Novel autophagic vacuolar myopathies: Phenotype and genotype features.

BACKGROUND: Autophagic vacuolar myopathies (AVMs) are an emerging group of heterogeneous myopathies sharing histopathological features on muscle pathology, in which autophagic vacuoles are the pathognomonic morphologic hallmarks. Glycogen storage disease type II (GSDII) caused by lysosomal acid α-glucosidase (GAA) deficiency is the best-characterised AVM. AIMS: This study aimed to investigate the mutational profiling of seven neuromuscular outpatients sharing clinical, myopathological and biochemical findings with AVMs. METHODS: We applied a diagnostic protocol, recently published by our research group for suspected late-onset GSDII (LO-GSDII), including counting PAS-positive lymphocytes on blood smears, dried blood spot (DBS)-GAA, muscle biopsy histological and immunofluorescence studies, GAA activity assay and expression studies on muscle homogenate, GAA sequencing, GAA multiplex ligation-dependent probe amplification (MLPA) and whole exome sequencing (WES). RESULTS: The patients had a limb girdle-like muscular pattern with persistent hyperCKaemia; vacuolated PAS-positive lymphocytes, glycogen accumulation and impaired autophagy at muscle biopsy. Decreased GAA activity was also measured. While GAA sequencing identified no pathogenic mutations, WES approach allowed us to identify for each patient an unexpected mutational pattern in genes cooperating in lysosomal-autophagic machinery, some of which have never been linked to human diseases. CONCLUSIONS: Our data suggest that reduced GAA activity may occur in any condition of impaired autophagy and that WES approach is advisable in all genetically undefined cases of autophagic myopathy. Therefore, deficiency of GAA activity and PAS-positive lymphocytes should be considered as AVM markers together with LC3/p62-positive autophagic vacuoles.

Vacuolated PAS-positive lymphocytes as an hallmark of Pompe disease and other myopathies related to impaired autophagy.

Autosomal recessive Pompe disease is a lysosomal disorder caused by mutations of the acid-α-glucosidase (GAA) gene. Deficiency of GAA enzyme leads to glycogen accumulation and autophagy impairment in cardiac and skeletal muscles, but also in lymphocytes. Since an effective therapy is available, a rapid, sensitive, and specific test is crucial to early identify affected subjects. Number of lymphocytes containing PAS-positive vacuoles was evaluated on blood films from 72 consecutive adult patients with hyperckemia and/or muscle weakness, 13 genetically confirmed late-onset-Pompe-disease (LOPD) and 13 of their offspring. GAA activity, measured on dried blood spot (DBS) in all patients inversely correlated with number of PAS-positive lymphocytes. More than 4 PAS-positive lymphocytes were found in 11 out of the 72 patients (6 new diagnosis of LOPD, 3 different glycogen storage myopathies, 1 glucose-6-phosphate dehydrogenase deficiency, 1 caveolinopathy), in all 13 LOPD patients and in the 13 LOPD offspring. These latter resulted to have all a single GAA mutation but low GAA levels. Immunostaining with the autophagy markers LC3 and p62 confirmed the autophagic nature of lymphocytes vacuoles. ROC curve assessment of PAS-positive lymphocytes disclosed 100% of sensitivity and 94% of specificity in recognizing both compound heterozygous and heterozygous GAA carriers. The other myopathies with more than 4 PAS-positive lymphocytes appeared to be all related to impaired autophagy, which seems to be responsible of PAS-positive vacuolated lymphocytes formation. Quantification of PAS-positive lymphocytes in blood films is useful to identify autophagic vacuolar myopathies and should be routinely used as first level test for Pompe disease.

Identification of the first dominant mutation of LAMA5 gene causing a complex multisystem syndrome due to dysfunction of the extracellular matrix.

