Homogeneity assessment of the SuperCam calibration targets onboard rover perseverance.

The SuperCam instrument, onboard the Perseverance rover (Mars 2020 mission) is designed to perform remote analysis on the Martian surface employing several spectroscopic techniques such as Laser Induced Breakdown Spectroscopy (LIBS), Time-Resolved Raman (TRR), Time-Resolved Fluorescence (TRF) and Visible and Infrared (VISIR) reflectance. In addition, SuperCam also acquires high-resolution images using a color remote micro-imager (RMI) as well as sounds with its microphone. SuperCam has three main subsystems, the Mast Unit (MU) where the laser for chemical analysis and collection optics are housed, the Body Unit (BU) where the different spectrometers are located inside the rover, and the SuperCam Calibration Target (SCCT) located on the rover's deck to facilitate calibration tests at similar ambient conditions as the analyzed samples. To perform adequate calibrations on Mars, the 22 mineral samples included in the complex SCCT assembly must have a very homogeneous distribution of major and minor elements. The analysis and verification of such homogeneity for the 5-6 replicates of the samples included in the SCCT has been the aim of this work. To verify the physic-chemical homogeneity of the calibration targets, micro Energy Dispersive X-ray Fluorescence (EDXRF) imaging was first used on the whole surface of the targets, then the relative abundances of the detected elements were computed on 20 randomly distributed areas of 100 × 100 µm. For those targets showing a positive Raman response, micro-Raman spectroscopy imaging was performed on the whole surface of the targets at a resolution of 100 × 100 µm. The %RSD values (percent of relative standard deviation of mean values) for the major elements measured with EDXRF were compared with similar values obtained by two independent LIBS set-ups at spot sizes of 300 µm in diameter. The statistical analysis showed which elements were homogeneously distributed in the 22 mineral targets of the SCCT, providing their uncertainty values for further calibration. Moreover, nine of the 22 targets showed a good Raman response and their mineral distributions were also studied. Those targets can be also used for calibration purposes of the Raman part of SuperCam using the wavenumbers of their main Raman bands proposed in this work.

SuperCam Calibration Targets: Design and Development.

SuperCam is a highly integrated remote-sensing instrumental suite for NASA's Mars 2020 mission. It consists of a co-aligned combination of Laser-Induced Breakdown Spectroscopy (LIBS), Time-Resolved Raman and Luminescence (TRR/L), Visible and Infrared Spectroscopy (VISIR), together with sound recording (MIC) and high-magnification imaging techniques (RMI). They provide information on the mineralogy, geochemistry and mineral context around the Perseverance Rover. The calibration of this complex suite is a major challenge. Not only does each technique require its own standards or references, their combination also introduces new requirements to obtain optimal scientific output. Elemental composition, molecular vibrational features, fluorescence, morphology and texture provide a full picture of the sample with spectral information that needs to be co-aligned, correlated, and individually calibrated. The resulting hardware includes different kinds of targets, each one covering different needs of the instrument. Standards for imaging calibration, geological samples for mineral identification and chemometric calculations or spectral references to calibrate and evaluate the health of the instrument, are all included in the SuperCam Calibration Target (SCCT). The system also includes a specifically designed assembly in which the samples are mounted. This hardware allows the targets to survive the harsh environmental conditions of the launch, cruise, landing and operation on Mars during the whole mission. Here we summarize the design, development, integration, verification and functional testing of the SCCT. This work includes some key results obtained to verify the scientific outcome of the SuperCam system.

Pre-launch radiometric calibration of the infrared spectrometer onboard SuperCam for the Mars2020 rover.

