by Keyword: binding

Lagunas, Anna, Belloir, Christine, Briand, Loïc, Gorostiza, Pau, Samitier, Josep, (2022). Determination of the nanoscale electrical properties of olfactory receptor hOR1A1 and their dependence on ligand binding: Towards the development of capacitance-operated odorant biosensors Biosensors & Bioelectronics 218, 114755

Fischer NG, Aparicio C, (2022). Junctional epithelium and hemidesmosomes: Tape and rivets for solving the “percutaneous device dilemma” in dental and other permanent implants Bioactive Materials 18, 178-198

The percutaneous device dilemma describes etiological factors, centered around the disrupted epithelial tissue surrounding non-remodelable devices, that contribute to rampant percutaneous device infection. Natural percutaneous organs, in particular their extracellular matrix mediating the “device”/epithelium interface, serve as exquisite examples to inspire longer lasting long-term percutaneous device design. For example, the tooth's imperviousness to infection is mediated by the epithelium directly surrounding it, the junctional epithelium (JE). The hallmark feature of JE is formation of hemidesmosomes, cell/matrix adhesive structures that attach surrounding oral gingiva to the tooth's enamel through a basement membrane. Here, the authors survey the multifaceted functions of the JE, emphasizing the role of the matrix, with a particular focus on hemidesmosomes and their five main components. The authors highlight the known (and unknown) effects dental implant – as a model percutaneous device – placement has on JE regeneration and synthesize this information for application to other percutaneous devices. The authors conclude with a summary of bioengineering strategies aimed at solving the percutaneous device dilemma and invigorating greater collaboration between clinicians, bioengineers, and matrix biologists. © 2022 The Authors

JTD Keywords: amino-acid-sequence, bioinspired surfaces, cell-secreted protein, growth-factor receptor, hemidesmosome, integrin beta-4 subunit, junctional epithelium, keratinocyte-derived chemokine, laminin-binding integrins, marginal bone loss, percutaneous implant, pressure wound therapy, soft-tissue integration, Bioinspired surfaces, Bullous-pemphigoid antigen, Hemidesmosome, Junctional epithelium, Percutaneous device, Percutaneous implant

Zamora RA, López-Ortiz M, Sales-Mateo M, Hu C, Croce R, Maniyara RA, Pruneri V, Giannotti MI, Gorostiza P, (2022). Light- and Redox-Dependent Force Spectroscopy Reveals that the Interaction between Plastocyanin and Plant Photosystem I Is Favored when One Partner Is Ready for Electron Transfer Acs Nano 16, 15155-15164

Photosynthesis is a fundamental process that converts photons into chemical energy, driven by large protein complexes at the thylakoid membranes of plants, cyanobacteria, and algae. In plants, water-soluble plastocyanin (Pc) is responsible for shuttling electrons between cytochrome b6f complex and the photosystem I (PSI) complex in the photosynthetic electron transport chain (PETC). For an efficient turnover, a transient complex must form between PSI and Pc in the PETC, which implies a balance between specificity and binding strength. Here, we studied the binding frequency and the unbinding force between suitably oriented plant PSI and Pc under redox control using single molecule force spectroscopy (SMFS). The binding frequency (observation of binding-unbinding events) between PSI and Pc depends on their respective redox states. The interaction between PSI and Pc is independent of the redox state of PSI when Pc is reduced, and it is disfavored in the dark (reduced P700) when Pc is oxidized. The frequency of interaction between PSI and Pc is higher when at least one of the partners is in a redox state ready for electron transfer (ET), and the post-ET situation (PSIRed-PcOx) leads to lower binding. In addition, we show that the binding of ET-ready PcRed to PSI can be regulated externally by Mg2+ ions in solution.

