Non-Coding Keratin Variants Associate with Liver Fibrosis Progression in Patients with Hemochromatosis

Background: Keratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with endstage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis. Methods: The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCRamplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previouslygenerated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed. Results: We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. I

Hepcidin knockout mice as a model of iron-overload associated liver disease

Hepcidin is the central regulatory hormone of iron metabolism. Disrupted hepcidin signalling is seen in hereditary hemochromatosis (HH) which leads to iron overload. While the association between iron overload and development of end-stage liver disease is well established, the precise underlying mechanisms remain to be defined. To develop iron overload model similar to human HH, we analyzed hepcidin knockout (KO) and wild type (WT) mice were fed with iron-rich diet for 5 or 11 months and compared them to age matched mice kept on standard chow. Harvested livers and serum samples were used for evaluation of liver injury and fibrosis. To determine the iron localization, a subcellular fractionation and electron microscopy was performed. Hepcidin KOs kept on standard diet developed spontaneous hepatic iron overload, which was even more pronounced in KOs fed iron-rich chow who reached levels similar to the ones observed in HH patients. Elevated serum liver enzymes, serum iron levels, mild hepatocellular inflammation and apoptosis were observed in hepcidin KOs fed iron-rich diet. After 11 months of iron-rich chow, hepcidin KOs developed moderate liver fibrosis. The liver injury was accompanied by a marked lysosomal iron overload and lysosomal fragility with release of cathepsins into the cytoplasm. Increased LC3-II and p62 levels pointed towards an activated autophagy which likely contributes to the lysosomal iron overload. On the other hand, large indigestible iron complexes were found in hepcidin knockout mice fed iron-rich diet thereby suggesting a defect in protein degradation as it is observed in lysosomal storage diseases. In conclusion, hepcidin KO mice represent an attractive animal model which mimics both iron overload and associated liver injury observed in humans with HH

Iron Oxide Nanoparticle-Induced Autophagic Flux Is Regulated by Interplay between p53-mTOR Axis and Bcl-2 Signaling in Hepatic Cells

Iron oxide-based nanoparticles have been repeatedly shown to affect lysosomal-mediated signaling. Recently, nanoparticles have demonstrated an ability to modulate autophagic flux via lysosome-dependent signaling. However, the precise underlying mechanisms of such modulation as well as the impact of cellular genetic background remain enigmatic. In this study, we investigated how lysosomal-mediated signaling is affected by iron oxide nanoparticle uptake in three distinct hepatic cell lines. We found that nanoparticle-induced lysosomal dysfunction alters sub-cellular localization of pmTOR and p53 proteins. Our data indicate that alterations in the sub-cellular localization of p53 protein induced by nanoparticle greatly affect the autophagic flux. We found that cells with high levels of Bcl-2 are insensitive to autophagy initiated by nanoparticles. Altogether, our data identify lysosomes as a central hub that control nanoparticle-mediated responses in hepatic cells. Our results provide an important fundamental background for the future development of targeted nanoparticle-based therapies

Targeting the mTOR Signaling Pathway Utilizing Nanoparticles: A Critical Overview

Proteins of the mammalian target of rapamycin (mTOR) signaling axis are overexpressed or mutated in cancers. However, clinical inhibition of mTOR signaling as a therapeutic strategy in oncology shows rather limited progress. Nanoparticle-based mTOR targeted therapy proposes an attractive therapeutic option for various types of cancers. Along with the progress in the biomedical applications of nanoparticles, we start to realize the challenges and opportunities that lie ahead. Here, we critically analyze the current literature on the modulation of mTOR activity by nanoparticles, demonstrate the complexity of cellular responses to functionalized nanoparticles, and underline challenges lying in the identification of the molecular mechanisms of mTOR signaling affected by nanoparticles. We propose the idea that subcytotoxic doses of nanoparticles could be relevant for the induction of subcellular structural changes with possible involvement of mTORC1 signaling. The evaluation of the mechanisms and therapeutic effects of nanoparticle-based mTOR modulation will provide fundamental knowledge which could help in developing safe and efficient nano-therapeutics

Expression of Interferons Lambda 3 and 4 Induces Identical Response in Human Liver Cell Lines Depending Exclusively on Canonical Signaling

Lambda interferons mediate antiviral immunity by inducing interferon-stimulated genes (ISGs) in epithelial tissues. A common variant rs368234815TT/∆G creating functional gene from an IFNL4 pseudogene is associated with the expression of major ISGs in the liver but impaired clearance of hepatitis C. To explain this, we compared Halo-tagged and non-tagged IFNL3 and IFNL4 signaling in liver-derived cell lines. Transfection with non-tagged IFNL3, non-tagged IFNL4 and Halo-tagged IFNL4 led to a similar degree of JAK-STAT activation and ISG induction; however, the response to transfection with Halo-tagged IFNL3 was lower and delayed. Transfection with non-tagged IFNL3 or IFNL4 induced no transcriptome change in the cells lacking either IL10R2 or IFNLR1 receptor subunits. Cytosolic overexpression of signal peptide-lacking IFNL3 or IFNL4 in wild type cells did not interfere with JAK-STAT signaling triggered by interferons in the medium. Finally, expression profile changes induced by transfection with non-tagged IFNL3 and IFNL4 were highly similar. These data do not support the hypothesis about IFNL4-specific non-canonical signaling and point out that functional studies conducted with tagged interferons should be interpreted with caution

Keratin 8 variants are infrequent in patients with alcohol-related liver cirrhosis and do not associate with development of hepatocellular carcinoma

Abstract Background Keratins 8/18 (K8/K18) are established hepatoprotective proteins and K8/K18 variants predispose to development and adverse outcome of multiple liver disorders. The importance of K8/K18 in alcoholic liver disease as well as in established cirrhosis remains unknown. Methods We analyzed the K8 mutational hot-spots in 261 prospectively followed-up patients with alcoholic cirrhosis (mean follow-up 65 months). PCR-amplified samples were pre-screened by denaturing high-performance liquid chromatography and conspicuous samples were sequenced. Results 67 patients developed hepatocellular carcinoma (HCC) and 133 died. Fourteen patients harbored amino-acid-altering K8 variants (5xG62C, 8xR341H). The presence of K8 variants did not associate with development of HCC (log-rank=0.5) or death (log-rank=0.7) and no significant associations were obtained for the single K8 variants after a correction for multiple testing was performed. Conclusions Keratin variants are expressed in a low percentage of patients with alcoholic cirrhosis and do not influence HCC development. Further studies conducted in larger prospective cohorts are needed to find out whether presence of K8 R341H variant predispose to non-HCC-related liver mortality.</p

Аналіз спектральних характеристик української та декількох європейських мов

Для вирішення ряду задач науково-технічного, медичного та лінгвістичного спрямування існує потреба встановлення акустичних особливостей української мови у порівнянні з європейськими мовами. Дослідження виконується на основі найбільш об’єктивної акустичної характеристик мовного сигналу — усередненої спектральної густини потужності. Одержані спектральні характеристики української, російської, польської, італійської та англійської мов шляхом обробки звукових сигналів аудіозаписів публіцистичного та художнього стилів мовлення. Виконане порівняння усереднених за стилями спектрів іноземних мов із відповідними характеристиками української мови. Проаналізовані спільні та відмінні риси цих характеристик у різних частотних областях спектру