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Spinal cord injury produces muscle wasting, which is especially severe after the complete and permanent damage of lower motor neurons that occurs in complete Cauda Equina Syndrome. Even in this worst-case scenario, we have shown that permanently denervated Quadriceps muscle can be rescued by surface Functional Electrical Stimulation and a purpose designed home-based rehabilitation regime. Here, our aim is to show that the effects are extended to both antagonist muscles and the skin of the thighs. Before and after 2 years of electrical stimulation, mass and structure of Quadriceps and Hamstrings muscles were quantitated by force measurements. Muscle gross cross section were evaluated using color computed tomography, muscle and skin biopsies by quantitative histology and immunohistochemistry. The treatment produced: a) an increase in cross-sectional area of stimulated muscles; b) an increase in muscle fiber mean diameter; c) improvements in ultrastructural organization; and d) increased force output during electrical stimulation. The recovery of Quadriceps muscle force was sufficient to allow 25% of the compliant subjects to perform stand-up and step-in place trainings. Improvements are extended to hamstring muscles and skin. Indeed, the cushioning effect provided by recovered tissues is a major clinical benefit. It is our hope that, with or without our advice, trials may start soon in Europe and Russia to provide persons-in-need the help they deserve.

The marathon is the most classic Olympic running event. In several cities worldwide it has become very popular with participation increasing during the last 20 years, particularly by Master Athletes. There are evidences that long-distance running could provide considerable health benefits for older runners, specifically risk reduction of cardiovascular diseases, cancer, diabetes, depression, and falls. Several studies have focused on the distribution of participants and their performance on famous marathons such as those of Berlin, Boston and New York. In this preliminary study we have analyzed data from several editions of the Venice marathon, a famous Italian race that attracts people from every corner of the world. The Venice marathon is listed in Abbott World Marathon Majors Wanda Age Group World Ranking and is Bronze Label certificated by IAAF, and Gold Label by FIDAL. The marathon starts outside Venice near Stra, then runs along the Brenta Riviera to Venice where the runners cross the canals over floating bridges set up for the race. For this study we analyzed data of the Venice marathon describing gender distribution in 17 editions (2003-2019), but groups of age-categories and their nationality only in 13 editions from 2007 to 2019. The analysis shows a steady increase in female participation, from 2003 to 2019.

Paolo Gava, (Conegliano, Treviso, September 1, 1946 – Stra, Venezia, Italy, July 19, 2021) was a sustainable resources engineer, who worked in Italy, France and England, leading research programs well before the current international interest in countering global warming. Passionate about Tango, Paolo kept himself in shape for many decades by running or pedaling or roller-skating, after years of training as a semi-professional athlete, competing and winning Italian and European short distance races in the Master classes. Then, Paolo applied his engineering skills to optimize comparisons between the results of the different Classes of Master Athletes, questioning the rules used by Italian and World Master Sports Associations. Friendly discussing during an after-dinner, he shocked us claiming that, in absence of diseases and trauma (Early Aging), the aging decay is a linear process from 30 to 110 years. Under our friendly pressure he was able to publish his first biomedical article, detailing his mathematical approaches and results in a 2015 issue of Experimental Aging Research, titled: Age-associated power decline from running, jumping and throwing male master world records. To honor his other legacies during his last six years of life, we add here further examples of Paolo's scientific studies and his relationships with senior colleagues and young students of sports and aging sciences.

Skeletal muscle atrophy may occur with disease, injury, decreased muscle use, starvation, and normal aging. No reliably effective treatments for atrophy are available, thus research into the mechanisms contributing to muscle loss is essential. The ERG1A K+ channel contributes to muscle loss by increasing ubiquitin proteasome proteolysis (UPP) in the skeletal muscle of both unweighted and cachectic mice. Because the mechanisms which produce atrophy vary based upon the initiating factor, here we investigate atrophy produced by denervation. Using immunohistochemistry and immunoblots, we demonstrate that ERG1A protein abundance increases significantly in the Gastrocnemius muscle of rodents 7 days after both sciatic nerve transection and hind limb unweighting. Further, we reveal that ectopic expression of a Merg1a encoded plasmid in normal mouse Gastrocnemius muscle has no effect on activity of the NFκB transcription factor family, a group of proteins which contribute to muscle atrophy by modulation of the UPP. Further, although NFκB activity increases significantly after denervation, we show that expression of a plasmid encoding a dominant negative Merg1a mutant in Gastrocnemius muscle prior to denervation, has no effect on NFκB activity. Thus, although the ERG1A K+ channel increases UPP, it does not do so through modulation of NFκB transcription factors.

