamers with one sole conformation. The aptamers had an advantage in ensuring the uniqueness of aptamer targeting. This semirational design and multilevel screening strategy provide a reference for the screening of other aptamers.

Glucosidase is one of the most important hydrolases in the carbohydrate metabolism pathway in human body. It is noteworthy that glucosidase was raising high attention in numerous researches, attributed to its functional values and biological activities. The expression, secretion, extraction, purification, and quantitative analysis of glucosidase occupies an important position in the research of glucosidase. The Pichia pastoris expression system has been developed for 50 years. With its unique advantages, the expression system has successfully expressed hundreds of heterologous proteins. This article briefly describes the process of high-efficiency expression, extraction and purification of glucosidase in the P. pastoris expression system, focusing on the expression, separation and purification of glucosidase in P. pastoris, as well as the quantitative and activity analysis methods of glucosidase. Affinity chromatography is used for the separation and purification of crude enzyme solution, and UV–visible spectroscopy can be used for enzyme quantification and activity analysis.

Vascular endothelial growth factor (VEGF165) is a highly specific vascular endothelial growth factor that can be used to treat many cardiovascular diseases. The development of anti-tumor drugs and disease detection reagents requires highly pure VEGF165 (at least 95% purity). To date, the methods for heterologous expression and purification of VEGF165 require multiple purification steps, but the product purity remains to be low. In this study, we optimized the codons of the human VEGF165 gene (vegf165) according to the yeast codon preference. Based on the Pichia pastoris BBPB vector, we used the Biobrick method to construct a five-copy rhVEGF165 recombinant expression vector using Pgap as the promoter. In addition, a histidine tag was added to the vector. Facilitated by the His tag and the heparin-binding domain of VEGF165, we were able to obtain highly pure rhVEGF165 (purity98%) protein using two-step affinity chromatography. The purified rhVEGF165 was biologically active, and reached a concentration of 0.45 mg/mL. The new design of the expression vector enables production of active and highly pure rhVEGF165 ) in a simplified purification process, the purity of the biologically active natural VEGF165 reached the highest reported to date.

Background: Jianpi huoxue decoction (JHD), a Chinese herbal formula, is commonly applied in clinical practice for treatment of alcoholic liver disease (ALD) in China. Involvement of intestinal barrier function is increasingly recognized in the pathogenesis and progression of ALD. The aim of this study was to explore whether JHD on potentially protects intestinal barrier function in ALD rats. Methods: Sprague-Dawley rats were randomly divided into three groups. Model (alcohol-fed) and JHD groups were fed Lieber-DeCarli liquid diets while rats in the control group were fed isocaloric maltose dextrin for six weeks. In the last three weeks of the respective diets, rats were intragastrically administrated JHD. Each rat received a gavage administration of lipopolysaccharide (LPS) before sacrifice. Gut leakage was evaluated by measuring LPS in the portal blood. Pathological changes in ileum tissue were observed by hematoxylin and eosin (H&E) staining. Ultrastructural changes of the intestinal tract were observed by transmission electron microscopy (TEM). The tight junction (TJ) proteins ZO-1 and occludin were assessed by western blot and immunofluorescence. Myosin light chain (MLC) phosphorylation, a key regulator of TJs, was also assessed by western blot.Results: JHD administration attenuated Lieber-DeCarli liquid diet-induced hepatic steatosis and inflammation. Alcohol feeding increased portal vein plasma LPS levels in the model group. This deleterious effect was decreased in the JHD-treated group. Moreover, ileum tissue H&E staining demonstrated that JHD diminished intestinal inflammation. In addition, TEM indicated that JHD improved focal loosely packed microvilli. Furthermore, expression of ZO-1 and occludin decreased in the ileum tract of model rats. Finally, an increased level of p-MLC in the model group was also observed. JHD treatment reversed phosphorylation of MLC in the ileum.Conclusions: The above results suggest that JHD has potential value in the prevention and treatment of ALD in rats; its mechanism of action may be closely related to its effect on improving intestinal barrier function and attenuating gut leakiness.

