Publications

Microbial biofilms are complex three-dimensional structures where sessile microbes are embedded in a polymeric extracellular matrix. Their resistance toward the host immune system as well as to a diverse range of antimicrobial treatments poses a serious health and development threat, being in the top 10 global public health threats declared by the World Health Organization. In an effort to combat biofilm-related microbial infections, several strategies have been developed to independently eliminate biofilms or to complement conventional antibiotic therapies. However, their limitations leave room for other treatment alternatives, where the application of nanotechnology to biofilm eradication has gained significant relevance in recent years. Their small size, penetration efficiency, and the design flexibility that they present makes them a promising alternative for biofilm infection treatment, although they also present set-backs. This review aims to describe the main possibilities and limitations of nanomedicine against biofilms, while covering the main aspects of biofilm formation and study, and the current therapies for biofilm treatment. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

JTD Keywords: Anti-bacterial agents, Anti-infective agents, Antiinfective agent, Antimicrobial, Antimicrobials, Antimicrobials,bacteria,biofilm,infectious diseases,microorganism, Bacteria, Biofilm, Biofilm infections, Biofilms, Complex three dimensional structures, Diseases, Diverse range, Drug-delivery systems,in-vitro,cellular toxicity,nanoparticles,penetration,model,biocompatibility,perspectives,hyperthermia,diagnosi, Extracellular matrices, Global public health, Health risks, Infectious disease, Infectious diseases, Medical nanotechnology, Microbial biofilm, Microorganisms, Nanomedicine, Polymer, Polymers, Regulatory issues, Roadmap

The performance of single-chain polymeric nanoparticles (SCPNs) in biomedical applications highly depends on their conformational stability in cellular environments. Until now, such stability studies are limited to 2D cell culture models, which do not recapitulate the 3D tumor microenvironment well. Here, a microfluidic tumor-on-a-chip model is introduced that recreates the tumor milieu and allows in-depth insights into the diffusion, cellular uptake, and stability of SCPNs. The chip contains Matrigel/collagen-hyaluronic acid as extracellular matrix (ECM) models and is seeded with cancer cell MCF7 spheroids. With this 3D platform, it is assessed how the polymer's microstructure affects the SCPN's behavior when crossing the ECM, and evaluates SCPN internalization in 3D cancer cells. A library of SCPNs varying in microstructure is prepared. All SCPNs show efficient ECM penetration but their cellular uptake/stability behavior depends on the microstructure. Glucose-based nanoparticles display the highest spheroid uptake, followed by charged nanoparticles. Charged nanoparticles possess an open conformation while nanoparticles stabilized by internal hydrogen bonding retain a folded structure inside the tumor spheroids. The 3D microfluidic tumor-on-a-chip platform is an efficient tool to elucidate the interplay between polymer microstructure and SCPN's stability, a key factor for the rational design of nanoparticles for targeted biological applications.© 2024 The Authors. Small Methods published by Wiley-VCH GmbH.

JTD Keywords: 3d cancer cell uptake, Cancer cells, Cell culture, Cell uptake, Cellular uptake, Diseases, Ecm penetration, Extracellular matrices, Extracellular matrix penetration, Functional polymers, Hydrogen bonds, Medical applications, Microfluidics, Microstructure, Nanoparticles, Polymeric nanoparticles, Scpns, Single chains, Single-chain polymeric nanoparticle, Stability, Tumor-on-a-chip, Tumors

During embryogenesis, the mammalian kidney arises because of reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (MM), driving UB branching and nephron induction. These morphogenetic processes involve a series of cellular rearrangements that are tightly controlled by gene regulatory networks and signaling cascades. Here, we discuss how kidney developmental studies have informed the definition of procedures to obtain kidney organoids from human pluripotent stem cells (hPSCs). Moreover, bioengineering techniques have emerged as potential solutions to externally impose controlled microenvironments for organoid generation from hPSCs. Next, we summarize some of these advances with major focus On recent works merging hPSC-derived kidney organoids (hPSC-kidney organoids) with organ-on-chip to develop robust models for drug discovery and disease modeling applications. We foresee that, in the near future, coupling of different organoid models through bioengineering approaches will help advancing to recreate organ-to-organ crosstalk to increase our understanding on kidney disease progression in the human context and search for new therapeutics.Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.

JTD Keywords: Animal, Animals, Bioengineering, Cell differentiation, Embryo development, Embryology, Embryonic structures, Gene regulatory network, Human, Humans, Kidney, Kidney development, Kidney mesenchyme cell, Kidney organoid, Mammal, Mammals, Mesenchyme, Metanephric mesenchyme, Microenvironment, Nephron, Nephrons, Organoid, Organoids, Physiology, Pluripotent stem cell, Pluripotent stem cells, Review, Signal transduction, Ureteric bud

The accurate spatial segregation into distinct phases within cell membranes coordinates vital biochemical processes and functionalities in living organisms. One of nature's strategies to localize reactivity is the formation of dynamic raft domains. Most raft models rely on liquid-ordered L-0 phases in a liquid-disordered L-d phase lacking correlation and remaining static, often necessitating external agents for phase separation. Here, we introduce a synthetic system of bicomponent glycodendrimersomes coassembled from Janus dendrimers and Janus glycodendrimers (JGDs), where lactose-lactose interactions exclusively drive lateral organization. This mechanism results in modulated phases across two length scales, yielding raft-like microdomains featuring nanoarrays at the nanoscale. By varying the density of lactose and molecular architecture of JGDs, the nanoarray type and size, shape, and spacing of the domains were controlled. Our findings offer insight into the potential primordial origins of rudimentary raft domains and highlight the crucial role of glycans within the glycocalyx.

JTD Keywords: Article, Artificial cells, Atomic force microscopy, Bicomponents, Bilayer, Bilayer membrane, Biochemical functionality, Biochemical process, Biological-membranes, Cell component, Cell membrane, Cellular parameters, Chemical interaction, Chemical structure, Chemistry, Cytology, Defined janus glycodendrimers, Dehydration, Dendrimer, Dendrimers, Dilution, Dimer, External agents, Fourier transform, Giant vesicles, Glycan, Glycans, Glycocalyx, Glycodendrimers, Janus dendrimer, Janus glycodendrimer, Lactose, Lateral organization, Lectin, Lipid rafts, Living organisms, Membrane damage, Membrane microdomain, Membrane microdomains, Membrane structure, Metabolism, Modulated phases, Molecule, Monomer, Nanoarrays, Oligosaccharide, Organization, Periodicity, Phase separation, Phase-separation, Phospholipids, Polysaccharide, Polysaccharides, Raft like domain, Relative humidity, Spatial segregation, Structure analysis, Sugars, Synthetic systems, Tetramer, Unclassified drug, Unilamellar vesicles, Water

Charge exchange is the fundamental process that sustains cellular respiration and photosynthesis by shuttling electrons in a cascade of electron transfer (ET) steps between redox cofactors. While intraprotein charge exchange is well characterized in protein complexes bearing multiple redox sites, interprotein processes are less understood due to the lack of suitable experimental approaches and the dynamic nature of the interactions. Proteins constrained between electrodes are known to support electron transport (ETp) through the protein matrix even without redox cofactors, as the charges housed by the redox sites in ET are furnished by the electrodes. However, it is unknown whether protein ETp mechanisms apply to the interprotein medium present under physiological conditions. We study interprotein charge exchange between plant photosystem I (PSI) and its soluble redox partner plastocyanin (Pc) and address the role of the Pc copper center. Using electrochemical scanning tunneling spectroscopy (ECSTS) current-distance and blinking measurements, we quantify the spatial span of charge exchange between individual Pc/PSI pairs and ETp through transient Pc/PSI complexes. Pc devoid of the redox center (Pcapo) can exchange charge with PSI at longer distances than with the copper ion (Pcholo). Conductance bursts associated with Pcapo/PSI complex formation are higher than in Pcholo/PSI. Thus, copper ions are not required for long-distance Pc/PSI ETp but regulate its spatial span and conductance. Our results suggest that the redox center that carries the charge in Pc is not necessary to exchange it in interprotein ET through the aqueous solution and question the canonical view of tight complex binding between redox protein partners.

JTD Keywords: azurin, binding, blinking, crystal-structure, cupredoxin, current distance spectroscopy, electrochemical tunneling microscopy, proteinconductance, reduction, single metalloprotein, single molecule measurements, site, spectroscopy, Blinking, Cupredoxin, Current distance spectroscopy, Electrochemical tunneling microscopy, Interprotein electron transfer, Protein conductance, Single molecule measurements, State electron-transport

The intestine is a complex tissue with a characteristic three-dimensional (3D) crypt-villus architecture, which plays a key role in the intestinal function. This function is also regulated by the intestinal stroma that actively supports the intestinal epithelium, maintaining the homeostasis of the tissue. Efforts to account for the 3D complex structure of the intestinal tissue have been focused mainly in mimicking the epithelial barrier, while solutions to include the stromal compartment are scarce and unpractical to be used in routine experiments. Here we demonstrate that by employing an optimized bioink formulation and the suitable printing parameters it is possible to produce fibroblast-laden crypt-villus structures by means of digital light projection stereolithography (DLP-SLA). This process provides excellent cell viability, accurate spatial resolution, and high printing throughput, resulting in a robust biofabrication approach that yields functional gut mucosa tissues compatible with conventional testing techniques.Copyright © 2023 Elsevier B.V. All rights reserved.

JTD Keywords: 3d microstructure, barrier, cells, epithelial-stromal interactions, gelma-pegda soft hydrogels, growth, hydrogel, intestinal mucosa model, methacrylamide, microfabrication, proliferation, scaffold, stereolithography, 3d bioprinting, 3d microstructure, Epithelial-stromal interactions, Fibroblasts, Gelma-pegda soft hydrogels, Intestinal mucosa model

Gap junction channels (GJCs) mediate intercellular communication by connecting two neighbouring cells and enabling direct exchange of ions and small molecules. Cell coupling via connexin-43 (Cx43) GJCs is important in a wide range of cellular processes in health and disease (Churko and Laird, 2013; Liang et al., 2020; Poelzing and Rosenbaum, 2004), yet the structural basis of Cx43 function and regulation has not been determined until now. Here, we describe the structure of a human Cx43 GJC solved by cryo-EM and single particle analysis at 2.26 Å resolution. The pore region of Cx43 GJC features several lipid-like densities per Cx43 monomer, located close to a putative lateral access site at the monomer boundary. We found a previously undescribed conformation on the cytosolic side of the pore, formed by the N-terminal domain and the transmembrane helix 2 of Cx43 and stabilized by a small molecule. Structures of the Cx43 GJC and hemichannels (HCs) in nanodiscs reveal a similar gate arrangement. The features of the Cx43 GJC and HC cryo-EM maps and the channel properties revealed by molecular dynamics simulations suggest that the captured states of Cx43 are consistent with a closed state.© 2023, Qi, Acosta Gutierrez et al.

JTD Keywords: cryo-em, dehydroepiandrosterone dhea, expression, gap junction channel, gene, gja1 mutations, hemichannel, membrane protein, phenotype, protein, structure, system, visualization, Biochemistry, Chemical biology, Connexin-43, Cryo-em, Gap junction channel, Hemichannel, Human, Membrane protein, Molecular biophysics, Oculodentodigital dysplasia, Structural biology, Structure

Live cell actin visualization is fundamental for exploring cellular motility, cytokinesis, intracellular transport, and other correlated functions. The current imaging techniques that allow imaging of actin in its native environment are optical and electron microscopy. Such imaging techniques offer high enough resolution to investigate the ultrastructure of actin however they come at the expense of actin integrity. Inspired by the lack of suitable probes that preserve actin's integrity, we designed a cyclometalated Ir (III) complex that interacts with live cells and displays light switch behaviour upon specific actin binding. The exceptional photophysical properties of the proposed probe allow unprecedented resolution of cytoskeleton ultrastructures under stimulated emission depletion (STED) super-resolution nanoscopy. Moreover, the Ir complex enables the capability of visualizing actin polymers and periodicity under correlative light electron microscopy (CLEM) and liquid-phase electron microscopy (LPEM) at similar to 8 nm resolution.

JTD Keywords: Actin dynamics, Actin targeting, Adhesion, Cells, Clem, Fluorescent, Iridium (iii) complex, Lead, Light, Lpem, Super-resolution ultrastructures

Previously, functional coatings on 3D-printed titanium implants were developed to improve their biointegration by separately incorporating Ga and Ag on the biomaterial surface. Now, a thermochemical treatment modification is proposed to study the effect of their simultaneous incorporation. Different concentrations of AgNO3 and Ga(NO3)3 are evaluated, and the obtained surfaces are completely characterized. Ion release, cytotoxicity, and bioactivity studies complement the characterization. The provided antibacterial effect of the surfaces is analyzed, and cell response is assessed by the study of SaOS-2 cell adhesion, proliferation, and differentiation. The Ti surface doping is confirmed by the formation of Ga-containing Ca titanates and nanoparticles of metallic Ag within the titanate coating. The surfaces generated with all combinations of AgNO3 and Ga(NO3)3 concentrations show bioactivity. The bacterial assay confirms a strong bactericidal impact achieved by the effect of both Ga and Ag present on the surface, especially for Pseudomonas aeruginosa, one of the main pathogens involved in orthopedic implant failures. SaOS-2 cells adhere and proliferate on the Ga/Ag-doped Ti surfaces, and the presence of gallium favors cell differentiation. The dual effect of both metallic agents doping the titanium surface provides bioactivity while protecting the biomaterial from the most frequent pathogens in implantology.

