Genetic diagnosis as a tool for personalized treatment of Duchenne muscular dystrophy

Accurate definition of genetic mutations causing Duchenne muscular dystrophy (DMD) has always been relevant in order to provide genetic counseling to patients and families, and helps to establish the prognosis in the case where the distinction between Duchenne, Becker, or intermediate muscular dystrophy is not obvious. As molecular treatments aimed at dystrophin restoration in DMD are increasingly available as commercialized drugs or within clinical trials, genetic diagnosis has become an indispensable tool in order to determine eligibility for these treatments. DMD patients in which multiplex ligation-dependent probe amplification (MLPA) or similar techniques show a deletion suitable to exon skipping of exons 44, 45, 51, or 53, may be currently treated with AONs targeting these exons, in the context of clinical trials, or, as is the case for exon 51 skipping in the United States, with the first commercialized drug (eteplirsen). Patients who test negative at MLPA, but in whom DMD gene sequencing shows a nonsense mutation, may be amenable for treatment with stop codon readthrough compounds such as ataluren. Novel molecular approaches such as CRISPR-Cas9 targeting of specific DMD mutations are still in the preclinical stages, but appear promising. In conclusion, an accurate genetic diagnosis represents the entrance into a new scenario of personalized medicine in DMD

Molecular bases of phenotypic and clinical variability in Duchenne and Becker muscular dystrophy

Le distrofinopatie sono un gruppo di malattie legate al cromosoma X in modalit\ue0 recessiva, dovute a mutazioni nel gene DMD. La forma pi\uf9 severa, la distrofia muscolare di Duchenne (DMD), \ue8 causata da mutazioni troncanti, che provocano una completa assenza della proteina distrofina nel muscolo scheletrico e cardiaco. Al contrario, la forma allelica pi\uf9 mite, nota come distrofia muscolare di Becker (BMD), \ue8 causata da mutazioni che rispettano la cornice di lettura del gene, dando origine a distrofina quantitativamente e/o qualitativamente alterata. La DMD \ue8 una malattia devastante, caratterizzata da una progressiva degenerazione del tessuto muscolare, con deficit di forza e disabilit\ue0 sin dall'infanzia. La storia naturale \ue8 caratterizzata da perdita della deambulazione autonoma attorno all'et\ue0 di 10 - 15 anni, e da ridotta aspettativa di vita a causa di complicanze respiratorie e cardiache nei giovani adulti. I glucocorticoidi (GC) possono ritardare la progressione della malattia, ma una cura definitiva non \ue8 ancora disponibile. Fra i trattamenti molecolari innovativi pi\uf9 promettenti, annoveriamo gli oligonucleotidi antisenso (AON) che inducono l\u2019\u201cexon skipping\u201d di specifici esoni nelle mutazioni \u201cout-of-frame\u201d, e i composti che inducono il \u201creadthrough\u201d dei codoni di stop prematuri. Vi \ue8 una rilevante variabilit\ue0 nella severit\ue0 e velocit\ue0 di progressione del deficit di forza e della degenerazione del tessuto muscolare nella DMD, che non si spiega, se non in piccola parte, in base alle diverse mutazioni patogenetiche, dal momento che tutti i pazienti presentano una completa o quasi completa assenza di distrofina. Recentemente, il nostro gruppo e altri autori hanno descritto modificatori genetici della DMD, cio\ue8 polimorfismi di singolo nucleotide (SNP) associati a espressivit\ue0 pi\uf9 o meno severa del fenotipo DMD: lo SNP rs28357094 nel promotere del gene SPP1, codificante per la citochina osteopontina, e un aplotipo codificante nel gene LTBP4 (Transforming growth factor \u3b2-Binding Protein 4). Queste varianti modulano l\u2019espressione (SPP1) o alterano la sequenza aminoacidica (LTBP4) delle rispettive proteine, entrambe le quali sono coinvolte in vie di segnale pro-infiammatorie e pro-fibrotiche. Questi geni sono stati identificati come modificatori candidati con approcci diversi (rispettivamente, studi di profili di espressione e mappatura genomica di un modello murino). Il primo obiettivo di questa tesi \ue8 stato di ottenere una validazione indipendente dell\u2019associazione genetica dello SNP rs28357094 nel gene SPP1 e dell'aplotipo LTBP4 con l\u2019et\ue0 di perdita della deambulazione nella DMD. Questo risultato \ue8 stato conseguito utilizzando dati raccolti nel Duchenne Natural History Study del Cooperative International Neuromuscular Research Group (CINRG-DNHS), condotto su 340 pazienti DMD in 20 Centri in tutto il mondo. In questa coorte, l\u2019allele minore G dello SNP rs28357094 era associato a un prolungamento della deambulazione di 2 anni nei partecipanti al CINRG-DNHS che erano stati trattati con GC (p < 0.05), ma a nessun effetto nei partecipanti non trattati. Ci\uf2 suggerisce che questo SNP potrebbe essere un biomarcatore farmacodinamico di risposta ai GC. L'aplotipo omozigote \u201cIAAM\u201d di LTBP4, invece, era associato a un prolungamento della deambulazione di 2 anni nei partecipanti di origine europea (p < 0.05), ma non nell'intera coorte multietnica CINRG-DNHS, evidenziando la rilevanza della stratificazione di popolazione negli studi sui modificatori genetici. Il secondo obiettivo \ue8 stato rivolto alla identificazione di eventuali associazioni fra specifiche mutazioni DMD e perdita della deambulazione nel CINRG-DNHS. [\u2026