Acute kidney injury is a clinical syndrome with both high morbidity and mortality. However, the underlying molecular mechanism of AKI is still largely unknown. The role of SENP1 in AKI is unclear, while one of its substrates, HIF-1α possesses nephroprotective effect in AKI. Herein, this study aimed to reveal the role of SENP1/HIF-1α axis in AKI by using both cell and animal models.We investigated the effects of AKI on SENP1 expression using clinical samples, and cisplatin-induced AKI model based on mice or HK-2 cells. The influence of SENP1 knockdown or over-expression on cisplatin-induced AKI was studied in vitro and in vivo. Following the exploration of the change in HIF-1α expression brought by AKI, the synergistic effects of SENP1 knockdown and HIF-1α over-expression on AKI were examined.The results showed the up-regulation of SENP1 in clinical specimens, as well as cell and animal models. The knockdown or over-expression of SENP1 in HK-2 cells could promote or inhibit AKI through regulating cell apoptosis, respectively. Moreover, SENP1we showed that SENP1 provided protection for kidney in AKI via regulating cell apoptosis and through the regulation of HIF-1α. This study could benefit for the understanding of the pathogenesis of AKI and provide potential therapeutic target for AKI treatment.

Sequence-indexed insertional libraries are important resources for functional gene study in model plants. However, the maize (Zea mays) UniformMu library covers only 36% of the annotated maize genes. Here, we generated a new sequence-indexed maize Mutator insertional library named ChinaMu through high-throughput sequencing of enriched Mu-tagged sequences. A total of 2,581 Mu F2 lines were analyzed, and 311,924 nonredundant Mu insertion sites were obtained. Based on experimental validation, ChinaMu contains about 97,000 germinal Mu insertions, about twice as many as UniformMu. About two-thirds (66,565) of the insertions are high-quality germinal insertions (positive rate > 90%), 89.6% of which are located in genic regions. Furthermore, 45.7% (20,244) of the 44,300 annotated maize genes are effectively tagged and about two-thirds (13,425) of these genes harbor multiple insertions. We tested the utility of ChinaMu using pentatricopeptide repeat (PPR) genes. For published PPR genes with defective kernel phenotypes, 17 out of 20 were tagged, 11 of which had the previously reported mutant phenotype. For 16 unstudied PPR genes with both Mu insertions and defective kernel phenotypes, 6 contained insertions that cosegregated with the mutant phenotype. Our sequence-indexed Mu insertional library provides an important resource for functional genomics study in maize.

The rare earth doped NiZnCo ferrites were prepared by the sol-gel auto-combustion method. X-ray diffraction, scanning electron microscopy, infrared spectroscopy, ultraviolet visible spectrophotometer and vibrating sample magnetometer were used to characterize the microstructure, optical and magnetic properties of rare earth doped NiZnCo ferrite. The results show that with the increase of rare earth doping amount, the lattice parameters of NiZnCo ferrite increase firstly and then decrease, the grain size decreases monotonously. And the second phase appears when the La and Gd doping amount is 0.06 and 0.1, respectively. The morphology of rare earth doped ferrites is relatively homogeneous sphere. The cations are redistributed in the crystal lattice. When the amount of dopant is small, the rare earth ions occupy both A-site and B-site. Increasing dopants, the rare earth ions occupy the B-sites. The critical content is 0.02 for La dopant, while it is 0.06 for Gd. La doping results in broadening of the band gap; Doping Gd results in narrowing of the band gap. The saturation magnetization decreased linearly from 108 emu/g to 43 emu/g and 85 emu/g, respectively, with the increase of Gd and La doping. The faster downtrend for Gd doped ferrites is due to its greater solubility.

Keypoint-based methods achieve increasing attention and competitive performance in the field of object detection. In this paper, we propose a new keypoint-based object detection method in order to better locate center keypoints of objects and adaptively combine keypoints to obtain more accurate bounding boxes. Specifically, to better locate center keypoints of objects, we aggregate boundary information by adding the center pooling operation to the original center keypoints prediction branch. The boundary information is the location of object boundary which is more easier to predict than object center. Furthermore, to obtain more accurate bounding boxes, we propose an adaptive keypoint combination algorithm to map all keypoints back to the original image so that the keypoints are combined with less localization errors. Experiments have demonstrated the effectiveness of the our proposed methods.

