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Melittin peptide kills Trypanosoma cruzi parasites by inducing different cell death pathways.

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Antimicrobial peptides (AMPs) are components of the innate immune response that represent desirable alternatives to conventional pharmaceuticals, as they have a fast mode of action, a low likelihood of resistance development and can act in conjunction with existing drug regimens. AMPs exhibit strong inhibitory activity against both Gram-positive and Gram-negative bacteria, fungi, viruses, metazoans and other parasites, such as the protozoan Leishmania. Melittin is a naturally occurring AMP, which comprises 40-50% of the dry weight of Apis mellifera venom. Our group has recently shown that crude A. mellifera venom is lethal to Trypanosoma cruzi, the Chagas disease etiologic agent, and generates a variety of cell death phenotypes among treated parasites. Here, we demonstrate that the melittin affected all of T. cruzi developmental forms, including the intracellular amastigotes. The ultrastructural changes induced by melittin suggested the occurrence of different programmed cell death pathways, as was observed in A. mellifera-treated parasites. Autophagic cell death appeared to be the main death mechanism in epimastigotes. In contrast, melittin-treated trypomastigotes appeared to be dying via an apoptotic mechanism. Our findings confirm the great potential of AMPs, including melittin, as a potential source of new drugs for the treatment of neglected diseases, such as Chagas disease.

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TRP and ASIC channels mediate the antinociceptive effect of citronellyl acetate.

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Background Citronellyl acetate (CAT), a monoterpene product of the secondary metabolism of plants, has been shown in the literature to possess several different biological activities. However, no antinociceptive abilities have yet been discussed. Here, we used acute pain animal models to describe the antinociceptive action of CAT. Methods The acetic acid-induced writhing test and the paw-licking test, in which paw licking was induced by glutamate and formalin, were performed to evaluate the antinociceptive action of CAT and to determine the involvement of PKC, PKA, TRPV1, TRPA1, TRPM8 and ASIC in its antinociceptive mechanism. To do so, we induced paw-linking using agonists. Results CAT was administered intragastrically (25, 50, 75, 100 and 200mg/kg), and the two higher doses caused antinociceptive effects in the acetic acid model; the highest dose reduced pain for 4h after it was administered (200mg/kg). In the formalin test, two doses of CAT promoted antinociception in both the early and later phases of the test. The glutamate test showed that its receptors are involved in the antinociceptive mechanism of CAT. Pretreatment with CAT did not alter locomotor activity or motor coordination. In an investigation into the participation of TRP channels and ASICs in CAT's antinociceptive mechanism, we used capsaicin (2.2μg/paw), cinnamaldehyde (10mmol/paw), menthol (1.2mmol/paw) and acidified saline (2% acetic acid, pH 1.98). The results showed that TRPV1, TRPM8 and ASIC, but not TRPA1, are involved in the antinociceptive mechanism. Finally, the involvement of PKC and PKA was also studied, and we showed that both play a role in the antinociceptive mechanism of CAT. Conclusion The results of this work contribute information regarding the antinociceptive properties of CAT on acute pain and show that, at least in part, TRPV1, TRPM8, ASIC, glutamate receptors, PKC and PKA participate in CAT's antinociceptive mechanism.

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Poly(N-isopropylacrylamide-co-hydroxyethylacrylamide) thermosensitive microspheres: The size of microgels dictates the pulsatile release mechanism.

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Poly(N-isopropylacrylamide-co-N-hydroxyethylacrylamide) (poly(NIPAAm-co-HEAAm)) was prepared as a new thermosensitive copolymer possessing a sharp phase transition around the human body temperature. The effect of the copolymer concentration on the lower critical solution temperature (LCST) was determined under physiological conditions by cloud point (CP) and differential scanning calorimetric (DSC) methods. Then, thermosensitive microspheres were prepared from preformed copolymers by chemical cross-linking of hydroxyl groups with glutaraldehyde at a temperature situated slightly below LCST of the copolymer solution. The volume phase transition temperature (VPTT) of corresponding cross-linked microspheres was determined from swelling degree-temperature curve. The microspheres were loaded with model drug indomethacin by the solvent evaporation method. The DSC analysis proved that the drug is molecularly dispersed in the polymer network. Finally, the influence of the microsphere size on drug release was investigated. It was established that microspheres with the diameter ranging between 5 and 60μm release the drug with almost the same rate below (in the swollen state) and above the VPTT (in the collapsed state). On the contrary, microspheres with the diameter ranging between 125 and 220μm release a significantly higher amount of indomethacin below than above the VPTT. This different behavior is enough to assure a pulsatile release mechanism when the temperature changes cyclically below and above the VPTT. However, both small and large microspheres release a large amount of the drug during the collapsing process.

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Carboxymethyl starch and lecithin complex as matrix for targeted drug delivery: I. Monolithic Mesalamine forms for colon delivery.

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For drugs expected to act locally in the colon, and for successful treatment, a delivery device is necessary, in order to limit the systemic absorption which decreases effectiveness and causes important side effects. Various delayed release systems are currently commercialized; most of them based on pH-dependent release which is sensitive to gastrointestinal pH variation. This study proposes a novel excipient for colon delivery. This new preparation consists in the complexation between carboxymethyl starch (CMS) and Lecithin (L). As opposed to existing excipients, the new complex is pH-independent, inexpensive, and easy to manufacture and allows a high drug loading. FTIR, X-ray, and SEM structural analysis all support the hypothesis of the formation of a complex. By minor variation of the excipient content within the tablet, it is possible to modulate the release time and delivery at specific sites of the gastrointestinal tract. This study opens the door to a new pH-independent delivery system for mesalamine targeted administration. Our novel formulation fits well with the posology of mesalamine, used in the treatment of Inflammatory Bowel Disease (IBD), which requires repeated administrations (1g orally four times a day) to maintain a good quality of life.

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Crystallization of poly(ethylene oxide) with acetaminophen - A study on solubility, spherulitic growth, and morphology.

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A simple, sensitive, efficient, and novel method analyzing the number of spherulitic nuclei was proposed to estimate the solubility of a model drug acetaminophen (APAP) in poly(ethylene oxide) (PEO). At high crystallization temperature (323K), 10% APAP-PEO had the same low number of spherulitic nuclei as pure PEO, indicating that APAP and PEO were fully miscible. At low crystallization temperature (303K), the number of nuclei for 10% APAP-PEO was significantly higher, suggesting that APAP was oversaturated and therefore recrystallized and acted as a nucleating agent. Based on the results obtained, the solubility of APAP in PEO is possibly between the concentration of 0.1% and 1% at 303K. The spherulitic growth rate G of PEO was found to decrease with increasing APAP concentration, suggesting that APAP is most likely functioning as a chemical defect and is either rejected from or included in the PEO crystals during chain folding. APAP could possibly locate in the inter-spherulitic, inter-fibrillar, inter-lamellar, or intra-lamellar regions of PEO. At 323K, the morphology of 10% APAP-PEO is more dendritic than spherulitic with large unfilled space in between dendrites and spherulites, which is a sign of one or the combination of the four modes of segregation. An extensive spherulitic nucleation and growth kinetics study using the classical theoretical relationships, for example, the Hoffman-Lauritzen (HL) and Avrami theories, was conducted. Both microscopic and differential scanning calorimetric (DSC) analysis yielded similar values for the nucleation constant Kg as well as the fold surface free energy σe and work of chain folding q. The values of σe and q increased with APAP concentration, indicating that the chain folding of PEO was hindered by APAP.

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Hyaluronic acid / chitosan multilayer coatings on neuronal implants for localized delivery of siRNA nanoplexes.

