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Streptococcus pyogenes (group A Streptococcus, GAS) is a human pathogen that causes diseases of various intensity, from mild strep throat to life threatening invasive infections and postinfectional sequelae. S. pyogenes encodes multiple, often phage encoded, virulence factors and their presence is related to severity of the disease. Acquisition of mobile genetic elements, carrying virulence factors, as phages or ICEs (integrative and cojugative elements) has been shown previously to promote selection of virulent clones. We designed the system of eight low volume multi- and one singleplex PCR reactions to detect genes encoding twenty virulence factors (spd3, sdc, sdaB, sdaD, speB, spyCEP, scpA, mac, sic, speL, K, M, C, I, A, H, G, J, smeZ and ssa) and twenty one phage and ICE integration sites described so far for S. pyogenes. Classification of strains based on the phage and virulence factors absence or presence, correlates with PFGE MLST and emm typing results. We developed a novel, fast and cost effective system that can be used to detect GAS virulence factors. Moreover, this system may become an alternative and effective system to differentiate between GAS strains.
INTRODUCTION: Epilepsy is one of the most common neurological disorders in humans. Precise pathogenesis of epilepsy is complex and unclear. The Matrix Metalloproteinase-9 (MMP-9) is a proepileptic protein involved in a formation of aberrant brain neuronal networks during epileptogenesis, what finally leads to the development of seizures. Despite of its essential role in etiology of epilepsy, regulation of the MMP-9 expression during epileptogenesis is almost unknown. Similarly, completely obscured is a dependence of the MMP-9 expression on the mRNA stabilization mechanisms in the epilepsy. AIM(S): Our goal was to determine mechanisms responsible for the MMP-9 mRNA stability changes occurring in the rat hippocampus during epileptogenesis. METHOD(S): We used two models to study the mRNA stabilization-dependent regulation of MMP-9 during epileptogenesis: the pentylenotetrazole (PTZ)-dependent kindling in rats (in vivo pharmacological model of epileptogenesis) and the generation of the spontaneous recurrent epileptform discharges (SREDs) in cultured rat hippocampal neurons (in vitro model of epilepsy). RESULTS: Considering the MMP-9 mRNA expression profile and results obtained using the RNA degradation assay, we observed significant stabilization of the MMP-9 mRNA during epileptogenesis, and corresponding to this phenomenon, a gradual upregulation of its hippocampal mRNA expression during epileptogenesis. Interestingly, our data collected with REMSA supershift assays, RIPA, protein mass spectrometry as well as functional HuR overexpression and depletion studies have showed that HuR directly binds to the ARE1 and ARE4 sites in the 3’UTR of MMP-9 mRNA and therefore stabilize MMP-9 mRNA. CONCLUSIONS: The epileptogenesis-evoked upregulation of MMP-9 expression in the rat hippocampus is clearly and strongly dependent on its mRNA stabilization mediated by HuR action related to its direct binding to the ARE1 and ARE4 sites in the 3’UTR of MMP-9 mRNA. FINANCIAL SUPPORT: This work was supported by the Polish National Science Centre grant no. 2012/05/B/ N23/01943.
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