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In order to improve the effectiveness of the network prediction result of rice protein interaction, the network prediction method of rice protein interaction based on gray BP neural network has been proposed. Firstly, a series of key features about interaction sites of rice protein such as the series spectrum, conserved weight, entropy value, accessible exterior product of compound and sequence rate, etc., should be extracted firstly. Then the gray BP neural network and their integration will be applied to the training and test of these sample set. Ten times of cross validation are applied to the training and test, and four groups of feature combinations of rice protein interaction with comparability are created. As for each time of the addition of new features in the experiment, the accurate prediction rate would be improved, especially in case of the addition of exterior product and sequence rate features, the accuracy can be improved greatly, which means that in case the combination of multiple features is adopted, the method of predicting the interaction of rice protein by combining the gray BP neural network algorithm is accurate and effective.

The monitoring of heavy metal ions (HMIs) in rice phloem sap is of great significance for studying the migration and accumulation of HMIs in rice plants and, ultimately, for human health. However, achieving sensitive and selective electroanalysis of HMIs in such complex real samples is a huge challenge due to severe interference. In this work, a sensitive anti-interference sensing interface for the analysis of Pb2+ in rice phloem sap is fabricated based on glutathione-functionalized gold/multi-walled carbon nanotubes (MWCNTs-GSH-Au-GSH). Combining the selective capturing abilities of glutathione and the electrocatalytic abilities of small sized gold nanoparticles (mean diameter: 3.1 nm) toward Pb2+, the sensitivity of MWCNTs-GSH-Au-GSH is as high as 1122.8 μA μM−1 cm−2 without interference from other small ions, sucrose, ATP, amino acids, plant hormones, and so on. The limit of detection (0.01 μM) satisfactorily meets the requirements for sensing trace Pb2+. Importantly, a MWCNTs-GSH-Au-GSH electrode exhibited excellent stability over a test period of 6 months. In addition, the developed sensor shows high reproducibility, which is checked using ten different MWCNTs-GSH-Au-GSH electrodes. Based on the above advantages, the MWCNTs-GSH-Au-GSH electrode can be suitably applied to the detection of Pb2+ in rice phloem sap. The mechanism behind the high selectivity and sensitivity of the MWCNTs-GSH-Au-GSH electrode toward Pb2+ is clearly investigated via X-ray photoelectron spectroscopy (XPS) and extended X-ray absorption fine structure (EXAFS) studies.

A hyaluronic acid-coated gold nanoparticle (HA-AuNPs) was synthesized and further employed for hyaluronidase (Hyal) assay and for screening effective inhibitors. The negatively charged hyaluronate on the surface of AuNP enhanced the solubility and stability of colloidal HA-AuNPs in aqueous solution. The HA-AuNPs in the solution possess a characteristic absorption band at 518 nm, whereas at the aggregation form, the band gradually red-shifts to 630 nm in various concentration of NaCl solution. The NaCl-induced red-shift absorption became steady when NaCl concentration reached 375 mM or higher concentration. In an enzymatic assay, Hyal promotes the hydrolysis of hyaluronate on AuNPs to produce more negatively charged HA oligomers, which can increase the surface shielding of AuNPs and to be released into solution to bind with sodium ion from NaCl. Both effects can largely prevent the aggregation of AuNPs. With this feature, inhibitors of Hyal can be effectively screened by monitoring the absorption change at 630 nm. Several effective inhibitors were found from a chemical library containing 2000 compounds. Thioguanine, harmine, and phenylbiguanide were selected for IC 50 measurement using this newly designed method with values of 4.5, 13.3, and 28.7 μM, respectively. The same concept can be easily applied to the case of other polysaccharide degrading enzyme, such as chitosanase, condroitinase, haparinase, etc.