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Binding, stabilizing and promoting cellular uptake of siRNA are all critical efforts in creating matrices for the localized delivery of siRNA molecules to target cells. In this study, we describe the generation of chitosan imidazole/siRNA nanoplexes (NPs) embedded in nano scope polyelectrolyte multilayers (PEMs) composed of hyaluronic acid and chitosan for sustained and localized drug delivery. Regular PEM build-up, successful integration of NPs and controlled release under physiological conditions were shown. Biological efficacy was evaluated in neuronal cell culture concerning cell adhesion, viability, NPs uptake and gene silencing. The additionally shown biological functionalization of neuronal implants possesses potential for future applications in the field of regenerative medicine and treatment of spinal cord injuries.

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Medicinal plants of genus Curculigo: Traditional uses and a phytochemical and ethnopharmacological review.

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ETHNOPHARMACOLOGICAL RELEVANCE: In the genus Curculigo, Curculigo orchioides Gaertn, Curculigo capitulata (Lour) O. Ktze and Curculigo pilosa (Schumach. & Thonn.) Engl are often used in traditional medicine. Curculigo orchioides is used for the treatment of impotence, limb limpness, arthritis of the lumbar and knee joints, and watery diarrhea in traditional Chinese medicine, and also used as a potent immunomodulator and aphrodisiac in the Ayurvedic medical system. Curculigo capitulata is used for the treatment of consumptive cough, kidney asthenia, impotence and spermatorrhea, hemorrhoids, asthma, jaundice, diarrhea, colic and gonorrhea in traditional Chinese and India medicine, and to treat urinary tract infection, acute renal pelvis and nephritis, nephritis-edema, cystitis, nephrolithiasis, hypertension and rheumatic arthritis in traditional Dai medicine. Curculigo pilosa are applied to treat gastrointestinal and heart diseases in Africa. AIM OF THE REVIEW: This review aims to exhibit up-to-date and comprehensive information about traditional uses, phytochemistry, pharmacology and toxicology of medicinal plants in the genus Curculigo, and has an insight into the opportunities for the future research and development of Curculigo plant. METHODS: A bibliographic investigation was performed by analyzing the information available on Curculigo plant from worldwide accepted scientific databases (Pubmed, Scopus and Web of Science, SciFinder, Google Scholar, Yahoo). Furthermore, information also was obtained from some local and foreign books on ethnobotany and ethnomedicines. RESULTS: Curculigo orchioides, Curculigo capitulata and Curculigo pilosa have been used as traditional medicine to treat kinds of diseases such as impotence, limb limpness, gastrointestinal and heart diseases, etc. Phytochemical investigation of eight species of the genus Curculigo has resulted in identification of more than 110 compounds. The content of curculigoside is used as an indicator to evaluate the quality of rhizome of Curculigo orchioides. The medicinal plants have showed a wide spectrum pharmacological activities, including adaptive, immunostimulatory, taste-modifying and sweet-tasting, antioxidant, mast cell stabilization, antihistaminic and antiasthmatic, hepatoprotective and neuroprotective activity. Toxicological test indicated that Curculigo orchioides at the dose of 120g/kg after administrating rats for 180 days may cause injury of liver and kidney. CONCLUSION: The medicinal plants of genus Curculigo have emerged as a good source of the traditional medicines. Some uses of these plants in the traditional medicines have been validated by pharmacological investigation. However, the mechanism of their actions should be further elucidated; the particular constituent responsible for toxicity should be isolated and identified, and the target tissue and mechanism of toxic ingredients also deserve to be further investigated; more reference substances should be prepared, and sophisticated analytical technologies should be developed to comprehensively assess the quality of Curculigo herbs. These investigations will be helpful for further utilization of the plants of genus Curculigo.

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Anti-inflammatory activity of hexane extracts from bones and internal organs of Anguilla japonica suppresses cyclooxygenase-2-dependent prostaglandin D2 generation in mast cells and anaphylaxis in mice.

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The purpose of this study is to investigate the effects of n-hexane extracts from bones and internal organs of Japanese eel, Anguilla japonica (HEE), on cyclooxygenase-2 (COX-2)-dependent prostaglandin D2 (PGD2) generation in stem cell factor (SCF), IL-10, plus LPS-induced mouse bone marrow-derived mast cells (BMMCs) and on passive cutaneous anaphylaxis (PCA) in mice. HEE suppressed SCF/IL-10/LPS-induced PGD2 generation, and concomitantly reduced COX-2 protein expression dose-dependently. To understand the mechanistic basis for the inhibition of PGD2 generation by HEE, we examined the effects of HEE on upstream signaling pathways essential for COX-2 induction. HEE was found to inhibit the translocation of nuclear factor-κB (NF-κB) p65 subunit to the nucleus and its DNA-binding ability through the inhibition of TAK1, IKK and IκB phosphorylation. Furthermore, HEE also attenuated mitogen-activated protein kinase (MAPK)-mediated regulation of DNA binding of activator protein-1 (AP-1). Moreover, oral administration of HEE inhibited anti-dinitrophenyl (DNP) IgE-induced PCA in a dose dependent manner. Taken together, the present study provides new insights into the anti-inflammatory activity of HEE, which could be a promising candidate to be used for an inflammatory therapy.

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Vinblastine-induced apoptosis of melanoma cells is mediated by Ras homologous A protein (Rho A) via mitochondrial and non-mitochondrial-dependent mechanisms.

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Gene expression profiling of three different stressors in the water flea Daphnia magna.

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Microarrays are an ideal tool to screen for differences in gene expression of thousands of genes simultaneously. However, often commercial arrays are not available. In this study, we performed microarray analyses to evaluate patterns of gene transcription following exposure to two natural and one anthropogenic stressor. cDNA microarrays compiled of three life stage specific and three stressor-specific EST libraries, yielding 1734 different EST sequences, were used. We exposed juveniles of the water flea Daphnia magna for 48, 96 and 144 h to three stressors known to exert strong selection in natural populations of this species i.e. a sublethal concentration of the pesticide carbaryl, infective spores of the endoparasite Pasteuria ramosa, and fish predation risk mimicked by exposure to fish kairomones. A total of 148 gene fragments were differentially expressed compared to the control. Based on a PCA, the exposure treatments were separated into two main groups based on the extent of the transcriptional response: a low and a high (144 h of fish or carbaryl exposure and 96 h of parasite exposure) stress group. Firstly, we observed a general stress-related transcriptional expression profile independent of the treatment characterized by repression of transcripts involved in transcription, translation, signal transduction and energy metabolism. Secondly, we observed treatment-specific responses including signs of migration to deeper water layers in response to fish predation, structural challenge of the cuticle in response to carbaryl exposure, and disturbance of the ATP production in parasite exposure. A third important conclusion is that transcription expression patterns exhibit stress-specific changes over time. Parasite exposure shows the most differentially expressed gene fragments after 96 h. The peak of differentially expressed transcripts came only after 144 h of fish exposure, while carbaryl exposure induced a more stable number of differently expressed gene fragments over time.

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Structure-Property Investigations of Substituted Triarylamines and Their Applications as Fluorescent pH Sensors.

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Fourteen triphenylamine derivatives functionalized with fluorophenyl, methoxyphenyl, and pyridinyl groups as respective donors and acceptors were synthesized and characterized. Their photophysical properties were systematically investigated in various solvents with different polarities. The solvent-dependent Stokes shifts of these compounds were observed and analyzed by the Lippert-Mataga equation. The synthesized compounds, especially tris(4-(pyridin-4-yl)phenyl) amine, presented pH-dependent absorptions and emissions, indicating that these compounds might be used as pH sensors.

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Twisted Signatures of GC-Biased Gene Conversion Embedded in an Evolutionary Stable Karyotype.