JTD Keywords: 3d-printing, agent, antibacterial activity, bioactive ti, biomaterials, coatings, competition, cu, gallium, glasses, ions, metal, porous structures, promote osseointegration, silver, titanium implants, In-vitro, Porous structures, Titanium implants

JTD Keywords: deep generative models, eatris european infrastructure for translational medicine, integrative omics, machine learning, multi-omics, personalized medicine, Deep generative models, Eatris european infrastructure for translational medicine, Integrative omics, Machine learning, Multi-omics, Personalized medicine, Protein protein interaction (ppi) network

Actual polymeric wound closure devices are not optimal for load-bearing applications due to the low mechanical properties and the risk of inflammation and bacterial infection mainly produced by multifil-ament and braided configurations. Biodegradable metallic Zn alloys are promising materials candidates; however, mechanical performance, corrosion behaviour, and biological response should be controlled in order to inhibit the risk of inflammation and bacterial infection. To this end, a Zn-2Ag (2 wt% Ag) alloy was processed by ECAP to evaluate the concurrent combined effect of grain refinement and Ag alloying on biodegradation and antibacterial activity. Two ECAP cycles were successfully applied to a Zn-2Ag alloy obtaining a homogeneous ultra-fine-grained structure in which nanoindentation maps suggested isotro-pic mechanical properties. Lower UTS and YS with higher elongation was reported after ECAP with similar corrosion rates as before processing. ECAP processed samples showed a homogeneous Ag+ release below the minimum inhibitory concentration for S. Aureus and no antibacterial effect was observed by diffusion. As expected, the presence of Ag in Zn-Ag alloys reduced bacterial attachment. Nevertheless, ECAP processed Zn-2Ag provided an excellent antibacterial activity after 3 h probably caused by the uniformly degraded and thus, non- stable, surface observed after bacterial adhesion.(c) 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

JTD Keywords: Behavior, Binary alloys, Biodegradable zinc-alloys, Biomaterials, Equal channel angular pressing, Grain-refinement, In-vitro degradation, Mg, Microstructure, Nanoindentation, Progress, Staphylococcus-aureus, Temperature superplasticity, Ultrafine-grained materials, Zinc alloys, Zn alloys

Supramolecular systems chemistry has been an area of active research to develop nanomaterials with life-like functions. Progress in systems chemistry relies on our ability to probe the nanostructure formation in solution. Often visualizing the dynamics of nanostructures which transform over time is a formidable challenge. This necessitates a paradigm shift from dry sample imaging towards solution-based techniques. We review the application of state-of-the-art techniques for real-time, in situ visualization of dynamic self-assembly processes. We present how solution-based techniques namely optical super-resolution microscopy, solution-state atomic force microscopy, liquid-phase transmission electron microscopy, molecular dynamics simulations and other emerging techniques are revolutionizing our understanding of active and adaptive nanomaterials with life-like functions. This Review provides the visualization toolbox and futuristic vision to tap the potential of dynamic nanomaterials.© 2022 Wiley-VCH GmbH.

JTD Keywords: electron-microscopy, fluorescence microscopy, in-situ, mechanical-properties, molecular simulations, nanostructures, polymerization, polymers, stimulated-emission, super-resolution microscopy, supramolecular chemistry, systems chemistry, water, Atomic-force microscopy, Liquid tem, Nanostructures, Super-resolution microscopy, Supramolecular chemistry, Systems chemistry

Human induced pluripotent stem cell (hiPSC) technologies offer a unique resource for modeling neurological diseases. However, iPSC models are fraught with technical limitations including abnormal aggregation and inefficient maturation of differentiated neurons. These problems are in part due to the absence of synergistic cues of the native extracellular matrix (ECM). We report on the use of three artificial ECMs based on peptide amphiphile (PA) supramolecular nanofibers. All nanofibers display the laminin-derived IKVAV signal on their surface but differ in the nature of their non-bioactive domains. We find that nanofibers with greater intensity of internal supramolecular motion have enhanced bioactivity toward hiPSC-derived motor and cortical neurons. Proteomic, biochemical, and functional assays reveal that highly mobile PA scaffolds caused enhanced β1-integrin pathway activation, reduced aggregation, increased arborization, and matured electrophysiological activity of neurons. Our work highlights the importance of designing biomimetic ECMs to study the development, function, and dysfunction of human neurons.Copyright © 2022 Elsevier Inc. All rights reserved.

JTD Keywords: differentiation, force-field, laminin, migration, nanostructures, peptide amphiphiles, spinal-cord, statistical-model, supramolecular materials, Coarse-grained model, Dynamics, Extracellular matrix, Ikvav, Ipsc-derived neurons, Laminin, Neuronal maturation, Peptide amphiphiles, Supramolecular motion, Supramolecular nanofibers

Tissue engineering is nowadays a powerful tool to restore damaged tissues and recover their normal functionality. Advantages over other current methods are well established, although a continuous evolution is still necessary to improve the final performance and the range of applications. Trends are nowadays focused on the development of multifunctional scaffolds with hierarchical structures and the capability to render a sustained delivery of bioactive molecules under an appropriate stimulus. Nanocomposites incorporating hydroxyapatite nanoparticles (HAp NPs) have a predominant role in bone tissue regeneration due to their high capacity to enhance osteoinduction, osteoconduction, and osteointegration, as well as their encapsulation efficiency and protection capability of bioactive agents. Selection of appropriated polymeric matrices is fundamental and consequently great efforts have been invested to increase the range of properties of available materials through copolymerization, blending, or combining structures constituted by different materials. Scaffolds can be obtained from different processes that differ in characteristics, such as texture or porosity. Probably, electrospinning has the greater relevance, since the obtained nanofiber membranes have a great similarity with the extracellular matrix and, in addition, they can easily incorporate functional and bioactive compounds. Coaxial and emulsion electrospinning processes appear ideal to generate complex systems able to incorporate highly different agents. The present review is mainly focused on the recent works performed with Hap-loaded scaffolds having at least one structural layer composed of core/shell nanofibers.

JTD Keywords: bone tissue, coaxial electrospinning, composite nanofibers, drug-release behavior, emulsion electrospinning, hydroxyapatite, in-vitro evaluation, mechanical-properties, osteogenic differentiation, pickering emulsions, protein adsorption, structured scaffolds, surface-initiated polymerization, tissue regeneration, Bone tissue, Coaxial electrospinning, Emulsion electrospinning, Hydroxyapatite, Multifunctional scaffolds, Poly(3-hydroxybutyrate) phb patches, Tissue regeneration

Multiplexed assays of variant effects (MAVEs) guide clinical variant interpretation and reveal disease mechanisms. To date, MAVEs have focussed on a single mutation type-amino acid (AA) substitutions-despite the diversity of coding variants that cause disease. Here we use Deep Indel Mutagenesis (DIM) to generate a comprehensive atlas of diverse variant effects for a disease protein, the amyloid beta (Aβ) peptide that aggregates in Alzheimer's disease (AD) and is mutated in familial AD (fAD). The atlas identifies known fAD mutations and reveals that many variants beyond substitutions accelerate Aβ aggregation and are likely to be pathogenic. Truncations, substitutions, insertions, single- and internal multi-AA deletions differ in their propensity to enhance or impair aggregation, but likely pathogenic variants from all classes are highly enriched in the polar N-terminal region of Aβ. This comparative atlas highlights the importance of including diverse mutation types in MAVEs and provides important mechanistic insights into amyloid nucleation.© 2022. The Author(s).

JTD Keywords: amyloid-beta(1-42), determinants, disease, mutants, protein, secondary nucleation, Atomic-resolution structure

Hydroxyapatite nanoparticles are popular tools in bone regeneration, but they have also been used for gene delivery and as anticancer drugs. Understanding their mechanism of action, particularly for the latter application, is crucial to predict their toxicity. To this end, we aimed to elucidate the importance of nanoparticle membrane interactions in the cytotoxicity of MG-63 cells using two different types of nanoparticles. In addition, conventional techniques for studying nanoparticle internalisation were evaluated and compared with newer and less exploited approaches. Hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles were used as suspensions or compacted as specular discs. Comparison between cells seeded on the discs and those supplemented with the nanoparticles allowed direct interaction of the cell membrane with the material to be ruled out as the main mechanism of toxicity. In addition, standard techniques such as flow cytometry were inconclusive when used to assess nanoparticles toxicity. Interestingly, the use of intracellular calcium fluorescent probes revealed the presence of a high number of calcium-rich vesicles after nanoparticle supplementation in cell culture. These structures could not be detected by transmission electron microscopy due to their liquid content. However, by using cryo-soft X-ray imaging, which was used to visualise the cellular ultrastructure without further treatment other than vitrification and to quantify the linear absorption coefficient of each organelle, it was possible to identify them as multivesicular bodies, potentially acting as calcium stores. In the study, an advanced state of degradation of the hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles within MG-63 cells was observed. Overall, we demonstrate that the combination of fluorescent calcium probes together with cryo-SXT is an excellent approach to investigate intracellular calcium, especially when found in its soluble form.Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.

JTD Keywords: adsorption, cryo-soft x-ray tomography, cytotoxicity, expression, flow cytometry, internalisation, intracellular calcium, magnesium, nano, nanomaterials, nanoparticles, proliferation, protein corona, ultrastructure, Calcium-phosphate nanoparticles, Cryo-soft x-ray tomography, Flow cytometry, Hydroxyapatite, Internalisation, Intracellular calcium, Nanoparticles

The synthesis and study of the tripeptide Arg-Gly-Asp (RGD), the binding site of different extracellular matrix proteins, e.g., fibronectin and vitronectin, has allowed the production of a wide range of cell adhesive surfaces. Although the surface density and spacing of the RGD peptide at the nanoscale have already shown a significant influence on cell adhesion, the impact of its hierarchical nanostructure is still rather unexplored. Accordingly, a versatile colloidal system named quatsomes, based on fluid nanovesicles formed by the self-assembling of cholesterol and surfactant molecules, has been devised as a novel template to achieve hierarchical nanostructures of the RGD peptide. To this end, RGD was anchored on the vesicle's fluid membrane of quatsomes, and the RGD-functionalized nanovesicles were covalently anchored to planar gold surfaces, forming a state of quasi-suspension, through a long poly(ethylene glycol) (PEG) chain with a thiol termination. An underlying self-assembled monolayer (SAM) of a shorter PEG was introduced for vesicle stabilization and to avoid unspecific cell adhesion. In comparison with substrates featuring a homogeneous distribution of RGD peptides, the resulting hierarchical nanoarchitectonic dramatically enhanced cell adhesion, despite lower overall RGD molecules on the surface. The new versatile platform was thoroughly characterized using a multitechnique approach, proving its enhanced performance. These findings open new methods for the hierarchical immobilization of biomolecules on surfaces using quatsomes as a robust and novel tissue engineering strategy.

JTD Keywords: activation, arg-gly-asp (rgd), cell adhesion, extracellular-matrix, growth, integrins, ligands, nanopatterns, quatsomes, scaffolds, self-assembled monolayers, surface engineering, tissue engineering, Arg-gly-asp (rgd), Cell adhesion, Integrins, Nano-structured surfaces, Nanovesicles, Quatsomes, Self-assembled monolayers, Surface engineering, Tissue engineering

The emerging field of synthetic developmental biology proposes bottom-up approaches to examine the contribution of each cellular process to complex morphogenesis. However, the shortage of tools to manipulate three-dimensional (3D) shapes of mammalian tissues hinders the progress of the field. Here we report the development of OptoShroom3, an optogenetic tool that achieves fast spatiotemporal control of apical constriction in mammalian epithelia. Activation of OptoShroom3 through illumination in an epithelial Madin-Darby Canine Kidney (MDCK) cell sheet reduces the apical surface of the stimulated cells and causes displacements in the adjacent regions. Light-induced apical constriction provokes the folding of epithelial cell colonies on soft gels. Its application to murine and human neural organoids leads to thickening of neuroepithelia, apical lumen reduction in optic vesicles, and flattening in neuroectodermal tissues. These results show that spatiotemporal control of apical constriction can trigger several types of 3D deformation depending on the initial tissue context.© 2022. The Author(s).

JTD Keywords: build, developmental biology, disease, light, localization, multicellular structures, organization, plate, shroom, Epithelial-cell shape

Peptide derivatives and, most specifically, their self-assembled supramolecular structures are being considered in the design of novel biofunctional materials. Although the self-assembly of triphenylalanine homopeptides has been found to be more versatile than that of homopeptides containing an even number of residues (i.e. diphe-nylalanine and tetraphenylalanine), only uncapped triphenylalanine (FFF) and a highly aromatic analog blocked at both the N-and C-termini with fluorenyl-containing groups (Fmoc-FFF-OFm), have been deeply studied before. In this work, we have examined the self-assembly of a triphenylalanine derivative bearing 9-fluorenylme-thyloxycarbonyl and benzyl ester end-capping groups at the N-and C-termini, respectively (Fmoc-FFF-OBzl). The antiparallel arrangement clearly dominates in beta-sheets formed by Fmoc-FFF-OBzl, whereas the parallel and antiparallel dispositions are almost isoenergetic in Fmoc-FFF-OFm beta-sheets and the parallel one is slightly favored for FFF. The effects of both the peptide concentration and the mediu m on the self-assembly process have been examined considering Fmoc-FFF-OBzl solutions in a wide variety of solvent:co-solvent mixtures. In addi-tion, Fmoc-FFF-OBzl supramolecular structures have been compared to those obtained for FFF and Fmoc-FFF-OFm under identical experimental conditions. The strength of pi-pi stacking interactions involving the end-capping groups plays a crucial role in the nucleation and growth of supramolecular structures, which de-termines the resulting morphology. Finally, the influence of a non-invasive external stimulus, ultrasounds, on the nucleation and growth of supramolecular structures has been examined. Overall, FFF-based peptides provide a wide range of supramolecular structures that can be of interest in the biotechnological field.

JTD Keywords: aromatic interactions, beta-sheet, hierarchical structures, phenylalanine homopeptides, supramolecular structures, Amino-acids, Aromatic interactions, Beta-sheet, Fmoc, Hierarchical struc tures, Hydrogels, Phenylalanine homopeptides, Solvent, Spectroscopy, Supramolecular structures, Triphenylalanine

3DNA holds promise as a carrier for drugs that can be intercalated into its core or linked to surface arms. Coupling 3DNA to an antibody targeting intercellular adhesion molecule 1 (ICAM-1) results in high lung-specific biodistributions in vivo. While the role of individual parameters on ICAM-1 targeting has been studied for other nanocarriers, it has never been examined for 3DNA or in a manner capable of revealing the hierarchic interplay among said parameters. In this study, we used 2-layer vs. 4-layer anti-ICAM 3DNA and radiotracing to examine biodistribution in mice. We found that, below saturating conditions and within the ranges tested, the density of targeting antibodies on 3DNA is the most relevant parameter driving lung targeting over liver clearance, compared to the number of antibodies per carrier, total antibody dose, 3DNA dose, 3DNA size, or the administered concentration, which influenced the dose in organs but not the lung specific-over-liver clearance ratio. Data predicts that lung-specific delivery of intercalating (core loaded) drugs can be tuned using this biodistribution pattern, while that of arm-linked (surface loaded) drugs requires a careful parametric balance because increasing anti-ICAM density reduces the number of 3DNA arms available for drug loading.