Near-infrared spectroscopy has become a well-known remote sensing technique for the surface characterization of planetary objects. Among them, Mars was observed in the past by three imaging spectrometers from orbit. The Infrared Spectrometer/SuperCam instrument performs near-infrared spectroscopy from the martian surface for the first time, with a 1.15 mrad field of view, in the 1.3 µm-2.6 µm range, enabling the identification of a variety of mafic and altered minerals. Before integration aboard the rover, the spectrometer underwent a calibration campaign. Here, we report the radiometric and linearity responses of the instrument, including the optical and thermal setups used to perform them over its nominal range of operations, in terms of instrument detector temperatures and spectral range. These responses were constrained by accuracy requirements (20% in absolute radiometry, 1% in relative). The derived instrument transfer function fits within these requirements (<15% in absolute and <0.8% in relative) and shall be used to calculate the expected instrumental signal-to-noise ratio for typical observation scenarios of mineral mixtures expected to be found in the Jezero crater, and ultimately to retrieve the spectral properties of the regions of interest observed by the rover.

CROCC-mutated rhabdoid colorectal carcinoma showing in intercellular spaces lamellipodia and cellular projections revealed by electron microscopy.

BACKGROUND: Rhabdoid colorectal carcinoma (RC) is a rare lesion localized to the proximal colon of patients with a mean age at diagnosis of around 70 years. This tumor shows an aggressive behavior with an overall survival period shorter than 12 months. The diagnostic hallmark is the presence of rhabdoid cells. Alterations in chromatin remodeling (SMARCB1) and in the centrosome structure (CROCC) are reported in RC usually BRAFmut and MSI-H. RKO intestinal neoplastic cells culture (BRAFmut, SMARCB1wt, MSI-H) with CROCC knockdown exhibit rhabdoid features and develop prominent projections from the edge of the cell. METHODS: Here, we investigated two cases of CROCCmutSMARCB1wt RC by scanning and transmission electron microscopy (SEM, TEM). RESULTS: TEM confirmed the diagnostic presence of intermediate cytoplasmic filaments and nucleolar margination. SEM showed cellular protrusions (lamellipodia) in the intercellular spaces not evident at light microscopy. CONCLUSIONS: These protrusions CROCC-related might represent the pathogenetic mechanism underlying the rhabdoid aggressive behavior, independently of tumor staging. To our knowledge, the SEM technique was applied in the study of this neoplasm for the first time.

ALDH2 Activity Reduces Mitochondrial Oxygen Reserve Capacity in Endothelial Cells and Induces Senescence Properties.

Endothelial cells (ECs) are dynamic cells that turn from growth into senescence, the latter being associated with cellular dysfunction, altered metabolism, and age-related cardiovascular diseases. Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme metabolizing acetaldehyde and other toxic aldehydes, such as 4-hydroxynonenal (4-HNE). In conditions in which lipid peroxidation products and reactive oxygen species (ROS) are accumulated, ECs become dysfunctional and significantly contribute to the progression of vascular-dependent diseases. The aim of the present study has been to investigate whether inhibition of ALDH2 alters endothelial functions together with the impairment of bioenergetic functions, accelerating the acquisition of a senescent phenotype. HUVECs transfected with siRNA targeting ALDH2 or treated with daidzin, an ALDH2 inhibitor, were used in this study. We observed an alteration in cell morphology associated with endothelial dysfunctions. Loss of ALDH2 reduced cell proliferation and migration and increased paracellular permeability. To assess bioenergetic function in intact ECs, extracellular flux analysis was carried out to establish oxygen consumption rates (OCR). We observed a decrease in mitochondrial respiration and reserve capacity that coincided with SA-ß-Gal accumulation and an increase in p21 and p53 expression in siALDH2 or daidzin-treated HUVECs. Treatment with N-acetyl-L-cysteine (NAC) reduced endothelial dysfunctions mediated by siALDH2, indicating that oxidative stress downstream to siALDH2 plays an instrumental role. Our results highlight that ALDH2 impairment accelerates the acquisition of a premature senescent phenotype, a change likely to be associated with the observed reduction of mitochondrial respiration and reserve capacity.

Filamentous fungi associated with natural infection of noble rot on withered grapes.