JTD Keywords: architecture, binding-site, complexes, ferredoxin, force spectroscopy, induced structural-changes, interprotein electron transfer, light-dependent interaction, mg2+ concentration, photosystem i, plastocyanin, probe, recognition, reduction, Force spectroscopy, Interprotein electron transfer, Light-dependent interaction, Photosynthetic reaction-center, Photosystem i, Plastocyanin, Single molecule measurements

Acosta-Gutiérrez, Silvia, Matias, Diana, Avila-Olias, Milagros, Gouveia, Virginia M., Scarpa, Edoardo, Forth, Joe, Contini, Claudia, Duro-Castano, Aroa, Rizzello, Loris, Battaglia, Giuseppe, (2022). A Multiscale Study of Phosphorylcholine Driven Cellular Phenotypic Targeting Acs Central Science 8, 891-904

Varea, Olga, Guinovart, Joan J, Duran, Jordi, (2022). Malin restoration as proof of concept for gene therapy for Lafora disease Brain Commun 4, fcac168

Abstract Lafora disease is a fatal neurodegenerative childhood dementia caused by loss-of-function mutations in either the laforin or malin gene. The hallmark of the disease is the accumulation of abnormal glycogen aggregates known as Lafora bodies (LBs) in the brain and other tissues. These aggregates are responsible for the pathological features of the disease. As a monogenic disorder, Lafora disease is a good candidate for gene therapy-based approaches. However, most patients are diagnosed after the appearance of the first symptoms and thus when LBs are already present in the brain. In this context, it was not clear whether the restoration of a normal copy of the defective gene (either laforin or malin) would prove effective. Here we evaluated the effect of restoring malin in a malin-deficient mouse model of Lafora disease as a proof of concept for gene replacement therapy. To this end, we generated a malin-deficient mouse in which malin expression can be induced at a certain time. Our results reveal that malin restoration at an advanced stage of the disease arrests the accumulation of LBs in brain and muscle, induces the degradation of laforin and glycogen synthase bound to the aggregates, and ameliorates neuroinflammation. These results identify malin restoration as the first therapeutic strategy to show effectiveness when applied at advanced stages of Lafora disease.

JTD Keywords: accumulation, gene therapy, glycogen, lafora disease, neurodegeneration, neuroinflammation, neurons, targets, Carbohydrate-binding domain, Glycogen

Hernández F, Ferrer I, Pérez M, Zabala JC, Del Rio JA, Avila J, (2022). Tau Aggregation Neuroscience , S0306-2

Here we revisit tau protein aggregation at primary, secondary, tertiary and quaternary structures. In addition, the presence of non-aggregated tau protein, which has been recently discovered, is also commented on.Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.

JTD Keywords: Microtubule-binding repeats, Tau, Tau conformations, W-tau isoform

Almici E, Chiappini V, López-Márquez A, Badosa C, Blázquez B, Caballero D, Montero J, Natera-de Benito D, Nascimento A, Roldán M, Lagunas A, Jiménez-Mallebrera C, Samitier J, (2022). Personalized in vitro Extracellular Matrix Models of Collagen VI-Related Muscular Dystrophies Frontiers In Bioengineering And Biotechnology 10, 851825

Collagen VI-related dystrophies (COL6-RDs) are a group of rare congenital neuromuscular dystrophies that represent a continuum of overlapping clinical phenotypes that go from the milder Bethlem myopathy (BM) to the severe Ullrich congenital muscular dystrophy, for which there is no effective treatment. Mutations in one of the three Collagen VI genes alter the incorporation of this protein into the extracellular matrix (ECM), affecting the assembly and the structural integrity of the whole fibrillar network. Clinical hallmarks of COL6-RDs are secondary to the ECM disruption and include muscle weakness, proximal joint contractures, and distal hyperlaxity. Although some traits have been identified in patients’ ECMs, a correlation between the ECM features and the clinical phenotype has not been established, mainly due to the lack of predictive and reliable models of the pathology. Herein, we engineered a new personalized pre-clinical model of COL6-RDs using cell-derived matrices (CDMs) technology to better recapitulate the complexity of the native scenario. We found that CDMs from COL6-RD patients presented alterations in ECM structure and composition, showing a significantly decreased Collagen VI secretion, especially in the more severe phenotypes, and a decrease in Fibrillin-1 inclusion. Next, we examined the Collagen VI-mediated deposition of Fibronectin in the ECM, finding a higher alignment, length, width, and straightness than in patients with COL6-RDs. Overall, these results indicate that CDMs models are promising tools to explore the alterations that arise in the composition and fibrillar architecture due to mutations in Collagen VI genes, especially in early stages of matrix organization. Ultimately, CDMs derived from COL6-RD patients may become relevant pre-clinical models, which may help identifying novel biomarkers to be employed in the clinics and to investigate novel therapeutic targets and treatments. Copyright © 2022 Almici, Chiappini, López-Márquez, Badosa, Blázquez, Caballero, Montero, Natera-de Benito, Nascimento, Roldán, Lagunas, Jiménez-Mallebrera and Samitier.

