A proteomic study on off-target effects of therapeutic oligonucleotides

Ein Schwerpunkt der modernen pharmazeutischen Forschung liegt in der Erforschung sicherer und effizienter Methoden zur Hemmung bestimmter Gene im menschlichen Körper, die mit schwerwiegenden Krankheiten in Verbindung gebracht werden. In diesem Zusammenhang haben sogenannte „gene-silencing“ Therapeutika, also Substanzen, die spezifische Gensequenzen blockieren können, wie zum Beispiel Antisense-Oligonukleotide (ASOs) oder kleine hemmende RNA-Strukturen (siRNAs), großes Potential als potentielle neue Wirkstoffe. Trotz vielversprechender Ansätze und bisher einer Substanz dieser Klasse auf dem Markt, ist die erfolgreiche klinische Entwicklung dieser Therapeutika noch mit zahlreichen offenen Fragestellungen behaftet, wie etwa hinsichtlich der Stabilität, der Aufnahme in die entsprechenden kranken Zellen sowie unspezifischer Effekte mancher Substanzen. Die meisten Antisense-Oligonukleotide, die in höheren klinischen Studien getestet werden, gehören zur Gruppe der Phosphorthioate, wie zum Beispiel Oblimersen (Genasense, G3139), das gegen verschiedene Tumorarten, unter anderem gegen Melanome, getestet wird. Dieses 18 Basenpaar lange Oligonukleotid hemmt spezifisch ein Protein namens Bcl-2, das eine Schlüsselfunktion in der mitochondrialen Apoptose, also im programmierten Zelltod, spielt. Der genaue Wirkmechanismus von Oblimersen ist noch nicht völlig geklärt. Es wird aber davon ausgegangen, dass die Bcl-2 Hemmung nicht der einzige Mechanismus ist, mit dem Oblimersen eine (Re-)Sensibilisierung von Melanomzellen gegenüber Chemotherapie erreicht [Stein et al., 2009]. Das Hauptziel dieser Arbeit bestand darin, potentielle unspezifische Effekte am Proteom von Melanomzellen, verursacht durch Oblimersen und durch eine ebenfalls gegen Bcl-2 gerichtete siRNA, mit Hilfe einer validierten Proteomuntersuchung dar zu stellen und zu vergleichen. Benötigte ASOs und siRNAs wurden synthetisiert. Anschließend wurde die humane Melanomzelllinie 607B, die das anti-apoptotische Zielprotein Bcl-2 über-exprimiert, damit transfiziert. Obwohl beide Sequenzen das Zielprotein effektiv herunter regulierten, konnte nach 48 Stunden Transfektion nur bei Oblimersen-behandelten Zellen eine Apoptoseinduktion festgestellt werden. Basierend auf derselben 75 prozentigen Bcl-2 Hemmung, wurden die unterschiedlich behandelten Zelllysate mittels zwei-dimensionaler Gelelektrophorese und anschließender Software-gestützter Bildanalyse untersucht. Dazu mußten im Vorfeld verschiedene Parameter, unter anderem die Auftrennungs- und Färbebedingungen, optimiert und die bioanalytischen Methoden validiert werden. Da die verwendete Zelllinie auf Proteinebene noch nicht charakterisiert worden war, wurde eine Referenzkarte des Proteommusters erstellt. Mit Hilfe der DIGE Technologie konnte eine Beeinflussung der Genexpression von 22 Proteinen nach der Behandlung mit Oblimersen und von 2 Proteinen nach der Behandlung mit der siRNA festgestellt werden. Dies deutet darauf hin, dass die verwendete siRNA target-spezifischer wirkt als das Phosphorthioat. Die in ihrer Expression beeinflußten Proteine wurden im Anschluß mittels „in-gel“ Verdau und MALDI-rTOF-MS eindeutig identifiziert. Unter ihnen befanden sich Zytoskelett-Proteine, Chaperone, Transportproteine, glykolytische und andere Enzyme, Proteine, die in die Ca2+-Regulierung oder in die Redox-Regulierung der Zelle involviert waren, und auch andere Proteine, wie zum Beispiel Galectin-1. Viele dieser Proteine stehen mit Apoptose oder Therapie-Resistenzentwicklung in Verbindung. Das interessanteste Ergebnis der Studie war jedoch die beobachtete Expressionsreduktion glykolytischer Enzyme nach der Behandlung mit Oblimersen, welche auf eine teilweise Umkehr des sogenannten Warburg Effekts hinwies [Warburg et al., 1930]. In derselben Proteomuntersuchung wurden ähnliche Ergebnisse mit Phosphorthioat-modifizierten Kontrollsequenzen erreicht, die gegen andere, nicht mit Apoptose in Zusammenhang stehende, Gene gerichtet waren. Die präsentierten Ergebnisse unterstützen die Theorie, dass die bekannte Bcl-2 Reduktion nicht der einzige essentielle Wirkungsmechanismus