Synthesis and Characterization of Novel Resveratrol Butyrate Esters That Have the Ability to Prevent Fat Accumulation in a Liver Cell Culture Model.

To facilitate broad applications and enhance bioactivity, resveratrol was esterified to resveratrol butyrate esters (RBE). Esterification with butyric acid was conducted by the Steglich esterification method at room temperature with N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) and 4-dimethyl aminopyridine (DMAP). Our experiments demonstrated the synthesis of RBE through EDC- and DMAP-facilitated esterification was successful and that the FTIR spectra of RBE revealed absorption (1751 cm-1) in the ester region. 13C-NMR spectrum of RBE showed a peak at 171 ppm corresponding to the ester group and peaks between 1700 and 1600 cm-1 in the FTIR spectra. RBE treatment (25 or 50 µM) decreased oleic acid-induced lipid accumulation in HepG2 cells. This effect was stronger than that of resveratrol and mediated through the downregulation of p-ACC and SREBP-2 expression. This is the first study demonstrating RBE could be synthesized by the Steglich method and that resulting RBE could inhibit lipid accumulation in HepG2 cells. These results suggest that RBE could potentially serve as functional food ingredients and supplements for health promotion.

Stability Designs of Cell Membrane Cloaked Magnetic Carbon Nanotubes for Improved Life Span in Screening Drug Leads.

Convenient strategies to provide natural cell membranes (CMs)-camouflaged nanomaterials with enhanced stability would prompt the advancement of CMs-coated biomimetic technology and expand the application of these emerging nanomaterials. Herein, we have developed stability-enhanced CMs-camouflaged magnetic carbon nanotubes (MCNTs) to screen drug leads from traditional Chinese medicine (TCMs) that target membrane receptors. By modifying MCNTs with N-ethyl-N'-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), the resulting covalent immobilized CMs-camouflaged MCNTs have improved stability, where the losing amount (20 mg g-1) was significantly decreased compared with that of the unimmobilized materials (40 mg g-1). The high expression ephrinb2/HEK293 cell lines were used to camouflage the EDC/NHS modified MCNTs (CMCNTs) to endow it with drug-screening sites. Moreover, with inherited properties from CMs, ephrinb2/HEK293 CMs-camouflaged CMCNTs possessed good binding capacity and selectivity, and three potential drug leads as mesaconine, deltaline, and 13-dehydroxyindine were screened from Aconitum carmichaeli Debx. The pharmacological assays indicated that mesaconine and 13-dehydroxyindine could inhibit cancer cell growth by targeting ephrinb2. As a result, this surface engineering method not only offers an insight into fabrication of stabilized CMs-coated nanomaterials but also inspires more brilliant work in the future and paves the way for the biomimetic functional modification of CNTs for a variety of applications.

Chitosan coated calcium alginate beads for covalent immobilization of acrylamidase: Process parameters and removal of acrylamide from coffee.

This study reports on removal of acrylamide from roasted coffee by acrylamidase from Cupriavidus oxalaticus ICTDB921. Chitosan coated calcium alginate beads were functionalized with citric acid as nontoxic cross linker and activated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (1.66:1 w/w) for covalent immobilization of acrylamidase. The optimum beads were obtained using 5% sodium alginate, 1.5% chitosan, and 0.6 mol/L citric acid. The beads prepared at each step were characterized by FTIR and SEM. Coating of chitosan matrix on calcium alginate beads enhanced the mechanical stability over that of calcium alginate and/or chitosan. The immobilized acrylamidase showed optimum pH/temperature of 8.5/65 °C, improved pH/thermal/shelf stability, and retained 80% activity after four cycles. Haldane model could describe the degradation kinetics of acrylamide in batch study. In packed bed column, a bed height, feed flow rate and inlet acrylamide concentration of 20 cm, 1 mL/min, and 100 mg/L gave best results.

Acetylcholinesterase biosensor for inhibitor measurements based on glassy carbon electrode modified with carbon black and pillar[5]arene.

New acetylcholinesterase (AChE) biosensor based on unsubstituted pillar[5]arene (P[5]A) as electron mediator was developed and successfully used for highly sensitive detection of organophosphate and carbamate pesticides. The AChE from electric eel was immobilized by carbodiimide binding on carbon black (CB) placed on glassy carbon electrode. The working potential of 200mV was obtained in chronoamperometric mode with the measurement time of 180 s providing best inter-biosensors precision of the results. The AChE biosensor developed made it possible to detect 1×10(-11)-1×10(-6) M of malaoxon, 1×10(-8)-7×10(-6) M of methyl-paraoxon, 1×10(-10)-2×10(-6) M of carbofuran and 7×10(-9)-1×10(-5) M of aldicarb with 10 min incubation. The limits of detection were 4×10(-12), 5×10(-9), 2×10(-11) and 6×10(-10) M, respectively. The AChE biosensor was tested in the analysis of pesticide residuals in spiked samples of peanut and beetroot. The protecting effect of P[5]A derivative bearing quaternary ammonia groups on malaoxon inhibition was shown.

