Publications

Mesenchymal condensation is a prevalent morphogenetic transition that is essential in chondrogenesis. However, the current understanding of condensation mechanisms is limited. In vivo, progenitor cells directionally migrate from the surrounding loose mesenchyme towards regions of increasing matrix adherence (the condensation centers), which is accompanied by the upregulation of fibronectin. Here, we focused on the mechanisms of cell migration during mesenchymal cell condensation and the effects of matrix adherence. Dendrimer-based nanopatterns of the cell-adhesive peptide arginine-glycine-aspartic acid (RGD), which is present in fibronectin, were used to regulate substrate adhesion. We recorded collective and single-cell migration of mesenchymal stem cells, under chondrogenic induction, using live-cell imaging. Our results show that the cell migration mode of single cells depends on substrate adhesiveness, and that cell directionality controls cell condensation and the fusion of condensates. Inhibition experiments revealed that cell-cell interactions mediated by N-cadherin (also known as CDH2) are also pivotal for directional migration of cell condensates by maintaining cell-cell cohesion, thus suggesting a fine interplay between cell-matrix and cell-cell adhesions. Our results shed light on the role of cell interactions with a fibronectin-depositing matrix during chondrogenesis in vitro, with possible applications in regenerative medicine. This article has an associated First Person interview with the first author of the paper.© 2022. Published by The Company of Biologists Ltd.

JTD Keywords: alpha-v-beta-3, arginine-glycine-aspartic acid, chondrogenesis, dynamics, expression, fibronectin, gastrulation, involvement, mechanisms, mesenchymal condensation, model, nanopatterned substrates, rgd, Arginine-glycine-aspartic acid, Cell migration, Chondrogenesis, Mesenchymal condensation, N-cadherin, Nanopatterned substrates, Rgd

Aim: To unveil the influence of cell-matrix adhesions in the establishment of gap junction intercellular communication (GJIC) during cell condensation in chondrogenesis. Materials & methods: Previously developed nanopatterns of the cell adhesive ligand arginine-glycine-aspartic acid were used as cell culture substrates to control cell adhesion at the nanoscale. In vitro chondrogenesis of mesenchymal stem cells was conducted on the nanopatterns. Cohesion and GJIC were evaluated in cell condensates. Results: Mechanical stability and GJIC are enhanced by a nanopattern configuration in which 90% of the surface area presents adhesion sites separated less than 70 nm, thus providing an onset for cell signaling. Conclusion: Cell-matrix adhesions regulate GJIC of mesenchymal cell condensates during in vitro chondrogenesis from a threshold configuration at the nanoscale.

JTD Keywords: arginine-glycine-aspartic acid, arginine–glycine–aspartic acid, cell adhesion, condensation, dendrimer-based nanopatterning, gap junction intercellular communication, Actin, Adhesion, Arginine-glycine-aspartic acid, Cell adhesion, Collagen, Condensation, Connexin-43, Dendrimer-based nanopatterning, Dynamics, Extracellular-matrix, Fibronectin, Gap junction intercellular communication, Mesenchymal stem cells, Permeability, Phenotype, Vinculin

Contact-based antimicrobials, as antibiotic-free technologies that use non-specific interactions with bacterial cells to exert antimicrobial activity, are a prospective solution in fighting the global issue of bacterial resistance. A very simplified approach to their design considers the direct bonding of cationic guanidine-containing amino acids to the surface of nano-gold carriers. The structure enables antimicrobial activity due to a high density of cationic surface charges. This opens a set of novel questions that are important for their effective engineering, particularly regarding (i) chemistry and events that take place at the interface between NPs and cells, (ii) the direct influence of a charge (and its change) on interactions with bacterial and mammalian cells, and (iii) the stability of structures (and their antimicrobial activity) in the presence of enzymes, which are addressed in this paper. Because of the ability of amino acid-functionalized nano-gold to retain structural and functional activity, even after exposure to a range of physicochemical stimuli, they provide an excellent nanotechnological platform for designing highly effective contact-based antimicrobials and their applications.

