Extracellular Collagen VI Has Prosurvival and Autophagy Instructive Properties in Mouse Fibroblasts

Collagen VI (ColVI) is an abundant and distinctive extracellular matrix protein secreted by fibroblasts in different tissues. Human diseases linked to mutations on ColVI genes are primarily affecting skeletal muscle due to non-cell autonomous myofiber defects. To date, it is not known whether and how fibroblast homeostasis is affected by ColVI deficiency, a critical missing information as this may strengthen the use of patients’ fibroblasts for preclinical purposes. Here, we established primary and immortalized fibroblast cultures from ColVI null (Col6a1-/-) mice, the animal model of ColVI-related diseases. We found that, under nutrient-stringent condition, lack of ColVI affects fibroblast survival, leading to increased apoptosis. Moreover, Col6a1-/- fibroblasts display defects in the autophagy/lysosome machinery, with impaired clearance of autophagosomes and failure of Parkin-dependent mitophagy. Col6a1-/- fibroblasts also show an increased activation of the Akt/mTOR pathway, compatible with the autophagy impairment, and adhesion onto purified ColVI elicits a major effect on the autophagic flux. Our findings reveal that ColVI ablation in fibroblasts impacts on autophagy regulation and cell survival, thus pointing at the new concept that this cell type may contribute to the pathological features of ColVI-related diseases

Collagen VI–NG2 axis in human tendon fibroblasts under conditions mimicking injury response

In response to injury, tendon fibroblasts are activated, migrate to the wound, and contribute to tissue repair by producing and organizing the extracellular matrix. Collagen VI is a microfibrillar collagen enriched in the pericellular matrix of tendon fibroblasts with a potential regulatory role in tendon repair mechanism. We investigated the molecular basis of the interaction between collagen VI and the cell membrane both in tissue sections and fibroblast cultures of human tendon, and analyzed the deposition of collagen VI during migration and myofibroblast trans-differentiation, two crucial events for tendon repair. Tendon fibroblast displayed a collagen VI microfibrillar network closely associated with the cell surface. Binding of collagen VI with the cell membrane was mediated by NG2 proteoglycan, as demonstrated by in vitro perturbation of collagen VI–NG2 interaction with a NG2-blocking antibody. Cultures subjected to wound healing scratch assay displayed collagen VI–NG2 complexes at the trailing edge of migrating cells, suggesting a potential role in cell migration. In fact, the addition of a NG2-blocking antibody led to an impairment of cell polarization and delay of wound closure. Similar results were obtained after in vitro perturbation of collagen VI extracellular assembly with the 3C4 anti-collagen VI antibody and in collagen VI-deficient tendon cultures of a Ullrich congenital muscular dystrophy patient carrying mutations in COL6A2 gene. Moreover, in vitro treatment with transforming growth factor β1 (TGFβ1) induced a dramatic reduction of NG2 expression, both at protein and mRNA transcript level, and the impairment of collagen VI association with the cell membrane. Instead, collagen VI was still detectable in the extracellular matrix in association with ED-A fibronectin and collagen I, which were strongly induced by TGFβ1 treatment. Our findings reveal a critical role of the NG2 proteoglycan for the binding of collagen VI to the surface of tendon fibroblasts. By interacting with NG2 proteoglycan and other extracellular matrix proteins, collagen VI regulates fibroblasts behavior and the assembly of tendon matrix, thereby playing a crucial role in tendon repair

A major glacial-interglacial change in aeolian dust composition inferred from Rare Earth Elements in Antarctic ice

We present the first Rare Earth Elements (REE) concentration record determined in 294 sections of an Antarctic ice core (EPICA Dome C), covering a period from 2.9 to 33.7 kyr BP. REE allow a detailed quantitative evaluation of aeolian dust composition because of the large number of variables (i.e. 14 elements). REE concentrations match the particulate dust concentration profile over this period and show a homogeneous crustal-like composition during the last glacial stage (LGS), with only a slight enrichment in medium REE. This signature is consistent with the persistent fallout of a mixture of dust from heterogeneous sources located in different areas or within the same region (e.g. South America). Starting at ∼15 kyr BP, there was a major change in dust composition, the variable character of which persisted throughout the Holocene. This varying signature may highlight the alternation of single dust contributions from different sources during the Holocene. We observe that the frequent changes in REE composition at the onset of the Holocene (10-13.5 kyr BP) are linked to dust size and in turn to wind strength and/or the path of the atmospheric trajectory. This may indicate that atmospheric circulation dictated the composition of the dust fallout to East Antarctica at that time. Although the dust concentrations remained fairly low, a notable return towards more glacial dust characteristics is recorded between 7.5 and 8.3 kyr BP. This happened concomitantly with a widespread cold event around 8 kyr BP that was 400-600 years long and suggests a moderate reactivation of the dust emission from the same potential source areas of the LGS