Hepatic nuclear factor 4 alpha (HNF4α) drives the expression of apolipoprotein B (ApoB), microsomal triglyceride transfer protein (MTP) and phospholipase A2 G12B (PLA2G12B), governing hepatic very-low-density lipoprotein (VLDL) production and secretion. Andrographolide (AP) is a major constituent isolated from Andrographis paniculata. We found that AP can disrupt the interaction between HNF4α and its coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α). Virtual docking and mutational analysis indicated that arginine 235 of HNF4α is essential for binding to AP. As a consequence of antagonizing the activity of HNF4α, AP suppresses the expression of ApoB, MTP and PLA2G12B and reduces the rate of hepatic VLDL secretion in vivo. AP additionally reduced gluconeogenesis via down-regulating the expression of HNF4α target genes phosphoenolpyruvate carboxykinase (Pepck) and glucose-6-phosphatase (G6pc). Collectively, our results suggest that AP affects liver function via modulating the transcriptional activity of HNF4α.

Li-doped In2O3 nanoparticles with room temperature d0 ferromagnetism were prepared by a sol-gel method. X-ray diffraction, X-ray photoelectron spectroscopy and photoluminescence were carried out to investigate the effects of Li incorporation on the lattice defects. As the content of Li increases, non-monotonic changes in shifts of XRD peak (2 2 2) and the intensity ratios of indium vacancies related photoluminescence peak (PII) with respect to oxygen vacancies related peak (PI) are observed. Results show that at low doping level (≤2 at.%) Li prefers to occupy In sites, while with further doping the interstitial sites are more favorable for Li. Combined with the consistent non-monotonic change in saturation magnetization, we think that indium vacancies resulting from Li-doping play an important role in inducing d0 ferromagnetism in our Li-doped In2O3 nanoparticles, and the FM coupling is mainly mediated by the LiIn-ONN-VIn-ONN-LiIn chains.

Caprock-sealing capability affected by fault activity is an important problem that needs to be resolved in hydrocarbon accumulation analysis, especially for rifted basins. In this article, a quantitative model based on the mechanical properties estimated by geophysical logging was first established to characterize the brittle-ductile transition of mudstone caprocks. By establishing a burial depth reconstruction model, the brittle-ductile stage of the mudstone caprock at the termination time of fault activity (TTFA) was restored. Moreover, the caprock juxtaposition thickness (CJT) for brittle rock and the shale smear factor (SSF) for brittle-ductile rock were selected to quantitatively describe the sealing capacity of caprock controlled by faults. These mathematical methods were finally applied to a case study of the K gasfield in the Xihu Depression, East China Sea Basin. Compared with the sealing characterization based on the current mechanical properties, the brittle-ductile reconstruction at the TTFA and matching sealing evaluation better describe the effectiveness of the mudstone caprock. Additionally, the quantified sealing capacity and corresponding hydrocarbon response indicate that a CJT value of 56.1 m and an SSF value of 3.0 can be the threshold for caprock sealing and nonsealing in the study area. As a result, caprock with poor sealing led to vertical hydrocarbon leakage in the structural low zone and structural high zone, while strong sealing caprock located in the structural center zone effectively prevented the upward migration of hydrocarbons, thereby controlling the vertical hydrocarbon distribution in the K gasfield. In general, the evaluation accuracy of caprock sealing predominantly relies on the geological input to the model, especially the fluid pressure, rock mechanical parameters, and caprock and fault interpretation results, and it is also affected by further uncertainties arising from the sealing threshold definition and structural uplift.

Abnormal pressure is of great significance for hydrocarbon generation, migration and accumulation. The tight gas reservoirs in the Middle Jurassic Shaximiao Formation of the Sichuan Basin are characterized by considerable underpressure, which origin and evolution are still unclear. In this study, the pressure distribution was characterized by repeat formation test and Bowers’ method using sonic and density well log data. The origins of underpressure were analysed by geological analysis and quantitative calculation. Basin modelling method was also applied for pressure evolutionary process reconstruction. The results showed that underpressure developed at depths of 1900–2300 m with pressure value of 9.24–17.19 MPa and the pressure coefficients of 0.45–0.89. Laterally, the pressure coefficient showed a decreasing tendency from the western to the eastern portion of the study area, which was characterized by considerable underpressure. The results of the underpressure origin analysis indicated that the elastic rebound and temperature reduction led to pressure reductions of approximately 12–15 and 3–4 MPa, respectively. However, the source rocks of Lower Jurassic Ziliujing Formation and Upper Triassic Xujiahe Formation provided pressure increasing by hydrocarbon generation as two of the main sources in the Sichuan Basin. The gas generating process in the source rocks of the Xujiahe Formation increased the pressure by more than 20 MPa which compensated for the pressure decrease in the western part of the Central Sichuan Basin. However, the gas generation of the Ziliujing Formation only provided less than 10 MPa of pressure compensation for the eastern part, which was then characterized by widely distributed underpressure. Consequently, the western and eastern portions of the Central Sichuan Basin underwent different pressure evolution processes, which lead to different pressure distributions at present. This study provided a basis for underpressure analysis of gas reservoirs in quality and quantity in the Central Sichuan Basin and may be helpful for similar gas fields worldwide.

