Search

Die Erforschung von unbekannten Himmelsphänomenen, die heute allgemeiner als "Unidentified Aerial Phenomena" (UAP) bezeichnet und früher Unidentified Flying Object (UFO) genannt wurden, haben das Potential zu neuen, bedeutenden wissenschaftlichen Erkenntnissen zu führen, sollte sich herausstellen, dass die Ursachen auf neue, bisher unbekannte Phänomene zurückzuführen sind. Bisher wurden diese Phänomene in der Wissenschaft allerdings kaum beachtet, weshalb es nur wenige wissenschaftlich fundierte Daten und Erkenntnisse dazu gibt. Die bloße Existenz von UAP oder UFO wird spätestens seit den offiziellen Bekanntmachungen im Sommer 2021 in den USA kaum noch in Zweifel gezogen. Die Phänomene zeigen zu einem kleinen, aber nicht zu ignorierenden Teil, sehr ungewöhnliche Eigenschaften wie extreme Flugcharakteristika, die teilweise scheinbar gleichzeitig durch verschiedene Sensoren bestätigt sind. Diese können nicht immer auf herkömmliche Ursachen zurückgeführt werden. Es geht sogar so weit, dass Sie in manchen Fällen scheinbar in Widerspruch zu physikalischen Naturgesetzen stehen. Sollte es sich dabei nicht ausnahmslos um Sensorartefakte, Fehlinterpretationen oder ähnliches handeln, sondern wirklich um physikalische Phänomene oder Objekte, müssten ganz neue Hypothesen und Theorien entwickelt werden. Dies könnte zur Entdeckung von neuen, bisher völlig unbekannten Naturphänomenen oder sogar zu ungeahnten Paradigmenwechsel im Verständnis unserer Welt führen. Nicht komplett auszuschließen wäre darüber hinaus die extraterrestrische Hypothese, wonach die Ursachen der Phänomene teilweise auf Signaturen technologisch fortgeschrittener außerirdischer Intelligenzen zurückzuführen sein könnten. Sollte sich diese Hypothese bewahrheiten, wäre es die bedeutendste Entdeckung der Menschheitsgeschichte mit weitreichenden Folgen für die Wissenschaft und Gesellschaft. Obwohl die Phänomene seit über 70 Jahren beobachtet werden, gibt es aufgrund der Stigmatisierung des Themas an sich, dem Fehlen von begründeten Hypothesen, der nicht Vorhersehbarkeit des Auftretens, der Sicherheitsaspekte und vor allem dem Fehlen einer soliden Datenbasis, kaum wissenschaftliche Untersuchungen. Es ist deswegen unerlässlich, zunächst eine verlässliche Datenbasis zu schaffen, um die Phänomene überhaupt erst untersuchen zu können. An der Professur für Raumfahrttechnik der Julius-Maximilian Universität Würzburg wird seit 2008 an diesem Forschungsschwerpunkt gearbeitet. Der Schwerpunkt liegt auf der Entwicklung und dem Betrieb von neuartigen, dem flüchtigen Problem angepassten, intelligenten Sensorsystemen. Dazu sollen die Daten von verschiedenen Typen von abbildenden und nicht abbildenden Sensoren aus unterschiedlichen Wellenlängenbereichen kombiniert und ausgewertet werden. Einer der Schwerpunkte liegt in der Software, die eine effiziente und KI-gestützte Klassifizierung an den vernetzten Beobachtungsstationen in nahezu Echtzeit ermöglichen und die Falschalarmrate verringern soll. Die an ausgewählten Regionen aufgestellten Beobachtungsstationen können dann in der Folge mit hoher geometrischer, radiometrischer und temporaler Auflösung Daten über das Phänomen liefern, was systematische Analysen ermöglichen soll.

Der Zusammenschluss mehrerer miniaturisierter Sternsensoren bietet in der richtigen Konfiguration eine stark erhöhte Verfügbarkeit an genauen Lagedaten. Das begrenzte Bauvolumen auf einem CubeSat stellt dabei eine Herausforderung dar. Die Verwendung des TunaCan-Slots, verbunden mit dem Einsatz der gegebenen hohen Sensorredundanz, ermöglicht allerdings die Realisierung eines solchen Verbundes, während die Anforderungen im Rahmen einer CubeSat-Mission bleiben. Die internen Komponenten müssen selbständig miteinander arbeiten, um die funktionale Redundanz zu bewerkstelligen. Dazu zählt sowohl die Software als auch die entsprechend entworfene Elektronik. In diesem Paper wird solch ein Konzept ausgearbeitet und die Implementierung aufgeführt. Die interne Arbeitsweise zur Redundanzsteuerung und der Datenverarbeitung wird erläutert und die Validierung zum Einsatz im Orbit in Aussicht gestellt.

Motivated by advances in flexible electronic technologies and by the endeavour to develop non-destructive testing methods, this article analyses the capability of computational multiscale formulations to predict the influence of microscale cracks on effective macroscopic electrical and mechanical material properties. To this end, thin metal films under mechanical load are experimentally analysed by using in-situ confocal laser scanning microscopy (CLSM) and in-situ four point probe resistance measurements. Image processing techniques are then used to generate representative volume elements from the laser intensity images. These discrete representations of the crack pattern at the microscale serve as the basis for the calculation of effective macroscopic electrical conductivity and mechanical stiffness tensors by means of computational homogenisation approaches. A comparison of simulation results with experimental electrical resistance measurements and a detailed study of fundamental numerical properties demonstrates the applicability of the proposed approach. In particular, the (numerical) errors that are induced by the representative volume element size and by the finite element discretisation are studied, and the influence of the filter that is used in the generation process of the representative volume element is analysed., International journal of fracture;Bd 231. 2021, H. 2, S. 223-242

Byron P Vaughn,1 Thomas Kaiser,2,3 Christopher Staley,2,3 Matthew J Hamilton,2 Jon Reich,1 Carolyn Graiziger,1 Stephanie Singroy,2 Amanda J Kabage,1 Michael J Sadowsky,2,4,5 Alexander Khoruts1,2 1Inflammatory Bowel Program, Division of Gastroenterology, Hepatology and Nutrition, University of Minnesota, Minneapolis, MN, USA; 2BioTechnology Institute, University of Minnesota, St Paul, MN, USA; 3Department of Surgery, University of Minnesota, Minneapolis, MN, USA; 4Department of Soil, Water, and Climate, University of Minnesota, St Paul, MN, USA; 5Department of Plant and Microbial Biology, University of Minnesota, St Paul, MN, USA Introduction: Inflammatory bowel disease (IBD) is thought to arise from an abnormal immune response to the gut microbiota. IBD is associated with altered intestinal microbial community structure and functionality, which may contribute to inflammation and complications such as colon cancer and liver disease. Primary sclerosing cholangitis (PSC) is associated with IBD and markedly increases the risk of colon cancer. We hypothesized that secondary bile acids, which are products of microbial metabolism, are increased in PSC patients.Aim: Here, we profiled the fecal bile acid composition and gut microbiota of participants with IBD and PSC, as well as healthy participants. Additionally, we tested the effects of vancomycin, a proposed treatment for PSC, on gut microbiota and fecal bile acid composition in participants with IBD and PSC.Methods: Fecal samples were collected from patients with IBD, IBD/PSC and healthy controls and fecal bile acids and DNA for microbiota analysis were extracted. Fecal bile acids were averaged over a seven-day period. For subjects with IBD/PSC, oral vancomycin 500mg twice a day was administered and fecal samples were collected for up to eleven weeks.Results: Participants with IBD and PSC had less fecal microbial diversity at baseline relative to controls. While there was some evidence of altered conversion of cholic acid to deoxycholic acid, no substantial differences were found in the fecal bile acid profiles of patients with IBD and PSC (n=7) compared to IBD alone (n=8) or healthy controls (n=8). Oral vancomycin was a potent inhibitor of secondary bile acid production in participants with IBD and PSC, particularly deoxycholic acid, although no changes in liver biochemistry patterns were noted over a two week period.Conclusion: In this pilot study, bile acid profiles were overall similar among patients with IBD and PSC, IBD alone, and healthy controls. Microbiota diversity was reduced in those with PSC and IBD compared to IBD alone or healthy controls. Keywords: vancomycin, Crohn’s disease, ulcerative colitis

BACKGROUND & AIMS: A better understanding of prognostic factors in ulcerative colitis (UC) could improve patient management and reduce complications. We aimed to identify evidence-based predictors for outcomes in pediatric UC, which may be used to optimize treatment algorithms. METHODS: Potential outcomes worthy of prediction in UC were determined by surveying 202 experts in pediatric UC. A systematic review of the literature, with selected meta-analysis, was performed to identify studies that investigated predictors for these outcomes. Multiple national and international meetings were held to reach consensus on evidence-based statements. RESULTS: Consensus was reached on 31 statements regarding predictors of colectomy, acute severe colitis (ASC), chronically active pediatric UC, cancer and mortality. At diagnosis, disease extent (6 studies, N = 627; P = .035), Pediatric Ulcerative Colitis Activity Index score (4 studies, n = 318; P 10 years), male sex, and younger age at diagnosis. CONCLUSIONS: These evidence-based consensus statements offer predictions to be considered for a personalized medicine approach in treating pediatric UC.

BACKGROUND & AIMS: A better understanding of prognostic factors within the heterogeneous spectrum of pediatric Crohn's disease (CD) should improve patient management and reduce complications. We aimed to identify evidence-based predictors of outcomes with the goal of optimizing individual patient management. METHODS: Asurvey of 202 experts in pediatric CD identified and prioritized adverse outcomes to be avoided. A systematic review of the literature with meta-analysis, when possible, was performed to identify clinical studies that investigated predictors of these outcomes. Multiple national and international face-to-face meetings were held to draft consensus statements based on the published evidence. RESULTS: Consensus was reached on 27 statements regarding prognostic factors for surgery, complications, chronically active pediatric CD, and hospitalization. Prognostic factors for surgery included CD diagnosis during adolescence, growth impairment, NOD2/CARD15 polymorphisms, disease behavior, and positive anti-Saccharomyces cerevisiae antibody status. Isolated colonic disease was associated with fewer surgeries. Older age at presentation, small bowel disease, serology (anti-Saccharomyces cerevisiae antibody, antiflagellin, and OmpC), NOD2/CARD15 polymorphisms, perianal disease, and ethnicity were risk factors for penetrating (B3) and/or stenotic disease (B2). Male sex, young age at onset, small bowel disease, more active disease, and diagnostic delay may be associated with growth impairment. Malnutrition and higher disease activity were associated with reduced bone density. CONCLUSIONS: These evidence-based consensus statements offer insight into predictors of poor outcomes in pediatric CD and are valuable when developing treatment algorithms and planning future studies. Targeted longitudinal studies are needed to further characterize prognostic factors in pediatric CD and to evaluate the impact of treatment algorithms tailored to individual patient risk.

This manuscript reviews the primary and secondary endpoints of pivotal phase III trials with immunomodulatory drugs in multiple sclerosis (MS). Considering the limitations of previous trial designs, we propose new standards for the planning of clinical trials, taking into account latest insights into MS pathophysiology and patient-relevant aspects. Using a systematic overview of published phase III (pivotal) trials performed as part of application for drug market approval, we evaluate the following characteristics: trial duration, number of trial participants, comparators, and endpoints (primary, secondary, magnetic resonance imaging outcome, and patient-reported outcomes). From a patient perspective, the primary and secondary endpoints of clinical trials are only partially relevant. High-quality trial data pertaining to efficacy and safety that stretch beyond the time frame of pivotal trials are almost non-existent. Understanding of long-term benefits and risks of disease-modifying MS therapy is largely lacking. Concrete proposals for the trial designs of relapsing (remitting) multiple sclerosis/clinically isolated syndrome, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis (e.g., study duration, mechanism of action, and choice of endpoints) are presented based on the results of the systematic overview. Given the increasing number of available immunotherapies, the therapeutic strategy in MS has shifted from a mere "relapse-prevention" approach to a personalized provision of medical care as to the choice of the appropriate drugs and their sequential application over the course of the disease. This personalized provision takes patient preferences as well as disease-related factors into consideration such as objective clinical and radiographic findings but also very burdensome symptoms such as fatigue, depression, and cognitive impairment. Future trial designs in MS will have to assign higher relevance to these patient-reported outcomes and will also have to implement surrogate measures that can serve as predictive markers for individual treatment response to new and investigational immunotherapies. This is an indispensable prerequisite to maximize the benefit of individual patients when participating in clinical trials. Moreover, such appropriate trial designs and suitable enrolment criteria that correspond to the mode of action of the study drug will facilitate targeted prevention of adverse events, thus mitigating risks for individual study participants.

