scholarly journals Thyrotropin cross-links to the thyrotropin receptor through both the α and β subunits

1986 ◽  
Vol 235 (3) ◽  
pp. 879-882 ◽  
Author(s):  
P R Buckland ◽  
C R Rickards ◽  
R D Howells ◽  
B R Smith
Keyword(s):  
Tsh Receptor ◽  
Alpha Subunit ◽  
Receptor Interaction ◽  
Cross Linking ◽  
Thyrotropin Receptor ◽  
Cross Link ◽  
Receptor Binding Site ◽  
Form Part ◽  
Cross Links

We have recently shown that the beta subunit of thyrotropin (TSH) can be cross-linked to the TSH receptor [Buckland, Strickland, Pierce & Rees Smith (1985) Endocrinology (Baltimore) 116, 2122-2124; Buckland, Strickland & Rees Smith (1985) Biochem. Soc. Trans. 13, 942-943]. We failed, however, to cross-link the alpha subunit to the receptor, leaving the role of this subunit in the TSH-TSH-receptor interaction uncertain. We now report the successful cross-linking of the TSH alpha subunit to the receptor by the use of two different cross-linking reagents. Our studies suggest therefore that both subunits of TSH form part of the hormone's receptor-binding site.

EXPERIMENTAL VALIDATION OF THE CHARLESBY AND HORIKX MODELS APPLIED TO DE-VULCANIZATION OF SULFUR AND PEROXIDE VULCANIZATES OF NR AND EPDM

2016 ◽  
Vol 89 (4) ◽  
pp. 671-688 ◽  
Author(s):  
M. A. L. Verbruggen ◽  
L. van der Does ◽  
W. K. Dierkes ◽  
J. W. M. Noordermeer
Keyword(s):  
Experimental Validation ◽  
Main Chain ◽  
Sol Gel ◽  
Chain Scission ◽  
Cross Linking ◽  
Cross Link ◽  
Practical Reality ◽  
Cross Links ◽  
The Cross ◽  
Theoretical Considerations

ABSTRACT The theoretical model developed by Charlesby to quantify the balance between cross-links creation of polymers and chain scission during radiation cross-linking and further modifications by Horikx to describe network breakdown from aging were merged to characterize the balance of both types of scission on the development of the sol content during de-vulcanization of rubber networks. There are, however, disturbing factors in these theoretical considerations vis-à-vis practical reality. Sulfur- and peroxide-cured NR and EPDM vulcanizates were de-vulcanized under conditions of selective cross-link and random main-chain scissions. Cross-link scission was obtained using thiol-amine reagents for selective cleavage of sulfur cross-links. Random main-chain scission was achieved by heating peroxide vulcanizates of NR with diphenyldisulfide, a method commonly employed for NR reclaiming. An important factor in the analyses of these experiments is the cross-linking index. Its value must be calculated using the sol fraction of the cross-linked network before de-vulcanization to obtain reliable results. The values for the cross-linking index calculated with sol-gel data before de-vulcanization appear to fit the experimentally determined modes of network scission during de-vulcanization very well. This study confirms that the treatment of de-vulcanization data with the merged Charlesby and Horikx models can be used satisfactorily to characterize the de-vulcanization of NR and EPDM vulcanizates.

Role of the FAD-dependent polyamine oxidase in the selective formation of N1,N8-bis(γ- glutamyl)spermidine protein cross-links1

2007 ◽  
Vol 35 (2) ◽  
pp. 396-400 ◽  
Author(s):  
A. Lentini ◽  
P. Mattioli ◽  
B. Provenzano ◽  
A. Abbruzzese ◽  
M. Caraglia ◽  
...  
Keyword(s):  
Stratum Corneum ◽  
Polyamine Oxidase ◽  
Polyamine Metabolism ◽  
Suitable Model ◽  
Cross Link ◽  
Cellular Mechanisms ◽  
Preferential Formation ◽  
Cross Links ◽  
Selective Formation

