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Enzymatic Digestion (enzymatic + digestion)
Selected AbstractsEnzymatic digestion of liquid hot water pretreated hybrid poplarBIOTECHNOLOGY PROGRESS, Issue 2 2009Youngmi Kim Abstract Liquid hot (LHW) water pretreatment (LHW) of lignocellulosic material enhances enzymatic conversion of cellulose to glucose by solubilizing hemicellulose fraction of the biomass, while leaving the cellulose more reactive and accessible to cellulase enzymes. Within the range of pretreatment conditions tested in this study, the optimized LHW pretreatment conditions for a 15% (wt/vol) slurry of hybrid poplar were found to be 200oC, 10 min, which resulted in the highest fermentable sugar yield with minimal formation of sugar decomposition products during the pretreatment. The LHW pretreatment solubilized 62% of hemicellulose as soluble oligomers. Hot-washing of the pretreated poplar slurry increased the efficiency of hydrolysis by doubling the yield of glucose for a given enzyme dose. The 15% (wt/vol) slurry of hybrid poplar, pretreated at the optimal conditions and hot-washed, resulted in 54% glucose yield by 15 FPU cellulase per gram glucan after 120 h. The hydrolysate contained 56 g/L glucose and 12 g/L xylose. The effect of cellulase loading on the enzymatic digestibility of the pretreated poplar is also reported. Total monomeric sugar yield (glucose and xylose) reached 67% after 72 h of hydrolysis when 40 FPU cellulase per gram glucan were used. An overall mass balance of the poplar-to-ethanol process was established based on the experimentally determined composition and hydrolysis efficiencies of the liquid hot water pretreated poplar. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Stress-related RNase PR-10c is post-translationally modified by glutathione in birchPLANT CELL & ENVIRONMENT, Issue 6 2002K. M. Koistinen Abstract The PR-10c (previously termed as Bet v 1-Sc3) protein of birch belongs to the family of intracellular pathogenesis-related proteins. The high-performance liquid chromatography electrospray ionization ion trap mass spectrometry (HPLC-ESI-MS) analysis of PR-10c-His fusion protein, produced in Escherichia coli, revealed three major peaks and masses. Enzymatic digestions and HPLC-ESI-MS and matrix assisted laser desorption/ionization , time of flight mass spectrometry (MALDI-TOF-MS) analyses of each fraction indicated that PR-10c-His protein is post-translationally modified by carbamylation and S-glutathiolation. Carbamylation was localized into the N-terminal end of PR-10c-His and does not represent a biologically significant modification. The possible nuclease activity of PR-10c was analysed with S-glutathiolated and reduced fractions of PR-10c-His fusion protein. Both forms of PR-10c-His as well as the dimeric form of the protein possess RNase activity which is capable of digesting different RNA substrates. None of the fractions showed activity against single- or double-stranded DNA. The MALDI-TOF-MS analysis of PR-10c polypeptide extracted from zinc-exposed birch roots showed that the protein is post-translationally modified by glutathione (, -Glu-Cys-Gly) also in vivo. The S-glutathiolated cysteine residue of PR-10c is not conserved among Bet v 1 homologous proteins and is also unique in the PR-10 family. As far as we know this is the first observation of S-glutathiolation in plants, or any post-translational modification in the PR-10 family of proteins. [source] Development of a simulated earthworm gut for determining bioaccessible arsenic, copper, and zinc from soil,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 7 2009Wai K. Ma Abstract Soil physicochemical characteristics and contamination levels alter the bioavailability of metals to terrestrialinvertebrates. Current laboratory-derived benchmark concentrations used to estimate risk do not take into account site-specific conditions, such as contaminant sequestration, and site-specific risk assessment requires a battery of time-consuming and costly toxicity tests. The development of an in vitro simulator for earthworm bioaccessibility would significantly shorten analytical time and enable site managers to focus on areas of greatest concern. The simulated earthworm gut (SEG) was developed to measure the bioaccessibility of metals in soil to earthworms by mimicking the gastrointestinal fluid composition of earthworms. Three formulations of the SEG (enzymes, microbial culture, enzymes and microbial culture) were developed and used to digest field soils from a former industrial site with varying physicochemical characteristics and contamination levels. Formulations containing enzymes released between two to 10 times more arsenic, copper, and zinc from contaminated soils compared with control and 0.01 M CaCl2 extractions. Metal concentrations in extracts from SEG formulation with microbial culture alone were not different from values for chemical extractions. The mechanism for greater bioaccessible metal concentrations from enzyme-treated soils is uncertain, but it is postulated that enzymatic digestion of soil organic matter might release sequestered metal. The relevance of these SEG results will need validation through further comparison and correlation with bioaccumulation tests, alternative chemical extraction tests, and a battery of chronic toxicity tests with invertebrates and plants. [source] Evidence for intestinal chloride secretionEXPERIMENTAL PHYSIOLOGY, Issue 4 2010Michael Murek Intestinal fluid secretion is pivotal in the creation of an ideal environment for effective enzymatic digestion, nutrient absorption and stool movement. Since fluid cannot be actively secreted into the gut, this process is dependent on an osmotic gradient, which is mainly created by chloride transport by the enterocyte. A pathological dysbalance between fluid secretion and absorption leads to obstruction or potentially fatal diarrhoea. This article reviews the widely accepted model of intestinal chloride secretion with an emphasis on the molecular players involved in this tightly regulated process. [source] Association of vitamin D receptor genotypes with early onset rheumatoid arthritisINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 1 2001J. R. Garcia-Lozano The presence of certain vitamin D receptor (VDR) genotypes has been associated with low bone mineral density (BMD) in elderly populations as well as with accelerated bone loss in patients with rheumatoid arthritis (RA). In the present study, VDR genotypes from 120 Spanish patients with RA were investigated. Three VDR gene polymorphisms (BsmI, ApaI and TaqI) were investigated using polymerase chain reaction followed by enzymatic digestion. The distributions of VDR allelic frequencies were similar in patients and controls and