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Introduction: This study examined the efficacy of a therapy based on a combination of Platelet Rich Plasma and hydroxyapatite nanoparticles in a severe clinical case involving a young Rottweiler with a complex spiral fracture of the tibia., Method: Following a worsening of the lesion after traditional surgical intervention, the subject was treated with the combined therapy. X-rays were taken at the following stages: immediately post-surgery, four weeks post-surgery, and 10 days post-treatment. Fracture gap and callus density measurements were obtained using ImageJ analysis, allowing for a detailed quantitative assessment of bone regeneration over time., Results: Post-operative radiographs indicated a clinical worsening of the fracture, revealing an increased fracture gap due to bone loss. However, significant improvements were observed ten days following the treatment, with a marked reduction in fracture gaps and increased callus density. These results demonstrated a notable acceleration in bone healing and callus formation compared to typical recovery times for similar lesions., Conclusion: The method showed potential for enhancing osteogenic regeneration, facilitating faster healing of serious orthopedic injuries compared to traditional methods., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Barbaro, Marconi, Innocenzi, Altigeri, Zepparoni, Monteleone, Alimonti, Marcoccia, Ghisellini, Rando, Ottoboni, Rau, Eggenhöffner and Scicluna.)

Subchondral bone cysts in horses represent one of the main causes of lameness that can occur in different anatomical locations. The study describes the treatment in regenerative therapy of the intracystic implantation of adipose tissue mesenchymal stromal cells (AMSCs) included in platelet-rich plasma (PRP). The ability of AMSCs to differentiate in osteogenic cells was tested in vitro and in vivo. Given the aim to investigate the application of AMSCs in bone defects and orthopedic pathologies in horses, a four-year-old male thoroughbred racing horse that had never raced before was treated for lameness of the left hind leg caused by a cyst of the medial femoral condyle. The horse underwent a new surgery performed with an arthroscopic approach in which the cystic cavity was filled with AMSCs contained in the PRP. Radiographs were taken 3, 5, and 10 months after the surgery to assess the development of newly regenerated bone tissue in the gap left by the cyst. Twelve months after the operation and after six months of regular daily training, the horse did not show any symptoms of lameness and started a racing career. According to the study, the use of AMSCs and PRP suggests promising benefits for treating subchondral bone cysts.

The possibility of improving dental restorative materials is investigated through the addition of two different types of fillers to a polymeric resin. These fillers, consisting of porous alumina and TiO 2 nanotubes, are compared based on their common physicochemical properties on the nanometric scale. The aim was to characterize and compare the surface morphological properties of composite resins with different types of fillers using analytical techniques. Moreover, ways to optimize the mechanical, surface, and aesthetic properties of reinforced polymer composites are discussed for applications in dental treatments. Filler-reinforced polymer composites are the most widely used materials in curing dental pathologies, although it remains necessary to optimize properties such as mechanical resistance, surface characteristics, and biocompatibility. Anodized porous alumina nanoparticles prepared by electrochemical anodization offer a route to improve mechanical properties and biocompatibility as well as to allow for the controlled release of bioactive molecules that can promote tissue integration and regeneration. The inclusion of TiO 2 nanotubes prepared by hydrothermal treatment in the resin matrix promotes the improvement of mechanical and physical properties such as strength, stiffness, and hardness, as well as aesthetic properties such as color stability and translucency. The surface morphological properties of composite resins with anodized porous alumina and TiO 2 nanotube fillers were characterized by Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM), and X-ray chemical analysis. In addition, the stress-strain behavior of the two composite resins is examined in comparison with enamel and dentin.

