The galectin-3/RAGE dyad modulates vascular osteogenesis in atherosclerosis

Vascular calcification correlates with inflammation and plaque instability in a dual manner, depending on the spotty/granular (micro) or sheet-like/lamellated (macro) pattern of calcification. Modified lipoproteins trigger both inflammation and calcification via receptors for advanced lipoxidation/glycation endproducts (ALEs/AGEs). This study compared the roles of galectin-3 and receptor for AGEs (RAGE), two ALEs/AGEs-receptors with diverging effects on inflammation and bone metabolism, in the process of vascular calcification. We evaluated galectin-3 and RAGE expression/localization in 62 human carotid plaques and its relation to calcification pattern, plaque phenotype, and markers of inflammation and vascular osteogenesis; and the effect of galectin-3 ablation and/or exposure to an ALE/AGE on vascular smooth muscle cell (VSMC) osteogenic differentiation. While RAGE co-localized with inflammatory cells in unstable regions with microcalcification, galectin-3 was expressed also by VSMCs, especially in macrocalcified areas, where it co-localized with alkaline phosphatase. Expression of galectin-3 and osteogenic markers was higher in macrocalcified plaques, whereas the opposite occurred for RAGE and inflammatory markers. Galectin-3-deficient VSMCs exhibited defective osteogenic differentiation, as shown by altered expression of osteogenic transcription factors and proteins, blunted activation of pro-osteoblastogenic Wnt/β-catenin signalling and proliferation, enhanced apoptosis, and disorganized mineralization. These abnormalities were associated with RAGE up-regulation, but were only in part prevented by RAGE silencing, and were partially mimicked or exacerbated by treatment with an AGE/ALE. These data indicate a novel molecular mechanism by which galectin-3 and RAGE modulate in divergent ways, not only inflammation, but also vascular osteogenesis, by modulating Wnt/β-catenin signalling, and independently of ALEs/AGEs

Oligodendroglioma cells lack glutamine synthetase and are auxotrophic for glutamine, but do not depend on glutamine anaplerosis for growth

In cells derived from several types of cancer, a transcriptional program drives high consumption of glutamine (Gln), which is used for anaplerosis, leading to a metabolic addiction for the amino acid. Low or absent expression of Glutamine Synthetase (GS), the only enzyme that catalyzes de novo Gln synthesis, has been considered a marker of Gln-addicted cancers. In this study, two human cell lines derived from brain tumors with oligodendroglioma features, HOG and Hs683, have been shown to be GS-negative. Viability of both lines depends from extracellular Gln with EC of 0.175 ± 0.056 mM (Hs683) and 0.086 ± 0.043 mM (HOG), thus suggesting that small amounts of extracellular Gln are sufficient for OD cell growth. Gln starvation does not significantly affect the cell content of anaplerotic substrates, which, consistently, are not able to rescue cell growth, but causes hindrance of the Wnt/β-catenin pathway and protein synthesis attenuation, which is mitigated by transient GS expression. Gln transport inhibitors cause partial depletion of intracellular Gln and cell growth inhibition, but do not lower cell viability. Therefore, GS-negative human oligodendroglioma cells are Gln-auxotrophic but do not use the amino acid for anaplerosis and, hence, are not Gln addicted, exhibiting only limited Gln requirements for survival and growth

Intima neovascularization studied on epoxy-resin semithin sections in a rare case of carotid recurrent in-stent restenosis

