Identification and Functional Assessment of Novel Neuromuscular Disease-Causing Genes

Inherited neuromuscular diseases comprise a highly heterogeneous group of disorders characterized by the impairment of the neural structures or motor unit components responsible for the generation of movement. While as single gene-associated disorder the majority of them are rare, taken together their estimated prevalence reaches 1 – 3 cases / 1000 individuals. Due to their elevated morbidity and mortality, they represent a significant health burden for the affected individuals, their families, and the healthcare systems. Moreover, their clinical and genetic heterogeneity makes their diagnosis a long and complex process, which often requires specialized diagnostic procedures and poses a challenge in about half of the cases. However, thanks to decreasing costs and increased availability of next-generation sequencing technologies, the last years had witnessed a rise in the number of novel genes associated to neuromuscular disorders. In this study, we identified three novel neuromuscular disease-causing genes: PIEZO2, whose biallelic loss-of-function mutations cause distal arthrogryposis with impaired proprioception and touch; VAMP1, whose biallelic loss-of-function mutations cause a novel presynaptic congenital myasthenic syndrome; CAPRIN1, whose specific p.Pro512Leu mutation causes a neurodegenerative disorder characterized by ataxia and muscle weakness. For PIEZO2, we identified biallelic loss-of-function mutations using exome sequencing, SNPchip-based linkage analysis, DNA microarray, and Sanger sequencing in ten affected individuals of four independent families showing arthrogryposis, hypotonia, respiratory insufficiency at birth, scoliosis, and delayed motor development. This phenotype is clearly distinct from distal arthrogryposis with ocular anomalies which characterize the autosomal dominant distal arthrogryposis 3 (DA3), distal arthrogryposis 5 (DA5), and Marden-Walker syndrome (MWKS). While these disorders are caused by heterozygous gain-of-function mutations in PIEZO2, the novel reported mutations result in the loss of PIEZO2, since they lead to nonsense-mediated mRNA decay in patient-derived fibroblast cell lines. PIEZO2 is a mechanosensitive ion channel playing a major role in light-touch sensation and proprioception. Mice ubiquitously depleted of PIEZO2 die postnatally because of respiratory distress, while individuals lacking PIEZO2 develop a neuromuscular disorder, likely due to the loss of proprioception inputs in muscles. For VAMP1, we identified biallelic loss-of-function mutations using exome or genome sequencing in two pairs of siblings from two independent families affected by a novel congenital myasthenic syndrome. Electrodiagnostic examination showed severely low compound muscle action potentials and presynaptic impairment. The two described homozygous mutations are a frameshift and a missense mutation of a highly conserved residue, therefore are likely to result in the loss of VAMP1 function. Indeed, the phenotype is resembled by VAMP1lew/lew mice, which carry a homozygous VAMP1 truncating mutation and show neurophysiological features of presynaptic impairment. For CAPRIN1, we identified the identical de novo c.1535C>T (p.Pro512Leu) missense variant using trio exome sequencing in two unrelated individuals displaying early-onset ataxia, dysarthria, cognitive decline and muscle weakness. This mutation causes the substitution of a highly conserved residue and in silico tools predict an increase in the protein aggregation propensity. Overexpression of CAPRIN1-P512L caused the formation of insoluble ubiquitinated aggregates, sequestrating proteins associated with neurodegenerative disorders, such as ATXN2, GEMIN5, SNRNP200, and SNCA. Upon differentiation in cortical neurons of induced pluripotent stem cell (iPSC) lines where the CAPRIN1-P512L was introduced via CRISPR/Cas9, reduced neuronal activity and altered stress granules dynamics were observed in the lines harboring the mutation. Moreover, nano-differential scanning fluorimetry revealed that CAPRIN1-P512L adopts an extended conformation, and fluorescence microscopy demonstrated that RNA greatly enhances its aggregation in vitro. Taken together, this study associates: (1) biallelic loss-of-function mutations in PIEZO2 with the autosomal recessive distal arthrogryposis with impaired proprioception and touch; (2) biallelic loss-of-function mutations in VAMP1 with an autosomal recessive presynaptic congenital myasthenic syndrome; (3) a recurrent de novo p.Pro512Leu mutation of CAPRIN1 with a neurodegenerative disorder characterized by ataxia and muscle weakness

