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Fraioli, R., Dashnyam, K., Kim, J. H., Perez, R. A., Kim, H. W., Gil, J., Ginebra, M. P., Manero, J. M., Mas-Moruno, C., (2016). Surface guidance of stem cell behavior: Chemically tailored co-presentation of integrin-binding peptides stimulates osteogenic differentiation in vitro and bone formation in vivo Acta Biomaterialia 43, 269-281

Surface modification stands out as a versatile technique to create instructive biomaterials that are able to actively direct stem cell fate. Chemical functionalization of titanium has been used in this work to stimulate the differentiation of human mesenchymal stem cells (hMSCs) into the osteoblastic lineage, by covalently anchoring a synthetic double-branched molecule (PTF) to the metal that allows a finely controlled presentation of peptidic motifs. In detail, the effect of the RGD adhesive peptide and its synergy motif PHSRN is studied, comparing a random distribution of the two peptides with the chemically-tailored disposition within the custom made synthetic platform, which mimics the interspacing between the motifs observed in fibronectin. Contact angle measurement and XPS analysis are used to prove the efficiency of functionalization. We demonstrate that, by rationally designing ligands, stem cell response can be efficiently guided towards the osteogenic phenotype: In vitro, PTF-functionalized surfaces support hMSCs adhesion, with higher cell area and formation of focal contacts, expression of the integrin receptor α5β1 and the osteogenic marker Runx2, and deposition a highly mineralized matrix, reaching values of mineralization comparable to fibronectin. Our strategy is also demonstrated to be efficient in promoting new bone growth in vivo in a rat calvarial defect. These results highlight the efficacy of chemical control over the presentation of bioactive peptides; such systems may be used to engineer bioactive surfaces with improved osseointegrative properties, or can be easily tuned to generate multi-functional coatings requiring a tailored disposition of the peptidic motifs. Statement of significance Organic coatings have been proposed as a solution to foster osseointegration of orthopedic implants. Among them, extracellular matrix-derived peptide motifs are an interesting biomimetic strategy to harness cell-surface interactions. Nonetheless, the combination of multiple peptide motifs in a controlled manner is essential to achieve receptor specificity and fully exploit the potentiality of synthetic peptides. Herein, we covalently graft to titanium a double branched molecule to guide stem cell fate in vitro and generate an osseoinductive titanium surface in vivo. Such synthetic ligand allows for the simultaneous presentation of two bioactive motifs, thus is ideal to test the effect of synergic sequences, such as RGD and PHSRN, and is a clear example of the versatility and feasibility of rationally designed biomolecules.

Keywords: hMSCs, Integrin-binding peptides, Osseointegration, RGD-PHSRN, Titanium

Cuervo, A., Dans, P. D., Carrascosa, J. L., Orozco, M., Gomila, G., Fumagalli, L., (2014). Direct measurement of the dielectric polarization properties of DNA Proceedings of the National Academy of Sciences of the United States of America 111, (35), E3624-E3630

The electric polarizability of DNA, represented by the dielectric constant, is a key intrinsic property that modulates DNA interaction with effector proteins. Surprisingly, it has so far remained unknown owing to the lack of experimental tools able to access it. Here, we experimentally resolved it by detecting the ultraweak polarization forces of DNA inside single T7 bacteriophages particles using electrostatic force microscopy. In contrast to the common assumption of low-polarizable behavior like proteins (εr ~ 2–4), we found that the DNA dielectric constant is ~ 8, considerably higher than the value of ~ 3 found for capsid proteins. State-of-the-art molecular dynamic simulations confirm the experimental findings, which result in sensibly decreased DNA interaction free energy than normally predicted by Poisson–Boltzmann methods. Our findings reveal a property at the basis of DNA structure and functions that is needed for realistic theoretical descriptions, and illustrate the synergetic power of scanning probe microscopy and theoretical computation techniques.

