Collagen and related research最新文献

{"title":"Differential Regulation by Glucocorticoids of Mouse Proα2(I) and Proα1(III) DNA-Binding Proteins","authors":"Colleen Sweeney,&nbsp;Kenneth R. Cutroneo","doi":"10.1016/S0174-173X(88)80041-4","DOIUrl":"10.1016/S0174-173X(88)80041-4","url":null,"abstract":"<div><p>Multiple procollagen-specific DNA-binding proteins were identified for the α2(I) and the αl(III) procollagen promotor containing gene fragments. The proteins are genespecific, differing in their relative molecular weights and relative binding specificities. A major finding was the increased DNA-binding with specificity for the procollagen promoter containing DNAs by several nonhistone nuclear proteins in mouse embryo fibroblasts treated with dexamethasone. The previously reported coordinate decrease of type I and type III procollagens by glucocorticoids may involve differential regulation by glucocorticoids of procollagen gene specific DNA-binding proteins.</p></div>","PeriodicalId":77694,"journal":{"name":"Collagen and related research","volume":"8 3","pages":"Pages 209-219"},"PeriodicalIF":0.0,"publicationDate":"1988-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0174-173X(88)80041-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14521314","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Human Arthritic Synovial Fluid Influences Proteoglycan Biosynthesis and Degradation in Organ Culture of Bovine Nasal Cartilage","authors":"Tore Saxne ,&nbsp;Dick Heinegård ,&nbsp;Frank A. Wollheim","doi":"10.1016/S0174-173X(88)80043-8","DOIUrl":"10.1016/S0174-173X(88)80043-8","url":null,"abstract":"<div><p>The influence of synovial fluid and serum from patients with inflammatory joint disease on proteoglycan metabolism was studied in organ culture of bovine nasal cartilage. Proteoglycan biosynthesis, i.e. incorporation of [³⁵S] -sulphate, was reduced after addition of synovial fluid from rheumatoid arthritis and reactive arthritis patients. Also some rheumatoid arthritis sera but no reactive arthritis serum reduced the biosynthesis compared to control sera. Proteoglycan degradation, i.e. release of proteoglycans prelabelled with [³⁵S] -sulphate, as well as release of proteoglycans determined by.chemical methods, was highest under the influence of rheumatoid arthritis synovial fluid. This effect appears to represent an activity truly stimulating degradation, since added control serum did not prevent the effect. The lowest proteoglycan degradation was observed in culture medium only. Addition of synovial fluid compared to addition of control serum did not increase proteoglycan degradation in freeze-killed cartilage indicating that the effect requires living cells.</p><p>The findings are consistent with the presence in synovial fluid of mediators stimulating the chondrocytes both to activate proteoglycan degradation and to reduce proteoglycan biosynthesis.</p></div>","PeriodicalId":77694,"journal":{"name":"Collagen and related research","volume":"8 3","pages":"Pages 233-247"},"PeriodicalIF":0.0,"publicationDate":"1988-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0174-173X(88)80043-8","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14521316","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of Small Collagenous Proteins with Properties of Procollagen α1 (I) pN-Propeptide in Fetal Porcine Calvarial Bone","authors":"Harvey A. Goldberg,&nbsp;Masao Maeno,&nbsp;Carmelo Domenicucci,&nbsp;Qi Zhang,&nbsp;Jaro Sodek","doi":"10.1016/S0174-173X(88)80039-6","DOIUrl":"10.1016/S0174-173X(88)80039-6","url":null,"abstract":"<div><p>Several small collagenous apatite binding (SCAB) proteins have been extracted from the mineralized matrix of fetal porcine calvarial bone. One protein (SCAB 3), released on demineralization of bone with 0.5 M EDTA, appears to represent the a1 pN-propeptide that is normally released during proteolytic processing of type I procollagen. The 28 Kd protein, which stains blue with “Stains-all”, is reduced to a 19 Kd fragment by bacterial collagenase digestion, but is not susceptible to cyanogen bromide. The amino acid composition, blocked amino-terminus and immunological properties are all consistent with properties of α1 (I) pN-propeptide. Fractionation on hydroxylapatite in the presence of urea has revealed a nonbinding (SCAB 3a) and a binding (SCAB 3b) form. Extraction of the demineralized matrix of bone with 4 M GuHCl revealed a third form (G2-28) which was similar to SCAB 3a on hydroxylapatite chromatography but showed differences on FPLC “Mono Q” resin. The occurrence of these different forms of pNpropeptide in bone may be of significance in collagen fibril-associated hydroxylapatite formation and in the regulation of osteoblastic function during bone resorption.