therefore no influence of VDR polymorphisms on rheumatoid arthritis susceptibility could be demonstrated. However, in an analysis of the clinical features of the different VDR-related genetic subgroups, the BB/tt genotype, defined by the BsmI and TaqI restriction site polymorphisms, was identified to be weakly associated with an early onset RA in female patients. This VDR genotype has been associated with a low BMD level in various studies. When patients were stratified according to the presence of the shared HLA epitope SE, it was found that SE + female patients bearing the BB/tt genotype showed the earliest disease onset. The mechanisms by which the VDR polymorphism is associated with RA is unknown, but they could be related to the immunoregulatory properties of vitamin D. [source] Hyaluronate-heparin conjugate gels for the delivery of basic fibroblast growth factor (FGF-2)JOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 1 2002Lin-Shu Liu Abstract The stability and activity of recombinant growth factors administered locally for the repair of damaged tissue can be directly influenced by the physical structure and chemical composition of the delivery matrix. This study describes a novel basic fibroblast growth factor-2 (FGF-2) delivery system synthesized by the conjugation of a structure-stabilizing polymer, hyaluronate (HA), with a sulfated glycosaminoglycan, heparin (HP), that has inherent specific binding sites for members of the FGF family. The biopolymers were formed via stable amine or labile imine bonds by coupling amine-modified HA with oxidized heparin. The addition of recombinant human FGF-2 resulted in the rapid binding of FGF-2 to the heparin segment of the hyaluronate-heparin (HAHP) conjugate. The FGF-2 was released in vitro from the imine-bonded (HAHPi) gels in the form of FGF-2-heparin complexes through the hydrolysis of the imine bonds. In contrast, the release of growth factor from the more stable amine-bonded (HAHPa) gels required treatment with free heparin or enzymatic digestion of the hyaluronate segment. Functional analysis of the released FGF-2 showed that the HAHP conjugate gels increased both the stability and activity of the growth factor. © 2002 Wiley Periodicals, Inc. J Biomed Mater Res 62: 128,135, 2002 [source] Comparative Pharmacology of Guinea Pig Cardiac Myocyte and Cloned hERG (IKr) ChannelJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 11 2004CHRISTINA DAVIE Ph.D. Introduction: This study used whole-cell, patch clamp techniques on isolated guinea pig ventricular myocytes and HEK293 cells expressing cloned human ether-a-go-go-related gene (hERG) to examine the action of drugs causing QT interval prolongation and torsades de pointes (TdP) in man. Similarities and important differences in drug actions on cardiac myocytes and cloned hERG IKr channels were established. Qualitative actions of the drugs on cardiac myocytes corresponded with results obtained from Purkinje fibers and measurement of QT interval prolongation in animal and human telemetry studies. Methods and Results: Adult guinea pig ventricular myocytes were isolated by enzymatic digestion. Cells were continuously perfused with Tyrode's solution at 33,35°C. Recordings were made using the whole-cell, patch clamp technique. Action potentials (APs) were elicited under current clamp. Voltage clamp was used to study the effect of drugs on IKr (rapidly activating delayed rectifier potassium current), INa (sodium current), and ICa (L-type calcium current). Dofetilide increased the myocyte action potential duration (APD) in a concentration-dependent manner, with a pIC50 of 7.3. Dofetilide 1 ,M elicited early afterdepolarizations (EADs) but had little affect on ICa or INa. E-4031 increased APD in a concentration-dependent manner, with a pIC50 of 7.2. In contrast, 10 ,M loratadine, desloratadine, and cetirizine had little effect on APD or IKr. Interestingly, cisapride displayed a biphasic effect on myocyte APD and inhibited ICa at 1 ,M. Even at this high concentration, cisapride did not elicit EADs. A number of AstraZeneca compounds were tested on cardiac myocytes, revealing a mixture of drug actions that were not observed in hERG currents in HEK293 cells. One compound, particularly AR-C0X, was a potent blocker of myocyte AP (pIC50 of 8.4). AR-C0X also elicited EADs in cardiac myocytes. The potencies of the same set of drugs on the cloned hERG channel also were assessed. The pIC50 values for dofetilide, E-4031, terfenadine, loratadine, desloratadine, and cetirizine were 6.8, 7.1, 7.3, 5.1, 5.2, and <4, respectively. Elevation of temperature from 22 to 35°C significantly enhanced the current kinetics and amplitudes of hERG currents and resulted in approximately fivefold increase in E-4031 potency. Conclusion: Our study demonstrates the advantages of cardiac myocytes over heterologously expressed hERG channels in predicting QT interval prolongation and TdP in man. The potencies of some drugs in cardiac myocytes were similar to hERG, but only myocytes were able to detect important changes in APD characteristics and display EADs predictive of arrhythmia development. We observed similar qualitative drug profiles in cardiac myocytes, dog Purkinje fibers, and animal and human telemetry studies. Therefore, isolated native cardiac myocytes are a better predictor of drug-induced QT prolongation and TdP than heterologously expressed hERG channels. Isolated cardiac myocytes, when used with high-throughput patch clamp instruments, may have an important role in screening potential cardiotoxic compounds in the early phase of drug discovery. This would significantly reduce the attrition rate of drugs entering preclinical and/or clinical development. The current kinetics and amplitudes of the cloned hERG channel were profoundly affected by temperature, significantly altering the potency of one drug (E-4031). This finding cautions against routine drug testing at room temperature compared to physiologic temperature when using the cloned hERG channel. [source] Immunophenotypic analysis of human articular chondrocytes: Changes in surface markers associated with cell expansion in monolayer cultureJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2005Jose Diaz-Romero Cartilage tissue engineering relies on in vitro expansion of primary chondrocytes. Monolayer is the chosen culture model for chondrocyte expansion because in this system the proliferative capacity of chondrocytes is substantially higher compared to non-adherent systems. However, human articular chondrocytes (HACs) cultured as monolayers undergo changes in phenotype and gene expression known as "dedifferentiation." To gain a better understanding of the cellular mechanisms involved in the dedifferentiation process, our research focused on the characterization