Keywords: Atomic force microscopy, Atomistic simulations, DNA packaging, DNA-ligand binding, Poisson-Boltzmann equation, capsid protein, DNA, double stranded DNA, amino acid composition, article, atomic force microscopy, bacteriophage, bacteriophage T7, dielectric constant, dipole, DNA binding, DNA packaging, DNA structure, electron microscopy, ligand binding, nonhuman, polarization, priority journal, protein analysis, protein DNA interaction, scanning probe microscopy, static electricity, virion, virus capsid, virus particle, atomic force microscopy, atomistic simulations, DNA packaging, DNA-ligand binding, Poisson-Boltzmann equation, Bacteriophage T7, Capsid, Cations, Dielectric Spectroscopy, DNA, DNA, Viral, DNA-Binding Proteins, Electrochemical Techniques, Ligands, Microscopy, Atomic Force, Models, Chemical, Nuclear Proteins

Marques, J., Moles, E., Urbán, P., Prohens, R., Busquets, M. A., Sevrin, C., Grandfils, C., Fernàndez-Busquets, X., (2014). Application of heparin as a dual agent with antimalarial and liposome targeting activities toward Plasmodium-infected red blood cells Nanomedicine: Nanotechnology, Biology, and Medicine 10, (8), 1719-1728

Heparin had been demonstrated to have antimalarial activity and specific binding affinity for Plasmodium-infected red blood cells (pRBCs) vs. non-infected erythrocytes. Here we have explored if both properties could be joined into a drug delivery strategy where heparin would have a dual role as antimalarial and as a targeting element of drug-loaded nanoparticles. Confocal fluorescence and transmission electron microscopy data show that after 30. min of being added to living pRBCs fluorescein-labeled heparin colocalizes with the intracellular parasites. Heparin electrostatically adsorbed onto positively charged liposomes containing the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane and loaded with the antimalarial drug primaquine was capable of increasing three-fold the activity of encapsulated drug in Plasmodium falciparum cultures. At concentrations below those inducing anticoagulation of mouse blood in vivo, parasiticidal activity was found to be the additive result of the separate activities of free heparin as antimalarial and of liposome-bound heparin as targeting element for encapsulated primaquine. From the Clinical Editor: Malaria remains an enormous global public health concern. In this study, a novel functionalized heparin formulation used as drug delivery agent for primaquine was demonstrated to result in threefold increased drug activity in cell cultures, and in a murine model it was able to provide these benefits in concentrations below what would be required for anticoagulation. Further studies are needed determine if this approach is applicable in the human disease as well.

Keywords: Heparin, Liposomes, Malaria, Plasmodium, Targeted drug delivery, Heparin, Malaria, Plasmodium, Red blood cell, Targeted drug delivery, Liposomes, 1,2 dioleoyl 3 trimethylammoniopropane, fluorescein, heparin, liposome, nanoparticle, primaquine, adsorption, animal experiment, anticoagulation, antimalarial activity, Article, binding affinity, confocal microscopy, controlled study, drug targeting, encapsulation, erythrocyte, female, fluorescence microscopy, human, human cell, in vivo study, liposomal delivery, mouse, nonhuman, Plasmodium falciparum, transmission electron microscopy

Penon, O., Novo, S., Duran, S., Ibanez, E., Nogues, C., Samitier, J., Duch, M., Plaza, J. A., Perez-Garcia, L., (2012). Efficient biofunctionalization of polysilicon barcodes for adhesion to the zona pellucida of mouse embryos Bioconjugate Chemistry , 23, (12), 2392-2402

