A fractal nature for polymerized laminin

Polylaminin (polyLM) is a non-covalent acid-induced nano- and micro-structured polymer of the protein laminin displaying distinguished biological properties. Polylaminin stimulates neuritogenesis beyond the levels achieved by ordinary laminin and has been shown to promote axonal regeneration in animal models of spinal cord injury. Here we used confocal fluorescence microscopy (CFM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize its three-dimensional structure. Renderization of confocal optical slices of immunostained polyLM revealed the aspect of a loose flocculated meshwork, which was homogeneously stained by the antibody. On the other hand, an ordinary matrix obtained upon adsorption of laminin in neutral pH (LM) was constituted of bulky protein aggregates whose interior was not accessible to the same anti-laminin antibody. SEM and AFM analyses revealed that the seed unit of polyLM was a flat polygon formed in solution whereas the seed structure of LM was highly heterogeneous, intercalating rod-like, spherical and thin spread lamellar deposits. As polyLM was visualized at progressively increasing magnifications, we observed that the morphology of the polymer was alike independently of the magnification used for the observation. A search for the Hausdorff dimension in images of the two matrices showed that polyLM, but not LM, presented fractal dimensions of 1.55, 1.62 and 1.70 after 1, 8 and 12 hours of adsorption, respectively. Data in the present work suggest that the intrinsic fractal nature of polymerized laminin can be the structural basis for the fractal-like organization of basement membranes in the neurogenic niches of the central nervous system.This work was supported by a grant from the Brazilian National Research Council (CNPq; 476772/2008-7) to TCS. MSS acknowledges support from the European Research Council through ERC - 306990. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Hochman Méndez, C.; Cantini ., M.; Moratal Pérez, D.; Salmerón Sánchez, M.; Coelho-Sampaio, T. (2014). A fractal nature for polymerized laminin. PLoS ONE. 9(10):109388-1-109388-11. https://doi.org/10.1371/journal.pone.0109388S109388-1109388-11910Durbeej, M. (2009). Laminins. Cell and Tissue Research, 339(1), 259-268. doi:10.1007/s00441-009-0838-2Miner, J. H., & Yurchenco, P. D. (2004). LAMININ FUNCTIONS IN TISSUE MORPHOGENESIS. Annual Review of Cell and Developmental Biology, 20(1), 255-284. doi:10.1146/annurev.cellbio.20.010403.094555Yurchenco, P. D. (2010). Basement Membranes: Cell Scaffoldings and Signaling Platforms. Cold Spring Harbor Perspectives in Biology, 3(2), a004911-a004911. doi:10.1101/cshperspect.a004911Hohenester, E., & Yurchenco, P. D. (2013). Laminins in basement membrane assembly. Cell Adhesion & Migration, 7(1), 56-63. doi:10.4161/cam.21831Freire, E., & Coelho-Sampaio, T. (2000). Self-assembly of Laminin Induced by Acidic pH. Journal of Biological Chemistry, 275(2), 817-822. doi:10.1074/jbc.275.2.817Freire, E., Sant’Ana Barroso, M. M., Klier, R. N., & Coelho-Sampaio, T. (2011). Biocompatibility and Structural Stability of a Laminin Biopolymer. Macromolecular Bioscience, 12(1), 67-74. doi:10.1002/mabi.201100125Freire, E. (2002). Structure of laminin