1,151 research outputs found
Structural studies of a Tremella fuciformis mushroom lectin: a novel protein with antitumoral properties
No full textLectins are carbohydrate-binding proteins of non-immune origin, widely distributed in all the living organisms. They play a role in different biological processes and, due to their carbohydrate specific binding, some lectins have been found to possess antitumoral properties. A particular case is the recognition of the T-antigenic determinant (Galβ1-3GalNAcα) on the malignant cell surfaces, which is present in 90% of human carcinomas. Upon binding to the carbohydrate, some lectins can cause apoptosis, cytotoxicity, inhibition of tumour growth, thus preventing the proliferation of tumoral cells. Considering the fact that these carbohydrates are masked on healthy cells, the highly specific carbohydrate-lectin interaction can be exploited to target only malignant cells. Although the function and the biological properties of several lectins have been determined, there are still many lectins that remain to be structurally and functionally characterized. As reported in the literature, some Tremella fuciformis proteins have been investigated for their potential therapeutical properties and in the light of this, the crude extract of this fungus was examined to assess the presence of lectins. A lectin of 11 KDa, named TFL, was isolated and purified from the dried fruiting bodies and used for testing several crystal screening conditions. The best crystals were grown in 0.1 M TRIS pH 8.5, 1.5 potassium phosphate dibasic, 1 % DMSO and the final data sets were collected at the ESRF of Grenoble, revealing the three-dimensional structure of the protein. TFL belongs to the space group P1211 and the cell parameters of the crystal are the following: a = 61.62 Å, b = 61.83 Å, c = 67.84 Å, with β = 106.87 °. The protein is a monomer, composed of six β-sheets (from A to F) that arrange to form the so called “β-barrel”. In addition, two α-helixes, named H1 and H2 can be recognized in the structure, which is also stabilized by the presence of two disulphide bridges that connect Cys 54 to Cys 70 and Cys 97 to Cys 100, respectively. Thermal protein stability was analyzed by means of differential scanning calorimetry, revealing that TFL does not undergo a two-state unfolding process and its denaturation is reversible, a feature that has been rarely observed for lectins before. In addition, chemical and pH-induced unfolding were investigated using fluorescence spectroscopy, highlighting the high stability of TFL in a wide range of conditions. Exploiting isothermal titration calorimetry, more information about sugar-binding were acquired, identifying N-acetylgalactosamine as the best candidate, which has micromolar affinity for TFL. It has also been observed that Tremella fuciformis lectin shows no cytotoxicity on malignant and healthy cells and interestingly, it seems to reduce skin malignant cells migration and to have a positive effect in the upregulation of certain genes involved in cancer arresting (SBPP1). In addition, immunomodulatory activity of TFL was analyzed by means of ELISA essay, resulting in the up-regulation of the most important inflammatory markers (IL-6, TNFα), that could be usefully exploited in order to kill intracellular microorganisms and to help the human body to develop a stronger tumoral resistance. In conclusion, this work provides a new and interesting insight into cancer treatment, being an essential prerequisite for future in vitro and in vivo experiments. The encouraging results obtained so far, alongside with the fact that TFL has been produced also heterologously in E.coli, pave the way for further studies, including the feasibility to perform mutagenesis and to explore the possibility to further encapsulate the protein inside tailor-made nanoparticles, with the aim of reducing the amount of protein used, increasing its therapeutical efficiency, and preventing side effects
Pegylated silica nanoparticles: cytotoxicity and macrophage uptake
No full textHere, we present a thorough study of pegylated silica nanoparticle (SNP) interaction with different biological environments. The SNPs have a mean diameter of about 40Â nm and are coated with polyethylene glycol (PEG) of different molecular weights. The physicochemical characterization of SNPs allowed the confirmation of the binding of PEG chains to the silica surface, the reproducibility of the synthesis and the narrow size-dispersion. In view of clarifying the SNP interaction with biological environments, we first assessed the SNP reactivity after the incubation with two cell lines (macrophages RAW 264.7 and primary human fibroblasts), observing a reduced toxicity of pegylated SNPs compared to the bare ones. Then, we investigated the effect of the protein adsorption on the SNP surface using the model serum protein, bovine serum albumin (BSA). We found that the protein adsorption takes place more heavily on poorly pegylated SNPs, promoting the uptake of the latter by macrophages and leading to an increased mortality of these cells. To better understand this mechanism by means of flow cytometry, the dye Ru(bpy)3Cl2 was incorporated in the SNPs. The overall results highlight the SNP potentialities as a drug delivery system, thanks to the low interactions with the macrophages