A Novel MAC Protocol for Low Datarate Cooperative Mobile Robot Teams

Cooperative mobile robot applications enable robots to perform tasks that are more complex than those that each single robot can perform alone. In this application context, communication networks play a very important role, as they have to cope with strict requirements (e.g., in terms of mobility, reliability, and bounded latencies). Recent cooperative robot applications foresee the support of low datarate communication technologies, that provide, among other benefits, lower energy consumption and easy integration with Wireless Sensor Networks (WSNs). Unfortunately, the state-of-the-art solutions either entail high costs and complexity or are not suitable for low data rate communications. Consequently, novel solutions for cooperating robots are required. For this reason, this paper presents RoboMAC, a new MAC protocol for mobile cooperating robots that enables the integration of robots with WSNs, supports mobility and real-time communications, and provides high scalability. The paper also presents a proof-of-concept implementation that proves the feasibility of the RoboMAC protocol on COTS devices

Priority-Based Bandwidth Management in Virtualized Software-Defined Networks

In Industrial Internet of Things (IoT) applications, when the network size increases and different types of flows share the bandwidth, the demand for flexible and efficient management of the communication network is compelling. In these scenarios, under varying workload and flow priorities, the combined use of Software-Defined Networking (SDN) and Network Virtualization (NV) is a promising solution, as such techniques allow to reduce the network management complexity. This work presents the PrioSDN Resource Manager (PrioSDN_RM), a resource management mechanism based on admission control for virtualized SDN-based networks. The proposed combination imposes bounds on the resource utilization for the virtual slices, which therefore share the network links, while maintaining isolation from each other. The presented approach exploits a priority-based runtime bandwidth distribution mechanism to dynamically react to load changes (e.g., due to alarms). The paper describes the design of the approach and provides experimental results obtained on a real testbed

Molecular bases of phenotypic and clinical variability in Duchenne and Becker muscular dystrophy

