Exploration of Succinimide Derivative as a Multi-Target, Anti-Diabetic Agent: In Vitro and In Vivo Approaches.

Diabetes mellitus (DM) is counted among one of the leading challenges in the recent era, and it is a life-threatening disorder. Compound 4-hydroxy 3-methoxy phenylacetone (compound 1) was previously isolated from Polygonum aviculare. This compound was reacted with N-benzylmaleimide to synthesize the targeted compound 3. The purpose of this research is to exhibit our developed compound 3's ability to concurrently inhibit many targets that are responsible for hyperglycemia. Compound 3 was capable of inhibiting α-amylase, α-glucosidase, and protein tyrosine phosphatase 1 B. Even so, outstanding in vitro inhibition was shown by the compound against dipeptidyl peptidase-4 (DPP-4) with an IC50 value of 0.07 µM. Additionally, by using DPPH in the antioxidant activity, it exhibited good antioxidant potential. Similarly, in the in vivo activity, the experimental mice proved to be safe by treatment with compound 3. After 21 days of examination, the compound 3 activity pattern was found to be effective in experimental mice. Compound 3 decreased the excess peak of total triglycerides, total cholesterol, AST, ALT, ALP, LDL, BUN, and creatinine in the STZ-induced diabetic mice. Likewise, the histopathology of the kidneys, liver, and pancreas of the treated animals was also evaluated. Overall, the succinimde moiety, such as compound 3, can affect several targets simultaneously, and, finally, we were successful in synthesizing a multi-targeted preclinical therapy.

Demulsification of amphiphilic gemini ionic liquids and its demulsification mechanism.

This work aims to prepare two new amphiphilic and interfacial active gemini ionic liquids to treat crude oil and investigates its demulsification mechanism. Tetraethylene glycol was pretreated with thionyl chloride and used as a linker to connect succinimide or phthalimide, and then reacted with dodecyl benzene sulphonic acid to obtain the corresponding amphiphilic and interfacial active gemini ionic liquid STA or PTA, respectively. 1H nuclear magnetic resonance spectroscopy (1HNMR) and Fourier-transform infrared spectroscopy (FTIR) was used to determine the chemical structures. The demulsification tests showed the demulsification efficiency with 150 mg/L of STA or PTA at 60 °C for 30 min was 99.89% and 99.79%, respectively. Furthermore, the demulsification mechanism of STA and PTA were studied and the prominent demulsification ability of STA and PTA were attributed to the better interfacial activity and amphipathy which could destroy the asphaltenes interfacial film. These results showed that STA and PTA had excellent demulsification efficiency, which promised application in petroleum industry.

Synthesis, Molecular Docking, and Preclinical Evaluation of a New Succinimide Derivative for Cardioprotective, Hepatoprotective and Lipid-Lowering Effects.

Cardiac and hepatotoxicities are major concerns in the development of new drugs. Better alternatives to other treatments are being sought to protect these vital organs from the toxicities of these pharmaceuticals. In this regard, a preclinical study is designed to investigate the histopathological effects of a new succinimide derivative (Comp-1) on myocardial and liver tissues, and the biochemical effects on selected cardiac biomarkers, hepatic enzymes, and lipid profiles. For this, an initially lethal/toxic dose was determined, followed by a grouping of selected albino rats into five groups (each group had n = 6). The control group received daily oral saline for 8 days. The 5-FU (5-Fluorouracil) group received oral saline daily for 8 days, added with the administration of a single dose of 5-FU (150 mg/kg I.P.) on day 5 of the study. The atenolol group received oral atenolol (20 mg/kg) for 8 days and 5-FU (150 mg/kg I.P.) on day 5 of the protocol. Similarly, two groups of rats treated with test compound (Comp-1) were administered with 5 mg/kg I.P. and 10 mg/kg I.P. for 8 days, followed by 5-FU (150 mg/kg I.P.) on day 5. Toxicity induced by 5-FU was manifested by increases in the serum creatinine kinase myocardial band (CK-MB), troponin I (cTnI) and lactate dehydrogenase (LDH), lipid profile, and selected liver enzymes, including ALP (alkaline phosphatase), ALT (alanine transaminase), AST (aspartate aminotransferase), BT (bilirubin total), and BD (direct bilirubin). These biomarkers were highly significantly decreased after the administration of the mentioned doses of the test compound (5 mg/kg and 10 mg/kg). Similarly, histological examination revealed cardiac and hepatic tissue toxicity by 5-FU. However, those toxic effects were also significantly recovered/improved after the administration of Comp-1 at the said doses. This derivative showed dose-dependent effects and was most effective at a dose of 10 mg/kg body weight. Binding energy data computed via docking simulations revealed that our compound interacts toward the human beta2-adrenergic G protein-coupled receptor (S = -7.89 kcal/mol) with a slight stronger affinity than the calcium channel T-type (S = -7.07 kcal/mol). In conclusion, the histological and biochemical results showed that the test compound (Comp-1) had prominent cardioprotective, hepatoprotective, and lipolytic effects against 5-FU-induced toxicity in the subjected animal model.