Purification and structural studies of a Tremella fuciformis mushroom lectin
No full textLectins are carbohydrate-binding proteins or glycoproteins of non-immune origine widely distributed in living organisms including animals, plants and fungi. They play a role in different biological processes mediating cellular signaling, differentiation, tissue metastasis and host-pathogen interactions. Moreover they serve as storage proteins, are fundamental during fungi and plant morphogenesis and development and take part into their defense processes [1].Thanks to their carbohydrate specific binding, some lectins are able to recognize, in a reversible way, the sugar moieties on the erythrocytes cell surface (N-acetylgalactosamine, D-galactosamine), causing a phenomena called hemagglutination. Furthermore some lectins have been found to possess antitumoral properties [2]. Specifically they recognize the Tn-antigenic determinant (Galβ1-3GalNAcα) on the malignant cells surface causing apoptosis, cytotoxicity, inhibition of tumor growth and preventing the proliferation of tumor cells. Considering the fact that this kind of residues are masked on healthy cells, the highly specific carbohydrate-lectin interaction can be exploited to target only malignant cells, also because the Tn-antigen is the most specific human cancer-associated structure, expressed in about 90% of the human carcinomas.For the reasons described above, during the last decades lectins have been extensively investigated for their potential therapeautical effects and biotechonological applications, especially fungal lectins which have unique carbohydrate specificities. However, altough the function and the biological properties of many lectins have been determined, their structural characterization lags behind.As reported in the literature, some Tremella fuciformis proteins have been investigated for their potential therapeutical properties and have shown to possess anticancer, anti-inflammatory, antioxidant and neuroprotective activities. In the light of above the crude extract proteins have been checked to assess the presence of lectins [3]. To this purpose, the mushrooms dried fruiting bodies of Tremella fuciformis were homogenized and extracted in a phospate buffer at 4°C and neutral pH. The crude extract was then precipitated using a high concentration of (NH4)2SO4 and dyalised against TRIS buffer in order to remove the precipitant. A lectin was eluted from a hog gastric mucin affinity column and purified first with a DEAE-cellulose column and then with a size exclusion SEPHACRYL G-100 column. An electrophoresis gel was required to precisely define the lectin molecular weight, which is 22 kDa. The purified lectin has been used for testing several crystal screening conditions
Structural and biophysical studies on the lectin domain of GalNAc-T6 for therapeutic applications
No full textThe expression of glycoproteins containing immature truncated O-glycans such as the Thomsen-Friedenreich antigen (Ser/Thr-O-Galβ1–3GalNAc; T-antigen) and the Lewis antigen (sialyl-T-antigen) is a characteristic feature observed on almost all malignant epithelial cells. Therefore, there is a particular interest in their application not only as prognostic markers but also as therapeutic targets [1]. These antigens can be recognized by lectins, a group of highly specific carbohydrate-binding proteins that have been proposed as useful tools for antitumor drug-targeting [2].The three-dimensional structure of several lectins with antitumor properties has been determined in our laboratory by X-ray crystallography. N-α-acetylgalactosaminyltransferase-6 (GalNAc-T6) is an enzyme present also in humans which contains a catalytic domain and a lectin domain with a binding site for N-acetylgalactosamine (GalNAc), one of the saccharides exposed by cancer cells (Tn-antigen). Unlike other lectins with these properties, the lectin domain of GalNAc-T6 presents a structural fold found also in other human proteins, unlocking the opportunity to use protein engineering tools to design new anticancer therapeutics [3]. The three-dimensional structure of