Dystrophinopathies are a group of X-linked recessive neuromuscular disorders due to mutations in the DMD gene. Truncating mutations, causing dystrophin absence in skeletal and cardiac muscle, cause the more severe form of dystrophinopathy, Duchenne muscular dystrophy (DMD). Conversely, mutations which respect the open reading frame, and give rise to quantitatively or qualitatively altered dystrophin, cause the milder allelic variant known as Becker muscular dystrophy (BMD). DMD is a devastating disorder. Progressive muscle wasting and weakness causes disability since childhood, and the natural history is characterized by loss of independent ambulation (LoA) around 10 - 15 years of age, and reduced life expectancy because of respiratory and cardiac complications in young adults. Glucocorticoid corticosteroids (GCs) might delay disease progression, and there are promising novel molecular treatments, but a definitive cure remains elusive. Promising molecular treatments include antisense oligonucleotides (AONs) inducing exon skipping in out-of-frame deletions and premature stop codon readthrough compounds. There is relevant variability in the severity and rate of progression of muscle wasting and weakness in DMD, which is not explained, if not in a minor proportion, by the disease-causing mutation, as all DMD patients have a complete, or near to complete dystrophin defect. Recently, our group and other authors have described genetic modifiers of DMD, i.e. common single nucleotide polymorphisms (SNPs) associated to more or less severe DMD expressivity. These include rs28357094, a SNP in the promoter of the SPP1 gene, enconding the cytokine osteopontin, and a coding haplotype in the LTBP4 gene, enconding Latent Transforming growth factor β-Binding Protein 4. These variants modulate the expression (SPP1), or alter the aminoacid sequence (for LTBP4) of corresponding proteins, both of which are involved in inflammatory and pro-fibrotic pathways. They were both identified by candidate gene approaches (respectively, expression profiling studies and a murine genome scan). The first aim of this thesis was to provide an independent validation of the genetic association of the SPP1 rs28357094 SNP and the LTBP4 haplotype with age at LoA in DMD. This was achieved using data from the Cooperative International Neuromuscular Research Group Duchenne Natural History Study (CINRG-DNHS) of 340 DMD patients from 20 worldwide Centers. In this population, the minor allele G at rs28357094 was associated to a 2-year delay of LoA in CINRG-DNHS participants who had been treated with glucocorticoids (p < 0.05), and no significant effect in untreated patients, suggesting that the SNP might be a pharmacodynamic biomarker of GC response. Furthermore, the homozygous LTBP4 haplotype “IAAM” was associated to a 2-year delay of LoA in participants of European descent (p < 0.05), but not in the whole multi-ethinc CINRG-DNHS cohort, highlighting the relevance of population stratification in genetic modifier studies. The second aim was to test for associations between specific DMD mutations and age at LoA in the CINRG-DNHS. We confirmed previous reports that deletions bordering exon 44 (and thus amenable to AON treatment for skipping of this exon), as well as the deletion of exons 3-7, were associated to later LoA (p < 0.01 and < 0.05 respectively). These findings have repercussions on clinical trial design and prognosis. A third aim was to study age at LoA as a long-term outcome of several different GC regimens currently adopted in DMD. In this observational study, we found that the use of daily deflazacort was associated to 2.7-year later LoA than daily prednisone (p < 0.001), an unexpected finding that may be confirmed by ongoing randomized trials. As a fourth aim, we genotyped 175/340 CINRG-DNHS participants with an Exome Chip, including thousands of functional (regulatory or coding) variants, and performed a genome-wide association study (GWAS) of age at LoA in a subgroup of 109 unrelated participants of European ancestry. While no SNP surpassed the Bonferroni-corrected significance threshold, we performed a hypothesis-driven prioritization of findings, focused on inflammatory and pro-fibrotic pathways, and identified a hit in a gene involved in inflammation and cell-mediated immunity. The GWAS association of earlier LoA with the minor allele at the identified locus (p < 9.9*10-5) was validated in a collaborative cohort of 660 DMD patients from the University of Padova, the European Bio-NMD network, and the United Dystrophinopathy Project in the USA (p < 0.05). Finally, the fifth aim focused on BMD, which features a milder, but even more variable clinical picture than DMD. In BMD, most patients have in-frame deletions leading to internally deleted dystrophin protein. We quantified dystrophin by Western Blot, performed a retrospective study of LoA and loss of the ability to run, and a 1-year longitudinal study of motor function (6 Minute Walk Test [6MWT], North Star Ambulatory Assessment [NSAA], timed function tests [TFTs]) in 69 BMD patients at the University of Padova. We found that deletions bordering exon 45 were associated with frequent loss of the ability to run, risk of LoA in adults, and overt muscle weakness; while some deletion groups, like those bordering exon 51, or limited to exon 48, were preserved from these signs of disease progression. This is relevant not only to BMD prognosis and genetic counseling, but also to outcomes of exon skipping AON treatments which aim to reproduce the same deletions at the transcript level in DMD. Furthermore, we observed that NSAA and 6MWT, which we mutuated from DMD studies and had not been applied to BMD, were feasible, clinically meaningful, and able to identify disease progression at 1 year, suggesting their adequacy as outcome measure for future BMD clinical trials. Altogether, the work presented here provides novel insights into the mechanisms of phenotypic and clinical variability in dystrophinopathy, which will be useful in delivering improved care for these disabling diseases

Evidence for ACTN3 as a genetic modifier of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is characterized by muscle degeneration and progressive weakness. There is considerable inter-patient variability in disease onset and progression, which can confound the results of clinical trials. Here we show that a common null polymorphism (R577X) in ACTN3 results in significantly reduced muscle strength and a longer 10\u2009m walk test time in young, ambulant patients with DMD; both of which are primary outcome measures in clinical trials. We have developed a double knockout mouse model, which also shows reduced muscle strength, but is protected from stretch-induced eccentric damage with age. This suggests that \u3b1-actinin-3 deficiency reduces muscle performance at baseline, but ameliorates the progression of dystrophic pathology. Mechanistically, we show that \u3b1-actinin-3 deficiency triggers an increase in oxidative muscle metabolism through activation of calcineurin, which likely confers the protective effect. Our studies suggest that ACTN3 R577X genotype is a modifier of clinical phenotype in DMD patients

Effects of grazer diversity on marine microphytobenthic biofilm: a ‘tug of war’ between complementarity and competition

