Focus on:  Hide Contributions
Back to Conference View
Europe/Rome English Login

I° Conference on Crystallography, Structural Chemistry and Biosystems

→ Europe/Rome
Catania
Cinzia Giannini (CNR - Istituto di Cristallografia) , Michele Saviano (CNR - IC)
Description

On behalf of the Advisory Council of the Institute of Crystallography, it is my great pleasure to announce that the next conference on Crystallography, Structural Chemistry and Biosystems (CSCB) will be held during October 04-06, 2021 in Catania.

The CSCB meeting aims to share the most recent and advanced research in the field of structural and functional chemistry and biology conducted in our institute. Such an important event will be a great opportunity to promote new collaborations and research projects among all the scientists belonging to IC research units of Bari, Catania, Roma and Trieste.

The CSCB conference will also provide the chance for academic and industrial networks to appreciate the various types of know-how and multidisciplinary competencies in the fields of Nanomedicine, Biosciences, Chemistry and Structural Biology, Applied Chemistry and/or Physics and Material Science and discover the great potential of these topics in both basic and applied research.

Looking forward to seeing you in Catania

Best regards,

Cinzia Giannini

timetable.pdf
Usefull information
Contact
  • Monday, 4 October
    • 14:00 → 15:00
      Registration
    • 15:00 → 15:30
      Opening
      • 15:00
        Opening Remarks 5m
        Speakers: Dr Cinzia Giannini (CNR - Istituto di Cristallografia) , Prof. Lidia Armelao (CNR - Dipartimento di Scienze Chimiche e Tecnologie dei Materiali)
      • 15:05
        Nuove opportunità di finanziamento della ricerca 25m
        Speaker: Prof. Luigi Nicolais (Università di Napoli)
    • 15:30 → 16:30
      Session: 1
      Convener: Dr Michele Saviano (CNR - Istituto di Cristallografia)
      • 15:30
        Supra and Sub Molecular Investigation of Pathologic Tissues by X-Ray Scanning Microscopy 15m

        X-ray Small and Wide Scattering scanning microscopies have been adopted to inspect morphological and structural properties of collagen-based tissues at the atomic and nano scale1. Examples will be discussed on specific pathologies:

        • osteoarthritis of the hip, also named osteoarthrosis of the hip or coxarthrosis, which is a chronic degenerative disorder of the hip joint, causing growing articular pain that can bring the patient to lifestyle limitations until surgical intervention is needed2
        • keratoconus, a pathology affecting cornea, which causes progressive thinning of the stroma and consequently abnormal curvature, inducing irregular astigmatism and myopia, corneal fibrosis, and distortion of vision, due to the modification in the organization of the corneal collagen3
        • abdominal aortic aneurysm, that occurs in the major artery from the hearth that supplies blood to the abdomen, and popliteal aneurysm, that takes place in the legs, behind the knees, characterized by alteration of collagen structure into vessel’s wall of the aneurysm tissues, heterogeneous grade of inflammation related to infiltrating cells and extracellular matrix changes, in particular disruption of elastic fibers, fibrosis and calcifications4
        • diabetes mellitus, a metabolic disorder characterized by high blood sugar levels over a prolonged period due to defects in insulin action or secretion, which causes collagen to have a fixed orientation, stiffen the tissue and is likely to disrupt the normal cell interactions5.

        References

        1. Giannini C., Altamura D., Aresta B. M., Sibillano T., Siliqi D., De Caro L. Book Title : Synthesis and characterization of inorganic micro and nano-materials. Edited by: A. Dibenedetto, M. Aresta, Published 2013 by De Gruyter
        2. Giannini C., Siliqi D., Ladisa M., Altamura D., Diaz A., Beraudi A., Sibillano T., De Caro L., Stea S., Baruffaldi F. and Bunk O. J. Appl. Cryst. 2014, 47, 110-117
        3. Sibillano T., De Caro L., Scattarella F., Scarcelli G., Siliqi D., Altamura D., Liebi M., Ladisa M., Bunk O. and Giannini, C.. J. Appl. Cryst. 2016, 49, 1240-1244
        4. Giannini C., Ladisa M., Lutz-Bueno V., Terzi A., Ramella M., Fusaro L., Altamura D., Siliqi D., Sibillano T., Diaz A., Boccafoschi F. and Bunk O. IUCrJ 2019, 6(2), 267-276
        5. Giannini C., Terzi A., Fusaro L., Sibillano T., Diaz A., Ramella M., Lutz-Bueno V., Boccafoschi F. and Bunk O. IuCrJ 2021 8(4), 621-632.
        Speaker: Dr Cinzia Giannini (CNR - Istituto di Cristallografia)
        Abstract_CinziaGiannini.docx
      • 15:45
        Structure-based prediction of hERG-related cardiotoxicity: a benchmark study 15m

        Drug-induced blockade of the human ether-à-go-go-related gene (hERG) channel is today considered the main cause of cardiotoxicity in post-marketing surveillance. Hence, several ligand-based approaches were developed in the last years and are currently employed in the early stages of a drug discovery process for in silico cardiac safety assessment of drug candidates. The first structure-based classifiers able to discern hERG binders from non-binders will be presented. LASSO regularized Support Vector Machines were applied to integrate docking scores and protein-ligand interaction fingerprints. 396 models were trained and validated based on: i) high-quality experimental bioactivity information returned by 8,337 curated compounds extracted from ChEMBL (version 251) and ii) structural predictor data. Molecular docking simulations were performed by using GLIDE and GOLD software programs and four different hERG structural models, namely the recently published structures obtained by cryo-electron microscopy (PDB codes: 5VA12 and 7CN13) and two published homology models selected for comparison. Interestingly, some classifiers return performances comparable to ligand-based models in terms of accuracy (AUCMAX = 0.86±0.01) and negative predictive values (NPVMAX = 0.81±0.01) thus putting forward the herein presented computational workflow as a valuable tool for predicting hERG-related cardiotoxicity without the limitations of ligand-based models, typically affected by low interpretability and a limited applicability domain. From a methodological point of view, the study represents the first effort to develop classifiers integrating docking scores and protein-ligand interaction fingerprints, an approach ensuring significantly better accuracies than those returned by classifiers based on docking scores only (p < 0.05). Finally, the study highlights the importance of using hERG structural models accounting for ligand-induced fit effects and allowed us to select the best performing protein conformation to be employed for a reliable structure-based prediction of hERG-related cardiotoxicity.

        References
        1. Davies, M.; Nowotka, M.; Papadatos, G.; Dedman, N.; Gaulton, A.; Atkinson, F.; Bellis, L.; Overington, J. P. ChEMBL Web Services: Streamlining Access to Drug Discovery Data and Utilities. Nucleic Acids Res 2015, 43 (W1), W612–W620
        2. Wang, W.; MacKinnon, R. Cryo-EM Structure of the Open Human Ether-à-Go-Go-Related K+ Channel HERG. Cell 2017, 169 (3), 422-430
        3. Asai, T.; Adachi, N.; Moriya, T.; Oki, H.; Maru, T.; Kawasaki, M.; Suzuki, K.; Chen, S.; Ishii, R.; Yonemori, K.; Igaki, S.; Yasuda, S.; Ogasawara, S.; Senda, T.; Murata, T. Cryo-EM Structure of K+-Bound HERG Channel Complexed with the Blocker Astemizole. Structure 2021, 29 (3), 203-212.

        Speaker: Dr Giuseppe Felice Mangiatordi (CNR - Istituto di Cristallografia)
        Abstract_Mangiatordi_2021.docx
      • 16:00
        Structural Insights into the Intracellular Region of the Human Magnesium Transport Mediator CNNM4 15m

        The four member family of “Cyclin and Cystathionine-synthase (CBS) domain divalent metal cation transport mediators”, CNNMs, are the least-studied mammalian magnesium transport mediators. CNNM4 is abundant in the brain and the intestinal tract, and its abnormal activity causes Jalili Syndrome. Recent findings show that suppression of CNNM4 in mice promotes malignant progression of intestinal polyps and is linked to infertility. The association of CNNM4 with phosphatases of the regenerating liver, PRLs, abrogates its Mg2+-efflux capacity, thus resulting in an increased intracellular Mg2+ concentration that favors tumor growth. Here we present the crystal structures of the two independent intracellular domains of human CNNM4, i.e., the Bateman module and the cyclic nucleotide binding-like domain (cNMP)1. We also derive, by Small Angle X ray Scattering (SAXS) as shown if Figure 1, a model structure for the full intracellular region in the absence and presence of MgATP and the oncogenic interacting partner, PRL-11. We find that only the Bateman module interacts with ATP and Mg2+, at non-overlapping sites facilitating their positive cooperativity. Furthermore, both domains dimerize autonomously, where the cNMP domain dimer forms a rigid cleft to restrict the Mg2+ induced sliding of the inserting CBS1 motives of the Bateman module, from a twisted to a flat disk shaped dimer.

        References
        1. Giménez-Mascarell, P.; Oyenarte, I.; González-Recio, I.; Fernández-Rodríguez, C.; Corral-Rodríguez, M.Á.; Campos-Zarraga, I.; Simón, J.; Kostantin, E.; Hardy, S.; Díaz Quintana, A.; Zubillaga Lizeaga, M.; Merino, N.; Diercks, T.; Blanco, F.J.; Díaz Moreno, I.; Martínez-Chantar, M.L.; Tremblay, M.L.; Müller, D.; Siliqi, D.; Martínez-Cruz, L.A. Structural Insights into the Intracellular Region of the Human Magnesium Transport Mediator CNNM4. Int. J. Mol. Sci. 2019, 20, 6279.

        Speaker: Dr Dritan Siliqi (CNR - Istituto di Cristallografia)
        Abstract_siliqi.pdf
      • 16:15
        Synthesis of Porphyrin-Peptide conjugates and interaction with Aβ42: potential use of the systems as theranostic agents in Alzheimer’s disease 15m

        Alzheimer’s disease (AD) is the most common cause of senile dementia affecting more than 50 million people worldwide. AD is a disorder of the central nervous system, clinically characterized by progressive loss of memory and other cognitive skills. Unfortunately, there is no cure for AD.
        The major pathophysiological hallmark is the formation of amyloid deposits with a common β-sheet structure1. Amyloid plaques are the results of a long process initiated with the seeding and production of smaller aggregated soluble forms of Aβ called oligomers. They are the underlying toxic species responsible for synaptic dysfunction in the brains of AD patients.
        Our research focus on development of new compounds able to interfere with the early stages of the aggregation process of the Aβ peptides. Our compounds could be of interest as potential drug candidates AD therapy. We designed and synthesized a series of di-functional systems explicating synergic and/or additive actions to counteract the adverse effects of Aβ aggregated forms2, 3.
        In particular, we covalently linked a cationic metallo-porphyrin with the well-known Aβ-recognizing KLVFF amino acid sequence4. The peptide conjugation to the porphyrin macrocyclic was first accomplished via the formation of an amide. A second generation was obtained in good yields using a click chemistry approach.
        In this communication, we describe the different synthesis approach and the ability of the conjugated porphyrin peptides to interact with Aβ, together with the role of metal ions (Zn or Cu) at the core of porphyrin macrocycle in assisting the recognition process.5
        The interaction between the peptide conjugate and Aβ was studied by using an array of different biophysical techniques including Dynamic Light Scattering (DLS), far-UV, Circular Dicroism (CD), Fluorescence spectroscopy and MALDI-TOF-MS.

        References
        1. Tay W. M., Huang D., Rosenberry T. L., Paravastu A. K., J. Mol. Biol. 2013, 425, 2494–2508.
        2. De Bona P., Giuffrida M. L., Caraci F., Copani A., Pignataro B., Attanasio F., Cataldo S., Pappalardo G., Rizzarelli E., Journal of peptide science: an official publication of the European Peptide Society, 2009, 15(3), 220-228
        3. Sinopoli A., Giuffrida A., Tomasello M.F., Giuffrida M.L., Leone M., Attanasio F., Caraci F., De Bona P., Naletova I., Saviano M., Copani A., Pappalardo G., Rizzarelli E.,ChemBioChem, 2016, 17(16), 1541-1549.
        4. Villari V., Tosto R., Di Natale G., Sinopoli A., Tomasello M., Lazzaro S., Micali N., Pappalardo G., Chem Select, 2017, 2 (28), 9122-9127

        Speaker: Dr Rita Tosto (CNR - Istituto di Cristallografia)
        Abstract_finale.docx
    • 16:30 → 17:00
      Coffe break 30m
    • 17:00 → 18:15
      Session: 2
      Convener: Dr Francesco Capitelli (CNR - Istituto di Cristallografia)
      • 17:00
        PNAs: a versatile synthetic tool in miRNAs targeting 15m

        MiRNAs, small, highly conserved non coding RNAs, directly regulate more than 60% of the entire human mRNAs.1 They are involved in the regulation of many biological processes as well as their dysregulation can lead to various human diseases.2 As a result, miRNAs represent an attractive class of molecules in drug development and diagnostic. Peptide Nucleic Acid (PNA)3 is an unnatural mimic of DNA/RNA made of a pseudo peptide backbone of N-aminoethyl glycine (aeg) unit instead of the sugar phosphate one. PNA showed high affinity/specificity binding to DNA/RNA and demonstrated excellent enzymatic resistance. We will show the employment of PNAs based molecules in two case studies: a) PNAs as a new class of analogues of tumor suppressive miRNA-34a4 b) PNAs as smart probes for multiple detection of circulating miRNAs.

        In the first case, through an silico and experimental integrated approach new PNA based analogues of different length of tumor suppressive miRNA-34a, were realized (Figure 1A). Their interaction with two binding sites on the target MYCN mRNA was investigated by molecular dynamics simulation and spectroscopic techniques (CD, UV, NMR). Intake assay and confocal microscopy of PNA sequences were also carried out in vitro on neuroblastoma Kelly cells. PNA/RNA hetero duplexes, despite the presence of multiple mismatches, showed very interesting features in terms of stability/affinity as well as of cellular uptake. In the second case PNA based smart probes (fluorescent and electrochemical), were designed, synthesized and characterized by CD, UV, fluorescence spectroscopy. The smart probes were developed for the early diagnosis of pancreatic cancer and coeliac disease, for which a prompt and non-invasive diagnosis methodology is still missing.

        References

        1. Bartel DP, Cell. 2009,136, 215–233.
        2. Kasinski AL, Slack FJ, Nat Rev Cancer. 2011;11, 849–864
        3. Nielsen PE, Eghoim M, Berg RH, Buchardt O. Science,1991; 254:1497–1500.
        4. Piacenti V. et al. Bioorganic Chemistry, 2019,91, 103165.
        Speaker: Dr Maria Moccia (CNR - Istituto di Cristallografia)
        Abstract_Moccia.docx
      • 17:15
        Visible light activeTiO2/Au nanorods for photocatalytic environmental remediation 15m

        Nanostructured materials exhibit outstanding size/shape dependent properties that make them extremely promising in several application fields. Fundamental features that include (but are not limited to) their optical response, thermodynamic behaviour, plasmonic, magnetic and catalytic properties can be modulated by varying nanocrystal size and shape, without altering their chemical composition.1 Furthermore, synthesis routes as well as characterization techniques have rapidly evolved thus enabling to rationally design, synthesize, process and organise nanomaterials for specific application fields.1 In the area of environmental remediation, nanomaterial with photocatalytic properties are gaining increasing attention. In particular, metal/semiconductor thanks to their original photocatalytic properties can be exploited to address several environmental concerns. Indeed, the nanometal moiety can induce the photoactivation of the nanocomposite under visible light irradiation because of several effects, promoted by the Surface Plasmon Resonance (SPR) phenomenon that is strongly size and shape dependent.2 The present work, aims at synthesizing a visible light active plasmonic photocatalyst on a gram scale by means of promptly scalable procedure. The synthesis of TiO2/Au NRs hybrid nanocomposites has been performed and their photocatalytic properties investigated. The choice of Au NRs arises from their unique plasmonic properties that enable, to efficiently exploit the UV, Visible and NIR ranges of the solar spectrum. The TiO2/AuNRs nanocomposites have been prepared by a conventional co-precipitation technique and their textural and structural properties have been investigated. The photocatalytic efficiency of the TiO2/AuNRs has been evaluated by carrying out a set of experiments including the photodegradation reactions under UV light irradiation of an organic model compound, Methylene Blue, the photodegradation of the Nalidixic acid as a model real pollutant under visible light irradiation. Finally, the TiO2/AuNRs calcinated at 450°C resulted to be the most efficient photocatalyst among the investigated nanocomposites, thus holding a great promise as a versatile candidate for real scale applications.3

        Speaker: Dr Francesca Petronella (CNR - Istituto di Cristallografia)
        Petronella-Abstract-ICSCB_Conference.docx
      • 17:30
        A survey of different approaches using ESI Mass Spectrometry for the characterization of metal binding sites in amyloid peptide fragments. 15m

        Neurodegenerative disorders (NDs) such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and prion diseases are some of the most common forms of age-related diseases. Even if pathogenesis of these neurodegenerative diseases remains unclear, increasing evidences point out a common critical molecular process involving the assembly of various aggregated protein with a β-sheet conformation, termed amyloids.[1] The inhibition of this process could be a viable therapeutic strategy for the treatment of neurodegenerative diseases. Metal ions, especially copper, zinc and iron play a very important roles in neurodegeneration having impact on both protein structure and oxidative stress.[2] For a better comprehension of the structural features of the metal-amyloid protein complexes, our previous studies aimed at determining the stoichiometry, the affinity and the location of metal binding sites as well as the coordination environment around the metal ions.[3-6] In particular, potentiometric investigations were used to determine the stoichiometry and quantify the metal binding affinity of protein and/or peptide complex species. Moreover, the speciations resulting from thermodynamic data were coupled with spectroscopic information (UV–Vis, CD, ESR, NMR, and MS) in order to clarify the binding modes of various species. One of the main hindrances in amyloid protein investigation concerns the low peptide solubility at the concentrations needed to perform potentiometric titrations and spectroscopic studies. In this contest, the high sensitivity of mass spectrometry may overcome this limit. In this presentation, i will discuss the different mass spectrometry approach used in the characterization of metal ion complexes with different amyloid peptides such as prion,[4] Aβ,[5] Tau[6] and IAPP fragments. In particular, high-resolution ESI-MS assignments can be used to obtain direct information on stoichiometry of different metal complex species existing in dilute solutions. Tandem mass spectrometry investigations provides additional information regarding the metal interaction with amyloid peptide fragments. In particular, HCD (High energy Collision Dissociation) of copper(II) complexes, selected as precursor ions, provides interesting information about the amino acid residues involved in the copper(II) coordination. In order to evaluate the involvement of specific aminoacid residues in the formation of metal complexes, i will show some examples of limited proteolysis experiments together with ESI mass spectrometric analysis of peptide complexes.[4, 5]

        Speaker: Dr Giuseppe Di Natale (CNR - Istituto di Cristallografia)
        Abstract-Di_Natale_Giuseppe.doc
      • 17:45
        The Interdepartmental S&T Foresight Project 15m

        WHY FORESIGHT?