of the surface molecule phenotype of HACs in monolayer culture. Adult HACs were isolated by enzymatic digestion of cartilage samples obtained post-mortem. HACs cultured in monolayer for different time periods were analyzed by flow cytometry for the expression of cell surface markers with a panel of 52 antibodies. Our results show that HACs express surface molecules belonging to different categories: integrins and other adhesion molecules (CD49a, CD49b, CD49c, CD49e, CD49f, CD51/61, CD54, CD106, CD166, CD58, CD44), tetraspanins (CD9, CD63, CD81, CD82, CD151), receptors (CD105, CD119, CD130, CD140a, CD221, CD95, CD120a, CD71, CD14), ectoenzymes (CD10, CD26), and other surface molecules (CD90, CD99). Moreover, differential expression of certain markers in monolayer culture was identified. Up-regulation of markers on HACs regarded as distinctive for mesenchymal stem cells (CD10, CD90, CD105, CD166) during monolayer culture suggested that dedifferentiation leads to reversion to a primitive phenotype. This study contributes to the definition of HAC phenotype, and provides new potential markers to characterize chondrocyte differentiation stage in the context of tissue engineering applications. © 2004 Wiley-Liss, Inc. [source] Protein identification via ion-trap collision-induced dissociation and examination of low-mass product ionsJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2008Jeremiah J. Bowers Abstract A whole-protein tandem mass spectrometry approach for protein identification based on precursor ion charge state concentration via ion/ion reactions, ion-trap collisional activation, ion/ion proton-transfer reactions involving the product ions, and mass analysis over a narrow m/z range (up to m/z 2000) is described and evaluated. The experiments were carried out with a commercially available electrospray ion-trap instrument that has been modified to allow for ion/ion reactions. Reaction conditions and the approach to searching protein databases were developed with the assumption that the resolving power of the mass analyzer is insufficient to distinguish charge states on the basis of the isotope spacings. Ions derived from several charge states of cytochrome c, myoglobin, ribonuclease A, and ubiquitin were used to evaluate the approach for protein identification and to develop a two-step procedure to database searching to optimize specificity. The approach developed with the model proteins was then applied to whole cell lysate fractions of Saccharomyces cerevisiae. The results are illustrated with examples of assignments made for three a priori unknown proteins, each selected randomly from a lysate fraction. Two of the three proteins were assigned to species present in the database, whereas one did not match well any database entry. The combination of the mass measurement and the product ion masses suggested the possibility for the oxidation of two methionine residues of a protein in the database. The examples show that this limited whole-protein characterization approach can provide insights that might otherwise be lacking with approaches based on complete enzymatic digestion. Copyright © 2007 John Wiley & Sons, Ltd. [source] Differentiation of sulfate and phosphate by H/D exchange mass spectrometry: application to isoflavoneJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 11 2004Akira Kanakubo Abstract Often phosphorylation or sulfation is an important step which occurs in the signal transduction and cascade of metabolic pathways. Some natural products and metabolites contain one or more sulfate or phosphate groups. Isoflavone sulfate has been identified from high-resolution mass spectrometry (HRMS) and enzymatic digestion by sulfatase. We previously reported the new water-soluble isoflavone analogs, daidzein 7- O -phosphate and genistein 7- O -phosphate, which were surprisingly hydrolyzed by sulfatase. In this previous study, we could not determine the phosphate from the results of HRMS and enzymatic digestion, that is, HRMS and enzymatic digestion did not provide clear evidence. In this case, we drew conclusions from NMR analysis. HRMS has been ineffective with a regular fast atom bombardment (FAB) mass spectrometer to distinguish between phosphate and sulfate since the mass difference is only 0.009 mass units. There was, however, no conventional method of microanalysis to distinguish phosphate from sulfate owing to the same nominal mass. It is still very difficult to determine by negative FABMS [OP(O)(OH)2] = 80 and [OS(O)2OH] = 80. In this paper, we report a method to distinguish between these groups by using a popular low-resolution mass instrument; thus, phosphate and sulfate were measured by H/D exchange mass spectrometry at the picomole level to differentiate [OP(O)(OD)2] = 82 and [OS(O)2OD] = 81, respectively. This method is applicable not only to the isoflavone, but also to other phospho and sulfo compounds. Copyright © 2004 John Wiley & Sons, Ltd. [source] Negative and positive ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and positive ion nano-electrospray ionization quadrupole ion trap mass spectrometry of peptidoglycan fragments isolated from various Bacillus speciesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 2 2001Gerold Bacher Abstract A general approach for the detailed characterization of sodium borohydride-reduced peptidoglycan fragments (syn. muropeptides), produced by muramidase digestion of the purified sacculus isolated from Bacillus subtilis (vegetative cell form of the wild type and a dacA mutant) and Bacillus megaterium (endospore form), is outlined based on UV matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nano-electrospray ionization (nESI) quadrupole ion trap (QIT) mass spectrometry (MS). After enzymatic digestion and reduction of the resulting muropeptides, the complex glycopeptide mixture was separated and fractionated by reversed-phase high-performance liquid chromatography. Prior to mass spectrometric analysis, the muropeptide samples were subjected to a desalting step and an aliquot was taken for amino acid analysis. Initial molecular mass determination of these peptidoglycan fragments (ranging from monomeric to tetrameric muropeptides) was performed by positive and negative ion MALDI-MS using the thin-layer technique with the matrix ,-cyano-4-hydroxycinnamic acid. The results demonstrated that for the fast molecular mass determination of large sample numbers in the 0.8,10 pmol range and with a mass accuracy of ±0.07%, negative ion MALDI-MS in the linear TOF mode is the method of choice. After this kind of muropeptide screening often a detailed primary structural analysis is required owing to ambiguous data. Structural data could be obtained from peptidoglycan monomers by post-source decay (PSD) fragment ion