JTD Keywords: 3dna nanocarrier, acid sphingomyelinase, antibody, carrier design parameters, carriers, dna nanostructures, doxorubicin, drug type, icam-1, inflammation, lung targeting, multiparametric hierarchy, nanoparticles, size, 3dna nanocarrier, Intracellular delivery, Multiparametric hierarchy

The influence of the properties of different solid substrates on the tethering of two antibodies, IgG1-CR3022 and IgG1-S309, which were specifically engineered for the detection of SARS-CoV-2, has been examined at the molecular level using conventional and accelerated Molecular Dynamics (cMD and aMD, respectively). Two surfaces with very different properties and widely used in immunosensors for diagnosis, amorphous silica and the most stable facet of the face-centered cubic gold structure, have been considered. The effects of such surfaces on the structure and orientation of the immobilized antibodies have been determined by quantifying the tilt and hinge angles that describe the orientation and shape of the antibody, respectively, and the dihedrals that measure the relative position of the antibody arms with respect to the surface. Results show that the interactions with amorphous silica, which are mainly electrostatic due to the charged nature of the surface, help to preserve the orientation and structure of the antibodies, especially of the IgG1-CR3022, indicating that the primary sequence of those antibodies also plays some role. Instead, short-range van der Waals interactions with the inert gold surface cause a higher degree tilting and fraying of the antibodies with respect to amorphous silica. The interactions between the antibodies and the surface also affect the correlation among the different angles and dihedrals, which increases with their strength. Overall, results explain why amorphous silica substrates are frequently used to immobilize antibodies in immunosensors. © 2022 The Authors

JTD Keywords: amorphous silica, antibody immobilization, enzyme, gol d, gold, immobilization, immunosensor, molecu l a r dynamics, molecular dynamics, protein adsorption, sars-cov-2 immunosensor, simulations, spike protein, surface interactions, target, vaccine, Amorphous silica, Antibodies, Antibody engineering, Antibody immobilization, Antibody structure, Article, Chemical detection, Computer model, Controlled study, Dihedral angle, Gold, In-silico, Molecular dynamics, Molecular levels, Molecular-dynamics, Nonhuman, Property, Sars, Sars-cov-2 immunosensor, Severe acute respiratory syndrome coronavirus 2, Silica, Silico studies, Silicon dioxide, Solid substrates, Structure analysis, Substrate chemistry, Substrates, Van der waals forces, Virus detection

Visual impairments are a critical medical hurdle to be addressed in modern society. Müller glia (MG) have regenerative potential in the retina in lower vertebrates, but not in mammals. However, in mice, in vivo cell fusion between MG and adult stem cells forms hybrids that can partially regenerate ablated neurons.We used organotypic cultures of human retina and preparations of dissociated cells to test the hypothesis that cell fusion between human MG and adult stem cells can induce neuronal regeneration in human systems. Moreover, we established a microinjection system for transplanting human retinal organoids to demonstrate hybrid differentiation.We first found that cell fusion occurs between MG and adult stem cells, in organotypic cultures of human retina as well as in cell cultures. Next, we showed that the resulting hybrids can differentiate and acquire a proto-neural electrophysiology profile when the Wnt/beta-catenin pathway is activated in the adult stem cells prior fusion. Finally, we demonstrated the engraftment and differentiation of these hybrids into human retinal organoids.We show fusion between human MG and adult stem cells, and demonstrate that the resulting hybrid cells can differentiate towards neural fate in human model systems. Our results suggest that cell fusion-mediated therapy is a potential regenerative approach for treating human retinal dystrophies.This work was supported by La Caixa Health (HR17-00231), Velux Stiftung (976a) and the Ministerio de Ciencia e Innovación, (BFU2017-86760-P) (AEI/FEDER, UE), AGAUR (2017 SGR 689, 2017 SGR 926).Published by Elsevier B.V.

JTD Keywords: cell fusion, expression, fusion, ganglion-cells, in-vitro, mouse, müller glia, neural differentiation, organoids, regeneration, retina regeneration, stem cells, stromal cells, transplantation, 4',6 diamidino 2 phenylindole, 5' nucleotidase, Agarose, Alcohol, Arpe-19 cell line, Article, Beta catenin, Beta tubulin, Bone-marrow-cells, Bromophenol blue, Buffer, Calcium cell level, Calcium phosphate, Calretinin, Canonical wnt signaling, Cd34 antigen, Cell culture, Cell fusion, Cell viability, Coculture, Complementary dna, Confocal microscopy, Cornea transplantation, Cryopreservation, Cryoprotection, Crystal structure, Current clamp technique, Dimethyl sulfoxide, Dodecyl sulfate sodium, Edetic acid, Electrophysiology, Endoglin, Fetal bovine serum, Fibroblast growth factor 2, Flow cytometry, Fluorescence activated cell sorting, Fluorescence intensity, Glyceraldehyde 3 phosphate dehydrogenase, Glycerol, Glycine, Hoe 33342, Immunofluorescence, Immunohistochemistry, Incubation time, Interleukin 1beta, Lentivirus vector, Matrigel, Mercaptoethanol, Microinjection, Mueller cell, Müller glia, N methyl dextro aspartic acid, Nerve cell differentiation, Neural differentiation, Nitrogen, Nonhuman, Organoids, Paraffin, Paraffin embedding, Paraformaldehyde, Patch clamp technique, Penicillin derivative, Phenolsulfonphthalein, Phenotype, Phosphate buffered saline, Phosphoprotein phosphatase inhibitor, Polyacrylamide gel electrophoresis, Potassium chloride, Povidone iodine, Promoter region, Proteinase inhibitor, Real time polymerase chain reaction, Receptor type tyrosine protein phosphatase c, Restriction endonuclease, Retina, Retina dystrophy, Retina regeneration, Retinol, Rhodopsin, Rna extraction, Stem cell, Stem cells, Subcutaneous fat, Tunel assay, Visual impairment, Western blotting

The characterization of newly synthesized materials is a cornerstone of all chemistry and nanotechnology laboratories. For this purpose, a wide array of analytical techniques have been standardized and are used routinely by laboratories across the globe. With these methods we can understand the structure, dynamics and function of novel molecular architectures and their relations with the desired performance, guiding the development of the next generation of materials. Moreover, one of the challenges in materials chemistry is the lack of reproducibility due to improper publishing of the sample preparation protocol. In this context, the recent adoption of the reporting standard MIRIBEL (Minimum Information Reporting in Bio–Nano Experimental Literature) for material characterization and details of experimental protocols aims to provide complete, reproducible and reliable sample preparation for the scientific community. Thus, MIRIBEL should be immediately adopted in publications by scientific journals to overcome this challenge. Besides current standard spectroscopy and microscopy techniques, there is a constant development of novel technologies that aim to help chemists unveil the structure of complex materials. Among them super-resolution microscopy (SRM), an optical technique that bypasses the diffraction limit of light, has facilitated the study of synthetic materials with multicolor ability and minimal invasiveness at nanometric resolution. Although still in its infancy, the potential of SRM to unveil the structure, dynamics and function of complex synthetic architectures has been highlighted in pioneering reports during the last few years. Currently, SRM is a sophisticated technique with many challenges in sample preparation, data analysis, environmental control and automation, and moreover the instrumentation is still expensive. Therefore, SRM is currently limited to expert users and is not implemented in characterization routines. This perspective discusses the potential of SRM to transition from a niche technique to a standard routine method for material characterization. We propose a roadmap for the necessary developments required for this purpose based on a collaborative effort from scientists and engineers across disciplines.

JTD Keywords: blinking, fluorophore, intramolecular spirocyclization, localization, nanoparticles, resolution limit, reveals, single-molecule fluorescence, stimulated-emission, Characterization techniques, Diffraction, Distributed computer systems, Environmental management, Information reporting, Material chemistry, Materials characterization, Minimum information, Optical reconstruction microscopy, Optical resolving power, Sample preparation, Structure dynamics, Structure functions, Super-resolution microscopy, Synthesized materials

The mechanical signals sensed by the alveolar cells through the changes in the local matrix stiffness of the extracellular matrix (ECM) are determinant for regulating cellular functions. Therefore, the study of the mechanical response of lung tissue becomes a fundamental aspect in order to further understand the mechanosensing signals perceived by the cells in the alveoli. This study is focused on the development of a finite element (FE) model of a decellularized rat lung tissue strip, which reproduces accurately the mechanical behaviour observed in the experiments by means of a tensile test. For simulating the complex structure of the lung parenchyma, which consists of a heterogeneous and non-uniform network of thin-walled alveoli, a 3D model based on a Voronoi tessellation is developed. This Voronoi-based model is considered very suitable for recreating the geometry of cellular materials with randomly distributed polygons like in the lung tissue. The material model used in the mechanical simulations of the lung tissue was characterized experimentally by means of AFM tests in order to evaluate the lung tissue stiffness on the micro scale. Thus, in this study, the micro (AFM test) and the macro scale (tensile test) mechanical behaviour are linked through the mechanical simulation with the 3D FE model based on Voronoi tessellation. Finally, a micro-mechanical FE-based model is generated from the Voronoi diagram for studying the stiffness sensed by the alveolar cells in function of two independent factors: the stretch level of the lung tissue and the geometrical position of the cells on the extracellular matrix (ECM), distinguishing between pneumocyte type I and type II. We conclude that the position of the cells within the alveolus has a great influence on the local stiffness perceived by the cells. Alveolar cells located at the corners of the alveolus, mainly type II pneumocytes, perceive a much higher stiffness than those located in the flat areas of the alveoli, which correspond to type I pneumocytes. However, the high stiffness, due to the macroscopic lung tissue stretch, affects both cells in a very similar form, thus no significant differences between them have been observed. © 2021 The Authors

JTD Keywords: rat, scaffolds, stiffness, Afm, Animal cell, Animal experiment, Animal model, Animal tissue, Article, Biological organs, Cell function, Cells, Computational geometry, Cytology, Extracellular matrices, Extracellular matrix, Extracellular-matrix, Geometry, High stiffness, Human, Lung alveolus cell type 1, Lung alveolus cell type 2, Lung parenchyma, Lung tissue, Male, Mechanical behavior, Mechanical modeling, Mechanical simulations, Mechanosensing, Model-based opc, Nonhuman, Physical model, Rat, Rigidity, Stiffness, Stiffness matrix, Tensile testing, Thin walled structures, Three dimensional finite element analysis, Tissue, Type ii, Voronoi tessellations

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

Over the last years, optical biosensors based on plasmonic nanomaterials have gained great scientific interest due to their unquestionable advantages compared to other biosensing technologies. They can achieve sensitive, direct, and label-free analysis with exceptional potential for multiplexing and miniaturization. Recently, it has been demonstrated the potential of using optical discs as high throughput nanotemplates for the development of plasmonic biosensors in a cost-effective way. This work is a pilot study focused on the development of an integrated plasmonic biosensor for the monitoring of cell adhesion and growth of human retinal pigmented cell line (ARPE-19) under different media conditions (0 and 2% of FBS). We observed an increase of the plasmonic band displacement under 2% FBS compared to 0% conditions over time (1, 3, and 5 h). These preliminary results show that the proposed plasmonic biosensing approach is a direct, non-destructive, and real-time tool that could be employed in the study of living cells behavior and culture conditions. Furthermore, this setup could assess the viability of the cells and their growth over time with low variability between the technical replicates improving the experimental replicability.

JTD Keywords: cell confluency, cell culture, nanocrystals, optical biosensor, Adhesion monitoring, Biosensing, Biosensors, Cell adhesion, Cell confluency, Cell culture, Cells, Condition, Cost effectiveness, Disposables, Nano-structured, Nanocrystals, Optical bio-sensors, Optical biosensor, Plasmonic biosensors, Plasmonic nanostructures, Plasmonics, Polylysine

In the recent decades, zinc (Zn) and its alloys have been drawing attention as promising candidates for bioresorbable cardiovascular stents due to its degradation rate more suitable than magnesium (Mg) and iron (Fe) alloys. However, its mechanical properties need to be improved in order to meet the criteria for vascular stents. This work investigates the mechanical properties, biodegradability and biocompatibility of Zn-Mg and Zn-Cu alloys in order to determine a proper alloy composition for optimal stent performance. Nanoindentation measurements are performed to characterize the mechanical properties at the nanoscale as a function of the Zn microstructure variations induced by alloying. The biodegradation mechanisms are discussed and correlated to microstructure, mechanical performance and bacterial/cell response. Addition of Mg or Cu alloying elements refined the microstructure of Zn and enhanced yield strength (YS) and ultimate tensile strength (UTS) proportional to the volume fraction of secondary phases. Zn-1Mg showed the higher YS and UTS and better performance in terms of degradation stability in Hanks’ solution. Zn-Cu alloys presented an antibacterial effect for S. aureus controlled by diffusion mechanisms and by contact. Biocompatibility was dependent on the degradation rate and the nature of the corrosion products.

JTD Keywords: behavior, biocompatibility, biodegradable metals, bioresorbable metals, bioresorbable scaffold, copper, corrosion properties, elastic-modulus, galvanic corrosion, microstructure, nanoindentation, redox homeostasis, zinc, Biocompatibility, Bioresorbable metals, Galvanic corrosion, Nanoindentation, Room-temperature superplasticity, Zinc alloys

3D printing is a competitive manufacturing technology, which has opened up new possibilities for the fabrication of complex ceramic structures and customised parts. Extrusion-based technologies, also known as direct ink writing (DIW) or robocasting, are amongst the most used for ceramic materials. In them, the rheological properties of the ink play a crucial role, determining both the extrudability of the paste and the shape fidelity of the printed parts. However, comprehensive rheological studies of printable ceramic inks are scarce and may be difficult to understand for non-specialists. The aim of this review is to provide an overview of the main types of ceramic ink formulations developed for DIW and a detailed description of the more relevant rheological tests for assessing the printability of ceramic pastes. Moreover, the key rheological parameters are identified and linked to printability aspects, including the values reported in the literature for different ink compositions.