JTD Keywords: alpha-3 chain, binding, collagen vi related muscular dystrophy, decellularisation, decellularized matrices, deficiency, expression, fibroblasts, fibronectin, in vitro model, patient-derived ecms, skeletal-muscle, ullrich, Cell-derived matrices, Collagen, Collagen vi related muscular dystrophy, Decellularisation, Decellularization, Extracellular matrices, Extracellular matrix, Genes, In vitro model, In-vitro, In-vitro models, Matrix, Matrix model, Muscular dystrophy, Pathology, Patient-derived ecm, Patient-derived ecms, Pre-clinical

Marte L, Boronat S, Barrios R, Barcons-Simon A, Bolognesi B, Cabrera M, Ayté J, Hidalgo E, (2022). Expression of Huntingtin and TDP-43 Derivatives in Fission Yeast Can Cause Both Beneficial and Toxic Effects International Journal Of Molecular Sciences 23, 3950

Many neurodegenerative disorders display protein aggregation as a hallmark, Huntingtin and TDP-43 aggregates being characteristic of Huntington disease and amyotrophic lateral sclerosis, respectively. However, whether these aggregates cause the diseases, are secondary by-products, or even have protective effects, is a matter of debate. Mutations in both human proteins can modulate the structure, number and type of aggregates, as well as their toxicity. To study the role of protein aggregates in cellular fitness, we have expressed in a highly tractable unicellular model different variants of Huntingtin and TDP-43. They each display specific patterns of aggregation and toxicity, even though in both cases proteins have to be very highly expressed to affect cell fitness. The aggregation properties of Huntingtin, but not of TDP-43, are affected by chaperones such as Hsp104 and the Hsp40 couple Mas5, suggesting that the TDP-43, but not Huntingtin, derivatives have intrinsic aggregation propensity. Importantly, expression of the aggregating form of Huntingtin causes a significant extension of fission yeast lifespan, probably as a consequence of kidnapping chaperones required for maintaining stress responses off. Our study demonstrates that in general these prion-like proteins do not cause toxicity under normal conditions, and in fact they can protect cells through indirect mechanisms which up-regulate cellular defense pathways. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: Chaperone, Chemistry, Dna binding protein, Dna-binding proteins, Fission yeast, Genetics, Human, Humans, Huntingtin, Metabolism, Molecular chaperones, Neurodegenerative diseases, Prion, Prions, Protein aggregate, Protein aggregates, Protein aggregation, Schizosaccharomyces, Tdp-43

Bertran, Oscar, Martí, Didac, Torras, Juan, Turon, Pau, Alemán, Carlos, (2022). Computer simulations on oxidative stress-induced reactions in SARS-CoV-2 spike glycoprotein: a multi-scale approach Molecular Diversity ,

Abstract Oxidative stress, which occurs when an organism is exposed to an adverse stimulus that results in a misbalance of antioxidant and pro-oxidants species, is the common denominator of diseases considered as a risk factor for SARS-CoV-2 lethality. Indeed, reactive oxygen species caused by oxidative stress have been related to many virus pathogenicity. In this work, simulations have been performed on the receptor binding domain of SARS-CoV-2 spike glycoprotein to study what residues are more susceptible to be attacked by ·OH, which is one of the most reactive radicals associated to oxidative stress. The results indicate that isoleucine (ILE) probably plays a crucial role in modification processes driven by radicals. Accordingly, QM/MM-MD simulations have been conducted to study both the ·OH-mediated hydrogen abstraction of ILE residues and the induced modification of the resulting ILE radical through hydroxylation or nitrosylation reactions. All in all, in silico studies show the importance of the chemical environment triggered by oxidative stress on the modifications of the virus, which is expected to help for foreseeing the identification or development of antioxidants as therapeutic drugs. Graphic abstract