analysis, but not from dimers or higher oligomers and not with the necessary sensitivity. Multistage collision-induced dissociation (CID) experiments performed on an nESI-QIT instrument were found to be the superior method for structural characterization of not only monomeric but also of dimeric and trimeric muropeptides. Up to MS4 experiments were sometimes necessary to obtain unambiguous structural information. Three examples are presented: (a) CID MSn (n = 2,4) of a peptidoglycan monomer (disaccharide-tripeptide) isolated from B. subtilis (wild type, vegetative cell form), (b) CID MSn (n = 2,4) of a peptidoglycan dimer (bis-disaccharide-tetrapentapeptide) obtained from a B. subtilis mutant (vegetative cell form) and (c) CID MS2 of a peptidoglycan trimer (a linear hexasaccharide with two peptide side chains) isolated from the spore cortex of B. megaterium. All MSn experiments were performed on singly charged precursor ions and the MS2 spectra were dominated by fragments derived from interglycosidic bond cleavages. MS3 and MS4 spectra exhibited mainly peptide moiety fragment ions. In case of the bis-disaccharide-tetrapentapeptide, the peptide branching point could be determined based on MS3 and MS4 spectra. The results demonstrate the utility of nESI-QIT-MS towards the facile determination of the glycan sequence, the peptide linkage and the peptide sequence and branching of purified muropeptides (monomeric up to trimeric forms). The wealth of structural information generated by nESI-QIT-MSn is unsurpassed by any other individual technique. Copyright © 2001 John Wiley & Sons, Ltd. [source] Identification of three novel peptides isolated from the venom of the neotropical social wasp Polistes major majorJOURNAL OF PEPTIDE SCIENCE, Issue 7 2007Václav, ovský Abstract Three novel peptides designated as PMM1, PMM2, and PMM3 were isolated and characterized from the venom of the social wasp Polistes major major, one of the most common wasps in the Dominican Republic. By Edman degradation, and MALDI-TOF and ESI-QTOF mass spectrometry, the primary sequences of these peptides were established as follows: PMM1, H-Lys-Arg-Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH (1357.77 Da); PMM2, H-Ile-Asn-Trp-Lys-Lys-Ile-Ala-Ser-Ile-Gly-Lys-Glu-Val-Leu-Lys-Ala-Leu-NH2 (1909.19 Da); and PMM3, H-Phe-Leu-Ser-Ala-Leu-Leu-Gly-Met-Leu-Lys-Asn-Leu-NH2 (1317.78 Da). The suggested sequences were confirmed by MS analysis of peptide fragments obtained by enzymatic digestion. The peptide PMM1 is a lysyl-arginyl-Thr6 -bradykinine that belongs to the wasp kinins group. The sequence of the PMM2 peptide is unique; it resembles somewhat the tetradecapeptide amides of the mastoparan group; however, the chain is extended by three additional amino acid residues. The sequence of PMM3 dodecapeptide is homologous to the peptides of the wasp chemotactic group. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [source] Identification of the nitration site of insulin by peroxynitriteJOURNAL OF PEPTIDE SCIENCE, Issue 3 2007Quan Chi Abstract Our previous investigation indicated that insulin can be nitrated by peroxynitrite in vitro. In this study, the preferential nitration site of the four tyrosine residues in insulin molecule was confirmed. Mononitrated and dinitrated insulins were purified by RP-HPLC. Following reduction of insulin disulfide bridges, Native-PAGE indicated that A-chain was preferentially nitrated. Combination of enzymatic digestion of mononitrated insulin with endoproteinase Glu-C, mass spectrometry confirmed that Tyr-A14 was the preferential nitration site when insulin was treated with peroxynitrite. Tyr-A19, maybe, was the next preferential nitration site. According to the crystal structure, Tyr-B26 between the two tyrosine residues in insulin B-chain was likely easier to be nitrated by peroxynitrite. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd. [source] In vitro determination of digestible and unavailable protein in edible seaweedsJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 15 2002Isabel Goñi Abstract Edible seaweeds are considered a complementary source of food protein for human and animal nutrition. The physiological effects of seaweed protein depend on the degree of enzymatic digestion of protein in the small intestine and bacterial fermentation in the large intestine. The objective of this work was to estimate total, digestible, fermentable and unavailable protein in some red and brown seaweeds. Brown seaweeds Fucus vesiculosus, Laminaria digitata and Undaria pinnatifida and red seaweeds Chondrus crispus and Porphyra tenera were treated with pepsin and pancreatin to separate digestible protein. Residues containing indigestible protein were inoculated for 24,h with rat caecal droppings, and protein contents were evaluated in the non-fermented residue. Protein content in the seaweeds ranged from 8.9 to 25% of dry matter. Digestible protein was the major protein fraction (69%) only in P tenera; in the other seaweeds, this fraction ranged from 15 to 45%. Significant amounts of unavailable protein were found in all samples (2,24%). The distribution of total protein among the three fractions, ie digestible, fermentable and unavailable protein, could yield information about the physiological and metabolic consequences of the intake of seaweed proteins. © 2002 Society of Chemical Industry [source] Quantitative assessment of chondrocyte viability after laser mediated reshaping: A novel application of flow cytometryLASERS IN SURGERY AND MEDICINE, Issue 1 2003Alexandre Rasouli BS Abstract Background and Objectives Lasers can be used to reshape cartilage by accelerating mechanical stress relaxation. In this study, fluorescent differential cell viability staining and flow cytometry were used to determine chondrocyte viability following laser heating. Study Design/Materials and Methods Porcine septal cartilages were irradiated with an Nd:YAG laser (,,= 1.32 ,m, 25 W/cm2) while surface temperature, stress relaxation, and diffuse reflectance were recorded. Each slab received one, two, or three laser exposures (respective exposure times of 6.7, 7.2, 10 seconds). Irradiated samples were then divided into two groups analyzed immediately and at 5 days following laser exposure. Chondrocytes were isolated following serial enzymatic digestion, and stained using SYTO®/DEAD RedÔ (Molecular Probes, Eugene, OR). A flow cytometer was then used to detect differential cell fluorescence; size; granularity; and the number of live cells, dead cells, and post-irradiation debris in each treatment population. Results Nearly 60% of chondrocytes from reshaped cartilage samples isolated shortly after one irradiation, were viable while non-irradiated controls were 100% viable. Specimens irradiated two or three times demonstrated