von Oblimersen ist. Eine unspezifische Apoptoseinduktion, die wahrscheinlich durch eine Blockade des VDAC-Proteins („voltage-dependent anion channel“) [Stein et al., 2008] durch das Phosphorthioat verursacht wird, dürfte eine weitere entscheidende Rolle spielen. Da die erwähnte teilweise Umkehr des Warburg Effektes auch mit den Kontrolloligonukleotiden beobachtet wurde, ist es sehr wahrscheinlich, dass diese durch die Phosphorthioatmodifikation und nicht nur durch die Bcl-2 Hemmung verursacht wird.One focus of modern pharmaceutical research is the investigation of safe and efficacious ways of specifically silencing important targets that have been identified to play key roles in severe diseases. In this context gene silencing approaches, including antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), have great potential as therapeutics. Despite this promising concept and the success of so far one approved product, the clinical development of antisense agents is challenged with issues regarding site-specific delivery, stability and off-target effects. Most antisense agents in late-stage clinical development belong to the group of phosphorothioates, such as Oblimersen (Genasense, G3139). The 18mer phosphorothioate is directed against Bcl-2, a key protein of the mitochondrial apoptotic pathway, and is clinically tested against a variety of different cancers including melanoma. The working mechanism of Oblimersen is poorly understood, since Bcl-2 silencing is supposed not to be the sole mechanism for chemosensitization in melanoma cells [Stein et al., 2009]. The aim of this work was the establishment of a validated proteomic approach in order to monitor and compare potential off-target effects of the phosphorothioate Oblimersen and a bcl-2-targeted siRNA on the protein level. Required ASOs and siRNAs were synthesized and transfected to the human melanoma cell line 607B, which over-expressed the anti-apoptotic target protein Bcl-2. Although both sequences successfully down-regulated Bcl-2, only Oblimersen induced apoptosis in 607B cells after 48 hours of transfection as determined by annexin staining and caspase assay. Based on an equal target down-regulation of 75%, differently treated cell lysates were investigated by two-dimensional gel electrophoresis and subsequent software-based image analysis. Therefore optimization of two-dimensional separation and staining conditions, as well as the validation of bioanalytical methods had to be performed in order to obtain reliable quantitative proteomic data. Since the used cell line had not been characterized on the protein level before, a protein reference map was established. The differential expression of 22 proteins after Oblimersen-treatment and of 2 proteins after siRNA-treatment, monitored by DIGE technology, suggested a higher target specificity for the used siRNA compared to the tested phosphorothioate. Changed proteins, which were unambiguously identified by in-gel digestion and subsequent MALDI-rTOF-MS, included cytoskeletal proteins, chaperons, transport proteins, glycolytic and other enzymes, proteins involved in Ca2+-signaling and in redox-regulation as well as other proteins, such as galectin-1. Many of these proteins are linked to apoptosis or drug resistance. The most interesting result, however, was the observed reduction in the expression of glycolytic enzymes after Oblimersen treatment, indicating a partial reversal of the Warburg effect [Warburg et al., 1930]. Using the same proteomic approach similar effects were obtained with control phosphorothioate-modified oligonucleotides targeted at genes not involved in apoptosis. Presented findings support the theory that the Bcl-2 reduction is not the only essential molecular effect of Oblimersen. An unspecific apoptosis induction, possibly caused by a blockage of the voltage-dependent anion channel (VDAC) [Stein et al., 2008] by the phosphorothioate, seems to play a further key role. Since the discussed partial reversal of the Warburg effect has been observed also with control sequences, it seems to be caused by the phosphorothioate chemistry and not exclusively by bcl-2 targeted gene silencing