BACKGROUND: The laminin alpha 5 gene (LAMA5) plays a master role in the maintenance and function of the extracellular matrix (ECM) in mammalian tissues, which is critical in developmental patterning, stem cell niches, cancer and genetic diseases. Its mutations have never been reported in human disease so far. The aim of this study was to associate the first mutation in LAMA5 gene to a novel multisystem syndrome. METHODS: A detailed characterisation of a three-generation family, including clinical, biochemical, instrumental and morphological analysis, together with genetics and expression (WES and RNAseq) studies, was performed. RESULTS: The heterozygous LAMA5 mutation c.9418G>A (p.V3140M) was associated with skin anomalies, impaired scarring, night blindness, muscle weakness, osteoarthritis, joint and internal organs ligaments laxity, malabsorption syndrome and hypothyroidism. We demonstrated that the mutation alters the amount of LAMA5 peptides likely derived from protein cleavage and perturbs the activation of the epithelial-mesenchymal signalling, producing an unbalanced expression of Sonic hedgehog and GLI1, which are upregulated in cells from affected individuals, and of ECM proteins (COL1A1, MMP1 and MMP3), which are strongly inhibited. Studies carried out using human skin biopsies showed alteration of dermal papilla with a reduction of the germinative layer and an early arrest of hair follicle downgrowth. The knock-in mouse model, generated in our laboratory, shows similar changes in the tissues studied so far. CONCLUSIONS: This is the first report of a disease phenotype associated with LAMA5 mutation in humans.

Laser scanner and UAV digital photogrammetry as support tools for cosmic-ray muon radiography applications: an archaeological case study from Italy.

The use of light detection and ranging technologies, i.e. terrestrial laser scanner (TLS), airborne laser scanner (ALS) and mobile laser scanner (MLS), together with the unmanned aerial vehicles digital photogrammetry (UAV-DP) and satellite data are proving to be fundamental tools to carry out reliable muographic measurement campaigns. The main purpose of this paper is to propose a workflow to correctly plan and exploit these types of data for muon radiography aims. To this end, a real case study is presented: searching for hidden tombs in the Etruscan necropolis of Palazzone (Umbria, Italy). A high-resolution digital elevation model (DEM) and three-dimensional models of the ground surface/sub-surface of the study area were created by merging data obtained using different survey methods to achieve the most accurate three-dimensional environment. Indeed, the simulated muon flux transmission used to infer relative transmission values, and the estimated density distribution, depends on the reliability of the three-dimensional reconstructed ground surface model. The aim of this study is to provide knowledge on the use of TLS and UAV-DP data and GPS-acquired points within the transmission-based muography process and how these data could improve or worsen the muon imaging results. Moreover, this study confirmed that muography applications require a multidisciplinary approach.

Three-dimensional muon imaging of cavities inside the Temperino mine (Italy).

Muon radiography (muography) is an imaging technique based on atmospheric muon absorption in matter that allows to obtain two and three-dimensional images of internal details of hidden objects or structures. The technique relies on atmospheric muon flux measurements performed around and underneath the object under examination. It is a non-invasive and passive technique and thus can be thought of as a valid alternative to common prospecting techniques used in archaeological, geological and civil security fields. This paper describes muon radiography measurements, in the context of archaeological and geological studies carried out at the Temperino mine (LI, Tuscany, Italy), for the search and three-dimensional visualisation of cavities. This mine has been exploited since Etruscan times until recently (1973), and is now an active tourist attraction with public access to the tunnels. Apart from the archaeological interest, the importance of mapping the cavities within this mine lies in identifying the areas where the extraction ores were found and also in the safety issues arising from the tourist presence inside the mine. The three-dimensional imaging is achieved with two different algorithms: one involving a triangulation of two or more measurements at different locations; the other, an innovative technique used here for the first time, is based on the back-projections of reconstructed muon tracks. The latter requires only a single muographic data tacking and is to be preferred in applications where more than one site location can be difficult to access. Finally the quality of the three-dimensional muographic imaging was evaluated by comparing the results with the laser scan profiles obtained for some known cavities within the Temperino mine.

Exploring the Effects of Osteopathic Manipulative Treatment on Autonomic Function Through the Lens of Heart Rate Variability.

