The biochemical properties of muscle extracellular matrix are essential for stem cell adhesion, motility, proliferation and myogenic development. Recombinant elastin-like polypeptides are synthetic ...polypeptides that, besides maintaining some properties of the native protein, can be tailored by fusing bioactive sequences to their C-terminal. Our laboratory synthesized several Human Elastin-Like Polypeptides (HELP) derived from the sequence of human tropoelastin. Here, we developed a novel HELP family member by fusing the elastin-like backbone to the sequence of human Epidermal Growth Factor. We employed this synthetic protein, named HEGF, either alone or in combination with other proteins of the HELP family carrying RGD-integrin binding sites, as adhesion substrate for C2C12 myoblasts and satellite cells primary cultures. Adhesion of myoblasts to HEGF-based substrates induced scattering, decreased adhesion and cytoskeleton assembly; the concomitant presence of the RGD motifs potentiated all these effects. Recombinant substrates induced myoblasts proliferation, differentiation and the development of multinucleated myotubes, thus favoring myoblasts expansion and preserving their myogenic potential. The effects induced by adhesion substrates were inhibited by AG82 (Tyrphostin 25) and herbimycin A, indicating their dependence on the activation of both the EGF receptor and the tyrosine kinase c-src. Finally, HEGF increased the number of muscle stem cells (satellite cells) derived from isolated muscle fibers in culture, thus highlighting its potential as a novel substrate for skeletal muscle regeneration strategies.
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•The role of EGF on myogenesis is still debated.•Substrate-immobilized EGF increases myoblasts motility, proliferation, differentiation.•Soluble EGF failed to induce the same effects.•Adsorbed RGD sequences potentiate the effects of EGF on myogenesis.•Immobilized EGF induces the myogenic activation of satellite cells from isolated muscle fibers.•The effects of combined EGF and RGD immobilization can be exploited in skeletal muscle regeneration strategies.
Bilirubin is a toxicological biomarker for hemolysis and liver diseases. The current automated diazo method used in clinical chemistry has limited applicability in rodent models and cannot be used in ...small animals relevant to toxicology, microphysiological systems, cell cultures, and kinetic studies. Here, we present a versatile fluorometric method for nanoscale analysis of bilirubin based on its highly specific binding to the recombinant bifunctional protein HELP–UnaG (HUG). The assay is sensitive (LoQ = 1.1 nM), accurate (4.5% relative standard error), and remarkably robust, allowing analysis at pH 7.4–9.5, T = 25–37 °C, in various buffers, and in the presence of 0.4–4 mg × L−1 serum albumin or 30% DMSO. It allows repeated measurements of bilirubinemia in murine models and small animals, fostering the 3Rs principle. The assay determines bilirubin in human plasma with a relative standard error of 6.7% at values that correlate and agree with the standard diazo method. Furthermore, it detects differences in human bilirubinemia related to sex and UGT1A1 polymorphisms, thus demonstrating its suitability for the uniform assessment of bilirubin at the nanoscale in translational and precision medicine.
•Forced displacement is not a mere side-effect of conflict.•Price shocks generate a higher level of forced displacement in conflict settings.•Forced displacement takes place in districts with more ...labor-intensive commodities•When armed actors collude with the government, forced displacement is more frequent.
Do economic incentives explain forced displacement during conflict? This paper examines this question in Colombia, which has had one of the world’s most acute situations of internal displacement associated with conflict. Using data on the price of bananas along with data on historical levels of production, I find that price increases generate more forced displacement in municipalities more suitable to produce this good. However, I also show that this effect is concentrated in the period in which paramilitary power and operations reached an all-time peak. Additional evidence shows that land concentration among the rich has increased substantially in districts that produce these goods. These findings are consistent with extensive qualitative evidence that documents the link between economic interests and local political actors who collude with illegal armed groups to forcibly displace locals and appropriate their land, especially in areas with more informal land tenure systems, like those where bananas are grown more frequently.
In contrast to many antimicrobial peptides, members of the proline-rich group of antimicrobial peptides inactivate Gram-negative bacteria by a non-lytic mechanism. Several lines of evidence indicate ...that they are internalized into bacteria and their activity mediated by interaction with unknown cellular components. With the aim of identifying such interactors, we selected mutagenized Escherichia coli clones resistant to the proline-rich Bac7(1-35) peptide and analysed genes responsible for conferring resistance, whose products may thus be involved in the peptide's mode of action. We isolated a number of genomic regions bearing such genes, and one in particular coding for SbmA, an inner membrane protein predicted to be part of an ABC transporter. An E. coli strain carrying a point mutation in sbmA, as well as other sbmA-null mutants, in fact showed resistance to several proline-rich peptides but not to representative membranolytic peptides. Use of fluorescently labelled Bac7(1-35) confirmed that resistance correlated with a decreased ability to internalize the peptide, suggesting that a bacterial protein, SbmA, is necessary for the transport of, and for susceptibility to, proline-rich antimicrobial peptides of eukaryotic origin.
