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Transthyretin (TTR) amyloidoses are familial or sporadic degenerative conditions that often feature heavy cardiac involvement. Presently, no effective pharmacological therapy for TTR amyloidoses is available, mostly due to a substantial lack of knowledge about both the molecular mechanisms of TTR aggregation in tissue and the ensuing functional and viability modifications that occur in aggregate-exposed cells. TTR amyloidoses are of particular interest regarding the relation between functional and viability impairment in aggregate-exposed excitable cells such as peripheral neurons and cardiomyocytes. In particular, the latter cells provide an opportunity to investigate in parallel the electrophysiological and biochemical modifications that take place when the cells are exposed for various lengths of time to variously aggregated wild-type TTR, a condition that characterizes senile systemic amyloidosis. In this study, we investigated biochemical and electrophysiological modifications in cardiomyocytes exposed to amyloid oligomers or fibrils of wild-type TTR or to its T4-stabilized form, which resists tetramer disassembly, misfolding, and aggregation. Amyloid TTR cytotoxicity results in mitochondrial potential modification, oxidative stress, deregulation of cytoplasmic Ca2+ levels, and Ca2+ cycling. The altered intracellular Ca2+ cycling causes a prolongation of the action potential, as determined by whole-cell recordings of action potentials on isolated mouse ventricular myocytes, which may contribute to the development of cellular arrhythmias and conduction alterations often seen in patients with TTR amyloidosis. Our data add information about the biochemical, functional, and viability alterations that occur in cardiomyocytes exposed to aggregated TTR, and provide clues as to the molecular and physiological basis of heart dysfunction in sporadic senile systemic amyloidosis and familial amyloid cardiomyopathy forms of TTR amyloidoses.

Transthyretin (TTR) is involved in a subset of familial or sporadic amyloid diseases including senile systemic amyloidosis (SSA), familial amyloid polyneuropathy and cardiomyopathy (FAP/FAC) for which no effective therapy has been found yet. These conditions are characterized by extracellular deposits primarily found in the heart parenchyma and in peripheral nerves whose main component are amyloid fibrils, presently considered the main culprits of cell sufferance. The latter are polymeric assemblies grown from misfolded TTR, either wt or carrying one out of many identified mutations. The recent introduction in the clinical practice of synthetic TTR-stabilizing molecules that reduce protein aggregation provides the rationale to search natural effective molecules able to interfere with TTR amyloid aggregation by hindering the appearance of toxic species or by favoring the growth of harmless aggregates. Here we carried out an in depth biophysical and morphological study on the molecular features of the aggregation of wt- and L55P-TTR involved in SSA or FAP/FAC, respectively, and on the interference with fibril aggregation, stability and toxicity to cardiac HL-1 cells to demonstrate the ability of Oleuropein aglycone (OleA), the main phenolic component of the extra virgin olive oil. We describe the molecular basis of such interference and the resulting reduction of TTR amyloid aggregate cytotoxicity. Our data offer the possibility to validate and optimize the use of OleA or its molecular scaffold to rationally design promising drugs against TTR-related pathologies that could enter a clinical experimental phase.

Lipases resistant to inhibition and denaturation by methanol are valuable tools for biotechnological applications, in particular for biofuel production. Microbial lipases have attracted a great deal of interest because of their stability at high concentrations of organic solvents. Burkholderia cepacia lipase (BCL) is tested here for robustness towards methanol in terms of conformational stability and catalytic activity in transesterification assays. This lipase turns out to be even more tolerant than the homologous and better characterized enzyme from Burkholderia glumae. BCL unfolding transition, as monitored by far-UV circular dichroism (CD) and intrinsic fluorescence, displays a Tm above 60°C in the presence of 50% methanol. The protein unfolds at low pH, and the organic solvent affects the nature of the denatured state under acidic conditions. The protein performs well in transesterification assays upon prolonged incubations at high methanol concentrations. BCL is highly tolerant to methanol and displays particularly high conformational stability under conditions employed for transesterification reactions. These features depict BCL as a promising enzyme for biofuel industry.