        The Science and Technology Foresight Project has carried out initiatives of great relevance, in order to define research strategies able to address crucial social problems related to energy, food, health, water, as well as the cross-sectoral topic of breakthrough innovative materials, complexity of systems and data science. Both, the holistic approach applied to the analysis of the topics, as well as the innovative format of the invitation only workshops, enticed the participation of internationally acknowledged experts. This framework guaranteed all participants the necessary conditions to carry out an open interactive debate, consolidating a collective intelligence, which assisted in achieving a consensus on research priorities, knowledge gaps, and funding needs.

        ACTIVITY

        The Foresight Project has involved the international scientific community in the effort of identifying innovative, scientifically based, medium/long term solutions of problems within the four areas mentioned above. The current social needs have been identified as the drivers for change and the proposed solutions must address them. Each problem is a big challenge by itself, and it becomes even more substantial when considering that all issues are connected. Looking for a proper identification of both the relevant features of each problem, as well as the current knowledge gaps, which must be overcome, we adopted a systems approach. The goal is to move beyond a linear view of cause and effect, towards the development of a systemic and integrated view.

        Speaker: Dr Cecilia Bartolucci (CNR - Istituto di Cristallografia)
        Abstract_Bartolucci.docx
        Abstract Catania Cecilia Bartolucci-1.docx
      • 18:00
        A year in Antarctica 15m

        More than 1000km from the coast and at 3200m of altitude, the Concordia base is one of the 3 bases in the middle of the Antarctic plateau.
        Temperatures that go down to -80 degrees have a great effect on activities.
        During the long 4-month winter night a small group of people maintain the base and carry out measures and observers.
        the National Research Program in Antarctica, PNRA, and the French Polar Institute, IPEV, are the bodies for the management and implementation of scientific and technological research programs in Concordia.
        Sensations and observers in the Italian-French station of Concordia during the winterover 2019. Isolation and activities of the next 2022 campaign.
        What does it mean to work in an extreme environment.

        reference: https://www.ilmessaggero.it/scienza/la_spedizione_italo_francese_in_antartide-4370008.html

        Speaker: Mr Massimiliano Catricalà (CNR - Istituto di Cristallografia)
    • 18:15 → 19:00
      ePoster session: X-ray approaches
      Convener: Dr Francesco Bellia (CNR - Istituto di Cristallografia)
      • 18:15
        X-ray multilevel investigation of collagen in tissue engineering and pathology 5m

        Type I collagen is the main fibril-forming protein of the extracellular matrix (ECM), that provides mechanical support to tissues and organs. The protein distribution and organization is tissue-specific, depending on the bio-mechanical function of the tissue itself. From the molecular order, up to supramolecular scale, type I collagen is organized in triple helices assembled in fibrils and fibers, in accordance with a liquid crystalline arrangement at nanoscale. Thanks to its hierarchical structure and functional domains, collagen supplies physical support to cells attachment and growth, influencing tissue development. Thus its biocompatibility, bioactivity and biodegradability make this protein so attractive in tissue engineering, as biomaterial for implantable medical devices. Type I collagen can be extracted from different collagen-rich tissues of distinct animal species (bovine, equine, fish etc.) by chemical and/or enzymatic processes, that lead to structural alteration of the fibrillary arrangement. Since most of the structural features of type I collagen were assessed by classic X-rays investigations during last decades1,2, in our studies we demonstrate the worthwhile contribution of Wide and Small Angle X ray Scattering (WAXS, SAXS)3 techniques in the structural evaluation of sub and supramolecular changes of the protein, during the biomaterial fabrication steps from fresh collagen-rich tissues (bovine dermis, equine tendon, fish skin) to the final scaffolds. The evidences show the impact of processing conditions on both molecular scale and fibrillary arrangement at nanoscale. Moreover, the demonstration that further manufacturing protocols deeply affect the features of the biomaterial itself, allow to screen the suitable protocols according to the tissue to regenerate4,5.
        The X-ray structural evaluation of collagen has been proved to be useful when applied not only to the biomaterials of engineered tissues, but even when directly applied to pathologic tissues. In particular it was demonstrated the possibility to assess the morphological alteration of collagen through scanning X-ray microdiffraction (SAXS/WAXS), when the tissue is placed under specific pathologic conditions, such as high glucose concentration typical of diabetes. The study permitted to characterize intra and intermolecular parameters alteration of collagen network with picometer precision, opening the possibility to inspect the effect of diabetes on the connectives and the impact of therapies on them6.

        Speaker: Dr Alberta Terzi (CNR - Istituto di Cristallografia)
        Abstract_TerziAlberta.docx
      • 18:20
        Wide Angle and Small Angle X-ray Scattering for the structural characterization of fibers 5m

        Over the years, there has been a growing interest in the development of nanofibrous structures with natural (collagen, silk fibroin, elastin, chitosan, alginate) and synthetic (polylactic acid - PLA, poly-lactic-co-glycolic acid – PLGA, polycaprolactone – PCL) polymers as fibrous structures. Thanks to tunable specific features linked to their nanostructure, the application of nanofibers in several fields has shown to be suitable for the solution of practical problems. Wide-Angle (WAXS) and Small-Angle (SAXS) X-ray Scattering are two techniques available at the MicroImaging Laboratory (XMI-Lab) of the Institute of Crystallography in Bari1 equipped with by a table top high brilliance (synchrotron class) X-ray micro-source. Here, we report our work on natural polymeric fibers such as type I collagen2,3, silk fibroin from Bombix Mori, cellulose4 but also on engineered fibers, such as peptide-based supramolecular architectures5.
        .

        Speaker: Dr Teresa Sibillano (CNR - Istituto di Cristallografia)
        Abstract_SibillanoTeresa.docx
      • 18:25
        Molecular Replacement, new procedures and applications 5m

        Molecular Replacement is the most used technique to solve macromolecules; an original algorithm called REMO09 (Caliandro et al., 2009) has been developed and implemented in the software produced by Institute of Crystallography. In order to get a final model as complete and refined as possible, we have developed a pipeline based on the synergy between our phase refinement algorithms (Burla et al., 2017) and some Automated Model Building programs (AMB) distributed by different scientific teams. Among them we have selected the program Buccaneer (Cowtan, 2006), a well known fast and efficient automatic model building program, using it also as a tool for phase refinement: indeed input phases are used for calculating electron density maps which are interpreted in terms of molecular model, from which new phase estimates may be obtained in a cyclic way. This procedure, called CAB (Burla et al., 2018), has been implemented in a modified version of Sir2014 (Burla et al., 2015).
        CAB has been tested on 81 protein structures, solved via Molecular Replacement, anomalous dispersion and ab initio methods. As it is usually done the phases so obtained were submitted to phase refinement and then they have been used as input for CAB. The experimental results were compared with those obtained with the use of Buccaneer alone: it is shown that CAB improves Buccaneer results, both in completeness and in accuracy.
        The work is in progress in order to apply such a philosopy to DNA/RNA structures using Nautilus (Cowtan, 1994) as AMB program.
        An example of application to a protein of Molecular Replacement + CAB will be presented.

        Acknowledgement:
        The Italian Ministry for Education and Research (MIUR) is kindly acknowledged for financial support through Project PRIN 2017 (grant number 2017WBZFHL).

        References
        1. Caliandro, R., Carrozzini, B., Cascarano, G.L., Giacovazzo, C. Mazzone, A. & Siliqi, D. (2009). Acta Cryst. A65, 512–527,
        2. Burla, M.C., Caliandro, R., Carrozzini, B., Cuocci, C., Cascarano, G.L., Giacovazzo, C., Mallamo, M., Mazzone, A., Polidori, G. (2015). J. Appl. Cryst. 48, 306-309
        3. Burla, M.C., Cascarano, G.L., Giacovazzo, C. & Polidori, G. (2017). Acta Cryst. D73, 877–888
        4. Burla, M.C., Carrozzini, B., Cascarano, G.L., Giacovazzo, C. & Polidori, G. (2018). Acta Cryst. D74, 1096-1104
        5. Burla, M.C., Carrozzini, B., Cascarano, G.L., Giacovazzo, C. and Polidori, G. (2020) Acta Cryst. D76, 9-18
        6. Cowtan K. (2006) Acta Cryst. D62,1002-1011

        Speaker: Dr Giovanni Luca Cascarano (CNR - Istituto di Cristallografia)
        Abstract-Catania-Cascarano.docx
      • 18:30
        Structural characterization of halide perovskites by X-ray measurements and advanced analysis 5m

        The advent of new-generation X-ray sources as well as more sensitive and fast detectors discloses the possibility of deeper static and dynamic structural investigations. X-ray powder diffraction (XPD) and pair distribution function (PDF) measurements are sensitive to long and short-range order, which can be modelled by fitting procedures. Subtle structural changes induced in situ by varying external parameters (temperature, light) can be also detected by processing efficiently multiple measurements. In this case, the traditional approach to fit each measurement independently can be coupled with the new approach to apply multivariate methods to the whole dataset.1 Single and multiple XPD and PDF measurements have been here analyzed for the high-sensitivity structural characterization of halide perovskites. We have investigated the role played by cyclodextrins (CD) to generate a hybrid perovskite-soft material, demonstrating that the interaction between CDs and perovskite precursors leads to the formation of a supramolecular organic–inorganic hybrid framework that modifies solution chemistry and properties of the perovskite film.2 The multivariate analysis approach has been applied to in situ experiments to reveal under illumination the reversible generation of paramagnetic Pb3+ defects in CH3NH3PbI3 perovskite (Figure 1),3 and to get new insights into its tetragonal-to-cubic phase transition under temperature changes.4

        Speaker: Dr Rocco Caliandro (CNR - Istituto di Cristallografia)
        Abstract2.docx
      • 18:35
        The Structure of the Pro-domain of Mouse proNGF in Contact with the NGF Domain 5m

        Nerve growth factor (NGF) is an important neurotrophic factor involved in the regulation of cell differentiation and survival of target neurons. Expressed as a proNGF precursor, NGF is matured by furin-mediated protease cleavage.
        Increasing evidence suggests that NGF and proNGF have distinct functional roles. While the structure of mature NGF is available, little is known about that of the pro-domain because of its dynamical structural features.
        We exploited an ad hoc hybrid strategy based on nuclear magnetic resonance and modeling validated by small-angle X-ray scattering to gain novel insights on the pro-domain, both in isolation and in the context of proNGF(1).
        We show that the isolated pro-domain is intrinsically unstructured but forms transient intramolecular contacts with mature NGF and has per se the ability to induce growth cone collapse, indicating functional independence.
        Our data represent an important step toward the structural and functional characterization of the properties of proNGF.

        References
        (1) R. Yan, H. Yalinca, F. Paoletti, F. Gobbo, L. Marchetti, A. Kuzmanic, D. Lamba, F.L. Gervasio, P.V. Konarev, A. Cattaneo, A Structure. 2019, 27, 78-89.

        Speaker: Dr Doriano Lamba (CNR - Istituto di Cristallografia)
        DLamba_Abstract.docx
      • 18:40
        SUNBIM: a package for X-ray imaging of nano- and biomaterials using SAXS, WAXS, GISAXS and GIWAXS techniques 5m

        SUNBIM (supramolecular and submolecular nano- and biomaterials X-ray imaging) [1] is a suite of integrated programs which, through a user-friendly graphical interface, are optimized to perform a number of functions, such as: centering, q-scale calibration, two-dimensional to one-dimensional folding of small- and wide-angle X-ray scattering (SAXS/WAXS) data, also in grazing-incidence (GISAXS/GIWAXS), indexing of two-dimensional GISAXS frames and extraction of one-dimensional GISAXS profiles along specific cuts, quantitative scanning microscopy.
        SUNBIM consists of five main programs:
        (1) Calibration package, a set of functions allow one to find all of the geometrical parameters needed to extract a one-dimensional profile out of a two-dimensional image;
        (2) Batch Script & 2D Mesh Composite, to prepare batch script files (ASCII files) to run a sequential acquisition of two-dimensional frames (in scanning mode) and to perform a composite of the as-collected two-dimensional SAXS frames into a single image;
        (3) Multi-scan SAXS and WAXS data analysis, to fold each two-dimensional frame of the mesh into a one-dimensional profile and extract all the relevant features of the sample with a multi-modal imaging approach [2];
        (4) Single-scan (GI)SAXS and (GI)WAXS data analysis, to calibrate and fold the two-dimensional data, in order to extract relevant information from the experimental data and to fold 2D data into 1D profiles;
        (5) One-D Data Analysis Manager, to manage with one dimensional profiles and import, trigger, save and export plots.
        SUNBIM combines in the same package both originally developed algorithms (denoising, beam centering etc.) and reliable methods documented in the literature (multi-modal imaging, GISAXS three-dimensional frame indexing). SUNBIM is developed in the MATLAB language and it will be distributed free of charge to the academic user (downloadable after a valid registration from http://www.ba.ic.cnr.it/softwareic/sunbim/).

        References
        1. D. Siliqi, L. De Caro, M. Ladisa, F. Scattarella, A. Mazzone, D. Altamura, T. Sibillano and C. Giannini,. J. Appl. Cryst. 2016, 49, 1107–1114
        2. O. Bunk, M. Bech, T. H. Jensen, R. Feidenhans’l, T. Binderup, A. Menzel & F. Pfeiffer, New J. Phys. 2009, 11, 123016

        Speaker: Dr Teresa Sibillano (CNR - Istituto di Cristallografia)
        Abstract_CSCB_Sattarella.docx
      • 18:45
        Crystallographic investigation on whole anti-CD20 mAb 5m

        Molecules in crystal form represent nowadays one of the preferred formulation for bio-therapeutics. In addition, protein crystallization ensures high stability and a high purity level of the active ingredients and it is potentially able to reduce downstream manufacturing costs by improving the efficiency of the biomolecule purification step with respect to conventional chromatographic methods [1,2]. However, this process is often not trivial, particularly in case monoclonal antibodies (mAbs), which are recalcitrant to form ordinate solid state due to their flexibility and complex structure.
        mAbs are Y-shaped molecules, whose arms are made of one Fc-domain and two Fab-domains loosely connected by a very flexible polypeptide chain. Such great flexibility prevents mAb crystallization, making really difficult to get their structure determination at atomic resolution. This is the case of anti-cluster of differentiation 20 (anti-CD20), a mAb belonging to IgG1 family that is used in autoimmune disease therapy and against CD20-expressing non-Hodgkin's lymphoma [3]. Although anti- CD20 is the first antibody approved for clinical use and one of the most sold antibodies for therapeutic purposes, it shows a low reproducibility of the published crystallization conditions and its crystal structure is still not available.
        Recently, many efforts were made to optimize crystallization conditions for producing anti-CD20 crystals in terms of yield, starting material, speed, and crystal quality [4]. Due to the diffraction quality of mAb crystals obtained applying the meso batch crystallization protocol, low resolution methods for powder diffraction data were applied and exploited to assess that crystals were formed by the antibody molecules. The size of the whole mAb crystal cell, and its crystallographic symmetry, were also reasonably extrapolated from powder X-ray diffraction patterns. This study represents the first crystallographic characterization of the whole anti-CD20 mAb [4], and is an important step toward the unraveling of its full-length crystal structure.

        Acknowledgments
        The authors would like to thank the European Union’s Horizon 2020, Future and Emerging Technologies programme FET-OPEN, for funding this work within the AMECRYS project (http://www.amecrys-project.eu/) under grant agreement no. 712965.

        References
        [1] Yang H., Chen W., Peczulis P., Heng J.Y.Y., Cryst. Growth Des. 2019, 19, 983–991.
        [2] Govada L., Chayen N.E., Crystals 2019, 9, 106.
        [3] Pescovitz M.D., Am. J. Transplant. 2006, 6, 859–866.
        [4] Yang H., Belviso B.D., Li X., Chen W., Mastropietro T.F., Di Profio G., Caliandro R., Heng J.Y.Y., Crystals 2019, 9, 230.

        Speaker: Dr Benedetta Carrozzini (CNR - Istituto di Cristallografia)
        CSCB_abstact_Carrozzini.docx
      • 18:50
        Single-crystal synchrotron X-ray diffraction study of new anthracene derivative compounds 5m

        Anthracene derivative compounds have recently known an increasing scientific interest thanks to their unique physical properties (i.e., bright luminescence and large charge mobility)[1], making them ideal candidates for applications in new optoelectronic devices.
        A structural study of new anthracene derivative compounds was carried out by single-crystal synchrotron X-ray diffraction to have insights on the structure-property relationships, characterize the crystal packing and the main aromatic interactions, and detect the presence of stacking arrangements (see, for example, Figure 1).
        Synchrotron data were collected at room temperature at the Swiss Light Source (SLS), Villigen, Switzerland, at the beamline X06DA-PXIII[2]. Structure solution was carried out by Direct Methods using SIR2019[3] and refined by SHELXL2014/7[4]. All non-hydrogen atoms were refined anisotropically; the carbon-bound H atoms were placed on geometrically calculated positions and refined using a riding-model approximation.
        The main crystallographic results are here presented.

        References
        1. A. Camposeo, A., D.B. Granger, S.R. Parkin, D. Altamura, C. Giannini, J.E. Anthony, D. Pisignano Chem. Mater. 2019, 31, 1775-1783.
        2. X06DA-PXIII, http://www.psi.ch/sls/pxiii/.
        3. M.C. Burla, R. Caliandro, B. Carrozzini, G.L. Cascarano, C. Cuocci, C. Giacovazzo, M. Mallamo, A. Mazzone, G. Polidori J. Appl. Cryst. 2015, 48, 306-309.
        4. G.M. Sheldrick Acta Cryst. 2015, A71, 3-8.
        5. C.R. Martinez, B.L. Iverson Chem. Sci. 2012, 3, 2191-2201.

