The Compartmentalisation of Phosphorylated Free Oligosaccharides in Cells from a CDG Ig Patient Reveals a Novel ER-to-Cytosol Translocation Process

BACKGROUND: Biosynthesis of the dolichol linked oligosaccharide (DLO) required for protein N-glycosylation starts on the cytoplasmic face of the ER to give Man(5)GlcNAc(2)-PP-dolichol, which then flips into the ER for further glycosylation yielding mature DLO (Glc(3)Man(9)GlcNAc(2)-PP-dolichol). After transfer of Glc(3)Man(9)GlcNAc(2) onto protein, dolichol-PP is recycled to dolichol-P and reused for DLO biosynthesis. Because de novo dolichol synthesis is slow, dolichol recycling is rate limiting for protein glycosylation. Immature DLO intermediates may also be recycled by pyrophosphatase-mediated cleavage to yield dolichol-P and phosphorylated oligosaccharides (fOSGN2-P). Here, we examine fOSGN2-P generation in cells from patients with type I Congenital Disorders of Glycosylation (CDG I) in which defects in the dolichol cycle cause accumulation of immature DLO intermediates and protein hypoglycosylation. METHODS AND PRINCIPAL FINDINGS: In EBV-transformed lymphoblastoid cells from CDG I patients and normal subjects a correlation exists between the quantities of metabolically radiolabeled fOSGN2-P and truncated DLO intermediates only when these two classes of compounds possess 7 or less hexose residues. Larger fOSGN2-P were difficult to detect despite an abundance of more fully mannosylated and glucosylated DLO. When CDG Ig cells, which accumulate Man(7)GlcNAc(2)-PP-dolichol, are permeabilised so that vesicular transport and protein synthesis are abolished, the DLO pool required for Man(7)GlcNAc(2)-P generation could be depleted by adding exogenous glycosylation acceptor peptide. Under conditions where a glycotripeptide and neutral free oligosaccharides remain predominantly in the lumen of the ER, Man(7)GlcNAc(2)-P appears in the cytosol without detectable generation of ER luminal Man(7)GlcNAc(2)-P. CONCLUSIONS AND SIGNIFICANCE: The DLO pools required for N-glycosylation and fOSGN2-P generation are functionally linked and this substantiates the hypothesis that pyrophosphatase-mediated cleavage of DLO intermediates yields recyclable dolichol-P. The kinetics of cytosolic fOSGN2-P generation from a luminally-generated DLO intermediate demonstrate the presence of a previously undetected ER-to-cytosol translocation process for either fOSGN2-P or DLO

Use of toxicogenomics in drug safety evaluation: Current status and an industry perspective

Toxicogenomics held great promise as an approach to enable early detection of toxicities induced by xenobiotics; however, there remain questions regarding the impact of the discipline on pharmaceutical nonclinical safety assessment. To understand the current state of toxicogenomics in the sector, an industry group surveyed companies to determine the frequency of toxicogenomics use in in vivo studies at various stages of drug discovery and development and to assess how toxicogenomics use has evolved over time. Survey data were compiled during 2016 from thirteen pharmaceutical companies. Toxicogenomic analyses were infrequently conducted in the development phase and when performed were done to address specific mechanistic questions. Prior to development, toxicogenomics use was more frequent; however, there were significant differences in approaches among companies. Across all phases, gaining mechanistic insight was the most frequent reason cited for pursing toxicogenomics with few companies using toxicogenomics to predict toxicities. These data were consistent with the commentary submitted in response to survey questions asking companies to describe the evolution of their toxicogenomics strategy. Overall, these survey data indicate that toxicogenomics is not widely used as a predictive tool in the pharmaceutical industry but is used regularly by some companies and serves a broader role in mechanistic investigations and as a complement to other technologies

Cardiac spheroids as promising in vitro models to study the human heart microenvironment

Three-dimensional in vitro cell systems are a promising alternative to animals to study cardiac biology and disease. We have generated three-dimensional in vitro models of the human heart (“cardiac spheroids”, CSs) by co-culturing human primary or iPSC-derived cardiomyocytes, endothelial cells and fibroblasts at ratios approximating those present in vivo. The cellular organisation, extracellular matrix and microvascular network mimic human heart tissue. These spheroids have been employed to investigate the dose-limiting cardiotoxicity of the common anti-cancer drug doxorubicin. Viability/cytotoxicity assays indicate dose-dependent cytotoxic effects, which are inhibited by the nitric oxide synthase (NOS) inhibitor L-NIO, and genetic inhibition of endothelial NOS, implicating peroxynitrous acid as a key damaging agent. These data indicate that CSs mimic important features of human heart morphology, biochemistry and pharmacology in vitro, offering a promising alternative to animals and standard cell cultures with regard to mechanistic insights and prediction of toxic effects in human heart tissue

Epithelial cells as active player in fibrosis: findings from an in vitro model.

