Routinely frozen biopsies of human skeletal muscle are suitable for morphological and immunocytochemical analyses at transmission electron microscopy

The aim of the present investigation was to evaluate whether routinely frozen biopsies of human skeletal muscle may be suitable for morphological and immunocytochemical analyses at transmission electron microscopy. The fixation/embedding protocols we successfully used for decades to process fresh mammalian tissues have been applied to frozen muscle biopsies stored for one to four years in liquid nitrogen. After 2.5% glutaraldehyde -2% paraformaldehyde - 1% OsO4 fixation and embedding in epoxy resin, the ultrastructural morphology of myofibres and satellite cells as well as of their organelles and inclusions proved to be well preserved. As expected, after 4% paraformaldehyde - 0.5% glutaraldehyde fixation and embedding in LR White resin, the morphology of membrane-bounded organelles was relatively poor, although myofibrillar and sarcomeric organization was still recognizable. On the contrary, the myonuclei were excellently preserved and, after conventional staining with uranyl acetate, showed an EDTA-like effect, i.e. the bleaching of condensed chromatin, which allows the visualization of RNP-containing structures. These samples proved to be suitable for immunocytochemical analyses of both cytoskeletal and nuclear components, whereas the poor mitochondrial preservation makes unreliable any in situ investigation on these organelles

Ribonuclear inclusions as biomarker of myotonic dystrophy type 2, even in improperly frozen or defrozen skeletal muscle biopsies

Myotonic dystrophy type 2 (DM2) is a dominantly inherited disorder caused by a CCTG repeat expansion in intron 1 of ZNF9 gene. The size and the somatic instability of DM2 expansion complicate the molecular diagnosis of DM2. In situ hybridization represents a rapid and sensitive method to obtain a definitive diagnosis in few hours, since it allows the direct visualization of the mutant mRNA foci on skeletal muscle sections. This approach makes the muscle biopsy an important tool for definitive diagnosis of DM2. Consequently, a rapid freezing at ultra cold temperature and a good storage of muscle specimens are essential to avoid morphologic alterations and nucleic acids degradation. However incorrect freezing or thawing may accidentally occur. In this work we report that fluorescence in situ hybridization may be applied on improperly frozen or inappropriately stored muscle biopsies since foci of mutant mRNA are well preserved and can still be detected in muscle sections no more useful for histopathological evaluation

Biomolecular identification of (CCTG)n mutation in myotonic dystrophy type 2 (DM2) by FISH on muscle biopsy

Myotonic dystrophy type 2 (DM2) is a dominantly inherited disorder with multisystemic clinical features, caused by a CCTG repeat expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene. The mutant transcripts are retained in the nucleus forming multiple discrete foci also called ribonuclear inclusions. The size and the somatic instability of DM2 expansion complicate the molecular diagnosis of DM2. In our study fluorescence-labeled CAGG-repeat oligonucleotides were hybridized to muscle biopsies to investigate if fluorescence in situ hybridization (FISH), a relatively quick and simple procedure, could be used as a method to diagnose DM2. When FISH was performed with (CAGG)5 probe, nuclear foci of mutant RNA were present in all genetically confirmed DM2 patients (n = 17) and absent in all patients with myotonic dystrophy type 1 (DM1; n = 5) or with other muscular disease (n = 17) used as controls. In contrast, foci were observed both in DM1 and DM2 myonuclei when muscle tissue were hybridized with (CAG)6CA probe indicating that this probe is not specific for DM2 identification. The consistent detection of ribonuclear inclusions in DM2 muscles and their absence in DM1, in agreement with the clinical diagnosis and with leukocyte (CCTG)n expansion, suggests that fluorescence in situ hybridization using (CAGG)5 probes, may be a specific method to distinguish between DM1 and DM2. Moreover, the procedure is simple, and readily applicable in any pathology laboratory

Measurements and optimization of the light yield of a TeO2_2 crystal

Bolometers have proven to be good instruments to search for rare processes because of their excellent energy resolution and their extremely low intrinsic background. In this kind of detectors, the capability of discriminating alpha particles from electrons represents an important aspect for the background reduction. One possibility for obtaining such a discrimination is provided by the detection of the Cherenkov light which, at the low energies of the natural radioactivity, is only emitted by electrons. This paper describes the method developed to evaluate the amount of light produced by a crystal of TeO$_2$ when hit by a 511 keV photon. The experimental measurements and the results of a detailed simulation of the crystal and the readout system are shown and compared. A light yield of about 52 Cherenkov photons per deposited MeV was measured. The effect of wrapping the crystal with a PTFE layer, with the aim of maximizing the light collection, is also presented