        Speaker: Dr Davide Altamura (CNR - Istituto di Cristallografia)
        Moliterni_Anna_poster.docx
  • Tuesday, 5 October
    • 09:00 → 10:30
      Session: 1
      Convener: Dr Dritan Siliqi (CNR - Istituto di Cristallografia)
      • 09:00
        Advanced X-ray Characterization of Hierarchical Systems: from Biotissues to Electronic Devices 15m

        Label-free Multiscale X-ray Imaging can be performed by Table-Top equipment, exploiting both Absorption (usually referred to as X-ray Transmission Microscopy “XTM”) and Scattering contrast. X-ray scattering techniques can provide a large amount of structural and morphological information, both at the atomic and nano-scale, and are thus particularly suited to study composite/nanostructured materials. The crystalline components are mainly studied by Wide Angle X-ray Scattering (WAXS), providing information on the crystallinity and crystalline phases, as well as on possible texture. The nanoscale structure/morphology can be assessed based on the Small Angle X-ray Scattering (SAXS) signal, and related to the possible crystallinity through combined SAXS/WAXS mapping. SAXS/WAXS/XT Microscopy is particularly suited to study biological tissues (or any structured material) with nano and/or atomic scale periodicity, such as calcified healthy or pathological tissues.1 In the biomedical field, this approach allows for example to study mineralized bio-scaffolds, or reveal osteogenic differentiation of stem cells through the analysis of nanocrystalline differentiation products.2 The availability of high brilliance X-ray micro-sources for laboratory equipment allows nowadays to perform the aforesaid advanced X-ray characterization, in both transmission and reflection geometries, in the home laboratory, being such X-ray sources considered as “synchrotron-class”. Moreover, suitable data treatment can further enhance the performances of the experimental equipment, returning in several cases results comparable to those obtained at a synchrotron beamline. A in-house developed package (SUNBIM) for the collection and analysis of X-ray microscopies with absorption and/or diffraction contrast, as well as data reduction for transmission and reflection geometries is freely available at http://www.ba.ic.cnr.it/softwareic/sunbimweb/, and constantly updated. The synchrotron-class micro-source combined with the SAXS/WAXS system installed at IC-Bari (XMI-L@b) has been successfully applied to the study of free-standing bio- and composite materials,3 as well as to polymer nanofibers for optoelectronic devices. Moreover, it has been successfully applied in grazing incidence reflection geometry (GISAXS/GIWAXS) for the study of several nanosttructured films for advanced electronic applications, in particular based on 2D nanocrystals.

        References

        1. C. Giannini, M. Ladisa, V. Lutz-Bueno, A. Terzi, M. Ramella, L. Fusaro, D. Altamura, D. Siliqi, T. Sibillano, A. Diaz, F. Boccafoschi and O. Bunk. IUCrJ. Volume 6, Part 2, 2019, 267-276.
        2. Procopio, A.; Malucelli, E.; Pacureanu, A.; Cappadone, C.; Farruggia, G.; Sargenti, A.; Castiglioni, S.; Altamura, D.; Sorrentino, A.; Giannini, C.; Pereiro, E.; Cloetens, P.; Maier, J.; Iotti, S. ACS Cent. Sci. 2019, 5, 1449-1460.
        3. F. Scattarella, E. Altamura, P. Albanese, Dritan Siliqi, M. Ladisa, F. Mavelli, C. Giannini and D. Altamura. Table-top combined scanning X-ray small angle scattering and transmission microscopies of lipid vesicles dispersed in free-standing gel. RSC Adv., 2021, 11, 484.
        Speaker: Dr Davide Altamura (CNR - Istituto di Cristallografia)
        Abstract_Altamura.docx
      • 09:15
        Structural Basis for Inhibition of Copper Trafficking by Platinum Anticancer Drugs 15m

        Proteins involved in copper transport and regulation, such as the human Antioxidant 1 copper chaperone (Atox1), are able to mediate the cellular uptake, sequestration, and efflux from cell of platinum-based drugs, affecting their anticancer activity.1 X-ray crystallographic investigations have disclosed the Pt binding sites of Atox1, and shown that the metal binding site of the Atox1 dimer is partially occupied by a Pt2+ ion, with Cu+ ions completing the site occupancy.2 Thus, the platinum ion is able to replace partially the copper ion, leading to a disruption of the delicate process that regulates copper level in the cell. A further recently published study has revealed the structural mechanisms underlying the interaction between Pt-based drugs and two proteins involved in Cu trafficking (Atox1 and the first domain of Menkes ATPase, Mnk1).3 Crystallographic and nuclear magnetic resonance investigations demonstrated that the kinetic inertness of the Pt(II) derivative imparts a bullet time effect to the fast process of copper exchange between Atox1 and Mnk1, by freezing the Cu(I) ion or hijacking it to glutathione, a physiological antioxidant in the biological matrix. Thus, the anticancer drugs cisplatin and oxaliplatin can interfere with the rapid exchange of Cu between Atox1 and Mnk1, with possible consequences on cancer cell viability and migration.

        Acknowledgement
        The Italian Ministry for Education and Research (MIUR) is kindly acknowledged for financial support through Project PRIN 2017 (grant number 2017WBZFHL).

        Speaker: Dr Rocco Caliandro (CNR - Istituto di Cristallografia)
        Abstract1.docx
      • 09:30
        Design and fabrication of biosensors for environmental and food safety monitoring. 15m

        Modern environmental and food monitoring practices require reliable, facile, and on-site detection of hazardous compounds potentially affecting human health. By satisfying these requirements, sensors represent complementary screening tools with a lower impact on the environment compared to conventional chromatographic-based methods. Specifically, biosensor technology offers the possibility to combine and tailor the selectivity of biological recognition elements to the sensitivity of transducing platforms giving rise to analytical devices providing specific situation awareness. The exploitation of disposable scree-printed electrodes and microfluidic systems prompted the development of miniaturized, portable systems, while the integration of nanostructured materials enabled to improve selectivity and performance of electrical detectors. Besides these advancements, keeping the proper functionality of the biological component in a hostile environment, such as solid-state devices, is still a challenge.
        In the quest to identify physiological parameters correlated with more efficient phenotypes, we studied the structure/function/dynamics relationships occurring in microalgae strains hosting single aminoacidic substitutions in the photosystem II D1 protein. The mutants were produced by combining in silico studies with an in vitro directed evolution approach followed by site-directed mutagenesis experiments1,2. Biophysical and physiological studies enabled the identification of phenotypes having enhanced stability and photosynthetic performance under stressful conditions, and improved sensitivity to different classes of environmental contaminants3. As a further step, we set-up immobilization procedures to preserve the activity of algal cells on screen-printed electrodes4 and enhanced the electrochemical response by exploiting bi-metallic nanoparticles. Finally, neutron spectroscopy studies on algal mutants shed a light on dynamics properties of photosynthetic reaction centre underlying an improved biosensor performance5,6.
        The construction of amperometric photosinthesis-based biosensors will be presented alongside the development of a label-free impedimetric immunosensor for the detection of micotoxins7, that will be included in a novel multitransduction electrochemical biosensoristic platform. This work was supported by the Lazio project FACILE, n. 85-2017-15256.

        References
        1. Rea et al. Prot Sci 2009, 18, 2139.
        2. Rea et al. PloS ONE 2011, 6:e16216.
        3. Lambreva et al. PLoS ONE 2013, 8:e61851.
        4. Husu et al. Sens Actuators B Chem 2013, 185, 32.
        5. Antonacci et al. Sci Rep 2018, 8 (1), 14745.
        6. Russo et al. Biophys Journal 2019, 116, 1759.
        7. Cancelliere et al. Appl Surf Sci 2021, 567, 150791.

        Speaker: Dr Giuseppina Rea (CNR - IC)
        Rea_CSCB_Abstract_Catania_2020.docx
      • 09:45
        Multiple scale characterisation of ultrasmall nanocrystals by X-ray Total Scattering 15m

        Fundamental aspects and an overview of applications of cutting-edge nanocrystallography tools grounded on Wide Angle X-ray Total Scattering (WAXTS) techniques (mainly synchrotron-based) and the use of the Debye Scattering Equation (DSE) for data modeling in reciprocal space will be presented. [1,2]
        The approach overcome the limits of conventional diffraction-based methods for characterizing engineered ultrasmall nanocrystals and, by exploiting the full (Bragg and diffuse scattering) information in the experimental data, is able to quantitatively recover atomic and nanometer scales properties (structural distortions, defects of various kind, lattice strains, surface relaxation, size, morphology and their relative dispersion) suitably encoded in the same atomistic model. Combining WAXTS with Small Angle X-ray scattering (SAXS) data fruitfully exploits their complementarity for a more robust, quantitative multiple-scale analysis and/or for disentangling size/shape from structural defects-induced effects.

        Acknowledgement: The Italian Ministry for Education and Research is kindly acknowledged for partial financial support through the Project PRIN 2017 (grant no. 2017L8WW48).

        [1] A. Cervellino, R. Frison, N. Masciocchi, A. Guagliardi. X-ray Powder Diffraction Characterization of Nanomaterials, in X-ray and Neutron Techniques for Nanomaterials Characterization, 2016, C.S.S.R Kumar (Ed.), Springer-Verlag Berlin, DOI 10.1007/978-3-662-48606-1_10
        [2] A. Cervellino, R. Frison, F. Bertolotti, A. Guagliardi. J. Appl. Cryst. 2015, 48, 2026-2032.
        [3] F. Bertolotti, D. N. Dirin, M. Ibanez, F. Krumeich, A. Cervellino, R. Frison, O. Voznyy, E. H. Sargent, A. Guagliardi, N. Masciocchi, Nat. Mater. 2016, 15, 987-994.
        [4] F. Bertolotti, L. Protesescu, M. V. Kovalenko, S. Yakunin, A. Cervellino, S. J. L. Billinge, M. W. Terban, J.S. Pedersen, N. Masciocchi, A. Guagliardi. ACS nano 2017, 11, 3819
        [5] D. Moscheni, F. Bertolotti, L. Piveteau, L. Protesescu, D. N. Dirin, M. V. Kovalenko, A. Cervellino, J.S. Pedersen, N. Masciocchi, A. Guagliardi, ACS nano 2018, 12, 12558-12570.
        [6] F. Bertolotti, G. Nedelcu, A. Vivani, A. Cervellino, N. Masciocchi, A. Guagliardi, M. V. Kovalenko, ACS nano 2019, 13, 14294-14307.
        [7] J.M. Delgado-López, R. Frison, A. Cervellino, J. G. Morales, A Guagliardi, N. Masciocchi. Adv. Funct. Mater. 2014, 24, 1090-1099.
        [8] R. Frison, G. Cernuto, A. Cervellino, O. Zaharko, G. M. Colonna, A Guagliardi, N. Masciocchi. Chem. Mater. 2013, 25, 4820-4827.

        Speaker: Dr Antonella Guagliardi (CNR - Istituto di Cristallografia)
      • 10:00
        The XRD1 Synchrotron hard X-ray light source beamline as a tool for chemistry and science and technology of materials. 15m

        The Italian synchrotron Elettra was one of the first 3rd generation accumulation rings designed and built with the aim of producing photon beams to be used in radiation-matter interaction experiments, in which photons constitute a probe to investigate electronic, magnetic and structural properties of matter in solid, liquid and gaseous state. Elettra houses four beamlines dedicated to crystallography, and of these the first to be designed and built, in partnership with the CNR, was XRD11. This beamline, originally designed for X-ray diffraction experiments in macromolecular crystallography, has shown over the years to be very versatile and able to adapt to a great variety of geometries and experimental techniques2. Its source is a permanent magnet wiggler that offers a continuous range of emission from 4 to 21 keV, its experimental station is equipped with a two-dimensional high dynamic range X-ray detector coupled to a diffractometer for alignment and rotation about various axes of the samples in the beam. At the experimental station, it is possible to perform X-ray diffraction experiments ranging from macromolecular crystallography to studies of surfaces and thin films in grazing incidence, from powder diffraction to the use of low energies for solving the phase problem. Here a brief presentation of the experimental station is given along with some examples of investigations in materials sciences performed there, in particular for applications in electronic (organic semiconductors) energy conversion (solar cell prototypes), food science (ex-situ DSC-XRD combined investigations of lipidic matrices) and of phase transitions and structural properties of high Tc superconductors.

        References

        1. Bernstorff, S.; Busetto, E.; Gramaccioni, C.; Lausi, A.; Olivi, L.; Zanini, F.; Savoia, A.; Colapietro, M.; Portalone, G.; Camalli, M.; Pifferi, A.; Spagna, R.; Barba, L.; Cassetta, A. The Macromolecular Crystallography Beamline at ELETTRA. Review of Scientific Instruments 1995, 66 (2), 1661–1664.
2. Lausi, A.; Polentarutti, M.; Onesti, S.; Plaisier, J. R.; Busetto, E.; Bais, G.; Barba, L.; Cassetta, A.; Campi, G.; Lamba, D.; Pifferi, A.; Mande, S. C.; Sarma, D. D.; Sharma, S. M.; Paolucci, G. Status of the Crystallography Beamlines at Elettra. Eur. Phys. J.  Plus 2015, 130 (3), 43.
        Speaker: Dr Luisa Barba (CNR - Istituto di Cristallografia)
      • 10:15
        Functional disorder in complex materials 15m

        Structural and electronic disorder at nano and mesoscale plays an important role in the functionality of complex materials. Here local heterogeneity and weak interactions developing between structural units cause dynamical spatio-temporal configurations with dynamical correlated disorder. Visualizing these configurations is fundamental for understanding the physical properties of complex matter and requires advanced experimental methodologies. We discuss the connections between the dynamical correlated disorder and functionality in different fields from material science to biology.

        Speaker: Dr Gaetano Campi (CNR - Istituto di Cristallografia)
    • 10:30 → 11:00
      Coffe break 30m
    • 11:00 → 12:40
      Session: 2
      Convener: Dr Danilo Milardi (CNR - Istituto di Cristallografia)
      • 11:00
        Disclosing the Major Structural Determinants Essential for Proneurotrophins Biological Functions 15m

        Nerve Growth Factor, Brain-Derived Neurotrophic Factor, Neurotrophin 3 and Neurotrophin 4 play a range of crucial functions in the developing and adult peripheral and central nervous systems. Initially synthesized as precursors, named proneurotrophins (proNTs), that are cleaved to release C-terminal mature forms, they act through two types of receptors, the specific Trk receptors and the pan-neurotrophin receptor p75NTR, to promote differentiation, neuronal survival, synaptogenesis and modulating synaptic plasticity. Recently, all the proNTs but proNT4 have been demonstrated to be not just inactive precursors, but biologically active signaling ligands that mediate opposing actions in fundamental aspects of the nervous system with respect to the mature counterparts through dual receptor complexes formation, involving a member of the VPS10 family and p75NTR. Despite the functional relevance, the molecular determinants underpinning the interactions between the pro-domains and their receptors are still elusive probably due to their intrinsically disordered nature. Here we present an evolutionary approach coupled to an experimental study aiming to uncover the structural and dynamical basis of the biological functions displayed by proNGF, proBDNF and proNT3 but missing in proNT4. A bioinformatic analysis allowed elucidating the functional adaptability of the proNTs family in vertebrates, identifying conserved key structural features. The combined biochemical and SAXS experiments shed lights on the structure and dynamic behavior of the human proNTs in solution, giving insights on the evolutionary conserved structural motifs, essential for the multifaceted roles of proNTs in physiological as well as in pathological contexts [1].

        References:

        [1] Covaceusach S. Peche Y.L, Konarev P.V., Lamba D. A combined evolutionary and structural approach to disclose the primary structural determinants essential for proneurotrophins biological functions. Comput. Struct. Biotechnol. J. (2021) May 13;19:2891-2904. doi: 10.1016/j.csbj.2021.05.007.

        Speaker: Dr Sonia Covaceuszach (CNR - Istituto di Cristallografia)
        10-Abstract-DG.docx
      • 11:15
        Solving polycrystalline structures: from data collection to the development of innovative crystallographic methods 15m

        Although the crystal structure solution from powder diffraction data is limited respect to the single crystal case, its interest has surprisingly increased in the last twenty-five years. The difficult chance to get a correct interpretation of the experimental powder diffraction pattern, due to peak overlap, background estimation, and preferred orientation, is addressed with improvement in modern instrumentation, as well as development of innovative theories and software.
        Our research activity is mainly devoted to the solution by powder diffraction data of organic, inorganic, metal-organic crystal structures of different scientific and technological interest.
        The process of structure solution starts from powder diffraction data collection by a Rigaku laboratory diffractometer (18Kw) equipped with an asymmetric Ge(111) monochromator and a silicon strip detector.
        The core of the research activity is developing advanced methodological approaches and computational abilities aiming at successfully and automatically carrying out the full pathway of the solution process, from the indexation to the Rietveld refinement1.
        Regarding the methodologies, we develop computational approaches using Direct Methods, working in the reciprocal space, and Simulated Annealing, working in the real space and both2. They have differences: Direct Methods are fast and require minimal information, depend on the efficiency of the pattern decomposition, on the experimental resolution and structure complexity. Simulated Annealing methods can be very slow and need additional information on the expected molecular geometry, are independent from the accuracy of the extracted structure factor moduli and do not need atomic resolution.
        The decision on what should be the best solution strategy to apply, mainly depends on data quality, experimental resolution and peak overlap; structure complexity and/or degrees of freedom of the structure2.
        The new methodological and computational procedures are implemented in the software EXPO3, free for academic institution, based on a user-friendly graphic interface, able to adopt default and non-default solution strategies, all innovative approaches for attaining the complete and correct crystal structure solution.
        The different requested conditions and the most appropriate algorithm to apply for a successful powder solution by Direct Methods and Simulated Annealing with some examples, will be discussed.
        References
        1. International Tables for Crystallography, Volume H, Powder Diffraction; C.J. Gilmore, J.A. Kaduk, H. Schenk Eds., Wiley: New York, 2019, 978-1-118-41628-0
        2. A. Altomare, N. Corriero, C. Cuocci, A. Falcicchio, R. Rizzi. (2019). Crystals, 2020, 10(1), 16.
        3. A. Altomare, C. Cuocci, C. Giacovazzo, A. Moliterni, R. Rizzi, N. Corriero, A. Falcicchio. J. Appl. Cryst. 2013, 46, 1231.