The NixZnyMn1-x-yFe2O4 ferrite ceramics were prepared by the sol–gel auto-combustion method. X-ray diffraction, scanning electron microscopy, infrared spectroscopy, vibrating sample magnetometer and vector network analyzer were used to characterize the microstructure and magnetic properties of the samples. The results show that only cubic spinel structure was detected by XRD in all sintered samples except the sample Zn0.4Mn0.6Fe2O4. The lattice parameter decreases monotonously with Ni content and simply increases with Mn content, which is stemmed from the change of ionic radii. The saturation magnetism increases when Ni content x is less than 0.4. The initial permeability has the largest value at x = 0.2, which fits in with the mechanism of domain wall motion. The Curie temperature increases with the increase of x due to the enhanced A-B interaction. The highest Tc is 505℃ for Ni0.8Zn0.2Fe2O4 ferrite. The effect of adding Mn elements on the magnetic properties depends on the content of Zn. When y is 0.2, the magnetic parameters (Ms and μi) increase with Mn content. However, when y is 0.4, they decrease because of redistribution of cations and the increase of α-Fe2O3. In addition, all samples with Ni elements show strong electromagnetic wave absorption capability in 2–13 GHz range, the minimum RF is −25.36 dB at 4.74 GHz for the Ni0.6Zn0.2Mn0.2Fe2O4 samples.

Ce9Fe91films were fabricated at different temperature by Rf magnetron sputtering. The static and dynamic magnetic properties of these films have been investigated in details. The results reveal that the two films prepared at 293 K and 623 K with the critical film thickness exhibit weak stripe domains and a small perpendicular anisotropy, and their correlated dynamic permeability spectra show one resonance peak, implying the coherent precession of magnetization at such critical thickness. However, the film prepared at 523 K possesses an in-plane uniaxial anisotropy and lower coercive field. And the permeability spectrum displays two peaks, which can be ascribed to the coexistence of the rotatable anisotropy and in-plane uniaxial anisotropy.

This work investigates the high frequency characteristics of [FeCoB/ZnO]50 multilayers with different ZnO thickness. The results reveal that the Acher’s limit of [FeCoB/ZnO]50 multilayers can be modulated by medium thickness. Increasing medium layer thickness is favorable for breaking through the Acher’s limit. It is found that the differences of Acher’s limit between multilayers and single layers are caused by magnetic interface anisotropy related to interface roughness and some unknown factor.

Cubic bixbyite In2O3 nanoparticles with room temperature d0 ferromagnetism were prepared by sol-gel method with the air annealing temperature ranging from 500 to 900 °C. X-ray diffraction, X-ray photoelectron spectroscopy, Raman-scattering and photoluminescence were carried out to demonstrate the presence of oxygen vacancies. The lattice constant, the atomic ratio of crystal O and In, the Raman peak at 369 cm−1, the PL emission peak at 396 nm and the saturation magnetization of d0 ferromagnetism all had a consistent non-monotonic change with the increasing annealing temperature. With further considering the relation between the grain size and the distribution of oxygen vacancies, we think that d0 ferromagnetism in our samples is directly related with the singly charged oxygen vacancies at the surface of In2O3 nanoparticles.

Beta-glucosidase (BGL1) is widely used in animal feed industries. However, degradation caused by digestive enzymes in the intestine hampers its application. Improving the resistance of feed enzymes against proteases is crucial in livestock farming. To improve the resistance of beta-glucosidase against pepsin and trypsin, a rational molecular design based on the inhibition of bound-state formation and secondary design was developed. The strategy includes: (1) prediction of the interaction surface of the pepsin-BGL1 complex structure, (2) prediction of key amino acids affecting the formation of the complex, (3) optimization of pepsin-resistant mutants by structural evaluation, (4) secondary molecular design based on pepsin-resistant mutants, and optimization of pepsin and trypsin-resistant mutants. Two BGL1 protein mutants (BGL1Q627C and BGL1Q627C/R543H/R646W) were constructed, and then mutated and wild-type BGL1s were expressed in Pichia pastoris. The half-life of BGL1Q627C and BGL1Q627C/R543H/R646W were 1.36 and 1.51 times that of the wild type upon pepsin exposure, respectively. For trypsin resistance, the half-life were 0.93 and 1.53 times that of the wild type, respectively. Compare to those of the wild type, most of the basic enzymatic properties of both mutants were not significantly changed except for increased Michaelis constants. The rational design method can be used as a guide for modifying other feed enzymes.