</p></div>","PeriodicalId":77694,"journal":{"name":"Collagen and related research","volume":"8 3","pages":"Pages 187-197"},"PeriodicalIF":0.0,"publicationDate":"1988-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0174-173X(88)80039-6","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14522060","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Influence of Heparin on the Wound Healing Response to Collagen Implants in vivo","authors":"John M. Mcpherson ,&nbsp;Philip W. Ledger ,&nbsp;George Ksander ,&nbsp;Steven J. Sawamura ,&nbsp;Annemarie Conti ,&nbsp;Steven Kincaid ,&nbsp;Dov Michaeli ,&nbsp;Richard A.F. Clark","doi":"10.1016/S0174-173X(88)80037-2","DOIUrl":"10.1016/S0174-173X(88)80037-2","url":null,"abstract":"<div><p>The biologic response to fibrillar collagen (collagen) and fibrillar collagen plus heparin(collagen/heparin) implants have been compared in the rat subcutaneous and guinea pig dermal wound models. The reconstituted bovine dermal collagen implants were injected subcutaneously in rats at concentrations ranging from 18 to 30 mg/ml and in volumes ranging from 0.5 to 1.0 ml. The biologic response to the collagen implants alone was characterized by a transient invasion of a modest number of inflammatory cells within the first three days of implantation that was followed by limited fibroblast invasion into the peripheral 1/3 of the implant during the course of the next three to four weeks. Occasionally, blood vessels were observed to invade the peripheral regions of the implant. The degree (number) and extent (depth) of cell invasion were inversely related to initial collagen implant concentration. Addition of heparin (0.3-20 μg/mg collagen) to these implants resulted in a significant dose-dependent increase in the degree and extent of fibroblast invasion. Radiolabeling studies showed that the collagen and collagen/heparin implants were cleared from the subcutis at identical rates. Implantation of these formulations in a guinea pig dermal wound model was also performed, using a semi-occlusive wound dressing (Opsite) to maintain the implant in the wound site. The fibrillar collagen implant alone was pushed upward by developing granulation tissue at the base of the wound and served as a support for epidermal cell migration, proliferation, and differentiation as wound closure proceeded. The implant was slowly invaded and turned over as granulation tissue developed from the base and margins of the wound bed. The inclusion of heparin in these implants resulted in a significantly different pattern of wound healing. The collagen/heparin implants histologically presented a more broken-up or porous appearance following implantation, which was associated with a greater degree of penetration of developing granulation tissue into the implant itself as compared to the collagen implants. Radiolabeling studies revealed that clearance rates of implants with and without heparin from wound sites were similar, as noted in the rat subcutis. Laser doppler flowmetry studies suggested that the heparin-containing implants were more vascular than control wound sites or sites treated with collagen alone.</p></div>","PeriodicalId":77694,"journal":{"name":"Collagen and related research","volume":"8 1","pages":"Pages 83-100"},"PeriodicalIF":0.0,"publicationDate":"1988-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0174-173X(88)80037-2","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14256943","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Collagen Remodelling in Rat Periodontal Tissues: Compensation for Precursor Reutilization Confirms Rapid Turnover of Collagen","authors":"Jaro Sodek ,&nbsp;Jack M. Ferrier","doi":"10.1016/S0174-173X(88)80032-3","DOIUrl":"10.1016/S0174-173X(88)80032-3","url":null,"abstract":"<div><p>Measurement of collagen turnover is complicated by the reutilization of isotopicprecursors used to label the collagen. In an earlier study a novel approach was used to circumvent the problems of precursor recycling and unusually short half-lives were determined for collagen in