Structural and biophysical studies on the lectin domain of GalNAc-T6 for therapeutic applications

The expression of glycoproteins containing immature truncated O-glycans such as the Thomsen-Friedenreich antigen (Ser/Thr-O-Gal\u3b21\u20133GalNAc; 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-\u3b1-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 \uc5 (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\u2013510. [2] Sharon, N., and Lis, H. Lectins: from hemagglutinins to biological recognition molecules. A historical overview. Glycobiology. 2004. 14: 53\u201362. [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\u2013328

The Paradigms of Industry 4.0 and Circular Economy as Enabling Drivers for the Competitiveness of Businesses and Territories: The Case of an Italian Ceramic Tiles Manufacturing Company

Sustainable development and the circular economy are two important issues for the future and the competitiveness of businesses. The programs for the integration of sustainability into industrial activities include the reconfiguration of production processes with a view to reducing their impact on the natural system, the development of new eco-sustainable products and the redesign of the business model. This paradigm shift requires the participation and commitment of different stakeholder groups and industry can completely redesign supply chains, aiming at resource efficiency and circularity. Developments in key ICT technologies, such as the Internet of Things (IoT), help this systemic transition. This paper explores the phases of the transition from a linear to a circular economy and proposes a procedure for introducing the principles of sustainability (environmental, economic and social) in a manufacturing environment, through the design of a new Circular Business Model (CBM). The new procedure has been tested and validated in an Italian company producing ceramic tiles, using the digitalization of the production processes of the Industry 4.0 environment, to implement the impact assessment tools (LCA\u2014Life Cycle Assessment, LCC\u2014Life Cycle Costing and S-LCA\u2014Social Life Cycle Assessment) and the business intelligence systems to provide appropriate sustainability performance indicators essential for the definition of the new CBM

Structural studies of human acidic fibroblast growth factor mutants to be used in anticancer therapy

Lectins are carbohydrate-binding proteins present ubiquitously in nature. They play a role in biological recognition phenomena involving cells and proteins. The interaction lectin-carbohydrate is highly specific, and can be exploited for the development of nanoparticles containing on their surface specific lectins that are directed to carbohydrate residues present only on malignant cells and absent on healthy ones [1].Lectins have been found to possess several anticancer properties and they are proposed as therapeutic agents, binding to cancer cell membrane receptors, causing cytotoxicity, apoptosis and inhibition of tumor growth. Some lectins are able to prevent the proliferation of malignant tumor cells because they recognize the T-antigen (Gal \u3b2 1\u20133GalNAc) found specifically on the surface of tumor cells [2]. The main problem is that their use as a detection agent for the T-antigen in clinical studies is not possible because the immune system can recognize them as foreign molecules and develop an immune response.Previous studies in our laboratory have characterized a lectin found in Boletus edulis mushrooms called BEL \u3b2-trefoil which has antiproliferative activity on tumor cell lines, because it contains three binding sites for the T-antigen. Unlike other lectins with this property, BEL \u3b2-trefoil shows structural homology with a human protein, acidic Fibroblast Growth Factor (FGF1) [3]. Superposition of the two structures suggests that the human protein could be mutated to contain at least one of the binding sites for the T-antigen. Such mutations should create in FGF1 the potential capacity of recognizing tumor cells with less immunogenicity than the fungal protein.FGF1 is a mitogenic and chemotactic protein that mediates cellular functions by binding to transmembrane receptors, which are activated by ligand-induced dimerization requiring heparin as co-receptor.To reach our purpose FGF1 cDNA was cloned into a bacterial plasmid and then mutated in two positions to prevent its binding to the natural receptor, thus suppressing its physiological activity. Loss of function was tested in fibroblast growth tests and then site-directed mutagenesis was performed in three specific positions to produce an FGF1 capable to bind T-antigen. Ligand-protein binding affinity was measured using fluorimetric and isothermal titration calorimetric techniques. Attempts to crystalize the mutants of FGF1 were made using the hanging drop technique with the final aim to carry out their structural characterization by X-ray diffraction analysis of the crystals

Crystallographic studies on carp Fishelectin (FEL)