substrate modulates cellular signaling for neuritogenesis. Journal of Cell Science, 115(24), 4867-4876. doi:10.1242/jcs.00173Hochman-Mendez, C., Lacerda de Menezes, J. R., Sholl-Franco, A., & Coelho-Sampaio, T. (2013). Polylaminin recognition by retinal cells. Journal of Neuroscience Research, 92(1), 24-34. doi:10.1002/jnr.23298Menezes, K., Ricardo Lacerda de Menezes, J., Assis Nascimento, M., de Siqueira Santos, R., & Coelho-Sampaio, T. (2010). Polylaminin, a polymeric form of laminin, promotes regeneration after spinal cord injury. The FASEB Journal, 24(11), 4513-4522. doi:10.1096/fj.10-157628Barroso, M. M. S., Freire, E., Limaverde, G. S. C. S., Rocha, G. M., Batista, E. J. O., Weissmüller, G., … Coelho-Sampaio, T. (2008). Artificial Laminin Polymers Assembled in Acidic pH Mimic Basement Membrane Organization. Journal of Biological Chemistry, 283(17), 11714-11720. doi:10.1074/jbc.m709301200Freire, E. (2004). Sialic acid residues on astrocytes regulate neuritogenesis by controlling the assembly of laminin matrices. Journal of Cell Science, 117(18), 4067-4076. doi:10.1242/jcs.01276Hausdorff, F. (1918). Dimension und �u�eres Ma�. Mathematische Annalen, 79(1-2), 157-179. doi:10.1007/bf01457179Soille, P., & Rivest, J.-F. (1996). On the Validity of Fractal Dimension Measurements in Image Analysis. Journal of Visual Communication and Image Representation, 7(3), 217-229. doi:10.1006/jvci.1996.0020Theiler, J. (1990). Estimating fractal dimension. Journal of the Optical Society of America A, 7(6), 1055. doi:10.1364/josaa.7.001055Otsu, N. (1979). A Threshold Selection Method from Gray-Level Histograms. IEEE Transactions on Systems, Man, and Cybernetics, 9(1), 62-66. doi:10.1109/tsmc.1979.4310076Iranfar, H., Rajabi, O., Salari, R., & Chamani, J. (2012). Probing the Interaction of Human Serum Albumin with Ciprofloxacin in the Presence of Silver Nanoparticles of Three Sizes: Multispectroscopic and ζ Potential Investigation. The Journal of Physical Chemistry B, 116(6), 1951-1964. doi:10.1021/jp210685qPalmero, C. Y., Miranda-Alves, L., Sant’Ana Barroso, M. M., Souza, E. C. L., Machado, D. E., Palumbo-Junior, A., … Nasciutti, L. E. (2013). The follicular thyroid cell line PCCL3 responds differently to laminin and to polylaminin, a polymer of laminin assembled in acidic pH. Molecular and Cellular Endocrinology, 376(1-2), 12-22. doi:10.1016/j.mce.2013.05.020Behrens, D. T., Villone, D., Koch, M., Brunner, G., Sorokin, L., Robenek, H., … Hansen, U. (2012). The Epidermal Basement Membrane Is a Composite of Separate Laminin- or Collagen IV-containing Networks Connected by Aggregated Perlecan, but Not by Nidogens. Journal of Biological Chemistry, 287(22), 18700-18709. doi:10.1074/jbc.m111.336073Colognato, H., Winkelmann, D. A., & Yurchenco, P. D. (1999). Laminin Polymerization Induces a Receptor–Cytoskeleton Network. The Journal of Cell Biology, 145(3), 619-631. doi:10.1083/jcb.145.3.619Liesi, P., & Silver, J. (1988). Is astrocyte laminin involved in axon guidance in the mammalian CNS? Developmental Biology, 130(2), 774-785. doi:10.1016/0012-1606(88)90366-1Zhou, F. C. (1990). Four patterns of laminin-immunoreactive structure in developing rat brain. Developmental Brain Research, 55(2), 