Chitinases hydrolyze chitin, an insoluble linear polymer of N-acetyl-d-glucosamine (NAG)n, into nutrient sources. Bacillus cereus NCTU2 chitinase (ChiNCTU2) predominantly produces chitobioses and belongs to glycoside hydrolase family 18. The crystal structure of wild-type ChiNCTU2 comprises only a catalytic domain, unlike other chitinases that are equipped with additional chitin binding and insertion domains to bind substrates into the active site. Lacking chitin binding and chitin insertion domains, ChiNCTU2 utilizes two dynamic loops (Gly-67—Thr-69 and Ile-106–Val-112) to interact with (NAG)n, generating novel substrate binding and distortion for catalysis. Gln-109 is crucial for direct binding with substrates, leading to conformational changes of two loops with a maximum shift of ∼4.6 Å along the binding cleft. The structures of E145Q, E145Q/Y227F, and E145G/Y227F mutants complexed with (NAG)n reveal (NAG)2, (NAG)2, and (NAG)4 in the active site, respectively, implying various stages of reaction: before hydrolysis, E145G/Y227F with (NAG)4; in an intermediate state, E145Q/Y227F with a boat-form NAG at the −1 subsite, −1-(NAG); after hydrolysis, E145Q with a chair form −1-(NAG). Several residues were confirmed to play catalytic roles: Glu-145 in cleavage of the glycosidic bond between −1-(NAG) and +1-(NAG); Tyr-227 in the conformational change of −1-(NAG); Asp-143 and Gln-225 in stabilizing the conformation of −1-(NAG). Additionally, Glu-190 acts in the process of product release, and Tyr-193 coordinates with water for catalysis. Residues Asp-143, E145Q, Glu-190, and Tyr-193 exhibit multiple conformations for functions. The inhibitors zinc ions and cyclo-(l-His-l-Pro) are located at various positions and confirm the catalytic-site topology. Together with kinetics analyses of related mutants, the structures of ChiNCTU2 and its mutant complexes with (NAG)n provide new insights into its substrate binding and the mechanistic action.

Protein purification generally requires many steps of column chromatography that typically involve ion-exchange, hydrophobic-interaction and gel-filtration separations. More sophisticated purification of protein might be achieved through an application of affinity binding on a functionalized gel such as a nickel column, glutathione-modified column, maltose-modified gel column or others. Of several drawbacks existing in these methods, fusion proteins are commonly obtained, protease digestion might be necessary to remove the fusion moiety; a costly gel is employed for affinity binding, etc. Here we report that an expression vector derived from pREST was constructed to compose the gene of the chitin-binding protein (CBP) and the nucleotide sequence of the (EAAAK)(5) peptide linker following restriction sites for target gene insertion. Fusion proteins were expressed with E. coli and purified with a chitin column. The (EAAAK)(5) linker is shown to possess a pH-dependent auto-cleavage feature. In the range pH 6-7, the target protein becomes automatically released from the fusion protein without proteolytic treatment. Although the mechanism of this auto-cleavage property of an (EAAAK)(5) linker is unclear, this feature has been successfully employed for many cases of protein purification without the tag of a fusion protein.

The gene of Serratia marcescens chitinase A (chiA) was cloned by PCR. The complete gene was constructed into a pRSET vector and expressed in Escherichia coli. The recombinant enzyme was purified to > 90% homogeneity by hydrophobic interaction chromatography followed by ion-exchange separation. Measured with an electrospray-ionization mass spectrometer, the molecular mass of the protein was 58,607 Da, consistent with a theoretical calculation of the deduced protein without the signal peptide. The recombinant enzyme was characterized and tested for the preparation of chitobiose. In general, the recombinant Chtinase A exhibited an exo-type catalytic activity toward colloidal chitin and released both N-acetylglucosamine and N,N-diacetyl chitobiose as products. After extensive testing, we produced N,N-diacetyl chitobiose as the predominant product when the enzymatic reaction was performed in sodium acetate buffer at pH 5.5; under such conditions, an enzymatic process is established for the production of the disaccharide on a 100-g scale.

Ipomoea aquatica with low-energy N+ ion implantation was used for the removal of both nitrogen and phosphorus from the eutrophic Chaohu Lake, China. The biomass growth, nitrate reductase and peroxidase activities of the implanted I. aquatica were found to be higher than those of I. aquatica without ion implantation. Higher NO3-N and PO4-P removal efficiencies were obtained for the I. aquatica irradiation at 25 keV, 3.9 x 10(16) N+ ions/cm(2) and 20 keV 5.2 x 10(16) N+ ions/cm(2), respectively (p < 0.05). Moreover, the nitrogen and phosphorus contents in the plant biomass with ion implantation were also greater than those of the controls. I. aquatica with ion implantation was directly responsible for 51-68% N removal and 54-71% P removal in the three experiments. The results further confirm that the ion implantation could enhance the growth potential of I. aquatica in real eutrophic water and increase its nutrient removal efficiency. Thus, the low-energy ion implantation for aquatic plants could be considered as an approach for in situ phytoremediation and bioremediation of eutrophic waters.

The aim of this study was to investigate the use of water spinach (Ipomoea aquatica Forsk.) with N(+) ion-beam implantation for removal of nutrient species from eutrophic water. The mutated water spinach was grown on floating beds, and growth chambers were used to examine the growth of three cultivars of water spinach with ion implantation for 14 days in simulated eutrophic water at both high and low nitrogen levels. The specific weight growth rates of three cultivars of water spinach with ion implantation were significantly higher than the control, and their NO(3)-N and NH(4)-N removal efficiencies were also greater than those of the control. Furthermore, compared with the control, the nitrogen contents in the plant biomass with ion implantation were higher as well.