Formation of Volatile Heterocyclic Compounds and Open-Chain Amides of Theanine in Model Systems with Glucose, Tea Leaves, and Tea Extract under Tea-Roasting Conditions.

Theanine is a non-proteinogenic amino acid found in the tea plant Camellia sinensis. At an elevated temperature (>90 °C), it released two major volatile compounds 1-ethyl-1,5-dihydro-2H-pyrrol-2-one and N-ethylsuccinimide. Other products were identified, including 10 pyrroles and 12 amides/imides. The formation of the two major compounds was proposed to be initiated by the deamination of theanine and through the intermediate α-keto acid. In the presence of glucose, the two major products and many other volatiles from theanine thermal degradation were accelerated and further Maillard reactions occurred. A total of 56 compounds were identified in the model system of theanine and glucose, including 12 amides/imides, 16 pyrazines, 16 pyrroles and other N-heterocycles, and 12 furans and other O-heterocycles. Although most of the reaction products were detected in tea leaves and in their aqueous extract with or without the addition of theanine under the same experiment conditions, imides and amides were considerably suppressed, left only minute amounts, or were even no longer detectable. Pyrazines and pyrroles were also shown at reduced concentrations as a result of the interaction with tea components but to a lesser extent. A total of 16 and 12 pyrazines were identified in the theanine/glucose reaction system and tea leaves/aqueous extract after roasting, respectively. The results indicated that pyrazines and other main volatiles in roasted tea leaves were formed from the Maillard reactions of the aqueous fraction of tea leaves. Theanine participated in the formation of pyrazines in tea leaves under roasting conditions.

Nose-to-brain delivery of borneol modified tanshinone IIA nanoparticles in prevention of cerebral ischemia/reperfusion injury.

Targeted treatment of cerebral ischemia/reperfusion injury (CIRI) remains a problem due to the difficulty in drug delivery across the blood-brain barrier (BBB). In this study, we developed Bo-TSA-NP, a novel tanshinone IIA (TSA) loaded nanoparticles modified by borneol, which has long been proved with the ability to enhance other drugs' transport across the BBB. The Bo-TSA-NP, with a particle size of about 160 nm, drug loading of 3.6%, showed sustained release and P-glycoprotein (P-gp) inhibition property. It demonstrated a significantly higher uptake by 16HBE cells in vitro through the clathrin/caveolae-mediated endocytosis and micropinocytosis. Following intranasal (IN) administration, Bo-TSA-NP significantly improved the preventive effect on a rat model of CIRI with improved neurological scores, decreased cerebral infarction areas and a reduced content of malondialdehyde (MDA) and increased activity of superoxide dismutase (SOD) in rat brain. In conclusion, these results indicate that Bo-TSA-NP is a promising nose-to-brain delivery system that can enhance the prevention effect of TSA on CIRI.

Tracking the Behavior of Monoclonal Antibody Product Quality Attributes Using a Multi-Attribute Method Workflow.

