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Up-conversion luminescence in rare earth ions (REs) doped nanoparticles has attracted considerable research attention for the promising applications in solid-state lasers, three-dimensional displays, solar cells, biological imaging, and so forth. However, there have been no reports on REs doped nanoparticles to investigate their polarized energy transfer up-conversion, especially for single particle. Herein, the polarized energy transfer up-conversion from REs doped fluoride nanorods is demonstrated in a single particle spectroscopy mode for the first time. Unique luminescent phenomena, for example, sharp energy level split and singlet-to-triplet transitions at room temperature, multiple discrete luminescence intensity periodic variation with polarization direction, are observed upon excitation with 980 nm linearly polarized laser. Furthermore, nanorods with the controllable aspect ratio and symmetry are fabricated for analysis of the mechanism of polarization anisotropy. The comparative experiments suggest that intraions transition properties and crystal local symmetry dominate the polarization anisotropy, which is also confirmed by density functional theory calculations. Taking advantage of the REs based up-conversion, potential application in polarized microscopic multi-information transportation is suggested for the polarization anisotropy from REs doped fluoride single nanorod or nanorod array.

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Stability of sulforaphane for topical formulation.

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Abstract Context: Sulforaphane (SFN) is a natural compound that has been investigated as a chemopreventive agent. SFN has been shown to inhibit the activator-protein-1 (AP-1) transcription factor and may be effective for inhibition of ultraviolet (UV) induced skin carcinogenesis. This study was designed to investigate the stability of SFN as a function of pH, temperature and in various solvents and formulations. Materials and methods: Stability was analyzed using high-performance liquid chromatography. A potential lead formulation was identified and evaluated in vivo. Results: SFN was determined to undergo apparent first-order degradation kinetics for the conditions explored. It was observed that SFN undergoes base catalyzed degradation. Buffer species and solvent type impacts stability as well. SFN was found to be very sensitive to temperature with degradation rate changing by a factor of nearly 3.1 for every 10 °C change in temperature (at pH 4.0). SFN completely degraded after 30 days in a conventional pharmaceutical cream formulation. Improved stability was observed in organic formulation components. Stability studies were conducted on two nonaqueous topical formulations: a polyethylene glycol (PEG) ointment base and an organic oleaginous base. Conclusion: Topically applied SFN in the PEG base formulation significantly reduced AP-1 activation after UV stimulation in the skin of a transgenic mouse model, indicating that SFN in this formulation retains efficacy in vivo.

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DNA Interactions in Crowded Nanopores.

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The motion of DNA in crowded environments is a common theme in physics and biology. Examples include gel electrophoresis and the self-interaction of DNA within cells and viral capsids. Here we study the interaction of multiple DNA molecules within a nanopore by tethering the DNA to a bead held in a laser optical trap to produce a "molecular tug-of-war". We measure this tether force as a function of the number of DNA molecules in the pore and show that the force per molecule decreases with the number of molecules. A simple scaling argument based on a mean field theory of the hydrodynamic interactions between multiple DNA strands explains our observations. At high salt concentrations, when the Debye length approaches the size of the counterions, the force per molecule becomes essentially independent of the number of molecules. We attribute this to a sharp decrease in electroosmotic flow which makes the hydrodynamic interactions ineffective.

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Spontaneous partition of carbon nanotubes in polymer-modified aqueous phases.

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The distribution of nanoparticles in different aqueous environments is a fundamental problem underlying a number of processes, ranging from biomedical applications of nanoparticles to their effects on the environment, health, and safety. Here, we study distribution of carbon nanotubes (CNTs) in two immiscible aqueous phases formed by the addition of polyethylene glycol (PEG) and dextran. This well-defined model system exhibits a strikingly robust phenomenon: CNTs spontaneously partition between the PEG- and the dextran-rich phases according to nanotube's diameter and metallicity. Thermodynamic analysis suggests that this chirality-dependent partition is determined by nanotube's intrinsic hydrophobicity and reveals two distinct regimes in hydrophobicity-chirality relation: a small diameter (<1 nm) regime, where curvature effect makes larger diameter tubes more hydrophobic than small diameter ones, and a large diameter (>1.2 nm) regime, where nanotube's polarizability renders semiconducting tubes more hydrophobic than metallic ones. These findings reveal a general rule governing CNT behaviors in aqueous phase and provide an extremely simple way to achieve spatial separation of CNTs by their electronic structures.

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Protein kinase inhibitor design by targeting the Asp-Phe-Gly (DFG) motif: the role of the DFG motif in the design of epidermal growth factor receptor inhibitors.

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The Asp-Phe-Gly (DFG) motif plays an important role in the regulation of kinase activity. Structure-based drug design was performed to design compounds able to interact with the DFG motif; epidermal growth factor receptor (EGFR) was selected as an example. Structural insights obtained from the EGFR/2a complex suggested that an extension from the meta-position on the phenyl group (ring-5) would improve interactions with the DFG motif. Indeed, introduction of an N,N-dimethylamino tail resulted in 4b, which showed almost 50-fold improvement in inhibition compared to 2a. Structural studies confirmed this N,N-dimethylamino tail moved towards the DFG motif to form a salt bridge with the side chain of Asp831. That the interactions with the DFG motif greatly contribute to the potency of 4b is strongly evidenced by synthesizing and testing compounds 2a, 3g and 4f: when the charge interactions are absent, the inhibitory activity decreased significantly.

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Quantitative analysis of global phosphorylation changes with high-resolution tandem mass spectrometry and stable isotopic labeling.

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Quantitative measurement of specific protein phosphorylation sites is a primary interest of biologists, as site-specific phosphorylation information provides insights into cell signaling networks and cellular dynamics at a system level. Over the last decade, selective phosphopeptide enrichment methods including IMAC and metal oxides (TiO2 and ZrO2) have been developed and greatly facilitate large scale phosphoproteome analysis of various cells, tissues and living organisms, in combination with modern mass spectrometers featuring high mass accuracy and high mass resolution. Various quantification strategies have been applied to detecting relative changes in expression of proteins, peptides, and specific modifications between samples. The combination of mass spectrometry-based phosphoproteome analysis with quantification strategies provides a straightforward and unbiased method to identify and quantify site-specific phosphorylation. We describe common strategies for mass spectrometric analysis of stable isotope labeled samples, as well as two widely applied phosphopeptide enrichment methods based on IMAC(NTA-Fe(3+)) and metal oxide (ZrO2). Instrumental configurations for on-line LC-tandem mass spectrometric analysis and parameters of conventional bioinformatic analysis of large data sets are also considered for confident identification, localization, and reliable quantification of site-specific phosphorylation.

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Molecular Mechanisms of the antitumor activity of SB225002: A novel microtubule inhibitor.

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SB225002 (SB) is an IL-8 receptor B (IL-8RB) antagonist that has previously been shown to inhibit IL-8-based cancer cell invasion, and to possess in vivo anti-inflammatory and anti-nociceptive effects. The present study presented an evidence for the cell cycle-targeting activity of SB in a panel of p53-mutant human cancer cell lines of different origin, and investigated the underlying molecular mechanisms. A combination of cell cycle analysis, immunocytometry, immunoblotting, and RNA interference revealed that SB induced a BubR1-dependent mitotic arrest. Mechanistically, SB was shown to possess a microtubule destabilizing activity evidenced by hyperphosphorylation of Bcl2 and BclxL, suppression of microtubule polymerization and induction of a prometaphase arrest. Molecular docking studies suggested that SB has a good affinity towards vinblastine-binding site on β-tubulin subunit. Of note, SB265610 which is a close structural analogue of SB225002 with a potent IL-8RB antagonistic activity did not exhibit a similar antimitotic activity. Importantly, in P-glycoprotein overexpressing NCI/Adr-Res cells the antitumor activity of SB was unaffected by multidrug resistance. Interestingly, the mechanisms of SB-induced cell death were cell-line dependent, where in invasive hepatocellular carcinoma HLE cells the significant contribution of BAK-dependent mitochondrial apoptosis was demonstrated. Conversely, SB activated p38 MAPK signaling in colorectal adenocarcinoma cells SW480, and pharmacologic inhibition of p38 MAPK activity revealed its key role in mediating SB-induced caspase-independent cell death. In summary, the present study introduced SB as a promising antitumor agent which has the potential to exert its activity through dual mechanisms involving microtubules targeting and interference with IL-8-drivin cancer progression.

