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Sample records for biofilm extracellular matrix

  1. Extracellular DNA as matrix component in microbial biofilms

    DEFF Research Database (Denmark)

    Chiang, Wen-Chi; Tolker-Nielsen, Tim

    2010-01-01

    Bacteria in nature primarily live in surface-associated communities commonly known as biofilms. Because bacteria in biofilms, in many cases, display tolerance to host immune systems, antibiotics, and biocides, they are often difficult or impossible to eradicate. Biofilm formation, therefore, leads...... to various persistent infections in humans and animals, and to a variety of complications in industry, where solid–water interfaces occur. Knowledge about the molecular mechanisms involved in biofilm formation is necessary for creating strategies to control biofilms. Recent studies have shown...... that extracellular DNA is an important component of the extracellular matrix of microbial biofilms. The present chapter is focussed on extracellular DNA as matrix component in biofilms formed by Pseudomonas aeruginosa as an example from the Gram-negative bacteria, and Streptococcus and Staphylococcus as examples...

  2. The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps.

    Science.gov (United States)

    Johnson, Chad J; Cabezas-Olcoz, Jonathan; Kernien, John F; Wang, Steven X; Beebe, David J; Huttenlocher, Anna; Ansari, Hamayail; Nett, Jeniel E

    2016-09-01

    Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix.

  3. Extracellular DNA and lipoteichoic acids interact with exopolysaccharides in the extracellular matrix of Streptococcus mutans biofilms

    Science.gov (United States)

    Castillo Pedraza, Midian C.; Novais, Tatiana F.; Faustoferri, Roberta C.; Quivey, Robert G.; Terekhov, Anton; Hamaker, Bruce R.; Klein, Marlise I.

    2018-01-01

    Streptococcus mutans -derived exopolysaccharides are virulence determinants in the matrix of biofilms that cause caries. Extracellular DNA (eDNA) and lipoteichoic acid (LTA) are found in cariogenic biofilms, but their functions are unclear. Therefore, strains of S. mutans carrying single deletions that would modulate matrix components were used: eDNA – ΔlytS and ΔlytT; LTA – ΔdltA and ΔdltD; and insoluble exopolysaccharide – ΔgtfB. Single-species (parental strain S. mutans UA159 or individual mutant strains) and mixed-species (UA159 or mutant strain, Actinomyces naeslundii and Streptococcus gordonii) biofilms were evaluated. Distinct amounts of matrix components were detected, depending on the inactivated gene. eDNA was found to be cooperative with exopolysaccharide in early phases, while LTA played a larger role in the later phases of biofilm development. The architecture of mutant strains biofilms was distinct (vs UA159), demonstrating that eDNA and LTA influence exopolysaccharide distribution and microcolony organization. Thus, eDNA and LTA may shape exopolysaccharide structure, affecting strategies for controlling pathogenic biofilms. PMID:28946780

  4. Extracellular DNA and lipoteichoic acids interact with exopolysaccharides in the extracellular matrix of Streptococcus mutans biofilms.

    Science.gov (United States)

    Castillo Pedraza, Midian C; Novais, Tatiana F; Faustoferri, Roberta C; Quivey, Robert G; Terekhov, Anton; Hamaker, Bruce R; Klein, Marlise I

    2017-10-01

    Streptococcus mutans-derived exopolysaccharides are virulence determinants in the matrix of biofilms that cause caries. Extracellular DNA (eDNA) and lipoteichoic acid (LTA) are found in cariogenic biofilms, but their functions are unclear. Therefore, strains of S. mutans carrying single deletions that would modulate matrix components were used: eDNA - ∆lytS and ∆lytT; LTA - ∆dltA and ∆dltD; and insoluble exopolysaccharide - ΔgtfB. Single-species (parental strain S. mutans UA159 or individual mutant strains) and mixed-species (UA159 or mutant strain, Actinomyces naeslundii and Streptococcus gordonii) biofilms were evaluated. Distinct amounts of matrix components were detected, depending on the inactivated gene. eDNA was found to be cooperative with exopolysaccharide in early phases, while LTA played a larger role in the later phases of biofilm development. The architecture of mutant strains biofilms was distinct (vs UA159), demonstrating that eDNA and LTA influence exopolysaccharide distribution and microcolony organization. Thus, eDNA and LTA may shape exopolysaccharide structure, affecting strategies for controlling pathogenic biofilms.

  5. Extracellular-matrix-mediated osmotic pressure drives Vibrio cholerae biofilm expansion and cheater exclusion

    OpenAIRE

    Yan, Jing; Nadell, Carey D.; Stone, Howard A.; Wingreen, Ned S.; Bassler, Bonnie L.

    2017-01-01

    Biofilms, surface-attached communities of bacteria encased in an extracellular matrix, are a major mode of bacterial life. How the material properties of the matrix contribute to biofilm growth and robustness is largely unexplored, in particular in response to environmental perturbations such as changes in osmotic pressure. Here, using Vibrio cholerae as our model organism, we show that during active cell growth, matrix production enables biofilm-dwelling bacterial cells to establish an osmot...

  6. Extracellular-matrix-mediated osmotic pressure drives Vibrio cholerae biofilm expansion and cheater exclusion.

    Science.gov (United States)

    Yan, Jing; Nadell, Carey D; Stone, Howard A; Wingreen, Ned S; Bassler, Bonnie L

    2017-08-23

    Biofilms, surface-attached communities of bacteria encased in an extracellular matrix, are a major mode of bacterial life. How the material properties of the matrix contribute to biofilm growth and robustness is largely unexplored, in particular in response to environmental perturbations such as changes in osmotic pressure. Here, using Vibrio cholerae as our model organism, we show that during active cell growth, matrix production enables biofilm-dwelling bacterial cells to establish an osmotic pressure difference between the biofilm and the external environment. This pressure difference promotes biofilm expansion on nutritious surfaces by physically swelling the colony, which enhances nutrient uptake, and enables matrix-producing cells to outcompete non-matrix-producing cheaters via physical exclusion. Osmotic pressure together with crosslinking of the matrix also controls the growth of submerged biofilms and their susceptibility to invasion by planktonic cells. As the basic physicochemical principles of matrix crosslinking and osmotic swelling are universal, our findings may have implications for other biofilm-forming bacterial species.Most bacteria live in biofilms, surface-attached communities encased in an extracellular matrix. Here, Yan et al. show that matrix production in Vibrio cholerae increases the osmotic pressure within the biofilm, promoting biofilm expansion and physical exclusion of non-matrix producing cheaters.

  7. Escherichia coli biofilms have an organized and complex extracellular matrix structure.

    Science.gov (United States)

    Hung, Chia; Zhou, Yizhou; Pinkner, Jerome S; Dodson, Karen W; Crowley, Jan R; Heuser, John; Chapman, Matthew R; Hadjifrangiskou, Maria; Henderson, Jeffrey P; Hultgren, Scott J

    2013-09-10

    Bacterial biofilms are ubiquitous in nature, and their resilience is derived in part from a complex extracellular matrix that can be tailored to meet environmental demands. Although common developmental stages leading to biofilm formation have been described, how the extracellular components are organized to allow three-dimensional biofilm development is not well understood. Here we show that uropathogenic Escherichia coli (UPEC) strains produce a biofilm with a highly ordered and complex extracellular matrix (ECM). We used electron microscopy (EM) techniques to image floating biofilms (pellicles) formed by UPEC. EM revealed intricately constructed substructures within the ECM that encase individual, spatially segregated bacteria with a distinctive morphology. Mutational and biochemical analyses of these biofilms confirmed curli as a major matrix component and revealed important roles for cellulose, flagella, and type 1 pili in pellicle integrity and ECM infrastructure. Collectively, the findings of this study elucidated that UPEC pellicles have a highly organized ultrastructure that varies spatially across the multicellular community. Bacteria can form biofilms in diverse niches, including abiotic surfaces, living cells, and at the air-liquid interface of liquid media. Encasing these cellular communities is a self-produced extracellular matrix (ECM) that can be composed of proteins, polysaccharides, and nucleic acids. The ECM protects biofilm bacteria from environmental insults and also makes the dissolution of biofilms very challenging. As a result, formation of biofilms within humans (during infection) or on industrial material (such as water pipes) has detrimental and costly effects. In order to combat bacterial biofilms, a better understanding of components required for biofilm formation and the ECM is required. This study defined the ECM composition and architecture of floating pellicle biofilms formed by Escherichia coli.

  8. Biofilm-specific extracellular matrix proteins of non-typeable Haemophilus influenzae

    Science.gov (United States)

    Wu, Siva; Baum, Marc M.; Kerwin, James; Guerrero-Given, Debbie; Webster, Simon; Schaudinn, Christoph; VanderVelde, David; Webster, Paul

    2014-01-01

    Non-typeable Haemophilus influenzae (NTHi), a human respiratory tract pathogen can form colony biofilms in vitro. Bacterial cells and the amorphous extracellular matrix (ECM) constituting the biofilm can be separated using sonication. The ECM from 24 hr and 96 hr NTHi biofilms contained polysaccharides and proteinaceous components as detected by NMR and FTIR spectroscopy. More conventional chemical assays on the biofilm ECM confirmed the presence of these components and also DNA. Proteomics revealed eighteen proteins present in biofilm ECM that were not detected in planktonic bacteria. One ECM protein was unique to 24 hr biofilms, two were found only in 96 hr biofilms, and fifteen were present in the ECM of both 24 hr and 96 hr NTHi biofilms. All proteins identified were either associated with bacterial membranes or were cytoplasmic proteins. Immunocytochemistry showed two of the identified proteins, a DNA-directed RNA polymerase and the outer membrane protein OMP P2, associated with bacteria and biofilm ECM. Identification of biofilm-specific proteins present in immature biofilms is an important step in understanding the in vitro process of NTHi biofilm formation. The presence of a cytoplasmic protein and a membrane protein in the biofilm ECM of immature NTHi biofilms suggests that bacterial cell lysis may be a feature of early biofilm formation. PMID:24942343

  9. Extracellular matrix assembly in extreme acidic eukaryotic biofilms and their possible implications in heavy metal adsorption

    Energy Technology Data Exchange (ETDEWEB)

    Aguilera, Angeles [Centro de Astrobiologia (INTA-CSIC), Carretera de Ajalvir Km 4, Torrejon de Ardoz, 28850 Madrid (Spain)], E-mail: aguileraba@inta.es; Souza-Egipsy, Virginia [Centro de Astrobiologia (INTA-CSIC), Carretera de Ajalvir Km 4, Torrejon de Ardoz, 28850 Madrid (Spain); San Martin-Uriz, Patxi [Centro de Biologia Molecular (UAM-CSIC), Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); Amils, Ricardo [Centro de Astrobiologia (INTA-CSIC), Carretera de Ajalvir Km 4, Torrejon de Ardoz, 28850 Madrid (Spain); Centro de Biologia Molecular (UAM-CSIC), Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain)

    2008-07-30

    To evaluate the importance of the extracellular matrix in relation to heavy metal binding capacity in extreme acidic environments, the extracellular polymeric substances (EPS) composition of 12 biofilms isolated from Rio Tinto (SW, Spain) was analyzed. Each biofilm was composed mainly by one or two species of eukaryotes, although other microorganisms were present. EPS ranged from 130 to 439 mg g{sup -1} biofilm dry weight, representing between 15% and the 40% of the total biofilm dry weight (DW). Statistically significant differences (p < 0.05) were found in the amount of total EPS extracted from biofilms dominated by the same organism at different sampling points. The amount of EPS varied among different biofilms collected from the same sampling location. Colloidal EPS ranged from 42 to 313 mg g{sup -1} dry weight; 10% to 30% of the total biofilm dry weight. Capsular EPS ranged from 50 to 318 mg g{sup -1} dry weight; 5% to 30% of the total biofilm dry weight. Seven of the 12 biofilms showed higher amounts of capsular than colloidal EPS (p < 0.05). Total amount of EPS decreased when total cell numbers and pH increased. There was a positive correlation between EPS concentration and heavy metal concentration in the water. Observations by low temperature scanning electron microscopy (LTSEM) revealed the mineral adsorption in the matrix of EPS and onto the cell walls. EPS in all biofilms were primarily composed of carbohydrates, heavy metals and humic acid, plus small quantities of proteins and DNA. After carbohydrates, heavy metals were the second main constituents of the extracellular matrix. Their total concentrations ranged from 3 to 32 mg g{sup -1} biofilm dry weight, reaching up to 16% of the total composition. In general, the heavy metal composition of the EPS extracted from the biofilms closely resembled the metal composition of the water from which the biofilms were collected.

  10. Extracellular matrix assembly in extreme acidic eukaryotic biofilms and their possible implications in heavy metal adsorption

    International Nuclear Information System (INIS)

    Aguilera, Angeles; Souza-Egipsy, Virginia; San Martin-Uriz, Patxi; Amils, Ricardo

    2008-01-01

    To evaluate the importance of the extracellular matrix in relation to heavy metal binding capacity in extreme acidic environments, the extracellular polymeric substances (EPS) composition of 12 biofilms isolated from Rio Tinto (SW, Spain) was analyzed. Each biofilm was composed mainly by one or two species of eukaryotes, although other microorganisms were present. EPS ranged from 130 to 439 mg g -1 biofilm dry weight, representing between 15% and the 40% of the total biofilm dry weight (DW). Statistically significant differences (p -1 dry weight; 10% to 30% of the total biofilm dry weight. Capsular EPS ranged from 50 to 318 mg g -1 dry weight; 5% to 30% of the total biofilm dry weight. Seven of the 12 biofilms showed higher amounts of capsular than colloidal EPS (p -1 biofilm dry weight, reaching up to 16% of the total composition. In general, the heavy metal composition of the EPS extracted from the biofilms closely resembled the metal composition of the water from which the biofilms were collected

  11. Extracellular DNA as matrix component in microbial biofilms

    DEFF Research Database (Denmark)

    Chiang, Wen-Chi; Tolker-Nielsen, Tim

    2010-01-01

    Bacteria in nature primarily live in surface-associated communities commonly known as biofilms. Because bacteria in biofilms, in many cases, display tolerance to host immune systems, antibiotics, and biocides, they are often difficult or impossible to eradicate. Biofilm formation, therefore, leads...

  12. Extracellular matrix influence in Streptococcus mutans gene expression in a cariogenic biofilm.

    Science.gov (United States)

    Florez Salamanca, E J; Klein, M I

    2018-04-01

    Caries etiology is biofilm-diet-dependent. Biofilms are highly dynamic and structured microbial communities enmeshed in a three-dimensional extracellular matrix. The study evaluated the expression dynamics of Streptococcus mutans genes associated with exopolysaccharides (EPS) (gtfBCD, gbpB, dexA), lipoteichoic acids (LTA) (dltABCD, SMU_775c) and extracellular DNA (eDNA) (lytST, lrgAB, ccpA) during matrix development within a mixed-species biofilm of S. mutans, Actinomyces naeslundii and Streptococcus gordonii. Mixed-species biofilms using S. mutans strains UA159 or ΔgtfB formed on saliva-coated hydroxyapatite discs were submitted to a nutritional challenge (providing an abundance of sucrose and starch). Biofilms were removed at eight developmental stages for gene expression analysis by quantitative polymerase chain reaction. The pH of spent culture media remained acidic throughout the experimental periods, being lower after sucrose and starch exposure. All genes were expressed at all biofilm developmental phases. EPS- and LTA-associated genes had a similar expression profile for both biofilms, presenting lower levels of expression at 67, 91 and 115 hours and a peak of expression at 55 hours, but having distinct expression magnitudes, with lower values for ΔgtfB (eg, fold-difference of ~382 for gtfC and ~16 for dltB at 43 hours). The eDNA-associated genes presented different dynamics of expression between both strains. In UA159 biofilms lrgA and lrgB genes were highly expressed at 29 hours (which were ~13 and ~5.4 times vs ΔgtfB, respectively), whereas in ΔgtfB biofilms an inverse relationship between lytS and lrgA and lrgB expression was detected. Therefore, the deletion of gtfB influences dynamics and magnitude of expression of genes associated with matrix main components. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix.

    Science.gov (United States)

    Jennings, Laura K; Storek, Kelly M; Ledvina, Hannah E; Coulon, Charlène; Marmont, Lindsey S; Sadovskaya, Irina; Secor, Patrick R; Tseng, Boo Shan; Scian, Michele; Filloux, Alain; Wozniak, Daniel J; Howell, P Lynne; Parsek, Matthew R

    2015-09-08

    Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel's chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1→4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine. Guided by the knowledge of Pel's sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components.

  14. DNA builds and strengthens the extracellular matrix in Myxococcus xanthus biofilms by interacting with exopolysaccharides.

    Directory of Open Access Journals (Sweden)

    Wei Hu

    Full Text Available One intriguing discovery in modern microbiology is the extensive presence of extracellular DNA (eDNA within biofilms of various bacterial species. Although several biological functions have been suggested for eDNA, including involvement in biofilm formation, the detailed mechanism of eDNA integration into biofilm architecture is still poorly understood. In the biofilms formed by Myxococcus xanthus, a Gram-negative soil bacterium with complex morphogenesis and social behaviors, DNA was found within both extracted and native extracellular matrices (ECM. Further examination revealed that these eDNA molecules formed well organized structures that were similar in appearance to the organization of exopolysaccharides (EPS in ECM. Biochemical and image analyses confirmed that eDNA bound to and colocalized with EPS within the ECM of starvation biofilms and fruiting bodies. In addition, ECM containing eDNA exhibited greater physical strength and biological stress resistance compared to DNase I treated ECM. Taken together, these findings demonstrate that DNA interacts with EPS and strengthens biofilm structures in M. xanthus.

  15. Quantitative proteomic analysis of extracellular matrix extracted from mono- and dual-species biofilms of Fusobacterium nucleatum and Porphyromonas gingivalis.

    Science.gov (United States)

    Mohammed, Marwan Mansoor Ali; Pettersen, Veronika Kuchařová; Nerland, Audun H; Wiker, Harald G; Bakken, Vidar

    2017-04-01

    The Gram-negative bacteria Fusobacterium nucleatum and Porphyromonas gingivalis are members of a complex dental biofilm associated with periodontal disease. In this study, we cultured F. nucleatum and P. gingivalis as mono- and dual-species biofilms, and analyzed the protein composition of the biofilms extracellular polymeric matrix (EPM) by high-resolution liquid chromatography-tandem mass spectrometry. Label-free quantitative proteomic analysis was used for identification of proteins and sequence-based functional characterization for their classification and prediction of possible roles in EPM. We identified 542, 93 and 280 proteins in the matrix of F. nucleatum, P. gingivalis, and the dual-species biofilm, respectively. Nearly 70% of all EPM proteins in the dual-species biofilm originated from F. nucleatum, and a majority of these were cytoplasmic proteins, suggesting an enhanced lysis of F. nucleatum cells. The proteomic analysis also indicated an interaction between the two species: 22 F. nucleatum proteins showed differential levels between the mono and dual-species EPMs, and 11 proteins (8 and 3 from F. nucleatum and P. gingivalis, respectively) were exclusively detected in the dual-species EPM. Oxidoreductases and chaperones were among the most abundant proteins identified in all three EPMs. The biofilm matrices in addition contained several known and hypothetical virulence proteins, which can mediate adhesion to the host cells and disintegration of the periodontal tissues. This study demonstrated that the biofilm matrix of two important periodontal pathogens consists of a multitude of proteins whose amounts and functionalities vary largely. Relatively high levels of several of the detected proteins might facilitate their potential use as targets for the inhibition of biofilm development. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Effect of secondary metabolite of Actinidia deliciosa on the biofilm and extra-cellular matrix components of Acinetobacter baumannii.

    Science.gov (United States)

    Tiwari, Vishvanath; Tiwari, Deepika; Patel, Varsha; Tiwari, Monalisa

    2017-09-01

    Acinetobacter baumannii, opportunistic nosocomial pathogen, increases gradually in the clinical setup. The high level of resistance mechanisms acquired by these bacteria makes their eradication difficult and biofilm formation is one of them. Biofilm comprises of closely packed bacterial population crowded together by extra-cellular matrix (ECM). ECM contains bacterial secreted polymers such as exopolysaccharides (EPS), proteins and extracellular-DNA (e-DNA) and rarely amyloidogenic proteins. Biofilm offers protection of underlying bacterial population against chemotherapeutic agents and host immune system. Therefore, present efforts are focused to find a novel therapeutic that targets biofilm-associated infections. Plants are used as a natural therapeutic for numerous ailments. In order to find an alternative of the available antibacterial drugs, we have focused on the natural herbal active compounds. In this study, we have extracted active compounds from various medicinal plants and screened its anti-biofilm activity against carbapenem resistant strain of A. baumannii. Results showed that polar extract of kiwi (Actinidia deliciosa) and clove (Syzygium aromaticum) exhibit effective anti-biofilm activity. These two plants were also used for their phytochemical screening and TLC profiling to find out the constituting secondary metabolites. Actinidia deliciosa extract contains an alkaloid (sanquinarine) as well as a flavonoid (hydroxyflavone). Anti-biofilm effect of this extract on the ECM of A. baumannii showed that it reduces EPS, protein and eDNA contents in the ECM. Proteins of ECM have also shown to form amyloid like structure, which was evident from its interaction with the Congo Red. CFU counting after Actinidia deliciosa extract treatment also supported the results. Therefore, it can be concluded that polar extract of A. deliciosa can be used to find suitable alternative therapeutic to control biofilm formation by carbapenem resistant strain of

  17. KinD is a checkpoint protein linking spore formation to extracellular-matrix production in Bacillus subtilis biofilms.

    Science.gov (United States)

    Aguilar, Claudio; Vlamakis, Hera; Guzman, Alejandra; Losick, Richard; Kolter, Roberto

    2010-05-18

    Bacillus subtilis cells form multicellular biofilm communities in which spatiotemporal regulation of gene expression occurs, leading to differentiation of multiple coexisting cell types. These cell types include matrix-producing and sporulating cells. Extracellular matrix production and sporulation are linked in that a mutant unable to produce matrix is delayed for sporulation. Here, we show that the delay in sporulation is not due to a growth advantage of the matrix-deficient mutant under these conditions. Instead, we show that the link between matrix production and sporulation is through the Spo0A signaling pathway. Both processes are regulated by the phosphorylated form of the master transcriptional regulator Spo0A. When cells have low levels of phosphorylated Spo0A (Spo0A~P), matrix genes are expressed; however, at higher levels of Spo0A~P, sporulation commences. We have found that Spo0A~P levels are maintained at low levels in the matrix-deficient mutant, thereby delaying expression of sporulation-specific genes. This is due to the activity of one of the components of the Spo0A phosphotransfer network, KinD. A deletion of kinD suppresses the sporulation defect of matrix mutants, while its overproduction delays sporulation. Our data indicate that KinD displays a dual role as a phosphatase or a kinase and that its activity is linked to the presence of extracellular matrix in the biofilms. We propose a novel role for KinD in biofilms as a checkpoint protein that regulates the onset of sporulation by inhibiting the activity of Spo0A until matrix, or a component therein, is sensed.

  18. Confocal microscopy imaging of the biofilm matrix

    DEFF Research Database (Denmark)

    Schlafer, Sebastian; Meyer, Rikke L

    2017-01-01

    The extracellular matrix is an integral part of microbial biofilms and an important field of research. Confocal laser scanning microscopy is a valuable tool for the study of biofilms, and in particular of the biofilm matrix, as it allows real-time visualization of fully hydrated, living specimens...... the concentration of solutes and the diffusive properties of the biofilm matrix....

  19. In situ analysis of Bacillus licheniformis biofilms: amyloid-like polymers and eDNA are involved in the adherence and aggregation of the extracellular matrix.

    Science.gov (United States)

    Randrianjatovo-Gbalou, I; Rouquette, P; Lefebvre, D; Girbal-Neuhauser, E; Marcato-Romain, C-E

    2017-05-01

    This study attempts to determine which of the exopolymeric substances are involved in the adherence and aggregation of a Bacillus licheniformis biofilm. The involvement of extracellular proteins and eDNA were particularly investigated using DNase and proteinase K treatment. The permeability of the biofilms increased fivefold after DNase I treatment. The quantification of the matrix components showed that, irrespective to the enzyme tested, eDNA and amyloid-like polymers were removed simultaneously. Size-exclusion chromatography analyses supported these observations and revealed the presence of associated nucleic acid and protein complexes in the biofilm lysates. These data suggest that some extracellular DNA and amyloid-like proteins were closely interlaced within the matrix. Finally, confocal laser scanning microscopy imaging gave supplementary clues about the 3D organization of the biofilms, confirming that eDNA and exoproteins were essentially layered under and around the bacterial cells, whereas the amyloid-like fractions were homogeneously distributed within the matrix. These results confirm that some DNA-amyloid complexes play a key role in the modulation of the mechanical resistance of B. licheniformis biofilms. The study highlights the need to consider the whole structure of biofilms and to target the interactions between matrix components. A better understanding of B. licheniformis biofilm physiology and the structural organization of the matrix will strengthen strategies of biofilm control. © 2017 The Society for Applied Microbiology.

  20. Flo11p, drug efflux pumps, and the extracellular matrix cooperate to form biofilm yeast colonies

    Czech Academy of Sciences Publication Activity Database

    Váchová, Libuše; Šťovíček, V.; Hlaváček, Otakar; Chernyavskiy, Oleksandr; Štěpánek, L.; Kubínová, Lucie; Palková, Z.

    2011-01-01

    Roč. 194, č. 5 (2011), s. 679-687 ISSN 0021-9525 R&D Projects: GA ČR GA204/08/0718; GA MŠk(CZ) LC531; GA MŠk(CZ) LC06063 Institutional research plan: CEZ:AV0Z50200510; CEZ:AV0Z50110509 Keywords : CANDIDA-ALBICANS BIOFILMS * SACCHAROMYCES-CEREVISIAE * ABC TRANSPORTERS Subject RIV: EE - Microbiology, Virology Impact factor: 10.264, year: 2011

  1. A novel technique using potassium permanganate and reflectance confocal microscopy to image biofilm extracellular polymeric matrix reveals non-eDNA networks in Pseudomonas aeruginosa biofilms.

    Science.gov (United States)

    Swearingen, Matthew C; Mehta, Ajeet; Mehta, Amar; Nistico, Laura; Hill, Preston J; Falzarano, Anthony R; Wozniak, Daniel J; Hall-Stoodley, Luanne; Stoodley, Paul

    2016-02-01

    Biofilms are etiologically important in the development of chronic medical and dental infections. The biofilm extracellular polymeric substance (EPS) determines biofilm structure and allows bacteria in biofilms to adapt to changes in mechanical loads such as fluid shear. However, EPS components are difficult to visualize microscopically because of their low density and molecular complexity. Here, we tested potassium permanganate, KMnO4, for use as a non-specific EPS contrast-enhancing stain using confocal laser scanning microscopy in reflectance mode. We demonstrate that KMnO4 reacted with EPS components of various strains of Pseudomonas, Staphylococcus and Streptococcus, yielding brown MnO2 precipitate deposition on the EPS, which was quantifiable using data from the laser reflection detector. Furthermore, the MnO2 signal could be quantified in combination with fluorescent nucleic acid staining. COMSTAT image analysis indicated that KMnO4 staining increased the estimated biovolume over that determined by nucleic acid staining alone for all strains tested, and revealed non-eDNA EPS networks in Pseudomonas aeruginosa biofilm. In vitro and in vivo testing indicated that KMnO4 reacted with poly-N-acetylglucosamine and Pseudomonas Pel polysaccharide, but did not react strongly with DNA or alginate. KMnO4 staining may have application as a research tool and for diagnostic potential for biofilms in clinical samples. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Pseudomonas biofilm matrix composition and niche biology

    Science.gov (United States)

    Mann, Ethan E.; Wozniak, Daniel J.

    2014-01-01

    Biofilms are a predominant form of growth for bacteria in the environment and in the clinic. Critical for biofilm development are adherence, proliferation, and dispersion phases. Each of these stages includes reinforcement by, or modulation of, the extracellular matrix. Pseudomonas aeruginosa has been a model organism for the study of biofilm formation. Additionally, other Pseudomonas species utilize biofilm formation during plant colonization and environmental persistence. Pseudomonads produce several biofilm matrix molecules, including polysaccharides, nucleic acids, and proteins. Accessory matrix components shown to aid biofilm formation and adaptability under varying conditions are also produced by pseudomonads. Adaptation facilitated by biofilm formation allows for selection of genetic variants with unique and distinguishable colony morphology. Examples include rugose small-colony variants and wrinkly spreaders (WS), which over produce Psl/Pel or cellulose, respectively, and mucoid bacteria that over produce alginate. The well-documented emergence of these variants suggests that pseudomonads take advantage of matrix-building subpopulations conferring specific benefits for the entire population. This review will focus on various polysaccharides as well as additional Pseudomonas biofilm matrix components. Discussions will center on structure–function relationships, regulation, and the role of individual matrix molecules in niche biology. PMID:22212072

  3. Microscopic monitoring of extracellular pH in dental biofilms

    DEFF Research Database (Denmark)

    Schlafer, Sebastian; Garcia, Javier; Greve, Matilde

    pH in dental biofilm is a key virulence factor for the development of caries lesions. The complex three-dimensional architecture of dental biofilms leads to steep gradients of nutrients and metabolites, including organic acids, across the biofilm. For decades, measuring pH in dental biofilm has...... been limited to monitoring bulk pH with electrodes. Although pH microelectrodes with a better spatial resolution have been developed, they do not permit to monitor horizontal pH gradients in real-time. Quantitative fluorescent microscopic techniques, such as fluorescence lifetime imaging or pH...... ratiometry, can be employed to map the pH landscape in dental biofilm with more detail. However, when pH sensitive fluorescent probes are used to visualize pH in biofilms, it is crucial to differentiate between extracellular and intracellular pH. Intracellular microbial pH and pH in the extracellular matrix...

  4. Extracellular DNA Contributes to Dental Biofilm Stability

    DEFF Research Database (Denmark)

    Schlafer, Sebastian; Meyer, Rikke Louise; Dige, Irene

    2017-01-01

    dental biofilms. This study aimed to determine whether eDNA was part of the matrix in biofilms grown in situ in the absence of sucrose and whether treatment with DNase dispersed biofilms grown for 2.5, 5, 7.5, 16.5, or 24 h. Three hundred biofilms from 10 study participants were collected and treated...... the amount of biofilm in very early stages of growth (up to 7.5 h), but the treatment effect decreased with increasing biofilm age. This study proves the involvement of eDNA in dental biofilm formation and its importance for biofilm stability in the earliest stages. Further research is required to uncover...

  5. Extracellular DNA Shields against Aminoglycosides in Pseudomonas aeruginosa Biofilms

    DEFF Research Database (Denmark)

    Chiang, Wen-Chi; Nilsson, Martin; Jensen, Peter Østrup

    2013-01-01

    Within recent years, it has been established that extracellular DNA is a key constituent of the matrix of microbial biofilms. In addition, it has recently been demonstrated that DNA binds positively charged antimicrobials such as aminoglycosides and antimicrobial peptides. In the present study, we...... provide evidence that extracellular DNA shields against aminoglycosides in Pseudomonas aeruginosa biofilms. We show that exogenously supplemented DNA integrates into P. aeruginosa biofilms and increases their tolerance toward aminoglycosides. We provide evidence that biofilms formed by a DNA release......-deficient P. aeruginosa quorum-sensing mutant are more susceptible to aminoglycoside treatment than wild-type biofilms but become rescued from the detrimental action of aminoglycosides upon supplementation with exogenous DNA. Furthermore, we demonstrate that exposure to lysed polymorphonuclear leukocytes...

  6. Bacterial Extracellular Polysaccharides Involved in Biofilm Formation

    Directory of Open Access Journals (Sweden)

    Elena P. Ivanova

    2009-07-01

    Full Text Available Extracellular polymeric substances (EPS produced by microorganisms are a complex mixture of biopolymers primarily consisting of polysaccharides, as well as proteins, nucleic acids, lipids and humic substances. EPS make up the intercellular space of microbial aggregates and form the structure and architecture of the biofilm matrix. The key functions of EPS comprise the mediation of the initial attachment of cells to different substrata and protection against environmental stress and dehydration. The aim of this review is to present a summary of the current status of the research into the role of EPS in bacterial attachment followed by biofilm formation. The latter has a profound impact on an array of biomedical, biotechnology and industrial fields including pharmaceutical and surgical applications, food engineering, bioremediation and biohydrometallurgy. The diverse structural variations of EPS produced by bacteria of different taxonomic lineages, together with examples of biotechnological applications, are discussed. Finally, a range of novel techniques that can be used in studies involving biofilm-specific polysaccharides is discussed.

  7. Extracellular matrix structure.

    Science.gov (United States)

    Theocharis, Achilleas D; Skandalis, Spyros S; Gialeli, Chrysostomi; Karamanos, Nikos K

    2016-02-01

    Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network composed of collagens, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminins, and several other glycoproteins. Matrix components bind each other as well as cell adhesion receptors forming a complex network into which cells reside in all tissues and organs. Cell surface receptors transduce signals into cells from ECM, which regulate diverse cellular functions, such as survival, growth, migration, and differentiation, and are vital for maintaining normal homeostasis. ECM is a highly dynamic structural network that continuously undergoes remodeling mediated by several matrix-degrading enzymes during normal and pathological conditions. Deregulation of ECM composition and structure is associated with the development and progression of several pathologic conditions. This article emphasizes in the complex ECM structure as to provide a better understanding of its dynamic structural and functional multipotency. Where relevant, the implication of the various families of ECM macromolecules in health and disease is also presented. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Role of Extracellular DNA during Biofilm Formation by Listeria monocytogenes

    DEFF Research Database (Denmark)

    Harmsen, Morten; Lappann, Martin; Knøchel, S

    2010-01-01

    (eDNA) may be the only central component of the biofilm matrix and that it is necessary for both initial attachment and early biofilm formation for 41 L. monocytogenes strains that were tested. DNase I treatment resulted in dispersal of biofilms, not only in microtiter tray assays but also in flow......Listeria monocytogenes is a food-borne pathogen that is capable of living in harsh environments. It is believed to do this by forming biofilms, which are surface-associated multicellular structures encased in a self-produced matrix. In this paper we show that in L. monocytogenes extracellular DNA...... cell biofilm assays. However, it was also demonstrated that in a culture without eDNA, neither Listeria genomic DNA nor salmon sperm DNA by itself could restore the capacity to adhere. A search for additional necessary components revealed that peptidoglycan (PG), specifically N-acetylglucosamine (NAG...

  9. The Exopolysaccharide Matrix: A Virulence Determinant of Cariogenic Biofilm

    OpenAIRE

    Koo, H.; Falsetta, M.L.; Klein, M.I.

    2013-01-01

    Many infectious diseases in humans are caused or exacerbated by biofilms. Dental caries is a prime example of a biofilm-dependent disease, resulting from interactions of microorganisms, host factors, and diet (sugars), which modulate the dynamic formation of biofilms on tooth surfaces. All biofilms have a microbial-derived extracellular matrix as an essential constituent. The exopolysaccharides formed through interactions between sucrose- (and starch-) and Streptococcus mutans-derived exoenzy...

  10. Staphylococcus aureus biofilm removal by targeting biofilm-associated extracellular proteins

    Directory of Open Access Journals (Sweden)

    Sudhir K Shukla

    2017-01-01

    Methods: Biofilm assay was done in 96-well microtitre plate to evaluate the effect of proteinase K on biofilms of bovine mastitis S. Aureus isolates. Extracellular polymeric substances were extracted and evaluated for their composition (protein, polysaccharides and extracellular DNA, before and after the proteinase K treatment. Results: Biofilm assay showed that 2 μg/ml proteinase K significantly inhibited biofilm development in bap-positive S. aureus V329 as well as other S. aureus isolates (SA7, SA10, SA33, SA352, but not in bap-mutant M556 and SA392 (a weak biofilm-producing strain. Proteinase K treatment on S. aureus planktonic cells showed that there was no inhibition of planktonic growth up to 32 μg/ml of proteinase K. Proteinase K treatment on 24 h old preformed biofilms showed an enhanced dispersion of bap-positive V329 and SA7, SA10, SA33 and SA352 biofilms; however, proteinase K did not affect the bap-mutant S. aureus M556 and SA392 biofilms. Biofilm compositions study before and after proteinase K treatment indicated that Bap might also be involved in eDNA retention in the biofilm matrix that aids in biofilm stability. When proteinase K was used in combination with antibiotics, a synergistic effect in antibiotic efficacy was observed against all biofilm-forming S. aureus isolates. Interpretation & conclusions: Proteinase K inhibited biofilms growth in S. aureus bovine mastitis isolates but did not affect their planktonic growth. An enhanced dispersion of preformed S. aureus biofilms was observed on proteinase K treatment. Proteinase K treatment with antibiotics showed a synergistic effect against S. aureus biofilms. The study suggests that dispersing S. aureus by protease can be of use while devising strategies againstS. aureus biofilms.

  11. Assembly and development of the Pseudomonas aeruginosa biofilm matrix.

    Directory of Open Access Journals (Sweden)

    Luyan Ma

    2009-03-01

    Full Text Available Virtually all cells living in multicellular structures such as tissues and organs are encased in an extracellular matrix. One of the most important features of a biofilm is the extracellular polymeric substance that functions as a matrix, holding bacterial cells together. Yet very little is known about how the matrix forms or how matrix components encase bacteria during biofilm development. Pseudomonas aeruginosa forms environmentally and clinically relevant biofilms and is a paradigm organism for the study of biofilms. The extracellular polymeric substance of P. aeruginosa biofilms is an ill-defined mix of polysaccharides, nucleic acids, and proteins. Here, we directly visualize the product of the polysaccharide synthesis locus (Psl exopolysaccharide at different stages of biofilm development. During attachment, Psl is anchored on the cell surface in a helical pattern. This promotes cell-cell interactions and assembly of a matrix, which holds bacteria in the biofilm and on the surface. Chemical dissociation of Psl from the bacterial surface disrupted the Psl matrix as well as the biofilm structure. During biofilm maturation, Psl accumulates on the periphery of 3-D-structured microcolonies, resulting in a Psl matrix-free cavity in the microcolony center. At the dispersion stage, swimming cells appear in this matrix cavity. Dead cells and extracellular DNA (eDNA are also concentrated in the Psl matrix-free area. Deletion of genes that control cell death and autolysis affects the formation of the matrix cavity and microcolony dispersion. These data provide a mechanism for how P. aeruginosa builds a matrix and subsequently a cavity to free a portion of cells for seeding dispersal. Direct visualization reveals that Psl is a key scaffolding matrix component and opens up avenues for therapeutics of biofilm-related complications.

  12. Activation of phagocytic cells by Staphylococcus epidermidis biofilms: effects of extracellular matrix proteins and the bacterial stress protein GroEL on netosis and MRP-14 release.

    Science.gov (United States)

    Dapunt, Ulrike; Gaida, Matthias M; Meyle, Eva; Prior, Birgit; Hänsch, Gertrud M

    2016-07-01

    The recognition and phagocytosis of free-swimming (planktonic) bacteria by polymorphonuclear neutrophils have been investigated in depth. However, less is known about the neutrophil response towards bacterial biofilms. Our previous work demonstrated that neutrophils recognize activating entities within the extracellular polymeric substance (EPS) of biofilms (the bacterial heat shock protein GroEL) and that this process does not require opsonization. Aim of this study was to evaluate the release of DNA by neutrophils in response to biofilms, as well as the release of the inflammatory cytokine MRP-14. Neutrophils were stimulated with Staphylococcus epidermidis biofilms, planktonic bacteria, extracted EPS and GroEL. Release of DNA and of MRP-14 was evaluated. Furthermore, tissue samples from patients suffering from biofilm infections were collected and evaluated by histology. MRP-14 concentration in blood samples was measured. We were able to show that biofilms, the EPS and GroEL induce DNA release. MRP-14 was only released after stimulation with EPS, not GroEL. Histology of tissue samples revealed MRP-14 positive cells in association with neutrophil infiltration and MRP-14 concentration was elevated in blood samples of patients suffering from biofilm infections. Our data demonstrate that neutrophil-activating entities are present in the EPS and that GroEL induces DNA release by neutrophils. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Biofilms.

    Science.gov (United States)

    López, Daniel; Vlamakis, Hera; Kolter, Roberto

    2010-07-01

    The ability to form biofilms is a universal attribute of bacteria. Biofilms are multicellular communities held together by a self-produced extracellular matrix. The mechanisms that different bacteria employ to form biofilms vary, frequently depending on environmental conditions and specific strain attributes. In this review, we emphasize four well-studied model systems to give an overview of how several organisms form biofilms: Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. Using these bacteria as examples, we discuss the key features of biofilms as well as mechanisms by which extracellular signals trigger biofilm formation.

  14. Artificial biofilms establish the role of matrix interactions in staphylococcal biofilm assembly and disassembly

    Science.gov (United States)

    Stewart, Elizabeth J.; Ganesan, Mahesh; Younger, John G.; Solomon, Michael J.

    2015-01-01

    We demonstrate that the microstructural and mechanical properties of bacterial biofilms can be created through colloidal self-assembly of cells and polymers, and thereby link the complex material properties of biofilms to well understood colloidal and polymeric behaviors. This finding is applied to soften and disassemble staphylococcal biofilms through pH changes. Bacterial biofilms are viscoelastic, structured communities of cells encapsulated in an extracellular polymeric substance (EPS) comprised of polysaccharides, proteins, and DNA. Although the identity and abundance of EPS macromolecules are known, how these matrix materials interact with themselves and bacterial cells to generate biofilm morphology and mechanics is not understood. Here, we find that the colloidal self-assembly of Staphylococcus epidermidis RP62A cells and polysaccharides into viscoelastic biofilms is driven by thermodynamic phase instability of EPS. pH conditions that induce phase instability of chitosan produce artificial S. epidermidis biofilms whose mechanics match natural S. epidermidis biofilms. Furthermore, pH-induced solubilization of the matrix triggers disassembly in both artificial and natural S. epidermidis biofilms. This pH-induced disassembly occurs in biofilms formed by five additional staphylococcal strains, including three clinical isolates. Our findings suggest that colloidal self-assembly of cells and matrix polymers produces biofilm viscoelasticity and that biofilm control strategies can exploit this mechanism. PMID:26272750

  15. Biofilms

    OpenAIRE

    López, Daniel; Vlamakis, Hera; Kolter, Roberto

    2010-01-01

    The ability to form biofilms is a universal attribute of bacteria. Biofilms are multicellular communities held together by a self-produced extracellular matrix. The mechanisms that different bacteria employ to form biofilms vary, frequently depending on environmental conditions and specific strain attributes. In this review, we emphasize four well-studied model systems to give an overview of how several organisms form biofilms: Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and ...

  16. Extracellular DNA facilitates the formation of functional amyloids in Staphylococcus aureus biofilms.

    Science.gov (United States)

    Schwartz, Kelly; Ganesan, Mahesh; Payne, David E; Solomon, Michael J; Boles, Blaise R

    2016-01-01

    Persistent staphylococcal infections often involve surface-associated communities called biofilms. Staphylococcus aureus biofilm development is mediated by the co-ordinated production of the biofilm matrix, which can be composed of polysaccharides, extracellular DNA (eDNA) and proteins including amyloid fibers. The nature of the interactions between matrix components, and how these interactions contribute to the formation of matrix, remain unclear. Here we show that the presence of eDNA in S. aureus biofilms promotes the formation of amyloid fibers. Conditions or mutants that do not generate eDNA result in lack of amyloids during biofilm growth despite the amyloidogeneic subunits, phenol soluble modulin peptides, being produced. In vitro studies revealed that the presence of DNA promotes amyloid formation by PSM peptides. Thus, this work exposes a previously unacknowledged interaction between biofilm matrix components that furthers our understanding of functional amyloid formation and S. aureus biofilm biology. © 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  17. Neutrophil extracellular trap formation in supragingival biofilms.

    Science.gov (United States)

    Hirschfeld, Josefine; Dommisch, Henrik; Skora, Philipp; Horvath, Gabor; Latz, Eicke; Hoerauf, Achim; Waller, Tobias; Kawai, Toshihisa; Jepsen, Søren; Deschner, James; Bekeredjian-Ding, Isabelle

    2015-01-01

    Oral biofilms are the causative agents of the highly prevalent oral diseases periodontitis and caries. Additionally, the host immune response is thought to play a critical role in disease onset. Neutrophils are known to be a key host response factor to bacterial challenge on host surfaces. Release of neutrophil extracellular traps (NETs) as a novel antimicrobial defense strategy has gained increasing attention in the past years. Here, we investigated the influx of neutrophils into the dental plaque and the ability of oral bacteria to trigger intra-biofilm release of NETs and intracellular proteins. Supragingival biofilms and whole saliva were sampled from systemically healthy subjects participating in an experimental gingivitis study. Biofilms were analysed by immunofluorescence followed by confocal and fluorescence microscopy. Moreover, concentrations of cytokines and immune-associated proteins in biofilm suspensions and saliva were assessed by ELISA. Neutrophils obtained from blood were stimulated with twelve bacterial species isolated from cultured biofilms or with lipopolysaccharide to monitor NET formation. Neutrophils, NETs, neutrophil-associated proteins (myeloperoxidase, elastase-2, cathepsin G, cathelicidin LL-37), interleukin-8, interleukin-1β and tumor necrosis factor were detected within plaque samples and saliva. All tested bacterial species as well as the polymicrobial samples isolated from the plaque of each donor induced release of NETs and interleukin-8. The degree of NET formation varied among different subjects and did not correlate with plaque scores or clinical signs of local inflammation. Our findings indicate that neutrophils are attracted towards dental biofilms, in which they become incorporated and where they are stimulated by microbes to release NETs and immunostimulatory proteins. Thus, neutrophils and NETs may be involved in host biofilm control, although their specific role needs to be further elucidated. Moreover, inter

  18. Streptococcus suis Serotype 2 Biofilms Inhibit the Formation of Neutrophil Extracellular Traps.

    Science.gov (United States)

    Ma, Fang; Yi, Li; Yu, Ningwei; Wang, Guangyu; Ma, Zhe; Lin, Huixing; Fan, Hongjie

    2017-01-01

    Invasive infections caused by Streptococcus suis serotype 2 (SS2) has emerged as a clinical problem in recent years. Neutrophil extracellular traps (NETs) are an important mechanism for the trapping and killing of pathogens that are resistant to phagocytosis. Biofilm formation can protect bacteria from being killed by phagocytes. Until now, there have only been a few studies that focused on the interactions between bacterial biofilms and NETs. SS2 in both a biofilm state and a planktonic cell state were incubated with phagocytes and NETs, and bacterial survival was assessed. DNase I and cytochalasin B were used to degrade NET DNA or suppress phagocytosis, respectively. Extracellular DNA was stained with impermeable fluorescent dye to quantify NET formation. Biofilm formation increased up to 6-fold in the presence of neutrophils, and biofilms were identified in murine tissue. Both planktonic and biofilm cells induced neutrophils chemotaxis to the infection site, with neutrophils increasing by 85.1 and 73.8%, respectively. The bacteria in biofilms were not phagocytized. The bactericidal efficacy of NETs on the biofilms and planktonic cells were equal; however, the biofilm extracellular matrix can inhibit NET release. Although biofilms inhibit NETs release, NETs appear to be an important mechanism to eliminate SS2 biofilms. This knowledge advances the understanding of biofilms and may aid in the development of treatments for persistent infections with a biofilm component.

  19. Extracellular DNA contributes to dental biofilm formation: An ex vivo study

    DEFF Research Database (Denmark)

    Schlafer, Sebastian; Meyer, Rikke Louise; Dige, Irene

    2016-01-01

    The extracellular matrix of dental biofilms plays an important role during caries development. It increases the mechanical stability of the biofilm, it prevents desiccation, it serves as a reservoir for nutrients and it contributes to the long-term preservation of acidic microenvironments. Research...

  20. Extracellular DNases of Ralstonia solanacearum modulate biofilms and facilitate bacterial wilt virulence.

    Science.gov (United States)

    Minh Tran, Tuan; MacIntyre, April; Khokhani, Devanshi; Hawes, Martha; Allen, Caitilyn

    2016-11-01

    Ralstonia solanacearum is a soil-borne vascular pathogen that colonizes plant xylem vessels, a flowing, low-nutrient habitat where biofilms could be adaptive. Ralstonia solanacearum forms biofilm in vitro, but it was not known if the pathogen benefits from biofilms during infection. Scanning electron microscopy revealed that during tomato infection, R. solanacearum forms biofilm-like masses in xylem vessels. These aggregates contain bacteria embedded in a matrix including chromatin-like fibres commonly observed in other bacterial biofilms. Chemical and enzymatic assays demonstrated that the bacterium releases extracellular DNA in culture and that DNA is an integral component of the biofilm matrix. An R. solanacearum mutant lacking the pathogen's two extracellular nucleases (exDNases) formed non-spreading colonies and abnormally thick biofilms in vitro. The biofilms formed by the exDNase mutant in planta contained more and thicker fibres. This mutant was also reduced in virulence on tomato plants and did not spread in tomato stems as well as the wild-type strain, suggesting that these exDNases facilitate biofilm maturation and bacterial dispersal. To our knowledge, this is the first demonstration that R. solanacearum forms biofilms in plant xylem vessels, and the first documentation that plant pathogens use DNases to modulate their biofilm structure for systemic spread and virulence. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  1. The exopolysaccharide matrix: a virulence determinant of cariogenic biofilm.

    Science.gov (United States)

    Koo, H; Falsetta, M L; Klein, M I

    2013-12-01

    Many infectious diseases in humans are caused or exacerbated by biofilms. Dental caries is a prime example of a biofilm-dependent disease, resulting from interactions of microorganisms, host factors, and diet (sugars), which modulate the dynamic formation of biofilms on tooth surfaces. All biofilms have a microbial-derived extracellular matrix as an essential constituent. The exopolysaccharides formed through interactions between sucrose- (and starch-) and Streptococcus mutans-derived exoenzymes present in the pellicle and on microbial surfaces (including non-mutans) provide binding sites for cariogenic and other organisms. The polymers formed in situ enmesh the microorganisms while forming a matrix facilitating the assembly of three-dimensional (3D) multicellular structures that encompass a series of microenvironments and are firmly attached to teeth. The metabolic activity of microbes embedded in this exopolysaccharide-rich and diffusion-limiting matrix leads to acidification of the milieu and, eventually, acid-dissolution of enamel. Here, we discuss recent advances concerning spatio-temporal development of the exopolysaccharide matrix and its essential role in the pathogenesis of dental caries. We focus on how the matrix serves as a 3D scaffold for biofilm assembly while creating spatial heterogeneities and low-pH microenvironments/niches. Further understanding on how the matrix modulates microbial activity and virulence expression could lead to new approaches to control cariogenic biofilms.

  2. Extracellular DNA is essential for maintaining Bordetella biofilm integrity on abiotic surfaces and in the upper respiratory tract of mice.

    Directory of Open Access Journals (Sweden)

    Matt S Conover

    2011-02-01

    Full Text Available Bacteria form complex and highly elaborate surface adherent communities known as biofilms which are held together by a self-produced extracellular matrix. We have previously shown that by adopting a biofilm mode of existence in vivo, the gram negative bacterial pathogens Bordetella bronchiseptica and Bordetella pertussis are able to efficiently colonize and persist in the mammalian respiratory tract. In general, the bacterial biofilm matrix includes polysaccharides, proteins and extracellular DNA (eDNA. In this report, we investigated the function of DNA in Bordetella biofilm development. We show that DNA is a significant component of Bordetella biofilm matrix. Addition of DNase I at the initiation of biofilm growth inhibited biofilm formation. Treatment of pre-established mature biofilms formed under both static and flow conditions with DNase I led to a disruption of the biofilm biomass. We next investigated whether eDNA played a role in biofilms formed in the mouse respiratory tract. DNase I treatment of nasal biofilms caused considerable dissolution of the biofilm biomass. In conclusion, these results suggest that eDNA is a crucial structural matrix component of both in vitro and in vivo formed Bordetella biofilms. This is the first evidence for the ability of DNase I to disrupt bacterial biofilms formed on host organs.

  3. A Candida biofilm-induced pathway for matrix glucan delivery: implications for drug resistance.

    Directory of Open Access Journals (Sweden)

    Heather T Taff

    Full Text Available Extracellular polysaccharides are key constituents of the biofilm matrix of many microorganisms. One critical carbohydrate component of Candida albicans biofilms, β-1,3 glucan, has been linked to biofilm protection from antifungal agents. In this study, we identify three glucan modification enzymes that function to deliver glucan from the cell to the extracellular matrix. These enzymes include two predicted glucan transferases and an exo-glucanase, encoded by BGL2, PHR1, and XOG1, respectively. We show that the enzymes are crucial for both delivery of β-1,3 glucan to the biofilm matrix and for accumulation of mature matrix biomass. The enzymes do not appear to impact cell wall glucan content of biofilm cells, nor are they necessary for filamentation or biofilm formation. We demonstrate that mutants lacking these genes exhibit enhanced susceptibility to the commonly used antifungal, fluconazole, during biofilm growth only. Transcriptional analysis and biofilm phenotypes of strains with multiple mutations suggest that these enzymes act in a complementary fashion to distribute matrix downstream of the primary β-1,3 glucan synthase encoded by FKS1. Furthermore, our observations suggest that this matrix delivery pathway works independently from the C. albicans ZAP1 matrix formation regulatory pathway. These glucan modification enzymes appear to play a biofilm-specific role in mediating the delivery and organization of mature biofilm matrix. We propose that the discovery of inhibitors for these enzymes would provide promising anti-biofilm therapeutics.

  4. Mycobacterium avium Possesses Extracellular DNA that Contributes to Biofilm Formation, Structural Integrity, and Tolerance to Antibiotics.

    Directory of Open Access Journals (Sweden)

    Sasha J Rose

    Full Text Available Mycobacterium avium subsp. hominissuis is an opportunistic pathogen that is associated with biofilm-related infections of the respiratory tract and is difficult to treat. In recent years, extracellular DNA (eDNA has been found to be a major component of bacterial biofilms, including many pathogens involved in biofilm-associated infections. To date, eDNA has not been described as a component of mycobacterial biofilms. In this study, we identified and characterized eDNA in a high biofilm-producing strain of Mycobacterium avium subsp. hominissuis (MAH. In addition, we surveyed for presence of eDNA in various MAH strains and other nontuberculous mycobacteria. Biofilms of MAH A5 (high biofilm-producing strain and MAH 104 (reference strain were established at 22°C and 37°C on abiotic surfaces. Acellular biofilm matrix and supernatant from MAH A5 7 day-old biofilms both possess abundant eDNA, however very little eDNA was found in MAH 104 biofilms. A survey of MAH clinical isolates and other clinically relevant nontuberculous mycobacterial species revealed many species and strains that also produce eDNA. RAPD analysis demonstrated that eDNA resembles genomic DNA. Treatment with DNase I reduced the biomass of MAH A5 biofilms when added upon biofilm formation or to an already established biofilm both on abiotic surfaces and on top of human pharyngeal epithelial cells. Furthermore, co-treatment of an established biofilm with DNase 1 and either moxifloxacin or clarithromycin significantly increased the susceptibility of the bacteria within the biofilm to these clinically used antimicrobials. Collectively, our results describe an additional matrix component of mycobacterial biofilms and a potential new target to help treat biofilm-associated nontuberculous mycobacterial infections.

  5. Nutrient depletion in Bacillus subtilis biofilms triggers matrix production

    International Nuclear Information System (INIS)

    Zhang, Wenbo; Seminara, Agnese; Suaris, Melanie; Angelini, Thomas E; Brenner, Michael P; Weitz, David A

    2014-01-01

    Many types of bacteria form colonies that grow into physically robust and strongly adhesive aggregates known as biofilms. A distinguishing characteristic of bacterial biofilms is an extracellular polymeric substance (EPS) matrix that encases the cells and provides physical integrity to the colony. The EPS matrix consists of a large amount of polysaccharide, as well as protein filaments, DNA and degraded cellular materials. The genetic pathways that control the transformation of a colony into a biofilm have been widely studied, and yield a spatiotemporal heterogeneity in EPS production. Spatial gradients in metabolites parallel this heterogeneity in EPS, but nutrient concentration as an underlying physiological initiator of EPS production has not been explored. Here, we study the role of nutrient depletion in EPS production in Bacillus subtilis biofilms. By monitoring simultaneously biofilm size and matrix production, we find that EPS production increases at a critical colony thickness that depends on the initial amount of carbon sources in the medium. Through studies of individual cells in liquid culture we find that EPS production can be triggered at the single-cell level by reducing nutrient concentration. To connect the single-cell assays with conditions in the biofilm, we calculate carbon concentration with a model for the reaction and diffusion of nutrients in the biofilm. This model predicts the relationship between the initial concentration of carbon and the thickness of the colony at the point of internal nutrient deprivation. (paper)

  6. A major protein component of the Bacillus subtilis biofilm matrix.

    Science.gov (United States)

    Branda, Steven S; Chu, Frances; Kearns, Daniel B; Losick, Richard; Kolter, Roberto

    2006-02-01

    Microbes construct structurally complex multicellular communities (biofilms) through production of an extracellular matrix. Here we present evidence from scanning electron microscopy showing that a wild strain of the Gram positive bacterium Bacillus subtilis builds such a matrix. Genetic, biochemical and cytological evidence indicates that the matrix is composed predominantly of a protein component, TasA, and an exopolysaccharide component. The absence of TasA or the exopolysaccharide resulted in a residual matrix, while the absence of both components led to complete failure to form complex multicellular communities. Extracellular complementation experiments revealed that a functional matrix can be assembled even when TasA and the exopolysaccharide are produced by different cells, reinforcing the view that the components contribute to matrix formation in an extracellular manner. Having defined the major components of the biofilm matrix and the control of their synthesis by the global regulator SinR, we present a working model for how B. subtilis switches between nomadic and sedentary lifestyles.

  7. Electrochemical roles of extracellular polymeric substances in biofilms

    DEFF Research Database (Denmark)

    Xiao, Yong; Zhao, Feng

    2017-01-01

    Most microbial cells in nature are surrounded by extracellular polymeric substances (EPS), which are fundamental components and determine the physiochemical properties of a biofilm. This review highlights the EPS properties of conductivity and redox ability from an electrochemical perspective, em...

  8. A Biofilm Matrix-Associated Protease Inhibitor Protects Pseudomonas aeruginosa from Proteolytic Attack.

    Science.gov (United States)

    Tseng, Boo Shan; Reichhardt, Courtney; Merrihew, Gennifer E; Araujo-Hernandez, Sophia A; Harrison, Joe J; MacCoss, Michael J; Parsek, Matthew R

    2018-04-10

    Pseudomonas aeruginosa produces an extracellular biofilm matrix that consists of nucleic acids, exopolysaccharides, lipid vesicles, and proteins. In general, the protein component of the biofilm matrix is poorly defined and understudied relative to the other major matrix constituents. While matrix proteins have been suggested to provide many functions to the biofilm, only proteins that play a structural role have been characterized thus far. Here we identify proteins enriched in the matrix of P. aeruginosa biofilms. We then focused on a candidate matrix protein, the serine protease inhibitor ecotin (PA2755). This protein is able to inhibit neutrophil elastase, a bactericidal enzyme produced by the host immune system during P. aeruginosa biofilm infections. We show that ecotin binds to the key biofilm matrix exopolysaccharide Psl and that it can inhibit neutrophil elastase when associated with Psl. Finally, we show that ecotin protects both planktonic and biofilm P. aeruginosa cells from neutrophil elastase-mediated killing. This may represent a novel mechanism of protection for biofilms to increase their tolerance against the innate immune response. IMPORTANCE Proteins associated with the extracellular matrix of bacterial aggregates called biofilms have long been suggested to provide many important functions to the community. To date, however, only proteins that provide structural roles have been described, and few matrix-associated proteins have been identified. We developed a method to identify matrix proteins and characterized one. We show that this protein, when associated with the biofilm matrix, can inhibit a bactericidal enzyme produced by the immune system during infection and protect biofilm cells from death induced by the enzyme. This may represent a novel mechanism of protection for biofilms, further increasing their tolerance against the immune response. Together, our results are the first to show a nonstructural function for a confirmed matrix

  9. Visualizing the dental biofilm matrix by means of fluorescence lectin-binding analysis

    DEFF Research Database (Denmark)

    Tawakoli, Pune Nina; Neu, Thomas R; Busck, Mette Marie

    2017-01-01

    lectins to visualize and quantify extracellular glycoconjugates in dental biofilms. Lectin binding was screened on pooled supragingival biofilm samples collected from 76 subjects using confocal microscopy. FLBA was then performed with 10 selected lectins on biofilms grown in situ for 48 h in the absence......The extracellular matrix is a poorly studied, yet important component of dental biofilms. Fluorescence lectin-binding analysis (FLBA) is a powerful tool to characterize glycoconjugates in the biofilm matrix. This study aimed to systematically investigate the ability of 75 fluorescently labeled......-biofilms: Aleuria aurantia (AAL), Calystega sepiem (Calsepa), Lycopersicon esculentum (LEA), Morniga-G (MNA-G) and Helix pomatia (HPA). No significant correlation between the binding of specific lectins and bacterial composition was found. Fluorescently labeled lectins enable the visualization of glycoconjugates...

  10. Integrins and extracellular matrix in mechanotransduction

    Directory of Open Access Journals (Sweden)

    Ramage L

    2011-12-01

    Full Text Available Lindsay RamageQueen’s Medical Research Institute, University of Edinburgh, Edinburgh, UKAbstract: Integrins are a family of cell surface receptors which mediate cell–matrix and cell–cell adhesions. Among other functions they provide an important mechanical link between the cells external and intracellular environments while the adhesions that they form also have critical roles in cellular signal-transduction. Cell–matrix contacts occur at zones in the cell surface where adhesion receptors cluster and when activated the receptors bind to ligands in the extracellular matrix. The extracellular matrix surrounds the cells of tissues and forms the structural support of tissue which is particularly important in connective tissues. Cells attach to the extracellular matrix through specific cell-surface receptors and molecules including integrins and transmembrane proteoglycans. Integrins work alongside other proteins such as cadherins, immunoglobulin superfamily cell adhesion molecules, selectins, and syndecans to mediate cell–cell and cell–matrix interactions and communication. Activation of adhesion receptors triggers the formation of matrix contacts in which bound matrix components, adhesion receptors, and associated intracellular cytoskeletal and signaling molecules form large functional, localized multiprotein complexes. Cell–matrix contacts are important in a variety of different cell and tissue properties including embryonic development, inflammatory responses, wound healing, and adult tissue homeostasis. This review summarizes the roles and functions of integrins and extracellular matrix proteins in mechanotransduction.Keywords: ligand binding, α subunit, ß subunit, focal adhesion, cell differentiation, mechanical loading, cell–matrix interaction

  11. sarA negatively regulates Staphylococcus epidermidis biofilm formation by modulating expression of 1 MDa extracellular matrix binding protein and autolysis‐dependent release of eDNA

    DEFF Research Database (Denmark)

    Christner, Martin; Heinze, Constanze; Busch, Michael

    2012-01-01

    to biofilm formation in mutant 1585ΔsarA. Increased eDNA amounts indirectly resulted from upregulation of metalloprotease SepA, leading to boosted processing of autolysin AtlE, in turn inducing augmented autolysis and release of eDNA. Hence, this study identifies sarA as a negative regulator of Embp‐ and e...

  12. In vitro colonization of the muscle extracellular matrix components by Escherichia coli O157:H7: the influence of growth medium, temperature and pH on initial adhesion and induction of biofilm formation by collagens I and III.

    Directory of Open Access Journals (Sweden)

    Caroline Chagnot

    Full Text Available Enterohemorrhagic Escherichia coli (EHEC O157:H7 are responsible for repeated food-poisoning cases often caused by contaminated burgers. EHEC infection is predominantly a pediatric illness, which can lead to life-threatening diseases. Ruminants are the main natural reservoir for EHEC and food contamination almost always originates from faecal contamination. In beef meat products, primary bacterial contamination occurs at the dehiding stage of slaughtering. The extracellular matrix (ECM is the most exposed part of the skeletal muscles in beef carcasses. Investigating the adhesion to the main muscle fibrous ECM proteins, insoluble fibronectin, collagen I, III and IV, laminin-α2 and elastin, results demonstrated that the preceding growth conditions had a great influence on subsequent bacterial attachment. In the tested experimental conditions, maximal adhesion to fibril-forming collagens I or III occurred at 25°C and pH 7. Once initially adhered, exposure to lower temperatures, as applied to meat during cutting and storage, or acidification, as in the course of post-mortem physiological modifications of muscle, had no effect on detachment, except at pHu. In addition, dense biofilm formation occurred on immobilized collagen I or III and was induced in growth medium supplemented with collagen I in solution. From this first comprehensive investigation of EHEC adhesion to ECM proteins with respect to muscle biology and meat processing, new research directions for the development of innovative practices to minimize the risk of meat contamination are further discussed.

  13. Low Concentrations of Vitamin C Reduce the Synthesis of Extracellular Polymers and Destabilize Bacterial Biofilms

    KAUST Repository

    Pandit, Santosh

    2017-12-26

    Extracellular polymeric substances (EPS) produced by bacteria form a matrix supporting the complex three-dimensional architecture of biofilms. This EPS matrix is primarily composed of polysaccharides, proteins and extracellular DNA. In addition to supporting the community structure, the EPS matrix protects bacterial biofilms from the environment. Specifically, it shields the bacterial cells inside the biofilm, by preventing antimicrobial agents from getting in contact with them, thereby reducing their killing effect. New strategies for disrupting the formation of the EPS matrix can therefore lead to a more efficient use of existing antimicrobials. Here we examined the mechanism of the known effect of vitamin C (sodium ascorbate) on enhancing the activity of various antibacterial agents. Our quantitative proteomics analysis shows that non-lethal concentrations of vitamin C inhibit bacterial quorum sensing and other regulatory mechanisms underpinning biofilm development. As a result, the EPS biosynthesis in reduced, and especially the polysaccharide component of the matrix is depleted. Once the EPS content is reduced beyond a critical point, bacterial cells get fully exposed to the medium. At this stage, the cells are more susceptible to killing, either by vitamin C-induced oxidative stress as reported here, or by other antimicrobials or treatments.

  14. Low Concentrations of Vitamin C Reduce the Synthesis of Extracellular Polymers and Destabilize Bacterial Biofilms

    KAUST Repository

    Pandit, Santosh; Ravikumar, Vaishnavi; Abdel-Haleem, Alyaa M.; Derouiche, Abderahmane; Mokkapati, V. R. S. S.; Sihlbom, Carina; Mineta, Katsuhiko; Gojobori, Takashi; Gao, Xin; Westerlund, Fredrik; Mijakovic, Ivan

    2017-01-01

    Extracellular polymeric substances (EPS) produced by bacteria form a matrix supporting the complex three-dimensional architecture of biofilms. This EPS matrix is primarily composed of polysaccharides, proteins and extracellular DNA. In addition to supporting the community structure, the EPS matrix protects bacterial biofilms from the environment. Specifically, it shields the bacterial cells inside the biofilm, by preventing antimicrobial agents from getting in contact with them, thereby reducing their killing effect. New strategies for disrupting the formation of the EPS matrix can therefore lead to a more efficient use of existing antimicrobials. Here we examined the mechanism of the known effect of vitamin C (sodium ascorbate) on enhancing the activity of various antibacterial agents. Our quantitative proteomics analysis shows that non-lethal concentrations of vitamin C inhibit bacterial quorum sensing and other regulatory mechanisms underpinning biofilm development. As a result, the EPS biosynthesis in reduced, and especially the polysaccharide component of the matrix is depleted. Once the EPS content is reduced beyond a critical point, bacterial cells get fully exposed to the medium. At this stage, the cells are more susceptible to killing, either by vitamin C-induced oxidative stress as reported here, or by other antimicrobials or treatments.

  15. Extracellular matrix component signaling in cancer

    DEFF Research Database (Denmark)

    Multhaupt, Hinke A. B.; Leitinger, Birgit; Gullberg, Donald

    2016-01-01

    Cell responses to the extracellular matrix depend on specific signaling events. These are important from early development, through differentiation and tissue homeostasis, immune surveillance, and disease pathogenesis. Signaling not only regulates cell adhesion cytoskeletal organization and motil...... as well as matrix constitution and protein crosslinking. Here we summarize roles of the three major matrix receptor types, with emphasis on how they function in tumor progression. [on SciFinder(R)]...

  16. Silver colloidal nanoparticles: effect on matrix composition and structure of Candida albicans and Candida glabrata biofilms.

    Science.gov (United States)

    Monteiro, D R; Silva, S; Negri, M; Gorup, L F; de Camargo, E R; Oliveira, R; Barbosa, D B; Henriques, M

    2013-04-01

    The aim of this study was to assess the effect of different silver nanoparticles (SN) concentrations on the matrix composition and structure of Candida albicans and Candida glabrata biofilms. Candida biofilms were developed in 6-well microtiter plates during 48 h. After, these biofilms were exposed to 13.5 or 54 μg SN ml(-1) for 24 h. Then, extracellular matrices were extracted from biofilms and analysed chemically in terms of proteins, carbohydrates and DNA. To investigate the biofilm structure, scanning electron microscopy (SEM) and epifluorescence microscopy were used. SN interfered with the matrix composition of Candida biofilms tested in terms of protein, carbohydrate and DNA, except for the protein content of C. albicans biofilm. By SEM, Candida biofilms treated with SN revealed structural differences, when compared with the control groups. Further, SN showed a trend of agglomeration within the biofilms. Epifluorescence microscopy images suggest that SN induced damage on cell walls of the Candida isolates tested. In general, irrespective of concentration, SN affected the matrix composition and structure of Candida biofilms and these findings may be related to the mechanisms of biocide action of SN. This study reveals new insights about the behaviour of SN when in contact with Candida biofilms. SN may contribute to the development of therapies to prevent or control Candida infections. © 2012 The Society for Applied Microbiology.

  17. Extracellular DNA Release Acts as an Antifungal Resistance Mechanism in Mature Aspergillus fumigatus Biofilms

    Science.gov (United States)

    Rajendran, Ranjith; Williams, Craig; Lappin, David F.; Millington, Owain; Martins, Margarida

    2013-01-01

    Aspergillus fumigatus has been shown to form biofilms that are associated with adaptive antifungal resistance mechanisms. These include multidrug efflux pumps, heat shock proteins, and extracellular matrix (ECM). ECM is a key structural and protective component of microbial biofilms and in bacteria has been shown to contain extracellular DNA (eDNA). We therefore hypothesized that A. fumigatus biofilms also possess eDNA as part of the ECM, conferring a functional role. Fluorescence microscopy and quantitative PCR analyses demonstrated the presence of eDNA, which was released phase dependently (8 autolysis, were significantly upregulated as the biofilm matured and that inhibition of chitinases affected biofilm growth and stability, indicating mechanistically that autolysis was possibly involved. Finally, using checkerboard assays, it was shown that combinational treatment of biofilms with DNase plus amphotericin B and caspofungin significantly improved antifungal susceptibility. Collectively, these data show that eDNA is an important structural component of A. fumigatus ECM that is released through autolysis, which is important for protection from environmental stresses, including antifungal therapy. PMID:23314962

  18. Extracellular matrix formation enhances the ability of Streptococcus pneumoniae to cause invasive disease.

    Directory of Open Access Journals (Sweden)

    Claudia Trappetti

    Full Text Available During infection, pneumococci exist mainly in sessile biofilms rather than in planktonic form, except during sepsis. However, relatively little is known about how biofilms contribute to pneumococcal pathogenesis. Here, we carried out a biofilm assay on opaque and transparent variants of a clinical serotype 19F strain WCH159. After 4 days incubation, scanning electron microscopy revealed that opaque biofilm bacteria produced an extracellular matrix, whereas the transparent variant did not. The opaque biofilm-derived bacteria translocated from the nasopharynx to the lungs and brain of mice, and showed 100-fold greater in vitro adherence to A549 cells than transparent bacteria. Microarray analysis of planktonic and sessile bacteria from transparent and opaque variants showed differential gene expression in two operons: the lic operon, which is involved in choline uptake, and in the two-component system, ciaRH. Mutants of these genes did not form an extracellular matrix, could not translocate from the nasopharynx to the lungs or the brain, and adhered poorly to A549 cells. We conclude that only the opaque phenotype is able to form extracellular matrix, and that the lic operon and ciaRH contribute to this process. We propose that during infection, extracellular matrix formation enhances the ability of pneumococci to cause invasive disease.