Cell tracking is an emergent area in nano-biotechnology, promising the study of individual cells or the identification of populations of cultured cells. In our approach, microtools designed for extracellular tagging are prepared, because using biofunctionalized polysilicon barcodes to tag cell membranes externally avoids the inconveniences of cell internalization. The crucial covalent biofunctionalization process determining the ultimate functionality was studied in order to find the optimum conditions to link a biomolecule to a polysilicon barcode surface using a self-assembled monolayer (SAM) as the connector. Specifically, a lectin (wheat germ agglutinin, WGA) was used because of its capacity to recognize some specific carbohydrates present on the surface of most mammalian cells. Self-assembled monolayers were prepared on polysilicon surfaces including aldehyde groups as terminal functions to study the suitability of their covalent chemical bonding to WGA. Some parameters, such as the polysilicon surface roughness or the concentration of WGA, proved to be crucial for successful biofunctionalization and bioactivity. The SAMs were characterized by contact angle measurements, time-of-flight secondary ion mass spectrometry (TOF-SIMS), laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS), and atomic force microscopy (AFM). The biofunctionalization step was also characterized by fluorescence microscopy and, in the case of barcodes, by adhesion experiments to the zona pellucida of mouse embryos. These experiments showed high barcode retention rates after 96 h of culture as well as high embryo viability to the blastocyst stage, indicating the robustness of the biofunctionalization and, therefore, the potential of these new microtools to be used for cell tagging.

Keywords: Self-assembled monolayers, Wheat-germ-agglutinin, Protein immobilization strategies, Mass-spectrometry, Cell-surface, Petide, Binding, Identifications, Nanoparticles, Recognition

Tort, N., Salvador, J. P., Avino, A., Eritja, R., Comelles, J., Martinez, E., Samitier, J., Marco, M. P., (2012). Synthesis of steroid-oligonucleotide conjugates for a DNA site-encoded SPR immunosensor Bioconjugate Chemistry , 23, (11), 2183-2191

The excellent self-assembling properties of DNA and the excellent specificity of the antibodies to detect analytes of small molecular weight under competitive conditions have been combined in this study. Three oligonucleotide sequences (N(1)up, N(2)up, and N(3)up) have been covalently attached to three steroidal haptens (8, hG, and 13) of three anabolic-androgenic steroids (AAS), stanozolol (ST), tetrahydrogestrinone (THG), and boldenone (B), respectively. The synthesis of steroid oligonucleotide conjugates has been performed by the reaction of oligonucleotides carrying amino groups with carboxyl acid derivatives of steroidal haptens. Due to the chemical nature of the steroid derivatives, two methods for coupling the haptens and the ssDNA have been studied: a solid-phase coupling strategy and a solution-phase coupling strategy. Specific antibodies against ST, THG, and B have been used in this study to asses the possibility of using the self-assembling properties of the DNA to prepare biofunctional SPR gold chips based on the immobilization of haptens, by hybridization with the complementary oligonucleotide strands possessing SH groups previously immobilized. The capture of the steroid oligonucleotide conjugates and subsequent binding of the specific antibodies can be monitored on the sensogram due to variations produced on the refractive index on top of the gold chip. The resulting steroid oligonucleotide conjugates retain the hybridization and specific binding properties of oligonucleotides and haptens as demonstrated by thermal denaturation experiments and surface plasmon resonance (SPR).

Keywords: Directed protein immobilization, Plasmon resonance biosensor, Self-assembled monolayers, Label-free, Serum samples, Assay, Immunoassays, Antibodies, Progress, Binding

Caballero, D., Martinez, E., Bausells, J., Errachid, A., Samitier, J., (2012). Impedimetric immunosensor for human serum albumin detection on a direct aldehyde-functionalized silicon nitride surface Analytica Chimica Acta 720, 43-48

In this work we report the fabrication and characterization of a label-free impedimetric immunosensor based on a silicon nitride (Si 3N 4) surface for the specific detection of human serum albumin (HSA) proteins. Silicon nitride provides several advantages compared with other materials commonly used, such as gold, and in particular in solid-state physics for electronic-based biosensors. However, few Si 3N 4-based biosensors have been developed; the lack of an efficient and direct protocol for the integration of biological elements with silicon-based substrates is still one of its the main drawbacks. Here, we use a direct functionalization method for the direct covalent binding of monoclonal anti-HSA antibodies on an aldehyde-functionalized Si-p/SiO 2/Si 3N 4 structure. This methodology, in contrast with most of the protocols reported in literature, requires less chemical reagents, it is less time-consuming and it does not need any chemical activation. The detection capability of the immunosensor was tested by performing non-faradaic electrochemical impedance spectroscopy (EIS) measurements for the specific detection of HSA proteins. Protein concentrations within the linear range of 10 -13-10 -7M were detected, showing a sensitivity of 0.128ΩμM -1 and a limit of detection of 10 -14M. The specificity of the sensor was also addressed by studying the interferences with a similar protein, bovine serum albumin. The results obtained show that the antibodies were efficiently immobilized and the proteins detected specifically, thus, establishing the basis and the potential applicability of the developed silicon nitride-based immunosensor for the detection of proteins in real and more complex samples.

