Bedeutung des "Interferon Stimulated Gene 12" (ISG12) in der Enstehung von atherosklerotischen Plaques in LDL Rezeptor-defizienten Mäusen

Atherosklerose-bedingte kardiovaskuläre Erkrankungen sind die häufigste Todesursache in Österreich. Eine wichtige Ursache für die Entstehung atherosklerotischer Läsionen liegt in einer zu fettreichen Ernährung und der daraus resultierenden Hypercholesterinämie. Makrophagen spielen eine entscheidende Rolle in der Entstehung atherosklerotischer Plaques. Sie nehmen cholesterol-hältige Lipoproteine auf, wandeln sich in Schaumzellen um und tragen durch die Interaktion mit Zellen des Immunsystems zur Entstehung eines lokalen, chronisch entzündlichen Prozesses bei. Für die Entstehung und Propagation dieser inflammatorischen Reaktion spielen Zytokine, die zur Attraktion und Aktivierung von Entzündungszellen führen, eine wichtige Rolle. Interferon γ (IFNγ) ist ein Zytokin, das von T-Zellen sezerniert wird und die Funktion von Makrophagen moduliert. Es nimmt auf die Genexpression in den Makrophagen Einfluß und trägt so zum pro-inflammatorischen Phänotyp der Makrophagen bei. Bisher ist jedoch nur wenig über die Rolle von IFNγ-abhängigen Molekülen in Makrophagen und ihre Bedeutung für die Atherogenese bekannt. Im Rahmen der vorliegenden Arbeit wurde die Bedeutung von „interferon-stimulated gene 12“ (ISG12), einem kleinen IFNγ-induzierbaren Molekül, in der Atherogenese untersucht. ISG12 ist an der Zellkernmembran lokalisiert und fördert den nukleären Export von anti-inflammatorischen („protektiven“) Transkriptionsfaktoren wie PPARγ und NR4A1. Dadurch könnte ISG12 zur Förderung der lokalen Entzündungsreaktion durch IFNγ und in der Folge zur Entstehung von atherosklerotischen Plaques beitragen. Um die Rolle von ISG12 in Makrophagen zu untersuchen, wurde ein Mausmodell verwendet, in dem LDL-Rezeptor–/– Mäuse mit Knochenmark von ISG12-defizienten Mäusen transplantiert wurden. Nach einer dreimonatige Fettdiät wurde die Aorta der Tiere mikrochirurgisch präpariert, longitudinal aufgeschnitten und die atherosklerotischen Plaques durch eine Sudan IV Färbung sichtbar gemacht. Die Quantifizierung der Plaquefläche zeigte, dass die LDLR–/– Mäusen, welche mit ISG12-defizientem Knochenmark transplantiert worden waren, signifikant weniger atherosklerotische Plaques entwickelten als die Tiere der Kontrollgruppe. In einem zweiten Schritt wurde das Volumen der atherosklerotischen Läsionen an der Herzbasis anhand von Serienschnitten quantifiziert. Auch mit dieser Technik zeigte sich, dass die ISG12-Defizienz in den Makrophagen die Entwicklung der atherosklerotischen Plaques hemmt. Die Resultate dieser Studie zeigen, dass das in Makrophagen exprimierte ISG12 Protein zur Atherogenese beiträgt und legen nahe, dass ISG12 ein wichtiges pro-inflammatorisches Molekül in den Makrophagen sein könnte.Atherosclerotic cardiovascular disease is the main cause of death in Austria. One important risk factor for developing atherosclerotic lesions is a diet which is high in fat and results in hypercholesterolemia. Macrophages play a decisive role in the development of atherosclerotic lesions. They take up cholesterol-rich lipoproteins, transform into foam cells and interact with cells of the immune system to establish a local chronic inflammatory process. An important factor for the onset and propagation of this inflammatory reaction is the generation of cytokines which attract and activate cells of the immune system. Interferon-gamma (IFNgamma) is a T cell cytokine which modulates the function of macrophages. Binding of IFNgamma to its receptor leads to an alteration of gene expression patterns in macrophages and thus contributes to the pro-inflammatory phenotype of these cells observed in atherosclerotic plaques. However, so far, little is known about the relevance of IFNgamma-dependent molecules for atherogenesis and their specific role in macrophages. In this study, we investigated the role of “interferon-stimulated gene 12”, a small IFNgamma-inducible molecule, in atherogenesis. ISG12 is located at the nuclear envelope and promotes the nuclear export of anti-inflammatory (“protective”) transcription factors such as PPARgamma and NR4A1. Upregulation of ISG12 in macrophages could therefore contribute to IFNgamma-mediated inflammation and formation of atherosclerotic plaques. To investigate the role of ISG12 in macrophages, we transplanted LDL-receptor─/─ mice with bone marrow from ISG12-deficient mice. After transplantation, mice received a high fat diet for three months. Then, the aorta was microdissected, opened longitudinally and athersclerotic plaques were made visible by Sudan IV staining. Quantification of the plaque size showed that LDL-receptor─/─ mice transplanted with ISG12-deficient bone marrow developed significantly less atherosclerotic plaques than animals in the control group. In a second step, the volume of atherosclerotic lesions at the aortic origin was quantified on serial cross-sections. Again, we found that ISG12-deficiency in macrophages exerts a protective effect and inhibits the development of atherosclerotic plaques. The results of our study show that ISG12 expressed in macrophages contributes to atherogenesis. Moreover, our data suggest that ISG12 could represent an important IFNgamma-dependent proinflammatory molecule in macrophages