Auseinandersetzung mit der Entnazifizierung in der Memoirenliteratur

Die Auseinandersetzung mit der Entnazifizierung in der Memoirenliteratur. Wie ging der Entnazifizierungsprozess realpolitisch über die Bühne, bzw. welchen Zeitraum nahm er in Anspruch? Welche Rolle spielte dabei der Umstand, dass Österreich zumindest am Beginn der Nachkriegszeit nicht über volle politische Souveränität verfügte (Besatzungszonen)? Angeblich wurden die ehemaligen Eliten ausgetauscht, aber war das praktisch überhaupt möglich, wenn man den wirtschaftlichen Aufstieg des Landes nicht gefährden wollte? Der erste Abschnitt widmet sich der Geschichte und Kontinuität der politischen Lager Österreichs und greift damit ein Thema auf, das sich auch in der Memoirenliteratur inhaltlich widerspiegelt. Daran anschließend werden besonders exponierte Persönlichkeiten der verwendeten Memoirenliteratur verschiedener politischer Richtungen – darunter auch die Gründungsmitglieder des VdU – näher vorgestellt. In Folge soll ein Überblick über die Entnazifizierungsmaßnahmen in Österreich gegeben werden, dabei sollen die Probleme hinsichtlich der juristischen Beschlussfassung und der realpolitischen Durchführung der Entnazifizierungsmaßnahmen näher durchleuchtet werden. Das vierte Kapitel beschäftigt sich mit der Gründung des VdU und der sich daraus entwickelnden politischen Dynamik im Hinblick auf die Wiederintegration ehemaliger Nationalsozialisten. Am Ende der Arbeit werden die Schwerpunkte und Perspektiven der ausgewählten Memoiren nochmals zusammenfassend resümiert, um etwaige Tendenzen herauszufiltern. Der Zeitrahmen dieser Arbeit erstreckt sich von 1945 bis 1956, also von der Gründung der Provisorischen Regierung Renner bis zu der Übernahme des VdU durch die den nationalen Flügel in der Partei und der Gründung der FPÖ

Tn6188 - A Novel Transposon in Listeria monocytogenes Responsible for Tolerance to Benzalkonium Chloride

peer-reviewedControlling the food-borne pathogen Listeria (L.) monocytogenes is of great importance from a food safety perspective, and thus for human health. The consequences of failures in this regard have been exemplified by recent large listeriosis outbreaks in the USA and Europe. It is thus particularly notable that tolerance to quaternary ammonium compounds such as benzalkonium chloride (BC) has been observed in many L. monocytogenes strains. However, the molecular determinants and mechanisms of BC tolerance of L. monocytogenes are still largely unknown. Here we describe Tn6188, a novel transposon in L. monocytogenes conferring tolerance to BC. Tn6188 is related to Tn554 from Staphylococcus (S.) aureus and other Tn554-like transposons such as Tn558, Tn559 and Tn5406 found in various Firmicutes. Tn6188 comprises 5117 bp, is integrated chromosomally within the radC gene and consists of three transposase genes (tnpABC) as well as genes encoding a putative transcriptional regulator and QacH, a small multidrug resistance protein family (SMR) transporter putatively associated with export of BC that shows high amino acid identity to Smr/QacC from S. aureus and to EmrE from Escherichia coli. We screened 91 L. monocytogenes strains for the presence of Tn6188 by PCR and found Tn6188 in 10 of the analyzed strains. These isolates were from food and food processing environments and predominantly from serovar 1/2a. L. monocytogenes strains harboring Tn6188 had significantly higher BC minimum inhibitory concentrations (MICs) (28.5 ± 4.7 mg/l) than strains without Tn6188 (14 ± 3.2 mg/l). Using quantitative reverse transcriptase PCR we could show a significant increase in qacH expression in the presence of BC. QacH deletion mutants were generated in two L. monocytogenes strains and growth analysis revealed that ΔqacH strains had lower BC MICs than wildtype strains. In conclusion, our results provide evidence that Tn6188 is responsible for BC tolerance in various L. monocytogenes strains.This work was supported by a grant from the Austrian Science Fund (FWF, http://www.fwf.ac.at/) to SSE (grant no. P22703‐B17), by the European Union funded integrated project BIOTRACER (contract no. 036272) under the 6th RTD framework and by the EU grant FP7‐KBBE‐2010‐4 (FOODSEG)

Molecular Epidemiology of Invasive Listeriosis due to Listeria monocytogenes in a Spanish Hospital over a Nine-Year Study Period, 2006–2014