The multi-attribute method (MAM) is a liquid chromatography-mass spectrometry based method that is used to directly characterize and monitor many product quality attributes and impurities on biotherapeutics, most commonly at the peptide level. It utilizes high-resolution accurate mass spectral data which are analyzed in an automated fashion. MAM is a promising approach that is intended to replace or supplement several conventional assays with a single LC-MS analysis and can be implemented in a Current Good Manufacturing Practice environment. MAM provides accurate site-specific quantitation information on targeted attributes and the nontargeted new peak detection function allows to detect new peaks as impurities, modifications, or sequence variants when comparing to a reference sample. The high resolution MAM workflow was applied here for three independent case studies. First, to monitor the behavior of monoclonal antibody product quality attributes over the course of a 12-day cell culture experiment providing an insight into the behavior and dynamics of product attributes throughout the process. Second, the workflow was applied to test the purity and identity of a product through analysis of samples spiked with host cell proteins. Third, through the comparison of a drug product and a biosimilar with known sequence variants. The three case studies presented here, clearly demonstrate the robustness and accuracy of the MAM workflow that implies suitability for deployment in the regulated environment.

Influence of gelatin and collagen incorporation on peroxidase-mediated injectable pectin-based hydrogel and bioactivity of fibroblasts.

Pectin has recently attracted increasing attention for biomedical and pharmaceutical applications. Due to the lack of adhesion molecules in polysaccharides, phenolic hydroxyl conjugated gelatin was added to enzymatically-gellable peroxidase-modified pectin derivative and compared with phenolic hydroxyl -pectin/collagen. Both pectin and gelatin were modified by tyramine hydrochloride in the presence of EDC/NHS. The phenolic hydroxyl -pectin/phenolic hydroxyl -gelatin, phenolic hydroxyl-pectin/collagen, and phenolic hydroxyl -pectin hydrogels were prepared using horseradish peroxidase and hydrogen peroxide. The hydrogels were characterized by gelation time analysis. Morphology, enzymatic biodegradation, mechanical and swelling properties as well as water vapor transmission rate were also evaluated. Fibroblasts were cultured for 7 days, and the survival rate was evaluated using conventional MTT assay. Hydrogels composed of Ph-pectin/Ph-gelatin showed decreased biodegradation rate, and WVTR and further improved mechanical performance in comparison with other groups. Both phenolic hydroxyl -pectin/collagen and phenolic hydroxyl -pectin/phenolic hydroxyl -gelatin hydrogels exhibited porous structures. The hydrogels composed of collagen promoted cell survival rate 1.4 and 3.5 times compared to phenolic hydroxyl -gelatin and phenolic hydroxyl -pectin based hydrogels at the end of 7 days, respectively (p < 0.001). The study demonstrated the potential of enzymatically-gellable pectin-based hydrogels as cost-effective frameworks for use in tissue engineering applications.

Internal cross-linked polymeric nanoparticles with dual sensitivity for combination therapy of muscle-invasive bladder cancer.

