Gapmer Antisense Oligonucleotides Suppress the Mutant Allele of COL6A3 and Restore Functional Protein in Ullrich Muscular Dystrophy

Dominant-negative mutations in the genes that encode the three major a chains of collagen type VI, COL6A1, COL6A2, and COL6A3, account for more than 50% of Ullrich congenital muscular dystrophy patients and nearly all Bethlem myopathy patients. Gapmer antisense oligonucleotides (AONs) are usually used for gene silencing by stimulating RNA cleavage through the recruitment of an endogenous endonuclease known as RNase H to cleave the RNA strand of a DNA-RNA duplex. In this study, we exploited the application of the allele-specific silencing approach by gapmer AON as a potential therapy for Collagen-VI-related congenital muscular dystrophy (COL6-CMD). A series of AONs were designed to selectively target an 18-nt heterozygous genomic deletion in exon 15 of COL6A3 at the mRNA and pre-mRNA level. We showed that gapmer AONs can selectively suppress the expression of mutant transcripts at both pre-mRNA and mRNA levels, and that the latter strategy had a far stronger efficiency than the former. More importantly, we found that silencing of the mutant transcripts by gapmer AONs increased the deposition of collagen VI protein into the extracellular matrix, thus restoring functional protein production. Our findings provide a clear proof of concept for AON allele-specific silencing as a therapeutic approach for COL6-CM

Changes in MEG resting-state networks are related to cognitive decline in type 1 diabetes mellitus patients

OBJECTIVE: Integrity of resting-state functional brain networks (RSNs) is important for proper cognitive functioning. In type 1 diabetes mellitus (T1DM) cognitive decrements are commonly observed, possibly due to alterations in RSNs, which may vary according to microvascular complication status. Thus, we tested the hypothesis that functional connectivity in RSNs differs according to clinical status and correlates with cognition in T1DM patients, using an unbiased approach with high spatio-temporal resolution functional network.; METHODS: Resting-state magnetoencephalographic (MEG) data for T1DM patients with (n=42) and without (n=41) microvascular complications and 33 healthy participants were recorded. MEG time-series at source level were reconstructed using a recently developed atlas-based beamformer. Functional connectivity within classical frequency bands, estimated by the phase lag index (PLI), was calculated within eight commonly found RSNs. Neuropsychological tests were used to assess cognitive performance, and the relation with RSNs was evaluated.; RESULTS: Significant differences in terms of RSN functional connectivity between the three groups were observed in the lower alpha band, in the default-mode (DMN), executive control (ECN) and sensorimotor (SMN) RSNs. T1DM patients with microvascular complications showed the weakest functional connectivity in these networks relative to the other groups. For DMN, functional connectivity was higher in patients without microangiopathy relative to controls (all p<0.05). General cognitive performance for both patient groups was worse compared with healthy controls. Lower DMN alpha band functional connectivity correlated with poorer general cognitive ability in patients with microvascular complications.; DISCUSSION: Altered RSN functional connectivity was found in T1DM patients depending on clinical status. Lower DMN functional connectivity was related to poorer cognitive functioning. These results indicate that functional connectivity may play a key role in T1DM-related cognitive dysfunction

Effectiveness and Limitations of Unsupervised Home-Based Balance Rehabilitation with Nintendo Wii in People with Multiple Sclerosis

Balance training represents a critical part of the rehabilitation process of individuals living with multiple sclerosis (MS) since impaired postural control is a distinctive symptom of the disease. In recent years, the use of the Nintendo Wii system has become widespread among rehabilitation specialists for this purpose, but few studies have verified the effectiveness of such an approach using quantitative measures of balance. In this study, we analyzed the postural sway features of a cohort of twenty-seven individuals with MS before and after 5 weeks of unsupervised home-based balance training with the Wii system. Center of pressure (COP) time-series were recorded using a pressure platform and processed to calculate sway area, COP path length, displacements, and velocities in mediolateral (ML) and anteroposterior (AP) directions. Although the results show a significant reduction in sway area, COP displacements, and velocity, such improvements are essentially restricted to the ML direction, as the Wii platform appears to properly stimulate the postural control system in the frontal plane but not in the sagittal one. Available Wii games, although somewhat beneficial, appear not fully suitable for rehabilitation in MS owing to scarce flexibility and adaptability to MS needs and thus specific software should be developed

Large-scale lateral saturated soil hydraulic conductivity as a metric for the connectivity of subsurface flow paths at hillslope scale

