Publications

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

A bottom-up approach to fabricating monodisperse, two-component polymersomes that possess phase-separated ("patchy") chemical topology is presented. This approach is compared with already-existing top-down preparation methods for patchy polymer vesicles, such as film rehydration. These findings demonstrate a bottom-up, solvent-switch self-assembly approach that produces a high yield of nanoparticles of the target size, morphology, and surface topology for drug delivery applications, in this case patchy polymersomes of a diameter of ≈50 nm. In addition, an image processing algorithm to automatically calculate polymersome size distributions from transmission electron microscope images based on a series of pre-processing steps, image segmentation, and round object identification is presented.© 2023 Wiley-VCH GmbH.

JTD Keywords: assemblies, copolymers, evolution, membranes, micelles, ph, phase separation, polymersomes, rafts, self-assembly, size, vesicles, Cell biology, Drug delivery, Phase separation, Polymersomes, Self-assembly, Vesicles

Type 1 Diabetes results from autoimmune response elicited against β-cell antigens. Nowadays, insulin injections remain the leading therapeutic option. However, injection treatment fails to emulate the highly dynamic insulin release that β-cells provide. 3D cell-laden microspheres have been proposed during the last years as a major platform for bioengineering insulin-secreting constructs for tissue graft implantation and a model for in vitro drug screening platforms. Current microsphere fabrication technologies have several drawbacks: the need for an oil phase containing surfactants, diameter inconsistency of the microspheres, and high time-consuming processes. These technologies have widely used alginate for its rapid gelation, high processability, and low cost. However, its low biocompatible properties do not provide effective cell attachment. This study proposes a high-throughput methodology using a 3D bioprinter that employs an ECM-like microenvironment for effective cell-laden microsphere production to overcome these limitations. Crosslinking the resulting microspheres with tannic acid prevents collagenase degradation and enhances spherical structural consistency while allowing the diffusion of nutrients and oxygen. The approach allows customization of microsphere diameter with extremely low variability. In conclusion, a novel bio-printing procedure is developed to fabricate large amounts of reproducible microspheres capable of secreting insulin in response to extracellular glucose stimuli.© 2022 The Authors. Advanced Materials Technologies published by Wiley‐VCH GmbH.

JTD Keywords: 3d bioprinter, beta-cell, biomaterial, collagen, encapsulation, mechanics, microspheres, survival, 3d bioprinter, ?-cell, Advanced material technologies, Biocompatibility, Cell encapsulations, Cells, Collagen, Cross-linking, Cytology, Drug delivery, Encapsulation, Fabrication, Flavonoids, Gelation, In-vitro, Insulin injections, Insulin release, Microspheres, Tannic acid, Tannins, Throughput, Tissue grafts, Type 1 diabetes, Β‐cell

Tau hyperphosphorylation is the first step of neurofibrillary tangle (NFT) formation. In the present study, samples of the entorhinal cortex (EC) and frontal cortex area 8 (FC) of cases with NFT pathology classified as stages I–II, III–IV, and V–VI without comorbidities, and of middle-aged (MA) individuals with no NFT pathology, were analyzed by conventional label-free and SWATH-MS (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) to assess the (phospho)proteomes. The total number of identified dysregulated phosphoproteins was 214 in the EC, 65 of which were dysregulated at the first stages (I–II) of NFT pathology; 167 phosphoproteins were dysregulated in the FC, 81 of them at stages I–II of NFT pathology. A large percentage of dysregulated phosphoproteins were identified in the two regions and at different stages of NFT progression. The main group of dysregulated phosphoproteins was made up of components of the membranes, cytoskeleton, synapses, proteins linked to membrane transport and ion channels, and kinases. The present results show abnormal phosphorylation of proteins at the first stages of NFT pathology in the elderly (in individuals clinically considered representative of normal aging) and sporadic Alzheimer's disease (sAD). Dysregulated protein phosphorylation in the FC precedes the formation of NFTs and SPs. The most active period of dysregulated phosphorylation is at stages III–IV when a subpopulation of individuals might be clinically categorized as suffering from mild cognitive impairment which is a preceding determinant stage in the progression to dementia. Altered phosphorylation of selected proteins, carried out by activation of several kinases, may alter membrane and cytoskeletal functions, among them synaptic transmission and membrane/cytoskeleton signaling. Besides their implications in sAD, the present observations suggest a molecular substrate for “benign” cognitive deterioration in “normal” brain aging.