Persons suffering with systemic neuromuscular disorders or chronic organ failures, spend less time for daily physical activity, aggravating their mobility impairments. From 2020, patients at risk are also older adults, who, though negative for the SARS-Cov-2 infection, suffer with a fatigue syndrome due to home restriction/quarantine. Besides eventual psycological managements, it could be useful to offer to these patients a rehabilitation workouts easy to learn and to independently repeat at home (Full-Body In-Bed Gym). Inspired by the proven capability to recover skeletal muscle contractility and strength by home-based volitional exercises and functional electrical stimulation (FES), we suggest for this fatigue syndrome a 10-20 min long daily routine of easy and safe physical exercises that may recover from muscle weakness the main 400 skeletal muscles used for every-day activities. Leg muscles could be trained also by an adjunctive neuro-muscular electrical stimulation (NMES) in frail old persons. Many of the exercises could be performed in bed (Full-Body in-Bed Gym), thus hospitalized patients can learn this light training before leaving the hospital. Full-Body in-Bed Gym is, indeed, an extension of well-established cardiovascular-ventilation rehabilitation training performed by patients after heavy surgery. Blood pressure readings, monitored before and after daily routine of Full-Body in-Bed Gym, demonstrate a transient decrease in peripheral resistance due to increased blood flow to major body muscles. Continued regularly, Full-Body in-Bed Gym may help maintaining independence of frail people, including those suffering with the fatigue syndrome related to the restrictions/quarantine imposed to the general population during the COVID-19 pandemic.

Mobility-impaired persons, either very old or younger but suffering with systemic neuromuscular disorders or chronic organ failures, spend small amounts of time for daily physical activity, contributing to aggravate their poor mobility by resting muscle atrophy. Sooner or later the limitations to their mobility enforce them to bed and to more frequent hospitalizations. We include among these patients at risk those who are negative for the SARS-COV-2 infection, but suffering with COVID-19 pandemic syndrome. Beside managements of psychological symptoms, it is mandatory to offer to the last group physical rehabilitation approaches easy to learn and self-managed at home. Inspired by the proven capability to recover skeletal muscle contractility and strength by home-based volitional exercises and functional electrical stimulation, we suggest also for chronic COVID-19 pandemic syndrome a 10–20 min long daily routine of easy and safe physical exercises that can activate, and recover from weakness, the main 400 skeletal muscles used for every-day mobility activities. Persons can do many of them in bed (Full-Body in-Bed Gym), and hospitalized patients can learn this light training before leaving the hospital. It is, indeed, an extension of well-established cardiovascular-respiratory rehabilitation training performed after heavy surgical interventions. Blood pressure readings, monitored before and after daily routine, demonstrate a transient decrease in peripheral resistance due to increased blood flow of many muscles. Continued regularly, Full-Body in-Bed Gym may help maintaining independence of frail people, including those suffering with the COVID-19 pandemic syndrome.

All progressive muscle contractile impairments, including advanced age-related muscle power decline, need permanent management. Most elderly persons, in particular octogenarians, spend small amounts of time in daily physical activity, resulting in a decline in body condition with more and more frequent hospitalizations and finally potentially forcing them to bed permanently. Further several neurological injuries, which are even more acutely debilitating than those problems related to aging, are responsible for early limitation of mobility. Inspired by the proven capability to recover skeletal muscle contractility and strength by home-based functional electrical stimulation (h-bFES) in both elderly and SCI patients, we suggest that the elderly and early aging patients participate in hbFES and add a 20 min daily routine of 12 easy and safe physical exercises, namely home-based Full-Body In-Bed Gym. Continued regularly, h-bFES and the Full-Body In-Bed Gym will help to maintain the independence of frail older people and may reduce the risks of serious consequences of accidental falls and pressure sore complications.

To evaluate progression of skin atrophy during 8 years of complete Conus-Cauda Syndrome and its recovery after 2 years of surface Functional Electrical Stimulation a cohort study was organized and implemented. Functional assessments, tissue biopsies, and follow-up were performed at the Wilhelminenspital, Vienna, Austria; skin histology and immunohistochemistry at the University of Padova, Italy on 13 spinal cord injury persons suffering up to 10 years of complete conus/cauda syndrome. Skin biopsies (n. 52) of both legs were analyzed before and after 2 years of home-based Functional Electrical Stimulation delivered by large anatomically shaped surface electrodes placed on the skin of the anterior thigh. Using quantitative histology we analyzed: 1. Epidermis atrophy by thickness and by area; 2. Skin flattening by computing papillae per mm and Interdigitation Index of dermal-epidermal junctions; 3. Presence of Langerhans cells. Linear regression analyses show that epidermal atrophy and flattening worsen with increasing years post- spinal cord injury and that 2 years of skin electrostimulation by large anatomically shaped electrodes reverses skin changes (pre-functional Electrical Stimulation vs post-functional Electrical Stimulation: thickness 39%, P