Organic micropollutants in wet and dry depositions in the Venice Lagoon

Atmospheric transport is an important route by which pollutants are conveyed from the continents to both coastal and open sea. The role of aerosol deposition in the transport of polycyclic aromatic hydrocarbons (PAHs), polychlorobiphenyls (PCBs) and polybromodiphenyls ethers (PBDEs) to water and soil systems has been evaluated by measuring their concentrations in wet and dry depositions to the Venice Lagoon. The organic micropollutant flux data indicate that they contribute to the total deposition flux in different ways through wet and dry deposition, showing that the prevalent contribution derives from wet deposition. The fluxes calculated for PBDEs, showed the prevalence of 47, 99, 100 and 183 congeners, both in dry and wet fluxes. With regard to PCBs, the flux of PPCB for wet deposition is in the same order of magnitude of the diffusive flux at the air–water interface. The PAH fluxes obtained in the present study are similar to those obtained in previous studies on the atmospheric bulk deposition to the Venice Lagoon. The ratios between Phe/Ant and Fl/Py indicate that the pollutants sources are pyrolytic, deriving from combustion fuels

Prognostic Factors for Tumor Recurrence after a 12-Year, Single-Center Experience of Liver Transplantations in Patients with Hepatocellular Carcinoma

Background. Factors affecting outcomes after orthotopic liver transplantation (OLT) for hepatocellular carcinoma (HCC) have been extensively studied, but some of them have only recently been discovered or reassessed. Methods. We analyzed classical and more recently emerging variables with a hypothetical impact on recurrence-free survival (RFS) in a single-center series of 283 patients transplanted for HCC between 1997 and 2009. Results. Five-year patient survival and RFS were 75% and 86%, respectively. Thirty-four (12%) patients had HCC recurrence. Elevated preoperative alpha-fetoprotein (AFP) levels, preoperative treatments of HCC, unfulfilled Milan and up-to-seven criteria at final histology, poor tumor differentiation, and tumor microvascular invasion negatively affected RFS by univariate analysis. Milan and up-to-seven criteria applied preoperatively, and the use of m-TOR inhibitors did not reach statistical significance. Cox's proportional hazard model showed that only elevated AFP levels (Odds Ratio = 2.88; 95% C.I. = 1.43–5.80; P = .003), preoperative tumor treatments (Odds Ratio = 4.84; 95% C.I. = 1.42–16.42; P = .01), and microvascular invasion (Odds Ratio = 4.82; 95% C.I. = 1.87–12.41; P = .001) were predictors of lower RFS. Conclusions. Biological aggressiveness and preoperative tumor treatment, rather than traditional and expanded dimensional criteria, conditioned the outcomes in patients transplanted for HCC

Greenland Snow Evidence of Large Scale Atmospheric Contamination for Platinum, Palladium, and Rhodium

Since 1976 in the United States, Canada, and Japan, and later in other countries, the exhaust system of gasoline powered cars has been equipped with catalytic converters containing Pt and/or Pd and/or Rh. This has resulted in a very significant decrease in urban air pollution for various chemical species such as NOx, CO, and hydrocarbons. There has however been concern that their ever increasing use might lead to Platinum Group Metals (PGMs) becoming widely dispersed in the environment. From the analysis of Pt, Pd, and Rh in central Greenland recent snow and ancient ice using the ultrasensitive inductively coupled plasma sector field mass spectrometry technique, we show here that the concentrations of these metals in snow dated from the mid 1990s are indeed 40−120 times higher than in ice dated from 7000 years ago. The fact that such an increase is observed far away from populated areas at a high altitude location indicates there is now a large scale contamination of the troposphere of the Northern Hemisphere for PGMs. Pt/Rh mass ratio in the most recent snow samples is close to the same ratio documented for catalytic converter exhausts in a recent study, which suggests that a large fraction of the recent increase for Pt and Rh might originate from automobile catalytic converters