JTD Keywords: (phospho)proteomics, alzheimer's disease, amyloid-beta, association guidelines, brain aging, cytoskeleton, frontal-cortex, kinases, lipid rafts, membranes, national institute, neuropathologic assessment, pathological process, protein phosphorylation, synapse pathology, synapses, tau, tau pathology, (phospho)proteomics, Age-related tauopathy, Alzheimer's disease, Brain aging, Cytoskeleton, Kinases, Membranes, Protein phosphorylation, Synapses, Tau

Membrane fission triggered by the endosomal sorting complex required for transport (ESCRT) is an important process observed in several pathogenic and non-pathogenic cellular events. From a synthetic-biology viewpoint, ESCRT proteins represent an interesting machinery for the construction of cell mimetic sub-compartments produced by fission. Since their discovery, the studies on ESCRT-III-mediated action, have mainly focused on protein dynamics, ignoring the role of lipid organization and membrane phase state. Recently, it has been suggested that membrane buds formed by the action of ESCRT-III are generated from transient microdomains in endosomal membranes. However, the interplay between membrane domain formation and ESCRT remodeling pathways has not been investigated. Here, giant unilamellar vesicles made of ternary lipid mixtures, either homogeneous in phase or exhibiting liquid-ordered/liquid-disordered phase coexistence, were employed as a model membrane system. These vesicles were incubated with purified recombinant ESCRT-III proteins from the parasite Entamoeba histolytica. In homogeneous membranes, we observe that EhVps32 can trigger domain formation while EhVps20 preferentially co-localizes in the liquid disordered phase. The addition of EhVps24 appears to induce the formation of intraluminal vesicles produced from the liquid-ordered phase. In phase separated membranes, the intraluminal vesicles are also generated from the liquid-ordered phase and presumably emerge from the phase boundary region. Our findings reinforce the hypothesis that ESCRT-mediated remodeling depends on the membrane phase state. Furthermore, the obtained results point to a potential synthetic biology approach for establishing eukaryotic mimics of artificial cells with microcompartments of specific membrane composition, which can also differ from that of the mother vesicle.

JTD Keywords: cell-membranes, coexistence, complex, escrt-iii, fission, guvs, lipid domains, lipid rafts, membrane fission, microcompartments, microscopy, phase separation, plasma-membrane, protein microarrays, structural basis, ternary mixtures, Escrt-iii, Giant unilamellar vesicles, Guvs, Lipid domains, Membrane fission, Microcompartments, Phase separation, Ternary mixtures

We investigate the effects of Cholesterol (Chol) in the structural and nanomechanical properties of binary and ternary spin-coated single lipid bilayers made of Dioleoylphosphatidylcholine (DOPC) and Sphingomyelin (SM) in dry conditions. We show that for the DOPC/Chol bilayers, Chol induces an initial increase of the bilayer thickness, followed by decrease for concentrations above 30% Chol. The mechanical properties, instead, appear practically insensitive to the Chol content. For the SM/Chol bilayers we have observed both the thinning of the bilayer and the decrease of the force necessary to break it for Chol content above 40. mol%. In both binary mixtures phase separation is not observed. For ternary single bilayers of DOPC/SM/Chol, Chol induces phase segregation and the formation of domains resembling lipid rafts. The domains show a thickness and mechanical response clearly distinct from the surrounding phase and dependent on the relative Chol content. Based on the results obtained for the binary mixtures, DOPC- and SM-enriched domains can be identified. We highlight that many of the effects of Chol reported here for the dry multicomponent single lipid bilayers resemble closely those observed in hydrated bilayers, thus offering an additional insight into their properties.

JTD Keywords: AFM, Air-stable lipid layer, Force spectroscopy, Lipid raft, Spin-coating

Near-field optical microscopy is a technique not limited by the laws of diffraction that enables simultaneous high-resolution fluorescence and topographic measurements at the nanometer scale. This chapter highlights the intrinsic advantages of near-field optics in the study of cellular structures. The first part of the chapter lays the foundations of the near-field concept and technical implementation of near-field scanning optical microscopy (NSOM), whereas the second part of the chapter focuses on applications of NSOM to the study of model membranes and cellular structures on the plasma membrane. The last part of the chapter discusses further directions of near-field optics, including optical antennas and fluorescence correlation spectroscopy approaches in the near-field regime.