        Speaker: Dr Rosanna Rizzi (CNR - Istituto di Cristallografia)
        abstract_Catania_Rizzi_Rosanna.docx
      • 11:30
        The role of stereochemistry in the inhibition of Aβ Amyloid growth and toxicity by silybins. 15m

        The self-assembling of the amyloid β (Aβ) is considered an hallmark in the pathogenesis of Alzheimer’s disease (AD). Many efforts have been devoted in designing molecules able to halt disease progression by inhibiting Aβ self-assembly. We combine biophysical, biochemical and computational techniques to investigate the capacity of four optically pure components of the natural product silymarin (silybin A, silybin B, 2,3-dehydrosilybin A, 2,3-dehydrosilybin B) to inhibit Aβ aggregation. TEM analysis demonstrated that all the four investigated flavonoids prevent the formation of mature fibrils, however ThT assays, WB and AFM investigations showed that only silybin B was able inhibit the formation of small protofiber (considered the most toxic species) diverting the aggregation toward the formation of large amorphous aggregates. By using molecular dynamics (MD) simulations we observed that silybin B interacts mainly with the C-terminal hydrophobic segment 35MVGGVV40 of Aβ40 and the peptide conformation remains predominantly unstructured along all the simulations. By contrast, silybin A interacts preferentially with the segments 17LVFF20 and 27NKGAII32 of Aβ40 which shows a high tendency to form bend, turn, and β-sheet conformation in and around these two domains. Both 2,3-dehydrosilybin enantiomers bind preferentially the segment 17LVFF20 but lead to the formation of different small-sized, ThT-positive Aβ aggregates. Finally, in vivo studies in a transgenic Caenorhabditis elegans strain expressing human Aβ indicated that silybin B is the most effective of the four compounds in counteracting Aβ proteotoxicity. This study underscores the pivotal role of stereochemistry in determining the neuroprotective potential of silybins and points to silybin B as a promising lead compound for further development in anti-AD therapeutics.

        Speaker: Dr Michele F.M. Sciacca (CNR - Istituto di Cristallografia)
        Abstract_MFM_Sciacca.docx
      • 11:45
        Structural investigation of calcium phosphates 15m

        Calcium phosphates (CaP) materials, employed in different biomedical applications because of their density very close to those of CaP mineralized phases of human bones, are largely investigated in many scientific fields [1]. The most known CaP are tricalcium phosphate Ca3(PO4)2 (TCP) and hydroxyapatite Ca5(PO4)3OH (HAp). In the present work, we investigated two sets of substituted β-TCP materials, coming from solid-state reactions at T = 1200°, respectively with rare earths cations (RE) [2], and with divalent transition metals (Mn, Ni, Cu) [3]. All TCP were subject to a multi-methodological crystal chemical investigation based on SEM-EDS microscopy, powder X-ray diffraction (PXRD), FTIR, Raman and luminescence spectroscopies. Investigation was completed with the structural refinement (Rietveld method). SEM morphological analyses revealed the presence of subspherical microcrystalline aggregates. Refinement of site occupancies showed the tendency of RE cations to replace Ca in the largest structural sites; in the smaller octahedral M5 site, low RE (La→Gd) are not present, contrary to high RE (Dy→Lu) present in this site, while divalent cations showed a strong tendency to occupy this energetically favourable site. FTIR and Raman spectra show slight band shifts of the phosphate modes correlated to the evolving size of the replacing element, as well as strong luminescence properties were found in many RE- TPC (Eu and Gd phases) and in Mn-TCP.
        HAp is the primary mineral component of human bones and teeth. We investigated HAp, coming from high-temperature syntheses, doped with different cations at different concentrations, e.g. bismuth-HAp as possible biomaterial [4], and RE (Eu, Gd)-HAp as phosphor materials. Multi-methodological characterization was completed by dielectric measurements for Bi-HAp: morphological analysis revealed a decrease in crystallite size by bismuth-addition in HAp lattice, while the relative permittivity, dielectric loss and alternating current conductivity change with increasing frequency, the alternating conductivity gradually decreases with the addition of Bi. For (Eu, Gd)-HAp, FTIR spectra showed slight band shifts of the phosphate modes correlated to the evolving size of the replacing cation, while significant luminescence properties were found.

        References
        1. S. Dorozkhin. Ceram. Int. 2015, 41, 13913-13966.
        2. F. Capitelli, M. Rossi, A. El Khouri, M. Elaatmani, N. Corriero, A. Sodo, G. Della Ventura, J. Rare Earths 2018, 36, 1162-1168.
        3. A. Altomare, R. Rizzi, M. Rossi, A. El Khouri, M. Elaatmani, V. Paterlini, G. Della Ventura, F. Capitelli, Crystals, 2019, 9, 288.
        4. A. El Khouri, A. Zegzouti, M. Elaatmani, F. Capitelli, Inorg. Chem. Commun. 2019, 110, 107568.

        Speaker: Dr Francesco Capitelli (CNR - Istituto di Cristallografia)
        Abstract_Catania_2020.docx
      • 12:00
        Natural polysaccharides from microalgae for the protection of cultural heritage 15m

        Cultural heritage, whatever their composition and location (indoor or outdoor), are susceptible to deterioration triggered by several factors, such as thermo-hygrometric conditions, mechanical stress, electromagnetic radiation and biodeterioration. One of the main challenges in this sector is the promotion of innovative products for the restoration/protection of artistic value surfaces.
        In this context, we propose the development of a new product based on polysaccharides, extracted from unicellular algae, compatible with ancient materials and not harmful to humans and the environment. In particular, the interesting qualities of algal polysaccharides have been recently described, for their significant antioxidant, antifungal, cosmeceutical and nutraceutical activities in the medical, agri-food and cosmetics fields, but still little is known in the cultural heritage sector. Hence, the goal of our research is to utilize the microalgal polysaccharidic extract to foster an innovative restoration and conservation of artistic work (paper, wooden and mortar artworks), promoting mechanical restoration, as well as hindrance the onset of biotic colonization.
        For these reasons, specific strains of microalgae (wild type and mutants) were selected and grown to induce a natural accumulation of carbohydrates. Furthermore, an extraction protocol of the polysaccharide mixture was optimized and its composition determined by FT-IR, NMR, elemental chemical analysis, as well as its antioxidant capacity. Moreover, antifungal and antibacterial capacities on organic and inorganic materials will be tested. The consolidating and regenerative properties of polysaccharides will be analysed over the time by MO, FTIR, SEM-EDS, imaging UV, UV-VIS-NIR, in comparison with conventional treatments.

        Acknowledgments

        This work is supported by POR FESR Progetti Gruppi di Ricerca Lazio Innova NYMPHA Project A0375-2020-36489.

        Speaker: Dr Amina Antonacci (CNR - Istituto di Cristallografia)
        Antonacci_and_Scognamiglio_Catania_2020_jan_15.docx
      • 12:15
        GHK-Hyaluronic acid conjugates affect the wound closure in the presence of copper ions 15m

        Wound healing is a complex, efficient and highly regulated biological process that consists of four phases: hemostasis, inflammation, proliferation and migration of cells [1]. Copper-dependent stimulation of vessel formation during the wound healing has been mainly attributed to its regulation of vascular endothelial growth factor (VEGF) and angiogenin [2]. The human copper-binding peptide GHK (glycyl-l-histidyl-l-lysine) is a small, naturally occurring tri-peptide present in human plasma that can be released from tissues in the case of an injury and is able to control the fibrinogen biosynthesis in liver tissue [3,4]. Most authors attribute effects of GHK to its ability to bind copper (II) ions that can affect not only the copper metabolism but also regulate a number of human genes [5]. Hyaluronic acid (HA) is currently used in tissue regeneration either alone or conjugated with bioactive molecules [6,7]. 200 kDa HA affects tissue regeneration and pro-angiogenic and wound closure processes.

        In the present work, we report on the protective and regenerative actions of the HA-GHK conjugate on mouse embryonic fibroblasts cell line (NIH/3T3) in the presence and in the absence of Cu(II) ion. Dose-response experiments show no significant effect on cell viability/proliferation with or without 1 µM copper. The effect of HA-GHK conjugates on wound healing was investigated; as expected, HA200 treatment slightly increases the wound closure, while the addition of HA-GHK with different % of GHK loading results in higher % of wound closure than with HA200. Addition of 1 µM copper or 50 µM copper chelator BCS, modify this effect. Altogether, our findings pinpoint that GHK-HA is a good candidate as new molecular entity in wound healing and skin repairing.

        [1] Guo S and Dipietro LA, Factors affecting wound healing. J Dent Res. 2010; 89(3):219-29
        [2] Kornblatt AP et al J of Inorg Biochem. 2016; 161:1-8
        [3] Pickart L. J of Biomat Sci. Polymer edition. 2008; 19: 969-988
        [4] Pickart L et al. Oxidative medicine and cellular longevity; 2012: 324832
        [5] Pickart L et al. BioMed Res. Int., 2014; 151479
        [6] Neuman MG et al. Hyaluronic acid and wound healing. J Pharm & Pharmac Sci. 2015; 18:53.
        [7] Sciuto S et al. Derivatives obtained from hyaluronic acid and carnosine. 2015.
        PCT/IB/2015/055782.

        Speaker: Dr Irina Naletova (CNR - Istituto di Cristallografia)
    • 12:40 → 14:00
      Lunch 1h 20m
    • 14:00 → 15:00
      ePoster session: Life Science
      Convener: Dr Francesco Bellia (CNR - Istituto di Cristallografia)
      • 14:00
        VEGF bioactive fragments and their copper (II) complexes in angiogenesis 5m

        Angiogenesis plays a crucial role in numerous physiological and pathological phenomena and consists in a multi-regulated process in which many factors are involved, including growth factors and metals. In particular, Copper stimulation of growth factors, such as vascular endothelial growth factor (VEGF), is involved in the formation of new blood vessels from pre-existing ones1. Although the angiogenic process is intensely investigated the role played by copper(II) ions in angiogenic mechanisms is still unclear. Certainly, their ability to modulate molecular mechanisms involved in angiogenesis plays a crucial role in numerous diseases, including cancer and cardiovascular disease. Within this frame, new classes of metal-binding peptides have been designed and studied, as ligands able to modulate angiogenesis2. Recently, we reported the pro-apoptotic activity of VEGF73-1013, the protein fragment directly involved in the VEGF receptor (VEGFR2) interaction. This peptide combines the properties of beta hairpin sequence 73-85, rich in hydrophobic residues, with the sequence 85-101, characterized by the presence of three histidines, known sites of anchoring for Cu(II).
        In order to establish a relationship between the structure of the ligand fragment and the cytotoxic activity of the resulting copper(II) complexes, specific structural modifications were introduced to VEGF73-101 fragment, obtaining two singly mutated peptides.
        Herewith we extended the study of peptide VEGF73-101 to deepen its apoptotic activity on HUVEC cells in comparison with the singly mutated peptides, with particular attention to the ionophoric behavior hypothesized. To this aim, we studied the effects of these peptides and of their copper (II) complexes on membrane model system, the cellular uptake and the related anti-proliferative activity on HUVEC cells, by MTS and cytofluorimetry analysis.
        In addition, spectroscopic UV-Vis, CD-Vis and spectrometric ESI-MS measurements to characterize the metal coordination mode of peptides, were reported. The obtained results suggest that VEGF73-101, is an effective copper ionophore, and represents a promising tool in the regulation of angiogenetic pathways.

        References
        1. Finney L, Vogt S, Fukai T, Glesne D, 2009, 88-94
        2. Henninot A, Collins J C, Nuss J M, J. Med. Chem., 2018, 61, 1382−1414
        3. Grasso G, Santoro A M, Magrì A, La Mendola D, Tomasello M F, Zimbone S, Rizzarelli E, J. Inorg.Biochem 2016, 159, 149–158

        Speakers: Dr Stefania Zimbone (CNR - Istituto di Cristallografia) , Prof. Enrico Rizzarelli (CNR - Istituto di Cristallografia)
        AbstractZimboneCorretto.docx
      • 14:05
        Closing or opening proteasome doors by porphyrins. 5m

        The role of proteasome in the regulation of all cellular functions, is so relevant that its modulation became a useful therapeutic strategy for a large variety of diseases (1). Besides the potential clinical usefulness, proteasome regulators provide interesting and important tools for cell and molecular studies. Some years ago we have proposed cationic porphyrins as a new class of proteasome inhibitors (2), extending a new regulatory function to the broad-spectrum activities of these “multi-purpose” molecules. The external face of the 20S CP, the alpha ring, has its own regulation system consisting of dynamic gate that constantly switches between closed and open state. These physiological receptorial regions of canonical regulatory particles are characterized by a regular arrangement of charged aminoacids that represent a sort of “electrostatic code” regulating the “state” of the gate; the peripheral porphyrin charges represent a key able to interfere with the gate “door lock” (3). Thus, porphyrins behave as gatekeepers of 20S CP (4), either inducing a partial gate occlusion (e.g., H2T4) or, allosterically, triggering a conformational change that affect the open-closed equilibrium (e.g., pTMPyPP4) (3). Finally, in the case of tricationic porphyrin Tris-T4, 20S CP activation has been observed, as the result of a new proteasome functional state characterized by a much higher substrate affinity and a higher catalytic efficiency. According to our hypothesis, supported by NMR and computational data, the h20S activation observed upon Tris-T4 binding, might simulate to some extent the allosteric activation by regulatory proteins. These results coupled with porphyrin's versatile chemistry, position porphyrins as a novel class of CP conformational modulators of proteasome with a significant pharmacological potential.
        References
        1. Finley D., et al; Trends in Bioch. Sci, 2016, 41(1),77-93.
        2. Santoro A.M. et al.; J. Am. Chem. Soc., 2012, 134, 10451-57.
        3. Cunsolo A., et. al. Scientific Reports 2017, 7: 17098.
        4. Santoro A.M., et al. Chemical Science, 2016, 7, 1286-97.
        5. Santoro A.M. et al., submitted for pubblication.

        Speaker: Dr Anna Maria Santoro (CNR - Istituto di Cristallografia)
        Abstract_SantoroCatania.docx
      • 14:10
        Hyaluronan-carnosine conjugates inhibit Amyloid-β aggregation and toxicity 5m

        Hyaluronic acid (Hy) is a glycosaminoglycan widely distributed in humans and it is the main component of the extracellular matrix1. The wide range of physiological functions includes hydration and turgidity maintenance of tissue, extracellular matrix structure, regulation of innate immunity, and protection and lubrication of joints. The molecular and mechanical properties make this biocompatible polymer very used for several applications, such as tissue engineering, treatment of osteoarthritis and drug targeting2,3.
        The potential applications of Hy has been recently widen by the conjugation with carnosine (Car), a multifuntional dipeptide widely distributed in several animal species4. Also this dipeptide exerts a variety of physiological properties, including antioxidant, antiglycating, and antiaggregant abilities5. he biocojugation of carnosine effectively inhibits the rapid dipeptide degradation in serum catalysed by carnosinase6, making the new HyCar derivative7 a promising compound to better treat several diseases and/or prevent their onset.
        Based on these data, a series of Hy-Car derivatives have been synthesized and patented by us in recent years, by using two different molecular weights (200 and 700 kDa) and different loading percentages of carnosine. All of them have been structurally characterized. The ability of the HyCar derivatives to inhibit the aggregation of the amyloid-β peptide (whose dyshomeostasis is involved in the onset of Alzheimer’s disease) and the relative toxicity has been tested by using several assays and methodological approaches.

        References
        1. Fraser J. R. E., Laurent T. C., Laurent U. B. G., J. Int. Med. 1997, 242, 27–33.
        2. Kogan G., Šoltés L., Stern R., Gemeiner P., Biotechnol. Lett. 2007, 29, 17-25.
        3. Yadav A.K., Mishra P., Agrawal G.P., J. Drug Target. 2008, 16, 91-107.
        4. Boldyrev A.A., Aldini G., Derave W.. Physiol. Rev. 2013, 93, 1803-1845.
        5. Bellia F., Vecchio G., Cuzzocrea S., Calabrese V., Rizzarelli E., Mol. Aspects Med., 2011, 32, 258-266.
        6. Bellia F., Vecchio G., Rizzarelli E., Amino Acids 2012, 43, 153-163.
        7. Sciuto S., Greco V., Rizzarelli E., Bellia F., Lanza V., Vaccaro S., Messina L., WO2016016847A1.

        Speaker: Dr Francesco Bellia (CNR - Istituto di Cristallografia)
        Rizzarelli_Poster.docx
      • 14:15
        Intrinsically disordered proteins (IDP) and metal ions: the case of amylin (HiAPP) 5m

        Intrinsically disordered proteins (IDPs) do not have rigid 3D structures, showing changes in their folding depending on the environment or ligands. Intrinsically disordered proteins are widely spread in eukaryotic genomes, and these proteins participate in many cell regulatory metabolism processes. Some IDPs, when aberrantly folded, can be the cause of some diseases such as Alzheimer′s, Parkinson′s, diabetes and prionic, among others. In these diseases, there are modifications in parts of the protein or in its entirety. A common conformational variation of these IDPs is misfolding and aggregation, forming, for instance, neurotoxic amyloid plaques. Some metal ions play an important role in the aggregation processes of these IDPs as showed in several papers present in literature.
        Amylin is a 37-residue peptide hormone produced by the islet β-cells of pancreas and the formation of amylin aggregates is strongly associated with b-cell degeneration in type 2 diabetes, as demonstrated by more than 95% of patients exhibiting amylin amyloid upon autopsy. It is widely recognized that metal ions such as copper(II) and zinc(II) have been implicated in the aggregation process of amylin.1,2
        Here we reported our experimental studies (thermodynamic and spectroscopic) on the interaction between Cu(II) and Zn(II) with several isoforms of amylin (human, porcine, murine) and their fragments.3,4

        References
        1. G. Pappalardo, D. Milardi, A. Magri, F. Attanasio, G. Impellizzeri, C. La Rosa, D. Grasso, E. Rizzarelli Chem.–Eur. J., 2007, 13, 10204-10215.
        2. A. Sinopoli, A. Magrì, D. Milardi, M. Pappalardo, P. Pucci, A. Flagiello, J. J. Titman, V. G. Nicoletti, G. Caruso, G. Pappalardo, Metallomics, 2014, 6, 1841-1852.
        3. A. Magrì, D. La Mendola, V. G. Nicoletti, G. Pappalardo, E. Rizzarelli, Chem.–Eur. J., 2016, 22, 13287–13300.
        4. A. Magrì, A. Pietropaolo, G. Tabbì, D. La Mendola, E. Rizzarelli, Chem.–Eur. J., 2017, 23, 17898-17902.