191-201. doi:10.1016/0165-3806(90)90200-iGarcia-Abreu, J., Cavalcante, L. A., & Neto, V. M. (1995). Differential patterns of laminin expression in lateral and medial midbrain glia. NeuroReport, 6(5), 761-764. doi:10.1097/00001756-199503270-00014Kazanis, I., & ffrench-Constant, C. (2011). Extracellular matrix and the neural stem cell niche. Developmental Neurobiology, 71(11), 1006-1017. doi:10.1002/dneu.20970Mercier F, Schnack J, Chaumet MSG (2011) Chapter 4 Fractones: home and conductors of the neural stem cell niche. In: Seki, T., Sawamoto, K., Parent, J. M., Alvarez-Buylla, A., (Eds.) Neurogenesis in the adult brain I: neurobiology. Springer. pp 109–133.CAVALCANTIADAM, E., MICOULET, A., BLUMMEL, J., AUERNHEIMER, J., KESSLER, H., & SPATZ, J. (2006). Lateral spacing of integrin ligands influences cell spreading and focal adhesion assembly. European Journal of Cell Biology, 85(3-4), 219-224. doi:10.1016/j.ejcb.2005.09.011Frith, J. E., Mills, R. J., & Cooper-White, J. J. (2012). Lateral spacing of adhesion peptides influences human mesenchymal stem cell behaviour. Journal of Cell Science, 125(2), 317-327. doi:10.1242/jcs.087916Hernández, J. C. R., Salmerón Sánchez, M., Soria, J. M., Gómez Ribelles, J. L., & Monleón Pradas, M. (2007). Substrate Chemistry-Dependent Conformations of Single Laminin Molecules on Polymer Surfaces are Revealed by the Phase Signal of Atomic Force Microscopy. Biophysical Journal, 93(1), 202-207. doi:10.1529/biophysj.106.102491Douet, V., Kerever, A., Arikawa-Hirasawa, E., & Mercier, F. (2013). Fractone-heparan sulphates mediate FGF-2 stimulation of cell proliferation in the adult subventricular zone. Cell Proliferation, 46(2), 137-145. doi:10.1111/cpr.12023Nikolova, G., Strilic, B., & Lammert, E. (2007). The vascular niche and its basement membrane. Trends in Cell Biology, 17(1), 19-25. doi:10.1016/j.tcb.2006.11.005Yurchenco, P. D., Amenta, P. S., & Patton, B. L. (2004). Basement membrane assembly, stability and activities observed through a developmental lens. Matrix Biology, 22(7), 521-538. doi:10.1016/j.matbio.2003.10.006Nikolova, G., Jabs, N., Konstantinova, I., Domogatskaya, A., Tryggvason, K., Sorokin, L., … Lammert, E. (2006). The Vascular Basement Membrane: A Niche for Insulin Gene Expression and β Cell Proliferation. Developmental Cell, 10(3), 397-405. doi:10.1016/j.devcel.2006.01.015Qu, H., Liu, X., Ni, Y., Jiang, Y., Feng, X., Xiao, J., … Zheng, C. (2014). Laminin 411 acts as a potent inducer of umbilical cord mesenchymal stem cell differentiation into insulin-producing cells. Journal of Translational Medicine, 12(1), 135. doi:10.1186/1479-5876-12-135Kanatsu-Shinohara, M., & Shinohara, T. (2013). Spermatogonial Stem Cell Self-Renewal and Development. Annual Review of Cell and Developmental Biology, 29(1), 163-187. doi:10.1146/annurev-cellbio-101512-122353Lander, A. D., Kimble, J., Clevers, H., Fuchs, E., Montarras, D., Buckingham, M., … Oskarsson, T. (2012). What does the concept of the stem cell niche really mean today? BMC Biology, 10(1). doi:10.1186/1741-7007-10-19Loulier, K., Lathia, J. D., Marthiens, V., Relucio, J., Mughal, M. R., Tang, S.-C., … ffrench-Constant, C. (2009). β1 Integrin Maintains Integrity of the Embryonic Neocortical Stem Cell Niche. PLoS Biology, 7(8), e1000176. doi:10.1371/journal.pbio.100017

