The non-structural protein 3 (NS3) of hepatitis C virus (HCV) is a highly promising target for anti-HCV therapy because of its multiple enzymatic activities, such as RNAstimulated nucleoside triphosphatase, RNA helicase and serine protease. The helicase domain of NS3 as well as domain 2 of the helicase were expressed in a baculovirus system to obtain in high yield active proteins for prospective studies of complexes of the helicase with its inhibitors. A novel direct fluorometric test of helicase activity with a quenched DNA substrate, 3 0 labeled with a Cy3 dye and 5 0 labeled with a Black Hole Quencher, was developed and optimal reaction conditions established. This test based on fluorescence resonance energy transfer is simple and fast. It allows for direct measurements of enzyme activity, circumventing laborious and complicated radioactive techniques that are poorly reproducible. The results obtained encourage us to propose this new fluorescent assay as a method enabling high throughput screening of anti-helicase compounds.

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Enterobacter aerogenes LU2 was isolated from cow rumen and recognized as a potential succinic acid producer in our previous study. Here, we present the first complete genome sequence of this new, wild strain and report its basic genetic features from a biotechnological perspective. The MinION singlemolecule nanopore sequencer supported by the Illumina MiSeq platform yielded a circular 5,062,651 bp chromosome with a GC content of 55% that lacked plasmids. A total of 4,986 genes, including 4,741 protein-coding genes, 22 rRNA-, 86 tRNA-, and 10 ncRNA-encoding genes and 127 pseudogenes, were predicted. The genome features of the studied strain and other Enterobacteriaceae strains were compared. Functional studies on the genome content, metabolic pathways, growth, and carbon transport and utilization were performed. The genomic analysis indicates that succinic acid can be produced by the LU2 strain through the reductive branch of the tricarboxylic acid cycle (TCA) and the glyoxylate pathway. Antibiotic resistance genes were determined, and the potential for bacteriocin production was verified. Furthermore, one intact prophage region of length ~31,9 kb, 47 genomic islands (GIs) and many insertion sequences (ISs) as well as tandem repeats (TRs) were identified. No clustered regularly interspaced short palindromic repeats (CRISPRs) were found. Finally, comparative genome analysis with well-known succinic acid producers was conducted. The genome sequence illustrates that the LU2 strain has several desirable traits, which confirm its potential to be a highly efficient platform for the production of bulk chemicals. Enterobacter aerogenes LU2 is a gram-negative, wild bacterium that was isolated from cow rumen as a part of environmental screening for succinic acid-producing bacteria identification. Succinic acid (SA) plays an important role as a metabolic intermediate in the rumen by increasing propionate production, a crucial energy source for the ruminant 1,2. Anaerobic conditions and the presence of carbon dioxide, methane and trace amounts of hydrogen create an excellent environment for the biosynthesis of succinate by some bacteria existing in the rumen 3. In reports of the U.S. Department of Energy (DOE) from 2004 and 2010, SA was recognized as one of the top 10 most promising C4-building chemical platforms for the production of high-value commodity and specialty chemicals with great industrial potential 2,4. Succinate is widely applied as an additive in food, pharmaceuticals, detergents, solvents, and surfactants as well as in biodegradable polymer production 5,6. Until recently, industrial production of succinate was based on chemical synthesis. However, petroleum-based production of SA from n-butane through maleic anhydrate requires the use of high pressure, high temperature and costly catalysts 7. Therefore, because of the current global trend regarding sustainable development, including the support of green technologies, rational waste biomass management and pollution-reducing standards, the bio-based production of...

Th17 cells are important players in host defense against pathogens such as Staphylococcus aureus, Candida albicans, and Bacillus anthracis. Th17 cell-mediated inflammation, under certain conditions in which balance in the immune system is disrupted, is the underlying pathogenic mechanism of certain autoimmune disorders, e.g., rheumatoid arthritis, Graves’ disease, multiple sclerosis, and psoriasis. In the present study, using transcriptomic profiling, we selected genes and analyzed the expression of these genes to find potential novel markers of Th17 lymphocytes. We found that APOD (apolipoprotein D); C1QL1 (complement component 1, Q subcomponent-like protein 1); and CTSL (cathepsin L) are expressed at significantly higher mRNA and protein levels in Th17 cells than in the Th1, Th2, and Treg subtypes. Interestingly, these genes and the proteins they encode are well associated with the function of Th17 cells, as these cells produce inflammation, which is linked with atherosclerosis and angiogenesis. Furthermore, we found that high expression of these genes in Th17 cells is associated with the acetylation of H2BK12 within their promoters. Thus, our results provide new information regarding this cell type. Based on these results, we also hope to better identify pathological conditions of clinical significance caused by Th17 cells.

Hepatitis C virus (HCV) infections represent one of the major and still unresolved health problems because current therapy is effective in only 50-80% of cases, depending on viral genotype. A large group of amidinoanthracyclines, with decreased acute toxicity and cardiotoxicity compared to the parent antibiotics, was tested in a high-throughput fluorometric HCV helicase assay. Here, we report the selection of more than 50 potent inhibitors of helicase activity that inhibit the enzyme with IC(50) values in the range of 0.03- 10 mum; four of these compounds are the most potent inhibitors of helicase activity described in the literature. The activity of these inhibitors is highly dependent on their chemical structure, mainly on the substituent at the amidino carbon atom and on the orientation of the hydroxyl group at the 4 inch position of the daunosamine moiety. The most effective compounds act not solely via intercalation into the double-stranded DNA substrate, but also compete with the enzyme for access to the substrate, impeding formation of the active helicase complex. Selected amidinoanthracyclines were tested in the subgenomic HCV replicon system. These experiments confirmed the antiviral activity of two selected inhibitors (EC(50) values below 0.2 mum with selectivity indices of 19 and 33) and proved that they may be considered as potential anti-HCV drugs.