Lateral saturated soil hydraulic conductivity, Ks,l, is the soil property governing subsurface water transfer in hillslopes, and the key parameter in many numerical models simulating hydrological processes at the hillslope and catchment scales. Likewise, the hydrological connectivity of the lateral flow paths plays a significant role in determining the rate of the subsurface flow at various spatial scales. This study investigates the relationship between Ks,l and hydrological connectivity at the hillslope spatial scale. Ks,l was determined by the subsurface flow rates intercepted by drains and water table depths observed in a well network. The hydrological connectivity was evaluated by the synchronicity among water table peaks, and between these and the peaks of the drained flow. Rainfall and soil moisture were used to investigate the influence of the transient hydrological soil condition on connectivity and Ks,l. As the synchronicity of the water table response between wells increased, the lag times between the peaks of water levels and those of the drained subsurface flow decreased. Moreover, the most synchronic water table rises determined the highest drainage rates. The relationships between Ks,l and water table depths were highly non-linear, with a sharp increase in the values for water table levels close to the soil surface. Estimated Ks,l values for the full saturated soil were in the order of thousands of mm h−1, suggesting the activation of macropores in the root zone. The Ks,l values determined at the peak of the drainage events were correlated with the indicators of synchronicity. The sum of cumulative rainfall and antecedent soil moisture was correlated with the connectivity indicators and Ks,l. We suggest that, for simulating realistic processes at the hillslope scale, the hydrological connectivity could be implicitly considered in hydrological modelling through an evaluation of Ks,l at the same spatial scale

SCHEMA SPERIMENTALE PER LA STIMA DELLA CONDUCIBILITÀ IDRAULICA LATERALE ALLA SCALA DI VERSANTE

La conducibilità idraulica alla saturazione, KS, è un parametro fondamentale per la simulazione del flusso idrico nel suolo. La memoria illustra un approccio sperimentale per la stima della KS laterale alla scala di versante. La metodologia proposta è stata applicata in due aree contigue ricoperte, rispettivamente, da macchia mediterranea e prato. Sono stati registrati i livelli di falda e i deflussi idrici sottosuperficiali, e queste misure sono state usate per il calcolo della KS del suolo tramite l’equazione di Darcy. Il monitoraggio è stato effettuato durante il periodo piovoso compreso tra gennaio e giugno 2014. In aprile, inoltre, sono state eseguite prove di pioggia artificiale con intensità di 30 e 70 mm h-1. Durante le precipitazioni naturali, il valore massimo di KS stimato nel prato è stato di 2870 mm h-1, mentre è stato pari a 2400 mm h-1 nel corso delle prove di saturazione artificiale. Il valore massimo di KS pari 4000 mm h-1 è stato ottenuto, nella macchia, durante le prove di pioggia artificiale. Per contro, valori minori di KS sono stati ottenuti durante le piogge naturali a causa delle basse portate sottosuperficiali registrate. La metodologia proposta è risultata idonea per la stima di valori della conducibilità idraulica rappresentativi per le aree di interesse. Tali informazioni potranno garantire una maggiore attendibilità della modellizzazione dei processi idrologici a scala di versante e di bacino

Reshaping cortical connectivity in traumatic spinal cord injury: a novel effect of hyperbaric oxygen therapy

Introduction: Spinal cord injuries (SCIs) represent a severe neuro-traumatic occurrence and an excruciating social burden. Though the hyperbaric oxygen (HBO2) has been credited as a first line therapeutic resource for SCIs, its mechanism of action in the spine is only partially known, while the impingement upon other areas of the nervous system deserves additional investigation. In this study we deem to describe a novel effect of HBO2 in a subject affected by SCI who, along with the clinical improvement, showed a reshaped connectivity in cortical sensory-motor areas. Case presentation: A 45 years male presenting severe sensory-motor symptoms following a spinal lesion partially involving the C1 segment was successfully treated with HBO2 cycles. After the dramatic improvement reflected by an excellent optimization of the single performances, it has been investigated whether this result would reveal not only an intrinsic effect upon the spinal cord, but also a better connectivity strength in sensory-motor cortical regions. The results obtained by implementing EEG recordings with EEGLAB auto regressive vector plugins indeed suggest a substantial reshaping of cortico-cortical connectivity after HBO2. Discussion: These results show a correlation between positive clinical evolution and a new modulation of cortical connectivity. Though further clinical investigations would clarify as to whether HBO2 might be directly or epiphenomenally involved in this aspect of the network architecture, our report suggests that a comparison between clinical results and the study of brain connectivity represent a holistic approach in investigating the physiopathology of SCIs and in monitoring the treatment

Differential effects of phytotherapic preparations in the hSOD1 Drosophila melanogaster model of ALS