Our previous studies have shown that severely atrophic Quadriceps muscles of spinal cord injury (SCI) persons suffering with complete conus and cauda equina syndrome, and thus with permanent denervation-induced atrophy and degeneration of muscle, were almost completely rescued to normal size after two years of home based Functional Electrical Stimulation (hbFES). Since large surface electrodes were used to stimulate the denervated thigh muscles, we wanted to know if the skin was affected by this peculiar long-term treatment. Indeed, we demonstrated by two approaches that the epidermis decreases in thickness in the long term denervated persons, while it increased to almost pre-SCI values in hbFES compliant SCI persons. Here we report data of morphometry of skin biopsies from both legs of 18 SCI persons, harvested at enrolment in the Project RISE, to test if the Interdigitation Index, a simple measurement of the epidermal‐dermal junction, may provide a further precise quantitative evidence of the flattening of the skin in those SCI persons. The Interdigitation Index of the 36 skin biopsies shows a higly significant linear correlation with the years of SCI (p < 0.001). Furthermore, when the 18 SCI persons are divided in two groups (1 to 3.9 versus 4.1 to 8.0 years from SCI, respectively) and the data are compared, the later Group presents a statistically significant -22% decrease (p, 0.029) of the Interdigitation Index. On the other hand counting the papille do not provide the same strong evidence. In conclusion, the Interdigitation Index is an additional sound quantitative structural biomarker of skin atrophy and flattening occurring in SCI. The result correlates with the much severe extent of atrophy of the permanently denervated thigh muscles, as determined at both macro and microscopic levels.We are confident that the Interdigitation Index will provide sound evidence that the effects of hbFES, we previously reported on skeletal muscle and epidermis thickness, will be extended to the dermal layer of the skin, suggesting a coordinated negative effects of SCI on skeletal muscle and skin, and an improvement of both tissues after hbFES. Incoming analyses will be extended to basal lamina, collagene types, elastic fibers and skin annexes in the subcutaneous layer.

Our previous studies have shown that severely atrophic Quadriceps muscles of spinal cord injury (SCI) patients suffering with complete conus and cauda equina lesions, and thus with permanent denervation-induced atrophy and degeneration of muscle fibers, were almost completely rescued to normal size after two years of home-based Functional Electrical Stimulation (h-bFES). Since we used large surface electrodes to stimulate the thigh muscles, we wanted to know if the skin was affected by long-term treatment. Here we report preliminary data of morphometry of skin biopsies harvested from legs of 3 SCI patients before and after two years of h-bFES to determine the total area of epidermis in transverse skin sections. By this approach we support our recently published results obtained randomly measuring skin thickness in the same biopsies after H-E stain. The skin biopsies data of three subjects, taken together, present indeed a statistically significant 30% increase in the area of the epidermis after two years of h-bFES. In conclusion, we confirm a long term positive modulation of electrostimulated epidermis, that correlates with the impressive improvements of the FES-induced muscle strength and bulk, and of the size of the muscle fibers after 2-years of h-bFES.

After spinal cord injury (SCI), patients spend daily several hours in wheelchairs, sitting on their hamstring muscles. SCI causes muscle atrophy and wasting, which is especially severe after complete and permanent damage to lower motor neurons. A European Union (EU)-supported work demonstrates that electrical fields produced by large electrodes and purpose-developed electrical stimulators recover both quadriceps and hamstring muscles, producing a cushioning effect capable of benefitting SCI patients, even in the worst case of complete and long-term lower motor neuron denervation of leg muscles. We reported that 20 out of 25 patients completed a 2-year h-bFES program, which resulted in (1) a 35% increase in cross-sectional area of the quadriceps muscles (P < 0.001), (2) a 75% increase in mean diameter of quadriceps muscle fibers (P < 0.001), and (3) improvement of the ultrastructural organization of contractile machinery and of the Ca2+-handling system. Though not expected, after 2 years during which the 20 subjects performed 5 days per week h-bFES of the atrophic quadriceps muscles, the CT cross-sectional area of the hamstring muscles also augmented, increasing from 26.9+/−8.4 (cm2) to 30.7+/−9.8 (cm2), representing a significant (p ≤ 0.05) 15% increase. Here we show by quantitative muscle color computed tomography (QMC-CT) that h-bFES-induced tissue improvements are present also in the hamstring muscles: a once supposed drawback (lack of specificity of muscle activation by large surface electrodes) is responsible for a major positive clinical effect. Interestingly, 2 years of home-based FES by large surface electrodes reversed also the denervation-induced skin atrophy, increasing epidermis thickness. Finally, we would like to attract attention of the readers to quantitative muscle color computed tomography (QMC-CT), a sensitive quantitative imaging analysis of anatomically defined skeletal muscles introduced by our group to monitor atrophy/degeneration of skeletal muscle tissue. Worldwide acceptance of QMC-CT will provide physicians an improved tool to quantitate skeletal muscle atrophy/degeneration before and during rehabilitation strategies so that therapy for mobility-impaired persons can be better prescribed, evaluated, and altered where needed.

