Single amino acid substitution of VP1 N17D or VP2 H145Y confers acid-resistant phenotype of type Asia1 foot-and-mouth disease virus

Infection by foot-and-mouth disease virus(FMDV) is triggered by the acidic pH in endosomes after virus uptake by receptor-mediated endocytosis. However, dissociation of the FMDV 146S particle in mildly acidic conditions renders inactivated foot-and-mouth disease(FMD) vaccines much less effective. Type Asia1 FMDV mutants with increased resistance to acid inactivation were selected to study the molecular basis of viral resistance to acid-induced disassembly and improve the acid stability of FMDV. Sequencing of capsid-coding regions revealed four amino acid replacements(VP1 N17D, VP2 H145Y, VP2 G192D, and VP3 K153E) in the viral population of the acid-selected 10th passage. We performed single or combined mutagenesis using a reverse genetic system, and our results provide direct experimental evidence that VP2 H145Y or VP1 N17D substitution confers an acid-resistant phenotype to type Asia1 FMDV.

The Effect of the Extracts of Endophytic Fungi on Pancreatic a-Amylase Activity

Inhibitors of pancreatic a-amylase offer an effective strategy to lower the levels of postprandial hyperglycemia by controlof starch breakdown. Among 86 fungal endophytes isolated from 15 medicinal plants Aspergillus terreus-AFl04S, Aspergillusegypticus-HT166S and Penicillium sp.-CC200 exhibited strong pancreatic amylase inhibitory potential were selected. Endophyteswere subjected to ethyl acetate extraction and tested for a-amylase inhibition, in order to assess and evaluate their inhibitory potentialon pancreatic a-amylase. Analysis showed concentration dependent enzyme inhibition up to 83% with half inhibition （IC50） valuesfor less 25 mg.mL1, which is lower than acarbose as control. It was observed 3-fold increasing of Vmax and maintenance Km atcontrol level in the presence of extracts A. terreus-AFl04S and Penicillium sp.-CC200, while in presence of extract A.egypticus-HT166S Km was doubled, and Vmax was maintained at the control level. Kinetic studies allow proposing the competitivemode of a-amylase inhibition by extracts A. egypticus-HT166S and uncompetitive inhibition by extracts A, terreus-AFl04S andPenicillium sp.-CC200.

Ultrasonic Extraction of Polysaccharides from Laminaria japonica and Their Antioxidative and Glycosidase Inhibitory Activities

In the present study, ultrasonic extraction technique （UET） is used to improve the yield of polysaccharides from Lami- naria japonica （LJPs）. And their antioxidative as well as glycosidase inhibitory activities are investigated. Box-Behnken design （BBD） combined with response surface methodology （RSM） is applied to optimize ultrasonic extraction for polysaccharides. The optimized conditions are obtained as extraction time at 54 min, ultrasonic power at 1050 W, extraction temperature at 80℃ and ratio of material to solvent at 1：50 （g mL-1）. Under these optimal ultrasonic extraction conditions, an actual experimental yield （5.75% ＋ 0.3%） is close to the predicted result （5.67%） with no significant difference （P〉0.05）. Vitro antioxidative and glycosidase inhibitory activities tests indicate that the crude polysaccharides （LJP） and two major ethanol precipitated fractions （LJP1 and LJP2） are in a concentration-dependent manner. LJP2 （30%-60% ethanol precipitated polysaccharides） possesses the strongest α-glucosidase in- hibitory activity and moderate scavenging activity against hydroxyl radicals （66.09% ±2.19%, 3.0 mg mL-l）. Also, the inhibitory activity against a-glucosidase （59.08% ± 3.79%, 5.0 mg mL-1） is close to that of acarbose （63.99% ± 3.27%, 5.0 mg mL-l）. LJP 1 （30% ethanol precipitated polysaccharides） exhibits the strongest scavenging activity against hydroxyl radicals （99.80%q-0.00%, 3.0mg mL-1） and moderate a-glucosidase inhibitory activity （47.76%± 1.92%, 5.0 mgmL-1）. LJP shows the most remarkable DPPH scav- enging activity （66.20%±0.11%, 5.0mgmL-1） but weakest a-glucosidase inhibitory activity （37.77%±1.30%, 5.0mgmL-1）. How- ever, all these LJPs exert weak inhibitory effects against a-amylase. These results show that UET is an effective method for extract- ing bioactive polysaccharides from seaweed materials. LJP 1 and LJP2 can be developed as a potential ingredient in hypoglycemic agents or functional food for the management of diabetes. This study provides scientific evidence and advances in the preparation technology and a hypoglycemic activities evaluation method for seaweed polysaccharides, especially glycosidase inhibition in com- bination with an antioxidative activity evaluation method.