Enhanced brain delivery of deferasirox-lactoferrin conjugates for iron chelation therapy in neurodegenerative disorders: in vitro and in vivo studies.

Oxidative stress associated cell damage is one of the key factors in neurodegeneration development and is highly related to the presence of transition metal ions including iron. Herein, deferasirox, a high affinity iron chelator, was conjugated to lactoferrin molecules by carbodiimide mediated coupling reaction to create a novel drug delivery system with higher brain permeability through receptor mediated transcytosis. Each lactoferrin molecule was averagely attached to 4 to 6 deferasirox molecules resulting in water-soluble conjugated nanostructures which were purified and characterized. Neuroprotective effects of lactoferrin conjugated nanostructures and their cellular uptake were evaluated in differentiated PC12 cell line, and the molecular mechanisms involved in such neuroprotection were elucidated. Lactoferrin conjugates were able to interfere in apoptotic caspase cascade by affecting the expression level of caspase-3, PARP, Bax and Bcl-2. Furthermore, an elevation in the expression level of autophagy markers including Atg7, Atg12-Atg5 and LC3-II/LC3-I ratio was observed. Intraperitoneal injection of lactoferrin conjugates was able to significantly attenuate learning deficits induced by beta amyloid injection in a rat model of Alzheimer's disease, which further confirms a potential neuroprotective effect for lactoferrin conjugated deferasirox in neurodegenerative disorder management through metal chelation therapy.

Simple colorimetric bacterial detection and high-throughput drug screening based on a graphene-enzyme complex.

A simple, colorimetric, sensitive, cost-effective and high-throughput system based on a positively charged graphene oxide-enzyme complex was developed for bacterial detection and drug screening.

Surface-modified ZnSe waveguides for label-free infrared attenuated total reflection detection of DNA hybridization.

This communication presents a novel label-free biosensing method to monitor DNA hybridization via infrared attenuated total reflection (IR-ATR) spectroscopy using surface-modified ZnSe waveguides. Well-defined carboxyl-terminated monolayers were formed at H-terminated ZnSe by direct photochemical activation. Chemical activation of the acidic function was obtained by using succinimide/carbodiimide linkers. The sequential surface modification reactions were characterized by XPS and IR-ATR spectroscopy. Finally, a single stranded DNA probe with a C6-NH(2) 5' modifier was coupled to the ester-terminated surface via peptide bonding, and the hybridization of the immobilized DNA sequence with its complementary strand was directly evaluated by IR-ATR spectroscopy in the mid-infrared (MIR) spectral regime (3-20 µm) without requiring an additional label. A shift of the vibrational modes corresponding to the phosphodiester and deoxyribose structures of the DNA backbone was observed. Hence, this approach substantiates a novel strategy for label-free DNA detection utilizing mid-infrared spectroscopy as the optical sensing platform.

Highly efficient electroosmotic flow through functionalized carbon nanotube membranes.

Carbon nanotube membranes with inner diameter ranging from 1.5-7 nm were examined for enhanced electroosmotic flow. After functionalization via electrochemical diazonium grafting and carbodiimide coupling reaction, it was found that neutral caffeine molecules can be efficiently pumped via electroosmosis. An electroosmotic velocity as high as 0.16 cm s(-1) V(-1) has been observed. Power efficiencies were 25-110 fold improved compared to related nanoporous materials, which has important applications in chemical separations and compact medical devices. Nearly ideal electroosmotic flow was seen in the case where the mobile cation diameter nearly matched the inner diameter of the single-walled carbon nanotube resulting in a condition of using one ion is to pump one neutral molecule at equivalent concentrations.

Single conducting polymer nanowire chemiresistive label-free immunosensor for cancer biomarker.

A simple and cost-effective, all-electrochemical method to fabricate and assemble single conducting polymer nanowire based biosensors was developed. Polypyrrole (Ppy) nanowires were synthesized by electrochemical polymerization using an alumina template. The single-nanowire chemoresistive sensor device was assembled using ac dielectrophoretic alignment followed by maskless anchoring on a pair of gold electrodes separated by 3 microm. To establish an efficient covalent surface biofunctionalization route, glutaraldehyde (GA) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) chemistries were compared. EDC was established to be the most effective chemistry and was used to surface-functionalize a single Ppy nanowire with cancer antigen (CA 125) antibody to fabricate a nanoimmunosensor for CA 125 biomarker detection and quantification. The immunosensor had excellent sensitivity with a lower detection limit of 1 U/mL CA 125 and dynamic range up to 1000 U/mL in 10 mM phosphate buffer. Furthermore, there was no loss of performance upon exposure to CA 125 in spiked human blood plasma. This demonstrates the clinical importance of these sensors for cancer marker detection with cost benefits and great portability for diagnosis of patients at the point of care.

Pectin-cysteine conjugate: synthesis and in-vitro evaluation of its potential for drug delivery.