  19. The Candida albicans Biofilm Matrix: Composition, Structure and Function.

    Science.gov (United States)

    Pierce, Christopher G; Vila, Taissa; Romo, Jesus A; Montelongo-Jauregui, Daniel; Wall, Gina; Ramasubramanian, Anand; Lopez-Ribot, Jose L

    2017-03-01

    A majority of infections caused by Candida albicans -the most frequent fungal pathogen-are associated with biofilm formation. A salient feature of C. albicans biofilms is the presence of the biofilm matrix. This matrix is composed of exopolymeric materials secreted by sessile cells within the biofilm, in which all classes of macromolecules are represented, and provides protection against environmental challenges. In this review, we summarize the knowledge accumulated during the last two decades on the composition, structure, and function of the C. albicans biofilm matrix. Knowledge of the matrix components, its structure, and function will help pave the way to novel strategies to combat C. albicans biofilm infections.

  20. Prevention of biofilm formation and removal of existing biofilms by extracellular DNases of Campylobacter jejuni.

    Science.gov (United States)

    Brown, Helen L; Reuter, Mark; Hanman, Kate; Betts, Roy P; van Vliet, Arnoud H M

    2015-01-01

    The fastidious nature of the foodborne bacterial pathogen Campylobacter jejuni contrasts with its ability to survive in the food chain. The formation of biofilms, or the integration into existing biofilms by C. jejuni, is thought to contribute to food chain survival. As extracellular DNA (eDNA) has previously been proposed to play a role in C. jejuni biofilms, we have investigated the role of extracellular DNases (eDNases) produced by C. jejuni in biofilm formation. A search of 2791 C. jejuni genomes highlighted that almost half of C. jejuni genomes contains at least one eDNase gene, but only a minority of isolates contains two or three of these eDNase genes, such as C. jejuni strain RM1221 which contains the cje0256, cje0566 and cje1441 eDNase genes. Strain RM1221 did not form biofilms, whereas the eDNase-negative strains NCTC 11168 and 81116 did. Incubation of pre-formed biofilms of NCTC 11168 with live C. jejuni RM1221 or with spent medium from a RM1221 culture resulted in removal of the biofilm. Inactivation of the cje1441 eDNase gene in strain RM1221 restored biofilm formation, and made the mutant unable to degrade biofilms of strain NCTC 11168. Finally, C. jejuni strain RM1221 was able to degrade genomic DNA from C. jejuni NCTC 11168, 81116 and RM1221, whereas strain NCTC 11168 and the RM1221 cje1441 mutant were unable to do so. This was mirrored by an absence of eDNA in overnight cultures of C. jejuni RM1221. This suggests that the activity of eDNases in C. jejuni affects biofilm formation and is not conducive to a biofilm lifestyle. These eDNases do however have a potential role in controlling biofilm formation by C. jejuni strains in food chain relevant environments.

  1. Flow environment and matrix structure interact to determine spatial competition in Pseudomonas aeruginosa biofilms.

    Science.gov (United States)

    Nadell, Carey D; Ricaurte, Deirdre; Yan, Jing; Drescher, Knut; Bassler, Bonnie L

    2017-01-13

    Bacteria often live in biofilms, which are microbial communities surrounded by a secreted extracellular matrix. Here, we demonstrate that hydrodynamic flow and matrix organization interact to shape competitive dynamics in Pseudomonas aeruginosa biofilms. Irrespective of initial frequency, in competition with matrix mutants, wild-type cells always increase in relative abundance in planar microfluidic devices under simple flow regimes. By contrast, in microenvironments with complex, irregular flow profiles - which are common in natural environments - wild-type matrix-producing and isogenic non-producing strains can coexist. This result stems from local obstruction of flow by wild-type matrix producers, which generates regions of near-zero shear that allow matrix mutants to locally accumulate. Our findings connect the evolutionary stability of matrix production with the hydrodynamics and spatial structure of the surrounding environment, providing a potential explanation for the variation in biofilm matrix secretion observed among bacteria in natural environments.

  2. Characterization of the Vibrio cholerae extracellular matrix: a top-down solid-state NMR approach.

    Science.gov (United States)

    Reichhardt, Courtney; Fong, Jiunn C N; Yildiz, Fitnat; Cegelski, Lynette

    2015-01-01

    Bacterial biofilms are communities of bacterial cells surrounded by a self-secreted extracellular matrix. Biofilm formation by Vibrio cholerae, the human pathogen responsible for cholera, contributes to its environmental survival and infectivity. Important genetic and molecular requirements have been identified for V. cholerae biofilm formation, yet a compositional accounting of these parts in the intact biofilm or extracellular matrix has not been described. As insoluble and non-crystalline assemblies, determinations of biofilm composition pose a challenge to conventional biochemical and biophysical analyses. The V. cholerae extracellular matrix composition is particularly complex with several proteins, complex polysaccharides, and other biomolecules having been identified as matrix parts. We developed a new top-down solid-state NMR approach to spectroscopically assign and quantify the carbon pools of the intact V. cholerae extracellular matrix using ¹³C CPMAS and ¹³C{(¹⁵N}, ¹⁵N{³¹P}, and ¹³C{³¹P}REDOR. General sugar, lipid, and amino acid pools were first profiled and then further annotated and quantified as specific carbon types, including carbonyls, amides, glycyl carbons, and anomerics. In addition, ¹⁵N profiling revealed a large amine pool relative to amide contributions, reflecting the prevalence of molecular modifications with free amine groups. Our top-down approach could be implemented immediately to examine the extracellular matrix from mutant strains that might alter polysaccharide production or lipid release beyond the cell surface; or to monitor changes that may accompany environmental variations and stressors such as altered nutrient composition, oxidative stress or antibiotics. More generally, our analysis has demonstrated that solid-state NMR is a valuable tool to characterize complex biofilm systems. Copyright © 2014. Published by Elsevier B.V.

  3. Extracellular matrix and tissue engineering applications

    NARCIS (Netherlands)

    Fernandes, H.A.M.; Moroni, Lorenzo; van Blitterswijk, Clemens; de Boer, Jan

    2009-01-01

    The extracellular matrix is a key component during regeneration and maintenance of tissues and organs, and it therefore plays a critical role in successful tissue engineering as well. Tissue engineers should recognise that engineering technology can be deduced from natural repair processes. Due to

  4. Spatiotemporal distribution of different extracellular polymeric substances and filamentation mediate Xylella fastidiosa adhesion and biofilm formation.

    Science.gov (United States)

    Janissen, Richard; Murillo, Duber M; Niza, Barbara; Sahoo, Prasana K; Nobrega, Marcelo M; Cesar, Carlos L; Temperini, Marcia L A; Carvalho, Hernandes F; de Souza, Alessandra A; Cotta, Monica A

    2015-04-20

    Microorganism pathogenicity strongly relies on the generation of multicellular assemblies, called biofilms. Understanding their organization can unveil vulnerabilities leading to potential treatments; spatially and temporally-resolved comprehensive experimental characterization can provide new details of biofilm formation, and possibly new targets for disease control. Here, biofilm formation of economically important phytopathogen Xylella fastidiosa was analyzed at single-cell resolution using nanometer-resolution spectro-microscopy techniques, addressing the role of different types of extracellular polymeric substances (EPS) at each stage of the entire bacterial life cycle. Single cell adhesion is caused by unspecific electrostatic interactions through proteins at the cell polar region, where EPS accumulation is required for more firmly-attached, irreversibly adhered cells. Subsequently, bacteria form clusters, which are embedded in secreted loosely-bound EPS, and bridged by up to ten-fold elongated cells that form the biofilm framework. During biofilm maturation, soluble EPS forms a filamentous matrix that facilitates cell adhesion and provides mechanical support, while the biofilm keeps anchored by few cells. This floating architecture maximizes nutrient distribution while allowing detachment upon larger shear stresses; it thus complies with biological requirements of the bacteria life cycle. Using new approaches, our findings provide insights regarding different aspects of the adhesion process of X. fastidiosa and biofilm formation.

  5. Biofilm extracellular DNA enhances mixed species biofilms of Staphylococcus epidermidis and Candida albicans.

    Science.gov (United States)

    Pammi, Mohan; Liang, Rong; Hicks, John; Mistretta, Toni-Ann; Versalovic, James

    2013-11-14

    Polymicrobial infections are responsible for significant mortality and morbidity in adults and children. Staphylococcus epidermidis and Candida albicans are the most frequent combination of organisms isolated from polymicrobial infections. Vascular indwelling catheters are sites for mixed species biofilm formation and pose a significant risk for polymicrobial infections. We hypothesized that enhancement of biofilms in a mixed species environment increases patient mortality and morbidity. Mixed species biofilms of S. epidermidis and C. albicans were evaluated in vitro and in a subcutaneous catheter infection model in vivo. Mixed species biofilms were enhanced compared to single species biofilms of either S. epidermidis or C. albicans. A mixed species environment increased catheter infection and increased dissemination of S. epidermidis in mice. Microarrays were used to explore differential gene expression of S. epidermidis in the mixed species biofilms. In mixed species biofilms, compared to single species S. epidermidis biofilms, 2.7% of S. epidermidis genes were upregulated and 6% were down regulated. Staphylococcal autolysis repressors lrgA and lrgB were down regulated 36-fold and 27-fold respectively. The role of biofilm extracellular DNA was investigated by quantitation and by evaluating the effects of DNAse in a concentration and time dependent manner. S. epidermidis specific eDNA was increased in mixed species biofilms and further confirmed by degradation with DNAse. Mixed-species biofilms are enhanced and associated with increased S. epidermidis-specific eDNA in vitro and greater systemic dissemination of S. epidermidis in vivo. Down regulation of the lrg operon, a repressor of autolysis, associated with increased eDNA suggests a possible role for bacterial autolysis in mixed species biofilms. Enhancement and systemic dissemination of S. epidermidis may explain adverse outcomes after clinical polymicrobial infections of S. epidermidis and C. albicans.

  6. Biofilm extracellular DNA enhances mixed species biofilms of Staphylococcus epidermidis and Candida albicans

    Science.gov (United States)

    2013-01-01

    Background Polymicrobial infections are responsible for significant mortality and morbidity in adults and children. Staphylococcus epidermidis and Candida albicans are the most frequent combination of organisms isolated from polymicrobial infections. Vascular indwelling catheters are sites for mixed species biofilm formation and pose a significant risk for polymicrobial infections. We hypothesized that enhancement of biofilms in a mixed species environment increases patient mortality and morbidity. Results Mixed species biofilms of S. epidermidis and C. albicans were evaluated in vitro and in a subcutaneous catheter infection model in vivo. Mixed species biofilms were enhanced compared to single species biofilms of either S. epidermidis or C. albicans. A mixed species environment increased catheter infection and increased dissemination of S. epidermidis in mice. Microarrays were used to explore differential gene expression of S. epidermidis in the mixed species biofilms. In mixed species biofilms, compared to single species S. epidermidis biofilms, 2.7% of S. epidermidis genes were upregulated and 6% were down regulated. Staphylococcal autolysis repressors lrgA and lrgB were down regulated 36-fold and 27-fold respectively. The role of biofilm extracellular DNA was investigated by quantitation and by evaluating the effects of DNAse in a concentration and time dependent manner. S. epidermidis specific eDNA was increased in mixed species biofilms and further confirmed by degradation with DNAse. Conclusions Mixed-species biofilms are enhanced and associated with increased S. epidermidis-specific eDNA in vitro and greater systemic dissemination of S. epidermidis in vivo. Down regulation of the lrg operon, a repressor of autolysis, associated with increased eDNA suggests a possible role for bacterial autolysis in mixed species biofilms. Enhancement and systemic dissemination of S. epidermidis may explain adverse outcomes after clinical polymicrobial infections of S

  7. Shaping Synapses by the Neural Extracellular Matrix

    Directory of Open Access Journals (Sweden)

    Maura Ferrer-Ferrer

    2018-05-01

    Full Text Available Accumulating data support the importance of interactions between pre- and postsynaptic neuronal elements with astroglial processes and extracellular matrix (ECM for formation and plasticity of chemical synapses, and thus validate the concept of a tetrapartite synapse. Here we outline the major mechanisms driving: (i synaptogenesis by secreted extracellular scaffolding molecules, like thrombospondins (TSPs, neuronal pentraxins (NPs and cerebellins, which respectively promote presynaptic, postsynaptic differentiation or both; (ii maturation of synapses via reelin and integrin ligands-mediated signaling; and (iii regulation of synaptic plasticity by ECM-dependent control of induction and consolidation of new synaptic configurations. Particularly, we focused on potential importance of activity-dependent concerted activation of multiple extracellular proteases, such as ADAMTS4/5/15, MMP9 and neurotrypsin, for permissive and instructive events in synaptic remodeling through localized degradation of perisynaptic ECM and generation of proteolytic fragments as inducers of synaptic plasticity.

  8. Involvement of extracellular matrix constituents in breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Lochter, Andre; Bissell, Mina J

    1995-06-01

    It has recently been established that the extracellular matrix is required for normal functional differentiation of mammary epithelia not only in culture, but also in vivo. The mechanisms by which extracellular matrix affects differentiation, as well as the nature of extracellular matrix constituents which have major impacts on mammary gland function, have only now begun to be dissected. The intricate variety of extracellular matrix-mediated events and the remarkable degree of plasticity of extracellular matrix structure and composition at virtually all times during ontogeny, make such studies difficult. Similarly, during carcinogenesis, the extracellular matrix undergoes gross alterations, the consequences of which are not yet precisely understood. Nevertheless, an increasing amount of data suggests that the extracellular matrix and extracellular matrix-receptors might participate in the control of most, if not all, of the successive stages of breast tumors, from appearance to progression and metastasis.

  9. The biofilm matrix destabilizers, EDTA and DNaseI, enhance the susceptibility of nontypeable Hemophilus influenzae biofilms to treatment with ampicillin and ciprofloxacin.

    Science.gov (United States)

    Cavaliere, Rosalia; Ball, Jessica L; Turnbull, Lynne; Whitchurch, Cynthia B

    2014-08-01

    Nontypeable Hemophilus influenzae (NTHi) is a Gram-negative bacterial pathogen that causes chronic biofilm infections of the ears and airways. The biofilm matrix provides structural integrity to the biofilm and protects biofilm cells from antibiotic exposure by reducing penetration of antimicrobial compounds into the biofilm. Extracellular DNA (eDNA) has been found to be a major matrix component of biofilms formed by many species of Gram-positive and Gram-negative bacteria, including NTHi. Interestingly, the cation chelator ethylenediaminetetra-acetic acid (EDTA) has been shown to reduce the matrix strength of biofilms of several bacterial species as well as to have bactericidal activity against various pathogens. EDTA exerts its antimicrobial activity by chelating divalent cations necessary for growth and membrane stability and by destabilizing the matrix thus enhancing the detachment of bacterial cells from the biofilm. In this study, we have explored the role of divalent cations in NTHi biofilm development and stability. We have utilized in vitro static and continuous flow models of biofilm development by NTHi to demonstrate that magnesium cations enhance biofilm formation by NTHi. We found that the divalent cation chelator EDTA is effective at both preventing NTHi biofilm formation and at treating established NTHi biofilms. Furthermore, we found that the matrix destablilizers EDTA and DNaseI increase the susceptibility of NTHi biofilms to ampicillin and ciprofloxacin. Our observations indicate that DNaseI and EDTA enhance the efficacy of antibiotic treatment of NTHi biofilms. These observations may lead to new strategies that will improve the treatment options available to patients with chronic NTHi infections. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  10. Regulation of corneal stroma extracellular matrix assembly.

    Science.gov (United States)

    Chen, Shoujun; Mienaltowski, Michael J; Birk, David E

    2015-04-01

    The transparent cornea is the major refractive element of the eye. A finely controlled assembly of the stromal extracellular matrix is critical to corneal function, as well as in establishing the appropriate mechanical stability required to maintain corneal shape and curvature. In the stroma, homogeneous, small diameter collagen fibrils, regularly packed with a highly ordered hierarchical organization, are essential for function. This review focuses on corneal stroma assembly and the regulation of collagen fibrillogenesis. Corneal collagen fibrillogenesis involves multiple molecules interacting in sequential steps, as well as interactions between keratocytes and stroma matrix components. The stroma has the highest collagen V:I ratio in the body. Collagen V regulates the nucleation of protofibril assembly, thus controlling the number of fibrils and assembly of smaller diameter fibrils in the stroma. The corneal stroma is also enriched in small leucine-rich proteoglycans (SLRPs) that cooperate in a temporal and spatial manner to regulate linear and lateral collagen fibril growth. In addition, the fibril-associated collagens (FACITs) such as collagen XII and collagen XIV have roles in the regulation of fibril packing and inter-lamellar interactions. A communicating keratocyte network contributes to the overall and long-range regulation of stromal extracellular matrix assembly, by creating micro-domains where the sequential steps in stromal matrix assembly are controlled. Keratocytes control the synthesis of extracellular matrix components, which interact with the keratocytes dynamically to coordinate the regulatory steps into a cohesive process. Mutations or deficiencies in stromal regulatory molecules result in altered interactions and deficiencies in both transparency and refraction, leading to corneal stroma pathobiology such as stromal dystrophies, cornea plana and keratoconus. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. In-situ, time-lapse study of extracellular polymeric substance discharge in Streptococcus mutans biofilm.

    Science.gov (United States)

    Liu, Bernard Haochih; Yu, Li-Chieh

    2017-02-01

    Streptococcus mutans is one of the main pathogens that cause tooth decay. By metabolizing carbohydrates, S. mutans emits extracellular polymeric substance (EPS) that adheres to the tooth surface and forms layers of biofilm. Periodontal disease occurs due to the low pH environment created by S. mutans biofilm, and such an acidic environment gradually erodes tooth enamel. Since the existence of EPS is essential in the formation of biofilm, the in-situ investigation of its generation and distribution in real time is the key to the control and suppression of S. mutans biofilm. Prior studies of the biofilm formation process by fluorescence microscope, scanning electron microscope, or spectroscope have roughly divided the mechanism into three stages: (1) initial attachment; (2) microcolonies; and (3) maturation. However, these analytical methods are incapable to observe real-time changes in different locations of the extracellular matrix, and to analyze mechanical properties for single bacteria in micro and nanoscale. Since atomic force microscopy (AFM) operates by precise control of tip-sample interaction forces in liquid and in air, living microorganisms can be analyzed under near-physiological conditions. Thus, analytical techniques based on AFM constitute powerful tools for the study of biological samples, both qualitatively and quantitatively. In this study, we used AFM to quantitatively track the changes of multiple nanomechanical properties of S. mutans, including dissipation energy, adhesion force, deformation, and elastic modulus at different metabolic stages. The data revealed that the bacterial extracellular matrix has a gradient distribution in stickiness, in which different stickiness indicates the variation of EPS compositions, freshness, and metabolic stages. In-situ, time-lapse AFM images showed the local generation and distribution of EPS at different times, in which the highest adhesion distributed along sides of the S. mutans cells. Through time

  12. Biofilm extracellular polysaccharides degradation during starvation and enamel demineralization.

    Directory of Open Access Journals (Sweden)

    Bárbara Emanoele Costa Oliveira

    Full Text Available This study was conducted to evaluate if extracellular polysaccharides (EPS are used by Streptococcus mutans (Sm biofilm during night starvation, contributing to enamel demineralization increasing occurred during daily sugar exposure. Sm biofilms were formed during 5 days on bovine enamel slabs of known surface hardness (SH. The biofilms were exposed to sucrose 10% or glucose + fructose 10.5% (carbohydrates that differ on EPS formation, 8x/day but were maintained in starvation during the night. Biofilm samples were harvested during two moments, on the end of the 4th day and in the morning of the 5th day, conditions of sugar abundance and starvation, respectively. The slabs were also collected to evaluate the percentage of surface hardness loss (%SHL. The biofilms were analyzed for EPS soluble and insoluble and intracellular polysaccharides (IPS, viable bacteria (CFU, biofilm architecture and biomass. pH, calcium and acid concentration were determined in the culture medium. The data were analyzed by two-way ANOVA followed by Tukey's test or Student's t-test. The effect of the factor carbohydrate treatment for polysaccharide analysis was significant (p 0.05. Larger amounts of soluble and insoluble EPS and IPS were formed in the sucrose group when compared to glucose + fructose group (p < 0.05, but they were not metabolized during starvation time (S-EPS, p = 0.93; I-EPS, p = 0.11; and IPS = 0.96. Greater enamel %SHL was also found for the sucrose group (p < 0.05 but the demineralization did not increase during starvation (p = 0.09. In conclusion, the findings suggest that EPS metabolization by S. mutans during night starvation do not contribute to increase enamel demineralization occurred during the daily abundance of sugar.

  13. Distinct roles of extracellular polymeric substances in Pseudomonas aeruginosa biofilm development

    DEFF Research Database (Denmark)

    Yang, Liang; Hu, Yifan; Liu, Yang

    2011-01-01

    Bacteria form surface attached biofilm communities as one of the most important survival strategies in nature. Biofilms consist of water, bacterial cells and a wide range of self‐generated extracellular polymeric substances (EPS). Biofilm formation is a dynamic self‐assembly process and several d...... polysaccharide is more important than Pel polysaccharide in P. aeruginosa PAO1 biofilm formation and antibiotic resistance. Our study thus suggests that different EPS materials play distinct roles during bacterial biofilm formation.......Bacteria form surface attached biofilm communities as one of the most important survival strategies in nature. Biofilms consist of water, bacterial cells and a wide range of self‐generated extracellular polymeric substances (EPS). Biofilm formation is a dynamic self‐assembly process and several...... distinguishable stages are observed during bacterial biofilm development. Biofilm formation is shown to be coordinated by EPS production, cell migration, subpopulation differentiation and interactions. However, the ways these different factors affect each other and contribute to community structural...

  14. Poly-N-acetylglucosamine matrix polysaccharide impedes fluid convection and transport of the cationic surfactant cetylpyridinium chloride through bacterial biofilms.

    Science.gov (United States)

    Ganeshnarayan, Krishnaraj; Shah, Suhagi M; Libera, Matthew R; Santostefano, Anthony; Kaplan, Jeffrey B

    2009-03-01

    Biofilms are composed of bacterial cells encased in a self-synthesized, extracellular polymeric matrix. Poly-beta(1,6)-N-acetyl-d-glucosamine (PNAG) is a major biofilm matrix component in phylogenetically diverse bacteria. In this study we investigated the physical and chemical properties of the PNAG matrix in biofilms produced in vitro by the gram-negative porcine respiratory pathogen Actinobacillus pleuropneumoniae and the gram-positive device-associated pathogen Staphylococcus epidermidis. The effect of PNAG on bulk fluid flow was determined by measuring the rate of fluid convection through biofilms cultured in centrifugal filter devices. The rate of fluid convection was significantly higher in biofilms cultured in the presence of the PNAG-degrading enzyme dispersin B than in biofilms cultured without the enzyme, indicating that PNAG decreases bulk fluid flow. PNAG also blocked transport of the quaternary ammonium compound cetylpyridinium chloride (CPC) through the biofilms. Binding of CPC to biofilms further impeded fluid convection and blocked transport of the azo dye Allura red. Bioactive CPC was efficiently eluted from biofilms by treatment with 1 M sodium chloride. Taken together, these findings suggest that CPC reacts directly with the PNAG matrix and alters its physical and chemical properties. Our results indicate that PNAG plays an important role in controlling the physiological state of biofilms and may contribute to additional biofilm-associated processes such as biocide resistance.

  15. Molecular Determinants of the Thickened Matrix in a Dual-Species Pseudomonas aeruginosa and Enterococcus faecalis Biofilm.

    Science.gov (United States)

    Lee, Keehoon; Lee, Kang-Mu; Kim, Donggeun; Yoon, Sang Sun

    2017-11-01

    Biofilms are microbial communities that inhabit various surfaces and are surrounded by extracellular matrices (ECMs). Clinical microbiologists have shown that the majority of chronic infections are caused by biofilms, following the introduction of the first biofilm infection model by J. W. Costerton and colleagues (J. Lam, R. Chan, K. Lam, and J. W. Costerton, Infect Immun 28:546-556, 1980). However, treatments for chronic biofilm infections are still limited to surgical removal of the infected sites. Pseudomonas aeruginosa and Enterococcus faecalis are two frequently identified bacterial species in biofilm infections; nevertheless, the interactions between these two species, especially during biofilm growth, are not clearly understood. In this study, we observed phenotypic changes in a dual-species biofilm of P. aeruginosa and E. faecalis , including a dramatic increase in biofilm matrix thickness. For clear elucidation of the spatial distribution of the dual-species biofilm, P. aeruginosa and E. faecalis were labeled with red and green fluorescence, respectively. E. faecalis was located at the lower part of the dual-species biofilm, while P. aeruginosa developed a structured biofilm on the upper part. Mutants with altered exopolysaccharide (EPS) productions were constructed in order to determine the molecular basis for the synergistic effect of the dual-species biofilm. Increased biofilm matrix thickness was associated with EPSs, not extracellular DNA. In particular, Pel and Psl contributed to interspecies and intraspecies interactions, respectively, in the dual-species P. aeruginosa and E. faecalis biofilm. Accordingly, targeting Pel and Psl might be an effective part of eradicating P. aeruginosa polymicrobial biofilms. IMPORTANCE Chronic infection is a serious problem in the medical field. Scientists have observed that chronic infections are closely associated with biofilms, and the vast majority of infection-causing biofilms are polymicrobial. Many studies

  16. A Rhizobium leguminosarum CHDL- (Cadherin-Like-) Lectin Participates in Assembly and Remodeling of the Biofilm Matrix

    DEFF Research Database (Denmark)

    Vozza, Nicolás F.; Abdian, Patricia L; Russo, Daniela M

    2016-01-01

    In natural environments most bacteria live in multicellular structures called biofilms. These cell aggregates are enclosed in a self-produced polymeric extracellular matrix, which protects the cells, provides mechanical stability and mediates cellular cohesion and adhesion to surfaces. Although...... important advances were made in the identification of the genetic and extracellular factors required for biofilm formation, the mechanisms leading to biofilm matrix assembly, and the roles of extracellular proteins in these processes are still poorly understood. The symbiont Rhizobium leguminosarum requires...... the synthesis of the acidic exopolysaccharide and the PrsDE secretion system to develop a mature biofilm. PrsDE is responsible for the secretion of the Rap family of proteins that share one or two Ra/CHDL (cadherin-like-) domains. RapA2 is a calcium-dependent lectin with a cadherin-like β sheet structure...

  17. Light Regimes Shape Utilization of Extracellular Organic C and N in a Cyanobacterial Biofilm

    Energy Technology Data Exchange (ETDEWEB)

    Stuart, Rhona K.; Mayali, Xavier; Boaro, Amy A.; Zemla, Adam; Everroad, R. Craig; Nilson, Daniel; Weber, Peter K.; Lipton, Mary; Bebout, Brad M.; Pett-Ridge, Jennifer; Thelen, Michael P.

    2016-06-28

    >IMPORTANCECyanobacteria are globally distributed primary producers, and the fate of their fixed C influences microbial biogeochemical cycling. This fate is complicated by cyanobacterial degradation and assimilation of organic matter, but because cyanobacteria are assumed to be poor competitors for organic matter consumption, regulation of this process is not well tested. In mats and biofilms, this is especially relevant because cyanobacteria produce an extensive organic extracellular matrix, providing the community with a rich source of nutrients. Light is a well-known regulator of cyanobacterial metabolism, so we characterized the effects of light availability on the incorporation of organic matter. Using stable isotope tracing at the single-cell level, we quantified photoautotroph assimilation under different metabolic conditions and integrated the results with proteomics to elucidate metabolic status. We found that cyanobacteria effectively compete for organic matter in the light and the dark and that nutrient requirements and community interactions contribute to cycling of extracellular organic matter.

  18. Two genetic loci produce distinct carbohydrate-rich structural components of the Pseudomonas aeruginosa biofilm matrix.

    Science.gov (United States)

    Friedman, Lisa; Kolter, Roberto

    2004-07-01

    Pseudomonas aeruginosa forms biofilms, which are cellular aggregates encased in an extracellular matrix. Molecular genetics studies of three common autoaggregative phenotypes, namely wrinkled colonies, pellicles, and solid-surface-associated biofilms, led to the identification of two loci, pel and psl, that are involved in the production of carbohydrate-rich components of the biofilm matrix. The pel gene cluster is involved in the production of a glucose-rich matrix material in P. aeruginosa strain PA14 (L. Friedman and R. Kolter, Mol. Microbiol. 51:675-690, 2004). Here we investigate the role of the pel gene cluster in P. aeruginosa strain ZK2870 and identify a second genetic locus, termed psl, involved in the production of a mannose-rich matrix material. The 11 predicted protein products of the psl genes are homologous to proteins involved in carbohydrate processing. P. aeruginosa is thus able to produce two distinct carbohydrate-rich matrix materials. Either carbohydrate-rich matrix component appears to be sufficient for mature biofilm formation, and at least one of them is required for mature biofilm formation in P. aeruginosa strains PA14 and ZK2870. Copyright 2004 American Society for Microbiology

  19. Why regenerative medicine needs an extracellular matrix.

    Science.gov (United States)

    Prestwich, Glenn D; Healy, Kevin E

    2015-01-01

    Regenerative medicine is now coming of age. Many attempts at cell therapy have failed to show significant efficacy, and the umbrella term 'stem cell therapy' is perceived in some quarters as hype or just expensive and unnecessary medical tourism. Here we present a short editorial in three parts. First, we examine the importance of using a semisynthetic extracellular matrix (ECM) mimetic, or sECM, to deliver and retain therapeutic cells at the site of administration. Second, we describe one approach in which biophysical and biochemical properties are tailored to each tissue type, which we call "design for optimal functionality." Third, we describe an alternative approach to sECM design and implementation, called "design for simplicity," in which a deconstructed, minimalist sECM is employed and biology is allowed to perform the customization in situ. We opine that an sECM, whether minimal or instructive, is an essential contributor to improve the outcomes of cell-based therapies.

  20. Extracellular matrix fluctuations during early embryogenesis

    International Nuclear Information System (INIS)

    Szabó, A; Rupp, P A; Rongish, B J; Little, C D; Czirók, A

    2011-01-01

    Extracellular matrix (ECM) movements and rearrangements were studied in avian embryos during early stages of development. We show that the ECM moves as a composite material, whereby distinct molecular components as well as spatially separated layers exhibit similar displacements. Using scanning wide field and confocal microscopy we show that the velocity field of ECM displacement is smooth in space and that ECM movements are correlated even at locations separated by several hundred micrometers. Velocity vectors, however, strongly fluctuate in time. The autocorrelation time of the velocity fluctuations is less than a minute. Suppression of the fluctuations yields a persistent movement pattern that is shared among embryos at equivalent stages of development. The high resolution of the velocity fields allows a detailed spatio-temporal characterization of important morphogenetic processes, especially tissue dynamics surrounding the embryonic organizer (Hensen's node)

  1. Extracellular Matrix Molecules Facilitating Vascular Biointegration

    Directory of Open Access Journals (Sweden)

    Martin K.C. Ng

    2012-08-01

    Full Text Available All vascular implants, including stents, heart valves and graft materials exhibit suboptimal biocompatibility that significantly reduces their clinical efficacy. A range of biomolecules in the subendothelial space have been shown to play critical roles in local regulation of thrombosis, endothelial growth and smooth muscle cell proliferation, making these attractive candidates for modulation of vascular device biointegration. However, classically used biomaterial coatings, such as fibronectin and laminin, modulate only one of these components; enhancing endothelial cell attachment, but also activating platelets and triggering thrombosis. This review examines a subset of extracellular matrix molecules that have demonstrated multi-faceted vascular compatibility and accordingly are promising candidates to improve the biointegration of vascular biomaterials.

  2. Natural antigenic differences in the functionally equivalent extracellular DNABII proteins of bacterial biofilms provide a means for targeted biofilm therapeutics

    Science.gov (United States)

    Rocco, Christopher J.; Davey, Mary Ellen; Bakaletz, Lauren O.; Goodman, Steven D.

    2016-01-01

    SUMMARY Bacteria that persist in the oral cavity exist within complex biofilm communities. A hallmark of biofilms is the presence of an extracellular polymeric substance (EPS), which consists of polysaccharides, extracellular DNA (eDNA), and proteins, including the DNABII family of proteins. The removal of DNABII proteins from a biofilm results in the loss of structural integrity of the eDNA and the collapse of the biofilm structure. We examined the role of DNABII proteins in the biofilm structure of the periodontal pathogen Porphyromonas gingivalis and the oral commensal Streptococcus gordonii. Co-aggregation with oral streptococci is thought to facilitate the establishment of P. gingivalis within the biofilm community. We demonstrate that DNABII proteins are present in the EPS of both S. gordonii and P. gingivalis biofilms, and that these biofilms can be disrupted through the addition of antisera derived against their respective DNABII proteins. We provide evidence that both eDNA and DNABII proteins are limiting in S. gordonii but not in P. gingivalis biofilms. In addition, these proteins are capable of complementing one another functionally. We also found that while antisera derived against most DNABII proteins are capable of binding a wide variety of DNABII proteins, the P. gingivalis DNABII proteins are antigenically distinct. The presence of DNABII proteins in the EPS of these biofilms and the antigenic uniqueness of the P. gingivalis proteins provide an opportunity to develop therapies that are targeted to remove P. gingivalis and biofilms that contain P. gingivalis from the oral cavity. PMID:26988714

  3. Extracellular dextran and DNA affect the formation of Enterococcus faecalis biofilms and their susceptibility to 2% chlorhexidine.

    Science.gov (United States)

    Li, Weilan; Liu, Hongyan; Xu, Qiong

    2012-07-01

    Enterococcus faecalis is frequently recovered from root-filled teeth with refractory apical periodontitis. The ability of E. faecalis to form a matrix-encased biofilm contributes to its pathogenicity; however, the role of extracellular dextran and DNA in biofilm formation and its effect on the susceptibility of the biofilm to chlorhexidine remains poorly understood. E. faecalis biofilms were incubated on dentin blocks. The effect of a dextran-degrading enzyme (dextranase) and DNase I on the adhesion of E. faecalis to dentin was measured using the colony-forming unit (CFU) counting method. CFU assays and confocal laser scanning microscopy were used to investigate the influence of dextranase and DNase I on the antimicrobial activity of 2% chlorhexidine. The CFU count assays indicated that the formation of biofilms by E. faecalis was reduced in cells treated with dextranase or DNase I compared with that in untreated cells (P biofilms with dextranase or DNase I effectively sensitized the biofilms to 2% chlorhexidine (P biofilms to 2% chlorhexidine. Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  4. Tumorigenic Potential of Extracellular Matrix Metalloproteinase Inducer

    Science.gov (United States)

    Zucker, Stanley; Hymowitz, Michelle; Rollo, Ellen E.; Mann, Richard; Conner, Cathleen E.; Cao, Jian; Foda, Hussein D.; Tompkins, David C.; Toole, Bryan P.

    2001-01-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN), a glycoprotein present on the cancer cell plasma membrane, enhances fibroblast synthesis of matrix metalloproteinases (MMPs). The demonstration that peritumoral fibroblasts synthesize most of the MMPs in human tumors rather than the cancer cells themselves has ignited interest in the role of EMMPRIN in tumor dissemination. In this report we have demonstrated a role for EMMPRIN in cancer progression. Human MDA-MB-436 breast cancer cells, which are tumorigenic but slow growing in vivo, were transfected with EMMPRIN cDNA and injected orthotopically into mammary tissue of female NCr nu/nu mice. Green fluorescent protein was used to visualize metastases. In three experiments, breast cancer cell clones transfected with EMMPRIN cDNA were considerably more tumorigenic and invasive than plasmid-transfected cancer cells. Increased gelatinase A and gelatinase B expression (demonstrated by in situ hybridization and gelatin substrate zymography) was demonstrated in EMMPRIN-enhanced tumors. In contrast to de novo breast cancers in humans, human tumors transplanted into mice elicited minimal stromal or inflammatory cell reactions. Based on these experimental studies and our previous demonstration that EMMPRIN is prominently displayed in human cancer tissue, we propose that EMMPRIN plays an important role in cancer progression by increasing synthesis of MMPs. PMID:11395366

  5. In Situ Molecular Imaging of the Biofilm and Its Matrix

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Yuanzhao; Zhou, Yufan; Yao, Juan; Szymanski, Craig J.; Fredrickson, Jim K.; Shi, Liang; Cao, B.; Zhu, Zihua; Yu, Xiao-Ying

    2016-11-15

    Molecular mapping of live biofilms at submicron resolution presents a grand challenge. Here, we present the first chemical mapping results of biofilm extracellular polymeric sub-stance (EPS) components in biofilms using correlative imaging be-tween super resolution florescence microscopy and liquid time-of-flight secondary ion mass spectrometry (ToF-SIMS). Shewanella oneidensis is used as a model organism. Heavy metal anions chro-mate (Cr2O72-) consisting of chromium Cr (VI) was a model envi-ronmental stressor used to treat the biofilms. Of particular interest, biologically relevant water clusters have been first observed in the biofilms. Characteristic fragments of biofilm matrix components such as proteins, polysaccharides, and lipids can be spatially im-aged. Furthermore, characteristic fatty acids (e.g., palmitic acid), quinolone signal, and riboflavin fragments are found to respond af-ter the biofilm is treated with Cr (VI), leading to biofilm dispersion. Significant changes in water clusters and quorum sensing signals indicative of intercellular communication in the aqueous environ-ment are observed, suggesting that they might result in fatty acid synthesis and inhibit riboflavin production. The Cr (VI) reduction seems to follow the Mtr pathway leading to Cr (III) formation. Our approach potentially opens a new avenue for mechanistic insight of microbial community processes and communications using in situ imaging mass spectrometry and superresolution optical micros-copy.

  6. Extracellular matrix dynamics during vertebrate axis formation.

    Science.gov (United States)

    Czirók, András; Rongish, Brenda J; Little, Charles D

    2004-04-01

    The first evidence for the dynamics of in vivo extracellular matrix (ECM) pattern formation during embryogenesis is presented below. Fibrillin 2 filaments were tracked for 12 h throughout the avian intraembryonic mesoderm using automated light microscopy and algorithms of our design. The data show that these ECM filaments have a reproducible morphogenic destiny that is characterized by directed transport. Fibrillin 2 particles initially deposited in the segmental plate mesoderm are translocated along an unexpected trajectory where they eventually polymerize into an intricate scaffold of cables parallel to the anterior-posterior axis. The cables coalesce near the midline before the appearance of the next-formed somite. Moreover, the ECM filaments define global tissue movements with high precision because the filaments act as passive motion tracers. Quantification of individual and collective filament "behaviors" establish fate maps, trajectories, and velocities. These data reveal a caudally propagating traveling wave pattern in the morphogenetic movements of early axis formation. We conjecture that within vertebrate embryos, long-range mechanical tension fields are coupled to both large-scale patterning and local organization of the ECM. Thus, physical forces or stress fields are essential requirements for executing an emergent developmental pattern-in this case, paraxial fibrillin cable assembly.

  7. Peroxidase enzymes regulate collagen extracellular matrix biosynthesis.

    Science.gov (United States)

    DeNichilo, Mark O; Panagopoulos, Vasilios; Rayner, Timothy E; Borowicz, Romana A; Greenwood, John E; Evdokiou, Andreas

    2015-05-01

    Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes often physically associated with fibrotic tissue and cancer in various organs, without any direct involvement in promoting fibroblast recruitment and extracellular matrix (ECM) biosynthesis at these sites. We report herein novel findings that show peroxidase enzymes possess a well-conserved profibrogenic capacity to stimulate the migration of fibroblastic cells and promote their ability to secrete collagenous proteins to generate a functional ECM both in vitro and in vivo. Mechanistic studies conducted using cultured fibroblasts show that these cells are capable of rapidly binding and internalizing both myeloperoxidase and eosinophil peroxidase. Peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl 4-hydroxylase-dependent manner that does not require ascorbic acid. This response was blocked by the irreversible myeloperoxidase inhibitor 4-amino-benzoic acid hydrazide, indicating peroxidase catalytic activity is essential for collagen biosynthesis. These results suggest that peroxidase enzymes, such as myeloperoxidase and eosinophil peroxidase, may play a fundamental role in regulating the recruitment of fibroblast and the biosynthesis of collagen ECM at sites of normal tissue repair and fibrosis, with enormous implications for many disease states where infiltrating inflammatory cells deposit peroxidases. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  8. Towards integrating extracellular matrix and immunological pathways.

    Science.gov (United States)

    Boyd, David F; Thomas, Paul G

    2017-10-01

    The extracellular matrix (ECM) is a complex and dynamic structure made up of an estimated 300 different proteins. The ECM is also a rich source of cytokines and growth factors in addition to numerous bioactive ECM degradation products that influence cell migration, proliferation, and differentiation. The ECM is constantly being remodeled during homeostasis and in a wide range of pathological contexts. Changes in the ECM modulate immune responses, which in turn regulate repair and regeneration of tissues. Here, we review the many components of the ECM, enzymes involved in ECM remodeling, and the signals that feed into immunological pathways in the context of a dynamic ECM. We highlight studies that have taken an integrative approach to studying immune responses in the context of the ECM and studies that use novel proteomic strategies. Finally, we discuss research challenges relevant to the integration of immune and ECM networks and propose experimental and translational approaches to resolve these issues. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Force spectroscopy of hepatocytic extracellular matrix components

    Energy Technology Data Exchange (ETDEWEB)

    Yongsunthon, R., E-mail: YongsuntR@Corning.com [Corning Incorporated, SP-FR-01, R1S32D, Corning, NY 14831 (United States); Baker, W.A.; Bryhan, M.D.; Baker, D.E.; Chang, T.; Petzold, O.N.; Walczak, W.J.; Liu, J.; Faris, R.A.; Senaratne, W.; Seeley, L.A.; Youngman, R.E. [Corning Incorporated, SP-FR-01, R1S32D, Corning, NY 14831 (United States)

    2009-07-15

    We present atomic force microscopy and force spectroscopy data of live hepatocytes (HEPG2/C3A liver cell line) grown in Eagle's Minimum Essential Medium, a complex solution of salts and amino acids commonly used for cell culture. Contact-mode imaging and force spectroscopy of this system allowed correlation of cell morphology and extracellular matrix (ECM) properties with substrate properties. Force spectroscopy analysis of cellular 'footprints' indicated that the cells secrete large polymers (e.g., 3.5 {mu}m contour length and estimated MW 1000 kDa) onto their substrate surface. Although definitive identification of the polymers has not yet been achieved, fluorescent-labeled antibody staining has specified the presence of ECM proteins such as collagen and laminin in the cellular footprints. The stretched polymers appear to be much larger than single molecules of known ECM components, such as collagen and heparan sulfate proteoglycan, thus suggesting that the cells create larger entangled, macromolecular structures from smaller components. There is strong evidence which suggests that the composition of the ECM is greatly influenced by the hydrophobicity of the substrate surface, with preferential production and/or adsorption of larger macromolecules on hydrophobic surfaces.

  10. The extracellular matrix of plants: Molecular, cellular and developmental biology

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-12-31

    A symposium entitled ``The Extracellular Matrix of Plants: Molecular, Cellular and Developmental Biology was held in Tamarron, Colorado, March 15--21, 1996. The following topics were explored in addresses by 43 speakers: structure and biochemistry of cell walls; biochemistry, molecular biology and biosynthesis of lignin; secretory pathway and synthesis of glycoproteins; biosynthesis of matrix polysaccharides, callose and cellulose; role of the extracellular matrix in plant growth and development; plant cell walls in symbiosis and pathogenesis.

  11. Pseudomonas aeruginosa extracellular products inhibit staphylococcal growth, and disrupt established biofilms produced by Staphylococcus epidermidis

    DEFF Research Database (Denmark)

    Qin, Zhiqiang; Yang, Liang; Qu, Di

    2009-01-01

    Multiple bacterial species often coexist as communities, and compete for environmental resources. Here, we describe how an opportunistic pathogen, Pseudomonas aeruginosa, uses extracellular products to interact with the nosocomial pathogen Staphylococcus epidermidis. S. epidermidis biofilms and p...... of a novel strategy for controlling S. epidermidis biofilms....

  12. Effects of ionizing radiation on extracellular matrix

    Energy Technology Data Exchange (ETDEWEB)

    Mohamed, F. [School of Physics, University of Exeter, Exeter EX44QL (United Kingdom)], E-mail: f.mohamed@ex.ac.uk; Bradley, D.A. [Department of Physics, University of Surrey, Guildford GU72XH (United Kingdom); Winlove, C.P. [School of Physics, University of Exeter, Exeter EX44QL (United Kingdom)

    2007-09-21

    The extracellular matrix is a ubiquitous and important component of tissues. We investigated the effects of ionizing radiation on the physical properties of its principal macromolecular components, pericardial collagen, ligament elastin and hyaluronan, a representative glycosaminoglycan. Samples were exposed to X-rays from an electron linear accelerator in the range of 10-100 Gy to cover the range of irradiation exposure during radiotherapy. A uniaxial mechanical testing protocol was used to characterize the fibrous proteins. For pericardial tissue the major change was an increase in the elastic modulus in the toe region of the curve ({<=}20% strain), from 23{+-}18 kPa for controls to 57{+-}22 kPa at a dose of 10 Gy (p=0.01, {alpha}=0.05). At larger strain ({>=}20% strain), the elastic modulus in the linear region decreased from 1.92{+-}0.70 MPa for control pericardium tissue to 1.31{+-}0.56 MPa (p=0.01, {alpha}=0.05) for 10 Gy X-irradiated sample. Similar observations have been made previously on tendon collagen at larger strains. For elastin, the stress-strain relationship was linear up to 30% strain, but the elastic modulus decreased significantly with irradiation (controls 626{+-}65 kPa, irradiated 474{+-}121 kPa (p=0.02, {alpha}=0.05), at 10 Gy X-irradiation). The results suggest that for collagen the primary effect of irradiation is generation of additional cross-links, while for elastin chain scissions are important. The viscosity of HA (at 1.25% w/v and 0.125% w/v) was measured by both cone and plate and capillary viscometry, the former providing measurement at uniform shear rate and the latter providing a more sensitive indication of changes at low viscosity. Both techniques revealed a dose-dependent reduction in viscosity (from 3400{+-}194 cP for controls to 1500{+-}88 cP at a shear rate of 2 s{sup -1} and dose of 75 Gy), again suggesting depolymerization.

  13. Effects of ionizing radiation on extracellular matrix

    International Nuclear Information System (INIS)

    Mohamed, F.; Bradley, D.A.; Winlove, C.P.

    2007-01-01

    The extracellular matrix is a ubiquitous and important component of tissues. We investigated the effects of ionizing radiation on the physical properties of its principal macromolecular components, pericardial collagen, ligament elastin and hyaluronan, a representative glycosaminoglycan. Samples were exposed to X-rays from an electron linear accelerator in the range of 10-100 Gy to cover the range of irradiation exposure during radiotherapy. A uniaxial mechanical testing protocol was used to characterize the fibrous proteins. For pericardial tissue the major change was an increase in the elastic modulus in the toe region of the curve (≤20% strain), from 23±18 kPa for controls to 57±22 kPa at a dose of 10 Gy (p=0.01, α=0.05). At larger strain (≥20% strain), the elastic modulus in the linear region decreased from 1.92±0.70 MPa for control pericardium tissue to 1.31±0.56 MPa (p=0.01, α=0.05) for 10 Gy X-irradiated sample. Similar observations have been made previously on tendon collagen at larger strains. For elastin, the stress-strain relationship was linear up to 30% strain, but the elastic modulus decreased significantly with irradiation (controls 626±65 kPa, irradiated 474±121 kPa (p=0.02, α=0.05), at 10 Gy X-irradiation). The results suggest that for collagen the primary effect of irradiation is generation of additional cross-links, while for elastin chain scissions are important. The viscosity of HA (at 1.25% w/v and 0.125% w/v) was measured by both cone and plate and capillary viscometry, the former providing measurement at uniform shear rate and the latter providing a more sensitive indication of changes at low viscosity. Both techniques revealed a dose-dependent reduction in viscosity (from 3400±194 cP for controls to 1500±88 cP at a shear rate of 2 s -1 and dose of 75 Gy), again suggesting depolymerization

  14. Extracellular matrix as a driver for lung regeneration.

    Science.gov (United States)

    Balestrini, Jenna L; Niklason, Laura E

    2015-03-01

    Extracellular matrix has manifold roles in tissue mechanics, guidance of cellular behavior, developmental biology, and regenerative medicine. Over the past several decades, various pre-clinical and clinical studies have shown that many connective tissues may be replaced and/or regenerated using suitable extracellular matrix scaffolds. More recently, decellularization of lung tissue has shown that gentle removal of cells can leave behind a "footprint" within the matrix that may guide cellular adhesion, differentiation and homing following cellular repopulation. Fundamental issues like understanding matrix composition and micro-mechanics remain difficult to tackle, largely because of a lack of available assays and tools for systematically characterizing intact matrix from tissues and organs. This review will critically examine the role of engineered and native extracellular matrix in tissue and lung regeneration, and provide insights into directions for future research and translation.

  15. Extracellular matrix scaffolds for cartilage and bone regeneration

    NARCIS (Netherlands)

    Benders, K.E.M.; van Weeren, P.R.; Badylak, S.F.; Saris, Daniël B.F.; Dhert, W.J.A.; Malda, J.

    2013-01-01

    Regenerative medicine approaches based on decellularized extracellular matrix (ECM) scaffolds and tissues are rapidly expanding. The rationale for using ECM as a natural biomaterial is the presence of bioactive molecules that drive tissue homeostasis and regeneration. Moreover, appropriately

  16. In vivo extracellular matrix protein expression by human periodontal ...

    African Journals Online (AJOL)

    ONOS

    2010-08-23

    Aug 23, 2010 ... Extracellular matrix proteins (ECM) are described as molecular regulators of these events. ..... zation and adhesive interaction of cells (Yamada, 1983). .... periodontal ligament fibroblasts after simulation of orthodontic force.

  17. The danger signal extracellular ATP is an inducer of Fusobacterium nucleatum biofilm dispersal

    Directory of Open Access Journals (Sweden)

    Qinfeng Ding

    2016-11-01

    Full Text Available Plaque biofilm is the primary etiological agent of periodontal disease. Biofilm formation progresses through multiple developmental stages beginning with bacterial attachment to a surface, followed by development of microcolonies and finally detachment and dispersal from a mature biofilm as free planktonic bacteria. Tissue damage arising from inflammatory response to biofilm is one of the hallmark features of periodontal disease. A consequence of tissue damage is the release of ATP from within the cell into the extracellular space. Extracellular ATP (eATP is an example of a danger associated molecular pattern (DAMP employed by mammalian cells to elicit inflammatory and damage healing responses. Although the roles of eATP as a signaling molecule in multi-cellular organisms have been relatively well studied, exogenous ATP also influences bacteria biofilm formation. Since plaque biofilms are continuously exposed to various stresses including exposure to the host damage factors eATP, we hypothesized that eATP, in addition to eliciting inflammation could potentially influence the biofilm lifecycle of periodontal associated bacteria. We found that eATP rather than nutritional factors or oxidative stress induced dispersal of Fusobacterium nucleatum, an organism associated with periodontal disease. eATP induced biofilm dispersal through chelating metal ions present in biofilm. Dispersed F. nucleatum biofilm, regardless of natural or induced dispersal by exogenous ATP, were significantly more adhesive and invasive compared to planktonic or biofilm counterparts, and correspondingly activated significantly more pro-inflammatory cytokine production in infected periodontal fibroblasts. Dispersed F. nucleatum also exhibited significantly higher expression of fadA, a virulence factor implicated in adhesion and invasion, compared to planktonic or biofilm bacteria. This study revealed for the first time that periodontal bacterium is capable of co-opting eATP, a

  18. Chemical Analysis of Cellular and Extracellular Carbohydrates of a Biofilm-Forming Strain Pseudomonas aeruginosa PA14

    Science.gov (United States)

    Coulon, Charlène; Vinogradov, Evgeny; Filloux, Alain; Sadovskaya, Irina

    2010-01-01

    Background Pseudomonas aeruginosa is a Gram-negative bacterium and an opportunistic pathogen, which causes persisting life-threatening infections in cystic fibrosis (CF) patients. Biofilm mode of growth facilitates its survival in a variety of environments. Most P. aeruginosa isolates, including the non-mucoid laboratory strain PA14, are able to form a thick pellicle, which results in a surface-associated biofilm at the air-liquid (A–L) interface in standing liquid cultures. Exopolysaccharides (EPS) are considered as key components in the formation of this biofilm pellicle. In the non-mucoid P. aeruginosa strain PA14, the “scaffolding” polysaccharides of the biofilm matrix, and the molecules responsible for the structural integrity of rigid A–L biofilm have not been identified. Moreover, the role of LPS in this process is unclear, and the chemical structure of the LPS O-antigen of PA14 has not yet been elucidated. Principal Findings In the present work we carried out a systematic analysis of cellular and extracellular (EC) carbohydrates of P. aeruginosa PA14. We also elucidated the chemical structure of the LPS O-antigen by chemical methods and 2-D NMR spectroscopy. Our results showed that it is composed of linear trisaccharide repeating units, identical to those described for P. aeruginosa Lanýi type O:2a,c (Lanýi-Bergman O-serogroup 10a, 10c; IATS serotype 19) and having the following structure: -4)-α-L-GalNAcA-(1–3)-α-D-QuiNAc-(1–3)- α-L-Rha-(1-. Furthermore, an EC O-antigen polysaccharide (EC O-PS) and the glycerol-phosphorylated cyclic β-(1,3)-glucans were identified in the culture supernatant of PA14, grown statically in minimal medium. Finally, the extracellular matrix of the thick biofilm formed at the A-L interface contained, in addition to eDNA, important quantities (at least ∼20% of dry weight) of LPS-like material. Conclusions We characterized the chemical structure of the LPS O-antigen and showed that the O-antigen polysaccharide is

  19. Ratiometric Imaging of Extracellular pH in Dental Biofilms

    DEFF Research Database (Denmark)

    Schlafer, Sebastian; Dige, Irene

    2016-01-01

    The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces...... the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms...... allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6...

  20. Fragmentation of extracellular matrix by hypochlorous acid

    DEFF Research Database (Denmark)

    Woods, Alan A; Davies, Michael Jonathan

    2003-01-01

    /chloramide decomposition, with copper and iron ions being effective catalysts, and decreased by compounds which scavenge chloramines/chloramides, or species derived from them. The effect of such matrix modifications on cellular behaviour is poorly understood, though it is known that changes in matrix materials can have...... profound effects on cell adhesion, proliferation, growth and phenotype. The observed matrix modifications reported here may therefore modulate cellular behaviour in diseases such as atherosclerosis where MPO-derived oxidants are generated....

  1. Paparan zat besi pada ekspresi protein spesifik extracellular polymeric substance biofilm Aggregatibacter actinomycetemcomitans

    Directory of Open Access Journals (Sweden)

    Marchella Hendrayanti W

    2014-06-01

    Full Text Available Background: The study of biofilms bacteria could be an alternative of preventive treatment in reducing prevalence of aggressive periodontitis in the community, because biofilm protects the bacteria from environmental conditions, including the attack of immune system and antimicrobial. Aggregatibacter actinomycetemcomitans is a major cause of bacterial aggressive periodontitis. Purpose: This study aims to examine the iron exposure to specific protein expression of extracellular polymeric substance (EPS of Aggregatibacter actinomycetemcomitans biofilm. Methods: Protein containing EPS biofilm was isolated from cultures of A.actinomycetemcomitans. The protein was processed through several procedures: electrophoresis , electroelution , immunization of rabbits , serum isolation , and purification of antibodies. After the Western blotting procedure the antibody was used. Protein containing EPS biofilms exposed to iron, then once again isolated from cultures of A. actinomycetemcomitans. The electrophoresis and Western blotting were done on the isolated protein. Results: The result showed that the the expression of specific proteins in EPS biofilm decreased in response to iron exposure. Conclusions: Iron exposure could influenced the specific protein expression in EPS biofilm of Aggregatibacter actinomycetemcomitans.Latar belakang: Penelitian terhadap bakteri biofilm dapat menjadi alternatif perawatan preventif dalam menurunkan prevalensi periodontitis agresif di masyarakat, karena biofilm melindungi bakteri terhadap kondisi lingkungan, termasuk serangan sistem imun dan antimikroba. Aggregatibacter actinomycetemcomitans merupakan bakteri penyebab utama periodontitis agresif. Tujuan: Studi ini bertujuan meneliti paparan zat besi terhadap ekspresi protein spesifik extracellular polymeric substance (EPS Aggregatibacter actinomycetemcomitans. Metode: Protein yang mengandung EPS biofilm diisolasi dari kultur A. actinomycetemcomitans. Protein yang diisolasi

  2. Inhibitory effects of extracellular polymeric substances on ofloxacin sorption by natural biofilms.

    Science.gov (United States)

    Zhang, Liwen; Dong, Deming; Hua, Xiuyi; Guo, Zhiyong

    2018-06-01

    Natural biofilms have strong affinities for organic contaminants, and their extracellular polymeric substances (EPS) have been thought to control the sorption process. However, the role of EPS in the sorption of antibiotics, an emerging concern, is poorly understood. Here, soluble (SEPS) and bound EPS (BEPS) were extracted from intact biofilms incubated at different lengths of time to obtain SEPS- and BEPS-free biofilms. Batch sorption experiments and infrared spectroscopy were used to investigate the role of EPS in the sorption of ofloxacin (OFL) by natural biofilms. The sorption capacities of OFL onto intact biofilms were lower than that those onto SEPS-free and BEPS-free biofilms. Partition and Langmuir adsorption contributed to the sorption of OFL onto these biofilms. SEPS and BEPS suppressed partitioning of OFL into biofilm organic matter. Meanwhile, the formation of hydrogen bonds could affect the Langmuir adsorption of OFL onto BEPS-free biofilms. These sorption mechanisms occurred simultaneously and enhanced the sorption capacities of biofilms after EPS removal. The information obtained in this study is beneficial for understanding the interaction mechanisms between antibiotics and natural biofilms. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Integration of concepts: cardiac extracellular matrix remodeling after myocardial infarction

    NARCIS (Netherlands)

    Cleutjens, Jack P. M.; Creemers, Esther E. J. M.

    2002-01-01

    The cardiac extracellular matrix consists of a three-dimensional structural network of interstitial collagens to which other matrix components are attached. The main physiological functions of this network are to retain tissue integrity and cardiac pump function. Collagen deposition is controlled

  4. Binding of matrix metalloproteinase inhibitors to extracellular matrix: 3D-QSAR analysis.

    Science.gov (United States)

    Zhang, Yufen; Lukacova, Viera; Bartus, Vladimir; Nie, Xiaoping; Sun, Guorong; Manivannan, Ethirajan; Ghorpade, Sandeep R; Jin, Xiaomin; Manyem, Shankar; Sibi, Mukund P; Cook, Gregory R; Balaz, Stefan

    2008-10-01

    Binding to the extracellular matrix, one of the most abundant human protein complexes, significantly affects drug disposition. Specifically, the interactions with extracellular matrix determine the free concentrations of small molecules acting in tissues, including signaling peptides, inhibitors of tissue remodeling enzymes such as matrix metalloproteinases, and other drug candidates. The nature of extracellular matrix binding was elucidated for 63 matrix metalloproteinase inhibitors, for which the association constants to an extracellular matrix mimic were reported here. The data did not correlate with lipophilicity as a common determinant of structure-nonspecific, orientation-averaged binding. A hypothetical structure of the binding site of the solidified extracellular matrix surrogate was analyzed using the Comparative Molecular Field Analysis, which needed to be applied in our multi-mode variant. This fact indicates that the compounds bind to extracellular matrix in multiple modes, which cannot be considered as completely orientation-averaged and exhibit structural dependence. The novel comparative molecular field analysis models, exhibiting satisfactory descriptive and predictive abilities, are suitable for prediction of the extracellular matrix binding for the untested chemicals, which are within applicability domains. The results contribute to a better prediction of the pharmacokinetic parameters such as the distribution volume and the tissue-blood partition coefficients, in addition to a more imminent benefit for the development of more effective matrix metalloproteinase inhibitors.

  5. Ratiometric Imaging of Extracellular pH in Dental Biofilms Using C-SNARF-4

    DEFF Research Database (Denmark)

    Dige, Irene

    pH in dental biofilms plays a central role for the development of caries lesions. For decades, pH measurements in biofilms have been limited to recording pH with electrodes/microelectrodes that do not permit monitoring horizontal pH gradients in biofilms in real-time. Quantitative fluorescent...... microscopy can overcome these problems. Objective: The aim of this demonstration study was to monitor extracellular biofilm pH microscopically with the ratiometric pH-sensitive dye C-SNARF-4 in in-situ-grown dental biofilms. Methods: Using confocal microscopy, the dye C-SNARF-4 was employed both as p...... the microscopic images in order to exclusively determine extracellular pH. We monitored the pH drop at the biofilm-substratum interface in six microscopic fields of view per biofilm for 1h after exposure to 0.4% glucose. Results: Extracellular pH dropped rapidly in all specimens. In both individuals, analysis...

  6. Identification of Bicarbonate as a Trigger and Genes Involved with Extracellular DNA Export in Mycobacterial Biofilms

    Directory of Open Access Journals (Sweden)

    Sasha J. Rose

    2016-12-01

    Full Text Available Extracellular DNA (eDNA is an integral biofilm matrix component of numerous pathogens, including nontuberculous mycobacteria (NTM. Cell lysis is the source of eDNA in certain bacteria, but the source of eDNA remains unidentified for NTM, as well as for other eDNA-containing bacterial species. In this study, conditions affecting eDNA export were examined, and genes involved with the eDNA export mechanism were identified. After a method for monitoring eDNA in real time in undisturbed biofilms was established, different conditions affecting eDNA were investigated. Bicarbonate positively influenced eDNA export in a pH-independent manner in Mycobacterium avium, M. abscessus, and M. chelonae. The surface-exposed proteome of M. avium in eDNA-containing biofilms revealed abundant carbonic anhydrases. Chemical inhibition of carbonic anhydrases with ethoxzolamide significantly reduced eDNA export. An unbiased transposon mutant library screen for eDNA export in M. avium identified many severely eDNA-attenuated mutants, including one not expressing a unique FtsK/SpoIIIE-like DNA-transporting pore, two with inactivation of carbonic anhydrases, and nine with inactivation of genes belonging to a unique genomic region, as well as numerous mutants involved in metabolism and energy production. Complementation of nine mutants that included the FtsK/SpoIIIE and carbonic anhydrase significantly restored eDNA export. Interestingly, several attenuated eDNA mutants have mutations in genes encoding proteins that were found with the surface proteomics, and many more mutations are localized in operons potentially encoding surface proteins. Collectively, our data strengthen the evidence of eDNA export being an active mechanism that is activated by the bacterium responding to bicarbonate.

  7. Syndecans as receptors and organizers of the extracellular matrix

    DEFF Research Database (Denmark)

    Xian, Xiaojie; Gopal, Sandeep; Couchman, John

    2009-01-01

    , the collagens and glycoproteins of the extracellular matrix are prominent. Frequently, they do so in conjunction with other receptors, most notably the integrins. For this reason, they are often referred to as "co-receptors". However, just as with integrins, syndecans can interact with actin-associated proteins...... and signalling molecules, such as protein kinases. Some aspects of syndecan signalling are understood but much remains to be learned. The functions of syndecans in regulating cell adhesion and extracellular matrix assembly are described here. Evidence from null mice suggests that syndecans have roles...

  8. TOL plasmid carriage enhances biofilm formation and increases extracellular DNA content in Pseudomonas putida KT2440

    DEFF Research Database (Denmark)

    D'Alvise, Paul; Sjoholm, O.R.; Yankelevich, T.

    2010-01-01

    laser scanning microscopy. The TOL-carrying strains formed pellicles and thick biofilms, whereas the same strains without the plasmid displayed little adherent growth. Microscopy using fluorescent nucleic acid-specific stains revealed differences in the production of extracellular polymeric substances......: TOL carriage leads to more extracellular DNA (eDNA) in pellicles and biofilms. Pellicles were dissolved by DNase I treatment. Enhanced cell lysis due to plasmid carriage was ruled out as the mechanism for eDNA release. We report, for the first time, that carriage of a conjugative plasmid leads...

  9. Ciliates as engineers of phototrophic biofilms

    NARCIS (Netherlands)

    Weerman, Ellen J.; van der Geest, Harm G.; van der Meulen, Myra D.; Manders, Erik M. M.; van de Koppel, Johan; Herman, Peter M. J.; Admiraal, Wim

    1. Phototrophic biofilms consist of a matrix of phototrophs, non-photosynthetic bacteria and extracellular polymeric substances (EPS) which is spatially structured. Despite widespread exploitation of algae and bacteria within phototrophic biofilms, for example by protozoans, the 'engineering'

  10. An immunofluorescence assay for extracellular matrix components highlights the role of epithelial cells in producing a stable, fibrillar extracellular matrix

    Directory of Open Access Journals (Sweden)

    Omar S. Qureshi

    2017-10-01

    Full Text Available Activated fibroblasts are considered major drivers of fibrotic disease progression through the production of excessive extracellular matrix (ECM in response to signals from damaged epithelial and inflammatory cells. Nevertheless, epithelial cells are capable of expressing components of the ECM, cross-linking enzymes that increase its stability and are sensitive to factors involved in the early stages of fibrosis. We therefore wanted to test the hypothesis that epithelial cells can deposit ECM in response to stimulation in a comparable manner to fibroblasts. We performed immunofluorescence analysis of components of stable, mature extracellular matrix produced by primary human renal proximal tubular epithelial cells and renal fibroblasts in response to cytokine stimulation. Whilst fibroblasts produced a higher basal level of extracellular matrix components, epithelial cells were able to deposit significant levels of fibronectin, collagen I, III and IV in response to cytokine stimulation. In response to hypoxia, epithelial cells showed an increase in collagen IV deposition but not in response to the acute stress stimuli aristolochic acid or hydrogen peroxide. When epithelial cells were in co-culture with fibroblasts we observed significant increases in the level of matrix deposition which could be reduced by transforming growth factor beta (TGF-β blockade. Our results highlight the role of epithelial cells acting as efficient producers of stable extracellular matrix which could contribute to renal tubule thickening in fibrosis.

  11. Extracellular matrix proteins: a positive feedback loop in lung fibrosis?

    NARCIS (Netherlands)

    Blaauboer, M.E.; van Boeijen, F.R.; Emson, C.L.; Turner, S.M.; Zandieh-Doulabi, B.; Hanemaaijer, R.; Smit, T.H.; Stoop, R.; Everts, V.

    2014-01-01

    Lung fibrosis is characterized by excessive deposition of extracellular matrix. This not only affects tissue architecture and function, but it also influences fibroblast behavior and thus disease progression. Here we describe the expression of elastin, type V collagen and tenascin C during the

  12. Extracellular matrix proteins: A positive feedback loop in lung fibrosis?

    NARCIS (Netherlands)

    Blaauboer, M.E.; Boeijen, F.R.; Emson, C.L.; Turner, S.M.; Zandieh-Doulabi, B.; Hanemaaijer, R.; Smit, T.H.; Stoop, R.; Everts, V.

    2014-01-01

    Lung fibrosis is characterized by excessive deposition of extracellular matrix. This not only affects tissue architecture and function, but it also influences fibroblast behavior and thus disease progression. Here we describe the expression of elastin, type V collagen and tenascin C during the

  13. In vivo extracellular matrix protein expression by human periodontal ...

    African Journals Online (AJOL)

    It is well known that the orthodontic force applied to teeth generates a series of events that remodel the periodontal ligament (PDL). Extracellular matrix proteins (ECM) are described as molecular regulators of these events. However, the exact contribution of these proteins in human PDL modeling by orthodontic force ...

  14. Control of extracellular matrix assembly by syndecan-2 proteoglycan

    DEFF Research Database (Denmark)

    Klass, C M; Couchman, J R; Woods, A

    2000-01-01

    Extracellular matrix (ECM) deposition and organization is maintained by transmembrane signaling and integrins play major roles. We now show that a second transmembrane component, syndecan-2 heparan sulfate proteoglycan, is pivotal in matrix assembly. Chinese Hamster Ovary (CHO) cells were stably...... to rearrange laminin or fibronectin substrates into fibrils and to bind exogenous fibronectin. Transfection of activated alphaIIbalphaLdeltabeta3 integrin into alpha(5)-deficient CHO B2 cells resulted in reestablishment of the previously lost fibronectin matrix. However, cotransfection of this cell line with S...

  15. How does the extracellular matrix direct gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Bissell, M J; Hall, H G; Parry, G

    1982-01-01

    Based on the existing literature, a model is presented that postulates a ''dynamic reciprocity'' between the extracellular matrix (ECM) on the one hand and the cytoskeleton and the nuclear matrix on the other hand. The ECM is postulated to exert physical and chemical influences on the geometry and the biochemistry of the cell via transmembrane receptors so as to alter the pattern of gene expression by changing the association of the cytoskeleton with mRNA and the interaction of the chromatin with the nuclear matrix. This, in turn, would affect the ECM, which would affect the cell.

  16. Isolation of Extracellular Polymeric Substances from Biofilms of the Thermoacidophilic Archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Jachlewski, Silke; Jachlewski, Witold D; Linne, Uwe; Bräsen, Christopher; Wingender, Jost; Siebers, Bettina

    2015-01-01

    Extracellular polymeric substances (EPS) are the major structural and functional components of microbial biofilms. The aim of this study was to establish a method for EPS isolation from biofilms of the thermoacidophilic archaeon, Sulfolobus acidocaldarius, as a basis for EPS analysis. Biofilms of S. acidocaldarius were cultivated on the surface of gellan gum-solidified Brock medium at 78°C for 4 days. Five EPS extraction methods were compared, including shaking of biofilm suspensions in phosphate buffer, cation-exchange resin (CER) extraction, and stirring with addition of EDTA, crown ether, or NaOH. With respect to EPS yield, impact on cell viability, and compatibility with subsequent biochemical analysis, the CER extraction method was found to be the best suited isolation procedure resulting in the detection of carbohydrates and proteins as the major constituents and DNA as a minor component of the EPS. Culturability of CER-treated cells was not impaired. Analysis of the extracellular proteome using two-dimensional gel electrophoresis resulted in the detection of several hundreds of protein spots, mainly with molecular masses of 25-116 kDa and pI values of 5-8. Identification of proteins suggested a cytoplasmic origin for many of these proteins, possibly released via membrane vesicles or biofilm-inherent cell lysis during biofilm maturation. Functional analysis of EPS proteins, using fluorogenic substrates as well as zymography, demonstrated the activity of diverse enzyme classes, such as proteases, lipases, esterases, phosphatases, and glucosidases. In conclusion, the CER extraction method, as previously applied to bacterial biofilms, also represents a suitable method for isolation of water soluble EPS from the archaeal biofilms of S. acidocaldarius, allowing the investigation of composition and function of EPS components in these types of biofilms.