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Wogonoside displays anti-inflammatory effects through modulating inflammatory mediator expression using RAW264.7 cells.

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ETHNOPHARMACOLOGICAL RELEVANCE: The root of Scutellaria baicalensis Georgi, also called Huangqin in China, is an herbal-based nutraceutical which is usually used in Chinese medicated diet (CMD). As an abundant ingredient in Huangqin, wogonoside is a flavonoid glycoside. The present work investigated the anti-inflammatory activities of wogonoside in lipopolysaccharides (LPS)-induced RAW264.7 cells. MATERIALS AND METHODS: RAW264.7 cells were used. The inhibition of wogonoside against nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in LPS-induced RAW264.7 cells were measured. Additionally, the effects of wogonoside on mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), TNF-α and IL-6 were also investigated. RESULTS AND DISCUSSION: Wogonoside not only dose-dependently decreased the production of inflammatory mediators including NO and PGE2 but also inhibited the release of pro-inflammatory cytokines including TNF-α and IL-6 in LPS-induced RAW264.7 cells. Furthermore, wogonoside possessed significantly in vitro inhibitory effects on the gene expression of iNOS, COX2, TNF-α and IL-6. CONCLUSION: These results suggest that wogonoside may be used as a functional food component for prevention and treatment of inflammation.

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Assuring Safety Without Animal Testing: The Case for the Human Testis In Vitro.

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From 15-17 June 2011, a dedicated workhop was held on the subject of in vitro models for mammalian spermatogenesis and their applications in toxicological hazard and risk assessment. The workshop was sponsored by the Dutch ASAT initiative (Assuring Safety without Animal Testing), which aims at promoting innovative approaches towards toxicological hazard and risk assessment on the basis of human and in vitro data, and replacement of animal studies. Participants addressed the state of the art regarding human and animal evidence for compound mediated testicular toxicity, reviewed existing alternative assay models, and brainstormed about future approaches, specifically considering tissue engineering. The workshop recognized the specific complexity of testicular function exemplified by dedicated cell types with distinct functionalities, as well as different cell compartments in terms of microenvironment and extracellular matrix components. This complexity hampers quick results in the realm of alternative models. Nevertheless, progress has been achieved in recent years, and innovative approaches in tissue engineering may open new avenues for mimicking testicular function in vitro. Although feasible, significant investment is deemed essential to be able to bring new ideas into practice in the laboratory. For the advancement of in vitro testicular toxicity testing, one of the most sensitive end points in regulatory reproductive toxicity testing, such an investment is highly desirable.

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Relations between stimulation of mesolimbic dopamine and place conditioning in rats produced by cocaine or drugs that are tolerant to dopamine transporter conformational change.

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RATIONALE: Dopamine transporter (DAT) conformation plays a role in the effectiveness of cocaine-like and other DAT inhibitors. Cocaine-like stimulants are intolerant to DAT conformation changes having decreased potency in cells transfected with DAT constructs that face the cytosol compared to wild-type DAT. In contrast, analogs of benztropine (BZT) are among compounds that are less affected by DAT conformational change. METHODS: We compared the displacement of radioligand binding to various mammalian CNS sites, acute stimulation of accumbens shell dopamine levels, and place conditioning in rats among cocaine and four BZT analogs with Cl substitutions on the diphenyl-ether system including two with carboalkoxy substitutions at the 2-position of the tropane ring. RESULTS: Binding assays confirmed high-affinity and selectivity for the DAT with the BZT analogs which also produced significant stimulation of mesolimbic dopamine efflux. Because BZT analogs produced temporal patterns of extracellular dopamine levels different from those by cocaine (3-10 mg/kg, i.p.), the place conditioning produced by BZT analogs and cocaine was compared at doses and times at which both the increase in dopamine levels and rates of increase were similar to those produced by an effective dose of cocaine. Despite this equilibration, none of the BZT analogs tested produced significant place conditioning. CONCLUSIONS: The present results extend previous findings suggesting that cocaine-like actions are dependent on a binding equilibrium that favors the outward conformational state of the DAT. In contrast, BZT analogs with reduced dependence on DAT conformation have reduced cocaine-like behavioral effects and may prove useful in development of medications for stimulant abuse.

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The von Hippel-Lindau Protein pVHL Inhibits Ribosome Biogenesis and Protein Synthesis.

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pVHL, product of von Hippel-Lindau (VHL) tumor suppressor gene, functions as the substrate recognition component of an E3-ubiquitin ligase complex that targets hypoxia inducible factor alpha (HIFalpha) for ubiquitination and degradation. Besides HIFalpha, pVHL also interacts with other proteins and has multiple functions. Here, we report that pVHL inhibits ribosome biogenesis and protein synthesis. We find that pVHL associates with the 40S ribosomal protein S3 (RPS3) but does not target it for destruction. Rather, the pVHL-RPS3 association interferes with the interaction between RPS3 and RPS2. Expression of pVHL also leads to nuclear retention of pre-40S ribosomal subunits, diminishing polysomes and 18S rRNA levels. We also demonstrate that pVHL suppresses both cap-dependent and cap-independent protein synthesis. Our findings unravel a novel function of pVHL and provide insight into the regulation of ribosome biogenesis by the tumor suppressor pVHL.

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Decreased Permeability Surface Area for Glucose in Obese Women with Postprandial Hyperglycemia: No Effect of Phosphodiesterase-5 (PDE-5) Inhibition.

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Insulin-mediated microvascular recruitment is recognized as a potential mechanism contributing to insulin resistance. In this study, we compared a marker of microvascular function, the permeability surface area for glucose (PSglu), and forearm glucose uptake after an OGTT in obese women with impaired glucose metabolism and healthy lean nondiabetic women, with the aim to characterize whether decreased permeability surface area for glucose or decreased glucose uptake may contribute to postprandial hyperglycemia in the obese group. In addition, we evaluated whether the phosphodiesterase-5 (PDE-5) inhibitor tadalafil, in a randomized double blind placebo controlled design, might attenuate postprandial glucose levels in obese women. For these purposes, intramuscular microdialysis, blood sampling from arterial and venous blood of the forearm, and measurements of forearm blood flow were performed. The results showed an impaired permeability surface area for glucose (IAUC PSglu 31±13 vs. 124±31; p<0.05) in obese when compared with lean participants, but no differences in forearm glucose uptake appeared between the groups. Furthermore, a single dose of tadalafil 10 mg showed no improvement of the permeability surface area for glucose, glucose uptake, or circulating glucose levels in obese participants. In conclusion, the postprandial PSglu response was impaired in obese women showing postprandial hyperglycemia, indicating a compromised microcirculation. However, we were unable to demonstrate any acute effect on either vascular function or glucose uptake of the phosphodiesterase-5 (PDE-5) inhibitor tadalafil.

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Dietary Flaxseed Oil Supplementation Mitigates the Effect of Lead on the Enzymes of Carbohydrate Metabolism, Brush Border Membrane, and Oxidative Stress in Rat Kidney Tissues.