Introduction. Restenosis after carotid endarterectomy (CE) has been described in 8-19% of patients (1). In-stent  restenosis (ISR) after stenting has been reported in a few cases (2). Here we report an extremely rare case of recurrent ISR after CE and wallstent.  Materials and methods. An atherosclerothic plaque belonging to a patient who underwent recurrent ISR after both CE and wallstent has been studied. Semithin sections (0,850 µm thick) obtained from epoxy-resin embedded samples were stained as reported (3). Light microscopy observations were performed at magnifications of 400X and 1000X, within an area of 3,8mm2 corresponding to 10 microscopic field at 400X. The morphological characteristics considered were: cells number and type; presence, number and type of neovessels; calcifications, and inflammatory cells. Results. The plaque appeared mostly fibrous, with an internal rupture and hemorrhagy. Very few foam cells (N= 12/3,8mm2) were observed whereas vascular smooth muscle cells (VSMCs) were abundant (n=160/3,8mm2) and often entrapped in a thick coat of self-secreted collagen. Lipid laden VSMCs were present (n=18/3,8mm2); three of them showed intracellular microcalcifications. Marked signs of inflammation were found, macrophage (n=50/3,8mm2), mast cells (n=8/3,8mm2), lymphocytes (n=5/3,8mm2) as well as granulocytes (n=4/3,8mm2). There was a marked neovascularization, 24 sections of circular regular vessel and 6 sections of flattened but patent vessels were found in 3,8mm2. Neovessels had intact endothelium and likely derived from the underneath media. Discussion. Histopathological diagnosis is usually carried out on paraffin-embedded 4µm-thick sections. The observation of semithin sections has allowed us to achieve a better morphological characterization than that routinely obtained. The cellular population observed is that of an active remodelling plaque, susceptible of rupture, both for the presence of mast cells and extensive neovascularization. Mast cells secrete effector molecules that affect endothelial cells and VSMCs activity, the presence of neovessels weakens the plaque, thus producing cleavage planes. 1. Counsell et al., 2011. Carotid stenting for restenosis after endarterectomy. Cardiovasc Intervent Radiol. 34(3):488-92. 2. Willfort-Ehringer et al., 2002. Single-center experience with carotid stent restenosis. J Endovasc Ther. 9(3):299-307. 3. Relucenti et al., 2010. Detecting microcalcifications in atherosclerotic plaques by a simple trichromic staining method for epoxy embedded carotid endarterectomies. Eur J Histochem. 54(3):e33

The TLR4/NFκB-Dependent Inflammatory Response Activated by LPS Is Inhibited in Human Macrophages Pre-Exposed to Amorphous Silica Nanoparticles

Amorphous silica nanoparticles (ASNP) are present in a variety of products and their biological effects are actively investigated. Although several studies have documented pro-inflammatory effects of ASNP, the possibility that they also modify the response of innate immunity cells to natural activators has not been thoroughly investigated. Here, we study the effects of pyrogenic ASNP on the LPS-dependent activation of human macrophages differentiated from peripheral blood monocytes. In macrophages, 24 h of pre-exposure to non-cytotoxic doses of ASNP markedly inhibited the LPS-dependent induction of pro-inflammatory (TNFα, IL-6) and anti-inflammatory cytokines (IL-10). The inhibitory effect was associated with the suppression of NFκB activation and the increased intracellular sequestration of the TLR4 receptor. The late induction of glutamine synthetase (GS) by LPS was also prevented by pre-exposure to ASNP, while GS silencing did not interfere with cytokine secretion. It is concluded that (i) macrophages exposed to ASNP are less sensitive to LPS-dependent activation and (ii) GS induction by LPS is likely secondary to the stimulation of cytokine secretion. The observed interference with LPS effects may point to a dampening of the acute inflammatory response after exposure to ASNP in humans

Pulsed laser deposition of a dense and uniform Au nanoparticles layer for surface plasmon enhanced efficiency hybrid solar cells

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Microscopy methods for biofilm imaging: focus on SEM and VP-SEM pros and cons

Several imaging methodologies have been used in biofilm studies, contributing to deep- ening the knowledge on their structure. This review illustrates the most widely used microscopy techniques in biofilm investigations, focusing on traditional and innovative scanning electron mi- croscopy techniques such as scanning electron microscopy (SEM), variable pressure SEM (VP-SEM), environmental SEM (ESEM), and the more recent ambiental SEM (ASEM), ending with the cutting edge Cryo-SEM and focused ion beam SEM (FIB SEM), highlighting the pros and cons of several methods with particular emphasis on conventional SEM and VP-SEM. As each technique has its own advantages and disadvantages, the choice of the most appropriate method must be done carefully, based on the specific aim of the study. The evaluation of the drug effects on biofilm requires imaging methods that show the most detailed ultrastructural features of the biofilm. In this kind of research, the use of scanning electron microscopy with customized protocols such as osmium tetroxide (OsO4), ruthenium red (RR), tannic acid (TA) staining, and ionic liquid (IL) treatment is unrivalled for its image quality, magnification, resolution, minimal sample loss, and actual sample structure preserva- tion. The combined use of innovative SEM protocols and 3-D image analysis software will allow for quantitative data from SEM images to be extracted; in this way, data from images of samples that have undergone different antibiofilm treatments can be compared

