In situ immunofluorescent staining of autophagy in muscle stem cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and agerelated muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration

Investigation of genomic DNA methylation by ultraviolet resonant Raman spectroscopy

Cytosine plays a preeminent role in DNA methylation, an epigenetic mechanism that regulates gene expression, the misregulation of which can lead to severe diseases. Several methods are nowadays employed for assessing the global DNA methylation levels, but none of them combines simplicity, high sensitivity, and low operating costs to be translated into clinical applications. Ultraviolet (UV) resonant Raman measurements at excitation wavelengths of 272 nm, 260 nm, 250 nm, and 228 nm have been carried out on isolated deoxynucleoside triphosphates (dNTPs), on a dNTP mixture as well as on genomic DNA (gDNA) samples, commercial from salmon sperm and non-commercial from B16 murine melanoma cell line. The 228 nm excitation wavelength was identified as the most suitable energy for enhancing cytosine signals over the other DNA bases. The UV Raman measurements performed at this excitation wavelength on hyper-methylated and hypo-methylated DNA from Jurkat leukemic T-cell line have revealed significant spectral differences with respect to gDNA isolated from salmon sperm and mouse melanoma B16 cells. This demonstrates how the proper choice of the excitation wavelength, combined with optimized extraction protocols, makes UV Raman spectroscopy a suitable technique for highlighting the chemical modifications undergone by cytosine nucleotides in gDNA upon hyper- and hypo-methylation events

Inflammatory bowel disease: New insights into the interplay between environmental factors and pparγ

The pathophysiological processes of inflammatory bowel diseases (IBDs), i.e., Crohn’s disease (CD) and ulcerative colitis (UC), are still not completely understood. The exact etiology re-mains unknown, but it is well established that the pathogenesis of the inflammatory lesions is due to a dysregulation of the gut immune system resulting in over-production of pro-inflammatory cy-tokines. Increasing evidence underlines the involvement of both environmental and genetic factors. Regarding the environment, the microbiota seems to play a crucial role. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that exert pleiotropic effects on glucose homeo-stasis, lipid metabolism, inflammatory/immune processes, cell proliferation, and fibrosis. Further-more, PPARs modulate interactions with several environmental factors, including microbiota. A significantly impaired PPARγ expression was observed in UC patients’ colonic epithelial cells, suggesting that the disruption of PPARγ signaling may represent a critical step of the IBD pathogenesis. This paper will focus on the role of PPARγ in the interaction between environmental factors and IBD, and it will analyze the most suitable in vitro and in vivo models available to better study these relationships

Allele specific CRISPR/Cas9 editing of dominant Epidermolysis Bullosa Simplex in human epidermal stem cells

: Epidermolysis Bullosa Simplex (EBS) is a rare skin disease inherited mostly in an autosomal dominant manner. Patients display a skin fragility that leads to blisters and erosions caused by minor mechanical trauma. EBS phenotypic and genotypic variants are caused by genetic defects in intracellular proteins whose function is to provide the attachment of basal keratinocytes to the basement membrane zone and most of EBS cases display mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes. Besides palliative treatments, there is still no long-lasting effective cure to correct the mutant gene and abolish dominant negative effect of the pathogenic protein over its wild-type counterpart. Here, we propose a molecular strategy for EBS01 patient's keratinocytes carrying a monoallelic c.475/495del21 mutation in KRT14 exon1. Through the CRISPR/Cas9 system we performed a specific cleavage only on the mutant allele and restore a normal cellular phenotype and a correct intermediate filament network, without affecting the epidermal stem cell, referred to as holoclones, which play a crucial role in epidermal regeneration

Bisimulation of Labeled State-to-Function Transition Systems of Stochastic Process Languages

Labeled state-to-function transition systems, FuTS for short, admit multiple transition schemes from states to functions of finite support over general semirings. As such they constitute a convenient modeling instrument to deal with stochastic process languages. In this paper, the notion of bisimulation induced by a FuTS is proposed and a correspondence result is proven stating that FuTS-bisimulation coincides with the behavioral equivalence of the associated functor. As generic examples, the concrete existing equivalences for the core of the process algebras ACP, PEPA and IMC are related to the bisimulation of specific FuTS, providing via the correspondence result coalgebraic justification of the equivalences of these calculi.Comment: In Proceedings ACCAT 2012, arXiv:1208.430

Study protocol: Investigating the feasibility of a hybrid delivery of home-based cluster set resistance training for individuals previously treated for lung cancer