Hintergrund/Fragestellung: Die Suche in Studienregistern ist ein wichtiges Instrument zur Kontrolle von Publikationsbias und Standard bei der Erstellung von Cochrane Reviews. Es ist jedoch unklar, welchen Stellenwert die Studienregistersuche in Cochrane Reviews hat und wie Reviewautoren mit Studien[zum vollständigen Text gelangen Sie über die oben angegebene URL], EbM und Digitale Transformation in der Medizin; 20. Jahrestagung des Deutschen Netzwerks Evidenzbasierte Medizin

Francia, Bd. 34, Nr. 2 (2007)

The ability of squeezing and guiding light in nanoscale plasmonic waveguides makes them especially interesting for photonic circuits. In spite of reported realizations of plasmonic waveguides, experimental studies on the content of plasmonic modes and mode-selective excitation methods are rare. We apply here a Dual-SNOM technique, incorporating two aperture scanning near-field optical microscopes, for simultaneous near-field excitation and detection of plasmonic modes in gold strip waveguides. Depending on the waveguide width, either a single waveguide mode or a beating pattern of several modes is observed. The relative excitation strengths of the individual modes in multi-mode waveguides are shown to be controllable by the lateral position of the excitation tip. The excitation coefficients are described by an analytical model and the results are fully corroborated by analytical calculations and full-wave numerical simulations. The Dual-SNOM technique provides a "non-invasive" method of local excitation and detection of photonic modes thus making it a valuable tool for in situ characterization of complex photonic micro-and nanostructures.

Malgorzata Banys,1,2 Andreas D Hartkopf,1 Natalia Krawczyk,1 Tatjana Kaiser,1 Franziska Meier-Stiegen,1 Tanja Fehm,1 Hans Neubauer11Department of Obstetrics and Gynecology, University of Tuebingen, Tuebingen, Germany; 2Department of Obstetrics and Gynecology, Marienkrankenhaus Hamburg, Hamburg, GermanyAbstract: Tumor dormancy describes a prolonged quiescent state in which tumor cells are present, but disease progression is not yet clinically apparent. Breast cancer is especially known for long asymptomatic periods, up to 25 years, with no evidence of the disease, followed by a relapse. Factors that determine the cell's decision to enter a dormant state and that control its duration remain unclear. In recent years, considerable progress has been made in understanding how tumor cells circulating in the blood interact and extravasate into secondary sites and which factors might determine whether these cells survive, remain dormant, or become macrometastases. The mechanisms of tumor cell dormancy are still not clear. Two different hypotheses are currently discussed: tumor cells persist either by completely withdrawing from the cell cycle or by continuing to proliferate at a slow rate that is counterbalanced by cell death. Because dormant disseminated tumor cells may be the founders of metastasis, one hypothesis is that dormant tumor cells, or at least a fraction of them, share stem cell-like characteristics that may be responsible for their long half-lives and their suggested resistance to standard chemotherapy. Therefore, knowledge of the biology of tumor cell dormancy may be the basis from which to develop innovative targeted therapies to control or eliminate this tumor cell fraction. In this review, we discuss biological mechanisms and clinical implications of tumor dormancy in breast cancer patients.Keywords: tumor dormancy, disseminated tumor cell, circulating tumor cell, targeted therapy

Tl1Ba2Ca2Cu3Ox thin films, with and without Bi-substitution, have been fabricated by in-situ laser ablation on (001) SrTiO3 and (001) LaAlO3 substrates. Using transmission electron microscopy and allied techniques, the films have been characterised in cross-section. In the case of films grown without Bi-substitution, heavily defected microstructures with a-axis oriented grains and stacking-type planar defects resulting in discontinuous a-b planes have been observed. Films fabricated with Bi-substitution have highly defected micro structure s within about 150 nm of the substrate, with considerably less faulting further from the substrate where the bulk of the superconducting currents flow and the microwave properties are primarily determined. Bi-substituted films have been fabricated with critical current density values up to 8 X 105 A cm-2 (77 K) and surface resistances values as low as 56 mΩ (77 K, 87 GHz), which is equivalent to 0.74 mΩ at 10 GHz, assuming a frequency-squared relationship. © 1998 Elsevier Science B.V. All rights reserved.