Keywords: Aldehyde, Electrochemical impedance spectroscopy, Human serum albumin, Immunosensor, Silicon nitride, Bovine serum albumins, Chemical reagents, Complex samples, Covalent binding, Detection capability, Electrochemical impedance, Electrochemical impedance spectroscopy measurements, Functionalizations, Human serum albumins, Impedimetric immunosensors, Label free, Limit of detection, Linear range, Protein concentrations, Silicon-based, Specific detection, Aldehydes

Valle-Delgado, J. J., Liepina, I., Lapidus, D., Sabaté, R., Ventura, S., Samitier, J., Fernàndez-Busquets, X., (2012). Self-assembly of human amylin-derived peptides studied by atomic force microscopy and single molecule force spectroscopy Soft Matter , 8, (4), 1234-1242

The self-assembly of peptides and proteins into amyloid fibrils of nanometric thickness and up to several micrometres in length, a phenomenon widely observed in biological systems, has recently aroused a growing interest in nanotechnology and nanomedicine. Here we have applied atomic force microscopy and single molecule force spectroscopy to study the amyloidogenesis of a peptide derived from human amylin and of its reverse sequence. The spontaneous formation of protofibrils and their orientation along well-defined directions on graphite and DMSO-coated graphite substrates make the studied peptides interesting candidates for nanotechnological applications. The measured binding forces between peptides correlate with the number of hydrogen bonds between individual peptides inside the fibril structure according to molecular dynamics simulations.

Keywords: Amyloid fibril, Amyloidogenesis, Binding forces, Fibril structure, Graphite substrate, Molecular dynamics simulations, Nanometrics, Protofibrils, Single molecule force spectroscopy, Spontaneous formation, Atomic force microscopy, Atomic spectroscopy, Graphite, Hydrogen bonds, Medical nanotechnology, Molecular dynamics, Molecular physics, Self assembly, Thickness measurement, Peptides

Redondo-Morata, Lorena, Oncins, Gerard, Sanz, Fausto, (2012). Force spectroscopy reveals the effect of different ions in the nanomechanical behavior of phospholipid model membranes: The case of potassium cation Biophysical Journal , 102, (1), 66-74

How do metal cations affect the stability and structure of phospholipid bilayers? What role does ion binding play in the insertion of proteins and the overall mechanical stability of biological membranes? Investigators have used different theoretical and microscopic approaches to study the mechanical properties of lipid bilayers. Although they are crucial for such studies, molecular-dynamics simulations cannot yet span the complexity of biological membranes. In addition, there are still some experimental difficulties when it comes to testing the ion binding to lipid bilayers in an accurate way. Hence, there is a need to establish a new approach from the perspective of the nanometric scale, where most of the specific molecular phenomena take place. Atomic force microscopy has become an essential tool for examining the structure and behavior of lipid bilayers. In this work, we used force spectroscopy to quantitatively characterize nanomechanical resistance as a function of the electrolyte composition by means of a reliable molecular fingerprint that reveals itself as a repetitive jump in the approaching force curve. By systematically probing a set of bilayers of different composition immersed in electrolytes composed of a variety of monovalent and divalent metal cations, we were able to obtain a wealth of information showing that each ion makes an independent and important contribution to the gross mechanical resistance and its plastic properties. This work addresses the need to assess the effects of different ions on the structure of phospholipid membranes, and opens new avenues for characterizing the (nano)mechanical stability of membranes.