Inclusion bodies (IBs) are protein-based nanoparticles formed in Escherichia coli through stereospecific aggregation processes during the overexpression of recombinant proteins. In the last years, it has been shown that IBs can be used as nanostructured biomaterials to stimulate mammalian cell attachment, proliferation, and differentiation. In addition, these nanoparticles have also been explored as natural delivery systems for protein replacement therapies. Although the production of these protein-based nanomaterials in E. coli is economically viable, important safety concerns related to the presence of endotoxins in the products derived from this microorganism need to be addressed. Lactic acid bacteria (LAB) are a group of food-grade microorganisms that have been classified as safe by biologically regulatory agencies. In this context, we have demonstrated herein, for the first time, the production of fully functional, IB-like protein nanoparticles in LAB. These nanoparticles have been fully characterized using a wide range of techniques, including field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy, zymography, cytometry, confocal microscopy, and wettability and cell coverage measurements. Our results allow us to conclude that these materials share the main physico-chemical characteristics with IBs from E. coli and moreover are devoid of any harmful endotoxin contaminant. These findings reveal a new platform for the production of protein-based safe products with high pharmaceutical interest. Statement of Significance The development of both natural and synthetic biomaterials for biomedical applications is a field in constant development. In this context, E. coli is a bacteria that has been widely studied for its ability to naturally produce functional biomaterials with broad biomedical uses. Despite being effective, products derived from this spe

Poly(2-ethyl-2-oxazoline) thin coatings were deposited on different substrates by means of low pressure plasma polymerization. The stability of the thin films was evaluated by immersion in different aqueous environments (Phosphate Buffer Saline (PBS) at 37 °C, sodium dodecyl sulfate (SDS) solutions, aqueous solutions with different pH) and was found to be dependent on the deposition conditions. Chemical characterization of the coatings deposited with different process parameters was performed by means of Attenuated Total Reflectance Fourier Transform Infrared (FTIR/ATR) spectroscopy and Nuclear Magnetic Resonance (NMR) spectroscopy. Partial retention of the oxazoline ring was assessed also for the stable coatings. Noteworthy, the coatings displayed an intrinsic fluorescence with an emission maximum around 534–542 nm. The reactivity of the plasma deposited coatings towards carboxylic, amino- and hydroxyl groups was investigated through selected reactions with poly(acrylic acid), amino-terminated polyethylene glycol and zonyl FSO-100 fluorosurfactant. Results indicate strong reactivity with COOH groups, probably as a consequence of the partial retention of the oxazoline ring. As a result, it is suggested that stable coatings can be exploited for the covalent immobilization of COOH-containing proteins or biomolecules, opening a wide range of potential applications.

An artificial aggrecan-like proteoglycan has been designed and synthesized in vitro. At variance with natural proteoglycans, whose glycosaminoglycan chains are always O-linked via a tetrasaccharide bridge to the serine residues of a specific protein core, the present structure consists of chondroitin-6-sulfate chains directly bound to the lysine and hydroxylysine residues of a collagen molecule backbone. The resulting macromolecule has been characterized by histochemistry, atomic force microscopy and FTIR. The number of variables involved (e.g., length and type of the collagen backbone, glycosaminoglycan species, sulfation type and pattern, molecular weight, number and length of side chains, etc.) makes possible to conceive an almost endless variety of artificial proteoglycans, each precisely tailored to a specific functional role. In addition to their use as biomaterials, glycated collagens interact with cells in complex ways and a previous study has already shown the ability of a glycated collagen to redirect fibroblastoma cells from proliferation to differentiation. The research is still underway.

Inclusion bodies (IBs) are protein-based nanoparticles formed in Escherichia coli through stereospecific aggregation processes during the overexpression of recombinant proteins. In the last years, it has been shown that IBs can be used as nanostructured biomaterials to stimulate mammalian cell attachment, proliferation, and differentiation. In addition, these nanoparticles have also been explored as natural delivery systems for protein replacement therapies. Although the production of these protein-based nanomaterials in E. coli is economically viable, important safety concerns related to the presence of endotoxins in the products derived from this microorganism need to be addressed. Lactic acid bacteria (LAB) are a group of food-grade microorganisms that have been classified as safe by biologically regulatory agencies. In this context, we have demonstrated herein, for the first time, the production of fully functional, IB-like protein nanoparticles in LAB. These nanoparticles have been fully characterized using a wide range of techniques, including field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy, zymography, cytometry, confocal microscopy, and wettability and cell coverage measurements. Our results allow us to conclude that these materials share the main physico-chemical characteristics with IBs from E. coli and moreover are devoid of any harmful endotoxin contaminant. These findings reveal a new platform for the production of protein-based safe products with high pharmaceutical interest., Peer Reviewed, Postprint (published version)