Control of the chemiluminescence spectrum with porous Bragg mirrors

Tunable, battery free light emission is demonstrated in a solid state device that is compatible with lab on a chip technology and easily fabricated via solution processing techniques. A porous one dimensional (1D) photonic crystal (also called Bragg stack or mirror) is infiltrated by chemiluminescence rubrene-based reagents. The Bragg mirror has been designed to have the photonic band gap overlapping with the emission spectrum of rubrene. The chemiluminescence reaction occurs in the intrapores of the photonic crystal and the emission spectrum of the dye is modulated according to the photonic band gap position. This is a compact, powerless emitting source that can be exploited in disposable photonic chip for sensing and point of care applications.Comment: 8 pages, 3 figure

Automated Monitoring of Soil Respiration: an Improved Automatic Chamber System

We designed, constructed and tested an automated chamber system for continuously monitoring soil respiration. Our objective was to design a system that would permit monitoring of CO2 efflux rates over long time periods without altering microclimate inside the chamber. The measuring principle is based on the measurement of the increase in CO2 concentration within an automated chamber in a fixed amount of time using a non linear regression method. The chamber operates by closing over the soil in response to a control signal and remains closed for a fixed amount of time. In this way, the chamber allows normal drying and wetting of the soil between measurements. We report results that show the reliability of soil CO2 efflux measurements in comparison with Li-Cor 6400. The system holds great potential for long term continuous measurements campaigns in natural environments

Temporal evolution of carbon stocks, fluxes and carbon balance in pedunculate oak chronosequence under close-to-nature forest management

Under current environmental changes, forest management is challenged to foster contrasting benefits from forests, such as continuous wood supply while preserving biomass production, biodiversity conservation, and contribution to climate change mitigation through atmospheric carbon sequestration. Although being found as globally important, estimates of long-term forest C balance are still highly uncertain. In this context, the chronosequence experiments (space-for-time substitution) might fill this gap in even-aged forests, as they represent an approach that enables the assessment of forest net C balance in the long term. In this research, we explored the dynamics of C stocks and fluxes in different forest pools throughout the rotation period (140 years) of a Pedunculate oak (Quercus robur L.) forest in Croatia. For this purpose, we selected a chronosequence that was made up of seven forest stands with different age (5, 13, 38, 53, 68, 108, and 138 years). To address the issues of uncertainty in C balance estimates, we compared net ecosystem carbon balance (NECB) estimated while using two different approaches, which we name pool-change (from C stocks) approach and component-flux (from C fluxes) approach. Overall, the pool-change approach showed higher NECB estimate, with the greatest difference being observed in younger stands (<50 years). Component-flux approach showed significantly higher uncertainty. Throughout the rotation period, managed pedunculate oak stands become a C sink early in their development phase, between the age of 13 and 35 years according to pool-change and component-flux approach, respectively. During the 140 years, oak forest provided 187.2 Mg C ha−1 (604 m3 ha−1) through thinnings and 147.9 Mg C ha−1 (477 m3 ha−1) in the final cut, while preserving, on average, 88.9 Mg C ha−1 in mineral soil down to 40 cm, 18.2 Mg C ha−1 in dead wood, and 6.0 Mg C ha−1 in the forest floor. Soil C stocks in our chronosequence did not show any age-related trend, indicating that current management practice has no negative effect on soil C stocks. Finally, under current close-to-nature forest management, Pedunculate oak forest showed to be sustainable in providing both economic and ecological ecosystem services

Sintesi di complessi poliossometallati ibridi e multifunzionali

Polyoxometalates (POMs) are discrete polyanionic oxides that shows a wide variety of applications in material science, nanomedicine and catalysis. This thesis describes the synthesis and characterization of a small library of novel polyoxometalate-based hybrids, and includes the preliminary investigations about their possible applications. The preparation of the complexes was obtained through the bis-functionalization of the divacant Keggin polyanion [γ-SiW10O36]8-, which was selected as a model compound for its reproducible synthesis and stability. The decoration of this POM was obtained in one or two steps, and involved the reaction of trialkoxy organosilanes or organo phosphonic acids with the reactive oxygen atoms of the POM vacancy. The POM was thus decorated with the following pendants: -luminescent units (dansyl, pyrene, fluoresceine), to obtain POMs with sensing capabilities in the presence of ions (Cu2+) or apolar molecules (C60), as well as with promising applications in fluorescence imaging, for cell trafficking studies. -bioactive molecules (avidin, folic acid, tryptophan), to be used for the recognition of small molecules or biological targets, with potential application in oxidation catalysis and medicine. In all cases, the successful functionalization was confirmed by means of proton, carbon and heteronuclear NMR (P, Si, W), and ESI-MS and FTIR. The spectral features of the molecular hybrids, and the interactions between the POMs and target molecules/ions were highlighted by fluorescence and UV-Vis spectroscopy. Chiral derivative were also studied by circular dichroism.openTesi 3797Tesi non accessibile sino al 04/04/2014 per motivi correlati alla proprieta' intellettuale