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The genomes of many vertebrates show a characteristic heterogeneous distribution of GC content, the so-called GC isochore structure. The origin of isochores has been explained via the mechanism of GC-biased gene conversion (gBGC). However, although the isochore structure is declining in many mammalian genomes, the heterogeneity in GC content is being reinforced in the avian genome. Despite this discrepancy, which remains unexplained, examinations of individual substitution frequencies in mammals and birds are both consistent with the gBGC model of isochore evolution. On the other hand, a negative correlation between substitution and recombination rate found in the chicken genome is inconsistent with the gBGC model. It should therefore be important to consider along with gBGC other consequences of recombination on the origin and fate of mutations, as well as to account for relationships between recombination rate and other genomic features. We therefore developed an analytical model to describe the substitution patterns found in the chicken genome, and further investigated the relationships between substitution patterns and several genomic features in a rigorous statistical framework. Our analysis indicates that GC content itself, either directly or indirectly via interrelations to other genomic features, has an impact on the substitution pattern. Further, we suggest that this phenomenon is particularly visible in avian genomes due to their unusually low rate of chromosomal evolution. Because of this, interrelations between GC content and other genomic features are being reinforced, and are as such more pronounced in avian genomes as compared with other vertebrate genomes with a less stable karyotype.

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Combinatorial Treatment of Tart Cherry Extract and Essential Fatty Acids Reduces Cognitive Impairments and Inflammation in the mu-p75 Saporin-Induced Mouse Model of Alzheimer's Disease.

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Abstract Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects more than five million Americans and is characterized by a progressive loss of memory, loss of cholinergic neurons in the basal forebrain, formation of amyloid plaques and neurofibrillary tangles, and an increase in oxidative stress. Recent studies indicate that dietary supplements of antioxidants and omega-3 and omega-6 fatty acids may reduce the cognitive deficits in AD patients. The current study tested a combinatorial treatment of antioxidants from tart cherry extract and essential fatty acids from Nordic fish and emu oils for reducing cognitive deficits in the mu-p75 saporin (SAP)-induced mouse model of AD. Mice were given daily gavage treatments of Cerise(®) Total-Body-Rhythm™ (TBR; containing tart cherry extract, Nordic fish oil, and refined emu oil) or vehicle (methylcellulose) for 2 weeks before intracerebroventricular injections of the cholinergic toxin, mu-p75 SAP, or phosphate-buffered saline. The TBR treatments continued for an additional 17 days, when the mice were tested on a battery of cognitive and motor tasks. Results indicate that TBR decreased the SAP-induced cognitive deficits assessed by the object-recognition, place-recognition, and Morris-water-maze tasks. Histological examination of the brain tissue indicated that TBR protected against SAP-induced inflammatory response and loss of cholinergic neurons in the area around the medial septum. These findings indicate that TBR has the potential to serve as an adjunctive treatment which may help reduce the severity of cognitive deficits in disorders involving cholinergic deficits, such as AD.

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Zhizhu decoction promotes gastric emptying and protects the gastric mucosa.

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Abstract The aim of this study was to evaluate the effects of the Zhizhu decoction on gastric emptying and gastric mucosal protection. The Zhizhu decoction is composed of Aurantii fructus and Atractylodes macrocephala Rhizoma. Results showed that oral administration of the Zhizhu decoction accelerated gastric emptying in mouse and protected gastric mucosa from ethanol-induced ulcers in rat. Our investigations demonstrated that the Zhizhu decoction accelerated gastric emptying, at least in part, by activating the muscarinic and 5-HT3 receptors. The gastroprotective effect is involved in its antioxidant effects and increased vascular endothelial growth factor expression.

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The immunomodulation effect of aronia extract lacks association with its antioxidant anthocyanins.

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Abstract Polyphenols comprise a diverse group of molecules with antioxidative and anti-inflammatory activities. To compare the antioxidative and anti-inflammatory capacity of Aronia melanocarpa berries (chokeberries), recognized for their high content of anthocyanins, a noncytotoxic isolation method was developed to obtain high-purity anthocyanins in the extract. The antioxidative activity of the extract, the anthocyanin-rich fraction (AF) was determined by 1,1-diphenyl-2-picrylhydrazyl radical and ferric-reducing ability of plasma along with resveratrol as a reference. The immunomodulation properties were assessed in lipopolysaccharide (LPS)-stimulated human monocytes mono mac 6. The isolated AF, containing six different anthocyanins, exhibited a stronger antioxidative capacity compared to resveratrol. Resveratrol enhanced tumor necrosis factor-α and reduced interleukin-10 (IL-10) production by LPS, whereas AF only had a slight effect in reducing IL-10. These results demonstrated that there was no major relationship between the antioxidative effect and immunomodulation capacities of AF and resveratrol. The immunomodulatory activity of the extract is associated with bioactive compounds in Aronia other than its anthocyanins.

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Novel sulfonamide compounds for inhibition of metastatic tumor growth (WO2012021963).

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A series of novel ureido-sulfonamides was prepared by reaction of aminobenzenesulfonamide with alkyl/aryl isocyanate. These compounds are claimed for use as therapeutic agents of metastatic tumors, which are poorly responsive to classical chemotherapies and constitute a conceptually novel approach for cancer treatment.

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High glucose, insulin and free fatty acid concentrations synergistically enhance perilipin 3 expression and lipid accumulation in macrophages.

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OBJECTIVE: Perilipin (PLIN) 3, an intracellular lipid droplet (LD)-associated protein, is implicated in foam cell formation. Since metabolic derangements found in metabolic syndrome, such as high serum levels of glucose, insulin and free fatty acids (FFAs), are major risk factors promoting atherosclerosis, we investigated whether PLIN3 expression is affected by glucose, insulin and oleic acid (OA) using RAW264.7 cells. METHODS: Real-time PCR and Western blotting were performed to detect PLIN3 or PLIN2 expression. Oil-red O staining and Lipid Analysis were employed to measure cellular content of triacylglycerides (TAG) and cholesterol. RESULTS: PLIN3 mRNA was stimulated by high glucose or insulin concentrations individually, but not by OA. A combination of any two factors did not enhance PLIN3 expression any more than that evoked by glucose alone at 24h. Interestingly, however, simultaneous addition of all three factors synergistically enhanced the PLIN3 expression. This synergistic effect was not apparent for PLIN2 mRNA expression. Inhibitors of Src family tyrosine kinase and/or phosphatidylinositol 3-kinase, both of which are activated by insulin and FFA signaling, partially suppressed PLIN3 expression induced by the combination of the three factors. While simultaneous addition of glucose, insulin and OA remarkably increased the cellular content of TAG and cholesterol, knocking-down of PLIN3 predominantly reduced TAG content. CONCLUSIONS: These results indicate that PLIN3 expression is synergistically stimulated by high glucose, insulin and FFA concentrations, in parallel with TAG accumulation in macrophages. This finding raises new evidence of PLIN3 involvement in conversion of macrophages into foam cells.

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(1)H NMR-based metabonomic analysis of the serum and urine of rats following subchronic exposure to dichlorvos, deltamethrin, or a combination of these two pesticides.

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Identification of active compounds from Caesalpinia sappan L. extracts suppressing IL-6 production in RAW 264.7 cells by PLS.

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ETHNOPHARMACOLOGICAL RELEVANCE: Caesalpinia sappan L. is distributed in Southeast Asia and also used as herbal medicine for the treatment of various diseases such as burning sensations, leprosy, dysentery, osteoarthritis and rheumatoid arthritis (RA). The overproduction of IL-6 plays an important role in the prognosis of RA, but the active compounds from the extracts of Caesalpinia sappan L. suppressing IL-6 production remain unknown. AIMS OF THE STUDY: Identifying the main active compounds of Caesalpinia sappan L. extracts inhibiting the IL-6 production in LPS-stimulated RAW 264.7 cells by partial least squares (PLS). MATERIALS AND METHODS: Sixty-four samples with different proportions of compounds were prepared from Caesalpinia sappan L. by supercritical CO2 fluid extraction (SCFE) and refluxing. Each of 64 samples was applied to RAW 264.7 cells with LPS to evaluate whether IL-6 production by LPS is affected by addition of each sample. The IL-6 production in medium was determined by ELISA and the inhibitory activity of each sample was analyzed. In addition, the fingerprints of these 64 samples were also established by ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-MS). We used the PLS, a simplified method, to evaluate the results from IL-6 production and fingerprints. RESULTS: Each of 64 samples markedly suppressed LPS-induced IL-6 production in RAW cells. The fingerprints by UPLC-MS clearly revealed variations among 64 samples produced in different extract conditions. The PLS analysis with IL-6 production and fingerprints by UPLC-MS suggested that the peaks 71, 93, 150, 157, 168 have more influence on the inhibitory activity of Caesalpinia sappan L. extracts. The peaks 71, 93, 150 are likely representing sappanone A, protosappanin E and neoprotosappanin, respectively. The peaks 157 and 168 are still at large. CONCLUSION: This is the first report that sappanone A, protosappanin E, neoprotosappanin and two unidentified compounds can be considered as possible active compounds that might inhibit IL-6 production. Further studies are needed to confirm the effectiveness of these five compounds on IL-6 production and possible mechanism.

