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Multiple ssRNA viruses which infect bacteria, plants or humans use RNA Packaging Signal (PS)-mediated regulation during assembly to package their genomes faithfully and efficiently [1], [2], [3], [4], [5]. PSs typically comprise short nucleotide recognition motifs, most often presented in the unpaired region of RNA stem-loops, and often bind their cognate coat proteins (CPs) with nanomolar affinity. PSs identified to date are resilient in the face of the typical error prone replication of their virus-coded polymerases, making them potential drug targets [1], [2], [3]. An immobilised array of small molecular weight, drug-like compounds was panned [6] against a fluorescently-labelled oligonucleotide encompassing the most conserved Hepatitis B Virus (HBV) PS, PS1 [3], known to be a major determinant in nucleocapsid formation [7]. This identified > 70 compounds that bind PS1 uniquely in the array. The commercially available 66 of these were tested for their potential effect(s) on HBV nucleocapsid-like particle (NCP) assembly in vitro [8], which identified potent assembly inhibitors. Here, we describe a high-throughput screen for such effects using employing fluorescence anisotropy in a 96-well microplate format. HBV genomic RNAs (gRNA) and short oligonucleotides encompassing PS1 were 5ʹ labelled with an Alexa Fluor 488 dye. Excess (with respect to stoichiometric T=4 NCP formation [9]) HBV core protein (Cp) dimers were titrated robotically into solutions containing each of these RNAs stepwise, using a Biomek 4000 liquid handling robot. The anisotropy values of these mixtures were monitored using a POLARstar microplate reader. NCP-like structures were challenged with RNase A to identify reactions that did not result in complete NCP formation [10]. The results imply that ∼50 % of the compounds prevent complete NCP formation, highlighting both PS-meditated assembly and the PS-binding compounds as potential directly-acting anti-virals with a novel molecular target. Importantly, this method allows high-throughput in vitro screening for assembly inhibitors in this major human pathogen.

RNA sequences/motifs dispersed across the genome of Hepatitis B Virus regulate formation of nucleocapsid-like particles (NCPs) by core protein (Cp) in vitro, in an epsilon/polymerase-independent fashion. These multiple RNA Packaging Signals (PSs) can each form stem-loops encompassing a Cp-recognition motif, -RGAG-, in their loops. Drug-like molecules that bind the most important of these PS sites for NCP assembly regulation with nanomolar affinities, were identified by screening an immobilized ligand library with a fluorescently-labelled, RNA oligonucleotide encompassing this sequence. Sixty-six of these “hits”, with affinities ranging from low nanomolar to high micromolar, were purchased as non-immobilized versions. Their affinities for PSs and effects on NCP assembly were determined in vitro by Surface Plasmon Resonance. High-affinity ligand binding is dependent on the presence of an -RGAG- motif within the loop of the PS, consistent with ligand cross-binding between PS sites. Simple structure–activity relationships show that it is also dependent on the presence of specific functional groups in these ligands. Some compounds are potent inhibitors of in vitro NCP assembly at nanomolar concentrations. Despite appropriate logP values, these ligands do not inhibit HBV replication in cell culture. However, modelling confirms the potential of using PS-binding ligands to target NCP assembly as a novel anti-viral strategy. This also allows for computational exploration of potential synergic effects between anti-viral ligands directed at distinct molecular targets in vivo. HBV PS-regulated assembly can be dysregulated by novel small molecule RNA-binding ligands opening a novel target for developing directly-acting anti-virals against this major pathogen.

Background: Thyme (Thymus vulgaris L.) is a valuable medicinal plant which has many secondary metabolites such as thymol and carvacrol. Objective: This study was done to evaluation of cold stress effect on gene expression in thymol biosynthesis pathway including DXR، HMGR and TPS1. Methods: The effect of cold stress in the time period of 0, 3, 12, 24 and 48 hours on gene expression in thymol biosynthesis pathway including DXR, HMGR and TPS1 was evaluated by RT-PCR. This study was done on base of complete randomized design (CRD) in Institute of Medicinal Plants, ACECR, Karaj, Iran. Results: Results showed that the cold stress had significantly effect on expression of studied genes. The highest expression of HMGR gene was observed within 3 hours after cold treatment (1.57 times more than control treatment). The expression of DXR and TPS1 genes were reduced in different periods of cold stress. Conclusion: In general, the genes expression of thymol and carvacrol biosynthesis pathway including DXR, HMGR and TPS1 were significantly changed by cold stress.

Background: Distal radius fracture is a common fracture in elderly. It has well established that vitamin D deficiency and hip fractures are related. Some studies have claimed such a relation for distal radius fractures. Methods: In this matched case-control study conducted from June 2015 to April 2016, patients aged beyond 50 years presenting to Alzahra hospital, Isfahan, Iran, with a distal radius fracture were enrolled. The serum levels of 25(OH) vitamin D, calcium and phosphorus were measured and then data were compared to control group. Findings: During this study, 130 people including 65 cases and 65 controls in each group composed of 30 men and 35 women were studied. Mean level of serum vitamin D was significantly lower in case group; such a difference was also seen in affected men compared to control ones. There was no significant difference in vitamin D levels between case and control women. Mean serum levels of calcium and phosphorus were significantly higher in case group. Conclusion: The results show a direct correlation between low serum vitamin D level and the incidence of distal radius fractures. More studies are needed to assess whether the correction of this lack of vitamin D leads to a decrease in distal radius fracture and other future fractures.

Objectives: One of the most effective methods to describe speech disorders is the measurement of speech intelligibility. The speech intelligibility indicates the extent of acoustic signals that correctly speaker produces and hearer receives. The purpose of this study was to investigate the speech intelligibility in the Persian children with Down syndrome, age range was 3 to 5 years, who had spoken Persian. Methods: this cross- sectional study investigates 12 children (6 girls and 6 boys) with Down syndrome who had referred to speech therapy clinic in Hamadan city and 12 normal children (6 girls and 6 boys) who went to the kindergarten in Hamadan city. The pictures of speech intelligibility test (in Persian language) were used to collect speech samples of participants. The participant’s voice was recorded by voice recorder and was investigated in two age groups. Results: The results of this study indicated the means of speech intelligibility was 92.25 for normal children and 35.08 for children with Down syndrome. The correlation between age and speech intelligibility for normal children was 0.866 and for children with Down syndrome was 0.352. The mean of speech intelligibility 2 for normal boys was 93 and for normal girls 91.5 and for boys with Down syndrome 34.66 and for girls with Down syndrome 35.5. Discussion: The difference between normal children and children with Down syndrome was Significant. One of the factors that affects speech intelligibility for children with Down syndrome is difficulty with voluntarily programming, combining, organizing, and sequencing the movements necessary for speech.

Peripheral-Nerve-Society Meeting, Sitges, SPAIN, JUL 08-12, 2017; International audience

The human erythrocyte is a rich raw material for the purification of Cu-Zn superoxide dismutase (SOD). We applied a simple and rapid procedure for the purification of SOD from human erythrocytes by ion exchange chromatography. The purified SOD had a specific activity of 2285.6 u/mg protein and gave a single band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) and each of its to subunit has a molecular weight about 18600 daltons (SOD molecular weight is 37200 daltons).The physicochemical properties of the enzyme obtained by this method are identical to those of the native protein.This procedure appears, therefore, to be a convenient and easily method for isolating this enzyme.