Cardiac Glycosides Induce Cell Death in Human Cells by Inhibiting General Protein Synthesis

BACKGROUND: Cardiac glycosides are Na(+)/K(+)-pump inhibitors widely used to treat heart failure. They are also highly cytotoxic, and studies have suggested specific anti-tumor activity leading to current clinical trials in cancer patients. However, a definitive demonstration of this putative anti-cancer activity and the underlying molecular mechanism has remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: Using an unbiased transcriptomics approach, we found that cardiac glycosides inhibit general protein synthesis. Protein synthesis inhibition and cytotoxicity were not specific for cancer cells as they were observed in both primary and cancer cell lines. These effects were dependent on the Na(+)/K(+)-pump as they were rescued by expression of a cardiac glycoside-resistant Na(+)/K(+)-pump. Unlike human cells, rodent cells are largely resistant to cardiac glycosides in vitro and mice were found to tolerate extremely high levels. CONCLUSIONS/SIGNIFICANCE: The physiological difference between human and mouse explains the previously observed sensitivity of human cancer cells in mouse xenograft experiments. Thus, published mouse xenograft models used to support anti-tumor activity for these drugs require reevaluation. Our finding that cardiac glycosides inhibit protein synthesis provides a mechanism for the cytotoxicity of CGs and raises concerns about ongoing clinical trials to test CGs as anti-cancer agents in humans

Transcriptome analysis of human cancer reveals a functional role of Heme Oxygenase-1 in tumor cell adhesion

<p>Abstract</p> <p>Background</p> <p>Heme Oxygenase-1 (HO-1) is expressed in many cancers and promotes growth and survival of neoplastic cells. Recently, HO-1 has been implicated in tumor cell invasion and metastasis. However, the molecular mechanisms underlying these biologic effects of HO-1 remain largely unknown. To identify a common mechanism of action of HO-1 in cancer, we determined the global effect of HO-1 on the transcriptome of multiple tumor entities and identified a universal HO-1-associated gene expression signature.</p> <p>Results</p> <p>Genome-wide expression profiling of Heme Oxygenase-1 expressing versus HO-1 silenced BeWo choriocarcinoma cells as well as a comparative meta-profiling of the preexisting expression database of 190 human tumors of 14 independent cancer types led to the identification of 14 genes, the expression of which correlated strongly and universally with that of HO-1 (P = 0.00002). These genes included regulators of cell plasticity and extracellular matrix (ECM) remodeling (MMP2, ADAM8, TGFB1, BGN, COL21A1, PXDN), signaling (CRIP2, MICB), amino acid transport and glycosylation (SLC7A1 and ST3GAL2), estrogen and phospholipid biosynthesis (AGPAT2 and HSD17B1), protein stabilization (IFI30), and phosphorylation (ALPPL2). We selected PXDN, an adhesion molecule involved in ECM formation, for further analysis and functional characterization. Immunofluorescence and Western blotting confirmed the positive correlation of expression of PXDN and HO-1 in BeWo cancer cells as well as co-localization of these two proteins in invasive extravillous trophoblast cells. Modulation of HO-1 expression in both loss-of and gain-of function cell models (BeWo and 607B melanoma cells, respectively) demonstrated a direct relationship of HO-1 expression with cell adhesion to Fibronectin and Laminin coated wells. The adhesion-promoting effects of HO-1 were dependent on PXDN expression, as loss of PXDN in HO-1 expressing BeWo and 607B cells led to reduced cell attachment to Laminin and Fibronectin coated wells.</p> <p>Conclusions</p> <p>Collectively, our results show that HO-1 expression determines a distinct 'molecular signature' in cancer cells, which is enriched in genes associated with tumorigenesis. The protein network downstream of HO-1 modulates adhesion, signaling, transport, and other critical cellular functions of neoplastic cells and thus promotes tumor cell growth and dissemination.</p