DIGITAL MODELLING AND ANALYSIS OF MASONRY VAULTS

The focus of this paper is to discuss the peculiarities of the digital modelling of masonry cross vaults. The proposed methodological approach for a multidisciplinary study of masonry vaults will be focused on: geometrical survey, crack pattern, geometrical modelling, and safety assessment based on structural analysis.Automatic reconstruction procedures recently proposed in the literature for ribbed masonry vaults, are used to overcome time-consuming modelling tasks. The created digital models take into consideration the different aspects of the three-dimensional geometry, the internal divisions with variations of material properties in different parts, and a suitable discretization for finite element analysis. Moreover, the same model can be used in BIM (Building Information Modelling), judged as a suitable environment in which to combine different aspects of the restoration works such as: documentation, intervention design, and the data system in an unique model. The same model is then used to perform finite element analysis. Each of these aspects is clarified with examples of different vaults typology coming from case studies as that of the church of St. Bassiano in Pizzighettone (Cremona) or the notable Milan Cathedral.</p

Cultured myoblasts from patients affected by myotonic dystrophy type 2 exhibit senescence-related features: ultrastructural evidence

Myotonic dystrophy type 2 (DM2) is an autosomal dominant disorder caused by the expansion of the tetranucleotidic repeat (CCTG)n in the first intron of the Zinc Finger Protein-9 gene. In DM2 tissues, the expanded mutant transcripts accumulate in nuclear focal aggregates where splicing factors are sequestered, thus affecting mRNA processing. Interestingly, the ultrastructural alterations in the splicing machinery observed in the myonuclei of DM2 skeletal muscles are reminiscent of the nuclear changes occurring in age-related muscle atrophy. Here, we investigated in vitro structural and functional features of satellite cell-derived myoblasts from biceps brachii, in the attempt to investigate cell senescence indices in DM2 patients by ultrastructural cytochemistry. We observed that in satellite cell-derived DM2 myoblasts, cell-senescence alterations such as cytoplasmic vacuolization, reduction of the proteosynthetic apparatus, accumulation of heterochromatin and impairment of the pre-mRNA maturation pathways occur earlier than in myoblasts from healthy patients. These results, together with preliminary in vitro observations on the early onset of defective structural features in DM2 myoblast derived-myotubes, suggest that the regeneration capability of DM2 satellite cells may be impaired, thus contributing to the muscular dystrophy in DM2 patients

Overexpression of CUGBP1 in skeletal muscle from adult classic myotonic dystrophy type 1 but not from myotonic dystrophy type 2

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are progressive multisystemic disorders caused by similar mutations at two different genetic loci. The common key feature of DM pathogenesis is nuclear accumulation of mutant RNA which causes aberrant alternative splicing of specific pre-mRNAs by altering the functions of two RNA binding proteins, MBNL1 and CUGBP1. However, DM1 and DM2 show disease-specific features that make them clearly separate diseases suggesting that other cellular and molecular pathways may be involved. In this study we have analysed the histopathological, and biomolecular features of skeletal muscle biopsies from DM1 and DM2 patients in relation to presenting phenotypes to better define the molecular pathogenesis. Particularly, the expression of CUGBP1 protein has been examined to clarify if this factor may act as modifier of disease-specific manifestations in DM. The results indicate that the splicing and muscle pathological alterations observed are related to the clinical phenotype both in DM1 and in DM2 and that CUGBP1 seems to play a role in classic DM1 but not in DM2. In conclusion, our results indicate that multisystemic disease spectrum of DM pathologies may not be explained only by spliceopathy thus confirming that the molecular pathomechanism of DM is more complex than that actually suggested

Prediction of the service life of brick/stone masonry damaged by salt crystallisation: application of a stochastic model

An attempt has been made within a EC Contract, to establish the maximum salt content in brick and stone masonry, below which the surface protection treatments do not fail. Crystallisation tests were carried out on treated and untreated brick and limestone masonry specimens. A large number of tests were previously carried out on the single units used for the masonry specimens. Salt solutions with two low concentrations of sodium sulphate were inserted in masonry wallettes treated with a water based water repellent or with a consolidant. On the basis of the recorded experimental data, a suitable damage parameter describing the material deterioration process has been chosen. The parameter assumed is the loss of surface material. The deterioration process could be interpreted as a stochastic process L(t,l), function of time t and damage l. In this way, for differen t damage levels l it is possible to build the fragility curve. By using this approach the magnitude of the expected damage over time and the occurrence time of it can be predicted. The results will allow for the investigation on the durability of materials with respect to the prediction treatments and on the decay process of single and composite materials

Characterization of the KID-Based Light Detectors of CALDER

The aim of the Cryogenic wide-Area Light Detectors with Excellent Resolution (CALDER) project is the development of light detectors with active area of $5\times5$ cm$^2$ and noise energy resolution smaller than 20 eV RMS, implementing phonon-mediated kinetic inductance detectors. The detectors are developed to improve the background suppression in large-mass bolometric experiments such as CUORE, via the double read-out of the light and the heat released by particles interacting in the bolometers. In this work, we present the characterization of the first light detectors developed by CALDER. We describe the analysis tools to evaluate the resonator parameters (resonant frequency and quality factors) taking into account simultaneously all the resonance distortions introduced by the read-out chain (as the feed-line impedance and its mismatch) and by the power stored in the resonator itself. We detail the method for the selection of the optimal point for the detector operation (maximizing the signal-to-noise ratio). Finally, we present the response of the detector to optical pulses in the energy range of 0-30 keV