Analyzed the data: MVA NAA-D VD CM. Wrote the manuscript: MVA NAA-D VD SJK. All authors read and approved the final manuscript.”
“Background

Avian influenza remains a serious threat to poultry and human health. From December 2003 to April 2013, more than 600 human infections and 374 deaths have been reported to the World Health Organization [1]. Outbreaks of H5N1 in poultry swept from EPZ015938 molecular weight Southeast Asia to many parts of the world. To date, there is still no sign that the epidemic is under control. While it has been well documented that infection with H5N1 results in high mortality in humans [2–5], the cellular pathway leading to such adverse outcome is unknown. Vorinostat supplier The naive host immune system cannot be the sole explanation as infection of other avian influenza viruses, e.g. H9N2, only results in mild infections [6]. While the predilection of H5N1

towards cells in the lower respiratory tract contributes to the development of severe pneumonia [7], the available clinico-pathological evidence indicates that the infected patients progress to multi-organ failure early in the course of illness, and the CRT0066101 in vitro degree of organ failure is out of proportion to the involvement of infection [8–10]. Cytokine storm and reactive haemophagocytic syndrome are the key features that distinguish H5N1 infection from severe seasonal influenza. These indirect mechanisms seem to play an even more important role than direct cell killing due to lytic viral infection. MiRNAs, a new class of endogenous, 18–23 nucleotide long noncoding and single-stranded RNAs, were recently discovered in both animals and plants. They trigger translational repression and/or mRNA degradation mostly through complementary binding to the 3′UTR of target mRNAs. Studies have shown that miRNAs can regulate a wide array of biological processes such as cell proliferation, differentiation, and apoptosis [11–14]. Given the nature of viruses,

being intracellular parasites and using Phosphatidylethanolamine N-methyltransferase the cellular machinery for their survival and replication, the success of the virus essentially depends on its ability to effectively and efficiently use the host machinery to propagate itself. This dependence on the host also makes it susceptible to the host gene-regulatory mechanisms, i.e. the host miRNAs may also have direct or indirect regulatory role on viral mRNAs expression. Recently, several reports indicated that miRNAs can target influenza viruses and regulate influenza virus replication. In one report, 36 pig-encoded miRNAs and 22 human-encoded miRNAs were found to have putative targets in swine influenza virus and Swine-Origin 2009 A/H1N1 influenza virus genes, respectively [15]. In another report, results showed that miR-323, miR-491 and miR-654 could inhibit replication of H1N1 influenza A virus through binding to the conserved region of the PB1 gene [16]. These miRNAs could downregulate PB1 expression through mRNA degradation instead of translation repression [16].

monocytogenes strains without the need for additional genetic manipulations to introduce the nisRK genes into the chromosome of each strain. Plasmid pAKB, a derivative of plasmid pNZ8048 carrying the nisA promoter, was constructed for the planned overexpression experiments. To construct this plasmid, BB-94 solubility dmso a cassette comprised of the nisRK genes cloned downstream of the L. monocytogenes hly promoter was introduced into pNZ8048 to ensure efficient expression of these genes in L. monocytogenes [15]. The lmo1438 gene was then cloned downstream

of the Pnis promoter in pAKB to produce plasmid pAKB-lmo1438. Before starting the experiments on overexpression of the lmo1438 gene, the susceptibility of L. monocytogenes to nisin was examined, since nisin is an inducer of the NICE system but it can affect or inhibit the growth of L. monocytogenes when used at high concentrations. The level of nisin required to completely inhibit the growth of L. monocytogenes EGD and of L. monocytogenes carrying the pAKB plasmid lacking an Necrostatin-1 ic50 insert (used as a negative control in subsequent experiments) was over ten times higher than the concentration used previously VX-680 purchase to induce

the NICE system in L. monocytogenes [15]. Furthermore, growth curves were plotted for L. monocytogenes pAKB grown in the presence of different concentrations of nisin as well as in the absence of this inducer to determine the concentration of nisin that has no effect on growth. These preliminary experiments showed that 15 μg/ml was the maximum concentration of nisin that did not cause any changes in the growth rate of the control strain. At higher nisin concentrations, including that used previously (45 Florfenicol μg/ml) to induce NICE in L. monocytogenes [15], a slight reduction in the growth rate of L. monocytogenes pAKB was observed during the exponential phase, compared to growth in the absence of nisin. The differences between the optimal

nisin concentrations for growth and induction determined here and those established by Cotter et al. [15] may be due to the differential susceptibility of the strains EGD and LO28 to this peptide. To confirm that nisin induced overexpression of the lmo1438 gene in L. monocytogenes pAKB-lmo1438, the cell membrane proteins of this strain and the control strain were analyzed. SDS-PAGE of isolated membrane proteins revealed the presence of an additional protein in L. monocytogenes pAKB-lmo1438 grown in the presence of 15 μg/ml nisin (Figure 1). The estimated mass of this additional protein was approximately 80 kDa, which corresponds to the predicted mass of Lmo1438 (79.9 kDa). The additional protein was detected at both 2 and 24 h following induction, but it was not observed when L. monocytogenes pAKB-lmo1438 was grown in the absence of nisin (data not shown). Figure 1 Overexpression of the lmo1438 gene in L. monocytogenes. Membrane proteins were isolated from L. monocytogenes pAKB (lane 1) and L.