Kidney fibrosis, a scarring of the tubulo-interstitial space, is due to activation of interstitial myofibroblasts recruited locally or systemically with consecutive extracellular matrix deposition. Newly published clinical studies correlating acute kidney injury (AKI) to chronic kidney disease (CKD) challenge this pathological concept putting tubular epithelial cells into the spotlight. In this work we investigated the role of epithelial cells in fibrosis using a simple controlled in vitro system. An epithelial/mesenchymal 3D cell culture model composed of human proximal renal tubular cells and fibroblasts was challenged with toxic doses of Cisplatin, thus injuring epithelial cells. RT-PCR for classical fibrotic markers was performed on fibroblasts to assess their modulation toward an activated myofibroblast phenotype in presence or absence of that stimulus. Epithelial cell lesion triggered a phenotypical modulation of fibroblasts toward activated myofibroblasts as assessed by main fibrotic marker analysis. Uninjured 3D cell culture as well as fibroblasts alone treated with toxic stimulus in the absence of epithelial cells were used as control. Our results, with the caveats due to the limited, but highly controllable and reproducible in vitro approach, suggest that epithelial cells can control and regulate fibroblast phenotype. Therefore they emerge as relevant target cells for the development of new preventive anti-fibrotic therapeutic approaches

Epithelial cell injury characterization (upper panel) and fibroblast activation (lower panel) in an <i>in vitro</i> reconstructed microenvironment.

<p>(<b>A</b>) Scheme of the reconstructed microenvironment and workflow analysis of the cisplatin-injured proximal tubular epithelial cells HKC-8 cells and of the WS-1 dermal fibroblasts. (<b>B</b>) Cell viability and (<b>C</b>) apoptosis analysis. Cisplatin-treated proximal tubular epithelial cells HKC-8 cells showed decreased cell viability and increased apoptosis. (<b>D</b>). Cell cycle analysis showed that HKC-8 cells treated with cisplatin high dose (40 µM) were blocked in G2/M phase at 24, 48 and 72 h, whereas cells treated with the low dose (20 µM) reverted at 72 h to a condition similar to control. Cytokine release analysis with (<b>E</b>) IL-6 and (<b>F</b>) RANTES levels. Cisplatin-treated HKC-8 cells produced increased amounts of IL-6 and RANTES. (<b>G</b>) Gene-level analysis results for selected genes showing a stronger response to Ciplatin high dose (CisHigh) than to Ciplatin low dose (CisLow). Expression levels on a logarithmic scale are shown as a heat map: no detectable expression is indicated by black color, increasing expression levels are indicated by brighter shades of yellow. Note that several genes show up twice in the figure because they are represented by multiple probes on the Illumina chip. While the measured values do not necessarily agree, the overall trend of up-regulation is the same. (<b>H</b>) Gene-level analysis was complemented by a network-level approach using Gene Set Enrichment Analysis against the Pathway Commons collection of gene regulatory networks (<a href="http://www.pathwaycommons.org" target="_blank">www.pathwaycommons.org</a>). Cisplatin treated cells (L: low, H: high) were compared to controls (C), and renal clear cell carcinoma (RCC) cells were compared to “normal adjacent” tissue (GEO accession number GSE781; as this data set is based on a different expression array technology, we did not compare expression levels of individual genes for this analysis). The heat map shows FDR-corrected q values on a logarithmic scale for up-regulated (red shades) and down-regulated networks (green shades), black indicating no change. An FDR-corrected q value of 0.01 corresponds to an absolute score of 4.6 on this scale. Please, note that the RCC dataset (last column) does not imply any involvement of the networks shown here. (<b>I–L</b>) RT-PCR analysis and mRNA levels of the (<b>I</b>) <i>Acta2</i> gene (encoding alpha smooth muscle actin) (<b>J</b>) <i>TGF-b1</i>gene (encoding transforming growth factor beta 1), (<b>K</b>) <i>COL1A1</i> gene (encoding collagen-1α1) and (<b>L</b>) ID-1 gene (encoding Inhibitor of differentiation 1). Retrieved WS-1 dermal fibroblasts showed increased level for key fibrotic markers α-SMA, TGF-β1 and Collagen 1α1 and decreased level of ID-1 when epithelial cells HK-C8 cells were layered on top. Gene expression profile for the same gene in absence of HK-C8 cells can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056575#pone.0056575.s002" target="_blank">Figure S2B</a>-E. n.s. = not statistically different, * = p<0.05, ** = p<0.001.</p

Combining In Vivo and Organotypic In Vitro Approaches to Assess the Human Relevance of Basimglurant (RG7090), a Potential CAR Activator

ISSN:1096-6080ISSN:1096-092

STAT3 Gain of Function: A New Kid on the Block in Interstitial Lung Diseases

International audienceA 5-year-old girl with failure to thrive and multiorgan disease was referred to our center for chronic hypoxemia. On evaluation, we noted tachypnea (respiratory rate 35/min), supraclavicular retractions, median diurnal oxygen saturation as measured by pulse oximetry (Sp O 2) = 91.7% at rest, percentage of time below Sp O 2 90% at 26% during sleep, and clubbing. A computed tomography scan showed diffuse interstitial lung disease (Figure 1). Spirometry was normal (TLC, 83% of predicted; FEV 1 , 83% of predicted; FEV 1 /FVC, 98%; and forced expiratory flow, midexpiratory phase, 142% of predicted), but it was not possible to measure DL CO