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#28098753   2017/01/18 Save this To Up

Reagent-Less and Robust Biosensor for Direct Determination of Lactate in Food Samples.

Lactic acid is a relevant analyte in the food industry, since it affects the flavor, freshness, and storage quality of several products, such as milk and dairy products, juices, or wines. It is the product of lactose or malo-lactic fermentation. In this work, we developed a lactate biosensor based on the immobilization of lactate oxidase (LOx) onto N,N'-Bis(3,4-dihydroxybenzylidene) -1,2-diaminobenzene Schiff base tetradentate ligand-modified gold nanoparticles (3,4DHS-AuNPs) deposited onto screen-printed carbon electrodes, which exhibit a potent electrocatalytic effect towards hydrogen peroxide oxidation/reduction. 3,4DHS-AuNPs were synthesized within a unique reaction step, in which 3,4DHS acts as reducing/capping/modifier agent for the generation of stable colloidal suspensions of Schiff base ligand-AuNPs assemblies of controlled size. The ligand-in addition to its reduction action-provides a robust coating to gold nanoparticles and a catalytic function. Lactate oxidase (LOx) catalyzes the conversion of l-lactate to pyruvate in the presence of oxygen, producing hydrogen peroxide, which is catalytically oxidized at 3,4DHS-AuNPs modified screen-printed carbon electrodes at +0.2 V. The measured electrocatalytic current is directly proportional to the concentration of peroxide, which is related to the amount of lactate present in the sample. The developed biosensor shows a detection limit of 2.6 μM lactate and a sensitivity of 5.1 ± 0.1 μA·mM(-1). The utility of the device has been demonstrated by the determination of the lactate content in different matrixes (white wine, beer, and yogurt). The obtained results compare well to those obtained using a standard enzymatic-spectrophotometric assay kit.

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#26204835   2015/07/24 Save this To Up

14-3-3γ Regulates Lipopolysaccharide-Induced Inflammatory Responses and Lactation in Dairy Cow Mammary Epithelial Cells by Inhibiting NF-κB and MAPKs and Up-Regulating mTOR Signaling.

As a protective factor for lipopolysaccharide (LPS)-induced injury, 14-3-3γ has been the subject of recent research. Nevertheless, whether 14-3-3γ can regulate lactation in dairy cow mammary epithelial cells (DCMECs) induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3γ in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3γ overexpression significantly inhibited the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS). Enzyme-linked immunosorbent assay (ELISA) analysis revealed that 14-3-3γ overexpression also suppressed the production of TNF-α and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3γ overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3γ overexpression promoted the secretion of triglycerides and lactose and the synthesis of β-casein. Furthermore, the expression of genes relevant to nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPKs) and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3γ overexpression inactivated the NF-κB and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK) and inhibitor of NF-κB (IκB) phosphorylation levels, as well as by inhibiting NF-κB translocation. Meanwhile, 14-3-3γ overexpression enhanced the expression levels of β-casein, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), serine/threonine protein kinase Akt 1 (AKT1), sterol regulatory element binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor gamma (PPARγ). These results suggest that 14-3-3γ was able to attenuate the LPS-induced inflammatory responses and promote proliferation and lactation in LPS-induced DCMECs by inhibiting the activation of the NF-κB and MAPK signaling pathways and up-regulating mTOR signaling pathways to protect against LPS-induced injury.

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#25262408   2014/12/08 Save this To Up

Coat formation of surface-active proteins on aqueous surfaces during drying.

Segregation of the protein bovine serum albumin (BSA) and lactose in thin aqueous films during drying was investigated by examining the composition of the dried films using inverse micro Raman spectroscopy (IMRS) and X-ray photoelectron spectroscopy (XPS) sputter-depth profiling. The composition was uniform through the thickness of the dried films except within a 10nm region at the exposed surface where BSA had accumulated, most likely due to its surface activity. The thickness of the BSA layer was similar to the diameter of a BSA molecule, which suggests that a single monolayer of BSA adsorbed at the exposed surface. The BSA surface concentration of the dried films was constant over a wide range of BSA bulk concentrations, indicating that the aqueous surface became saturated with BSA during drying. The BSA surface layer of order 10nm was significantly thinner than the film thickness of order 10 μm, which implies that BSA formed a surface coating rather than a shell, and thus lent no structural rigidity to the film.