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Commonly used metal and crystalline salts in South African traditional medicine.

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ETHNOPHARMACOLOGICAL RELEVANCE: Traditional medicines in the form of plants, animals and/or minerals are used by millions of South Africans. There is currently no data regarding the commonly used mineral elements thus the potential benefits or hazards of such products remain unclear. MATERIALS AND METHODS: Metal and crystalline salts were purchased from a rural market (Nongoma, Zululand, South Africa). Information regarding the colloquial name, price and weight was recorded. Energy dispersive X-ray spectroscopy (EDX) was used to quantatively determine the unknown salts. RESULTS: Six widely available salts were analyzed. Ndonya, as it is colloquially known, refers to two products which look identical to the untrained eye-one is dyed table salt and the other is hexavalent chromium. A further product used medicinally, although not widely available, was identified as iron chromite ore. CONCLUSIONS: The array of substances documented, ranging from benign to carcinogenic, stresses the importance of documenting components used in traditional medicine and confirms the necessity to regulate South Africa traditional medicine. Healthcare workers should be aware of the complexities of using such metal salt.

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Real-time imaging and kinetics measurements of focused ultrasound-induced extravasation in skeletal muscle using SPECT/CT.

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Drugs need to overcome several biological barriers such as the endothelium and cellular membranes in order to reach their target. Promising new therapeutics, many of which are charged and macromolecular, are not able to passively extravasate, let alone cross cell membranes, and stay mainly in the blood pool upon intravenous injection until clearance. Using focused ultrasound (fUS) in combination with circulating microbubbles (MBs) leads to temporary localized tissue permeabilization allowing extravasation of (macro) molecules from the vascular system. Thus, fUS is a promising approach for localized drug delivery. However, little is known about the permeabilization kinetics in skeletal muscle. In this study, we used single photon emission computed tomography (SPECT) to characterize the kinetics of extravasation of (111)In-labeled bovine serum albumin (BSA), a model macromolecular drug, in muscle treated with fUS and MBs. The same fUS protocol was applied to 6 groups of mice with different times, ∆t, between fUS application and BSA injection (∆t=-10, 2.5, 10, 30, 60, 90min) followed by SPECT imaging. For ∆t≤30min we observed an exponential accumulation of activity in an area of the treated muscle which extended to a volume larger than the fUS pattern with highest accumulation for short waiting times ∆t. The extent of extravasation decreased exponentially with increasing ∆t, with a calculated half-life of ca. 21min, defining the time window of extravasation. The same treatment without MBs did not induce extravasation of BSA thus supporting MBs and drug co-injection strategies. These results provide essential information for the development of fUS based strategies for localized drug delivery.

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Comparative study on transcriptional activity of 17 parabens mediated by estrogen receptor α and β and androgen receptor.

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The structure-activity relationships of parabens which are widely used as preservatives for transcriptional activities mediated by human estrogen receptor α (hERα), hERβ and androgen receptor (hAR) were investigated. Fourteen of 17 parabens exhibited hERα and/or hERβ agonistic activity at concentrations of ⩽1×10(-5)M, whereas none of the 17 parabens showed AR agonistic or antagonistic activity. Among 12 parabens with linear alkyl chains ranging in length from C1 to C12, heptylparaben (C7) and pentylparaben (C5) showed the most potent ERα and ERβ agonistic activity in the order of 10(-7)M and 10(-8)M, respectively, and the activities decreased in a stepwise manner as the alkyl chain was shortened to C1 or lengthened to C12. Most parabens showing estrogenic activity exhibited ERβ-agonistic activity at lower concentrations than those inducing ERα-agonistic activity. The estrogenic activity of butylparaben was markedly decreased by incubation with rat liver microsomes, and the decrease of activity was blocked by a carboxylesterase inhibitor. These results indicate that parabens are selective agonists for ERβ over ERα; their interactions with ERα/β are dependent on the size and bulkiness of the alkyl groups; and they are metabolized by carboxylesterases, leading to attenuation of their estrogenic activity.

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DICO, a novel nonaromatic B-ring flavonoid, induces G2/M cell cycle arrest and apoptosis in human hepatoma cells.

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DICO was a novel nonaromatic B-ring flavonoid obtained from Macrothelypteris torresiana. In the present work, we investigated the antitumor activity and the antineoplastic mechanism of DICO. Our study showed that DICO inhibited the growth of HepG2 cells in dose and time-dependent manners. As well as DICO induced G2/M cell cycle arrest and apoptosis via a ROS-mediated mitochondrial pathway. Western blot assay demonstrated that DICO decreased Bcl-2 level and induced Bax translocation to cause cytochrome c release. Subsequently, caspase-9 and caspase-3 were activated. Meanwhile, the alterations of cyclin A and B1, p-CDK1 and p-cdc25c levels were also observed in response to DICO treatment. Taken together, DICO displayed a significant antitumor effect through G2/M cell cycle arrest and apoptosis induction, which suggested DICO might have therapeutic potential against tumors.

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Chloroquine causes similar electroretinogram modifications, neuronal phospholipidosis and marked impairment of synaptic vesicle transport in Albino and Pigmented Rats.

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Retinal toxicity of chloroquine has been known for several years, but the mechanism(s) of toxicity remain controversial; some author support the idea that the binding of chloroquine to melanin pigments in the retinal pigmented epithelium (RPE) play a major toxic role by concentrating the drug in the eye. In our study, 12 albinos Sprague-Dawley (SD) and 12 pigmented Brown Norway (BN) rats were treated orally for 3 months with chloroquine to compare functional and pathological findings. On Flash electroretinograms (ERG) performed in scotopic conditions, similar and progressive (time-dependent) delayed onset and decreased amplitudes of oscillatory potentials (from Day 71) and b-waves (on Day 92) were identified in both BN and SD rats. In both strains, identical morphological changes consisted of neuronal phospholipidosis associated with UV auto-fluorescence without evidence of retinal degeneration and gliosis; the RPE did not show any morphological lesions or autofluorescence. IHC analyses demonstrated a decrease in GABA expression in the inner nuclear layer. In addition, a marked accumulation of synaptic vesicles coupled with a marked disruption of neurofilaments in the optic nerve fibers was identified. In conclusion, ERG observations were very similar to those described in humans. Comparable ERG modifications, histopathology and immunohistochemistry findings were observed in the retina of both rat strains suggesting that melanin pigment is unlikely involved. chloroquine-induced impairment of synaptic vesicle transport, likely related to disruption of neurofilaments was identified and non-previously reported. This new mechanism of toxicity may also be responsible for the burry vision described in humans chronically treated with chloroquine.

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Thiazolidin-4-one and thiazinan-4-one derivatives analogous to rosiglitazone as potential antihyperglycemic and antidyslipidemic agents.