  17. Isolation of extracellular polymeric substances from biofilms of the thermoacidophilic archaeon Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Silke eJachlewski

    2015-08-01

    Full Text Available Extracellular polymeric substances (EPS are the major structural and functional components of microbial biofilms. The aim of this study was to establish a method for EPS isolation from biofilms of the thermoacidophilic archaeon Sulfolobus acidocaldarius as a basis for EPS analysis. Biofilms of S. acidocaldarius were cultivated on the surface of gellan gum-solidified Brock medium at 78 °C for 4 days. Five EPS extraction methods were compared, including shaking of biofilm suspensions in phosphate buffer, cation-exchange resin (CER extraction and stirring with addition of EDTA, crown ether or NaOH. With respect to EPS yield, impact on cell viability and compatibility with subsequent biochemical analysis, the CER extraction method was found to be the best suited isolation procedure resulting in the detection of carbohydrates and proteins as the major constituents and DNA as a minor component of the EPS. Culturability of CER-treated cells was not impaired. Analysis of the extracellular proteome using two-dimensional gel electrophoresis resulted in the detection of several hundredshundred of protein spots, mainly with molecular masses of 25 kDa to 116 kDa and pI values of 5 to 8. Identification of proteins suggested a cytoplasmic origin for many of these proteins, possibly released via membrane vesicles or biofilm-inherent cell lysis during biofilm maturation. Functional analysis of EPS proteins, using fluorogenic substrates as well as zymography, demonstrated the activity of diverse groups of enzymes such as proteases, lipases, esterases, phosphatases and glucosidases. In conclusion, the CER extraction method, as previously applied to bacterial biofilms, also represents a suitable method for isolation of water soluble EPS from the archaeal biofilms of S. acidocaldarius, allowing the investigation of composition and function of EPS components in these types of biofilms.

  18. Specialisation of extracellular matrix for function in tendons and ligaments

    Science.gov (United States)

    Birch, Helen L.; Thorpe, Chavaunne T.; Rumian, Adam P.

    2013-01-01

    Summary Tendons and ligaments are similar structures in terms of their composition, organisation and mechanical properties. The distinction between them stems from their anatomical location; tendons form a link between muscle and bone while ligaments link bones to bones. A range of overlapping functions can be assigned to tendon and ligaments and each structure has specific mechanical properties which appear to be suited for particular in vivo function. The extracellular matrix in tendon and ligament varies in accordance with function, providing appropriate mechanical properties. The most useful framework in which to consider extracellular matrix differences therefore is that of function rather than anatomical location. In this review we discuss what is known about the relationship between functional requirements, structural properties from molecular to gross level, cellular gene expression and matrix turnover. The relevance of this information is considered by reviewing clinical aspects of tendon and ligament repair and reconstructive procedures. PMID:23885341

  19. Extracellular matrix in canine mammary tumors with special focus on versican, a versatile extracellular proteoglycan

    NARCIS (Netherlands)

    Erdélyi, Ildikó

    2006-01-01

    The extracellular matrix (ECM) research has become fundamental to understand cancer. This thesis focuses on the exploration of ECM composition and organization in canine mammary tumors, with a special interest in the large chondroitin-sulfate proteoglycan (PG), versican. Chapter 1 gives an

  20. Extracellular DNA formation during biofilm development by freshwater bacteria

    DEFF Research Database (Denmark)

    Tang, Lone; Schramm, Andreas; Revsbech, Niels Peter

    2011-01-01

    a transient peak at 6 hours, and in Rheinheimera the concentration peaked at 12 hours and remained high. Interestingly, the Rheinheimera biofilm dispersed immediately after the eDNA concentration peaked. The antimicrobial effect of eDNA was tested in growth experiments, and Rheinheimera was strongly affected...

  1. Roles of Extracellular Polysaccharides and Biofilm Formation in Heavy Metal Resistance of Rhizobia

    OpenAIRE

    Natalia Nocelli; Pablo C. Bogino; Erika Banchio; Walter Giordano

    2016-01-01

    Bacterial surface components and extracellular compounds, particularly flagella, lipopolysaccharides (LPSs), and exopolysaccharides (EPSs), in combination with environmental signals and quorum-sensing signals, play crucial roles in bacterial autoaggregation, biofilm development, survival, and host colonization. The nitrogen-fixing species Sinorhizobium meliloti (S. meliloti) produces two symbiosis-promoting EPSs: succinoglycan (or EPS I) and galactoglucan (or EPS II). Studies of the S. melilo...

  2. Extracellular matrix components direct porcine muscle stem cell behavior

    International Nuclear Information System (INIS)

    Wilschut, Karlijn J.; Haagsman, Henk P.; Roelen, Bernard A.J.

    2010-01-01

    In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatin and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.

  3. Extracellular matrix components direct porcine muscle stem cell behavior

    Energy Technology Data Exchange (ETDEWEB)

    Wilschut, Karlijn J. [Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 104, 3584 CM, Utrecht (Netherlands); Haagsman, Henk P. [Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL, Utrecht (Netherlands); Roelen, Bernard A.J., E-mail: b.a.j.roelen@uu.nl [Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 104, 3584 CM, Utrecht (Netherlands)

    2010-02-01

    In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatin and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.

  4. The Extracellular Matrix Regulates Granuloma Necrosis in Tuberculosis.

    Science.gov (United States)

    Al Shammari, Basim; Shiomi, Takayuki; Tezera, Liku; Bielecka, Magdalena K; Workman, Victoria; Sathyamoorthy, Tarangini; Mauri, Francesco; Jayasinghe, Suwan N; Robertson, Brian D; D'Armiento, Jeanine; Friedland, Jon S; Elkington, Paul T

    2015-08-01

    A central tenet of tuberculosis pathogenesis is that caseous necrosis leads to extracellular matrix destruction and bacterial transmission. We reconsider the underlying mechanism of tuberculosis pathology and demonstrate that collagen destruction may be a critical initial event, causing caseous necrosis as opposed to resulting from it. In human tuberculosis granulomas, regions of extracellular matrix destruction map to areas of caseous necrosis. In mice, transgenic expression of human matrix metalloproteinase 1 causes caseous necrosis, the pathological hallmark of human tuberculosis. Collagen destruction is the principal pathological difference between humanised mice and wild-type mice with tuberculosis, whereas the release of proinflammatory cytokines does not differ, demonstrating that collagen breakdown may lead to cell death and caseation. To investigate this hypothesis, we developed a 3-dimensional cell culture model of tuberculosis granuloma formation, using bioelectrospray technology. Collagen improved survival of Mycobacterium tuberculosis-infected cells analyzed on the basis of a lactate dehydrogenase release assay, propidium iodide staining, and measurement of the total number of viable cells. Taken together, these findings suggest that collagen destruction is an initial event in tuberculosis immunopathology, leading to caseous necrosis and compromising the immune response, revealing a previously unappreciated role for the extracellular matrix in regulating the host-pathogen interaction. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  5. Extracellular polymeric substances affect the responses of multi-species biofilms in the presence of sulfamethizole.

    Science.gov (United States)

    Wang, Longfei; Li, Yi; Wang, Li; Zhang, Huanjun; Zhu, Mengjie; Zhang, Peisheng; Zhu, Xiaoxiao

    2018-04-01

    The occurrence and transportation of antibiotics in biofilms from natural and engineered sources have attracted increasing interests. Nevertheless, the effects of extracellular polymeric substances (EPS) on the responses of biofilms to the exposure to antibiotics are not clear. In this study, the effects of EPS on the sorption and biological responses to one representative antibiotic, sulfamethizole (STZ), in model biofilms were investigated. Proteins dominated the interactions between the EPS and the STZ and the EPS from a moving bed biofilm reactor exhibited the strongest interaction with the STZ. The EPS served as important reservoirs for the STZ and the tested biofilms all showed reduced sorption capacities for the STZ after the EPS were extracted. The respiratory rates and typical enzymatic activities were reduced after the EPS were extracted. High-throughput 16S rRNA gene sequencing results confirmed that the bacterial community in the biofilm without the EPS was more vulnerable to antibiotic shock as indicated by the community diversity and richness indices. A greater increase in the abundance of susceptible species was observed in the natural biofilm. The results comprehensively suggested that the EPS played important role in biosorption of STZ and alleviated the direct damage of the antibiotic to the cells; in addition the extent of the bacterial community response was associated with the origins of the biofilms. Our study provided details on the responses of multi-species biofilms to the exposure to an antibiotic and highlighted the role of the EPS in interacting with the antibiotic, thereby providing a deeper understanding of the bioremediation of antibiotics in real-life natural and engineered biofilm systems. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Enterococcus faecium biofilm formation: identification of major autolysin AtlAEfm, associated Acm surface localization, and AtlAEfm-independent extracellular DNA Release.

    Science.gov (United States)

    Paganelli, Fernanda L; Willems, Rob J L; Jansen, Pamela; Hendrickx, Antoni; Zhang, Xinglin; Bonten, Marc J M; Leavis, Helen L

    2013-04-16

    Enterococcus faecium is an important multidrug-resistant nosocomial pathogen causing biofilm-mediated infections in patients with medical devices. Insight into E. faecium biofilm pathogenesis is pivotal for the development of new strategies to prevent and treat these infections. In several bacteria, a major autolysin is essential for extracellular DNA (eDNA) release in the biofilm matrix, contributing to biofilm attachment and stability. In this study, we identified and functionally characterized the major autolysin of E. faecium E1162 by a bioinformatic genome screen followed by insertional gene disruption of six putative autolysin genes. Insertional inactivation of locus tag EfmE1162_2692 resulted in resistance to lysis, reduced eDNA release, deficient cell attachment, decreased biofilm, decreased cell wall hydrolysis, and significant chaining compared to that of the wild type. Therefore, locus tag EfmE1162_2692 was considered the major autolysin in E. faecium and renamed atlAEfm. In addition, AtlAEfm was implicated in cell surface exposure of Acm, a virulence factor in E. faecium, and thereby facilitates binding to collagen types I and IV. This is a novel feature of enterococcal autolysins not described previously. Furthermore, we identified (and localized) autolysin-independent DNA release in E. faecium that contributes to cell-cell interactions in the atlAEfm mutant and is important for cell separation. In conclusion, AtlAEfm is the major autolysin in E. faecium and contributes to biofilm stability and Acm localization, making AtlAEfm a promising target for treatment of E. faecium biofilm-mediated infections. IMPORTANCE Nosocomial infections caused by Enterococcus faecium have rapidly increased, and treatment options have become more limited. This is due not only to increasing resistance to antibiotics but also to biofilm-associated infections. DNA is released in biofilm matrix via cell lysis, caused by autolysin, and acts as a matrix stabilizer. In this study

  7. The extracellular matrix and altered diffusion in focal cortical dysplasia

    Czech Academy of Sciences Publication Activity Database

    Homola, Aleš; Vargová, Lýdia; Cicanič, Michal; Zámečník, J.; Marusič, P.; Kršek, P.; Syková, Eva

    2011-01-01

    Roč. 59, S1 (2011), S106-S106 ISSN 0894-1491. [European meeting on Glia l Cells in Health and Disease /10./. 13.09.2011-17.09.2011, Prague] R&D Projects: GA MŠk 1M0538; GA ČR GA309/09/1597 Institutional research plan: CEZ:AV0Z50390703 Keywords : focal cortical dysplasia * diffusion * extracellular matrix Subject RIV: FH - Neurology

  8. Extracellular matrix organization modulates fibroblast growth and growth factor responsiveness.

    Science.gov (United States)

    Nakagawa, S; Pawelek, P; Grinnell, F

    1989-06-01

    To learn more about the relationship between extracellular matrix organization, cell shape, and cell growth control, we studied DNA synthesis by fibroblasts in collagen gels that were either attached to culture dishes or floating in culture medium during gel contraction. After 4 days of contraction, the collagen density (initially 1.5 mg/ml) reached 22 mg/ml in attached gels and 55 mg/ml in floating gels. After contraction, attached collagen gels were well organized; collagen fibrils were aligned in the plane of cell spreading; and fibroblasts had an elongated, bipolar morphology. Floating collagen gels, however, were unorganized; collagen fibrils were arranged randomly; and fibroblasts had a stellate morphology. DNA synthesis by fibroblasts in contracted collagen gels was suppressed if the gels were floating in medium but not if the gels were attached, and inhibition was independent of the extent of gel contraction. Therefore, growth of fibroblasts in contracted collagen gels could be regulated by differences in extracellular matrix organization and cell shape independently of extracellular matrix density. We also compared the responses of fibroblasts in contracted collagen gels and monolayer culture to peptide growth factors including fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta, and interleukin 1. Cells in floating collagen gels were generally unresponsive to any of the growth factors. Cells in attached collagen gels and monolayer culture were affected similarly by fibroblast growth factor but not by the others. Our results indicate that extracellular matrix organization influenced not only cell growth, but also fibroblast responsiveness to peptide growth factors.

  9. Intermolecular interactions of thrombospondins drive their accumulation in extracellular matrix

    OpenAIRE

    Kim, Dae Joong; Christofidou, Elena D.; Keene, Douglas R.; Hassan Milde, Marwah; Adams, Josephine C.

    2015-01-01

    Thrombospondins participate in many aspects of tissue organization in adult tissue homeostasis, and their dysregulation contributes to pathological processes such as fibrosis and tumor progression. The incorporation of thrombospondins into extracellular matrix (ECM) as discrete puncta has been documented in various tissue and cell biological contexts, yet the underlying mechanisms remain poorly understood. We find that collagen fibrils are disorganized in multiple tissues of Thbs1 −/− mice. I...

  10. Accelerated extracellular matrix turnover during exacerbations of COPD

    DEFF Research Database (Denmark)

    Sand, Jannie M B; Knox, Alan J; Lange, Peter

    2015-01-01

    progression. Extracellular matrix (ECM) turnover reflects activity in tissues and consequently assessment of ECM turnover may serve as biomarkers of disease activity. We hypothesized that the turnover of lung ECM proteins were altered during exacerbations of COPD. METHODS: 69 patients with COPD hospitalised...... of circulating fragments of structural proteins, which may serve as markers of disease activity. This suggests that patients with COPD have accelerated ECM turnover during exacerbations which may be related to disease progression....

  11. "Tipping" extracellular matrix remodeling towards regression of liver fibrosis

    DEFF Research Database (Denmark)

    Magdaleno, Fernando; Schierwagen, Robert; Uschner, Frank E

    2018-01-01

    Fibrosis development was initially conceived as an incessant progressive condition. Nowadays, it has become evident that fibrotic tissue undergoes a continuous two-way process: fibrogenesis and fibrinolysis, characterizing the remodeling of extracellular matrix (ECM). However, in established...... fibrosis, this two-way process is tipped towards fibrogenesis and this leads to a self-perpetuating accumulation of ECM, a distinct metabolic unit, together with other cells and processes promoting fibrosis deposition. Several mechanisms promote fibrosis regression, such as degradation of ECM, infiltration...

  12. DMPD: Fragments of extracellular matrix as mediators of inflammation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18243041 Fragments of extracellular matrix as mediators of inflammation. Adair-Kirk...l) Show Fragments of extracellular matrix as mediators of inflammation. PubmedID 18243041 Title Fragments of... extracellular matrix as mediators of inflammation. Authors Adair-Kirk TL, Senior

  13. Incorporation of Tenascin-C into the Extracellular Matrix by Periostin Underlies an Extracellular Meshwork Architecture*

    OpenAIRE

    Kii, Isao; Nishiyama, Takashi; Li, Minqi; Matsumoto, Ken-ichi; Saito, Mitsuru; Amizuka, Norio; Kudo, Akira

    2009-01-01

    Extracellular matrix (ECM) underlies a complicated multicellular architecture that is subjected to significant forces from mechanical environment. Although various components of the ECM have been enumerated, mechanisms that evolve the sophisticated ECM architecture remain to be addressed. Here we show that periostin, a matricellular protein, promotes incorporation of tenascin-C into the ECM and organizes a meshwork architecture of the ECM. We found that both periostin null mice and tenascin-C...

  14. Intestinal Stem Cell Niche: The Extracellular Matrix and Cellular Components

    Directory of Open Access Journals (Sweden)

    Laween Meran

    2017-01-01

    Full Text Available The intestinal epithelium comprises a monolayer of polarised columnar cells organised along the crypt-villus axis. Intestinal stem cells reside at the base of crypts and are constantly nourished by their surrounding niche for maintenance, self-renewal, and differentiation. The cellular microenvironment including the adjacent Paneth cells, stromal cells, smooth muscle cells, and neural cells as well as the extracellular matrix together constitute the intestinal stem cell niche. A dynamic regulatory network exists among the epithelium, stromal cells, and the matrix via complex signal transduction to maintain tissue homeostasis. Dysregulation of these biological or mechanical signals could potentially lead to intestinal injury and disease. In this review, we discuss the role of different intestinal stem cell niche components and dissect the interaction between dynamic matrix factors and regulatory signalling during intestinal stem cell homeostasis.

  15. Association of Bordetella dermonecrotic toxin with the extracellular matrix

    Directory of Open Access Journals (Sweden)

    Miyake Masami

    2010-09-01

    Full Text Available Abstract Background Bordetella dermonecrotic toxin (DNT causes the turbinate atrophy in swine atrophic rhinitis, caused by a Bordetella bronchiseptica infection of pigs, by inhibiting osteoblastic differentiation. The toxin is not actively secreted from the bacteria, and is presumed to be present in only small amounts in infected areas. How such small amounts can affect target tissues is unknown. Results Fluorescence microscopy revealed that DNT associated with a fibrillar structure developed on cultured cells. A cellular component cross-linked with DNT conjugated with a cross-linker was identified as fibronectin by mass spectrometry. Colocalization of the fibronectin network on the cells with DNT was also observed by fluorescence microscope. Several lines of evidence suggested that DNT interacts with fibronectin not directly, but through another cellular component that remains to be identified. The colocalization was observed in not only DNT-sensitive cells but also insensitive cells, indicating that the fibronectin network neither serves as a receptor for the toxin nor is involved in the intoxicating procedures. The fibronectin network-associated toxin was easily liberated when the concentration of toxin in the local environment decreased, and was still active. Conclusions Components in the extracellular matrix are known to regulate activities of various growth factors by binding and liberating them in response to alterations in the extracellular environment. Similarly, the fibronectin-based extracellular matrix may function as a temporary storage system for DNT, enabling small amounts of the toxin to efficiently affect target tissues or cells.

  16. Conservation and Divergence in the Candida Species Biofilm Matrix Mannan-Glucan Complex Structure, Function, and Genetic Control.

    Science.gov (United States)

    Dominguez, Eddie; Zarnowski, Robert; Sanchez, Hiram; Covelli, Antonio S; Westler, William M; Azadi, Parastoo; Nett, Jeniel; Mitchell, Aaron P; Andes, David R

    2018-04-03

    Candida biofilms resist the effects of available antifungal therapies. Prior studies with Candida albicans biofilms show that an extracellular matrix mannan-glucan complex (MGCx) contributes to antifungal sequestration, leading to drug resistance. Here we implement biochemical, pharmacological, and genetic approaches to explore a similar mechanism of resistance for the three most common clinically encountered non- albicans Candida species (NAC). Our findings reveal that each Candida species biofilm synthesizes a mannan-glucan complex and that the antifungal-protective function of this complex is conserved. Structural similarities extended primarily to the polysaccharide backbone (α-1,6-mannan and β-1,6-glucan). Surprisingly, biochemical analysis uncovered stark differences in the branching side chains of the MGCx among the species. Consistent with the structural analysis, similarities in the genetic control of MGCx production for each Candida species also appeared limited to the synthesis of the polysaccharide backbone. Each species appears to employ a unique subset of modification enzymes for MGCx synthesis, likely accounting for the observed side chain diversity. Our results argue for the conservation of matrix function among Candida spp. While biogenesis is preserved at the level of the mannan-glucan complex backbone, divergence emerges for construction of branching side chains. Thus, the MGCx backbone represents an ideal drug target for effective pan- Candida species biofilm therapy. IMPORTANCE Candida species, the most common fungal pathogens, frequently grow as a biofilm. These adherent communities tolerate extremely high concentrations of antifungal agents, due in large part, to a protective extracellular matrix. The present studies define the structural, functional, and genetic similarities and differences in the biofilm matrix from the four most common Candida species. Each species synthesizes an extracellular mannan-glucan complex (MGCx) which

  17. Nonleachable Imidazolium-Incorporated Composite for Disruption of Bacterial Clustering, Exopolysaccharide-Matrix Assembly, and Enhanced Biofilm Removal.

    Science.gov (United States)

    Hwang, Geelsu; Koltisko, Bernard; Jin, Xiaoming; Koo, Hyun

    2017-11-08

    Surface-grown bacteria and production of an extracellular polymeric matrix modulate the assembly of highly cohesive and firmly attached biofilms, making them difficult to remove from solid surfaces. Inhibition of cell growth and inactivation of matrix-producing bacteria can impair biofilm formation and facilitate removal. Here, we developed a novel nonleachable antibacterial composite with potent antibiofilm activity by directly incorporating polymerizable imidazolium-containing resin (antibacterial resin with carbonate linkage; ABR-C) into a methacrylate-based scaffold (ABR-modified composite; ABR-MC) using an efficient yet simplified chemistry. Low-dose inclusion of imidazolium moiety (∼2 wt %) resulted in bioactivity with minimal cytotoxicity without compromising mechanical integrity of the restorative material. The antibiofilm properties of ABR-MC were assessed using an exopolysaccharide-matrix-producing (EPS-matrix-producing) oral pathogen (Streptococcus mutans) in an experimental biofilm model. Using high-resolution confocal fluorescence imaging and biophysical methods, we observed remarkable disruption of bacterial accumulation and defective 3D matrix structure on the surface of ABR-MC. Specifically, the antibacterial composite impaired the ability of S. mutans to form organized bacterial clusters on the surface, resulting in altered biofilm architecture with sparse cell accumulation and reduced amounts of EPS matrix (versus control composite). Biofilm topology analyses on the control composite revealed a highly organized and weblike EPS structure that tethers the bacterial clusters to each other and to the surface, forming a highly cohesive unit. In contrast, such a structured matrix was absent on the surface of ABR-MC with mostly sparse and amorphous EPS, indicating disruption in the biofilm physical stability. Consistent with lack of structural organization, the defective biofilm on the surface of ABR-MC was readily detached when subjected to low shear

  18. Inhibition of Streptococcus mutans biofilm formation, extracellular polysaccharide production, and virulence by an oxazole derivative.

    Science.gov (United States)

    Chen, Lulu; Ren, Zhi; Zhou, Xuedong; Zeng, Jumei; Zou, Jing; Li, Yuqing

    2016-01-01

    Dental caries, a biofilm-related oral disease, is a result of disruption of the microbial ecological balance in the oral environment. Streptococcus mutans, which is one of the primary cariogenic bacteria, produces glucosyltransferases (Gtfs) that synthesize extracellular polysaccharides (EPSs). The EPSs, especially water-insoluble glucans, contribute to the formation of dental plaque, biofilm stability, and structural integrity, by allowing bacteria to adhere to tooth surfaces and supplying the bacteria with protection against noxious stimuli and other environmental attacks. The identification of novel alternatives that selectively inhibit cariogenic organisms without suppressing oral microbial residents is required. The goal of the current study is to investigate the influence of an oxazole derivative on S. mutans biofilm formation and the development of dental caries in rats, given that oxazole and its derivatives often exhibit extensive and pharmacologically important biological activities. Our data shows that one particular oxazole derivative, named 5H6, inhibited the formation of S. mutans biofilms and prevented synthesis of extracellular polysaccharides by antagonizing Gtfs in vitro, without affecting the growth of the bacteria. In addition, topical applications with the inhibitor resulted in diminished incidence and severity of both smooth and sulcal surface caries in vivo with a lower percentage of S. mutans in the animals' dental plaque compared to the control group (P mutans.

  19. Does Extracellular DNA Production Vary in Staphylococcal Biofilms Isolated From Infected Implants versus Controls?

    Science.gov (United States)

    Zatorska, Beata; Groger, Marion; Moser, Doris; Diab-Elschahawi, Magda; Lusignani, Luigi Segagni; Presterl, Elisabeth

    2017-08-01

    Prosthetic implant infections caused by Staphylococcus aureus and epidermidis are major challenges for early diagnosis and treatment owing to biofilm formation on the implant surface. Extracellular DNA (eDNA) is actively excreted from bacterial cells in biofilms, contributing to biofilm stability, and may offer promise in the detection or treatment of such infections. (1) Does DNA structure change during biofilm formation? (2) Are there time-dependent differences in eDNA production during biofilm formation? (3) Is there differential eDNA production between clinical and control Staphylococcal isolates? (4) Is eDNA production correlated to biofilm thickness? We investigated eDNA presence during biofilm formation in 60 clinical and 30 control isolates of S aureus and S epidermidis. The clinical isolates were isolated from patients with infections of orthopaedic prostheses and implants: 30 from infected hip prostheses and 30 from infected knee prostheses. The control isolates were taken from healthy volunteers who had not been exposed to antibiotics and a hospital environment during the previous 3 and 12 months, respectively. Control S epidermidis was isolated from the skin of the antecubital fossa, and control S aureus was isolated from the nares. For the biofilm experiments the following methods were used to detect eDNA: (1) fluorescent staining with 4',6-diamidino-2-phenylindole (DAPI), (2) eDNA extraction using a commercial kit, and (3) confocal laser scanning microscopy for 24-hour biofilm observation using propidium iodide and concanavalin-A staining; TOTO ® -1 and SYTO ® 60 staining were used for observation and quantification of eDNA after 6 and 24 hours of biofilm formation. Additionally antibiotic resistance was described. eDNA production as observed by confocal laser scanning microscopy was greater in clinical isolates than controls (clinical isolates mean ± SD: 1.84% ± 1.31%; control mean ± SD: 1.17% ± 1.37%; p biofilm formation. After 24 hours, the

  20. Fibroblasts and the extracellular matrix in right ventricular disease.

    Science.gov (United States)

    Frangogiannis, Nikolaos G

    2017-10-01

    Right ventricular failure predicts adverse outcome in patients with pulmonary hypertension (PH), and in subjects with left ventricular heart failure and is associated with interstitial fibrosis. This review manuscript discusses the cellular effectors and molecular mechanisms implicated in right ventricular fibrosis. The right ventricular interstitium contains vascular cells, fibroblasts, and immune cells, enmeshed in a collagen-based matrix. Right ventricular pressure overload in PH is associated with the expansion of the fibroblast population, myofibroblast activation, and secretion of extracellular matrix proteins. Mechanosensitive transduction of adrenergic signalling and stimulation of the renin-angiotensin-aldosterone cascade trigger the activation of right ventricular fibroblasts. Inflammatory cytokines and chemokines may contribute to expansion and activation of macrophages that may serve as a source of fibrogenic growth factors, such as transforming growth factor (TGF)-β. Endothelin-1, TGF-βs, and matricellular proteins co-operate to activate cardiac myofibroblasts, and promote synthesis of matrix proteins. In comparison with the left ventricle, the RV tolerates well volume overload and ischemia; whether the right ventricular interstitial cells and matrix are implicated in these favourable responses remains unknown. Expansion of fibroblasts and extracellular matrix protein deposition are prominent features of arrhythmogenic right ventricular cardiomyopathies and may be implicated in the pathogenesis of arrhythmic events. Prevailing conceptual paradigms on right ventricular remodelling are based on extrapolation of findings in models of left ventricular injury. Considering the unique embryologic, morphological, and physiologic properties of the RV and the clinical significance of right ventricular failure, there is a need further to dissect RV-specific mechanisms of fibrosis and interstitial remodelling. Published on behalf of the European Society of

  1. Biglycan fragmentation in pathologies associated with extracellular matrix remodeling by matrix metalloproteinases

    DEFF Research Database (Denmark)

    Genovese, Federica; Barascuk, Natasha; Larsen, Lise Skakkebæk

    2013-01-01

    The proteoglycan biglycan (BGN) is involved in collagen fibril assembly and its fragmentation is likely to be associated with collagen turnover during the pathogenesis of diseases which involve dysregulated extracellular matrix remodeling (ECMR), such as rheumatoid arthritis (RA) and liver fibrosis...

  2. Extracellular matrix and its receptors in Drosophila neural development

    Science.gov (United States)

    Broadie, Kendal; Baumgartner, Stefan; Prokop, Andreas

    2011-01-01

    Extracellular matrix (ECM) and matrix receptors are intimately involved in most biological processes. The ECM plays fundamental developmental and physiological roles in health and disease, including processes underlying the development, maintenance and regeneration of the nervous system. To understand the principles of ECM-mediated functions in the nervous system, genetic model organisms like Drosophila provide simple, malleable and powerful experimental platforms. This article provides an overview of ECM proteins and receptors in Drosophila. It then focuses on their roles during three progressive phases of neural development: 1) neural progenitor proliferation, 2) axonal growth and pathfinding and 3) synapse formation and function. Each section highlights known ECM and ECM-receptor components and recent studies done in mutant conditions to reveal their in vivo functions, all illustrating the enormous opportunities provided when merging work on the nervous system with systematic research into ECM-related gene functions. PMID:21688401

  3. Streptococcus pyogenes degrades extracellular matrix in chondrocytes via MMP-13

    International Nuclear Information System (INIS)

    Sakurai, Atsuo; Okahashi, Nobuo; Maruyama, Fumito; Ooshima, Takashi; Hamada, Shigeyuki; Nakagawa, Ichiro

    2008-01-01

    Group A streptococcus (GAS) causes a wide range of human diseases, including bacterial arthritis. The pathogenesis of arthritis is characterized by synovial proliferation and the destruction of cartilage and subchondral bone in joints. We report here that GAS strain JRS4 invaded a chondrogenic cell line ATDC5 and induced the degradation of the extracellular matrix (ECM), whereas an isogenic mutant of JRS4 lacking a fibronectin-binding protein, SAM1, failed to invade the chondrocytes or degrade the ECM. Reverse transcription-PCR and Western blot analysis revealed that the expression of matrix metalloproteinase (MMP)-13 was strongly elevated during the infection with GAS. A reporter assay revealed that the activation of the AP-1 transcription factor and the phosphorylation of c-Jun terminal kinase participated in MMP-13 expression. These results suggest that MMP-13 plays an important role in the destruction of infected joints during the development of septic arthritis

  4. Mifepristone inhibits extracellular matrix formation in uterine leiomyoma.

    Science.gov (United States)

    Patel, Amrita; Malik, Minnie; Britten, Joy; Cox, Jeris; Catherino, William H

    2016-04-01

    To characterize the efficacy of mifepristone treatment on extracellular matrix (ECM) production in leiomyomas. Laboratory study. University research laboratory. None. Treatment of human immortalized two-dimensional (2D) and three-dimensional (3D) leiomyoma and myometrial cells with mifepristone and the progestin promegestone (R5020). Expression of COL1A1, fibronectin, versican variant V0, and dermatopontin in treated leiomyoma cells by Western blot analysis and confirmatory immunohistochemistry staining of treated 3D cultures. Treatment with progestin stimulated production of COL1A1, fibronectin, versican, and dermatopontin. Mifepristone treatment inhibited protein production of these genes, most notably with versican expression. Combination treatment with both the agonist and antagonist further inhibited protein expression of these genes. Immunohistochemistry performed on 3D cultures demonstrated generalized inhibition of ECM protein concentration. Our study demonstrated that the progesterone agonist R5020 directly stimulated extracellular matrix components COL1A1, fibronectin, versican, and dermatopontin production in human leiomyoma cells. Progesterone antagonist mifepristone decreased protein production of these genes to levels comparable with untreated leiomyoma cells. Published by Elsevier Inc.

  5. Syndecans as receptors and organizers of the extracellular matrix.

    Science.gov (United States)

    Xian, Xiaojie; Gopal, Sandeep; Couchman, John R

    2010-01-01

    Syndecans are type I transmembrane proteins having a core protein modified with glycosaminoglycan chains, most commonly heparan sulphate. They are an ancient group of molecules, present in invertebrates and vertebrates. Among the plethora of molecules that can interact with heparan sulphate, the collagens and glycoproteins of the extracellular matrix are prominent. Frequently, they do so in conjunction with other receptors, most notably the integrins. For this reason, they are often referred to as "co-receptors". However, just as with integrins, syndecans can interact with actin-associated proteins and signalling molecules, such as protein kinases. Some aspects of syndecan signalling are understood but much remains to be learned. The functions of syndecans in regulating cell adhesion and extracellular matrix assembly are described here. Evidence from null mice suggests that syndecans have roles in postnatal tissue repair, inflammation and tumour progression. Developmental deficits in lower vertebrates in which syndecans are eliminated are also informative and suggest that, in mammals, redundancy is a key issue.

  6. Wetting and dewetting of extracellular matrix and glycocalix models

    International Nuclear Information System (INIS)

    Tanaka, Motomu; Rehfeldt, Florian; Schneider, Matthias F; Mathe, Gerald; Albersdoerfer, Antero; Neumaier, Klaus R; Purrucker, Oliver; Sackmann, Erich

    2005-01-01

    In this paper, we study wetting and dewetting of hydrated biopolymer layers mediating cell-cell and cell-tissue contacts, called the extracellular matrix and cell surface glycocalix, by the combination of various physical techniques. Here, the sum of the net effects of the various interfacial forces, which is referred to as the disjoining pressure, is used as a semi-quantitative measure to describe the thermodynamics of hydrated interlayers. The disjoining pressure can be measured by applying external forces to maintain the equilibrium distance between two parallel surfaces (in biology, two neighbouring plasma membranes). Using artificial models of the extracellular matrix and glycocalix, we describe stable cell-cell contacts in terms of the wetting (or spreading) of complex fluids on polymer surfaces. In fact, the adjustment of the wetting interaction via thin hydrating layers enables us to transform three-dimensional cell membranes into quasi-two-dimensional films on macroscopically large surfaces. Fine-tuning of local wetting conditions at the interface further allows for the selective wetting of native cell membranes on microstructured polysaccharide films, which has a large potential for individual detection of biological functions in confined geometries

  7. Extracellular matrix hydrogels from decellularized tissues: Structure and function.

    Science.gov (United States)

    Saldin, Lindsey T; Cramer, Madeline C; Velankar, Sachin S; White, Lisa J; Badylak, Stephen F

    2017-02-01

    Extracellular matrix (ECM) bioscaffolds prepared from decellularized tissues have been used to facilitate constructive and functional tissue remodeling in a variety of clinical applications. The discovery that these ECM materials could be solubilized and subsequently manipulated to form hydrogels expanded their potential in vitro and in vivo utility; i.e. as culture substrates comparable to collagen or Matrigel, and as injectable materials that fill irregularly-shaped defects. The mechanisms by which ECM hydrogels direct cell behavior and influence remodeling outcomes are only partially understood, but likely include structural and biological signals retained from the native source tissue. The present review describes the utility, formation, and physical and biological characterization of ECM hydrogels. Two examples of clinical application are presented to demonstrate in vivo utility of ECM hydrogels in different organ systems. Finally, new research directions and clinical translation of ECM hydrogels are discussed. More than 70 papers have been published on extracellular matrix (ECM) hydrogels created from source tissue in almost every organ system. The present manuscript represents a review of ECM hydrogels and attempts to identify structure-function relationships that influence the tissue remodeling outcomes and gaps in the understanding thereof. There is a Phase 1 clinical trial now in progress for an ECM hydrogel. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  8. Niche Extracellular Matrix Components and Their Influence on HSC.

    Science.gov (United States)

    Domingues, Mélanie J; Cao, Huimin; Heazlewood, Shen Y; Cao, Benjamin; Nilsson, Susan K

    2017-08-01

    Maintenance of hematopoietic stem cells (HSC) takes place in a highly specialized microenvironment within the bone marrow. Technological improvements, especially in the field of in vivo imaging, have helped unravel the complexity of the niche microenvironment and have completely changed the classical concept from what was previously believed to be a static supportive platform, to a dynamic microenvironment tightly regulating HSC homeostasis through the complex interplay between diverse cell types, secreted factors, extracellular matrix molecules, and the expression of different transmembrane receptors. To add to the complexity, non-protein based metabolites have also been recognized as a component of the bone marrow niche. The objective of this review is to discuss the current understanding on how the different extracellular matrix components of the niche regulate HSC fate, both during embryonic development and in adulthood. Special attention will be provided to the description of non-protein metabolites, such as lipids and metal ions, which contribute to the regulation of HSC behavior. J. Cell. Biochem. 118: 1984-1993, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  9. Specific extracellular matrix remodeling signature of colon hepatic metastases.

    Directory of Open Access Journals (Sweden)

    Maguy Del Rio

    Full Text Available To identify genes implicated in metastatic colonization of the liver in colorectal cancer, we collected pairs of primary tumors and hepatic metastases before chemotherapy in 13 patients. We compared mRNA expression in the pairs of patients to identify genes deregulated during metastatic evolution. We then validated the identified genes using data obtained by different groups. The 33-gene signature was able to classify 87% of hepatic metastases, 98% of primary tumors, 97% of normal colon mucosa, and 95% of normal liver tissues in six datasets obtained using five different microarray platforms. The identified genes are specific to colon cancer and hepatic metastases since other metastatic locations and hepatic metastases originating from breast cancer were not classified by the signature. Gene Ontology term analysis showed that 50% of the genes are implicated in extracellular matrix remodeling, and more precisely in cell adhesion, extracellular matrix organization and angiogenesis. Because of the high efficiency of the signature to classify colon hepatic metastases, the identified genes represent promising targets to develop new therapies that will specifically affect hepatic metastasis microenvironment.

  10. Regulation of pituitary hormones and cell proliferation by components of the extracellular matrix

    Directory of Open Access Journals (Sweden)

    M. Paez-Pereda

    2005-10-01

    Full Text Available The extracellular matrix is a three-dimensional network of proteins, glycosaminoglycans and other macromolecules. It has a structural support function as well as a role in cell adhesion, migration, proliferation, differentiation, and survival. The extracellular matrix conveys signals through membrane receptors called integrins and plays an important role in pituitary physiology and tumorigenesis. There is a differential expression of extracellular matrix components and integrins during the pituitary development in the embryo and during tumorigenesis in the adult. Different extracellular matrix components regulate adrenocorticotropin at the level of the proopiomelanocortin gene transcription. The extracellular matrix also controls the proliferation of adrenocorticotropin-secreting tumor cells. On the other hand, laminin regulates the production of prolactin. Laminin has a dynamic pattern of expression during prolactinoma development with lower levels in the early pituitary hyperplasia and a strong reduction in fully grown prolactinomas. Therefore, the expression of extracellular matrix components plays a role in pituitary tumorigenesis. On the other hand, the remodeling of the extracellular matrix affects pituitary cell proliferation. Matrix metalloproteinase activity is very high in all types of human pituitary adenomas. Matrix metalloproteinase secreted by pituitary cells can release growth factors from the extracellular matrix that, in turn, control pituitary cell proliferation and hormone secretion. In summary, the differential expression of extracellular matrix components, integrins and matrix metalloproteinase contributes to the control of pituitary hormone production and cell proliferation during tumorigenesis.

  11. Cartilage extracellular matrix as a biomaterial for cartilage regeneration.

    Science.gov (United States)

    Kiyotake, Emi A; Beck, Emily C; Detamore, Michael S

    2016-11-01

    The extracellular matrix (ECM) of various tissues possesses the model characteristics that biomaterials for tissue engineering strive to mimic; however, owing to the intricate hierarchical nature of the ECM, it has yet to be fully characterized and synthetically fabricated. Cartilage repair remains a challenge because the intrinsic properties that enable its durability and long-lasting function also impede regeneration. In the last decade, cartilage ECM has emerged as a promising biomaterial for regenerating cartilage, partly because of its potentially chondroinductive nature. As this research area of cartilage matrix-based biomaterials emerged, investigators facing similar challenges consequently developed convergent solutions in constructing robust and bioactive scaffolds. This review discusses the challenges, emerging trends, and future directions of cartilage ECM scaffolds, including a comparison between two different forms of cartilage matrix: decellularized cartilage (DCC) and devitalized cartilage (DVC). To overcome the low permeability of cartilage matrix, physical fragmentation greatly enhances decellularization, although the process itself may reduce the chondroinductivity of fabricated scaffolds. The less complex processing of a scaffold composed of DVC, which has not been decellularized, appears to have translational advantages and potential chondroinductive and mechanical advantages over DCC, without detrimental immunogenicity, to ultimately enhance cartilage repair in a clinically relevant way. © 2016 New York Academy of Sciences.

  12. Modulating bacterial and gut mucosal interactions with engineered biofilm matrix proteins.

    Science.gov (United States)

    Duraj-Thatte, Anna M; Praveschotinunt, Pichet; Nash, Trevor R; Ward, Frederick R; Joshi, Neel S

    2018-02-22

    Extracellular appendages play a significant role in mediating communication between bacteria and their host. Curli fibers are a class of bacterial fimbria that is highly amenable to engineering. We demonstrate the use of engineered curli fibers to rationally program interactions between bacteria and components of the mucosal epithelium. Commensal E. coli strains were engineered to produce recombinant curli fibers fused to the trefoil family of human cytokines. Biofilms formed from these strains bound more mucins than those producing wild-type curli fibers, and modulated mucin rheology as well. When treated with bacteria producing the curli-trefoil fusions mammalian cells behaved identically in terms of their migration behavior as when they were treated with the corresponding soluble trefoil factors. Overall, this demonstrates the potential utility of curli fibers as a scaffold for the display of bioactive domains and an untapped approach to rationally modulating host-microbe interactions using bacterial matrix proteins.

  13. Platelet activation by extracellular matrix proteins in haemostasis and thrombosis.

    Science.gov (United States)

    Watson, Steve P

    2009-01-01

    The prevention of excessive blood loss to avoid fatal haemorrhage is a pivotal process for all organisms possessing a circulatory system. Increased circulating blood volume and pressure, as required in larger animals, make this process all the more important and challenging. It is essential to have a powerful and rapid system to detect damage and generate an effective seal, and which is also exquisitely regulated to prevent unwanted, excessive or systemic activation so as to avoid blockage of vessels. Thus, a highly specialised and efficient haemostatic system has evolved that consists of cellular (platelets) and protein (coagulation factors) components. Importantly, this is able to support haemostasis in both the low shear environment of the venous system and the high shear environment of the arterial system. Endothelial cells, lining the entire circulation system, play a crucial role in the delicate balance between activation and inhibition of the haemostatic system. An intact and healthy endothelium supports blood flow by preventing attachment of cells and proteins which is required for initiation of coagulation and platelet activation. Endothelial cells produce and release the two powerful soluble inhibitors of platelet activation, nitric oxide and prostacyclin, and express high levels of CD39 which rapidly metabolises the major platelet feedback agonist, ADP. This antithrombotic environment however can rapidly change following activation or removal of endothelial cells through injury or rupture of atherosclerotic plaques. Loss of endothelial cells exposes the subendothelial extracellular matrix which creates strong signals for activation of the haemostatic system including powerful platelet adhesion and activation. Quantitative and qualitative changes in the composition of the subendothelial extracellular matrix influence these prothrombotic characteristics with life threatening thrombotic and bleeding complications, as illustrated by formation of

  14. [Inhibitory proteins of neuritic regeneration in the extracellular matrix: structure, molecular interactions and their functions. Mechanisms of extracellular balance].

    Science.gov (United States)

    Vargas, Javier; Uribe-Escamilla, Rebeca; Alfaro-Rodríguez, Alfonso

    2013-01-01

    After injury of the central nervous system (CNS) in higher vertebrates, neurons neither grow nor reconnect with their targets because their axons or dendrites cannot regenerate within the injured site. In the CNS, the signal from the environment regulating neurite regeneration is not exclusively generated by one molecular group. This signal is generated by the interaction of various types of molecules such as extracellular matrix proteins, soluble factors and surface membrane molecules; all these elements interact with one another generating the matrix's biological state: the extracellular balance. Proteins in the balanced extracellular matrix, support and promote cellular physiological states, including neuritic regeneration. We have reviewed three types of proteins of the extracellular matrix possessing an inhibitory effect and that are determinant of neuritic regeneration failure in the CNS: chondroitin sulfate proteoglycans, keratan sulfate proteoglycans and tenascin. We also review some of the mechanisms involved in the balance of extracellular proteins such as isomerization, epimerization, sulfation and glycosylation as well as the assemblage of the extracellular matrix, the interaction between the matrix and soluble factors and its proteolytic degradation. In the final section, we have presented some examples of the matrix's role in development and in tumor propagation.

  15. Biochemical and biomechanical properties of the pacemaking sinoatrial node extracellular matrix are distinct from contractile left ventricular matrix.

    Directory of Open Access Journals (Sweden)

    Jessica M Gluck

    Full Text Available Extracellular matrix plays a role in differentiation and phenotype development of its resident cells. Although cardiac extracellular matrix from the contractile tissues has been studied and utilized in tissue engineering, extracellular matrix properties of the pacemaking sinoatrial node are largely unknown. In this study, the biomechanical properties and biochemical composition and distribution of extracellular matrix in the sinoatrial node were investigated relative to the left ventricle. Extracellular matrix of the sinoatrial node was found to be overall stiffer than that of the left ventricle and highly heterogeneous with interstitial regions composed of predominantly fibrillar collagens and rich in elastin. The extracellular matrix protein distribution suggests that resident pacemaking cardiomyocytes are enclosed in fibrillar collagens that can withstand greater tensile strength while the surrounding elastin-rich regions may undergo deformation to reduce the mechanical strain in these cells. Moreover, basement membrane-associated adhesion proteins that are ligands for integrins were of low abundance in the sinoatrial node, which may decrease force transduction in the pacemaking cardiomyocytes. In contrast to extracellular matrix of the left ventricle, extracellular matrix of the sinoatrial node may reduce mechanical strain and force transduction in pacemaking cardiomyocytes. These findings provide the criteria for a suitable matrix scaffold for engineering biopacemakers.

  16. Synthetic osteogenic extracellular matrix formed by coated silicon dioxide nanosprings

    Directory of Open Access Journals (Sweden)

    Hass Jamie L

    2012-01-01

    Full Text Available Abstract Background The design of biomimetic materials that parallel the morphology and biology of extracellular matrixes is key to the ability to grow functional tissues in vitro and to enhance the integration of biomaterial implants into existing tissues in vivo. Special attention has been put into mimicking the nanostructures of the extracellular matrix of bone, as there is a need to find biomaterials that can enhance the bonding between orthopedic devices and this tissue. Methods We have tested the ability of normal human osteoblasts to propagate and differentiate on silicon dioxide nanosprings, which can be easily grown on practically any surface. In addition, we tested different metals and metal alloys as coats for the nanosprings in tissue culture experiments with bone cells. Results Normal human osteoblasts grown on coated nanosprings exhibited an enhanced rate of propagation, differentiation into bone forming cells and mineralization. While osteoblasts did not attach effectively to bare nanowires grown on glass, these cells propagated successfully on nanosprings coated with titanium oxide and gold. We observed a 270 fold increase in the division rate of osteoblasts when grow on titanium/gold coated nanosprings. This effect was shown to be dependent on the nanosprings, as the coating by themselves did not alter the growth rate of osteoblast. We also observed that titanium/zinc/gold coated nanosprings increased the levels of osteoblast production of alkaline phosphatase seven folds. This result indicates that osteoblasts grown on this metal alloy coated nanosprings are differentiating to mature bone making cells. Consistent with this hypothesis, we showed that osteoblasts grown on the same metal alloy coated nanosprings have an enhanced ability to deposit calcium salt. Conclusion We have established that metal/metal alloy coated silicon dioxide nanosprings can be used as a biomimetic material paralleling the morphology and biology of

  17. A Nuclease from Streptococcus mutans Facilitates Biofilm Dispersal and Escape from Killing by Neutrophil Extracellular Traps.

    Science.gov (United States)

    Liu, Jia; Sun, Luping; Liu, Wei; Guo, Lihong; Liu, Zhaohui; Wei, Xi; Ling, Junqi

    2017-01-01

    Streptococcus mutans is the primary etiologic agent of dental caries and occasionally infective endocarditis, with the ability to form biofilms and disperse cells into distal sites to exacerbate and spread infection. In this study, we identified a nuclease (DeoC) as a S. mutans biofilm dispersal modulating factor through microarray analysis. In vitro assays revealed a dispersal defect of a deoC deletion mutant, and functional studies with purified protein were indicative of the biofilm dispersal activity of DeoC. Neutrophils are a key host response factor restraining bacterial spreading through the formation of neutrophil extracellular traps (NETs), which consist of a nuclear DNA backbone associated with antimicrobial peptides. Therefore, we hypothesized that the dispersed S. mutans might utilize DeoC to degrade NETs and escape killing by the immune system. It was found that S. mutans induced NET formation upon contact with neutrophils, while the presence of NETs in turn enhanced the deoC expression of S. mutans . Fluorescence microscopy inspection showed that deoC deletion resulted in a decreased NET degradation ability of S. mutans and enhanced susceptibility to neutrophil killing. Data obtained from this study assigned two important roles for DeoC in S. mutans : contributing to the spread of infection through mediating biofilm dispersal, and facilitating the escape of S. mutans from neutrophil killing through NET degradation.

  18. Cell stiffness, contractile stress and the role of extracellular matrix

    International Nuclear Information System (INIS)

    An, Steven S.; Kim, Jina; Ahn, Kwangmi; Trepat, Xavier; Drake, Kenneth J.; Kumar, Sarvesh; Ling, Guoyu; Purington, Carolyn; Rangasamy, Tirumalai; Kensler, Thomas W.; Mitzner, Wayne; Fredberg, Jeffrey J.; Biswal, Shyam

    2009-01-01

    Here we have assessed the effects of extracellular matrix (ECM) composition and rigidity on mechanical properties of the human airway smooth muscle (ASM) cell. Cell stiffness and contractile stress showed appreciable changes from the most relaxed state to the most contracted state: we refer to the maximal range of these changes as the cell contractile scope. The contractile scope was least when the cell was adherent upon collagen V, followed by collagen IV, laminin, and collagen I, and greatest for fibronectin. Regardless of ECM composition, upon adherence to increasingly rigid substrates, the ASM cell positively regulated expression of antioxidant genes in the glutathione pathway and heme oxygenase, and disruption of a redox-sensitive transcription factor, nuclear erythroid 2 p45-related factor (Nrf2), culminated in greater contractile scope. These findings provide biophysical evidence that ECM differentially modulates muscle contractility and, for the first time, demonstrate a link between muscle contractility and Nrf2-directed responses.

  19. Targeting the extracellular matrix to disrupt cancer progression

    Directory of Open Access Journals (Sweden)

    Freja Albjerg Venning

    2015-10-01

    Full Text Available Metastatic complications are responsible for more than 90% of cancer related deaths. The progression from an isolated tumor to disseminated metastatic disease is a multi-step process, with each step involving intricate cross-talk between the cancer cells and their non-cellular surroundings, the extracellular matrix (ECM. Many ECM proteins are significantly de-regulated during the progression of cancer, causing both biochemical and biomechanical changes that together promote the metastatic cascade. In this review, the influence of several ECM proteins on these multiple steps of cancer spread is summarized. In addition, we highlight the promising (pre-clinical data showing benefits of targeting these ECM macromolecules to prevent cancer progression.

  20. Extracellular matrix adaptation of tendon and skeletal muscle to exercise

    DEFF Research Database (Denmark)

    Kjaer, Michael; Magnusson, Peter; Krogsgaard, Michael

    2006-01-01

    The extracellular matrix (ECM) of connective tissues enables linking to other tissues, and plays a key role in force transmission and tissue structure maintenance in tendons, ligaments, bone and muscle. ECM turnover is influenced by physical activity, and both collagen synthesis and metalloprotease......-beta and IL-6 is enhanced following exercise. For tendons, metabolic activity (e.g. detected by positron emission tomography scanning), circulatory responses (e.g. as measured by near-infrared spectroscopy and dye dilution) and collagen turnover are markedly increased after exercise. Tendon blood flow...... is regulated by cyclooxygenase-2 (COX-2)-mediated pathways, and glucose uptake is regulated by specific pathways in tendons that differ from those in skeletal muscle. Chronic loading in the form of physical training leads both to increased collagen turnover as well as to some degree of net collagen synthesis...

  1. Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix.

    Science.gov (United States)

    Piotrowski-Daspit, Alexandra S; Nelson, Celeste M

    2016-07-10

    The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

  2. Cell stiffness, contractile stress and the role of extracellular matrix

    Energy Technology Data Exchange (ETDEWEB)

    An, Steven S., E-mail: san@jhsph.edu [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Kim, Jina [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Ahn, Kwangmi [Division of Biostatistics, Penn State College of Medicine, Hershey, PA 17033 (United States); Trepat, Xavier [CIBER, Enfermedades Respiratorias, 07110 Bunyola (Spain); Drake, Kenneth J. [Division of Molecular and Integrative Physiological Sciences, Harvard School of Public Health, Boston, MA 02115 (United States); Kumar, Sarvesh; Ling, Guoyu; Purington, Carolyn; Rangasamy, Tirumalai; Kensler, Thomas W.; Mitzner, Wayne [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Fredberg, Jeffrey J. [Division of Molecular and Integrative Physiological Sciences, Harvard School of Public Health, Boston, MA 02115 (United States); Biswal, Shyam [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Division of Pulmonary and Critical Care Medicine, Johns Hopkins School of Medicine, Baltimore, MD 21205 (United States)

    2009-05-15

    Here we have assessed the effects of extracellular matrix (ECM) composition and rigidity on mechanical properties of the human airway smooth muscle (ASM) cell. Cell stiffness and contractile stress showed appreciable changes from the most relaxed state to the most contracted state: we refer to the maximal range of these changes as the cell contractile scope. The contractile scope was least when the cell was adherent upon collagen V, followed by collagen IV, laminin, and collagen I, and greatest for fibronectin. Regardless of ECM composition, upon adherence to increasingly rigid substrates, the ASM cell positively regulated expression of antioxidant genes in the glutathione pathway and heme oxygenase, and disruption of a redox-sensitive transcription factor, nuclear erythroid 2 p45-related factor (Nrf2), culminated in greater contractile scope. These findings provide biophysical evidence that ECM differentially modulates muscle contractility and, for the first time, demonstrate a link between muscle contractility and Nrf2-directed responses.

  3. The Role of Extracellular Matrix Quality in Pulmonary Fibrosis

    DEFF Research Database (Denmark)

    Kristensen, Jacob Hull; Karsdal, Morten Asser; Genovese, Federica

    2014-01-01

    This review discusses the role of extracellular matrix (ECM) quality in the pathogenesis of pulmonary fibrosis (PF). In PF, the highly ordered structure of collagens and elastin within the ECM of the lung is severely disrupted and lacks its original tissue quality. Discussions about the ECM have...... focused on the role of protein quantity in relation to the progression of PF, while the importance of lung ECM quality, defined by the levels of ECM protein modifications and by the protein distribution in lung tissue, has not been properly addressed. The quality and function of proteins may be altered...... by different post-translational modifications (PTMs), such as crosslinking, proteolytic cleavage, citrullination, misfolding and glycosylation. This paper is the first to review key data from the literature related to the lung ECM at the molecular level, relate these to changes observed at a macroscopic level...

  4. Extracellular matrix alterations in human corneas with bullous keratopathy

    DEFF Research Database (Denmark)

    Ljubimov, A V; Burgeson, R E; Butkowski, R J

    1996-01-01

    PURPOSE. To uncover abnormalities of extracellular matrix (ECM) distribution in human corneas with pseudophakic and aphakic bullous keratopathy (PBK/ABK). METHODS. Indirect immunofluorescence with antibodies to 27 ECM components was used on frozen sections of 14 normal and 20 PBK/ABK corneas...... in some cases, correlated with decreased visual acuity. In normal central corneas, tenascin was never found. Other major ECM abnormalities in PBK/ABK corneas compared to normals included: discontinuous epithelial BM straining for laminin-1 (alpha 1 beta 1 gamma 1), entactin/nidogen and fibronectin......; accumulation of fibronectin and alpha 1-alpha 2 type IV collagen on the endothelial face of the Descemet's membrane; and abnormal deposition of stromal ECM (tenascin, fibronectin, decorin, types I, III, V, VI, VIII, XII, XIV collagen) and BM components (type IV, collagen, perlecan, bamacan, laminin-1, entactin...

  5. Cells involved in extracellular matrix remodeling after acute myocardial infarction

    Energy Technology Data Exchange (ETDEWEB)

    Garcia, Larissa Ferraz [Faculdade de Medicina do ABC, Santo André, SP (Brazil); Mataveli, Fábio D’Aguiar [Universidade Federal de São Paulo, São Paulo, SP (Brazil); Mader, Ana Maria Amaral Antônio; Theodoro, Thérèse Rachell [Faculdade de Medicina do ABC, Santo André, SP (Brazil); Justo, Giselle Zenker; Pinhal, Maria Aparecida da Silva [Universidade Federal de São Paulo, São Paulo, SP (Brazil)

    2015-07-01

    Evaluate the effects of VEGF{sub 165} gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF{sub 165} treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF{sub 165}. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF{sub 165}, suggesting greater tissue differentiation. The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF{sub 165} seems to provide a protective effect in the treatment of acute myocardial infarct.

  6. Cells involved in extracellular matrix remodeling after acute myocardial infarction

    International Nuclear Information System (INIS)

    Garcia, Larissa Ferraz; Mataveli, Fábio D’Aguiar; Mader, Ana Maria Amaral Antônio; Theodoro, Thérèse Rachell; Justo, Giselle Zenker; Pinhal, Maria Aparecida da Silva

    2015-01-01

    Evaluate the effects of VEGF_1_6_5 gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF_1_6_5 treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF_1_6_5. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF_1_6_5, suggesting greater tissue differentiation. The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF_1_6_5 seems to provide a protective effect in the treatment of acute myocardial infarct

  7. Pancreatic morphogenesis and extracellular matrix organization during rat development.

    Science.gov (United States)

    Hisaoka, M; Haratake, J; Hashimoto, H

    1993-07-01

    We investigated the rat pancreatic morphology at various developmental stages ranging from 12 days of gestation to the neonatal stage, with special emphasis on alterations in extracellular matrix organization in vivo. The rat pancreatic development in utero could be divided into four representative stages as follows: (1) initial epithelial buds (12 days of gestation), (2) elongated and branching epithelium (13-14 days), (3) tubular structure (15-16 days), and (4) acinar structure (17 days or more). Ultrastructurally, the fetal and neonatal pancreata were almost constantly encompassed by continuous basal lamina, except for the earliest stage, in which minute disruptions of basal lamina were observed. Through the disruption, the direct epithelial-mesenchymal contact was formed between an endocrine cell and an adjacent mesenchymal cell, which implied epithelial-mesenchymal interactions in processes of endocrine cell differentiation. Collagen fibrils were frequently accumulated at the cleft (branchpoint) of the branching epithelium during the second and third stages mentioned above. Immunohistochemically, fibronectin and collagen type-I were localized particularly beside the neck (narrow part) or cleft of the pancreatic epithelium at these stages, although continuous linear localization of these matrices was noted around the initial pancreatic bud. This was in contrast to invariable linear localization of laminin and collagen type-IV at the epithelial/mesenchymal interface throughout the pancreatic development. Diffuse fibrillar localization of fibronectin and collagen type-I in the mesenchyme was pronounced at the later stages and after birth. Collagen type-III was only focally detectable around the pancreatic epithelium from the second stage, and its distinct localization was noted in the interlobular connective tissue after birth. Thus, chronological changes in extracellular matrix organization seemed to be closely related to morphogenetic processes of the rat

  8. Micromechanical anisotropy and heterogeneity of the meniscus extracellular matrix.

    Science.gov (United States)

    Li, Qing; Qu, Feini; Han, Biao; Wang, Chao; Li, Hao; Mauck, Robert L; Han, Lin

    2017-05-01

    To understand how the complex biomechanical functions of the meniscus are endowed by the nanostructure of its extracellular matrix (ECM), we studied the anisotropy and heterogeneity in the micromechanical properties of the meniscus ECM. We used atomic force microscopy (AFM) to quantify the time-dependent mechanical properties of juvenile bovine meniscus at deformation length scales corresponding to the diameters of collagen fibrils. At this scale, anisotropy in the elastic modulus of the circumferential fibers, the major ECM structural unit, can be attributed to differences in fibril deformation modes: uncrimping when normal to the fiber axis, and laterally constrained compression when parallel to the fiber axis. Heterogeneity among different structural units is mainly associated with their variations in microscale fiber orientation, while heterogeneity across anatomical zones is due to alterations in collagen fibril diameter and alignment at the nanoscale. Unlike the elastic modulus, the time-dependent properties are more homogeneous and isotropic throughout the ECM. These results enable a detailed understanding of the meniscus structure-mechanics at the nanoscale, and can serve as a benchmark for understanding meniscus biomechanical functions, documenting disease progression and designing tissue repair strategies. Meniscal damage is a common cause of joint injury, which can lead to the development of post-traumatic osteoarthritis among young adults. Restoration of meniscus function requires repairing its highly heterogeneous and complex extracellular matrix. Employing AFM, this study quantifies the anisotropic and heterogeneous features of the meniscus ECM structure and mechanics. The micromechanical properties are interpreted within the context of the collagen fibril nanostructure and its variation with tissue anatomical locations. These results provide a fundamental structure-mechanics knowledge benchmark, against which, repair and regeneration strategies can

  9. Engineering a collagen matrix that replicates the biological properties of native extracellular matrix.

    Science.gov (United States)

    Nam, Kwangwoo; Sakai, Yuuki; Funamoto, Seiichi; Kimura, Tsuyoshi; Kishida, Akio

    2011-01-01

    In this study, we aimed to replicate the function of native tissues that can be used in tissue engineering and regenerative medicine. The key to such replication is the preparation of an artificial collagen matrix that possesses a structure resembling that of the extracellular matrix. We, therefore, prepared a collagen matrix by fibrillogenesis in a NaCl/Na(2)HPO(4) aqueous solution using a dialysis cassette and investigated its biological behavior in vitro and in vivo. The in vitro cell adhesion and proliferation did not show any significant differences. The degradation rate in the living body could be controlled according to the preparation condition, where the collagen matrix with high water content (F-collagen matrix, >98%) showed fast degradation and collagen matrix with lower water content (T-collagen matrix, >80%) showed no degradation for 8 weeks. The degradation did not affect the inflammatory response at all and relatively faster wound healing response was observed. Comparing this result with that of collagen gel and decellularized cornea, it can be concluded that the structural factor is very important and no cell abnormal behavior would be observed for quaternary structured collagen matrix.

  10. Quantifying biostabilisation effects of biofilm-secreted and extracted extracellular polymeric substances (EPSs) on sandy substrate

    Science.gov (United States)

    van de Lageweg, Wietse I.; McLelland, Stuart J.; Parsons, Daniel R.

    2018-03-01

    Microbial assemblages (biofilms) preferentially develop at water-sediment interfaces and are known to have a considerable influence on sediment stability and erodibility. There is potential for significant impacts on sediment transport and morphodynamics, and hence on the longer-term evolution of coastal and fluvial environments. However, the biostabilisation effects remain poorly understood and quantified due to the inherent complexity of biofilms and the large spatial and temporal (i.e. seasonality) variations involved. Here, we use controlled laboratory tests to systematically quantify the effects of natural biofilm colonisation as well as extracted extracellular polymeric substances (EPSs) on sediment stability. Extracted EPSs may be useful to simulate biofilm-mediated biostabilisation and potentially provide a method of speeding up timescales of physical modelling experiments investigating biostabilisation effects. We find a mean biostabilisation effect due to natural biofilm colonisation and development of almost 4 times that of the uncolonised sand. The presented cumulative probability distribution of measured critical threshold for erosion of colonised sand reflects the large spatial and temporal variations generally seen in natural biostabilised environments. For identical sand, engineered sediment stability from the addition of extracted EPSs compares well across the measured range of the critical threshold for erosion and behaves in a linear and predictable fashion. Yet, the effectiveness of extracted EPSs to stabilise sediment is sensitive to the preparation procedure, time after application and environmental conditions such as salinity, pH and temperature. These findings are expected to improve biophysical experimental models in fluvial and coastal environments and provide much-needed quantification of biostabilisation to improve predictions of sediment dynamics in aquatic ecosystems.

  11. Giving structure to the biofilm matrix: an overview of individual strategies and emerging common themes.

    Science.gov (United States)

    Hobley, Laura; Harkins, Catriona; MacPhee, Cait E; Stanley-Wall, Nicola R

    2015-09-01

    Biofilms are communities of microbial cells that underpin diverse processes including sewage bioremediation, plant growth promotion, chronic infections and industrial biofouling. The cells resident in the biofilm are encased within a self-produced exopolymeric matrix that commonly comprises lipids, proteins that frequently exhibit amyloid-like properties, eDNA and exopolysaccharides. This matrix fulfils a variety of functions for the community, from providing structural rigidity and protection from the external environment to controlling gene regulation and nutrient adsorption. Critical to the development of novel strategies to control biofilm infections, or the capability to capitalize on the power of biofilm formation for industrial and biotechnological uses, is an in-depth knowledge of the biofilm matrix. This is with respect to the structure of the individual components, the nature of the interactions between the molecules and the three-dimensional spatial organization. We highlight recent advances in the understanding of the structural and functional role that carbohydrates and proteins play within the biofilm matrix to provide three-dimensional architectural integrity and functionality to the biofilm community. We highlight, where relevant, experimental techniques that are allowing the boundaries of our understanding of the biofilm matrix to be extended using Escherichia coli, Staphylococcus aureus, Vibrio cholerae, and Bacillus subtilis as exemplars. © FEMS 2015.

  12. Incorporation of Tenascin-C into the Extracellular Matrix by Periostin Underlies an Extracellular Meshwork Architecture*

    Science.gov (United States)

    Kii, Isao; Nishiyama, Takashi; Li, Minqi; Matsumoto, Ken-ichi; Saito, Mitsuru; Amizuka, Norio; Kudo, Akira

    2010-01-01

    Extracellular matrix (ECM) underlies a complicated multicellular architecture that is subjected to significant forces from mechanical environment. Although various components of the ECM have been enumerated, mechanisms that evolve the sophisticated ECM architecture remain to be addressed. Here we show that periostin, a matricellular protein, promotes incorporation of tenascin-C into the ECM and organizes a meshwork architecture of the ECM. We found that both periostin null mice and tenascin-C null mice exhibited a similar phenotype, confined tibial periostitis, which possibly corresponds to medial tibial stress syndrome in human sports injuries. Periostin possessed adjacent domains that bind to tenascin-C and the other ECM protein: fibronectin and type I collagen, respectively. These adjacent domains functioned as a bridge between tenascin-C and the ECM, which increased deposition of tenascin-C on the ECM. The deposition of hexabrachions of tenascin-C may stabilize bifurcations of the ECM fibrils, which is integrated into the extracellular meshwork architecture. This study suggests a role for periostin in adaptation of the ECM architecture in the mechanical environment. PMID:19887451

  13. Incorporation of tenascin-C into the extracellular matrix by periostin underlies an extracellular meshwork architecture.

    Science.gov (United States)

    Kii, Isao; Nishiyama, Takashi; Li, Minqi; Matsumoto, Ken-Ichi; Saito, Mitsuru; Amizuka, Norio; Kudo, Akira

    2010-01-15

    Extracellular matrix (ECM) underlies a complicated multicellular architecture that is subjected to significant forces from mechanical environment. Although various components of the ECM have been enumerated, mechanisms that evolve the sophisticated ECM architecture remain to be addressed. Here we show that periostin, a matricellular protein, promotes incorporation of tenascin-C into the ECM and organizes a meshwork architecture of the ECM. We found that both periostin null mice and tenascin-C null mice exhibited a similar phenotype, confined tibial periostitis, which possibly corresponds to medial tibial stress syndrome in human sports injuries. Periostin possessed adjacent domains that bind to tenascin-C and the other ECM protein: fibronectin and type I collagen, respectively. These adjacent domains functioned as a bridge between tenascin-C and the ECM, which increased deposition of tenascin-C on the ECM. The deposition of hexabrachions of tenascin-C may stabilize bifurcations of the ECM fibrils, which is integrated into the extracellular meshwork architecture. This study suggests a role for periostin in adaptation of the ECM architecture in the mechanical environment.

  14. Expression of extracellular matrix metalloproteinase inducer in odontogenic cysts.

    Science.gov (United States)

    Ali, Mohammad Abdulhadi Abbas

    2008-08-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN) is known to induce matrix metalloproteinase (MMP) production. The expression of EMMPRIN in odontogenic cysts has not been previously studied. This study was done to determine the presence and the variability of EMMPRIN expression in various types of odontogenic cysts. An immunohistochemical study using a polyclonal anti-EMMPRIN antibody was done using 48 odontogenic cyst cases: 13 odontogenic keratocysts (OKCs), 18 dentigerous cysts (DCs), and 17 periapical cysts (PAs). Twelve cases of normal dental follicles (DFs) were also included in this study for comparison. EMMPRIN immunoreactivity was detected in all of the cysts and DFs studied. In odontogenic cysts, EMMPRIN immunoreactivity was generally higher in basal cells than in suprabasal cells. The overall EMMPRIN expression in the epithelial lining of the 3 different types of odontogenic cyst was significantly higher than in the DFs. Overall EMMPRIN expression was also found to be significantly higher in the epithelial lining of OKCs than in the other types of cysts. This study confirmed that EMMPRIN is present in odontogenic cysts and DFs. The higher EMMPRIN expression in OKCs suggests that it may be involved in the aggressive behavior of this type of cyst.

  15. Collagen and related extracellular matrix proteins in atherosclerotic plaque development.

    Science.gov (United States)

    Shami, Annelie; Gonçalves, Isabel; Hultgårdh-Nilsson, Anna

    2014-10-01

    The structure, composition and turnover of the extracellular matrix (ECM) as well as cell-matrix interactions are crucial in the developing atherosclerotic plaque. There is a need for further insight into specific proteins in the ECM and their functions in the developing plaque, and during the last few years a number of publications have highlighted this very important field of research. These novel findings will be addressed in the present review. This review covers literature focused on collagen and ECM proteins interacting with collagen, and what their roles may be in plaque development. Acute myocardial infarction and stroke are common diseases that cause disability and mortality, and the underlying mechanism is often the rupture of a vulnerable atherosclerotic plaque. The vascular ECM and the tissue repair in the atherosclerotic lesion are important players in plaque progression. Understanding how specific proteins in the ECM interact with cells in the plaque and affect the fate of the plaque can lead to new treatments for cardiovascular disease.

  16. Domain organizations of modular extracellular matrix proteins and their evolution.

    Science.gov (United States)

    Engel, J

    1996-11-01

    Multidomain proteins which are composed of modular units are a rather recent invention of evolution. Domains are defined as autonomously folding regions of a protein, and many of them are similar in sequence and structure, indicating common ancestry. Their modular nature is emphasized by frequent repetitions in identical or in different proteins and by a large number of different combinations with other domains. The extracellular matrix is perhaps the largest biological system composed of modular mosaic proteins, and its astonishing complexity and diversity are based on them. A cluster of minireviews on modular proteins is being published in Matrix Biology. These deal with the evolution of modular proteins, the three-dimensional structure of domains and the ways in which these interact in a multidomain protein. They discuss structure-function relationships in calcium binding domains, collagen helices, alpha-helical coiled-coil domains and C-lectins. The present minireview is focused on some general aspects and serves as an introduction to the cluster.

