Increased yield of 2-O-α-d-glucopyranosyl-l-ascorbic acid synthesis by α-glucosidase using rational design that regulating the ground state of enzyme and substrate complex.

BACKGROUND: 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.

Biphasic alcoholysis coupled with high-speed countercurrent chromatography for high performance on separating phorbol from Croton tiglium Linn extracts.

Phorbol is a tetracyclic diterpenoid found in Euphorbia tirucalli, Croton tiglium, and Rehmannia glutinosa, and is nuclear of various phorbol esters. The rapid obtaining of phorbol with high purity highly contributes to its application, such as synthesizing phorbol esters with designable side chains and particular therapeutic efficacy. This study introduced a biphasic alcoholysis method for obtaining phorbol from croton oil by using polarity imparity organic solvents in both phases and established a high-speed countercurrent chromatography method for simultaneous separation and purification of phorbol. The optimized operation conditions of biphasic alcoholysis were a reaction time of 91 min, a temperature of 14°C, and a croton oil-methanol ratio of 1:30 (g:ml). The phorbol during the biphasic alcoholysis was 3.2-fold higher in content than that obtained in conventional monophasic alcoholysis. The optimized high-speed countercurrent chromatography method was using the ethyl acetate/n-butyl alcohol/water at 4.7:0.3:5 (v:v:v) with Na2 SO4 at 0.36 g/10 ml as the solvent system, using the mobile phase flow rate of 2 ml/min, the revolution of 800 r/min, under which the retention of the stationary phase was achieved at 72.83%. The crystallized phorbol following high-speed countercurrent chromatography was obtained as high purity of 94%.

Near-infrared reflectance spectroscopy-based fast versicolorin A detection in maize for early aflatoxin warning and safety sorting.

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.

Predictive model of aflatoxin contamination risk associated with granary-stored corn with versicolorin A monitoring and logistic regression.

Predictions of aflatoxin (AF) in grain at post-harvest can be useful for ensuring the safety of stored grain. Versicolorin (Ver) A, a precursor of AFB1, can serve as an early indicator of AF contamination, even when AFs themselves are present at undetectable levels. In the current research, we developed a probabilistic model based on logistic regression and Ver A levels to estimate the risk of AF contamination in stored corn. Moisture content, aflatoxigenic fungal load, and initial and maximum values of Ver A in the first three sampling cycles were experimentally determined as the four important factors for the probabilistic model. Both internal and external model validations were shown to be high at 96.4% and 93.3%, respectively. For high-risk samples, a precise model was developed to predict the maximum period of safe storage, which can be useful for decision-making by the stakeholders in feed and food supply chain. Our findings provide a basis tool for establishing an early warning system for AF contamination in granaries, which can improve global food safety.

Versicolorin A is a potential indicator of aflatoxin contamination in the granary-stored corn.

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 <5 µg/kg (EC)). Statistical analyses demonstrated that aflatoxin levels after both actual storage and safe storage (AFB1˂5µg/kg) times are significantly correlated with the Ver A levels and the changes in Ver A levels (ΔVer A). Both high and variable Ver A levels were indicative of the vigorous metabolic activity of aflatoxigenic fungi. In contrast, steady Ver A levels showed that aflatoxin production by the fungi was not active. Monitoring Ver A levels and their changes may allow an earlier detection of harmful aflatoxin contamination in the stored grain. Additionally, the toxicity of Ver A should be further examined. The results of our study indicate that the monitoring of Ver A levels, even when the AFB1 levels are very low, may increase the safety of grain consumption, especially considering Ver A toxicity.

The furofuran-ring selectivity, hydrogen peroxide-production and low Km value are the three elements for highly effective detoxification of aflatoxin oxidase.

AFO (aflatoxin oxidase), an enzyme from Armillariella tabescens previously named aflatoxin detoxifizyme, exhibits oxidative detoxification activity toward aflatoxin B1 and sterigmatocystin. Bioinformatics reveals that AFO is a newly discovered oxidase because AFO does not share any significant similarities with any known oxidase. It is critically important to understand how AFO acts on aflatoxin B1. In this study, in addition to aflatoxin B1 (AFB1) and sterigmatocystin (ST), five other chemicals that have furan or pyran structures were investigated. The results indicated that in addition to AFB1 and ST, AFO is also able to act on versicolorin A, 3,4-dihydro-2H-pyran and furan. These results suggested that 8,9-unsaturated carboncarbon bond of aflatoxin B1 is the potential reactive site for AFO. Further findings indicated that the action of AFO is oxygen-dependent and hydrogen peroxide-producing. The simultaneously produced-hydrogen peroxide possibly plays the essential role in detoxification of AFO. In addition, the extremely low Km value of 0.33 µmol/l for AFO-AFB1 and 0.11 µmol/l for AFO-ST signifies that AFO is highly selective for AFB1 as well as ST.

Radiation-induced sarcoma in patients with nasopharyngeal carcinoma: a single-institution study.

BACKGROUND: The increasing incidence of radiation-induced sarcoma (RIS) has become a significant problem that can limit long-term survival. The objective of the current study was to analyze the clinicopathologic characteristics, treatment outcomes, and prognostic factors of RIS after radiotherapy for nasopharyngeal carcinoma (NPC). METHODS: Institutional electronic medical records of patients with NPC who received definitive radiotherapy between February 1964 and 2003 were reviewed. Fifty-three patients who developed RIS and fulfilled the study criteria were included. RESULTS: The median follow-up after a diagnosis of RIS was 15.5 months (range, 0.4-90.3 months), and the median latency between radiotherapy for NPC and an RIS diagnosis was 9.3 years (range, 3.2-26.6 years). Fibrosarcoma was the most frequent histologic type observed, followed by osteosarcoma, and malignant fibrous histiocytoma. The 3-year overall survival (OS) rate for 49 patients who received treatment was 32.4%, and the median survival was 21.2 months (95% confidence interval, 8.7-33.8 months). The median OS was 41.3 months, 8.4 months, and 11 months for the complete resection group, the incomplete resection group, and the chemotherapy group, respectively (P<.0001). The only independent predictive factor that was associated with better OS was complete surgical resection. CONCLUSIONS: This retrospective study confirmed the rarity and poor prognosis of RIS in patients with NPC. Complete surgical resection was a significant prognostic factor for survival. The authors concluded that long-term follow-up is necessary for the early detection of RIS in patients with NPC.