JTD Keywords: atom abstraction, damage, density functionals, hydrogen abstraction, isoleucine, molecular dynamics, pathogenesis, protein, reactive oxygen species, receptor binding domain, residues, spike protein, Amino-acids, Hydrogen abstraction, Isoleucine, Molecular dynamics, Reactive oxygen species, Receptor binding domain, Spike protein

Martí, Didac, Alemán, Carlos, Ainsley, Jon, Ahumada, Oscar, Torras, Juan, (2022). IgG1-b12–HIV-gp120 Interface in Solution: A Computational Study Journal Of Chemical Information And Modeling 62, 359-371

The use of broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) has been shown to be a promising therapeutic modality in the prevention of HIV infection. Understanding the b12-gp120 binding mechanism under physiological conditions may assist the development of more broadly effective antibodies. In this work, the main conformations and interactions between the receptor-binding domain (RBD) of spike glycoprotein gp120 of HIV-1 and the IgG1-b12 mAb are studied. Accelerated molecular dynamics (aMD) and ab initio hybrid molecular dynamics have been combined to determine the most persistent interactions between the most populated conformations of the antibody-antigen complex under physiological conditions. The results show the most persistent receptor-binding mapping in the conformations of the antibody-antigen interface in solution. The binding-free-energy decomposition reveals a small enhancement in the contribution played by the CDR-H3 region to the b12-gp120 interface compared to the crystal structure.

JTD Keywords: antibody, complex, functionals, gp120 envelope glycoprotein, hiv, immunodeficiency-virus, noncovalent interactions, simulations, software integration, Ab initio, Accelerated molecular dynamics, Accelerated molecular-dynamics, Antibodies, Antigens, Binding energy, Binding mechanisms, Computational studies, Crystal structure, Diseases, Free energy, Hiv infection, Human immunodeficiency virus, Molecular dynamics, Neutralizing antibodies, Physiological condition, Physiology, Receptor-binding domains, Therapeutic modality, Viruses

Riera, Roger, Hogervorst, Tim P., Doelman, Ward, Ni, Yan, Pujals, Silvia, Bolli, Evangelia, Codée, Jeroen DC., van Kasteren, Sander I., Albertazzi, Lorenzo, (2021). Single-molecule imaging of glycan–lectin interactions on cells with Glyco-PAINT Nature Chemical Biology 17, 1281-+

Most lectins bind carbohydrate ligands with relatively low affinity, making the identification of optimal ligands challenging. Here we introduce a point accumulation in nanoscale topography (PAINT) super-resolution microscopy method to capture weak glycan-lectin interactions at the single-molecule level in living cells (Glyco-PAINT). Glyco-PAINT exploits weak and reversible sugar binding to directly achieve single-molecule detection and quantification in cells and is used to establish the relative kon and koff rates of a synthesized library of carbohydrate-based probes, as well as the diffusion coefficient of the receptor-sugar complex. Uptake of ligands correlates with their binding affinity and residence time to establish structure-function relations for various synthetic glycans. We reveal how sugar multivalency and presentation geometry can be optimized for binding and internalization. Overall, Glyco-PAINT represents a powerful approach to study weak glycan-lectin interactions on the surface of living cells, one that can be potentially extended to a variety of lectin-sugar interactions.© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.

JTD Keywords: dc-sign, density, dimerization, endocytosis, lateral mobility, ligand-binding, mannose receptor, proteins, recognition, Animal, Animals, Cell membrane, Cell membrane permeability, Chemistry, Cho cell line, Cho cells, Cricetulus, Cysteine-rich domain, Kinetics, Lectin, Lectins, Ligand, Ligands, Molecular library, Multivariate analysis, Polysaccharide, Polysaccharides, Procedures, Protein binding, Single molecule imaging, Small molecule libraries, Structure activity relation, Structure-activity relationship