GalNAc-T6 has not been determined so far, neither has been its substrate specificity. Therefore, the production of a recombinant form containing only the lectin domain can contribute to these two critical points that need to be considered to evaluate its possible use in cancer therapies. The lectin domain of this enzyme was expressed by cloning the C-terminal portion of the DNA coding sequence and introducing it into Pichia pastoris for its recombinant production. Biophysical methods such as spectrofluorimetry and isothermal titration calorimetry were used to analyze the ability of the engineered protein to bind the T-antigen monosaccharides. The binding dissociation constant (Kd) of the protein-carbohydrate interaction was determined. The stability of the protein was also studied through its thermodynamic parameters of unfolding using differential scanning calorimetry. Crystallization screenings were set up using a broad variety of precipitants in order to produce crystals to be used to study the three-dimensional structure of the engineered protein using X-ray diffraction. The crystals that were grown were taken to the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to carry out the diffraction experiments. Although we were able to collect data up to a resolution of 2.8 Å (854,648 reflections) all the crystals we have examined so far were found to be twinned making the assignment of a definitive space group uncertain. We are currently working on correcting this problem using both the appropriate software and attempting to grow better crystals. Our goal is to produce an engineered human protein that specifically recognizes cancer specific carbohydrates and is thus suitable for protein therapeutics applied in drug-delivery methods for cancer treatment. The present structural and biophysical data are the prerequisite for future studies regarding the biological and clinical properties of the lectin. [1] Stowell, S. R. Tongzhong J. and Cummings R. D. Protein Glycosylation in Cancer. Annu Rev Pathol 2015. 10: 473–510. [2] Sharon, N., and Lis, H. Lectins: from hemagglutinins to biological recognition molecules. A historical overview. Glycobiology. 2004. 14: 53–62. [3] Berois, N., Mazal, D. et al. UDP-N-Acetyl-D-Galactosamine: N-acetylgalactosaminyltransferase-6 as a New Immunohistochemical Breast Cancer Marker. Journal of Histochemistry & Cytochemistry. 2006. 54(3): 317–328
Giulia Veronica Varisco
No full textThe headword explains the biography and the contribution of the author Giulia Varisco to the children's literatur
Ytterbium Disilicate/Monosilicate Multilayer Environmental Barrier Coatings: Influence of Atmospheric Plasma Spray Parameters on Composition and Microstructure
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Ytterbium Disilicate/Monosilicate Multilayer Environmental Barrier Coatings: Influence of Atmospheric Plasma Spray Parameters on Composition and Microstructure
by Giulia Di Iorio,Laura Paglia *ORCID,Giulia PedrizzettiORCID,Virgilio GenovaORCID,Francesco MarraORCID,Cecilia BartuliORCID andGiovanni PulciORCID
INSTM Reference Laboratory for Materials and Surface Engineering, Sapienza University of Rome, Eudossiana 18, 00184 Rome, Italy
*
Author to whom correspondence should be addressed.
Coatings 2023, 13(9), 1602; https://doi.org/10.3390/coatings13091602
Original submission received: 10 August 2023 / Revised: 31 August 2023 / Accepted: 11 September 2023 / Published: 13 September 2023
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Abstract
SiC/SiC ceramic matrix composites (SiCf/SiC CMCs) are regarded as the new materials for the hot-section components of aircraft gas turbine engines, since they have one-third of the density of metallic superalloys, a higher temperature capability, good mechanical strength, and excellent thermal shock resistance. However, high-temperature water-vapor-rich combustion gases can induce severe surface recession phenomena in SiC/SiC leading to component failure. For this reason, it is necessary to design protective coatings, i.e., environmental barrier coatings (EBCs), able to protect the SiC/SiC surface in combustion environments. In the present work, ytterbium monosilicate (Yb2SiO5), stable when exposed to water vapor at high temperatures, and ytterbium disilicate (Yb2Si2O7), characterized by a thermal expansion coefficient closer to that of the substrate, were selected for a multilayer EBC system. EBCs were processed using the atmospheric plasma spray (APS) technique. A set of deposition parameters were tested, varying the power of the torch, and the composition and microstructure of the deposited coatings were studied in terms of porosity, crack density, and post-deposition phase retention by performing SEM, EDS, and XRD analysis. The results allow for the definition of the influence of deposition parameters on the final properties of multilayer EBC coatings