Recently photoassimilated carbon and fungus-delivered nitrogen are spatially correlated in the ectomycorrhizal tissue of Fagus sylvatica

Ectomycorrhizal plants trade plant‐assimilated carbon for soil nutrients with their fungal partners. The underlying mechanisms, however, are not fully understood. Here we investigate the exchange of carbon for nitrogen in the ectomycorrhizal symbiosis of Fagus sylvatica across different spatial scales from the root system to the cellular level. We provided (15)N‐labelled nitrogen to mycorrhizal hyphae associated with one half of the root system of young beech trees, while exposing plants to a (13)CO(2) atmosphere. We analysed the short‐term distribution of (13)C and (15)N in the root system with isotope‐ratio mass spectrometry, and at the cellular scale within a mycorrhizal root tip with nanoscale secondary ion mass spectrometry (NanoSIMS). At the root system scale, plants did not allocate more (13)C to root parts that received more (15)N. Nanoscale secondary ion mass spectrometry imaging, however, revealed a highly heterogenous, and spatially significantly correlated distribution of (13)C and (15)N at the cellular scale. Our results indicate that, on a coarse scale, plants do not allocate a larger proportion of photoassimilated C to root parts associated with N‐delivering ectomycorrhizal fungi. Within the ectomycorrhizal tissue, however, recently plant‐assimilated C and fungus‐delivered N were spatially strongly coupled. Here, NanoSIMS visualisation provides an initial insight into the regulation of ectomycorrhizal C and N exchange at the microscale

Signal Peptide Peptidase-Like 2c (SPPL2c) impairs vesicular transport and cleavage of SNARE proteins

Members of the GxGD-type intramembrane aspartyl proteases have emerged as key players not only in fundamental cellular processes such as B-cell development or protein glycosylation, but also in development of pathologies, such as Alzheimer's disease or hepatitis virus infections. However, one member of this protease family, signal peptide peptidase-like 2c (SPPL2c), remains orphan and its capability of proteolysis as well as its physiological function is still enigmatic. Here, we demonstrate that SPPL2c is catalytically active and identify a variety of SPPL2c candidate substrates using proteomics. The majority of the SPPL2c candidate substrates cluster to the biological process of vesicular trafficking. Analysis of selected SNARE proteins reveals proteolytic processing by SPPL2c that impairs vesicular transport and causes retention of cargo proteins in the endoplasmic reticulum. As a consequence, the integrity of subcellular compartments, in particular the Golgi, is disturbed. Together with a strikingly high physiological SPPL2c expression in testis, our data suggest involvement of SPPL2c in acrosome formation during spermatogenesis

Adolescent well-being and learning in times of COVID-19-A multi-country study of basic psychological need satisfaction, learning behavior, and the mediating roles of positive emotion and intrinsic motivation

The sudden switch to distance education to contain the outbreak of the COVID-19 pandemic has fundamentally altered adolescents' lives around the globe. The present research aims to identify psychological characteristics that relate to adolescents' well-being in terms of positive emotion and intrinsic learning motivation, and key characteristics of their learning behavior in a situation of unplanned, involuntary distance education. Following Self-Determination Theory, experienced competence, autonomy, and relatedness were assumed to relate to active learning behavior (i.e., engagement and persistence), and negatively relate to passive learning behavior (i.e., procrastination), mediated via positive emotion and intrinsic learning motivation. Data were collected via online questionnaires in altogether eight countries from Europe, Asia, and North America (N = 25,305) and comparable results across countries were expected. Experienced competence was consistently found to relate to positive emotion and intrinsic learning motivation, and, in turn, active learning behavior in terms of engagement and persistence. The study results further highlight the role of perceived relatedness for positive emotion. The high proportions of explained variance speak in favor of taking these central results into account when designing distance education in times of COVID-19.Peer reviewe