        Speakers: Dr Antonio Magrì (CNR - Istituto di Cristallografia) , Dr Enrico Rizzarelli (CNR - Istituto di Cristallografia)
        Abstract_Magri_et_al.docx
      • 14:20
        Antioxidant properties of Hyaluronate–Carnosine bioconjugates and their copper(II) complexes 5m

        Hyaluronic acid (Hy), is a polyanionic linear nonsulfated glycosaminoglycan (GAG)1. It is widely distributed throughout mammalian cells and tissues and its biomechanical and biochemical properties support its involvement in myriad physiological functions, including hydration and turgidity maintenance of tissue, extracellular matrix structure, regulation of innate immunity, and protection and lubrication of joints. Due to this versatility, Hy represents a promising bio-indicator of pathophysiology and inflammation, and has consequently been targeted for disease-specific diagnostics2. Moreover, the excellent biocompatibility makes Hy useful as a drug delivery system, to which pharmacologically active compounds can be covalently conjugated3.
        Carnosine (Car) is a multifuntional dipeptide widely distributed in several animal species4. The variety of physiological properties, including antioxidant, antiglycating, antiaggregant and metal binding abilities, account for the distribution in several tissues and the relative high concentration reached in many cerebral areas5. The promising role as a drug is limited by the rapid degradation in serum catalyzed by carnosinase. The bioconjugation of Car through the amino on the carboxylic groups has been proposed as a promising strategy to overcome this limitation6.
        Based on these data, a series of Hy-Car derivatives have been synthesized and patented by us in recent years7, by using two different molecular weights (200 and 700 kDa) and different loading percentages of carnosine. All of them have been structurally characterized. The antioxidant property of the HyCar derivatives and that of their copper(II) complexes has been tested by using several assays and methodological approaches.

        References
        1. Fraser J. R. E., Laurent T. C., Laurent U. B. G., J. Int. Med. 1997, 242, 27–33.
        2. Kogan G., Šoltés L., Stern R., Gemeiner P., Biotechnol. Lett. 2007, 29, 17-25.
        3. Yadav A.K., Mishra P., Agrawal G.P., J. Drug Target. 2008, 16, 91-107.
        4. Boldyrev A.A., Aldini G., Derave W.. Physiol. Rev. 2013, 93, 1803-1845.
        5. Bellia F., Vecchio G., Cuzzocrea S., Calabrese V., Rizzarelli E., Mol. Aspects Med., 2011, 32, 258-266.
        6. Bellia F., Vecchio G., Rizzarelli E., Amino Acids 2012, 43, 153-163.
        7. Sciuto S., Greco V., Rizzarelli E., Bellia F., Lanza V., Vaccaro S., Messina L., WO2016016847A1.

        Speaker: Dr Eleonora Bocchieri (CNR - Istituto di Cristallografia)
        Bocchieri_Poster.docx
      • 14:25
        Structural determinants of quorum sensing specificity vs promiscuity 5m

        Quorum sensing (QS) is a bacterial cell-to-cell communication mechanism based on the production and detection of chemical signals called autoinducers (AIs) to regulate gene expression in response to changes in population density. QS allows bacteria to behave as a cohesive group under differing environments by means of microbe–microbe and host–microbe interactions1. Interestingly, bacteria employs QS not only to coordinate collective behaviors such as bioluminescence production, but also to synchronize microbial activities essential for infection and survival in the host, such as biofilm formation, genetic exchange and virulence factor expression. This key role in bacterial pathogenicity suggests that quenching microbial QS (namely quorum quenching2) can be a promising disease control strategy, aiming at the attenuation of virulence, instead of imposing a ‘life-or-death’ selection pressure, in order to contain infectious diseases and to prevent antibiotic resistance in microbial communities.
        In Gram-negative bacteria, AI molecules are mainly comprised of N-acyl homoserine lactones (AHLs) that differ in the length, in the saturation levels and the oxidation states of the acylic tail. Generally, AHL QS involves two regulatory proteins encoded in the same operon, a member of the LuxI family of AHL synthase and a member of the LuxR family of AHL-responsive transcriptional regulator. Recently several studies and the sequencing of an increasing number of bacterial genomes have evidenced the widespread distribution of luxR-type genes that are unpaired to a cognate luxI (therefore defined solos), which are considered major players in bacterial cell–cell communication, not only in intra-species but also in inter-species and inter-kingdom signaling.
        All LuxR regulators are composed of two distinct domains: an N-terminal ligand binding domain (LBD) and a C-terminal DNA-binding domain. While most QS LuxR-type receptors display high specificity to the AHL ligand produced by their cognate LuxI-type synthetase, others are quite promiscuous in signal detection. The structural basis of the different degree of specificity of these pharmacological targets is still poorly understood.
        Our study is focused on the structural characterization of a recently described LuxR solo, LoxR from Kosakonia KO348. We demonstrated that it is a very promiscuous LuxR whose ensemble of conformations is dependent on the type of the bound AHL. The solution of its crystal structure in complex with a long acylic chain AHL together with the structural information on promiscuous and specific members of LuxR family in the PDB allowed to elucidate the structural determinants mediating LuxR specificity towards AHLs with long acylic chains.

        Speaker: Dr Sonia Covaceuszach (CNR - Istituto di Cristallografia)
        2020-Abstract-QS-1.docx
      • 14:30
        A painful dance between Insulin degrading enzyme and two substrates: Insulin and OFQ/N 5m

        Neurodegenerative disorders are mainly protein aggregation diseases widespread in the elderly, yet onsets in youngsters are not unheard of. The complexity also arises from their association with each other. For example, a linkage was established between Alzheimer’s disease (AD) and Diabetes mellitus type 2 (T2DM). Studies have shown the increased prevalence of dementia in diabetic vs. non diabetic patients. Cognitive dysfunction is associated with hyperglycemia, hypoglycemia and insulin resistance all of which are factors associated with T2DM. Both diseases decrease the quality of life and impose a huge financial burden on the healthcare system (1).

        The Insulin degrading enzyme (IDE) is a zinc metallopeptidase (2,3) that targets proteins with a size limit of 40-50 amino acid residues. This enzyme is involved in protein clearance which is a crucial step in avoiding the pathological consequences of protein aggregation. IDE is linked to T2DM (4) as it mainly targets insulin, but it is also able to degrade other endogenous small peptides. One of them is Nociceptin/orphanin (OFQ/N) (5,6) , a 17 amino acid neuropeptide involved in pain transmission and brain disorders such as AD. Interestingly, amyloid beta (Aβ) one of the main proteins linked to AD is also degraded by IDE (7). Hence, a possible indirect link between T2DM and AD can be established through studies of IDE, insulin and OFQ/N.
        In this study, mass spectrometric approaches were utilized to establish the hydrolysis pattern of OFQ/N1-16 peptide in the presence of IDE. Moreover, the effect of the insulin co-incubation on the hydrolysis of OFQ/N1-16 peptide was monitored. The experiment revealed the clear preference of insulin over OFQ/N as a substrate of IDE, and the potential role of T2DM in pain transmission.

        Speaker: Dr Iklas Mohamed Mohamud Ahmed (CNR - Istituto di Cristallografia)
        Abstract_Mohamud.docx
      • 14:35
        A re-investigation of copper coordination mode in the N-terminal 1-14 fragment of human Ctr1 protein. 5m

        Copper (Cu) is an essential micronutrient for most organisms and serves mainly as a redox-active catalytic centre in enzyme cycling between Cu+ and Cu2+. In mammalian cells the membrane transporter Ctr1 regulates the import of Cu into the cytosol (Fig.1). Even though the Cu is imported by Ctr1 as Cu+ , it may be transferred as Cu2+ to the high-affinity N-terminal Cu2+ binding site of the h-Ctr1 extracellular domain (ATCUN) and then reduced, for instance by ascorbate or a STEAP reductase on the cell membrane [1]. The critical nature of Ctr1 in human health has spurred interest in structure and function; however details on Ctr1- dependent Cu uptake and transport have to be elucidate [2]. Several studies on Ctr1 model peptides shed light on the identity of the Cu uptake through the extracellular binding site motif, but unresolved questions are yet opened [3]. We examinated the copper coordination mode of the Ctr11-14 fragment, a most used Ctr1 model peptide, in the presence of an excess of copper (II) (1:2 peptide/copper ratio). Taking in consideration that silver ion may adopt the typical Cu+ coordination modes we used Ag+ to study the possible formation of ternary complex species with Cu2+ . A combined potentiometric, spectroscopic and redox study, revealed that, at physiological pH, no ternary complex species are present, suggesting a crucial involvment of the His 3 as an anchoring point in the coordination of the Cu2+ and/or Ag+

        Speaker: Dr Francesco Attanasio (CNR - Istituto di Cristallografia)
      • 14:40
        Cyclic synthetic peptide mimicking NGF enhances PC12 differentiation and BDNF/VEGF expression 5m

        The neurotrophins (NTs) are a structurally and functionally related family of growth factors that regulate cell survival, differentiation, neurite outgrowth and regeneration, and synaptic plasticity in both the central and the peripheral nervous system. NTs include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5, -6, -7 [1]. A few small molecules have been designed and synthesized to achieve the functional and structural mimicry of NTs [2; 3; 4]. Cyclic peptides have gained increasing attention in recent years as an alternative scaffold. The structural constraints provided by cyclization not only helps to resist to a degradation by proteases but also facilitates passage through the cell membrane, thus broadening the potential use of cyclic peptides beyond extracellular targets to include intracellular targets [5].
        In this work we characterized cyclic peptide of N-terminal domain of NGF. The biological properties of this peptide were tested on the PC12 cell line. Our results demonstrate that peptides activate phosphorylation of TrkA, VEGFR1 and VEGFR2 receptors, induce the phosphorylation of CREB, cells differentiation and BDNF, VEGF protein release. It is well known that metal ions may affect the neurotrophin activity and that the alteration of copper homeostasis is a prominent factor in the development of neurological pathologies. Incubation of cells with peptide and copper chelator BCS leads to markedly decrease the effect of peptide. This indicates that the activation of BDNF and VEGF protein release by the peptide is not strictly related to Trks recognition but also includes pathways that are some how linked to copper.

        Speaker: Dr Irina Naletova (CNR - Istituto di Cristallografia)
    • 15:00 → 16:15
      Session: 3
      Convener: Dr Rocco Caliandro (CNR - Istituto di Cristallografia)
      • 15:00
        Rescuing proteostasis by small molecules 15m

        Many apparently unrelated diseases, including Alzheimer`s Disease (AD), Parkinson Disease (PD) and type II diabetes mellitus (T2DM), result from protein misfolding and abnormal accumulation of toxic amyloid deposits in affected tissues. According to the “Amyloid Hypothesis”, targeting protein misfolding and self-assembly into toxic amyloid aggregates would prevent the diseases. However, the failure of all clinical trials focusing on anti-aggregating drugs has clearly demonstrated that a deeper understanding of the mechanisms involved in proteome maintenance is needed.
        Our group focuses on the intertwined biochemical mechanisms that control protein homeostasis (proteostasis). We employ an interdisciplinary approach to screen small molecules (e.g. natural compounds, bioconjugates, and repurposed drugs) for their ability to restore proteome integrity by a multi-target strategy. Our work spans from fundamental topics related to thermodynamics of protein stability, amyloid aggregation, and ligand-protein interactions to applications in medicinal chemistry focusing on the development of bioactive compounds, involving chemically tailored contacts between lipids, proteins, nucleic acids, small molecules, and metal ions.1,3

        References
        1. F. Bellia, V. Lanza, S. Garcıa-Vinuales, I. M. M. Ahmed, A. Pietropaolo, C. Iacobucci, G. Malgieri, G. D'Abrosca, R. Fattorusso, V. G. Nicoletti, D. Sbardella, G. R. Tundo, M. Coletta, L. Pirone, E. Pedone, D. Calcagno, G. Grasso and D. Milardi, Chem. Sci. 2019, 10, 2732-2742.
        2. D. Milardi, E. Gazit, S. E. Radford, Y. Xu, R. U. Gallardo, A. Caflisch, G. T. Westermark, P. Westermark, C. La Rosa and A. Ramamoorthy, Chem. Rev. 2021, 121, 1845−1893.
        3. G. Grasso, A.M. Santoro, V. Lanza, D. Sbardella, G.R. Tundo, C. Ciaccio, S. Marini, M. Coletta, D Milardi, Coord. Chem. Rev., 2017, 347, 1–22.

        Speaker: Dr Danilo Milardi (CNR - Istituto di Cristallografia)
        Abstract_Danilo_Milardi_2021.docx
      • 15:15
        Cross talk between Amyloid ß peptides and Ubiquitin: new perspective in Alzheimer’s disease 15m

        Alzheimer's disease (AD), the most common form of dementia worldwide, is an age-related, fatal neurodegenerative disorder. A hallmark of AD is the presence of extracellular proteinaceous deposits (senile plaques) in the brain of affected people.[1] The prevalent component of senile plaques are β-amyloid (Aβ) peptides but it has been underlined the presence of ubiquitin. A reduced Ubiquitin Proteasome System (UPS) activity has been found in patients affected by AD and many reports suggest that the UPS malfunction plays a significant role in Aβ accumulation and, in turn, in AD progress.
        Here we set out to test whether Ub may bind the Aβ peptide and have any effect on its physiological clearance pathways.
        We demonstrated that Aβ40 binds Ub with a 1:1 stochiometry and Kd in the low micromolar range, using an integrated array of MALDI-TOF/UPLC-HRMS, fluorescence, NMR, SPR and molecular dynamics studies.
        In particular, we show that the N-terminal domain of Aβ peptide (through residues D1, E3 and R5) interacts with the C-terminal tail of Ub (involving residues K63 and E64), inducing the central region of Aβ (14HQKLVFFAEDVGSNK28) to adopt a mixed α-helix/β-turn structure. In neuroblastoma cell lysates, we have shown that Aβ competitively binds Ub also in the presence of the entire pool of cytosolic Ub binding proteins. Ub-bound Aβ has a lower tendency to aggregate into amyloid-like fibrils and is more slowly degraded by the Insulin degrading Enzyme (IDE). Finally, we observe that the water soluble fragment Aβ1-16 significantly inhibits Ub chain growth reactions.
        These results point out how the non-covalent interaction between Aβ peptides and Ub may have relevant effects on the regulation of the upstream events of the UPS.

        Speaker: Dr Valeria Lanza (CNR - Istituto di Cristallografia)
        Lanza_CSCB2020.docx
      • 15:30
        Software for the Characterization of Polycrystalline Compounds 15m

        Innovative methodologies have been developed and implemented in computer programs aiming at providing efficient computational tools for a wide range of applications in the field of the characterization of polycrystalline materials. The software, based on sound theoretical principles, developed according to the most recent and advanced programming languages and supported by a high level of automatism which makes its use simple also to non-expert in crystallographic knowledge, is worldwide used.
        The developed software are:
        EXPO1: a computing program devoted to structure solution of materials available in the form of microcrystalline powder, by using X-ray diffraction data. The chemical formula and the experimental diffraction profile are the only information necessary to run it. EXPO is able to investigate small molecules, inorganic, organic and metal-organic. It can execute, in a complete automatic way, all the steps characterizing the structure solution process and consisting of: 1) identification of crystal cell parameters; 2) determination of space group; 3) solution of crystal structure by locating the correct atom positions; 4) refinement of the final crystal model.
        QUALX2: a software for identifying the crystal phase(s) present in a powder sample. It automatically performs: 1) the location of peaks in the experimental diffraction profile, 2) the subtraction of the background noise, 3) the search in crystal phase databanks. At the end of the process, QUALX brings out the chemical phase(s) which best match the peaks in the experimental pattern. In addition, it can query two databases: one commercial (PDF-2) and one free (POW_COD).
        OChemDb3(Open Chemistry Database): a new free web portal which has been developed for assisting in the crystal structure determination process by searching and analyzing crystal chemical information of organic and inorganic compounds. OChemDb is dedicated to collect, to make available by statistical tools and to manage crystal-chemical information coming from the CIF files contained in the Crystallography Open Database (COD, http://www.crystallography.net/cod/). OChemDb can be used for searching and analyzing crystal-chemical information (bond distance, bond angle, space group) of structures already solved, to be used for different scientific purposes. It provides statistics on desired distances, bond angles, torsion angles and space groups. OChemDb uses a suitably designed database of solved crystal structures. The use of OChemDb requires only a web browser and an internet connection.
        For each software, examples of challenging applications by users around the world covering a wide range of scientific interests will be presented.

        Speaker: Dr Nicola Corriero (CNR - Istituto di Cristallografia)
        Catania.docx
      • 15:45
        Competitive effectors of alpha-synuclein 15m

        Parkinson’s disease (PD) and α-synucleinopathies are characterized by the progressive loss of neuronal cells and the decline of cognitive and motor functions. Biochemical and neuropathological evidence supports the role of oxidative stress, metal dyshomeostasis and α-synuclein (αSyn, a presynaptic and intrinsically disordered protein), in the development of these disorders. Mounting evidence suggests that the aggregation of αSyn is a crucial event in the pathogenesis of α-synucleinopathies.
        Metal-protein interactions play an important role in αSyn aggregation and might represent a link between the pathological processes of protein aggregation, oxidative damage, and neural death. High Copper concentration is detected the cerebrospinal fluid of PD patients, as well as in the Lewy bodies, the intracellular aggregates of αSyn. Moreover, Copper regulates αSyn intracellular localization and cytotoxicity.
        Lipoxidation and carbonylation have also been observed in neurodegenerative diseases. αSyn seems to induce lipid peroxidation and, conversely, αSyn carbonylation has been found in PD. Lipoxidation leads to the formation of the so-called Reactive Carbonyl Species (RCS); in particular, acrolein (ACR) and 4-hydroxy-nonenal (HNE) have been reported to affect the aggregation process of αSyn. The adducts ACR-αSyn have been less explored and characterized. Moreover, the interplay between ACR, copper, and αSyn has never been investigated.
        Therefore, we explored more thoroughly the dose- and time-dependent effects of ACR on αSyn using an approach based on Ultra Performance Liquid Chromatography coupled with High-Resolution Mass spectrometry. Moreover, we evaluated the effects of Cu2+ ions on these chemical modifications, and the influence of His carbonylation on Cu2+-binding. Finally, we investigated the effects of ACR and Cu2+ ions on αSyn aggregation by a fluorescence assay and dynamic light scattering (DLS).