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A number of thiazolidin-4-one and thiazinan-4-one derivatives were prepared by three component condensation in one pot reaction method. These compounds were evaluated for anti-hyperglycemic activity by in vitro and in vivo assay systems. The compounds with thiazolidin-4-one and thiazinan-4-one moieties exhibited significant anti-hyperglycemic activity. A few compounds (3a, 3b, 4a and 4b) have exhibited both anti-hyperglycemic and anti-dyslipidemic activities. Among them the thiazinan-4-one derivative 4a showed maximal (45%) improvement in oral glucose tolerance test in db/db mice at 30 mg/kg oral dose.

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Synthesis and biological evaluation of novel aliphatic amido-quaternary ammonium salts for anticancer chemotherapy: Part II.

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A series of novel aliphatic amido-quaternary ammonium salts were synthesized and evaluated for their anticancer effects involving induction of RhoB. Most of these compounds, featuring open-ring forms of aliphatic amido-quaternary ammonium salts, exhibited potent anti-proliferative activities in human cancer cell lines, including PC-3, NUGC-3, MDA-MB-231, ACHN, HCT-15, and NCI-H23. In further evaluation, the representative compound N,N-diethyl-N-(2-(N-methyltetradecanamido)ethyl)prop-2-en-1-aminium bromide (3b) exhibited potent pro-apoptotic activity, through RhoB activation, in HeLa cells.

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Identification of novel FLT3 kinase inhibitors.

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FLT3 and PDGFR tyrosine kinases are important targets for therapy of different types of leukemia. Several FLT3/PDGFR inhibitors are currently under clinical investigation for combination with standard therapy for treatment of acute myeloid leukemia (AML), however these agents only induce partial remission and development of resistance has been reported. In this work we describe the identification of potent and novel dual FLT3/PDGFR inhibitors that resulted from our efforts to screen a library of 25,607 small molecules against the FLT3 dependent cell line MOLM-13 and the PDGFR dependent cell line EOL-1. This effort led to the identification of five compounds that were confirmed to be active on additional FLT3 dependent cell lines (cellular EC50 values between 35 and 700 nM), while having no significant effect on 24 other tyrosine kinases.

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Preparation and antimalarial activity of semisynthetic lycorenine derivatives.

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A set of twenty one lycorenine derivatives has been prepared from the alkaloid hippeastrine (1). The modifications performed on hippeastrine included some functional group transformations, structural simplification and preparation of dimers. All alkaloids were tested as potential antimalarial agents, being the hippeastrine dimers the most active compounds.

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Transport of gabapentin by LAT1 (SLC7A5).

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Gabapentin is used in the treatment of epilepsy and neuropathic pain. Gabapentin has high and saturable permeability across the BBB, but no mechanistic studies underpinning this process have been reported. The aim of the current study was to investigate the transport of gabapentin in a model of the BBB, identify the important drug transporter(s) and to use mathematical modelling to quantify the processes involved. A human brain endothelial cell line (hCMEC/D3) was utilised as an in-vitro model of the BBB. Uptake of radiolabeled gabapentin into cells in the presence of chemical inhibitors, siRNA or overexpressed drug transporters of interest was investigated. Gabapentin was demonstrated to be a LAT1 substrate in brain endothelial cells (LAT1-process; Km=530μM and Vmax=7039pmoles/million cells/min versus other-processes; Km=923μM and Vmax=3656pmoles/million cells/min) and in transfected HEK 293 LAT1 cells (LAT1-process; Km=217μM and Vmax=5192pmoles/million cells/min versus otherprocesses; Km=1546μM and Vmax=3375pmoles/million cells/min). At physiological concentrations of gabapentin, LAT1 mediated transport was 3 or ~10-fold higher than the other transport processes in the two systems, respectively, demonstrating clear selectivity for gabapentin. In-silico structural homology modelling confirmed that LAT1 could have the LeuT conserved fold and functions by the alternative access mechanism. Mathematical modelling of this mechanism revealed revised significance of Vmax and Km so that a low Km may not necessarily imply a high affinity transport process. Gabapentin was negative for OCT like transport and LAT2 activity in the hCMEC/D3 and OCT1 transfected cells. Our data shows that gabapentin is a substrate for the influx transporter LAT1 at therapeutic concentrations.

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Single-photon sources: non-blinking single-photon generation with anisotropic colloidal nanocrystals: towards room-temperature, efficient, colloidal quantum sources (adv. Mater. 14/2013).

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High-quality core/shell CdSe/CdS colloidal nanocrystals are demonstrated to be efficient sources of non-classical light. As shown by Ferruccio Pisanello and co-workers on page 1974, the intrinsic anisotropy of these nanoparticles allows an independent tuning of shell length and thickness, resulting in a full control of photon statistics. This can be exploited to obtain non-blinking, room-temperature single-photon generation, thus bringing colloidal nanocrystals one step closer to non-classical light sources for quantum applications.

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Light-Emitting Field-Effect Transistors Having Combined Organic Semiconductor and Metal Oxide Layers.

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A new organic light-emitting field-effect transistor characterized by a metal oxide layer inserted between the organic layer and the gate insulator is proposed. The metal oxide is indirectly connected with source and drain electrodes through the organic layer. Upon increasing the potential difference between the source and drain electrodes, the emission becomes exceedingly strong and the emission region encompasses the whole channel zone.

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Patterned Growth of Crystalline Organic Heterostructures.

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Organic droplet epitaxy is presented as a method for growing nanopatterned crystalline heterostructures, thanks to the transport of molecules of an amorphous first-layer on top of a crystalline second-layer, where they form an epitaxial interface. Such heterostructures may be transferred to any substrates, raising particular interest for applications (e.g., for organic photovoltaics), where crystallinity and nanopatterning constitute well recognized advantages.

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Decreased consumption of sweet fluids in mu opioid receptor knockout mice: a microstructural analysis of licking behavior.

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RATIONALE: Evidence suggests that the palatability of food (i.e., the hedonic impact produced by its sensory features) can promote feeding and may underlie compulsive eating, leading to obesity. Pharmacological studies implicate opioid transmission in the hedonic control of feeding, though these studies often rely on agents lacking specificity for particular opioid receptors. OBJECTIVES: Here, we investigated the role of mu opioid receptors (MORs) specifically in determining hedonic responses to palatable sweet stimuli. METHODS: In Experiment 1, licking microstructure when consuming sucrose solution (2 to 20 %) was compared in MOR knockout and wildtype mice as a function of sucrose concentration and level of food deprivation. In Experiment 2, a similar examination was conducted using the palatable but calorie-free stimulus sucralose (0.001 to 1 %), allowing study of licking behavior independent of homeostatic variables. RESULTS: In Experiment 1, MOR knockout mice exhibited several alterations in sucrose licking. Although wildtype mice exhibited a twofold increase in the burst length when food deprived, relative to the nondeprived test, this aspect of sucrose licking was generally insensitive to manipulations of food deprivation for MOR knockout mice. Furthermore, during concentration testing, their rate of sucrose licking was less than half that of wildtype mice. During sucralose testing (Experiment 2), MOR knockout mice licked at approximately half the wildtype rate, providing more direct evidence that MOR knockout mice were impaired in processing stimulus palatability. CONCLUSIONS: These results suggest that transmission through MORs mediates hedonic responses to palatable stimuli, and therefore likely contributes to normal and pathological eating.

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Non-classical Pharmacology of the Dopamine Transporter: Atypical Inhibitors, Allosteric Modulators and Partial Substrates.

