Muscle biopsy and cell cultures: potential diagnostic tools in hereditary skeletal muscle channelopathies.

Hereditary muscle channelopathies are caused by dominant mutations in the genes encoding for subunits of muscle voltage- gated ion channels. Point mutations on the human skeletal muscle Na+ channel (Nav1.4) give rise to hyperkalemic periodic paralysis, potassium aggravated myotonia, paramyotonia congenita and hypokalemic periodic paralysis type 2. Point mutations on the human skeletal muscle Ca2+ channel give rise to hypokalemic periodic paralysis and malignant hyperthermia. Point mutations in the human skeletal chloride channel ClC-1 give rise to myotonia congenita. Point mutations in the inwardly rectifying K+ channel Kir2.1 give rise to a syndrome characterized by periodic paralysis, severe cardiac arrhythmias and skeletal alterations (Andersen's syndrome). Involvement of the same ion channel can thus give rise to different phenotypes. In addition, the same mutation can lead to different phenotypes or similar phenotypes can be caused by different mutations on the same or on different channel subtypes. Bearing in mind, the complexity of this field, the growing number of potential channelopathies (such as the myotonic dystrophies), and the time and cost of the genetic procedures, before a biomolecular approach is addressed, it is mandatory to apply strict diagnostic protocols to screen the patients. In this study we propose a protocol to be applied in the diagnosis of the hereditary muscle channelopathies and we demonstrate that muscle biopsy studies and muscle cell cultures may significantly contribute towards the correct diagnosis of the channel involved. DNAbased diagnosis is now a reality for many of the channelopathies. This has obvious genetic counselling, prognostic and therapeutic implications

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

Noises investigations and image denoising in femtosecond stimulated Raman scattering microscopy

In the literature of SRS microscopy, the hardware characterization usually remains separate from the image processing. In this article, we consider both these aspects and statistical properties analysis of image noise, which plays the vital role of joining links between them. Firstly, we perform hardware characterization by systematic measurements of noise sources, demonstrating that our in-house built microscope is shot noise limited. Secondly, we analyze the statistical properties of the overall image noise, and we prove that the noise distribution can be dependent on image direction, whose origin is the use of a lock-in time constant longer than pixel dwell time. Finally, we compare the performances of two widespread general algorithms, that is, singular value decomposition and discrete wavelet transform, with a method, that is, singular spectrum analysis (SSA), which has been adapted for stimulated Raman scattering images. In order to validate our algorithms, in our investigations lipids droplets have been used and we demonstrate that the adapted SSA method provides an improvement in image denoising

Reinitiation of protein synthesis in Escherichia coli can be induced by mRNA cis-elements unrelated to canonical translation initiation signals

AbstractIn Eubacteria, de novo translation of some internal cistrons may be inefficient or impossible unless the 5′ neighboring cistron is also translated (translational coupling). Translation reinitiation is an extreme case of translational coupling in which translation of a message depends entirely on the presence of a nearby terminating ribosome. In this work, the characteristics of mRNA cis-elements inducing the reinitiation process in Escherichia coli have been investigated using a combinatorial approach. A number of novel translational reinitiation sequences (TRSs) were thus identified, which show a wide range of reinitiation activities fully dependent on a translational coupling event and unrelated to the presence/absence of secondary structure or mRNA stability. Moreover, some of the isolated TRSs are similar to intercistronic sequences present in the E. coli genome

Sperm motility and fertilization performance of Nodipecten nodosus (L., 1758) exposed at two different cryoprotectants

Cryopreservation is a valuable tool for aquaculture as it provides continuous seed production, regardless of the spawning season of the brood stock. The selection of a suitable cryoprotectant with low toxicity and high water solubility is important to avoid membrane injuries and intracellular ice crystallization. This study has been aimed at the assessment of the toxic effects of two usually applied cryoprotectants, 1-2 propylene glycol (PG) and methanol (MetOH), on spermatozoa of the of lion-paw scallop Nodipecten nodosus, by evaluating the sperm motility and the development of D larvae after fertilization procedure. Sperm was exposed at room temperature (22°C) for 10, 20 and 30 min to different concentration ranges of two cryoprotectants. Regarding the sperm motility, PG5%, PG7%, MetOH4% and MetOH6% did not show differences compared to control (semen incubated in seawater) (P < 0.05). The development of D larvae was not affected by the exposition to PG5%, MetOH 4% and MetOH 6%. These results indicate the potential use of both cryoprotectants for cryopreservation procedures

Production and covalent immobilisation of the recombinant bacterial carbonic anhydrase (SspCA) onto magnetic nanoparticles

Carbonic anhydrases (CAs; EC 4.2.1.1) are metalloenzymes with a pivotal potential role in the biomimetic CO2 capture process (CCP) because these biocatalysts catalyse the simple but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons in all life kingdoms. The CAs are among the fastest known enzymes, with kcat values of up to 106 s!1 for some members of the superfamily, providing thus advantages when compared with other CCP methods, as they are specific for CO2. Thermostable CAs might be used in CCP technology because of their ability to perform catalysis in operatively hard conditions, typical of the industrial processes. Moreover, the improvement of the enzyme stability and its reuse are important for lowering the costs. These aspects can be overcome by immobilising the enzyme on a specific support. We report in this article that the recombinant thermostable SspCA (a-CA) from the thermophilic bacterium Sulfurihydrogenibium yellowstonense can been heterologously produced by a highdensity fermentation of Escherichia coli cultures, and covalently immobilised onto the surface of magnetic Fe3O4 nanoparticles (MNP) via carbodiimide activation reactions. Our results demonstrate that using a benchtop bioprocess station and strategies for optimising the bacterial growth, it is possible to produce at low cost a large amount SspCA. Furthermore, the enzyme stability and storage greatly increased through the immobilisation, as SspCA bound to MNP could be recovered from the reaction mixture by simply using a magnet or an electromagnetic field, due to the strong ferromagnetic properties of Fe3O4

