Analysis of the Effect of N555 Mutations on the Product Specificity of Dextransucrase Using Caffeic Acid Phenethyl Ester as an Acceptor Substrate.

Bioglycosylation is an efficient strategy to improve biological activities and physicochemical properties of natural compounds to develop structural modifications of drugs. In this study, an N555 residue was identified as a candidate for site-directed mutagenesis through sequence alignment with GTF180ΔN. Caffeic acid phenethyl ester (CAPE) was used as an acceptor substrate. Two generated mutants, N555Q and N555E, demonstrated significant specificity of distribution of products. Under identical conditions, the conversion rates of diglycoside products (CAPE-2G) generated by the N555E (80.8%) and N555Q (84.5%) mutants were 3.30- and 3.46-fold higher than those generated by the original enzyme (24.4%). The structural simulation results demonstrated that a new hydrogen bond was formed between the N555 residue and CAPE, and the N555 residue was closely related to substrate elongation. These results provide a reference for subsequent studies. Suitable mutants for transfer of diglycosides have important application potential in the food and pharmaceutical industries.

Transglycosylation Improved Caffeic Acid Phenethyl Ester Anti-Inflammatory Activity and Water Solubility by Leuconostoc mesenteroides Dextransucrase.

Bioglycosylation is an efficient strategy to improve the biological activity and physicochemical properties of natural compounds for therapeutic drug development. In this study, two caffeic acid phenethyl ester (CAPE) glucosides (G-CAPE and 2G-CAPE) were synthesized by transglycosylation with dextransucrase from Leuconostoc mesenteroides 0326 with CAPE as an acceptor and sucrose as a donor. The products were purified and the structures were characterized. The physicochemical properties, anti-inflammatory activity, and cytotoxicity of the two CAPE glucosides were measured. The water solubility of G-CAPE and 2G-CAPE is 35 and 90 times higher, respectively, than that of CAPE. Compared to CAPE, the monoglycoside product showed superior anti-inflammatory effects, and its inhibition rate of NO, IF-6, and TNF-α is 93.4%, 76.81%, and 56.58% in RAW 264.7 macrophages, respectively, at 20 µM. Also, the cytotoxicity of both products was significantly improved. These glycosylation-modified CAPEs circumvent some of the flaws in CAPE application in anti-inflammatory drugs.

Portable optical aptasensor for rapid detection of mycotoxin with a reversible ligand-grafted biosensing surface.

As food safety is gaining prominence as a global issue, the demand for developing rapid, simple, on-site, accurate and low-cost biosensor technologies will continue to grow. This study demonstrates an evanescent wave optical aptasensor with a reversible ligand-grafted biosensing surface for rapid, sensitive and highly selective detection of ochratoxin A (OTA) in food. In this system, the OTA molecules were covalently immobilized onto the surface of an optical fiber using glutaraldehyde and ethylenediamine as space linkers. An integrated evanescent wave all-fiber (EWA) biosensing platform was developed for investigating the binding kinetics between the tethered ligand and free OTA-aptamer, the performance of the aptamer-based bioassay and the reversibility of biosensing surface. The affinity constant (Ka) of aptamer with tethered OTA was measured to be 2.2 × 10(8)M(-1) based on the EWA biosensing platform. With a competitive detection mode, the quantification of OTA over concentration ranges from 0.73 µg L(-1) to 12.50 µg L(-1) with a detection limit of 0.39 µg L(-1). The performance of the aptasensor with other interfering mycotoxins and spiked real wheat samples shows high specificity and selectivity, good recovery, precision, and accuracy, indicating that it can be applied for on-site, inexpensive and easy-to-use monitoring of OTA in real samples. Moreover, since the organic ligands are grafted onto the fiber surface, this strategy may avoid the potential disadvantages caused by immobilizing the nucleic acid biomolecules, such as weak restoration to the original DNA conformation after repeated uses.

A reusable aptamer-based evanescent wave all-fiber biosensor for highly sensitive detection of Ochratoxin A.

Although aptamer-based biosensors have attracted ever-increasing attentions and found potential applications in a wide range of areas, they usually adopted the assay protocol of immobilizing DNA probe (e.g., aptamer, aptamer-complementary oligonucleotides) on a solid sensing surface, making it critical and challengeable to keep the integration of nucleic acid surface during the regeneration and the restoration to its original DNA probe form after repeated uses. In order to address the issue, we report a novel aptamer-based biosensing strategy based on an evanescent wave all-fiber (EWA) platform. In a simple target capturing step using aptamer-functionalized magnetic microbeads, signal probes conjugated with streptavidin are released and further detected by a EWA biosensor via a facial dethiobiotin-streptavidin recognition. Apart from the inherent advantages of aptamer-based evanescent wave biosensors (e.g. target versatility, sensitivity, selectivity and portability), the proposed strategy exhibits a high stability and remarkable reusability over other aptasensors. Under the optimized conditions, the typical calibration curve obtained for Ochratoxin A has a detection limit of 3nM with a linear response ranging from 6nM to 500nM. The dethiobiotin-streptavidin sensing surface instead of the traditional nucleic acid one can be reused for over 300 times without losing sensitivity.