The osteopathic community has long hypothesized that the autonomic nervous system (ANS) represents one of the putative substrates through which osteopathic manipulative treatment (OMT) can improve body functions that have been altered by musculoskeletal alterations. Heart rate variability (HRV) is an important physiological measure of cardiac ANS activity. Emerging evidence suggests that OMT is associated with HRV changes that (i) are indicative of a larger cardiac vagal modulation, (ii) are independent from the part of the body needing treatment, (iii) occur even in the absence of musculoskeletal alterations. Yet, many questions remain unanswered, the duration of these effects and the specificity of HRV responses to different OMT techniques being perhaps the most critical. Therefore, this paper discusses prospects for future applications of HRV for the study of the influence of OMT on ANS function. Moreover, based on existing studies and preliminary data on the effects of OMT on HRV in specific pathological (hypertension) and physiological (stress exposure and recovery from sport competition) conditions that are commonly associated with increased sympathetic and/or decreased vagal activity, we propose that HRV analysis could be exploited to evaluate the effectiveness of OMT as a preventive or complementary strategy in clinical and non-clinical conditions characterized by ANS imbalance.

Teaching Computer Graphics Through a Digital Humanities Project.

Teaching computer graphics in an effective way is a challenging task. Different approaches are proposed in the literature, some focusing on traditional teacher-led lectures, others on interactive teaching methods. This paper describes an experiential learning approach applied in a university-level course in computer engineering. Students were involved in the realization of a digital humanities project, the 3-D modeling of the city of Pavia, Italy, as it was in the 16th century. Traditional lectures introduced key theoretical elements, but students were subsequently given specific assignments and retrieved additional content when completing the tasks assigned to them under the overall guidance of their supervisors.

First study on the peptidergic innervation of the brain superior sagittal sinus in humans.

The superior sagittal sinus (SSS) of the mammalian brain is a pain-sensitive intracranial vessel thought to play a role in the pathogenesis of migraine headaches. Here, we aimed to investigate the presence and the potential co-localization of some neurotransmitters in the human SSS. Immunohistochemical and double-labeling immunofluorescence analyses were applied to paraformaldehyde-fixed, paraffin-embedded, coronal sections of the SSS. Protein extraction and Western blotting technique were performed on the same material to confirm the morphological data. Our results showed nerve fibers clustered mainly in large bundles tracking parallel to the longitudinal axis of the sinus, close in proximity to the vascular endothelium. Smaller fascicles of fibers encircled the vascular lumen in a spiral fashion, extending through the subendothelial connective tissue. Isolated nerve fibers were observed around the openings of bridging veins in the sinus or around small vessels extending into the perisinusal dura. The neurotransmitters calcitonin gene related peptide (CGRP), substance P (SP), neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), tyrosine hydroxylase (TH), and neuropeptide Y (NPY) were found in parietal nerve structures, distributed all along the length of the SSS. Overall, CGRP- and TH-containing nerve fibers were the most abundant. Neurotransmitters co-localized in the same fibers in the following pairs: CGRP/SP, CGRP/NOS, CGRP/VIP, and TH/NPY. Western blotting analysis confirmed the presence of such neurosubstances in the SSS wall. Overall our data provide the first evidence of the presence and co-localization of critical neurotransmitters in the SSS of the human brain, thus contributing to a better understanding of the sinus functional role.

A novel GBE1 mutation and features of polyglucosan bodies autophagy in adult polyglucosan body disease.

We report the clinical, neuro-imaging, pathological and biochemical features of an Italian family in which two siblings have the Adult Polyglucosan Body Disease (APBD). APBD is a rare autosomal recessive disorder characterized by a gradually progressive involvement of both the central and peripheral nervous systems caused by the deficiency of the glycogen branching enzyme (GBE1). The two affected siblings, a 64-year-old man and his 67-year-old sister who had complained of urinary urgency and sporadic incontinence and also progressive gait difficulty for 6 and 7 years respectively, had severely impaired deep sensations on direct examination and a moderately severe symmetrical, axonal sensory-motor neuropathy on electrophysiological testing. GBE1 activity was below 25% of the normal rate in leukocytes and sural nerves. The siblings were homozygous for the novel GBE1 mutation p.N541D. All other members of the pedigree are heterozygous and manifest no symptoms, even in the very elderly. The affected siblings showed polyglucosan bodies (PBs) included within non-myelinating Schwann cells and within lymphocyte vesicles, which were positive for the autophagy markers P62 and LC3-II at immunofluorescence microscopy.