[Determination of steviol in Stevia Rebaudiana leaves by near infrared spectroscopy].

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.

[Quantitative analysis of contents in compound fertilizer and application research using near infrared reflectance spectroscopy].

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.

[Clinical efficacy of adjuvant therapy with glucocorticoids in children with lobar pneumonia caused by Mycoplasma pneumoniae].

OBJECTIVE: To study the clinical efficacy of adjuvant therapy with glucocorticoids in children with lobar pneumonia caused by Mycoplasma pneumoniae. METHODS: 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. RESULTS: 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 (P<0.05). After treatment, the two groups showed improvements in serum CRP, ESR, IL-2, and IL-6 (P<0.05), but the hormone treatment group showed significantly more improvement (P<0.05). 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. CONCLUSIONS: 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.

Recent progress in the development of nanomaterials targeting multiple cancer metabolic pathways: a review of mechanistic approaches for cancer treatment.

Cancer is a very heterogeneous disease, and uncontrolled cell division is the main characteristic of cancer. Cancerous cells need a high nutrition intake to enable aberrant growth and survival. To do so, cancer cells modify metabolic pathways to produce energy and anabolic precursors and preserve redox balance. Due to the importance of metabolic pathways in tumor growth and malignant transformation, metabolic pathways have also been given promising perspectives for cancer treatment, providing more effective treatment strategies, and target-specific with minimum side effects. Metabolism-based therapeutic nanomaterials for targeted cancer treatment are a promising option. Numerous types of nanoparticles (NPs) are employed in the research and analysis of various cancer therapies. The current review focuses on cutting-edge strategies and current cancer therapy methods based on nanomaterials that target various cancer metabolisms. Additionally, it highlighted the primacy of NPs-based cancer therapies over traditional ones, the challenges, and the future potential.

Thinning or Opening: A Randomized Sibling-Embryo Pilot Trial on the Efficacy of Two Laser-Assisted Hatching Modes During the Extended Culture of Highly Fragmented Cleavage Embryos.

A randomized sibling-embryo pilot trial investigated whether two ways of laser-assisted hatching result in different blastulation and clinical outcomes after extended in vitro culture process of highly fragmented day-3 cleavage embryos. From 92 couples, a total of 315 highly fragmented day-3 embryos (the fragmentation >25%) were recruited and randomized into laser-assisted zona thinning (LAT, n=157) and opening (LAO, n=158) groups, and then underwent a blastocyst culture in vitro. The main endpoint measurements including blastocyst formation and grading as well as the clinical pregnancy after blastocyst transfer were obtained during the treatment procedure of in vitro fertilization and embryo transfer, and then analyzed with generalized estimating equation (GEE) and/or time-to blastocyst analysis models. A total of 166 day-3 embryos developed into blastocyst stage (52.70%), of which 97 were viable blastocysts (30.79%), and 42 top-quality ones (13.33%). LAT did not have any inferior or superior to LAO in the endpoints of either total, viable, top-quality or hatched blastocyst formation, with the ORs (95%CI) from GEE model as 0.89 (0.55-1.45), 0.71 (0.42-1.21), 1.12 (0.56-2.25) and 0.68 (0.42-1.12) respectively for LAT treatment. And the time-to-blastocyst analysis showed a similar result. Additionally, no difference in clinical outcomes after blastocyst transfer was found between the two groups. The author concluded that when applying the LAHs during the extended culture of highly fragmented embryos, both LAT and LAO can generate a promising clinical outcome, and the LAT operation be equivalent to the LAO. Future well-designed, multiple-center, larger-sample investigations are required to ascertain above conclusion.

Crystal structures of Bacillus cereus NCTU2 chitinase complexes with chitooligomers reveal novel substrate binding for catalysis: a chitinase without chitin binding and insertion domains.

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 Šalong 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.

Enhanced nitrogen and phosphorus removal from eutrophic lake water by Ipomoea aquatica with low-energy ion implantation.

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.

Nitrogen removal from eutrophic water by floating-bed-grown water spinach (Ipomoea aquatica Forsk.) with ion implantation.

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.

Purification, crystallization and preliminary X-ray crystallographic analysis of chitinase from Bacillus cereus NCTU2.

Chitinases (EC 3.2.1.14) are found in a broad range of organisms, including bacteria, fungi and higher plants, and play different roles depending on their origin. A chitinase from Bacillus cereus NCTU2 (ChiNCTU2) capable of hydrolyzing chitin as a carbon and nitrogen nutrient has been identified as a member of the family 18 glycoside hydrolases. ChiNCTU2 of molecular weight 36 kDa has been crystallized using the hanging-drop vapour-diffusion method. According to the diffraction of chitinase crystals at 1.10 A resolution, the crystal belongs to space group P2(1), with unit-cell parameters a = 50.79, b = 48.79, c = 66.87 A, beta = 99.31 degrees . Preliminary analysis indicates there is one chitinase molecule in the asymmetric unit, with a solvent content of 43.4%.

Overexpression of an outer membrane protein associated with decreased susceptibility to carbapenems in Proteus mirabilis.

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.

Enhancement of nitrogen and phosphorus removal from eutrophic water by economic plant annual ryegrass (Lolium multiflorum) with ion implantation.

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.

Protein purification involving a unique auto-cleavage feature of a repeated EAAAK peptide.

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.

Enhanced enzymatic saccharification of rice straw by microwave pretreatment.

In this study, Box-Behnken design and response surface methodology were employed to plan experiments and optimize the microwave pretreatment of rice straw. Experimental results show that microwave intensity (MI), irradiation time (IT) and substrate concentration (SC) were main factors governing the enzymatic saccharification of rice straw. The maximal efficiencies of cellulose, hemicellulose and total saccharification were respectively increased by 30.6%, 43.3% and 30.3% under the optimal conditions of MI 680 W, IT 24 min and SC 75 g/L. The chemical composition analysis of straw further confirmed that microwave pretreatment could disrupt the silicified waxy surface, break down the lignin-hemicellulose complex and partially remove silicon and lignin.

Structural simulation and protein engineering to convert an endo-chitosanase to an exo-chitosanase.

To obtain an enzyme for the production of chito-disaccharides (GlcN(2)) by converting endo-chitosanase to exo-chitosanase, we chose an endo-chitosanase from Bacillus circulans MH-K1 (Csn) as the candidate for protein engineering. Using molecular modeling, two peptides with five amino acids (PCLGG) and six amino acids (SRTCKP) were designed and inserted after the positions of D(115) and T(222) of Csn, respectively. The inserted fragments are expected to form loops that might protrude from opposite walls of the substrate-binding cleft, thus forming a 'roof' over the catalytic site that might alter the product specificity. The chimeric chitosanase (Chim-Csn) and wild-type chitosanase (WT-Csn) were both over-expressed in Escherichia coli and purified nearly to homogeneity. The products formed from chitosan were analyzed by ESI-MS (electrospray ionization-mass spectrometry). A mixture of GlcN(2), GlcN(3) and GlcN(4) was obtained with WT-Csn, whereas Chim-Csn formed, with a smaller catalytic rate (3% of WT-Csn activity), GlcN(2) as the dominant product. Measurements of viscosity showed that, with similar amounts of enzyme activity, Chim-Csn catalyzed the hydrolysis of chitosan with a smaller rate of viscosity decrease than WT-Csn. The results indicate that, on inserting two surface loops, the endo-type chitosanase was converted into an exo-type chitosanase, which to our knowledge is the first chitosanase that releases GlcN(2) from chitosan as the dominant product.

Exploration of glycosyl hydrolase family 75, a chitosanase from Aspergillus fumigatus.

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.