Protein-bound γ-glutamylpolyamines have highlighted a new pathway in polyamine metabolism. Human foreskin keratinocytes offer a suitable model for this study. Indeed, they develop polymerized envelopes, as they differentiate, rich in ϵ-(γ-glutamyl)lysine and N1,N8-bis(γ-glutamyl)spermidine cross-links. We have found that the selective oxidation of N1-(γ-glutamyl)spermidine and N-(γ-glutamyl)spermine by FAD-dependent polyamine oxidase (PAO) may be one of the cellular mechanisms regulating the preferential formation of a sterically defined bis(γ-glutamyl)spermidine cross-link. The significance of this finding is unknown, but it suggests that the target of this PAO-modulation is to achieve the biochemical prerequisite for production of a normal epidermal stratum corneum.

scholarly journals Two DD-Carboxypeptidases fromMycobacterium smegmatisAffect Cell Surface Properties through Regulation of Peptidoglycan Cross-Linking and Glycopeptidolipids

2018 ◽  
Vol 200 (14) ◽  
Author(s):  
Satya Deo Pandey ◽  
Shilpa Pal ◽  
Ganesh Kumar N ◽  
Ankita Bansal ◽  
Sathi Mallick ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Cross Linking ◽  
Surface Lipid ◽  
Cross Link ◽  
Content Type ◽  
Murine Macrophages ◽  
Cross Links ◽  
Link Formation

ABSTRACTDuring the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between twomeso-diaminopimelic acids [meso-DAPs]) and 4-3 cross-links (betweend-Ala andmeso-DAP), though there is a predominance (60 to 80%) of 3-3 cross-links. Thedd-carboxypeptidases (dd-CPases) act on pentapeptides to generate tetrapeptides that are used byld-transpeptidases as substrates to form 3-3 cross-links. Therefore,dd-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology ofdd-CPases in mycobacteria is relatively unexplored. In this study, we deleted twodd-CPase genes,msmeg_2433andmsmeg_2432, both individually and in combination, fromMycobacterium smegmatismc2155. Though the singledd-CPase gene deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double-deletion mutant,viz., a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced, and susceptibility to β-lactams and antitubercular agents was enhanced. Moreover, the survival rate of the double mutant within murine macrophages was higher than that of the parent. Interestingly, the complementation with any one of thedd-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3 to 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation, drug susceptibility, and subsistence of the cells within macrophages.IMPORTANCEThe glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. Thedd-CPases generate tetrapeptides by acting on the pentapeptides, andld-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. In this study, we showed that simultaneous deletions of twodd-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of glycopeptidolipids (significant surface lipid present in many nontuberculous mycobacteria, includingMycobacterium smegmatis) and affect other physiological parameters, like cell morphology, growth rate, biofilm formation, antibiotic susceptibility, and survival within murine macrophages. Thus, unraveling the physiology ofdd-CPases might help us design antimycobacterial therapeutics in the future.

scholarly journals Overhydroxylation of Lysine of Collagen Increases Uterine Fibroids Proliferation: Roles of Lysyl Hydroxylases, Lysyl Oxidases, and Matrix Metalloproteinases

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
Marwa Kamel ◽  
Mohamed Wagih ◽  
Gokhan S. Kilic ◽  
Concepcion R. Diaz-Arrastia ◽  
Mohamed A. Baraka ◽  
...  
Keyword(s):  
Matrix Metalloproteinases ◽  
Treatment Options ◽  
Uterine Fibroids ◽  
Cross Linking ◽  
Ki 67 ◽  
Subsequent Formation ◽  
Lysyl Oxidases ◽  
Cross Links ◽  
Collagen Cross Linking