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Lead is a heavy metal widely distributed in the environment. Lead is a ubiquitous environmental toxin that is capable of causing numerous acute and chronic illnesses. Human and animal exposure demonstrates that lead is nephrotoxic. However, attempts to reduce lead-induced nephrotoxicity were not found suitable for clinical use. Recently, flaxseed oil (FXO), a rich source of ω-3 fatty acids and lignans, has been shown to prevent/reduce the progression of certain types of cardiovascular and renal disorders. In view of this, the present study investigates the protective effect of FXO on lead acetate (PbAc)-induced renal damage. Rats were pre-fed normal diet and the diet rich in FXO for 14 days, and then, four doses of lead acetate (25 mg/kg body weight) were administered intraperitoneally while still on diet. Various serum parameters, enzymes of carbohydrate metabolism, brush border membrane (BBM), and oxidative stress were analyzed in rat kidney. PbAc nephrotoxicity was characterized by increased serum creatinine and blood urea nitrogen. PbAc increased the activities of lactate dehydrogenase and NADP-malic enzyme, whereas it decreased malate and glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, fructose-1, 6-bisphosphatase, and BBM enzyme activities. PbAc caused oxidant/antioxidant imbalances as reflected by increased lipid peroxidation and decreased activities of superoxide dismutase, glutathione peroxidase, and catalase. In contrast, FXO alone enhanced the enzyme activities of carbohydrate metabolism, BBM, and antioxidant defense system. FXO feeding to PbAc-treated rats markedly enhanced resistance to PbAc-elicited deleterious effects. In conclusion, dietary FXO supplementation ameliorated PbAc-induced specific metabolic alterations and oxidative damage by empowering antioxidant defense mechanism and improving BBM integrity and energy metabolism.

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Topological Dangling Bonds with Large Spin Splitting and Enhanced Spin Polarization on the Surfaces of Bi2Se3.

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We investigate the topological surface state properties at various surface cleaves in the topological insulator Bi2Se3, via first principles calculations and scanning tunneling microscopy/spectroscopy (STM/STS). While the typical surface termination occurs between two quintuple layers, we report the existence of a surface termination within a single quintuple layer where dangling bonds form with giant spin splitting owing to strong spin-orbit coupling. Unlike Rashba split states in a 2D electron gas, these states are constrained by the band topology of the host insulator with topological properties similar to the typical topological surface state, and thereby offer an alternative candidate for spintronics usage. We name these new states "topological dangling-bond states". The degree of the spin polarization of these states is greatly enhanced. Since dangling bonds are more chemically reactive, the observed topological dangling-bond states provide a new avenue for manipulating band dispersions and spin-textures by adsorbed atoms or molecules.

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Efficient cell and cell-sheet harvesting based on smart surfaces coated with a multifunctional and self-organizing Elastin-Like Recombinamer.

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A wide range of smart surfaces with novel properties relevant for biomedical applications have been developed recently. Herein we focus on thermoresponsive surfaces that switch between cell-adherent and non-adherent states, and their applications for cell harvesting. These smart surfaces are obtained by covalently coupling a tailored Elastin-Like Recombinamer onto glass surfaces by means of the well-known and widely applied Click Chemistry methodology. The resulting recombinamer-functionalized surfaces have been characterized by means of water contact angle measurements, XPS and TOF-SIMS. A cell-based analysis of these surfaces with human fibroblasts showed a high degree of adhesion to the surface in its adherent state (37ºC), thus promoting cell viability and proliferation. A temperature decrease triggers reorganization of the recombinamer, thus markedly increasing the number of non-adherent domains and masking the adherent ones. This process allows a specific and efficient temporal control of cell adhesion and cell detachment. After determination of the properties required for a suitable cell-harvesting system, optimization of the process allows single cells or cell sheets from at least two types of cells (HFF-1 and ADSCs) to be rapidly harvested.

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Aromatic glycosides from the flower buds of Lonicera japonica.

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Six new glycosides (1-6) have been isolated from the flower buds of Lonicera japonica. Their structures including the absolute configurations were determined by spectroscopic and chemical methods as ( - )-2-hydroxy-5-methoxybenzoic acid 2-O-β-d-(6-O-benzoyl)-glucopyranoside (1), ( - )-4-hydroxy-3,5-dimethoxybenzoic acid 4-O-β-d-(6-O-benzoyl)-glucopyranoside (2), ( - )-(E)-3,5-dimethoxyphenylpropenoic acid 4-O-β-d-(6-O-benzoyl)-glucopyranoside (3), ( - )-(7S,8R)-(4-hydroxyphenylglycerol 9-O-β-d-[6-O-(E)-4-hydroxy-3,5-dimethoxyphenylpropenoyl]-glucopyranoside (4), ( - )-(7S,8R)-(4-hydroxy-3-methoxyphenylglycerol 9-O-β-d-[6-O-(E)-4-hydroxy-3,5-dimethoxyphenylpropenoyl]-glucopyranoside (5), and ( - )-4-hydroxy-3-methoxyphenol β-d-{6-O-[4-O-(7S,8R)-(4-hydroxy-3-methoxyphenylglycerol-8-yl)-3-methoxybenzoyl]}-glucopyranoside (6), respectively.

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Sub-nanomolar Detection of Prostate Specific Membrane Antigen in Synthetic Urine by Synergistic, Dual Ligand Phage.

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The sensitive detection of cancer biomarkers in urine could revolutionize cancer diagnosis and treatment. Such detectors must be inexpensive, easy to interpret, and sensitive. This report describes a bioaffinity matrix of viruses integrated into PEDOT films for electrochemical sensing of prostate specific membrane antigen (PSMA), a prostate cancer biomarker. High sensitivity to PSMA resulted from synergistic action by two different ligands to PSMA on the same phage particle. One ligand was chemically synthesized and wrapped around the phage, and the second was genetically encoded. The dual ligands result in a bidentate binder with high copy, dense ligand display for enhanced PSMA detection through a chelate-based, avidity effect. Biosensing with virus-PEDOT films provides a 100 pM limit of detection for PSMA in synthetic urine without requiring enzymatic or other amplification.

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Oxidative damage and genotoxic effect in mice caused by sub-chronic exposure to low-dose volatile organic compounds.

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Surgical resection of brain metastases-impact on neurological outcome.

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Brain metastases (BM) develop in about 30% of all cancer patients. Surgery plays an important role in confirming neuropathological diagnosis, relieving mass effects and improving the neurological status. To select patients with the highest benefit from surgical resection, prognostic indices (RPA, GPA) have been formulated which are solely focused on survival without considering neurological improvement. In this study we analyzed the impact of surgical resection on the neurological status in addition to overall survival in 206 BM patients. Surgical mortality and morbidity was 0.0% and 10.3% respectively. New neurologic deficits occurred in 6.3% of all patients. The median overall survival was 6.3 months. Poor RPA class and short time interval between diagnosis of cancer and the occurrence of BM were independent factors predictive for poor survival. Improvement of neurological performance was achieved in 56.8% of all patients, with the highest improvement rate seen in patients presenting with increased intracranial pressure and hemiparesis. Notably, the neurological benefits were independent from RPA class. In conclusion, surgical resection leads to significant neurological improvement despite poor RPA class and short overall survival. Considering the low mortality and morbidity rates, resection should be considered as a valid option to increase neurological function and quality of life for patients with BM.

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Molecular sensing: modulating molecular conduction through intermolecular interactions.

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We observe changes in the molecular conductivity of individual oligophenylene-vinylene (OPV) molecules due to interactions with small aromatic molecules. Fluorescence experiments were correlated with scanning tunneling microscopy measurements in order to determine the origin of the observed effect. Both nitrobenzene and 1,4-dinitrobenzene decreased fluorescence intensity and molecular conductivity, while toluene had no effect. The observed changes in the fluorescence and conduction of OPV correlate well with the electron withdrawing ability of the interacting aromatic molecules. These results demonstrate the potential usefulness of OPV as a sensor for aromatic compounds containing electron withdrawing groups.