The present study was aimed at characterizing the effects of Withania somnifera (Wse) and Mucuna pruriens (Mpe) on a Drosophila melanogaster model for Amyotrophic Lateral Sclerosis (ALS). In particular, the effects of Wse and Mpe were assessed following feeding the flies selectively overexpressing the wild human copper, zinc-superoxide dismutase (hSOD1-gain-of-function) in Drosophila motoneurons. Although ALS-hSOD1 mutants showed no impairment in life span, with respect to GAL4 controls, the results revealed impairment of climbing behaviour, muscle electrophysiological parameters (latency and amplitude of ePSPs) as well as thoracic ganglia mitochondrial functions. Interestingly, Wse treatment significantly increased lifespan of hSDO1 while Mpe had not effect. Conversely, both Wse and Mpe significantly rescued climbing impairment, and also latency and amplitude of ePSPs as well as failure responses to high frequency DLM stimulation. Finally, mitochondrial alterations were any more present in Wse-but not in Mpe-Treated hSOD1 mutants. Hence, given the role of inflammation in the development of ALS, the high translational impact of the model, the known anti-inflammatory properties of these extracts, and the viability of their clinical use, these results suggest that the application of Wse and Mpe might represent a valuable pharmacological strategy to counteract the progression of ALS and related symptom

(1)H-NMR analysis provides a metabolomic profile of patients with multiple sclerosis

OBJECTIVE: To investigate the metabolomic profiles of patients with multiple sclerosis (MS) and to define the metabolic pathways potentially related to MS pathogenesis. METHODS: Plasma samples from 73 patients with MS (therapy-free for at least 90 days) and 88 healthy controls (HC) were analyzed by (1)H-NMR spectroscopy. Data analysis was conducted with principal components analysis followed by a supervised analysis (orthogonal partial least squares discriminant analysis [OPLS-DA]). The metabolites were identified and quantified using Chenomx software, and the receiver operating characteristic (ROC) curves were calculated. RESULTS: The model obtained with the OPLS-DA identified predictive metabolic differences between the patients with MS and HC (R2X = 0.615, R2Y = 0.619, Q2 = 0.476; p < 0.001). The differential metabolites included glucose, 5-OH-tryptophan, and tryptophan, which were lower in the MS group, and 3-OH-butyrate, acetoacetate, acetone, alanine, and choline, which were higher in the MS group. The suitability of the model was evaluated using an external set of samples. The values returned by the model were used to build the corresponding ROC curve (area under the curve of 0.98). CONCLUSION: NMR metabolomic analysis was able to discriminate different metabolic profiles in patients with MS compared with HC. With the exception of choline, the main metabolic changes could be connected to 2 different metabolic pathways: tryptophan metabolism and energy metabolism. Metabolomics appears to represent a promising noninvasive approach for the study of M

EEG functional network topology is associated with disability in patients with amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS) is one of the most severe neurodegenerative diseases, which is known to affect upper and lower motor neurons. In contrast to the classical tenet that ALS represents the outcome of extensive and progressive impairment of a fixed set of motor connections, recent neuroimaging findings suggest that the disease spreads along vast non-motor connections. Here, we hypothesised that functional network topology is perturbed in ALS, and that this reorganization is associated with disability. We tested this hypothesis in 21 patients affected by ALS at several stages of impairment using resting-state electroencephalography (EEG) and compared the results to 16 age-matched healthy controls. We estimated functional connectivity using the Phase Lag Index (PLI), and characterized the network topology using the minimum spanning tree (MST). We found a significant difference between groups in terms of MST dissimilarity and MST leaf fraction in the beta band. Moreover, some MST parameters (leaf, hierarchy and kappa) significantly correlated with disability. These findings suggest that the topology of resting-state functional networks in ALS is affected by the disease in relation to disability. EEG network analysis may be of help in monitoring and evaluating the clinical status of ALS patients

FOOD SAFETY AND PROCESS HYGIENE CRITERIONS ON SHEEP CARCASSES

The hygienic status and the presence of some pathogens (Staphylococcus aureus, Listeria monocytogenes e Salmonella spp.) at slaughterhouses was evaluated in different matrix of sheep and lambs (carcass surface, faeces, fleeces and mesenteric lymph nodes) according to the Com. Reg. (EC) No 2073/2005. The 48% of sheep and 68.9% of lamb sampled carcasses resulted allocated into the marginal category for Aerobic colony count, while the 28% and 42.2% respectively were allocated into unacceptable category for Enterobacteriaceae. S.aureus was isolated more frequently in fleeces (11.5%), carcasses (12.6%) of lambs than sheep. L. monocytogenes was found in fleeces and carcass of two sheep and in faeces of four lambs, while Salmonella spp. was detected only in sheep carcasses of a single plant