Distance learning in higher education during COVID-19 : The role of basic psychological needs and intrinsic motivation for persistence and procrastination–a multi-country study

Due to the COVID-19 pandemic, higher educational institutions worldwide switched to emergency distance learning in early 2020. The less structured environment of distance learning forced students to regulate their learning and motivation more independently. According to self-determination theory (SDT), satisfaction of the three basic psychological needs for autonomy, competence and social relatedness affects intrinsic motivation, which in turn relates to more active or passive learning behavior. As the social context plays a major role for basic need satisfaction, distance learning may impair basic need satisfaction and thus intrinsic motivation and learning behavior. The aim of this study was to investigate the relationship between basic need satisfaction and procrastination and persistence in the context of emergency distance learning during the COVID-19 pandemic in a cross-sectional study. We also investigated the mediating role of intrinsic motivation in this relationship. Furthermore, to test the universal importance of SDT for intrinsic motivation and learning behavior under these circumstances in different countries, we collected data in Europe, Asia and North America. A total of N = 15,462 participants from Albania, Austria, China, Croatia, Estonia, Finland, Germany, Iceland, Japan, Kosovo, Lithuania, Poland, Malta, North Macedonia, Romania, Sweden, and the US answered questions regarding perceived competence, autonomy, social relatedness, intrinsic motivation, procrastination, persistence, and sociodemographic background. Our results support SDT’s claim of universality regarding the relation between basic psychological need fulfilment, intrinsic motivation, procrastination, and persistence. However, whereas perceived competence had the highest direct effect on procrastination and persistence, social relatedness was mainly influential via intrinsic motivation.Peer reviewe

Rapid Transfer of Plant Photosynthates to Soil Bacteria via Ectomycorrhizal Hyphae and Its Interaction With Nitrogen Availability

Plant roots release recent photosynthates into the rhizosphere, accelerating decomposition of organic matter by saprotrophic soil microbes (“rhizosphere priming effect”) which consequently increases nutrient availability for plants. However, about 90% of all higher plant species are mycorrhizal, transferring a significant fraction of their photosynthates directly to their fungal partners. Whether mycorrhizal fungi pass on plant-derived carbon (C) to bacteria in root-distant soil areas, i.e., incite a “hyphosphere priming effect,” is not known. Experimental evidence for C transfer from mycorrhizal hyphae to soil bacteria is limited, especially for ectomycorrhizal systems. As ectomycorrhizal fungi possess enzymatic capabilities to degrade organic matter themselves, it remains unclear whether they cooperate with soil bacteria by providing photosynthates, or compete for available nutrients. To investigate a possible C transfer from ectomycorrhizal hyphae to soil bacteria, and its response to changing nutrient availability, we planted young beech trees (Fagus sylvatica) into “split-root” boxes, dividing their root systems into two disconnected soil compartments. Each of these compartments was separated from a litter compartment by a mesh penetrable for fungal hyphae, but not for roots. Plants were exposed to a 13C-CO2-labeled atmosphere, while 15N-labeled ammonium and amino acids were added to one side of the split-root system. We found a rapid transfer of recent photosynthates via ectomycorrhizal hyphae to bacteria in root-distant soil areas. Fungal and bacterial phospholipid fatty acid (PLFA) biomarkers were significantly enriched in hyphae-exclusive compartments 24 h after 13C-CO2-labeling. Isotope imaging with nanometer-scale secondary ion mass spectrometry (NanoSIMS) allowed for the first time in situ visualization of plant-derived C and N taken up by an extraradical fungal hypha, and in microbial cells thriving on hyphal surfaces. When N was added to the litter compartments, bacterial biomass, and the amount of incorporated 13C strongly declined. Interestingly, this effect was also observed in adjacent soil compartments where added N was only available for bacteria through hyphal transport, indicating that ectomycorrhizal fungi were acting on soil bacteria. Together, our results demonstrate that (i) ectomycorrhizal hyphae rapidly transfer plant-derived C to bacterial communities in root-distant areas, and (ii) this transfer promptly responds to changing soil nutrient conditions