adult rat periodontal tissues (Sodek, 1977). To verify these results we have used an alternate procedure devised by Poole (1971) in which the decay profile for the radiolabelled protein is corrected in accordance with the decay of the radiolabelled precursor. In this manner real half-lives for mature, neutral salt-insoluble collagen were determined as 3 days in the molar periodontal ligament, 6 days in the continuously erupting incisor ligament and approximately 10 days in the lamina propria of the gingiva, compared to apparent half-lives for these tissues of 6, 12 and approximately 20 days, respectively. The values calculated for actual half-lives are, therefore, approximately two-fold faster than values determined without compensating for reutilization, a difference that is in agreement with other protein turnover studies in which the effects of precursor reutilization have been measured. Although the real half-lives determined in this study indicate turnover rates for the periodontal tissues that are slightly slower than reported previously, the relative differences between the tissues in the rates of collagen turnover are similar. Moreover, the study confirms the existence of a remarkably high rate of collagen remodelling in these tissues.</p></div>","PeriodicalId":77694,"journal":{"name":"Collagen and related research","volume":"8 1","pages":"Pages 11-21"},"PeriodicalIF":0.0,"publicationDate":"1988-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0174-173X(88)80032-3","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14470515","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative Evaluation of the Factors Affecting the Process of Fibroblast-Mediated Collagen Gel Contraction by Separating the Process into Three Phases","authors":"Toshio Nishiyama ,&nbsp;Naoki Tominaga ,&nbsp;Keisuke Nakajima ,&nbsp;Toshihiko Hayashi","doi":"10.1016/S0174-173X(88)80045-1","DOIUrl":"10.1016/S0174-173X(88)80045-1","url":null,"abstract":"<div><p>Kinetics of collagen gel contraction by fibroblasts cultured <em>in vitro</em> was examined in detail for quantitative analysis. The process of collagen gel contraction was not expressed by a simple function of time. It appeared to consist of three distinct phases; a lag phase before the initiation of contraction, a rapid contraction phase and a slow contraction phase. Factors affecting the gel contraction can be classified into four groups. The first group includes increase in cell number, in culture temperature or in serum concentration, which strengthened the contraction in all the three phases, suggesting that they affected cellular activity particularly in interacting with collagen. The second group repressed the later two phases of contraction but not the first lag phase, typically increase in collagen concentration and a low dose of nocodazole or colcemid. Increasing population doubling levels of fibroblasts belongs to the third group which caused a reduced lag time but no change in the later two phases. Cytochalasin D at a low dose (0.03–0.1 µg/ml) is another example of the third group which shortened the lag time. The last group did not change the contraction curves. Donor age of fibroblasts isolated from the skin is an example of this group. The rate of rapid contraction in the second phase was always found to be closely correlated with the degree of contraction at the end of the third phase, in a whole set of the factors above mentioned. The results suggest that the extent of the later two phases might be a reflection of the same cellular activity, particularly cytokinetical one. The lag time is directly related to the time for cells to become elongate in shape as observed by using the video-microscopy, suggesting that the lag phase is also governed by cytokinetical activity. The two cytokinetical activities are closely related, but may be distinct, since the factors affecting the collagen gel contraction can be differentiated into four groups.