Effect of chronic alcohol ingestion on the progression of periodontitis induced in Fisher-344 rats

Objective: Understand the effect of chronic alcohol on the progression of periodontitis induced in Fischer-344 rats.Methods: For the study, 22 Fischer-344 rats, two months old were used, divided into groups: alcohol (n=8), ligature (n=7) and control (n=7). On the first day, the animals in the alcohol group were exposed to ingestion of a water solution containing 20% alcohol (size/size), up to day 90. After thirty days from the beginning of the experiment, the animals in the alcohol group and the ligature group were submitted to the placement of a silk thread around the right maxillary second molar. Nothing was performed on the left side, serving as control. All the groups were submitted to euthanasia 60 days after ligature placement. To assess the destruction of periodontitis, a radiographic exam was used to measure the destruction of bone height. Results: The results of the study showed that on the side in which periodontitis was induced, the group that ingested alcohol suffered an increase in destruction, with statistical differences when compared with the ligature and control groups and increased bone destruction in the ligature group when compared to control. Conclusion: Within the limitations of the study, it was concluded that chronic alcohol consumption by Fischer-344 rats led to greater progression of induced periodontitis

Teaching with emerging experience: to develop leadership and innovation skills

This paper is based on a hands-on session proposed at ALE 2009 Conference (Active Learning for Engineering Education). It presents a teaching practice used at Ecole Centrale Paris (ECP) to foster engineering students’ leadership and innovation skills and to help them going through major paradigm shifts. Considering a class as a human complex system, teachers work in a posture of facilitators or coaches who orchestrate a learning process. Instead of transmitting knowledge as traditional professors, they focus on the relationships in the classroom and the experience emerging from them.Peer Reviewe

Biocompatibility of different intracanal medications in rat bucal submucosa tissue

The aim of this study was to analyze the buccal tissue responses of Wistar rats to 2% chlorhexidine solution, calcium hydroxide and the association of both products. For this purpose, 30 specimens were randomly implanted in the filtrum of the four upper and lower hemiarches with a polyethylene tube containing one of the following substances: 2% chlorhexidine solution, calcium hydroxide and 2% chlorhexidine solution (test groups); calcium hydroxide and distilled water and distilled water (control groups). Ten rats each were distributed according to time interval of evaluation at 7, 15 and 30 days. The histological sections were stained with Harris hematoxylin and eosin. Analysis was performed with an optical microscope at x100, x200 and x400 magnifications by an expert examiner blinded to the materials. The sections were classified by scores attributed to inflammatory events and by a ranking determined according to the severity of the inflammation. The results of the inflammatory events and severity ranking were submitted to the Kruskal-Wallis test at a 0.05 level of significance. No statistically significant difference occurred among the tested materials; however, all materials showed a decreased of severity with respect to longer time intervals

Molecular dynamics simulations provide insights into the substrate specificity of FAOX family members

Enzymatic assays based on Fructosyl Amino Acid Oxidases (FAOX) represent a potential, rapid and economical strategy to measure glycated hemoglobin (HbA1c), which is in turn a reliable method to monitor the insurgence and the development of diabetes mellitus. However, the engineering of naturally occurring FAOX to specifically recognize fructosyl-valine (the glycated N-terminal residue of HbA1c) has been hindered by the paucity of information on the tridimensional structures and catalytic residues of the different FAOX that exist in nature, and in general on the molecular mechanisms that regulate specificity in this class of enzymes. In this study, we use molecular dynamics simulations and advanced modeling techniques to investigate five different relevant wild-type FAOX (Amadoriase I, Amadoriase II, PnFPOX, FPOX-E and N1-1-FAOD) in order to elucidate the molecular mechanisms that drive their specificity towards polar and nonpolar substrates. Specifically, we compare these five different FAOX in terms of overall folding, ligand entry tunnels, ligand binding residues and ligand binding energies. Our work will contribute to future enzyme structure modifications aimed at the rational design of novel biosensors for the monitoring of blood glucose levels