  17. Towards rebuilding vaginal support utilizing an extracellular matrix bioscaffold.

    Science.gov (United States)

    Liang, Rui; Knight, Katrina; Easley, Deanna; Palcsey, Stacy; Abramowitch, Steven; Moalli, Pamela A

    2017-07-15

    As an alternative to polypropylene mesh, we explored an extracellular matrix (ECM) bioscaffold derived from urinary bladder matrix (MatriStem™) in the repair of vaginal prolapse. We aimed to restore disrupted vaginal support simulating application via transvaginal and transabdominal approaches in a macaque model focusing on the impact on vaginal structure, function, and the host immune response. In 16 macaques, after laparotomy, the uterosacral ligaments and paravaginal attachments to pelvic side wall were completely transected (IACUC# 13081928). 6-ply MatriStem was cut into posterior and anterior templates with a portion covering the vagina and arms simulating uterosacral ligaments and paravaginal attachments, respectively. After surgically exposing the correct anatomical sites, in 8 animals, a vaginal incision was made on the anterior and posterior vagina and the respective scaffolds were passed into the vagina via these incisions (transvaginal insertion) prior to placement. The remaining 8 animals underwent the same surgery without vaginal incisions (transabdominal insertion). Three months post implantation, firm tissue bands extending from vagina to pelvic side wall appeared in both MatriStem groups. Experimental endpoints examining impact of MatriStem on the vagina demonstrated that vaginal biochemical and biomechanical parameters, smooth muscle thickness and contractility, and immune responses were similar in the MatriStem no incision group and sham-operated controls. In the MatriStem incision group, a 41% decrease in vaginal stiffness (P=0.042), a 22% decrease in collagen content (P=0.008) and a 25% increase in collagen subtypes III/I was observed vs. Sham. Active MMP2 was increased in both Matristem groups vs. Sham (both P=0.002). This study presents a novel application of ECM bioscaffolds as a first step towards the rebuilding of vaginal support. Pelvic organ prolapse is a common condition related to failure of the supportive soft tissues of the vagina

  18. Raffinose Induces Biofilm Formation by Streptococcus mutans in Low Concentrations of Sucrose by Increasing Production of Extracellular DNA and Fructan.

    Science.gov (United States)

    Nagasawa, Ryo; Sato, Tsutomu; Senpuku, Hidenobu

    2017-08-01

    Streptococcus mutans is the primary etiological agent of dental caries and causes tooth decay by forming a firmly attached biofilm on tooth surfaces. Biofilm formation is induced by the presence of sucrose, which is a substrate for the synthesis of extracellular polysaccharides but not in the presence of oligosaccharides. Nonetheless, in this study, we found that raffinose, which is an oligosaccharide with an intestinal regulatory function and antiallergic effect, induced biofilm formation by S. mutans in a mixed culture with sucrose, which was at concentrations less than those required to induce biofilm formation directly. We analyzed the possible mechanism behind the small requirement for sucrose for biofilm formation in the presence of raffinose. Our results suggested that sucrose contributed to an increase in bacterial cell surface hydrophobicity and biofilm formation. Next, we examined how the effects of raffinose interacted with the effects of sucrose for biofilm formation. We showed that the presence of raffinose induced fructan synthesis by fructosyltransferase and aggregated extracellular DNA (eDNA, which is probably genomic DNA released from dead cells) into the biofilm. eDNA seemed to be important for biofilm formation, because the degradation of DNA by DNase I resulted in a significant reduction in biofilm formation. When assessing the role of fructan in biofilm formation, we found that fructan enhanced eDNA-dependent cell aggregation. Therefore, our results show that raffinose and sucrose have cooperative effects and that this induction of biofilm formation depends on supportive elements that mainly consist of eDNA and fructan. IMPORTANCE The sucrose-dependent mechanism of biofilm formation in Streptococcus mutans has been studied extensively. Nonetheless, the effects of carbohydrates other than sucrose are inadequately understood. Our findings concerning raffinose advance the understanding of the mechanism underlying the joint effects of sucrose and

  19. Iron Triggers λSo Prophage Induction and Release of Extracellular DNA in Shewanella oneidensis MR-1 Biofilms

    OpenAIRE

    Binnenkade, Lucas; Teichmann, Laura; Thormann, Kai M.

    2014-01-01

    Prophages are ubiquitous elements within bacterial chromosomes and affect host physiology and ecology in multiple ways. We have previously demonstrated that phage-induced lysis is required for extracellular DNA (eDNA) release and normal biofilm formation in Shewanella oneidensis MR-1. Here, we investigated the regulatory mechanisms of prophage λSo spatiotemporal induction in biofilms. To this end, we used a functional fluorescence fusion to monitor λSo activation in various mutant backgrounds...

  20. Quorum-sensing regulation of the biofilm matrix genes (pel) of Pseudomonas aeruginosa.

    Science.gov (United States)

    Sakuragi, Yumiko; Kolter, Roberto

    2007-07-01

    Quorum sensing (QS) has been previously shown to play an important role in the development of Pseudomonas aeruginosa biofilms (D. G. Davies et al., Science 280:295-298, 1998). Although QS regulation of swarming and DNA release has been shown to play important roles in biofilm development, regulation of genes directly involved in biosynthesis of biofilm matrix has not been described. Here, transcription of the pel operon, essential for the production of a glucose-rich matrix exopolysaccharide, is shown to be greatly reduced in lasI and rhlI mutants. Chemical complementation of the lasI mutant with 3-oxo-dodecanoyl homoserine lactone restores pel transcription to the wild-type level and biofilm formation ability. These findings thus connect QS signaling and transcription of genes responsible for biofilm matrix biosynthesis.

  1. A novel assay for extracellular matrix remodeling associated with liver fibrosis

    DEFF Research Database (Denmark)

    Barascuk, N; Veidal, S S; Larsen, L

    2010-01-01

    Accumulation of extracellular matrix (ECM) components and increased matrix-metalloprotease (MMPs) activity are hallmarks of fibrosis. We developed an ELISA for quantification of MMP-9 derived collagen type III (CO3) degradation.......Accumulation of extracellular matrix (ECM) components and increased matrix-metalloprotease (MMPs) activity are hallmarks of fibrosis. We developed an ELISA for quantification of MMP-9 derived collagen type III (CO3) degradation....

  2. Basic Components of Vascular Connective Tissue and Extracellular Matrix.

    Science.gov (United States)

    Halper, Jaroslava

    2018-01-01

    Though the composition of the three layers constituting the blood vessel wall varies among the different types of blood vessels, and some layers may even be missing in capillaries, certain basic components, and properties are shared by all blood vessels, though each histologically distinct layer contains a unique complement of extracellular components, growth factors and cytokines, and cell types as well. The structure and composition of vessel layers informs and is informed by the function of the particular blood vessel. The adaptation of the composition and the resulting function of the extracellular matrix (ECM) to changes in circulation/blood flow and a variety of other extravascular stimuli can be characterized as remodeling spearheaded by vascular cells. There is a surprising amount of cell traffic among the three layers. It starts with endothelial cell mediated transmigration of inflammatory cells from the bloodstream into the subendothelium, and then into tissue adjoining the blood vessel. Smooth muscle cells and a variety of adventitial cells reside in tunica media and tunica externa, respectively. The latter cells are a mixture of progenitor/stem cells, fibroblasts, myofibroblasts, pericytes, macrophages, and dendritic cells and respond to endothelial injury by transdifferentiation as they travel into the two inner layers, intima and media for corrective mission in the ECM composition. This chapter addresses the role of various vascular cell types and ECM components synthesized by them in maintenance of normal structure and in their contribution to major pathological processes, such as atherosclerosis, organ fibrosis, and diabetic retinopathy. © 2018 Elsevier Inc. All rights reserved.

  3. Contribution of Extracellular Polymeric Substances from Shewanella sp. HRCR-1 Biofilms to U(VI) Immobilization

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Bin; Ahmed, B.; Kennedy, David W.; Wang, Zheming; Shi, Liang; Marshall, Matthew J.; Fredrickson, Jim K.; Isern, Nancy G.; Majors, Paul D.; Beyenal, Haluk

    2011-06-05

    The goal of this study was to quantify the contribution of extracellular polymeric substances (EPS) in U(VI) immobilization by Shewanella sp. HRCR-1. Through comparison of U(VI) immobilization using cells with bound EPS (bEPS) and cells without EPS, we showed that i) bEPS from Shewanella sp. HRCR-1 biofilms contributed significantly to U(VI) immobilization, especially at low initial U(VI) concentrations, through both sorption and reduction; ii) bEPS could be considered as a functional extension of the cells for U(VI) immobilization and they likely play more important roles at initial U(VI) concentrations; and iii) U(VI) reduction efficiency was found to be dependent upon initial U(VI) concentration and the efficiency decreased at lower concentrations. To quantify relative contribution of sorption and reduction in U(VI) immobilization by EPS fractions, we isolated loosely associated EPS (laEPS) and bEPS from Shewanella sp. HRCR-1 biofilms grown in a hollow fiber membrane biofilm reactor and tested their reactivity with U(V). We found that, when in reduced form, the isolated cell-free EPS fractions could reduce U(VI). Polysaccharides in the EPS likely contributed to U(VI) sorption and dominated reactivity of laEPS while redox active components (e.g., outer membrane c-type cytochromes), especially in bEPS, might facilitate U(VI) reduction.

  4. Contribution of extracellular polymeric substances from Shewanella sp. HRCR-1 biofilms to U(VI) immobilization.

    Science.gov (United States)

    Cao, Bin; Ahmed, Bulbul; Kennedy, David W; Wang, Zheming; Shi, Liang; Marshall, Matthew J; Fredrickson, Jim K; Isern, Nancy G; Majors, Paul D; Beyenal, Haluk

    2011-07-01

    The goal of this study was to quantify the contribution of extracellular polymeric substances (EPS) to U(VI) immobilization by Shewanella sp. HRCR-1. Through comparison of U(VI) immobilization using cells with bound EPS (bEPS) and cells with minimal EPS, we show that (i) bEPS from Shewanella sp. HRCR-1 biofilms contribute significantly to U(VI) immobilization, especially at low initial U(VI) concentrations, through both sorption and reduction; (ii) bEPS can be considered a functional extension of the cells for U(VI) immobilization and they likely play more important roles at lower initial U(VI) concentrations; and (iii) the U(VI) reduction efficiency is dependent upon the initial U(VI) concentration and decreases at lower concentrations. To quantify the relative contributions of sorption and reduction to U(VI) immobilization by EPS fractions, we isolated loosely associated EPS (laEPS) and bEPS from Shewanella sp. HRCR-1 biofilms grown in a hollow fiber membrane biofilm reactor and tested their reactivity with U(VI). We found that, when reduced, the isolated cell-free EPS fractions could reduce U(VI). Polysaccharides in the EPS likely contributed to U(VI) sorption and dominated the reactivity of laEPS, while redox active components (e.g., outer membrane c-type cytochromes), especially in bEPS, possibly facilitated U(VI) reduction.

  5. The dynamic extracellular matrix: intervention strategies during heart failure and atherosclerosis

    NARCIS (Netherlands)

    Heeneman, Sylvia; Cleutjens, Jack P.; Faber, Birgit C.; Creemers, Esther E.; van Suylen, Robert-Jan; Lutgens, Esther; Cleutjens, Kitty B.; Daemen, Mat J.

    2003-01-01

    The extracellular matrix is no longer seen as the static embedding in which cells reside; it has been shown to be involved in cell proliferation, migration and cell-cell interactions. Turnover of the different extracellular matrix components is an active process with multiple levels of regulation.

  6. Biofilm Matrix Composition Affects the Susceptibility of Food Associated Staphylococci to Cleaning and Disinfection Agents

    Science.gov (United States)

    Fagerlund, Annette; Langsrud, Solveig; Heir, Even; Mikkelsen, Maria I.; Møretrø, Trond

    2016-01-01

    Staphylococci are frequently isolated from food processing environments, and it has been speculated whether survival after cleaning and disinfection with benzalkonium chloride (BC)-containing disinfectants is due to biofilm formation, matrix composition, or BC efflux mechanisms. Out of 35 food associated staphylococci, eight produced biofilm in a microtiter plate assay and were identified as Staphylococcus capitis (2), S. cohnii, S. epidermidis, S. lentus (2), and S. saprophyticus (2). The eight biofilm producing strains were characterized using whole genome sequencing. Three of these strains contained the ica operon responsible for production of a polysaccharide matrix, and formed a biofilm which was detached upon exposure to the polysaccharide degrading enzyme Dispersin B, but not Proteinase K or trypsin. These strains were more tolerant to the lethal effect of BC both in suspension and biofilm than the remaining five biofilm producing strains. The five BC susceptible strains were characterized by lack of the ica operon, and their biofilms were detached by Proteinase K or trypsin, but not Dispersin B, indicating that proteins were major structural components of their biofilm matrix. Several novel cell wall anchored repeat domain proteins with domain structures similar to that of MSCRAMM adhesins were identified in the genomes of these strains, potentially representing novel mechanisms of ica-independent biofilm accumulation. Biofilms from all strains showed similar levels of detachment after exposure to alkaline chlorine, which is used for cleaning in the food industry. Strains with qac genes encoding BC efflux pumps could grow at higher concentrations of BC than strains without these genes, but no differences were observed at biocidal concentrations. In conclusion, the biofilm matrix of food associated staphylococci varies with respect to protein or polysaccharide nature, and this may affect the sensitivity toward a commonly used disinfectant. PMID:27375578

  7. Biofilm matrix composition affects the susceptibility of food associated staphylococci to cleaning and disinfection agents

    Directory of Open Access Journals (Sweden)

    Annette eFagerlund

    2016-06-01

    Full Text Available Staphylococci are frequently isolated from food processing environments, and it has been speculated whether survival after cleaning and disinfection with benzalkonium chloride-containing disinfectants is due to biofilm formation, matrix composition or benzalkonium chloride efflux mechanisms. Out of 35 food associated staphylococci, eight produced biofilm in a microtiter plate assay and were identified as Staphylococcus capitis (2, S. cohnii, S. epidermidis, S. lentus (2, and S. saprophyticus (2. The eight biofilm producing strains were characterized using whole genome sequencing. Three of these strains contained the ica operon responsible for production of a polysaccharide matrix, and formed a biofilm which was detached upon exposure to the polysaccharide degrading enzyme Dispersin B, but not Proteinase K or trypsin. These strains were more tolerant to the lethal effect of benzalkonium chloride both in suspension and biofilm than the remaining five biofilm producing strains. The five benzalkonium chloride susceptible strains were characterized by lack of the ica operon, and their biofilms were detached by Proteinase K or trypsin, but not Dispersin B, indicating that proteins were major structural components of their biofilm matrix. Several novel cell wall anchored repeat domain proteins with domain structures similar to that of MSCRAMM adhesins were identified in the genomes of these strains, potentially representing novel mechanisms of ica-independent biofilm accumulation. Biofilms from all strains showed similar levels of detachment after exposure to alkaline chlorine, which is used for cleaning in the food industry. Strains with qac genes encoding benzalkonium chloride efflux pumps could grow at higher concentrations of benzalkonium chloride than strains without these genes, but no differences were observed at biocidal concentrations. In conclusion, the biofilm matrix of food associated staphylococci varies with respect to protein or

  8. Spermidine promotes Bacillus subtilis biofilm formation by activating expression of the matrix regulator slrR.

    Science.gov (United States)

    Hobley, Laura; Li, Bin; Wood, Jennifer L; Kim, Sok Ho; Naidoo, Jacinth; Ferreira, Ana Sofia; Khomutov, Maxim; Khomutov, Alexey; Stanley-Wall, Nicola R; Michael, Anthony J

    2017-07-21

    Ubiquitous polyamine spermidine is not required for normal planktonic growth of Bacillus subtilis but is essential for robust biofilm formation. However, the structural features of spermidine required for B. subtilis biofilm formation are unknown and so are the molecular mechanisms of spermidine-stimulated biofilm development. We report here that in a spermidine-deficient B. subtilis mutant, the structural analogue norspermidine, but not homospermidine, restored biofilm formation. Intracellular biosynthesis of another spermidine analogue, aminopropylcadaverine, from exogenously supplied homoagmatine also restored biofilm formation. The differential ability of C-methylated spermidine analogues to functionally replace spermidine in biofilm formation indicated that the aminopropyl moiety of spermidine is more sensitive to C -methylation, which it is essential for biofilm formation, but that the length and symmetry of the molecule is not critical. Transcriptomic analysis of a spermidine-depleted B. subtilis speD mutant uncovered a nitrogen-, methionine-, and S -adenosylmethionine-sufficiency response, resulting in repression of gene expression related to purine catabolism, methionine and S -adenosylmethionine biosynthesis and methionine salvage, and signs of altered membrane status. Consistent with the spermidine requirement in biofilm formation, single-cell analysis of this mutant indicated reduced expression of the operons for production of the exopolysaccharide and TasA protein biofilm matrix components and SinR antagonist slrR Deletion of sinR or ectopic expression of slrR in the spermidine-deficient Δ speD background restored biofilm formation, indicating that spermidine is required for expression of the biofilm regulator slrR Our results indicate that spermidine functions in biofilm development by activating transcription of the biofilm matrix exopolysaccharide and TasA operons through the regulator slrR . © 2017 by The American Society for Biochemistry and

  9. Astrocytes as a source for Extracellular matrix molecules and cytokines

    Directory of Open Access Journals (Sweden)

    Stefan eWiese

    2012-06-01

    Full Text Available Research of the past 25 years has shown that astrocytes do more than participating and building up the blood brain barrier and detoxify the active synapse by reuptake of neurotransmitters and ions. Indeed, astrocytes express neurotransmitter receptors and, as a consequence, respond to stimuli. Deeper knowledge of the differentiation processes during development of the central nervous system (CNS might help explaining and even help treating neurological diseases like Alzheimer’s disease, Amyotrophic lateral sclerosis (ALS and psychiatric disorders in which astrocytes have been shown to play a role. Astrocytes and oligodendrocytes develop from a multipotent stem cell that prior to this has produced primarily neuronal precursor cells. This switch towards the more astroglial differentiation is regulated by a change in receptor composition on the cell surface and responsiveness of the respective trophic factors Fibroblast growth factor (FGF and Epidermal growth factor (EGF. The glial precursor cell is driven into the astroglial direction by signaling molecules like Ciliary neurotrophic factor (CNTF, Bone Morphogenetic Proteins (BMPs, and EGF. However, the early astrocytes influence their environment not only by releasing and responding to diverse soluble factors but also express a wide range of extracellular matrix (ECM molecules, in particular proteoglycans of the lectican family and tenascins. Lately these ECM molecules have been shown to participate in glial development. In this regard, especially the matrix protein Tenascin C (Tnc proved to be an important regulator of astrocyte precursor cell proliferation and migration during spinal cord development. On the other hand, ECM molecules expressed by reactive astrocytes are also known to act mostly in an inhibitory fashion under pathophysiological conditions. In this regard, we further summarize recent data concerning the role of chondroitin sulfate proteoglycans and Tnc under pathological

  10. Roles of Extracellular Polysaccharides and Biofilm Formation in Heavy Metal Resistance of Rhizobia

    Directory of Open Access Journals (Sweden)

    Natalia Nocelli

    2016-05-01

    Full Text Available Bacterial surface components and extracellular compounds, particularly flagella, lipopolysaccharides (LPSs, and exopolysaccharides (EPSs, in combination with environmental signals and quorum-sensing signals, play crucial roles in bacterial autoaggregation, biofilm development, survival, and host colonization. The nitrogen-fixing species Sinorhizobium meliloti (S. meliloti produces two symbiosis-promoting EPSs: succinoglycan (or EPS I and galactoglucan (or EPS II. Studies of the S. meliloti/alfalfa symbiosis model system have revealed numerous biological functions of EPSs, including host specificity, participation in early stages of host plant infection, signaling molecule during plant development, and (most importantly protection from environmental stresses. We evaluated functions of EPSs in bacterial resistance to heavy metals and metalloids, which are known to affect various biological processes. Heavy metal resistance, biofilm production, and co-culture were tested in the context of previous studies by our group. A range of mercury (Hg II and arsenic (As III concentrations were applied to S. meliloti wild type strain and to mutant strains defective in EPS I and EPS II. The EPS production mutants were generally most sensitive to the metals. Our findings suggest that EPSs are necessary for the protection of bacteria from either Hg (II or As (III stress. Previous studies have described a pump in S. meliloti that causes efflux of arsenic from cells to surrounding culture medium, thereby protecting them from this type of chemical stress. The presence of heavy metals or metalloids in culture medium had no apparent effect on formation of biofilm, in contrast to previous reports that biofilm formation helps protect various microorganism species from adverse environmental conditions. In co-culture experiments, EPS-producing heavy metal resistant strains exerted a protective effect on AEPS-non-producing, heavy metal-sensitive strains; a phenomenon

  11. Single particle tracking reveals spatial and dynamic organization of the Escherichia coli biofilm matrix

    International Nuclear Information System (INIS)

    Birjiniuk, Alona; Doyle, Patrick S; Billings, Nicole; Ribbeck, Katharina; Nance, Elizabeth; Hanes, Justin

    2014-01-01

    Biofilms are communities of surface-adherent bacteria surrounded by secreted polymers known as the extracellular polymeric substance. Biofilms are harmful in many industries, and thus it is of great interest to understand their mechanical properties and structure to determine ways to destabilize them. By performing single particle tracking with beads of varying surface functionalization it was found that charge interactions play a key role in mediating mobility within biofilms. With a combination of single particle tracking and microrheological concepts, it was found that Escherichia coli biofilms display height dependent charge density that evolves over time. Statistical analyses of bead trajectories and confocal microscopy showed inter-connecting micron scale channels that penetrate throughout the biofilm, which may be important for nutrient transfer through the system. This methodology provides significant insight into a particular biofilm system and can be applied to many others to provide comparisons of biofilm structure. The elucidation of structure provides evidence for the permeability of biofilms to microscale objects, and the ability of a biofilm to mature and change properties over time. (paper)

  12. Extracellular matrix in lung development, homeostasis and disease.

    Science.gov (United States)

    Zhou, Yong; Horowitz, Jeffrey C; Naba, Alexandra; Ambalavanan, Namasivayam; Atabai, Kamran; Balestrini, Jenna; Bitterman, Peter B; Corley, Richard A; Ding, Bi-Sen; Engler, Adam J; Hansen, Kirk C; Hagood, James S; Kheradmand, Farrah; Lin, Qing S; Neptune, Enid; Niklason, Laura; Ortiz, Luis A; Parks, William C; Tschumperlin, Daniel J; White, Eric S; Chapman, Harold A; Thannickal, Victor J

    2018-03-08

    The lung's unique extracellular matrix (ECM), while providing structural support for cells, is critical in the regulation of developmental organogenesis, homeostasis and injury-repair responses. The ECM, via biochemical or biomechanical cues, regulates diverse cell functions, fate and phenotype. The composition and function of lung ECM become markedly deranged in pathological tissue remodeling. ECM-based therapeutics and bioengineering approaches represent promising novel strategies for regeneration/repair of the lung and treatment of chronic lung diseases. In this review, we assess the current state of lung ECM biology, including fundamental advances in ECM composition, dynamics, topography, and biomechanics; the role of the ECM in normal and aberrant lung development, adult lung diseases and autoimmunity; and ECM in the regulation of the stem cell niche. We identify opportunities to advance the field of lung ECM biology and provide a set recommendations for research priorities to advance knowledge that would inform novel approaches to the pathogenesis, diagnosis, and treatment of chronic lung diseases. Copyright © 2017. Published by Elsevier B.V.

  13. Biomarkers of the extracellular matrix and of collagen fragments.

    Science.gov (United States)

    Chalikias, Georgios K; Tziakas, Dimitrios N

    2015-03-30

    A great body of evidence has shown that extracellular matrix (ECM) alterations are present in the major types of cardiac diseases: ischemic heart disease, heart disease associated with pressure overload, heart disease associated with volume overload, and intrinsic myocardial disease or cardiomyopathy. Collagen, type I and III, is the principal structural protein found in the myocardium and its pro- or telopeptides are released into the circulation during the course of cardiovascular diseases. Therefore, these peptides may reflect collagen synthesis and break-down and also represent a much more useful tool to address ECM changes from a distance. Clinical trials have been performed during recent years to examine the usage of these peptides as diagnostic or prognostic biomarkers in heart failure (HF) patients. This review aims to summarize published data concerning cardiac ECM and its circulating biomarkers. Studies that focused on collagen metabolism related biomarkers in patients with HF are analyzed. Finally, limitations associated with the clinical use of the aforementioned biomarkers are also discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Extracellular Matrix as a Regulator of Epidermal Stem Cell Fate.

    Science.gov (United States)

    Chermnykh, Elina; Kalabusheva, Ekaterina; Vorotelyak, Ekaterina

    2018-03-27

    Epidermal stem cells reside within the specific anatomic location, called niche, which is a microenvironment that interacts with stem cells to regulate their fate. Regulation of many important processes, including maintenance of stem cell quiescence, self-renewal, and homeostasis, as well as the regulation of division and differentiation, are common functions of the stem cell niche. As it was shown in multiple studies, extracellular matrix (ECM) contributes a lot to stem cell niches in various tissues, including that of skin. In epidermis, ECM is represented, primarily, by a highly specialized ECM structure, basement membrane (BM), which separates the epidermal and dermal compartments. Epidermal stem cells contact with BM, but when they lose the contact and migrate to the overlying layers, they undergo terminal differentiation. When considering all of these factors, ECM is of fundamental importance in regulating epidermal stem cells maintenance, proper mobilization, and differentiation. Here, we summarize the remarkable progress that has recently been made in the research of ECM role in regulating epidermal stem cell fate, paying special attention to the hair follicle stem cell niche. We show that the destruction of ECM components impairs epidermal stem cell morphogenesis and homeostasis. A deep understanding of ECM molecular structure as well as the development of in vitro system for stem cell maintaining by ECM proteins may bring us to developing new approaches for regenerative medicine.

  15. Redox Signaling in Diabetic Wound Healing Regulates Extracellular Matrix Deposition.

    Science.gov (United States)

    Kunkemoeller, Britta; Kyriakides, Themis R

    2017-10-20

    Impaired wound healing is a major complication of diabetes, and can lead to development of chronic foot ulcers in a significant number of patients. Despite the danger posed by poor healing, very few specific therapies exist, leaving patients at risk of hospitalization, amputation, and further decline in overall health. Recent Advances: Redox signaling is a key regulator of wound healing, especially through its influence on the extracellular matrix (ECM). Normal redox signaling is disrupted in diabetes leading to several pathological mechanisms that alter the balance between reactive oxygen species (ROS) generation and scavenging. Importantly, pathological oxidative stress can alter ECM structure and function. There is limited understanding of the specific role of altered redox signaling in the diabetic wound, although there is evidence that ROS are involved in the underlying pathology. Preclinical studies of antioxidant-based therapies for diabetic wound healing have yielded promising results. Redox-based therapeutics constitute a novel approach for the treatment of wounds in diabetes patients that deserve further investigation. Antioxid. Redox Signal. 27, 823-838.

  16. Filamentation and spatiotemporal distribution of extracellular polymeric substances: role on X.fastidiosa single cell adhesion and biofilm formation (Conference Presentation)

    Science.gov (United States)

    Janissen, Richard; Murillo, Duber M.; Niza, Barbara; Sahoo, Prasana K.; Monteiro, Moniellen P.; César, Carlos L.; Carvalho, Hernandes F.; de Souza, Alessandra A.; Cotta, Monica A.

    2016-04-01

    Biofilms can be defined as a community of microorganisms attached to a surface, living embedded in a self- produced matrix of hydrated extracellular polymeric substances (EPS) which comprises most of the biofilm mass. We have recently used an extensive pool of microscopy techniques (confocal fluorescence, electron and scanning probe microscopies) at the micro and nanoscales in order to create a detailed temporal observation of Xylella fastidiosa biofilm formation, using both wild type strain and Green Fluorescent Protein (GFP)-modified cells of this citrus phytopathogen. We have identified three different EPS compositions, as well as their spatial and temporal distribution from single cell to mature biofilm formation stages. In the initial adhesion stage, soluble-EPS (S-EPS) accumulates at cell polar regions and forms a surface layer which facilitates irreversible cell attachment and cell cluster formation. These small clusters are subsequently connected by filamentous cells; further S-EPS surface coverage facilitates cell attachment and form filaments, leading to a floating framework of mature biofilms. The important role of EPS in X.fastidiosa biology was further investigated by imunolabelling experiments to detect the distribution of XadA1 adhesin, which is expressed in early stages of biofilm formation and released in outer membrane vesicles. This protein is located mainly in S-EPS covered areas, as well as on the filaments, indicating a molecular pathway to the enhanced cell attachment previously observed. These results suggest that S-EPS may thus represent an important target for disease control, slow plant colonization by the bacteria, keeping the plant more productive in the field.

  17. Characteristics of Alcian-blue Dye Adsorption of Natural Biofilm Matrix

    Science.gov (United States)

    Kurniawan, A.; Yamamoto, T.; Sukandar; Guntur

    2018-01-01

    In this study, natural biofilm matrices formed on stones have been used for the adsorption of Alcian blue dye. Alcian blue is a member of polyvalent basic dyes that largely used from laboratory until industrial dying purposes. The adsorption of the dye onto the biofilm matrix has been carried out at different experimental conditions such as adsorption isotherm and kinetic of adsorption. The electric charge properties of biofilm matrix and its changes related to the adsorption of Alcian blue have been also investigated. Moreover, the results of Alcian blue adsorption to the biofilm were compared to those onto the acidic and neutral resin. The kinetics of adsorption result showed that the adsorption of the Alcian blue dye reached to a maximum adsorption amount within 60 minutes. The adsorption amount of Alcian blue to biofilm increased monotonously, and the maximum adsorption amount was greater compared to the resins. On the contrary, Alcian blue did not attach to the neutral resin having no electric charge. It seems that Alcian blue attached to the acidic resins due to electrostatic attractive force, and the same seems to be the case for adsorption of Alcian blue to biofilm. The adsorption of Alcian blue to the biofilm and acidic resins fitted to Langmuir type indicates that the binding of Alcian blue to the biofilm and acidic resins occurred in a monolayer like form. The maximum adsorption amount of Alcian blue on the biofilm (0.24 mmol/dry-g) was greater than those of acidic resin (0.025 mmol/dry-g). This indicates that the biofilm has many more sites for Alcian blue attachment than acidic resins. According to the result of this study, the biofilm matrix can be a good adsorbent for dye such as Alcian blue or other dyes that causing hazards in nature.

  18. Commensal Protection of Staphylococcus aureus against Antimicrobials by Candida albicans Biofilm Matrix

    Science.gov (United States)

    Kong, Eric F.; Tsui, Christina; Kucharíková, Sona; Andes, David

    2016-01-01

    ABSTRACT Biofilm-associated polymicrobial infections, particularly those involving fungi and bacteria, are responsible for significant morbidity and mortality and tend to be challenging to treat. Candida albicans and Staphylococcus aureus specifically are considered leading opportunistic fungal and bacterial pathogens, respectively, mainly due to their ability to form biofilms on catheters and indwelling medical devices. However, the impact of mixed-species biofilm growth on therapy remains largely understudied. In this study, we investigated the influence of C. albicans secreted cell wall polysaccharides on the response of S. aureus to antibacterial agents in biofilm. Results demonstrated significantly enhanced tolerance for S. aureus to drugs in the presence of C. albicans or its secreted cell wall polysaccharide material. Fluorescence confocal time-lapse microscopy revealed impairment of drug diffusion through the mixed biofilm matrix. Using C. albicans mutant strains with modulated cell wall polysaccharide expression, exogenous supplementation, and enzymatic degradation, the C. albicans-secreted β-1,3-glucan cell wall component was identified as the key matrix constituent providing the bacteria with enhanced drug tolerance. Further, antibody labeling demonstrated rapid coating of the bacteria by the C. albicans matrix material. Importantly, via its effect on the fungal biofilm matrix, the antifungal caspofungin sensitized the bacteria to the drugs. Understanding such symbiotic interactions with clinical relevance between microbial species in biofilms will greatly aid in overcoming the limitations of current therapies and in defining potential new targets for treating polymicrobial infections. PMID:27729510

  19. The extracellular matrix deposited by asthmatic airway smooth muscle cells in a resting state reflects a healthy matrix

    NARCIS (Netherlands)

    Harkness, Louise; Ashton, Anthony; Burgess, Janette

    2015-01-01

    Introduction: The remodelled asthmatic airway features an altered extracellular matrix (ECM) & increased vasculature. Previous studies found asthmatic (A) airway smooth muscle cells (ASMCs) to deposit an ECM with enhanced bioactivity. These studies however investigated ECM deposited in the presence

  20. Intracellular Calreticulin Regulates Multiple Steps in Fibrillar Collagen Expression, Trafficking, and Processing into the Extracellular Matrix*

    OpenAIRE

    Van Duyn Graham, Lauren; Sweetwyne, Mariya T.; Pallero, Manuel A.; Murphy-Ullrich, Joanne E.

    2009-01-01

    Calreticulin (CRT), a chaperone and Ca2+ regulator, enhances wound healing, and its expression correlates with fibrosis in animal models, suggesting that CRT regulates production of the extracellular matrix. However, direct regulation of collagen matrix by CRT has not been previously demonstrated. We investigated the role of CRT in the regulation of fibrillar collagen expression, secretion, processing, and deposition in the extracellular matrix by fibroblasts. Mouse embryonic fibroblasts defi...

  1. Withania somnifera attenuates acid production, acid tolerance and extra-cellular polysaccharide formation of Streptococcus mutans biofilms.

    Science.gov (United States)

    Pandit, Santosh; Song, Kwang-Yeob; Jeon, Jae-Gyu

    2014-01-01

    Withania somnifera (Ashwagandha) is a plant of the Solanaceae family. It has been widely used as a remedy for a variety of ailments in India and Nepal. The plant has also been used as a controlling agent for dental diseases. The aim of the present study was to evaluate the activity of the methanol extract of W. somnifera against the physiological ability of cariogenic biofilms and to identify the components of the extract. To determine the activity of the extract, assays for sucrose-dependent bacterial adherence, glycolytic acid production, acid tolerance, and extracellular polysaccharide formation were performed using Streptococcus mutans biofilms. The viability change of S. mutans biofilms cells was also determined. A phytochemical analysis of the extract was performed using TLC and LC/MS/MS. The extract showed inhibitory effects on sucrose-dependent bacterial adherence (≥ 100 μg/ml), glycolytic acid production (≥ 300 μg/ml), acid tolerance (≥ 300 μg/ml), and extracellular polysaccharide formation (≥ 300 μg/ml) of S. mutans biofilms. However, the extract did not alter the viability of S. mutans biofilms cells in all concentrations tested. Based on the phytochemical analysis, the activity of the extract may be related to the presence of alkaloids, anthrones, coumarines, anthraquinones, terpenoids, flavonoids, and steroid lactones (withanolide A, withaferin A, withanolide B, withanoside IV, and 12-deoxy withastramonolide). These data indicate that W. somnifera may be a potential agent for restraining the physiological ability of cariogenic biofilms.

  2. Microbial ecology of phototrophic biofilms

    NARCIS (Netherlands)

    Roeselers, G.

    2007-01-01

    Biofilms are layered structures of microbial cells and an extracellular matrix of polymeric substances, associated with surfaces and interfaces. Biofilms trap nutrients for growth of the enclosed microbial community and help prevent detachment of cells from surfaces in flowing systems. Phototrophic

  3. Helicobacter pylori ATCC 43629/NCTC 11639 Outer Membrane Vesicles (OMVs) from Biofilm and Planktonic Phase Associated with Extracellular DNA (eDNA)

    Science.gov (United States)

    Grande, Rossella; Di Marcantonio, Maria C.; Robuffo, Iole; Pompilio, Arianna; Celia, Christian; Di Marzio, Luisa; Paolino, Donatella; Codagnone, Marilina; Muraro, Raffaella; Stoodley, Paul; Hall-Stoodley, Luanne; Mincione, Gabriella

    2015-01-01

    Helicobacter pylori persistence is associated with its capacity to develop biofilms as a response to changing environmental conditions and stress. Extracellular DNA (eDNA) is a component of H. pylori biofilm matrix but the lack of DNase I activity supports the hypothesis that eDNA might be protected by other extracellular polymeric substances (EPS) and/or Outer Membrane Vesicles (OMVs), which bleb from the bacteria surface during growth. The aim of the present study was to both identify the eDNA presence on OMVs segregated from H. pylori ATCC 43629/NCTC 11639 biofilm (bOMVs) and its planktonic phase (pOMVs) and to characterize the physical-chemical properties of the OMVs. The presence of eDNA in bOMVs and pOMVs was initially carried out using DNase I-gold complex labeling and Transmission Electron Microscope analysis (TEM). bOMVs and pOMVs were further isolated and physical-chemical characterization carried out using dynamic light scattering (DLS) analysis. eDNA associated with OMVs was detected and quantified using a PicoGreen spectrophotometer assay, while its extraction was performed with a DNA Kit. TEM images showed that eDNA was mainly associated with the OMV membrane surfaces; while PicoGreen staining showed a four-fold increase of dsDNA in bOMVs compared with pOMVs. The eDNA extracted from OMVs was visualized using gel electrophoresis. DLS analysis indicated that both planktonic and biofilm H. pylori phenotypes generated vesicles, with a broad distribution of sizes on the nanometer scale. The DLS aggregation assay suggested that eDNA may play a role in the aggregation of OMVs, in the biofilm phenotype. Moreover, the eDNA associated with vesicle membrane may impede DNase I activity on H. pylori biofilms. These results suggest that OMVs derived from the H. pylori biofilm phenotype may play a structural role by preventing eDNA degradation by nucleases, providing a bridging function between eDNA strands on OMV surfaces and promoting aggregation. PMID:26733944

  4. The emerging role of skeletal muscle extracellular matrix remodelling in obesity and exercise.

    Science.gov (United States)

    Martinez-Huenchullan, S; McLennan, S V; Verhoeven, A; Twigg, S M; Tam, C S

    2017-07-01

    Skeletal muscle extracellular matrix remodelling has been proposed as a new feature associated with obesity and metabolic dysfunction. Exercise training improves muscle function in obesity, which may be mediated by regulatory effects on the muscle extracellular matrix. This review examined available literature on skeletal muscle extracellular matrix remodelling during obesity and the effects of exercise. A non-systematic literature review was performed on PubMed of publications from 1970 to 2015. A total of 37 studies from humans and animals were retained. Studies reported overall increases in gene and protein expression of different types of collagen, growth factors and enzymatic regulators of the skeletal muscle extracellular matrix in obesity. Only two studies investigated the effects of exercise on skeletal muscle extracellular matrix during obesity, with both suggesting a regulatory effect of exercise. The effects of exercise on muscle extracellular matrix seem to be influenced by the duration and type of exercise training with variable effects from a single session compared with a longer duration of exercise. More studies are needed to elucidate the mechanisms behind skeletal muscle extracellular matrix remodelling during obesity and the effects of exercise. © 2017 World Obesity Federation.

  5. Biofouling of reverse osmosis membranes: effects of cleaning on biofilm microbial communities, membrane performance, and adherence of extracellular polymeric substances.

    Science.gov (United States)

    Al Ashhab, Ashraf; Sweity, Amer; Bayramoglu, Bihter; Herzberg, Moshe; Gillor, Osnat

    2017-05-01

    Laboratory-scale reverse osmosis (RO) flat-sheet systems were used with two parallel flow cells, one treated with cleaning agents and a control (ie undisturbed). The cleaning efforts increased the affinity of extracellular polymeric substances (EPS) to the RO membrane and altered the biofilm surface structure. Analysis of the membrane biofilm community composition revealed the dominance of Proteobacteria. However, within the phylum Proteobacteria, γ-Proteobacteria dominated the cleaned membrane biofilm, while β-Proteobacteria dominated the control biofilm. The composition of the fungal phyla was also altered by cleaning, with enhancement of Ascomycota and suppression of Basidiomycota. The results suggest that repeated cleaning cycles select for microbial groups that strongly attach to the RO membrane surface by producing rigid and adhesive EPS that hampers membrane performance.

  6. Susceptibility patterns and the role of extracellular DNA in Staphylococcus epidermidis biofilm resistance to physico-chemical stress exposure.

    Science.gov (United States)

    Olwal, Charles Ochieng'; Ang'ienda, Paul Oyieng'; Onyango, David Miruka; Ochiel, Daniel Otieno

    2018-05-02

    Over 65% of human infections are ascribed to bacterial biofilms that are often highly resistant to antibiotics and host immunity. Staphylococcus epidermidis is the predominant cause of recurrent nosocomial and biofilm-related infections. However, the susceptibility patterns of S. epidermidis biofilms to physico-chemical stress induced by commonly recommended disinfectants [(heat, sodium chloride (NaCl), sodium hypochlorite (NaOCl) and hydrogen peroxide (H 2 O 2 )] in domestic and human healthcare settings remains largely unknown. Further, the molecular mechanisms of bacterial biofilms resistance to the physico-chemical stresses remain unclear. Growing evidence demonstrates that extracellular DNA (eDNA) protects bacterial biofilms against antibiotics. However, the role of eDNA as a potential mechanism underlying S. epidermidis biofilms resistance to physico-chemical stress exposure is yet to be understood. Therefore, this study aimed to evaluate the susceptibility patterns of and eDNA release by S. epidermidis biofilm and planktonic cells to physico-chemical stress exposure. S. epidermidis biofilms exposed to physico-chemical stress conditions commonly recommended for disinfection [heat (60 °C), 1.72 M NaCl, solution containing 150 μL of waterguard (0.178 M NaOCl) in 1 L of water or 1.77 M H 2 O 2 ] for 30 and 60 min exhibited lower log reductions of CFU/mL than the corresponding planktonic cells (p chemical stress induced by the four commonly recommended disinfectants than the analogous planktonic cells. Further, S. epidermidis biofilms enhanced eDNA release in response to the sub-lethal heat and oxidative stress exposure than the corresponding planktonic cells suggesting a role of eDNA in biofilms resistance to the physico-chemical stresses.

  7. Glia and extracellular matrix changes affect extracellular diffusion and volume transmission in the brain in health and disease

    Czech Academy of Sciences Publication Activity Database

    Vargová, Lýdia; Syková, Eva

    2011-01-01

    Roč. 59, S1 (2011), S38 ISSN 0894-1491. [European meeting on Glia l Cells in Health and Disease /10./. 13.09.2011-17.09.2011, Prague] Institutional research plan: CEZ:AV0Z50390512; CEZ:AV0Z50390703 Keywords : diffusion * extracellular matrix * extrasynaptic transmission Subject RIV: FH - Neurology

  8. Atomic Force Microscopy (AFM) for In-Situ Biofilm Surface Characterization during Free Chlorine and Monochloramine Exposure

    Science.gov (United States)

    Drinking water distribution system biofilm are attached to pipe walls and found in sediments. These biofilms are complex and contain a variety of microorganisms embedded in a matrix with extracellular polymeric substances (EPS), providing protection from disinfection. Without pro...

  9. Streptococcus mutans Extracellular DNA Is Upregulated during Growth in Biofilms, Actively Released via Membrane Vesicles, and Influenced by Components of the Protein Secretion Machinery

    Science.gov (United States)

    Liao, Sumei; Klein, Marlise I.; Heim, Kyle P.; Fan, Yuwei; Bitoun, Jacob P.; Ahn, San-Joon; Burne, Robert A.; Koo, Hyun; Brady, L. Jeannine

    2014-01-01

    Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA. PMID:24748612

  10. Adapted Boolean network models for extracellular matrix formation

    Directory of Open Access Journals (Sweden)

    Wollbold Johannes

    2009-07-01

    Full Text Available Abstract Background Due to the rapid data accumulation on pathogenesis and progression of chronic inflammation, there is an increasing demand for approaches to analyse the underlying regulatory networks. For example, rheumatoid arthritis (RA is a chronic inflammatory disease, characterised by joint destruction and perpetuated by activated synovial fibroblasts (SFB. These abnormally express and/or secrete pro-inflammatory cytokines, collagens causing joint fibrosis, or tissue-degrading enzymes resulting in destruction of the extra-cellular matrix (ECM. We applied three methods to analyse ECM regulation: data discretisation to filter out noise and to reduce complexity, Boolean network construction to implement logic relationships, and formal concept analysis (FCA for the formation of minimal, but complete rule sets from the data. Results First, we extracted literature information to develop an interaction network containing 18 genes representing ECM formation and destruction. Subsequently, we constructed an asynchronous Boolean network with biologically plausible time intervals for mRNA and protein production, secretion, and inactivation. Experimental gene expression data was obtained from SFB stimulated by TGFβ1 or by TNFα and discretised thereafter. The Boolean functions of the initial network were improved iteratively by the comparison of the simulation runs to the experimental data and by exploitation of expert knowledge. This resulted in adapted networks for both cytokine stimulation conditions. The simulations were further analysed by the attribute exploration algorithm of FCA, integrating the observed time series in a fine-tuned and automated manner. The resulting temporal rules yielded new contributions to controversially discussed aspects of fibroblast biology (e.g., considerable expression of TNF and MMP9 by fibroblasts stimulation and corroborated previously known facts (e.g., co-expression of collagens and MMPs after TNF

  11. Role of the extracellular matrix during neural crest cell migration.

    Science.gov (United States)

    Perris, R; Perissinotto, D

    2000-07-01

    Once specified to become neural crest (NC), cells occupying the dorsal portion of the neural tube disrupt their cadherin-mediated cell-cell contacts, acquire motile properties, and embark upon an extensive migration through the embryo to reach their ultimate phenotype-specific sites. The understanding of how this movement is regulated is still rather fragmentary due to the complexity of the cellular and molecular interactions involved. An additional intricate aspect of the regulation of NC cell movement is that the timings, modes and patterns of NC cell migration are intimately associated with the concomitant phenotypic diversification that cells undergo during their migratory phase and the fact that these changes modulate the way that moving cells interact with their microenvironment. To date, two interplaying mechanisms appear central for the guidance of the migrating NC cells through the embryo: one involves secreted signalling molecules acting through their cognate protein kinase/phosphatase-type receptors and the other is contributed by the multivalent interactions of the cells with their surrounding extracellular matrix (ECM). The latter ones seem fundamental in light of the central morphogenetic role played by the intracellular signals transduced through the cytoskeleton upon integrin ligation, and the convergence of these signalling cascades with those triggered by cadherins, survival/growth factor receptors, gap junctional communications, and stretch-activated calcium channels. The elucidation of the importance of the ECM during NC cell movement is presently favoured by the augmenting knowledge about the macromolecular structure of the specific ECM assembled during NC development and the functional assaying of its individual constituents via molecular and genetic manipulations. Collectively, these data propose that NC cell migration may be governed by time- and space-dependent alterations in the expression of inhibitory ECM components; the relative ratio

  12. Supercritical carbon dioxide extracted extracellular matrix material from adipose tissue

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jun Kit; Luo, Baiwen; Guneta, Vipra [School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 (Singapore); Li, Liang; Foo, Selin Ee Min [School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Dai, Yun; Tan, Timothy Thatt Yang [School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459 (Singapore); Tan, Nguan Soon [School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, 61 Biopolis Drive, Proteos, Singapore 138673 (Singapore); KK Research Centre, KK Women' s and Children' s Hospital, 100 Bukit Timah Road, Singapore 229899 (Singapore); Choong, Cleo, E-mail: cleochoong@ntu.edu.sg [School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 (Singapore); KK Research Centre, KK Women' s and Children' s Hospital, 100 Bukit Timah Road, Singapore 229899 (Singapore); Wong, Marcus Thien Chong [Plastic, Reconstructive & Aesthetic Surgery, Tan Tock Seng Hospital, 11 Jalan Tan Tock Seng, Singapore 308433 (Singapore)

    2017-06-01

    Adipose tissue is a rich source of extracellular matrix (ECM) material that can be isolated by delipidating and decellularizing the tissue. However, the current delipidation and decellularization methods either involve tedious and lengthy processes or require toxic chemicals, which may result in the elimination of vital proteins and growth factors found in the ECM. Hence, an alternative delipidation and decellularization method for adipose tissue was developed using supercritical carbon dioxide (SC-CO{sub 2}) that eliminates the need of any harsh chemicals and also reduces the amount of processing time required. The resultant SC-CO{sub 2}-treated ECM material showed an absence of nuclear content but the preservation of key proteins such as collagen Type I, collagen Type III, collagen Type IV, elastin, fibronectin and laminin. In addition, other biological factors such as glycosaminoglycans (GAGs) and growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were also retained. Subsequently, the resulting SC-CO{sub 2}-treated ECM material was used as a bioactive coating on tissue culture plastic (TCP). Four different cell types including adipose tissue-derived mesenchymal stem cells (ASCs), human umbilical vein endothelial cells (HUVECs), immortalized human keratinocyte (HaCaT) cells and human monocytic leukemia cells (THP-1) were used in this study to show that the SC-CO{sub 2}-treated ECM coating can be potentially used for various biomedical applications. The SC-CO{sub 2}-treated ECM material showed improved cell-material interactions for all cell types tested. In addition, in vitro scratch wound assay using HaCaT cells showed that the presence of SC-CO{sub 2}-treated ECM material enhanced keratinocyte migration whilst the in vitro cellular studies using THP-1-derived macrophages showed that the SC-CO{sub 2}-treated ECM material did not evoke pro-inflammatory responses from the THP-1-derived macrophages. Overall

  13. Supercritical carbon dioxide extracted extracellular matrix material from adipose tissue.

    Science.gov (United States)

    Wang, Jun Kit; Luo, Baiwen; Guneta, Vipra; Li, Liang; Foo, Selin Ee Min; Dai, Yun; Tan, Timothy Thatt Yang; Tan, Nguan Soon; Choong, Cleo; Wong, Marcus Thien Chong

    2017-06-01

    Adipose tissue is a rich source of extracellular matrix (ECM) material that can be isolated by delipidating and decellularizing the tissue. However, the current delipidation and decellularization methods either involve tedious and lengthy processes or require toxic chemicals, which may result in the elimination of vital proteins and growth factors found in the ECM. Hence, an alternative delipidation and decellularization method for adipose tissue was developed using supercritical carbon dioxide (SC-CO 2 ) that eliminates the need of any harsh chemicals and also reduces the amount of processing time required. The resultant SC-CO 2 -treated ECM material showed an absence of nuclear content but the preservation of key proteins such as collagen Type I, collagen Type III, collagen Type IV, elastin, fibronectin and laminin. In addition, other biological factors such as glycosaminoglycans (GAGs) and growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were also retained. Subsequently, the resulting SC-CO 2 -treated ECM material was used as a bioactive coating on tissue culture plastic (TCP). Four different cell types including adipose tissue-derived mesenchymal stem cells (ASCs), human umbilical vein endothelial cells (HUVECs), immortalized human keratinocyte (HaCaT) cells and human monocytic leukemia cells (THP-1) were used in this study to show that the SC-CO 2 -treated ECM coating can be potentially used for various biomedical applications. The SC-CO 2 -treated ECM material showed improved cell-material interactions for all cell types tested. In addition, in vitro scratch wound assay using HaCaT cells showed that the presence of SC-CO 2 -treated ECM material enhanced keratinocyte migration whilst the in vitro cellular studies using THP-1-derived macrophages showed that the SC-CO 2 -treated ECM material did not evoke pro-inflammatory responses from the THP-1-derived macrophages. Overall, this study shows the efficacy

  14. Adaptive evolution of the matrix extracellular phosphoglycoprotein in mammals

    Directory of Open Access Journals (Sweden)

    Machado João

    2011-11-01

    Full Text Available Abstract Background Matrix extracellular phosphoglycoprotein (MEPE belongs to a family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs that play a key role in skeleton development, particularly in mineralization, phosphate regulation and osteogenesis. MEPE associated disorders cause various physiological effects, such as loss of bone mass, tumors and disruption of renal function (hypophosphatemia. The study of this developmental gene from an evolutionary perspective could provide valuable insights on the adaptive diversification of morphological phenotypes in vertebrates. Results Here we studied the adaptive evolution of the MEPE gene in 26 Eutherian mammals and three birds. The comparative genomic analyses revealed a high degree of evolutionary conservation of some coding and non-coding regions of the MEPE gene across mammals indicating a possible regulatory or functional role likely related with mineralization and/or phosphate regulation. However, the majority of the coding region had a fast evolutionary rate, particularly within the largest exon (1467 bp. Rodentia and Scandentia had distinct substitution rates with an increased accumulation of both synonymous and non-synonymous mutations compared with other mammalian lineages. Characteristics of the gene (e.g. biochemical, evolutionary rate, and intronic conservation differed greatly among lineages of the eight mammalian orders. We identified 20 sites with significant positive selection signatures (codon and protein level outside the main regulatory motifs (dentonin and ASARM suggestive of an adaptive role. Conversely, we find three sites under selection in the signal peptide and one in the ASARM motif that were supported by at least one selection model. The MEPE protein tends to accumulate amino acids promoting disorder and potential phosphorylation targets. Conclusion MEPE shows a high number of selection signatures, revealing the crucial role of positive selection in the

  15. Supercritical carbon dioxide extracted extracellular matrix material from adipose tissue

    International Nuclear Information System (INIS)

    Wang, Jun Kit; Luo, Baiwen; Guneta, Vipra; Li, Liang; Foo, Selin Ee Min; Dai, Yun; Tan, Timothy Thatt Yang; Tan, Nguan Soon; Choong, Cleo; Wong, Marcus Thien Chong

    2017-01-01

    Adipose tissue is a rich source of extracellular matrix (ECM) material that can be isolated by delipidating and decellularizing the tissue. However, the current delipidation and decellularization methods either involve tedious and lengthy processes or require toxic chemicals, which may result in the elimination of vital proteins and growth factors found in the ECM. Hence, an alternative delipidation and decellularization method for adipose tissue was developed using supercritical carbon dioxide (SC-CO 2 ) that eliminates the need of any harsh chemicals and also reduces the amount of processing time required. The resultant SC-CO 2 -treated ECM material showed an absence of nuclear content but the preservation of key proteins such as collagen Type I, collagen Type III, collagen Type IV, elastin, fibronectin and laminin. In addition, other biological factors such as glycosaminoglycans (GAGs) and growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were also retained. Subsequently, the resulting SC-CO 2 -treated ECM material was used as a bioactive coating on tissue culture plastic (TCP). Four different cell types including adipose tissue-derived mesenchymal stem cells (ASCs), human umbilical vein endothelial cells (HUVECs), immortalized human keratinocyte (HaCaT) cells and human monocytic leukemia cells (THP-1) were used in this study to show that the SC-CO 2 -treated ECM coating can be potentially used for various biomedical applications. The SC-CO 2 -treated ECM material showed improved cell-material interactions for all cell types tested. In addition, in vitro scratch wound assay using HaCaT cells showed that the presence of SC-CO 2 -treated ECM material enhanced keratinocyte migration whilst the in vitro cellular studies using THP-1-derived macrophages showed that the SC-CO 2 -treated ECM material did not evoke pro-inflammatory responses from the THP-1-derived macrophages. Overall, this study shows the efficacy

  16. Effect of Electrolyzed Water on the Disinfection of Bacillus cereus Biofilms: The Mechanism of Enhanced Resistance of Sessile Cells in the Biofilm Matrix.

    Science.gov (United States)

    Hussain, Mohammad Shakhawat; Kwon, Minyeong; Tango, Charles Nkufi; Oh, Deog Hwan

    2018-05-01

    This study examined the disinfection efficacy and mechanism of electrolyzed water (EW) on Bacillus cereus biofilms. B. cereus strains, ATCC 14579 and Korean Collection for Type Cultures (KCTC) 13153 biofilms, were formed on stainless steel (SS) and plastic slide (PS) coupons. Mature biofilms were treated with slightly acidic EW (SAEW), acidic EW (AEW), and basic EW (BEW). SAEW (available chlorine concentration, 25 ± 1.31 mg L -1 ; pH 5.71 ± 0.16; and oxidation reduction potential, 818 to 855 mV) reduced ATCC 14579 biofilms on plastic slides to below the detection limit within 30 s. However, biofilms on SS coupons showed a higher resistance to the SAEW treatment. When the disinfection activities of three types of EW on biofilms were compared, AEW showed a higher bactericidal activity, followed by SAEW and BEW. In contrast, BEW showed a significantly ( P biofilm dispersal activity than AEW and SAEW. SAEW disinfection of the B. cereus biofilms was due to the disruption of the B. cereus plasma membrane. The higher resistance of biofilms formed on the SS coupon might be due to the higher number of attached cells and extracellular polymeric substances formation that reacts with the active chlorine ions, such as hypochlorous acid and hypochlorite ion of SAEW, which decreased the disinfection efficacy of SAEW. This study showed that the EW treatment effectively disinfected B. cereus biofilms, providing insight into the potential use of EW in the food processing industry to control the biofilm formation of B. cereus.

  17. Material properties of biofilms – key methods for understanding permeability and mechanics

    Science.gov (United States)

    Billings, Nicole; Birjiniuk, Alona; Samad, Tahoura S.; Doyle, Patrick S.; Ribbeck, Katharina

    2015-01-01

    Microorganisms can form biofilms, which are multicellular communities surrounded by a hydrated extracellular matrix of polymers. Central properties of the biofilm are governed by this extracellular matrix, which provides mechanical stability to the three-dimensional biofilm structure, regulates the ability of the biofilm to adhere to surfaces, and determines the ability of the biofilm to adsorb gasses, solutes, and foreign cells. Despite their critical relevance for understanding and eliminating of biofilms, the materials properties of the extracellular matrix are understudied. Here, we offer the reader a guide to current technologies that can be utilized to specifically assess the permeability and mechanical properties of the biofilm matrix and its interacting components. In particular, we highlight technological advances in instrumentation and interactions between multiple disciplines that have broadened the spectrum of methods available to conduct these studies. We review pioneering work that furthers our understanding of the material properties of biofilms. PMID:25719969

  18. Extracellular matrix organization in developing muscle: correlation with acetylcholine receptor aggregates.

    Science.gov (United States)

    Bayne, E K; Anderson, M J; Fambrough, D M

    1984-10-01

    Monoclonal antibodies recognizing laminin, heparan sulfate proteoglycan, fibronectin, and two apparently novel connective tissue components have been used to examine the organization of extracellular matrix of skeletal muscle in vivo and in vitro. Four of the five monoclonal antibodies are described for the first time here. Immunocytochemical experiments with frozen-sectioned muscle demonstrated that both the heparan sulfate proteoglycan and laminin exhibited staining patterns identical to that expected for components of the basal lamina. In contrast, the remaining matrix constituents were detected in all regions of muscle connective tissue: the endomysium, perimysium, and epimysium. Embryonic muscle cells developing in culture elaborated an extracellular matrix, each antigen exhibiting a unique distribution. Of particular interest was the organization of extracellular matrix on myotubes: the build-up of matrix components was most apparent in plaques overlying clusters of an integral membrane protein, the acetylcholine receptor (AChR). The heparan sulfate proteoglycan was concentrated at virtually all AChR clusters and showed a remarkable level of congruence with receptor organization; laminin was detected at 70-95% of AChR clusters but often was not completely co-distributed with AChR within the cluster; fibronectin and the two other extracellular matrix antigens occurred at approximately 20, 8, and 2% of the AChR clusters, respectively, and showed little or no congruence with AChR. From observations on the distribution of extracellular matrix components in tissue cultured fibroblasts and myogenic cells, several ideas about the organization of extracellular matrix are suggested. (a) Congruence between AChR clusters and heparan sulfate proteoglycan suggests the existence of some linkage between the two molecules, possibly important for regulation of AChR distribution within the muscle membrane. (b) The qualitatively different patterns of extracellular matrix

  19. The matrikine N-α-PGP couples extracellular matrix fragmentation to endothelial permeability

    NARCIS (Netherlands)

    Hahn, Cornelia S; Scott, David W; Xu, Xin; Roda, Mojtaba Abdul; Payne, Gregory A; Wells, J Michael; Viera, Liliana; Winstead, Colleen J; Bratcher, Preston; Sparidans, Rolf W; Redegeld, Frank A; Jackson, Patricia L; Folkerts, Gert; Blalock, J Edwin; Patel, Rakesh P; Gaggar, Amit

    2015-01-01

    The compartmentalization and transport of proteins and solutes across the endothelium is a critical biologic function altered during inflammation and disease, leading to pathology in multiple disorders. The impact of tissue damage and subsequent extracellular matrix (ECM) fragmentation in regulating

  20. FK506 protects against articular cartilage collagenous extra-cellular matrix degradation

    NARCIS (Netherlands)

    M. Siebelt (Michiel); A.E. van der Windt (Anna); H.C. Groen (Harald); M. Sandker (Marjan); J.H. Waarsing (Jan); C. Müller (Cristina); M. de Jong (Marcel); H. Jahr (Holger); H.H. Weinans (Harrie)

    2014-01-01

    textabstractObjective: Osteoarthritis (OA) is a non-rheumatologic joint disease characterized by progressive degeneration of the cartilage extra-cellular matrix (ECM), enhanced subchondral bone remodeling, activation of synovial macrophages and osteophyte growth. Inhibition of calcineurin (Cn)

  1. hMSCs Cultured on Plant-Derived Tissue Engineering Extracellular Matrix in a Microgravity Environment

    Data.gov (United States)

    National Aeronautics and Space Administration — The objective of this proposal is to fabricate an all plant-derived renewable, biodegradable complete mimic of the bone extracellular matrix (ECM). For the first...

  2. Fibulin-1 is a marker for arterial extracellular matrix alterations in type 2 diabetes

    DEFF Research Database (Denmark)

    Cangemi, Claudia; Skov, Vibe; Poulsen, Michael Kjaer

    2011-01-01

    Extracellular matrix alterations are important elements in the arterial changes seen in diabetes, being associated with increased vascular stiffness and the development of cardiovascular diseases. However, no biomarkers for diabetes-related arterial changes have been defined.......Extracellular matrix alterations are important elements in the arterial changes seen in diabetes, being associated with increased vascular stiffness and the development of cardiovascular diseases. However, no biomarkers for diabetes-related arterial changes have been defined....

  3. A cathelicidin-2-derived peptide effectively impairs Staphylococcus epidermidis biofilms

    NARCIS (Netherlands)

    Molhoek, E.M.; van Dijk, A.; Veldhuizen, E.J.A.; Haagsman, H.P.; Bikker, F.J.

    2011-01-01

    Staphylococcus epidermidis is a major cause of nosocomial infections owing to its ability to form biofilms on the surface of medical devices. Biofilms are surface-adhered bacterial communities. In mature biofilms these communities are encased in an extracellular matrix composed of bacterial

  4. The extracellular matrix: Structure, composition, age-related differences, tools for analysis and applications for tissue engineering.

    Science.gov (United States)

    Kular, Jaspreet K; Basu, Shouvik; Sharma, Ram I

    2014-01-01

    The extracellular matrix is a structural support network made up of diverse proteins, sugars and other components. It influences a wide number of cellular processes including migration, wound healing and differentiation, all of which is of particular interest to researchers in the field of tissue engineering. Understanding the composition and structure of the extracellular matrix will aid in exploring the ways the extracellular matrix can be utilised in tissue engineering applications especially as a scaffold. This review summarises the current knowledge of the composition, structure and functions of the extracellular matrix and introduces the effect of ageing on extracellular matrix remodelling and its contribution to cellular functions. Additionally, the current analytical technologies to study the extracellular matrix and extracellular matrix-related cellular processes are also reviewed.

  5. Expressions of matrix metalloproteinase-2 and extracellular matrix metalloproteinase inducer in retinoblastoma

    Directory of Open Access Journals (Sweden)

    Yu-Hong Cheng

    2015-07-01

    Full Text Available AIM: To investigate expressions of matrix metalloproteinase-2(MMP-2and extracellular matrix metalloproteinase inducer(EMMPRINin retinoblastoma(Rband the relationships between MMP-2, EMMPRIN and tumor development.METHODS:Immunohistochemical technique was used to detect expressions of MMP-2 and EMMPRIN in 39 cases of paraffin embedded Rb samples. Quantitative analysis of expressions of MMP-2 and EMMPRIN were assessed by measuring the mean gray scale of Rb tissue with LEICA IM50 Color Pathologic Analysis System. The differences of expressions of MMP-2 and EMMPRIN in each clinical and pathological stage were statistically analyzed, and the same step was also undertaken to study the relationship between Rb with MMP-2 positive expression and that with EMMPRIN positive expression.RESULTS: The positive expression rate of MMP-2 was 90%(Gray value: 109.64±14.52; 35/39, and that of EMMPRIN was 85%(Gray value: 108.01±13.60; 33/39. The expressions of MMP-2 and EMMPRIN were significantly higher in tumors of glaucomatous stage(Gray value: 108.21±11.47 and 107.56±14.32than those in intraocular stage(Gray value: 121.13±11.32 and 119.34±12.66; PPPPPPCONCLUSION: The positive expression levels of MMP-2 and EMMPRIN may correlate with tumor infiltration and metastasis.

  6. Extracellular matrix remodeling and matrix metalloproteinase inhibition in visceral adipose during weight cycling in mice.

    Science.gov (United States)

    Caria, Cíntia Rabelo E Paiva; Gotardo, Érica Martins Ferreira; Santos, Paola Souza; Acedo, Simone Coghetto; de Morais, Thainá Rodrigues; Ribeiro, Marcelo Lima; Gambero, Alessandra

    2017-10-15

    Extracellular matrix (ECM) remodeling is necessary for a health adipose tissue (AT) expansion and also has a role during weight loss. We investigate the ECM alteration during weight cycling (WC) in mice and the role of matrix metalloproteinases (MMPs) was assessed using GM6001, an MMP inhibitor, during weight loss (WL). Obesity was induced in mice by a high-fat diet. Obese mice were subject to caloric restriction for WL followed by reintroduction to high-fat diet for weight regain (WR), resulting in a WC protocol. In addition, mice were treated with GM6001 during WL period and the effects were observed after WR. Activity and expression of MMPs was intense during WL. MMP inhibition during WL results in inflammation and collagen content reduction. MMP inhibition during WL period interferes with the period of subsequent expansion of AT resulting in improvements in local inflammation and systemic metabolic alterations induced by obesity. Our results suggest that MMPs inhibition could be an interesting target to improve adipose tissue inflammation during WL and to support weight cyclers. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Extracellular Matrix components regulate cellular polarity and tissue structure in the developing and mature Retina

    Directory of Open Access Journals (Sweden)

    Shweta Varshney

    2015-01-01

    Full Text Available While genetic networks and other intrinsic mechanisms regulate much of retinal development, interactions with the extracellular environment shape these networks and modify their output. The present review has focused on the role of one family of extracellular matrix molecules and their signaling pathways in retinal development. In addition to their effects on the developing retina, laminins play a role in maintaining Müller cell polarity and compartmentalization, thereby contributing to retinal homeostasis. This article which is intended for the clinical audience, reviews the fundamentals of retinal development, extracellular matrix organization and the role of laminins in retinal development. The role of laminin in cortical development is also briefly discussed.

  8. SPARC regulates extracellular matrix organization through its modulation of integrin-linked kinase activity.

    Science.gov (United States)

    Barker, Thomas H; Baneyx, Gretchen; Cardó-Vila, Marina; Workman, Gail A; Weaver, Matt; Menon, Priya M; Dedhar, Shoukat; Rempel, Sandra A; Arap, Wadih; Pasqualini, Renata; Vogel, Viola; Sage, E Helene

    2005-10-28

    SPARC, a 32-kDa matricellular glycoprotein, mediates interactions between cells and their extracellular matrix, and targeted deletion of Sparc results in compromised extracellular matrix in mice. Fibronectin matrix provides provisional tissue scaffolding during development and wound healing and is essential for the stabilization of mature extracellular matrix. Herein, we report that SPARC expression does not significantly affect fibronectin-induced cell spreading but enhances fibronectin-induced stress fiber formation and cell-mediated partial unfolding of fibronectin molecules, an essential process in fibronectin matrix assembly. By phage display, we identify integrin-linked kinase as a potential binding partner of SPARC and verify the interaction by co-immunoprecipitation and colocalization in vitro. Cells lacking SPARC exhibit diminished fibronectin-induced integrin-linked kinase activation and integrin-linked kinase-dependent cell-contractile signaling. Furthermore, induced expression of SPARC in SPARC-null fibroblasts restores fibronectin-induced integrin-linked kinase activation, downstream signaling, and fibronectin unfolding. These data further confirm the function of SPARC in extracellular matrix organization and identify a novel mechanism by which SPARC regulates extracellular matrix assembly.

  9. Biofilms of vaginal Lactobacillus reuteri CRL 1324 and Lactobacillus rhamnosus CRL 1332: kinetics of formation and matrix characterization.

    Science.gov (United States)

    Leccese Terraf, María Cecilia; Juárez Tomás, María Silvina; Rault, Lucie; Le Loir, Yves; Even, Sergine; Nader-Macías, María Elena Fátima

    2016-09-01

    Adhesion and biofilm formation are strain properties that reportedly contribute to the permanence of lactobacilli in the human vagina. The kinetics of biofilm formation and the chemical nature of the biofilm matrix formed by Lactobacillus reuteri CRL (Centro de Referencia para Lactobacilos Culture Collection) 1324 and Lactobacillus rhamnosus CRL 1332, vaginal beneficial strains, were evaluated in this work. Crystal violet-stained microplate assay and techniques of epifluorescence, electron and confocal microscopy were applied. The highest density and complexity of biofilms of both vaginal lactobacilli were observed at 72 h of incubation. Protease, proteinase K, α-chymotrypsin and trypsin treatments efficiently detached L. reuteri CRL 1324 biofilm that was also partially affected by α-amylase. However, L. rhamnosus CRL 1332 biofilm was slightly affected by protease, proteinase K and α-amylase. Confocal microscopy revealed greater amount of polysaccharides in L. rhamnosus CRL 1332 biofilm matrix than in L. reuteri CRL 1324 biofilm matrix. The results indicate that proteins are one of the main components of the L. reuteri CRL 1324 biofilm, while the biofilm matrix of L. rhamnosus CRL 1332 is composed of carbohydrates and proteins. The results obtained support the knowledge, understanding and characterization of two biofilm-forming vaginal Lactobacillus strains.

  10. Cranberry Flavonoids Modulate Cariogenic Properties of Mixed-Species Biofilm through Exopolysaccharides-Matrix Disruption.

    Directory of Open Access Journals (Sweden)

    Dongyeop Kim

    Full Text Available The exopolysaccharides (EPS produced by Streptococcus mutans-derived glucosyltransferases (Gtfs are essential virulence factors associated with the initiation of cariogenic biofilms. EPS forms the core of the biofilm matrix-scaffold, providing mechanical stability while facilitating the creation of localized acidic microenvironments. Cranberry flavonoids, such as A-type proanthocyanidins (PACs and myricetin, have been shown to inhibit the activity of Gtfs and EPS-mediated bacterial adhesion without killing the organisms. Here, we investigated whether a combination of cranberry flavonoids disrupts EPS accumulation and S. mutans survival using a mixed-species biofilm model under cariogenic conditions. We also assessed the impact of cranberry flavonoids on mechanical stability and the in situ pH at the biofilm-apatite interface. Topical application of an optimized combination of PACs oligomers (100-300 μM with myricetin (2 mM twice daily was used to simulate treatment regimen experienced clinically. Treatments with cranberry flavonoids effectively reduced the insoluble EPS content (>80% reduction vs. vehicle-control; p<0.001, while hindering S. mutans outgrowth within mixed-species biofilms. As a result, the 3D architecture of cranberry-treated biofilms was severely compromised, showing a defective EPS-matrix and failure to develop microcolonies on the saliva-coated hydroxyapatite (sHA surface. Furthermore, topical applications of cranberry flavonoids significantly weaken the mechanical stability of the biofilms; nearly 90% of the biofilm was removed from sHA surface after exposure to a shear stress of 0.449 N/m2 (vs. 36% removal in vehicle-treated biofilms. Importantly, in situ pH measurements in cranberry-treated biofilms showed significantly higher pH values (5.2 ± 0.1 at the biofilm-apatite interface vs. vehicle-treated biofilms (4.6 ± 0.1. Altogether, the data provide important insights on how cranberry flavonoids treatments modulate

  11. Astrocytes and extracellular matrix in extrasynaptic volume transmission

    Czech Academy of Sciences Publication Activity Database

    Vargová, Lýdia; Syková, Eva

    2014-01-01

    Roč. 369, č. 1654 (2014) ISSN 0962-8436 R&D Projects: GA ČR GA13-11867S; GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:68378041 Keywords : extracellular space * diffusion * astrocytes Subject RIV: FH - Neurology Impact factor: 7.055, year: 2014

  12. Pseudomonas aeruginosa Biofilm, a Programmed Bacterial Life for Fitness.

    Science.gov (United States)

    Lee, Keehoon; Yoon, Sang Sun

    2017-06-28

    A biofilm is a community of microbes that typically inhabit on surfaces and are encased in an extracellular matrix. Biofilms display very dissimilar characteristics to their planktonic counterparts. Biofilms are ubiquitous in the environment and influence our lives tremendously in both positive and negative ways. Pseudomonas aeruginosa is a bacterium known to produce robust biofilms. P. aeruginosa biofilms cause severe problems in immunocompromised patients, including those with cystic fibrosis or wound infection. Moreover, the unique biofilm properties further complicate the eradication of the biofilm infection, leading to the development of chronic infections. In this review, we discuss the history of biofilm research and general characteristics of bacterial biofilms. Then, distinct features pertaining to each stage of P. aeruginosa biofilm development are highlighted. Furthermore, infections caused by biofilms on their own or in association with other bacterial species ( i.e. , multispecies biofilms) are discussed in detail.