Cartilage injury defects in animals and humans result in the development of osteoarthritis and the progression of joint deterioration. Cell isolation from equine hyaline cartilage and evaluation of their ability to repair equine joint cartilage injuries establish a new experimental protocol for an alternative approach to osteochondral lesions treatment. Chondrocytes (CCs), isolated from the autologous cartilage of the trachea, grown in the laboratory, and subsequently arthroscopically implanted into the lesion site, were used to regenerate a chondral lesion of the carpal joint of a horse. Biopsies of the treated cartilage taken after 8 and 13 months of implantation for histological and immunohistochemical evaluation of the tissue demonstrate that the tissue was still immature 8 months after implantation, while at 13 months it was organized almost similarly to the original hyaline cartilage. Finally, a tissue perfectly comparable to native articular cartilage was detected 24 months after implantation. Histological investigations demonstrate the progressive maturation of the hyaline cartilage at the site of the lesion. The hyaline type of tracheal cartilage, used as a source of CCs, allows for the repair of joint cartilage injuries through the neosynthesis of hyaline cartilage that presents characteristics identical to the articular cartilage of the original tissue.

The study briefly recalls the evolution and crisis of the theory and thought of John Maynard Keynes with the rise and progressive dominance of the neoliberalism paradigm. The exercise has been made for evaluating the effects of such process for the sustainability of the global economy and society. In this view, we explored how Keynes' contribution could be useful for the global economy in building a new paradigm of socio-economic development underpinning the transition to circular economy (CE). We also evaluated the adoption of the Global Green New Deal including a case study of Italy for the purpose of suggesting how that topical political programme can be key in the CE transition. Given the urgency of environmental problems, we underline the importance of the adoption of Keynesian expansionary "green mission oriented" fiscal policies with the purpose of allowing the triggering of a virtuous circle of sustainable welfare involving the Green New deal and the transition to CE. At the basis of such virtuous circle, we propose a new paradigm based on a revisited Keynesian paradigm and models of economy within the framework of Genovesi's "civil economy" that entails an active role and responsibility of all the societal actors (consumers, companies and institutions). In this view, the "spirit" of Keynes in the economy, policy and society could be appreciated once more and be extremely useful along with other scholars' contributions in accelerating the CE transition and a more sustainable development., Competing Interests: The authors declare no competing interests., (© The Author(s) 2021.)

We have devised a supramolecular edifice involving His-tagged protein A and antibodies to yield surface immobilized, uniformly oriented, IgG-type, antibody layers with F ab fragments exposed off an electrode surface. We demonstrate here that we can affect the conformation of IgGs, likely pushing/pulling electrostatically F ab fragments towards/from the electrode surface. A potential difference between electrode and solution acts on IgGs' charged aminoacids modulating the accessibility of the specific recognition regions of F ab fragments by antigens in solution. Consequently, antibody-antigen affinity is affected by the sign of the applied potential: a positive potential enables an effective capture of antigens; a negative one pulls the fragments towards the electrode, where steric hindrance caused by neighboring molecules largely hampers the capture of antigens. Different experimental techniques (electrochemical quartz crystal microbalance, electrochemical impedance spectroscopy, fluorescence confocal microscopy and electrochemical atomic force spectroscopy) were used to evaluate binding kinetics, surface coverage, effect of the applied electric field on IgGs, and role of charged residues on the phenomenon described. These findings expand the concept of electrical control of biological reactions and can be used to gate electrically specific recognition reactions with impact in biosensors, bioactuators, smart biodevices, nanomedicine, and fundamental studies related to chemical reaction kinetics.

In this work, a new biomaterial resulting from the isolation of octopus rhodopsin (OR) starting from octopus photoreceptor membranes is presented. Mass spectroscopic characterization was employed in order to verify the presence of rhodopsin in the extract. Photoreversibility and photochromic properties were investigated using spectrophotometric measurements and pulsed light. Thin films of OR were realized using the gel-matrix entrapment method in polyvinyl alcohol solution. The results indicate that the photoreversibility and the photostability of the OR in gel-matrices are maintained. Several measurements were performed to test the stability of the resulting biomaterial in time and at room temperature. Preliminary tests demonstrate that the photoreversibility and the photostability are still found after few days from the biomaterial preparation and after the exposure for several hours at room temperature.

