Mimicking sarcolemmal damage in vitro: a contractile 3D model of skeletal muscle for drug testing in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most prevalent neuromuscular disease diagnosed in childhood. It is a progressive and wasting disease, characterized by a degeneration of skeletal and cardiac muscles caused by the lack of dystrophin protein. The absence of this crucial structural protein leads to sarcolemmal fragility, resulting in muscle fiber damage during contraction. Despite ongoing efforts, there is no cure available for DMD patients. One of the primary challenges is the limited efficacy of current preclinical tools, which fail in modeling the biological complexity of the disease. Human-based three-dimensional (3D) cell culture methods appear as a novel approach to accelerate preclinical research by enhancing the reproduction of pathophysiological processes in skeletal muscle. In this work, we developed a patient-derived functional 3D skeletal muscle model of DMD that reproduces the sarcolemmal damage found in the native DMD muscle. These bioengineered skeletal muscle tissues exhibit contractile functionality, as they responded to electrical pulse stimulation. Sustained contractile regimes induced the loss of myotube integrity, mirroring the pathological myotube breakdown inherent in DMD due to sarcolemmal instability. Moreover, damaged DMD tissues showed disease functional phenotypes, such as tetanic fatigue. We also evaluated the therapeutic effect of utrophin upregulator drug candidates on the functionality of the skeletal muscle tissues, thus providing deeper insight into the real impact of these treatments. Overall, our findings underscore the potential of bioengineered 3D skeletal muscle technology to advance DMD research and facilitate the development of novel therapies for DMD and related neuromuscular disorders

Evolutionary history of Podarcis tiliguerta on Corsica and Sardinia.

BACKGROUND: Podarcis tiliguerta is a wall lizard endemic to the Mediterranean islands of Corsica and Sardinia. Previous findings of high mtDNA and morphological diversity have led to the suggestion that it may represent a species complex. Here, we analysed mitochondrial and nuclear markers (mtDNA, 3110 bp; 6 nDNA loci, 3961 bp) in P. tiliguerta sampled from thirty-two localities across Corsica and Sardinia. RESULTS: We find much greater intraspecific genetic divergence than between sister species of other Mediterranean island Podarcis, i.e., between P. lilfordi and P. pityusensis. We detected three mtDNA clusters in Corsica (North, South-East and South-West) and either two or three in Sardinia (North vs. South) depending on the clustering method. Only one or two nDNA groups were identified within each main island (again, depending on the method). A Bayesian time-calibrated multispecies coalescent tree was obtained from mtDNA and provided statistical support for a Miocene origin of the species (13.87 Ma, 95% HPD: 18.30-10.77 Ma). The posterior mean divergence time for the Corsican and Sardinian lineages was 12.75 Ma ago (95% HPD: 16.94-9.04 Ma). CONCLUSION: The results support the evolutionary distinctiveness of Corsican and Sardinian populations and also indicate a lack of post-divergence migration despite periods of contact being possible. Further to this, species delimitation analyses of Corsican and Sardinian lineages provided statistical support for their recognition as distinct (sister) taxa. Our results provide new insights into the biogeography of the Mediterranean biodiversity hotspot, and contribute important findings relevant to the systematics and evolution of this speciose lizard genus

On the conservation of the slow conformational dynamics within the amino acid kinase family: NAGK the paradigm

N-Acetyl-L-Glutamate Kinase (NAGK) is the structural paradigm for examining the catalytic mechanisms and dynamics of amino acid kinase family members. Given that the slow conformational dynamics of the NAGK (at the microseconds time scale or slower) may be rate-limiting, it is of importance to assess the mechanisms of the most cooperative modes of motion intrinsically accessible to this enzyme. Here, we present the results from normal mode analysis using an elastic network model representation, which shows that the conformational mechanisms for substrate binding by NAGK strongly correlate with the intrinsic dynamics of the enzyme in the unbound form. We further analyzed the potential mechanisms of allosteric signalling within NAGK using a Markov model for network communication. Comparative analysis of the dynamics of family members strongly suggests that the low-frequency modes of motion and the associated intramolecular couplings that establish signal transduction are highly conserved among family members, in support of the paradigm sequence→structure→dynamics→function © 2010 Marcos et al

CNVassoc: Association analysis of CNV data using R

Background: Copy number variants (CNV) are a potentially important component of the genetic contribution to risk of common complex diseases. Analysis of the association between CNVs and disease requires that uncertainty in CNV copy-number calls, which can be substantial, be taken into account; failure to consider this uncertainty can lead to biased results. Therefore, there is a need to develop and use appropriate statistical tools. To address this issue, we have developed CNVassoc, an R package for carrying out association analysis of common copy number variants in population-based studies. This package includes functions for testing for association with different classes of response variables (e.g. class status, censored data, counts) under a series of study designs (case-control, cohort, etc) and inheritance models, adjusting for covariates. The package includes functions for inferring copy number (CNV genotype calling), but can also accept copy number data generated by other algorithms (e.g. CANARY, CGHcall, IMPUTE). Results: Here we present a new R package, CNVassoc, that can deal with different types of CNV arising from different platforms such as MLPA o aCGH. Through a real data example we illustrate that our method is able to incorporate uncertainty in the association process. We also show how our package can also be useful when analyzing imputed data when analyzing imputed SNPs. Through a simulation study we show that CNVassoc outperforms CNVtools in terms of computing time as well as in convergence failure rate. Conclusions: We provide a package that outperforms the existing ones in terms of modelling flexibility, power, convergence rate, ease of covariate adjustment, and requirements for sample size and signal quality. Therefore, we offer CNVassoc as a method for routine use in CNV association studiesThis work has been supported by the Spanish Ministry of Science and Innovation (MTM2008-02457 to JRG, BIO2009-12458 to RD-U and statistical genetics network MTM2010-09526-E (subprograma MTM) to JRG, IS, GL and RD-U). GL is supported by the Juan de la Cierva Program of the Spanish Ministry of Science and Innovation