  13. Novel serological neo-epitope markers of extracellular matrix proteins for the detection of portal hypertension

    DEFF Research Database (Denmark)

    Leeming, Diana Julie; Karsdal, M A; Byrjalsen, I

    2013-01-01

    The hepatic venous pressure gradient (HVPG) is an invasive, but important diagnostic and prognostic marker in cirrhosis with portal hypertension (PHT). During cirrhosis, remodelling of fibrotic tissue by matrix metalloproteinases (MMPs) is a permanent process generating small fragments of degrade...... extracellular matrix (ECM) proteins known as neoepitopes, which are then released into the circulation....

  14. Pseudomonas aeruginosa biofilm infections

    DEFF Research Database (Denmark)

    Tolker-Nielsen, Tim

    2014-01-01

    Bacteria in natural, industrial and clinical settings predominantly live in biofilms, i.e., sessile structured microbial communities encased in self-produced extracellular matrix material. One of the most important characteristics of microbial biofilms is that the resident bacteria display...... a remarkable increased tolerance toward antimicrobial attack. Biofilms formed by opportunistic pathogenic bacteria are involved in devastating persistent medical device-associated infections, and chronic infections in individuals who are immune-compromised or otherwise impaired in the host defense. Because...... the use of conventional antimicrobial compounds in many cases cannot eradicate biofilms, there is an urgent need to develop alternative measures to combat biofilm infections. The present review is focussed on the important opportunistic pathogen and biofilm model organism Pseudomonas aeruginosa. Initially...

  15. Extracellular matrix metalloproteinase inducer (EMMPRIN) remodels the extracellular matrix through enhancing matrix metalloproteinases (MMPs) and inhibiting tissue inhibitors of MMPs expression in HPV-positive cervical cancer cells.

    Science.gov (United States)

    Xu, Q; Cao, X; Pan, J; Ye, Y; Xie, Y; Ohara, N; Ji, H

    2015-01-01

    PUPOSE OF INVESTIGATION: To study the expression of extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), and tissue inhibitors of MMP (TIMPs) in uterine cervical cancer cell lines in vitro. EMMPRIN, MMPs, and TIMPs expression were assessed by Western blot and real-time RT-PCR from cervical carcinoma SiHa, HeLa, and C33-A cells. EMMPRIN recombinant significantly increased MMP-2, MMP-9 protein and mRNA expression in SiHa and Hela cells, but not in C33-A cells by Western blot analysis and real-time RT-PCR. EMMPRIN recombinant significantly inhibited TIMP-1 protein and mRNA levels in SiHa and Hela cells, but not in C33-A cells. There was no difference on the TIMP-2 expression in those cells with the treatment of EMMPRIN recombinant. EMMPRIN RNAi decreased MMP-2 and MMP-9 and increased TIMP-1 expression in SiHa and HeLa cells, but not in C33-A cells. There was no change on the expression of TIMP-2 mRNA levels in SiHa, HeLa and C33-A cells transfected with siEMMPRIN. EMMPRIN may induce MMP-2 and MMP-9, and downregulate TIMP-1 in HPV-positive cervical cancer cells in vitro.

  16. Extracellular matrix-derived hydrogels for dental stem cell delivery

    OpenAIRE

    Viswanath, Aiswarya; Vanacker, Julie; Germain, Loic; Leprince, Julien G.; Diogenes, Anibal; Shakesheff, Kevin M.; White, Lisa J.; des Rieux, Anne

    2016-01-01

    Decellularised mammalian extracellular matrices (ECM) have been widely accepted as an ideal substrate for repair and remodelling of numerous tissues in clinical and pre-clinical studies. Recent studies have demonstrated the ability of ECM scaffolds derived from site-specific homologous tissues to direct cell differentiation. The present study investigated the suitability of hydrogels derived from different source tissues: bone, spinal cord and dentine, as suitable carriers to deliver human ap...

  17. Angiogenic Type I Collagen Extracellular Matrix Integrated with Recombinant Bacteriophages Displaying Vascular Endothelial Growth Factors.

    Science.gov (United States)

    Yoon, Junghyo; Korkmaz Zirpel, Nuriye; Park, Hyun-Ji; Han, Sewoon; Hwang, Kyung Hoon; Shin, Jisoo; Cho, Seung-Woo; Nam, Chang-Hoon; Chung, Seok

    2016-01-21

    Here, a growth-factor-integrated natural extracellular matrix of type I collagen is presented that induces angiogenesis. The developed matrix adapts type I collagen nanofibers integrated with synthetic colloidal particles of recombinant bacteriophages that display vascular endothelial growth factor (VEGF). The integration is achieved during or after gelation of the type I collagen and the matrix enables spatial delivery of VEGF into a desired region. Endothelial cells that contact the VEGF are found to invade into the matrix to form tube-like structures both in vitro and in vivo, proving the angiogenic potential of the matrix. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Ciliates as engineers of phototrophic biofilms.

    NARCIS (Netherlands)

    Weerman, E.J.; van der Geest, H.G.; van der Meulen, M.D; Manders, E.M.M.; van de Koppel, J.; Herman, P.M.J.; Admiraal, W.

    2011-01-01

    1. Phototrophic biofilms consist of a matrix of phototrophs, non-photosynthetic bacteria and extracellular polymeric substances (EPS) which is spatially structured. Despite widespread exploitation of algae and bacteria within phototrophic biofilms, for example by protozoans, the ‘engineering’

  19. Ciliates as engineers of phototrophic biofilms.

    NARCIS (Netherlands)

    Weerman, E.J.; Geest, H.G.; Meulen, M.D.; Manders, E.M.M.; Van de Koppel, J.; Herman, P.M.J.; Admiraal, W.

    2011-01-01

    1.Phototrophic biofilms consist of a matrix of phototrophs, non-photosynthetic bacteria and extracellular polymeric substances (EPS) which is spatially structured. Despite widespread exploitation of algae and bacteria within phototrophic biofilms, for example by protozoans, the ‘engineering’ effects

  20. Anisotropic properties of the enamel organic extracellular matrix.

    Science.gov (United States)

    do Espírito Santo, Alexandre R; Novaes, Pedro D; Line, Sérgio R P

    2006-05-01

    Enamel biosynthesis is initiated by the secretion, processing, and self-assembly of a complex mixture of proteins. This supramolecular ensemble controls the nucleation of the crystalline mineral phase. The detection of anisotropic properties by polarizing microscopy has been extensively used to detect macromolecular organizations in ordinary histological sections. The aim of this work was to study the birefringence of enamel organic matrix during the development of rat molar and incisor teeth. Incisor and molar teeth of rats were fixed in 2% paraformaldehyde/0.5% glutaraldehyde in 0.2 M phosphate-buffered saline (PBS), pH 7.2, and decalcified in 5% nitric acid/4% formaldehyde. After paraffin embedding, 5-microm-thick sections were obtained, treated with xylene, and hydrated. Form birefringence curves were obtained after measuring optical retardations in imbibing media, with different refractive indices. Our observations showed that enamel organic matrix of rat incisor and molar teeth is strongly birefringent, presenting an ordered supramolecular structure. The birefringence starts during the early secretion phase and disappears at the maturation phase. The analysis of enamel organic matrix birefringence may be used to detect the effects of genetic and environmental factors on the supramolecular orientation of enamel matrix and their effects on the structure of mature enamel.

  1. The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules.

    Science.gov (United States)

    Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

    2006-05-01

    Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including adherence and immunomodulation, thus contributing to S. aureus pathogenesis. Eap binding to host macromolecules is unusually promiscuous and includes matrix or matricellular proteins as well as plasma proteins. The structural basis of this promiscuity is poorly understood. Here, we show that in spite of the preferential location of the binding epitopes within triple helical regions in some collagens there is a striking specificity of Eap binding to different collagen types. Collagen I, but not collagen II, is a binding substrate in monomolecular form. However, collagen I is virtually unrecognized by Eap when incorporated into banded fibrils. By contrast, microfibrils containing collagen VI as well as basement membrane-associated networks containing collagen IV, or aggregates containing fibronectin bound Eap as effectively as the monomeric proteins. Therefore, Eap-binding to extracellular matrix ligands is promiscuous at the molecular level but not indiscriminate with respect to supramolecular structures containing the same macromolecules. In addition, Eap bound to banded fibrils after their partial disintegration by matrix-degrading proteinases, including matrix metalloproteinase 1. Therefore, adherence to matrix suprastructures by S. aureus can be supported by inflammatory reactions.

  2. Regulation of extracellular matrix vesicles via rapid responses to steroid hormones during endochondral bone formation.

    Science.gov (United States)

    Asmussen, Niels; Lin, Zhao; McClure, Michael J; Schwartz, Zvi; Boyan, Barbara D

    2017-12-09

    Endochondral bone formation is a precise and highly ordered process whose exact regulatory framework is still being elucidated. Multiple regulatory pathways are known to be involved. In some cases, regulation impacts gene expression, resulting in changes in chondrocyte phenotypic expression and extracellular matrix synthesis. Rapid regulatory mechanisms are also involved, resulting in release of enzymes, factors and micro RNAs stored in extracellular matrisomes called matrix vesicles. Vitamin D metabolites modulate endochondral development via both genomic and rapid membrane-associated signaling pathways. 1α,25-dihydroxyvitamin D3 [1α,25(OH) 2 D 3 ] acts through the vitamin D receptor (VDR) and a membrane associated receptor, protein disulfide isomerase A3 (PDIA3). 24R,25-dihydroxyvitamin D3 [24R,25(OH) 2 D 3 ] affects primarily chondrocytes in the resting zone (RC) of the growth plate, whereas 1α,25(OH) 2 D 3 affects cells in the prehypertrophic and upper hypertrophic cell zones (GC). This includes genomically directing the cells to produce matrix vesicles with zone specific characteristics. In addition, vitamin D metabolites produced by the cells interact directly with the matrix vesicle membrane via rapid signal transduction pathways, modulating their activity in the matrix. The matrix vesicle payload is able to rapidly impact the extracellular matrix via matrix processing enzymes as well as providing a feedback mechanism to the cells themselves via the contained micro RNAs. Copyright © 2017. Published by Elsevier Inc.

  3. Tissue architecture and breast cancer: the role of extracellular matrix and steroid hormones

    Science.gov (United States)

    Hansen, R K; Bissell, M J

    2010-01-01

    The changes in tissue architecture that accompany the development of breast cancer have been the focus of investigations aimed at developing new cancer therapeutics. As we learn more about the normal mammary gland, we have begun to understand the complex signaling pathways underlying the dramatic shifts in the structure and function of breast tissue. Integrin-, growth factor-, and steroid hormone-signaling pathways all play an important part in maintaining tissue architecture; disruption of the delicate balance of signaling results in dramatic changes in the way cells interact with each other and with the extracellular matrix, leading to breast cancer. The extracellular matrix itself plays a central role in coordinating these signaling processes. In this review, we consider the interrelationships between the extracellular matrix, integrins, growth factors, and steroid hormones in mammary gland development and function. PMID:10903527

  4. Adherence of extracellular matrix components to modified surfaces of titanium alloys

    International Nuclear Information System (INIS)

    Stelzer, C; Uhlmann, E; Meinke, M; Lademann, J; Hansen, U

    2009-01-01

    The adherence of biological materials on metal surfaces is of special importance in biology and medicine. The underlying interactions between surface and biological materials (e.g. extracellular matrix components or cells) are responsible for the application as a medical device. Numerous products are made of pure titanium and titanium alloys. This paper shows the influence of a laser production technology on machined surfaces of TiAl 6 V 4 and the resulting adherence of biological material on the basis of the surface characterisation. In this study, different machined TiAl 6 V 4 surfaces were used for coatings with extracellular matrix components. For this process, different coating with collagen I monomers and a complex mixture of extracellular matrix proteins derived from the dermal-epidermal basement membrane zone were analysed. The efficiency of the coating was analysed by different methods and the results are presented in this paper

  5. Extracellular matrix remodeling in patients with ischemic chronic heart failure with preserved ejection fraction

    Directory of Open Access Journals (Sweden)

    V. D. Syvolap

    2015-04-01

    Full Text Available Aim. To identify features, relationships between parameters of the extracellular matrix and renal function in 110 patients with ischemic chronic heart failure the activity of collagen metabolism markers (MMP-9, TIMP-1, PICP, cystatin C, structural and functional parameters of the heart were studied using ELISA, echocardiography. Results. It was established that imbalance in the system MMP/TIMP in ischemic heart failure with preserved left ventricular ejection fraction leads to disruption of the extracellular matrix structural functional sufficiency, increases functional failure and is associated with impaired renal function. Conclusion. Correlation analysis showed significant relationships between MMP/TIMP and GFR, cystatin C, indicating that the significant role of extracellular matrix imbalance in the development of renal dysfunction in patients with ischemic chronic heart failure.

  6. Tissue architecture and breast cancer: the role of extracellular matrix and steroid hormones

    Energy Technology Data Exchange (ETDEWEB)

    Hansen, R K; Bissell, M J

    2000-06-01

    The changes in tissue architecture that accompany the development of breast cancer have been the focus of investigations aimed at developing new cancer therapeutics. As we learn more about the normal mammary gland, we have begun to understand the complex signaling pathways underlying the dramatic shifts in the structure and function of breast tissue. Integrin-, growth factor-, and steroid hormone-signaling pathways all play an important part in maintaining tissue architecture; disruption of the delicate balance of signaling results in dramatic changes in the way cells interact with each other and with the extracellular matrix, leading to breast cancer. The extracellular matrix itself plays a central role in coordinating these signaling processes. In this review, we consider the interrelationships between the extracellular matrix, integrins, growth factors, and steroid hormones in mammary gland development and function.

  7. New Technologies for Studying Biofilms

    Science.gov (United States)

    FRANKLIN, MICHAEL J.; CHANG, CONNIE; AKIYAMA, TATSUYA; BOTHNER, BRIAN

    2016-01-01

    Bacteria have traditionally been studied as single-cell organisms. In laboratory settings, aerobic bacteria are usually cultured in aerated flasks, where the cells are considered essentially homogenous. However, in many natural environments, bacteria and other microorganisms grow in mixed communities, often associated with surfaces. Biofilms are comprised of surface-associated microorganisms, their extracellular matrix material, and environmental chemicals that have adsorbed to the bacteria or their matrix material. While this definition of a biofilm is fairly simple, biofilms are complex and dynamic. Our understanding of the activities of individual biofilm cells and whole biofilm systems has developed rapidly, due in part to advances in molecular, analytical, and imaging tools and the miniaturization of tools designed to characterize biofilms at the enzyme level, cellular level, and systems level. PMID:26350329

  8. A Collagen-based Scaffold Delivering Exogenous MicroRNA-29B to Modulate Extracellular Matrix Remodeling

    OpenAIRE

    Monaghan, Michael; Browne, Shane; Schenke-Layland, Katja; Pandit, Abhay

    2014-01-01

    Directing appropriate extracellular matrix remodeling is a key aim of regenerative medicine strategies. Thus, antifibrotic interfering RNA (RNAi) therapy with exogenous microRNA (miR)-29B was proposed as a method to modulate extracellular matrix remodeling following cutaneous injury. It was hypothesized that delivery of miR-29B from a collagen scaffold will efficiently modulate the extracellular matrix remodeling response and reduce maladaptive remodeling such as aggressive deposition of coll...

  9. Depressed immune surveillance against cancer: role of deficient T cell: extracellular matrix interactions.

    Science.gov (United States)

    Górski, A; Castronovo, V; Stepień-Sopniewska, B; Grieb, P; Ryba, M; Mrowiec, T; Korczak-Kowalska, G; Wierzbicki, P; Matysiak, W; Dybowska, B

    1994-07-01

    Although T cells infiltrate malignant tumors, the local immune response is usually inefficient and tumors escape destruction. While extracellular matrix proteins strongly costimulate T cell responses in normal individuals, our studies indicate that peripheral blood T cells from cancer patients and tumor infiltrating cells respond poorly or are resistant to stimulative signals mediated by collagen I and IV and fibronectin. Moreover, the adhesive properties of cancer T cells are markedly depressed. Those functional deficiencies are paralleled by variable deficits in integrin and non-integrin T cell receptors for extracellular matrix. Immunotherapy with BCG causes a dramatic but transient increase in T cell: ECM interactions.

  10. Iron triggers λSo prophage induction and release of extracellular DNA in Shewanella oneidensis MR-1 biofilms.

    Science.gov (United States)

    Binnenkade, Lucas; Teichmann, Laura; Thormann, Kai M

    2014-09-01

    Prophages are ubiquitous elements within bacterial chromosomes and affect host physiology and ecology in multiple ways. We have previously demonstrated that phage-induced lysis is required for extracellular DNA (eDNA) release and normal biofilm formation in Shewanella oneidensis MR-1. Here, we investigated the regulatory mechanisms of prophage λSo spatiotemporal induction in biofilms. To this end, we used a functional fluorescence fusion to monitor λSo activation in various mutant backgrounds and in response to different physiological conditions. λSo induction occurred mainly in a subpopulation of filamentous cells in a strictly RecA-dependent manner, implicating oxidative stress-induced DNA damage as the major trigger. Accordingly, mutants affected in the oxidative stress response (ΔoxyR) or iron homeostasis (Δfur) displayed drastically increased levels of phage induction and abnormal biofilm formation, while planktonic cells were not or only marginally affected. To further investigate the role of oxidative stress, we performed a mutant screen and identified two independent amino acid substitutions in OxyR (T104N and L197P) that suppress induction of λSo by hydrogen peroxide (H2O2). However, λSo induction was not suppressed in biofilms formed by both mutants, suggesting a minor role of intracellular H2O2 in this process. In contrast, addition of iron to biofilms strongly enhanced λSo induction and eDNA release, while both processes were significantly suppressed at low iron levels, strongly indicating that iron is the limiting factor. We conclude that uptake of iron during biofilm formation triggers λSo-mediated lysis of a subpopulation of cells, likely by an increase in iron-mediated DNA damage sensed by RecA. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  11. A collagen-based scaffold delivering exogenous microrna-29B to modulate extracellular matrix remodeling.

    Science.gov (United States)

    Monaghan, Michael; Browne, Shane; Schenke-Layland, Katja; Pandit, Abhay

    2014-04-01

    Directing appropriate extracellular matrix remodeling is a key aim of regenerative medicine strategies. Thus, antifibrotic interfering RNA (RNAi) therapy with exogenous microRNA (miR)-29B was proposed as a method to modulate extracellular matrix remodeling following cutaneous injury. It was hypothesized that delivery of miR-29B from a collagen scaffold will efficiently modulate the extracellular matrix remodeling response and reduce maladaptive remodeling such as aggressive deposition of collagen type I after injury. The release of RNA from the scaffold was assessed and its ability to silence collagen type I and collagen type III expression was evaluated in vitro. When primary fibroblasts were cultured with scaffolds doped with miR-29B, reduced levels of collagen type I and collagen type III mRNA expression were observed for up to 2 weeks of culture. When the scaffolds were applied to full thickness wounds in vivo, reduced wound contraction, improved collagen type III/I ratios and a significantly higher matrix metalloproteinase (MMP)-8: tissue inhibitor of metalloproteinase (TIMP)-1 ratio were detected when the scaffolds were functionalized with miR-29B. Furthermore, these effects were significantly influenced by the dose of miR-29B in the collagen scaffold (0.5 versus 5 μg). This study shows a potential of combining exogenous miRs with collagen scaffolds to improve extracellular matrix remodeling following injury.

  12. Extracellular matrix organization in various regions of rat brain grey matter.

    Science.gov (United States)

    Brückner, G; Härtig, W; Kacza, J; Seeger, J; Welt, K; Brauer, K

    1996-05-01

    Previous studies revealed the concentration of extracellular matrix proteoglycans in the so-called perineuronal nets on the one hand and in certain zones of the neuropil on the other. This nonhomogeneous distribution suggested a non-random chemical and spatial heterogeneity of the extracellular space. In the present investigation, regions dominated by one of both distribution patterns, i.e. piriform and parietal cortex, reticular thalamic nucleus, medial septum/diagonal band complex and cerebellar nuclei, were selected for correlative light and electron microscopic analysis. The labelling was performed by the use of the N-acetylgalactosamine-binding plant lectin Wisteria floribunda agglutinin visualized by peroxidase staining and additionally by photoconversion of red carbocyanine fluorescence labelling for electron microscopy. The intense labelling of the neuropil of a superficial piriform region, presumably identical with sublayer Ia, was confined to a fine meshwork spreading over the extracellular space between non-myelinated axons, dendrites and glial profiles. In the reticular thalamic nucleus the neuronal cell bodies were embedded in zones of labelled neuropil. In contrast to these patterns, the labelled extracellular matrix in different cortical layers and in the other subcortical regions was concentrated in perineuronal nets as large accumulations at surface areas of the neuronal perikarya and dendrites and the attached presynaptic boutons. Astrocytic processes usually were separated from the neuronal surface by the interposed extracellular material. Despite a great variability, the width of the extracellular space containing the labelled matrix components in all perineuronal nets appeared to be considerably larger than that in the labelled zones of neuropil and the non-labelled microenvironment of other neurons. Our results support the view that differences expressed in topographical and spatial peculiarities of the extracellular matrix constituents are

  13. Extracellular matrix-derived hydrogels for dental stem cell delivery.

    Science.gov (United States)

    Viswanath, Aiswarya; Vanacker, Julie; Germain, Loïc; Leprince, Julian G; Diogenes, Anibal; Shakesheff, Kevin M; White, Lisa J; des Rieux, Anne

    2017-01-01

    Decellularized mammalian extracellular matrices (ECM) have been widely accepted as an ideal substrate for repair and remodelling of numerous tissues in clinical and pre-clinical studies. Recent studies have demonstrated the ability of ECM scaffolds derived from site-specific homologous tissues to direct cell differentiation. The present study investigated the suitability of hydrogels derived from different source tissues: bone, spinal cord and dentine, as suitable carriers to deliver human apical papilla derived mesenchymal stem cells (SCAP) for spinal cord regeneration. Bone, spinal cord, and dentine ECM hydrogels exhibited distinct structural, mechanical, and biological characteristics. All three hydrogels supported SCAP viability and proliferation. However, only spinal cord and bone derived hydrogels promoted the expression of neural lineage markers. The specific environment of ECM scaffolds significantly affected the differentiation of SCAP to a neural lineage, with stronger responses observed with spinal cord ECM hydrogels, suggesting that site-specific tissues are more likely to facilitate optimal stem cell behavior for constructive spinal cord regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 319-328, 2017. © 2016 Wiley Periodicals, Inc.

  14. Degenerated human intervertebral discs contain autoantibodies against extracellular matrix proteins

    Directory of Open Access Journals (Sweden)

    S Capossela

    2014-04-01

    Full Text Available Degeneration of intervertebral discs (IVDs is associated with back pain and elevated levels of inflammatory cells. It has been hypothesised that discogenic pain is a direct result of vascular and neural ingrowth along annulus fissures, which may expose the avascular nucleus pulposus (NP to the systemic circulation and induce an autoimmune reaction. In this study, we confirmed our previous observation of antibodies in human degenerated and post-traumatic IVDs cultured in vitro. We hypothesised that the presence of antibodies was due to an autoimmune reaction against specific proteins of the disc. Furthermore we identified antigens which possibly trigger an autoimmune response in degenerative disc diseases. We demonstrated that degenerated and post-traumatic IVDs contain IgG antibodies against typical extracellular proteins of the disc, particularly proteins of the NP. We identified IgGs against collagen type II and aggrecan, confirming an autoimmune reaction against the normally immune privileged NP. We also found specific IgGs against collagens types I and V, but not against collagen type III. In conclusion, this study confirmed the association between disc degeneration and autoimmunity, and may open the avenue for future studies on developing prognostic, diagnostic and therapy-monitoring markers for degenerative disc diseases.

  15. Pattern formation in Pseudomonas aeruginosa biofilms

    DEFF Research Database (Denmark)

    Parsek, Matthew R.; Tolker-Nielsen, Tim

    2008-01-01

    Bacteria are capable of forming elaborate multicellular communities called biofilms. Pattern formation in biofilms depends on cell proliferation and cellular migration in response to the available nutrients and other external cues, as well as on self-generated intercellular signal molecules...... and the production of an extracellular matrix that serves as a structural 'scaffolding' for the biofilm cells. Pattern formation in biofilms allows cells to position themselves favorably within nutrient gradients and enables buildup and maintenance of physiologically distinct subpopulations, which facilitates...... survival of one or more subpopulations upon environmental insult, and therefore plays an important role in the innate tolerance displayed by biofilms toward adverse conditions....

  16. Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink

    Science.gov (United States)

    Pati, Falguni; Jang, Jinah; Ha, Dong-Heon; Won Kim, Sung; Rhie, Jong-Won; Shim, Jin-Hyung; Kim, Deok-Ho; Cho, Dong-Woo

    2014-06-01

    The ability to print and pattern all the components that make up a tissue (cells and matrix materials) in three dimensions to generate structures similar to tissues is an exciting prospect of bioprinting. However, the majority of the matrix materials used so far for bioprinting cannot represent the complexity of natural extracellular matrix (ECM) and thus are unable to reconstitute the intrinsic cellular morphologies and functions. Here, we develop a method for the bioprinting of cell-laden constructs with novel decellularized extracellular matrix (dECM) bioink capable of providing an optimized microenvironment conducive to the growth of three-dimensional structured tissue. We show the versatility and flexibility of the developed bioprinting process using tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function. We achieve high cell viability and functionality of the printed dECM structures using our bioprinting method.

  17. Extracellular matrix of smooth muscle cells: interaction of collagen type V with heparan sulfate proteoglycan

    International Nuclear Information System (INIS)

    Gay, S.; Hoeoek, M.; Gay, R.E.; Magargal, W.W.; Reynertson, R.H.

    1986-01-01

    Alteration in the extracellular matrix produced by smooth muscle cells may play a role in the development of atherosclerotic lesions. Consequently the authors have initiated studies on the structural organization of the extracellular matrix produced by cultured smooth muscle cells. Immunohisotological examination of this matrix using well-characterized mono- and polyclonal antibodies showed a partial codistribution of heparan sulfate (HS) proteoglycans with a number of different matrix components including collagen types I, III, IV, V and VI, laminin and fibronectin. Subsequent binding studies between isolated matrix proteins and HS showed that the polysaccharide interacts strongly with type V collagen and to a lesser extent with fibronectin as well as collagen types III and VI. The interaction between type V and HS was readily inhibited by heparin and highly sulfated HS but not be dermatan sulfate, chondroitin sulfate or HS with a low sulfate content. Furthermore, [ 35 S]-HS proteoglycans isolated from cultured smooth muscle cells could be adsorbed on a column of sepharose conjugated with native type V collagen and eluted in a salt gradient. Hence, the interaction between type V and HS may play a major part in stabilizing the extracellular matrix of the vessel wall

  18. Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies.

    Science.gov (United States)

    Kumar, Pramod; Satyam, Abhigyan; Fan, Xingliang; Collin, Estelle; Rochev, Yury; Rodriguez, Brian J; Gorelov, Alexander; Dillon, Simon; Joshi, Lokesh; Raghunath, Michael; Pandit, Abhay; Zeugolis, Dimitrios I

    2015-03-04

    Therapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex vivo microenvironments, prolonged culture time is required to develop an extracellular matrix-rich implantable device. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 h in culture, even at low serum concentration. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture. Morphological, histological, gene and protein analysis assays demonstrated maintenance of tissue-specific function. Macromolecular crowding opens new avenues for a more rational design in engineering of clinically relevant tissue modules in vitro.

  19. Macromolecular crowding directs extracellular matrix organization and mesenchymal stem cell behavior.

    Directory of Open Access Journals (Sweden)

    Adam S Zeiger

    Full Text Available Microenvironments of biological cells are dominated in vivo by macromolecular crowding and resultant excluded volume effects. This feature is absent in dilute in vitro cell culture. Here, we induced macromolecular crowding in vitro by using synthetic macromolecular globules of nm-scale radius at physiological levels of fractional volume occupancy. We quantified the impact of induced crowding on the extracellular and intracellular protein organization of human mesenchymal stem cells (MSCs via immunocytochemistry, atomic force microscopy (AFM, and AFM-enabled nanoindentation. Macromolecular crowding in extracellular culture media directly induced supramolecular assembly and alignment of extracellular matrix proteins deposited by cells, which in turn increased alignment of the intracellular actin cytoskeleton. The resulting cell-matrix reciprocity further affected adhesion, proliferation, and migration behavior of MSCs. Macromolecular crowding can thus aid the design of more physiologically relevant in vitro studies and devices for MSCs and other cells, by increasing the fidelity between materials synthesized by cells in vivo and in vitro.

  20. Macromolecular crowding directs extracellular matrix organization and mesenchymal stem cell behavior.

    Science.gov (United States)

    Zeiger, Adam S; Loe, Felicia C; Li, Ran; Raghunath, Michael; Van Vliet, Krystyn J

    2012-01-01

    Microenvironments of biological cells are dominated in vivo by macromolecular crowding and resultant excluded volume effects. This feature is absent in dilute in vitro cell culture. Here, we induced macromolecular crowding in vitro by using synthetic macromolecular globules of nm-scale radius at physiological levels of fractional volume occupancy. We quantified the impact of induced crowding on the extracellular and intracellular protein organization of human mesenchymal stem cells (MSCs) via immunocytochemistry, atomic force microscopy (AFM), and AFM-enabled nanoindentation. Macromolecular crowding in extracellular culture media directly induced supramolecular assembly and alignment of extracellular matrix proteins deposited by cells, which in turn increased alignment of the intracellular actin cytoskeleton. The resulting cell-matrix reciprocity further affected adhesion, proliferation, and migration behavior of MSCs. Macromolecular crowding can thus aid the design of more physiologically relevant in vitro studies and devices for MSCs and other cells, by increasing the fidelity between materials synthesized by cells in vivo and in vitro.

  1. Proteoglycan changes in the extracellular matrix of lung tissue from patients with pulmonary emphysema

    NARCIS (Netherlands)

    van Straaten, JFM; Coers, W; Noordhoek, JA; Flipsen, JTM; Kauffman, HF; Timens, W; Postma, DS

    To characterize the changes in the extracellular matrix in smoking-related pulmonary emphysema, we undertook immunohistochemical studies in lung tissues from controls (n = 7), from patients with mild (n = 11) and severe (n = 8) emphysema, and from patients with lung fibrosis (n = 6). We studied

  2. Involutional ectropion and entropion: clinicopathologic correlation between horizontal eyelid laxity and eyelid extracellular matrix.

    Science.gov (United States)

    Damasceno, Renato Wendell; Osaki, Midori Hentona; Dantas, Paulo Elias Correa; Belfort, Rubens

    2011-01-01

    To investigate the clinicopathologic correlation between horizontal eyelid laxity and extracellular matrix components, such as collagen and elastic fibers, in involutional ectropion and entropion. Another goal was to compare the differences between involutional ectropion and entropion in regard to extracellular matrix content using computer-assisted morphometry. This clinicopathologic study included 20 consecutive patients with involutional ectropion (group 1) and 20 consecutive patients with involutional entropion (group 2). The pinch test was performed to measure horizontal eyelid laxity in both groups. Full-thickness eyelid biopsy specimens were examined by light microscopy and computer-assisted morphometry. The Mann-Whitney U test, the Pearson chi-square test, the Pearson correlation coefficient calculation, and a linear regression analysis were performed. All sections of specimens from patients in groups 1 and 2 revealed abnormal collagen and elastic fibers. The Pearson correlation coefficient revealed a significant negative correlation between horizontal eyelid laxity and extracellular matrix content in the eyelid skin, the pretarsal orbicularis oculi muscle, the perimeibomian tarsal stroma, and the intermeibomian tarsal stroma. Linear regression demonstrated that horizontal eyelid laxity is dependent upon extracellular matrix components in all eyelid regions. Collagen fiber content was significantly increased in specimens from patients in group 1 compared with specimens from patients in group 2. The present findings suggest that a reduction of collagen and elastic fibers may contribute to the development of excessive horizontal eyelid laxity in patients with involutional ectropion and entropion of the lower eyelid.

  3. Topical application of amelogenin extracellular matrix protein in non-healing venous ulcers

    Directory of Open Access Journals (Sweden)

    Burçin Abud

    2014-12-01

    Full Text Available Background and Design: Treatment of chronic venous ulcers of the lower extremity is still an important difficulty. The principal treatment of these ulcers includes compression therapy, local wound care and surgery. Unresponsiveness to these standard treatments is a frequent situation with negative effects on life quality and reductions in personal productivity. Therefore, there is a need for new applications to increase the effectiveness of treatment in treatment-resistant cases. In the present study, we retrospectively evaluated the results of topical application of amelogenin extracellular matrix protein in resistant venous ulcers. Materials and Methods: We analyzed the records of patients with treatment-resistant venous ulceration who were treated with amelogenin extracellular matrix protein between June 2011 and December 2012.. Results: 26 patients (21 male and 5 female with a total number of 28 ulcers (24 patients with 1 ulcer, 2 patients with two ulcers were evaluated. The patients were treated with topically applied amelogenin extracellular matrix protein and regional four bandage compression. Bandages were changed weekly. Each cure continued for six weeks. In fourteen patients (15 ulcers, we observed a complete healing by the end of the first cure. In another twelve cases (13 ulcers, the same period resulted with a reduction in wound diameter. We continued to the second cure for these patients. By the end of the second cure, complete healing was achieved in five cases (6 ulcers. Conclusion: Topical application of amelogenin extracellular matrix protein may be considered as an effective therapeutic choice for refractory venous ulcers.

  4. Impact of TGF-β inhibition during acute exercise on Achilles tendon extracellular matrix

    DEFF Research Database (Denmark)

    Potter, Ross M; Huynh, Richard T; Volper, Brent D

    2017-01-01

    The purpose of this study was to evaluate the role of TGF-β1in regulating tendon extracellular matrix after acute exercise. Wistar rats exercised (n = 15) on a treadmill for four consecutive days (60 min/day) or maintained normal cage activity. After each exercise bout, the peritendinous space of...

  5. Adherence of Staphylococci to plastic, mesothelial cells and mesothelial extracellular matrix

    NARCIS (Netherlands)

    Betjes, M. G.; Tuk, C. W.; Struijk, D. G.; Krediet, R. T.; Arisz, L.; Beelen, R. H.

    1992-01-01

    In this study we have investigated whether mesothelial cells (MC) and mesothelial extracellular matrix (ECM) are suitable substrates for the adherence of Staphylococci. Mesothelial cells were isolated from the peritoneal dialysis effluent by making use of their lack of Fc-receptors and capacity to

  6. IMMUNOHISTOCHEMICAL STUDY OF EXTRACELLULAR-MATRIX IN ACUTE GALACTOSAMINE HEPATITIS IN RATS

    NARCIS (Netherlands)

    JONKER, AM; DIJKHUIS, FWJ; BOES, A; HARDONK, MJ

    A single injection of D-galactosamine hydrochloride induces acute self-limiting liver disease in rats that morphologically resembles drug-induced hepatitis in human beings. In this immunohistochemical study we examined the localization and expression of the hepatic extracellular matrix components

  7. Collagen fiber alignment and biaxial mechanical behavior of porcine urinary bladder derived extracellular matrix

    NARCIS (Netherlands)

    Gilbert, Thomas W.; Wognum, Silvia; Joyce, Erinn M.; Freytes, Donald O.; Sacks, Michael S.; Badylak, Stephen F.

    2008-01-01

    The collagen fiber alignment and biomechanical behavior of naturally occurring extracellular matrix (ECM) scaffolds are important considerations for the design of medical devices from these materials. Both should be considered in order to produce a device to meet tissue specific mechanical

  8. Leg ulcer treatment outcomes with new ovine collagen extracellular matrix dressing: a retrospective case series.

    Science.gov (United States)

    Bohn, Gregory A; Gass, Kimberly

    2014-10-01

    The purpose of this study was to describe the rate of closure observed in venous leg ulcers during treatment with ovine collagen extracellular matrix dressings and compression. Fourteen patients with 23 wounds were retrospectively evaluated with respect to healing rates, time to closure, and weekly facility charge fees.

  9. Plasticity of the actin cytoskeleton in response to extracellular matrix nanostructure and dimensionality

    NARCIS (Netherlands)

    Starke, J.; Wehrle-Haller, B.; Friedl, P.

    2014-01-01

    Mobile cells discriminate and adapt to mechanosensory input from extracellular matrix (ECM) topographies to undergo actin-based polarization, shape change and migration. We tested 'cell-intrinsic' and adaptive components of actin-based cell migration in response to widely used in vitro

  10. The extracellular matrix - the under-recognized element in lung disease?

    NARCIS (Netherlands)

    Burgess, Janette K.; Mauad, Thais; Tjin, Gavin; Karlsson, Jenny C.; Westergren-Thorsson, Gunilla

    2016-01-01

    The lung is composed of airways and lung parenchyma, and the extracellular matrix (ECM) contains the main building blocks of both components. The ECM provides physical support and stability to the lung, and as such it has in the past been regarded as an inert structure. More recent research has

  11. A dual role of extracellular DNA during biofilm formation of Neisseria meningitidis

    DEFF Research Database (Denmark)

    Lappann, M.; Claus, H.; van Alen, T.

    2010-01-01

    formation, whereas biofilm formation of cc with low point prevalence (ST-8 cc and ST-11 cc) was eDNA-independent. For initial biofilm formation, a ST-32 cc type strain, but not a ST-11 type strain, utilized eDNA. The release of eDNA was mediated by lytic transglycosylase and cytoplasmic N......-acetylmuramyl-l-alanine amidase genes. In late biofilms, outer membrane phospholipase A-dependent autolysis, which was observed in most cc, but not in ST-8 and ST-11 strains, was required for shear force resistance of microcolonies. Taken together, N. meningitidis evolved two different biofilm formation strategies, an e....... On the contrary, spreaders (ST-11 and ST-8 cc) are unable to use eDNA for biofilm formation and might compensate for poor colonization properties by high transmission rates....

  12. Biofilms from Klebsiella pneumoniae: Matrix Polysaccharide Structure and Interactions with Antimicrobial Peptides.

    Science.gov (United States)

    Benincasa, Monica; Lagatolla, Cristina; Dolzani, Lucilla; Milan, Annalisa; Pacor, Sabrina; Liut, Gianfranco; Tossi, Alessandro; Cescutti, Paola; Rizzo, Roberto

    2016-08-10

    Biofilm matrices of two Klebsiella pneumoniae clinical isolates, KpTs101 and KpTs113, were investigated for their polysaccharide composition and protective effects against antimicrobial peptides. Both strains were good biofilm producers, with KpTs113 forming flocs with very low adhesive properties to supports. Matrix exopolysaccharides were isolated and their monosaccharide composition and glycosidic linkage types were defined. KpTs101 polysaccharide is neutral and composed only of galactose, in both pyranose and furanose ring configurations. Conversely, KpTs113 polysaccharide is anionic due to glucuronic acid units, and also contains glucose and mannose residues. The susceptibility of the two strains to two bovine cathelicidin antimicrobial peptides, BMAP-27 and Bac7(1-35), was assessed using both planktonic cultures and biofilms. Biofilm matrices exerted a relevant protection against both antimicrobials, which act with quite different mechanisms. Similar protection was also detected when antimicrobial peptides were tested against planktonic bacteria in the presence of the polysaccharides extracted from KpTs101 and KpTs113 biofilms, suggesting sequestering adduct formation with antimicrobials. Circular dichroism experiments on BMAP-27 in the presence of increasing amounts of either polysaccharide confirmed their ability to interact with the peptide and induce an α-helical conformation.

  13. Differential effect of extracellular matrix derived from papillary and reticular fibroblasts on epidermal development in vitro.

    Science.gov (United States)

    Janson, David; Rietveld, Marion; Mahé, Christian; Saintigny, Gaëlle; El Ghalbzouri, Abdoelwaheb

    2017-06-01

    Papillary and reticular fibroblasts have different effects on keratinocyte proliferation and differentiation. The aim of this study was to investigate whether these effects are caused by differential secretion of soluble factors or by differential generation of extracellular matrix from papillary and reticular fibroblasts. To study the effect of soluble factors, keratinocyte monolayer cultures were grown in papillary or reticular fibroblast-conditioned medium. To study the effect of extracellular matrix, keratinocytes were grown on papillary or reticular-derived matrix. Conditioned medium from papillary or reticular fibroblasts did not differentially affect keratinocyte viability or epidermal development. However, keratinocyte viability was increased when grown on matrix derived from papillary, compared with reticular, fibroblasts. In addition, the longevity of the epidermis was increased when cultured on papillary fibroblast-derived matrix skin equivalents compared with reticular-derived matrix skin equivalents. The findings indicate that the matrix secreted by papillary and reticular fibroblasts is the main causal factor to account for the differences in keratinocyte growth and viability observed in our study. Differences in response to soluble factors between both populations were less significant. Matrix components specific to the papillary dermis may account for the preferential growth of keratinocytes on papillary dermis.

  14. Extracellular Matrix Molecular Remodeling in Human Liver Fibrosis Evolution.

    Directory of Open Access Journals (Sweden)

    Andrea Baiocchini

    Full Text Available Chronic liver damage leads to pathological accumulation of ECM proteins (liver fibrosis. Comprehensive characterization of the human ECM molecular composition is essential for gaining insights into the mechanisms of liver disease. To date, studies of ECM remodeling in human liver diseases have been hampered by the unavailability of purified ECM. Here, we developed a decellularization method to purify ECM scaffolds from human liver tissues. Histological and electron microscopy analyses demonstrated that the ECM scaffolds, devoid of plasma and cellular components, preserved the three-dimensional ECM structure and zonal distribution of ECM components. This method has been then applied on 57 liver biopsies of HCV-infected patients at different stages of liver fibrosis according to METAVIR classification. Label-free nLC-MS/MS proteomics and computation biology were performed to analyze the ECM molecular composition in liver fibrosis progression, thus unveiling protein expression signatures specific for the HCV-related liver fibrotic stages. In particular, the ECM molecular composition of liver fibrosis was found to involve dynamic changes in matrix stiffness, flexibility and density related to the dysregulation of predominant collagen, elastic fibers and minor components with both structural and signaling properties. This study contributes to the understanding of the molecular bases underlying ECM remodeling in liver fibrosis and suggests new molecular targets for fibrolytic strategies.

  15. Extracellular Matrix Hydrogel Derived from Human Umbilical Cord as a Scaffold for Neural Tissue Repair and Its Comparison with Extracellular Matrix from Porcine Tissues

    Czech Academy of Sciences Publication Activity Database

    Kočí, Zuzana; Výborný, Karel; Dubišová, Jana; Vacková, Irena; Jäger, Aleš; Lunov, Oleg; Jiráková, Klára; Kubinová, Šárka

    2017-01-01

    Roč. 23, č. 6 (2017), s. 333-345 ISSN 1937-3384 R&D Projects: GA ČR(CZ) GA15-01396S; GA MŠk(CZ) LO1309; GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) EF15_003/0000419 Grant - others:AV ČR(CZ) Fellowship J. E. Purkyně Institutional support: RVO:68378041 ; RVO:68378271 Keywords : extracellular matrix * hydrogel * umbilical cord Subject RIV: FH - Neurology; EB - Genetics ; Molecular Biology (FZU-D) OBOR OECD: Neurosciences (including psychophysiology; Biophysics (FZU-D)

  16. Inhibiting extracellular matrix metalloproteinase inducer maybe beneficial for diminishing the atherosclerotic plaque instability

    Directory of Open Access Journals (Sweden)

    Xie S

    2009-01-01

    Full Text Available Atherosclerotic plaque rupture and local thrombosis activation in the artery cause acute serious incidents such as acute coronary syndrome and stroke. The exact mechanism of plaque rupture remains unclear but excessive degradation of the extracellular matrix scaffold by matrix-degrading metalloproteinases (MMPs has been implicated as one of the major molecular mechanisms in this process. Convincing evidence is available to prove that extracellular matrix metalloproteinase inducer (EMMPRIN induces MMP expression and is involved in the inflammatory responses in the artery wall. The inflammation and MMPs have been shown to play a critical role for atherosclerotic lesion development and progression. More recent data showed that increased EMMPRIN expression was associated with vulnerable atherosclerotic lesions. Therefore, we speculate that EMMPRIN may be pivotal for atherosclerotic plaque instability, and hence inhibition of EMMPRIN expression could be a promising approach for the prevention or treatment of atheroma instability.

  17. Rat hair follicle dermal papillae have an extracellular matrix containing basement membrane components

    DEFF Research Database (Denmark)

    Couchman, J R

    1986-01-01

    , to be replaced by synthesis of other components including type I and III collagens. It seems likely therefore that the dermal papilla cells in vivo synthesize a basement membrane type of extracellular matrix, although a contribution from epithelial, and in some cases capillary endothelial, cells cannot be ruled......Dermal papillae are small mesenchymally derived zones at the bases of hair follicles which have an important role in hair morphogenesis in the embryo and control of the hair growth cycle in postnatal mammals. The cells of the papilla are enmeshed in a dense extracellular matrix which undergoes...... extensive changes in concert with the hair cycle. Here it is shown that this matrix in anagen pelage follicles of postnatal rats contains an abundance of basement membrane components rather than dermal components such as interstitial collagens. In particular, type IV collagen, laminin, and basement membrane...

  18. Candida Biofilms: Development, Architecture, and Resistance

    Science.gov (United States)

    CHANDRA, JYOTSNA; MUKHERJEE, PRANAB K.

    2015-01-01

    Intravascular device–related infections are often associated with biofilms (microbial communities encased within a polysaccharide-rich extracellular matrix) formed by pathogens on the surfaces of these devices. Candida species are the most common fungi isolated from catheter-, denture-, and voice prosthesis–associated infections and also are commonly isolated from contact lens–related infections (e.g., fungal keratitis). These biofilms exhibit decreased susceptibility to most antimicrobial agents, which contributes to the persistence of infection. Recent technological advances have facilitated the development of novel approaches to investigate the formation of biofilms and identify specific markers for biofilms. These studies have provided extensive knowledge of the effect of different variables, including growth time, nutrients, and physiological conditions, on biofilm formation, morphology, and architecture. In this article, we will focus on fungal biofilms (mainly Candida biofilms) and provide an update on the development, architecture, and resistance mechanisms of biofilms. PMID:26350306

  19. Lipase biofilm deposited by Matrix Assisted Pulsed Laser Evaporation technique

    International Nuclear Information System (INIS)

    Aronne, Antonio; Bloisi, Francesco; Calabria, Raffaela; Califano, Valeria; Depero, Laura E.; Fanelli, Esther; Federici, Stefania; Massoli, Patrizio; Vicari, Luciano R.M.

    2015-01-01

    Highlights: • A lipase film was deposited with Matrix Assisted Pulsed Laser Evaporation technique. • FTIR spectra show that laser irradiation do not damage lipase molecule. • Laser fluence controls the characteristics of complex structure generated by MAPLE. - Abstract: Lipase is an enzyme that finds application in biodiesel production and for detection of esters and triglycerides in biosensors. Matrix Assisted Pulsed Laser Evaporation (MAPLE), a technique derived from Pulsed Laser Deposition (PLD) for deposition of undamaged biomolecules or polymers, is characterized by the use of a frozen target obtained from a solution/suspension of the guest material (to be deposited) in a volatile matrix (solvent). The presence of the solvent avoids or at least reduces the potential damage of guest molecules by laser radiation but only the guest material reaches the substrate in an essentially solvent-free deposition. MAPLE can be used for enzymes immobilization, essential for industrial application, allowing the development of continuous processes, an easier separation of products, the reuse of the catalyst and, in some cases, enhancing enzyme properties (pH, temperature stability, etc.) and catalytic activity in non-aqueous media. Here we show that MAPLE technique can be used to deposit undamaged lipase and that the complex structure (due to droplets generated during extraction from target) of the deposited material can be controlled by changing the laser beam fluence

  20. Lipase biofilm deposited by Matrix Assisted Pulsed Laser Evaporation technique

    Energy Technology Data Exchange (ETDEWEB)

    Aronne, Antonio [Department of Chemical Engineering, Materials and Industrial Production, University of Naples “Federico II”, Napoli (Italy); Bloisi, Francesco, E-mail: bloisi@na.infn.it [SPIN – CNR, Naples (Italy); Department of Physics, University of Naples “Federico II”, Napoli (Italy); Calabria, Raffaela; Califano, Valeria [Istituto Motori – CNR, Naples (Italy); Depero, Laura E. [Department of Mechanical and Industrial Engineering, University of Brescia, Brescia (Italy); Fanelli, Esther [Department of Chemical Engineering, Materials and Industrial Production, University of Naples “Federico II”, Napoli (Italy); Federici, Stefania [Department of Mechanical and Industrial Engineering, University of Brescia, Brescia (Italy); Massoli, Patrizio [Istituto Motori – CNR, Naples (Italy); Vicari, Luciano R.M. [SPIN – CNR, Naples (Italy); Department of Physics, University of Naples “Federico II”, Napoli (Italy)

    2015-05-01

    Highlights: • A lipase film was deposited with Matrix Assisted Pulsed Laser Evaporation technique. • FTIR spectra show that laser irradiation do not damage lipase molecule. • Laser fluence controls the characteristics of complex structure generated by MAPLE. - Abstract: Lipase is an enzyme that finds application in biodiesel production and for detection of esters and triglycerides in biosensors. Matrix Assisted Pulsed Laser Evaporation (MAPLE), a technique derived from Pulsed Laser Deposition (PLD) for deposition of undamaged biomolecules or polymers, is characterized by the use of a frozen target obtained from a solution/suspension of the guest material (to be deposited) in a volatile matrix (solvent). The presence of the solvent avoids or at least reduces the potential damage of guest molecules by laser radiation but only the guest material reaches the substrate in an essentially solvent-free deposition. MAPLE can be used for enzymes immobilization, essential for industrial application, allowing the development of continuous processes, an easier separation of products, the reuse of the catalyst and, in some cases, enhancing enzyme properties (pH, temperature stability, etc.) and catalytic activity in non-aqueous media. Here we show that MAPLE technique can be used to deposit undamaged lipase and that the complex structure (due to droplets generated during extraction from target) of the deposited material can be controlled by changing the laser beam fluence.

  1. Fractional Excretion of Survivin, Extracellular Matrix Metalloproteinase Inducer, and Matrix Metalloproteinase 7 in Children with Chronic Kidney Disease

    Directory of Open Access Journals (Sweden)

    Agnieszka Bargenda

    2016-07-01

    Full Text Available Background: Epithelial–mesenchymal transition (EMT is defined as a transformation of tubular epithelial cells into mesenchymal ones. These cells migrate through the extracellular matrix and change into active myofibroblasts, which are responsible for excessive matrix deposition. Such changes may lead to tubular dysfunction and fibrosis of the renal parenchyma, characteristic of chronic kidney disease (CKD. However, there are no data on potential EMT markers in children with CKD. The aim of our study was to assess the usefulness of fractional excretion (FE of survivin, E-cadherin, extracellular matrix metalloproteinase inducer (EMMPRIN, matrix metalloproteinase (MMP7, and transforming growth factor beta 1 (TGF-β1 as potential markers of CKD-related complications such as tubular damage and fibrosis. Methods: Forty-one pre-dialysis children with CKD Stages 3–5 and 23 age-matched controls were enrolled in the study. The serum and urine concentrations of analysed parameters were assessed by an enzyme-linked immunosorbent assay test. Results: Tubular reabsorption of all analysed parameters was >99% in the control group. All FE values rose significantly in children with CKD, yet they remained 1%. Conclusions: FE of the examined markers may become a useful tool in the assessment of tubular dysfunction during the course of CKD. The FE of survivin, EMMPRIN, and MMP7 warrant further research as potential independent markers of kidney-specific EMT.

  2. Bacteriophages and Biofilms

    Directory of Open Access Journals (Sweden)

    David R. Harper

    2014-06-01

    Full Text Available Biofilms are an extremely common adaptation, allowing bacteria to colonize hostile environments. They present unique problems for antibiotics and biocides, both due to the nature of the extracellular matrix and to the presence within the biofilm of metabolically inactive persister cells. Such chemicals can be highly effective against planktonic bacterial cells, while being essentially ineffective against biofilms. By contrast, bacteriophages seem to have a greater ability to target this common form of bacterial growth. The high numbers of bacteria present within biofilms actually facilitate the action of bacteriophages by allowing rapid and efficient infection of the host and consequent amplification of the bacteriophage. Bacteriophages also have a number of properties that make biofilms susceptible to their action. They are known to produce (or to be able to induce enzymes that degrade the extracellular matrix. They are also able to infect persister cells, remaining dormant within them, but re-activating when they become metabolically active. Some cultured biofilms also seem better able to support the replication of bacteriophages than comparable planktonic systems. It is perhaps unsurprising that bacteriophages, as the natural predators of bacteria, have the ability to target this common form of bacterial life.

  3. Quantitative proteomics reveals altered expression of extracellular matrix related proteins of human primary dermal fibroblasts in response to sulfated hyaluronan and collagen applied as artificial extracellular matrix.

    Science.gov (United States)

    Müller, Stephan A; van der Smissen, Anja; von Feilitzsch, Margarete; Anderegg, Ulf; Kalkhof, Stefan; von Bergen, Martin

    2012-12-01

    Fibroblasts are the main matrix producing cells of the dermis and are also strongly regulated by their matrix environment which can be used to improve and guide skin wound healing processes. Here, we systematically investigated the molecular effects on primary dermal fibroblasts in response to high-sulfated hyaluronan [HA] (hsHA) by quantitative proteomics. The comparison of non- and high-sulfated HA revealed regulation of 84 of more than 1,200 quantified proteins. Based on gene enrichment we found that sulfation of HA alters extracellular matrix remodeling. The collagen degrading enzymes cathepsin K, matrix metalloproteinases-2 and -14 were found to be down-regulated on hsHA. Additionally protein expression of thrombospondin-1, decorin, collagen types I and XII were reduced, whereas the expression of trophoblast glycoprotein and collagen type VI were slightly increased. This study demonstrates that global proteomics provides a valuable tool for revealing proteins involved in molecular effects of growth substrates for further material optimization.

  4. Interactions between Skeletal Muscle Myoblasts and their Extracellular Matrix Revealed by a Serum Free Culture System.

    Science.gov (United States)

    Chaturvedi, Vishal; Dye, Danielle E; Kinnear, Beverley F; van Kuppevelt, Toin H; Grounds, Miranda D; Coombe, Deirdre R

    2015-01-01

    Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates.

  5. The extracellular matrix of the lung and its role in edema formation

    Directory of Open Access Journals (Sweden)

    Paolo Pelosi

    2007-06-01

    Full Text Available The extracellular matrix is composed of a three-dimensional fiber mesh filled with different macromolecules such as: collagen (mainly type I and III, elastin, glycosaminoglycans, and proteoglycans. In the lung, the extracellular matrix has several functions which provide: 1 mechanical tensile and compressive strength and elasticity, 2 low mechanical tissue compliance contributing to the maintenance of normal interstitial fluid dynamics, 3 low resistive pathway for an effective gas exchange, d control of cell behavior by the binding of growth factors, chemokines, cytokines and the interaction with cell-surface receptors, and e tissue repair and remodeling. Fragmentation and disorganization of extracellular matrix components comprises the protective role of the extracellular matrix, leading to interstitial and eventually severe lung edema. Thus, once conditions of increased microvascular filtration are established, matrix remodeling proceeds fairly rapidly due to the activation of proteases. Conversely, a massive matrix deposition of collagen fiber decreases interstitial compliance and therefore makes the tissue safety factor stronger. As a result, changes in lung extracellular matrix significantly affect edema formation and distribution in the lung.A matriz extracelular é um aglomerado tridimensional demacromoléculas composta por: fibras colágenas (principalmente, tipos I e III, elastina, glicosaminoglicanos e proteoglicanos. No pulmão, a matriz extracelular tem várias funções, tais como: 1 promover estresse tensil e elasticidade tecidual, 2 contribuir para a manutenção da dinâmica de fluidos no interstício, 3 propiciar efetiva troca gasosa, 4 controlar a função celular através de sua ligação com fatores de crescimento, quimiocinas, citocinas e interação com receptores de superfície, e 5 remodelamento e reparo tecidual. A fragmentação e a desorganização da matriz extracelular pode acarretar edema intersticial e

  6. Endocytosis of collagen by hepatic stellate cells regulates extracellular matrix dynamics.

    Science.gov (United States)

    Bi, Yan; Mukhopadhyay, Dhriti; Drinane, Mary; Ji, Baoan; Li, Xing; Cao, Sheng; Shah, Vijay H

    2014-10-01

    Hepatic stellate cells (HSCs) generate matrix, which in turn may also regulate HSCs function during liver fibrosis. We hypothesized that HSCs may endocytose matrix proteins to sense and respond to changes in microenvironment. Primary human HSCs, LX2, or mouse embryonic fibroblasts (MEFs) [wild-type; c-abl(-/-); or Yes, Src, and Fyn knockout mice (YSF(-/-))] were incubated with fluorescent-labeled collagen or gelatin. Fluorescence-activated cell sorting analysis and confocal microscopy were used for measuring cellular internalization of matrix proteins. Targeted PCR array and quantitative real-time PCR were used to evaluate gene expression changes. HSCs and LX2 cells endocytose collagens in a concentration- and time-dependent manner. Endocytosed collagen colocalized with Dextran 10K, a marker of macropinocytosis, and 5-ethylisopropyl amiloride, an inhibitor of macropinocytosis, reduced collagen internalization by 46%. Cytochalasin D and ML7 blocked collagen internalization by 47% and 45%, respectively, indicating that actin and myosin are critical for collagen endocytosis. Wortmannin and AKT inhibitor blocked collagen internalization by 70% and 89%, respectively, indicating that matrix macropinocytosis requires phosphoinositide-3-kinase (PI3K)/AKT signaling. Overexpression of dominant-negative dynamin-2 K44A blocked matrix internalization by 77%, indicating a role for dynamin-2 in matrix macropinocytosis. Whereas c-abl(-/-) MEF showed impaired matrix endocytosis, YSF(-/-) MEF surprisingly showed increased matrix endocytosis. It was also associated with complex gene regulations that related with matrix dynamics, including increased matrix metalloproteinase 9 (MMP-9) mRNA levels and zymographic activity. HSCs endocytose matrix proteins through macropinocytosis that requires a signaling network composed of PI3K/AKT, dynamin-2, and c-abl. Interaction with extracellular matrix regulates matrix dynamics through modulating multiple gene expressions including MMP-9

  7. Proteomics of Fuchs' Endothelial Corneal Dystrophy support that the extracellular matrix of Descemet's membrane is disordered

    DEFF Research Database (Denmark)

    Poulsen, Ebbe Toftgaard; Dyrlund, Thomas F; Runager, Kasper

    2014-01-01

    Fuchs' endothelial corneal dystrophy (FECD) is a major corneal disorder affecting the innermost part of the cornea, leading to visual impairment. As the morphological changes in FECD are mainly observed in the extracellular matrix of the Descemet's membrane/endothelial layer we determined...... that the morphological changes observed in FECD is caused in part by an aberrant assembly of the extracellular matrix within the Descemet's membrane/endothelial layer......., respectively, of which 10 were significantly regulated. The results indicated that the level of type VIII collagen was unaltered even though the protein previously has been implicated in familial early onset forms of the disease. Using the second relative quantitation method iTRAQ we identified 22...

  8. Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope

    Science.gov (United States)

    Wang, Han-Wei; Le, Thuc T.; Cheng, Ji-Xin

    2008-04-01

    A multimodal nonlinear optical imaging system that integrates coherent anti-Stokes Raman scattering (CARS), sum-frequency generation (SFG), and two-photon excitation fluorescence (TPEF) on the same platform was developed and applied to visualize single cells and extracellular matrix in fresh carotid arteries. CARS signals arising from CH 2-rich membranes allowed visualization of endothelial cells and smooth muscle cells of the arterial wall. Additionally, CARS microscopy allowed vibrational imaging of elastin and collagen fibrils which are also rich in CH 2 bonds. The extracellular matrix organization was further confirmed by TPEF signals arising from elastin's autofluorescence and SFG signals arising from collagen fibrils' non-centrosymmetric structure. Label-free imaging of significant components of arterial tissues suggests the potential application of multimodal nonlinear optical microscopy to monitor onset and progression of arterial diseases.

  9. Extracellular Matrix in Plants and Animals: Hooks and Locks for Viruses

    Directory of Open Access Journals (Sweden)

    Livia Stavolone

    2017-09-01

    Full Text Available The extracellular matrix (ECM of animal and plants cells plays important roles in viral diseases. While in animal cells extracellular matrix components can be exploited by viruses for recognition, attachment and entry, the plant cell wall acts as a physical barrier to viral entry and adds a higher level of difficulty to intercellular movement of viruses. Interestingly, both in plant and animal systems, ECM can be strongly remodeled during virus infection, and the understanding of remodeling mechanisms and molecular players offers new perspectives for therapeutic intervention. This review focuses on the different roles played by the ECM in plant and animal hosts during virus infection with special emphasis on the similarities and differences. Possible biotechnological applications aimed at improving viral resistance are discussed.

  10. Sprifermin (rhFGF18) modulates extracellular matrix turnover in cartilage explants ex vivo

    DEFF Research Database (Denmark)

    Reker, Ditte; Kjelgaard-Petersen, Cecilie Freja; Siebuhr, Anne Sofie

    2017-01-01

    Background: Sprifermin (recombinant human fibroblast growth factor 18) is in clinical development as a potential disease-modifying osteoarthritis drug (DMOAD). In vitro studies have shown that cartilage regenerative properties of sprifermin involve chondrocyte proliferation and extracellular matrix...... or placebo at weekly intervals, similar to the dosing regimen used in clinical trials. Pre-culturing with oncostatin M and tumour necrosis factor-a, was also used to induce an inflammatory state before treatment. Metabolic activity was measured using AlamarBlue, and chondrocyte proliferation was visualized...... aggrecanase activity. Results: Sprifermin was able to reach the chondrocytes through the extracellular matrix, as it increased cell proliferation and metabolic activity of explants. ProC2 and CS846 was dose-dependently increased (P

  11. Bacterial biofilm and associated infections

    Directory of Open Access Journals (Sweden)

    Muhsin Jamal

    2018-01-01

    Full Text Available Microscopic entities, microorganisms that drastically affect human health need to be thoroughly investigated. A biofilm is an architectural colony of microorganisms, within a matrix of extracellular polymeric substance that they produce. Biofilm contains microbial cells adherent to one-another and to a static surface (living or non-living. Bacterial biofilms are usually pathogenic in nature and can cause nosocomial infections. The National Institutes of Health (NIH revealed that among all microbial and chronic infections, 65% and 80%, respectively, are associated with biofilm formation. The process of biofilm formation consists of many steps, starting with attachment to a living or non-living surface that will lead to formation of micro-colony, giving rise to three-dimensional structures and ending up, after maturation, with detachment. During formation of biofilm several species of bacteria communicate with one another, employing quorum sensing. In general, bacterial biofilms show resistance against human immune system, as well as against antibiotics. Health related concerns speak loud due to the biofilm potential to cause diseases, utilizing both device-related and non-device-related infections. In summary, the understanding of bacterial biofilm is important to manage and/or to eradicate biofilm-related diseases. The current review is, therefore, an effort to encompass the current concepts in biofilm formation and its implications in human health and disease.

  12. The ECM-Cell Interaction of Cartilage Extracellular Matrix on Chondrocytes

    Directory of Open Access Journals (Sweden)

    Yue Gao

    2014-01-01

    Full Text Available Cartilage extracellular matrix (ECM is composed primarily of the network type II collagen (COLII and an interlocking mesh of fibrous proteins and proteoglycans (PGs, hyaluronic acid (HA, and chondroitin sulfate (CS. Articular cartilage ECM plays a crucial role in regulating chondrocyte metabolism and functions, such as organized cytoskeleton through integrin-mediated signaling via cell-matrix interaction. Cell signaling through integrins regulates several chondrocyte functions, including differentiation, metabolism, matrix remodeling, responses to mechanical stimulation, and cell survival. The major signaling pathways that regulate chondrogenesis have been identified as wnt signal, nitric oxide (NO signal, protein kinase C (PKC, and retinoic acid (RA signal. Integrins are a large family of molecules that are central regulators in multicellular biology. They orchestrate cell-cell and cell-matrix adhesive interactions from embryonic development to mature tissue function. In this review, we emphasize the signaling molecule effect and the biomechanics effect of cartilage ECM on chondrogenesis.

  13. Bral1: "Superglue" for the extracellular matrix in the brain white matter.

    Czech Academy of Sciences Publication Activity Database

    Cicanič, Michal; Syková, Eva; Vargová, Lýdia

    2012-01-01

    Roč. 44, č. 4 (2012), s. 596-599 ISSN 1357-2725 R&D Projects: GA ČR GA309/09/1597; GA ČR(CZ) GAP304/11/0184 Institutional research plan: CEZ:AV0Z50390703 Institutional support: RVO:68378041 Keywords : extracellular matrix * link proteins * signal transmission Subject RIV: FH - Neurology Impact factor: 4.152, year: 2012

  14. The Biological Role of Hyaluronan-Rich Oocyte-Cumulus Extracellular Matrix in Female Reproduction

    Czech Academy of Sciences Publication Activity Database

    Nagyová, Eva

    2018-01-01

    Roč. 19, č. 1 (2018), č. článku 283. E-ISSN 1422-0067 R&D Projects: GA MŠk EF15_003/0000460 Institutional support: RVO:67985904 Keywords : extracellular matrix * hyaluronan * inter-alpha-trypsin inhibitor Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 3.226, year: 2016

  15. IMMUNE RESPONSE TO EXTRACELLULAR MATRIX COLLAGEN IN CHRONIC HEPATITIS C INDUCED LIVER FIBROSIS

    OpenAIRE

    Borg, Brian B.; Seetharam, Anil; Subramanian, Vijay; Ilias, Haseeb; Lisker–Melman, Mauricio; Korenblat, Kevin; Anderson, Christopher; Shenoy, Surendra; Chapman, William C.; Crippin, Jeffrey S.; Mohanakumar, Thalachallour

    2011-01-01

    Hepatitis C Virus (HCV) infection and recurrence post-transplant (OLT) is associated with extracellular matrix (ECM) components remodeling, particularly collagen (Col), leading to fibrosis. Our aim was to determine whether development of antibodies (Abs) to self antigen Col in HCV infection correlates with fibrosis stage and peripheral cytokine response. Chronic HCV patients, those with recurrence after OLT undergoing biopsy and healthy control subjects were enrolled. HCV subjects (n=70) were...

  16. Hyaluronan and hyaluronectin in the extracellular matrix of human brain tumour stroma.

    Science.gov (United States)

    Delpech, B; Maingonnat, C; Girard, N; Chauzy, C; Maunoury, R; Olivier, A; Tayot, J; Creissard, P

    1993-01-01

    Hyaluronan (HA) and the hyaluronan-binding glycoprotein hyaluronectin (HN) were measured in 23 gliomas and 8 meningiomas and their location was revisited in 35 tumours. A clear-cut difference was found in the HN/HA ratio values of glioblastomas (below 0.5) and that of astrocytomas (above 0.5 P edification of the extracellular matrix. In meningiomas only the stroma would be responsible for HA and HN production.

  17. In vivo xenogeneic scaffold fate is determined by residual antigenicity and extracellular matrix preservation

    OpenAIRE

    Wong, Maelene L.; Wong, Janelle L.; Vapniarsky, Natalia; Griffiths, Leigh G.

    2016-01-01

    The immunological potential of animal-derived tissues and organs is the critical hurdle to increasing their clinical implementation. Glutaraldehyde-fixation cross-links proteins in xenogeneic tissues (e.g., bovine pericardium) to delay immune rejection, but also compromises the regenerative potential of the resultant biomaterial. Unfixed xenogeneic biomaterials in which xenoantigenicity has been ameliorated and native extracellular matrix (ECM) architecture has been maintained have the potent...

  18. A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix

    OpenAIRE

    Hui Liang; Xiaoran Li; Bin Wang; Bing Chen; Yannan Zhao; Jie Sun; Yan Zhuang; Jiajia Shi; He Shen; Zhijun Zhang; Jianwu Dai

    2016-01-01

    Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of ...

  19. Structural, biochemical, cellular, and functional changes in skeletal muscle extracellular matrix with aging

    DEFF Research Database (Denmark)

    Kragstrup, T W; Kjaer, M; Mackey, A L

    2011-01-01

    The extracellular matrix (ECM) of skeletal muscle is critical for force transmission and for the passive elastic response of skeletal muscle. Structural, biochemical, cellular, and functional changes in skeletal muscle ECM contribute to the deterioration in muscle mechanical properties with aging....... Structural changes include an increase in the collagen concentration, a change in the elastic fiber system, and an increase in fat infiltration of skeletal muscle. Biochemical changes include a decreased turnover of collagen with potential accumulation of enzymatically mediated collagen cross...

  20. Sequential changes in histologic pattern and extracellular matrix deposition during the healing of chronic venous ulcers.

    OpenAIRE

    Herrick, S. E.; Sloan, P.; McGurk, M.; Freak, L.; McCollum, C. N.; Ferguson, M. W.

    1992-01-01

    As part of a major clinical trial, sequential biopsies were taken from the margins of venous leg ulcers during their healing. The changing patterns of tissue architecture and extracellular matrix synthesis during healing were documented histologically and immunocytochemically. Initial biopsies were similar in appearance: prominent fibrin cuffs, variable inflammation, hemosiderin, and red blood cell extravasation. So called "fibrin cuffs" were highly organized structures composed of laminin, f...

  1. Combating biofilms

    DEFF Research Database (Denmark)

    Yang, Liang; Liu, Yang; Wu, Hong

    2012-01-01

    Biofilms are complex microbial communities consisting of microcolonies embedded in a matrix of self-produced polymer substances. Biofilm cells show much greater resistance to environmental challenges including antimicrobial agents than their free-living counterparts. The biofilm mode of life...... is believed to significantly contribute to successful microbial survival in hostile environments. Conventional treatment, disinfection and cleaning strategies do not proficiently deal with biofilm-related problems, such as persistent infections and contamination of food production facilities. In this review......, strategies to control biofilms are discussed, including those of inhibition of microbial attachment, interference of biofilm structure development and differentiation, killing of biofilm cells and induction of biofilm dispersion....