The role of the extracellular matrix (ECM) in uterine fibroids (UF) has recently been appreciated. Overhydroxylation of lysine residues and the subsequent formation of hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) cross-links underlie the ECM stiffness and profoundly affect tumor progression. The aim of the current study was to investigate the relationship between ECM of UF, collagen and collagen cross-linking enzymes [lysyl hydroxylases (LH) and lysyl oxidases (LOX)], and the development and progression of UF. Our results indicated that hydroxyl lysine (Hyl) and HP cross-links are significantly higher in UF compared to the normal myometrial tissues accompanied by increased expression of LH (LH2b) and LOX. Also, increased resistance to matrix metalloproteinases (MMP) proteolytic degradation activity was observed. Furthermore, the extent of collagen cross-links was positively correlated with the expression of myofibroblast marker (α-SMA), growth-promoting markers (PCNA; pERK1/2;FAKpY397; Ki-67; and Cyclin D1), and the size of UF. In conclusion, our study defines the role of overhydroxylation of collagen and collagen cross-linking enzymes in modulating UF cell proliferation, differentiation, and resistance to MMP. These effects can establish microenvironment conducive for UF progression and thus represent potential target treatment options of UF.

scholarly journals Cross-linking modifications of HDL apoproteins by oxidized phospholipids: structural characterization, in vivo detection, and functional implications

2020 ◽  
Vol 295 (7) ◽  
pp. 1973-1984
Author(s):  
Detao Gao ◽  
Mohammad Z. Ashraf ◽  
Lifang Zhang ◽  
Niladri Kar ◽  
Tatiana V. Byzova ◽  
...  
Keyword(s):  
Density Lipoprotein ◽  
Cross Linking ◽  
Oxidized Phospholipids ◽  
Cross Link ◽  
In Vivo Detection ◽  
Lysine Residues ◽  
Cross Links ◽  
Human Atheroma

Apolipoprotein A-I (apoA-I) is cross-linked and dysfunctional in human atheroma. Although multiple mechanisms of apoA-I cross-linking have been demonstrated in vitro, the in vivo mechanisms of cross-linking are not well-established. We have recently demonstrated the highly selective and efficient modification of high-density lipoprotein (HDL) apoproteins by endogenous oxidized phospholipids (oxPLs), including γ-ketoalkenal phospholipids. In the current study, we report that γ-ketoalkenal phospholipids effectively cross-link apoproteins in HDL. We further demonstrate that cross-linking impairs the cholesterol efflux mediated by apoA-I or HDL3 in vitro and in vivo. Using LC-MS/MS analysis, we analyzed the pattern of apoprotein cross-linking in isolated human HDL either by synthetic γ-ketoalkenal phospholipids or by oxPLs generated during HDL oxidation in plasma by the physiologically relevant MPO-H2O2-NO2− system. We found that five histidine residues in helices 5–8 of apoA-I are preferably cross-linked by oxPLs, forming stable pyrrole adducts with lysine residues in the helices 3–4 of another apoA-I or in the central domain of apoA-II. We also identified cross-links of apoA-I and apoA-II with two minor HDL apoproteins, apoA-IV and apoE. We detected a similar pattern of apoprotein cross-linking in oxidized murine HDL. We further detected oxPL cross-link adducts of HDL apoproteins in plasma and aorta of hyperlipidemic LDLR−/− mice, including cross-link adducts of apoA-I His-165–apoA-I Lys-93, apoA-I His-154–apoA-I Lys-105, apoA-I His-154–apoA-IV Lys-149, and apoA-II Lys-30–apoE His-227. These findings suggest an important mechanism that contributes to the loss of HDL's atheroprotective function in vivo.

Mechanistic and Pharmacodynamic Studies of Adct-301, a Pyrrolobenzodiazepine (PBD) Dimer-Containing Antibody Drug Conjugate (ADC) Targeting CD25-Expressing Hematological Malignancies

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1559-1559 ◽  
Author(s):  
Michael J. Flynn ◽  
Patrick H. van Berkel ◽  
Francesca Zammarchi ◽  
Peter C. Tyrer ◽  
Ayse U. Akarca ◽  
...  
Keyword(s):  
Cell Surface ◽  
Clinical Development ◽  
Cross Linking ◽  
Cross Link ◽  
Employment Equity ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Equity Ownership ◽  
Dose Dependent ◽  
Link Formation