We investigated the pathogenicity, invasiveness, and genetic relatedness of 17 clinical Listeria monocytogenes stains isolated over a period of nine years (2006–2014). All isolates were phenotypically characterised and growth patterns were determined. The antimicrobial susceptibility of L. monocytogenes isolates was determined in E-tests. Invasion assays were performed with epithelial HeLa cells. Finally, L. monocytogenes isolates were subtyped by PFGE and MLST. All isolates had similar phenotypic characteristics (β-haemolysis and lecithinase activity), and three types of growth curve were observed. Bacterial recovery rates after invasion assays ranged from 0.09% to 7.26% (1.62 ± 0.46). MLST identified 11 sequence types (STs), and 14 PFGE profiles were obtained, indicating a high degree of genetic diversity. Genetic studies unequivocally revealed the occurrence of one outbreak of listeriosis in humans that had not previously been reported. This outbreak occurred in October 2009 and affected three patients from neighbouring towns. In conclusion, the molecular epidemiological analysis clearly revealed a cluster (three human cases, all ST1) of not previously reported listeriosis cases in northwestern Spain. Our findings indicate that molecular subtyping, in combination with epidemiological case analysis, is essential and should be implemented in routine diagnosis, to improve the tracing of the sources of outbreaks.Gerencia Regional de Salud, Junta de Castilla y Le´on, Spain, research project GRS 698/A/2011 and the EU 7th Framework Programme through the PROMISE project (Project no. 265877)

Editorial: Technologies to address risk assessment, food safety and public health in food production chain

Access to sufficient, safe and nutritious food is a basic human right and is referred to as food security. To ensure this right, governments should develop and support effective and efficient prevention and control strategies along the food production chain. This should be based on integrated approach, from farm-to-fork, supported by risk assessment, risk-based food safety management and risk communication to consumers on major public health hazards. The continuous and rapid development of new technologies to detect and control food safety hazards, together with the introduction of digital innovations in the food chain, mark the 4th industrial revolution. The food chain is not exempted from these changes. In light of food security needs and current global challenges, such as population growth and urbanization, international livestock and food trade, climate change, biodiversity loss, decrease of arable land, food waste, and greenhouse gas emissions there is a strong need to transform agri-food systems. Such transformation should enable a more climate resilient and sustainable food value chain

Assessment of microbial quality in poultry drinking water on farms in Austria

The quality of poultry drinking water has a significant effect on broiler health and performance. This study conducted an analysis of aerobic mesophilic counts (AMC), Enterobacteriaceae (EB), Pseudomonadaceae (PS), and screened for the presence of Campylobacter spp. in water samples collected from a total of 14 farms in Austria, with either a public or private water source. The efficacy of two water line treatment methods was evaluated: a chemical treatment of the water lines with 4.0 ppm ClO2 (T1) and a combined chemical (4.0 ppm active ClO2 and 3.0% peracetic acid) and mechanical treatment (purging of the water lines with a high-pressure air pump; T2). However, both the T1 and T2 treatments failed to reduce the AMC counts below the maximum acceptable microbial limit of 4.0 log10 CFU/ml in water samples. In addition, no significant reduction in EB and PS counts was observed in water samples after either T1 or T2 water line treatment. The water samples showed a high level of microbial diversity with 18 to 26 different genera. The genus Pseudomonas was most frequently isolated across all poultry farms, while Campylobacter jejuni was identified in a single sample collected before water line treatment. Isolate analysis revealed the presence of opportunistic pathogens in water samples both before (T1 43.1%, T2 30.9%) and after (T1 36.3%, T2 33.3%) water line treatment. Opportunistic pathogens belonging to genera including Pseudomonas spp., Stenotrophomonas spp., and Ochrobactrum spp., were most frequently isolated from poultry drinking water. These isolates exhibited multidrug resistance and resistance phenotypes to antimicrobials commonly used in Austrian poultry farms. The findings of this study emphasize the potential risk of exposure to opportunistic pathogens for poultry and personnel, underscoring the importance of efficient water line management

Detection of crenosoma spp., angiostrongylus vasorum and aelurostrongylus abstrusus in gastropods in Eastern Austria

Canine and feline cardiorespiratory parasites are of utmost relevance in veterinary medicine. Key epizootiological information on major pet metastrongyloids, i.e., Angiostrongylus vasorum and Crenosoma vulpis infecting dogs, and Aelurostrongylus abstrusus and Troglostrongylus brevior infecting cats, is missing from Austria. This study investigated their occurrence in 1320 gastropods collected in the Austrian provinces of Styria, Burgenland, Lower Austria, and in metropolitan Vienna. Metastrongyloid larvae were microscopically detected in 25 samples, and sequence analysis confirmed the presence of metastrongyloids in nine samples, i.e., A. vasorum in one slug (Arion vulgaris) (0.07%), C. vulpis in five slugs (one Limax maximus and four A. vulgaris) (0.4%), A. abstrusus in two A. vulgaris (0.17%), and the hedgehog lungworm Crenosoma striatum was detected in one A. vulgaris. The present study confirms the enzooticity of major cardiorespiratory nematodes in Austria and that canine and feline populations are at risk of infection