In recent years, antimicrobial peptides (AMPs) have enjoyed a renaissance, as the world is currently facing an emergency in terms of severe infections that evade antibiotics' treatment. This is due ...to the increasing emergence and spread of resistance mechanisms. Covalent conjugation with polymers is an interesting strategy to modulate the pharmacokinetic profile of AMPs and enhance their biocompatibility profile. It can also be an effective approach to develop active coatings for medical implants and devices, and to avoid biofilm formation on their surface. In this concise review, we focus on the last 5 years' progress in this area, pertaining in particular to AMPs that contain d-amino acids, as well as their role, and the advantages that may arise from their introduction into AMPs.
Stimuli-responsive hydrogel matrices have attracted great attention in biomedical and biotechnological fields for controlled delivery of bioactive compounds, as well as a vehicle for therapeutic cell ...spreading. Elastin-derived biomimetic polypeptides are recombinant macromolecules suitable for the realization of smart biomaterials. In this study, we explored the potential of an elastin biomimetic matrix to realize proteolytic stimuli-responsive systems to control the release of substances. Our approach showed that this matrix was susceptible to elastolytic degradation, and it has been successfully employed to obtain an efficient delivery of a model protein. This setup will constitute a therapeutic agent delivery platform to realize devices capable of responding and interacting with biological systems at the molecular level.
There is an increasing interest towards the development of new antimicrobial coatings, especially in light of the emergence of antimicrobial resistance (AMR) towards common antibiotics. ...Cyclodipeptides (CDPs) or diketopiperazines (DKPs) are attractive candidates for their ability to self-assemble into supramolecular polymers and yield gel coatings that do not persist in the environment. In this work, we compare the antimicrobial cyclo(Leu-Phe) with its heterochiral analogs cyclo(D-Leu-L-Phe) and cyclo(L-Leu-D-Phe), as well as cyclo(L-Phe-D-Phe), for their ability to gel. The compounds were synthesized, purified by HPLC, and characterized by 1H-NMR, 13C-NMR, and ESI-MS. Single-crystal X-ray diffraction (XRD) revealed details of the intermolecular interactions within the supramolecular polymers. The DKPs were then tested for their cytocompatibility on fibroblast cells and for their antimicrobial activity on S. aureus. Overall, DKPs displayed good cytocompatibility and very mild antimicrobial activity, which requires improvement towards applications.
Stimuli-responsive hydrogel matrices are inspiring manifold applications in controlled delivery of bioactive compounds. Elastin-derived polypeptides form hydrogel matrices that may release bioactive ...moieties as a function of local increase of active elastases, as it would occur in several processes like inflammation. In view of the development of a patch for healing wounds, recombinant elastin-based polypeptides were combined with a proteolysis-resistant scaffold, made of electrospun poly-L-lactic acid (PLLA) fibers. The results of this study demonstrated the compatibility of these two components. An efficient procedure to obtain a composite material retaining the main features of each component was established. The release of the elastin moiety was monitored by means of a simple protocol. Our data showed that electrospun PLLA can form a composite with fusion proteins bound to elastin-derived polypeptides. Therefore, our approach allows designing a therapeutic agent delivery platform to realize devices capable of responding and interacting with biological systems at the molecular level.
Cyclodipeptides (CDPs) or diketopiperazines (DKPs) are often found in nature and in foodstuff and beverages and have attracted great interest for their bioactivities, biocompatibility, and ...biodegradability. In the laboratory, they can be prepared by green procedures, such as microwave-assisted cyclization of linear dipeptides in water, as performed in this study. In particular, five CDPs were prepared and characterized by a variety of methods, including NMR and ESI-MS spectroscopies and single-crystal X-ray diffraction (XRD), and their cytocompatibility and anti-aging activity was tested in vitro, as well as their ability to penetrate the different layers of the skin. Although their mechanism of action remains to be elucidated, this proof-of-concept study lays the basis for their future use in anti-age cosmetic applications.
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