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In vitro effect of N-acetylcysteine on hepatocyte injury caused by dichlorodiphenyltrichloroethane and its metabolites.

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The organochlorine pesticide, dichlorodiphenyltrichloroethane (DDT), is still used to combat the spread of malaria in several developing countries despite its accumulation and known hepatotoxic effects that have been demonstrated both in vitro and in vivo. N-Acetylcysteine (NAC) is a recognized hepatoprotective agent that has been reported to reduce hepatotoxicity initiated by many different compounds. The aim of this study was to determine whether NAC could counter in vitro hepatocyte injury induced by DDT or its two major metabolites, dichlorodiphenyldichloroethylene and dichlorodiphenyldichloroethane. HepG2 cell cultures were used to assess the following parameters of toxicity: cellular viability, intracellular levels of reactive oxygen species (ROS), mitochondrial membrane potential and initiation of apoptosis. None of the three test compounds induced ROS generation, yet exposure to any of the three compounds produced mitochondrial hyperpolarization, which was countered by NAC pretreatment. All three test compounds also induced apoptotic cell death, which was inhibited by NAC. Despite NAC counteracting some adverse intracellular changes due to organochlorine exposure, it appeared to aggravate the cytotoxic effects of the organochlorine compounds at low test concentrations. As the same outcome may also occur in vivo, results from the present study raise concern about the use of NAC as treatment for DDT-induced hepatotoxicity.

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Improving the default data analysis workflow for large autoimmune biomarker discovery studies with protoarrays.

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Contemporary protein microarrays like the ProtoArray® are used for autoimmune antibody screening studies to discover biomarker panels. For ProtoArray data analysis the software Prospector and a default workflow are suggested by the manufacturer. While analyzing a large data set of a discovery study for diagnostic biomarkers of the Parkinson's Disease ("ParkCHIP") we have revealed the need for distinct improvements of the suggested workflow concerning raw data acquisition, normalization and pre-selection method availability, batch effects, feature selection, and feature validation. In this work appropriate improvements of the default workflow are proposed and it is shown that completely automatic data acquisition as a batch, a re-implementation of Prospector's pre-selection method, multivariate or hybrid feature selection, and validation of the selected protein panel using an independent test set define in combination an improved workflow for large studies. This article is protected by copyright. All rights reserved.

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Elucidation of in situ polycyclic aromatic hydrocarbon degradation by functional metaproteomics (protein-SIP).

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Current knowledge of the physiology and phylogeny of polycyclic aromatic hydrocarbon (PAH) degrading bacteria often relies on laboratory enrichments and isolations. In the present study, in situ microcosms consisting of activated carbon pellets (BACTRAP®s) were loaded with either (13) C-naphthalene or (13) C-fluorene and were subsequently exposed in the contaminant source and plume fringe region of an PAH contaminated aquifer. Metaproteomic analysis and protein-SIP revealed Burkholderiales, Actinomycetales and Rhizobiales as the most active microorganisms in the groundwater communities. Proteins identified of the naphthalene degradation pathway showed a relative (13) C isotope abundance of approximately 50 atom% demonstrating that the identified naphthalene degrading bacteria gained at least 80% of their carbon by PAH degradation. Although the microbial community grown on the fluorene-BACTRAPs showed a structure similar to the naphthalene-BACTRAPs, the identification of fluorene degraders and degradation pathways failed in situ. In complementary laboratory microcosms, a clear enrichment in proteins related to Rhodococcus and possible fluorene degradation enzymes was observed. This result demonstrates the impact of laboratory conditions on microbial community structure and activity of certain species and underlines the need on in situ exploration of microbial community functions. In situ microcosms in combination with protein-SIP may be a significant tool for in situ identification of metabolic key players as well as degradation pathways. This article is protected by copyright. All rights reserved.

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Folate-Conjugated PEG on Single Walled Carbon Nanotubes for Targeting Delivery of Doxorubicin to Cancer Cells.

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A highly effective drug carrier is constructed by coating folic acid-terminated poly(ethylene glycol) (PEG-FA) on single walled carbon nanotubes (SWNTs) in a facile non-covalent method. The anti-cancer drug, doxorubicin (DOX), is further loaded on the surface of SWNTs at a very high loading efficiency, 149.3 ± 4.1%. The drug system (DOX/PEG-FA/SWNTs) exhibits excellent stability under neutral pH conditions such as serum, but dramatically releases DOX at reduced pH typical of the tumour environment and intracellular lysosomes and endosomes. With the help of FA, DOX/PEG-FA/SWNTs tend to selectively attach onto cancer cells and enter the lysosomes or endosomes by clathrin-mediated endocytosis. This can greatly improve the pharmaceutical efficiency and reduce potential side effects.

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Physiotherapeutic approach in early and late post-menopausal Brazilian women.

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Abstract To evaluate changes in joints after physiotherapy in post-menopausal women, specifically to identify clinical responses to the measurements of flexibility, functional capacity and joint pain in early and late post-menopausal women at a multi-disciplinary health education programme. A total of 69 women participated in the Integral Program for the Attention to Climacteric Women at the Department of Gynecology - Federal University of Sao Paulo and were sorted into two groups of early (n = 32) and late (n = 37) post-menopause. The average age of menopause was 47.9 ± 5.6 years. The Blatt Kupperman Menopausal Index scores for the early (baseline = 12.8 ± 6.1) and late (baseline = 14.1 ± 7.7) post-menopausal groups after the programme were 8.4 ± 7.1 and 9.4 ± 8.1, respectively. Both groups presented improvements regarding functional capacity (p < 0.01) and complaints of pain (p < 0.001) after the intervention. The group of early post-menopausal women had better flexibility for hip flexion (p < 0.001), and the late post-menopausal group showed greater improvement in shoulder flexion (p < 0.001), extension (p < 0.001) and elbow flexion (p < 0.001). After multi-disciplinary approach, both early and late post-menopausal groups experienced decrease in intensity of climacteric symptoms, reduction in pain intensity and improvement in functional capacity, but the flexibility was different between both the groups.

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Structural identification of (1→6)-α-D-glucan, a key responsible for the health benefits of longan, and evaluation of anticancer activity.

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Longan is a delicious subtropical fruit with great health-beneficial effects. It has been utilised for disease prevention and health care since ancient age. To explore the chemicals responsible for the health benefits, water-soluble polysaccharides were extracted from longan flesh in this work. A pure polysaccharide (LPS1) was obtained through column purification. Analysis by gas chromatography showed LPS1 was a homopolysaccharide of glucose with glycosidic linkage of →6)-D-Glc-(1→. Nuclear magnetic resonance (NMR ) spectra indicated that the configuration of anomeric carbon in glucose residual was α-form. The polysaccharide structure was further confirmed to be (1→6)-α-D-glucan by chemcial shift of C6. The molecular weight of LPS1 was calculated to be 108 kDa, which had 661 glucose residuals. Anticancer assay showed that LPS1 had anticancer activity against the growth of HepG2 cells to a certain extent. However, it did not show any cytotoxicity against MCF-7 breast cancer cells.

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Hybrid Molecule from O(2)-(2,4-Dinitrophenyl)diazeniumdiolate and Oleanolic Acid: a GSTπ Activated Nitric Oxide Prodrug with Selective Anti-human Hepatocellular Carcinoma Activity and Improved Stability.