This study was aimed at improving certain properties of pectin by introduction of thiol moieties on the polymer. Thiolated pectin was synthesized by covalent attachment of cysteine. Pectin-cysteine conjugate was evaluated for its ability to be degraded by pectinolytic enzyme. The toxicity profile of the thiolated polymer in Caco-2-cells, its permeation enhancing effect and its mucoadhesive and swelling properties were studied. Moreover insulin-loaded hydrogel beads of the new polymer were examined for their stability in simulated gastrointestinal conditions and their drug release profile. The new polymer displayed 892.27 +/- 68.68 micromol thiol groups immobilized per g polymer, and proved to have retained its biodegradability, upon addition of Pectinex Ultra SPL in-vitro, determined by viscosity measurements and titration method. Pectin-cysteine showed no severe toxicity in Caco-2 cells, as tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Moreover, the synthesized polymer exhibited a relative permeation enhancement ratio of 1.61 for sodium fluorescein, compared to unmodified pectin. Pectin-cysteine conjugate exhibited approximately 5-fold increased in in-vitro adhesion duration and significantly improved cohesive properties. Zinc pectin-cysteine beads showed improved stability in simulated gastrointestinal media; however, insulin release from these beads followed the same profile as unmodified zinc pectinate beads. Due to favourable safety and biodegradability profile, and improved cohesive and permeation-enhancing properties, pectin-cysteine might be a promising excipient in various transmucosal drug delivery systems.

Preparation of a monoclonal antibody to N(epsilon)-(Hexanonyl)lysine: application to the evaluation of protective effects of flavonoid supplementation against exercise-induced oxidative stress in rat skeletal muscle.

The monoclonal antibody to N(epsilon)-(hexanonyl)lysine (HEL), a novel adduct formed by the reaction of linoleic acid hydroperoxide and lysine, has been prepared and characterized. The obtained antibody specifically recognized the HEL moiety. Using the monoclonal antibody, we evaluated the protective effects of feeding eriocitrin, which is one of flavonoids in lemon fruit, on oxidative modification induced by exercise in rats. The supplementation of eriocitrin significantly suppressed the increase in HEL in the skeletal muscle by exercise. The result suggests that the determination of HEL may be a good method for evaluation of the protective effect of beneficial food factors against oxidative stress.

Crosslinking of hyaluronic acid with water-soluble carbodiimide.

Hyaluronic acid (HA) was chemically crosslinked with a water-soluble carbodiimide (WSC) to produce low-water-content films when brought into contact with water. The crosslinking reaction was performed in two different ways; one was by using HA films and the other by casting HA solutions. Both methods produced water-insoluble HA films. The lowest water content of the crosslinked HA films subjected to swelling with water was 60 wt % at 37 degrees C, which was lower than any reported values. Infrared spectra of the crosslinked films suggested that intermolecular formation of ester bonds between the hydroxyl and carboxyl groups belonging to different polysaccharide molecules led to crosslinking. For comparison, pectin which possesses hydroxyl and carboxyl groups in one molecule, similar to HA, was subjected to crosslinking with WSC. The finding on pectin also supported ester formation between different polysaccharide molecules. The crosslinking of HA film with WSC in the presence of L-lysine methyl ester prolonged the in vivo degradation of HA film, probably because of amide bond formation as the crosslink.

New biotinylating reagent utilizing carbodiimide function.

A biotin derivative having a carbodiimide function (fig.1 Carbo-biotin) was synthesized and reacted with DNAs. This compound was used to label biotin to DNAs, by reaction of carbodiimide group and nucleotides as they form stable adducts. Our experiments showed that it is able to bind to both single and double stranded DNAs. The DNA oligomer labeled with this reagent was shown to hybridize to complementary DNA chain. From our results, this compound is expected to be a useful biotinylating reagent for non-radioactive detection.

Binding of ferredoxin to ferredoxin:NADP+ oxidoreductase: the role of carboxyl groups, electrostatic surface potential, and molecular dipole moment.

The small, soluble, (2Fe-2S)-containing protein ferredoxin (Fd) mediates electron transfer from the chloroplast photosystem I to ferredoxin: NADP+ oxidoreductase (FNR), a flavoenzyme located on the stromal side of the thylakoid membrane. Ferredoxin and FNR form a 1:1 complex, which is stabilized by electrostatic interactions between acidic residues of Fd and basic residues of FNR. We have used differential chemical modification of Fd to locate aspartic and glutamic acid residues at the intermolecular interface of the Fd:FNR complex (both proteins from spinach). Carboxyl groups of free and FNR-bound Fd were amidated with carbodiimide/2-aminoethane sulfonic acid (taurine). The differential reactivity of carboxyl groups was assessed by double isotope labeling. Residues protected in the Fd:FNR complex were D-26, E-29, E-30, D-34, D-65, and D-66. The protected residues belong to two domains of negative electrostatic surface potential on either side of the iron-sulfur cluster. The negative end of the molecular dipole moment vector of Fd (377 Debye) is close to the iron-sulfur cluster, in the center of the area demarcated by the protected carboxyl groups. The molecular dipole moment and the asymmetric surface potential may help to orient Fd in the reaction with FNR. In support, we find complementary domains of positive electrostatic potential on either side of the FAD redox center of FNR. The results allow a binding model for the Fd:FNR complex to be constructed.