A few years ago we isolated and sequenced a novel glycoprotein present in the eggs of the carp (Cyprinus carpio). The protein, that binds to a Sepharose 4B matrix column and can be eluted with 0.4 M N-acetyl glucosamine, behaves like a lectin of molecular mass 26686 Da. On the basis of DLS experiments the lectin is present in solution as a stable dimer. We have determined its 238 amino acid long sequence, the position of its 4 disulfide bridges and the structure of its single N-linked carbohydrate chain. The lectin shows a very low agglutinating activity for human A-type erythrocytes and interacts with both Gram positive and negative bacteria, these last interactions are inhibited by N-acetyl glucosamine. A data base search shows that its amino acid sequence is significantly similar to that of the members of an invertebrate lectin family that includes tachylectin-1, present in the amebocytes of the horseshoe crab Tachypleus tridentatus, and known to participate in the innate defense system of this species and two other lectins, characterized in the plasmodium Physarum polycephalum, that are called Tectonins I and II and are located in the external surface of the plasma membrane. We have proposed the name fishelectins (by analogy with tachylectins) for this new vertebrate protein family. Homologous genes are present in other bony fish. The carp protein has 85 % identity with a gene expressed in the crucian carp (Carassius auratus gibelio) and 78 % identity with a gene in the cDNA library of the zebrafish (Danio rerio). We have prepared three different crystal forms of the apo protein and two of co-crystals with N-acetyl glucosamine. The orthorhombic form of the apoprotein belongs to space group P212121 and was solved first using the MIR method. Our poster will present the data collection statistics of the best apo and holo forms of the lectin and the refinement statistics of the holo form and will discuss the structure and similarity of this newly identified family of vertebrate proteins to a well known invertebrate protein family

Crystallographic studies on carp Fishelectin (FEL)

A few years ago we isolated and sequenced a novel glycoprotein present in the eggs of the carp (Cyprinus carpio) (1). The protein, that binds to a Sepharose 4B matrix column and can be eluted with 0.4 M N-acetyl glucosamine, behaves like a lectin of molecular mass 26686 Da. On the basis of DLS experiments the lectin is present in solution as a stable dimer. We have determined its 238 amino acid long sequence, the position of its 4 disulfide bridges and the structure of its single N-linked carbohydrate chain. The lectin shows a very low agglutinating activity for human A-type erythrocytes and interacts with both Gram positive and negative bacteria, these last interactions are inhibited by N-acetyl glucosamine. A data base search shows that its amino acid sequence is significantly similar to that of the members of an invertebrate lectin family that includes tachylectin-1, present in the amebocytes of the horseshoe crab Tachypleus tridentatus, and known to participate in the innate defense system of this species (2.3) and two other lectins, characterized in the plasmodium Physarum polycephalum, that are called Tectonins I and II and are located in the external surface of the plasma membrane (4). We have proposed the name fishelectins (by analogy with tachylectins) for this new vertebrate protein family. Homologous genes are present in other bony fish. The carp protein has 85 % identity with a gene expressed in the crucian carp (Carassius auratus gibelio) (5) and 78 % identity with a gene in the cDNA library of the zebrafish (Danio rerio). We have prepared three different crystal forms of the apo protein and two of co-crystals with N-acetyl glucosamine. The orthorhombic form of the apoprotein belongs to space group P212121 and was solved first using the MIR method. Our poster will present the data collection statistics of the best apo and holo forms of the lectin and the refinement statistics of the holo form and will discuss the structure and similarity of this newly identified family of vertebrate proteins to a well known invertebrate protein family

Cosmology with the submillimetre galaxies magnification bias: Proof of concept

Context. As recently demonstrated, high-z submillimetre galaxies (SMGs) are the perfect background sample for tracing the mass density profiles of galaxies and clusters (baryonic and dark matter) and their time-evolution through gravitational lensing. Their magnification bias, a weak gravitational lensing eect, is a powerful tool for constraining the free parameters of a halo occupation distribution (HOD) model and potentially also some of the main cosmological parameters. Aims. The aim of this work is to test the capability of the magnification bias produced on high-z SMGs as a cosmological probe. We exploit cross-correlation data to constrain not only astrophysical parameters (Mmin, M1, and ), but also some of the cosmological ones (m, 8, and H0) for this proof of concept. Methods. The measured cross-correlation function between a foreground sample of GAMA galaxies with spectroscopic redshifts in the range 0.2 < z < 0.8 and a background sample of H-ATLAS galaxies with photometric redshifts >1.2 is modelled using the traditional halo model description that depends on HOD and cosmological parameters. These parameters are then estimated by performing a Markov chain Monte Carlo analysis using dierent sets of priors to test the robustness of the results and to study the performance of this novel observable with the current set of data. Results. With our current results, m and H0 cannot be well constrained. However, we can set a lower limit of >0.24 at 95% confidence level (CL) on m and we see a slight trend towards H0 > 70 values. For our constraints on 8 we obtain only a tentative peak around 0.75, but an interesting upper limit of 8 . 1 at 95% CL.We also study the possibility to derive better constraints by imposing more restrictive priors on the astrophysical parameters