increasing amounts of cellular debris along with a reduction in chondrocyte viability: 31 and 16% after two and three exposures, respectively. In those samples maintained in culture medium and assayed 5 days after irradiation, viability was reduced by 28,88%, with the least amount of deterioration in untreated and singly irradiated samples. Conclusions Functional fluorescent dyes combined with flow cytometric analysis successfully determines the effect of laser irradiation on the viability of reshaped cartilage. Lasers Surg. Med. 32:3,9,2003. © 2003 Wiley-Liss, Inc. [source] Off-line liquid chromatography-MALDI by with various matrices and tandem mass spectrometry for analysis of glycated human serum albumin tryptic peptidesMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 4 2007Annunziata Lapolla Abstract Advanced glycation end-product (AGE)/peptides, arising from in vivo digestion of glycated proteins, are biologically important compounds, due to their reactivity against circulating and tissue proteins. For information on their possible structure, in vitro glycation of HSA and its further enzymatic digestion were performed. The resulting digestion product mixture was analysed directly by MALDI MS with various matrices [2,5-dihydroxy benzoic acid (DHB) and ,-cyano-4-hydroxy cinnamic acid (CHCA)]. Alternatively, offline microbore LC prior to MALDI analysis was used, and showed that 63% of the free amino groups prone to glycation are modified, indicating the contemporary presence of unglycated peptides. This result proves that, regardless of the high glucose concentration employed for HSA incubation, glycation does not go to completion. Further studies showed that the collisionally activated decomposition of singly charged glycated peptides leads to specific fragmentation pathways, all related to the condensed glucose molecule. These unique product ions can be used as effective markers to establish the presence of a glucose molecule within a peptide ion. [source] Functional protease-activated receptors in the dorsal motor nucleus of the vagusNEUROGASTROENTEROLOGY & MOTILITY, Issue 4 2010H. Wang Abstract Background, Protease-activated receptors (PARs), a family member of G-protein coupled receptors, are present and functionally active in a wide variety of cells. The object of this study was to demonstrate the presence and function of PAR-1 and PAR-2 in the dorsal motor nucleus of the vagus (DMV). Methods, DMNV neurons were isolated from neonatal rat brainstems using micro-dissection and enzymatic digestion. Neurons were cultured in Neurobasal medium A containing 2% B27 supplement. Intracellular calcium concentration ([Ca2 + ]i) was measured using fura-2 based microspectrometry. Expression of PARs was detected by RT-PCR and immunofluorescent staining. Key Result, Thrombin and PAR-1 agonist peptide activate PAR-1 with a maximum change in [Ca2 + ]i expressed as ,F/F0 of 229 ± 14% and 137 ± 7%, respectively. Trypsin and PAR-2 agonist peptide activate PAR-2 with a maximum ,F/F0 change of 258 ± 12% and 242 ± 10%, respectively. Inhibition of phospholipase C (PLC) by U73312 (1 ,m) decreased the maximal change in ,F/F0 induced by PAR-1 activation from 140 ± 17% to 21 ± 3%, while the PAR-2-mediated maximal change in ,F/F0 decreased from 185 ± 21% to 19 ± 6%. Blockade of IP3 receptor with 2APB inhibited the maximal change in ,F/F0 due to PAR-1 and PAR-2 activation by 72 ± 13% and 71 ± 20% respectively. PAR-1 immnuoreactivity was present in DMV neurons. Increase in transcripts for PAR-1 and PAR-2 were detected in DMV tissues derived from IBD rats relative to control animals. Conclusions & Inferences, Our results indicate that PAR-1 and PAR-2 are present in the DMV neurons, and their activation leads to increases in intracellular calcium via signal transduction mechanism that involves activation of PLC and the production of IP3. [source] Saliva and gastrointestinal functions of taste, mastication, swallowing and digestionORAL DISEASES, Issue 3 2002AM Pedersen Saliva has multiple essential functions in relation to the digestive process taking place in the upper parts of the gastrointestinal (GI) tract. This paper reviews the role of human saliva and its compositional elements in relation to the GI functions of taste, mastication, bolus formation, enzymatic digestion, and swallowing. The indirect function of saliva in the digestive process that includes maintenance of an intact dentition and mucosa is also reviewed. Finally, pathophysiological considerations of salivary dysfunction in relation to some GI functions are considered. [source] A method for the selective isolation and enrichment of carrier protein-bound low-molecular weight proteins and peptides in the bloodPROTEOMICS - CLINICAL APPLICATIONS, Issue 2 2007Serena Camerini Abstract The low molecular weight (LMW) region of the circulatory proteome, thought to contain a rich source of biomarkers, resides in vivo, in a complexed state with larger, highly abundant resident proteins. Consequently, serum fractionation approaches that deplete the high-abundance proteins under native conditions will remove much of the LMW proteome. We describe a new strategy to systematically collect, isolate and enrich the LMW molecules that would be otherwise eliminated during the depletion of high-abundance circulatory proteins based on continuous elution electrophoresis. We employ strong denaturing conditions to disrupt association with the high-abundance carrier proteins followed by fractionation and removal of SDS. Under denaturation, the LMW molecules were effectively stripped from the highly abundant carrier proteins. We then removed the SDS by ion exchange matrix sequestration and concentrated the fractions. The outcome is a series of SDS-free fractions of LMW molecules. The isolated fractions were then analyzed by enzymatic digestion followed by LC-MS/MS analysis. The yield of multiple peptide hits as well as the total number of identifications significantly increased (50%) compared to unfractionated serum. The method yielded a 30% higher number of low-abundance serum proteins compared to direct sequencing of unfractionated serum. [source] Cysteine-capped ZnSe quantum dots as affinity and accelerating probes for microwave enzymatic digestion of proteins via direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysisRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 15 2009Lokesh A. Shastri Fluorescent semiconductor quantum dots (QDs) exhibit great potential and capability for many biological and biochemical applications. We report a simple strategy for the synthesis of aqueous stable ZnSe QDs by using cysteine as the capping agent (ZnSe-Cys QDs). The ZnSe QDs can act as affinity probes to enrich peptides and proteins via direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis. This nanoprobe could significantly enhance protein signals (insulin, ubiquitin, cytochrome c, myoglobin and lysozyme) in MALDI-TOFMS by 2.5,12 times compared with the traditional method. Additionally, the ZnSe-Cys QDs can be applied as heat absorbers (as accelerating probes) to speed up microwave-assisted enzymatic digestion reactions and also as affinity probes to enrich lysozyme-digested products in MALDI-TOFMS. Furthermore, after the enrichment experiments, the solutions of ZnSe-Cys QDs mixed with proteins can be directly deposited onto the MALDI plates for rapid analysis. This approach shows a simple, rapid, efficient and straightforward method for direct analysis of proteins or peptides by MALDI-TOFMS without the requirement for further time-consuming separation processes, tedious washing steps or laborious purification procedures. The present study has demonstrated that ZnSe-Cys QDs are reliable and potential materials for rapid, selective separation and enrichment of proteins as well as accelerating probes for microwave-digested reactions for proteins than the regular MALDI-MS tools. Additionally, we also believe that this work may also inspire investigations for applications of QDs in the field of MALDI-MS for proteomics. Copyright © 2009 John Wiley & Sons, Ltd. [source] Simultaneous detection of five different 2-hydroxyethyl-DNA adducts formed by ethylene oxide exposure, using a high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry assayRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 1 2008Elaine M. Tompkins A method has been developed for the simultaneous detection and quantitation of five different 2-hydroxyethyl-DNA (HE-DNA) adducts that could be formed as a result of exposure to ethylene oxide (EO). In addition to the major N7-HE-guanine (N7-HEG) adducts this assay can also measure the less prevalent but potentially more biologically significant N1-HE-2,-deoxyadenosine (N1-HEdA), O6 -HE-2,-deoxyguanosine (O6 -HEdG), N6 -HE-2,-deoxyadenosine (N6 -HEdA) and N3-HE-2,-deoxyuridine adducts (N3-HEdU). The method involves the isolation of HE adducts from the unmodified nucleosides by either neutral thermal hydrolysis or enzymatic digestion, followed by high-performance liquid chromatographic (HPLC) purification, before detection and quantification by liquid chromatography tandem mass spectrometry (LC/MS/MS) using selective reaction monitoring (SRM). The limits of detection were in the range 0.5,25,fmol for each individual adduct, making this one of the most sensitive assays available for the detection of N7-HEG. To illustrate the possible applications of the assay, it has been employed in the measurement of endogenous/background and EO-induced HE adducts in a variety of DNA samples. Copyright © 2007 John Wiley & Sons, Ltd. [source] A Novel In Vitro Model of Canine Malignant HemangioendotheliomaANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 2005D. Kühn Introduction and Aim:, Canine malignant haemangioendothelioma is an aggressive neoplasia that affects mostly older dogs of large breeds with a strong predilection for the spleen, liver, heart and skin. The tumour originates in the vascular endothelium and consists of transformed cells forming large and leaky vessel-like structures. Prognosis is poor because surgery and chemotherapy have limited success in prolonging survival times and increasing quality of patients. A new strategy to treat this malignancy could be anti-angiogenic therapy based on the inhibition of proliferation, migration and three-dimensional organization of transformed cells. In order to reduce animal experiments, in vitro -models are required to test the safety and efficacy of anti-angiogenic drugs. So far only few models of angiogenesis are available using mostly human, rodent and bovine cells. Therefore, the aim of our study was to establish an in vitro model of canine haemangioendothelioma. Materials and Methods:, Tumours were collected from dogs during surgery or immediately after euthanasia. Isolation of cells was done from different areas of the tumours and by enzymatic digestion of the tissue. Cells were incubated in culture media with and without endothelial growth factors. Cells were characterized by lectin histochemistry using Dolichos biflorus agglutinin, Ulex europaeus agglutinin and Bandeiraea simplicifolia agglutinin I. Moreover, RT-PCR (polymerase chain reaction) was employed to investigate the expression of vascular endothelial growth factor (VEGF) and its endothelium-specific receptors VEGF-R1 and -R2. Results and Conclusions:, Different populations of cells were isolated and cultured successfully from canine malignant haemangioendothelioma. Cells show characteristics of microvascular endothelial cells of an angiogenic phenotype, i.e. the formation of spheroids and tube-like structures as well as strong labelling for Bandeiraea simplicifolia agglutinin I. Thus, morphological and glycohistochemical results confirm the vascular character of the cells isolated. RT-PCR showed expression of VEGF. However, endothelium-specific VEGF receptors were not expressed. Loss of typical receptors is common in cancer and may correlate with increased tumour dedifferentiation. [source] MtDNA from extinct Tainos and the peopling of the CaribbeanANNALS OF HUMAN GENETICS, Issue 2 2001C. LALUEZA-FOX Tainos and Caribs were the inhabitants of the Caribbean when Columbus reached the Americas; both human groups became extinct soon after contact, decimated by the Spaniards and the diseases they brought. Samples belonging to pre-Columbian Taino Indians from the La Caleta site (Dominican Republic) have been analyzed, in order to ascertain the genetic affinities of these groups in relation to present-day Amerinds, and to reconstruct the genetic and demographic events that took place during the peopling of the Caribbean. Twenty-seven bone samples were extracted and analyzed for mtDNA variation. The four major Amerindian mtDNA lineages were screened through amplification of the specific marker regions and restriction enzymatic digestion, when needed. The HVRI of the control region was amplified with four sets of overlapping primers and sequenced in 19 of the samples. Both restriction enzyme and sequencing results suggest that only two (C and D) of the major mtDNA lineages were present in the sample: 18 individuals (75%) belonged to the C haplogroup, and 6 (25%) to the D haplogroup. Sequences display specific substitutions that are known to correlate with each haplogroup, a fact that helped to reject the possibility of European DNA contamination. A low rate of Taq misincorporations due to template damage was estimated from the cloning and sequencing of different PCR products of one of the samples. High frequencies of C and D haplogroups