Iron(III) modification of Bacillus subtilis membranes provides record sorption capacity for arsenic and endows unusual selectivity for As(V).

Bacillus subtilis is a spore forming bacterium that takes up both inorganic As(III) and As(V). Incubating the bacteria with Fe(III) causes iron uptake (up to ∼0.5% w/w), and some of the iron attaches to the cell membrane as hydrous ferric oxide (HFO) with additional HFO as a separate phase. Remarkably, 30% of the Bacillus subtilis cells remain viable after treatment by 8 mM Fe(III). At pH 3, upon metalation, As(III) binding capacity becomes ∼0, while that for As(V) increases more than three times, offering an unusual high selectivity for As(V) against As(III). At pH 10 both arsenic forms are sorbed, the As(V) sorption capacity of the ferrated Bacillus subtilis is at least of 11 times higher than that of the native bacteria. At pH 8 (close to pH of most natural water), the arsenic binding capacity per mole iron for the ferrated bacteria is greater than those reported for any iron containing sorbent. A sensitive arsenic speciation approach is thus developed based on the binding of inorganic arsenic species by the ferrated bacteria and its unusual high selectivity toward As(V) at low pH.

[Prevalence of peripheral vascular disease in diabetics and impaired glucose regulation subjects: a community-based study].

OBJECTIVE: To investigate the prevalence of peripheral vascular disease (PVD) in patients with diabetes mellitus (DM) and impaired glucose regulation (IGR). METHODS: Follow-up 5 years after the previous epidemiologic study of metabolic syndrome in Chaoyang community, Shanghai, 2000 - 2001 was conducted among 717 patients with DM or IGR. Questionnaire survey about general status and DM-related history and behaviors, including smoking, was carried out. Physical examination was made. Ankle/brachial index (A/BI) was measured. Intermittent claudication was surveyed by Edinburgh claudication questionnaire (ECQ). Blood sugar and blood lipid were detected. 427 patients, 210 males and 217 females, aged 67.3 +/- 14.0 (21 - 104), with intact data were enrolled into the analysis. RESULTS: The overall prevalence of PVD was 12.2% in the hyperglycemic population. The prevalence of PVD in the diabetics was 15.1%, significantly higher than that of the IGP subjects (7.7%, P = 0.022). The rate of intermittent claudication in those with the A/BI

[Chronic renal disease in diabetic and prediabetic subjects: a community-based study in Shanghai].

OBJECTIVE: The aim of the present study was to assess the prevalence and risk factors of chronic renal disease in hyperglycemic population of Shanghai Caoyang Community. METHODS: Microalbuminuria was determined by measuring urinary albumin-to-creatinine ratio (ACR) and glomerular filtration rate (GFR) was estimated from fasting serum creatinine. RESULTS: A total of 406 Shanghai Chinese, with the average age of 67.5 +/- 13.8 years (244 with diabetes mellitus and 162 with impaired glucose regulation) from the established hyperglycemic cohort were included. (1) The prevalence of microalbuminuria was 20.9% and 10.5% in the subjects with diabetes and impaired glucose regulation (IGR); (2) The prevalence of a cGFR >or= 60 and < 90, >or= 30 and < 60, < 30 mlxmin(-1)x(1.73 m(2))(-1) were 41.6%, 37.0%, 1.2% respectively in the patients with diabetes, and 34.2%, 47.2%, 1.9% in the patients with IGR. Impaired renal function was 38.2% and 49.1% respectively in the subjects with diabetes and IGR; (3) The prevalence of microalbuminuria was significantly higher in the diabetic patients with hypertension, central obesity, dyslipidemia, history of cardiovascular disease or selinity. Systolic blood pressure, waist circumstance, fasting plasma glucose and history of cardiovascular disease were all independently associated with hyperglycemic microalbuminuria; (4) cGFR was diminished with increased age and the impaired renal function was more frequent in the patients with hypertension (48.5%). There was a significant positive correlation between a diminished cGFR and increasing levels of ACR after the patients with macroalbuminuria were deleted and adjusted age. Serum creatinine, age and systolic blood pressure were independently associated with diabetic cGFR < 60 ml/min/1.73 m(2). CONCLUSIONS: The high prevalence of microalbuminuria and impaired renal function in the hyperglycemic population of Caoyang Community underlines the need for cost-effective programs for the detection of chronic renal disease, and approaches to screen it in the hyperglycemic patients should incorporate assessment of GFR in addition to monitoring urine albumin excretion.