Abstract ADCT-301, currently in Phase I clinical trial, is an ADC composed of a recombinant human IgG1, HuMax®-TAC against human IL-2R-α (CD25) conjugated through a cleavable linker to a PBD dimer warhead with a drug-antibody ratio of 2.3. In vitro and ex vivo, ADCT-301 binds human CD25 with picomolar affinity. ADCT-301 has highly potent and targeted cytotoxicity against a panel of human lymphoma cell lines. On release, PBD dimers bind in the DNA minor groove and exert their cytotoxic action via the formation of DNA interstrand cross-links. In vivo, ADCT-301 demonstrates dose-dependent antitumor activity against subcutaneous and disseminated lymphoma models. For example, in the Karpas 299 xenograft model, 10/10 tumor-free survivors are observed following a single dose of 0.5 mg/kg, whereas Adcetris® gives only a modest delay in mean tumor growth at 0.5 mg/kg, despite this tumor expressing three-fold higher target antigen levels for this drug. The current study aimed to further define the mechanism of action of ADCT-301 and validate pharmacodynamic assays for clinical development. In Karpas 299 cells, evidence for internalization of ADCT-301 was shown by a reduction of CD25 molecules on the cell surface over the first three hours post-treatment followed by a return to pre-treatment levels by 16 hours. This is consistent with the documented rapid recycling of CD25 to the membrane after exposure to IL-2 (Hemar et al Journal of Cell Biology 1995). Furthermore, ADCT-301 on the cell surface declined by >70% over four hours. Following a two-hour exposure to ADCT-301, DNA interstrand cross-linking, measured using a modification of the single cell gel electrophoresis (comet) assay, reached a peak between 4 and 8 hours after which cross-links persisted up to 36 hours. In contrast, the peak of cross-link formation for an equimolar concentration of warhead was immediately following drug exposure and a non-targeted PBD-containing ADC did not produce crosslinks in these cells. A strong correlation (r = 0.97) between loss of viability and DNA cross-link formation provides support for this DNA damage being the critical initiating mechanism of cytotoxicity of ADCT-301. We have previously shown that PBD-induced DNA interstrand cross-links elicit a robust, but delayed γ-H2AX response (Wu et al Clinical Cancer Research 2013). In Karpas 299 cells phosphorylation of H2AX was observed 24 hours after a two-hour exposure to sub-GI50 concentrations of ADCT-301. In these cells continuous exposure to ADCT-301 resulted in a dose-dependent G2/M arrest, peaking at 48 hours, later than for the naked warhead. The peak of the early apoptosis marker annexin-V on the cell surface of Karpas 299 cells was observed between 60 and 72 hours and maximal loss of viability was at 96 hours. Significant bystander killing of CD25-negative human Burkitt's lymphoma-derived Ramos cells was demonstrated for ADCT-301 both by co-culture experiments with CD25-positive Karpas 299 cells, and by media transfer from Karpas 299 cells treated with ADCT-301. This is important as many lymphomas are heterogeneous in their CD25 expression profile (Strauchen et al American Journal of Pathology 1987). In SCID mice with Karpas 299 subcutaneous tumors a single dose of ADCT-301 was administered at 0.2 or 0.6 mg/kg. 24 hours after treatment, excised tumors showed a dose proportional increase in intensity of membrane and cytoplasmic staining by an anti-PBD payload antibody. Cross-linking was determined as 23% (0.2 mg/kg) vs 49% (0.6 mg/kg) (p ≤ 0.01) reduction in Tail Moment using the comet assay and dose-dependent γ-H2AX formation measured by immunohistochemistry was observed. No cross-linking was observed in matched lymphocyte samples. These data confirm the mechanism of cell killing of ADCT-301 and provide relevant pharmacodynamic assays for use in the clinical development of PBD-based ADCs. Disclosures Flynn: Spirogen/Medimmune: Employment. van Berkel:ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zammarchi:ADC Therapeutics: Employment. Tyrer:Spirogen/Medimmune: Employment. Williams:Spirogen/Medimmune: Employment. Howard:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Patents & Royalties. Hartley:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Chemistry of collagen cross-linking: biochemical changes in collagen during the partial mineralization of turkey leg tendon

