Budget impact analysis of universal rotavirus vaccination in the Local Health Unit 11 Empoli, Tuscany, Italy

Background. Rotavirus (RV) infection is the first cause of acute viral gastroenteritis in children under five years of age all over the world; it mainly affects children between six and 24 months of age and can cause serious acute diarrhoea and dehydration. The aim of this study is to perform the budget impact analysis of universal rotavirus vaccination in the Local Health Unit (LHU) 11 Empoli, Tuscany, Italy.Methods. An ad hoc mathematical simulation model was developed to evaluate the budget impact analysis of 5-years universal rotavirus vaccination. Particularly, incidence of rotavirus gastroenteritis (RVGE), hospitalizations, nosocomial diarrhoea, medical consultations, prescriptions and accesses to emergency department were taken into account in the analysis. The direct medical costs due to RV diarrhoea and the costs of vaccination campaign were considered as the main outcome measures in the study.Results. The adoption of universal rotavirus vaccination campaign for five years in the LHU 11 Empoli results in relevant savings due to the health cares avoided. These savings would overlapped the costs of vaccination yet from the second year after the introduction of vaccination. The saving for the Health Service would be 1.5 million Euro after five years of campaign.Conclusions. Universal vaccination against rotavirus results clinically and economically favourable for both the Health Service and the Society perspectives

Citron Kinase Deficiency Leads to Chromosomal Instability and TP53-Sensitive Microcephaly

Mutations in citron (CIT), leading to loss or inactivation of the citron kinase protein (CITK), cause primary microcephaly in humans and rodents, associated with cytokinesis failure and apoptosis in neural progenitors. We show that CITK loss induces DNA damage accumulation and chromosomal instability in both mammals and Drosophila. CITK-deficient cells display "spontaneous" DNA damage, increased sensitivity to ionizing radiation, and defective recovery from radiation-induced DNA lesions. In CITK-deficient cells, DNA double-strand breaks increase independently of cytokinesis failure. Recruitment of RAD51 to DNA damage foci is compromised by CITK loss, and CITK physically interacts with RAD51, suggesting an involvement of CITK in homologous recombination. Consistent with this scenario, in doubly CitK and Trp53 mutant mice, neural progenitor cell death is dramatically reduced; moreover, clinical and neuroanatomical phenotypes are remarkably improved. Our results underscore a crucial role of CIT in the maintenance of genomic integrity during brain development

ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules.

Correct orientation of cell division is considered an important factor for the achievement of normal brain size, as mutations in genes that affect this process are among the leading causes of microcephaly. Abnormal spindle orientation is associated with reduction of the neuronal progenitor symmetric divisions, premature cell cycle exit, and reduced neurogenesis. This mechanism has been involved in microcephaly resulting from mutation of ASPM, the most frequently affected gene in autosomal recessive human primary microcephaly (MCPH), but it is presently unknown how ASPM regulates spindle orientation. In this report, we show that ASPM may control spindle positioning by interacting with citron kinase (CITK), a protein whose loss is also responsible for severe microcephaly in mammals. We show that the absence of CITK leads to abnormal spindle orientation in mammals and insects. In mouse cortical development, this phenotype correlates with increased production of basal progenitors. ASPM is required to recruit CITK at the spindle, and CITK overexpression rescues ASPM phenotype. ASPM and CITK affect the organization of astral microtubules (MT), and low doses of MT-stabilizing drug revert the spindle orientation phenotype produced by their knockdown. Finally, CITK regulates both astral-MT nucleation and stability. Our results provide a functional link between two established microcephaly proteins

Citron Kinase Deficiency Leads to Chromosomal Instability and TP53-Sensitive Microcephaly

Mutations in citron (CIT), leading to loss or inactivation of the citron kinase protein (CITK), cause primary microcephaly in humans and rodents, associated with cytokinesis failure and apoptosis in neural progenitors. We show that CITK loss induces DNA damage accumulation and chromosomal instability in both mammals and Drosophila. CITK-deficient cells display “spontaneous” DNA damage, increased sensitivity to ionizing radiation, and defective recovery from radiation-induced DNA lesions. In CITK-deficient cells, DNA double-strand breaks increase independently of cytokinesis failure. Recruitment of RAD51 to DNA damage foci is compromised by CITK loss, and CITK physically interacts with RAD51, suggesting an involvement of CITK in homologous recombination. Consistent with this scenario, in doubly CitK and Trp53 mutant mice, neural progenitor cell death is dramatically reduced; moreover, clinical and neuroanatomical phenotypes are remarkably improved. Our results underscore a crucial role of CIT in the maintenance of genomic integrity during brain development

Mg2+-dependent conformational equilibria in CorA and an integrated view on transport regulation

The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg2+ channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR). We find that neither the Mg2+-bound closed structure nor the Mg2+-free open forms are sufficient to explain the average conformation of CorA. Our data support the presence of conformational equilibria between multiple states, and we further find a variation in the behaviour of the backbone dynamics with and without Mg2+. We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg2+ but that conducting states become more populated in Mg2+-free conditions. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA.Peer reviewe