Keywords: Molecular-dynamics simulation, Liquid expanded monolayers, Lipid-bilayers, Hofmeister series, Monovalent salt, Phosphatidylcholine, Microscopy, Binding, Surfaces, NaCl

Cordeiro, T. N., Schmidt, H., Madrid, C., Juarez, A., Bernado, P., Griesinger, C., Garcia, J., Pons, M., (2011). Indirect DNA readout by an H-NS related protein: Structure of the DNA complex of the C-terminal domain of Ler PLoS Pathogens Plos Pathogens , 7, (11), 12

Ler, a member of the H-NS protein family, is the master regulator of the LEE pathogenicity island in virulent Escherichia coli strains. Here, we determined the structure of a complex between the DNA-binding domain of Ler (CT-Ler) and a 15-mer DNA duplex. CT-Ler recognizes a preexisting structural pattern in the DNA minor groove formed by two consecutive regions which are narrower and wider, respectively, compared with standard B-DNA. The compressed region, associated with an AT-tract, is sensed by the side chain of Arg90, whose mutation abolishes the capacity of Ler to bind DNA. The expanded groove allows the approach of the loop in which Arg90 is located. This is the first report of an experimental structure of a DNA complex that includes a protein belonging to the H-NS family. The indirect readout mechanism not only explains the capacity of H-NS and other H-NS family members to modulate the expression of a large number of genes but also the origin of the specificity displayed by Ler. Our results point to a general mechanism by which horizontally acquired genes may be specifically recognized by members of the H-NS family.

Keywords: Enteropathogenic escherichia-coli, Nucleoid-associated protein, Nmr structure determination, Encoded regulator ler, Controls expression, Binding domain

Harder, A., Walhorn, V., Dierks, T., Fernàndez-Busquets, X., Anselmetti, D., (2010). Single-molecule force spectroscopy of cartilage aggrecan self-adhesion Biophysical Journal , 99, (10), 3498-3504

We investigated self-adhesion between highly negatively charged aggrecan macromolecules extracted from bovine cartilage extracellular matrix by performing atomic force microscopy (AFM) imaging and single-molecule force spectroscopy (SMFS) in saline solutions. By controlling the density of aggrecan molecules on both the gold substrate and the gold-coated tip surface at submonolayer densities, we were able to detect and quantify the Ca2+-dependent homodimeric interaction between individual aggrecan molecules at the single-molecule level. We found a typical nonlinear sawtooth profile in the AFM force-versus-distance curves with a molecular persistence length of I-p = 0.31 +/- 0.04 nm. This is attributed to the stepwise dissociation of individual glycosaminoglycan (GAG) side chains in aggrecans, which is very similar to the known force fingerprints of other cell adhesion proteoglycan systems. After studying the GAG-GAG dissociation in a dynamic, loading-rate-dependent manner (dynamic SMFS) and analyzing the data according to the stochastic Bell-Evans model for a thermally activated decay of a metastable state under an external force, we estimated for the single glycan interaction a mean lifetime of tau = 7.9 +/- 4.9 s and a reaction bond length of x(beta) = 0.31 +/- 0.08 nm. Whereas the x(beta)-value compares well with values from other cell adhesion carbohydrate recognition motifs in evolutionary distant marine sponge proteoglycans, the rather short GAG interaction lifetime reflects high intermolecular dynamics within aggrecan complexes, which may be relevant for the viscoelastic properties of cartilage tissue.

Keywords: Bovine nasal cartilage, Articular-cartilage, Sinorhizobium-meliloti, Proteoglycan, Microscopy, DNA, Macromolecules, Binding, Protein, Glycosaminoglycans

Pairo, E., Marco, S., Perera, A., (2010). A subspace method for the detection of transcription factor binding sites BIOINFORMATICS 2010. Proceedings of the First International Conference on Bioinformatics BIOINFORMATICS 2010. First International Conference on Bioinformatics (ed. Fred, A., Filipe, J., Gamboa, H.), INSTICC Press (Valencia, Spain) , 102-107

Transcription Factor binding sites are short and degenerate sequences, located mostly at the promoter of the gene, where some proteins bind in order to regulate transcription. Locating these sequences is an important issue, and many experimental and computational methods have been developed. Algorithms to search binding sites are usually based on Position Specific Scoring Matrices (PSSM), where each position is treated independently. Mapping symbolical DNA to numerical sequences, a detector has been built with a Principal Component Analysis of the numerical sequences, taking into account covariances between positions. When a treatment of missing values is incorporated the Q-residuals detector, based on PCA, performs better than a PSSM algorithm. The performance on the detector depends on the estimation of missing values and the percentage of missing values considered in the model.