JTD Keywords: agents, antibiotic-free technology, arginine, charged amino acids, contact-based antimicrobials, discovery, enzyme-resistant antimicrobials, functionalized gold, peptides, polymers, resistant, Antibiotic-free technology, Charged amino acids, Contact-based antimicrobials, Enzyme-resistant antimicrobials, Functionalized gold, Nanoparticles

Enteroaggregative Escherichia coli (EAEC) strains are one of the diarrheagenic pathotypes. EAEC strains harbor a virulence plasmid (pAA2) that encodes, among other virulence determinants, the aggR gene. The expression of the AggR protein leads to the expression of several virulence determinants in both plasmids and chromosomes. In this work, we describe a novel mechanism that influences AggR expression. Because of the absence of a Rho-independent terminator in the 3?UTR, aggR transcripts extend far beyond the aggR ORF. These transcripts are prone to PNPase-mediated degradation. Structural alterations in the 3?UTR result in increased aggR transcript stability, leading to increased AggR levels. We therefore investigated the effect of increased AggR levels on EAEC virulence. Upon finding the previously described AggR-dependent virulence factors, we detected novel AggR-regulated genes that may play relevant roles in EAEC virulence. Mutants exhibiting high AggR levels because of structural alterations in the aggR 3?UTR show increased mobility and increased pAA2 conjugation frequency. Furthermore, among the genes exhibiting increased fold change values, we could identify those of metabolic pathways that promote increased degradation of arginine, fatty acids and gamma-aminobutyric acid (GABA), respectively. In this paper, we discuss how the AggR-dependent increase in specific metabolic pathways activity may contribute to EAEC virulence.

JTD Keywords: aggregative adherence, arginine metabolism, biofilm formation, escherichia-coli, gene-expression, messenger-rna, operon, persistent diarrhea, untranslated region, Fimbria-i expression

The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway involves a three-step cascade of kinases that transduce signals and promote processes such as cell growth, development, and apoptosis. An aberrant response of this pathway is related to the proliferation of cell diseases and tumors. By using simulation modeling, we document that the protein arginine methyltransferase 5 (PRMT5) modulates the MAPK pathway and thus avoids an aberrant behavior. PRMT5 methylates the Raf kinase, reducing its catalytic activity and thereby, reducing the activation of ERK in time and amplitude. Two minimal computational models of the epidermal growth factor (EGF)-Ras-ERK MAPK pathway influenced by PRMT5 were proposed: a first model in which PRMT5 is activated by EGF and a second one in which PRMT5 is stimulated by the cascade response. The reported results show that PRMT5 reduces the time duration and the expression of the activated ERK in both cases, but only in the first model PRMT5 limits the EGF range that generates an ERK activation. Based on our data, we propose the protein PRMT5 as a regulatory factor to develop strategies to fight against an excessive activity of the MAPK pathway, which could be of use in chronic diseases and cancer.

JTD Keywords: cancer, cell response modulation, computational model, egf-ras-erk signaling route, mapk pathway, methylation, Arginine methyltransferase 5, Cancer, Cell response modulation, Colorectal-cancer, Computational model, Egf-ras-erk signaling route, Epidermal-growth-factor, Factor receptor, Histone h3, Kinase cascade, Mapk pathway, Methylation, Negative-feedback, Pc12 cells, Prmt5, Protein, Signal-transduction