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A series of hybrids from O(2)-(2,4-dinitrophenyl)diazeniumdiolate and oleanolic acid (OA) were designed, synthesized, and biologically evaluated as novel nitric oxide (NO) releasing prodrugs which could be activated by glutathione S-transferase π (GSTπ) overexpressed in a number of cancer cells. It was discovered that the most active compound 21 released high levels of NO selectively in HCC cells but not in the normal cells, and exhibited potent antiproliferative activity in vitro as well as remarkable tumor-retarding effects in vivo. Compared with the reported GSTπ-activated prodrugs JS-K and PABA/NO, 21 exhibited remarkably improved stability in the absence of GSTπ. Importantly, the decomposition of 21 occurred in the presence of GSTπ, and was much more effective than in GSTα. Additionally, 21 induced HepG2 cells apoptosis by arresting cell cycle at G2/M phase, activating both the mitochondria-mediated pathway and the MAPKs pathway, as well as enhancing the intracellular production of ROS.

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Rationale for the higher reactivity of interfacial sites in methanol decomposition on Au13/TiO2(110).

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Cadmium-Based Quantum Dot Induced Autophagy Formation for Cell Survival via Oxidative Stress.

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Quantum dots (QDs) are one of most utilized nanomaterials in nanocrystalline semiconductors. QDs emit near-infrared fluorescence and can be applied as probes for detecting vasculature and imaging in biological systems. Since QDs have potential in clinical application, the toxicity of QDs needs to be carefully evaluated. In our present study, we elucidate the cytotoxic mechanisms of QDs using a mouse renal adenocarcinoma (RAG) cell line. QDs in RAG cells increased intracellular reactive oxygen species (ROS) levels and induced autophagy at 6 h, leading to subsequent apoptosis at 24 h. QDs entered the cells and were located within the endoplasmic reticulum (ER), endosome, and lysosome at 6 h and endosome, lysosome, and mitochondria at 24 h. However, QDs only affected mitochondrial function and did not induce ER stress. N-Acetylcysteine, an antioxidant agent, reduced intracellular ROS levels and decreased QD-induced autophagy but enhanced QD-induced cell death. Moreover, 3-methylamphetamine (an autophagy inhibitor) also reduced the cell viability in QD-treated cells. These findings suggest that ROS plays an essential role in the regulation of QD-induced autophagy, which subsequently enhances cell survival. Taken together, these results suggest that oxidative stress-induced autophagy is a defense/survival mechanism against the cytotoxicity of QD.

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MicroRNA-32 (miR-32) regulates phosphatase and tensin homologue (PTEN) expression and promotes growth, migration, and invasion in colorectal carcinoma cells.

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BACKGROUND: Colorectal carcinoma (CRC) is one of the leading causes of cancer-related mortality worldwide. MicroRNAs (miRNAs, miRs) play important roles in carcinogenesis. MiR-32 has been shown to be upregulated in CRC. In this study, we identified the potential effects of miR-32 on some important biological properties of CRC cells, and clarified the regulation of PTEN by miR-32. METHODS: The effect of miR-32 on PTEN expression was assessed in CRC cell lines with miR-32 mimics/inhibitor to increase/decrease miR-32 expression. Furthermore, the roles of miR-32 in regulating CRC cells biological properties were analyzed with miR-32 mimics/inhibitor-transfected cells. The 3[prime]-untranslated region (3[prime]-UTR) of PTEN combined with miR-32 was verified by dual-luciferase reporter assay. RESULTS: Gain-of-function and loss-of-function studies showed that overexpression of miR-32 promoted SW480 cell proliferation, migration, and invasion, reduced apoptosis, and resulted in downregulation of PTEN at a posttranscriptional level. However, miR-32 knock-down inhibited these processes in HCT-116 cells and enhanced the expression of PTEN protein. In addition, we further identified PTEN as the functional downstream target of miR-32 by directly targeting the 3[prime]-UTR of PTEN. CONCLUSIONS: Our results demonstrated that miR-32 was involved in tumorigenesis of CRC at least in part by suppression of PTEN.

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Structure--activity relationships and in silico models of P-glycoprotein (ABCB1) inhibitors.

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Abstract 1. The efflux pump p-glycoprotein (P-gp/ABCB1) has received enormous attention in drug (xenobiotic) disposition due to its role in modulation of the drug availability and in protection of sensitive organs. 2. P-gp mediated efflux is one of main mechanisms for multidrug resistance in cancer cells. A main approach to reverse the resistance and restore the drug efficacy is to use specific inhibitors of P-gp that suppress the efflux activity. 3. This review summarizes the binding capabilities of known chemical inhibitors based on the analyses of structure-activity relationships, and computational modeling of the inhibitors as well as the binding site of P-gp protein. 4. The molecular models will facilitate the design of lead inhibitors as drug candidates. Also, it helps scientists in early drug discovery phase to synthesize chemical series with better understanding of their P-gp binding liabilities.

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Nasal dosimetry of inspired naphthalene vapor in the male and female B6C3F1 mouse.

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Naphthalene vapor is a nasal cytotoxicant in the rat and mouse but is a nasal carcinogen in only the rat. Inhalation dosimetry is a critical aspect of the inhalation toxicology of inspired vapors and may contribute to the species differences in the nasal response. To define the nasal dosimetry of naphthalene in the B6C3F1 male and female mouse, uptake of naphthalene vapor was measured in the surgically isolated upper respiratory tract (URT) at inspiratory flow rates of 25 or 50ml/min. Uptake was measured at multiple concentrations (0.5, 3, 10, 30ppm) in controls and mice treated with the cytochrome P450 inhibitor 5-phenyl-1-pentyne. In both sexes, URT uptake efficiency was strongly concentration dependent averaging 90% at 0.5ppm compared to 50% at 30ppm (25ml/min flow rate), indicating saturable processes were involved. Both uptake efficiency and the concentration dependence of uptake were significantly diminished by 5-phenyl-1-pentyne indicating inspired naphthalene vapor is extensively metabolized in the mouse nose with saturation of metabolism occurring at the higher concentrations. A hybrid computational fluid dynamic physiologically based pharmacokinetic model was developed for nasal dosimetry. This model accurately predicted the observed URT uptake efficiencies. Overall, the high URT uptake efficiency of naphthalene in the mouse nose indicates the absence of a tumorigenic response is not attributable to low delivered dose rates in this species.

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Exposure to DEHP decreased four fatty acid levels in plasma of prepartum mice.

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Maternal exposure to di(2-ethylhexyl) phthalate (DEHP) decreased the plasma triglyceride in prepartum mice. To identify the fatty acid (FA) species involved and to understand the underlying mechanisms, pregnant Sv/129 wild-type (mPPARα), peroxisome proliferator-activated receptor α-null (Pparα-null) and humanized PPARα (hPPARα) mice were treated with diets containing 0%, 0.01%, 0.05% or 0.1% DEHP. Dams were dissected on gestational day 18 together with fetuses, and on postnatal day 2 together with newborns. n-3/n-6 polyunsaturated, saturated, and monounsaturated FAs in maternal plasma and in liver of wild-type offspring, and representative enzymes for FA desaturation and elongation in maternal liver, were measured. The plasma levels of linoleic acid, α-linolenic acid, palmitic acid and oleic acid were higher in the pregnant control mPPARa mice than in Ppara-null and hPPARa mice. DEHP exposure significantly decreased the levels of these four FAs only in pregnant mPPARα mice. Plasma levels of many FAs were higher in pregnant mice than in postpartum ones in a genotype-independent manner, while it was lower in the livers of fetuses than pups. DEHP exposure slightly increased hepatic arachidonic acid, α-linolenic acid, palmitoleic acid and oleic acid in fetuses, but not in pups. However, DEHP exposure did not clearly influence FA desaturase 1 and 2 nor elongase 2 and 5 expressions in the liver of all maternal mice. Taken together, the levels of plasma four FAs with shorter carbon chains were higher in pregnant mPPARα mice than in other genotypes, and DEHP exposure decreased these specific FA concentrations only in mPPARα mice, similarly to triglyceride levels.