        Speaker: Dr Francesco Bellia (CNR - IC)
        Bellia_F_abstract.docx
      • 16:00
        Antimicrobial peptides (AMPs): Analysis, synthetic design and biological analysis. 15m

        The increase of resistance to antibiotics [1] , also due to a systematic and widespread misuse and abuse of these drugs, is a tremendous problem of healthcare systems and society. Multiple resistance to antibiotics is a global threat aggravated by the lack of novel alternative and effective therapeutic agents [2] . The most worrying multidrug-resistant pathogens are listed by the World Health Organization under the acronym “ESKAPE” [3] , (i.e., Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) as needing urgent and prompt discovery of new antimicrobials.
        Antimicrobial peptides (AMPs) are potentially suitable to alternatives to conventional antibiotics [4] or effective adjuvant drugs allowing conventional antibiotics to overcome resistance [2,5] . In this scenario, our research activity focuses on identifying, analyzing, synthesizing and testing new AMPs of natural origin, and their optimized synthetic variants.
        Here, we present a hierarchical approach applied to Taenia solium peptides (TSO8), whose sequence is compared with other native AMPs, analyzed to determine potential active fragments, and then synthesized to obtain preliminary functional (MIC, cytotoxicity) and structural (CD) characteristics.

        Speaker: Dr Andrea Caporale (CNR - Istituto di Cristllografia)
    • 16:15 → 17:00
      Break 45m
    • 17:00 → 18:40
      Session: 4
      Convener: Dr Sonia Covaceuszach (CNR - Istituto di Cristallografia)
      • 17:00
        Free radicals induced DNA damage: Chemical, Analytical, Biological, and Diagnostic Aspects 15m

        DNA damage caused by free-radicals includes a large variety of base and sugar lesions leading to DNA breaks
        and DNA-protein cross-links. Among free radicals, the diffusible hydroxyl radicals (HO · ) are key oxidant species
        responsible for reacting with DNA either by hydrogen abstraction from 2-deoxyribose units or by addition to the base
        moieties. The purine 5’,8-cyclo-2’-deoxynucleosides (cPu) are an important class of lesions exclusively generated by
        the HO · attack to DNA purine nucleotides, forming C5’ radicals followed by an internal cyclization giving 5’,8-cyclo-2’-
        deoxyadenosine (cdA) and 5’,8-cyclo-2’-deoxyguanosine (cdG) identified as products, in two possible diastereomeric
        forms, 5’R and 5’S (Fig. 1). 1,2

        These lesions are exclusively repaired by the Nucleotide Excision Repair pathway (NER) with low efficiency in comparison with other bulky DNA lesions.3

        Nowadays cPu are used as radical stress biomarkers of DNA damage thanks to their specific formation mechanism and their chemical stability during work-up.
        The diastereomeric 5’S- and 5’R-cPu are discussed in terms of:
        (i) Synthetic and analytical protocols for the availability and characterization of the diastereoisomeric 5′S- and 5′R-cPu lesions, also as isotopic labelled references;
        (ii) Physical-chemical studies on specific oligonucleotide models as: MD simulations, NMR, Melting Temperature;
        (iii) Biological and Clinical studies for the investigation of the relationships between the levels of lesions and human health, disease, and aging is a matter of investigation.4

        References

        1 C. Chatgilialoglu, C. Ferreri, N. E. Geacintov, M. G. Krokidis, Y. Liu, A. Masi, V. Shafirovich, M. A. Terzidis, P. S.
        Tsegay, 5’,8-cyclopurine lesions in dna damage: Chemical, analytical, biological, and diagnostic significance, Cells
        8 (6) (2019) 513.
        2 C. Chatgilialoglu, L. A. Eriksson, M. G. Krokidis, A. Masi, S. Wang, R. Zhang, Oxygen dependent purine lesions
        in double-stranded oligodeoxynucleotides: Kinetic and computational studies highlight the mechanism for 5’, 8-cyclopurine formation, Journal of the American Chemical Society 142 (12) (2020) 5825–5833.
        3. Kropachev K.; Ding S.; Terzidis M.A.; Masi A.; Liu Z.; Cai Y.; Kolbanovskiy M.; Chatgilialoglu C.; Broyde S.; Nicholas E. Geacintov N.E.; Shafirovich V. Nucleic Acids Res., 2014, 42, 5020
        4. Masi, A.; Fortini, P.; Krokidis, M.G.; Romeo, E.F.; Bascietto, C.; De Angelis, P.; Guglielmi, V.; Chatgilialoglu, C. Redox Biol. 2020, 34, 101562.

        Speaker: Dr Annalisa Masi (CNR - Istituto di Cristallografia)
        Abstract_A.MASI- Conferenza di Istituto.pdf
      • 17:15
        Carnosine and trehalose-carnosine tunesthe activity and expression of endogenous protection factors and their crosstalk with metal homeostasis 15m

        Carnosine (β-alanyl-L-histidine) is a natural dipeptide widely distributed in mammalian tissues and presented at high concentrations (0.7–2.0mM) in the brain1. As reported previously, carnosine augmented the secretion and expression of various neurotrophic factors (for example, BDNF), leading to the induction of neurite growth in SY-SY5Y cells2. Moreover, carnosine glial release and neuronal utilization in CNS have been described3; carnosine intercepts the regulatory routes of Cu homeostasis in nervous cells and tissues. Cu dysregulation imply the oxidative stress, free-radical production and functional impairment leading to neurodegeneration. Barca et al showed that the extracellular carnosine exposure influenced intracellular Cu entry and affected the key Cu-sensing system (SP1 and CTR1) 4. On this basis, carnosine, its derivate with trehalose and potential role of copper ions were investigated in the present study. First of all, we demonstrate that trehalose-carnosine crosses the cell membrane better than carnosine and its translocation does not depend on copper ions. On the next step, we analyzed a role of carnosine and its derivative in the modulation of CREB functions in the normal and in the copper ions deprivation conditions. Previously, it has been shown that carnosine and copper alone induce CREB phosphorylation 5, 6. Here we found that 30 min of PC12 cells incubation with trehalose-carnosine stimulates CREB phosphorylation more than carnosine alone and the level of phospho-CREB depends on the presence of copper ions in the medium. To compare the influence of trehalose-carnosine and carnosine alone on copper homeostasis, a measure of the copper transporter CTR1 and transcriptional factor SP1 expression in culture of PC12 cells was carried out.

        References
        1. AR Hipkiss et al. Ann. N. Y. Acad. Sci. (1998) 20;854:37-53.
        2. K Kadooka et al. J of Functional Foods (2015) 13;32-37.
        3. K Bauer, Neurochem Res. (2005) 30(10):1339-45.
        4. A Barca et al. Am J Physiol Cell Physiol. (2019) 316(2):C235-C245.
        5. K Fujii et al. Cytotechnology (2017) 69(3):523-527.
        6. I Naletova et al (2019) Cells. (2019) 8(4). pii: E301

        Speaker: Dr Francesco Attanasio (CNR - Istituto di Cristallografia)
        Abstract-Attanasio.doc
      • 17:30
        Insights into the PPARγ phosphorylation and its inhibition mechanism 15m

        Peroxisome proliferator-activated receptor gamma Ligand Binding Domain (PPARγ-LBD) represents a key
        target for the treatment of type II diabetes and metabolic syndrome. This receptor is the target of
        thiazolidinediones, a class of antidiabetic drugs, which improve insulin sensitization and regulate glycemia in
        type 2 diabetes. Unfortunately, despite the beneficial effects of synthetic drugs, their use is associated with
        serious undesirable side effects1,2 related to their agonism. By contrast, a promising activation-independent
        mechanism that involves the inhibition of cyclin-dependent kinase 5 (Cdk5)-mediated PPARγ
        phosphorylation (CMPF) has been related to the insulin-sensitizing effects induced by these drugs3,4. For this
        reason, the search for new inhibitors of CMPF represents an opportunity for the development of an improved
        generation of anti-diabetic drugs acting through this nuclear receptor. Thus, with the aim to identify novel
        drug-like inhibitors of CMPF capable of interacting with PPARγ but that lack agonist properties we adopted
        a multi-disciplinary approach, including protein-protein docking, X-ray, NMR, HDX, MD simulations and
        site-directed mutagenesis to investigate conformational changes in PPARγ that impair the ability of Cdk5 to
        interact with this nuclear receptor and hence inhibit its phosphorylation.

        Speaker: Dr Roberta Montanari (CNR - Istituto di Cristallografia)
        Abstract_MontanariR.pdf
      • 17:45
        Peptide therapeutics: β-sheet breaker conjugates for neuroprotection and amyloid detection. 15m

        Peptides may represent an opportunity for therapeutic intervention that closely mimics natural pathways.1 As a unique class of pharmaceutical compounds peptides are endowed with biochemically and therapeutically distinct properties with respect to small molecules or proteins. Peptides’ favourable pharmaceutical properties include high specificity and potency for their target, minimal potential for drug-drug interactions, lack of accumulation in tissues, and effectively metabolized by endogenous enzymes to non-toxic metabolites. On the other hand, peptide therapeutics may suffer unfavourable pharmaceutical drawbacks. These comprise instability and short duration of action, inability to cross cellular membranes, and potential for immunogenicity. Recent research in peptide chemistry has made advances to resolve these critical aspects. In particular, bio-conjugation can prolong peptides‘ plasma stability by preventing their degradation by exopeptidases. Bio-conjugated peptides play an important role in several fields of biomolecular and medicinal chemistry.2 In particular, peptide-based epitopes with covalently attached other moieties able to explicate additional or complementary functions, including BBB permeation, metal chelation or aggregates disassembling, targeted imaging and treatment, hold a promising potential for applications in Alzheimer’s disease (AD). AD has been proving to be extraordinarily refractory to any attempt aimed at halting or slowing the progression of the disease. In our laboratory, we have been synthesizing a variety of small peptides bio-conjugates differing by the peptide epitope or the conjugated scaffold.3,4 A range of molecular details, together with measured biological effects, have been listed with these systems, all of them accounting for the observed neuroprotection against the toxic insult induced by Aβ aggregation in primary cortical neurons. In this communication an overview of the design principles of the peptide conjugates, their neuroprotective activity and their capability in detecting Aβ peptide in solution are described in terms of potential use of these compounds as theranostic agents and for the targeted drug delivery.

        Speaker: Dr Giuseppe Pappalardo (CNR - IC)
        Abstract_Pappalardo_G.docx
      • 18:00
        Reverse engineering and 3D additive manufacturing from research to the clinical assistance 15m

        Introduction
        This contribution aims to present the design and implementation of an innovative image treatment and 3D printing lab for the production of customized medical devices within a hospital facility.
        Methods and Materials
        In 2016 a charity from Bank of Italy to Fondazione Santobono Pausilipon (active in assistance, research, training and improvement of the quality of life of pediatric patients) was spent in a project to create in reverse engineering and 3D printing personalised plastic casts to be experimented at Orthopaedics and Traumatology Dep. of the Santobono Pediatric Hospital. To develop this study the know-how and multidisciplinary competences from different CNR Institutes (IC now ICCSB, IBB, IPCB) has been involved to produce the proof of concept and the consequent clinical application. Following the design phase1 in 2017 the Ethics Committee authorized a clinical trial of the developed device on 40 patients2. In the meanwhile the project was awarded by Italian Convention of Public Health (ForimPA Sanità). The success of clinical trial3 brought researcher and hospital management to awareness that the scientific outcomes are ready to be transferred to the real clinical use.
        This led to the establishment of an innovative company (Santobono Innovatin srl) operating within the Santobono-Pausilipon Foundation in wich CNR was consultant to optimize manufacturing workflow. The company structured two models of 3D printed devices ad submitted itself to the certification process for the production of medical devices. In June 2019 the ISO 13485 certification and the exposure on market of public companies (MEPA).
        Conclusion
        The collaboration between researcher from CNR and clinical practitioners inside a health facility produces real innovations and represent a great opportunity to promote new collaborations and research projects among stakeholders. Indeed the introduction of image processing, reverse engineering and 3D printing techniques is rapidly spreading to exploit the advantages given by the design flexibility and low-cost production of prostheses, medical devices and anatomical models.
        References
        1. Clemente F et al. I Congresso IDBN, Bologna 2017, Biomodello 8001-08
        2. Ethics Commetee AA.OO.R.N “A.Cardarelli/Santobono Pausilipon, clinical trial:. FSP1/2016 and Italian Ministry of Health, clinical trial: 0002535-13/01/2017-DGDMF-MDS-P.
        3. Guida P et al., Jour Ped Orthop Part B, 28 (3), 2019, pp: 248-255

        Speaker: Dr Fabrizio Clemente (CNR - Istituto di Cristallografia)
        Abstract_Clemente_3D.docx
      • 18:15
        Privacy @CNR: panoramica generale su argomento, organizzazione e adempimenti 15m

        Con la entrata in vigore del Regolamento generale sulla protezione dei dati UE 2016/679 (RGPD), il CNR ha delineato il proprio assetto organizzativo per la gestione degli adempimenti da questo previsti e posto in essere le attività da svolgere, con il fine di assicurare l’applicazione delle disposizioni in materia di protezione dei dati personali.
        Si daranno i principali riferimenti normativi sull’argomento, si riporterà l’assetto organizzativo che si è dato il CNR e si metteranno in evidenza gli adempimenti cui sono tenuti gli Istituti, con particolare riferimento alla istituzione del “Registro delle attività di trattamento dei dati” e suo aggiornamento.
        L’aggiornamento deve avvenire prima di avviare un trattamento, sia esso per finalità gestionali-amministrative che di ricerca. Tutte le attività condotte in Istituto sono quindi interessate.
        L’obiettivo è quello di informare e sensibilizzare il personale sulla tematica. Considerata la trasversalità dell’argomento, è necessaria una ottimale collaborazione tra tutti i ruoli e profili presenti in Istituto.

        Speaker: Dr Caterina Chiarella (CNR - Istituto di Cristallografia)
    • 18:40 → 19:00
      ePoster session: Software and Tools
      Convener: Dr Francesco Bellia (CNR - Istituto di Cristallografia)
      • 18:40
        Ceftazidime and other cephalosporins inhibit bacterial NAD biosynthesis by targeting NadD enzymes. Implication for the treatment of chronic infections. 5m

        It is known since 40’s that the rate of killing by antibiotic is in strict correlation with the bacterial growth rate, and that for non growing bacteria antibiotics result ineffective. The ability of some bacteria to enter and endure in a highly depressed non-replicating metabolic state has been recognize as a frequent cause of failure in the treatment of chronic infections, and the need for new targets whose inhibition kills both replicating and non-replicating bacteria has been highlighted [1].
        Genomics-based reconstruction and experimental data showed that in bacteria the last two steps of NAD biosynthesis from a nicotinate mononucleotide (NaMN) precursor via nicotinate adenine dinucleotide (NaAD) intermediate are irreplaceable. The respective enzymes, NaMN adenyltransferase (NadD) and NAD-synthase (NadE), are conserved in most bacterial species, being quite distant from their human counterparts in term both of kinetic properties and structural features[18]. The inhibition of NadD enzymes was shown to kill bacteria both in the replicating and non-replicating state [2,3], validating NadD as a target for the development of new antibiotics.
        We report on a structure-based virtual screening which indicated some cephalosporins as potential inhibitors of NadD enzymes. Ceftazidime, and other third generation cephalosporins, were confirmed to inhibit NadD of M. tuberculosis, E. coli and P. aeruginosa with Ki in the μM range. It was also found that Ceftazidime and Cefquinome speed-up the cell death rate in non replicating cultures of E. coli and P. aeruginosa. The same cultures were insensitive to high concentrations of Ampicillin and other cephalosporins which do not inhibit NadD. Showing that the observed toxicity depends on the impairement of NAD biosynthesis by Ceftazidime and Cefquinome. Altough their potency is probably not sufficient to suggest an alternative clinical use of these drugs, they might serve as lead compound to develop better NadD inhibitors and possibly new antibiotics.

        Speaker: Dr Alessandro Pesaresi (CNR - Istituto di Cristallografia)
        Abstract_IC-Catanaia_Pesaresi.pdf
      • 18:45
        Geant4, a Monte-Carlo simulation tool for nuclear medicine and molecular radiotherapy 5m

        Understanding of how radiation emitted by a particular radiotracer distributed in various organs, deposits energy in different tissues through various mechanisms of radiation interaction with matter, relies on the correct description of the chemical composition of the scattering medium and the involved radiation transport processes. Monte-Carlo (MC) methods allow the simulation of the interaction between particle and matter thus providing a fundamental method to study the physics of nuclear medicine, radiology, and radiation therapy.
        The concepts of deposited energy and absorbed dose are of particular interest not only for radiotherapy applications[1] but also for imaging applications[2] involving ionizing radiations.
        The accurate assessment of the absorbed dose distribution throughout the organs and tissues of interest is required in radiation therapy (RT) treatment planning whatever the RT approach (e.g., using photons, electrons, protons, carbon beams, radioisotopes) and the different delivery conditions (broad beam, pencil beam, scanning, rotational, brachytherapy, and targeted radionuclide therapy).
        In diagnostic imaging applications involving ionizing radiation (e.g., computed tomography, positron emission tomography, or single-photon emission tomography) the assessment of the absorbed doses is important to better analyze the risk-benefit of the procedure.
        Hence the need for a MC simulation platform supporting radiation transport modeling for imaging and dosimetry applications. Among the MC simulation tools that have been developed for imaging or dosimetry, the GEANT4[3] toolkit will be presented. It is a powerful tool that allows simulating the interactions between particles and matter, while different geant4 projects provide additional high-level features to facilitate the design of GEANT4-based simulations.