After spinal cord injury (SCI), patients spend daily several hours in wheelchairs, sitting on their hamstring muscles. SCI causes muscle atrophy and wasting, which is especially severe after complete and permanent damage to lower motor neurons. A European Union (EU)-supported work demonstrates that electrical fields produced by large electrodes and purpose-developed electrical stimulators recover both quadriceps and hamstring muscles, producing a cushioning effect capable of benefitting SCI patients, even in the worst case of complete and long-term lower motor neuron denervation of leg muscles. We reported that 20 out of 25 patients completed a 2-year h-bFES program, which resulted in (1) a 35% increase in cross-sectional area of the quadriceps muscles (P 0.001), (2) a 75% increase in mean diameter of quadriceps muscle fibers (P 0.001), and (3) improvement of the ultrastructural organization of contractile machinery and of the Ca2+-handling system. Though not expected, after 2 years during which the 20 subjects performed 5 days per week h-bFES of the atrophic quadriceps muscles, the CT cross-sectional area of the hamstring muscles also augmented, increasing from 26.9+/-8.4 (cm

The sternomastoid (SM) muscle in rodents presents a peculiar distribution of fiber types with a steep gradient from the ventral, superficial, white portion to the dorsal, deep, red region, where muscle spindles are restricted. Cross section of the medial longitudinal third of the rat SM contains around 10,000 muscle fibers with a mean diameter of 51.28±12.62 (μm +/- SD). Transverse sections stained by Succinate Dehydrogenase (SDH) reaction clearly presents two distinct regions: the dorsal deep red portion encompassing a 40% cross section area contains a high percentage of packed SDH-positive muscle fibers, and the ventral superficial region which contains mainly SDH-negative muscle fibers. Indeed, the ventral superficial region of the rat SM muscle contains mainly fast 2B muscle fibers. These acidic ATPase pH 4.3-negative and SDH-negative 2B muscle fibers are the largest of the SM muscle, while the acidic ATPase pH 4.3-positive and SDH-positive Type 1 muscle fibers are the smallest. Here we show that in thin transverse cryosections only 2 or 3 muscle spindle are observed in the central part of the dorsal deep red portion of the SM muscle. Azan Mallory stained sections allow at the same time to count the spindles and to evaluate aging fibrosis of the skeletal muscle tissue. Though restricted in the muscle red region, SM spindles are embedded in perimysium, whose changes may influence their reflex activity. Our findings confirm that any comparisons of changes in number and percentage of muscle spindles and muscle fibers of the rat SM muscle will require morphometry of the whole muscle cross-section. Muscle biopsies of SM muscle from large mammals will only provide partial data on the size of the different types of muscle fibers biased by sampling. Nonetheless, histology of muscle tissue continue to provide practical and low-cost quantitative data to follow-up translational studies in rodents and beyond.

Our studies have shown that atrophic Quadriceps muscles from spinal cord injury patients suffering with permanent denervation-induced atrophy and degeneration of muscle fibers, were almost completely rescued to normal size after two years of home-based functional electrical stimulation (h-bFES). Because we used surface electrodes to stimulate the muscle, we wanted to know how the skin was affected by the treatments. Here, we report preliminary data from histological morphometry of Hematoxylin-Eosin-stained paraffin-embedded skin sections harvested from the legs of three SCI patients before and after two years of h-bFES. Despite the heterogeneity of gender and time from SCI, comparing pre vs post h-bFES in these three SCI patients, the data show that: (1) In one subject skin biopsies from both the right and left leg experienced a statistically significant increase in thickness of the epidermis after two years of H-bFES; (2) In the other two subjects, one leg showed a significant increase in epidermis thickness, while in the other leg there was either small positive or negative non-significant changes in epidermis thickness; and (3) more importantly, comparison of grouped data from the three subjects shows that there was a significant 28% increase in the thickness of the epidermis in response to two years of h-bFES rehabilitation. In conclusion, the three educational cases show a long-term positive modulation of epidermis thickness after two years of h-bFES, thus extending to skin the positive results previously demonstrated in skeletal muscle, specifically, a substantial recovery of muscle mass and contractile function after long-term h-bFES.

Screening nanoparticle toxicity directly on cell culture can be a fast and cheap technique. Nevertheless, to obtain results in accordance with those observed in live animals, the conditions in which cells are cultivated should resemble the one encountered in live systems. Microfluidic devices offer the possibility to satisfy this requirement, in particular with endothelial cell lines, because they are capable to reproduce the flowing media and shear stress experienced by these cell lines in vivo. In this work, we exploit a microfluidic device to observe how human umbilical vein endothelial cells (HUVEC) viability changes when subject to a continuous flow of culture medium, in which spherical citrate-stabilized gold nanoparticles of different sizes and at varying doses are investigated. For comparison, the same experiments are also run in multiwells where the cells do not experience the shear stress induced by the flowing medium. We discuss the results considering the influence of mode of exposure and nanoparticle size (24 and 13 nm). We observed that gold nanoparticles show a lower toxicity under flow conditions with respect to static and the HUVEC viability decreases as the nanoparticle surface area per unit volume increases, regardless of size. Electronic supplementary material The online version of this article (10.1007/s11051-017-3993-5) contains supplementary material, which is available to authorized users.