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The dopamine transporter (DAT) is a sodium-coupled symporter protein responsible for modulating the concentration of extraneuronal dopamine in the brain. The DAT is a principle target of various psychostimulant, nootropic and antidepressant drugs, as well as certain drugs used recreationally, including the notoriously addictive stimulant cocaine. DAT ligands have traditionally been divided into two categories: cocaine-like inhibitors and amphetamine-like substrates. Whereas inhibitors block monoamine uptake by the DAT, but are not translocated across the membrane, substrates are actively translocated and trigger DAT-mediated release of dopamine by reversal of the translocation cycle. As both inhibitors and substrates increase extraneuronal dopamine levels, it is often assumed that all DAT ligands posses an addictive liability equivalent to cocaine. However, the recent development of novel 'atypical' inhibitors and 'partial substrate' ligands with reduced or even a complete lack of cocaine-like rewarding effects suggests that addictiveness is not a constant property of DAT-affecting compounds. These atypical ligands do not conform to the classical preconception that all DAT inhibitors (or substrates) are functionally and mechanistically alike. Instead, they suggest the possibility that the DAT exhibits some of the ligand-specific 'pleiotropic' functional qualities inherent to G-protein-coupled receptors. That is, ligands with different chemical structures induce specific conformational changes in the transporter protein, which can be differentially transduced by the cell, ultimately eliciting unique behavioral and psychical effects. The present overview discusses compounds with conformation-specific activity, useful not only as tools for studying the mechanics of dopamine transport, but also as leads for medication development in addictive disorders.

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Molecular Identification and Characterization of an Essential Pyruvate Transporter from Trypanosoma brucei.

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Pyruvate export is an essential physiological process for the bloodstream form of T. brucei as the parasite would otherwise accumulate this end product of glucose metabolism to toxic levels. In the studies reported here genetic complementation in Saccharomyces cerevisiae has been employed to identify a gene (TbPT0) that encodes this vital pyruvate transporter from T. brucei. Expression of TbPT0 in S. cerevisiae reveals that TbPT0 is a high affinity pyruvate transporter. TbPT0 belongs to a clustered multigene family consisting of five members, whose expression is up-regulated in the bloodstream form. Interestingly, TbPT family permeases are related to polytopic proteins from plants but not to characterized monocarboxylate transporters from mammals. Remarkably, inhibition of the TbPT gene family expression in bloodstream parasites by RNAi is lethal, confirming the physiological relevance of these transporters. The discovery of TbPT0 reveals for the first time the identity of the essential pyruvate transporter and provides a potential drug target against the mammalian life cycle stage of T. brucei.

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DNA repair and cytotoxic drugs: the potential role of RAD51 in clinical outcome of non-small-cell lung cancer patients.

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Many of the cytotoxic drugs used in the treatment of non-small-cell lung carcinoma patients can interfere with DNA activity and the definition of an individual DNA repair profile could be a key strategy to achieve better response to chemotherapeutic treatment. Although DNA repair mechanisms are important factors in the prevention of carcinogenesis, these molecular pathways are also involved in therapy response. RAD51 is a crucial element in DNA repair by homologous recombination and has been shown to interfere with the prognosis of patients treated with chemoradiotherapy. There is increasing evidence that genetic polymorphisms in repair enzymes can influence DNA repair capacity and, consequently, affect chemotherapy efficacy. We conducted this review to show the possible influence of the RAD51 genetic variants in damage repair capacity and treatment response in non-small-cell lung carcinoma patients.

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Extracellular Signal-Regulated Kinases (ERK) Inhibitors from Aristolochia yunnanensis.

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Six new sesquiterpenoids, aristoyunnolins A-F (1-6), an artifact of isolation [7-O-ethyl madolin W (7)], and 12 known analogues were isolated from stems of Aristolochia yunnanensis. The structures were determined by combined chemical and spectral methods, and the absolute configurations of compounds 2, 3, 5-7, 9, 14, and 17 were determined by the modified Mosher's method and CD analysis. Compounds 1-19 were screened using a bioassay system designed to evaluate the effect on mitogen-activated protein kinases (MAPKs) signaling pathways. Among three MAPKs (ERK1/2, JNK, and p38), compounds 1, 4, 10-13, 16, 18, and 19 exhibited selective inhibition of the phosphorylation of ERK1/2. Compounds 16 and 19 were more active than the positive control PD98059, a known inhibitor of the ERK1/2 signaling pathway.

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Different levels of prenatal zinc and selenium had different effects on neonatal neurobehavioral development.

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Either deficient or excessive of essential nutrients had adverse effects. Effects of different levels of prenatal zinc (Zn) and selenium (Se) on fetal neurobehavioral development remain unclear. To determine the effects of different cord serum levels of Zn and Se on neurobehavioral development in neonates and to explore possible threshold level of Zn and Se based on fetal neurodevelopment, we conducted this epidemiological research. In the multi-center study, we investigated these questions in 927 mother-newborn pairs in Shanghai, China, from 2008 through 2009. Umbilical cord serum concentrations of Zn and Se were measured and Neonatal Behavioral Neurological Assessment (NBNA) tests were conducted. The median cord serum Zn and Se concentrations were 794.3μg/L and 63.1μg/L, respectively. A nonlinear relationship was observed between cord serum Zn and NBNA after adjusting for potential confounders. NBNA score decreased with increasing Zn levels after 794.3μg/L (adjusted β=-3.0, 95% CI: -3.6 to -2.4, p<0.001). Additionally, an invert U-shape with a threshold Se of 100μg/L was observed between cord serum Se and NBNA. The adjusted regression coefficient was 4.4 (95% CI: 3.6-5.2, p<0.001) for Se<100μg/L while -3.6 (95% CI: -6.1 to -1.1, p<0.01) for Se≥100μg/L. Of the 927 infants, 50% had a high level Zn (≥794.3μg/L) and 8.6% had a high level Se (≥100μg/L). High levels of both Zn and Se mainly had adverse effects on behavior and passive tone (p<0.001). Taken together, our study suggested that a threshold of cord blood Zn and Se was existed for fetal neurodevelopment and the prevalence of excessive Zn was high. Thus, the supplementation of Zn during pregnancy should be considered with caution in Shanghai, China.

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Substituted imidazopyridazines are potent and selective inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1).

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A series of imidazopyridazines which are potent inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) was identified from a high-throughput screen against the isolated enzyme. Subsequent exploration of the SAR and optimisation has yielded leading members which show promising in vitro anti-parasite activity along with good in vitro ADME and selectivity against human kinases. Initial in vivo testing has revealed good oral bioavailability in a mouse PK study and modest in vivo efficacy in a Plasmodium berghei mouse model of malaria.

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Development and evaluation of novel solid nanodispersion system for oral delivery of poorly water-soluble drugs.

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The aim of the present study was to develop and evaluate a novel drug solubilization platform (so-called solid nanodispersion) prepared by a simple co-grinding and solvent-free process. Using structurally diverse model compounds from the Pfizer drug library, including ingliforib, furosemide and celecoxib, we successfully prepared stable solid nanodispersions (SNDs) without the use of solvent or heat. Stable colloidal particles (<350nm) containing drug, polyvinylpyrrolidone (PVP) K12 and sodium dodecyl sulfate (SDS) in 1:2.75:0.25 ratio were produced after 2h of co-grinding. The composition and particle size of SNDs were optimized by varying the grinding media size, powder-to-grinding media ratio, milling speed and milling time. The resulting formulations contained crystalline drug and were stable at room temperature for over one month. Greater than 80% of the drug was released from the SND in less than 30min, with sustained supersaturation over 4h. Using furosemide (BCS class IV compound) as a model compound, we conducted transport studies with Madin-Darby canine kidney cells transfected with human MDR1 gene (MDCK/MDR1), followed by pharmacokinetics studies in rats. Results showed that the SND formulation enhanced the absorptive flux of furosemide by more than 3-fold. In the pharmacokinetics studies, the SND formulation increased Cmax and AUC of furosemide by 36.6 and 43.2 fold respectively, relative to Methocel formulation. Interestingly, physical mixture containing furosemide, PVP K12 and SDS produced a similar level of oral exposure as the SNDs, albeit with a longer Tmax than the SND formulation. The results suggest that PVP K12 and SDS were able to increase the furosemide free fraction available for oral absorption. Low solubility, poor permeability, and high first-pass effect of furosemide may also have produced the effect that small improvements in solubilization resulted in significant potentiation of the oral exposure of the physical mixture. However the use of a physical mixture of drug, polymer and surfactant, to increase drug bioavailability cannot be generalized to all drugs. There are only a few reported cases of such phenomenon. While SNDs may not be the only option to solubilize compounds in every case, SNDs are expected to be applicable to a broader chemical space of pharmaceutical compounds compared to a physical mixture. Ultimately, formulation scientists have to exercise judgment in choosing the appropriate formulation strategy for the compound of interest. SNDs represent a significant improvement over current enabling technologies such as nanocrystal and spray-dried dispersion technologies, in that SNDs are simple, do not require solvent or heat, are applicable to a structurally diverse chemical space, and are amenable to the development of solid dosage forms.