The effects of noble rot infection of grapes on the characteristics of different types of wine, including Italian passito wine, are well known. Nevertheless, there is still little information on filamentous fungi associated with noble-rotten grapes. In this study, withered Garganega grapes for passito wine production, naturally infected by noble rot, were analyzed and compared to sound grapes. Skin morphology and fungal population on berry surfaces were analyzed. Scanning electron microscopy analysis revealed microcracks, germination conidia and branched hyphae on noble-rotten berries. Penicillium, Aureobasidium and Cladosporium were the most frequent genera present. Analysis of single berries displayed higher heterogeneity of epiphytic fungi in those infected by noble-rot than in sound berries. Penicillium adametzoides, Cladosporium cladospoirioides and Coniochaeta polymorpha were recovered. These, to the best of our knowledge, had never been previously isolated from withered grapes and, for C. polymorpha, from grapevine. This study provided novel data on noble rot mycobiota and suggests that fungi that co-habit with B. cinerea could have an important role on grape and wine quality.

Diagnosis of electrocution: The application of scanning electron microscope and energy-dispersive X-ray spectroscopy in five cases.

INTRODUCTION: Deaths from electricity, generally, do not have specific findings at the autopsy. The diagnosis is commonly based on the circumstances of the death and the morphologic findings, above all the current mark. Yet, the skin injury due to an electrocution and other kinds of thermal injuries often cannot be differentiated with certainty. Therefore, there is a great interest in finding specific markers of electrocution. The search for the metallization of the skin through Scanning Electron Microscope equipped with Energy Dispersive X-Ray Spectroscopy (EDS) probe is of special importance in order to achieve a definite diagnosis in case of suspected electrocution. MATERIALS AND METHODS: We selected five cases in which the electrocution was extremely likely considering the circumstances of the death. In each case a forensic autopsy was performed. Then, the skin specimens were stained with Hematoxylin Eosin and Perls. On the other hand, the skin lesions were examined with a scanning electron microscope equipped with EDS probe in order to evaluate the morphological ultrastructural features and the presence of deposits on the surface of the skin. RESULTS: The typical skin injury of the electrocution (current mark) were macroscopically detected in all of the cases. The microscopic examination of the skin lesions revealed the typical spherical vacuoles in the horny layer and, in the epidermis, the elongation of the cell nuclei as well as necrosis. Perls staining was negative in 4 out 6 cases. Ultrastructural morphology revealed the evident vacuolization of the horny layer, elongation of epidermic cells, coagulation of the elastic fibers. EDS-MICROANALYSIS: In the specimens collected from the site of contact with the conductor of case 1 and 2, the presence of the Kα peaks of iron was detected. In the corresponding specimens taken from cases 2, 4, 5 the microanalysis showed the Kα peaks of titanium. In case 3, titanium and carbon were found. CONCLUSIONS: In the suspicion of electrocution, the integrated use of different tools is recommended, including macroscopic observation, H&E staining, iron-specific staining, scanning electron microscopy and EDS microanalysis. Only the careful interpretation of the results provided by all these methods can allow the pathologist to correctly identify the cause of the death. Particularly, the present study suggests that the microanalysis (SEM-EDS) represents a very useful tool for the diagnosis of electrocution, allowing the detection and the identification of the metals embedded in the skin and their evaluation in the context of the ultrastructural morphology.

Application of a LC-MS/MS method for evaluating lung penetration of tobramycin in rats by microdialysis.

A bioanalytical LC-MS/MS method was developed and validated to determine tobramycin in plasma and lung microdialysate samples. Tobramycin was separated using a C18 column and a mobile phase consisting of water and acetonitrile, both with 10mM of heptafluorobutyric acid, eluted as gradient. Apramycin was used as internal standard (IS) for plasma samples. Drugs were monitored using electrospray ionization operating on positive mode (ESI+) monitoring the transitions 468.2>163.3 m/z for tobramycin and 540.3>217.2 m/z for IS. The method showed linearity in the concentration range of 0.1-50µgmL-1 for microdialysate and 0.5-100µgmL-1 for plasma with coefficients of determination ≥0.991. The inter- and intra-day precision, the accuracy and the stability of the drug in different conditions were in accordance with the limits established by US Food and Drug Administration guideline. The concentrations of tobramycin in plasma and lung microdialysate, determined using CMA/20 probes and a Ringer solution at a flow rate of 1µLmin-1, were evaluated in healthy Wistar rats after a 10mgkg-1 i.v. bolus dose. Samples were harvested up to 12h post-dose. Before animal's experiments, probe recovery was determined by dialysis and retrodialysis in vitro and by retrodialysis in vivo. Probes recovery was independent of drug concentration and method of determination. In vivo recovery was 27.74±7.70%. Pharmacokinetic parameters were estimated by non-compartmental analysis using the software Phoenix®. The estimated area under the curve (AUC0-∞) was 128±19µghmL-1 and 105±12µghmL-1 for plasma and lung, respectively. Tobramycin plasma clearance was 0.07±0.01L/h/kg and the volume of distribution was 0.49±0.09L/kg. Elimination half-life in plasma was 4.4±0.7h and in lung, 4.2±0.56h. The free tissue/free plasma AUC0-∞ ratio was 0.94. This is the first study showing a validated method to quantify tobramycin in microdialysate samples and to evaluate the lung interstitial concentration of the drug.