The term ‘visceral fascia’ is a general term used to describe the fascia lying immediately beneath the mesothelium of the serosa, together with that immediately surrounding the viscera, but there are many types of visceral fasciae. The aim of this paper was to identify the features they have in common and their specialisations. The visceral fascia of the abdomen (corresponding to the connective tissue lying immediately beneath the mesothelium of the parietal peritoneum), thorax (corresponding to the connective tissue lying immediately beneath the mesothelium of the parietal pleura), lung (corresponding to the connective tissue under the mesothelium of the visceral pleura), liver (corresponding to the connective tissue under the mesothelium of the visceral peritoneum), kidney (corresponding to the Gerota fascia), the oesophagus (corresponding to its adventitia) and heart (corresponding to the fibrous layer of the pericardial sac) from eight fresh cadavers were sampled and analysed with histological and immunohistochemical stains to evaluate collagen and elastic components and innervation. Although the visceral fasciae make up a well‐defined layer of connective tissue, the thickness, percentage of elastic fibres and innervation vary among the different viscera. In particular, the fascia of the lung has a mean thickness of 134 μm (± 21), that of heart 792 μm (± 132), oesophagus 105 μm (± 10), liver 131 μm (± 18), Gerota fascia 1009 μm (± 105) and the visceral fascia of the abdomen 987 μm (± 90). The greatest number of elastic fibres (9.79%) was found in the adventitia of the oesophagus. The connective layers lying immediately outside the mesothelium of the pleura and peritoneum also have many elastic fibres (4.98% and 4.52%, respectively), whereas the pericardium and Gerota fascia have few (0.27% and 1.38%). In the pleura, peritoneum and adventitia of the oesophagus, elastic fibres form a well‐defined layer, corresponding to the elastic lamina, while in the other cases they are thinner and scattered in the connective tissue. Collagen fibres also show precise spatial organisation, being arranged in several layers. In each layer, all the fibrous bundles are parallel with each other, but change direction among layers. Loose connective tissue rich in elastic fibres is found between contiguous fibrous layers. Unmyelinated nerve fibres were found in all samples, but myelinated fibres were only found in some fasciae, such as those of the liver and heart, and the visceral fascia of the abdomen. According to these findings, we propose distinguishing the visceral fasciae into two large groups. The first group includes all the fasciae closely related to the individual organ and giving shape to it, supporting the parenchyma; these are thin, elastic and very well innervated. The second group comprises all the fibrous sheets forming the compartments for the organs and also connecting the internal organs to the musculoskeletal system. These fasciae are thick, less elastic and less innervated, but they contain larger and myelinated nerves. We propose to call the first type of fasciae ‘investing fasciae’, and the second type ‘insertional fasciae’.

Toxicity of gold nanoparticles towards human endothelial cells is observed under static and flow conditions by means of a microfluidic device which mimics the in vivo environmental conditions for this type of cells.

Conflicting results have been obtained by clinical studies investigating the effect of liver cirrhosis on enzyme induction. Because ethical concerns do not give consent for methodologically rigorous studies in humans, we addressed this question by examining the effect of the prototypical inducer dexamethasone (DEX) on the pregnane X receptor (PXR)-mediated induction of CYP3A1 and 3A2 in a validated animal model of liver cirrhosis obtained by exposure of rats to carbon tetrachloride. For this purpose, we assessed mRNA levels, protein expressions, and enzymatic activities of both CYP3A enzymes, as well as mRNA and protein expressions of PXR in rat populations rigorously stratified according to the severity of liver insufficiency. Constitutive mRNA and protein expressions of CYP3A1 and CYP3A2 and their basal enzyme activities were not affected by liver dysfunction. DEX treatment markedly increased steady-state mRNA level, protein content, and enzymatic activity of CYP3A1 in healthy and cirrhotic rats, irrespective of the degree of liver dysfunction. On the contrary, the inducing effect of DEX on gene and protein expressions and enzyme activity of CYP3A2 was preserved in moderate liver insufficiency, whereas it was greatly curtailed when liver insufficiency became severe. mRNA and protein expressions of PXR were neither reduced by liver dysfunction nor increased by DEX treatment. These results indicate that even the inducibility of cytochrome P450 isoforms under the transcriptional control of the same nuclear receptor may be differentially affected by cirrhosis and may partly explain why conflicting results were obtained by human studies.

Although it is now recognized that women suffer from myofascial pain to a greater extent than men, and that the muscular fasciae can respond to hormonal stimuli, thanks to the expression of sex hormone receptors, how the fasciae can modify their structure under hormonal stimulation is not clear. In this work, an immunocytochemical analysis of collagen-I, collagen-III and fibrillin were carried out on fibroblasts isolated from human fascia lata after in vitro treatment with various levels of sex hormones β-estradiol and/or relaxin-1, according to the phases of a woman’s period (follicular, periovulatory, luteal, post-menopausal phases and pregnancy). This study demonstrates for the first time that fascial cells can modulate the production of some components of the extracellular matrix according to hormone levels, when treated with β-estradiol: collagen-I falls from 6% of positivity in the follicular phase to 1.9 in the periovulatory phase. However, after the addition of relaxin-1 to the cell culture, the production of extracellular matrix decreased and remained at the same level (1.7% of collagen-I, at both follicular and periovulatory levels of hormones). These results confirm the antifibrotic function of relaxin-1, thanks to its ability to reduce matrix synthesis. They are also a first step in our understanding of how some hormonal dysfunctions in women can cause a dysregulation of extracellular matrix production in fasciae.