Muscle regeneration is a multistep process that is regulated by a restricted number of transcription factors whose activity is modulated at multiple levels. However, how different layers of regulation are coordinated to promote adult myogenesis is not yet understood. Here we show that the MEF2C transcription factor controls multiple steps of muscle regeneration, including myogenic progression of satellite cells and muscle maturation of newly generated myofibers, exhibiting multiple functions that depend on alternative splicing and post-translational modifications. Inclusion of the α1 exon in Mef2c transcripts is upregulated in proliferating mouse satellite cells and in the early phases of muscle regeneration. The encoded MEF2Cα1 isoform stimulates expansion of primary myoblasts ex vivo and in vivo. The pro-proliferative activity of MEF2C is mediated by phosphorylation of two phosphoserines located in exon α1. Subsequent terminal differentiation and growth of newly formed myofibers are promoted by dephosphorylated MEF2Cα1 and MEF2Cα2. Our results thus reveal an important role for regulatory interactions between alternative splicing and post translational modifications of a single transcription factor in the control of the multilayered regulatory programs required for adult myogenesis., Skeletal muscle exhibits a high capacity to regenerate, mainly due to the ability of satellite cells to replicate and differentiate in response to stimuli. Epigenetic control is effective at multiple steps of this process. The chromatin remodeling factor, HDAC4, is up-regulated in skeletal muscle upon injury, suggesting a role for this protein in muscle regeneration. With the aim to elucidate the role of HDAC4 in satellite cells and skeletal muscle regeneration, we generated inducible mice lacking HDAC4 in Pax7+ cells (HDAC4 KO mice). Despite having similar amount of satellite cells, HDAC4 KO mice show impaired muscle regeneration in vivo, and compromised satellite cell proliferation and differentiation in vitro. HDAC4 deletion in satellite cells is sufficient to block their differentiation, not acting via soluble factors, and possibly through the inhibition of Pax7 expression. The molecular mechanisms underling compromised muscle regeneration in HDAC4 KO mice are currently under investigation. All together, these data delineate the importance of HDAC4 in satellite cell differentiation and suggest a protective role of HDAC4 in response to muscle damage., The adult skeletal muscle has the ability to self-renew and repair in response to increased workload, stress conditions or limited damage. These properties rely on an array of different progenitor cell populations. While satellite cells play a central role in muscle regeneration, a variety of other mononuclear progenitor cells, either resident in the muscle or recruited from the blood stream, contribute to the complex crosstalk leading to muscle repair. In pathological conditions or with aging, the relative abundance and the activation stage of the different cell populations in the myogenic stem cell compartment vary. The ability to probe the heterogeneity and the dynamic of the muscle tissue is fundamental to achieve a complete understanding of muscle regeneration. To this end we have invested in a novel approach exploiting mass cytometry technology (CyTOF2 platform). CyTOF technology enables probing single cell events, by labelling intracellular and cell surface markers with up to 40 antibodies tagged with stable heavy metal isotopes. The sharp mass peaks obtained by TOF inductively coupled plasma mass spectrometry eliminates the problems of spectra overlap typical of fluorescence based flow cytometry. I will describe the panel of tagged antibodies that I have developed to characterize the heterogeneous muscle mononuclear cell populations and the advantages and limitations of mass cytometry. In addition I will present preliminary data on the dynamic of cell populations under different conditions and stimuli., The mechanisms that regulate skeletal muscle development involve the coordinated activity of transcription factors (TFs) and a precise timing of gene expression patterns. NF-Y is a heterotrimeric TF with a pioneer role in the transcriptional and epigenetic regulation of promoters containing the CCAAT-box. NF-Y activates the expression of various genes related to the cell cycle, particularly genes of the G2/M phase. NF-YA, the regulatory DNA-binding subunit of the complex, is expressed in proliferating myoblasts and down-regulated during terminal differentiation. The NF-YA gene encodes for two alternatively spliced isoforms, namely NF-YAs and NF-YAl, which are not functionally identical. Using mouse C2C12 cells, we provide evidence of a different role for NF-YA variants in the myogenic program. While NF-YAs enhances myoblasts proliferation, NF-YAl boosts their differentiation by up-regulating the transcription of novel target genes, among which Mef2D, Sixs and Cdkn1C, which are known to be involved in the differentiation program. We further demonstrate that NF-YA is expressed in resident stem cells (SCs) and the two isoforms are transcribed at different levels during SCs activation and differentiation. The inhibition of NF-Y activity impairs both proliferation and differentiation of SCs and the overexpression of the two NF-YA isoforms differentially affects their fate., Sarcopenia is the age-related loss of muscle leading to loss of muscle power, which in the end results in frailty and disability. At molecular level, sarcopenia is characterized by insufficient antioxidant defense mechanism, increased oxidative stress and altered function of respiratory chain. It has been hypothesized that the accumulation of oxidative stress is also related to an impaired regeneration cooperating to the atrophic state that characterizes muscle ageing. To the purpose, we investigated the myogenic process in satellite cells, the skeletal muscle stem cells, as myoblasts and myotubes collected by human Vastus Lateralis skeletal muscle of young and old subjects through needle-biopsies. To measure both the O2- and ROS level we used NBT and H2DCF-DA assays revealing higher concentration in elderly myoblasts compared to young ones. To evaluate if mitochondria are damaged by ROS we measured mitochondrial transmembrane potential after an oxidant insult as H2O2. We found that in elderly myoblasts mitochondrial transmembrane potential decreases much more than in young ones probably due to their lower endogenous antioxidant abilities. Specifically, MitoSOX™ Red reagent for direct measurements of O2- in mitochondria revealed that in elderly myoblasts O2- production is increased respect to young ones and the result is worsened in myotubes. Furthermore, the upregulation of the atrophic and ubiquitin-proteasome pathways together with a dysregulation of the proliferative one revealed an alteration at gene expression level in elderly myoblasts vs young ones. Overall our data confirm that oxidative stress impairs muscle regeneration in elderly subjects., Skeletal muscle is a complex structure endowed with extreme regenerative capability; this ability relies on the orchestrated interplay between different muscle populations that reside within the tissue. Functional changes occurring at the microenvironmental level during aging or pathological conditions however interfere with this ability leading to fibrosis and fat infiltration. Despite a large body of work still we are far from completely understanding these changes; even when genetic cause is known (e.g. Duchenne muscular Dystrophy) we are still unable to pin-point the steps that lead from the molecular cause to the outcome of the disease. The main reason for this bottleneck is that our knowledge has been limited so far by the lack of technical tools to dissect the heterogeneity of these populations. The use of bulk-scale methods able only to provide averaged information has frustrated our effort to characterize those pathological changes leaving those dysfunctional, disease-specific subpopulation to remain hidden within the bulk. Here we present a novel approach based on single cell mass spectrometry to study the populations that reside in the muscle. Using Cytof technology we would profile at single cell resolution the muscle resident populations during aging and in diseased state. This would allow us to identify dysfunctional subsets involved in the regeneration defect. This study would not only shed light on the mechanisms underpinning muscle regeneration but would provide a solid ground for the future identification of diagnostic biomarkers through the study of disease specific subpopulations., Hypertrophy and dystrophy are distinct, yet linked processes that remodel both skeletal and cardiac muscles in physiological or pathological settings. Not only is hypertrophy important during development, but it also plays major role upon acute or chronic damage. Muscular dystrophies (MDs) cause progressive degeneration and loss of functionality in both striated muscle types. In MD patients and animal models, an initial hypertrophic response occurs, with contrasting effects on skeletal and cardiac muscle. Recently it has been established that muscle fibers secrete exosomes, whose cargo acts as endocrine signals during myogenesis. We aim at deciphering the exosomal information guiding hypertrophy/dystrophy in both muscle in order to establish a new strategy based on miRNA modulation for novel myogenic regeneration. We performed ex vivo exosome analysis comparing age-matched WT, Sgcb-null (dystrophic), and MAGIC-F1+/+ (hypertrophic) mice. We detected several differentially regulated miRNAs, virtually relevant for striated muscle remodeling and de-/re-generation. We have preliminary results on the effects of ex vivo exosomes on cell types relevant for skeletal and cardiac muscle analysis. Moreover we are currently investigating the uptake routes of exosomes in both muscle types. In the future we will rely on miRNA-sequencing of ex vivo exosomes, to identify key mRNA/miRNA distinctive signatures by means of an high-throughput approach and place our ongoing results into a genome-wide setting. As a final goal, the hypertrophic/dystrophic signatures and tissue-specific information will further be integrated to establish skeletal- and cardiac-enhancing cocktails to selectively improve the regenerative outcome of patient-specific progenitors in vivo, into a xenograft-permissive murine model., Duchenne muscular dystrophy (DMD) is the most common, lethal, inherited myopathy, which results in muscle degeneration. In this work, we aimed at developing an innovative 3D satellite cell niche derived from human induced pluripotent stem cells (hiPSC) within their native sublaminal position in an engineered human skeletal muscle myofiber. One of the main limitations of cell therapy for DMD is the high number of myogenic cells required and the efficiency of engraftment in vivo. hiPSC ensure large amount of cells and the possibility of derive patient-specific cells, but obtaining myogenic cells in vitro from hiPSC is difficult and the yield is low. In this work, we induced the myogenic differentiation of hiPSC through multiple transfection of modified mRNA of the master transcription factors MYOD, PAX3 and PAX7. To this aim, we exploited a microfluidic platform that allows the downscaling of the process for performing cost-effective, multiparametric and highthroughput experimental investigations. We optimized the protocol for transfecting hiPSC colonies leading to a transfection efficiency of 60% per single transfection. After multiple transfections, exogenous MyoD is expressed in 95% of the cells and endogenous expression of desmin and myosin heavy chain was observed (4 days after the last transfection). Ongoing experiments are extending these results to Pax3 and Pax7. Another key factor for a successful cell therapy is the cell delivery. In this sight, we developed a 3D poly-acrilammide/hyaluronic acid hydrogel (HY) scaffold and optimized its biochemical and mechanical properties in order to sustain the myogenic differentiation of human primary myoblasts and to reproduce the protective microenvironment of the satellite cell niche. The scaffold was designed in order to control the cell topology: 3D parallel micro-channels (80-160 μm in diameter, 10-15 mm long) were produced inside the scaffold and functionalized with ECM proteins. To reproduce the physiological mechanobiology, HY chemical composition was optimized in order to obtain a soft scaffold with physiological elastic modulus, E≈12±4kPa. Human primary myoblasts were used to optimize the seeding, culture and differentiation protocols. At 10 days, we observed tightly packed myotubes bundles, expressing myosin heavy chain, α-actinin and dystrophin. We are now integrating hESC-derived myoblast and we observed differentiation into myoubes and expression of myosin heavy chai, α-actinin and desmin.We hypothesize that such engineered niche will provide, upon in vivo implantation, satellite cells able to regenerate the damaged muscle of DMD patients, and reconstitute the stem cell pool for future muscle damages. On the other hand, our 3D niche could be exploited as an in vitro tool to study the biology of the niche itself, the mechanism of the pathology or as a tool for testing new drugs and therapies in a personalized manner., Skeletal muscle regeneration is mediated by a complex crosstalk between various resident mononucleated cell populations. These cell interactions after fiber damage or stress are finely regulated in time and space. Satellite cells, skeletal muscle stem cells, play a pivotal role during regeneration being the main source of new myoblasts. However, their activation, proliferation and differentiation relies on environmental cues shaped by cell populations such as macrophages, pericytes, and fibro-adipogenic progenitors (FAPs). FAPs have a leading role in the regeneration process since they promote myotube formation by positively regulating satellite cell differentiation. However, in pathological conditions, such as muscular dystropies, these cells play a negative role since they are responsible for fibrosis and fatty tissue accumulation. In in vitro experiments we have observed an improvement in the maturation of myotubes derived from satellite cells, when co-cultured with FAPs. Furthermore, we have also observed that direct contact of these two cell populations inhibits adipogenic differentiation of FAPs while in the transwell system this inhibition does not occur. Even though there is a clear interaction between these two populations, it has not been thoroughly characterized yet. Thus exploiting Luminex technology we are aiming at identifying molecules affecting the differentiation process of these two cell types focusing on cytokines, chemokines and growth factors. In addition we are planning to include in these studies macrophages and pericytes in order to obtain a more complete picture of molecular networks involved in myogenesis and finally build a cell-cell interaction model of skeletal muscle regeneration., Electrical stimulation (ES) of skeletal muscle has been proposed to mimic the beneficial effects of physical training and to counteract the muscle atrophy associated with reduced motor activity. If properly used, it can be a potent tool to increase strength and endurance in patients affected by muscle weakness due to ageing or prolonged debilitating illness. However, classical ES exhibits several limitations, such as the unpleasant symptoms due to pulse strength and the occurrence of muscle fatigue. The most appropriate parameters of stimulation, such as intensity, frequency and pulse duration, are still under debate. Field ESs were given to mouse skeletal myotubes in culture. Changes in membrane potential were detected by perforated patch recordings and calcium dynamics was followed using fluorescent indicators. Different patterns of ES were tested. Tetanic high frequency stimulation at 45 Hz induced voltage changes invariably characterized by failures, and discontinuous firing preceding the complete disappearance of the electrical activity, whereas low-frequency stimulations at 1 Hz more efficiently elicited single action potentials. An innovative “noisy” waveform ES pattern was tested, obtained from a segment of electromyogram recording, sampled from a limb muscle of adult volunteers during the execution of a rhythmic motor activity. Using half of the intensity of stimulation employed for more stereotyped ES patterns, it was found to be more efficient in inducing repetitive cell firing, calcium transients and cell twitching. We suggest this approach as a new strategy for the design of new electrical devices able to provide a therapy option for injured muscles in human patients., Muscle contraction is generated by cyclical interactions of myosin heads with actin filaments to form the actomyosin complex. The stable configurations of the actomyosin complex have been described in detail, but whether in vivo, at physiological temperatures, these configurations are fixed to the ones observed in cryomicroscopy (at low temperature) or undergo thermal oscillations is unknown and not generally considered in mathematical modeling. By comparing three mathematical models, we analyze how this intrinsic property of the actomyosin complex affects muscle contraction at three level; namely, single cross-bridge, single fiber and organ levels, in a ceteris paribus analysis. We observed that state fluctuations allow the lever arm of myosin to easily and dynamically explore all possible minima in the energy landscape, generating several backward and forward jumps between states during the lifetime of the actomyosin complex, whereas the rigid case is characterized by fewer force generating events. Therefore, dynamical oscillations enable an efficient contraction mechanism, in which a higher force is sustained