The objective of the present study is to develop a method for rapid determination of the content of stevioside (ST) and rebaudioside A (RA) in Stevia Rebaudiana leaves. One hundred and five samples of stevia from different areas containing ST of 0.27%-1.40% and RA of 0.61%-3.98% were used. The 105 groups' NIRS diagram was processed by different methods including subtracting a straight line (SLS), multiplicative scatter correction (MSC), first derivative (FD), second derivative (SD) and so on, and then all data were analyzed by partial least square (PLS). The study showed that SLS can be used to extracted spectra information thoroughly to analyze the contents of ST, the correlation coefficients of calibration (Re), the root-mean-square errors of calibration (RMSEC) and prediction (RMSEP), and the residual predictive deviation (RPD) were 0.986, 0.341, 1.00 and 2.8, respectively. The correlation coefficients of RA was 0.967, RMSEC was 1.50, RMSEP was 1.98 and RPD was 4.17. The results indicated that near infrared spectroscopy (NIRS) technique offers effective quantitative capability for ST and RA in Stevia Rebaudiana leaves. Then the model of stevia dried leaves was used to compare with the stevia powder near infrared model whose correlation coefficients of ST was 0.986, RMSEC was 0.32, RMSEP was 0.601 and RPD was 2.86 and the correlation coefficients of RA was 0.968, RMSEC was 1.50, RMSEP was 1.48 and RPD was 4.2. The result showed that there was no significant difference between the model of dried leaves and that of the powders. However, the dried leaves NIR model reduces the unnecessary the steps of drying and grinding in the actual detection process, saving the time and reducing the workload.

A powerful endo-chitosanase (CSN) previously described for a large scale preparation of chito-oligosaccharides (Cheng, C.-Y., and Li, Y.-K. (2000) Biotechnol. Appl. Biochem. 32, 197-203) was cloned from Aspergillus fumigatus and further identified as a member of glycosyl hydrolase family 75. We report here a study of gene expression, functional characterization, and mutation analysis of this enzyme. Gene cloning was accomplished by reverse transcription-PCR and inverse PCR. Within the 1382-bp Aspergillus gene (GenBank accession number AY190324), two introns (67 and 82 bp) and an open reading frame encoding a 238-residue protein containing a 17-residue signal peptide were characterized. The recombinant mature protein was overexpressed as an inclusion body in Escherichia coli, rescued by treatment with 5 m urea, and subsequently purified by cation exchange chromatography. A time course 1H NMR study on the enzymatic formation of chito-oligosaccharides confirmed that this A. fumigatus CSN is an inverting enzyme. Tandem mass spectrum analysis of the enzymatic hydrolysate revealed that the recombinant CSN can cleave linkages of GlcNAc-GlcN and GlcN-GlcN in its substrate, suggesting that it is a subclass I chitosanase. In addition, an extensive site-directed mutagenesis study on 10 conserved carboxylic amino acids of glycosyl hydrolase family 75 was performed. This showed that among these various mutants, D160N and E169Q lost nearly all activity. Further investigation using circular dichroism measurements of D160N, E169Q, wild-type CSN, and other active mutants showed similar spectra, indicating that the loss of enzymatic activity in D160N and E169Q was not because of changes in protein structure but was caused by loss of the catalytic essential residue. We conclude that Asp160 and Glu169 are the essential residues for the action of A. fumigatus endo-chitosanase.

This paper summarizes the development of the research and application of ion implantation biotechnology on plant, animal and microorganism varieties Improvement, and it also includes the contemporary effects and utilization of ion implantation, and the application of ion implantation on trans-gene plant. In 1980s, cooperated with Anhui Academy of Agriculture Sciences, the ion implantation was applied for crops’ improvement by Plasma Physics Research Institute of the Chinese Academy of Sciences, and made greater progress in both practice and theory, which attracted many scholars inside and outside. By using ion implantation, extensive researches had been made in different materials including plant, animal and microorganism on different levels. With combination of physics and biology, as a new cross subject and a unique technology system, the ion implantation biotechnology came into being, and created more dramatically economic and social benefit in crops improvement by transferring exogenous gene. This paper summarizes the development of the research and application of ion implantation biotechnology.

In the present study, a new approach to fast determining the content of urea, biuret and moisture in compound fertilizer composed of urea, ammonium dihydrogenphosphate and potassium chloride was proposed by using near infrared diffuse reflectance spectroscopy. After preprocessing the original spectrum, partial least squares (PLS) models of urea, biuret and moisture were built with the R2 values of 0.9861, 0.9770 and 0.9713 respectively, the root mean square errors of cross validation were 2.59, 0.38, 0.132 respectively. And the prediction correlation factors were 0.9733, 0.9215 and 0.9679 respectively. The authors detected six kinds of compound fertilizer in market for the model verification, the correlation factors were 0.9237, 0.9786 and 0.9874 respectively. The data implied that the new method can be used for situ quality control in the production process of compound fertilizer.