Grob, M, Anselmetti, D, Fernandez-Busquets, X, (2021). In memory of Max Burger Journal Of Cellular Biochemistry 122, 1259-1261

Barbero-Castillo A, Riefolo F, Matera C, Caldas-Martínez S, Mateos-Aparicio P, Weinert JF, Garrido-Charles A, Claro E, Sanchez-Vives MV, Gorostiza P, (2021). Control of Brain State Transitions with a Photoswitchable Muscarinic Agonist Advanced Science 8, 2005027

The ability to control neural activity is essential for research not only in basic neuroscience, as spatiotemporal control of activity is a fundamental experimental tool, but also in clinical neurology for therapeutic brain interventions. Transcranial-magnetic, ultrasound, and alternating/direct current (AC/DC) stimulation are some available means of spatiotemporal controlled neuromodulation. There is also light-mediated control, such as optogenetics, which has revolutionized neuroscience research, yet its clinical translation is hampered by the need for gene manipulation. As a drug-based light-mediated control, the effect of a photoswitchable muscarinic agonist (Phthalimide-Azo-Iper (PAI)) on a brain network is evaluated in this study. First, the conditions to manipulate M2 muscarinic receptors with light in the experimental setup are determined. Next, physiological synchronous emergent cortical activity consisting of slow oscillations-as in slow wave sleep-is transformed into a higher frequency pattern in the cerebral cortex, both in vitro and in vivo, as a consequence of PAI activation with light. These results open the way to study cholinergic neuromodulation and to control spatiotemporal patterns of activity in different brain states, their transitions, and their links to cognition and behavior. The approach can be applied to different organisms and does not require genetic manipulation, which would make it translational to humans.

JTD Keywords: brain states, light-mediated control, muscarinic acetylcholine receptors, neuromodulation, Activation, Alternating/direct currents, Basal forebrain, Brain, Brain states, Clinical research, Clinical translation, Controlled drug delivery, Cortex, Forebrain cholinergic system, Genetic manipulations, Higher frequencies, Hz oscillation, Light‐, Light-mediated control, Mediated control, Muscarinic acetylcholine receptors, Muscarinic agonists, Muscarinic receptor, Neurology, Neuromodulation, Neurons, Noradrenergic modulation, Parvalbumin-positive interneurons, Photopharmacology, Receptor-binding, Slow, Spatiotemporal control, Spatiotemporal patterns

Riefolo, F, Sortino, R, Matera, C, Claro, E, Preda, B, Vitiello, S, Traserra, S, Jimenez, M, Gorostiza, P, (2021). Rational Design of Photochromic Analogues of Tricyclic Drugs Journal Of Medicinal Chemistry 64, 9259-9270

Tricyclic chemical structures are the core of many important drugs targeting all neurotransmitter pathways. These medicines enable effective therapies to treat from peptic ulcer disease to psychiatric disorders. However, when administered systemically, they cause serious adverse effects that limit their use. To obtain localized and on-demand pharmacological action using light, we have designed photoisomerizable ligands based on azobenzene that mimic the tricyclic chemical structure and display reversibly controlled activity. Pseudo-analogues of the tricyclic antagonist pirenzepine demonstrate that this is an effective strategy in muscarinic acetylcholine receptors, showing stronger inhibition upon illumination both in vitro and in cardiac atria ex vivo. Despite the applied chemical modifications to make pirenzepine derivatives sensitive to light stimuli, the most potent candidate of the set, cryptozepine-2, maintained a moderate but promising M-1 vs M-2 subtype selectivity. These photoswitchable crypto-azologs of tricyclic drugs might open a general way to spatiotemporally target their therapeutic action while reducing their systemic toxicity and adverse effects.