Proteins represent versatile building blocks for the realisation of nanostructured materials to be applied in the nanobiotechnological field. The Langmuir-Blodgett (LB) technique was utilised as a means to develop nanobiodevices based on protein molecules. Namely, engineered Cytochrome P450 thin films were fabricated and characterised. The possibility to employ LB-based protein structures to use in biosensors has been exploited. The characterisation process was performed in order to verify the best working parameters. As a first step' the protein films were studied at the air-water interface and then transferred into a solid support for further characterisation. The films were characterised by different techniques such as: UV-vis spectroscopy, nanogravimetry, atomic force microscopy and biochemical assays. The results showed that it was possible to form active cytochrome P450s nanostructures by the LB technique.

Cytochrome P450scc, mitochondrial adrenodoxin (Adx), and adrenodoxin reductase (AdR) are an essential components in a steroid hydroxylation system. In particular, mytochondrial cytochrome P450scc enzyme catalyses the first step in steroid hormones biosynthesis, represented by the conversion of cholesterol to pregnenolone. In order to study the effect of single mutations on the Adx binding a model of bovine cytochrome P450scc, previously optimized by molecular modeling, was utilized. It was hypothesized by molecular docking that two residues (Asp405 and Glu124) are involved in Adx binding. By site-directed mutagenesis, two mutants of cytochrome P450scc (Asp405Ile and Glu124Ile) expressed in Escherichia coli, were realized by replacing with isoleucines. The site-directed mutations effect on Adx binding was evaluated by differential spectral titration. The apparent dissociation constant values for Asp405Ile and Glu124Ile cytochrome P450scc show that the mutated residues seem to be at the interaction domain with Adx or at least close to it, as predicted by molecular modeling study. Finally, the engineered enzymes were characterized by biochemical and biophysical techniques such as circular dichroism (CD), UV/Vis spectroscopy, and electrochemical analysis.

As a result of the very attractive pleiotropic properties of the heme-enzymes, three P450 cytochrome isoforms (P4501A2, P4502B4, P450SCC) have been utilized to identify a general optimal procedure to biodevice assembly for sensing a wide range of organic substances. The Langmuir-Blodgett films appears to yield the best stable working conditions as shown by UV-vis spectrophotometry, nanogravimetry, circular dichroism, and electrochemical characterization, to identify the ordered nanostructures of P450 cytochromes optimal for clozapine, styrene, and cholesterol sensing. Only in the presence of low purity grade protein, as in the case of P4501A2, a gel-matrix was needed to warrant the optimal clozapine sensing. By the combination of proper immobilization, transducer and nanostructured mutants of high-grade stable and selective P450-based sensors appear capable to detect the interaction with a wide range of organic substrates such as fatty acids, drugs, and toxic compounds.

Molecular modeling and protein engineering were synergically employed to improve the fabrication of cytochrome P450scc mutant nanostructures for biodevice assembly. The optimization of protein three-dimensional structure by molecular modeling was performed using two models: in vacuum and simulating the presence of a polar solvent. Calculations were performed on a model to predict a P450scc mutant which could improve the process of molecules' immobilization onto solid supports. Engineerized cytochrome P450scc thin films were prepared and characterized by various biophysical techniques such as pi-A isotherms, surface potential measurements, Brewster angle microscopy, UV-vis spectroscopy, circular dichroism, nanogravimetry, and electrochemical analysis. This paper takes into consideration biomolecules modified by protein engineering that represent a new and powerful approach for obtaining synthetic simpler artificial structures with new or improved properties (i.e., specificity, stability, sensitivity, etc.) useful for biosensors development.

Cytochrome P450sccK201E, mutated form of cytochrome P450scc native recombinant (P450sccNR), was employed to study the enzyme-substrate interaction. The detection of the cholesterol was performed by electrochemical method using cyclic voltammetry (CV) and chronoamperometry measurements. The biochemical analysis was realized to observe the electrochemical responses of the engineerized enzyme to three different forms of cholesterol: free, low-density lipoprotein (LDL) and high-density lipoproteins (HDL). Compared to cytochrome P450sccNR, the cytochrome P450sccK201E displays a different behavior in the interaction with the substrate detection. The results show that the engineerized enzyme can be utilized for the cholesterol detection in biosensor field.