by fewer attached cross-bridges., Mammalian neuromuscular junctions (NMJs) undergo a postnatal topological transformation from a simple oval plaque to a complex branch-shaped structure often called a “pretzel”. Although abnormalities in NMJ maturation and/or maintenance are frequently observed in neuromuscular disorders, such as congenital myasthenic syndromes (CMSs), the mechanisms that govern synaptic developmental remodeling are poorly understood. It was reported that myotubes, when cultured aneurally on laminin-coated surfaces, form complex postsynaptic machinery, which resembles that at the NMJ. Interestingly, these assemblies of postsynaptic machinery undergo similar stages in developmental remodeling from “plaques” to “pretzels” as those formed in vivo. We have recently demonstrated that podosomes, actin-rich adhesive organelles, promote the remodeling process in cultured myotubes and showed a key role of one podosome component, Amotl2. We now provide evidence that several other known podosome-associated proteins are present at the NMJ in vivo and are located to the sites of synaptic remodeling. Additionally, we identified proteins that interact with Amotl2 in muscle cells. We show that two of them: Rassf8 and Homer1, together with other podosome components, are concentrated at postsynaptic areas of NMJs in the indentations between the AChR-rich branches. Our results provide further support for the hypothesis that podosome-like organelles are involved in synapse remodeling and that Rassf8 and Homer1 may regulate this process., Depletion of calcium (Ca2+) from intracellular stores (endoplasmic reticulum, ER), triggers Ca2+ entry across the plasma membrane, a process known as store-operated Ca2+ entry (SOCE). SOCE is mediated by the interaction between STIM1 (stromal interaction molecule 1), which functions as the Ca2+ sensor in the ER, and Ca2+ permeable Orai1 channels in the external membrane. In skeletal muscle, SOCE is the primary mechanism of Ca2+ entry during repetitive activity, a crucial step that prevents/delays fatigue. Despite the importance of this mechanism for proper muscle function during sustained activity, the subcellular sites for SOCE in skeletal fibers have not been identified. Here we show that prolonged muscle activity (treadmill running in mice) drives the formation of previously unidentified intracellular junctions between the sarcoplasmic reticulum (SR) and extensions of the external transverse tubule (TT) membrane. The activity-dependent formation of these unique SR-TT junctions reflects a striking and unexpected remodeling of the existing sarcotubular system at the I band of the sarcomere. Using immunochemistry and immuno-gold labeling we also demonstrate that these newly formed, activity-driven junctions contain the molecular machinery known to mediate SOCE in muscle: STIM1 Ca2+ sensor proteins in the SR, already present in the I band in control conditions, and Ca2+- permeable Orai1 channels, which move into the I band with TTs during prolonged muscle activity. Thus, we refer to these junctions as Ca2+ Entry Units, the first new, molecularly defined subcellular structure in skeletal muscle in over 30 years., The loss of connection between muscle and nerve is a crucial biological mechanism involved in Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease associated with motor neuron degeneration, muscle atrophy and paralysis [1]. Recent studies showed the primary role of the skeletal muscle in the pathogenesis of the disease, pointing out the key role of the communication between muscle and nerve. In this context, we developed a protocol to measure, ex vivo, the neuromuscular junction (NMJ) functionality [2]. The experimental technique is based on the comparison between the muscle contractile response elicited by membrane stimulation and the response evoked by nerve stimulation. Since this latter stimulation bypasses the neuronal signalling, any difference between the two responses may be related to NMJ alterations. In particular, we started studying the Soleus-sciatic nerve preparation of one of the most studied ALS animal models, the SOD1G93A mouse [3], with the particular aim of following the pathology’s progress. We observed that the first functional alterations begin at 90 days of age, with an intrinsic damage of the muscle and defects in NMJ functionality who increase until the end-stage of the disease. Subsequently, we approached the study of the MLC/SOD1G93A mouse model, in which superoxyde dismutase-1 mutated gene is expressed exclusively in the skeletal muscle [4]. Our preliminary results highlighted defects in soleus muscle and NMJs functionality in MLC/SOD1G93A mouse model, compared to the wild type, suggesting a direct muscle impairment. Further analysis on this model will provide useful information about the NMJ alterations directly related to oxidative stress on skeletal muscles., Myosin is an abundant ATPase protein. It is estimated that 10% of muscle tissue weight is myosin. Due to its abundance, myosin can be a good target to raise basal metabolic rate in animals. A new low-ATP-consumption state of myosin has recently been proposed (1, 2). This new state has been called the “Super Relaxed State (SRX)” of Myosin. Structural evidence for the SRX state have recently been published showing a close complex formed by the two-myosin heads (3). It is characterized by an ATPase time constant in the order of 300 seconds versus the 15 seconds for the so-called “Disordered Relax State” (DRX)(1,2). The idea is that behind that large number of “dormant” ATPases, there is the key to raise basal metabolism in a physiological way. The amount of myosin in the SRX state is estimated to be approximately 60% of the total. Switching of the myosin heads from the SRX state to the DRX state is regulated by phosphorylation in a cooperativeness-driven-equilibrium. Controlling this equilibrium may lead to an increase in basal metabolism that would consume an additional energy of up to 1000 Kcal/day. We studied the effect of several Regulatory Light Chain mutants on the SRX state and we applied this information to the development of a high throughput screen. We are searching a molecule that is able to destabilize the SRX state in skeletal muscle fibers. We screened 2000 compound of an FDA approved library. Potential lead compounds will be discussed, The muscle cell is a biological machine where steady force and shortening are generated by arrays of the motor protein myosin II pulling the actin filament towards the centre of the sarcomere (the ~2 μm long structural unit of muscle) during cyclical ATP-driven interactions. The fraction of the time of the ATP hydrolysis cycle that myosin II spends attached to actin depends on the sarcomere load and at low load can be as small as 0.02. The array-type arrangement of the motors enhances and makes steady the production of force and shortening, but has so far limited the investigation of mechanics, energetics and structural dynamics of this collective motor to top-down approaches, such as single-cell mechanics and X-ray diffraction (Piazzesi et al. Cell 131:784-795, 2007). The laser trap technique in the Three Bead Assay (TBA) configuration allowed the recording of single actin-myosin interaction in vitro, but only when the duration of attachment was increased by reducing the ATP concentration to a few tens of micromolar (two orders of magnitude lower than that in situ in physiological conditions). In this project we use an alternative approach consisting in assembling molecular motor proteins on a nanostructured support to generate a synthetic sarcomere-scale machine, the mechanical output of which is measured with a double laser optical tweezers apparatus (Bianco et al. Biophys. J. 101:866-874, 2011). The shape, the material and the coating of the support carrying the motor array have been optimised using a preliminary version of the machine consisting of an ensemble of motor proteins randomly adsorbed on a flat surface and brought to interact with an actin filament attached to the trapped bead with the correct polarity. Tests on the density and the disposition of the myosin motors on the surface have been done using AFM. The most reproducible results have been obtained when the support for the motor ensemble is the lateral surface of a chemically etched single mode optical fibre (diameter 4 µm). In solution with physiological [ATP] (2 mM), the ensemble drives 350 nm of actin filament sliding developing a steady force of 50 pN. Supported by Italian Institute of Technology-SEED, project MYOMAC (Genova) and PRIN-MIUR, Italy., Skeletal muscle atrophy is caused by several and heterogeneous conditions, such as cancer (cachexia), neuromuscular disorders and aging. In most types of skeletal muscle atrophy overall rates of protein synthesis are suppressed, protein degradation is consistently elevated and atrogenes, such as the ubiquitin ligase Atrogin-1/MAFbx, are up-regulated. Sphingolipids represent a class of bioactive molecules capable of modulating the destiny of many cell types, including skeletal muscle cells. In particular, we and others have shown that sphingosine 1-phosphate (S1P), formed by sphingosine kinase (SphK), is able to act as trophic and morphogenic factor in myoblasts. Here, we report that the inhibition of SphK1 by specific gene silencing or pharmacological inhibition drastically reduced myotube size and myonuclei number, and increased Atrogin-1/MAFbx expression. Notably, the atrophic phenotype of C2C12 myotubes treated with dexamethasone and of muscle fibers obtained from cachectic mice inoculated with C26 adenocarcinoma, was characterized by increased expression of Atrogin-1/MAFbx and reduced levels of active SphK1. In addition, we found that C2C12 muscle cell atrophy was accomplished by changes in the pattern of expression of S1P receptor subtypes (S1PRs) and treatment of myotubes with S1P was able to prevent Dexa-induced atrophic marker expression. Finally, by using specific S1PR agonists and antagonists, we extended the investigation on the role played by S1PRs in the control of Atrogin-1/MAFbx expression. Altogether, these findings provide the first evidence that S1P/SphK1/S1PR axis acts as a molecular regulator of skeletal muscle atrophy, thereby representing a new possible target for therapy in many physiological and pathological conditions., Skeletal muscle is a dynamic tissue that can respond to external stimuli through both anabolic and catabolic processes. In a variety of conditions, including immobilization, AIDS and neuromuscular disorders, skeletal muscle mass is decreased (atrophy). Upon denervation or disuse, skeletal muscle undergoes atrophy, leading to reduced size of myofibers, impaired contractile and metabolic activities. Previous studies have identified key molecular pathways leading to protein breakdown and degradation of sarcomeric proteins; yet, it remains a gap of knowledge on whether muscle resident cell populations can regulate the response of muscle to atrophic stimuli. Indeed, the recent identification of muscle-derived interstitial cells, named fibro-adipogenic progenitors, that can adopt multiple lineages and contribute, either directly o indirectly, to muscle regeneration (Joe et al,2010; Uezumi et al,2010) indicates a previously unrecognized complexity in the regulation of muscle homeostasis (Saccone et al,2014). We have discovered an unexpected key role of specific muscle-derived mononuclear cells in the pathogenesis of muscle atrophy. The characterization of the mechanism by which these cells contribute to the loss of muscle mass may lead to the identification of new therapeutic targets to counteract muscolar atrophy., PGC-1α overexpression is able to protect skeletal muscle from fasting or denervation-induced atrophy (1) and to improve sarcopenia in old mice (2). Consistently, in the skeletal muscle of cachectic tumor-bearing mice, PGC-1α expression is reduced (3), in Association with the accumulation of PAX7+ cells, which is suggestive of an impairment of myogenesis (4). Preliminary observations obtained in mice overexpressing PGC-1α specifically in the skeletal muscle show that the number of CD34+/Sca1+ cells, both integrin-α7 positive (satellite cells) and negative (other myogenic precursors), was higher in the muscle of transgenic (TG) mice than in those of wild-type (wt) animals. Not only, myotubes originating from TG-derived myogenic precursors were increased in both number and size in comparison to those obtained from wt progenitors. Aim of the present study was to investigate if PGC-1α overexpression can improve the regenerative capacity in the muscle of tumor (C26)-bearing animals after chemically-induced injury. BaCl2 (30 μl, 1.2% w/v) was injected in the tibialis anterior muscle the day after tumor implantation. Thirteen days after injury, both wt and TG controls almost completely recovered the initial myofiber cross sectional area (CSA; 70% of uninjured muscle). By contrast, CSA recovery was markedly delayed in wt or TG tumor-bearing mice (30% of uninjured muscle). Such a lack of CSA rescue in TG C26 hosts occurred despite TG mice constitutively possess a number of myogenic precursors higher than wt animals. As an estimate of mitochondria number, cytochrome c expression was evaluated. The results show that cytochrome c levels were significantly reduced in the regenerating muscle of wt C26 hosts, while remained comparable to those of uninjured muscle in BaCl2-treated TG tumor bearers. Previous observations showed that inhibition of ERK activity improved muscle wasting and myogenesis in the C26 hosts (4). In this regard, muscle pERK levels were significantly lower in TG tumor bearers than in wt C26 hosts. In conclusion, the present study shows that PGC-1α overexpression in the regenerating muscle of tumor hosts resulted in improved mitochondrial mass, and likely, oxidative capacity, and in reduced pERK levels, however without obtaining a significant CSA rescue. These observations suggest that while PGC1α overexpression exerts positive effects on tumor-induced derangements at the molecular level, it does not appear able to impinge on the multifactorial nature of muscle wasting., Cancer cachexia is a life-threatening syndrome that affects most patients with advanced cancers and involves severe body weight loss, with rapid depletion of skeletal muscle. No effective treatment is available. We analyzed microarray datasets to identify a subset of genes whose expression is specifically altered in cachectic muscles of Yoshida hepatoma-bearing rodents, but not in those with diabetes, disuse, uremia or fasting. By Ingenuity Pathways Analysis, we found three genes belonging to the CXCR4 pathway downregulated only in muscles atrophying because of cancer: SDF1, PAK1 and ADCY7. Consistently, we show that the expression of all SDF1 isoforms declines also in Tibialis Anterior from cachectic mice bearing colon adenocarcinoma or renal cancer and anti-cachexia drugs such as sunitinib restore it. Overexpressing genes of this pathway (i.e. SDF1 or CXCR4) in cachectic muscles increases the fiber area by 20%, partially protecting them from wasting. The mechanisms behind this muscle preservation during cachexia include both reduced degradation of long-lived proteins, by either SDF1α or SDF1β on atrophying myotubes, and increased protein synthesis, mainly by SDF1α. However, inhibiting CXCR4 signaling with the antagonist AMD3100 does not affect protein homeostasis in atrophying myotubes at all, whereas normal myotubes treated with AMD3100 display decreased diameter in a time- and dose-dependent manner, until a plateau. This further confirms the involvement of a saturable pathway (i.e. CXCR4). Overall, these findings support the idea that activating the CXCR4 pathway in muscle suppresses the deleterious wasting associated with cancer., Cancer cachexia is a systemic syndrome that consists of a dramatic weight loss with rapid muscle depletion due to enhanced protein degradation, irrespective of food intake. Remarkably, 50% of advanced cancer patients are affected by cachexia, which accounts for approximately 20% of cancer deaths. No therapy is available. Interestingly, females are more resistant to cancer cachexia than males. We analyzed previous microarray datasets to identify genes whose expression is specifically altered in cachectic muscles of Yoshida hepatoma-bearing male rodents. Among these genes, we found that apelin was drastically downregulated to 8% of controls in cachectic gastrocnemius muscles (with 14% of weight loss) from male rats bearing Yoshida hepatoma for 5 days. We confirmed by Q-PCR that apelin was downregulated to 45% and 2% of controls also in Tibialis Anterior (TA) muscles in Lewis Lung Carcinoma and in Colon Adenocarcinoma 26 (C26)-bearing mice, respectively. Moreover, in TA from C26-bearing mice also the expression of apelin receptor (APJ), a member of G-protein coupled receptors, was reduced to 16%. Q-PCR analysis further confirmed that apelin downregulation occurred at all stages of cancer cachexia of C26-bearing male mice, while the expression of APJ was significantly reduced to 30% of controls only in early cachectic mice with less than 14% of body weight loss. Since apelin is expressed on X chromosome both in humans and mice and it is not downregulated in muscles from C26-bearing female mice, we believe that apelin could be a good candidate to explain the gender difference of cancer cachexia., Cachexia is a syndrome frequently occurring in cancer patients. It is characterized by body and skeletal muscle wasting and by metabolic abnormalities. These latter are mediated, partially at least, by humoral factors. Energy balance perturbations also contribute to the onset of cachexia. In this regard, impaired mitochondrial functions and altered energy expenditure likely play a role. Recent observations suggest that in addition to classical humoral factors such as hormones