JTD Keywords: Binding, M1, Pirenzepine, Rat-brain, Receptor

Oliver-Cervelló L, Martin-Gómez H, Reyes L, Noureddine F, Ada Cavalcanti-Adam E, Ginebra MP, Mas-Moruno C, (2021). An Engineered Biomimetic Peptide Regulates Cell Behavior by Synergistic Integrin and Growth Factor Signaling Advanced Healthcare Materials 10, 2001757

© 2020 Wiley-VCH GmbH Recreating the healing microenvironment is essential to regulate cell–material interactions and ensure the integration of biomaterials. To repair bone, such bioactivity can be achieved by mimicking its extracellular matrix (ECM) and by stimulating integrin and growth factor (GF) signaling. However, current approaches relying on the use of GFs, such as bone morphogenetic protein 2 (BMP-2), entail clinical risks. Here, a biomimetic peptide integrating the RGD cell adhesive sequence and the osteogenic DWIVA motif derived from the wrist epitope of BMP-2 is presented. The approach offers the advantage of having a spatial control over the single binding of integrins and BMP receptors. Such multifunctional platform is designed to incorporate 3,4-dihydroxyphenylalanine to bind metallic oxides with high affinity in a one step process. Functionalization of glass substrates with the engineered peptide is characterized by physicochemical methods, proving a successful surface modification. The biomimetic interfaces significantly improve the adhesion of C2C12 cells, inhibit myotube formation, and activate the BMP-dependent signaling via p38. These effects are not observed on surfaces displaying only one bioactive motif, a mixture of both motifs or soluble DWIVA. These data prove the biological potential of recreating the ECM and engaging in integrin and GF crosstalk via molecular-based mimics.

JTD Keywords: binding, biomaterials, biomimetic peptides, bone, cell adhesion, cell differentiation, differentiation, dwiva, multifunctional coatings, osseointegration, osteoblasts, rgd, surface, surface functionalization, Biomimetic peptides, Cell adhesion, Cell differentiation, Dwiva, Matrix-bound bmp-2, Rgd, Surface functionalization

Badia M, Bolognesi B, (2021). Assembling the right type of switch: Protein condensation to signal cell death Current Opinion In Cell Biology 69, 55-61

© 2020 Elsevier Ltd Protein phase transitions are particularly amenable for cell signalling as these highly cooperative processes allow cells to make binary decisions in response to relatively small intracellular changes. The different processes of condensate formation and the distinct material properties of the resulting condensates provide a dictionary to modulate a range of decisions on cell fate. We argue that, on the one hand, the reversibility of liquid demixing offers a chance to arrest cell growth under specific circumstances. On the other hand, the transition to amyloids is better suited for terminal decisions such as those leading to apoptosis and necrosis. Here, we review recent examples of both scenarios, highlighting how mutations in signalling proteins affect the formation of biomolecular condensates with drastic effects on cell survival.

JTD Keywords: amyloid, cell death, deep mutagenesis, llps, rna-binding proteins, Amyloid, Cell death, Deep mutagenesis, Llps, Rna-binding proteins

Woythe L, Tito NB, Albertazzi L, (2021). A quantitative view on multivalent nanomedicine targeting Advanced Drug Delivery Reviews 169, 1-21

© 2020 The Authors Although the concept of selective delivery has been postulated over 100 years ago, no targeted nanomedicine has been clinically approved so far. Nanoparticles modified with targeting ligands to promote the selective delivery of therapeutics towards a specific cell population have been extensively reported. However, the rational design of selective particles is still challenging. One of the main reasons for this is the lack of quantitative theoretical and experimental understanding of the interactions involved in cell targeting. In this review, we discuss new theoretical models and experimental methods that provide a quantitative view of targeting. We show the new advancements in multivalency theory enabling the rational design of super-selective nanoparticles. Furthermore, we present the innovative approaches to obtain key targeting parameters at the single-cell and single molecule level and their role in the design of targeting nanoparticles. We believe that the combination of new theoretical multivalent design and experimental methods to quantify receptors and ligands aids in the rational design and clinical translation of targeted nanomedicines.