</p></div>","PeriodicalId":77694,"journal":{"name":"Collagen and related research","volume":"8 3","pages":"Pages 259-273"},"PeriodicalIF":0.0,"publicationDate":"1988-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0174-173X(88)80045-1","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14521318","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Interleukin-1-Induced Changes in Extracellular Glycosaminoglycan Composition of Cutaneous Scar-Derived Fibroblasts in Culture","authors":"Rebecca E. Bronson,&nbsp;Jacqueline G. Argenta,&nbsp;Charles N. Bertolami","doi":"10.1016/S0174-173X(88)80040-2","DOIUrl":"10.1016/S0174-173X(88)80040-2","url":null,"abstract":"<div><p>Fibroblast cultures established from explants of mature scar and skin tissue were analyzed with regard to extracellular glycosaminoglycan (GAG) composition and response to interleukin-1 (IL-1). Following a serum-free 48 hour label with [3H]glucosamine, pericellular and medium GAGs were isolated by precipitation with cetylpyridinium chloride (CPC) and analyzed by cellulose acetate electrophoresis. In addition, susceptibility of the precipitates to <em>Streptomyces</em> hyaluronidase, chondroitinase ABC and heparitinase was determined. Labeled conditioned medium from the scarderived cells contained both dermatan sulfate (DS) and hyaluronate (HA), as compared to medium from the control (skin-derived) cells which contained predominantly DS. IL1 induced the appearance of chondroitin 4-sulfate (C4-S) in the medium of the scar cells with no concurrent effect on either DS or HA, and increased the amount of HA in the medium fraction of normal skin cells. The pericellular fraction of the scar-derived cells contained chondroitin 6-sulfate (C6-S) and DS; addition of IL-1 resulted in a shift from DS to heparan sulfate (HS), and the emergence of a pericellular GAG profile similar to that of normal dermal fibroblasts.</p></div>","PeriodicalId":77694,"journal":{"name":"Collagen and related research","volume":"8 3","pages":"Pages 199-208"},"PeriodicalIF":0.0,"publicationDate":"1988-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0174-173X(88)80040-2","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14266625","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Immunohistochemical Micromethods for the Measurement of Specific Collagen Types in Human Liver Biopsies","authors":"S. Guerret ,&nbsp;M. Rojkind ,&nbsp;M. Druguet ,&nbsp;M. Chevallier ,&nbsp;J.A. Grimaud","doi":"10.1016/S0174-173X(88)80044-X","DOIUrl":"10.1016/S0174-173X(88)80044-X","url":null,"abstract":"<div><p>Micromethods are needed for measuring the amount of collagen and other matricial proteins in human liver biopsies. Recently, a colorimetric method for the quantitative estimation of total collagen and protein has been developed. This method is sensitive, can be applied to fresh and fixed tissues and provides the absolute value of total collagen per section. Results are expressed as µg of collagen per mg of proteins or per section. We now report two micromethods for the determination of specific collagen types in fresh frozen liver sections. The first method is a direct method utilizing the tissue section as an ELISA plate. Results are expressed as the amount of color reaction read at 450 nm per slide. After cleaving the antigen antibody complex with dilute acetic acid, the amount of total collagen can be measured by the colorimetric microchemical method and the amount of specific collagen type is expressed as the amount of color reaction per µg of total collagen. The second method is an indirect method: the floating tissue section is incubated with an optimal amount of specific anti-collagen antibody and the residual amount of antibody present in the supernatant is measured in an ELISA plate coated with the specific collagen type. The amount of specific collagen type present in the tissue section is determined by comparing the optical deviation obtained with a standard curve prepared with known amounts of specific collagen, and values are expressed as µg of collagen per tissue sections. As with the direct method, the amount of total collagen per section can be measured by the colorimetric method and then, the amount of specific collagen type can be expressed as µg of specific collagen type per µg of total collagen.