Cytochromes P450 are a large superfamily of heme-thiolate enzymes involved in the metabolism of many different organic substrates such as drugs, fatty acids and toxic compounds. The aim of this work is to analyse the binding between the cytochrome P4501A2, in solution and in gel-matrix, and its substrate (clozapine), utilising voltammetric tests. The interaction measurements were carried out using two different screen printed electrodes (rhodium-graphite and graphite-riboflavin), and the results were compared. It was demonstrated that it is possible to realise a biosensor prototype to detect the presence of clozapine indirectly by chronoamperometry.

CK2 is an essential, ubiquitous, and highly pleiotropic protein kinase whose catalytic subunits (alpha and alpha') and holoenzyme (composed by two catalytic and two regulatory beta-subunits) are both constitutively active, a property that is suspected to contribute to its pathogenic potential. Extensive interactions between the N-terminal segment and the activation loop are suspected to underlie the high constitutive activity of the isolated catalytic subunit. Here we show that a number of point mutations (Tyr(26) --> Phe, Glu(180) --> Ala, Tyr(182) --> Phe) and deletions (Delta 2-6, Delta 2-12, Delta 2-18, Delta 2-24, Delta 2-30) expected to affect these interactions are more or less detrimental to catalytic activity of the alpha-subunit of human CK2, the deleted mutants Delta 2-24 and Delta 2-30 being nearly inactive under normal assay conditions. Kinetic analyses showed that impaired catalytic activity of mutants Delta 2-12, Delta 2-18, Delta 2-24, and Y182F is mainly accounted for by dramatic increases in the K(m) values for ATP, whereas a drop in K(cat) with K(m) values almost unchanged was found with mutants Y26F and E180A. Holoenzyme reconstitution restored the activity of mutants Delta 2-12, Delta 2-18, Y26F, E180A, and Y182F to wild type level and also conferred catalytic activity to the intrinsically inactive mutants, Delta 2-24 and Delta 2-30. These data demonstrate that specific interactions between the N-terminal segment and the activation loop are essential to provide a fully active conformation to the catalytic subunits of CK2; they also show that these interactions become dispensable upon formation of the holoenzyme, whose constitutive activity is conferred by the beta-subunit through a different mechanism.

A new matrix-integral part of the new DNA microarray instrumentation DNA analyzer (DNASER) is introduced based on a novel DNA patterning on the solid support surface. Such patterning found the way to modify a glass surface for a precise positioning of small droplets of aqueous DNA solutions, without special robots (arrayers), within the boundaries of the modified regions. The physically heterogeneous surface consists of highly hydrophilic spots surrounded by a highly hydrophobic area leading to the surface patterning needed for a DNA microarray: a matrix of hydrophilic spots properly activated for immobilization of oligonucleotides has been fabricated on absolutely passive hydrophobic surface. The optimal efficiency of the above functionalitation technology of a glass-substrate in obtaining DNA microarray was confirmed by the Cy3-dCTP-labeled DNA sample, as shown by charge-coupled device images of the DNASER previously described.

Recombinant human histone H4 (hH4) was produced in milligrams quantities in Escherichia coli, without altering the codons of the original cDNA sequence. The hH4 cDNA was subcloned into the pQE30 expression vector, in frame with a sequence encoding an N-terminal stretch of six histidine residues. Purification to electrophoretic homogeneity was obtained by nickel-chelating chromatography, followed by gel filtration. The final yield of the entire expression and purification process was about 1 mg of pure histone H4 per liter of bacterial culture. SDS-PAGE analysis showed for the recombinant H4 a molecular weight corresponding to the expected one (12,535 Da). Circular dichroism spectroscopy was used to estimate the secondary structural composition of recombinant histone, when it is isolated from the physiological core particle. It was observed that under these conditions histone H4 exhibits an altered secondary conformation. In order to induce the recombinant histone to assume a conformation more similar to the one measured when it is organized inside the nucleosome, we resuspended it in buffers at increasing ionic strengths and in the presence of different concentrations of trifluoroethanol. We tried also to mimic the physiological situation of histone H4 by adding an equimolar amount of a commercial DNA to the protein solution. Finally, an estimation of protein thermal stability was evaluated by spectropolarimetry., (Copyright 2002 Elsevier Science (USA).)