Despite the relevance of carbohydrates as cues in eliciting specific biological responses, the covalent surface modification of collagen-based matrices with small carbohydrate epitopes has been scarcely investigated. We report thereby the development of an efficient procedure for the chemoselective neoglycosylation of collagen matrices (patches) via a thiol-ene approach, between alkene-derived monosaccharides and the thiol-functionalized material surface. Synchrotron radiation-induced X-ray photoelectron spectroscopy (SR-XPS), Fourier transform-infrared (FT-IR), and enzyme-linked lectin assay (ELLA) confirmed the effectiveness of the collagen neoglycosylation. Preliminary biological evaluation in osteoarthritic models is reported. The proposed methodology can be extended to any thiolated surface for the development f smart biomaterials for innovative approaches in regenerative medicine

Epigallocatechin-3-gallate (EGCG) and tetracycline are two known inhibitors of amyloid aggregation able to counteract the fibrillation of most of the proteins involved in neurodegenerative diseases. We have recently investigated their effect on ataxin-3 (AT3), the polyglutamine-containing protein responsible for spinocerebellar ataxia type 3. We previously showed that EGCG and tetracycline can contrast the aggregation process and toxicity of expanded AT3, although by different mechanisms. Here, we have performed further experiments by using the sole Josephin domain (JD) to further elucidate the mechanism of action of the two compounds. By protein solubility assays and FTIR spectroscopy we have first observed that EGCG and tetracycline affect the JD aggregation essentially in the same way displayed when acting on the full-length expanded AT3. Then, by saturation transfer difference (STD) NMR experiments, we have shown that EGCG binds both the monomeric and the oligomeric JD form, whereas tetracycline can only interact with the oligomeric one. Surface plasmon resonance (SPR) analysis has confirmed the capability of the sole EGCG to bind monomeric JD, although with a KD value suggestive for a non-specific interaction. Our investigations provide new details on the JD interaction with EGCG and tetracycline, which could explain the different mechanisms by which the two compounds reduce the toxicity of AT3.

The overexpression of a recombinant protein in bacteria often leads to its deposition in cytoplasmic or periplasmic aggregates, called inclusion bodies (IBs). The polypeptide newly synthetized by the ribosome reaches its native conformation through folding intermediates that can be more prone to aggregation compared to the native form. IBs might contain proteolytic fragments of the recombinant protein, ribosomal components, and traces of phospholipids and nucleic acids, as well as a small fraction of polypeptides unrelated to the target protein. The relationship between the retention of residual native-like structure and IB formation was also investigated by Fourier transform infrared (FTIR) in the case of two recombinant E. coli strains expressing different levels of human interferon-alpha-2b. This chapter gives a brief overview of the main experimental methods applied to IB structural characterization: fluorescence spectroscopy and microscopy, flow cytometry, circular dichroism, FTIR spectroscopy, electron microscopies, atomic force microscopy, and x-ray fiber diffraction.

The polyglutamine (polyQ)-containing protein ataxin-3 (AT3) triggers the neurodegenerative disease spinocerebellar ataxia type 3 when its polyQ tract is expanded beyond a critical length. This results in protein aggregation and generation of toxic oligomers and fibrils. Currently, no effective treatment is available for such and other polyQ diseases. Therefore, plenty of investigations are being carried on to assess the mechanism of action and the therapeutic potential of anti-amyloid agents. Tetracycline and the polyphenol compound epigallocatechin-3-gallate (EGCG) have been shown to exert some effect in preventing fibrillogenesis of amyloidogenic proteins. Here, we have incubated an expanded AT3 variant with either compound to assess their effects on the aggregation pattern. The process was monitored by atomic force microscopy and FTIR spectroscopy. Whereas in the absence of any treatment AT3 gives rise to amyloid -rich fibrils, whose hallmark is the typical glutamine side-chain hydrogen bonding, when incubated in the presence of EGCG it generated soluble, SDS-resistant aggregates, much poorer in -sheets and devoid of any ordered side-chain hydrogen bonding. These are off-pathway species that persist until the latest incubation time and are virtually absent in the control sample. In contrast, tetracycline did not produce major alterations in the structural features of the aggregated species compared to the control, but substantially increased their solubility. Both compounds significantly reduced toxicity, as shown by the MTT assay in COS-7 cell line and in a transgenic Caenorhabditis elegans strain expressing in the nervous system an AT3 expanded variant in fusion with GFP.