Impact of jet-production data on the next-to-next-to-leading-order determination of HERAPDF2.0 parton distributions

The HERAPDF2.0 ensemble of parton distribution functions (PDFs) was introduced in 2015. The final stage is presented, a next-to-next-to-leading-order (NNLO) analysis of the HERA data on inclusive deep inelastic ep scattering together with jet data as published by the H1 and ZEUS collaborations. A perturbative QCD fit, simultaneously of αs(M2Z) and the PDFs, was performed with the result αs(M2Z)=0.1156±0.0011 (exp) +0.0001−0.0002 (model +parameterisation) ±0.0029 (scale). The PDF sets of HERAPDF2.0Jets NNLO were determined with separate fits using two fixed values of αs(M2Z), αs(M2Z)=0.1155 and 0.118, since the latter value was already chosen for the published HERAPDF2.0 NNLO analysis based on HERA inclusive DIS data only. The different sets of PDFs are presented, evaluated and compared. The consistency of the PDFs determined with and without the jet data demonstrates the consistency of HERA inclusive and jet-production cross-section data. The inclusion of the jet data reduced the uncertainty on the gluon PDF. Predictions based on the PDFs of HERAPDF2.0Jets NNLO give an excellent description of the jet-production data used as input

CMS physics technical design report : Addendum on high density QCD with heavy ions

Peer reviewe

Impact of jet-production data on the next-to-next-to-leading-order determination of HERAPDF2.0 parton distributions

International audienceThe HERAPDF2.0 ensemble of parton distribution functions (PDFs) was introduced in 2015. The final stage is presented, a next-to-next-to-leading-order (NNLO) analysis of the HERA data on inclusive deep inelastic ep scattering together with jet data as published by the H1 and ZEUS collaborations. A perturbative QCD fit, simultaneously of $\alpha _s(M_Z^2)$ and the PDFs, was performed with the result $\alpha _s(M_Z^2)= 0.1156 \pm 0.0011~\mathrm{(exp)}~ ^{+0.0001}_{-0.0002}~ \mathrm{(model}\mathrm{+ parameterisation)}~ \pm 0.0029~\mathrm{(scale)}$. The PDF sets of HERAPDF2.0Jets NNLO were determined with separate fits using two fixed values of $\alpha _s(M_Z^2)$, $\alpha _s(M_Z^2)=0.1155$ and 0.118, since the latter value was already chosen for the published HERAPDF2.0 NNLO analysis based on HERA inclusive DIS data only. The different sets of PDFs are presented, evaluated and compared. The consistency of the PDFs determined with and without the jet data demonstrates the consistency of HERA inclusive and jet-production cross-section data. The inclusion of the jet data reduced the uncertainty on the gluon PDF. Predictions based on the PDFs of HERAPDF2.0Jets NNLO give an excellent description of the jet-production data used as input

The microstructure of laminin-111 compensates for dystroglycan loss in mammary epithelial cells in downstream expression of milk proteins.

Laminin-111 (Ln-1), an extracellular matrix (ECM) glycoprotein found in the basement membrane of mammary gland epithelia, is essential for lactation. In mammary epithelial cells (MECs), dystroglycan (Dg) is believed to be necessary for polymerization of laminin-111 into networks., thus we asked whether correct polymerization could compensate for Dg loss. Artificially polymerized laminin-111 and the laminin-glycoprotein mix Matrigel, both formed branching, spread networks with fractal dimensions from 1.7 to 1.8, whereas laminin-111 in neutral buffers formed small aggregates without fractal properties (a fractal dimension of 2). In Dg knockout cells, either polymerized laminin-111 or Matrigel readily attached to the cell surface, whereas aggregated laminin-111 did not. In contrast, polymerized and aggregated laminin-111 bound similarly to Dg knock-ins. Both polymerized laminin-111 and Matrigel promoted cell rounding, clustering, formation of tight junctions, and expression of milk proteins, whereas aggregated Ln-1 did not attach to cells or promote functional differentiation. These findings support that the microstructure of Ln-1 networks in the basement membrane regulates mammary epithelial cell function

The microstructure of laminin-111 compensates for dystroglycan loss in mammary epithelial cells in downstream expression of milk proteins.

Laminin-111 (Ln-1), an extracellular matrix (ECM) glycoprotein found in the basement membrane of mammary gland epithelia, is essential for lactation. In mammary epithelial cells (MECs), dystroglycan (Dg) is believed to be necessary for polymerization of laminin-111 into networks., thus we asked whether correct polymerization could compensate for Dg loss. Artificially polymerized laminin-111 and the laminin-glycoprotein mix Matrigel, both formed branching, spread networks with fractal dimensions from 1.7 to 1.8, whereas laminin-111 in neutral buffers formed small aggregates without fractal properties (a fractal dimension of 2). In Dg knockout cells, either polymerized laminin-111 or Matrigel readily attached to the cell surface, whereas aggregated laminin-111 did not. In contrast, polymerized and aggregated laminin-111 bound similarly to Dg knock-ins. Both polymerized laminin-111 and Matrigel promoted cell rounding, clustering, formation of tight junctions, and expression of milk proteins, whereas aggregated Ln-1 did not attach to cells or promote functional differentiation. These findings support that the microstructure of Ln-1 networks in the basement membrane regulates mammary epithelial cell function