Keywords: Binding sites, BPCA, Missing values, Numerical DNA, Principal components analysis, Transcription factors

Banos, R. C., Vivero, A., Aznar, S., Garcia, J., Pons, M., Madrid, C., Juarez, A., (2009). Differential regulation of horizontally acquired and core genome genes by the bacterial modulator H-NS PLoS Genetics , 5, (6), 8

Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.

Keywords: 2A strain 2457T, Escherichia-Coli, Salmonella-Enterica, Protein, DNA, Expression, Binding, HHA, Shigella, Plasmid

Roca-Cusachs, P., Gauthier, N. C., del Rio, A., Sheetz, M. P., (2009). Clustering of alpha(5)beta(1) integrins determines adhesion strength whereas alpha(v)beta(3) and talin enable mechanotransduction Proceedings of the National Academy of Sciences of the United States of America 106, (38), 16245-16250

A key molecular link between cells and the extracellular matrix is the binding between fibronectin and integrins alpha(5)beta(1) and alpha(v)beta(3). However, the roles of these different integrins in establishing adhesion remain unclear. We tested the adhesion strength of fibronectin-integrin-cytoskeleton linkages by applying physiological nanonewton forces to fibronectin-coated magnetic beads bound to cells. We report that the clustering of fibronectin domains within 40 nm led to integrin alpha(5)beta(1) recruitment, and increased the ability to sustain force by over six-fold. This force was supported by alpha(5)beta(1) integrin clusters. Importantly, we did not detect a role of either integrin alpha(v)beta(3) or talin 1 or 2 in maintaining adhesion strength. Instead, these molecules enabled the connection to the cytoskeleton and reinforcement in response to an applied force. Thus, high matrix forces are primarily supported by clustered alpha(5)beta(1) integrins, while less stable links to alpha(v)beta(3) integrins initiate mechanotransduction, resulting in reinforcement of integrin-cytoskeleton linkages through talin-dependent bonds.

Keywords: Cell-adhesion, Mechanical force, Vinculin-binding, Fibronectin, Activation, Dynamics, Domain, Alpha-v-beta-3, Translocation, Bonds

Gugutkov, Dencho, Gonzalez-Garcia, Cristina, Rodriguez Hernandez, Jose Carlos, Altankov, George, Salmeron-Sanchez, Manuel, (2009). Biological activity of the substrate-induced fibronectin network: insight into the third dimension through electrospun fibers Langmuir , 25, (18), 10893-10900

Fibronectin (FN) fibrillogenesis is a cell-mediated process involving integrin activation that results in conformational changes of FN molecules and the organization of actin cytoskeleton. A similar process can be induced by some chemistries in the absence of cells, e.g., poly(ethyl acrylate) (PEA), which enhance FN-FN interactions leading to the formation of a biologically active network. Atomic force microscopy images of single FN molecules, at the early stages of adsorption on plane PEA, allow one to rationalize the process. Further, the role of the spatial organization of the FN network on the cellular response is investigated through its adsorption on electrospun fibers. Randomly oriented and aligned PEA fibers were prepared to mimic the three-dimensional organization of the extracellular matrix. The formation of the FN network on the PEA fibers but not on the supporting coverglass was confirmed. Fibroblasts aligned with oriented fibers, displayed extended morphology, developed linearly organized focal adhesion complexes, and matured actin filaments. Conversely, on random PEA fibers, cells acquired polygonal morphology with altered actin cytoskeleton but well-developed focal adhesions. Late FN matrix formation was also influenced: spatially organized FN matrix fibrils along the oriented PEA fibers and an altered arrangement on random ones.