BCR-ABL1-independent PI3Kinase activation causing imatinib-resistance

<p>Abstract</p> <p>Background</p> <p>The <it>BCR-ABL1 </it>translocation occurs in chronic myeloid leukemia (CML) and in 25% of cases with acute lymphoblastic leukemia (ALL). The advent of tyrosine kinase inhibitors (TKI) has fundamentally changed the treatment of CML. However, TKI are not equally effective for treating ALL. Furthermore, <it>de novo </it>or <it>secondary </it>TKI-resistance is a significant problem in CML. We screened a panel of <it>BCR-ABL1 </it>positive ALL and CML cell lines to find models for imatinib-resistance.</p> <p>Results</p> <p>Five of 19 <it>BCR-ABL1 </it>positive cell lines were resistant to imatinib-induced apoptosis (KCL-22, MHH-TALL1, NALM-1, SD-1, SUP-B15). None of the resistant cell lines carried mutations in the kinase domain of <it>BCR-ABL1 </it>and all showed resistance to second generation TKI, nilotinib or dasatinib. STAT5, ERK1/2 and the ribosomal S6 protein (RPS6) are <it>BCR-ABL1 </it>downstream effectors, and all three proteins are dephosphorylated by imatinib in sensitive cell lines. TKI-resistant phosphorylation of RPS6, but responsiveness as regards JAK/STAT5 and ERK1/2 signalling were characteristic for resistant cell lines. PI3K pathway inhibitors effected dephosphorylation of RPS6 in imatinib-resistant cell lines suggesting that an oncogene other than <it>BCR-ABL1 </it>might be responsible for activation of the PI3K/AKT1/mTOR pathway, which would explain the TKI resistance of these cells. We show that the TKI-resistant cell line KCL-22 carries a PI3Kα E545G mutation, a site critical for the constitutive activation of the PI3K/AKT1 pathway. Apoptosis in TKI-resistant cells could be induced by inhibition of AKT1, but not of mTOR.</p> <p>Conclusion</p> <p>We introduce five Philadelphia-chromosome positive cell lines as TKI-resistance models. None of these cell lines carries mutations in the kinase domain of <it>BCR-ABL1 </it>or other molecular aberrations previously indicted in the context of imatinib-resistance. These cell lines are unique as they dephosphorylate ERK1/2 and STAT5 after treatment with imatinib, while PI3K/AKT1/mTOR activity remains unaffected. Inhibition of AKT1 leads to apoptosis in the imatinib-resistant cell lines. In conclusion, Ph+ cell lines show a form of imatinib-resistance attributable to constitutive activation of the PI3K/AKT1 pathway. Mutations in <it>PIK3CA</it>, as observed in cell line KCL-22, or PI3K activating oncogenes may undelie TKI-resistance in these cell lines.</p

Distance learning in higher education during COVID-19: The role of basic psychological needs and intrinsic motivation for persistence and procrastination–a multi-country study

Due to the COVID-19 pandemic, higher educational institutions worldwide switched to emergency distance learning in early 2020. The less structured environment of distance learning forced students to regulate their learning and motivation more independently. According to self-determination theory (SDT), satisfaction of the three basic psychological needs for autonomy, competence and social relatedness affects intrinsic motivation, which in turn relates to more active or passive learning behavior. As the social context plays a major role for basic need satisfaction, distance learning may impair basic need satisfaction and thus intrinsic motivation and learning behavior. The aim of this study was to investigate the relationship between basic need satisfaction and procrastination and persistence in the context of emergency distance learning during the COVID-19 pandemic in a cross-sectional study. We also investigated the mediating role of intrinsic motivation in this relationship. Furthermore, to test the universal importance of SDT for intrinsic motivation and learning behavior under these circumstances in different countries, we collected data in Europe, Asia and North America. A total of N = 15,462 participants from Albania, Austria, China, Croatia, Estonia, Finland, Germany, Iceland, Japan, Kosovo, Lithuania, Poland, Malta, North Macedonia, Romania, Sweden, and the US answered questions regarding perceived competence, autonomy, social relatedness, intrinsic motivation, procrastination, persistence, and sociodemographic background. Our results support SDT’s claim of universality regarding the relation between basic psychological need fulfilment, intrinsic motivation, procrastination, and persistence. However, whereas perceived competence had the highest direct effect on procrastination and persistence, social relatedness was mainly influential via intrinsic motivation.</p