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#24534513   2014/03/24 Save this To Up

Technical note: optimization of lactose quantification based on coupled enzymatic reactions.

A colorimetric microplate-adapted lactose assay was developed to quantify lactose in dairy products. The assay was based on the coupled enzymatic reaction of β-galactosidase-glucose oxidase-horseradish peroxidase using Amplex red as detection probe. The assay showed good linearity in the range of 0.1 to 0.5mmol of lactose/L, with a limit of detection of 0.0433mmol/L and a limit of quantification of 0.1313mmol/L. The lactose assay at optimized conditions (5 U of β-galactosidase/mL, 5 U of glucose oxidase/mL, 1 U of horseradish peroxidase/mL, and 100μmol of Amplex red/L for 1h at 37°C in the dark) showed good correlation with a commercial lactose enzymatic kit with intraassay variation below 10% and interassay variations below 7.6%. The developed lactose microplate assay can be adopted as routine analysis for lactose determination in dairy products due to its relatively low cost compared with a commercial kit, relatively short reaction time, and high sensitivity and reproducibility.

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#24125616   2013/10/15 Save this To Up

[Assessment and application of a molecular diagnostic method on the detection of four types of diarrheagenic Escherichia coli].

To establish and evaluate a molecular diagnostic method for routine monitoring of four types of diarrheagenic Escherichia (E.) coli (DEC)and to study the distribution of four types of DEC isolated from diarrheal patients in Shanghai.

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#23607586   2013/04/23 Save this To Up

Development of a novel emergency hemostatic kit for severe hemorrhage.

Photocrosslinkable chitosan (Az-CH-LA) contains lactose moieties and photoreactive azide groups, and its viscous solution forms an insoluble hydrogel on exposure to UV irradiation. We previously developed an emergency hemostatic kit using the Az-CH-LA solution, calcium alginate, and a UV irradiation apparatus. However, a suitable UV irradiation apparatus is required to effectively convert the Az-CH-LA solution into a hydrogel, and power supply to use the UV irradiation apparatus may not always be available in a disaster area or battlefield. To address this problem, we produced a portable, battery-powered UV irradiation apparatus constituting a novel hemostatic kit for severe hemorrhage. When the hemostatic kit using the UV irradiation apparatus was examined using a rat model of severe hemorrhage, the survival rate increased up to 73%. Hematological values as markers of hemorrhage did not change significantly over the first 3 days. In this study, we describe the characteristics of a portable UV irradiation apparatus and its use in an emergency hemostatic kit prepared using Az-CH-LA and calcium alginate for severe hemorrhage.

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#23158148   2012/11/19 Save this To Up

Effect of kappa elastin on melanogenesis in A375 human melanoma cells and its related mechanism.

Elastin derived peptides can regulate melanocyte precursor development. Ultraviolet irradiation, infrared radiation and heat can increase the synthesis of tropoelastin in human skin epidermis. The aim of this study was to investigate whether the over expressed tropoelastin in epidermis has some role in melanogenesis of melanocytes.

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#20522272   2010/10/29 Save this To Up

Variation of human milk oligosaccharides in relation to milk groups and lactational periods.