        References
        1. N. Reynaert, S. Vandermarck, D. Schaart, W. Vanderzee, C. Vanvli-etvroegindeweij, M. Tomsej, J. Jansen, B. Heijmen, M. Coghe, andC. Dewagter, “Monte carlo treatment planning for photon and electronbeams,”Radiat. Phys. Chem.76, 643–686 (2007).
        2. M. J. Murphy, J. M. Balter, S. Balter, J. A. BenComo, I. J. Das, S. B. Jiang, C.-M. Ma, G. H. Olivera, R. F. Rodebaugh, K. J. Ruchala, H. Shirato, and F.-F. Yin, “The management of imaging dose during image-guided radiotherapy: Report of the AAPM Task Group 75” Med. Phys.34, 4041–4063 (2007).
        3. S. Agostinelli et al., “Geant4 - a simulation toolkit”, Nuclear Instruments and Methods in Physics Research A 506 (2003) 250-303

        Speaker: Dr Annamaria Mazzone (CNR - Istituto di Cristallografia)
        Anna_Maria_Mazzone.docx
      • 18:50
        ICT methods and tools for health 5m

        Introduction. An actual challenge of health systems is the practical implementation of ICT. It is useful to introduce new organizational models and guide decision makers towards an appropriate use of resources. Thanks to a strictly cooperation with healthcare units using ICT techniques and technologies, innovative applications have been studied, designed and implemented.
        Telemedicine. Telemedicine is the provision of healthcare services, through use of ICT. It has been proposed in a variety of health care fields with significant advantages: As a matter of fact today at national level there are patchworked telemedicine initiatives not well coordinated. The ICCSB has collaborated in the design of new teleconsultation, telemonitoring and teleassistance services1. These projects involve territorial and specialist hospitals from different regions. Particular experience dealt with teleconsultation and sharing of clinical documentation for second-opinion, As technology is beyond clinical practice, making operative telemedicine it is necessary building confidence in and acceptance of new services. Last but not least it is necessary to bring legal clarity in management sensitive data. In practice, the design of each project must consider functional and ethical requirements, organizational requirements, technological requirements, legal requirements (GDPR). Some active telemedicine services will be presented.
        Home care. A theme ancillary to telemedicine and care outside of hospital is related to the high intensity home care. The use of medical devices at patient’s home by personnel who are not healthcare practitioners introduces new potential risks. A methodological approach to investigate potential failures and define improvement actions to address the dangerous potential situations in home care has been developed2.
        Process Modelling. A third application regards healthcare process modelling and simulation. The use of software tools allows the representation of the system (As Is) to study the performances and the design of suggested variations (To Be) to predict the effect of designed changes. Application has been developer for home care and to study the emergency department optimization3.
        H2020 Project. An effective study on the security, privacy data protection in e-health has been carried out in the framework of the participation at H2020 KONFIDO project devoted to cross border data exange4.
        References
        1. Clemente et al. 20197th IEEE EHB Proceedings
        2. Clemente et al 2019 Heliyon 5 (12), e03034.
        3. Faiella et al, 2019 IFMBE Proceedings, 68 (1), pp. 567-572
        4. Nalin et al. 2019 Journal of Biomedical Informatics 94,103183

        Speaker: Dr Fabrizio Clemente (CNR - Istituto di Cristallografia)
        Abstract_Clemente_ICT.docx
  • Wednesday, 6 October
    • 09:00 → 10:20
      Session: 1
      Convener: Dr Roberta Montanari (CNR - Istituto di Cristallografia)
      • 09:00
        Toward Personalized Medicine: RGD-Peptide as Scaffold for the Comprehension of Structural Determinants for Integrin Specific Recognition 15m

        A great challenge in cancer therapy is the selective delivery of anti cancer agents or radiotherapeutic to tumour cells reducing the side effects on normal cells. To further improve delivery efficiency and cancer specificity, great efforts have been dedicated to the development of effective systems that can actively target tumours through the molecular recognition of unique cancer-specific “markers” that are overexpressed in the cancerous tissues.
        The use of peptides as targeting tools has been validated in a number of applications. Examples are radio-labelled peptides used to deliver radio-therapeutic doses to cancer tissues. In this case a cytotoxic entity, such as a radionuclide or a chemotherapeutic drug is driven by the peptide to the transformed cells with a higher efficacy than to normal cells. Typically such conjugates, at lower doses, have useful applications for diagnostic applications whereby they are used to visualize even small lesions due to the overexpression of tumour genes.
        In this lecture, we will present our most recent results on a project that aim at the rational design of targeting peptidomimetic ligands to specific tumour receptors. More specifically, the design of novel anticancer drugs and/or new class of diagnostic non invasive tracers as suitable tools forv3 -targeted therapy and imaging. Since integrins are involved in the regulation of physiological processes, as well as in pathological ones, the development of integrin subtype-selective antagonists is highly desirable. Integrins v3 and 51, key mediators of cell adhesion, differentiation, proliferation, angiogenesis and tumor growth, have been considered very promising targets for theranostic application. Due to the similarity of the RGD binding regions in these integrins, the development of small synthetic molecules with high activity and selectivity for the different subtypes is a tricky goal to pursue. In the last decade, we designed and characterized the chimeric molecule RGDechi, a peptide selective for v31. We demonstrated anti-adhesive and proapoptotic effects on tumor cells and antiangiogenic activity in vivo2-4. In addition, SPECT and PET imaging studies with 111In and 18F-labelled RGDechi in a xenograft model confirmed the ability of peptide to selectively visualize this integrin. Recently, NMR and computational analyses on cell membranes allowed a detailed understanding of v3/RGDechi recognition mechanism5. Herein we have investigated the capability of the peptide to bind 51 integrin and characterized the molecular determinants governing this interaction through a combined experimental and computational approach.
        The detailed comparison of RGDechi/51 structural model with that, previously, determined of RGDechi in complex with v3 has shown how the chimeric nature of the peptide renders the molecule an important scaffold to recognize integrins with different recognition modalities, providing insight on the structural requirements needed to design novel peptides selective for 51 to use in theranostics6.

        References
        1. Del Gatto, A. et al. J Med Chem., 2006, 49, 3416-3420
        2. Zannetti, A. et al. Clin Cancer Res., 2009 15, 5224-5233
        3. Santulli, G. et al. J Transl Med., 2011, 9, 7
        4. Pisano, M. et al. Anticancer Res., 2013, 33, 871-879.
        5. Farina B, et al. Chemistry, 2016, 22, 681-693.
        6. Farina B, et al. J Pept Sci, 2019, 18:e3166.

        Speaker: Dr Michele Saviano (CNR - Istituto di Cristallografia)
      • 09:15
        Protein Crystallization in Hydrated Deep Eutectic Solvents 15m

        Deep Eutectic Solvents (DESs) are mixtures of Lewis or Brønsted acids and bases whose melting point is much lower than that of the individual components.1 Several applications are known in the literature for such neoteric solvents which include the usage of DESs in extraction processes, in biodiesel production and purification, as proton conductors for fuel cells, in metal-, organo- and biocatalyzed transformations, as well as the in polishing procedures of metal surfaces.2 Ten years ago, it was found that deep eutectic mixtures spontaneously form in plants as a consequence of the accumulation in cells of metabolites like sugars, alcohols, amines, amino acids, and organic acids,3 an evidence that aroused the curiosity of the experts. Indeed, such these mixtures appeared to be an ideal solvent for the transport of macromolecules of diverse polarities inside the plants, hence they could be used as component of the intra cellular medium in synthetic biology. Thus, the quest for reliable structural information about the interactions between DESs and proteins is of the utmost importance and the first step towards such target is to obtain high quality crystals of protein molecules in the presence of DESs.
        Here, we show, for the first time, that protein molecules are able to crystallize in the presence of DESs.4 Our crystallization experiments have shown that hen-egg white lysozyme crystallizes according to the DES components and the hydration level. Successful crystallization have been achieved by using choline chloride:urea, choline chloride:glycerol, and choline chloride:glutamic acid eutectic mixtures at a 1:2 molar ratio. X-ray diffraction experiments have provided details about the binding sites of DES components on lysozyme protein surface and precise information about the intriguing non-covalent bond network by which protein, DES components and water molecules interact each other. A non-covalent bond network between DES components mediated by water molecules has also been unveiled. DESs appear to have negligible effects on protein conformation. On the other hand, DESs significantly reduce solvent evaporation from the crystallization drop, a key property to increase the dissolution time of the protein crystals. Moreover, DESs could tune protein solubility because it appears to affect hydration shell of the protein.
        Our results prove that high-quality protein crystals can be obtained in the presence of DESs and that such crystals can be used to get precise structural information about the interaction between DESs and proteins, which is still lacking in the literature. Moreover, the ability of DESs to protect protein crystals from their rapid dissolution (which has been observed in this work) could be exploited in biotechnological applications involving enzymes in crystalline form.

        References
        1. Benworth B Hansen et al., Chemical Review, 2021, 121, 1232
        2. a) Maan Hayyan et al., Fuel Processing Technology, 2009, 91, 116; b) Adeeb Hayyan et al., Industrial Crops and Products, 2013 46, 392; c) Habibi Emadaldin, Analytica Chimica Acta, 2013, 762, 61; d) Ghanemi Kamal et al., Analitical Methods, 2014, 6, 1774; e) Perna Filippo Maria et al., Current Opinion Green Sustainable. Chemistry, 2020, 21, 27; f) Cicco Luciana et al., Organic Biomolecular Chemistry 2021, 19, 2558
        3. a) Choi Young Hae et al., Plant Physiology, 2011, 156, 1701; b) Dai Yuntao et al., Analytica Chimica Acta, 2013, 766, 61
        4. Belviso Benny Danilo et al., ACS Sustainable Chemistry Eng, 2021, 9, 8435

        Speaker: Dr Benny Danilo Belviso (CNR - Istituto di Cristallografia)
        abstract_Belviso_final.docx
      • 09:30
        Surface Plasmon Resonance, a powerful tool for ligand binding investigation 15m

        Selection of promising, well characterized hits and leads is essential for success in the drug discovery process. Furthermore, information on the interaction of potential drug candidates with the targeted biomolecule is important as a basis for the understanding of more complex schemes. Binding assays are the type of assays that provide such information on affinity, kinetics and thermodynamics [1]. Biophysical binding assays are alternative tools in generating label free, high quality data on the interaction between a target and a potential drug candidate. Label free screening methods include mass spectroscopy, nuclear magnetic resonance spectroscopy, isothermal titration calorimetry (ITC) and biosensor.
        Biosensors based on optical detection principles are the type of sensors most often used. They offer a rapid way to determine relevant binding data without the need for labeling of the interacting molecules. These biosensors measure in real time the quantity of complex formed between a molecule immobilized on the sensor surface and a molecule in solution, determining small changes in refractive index induced at the interface upon binding. A huge number of different optical techniques to monitor such refractive index changes have already been introduced in the past and this number is still increasing. Among them, Surface Plasmon Resonance (SPR) is currently the most widely used due to the wide versatility and sensibility of the method.
        Here we present the results obtained using SPR with various types of assay involving different biological targets. In particular, kinetics data from single compound binding assay [2], surface competition assay [3] and screening of small compounds library [4] will be shown.

        References
        Marks et al. Methods Mol Biol 2002, 190:51-63.
        Capelli et al. Front Chem. 2020, 7:910
        Souvalidou et al. bioRxiv (Preprint) 2021
        Montanari et al. Sci Rep. 2016, 6:27658

        Speaker: Dr Davide Capelli (CNR - Istituto di Cristallografia)
        Abstract_Davide_Capelli.pdf
      • 09:45
        ICT solution for Scientific Communication and Learning 15m

        One of the institutional tasks of public research bodies is to promote the dissemination of knowledge through editorial initiatives and training courses. Technological progress, in the field of communication, and the diffusion of the Internet makes it possible for the scientific community to produce and distribute academic publishing products on its own. The self-publication of scientific content takes place through the use of IT platforms dedicated to the management of the editorial process of production 1 and distribution of both magazines and digital monographs. Specialized IT platforms for collaborative scientific writing2 are also available to complete the editorial process. With this platform it is possible to create scientific publishing products of high typographical quality in a collaborative way, multiple authors working simultaneously on the same document, even on different parts of the same. The software keeps track of all changes and also manages a workgroup chat. The results of the experimentation and the advantages found on the use of the IT platforms created will be exposed.

        Also for training, various IT solutions were tested to the creation of an integrated system of IT platforms to carry out e-learning. The results of Learning Contest Manager System3 and Web services integration will be exposed.

        Another area of ​​scientific communication is the organization and distribution of the contents of conferences and scientific seminars. Also in this area, a specific IT platform4,5 was tested with which to manage the entire organizational process of scientific events: from the collection of contributions, to the realization of the scientific program of the event and the management of registrations.

        References
        1. https://doi.org/10.30441/smart-elab.v12i0.217 (Indico)
        2. https://doi.org/10.30441/smart-elab.v8i0.196 (ShareLaTeX)
        3. https://doi.org/10.30441/smart-elab.v1i0.24 (Moodle)
        4. https://doi.org/10.30441/smart-elab.v1i0.27 (OJS)
        5. https://doi.org/10.30441/smart-elab.v13i0.222 (Jitsi)

        Speaker: Dr Guido Righini (CNR - Istituto di Cristallografia)
        ICT_solution_for_Scientific_Communication_and_Learning.pdf
      • 10:00
        The synthetic heptapeptide Semax, a fragment of the ACTH hormone, sustains differentiated neuroblastoma, by improving the cellular bioenergetic: investigations toward New Therapeutic Solutions 15m

        Semax is the active component of a drugs developed in Russia in the1982 and originally used as a treatment for brain hypoxia and ischemia, brain traumas, and to facilitate adaptive processes to extreme situations. Semax is now used as a nootropic for mental enhancement in healthy people and for treating many cognitive disorders. N-terminal fragments of the adrenocorticotropic hormone (ACTH), are well known for their potent neuroregenerative and cognitive activities1. The syntetic eptapeptide Semax consisting of the Met-Glu-His-Phe fragment of ACTH and the C-terminal tripeptide Pro-GlyPro, is devoid of any hormonal activity, but is still able to stimulate learning and memory formation in rodents and humans2. Further major advantages of Semax, is the higher resistance against enzymatic cleavage confered by the C-terminal PGP. Despite the widespread use, the cellular and molecular mechanisms underlying the action of Semax, are widely unknown. At the cellular level, Semax was shown to prevent the death of cultured neurons, and to increase the expression of neurotrophine and their receptors3, thus implying that Semax might modulate brain functions by affecting neurotrophin synthesis. We investigated about the mechanism by which Semax acts and describe the effects at the cellular level on the RA-differentiated SHSY5SY cells. Our findings indicate that Semax is able to modulate the BDNF expression, as well as TrkB and MCR4 receptors levels. Interestingly, cells differentiated in the presence of Semax are more resistant to different stresses, having a higher mitochondrial membrane potential and an increased mitochondrial mass compared to cells differentiated in RA only. In cultured hippocampal neurons, it has been found that BDNF influnces the mitochondrial function, a process dependent on the activation of the TrkB receptor and its downstream signaling pathways. We suggest that Semax affects cognitive brain functions by modulating the expression of the BDNF/trkB system. By increasing mitochondrial metabolism, Semax provide neurons with the ability to better exploit feuls, either under basal and stressful conditions, which results in the improvement of neurons viability as demostrate by the means of several experimental approaches. The finding that Semax modulating neurotrophins levels can alter mitochondrial oxidative efficiency, has important implications for neurodegenerative and psychiatric diseases. Therefore, the therapeutic potential of Semax is far from being exhausted and new indications for its application could be discovered by an in-depth study of its mechanism of action.
        References
        1. de Wied D.Eur J Pharmacol. (1999)
        2. Ashmarin, et al. (1997)
        3. Kolomin T., et al (2013)

        Speaker: Dr Marianna Flora Tomasello (CNR - Istituto di Cristallografia)
        Abstract_Semax_conf_istituto_IC.docx
    • 10:20 → 11:00
      Coffe break 40m
    • 11:00 → 13:00
      Session: 2
      Convener: Dr Cinzia Giannini (CNR - Istituto di Cristallografia)
      • 11:00
        In vitro and in vivo approaches to the study of Alzheimer’s disease 15m

        Alzheimer’s disease (AD) is a progressive brain disorder that slowly leads to memory loss and cognitive decline. It is considered the most common cause of dementia in the elderly population, with an incidence and related cost of medical care that are expected to increase in the next future. However, none of the pharmacologic treatments available today for AD dementia is able to stop the neurodegeneration that causes the common symptoms of the disease.
        This lack of effective cure has led to the need of new biomarkers to be used in early diagnosis and new molecular target to address interventions.
        We have recently focused our work on the study of HSP60 as a new potential target for AD. By the use of in vitro and in vivo techniques we found that AD conditions affect the expression level and localization of the protein, in primary neuronal cultures. We also observed that decreased level of HSP60 in neuronal cells, positively correlate with IGF-1Rs expression, which are known to be downregulated in AD post-mortem brains (Talbot et al., 2012).
        According to recent data of the literature, we investigated the modulation of HSP60 in an in vitro model of insulin resistance, in order to better understand its involvement in the downregulation of IGF1R observed in AD brain and in the other molecular mechanisms shared by Alzheimer’s and Type II diabetes (De La Monte et al 2012; Giuffrida et al., 2012).
        Starting from these new data and in the light of our previous results, the main point of this presentation, will be the relevance of IGF-1 receptor and its downstream effectors in AD progression, with particular focus on the experimental techniques commonly used by our group to study Alzheimer’s disease.

        References:

        1. Talbot K., Wang HY, J Clin Invest. 2012, 4, 1316-38.
        2. De La Monte SM., Front Biosci (Elite Ed), 2012, 4, 1582-1605.
        3. Giuffrida ML., Mol Neurobiol, 2012, 46, 605-13.

        Acknowledgements:

        We acknowledge financial support from MIUR, grant SIR RBS1148TJD to M.L. Giuffrida. S.Zimbone is supported by a SIR project post-doctoral contract

        Speaker: Dr Maria Laura Giuffrida (CNR - Istituto di Cristallografia)
        10-Abstract-Giuffrida_ML.docx
      • 11:15
        New insight into the physiological activities of Amyloid Beta monomers 15m

        Alzheimer’s disease (AD) is one of the most common form of dementia in the elderly, characterized by a progressive neurodegeneration associated with synaptic dysfunction, pathological accumulation of ß-amyloid (Aß) in plaques, and neuronal loss. The self-association of Aß monomers into soluble oligomers seems to be crucial for the development of neurotoxicity (Walsh and Selkoe, 2007).
        Some of the toxic effects of Aß are mediated by its adverse effect on neurotrophic factor expressions. In particular, Aβ oligomers have been found to decrease both phosphorylated CREB and BDNF mRNA in the neuroblastoma cell line, SH-SY5Y, suggesting that oligomeric Aβ could compromise neuronal functions in AD by downregulating BDNF (Garzon and Fahnestock, 2007). Accordingly, phosphorylated CREB and CREB-regulated BDNF are recently shown to be reduced in the brain of AD patients and Tg2576 mice (Pugazhenthi, 2011)
        We previously reported a neuroprotective activity of monomeric Aß involving the activation of a PI3K/Akt survival pathway (Giuffrida et al., 2009). Here we demonstrate that Aß monomers are specifically able to activate CREB, a converging point for mechanisms and pathways involved in memory formation (Teich et al., 2015). Our data suggest a new model whereby Aß monomers may preserve cognitive decline.

        References:

        1. Walsh DM, Selkoe DJ., J. Neurochem., 2007, 101, 1172–1184.
        2. Garzon DJ, Fahnestock M. J., Neurosci., 2007, 27, 2628-2635.
        3. Pugazhenthi S., et al., Mol. Neurodegener. 2011, 6,60.
        4. Giuffrida ML., Caraci F. et al., J. Neurosci. 2009, 29, 10582-10587.
        5. Teich AF., Nicholls RE. et al., Neurotherapeutics, 2015, 12, 29-41.