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In vivo and in vitro anti-inflammatory potential of pentahydroxy-pregn-14-ol, 20-one-β-d-thevetopyranoside in rats.

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Polyhydroxy pregnane glycoside (PPG), a steroidal glycoside was isolated from Wattakaka volubilis Linn. (Stap.f.). PPG was evaluated for in vivo and in vitro anti-inflammatory activity using acute inflammation and chronic model of inflammation in rats and LPS-induced RAW 264.7 macrophage cells. PPG seemed to be responsible for the anti-inflammatory activity in the studied models. PPG at dose level of both 5 and 10mg/kg significantly reduced the edema induced by the carrageenan in acute model of inflammation. It also showed significant anti-proliferative effect (dry pellet weight basis) in chronic model of inflammation. Cellular content of granuloma was measured by assaying activity of N-acetyl glucosaminidase (NAG) and total nucleic acid content. PPG at 5 and 10mg/kg significantly suppressed the cellular infiltration measured by total nucleic acid content. In contrast, NAG activity decreased over a period of 10 days resulting in inhibition of granuloma weight gain. PPG had a more effective response than the reference drug diclofenac sodium in both the models of inflammation. Wattakaka volubilis steroidal glycoside mixture (WVSM) and PPG (1-50μM) significantly inhibited the COX-2 and iNOS enzymes resulting in low levels of PGE2 and NO in LPS-induced RAW 264.7 macrophage cells. Hence the study supports the traditional use of Wattakaka volubilis and its constituent PPG in treatment of inflammatory disorders.

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DNA repair activity in fish and interest in ecotoxicology: A review.

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The knowledge of DNA repair in a target species is of first importance as it is the primary line of defense against genotoxicants, and a better knowledge of DNA repair capacity in fish could help to interpret genotoxicity data and/or assist in the choice of target species, developmental stage and tissues to focus on, both for environmental biomonitoring studies and DNA repair testing. This review focuses in a first part on what is presently known on a mechanistic basis, about the various DNA repair systems in fish, in vivo and in established cell lines. Data on base excision repair (BER), direct reversal with O(6)-alkylguanine transferase and double strand breaks repair, although rather scarce, are being reviewed, as well as nucleotide excision repair (NER) and photoreactivation repair (PER), which are by far the most studied repair mechanisms in fish. Most of these repair mechanisms seem to be strongly species and tissue dependent; they also depend on the developmental stage of the organisms. BER is efficient in vivo, although no data has been found on in vitro models. NER activity is quite low or even inexistent depending on the studies; however this lack is partly compensated by a strong PER activity, especially in early developmental stage. In a second part, a survey of the ecotoxicological studies integrating DNA repair as a parameter responding to single or mixture of contaminant is realized. Three main approaches are being used: the measurement of DNA repair gene expression after exposure, although it has not yet been clearly established whether gene expression is indicative of repair capacity; the monitoring of DNA damage removal by following DNA repair kinetics; and the modulation of DNA repair activity following exposure in situ, in order to assess the impact of exposure history on DNA repair capacity. Since all DNA repair processes are possible targets for environmental pollutants, we can also wonder at which extent such a modulation of repair capacities in fish could be the base for the development of new biomarkers of genotoxicity. Knowing the importance of the germ cell DNA integrity in the reproductive success of aquatic organisms, the DNA repair capacity of such cells deserve to be more studied, as well as DNA repair capacities of established fish cell lines. The limited amount of available data, which shows low/slow DNA repair capacities of fish cell lines compared with mammalian cell lines, concerned mainly the NER system; thus this point merits to be explored more deeply. Additionally, since some of the DNA repair systems appear more efficient in embryo larval stages, it would be of interest to consider embryonic cell lineages more closely.

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The Ras-GTPase activity of neurofibromin restrains ERK-dependent FGFR signaling during endochondral bone formation.

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The severe defects in growth plate development caused by chondrocyte extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) gain or loss-of-function suggest that tight spatial and temporal regulation of mitogen-activated protein kinase signaling is necessary to achieve harmonious growth plate elongation and structure. We provide here evidence that neurofibromin, via its Ras guanosine triphosphatase -activating activity, controls ERK1/2-dependent fibroblast growth factor receptor (FGFR) signaling in chondrocytes. We show first that neurofibromin is expressed in FGFR-positive prehypertrophic and hypertrophic chondrocytes during growth plate endochondral ossification. Using mice lacking neurofibromin 1 (Nf1) in type II collagen-expressing cells, (Nf1col2(-/-) mutant mice), we then show that lack of neurofibromin in post-mitotic chondrocytes triggers a number of phenotypes reminiscent of the ones observed in mice characterized by FGFR gain-of-function mutations. Those include dwarfism, constitutive ERK1/2 activation, strongly reduced Ihh expression and decreased chondrocyte proliferation and maturation, increased chondrocytic expression of Rankl, matrix metalloproteinase 9 (Mmp9) and Mmp13 and enhanced growth plate osteoclastogenesis, as well as increased sensitivity to caspase-9 mediated apoptosis. Using wildtype (WT) and Nf1(-/-) chondrocyte cultures in vitro, we show that FGF2 pulse-stimulation triggers rapid ERK1/2 phosphorylation in both genotypes, but that return to the basal level is delayed in Nf1(-/-) chondrocytes. Importantly, in vivo ERK1/2 inhibition by daily injection of a recombinant form of C-type natriuretic peptide to post-natal pups for 18 days was able to correct the short stature of Nf1col2(-/-) mice. Together, these results underscore the requirement of neurofibromin and ERK1/2 for normal endochondral bone formation and support the notion that neurofibromin, by restraining RAS-ERK1/2 signaling, is a negative regulator of FGFR signaling in differentiating chondrocytes.

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Stereoselective Glucuronidation of Ornidazole in Humans: Predominant Contribution of UDP-Glucuronosyltransferases 1A9 and 2B7.

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Ornidazole [R,S-1-chloro-3-(2-methyl-5-nitro-1H-imidazol-1-yl)propan-2-ol] is a chiral 5-nitroimidazole class antimicrobial agent. This study aimed to investigate the principal metabolic pathway of ornidazole in humans and identify the major enzymes involved. A total of 19 metabolites were identified in human urine collected from patients with hepatobiliary diseases after an intravenous drip infusion of 500 mg of racemic ornidazole. Stereoselective glucuronidation, followed by renal excretion, was the principal metabolic pathway of ornidazole in humans, accounting for 37.3% of the administered dose. Screening assays with 12 available human recombinant UDP-glucuronosyltransferases (UGTs) demonstrated that UGT1A9 was the predominant UGT isoform involved in R-ornidazole glucuronidation, whereas S-ornidazole glucuronidation was almost exclusively catalyzed by UGT2B7. Chemical inhibition study with niflumic acid and flurbiprofen supported these findings. Enzyme kinetic parameters were then determined in human liver microsomes (HLMs), human kidney microsomes (HKMs), UGT1A9 and 2B7. The Km values for UGT1A9 (15.6 ± 1.6 mM for R-ornidazole) and 2B7 (3.8 ± 0.9 mM for S-ornidazole) were quite similar to those determined in HLMs and HKMs (20.1 ± 1.4 and 17.7 ± 4.0 mM for R-ornidazole; 6.6 ± 1.3 and 3.2 ± 0.4 mM for S-ornidazole). The in vitro intrinsic clearance (CLint) ratios of S- and R-ornidazole were approximately 4.3 in HLMs and 6.5 in HKMs, respectively. The hepatic and renal clearances were estimated based on the well-stirred model. Overall, stereoselective glucuronidation was the principal metabolic pathway of ornidazole in humans. Furthermore, UGT1A9 and 2B7 were the predominant UGT isoforms responsible for R- and S-ornidazole glucuronidation in humans, respectively.

