JoussefHayek and Giuseppe Valacchi contributed equally to this work.Abbreviations: 4-HNE, 4-hydroxynonenal; ASD, autism spectrum disorder; ATP5A, ATP synthase subunit alpha, mitochondrial; COX4, cytochrome c oxidase subunit 4 isoform 1, mitochondrial; DMEM, Dulbecco's Modification of Eagle's Medium; DRP1, dynamin-1-like protein; ECAR, extracellular acidification rate; ETC, electron transport chain; FBS, fetal bovine serum; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; FIS1, mitochondrial fission 1 protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSH, glutathione; H 2 O 2 , hydrogen peroxide; HBSS, Hanks' balanced salt solution; HDCA1, histone deacetylase 1; HDL, high-density lipoproteins; HO-1, heme oxygenase 1; MFN1, mitofusin-1; MFN2, mitofusin-2; NADPH, nicotinamide adenine dinucleotide phosphate; NDUFB8, NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8, mitochondrial; NFκB, nuclear factor NF-kappa-B; NOX, NADPH (Nicotinamide adenine dinucleotide phosphate) oxidase; NPBI, nonprotein-bound iron; NQO1, NAD(P)H quinone dehydrogenase 1; Nrf2, nuclear factor erythroid 2-related factor 2; OCR, oxygen consuming ratio; OPA1, dynamin-like 120 kDa protein, mitochondrial; Parkin, E3 ubiquitin-protein ligase parkin; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PINK1, serine/threonine-protein kinase PINK1, mitochondrial; PMA, phorbol myristate acetate; PON-1, paraoxonase-1; PVDF, polyvinylidene difluoride; RFU, relative fluorescence units; RIPA, radio-immunoprecipitation assay; RLU, relative luminescence units; ROS, reactive oxygen species; SDHA, succinate dehydrogenase complex flavoprotein subunit A; SIRT3, sirtuin 3; SOD, superoxide dismutase; TBS-T, tris-buffered saline, 0.1% Tween 20; TEM, transmission electron microscopy; TOMM20, translocase of outer mitochondrial membrane 20; TRX, thioredoxin; TXNIP, thioredoxin-interacting protein; UCP2, mitochondrial uncoupling protein 2; UQCRC2, cytochrome b-c1 complex subunit 2, mitochondrial. AbstractAutism spectrum disorder (ASD) has been hypothesized to be a result of the interplay between genetic predisposition and increased vulnerability to early environmental insults. Mitochondrial dysfunctions appear also involved in ASD pathophysiology, but the mechanisms by which such alterations develop are not completely understood.Here, we analyzed ASD primary fibroblasts by measuring mitochondrial bioenergetics, ultrastructural and dynamic parameters to investigate the hypothesis that defects in these pathways could be interconnected phenomena responsible or consequence for the redox imbalance observed in ASD. High levels of 4-hydroxynonenal protein adducts together with increased NADPH (nicotinamide adenine dinucleotide phosphateoxidase) activity and mitochondrial superoxide production coupled with a compromised antioxidant response guided by a defective Nuclear Factor Erythroid 2-Related Factor 2 pathway confirmed an unbalanced redox homeostasis in ASD.Moreover, ASD fibroblasts showed overactive mitochondrial bioenerget...
(1) Background: The gastrointestinal tract (GI) tract is one of the main organs exposed to particulate matter (PM) directly through ingestion of contaminated food or indirectly through inhalation. Previous studies have investigated the effects of chronic PM exposure on intestinal epithelia in vitro using Caco−2 cells and in vivo using mice. In this study, we hypothesized that chronic PM exposure would increase epithelial permeability and decrease barrier function due to altered redox homeostasis, which alters levels and/or localization of barrier-associated proteins in human three-dimensional (3D) intestinal tissues. (2) Methods: Transepithelial electrical resistance (TEER) in tissues exposed to 50, 100, 150, 250, and 500 µg/cm2 of PM for 1 week and 2 weeks was analyzed. Levels and localization of tight junction proteins zonula occludens protein 1 (ZO−1) and claudin−1 and desmosome-associated desmocollin were analyzed using immunofluorescence. As a marker of oxidative stress, levels of 4-hydroxy-nonenal (4HNE) adducts were measured. (3) Results: No differences in TEER measurements were observed between exposed and un-exposed tissues. However, increased levels of 4HNE adducts in exposed tissues were observed. Additionally, decreased levels of ZO−1, claudin−1, and desmocollin were demonstrated. (4) Conclusion: These data suggest that chronic PM exposure results in an increase of oxidative stress; modified levels of barrier-associated proteins could possibly link to GI tract inflammatory conditions.
Osteomyelitis (OM) is an infectious disease of the bone primarily caused by the opportunistic pathogen Staphylococcus aureus (SA). This Gram-positive bacterium has evolved a number of strategies to evade the immune response and subvert bone homeostasis, yet the underlying mechanisms remain poorly understood. OM has been modeled in vitro to challenge pathogenetic hypotheses in controlled conditions, thus providing guidance and support to animal experimentation. In this regard, traditional 2D models of OM inherently lack the spatial complexity of bone architecture. Three-dimensional models of the disease overcome this limitation; however, they poorly reproduce composition and texture of the natural bone. Here, we developed a new 3D model of OM based on cocultures of SA and murine osteoblastic MC3T3-E1 cells on magnesium-doped hydroxyapatite/collagen I (MgHA/Col) scaffolds that closely recapitulate the bone extracellular matrix. In this model, matrix-dependent effects were observed in proliferation, gene transcription, protein expression, and cell–matrix interactions both of the osteoblastic cell line and of bacterium. Additionally, these had distinct metabolic and gene expression profiles, compared to conventional 2D settings, when grown on MgHA/Col scaffolds in separate monocultures. Our study points to MgHA/Col scaffolds as biocompatible and bioactive matrices and provides a novel and close-to-physiology tool to address the pathogenetic mechanisms of OM at the host–pathogen interface.
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