Reduced mitochondrial Ca(2+) transients stimulate autophagy in human fibroblasts carrying the 13514A>G mutation of the ND5 subunit of NADH dehydrogenase.

Mitochondrial disorders are a group of pathologies characterized by impairment of mitochondrial function mainly due to defects of the respiratory chain and consequent organellar energetics. This affects organs and tissues that require an efficient energy supply, such as brain and skeletal muscle. They are caused by mutations in both nuclear- and mitochondrial DNA (mtDNA)-encoded genes and their clinical manifestations show a great heterogeneity in terms of age of onset and severity, suggesting that patient-specific features are key determinants of the pathogenic process. In order to correlate the genetic defect to the clinical phenotype, we used a cell culture model consisting of fibroblasts derived from patients with different mutations in the mtDNA-encoded ND5 complex I subunit and with different severities of the illness. Interestingly, we found that cells from patients with the 13514A>G mutation, who manifested a relatively late onset and slower progression of the disease, display an increased autophagic flux when compared with fibroblasts from other patients or healthy donors. We characterized their mitochondrial phenotype by investigating organelle turnover, morphology, membrane potential and Ca(2+) homeostasis, demonstrating that mitochondrial quality control through mitophagy is upregulated in 13514A>G cells. This is due to a specific downregulation of mitochondrial Ca(2+) uptake that causes the stimulation of the autophagic machinery through the AMPK signaling axis. Genetic and pharmacological manipulation of mitochondrial Ca(2+) homeostasis can revert this phenotype, but concurrently decreases cell viability. This indicates that the higher mitochondrial turnover in complex I deficient cells with this specific mutation is a pro-survival compensatory mechanism that could contribute to the mild clinical phenotype of this patient.

Chromium-induced diffuse dermatitis with lymph node involvement resulting from Langerhans cell histiocytosis after metal-on-metal hip resurfacing.

Total hip arthroplasty (THA) is a highly effective surgical treatment for severe joint involvement. However, due to the release of metal ions in the blood, the patients who undergo hip replacement with metal-on-metal (MOM) bearings may develop signs of allergic skin disease. We report a case of a 60-year-old man who had received MOM hip resurfacing 5 years earlier for osteoarthritis. He presented with a 3-year history of diffuse dermatitis that did not respond to antihistamines and corticosteroids and also had elevated serum levels of chromium and cobalt. A patch test revealed chromium-sulfate hypersensitivity. A skin biopsy showed nonspecific perivascular lymphocytic infiltrate associated with histiocytes. A biopsy of an inguinal lymph node demonstrated large aggregates of Langerhans cells, suggesting type IV delayed-type hypersensitivity. The prosthesis was replaced using ceramic-on-ceramic bearings and the dermatitis resolved after 3 months. The lymph nodes decreased in volume and the serum chromium levels normalized within 24 months of revision surgery. The high levels of serum ions associated with the metal debris from MOM-THAs may induce sensitization and type IV hypersensitivity reactions. Replacing the prosthesis using alternative coupling surfaces is the only approach that has the capacity to resolve these symptoms. Physicians who are not familiar with this issue may misdiagnose systemic symptoms and provide inadequate treatment.