An approach to the surface modification of poly(lactic-co-glycolic acid) (PLGA) to render it adhesive to poly(amidoamine) (PAA) hydrogels, thus allowing fabrication of entirely biodegradable and biomimetic multilayered composite biomaterials with the PLGA film playing the role of reinforcing material, for instance imparting resistance to stitching, is N2/H2 plasma treatment of PLGA surfaces aimed at introducing amine groups and covalently immobilizing PAAs. Grafting of linear PAAs, demonstrated by XPS analysis, is reported first. Coherent PAA/PLGA composite hydrogels with embedded PLGA films can be obtained likewise. They are soft, elastic and resistant to osmotic shock. In contrast, hydrogels prepared from untreated PLGA films delaminate on swelling. Accessible hybrid PAA/PLGA materials may expand PLGA's biomedical applications

The synthesis of new dendrons and their immobilization on collagen patches via thiol–ene photoclick reaction, followed by chemoselective alkoxyamino–carbonyl conjugation to carbohydrates is presented. XPS, FTIR, and ELLA assays confirmed the effectiveness of the collagen multivalent neoglycosylation.

Despite the relevance of carbohydrates as cues in eliciting specific biological responses, glycans have been rarely exploited in the study of neuronal physiology. We report thereby the study of the effect of neoglucosylated collagen matrices on neuroblastoma F11 cell line behavior. Morphological and functional analysis clearly showed that neoglucosylated collagen matrices were able to drive cells to differentiate. These data show for the first time that F11 cells can be driven from proliferation to differentiation without the use of chemical differentiating agents. Our work may offer to cell biologists new opportunities to study neuronal cell differentiation mechanisms in a cell environment closer to physiological conditions.

Despite the relevance of carbohydrates as cues in eliciting specific biological responses, the covalent surface modification of collagen-based matrices with small carbohydrate epitopes has been scarcely investigated. We report thereby the development of an efficient procedure for the chemoselective neoglycosylation of collagen matrices (patches) via a thiol-ene approach, between alkene-derived monosaccharides and the thiol-functionalized material surface. SR-XPS, FTIR and ELLA assays confirmed the effectiveness of the collagen neoglycosylation. Preliminary biological evaluation in osteoarthritic models is reported. The proposed methodology can be extended to any thiolated surface for the development of smart biomaterials for innovative approaches in regenerative medicine.

Protein misfolding and aggregation in intracellular and extracellular spaces is regarded as a main marker of the presence of degenerative disorders such as amyloidoses. To elucidate the mechanisms of protein misfolding, the interaction of proteins with inorganic surfaces is of particular relevance, since surfaces displaying different wettability properties may represent model systems of the cell membrane. Here, we unveil the role of surface hydrophobicity/hydrophilicity in the misfolding of the Josephin domain (JD), a globular-shaped domain of ataxin-3, the protein responsible for the spinocerebellar ataxia type 3. By means of a combined experimental and theoretical approach based on atomic force microscopy, Fourier transform infrared spectroscopy and molecular dynamics simulations, we reveal changes in JD morphology and secondary structure elicited by the interaction with the hydrophobic gold substrate, but not by the hydrophilic mica. Our results demonstrate that the interaction with the gold surface triggers misfolding of the JD when it is in native-like configuration, while no structural modification is observed after the protein has undergone oligomerization. This raises the possibility that biological membranes would be unable to affect amyloid oligomeric structures and toxicity.

Methanol is often employed in biocatalysis with the purpose of increasing substrates solubility or as the acyl acceptor in transesterification reactions, but inhibitory effects are observed in several cases. We have studied the influence of methanol on the catalytic activity and on the conformation of the lipase from Burkholderia glumae, which is reported to be highly methanol tolerant if compared with other lipases. We detected highest activity in the presence of 50-70 % methanol. Under these conditions, however, the enzyme stability is perturbed, leading to gradual protein unfolding and finally to aggregation. These results surmise that, for this lipase, methanol-induced deactivation does not depend on inhibition of catalytic activity but rather on negative effects on the conformational stability of the catalyst. © 2013 Springer-Verlag Berlin Heidelberg.