JTD Keywords: 3-dimensional structures, behavior, deposition, direct ink writing, freeform fabrication, gelation, glass scaffolds, mechanical-properties, printability, rheology, robocasting, suspensions, 3d printing, Direct ink writing, Phosphate scaffolds, Printability, Rheology, Robocasting

Abstract The development of nanostructured plasmonic biosensors has been widely widespread in the last years, motivated by the potential benefits they can offer in integration, miniaturization, multiplexing opportunities, and enhanced performance label-free biodetection in a wide field of applications. Between them, engineering tissues represent a novel, challenging, and prolific application field for nanostructured plasmonic biosensors considering the previously described benefits and the low levels of secreted biomarkers (?pM–nM) to detect. Here, we present an integrated plasmonic nanocrystals-based biosensor using high throughput nanostructured polycarbonate substrates. Metallic film thickness and incident angle of light for reflectance measurements were optimized to enhance the detection of antibody–antigen biorecognition events using numerical simulations. We achieved an enhancement in biodetection up to 3× as the incident angle of light decreases, which can be related to shorter evanescent decay lengths. We achieved a high reproducibility between channels with a coefficient of variation below 2% in bulk refractive index measurements, demonstrating a high potential for multiplexed sensing. Finally, biosensing potential was demonstrated by the direct and label-free detection of interleukin-6 biomarker in undiluted cell culture media supernatants from bioengineered 3D skeletal muscle tissues stimulated with different concentrations of endotoxins achieving a limit of detection (LOD) of ? 0.03 ng/mL (1.4 pM).

JTD Keywords: assay, crystals, drug, label-free biosensing, molecules, plasmonic nanostructures, sensors, skeletal muscle, tissue engineering, Biodetection, Biomarkers, Biosensors, Cell culture, Cells, Chemical detection, Histology, Interleukin-6, Interleukin6 (il6), Label free, Label-free biosensing, Muscle, Nano-structured, Nanocrystals, Plasmonic nanocrystals, Plasmonic nanostructures, Plasmonics, Polycarbonate substrates, Polycarbonates, Refractive index, Sensitivity, Skeletal muscle, Tissue engineering, Tissues engineerings

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

One of the major problems faced by metallic implants is the high probability of bacterial infections, with significant consequences for the patient. In this work, a thermochemical treatment is proposed to obtain silver-doped calcium titanate coatings on the Ti surface to improve the bioactivity of porous 3D-printed Ti structures and simultaneously provide them with antibacterial properties. A complete characterization of the new coating, the study of the ion release and the analysis of its cytotoxicity were carried out together with evaluation of the natural apatite forming in simulated body fluid (SBF). Moreover, the antibacterial properties of the coatings were assessed against Pseudomona aeruginosa and Escherichia coli as gram-negative and Staphylococcus aureus and Staphylococcus epidermidis as gram-positive bacterial strains. Ag ions were integrated into the Ca titanate layer and Ag nanoparticles were formed within the entire 3D Ti surface. Ca and Ag ions were released from both porous and solid samples into the Hanks' solution for 48 h. The treated surfaces showed no cytotoxicity and an apatite layer precipitated on the entire porous surface when the samples were immersed in SBF. The release of Ag from the surface had a strong antibacterial effect and prevented bacterial adhesion and proliferation on the surface. Moreover, the nanostructured topography of the coating resulted also in a reduction of bacterial adhesion and proliferation, even in absence of Ag. In conclusion, the cost-effective approach here reported provided protection against the most predominant bacterial colonizers to the Ti porous implants, while maintaining their bioactivity.

JTD Keywords: 3d-printing, alkaline, antibacterial activity, arthroplasty, bacterial adhesion, biomaterials, generation, ions, nanoparticles, osseointegration, silver, surface-layer, titanium implants, toxicity, 3d-printing, Antibacterial activity, Biomaterials, Porous structures, Silver, Ti metal, Titanium implants

Biomimetic calcium-deficient hydroxyapatite (CDHA) as a bioactive material exhibits exceptional intrinsic osteoinductive and osteogenic properties because of its nanostructure and composition, which promote a favorable microenvironment. Its high reactivity has been hypothesized to play a relevant role in the in vivo performance, mediated by the interaction with the biological fluids, which is amplified by its high specific surface area. Paradoxically, this high reactivity is also behind the in vitro cytotoxicity of this material, especially pro-nounced in static conditions. The present work explores the structural and physicochemical changes that CDHA undergoes in contact with physiological fluids and to investigate its interaction with proteins. Calcium-deficient hydroxyapatite discs with different micro/nanostructures, coarse (C) and fine (F), were exposed to cell-free complete culture medium over extended periods of time: 1, 7, 14, 21, 28, and 50 days. Precipitate formation was not observed in any of the materials in contact with the physiological fluid, which would indicate that the ionic exchanges were linked to incorporation into the crystal structure of CDHA or in the hydrated layer. In fact, CDHA experienced a maturation process, with a progressive increase in crystallinity and the Ca/P ratio, accompanied by an uptake of Mg and a B-type carbonation process, with a gradual propagation into the core of the samples. However, the reactivity of biomimetic hydroxyapatite was highly dependent on the specific surface area and was amplified in nanosized needle-like crystal structures (F), whereas in coarse specimens the ionic exchanges were restricted to the surface, with low penetration in the material bulk. In addition to showing a higher protein adsorption on F substrates, the proteomics study revealed the existence of protein selectivity to-ward F or C microstructures, as well as the capability of CDHA, and more remarkably of F-CDHA, to concentrate specific proteins from the culture medium. Finally, a substantial improvement in the material's ability to support cell proliferation was observed after the CDHA maturation process.

JTD Keywords: calcium phosphates, ion exchange, nanostructure, protein adsorption, Biological-systems, Biomaterials, Biomimetic hydroxyapatites, Biomimetics, Bone-formation, Calcium deficient hydroxyapatite, Calcium phosphate, Calcium phosphates, Cell proliferation, Crystal structure, Crystallinity, Crystals structures, Culture medium, Growth, High reactivity, Hydroxyapatite, In-vitro, Ion exchange, Ionic exchange, Molecular biology, Nanocrystalline apatites, Nanostructure, Nanostructures, Octacalcium phosphate, Physicochemical studies, Physiological fluids, Physiology, Protein adsorption, Proteins, Proteomic studies, Raman spectroscopy, Serum-albumin, Specific surface area

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved. Cells sense mechanical forces from their environment, but the precise mechanical variable sensed by cells is unclear. Here, the authors show that cells can sense the rate of force application, known as the loading rate, with effects on YAP nuclear localization and cytoskeletal stiffness remodelling.

JTD Keywords: Actin cytoskeleton, Actin filament, Actin-filament, Adhesion, Animal, Animals, Atomic force microscopy, Breathing, Cell, Cell adhesion, Cell culture, Cell nucleus, Cells, cultured, Cytoplasm, Extracellular-matrix, Fibroblast, Fibroblasts, Fibronectin, Frequency, Gene knockdown, Gene knockdown techniques, Genetics, Germfree animal, Integrin, Intracellular signaling peptides and proteins, Knockout mouse, Lung, Male, Mechanotransduction, Mechanotransduction, cellular, Metabolism, Mice, Mice, knockout, Microscopy, atomic force, Mouse, Optical tweezers, Paxillin, Physiology, Primary cell culture, Pxn protein, mouse, Rat, Rats, Rats, sprague-dawley, Respiration, Signal peptide, Softening, Specific pathogen-free organisms, Sprague dawley rat, Stress, Substrate, Substrate rigidity, Talin, Talin protein, mouse, Tln2 protein, mouse, Traction, Transmission, Ultrastructure, Yap1 protein, rat

Polymer nanocomposite materials based on metallic nanowires are widely investigated as transparent and flexible electrodes or as stretchable conductors and dielectrics for biosensing. Here we show that Scanning Dielectric Microscopy (SDM) can map the depth distribution of metallic nanowires within the nanocomposites in a non-destructive way. This is achieved by a quantitative analysis of sub-surface electrostatic force microscopy measurements with finite-element numerical calculations. As an application we determined the three-dimensional spatial distribution of ?50 nm diameter silver nanowires in ?100 nm-250 nm thick gelatin films. The characterization is done both under dry ambient conditions, where gelatin shows a relatively low dielectric constant, ?r ? 5, and under humid ambient conditions, where its dielectric constant increases up to ?r ? 14. The present results show that SDM can be a valuable non-destructive subsurface characterization technique for nanowire-based nanocomposite materials, which can contribute to the optimization of these materials for applications in fields such as wearable electronics, solar cell technologies or printable electronics. © The Royal Society of Chemistry.

JTD Keywords: composite, constant, electrodes, mode, nanostructures, objects, progress, subsurface, tomography, Composite materials, Dielectric materials, Electric force microscopy, Electrostatic force, Force microscopy, Low dielectric constants, Nanocomposites, Numerical calculation, Polymer nanocomposite, Printable electronics, Scanning dielectric microscopy, Silver nanowires, Solar cell technology, Stretchable conductors, Subsurface characterizations, Transparent electrodes, Wearable technology

The human olfactory system remains one of the most challenging biological systems to replicate. Humans use it without thinking, where it can equally offer protection from harm and bring enjoyment in equal measure. It is the system’s ability to detect and analyze complex odors, without the need for specialized infra-structure, that is the envy of many scientists. The field of artificial olfaction has recruited and stimulated interdisciplinary research and commercial development for several applications that include malodor measurement, medical diagnostics, food and beverage quality, environment and security. Over the last century, innovative engineers and scientists have been focused on solving a range of problems associated with measurement and control of odor. The IEEE Sensors Journal has published Special Issues on olfaction in 2002 and 2012. Here we continue that coverage. In this article, we summarize early work in the 20th Century that served as the foundation upon which we have been building our odor-monitoring instrumental and measurement systems. We then examine the current state of the art that has been achieved over the last two decades as we have transitioned into the 21st Century. Much has been accomplished, but great progress is needed in sensor technology, system design, product manufacture and performance standards. In the final section, we predict levels of performance and ubiquitous applications that will be realized during in the mid to late 21st Century.

JTD Keywords: air-quality, breath analysis, calibration transfer, chemical sensor arrays, chemosensor arrays, drift compensation, electronic nose, gas sensors, headspace sampling, machine learning, machine olfaction, odor detection, plume structure, voc analysis, Artificial olfaction, Electrodes, Electronic nose, Electronic nose technology, Headspace sampling, Instruments, Machine learning, Machine olfaction, Monitoring, Odor detection, Olfactory, Sensor phenomena and characterization, Sensors, Temperature sensors, Voc analysis

BACKGROUND: The embryo implantation process is crucial for the correct establishment and progress of pregnancy. During implantation, the blastocyst trophectoderm cells attach to the epithelium of the endometrium, triggering intense cell-to-cell crosstalk that leads to trophoblast outgrowth, invasion of the endometrial tissue, and formation of the placenta. However, this process, which is vital for embryo and foetal development in utero, is still elusive to experimentation because of its inaccessibility. Experimental implantation is cumbersome and impractical in adult animal models and is inconceivable in humans. OBJECTIVE AND RATIONALE: A number of custom experimental solutions have been proposed to recreate different stages of the implantation process in vitro, by combining a human embryo (or a human embryo surrogate) and endometrial cells (or a surrogate for the endometrial tissue). In vitro models allow rapid high-throughput interrogation of embryos and cells, and efficient screening of molecules, such as cytokines, drugs, or transcription factors, that control embryo implantation and the receptivity of the endometrium. However, the broad selection of available in vitro systems makes it complicated to decide which system best fits the needs of a specific experiment or scientific question. To orient the reader, this review will explore the experimental options proposed in the literature, and classify them into amenable categories based on the embryo/cell pairs employed. The goal is to give an overview of the tools available to study the complex process of human embryo implantation, and explain the differences between them, including the advantages and disadvantages of each system. SEARCH METHODS: We performed a comprehensive review of the literature to come up with different categories that mimic the different stages of embryo implantation in vitro, ranging from initial blastocyst apposition to later stages of trophoblast invasion or gastrulation. We will also review recent breakthrough advances on stem cells and organoids, assembling embryo-like structures and endometrial tissues. OUTCOMES: We highlight the most relevant systems and describe the most significant experiments. We focus on in vitro systems that have contributed to the study of human reproduction by discovering molecules that control implantation, including hormones, signalling molecules, transcription factors and cytokines. WIDER IMPLICATIONS: The momentum of this field is growing thanks to the use of stem cells to build embryo-like structures and endometrial tissues, and the use of bioengineering to extend the life of embryos in culture. We propose to merge bioengineering methods derived from the fields of stem cells and reproduction to develop new systems covering a wider window of the implantation process.

JTD Keywords: in vitro models, blastocyst, blastocyst-like structures, early-pregnancy, endometrial cells, epidermal-growth-factor, gene-expression, implantation, in vitro models, in-vitro model, indian hedgehog, organoids, receptivity, self-organization, spheroids, trophoblast, trophoblast invasion, uterine receptivity, Blastocyst, Blastocyst-like structures, Early-pregnancy, Endometrial cells, Endometrial stromal cells, Epidermal-growth-factor, Gene-expression, Implantation, In vitro models, In-vitro model, Indian hedgehog, Organoids, Receptivity, Self-organization, Spheroids, Trophoblast, Trophoblast invasion, Uterine receptivity

Porous biodegradable scaffolds provide a physical substrate for cells allowing them to attach, proliferate and guide the formation of new tissues. A variety of techniques have been developed to fabricate tissue engineering (TE) scaffolds, among them the most relevant is the thermally-induced phase separation (TIPS). This technique has been widely used in recent years to fabricate three-dimensional (3D) TE scaffolds. Low production cost, simple experimental procedure and easy processability together with the capability to produce highly porous scaffolds with controllable architecture justify the popularity of TIPS. This paper provides a general overview of the TIPS methodology applied for the preparation of 3D porous TE scaffolds. The recent advances in the fabrication of porous scaffolds through this technique, in terms of technology and material selection, have been reviewed. In addition, how properties can be effectively modified to serve as ideal substrates for specific target cells has been specifically addressed. Additionally, examples are offered with re-spect to changes of TIPS procedure parameters, the combination of TIPS with other techniques and innovations in polymer or filler selection.