The post-adipocytic phase of the adipose cell cycle.

Subcutaneous white adipose tissue harvested by liposuction has been studied with the aim to understand how the adipocytes modify their morphology when subjected to the passage in a needle for liposuction and to cryopreservation. The work try to clarify the ultrastructural aspects of adipose tissue, in the conditions described before, examining samples of body fat employed in fat graft procedures, and samples after cryopreservation. Scanning and transmission electron microscopy show that the first event that occur in the adipocytes is a lesion of mild degree detectable early in the samples fixed immediately after liposuction. The sequence of events following the adipocyte stress appeared composed by different phases: plasmatic membrane interruption, loss of lipid charge, formation of cup-like adipocytes and formation of post-adipocytes (i.e. cells that survive to traumatic events and restart to internalize lipid droplets). In conclusion, the study suggests that the loss of lipid charge in adipose cell is an active process that can be due to a small hole in the cytoplasmic membrane with the preservation of a large part of the cytoplasmatic content and that at the end of the process of lipid extrusion the cell can maintain viability.

The biocompatibility of porous vs non-porous bone cements: a new methodological approach.

Composite cements have been shown to be biocompatible, bioactive, with good mechanical properties and capability to bind to the bone. Despite these interesting characteristic, in vivo studies on animal models are still incomplete and ultrastructural data are lacking. The acquisition of new ultrastructural data is hampered by uncertainties in the methods of preparation of histological samples due to the use of resins that melt methacrylate present in bone cement composition. A new porous acrylic cement composed of polymethylmetacrylate (PMMA) and ß-tricalciumphosphate (ß-TCP) was developed and tested on an animal model. The cement was implanted in femurs of 8 New Zealand White rabbits, which were observed for 8 weeks before their sacrifice. Histological samples were prepared with an infiltration process of LR white resin and then the specimens were studied by X-rays, histology and scanning electron microscopy (SEM). As a control, an acrylic standard cement, commonly used in clinical procedures, was chosen. Radiographic ultrastructural and histological exams have allowed finding an excellent biocompatibility of the new porous cement. The high degree of osteointegration was demonstrated by growth of neo-created bone tissue inside the cement sample. Local or systemic toxicity signs were not detected. The present work shows that the proposed procedure for the evaluation of biocompatibility, based on the use of LR white resin allows to make a thorough and objective assessment of the biocompatibility of porous and non-porous bone cements.

Monoamine oxidase inhibition prevents mitochondrial dysfunction and apoptosis in myoblasts from patients with collagen VI myopathies.

Although mitochondrial dysfunction and oxidative stress have been proposed to play a crucial role in several types of muscular dystrophy (MD), whether a causal link between these two alterations exists remains an open question. We have documented that mitochondrial dysfunction through opening of the permeability transition pore plays a key role in myoblasts from patients as well as in mouse models of MD, and that oxidative stress caused by monoamine oxidases (MAO) is involved in myofiber damage. In the present study we have tested whether MAO-dependent oxidative stress is a causal determinant of mitochondrial dysfunction and apoptosis in myoblasts from patients affected by collagen VI myopathies. We find that upon incubation with hydrogen peroxide or the MAO substrate tyramine myoblasts from patients upregulate MAO-B expression and display a significant rise in reactive oxygen species (ROS) levels, with concomitant mitochondrial depolarization. MAO inhibition by pargyline significantly reduced both ROS accumulation and mitochondrial dysfunction, and normalized the increased incidence of apoptosis in myoblasts from patients. Thus, MAO-dependent oxidative stress is causally related to mitochondrial dysfunction and cell death in myoblasts from patients affected by collagen VI myopathies, and inhibition of MAO should be explored as a potential treatment for these diseases.

Myotonic dystrophy protein kinase (DMPK) prevents ROS-induced cell death by assembling a hexokinase II-Src complex on the mitochondrial surface.