Cannabinoid receptors have been localized in the central and peripheral nervous system as well as on cells of the immune system, but recent studies on animal tissue gave evidence for the presence of cannabinoid receptors in different types of tissues. Their presence was supposed also in myofascial tissue, suggesting that the endocannabinoid system may help resolve myofascial trigger points and relieve symptoms of fibromyalgia. However, until now the expression of CB1 (cannabinoid receptor 1) and CB2 (cannabinoid receptor 2) in fasciae has not yet been established. Small samples of fascia were collected from volunteers patients during orthopedic surgery. For each sample were done a cell isolation, immunohistochemical investigation (CB1 and CB2 antibodies) and real time RT-PCR to detect the expression of CB1 and CB2. Both cannabinoid receptors are expressed in human fascia and in human fascial fibroblasts culture cells, although to a lesser extent than the control gene. We can assume that the expression of mRNA and protein of CB1 and CB2 receptors in fascial tissue are concentrated into the fibroblasts. This is the first demonstration that the fibroblasts of the muscular fasciae express CB1 and CB2. The presence of these receptors could help to provide a description of cannabinoid receptors distribution and to better explain the role of fasciae as pain generator and the efficacy of some fascial treatments. Indeed the endocannabinoid receptors of fascial fibroblasts can contribute to modulate the fascial fibrosis and inflammation.

Conformational protein diseases of the human central nervous system represent a subject that has crucial theoretical and medical implications. They include several important neurodegenerative diseases, such as Alzheimer's, Parkinson's, Huntington's and Creutzfeldt-Jacob's diseases, amyotrophic lateral sclerosis, and the tauopathies. They occur when soluble proteins undergo conformational rearrangements becoming capable of aggregate into β-sheets conformations leading to the production of insoluble complexes known as amyloid deposits, that accumulate and lead to neurons and glial cells death. Theoretical and experimental evidence indicates that a key role in the conformational changes leading to amyloid formation is played by short sequence stretches within a given protein. Thus, the identification of protein regions potentially involved in aggregate formation and the characterization of their properties are relevant questions in the study of conformational proteins diseases. To address these questions, bioinformatics methods might provide an important contribution, suggesting possible mechanisms of protein aggregation, and focusing and orienting the experimental work. Thus, in the first part of the present review bioinformatics methods specifically attempting to predict aggregation-prone regions in proteins will be briefly described. Furthermore, the results provided by the combined use of some of them to analyze a set of particularly important proteins involved in human degenerative diseases will be discussed.

The concept of intra-membrane receptor–receptor interactions (RRIs) between different types of G protein-coupled receptors (GPCRs) and evidence for their existence was introduced by Agnati and Fuxe in 1980/81 through the biochemical analysis of the effects of neuropeptides on the binding characteristics of monoamine receptors in membrane preparations from discrete brain regions and functional studies of the interactions between neuropeptides and monoamines in the control of specific functions such as motor control and arterial blood pressure control in animal models. Whether GPCRs can form high-order structures is still a topic of an intense debate. Increasing evidence, however, suggests that the hypothesis of the existence of high-order receptor oligomers is correct. A fundamental consequence of the view describing GPCRs as interacting structures, with the likely formation at the plasma membrane of receptor aggregates of multiple receptors (Receptor Mosaics) is that it is no longer possible to describe signal transduction simply as the result of the binding of the chemical signal to its receptor, but rather as the result of a filtering/integration of chemical signals by the Receptor Mosaics (RMs) and membrane-associated proteins. Thus, in parallel with experimental research, significant efforts were spent in bioinformatics and mathematical modelling. We review here the main approaches that have been used to assess the interaction interfaces allowing the assembly of GPCRs and to shed some light on the integrative functions emerging from the complex behaviour of these RMs. Particular attention was paid to the RMs generated by adenosine A2A, dopamine D2, cannabinoid CB1, and metabotropic glutamate mGlu5 receptors (A2A, D2, CB1 and mGlu5, respectively), and a possible approach to model the interplay between the D2–A2A–CB1 and D2–A2A–mGlu5 trimers is proposed. This article is part of a Special Issue entitled: “Adenosine Receptors”.