</p></div>","PeriodicalId":77694,"journal":{"name":"Collagen and related research","volume":"8 3","pages":"Pages 249-258"},"PeriodicalIF":0.0,"publicationDate":"1988-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0174-173X(88)80044-X","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14521317","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Decline of Fibroblast Chemotaxis with Age of Donor and Cell Passage Number","authors":"Adriana Albini ,&nbsp;Bertram Pontz ,&nbsp;Matthias Pulz ,&nbsp;Gabriella Allavena ,&nbsp;Hartwig Mensing ,&nbsp;Peter K. Müller","doi":"10.1016/S0174-173X(88)80033-5","DOIUrl":"10.1016/S0174-173X(88)80033-5","url":null,"abstract":"<div><p>Human dermal fibroblasts have a limited life span in culture, which is manifested by aprogressive decline of their proliferative activity. Here we show by the Boyden Chamber assay that the chemotactic response of human fibroblasts to fib rob last-conditioned medium and fibronectin declines during cellular aging in <em>vitro</em> and <em>in vivo</em>. The chemotactic response of human embryonic fibroblasts (HEF) declined progressively after the 25th passage. Virtually no chemotactic activity could be observed after the 40th passage in culture. Fibroblasts cultures from donors aged between 70-90 years had lost chemotactic activity by the 15th passage. Cells from patients suffering from progeroid syndromes of premature aging showed, even in early passages, a very low chemotactic response (20% of the HEF) and lost their chemotactic activity after a few subcultures. The response to the chemoattractant fibronectin also decreased with aging. Immuno-fluorescence studies indicated that the decline in chemotactic activity was accompanied by the formation of a thicker fibronectin network in the extracellular matrix of senescent human fibroblasts and progeroid cells than that observed in early passage embryonic cultures. Since fibroblast chemotaxis and synthesis of connective tissue components probably play an important role in tissue repair, our results could contribute to an understanding of age-related differences in the healing of skin wounds.</p></div>","PeriodicalId":77694,"journal":{"name":"Collagen and related research","volume":"8 1","pages":"Pages 23-37"},"PeriodicalIF":0.0,"publicationDate":"1988-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0174-173X(88)80033-5","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14407776","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Effects of Heparin on the Physicochemical Properties of Reconstituted Collagen","authors":"John M. Mcpherson ,&nbsp;Steven J. Sawamura ,&nbsp;Richard A. Condell ,&nbsp;Woonza Rhee ,&nbsp;Donald G. Wallace","doi":"10.1016/S0174-173X(88)80036-0","DOIUrl":"10.1016/S0174-173X(88)80036-0","url":null,"abstract":"<div><p>Pepsin-solubilized bovine dermal collagen was reconstituted in 0.02 M sodium phosphate(pH 7.2), concentrated to 30-40 mg/ml, and adjusted to physiological ionic strength by addition of sodium chloride. These preparations, at 4-15 °C, are fibrillar suspensions composed of fibrils of varying diameters and nonassociated molecules. Addition of heparin to these suspensions promoted a dose-dependent increase in average fibril diameter as measured by turbidimetry and electron microscopic analyses. These effects were relatively specific for heparin and heparin-like glycosaminoglycans. Chondroitin sulfate and hyaluronic acid had little or no effect on fibrillar diameters under these conditions, whereas dermatan sulfate had an intermediate effect on fibrillar reorganization. Differential scanning calorimetry revealed that addition of optimal concentrations of heparin generated fibrils of higher stability and that this effect was associated with the disappearance of structures of lower stability, including nonassociated molecules and thin fibrils. Light microscopic analyses of the fibrillar collagen/heparin matrix showed it to be a more open network of distinct collagen fibers than was observed with the fibrillar collagen preparation alone. Binding experiments indicated that heparin bound to fibrillar collagen in a saturable fashion with a Kd of approximately 4 × 10^-7 M. Creep experiments provided evidence that the addition of heparin to fibrillar collagen suspensions greatly reduces the gelation phenomenon that is normally observed when such suspensions are warmed to 37°C. These differences in fibrillar architecture may be in part responsible for differences noted in the biological response to fibrillar collagen and fibrillar collagen/heparin implants <em>in vivo</em> (McPherson et al., 1988).</p></div>","PeriodicalId":77694,"journal":{"name":"Collagen and related research","volume":"8 1","pages":"Pages 65-82"},"PeriodicalIF":0.0,"publicationDate":"1988-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0174-173X(88)80036-0","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14256942","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}