are more common in South American populations, a fact that points to that sub-continent as the homeland of the Taino ancestors, as previously suggested by linguistic and archaeological evidence. Sequence and haplogroup data show that the Tainos had a substantially reduced mtDNA diversity, which is indicative of an important founder effect during the colonization of the Caribbean Islands, assumed to have been a linear migratory movement from mainland South America following the chain configuration of the Antilles. [source] Induction of CCR2-dependent macrophage accumulation by oxidized phospholipids in the air-pouch model of inflammationARTHRITIS & RHEUMATISM, Issue 5 2009Alexandra Kadl Objective Macrophages are key players in the pathogenesis of rheumatoid synovitis as well as in atherosclerosis. To determine whether atherogenic oxidized phospholipids potentially contribute to synovial inflammation and subsequent monocyte/macrophage recruitment, we examined the effects of oxidized 1- palmitoyl-2-arachidonoyl- sn -3-glycero-phosphorylcholine (OxPAPC) on chemokine expression and leukocyte recruitment in a facsimile synovium in vivo using the murine air-pouch model. Methods Air pouches were raised by 2 injections of sterile air, and inflammation was induced by injecting either lipopolysaccharide (LPS) or OxPAPC into the pouch lumen. Inflammation was assessed by analysis of inflammatory gene expression using reverse transcription,polymerase chain reaction or immunohistochemical analysis, and leukocytes were quantified in the lavage fluid and in the pouch wall after staining with Giemsa or after enzymatic digestion followed by fluorescence-activated cell sorter analysis. Results Application of OxPAPC resulted in selective recruitment of monocyte/macrophages into the air-pouch wall, but not in the lumen. In contrast, LPS induced both monocyte and neutrophil accumulation in the pouch lumen as well as in the wall. LPS, but not OxPAPC, induced the expression of adhesion molecules E-selectin, P-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1. OxPAPC increased the expression of the CCR2 ligands monocyte chemotactic protein 1 (MCP-1), MCP-3, and MCP-5, as well as RANTES and growth-related oncogene , (GRO,), while it down-regulated the expression of CCR2 on macrophages. Moreover, oxidized phospholipid,induced macrophage accumulation was abrogated in CCR2,/, mice. Conclusion These data demonstrate that oxidized phospholipids trigger a type of inflammatory response that leads to selective macrophage accumulation in vivo, a process relevant for the pathogenesis of chronic inflammatory rheumatic diseases. [source] Mechanical Dissociation of Swine Liver to Produce Organoid Units for Tissue Engineering and In Vitro Disease ModelingARTIFICIAL ORGANS, Issue 1 2010Katayun Irani Abstract The complex intricate architecture of the liver is crucial to hepatic function. Standard protocols used for enzymatic digestion to isolate hepatocytes destroy tissue structure and result in significant loss of synthetic, metabolic, and detoxification processes. We describe a process using mechanical dissociation to generate hepatic organoids with preserved intrinsic tissue architecture from swine liver. Oxygen-supplemented perfusion culture better preserved organoid viability, morphology, serum protein synthesis, and urea production, compared with standard and oxygen-supplemented static culture. Hepatic organoids offer an alternative source for hepatic assist devices, engineered liver, disease modeling, and xenobiotic testing. [source] Emodin Inhibits Voltage-Dependent Potassium Current in Guinea Pig Gallbladder Smooth MuscleBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 3 2009Zhi-Xuan Wu We studied the effects of emodin on the contraction of gallbladder smooth muscle and voltage-dependent K+ current in gallbladder smooth muscle cells. Gallbladder muscle strips were obtained from adult guinea pigs and the resting tension was recorded. Gallbladder smooth muscle cells were isolated by enzymatic digestion, and K+ current was recorded by the whole-cell patch clamp method. Emodin increased the resting tension of gallbladder smooth muscle strips and inhibited voltage-dependent K+ current in a dose-dependent manner. When 10 µM emodin was applied to gallbladder smooth muscle cells for 3,6 min., the amplitude of voltage-dependent K+ current was decreased by 31.5 ± 0.5% at +40 mV, and this inhibitory effect mostly recovered after washout. The steady-state inactivation curves were shifted in a hyperpolarizing direction by emodin. In the presence of the protein kinase C inhibitors staurosporine and chelerythrine, the effect of emodin on voltage-dependent K+ current was significantly attenuated. In conclusion, emodin promotes gallbladder contraction, mainly by inhibiting voltage-dependent K+ current via the protein kinase C pathway. These findings provide theoretical foundation for the application of emodin in gallbladder motility disorders. [source] Access of cellulase to cellulose and lignin for poplar solids produced by leading pretreatment technologiesBIOTECHNOLOGY PROGRESS, Issue 3 2009Rajeev Kumar Abstract Adsorption of cellulase on solids resulting from pretreatment of poplar wood by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid (DA), flowthrough (FT), lime, and sulfur dioxide (SO2) and pure Avicel glucan was measured at 4°C, as were adsorption and desorption of cellulase and adsorption of ,-glucosidase for lignin left after enzymatic digestion of the solids from these pretreatments. From this, Langmuir adsorption parameters, cellulose accessibility to cellulase, and the effectiveness of cellulase adsorbed on poplar solids were estimated, and the effect of delignification on cellulase effectiveness was determined. Furthermore, Avicel hydrolysis inhibition by enzymatic and acid lignin of poplar solids was studied. Flowthrough pretreated solids showed the highest maximum cellulase adsorption capacity (,solids = 195 mg/g solid) followed by dilute acid (,solids = 170.0 mg/g solid) and lime pretreated solids (,solids = 150.8 mg/g solid), whereas controlled pH pretreated solids had the lowest (,solids = 56 mg/g solid). Lime pretreated solids also had the highest cellulose accessibility (,cellulose = 241 mg/g cellulose) followed by FT and DA. AFEX lignin had the lowest cellulase adsorption capacity (,lignin = 57 mg/g lignin) followed by dilute acid lignin (,lignin = 74 mg/g lignin). AFEX lignin also had the lowest ,-glucosidase capacity (,lignin = 66.6 mg/g lignin), while lignin from SO2 (,lignin = 320 mg/g lignin) followed by dilute acid had the highest (301 mg/g lignin). Furthermore, SO2 followed by dilute acid pretreated solids gave the highest cellulase effectiveness, but delignification enhanced cellulase