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The role of methyl-CpG binding protein 2 in liver fibrosis.

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Liver injure is induced by various insults such as alcohol abuse, if insults persistent, may result in the formation of liver fibrosis. Hepatic stellate cell (HSC) activation and transdifferentiation into hepatic myofibroblast, accompanied with potent pro-inflammatory and pro-fibrogenic activities and the down-regulation of anti-inflammatory anti-fibrogenic in gene expression in coordination with epigenetic modifications at the level of the chromatin structure, are pivotal events in liver fibrogenesis. In this review we focus on the role of the methyl-CpG binding protein 2 (MeCP2) transcriptional regulation of different target genes and the interaction MeCP2 with microRNAs (miRNAs) during liver fibrosis. In addition, we address different signaling pathways interacted with MeCP2 regulated HSC activation. Such approaches provide valuable insights into the potential targets of liver fibrosis, and are useful pointers for the development of future therapeutic strategies.

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Dabigatran for the Prevention of Stroke and Systemic Embolism in Atrial Fibrillation: A NICE Single Technology Appraisal.

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The National Institute for Health and Clinical Excellence (NICE) invited the manufacturer of dabigatran etexilate (Boehringer Ingelheim Ltd, UK) to submit evidence for the clinical and cost-effectiveness of this drug for the prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation (AF) as part of the NICE single technology appraisal process. The Centre for Reviews and Dissemination and the Centre for Health Economics at the University of York were commissioned to act as the evidence review group (ERG). This article presents a summary of the manufacturer's submission, the ERG report and the subsequent development of NICE guidance for the use of dabigatran within the UK National Health Service. Dabigatran was granted marketing authorisation by the European Medicines Agency for a sequential dosing regimen (DBG sequential), in which patients under 80 years are treated with dabigatran 150 mg twice daily (DBG150) and patients 80 years and over are given dabigatran 110 mg twice daily (DBG110). NICE decisions are bound by the marketing authorisation; therefore, the decision problem faced by the committee was whether the DBG sequential regimen was effective and cost-effective compared with warfarin or aspirin for patients with non-valvular AF and one or more risk factors. The RE-LY trial, a large multi-centre non-inferiority randomised clinical trial, was the primary source of clinical evidence. DBG150 was shown to be non-inferior, and subsequently superior to warfarin, for the primary outcome of all stroke/systemic embolism. DBG110 was found to be non-inferior to warfarin. Results were presented for a post hoc subgroup analysis for patients under and over 80 years of age, where DBG110 showed a statistically significant reduction of haemorrhagic stroke and intracranial haemorrhage in comparison to warfarin in patients over 80 years of age. This post hoc subgroup analysis by age was the basis for the licensed DBG sequential regimen. The economic evaluation compared the costs and outcomes of DBG110, DBG150 and DBG sequential against warfarin, aspirin, and aspirin plus clopidogrel. Across the three dosing regimens, dabigatran was associated with greater costs and better health outcomes than warfarin; however, DBG150 offered the most benefits and dominated DBG110 and DBG sequential (i.e. less costly and more effective). The cost-effectiveness of DBG150 was less favourable for patients well controlled on warfarin. In the first appraisal meeting, the committee issued a 'minded no' decision until additional analyses on the licensed DBG sequential regimen were presented by the manufacturer. These additional analyses indicated that the incremental cost-effectiveness ratio (ICER) of the DBG sequential regimen compared with warfarin ranged from £8,388 to £18,987 per quality-adjusted life year (QALY) gained depending on the level of monitoring costs assumed for warfarin. Patients on warfarin would need to be within therapeutic range 83-85 % of the time for the ICER to exceed £30,000 per additional QALY. Following consideration of the additional evidence and the responses from a large number of consultees and commentators, the committee recommended dabigatran as DBG sequential as an option for the prevention of stroke and systemic embolism in people with non-valvular AF with one or more risk factors for ischaemic stroke.

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Asymmetric zinc-catalyzed hydrosilylation of ketones and the effect of carboxylate on the enantioselectivity.

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Several chiral ligands containing (R,R)-diaminocyclohexane moieties and pyrrole, furan, or benzene have been synthesized. These ligands were tested in enantioselective zinc-catalyzed hydrosilylation reactions; excellent enantioselectivities were obtained when the ligands containing (R,R)-diaminocyclohexane moieties and furan rings were used. For comparison, zinc chloride combined with different potassium carboxylate salts and ligands were also tested for catalytic hydrosilylation reactions. Chirality 25:275-280, 2013. © 2013 Wiley Periodicals, Inc.

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Variability in P-Glycoprotein Inhibitory Potency (IC50) Using Various In Vitro Experimental Systems: Implications for Universal Digoxin DDI Risk Assessment Decision Criteria.

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A P-glycoprotein (P-gp) IC50 working group was established with twenty-three participating pharmaceutical and contract research laboratories and one academic institution to assess inter-laboratory variability in P-gp IC50 determinations. Each lab followed their in-house protocol to determine in vitro IC50 values for sixteen inhibitors using four different test systems: Caco-2 (11 labs), MDCKII-MDR1 (6 labs) and LLC-PK1-MDR1 (4 labs) cells, and membrane vesicles containing human P-gp (5 labs). For cell models, various equations to calculate remaining transport activity (e.g. efflux ratio, unidirectional flux, net secretory flux) were also evaluated. The difference in IC50 values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC50 values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC50 values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio based-equation generally resulted in several fold lower IC50 values compared to unidirectional or net-secretory flux equations. Statistical analysis indicated that variability in IC50 values was mainly due to inter-laboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC50 determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion paper and recommendations are provided.

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Mechanism of Fibrinogen Adsorption at Solid Substrates at lower pH.

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Adsorption of fibrinogen was theoretically studied using the three dimensional (3D) random sequential adsorption (RSA) model. Fibrinogen molecule shape was approximated by the bead model considering the presence of flexible side arms. Various cases were considered inter alia, the side-on adsorption mechanisms and the 3D side-on/end-on simultaneous adsorption mechanism. The latter mechanisms is pertinent to fibrinogen adsorption at lower pH where the entire molecule is positively charged. Extensive calculations enabled one to determine the jamming surface concentration (coverage) of molecules adsorbed under the side-on and end-on orientations as well as the total coverage. For the 3D model the maximum surface concentration was 7.29x103 μm-2 corresponding to the protein coverage of 4.12 mg m-2 (without considering hydration). Additionally, the surface blocking functions for the side-on and the 3D adsorption regimes were determined and analytically approximated for the entire range of coverage by the interpolating polynomials. Using these blocking functions, fibrinogen adsorption kinetics for diffusion controlled transport conditions was evaluated. Comparison of these theoretical results with experimental data was made. It was demonstrated that the 3D model properly reflects the maximum coverage of fibrinogen adsorbed on latex particles determined via the electrokinetic (electrophoretic mobility) and AFM measurements. Also, streaming potential measurements of fibrinogen adsorption kinetics on mica were successfully interpreted in terms of the 3D adsorption model. The theoretical results derived in this work have implications for basic science providing information on mechanisms of anisotropic protein adsorption. Key words: adsorption of fibrinogen, fibrinogen adsorption at lower pH, irreversible adsorption of fibrinogen, jamming coverage of fibrinogen, kinetics of fibrinogen adsorption, random sequential adsorption modeling of fibrinogen adsorption.

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Aggregation-Mediated Macromolecular Uptake by a Molecular Transporter.