To study the clinical efficacy of adjuvant therapy with glucocorticoids in children with lobar pneumonia caused by Mycoplasma pneumoniae.One hundred and eight children with lobar pneumonia caused by Mycoplasma pneumoniae were randomly divided into routine treatment and hormone treatment groups. Both groups were treated with azithromycin and other symptomatic therapies. In addition to the basic treatment, the hormone treatment group was given dexamethasone 0.25-0.3 mg/(kg·d) by intravenous drip until the body temperature was normal. Then given oral prednisone tablets 0.5-1 mg/(kg·d) (gradually reduced) for a total treatment course of 7-10 days. Before and after treatment pulmonary functions were examined, and serum C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), interleukin-2 (IL-2) and interleukin-6 (IL-6) were measured.The duration of fever, cough relief time and pulmonary shadow absorption time on chest X-ray were significantly shorter in the hormone treatment group than in the routine treatment group (P0.05). After treatment, the two groups showed improvements in serum CRP, ESR, IL-2, and IL-6 (P0.05), but the hormone treatment group showed significantly more improvement (P0.05). Varying degrees of mixed ventilation dysfunction were seen in the two groups before treatment, and hormone therapy significantly improved pulmonary function, especially promoting the recovery of small airway function.Adjuvant therapy with glucocorticoids can effectively alleviate clinical symptoms, promote the absorption of pulmonary inflammation, and improve pulmonary function in children with lobar pneumonia caused by Mycoplasma pneumoniae.

Severe eutrophication of surface water has been a major problem of increasing environmental concern worldwide. In the present study, economic plant annual ryegrass (Lolium multiflorum) was grown in floating mats as an economic plant-based treatment system to evaluate its potential after ion implantation for removing nutrients in simulated eutrophic water. The specific weight growth rate of L. multiflorum with ion implantation was significantly greater than that of the control, and the peroxidase, nitrate reductase, and acid phosphatase activities of the irradiated L. multiflorum were found to be greater than those plants without ion implantation. Higher total nitrogen (TN) and total phosphorus (TP) removal efficiencies were obtained for the L. multiflorum irradiated with 25 keV 5.2 × 10(16) N(+) ions/cm(2) and 30 keV 4.16 × 10(16) N(+) ions/cm(2), respectively (p 0.05). Furthermore, the nitrogen and phosphorus contents in the plant biomass with ion implantation were also greater than those in the control and were positively correlated with TN and TP supplied. L. multiflorum itself was directly responsible for 39-49 and 47-58 % of the overall N and P removal in the experiment, respectively. The research results suggested that ion implantation could become a promising approach for increasing phytoremediation efficiency of nutrients from eutrophic water by L. multiflorum.

Proteus mirabilis isolates commonly have decreased susceptibility to imipenem. Previously, we found P. mirabilis hfq mutant was more resistant to imipenem and an outer membrane protein (OMP) could be involved. Therefore, we investigated the role of this OMP in carbapenem susceptibility. By SDS-PAGE we found this OMP (named ImpR) was increased in hfq mutant and LC-MS/MS revealed it to be the homologue of Salmonella YbfM, which is a porin for chitobiose and subject to MicM (a small RNA) regulation. We demonstrated that ImpR overexpression resulted in increased carbapenem MICs in the laboratory strain and clinical isolates. Chitobiose induced expression of chb (a chitobiose utilization operon). Real-time RT-PCR and SDS-PAGE were performed to elucidate the relationship of hfq, impR, chb and MicM in P. mirabilis. We found MicM RNA was decreased in hfq mutant and chbBC-intergenic region (chbBC-IGR) overexpression strain (chbIGRov), while impR mRNA was increased in hfq mutant, micM mutant and chbIGRov strain. In addition, mutation of hfq or micM and overexpression of chbBC-IGR increased ImpR protein level. Accordingly, chitobiose made wild-type have higher levels of ImpR protein and are more resistant to carbapenems. Hfq- and MicM-complemented strains restored wild-type MICs. Mutation of both impR and hfq eliminated the increase in carbapenem MICs observed in hfq mutant and ImpR-complementation of hfq/impR double mutant resulted in MICs as hfq mutant, indicating that the ImpR-dependent decreased carbapenem susceptibility of hfq mutant. These indicate MicM was antisense to impR mRNA and was negatively-regulated by chbBC-IGR. Together, overexpression of ImpR contributed to the decreased carbapenem susceptibility in P. mirabilis.