The biological functions of myotonic dystrophy protein kinase (DMPK), a serine/threonine kinase whose gene mutations cause myotonic dystrophy type 1 (DM1), remain poorly understood. Several DMPK isoforms exist, and the long ones (DMPK-A/B/C/D) are associated with the mitochondria, where they exert unknown activities. We have studied the isoform A of DMPK, which we have found to be prevalently associated to the outer mitochondrial membrane. The kinase activity of mitochondrial DMPK protects cells from oxidative stress and from the ensuing opening of the mitochondrial permeability transition pore (PTP), which would otherwise irreversibly commit cells to death. We observe that DMPK (i) increases the mitochondrial localization of hexokinase II (HK II), (ii) forms a multimeric complex with HK II and with the active form of the tyrosine kinase Src, binding its SH3 domain and (iii) it is tyrosine-phosphorylated by Src. Both interaction among these proteins and tyrosine phosphorylation of DMPK are increased under oxidative stress, and Src inhibition selectively enhances death in DMPK-expressing cells after HK II detachment from the mitochondria. Down-modulation of DMPK abolishes the appearance of muscle markers in in vitro myogenesis, which is rescued by oxidant scavenging. Our data indicate that, together with HK II and Src, mitochondrial DMPK is part of a multimolecular complex endowed with antioxidant and pro-survival properties that could be relevant during the function and differentiation of muscle fibers.

Chemotherapeutic induction of mitochondrial oxidative stress activates GSK-3α/ß and Bax, leading to permeability transition pore opening and tumor cell death.

Survival of tumor cells is favored by mitochondrial changes that make death induction more difficult in a variety of stress conditions, such as exposure to chemotherapeutics. These changes are not fully characterized in tumor mitochondria, and include unbalance of the redox equilibrium, inhibition of permeability transition pore (PTP) opening through kinase signaling pathways and modulation of members of the Bcl-2 protein family. Here we show that a novel chemotherapeutic, the Gold(III)-dithiocarbamato complex AUL12, induces oxidative stress and tumor cell death both favoring PTP opening and activating the pro-apoptotic protein Bax of the Bcl-2 family. AUL12 inhibits the respiratory complex I and causes a rapid burst of mitochondrial superoxide levels, leading to activation of the mitochondrial fraction of GSK-3α/ß and to the ensuing phosphorylation of the mitochondrial chaperone cyclophilin D, which in turn facilitates PTP opening. In addition, following AUL12 treatment, Bax interacts with active GSK-3α/ß and translocates onto mitochondria, where it contributes to PTP induction and tumor cell death. These findings provide evidence that targeting the redox equilibrium maintained by mitochondria in tumor cells allows to hit crucial mechanisms that shield neoplasms from the toxicity of many anti-tumor strategies, and identify AUL12 as a promising chemotherapeutic compound.

A large outbreak of Opisthorchis felineus in Italy suggests that opisthorchiasis develops as a febrile eosinophilic syndrome with cholestasis rather than a hepatitis-like syndrome.

We describe the greatest Italian human acute opisthorchiasis outbreak acquired from eating raw tenches. Out of 52 people with suspected opisthorchiasis, 45 resulted in being infected. The most frequent symptoms and laboratory findings were fever, abdominal pain and eosinophilia. Seven tri-phasic computed tomography (CT) scans were done, showing multiple hypodense nodules with hyper-enhancement in the arterial phase. All patients took one day of praziquantel 25 mg/kg TID without failures. Reported symptoms suggested a febrile eosinophilic syndrome with cholestasis rather than a hepatitis-like syndrome. It seems common to find hepatic imaging alterations during acute opisthorchiasis: CT scan could be the most suitable imaging examination. Even if stool test remains the diagnostic gold standard, we found earlier positivity with the serum antibody test. Without previous freezing, the consumption of raw freshwater fish should be avoided.

Critical evaluation of the use of cell cultures for inclusion in clinical trials of patients affected by collagen VI myopathies.