3D numerical modeling of YSO accretion shocks

The dynamics of YSO accretion shocks is determined by radiative processes as well as the strength and structure of the magnetic field. A quasi-periodic emission signature is theoretically expected to be observed, but observations do not confirm any such pattern. In this work, we assume a uniform background field, in the regime of optically thin energy losses, and we study the multi-dimensional shock evolution in the presence of perturbations, i.e. clumps in the stream and an acoustic energy flux flowing at the base of the chromosphere. We perform 3D MHD simulations using the PLUTO code, modelling locally the impact of the infalling gas onto the chromosphere. We find that the structure and dynamics of the post-shock region is strongly dependent on the plasma-beta (thermal over magnetic pressure), different values of which may give distinguishable emission signatures, relevant for observations. In particular, a strong magnetic field effectively confines the plasma inside its flux tubes and leads to the formation of quasi-independent fibrils. The fibrils may oscillate out of phase and hence the sum of their contributions in the emission results in a smooth overall profile. On the contrary, a weak magnetic field is not found to have any significant effect on the shocked plasma and the turbulent hot slab that forms is found to retain its periodic signature

ALMA Lensing Cluster Survey: A strongly lensed multiply imaged dusty system at z ≥ 6

We report the discovery of an intrinsically faint, quintuply-imaged, dusty galaxy MACS0600-z6 at a redshift z = 6.07 viewed through the cluster MACSJ0600.1–2008 (z = 0.46). A ≃ 4σ dust detection is seen at 1.2mm as part of the ALMA Lensing Cluster Survey (ALCS), an on-going ALMA Large programme, and the redshift is secured via [C II] 158 μm emission described in a companion paper. In addition, spectroscopic follow-up with GMOS/Gemini-North shows a break in the galaxy’s spectrum, consistent with the Lyman break at that redshift. We use a detailed mass model of the cluster and infer a magnification μ ≳ 30 for the most magnified image of this galaxy, which provides an unprecedented opportunity to probe the physical properties of a sub-luminous galaxy at the end of cosmic reionization. Based on the spectral energy distribution, we infer lensing-corrected stellar and dust masses of 2.9-2.3+115 7 109 and 4.8-3.4+45 7 106 M☉, respectively, a star formation rate of 9.7-6.6+220 M☉ yr−1, an intrinsic size of 0.54-0.14+026 kpc, and a luminosity-weighted age of 200 \ub1 100 Myr. Strikingly, the dust production rate in this relatively young galaxy appears to be larger than that observed for equivalent, lower redshift sources. We discuss if this implies that early supernovae are more efficient dust producers and the consequences for using dust mass as a probe of earlier star formation

Corrigendum: Analysis of the common genetic component of large-vessel vasculitides through a meta-Immunochip strategy.

Giant cell arteritis (GCA) and Takayasu's arteritis (TAK) are major forms of large-vessel vasculitis (LVV) that share clinical features. To evaluate their genetic similarities, we analysed Immunochip genotyping data from 1,434 LVV patients and 3,814 unaffected controls. Genetic pleiotropy was also estimated. The HLA region harboured the main disease-specific associations. GCA was mostly associated with class II genes (HLA-DRB1/HLA-DQA1) whereas TAK was mostly associated with class I genes (HLA-B/MICA). Both the statistical significance and effect size of the HLA signals were considerably reduced in the cross-disease meta-analysis in comparison with the analysis of GCA and TAK separately. Consequently, no significant genetic correlation between these two diseases was observed when HLA variants were tested. Outside the HLA region, only one polymorphism located nearby the IL12B gene surpassed the study-wide significance threshold in the meta-analysis of the discovery datasets (rs755374, P\u2009=\u20097.54E-07; ORGCA\u2009=\u20091.19, ORTAK\u2009=\u20091.50). This marker was confirmed as novel GCA risk factor using four additional cohorts (PGCA\u2009=\u20095.52E-04, ORGCA\u2009=\u20091.16). Taken together, our results provide evidence of strong genetic differences between GCA and TAK in the HLA. Outside this region, common susceptibility factors were suggested, especially within the IL12B locus