effectiveness more for high pH than low pH pretreatments, suggesting that lignin impedes access of enzymes to xylan more than to glucan, which in turn affects glucan accessibility. In addition, lignin from enzymatic digestion of AFEX and dilute acid pretreated solids inhibited Avicel hydrolysis less than ARP and flowthrough lignin, whereas acid lignin from unpretreated poplar inhibited enzymes the most. Irreversible binding of cellulase to lignin varied with pretreatment type and desorption method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Cell-surface matrix proteins and sialic acids in cell-crystal adhesion; the effect of crystal binding on the viability of human CAKI-1 renal epithelial cellsBJU INTERNATIONAL, Issue 6 2003G. Kramer Objective To investigate the role of sialic acids and cellular matrix proteins as crystal-binding molecules in human calcium-oxalate nephrolithiasis. Materials and methods The well-defined human renal cancer cell line CAKI-1 was used a standard cell culture system. After enzymatic digestion of various cell surface molecules, the binding of ,2,6 (Sambucus nigra, SN-) and ,2,3 (Maackia amurensis, MA)-specific lectins to CAKI-1 cells was analysed. Simultaneously, the effect on adhesion and release of calcium oxalate monohydrate crystals was investigated (eight replicates). The effect of crystal adhesion on cell viability was assessed using Trypan blue exclusion (five replicates). Results Neuraminidase decreased MA-lectin binding of CAKI-1 cells by 39% (P < 0.05) but elevated SN-lectin binding by 812% (P < 0.05). Simultaneously, crystal binding to CAKI-1 cells was increased by 28% (P > 0.05). Pretreatment with collagenase type I, trypsin and dispase II reduced crystal-binding by 61,74% (P < 0.05) with no effect on sialic acid-specific lectin-binding. However, only collagenase type I and dispase (ratio 4 : 1) were also able to release crystals from their receptor-binding sites (P < 0.05). An increase in the number of cell surface-bound crystals correlated significantly with a decrease in cell viability (P < 0.05). Conclusions ,2,3-linked sialic acids protect cells from crystal-binding. Much greater SN-lectin binding associated with only moderately increased crystal binding argues against ,2,6-linked sialic acids as a main target structure of crystals. In contrast, collagen type I, type IV and/or fibronectin seem to be potent crystal-binding molecules on human renal epithelial cells, with collagen type I involved in a potential second step of crystal,cell interaction. [source] Long,term culture of multibacillary leprosy macrophages isolated from skin lesions: a new model to study Mycobacterium leprae,human cell interactionBRITISH JOURNAL OF DERMATOLOGY, Issue 2 2007D.F. Moura Summary Background, Leprosy is characterized by a disease spectrum having two polar clinical forms dependent on the presence or not of cell-mediated immunity. In the tuberculoid forms, granuloma-activated macrophages kill Mycobacterium leprae in conjunction with a Th1 response while, in multibacillary (MB) lesions, M. leprae nonactivated macrophages infiltrate the nerves and internal organs together with a Th2 response. The functional properties and activation pathways of macrophages isolated from patients with MB leprosy remain only partially understood. Objectives, To establish an ex vivo methodology capable of evaluating the activation pathways, grade and fate of cultured macrophages isolated from MB lesions. Methods, Skin biopsies from patients with borderline tuberculoid, bordeline lepromatous and lepromatous leprosy (LL) were characterized by immunohistochemistry and transcriptional analysis. To isolate inflammatory cells, a portion of the samples was submitted to enzymatic digestion. These same cells, maintained in culture for a minimum 7-day period, were characterized morphologically and via flow cytometry at different culture time points. Cytokine [interferon (IFN)-,, tumour necrosis factor (TNF)-, and interleukin (IL)-10] mRNA levels were quantified by real-time polymerase chain reaction and protein secretion in the culture supernatants was measured by enzyme-linked immunosorbent assay and the nitric oxide levels by Griess reagent. Results, RNA expression in tuberculoid and MB lesions showed the profile expected of characteristic Th1 and Th2 responses, respectively. The inflammatory cells in all biopsies were successfully isolated. Although the number of cells varied between biopsies, it was highest in LL biopsies. The frequency of isolated CD14+ and CD3+ cells measured by flow cytometry correlated with the percentages of macrophages and lymphocytes in the lesions. Throughout the culture period, CD68+ macrophages showed morphological changes. A progressive increase in cell number and reduction of infected cells were perceptible in the cultures. In contrast to the biopsies, TNF-,, IFN-, and IL-10 expression in the tuberculoid and MB leprosy cells in 24-h culture and the cytokine levels in the supernatants did not differ significantly. During the culture period, cytokine expression in the MB cells progressively declined, whereas, from days 1 to 7, nitrite levels progressively increased. After day 40, the remaining macrophages were able to ingest fluorescein isothiocyanate-labelled M. leprae. These data need to be confirmed. Conclusions, This study confirmed the feasibility of obtaining ex vivo macrophages from leprosy lesions and keeping them in long-term culture. This procedure may open new pathways to studying the interaction between M. leprae and human macrophages, which might, in turn, lead to the development of therapeutic tools capable of overcoming the specific anergy found in patients with MB leprosy. [source] Synthesis and biological activity of GnRH antagonists modified at position 3 with 3-(2-methoxy-5-pyridyl)-alanine,CHEMICAL BIOLOGY & DRUG DESIGN, Issue 2 2005M.P. Samant Abstract:, Degarelix is a potent very long-acting GnRH antagonist after subcutaneous administration. In this paper, we describe the synthesis of two analogs of degarelix incorporating racemic 3-(2-methoxy-5-pyridyl)-alanine (2-OMe-5Pal, 5) at position 3. The two diastereomers were separated by reverse-phase high-performance liquid chromatography (RP-HPLC) and the absolute stereochemistry at position 3 in the peptides was determined by enzymatic digestion with proteinase K. These analogs were tested in vitro for their ability to antagonize the GnRH receptor and in vivo for duration of action in a castrated male rat assay. Analog 7 with D2-OMe-5Pal was potent in vitro (IC50 = 5.22 nm); however, analog 8 with L2-OMe-5Pal at position 3 in degarelix lost potency as an antagonist of the human GnRH receptor (IC50 = 36.95 nm). Both the analogs were found to be short-acting in vivo. [source] | ||||||||||||||||