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Endocytosis is a key process in cellular delivery of macromolecules by molecular transporters, although the mechanism of internalization remains unclear. Here, we probe the cellular uptake of streptavidin using biotinylated guanidinoneomycin (biotinGNeo), a low molecular weight guanidinium-rich molecular transporter. Two distinct modes were explored: (i) incubation of cells with a preformed tetravalent streptavidin-(biotinGNeo)4 conjugate and (ii) preincubation of cells with the biotinGNeo before exposure to streptavidin. A significant enhancement in uptake was observed after preincubation with biotinGNeo. FRET studies showed that the enhanced uptake was accompanied by extensive aggregation of streptavidin on the cell surface. Because guanidinylated neomycin was previously found to exclusively bind to heparan sulfate, our observations suggest that heparan sulfate proteoglycan aggregation is a pivotal step for endocytic entry into cells by guanidinoglycosides. These observations put forward a practical and general pathway for the cellular delivery of diverse macromolecules.

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Study of Charge-Dependent Transport and Toxicity of Peptide-Functionalized Silver Nanoparticles Using Zebrafish Embryos and Single Nanoparticle Plasmonic Spectroscopy.

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Small molecule mediated proliferation of primary retinal pigment epithelial cells.

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Retinal pigment epithelial (RPE) cells form a monolayer adjacent to the retina and play a critical role in the visual light cycle. Degeneration of this layer results in vision loss, causing retinal disorders such as age-related macular degeneration. Cell transplant therapies exist to restore vision loss; however, risks associated with and an inadequate supply of donor cells have limited their therapeutic success. The identification of factors that proliferate RPE cells ex vivo could provide a renewable source of cells for the treatment of such disorders. We show that a small molecule (WS3) can reversibly proliferate primary RPE cells isolated from fetal and adult human donors. Following withdrawal of WS3, RPE cells differentiate into a functional monolayer, as exhibited by their expression of mature RPE genes and phagocytosis of photoreceptor outer segments. Furthermore, chemically expanded RPE cells preserve vision when transplanted into dystrophic Royal College of Surgeons (RCS) rats, a well-established model of retinal degeneration.

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Two new vinblastine-type N-oxide alkaloids from Catharanthus roseus.

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Two new vinblastine-type N-oxide alkaloids, 17-desacetoxyvinblastine [Formula: see text]-oxide (1) and 20'-deoxyvinblastine [Formula: see text]-oxide (2), were isolated from the leaves of Catharanthus roseus. The structures of 1 and 2 were established by the analysis of their nuclear magnetic resonance and HR-ESI-MS spectroscopic data. All alkaloids were evaluated for their cytotoxic activities against the human hepatocellular carcinoma (HepG2) cell line, human colorectal carcinoma (Lovo) cell line and human breast carcinoma (MCF-7) cell line by the MTT method in vitro, respectively. The results showed that cytotoxic activities of alkaloids 1 and 2 exhibited moderate inhibitory activity on the proliferation of three cancer cells.

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Mechanism-based design of a photo-activatable firefly Luciferase.

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We developed a photo-activatable firefly luciferase (fLuc) whose activation can be controlled by light. A photocaged lysine analogue was site-specifically incorporated into fLuc to replace its key catalytic lysine residue-Lys529, rendering fLuc inactive until light-triggered removal of the caging group. This photo-induced gain-of-luminescence provides a facile approach for assessing the photolysis efficiency of this valuable photosensitive lysine analogue within the context of its carrier protein in vitro and in living cells. We further took advantage of the spatial and temporal activation feature of pfLuc for intracellular measurement of labile ATP levels without impairment of a cell's physiology.

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The Histone Mark H3K36me3 Regulates Human DNA Mismatch Repair through Its Interaction with MutSα.

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DNA mismatch repair (MMR) ensures replication fidelity by correcting mismatches generated during DNA replication. Although human MMR has been reconstituted in vitro, how MMR occurs in vivo is unknown. Here, we show that an epigenetic histone mark, H3K36me3, is required in vivo to recruit the mismatch recognition protein hMutSα (hMSH2-hMSH6) onto chromatin through direct interactions with the hMSH6 PWWP domain. The abundance of H3K36me3 in G1 and early S phases ensures that hMutSα is enriched on chromatin before mispairs are introduced during DNA replication. Cells lacking the H3K36 trimethyltransferase SETD2 display microsatellite instability (MSI) and an elevated spontaneous mutation frequency, characteristic of MMR-deficient cells. This work reveals that a histone mark regulates MMR in human cells and explains the long-standing puzzle of MSI-positive cancer cells that lack detectable mutations in known MMR genes.

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Antiproliferative activity of 2,3-disubstituted indoles toward apoptosis-resistant cancers cells.

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Many types of cancer, including glioma, melanoma, NSCLC, among others, are resistant to apoptosis induction and poorly responsive to current therapies with propaptotic agents. We describe a series of 2,3-disubstituted indoles, which display cytostatic rather than cytotoxic effects in cancer cells, and serve as a new chemical scaffold to develop anticancer agents capable of combating apoptosis-resistant cancers associated with dismal prognoses.

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Synthesis and evaluation of Janus type nucleosides as potential HCV NS5B polymerase inhibitors.

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The synthesis of new ribo and 2'-β-C-methyl ribo Janus type nucleosides J-AA, J-AG and J-AU is reported along with their ability to block HCV and HIV replication. Their toxicity was also assessed in Huh7, human lymphocytes, CEM and Vero cells.

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The Demographic Transition Influences Variance in Fitness and Selection on Height and BMI in Rural Gambia.

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Recent human history is marked by demographic transitions characterized by declines in mortality and fertility [1]. By influencing the variance in those fitness components, demographic transitions can affect selection on other traits [2]. Parallel to changes in selection triggered by demography per se, relationships between fitness and anthropometric traits are also expected to change due to modification of the environment. Here we explore for the first time these two main evolutionary consequences of demographic transitions using a unique data set containing survival, fertility, and anthropometric data for thousands of women in rural Gambia from 1956-2010 [3]. We show how the demographic transition influenced directional selection on height and body mass index (BMI). We observed a change in selection for both traits mediated by variation in fertility: selection initially favored short females with high BMI values but shifted across the demographic transition to favor tall females with low BMI values. We demonstrate that these differences resulted both from changes in fitness variance that shape the strength of selection and from shifts in selective pressures triggered by environmental changes. These results suggest that demographic and environmental trends encountered by current human populations worldwide are likely to modify, but not stop, natural selection in humans.

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First demonstration that brain CYP2D-mediated opiate metabolic activation alters analgesia in vivo.

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The response to centrally-acting drugs is highly variable between individuals and does not always correlate with plasma drug levels. Drug-metabolizing CYP enzymes in the brain may contribute to this variability by affecting local drug and metabolite concentrations. CYP2D metabolizes codeine to the active morphine metabolite. We investigate the effect of inhibiting brain, and not liver, CYP2D activity on codeine-induced analgesia. Rats received intracerebroventricular injections of CYP2D inhibitors (20μg propranolol or 40μg propafenone) or vehicle controls. Compared to vehicle-pretreated rats, inhibitor-pretreated rats had: a) lower analgesia in the tail-flick test (p<0.05) and lower areas under the analgesia-time curve (p<0.02) within the first hour after 30mg/kg subcutaneous codeine, b) lower morphine concentrations and morphine to codeine ratios in the brain (p<0.02 and p<0.05, respectively), but not in plasma (p>0.6 and p>0.7, respectively), tested at 30min after 30mg/kg subcutaneous codeine, and c) lower morphine formation from codeine ex vivo by brain membranes (p<0.04), but not by liver microsomes (p>0.9). Analgesia trended toward a correlation with brain morphine concentrations (p=0.07) and correlated with brain morphine to codeine ratios (p<0.005), but not with plasma morphine concentrations (p>0.8) or plasma morphine to codeine ratios (p>0.8). Our findings suggest that brain CYP2D affects brain morphine levels after peripheral codeine administration, and may thereby alter codeine's therapeutic efficacy, side-effect profile and abuse liability. Brain CYPs are highly variable due to genetics, environmental factors and age, and may therefore contribute to interindividual variation in the response to centrally-acting drugs.