or cytokines, also tumor-derived microvesicles (MVs), circulating particles containing different molecules such as proteins, mRNAs and microRNAs, may contribute to derangements associated with cachexia (1). MVs were isolated by differential ultracentrifugation from the conditioned medium of LLC (Lewis Lung Carcinoma) cells and were quantified by a NanoSight apparatus. After five day culture in differentiation medium, C2C12 myotubes were treated for 24 h with LLC-derived MVs. In C2C12 myotubes tumor-derived MVs induce a reduction of PGC-1α, the master regulator of oxidative metabolisms and mitochondrial biogenesis, as well as of Cyt-c mRNA expression. These results are in agreement with previous observations showing decreased PGC1α expression in the skeletal muscle of cachectic mice. In myotubes oxygen consumption is significantly decreased while lactate levels in the culture medium are increased after treatment with MVs. BNIP3 mRNA expression is significantly increased, while no differences can be observed as for myotube size and mRNA expression of both Atrogin1 and MuRF-1, two muscle-specific ubiquitin ligases. These results suggest that tumor-derived MVs affect mitochondria in C2C12 cultures. The reduction of mitochondrial mass (decreased Cyt-c mRNA expression) and function is associated with down-regulation of PGC-1α expression and enhancement of selective autophagy (mitophagy). On the whole, MV-induced alterations could contribute to muscle wasting during cancer cachexia., High mobility group box 1 (HMGB1) is a nuclear protein that acts extracellularly as an alarmin to modulate inflammation and tissue repair by recruiting cells and promoting their migration and activation. Recently, we showed that HMGB1 orchestrates both processes by switching among mutually exclusive redox states. Fully reduced HMGB1 acts as a chemoattractant, whereas a disulfide bond makes it a proinflammatory cytokine and further cysteine oxidation by reactive oxygen species (ROS) abrogates both activities. The fully reduced HMGB1 is prevalent in the extracellular environment immediately after acute muscle injury, and disulfide- HMGB1 appears a few hours later. Thus, the generation of ROS during muscle damage might modulate the redox status of the protein and eventually limit its lifespan and functions. We created a mutant (3S-HMGB1) not susceptible to redox modifications and we evaluated its regenerative activity in a model of acute muscle injury induced by cardiotoxin. We demonstrated so far that HMGB1 has beneficial effects in skeletal muscle regeneration after acute injury by dramatically increasing the number of healthy fibers and the number of satellite cells and M2c macrophages, two cell types essential for muscle repair. Moreover, HMGB1 acts directly on primary myoblasts by inducing their migration and their fusion to form large myotubes. Remarkably, 3S-HMGB1 behaves as a superagonist of HMGB1 in vivo, suggesting that it is a promising candidate for muscle repair therapies. Our study will be extended to other models of muscle damage, in particular dystrophies, in order to evaluate the therapeutic potential of 3S-HMGB1 in chronic conditions., Atrophy is an active process controlled by specific signaling pathways and transcriptional programs. The identification of the precise signaling cascades that regulate muscle wasting remains poorly understood. The Ubiquitin Proteasome System (UPS) is one of the major systems that control protein breakdown during muscle wasting. The specificity of ubiquitin-dependent degradation derives from many E3s that recognize specific substrates. This work is focus on a novel muscle-specific circadian-rhythm dependent ubiquitin ligase named Asb2β. To dissect its role, we have generated muscle specific and tamoxifen-inducible muscle specific knock-out mice. We have characterized these knockout mice in physiological and in catabolic conditions. Asb2β defective muscles show normal muscle morphology and mitochondrial content but muscles display a fiber type switch and glycogen accumulation. Glucose tolerance test revealed an improved glucose uptake in knockout mice. Moreover, glycogen content dramatically decreased in Asb2β knockout mice during fasting. The changes in glucose homeostasis are Akt independent but TBC1D1and AS160 dependent. However, absence of nutrients triggers necrotic degeneration and appearance of abnormal mitochondria in Asb2β-null muscles. We have also started to characterize the tamoxifen-inducible knockout mice. Preliminary data show that acute inhibition of Asb2β induces a time dependent muscle growth. In conclusion, we have identified a novel muscle specific ubiquitin ligase, Asb2β, that plays an important role in glucose homeostasis and muscle hypertrophy., Aging is characterized by loss of skeletal muscle mass and function, condition known as sarcopenia. The mechanisms underlying sarcopenia are not completely understood, however a role for ectopic fat accumulation has been proposed. Skeletal muscle accumulates lipid in form of triglycerides within lipid droplets (LDs). LDs are characterized by the presence of Perilipins (Plins), that control lipid accumulation and metabolism under physiological and pathological conditions. In skeletal muscle one of the most representative is Plin2, particularly involved in lipid storage. However, the exact role of Plin2 is not still clear. We found that in human muscle the expression of Plin2 increases with aging and it is inversely associated with muscle mass and strength. Moreover, Plin2 expression is associated with atrophy-related genes, MuRF-1 and Atrogin, suggesting a role for Plin2 in muscle aging and atrophy. We also analysed the expression of Plin2 in adult mice where muscle atrophy was induced by denervation. Denervation of tibialis anterior muscle was compared with the contralateral intact side. After denervation, beside the expected increase of MuRF-1 and Atrogin, also Plin2 expression actually increases. This suggested that Plin2 expression is somehow associated with muscle atrophy. To support this hypothesis, we performed muscle-specific in vivo silencing experiments of Plin2. After 7 days from injection, a decrease of Plin2 was observed, and most interestingly the cross-sectional area (CSA) of Plin2-negative fibres resulted increased of about 30% with respect to Plin2-positive ones. As a whole, these data suggest that in skeletal muscle Plin2 is involved not only in muscle atrophy, but also in hypertrophy. Further studies are ongoing to better clarify this new role of Plin2 in skeletal muscle., The rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in people under 20 years of age. It can commonly arise anywhere in the body but the head and neck, the extremities and the genitourinary tract are predominant sites. Based on histology, RMS tumors are classified into two major subtypes, embryonal and alveolar, which also differ in the molecular pathogenesis of development. Despite these differences, the origin of aRMS and eRMS seems to be the same but the precise cell type that triggers RMS is still unclear. Some evidence supports the notion that skeletal muscle precursors, probably satellite cells, may initiate RMS. Alternative theories propose mesenchymal stem cells, or even cells belonging to the adipocyte lineage, as possible tumor-initiating cells. In order to shed light on the origin of eRMS, we adopted the KrasG12D/+Trp53Fl/Fl conditional mouse model. This model allows us to generate eRMS in the hind limb of mice by infecting them with an adenovirus vector carrying the CRE recombinase. In a first approach we want to describe and rationalize the changes in the tumor mass cell populations by analyzing the tumor at different stages of development by using flow and mass cytometry techniques. In a second approach we aim at identifying the cell population(s) that are responsible for initiating the tumor. To this end we induce the gene mutations that are responsible for rhabdomyosarcoma development by infecting, with the CRE recombinase adenovirus, isolated muscle mononucleate cell populations and monitor their ability to develop rhabdomyosarcoma tumorigenic properties in vitro., The purpose of this study was to investigate whether MURC/cavin-4, a plasma membrane and Z-line associated protein exhibiting an overlapping distribution with Caveolin-3 (Cav-3) in heart and muscle tissues, may be expressed and play a role in rhabdomyosarcoma (RMS), an aggressive myogenic tumor affecting childhood. We found MURC/cavin-4 to be expressed, often concurrently with Cav3, in mouse and human RMS, as demonstrated through in silico analysis of gene datasets and immunohistochemical analysis of tumor samples. In vitro expression studies carried out using human cell lines and primary mouse tumor cultures showed that expression levels of both MURC/cavin-4 and Cav-3, while being low or undetectable during cell proliferation, became robustly increased during myogenic differentiation, as detected via semi-quantitative RT-PCR and immunoblotting analysis. Furthermore, confocal microscopy analysis performed on human RD and RH30 cell lines confirmed that MURC/cavin-4 mostly marks differentiated cell elements, colocalizing at the cell surface with Cav-3 and labeling myosin heavy chain (MHC) expressing cells. Finally, MURC/cavin-4 silencing prevented the differentiation in the RD cell line, leading to morphological cell impairment characterized by depletion of myogenin, Cav-3 and MHC protein levels. Overall, our data suggest that MURC/cavin-4, especially in combination with Cav-3, may play a consistent role in the differentiation process of RMS., Rhabdomyosarcoma (RMS) is a childhood soft tissue tumor with broad expression of markers that are typically found in skeletal muscle. Cavin-1 is a recently discovered protein actively cooperating with Caveolin-1 (Cav-1) in the morphogenesis of caveolae and whose role in cancer is drawing increasing attention. Using a combined in silico and in vitro analysis here we show that Cavin-1 is expressed in myogenic RMS tumors as well as in human and primary mouse RMS cultures, exhibiting a broad subcellular localization, ranging from nuclei and cytosol to plasma membrane. In particular, the coexpression and plasma membrane interaction between Cavin-1 and Cav-1 characterized the proliferation of human and mouse RMS cell cultures, while a downregulation of their expression levels was observed during the myogenic differentiation. Knockdown of Cavin-1 or Cav-1 in the human RD and RH30 cells led to impairment of cell proliferation and migration. Moreover, loss of Cavin-1 in RD cells impaired the anchorage-independent cell growth in soft agar. While the loss of Cavin-1 did not affect the Cav-1 protein levels in RMS cells, Cav-1 overexpression and knockdown triggered a rise or depletion of Cavin-1 protein levels in RD cells, respectively, in turn reflecting on increased or decreased cell proliferation, migration and anchorage-independent cell growth. Collectively, these data indicate that the interaction between Cavin-1 and Cav-1 underlies the cell growth, migration and differentiation grade in myogenic tumors., Recently, it was shown that in yeast CK2-dependent phosphorylation of the mitochondrial import receptor Tom22 promotes biogenesis of the TOM translocase and is required for import of mitochondrial proteins. We asked whether CK2-dependent phosphorylation of TOM proteins also occurs in mammals. Using CK2β-deficient skeletal muscle lysates, we observed less phosphorylation of Tom22. Moreover, we confirmed CK2 phosphorylating residues serine 15 and threonine 43 of murine Tom22. Further, CK2-dependent phosphorylation of Tom22 changes its binding affinity for proteins need to be imported into mitochondria. In the absence of CK2 mitochondrial protein import is impaired in muscle fibers and mitochondria are dysfunctional. Pink1, a mitochondria health sensor and involved in Parkinson s disease, accumulates within mutant muscle cells, and labels removal of dysfunctional mitochondria by mitophagy and involvement of autophagic adaptor protein p62/SQSTM1. Consequently, the metabolism of oxidative muscle fibers in mutant muscles shifted towards glycolytic. As proof of concept, removal of aggregated p62/SQSTM1 by muscular in vivo electroporation of phosphomimetic Tom22 was successful. This is the first evidence for both, regulated protein import into mammalian mitochondria, and muscle weaknes due to a mitochondrial protein import defect., Canonical Wnt/β-catenin signaling plays a role in myogenic differentiation, but its role in adult muscle fibers is completely unknown. We approached canonical Wnt signaling in adult myofibers by well-known reporter Axin2-lacZ mice, monitoring X-Gal staining in muscle stem cells, in adult muscle fibers and at their neuromuscular synapse. In muscle stem cells, canonical Wnt signalling is absent in quiescent cells and 72 h proliferating cells. In adult muscle fibers, canonical Wnt signaling is strongly detectable by Axin2- and β-catenin-positive skeletal muscle fibers, where it is expressed only in fast fiber types with small cross-sectional areas. In these fibers, canonical Wnt signaling is active together with Hippo signaling members, YAP/TAZ and TEAD1. During differentiation of C2C12 cells, Axin2 increases together with the expression of TEAD1-target genes: CTGF, Ankrd1 and Cyr61. In cultured primary muscle cells, we observed Axin1 and Axin2 being involved in proliferation and myotube formation in a Wnt1 and Wnt3a dependent manner. We present a model how canonical Wnt/β-catenin signaling, together with YAP/TAZ and TEAD1, influences muscle fiber diameter in fiber-type specific manner., Muscle atrophy contributes to the poor prognosis of many pathophysiological conditions, but pharmacological therapies are still limited. Muscle activity leads to major swings in mitochondrial [Ca2+] which control aerobic metabolism, cell death and survival pathways. We have investigated in vivo the effects of mitochondrial Ca2+ homeostasis in skeletal muscle function and trophism, by overexpressing or silencing the Mitochondrial Calcium Uniporter (MCU). The results demonstrate that both in developing and in adult muscles MCU-dependent mitochondrial Ca2+ uptake has a marked trophic effect that does not depend on aerobic control, but impinges on two major hypertrophic pathways of skeletal muscle, PGC-1α4 and IGF1-AKT/PKB. In addition, MCU overexpression protects from denervation-induced atrophy. These data reveal a novel Ca2+-dependent organelle-to-nucleus signaling route, which links mitochondrial function to the control of muscle mass and may represent a possible pharmacological target in conditions of muscle loss., PKC (protein kinase C) family is composed by 3 subgroups: conventional, novel and atypical PKC. These kinases are involved in a large number of biological processes (such as protein synthesis, glucose metabolism, apoptosis). PKCzeta and PKClambda/iota belong to the atypical PKC subgroup and differ from conventional and novel PKCs for their activation mechanism. Indeed atypical PKCs are calcium and diacylglycerol (DAG) insensitive, while classical PKCs are activated by calcium and DAG, and novel PKCs are activated by DAG but not by calcium (1,2). Little is known on the role of PKCzeta on skeletal muscle homeostasis. Thus, we overexpressed this kinase by in vivo transient transfection. We observed a marked hypertrophy in PKCzeta positive myofibers compared to surrounding not transfected fibers. In addition PKCzeta overexpression protected muscle from denervation-induced atrophy. Next, we studied the effects of 3 different PKCzeta mutants on fiber size: 1) PKCzeta-DN (a dominant negative isoform carrying a point mutation on the ATP-binding site); 2) PKCzeta-ΔNPS (a costitutive active mutant); 3) PKCzeta-InLoop (a dominant negative isoform mutated in the activation loop). Surprisingly all these mutants cause muscle hypertrophy and protect from denervation-induced atrophy suggesting a possible kinase-independent mechanism of PKCzeta on skeletal muscle trophism., P38 mitogen activated protein kinases (MAPKs) are required at several stages during differentiation of muscle progenitor cells. P38 phosphorylation initially accompanies satellite cells activation and triggers asymmetric division. At a later stage, it orchestrates myoblast differentiation promoting myotube formation. The signals that trigger or modulate p38 activation during the differentiation process are still debated. Both cellJtoJcell contact and TNFα prime p38α/β phosphorylation and activation during myogenesis. Cdo, a multifunctional surface protein has been implicated in myogenesis. Following cellJtoJcell contact and ligation to cadherin, Cdo binds JLP and BnipJ2 which act as scaffolds for recruitment of p38α/β and Cdc42. The formation of the complex leads to activation of Cdc42, which is fundamental to promote p38α/β phosphorylation and myogenic differentiation. However, the phosphorylation cascade leading to p38α/β activation has not been elucidated. We focused on Pak1, a member of the p21 activated kinase family, which is activated by Cdc42. We have observed that treatment of differentiating myogenic progenitors (mesoangioblasts) with the Pak1 inhibitor IPAJ3 negatively modulates p38α/β phosphorylation and myogenin expression without affecting cell proliferation. This inhibition of the myogenic differentiation program results in a lower efficiency of myotube formation. We followed these observations in vivo by monitoring regeneration efficiency in mice treated with IPA-3 and we observed that mice treated with IPA-3 displayed a delayed recovery after cardiotoxin injury. These results suggest the Pak1 contributes to myogenic differentiation of progenitor cells in vitro and participates in muscle regeneration in vivo., Ambra1 (activating molecule in Beclin 1-regulated autophagy) is an adaptor protein involved in a plethora of cellular