Casein kinase-2 (CK2) probably is the most pleiotropic member of the protein kinase family, with more than 200 substrates known to date. Unlike the great majority of protein kinases, which are tightly regulated enzymes, CK2 is endowed with high constitutive activity, a feature that is suspected to underlie its oncogenic potential and possible implication in viral infections. This makes CK2 an attractive target for anti-neoplastic and antiviral drugs. Here, we present an overview of our present knowledge about CK2 inhibitors, with special reference to the information drawn from two recently solved crystal structures of CK2alpha in complex with emodin and with 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), this latter being the most specific CK2 inhibitor known to date. A comparison with a series of anthraquinone and xanthenone derivatives highlights the crucial relevance of the hydroxyl group at position 3 for inhibition by emodin, and discloses the possibility of increasing the inhibitory potency by placing an electron withdrawing group at position 5. We also present mutational data corroborating the relevance of two hydrophobic residues unique to CK2, Val66 and Ile174, for the interactions with emodin and TBB, but not with the flavonoid inhibitors quercetin and fisetin. In particular, the CK2alpha mutant V66A displays 27- and 11-fold higher IC(50) values with emodin and TBB, respectively, as compared with the wild-type, while the IC(50) value with quercetin is unchanged. The data presented pave the road toward the rational design of more potent and selective inhibitors of CK2 and the generation of CK2 mutants refractory to inhibition, useful to probe the implication of CK2 in specific cellular functions.

To shed light on the structural features underlying high constitutive activity of protein kinase CK2 a number of mutants of the human CK2alpha-subunit altered in the interactions between the N-terminal segment and the activation loop have been generated and shown to be defective in catalytic activity. In particular the truncated mutant delta2-12 displays under standard conditions an almost complete loss of catalytic activity accounted for by a dramatic rise in its Km forATP (from 10 to 206 microM) and a reduced Kcat. Such a drop in efficiency is paralleled by conformational disorganization, as judged from Superdex 75 gel filtration profile. Both catalytic properties and gel filtration behaviour similar to those of wild type CK2alpha were restored upon association with the regulatory beta-subunit, suggesting that constitutive activity is conferred to CK2alpha and to CK2 holoenzyme through different molecular mechanisms. In the holoenzyme an assumable release of tension at the backbone of Ala-193 (as seems to be indicated by a comparison of the crystal structures of maize CK2alpha alone vs. a CK2alpha-beta peptide complex) may result in the ability of the activation loop to adopt its proper conformation independently of interactions with the N-terminal segment.

The concept that the amino-terminal segment plays a role in conferring high basal activity to protein kinase CK2 alpha subunit has been validated by generating two mutants (Y26F and delta2-6) which are defective both in catalytic activity and in thermal stability. The additional finding that the activity of the two mutants is fully restored upon association with the regulatory beta subunit, in conjunction with the observation that synthetic peptides reproducing the N-terminal segment (1-30) and the activation loop (175-201) of CK2alpha counteract the functional effects of the C-terminal domain of the beta subunit, is consistent with a mechanism of activation of CK2 where the N-terminal domain of alpha and the C-terminal domain of beta play interchangeable roles.

We studied chromosome 1p loss of heterozygosity (1p-LOH) in 53 neuroblastomas (NBs) using 15 (CA)n repeat loci, which covered a region of 90 cM. We also assessed chromosome 1p36 deletion by fluorescence in situ hybridization (FISH) on interphase nuclei. 1p-LOH was found in 19 (36%, 95% confidence interval (CI) 23-50%) NBs. We detected interstitial and large deletion in both localized and disseminated tumours and in one tumour of a patient at stage 4S. Allelic loss was frequently observed in 1p36 and 1p32 regions. In patients older than 1 year of age (53 versus 13%, P < 0.002) we detected significant chromosome 1p deletion and it was associated with MYCN amplification (P = 0.001). Overall survival (OS) analysis showed that 1p-LOH is predictive of a poor outcome (odds ratio 16.5, 95% CI 5.4-50.9%); therefore, 1p-LOH should be regarded as an additional tumour progression marker in neuroblastoma.