JTD Keywords: Biological membranes, Cell membrane nanoscale compartmentalization, Cellular nanodomains, Fluorescence correlation spectroscopy in reduced volumes, Immunoreceptor imaging, Lipid rafts, Near-field scanning optical microscopy, Optical nano-antennas, Shear force imaging, Single molecule detection, Super-resolution microscopy

Apart from the paradigm that cell-biomaterials interaction depends on the adsorption of soluble adhesive proteins we anticipate that upon distinct conditions also other, less soluble ECM proteins such as collagens, associate with the biomaterials interface with consequences for cellular response that might be of significant bioengineering interest. Using atomic force microscopy (AFM) we seek to follow the nanoscale behavior of adsorbed type IV collagen (Col IV)-a unique multifunctional matrix protein involved in the organization of basement membranes (BMs) including vascular ones. We have previously shown that substratum wettability significantly affects Col IV adsorption pattern, and in turn alters endothelial cells interaction. Here we introduce two new model surfaces based on self-assembled monolayers (SAMs), a positively charged - NH(2), and negatively charged -COOH surface, to learn more about their particular effect on Col IV behavior. AFM studies revealed distinct pattern of Col IV assembly onto the two SAMs resembling different aspects of network-like structure or aggregates (suggesting altered protein conformation). Moreover, the amount of adsorbed FITC-labeled Col IV was quantified and showed about twice more protein on NH(2) substrata. Human umbilical vein endothelial cells attached less efficiently to Col IV adsorbed on negatively charged COOH surface judged by altered cell spreading, focal adhesions formation, and actin cytoskeleton development. Immunofluorescence studies also revealed better Col IV recognition by both alpha(1) and alpha(2) integrins on positively charged NH(2) substrata resulting in higher phosphorylated focal adhesion kinase recruitment in the focal adhesion complexes. On COOH surface, no integrin clustering was observed. Taken altogether these results, point to the possibility that combined NH(2) and Col IV functionalization may support endothelization of cardiovascular implants.

JTD Keywords: Collagen type IV, SAMs, AFM, Surface-induced protein assembly, Endothelial cells, Vascular grafts

Characterization of molecular dynamics on living cell membranes at the nanoscale is fundamental to unravel the mechanisms of membrane organization and compartmentalization. Here we demonstrate the feasibility of fluorescence correlation spectroscopy (FCS) based on the nanometric illumination of near-field scanning optical microscopy (NSOM) probes on intact living cells. NSOM-FCS applied to fluorescent lipid analogs allowed us to reveal details of the diffusion hidden by larger illumination areas. Moreover, the technique offers the unique, advantages of evanescent axial illumination and straightforward implementation of multiple color excitation. As such, NSOM-FCS represents a powerful tool to study a variety of dynamic processes occurring at the nanometer scale on cell membranes.

JTD Keywords: Mode wave-guides, Lipid rafts, Difussion, Organization, Aperture

For many years, it was believed that the laws of diffraction set a fundamental limit to the spatial resolution of conventional light microscopy. Major developments, especially in the past few years, have demonstrated that the diffraction barrier can be overcome both in the near- and far-field regime. Together with dynamic measurements, a wealth of new information is now emerging regarding the compartmentalization of cell membranes. In this review we focus on optical methods designed to explore the nanoscale architecture of the cell membrane, with a focal point on near-field optical microscopy (NSOM) as the first developed technique to provide truly optical super-resolution beyond the diffraction limit of light. Several examples illustrate the unique capabilities offered by NSOM and highlight its usefulness on cell membrane studies, complementing the palette of biophysical techniques available nowadays.

JTD Keywords: Membrane nanodomain, Lipid raft, Single molecule detection, Near-field scanning optical microscopy, Super-resolution optical microscopy

The use of injectable self-setting calcium phosphate cements or soluble glass granules represent two different strategies for bone regeneration, each with distinct advantages and potential applications. This study compares the in vivo behavior of two calcium phosphate cements and two phosphate glasses with different composition, microstructure and solubility, using autologous bone as a control, in a rabbit model. The implanted materials were alpha-tricalcium phosphate cement (cement H), calcium sodium potassium phosphate cement (cement R), and two phosphate glasses in the P2O5-CaO-Na2O and P2O5-CaO-Na2O-TiO2 systems. The four materials were osteoconductive, biocompatible and biodegradable. Radiological and histological studies demonstrated correct osteointegration and substitution of the implants by new bone. The reactivity of the different materials, which depends on their solubility, porosity and specific surface area, affected the resorption rate and bone formation mainly during the early stages of implantation, although this effect was weak. Thus, at 4 weeks the degradation was slightly higher in cements than in glasses, especially for cement R. However, after 12 weeks of implantation all materials showed a similar degradation degree and promoted bone neoformation equivalent to that of the control group.

JTD Keywords: Calcium phosphates, Calcium phosphate cements, Phosphate glasses, Bone grafts, Bone regenerations