Sequence Analysis, Kinetic Constants, and Anion Inhibition Profile of the Nacrein-Like Protein (CgiNAP2X1) from the Pacific Oyster Magallana gigas (Ex-Crassostrea gigas)

The carbonic anhydrase (CA, EC 4.2.1.1) superfamily of metalloenzymes catalyzes the hydration of carbon dioxide to bicarbonate and protons. The catalytically active form of these enzymes incorporates a metal hydroxide derivative, the formation of which is the rate-determining step of catalytic reaction, being affected by the transfer of a proton from a metal-coordinated water molecule to the environment. Here, we report the cloning, expression, and purification of a particular CA, i.e., nacrein-like protein encoded in the genome of the Pacific oyster Magallana gigas (previously known as Crassostrea gigas). Furthermore, the amino acid sequence, kinetic constants, and anion inhibition profile of the recombinant enzyme were investigated for the first time. The new protein, CgiNAP2X1, is highly effective as catalyst for the CO₂ hydration reaction, based on the measured kinetic parameters, i.e., kcat = 1.0 × 10⁶ s(-1) and kcat/KM = 1.2 × 10⁸ M(-1)·s(-1). CgiNAP2X1 has a putative signal peptide, which probably allows an extracellular localization of the protein. The inhibition data demonstrated that the best anion inhibitors of CgiNAP2X1 were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, which showed a micromolar affinity for this enzyme, with KIs in the range of 76-87 μM. These studies may add new information on the physiological role of the molluskan CAs in the biocalcification processes

The Turkey oak high forests in the Molise region (central Italy). Analysis of past silvicultural system and current management choices.

Aim of the work is to provide further knowledge on the silvicultural system applied to Quercus cerris hight forests in the Molise Region (Central Italy). An historical analysis, based on a number of forest management plans applied since 1940 referred to 19 municipalities and on other historical documents, is provided in the paper. Forest management has been traditionally applied in the Molise Region and therefore is at now possible to reconstruct in detail the management of the forests of Molise Region. The historical study has been integrated with the analysis of a chronosequence including four steps of stand development in a Turkey oak stand: the regeneration phase (1-2 yrs) - the unthinned young stand (46 yrs) - the unthinned adult stand (aged 60 to 100) - the mature stand (126 yrs). Mensurational surveys were carried out at each phase in order to characterize both stand structure and derive information on the silvicultural practices applied in the past, but not documented in the available papers. The stand age was determined by tree coring and count of annual rings. At the beginning of the last century, the silvicultural system to be applied in oak high forests wasn’t strictly defined and a particular kind of selection cutting was carried out. It was named taglio a salto per sezioni i.e. “compartment selection cutting”, partly leading back to a real selection cutting, partly to a shelterwood system. The use of the reported silvicultural system gave rise to irregular forest structures and led to management problems well-described in the management plans at the end of 1940s. Another consequence of the applied practices was the absence or the inadequate natural regeneration establishment. The contemporary unregulated practice of grazing the forest floor contributed to the unsuccessful regeneration and made the situation worse. The presence of an understorey layer made up by sproutings from Carpinus betulus stools clearcutting, was a further hindrance to the establishment of natural oak regeneration. In the fifties, the rate of forest management increased as a consequence of the coming up economic concern in sleeper production. The silvicultural shelterwood system was therefore codified. The system resulted to be more effective and adopted up to the present time. The traditional shelterwood system was based on a 90 yrs rotation length, three thinnings performed from the age of 25 up to 75, time of the seed cutting with a release of 90 trees per hectare. In the seventies, following the much more reduced and even null concern in wood production from oak high forests, practices into the same forests were suspended or postponed. At now, the renewed concern for fuelwood production, the only assortment marketable, as well as the increased attention paid to the multifunctional role of these forests, made again actual the problem of oak high forest management. The effectiveness of shelterwood system is the main outcome of the documental analysis performed. The study of the Carovilli’s chronosequence confirmed the close link between the current stand structures and the prescribed rules adopted in the past, in accordance with the management plans. The experimental trials in progress are now focused at defining an improvement of the shelterwood system practice as for the following issues: reduction of cutting areas; establishment of a more complex structural mosaic at neighbouring forest patches, setting up of a consistent release of seed trees i.e. number and dendrotypes to be selected at the purpose, with a special care to the crown cover to the forest floor, effective control of the understorey development due to the resprouting of Carpinus betulus stools, to enhance the establishment of oak natural regeneration

What is known in male gender differences, comorbidity and age for covid-19 pandemia? A narrative minireview

Background. On March 2020, WHO declares the world pandemic by COVID-19. In this report we report the COVID-19 infection, related to male gender, comorbidity and special population. Data resources. We describe the published studies by PubMed, Medscape and Scopus between December 2019 to May 2020. Keywords used: male/man gender, sex differences, COVID-19, comorbidity, diabetes, hypertension, elderly, pregnancy, children. Results. The elderly population and infants are a population at higher risk. The comorbidities are risk factors for the development of a more severe form of disease. There may be a sex predisposition to COVID-19 infection, with men more prone to be affected. 83.9% of COVID-19 patients with chronic kidney disease (CKD) and 57.3% of COVID-19 patients with liver diseases, have a severe disease. Conclusions. Older age, infants, male gender and comorbidity describe a crucial role for severity of COVID-19 disease. Future studies are need for the management of these patients