Aflatoxins (AFs) are potent carcinogens present in numerous crops. Access to accurate methods for evaluating contamination is a critical factor in aflatoxin risk assessment. Versicolorin A (Ver A), a precursor of aflatoxin B1 (AFB1), can be used as an indicator for the presence of AFB1, even when the AF is not yet detectable. Currently employed Ver A detection methods are expensive, time consuming, and difficult to apply to numerous samples. Herein, Ver A was detected via near-infrared spectroscopy. Both quantitative and two-grade sorting methods were set-up using the extreme gradient boosting algorithm coupled with a support vector machine. This two-tiered method obtained a root-mean-square error of prediction value of 3.57 μg/kg for the quantitative model, and an accuracy rate of 90.32% for the sorting approach. This novel method is rapid, accurate, solvent free, requires no sample pretreatment, and detects Ver A in maize, making it convenient for practical use.

The larger $(\mu _{\mathrm {s}} - 1)f_{\mathrm {r}}^{2}$ in the [Fe 45Co 45 B 10/ZnO] n multilayers than that in single-layer films was found. Large Acher’s limit resulted from the interface magnetic anisotropy of Fe 45Co 45 B 10/ZnO, and the effect of interface magnetic anisotropy increases with increasing the number (n) of magnetic layers. With increasing n, μ s decreases, H k increases, and f r of the multilayer increases from 2.7 to 6 GHz, which greatly broadens the working frequency range. The damping coefficient also increases with increasing n, so the magnetic loss increases with n, and it is introduced by the dispersion of the spins at the interface.

Highlight Proteomic analysis of developing maize protein bodies (PBs) reveals an unexpected diversity and complexity of PB proteins, and provides a roadmap for the transport and translation of mRNAs of zein genes, and the assembly of PBs., Prolamins, the major cereal seed storage proteins, are sequestered and accumulated in the lumen of the endoplasmic reticulum (ER), and are directly assembled into protein bodies (PBs). The content and composition of prolamins are the key determinants for protein quality and texture-related traits of the grain. Concomitantly, the PB-inducing fusion system provides an efficient target to produce therapeutic and industrial products in plants. However, the proteome of the native PB and the detailed mechanisms underlying its formation still need to be determined. We developed a method to isolate highly purified and intact PBs from developing maize endosperm and conducted proteomic analysis of intact PBs of zein, a class of prolamine protein found in maize. We thus identified 1756 proteins, which fall into five major categories: metabolic pathways, response to stimulus, transport, development, and growth, as well as regulation. By comparing the proteomes of crude and enriched extractions of PBs, we found substantial evidence for the following conclusions: (i) ribosomes, ER membranes, and the cytoskeleton are tightly associated with zein PBs, which form the peripheral border; (ii) zein RNAs are probably transported and localized to the PB–ER subdomain; and (iii) ER chaperones are essential for zein folding, quality control, and assembly into PBs. We futher confirmed that OPAQUE1 (O1) cannot directly interact with FLOURY1 (FL1) in yeast, suggesting that the interaction between myosins XI and DUF593-containing proteins is isoform-specific. This study provides a proteomic roadmap for dissecting zein PB biogenesis and reveals an unexpected diversity and complexity of proteins in PBs.