JTD Keywords: biodegradable polymer, composites, morphology, pore structure, porosity, processing parameters, thermally induced phase separation, Biodegradable polymer, Composites, Morphology, Pore structure, Porosity, Processing parameters, Thermally induced phase separation, Tissue engineering scaffold

© 2020 Hydrolytic degradation of poly(4-hydroxybutyrate) (P4HB) films has been studied considering media of different pH values (i.e., 3, 7 and 10) and temperatures (i.e., 37 and 55 °C). Enzymatic degradation has also been evaluated at physiological conditions using two different lipases: Pseudomonas cepacia and Rhizopus oryzae. Different bulk and surface erosion mechanisms with random chain scissions and successive removal of monomer units have been supported through weight loss measurements, molecular weight determinations by GPC and NMR spectroscopy and changes on thermal properties by DSC. Thermal annealing during exposure to different media and even degradation influenced on the melting temperature and crystallinity of samples, as well as on the lamellar geometrical parameters as evaluated by SAXS. Enzymatic degradation was ideal to selectively eliminate the amorphous regions and highlight the spherulitic morphology. Presence of ringed textures were therefore evident in bright field optical micrographs in addition to SEM images, namely observations under polarized light was not necessary to distinguish the presence of banded spherulites. Rhizopus oryzae was revealed to be the most suitable enzyme to crop out the P4HB spherulites that form part of the initial smooth surfaces of solvent casting films. After determining the appropriate activity and exposure time, the presence of rings constituted by cooperative C-shaped edge-on lamellae and flat-on lamellae was highlighted.

JTD Keywords: biodegradable polymers, enzymatic degradation, films, hydrolytic degradation, microstructure, thermal properties, Biodegradable polymers, Enzymatic degradation, Films, Hydrolytic degradation, Microstructure, Poly(4-hydroxybutyrate), Thermal properties

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

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

Blends with different ratios of polylactide and polyamide 6,10 (PA610) have been prepared by melt-mixing using a Brabender mixer equipment. Previously, a rheologically modified polylactide (PLAREx) was obtained through reactive extrusion using a multifunctional epoxide agent. It was expected that unreacted epoxy groups of PLAREx were able to improve the compatibility between the two polymers. SEM observations revealed a logical dependence of the morphology of immiscible phases with composition, and more interestingly a co-continuity at relatively low PA content (around 50%) was detected. This result contrasts with previous observations performed with non-modified PLA. Confined PA domains increased with the PA content and hardly crystallized at the typical crystallization temperature of the pure PA (195 °C). Synchrotron X-ray diffraction studies indicated that a PA crystallization at a lower temperature close to 120 °C was enhanced and led to a pseudohexagonal γ phase that differs from the characteristic layered structure of PA610. SAXS data revealed also that well differentiated lamellar entities could be assigned at both immiscible polymer phases. Clear differences were observed in the spherulitic morphologies attained under isothermal melt crystallization experiments. Results indicated that the texture of PLAREx spherulites was modified by the presence of PA. Compatibilization of PA molecules on the crystal lamellar boundaries of PLAREx led to an enhancement of the lamellar twisting frequency. Optical microscopy results also indicated that the crystal growth rate of PLAREx increased by the incorporation of PA, but in contrast this had an adverse effect on the nucleation process.

JTD Keywords: Crystal growth rate, Epoxy modified polylactide, Nucleation, Polyamide 6,10, Polyamide crystalline structure, Polyamide/polylactide blend morphology, Thermal properties

Gallium (Ga) has been recently proposed as a novel therapeutic agent, since it promotes bone formation and exhibits antibacterial properties. This work focuses on the optimization of a thermochemical treatment that incorporates Ga ions by the addition of the body-friendly Ga nitrate approved by the Food and Drug Administration. The objective was to simultaneously provide the inner and the outer surfaces of porous‑titanium surfaces obtained by 3D-printing with bioactivity and antibacterial properties. The apatite-forming ability of the coating, as well as the antibacterial activity and SaOS-2 cell adhesion, proliferation, differentiation and mineralization were evaluated and compared with untreated Ti surfaces. The characterization of the surfaces revealed the presence of a Ga-containing calcium titanate layer, which was non cytotoxic and in simulated body fluid produced a homogeneous apatite coating well adhered to the substrate. The formation of this apatite layer was accelerated with increasing Ga amounts present on the surface, resulting also in an increase in thickness. An initial quick release of Ga ion promoted the antibacterial effect against gram positive strains, especially for Pseudomonas aeruginosa, one of the most frequent resistant pathogens in nosocomial infections. SaOS-2 cells adhered and proliferated on the Ga-doped Ti surfaces, its presence contributed to cell differentiation and to considerably increase the mineralization levels. Thus, the developed multifunctional coatings could provide bioactivity to the porous Ti implants while protecting them from the most frequent gram-negative pathogens.

JTD Keywords: 3D-printing, Antibacterial activity, Biomaterials, Gallium, Porous structures, Titanium implants

A comprehensive study of progressively ground/polished 3Y-TZP was performed with the aim of better understanding the mechanisms driving the microstructural modifications observed after such procedures, and identifying the processing parameters leading to optimal microstructures (i.e. ageing-protective and damage-free). Gradually ground/polished surfaces were produced, yielding four different topographies of increasing roughness (grades 1–4) and two different textures (unidirectionally, U, and multidirectionally, M). Phase transformation, microstructure and residual stresses were investigated by means of advanced characterization techniques. It was found that low-roughness mildly ground/polished specimens (i.e. 2-M/U) presented a nanometric layer with the ageing-related protective features generally associated with coarsely ground specimens. A lower limit for grain refinement in terms of surface abrasion was also found, in which partial recrystallization took place (i.e. 1-M/U). A mathematical relation was established between average surface roughness (Sa), monoclinic volume fraction (Vm) and surface compressive residual stresses, demonstrating that if the processing parameters are controlled, both Vm and residual stresses can be predicted by the measurement of Sa.

JTD Keywords: Grinding, Microstructure, Phase transformation, Residual stresses, Zirconia

In this work, we study the mechanics of metamaterial sheets inspired by the pellicle of Euglenids. They are composed of interlocking elastic rods which can freely slide along their edges. We characterize the kinematics and the mechanics of these structures using the special Cosserat theory of rods and by assuming axisymmetric deformations of the tubular assembly. Through an asymptotic expansion, we investigate both structures that comprise a discrete number of rods and the limit case of a sheet composed by infinitely many rods. We apply our theoretical framework to investigate the stability of these structures in the presence of an axial load. Through a linear analysis, we compute the critical buckling force for both the discrete and the continuous case. For the latter, we also perform a numerical post-buckling analysis, studying the non-linear evolution of the bifurcation through finite elements simulations.

JTD Keywords: Biomimetic structures, Elastic structures, Helical rods, Mechanical instabilities, Metamaterials, Post-buckling analysis

Bone apatite consists of carbonated calcium-deficient hydroxyapatite (CDHA) nanocrystals. Biomimetic routes allow fabricating synthetic bone grafts that mimic biological apatite. In this work, we explored the role of two distinctive features of biomimetic apatites, namely, nanocrystal morphology (plate vs needle-like crystals) and carbonate content, on the bone regeneration potential of CDHA scaffolds in an in vivo canine model. Both ectopic bone formation and scaffold degradation were drastically affected by the nanocrystal morphology after intramuscular implantation. Fine-CDHA foams with needle-like nanocrystals, comparable in size to bone mineral, showed a markedly higher osteoinductive potential and a superior degradation than chemically identical coarse-CDHA foams with larger plate-shaped crystals. These findings correlated well with the superior bone-healing capacity showed by the fine-CDHA scaffolds when implanted intraosseously. Moreover, carbonate doping of CDHA, which resulted in small plate-shaped nanocrystals, accelerated both the intrinsic osteoinduction and the bone healing capacity, and significantly increased the cell-mediated resorption. These results suggest that tuning the chemical composition and the nanostructural features may allow the material to enter the physiological bone remodeling cycle, promoting a tight synchronization between scaffold degradation and bone formation.

JTD Keywords: Biomimetic, Calcium phosphate, Carbonated apatite, Foaming, Nanostructure, Osteogenesis, Osteoinduction

DNA nanostructures hold great potential for drug delivery. However, their specific targeting is often compromised by recognition by scavenger receptors involved in clearance. In our previous study in cell culture, we showed targeting specificity of a 180 nm, 4-layer DNA-built nanocarrier called 3DNA coupled with antibodies against intercellular adhesion molecule-1 (ICAM-1), a glycoprotein overexpressed in the lungs in many diseases. Here, we examined the biodistribution of various 3DNA formulations in mice. A formulation consisted of 3DNA whose outer-layer arms were hybridized to secondary antibody-oligonucleotide conjugates. Anchoring IgG on this formulation reduced circulation and kidney accumulation vs. non-anchored IgG, while increasing liver and spleen clearance, as expected for a nanocarrier. Anchoring anti-ICAM changed the biodistribution of this antibody similarly, yet this formulation specifically accumulated in the lungs, the main ICAM-1 target. Since lung targeting was modest (2-fold specificity index over IgG formulation), we pursued a second preparation involving direct hybridization of primary antibody-oligonucleotide conjugates to 3DNA. This formulation had prolonged stability in serum and showed a dramatic increase in lung distribution: the specificity index was 424-fold above a matching IgG formulation, 144-fold more specific than observed for PLGA nanoparticles of similar size, polydispersity, ζ-potential and antibody valency, and its lung accumulation increased with the number of anti-ICAM molecules per particle. Immunohistochemistry showed that anti-ICAM and 3DNA components colocalized in the lungs, specifically associating with endothelial markers, without apparent histological changes. The degree of in vivo targeting for anti-ICAM/3DNA-nanocarriers is unprecedented, for which this platform technology holds great potential to develop future therapeutic applications.

JTD Keywords: 3DNA, DNA nanostructure, Drug nanocarrier, Endothelial and lung targeting, ICAM-1, In vivo biodistribution

This paper deals with a concept for a reconfigurable structure bio-inspired by the cell wall architecture of euglenids, a family of unicellular protists, and based on the relative sliding of adjacent strips. Uniform sliding turns a cylinder resulting from the assembly of straight and parallel strips into a cylinder of smaller height and larger radius, in which the strips are deformed into a family of parallel helices. We examine the mechanics of this cylindrical assembly, in which the interlocking strips are allowed to slide freely at their junctions, and compute the external forces (axial force and axial torque at the two ends, or pressure on the lateral surface) necessary to drive and control the shape changes of the composite structure. Despite the simplicity of the structure, we find a remarkably complex mechanical behaviour that can be tuned by the spontaneous curvature or twist of the strips.

JTD Keywords: Bio-inspired structures, Euglenoid pellicle, Helical bundles, Morphing structures, Reconfigurable structures

Cannabis is the most used illicit substance in the world. As many countries are moving towards decriminalization, it is crucial to determine whether and how cannabis use affects human brain and behavior. The role of the cerebellum in cognition, emotion, learning, and addiction is increasingly recognized. Because of its high density in CB1 receptors, it is expected to be highly affected by cannabis use. The aim of this systematic review is to investigate how cannabis use affects cerebellar structure and function, as well as cerebellar-dependent behavioral tasks. Three databases were searched for peer-reviewed literature published until March 2018. We included studies that focused on cannabis effects on cerebellar structure, function, or cerebellar-dependent behavioral tasks. A total of 348 unique records were screened, and 40 studies were included in the qualitative synthesis. The most consistent findings include (1) increases in cerebellar gray matter volume after chronic cannabis use, (2) alteration of cerebellar resting state activity after acute or chronic use, and (3) deficits in memory, decision making, and associative learning. Age of onset and higher exposure to cannabis use were frequently associated with increased cannabis-induced alterations. Chronic cannabis use is associated with alterations in cerebellar structure and function, as well as with deficits in behavioral paradigms that involve the cerebellum (eg, eyeblink conditioning, memory, and decision making). Future studies should consider tobacco as confounding factor and use standardized methods for assessing cannabis use. Paradigms exploring the functional activity of the cerebellum may prove useful as monitoring tools of cannabis-induced impairment.

JTD Keywords: Behavior, Cannabis use, Cerebellum, Cognitive function, Structure

Emerging nanobiotechnologies can offer solutions to the current and future challenges in medicine. By covering topics from regenerative medicine, tissue engineering, drug delivery, bionanofabrication, and molecular biorecognition, this Special Issue aims to provide an update on the trends in nanomedicine and drug delivery using biomimetic approaches, and the development of novel biologically inspired devices for the safe and effective diagnosis, prevention, and treatment of disease.

JTD Keywords: Bioinspired nanotechnologies, Bionanofabrication, Bio-nano measurement and microscopy, Nanomaterials for biological and medical applications, Nanoassemblies, Nanostructured surfaces, Drug delivery, Nanobioelectronics, Integrated systems/nanobiosensors, Nanotoxicology, Graphene-based applications

The first steps of nickel electrodeposition in a deep eutectic solvent (DES) are analyzed in detail. Several substrates from glassy carbon to Pt(111) were investigated pointing out the surface sensitivity of the nucleation and growth mechanism. For that, cyclic voltammetry and chronoamperometry, in combination with scanning electron microscopy (SEM), were employed. X-ray diffraction (XRD) and atomic force microscopy (AFM) were used to more deeply analyze the Ni deposition on Pt substrates. In a 0.1 M NiCl2 + DES solution (at 70 °C), the nickel deposition on glassy carbon takes place within the potential limits of the electrode in the blank solution. Although, the electrochemical window of Pt|DES is considerably shorter than on glassy carbon|DES, it was still sufficient for the nickel deposition. On the Pt electrode, the negative potential limit was enlarged while the nickel deposit grew, likely because of the lower catalytic activity of the nickel toward the reduction of the DES. At lower overpotentials, different hydrogenated Ni structures were favored, most likely because of the DES co-reduction on the Pt substrate. Nanometric metallic nickel grains of rounded shape were obtained on any substrate, as evidenced by the FE-SEM. Passivation phenomena, related to the formation of Ni oxide and Ni hydroxylated species, were observed at high applied overpotentials. At low deposited charge, on Pt(111) the AFM measurements showed the formation of rounded nanometric particles of Ni, which rearranged and formed small triangular arrays at sufficiently low applied overpotential. This particle pattern was induced by the (111) orientation and related to surface sensitivity of the nickel deposition in DES. The present work provides deep insights into the Ni electrodeposition mechanism in the selected deep eutectic solvent.