processes. Studies in mice with a randomly mutated Ambra1 locus (Ambra1gt/gt) showed that this gene is essential for the development of the central nervous system. A recently published work by our team suggests that Ambra1 may also play a key role for muscle development in zebrafish and mouse. Indeed, Ambra1gt/gt E13.5 mouse embryos display severe defects of neck, tongue, dorsal and limb muscles, being characterized by increased cellularity and a marked disorganization of myofibers. To better clarify the role of Ambra1 in skeletal muscles, we generated mice with a floxed Ambra1 allele (Ambra1flox/flox). Ambra1flox/flox mice were then crossed with a CAG-Cre transgenic line, which express Cre recombinase in the oocytes, thus obtaining Ambra1+/- mice. Here we show that Ambra1–/– mice die at late developmental stages and display severe morphological defects, similar to Ambra1gt/gt embryos. Adult Ambra1+/- mice show an increased percentage of centrally nucleated fibers and a decreased proportion of oxidative fibers. Ambra1flox/flox mice were then bred with MLC-1f-Cre transgenic animals, which only express Cre recombinase in mature myofibers. Our preliminary data in adult Ambra1flox/flox;MLC-1f-Cre mice show a significant increase of centrally nucleated fibers, although we did not observe any overt defect of oxidative fibers. Altogether, our data suggest that Ambra1 is important for the development of skeletal muscle. Further studies in different muscles of Ambra1flox/flox;MLC-1f-Cre mice under different stress conditions will allow elucidating the role of this adaptor protein in myofiber homeostasis, Myopalladin (MYPN) is a striated muscle-specific sarcomeric protein belonging to a small family of actin-associated immunoglobulin-containing proteins. MYPN mutations have been identified in patients with dilated (DCM), hypertrophic, and restrictive cardiomyopathy. Furthermore, we identified three MYPN mutations in limb girdle muscular dystrophy (LGMD) patients with associated DCM. Within the sarcomeric Z-line, myopalladin binds to α-actinin, nebulin, and PDZ-LIM proteins. Furthermore, it is present in the nucleus and the I-band where it binds to the stress-inducible transcriptional cofactor CARP/Ankrd1, which, in turn, binds to the I-band region of titin, suggesting a role of MYPN in mechanosensing. In our preliminary studies, we found that MYPN can bind to and bundle filamentous actin, thereby promoting actin polymerization. Moreover, MYPN interacts with MRTF-A and strongly increases MRTF-A-mediated activation of serum response factor (SRF) signaling. In studies of MYPN knockout (MKO) mice, we found that MKO mice are significantly smaller compared to wildtype (WT) mice and have an about 30% reduction in skeletal muscle cross-sectional area (CSA) at all ages. Consistently, reduced differentiation rate and myotube width was observed in primary skeletal muscle cultures derived from MKO mice. Furthermore, studies of muscle performance in 2-month-old MKO mice showed reduced isometric force and power during isotonic shortening at any loads as a result of the reduced cross sectional area, whereas the force developed by each myosin molecular motor was unaffected. By up- and downhill treadmill running, MKO and WT mice performed similarly. However, while the performance of WT mice was unaffected following four consecutive days of downhill running, the performance of MKO mice decreased progressively and signs of muscle regeneration following muscle damage were observed. Consistent with a higher susceptibility to muscle damage, progressive Z-line widening was observed in MKO skeletal muscle from about 8 months of age. RNAseq revealed downregulation of actin isoforms and other SRF-target genes in MKO muscle both at 2 and 4 weeks of age, suggesting altered SRF signaling as a possible explanation for the reduced CSA in MKO mice., Impairment of autophagy in muscle leads to precocious ageing1. In particular, autophagy deficient mice are characterized by weakness are atrophy that are associated with alteration in Neuro Muscular Junction (NMJ) and loss of innervation. In order to investigate the cross-talk between muscle and nerve, we found that the expression of FGFBP1, a neurotrophic factor that is critical to preserve muscle-nerve interaction, is suppressed in muscle of autophagy deficient mice. FGFBP1 has been found to be regulated by miRNA206, the muscle-specific miRNA2. When we checked the level of miRNA206 expression, we found higher level of miRNA206 in serum of muscle specific autophagy deficient mice than in controls. Importantly, miRNA206 was detected in the heart of those mice. To understand whether autophagy deficient muscles released vesicles containing microRNAs, we analysed exosomes Quantitative RT-PCR analyses confirmed an increased expression of miRNA206 in purified exosomes from both denervated and autophagy deficient fibers. Moreover expression of BDNF in neurons treated with purified exosomes containing miRNA206 was down-regulated. This finding suggests a potential role of exosomes and miRNA206 in modulating synaptic plasticity. In order to mimic autophagy deficient mice condition, we systemically injected exosomes transfected with miRNA206 in wild-type animals. MiRNA206 was found in several tissues, in particular liver and heart. Moreover the treatment was sufficient to induce skeletal muscle atrophy and changes in the expression of several neurotrophic factors. These data support the role of exosome as a signaling mechanism that connects muscle with different tissues including motorneuron, heart and liver., Duchenne muscular dystrophy (DMD) is a genetic disease in which loss of functional dystrophin protein results in progressive skeletal muscle degeneration. Although the genetic defect is widely known, the mechanisms by which the absence of dystrophin leads to the complex pathophisiology of the disease is not completely understood. MiRNAs are small non coding RNA that are important regulatory elements for muscle development and function [1]. Altered levels of specific miRNAs were found in several muscular disorders, including DMD [2, 3]. In particular it has been identified a specific DMD-signature miRNAs that may serve as a marker for therapeutic purposes [4]. Moreover, in a recent work it has been defined a specific group of miRNAs strictly correlated to dystrophin levels and whose deregulated expression could explain several pathogenetic features of DMD [5]. Previously we have demonstrated that the local expression of mIGF-1 in mdx mice ameliorates the dystrophic phenotype reducing myonecrosis and upregulating survival pathways such as AKT pathway [6]. In this work, we show that a specific group of miRNAs, dystrophin-indipendent, are modulated by mIGF-1 expression. In particular, local expression of mIGF-1 promotes the modulation of miR-206 and miR-24 as well as muscle specific genes associated with maturation of regenerating muscle fibers and differentiation. These results indicates that local overexpression of the anabolic factor mIGF-1 in mdx mice ameliorates the dystrophic microenviroment modulating the expression of a specific group of miRNAs and inducing a partial rescue of the characteristic DMD-signature., Circular RNAs have been recently re-discovered as a large class of putative non-coding RNAs with a peculiar structure and poorly understood functions. Although their biogenesis, which proceeds via a back-splicing reaction, has been studied and dissected in the last years, their role in biologically relevant processes is still uncharacterized. Here, we performed expression profiling of circRNAs during in vitro differentiation of murine and human myoblasts, we selected and validated the expression of a subset of highly expressed, conserved circular RNAs and applied a high-content functional genomic screen in order to identify molecules that were able to impact on the differentiation process. We focused on three circRNAs whose down-regulation resulted in important phenotypes and further scrutinized one of them, named circ-ZNF609, with the aim of undestanding its molecular function. We found that circ-ZNF609 contains an open reading frame spanning from the native start codon of its host transcript and terminating at an in-frame stop codon that is created upon circularization. Circ-ZNF609 is associated to heavy polysomes and is translated into a 30-KDa peptide that is able to promote human myoblasts proliferation., Stem cells and regenerative medicine have greatly increased the expectations of the scientific community and the public for their potential in applications that aim at recovering or replacing injured, aged and diseased tissues. Nevertheless their clinical application is currently hindered by cell survival, inflammatory response, tissue engraftment, vascularization and efficient differentiation. Tissue engineering exploits biomaterials to improve stem cell engraftment and differentiation by mimicking organogenesis. Skeletal muscle tissue engineering is an up-and-coming biotechnology that could offer great potential, in the near future, for muscle repair. Reconstructing the skeletal muscle architecture and function is still a challenge requiring parallel alignment of myofibrils arranged into organized sarcomeres. We show that an “anatomical bioreactor-like” represented by the surface of mouse tibialis anterior muscle (TA), positively influences maturation and alignment of fibers derived from adult muscle stem/progenitor cells embedded into a poly-ethylene-glycol-fibrinogen (PF) hydrogel. This approach leads to the generation of an artificial normal muscle. Furthermore by the same approach we succeeded in replacing a complete mouse TA after massive muscle ablation, recovering morphology and function of the substituting artificial TA. Starting from these observations, we are now developing a novel approach for regeneration and/or reconstruction of skeletal muscle tissue segments human-like size in order to translate this technique to clinical application. For this purpose human derived muscle pericytes have been isolated from muscle biopsies and have been investigated for their myogenic potential. Moreover by exploiting the PF properties, we demonstrated the noteworthy potential of this cell population for human skeletal muscle tissue engineering., Histone deacetylases (HDACs) control the transcriptional networks underlying both muscle differentiation and progression of dystrophy. Considering that, HDAC inhibitors (HDACi) are important candidate drugs for pharmacological interventions in muscular dystrophies. Although the beneficial effects of HDACi in the treatment of muscular dystrophy are known, it remains to dissect the mechanism of action and the cellular mediators of these drugs. The goal of this project is to analyze the molecular mechanisms underlying the role of resident satellite cells and infiltrating macrophages in mediating the activity of HDACi ITF2357 (also referred to as Givinostat) in dystrophic muscle of mdx mice, the best animal model of Duchenne Muscular Dystrophy (DMD). We analyzed the dystrophic phenotype of mdx mice treated with Givinostat at different stages of disease, specifically 6, 12 and 36 weeks, corresponding to necrotic/inflammatory, regenerative and fibrotic stage, respectively. The histopathological and morphometric analyses show an amelioration of dystrophic phenotype with a significant increase of muscle fiber cross-sectional area and a consistent reduction of intramuscular fibrosis, surprisingly also at late stage of disease, suggest a positive outcome also in old mdx mice. Moreover, gene expression analysis of whole skeletal muscle and purified cell populations pointed out a modulation of fibrosis and inflammatory markers and fibroadipogenic differentiation. Overall, these data confirm the beneficial effects of Givinostat on dystrophic muscle and identify the involvement of macrophages in mediating Givinostat activity., Central Core Disease (CCD) and Malignant Hyperthermia (MH) are related disorders linked to mutations in the ryanodine receptopr-1 (RYR1) gene, encoding for the sarcoplasmic reticulum (SR) Ca2+ release channel. CCD is a congenital myopathy characterized by amorphous regions lacking mitochondrial activity (cores) in skeletal fibers. In humans, the RYR1-Y522S mutation is associated with MH and formation of structural cores. Skeletal fibers of knock-in RYR1Y522S/WT mice develop mitochondrial damage and cores, caused by excessive oxidative stress (Boncompagni et al. 2009 PNAS). We treated RYR1Y522S/WT mice with an antioxidant, N-acetylcysteine (NAC), provided ad-libitum in drinking water (1%w/v) for 2 months and verified reduction of oxidative stress: indeed, level of 3-nitrotyrosine was increased by 1.44-folds in RYR1Y522S/WT mice, but reduced to control levels after NAC treatment. Electron microscopy was used to assess mitochondrial integrity following NAC-treatment: a) mitochondria swelling and frequency of damaged mitochondria were both decreased (-24% and -10%, respectively); b) the number/100 µm2 of mitochondria (25.9 ± 0.7 vs 29.1 ± 0.6) and their proper association with the SR (+22%) were both increased. Using histological analysis, we also verified that NAC was effective in reducing the frequency of cores (-20% contracture cores; -30% unstructured cores). Finally, we evaluated muscle function in treated mice by grip strength test: NAC was able to improve muscle strength of about 80%. This work provides the bases for clinical trial as it demonstrate that NAC-administration prevents mitochondrial damage, development of cores, and improves muscle function in a mouse model of CCD., In humans, lethal hyperthermic episodes can be trigger by anesthetics (a disorder known as Malignant Hyperthermia, Susceptibility, MHS) and by high temperature and/or strenuous exercise (crises identified as Environmental/Exertional Heat Strokes, EHSs). The correlation between MHS and EHS is strongly supported by extensive work in animal models: indeed, both RYR1Y522S/WT knock-in and CASQ-1 knockout mice trigger similar lethal crises when exposed to both halothane and heat. Here we tested the following hypotheses: a) strenuous exercise is a stimulus capable to trigger EHS-lethal episodes; b) MHS and EHS share common molecular mechanisms underlying crises. When RYR1Y522S/WT and CASQ1-null mice were subjected to an exertional-stress (ES) protocol (executed on a treadmill placed in an environmental chamber), which was well tolerated by WT animals (0% of deaths), the mortality rate was dramatically increased (80% and 75%, respectively), with a rise in core temperature (hyperthermia) significantly higher than in WT at the end of the stimulus. During exertional-crises, most fibers from RYR1 Y522S/WT and CASQ1-null mice suffer severe structural damage (~99% and ~64% of fibers, respectively), indication of rhabdomyolysis. Importantly, pre-treatment of animals with azumolene (a more water soluble dantrolene analog, the only drug approved for treatment of MH crises in humans) almost completely abolished mortality rate in RYR1 Y522S/WT and CASQ1-null animals, by reducing hyperthermia, rhabdomyolysis, and Ca2+leak from the SR. All these results strongly suggest that EHS share common molecular mechanisms with anesthetic-induced MH episodes and that drugs used to treat classic MH should be considered for treatment of EHS., Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disorder characterized by progressive muscle degeneration due to lack of dystrophin, a protein essential for the integrity of sarcolemma during contraction. In DMD compensative degeneration/regeneration cycles determine a condition of chronic inflammation contributing to progressive muscle wasting. RAGE (receptor for advanced glycation end-products) is a multiligand receptor belonging to the immunoglobulin superfamily involved in physiological and pathological processes including inflammation and myogenesis [1]. RAGE is not expressed in adult muscle tissue, whereas it is expressed in regenerating myofibers during muscle regeneration [2,3], in dystrophic muscles and activated immune cells. To have information about the role of RAGE in the pathophysiology of DMD we generated a double mutant mdx/Ager–/– mouse lacking dystrophin and RAGE (Ager). We analyzed diaphragms and hind-limb muscles (i.e., tibialis anterior and quadriceps femoris) of mdx, mdx/Ager–/–, Ager–/– and WT mice at different ages (i.e., 2, 3, 4 and 5 weeks, and 3 and 6 months of age). We found that although the absence of RAGE in dystrophic mice did not affect the onset of the pathology, muscles of 5 week- and 6 month-old mdx/Ager–/– mice showed significantly reduced numbers of necrotic myofibers, and reduced areas of immune cell infiltrate compared with age-matched mdx mice. Also, muscles of mdx/Ager–/–mice showed strongly reduced expression of the marker of activated macrophages, MAC3, compared with mdx mice. Moreover, muscles of mdx/Ager–/– mice exhibited significantly reduced PAX7+ve and myogenin+ve cell numbers, pointing to a reduced recruitment of muscle precursor cells and a more efficient regeneration in dystrophic mice lacking RAGE. Our results suggest that RAGE has an important role in sustaining inflammatory and degenerative processes in dystrophic muscles, and that inhibition of RAGE expression/activity might represent a potential therapeutic approach in DMD patients., Ageing is associated to a dramatic increase in the incidence of heart failure, even if the existence of a real age-related cardiomyopathy remains controversial. As effective contraction and relaxation of cardiomyocytes also depends on Ca2+ supply to myofibrils (handled by calcium release units, CRUs) and on efficient ATP production (provided by mitochondria), in this study we performed a morphological study of cardiac cells in hearts from adult and old mice (4 months vs. ≥ 24 months of age) using confocal and electron microscopy. The analysis of CRUs indicates that