β-agonists are phenylethanolamines with different substituent groups on the aromatic ring and the terminal amino group which have the effect of nutrition redistribution and can accumulate in body tissues causing acute or chronic poisoning when consumed. Therefore, it is very important to establish a fast screening method for the detection of several kinds of β-agonists in food safety control. In this study, the aptamer-agonists (AP-Ago) has screened out by Isothermal Titration Calorimetric method. AP-Ago was a single-strand DNA with 22 base-pairs. The dissociation constant (Kd) to phenylethanolamine (PHL) was 3.34 × 10(-5)mol L(-1). The AP-Ago based electrode was constructed by self-assembling on gold electrode. A label-free electrochemical aptasensor was then developed with AP-Ago-based gold electrode, which was sensitive to phenylethanolamine(PHL), clenbuterol (CLB), ractopamine (RAC), salbutamol (SAL) and procaterol (PRO). The detection limits were 0.04 ng/mL (RAC), 0.35 pg/mL (CLB), 1.0 pg/mL (PHL), 0.53 pg/mL (SAL) and 1.73 pg/mL(PRO), respectively, The detection time was 15 min. The reproductivity of the mentioned aptasensor is good with RSD of 2.09%. Comparing with ELISA and HPLC on β-agonists detection in actual sample, this aptasensor is advantage of fewer steps and fast screen-detection of these five β-agonists or their mixtures. This study suggests that the aptasensor can be developed to a rapid screening means with multi-β-agonists (may be one or more) in sample.

A label free electrochemical (EC) aptasensor for ultrasensitive detection of ractopamine (RAC) was developed. A special immobilization media consisting of gold nanoparticles/poly dimethyl diallyl ammonium chloride-graphene composite (AuNPs/PDDA-GN) was utilized to improve conductivity and performance of the biosensor. The RAC aptamer was attached on AuNPs of the composite membrane via Au-S bond. The fabrication process of the EC aptasensor was characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The peak currents obtained by differential pulse voltammetry decreased linearly with the increasing of RAC concentrations and the sensor responds approximately logarithmically over a wide dynamic range of RAC concentration from 1.0 × 10(-12)mol/L to 1.0 × 10(-8)mol/L. The linear correlation coefficient of the developed aptasensor was 0.998, the limit of detection was 5.0 × 10(-13)mol/L. The proposed EC aptasensor displayed good stability, reproducibility and robust operation in animal urine. Particularly, the generality of the fabrication approach of electrochemical aptasensor is highlighted with a further example for illegal drugs detection via the aptamer identification.

The effect of ZnO layer thickness t on the structure, static magnetic properties and high frequency properties is systemically studied for [Fe 65 Co 35 /ZnO( t )] 50 multilayer films. The gradually increasing crystallinity of ZnO layer with increasing t is observed in the XRD patterns. The VSM measurements show good soft magnetic properties are obtained in these multilayer films, even though the ZnO layer is thick (up to 3.6 nm). For the typical sample with t =3.6 nm, an obvious in-plane anisotropy field and comparatively small coercivity are achieved, and high electric resistivity up to 12.6 mΩ cm is obtained. The measurement of permeability spectra indicates that the multilayers have an invariable ferromagnetic resonance frequency f r about 4.0 GHz; however, the initial permeability μ i can be adjusted from 48 to 136 by decreasing t from 3.6 nm to 1.0 nm. The phenomenon that f r is almost invariable is the result of the decreasing saturation magnetization 4πM s and the increasing dynamic anisotropy field H k-dyn with increasing t. For our samples, the real part ( μ‘ 2G ) of complex permeability at f =2.0 GHz remains a high value beyond 90, while the imaginary part ( μ“ 2G ) keeps a low value below 1.55, indicating that these multilayer films are promising for application in high frequency range.

Hepatocyte nuclear factor 4 alpha (HNF4α) regulates the expression of essential genes involved in very-low-density lipoprotein (VLDL) homeostasis and gluconeogenesis. 18β-glycyrrhetinic acid (GA) is an active ingredient of Glycyrrhiza uralensis an herbal medicine used for treating liver aliments. In this study, we established that GA functions as a partial antagonist of HNF4α through HNF4α-driven reporter luciferase assay and co-immunoprecipitation experiments with co-activator PGC1α. By virtual docking and site-directed mutagenesis analysis, we confirmed that serine 190 and arginine 235 of HNF4α are both essential for GA to exert its antagonistic action on HNF4α. Importantly, GA suppressed the expression of HNF4α target genes such as apolipoprotein B (ApoB), microsomal triglyceride transfer protein (MTP) and phospholipase A2 G12B (PLA2G12B) modulating hepatic VLDL secretion in mice fed on a high fat diet. In addition, GA also suppressed gluconeogenesis and ameliorated glucose intolerance via down-regulating the expression of HNF4α target genes glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase (Pepck). Furthermore, GA significantly lowered blood glucose and improved insulin resistance in db/db mice. In all, we established that GA acts as a partial HNF4α antagonist modulating lipid and carbohydrate metabolism.