JTD Keywords: binding-kinetics, biological identity, biomolecular corona, blood-brain-barrier, drug-delivery, gold nanoparticles, multivalency, nanotechnology, protein corona, quantitative characterization, rational design, super-selectivity, superresolution microscopy, tumor heterogeneity, Ligand-receptor interactions, Multivalency, Nanotechnology, Quantitative characterization, Rational design, Super-selectivity

Martí D, Torras J, Bertran O, Turon P, Alemán C, (2021). Temperature effect on the SARS-CoV-2: A molecular dynamics study of the spike homotrimeric glycoprotein Computational And Structural Biotechnology Journal 19, 1848-1862

Rapid spread of SARS-CoV-2 virus have boosted the need of knowledge about inactivation mechanisms to minimize the impact of COVID-19 pandemic. Recent studies have shown that SARS-CoV-2 virus can be disabled by heating, the exposure time for total inactivation depending on the reached temperature (e.g. more than 45 min at 329 K or less than 5 min at 373 K. In spite of recent crystallographic structures, little is known about the molecular changes induced by the temperature. Here, we unravel the molecular basis of the effect of the temperature over the SARS-CoV-2 spike glycoprotein, which is a homotrimer with three identical monomers, by executing atomistic molecular dynamics (MD) simulations at 298, 310, 324, 338, 358 and 373 K. Furthermore, both the closed down and open up conformational states, which affect the accessibility of receptor binding domain, have been considered. Our results suggest that the spike homotrimer undergoes drastic changes in the topology of the hydrogen bonding interactions and important changes on the secondary structure of the receptor binding domain (RBD), while electrostatic interactions (i.e. salt bridges) are mainly preserved. The proposed inactivation mechanism has important implications for engineering new approaches to fight the SARS-CoV-2 coronavirus, as for example, cleaving or reorganizing the hydrogen bonds through chaotropic agents or nanoparticles with local surface resonant plasmon effect.

JTD Keywords: atomistic simulations, coronaviruses, denaturation, homotrimeric protein, inactivation, proteins, receptor binding domain, salt bridges, simulation, thermal inactivation, virus spike, Atomistic simulations, Homotrimeric protein, Receptor binding domain, Secondary-structure, Thermal inactivation, Virus spike

Marti, D, Martin-Martinez, E, Torras, J, Bertran, O, Turon, P, Aleman, C, (2021). In silico antibody engineering for SARS-CoV-2 detection Computational And Structural Biotechnology Journal 19, 5525-5534

Engineered immunoglobulin-G molecules (IgGs) are of wide interest for the development of detection elements in protein-based biosensors with clinical applications. The strategy usually employed for the de novo design of such engineered IgGs consists on merging fragments of the three-dimensional structure of a native IgG, which is immobilized on the biosensor surface, and of an antibody with an exquisite target specificity and affinity. In this work conventional and accelerated classical molecular dynamics (cMD and aMD, respectively) simulations have been used to propose two IgG-like antibodies for COVID-19 detection. More specifically, the crystal structure of the IgG1 B12 antibody, which inactivates the human immunodeficiency virus-1, has been merged with the structure of the antibody CR3022 Fab tightly bounded to SARS-CoV-2 receptor-binding domain (RBD) and the structure of the 5309 antibody Fab fragment complexed with SARS-CoV-2 RBD. The two constructed antibodies, named IgG1-CR3022 and IgG1-S309, respectively, have been immobilized on a stable gold surface through a linker. Analyses of the influence of both the merging strategy and the substrate on the stability of the two constructs indicate that the IgG1-S309 antibody better preserves the neutralizing structure than the IgG1-CR3022 one. Overall, results indicate that the IgG1-S309 is appropriated for the generation of antibody based sensors for COVID-19 diagnosis. (C) 2021 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.

JTD Keywords: cr3022, igg1, molecular engineering, s309, Antibodies, Antibody engineering, Biosensors, Chemical detection, Clinical application, Cov, Cr3022, Crystal structure, Design, Diseases, Gold nanoparticles, Igg1, Igg1 antibody, Immobilization, Immunoglobulin g, Immunosensor, In-silico, Merging, Molecular dynamics, Molecular engineering, Orientation, Protein-based biosensors, Receptor-binding domains, S309, Sars, Sensor, Spike protein, Target, Vaccine, Viruses

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.

JTD 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.

JTD 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.

JTD 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.

JTD 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).

JTD 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.

JTD 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.

JTD 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.

JTD 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.

JTD 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.

JTD 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.

JTD 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.

JTD 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.

JTD 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.

JTD 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.

JTD 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.

JTD 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.

JTD 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.

JTD 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.

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