The study of the mercury cycle in polar regions: An international study in Ny-Alesund, Svalbard

Mercury (Hg) is a toxic pollutant and it can be strongly accumulated in the food chain, especially in Polar Regions. This paper presents a part of the work that has been on-going for 3-4 years in Ny-Alesund, Svalbard within the frame of an international collaboration. In Ny-Alesund in spring 2003, the atmospheric chemistry of mercury has been studied so as to better understand the formation of oxidized mercury species in the atmosphere that could be deposited onto snow surfaces. The role of snow as a potential source of mercury to the atmosphere or as a sink has also been approached to better understand the behavior of this metal. Chemical and biological processes seem to play a major role in Hg storage in snow. When melting, snow could be a major source of Hg into the various ecosystems and this toxin could therefore be accumulated into the food chain

Regulatory T cells from patients with end-stage organ disease can be isolated, expanded and cryopreserved according good manufacturing practice improving their function

Background Here, we isolated, expanded and functionally characterized regulatory T cells (Tregs) from patients with end stage kidney and liver disease, waiting for kidney/liver transplantation (KT/LT), with the aim to establish a suitable method to obtain large numbers of immunomodulatory cells for adoptive immunotherapy post-transplantation. Methods We first established a preclinical protocol for expansion/isolation of Tregs from peripheral blood of LT/KT patients. We then scaled up and optimized such protocol according to good manufacturing practice (GMP) to obtain high numbers of purified Tregs which were phenotypically and functionally characterized in vitro and in vivo in a xenogeneic acute graft-versus-host disease (aGVHD) mouse model. Specifically, immunodepressed mice (NOD-SCID-gamma KO mice) received human effector T cells with or without GMP-produced Tregs to prevent the onset of xenogeneic GVHD. Results Our small scale Treg isolation/expansion protocol generated functional Tregs. Interestingly, cryopreservation/thawing did not impair phenotype/function and DNA methylation pattern of FOXP3 gene of the expanded Tregs. Fully functional Tregs were also isolated/expanded from KT and LT patients according to GMP. In the mouse model, GMP Tregs from LT or KT patient proved to be safe and show a trend toward reduced lethality of acute GVHD. Conclusions These data demonstrate that expanded/thawed GMP-Tregs from patients with end-stage organ disease are fully functional in vitro. Moreover, their infusion is safe and results in a trend toward reduced lethality of acute GVHD in vivo, further supporting Tregs-based adoptive immunotherapy in solid organ transplantation

Assessment of a procedure to determine trace and major elements in Atmospheric Aerosol

The determination of trace elements in atmospheric particulate is affected by a number of problems that arise from some critical points such as the blank of the filters, sample heterogeneity and pre-analytical treatments. In the framework of a monitoring campaign conducted in the Venice Lagoon the analytical methodology for the determination of 20 trace elements (Al, As, Ca, Cd, Co, Cr, Cs, Cu, Fe, K, Li, Mg, Mn, Na, Ni, Pb, Rb, Sr, V, Zn) in atmospheric particulate samples by inductively coupled plasma quadrupole mass spectroscopy (ICP-QMS) has been optimized taking into account the individual critical points. Tests were carried out to estimate the blank contributions, and minimize the detection limit (LOD), measurements were also carried out to evaluate the accuracy and the repeatability. To obtain a complete dissolution of aerosol dust material and good recoveries of the elements, the acid mixture and the microwave assisted digestion program were optimized. The blank contributions from membrane filter manipulation and transportation prior to exposure were tested for the slotted and back filters by placing them on the sampling device for some minutes without air flowing to obtain field blanks (FBs). The contribution to the blank values of passive deposition and by contact with the samplers (quoted as campaign blanks, CBs) was measured by exposing the membranes throughout the sampling session (fifteen days) without any air flow. Instrumental ICP-QMS parameters were optimized and calibration curve intervals were selected on the basis of the necessity of simultaneous determination of the elements present at different levels of concentration. The limits of detection for each elements and the investigated method were suitable to determine the 20 elements reported above in the atmospheric aerosol fractionated in 6 classes ranging between 10 to 0.49 mm. It allows the determination of trace elements in aerosol in a large range of concentrations that can be observed in areas characterized by remarkable variability and regions with different levels of contamination