  2. Bacillus cereus growth and biofilm formation: the impact of substratum, iron sources, and transcriptional regulator Sigma 54

    NARCIS (Netherlands)

    Hayrapetyan, Hasmik

    2017-01-01

    Biofilms are surface-associated communities of microbial cells embedded in a matrix of extracellular polymers. It is generally accepted that the biofilm growth mode represents the most common lifestyle of microorganisms. Next to beneficial biofilms used in biotechnology applications, undesired

  3. Altered extracellular matrix remodeling and angiogenesis in sponge granulomas of thrombospondin 2-null mice.

    Science.gov (United States)

    Kyriakides, T R; Zhu, Y H; Yang, Z; Huynh, G; Bornstein, P

    2001-10-01

    The matricellular angiogenesis inhibitor, thrombospondin (TSP) 2, has been shown to be an important modulator of wound healing and the foreign body response. Specifically, TSP2-null mice display improved healing with minimal scarring and form well-vascularized foreign body capsules. In this study we performed subcutaneous implantation of sponges and investigated the resulting angiogenic and fibrogenic responses. Histological and immunohistochemical analysis of sponges, excised at 7, 14, and 21 days after implantation, revealed significant differences between TSP2-null and wild-type mice. Most notably, TSP2-null mice exhibited increased angiogenesis and fibrotic encapsulation of the sponge. However, invasion of dense tissue was compromised, even though its overall density was increased. Furthermore, histomorphometry and biochemical assays demonstrated a significant increase in the extracellular distribution of matrix metalloproteinase (MMP) 2, but no change in the levels of active transforming growth factor-beta(1). The alterations in neovascularization, dense tissue invasion, and MMP2 in TSP2-null mice coincided with the deposition of TSP2 in the extracellular matrix of wild-type animals. These observations support the proposed role of TSP2 as a modulator of angiogenesis and matrix remodeling during tissue repair. In addition, they provide in vivo evidence for a newly proposed function of TSP2 as a modulator of extracellular MMP2 levels.

  4. Collagen VI disorders: Insights on form and function in the extracellular matrix and beyond.

    Science.gov (United States)

    Lamandé, Shireen R; Bateman, John F

    2017-12-22

    Mutations in the three canonical collagen VI genes, COL6A1, COL6A2 and COL6A3, cause a spectrum of muscle disease from Bethlem myopathy at the mild end to the severe Ullrich congenital muscular dystrophy. Mutations can be either dominant or recessive and the resulting clinical severity is influenced by the way mutations impact the complex collagen VI assembly process. Most mutations are found towards the N-terminus of the triple helical collagenous domain and compromise extracellular microfibril assembly. Outside the triple helix collagen VI is highly polymorphic and discriminating mutations from rare benign changes remains a major diagnostic challenge. Collagen VI deficiency alters extracellular matrix structure and biomechanical properties and leads to increased apoptosis and oxidative stress, decreased autophagy, and impaired muscle regeneration. Therapies that target these downstream consequences have been tested in a collagen VI null mouse and also in small human trials where they show modest clinical efficacy. An important role for collagen VI in obesity, cancer and diabetes is emerging. A major barrier to developing effective therapies is the paucity of information about how collagen VI deficiency in the extracellular matrix signals the final downstream consequences - the receptors involved and the intracellular messengers await further characterization. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

  5. [Three-dimensional parallel collagen scaffold promotes tendon extracellular matrix formation].

    Science.gov (United States)

    Zheng, Zefeng; Shen, Weiliang; Le, Huihui; Dai, Xuesong; Ouyang, Hongwei; Chen, Weishan

    2016-03-01

    To investigate the effects of three-dimensional parallel collagen scaffold on the cell shape, arrangement and extracellular matrix formation of tendon stem cells. Parallel collagen scaffold was fabricated by unidirectional freezing technique, while random collagen scaffold was fabricated by freeze-drying technique. The effects of two scaffolds on cell shape and extracellular matrix formation were investigated in vitro by seeding tendon stem/progenitor cells and in vivo by ectopic implantation. Parallel and random collagen scaffolds were produced successfully. Parallel collagen scaffold was more akin to tendon than random collagen scaffold. Tendon stem/progenitor cells were spindle-shaped and unified orientated in parallel collagen scaffold, while cells on random collagen scaffold had disorder orientation. Two weeks after ectopic implantation, cells had nearly the same orientation with the collagen substance. In parallel collagen scaffold, cells had parallel arrangement, and more spindly cells were observed. By contrast, cells in random collagen scaffold were disorder. Parallel collagen scaffold can induce cells to be in spindly and parallel arrangement, and promote parallel extracellular matrix formation; while random collagen scaffold can induce cells in random arrangement. The results indicate that parallel collagen scaffold is an ideal structure to promote tendon repairing.

  6. Clinical Usage of an Extracellular, Collagen-rich Matrix: A Case Series.

    Science.gov (United States)

    AbouIssa, Abdelfatah; Mari, Walid; Simman, Richard

    2015-11-01

    OASIS Ultra (Smith and Nephew, St. Petersburg, FL) is an extracellular, collagen-rich matrix derived from submucosa of porcine intestine. It is composed of collagen type I, glycosaminoglycan, and proteoglycans. This extracellular matrix (ECM) differs from the single layer in thickness and offers ease of handling and application. It also stimulates cell migration and structural support, provides moisture environment, decreases inflammation, and induces cell proliferation and cellular attachments. In this case series, the authors present their experience with this product in various clinical scenarios. The authors used the product in a variety of wounds with different etiologies to test the clinical outcome of the ECM. This was an observational case series with prospective review of 6 different patients with different types of wounds who received treatment with the ECM during their treatment. The product was applied on the following types of wounds: chronic venous ulcer, nonhealing Achilles tendon vasculitic wound, Marjolin's ulcer, posttraumatic wound, stage IV sacral-coccygeal pressure wound, and complicated transmetatarsal amputation of gangrenous left forefoot diabetic wound. All of these wounds healed within the expected time periods and without complications. In general, healing was achieved in 4-16 weeks using 1-12 applications of the ECM. Wounds with different etiologies were successfully treated with an extracellular, collagen-rich matrix. By replacing the lost ECM to guide cellular growth and migration, this product did ultimately hasten the healing process.

  7. Hydraulic resistance of biofilms

    KAUST Repository

    Dreszer, C.; Vrouwenvelder, Johannes S.; Paulitsch-Fuchs, Astrid H.; Zwijnenburg, Arie; Kruithof, Joop C.; Flemming, Hans Curt

    2013-01-01

    resistance is very low compared to the expected biofilm resistance and, thus, biofilm resistance can be determined accurately. Transmembrane pressure drop was monitored. As biofilm parameters, thickness, total cell number, TOC, and extracellular polymeric

  8. TOL Plasmid Carriage Enhances Biofilm Formation and Increases Extracellular DNA Content in Pseudomonas Putida KT2440

    DEFF Research Database (Denmark)

    Smets, Barth F.; D'Alvise, Paul; Yankelovich, T.

    laser scanning microscopy. The TOL-carrying strains formed pellicles and thick biofilms, whereas the same strains without the plasmid displayed little adherent growth. Microscopy using fluorescent nucleic acid- specific stains (cytox orange, propidium iodide) revealed differences in production...... combined with specific cytostains; release of cytoplasmic material was assayed by a β-glucosidase assay. Enhanced cell lysis due to plasmid carriage was ruled out as the mechanism for eDNA release. We report, for the first time, that carriage of a conjugative plasmid leads to increased biofilm formation...

  9. Effects of Iron on DNA Release and Biofilm Development by Pseudomonas Aeruginosa

    DEFF Research Database (Denmark)

    Yang, Liang; Barken, Kim Bundvig; Skindersø, Mette Elena

    2007-01-01

    Extracellular DNA is one of the major matrix components in Pseudomonas aeruginosa biofilms. It functions as an intercellular connector and plays a role in stabilization of the biofilms. Evidence that DNA release in P. aeruginosa PAO1 biofilms is controlled by the las-rhl and pqs quorum-sensing sy......Extracellular DNA is one of the major matrix components in Pseudomonas aeruginosa biofilms. It functions as an intercellular connector and plays a role in stabilization of the biofilms. Evidence that DNA release in P. aeruginosa PAO1 biofilms is controlled by the las-rhl and pqs quorum......-sensing systems has been previously presented. This paper provides evidence that DNA release in P. aeruginosa PAO1 biofilms is also under iron regulation. Experiments involving cultivation of P. aeruginosa in microtitre trays suggested that pqs expression, DNA release and biofilm formation were favoured in media...

  10. Characterisation of Lactobacillus plantarum single and multi-strain biofilms

    NARCIS (Netherlands)

    Fernández Ramírez, Mónica D.

    2016-01-01

    Biofilms consist of microorganisms attached to a surface and embedded in a protective matrix of extracellular polymeric substances. Within a biofilm, micro-organisms are protected from harsh environmental conditions including those resulting from cleaning and disinfecting agents leading to food

  11. Host Proteins Determine MRSA Biofilm Structure and Integrity

    DEFF Research Database (Denmark)

    Dreier, Cindy; Nielsen, Astrid; Jørgensen, Nis Pedersen

    Human extracellular matrix (hECM) proteins aids the initial attachment and initiation of an infection, by specific binding to bacterial cell surface proteins. However, the importance of hECM proteins in structure, integrity and antibiotic resilience of a biofilm is unknown. This study aims...... to determine how specific hECM proteins affect S. aureus USA300 JE2 biofilms. Biofilms were grown in the presence of synovial fluid from rheumatoid arteritis patients to mimic in vivo conditions, where bacteria incorporate hECM proteins into the biofilm matrix. Difference in biofilm structure, with and without...... addition of hECM to growth media, was visualized by confocal laser scanning microscopy. Two enzymatic degradation experiments were used to study biofilm matrix composition and importance of hECM proteins: enzymatic removal of specific hECM proteins from growth media, before biofilm formation, and enzymatic...

  12. Facultative control of matrix production optimizes competitive fitness in Pseudomonas aeruginosa PA14 biofilm models.

    Science.gov (United States)

    Madsen, Jonas S; Lin, Yu-Cheng; Squyres, Georgia R; Price-Whelan, Alexa; de Santiago Torio, Ana; Song, Angela; Cornell, William C; Sørensen, Søren J; Xavier, Joao B; Dietrich, Lars E P

    2015-12-01

    As biofilms grow, resident cells inevitably face the challenge of resource limitation. In the opportunistic pathogen Pseudomonas aeruginosa PA14, electron acceptor availability affects matrix production and, as a result, biofilm morphogenesis. The secreted matrix polysaccharide Pel is required for pellicle formation and for colony wrinkling, two activities that promote access to O2. We examined the exploitability and evolvability of Pel production at the air-liquid interface (during pellicle formation) and on solid surfaces (during colony formation). Although Pel contributes to the developmental response to electron acceptor limitation in both biofilm formation regimes, we found variation in the exploitability of its production and necessity for competitive fitness between the two systems. The wild type showed a competitive advantage against a non-Pel-producing mutant in pellicles but no advantage in colonies. Adaptation to the pellicle environment selected for mutants with a competitive advantage against the wild type in pellicles but also caused a severe disadvantage in colonies, even in wrinkled colony centers. Evolution in the colony center produced divergent phenotypes, while adaptation to the colony edge produced mutants with clear competitive advantages against the wild type in this O2-replete niche. In general, the structurally heterogeneous colony environment promoted more diversification than the more homogeneous pellicle. These results suggest that the role of Pel in community structure formation in response to electron acceptor limitation is unique to specific biofilm models and that the facultative control of Pel production is required for PA14 to maintain optimum benefit in different types of communities. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. The role of microbial-produced extracellular polymeric matrix in the formation and survival of biological soil crusts

    Science.gov (United States)

    Rossi, Federico; Adessi, Alessandra; De Philippis, Roberto

    2016-04-01

    Biological soil crusts (BSCs) are complex communities commonly constituting organo-mineral layers in arid and semiarid environment having a major influence on these ecosystems (Belnap and Lange, 2001). They have high tolerance towards a-biotic stresses and fluctuations in moisture, illumination, salinity and nutrients. The plasticity exhibited by BSCs is hugely contributed by the presence of the extracellular polymeric matrix (EPM) that is synthesized by crustal organisms, notably cyanobacteria and microalgae. This polysaccharidic net plays key roles in biofilm relations with the surrounding constrained environment. Notably, EPM concurs in coping with water scarcity, freezing and salt stress; increases biolayers stability against erosion, and is involved in nutrient provision (Rossi and De Philippis, 2015). We conducted several investigations in a research area located in the Inner Mongolian desert (Inner Mongolia, China) where BSCs were induced over different sites through inoculation-based techniques performed in different years. Our studies were aimed at determining the role of EPM in BSC development and survival in such a hyper-arid system. This presentation will report the results concerning the role of EPM in water capture from non-rainfall sources, water maintenance at the topsoil, and in water infiltrability, the latter being a factor with important ecological implications. In additions we investigated the role of the matrix as a source of carbon for the crustal heterotrophs. Furthermore, EPM was extracted with methods optimized in our lab, aiming at removing tightly bound fractions and loosely bound fractions from BSCs having different ages. The fractions were analyzed in terms of monosaccharidic composition, and molecular weight (MW) distribution. We show how the relative amounts of uronic acids increase in the EPM with the age of the crusts, implying advantages for the community-water relations. In addition, we observed significant differences in MW

  14. The Interface between Fungal Biofilms and Innate Immunity

    Directory of Open Access Journals (Sweden)

    John F. Kernien

    2018-01-01

    Full Text Available Fungal biofilms are communities of adherent cells surrounded by an extracellular matrix. These biofilms are commonly found during infection caused by a variety of fungal pathogens. Clinically, biofilm infections can be extremely difficult to eradicate due to their resistance to antifungals and host defenses. Biofilm formation can protect fungal pathogens from many aspects of the innate immune system, including killing by neutrophils and monocytes. Altered immune recognition during this phase of growth is also evident by changes in the cytokine profiles of monocytes and macrophages exposed to biofilm. In this manuscript, we review the host response to fungal biofilms, focusing on how these structures are recognized by the innate immune system. Biofilms formed by Candida, Aspergillus, and Cryptococcus have received the most attention and are highlighted. We describe common themes involved in the resilience of fungal biofilms to host immunity and give examples of biofilm defenses that are pathogen-specific.

  15. Correlation between expression of extracellular matrix metalloproteinase inducer and matrix metalloproteinase-2 and cervical lymph node metastasis of nasopharyngeal carcinoma.

    Science.gov (United States)

    Huang, Tian; Chen, Mao-Huai; Wu, Ming-Yao; Wu, Xian-Ying

    2013-03-01

    We evaluated the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and matrix metalloproteinase-2 (MMP-2) in nasopharyngeal carcinoma (NPC) and studied their relationship with cervical lymph node metastasis. Immunohistochemical staining was used to detect the expression of EMMPRIN and MMP-2 in specimens from patients with chronic nasopharyngitis (CN), nonmetastastic NPC (NM-NPC), and lymph node-metastatic NPC (LNM-NPC). The rates of positive EMMPRIN expression in CN, NM-NPC, and LNM-NPC were 13.3%, 30.0%, and 66.7%, respectively. Significant differences were found between the rates in CN and LNM-NPC (p correlated (rs = 0.466; p <0.01). Nasopharyngeal carcinoma cells may attain enhanced metastastic capability through the expression of MMP-2 induced by EMMPRIN.

  16. Matrix metalloproteinases (MMPs), the main extracellular matrix (ECM) enzymes in collagen degradation, as a target for anticancer drugs.

    Science.gov (United States)

    Jabłońska-Trypuć, Agata; Matejczyk, Marzena; Rosochacki, Stanisław

    2016-01-01

    The main group of enzymes responsible for the collagen and other protein degradation in extracellular matrix (ECM) are matrix metalloproteinases (MMPs). Collagen is the main structural component of connective tissue and its degradation is a very important process in the development, morphogenesis, tissue remodeling, and repair. Typical structure of MMPs consists of several distinct domains. MMP family can be divided into six groups: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and other non-classified MMPs. MMPs and their inhibitors have multiple biological functions in all stages of cancer development: from initiation to outgrowth of clinically relevant metastases and likewise in apoptosis and angiogenesis. MMPs and their inhibitors are extensively examined as potential anticancer drugs. MMP inhibitors can be divided into two main groups: synthetic and natural inhibitors. Selected synthetic inhibitors are in clinical trials on humans, e.g. synthetic peptides, non-peptidic molecules, chemically modified tetracyclines, and bisphosphonates. Natural MMP inhibitors are mainly isoflavonoids and shark cartilage.

  17. The clinical impact of bacterial biofilms

    DEFF Research Database (Denmark)

    Høiby, Niels; Ciofu, Oana; Johansen, Helle Krogh

    2011-01-01

    Bacteria survive in nature by forming biofilms on surfaces and probably most, if not all, bacteria (and fungi) are capable of forming biofilms. A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and extracellular DNA....... Bacterial biofilms are resistant to antibiotics, disinfectant chemicals and to phagocytosis and other components of the innate and adaptive inflammatory defense system of the body. It is known, for example, that persistence of staphylococcal infections related to foreign bodies is due to biofilm formation....... Likewise, chronic Pseudomonas aeruginosa lung infections in cystic fibrosis patients are caused by biofilm growing mucoid strains. Gradients of nutrients and oxygen exist from the top to the bottom of biofilms and the bacterial cells located in nutrient poor areas have decreased metabolic activity...

  18. Analysis of the interaction of extracellular matrix and phenotype of bladder cancer cells

    International Nuclear Information System (INIS)

    Dozmorov, Mikhail G; Kyker, Kimberly D; Saban, Ricardo; Knowlton, Nicholas; Dozmorov, Igor; Centola, Michael B; Hurst, Robert E

    2006-01-01

    The extracellular matrix has a major effect upon the malignant properties of bladder cancer cells both in vitro in 3-dimensional culture and in vivo. Comparing gene expression of several bladder cancer cells lines grown under permissive and suppressive conditions in 3-dimensional growth on cancer-derived and normal-derived basement membrane gels respectively and on plastic in conventional tissue culture provides a model system for investigating the interaction of malignancy and extracellular matrix. Understanding how the extracellular matrix affects the phenotype of bladder cancer cells may provide important clues to identify new markers or targets for therapy. Five bladder cancer cell lines and one immortalized, but non-tumorigenic, urothelial line were grown on Matrigel, a cancer-derived ECM, on SISgel, a normal-derived ECM, and on plastic, where the only ECM is derived from the cells themselves. The transcriptomes were analyzed on an array of 1186 well-annotated cancer derived cDNAs containing most of the major pathways for malignancy. Hypervariable genes expressing more variability across cell lines than a set expressing technical variability were analyzed further. Expression values were clustered, and to identify genes most likely to represent biological factors, statistically over-represented ontologies and transcriptional regulatory elements were identified. Approximately 400 of the 1186 total genes were expressed 2 SD above background. Approximately 100 genes were hypervariable in cells grown on each ECM, but the pattern was different in each case. A core of 20 were identified as hypervariable under all 3 growth conditions, and 33 were hypervariable on both SISgel and Matrigel, but not on plastic. Clustering of the hypervariable genes showed very different patterns for the same 6 cell types on the different ECM. Even when loss of cell cycle regulation was identified, different genes were involved, depending on the ECM. Under the most permissive conditions

  19. The characteristics of extracellular polymeric substances and soluble microbial products in moving bed biofilm reactor-membrane bioreactor.

    Science.gov (United States)

    Duan, Liang; Jiang, Wei; Song, Yonghui; Xia, Siqing; Hermanowicz, Slawomir W

    2013-11-01

    The characteristics of extracellular polymeric substances (EPS) and soluble microbial products (SMP) in conventional membrane bioreactor (MBR) and in moving bed biofilm reactor-membrane bioreactors (MBBR-MBR) were investigated in long-term (170 days) experiments. The results showed that all reactors had high removal efficiency of ammonium and COD, despite very different fouling conditions. The MBBR-MBR with media fill ratio of 26.7% had much lower total membrane resistance and no obvious fouling were detected during the whole operation. In contrast, MBR and MBBR-MBR with lower and higher media fill experienced more significant fouling. Low fouling at optimum fill ratio may be due to the higher percentage of small molecular size (100 kDa) of EPS and SMP in the reactor. The composition of EPS and SMP affected fouling due to different O-H bonds in hydroxyl functional groups, and less polysaccharides and lipids. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. EcmPred: Prediction of extracellular matrix proteins based on random forest with maximum relevance minimum redundancy feature selection

    KAUST Repository

    Kandaswamy, Krishna Kumar Umar; Ganesan, Pugalenthi; Kalies, Kai Uwe; Hartmann, Enno; Martinetz, Thomas M.

    2013-01-01

    The extracellular matrix (ECM) is a major component of tissues of multicellular organisms. It consists of secreted macromolecules, mainly polysaccharides and glycoproteins. Malfunctions of ECM proteins lead to severe disorders such as marfan

  1. AtlA Mediates Extracellular DNA Release, Which Contributes to Streptococcus mutans Biofilm Formation in an Experimental Rat Model of Infective Endocarditis.

    Science.gov (United States)

    Jung, Chiau-Jing; Hsu, Ron-Bin; Shun, Chia-Tung; Hsu, Chih-Chieh; Chia, Jean-San

    2017-09-01

    Host factors, such as platelets, have been shown to enhance biofilm formation by oral commensal streptococci, inducing infective endocarditis (IE), but how bacterial components contribute to biofilm formation in vivo is still not clear. We demonstrated previously that an isogenic mutant strain of Streptococcus mutans deficient in autolysin AtlA (Δ atlA ) showed a reduced ability to cause vegetation in a rat model of bacterial endocarditis. However, the role of AtlA in bacterial biofilm formation is unclear. In this study, confocal laser scanning microscopy analysis showed that extracellular DNA (eDNA) was embedded in S. mutans GS5 floes during biofilm formation on damaged heart valves, but an Δ atlA strain could not form bacterial aggregates. Semiquantification of eDNA by PCR with bacterial 16S rRNA primers demonstrated that the Δ atlA mutant strain produced dramatically less eDNA than the wild type. Similar results were observed with in vitro biofilm models. The addition of polyanethol sulfonate, a chemical lysis inhibitor, revealed that eDNA release mediated by bacterial cell lysis is required for biofilm initiation and maturation in the wild-type strain. Supplementation of cultures with calcium ions reduced wild-type growth but increased eDNA release and biofilm mass. The effect of calcium ions on biofilm formation was abolished in Δ atlA cultures and by the addition of polyanethol sulfonate. The VicK sensor, but not CiaH, was found to be required for the induction of eDNA release or the stimulation of biofilm formation by calcium ions. These data suggest that calcium ion-regulated AtlA maturation mediates the release of eDNA by S. mutans , which contributes to biofilm formation in infective endocarditis. Copyright © 2017 American Society for Microbiology.

  2. Mechanical phenotyping of cells and extracellular matrix as grade and stage markers of lung tumor tissues.

    Science.gov (United States)

    Panzetta, Valeria; Musella, Ida; Rapa, Ida; Volante, Marco; Netti, Paolo A; Fusco, Sabato

    2017-07-15

    The mechanical cross-talk between cells and the extra-cellular matrix (ECM) regulates the properties, functions and healthiness of the tissues. When this is disturbed it changes the mechanical state of the tissue components, singularly or together, and cancer, along with other diseases, may start and progress. However, the bi-univocal mechanical interplay between cells and the ECM is still not properly understood. In this study we show how a microrheology technique gives us the opportunity to evaluate the mechanics of cells and the ECM at the same time. The mechanical phenotyping was performed on the surgically removed tissues of 10 patients affected by adenocarcinoma of the lung. A correlation between the mechanics and the grade and stage of the tumor was reported and compared to the mechanical characteristics of the healthy tissue. Our findings suggest a sort of asymmetric modification of the mechanical properties of the cells and the extra-cellular matrix in the tumor, being the more compliant cell even though it resides in a stiffer matrix. Overall, the simultaneous mechanical characterization of the tissues constituents (cells and ECM) provided new support for diagnosis and offered alternative points of analysis for cancer mechanobiology. When the integrity of the mechanical cross-talk between cells and the extra-cellular matrix is disturbed cancer, along with other diseases, may initiate and progress. Here, we show how a new technique gives the opportunity to evaluate the mechanics of cells and the ECM at the same time. It was applied on surgically removed tissues of 10 patients affected by adenocarcinoma of the lung and a correlation between the mechanics and the grade and stage of the tumor was reported and compared to the mechanical characteristics of the healthy tissue. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  3. Mitochondrial function in engineered cardiac tissues is regulated by extracellular matrix elasticity and tissue alignment.

    Science.gov (United States)

    Lyra-Leite, Davi M; Andres, Allen M; Petersen, Andrew P; Ariyasinghe, Nethika R; Cho, Nathan; Lee, Jezell A; Gottlieb, Roberta A; McCain, Megan L

    2017-10-01

    Mitochondria in cardiac myocytes are critical for generating ATP to meet the high metabolic demands associated with sarcomere shortening. Distinct remodeling of mitochondrial structure and function occur in cardiac myocytes in both developmental and pathological settings. However, the factors that underlie these changes are poorly understood. Because remodeling of tissue architecture and extracellular matrix (ECM) elasticity are also hallmarks of ventricular development and disease, we hypothesize that these environmental factors regulate mitochondrial function in cardiac myocytes. To test this, we developed a new procedure to transfer tunable polydimethylsiloxane disks microcontact-printed with fibronectin into cell culture microplates. We cultured Sprague-Dawley neonatal rat ventricular myocytes within the wells, which consistently formed tissues following the printed fibronectin, and measured oxygen consumption rate using a Seahorse extracellular flux analyzer. Our data indicate that parameters associated with baseline metabolism are predominantly regulated by ECM elasticity, whereas the ability of tissues to adapt to metabolic stress is regulated by both ECM elasticity and tissue alignment. Furthermore, bioenergetic health index, which reflects both the positive and negative aspects of oxygen consumption, was highest in aligned tissues on the most rigid substrate, suggesting that overall mitochondrial function is regulated by both ECM elasticity and tissue alignment. Our results demonstrate that mitochondrial function is regulated by both ECM elasticity and myofibril architecture in cardiac myocytes. This provides novel insight into how extracellular cues impact mitochondrial function in the context of cardiac development and disease. NEW & NOTEWORTHY A new methodology has been developed to measure O 2 consumption rates in engineered cardiac tissues with independent control over tissue alignment and matrix elasticity. This led to the findings that matrix

  4. Host-Parasite Interaction: Parasite-Derived and -Induced Proteases That Degrade Human Extracellular Matrix

    Directory of Open Access Journals (Sweden)

    Carolina Piña-Vázquez

    2012-01-01

    Full Text Available Parasitic protozoa are among the most important pathogens worldwide. Diseases such as malaria, leishmaniasis, amoebiasis, giardiasis, trichomoniasis, and trypanosomiasis affect millions of people. Humans are constantly threatened by infections caused by these pathogens. Parasites engage a plethora of surface and secreted molecules to attach to and enter mammalian cells. The secretion of lytic enzymes by parasites into host organs mediates critical interactions because of the invasion and destruction of interstitial tissues, enabling parasite migration to other sites within the hosts. Extracellular matrix is a complex, cross-linked structure that holds cells together in an organized assembly and that forms the basement membrane lining (basal lamina. The extracellular matrix represents a major barrier to parasites. Therefore, the evolution of mechanisms for connective-tissue degradation may be of great importance for parasite survival. Recent advances have been achieved in our understanding of the biochemistry and molecular biology of proteases from parasitic protozoa. The focus of this paper is to discuss the role of protozoan parasitic proteases in the degradation of host ECM proteins and the participation of these molecules as virulence factors. We divide the paper into two sections, extracellular and intracellular protozoa.

  5. Sticking together: building a biofilm the Bacillus subtilis way.

    Science.gov (United States)

    Vlamakis, Hera; Chai, Yunrong; Beauregard, Pascale; Losick, Richard; Kolter, Roberto

    2013-03-01

    Biofilms are ubiquitous communities of tightly associated bacteria encased in an extracellular matrix. Bacillus subtilis has long served as a robust model organism to examine the molecular mechanisms of biofilm formation, and a number of studies have revealed that this process is regulated by several integrated pathways. In this Review, we focus on the molecular mechanisms that control B. subtilis biofilm assembly, and then briefly summarize the current state of knowledge regarding biofilm disassembly. We also discuss recent progress that has expanded our understanding of B. subtilis biofilm formation on plant roots, which are a natural habitat for this soil bacterium.

  6. Chondrogenic properties of collagen type XI, a component of cartilage extracellular matrix.

    Science.gov (United States)

    Li, Ang; Wei, Yiyong; Hung, Clark; Vunjak-Novakovic, Gordana

    2018-08-01

    Cartilage extracellular matrix (ECM) has been used for promoting tissue engineering. However, the exact effects of ECM on chondrogenesis and the acting mechanisms are not well understood. In this study, we investigated the chondrogenic effects of cartilage ECM on human mesenchymal stem cells (MSCs) and identified the contributing molecular components. To this end, a preparation of articular cartilage ECM was supplemented to pellets of chondrogenically differentiating MSCs, pellets of human chondrocytes, and bovine articular cartilage explants to evaluate the effects on cell proliferation and the production of cartilaginous matrix. Selective enzymatic digestion and screening of ECM components were conducted to identify matrix molecules with chondrogenic properties. Cartilage ECM promoted MSC proliferation, production of cartilaginous matrix, and maturity of chondrogenic differentiation, and inhibited the hypertrophic differentiation of MSC-derived chondrocytes. Selective digestion of ECM components revealed a contributory role of collagens in promoting chondrogenesis. The screening of various collagen subtypes revealed strong chondrogenic effect of collagen type XI. Finally, collagen XI was found to promote production and inhibit degradation of cartilage matrix in human articular chondrocyte pellets and bovine articular cartilage explants. Our results indicate that cartilage ECM promotes chondrogenesis and inhibits hypertrophic differentiation in MSCs. Collagen type XI is the ECM component that has the strongest effects on enhancing the production and inhibiting the degradation of cartilage matrix. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Modeling the tumor extracellular matrix: Tissue engineering tools repurposed towards new frontiers in cancer biology.

    Science.gov (United States)

    Gill, Bartley J; West, Jennifer L

    2014-06-27

    Cancer progression is mediated by complex epigenetic, protein and structural influences. Critical among them are the biochemical, mechanical and architectural properties of the extracellular matrix (ECM). In recognition of the ECM's important role, cancer biologists have repurposed matrix mimetic culture systems first widely used by tissue engineers as new tools for in vitro study of tumor models. In this review we discuss the pathological changes in tumor ECM, the limitations of 2D culture on both traditional and polyacrylamide hydrogel surfaces in modeling these characteristics and advances in both naturally derived and synthetic scaffolds to facilitate more complex and controllable 3D cancer cell culture. Studies using naturally derived matrix materials like Matrigel and collagen have produced significant findings related to tumor morphogenesis and matrix invasion in a 3D environment and the mechanotransductive signaling that mediates key tumor-matrix interaction. However, lack of precise experimental control over important matrix factors in these matrices have increasingly led investigators to synthetic and semi-synthetic scaffolds that offer the engineering of specific ECM cues and the potential for more advanced experimental manipulations. Synthetic scaffolds composed of poly(ethylene glycol) (PEG), for example, facilitate highly biocompatible 3D culture, modular bioactive features like cell-mediated matrix degradation and complete independent control over matrix bioactivity and mechanics. Future work in PEG or similar reductionist synthetic matrix systems should enable the study of increasingly complex and dynamic tumor-ECM relationships in the hopes that accurate modeling of these relationships may reveal new cancer therapeutics targeting tumor progression and metastasis. © 2013 Published by Elsevier Ltd.

  8. Biocorrosion: towards understanding interactions between biofilms and metals.

    Science.gov (United States)

    Beech, Iwona B; Sunner, Jan

    2004-06-01

    The term microbially influenced corrosion, or biocorrosion, refers to the accelerated deterioration of metals owing to the presence of biofilms on their surfaces. The detailed mechanisms of biocorrosion are still poorly understood. Recent investigations into biocorrosion have focused on the influence of biomineralization processes taking place on metallic surfaces and the impact of extracellular enzymes, active within the biofilm matrix, on electrochemical reactions at the biofilm-metal interface.

  9. Bioprinting of 3D Tissue Models Using Decellularized Extracellular Matrix Bioink.

    Science.gov (United States)

    Pati, Falguni; Cho, Dong-Woo

    2017-01-01

    Bioprinting provides an exciting opportunity to print and pattern all the components that make up a tissue-cells and extracellular matrix (ECM) material-in three dimensions (3D) to generate tissue analogues. A large number of materials have been used for making bioinks; however, majority of them cannot represent the complexity of natural ECM and thus are unable to reconstitute the intrinsic cellular morphologies and functions. We present here a method for making of bioink from decellularized extracellular matrices (dECMs) and a protocol for bioprinting of cell-laden constructs with this novel bioink. The dECM bioink is capable of providing an optimized microenvironment that is conducive to the growth of 3D structured tissue. We have prepared bioinks from different tissues, including adipose, cartilage and heart tissues and achieved high cell viability and functionality of the bioprinted tissue structures using our novel bioink.

  10. Chondrocyte secreted CRTAC1: a glycosylated extracellular matrix molecule of human articular cartilage.

    Science.gov (United States)

    Steck, Eric; Bräun, Jessica; Pelttari, Karoliina; Kadel, Stephanie; Kalbacher, Hubert; Richter, Wiltrud

    2007-01-01

    Cartilage acidic protein 1 (CRTAC1), a novel human marker which allowed discrimination of human chondrocytes from osteoblasts and mesenchymal stem cells in culture was so far studied only on the RNA-level. We here describe its genomic organisation and detect a new brain expressed (CRTAC1-B) isoform resulting from alternate last exon usage which is highly conserved in vertebrates. In humans, we identify an exon sharing process with the neighbouring tail-to-tail orientated gene leading to CRTAC1-A. This isoform is produced by cultured human chondrocytes, localized in the extracellular matrix of articular cartilage and its secretion can be stimulated by BMP4. Of five putative O-glycosylation motifs in the last exon of CRTAC1-A, the most C-terminal one is modified according to exposure of serial C-terminal deletion mutants to the O-glycosylation inhibitor Benzyl-alpha-GalNAc. Both isoforms contain four FG-GAP repeat domains and an RGD integrin binding motif, suggesting cell-cell or cell-matrix interaction potential. In summary, CRTAC1 acquired an alternate last exon from the tail-to-tail oriented neighbouring gene in humans resulting in the glycosylated isoform CRTAC1-A which represents a new extracellular matrix molecule of articular cartilage.

  11. Extracellular matrix and growth factor engineering for controlled angiogenesis in regenerative medicine

    Directory of Open Access Journals (Sweden)

    Mikaël M Martino

    2015-04-01

    Full Text Available Blood vessel growth plays a key role in regenerative medicine, both to restore blood supply to ischemic tissues and to ensure rapid vascularization of clinical-size tissue-engineered grafts. For example, vascular endothelial growth factor (VEGF is the master regulator of physiological blood vessel growth and is one of the main molecular targets of therapeutic angiogenesis approaches. However, angiogenesis is a complex process and there is a need to develop rational therapeutic strategies based on a firm understanding of basic vascular biology principles, as evidenced by the disappointing results of initial clinical trials of angiogenic factor delivery. In particular, the spatial localization of angiogenic signals in the extracellular matrix is crucial to ensure the proper assembly and maturation of new vascular structures. Here we discuss the therapeutic implications of matrix interactions of angiogenic factors, with a special emphasis on VEGF, as well as provide an overview of current approaches, based on protein and biomaterial engineering that mimic the regulatory functions of extracellular matrix to optimize the signaling microenvironment of vascular growth factors.

  12. Gonadotropin-releasing hormone analogues inhibit leiomyoma extracellular matrix despite presence of gonadal hormones.

    Science.gov (United States)

    Malik, Minnie; Britten, Joy; Cox, Jeris; Patel, Amrita; Catherino, William H

    2016-01-01

    To determine the effect of GnRH analogues (GnRH-a) leuprolide acetate (LA) and cetrorelix acetate on gonadal hormone-regulated expression of extracellular matrix in uterine leiomyoma three-dimensional (3D) cultures. Laboratory study. University research laboratory. Women undergoing hysterectomy for symptomatic leiomyomas. The 3D cell cultures, protein analysis, Western blot, immunohistochemistry. Expression of extracellular matrix proteins, collagen 1, fibronectin, and versican in leiomyoma cells 3D cultures exposed to E2, P, LA, cetrorelix acetate, and combinations for 24- and 72-hour time points. The 3D leiomyoma cultures exposed to E2 for 24 hours demonstrated an increased expression of collagen-1 and fibronectin, which was maintained for up to 72 hours, a time point at which versican was up-regulated significantly. Although P up-regulated collagen-1 protein (1.29 ± 0.04) within 24 hours of exposure, significant increase in all extracellular matrix (ECM) proteins was observed when the gonadal hormones were used concomitantly. Significant decrease in the amount of ECM proteins was observed on use of GnRH-a, LA and cetrorelix, with 24-hour exposure. Both the compounds also significantly decreased ECM protein concentration despite the presence of E2 or both gonadal hormones. This study demonstrates that GnRH-a directly affect the gonadal hormone-regulated collagen-1, fibronectin, and versican production in their presence. These findings suggest that localized therapy with GnRH-a may inhibit leiomyoma growth even in the presence of endogenous gonadal hormone exposure, thereby providing a mechanism to eliminate the hypoestrogenic side effects associated with GnRH-a therapy. Published by Elsevier Inc.

  13. Fibronectin distribution in the extracellular matrix in the cells grown in deuterated media

    International Nuclear Information System (INIS)

    Buzgariu, Wanda; Caloianu, Maria; Moldovan, Lucia; Stefanescu, I.; Titescu, Gh.

    2003-01-01

    The aim of this work is the study of the influence of deuterated water upon the synthesis and organization of fibronectin (FN) in extracellular matrices. Changes were evidenced at the level of extracellular matrix in case of embryo fibroblast cultivation in media with different concentrations of heavy water (20%, 40% and 65%). FN was identified in the extracellular matrix by means of indirect immunocytochemical technique, using a secondary antibody coupled with peroxydase. In the presence of heavy water in culture medium, the arrangement and localization of cellular FN showed changes depending on the exposure time, D 2 O concentration in the medium and the FN polymerization step in the extra cellular matrix in correlation with the culture stage of the monolayer. The heavy water determined a strong reduction of the FN amount released by the cells. This reduction was most evident in the 65% D 2 O medium following a 5 day exposure. The FN distribution after 2 day exposure in an early stage with regards to the FN network formation in a the deuterated medium presented a FN pericellular distribution arranged in aggregates. The heavy water can act upon formation of FN fibrils immediately due to solvent role in the FN polymerization process but also indirectly through metabolic processes and so upon the protein synthesis and FN cellular secretion.The FN network arrangement in the cells cultivated in deuterated media as aggregates might be the effect of solvent role played by D 2 O while the quantitative reduction of FN results from perturbation of protein synthesis as well from biochemical synthesis reactions

  14. Suppression of ICE and Apoptosis in Mammary Epithelial Cells by Extracellular Matrix

    Energy Technology Data Exchange (ETDEWEB)

    Boudreau, Nancy; Sympson, C. J.; Werb, Zena; Bissell, Mina J.

    1994-12-01

    Apoptosis (programmed cell death) plays a major role in development and tissue regeneration. Basement membrane extracellular matrix (ECM), but not fibronectin or collagen, was shown to suppress apoptosis of mammary epithelial cells in tissue culture and in vivo. Apoptosis was induced by antibodies to beta 1 integrins or by overexpression of stromelysin-1, which degrades ECM. Expression of interleukin-1 beta converting enzyme (ICE) correlated with the loss of ECM, and inhibitors of ICE activity prevented apoptosis. These results suggest that ECM regulates apoptosis in mammary epithelial cells through an integrin-dependent negative regulation of ICE expression.

  15. Extracellular matrix metalloproteinase inducer enhances host resistance against pseudomonas aeruginosa infection through MAPK signaling pathway

    OpenAIRE

    Li, Yongwei; Chen, Lu; Wang, Chunxia; Chen, Jianshe; Zhang, Xiaoqian; Hu, Yue; Niu, Xiaobin; Pei, Dongxu; He, Zhiqiang; Bi, Yongyi

    2016-01-01

    This study aims to explore the role of extra-cellular matrix metalloproteinase inducer (EMMPRIN) in the drug resistance of the pseudomonas aeruginosa (PA). The BALB/c mice were transfected with PA, then the mice were infected with the siRNA of EMMPRIN to silence the EMMPRIN gene. The EMMPRIN mRNA and protein were detected by using RT-PCR and western blot, respectively. In order to examine the function of EMMPRIN in drug resistance of PA, the BALB/c and C57BL/6 mice were treated with EMMPRIN s...

  16. Of extracellular matrix, scaffolds, and signaling: Tissuearchitectureregulates development, homeostasis, and cancer

    Energy Technology Data Exchange (ETDEWEB)

    Nelson, Celeste M.; Bissell, Mina J.

    2006-03-09

    The microenvironment surrounding cells influences gene expression, such that a cell's behavior is largely determined by its interactions with the extracellular matrix, neighboring cells, and soluble cues released locally or by distant tissues. We describe the essential role of context and organ structure in directing mammary gland development and differentiated function, and in determining response to oncogenic insults including mutations. We expand on the concept of 'dynamic reciprocity' to present an integrated view of development, cancer, and aging, and posit that genes are like piano keys: while essential, it is the context that makes the music.

  17. VANGL2 interacts with integrin αv to regulate matrix metalloproteinase activity and cell adhesion to the extracellular matrix.

    Science.gov (United States)

    Jessen, Tammy N; Jessen, Jason R

    2017-12-15

    Planar cell polarity (PCP) proteins are implicated in a variety of morphogenetic processes including embryonic cell migration and potentially cancer progression. During zebrafish gastrulation, the transmembrane protein Vang-like 2 (VANGL2) is required for PCP and directed cell migration. These cell behaviors occur in the context of a fibrillar extracellular matrix (ECM). While it is thought that interactions with the ECM regulate cell migration, it is unclear how PCP proteins such as VANGL2 influence these events. Using an in vitro cell culture model system, we previously showed that human VANGL2 negatively regulates membrane type-1 matrix metalloproteinase (MMP14) and activation of secreted matrix metalloproteinase 2 (MMP2). Here, we investigated the functional relationship between VANGL2, integrin αvβ3, and MMP2 activation. We provide evidence that VANGL2 regulates cell surface integrin αvβ3 expression and adhesion to fibronectin, laminin, and vitronectin. Inhibition of MMP14/MMP2 activity suppressed the cell adhesion defect in VANGL2 knockdown cells. Furthermore, our data show that MMP14 and integrin αv are required for increased proteolysis by VANGL2 knockdown cells. Lastly, we have identified integrin αvβ3 as a novel VANGL2 binding partner. Together, these findings begin to dissect the molecular underpinnings of how VANGL2 regulates MMP activity and cell adhesion to the ECM. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Teaching the Extracellular Matrix and Introducing Online Databases within a Multidisciplinary Course with i-Cell-MATRIX: A Student-Centered Approach

    Science.gov (United States)

    Sousa, Joao Carlos; Costa, Manuel Joao; Palha, Joana Almeida

    2010-01-01

    The biochemistry and molecular biology of the extracellular matrix (ECM) is difficult to convey to students in a classroom setting in ways that capture their interest. The understanding of the matrix's roles in physiological and pathological conditions study will presumably be hampered by insufficient knowledge of its molecular structure.…

  19. An update on Pseudomonas aeruginosa biofilm formation, tolerance, and dispersal

    DEFF Research Database (Denmark)

    Harmsen, Morten; Yang, Liang; Pamp, Sünje Johanna

    2010-01-01

    We review the recent advances in the understanding of the Pseudomonas aeruginosa biofilm lifestyle from studies using in vitro laboratory setups such as flow chambers and microtiter trays. Recent work sheds light on the role of nutrients, motility, and quorum sensing in structure formation in P....... aeruginosa biofilms. The second messenger, c-di-GMP, is established as an important regulator of the synthesis of polysaccharide and protein components of the biofilm matrix. Extracellular DNA is shown to be an essential component of the biofilm matrix. It has become apparent that biofilm formation involves...... interactions between different subpopulations. The molecular mechanisms underlying the tolerance of biofilm bacteria to antimicrobial agents are beginning to be unraveled, and new knowledge has been obtained regarding the environmental cues and regulatory mechanisms involved in biofilm dispersal....

  20. Nonlinear mechanical response of the extracellular matrix: learning from articular cartilage

    Science.gov (United States)

    Kearns, Sarah; Das, Moumita

    2015-03-01

    We study the mechanical structure-function relations in the extracellular matrix (ECM) with focus on nonlinear shear and compression response. As a model system, our study focuses on the ECM in articular cartilage tissue which has two major mechanobiological components: a network of the biopolymer collagen that acts as a stiff, reinforcing matrix, and a flexible aggrecan network that facilitates deformability. We model this system as a double network hydrogel made of interpenetrating networks of stiff and flexible biopolymers respectively. We study the linear and nonlinear mechanical response of the model ECM to shear and compression forces using a combination of rigidity percolation theory and energy minimization approaches. Our results may provide useful insights into the design principles of the ECM as well as biomimetic hydrogels that are mechanically robust and can, at the same time, easily adapt to cues in their surroundings.

  1. Dynamics of extracellular matrix in ovarian follicles and corpora lutea of mice

    DEFF Research Database (Denmark)

    Irving-Rodgers, Helen F; Hummitzsch, Katja; Murdiyarso, Lydia S

    2009-01-01

    Despite the mouse being an important laboratory species, little is known about changes in its extracellular matrix (ECM) during follicle and corpora lutea formation and regression. Follicle development was induced in mice (29 days of age/experimental day 0) by injections of pregnant mare's serum...... and antral follicles. The focimatrix, a specialised matrix of the membrana granulosa, contained collagen type IV alpha1 and alpha2, laminin alpha1, beta1 and gamma1 chains, nidogens 1 and 2, perlecan and collagen type XVIII. In the corpora lutea, staining was restricted to capillary sub-endothelial basal...... gonadotrophin on days 0 and 1 and ovulation was induced by injection of human chorionic gonadotrophin on day 2. Ovaries were collected for immunohistochemistry (n=10 per group) on days 0, 2 and 5. Another group was mated and ovaries were examined on day 11 (n=7). Collagen type IV alpha1 and alpha2, laminin...

  2. Mechanistic understanding of nanoparticles' interactions with extracellular matrix: the cell and immune system.

    Science.gov (United States)

    Engin, Ayse Basak; Nikitovic, Dragana; Neagu, Monica; Henrich-Noack, Petra; Docea, Anca Oana; Shtilman, Mikhail I; Golokhvast, Kirill; Tsatsakis, Aristidis M

    2017-06-24

    Extracellular matrix (ECM) is an extraordinarily complex and unique meshwork composed of structural proteins and glycosaminoglycans. The ECM provides essential physical scaffolding for the cellular constituents, as well as contributes to crucial biochemical signaling. Importantly, ECM is an indispensable part of all biological barriers and substantially modulates the interchange of the nanotechnology products through these barriers. The interactions of the ECM with nanoparticles (NPs) depend on the morphological characteristics of intercellular matrix and on the physical characteristics of the NPs and may be either deleterious or beneficial. Importantly, an altered expression of ECM molecules ultimately affects all biological processes including inflammation. This review critically discusses the specific behavior of NPs that are within the ECM domain, and passing through the biological barriers. Furthermore, regenerative and toxicological aspects of nanomaterials are debated in terms of the immune cells-NPs interactions.

  3. The planar cell polarity protein VANGL2 coordinates remodeling of the extracellular matrix.

    Science.gov (United States)

    Williams, B Blairanne; Mundell, Nathan; Dunlap, Julie; Jessen, Jason

    2012-07-01

    Understanding how planar cell polarity (PCP) is established, maintained, and coordinated in migrating cell populations is an important area of research with implications for both embryonic morphogenesis and tumor cell invasion. We recently reported that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell surface level of membrane type-1 matrix metalloproteinase (MMP14 or MT1-MMP). Here, we further discuss these findings in terms of extracellular matrix (ECM) remodeling, cell migration, and zebrafish gastrulation. We also demonstrate that VANGL2 function impacts the focal degradation of ECM by human cancer cells including the formation or stability of invadopodia. Together, our findings implicate MMP14 as a downstream effector of VANGL2 signaling and suggest a model whereby the regulation of pericellular proteolysis is a fundamental aspect of PCP in migrating cells.

  4. Cell-extracellular matrix and cell-cell adhesion are linked by syndecan-4

    DEFF Research Database (Denmark)

    Pakideeri Karat, Sandeep Gopal; Multhaupt, Hinke A B; Pocock, Roger

    2017-01-01

    Cell-extracellular matrix (ECM) and cell-cell junctions that employ microfilaments are sites of tension. They are important for tissue repair, morphogenetic movements and can be emblematic of matrix contraction in fibrotic disease and the stroma of solid tumors. One cell surface receptor, syndecan...... calcium. While it is known that cell-ECM and cell-cell junctions may be linked, possible roles for syndecans in this process are not understood. Here we show that wild type primary fibroblasts and those lacking syndecan-4 utilize different cadherins in their adherens junctions and that tension is a major...... factor in this differential response. This corresponds to the reduced ability of fibroblasts lacking syndecan-4 to exert tension on the ECM and we now show that this may extend to reduced tension in cell-cell adhesion....

  5. Macrophage-mediated proteolytic remodeling of the extracellular matrix in atherosclerosis results in neoepitopes

    DEFF Research Database (Denmark)

    Skjøt-Arkil, Helene; Barascuk, Natasha; Register, Thomas

    2010-01-01

    in almost all stages of atherosclerosis, by both initiating atherosclerotic plaques and degrading them through the secretion of proteolytic enzymes leading to rupture. This review summarizes the literature on the role of macrophages and their proteolytic activity on proteins in the extracellular matrix (ECM......) of the atherosclerotic plaque with a view to suggest a novel approach for identification of vulnerable plaques and turnover by the use of a new type of biomarker. The PubMed database was searched using the terms macrophages, foam cells, atherosclerosis, CVD, ECM remodeling, biomarker, neoepitope, matrix...... of the constituents of the ECM of the atherosclerotic plaque. At present it is not clear which proteases play pivotal roles at distinct stages of pathogenesis, rather that the combined proteolytic potential with some proteases at early stages and other at later stages may result in plaque rupture. This macrophage...

  6. Levels of Circulating MMCN-151, a Degradation Product of Mimecan, Reflect Pathological Extracellular Matrix Remodeling in Apolipoprotein E Knockout Mice

    DEFF Research Database (Denmark)

    Barascuk, N; Vassiliadis, E; Zheng, Qiuju

    2011-01-01

    Arterial extracellular matrix (ECM) remodeling by matrix metalloproteinases (MMPs) is one of the major hallmarks of atherosclerosis. Mimecan, also known as osteoglycin has been implicated in the integrity of the ECM. This study assessed the validity of an enzyme-linked immunosorbent assay (ELISA...

  7. Modulation of cardiac myocyte phenotype in vitro by the composition and orientation of the extracellular matrix.

    Science.gov (United States)

    Simpson, D G; Terracio, L; Terracio, M; Price, R L; Turner, D C; Borg, T K

    1994-10-01

    Cellular phenotype is the result of a dynamic interaction between a cell's intrinsic genetic program and the morphogenetic signals that serve to modulate the extent to which that program is expressed. In the present study we have examined how morphogenetic information might be stored in the extracellular matrix (ECM) and communicated to the neonatal heart cell (NHC) by the cardiac alpha 1 beta 1 integrin molecule. A thin film of type I collagen (T1C) was prepared with a defined orientation. This was achieved by applying T1C to the peripheral edge of a 100 mm culture dish. The T1C was then drawn across the surface of the dish in a continuous stroke with a sterile cell scraper and allowed to polymerize. When NHCs were cultured on this substrate, they spread, as a population, along a common axis in parallel with the gel lattice and expressed an in vivo-like phenotype. Individual NHCs displayed an elongated, rod-like shape and disclosed parallel arrays of myofibrils. These phenotypic characteristics were maintained for at least 4 weeks in primary culture. The evolution of this tissue-like organizational pattern was dependent upon specific interactions between the NHCs and the collagen-based matrix that were mediated by the cardiac alpha 1 beta 1 integrin complex. This conclusion was supported by a variety of experimental results. Altering the tertiary structure of the matrix or blocking the extracellular domains of either the cardiac alpha 1 or beta 1 integrin chain inhibited the expression of the tissue-like pattern of organization. Neither cell-to-cell contact or contractile function were necessary to induce the formation of the rod-like cell shape. However, beating activity was necessary for the assembly of a well-differentiated myofibrillar apparatus. These data suggest that the cardiac alpha 1 beta 1 integrin complex serves to detect and transduce phenotypic information stored within the tertiary structure of the surrounding matrix.

  8. Disruption of microbial biofilms by an extracellular protein isolated from epibiotic tropical marine strain of Bacillus licheniformis.

    Directory of Open Access Journals (Sweden)

    Devendra H Dusane

    Full Text Available BACKGROUND: Marine epibiotic bacteria produce bioactive compounds effective against microbial biofilms. The study examines antibiofilm ability of a protein obtained from a tropical marine strain of Bacillus licheniformis D1. METHODOLOGY/PRINCIPAL FINDINGS: B. licheniformis strain D1 isolated from the surface of green mussel, Perna viridis showed antimicrobial activity against pathogenic Candida albicans BH, Pseudomonas aeruginosa PAO1 and biofouling Bacillus pumilus TiO1 cultures. The antimicrobial activity was lost after treatment with trypsin and proteinase K. The protein was purified by ultrafiltration and size-exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF analysis revealed the antimicrobial agent to be a 14 kDa protein designated as BL-DZ1. The protein was stable at 75°C for 30 min and over a pH range of 3.0 to 11.0. The sequence alignment of the MALDI-fingerprint showed homology with the NCBI entry for a hypothetical protein (BL00275 derived from B. licheniformis ATCC 14580 with the accession number gi52082584. The protein showed minimum inhibitory concentration (MIC value of 1.6 µg/ml against C. albicans. Against both P. aeruginosa and B. pumilus the MIC was 3.12 µg/ml. The protein inhibited microbial growth, decreased biofilm formation and dispersed pre-formed biofilms of the representative cultures in polystyrene microtiter plates and on glass surfaces. CONCLUSION/SIGNIFICANCE: We isolated a protein from a tropical marine strain of B. licheniformis, assigned a function to the hypothetical protein entry in the NCBI database and described its application as a potential antibiofilm agent.

  9. Disruption of Microbial Biofilms by an Extracellular Protein Isolated from Epibiotic Tropical Marine Strain of Bacillus licheniformis

    Science.gov (United States)

    Dusane, Devendra H.; Damare, Samir R.; Nancharaiah, Yarlagadda V.; Ramaiah, N.; Venugopalan, Vayalam P.; Kumar, Ameeta Ravi; Zinjarde, Smita S.

    2013-01-01

    Background Marine epibiotic bacteria produce bioactive compounds effective against microbial biofilms. The study examines antibiofilm ability of a protein obtained from a tropical marine strain of Bacillus licheniformis D1. Methodology/Principal Findings B. licheniformis strain D1 isolated from the surface of green mussel, Perna viridis showed antimicrobial activity against pathogenic Candida albicans BH, Pseudomonas aeruginosa PAO1 and biofouling Bacillus pumilus TiO1 cultures. The antimicrobial activity was lost after treatment with trypsin and proteinase K. The protein was purified by ultrafiltration and size-exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis revealed the antimicrobial agent to be a 14 kDa protein designated as BL-DZ1. The protein was stable at 75°C for 30 min and over a pH range of 3.0 to 11.0. The sequence alignment of the MALDI-fingerprint showed homology with the NCBI entry for a hypothetical protein (BL00275) derived from B. licheniformis ATCC 14580 with the accession number gi52082584. The protein showed minimum inhibitory concentration (MIC) value of 1.6 µg/ml against C. albicans. Against both P. aeruginosa and B. pumilus the MIC was 3.12 µg/ml. The protein inhibited microbial growth, decreased biofilm formation and dispersed pre-formed biofilms of the representative cultures in polystyrene microtiter plates and on glass surfaces. Conclusion/Significance We isolated a protein from a tropical marine strain of B. licheniformis, assigned a function to the hypothetical protein entry in the NCBI database and described its application as a potential antibiofilm agent. PMID:23691235

  10. Right ventricular function after repair of tetralogy of Fallot: a comparison between bovine pericardium and porcine small intestinal extracellular matrix.

    Science.gov (United States)

    Naik, Ronak; Johnson, Jason; Kumar, T K S; Philip, Ranjit; Boston, Umar; Knott-Craig, Christopher J

    2017-05-29

    The porcine small intestinal extracellular matrix reportedly has the potential to differentiate into viable myocardial cells. When used in tetralogy of Fallot repair, it may improve right ventricular function. We evaluated right ventricular function after repair of tetralogy of Fallot with extracellular matrix versus bovine pericardium. Subjects with non-transannular repair of tetralogy of Fallot with at least 1 year of follow-up were selected. The extracellular matrix and bovine pericardium groups were compared. We used three-dimensional right ventricular ejection fraction, right ventricle global longitudinal strain, and tricuspid annular plane systolic excursion to assess right ventricular function. The extracellular matrix group had 11 patients, whereas the bovine pericardium group had 10 patients. No differences between the groups were found regarding sex ratio, age at surgery, and cardiopulmonary bypass time. The follow-up period was 28±12.6 months in the extracellular matrix group and 50.05±17.6 months in the bovine pericardium group (p=0.001). The mean three-dimensional right ventricular ejection fraction (55.7±5.0% versus 55.3±5.2%, p=0.73), right ventricular global longitudinal strain (-18.5±3.0% versus -18.0±2.2%, p=0.44), and tricuspid annular plane systolic excursions (1.59±0.16 versus 1.59±0.2, p=0.93) were similar in the extracellular matrix group and in the bovine pericardium group, respectively. Right ventricular global longitudinal strain in healthy children is reported at -29±3% in literature. In a small cohort of the patients undergoing non-transannular repair of tetralogy of Fallot, there was no significant difference in right ventricular function between groups having extracellular matrix versus bovine pericardium patches followed-up for more than 1 year. Lower right ventricular longitudinal strain noted in both the groups compared to healthy children.

  11. Bubaline Cholecyst Derived Extracellular Matrix for Reconstruction of Full Thickness Skin Wounds in Rats

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    Poonam Shakya

    2016-01-01

    Full Text Available An acellular cholecyst derived extracellular matrix (b-CEM of bubaline origin was prepared using anionic biological detergent. Healing potential of b-CEM was compared with commercially available collagen sheet (b-CS and open wound (C in full thickness skin wounds in rats. Thirty-six clinically healthy adult Sprague Dawley rats of either sex were randomly divided into three equal groups. Under general anesthesia, a full thickness skin wound (20 × 20 mm2 was created on the dorsum of each rat. The defect in group I was kept as open wound and was taken as control. In group II, the defect was repaired with commercially available collagen sheet (b-CS. In group III, the defect was repaired with cholecyst derived extracellular matrix of bovine origin (b-CEM. Planimetry, wound contracture, and immunological and histological observations were carried out to evaluate healing process. Significantly (P<0.05 increased wound contraction was observed in b-CEM (III as compared to control (I and b-CS (II on day 21. Histologically, improved epithelization, neovascularization, fibroplasia, and best arranged collagen fibers were observed in b-CEM (III as early as on postimplantation day 21. These findings indicate that b-CEM have potential for biomedical applications for full thickness skin wound repair in rats.

  12. Expression of extracellular matrix proteins: tenascin-C, fibronectin and galectin-3 in prostatic adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Monika Ulamec

    2015-12-01

    Full Text Available Introduction: The interchanged stromal-epithelial relations and altered expression profiles of various extracellular matrix (ECM proteins creates a suitable microenvironment for cancer development and growth. We support the opinion that remodeling of the extracellular matrix (ECM plays an important role in the cancer progression. The aim of this study was to examine the expression of ECM proteins tenascin-C, fibronectin and galectin-3 in prostatic adenocarcinoma. Methods: Glands and surrounding stroma were analyzed in randomly selected specimens from 52 patients with prostate cancer and 28 patients with benign prostatic hyperplasia (BHP. To evaluate the intensity of tenascin-C, fibronectin and galectin-3 expression the percentage of positively immunostained stromal cells was examined.Results: Compared to BPH, stroma of prostatic adenocarcinoma showed statistically significant increase in tenascin-C expression (p<0.001, predominantly around neoplastic glands, while fibronectin (p=0.001 and galectin-3 (p<0.001 expression in the same area was decreased.Conclusions: Our study confirms changes in the expression of ECM proteins of prostate cancer which may have important role in the cancer development.

  13. Ultrastructure of the extracellular matrix of bovine dura mater, optic nerve sheath and sclera.

    Science.gov (United States)

    Raspanti, M; Marchini, M; Della Pasqua, V; Strocchi, R; Ruggeri, A

    1992-10-01

    The sclera, the outermost sheath of the optic nerve and the dura mater have been investigated histologically and ultrastructurally. Although these tissues appear very similar under the light microscope, being dense connective tissues mainly composed of collagen bundles and a limited amount of cells and elastic fibres, they exhibit subtle differences on electron microscopy. In the dura and sclera collagen appears in the form of large, nonuniform fibrils, similar to those commonly found in tendons, while in the optic nerve sheath the fibrils appear smaller and uniform, similar to those commonly observed in reticular tissues, vessel walls and skin. Freeze-fracture also reveals these fibrils to have different subfibrillar architectures, straight or helical, which correspond to 2 distinct forms of collagen fibril previously described (Raspanti et al. 1989). The other extracellular matrix components also vary with the particular collagen fibril structure. Despite their common embryological derivation, the dura mater, optic nerve sheath and sclera exhibit diversification of their extracellular matrix consistent with the mechanical loads to which these tissues are subjected. Our observations indicate that the outermost sheath of the optic nerve resembles the epineurium of peripheral nerves rather than the dura to which it is commonly likened.

  14. Science of Hyaluronic Acid Beyond Filling: Fibroblasts and Their Response to the Extracellular Matrix.

    Science.gov (United States)

    Landau, Marina; Fagien, Steven

    2015-11-01

    Loss of viscoelasticity is one of the primarily signs of skin aging, followed by appearance of visible wrinkles. Hyaluronic acid (HA)-based fillers are widely used to fill wrinkles and compensate for volume loss. Recent clinical observations demonstrate persistence of the filling effect longer than the biological availability of the filler. Stimulation of new collagen by cross-linked HA and up-regulation of elastin have been suggested as possible explanation to this observation and have been supported experimentally. Cross-linked HA substitutes for fragmented collagen in restoring extracellular matrix required for normal activity of fibroblasts, such as collagen and elastin production. To restore extracellular matrix efficiently, serial monthly treatments are required. Boosting of facial and nonfacial skin through fibroblast activation is a new indication for HA-based products. Injectable HA has also been recently registered in Europe as agents specific for the improvement of skin quality (Restylane Skinboosters). Further explanation of the possible mechanisms supported by long-term clinical examples is presented herein.

  15. Regulation of PDGFC signalling and extracellular matrix composition by FREM1 in mice

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    Fenny Wiradjaja

    2013-11-01

    Fras1-related extracellular matrix protein 1 (FREM1 is required for epidermal adhesion during embryogenesis, and mice lacking the gene develop fetal skin blisters and a range of other developmental defects. Mutations in members of the FRAS/FREM gene family cause diseases of the Fraser syndrome spectrum. Embryonic epidermal blistering is also observed in mice lacking PdgfC and its receptor, PDGFRα. In this article, we show that FREM1 binds to PDGFC and that this interaction regulates signalling downstream of PDGFRα. Fibroblasts from Frem1-mutant mice respond to PDGFC stimulation, but with a shorter duration and amplitude than do wild-type cells. Significantly, PDGFC-stimulated expression of the metalloproteinase inhibitor Timp1 is reduced in cells with Frem1 mutations, leading to reduced basement membrane collagen I deposition. These results show that the physical interaction of FREM1 with PDGFC can regulate remodelling of the extracellular matrix downstream of PDGFRα. We propose that loss of FREM1 function promotes epidermal blistering in Fraser syndrome as a consequence of reduced PDGFC activity, in addition to its stabilising role in the basement membrane.

  16. The effect of low-dose neutron irradiation on extracellular matrix

    International Nuclear Information System (INIS)

    Chen Tiehe; Lu Yongjie; Chai Mingsheng; Peng Wulin; Yang Yifang; Pan Yan; Chen Jinguo

    2003-01-01

    Projective: To study the effect of neutron irradiation on extracellular matrix. Methods: 120 male wistar rats were divided into four groups at random, and then exposed to neutron of 252 Cf-source at the doses of 0, 0.29, 0.62 and 1.20 Gy, respectively. After the exposure of 3 days, 1 month and 2 months, the rats were sacrificed and lung tissue specimens stored at -30 degree C. Hyaluronan, laminin, type III procollagen and type IV collagen in the lung tissue were detected by the method of radioimmunoassay. Results: The differences of the levels of hyaluronan in lung tissue among the groups were unsignificant. The levels of laminin in 0.29, 0.62 and 1.20 Gy groups after the 3-day exposure were remarkably different to those of the control group, and unable to recover completely even 2 months after the exposure. The levels of type IV collagen in higher three irradiated groups were all higher, but not significantly. The levels of type III procollagen in the early stage after exposure were higher, and later they lowered. Conclusion: The levels of some components of extracellular matrix in the lung tissue of rat can be changed by low-dose of neutron irradiation, but their variational modes and degrees depend on the dose of neutron irradiation and the length of period after exposure

  17. Extracellular matrix of dental pulp stem cells: Applications in pulp tissue engineering using somatic MSCs

    Directory of Open Access Journals (Sweden)

    Sriram eRavindran

    2014-01-01

    Full Text Available Dental Caries affects approximately 90% of the world’s population. At present, the clinical treatment for dental caries is root canal therapy. This treatment results in loss of tooth sensitivity and vitality. Tissue engineering can potentially solve this problem by enabling regeneration of a functional pulp tissue. Dental pulp stem cells (DPSCs have been shown to be an excellent source for pulp regeneration. However, limited availability of these cells hinders its potential for clinical translation. We have investigated the possibility of using somatic mesenchymal stem cells from other sources for dental pulp tissue regeneration using a biomimetic dental pulp extracellular matrix (ECM incorporated scaffold. Human periodontal ligament stem cells (PDLSCs and human bone marrow stromal cells (HMSCs were investigated for their ability to differentiate towards an odontogenic lineage. In vitro real-time PCR results coupled with histological and immunohistochemical examination of the explanted tissues confirmed the ability of PDLSCs and HMSCs to form a vascularized pulp-like tissue. These findings indicate that the dental pulp stem derived ECM scaffold stimulated odontogenic differentiation of PDLSCs and HMSCs without the need for exogenous addition of growth and differentiation factors. This study represents a translational perspective toward possible therapeutic application of using a combination of somatic stem cells and extracellular matrix for pulp regeneration.

  18. Early Dysregulation of Cell Adhesion and Extracellular Matrix Pathways in Breast Cancer Progression

    Science.gov (United States)

    Emery, Lyndsey A.; Tripathi, Anusri; King, Chialin; Kavanah, Maureen; Mendez, Jane; Stone, Michael D.; de las Morenas, Antonio; Sebastiani, Paola; Rosenberg, Carol L.

    2009-01-01

    Proliferative breast lesions, such as simple ductal hyperplasia (SH) and atypical ductal hyperplasia (ADH), are candidate precursors to ductal carcinoma in situ (DCIS) and invasive cancer. To better understand the relationship of breast lesions to more advanced disease, we used microdissection and DNA microarrays to profile the gene expression of patient-matched histologically normal (HN), ADH, and DCIS from 12 patients with estrogen receptor positive sporadic breast cancer. SH were profiled from a subset of cases. We found 837 differentially expressed genes between DCIS-HN and 447 between ADH-HN, with >90% of the ADH-HN genes also present among the DCIS-HN genes. Only 61 genes were identified between ADH-DCIS. Expression differences were reproduced in an independent cohort of patient-matched lesions by quantitative real-time PCR. Many breast cancer-related genes and pathways were dysregulated in ADH and maintained in DCIS. Particularly, cell adhesion and extracellular matrix interactions were overrepresented. Focal adhesion was the top pathway in each gene set. We conclude that ADH and DCIS share highly similar gene expression and are distinct from HN. In contrast, SH appear more similar to HN. These data provide genetic evidence that ADH (but not SH) are often precursors to cancer and suggest cancer-related genetic changes, particularly adhesion and extracellular matrix pathways, are dysregulated before invasion and even before malignancy is apparent. These findings could lead to novel risk stratification, prevention, and treatment approaches. PMID:19700746

  19. Alterations in the extracellular matrix organization associated with the reexpression of tumorigenicity in human cell hybrids.

    Science.gov (United States)

    Der, C J; Stanbridge, E J

    1980-10-15

    The expression of fibronectin on the cell surface was evaluated on a series of intraspecific human cell hybrids formed between HeLa and normal fibroblast strains. Although these hybrids continued to express many of the in vitro transformation properties of their corresponding tumorigenic HeLa parent, they were now unable to form tumors when inoculated into athymic nude mice. From these suppressed hybrid populations, rare tumorigenic segregant subpopulations arose which had regained their tumorigenic capacity. A comparison of the expression of fibronectin on the cell surface was made between these tumorigenic segregant cell lines and their corresponding non-tumorigenic HeLa/fibroblast hybrid. Following specific immunofluorescent staining for fibronectin, a striking alteration in the cell surface organization was observed to correspond with the reexpression of tumorigenicity in these hybrids. Tumorigenic HeLa/fibroblast hybrids were also significantly altered in both their cellular and colonial morphology. Double immunofluorescent staining to simultaneously visualize both surface fibronectin and collagen revealed that these two extracellular matrix proteins displayed an extensive degree of codistribution and expressed a coordinate shift in organization which correlated with the appearance of tumorigenic segregant hybrid populations. These observations are in agreement with the apparently close structural association between fibronectin and collagen and suggest that the organization of these two components in the extracellular matrix may be an important determinant for in vivo growth potential.

  20. Expression of extracellular matrix metalloproteinase inducer (EMMPRIN and its related extracellular matrix degrading enzymes in the endometrium during estrous cycle and early gestation in cattle

    Directory of Open Access Journals (Sweden)

    Hosoe Misa

    2010-06-01

    Full Text Available Abstract Background Extracellular matrix metalloproteinase inducer (EMMPRIN regulates several biological functions involving the modulation of cell behaviors via cell-cell and cell-matrix interactions. According to its diverse functions, we hypothesized that EMMPRIN may play an important role in endometrial remodeling and establishment of pregnancy in cow. Methods In this study, endometrial tissues from the cyclic cows during before ovulation, after ovulation and middle of estrous cycle; and pregnant endometrial tissues from Day 19 to 35 of gestation have been used. Expression of mRNA was analyzed by RT-PCR, qPCR and in situ hybridization whereas protein expression by immunohistochemistry and western blot analysis. Results EMMPRIN mRNA was expressed in both cyclic and pregnant endometrium and significantly higher in the endometrium at Day 35 of gestation than the cyclic endometrium. In Western blot analysis, an approximately 65 kDa band was detected in the endometrium, and approximately 51 kDa in the cultured bovine epithelial cells and BT-1 cells, respectively. Both in situ hybridization and immunohistochemistry data showed that EMMPRIN was primarily expressed in luminal and glandular epithelium with strong staining on Day 19 conceptus. At Day 19 of gestation, expression of EMMPRIN mRNA on luminal epithelium was decreased than that observed at middle of estrous cycle, however, on Day 30 of gestation, slightly increased expression was found at the site of placentation. Expression of matrix metalloproteinase-2 (MMP-2 and MMP-14 mRNA were mainly detected in stroma and their expression also decreased at Day 19 of gestation however it was also expressed at the site of placentation at Day 30 of gestation as observed for EMMPRIN. Expression of MMP-1 or -9 mRNA was very low and was below the detection limit in the cyclic and pregnant endometrium. Conclusion EMMPRIN from the luminal epithelium may regulate the expression of stromal MMP-2 and -14

  1. Mimicking the extracellular matrix with functionalized, metal-assembled collagen peptide scaffolds.

    Science.gov (United States)

    Hernandez-Gordillo, Victor; Chmielewski, Jean

    2014-08-01

    Natural and synthetic three-dimensional (3-D) scaffolds that mimic the microenvironment of the extracellular matrix (ECM), with growth factor storage/release and the display of cell adhesion signals, offer numerous advantages for regenerative medicine and in vitro morphogenesis and oncogenesis modeling. Here we report the design of collagen mimetic peptides (CMPs) that assemble into a highly crosslinked 3-D matrix in response to metal ion stimuli, that may be functionalized with His-tagged cargoes, such as green fluorescent protein (GFP-His8) and human epidermal growth factor (hEGF-His6). The bound hEGF-His6 was found to gradually release from the matrix in vitro and induce cell proliferation in the EGF-dependent cell line MCF10A. The additional incorporation of a cell adhesion sequence (RGDS) at the N-terminus of the CMP creates an environment that facilitated the organization of matrix-encapsulated MCF10A cells into spheroid structures, thus mimicking the ECM environment. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Naturally Occurring Extracellular Matrix Scaffolds for Dermal Regeneration: Do They Really Need Cells?