Mining the Mesenchymal Stromal Cell Secretome in Patients with Chronic Left Ventricular Dysfunction

Close examination of the initial results of cardiovascular cell therapy clinical trials indicates the importance of patient-specific differences on outcomes and the need to optimize or customize cell therapies. The fields of regenerative medicine and cell therapy have transitioned from using heterogeneous bone marrow mononuclear cells (BMMNCs) to mesenchymal stromal cells (MSCs), which are believed to elicit benefits through paracrine activity. Here, we examined MSCs from the BMMNCs of heart failure patients enrolled in the FOCUS-CCTRN trial. We sought to identify differences in MSCs between patients who improved and those who declined in heart function, regardless of treatment received. Although we did not observe differences in the cell profile of MSCs between groups, we did find significant differences in the MSC secretome profile between patients who improved or declined. We conclude that “mining” the MSC secretome may provide clues to better understand the impact of patient characteristics on outcomes after cell therapy and this knowledge can inform future cell therapy trials

Laminin as a Potent Substrate for Large-Scale Expansion of Human Induced Pluripotent Stem Cells in a Closed Cell Expansion System

The number of high-quality cells required for engineering an adult human-sized bioartificial organ is greater than one billion. Until the emergence of induced pluripotent stem cells (iPSCs), autologous cell sources of this magnitude and with the required complexity were not available. Growing this number of cells in a traditional 2D cell culture system requires extensive time, resources, and effort and does not always meet clinical requirements. The use of a closed cell culture system is an efficient and clinically applicable method that can be used to expand cells under controlled conditions. We aimed to use the Quantum Cell Expansion System (QES) as an iPSC monolayer-based expansion system. Human iPSCs were expanded (up to 14-fold) using the QES on two different coatings (laminin 521 (LN521) and vitronectin (VN)), and a karyotype analysis was performed. The cells were characterized for spontaneous differentiation and pluripotency by RT-PCR and flow cytometry. Our results demonstrated that the QES provides the necessary environment for exponential iPSC growth, reaching 689.75 × 106 ± 86.88 × 106 in less than 7 days using the LN521 coating with a population doubling level of 3.80 ± 0.19. The same result was not observed when VN was used as a coating. The cells maintained normal karyotype (46-XX), expressed pluripotency markers (OCT4, NANOG, LIN28, SOX2, REX1, DPPA4, NODAL, TDGFb, TERT3, and GDF), and expressed high levels of OCT4, SOX2, NANOG, SSEA4, TRA1-60, and TRA1-81. Spontaneous differentiation into ectoderm (NESTIN, TUBB3, and NEFH), mesoderm (MSX1, BMP4, and T), and endoderm (GATA6, AFP, and SOX17) lineages was detected by RT-PCR with both coating systems. We conclude that the QES maintains the stemness of iPSCs and is a promising platform to provide the number of cells necessary to recellularize small human-sized organ scaffolds for clinical purposes

Polymerized Laminin-521: A Feasible Substrate for Expanding Induced Pluripotent Stem Cells at a Low Protein Concentration

Laminins (LNs) play a central role in the self-assembly and maintenance of basement membranes and are involved in critical interactions between cells and other extracellular matrix (ECM) proteins. Among the defined, xeno-free ECM culture matrices, LNs—namely LN521—have emerged as promising coating systems for the large-scale expansion of induced pluripotent stem cells (iPSCs). The biologic activity of LNs is enhanced by their acidification-induced self-polymerization into a cell-associated network called polylaminin (polyLN), which can recapitulate the native-like polymeric array in a cell-free system. Here, we show for the first time to our knowledge that polyLN521 displays a native-like hexagonal-like structure and that, at basal and low concentrations, it permits the large-scale expansion of human iPSCs. Human iPSCs expanded with polyLN521 maintained the pluripotent state and showed no impairment of karyotype stability or telomere length. These results suggest that low-concentration polyLN521 is a stable and cost-effective coating for large-scale iPSC expansion