Dynamique de Protéines par RMN avec Rotation Rapide à l’Angle Magique

The aim of my thesis is to develop Magic-Angle Spinning Nuclear Magnetic Resonance (MAS NMR) to characterize structure and dynamics in complex biological samples, with a particular focus on the role of metal ions in enzymes and channels.MAS NMR is a powerful technique that allows to extract atomic level information, characterize broad timescales of motions, and investigate functional states in native-like sample conditions, a particularly important requirement e.g. for transmembrane proteins in lipid bilayers. Nonetheless, a number of bottlenecks prevents its widespread application in structural biology.In my work I developed and applied tailored techniques based on high magnetic fields (800 MHz and 1 GHz 1H Lamor frequency) and MAS probes with sub-mm diameter rotors spinning at rates above 100 kHz, which contributed to push forward the capability of this technique: i) by enlarging the molecular size of the systems that can be investigated with site specificity; ii) by reducing the requirements in terms of isotopic labeling, notably deuteration; iii) by speeding up the tedious processes of resonance assignment and acquisition of dynamical parameters; iv) by enriching the palette of measurable parameters connected to dynamics.All along this thesis, the methods were benchmarked on microcrystalline samples of the model domain GB1, and applied to Cu,Zn-superoxide dismutase (a dimeric 2x16 kDa Cu metalloenzyme) in functional microcrystalline form, as well as to two transmembrane channels reconstituted in lipid bilayers, bacterial CorA (a pentameric 5x40 kDa cation channel) and human Aqp-1 (tetrameric 4x25 kDa aquaporin-1 water channel). The data obtained shed new light on the relation between internal dynamics and function.L'objectif de cette thèse est de développer la Résonance Magnetique Nucléaire en Rotation à l'Angle Magique (MAS NMR) pour caractériser la structure et la dynamique d' échantillons biologiques complexes, avec un accent particulier sur le rôle des ions métalliques dans les enzymes et les canaux transmembranaires. MAS NMR est une technique performante qui permet d'extraire des informations au niveau atomique, de caractériser la dynamique sur de larges échelles de temps, et d'étudier les états fonctionnels dans des échantillons en conditions natives, une exigence particulièrement importante pour les protéines transmembranaires dans les bicouches lipidiques, par exemple. Néanmoins, un certain nombre de difficultés empêchent son application généralisée en biologie structurelle.Dans mon travail, j'ai développé et appliqué des techniques basées sur des champs magnétiques élevés (fréquence de Larmor du 1H 800 MHz et 1 GHz ) et des sondes MAS avec des rotors de diamètre inférieur au millimètre, tournant à des vitesses supérieures à 100 kHz, ce qui a contribué à faire progresser les capacités de cette technique : i) en augmentant la taille moléculaire des systèmes qui peuvent être étudiés avec un détail du niveau du résidu ; ii) en réduisant les exigences en termes de marquage isotopique, notamment la deutération ; iii) en accélérant les processus laborieux d'attribution de résonance et d'acquisition de paramètres dynamiques ; iv) en enrichissant la palette des paramètres mesurables liés à la dynamique. Tout au long de cette thèse, les méthodes ont été évaluées sur des échantillons microcristallins du domaine modèle GB1, et appliquées à la Cu,Zn-superoxyde dismutase (une métalloenzyme dimérique de Cu de 2x16 kDa) sous forme microcristalline et fonctionnelle, ainsi qu'à deux canaux transmembranaires reconstitués en bicouches lipidiques, CorA (un canal cationique pentamérique bactérien de 5x40 kDa) et l'Aqp-1 humaine (aquaporin-1, canal d'eau tétramérique de 4x25 kDa). Les résultats obtenus éclaircissent d'avantage la relation entre la dynamique interne et la fonction des protéines

Solid State NMR study of the interactions between a synthetic antimicrobial peptide and model cell membranes

This thesis work concerns the study of the interactions between the synthetic antimicrobial peptide (YI13C)2 and model cell membranes by Solid State Nuclear Magnetic Resonance (SSNMR) techniques. The strategy used for the study involved the preparation of POPC ( 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) model cell membranes and the application of a multi-nuclear and multi-technique solid state NMR approach to study the interactions between the components of the system at a molecular level. Both non aligned and mechanically aligned samples were prepared and studied. The observation of the nuclei 31P, 2H, 1H and the measurement of different spectral properties gave structural and dynamic information on both the polar region of the bilayers and the water contained in the phase. From the study of non aligned samples a perturbative effect of the peptide on the bilayer structure and on the dynamic properties of water molecules was shown. The dependence of the perturbation on the concentration of the peptide and on the hydration of the membranes was revealed. The exploitation of mechanical alignment gave further information on the perturbation mechanism. From a compared analysis of all the experimental results some hints on the model of action of the peptide in the system studied were obtained. In addition, the properties of differently purified samples of LPS (lipopolysaccharide) in aqueous environment were preliminarily studied by 31P SSNMR

Protein structural dynamics by Magic-Angle Spinning NMR

International audienceMagic-Angle Spinning (MAS) Nuclear Magnetic Resonance (NMR) is a fastdeveloping technique, capable of complementing solution NMR, X-ray crystallography and electron microscopy for the biophysical characterization of microcrystalline, poorly crystalline or disordered protein samples, such as enzymes, biomolecular assemblies, membraneembedded systems or fibrils. Beyond structures, MAS NMR is an ideal tool for investigation of dynamics, since it is unique in its ability to distinguish static and dynamic disorder, and to characterize not only amplitudes, but also timescales of motion. Building on seminal work on model proteins, the technique is now ripe for widespread application in structural biology. This review briefly summarizes the recent evolutions in biomolecular MAS NMR and accounts for the growing number of systems where this spectroscopy has provided a description of conformational dynamics over the very last few years