Keywords: AFM, Cell-adhesion, Dependent conformations, Hydrophobic surfaces, Extracellular-matrix, Bound fibronectin, Polymer surfaces, Integrin binding, Biocompatibility, Adsorption

Rico, P., Rodriguez Hernandez, J. C., Moratal, D., Altankov, G., Monleon Pradas, M., Salmeron-Sanchez, M., (2009). Substrate-induced assembly of fibronectin into networks. Influence of surface chemistry and effect on osteoblast adhesion Tissue Engineering Part A , 15, (00), 1-11

The influence of surface chemistry -substrates with controlled surface density of -OH groups- on fibronectin conformation and distribution is directly observed by Atomic Force Microscopy (AFM). FN fibrillogenesis, which is known to be a process triggered by interaction with integrins, is shown in our case to be induced by the substrate (in absence of cells), which is able to enhance FN-FN interactions leading to the formation of a protein network on the material surface. This phenomenon depends both on surface chemistry and protein concentration. The level of the FN fibrillogenesis was quantified by calculating the fractal dimension of the adsorbed protein from image analysis of the AFM results. The total amount of adsorbed FN is obtained by making use of a methodology which employs western-blotting combined with image analysis of the corresponding protein bands, with the lowest sensitivity threshold equal to 15 ng of adsorbed protein. Furthermore, FN adsorption is correlated to human osteoblast adhesion through morphology and actin cytoskeleton formation. Actin polymerization is in need of the formation of the protein network on the substrate's surface. Cell morphology is more rounded (as quantified by calculating the circularity of the cells by image analysis) the lower the degree of FN fibrillogenesis on the substrate.

Keywords: Cell-adhesion, Conformational-changes, Electron-microscopy, Protein adsorption, Fractal dimension, Integrin binding, Biocompatibility, Monolayers, Matrix, Fibrillogenesis

Pairo, E., Marco, S., Perera, A., (2009). A preliminary study on the detection of transcription factor binding sites Biosignals 2009: Proceedings of the International Conference on Bio-Inspired Systems and Signal Processing 2nd International Conference on Bio-Inspired Systems and Signal Processing (ed. Encarnacao, P., Veloso, A.), Insticc-Inst Syst Technologies Information Control & Communication (Oporto, Portugal) , 506-509

Transcription starts when multiple proteins, known as transcription factors recognize and bind to transcription start site in DNA sequences. Since mutation in transcription factor binding sites are known to underlie diseases it remains a major challenge to identify these binding sites. Conversion from symbolic DNA to numerical sequences and genome data make it possible to construct a detector based on a numerical analysis of DNA binding sites. A subspace model for the TFBS is built. TFBS will show a very small distance to this particular subspace. Using this distance binding sites are distinguished from random sequences and from genome data.

Keywords: Transcription factors, Binding sites, Principal components analysis

Rodriguez-Segui, S. A., Bucior, I., Burger, M. M., Errachid, A., Fernàndez-Busquets, X., (2009). Application of the quartz crystal microbalance to the study of multivalent carbohydrate-carbohydrate adhesion Sensor Letters 6th Maghreb-Europe Meeting on Materials and Their Applications for Devices and Physical, Chemical and Biological Sensors , AMER SCIENTIFIC PUBLISHERS (Rabat, Morocco) 7, (5), 782-787

Carbohydrate-carbohydrate interactions in cell adhesion are being increasingly explored as important players in cell-cell and cell-extracellular matrix interactions that are characterized by finelytuned on-off rates. The emerging field of glycomics requires the application of new methodologies to the study of the generally weak and multivalent carbohydrate binding sites. Here we use the quartz crystal microbalance (QCM) for the analysis of the self-binding activity of the g200 glycan, a molecule of marine sponge origin that is responsible for Ca2+-dependent species-specific cell adhesion. The QCM has the advantages over other highly sensitive techniques of having only one of the interacting partners bound to a surface, and of lacking microfluidics circuits prone to be clogged by self-aggregating glycans. Our results show that g200 self-interaction is negligible in the absence of Ca2+. Different association kinetics at low and high Ca2+ concentrations suggest the existence of two different Ca2+ binding sites in g200. Finally, the observation of a non-saturable binding indicates that g200 has more than one self-adhesion site per molecule. This work represents the first report to date using the QCM to study carbohydrate-carbohydrate interactions involved in cell adhesion.