JTD Keywords: AFM, Deep eutectic solvent, Glassy carbon, Nanostructures, Nickel electrodeposition, Platinum electrode, Pt(111), SEM, Surface sensitive

Metal oxide semiconductor (MOX) sensors are usually temperature-modulated and calibrated with multivariate models such as Partial Least Squares (PLS) to increase the inherent low selectivity of this technology. The multivariate sensor response patterns exhibit heteroscedastic and correlated noise, which suggests that maximum likelihood methods should outperform PLS. One contribution of this paper is the comparison between PLS and maximum likelihood principal components regression (MLPCR) in MOX sensors. PLS is often criticized by the lack of interpretability when the model complexity increases beyond the chemical rank of the problem. This happens in MOX sensors due to cross-sensitivities to interferences, such as temperature or humidity and non-linearity. Additionally, the estimation of fundamental figures of merit, such as the limit of detection (LOD), is still not standardized in multivariate models. Orthogonalization methods, such as Orthogonal Projection to Latent Structures (O-PLS), have been successfully applied in other fields to reduce the complexity of PLS models. In this work, we propose a LOD estimation method based on applying the well-accepted univariate LOD formulas to the scores of the first component of an orthogonal PLS model. The resulting LOD is compared to the multivariate LOD range derived from error-propagation. The methodology is applied to data extracted from temperature-modulated MOX sensors (FIS SB-500-12 and Figaro TGS 3870-A04), aiming at the detection of low concentrations of carbon monoxide in the presence of uncontrolled humidity (chemical noise). We found that PLS models were simpler and more accurate than MLPCR models. Average LOD values of 0.79 ppm (FIS) and 1.06 ppm (Figaro) were found using the approach described in this paper. These values were contained within the LOD ranges obtained with the error-propagation approach. The mean LOD increased to 1.13 ppm (FIS) and 1.59 ppm (Figaro) when considering validation samples collected two weeks after calibration, which represents a 43% and 46% degradation, respectively. The orthogonal score-plot was a very convenient tool to visualize MOX sensor data and to validate the LOD estimates.

JTD Keywords: Metal oxide sensors, Partial least squares, Orthogonal projection to latent structures, Maximum likelihood principal component regression, Limit of detection, Temperature modulation

Some biomaterials are osteoinductive, that is, they are able to trigger the osteogenic process by inducing the differentiation of mesenchymal stem cells to the osteogenic lineage. Although the underlying mechanism is still unclear, microporosity and specific surface area (SSA) have been identified as critical factors in material-associated osteoinduction. However, only sintered ceramics, which have a limited range of porosities and SSA, have been analyzed so far. In this work, we were able to extend these ranges to the nanoscale, through the foaming and 3D-printing of biomimetic calcium phosphates, thereby obtaining scaffolds with controlled micro- and nanoporosity and with tailored macropore architectures. Calcium-deficient hydroxyapatite (CDHA) scaffolds were evaluated after 6 and 12 weeks in an ectopic-implantation canine model and compared with two sintered ceramics, biphasic calcium phosphate and β-tricalcium phosphate. Only foams with spherical, concave macropores and not 3D-printed scaffolds with convex, prismatic macropores induced significant ectopic bone formation. Among them, biomimetic nanostructured CDHA produced the highest incidence of ectopic bone and accelerated bone formation when compared with conventional microstructured sintered calcium phosphates with the same macropore architecture. Moreover, they exhibited different bone formation patterns; in CDHA foams, the new ectopic bone progressively replaced the scaffold, whereas in sintered biphasic calcium phosphate scaffolds, bone was deposited on the surface of the material, progressively filling the pore space. In conclusion, this study demonstrates that the high reactivity of nanostructured biomimetic CDHA combined with a spherical, concave macroporosity allows the pushing of the osteoinduction potential beyond the limits of microstructured calcium phosphate ceramics.

JTD Keywords: 3D-printing, Calcium phosphate, Foaming, Nanostructure, Osteoinduction

Biological membranes mediate several biological processes that are directly associated with their physical properties but sometimes difficult to evaluate. Supported lipid bilayers (SLBs) are model systems widely used to characterize the structure of biological membranes. Cholesterol (Chol) plays an essential role in the modulation of membrane physical properties. It directly influences the order and mechanical stability of the lipid bilayers, and it is known to laterally segregate in rafts in the outer leaflet of the membrane together with sphingolipids (SLs). Atomic force microscope (AFM) is a powerful tool as it is capable to sense and apply forces with high accuracy, with distance and force resolution at the nanoscale, and in a controlled environment. AFM-based force spectroscopy (AFM-FS) has become a crucial technique to study the nanomechanical stability of SLBs by controlling the liquid media and the temperature variations. In this contribution, we review recent AFM and AFM-FS studies on the effect of Chol on the morphology and mechanical properties of model SLBs, including complex bilayers containing SLs. We also introduce a promising combination of AFM and X-ray (XR) techniques that allows for in situ characterization of dynamic processes, providing structural, morphological, and nanomechanical information

JTD Keywords: Atomic force microscopy, Force spectroscopy, Lipid membranes, Supported lipid bilayers, Nanomechanics, Cholesterol, Sphingolipids, Membrane structure, XR-AFM combination

Metabotropic glutamate receptors (mGluRs) are important drug targets because of their involvement in several neurological diseases. Among mGluRs, mGlu5 is a particularly high-profile target because its positive or negative allosteric modulation can potentially treat schizophrenia or anxiety and chronic pain, respectively. Here, we computationally and experimentally probe the functional binding of a novel photoswitchable mGlu5 NAM, termed alloswitch-1, which loses its NAM functionality under violet light. We show alloswitch-1 binds deep in the allosteric pocket in a similar fashion to mavoglurant, the co-crystallized NAM in the mGlu5 transmembrane domain crystal structure. Alloswitch-1, like NAM 2-Methyl-6-(phenylethynyl)pyridine (MPEP), is significantly affected by P655M mutation deep in the allosteric pocket, eradicating its functionality. In MD simulations, we show alloswitch-1 and MPEP stabilize the co-crystallized water molecule located at the bottom of the allosteric site that is seemingly characteristic of the inactive receptor state. Furthermore, both NAMs form H-bonds with S809 on helix 7, which may constitute an important stabilizing interaction for NAM-induced mGlu5 inactivation. Alloswitch-1, through isomerization of its amide group from trans to cis is able to form an additional interaction with N747 on helix 5. This may be an important interaction for amide-containing mGlu5 NAMs, helping to stabilize their binding in a potentially unusual cis-amide state. Simulated conformational switching of alloswitch-1 in silico suggests photoisomerization of its azo group from trans to cis may be possible within the allosteric pocket. However, photoexcited alloswitch-1 binds in an unstable fashion, breaking H-bonds with the protein and destabilizing the co-crystallized water molecule. This suggests photoswitching may have destabilizing effects on mGlu5 binding and functionality.

JTD Keywords: Allosteric modulation, Docking, Metabotropic glutamate receptor, Molecular dynamics, Mutation, Protein structure, Transmembrane domain

The main therapeutic and prophylactic tools against malaria have been locked for more than a century in the classical approaches of using drugs targeting metabolic processes of the causing agent, the protist Plasmodium spp., and of designing vaccines against chosen antigens found on the parasite’s surface. Given the extraordinary resources exhibited by Plasmodium to escape these traditional strategies, which have not been able to free humankind from the scourge of malaria despite much effort invested in them, new concepts have to be explored in order to advance toward eradication of the disease. In this context, amyloid-forming proteins and peptides found in the proteome of the pathogen should perhaps cease being regarded as mere anomalous molecules. Their likely functionality in the pathophysiology of Plasmodium calls for attention being paid to them as a possible Achilles’ heel of malaria. Here we will give an overview of Plasmodium-encoded amyloid-forming polypeptides as potential therapeutic targets and toxic elements, particularly in relation to cerebral malaria and the blood–brain barrier function. We will also discuss the recent finding that the genome of the parasite contains an astonishingly high proportion of prionogenic domains.

JTD Keywords: Amyloids, Intrinsically unstructured proteins, Malaria, Prions

It is known that cells respond strongly to microtopography. However, cellular mechanisms of response are unclear. Here, we study wild-type fibroblasts responding to 25 μm2 posts and compare their response to that of FAK-/- fibroblasts and fibroblasts with PMA treatment to stimulate protein kinase C (PKC) and the small g-protein Rac. FAK knockout cells modulated adhesion number and size in a similar way to cells on topography; that is, they used more, smaller adhesions, but migration was almost completely stalled demonstrating the importance of FAK signaling in contact guidance and adhesion turnover. Little similarity, however, was observed to PKC stimulated cells and cells on the topography. Interestingly, with PKC stimulation the cell nuclei became highly deformable bringing focus on these surfaces to the study of metastasis. Surfaces that aid the study of cellular migration are important in developing understanding of mechanisms of wound healing and repair in aligned tissues such as ligament and tendon.

JTD Keywords: Adhesion, Cell migration, Cell morphology, Focal adhesion kinase, Microstructures

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

Traditional Sleep Structure Indexes (TSSIs) are insufficient to identify patterns of altered sleep. TSSIs mainly account for absolute time measures, but different levels of state instability may lead to similar absolute time distribution. Therefore, sleep stability remains beyond the scope of TSSIs. However, recent studies suggest that sleep disorders may be rather influenced by a breakdown in the sleep-stage switching mechanisms. In this study, we propose a set of 11 Sleep Transition Indexes (STIs) that characterize sleep fragmentation and account for the state-stability governed by the ultradian, homeostatic and circadian rhythms. We demonstrate that most of the proposed STIs are potential markers of SAHS severity, while TSSIs are not. In addition, we provide a new framework to analyze sleep disorders from the direct perspective of sleep regulatory mechanisms. In particular, our results indicate that SAHS may be influenced by a dysregulation of homeostatic rhythms but not of ultradian or circadian rhythms.

JTD Keywords: SAHS, Sleep Transitions, Sleep Structure, Polysomnography, Hypnogram

The present study reports a novel approach for the design and fabrication of polylactic acid (PLA) microparticle-based scaffolds with microstructural properties suitable for bone and cartilage regeneration. Macroporous PLA scaffolds with controlled shape were fabricated by means of a semicontinuous process involving (1) microfluidic emulsification of a PLA/ethyl lactate solution (5% w/v) in a span 80/paraffin oil solution (3% v/v) followed by (2) particles coagulation/assembly in an acetone/water solution for the development of a continuous matrix. Porous scaffolds prepared from particles with monomodal or bimodal size distribution, overall porosity ranges from 93 to 96%, interparticles porosity from 41 to 54%, and static compression moduli from 0.3 to 1.4 MPa were manufactured by means of flow rate modulation of of the continuous phase during emulsion. The biological response of the scaffolds was assessed in vitro by using bone marrow-derived rat mesenchymal stem cells (MSCs). The results demonstrated the ability of the scaffolds to support the extensive and uniform three-dimensional adhesion, colonization, and proliferation of MSCs within the entire construct.

JTD Keywords: Green solvent, Microfluidic, Microstructure, Stem cells, Scaffold

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

Biosensors' research filed has clearly been changing towards the production of multifunctional and innovative design concepts to address the needs related with sensitivity and selectivity of the devices. More recently, waveguide biosensors, that do not require any label procedure to detect biomolecules adsorbed on its surface, have been pointed out as one of the most promising technologies for the production of biosensing devices with enhanced performance. Moreover the combination of optical and electrochemical measurements through the integration of transparent and conducting oxides in the multilayer structures can greatly enhance the biosensors' sensitivity. Furthermore, the integration of polymeric substrates may bring powerful advantages in comparison with silicon based ones. The biosensors will have a lower production costs being possible to disposable them after use ("one use sensor chip"). This research work represents a preliminary study about the influence of substrate temperature on the overall properties of ITO thin films deposited by DC magnetron sputtering onto 0,5 mm thick PMMA sheets.

JTD Keywords: ITO thin films, PMMA sheets, Waveguide biosensing devices, Biosensing devices, Conducting oxides, Dc magnetron sputtering, Electrochemical measurements, Enhanced performance, Innovative design, ITO thin films, Multilayer structures, Overall properties, PMMA sheets, Polymeric substrate, Production cost, Sensor chips, Silicon-based, Substrate temperature, Biosensors, Deposition, Design, Film preparation, Optical multilayers, Thin films, Vapor deposition, Waveguides, Substrates

In this study, polylactic acid (PLA) and polyethylene glycol (PEG) were combined with soluble CaP glass particles and processed by rapid prototyping to obtain fully biodegradable structures for Tissue Engineering applications. The obtained 3D biodegradable structures were characterized in terms of their architecture and mechanical properties. The scaffold morphology, internal micro-architecture and mechanical properties were evaluated using Scanning Electron Microscopy (SEM), micro-computed tomography (micro-CT) and mechanical testing, respectively. Well defined structures with pore size of 350-400μm (in the axial view), struts width of approximately 70-80μm, and a porosity ranging between 60-65% were obtained. The combination RP and PLA/PEG/CaP glass turned into promising fully degradable, mechanically stable, bioactive and biocompatible composite scaffolds for TE.

JTD Keywords: Axial view, Biodegradable composites, Composite scaffolds, Glass particles, Mechanically stable, Micro architectures, Micro computed tomography (micro-CT), Poly lactic acid, Scaffold morphology, Tissue engineering applications, Well-defined structures, Bioactive glass, Mechanical properties, Mechanical testing, Polyethylene glycols, Polymer blends, Rapid prototyping, Scaffolds (biology), Scanning electron microscopy, Computerized tomography

Electrospinning is one of the most versatile and effective tools to produce nanostructured fibers in the biomedical science fields. The nanofibrous structure with diameters from tens to hundreds of nanometers largely mimics the native extracellular matrix (ECM) of many tissues. Thus far, a range of compositions including polymers and ceramics and their composites/hybrids have been successfully applied for generating electrospun nanofibers. Different processing tools in electrospinning set-ups and assemblies are currently developed to tune the morphology and properties of nanofibers. Herein, we demonstrate the electrospinning process and the electrospun biomaterials for specific use in tissue regeneration with some examples, involving different material combinations and fiber morphologies.