Eradication of common pathogens at days 2, 3 and 4 of moxifloxacin therapy in patients with acute bacterial sinusitis

BACKGROUND: Acute bacterial sinusitis (ABS) is a common infection in clinical practice. Data on time to bacteriologic eradication after antimicrobial therapy are lacking for most agents, but are necessary in order to optimize therapy. This was a prospective, single-arm, open-label, multicenter study to determine the time to bacteriologic eradication in ABS patients (maxillary sinusitis) treated with moxifloxacin. METHODS: Adult patients with radiologically and clinically confirmed ABS received once-daily moxifloxacin 400 mg for 10 days. Middle meatus secretion sampling was performed using nasal endoscopy pre-therapy, and repeated on 3 consecutive days during treatment. Target enrollment was 30 bacteriologically evaluable patients (pre-therapy culture positive for Streptococcus pneumoniae, Haemophilus influenzae or Moraxella catarrhalis and evaluable cultures for at least Day 2 and Day 3 during therapy visits), including at least 10 each with S. pneumoniae or H. influenzae. RESULTS: Of 192 patients enrolled, 42 were bacteriologically evaluable, with 48 pathogens isolated. Moxifloxacin was started on Day 1. Baseline bacteria were eradicated in 35/42 (83.3%) patients by day 2, 42/42 (100%) patients by day 3, and 41/42 (97.6%) patients by day 4. In terms of individual pathogens, 12/18 S. pneumoniae, 22/23 H. influenzae and 7/7 M. catarrhalis were eradicated by day 2 (total 41/48; 85.4%), and 18/18 S. pneumoniae and 23/23 H. influenzae were eradicated by day 3. On Day 4, S. pneumoniae was isolated from a patient who had negative cultures on Days 2 and 3. Thus, the Day 4 eradication rate was 47/48 (97.9%). Clinical success was achieved in 36/38 (94.7%) patients at the test of cure visit. CONCLUSION: In patients with ABS (maxillary sinusitis), moxifloxacin 400 mg once daily for 10 days resulted in eradication of baseline bacteria in 83.3% of patients by Day 2, 100% by Day 3 and 97.6% by Day 4

Discovery of VHE Gamma Radiation from IC443 with the MAGIC Telescope

We report the detection of a new source of very high energy (VHE, E_gamma >= 100GeV) gamma-ray emission located close to the Galactic Plane, MAGIC J0616+225, which is spatially coincident with SNR IC443. The observations were carried out with the MAGIC telescope in the periods December 2005 - January 2006 and December 2006 - January 2007. Here we present results from this source, leading to a VHE gamma-ray signal with a statistical significance of 5.7 sigma in the 2006/7 data and a measured differential gamma-ray flux consistent with a power law, described as dN_gamma/(dA dt dE) = (1.0 +/- 0.2)*10^(-11)(E/0.4 TeV)^(-3.1 +/- 0.3) cm^(-2)s^(-1)TeV^(-1). We briefly discuss the observational technique used and the procedure implemented for the data analysis. The results are put in the perspective of the multiwavelength emission and the molecular environment found in the region of IC443.Comment: Accepted by ApJ Letter

Changes in Dynamics upon Oligomerization Regulate Substrate Binding and Allostery in Amino Acid Kinase Family Members

Oligomerization is a functional requirement for many proteins. The interfacial interactions and the overall packing geometry of the individual monomers are viewed as important determinants of the thermodynamic stability and allosteric regulation of oligomers. The present study focuses on the role of the interfacial interactions and overall contact topology in the dynamic features acquired in the oligomeric state. To this aim, the collective dynamics of enzymes belonging to the amino acid kinase family both in dimeric and hexameric forms are examined by means of an elastic network model, and the softest collective motions (i.e., lowest frequency or global modes of motions) favored by the overall architecture are analyzed. Notably, the lowest-frequency modes accessible to the individual subunits in the absence of multimerization are conserved to a large extent in the oligomer, suggesting that the oligomer takes advantage of the intrinsic dynamics of the individual monomers. At the same time, oligomerization stiffens the interfacial regions of the monomers and confers new cooperative modes that exploit the rigid-body translational and rotational degrees of freedom of the intact monomers. The present study sheds light on the mechanism of cooperative inhibition of hexameric N-acetyl-L-glutamate kinase by arginine and on the allosteric regulation of UMP kinases. It also highlights the significance of the particular quaternary design in selectively determining the oligomer dynamics congruent with required ligand-binding and allosteric activities