Collagen VI myopathies (Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM), and myosclerosis myopathy) share a common pathogenesis, that is, mitochondrial dysfunction due to deregulation of the permeability transition pore (PTP). This effect was first identified in the Col6a1(-/-) mouse model and then in muscle cell cultures from UCMD and BM patients; the normalizing effect of cyclosporin A (CsA) confirmed the pathogenic role of PTP opening. In order to determine whether mitochondrial performance can be used as a criterion for inclusion in clinical trials and as an outcome measure of the patient response to therapy, it is mandatory to establish whether mitochondrial dysfunction is conserved in primary cell cultures from UCMD and BM patients. In this study we report evidence that mitochondrial dysfunction and the consequent increase of apoptotic rate can be detected not only, as previously reported, in muscle, but also in fibroblast cell cultures established from muscle biopsies of collagen VI-related myopathic patients. However, the mitochondrial phenotype is no longer maintained after nine passages in culture. These data demonstrate that the dire consequences of mitochondrial dysfunction are not limited to myogenic cells, and that this parameter can be used as a suitable diagnostic criterion, provided that the cell culture conditions are carefully established.

The trochanteric fat pad.

Technological developments based on the use of autologous white adipose tissue (WAT) attracted attention to minor fat depots as possible sources of adipose tissue. In plastic surgery, the trochanteric fatty pad is one of the most used WAT depots for its location and organoleptic characteristics that make it particularly suitable for reconstructive procedures. Despite its wide use in clinic, the structure of this depot has never been studied in detail and it is not known if structural differences exist among trochanteric fat and other subcutaneous WAT depots. The present study was performed on trochanteric fat pad with the aim to clarify the morphology of its adipocytes, stroma and microcirculation, with particular reference to the stem niches. Histological and ultrastructural studies showed that the main peculiar feature of the trochanteric fat concerns its stromal component, which appears less dense than in the other subcutaneous WATs studied. The intra-parenchymal collagen stroma is poor and the extracellular compartment shows large spaces, filled with electron-light material, in which isolated collagen bundles are present. The adipocytes are wrapped in weak and easily detachable collagen baskets. These connective sheaths are very thin compared to the sheaths in other subcutaneous WAT depots. The capillaries are covered by large, long and thin elements surrounded by an external lamina; these perivascular cells are poor in organelles and mainly contain poly-ribosomes. In conclusion, when compared to other WAT deposits, the trochanteric fatty pad shows structural peculiarities in its stroma and microcirculation suggesting a high regenerative potential. Resistance, dissociability, microvascular weft and high regenerative potential make the trochanteric fatty pad a privileged source for harvesting in autologous WAT-based regenerative procedures.

The cyclophilin inhibitor Debio 025 normalizes mitochondrial function, muscle apoptosis and ultrastructural defects in Col6a1-/- myopathic mice.

BACKGROUND AND PURPOSE: We have investigated the therapeutic effects of the selective cyclophilin inhibitor D-MeAla(3)-EtVal(4)-cyclosporin (Debio 025) in myopathic Col6a1(-/-) mice, a model of muscular dystrophies due to defects of collagen VI. EXPERIMENTAL APPROACH: We studied calcineurin activity based on NFAT translocation; T cell activation based on expression of CD69 and CD25; propensity to open the permeability transition pore in mitochondria and skeletal muscle fibres based on the ability to retain Ca(2+) and on membrane potential, respectively; muscle ultrastructure by electronmicroscopy; and apoptotic rates by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assays in Col6a1(-/-) mice before after treatment with Debio 025. KEY RESULTS: Debio 025 did not inhibit calcineurin activity, yet it desensitizes the mitochondrial permeability transition pore in vivo. Treatment with Debio 025 prevented the mitochondrial dysfunction and normalized the apoptotic rates and ultrastructural lesions of myopathic Col6a1(-/-) mice. CONCLUSIONS AND IMPLICATIONS: Desensitization of the mitochondrial permeability transition pore can be achieved by selective inhibition of matrix cyclophilin D without inhibition of calcineurin, resulting in an effective therapy of Col6a1(-/-) myopathic mice. These findings provide an important proof of principle that collagen VI muscular dystrophies can be treated with Debio 025. They represent an essential step towards an effective therapy for Ullrich Congenital Muscular Dystrophy and Bethlem Myopathy, because Debio 025 does not expose patients to the potentially harmful effects of immunosuppression.