Extraskeletal Ewing's sarcoma: a report of 18 cases and literature review.

BACKGROUND AND OBJECTIVE: Extraskeletal Ewing's sarcoma (EES) is a rare, rapidly growing, round-cell, malignant tumor that can develop in the soft tissues at any location. This study was to analyze the clinical features, diagnosis and treatment of EES. METHODS: Clinical data of 18 patients with EES, treated at between Cancer Center of Sun Yat-sen University between 1995 and 2007, were analyzed. RESULTS: Of the 18 patients, 13 were male and 8 were female, aged from 8 months to 60 years. Twelve (66.7%) patients were between 5-25 years of age. Eight (44.4%) patients had tumors originated from low extremities.Sixteen patients had masses at their first visit. Sixteen patients were treated by the combined modality therapy, and 2 patients were treated by the single modality therapy. The 1-, 3- and 5- year actuarial survival rates were 82.4%, 64.2% and 32.1%, respectively. The presence of metastatic disease at the time of diagnosis and the mode of treatment were prognostic factors. CONCLUSIONS: EES is common in adolescent. It often manifests as a localized mass. The combined modality therapy is recommended for this disease. The presence of metastatic disease at the time of diagnosis and the mode of treatment are prognostic factors.

[The primary study on the detection of sterigmatocystin by biologic enzyme electrode modified with the multiwall carbon nanotubes].

Sterigmatocystin (ST), the secondary metabolite of many kinds of filamentous fungi, is a potent carcinogen structurally related to the aflatoxins (AFT). With similar chemical structure, sterigmatocystion behaves much the homogeneous properties to aflatoxins, both of these mycotoxins exhibit similar biological properties due to their bisfuranoid structure. Since the common, and even heavier pollution, found in foods and feeds-stuff, sterigmatocystion is more harmful than aflatoxins. The reported detection methods of sterigmatocystion included the Thin-layer Chromatography, the High-Performance-Liquid Chromatography, the Enzyme-Linked Immunosorbant Assay and the PCR detection to the toxic gene, however studies about both easy and inexpensive electro-chemical methods have not been found. Our previous studies had discovered that Sterigmatocystin (ST) exist similar sensitivity towards aflatoxin-detoxifizyme (ADTZ), which we had isolated from a fungus, as aflatoxin does. In this work, the preliminary study on electrochemical analysis and determination of ST with triplet electrode enzyme-biosensor system (Ag/AgCl as the reference electrode, Pt and Au as the pair and work electrode, respectively) was carried out. Multiwall-carbon-nanotube (MWNT) had been used to increase the electron transportation on electrode. In the research, the Au electrode was modified by MWNT-immobilized ADTZ, and then the voltammertric behavior of ST was studied by means of cyclic voltammogram analysis and different pulse analysis. Autoprobe CP Research Atomic Force Microscope and TECNAI 10 Transmission Electron Microscope, had been used to detect the MWNT as well as the surface of MWNT-modified ADTZ. The voltammertric behavior of ST was studied by means of cyclic voltammogram analysis and different pulse analysis. The results show that the red-ox peak potential of ST is at the point of -600 mV, the linear detection range is from 8.32 x 10(-5) to 66.56 x 10(-5) mg/mL, the detection limit is at 8.32 x 10(-5) mg/mL, and the response time is 10 seconds. This study provided a good basic work for further research.

[Characterization of immobilized aflatoxin-detoxizyme].

Aflatoxins, found in contaminated food, are potent hepatocarcinogen. The aflatoxin-detoxiczyme (ADTZ) isolated from the edible fungus Armillariella sp., detoxifies aflatoxin B1 (AFB1). This paper reports on the characterization of immobilized ADTZ using a hydrophobic adsorption method. The ADTZ was isolated from cryo-homogenated fungus, previously cultivated at 24 - 28 degrees C for 20 - 30 days, using n-alkyl amino-agar beads. Various adsorption conditions of the enzyme to n-alkyl or n-octyl amino-agar beads were carried out. The effects of enzyme immobilization on different alkyl amino-agar beads, at different pH values (5.5 - 7.5), at different temperature (20 - 40 degrees C) and at different salt concentrations were investigated. The enzyme activity was measured at OD360 by reacting 133.3 ng/mL of AFB1 at 30 degrees C for 30 min with the immobilized ADTZ. The Km value of the immobilized enzyme, determined using Schematic Linewearver-Burk plot, is 3.308 x 10(-3) mol/L, lower than that of free enzyme, which is 2.16 x 10(-6) mol/L. This indicated the affinity of the detoxiczyme to AFB1 decreased after immobilization. The immobilized enzyme activity in oil-phase (n-hexane) was also studied with different concentration of water. After the treatment of the immobilized ADTZ, the toxin no longer causes liver toxicity in the rat toxicity test, no longer causes mutagenicity in Ames test and is no longer toxic in the chicken embryo test. Results also indicated that the pH stability, the thermostability and the freezing stability of ADTZ were improved after the immobilization.