Efficient adsorption and desorption of Cu^(2+) by a novel acid-resistant magnetic weak acid resin

A novel magnetic weak acid resin NDMC-1 was prepared in the presence of methyl acrylate （MA）, divinylbenzene （DVB） and titanate coupling agent （TCA） coated γ-Fe203 particles. To evaluate the adsorption of Cu^2＋ on the obtained resin NDMC- 1, another two magnetic resins NDMC-0 （the precursor of NDMC-1 without hydrolyzation） and NDMO-1 （the synthesized weak acid resin using oleic acid coated γ-Fe2O3） were chosen for comparison. The results showed that the carboxyl groups were formed after hydrolyzation, and NDMC-1 exhibited a greater adsorption capacity to Cu2~. The desorption experiment demonstrated that the desorption ratio at pH 2 （95.14%） was greatly higher than pH 3 （25.97%）. Moreover, the magnetic resin NDMC-1 was proved to be stable at pH 2, extending the application of magnetic materials which were always considered to be acid-nor, resistant.

Enzymatic Degradation of Poly(butylenesuccinate)/Thermoplastic Starch Blend

The degradation of thermoplastic starch blend in the presence of commerciala-amylase and unpurified amylase of microbial origin was investigated.The blends consisting of thermoplastic starch and poly(butylene succinate)have potential use in packaging applications thus,it is essential to establish susceptibility to degradation.Molar mass loss,gravimetric weight loss,and molecular structure were evaluated.The changes in the surface were observed with scanning electron microscopy.It was confirmed that there was a significant difference in gravimetric weight loss between the blends degraded in two different solutions.Unpurified enzymes of microbial origin,produced by Rhizopus oryzae cultures decomposed analyzed materials more efficiently than purified commercial ones.Moreover,it was proved that in applied conditions,the molar mass of PBS fraction did not change significantly.

Inhibitory actions of Pseuderanthemum palatiferum(Nees) Radlk. leaf ethanolic extract and its phytochemicals against carbohydrate-digesting enzymes

Objective: To investigate the effects of the leaf ethanolic extract of Pseuderanthemum palatiferum(PPE) and its isolated phytochemicals, stigmasterol and sitosterol-3-O-b-Dglucopyranoside, against a-amylase and a-glucosidase enzyme activities both in vitro and in vivo.Methods: A concentration of maltose, which is a product released in a-amylase-catalyzing reaction, was used as an index of in vitro a-amylase activity. Meanwhile, in vitro a-glucosidase enzyme activity was indicated by the amount of liberated p-nitrophenol in a-glucosidase-catalyzing reaction. In vivo a-amylase and a-glucosidase enzyme activities were evaluated in the normal rats by using oral starch tolerance test and oral sucrose tolerance test, respectively.Results: PPE exerted a concentration-dependent inhibitory action against both aamylase and a-glucosidase in vitro with the IC_(50) values of(11.79 ± 8.10) mg/m L and(1.00 ± 0.11) mg/m L, respectively. Stigmasterol and sitosterol-3-O-b-D-glucopyranoside also exerted an in vitro a-amylase inhibition with the IC_(50) values of(59.41 ± 8.22) mg/m L and(111.19 ± 9.02) mg/m L, respectively. However, these phytochemicals did not produce a concentration-dependent inhibition against in vitro a-glucosidase activity. PPE and its isolated phytochemicals significantly decreased the blood glucose levels at t = 30 min in the oral starch tolerance test. From the sucrose tolerance test, only PPE but not its isolated phytochemicals significantly caused a depletion in the blood glucose levels at t = 30 min Conclusions: These results indicate an inhibitory action against carbohydrate-digesting enzymes as the anti-diabetic mechanism of action of PPE. Nonetheless, further clinical study is required to justify its role in the treatment of diabetes.