Correction to: Safety and Psychological Outcomes of Tandem t:Slim X2 Insulin Pump with Control-IQ Technology in Children, Adolescents, and Young Adults with Type 1 Diabetes: A Systematic Review
No full textThe second author name was incorrectly published as Giula Maria Smylie. The correct name is Giulia Marie Smylie. The original article has been corrected
TREATMENT OF SKELETAL DISEASES WITH A NATURALLY DERIVED ANTIOXIDANT AND BISPHOSPHONATE EMBEDDED NANOPARTICLES
No full textSkeletal disorders are degenerative diseases causing progressive disability and are becoming more and more prevalent in our society. Bone is a special tissue able to support load and stress and is continuously renewed thanks to bone turnover mediated by the well coordinated activity of osteoblasts and osteoclasts. Many metabolic bone diseases are characterized by an imbalance of the activity of bone cells and bone turnover that could be estimated through the evaluation of laboratory data analysis. In addition to metabolic disorders, bone tissue can be impairs by genetic diseases such as osteogenesis imperfect. In addition, Osteoarthritis (OA), the most prevalent musculoskeletal pathology, is predominantly characterized by the progressive degradation of articular cartilage due to an imbalance between anabolic and catabolic processes. Age-related changes that occur in articular are thought to represent a major risk factor for OA development. Therefore, skeletal diseases have been associated to defective differentiation pathways of progenitor stem cells (PSCs) to produce osteoblasts or chondrocytes. RUNX2 as well as SOX9 are transcription factors 202 responsible of commitment of PSCs in osteoblasts and chondrocytes respectively. In order to evaluate the possibility to affect and improve osteoblasts or chondrocytes commitment we assayed a naturally derived antioxidant and a bisphosphonate (clodronate) embedded nanoparticles in vivo (murine model) and in vitro (cell line) respectively. Antioxidant molecule was embedded into PLGA (poly lactic-co-glycolic acid) with the emulsion evaporation method. Bisphosphonate nanoparticles were prepared using chitosan and hyaluronic acid applying the ionotropic gelation method
Scrivere senza anestesia. La chiarezza di Giulia Niccolai
No full textIl saggio colloca storicamente la narratrice e poetessa Giulia Niccolai nel canone del Novecento letterario italiano discutendone poetica e cifre stilistiche. L'ampia analisi proposta tocca tutte le opere dell'autrice evidenziandone i legami intertestuali, anche tra poesia e narrativa, e i progressivi sviluppi in un arco cronologico esteso, tra anni Sessanta e primi anni Duemila. Lo studio coglie anche l'importanza dei riferimenti alle arti visive, in particolare alla fotografia, che Giulia Niccolai ha praticato in prima persona negli anni della Neoavanguardia, e alla pittura americana.The essay places the narrator and poet Giulia Niccolai in the canonical twentieth century Italian literary discussing her poetics and stylistic figures. The wide analysis proposed touches all the works of the author highlighting the intertextual links, also between poetry and narrative, and the progressive developments in an extended chronological period, between the Sixties and early Twenties. The study also captures the importance of references to the visual arts, especially photography, which Giulia Niccolai has practiced in the years of the Neo-avant-garde, and to American painting
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L'afrofuturismo tra Stati Uniti e Italia. Dalla memoria storica ai viaggi intergalattici per re-immaginare futuri postumani
No full textIn this article the author explores the cultural movement called Afrofuturism, tracing its origins, features, simbology, and its historical and political meanings. Starting from the (supposed) discoursive gap between African and African American people on one side and technology and science fiction on the other, this paradigm addresses themes related to the intersection of African Diaspora and African cultures with technology and science fiction, with the aim to recover the history of slavery and racism through the projections into alternative space-time contexts. In particular, the author explores the feminist afrofuturism perspective in order to examine the specific oppression and cultural production of African and African American women. In doing so, the author analyzes the development of afrofuturism in Italy, focusing on the work of the singer and beatmaker Karima 2G
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