In vitro assays that stimulate the formation of capillary-like structures by EC have become increasingly popular, because they allow the study of the EC's intrinsic ability to self-organize to form vascular-like patterns. Here we describe a widely applied protocol involving the use of basement membrane matrix (Matrigel) as a suitable environment to induce an angiogenic phenotype in cultured EC. EC differentiation on basement membrane matrix is a highly specific process, which recapitulates many steps in blood vessel formation and for this reason it is presently considered as a reliable in vitro tool to identify factors with potential antiangiogenic or pro-angiogenic properties. The morphological features of the obtained cell patterns can also be accurately quantified by computer-assisted image analysis and the main steps of such a procedure will be here outlined and discussed. The dynamics of in vitro EC self-organization is a complex biological process, involving a network of interactions between a high number of cells. For this reason, the combined use of in vitro experiments and computational modeling can represent a key approach to unravel how mechanical and chemical signaling by EC coordinates their organization into capillary-like tubes. Thus, a particularly helpful approach to modeling is also briefly described together with examples of its application.

In vitro assays that stimulate the formation of capillary-like structures by EC have become increasingly popular, because they allow the study of the EC’s intrinsic ability to self-organize to form vascular-like patterns. Here we describe a widely applied protocol involving the use of basement membrane matrix (Matrigel) as a suitable environment to induce an angiogenic phenotype in cultured EC. EC differentiation on basement membrane matrix is a highly specific process, which recapitulates many steps in blood vessel formation and for this reason it is presently considered as a reliable in vitro tool to identify factors with potential antiangiogenic or pro-angiogenic properties. The morphological features of the obtained cell patterns can also be accurately quantified by computer-assisted image analysis and the main steps of such a procedure will be here outlined and discussed. The dynamics of in vitro EC self-organization is a complex biological process, involving a network of interactions between a high number of cells. For this reason, the combined use of in vitro experiments and computational modeling can represent a key approach to unravel how mechanical and chemical signaling by EC coordinates their organization into capillary-like tubes. Thus, a particularly helpful approach to modeling is also briefly described together with examples of its application.

Orexins A and B are hypothalamic peptides, that act via two subtypes of receptors, named OX1-R and OX2-R. Rat and human adrenal cortexes are provided with both OX1-R and OX2-R, and we have previously shown that orexin-A, but not orexin-B, enhances glucocorticoid secretion from dispersed adrenocortical cells. Since OX1-Rs preferentially bind orexin-A and OX2-Rs are non-selective for both orexins, the hypothesis has been advanced that the secretagogue effect of orexin-A is exclusively mediated by the OX1-R. Here, we aimed to verify this contention and to gain insight into the signaling mechanism(s) underlying the secretagogue effect of orexins using primary cultures of rat and human adrenocortical cells. Reverse transcription-polymerase chain reaction showed that cultured cells, as freshly dispersed cells, expressed both OX1-R and OX2-R mRNAs. Orexin-A, but not orexin-B, concentration-dependently increased corticosterone and cortisol secretion from cultured rat and human adrenocortical cells, respectively. The blockade of OX1-Rs by selective antibodies abrogated the secretagogue effect of orexin-A, while the immuno-blockade of OX2-Rs was ineffective. The glucocorticoid response of cultured cells to orexin-A was annulled by the adenylate cyclase and protein kinase (PK) A inhibitors SQ-22536 and H-89, and unaffected by the phospholipase C and PKC inhibitors U-73122 and calphostin-C. Orexin-A, but not orexin-B, enhanced cyclic-AMP production from cultured cells, and did not alter inositol-3-phosphate release. Collectively, our present results allow us to conclude that orexins stimulate glucocorticoid secretion from rat and human adrenocortical cells, exclusively acting through OX1-Rs coupled to the adenylate cyclase/PKA-dependent signaling cascade.

One of the major applications of real time polymerase chain reaction (PCR) is relative quantification, where the expression of a target gene is determined as a ratio to a stably expressed reference gene, the so-called housekeeping gene. Glyceraldehyde-3-phosphate dehydrogenase (GAPD) is a glycolytic enzyme, which is active in all mammalian tissues and is frequently used as housekeeping gene in expression studies. The functional locus maps to human chromosome 12p13, but several GAPD-related sequences, including processed pseudogenes, GenBank homologous sequences and computationally predicted sequences are present along the human genome. Due to the high level of GAPD-related sequences it is very important to avoid genomic DNA amplification when GAPD is used as endogenous control in mRNA quantification. We have outlined a GAPD couple of primers that avoid any genomic DNA amplification for real time reverse transcription PCR applications by SYBR-Green Dye. These new designed primers are an useful and chip alternative to probe technologies, and can carry out specific and reproducible data in mRNA expression studies.