Acid-resistant ROS-responsive hyperbranched polythioether micelles for ulcerative colitis therapy

Drug-loaded micelles for oral administration are desired for its conve nience,low cost and flexibility,but current designs rely on introducing pH responsiveness,leaving problems like drug leakage and low accuracy of targeted delivery un-solved.Herein,we reported an acid-resistant ROS-responsive hyperbranched polythioether which can self-assemble into micellar structure and pass through the gastrointestinal tract without leaking drugs.At the inflammatory lesions,the thioester bonds are oxidized to sulphone groups to significantly increase the hydrophilicity in response to accumulated ROS species and efficiently release the encapsulated drugs.Animal experiments,including the evaluation of bodyweight,colon length,MPO activity,spleen index,histology and quantitative reverse transcription PCR,evidenced that the drug-loaded micelles have improved therapeutic efficiency compared to bare drug administration for the treatment of DSS-induced colitis in mice.This study provides an example of oral administrated micellar system can be extended for the treatment of other intestinal tract diseases.

Effect of Moisture Content on Melilotus suaveolens Seed Quality During Ultra-drying Storage

In order to enhance the seed storability and supply high-quality seeds for vegetation restoration in the arid and semi-arid regions in Northwest China,the effects of ultra-drying and accelerated aging on the physiological characterstics of Melilotus suaveolens seeds were studied.Melilotus suaveolens seeds were dried in a desiccator containing silica gel to 80,53,42,33,23 and 16 g·kg-1 of moisture contents（MC）,respectively.The parameters of the seed quality including germination energy（GE）,germination percentage（GP）,relative conductivity（RC）,dehydrogenase activity（DA） and α-amylase activity（AA） were determined after ultra-drying and accelerated aging.The results showed that ultra-dried seeds with 42 g·kg-1 of MC showed the minimum changes of GE and GP before and after seed aging.Moreover,ultra-dry seeds with 42 g·kg-1 of MC showed higher DA and AA,and lower RC than non-ultra-drying seeds.Therefore,ultra-drying to 42 g·kg-1 of MC was helpful for M.suaveolens seed storage.

A New Approach for the Measurement of Digestible Carbohydrates in Different Food Samples with HPLC-RI

This paper gives an impetus in quantifying digestible carbohydrates, which are important components of cereals, legumes and vegetables. The HPLC method was applied for the first time in India to estimate the digestible carbohydrates such as sugars and starches in different branded rice, legumes and vegetable samples which were procured from local market of twin cities of Hyderabad and Secunderabad in Andhra Pradesh State, India. In the present study, we estimated carbohydrates in vitro, mimicking the in vivo condition by incorporating enzymatic digestion. Among the rice varieties the analyzed total soluble sugars were in the range of 5.69% to 9.62%, vegetables 0.0% to 3.72% and legumes 0.04% to 0.98%. Soluble starches in rice samples ranged from 12.51% to 17.64%, in vegetables from 1.73% to 7.28% and in legumes from 23.76% to 38.71%. Insoluble starches in rice samples were observed to be bracketed in between 52.53% to 60.43%, where as in vegetables from 0.58% to 8.83% and in legumes from 16.13% to 29.22%. Concluding our observation, the total amount of starches and total sugars in rice fell in between 74.32% to 80.75%, in vegetables from 1.74% to 16.11%, and in legumes from 39.93% to 68.91%.

Achieving robust synthetic tolerance in industrial E.coli through negative auto-regulation of a DsrA-Hfq module

In industrial fermentation processes,microorganisms often encounter acid stress,which significantly impact their productivity.This study focused on the acid-resistant module composed of small RNA(sRNA)DsrA and the sRNA chaperone Hfq.Our previous study had shown that this module improved the cell growth of Escherichia coli MG1655 at low pH,but failed to obtain this desired phenotype in industrial strains.Here,we performed a quantitative analysis of DsrA-Hfq module to determine the optimal expression mode.We then assessed the po-tential of the CymR-based negative auto-regulation(NAR)circuit for industrial application,under different media,strains and pH levels.Growth assay at pH 4.5 revealed that NAR-05D04H circuit was the best acid-resistant circuit to improve the cell growth of E.coli MG1655.This circuit was robust and worked well in the industrial lysine-producing strain E.coli SCEcL3 at a starting pH of 6.8 and without pH control,resulting in a 250%increase in lysine titer and comparable biomass in shaking flask fermentation compared to the parent strain.This study showed the practical application of NAR circuit in regulating DsrA-Hfq module,effectively and robustly improving the acid tolerance of industrial strains,which provides a new approach for breeding in-dustrial strains with tolerance phenotype.