BACKGROUND: Chemotherapy is a standard cancer treatment which uses anti-cancer drugs to destroy or slow the growth of cancer cells. However, chemotherapy has limited therapeutic effects in bladder cancer. One of the reasons of this resistance to chemotherapy is that higher levels of glutathione in invasive bladder cancer cells. We have fabricated nanoparticles that respond to high concentrations of glutathione and near-infrared laser irradiation in order to increase the drug accumulation at the tumor sites and combine chemotherapy with photothermal therapy to overcome the challenges of bladder cancer treatment. METHODS: The DOX&IR780@PEG-PCL-SS NPs were prepared by co-precipitation method. We investigated the tumor targeting capability of NPs in vitro and in vivo. The orthotopic bladder cancer model in C57BL/6 mice was established for in vivo study and the photothermal effects and therapeutic efficacy of NPs were evaluated. RESULTS: The DOX&IR780@PEG-PCL-SS NPs were synthesized using internal cross-linking strategy to increase the stability of nanoparticles. Nanoparticles can be ingested by tumor cells in a short time. The DOX&IR780@PEG-PCL-SS NPs have dual sensitivity to high levels of glutathione in bladder cancer cells and near-infrared laser irradiation. Glutathione triggers chemical structural changes of nanoparticles and preliminarily releases drugs, Near-infrared laser irradiation can promote the complete release of the drugs from the nanoparticles and induce a photothermal effect, leading to destroying the tumor cells. Given the excellent tumor-targeting ability and negligible toxicity to normal tissue, DOX&IR780@PEG-PCL-SS NPs can greatly increase the concentration of the anti-cancer drugs in tumor cells. The mice treated with DOX&IR780@PEG-PCL-SS NPs have a significant reduction in tumor volume. The DOX&IR780@PEG-PCL-SS NPs can be tracked by in vivo imaging system and have good tumor targeting ability, to facilitate our assessment during the experiment. CONCLUSION: A nanoparticle delivery system with dual sensitivity to glutathione and near-infrared laser irradiation was developed for delivering IR780 and DOX. Chemo-photothermal synergistic therapy of both primary bladder cancer and their metastases was achieved using this advanced delivery system.

Characterization of a monoacylglycerol lipase in the medicinal leech, Hirudo verbana.

Endocannabinoids are a class of lipid neuromodulators found throughout the animal kingdom. Among the endocannabinoids, 2-arachydonoyl glycerol (2-AG) is the most prevalent endocannabinoid and monoacylglycerol lipase (MAGL) is a serine hydrolase primarily responsible for metabolizing 2-AG in mammals. In the medicinal leech, Hirudo verbana, 2-AG has been found to be an important and multi-functional modulator of synaptic transmission and behavior. However, very little is known about the molecular components of its synthesis and degradation. In this study we have identified cDNA in Hirudo that encodes a putative MAGL (HirMAGL). The encoded protein exhibits considerable sequence and structural conservation with mammalian forms of MAGL, especially in the catalytic triad that mediates 2-AG metabolism. Additionally, HirMAGL transcripts are detected in the Hirudo central nervous system. When expressed in HEK 293 cells HirMAGL segregates to the plasma membrane as expected. It also exhibits serine hydrolase activity that is blocked when a critical active site residue is mutated. HirMAGL also demonstrates the capacity to metabolize 2-AG and this capacity is also prevented when the active site is mutated. Finally, HirMAGL activity is inhibited by JZL184 and MJN110, specific inhibitors of mammalian MAGL. To our knowledge these findings represent the first characterization of an invertebrate form of MAGL and show that HirMAGL exhibits many of the same properties as mammalian MAGL's that are responsible for 2-AG metabolism.

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.

On the use of acridinium indicators for the chemiluminescent determination of the total antioxidant capacity of dietary supplements.

Acridinium salts, due to their chemiluminogenic properties, have found several applications in biomedical analysis as labels and indicators, where the assessment of emission intensity is used for the end-point detection. This work presents the use of chemiluminescent indicators in the form of selected acridinium esters in order to determine the antioxidant properties of exemplary formulations, namely quercetin, vitamin C and the dietary supplement, Apiextract. The principle of measurements is based on a change in the kinetics of emission decay derived from the acridinium cations in alkaline solutions of hydrogen peroxide in the presence of an antioxidant (the analyte). The proposed system makes a beneficial alternative to related methods, which mostly rely on the assessment of emission efficiency and use the luminometric standard luminol - due to superior parameters of acridinium chemiluminescence, among others - high temporary emission efficiency. The features of the proposed method are manifested by a shorter time period of analysis and lower background signals associated with the environmental influences, as compared to typical approaches. The chromatographic (RP-HPLC) analyses of the substrates and products generated during chemiluminogenic oxidation of acridinium cations under assay conditions are also presented.

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.

Inhibition of monoacylglycerol lipase, an anti-inflammatory and antifibrogenic strategy in the liver.