        Acknowledgements:

        We acknowledge financial support from MIUR, grant SIR RBS1148TJD to M.L. Giuffrida. S.Zimbone is supported by a SIR project post-doctoral contract.

        Speaker: Dr Stefania Zimbone (CNR - Istituto di Cristallografia)
        10-Abstract-Zimbone_Stefania.docx
      • 11:30
        CNR Hard X-ray diffraction activities at Elettra 2.0 15m

        Elettra, the Italian third generation synchrotron source, has been in operation since 1994 and is currently planning a major upgrade of both the storage ring and the beamlines in the framework of a project named Elettra 2.0. A new machine is foresaw, which the current beam parameters1 are very much improved, and planned to be completed in 2026, making Elettra competitive with the most up-to-date European synchrotron radiation facilities. While the new machine will be still operating at 2-0-2.4 GeV and with an unchanged ring diameter, the brilliance will be greatly increased, with a substantial gain for the entire Elettra users’ community. Indeed, such an improvement in machine performance will be significant also for all the X-ray diffraction beamlines2 currently operating at Elettra.
        CNR has been contributing to the scientific growth and technical development of the Elettra research infrastructure as the main institutional partner in the last 24 years. CNR staff and associates have been involved in a variety of different experimental techniques: from X-ray macromolecular crystallography to VUV photoemissions. The CNR community has been essential in supporting the Elettra users’ activities and extremely active in proposing novel experiments. In view of Elettra 2.0, the CNR community working at the Elettra beamlines have been proposing its strategic view of the Elettra evolution, which has been shared with the Elettra management and the entire scientific community.
        The research group of the Istituto di Cristallografia operating in Trieste has been involved in the development of the XRD1 crystallographic beamline since the very beginning of Elettra activities. XRD1 is a multipurpose diffraction facility that has been used for macromolecular and small-molecule crystallography as well as for grazing-incidence X-ray diffraction and X-ray scattering experiments. While XRD1 has been for a long time the one and only hard X-ray diffraction beamline at the facility, owing to a strong request from the respective users’ community, a number of more specialized X-ray diffraction beamlines have been recently established. With Elettra 2.0, the performances of the X-ray diffraction beamlines will be further strengthened making them more competitive to those already operational at various radiation sources. In view of the improved storage ring, we have been involved in the re-thinking of the X-ray diffraction beamlines at Elettra, especially the ones on which are focused the research activities of the CNR-IC group: XRD1 but also the macromolecular crystallography beamline currently denominated XRD2, developed in partnership with the IIS

        Speaker: Dr Alberto Cassetta (CNR - Istituto di Cristallografia)
        XRD1_Cassetta.docx
      • 11:45
        Algae-Based Biosensors 15m

        Biosensors are interesting devices arising from a synergistic combination of well-established scientific knowledge and cutting-edge technologies, including nanotechnology, biotechnology, and materials science. This cross-disciplinary approach actively contributes to the customization of algae-based biosensors with improved analytical performance (e.g. sensitivity, reproducibility, and fast response) in environmental and agrifood applications1-4. The main advantages in using whole algae cells and their sub-components as biorecognition elements relies on their adaptability to diverse environments, easy immobilizations procedures, and sensitivity towards specific target analytes. In addition, algae and their sub-components can be integrated into both optical and electrochemical transduction, allowing analysis in complex matrices with different turbidity and analytes concentrations. Moreover, extensive sample pre-treatments are not required, unless filtration or concentration to enhance the sensitivity.
        In this scenario, we present examples of biosensors designed exploiting Chlamydomonas reinhardtii algae, in combination with advanced sustainable materials (e.g. paper) and nanomaterials (e.g. carbon black), for the detection of diverse target analytes (e.g. photosynthetic herbicides and chemical weapons). An optical eco-designed paper-based device was developed for nanoencapsulated-atrazine detection, where algae were immobilised on a paper substrate soaked with agar thin film. This biosensor provided detection limit in the picomolar range in tap water, intriguing results on interference, recovery and stability studies5. Then, an amperometric biosensor combining nanomaterials with algae was realised for the detection of photosynthetic herbicides in river water. C. reinhardtii cells were immobilised on carbon black modified screen-printed electrodes and atrazine was detected in the nanomolar range, with satisfactory results of working/storage stability, as well as in the presence of interferents6. The algae versatility in biosensor applications was further proved with an optical bioassay for the detection of chemical warfare agent simulants for application in security sector7. The bioassay was tested towards bis-2-chloroethyl amine and 2-chloroethyl ethyl sulphide in the millimolar range, demonstrating capability to discriminate chemical weapons from herbicides, if present in the same water sample. More recently, a dual opto-electrochemical biosensing platform was realised based on 28 strains of C. reinhardtii, immobilised on paper-based screen-printed electrodes nanomodified with carbon black, for the detection of atrazine, terbuthylazine, and diuron in tap and surface waters with detection limits in the picomolar ranges8. Pathogen detection was also accomplished by exploiting CC125 strain of C. reinhardtii based electrochemical biosensor associated to a dual electro-optical transduction prototype ad hoc designed for algal photosynthetic process. Escherichia coli was exploited as case study pathogen to assess the algae capability to sense their presence in wastewater. Indeed, aerobic bacteria can promote algal growth and thus oxygen evolution by reducing the photosynthetic oxygen tension within the microenvironment of the algal cells. E. coli was analysed in a concentration range from 100 to 2000 CFU / 100 mL, and an increase of the current signals and thus of the oxygen evolution of 10 % was registered in the presence of 1000 CFU / 100 mL and 25 % in the presence of 2000 CFU / 100 mL of pathogen concentration. A detection limit of 92 CFU/mL was achieved (LOD = 3×sd/slope)9. Moreover, an optical biosensor based on D1 bioinspired peptidomimetics functionalised with quantum dots nanoparticles was designed for the detection of atrazine, a case study herbicide widely exploited in agriculture and often found in WWT. This biosensor showed excellent sensitivity toward the atrazine target, with detection limits in the μg/L concentration range, meeting the requirements of E.U. legislation10.

        References
        1. Antonacci A, et al. Trends in Analytical Chemistry, 2018, 98, 95-103.
        2. Antonacci A, Scognamiglio V. Trends in Biotechnology. 2020, 38 (3), 334-347
        3. Antonacci A, Scognamiglio V. TrAC Trends in Analytical Chemistry, 2019, 115, 100-109.
        4. Bartolucci C., et al. TrAC Trends in Analytical Chemistry, 2019, in press.
        5. Scognamiglio V, et al. Journal of Hazardous Materials, 2019, 373, 483-492.
        6. Attaallah R., et al. Biosensors and Bioelectronics, 2020, 159, 112203.
        7. Antonacci A., et al. 2018, Sensors and Actuators B: Chemical, 257, 658-665.
        8. Antonacci, A., et al. 2021, Journal of Nanobiotechnology, 19(1), 1-13.
        9. Antonacci et al. 2021, Water, in press within the Special Issue: https://www.mdpi.com/journal/water/special_issues/treated_urban_wastewater?fbclid=IwAR24tZP2IckrkfD4kuTQgLH3zogsJ0gTPuPaZt6zkVvE8PhI04zzRYVaSj8
        10. Giardi, M. T., et al. 2021, Talanta, 224, 121854.

        Speaker: Dr Viviana Scognamiglio (CNR - Istituto di Cristallografia)
      • 12:00
        Cockayne syndrome: a DNA repair defective syndrome with neurodegenerative features 15m

        Cockayne syndrome (CS) is a rare genetic progeroid disorder characterized by growth and development defects, severe cutaneous photosensitivity, cachectic dwarfism, progressive neurological dysfunction, and precocious aging. CS is due to mutations in two genes, CSA and CSB. Both gene products are involved in transcription-coupled repair, subpathway of nucleotide excision repair (NER). Although CS-defective cells show hypersensitivity to UV light, impaired repair of bulky DNA lesions, delayed recovery of RNA synthesis after UV-damage, and enhanced apoptosis after transcription blockage (reviewed in 1), patients with CS do not present increased cancer risk as expected in DNA repair defective syndromes. Furthermore, the characteristics of CS patients are hardly attributable to the NER impairment only, since some of them do not occur in XP-A patients, which show a complete NER deficiency (2).
        A growing body of evidence indicate that, besides the typical NER lesions, CS cells are defective in the repair of a broad range of DNA damage that may account for the clinical symptoms of CS (reviewed in 3). For instance, it is well established that, upon oxidative stress, CS cells accumulate oxidatively induced DNA damage. In particular, CS cells are defective in the repair of 8-oxoguanine, 5-hydroxycytosine and cyclopurines (4). Moreover, also a correct repair of DNA breaks is of potential great relevance for the clinical features of CS patients (defective DNA break repair is involved in neurodegeneration and neurogenesis) (5). We have showed that CS proteins have a role in single and double strand break repair (6). Moreover, we and others have shown that CS cells present increased levels of intracellular reactive oxygen species, an intense glycolytic metabolism and mitochondria abnormalities with excessive mitochondrial fragmentation due to hyper-phosphorylation of the mitochondrial fission protein (DRP1) (7-10).
        Since the hyper-phosphorylation of DRP1, we wondered if DRP1 inhibition was capable to rescue the CS pathological phenotype. When enzymatic activity of DRP1 is inhibited by MDVI, a mitochondrial fission inhibitor, the high ROS levels are reduced, and the dysfunctional mitochondrial, apoptotic phenotype of CS cells is recovered. Moreover, CS cells present a significant reduction of nitric oxide levels and interestingly, CS cells are characterized by an overexpression of the S-nitrosoglutathione reductase, suggesting that nitrosative stress might play a role in CS. All these preliminary data indicate that the modulation of enzymatic activity of DRP1 is critical in CS cells, suggesting DRP1 as a potential therapeutic target as well as its inhibitors as potential therapeutic tools.

        References
        1. D’Errico M, Pascucci B, Iorio E, Van Houten B, Dogliotti E. Mech Ageing Dev. 2013, 134, 261-269.
        2. Kraemer KH, Patronas NJ, Schiffmann R, Brooks BP, Tamura D, DiGiovanna JJ. Neuroscience. 2007, 145, 1388-1396.
        3. Pascucci B, D’Errico M, Parlanti E, Giovannini S, Dogliotti E. Biochemistry (Mosc). 2011, 76, 4-15.
        4. D’Errico M, Parlanti E, Teson M, Degan P, Lemma T, Calcagnile A, Iavarone I, Jaruga P, Ropolo M, et al., Oncogene. 2007, 26, 4336-4343.
        5. Katyal S, McKinnon PJ. Mech Ageing Dev. 2008, 129, 483-491.
        6. Barbara Pascucci, Alessandra Fragale, Veronica Marabitti, Giuseppe Leuzzi, et al., Oncotarget. 2018, 9, 11581-11591.
        7. Pascucci B, Lemma T, Iorio E, Giovannini S, Vaz B, Iavarone I, Calcagnile A, Narciso L, Degan P, Podo F, et al. Aging Cell. 2012, 11, 520-529.
        8. Scheibye-Knudsen M, Ramamoorthy M, Sykora P, Maynard S, Lin PC, Minor RK, Wilson DM 3rd, et al. J Exp Med. 2012, 209, 855-869.
        9. Fang EF, Scheibye-Knudsen M, Brace LE, Kassahun H, SenGupta T, Nilsen H, Mitchell JR, Croteau DL, Bohr VA. Cell. 2014, 157, 882-896.
        10. Pascucci B, D’Errico M, Romagnoli A, DeNuccio C, Savino M, Pietraforte D, Lanzafame M, et al., Oncotarget. 2016, 8, 102852-102867.

        Speaker: Dr Barbara Pascucci (CNR - Istituto di Cristallografia)
        Abstract_Pascucci.docx
      • 12:15
        3D electron and X-ray powder diffraction multi-technique approach for structural investigation of new challenging lead chalcohalide nanocrystals 15m

        The promising, fascinating, and unique optoelectronic properties of lead-halide and lead-chalcogenide nanocrystals motivated, during the last decade, an always increasing scientific interest. The excellent performances of CsPbX3 (X = Cl,Br,I) and PbE (E=S,Se) as nanocrystalline semiconductors naturally raised the attention about the potential advantages of lead-chalcohalides, namely compounds within the Pb-E-X ternary diagram. However, this ternary diagram is mostly unexplored, with only two metastable materials being known through high-pressure and temperature solid-state synthesis [1,2].
        In the presented work, the versatility of colloidal chemistry gave us access to a whole series of Pb-S-X compounds. At the same time, it posed the challenge of elucidating the structure of a yet unknown material, namely Pb4S3Br2, that was obtained in the form of small nanocrystals. This challenge was tackled by a combination of techniques: 3D electron diffraction (3D-ED) [3], X-ray powder diffraction (XRPD), and 3D electron tomography (3D-ET) [4]. The extremely small size of crystallites called for a synergic combination of such techniques, which ensured a successful structure solution and proved to be a useful approach for overcoming the limits related to each individual technique. First, 3D-ED provided a plausible structural model by the application of Direct Methods with SIR2019 [5]. Then, 3D-ET confirmed the extracted cell parameters, and helped ruling out the presence of geminates or heterostructures. Finally, the reliability of the structural model was tested by an ab-initio structure solution process by XRPD that was performed through EXPO2014 [6]. Due to the broad diffraction peaks typical of nanocrystals, the indexing process by XRPD found many plausible candidates. At this stage, the prior information on the cell parameters provided by 3D-ED and 3D-ET was essential. In addition, the prior knowledge on the space group identified by 3D-ED (i.e., Pnma) compensated the failure of the space group determination by XRPD induced by the unavoidable errors on the integrated intensities. The structure determined by XRPD and refined by Rietveld method via Fullprof [7] was very similar to the one provided by 3D-ED: the similarity between the two structure models further validated the structural solution. The equivalence of the two structural models was further proved via DFT calculations (see Figure 1) that, applied to both, converged to the same crystal structure.
        The main crystallographic results are here presented.

        [1] D. Ni, S. Guo, Z.S. Yang, K.M. Powderly, R.J. Cava Solid State Sci. 2019, 91, 49.
        [2] D. Ni, S. Guo, Z.S. Yang, K.M. Powderly, R. Zhong, R.J. Cava J. Solid State Chem. 2019, 280, 120982.
        [3] M. Gemmi, E. Mugnaioli, T.E. Gorelik, U. Kolb, L. Palatinus, P. Boullay, S Hovmöller, J.P. Abrahams ACS Central Science 2019, 5, 1315.
        [4] S. Toso, Q. A. Akkerman, B. Martin-Garcia, M. Prato, J. Zito, I. Infante, Z. Dang, A. Moliterni, C. Giannini, E. Bladt, I. Lobato, J. Ramade, S. Bals, J. Buha, D. Spirito, E. Mugnaioli, M. Gemmi, L. Manna J. Am. Chem. Soc. 2020, 142, 10198.
        [5] M.C. Burla, R. Caliandro, B. Carrozzini, G.L. Cascarano, C. Cuocci, C. Giacovazzo, M. Mallamo, A. Mazzone, G. Polidori J. Appl. Cryst. 2015, 48, 306.
        [6] A. Altomare, C. Cuocci, C. Giacovazzo, A. Moliterni, R. Rizzi, N. Corriero, A. Falcicchio, J. Appl. Cryst. 2013, 46, 1231.
        [7] J. Rodriguez-Carvajal, J. Gonzalez-Platas, T. Roisnel, N. A. Katcho, C. Frontera, L.C. Chapon, O. Baltuano FullProf Suite 2020, https://www.ill.eu/sites/fullprof.

        Speaker: Dr Anna Moliterni (CNR - Istituto di Cristallografia)
      • 12:30
        Kinetic analysis for the new enzymology 15m

        Enzyme kinetics relies on a bulky corpus of knowledge which has been consolidated along with more than 100 years of biochemical research. The studies on sucrose hydrolysis by invertase made by Leonor Michaelis and Maud Menten led to the publication in 1913 of their homonymous equation, regarded as the fundamental equation of biochemistry. Since then, enzymologists have devised many different ways to measure and analyze kinetic data, and many different enzyme or inhibition mechanisms were identified.
        The Michaelis-Menten equation and the relative “steady state” model however provide only an oversimplified description of the real enzyme behavior. The recent development of computer software for the global fitting of enzymatic reaction progress curves by numerical integration represents a fundamental breakthrough in the century-old field of enzymology. The resolution of kinetic mechanisms by computer simulation will foster a shift in the paradigm from the “steady state” to “transient state”, simplifying the study of complex mechanisms and pushing toward a next level understanding of enzyme catalysis.
        Here a brief introduction will be given on this new method for the study of enzyme kinetic.

        Speaker: Dr Alessandro Pesaresi (CNR - Istituto di Cristallografia)
      • 12:45
        IC-CNR activities in Cultural Heritage 15m

        Italy holds a huge concentration of historical and cultural heritage. Just in Rome area are present 47 archaeological sites, 180 museums and galleries, 13 villas and palaces (www.beniculturali.it). This amount of artworks needs to be studied and preserved. For more than 6 years, in the Montelibretti IC-CNR secondary location, we are working together with archaeologists, art historians and restorers, providing them analysis results aimed at the chemical-physical characterization of artworks materials and helping them to evaluate their state of conservation.
        The priceless value of artworks requires to perform the analysis using non-invasive and non-destructive methods such as the ones based on X-Rays (ex. X Rays Fluorescence, Diffraction and Radiography) and the ones based on IR or visible radiation (ex. Reflectometry, Raman and Visible Spectroscopy). Moreover often it isn’t possible to move artworks from the places where they are displayed or conserved, therefore is essential having portable instrumentation in order to perform the analysis in situ.
        We present some of last results obtained on paintings from Corsini and Barberini Galleries, “Eretum Chariot”, soil blocks from Civic Archaeological Museum of Fara in Sabina (Ri).

        References
        1. O.Tarquini, L.Pronti, E.G.Lorenzetti, A. C. Felici J. Cult. Herit. 2020, 43, 294–302
        2. L. Pronti, M. Romani, G.Verona-Rinati, O. Tarquini, F. Colao, M. Colapietro, A.Pifferi, M. Cestelli-Guidi and M. Marinelli Herit. 2019, 2, 2275–2286
        3. V. Forte; O. Tarquini; M. Botticelli; L. Medeghini Archeometry 2020, 62, 4, 712-730

        Speaker: Dr Ombretta Tarquini (CNR - Istituto di Cristallografia)
    • 13:00 → 13:15
      Concluding remarks
Powered by Indico v2.3.4