JTD Keywords: Ceramic, Composites, Electrospinning, Nanofi bers, Nanostructured fi bers, Polymer, Tissue regeneration

The relationship between molecular properties of odorants and neural activities is arguably one of the most important issues in olfaction and the rules governing this relationship are still not clear. In the olfactory bulb (OB), glomeruli relay olfactory information to second-order neurons which in turn project to cortical areas. We investigate relevance of odorant properties, spatial localization of glomerular coding sites, and size of coding zones in a dataset of 2-deoxyglucose images of glomeruli over the entire OB of the rat. We relate molecular properties to activation of glomeruli in the OB using a nonparametric statistical test and a support-vector machine classification study. Our method permits to systematically map the topographic representation of various classes of odorants in the OB. Our results suggest many localized coding sites for particular molecular properties and some molecular properties that could form the basis for a spatial map of olfactory information. We found that alkynes, alkanes, alkenes, and amines affect activation maps very strongly as compared to other properties and that amines, sulfur-containing compounds, and alkynes have small zones and high relevance to activation changes, while aromatics, alkanes, and carboxylics acid recruit very big zones in the dataset. Results suggest a local spatial encoding for molecular properties.

JTD Keywords: Molecular-receptive range, Odor, Olfactory bulb, Olfactory coding, Property-activity relationship, Structure-odor relationship

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

In natural tissues, the extracellular matrix composition, cell density and physiological properties are often non-homogeneous. Here we describe a model system, in which the distribution of cells throughout tissue engineering scaffolds after perfusion seeding can be influenced by the pore architecture of the scaffold. Two scaffold types, both with gyroid pore architectures, were designed and built by stereolithography: one with isotropic pore size (412 ± 13 [mu]m) and porosity (62 ± 1%), and another with a gradient in pore size (250-500 [mu]m) and porosity (35%-85%). Computational fluid flow modelling showed a uniform distribution of flow velocities and wall shear rates (15-24 s-1) for the isotropic architecture, and a gradient in the distribution of flow velocities and wall shear rates (12-38 s-1) for the other architecture. The distribution of cells throughout perfusion-seeded scaffolds was visualised by confocal microscopy. The highest densities of cells correlated with regions of the scaffolds where the pores were larger, and the fluid velocities and wall shear rates were the highest. Under the applied perfusion conditions, cell deposition is mainly determined by local wall shear stress, which, in turn, is strongly influenced by the architecture of the pore network of the scaffold.

JTD Keywords: Scaffolds, Microstructure, Cell adhesion, Confocal microscopy, Image analysis, Computational fluid dynamics

Bacillus anthracis is a severe mammalian pathogen encoding a class Ib ribonucleotide reductase (RNR). RNR is a universal enzyme that provides the four essential deoxyribonucleotides needed for DNA replication and repair. Almost all Bacillus spp. encode both class Ib and class III RNR operons, but the B. anthracis class III operon was reported to encode a pseudogene, and conceivably class Ib RNR is necessary for spore germination and proliferation of B. anthracis upon infection. The class Ib RNR operon in B. anthracis encodes genes for the catalytic NrdE protein, the tyrosyl radical metalloprotein NrdF, and the flavodoxin protein NrdI. The tyrosyl radical in NrdF is stabilized by an adjacent Mn(2)(III) site (Mn-NrdF) formed by the action of the NrdI protein or by a Fe(2)(III) site (Fe-NrdF) formed spontaneously from Fe(2+) and O(2). In this study, we show that the properties of B. anthracis Mn-NrdF and Fe-NrdF are in general similar for interaction with NrdE and NrdI. Intriguingly, the enzyme activity of Mn-NrdF was approximately an order of magnitude higher than that of Fe-NrdF in the presence of the class Ib-specific physiological reductant NrdH, strongly suggesting that the Mn-NrdF form is important in the life cycle of B. anthracis. Whether the Fe-NrdF form only exists in vitro or whether the NrdF protein in B. anthracis is a true cambialistic enzyme that can work with either manganese or iron remains to be established.

JTD Keywords: Escherichia-coli, Corynebacterium-ammoniagenes, Crystal-structure, Cofactor, Cubunit, Growth, Genes

The reason behind the entire development in silicon technology was band models in solid state physics. However, the theories postulated in order to give response to this phenomenon do not explain all kinds of materials. In a bid to overcome this limitation, we approach the problem from another point of view. In this work, the wave properties of the electrons from the external orbitals of the atoms and its diffraction patterns through the lattice structure of the material have been used to explain the band structure of metals, semiconductor and insulators. In order to probe this hypothesis, a simulation has been used and according to the relation between the lattice constant and the atomic diameter, the splitting of the bands have been observed for different kind of materials, showing a strong correlation between the simulation and the experimental results.

JTD Keywords: Electrical band structure, Band gap, Fraunhofer diffraction, Semiconductor, Insulator

The role of amyloid beta (A beta) peptide in the onset and progression of Alzheimer's disease is linked to the presence of soluble A beta species. Sulfated glycosaminoglycans (GAGs) promote A beta fibrillogenesis and reduce the toxicity of the peptide in neuronal cell cultures, but a satisfactory rationale to explain these effects at the molecular level has not been provided yet. We have used circular dichroism, Fourier transform infrared spectroscopy, fluorescence microscopy and spectroscopy, protease digestion, atomic force microscopy (AFM), and molecular dynamics simulations to characterize the association of the 42-residue fragment A beta(42) with sulfated GAGs, hyaluronan, chitosan, and poly(vinyl sulfate) (PVS). Our results indicate that the formation of stable A beta(42) fibrils is promoted by polymeric GAGs with negative charges placed in-frame with the 4.8-angstrom separating A beta(42) monomers within protofibrillar beta-sheets. Incubation of A beta(42) with excess sulfated GAGs and hyaluronan increased amyloid fibril content and resistance to proteolysis 2- to 5-fold, whereas in the presence of the cationic polysaccharide chitosan, A beta(42) fibrillar species were reduced by 25% and sensitivity to protease degradation increased similar to 3-fold. Fibrils of intermediate stability were obtained in the presence of PVS, an anionic polymer with more tightly packed charges than GAGs. Important structural differences between A beta(42) fibrils induced by PVS and A beta(42) fibrils obtained in the presence of GAGs and hyaluronan were observed by AFM, whereas mainly precursor protofibrillar forms were detected after incubation with chitosan. Computed binding energies per peptide from -11.2 to -13.5 kcal/mol were calculated for GAGs and PVS, whereas a significantly lower value of -7.4 kcal/mol was obtained for chitosan. Taken together, our data suggest a simple and straightforward mechanism to explain the role of GAGs as enhancers of the formation of insoluble A beta(42) fibrils trapping soluble toxic forms.

JTD Keywords: Alzheimer's disease, Amyloid fibril structure, Fibrillogenesis enhancers and inhibitors, Polysaccharides

Quantitative measurement of the low-frequency dielectric constants of thick insulators at the nanoscale is demonstrated utilizing ac electrostatic force microscopy combined with finite-element calculations based on a truncated cone with hemispherical apex probe geometry. The method is validated on muscovite mica, borosilicate glass, poly(ethylene naphthalate), and poly(methyl methacrylate). The dielectric constants obtained are essentially given by a nanometric volume located at the dielectric-air interface below the tip, independently of the substrate thickness, provided this is on the hundred micrometer-length scale, or larger.

JTD Keywords: Borosilicate glasses, Finite element analysis, Insulating thin films, Mica, Nanostructured materials, Permittivity, Polymers, Scanning probe microscopy

The small flavoprotein NrdI is an essential component of the class Ib ribonucleotide reductase system in many bacteria. NrdI interacts with the class Ib radical generating protein NrdF. It is suggested to be involved in the rescue of inactivated diferric centres or generation of active dimanganese centres in NrdF. Although NrdI bears a superficial resemblance to flavodoxin, its redox properties have been demonstrated to be strikingly different. In particular, NrdI is capable of two-electron reduction, whereas flavodoxins are exclusively one-electron reductants. This has been suggested to depend on a lesser destabilization of the negatively-charged hydroquinone state than in flavodoxins. We have determined the crystal structures of NrdI from Bacillus anthracis, the causative agent of anthrax, in the oxidized and semiquinone forms, at resolutions of 0.96 and 1.4 Å, respectively. These structures, coupled with analysis of all curated NrdI sequences, suggest that NrdI defines a new structural family within the flavodoxin superfamily. The conformational behaviour of NrdI in response to FMN reduction is very similar to that of flavodoxins, involving a peptide flip in a loop near the N5 atom of the flavin ring. However, NrdI is much less negatively charged than flavodoxins, which is expected to affect its redox properties significantly. Indeed, sequence analysis shows a remarkable spread in the predicted isoelectric points of NrdIs, from approximately pH 4–10. The implications of these observations for class Ib ribonucleotide reductase function are discussed.

JTD Keywords: Crystal structure, Flavin mononucleotide, Flavodoxin, NrdI, Ribonucleotide reductase

In this study we propose the correntropy function as a discriminative measure for detecting nonlinearities in the respiratory pattern of chronic heart failure (CHF) patients with periodic or nonperiodic breathing pattern (PB or nPB, respectively). The complexity seems to be reduced in CHF patients with higher risk level. Correntropy reflects information on both, statistical distribution and temporal structure of the underlying dataset. It is a suitable measure due to its capability to preserve nonlinear information. The null hypothesis considered is that the analyzed data is generated by a Gaussian linear stochastic process. Correntropy is used in a statistical test to reject the null hypothesis through surrogate data methods. Various parameters, derived from the correntropy and correntropy spectral density (CSD) to characterize the respiratory pattern, presented no significant differences when extracted from the iteratively refined amplitude adjusted Fourier transform (IAAFT) surrogate data. The ratio between the powers in the modulation and respiratory frequency bands R was significantly different in nPB patients, but not in PB patients, which reflects a higher presence of nonlinearities in nPB patients than in PB patients.

JTD Keywords: Practical, Theoretical or Mathematical, Experimental/cardiology diseases, Fourier transforms, Medical signal processing, Pattern classification, Pneumodynamics, Spectral analysis, Statistical analysis, Stochastic processes/ correntropy based nonlinearity test, Chronic heart failure, Correntropy function, Respiratory pattern nonlinearities, CHF patients, Nonperiodic breathing pattern, Dataset statistical distribution, Dataset temporal structure, Nonlinear information, Null hypothesis, Gaussian linear stochastic process, Statistical test, Correntropy spectral density, Iteratively refined amplitude adjusted Fourier transform, Surrogate data, Periodic breathing pattern

A technique for producing micrometer-scale structures over large, nonplanar chitosan surfaces is described. The technique makes use of the rheological characteristics (deformability) of the chitosan to create freestanding, three-dimensional scaffolds with controlled shapes, incorporating defined microtopography. The results of an investigation into the technical limits of molding different combinations of shapes and microtopographies are presented, highlighting the versatility of the technique when used irrespectively with inorganic or delicate organic moulds. The final, replicated scaffolds presented here are patterned with arrays of one-micrometer-tall microstructures over large areas. Structural integrity is characterized by the measurement of structural degradation. Human umbilical vein endothelial cells cultured on a tubular scaffold show that early cell growth is conditioned by the microtopography and indicate possible uses for the structures in biomedical applications. For those applications requiring improved chemical and mechanical resistance, the structures can be replicated in poly(dimethyl siloxane).

JTD Keywords: Biocompatible Materials/ chemistry, Cell Adhesion, Cell Culture Techniques/ methods, Cell Proliferation, Cells, Cultured, Chitosan/ chemistry, Crystallization/methods, Endothelial Cells/ cytology/ physiology, Humans, Materials Testing, Nanostructures/ chemistry/ ultrastructure, Nanotechnology/methods, Particle Size, Surface Properties, Tissue Engineering/methods

Methods for the accurate positioning of nanometric beads on a substrate have been developed over a number of years, and range from serial atomic force microscopy (AFM)techniques for single-bead positioning to parallel techniques for the positioning of large populations of beads in monolayer or multilayer architectures, typically from a liquid suspension. For example, topographic cues have been used for bead-based protein array production, although in this case, there is a random distribution of beads within the topography. Bead patterning has also been achieved in capillaries using a micromolding in capillaries (MIMIC) technique. Line patterns with micrometer widths are possible with this technique, achieving good multilayer organization. For monolayer bead patterning at micrometer dimensions, electrostatic forces and similar electrostatic assemblies using nanoxerography, as well as patterning by selective chemical functionalization, by transfer of particles from a liquid–liquid interface, and by subtracting top–down processes, are possible.

JTD Keywords: Microcontact stripping, Nanostructures, Poly(acrylic acid), Polystyrene, Surface patterning

New fabrication technologies and, in particular, new nanotechnologies have provided biomaterial and biomedical scientists with enormous possibilities when designing customized supports and scaffolds with controlled nanoscale topography and chemistry. The main issue now is how to effectively design these components and choose the appropriate combination of structure and chemistry to tailor towards applications as challenging and complex as stem cell differentiation. Occasionally, an incomplete knowledge of the fundamentals of biological differentiation process has hampered this issue. However, the recent technological advances in creating controlled cellular microenvironments can be seen as a powerful tool for furthering fundamental biology studies. This article reviews the main strategies followed to achieve solutions to this challenge, particularly emphasizing the working hypothesis followed by the authors to elucidate the mechanisms behind the observed effects of structured surfaces on cell behavior.