OBJECTIVE: Sustained inflammation originating from macrophages is a driving force of fibrosis progression and resolution. Monoacylglycerol lipase (MAGL) is the rate-limiting enzyme in the degradation of monoacylglycerols. It is a proinflammatory enzyme that metabolises 2-arachidonoylglycerol, an endocannabinoid receptor ligand, into arachidonic acid. Here, we investigated the impact of MAGL on inflammation and fibrosis during chronic liver injury. DESIGN: C57BL/6J mice and mice with global invalidation of MAGL (MAGL -/- ), or myeloid-specific deletion of either MAGL (MAGLMye-/-), ATG5 (ATGMye-/-) or CB2 (CB2Mye-/-), were used. Fibrosis was induced by repeated carbon tetrachloride (CCl4) injections or bile duct ligation (BDL). Studies were performed on peritoneal or bone marrow-derived macrophages and Kupffer cells. RESULTS: MAGL -/- or MAGLMye-/- mice exposed to CCl4 or subjected to BDL were more resistant to inflammation and fibrosis than wild-type counterparts. Therapeutic intervention with MJN110, an MAGL inhibitor, reduced hepatic macrophage number and inflammatory gene expression and slowed down fibrosis progression. MAGL inhibitors also accelerated fibrosis regression and increased Ly-6Clow macrophage number. Antifibrogenic effects exclusively relied on MAGL inhibition in macrophages, since MJN110 treatment of MAGLMye-/- BDL mice did not further decrease liver fibrosis. Cultured macrophages exposed to MJN110 or from MAGLMye-/- mice displayed reduced cytokine secretion. These effects were independent of the cannabinoid receptor 2, as they were preserved in CB2Mye-/- mice. They relied on macrophage autophagy, since anti-inflammatory and antifibrogenic effects of MJN110 were lost in ATG5Mye-/- BDL mice, and were associated with increased autophagic flux and autophagosome biosynthesis in macrophages when MAGL was pharmacologically or genetically inhibited. CONCLUSION: MAGL is an immunometabolic target in the liver. MAGL inhibitors may show promising antifibrogenic effects during chronic liver injury.

Salmonella Typhimurium Sensing Strategy Based on the Loop-Mediated Isothermal Amplification Using Retroreflective Janus Particle as a Nonspectroscopic Signaling Probe.

Loop-mediated isothermal amplification (LAMP) is a powerful gene amplification method, which has many advantages, including high specificity, sensitivity, and simple operation. However, quantitative analysis of the amplified target gene with the LAMP assay is very difficult. To overcome this limitation, we developed a novel biosensing platform for molecular diagnosis by integrating the LAMP method and retroreflective Janus particle (RJP) together. The final amplified products of the LAMP assay are dumbbell-shaped DNA structures, containing a single-stranded loop with two different sequences. Therefore, the concentration of the amplified products can be measured in a manner similar to the sandwich-type immunoassay. To carry out the sandwich-type molecular diagnostics using the LAMP product, two DNA probes, with complementary sequences to the loop-regions, were prepared and immobilized on both the sensing surface and the surface of the RJPs. When the amplified LAMP product was applied to the sensing surface, the surface-immobilized DNA probe hybridized to the loop-region of the LAMP product to form a double-stranded structure. When the DNA probe-conjugated RJPs were injected, the RJPs bound to the unreacted loop-region of the LAMP product. The number of RJPs bound to the loop-region of the LAMP product was proportional to the concentration of the amplified LAMP product, indicating that the concentration of the target gene can be quantitatively analyzed by counting the number of observed RJPs. Using the developed system, a highly sensitive and selective quantification of Salmonella was successfully performed with a detection limit of 102 CFU.

XPlex: An Effective, Multiplex Cross-Linking Chemistry for Acidic Residues.