1997 ◽  
Vol 322 (2) ◽  
pp. 535-542 ◽  
Author(s):  
Lynda KNOTT ◽  
John F. TARLTON ◽  
Allen J. BAILEY
Keyword(s):  
Collagen Matrix ◽  
Cross Linking ◽  
Cross Link ◽  
Lysine Residues ◽  
Collagen Mineralization ◽  
Cross Links ◽  
The Cross ◽  
Calcified Tissues ◽  
Turkey Leg Tendon ◽  
Collagen Cross Linking

With age, the proximal sections of turkey leg tendons become calcified, and this phenomenon has led to their use as a model for collagen mineralization. Mineralizing turkey leg tendon was used in this study to characterize further the composition and cross-linking of collagen in calcified tissues. The cross-link profiles of mineralizing collagen are significantly different from those of other collagenous matrices with characteristically low amounts of hydroxylysyl-pyridinoline and the presence of lysyl-pyridinoline and pyrrolic cross-links. However, the presence of the immature cross-link precursors previously reported in calcifying tissues was not supported in the present study, and was found to be due to the decalcification procedure using EDTA. Analysis of tendons from young birds demonstrated differences in the cross-link profile which indicated a higher level of hydroxylation of specific triple-helical lysines involved in cross-linking of the proximal tendon. This may be related to later calcification, suggesting that this part of the tendon is predestined to be calcified. The minimal changes in lysyl hydroxylation in both regions of the tendon with age were in contrast with the large changes in the cross-link profile, indicating differential hydroxylation of the helical and telopeptide lysine residues. Changes with age in the collagen matrix, its turnover and thermal properties in both the proximal and distal sections of the tendon clearly demonstrate that a new and modified matrix is formed throughout the tendon, and that a different type of matrix is formed at each site.

scholarly journals Characterization of Genipin-Modified Dentin Collagen

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Hiroko Nagaoka ◽  
Hideaki Nagaoka ◽  
Ricardo Walter ◽  
Lee W. Boushell ◽  
Patricia A. Miguez ◽  
...  
Keyword(s):  
Amino Acid ◽  
Lysyl Oxidase ◽  
Biomechanical Properties ◽  
Biochemical Properties ◽  
Cross Linking ◽  
Dependent Manner ◽  
Cross Link ◽  
Concentration Dependent Manner ◽  
Cross Links ◽  
Dentin Collagen

Application of biomodification techniques to dentin can improve its biochemical and biomechanical properties. Several collagen cross-linking agents have been reported to strengthen the mechanical properties of dentin. However, the characteristics of collagen that has undergone agent-induced biomodification are not well understood. The objective of this study was to analyze the effects of a natural cross-linking agent, genipin (GE), on dentin discoloration, collagen stability, and changes in amino acid composition and lysyl oxidase mediated natural collagen cross-links. Dentin collagen obtained from extracted bovine teeth was treated with three different concentrations of GE (0.01%, 0.1%, and 0.5%) for several treatment times (0–24 h). Changes in biochemical properties of NaB3H4-reduced collagen were characterized by amino acid and cross-link analyses. The treatment of dentin collagen with GE resulted in a concentration- and time-dependent pigmentation and stability against bacterial collagenase. The lysyl oxidase-mediated trivalent mature cross-link, pyridinoline, showed no difference among all groups while the major divalent immature cross-link, dehydro-dihydroxylysinonorleucine/its ketoamine in collagen treated with 0.5% GE for 24 h, significantly decreased compared to control (P< 0.05). The newly formed GE-induced cross-links most likely involve lysine and hydroxylysine residues of collagen in a concentration-dependent manner. Some of these cross-links appear to be reducible and stabilized with NaB3H4.

scholarly journals MaXLinker: Proteome-wide Cross-link Identifications with High Specificity and Sensitivity