couplons become shorter with age and that the number of CRUs/50 µm2 is decreased of about 24% (adults: 5.1±0.32; old: 3.9±0.19). Also mitochondria present structural modifications, with a significant increase in the percentage of organelles presenting severe alterations (3.5% vs. 16.5%). Importantly, both CRUs and mitochondria undergo a spatial re-organization with respect to sarcomeres/myofibrils: CRUs may be miss-oriented (longitudinal) or miss-placed (found at the A band instead of being correctly placed in proximity of Z-lines), while mitochondria are often grouped in an abnormal fashion. In addition, WB analysis shows that in aged mice, there is a significant reduced expression of junctophilin-2 (JP-2), a membrane protein involved in maintaining stability and morphometry of dyads. These age-related ultra-structural changes may underlie an inefficient supply of Ca2+ and ATP to contractile elements, providing a possible explanation for heart dysfunction associate to age., Progressive muscle degeneration followed by dilated cardiomyopathy is a hallmark of muscular dystrophy. Stem cell therapy is suggested to replace diseased by healthy myofibers, although so far we are faced by low efficiencies of migration, engraftment and differentiation of stem cells. Chemokines are signalling proteins guiding cell migration and have been shown to tightly regulate cardiac repair. We sought to determine which chemokines are expressed in a dystrophic heart that is undergoing cardiac remodelling. Therefor we analysed chemokine expression of Sarcoglycan-α (Sgcα) null, Sarcoglycan-β (Sgcβ) null and immunodeficient Sgcβ-null mice. High expression of all three monocyte-chemotactic proteins was observed, especially Ccl8 in both Sgcβ-null models and to a greater extent in Sgcα-null mice. Additionally, Fractalkine (Cx3cl1) was upregulated in both the immunocompetent and immunodeficient Sgcβ-null mice. In addition, we aim to evaluate the migration potential of cardiovascular progenitors derived from pluripotent stem cells in vitro, that have the potential to differentiate with high efficiency towards cardiomyocytes, smooth muscle cells and endothelial cells in vitro. We plan to test these cells for their in vivo differentiation and migration capacity towards the previously mentioned chemokines. This sheds perspective for an approachable mechanism, which could potentially improve stem cell homing towards the dystrophic myocardium., Cardiac dysfunction from cardiomyopathy is a frequent manifestation of muscular dystrophy. The primary defect common to most dystrophies involves the disruption of the dystrophin-glycoprotein complex (DGC) with subsequent sarcolemma instability and Ca2+ influx, inducing cellular necrosis. Defective Ca2+ uptake resulting from decreased expression and reduced activity of calcium-transporting ATPase (SERCA2a) and, recently, SERCA2a gene therapy has been demonstrated to mitigate dystrophic diseases. Our previous studies have demonstrated that the dystrophic phenotype observed in δ-sarcoglycan–null hamster is dramatically improved by a long-term dietary supplementation with flaxseeds (FS) (rich in n3-PUFAs), but the molecular mechanisms have not yet been fully understood. The present study was designed to test the hypothesis that FS enriched diet could regulate DGC and SERCA2a proteins that play an important structural and functional role in cardiomyocytes. Therefore, the levels of these proteins and mRNAs were analyzed in heart dystrophic hamsters fed with FS diet for long (five months) or short time (48 hours). Results showed that α- distroglycan, α-, β, γ-sarcoglycan and SERCA2a proteins were down-regulated in dystrophic hearts and FS-diet restored their normal expression pattern. The RT-PCR analysis showed that α-distroglycan, α-sarcoglycan and SERCA2a were up-regulated at the transcriptional level. Interestingly, the mRNAs increase was observed even when FS was administered for short periods suggesting the involvement of an epigenetic mechanism. Therefore, it seems plausible to consider the administration of plant-originated n-3 PUFAs as an adjuvant strategy for attenuating sarcolemma instability and defective Ca2+ uptake that represent major damages associated with dystrophic cardiomyopathies., Oxidative stress (OS) is an imbalance between the production of free radicals, in particular reactive oxygen species (ROS), and the ability of the cells to counteract them by antioxidant responses. ROS production in skeletal muscle occurs mainly in mitochondria, both following physiological stimuli (e.g. aging, physical exercise, or at birth) (1-3) and in response to pathological events, such as denervation (4). In all cases, high levels of ROS actively influence the maintenance of muscle homeostasis. Histone deacetylase 4 (HDAC4) is a member the class II of the HDAC superfamily that regulates many cellular processes (5-7). Following denervation, HDAC4 is upregulated in skeletal muscle: it induces muscle atrophy and represses reinnervation (8-9). Increased levels of ROS cause HDAC4 translocation from the nucleus to the cytoplasm, thus inducing the release of genes transcriptionally repressed by HDAC4(10). However, HDAC4 targets in skeletal muscle have not been discovered yet. In order to investigate the role of HDAC4 in response to OS in skeletal muscle, we use a mouse model with the selective deletion of HDAC4 in myogenin positive cells (HDAC4 mKO mice). HDAC4 mKO mice are viable and do not show gross abnormalities in skeletal muscle. We analyzed mice in two different conditions characterized by elevated OS: at birth and in adult mice following denervation. Molecular responses to oxidative stress are blunted in both newborn and adult HDAC4 mKO compared to control mice. Since elevated ROS contribute to mitochondrial damage and are important in redox signaling from the organelle to the rest of the cell, we analyzed mitochondrial ultrastructure. Both newborn and adult HDAC4 mKO muscles presented damaged mitochondria, altered mitochondrial dynamics and defects in myofiber organization. Our results indicate that HDAC4 is important in skeletal muscle to maintain muscle integrity and a proper response upon OS. Current studies are focused on the identification of HDAC4 targets in the OS response in skeletal muscle, Collagen VI (ColVI) is a major extracellular matrix component made of three genetically distinct α chains and abundantly deposited in the basement membrane of both skeletal muscles and peripheral nerves. Mutations in COL6A1, COL6A2 and COL6A3 genes are known to cause different forms of muscle diseases, including Bethlem myopathy, Ullrich congenital muscular dystrophy and myosclerosis myopathy. ColVI null (Col6a1–/–) mice display a myopathic phenotype characterized by latent mitochondrial dysfunction, spontaneous apoptosis, defective autophagy regulation and compromised muscle regeneration. We recently demonstrated that the absence of ColVI in peripheral nerves leads to hypermyelination, altered Remak bundles, sensory-motor functional deficits and decreased nerve conduction velocities, thus pointing at ColVI as a crucial molecule for peripheral nerve structure and function. Given the muscle and nerve defects displayed by Col6a1 null mice, we decided to explore the role of ColVI in the neuromuscular junction (NMJ). Our unpublished studies revealed that ColVI is indeed deposited at the synapse. Immunofluorescence analysis showed ColVI deposition in NMJs. Preliminary results revealed altered expression of synaptic genes and abnormal electrophysiological parameters in Col6a1–/– mice. These findings suggest a potential role for ColVI at the NMJ, and further studies will allow shedding new light on the roles of this extracellular matrix component in the nerve/muscle axis., Muscular dystrophies are non curable diseases. Recently, new strategies shed light to an increase of muscle regeneration. These strategies focus on epigenetic drugs. TSA (HDACi) achieve to enhance the regeneration rate in both mice and humans. However, new challenges stay on the horizon. Monitoring and controlling the changes of the treatment in muscle without invasive techniques are one of that’s. In our research we identified seven microRNAs differential expressed in FAPs population. FAPs are Key players of muscle regeneration under HDACi treatment. From these seven microRNA, miR-143 has been validated with qRT-PCR, and Chip techniques. This miR-143 form part of a cluster with miR-145 that locates into a long non coding RNA non characterized until that moment. The overexpression of this miR-143 turns FAPs into a non adipogenic phenotype, whereas the inhibition of it recovershe adipogenic behavior. Thus, in this work we are trying to characterize the role of this microRNA and their host gene to understand if it could be a good candidate to be used as marker during the treatment., The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. In recent years, it has been discovered that genomes of multicellular organisms are characterized by the pervasive expression of different types of non-coding RNAs (ncRNA) and, among them, long non-coding RNAs (IncRNAs). In particular the mammalian genome contains many thousands of lncRNAs, which have been proposed to be fundamental in the regulation of many biological processes such as cellular differentiation and show an aberrant regulation in a variety of diseases. A transcriptome analysis performed during in vitro murine muscle differentiation allowed us to identify a subset of new lncRNAs differentially expressed during myogenesis (1). These transcripts were classified on the basis of their expression in proliferating versus differentiated conditions, muscle-restricted activation and subcellular localization. We are now focusing on the characterization of a nuclear lncRNA conserved in human, lnc-405, up-regulated during differentiation, whose expression is cardiac and skeletal muscle restricted. To dissect its role in myogenesis, we performed loss of function experiments using LNA-Gapmers followed by a transcriptome analysis. This approach revealed a strong down-regulation of a subset of genes involved in fiber contraction, cell fusion and related to several cardiomyopathies. With the idea to better explain its crucial role during myogenesis, we are now focusing on the molecular mechanism by which lnc-405 exerts its function in the nucleus by RIP, ChIRP and RNA pull-down assays that are on going., The functional connection between muscle and nerve is affected in several neuromuscular diseases, including Amyotrophic Lateral Sclerosis (ALS) whose major pathological processes are motor neuron degeneration. However, other cells may be involved in the pathogenesis of ALS and open the possibility that alteration in skeletal muscle homeostasis represents one of the principal mediators of motor neuron degeneration. We have evidences that indicate that muscle selective expression of SOD1G93A mutant gene modulates, at the level of spinal cord of MLC/SOD1G93A mice, relevant mRNA and microRNA associated with myelin homeostasis. Our study provided insights into the pathophysiological interplay between muscle and nerve and supports the hypothesis that skeletal muscle is a source of signals that can affect the nervous system., Calsequestrin (CASQ) is the major protein of the sarcoplasmic reticulum of striated muscle that binds Ca2+ with high capacity and moderate affinity. CASQ exist as a monomer and polymers, depending on Ca2+ concentration. CASQ switches from an unfolded state to a folded monomer when the ionic strength increases allowing the formation of front-to-front first and then back-to-back interactions in higher Ca2+ concentrations. In humans, mutations in the cardiac isoform CASQ2 lead to catecholamine-induced polymorphic ventricular tachycardia. Recently we reported one mutation in the skeletal CASQ1 gene in a group of patients with a vacuolar myopathy characterized by the presence of inclusions containing CASQ1 and other SR proteins. The CASQ1 mutation (CASQ1D244G) affects one of the high-affinity Ca2+-binding sites of the protein and alters the kinetics of Ca2+ release in muscle fibers from patients. Expression of the CASQ1D244G in myotubes and in mouse fibers causes the appearance of SR vacuoles containing aggregates of the mutant CASQ1 protein that resemble those observed in patients. Studies of Ca2+ release showed an increase in Ca2+ storage in CASQ1WT COS-7 transfected cells whereas no increase was observed in CASQ1D244G. Moreover both CASQ1WT and CASQ1D244G were expressed in bacteria, purified and analysed for their ability to polymerize at increasing Ca2+ concentrations. The results obtained indicate that the CASQ1D244G protein polymerizes at lower Ca2+ levels and more rapidly than CASQ1WT. These results suggest that the CASQ1D244G mutation interferes with the correct process of Ca2+ -dependent protein polymerization causing altered intracellular calcium storage and the formation of protein aggregates., Muscle regeneration is dependent upon a complex interplay of different cell types in the muscle stem cell niche. In particular, the recently described population of interstitial fibro-adipogenic progenitors (FAPs) and satellite cells (MuSCs) establish a complex network of interactions to coordinate muscle regeneration. FAPs are able to promote satellite cell differentiation and compensate for muscle necrosis. In our recent studies, we demonstrated that FAPs are the key cellular mediators of the beneficial effect of HDAC inhibitors at early stages of Duchenne Muscular Dystrophy (DMD). Indeed, FAPs, from young mdx mice HDACi, induce myogenesis at expense of adipogenesis and enhance their ability to support MuSCs differentiation. Conversely, FAPs from old mdx mice are resistant to HDACi and repress MuSCs differentiation (Mozzetta et al., 2013; Saccone et al., 2014). Given the importance of the cross-talk between FAPs and MuSCs in DMD progression, we are currently deciphering the role of FAP-released extracellular vesicles (and in particular the exosomes - endosome derived vesicles) as mediators of the functional interactions between mononuclear cell types that contribute to muscle regeneration., Limb Girdle Muscular Dystrophies are rare genetic diseases, characterized by weakness and progressive muscular atrophy. A subfamily of LGMD2 regroups sarcoglycanopathies caused by mutations in genes coding for sarcoglycans. These transmembrane proteins are part of the dystrophin complex that protects muscle fibers against mechanical stress due to contraction. There is no treatment available for these diseases.In order to understand the molecular mechanisms implicated in sarcoglycanopathies and to identify new therapeutic targets, we are conducting two studies: 1 - SG mutants are not present at the muscle fiber membrane because they are retained in the endoplasmic reticulum by the quality control (ERQC) and they are prematurely degraded by the proteasome. To study the ERCQ pathways responsible for sarcoglycan disposal at molecular level, we first generated cell lines expressing clonally one SG mutant. Those clones are now used to investigate the SGs cellular trafficking mechanisms and then to test pharmacological compounds modulating ERCQ pathways. 2 – In these diseases, muscular atrophy affects limb muscles and infrequently head muscles. To investigate mechanisms underlying the fact that some muscles are more affected than other, we analyzed different muscles to search for molecular differences that may sign their relative sensitivity to the genetic defects. The content in micro-RNA of muscles from the limbs and face of Macaca fascicularis was explored. Experiments are in progress to analyze the function of identified micro-RNAs and to evaluate their therapeutic potential for sarcoglycanopathies. These projects will improve the knowledge on physio-pathological mechanisms of sarcoglycanopathies in order to identify new therapies for patients., Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common human myopathies and arises with progressive wasting of facial mimic muscles as well as upper arms and shoulder girdle muscles. In 95% of the cases, FSHD is associated with the copy number reduction of D4Z4 macrosatellite repeats at the subtelomeric region of chromosome 4 (4q35). This change is associated with an epigenetic deregulation of the region that ultimately leads to the de-repression of nearby genes, such as DUX4 and FRG1 that have been reported to contribute to the muscular dystrophic phenotype observed in FSHD patients. The chromatin-associated lncRNA DBE-T, encoded by the FSHD locus, has been shown to be one of the main players of such event, though the molecular mechanism has not been yet fully elucidated. DBE-T is preferentially expressed in FSHD patients where it favors the transcription of the 4q35 genes thanks to the recruitment of the histone methyl transferase of the Trithorax group of epigenetic activators ASH1L. Interestingly, through a structural/functional analysis, we have recognized several DBE-T functional domains that can be exploited as new molecular targets for therapeutic purposes. Specifically, we have identified a region and the molecular mechanism required for DBE-T tethering to the chromatin. In addition, we have mapped the minimal binding domains in ASH1L and DBE-T. Finally, we have highlighted a portion of DBE-T required to positively promote transcription. In agreement, a DBE-T mutant lacking this region is unable to trigger transcription. Currently, through proteomic approaches, we are investigating DBE-T protein partners that are specific for each DBE-T functional domain. Our goal is to identify unknown molecular players that, similarly to ASH1L, are recruited by DBE-T to the FSHD locus and can play a role in the disease. Overall, our study elucidates the molecular mechanism of DBE-T in FSHD and might unveil new therapeutic targets for the treatment of the disease.