Cross-linking/Mass spectrometry (XLMS) is a consolidated technique for structural characterization of proteins and protein complexes. Despite its success, the cross-linking chemistry currently used is mostly based on N-hydroxysuccinimide (NHS) esters, which react primarily with lysine residues. One way to expand the current applicability of XLMS into several new areas is to increase the number of cross-links obtainable for a target protein. We introduce a multiplex chemistry (denoted XPlex) that targets Asp, Glu, Lys, and Ser residues. XPlex can generate significantly more cross-links with reactions occurring at lower temperatures and enables targeting proteins that are not possible with NHS ester-based cross-linkers. We demonstrate the effectiveness of our approach in model proteins as well as a target Lys-poor protein, SalBIII. Identification of XPlex spectra requires a search engine capable of simultaneously considering multiple cross-linkers on the same run; to achieve this, we updated the SIM-XL search algorithm with a search mode tailored toward XPlex. In summary, we present a complete chemistry/computational solution for significantly increasing the number of possible distance constraints by mass spectrometry experiments, and thus, we are convinced that XPlex poses as a real complementary approach for structural proteomics studies.

Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic ß-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity.

Inositol pyrophosphates have emerged as important regulators of many critical cellular processes from vesicle trafficking and cytoskeletal rearrangement to telomere length regulation and apoptosis. We have previously demonstrated that 5-di-phosphoinositol pentakisphosphate, IP7, is at a high level in pancreatic ß-cells and is important for insulin exocytosis. To better understand IP7 regulation in ß-cells, we used an insulin secreting cell line, HIT-T15, to screen a number of different pharmacological inhibitors of inositide metabolism for their impact on cellular IP7. Although the inhibitors have diverse targets, they all perturbed IP7 levels. This made us suspicious that indirect, off-target effects of the inhibitors could be involved. It is known that IP7 levels are decreased by metabolic poisons. The fact that the inositol hexakisphosphate kinases (IP6Ks) have a high Km for ATP makes IP7 synthesis potentially vulnerable to ATP depletion. Furthermore, many kinase inhibitors are targeted to the ATP binding site of kinases, but given the similarity of such sites, high specificity is difficult to achieve. Here, we show that IP7 concentrations in HIT-T15 cells were reduced by inhibitors of PI3K (wortmannin, LY294002), PI4K (Phenylarsine Oxide, PAO), PLC (U73122) and the insulin receptor (HNMPA). Each of these inhibitors also decreased the ATP/ADP ratio. Thus reagents that compromise energy metabolism reduce IP7 indirectly. Additionally, PAO, U73122 and LY294002 also directly inhibited the activity of purified IP6K. These data are of particular concern for those studying signal transduction in pancreatic ß-cells, but also highlight the fact that employment of these inhibitors could have erroneously suggested the involvement of key signal transduction pathways in various cellular processes. Conversely, IP7's role in cellular signal transduction is likely to have been underestimated.

Preparation of lipid nanoparticles with high loading capacity and exceptional gastrointestinal stability for potential oral delivery applications.

Exploitation of lipid nanoparticles for oral delivery of nutrients and drugs is limited by their poor stability under gastrointestinal tract and low loading capacity, unless a high concentration of synthetic surfactants is formulated. The main objective of present study is to design a series of new formulations for solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) that are suitable for potential oral delivery applications, using natural biopolymers, i.e. sodium caseinate (NaCas) as emulsifier and pectin as coating, with minimal addition of a synthetic surfactant, Tween 80. Effects of pectin coating, concentration of Tween 80, thermal treatment (80°C for 30min), as well as two chemical cross-linkers on the particulate characteristics, stability, encapsulation efficiency, controlled release and drying feasibility were comprehensively investigated. The intermolecular interactions and cross-linking reactions were studied using Fourier transform infrared spectroscopy. Tween 80 at 0.15% (w/v) together with 0.15% (w/v) NaCas was proved effective to obtain stable cross-linked pectin-coated SLN (PSLN) under 200nm with high loading capacity for curcumin, while NLC prepared under the same condition failed to pass storage stability test. The 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) cross-linked PSLN exhibited superior characteristics than glutaradehyde (GA) cross-linked PSLN, especially for the stability and controlled release under simulated gastrointestinal conditions, with curcumin studied as a model compound. The feasibility of both nano spray drying and freeze-drying technologies were both investigated to transform of colloidal lipid nanoparticles into dry powders. Our results demonstrated a novel strategy to prepare small and homogenous SLN with exceptional GI stability and high loading capacity as a potential oral delivery system.