Background: Emerging concepts for designing innovative drugs (i.e., novel generations of antimicrobials) frequently include nanostructures, new materials, and nanoparticles (NPs). Along with numerous advantages, NPs bring limitations, partly because they can limit the analytical techniques used for their biological and in vivo validation. From that standpoint, designing innovative drug delivery systems requires advancements in the methods used for their testing and investigations. Considering the well-known ability of resazurin-based methods for rapid detection of bacterial metabolisms with very high sensitivity, in this work we report a novel optimization for tracking bacterial growth kinetics in the presence of NPs with specific characteristics, such as specific optical properties. Results: Arginine-functionalized gold composite (HAp/Au/arginine) NPs, used as the NP model for validation of the method, possess plasmonic properties and are characterized by intensive absorption in the UV/vis region with a surface plasmon resonance maximum at 540 nm. Due to the specific optical properties, the NP absorption intensively interferes with the light absorption measured during the evaluation of bacterial growth (optical density; OD600). The results confirm substantial nonspecific interference by NPs in the signal detected during a regular turbidity study used for tracking bacterial growth. Instead, during application of a resazurin-based method (Presto Blue), when a combination of absorption and fluorescence detection is applied, a substantial increase in the signal-to-noise ratio is obtained that leads to the improvement of the accuracy of the measurements as verified in three bacterial strains tested with different growth rates (E. coli, P. aeruginosa, and S. aureus). Conclusions: Here, we described a novel procedure that enables the kinetics of bacterial growth in the presence of NPs to be followed with high time resolution, high sensitivity, and without sampling during the kinetic study. We showed the applicability of the Presto Blue method for the case of HAp/Au/arginine NPs, which can be extended to various types of metallic NPs with similar characteristics. The method is a very easy, economical, and reliable option for testing NPs designed as novel antimicrobials.

JTD Keywords: Antimicrobial nanoparticles, Arginine-functionalized gold, Bacterial growth kinetics, Plasmonic nanoparticles, Presto Blue

Extracellular matrix remodeling plays a pivotal role during mesenchyme patterning into different lineages. Tension exerted from cell membrane receptors bound to extracellular matrix ligands is transmitted by the cytoskeleton to the cell nucleus inducing gene expression. Here, we used dendrimer-based arginine–glycine–aspartic acid (RGD) uneven nanopatterns, which allow the control of local surface adhesiveness at the nanoscale, to unveil the adhesive requirements of mesenchymal tenogenic and osteogenic commitments. Cell response was found to depend on the tension resulting from cell–substrate interactions, which affects nuclear morphology and is regulated by focal adhesion size and distribution.

JTD Keywords: Arginine–glycine–aspartic acid (RGD), Nanopattern, Mesenchymal stem cells, Tenogenesis, Osteogenesis, Cell nuclei, Focal adhesions

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

JTD Keywords: Bioengineering, Dendrimer, Nanopattern, Arginine-Glycine-Aspartic Acid (RGD), Atomic Force Microscopy (AFM), Cell Adhesion, Mesenchymal Stem Cells (Mscs), Chondrogenesis

Cell adhesion processes are governed by the nanoscale arrangement of the extracellular matrix (ECM), being more affected by local rather than global concentrations of cell adhesive ligands. In many cell-based studies, grafting of dendrimers on surfaces has shown the benefits of the local increase in concentration provided by the dendritic configuration, although the lack of any reported surface characterization has limited any direct correlation between dendrimer disposition and cell response. In order to establish a proper correlation, some control over dendrimer surface deposition is desirable. Here, dendrimer nanopatterning has been employed to address arginine-glycine-aspartic acid (RGD) density effects on cell adhesion. Nanopatterned surfaces were fully characterized by atomic force microscopy (AFM), scanning tunneling microscopy (STM) and X-ray photoelectron spectroscopy (XPS), showing that tunable distributions of cell adhesive ligands on the surface are obtained as a function of the initial dendrimer bulk concentration. Cell experiments showed a clear correlation with dendrimer surface layout: Substrates presenting regions of high local ligand density resulted in a higher percentage of adhered cells and a higher degree of maturation of focal adhesions (FAs). Therefore, dendrimer nanopatterning is presented as a suitable and controlled approach to address the effect of local ligand density on cell response. Moreover, due to the easy modification of dendrimer peripheral groups, dendrimer nanopatterning can be further extended to other ECM ligands having density effects on cells.

JTD Keywords: Arginine-glycine-aspartic acid, Atomic force microscopy, Cell adhesion, Dendrimer, Focal adhesions, Scanning tunneling microscopy