2019 ◽  
Vol 19 (3) ◽  
pp. 554-568 ◽  
Author(s):  
Kumar Yugandhar ◽  
Ting-Yi Wang ◽  
Alden King-Yung Leung ◽  
Michael Charles Lanz ◽  
Ievgen Motorykin ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Search Engine ◽  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Cross Linking ◽  
Interaction Patterns ◽  
Protein Protein Interactions ◽  
Cross Link ◽  
Wide Cross ◽  
Cross Links

Protein-protein interactions play a vital role in nearly all cellular functions. Hence, understanding their interaction patterns and three-dimensional structural conformations can provide crucial insights about various biological processes and underlying molecular mechanisms for many disease phenotypes. Cross-linking mass spectrometry (XL-MS) has the unique capability to detect protein-protein interactions at a large scale along with spatial constraints between interaction partners. The inception of MS-cleavable cross-linkers enabled the MS2-MS3 XL-MS acquisition strategy that provides cross-link information from both MS2 and MS3 level. However, the current cross-link search algorithm available for MS2-MS3 strategy follows a “MS2-centric” approach and suffers from a high rate of mis-identified cross-links. We demonstrate the problem using two new quality assessment metrics [“fraction of mis-identifications” (FMI) and “fraction of interprotein cross-links from known interactions” (FKI)]. We then address this problem, by designing a novel “MS3-centric” approach for cross-link identification and implementing it as a search engine named MaXLinker. MaXLinker outperforms the currently popular search engine with a lower mis-identification rate, and higher sensitivity and specificity. Moreover, we performed human proteome-wide cross-linking mass spectrometry using K562 cells. Employing MaXLinker, we identified a comprehensive set of 9319 unique cross-links at 1% false discovery rate, comprising 8051 intraprotein and 1268 interprotein cross-links. Finally, we experimentally validated the quality of a large number of novel interactions identified in our study, providing a conclusive evidence for MaXLinker's robust performance.

An ether-linked tetrafunctional acylating reagent and its cross-linking reactions with hemoglobin

1999 ◽  
Vol 77 (2) ◽  
pp. 271-279 ◽  
Author(s):  
Ronald Kluger ◽  
Krisztina Paal ◽  
J Gordon Adamson
Keyword(s):  
Peptide Mapping ◽  
Protein Molecule ◽  
Cross Linking ◽  
Cross Link ◽  
Ion Exchange Hplc ◽  
New Type ◽  
Cross Links ◽  
Modified Proteins ◽  
A Minor ◽  
Α Subunits

A new type of tetrafunctional reagent for cross-linking proteins has been prepared and used to modify human hemoglobin A. DPEE (1,2-bis{2-[3,5-bis(3,5-dibromosalicyloxycarbonyl) phenoxy]ethoxy}ethane)) has two separate pairs of reacting sites connected by a flexible tetraether chain. DPEE is capable of connecting a cross-link within a protein to another cross-link, either within the same protein molecule or between molecules. DPEE was readily prepared by esterification of a tetraether-linked bisphthalate (prepared by coupling of 1,2-bis(2-iodoethoxy)ethane and 5-hydroxyisophthalic acid). DPEE reacts with deoxy hemoglobin to produce a mixture of modified proteins. Ion-exchange HPLC was used to separate the modified proteins in the mixture. The most abundant products were selected for structural analysis, which used data from reverse-phase chromatography and tryptic peptide mapping. To prevent dissociation of the modified proteins during analysis, the products were further reacted with the bifunctional reagent, bis(3,5-dibromosalicyl) fumarate, which produces fumaryl cross-links between α-subunits. From peptide analysis of the separated products, the major modified protein from DPEE was identified as a novel species with four links within the same α2β2 tetramer. In addition, a minor product that involves cross-links in two different proteins was observed. These results imply that the reagent reacts primarily in a folded state within the protein.Key words: acylation: cross-linking, multifunctional, hemoglobin, reaction pattern.

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