When feeding enzymes are consumed by livestock and poultry, the stomach is the first organ to come in contact with feeding enzymes. They will face major hazards: acidolysis and enzymatic hydrolysis. Therefore, it is very important to improve the tolerance of feed enzymes to the gastric juice such as pepsin. However, the hydrolysis of amino acids by pepsin is not specific, so improving the resistance of enzymes to pepsin is a great challenge. In this paper, we have selected cellulase E4 isolated from Thermonospora fusca as our study object, which exhibits unique properties because of its endo- and exocellulase characteristics, good synergistic property, and has great potential application value as feed addictive. So we propose a strategy to improve pepsin resistance of cellulose E4, including the following steps: 1) predicting amino acids that affect the formation of transitional complexes between pepsin and E4; 2) selection of candidate mutation sites; 3) prediction of optimal mutants. The mutant of E4L691H/L800H was predicted the best, and then we expressed and tested the characteristics of E4L691H/L800H, the results showed that the E4L691H/L800H had 25% more protein residual than the wild-type after 1-hour digestion, and the enzymatic properties were consistent with those of the wild-type. This study provides a useful molecular improvement strategy for protein pepsin resistance.

The objective of this study was to evaluate the feasibility of the predictive monitoring of aflatoxin B1 (AFB1) under granary conditions, since mycotoxin contamination of the stored grain represents an important issue. Using the storage test, we investigated the relationship between versicolorin A (Ver A, an intermediate in AFB1 biosynthesis) levels and the levels of aflatoxigenic fungi, and their relationship with aflatoxin production. All samples, except for one, were found to be contaminated with aflatoxigenic fungi using PCR analyses, while their AFB1 levels were not detectable before the storage test using an enzyme-linked immunosorbent assay (ELISA) method with an LOD of 2 μg/kg. Aflatoxigenic fungi levels were analysed, as well as Ver A levels prior to the accumulation of AFB1 (Levels were ≥5 μg/kg; the permissible levels of AFB1 in corn intended for direct consumption are

Excellent soft magnetic properties in a wide x range with coercivity not exceeding 5.0 Oe were obtained successfully in the (Ni75Fe25) x (ZnO)1−x granular films which were fabricated by the magnetic alloy Ni75Fe25 and semiconductor ZnO. For the typical sample with x = 0.74, coercivities in hard and easy axes are 1.1 and 1.7 Oe, respectively, and the electric resistivity ρ reaches 425 μΩ cm. The dependence of complex permeability u = u ′−j u ″ on frequency f shows that the real part u ′ is more than 200 below 1.0 GHz and the ferromagnetic resonance frequency f r reaches 1.32 GHz, implying the promising for high-frequency application.

Aflatoxins (AFs) are secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus. The molds may contribute to pre-harvest aflatoxin contamination of susceptible crops. For the customer and food producer, a predictive model for aflatoxin detection is very desirable. Versicolorin A (VerA), which is the first precursor in the pathway of aflatoxin B1 (AFB1) biosynthesis, shares similar toxic group with the furofuran structure in aflatoxin B1. VerA exhibits a much lower teratogenic toxicity than AFB1 and may be used as a predictive indicator for aflatoxin B1 contamination of storage crops. Therefore, the development of a fast detection method for VerA is important. One of the randomly computer-generated aptamers of VerA was confirmed by isothermal titration calorimetry with Kd = 9.26 × 10−6 mol l−1. In addition, a simple and sensitive label-free aptasensor was developed for the electrochemical detection of VerA. According to the results from differential pulse voltammetry (DPV), a linear relationship existed between the log conc. of VerA (ranged from 0.01 to 100 ng ml−1) and the current (△Ip) with a limit of detection (LOD) of 10 pg ml−1. The resulting aptasensor exhibited good reproducibility for detecting VerA and stability after storage for 15 days at 4 °C with acceptable anti-interference against ZEN, OTA, DON, and FB1. When used in corn samples, the recoveries of VerA were determined to be in the range of 81.3%–104.4 %. Although with some intercross, result suggests that the obtained aptamer for VerA is potentially used as a sorbent for the preparation of solid-phase-extraction procedure to clean up food samples in conjunction with high-performance liquid chromatography analysis.