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Ethnopharmacological and bioactivity guided investigation of five TCM anticancer herbs.

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ETHNOPHARMACOLOGICAL RELEVANCE: Five herbs, Curcuma longa L. (CL), Scutellaria baicalensis Georgi (SBC), Scutellaria barbata D. Don (SBB), Hedyotis diffusa Willd. (HD) and Solanum nigrum L. (SN), are often prescribed in the polyherbal formulas for cancer treatment by traditional Chinese medicine (TCM) practitioners. The purpose of the present study was to identify important anticancer herbs used in TCM and carry out bioactivity-directed fractionation and isolation (BDFI) using six cancer cell lines as well as peripheral blood mononuclear cells (PBMCs), to identify constituents with anticancer activity but devoid of toxic effects against healthy immune cells. MATERIALS AND METHODS: Of 243 document anticancer TCM treatments, 199 anticancer TCM herbs were ranked by the number of literature reports for each herb. Five herbs were identified from the top 50 ranked herbs by at least two out of three TCM practitioners as frequently used in the TCM treatment of cancer. BDFI using MTS assay was applied to determine the active anticancer extracts, fractions, and finally discrete compounds. RESULTS: Five herbs were selected for study of their anticancer activities. The extracts of Curcuma longa L., Scutellaria barbata D. Don, and Hedyotis diffusa showed antiproliferative activity to various extents, extracts of Scutellaria baicalensis Georgi and Solanum nigrum L. showed little anticancer activity. Seven out of the 21 fractions obtained from Hedyotis diffusa showed anticancer activity. One new compound, ethyl 13(2) (S)-hydroxy-chlorophyllide a(1), along with 10 known compounds, i.e. 2-methyl-3-methoxyanthraquinone (2), 2-hydroxymethylanthraquinone(3), 2-hydroxy-3-methylanthraquinone(4), 2-hydroxymethy-1-hydroxyanthraquinone(5), 1-methoxy-2-hydroxyanthraquinone(6), 2-hydroxy-3-methyl-1-methoxyanthraquinone (7), oleanolic acid (8), ursolic acid (9), stigmasterol (10) and docosanoic acid (11), were isolated and identified. Compounds 2-6, 8 and 9 dose-dependently inhibited the cell viability of cancer cells within a concentration range of 1-200µM. Furthermore, compounds 2, 3, 5 and 9 showed significantly stronger inhibition of tested cancer cell lines than on that of PBMCs. CONCLUSION: This study identified anticancer herbs, extracts, fractions and eventually compounds from the documented anticancer TCM herbs by using BDFI. It also determined the antiproliferative activity in cancer and healthy immune cells of the isolated compounds from Hedyotis diffusa. The results will be useful in the validation of the clinical application of these herbs and the development of novel anticancer therapeutics.

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Production of Cyr61 protein is modulated by extracellular acidification and PI3K/Akt signaling in prostate carcinoma PC-3 cells.

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High expression of Cyr61, an extracellular cysteine-rich heparin-binding protein, has been associated with a malignant cell phenotype and poor outcome in prostate cancers. Although Cyr61 was found by us to be overproduced in androgen-independent PC-3 cells treated with N-acetylcysteine (NAC), its significance is still unclear. We therefore aimed to determine how and why Cyr61 protein is overexpressed in NAC-treated cells. Here, we found that Cyr61 protein level markedly increased in cells treated with NAC at high cell seeding density. Silencing of Cyr61 by siRNA induced enhanced activity of caspase-3/7, upregulation of the proapototic Bok, BimL and BimS, cleavage of apoptosis hallmarkers such as Bax, PARP and caspase-3, and downregulation of antiapoptotic Bcl2, Bcl-xL and Mcl-1 proteins. NAC treatment caused a reduction of extracellular medium pH to acidic and an increase in Akt phosphorylation, after which the replacement with NAC-free medium returned them to control levels within 24h. Acid stimulation increased the levels of Cyr61 and p-Akt proteins, whereas it suppressed the induction of proapoptotic and antiapoptotic proteins. Overall, our data indicate that PC-3 cells overproduce Cyr61 protein via activation of the PI3K/Akt signaling as a part of the survival mechanisms under the conditions causing extracellular acidity and further cytotoxicity.

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Comparative evaluation of oral and dermal cypermethrin exposure on antioxidant profile in Bubalus bubalis.

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Cypermethrin, a type II synthetic pyrethroid insecticide, @ 0.5mg/kg/day for 14 consecutive weeks produced mild signs of toxicity in buffalo calves. Significant changes were observed in various antioxidant parameters in blood. There was a marked increase in the extent of lipid peroxidation (33.9%) and enzymic activity of glutathione peroxidase (6.7%), superoxide dismutase (35.0%), catalase (43.7%), glutathione-S-transferase (64.4%), glutathione reductase (36.7%) and glucose-6-phosphate dehydrogenase (32.1%). A significant decrease in blood glutathione (16.7%), total antioxidant activity (45.4%) and vitamin E (40.8%) was observed and no significant effect was found on blood selenium levels. However, the extent of lipid peroxidation (42%) and the depletion of glutathione (28.8%) was greater after dermal sub-acute toxicity of cypermethrin (0.25%) for 14 consecutive days. Similarly, it was observed that the incline in the enzymic activity of glutathione peroxidase (29.7%), superoxide dismutase (38.3%) and glutathione reductase (38.3%) was higher in dermally cypermethrin exposed animals. Thus, the present investigation contemplates that oxidative stress is the important mechanisms involved in cypermethrin-induced toxicity and the oxidative insult produced by dermal route is more severe as compared to oral intoxication.

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Biological effects induced by BSA-stabilized silica nanoparticles in mammalian cell lines.

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Much of the concerns regarding engineered nanoparticle (NP) toxicity are based on knowledge from previous studies on particles in ambient air or occupational situations. E.g., the effects of exposure to silica dust particles have been studied intensely due to the carcinogenicity of crystalline silica. However, the increasing usage of engineered amorphous silica NPs has emphasized the need for further mechanistic insight to predict the consequences of exposure to the amorphous type of silica NPs. The present study focused on the in vitro biological effects following exposure to well-dispersed, BSA-stabilized, amorphous silica NPs whereas unmodified silica NPs where included for reasons of comparison. The cytotoxicity of the silica NPs was investigated in six different cell lines (A549, THP-1, CaCo-2, ASB-XIV, J-774A.1, and Colon-26) selected to explore the significance of organ and species sensitivity in vitro. Viability data demonstrated that macrophages were most sensitive to silica NP and interestingly, murine cell lines were generally found to be more sensitive than comparable human cell lines. Further studies were conducted in the human epithelial lung cell line, A549, to explore the molecular mechanism of silica toxicity. Generation of reactive oxygen species, one of the proposed toxicological mechanisms of NPs, was investigated in A549 cells by the dichlorofluorescin (DCF) assay to be significantly induced at NP concentrations above 113μg/mL. However, induction of oxidative stress related pathways was not found after silica NP exposure for 24h in gene array studies conducted in A549 cells at a relatively low NP concentration (EC20). Up-regulated genes (more than 2-fold) were primarily related to lipid metabolism and biosynthesis whereas down-regulated genes included several processes such as transcription, cell junction, extra cellular matrix (ECM)-receptor interaction and others. Thus, gene expression data proposes that several cellular processes other than oxidative stress could be affected by exposure to silica NPs.

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