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    A. M. Eweida

    2015-01-01

    Full Text Available The pronounced effect of extracellular matrix (ECM scaffolds in supporting tissue regeneration is related mainly to their maintained 3D structure and their bioactive components. These decellularized matrix scaffolds could be revitalized before grafting via adding stem cells, fibroblasts, or keratinocytes to promote wound healing. We reviewed the online published literature in the last five years for the studies that performed ECM revitalization and discussed the results of these studies and the related literature. Eighteen articles met the search criteria. Twelve studies included adding cells to acellular dermal matrix (ADM, 3 studies were on small intestinal mucosa (SIS, one study was on urinary bladder matrix (UBM, one study was on amniotic membrane, and one study included both SIS and ADM loaded constructs. We believe that, in chronic and difficult-to-heal wounds, revitalizing the ECM scaffolds would be beneficial to overcome the defective host tissue interaction. This belief still has to be verified by high quality randomised clinical trials, which are still lacking in literature.

  3. Direct comparison of phosphate uptake by adnate and loosely attached microalgae within an intact biofilm matrix

    International Nuclear Information System (INIS)

    Burkholder, J.M.; Wetzel, R.G.; Klomparens, K.L.

    1990-01-01

    We report a direct comparison of phosphate uptake by adnate and loosely attached microalgae in an intact biofilm matrix, with resolution at the level of individual cells. Track scanning electron microscope autoradiography enabled assay of [ 33 P]phosphate uptake from the overlying water by adnate algae left undisturbed on mature leaves of the macrophyte Potamogeton illinoensis or on artificial plant mimics. The epiphyte communities developed in either phosphate-poor or moderately phosphate-enriched water, and they were assayed on both natural and artificial plants. All adnate taxa examined from both natural and artificial plants in both habitats took up significantly less radiolabel when assayed beneath the overlying matrix than when they were exposed to the water upon removal of the overstory material. Track scanning electron microscope autoradiography and track light microscope autoradiography were intercalibrated to enable comparison of [ 33 P]phosphate uptake by adnate and loosely attached components of the epiphyte matrix. Loosley attached cells on substrata from both habitats took up significantly more radiolabel than did underlying adnate cells, indicating that access to phosphate supplies from the water depended on the position of microbial cells in the matrix. In this short-term assay, the adnate microalgae were relatively isolated from the water column nutrient source

  4. Preparation of a three-dimensional extracellular matrix by decellularization of rabbit livers

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    Gustavo A. Nari

    2013-03-01

    Full Text Available Introduction: the availability of transplantable livers is not sufficient to fulfill the current demand for grafts, with the search for therapeutic alternatives having generated different lines of research, one of which is the use of decellularized three-dimensional biological matrices and subsequent cell seeding to obtain a functional organ. Objective: to produce a decellularization protocol from rabbit liver to generate a three-dimensional matrix. Methods: a combination of physical, chemical (Triton X-100 and SDS and enzymatic agents to decellularize rabbit livers was used. After 68 h of retrograde perfusion, a decellularized translucent matrix was generated. To evaluate if the decellularization protocol was successful, with the extracellular matrix being preserved, we carried out histological (light microscopy and scanning electron microscopy and biochemical (DNA quantification studies. Results: the decellularization process was verified by macroscopic observation of the organ using macroscopic staining, which revealed a correct conservation of bile and vascular trees. A microscopic observation corroborated these macroscopic results, with the hematoxylin-eosin staining showing no cells or nuclear material and the presence of a portal triad. Wilde's staining demonstrated the conservation of reticulin fibers in the decellularized matrix. In addition, scanning electron microscopy revealed a preserved Glisson's capsule and a decellularized matrix, with the DNA quantification being less than 10 % in the decellularized liver compared to control. Finally, the time taken to develop the decellularization protocol was less than 96 hours. Conclusions: the proposed decellularization protocol was correct, and was verified by an absence of cells. The hepatic matrix had preserved vascular and bile ducts with a suitable three-dimensional architecture permitting further cell seeding.

  5. An investigation of the influence of extracellular matrix anisotropy and cell–matrix interactions on tissue architecture

    KAUST Repository

    Dyson, R. J.

    2015-09-02

    © 2015 Springer-Verlag Berlin Heidelberg Mechanical interactions between cells and the fibrous extracellular matrix (ECM) in which they reside play a key role in tissue development. Mechanical cues from the environment (such as stress, strain and fibre orientation) regulate a range of cell behaviours, including proliferation, differentiation and motility. In turn, the ECM structure is affected by cells exerting forces on the matrix which result in deformation and fibre realignment. In this paper we develop a mathematical model to investigate this mechanical feedback between cells and the ECM. We consider a three-phase mixture of collagen, culture medium and cells, and formulate a system of partial differential equations which represents conservation of mass and momentum for each phase. This modelling framework takes into account the anisotropic mechanical properties of the collagen gel arising from its fibrous microstructure. We also propose a cell–collagen interaction force which depends upon fibre orientation and collagen density. We use a combination of numerical and analytical techniques to study the influence of cell–ECM interactions on pattern formation in tissues. Our results illustrate the wide range of structures which may be formed, and how those that emerge depend upon the importance of cell–ECM interactions.

  6. Collagen XII and XIV, New Partners of Cartilage Oligomeric Matrix Protein in the Skin Extracellular Matrix Suprastructure*

    Science.gov (United States)

    Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R.; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate

    2012-01-01

    The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone. PMID:22573329

  7. Collagen XII and XIV, new partners of cartilage oligomeric matrix protein in the skin extracellular matrix suprastructure.

    Science.gov (United States)

    Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate

    2012-06-29

    The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone.

  8. An investigation of the influence of extracellular matrix anisotropy and cell–matrix interactions on tissue architecture

    KAUST Repository

    Dyson, R. J.; Green, J. E. F.; Whiteley, J. P.; Byrne, H. M.

    2015-01-01

    © 2015 Springer-Verlag Berlin Heidelberg Mechanical interactions between cells and the fibrous extracellular matrix (ECM) in which they reside play a key role in tissue development. Mechanical cues from the environment (such as stress, strain and fibre orientation) regulate a range of cell behaviours, including proliferation, differentiation and motility. In turn, the ECM structure is affected by cells exerting forces on the matrix which result in deformation and fibre realignment. In this paper we develop a mathematical model to investigate this mechanical feedback between cells and the ECM. We consider a three-phase mixture of collagen, culture medium and cells, and formulate a system of partial differential equations which represents conservation of mass and momentum for each phase. This modelling framework takes into account the anisotropic mechanical properties of the collagen gel arising from its fibrous microstructure. We also propose a cell–collagen interaction force which depends upon fibre orientation and collagen density. We use a combination of numerical and analytical techniques to study the influence of cell–ECM interactions on pattern formation in tissues. Our results illustrate the wide range of structures which may be formed, and how those that emerge depend upon the importance of cell–ECM interactions.

  9. Novel serological neo-epitope markers of extracellular matrix proteins for the detection of portal hypertension.

    Science.gov (United States)

    Leeming, D J; Karsdal, M A; Byrjalsen, I; Bendtsen, F; Trebicka, J; Nielsen, M J; Christiansen, C; Møller, S; Krag, A

    2013-11-01

    The hepatic venous pressure gradient (HVPG) is an invasive, but important diagnostic and prognostic marker in cirrhosis with portal hypertension (PHT). During cirrhosis, remodelling of fibrotic tissue by matrix metalloproteinases (MMPs) is a permanent process generating small fragments of degraded extracellular matrix (ECM) proteins known as neoepitopes, which are then released into the circulation. To investigate their potential as plasma markers for detection of PHT. Ninety-four patients with alcoholic cirrhosis and 20 liver-healthy controls were included. Clinical and laboratory data of the patients were collected. All patients received HVPG measurement with blood sampling. In these samples, the following degradation or formation markers were measured: C1M (type I-collagen), C3M and PRO-C3 (type III collagen), C4M and P4NP 7S (type IV collagen), C5M (type V collagen), C6M (type VI collagen), BGM (biglycan), ELM (elastin), CRPM (CRP). All ECM markers except for CRPM correlated significantly with HVPG. Interestingly, C4M, C5M and ELM levels were significantly higher in patients with HVPG >10 mmHg. Multiple regression analysis identified PRO-C3, C6M and ELM as significant determinants, while the models A and B including PRO-C3, ELM, C6M and model for end-stage liver disease (MELD) provided better description of PHT (r = 0.75, P models provided odds ratios of >100 for having clinical significant PHT. These novel non-invasive extracellular matrix markers reflect the degree of liver dysfunction. The different degrees of portal hypertension correlated with these circulating neoepitopes. Using a single blood sample, these neoepitopes in combination with MELD detect the level of portal hypertension. © 2013 The Authors. Alimentary Pharmacology and Therapeutics published by John Wiley & Sons Ltd.

  10. Neutrophil elastase processing of Gelatinase A is mediated by extracellular matrix

    Energy Technology Data Exchange (ETDEWEB)

    Rice, A.; Banda, M.J. [Univ. of California, San Franciso, CA (United States)

    1995-07-18

    Gelatinase A (72-kDa type IV collagenase) is a metalloproteinase that is expressed by many cells in culture and is overexpressed by some tumor cells. It has been suggested that the serine proteinase neutrophil elastase might play a role iii the posttranslational processing of gelatinase A and that noncatalytic interactions between gelatinase A and components of the extracellular matrix might alter potential processing pathways. These questions were addressed with the use of gelatin substrate zymography, gelatinolytic activity assays, and amino acid sequence analysis. We found that neutrophil elastase does proteolytically modify gelatinase A by cleaving at a number of sites within gelatinase A. Sequential treatment of gelatinase A with 4-aminophenylmercuric acetate (APMA) and neutrophil elastase yielded an active gelatinase with a 4-fold increase in gelatinolytic activity. The increased gelatinolytic activity correlated with that of a 40-kDa fragment of gelatinase A. Matrix components altered the proteolytic modifications in gelatinase A that were mediated by neutrophil elastase. In the absence of gelatin, neutrophil elastase destructively degraded gelatinase A by hydrolyzing at least two bonds within the fibronectin-like gelatin-binding domain of gelatinase A. In the presence of gelatin, these two inactivating cleavage sites were protected, and cleavage at a site within the hemopexin-like carboxyl-terminal domain resulted in a truncated yet active gelatinase. The results suggest a regulatory role for extracellular matrix molecules in stabilizing gelatinase A fragments and in altering the availability of sites susceptible to destructive proteolysis by neutrophil elastase. 32 refs., 10 figs.

  11. Faslodex inhibits estradiol-induced extracellular matrix dynamics and lung metastasis in a model of lymphangioleiomyomatosis.

    Science.gov (United States)

    Li, Chenggang; Zhou, Xiaobo; Sun, Yang; Zhang, Erik; Mancini, John D; Parkhitko, Andrey; Morrison, Tasha A; Silverman, Edwin K; Henske, Elizabeth P; Yu, Jane J

    2013-07-01

    Lymphangioleiomyomatosis (LAM) is a destructive lung disease primarily affecting women. Genetic studies indicate that LAM cells carry inactivating tuberous sclerosis complex (TSC)-2 mutations, and metastasize to the lung. We previously discovered that estradiol increases the metastasis of TSC2-deficient cells in mice carrying xenograft tumors. Here, we investigate the molecular basis underlying the estradiol-induced lung metastasis of TSC2-deficient cells, and test the efficacy of Faslodex (an estrogen receptor antagonist) in a preclinical model of LAM. We used a xenograft tumor model in which estradiol induces the lung metastasis of TSC2-deficient cells. We analyzed the impact of Faslodex on tumor size, the extracellular matrix organization, the expression of matrix metalloproteinase (MMP)-2, and lung metastasis. We also examined the effects of estradiol and Faslodex on MMP2 expression and activity in tuberin-deficient cells in vitro. Estradiol resulted in a marked reduction of Type IV collagen deposition in xenograft tumors, associated with 2-fold greater MMP2 concentrations compared with placebo-treated mice. Faslodex normalized the Type IV collagen changes in xenograft tumors, enhanced the survival of the mice, and completely blocked lung metastases. In vitro, estradiol enhanced MMP2 transcripts, protein accumulation, and activity. These estradiol-induced changes in MMP2 were blocked by Faslodex. In TSC2-deficient cells, estradiol increased MMP2 concentrations in vitro and in vivo, and induced extracellular matrix remodeling. Faslodex inhibits the estradiol-induced lung metastasis of TSC2-deficient cells. Targeting estrogen receptors with Faslodex may be of efficacy in the treatment of LAM.

  12. [Effect of electroacupuncture intervention on expression of extracellular matrix collagen and metabolic enzymes].

    Science.gov (United States)

    Liao, Jun; Zhang, Le; Ke, Mei-gui; Xu, Teng

    2013-12-01

    To observe the effect of electroacupuncture (EA) at "Dazhui" (GV 14) on the contents of extracellular matrix (ECM), collagen type II (COL-II), collagen type V (COL-V), matrix metalloproteinase (MMP)-13, tissue inhibitor of metalloproteinase (TIMP)-1 in rats with cervicovertebral disc degeneration so as to explore its mechanism underlying relief of intervertebral disc degeneration. A total of 28 SD rats were randomly divided into sham group (n = 7), model group (n = 7), EA group (n = 7) and medication group (n = 7). The model of cervical intervertebral disc degeneration was established by trans-section of the deep neck splenius, the longest muscles of head, neck costocervicalis, head semi-spinatus muscle, supraspinous ligament and interspinal ligaments of cervical 2-7 segments, etc. to produce imbalance between the dynamic and static force. EA was applied to "Dazhui" (GV 14) for 30 min, once daily for 28 days, with a 2 days' interval between two courses. Animals of the medication group were treated by oral administration of meloxicam tablets (0.75 mg/kg) once daily for 28 days, with a 2 days' interval between two courses. Immunohistochemistry was used to measure the expression of ECM, COL- II, COL-V, MMP-13 and TIMP-1 in the cervicovertebral disc tissue. Compared with the sham group, the expression levels of ECM and COL-II proteins in the cervicovertebral disc tissue were significantly decreased in the model group (P 0.05). EA of "Dazhui" (GV 14) can effectively regulate extracellular matrix system in rats with cervical intervertebral disc degeneration, which is possibly related to its effect in relieving cervical spondylosis.

  13. Faslodex Inhibits Estradiol-Induced Extracellular Matrix Dynamics and Lung Metastasis in a Model of Lymphangioleiomyomatosis

    Science.gov (United States)

    Li, Chenggang; Zhou, Xiaobo; Sun, Yang; Zhang, Erik; Mancini, John D.; Parkhitko, Andrey; Morrison, Tasha A.; Silverman, Edwin K.; Henske, Elizabeth P.

    2013-01-01

    Lymphangioleiomyomatosis (LAM) is a destructive lung disease primarily affecting women. Genetic studies indicate that LAM cells carry inactivating tuberous sclerosis complex (TSC)–2 mutations, and metastasize to the lung. We previously discovered that estradiol increases the metastasis of TSC2-deficient cells in mice carrying xenograft tumors. Here, we investigate the molecular basis underlying the estradiol-induced lung metastasis of TSC2-deficient cells, and test the efficacy of Faslodex (an estrogen receptor antagonist) in a preclinical model of LAM. We used a xenograft tumor model in which estradiol induces the lung metastasis of TSC2-deficient cells. We analyzed the impact of Faslodex on tumor size, the extracellular matrix organization, the expression of matrix metalloproteinase (MMP)–2, and lung metastasis. We also examined the effects of estradiol and Faslodex on MMP2 expression and activity in tuberin-deficient cells in vitro. Estradiol resulted in a marked reduction of Type IV collagen deposition in xenograft tumors, associated with 2-fold greater MMP2 concentrations compared with placebo-treated mice. Faslodex normalized the Type IV collagen changes in xenograft tumors, enhanced the survival of the mice, and completely blocked lung metastases. In vitro, estradiol enhanced MMP2 transcripts, protein accumulation, and activity. These estradiol-induced changes in MMP2 were blocked by Faslodex. In TSC2-deficient cells, estradiol increased MMP2 concentrations in vitro and in vivo, and induced extracellular matrix remodeling. Faslodex inhibits the estradiol-induced lung metastasis of TSC2-deficient cells. Targeting estrogen receptors with Faslodex may be of efficacy in the treatment of LAM. PMID:23526212

  14. Comparative proteomic analysis of extracellular proteins expressed by various clonal types of Staphylococcus aureus and during planktonic growth and biofilm development.

    Science.gov (United States)

    Atshan, Salman S; Shamsudin, Mariana N; Sekawi, Zamberi; Thian Lung, Leslie T; Barantalab, Fatemeh; Liew, Yun K; Alreshidi, Mateg Ali; Abduljaleel, Salwa A; Hamat, Rukman A

    2015-01-01

    Staphylococcus aureus is well known for its biofilm formation with rapid emergence of new clones circulating worldwide. The main objectives of the study were (1) to identify possible differences in protein expression among various and closely related clonal types of S. aureus, (2) to establish the differences in protein expression in terms of size of protein spots and its intensities between bacteria which are grown statically (biofilm formation) with that of under aeration and agitation, and (3) to compare the differences in protein expression as a function of time (in hours). In this study, we selected six clinical isolates comprising two similar (MRSA-527 and MRSA-524) and four different (MRSA-139, MSSA-12E, MSSA-22d, and MSSA-10E) types identified by spa typing, MLST and SCCmec typing. We performed 2D gel migration comparison. Also, two MRSA isolates (527 and 139) were selected to determine quantitative changes in the level of extracellular proteins at different biofilm growth time points of 12, 24, and 48 h. The study was done using a strategy that combines 2-DGE and LC-MS/MS analysis for absolute quantification and identification of the extracellular proteins. The 2DGE revealed that the proteomic profiles for the isolates belonging to the similar spa, MLST, and SCCmec types were still quite different. Among the extracellular proteins secreted at different time points of biofilm formation, significant changes in protein expression were observed at 48 h incubation as compared to the exponential growth at 12 h incubation. The main conclusion of the work is that the authors do observe differences among isolates, and growth conditions do influence the protein content at different time points of biofilm formation.

  15. High fidelity visualization of cell-to-cell variation and temporal dynamics in nascent extracellular matrix formation.

    Science.gov (United States)

    McLeod, Claire M; Mauck, Robert L

    2016-12-12

    Extracellular matrix dynamics are key to tissue morphogenesis, homeostasis, injury, and repair. The spatiotemporal organization of this matrix has profound biological implications, but is challenging to monitor using standard techniques. Here, we address these challenges by using noncanonical amino acid tagging to fluorescently label extracellular matrix synthesized in the presence of bio-orthogonal methionine analogs. This strategy labels matrix proteins with high resolution, without compromising their distribution or mechanical function. We demonstrate that the organization and temporal dynamics of the proteinaceous matrix depend on the biophysical features of the microenvironment, including the biomaterial scaffold and the niche constructed by cells themselves. Pulse labeling experiments reveal that, in immature constructs, nascent matrix is highly fibrous and interdigitates with pre-existing matrix, while in more developed constructs, nascent matrix lacks fibrous organization and is retained in the immediate pericellular space. Inhibition of collagen crosslinking increases matrix synthesis, but compromises matrix organization. Finally, these data demonstrate marked cell-to-cell heterogeneity amongst both chondrocytes and mesenchymal stem cells undergoing chondrogenesis. Collectively, these results introduce fluorescent noncanonical amino acid tagging as a strategy to investigate spatiotemporal matrix organization, and demonstrate its ability to identify differences in phenotype, microenvironment, and matrix assembly at the single cell level.

  16. Biofilm inhibitors that target amyloid proteins.

    Science.gov (United States)

    Romero, Diego; Sanabria-Valentín, Edgardo; Vlamakis, Hera; Kolter, Roberto

    2013-01-24

    Bacteria establish stable communities, known as biofilms, that are resistant to antimicrobials. Biofilm robustness is due to the presence of an extracellular matrix, which for several species-among them Bacillus subtilis-includes amyloid-like protein fibers. In this work, we show that B. subtilis biofilms can be a simple and reliable tool for screening of molecules with antiamyloid activity. We identified two molecules, AA-861 and parthenolide, which efficiently inhibited biofilms by preventing the formation of amyloid-like fibers. Parthenolide also disrupted pre-established biofilms. These molecules also impeded the formation of biofilms of other bacterial species that secrete amyloid proteins, such as Bacillus cereus and Escherichia coli. Furthermore, the identified molecules decreased the conversion of the yeast protein New1 to the prion state in a heterologous host, indicating the broad range of activity of the molecules. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Mechanical forces regulate the interactions of fibronectin and collagen I in extracellular matrix.

    Science.gov (United States)

    Kubow, Kristopher E; Vukmirovic, Radmila; Zhe, Lin; Klotzsch, Enrico; Smith, Michael L; Gourdon, Delphine; Luna, Sheila; Vogel, Viola

    2015-08-14

    Despite the crucial role of extracellular matrix (ECM) in directing cell fate in healthy and diseased tissues--particularly in development, wound healing, tissue regeneration and cancer--the mechanisms that direct the assembly and regulate hierarchical architectures of ECM are poorly understood. Collagen I matrix assembly in vivo requires active fibronectin (Fn) fibrillogenesis by cells. Here we exploit Fn-FRET probes as mechanical strain sensors and demonstrate that collagen I fibres preferentially co-localize with more-relaxed Fn fibrils in the ECM of fibroblasts in cell culture. Fibre stretch-assay studies reveal that collagen I's Fn-binding domain is responsible for the mechano-regulated interaction. Furthermore, we show that Fn-collagen interactions are reciprocal: relaxed Fn fibrils act as multivalent templates for collagen assembly, but once assembled, collagen fibres shield Fn fibres from being stretched by cellular traction forces. Thus, in addition to the well-recognized, force-regulated, cell-matrix interactions, forces also tune the interactions between different structural ECM components.

  18. Biosorption of Cadmium by Non-Toxic Extracellular Polymeric Substances (EPS Synthesized by Bacteria from Marine Intertidal Biofilms

    Directory of Open Access Journals (Sweden)

    Juan Carlos Camacho-Chab

    2018-02-01

    Full Text Available Cadmium is a major heavy metal found in polluted aquatic environments, mainly derived from industrial production processes. We evaluated the biosorption of solubilized Cd2+ using the extracellular polymeric substances (EPS produced by Bacillus sp. MC3B-22 and Microbacterium sp. MC3B-10 (Microbactan; these bacteria were originally isolated from intertidal biofilms off the coast of Campeche, Mexico. EPS were incubated with different concentrations of cadmium in ultrapure water. Residual Cd2+ concentrations were determined by Inductive Coupled Plasma-Optic Emission Spectrometry and the maximum sorption capacity (Qmax was calculated according to the Langmuir model. EPS were characterized by X-ray photoelectron spectroscopy (XPS before and after sorption. The Qmax of Cd2+ was 97 mg g−1 for Microbactan and 141 mg g−1 for MC3B-22 EPS, these adsorption levels being significantly higher than previously reported for other microbial EPS. In addition, XPS analysis revealed changes in structure of EPS after biosorption and showed that amino functional groups contributed to the binding of Cd2+, unlike other studies that show the carbohydrate fraction is responsible for this activity. This work expands the current view of bacterial species capable of synthesizing EPS with biosorbent potential for cadmium and provides evidence that different chemical moieties, other than carbohydrates, participate in this process.

  19. Mechanisms of Candida biofilm drug resistance

    Science.gov (United States)

    Taff, Heather T; Mitchell, Kaitlin F; Edward, Jessica A; Andes, David R

    2013-01-01

    Candida commonly adheres to implanted medical devices, growing as a resilient biofilm capable of withstanding extraordinarily high antifungal concentrations. As currently available antifungals have minimal activity against biofilms, new drugs to treat these recalcitrant infections are urgently needed. Recent investigations have begun to shed light on the mechanisms behind the profound resistance associated with the biofilm mode of growth. This resistance appears to be multifactorial, involving both mechanisms similar to conventional, planktonic antifungal resistance, such as increased efflux pump activity, as well as mechanisms specific to the biofilm lifestyle. A unique biofilm property is the production of an extracellular matrix. Two components of this material, β-glucan and extracellular DNA, promote biofilm resistance to multiple antifungals. Biofilm formation also engages several stress response pathways that impair the activity of azole drugs. Resistance within a biofilm is often heterogeneous, with the development of a subpopulation of resistant persister cells. In this article we review the molecular mechanisms underlying Candida biofilm antifungal resistance and their relative contributions during various growth phases. PMID:24059922

  20. Neutrophil extracellular traps and bacterial biofilms in middle ear effusion of children with recurrent acute otitis media--a potential treatment target.

    Directory of Open Access Journals (Sweden)

    Ruth B Thornton

    Full Text Available BACKGROUND: Bacteria persist within biofilms on the middle ear mucosa of children with recurrent and chronic otitis media however the mechanisms by which these develop remain to be elucidated. Biopsies can be difficult to obtain from children and their small size limits analysis. METHODS: In this study we aimed to investigate biofilm presence in middle ear effusion (MEE from children with recurrent acute otitis media (rAOM and to determine if these may represent infectious reservoirs similarly to those on the mucosa. We examined this through culture, viability staining and fluorescent in situ hybridisation (FISH to determine bacterial species present. Most MEEs had live bacteria present using viability staining (32/36 and all effusions had bacteria present using the universal FISH probe (26/26. Of these, 70% contained 2 or more otopathogenic species. Extensive DNA stranding was also present. This DNA was largely host derived, representing neutrophil extracellular traps (NETs within which live bacteria in biofilm formations were present. When treated with the recombinant human deoxyribonuclease 1, Dornase alfa, these strands were observed to fragment. CONCLUSIONS: Bacterial biofilms, composed of multiple live otopathogenic species can be demonstrated in the MEEs of children with rAOM and that these contain extensive DNA stranding from NETs. The NETs contribute to the viscosity of the effusion, potentially contributing to its failure to clear as well as biofilm development. Our data indicates that Dornase alfa can fragment these strands and may play a role in future chronic OM treatment.

  1. Biological conduits combining bone marrow mesenchymal stem cells and extracellular matrix to treat long-segment sciatic nerve defects

    Directory of Open Access Journals (Sweden)

    Yang Wang

    2015-01-01

    Full Text Available The transplantation of polylactic glycolic acid conduits combining bone marrow mesenchymal stem cells and extracellular matrix gel for the repair of sciatic nerve injury is effective in some respects, but few data comparing the biomechanical factors related to the sciatic nerve are available. In the present study, rabbit models of 10-mm sciatic nerve defects were prepared. The rabbit models were repaired with autologous nerve, a polylactic glycolic acid conduit + bone marrow mesenchymal stem cells, or a polylactic glycolic acid conduit + bone marrow mesenchymal stem cells + extracellular matrix gel. After 24 weeks, mechanical testing was performed to determine the stress relaxation and creep parameters. Following sciatic nerve injury, the magnitudes of the stress decrease and strain increase at 7,200 seconds were largest in the polylactic glycolic acid conduit + bone marrow mesenchymal stem cells + extracellular matrix gel group, followed by the polylactic glycolic acid conduit + bone marrow mesenchymal stem cells group, and then the autologous nerve group. Hematoxylin-eosin staining demonstrated that compared with the polylactic glycolic acid conduit + bone marrow mesenchymal stem cells group and the autologous nerve group, a more complete sciatic nerve regeneration was found, including good myelination, regularly arranged nerve fibers, and a completely degraded and resorbed conduit, in the polylactic glycolic acid conduit + bone marrow mesenchymal stem cells + extracellular matrix gel group. These results indicate that bridging 10-mm sciatic nerve defects with a polylactic glycolic acid conduit + bone marrow mesenchymal stem cells + extracellular matrix gel construct increases the stress relaxation under a constant strain, reducing anastomotic tension. Large elongations under a constant physiological load can limit the anastomotic opening and shift, which is beneficial for the regeneration and functional reconstruction of sciatic nerve. Better

  2. Extracellular matrix components influence DNA synthesis of rat hepatocytes in primary culture

    International Nuclear Information System (INIS)

    Sawada, N.; Tomomura, A.; Sattler, C.A.; Sattler, G.L.; Kleinman, H.K.; Pitot, H.C.

    1986-01-01

    The effects of several extracellular matrix components (EMCs) - fibronectin (Fn), laminin (Ln), type I (C-I) and type IV (C-IV) collagen - on DNA synthesis in rat hepatocytes in primary culture were examined by both quantitative scintillation spectrometry and autoradiography of [ 3 H]thymidine incorporation. Hepatocytes cultured on Fn showed the most active DNA synthesis initiated by epidermal growth factor (EGF) with decreasing levels of [ 3 H]thymidine uptake exhibited in the cell cultured on C-IV, C-I, and Ln, respectively. The decreasing level of DNA synthesis in hepatocytes cultured on Fn, C-IV, C-I, and Ln respectively was not influenced by cell density. The number of EGF receptors of hepatocytes was also not influenced by EMCs. These data suggest that EMCs modify hepatocyte DNA synthesis by means of post-EGF-receptor mechanisms which are regulated by both growth factors and cell density

  3. Extracellular matrix elasticity and topography: material-based cues that affect cell function via conserved mechanisms

    Science.gov (United States)

    Janson, Isaac A.; Putnam, Andrew J.

    2014-01-01

    Chemical, mechanical, and topographic extracellular matrix (ECM) cues have been extensively studied for their influence on cell behavior. These ECM cues alter cell adhesion, cell shape, and cell migration, and activate signal transduction pathways to influence gene expression, proliferation, and differentiation. ECM elasticity and topography, in particular, have emerged as material properties of intense focus based on strong evidence these physical cue can partially dictate stem cell differentiation. Cells generate forces to pull on their adhesive contacts, and these tractional forces appear to be a common element of cells’ responses to both elasticity and topography. This review focuses on recently published work that links ECM topography and mechanics and their influence on differentiation and other cell behaviors, We also highlight signaling pathways typically implicated in mechanotransduction that are (or may be) shared by cells subjected to topographic cues. Finally, we conclude with a brief discussion of the potential implications of these commonalities for cell based therapies and biomaterial design. PMID:24910444

  4. Recruitment of dental pulp cells by dentine and pulp extracellular matrix components.

    Science.gov (United States)

    Smith, J G; Smith, A J; Shelton, R M; Cooper, P R

    2012-11-01

    The present study aimed to determine whether dentine tissue and preparations of extracellular matrix (ECM) from pulp (pECM) and dentine (dECM), and breakdown products, influenced pulp cell migration. Chemotaxis transwell and agarose spot assays demonstrated that both dentine and pulp ECM molecules acted as chemoattractants for primary pulp cells. Chemoattractant activities of dECM and pECM were enhanced when subjected to acid and enzymatic breakdown, respectively. This enhanced activity following physiologically relevant breakdown may be pertinent to the disease environment. Pulp cell migration in response to dental ECMs was dependent on an active rho pathway. Recruited cells exhibited increased stem cell marker expression indicating that dental ECMs and their breakdown products selectively attract progenitor cells that contribute to repair processes. In conclusion, combined these results indicate that ECM molecules contribute to cell recruitment necessary for regeneration of the dentine-pulp complex after injury. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Mechanisms of redox metabolism and cancer cell survival during extracellular matrix detachment.

    Science.gov (United States)

    Hawk, Mark A; Schafer, Zachary T

    2018-01-16

    Non-transformed cells that become detached from the extracellular matrix (ECM) undergo dysregulation of redox homeostasis and cell death. In contrast, cancer cells often acquire the ability to mitigate programmed cell death pathways and recalibrate the redox balance to survive after ECM detachment, facilitating metastatic dissemination. Accordingly, recent studies of the mechanisms by which cancer cells overcome ECM detachment-induced metabolic alterations have focused on mechanisms in redox homeostasis. The insights into these mechanisms may inform the development of therapeutics that manipulate redox homeostasis to eliminate ECM-detached cancer cells. Here, we review how ECM-detached cancer cells balance redox metabolism for survival. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  6. In silico analysis suggests interaction between Ebola virus and the extracellular matrix

    Directory of Open Access Journals (Sweden)

    Veljko eVeljkovic

    2015-02-01

    Full Text Available The worst Ebola virus (EV outbreak in history has hit Liberia, Sierra Leone and Guinea hardest and the trendlines in this crisis are grave, and now represents global public health threat concern. Limited therapeutic and/or prophylactic options which are available for humans suffering from Ebola virus disease (EVD further complicate situation. Previous studies suggested that the EV glycoprotein (GP is the main determinant causing structural damage of endothelial cells that triggers the hemorrhagic diathesis, but molecular mechanisms underlying this phenomenon remains elusive. Using the informational spectrum method (ISM, a virtual spectroscopy method for analysis of the protein-protein interactions, the interaction of GP with endothelial extracellular matrix (ECM was investigated. Presented results of this in silico study suggest that Elastin Microfibril Interface Located Proteins (EMILINs are involved in interaction between GP and ECM. This finding could contribute to better understanding of EV/endothelium interaction and its role in pathogenesis, prevention and therapy of EVD.

  7. Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

    Science.gov (United States)

    Nishiyama, Keita; Nakamata, Koichi; Ueno, Shintaro; Terao, Akari; Aryantini, Ni Putu Desy; Sujaya, I Nengah; Fukuda, Kenji; Urashima, Tadasu; Yamamoto, Yuji; Mukai, Takao

    2015-01-01

    We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding.

  8. Cell-mediated fibre recruitment drives extracellular matrix mechanosensing in engineered fibrillar microenvironments

    Science.gov (United States)

    Baker, Brendon M.; Trappmann, Britta; Wang, William Y.; Sakar, Mahmut S.; Kim, Iris L.; Shenoy, Vivek B.; Burdick, Jason A.; Chen, Christopher S.

    2015-12-01

    To investigate how cells sense stiffness in settings structurally similar to native extracellular matrices, we designed a synthetic fibrous material with tunable mechanics and user-defined architecture. In contrast to flat hydrogel surfaces, these fibrous materials recapitulated cell-matrix interactions observed with collagen matrices including stellate cell morphologies, cell-mediated realignment of fibres, and bulk contraction of the material. Increasing the stiffness of flat hydrogel surfaces induced mesenchymal stem cell spreading and proliferation; however, increasing fibre stiffness instead suppressed spreading and proliferation for certain network architectures. Lower fibre stiffness permitted active cellular forces to recruit nearby fibres, dynamically increasing ligand density at the cell surface and promoting the formation of focal adhesions and related signalling. These studies demonstrate a departure from the well-described relationship between material stiffness and spreading established with hydrogel surfaces, and introduce fibre recruitment as a previously undescribed mechanism by which cells probe and respond to mechanics in fibrillar matrices.

  9. Electrospun polyvinyl alcohol–collagen–hydroxyapatite nanofibers: a biomimetic extracellular matrix for osteoblastic cells

    International Nuclear Information System (INIS)

    Song Wei; Shi Tong; Ren Weiping; Markel, David C; Wang Sunxi; Mao Guangzhao

    2012-01-01

    The failure of prosthesis after total joint replacement is due to the lack of early implant osseointegration. In this study polyvinyl alcohol–collagen–hydroxyapatite (PVA-Col-HA) electrospun nanofibrous meshes were fabricated as a biomimetic bone-like extracellular matrix for the modification of orthopedic prosthetic surfaces. In order to reinforce the PVA nanofibers, HA nanorods and Type I collagen were incorporated into the nanofibers. We investigated the morphology, biodegradability, mechanical properties and biocompatibility of the prepared nanofibers. Our results showed these inorganic–organic blended nanofibers to be degradable in vitro. The encapsulated nano-HA and collagen interacted with the PVA content, reinforcing the hydrolytic resistance and mechanical properties of nanofibers that provided longer lasting stability. The encapsulated nano-HA and collagen also enhanced the adhesion and proliferation of murine bone cells (MC3T3) in vitro. We propose the PVA-Col-HA nanofibers might be promising modifying materials on implant surfaces for orthopedic applications. (paper)

  10. A non-equilibrium thermodynamic model for tumor extracellular matrix with enzymatic degradation

    Science.gov (United States)

    Xue, Shi-Lei; Li, Bo; Feng, Xi-Qiao; Gao, Huajian

    2017-07-01

    The extracellular matrix (ECM) of a solid tumor not only affords scaffolding to support tumor architecture and integrity but also plays an essential role in tumor growth, invasion, metastasis, and therapeutics. In this paper, a non-equilibrium thermodynamic theory is established to study the chemo-mechanical behaviors of tumor ECM, which is modeled as a poroelastic polyelectrolyte consisting of a collagen network and proteoglycans. By using the principle of maximum energy dissipation rate, we deduce a set of governing equations for drug transport and mechanosensitive enzymatic degradation in ECM. The results reveal that osmosis is primarily responsible for the compression resistance of ECM. It is suggested that a well-designed ECM degradation can effectively modify the tumor microenvironment for improved efficiency of cancer therapy. The theoretical predictions show a good agreement with relevant experimental observations. This study aimed to deepen our understanding of tumor ECM may be conducive to novel anticancer strategies.

  11. NG2 proteoglycan increases mesangial cell proliferation and extracellular matrix production

    International Nuclear Information System (INIS)

    Xiong Jing; Wang Yang; Zhu, Zhonghua; Liu Jianshe; Wang Yumei; Zhang Chun; Hammes, Hans-Peter; Lang, Florian; Feng Yuxi

    2007-01-01

    As a membrane-spanning protein, NG2 chondroitin sulfate proteoglycan interacts with molecules on both sides of plasma membrane. The present study explored the role of NG2 in the pathogenesis of diabetic nephropathy. In the normal kidneys, NG2 was observed predominantly in glomerular mesangium, Bowman's capsule and interstitial vessels. Both mRNA and protein expression in kidneys was significantly higher in strepozotocin-induced diabetic rats than that in normal rats. In the cultured rat mesangial cell line HBZY-1, overexpression of NG2 promoted mesangial cell proliferation and extracellular matrix (ECM) production, such as type VI collagen and laminin. Furthermore, target knockdown of NG2 resulted in decreased cell proliferation and ECM formation. The observations suggest that NG2 is up-regulated in diabetic nephropathy. It actively participates in the development and progression of glomerulosclerosis by stimulating proliferation of mesangial cells and deposition of ECM

  12. Alterations in proteins of bone marrow extracellular matrix in undernourished mice

    Directory of Open Access Journals (Sweden)

    C.L. Vituri

    2000-08-01

    Full Text Available The objective of the present study was to determine the effect of protein malnutrition on the glycoprotein content of bone marrow extracellular matrix (ECM. Two-month-old male Swiss mice were submitted to protein malnutrition with a low-protein diet containing 4% casein as compared to 20% casein in the control diet. When the experimental group had attained a 20% loss of their original body weight, we extracted the ECM proteins from bone marrow with PBS buffer, and analyzed ECM samples by SDS-PAGE (7.5% and ECL Western blotting. Quantitative differences were observed between control and experimental groups. Bone marrow ECM from undernourished mice had greater amounts of extractable fibronectin (1.6-fold increase and laminin (4.8-fold increase when compared to the control group. These results suggest an association between fluctuations in the composition of the hematopoietic microenvironment and altered hematopoiesis observed in undernourished mice.

  13. Detection of HOCl-mediated protein oxidation products in the extracellular matrix of human atherosclerotic plaques

    DEFF Research Database (Denmark)

    Woods, Alan A; Linton, Stuart M; Davies, Michael Jonathan

    2003-01-01

    Oxidation is believed to play a role in atherosclerosis. Oxidized lipids, sterols and proteins have been detected in early, intermediate and advanced human lesions at elevated levels. The spectrum of oxidized side-chain products detected on proteins from homogenates of advanced human lesions has...... been interpreted in terms of the occurrence of two oxidative mechanisms, one involving oxygen-derived radicals catalysed by trace transition metal ions, and a second involving chlorinating species (HOCl or Cl2), generated by the haem enzyme myeloperoxidase (MPO). As MPO is released extracellularly...... for 83-96% of the total oxidized protein side-chain products detected in these plaques. Oxidation of matrix components extracted from healthy artery tissue, and model proteins, with reagent HOCl is shown to give rise to a similar pattern of products to those detected in advanced human lesions...

  14. Hypoxia-driven angiogenesis: role of tip cells and extracellular matrix scaffolding.

    Science.gov (United States)

    Germain, Stéphane; Monnot, Catherine; Muller, Laurent; Eichmann, Anne

    2010-05-01

    Angiogenesis is a highly coordinated tissue remodeling process leading to blood vessel formation. Hypoxia triggers angiogenesis via induction of expression of growth factors such as vascular endothelial growth factor (VEGF). VEGF instructs endothelial cells to form tip cells, which lead outgrowing capillary sprouts, whereas Notch signaling inhibits sprout formation. Basement membrane deposition and mechanical cues from the extracellular matrix (ECM) induced by hypoxia may participate to coordinated vessel sprouting in conjunction with the VEGF and Notch signaling pathways. Hypoxia regulates ECM composition, deposition, posttranslational modifications and rearrangement. In particular, hypoxia-driven vascular remodeling is dynamically regulated through modulation of ECM-modifying enzyme activities that eventually affect both matricellular proteins and growth factor availability. Better understanding of the complex interplay between endothelial cells and soluble growth factors and mechanical factors from the ECM will certainly have significant implications for understanding the regulation of developmental and pathological angiogenesis driven by hypoxia.

  15. Quantification of fibronectin as a method to assess ex vivo extracellular matrix remodeling

    DEFF Research Database (Denmark)

    Bager, Cecilie Liv; Gudmann, N.; Willumsen, N.

    2016-01-01

    Altered architecture, composition and quality of the extracellular matrix (ECM) are pathological hallmarks of several inflammatory and fibro-proliferative pathological processes such as osteoarthritis (OA), rheumatoid arthritis (RA), fibrosis and cancer. One of the most important components...... of the ECM is fibronectin. Fibronectin serves as an adhesion molecule anchoring cells to the underlying basement membrane through direct interaction with integrin receptors. Fibronectin hereby modulates the properties of the ECM and affects cellular processes. Quantification of fibronectin remodeling could...... therefore be used to assess the changes in the ECM that occur during progression of fibro-proliferative pathologies. Ex vivo models are becoming state-of-the-art tools to study ECM remodeling as the cellular composition and the organization of the ECM are preserved. Ex vivo models may therefore...

  16. Structural, biochemical, cellular, and functional changes in skeletal muscle extracellular matrix with aging

    DEFF Research Database (Denmark)

    Kragstrup, Tue Wenzel; Kjaer, M; Mackey, A L

    2011-01-01

    The extracellular matrix (ECM) of skeletal muscle is critical for force transmission and for the passive elastic response of skeletal muscle. Structural, biochemical, cellular, and functional changes in skeletal muscle ECM contribute to the deterioration in muscle mechanical properties with aging......-links and a buildup of advanced glycation end-product cross-links. Altered mechanotransduction, poorer activation of satellite cells, poorer chemotactic and delayed inflammatory responses, and a change in modulators of the ECM are important cellular changes. It is possible that the structural and biochemical changes...... in skeletal muscle ECM contribute to the increased stiffness and impairment in force generated by the contracting muscle fibers seen with aging. The cellular interactions provide and potentially coordinate an adaptation to mechanical loading and ensure successful regeneration after muscle injury. Some...

  17. Early extracellular matrix changes are associated with later development of bronchiolitis obliterans syndrome after lung transplantation

    DEFF Research Database (Denmark)

    Müller, Catharina; Andersson-Sjöland, Annika; Schultz, Hans Henrik

    2017-01-01

    are largely unknown. The aim of this study was to identify potential early changes in the extracellular matrix (ECM) in different compartments of the transplanted lung prior to the development of BOS. Methods: Transbronchial biopsies from a cohort of 58 lung transplantation patients at the Copenhagen...... and immunohistochemistry. Results: A time-specific and compartment-specific pattern of ECM changes was detected. Alveolar total collagen (p=0.0190) and small airway biglycan (p=0.0199) increased between 3 and 12 months after transplantation in patients developing BOS, while collagen type IV (p=0.0124) increased...... in patients without BOS. Patients with early-onset BOS mirrored this increase. Patients developing grade 3 BOS showed distinct ECM changes already at 3 months. Patients with BOS with treated acute rejections displayed reduced alveolar total collagen (p=0.0501) and small airway biglycan (p=0.0485) at 3 months...

  18. The Role of Structural Extracellular Matrix Proteins in Urothelial Bladder Cancer

    Directory of Open Access Journals (Sweden)

    Andrea Brunner

    2007-01-01

    Full Text Available The extracellular matrix (ECM plays a key role in the modulation of cancer cell invasion. In urothelial carcinoma of the bladder (UC the role of ECM proteins has been widely studied. The mechanisms, which are involved in the development of invasion, progression and generalization, are complex, depending on the interaction of ECM proteins with each other as well as with cancer cells. The following review will focus on the pathogenetic role and prognostic value of structural proteins, such as laminins, collagens, fi bronectin (FN, tenascin (Tn-C and thrombospondin 1 (TSP1 in UC. In addition, the role of integrins mediating the interaction of ECM molecules and cancer cells will be addressed, since integrin-mediated FN, Tn-C and TSP1 interactions seem to play an important role during tumor cell invasion and angiogenesis.

  19. Novel insights into the function and dynamics of extracellular matrix in liver fibrosis

    DEFF Research Database (Denmark)

    Karsdal, Morten A; Manon-Jensen, Tina; Genovese, Federica

    2015-01-01

    Emerging evidence suggests that altered components and posttranslational modifications of proteins in the extracellular matrix (ECM) may both initiate and drive disease progression. The ECM is a complex grid consisting of multiple proteins, most of which play a vital role in containing......) explore key structural and functional components of the ECM as exemplified by monogenetic disorders leading to severe pathologies, 2) discuss selected pathological posttranslational modifications of ECM proteins resulting in altered functional (signaling) properties from the original structural proteins......, and 3) discuss how these findings support the novel concept that an increasing number of components of the ECM harbor signaling functions that can modulate fibrotic liver disease. The ECM entails functions in addition to anchoring cells and modulating their migratory behavior. Key ECM components...

  20. Extracellular Matrix-Regulated Gene Expression RequiresCooperation of SWI/SNF and Transcription Factors

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ren; Spencer, Virginia A.; Bissell, Mina J.

    2006-05-25

    Extracellular cues play crucial roles in the transcriptional regulation of tissue-specific genes, but whether and how these signals lead to chromatin remodeling is not understood and subject to debate. Using chromatin immunoprecipitation (ChIP) assays and mammary-specific genes as models, we show here that extracellular matrix (ECM) molecules and prolactin cooperate to induce histone acetylation and binding of transcription factors and the SWI/SNF complex to the {beta}- and ?-casein promoters. Introduction of a dominant negative Brg1, an ATPase subunit of SWI/SNF complex, significantly reduced both {beta}- and ?-casein expression, suggesting that SWI/SNF-dependent chromatin remodeling is required for transcription of mammary-specific genes. ChIP analyses demonstrated that the ATPase activity of SWI/SNF is necessary for recruitment of RNA transcriptional machinery, but not for binding of transcription factors or for histone acetylation. Coimmunoprecipitation analyses showed that the SWI/SNF complex is associated with STAT5, C/EBP{beta}, and glucocorticoid receptor (GR). Thus, ECM- and prolactin-regulated transcription of the mammary-specific casein genes requires the concerted action of chromatin remodeling enzymes and transcription factors.

  1. Myogenic Progenitor Cells Control Extracellular Matrix Production by Fibroblasts during Skeletal Muscle Hypertrophy.

    Science.gov (United States)

    Fry, Christopher S; Kirby, Tyler J; Kosmac, Kate; McCarthy, John J; Peterson, Charlotte A

    2017-01-05

    Satellite cells, the predominant stem cell population in adult skeletal muscle, are activated in response to hypertrophic stimuli and give rise to myogenic progenitor cells (MPCs) within the extracellular matrix (ECM) that surrounds myofibers. This ECM is composed largely of collagens secreted by interstitial fibrogenic cells, which influence satellite cell activity and muscle repair during hypertrophy and aging. Here we show that MPCs interact with interstitial fibrogenic cells to ensure proper ECM deposition and optimal muscle remodeling in response to hypertrophic stimuli. MPC-dependent ECM remodeling during the first week of a growth stimulus is sufficient to ensure long-term myofiber hypertrophy. MPCs secrete exosomes containing miR-206, which represses Rrbp1, a master regulator of collagen biosynthesis, in fibrogenic cells to prevent excessive ECM deposition. These findings provide insights into how skeletal stem and progenitor cells interact with other cell types to actively regulate their extracellular environments for tissue maintenance and adaptation. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Simple and high yielding method for preparing tissue specific extracellular matrix coatings for cell culture.

    Science.gov (United States)

    DeQuach, Jessica A; Mezzano, Valeria; Miglani, Amar; Lange, Stephan; Keller, Gordon M; Sheikh, Farah; Christman, Karen L

    2010-09-27

    The native extracellular matrix (ECM) consists of a highly complex, tissue-specific network of proteins and polysaccharides, which help regulate many cellular functions. Despite the complex nature of the ECM, in vitro cell-based studies traditionally assess cell behavior on single ECM component substrates, which do not adequately mimic the in vivo extracellular milieu. We present a simple approach for developing naturally derived ECM coatings for cell culture that provide important tissue-specific cues unlike traditional cell culture coatings, thereby enabling the maturation of committed C2C12 skeletal myoblast progenitors and human embryonic stem cells differentiated into cardiomyocytes. Here we show that natural muscle-specific coatings can (i) be derived from decellularized, solubilized adult porcine muscle, (ii) contain a complex mixture of ECM components including polysaccharides, (iii) adsorb onto tissue culture plastic and (iv) promote cell maturation of committed muscle progenitor and stem cells. This versatile method can create tissue-specific ECM coatings, which offer a promising platform for cell culture to more closely mimic the mature in vivo ECM microenvironment.

  3. Undersulfation of proteoglycans and proteins alter C6 glioma cells proliferation, adhesion and extracellular matrix organization.

    Science.gov (United States)

    Mendes de Aguiar, Claudia B N; Garcez, Ricardo Castilho; Alvarez-Silva, Marcio; Trentin, Andréa Gonçalves

    2002-11-01

    Proteoglycans are considered to be important molecule in cell-microenvironment interactions. They are overexpressed in neoplastic cells modifying their growth and migration in hosts. In this work we verified that undersulfation of proteoglycans and other sulfated molecules, induced by sodium chlorate treatment, inhibited C6 glioma cells proliferation in a dose-dependent way. This effect was restored by the addition of exogenous heparin. We could not detect significant cell mortality in our culture condition. The treatment also impaired in a dose-dependent manner, C6 cell adhesion to extracellular matrix (ECM) proteins (collagen IV, laminin and fibronectin). In addition, sodium chlorate treatment altered C6 glioma cell morphology, from the fibroblast-like to a more rounded one. This effect was accompanied by increased synthesis of fibronectin and alterations in its extracellular network organization. However, we could not observe modifications on laminin organization and synthesis. The results suggest an important connection between sulfation degree with important tumor functions, such as proliferation and adhesion. We suggest that proteoglycans may modulate the glioma microenvironment network during tumor cell progression and invasion.

  4. Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

    International Nuclear Information System (INIS)

    Nakanishi, Jun; Nakayama, Hidekazu; Horiike, Yasuhiro; Yamaguchi, Kazuo; Garcia, Andres J

    2011-01-01

    The development of methods for the off-on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs) of three disulfide compounds containing (i) a photocleavable poly(ethylene glycol) (PEG), (ii) nitrilotriacetic acid (NTA) and (iii) hepta(ethylene glycol) (EG 7 ). Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag) sequences in its Ni 2+ -ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG 7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII 7-10 ) to the irradiated regions. In contrast, when bovine serum albumin-a major serum protein-was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII 7-10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

  5. Changes in muscle fiber contractility and extracellular matrix production during skeletal muscle hypertrophy.

    Science.gov (United States)

    Mendias, Christopher L; Schwartz, Andrew J; Grekin, Jeremy A; Gumucio, Jonathan P; Sugg, Kristoffer B

    2017-03-01

    Skeletal muscle can adapt to increased mechanical loads by undergoing hypertrophy. Transient reductions in whole muscle force production have been reported during the onset of hypertrophy, but contractile changes in individual muscle fibers have not been previously studied. Additionally, the extracellular matrix (ECM) stores and transmits forces from muscle fibers to tendons and bones, and determining how the ECM changes during hypertrophy is important in understanding the adaptation of muscle tissue to mechanical loading. Using the synergist ablation model, we sought to measure changes in muscle fiber contractility, collagen content, and cross-linking, and in the expression of several genes and activation of signaling proteins that regulate critical components of myogenesis and ECM synthesis and remodeling during muscle hypertrophy. Tissues were harvested 3, 7, and 28 days after induction of hypertrophy, and nonoverloaded rats served as controls. Muscle fiber specific force (sF o ), which is the maximum isometric force normalized to cross-sectional area, was reduced 3 and 7 days after the onset of mechanical overload, but returned to control levels by 28 days. Collagen abundance displayed a similar pattern of change. Nearly a quarter of the transcriptome changed over the course of overload, as well as the activation of signaling pathways related to hypertrophy and atrophy. Overall, this study provides insight into fundamental mechanisms of muscle and ECM growth, and indicates that although muscle fibers appear to have completed remodeling and regeneration 1 mo after synergist ablation, the ECM continues to be actively remodeling at this time point. NEW & NOTEWORTHY This study utilized a rat synergist ablation model to integrate changes in single muscle fiber contractility, extracellular matrix composition, activation of important signaling pathways in muscle adaption, and corresponding changes in the muscle transcriptome to provide novel insight into the basic

  6. The role of extracellular matrix in lateral transmission of force in skeletal muscle

    Science.gov (United States)

    Gao, Yingxin

    This dissertation describes the role of extracellular matrix (ECM) in the lateral transmission of force. It consists of an experimental studies of the ECM and mathematical modeling of lateral transmission of force. The effect of aging on the structural and mechanical properties of the epimysium of muscle of the rats were examined. No statistically significant differences were found in the ultrastructure, or the thickness of the epimysium. However, from the tensile stress-strain tests, it was found that the epimysium of muscles from old rats was much stiffer than that of the young rats. Based on these observations. It was concluded that the differences in the mechanical properties of the epimysium of the muscles from the old compared with young rats were not associated with the arrangement and size of collagen fibers in the epimysium. Consequently, other methods will be required to identify the structural bases of the mechanical differences. The stress-strain relationships for the epimysiums of the skeletal muscles from both the young and old rats were found to be nonlinear. A mathematical model was developed that showed that the nonlinear behavior results from the waviness and the reorientation of the collagen fibers in the epimysium. The ECM plays an important role in lateral transmission of force in skeletal muscle by providing shear stress between the muscle fibers or fascicles. A mathematical model was developed to investigate the mechanisms of lateral transmission. It was a modification of the shear lag theory for chopped fiber composite materials used in engineering applications. The modified shear lag theory includes an activation strain to account for muscle contraction and a myofibrils-endomysium interfaces that accounts for the molecular lateral linkages. The model was used to simulate the classic experiments of Street. It was demonstrated that lateral transmission of force in the skeletal muscle is affected by the mechanical and structural properties of

  7. Tailoring the properties of cholecyst-derived extracellular matrix using carbodiimide cross-linking.

    LENUS (Irish Health Repository)

    Burugapalli, Krishna

    2009-01-01

    Modulation of properties of extracellular matrix (ECM) based scaffolds is key for their application in the clinical setting. In the present study, cross-linking was used as a tool for tailoring the properties of cholecyst-derived extracellular matrix (CEM). CEM was cross-linked with varying cross-linking concentrations of N,N-(3-dimethyl aminopropyl)-N\\'-ethyl carbodiimide (EDC) in the presence of N-hydroxysuccinimide (NHS). Shrink temperature measurements and ATR-FT-IR spectra were used to determine the degree of cross-linking. The effect of cross-linking on degradation was tested using the collagenase assay. Uniaxial tensile properties and the ability to support fibroblasts were also evaluated as a function of cross-linking. Shrink temperature increased from 59 degrees C for non-cross-linked CEM to 78 degrees C for the highest EDC cross-linking concentration, while IR peak area ratios for the free -NH(2) group at 3290 cm(-1) to that of the amide I band at 1635 cm(-1) decreased with increasing EDC cross-linking concentration. Collagenase assay demonstrated that degradation rates for CEM can be tailored. EDC concentrations 0 to 0.0033 mmol\\/mg CEM were the cross-linking concentration range in which CEM showed varied susceptibility to collagenase degradation. Furthermore, cross-linking concentrations up to 0.1 mmol EDC\\/mg CEM did not have statistically significant effect on the uniaxial tensile strength, as well as morphology, viability and proliferation of fibroblasts on CEM. In conclusion, the degradation rates of CEM can be tailored using EDC-cross-linking, while maintaining the mechanical properties and the ability of CEM to support cells.

  8. The role of extracellular matrix metalloproteinase inducer (EMMPRIN) in the regulation of bovine endometrial cell functions.

    Science.gov (United States)

    Mishra, Birendra; Kizaki, Keiichiro; Sato, Takashi; Ito, Akira; Hashizume, Kazuyoshi

    2012-06-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell surface glycoprotein that stimulates the production of several matrix metalloproteinases (MMPs) for tissue remodeling. Previously, we detected EMMPRIN in the bovine endometrium, and it is mainly expressed in the luminal and glandular epithelium whereas MMPs are expressed in the underlying stroma. From this expression pattern, we hypothesized that EMMPRIN may regulate stromal MMPs in endometrial cell functions. To test this hypothesis, a coculture of epithelial and stromal cells was performed using a transwell system. In the coculture, epithelial cells were cultured on the insert membrane and stromal cell on the surface of well plates. Expression of stromal MMP-2 and MMP-14 was significantly higher in coculture with epithelial cell. Further, with the addition of anti-EMMPRIN antibody into the epithelial cell compartment, the expression of stromal EMMPRIN and MMP-2 and MMP-14 was decreased. To identify the active site of EMMPRIN for the augmentation of MMPs, EMMPRIN synthetic peptides that correspond to the extracellular loop domain-I (EM1, EM2, EM3, and EM4) were added into the epithelial cell compartment, and only EM2 at a higher dose interfered with EMMPRIN-mediated expression of MMP-14. Next, we examined the effects of progesterone and/or estrogen on the expression of EMMPRIN, MMP-2, and MMP-14. Progesterone (300 nM) significantly stimulated the expression of EMMPRIN but had no effects on any of the MMPs. These results suggest that EMMPRIN derived from epithelial cells regulates MMPs in the endometrium under progesterone-rich conditions and may thereby modulate bovine endometrial cell functions during gestation.

  9. Human Adipose Tissue Derived Extracellular Matrix and Methylcellulose Hydrogels Augments and Regenerates the Paralyzed Vocal Fold.

    Directory of Open Access Journals (Sweden)

    Dong Wook Kim

    Full Text Available Vocal fold paralysis results from various etiologies and can induce voice changes, swallowing complications, and issues with aspiration. Vocal fold paralysis is typically managed using injection laryngoplasty with fat or synthetic polymers. Injection with autologous fat has shown excellent biocompatibility. However, it has several disadvantages such as unpredictable resorption rate, morbidities associated with liposuction procedure which has to be done in operating room under general anesthesia. Human adipose-derived extracellular matrix (ECM grafts have been reported to form new adipose tissue and have greater biostability than autologous fat graft. Here, we present an injectable hydrogel that is constructed from adipose tissue derived soluble extracellular matrix (sECM and methylcellulose (MC for use in vocal fold augmentation. Human sECM derived from adipose tissue was extracted using two major steps-ECM was isolated from human adipose tissue and was subsequently solubilized. Injectable sECM/MC hydrogels were prepared by blending of sECM and MC. Sustained vocal fold augmentation and symmetric vocal fold vibration were accomplished by the sECM/MC hydrogel in paralyzed vocal fold which were confirmed by laryngoscope, histology and a high-speed imaging system. There were increased number of collagen fibers and fatty granules at the injection site without significant inflammation or fibrosis. Overall, these results indicate that the sECM/MC hydrogel can enhance vocal function in paralyzed vocal folds without early resorption and has potential as a promising material for injection laryngoplasty for stable vocal fold augmentation which can overcome the shortcomings of autologous fat such as unpredictable duration and morbidity associated with the fat harvest.

  10. Human Adipose Tissue Derived Extracellular Matrix and Methylcellulose Hydrogels Augments and Regenerates the Paralyzed Vocal Fold.

    Science.gov (United States)

    Kim, Dong Wook; Kim, Eun Ji; Kim, Eun Na; Sung, Myung Whun; Kwon, Tack-Kyun; Cho, Yong Woo; Kwon, Seong Keun

    2016-01-01

    Vocal fold paralysis results from various etiologies and can induce voice changes, swallowing complications, and issues with aspiration. Vocal fold paralysis is typically managed using injection laryngoplasty with fat or synthetic polymers. Injection with autologous fat has shown excellent biocompatibility. However, it has several disadvantages such as unpredictable resorption rate, morbidities associated with liposuction procedure which has to be done in operating room under general anesthesia. Human adipose-derived extracellular matrix (ECM) grafts have been reported to form new adipose tissue and have greater biostability than autologous fat graft. Here, we present an injectable hydrogel that is constructed from adipose tissue derived soluble extracellular matrix (sECM) and methylcellulose (MC) for use in vocal fold augmentation. Human sECM derived from adipose tissue was extracted using two major steps-ECM was isolated from human adipose tissue and was subsequently solubilized. Injectable sECM/MC hydrogels were prepared by blending of sECM and MC. Sustained vocal fold augmentation and symmetric vocal fold vibration were accomplished by the sECM/MC hydrogel in paralyzed vocal fold which were confirmed by laryngoscope, histology and a high-speed imaging system. There were increased number of collagen fibers and fatty granules at the injection site without significant inflammation or fibrosis. Overall, these results indicate that the sECM/MC hydrogel can enhance vocal function in paralyzed vocal folds without early resorption and has potential as a promising material for injection laryngoplasty for stable vocal fold augmentation which can overcome the shortcomings of autologous fat such as unpredictable duration and morbidity associated with the fat harvest.

  11. Regulation of extracellular matrix organization by BMP signaling in Caenorhabditis elegans.

    Science.gov (United States)

    Schultz, Robbie D; Bennett, Emily E; Ellis, E Ann; Gumienny, Tina L

    2014-01-01

    In mammals, Bone Morphogenetic Protein (BMP) pathway signaling is important for the growth and homeostasis of extracellular matrix, including basement membrane remodeling, scarring, and bone growth. A conserved BMP member in Caenorhabditis elegans, DBL-1, regulates body length in a dose-sensitive manner. Loss of DBL-1 pathway signaling also results in increased anesthetic sensitivity. However, the physiological basis of these pleiotropic phenotypes is largely unknown. We created a DBL-1 over-expressing strain and show that sensitivity to anesthetics is inversely related to the dose of DBL-1. Using pharmacological, genetic analyses, and a novel dye permeability assay for live, microwave-treated animals, we confirm that DBL-1 is required for the barrier function of the cuticle, a specialized extracellular matrix. We show that DBL-1 signaling is required to prevent animals from forming tail-entangled aggregates in liquid. Stripping lipids off the surface of wild-type animals recapitulates this phenotype. Finally, we find that DBL-1 signaling affects ultrastructure of the nematode cuticle in a dose-dependent manner, as surface lipid content and cuticular organization are disrupted in animals with genetically altered DBL-1 levels. We propose that the lipid layer coating the nematode cuticle normally prevents tail entanglement, and that reduction of this layer by loss of DBL-1 signaling promotes aggregation. This work provides a physiological mechanism that unites the DBL-1 signaling pathway roles of not only body size regulation and drug responsiveness, but also the novel Hoechst 33342 staining and aggregation phenotypes, through barrier function, content, and organization of the cuticle.

  12. Regulation of extracellular matrix organization by BMP signaling in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Robbie D Schultz

    Full Text Available In mammals, Bone Morphogenetic Protein (BMP pathway signaling is important for the growth and homeostasis of extracellular matrix, including basement membrane remodeling, scarring, and bone growth. A conserved BMP member in Caenorhabditis elegans, DBL-1, regulates body length in a dose-sensitive manner. Loss of DBL-1 pathway signaling also results in increased anesthetic sensitivity. However, the physiological basis of these pleiotropic phenotypes is largely unknown. We created a DBL-1 over-expressing strain and show that sensitivity to anesthetics is inversely related to the dose of DBL-1. Using pharmacological, genetic analyses, and a novel dye permeability assay for live, microwave-treated animals, we confirm that DBL-1 is required for the barrier function of the cuticle, a specialized extracellular matrix. We show that DBL-1 signaling is required to prevent animals from forming tail-entangled aggregates in liquid. Stripping lipids off the surface of wild-type animals recapitulates this phenotype. Finally, we find that DBL-1 signaling affects ultrastructure of the nematode cuticle in a dose-dependent manner, as surface lipid content and cuticular organization are disrupted in animals with genetically altered DBL-1 levels. We propose that the lipid layer coating the nematode cuticle normally prevents tail entanglement, and that reduction of this layer by loss of DBL-1 signaling promotes aggregation. This work provides a physiological mechanism that unites the DBL-1 signaling pathway roles of not only body size regulation and drug responsiveness, but also the novel Hoechst 33342 staining and aggregation phenotypes, through barrier function, content, and organization of the cuticle.

  13. Changes in extracellular matrix in subcutaneous small resistance arteries of patients with essential hypertension.

    Science.gov (United States)

    Favero, Gaia; Paini, Anna; De Ciuceis, Carolina; Rodella, Luigi F; Moretti, Enrico; Porteri, Enzo; Rossini, Claudia; Ministrini, Silvia; Solaini, Leonardo; Stefano, Caletti; Coschignano, Maria Antonietta; Brami, Valeria; Petelca, Alina; Nardin, Matteo; Valli, Ilenia; Tiberio, Guido A M; Bonomini, Francesca; Agabiti Rosei, Claudia; Portolani, Nazario; Rizzoni, Damiano; Rezzani, Rita

    2018-03-09

    In the development of hypertensive microvascular remodeling, a relevant role may be played by changes in extracellular matrix proteins. Aim of this study was the to evaluate some extracellular matrix components within the tunica media of subcutaneous small arteries in 9 normotensive subjects and 12 essential hypertensive patients, submitted to a biopsy of subcutaneous fat from the gluteal or the anterior abdominal region. Subcutaneous small resistance arteries were dissected and mounted on an isometric myograph, and the tunica media to internal lumen ratio was measured. In addition, fibronectin, laminin, transforming growth factor-beta-1 (TGF-β1) and emilin-1 contents within the tunica media were evaluated by immunofluorescence and relative immunomorphometrical analysis (immunopositivity % of area). The total collagen content and collagen subtypes within the tunica media were evaluated using both Sirius red staining (under polarized light) and immunofluorescence assay. Normotensive controls had less total and type III collagen in respect with hypertensive patients. Fibronectin and TGF-β1 tunica media content was significantly greater in essential hypertensive patients, compared with normotensive controls, while laminin and emilin-1 tunica media content was lesser in essential hypertensive patients, compared with normotensive controls. A significant correlation was observed between fibronectin tunica media content and media to lumen ratio. Our results indicate that, in small resistance arteries of patients with essential hypertension, a relevant fibrosis may be detected; fibronectin and TGF-β1 tunica media content is increased, while laminin and emilin-1 content is decreased; these changes might be involved in the development of small resistance artery remodeling in humans.

  14. Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

    Energy Technology Data Exchange (ETDEWEB)

    Nakanishi, Jun; Nakayama, Hidekazu; Horiike, Yasuhiro [World Premier International (WPI) Research Center Initiative, International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science - NIMS (Japan); Yamaguchi, Kazuo [Department of Chemistry, Faculty of Science and Research Institute for Photofunctionalized Materials, Kanagawa University (Japan); Garcia, Andres J, E-mail: NAKANISHI.Jun@nims.go.jp [Institute for Bioengineering and Bioscience, Woodruff School of Mechanical Engineering, Georgia Institute of Technology (United States)

    2011-08-15

    The development of methods for the off-on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs) of three disulfide compounds containing (i) a photocleavable poly(ethylene glycol) (PEG), (ii) nitrilotriacetic acid (NTA) and (iii) hepta(ethylene glycol) (EG{sub 7}). Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag) sequences in its Ni{sup 2+}-ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG{sub 7} underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII{sub 7-10}) to the irradiated regions. In contrast, when bovine serum albumin-a major serum protein-was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII{sub 7-10} was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

  15. Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

    Directory of Open Access Journals (Sweden)

    Jun Nakanishi, Hidekazu Nakayama, Kazuo Yamaguchi, Andres J Garcia and Yasuhiro Horiike

    2011-01-01

    Full Text Available The development of methods for the off–on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs of three disulfide compounds containing (i a photocleavable poly(ethylene glycol (PEG, (ii nitrilotriacetic acid (NTA and (iii hepta(ethylene glycol (EG7. Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag sequences in its Ni2+-ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII7–10 to the irradiated regions. In contrast, when bovine serum albumin—a major serum protein—was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII7-10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

  16. Direct Comparison of Physical Properties of Bacillus subtilis NCIB 3610 and B-1 Biofilms

    Science.gov (United States)

    Kesel, Sara; Grumbein, Stefan; Gümperlein, Ina; Tallawi, Marwa; Marel, Anna-Kristina

    2016-01-01

    Many bacteria form surface-attached communities known as biofilms. Due to the extreme resistance of these bacterial biofilms to antibiotics and mechanical stresses, biofilms are of growing interest not only in microbiology but also in medicine and industry. Previous studies have determined the extracellular polymeric substances present in the matrix of biofilms formed by Bacillus subtilis NCIB 3610. However, studies on the physical properties of biofilms formed by this strain are just emerging. In particular, quantitative data on the contributions of biofilm matrix biopolymers to these physical properties are lacking. Here, we quantitatively investigated three physical properties of B. subtilis NCIB 3610 biofilms: the surface roughness and stiffness and the bulk viscoelasticity of these biofilms. We show how specific biomolecules constituting the biofilm matrix formed by this strain contribute to those biofilm properties. In particular, we demonstrate that the surface roughness and surface elasticity of 1-day-old NCIB 3610 biofilms are strongly affected by the surface layer protein BslA. For a second strain, B. subtilis B-1, which forms biofilms containing mainly γ-polyglutamate, we found significantly different physical biofilm properties that are also differently affected by the commonly used antibacterial agent ethanol. We show that B-1 biofilms are protected from ethanol-induced changes in the biofilm's stiffness and that this protective effect can be transferred to NCIB 3610 biofilms by the sole addition of γ-polyglutamate to growing NCIB 3610 biofilms. Together, our results demonstrate the importance of specific biofilm matrix components for the distinct physical properties of B. subtilis biofilms. PMID:26873313

  17. Co-transfection of decorin and interleukin-10 modulates pro-fibrotic extracellular matrix gene expression in human tenocyte culture

    Science.gov (United States)

    Abbah, Sunny A.; Thomas, Dilip; Browne, Shane; O'Brien, Timothy; Pandit, Abhay; Zeugolis, Dimitrios I.