A new in vitro model system, adding advection and shear stress associated with a flowing medium, is proposed for the investigation of nanoparticles uptake and toxicity towards endothelial cells, since these processes are normally present when nanoparticles formulations are intravenously administered. In this model system, mechanical forces normally present in vivo, such as advection and shear stress were applied and carefully controlled by growing human umbilical vein endothelial cells inside a microfluidic device and continuously infusing gold nanoparticle (Au NPs) solution in the device. The tests performed in the microfluidic device were also run in multiwells, where no flow is present, so as to compare the two model systems and evaluate if gold nanoparticles toxicity differs under static and flow culture conditions. Full characterization of Au NPs in water and in culture medium was accomplished by standard methods. Two-photon fluorescence correlation spectroscopy was also employed to map the flow speed of Au NPs in the microfluidic device and characterize Au NPs before and after interactions with the cells. Au NPs uptake in both in vitro systems was investigated through electron and fluorescence microscopy and ICP-AES, and NPs toxicity measured through standard bio-analytical tests. Comparison between experiments run in multiwells and in microfluidic device plays a pivotal role for the investigation of nanoparticle-cell interaction and toxicity assessment: our work showed that administration of equal concentrations of Au NPs under flow conditions resulted in a reduced sedimentation of nanoparticle aggregates onto the cells and lower cytotoxicity with respect to experiments run in ordinary static conditions (multiwells).

Apoptosis represents the key mechanism for the removal of surplus, damaged, or aged cells, and deregulated apoptosis has been implicated in the etiology of diverse pathologies. There are two main pathways which are known to initiate apoptosis: the death receptor-dependent (extrinsic) pathway and the mitochondrial-dependent (intrinsic) pathway. In the intrinsic pathway, as a response to diverse signals from the cellular environment, a permeabilization of the mitochondrial outer membrane occurs, followed by the release of cytochrome c and the activation of the effector caspases, which leads to cell death. Recently, increased attention has been paid to the possible role of the protein neuroglobin, in the regulation of the apoptotic process, and data have been provided, demonstrating the ability of the protein to inhibit the intrinsic pathway of apoptosis by interacting with mitochondrial proteins. The molecular details of these interactions, however, remain largely undefined. In the present study, well recognized bioinformatics methods were applied to predict the possible interaction interfaces which the protein can exploit to interact with relevant proteins of the mitochondrial-dependent pathway of apoptosis. In the search for therapeutic approaches based on the modulation of apoptosis, such a computational prediction could represent a first, guiding step, for the design of strategies aimed at modulating these interactions, and tune the apoptotic process.

Microfluidic, the technology that manipulates small amount of fluids in microscale complex devices, has undergone a remarkable development during the last decade, by targeting a significant range of applications, including biological tests and single-cell analysis, and by displaying many advantages such as reduced reagent consumption, decreased costs and faster analysis. Furthermore, the introduction of microfluidic tools has revolutionized the study of vascular functions, because the controlled three-dimensional environment and the continuous perfusion provided by the microdevice allow simulating the physiological characteristics of the circulatory system. Researchers interested in the study of vascular physiology, however, are often hampered by the difficulty in handling reduced number of cells after growth in these devices. This work shows how to apply different protocols commonly used in biology, such as the immunofluorescence technique, to cells grown in reversibly-bound microfluidic devices, obtaining results comparable to those retrieved under static conditions in multiwells. In this way, we are able to combine the advantages of microfluidic, i.e., application of continuous flow and shear stress, with classical protocols for the study of endothelial cells.

Glucagon-receptor mRNA was detected by reverse transcription-polymerase chain reaction in three human pheochromocytomas, but not in four normal adrenal medullas. Quantitative autoradiography demonstrated the presence of abundant [ 125 I-Thyr 10 ]glucagon binding sites in pheochromocytomas, which were displaced by both cold glucagon and the glucagon receptor antagonist Des-His 1 [Glu 9 ]glucagon amide (GR-A). Adrenal medulla was weakly labeled, and the binding was not displaced by GR-A. Glucagon enhanced epinephrine and norepinephrine release by pheochromocytoma slices, minimal and maximal effective concentrations being 10 −8 M and 10 −6 M. Adrenomedullary slices evidenced a weak catecholamine response only to 10 −5 M glucagon. GR-A abolished the secretory response to glucagon of pheochromocytomas, but not of adrenal medullas. Collectively, these findings indicate that human pheochromocytomas, but not adrenal medulla, express glucagon receptors and possess a marked secretory response to glucagon, thereby providing the rationale to explain the specificity of the glucagon provocative test in the diagnosis of pheochromocytoma.

The role played by endothelin (ET-1) and its receptor subtypes A and B (ETA and ETB) in the functional regulation of human NCI-H295 adrenocortical carcinoma cells has been investigated. Reverse transcription-PCR with primers specific for prepro-ET-1, human ET-1 converting enzyme-1, ETA, and ETB complementary DNAs consistently demonstrated the expression of all genes in NCI-H295 cells. The presence of mature ET-1 and both its receptor subtypes was confirmed by immunocytochemistry and autoradiography, respectively. Aldosterone synthase (AS) messenger RNA was also detected in NCI-H295 cells, and AS gene expression was enhanced by both ET-1 and the specific ETB agonist IRL-1620; this effect was not inhibited by either the ETA antagonist BQ-123 or the ETB antagonist BQ-788. A clear-cut increase in the intracellular Ca2+ concentration in NCI-H295 cells in response to ETB, but not ETA, activation was observed. In light of these findings, the following conclusions can be drawn: 1) NCI-H295 cells possess an active...