New insights into the oenological significance of Candida zemplinina. Impact of selected autochthonous strains on the volatile profile of Apulian wines

In this investigation, we explored the oenological significance of Candida zemplinina (syn. Starmerella bacillaris) isolates from Apulian grape musts. Moreover, we provide the first evidence of the impact of different C. zemplinina strains on the wine aromatic properties tested as monocultures. We described the diversity of C. zemplinina strains isolated from grapes and the variability of ‘volatile’ phenotypes associated with this intraspecific variability. Thirty-three isolates were characterized at strain level by PCR-based approach and, among these, 16 strains were identified and then tested by microfermentation tests carried out in grape must. Analyzed strains were low producers of acetic acid and hydrogen sulphide, not able to decarboxylate a panel of representative amino acids, whereas they showed fructophilic character and significant glycerol production. Volatile profiles of produced wines were investigated by gas chromatography–mass spectrometry. The Odor Activity Values of all molecules were calculated and 12 compounds showed values above their odor thresholds. Two selected strains (35NC1 and 15PR1) could be considered as possible starter cultures since they were able to positively affect the sensory properties of obtained wine. This report firstly supplies evidence on the strain-specific impact of different C. zemplinina strains on the final aroma of produced wines

Three-Dimensional Self-Assembly of Networked Branched TiO2 Nanocrystal Scaffolds for Efficient Room-Temperature Processed Depleted Bulk Heterojunction Solar Cells

In this work, we report on 4% power conversion efficiency (PCE) depleted bulk heterojunction (DBH) solar cells based on a high-quality electrode with a three-dimensional nanoscale architecture purposely designed so as to maximize light absorption and charge collection. The newly conceived architecture comprises a mesoporous electron-collecting film made of networked anisotropic metal-oxide nanostructures, which accommodates visible-to-infrared light harvesting quantum dots within the recessed regions of its volume. The three-dimensional electrodes were self-assembled by spin-coating a solution of colloidal branched anatase TiO2 NCs (BNC), followed by photocatalytic removal of the native organic capping from their surface by a mild UV-light treatment and filling with small PbS NCs via infiltration. The PCE = 4% of our TiO2 BNC/PbS QD DBH solar cell features an enhancement of 84% over the performance obtained for a planar device fabricated under the same conditions. Overall, the DBH device fabrication procedure is entirely carried out under mild processing conditions at room temperature, thus holding promise for low-cost and large-scale manufacturing

Morphofunctional characterization of a rare extracranial internal carotid artery giant aneurism

Aneurisms of the extracranial tract of the internal carotid artery (EICAA) are extremely rare, accounting for 0,4% to 2% of all carotid procedures (El-Sabrout et al., 2000); in females incidence is 2-11 times lower than in males (Siablis et al., 2004). A giant EICAA (32 x 35 mm) at C3 level was studied in a woman aged 81. Samples were prepared for standard transmission electron microscopy. Semithin sections were stained according to Relucenti et al. (2010). Ultrathin sections were contrasted with uranyl acetate and lead citrate. Images revealed the presence of many microvessels just beneath the tunica intima. They were patent, often showed sprouts and pericytes. Endothelial cells appeared metabolically active, with euchromatin, nucleoli, membrane blebs and junctional complexes. Their basal membrane was thickened and sometimes multilaminated. Leukocytes adhering to the endothelium were observed. The internal elastic lamina was so fragmented that it was very difficult to recognize it. The tunica media showed vascular smooth muscle cells (VSMCs) arranged in bundles encircled by collagen-rich extracellular matrix (EM), as well as scattered in the EM. VSMCs had perinuclear organelles, dense bands and caveolae. Collagen fibrils were arranged in a twisted or coiled fashion. Elongated fibroblasts were scattered among VSMCs. The external elastic lamina was conserved. Literature on EICAA is almost exclusively clinical, so this morphofunctional study elucidates structural and ultrastructural changes in the aneurism wall that can contribute to the knowledge of aneurism etiopathogenesis