Keywords: Ca2+-dependent binding, Carbohydrate-carbohydrate interaction, Cell adhesion, Proteoglycan, Quartz crystal microbalance, Sponges

Banos, R. C., Pons, J. I., Madrid, C., Juarez, A., (2008). A global modulatory role for the Yersinia enterocolitica H-NS protein Microbiology , 154, (5), 1281-1289

The H-NS protein plays a significant role in the modulation of gene expression in Gram-negative bacteria. Whereas isolation and characterization of hns mutants in Escherichia coli, Salmonella and Shigella represented critical steps to gain insight into the modulatory role of H-NS, it has hitherto not been possible to isolate hns mutants in Yersinia. The hns mutation is considered to be deleterious in this genus. To study the modulatory role of H-NS in Yersinia we circumvented hns lethality by expressing in Y. enterocolitica a truncated H-NS protein known to exhibit anti-H-NS activity in E. coli (H-NST(EPEC)). Y. enterocolitica cells expressing H-NST(EPEC) showed an altered growth rate and several differences in the protein expression pattern, including the ProV protein, which is modulated by H-NS in other enteric bacteria. To further confirm that H-NST(EPEC) expression in Yersinia can be used to demonstrate H-NS-dependent regulation in this genus, we used this approach to show that H-NS modulates expression of the YmoA protein.

Keywords: Bacterial Proteins/biosynthesis/genetics/ physiology, DNA-Binding Proteins/biosynthesis/genetics/ physiology, Electrophoresis, Gel, Two-Dimensional, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Genes, Essential, Proteome/analysis, RNA, Bacterial/biosynthesis, RNA, Messenger/biosynthesis, Reverse Transcriptase Polymerase Chain Reaction, Sequence Deletion, Yersinia enterocolitica/chemistry/genetics/growth & development/ physiology

Manara, S., Paolucci, F., Palazzo, B., Marcaccio, M., Foresti, E., Tosi, G., Sabbatini, S., Sabatino, P., Altankov, G., Roveri, N., (2008). Electrochemically-assisted deposition of biomimetic hydroxyapatite-collagen coatings on titanium plate Inorganica Chimica Acta , 361, (6), 1634-1645

A biomimetic bone-like composite, made of self-assembled collagen fibrils and carbonate hydroxyapatite nanocrystals, has been performed by an electrochemically-assisted deposition on titanium plate. The electrolytic processes have been carried out using a single type I collagen molecules suspension in a diluted Ca(NO3)(2) and NH4H2PO4 solution at room temperature and applying a constant current for different periods of time. Using the same electrochemical conditions, carbonate hydroxyapatite nanocrystals or reconstituted collagen. brils coatings were obtained. The reconstituted collagen. brils, hydroxyapatite nanocrystals and collagen fibrils/apatite nanocrystals coatings have been characterized chemically, structurally and morphologically, as well as for their ability to bind fibronectin (FN). Fourier Transform Infrared microscopy has been used to map the topographic distribution of the coating components at different times of electrochemical deposition, allowing to single out the individual deposition steps. Moreover, roughness of Ti plate has been found to affect appreciably the nucleation region of the inorganic nanocrystals. Laser scanning confocal microscopy has been used to characterize the FN adsorption pattern on a synthetic biomimetic apatitic phase, which exhibits a higher affinity when it is inter-grown with the collagen fibrils. The results offer auspicious applications in the preparation of medical devices such as biomimetic bone-like composite-coated metallic implants.

Keywords: Hydroxyapatite-collagen coating, Electrochemically-assisted deposition, Micro-imaging FTIR spectroscopy, Laser scanning confocal microscopy, Biomimetic crystal growth, Fibronectin binding