Optical plasmonic antennas allow for localizing and enhancing light at the nanoscale. To enhance the application opportunities of optical antennas, their quality factor needs to be substantially improved. Here, we numerically and experimentally demonstrate that the resonance of a dipolar metallic disc antenna can be enhanced by a circular grating that obeys the Bragg condition. The supporting grating effectively collects energy from an extended spatial domain and guides it spectrally-selected into the central antenna, leading to a significantly enhanced field intensity at resonance. Accordingly, the quality factor of the antenna is enhanced by at least five times. The approach can be applied to other plasmonic systems, hence constituting an important ingredient to a future plasmonic tool box.

Relevanz: Seit Inkrafttreten des AMNOG ist eine Bewertung des Ausmaßes des Zusatznutzens neuer Arzneimittel gegenüber der zweckmäßigen Vergleichstherapie vorgeschrieben. Die Arzneimittel-Nutzenbewertungsverordnung sieht eine Einteilung in folgende Kategorien vor: erheblicher Zusatznutzen,[for full text, please go to the a.m. URL], Komplexe Interventionen – Entwicklung durch Austausch; 13. Jahrestagung des Deutschen Netzwerks Evidenzbasierte Medizin

International audience

Background The intertidal zone of seacoasts, being affected by the superimposed tidal, diurnal and lunar cycles, is temporally the most complex environment on earth. Many marine organisms exhibit lunar rhythms in reproductive behaviour and some show experimental evidence of endogenous control by a circalunar clock, the molecular and genetic basis of which is unexplored. We examined the genetic control of lunar and diurnal rhythmicity in the marine midge Clunio marinus (Chironomidae, Diptera), a species for which the correct timing of adult emergence is critical in natural populations. Results We crossed two strains of Clunio marinus that differ in the timing of the diurnal and lunar rhythms of emergence. The phenotype distribution of the segregating backcross progeny indicates polygenic control of the lunar emergence rhythm. Diurnal timing of emergence is also under genetic control, and is influenced by two unlinked genes with major effects. Furthermore, the lunar and diurnal timing of emergence is correlated in the backcross generation. We show that both the lunar emergence time and its correlation to the diurnal emergence time are adaptive for the species in its natural environment. Conclusions The correlation implies that the unlinked genes affecting lunar timing and the two unlinked genes affecting diurnal timing could be the same, providing an unexpectedly close interaction of the two clocks. Alternatively, the genes could be genetically linked in a two-by-two fashion, suggesting that evolution has shaped the genetic architecture to stabilize adaptive combinations of lunar and diurnal emergence times by tightening linkage. Our results, the first on genetic control of lunar rhythms, offer a new perspective to explore their molecular clockwork.

Das IQWiG ist ein unabhängiges wissenschaftliches Institut, das Nutzen und Schaden medizinischer Maßnahmen für Patient und Patientinnen untersucht. Hierzu werden systematische Übersichten erstellt, deren Grundlage eine systematische Literaturrecherche darstellt. Der Prozess der Studienselektion[for full text, please go to the a.m. URL], EbM & Individualisierte Medizin; 12. Jahrestagung des Deutschen Netzwerks Evidenzbasierte Medizin

Hintergrund: Das IQWiG untersucht u.a. den Nutzen von Arzneimitteln. Systematische Reviews (SR) sind dabei eine von mehreren Quellen zur Identifikation von Primärstudien. Bei der Suche nach SR wird i.d.R. auf validierte Suchfilter zurückgegriffen. Die Health Information Research Unit (HiRU)[for full text, please go to the a.m. URL], EbM & Individualisierte Medizin; 12. Jahrestagung des Deutschen Netzwerks Evidenzbasierte Medizin

The marine midge Clunio marinus (Chironomidae, Diptera) is characterized by a one-dimensional distribution along the European Atlantic coast, where its lunar and circadian emergence rhythms are genetically adapted to the local tidal regimes, resulting in a series of ‘temporal races’. Clunio marinus is restricted to rocky coasts and thus the temporal races occur in different rocky patches. We studied 10 populations of Clunio marinus from five different regions, spanning the major rocky mainland coasts from Spain to Norway, using amplified fragment length polymorphisms (AFLP), microsatellites and mitochondrial cytochrome oxidase I (COI) sequences. Star-like patterns of COI haplotypes within regions indicate postglacial colonization. A high degree of shared polymorphisms in AFLP markers suggests colonization from a single source, implying postglacial evolution of timing adaptations in relation to the local tidal regime. In contrast, no COI haplotypes are shared among regions. We hypothesize that different levels of differentiation of nuclear vs. mitochondrial markers in the source region were carried forward during postglacial expansion. Despite the recent origin of populations, all markers reveal distinct genetic differentiation between rocky coasts on a scale of 650–1150 km. Differentiation between rocky coasts is not correlated to timing adaptations, suggesting that geographic isolation is prevalent between rocky coasts and that this facilitated the evolution of local timing adaptations. At the same time there is little genetic differentiation within rocky coasts on a scale of 2–6 km; leaving open the possibility that within rocky coasts with large variation in tidal regimes, temporal adaptations evolved in the face of gene flow.

Hintergrund: Studienregister stellen eine potenzielle Quelle zur Identifikation relevanter Studien für systematische Übersichten dar. Insbesondere Studienregister der pharmazeutischen Industrie enthalten häufig auch Studienergebnisse. Das Ziel dieser Analyse ist es zu untersuchen,[for full text, please go to the a.m. URL], EbM – ein Gewinn für die Arzt-Patient-Beziehung?; Forum Medizin 21 der Paracelsus Medizinischen Privatuniversität & 11. EbM-Jahrestagung des Deutschen Netzwerks Evidenzbasierte Medizin

Mit “Intention to treat” (ITT) wird eine Strategie für die Auswertung von Patientendaten in randomisierten kontrollierten Studien (RCTs) (und auch nichtrandomisierten Studien (CCTs)) bezeichnet, bei der alle (randomisierten) Patienten ihrer Zuteilung entsprechend ausgewertet werden.[for full text, please go to the a.m. URL], Evidenz und Entscheidung: System unter Druck; 10. Jahrestagung des Deutschen Netzwerks Evidenzbasierte Medizin

Aim of this contribution is to illustrate the state of the art of smart antenna research from several perspectives. The bow is drawn from transmitter issues via channel measurements and modeling, receiver signal processing, network aspects, technological challenges towards first smart antenna applications and current status of standardization. Moreover, some future prospects of different disciplines in smart antenna research are given.

Die Qualitätssicherung beim Fertigen von Mikrooptik- und Mikromechanikkomponenten erfordert das Vermessen (mikro-)strukturierter Oberflächen mit Strukturtiefen bis zu einigen 100 Mikrometer mit Nanometerauflösung. Die vertikal scannende Weißlichtinterferometrie kann mit derartigen Anforderungen Schritt halten. Nur so wird das hoch auflösende Vermessen sowohl unterschiedlichst strukturierter Oberflächen, zum Beispiel spezieller mikrooptischer und -mechanischer Bauelemente, als auch glatter stetiger Oberflächen mit einem Messverfahren möglich.

U Spili kod Nakovane na Pelješcu otkriven je nastavak glavnog špiljskog kanala ukupne dužine 60 metara. Novootkriveni dio špilje krio je arheološko nalazište iz razdoblja helenizma, odnosno kasnog željeznog doba, netaknuto od njegovog napuštanja u 1. st. prije Krista. Središnji dio nalazišta potpuno je istražen tijekom prošlogodišnje istraživačke kampanje. Prikupljena arheološka građa nedvosmisleno svjedoči o tome da je Spila bila mjesto održavanja ritualnih gozbi i prinošenja zavjetnih darova pred stalagmitom falusoidnog oblika.

Nakovana Cave is that rarity in archaeological fieldwork, a sealed cavern that is at the same time an undisturbed archaeological site, discovered by archaeologists, not casual explorers. Excavations in Nakovana in 1999 and 2000 show that the cave's occupation spaned at least 8000 years. Its most spectacular component is a late Iron Age Illyrian sanctuary. Here, the evidence strongly suggests complex drinking rituals centered around a stalagmitic monument to male potency. In this paper, we introduce the cave and discuss the particular problems associated with its excavation and interpretation.

The Late Iron Age sanctuary at Nakovana Cave provides a unique window onto the world of the Illyrians, the indigenous inhabitants of Dalmatia in the 1st MBC. In that millennium, the Mediterranean was a cosmopolitan world, with people, ideas and objects in seeming constant motion. Such commerce had a transformative effect on all parties. Communities in the region were able to adopt or reject cultural items and/or practices from abroad in order to suit their particular strategic ends. Nakovana cave, with its large assemblage of Hellenistic and local pottery, found in an undisturbed context around its cultic monument, is discussed with respect to the construction of cultural identities on the margin of the expanding Greco-Roman world.