Background: Symptom burden remains a critical concern for individuals with non-small cell lung cancer (NSCLC) following the completion of treatment. The most common symptom clusters, dyspnea (shortness of breath) and fatigue, can contribute to physical decline, reductions in quality of life, and a higher risk of comorbidities and mortality. Dyspnea is a primary limiter of exercise capacity in individuals with lung cancer, resulting in exercise avoidance and an accelerated physical decline. As such, designing resistance training with cluster sets to mitigate symptoms of dyspnea and fatigue may result in improved exercise tolerance. Thus, maintaining the exercise stimulus via cluster sets, combined with improved tolerance of the exercise, could result in the maintenance of physical function and quality of life. The purpose of this study is to investigate the feasibility and preliminary efficacy of a hybrid-delivery home-based cluster-set resistance training program in individuals with NSCLC. Methods: Individuals with NSCLC (n = 15), within 12 months of completion of treatment, will be recruited to participate in this single-arm feasibility trial. Participants will complete 8 weeks of home-based resistance training designed to minimize dyspnea and fatigue. The hybrid delivery of the program will include supervised sessions in the participants’ home and virtual supervision via video conferencing. The primary outcome of feasibility will be quantified by recruitment rates, retention, acceptability, and intervention fidelity. Exploratory outcomes (dyspnea, fatigue, quality of life, physical function, and body composition) will be assessed pre- and post-intervention. Discussion: This study will provide important data on the feasibility of delivering this intervention and inform procedures for a future randomized controlled trial. Trial registration: Record not yet publi

A Proposal for a Multidisciplinary Integrated Oral Health Network for Patients Undergoing Major Orthopaedic Surgery (IOHN-OS).

The passing of the years of life physiologically leads to the accumulation of changes in tissues in the oral cavity, influencing dentition, chewing and swallowing mechanisms, and the oral microbiota. Some diseases and medications can aggravate oral symptoms and negatively influence eating behaviours, increasing the likelihood of becoming malnourished. This could make older individuals more vulnerable to complications when undergoing major orthopaedic surgery. Hidden infection foci in the oral cavity are a recognised cause of post-operative periprosthetic joint infections. Dysfunctional oral problems might also compromise feeding after surgery when good nutrition represents a fundamental aspect of a proper recovery. To manage these shortcomings, in this article, the authors hypothesise a multidisciplinary path of care named the Integrated Oral Health Network applied to major Orthopaedic Surgery (IOHN-OS). This peri-operative initiative would include pre-operative oral health screening and risk management by a dental team, patient education programmes before and after surgery, and bedside gerodontology actions like oral care and meal and eating support for fragile individuals. The IOHN-OS has the potential to reshape the concept of suitability for major orthopaedic surgery and generate momentum for designing community-based surveillance programmes that can keep the mouths of older subjects healthy for a long time

Mechanical Stability and Decohesion of Sol-Fel Hybrid Coatings on Metallic Substrates

ABSTRACT The mechanical properties and adhesion behaviour of coatings based on organically modified silanes and synthesized using sol-gel technology were assessed using nano-indentation and microtensile testing, respectively. The relationship between the film structure and its mechanical response is examined. It is shown that the mechanical properties (hardness and Young's modulus) of the coatings is influenced dramatically by the organic substituent and the presence of an oxide layer thermally grown on the substrate material prior to deposition plays an important role on the film/substrate adhesion behaviour. Keywords: Sol-gel, cracking, debonding, adhesion. INTRODUCTION The adhesion and mechanical stability of thin film coatings on substrates is increasingly becoming a key issue in device reliability as technology driven products demand smaller, thinner and more complex functional coatings. It is well known that the reliability of metallurgical protective coatings and microelectronic circuits containing multilayered thin film structures is strongly influenced by their interfacial properties, namely strength, fracture resistance and adhesion. Hence, characterizing and understanding the cracking evolution, debonding behavior and adhesion performance of thin films subject to external applied stresses is crucial when planning such applications. By using micro-mechanical in-situ tensile experiments it is possible to detect and analyze the critical conditions for cracking and debonding of the thin film Sol-gel hybrid coatings, also known as "ormosils" (organically modified silicates), up to several hundred nanometers thick are used to modify the functional behavior of the inorganic component for plastic and metal surfaces to confer wear/abrasion resistance and corrosion protection, respectively Characterization of the film cracking and debonding is achieved primarily by in-situ optical microscopy and subsequent scanning electron microscopy (SEM). Load-partial unload data from nanoindentation of the coatings on copper were used to determine the film properties and to provide data for future fracture and adhesion analyses. Implications concerning the influence of the thermal oxide layer on the interfacial adhesion behavior of the sol-gel coatings are discussed. EXPERIMENTAL Sol-gel coating solutions were prepared by adding a 0.01 M solution of HNO 3 to a mixture of the inorganic constituent tetraethylorthosilicate (TEOS) and the organic constituent glycidoxypropyltrimethoxysilane (GTMS) in ethanol. The solutions prepared were: (i) 100% TEO