Species loss is one of the most striking problems related to human-driven environmental changes. Nevertheless, biodiversity and ecosystem functioning experiments have mainly focused on primary producers, paying less attention to the consequences of changing diversity at higher trophic levels. We performed a field experiment using cage enclosures to test the effects of species richness, identity and density of gastropod grazers on the photosynthetic efficiency and biomass of intertidal biofilm on an exposed rocky shore in the northwest Mediterranean. The diversity and composition of intertidal grazers affected the photosynthetic efficiency of biofilm with only negligible effects on biomass. Individual species showed strong identity effects. In assemblages of 2 or more species, positive or negative complementarity effects occurred. The magnitude of the ecosystem response is expected to depend on the particular species assemblage and its density, which will determine whether niche partitioning or competition is the prevailing process. Grazer preference in specific components of biofilm, characterized by different photosynthetic efficiency and competitive abilities, might explain concomitant changes in photosynthetic efficiency and comparable levels in biomass among treatments. The effects of grazers declined following the natural trend of decreasing biomass of biofilm during the study period, highlighting the importance of considering temporal variability in the effects of biodiversity on ecosystem functioning. This work emphasizes the key role of species identity to predict effects on their resources and ecosystem functioning.This work was partially supported by the University of Pisa

Two vs. Four-Channel Sound Event Localization and Detection

Sound event localization and detection (SELD) systems estimate both the direction-of-arrival (DOA) and class of sound sources over time. In the DCASE 2022 SELD Challenge (Task 3), models are designed to operate in a 4-channel setting. While beneficial to further the development of SELD systems using a multichannel recording setup such as first-order Ambisonics (FOA), most consumer electronics devices rarely are able to record using more than two channels. For this reason, in this work we investigate the performance of the DCASE 2022 SELD baseline model using three audio input representations: FOA, binaural, and stereo. We perform a novel comparative analysis illustrating the effect of these audio input representations on SELD performance. Crucially, we show that binaural and stereo (i.e. 2-channel) audio-based SELD models are still able to localize and detect sound sources laterally quite well, despite overall performance degrading as less audio information is provided. Further, we segment our analysis by scenes containing varying degrees of sound source polyphony to better understand the effect of audio input representation on localization and detection performance as scene conditions become increasingly complex

Assessment of a Remote Sensing Energy Balance Methodology (SEBAL) Using Different Interpolation Methods to Determine Evapotranspiration in a Citrus Orchard

"(c) 2015 IEEE. Personal use of this material is permitted. Permission from IEEE must be obtained for all other users, including reprinting/ republishing this material for advertising or promotional purposes, creating new collective works for resale or redistribution to servers or lists, or reuse of any copyrighted components of this work in other works."A surface energy balance algorithm for land (SEBAL) for estimating evapotranspiration (ET) has been parameterized and tested in a 400-ha drip irrigated citrus orchard. Simultaneously, during three growing seasons, energy fluxes were measured using Eddy Covariance. Instantaneous fluxes obtained with SEBAL using 10 images from Landsat-5 were compared with the measured fluxes. The Perrier function was the best method for properly estimating the roughness momentum length for discontinuous canopies, as in citrus orchards. Crop height was estimated using LIDAR data. In general, SEBAL performed well for net radiation estimation but failed in soil heat flux estimation. Latent heat estimations from the SEBAL model had a relative root mean square error (rRMSE) of 0.06 when compared with measurements obtained by Eddy Covariance. Three procedures were tested for up-scaling the instantaneous ET estimates from SEBAL to daily ET values: 1) assuming the fraction between the actual ET and the reference ET is constant throughout the day; 2) using actual local crop coefficient curves; and 3) using an up-scaling factor where the fraction of hourly ET to daily ET equals the ratio of hourly to daily global solar radiation. This last method gave acceptable results for daily ET estimations (rRMSE = 0.09) and for 15day ET (rRMSE = 0.19), and its main advantage is that no local data are required. It is concluded that the SEBAL methodology can be successfully applied for determining actual ET, even in discontinuous citrus canopies. However, additional parameterizations of momentum roughness length were needed in order to obtain reliable ET determinations.This work was supported in part by MINECO project Rideco-Consolider CSD2006-0067 and in part by Interreg IV Sudoe project "Telerieg."Jiménez Bello, MÁ.; Castel, JR.; Testi, L.; Intrigliolo Molina, DS. (2015). Assessment of a Remote Sensing Energy Balance Methodology (SEBAL) Using Different Interpolation Methods to Determine Evapotranspiration in a Citrus Orchard. IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing. 8(4):1465-1477. https://doi.org/10.1109/JSTARS.2015.2418817S146514778

Dietary Fibers and Cardiometabolic Diseases

The high prevalence of cardiovascular disease (CVD) is largely attributable to the contemporary lifestyle that is often sedentary and includes a diet high in saturated fats and sugars and low ingestion of polyunsaturated fatty acids (PUFAs), fruit, vegetables, and fiber. Experimental data from both animals and humans suggest an association between increased dietary fiber (DF) intakes and improved plasma lipid profiles, including reduced low density lipoprotein cholesterol (LDL-C) concentrations. These observations underline that the intake of DF may protect against heart disease and stroke