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Ion Mobility Spectrometry-Mass Spectrometry Analysis for the Site of Aromatic Hydroxylation.

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Hydroxylated metabolites often retain the pharmacological activity of parent compound, and the position of hydroxylation determines the formation of chemically reactive intermediates such as quinones and analogues from para- and/or ortho-hydroxylation of phenols or arylamines. Therefore, the identification of exact position of hydroxylation is often required at the early development stage of new drug candidates. In many cases, liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides identical MS/MS spectra among isomeric hydroxylated metabolites, and therefore, it alone cannot unequivocally identify the exact position(s) of hydroxylation. Ion mobility spectrometry (IMS), integrated with LC-MS/MS, recently demonstrates the capability of separating isomeric species based on differences in their drift times from IMS, which are linearly proportional to the collision cross-section (CCS) reflecting physical size and shape. In the present study, a chemical derivatization of isomeric hydroxylated metabolites with 2-fluoro-N-methyl pyridinium p-toluenesulfonate was found to confer distinct theoretical CCS value on each isomer by forming corresponding N-methyl pyridine (NMP) derivative. The regression lines established by the comparison between theoretical CCS values and observed drift times from IMS for each set of parent compound (labetalol, ezetimibe, atorvastatin and warfarin) and its MS/MS product ions accurately and selectively projected the actual drift times of NMP derivatives of corresponding aromatic or isomeric hydroxylated metabolites. The established method was used for the accurate assignment of predominant formation of 2-hydroxylated metabolite from imipramine in NADPH- fortified human liver microsomes. The present application expands the versatility of LC-IMS-MS technique to the structure identification of isomeric hydroxylated metabolites at the early stage for drug development.

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Pharmacovigilance Using Clinical Notes.

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With increasing adoption of electronic health records (EHRs), there is an opportunity to use the free-text portion of EHRs for pharmacovigilance. We present novel methods that annotate the unstructured clinical notes and transform them into a deidentified patient-feature matrix encoded using medical terminologies. We demonstrate the use of the resulting high-throughput data for detecting drug-adverse event associations and adverse events associated with drug-drug interactions. We show that these methods flag adverse events early (in most cases before an official alert), allow filtering of spurious signals by adjusting for potential confounding, and compile prevalence information. We argue that analyzing large volumes of free-text clinical notes enables drug safety surveillance using a yet untapped data source. Such data mining can be used for hypothesis generation and for rapid analysis of suspected adverse event risk.Clinical Pharmacology & Therapeutics (2013); advance online publication 10 April 2013. doi:10.1038/clpt.2013.47.

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Attenuation of erythrocytic actylcholinesterase by antidepressants: Evidence in an in vitro experiment.

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The current study was aimed to scrutinize acetylcholinesterase (AchE) inhibitory profile of two antidepressants, diazepam and phenobarbitone. The experimental designed was based on Michaelis-Menten parameters (apparent Michaelis constant (aKm) and apparent maximum velocity (aVm)) that estimate inhibition (%) as well as the type of inhibition (mechanism). The results showed marked inhibition of AchE by diazepam and the values of aKm and aVm were 65.5% and 52.63%, respectively. These values suggested a competitive type of antagonism for diazepam. Similar trend of antagonism was shown by phenobarbitone when it was subjected to the challenge of AchE with aKm and aVm values of 51.99% and 71.80%, respectively. It is concluded that diazepam and phenobarbitone exhibited prominent AchE attenuation apart from their well-established antidepressant activity, which could be more useful in related diseased conditions.

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A Role for Cargo in the Activation of ADP-Ribosylation Factors (Arf) and Adaptor Recruitment.

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Gelation-induced visible supramolecular chiral recognition by fluorescent metal complexes of quinolinol-glutamide.

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Three metal complexes consisting of Li(+), Zn(2+), and Al(3+) and quinolinol-functionalized l-glutamides (HQLG), (abbreviated as LiHQLG, Zn(HQLG)2, and Al(HQLG)3) were found to form fluorescent metallogels in several organic solvents. In solution, these chiral complexes showed neither any CD signal in the chromophore region nor chiral recognition of the chiral species. However, upon gel formation, the supramolecular chirality emerged because of the self-assembled nanostructures, which provided an opportunity for the chiral recognition of enantiomeric ligands. The metallogels showed different fluorescence changes when they met with enantiomeric (R,R)- or (S,S)-1,2-diaminocyclohexane. Among them, the Al(HQLG)3 metallogels did not show any change whereas the LiHQLG gels exhibited the same decrease in fluorescence. Interestingly, the Zn(HQLG)2 gels showed obviously different fluorescent color with respect to (R,R)- and (S,S)-1,2-diaminocyclohexane, thus providing visible chiral recognition via the naked eye. Such different recognition ability was discussed on the basis of the assembled chiral nanostructures and the primary molecular structures of the metal complexes. It was shown that both of them played important roles in chiral recognition.

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JNK inhibitors as anti-inflammatory and neuroprotective agents.

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JNK is involved in a broad range of physiological processes. Several inflammatory and neurodegenerative diseases, such as multiple sclerosis, Alzheimer's and Parkinson's disease have been linked with the dysregulated JNK pathway. Research on disease models using the relevant knockout mice has highlighted the importance of specific JNK isoformsin-particular disorders and has stimulated further efforts in the drug-discovery area. However, most of the experimental evidence for the efficacy of JNK inhibition in animal models is from studies using JNK inhibitors, which are not isoform selective. Some of the more recent compounds exhibit good oral bioavailability, CNS penetration and selectivity against the rest of the kinome. Efforts to design isoform-selective inhibitors have produced a number of examples with various selectivity profiles. This article presents recent progress in this area and comment on the role of isoform selectivity for efficacy.

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Effects of 17α-ethynylestradiol-induced cholestasis on the pharmacokinetics of doxorubicin in rats: reduced biliary excretion and hepatic metabolism of doxorubicin.

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Abstract 1. Since the prevalent hormonal combination therapy with estrogen analogues in cancer patients has frequency and possibility to induce the cholestasis, the frequent combination therapy with 17α-ethynylestradiol (EE, an oral contraceptive) and doxorubicin (an anticancer drug) might be monitored in aspect of efficacy and safety. Doxorubicin is mainly excreted into the bile via P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (Mrp2) in hepatobiliary route and metabolized via cytochrome P450 (CYP) 3A subfamily. Also the hepatic Mrp2 (not P-gp) and CYP3A subfamily levels were reduced in EE-induced cholestatic (EEC) rats. Thus, we herein report the pharmacokinetic changes of doxorubicin with respect to the changes in its biliary excretion and hepatic metabolism in EEC rats. 2. The pharmacokinetic study of doxorubicin after intravenous administration of its hydrochloride was conducted along with the investigation of bile flow rate and hepatobiliary excretion of doxorubicin in control and EEC rats. 3. The significantly greater AUC (58.7% increase) of doxorubicin in EEC rats was due to the slower CL (32.9% decrease). The slower CL was due to the reduction of hepatic biliary excretion (67.0% decrease) and hepatic CYP3A subfamily-mediated metabolism (21.9% decrease) of doxorubicin. These results might have broader implications to understand the altered pharmacokinetics and/or pharmacologic effects of doxorubicin via biliary excretion and hepatic metabolism in experimental and clinical estrogen-induced cholestasis.

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Oxidation of CO by Nickel Oxide Clusters Revealed by Post Heating.

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