JTD Keywords: Cell pattering, Differentiation, Microcontact printing, Micropatterning, Microstructure, Nanoimprinting, Nanostructure, Stem cells

Polycrystalline Cu2O layers have been selectively grown by electrochemical anodization of polycrystalline Cu electrodes in an alkaline medium (pH 12.85). Uniform layers with thicknesses around 100 nm have been obtained. Using electrochemical impedance spectroscopy, it was concluded that the Cu2O films behave as a p-type semiconductor. The Mott-Schottky plot gives a value for the flat band potential of U-FB = -255 mV vs silver/silver chloride electrode (SSC), an estimated carrier density N-A = 6.1 x 10(17) cm(-3), and the space charge layer width was calculated to be W-SCL = 9 nm at a band bending of 120 mV. The electronic structure of the Cu vertical bar Cu2O vertical bar electrolyte interface was for the first time probed by in situ electrochemical tunneling spectroscopy. The use of in situ electrochemical scanning tunneling microscopy allows us to directly observed the valence band edge and determine its position against the absolute energy scale to be E-VB = -4.9 eV. Finally, we constructed a quantitative electronic diagram of the Cu vertical bar Cu2O vertical bar electrolyte interface, where the positions of the valence and conduction band edges are depicted, as well as the edge of the previously reported electronic subband.

JTD Keywords: 0.1 m NaOH, Electrochemical tunneling spectroscopy, Cuprous-oxide films, Anodic-oxidation, Electronic-structure, Alkaline-solution, Aqueous-solution, Initial-stages, Passive film, Thin-films

This work describes our studies on the molecular design of interfacial architectures suitable for DNA sensing which could resist non-specific binding of nanomaterials commonly used as labels for amplifying biorecognition events. We observed that the non-specific binding of bio-nanomaterials to surface-confined oligonucleotide strands is highly dependent on the characteristics of the interfacial architecture. Thiolated double stranded oligonucleotide arrays assembled on Au surfaces evidence significant fouling in the presence of nanoparticles (NPs) at the nanomolar level. The non-specific interaction between the oligonucleotide strands and the nanomaterials can be sensitively minimized by introducing streptavidin (SAv) as an underlayer conjugated to the DNA arrays. The role of the SAv layer was attributed to the significant hydrophilic repulsion between the SAv-modified surface and the nanomaterials in close proximity to the interface, thus conferring outstanding anti-fouling characteristics to the interfacial architecture. These results provide a simple and straightforward strategy to overcome the limitations introduced by the non-specific binding of labels to achieve reliable detection of DNA-based biorecognition events.

JTD Keywords: DNA/ analysis, Gold, Nanostructures/ chemistry, Oligonucleotide Array Sequence Analysis/ instrumentation, Oligonucleotides/ chemistry, Streptavidin/ chemistry, Sulfhydryl Compounds

Substrate topography, independently of substrate chemistry, has been reported to have significant effects on cell behaviour. Based on the use of fabrication techniques developed by the silicon microtechnology industry, numerous studies can now be found in the literature analyzing cell behaviour as to various micro- and nanofeatures such as lines, wells, holes and more. Most of these works have been found to relate the micro- and nano-sized topographical features with cell. orientation, migration, morphology and proliferation. In recent papers, even the influence of substrate nanotopography on cell gene expression and differentiation has been pointed out. However, despite the large number of papers published on this topic, significant general trends in cell behaviour are difficult to establish due to differences in cell type, substrate material, feature aspect-ratio, feature geometry and parameters measured. This paper intends to compile and review the relevant existing information on the behaviour of cells on micro- and nano-structured artificial substrates and analyze possible general behavioural trends.

JTD Keywords: Microstructure, Topography, Cell behaviour, Cell morphology, Cell orientation

Recent studies on 2D substrates have revealed the importance of surface properties in affecting cell behaviour. In particular, surface topography appears to influence and direct cell migration. The development of new technologies of hot embossing and micro-imprinting has made it possible to study cell interactions with controlled micro features and to determine how these features can affect cell behaviour. Several studies have been carried out on the effect of microstructures on cell adhesion, cell guidance and cell proliferation. However, there is still a lack of knowledge on how these features affect mesenchymal stem cell differentiation. This study was designed to evaluate whether highly controlled microstructures on PMMA could induce rMSC differentiation into an osteogenic lineage. Structured PMMA was seeded with rMSC and cell number; cell morphology and cell differentiation were evaluated. Results confirm that microstructures not only affect cell proliferation and alignment but also have a synergistic effect with osteogenic medium on rMSC differentiation into mature osteoblasts.

JTD Keywords: Mesenchymal stem cells, Osteoblasts, Topography, Microstructures

Cr(VI) sensitive polysiloxane membranes containing tributylphosphate (TBP) or trioctylphosphine oxide (TOPO) were characterized in this study. TBP and TOPO as carriers, have a high selectivity for Cr(VI). The Potentiometric response of EMIS (Electrolyte/Membrane/Insulator/Semiconductor) sensors presents a quasi-nernstian response for Cr2O2-7 exchange. The ion exchange is shown by X-ray photoelectron spectrometry (XPS), the binding energy of the Cr 2p1/2 peak corresponding to Cr(VI) and the atomic composition after exposure to Cr(VI) shows a factor 1.7 higher for silopreneTBP membrane. The conformational topography of both polymeric membranes was characterized by Atomic Force Microscopy (AFM), the exchange of Cr(VI) leading to a heterogeneous topographic state. Adhesion force measurements are also performed to study the properties of adhesion of both selective membranes with a non-functionalized Si AFM tip and with an OTS functionalized one to study the interactions between the tip and the membrane, in liquid before and after the exposure of the membrane to ion chromium. The presence of the ionophores does not practically change the adhesion force compared to pure polysiloxane, showing a good solubility of the ionophore and the orientation of the alkyl chains towards the polysiloxane surface. After the exchange with Cr(VI), the adhesion force decreases drastically due to the hydrophilic character of the surface, complex of Cr(VI) with the P-O groups of both ionophore being oriented towards the surface.

JTD Keywords: AFM, Electrolyte/membrane/insulator/semiconductor structures, Polysiloxane membrane, Xps

A fundamental aspect of the rapidly expanding medical care sector, bone repair continues to benefit from emerging technological developments. This text provides researchers and students with a comprehensive review of the materials science and engineering principles behind these developments. The first part reviews the fundamentals of bone repair and regeneration. Further chapters discuss the science and properties of biomaterials used in bone repair, including both metals and biocomposites. Final chapters analyze device considerations such as implant lifetime and failure, and discuss potential applications, as well as the ethical issues that continually confront researchers and clinicians.

JTD Keywords: Bone composition and structure, Biomechanical properties of bone, Bone damage and repair

The electrophysiological characterisation of cultured neurons is of paramount importance for drug discovery, safety pharmacology and basic research in the neurosciences. Technologies offering low cost, low technical complexity and potential for scalability towards high-throughput electrophysiology on in vitro neurons would be advantageous, in particular for screening purposes. Here we describe a plastic culture substrate supporting low-complexity multi-unit loose-patch recording and stimulation of developing networks while retaining manufacturability compatible with low-cost and large-scale production. Our hybrid polydimethylsilane (PDMS)-on-polystyrene structures include chambers (6 mm in diameter) and microchannels (25 mu m x 3.7 mu m 1 mm) serving as substrate-embedded recording pipettes. Somas are plated and retained in the chambers due to geometrical constraints and their processes grow along the microchannels, effectively establishing a loose-patch configuration without human intervention. We demonstrate that off-the-shelf voltage-clamp, current-clamp and extracellular amplifiers can be used to record and stimulate multi-unit activity with the aid of our dishes. Spikes up to 50 pA in voltage-clamp and 300 mu V in current-clamp modes are recorded in sparse and bursting activity patterns characteristic of 1 week-old hippocampal cultures. Moreover, spike sorting employing principal component analysis (PCA) confirms that single microchannels support the recording of multiple neurons. Overall, this work suggests a strategy to endow conventional culture plasticware with added functionality to enable cost-efficient network electrophysiology.

JTD Keywords: Electrophysiological characterisation, Cultured neurons, Polydimethylsilane (PDMS)-on-polystyrene structures

Surface topography is known to have an influence on osteoblast activity. However, in the case of bioactive materials, topographical changes can affect also ion exchange properties. This makes the problem more complex, since it is often difficult to separate the strictly topographical effects from the effects of ionic fluctuations in the medium. The scope of this paper is to analyze the simultaneous effect of topography and topography-mediated ion exchange on the initial cellular behavior of osteoblastic-like cells cultured on bioactive tissue engineering substrates. Two apatitic substrates with identical chemical composition but different micro/nanostructural features were obtained by low-temperature setting of a calcium phosphate cement. MG63 osteoblastic-like cells were cultured either in direct contact with the substrates or with their extracts. A strong and permanent decrease of calcium concentration in the culture medium, dependent on substrate topography, was detected. A major effect of the substrate microstructure on cell proliferation was observed, explained in part by the topography-mediated ion exchange, but not specifically by the ionic Ca(2+) fluctuations. Cell differentiation was strongly enhanced when cells were cultured on the finer substrate. This effect was not explained by the chemical modification of the medium, but rather suggested a strictly topographical effect.

JTD Keywords: Alkaline Phosphatase/metabolism, Bone Cements/pharmacology, Calcium/metabolism, Calcium Phosphates/pharmacology, Cell Adhesion/drug effects, Cell Differentiation/drug effects, Cell Proliferation/drug effects, Cell Shape/drug effects, Cells, Cultured, Culture Media, Durapatite/pharmacology, Humans, Interferometry, Ion Exchange, Materials Testing, Osteoblasts/ cytology/drug effects/enzymology/ultrastructure, Phosphorus/metabolism, Powders, Tissue Engineering, Tissue Scaffolds

The nanomechanical properties of Langmuir-Blodgett monolayers of arachidic acid extracted at surface pressures of 1, 15, and 35 mN/m and deposited on mica were investigated by atomic force microscopy, force spectroscopy, and lateral force microscopy. It was experimentally demonstrated that the arachidic acid molecular orientation depends on the extraction pressure. According to this, tilting angles of 50, 34, and 22 degrees with respect to the surface perpendicular were detected and identified as conformations that maximize van der Waals interactions between the arachidic acid alkyl chains. The vertical force needed to puncture the monolayers with the AFM tip strongly depends on the molecular tilting angles attained at different monolayer extraction surface pressures, obtaining values that range from 13.07 +/- 3.24 nN for 50 degrees to 22.94 +/- 5.49 nN for 22 degrees tilting angles. The different molecular interactions involved in the monolayer cohesion are discussed and quantitatively related to the experimental monolayer breakthrough forces. The friction measurements performed from low vertical forces up to monolayer disruption reveal the existence of three well-defined regimes: first, a low friction response due to the elastic deformation of the monolayer, which is followed by a sharp increase in the friction force due to the onset of a sudden plastic deformation. The last regime corresponds to the monolayer rupture and the contact between tip and substrate. The friction coefficient of the substrate is seen to depend on the monolayer extraction pressure, a fact that is discussed in terms of the relationship between the sample compactness and its rupture mechanism.

JTD Keywords: AFM, SAM, Reflection-absortion spectroscopy, Lipid-bilayers, Frictional-properies, Molecular-structure, Thermal behavior, Nanometer-scale, Chain-length, LB films

A technique for imparting micro- and nano-structured topography into the surface of freestanding thin sheets of chitosan is described. Both micro- and nanometric surface structures have been produced using soft lithography. The soft lithography method, based on solvent evaporation, has allowed structures similar to 60 nm tall and similar to 500 X 500 nm(2) to be produced on freestanding similar to 0.5 mm thick sheets of the polymer when cured at 293 K, and structures similar to 400 nm tall and 5 X 5 mu m(2) to be produced when cured at 283 K. Nonstructured chitosan thin sheets (similar to 200 mu m thick) show excellent optical transmission properties in the visible portion of the electromagnetic spectrum. The structured sheets can be used for applications where optical microscopic analysis is required, such as cell interaction experiments and tissue engineering.

JTD Keywords: Chitin/chitosan, Microstructure, Nanotopography, Polymerization, Soft lithography

Micro- and nanoscale protein patterns have been produced via a new contact printing method using a nanoimprint lithography apparatus. The main novelty of the technique is the use of poly(methyl methacrylate) (PMMA) instead of the commonly used poly(dimethylsiloxane) (PDMS) stamps. This avoids printing problems due to roof collapse, which limits the usable aspect ratio in microcontact printing to 10:1. The rigidity of the PMMA allows protein patterning using stamps with very high aspect ratios, up to 300 in this case. Conformal contact between the stamp and the substrate is achieved because of the homogeneous pressure applied via the nanoimprint lithography instrument, and it has allowed us to print lines of protein similar to 150 nm wide, at a 400 nm period. This technique, therefore, provides an excellent method for the direct printing of high-density sub-micrometer scale patterns, or, alternatively, micro-/nanopatterns spaced at large distances. The controlled production of these protein patterns is a key factor in biomedical applications such as cell-surface interaction experiments and tissue engineering.

JTD Keywords: Soft lithography, Cell-adhesion, Microstructures, Fabrication, Stability, Patterns

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JTD Keywords: Self-assembled monolayers, High-aspect-ratio, Dip-pen nanolithography, Langmuir-blodgett-films, Conducting polymer, Enzymes, Microstructures, Immunosensors, Fabrication, Stability

Despite its tremendous scientific and economic impact, the mechanism that triggers metal passive film breakdown in the presence of aggressive ions remains under discussion. We have studied the iron passive film in chloride media using X-ray photoelectron spectroscopy (XPS), electrochemical impedance spectroscopy and electrochemical tunneling spectroscopy (ECTS). Ex situ XPS reveal that the film consists exclusively of an Fe(III) oxide without chloride content. In situ ECTS has been used to build up conductance maps of the Fe electrode during its electrochemical oxidation in a borate buffer solution and its breakdown when the film is grown in the presence of chloride. This conductograms provide direct and in situ experimental evidence of chloride-induced surface states within the band gap of the oxide film (~3.3eV). These states enable new charge exchange pathways that allow hole capture at the surface of the n-type Fe(III) oxide. The blocking of VB processes that occurs in the iron passive film is no longer present in chloride media, and electrode corrosion can proceed through these new states. We propose a simple 3-step mechanism for the process, in which chloride anions form an oxidizing Fe(II) surface intermediate but do not participate directly in the reaction.

JTD Keywords: Electrochemical tunneling spectroscopy, Electronic band structure, Fe passive film, Aqueous chloride corrosion, Semiconductor decomposition, Interface states