    2016-02-01

    Extracellular matrix synthesis and remodelling are driven by increased activity of transforming growth factor beta 1 (TGF-β1). In tendon tissue repair, increased activity of TGF-β1 leads to progressive fibrosis. Decorin (DCN) and interleukin 10 (IL-10) antagonise pathological collagen synthesis by exerting a neutralising effect via downregulation of TGF-β1. Herein, we report that the delivery of DCN and IL-10 transgenes from a collagen hydrogel system supresses the constitutive expression of TGF-β1 and a range of pro-fibrotic extracellular matrix genes.

  18. Medical biofilms--nanotechnology approaches.

    Science.gov (United States)

    Neethirajan, Suresh; Clond, Morgan A; Vogt, Adam

    2014-10-01

    Biofilms are colonies of bacteria or fungi that adhere to a surface, protected by an extracellular polymer matrix composed of polysaccharides and extracellular DNA. They are highly complex and dynamic multicellular structures that resist traditional means of killing planktonic bacteria. Recent developments in nanotechnology provide novel approaches to preventing and dispersing biofilm infections, which are a leading cause of morbidity and mortality. Medical device infections are responsible for approximately 60% of hospital acquired infections. In the United States, the estimated cost of caring for healthcare-associated infections is approximately between $28 billion and $45 billion per year. In this review, we will discuss our current understanding of biofilm formation and degradation, its relevance to challenges in clinical practice, and new technological developments in nanotechnology that are designed to address these challenges.

  19. HGF potentiates extracellular matrix-driven migration of human myoblasts: involvement of matrix metalloproteinases and MAPK/ERK pathway.

    Science.gov (United States)

    González, Mariela Natacha; de Mello, Wallace; Butler-Browne, Gillian S; Silva-Barbosa, Suse Dayse; Mouly, Vincent; Savino, Wilson; Riederer, Ingo

    2017-10-10

    The hepatocyte growth factor (HGF) is required for the activation of muscle progenitor cells called satellite cells (SC), plays a role in the migration of proliferating SC (myoblasts), and is present as a soluble factor during muscle regeneration, along with extracellular matrix (ECM) molecules. In this study, we aimed at determining whether HGF is able to interact with ECM proteins, particularly laminin 111 and fibronectin, and to modulate human myoblast migration. We evaluated the expression of the HGF-receptor c-Met, laminin, and fibronectin receptors by immunoblotting, flow cytometry, or immunofluorescence and used Transwell assays to analyze myoblast migration on laminin 111 and fibronectin in the absence or presence of HGF. Zymography was used to check whether HGF could modulate the production of matrix metalloproteinases by human myoblasts, and the activation of MAPK/ERK pathways was evaluated by immunoblotting. We demonstrated that human myoblasts express c-Met, together with laminin and fibronectin receptors. We observed that human laminin 111 and fibronectin have a chemotactic effect on myoblast migration, and this was synergistically increased when low doses of HGF were added. We detected an increase in MMP-2 activity in myoblasts treated with HGF. Conversely, MMP-2 inhibition decreased the HGF-associated stimulation of cell migration triggered by laminin or fibronectin. HGF treatment also induced in human myoblasts activation of MAPK/ERK pathways, whose specific inhibition decreased the HGF-associated stimulus of cell migration triggered by laminin 111 or fibronectin. We demonstrate that HGF induces ERK phosphorylation and MMP production, thus stimulating human myoblast migration on ECM molecules. Conceptually, these data state that the mechanisms involved in the migration of human myoblasts comprise both soluble and insoluble moieties. This should be taken into account to optimize the design of therapeutic cell transplantation strategies by improving

  20. Role of Stroma-Derived Extracellular Matrix in Regulation of Growth and Hormonal Responsiveness of Normal and Cancerous Human Breast Epithelium

    National Research Council Canada - National Science Library

    Woodward, Terry

    1997-01-01

    Specific extracellular matrix (ECM) proteins and their cellular receptors (integrins) are required for normal mammary gland morphogenesis and differentiation, while their expression is dramatically altered during tumorigenesis...

  1. Dynamic interactions of neutrophils and biofilms

    Directory of Open Access Journals (Sweden)

    Josefine Hirschfeld

    2014-12-01

    Full Text Available Background: The majority of microbial infections in humans are biofilm-associated and difficult to treat, as biofilms are highly resistant to antimicrobial agents and protect themselves from external threats in various ways. Biofilms are tenaciously attached to surfaces and impede the ability of host defense molecules and cells to penetrate them. On the other hand, some biofilms are beneficial for the host and contain protective microorganisms. Microbes in biofilms express pathogen-associated molecular patterns and epitopes that can be recognized by innate immune cells and opsonins, leading to activation of neutrophils and other leukocytes. Neutrophils are part of the first line of defense and have multiple antimicrobial strategies allowing them to attack pathogenic biofilms. Objective/design: In this paper, interaction modes of neutrophils with biofilms are reviewed. Antimicrobial strategies of neutrophils and the counteractions of the biofilm communities, with special attention to oral biofilms, are presented. Moreover, possible adverse effects of neutrophil activity and their biofilm-promoting side effects are discussed. Results/conclusion: Biofilms are partially, but not entirely, protected against neutrophil assault, which include the processes of phagocytosis, degranulation, and formation of neutrophil extracellular traps. However, virulence factors of microorganisms, microbial composition, and properties of the extracellular matrix determine whether a biofilm and subsequent microbial spread can be controlled by neutrophils and other host defense factors. Besides, neutrophils may inadvertently contribute to the physical and ecological stability of biofilms by promoting selection of more resistant strains. Moreover, neutrophil enzymes can degrade collagen and other proteins and, as a result, cause harm to the host tissues. These parameters could be crucial factors in the onset of periodontal inflammation and the subsequent tissue breakdown.

  2. Physical, Spatial, and Molecular Aspects of Extracellular Matrix of In Vivo Niches and Artificial Scaffolds Relevant to Stem Cells Research

    Directory of Open Access Journals (Sweden)

    Maria Akhmanova

    2015-01-01

    Full Text Available Extracellular matrix can influence stem cell choices, such as self-renewal, quiescence, migration, proliferation, phenotype maintenance, differentiation, or apoptosis. Three aspects of extracellular matrix were extensively studied during the last decade: physical properties, spatial presentation of adhesive epitopes, and molecular complexity. Over 15 different parameters have been shown to influence stem cell choices. Physical aspects include stiffness (or elasticity, viscoelasticity, pore size, porosity, amplitude and frequency of static and dynamic deformations applied to the matrix. Spatial aspects include scaffold dimensionality (2D or 3D and thickness; cell polarity; area, shape, and microscale topography of cell adhesion surface; epitope concentration, epitope clustering characteristics (number of epitopes per cluster, spacing between epitopes within cluster, spacing between separate clusters, cluster patterns, and level of disorder in epitope arrangement, and nanotopography. Biochemical characteristics of natural extracellular matrix molecules regard diversity and structural complexity of matrix molecules, affinity and specificity of epitope interaction with cell receptors, role of non-affinity domains, complexity of supramolecular organization, and co-signaling by growth factors or matrix epitopes. Synergy between several matrix aspects enables stem cells to retain their function in vivo and may be a key to generation of long-term, robust, and effective in vitro stem cell culture systems.

  3. Disorganized collagen scaffold interferes with fibroblast mediated deposition of organized extracellular matrix in vitro.

    Science.gov (United States)

    Saeidi, Nima; Guo, Xiaoqing; Hutcheon, Audrey E K; Sander, Edward A; Bale, Shyam Sundar; Melotti, Suzanna A; Zieske, James D; Trinkaus-Randall, Vickery; Ruberti, Jeffrey W

    2012-10-01

    Many tissue engineering applications require the remodeling of a degradable scaffold either in vitro or in situ. Although inefficient remodeling or failure to fully remodel the temporary matrix can result in a poor clinical outcome, very few investigations have examined in detail, the interaction of regenerative cells with temporary scaffoldings. In a recent series of investigations, randomly oriented collagen gels were directly implanted into human corneal pockets and followed for 24 months. The resulting remodeling response exhibited a high degree of variability which likely reflects differing regenerative/synthetic capacity across patients. Given this variability, we hypothesize that a disorganized, degradable provisional scaffold could be disruptive to a uniform, organized reconstruction of stromal matrix. In this investigation, two established corneal stroma tissue engineering culture systems (collagen scaffold-based and scaffold-free) were compared to determine if the presence of the disorganized collagen gel influenced matrix production and organizational control exerted by primary human corneal fibroblast cells (PHCFCs). PHCFCs were cultured on thin disorganized reconstituted collagen substrate (RCS--five donors: average age 34.4) or on a bare polycarbonate membrane (five donors: average age 32.4 controls). The organization and morphology of the two culture systems were compared over the long-term at 4, 8, and 11/12 weeks. Construct thickness and extracellular matrix organization/alignment was tracked optically with bright field and differential interference contrast (DIC) microscopy. The details of cell/matrix morphology and cell/matrix interaction were examined with standard transmission, cuprolinic blue and quick-freeze/deep-etch electron microscopy. Both the scaffold-free and the collagen-based scaffold cultures produced organized arrays of collagen fibrils. However, at all time points, the amount of organized cell-derived matrix in the scaffold

  4. Antibiofilm Effect of DNase against Single and Mixed Species Biofilm

    Science.gov (United States)

    Sharma, Komal

    2018-01-01

    Biofilms are aggregates of microorganisms that coexist in socially coordinated micro-niche in a self-produced polymeric matrix on pre-conditioned surfaces. The biofilm matrix reduces the efficacy of antibiofilm strategies. DNase degrades the extracellular DNA (e-DNA) present in the matrix, rendering the matrix weak and susceptible to antimicrobials. In the current study, the effect of DNase I was evaluated during biofilm formation (pre-treatment), on preformed biofilms (post-treatment) and both (dual treatment). The DNase I pre-treatment was optimized for P. aeruginosa PAO1 (model biofilm organism) at 10 µg/mL and post-treatment at 10 µg/mL with 15 min of contact duration. Inclusion of Mg2+ alongside DNase I post-treatment resulted in 90% reduction in biofilm within only 5 min of contact time (irrespective of age of biofilm). On extension of these findings, DNase I was found to be less effective against mixed species biofilm than individual biofilms. DNase I can be used as potent antibiofilm agent and with further optimization can be effectively used for biofilm prevention and reduction in situ. PMID:29562719

  5. The Exopolysaccharide Matrix

    Science.gov (United States)

    Koo, H.; Falsetta, M.L.; Klein, M.I.

    2013-01-01

    Many infectious diseases in humans are caused or exacerbated by biofilms. Dental caries is a prime example of a biofilm-dependent disease, resulting from interactions of microorganisms, host factors, and diet (sugars), which modulate the dynamic formation of biofilms on tooth surfaces. All biofilms have a microbial-derived extracellular matrix as an essential constituent. The exopolysaccharides formed through interactions between sucrose- (and starch-) and Streptococcus mutans-derived exoenzymes present in the pellicle and on microbial surfaces (including non-mutans) provide binding sites for cariogenic and other organisms. The polymers formed in situ enmesh the microorganisms while forming a matrix facilitating the assembly of three-dimensional (3D) multicellular structures that encompass a series of microenvironments and are firmly attached to teeth. The metabolic activity of microbes embedded in this exopolysaccharide-rich and diffusion-limiting matrix leads to acidification of the milieu and, eventually, acid-dissolution of enamel. Here, we discuss recent advances concerning spatio-temporal development of the exopolysaccharide matrix and its essential role in the pathogenesis of dental caries. We focus on how the matrix serves as a 3D scaffold for biofilm assembly while creating spatial heterogeneities and low-pH microenvironments/niches. Further understanding on how the matrix modulates microbial activity and virulence expression could lead to new approaches to control cariogenic biofilms. PMID:24045647

  6. Extracellular matrix production by human osteoblasts cultured on biodegradable polymers applicable for tissue engineering.

    Science.gov (United States)

    El-Amin, S F; Lu, H H; Khan, Y; Burems, J; Mitchell, J; Tuan, R S; Laurencin, C T

    2003-03-01

    The nature of the extracellular matrix (ECM) is crucial in regulating cell functions via cell-matrix interactions, cytoskeletal organization, and integrin-mediated signaling. In bone, the ECM is composed of proteins such as collagen (CO), fibronectin (FN), laminin (LM), vitronectin (VN), osteopontin (OP) and osteonectin (ON). For bone tissue engineering, the ECM should also be considered in terms of its function in mediating cell adhesion to biomaterials. This study examined ECM production, cytoskeletal organization, and adhesion of primary human osteoblastic cells on biodegradable matrices applicable for tissue engineering, namely polylactic-co-glycolic acid 50:50 (PLAGA) and polylactic acid (PLA). We hypothesized that the osteocompatible, biodegradable polymer surfaces promote the production of bone-specific ECM proteins in a manner dependent on polymer composition. We first examined whether the PLAGA and PLA matrices could support human osteoblastic cell growth by measuring cell adhesion at 3, 6 and 12h post-plating. Adhesion on PLAGA was consistently higher than on PLA throughout the duration of the experiment, and comparable to tissue culture polystyrene (TCPS). ECM components, including CO, FN, LM, ON, OP and VN, produced on the surface of the polymers were quantified by ELISA and localized by immunofluorescence staining. All of these proteins were present at significantly higher levels on PLAGA compared to PLA or TCPS surfaces. On PLAGA, OP and ON were the most abundant ECM components, followed by CO, FN, VN and LN. Immunofluorescence revealed an extracellular distribution for CO and FN, whereas OP and ON were found both intracellularly as well as extracellularly on the polymer. In addition, the actin cytoskeletal network was more extensive in osteoblasts cultured on PLAGA than on PLA or TCPS. In summary, we found that osteoblasts plated on PLAGA adhered better to the substrate, produced higher levels of ECM molecules, and showed greater cytoskeletal

  7. Cartilaginous extracellular matrix-modified chitosan hydrogels for cartilage tissue engineering.

    Science.gov (United States)

    Choi, Bogyu; Kim, Soyon; Lin, Brian; Wu, Benjamin M; Lee, Min

    2014-11-26

    Cartilaginous extracellular matrix (ECM) components such as type-II collagen (Col II) and chondroitin sulfate (CS) play a crucial role in chondrogenesis. However, direct clinical use of natural Col II or CS as scaffolds for cartilage tissue engineering is limited by their instability and rapid enzymatic degradation. Here, we investigate the incorporation of Col II and CS into injectable chitosan hydrogels designed to gel upon initiation by exposure to visible blue light (VBL) in the presence of riboflavin. Unmodified chitosan hydrogel supported proliferation and deposition of cartilaginous ECM by encapsulated chondrocytes and mesenchymal stem cells. The incorporation of native Col II or CS into chitosan hydrogels further increased chondrogenesis. The incorporation of Col II, in particular, was found to be responsible for the enhanced cellular condensation and chondrogenesis observed in modified hydrogels. This was mediated by integrin α10 binding to Col II, increasing cell-matrix adhesion. These findings demonstrate the potential of cartilage ECM-modified chitosan hydrogels as biomaterials to promote cartilage regeneration.

  8. Enhance and Maintain Chondrogenesis of Synovial Fibroblasts by Cartilage Extracellular Matrix Protein Matrilins

    Science.gov (United States)

    Pei, Ming; Luo, Junming; Chen, Qian

    2008-01-01

    Summary Objective Cartilage-specific extracellular matrix (ECM) proteins have been proposed to play key roles in modulating cellular phenotypes during chondrogenesis of mesenchymal stem cells. Matrilin (MATN) 1 and 3 are among the most up-regulated ECM proteins during chondrogenesis. The aim of this study was to analyze their roles in chondrogenesis of mesenchymal fibroblasts from synovium. Methods Primary synovial fibroblasts (SFBs) were purified from porcine synovium and incubated in pellet culture for 18 days. Chondrogenesis of SFB was analyzed by histological staining with safranin-O/fast green, and by quantifying glycosaminoglycans with dimethylmethylene blue assay. The mRNA levels of chondrogenic markers including collagen II, aggrecan, and Sox 9 were quantified by real-time RT-PCR, while the protein levels of Col II and matrilins were determined by western blot analysis. Results SFBs underwent chondrogenesis after incubation with TGF-β1 for three days; however, this process was attenuated during the subsequent incubation period. Expression of a MATN1 or 3 cDNA maintained and further enhanced chondrogenesis of SFBs as shown by increased cartilaginous matrix areas, elevated amount of glycosaminoglycans, and stimulated expression of chondrogenic markers. Conclusion Our findings suggest a novel function for MATN1 and 3 to maintain and enhance chondrogenesis of mesenchymal fibroblasts initiated by TGF-β. Our results also support a critical role of cartilage-specific ECM proteins to modulate cellular phenotypes in the microenvironment during chondrogenic differentiation. PMID:18282772

  9. Enhancing and maintaining chondrogenesis of synovial fibroblasts by cartilage extracellular matrix protein matrilins.

    Science.gov (United States)

    Pei, M; Luo, J; Chen, Q

    2008-09-01

    Cartilage-specific extracellular matrix (ECM) proteins have been proposed to play key roles in modulating cellular phenotypes during chondrogenesis of mesenchymal stem cells. Matrilin (MATN)1 and MATN3 are among the most up-regulated ECM proteins during chondrogenesis. The aim of this study was to analyze their roles in chondrogenesis of mesenchymal fibroblasts from synovium. Primary synovial fibroblasts (SFBs) were purified from porcine synovium and incubated in pellet culture for 18 days. Chondrogenesis of SFB was analyzed by histological staining with safranin-O/fast green, and by quantifying glycosaminoglycans (GAG) with dimethylmethylene blue assay. The mRNA levels of chondrogenic markers including collagen II, aggrecan, and Sox 9 were quantified by real-time reverse transcription polymerase chain reaction, while the protein levels of Col II and MATNs were determined by western blot analysis. SFBs underwent chondrogenesis after incubation with transforming growth factor-beta1 (TGF-beta1) for 3 days; however, this process was attenuated during the subsequent incubation period. Expression of a Matn1 or Matn3 cDNA maintained and further enhanced chondrogenesis of SFBs as shown by increased cartilaginous matrix areas, elevated amount of GAG, and stimulated expression of chondrogenic markers. Our findings suggest a novel function for MATN1 and MATN3 to maintain and enhance chondrogenesis of mesenchymal fibroblasts initiated by TGF-beta. Our results also support a critical role of cartilage-specific ECM proteins to modulate cellular phenotypes in the microenvironment during chondrogenic differentiation.

  10. A computational model of in vitro angiogenesis based on extracellular matrix fibre orientation.

    Science.gov (United States)

    Edgar, Lowell T; Sibole, Scott C; Underwood, Clayton J; Guilkey, James E; Weiss, Jeffrey A

    2013-01-01

    Recent interest in the process of vascularisation within the biomedical community has motivated numerous new research efforts focusing on the process of angiogenesis. Although the role of chemical factors during angiogenesis has been well documented, the role of mechanical factors, such as the interaction between angiogenic vessels and the extracellular matrix, remains poorly understood. In vitro methods for studying angiogenesis exist; however, measurements available using such techniques often suffer from limited spatial and temporal resolutions. For this reason, computational models have been extensively employed to investigate various aspects of angiogenesis. This paper outlines the formulation and validation of a simple and robust computational model developed to accurately simulate angiogenesis based on length, branching and orientation morphometrics collected from vascularised tissue constructs. Microvessels were represented as a series of connected line segments. The morphology of the vessels was determined by a linear combination of the collagen fibre orientation, the vessel density gradient and a random walk component. Excellent agreement was observed between computational and experimental morphometric data over time. Computational predictions of microvessel orientation within an anisotropic matrix correlated well with experimental data. The accuracy of this modelling approach makes it a valuable platform for investigating the role of mechanical interactions during angiogenesis.

  11. Increased extracellular matrix metalloproteinase inducer (EMMPRIN) expression in the conjunctival epithelium exposed to antiglaucoma treatments.

    Science.gov (United States)

    Labbé, Antoine; Gabison, Eric; Brignole-Baudouin, Françoise; Riancho, Luisa; Menashi, Suzanne; Baudouin, Christophe

    2015-01-01

    To analyze the effect of preserved antiglaucoma eye drops on the expression of extracellular matrix (ECM) metalloproteinase inducer (EMMPRIN) in conjunctival epithelial cells. A total of 18 patients treated for primary open-angle glaucoma with benzalkonium chloride (BAK) preserved eye drops and eight age-matched controls were included in this study. Glaucoma patients were divided into two groups according to their daily exposure to BAK: high-exposure (HE) group and low-exposure (LE) group. HLA-DR and EMMPRIN were quantified on conjunctival impression cytology specimens using flow cytometry. In parallel, IOBA-NHC conjunctival epithelial cells were exposed to different BAK concentrations, in the presence or absence of cyclosporine A (CsA), and their total and surface expressions of EMMPRIN were assessed by flow cytometry and results are given in relative fluorescence intensities (RFIs). Compared to the control group (1.71 ± 0.39 RFI), EMMPRIN was significantly increased in the HE (4.19 ± 1.50 RFI, p EMMPRIN (R(2) = 0.875, p EMMPRIN, which was proportional to the concentration of BAK. The surface expression of EMMPRIN was inhibited by CsA. The increased expression of EMMPRIN in patients topically treated with multiple antiglaucoma BAK-preserved eye drops suggests a matrix metalloproteinase-related modification of conjunctival ECM remodeling. In vitro results suggest that CsA has the potential to limit BAK effects on EMMPRIN.

  12. Culture temperature affects redifferentiation and cartilaginous extracellular matrix formation in dedifferentiated human chondrocytes.

    Science.gov (United States)

    Ito, Akira; Aoyama, Tomoki; Iijima, Hirotaka; Tajino, Junichi; Nagai, Momoko; Yamaguchi, Shoki; Zhang, Xiangkai; Kuroki, Hiroshi

    2015-05-01

    To date, there have been few studies on how temperature affects the phenotype and metabolism of human chondrocytes. Thus, the purpose of this study was to elucidate the effects of culture temperature on chondrocyte redifferentiation and extracellular matrix (ECM) formation using dedifferentiated mature human chondrocytes in vitro. Dedifferentiated chondrocytes were cultured in a pellet culture system for up to 21 days. The pellets were randomly divided into three groups with different culture temperature (32, 37, and 41°C). Chondrocyte redifferentiation and ECM formation were evaluated by wet weight, messenger ribonucleic acid (mRNA), histological, and biochemical analyses. The results showed that the wet weight and the mRNA expressions of collagen type II A1 and cartilage oligomeric matrix protein at 37°C were higher than the corresponding values at 32°C. The histological and biochemical analyses revealed that the syntheses of type II collagen and proteoglycan were promoted at 37°C compared to those at 32°C, whereas they were considerably inhibited at 41°C. In conclusion, the results obtained herein indicated that temperature affects chondrocyte redifferentiation and ECM formation, and modulation of temperature might thus represent an advantageous means to regulate the phenotype and biosynthetic activity of chondrocytes. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  13. Distinct profile of vascular progenitor attachment to extracellular matrix proteins in cancer patients.

    Science.gov (United States)

    Labonté, Laura; Li, Yuhua; Addison, Christina L; Brand, Marjorie; Javidnia, Hedyeh; Corsten, Martin; Burns, Kevin; Allan, David S

    2012-04-01

    Vascular progenitor cells (VPCs) facilitate angiogenesis and initiate vascular repair by homing in on sites of damage and adhering to extracellular matrix (ECM) proteins. VPCs also contribute to tumor angiogenesis and induce angiogenic switching in sites of metastatic cancer. In this study, the binding of attaching cells in VPC clusters that form in vitro on specific ECM proteins was investigated. VPC cluster assays were performed in vitro on ECM proteins enriched in cancer cells and in remodelling tissue. Profiles of VPC clusters from patients with cancer were compared to healthy controls. The role of VEGF and integrin-specific binding of angiogenic attaching cells was addressed. VPC clusters from cancer patients were markedly increased on fibronectin relative to other ECM proteins tested, in contrast to VPC clusters from control subjects, which formed preferentially on laminin. Specific integrin-mediated binding of attaching cells in VPC clusters was matrix protein-dependent. Furthermore, cancer patients had elevated plasma VEGF levels compared to healthy controls and VEGF facilitated preferential VPC cluster formation on fibronectin. Incubating cells from healthy controls with VEGF induced a switch from the 'healthy' VPC binding profile to the profile observed in cancer patients with a marked increase in VPC cluster formation on fibronectin. The ECM proteins laminin and fibronectin support VPC cluster formation via specific integrins on attaching cells and can facilitate patterns of VPC cluster formation that are distinct in cancer patients. Larger studies, however, are needed to gain insight on how tumor angiogenesis may differ from normal repair processes.

  14. Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I

    International Nuclear Information System (INIS)

    Palmieri, D.; Valli, M.; Viglio, S.; Ferrari, N.; Ledda, B.; Volta, C.; Manduca, P.

    2010-01-01

    Extracellular matrix (ECM) plays a fundamental role in angiogenesis affecting endothelial cells proliferation, migration and differentiation. Vessels-like network formation in vitro is a reliable test to study the inductive effects of ECM on angiogenesis. Here we utilized matrix deposed by osteoblasts as substrate where the molecular and structural complexity of the endogenous ECM is preserved, to test if it induces vessel-like network formation by endothelial cells in vitro. ECM is more similar to the physiological substrate in vivo than other substrates previously utilized for these studies in vitro. Osteogenic ECM, prepared in vitro from mature osteoblasts at the phase of maximal deposition and glycosylation of collagen I, induces EAhy926, HUVEC, and HDMEC endothelial cells to form vessels-like structures and promotes the activation of metalloproteinase-2 (MMP-2); the functionality of the p-38/MAPK signaling pathway is required. Osteogenic ECM also induces a transient increase of CXCL12 and a decrease of the receptor CXCR4. The induction of vessel-like networks is dependent from proper glycosylation of collagens and does not occur on osteogenic ECMs if deglycosylated by -galactosidase or on less glycosylated ECMs derived from preosteoblasts and normal fibroblasts, while is sustained on ECM from osteogenesis imperfecta fibroblasts only when their mutation is associated with over-glycosylation of collagen type I. These data support that post-translational glycosylation has a role in the induction in endothelial cells in vitro of molecules conductive to self-organization in vessels-like structures.

  15. Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I

    Energy Technology Data Exchange (ETDEWEB)

    Palmieri, D. [Genetics, DIBIO, University of Genova, Corso Europa 26, 16132 Genova (Italy); Valli, M.; Viglio, S. [Department of Biochemistry, University of Pavia (Italy); Ferrari, N. [Istituto Nazionale per la ricerca sul Cancro, Genova (Italy); Ledda, B.; Volta, C. [Genetics, DIBIO, University of Genova, Corso Europa 26, 16132 Genova (Italy); Manduca, P., E-mail: man-via@unige.it [Genetics, DIBIO, University of Genova, Corso Europa 26, 16132 Genova (Italy)

    2010-03-10

    Extracellular matrix (ECM) plays a fundamental role in angiogenesis affecting endothelial cells proliferation, migration and differentiation. Vessels-like network formation in vitro is a reliable test to study the inductive effects of ECM on angiogenesis. Here we utilized matrix deposed by osteoblasts as substrate where the molecular and structural complexity of the endogenous ECM is preserved, to test if it induces vessel-like network formation by endothelial cells in vitro. ECM is more similar to the physiological substrate in vivo than other substrates previously utilized for these studies in vitro. Osteogenic ECM, prepared in vitro from mature osteoblasts at the phase of maximal deposition and glycosylation of collagen I, induces EAhy926, HUVEC, and HDMEC endothelial cells to form vessels-like structures and promotes the activation of metalloproteinase-2 (MMP-2); the functionality of the p-38/MAPK signaling pathway is required. Osteogenic ECM also induces a transient increase of CXCL12 and a decrease of the receptor CXCR4. The induction of vessel-like networks is dependent from proper glycosylation of collagens and does not occur on osteogenic ECMs if deglycosylated by -galactosidase or on less glycosylated ECMs derived from preosteoblasts and normal fibroblasts, while is sustained on ECM from osteogenesis imperfecta fibroblasts only when their mutation is associated with over-glycosylation of collagen type I. These data support that post-translational glycosylation has a role in the induction in endothelial cells in vitro of molecules conductive to self-organization in vessels-like structures.

  16. Assembly of fibronectin into the extracellular matrix of early and late passage human skin fibroblasts

    International Nuclear Information System (INIS)

    Mann, D.M.

    1987-01-01

    The specific binding of soluble 125 I-human plasma fibronectin ( 125 I-HFN-P) to confluent cultures of early and late passage human skin fibroblasts was investigated. Previous studies HFN-P bound to fibroblast cell layers indicated that HNF-P was present in the cultures in two separate pools, distinguishable on the basis of their solubility in 1% deoxycholate. Examination of the kinetics of 125 I-HFN-P binding to Pool I of early and late passage cultures revealed that both cultures required 2-4 h to approach steady-state conditions. Other kinetic studies showed that the rates of low of 125 I-HFN-P from either Pool I or Pool II were similar for both cultures. Further, Scatchard analysis revealed a single class of Pool I binding sites with apparent dissociation constants (K/sub d/) of 5.3 x 10 -8 M (early passage) and 4.2 x 10 -8 M (late passage). These results indicate that early and late passage cultures of human fibroblasts exhibit differences in the number of cell surface biding sites for soluble fibronectin, and in the extent to which they incorporate soluble fibronectin into the extracellular matrix. Parameters which affect the fibronectin matrix assembly system of human skin fibroblasts were also examined. In addition, several monoclonal anti-fibronectin antibodies were characterized and developed as experimental probes for fibronectin structure and function

  17. The endogenous fluorescence of fibroblast in collagen gels as indicator of stiffness of the extracellular matrix

    Science.gov (United States)

    Padilla-Martinez, J. P.; Ortega-Martinez, A.; Franco, W.

    2016-03-01

    The stiffness or rigidity of the extracellular matrix (ECM) regulates cell response. Established mechanical tests to measure stiffness, such as indentation and tensile tests, are invasive and destructive to the sample. Endogenous or native molecules to cells and ECM components, like tryptophan and cross-links of collagen, display fluorescence upon irradiation with ultraviolet light. Most likely, the concentration of these endogenous fluorophores changes as the stiffness of the ECM changes. In this work we investigate the endogenous fluorescence of collagen gels containing fibroblasts as a non-invasive non-destructive method to measure stiffness of the ECM. Human fibroblast cells were cultured in three-dimensional gels of type I collagen (50,000 cells/ml). This construct is a simple model of tissue contraction. During contraction, changes in the excitation-emission matrix (a fluorescence map in the 240-520/290-530 nm range) of constructs were measured with a spectrofluoremeter, and changes in stiffness were measured with a standard indentation test over 16 days. Results show that a progressive increase in fluorescence of the 290/340 nm excitation-emission pair correlates with a progressive increase in stiffness (r=0.9, α=0.5). The fluorescence of this excitation-emission pair is ascribed to tryptophan and variations in the fluorescence of this pair correlate with cellular proliferation. In this tissue model, the endogenous functional fluorescence of proliferating fibroblast cells is a biomechanical marker of stiffness of the ECM.

  18. Periostin is an extracellular matrix protein required for eruption of incisors in mice

    International Nuclear Information System (INIS)

    Kii, Isao; Amizuka, Norio; Minqi, Li; Kitajima, Satoshi; Saga, Yumiko; Kudo, Akira

    2006-01-01

    A characteristic tooth of rodents, the incisor continuously grows throughout life by the constant formation of dentin and enamel. Continuous eruption of the incisor is accompanied with formation of shear zone, in which the periodontal ligament is remodeled. Although the shear zone plays a role in the remodeling, its molecular biological aspect is barely understood. Here, we show that periostin is essential for formation of the shear zone. Periostin -/- mice showed an eruption disturbance of incisors. Histological observation revealed that deletion of periostin led to disappearance of the shear zone. Electron microscopy revealed that the disappearance of the shear zone resulted from a failure in digestion of collagen fibers in the periostin -/- mice. Furthermore, immunohistochemical analysis using anti-periostin antibodies demonstrated the restricted localization of periostin protein in the shear zone. Periostin is an extracellular matrix protein, and immunoelectron microscopy showed a close association of periostin with collagen fibrils in vivo. These results suggest that periostin functions in the remodeling of collagen matrix in the shear zone

  19. Methods for the visualization and analysis of extracellular matrix protein structure and degradation.

    Science.gov (United States)

    Leonard, Annemarie K; Loughran, Elizabeth A; Klymenko, Yuliya; Liu, Yueying; Kim, Oleg; Asem, Marwa; McAbee, Kevin; Ravosa, Matthew J; Stack, M Sharon

    2018-01-01

    This chapter highlights methods for visualization and analysis of extracellular matrix (ECM) proteins, with particular emphasis on collagen type I, the most abundant protein in mammals. Protocols described range from advanced imaging of complex in vivo matrices to simple biochemical analysis of individual ECM proteins. The first section of this chapter describes common methods to image ECM components and includes protocols for second harmonic generation, scanning electron microscopy, and several histological methods of ECM localization and degradation analysis, including immunohistochemistry, Trichrome staining, and in situ zymography. The second section of this chapter details both a common transwell invasion assay and a novel live imaging method to investigate cellular behavior with respect to collagen and other ECM proteins of interest. The final section consists of common electrophoresis-based biochemical methods that are used in analysis of ECM proteins. Use of the methods described herein will enable researchers to gain a greater understanding of the role of ECM structure and degradation in development and matrix-related diseases such as cancer and connective tissue disorders. © 2018 Elsevier Inc. All rights reserved.

  20. Attenuation of endocrine-exocrine pancreatic communication in type 2 diabetes: pancreatic extracellular matrix ultrastructural abnormalities.

    Science.gov (United States)

    Hayden, Melvin R; Patel, Kamlesh; Habibi, Javad; Gupta, Deepa; Tekwani, Seema S; Whaley-Connell, Adam; Sowers, James R

    2008-01-01

    Ultrastructural observations reveal a continuous interstitial matrix connection between the endocrine and exocrine pancreas, which is lost due to fibrosis in rodent models and humans with type 2 diabetes mellitus (T2DM). Widening of the islet-exocrine interface appears to result in loss of desmosomes and adherens junctions between islet and acinar cells and is associated with hypercellularity consisting of pericytes and inflammatory cells in T2DM pancreatic tissue. Organized fibrillar collagen was closely associated with pericytes, which are known to differentiate into myofibroblasts-pancreatic stellate cells. Of importance, some pericyte cellular processes traverse both the connecting islet-exocrine interface and the endoacinar interstitium of the exocrine pancreas. Loss of cellular paracrine communication and extracellular matrix remodeling fibrosis in young animal models and humans may result in a dysfunctional insulino-acinar-ductal-incretin gut hormone axis, resulting in pancreatic insufficiency and glucagon-like peptide deficiency, which are known to exist in prediabetes and overt T2DM in humans.

  1. Hypoxic stellate cells of pancreatic cancer stroma regulate extracellular matrix fiber organization and cancer cell motility.

    Science.gov (United States)

    Sada, Masafumi; Ohuchida, Kenoki; Horioka, Kohei; Okumura, Takashi; Moriyama, Taiki; Miyasaka, Yoshihiro; Ohtsuka, Takao; Mizumoto, Kazuhiro; Oda, Yoshinao; Nakamura, Masafumi

    2016-03-28

    Desmoplasia and hypoxia in pancreatic cancer mutually affect each other and create a tumor-supportive microenvironment. Here, we show that microenvironment remodeling by hypoxic pancreatic stellate cells (PSCs) promotes cancer cell motility through alteration of extracellular matrix (ECM) fiber architecture. Three-dimensional (3-D) matrices derived from PSCs under hypoxia exhibited highly organized parallel-patterned matrix fibers compared with 3-D matrices derived from PSCs under normoxia, and promoted cancer cell motility by inducing directional migration of cancer cells due to the parallel fiber architecture. Microarray analysis revealed that procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) in PSCs was the gene that potentially regulates ECM fiber architecture under hypoxia. Stromal PLOD2 expression in surgical specimens of pancreatic cancer was confirmed by immunohistochemistry. RNA interference-mediated knockdown of PLOD2 in PSCs blocked parallel fiber architecture of 3-D matrices, leading to decreased directional migration of cancer cells within the matrices. In conclusion, these findings indicate that hypoxia-induced PLOD2 expression in PSCs creates a permissive microenvironment for migration of cancer cells through architectural regulation of stromal ECM in pancreatic cancer. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  2. Genetic Background is a Key Determinant of Glomerular Extracellular Matrix Composition and Organization.

    Science.gov (United States)

    Randles, Michael J; Woolf, Adrian S; Huang, Jennifer L; Byron, Adam; Humphries, Jonathan D; Price, Karen L; Kolatsi-Joannou, Maria; Collinson, Sophie; Denny, Thomas; Knight, David; Mironov, Aleksandr; Starborg, Toby; Korstanje, Ron; Humphries, Martin J; Long, David A; Lennon, Rachel

    2015-12-01

    Glomerular disease often features altered histologic patterns of extracellular matrix (ECM). Despite this, the potential complexities of the glomerular ECM in both health and disease are poorly understood. To explore whether genetic background and sex determine glomerular ECM composition, we investigated two mouse strains, FVB and B6, using RNA microarrays of isolated glomeruli combined with proteomic glomerular ECM analyses. These studies, undertaken in healthy young adult animals, revealed unique strain- and sex-dependent glomerular ECM signatures, which correlated with variations in levels of albuminuria and known predisposition to progressive nephropathy. Among the variation, we observed changes in netrin 4, fibroblast growth factor 2, tenascin C, collagen 1, meprin 1-α, and meprin 1-β. Differences in protein abundance were validated by quantitative immunohistochemistry and Western blot analysis, and the collective differences were not explained by mutations in known ECM or glomerular disease genes. Within the distinct signatures, we discovered a core set of structural ECM proteins that form multiple protein-protein interactions and are conserved from mouse to man. Furthermore, we found striking ultrastructural changes in glomerular basement membranes in FVB mice. Pathway analysis of merged transcriptomic and proteomic datasets identified potential ECM regulatory pathways involving inhibition of matrix metalloproteases, liver X receptor/retinoid X receptor, nuclear factor erythroid 2-related factor 2, notch, and cyclin-dependent kinase 5. These pathways may therefore alter ECM and confer susceptibility to disease. Copyright © 2015 by the American Society of Nephrology.

  3. A study of extracellular matrix remodeling in aortic heart valves using a novel biaxial stretch bioreactor.

    Science.gov (United States)

    Lei, Ying; Masjedi, Shirin; Ferdous, Zannatul

    2017-11-01

    In aortic valves, biaxial cyclic stretch is known to modulate cell differentiation, extracellular matrix (ECM) synthesis and organization. We designed a novel bioreactor that can apply independent and precise stretch along radial and circumferential directions in a tissue culture environment. While this bioreactor can be used for either native or engineered tissues, this study determined matrix remodeling and strain distribution of aortic cusps after culturing under biaxial stretch for 14 days. The contents of collagen and glycosaminoglycans were determined using standard biochemical assays and compared with fresh controls. Strain fields in static cusps were more uniform than those in stretched cusps, which indicated degradation of the ECM fibers. The glycosaminoglycan content was significantly elevated in the static control as compared to fresh or stretched cusps, but no difference was observed in collagen content among the groups. The strain profile of freshly isolated fibrosa vs. ventricularis and left, right, and noncoronary cusps were also determined by Digital Image Correlation technique. Distinct strain patterns were observed under stretch on fibrosa and ventricularis sides and among the three cusps. This work highlights the critical role of the anisotropic ECM structure for proper functions of native aortic valves and the beneficial effects of biaxial stretch for maintenance of the native ECM structure. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Changes in p53 expression in mouse fibroblasts can modify motility and extracellular matrix organization.

    Science.gov (United States)

    Alexandrova, A; Ivanov, A; Chumakov, P; Kopnin, B; Vasiliev, J

    2000-11-23

    Effects of p53 expression on cell morphology and motility were studied using the derivatives of p53-null 10(1) mouse fibroblasts with tetracycline-regulated expression of exogenous human p53. Induction of p53 expression was accompanied by significant decrease in extracellular matrix (fibronectin) and reduction of matrix fibrils, diminution of the number and size of focal contacts, decrease of cell areas, establishment of more elongated cell shape and alterations of actin cytoskeleton (actin bundles became thinner, their number and size decreased). Expression of His175 and Gln22/ Ser23 p53 mutants caused no such effects. To study the influence of p53 expression on cell motility we used wound technique and videomicroscopy observation of single living cells. It was found that induction of p53 expression led to increase of lamellar activity of cell edge. However, in spite of enhanced lamellar activity p53-expressing cells migrated to shorter distance and filled the narrow wound in longer time as compared with their p53-null counterparts. Possible mechanisms of the influence of p53 expression on cell morphology and motility are discussed.

  5. Sticking together: building a biofilm the Bacillus subtilis way

    Science.gov (United States)

    Vlamakis, Hera; Chai, Yunrong; Beauregard, Pascale; Losick, Richard; Kolter, Roberto

    2014-01-01

    Preface Biofilms are ubiquitous communities of tightly associated bacteria encased in an extracellular matrix. Bacillus subtilis has long-served as a robust model organism to examine the molecular mechanisms of biofilm formation and a number of studies have revealed that this process is subject to a number of integrated regulatory pathways. In this Review, we focus on the molecular mechanisms controlling biofilm assembly and briefly summarize the current state of knowledge regarding their disassembly. We also discuss recent progress that has expanded our understanding of biofilm formation on plant roots, which are a natural habitat for this soil bacterium. PMID:23353768

  6. The regulation of growth and metabolism of kidney stem cell with regional specificity using extracellular matrix derived from kidney

    OpenAIRE

    O’Neill, John D.; Freytes, Donald O.; Anandappa, Annabelle; Oliver, Juan A.; Vunjak-Novakovic, Gordana

    2013-01-01

    Native extracellular matrix (ECM) that is secreted and maintained by resident cells is of great interest for cell culture and cell delivery. We hypothesized that specialized bioengineered niches for stem cells can be established using ECM-derived scaffolding materials. Kidney was selected as a model system because of the high regional diversification of renal tissue matrix. By preparing the ECM from three specialized regions of the kidney (cortex, medulla, and papilla; whole kidney, heart, an...

  7. Charakterisierung des Extracellular Matrix Metalloproteinase Inducer (EMMPRIN/CD147) auf Thrombozyten und Untersuchung zur funktionellen Relevanz bei der Arteriosklerose

    OpenAIRE

    Fischel, Sina

    2007-01-01

    Der „Extracellular Matrix Metalloproteinase Inducer“ EMMPRIN ist bisher im Wesentlichen bekannt aus der Tumorpathologie; er induziert in umliegenden Fibroblasten eine Aktivierung der Matrix Metalloproteinasen (MMPs). Die Beteiligung von EMMPRIN am arteriosklerotischen Geschehen konnte in früheren Untersuchungen durch den Nachweis der EMMPRIN-Expression in verschiedenen kardiovaskulären Zellen wie Monozyten, Endothelzellen und glatten Muskelzellen in der arteriosklerotischen Plaque erbrach...

  8. Susceptibility of Staphylococcus aureus biofilms to reactive discharge gases.

    Science.gov (United States)

    Traba, Christian; Liang, Jun F

    2011-08-01

    Formation of bacterial biofilms at solid-liquid interfaces creates numerous problems in both industrial and biomedical sciences. In this study, the susceptibility of Staphylococcus aureus biofilms to discharge gas generated from plasma was tested. It was found that despite distinct chemical/physical properties, discharge gases from oxygen, nitrogen, and argon demonstrated very potent and almost the same anti-biofilm activity. The bacterial cells in S. aureus biofilms were killed (>99.9%) by discharge gas within minutes of exposure. Under optimal experimental conditions, no bacteria and biofilm re-growth from discharge gas treated biofilms was found. Further studies revealed that the anti-biofilm activity of the discharge gas occurred by two distinct mechanisms: (1) killing bacteria in biofilms by causing severe cell membrane damage, and (2) damaging the extracellular polymeric matrix in the architecture of the biofilm to release biofilm from the surface of the solid substratum. Information gathered from this study provides an insight into the anti-biofilm mechanisms of plasma and confirms the applications of discharge gas in the treatment of biofilms and biofilm related bacterial infections.

  9. Extracellular matrix biomimicry for the creation of investigational and therapeutic devices.

    Science.gov (United States)

    Pellowe, Amanda S; Gonzalez, Anjelica L

    2016-01-01

    The extracellular matrix (ECM) is a web of fibrous proteins that serves as a scaffold for tissues and organs, and is important for maintaining homeostasis and facilitating cellular adhesion. Integrin transmembrane receptors are the primary adhesion molecules that anchor cells to the ECM, thus integrating cells with their microenvironments. Integrins play a critical role in facilitating cell-matrix interactions and promoting signal transduction, both from the cell to the ECM and vice versa, ultimately mediating cell behavior. For this reason, many advanced biomaterials employ biomimicry by replicating the form and function of fibrous ECM proteins. The ECM also acts as a reservoir for small molecules and growth factors, wherein fibrous proteins directly bind and present these bioactive moieties that facilitate cell activity. Therefore biomimicry can be enhanced by incorporating small molecules into ECM-like substrates. Biomimetic ECM materials have served as invaluable research tools for studying interactions between cells and the surrounding ECM, revealing that cell-matrix signaling is driven by mechanical forces, integrin engagement, and small molecules. Mimicking pathological ECMs has also elucidated disease specific cell behaviors. For example, biomimetic tumor microenvironments have been used to induce metastatic cell behaviors, and have thereby shown promise for in vitro cancer drug testing and targeting. Further, ECM-like substrates have been successfully employed for autologous cell recolonization for tissue engineering and wound healing. As we continue to learn more about the mechanical and biochemical characteristics of the ECM, these properties can be harnessed to develop new biomaterials, biomedical devices, and therapeutics. © 2015 Wiley Periodicals, Inc.

  10. Bioengineered Bruch's-like extracellular matrix promotes retinal pigment epithelial differentiation

    Directory of Open Access Journals (Sweden)

    Samuel McLenachan

    2017-07-01

    Full Text Available In the eye, the retinal pigment epithelium (RPE adheres to a complex protein matrix known as Bruch's membrane (BrM. The aim of this study was to provide enriched conditions for RPE cell culture through the production of a BrM-like matrix. Our hypothesis was that a human RPE cell line would deposit an extracellular matrix (ECM resembling BrM. The composition and structure of ECM deposited by ARPE19 cells (ARPE19-ECM was characterized. To produce ARPE19-ECM, ARPE19 cells were cultured in the presence dextran sulphate. ARPE19-ECM was decellularized using deoxycholate and characterized by immunostaining and western blot analysis. Primary human RPE and induced pluripotent stem cells were seeded onto ARPE19-ECM or geltrex coated surfaces and examined by microscopy or RT-PCR. Culture of ARPE19 cells with dextran sulphate promoted nuclear localization of SOX2, formation of tight junctions and deposition of ECM. ARPE19 cells deposited ECM proteins found in the inner layers of BrM, including fibronectin, vitronectin, collagens IV and V as well as laminin-alpha-5, but not those found in the middle elastic layer (elastin or the outer layers (collagen VI. ARPE19-ECM promoted pigmentation in human RPE and pluripotent stem cell cultures. Expression of RPE65 was significantly increased on ARPE19-ECM compared with geltrex in differentiating pluripotent stem cell cultures. ARPE19 cells deposit ECM with a composition and structure similar to BrM in the retina. Molecular cues present in ARPE19-ECM promote the acquisition and maintenance of the RPE phenotype. Together, these results demonstrate a simple method for generating a BrM-like surface for enriched RPE cell cultures.

  11. Differences in extracellular matrix proteins between Friesian horses with aortic rupture, unaffected Friesians and Warmblood horses.

    Science.gov (United States)

    Ploeg, M; Gröne, A; van de Lest, C H A; Saey, V; Duchateau, L; Wolsein, P; Chiers, K; Ducatelle, R; van Weeren, P R; de Bruijn, M; Delesalle, C

    2017-09-01

    Unlike in Warmblood horses, aortic rupture is quite common in Friesian horses, in which a hereditary trait is suspected. The aortic connective tissue in affected Friesians shows histological changes such as medial necrosis, elastic fibre fragmentation, mucoid material accumulation and fibrosis with aberrant collagen morphology. However, ultrastructural examination of the collagen fibres of the mid-thoracic aorta has been inconclusive in further elucidating the pathogenesis of the disease. To assess several extracellular matrix (ECM) components biochemically in order to explore a possible underlying breed-related systemic ECM defect in Friesians with aortic rupture. Cadaver study. Tissues from affected Friesians (n = 18), unaffected Friesians (n = 10) and Warmblood horses (n = 30) were compared. Samples were taken from the thoracic aorta at the level of the rupture site, from two locations caudal to the rupture and from the deep digital flexor tendon. Total collagen content, post-translational modifications of collagen formation including lysine hydroxylation, and hydroxylysylpyridinoline (HP), lysylpyridinoline (LP) and pyrrole cross-links were analysed. Additionally, elastin cross-links, glycosaminoglycan content and matrix metalloproteinase (MMP) activity were assessed. Significantly increased MMP activity and increased LP and HP cross-linking, lysine hydroxylation and elastin cross-linking were found at the site of rupture in affected Friesians. These changes may reflect processes involved in healing and aneurysm formation. Unaffected Friesians had less lysine hydroxylation and pyrrole cross-linking within the tendons compared with Warmblood horses. No differences in the matrix of the aorta were found between normal Warmbloods and Friesian horses. Small sample size. The differences in collagen parameters in tendon tissue may reflect differences in connective tissue metabolism between Friesians and Warmblood horses. © 2017 EVJ Ltd.

  12. Morphological and ultrastructural characteristics of extracellular matrix changes in oral squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Usha Agrawal

    2011-01-01

    Full Text Available Background: The biology of oral squamous cell carcinoma (OSCC, including its progression from dysplasia to carcinoma, "field effects", genetic changes in tumor associated mucosa (TAM and effect of matrix metalloproteinases in breaking down of matrix proteins to facilitate invasion, has been well documented. However, what remains to be done is to extrapolate this knowledge to improve patient care. Aim: The aim of this study was to observe the extracellular matrix (ECM changes with the routine histochemical stains available to most histopathologists. Materials and Methods: The study includes 72 cases of OSCC in which the tumor and adjacent normal appearing areas were sampled to study the ECM changes with hematoxylin and eosin (H and E and Verhoeff′s-Van Gieson elastic stain (VVG. Results: Basophilic fragmentation of collagen (H and E and clumped short elastic fibers (VVG were seen in 12 (16.7% cases. Of the remaining cases, 18 (25% had a dense lymphocytic infiltrate and had no demonstrable elastic fibers. Those cases with H and E changes were further studied and compared with normal mucosa for ultrastructural changes. The ultrastructural study demonstrated an increase in oxytalan, elaunin and elastic fibers and decrease in collagen fibers with some transformation changes associated with OSCCs and lymph node metastasis. Conclusion: Changes in transformation of collagen to elastic fibers and also the loss of both the fibers in areas of lymphocytic infiltration possibly indicate degradation of ECM fibers by factors released from the lymphocytes or tumor cells and the limiting effect on the tumor by ECM remodeling.

  13. The influence of an in vitro generated bone-like extracellular matrix on osteoblastic gene expression of marrow stromal cells.

    NARCIS (Netherlands)

    Pham, Q.P.; Kasper, F.K.; Baggett, L.S.; Raphael, R.M.; Jansen, J.A.; Mikos, A.G.

    2008-01-01

    The function and development of cells rely heavily on the signaling interactions with the surrounding extracellular matrix (ECM). Therefore, a tissue engineering scaffold should mimic native ECM to recreate the in vivo environment. Previously, we have shown that an in vitro generated ECM secreted by

  14. Organization of the expanded cumulus-extracellular matrix in preovulatory follicles: arole for inter-alpha-trypsin inhibitor.

    Czech Academy of Sciences Publication Activity Database

    Nagyová, Eva

    2015-01-01

    Roč. 49, č. 1 (2015), s. 37-45 ISSN 1210-0668 R&D Projects: GA ČR GA305/05/0960 Institutional support: RVO:67985904 Keywords : cumulus expansion * cumulus-extracellular matrix * hyaluronan Subject RIV: ED - Physiology

  15. Influence of the temporal deposition of extracellular matrix on the mechanical properties of tissue-engineered cartilage

    NARCIS (Netherlands)

    Khoshgoftar, M.; Wilson, W.; Ito, K.; Donkelaar, van C.C.

    2014-01-01

    Enhancement of the load-bearing capacity of tissue engineered (TE) cartilage is expected to improve the clinical outcome of implantations. Generally, cartilage TE studies aim to increase the total extracellular matrix (ECM) content to improve implant mechanical properties. Besides the ECM content,

  16. Investigating the role of the extracellular matrix on differentiation of human mesenchymal stem cells and MC3T3 cells

    NARCIS (Netherlands)

    Fernandes, H.A.M.; Dechering, Koen; Someren, Eugene; van Blitterswijk, Clemens; de Boer, Jan

    2008-01-01

    Human mesenchymal stem cells (hMSCs) are a promising cell source for bone tissue engineering, but due to their limited number and donor variation, other cell types are used to answer relevant questions in bone tissue engineering. Since the extracellular matrix (ECM) is a complex entity with

  17. Disentangling the multifactorial contributions of fibronectin, collagen and cyclic strain on MMP expression and extracellular matrix remodeling by fibroblasts

    NARCIS (Netherlands)

    Zhang, Y.; Lin, Z.; Foolen, J.; Schoen, I.; Santoro, A.; Zenobi-Wong, M.; Vogel, Viola

    2014-01-01

    Early wound healing is associated with fibroblasts assembling a provisional fibronectin-rich extracellular matrix (ECM), which is subsequently remodeled and interlaced by type I collagen. This exposes fibroblasts to time-variant sets of matrices during different stages of wound healing. Our goal was

  18. Extracellular matrix components supporting human islet function in alginate-based immunoprotective microcapsules for treatment of diabetes

    NARCIS (Netherlands)

    Llacua Carrasco, Luis; de Haan, Bart J; Smink, Sandra A; de Vos, Paul

    In the pancreas, extracellular matrix (ECM) components play an import role in providing mechanical and physiological support, and also contribute to the function of islets. These ECM-connections are damaged during islet-isolation from the pancreas and are not fully recovered after encapsulation and

  19. Extracellular matrix of the human aortic media: an ultrastructural histochemical and immunohistochemical study of the adult aortic media

    NARCIS (Netherlands)

    Dingemans, K. P.; Teeling, P.; Lagendijk, J. H.; Becker, A. E.

    2000-01-01

    Aortic distensability is the key to normal aortic function and relates to the lamellar unit in the media. However, the organization of the extracellular matrix components in these lamellar units, which are largely responsible for the distensability, is insufficiently known, especially in the human.

  20. Biomimetics of the extracellular matrix: an integrated three-dimensional fiber-hydrogel composite for cartilage tissue engineering

    NARCIS (Netherlands)

    Coburn, J.; Gibson, M.; Bandalini, P.A.; Laird, C.; Mao, H.Q.; Moroni, Lorenzo; Seliktar, D.; Elisseeff, J.H.

    2011-01-01

    The native extracellular matrix (ECM) consists of an integrated fibrous protein network and proteoglycan-based ground (hydrogel) substance. We designed a novel electrospinning technique to engineer a three dimensional fiber-hydrogel composite that mimics the native ECM structure, is injectable, and

  1. Proliferation and extracellular matrix synthesis of smooth muscle cells cultured from human coronary atherosclerotic and restenotic lesions

    NARCIS (Netherlands)

    D.C. MacLeod (Donald); B.H. Strauss (Bradley); J. Escaned (Javier); V.A.W.M. Umans (Victor); R-J. van Suylen (Robert-Jan); A. Verkerk (Anton); P.J. de Feyter (Pim); P.W.J.C. Serruys (Patrick); M. de Jong (Marcel)

    1994-01-01

    textabstractOBJECTIVES. The purpose of this study was to examine the proliferative capacity and extracellular matrix synthesis of human coronary plaque cells in vitro. BACKGROUND. Common to both primary atherosclerosis and restenosis are vascular smooth muscle cell proliferation and production of

  2. ADAM12 induces actin cytoskeleton and extracellular matrix reorganization during early adipocyte differentiation by regulating beta1 integrin function

    DEFF Research Database (Denmark)

    Kawaguchi, Nobuko; Sundberg, Christina; Kveiborg, Marie

    2003-01-01

    -100 from cells overexpressing ADAM12 than from control cells. Collectively, these results show that surface expression of ADAM12 impairs the function of beta1 integrins and, consequently, alters the organization of the actin cytoskeleton and extracellular matrix. These events may be necessary...

  3. Influence of tissue- and cell-scale extracellular matrix distribution on the mechanical properties of tissue-engineered cartilage

    NARCIS (Netherlands)

    Khoshgoftar, M.; Wilson, W.; Ito, K.; Donkelaar, C.C. van

    2013-01-01

    The insufficient load-bearing capacity of today's tissue- engineered (TE) cartilage limits its clinical application. Generally, cartilage TE studies aim to increase the extracellular matrix (ECM) content, as this is thought to determine the load-bearing properties of the cartilage. However, there

  4. Influence of tissue- and cell-scale extracellular matrix distribution on the mechanical properties of tissue engineered cartilage

    NARCIS (Netherlands)

    Khoshgoftar, M.; Wilson, W.; Ito, K.; Donkelaar, van C.C.

    2013-01-01

    The insufficient load-bearing capacity of today’s tissue- engineered (TE) cartilage limits its clinical application. Generally, cartilage TE studies aim to increase the extracellular matrix (ECM) content, as this is thought to determine the load-bearing properties of the cartilage. However, there

  5. Electric field stimulation through a substrate influences Schwann cell and extracellular matrix structure

    Science.gov (United States)

    Nguyen, Hieu T.; Wei, Claudia; Chow, Jacqueline K.; Nguy, Lindsey; Nguyen, Hieu K.; Schmidt, Christine E.

    2013-08-01

    Objective. Electric field (EF) stimulation has been used to cue cell growth for tissue engineering applications. In this study, we explore the electrical parameters and extracellular mechanisms that elicit changes in cell behavior when stimulated through the substrate. Approach. Rat Schwann cell morphology was compared when exposed to EF through the media or a conductive indium tin oxide substrate. Ionic and structural effects were then analyzed on Matrigel and collagen I, respectively. Main results. When stimulating through media, cells had greater alignment perpendicular to the EF with higher current densities (106 mA cm-2 at 245 mV mm-1), and reached maximum alignment within 8 h. Stimulation through the substrate with EF (up to 110 mV mm-1) did not affect Schwann cell orientation, however the EF caused extracellular matrix (ECM) coatings on substrates to peel away, suggesting EF can physically change the ECM. Applying alternating current (ac) 2-1000 Hz signals through the media or substrate both caused cells to flatten and protrude many processes, without preferential alignment. Matrigel exposed to a substrate EF of 10 mV mm-1 for 2 h had a greater calcium concentration near the cathode, but quickly dissipated when the EF was removed. Schwann cells seeded 7 d after gels were exposed to substrate EF still aligned perpendicular to the EF direction. Microscopy of collagen I exposed to substrate EF shows alignment and bundling of fibrils. Significance. These findings demonstrate EF exposure can control Schwann cell alignment and morphology, change ECM bulk/surface architecture, and align ECM structures.

  6. Age and SPARC Change the Extracellular Matrix Composition of the Left Ventricle

    Directory of Open Access Journals (Sweden)

    Lisandra E. de Castro Brás

    2014-01-01

    Full Text Available Secreted protein acidic and rich in cysteine (SPARC, a collagen-binding matricellular protein, has been implicated in procollagen processing and deposition. The aim of this study was to investigate age- and SPARC-dependent changes in protein composition of the cardiac extracellular matrix (ECM. We studied 6 groups of mice (n=4/group: young (4-5 months old, middle-aged (11-12 m.o., and old (18–29 m.o. C57BL/6J wild type (WT and SPARC null. The left ventricle (LV was decellularized to enrich for ECM proteins. Protein extracts were separated by SDS-PAGE, digested in-gel, and analyzed by HPLC-ESI-MS/MS. Relative quantification was performed by spectral counting, and changes in specific proteins were validated by immunoblotting. We identified 321 proteins, of which 44 proteins were extracellular proteins. Of these proteins, collagen III levels were lower in the old null mice compared to WT, suggestive of a role for SPARC in collagen deposition. Additionally, fibrillin showed a significant increase in the null middle-aged group, suggestive of increased microfibril deposition in the absence of SPARC. Collagen VI increased with age in both genotypes (>3-fold, while collagen IV showed increased age-associated levels only in the WT animals (4-fold, P<0.05. These changes may explain the previously reported age-associated increases in LV stiffness. In summary, our data suggest SPARC is a possible therapeutic target for aging induced LV dysfunction.

  7. Development of an acellular tumor extracellular matrix as a three-dimensional scaffold for tumor engineering.

    Directory of Open Access Journals (Sweden)

    Wei-Dong Lü

    Full Text Available Tumor engineering is defined as the construction of three-dimensional (3D tumors in vitro with tissue engineering approaches. The present 3D scaffolds for tumor engineering have several limitations in terms of structure and function. To get an ideal 3D scaffold for tumor culture, A549 human pulmonary adenocarcinoma cells were implanted into immunodeficient mice to establish xenotransplatation models. Tumors were retrieved at 30-day implantation and sliced into sheets. They were subsequently decellularized by four procedures. Two decellularization methods, Tris-Trypsin-Triton multi-step treatment and sodium dodecyl sulfate (SDS treatment, achieved complete cellular removal and thus were chosen for evaluation of histological and biochemical properties. Native tumor tissues were used as controls. Human breast cancer MCF-7 cells were cultured onto the two 3D scaffolds for further cell growth and growth factor secretion investigations, with the two-dimensional (2D culture and cells cultured onto the Matrigel scaffolds used as controls. Results showed that Tris-Trypsin-Triton multi-step treated tumor sheets had well-preserved extracellular matrix structures and components. Their porosity was increased but elastic modulus was decreased compared with the native tumor samples. They supported MCF-7 cell repopulation and proliferation, as well as expression of growth factors. When cultured within the Tris-Trypsin-Triton treated scaffold, A549 cells and human colorectal adenocarcinoma cells (SW-480 had similar behaviors to MCF-7 cells, but human esophageal squamous cell carcinoma cells (KYSE-510 had a relatively slow cell repopulation rate. This study provides evidence that Tris-Trypsin-Triton treated acellular tumor extracellular matrices are promising 3D scaffolds with ideal spatial arrangement, biomechanical properties and biocompatibility for improved modeling of 3D tumor microenvironments.

  8. Load-dependent extracellular matrix organization in atrioventricular heart valves: differences and similarities.

    Science.gov (United States)

    Alavi, S Hamed; Sinha, Aditi; Steward, Earl; Milliken, Jeffrey C; Kheradvar, Arash

    2015-07-15

    The extracellular matrix of the atrioventricular (AV) valves' leaflets has a key role in the ability of these valves to properly remodel in response to constantly varying physiological loads. While the loading on mitral and tricuspid valves is significantly different, no information is available on how collagen fibers change their orientation in response to these loads. This study delineates the effect of physiological loading on AV valves' leaflets microstructures using Second Harmonic Generation (SHG) microscopy. Fresh natural porcine tricuspid and mitral valves' leaflets (n = 12/valve type) were cut and prepared for the experiments. Histology and immunohistochemistry were performed to compare the microstructural differences between the valves. The specimens were imaged live during the relaxed, loading, and unloading phases using SHG microscopy. The images were analyzed with Fourier decomposition to mathematically seek changes in collagen fiber orientation. Despite the similarities in both AV valves as seen in the histology and immunohistochemistry data, the microstructural arrangement, especially the collagen fiber distribution and orientation in the stress-free condition, were found to be different. Uniaxial loading was dependent on the arrangement of the fibers in their relaxed mode, which led the fibers to reorient in-line with the load throughout the depth of the mitral leaflet but only to reorient in-line with the load in deeper layers of the tricuspid leaflet. Biaxial loading arranged the fibers in between the two principal axes of the stresses independently from their relaxed states. Unlike previous findings, this study concludes that the AV valves' three-dimensional extracellular fiber arrangement is significantly different in their stress-free and uniaxially loaded states; however, fiber rearrangement in response to the biaxial loading remains similar. Copyright © 2015 the American Physiological Society.

  9. Biofilm formation by Staphylococcus epidermidis on peritoneal dialysis catheters and the effects of extracellular products from Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Pihl, Maria; Arvidsson, Anna; Skepö, Marie

    2013-01-01

    Biofilm formation by Staphylococcus epidermidis is a cause of infections related to peritoneal dialysis (PD). We have used a PD catheter flow-cell model in combination with confocal scanning laser microscopy and atomic force microscopy to study biofilm formation by S. epidermidis. Adherence....... aeruginosa contain promising substances for the prevention and treatment of biofilm infections, although further work is required to identity more active components....

  10. Cell adhesion control by ion implantation into extra-cellular matrix

    International Nuclear Information System (INIS)

    Suzuki, Yoshiaki; Kusakabe, Masahiro; Kaibara, Makoto; Iwaki, Masaya; Sasabe, Hiroyuki; Nishisaka, Tsuyoshi

    1994-01-01

    Cell adhesion control of polymer surfaces by ion implantation into polymers and extra-cellular matrix has been studied by means of in vitro adhesion measurements of the carcinoma of the cervix (HeLa cell). The specimens used were polystyrene (PS), oxygen plasma treated polystyrene (PS-O), extra-cellular matrix (Collagen: Type I) coated polystyrene (PS-C), and gelatin coated polystyrene (PS-G). Ne + , Na + , and Ar + implantations were performed with a fluence of 1x10 15 ions/cm 2 at energies of 50, 100 and 150 keV. The chemical and physical structures of ion implanted specimens have been investigated by Fourier transform infrared spectroscopy (FT-IR-ATR), X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy. Ion implanted PS demonstrated a dramatic improvement of adhesion of HeLa cell. HeLa cell adhered only to ion implanted circular domains of a diameter about 0.1 mm on PS. By contrast, ion implanted PS-C, PS-G and PS-O domains inhibited the cell adhesion. These phenomena were observed on Ne + , Na + , and Ar + implanted specimens at energies of 50, 100, and 150 keV. Ion implantation broke the original chemical bonds to form new radicals such as =C=O, condensed rings, C-C, C-O and OH radical. Ion implanted PS had a large amount of new radicals compared with that of PS-C, PS-G and PS-O. Ion implantation broke NH and NH 3 bonds originating from amino acid in PS-C and PS-G. OH and =C=O caused by oxygen treatment in PS-O were also destroyed by ion implantation. It is concluded that cell adhesion to ion implanted PS was caused by carbon structure and new radicals induced by ion implantation. The inhibition of HeLa cell adhesion on PS-C, PS-G and PS-O was caused by the destruction of cell adhesion properties of amino acid, OH and =C=O by radiation effects. ((orig.))

  11. Extracellular matrix disruption is an early event in the pathogenesis of skeletal disease in mucopolysaccharidosis I.

    Science.gov (United States)

    Heppner, Jonathan M; Zaucke, Frank; Clarke, Lorne A

    2015-02-01

    Progressive skeletal and connective tissue disease represents a significant clinical burden in all of the mucopolysaccharidoses. Despite the introduction of enzyme replacement strategies for many of the mucopolysaccharidoses, symptomatology related to bone and joint disease appears to be recalcitrant to current therapies. In order to address these unmet medical needs a clearer understanding of skeletal and connective tissue disease pathogenesis is required. Historically the pathogenesis of the mucopolysaccharidoses has been assumed to directly relate to progressive storage of glycosaminoglycans. It is now apparent for many lysosomal storage disorders that more complex pathogenic mechanisms underlie patients' clinical symptoms. We have used proteomic and genome wide expression studies in the murine mucopolysaccharidosis I model to identify early pathogenic events occurring in micro-dissected growth plate tissue. Studies were conducted using 3 and 5-week-old mice thus representing a time at which no obvious morphological changes of bone or joints have taken place. An unbiased iTRAQ differential proteomic approach was used to identify candidates followed by validation with multiple reaction monitoring mass spectrometry and immunohistochemistry. These studies reveal significant decreases in six key structural and signaling extracellular matrix proteins; biglycan, fibromodulin, PRELP, type I collagen, lactotransferrin, and SERPINF1. Genome-wide expression studies in embryonic day 13.5 limb cartilage and 5 week growth plate cartilage followed by specific gene candidate qPCR studies in the 5week growth plate identified fourteen significantly deregulated mRNAs (Adamts12, Aspn, Chad, Col2a1, Col9a1, Hapln4, Lum, Matn1, Mmp3, Ogn, Omd, P4ha2, Prelp, and Rab32). The involvement of biglycan, PRELP and fibromodulin; all members of the small leucine repeat proteoglycan family is intriguing, as this protein family is implicated in the pathogenesis of late onset osteoarthritis

  12. Deposition of tropoelastin into the extracellular matrix requires a competent elastic fiber scaffold but not live cells.

    Science.gov (United States)

    Kozel, Beth A; Ciliberto, Christopher H; Mecham, Robert P

    2004-04-01

    The initial steps of elastic fiber assembly were investigated using an in vitro assembly model in which purified recombinant tropoelastin (rbTE) was added to cultures of live or dead cells. The ability of tropoelastin to associate with preexisting elastic fibers or microfibrils in the extracellular matrix was then assessed by immunofluorescence microscopy using species-specific tropoelastin antibodies. Results show that rbTE can associate with elastic fiber components in the absence of live cells through a process that does not depend on crosslink formation. Time course studies show a transformation of the deposited protein from an initial globular appearance early in culture to a more fibrous structure as the matrix matures. Deposition required the C-terminal region of tropoelastin and correlated with the presence of preexisting elastic fibers or microfibrils. Association of exogenously added tropoelastin to the cellular extracellular matrix was inhibited by the addition of heparan sulfate but not chondroitin sulfate sugars. Together, these results suggest that the matrix elaborated by the cell is sufficient for the initial deposition of tropoelastin in the extracellular space and that elastin assembly may be influenced by the composition of sulfated proteoglycans in the matrix.

  13. INCREASE OF GLYCOSAMINOGLYCANS AND METALLOPROTEINASES 2 AND 9 IN LIVER EXTRACELLULAR MATRIX ON EARLY STAGES OF EXTRAHEPATIC CHOLESTASIS

    Directory of Open Access Journals (Sweden)

    Pedro Luiz Rodrigues GUEDES

    2014-12-01

    Full Text Available Context Cholestasis produces hepatocellular injury, leukocyte infiltration, ductular cells proliferation and fibrosis of liver parenchyma by extracellular matrix replacement. Objective Analyze bile duct ligation effect upon glycosaminoglycans content and matrix metalloproteinase (MMPs activities. Methods Animals (6-8 weeks; n = 40 were euthanized 2, 7 or 14 days after bile duct ligation or Sham-surgery. Disease evolution was analyzed by body and liver weight, seric direct bilirubin, globulins, gamma glutamyl transpeptidase (GGT, alkaline phosphatase (Alk-P, alanine and aspartate aminotransferases (ALT and AST, tissue myeloperoxidase and MMP-9, pro MMP-2 and MMP-2 activities, histopathology and glycosaminoglycans content. Results Cholestasis c