Human milk oligosaccharides, representing the third largest fraction of human milk, have been assigned important protective functions for newborns acting as bifidogenic substrates or as inhibitory agents towards pathogens. Using high-pH anion-exchange chromatography and an enzyme test kit, twenty oligosaccharides and lactose were determined in milk samples of German women from days 3 to 90 postpartum. Twenty-two secretor mothers with Lewis blood group Le(a - b+) synthesised all twenty oligosaccharides, and could be assigned to milk group 1. Five non-secretor mothers (Le(a+b - )) produced all oligosaccharides with the exception of α1,2-fucosylated compounds (milk group 2), whereas three secretor mothers with blood type Le(a - b - ) lacked α1,4-fucosyloligosaccharides, corresponding to milk group 3. Secretor women of milk groups 1 and 3 synthesised significantly higher amounts of total neutral oligosaccharides and of several total core structures (e.g. lacto-N-tetraose) than non-secretor women. Generally, these oligosaccharides significantly decrease during the first 3 months postpartum. By comparing fucosyloligosaccharides within and among the three milk groups, insight into their biosynthesis could be gained. Six acidic oligosaccharides without fucose residues were detected in milk samples of all mothers. Regression analysis confirmed that total acidic oligosaccharides declined threefold during the study period. Milk samples corresponding to the three milk groups exhibited significant qualitative and quantitative differences during the first 3 months of lactation. It can be assumed that particularly milk of non-secretor women (milk group 2) exerts a modified biological protection in the babies in comparison with milks of secretors (groups 1 and 3).

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#20509693   2010/06/16 Save this To Up

An integrated amperometric biosensor for the determination of lactose in milk and dairy products.

An integrated amperometric biosensor for the determination of lactose is reported. The bioelectrode design is based on the use of a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode on which the enzymes beta-galactosidase (beta-Gal), glucose oxidase (GOD), peroxidase (HRP) and the mediator tetrathiafulvalene (TTF) are coimmobilized by a dialysis membrane. beta-Gal catalyzes the hydrolysis of lactose, and the produced glucose is catalytically oxidized to gluconic acid and H(2)O(2), which is reduced in the presence of HRP. This enzyme reaction is mediated by TTF, and the reduction of TTF(+) at 0.00 V (vs Ag/AgCl) gives rise to an amperometric signal proportional to the lactose concentration. The biosensor exhibits a good repeatability of the measurement carried out with the same biosensor, a good reproducibility of the responses obtained with different biosensors and a useful lifetime of 28 days. A linear calibration plot was obtained for lactose over the 1.5 x 10(-6) to 1.2 x 10(-4) M concentration range, with a limit of detection of 4.6 x 10(-7) M. The effect of potential interferents (sucrose, lactulose, fructose, arabinose, maltose, galactose, glucose and uric and ascorbic acids) on the biosensor response was evaluated. Furthermore, the bioelectrode exhibits a suitable performance in flow-injection systems in connection with amperometric detection. The developed biosensor was applied to the determination of lactose in milk and other foodstuffs (chocolate, butter, margarine, yogurt, cheese and mayonnaise), and the results obtained were validated by comparison with those provided by using a commercial enzyme test kit.

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#20166614   2010/02/19 Save this To Up

TaqMan Salmonella enterica Detection Kit. Performance Tested Method 020803.

Peanut butter spiked with Salmonella enterica ser. Typhimurium was prepared by an independent laboratory and sent to Applied Biosystems to determine the sensitivity and specificity of the TaqMan Salmonella enterica Detection Kit for detecting Salmonella in peanut butter. The samples were spiked at three levels: five no-spike (0 CFU/25 g); 20 low-spike (0.2 CFU/25 g); and 20 high-spike (2 CFU/25 g). They were coded to create a blind set of 45 samples. The samples were processed based on an unpaired test design that included enrichment in buffered peptone water for the candidate method and lactose broth for the reference method. In the candidate method, a 1 mL aliquot of enriched sample was extracted using PrepMan Ultra Sample Preparation Reagent; the sample was amplified on the Applied Biosystems 7500 real-time PCR system, and analyzed for detection of Salmonella using RapidFinder Version 1.0 software. All samples processed by the candidate method were confirmed by culture according to the U.S. Food and Drug Administration's Bacteriological Analytical Manual procedures. Sensitivity, specificity, and Chi-square analysis were calculated by combining candidate method results with those of the reference method that were collected by the independent laboratory. The TaqMan Salmonella enterica Detection Kit showed 40% sensitivity, 100% specificity, and a Chi-square value equal to 1.52. Chi-square analysis indicated the candidate method and the reference method were comparable. Although the candidate method sensitivity was only 40% when compared with the reference method (unpaired samples), the sensitivity was > 100% when the candidate method results were compared with those of the confirmation method (same sample enrichment).

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