Effect of Bioactive Primers on Bacterial-Induced Secondary Caries at the Tooth-Resin Interface.

Secondary caries at the tooth-resin interface is the primary reason for replacement of resin composite restorations. The tooth-resin interface is formed by the interlocking of resin material with hydroxyapatite crystals in enamel and collagen mesh structure in dentin. Efforts to strengthen the tooth-resin interface have identified chemical agents with dentin collagen cross-linking potential and antimicrobial activities. The purpose of the present study was to assess protective effects of bioactive primer against secondary caries development around enamel and dentin margins of class V restorations, using an in vitro bacterial caries model. Class V composite restorations were prepared on 60 bovine teeth (n=15) with pretreatment of the cavity walls with control buffer solution, an enriched fraction of grape seed extract (e-GSE), 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide/N-hydroxysuccinimide, or chlorhexidine digluconate. After incubating specimens in a bacterial model with Streptococcus mutans for four days, dentin and enamel were assessed by fluorescence microscopy. Results revealed that only the naturally occurring product, e-GSE, significantly inhibited the development of secondary caries immediately adjacent to the dentin-resin interface, as indicated by the caries inhibition zone. No inhibitory effects were observed in enamel margins. The results suggest that the incorporation of e-GSE into components of the adhesive system may inhibit secondary caries and potentially contribute to the protection of highly vulnerable dentin-resin margins.

Synthesis of ß-1,4-Linked Galactan Side-Chains of Rhamnogalacturonan†I.

The synthesis of linear- and (1→6)-branched ß-(1→4)-d-galactans, side-chains of the pectic polysaccharide rhamnogalacturonan I is described. The strategy relies on iterative couplings of n-pentenyl disaccharides followed by a late stage glycosylation of a common hexasaccharide core. Reaction with a covalent linker and immobilization on N-hydroxysuccinimide (NHS)-modified glass surfaces allows the generation of carbohydrate microarrays. The glycan arrays enable the study of protein-carbohydrate interactions in a high-throughput fashion, demonstrated herein with binding studies of mAbs and a CBM.

The Selective Monoacylglycerol Lipase Inhibitor MJN110 Produces Opioid-Sparing Effects in a Mouse Neuropathic Pain Model.

Serious clinical liabilities associated with the prescription of opiates for pain control include constipation, respiratory depression, pruritus, tolerance, abuse, and addiction. A recognized strategy to circumvent these side effects is to combine opioids with other antinociceptive agents. The combination of opiates with the primary active constituent of cannabis (Δ(9)-tetrahydrocannabinol) produces enhanced antinociceptive actions, suggesting that cannabinoid receptor agonists can be opioid sparing. Here, we tested whether elevating the endogenous cannabinoid 2-arachidonoylglycerol through the inhibition of its primary hydrolytic enzyme monoacylglycerol lipase (MAGL), will produce opioid-sparing effects in the mouse chronic constriction injury (CCI) of the sciatic nerve model of neuropathic pain. The dose-response relationships of i.p. administration of morphine and the selective MAGL inhibitor 2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate (MJN110) were tested alone and in combination at equieffective doses for reversal of CCI-induced mechanical allodynia and thermal hyperalgesia. The respective ED50 doses (95% confidence interval) of morphine and MJN110 were 2.4 (1.9-3.0) mg/kg and 0.43 (0.23-0.79) mg/kg. Isobolographic analysis of these drugs in combination revealed synergistic antiallodynic effects. Acute antinociceptive effects of the combination of morphine and MJN110 required µ-opioid, CB1, and CB2 receptors. This combination did not reduce gastric motility or produce subjective cannabimimetic effects in the drug discrimination assay. Importantly, combinations of MJN110 and morphine given repeatedly (i.e., twice a day for 6 days) continued to produce antiallodynic effects with no evidence of tolerance. Taken together, these findings suggest that MAGL inhibition produces opiate-sparing events with diminished tolerance, constipation, and cannabimimetic side effects.