Role of GNRA motif mutations within stem-loop V of internal ribosome entry segment in coxsackievirus B3 molecular attenuation

The lengthy 5' nontranslated region of coxsackievirus B3 (CVB3) forms a highly ordered secondary structure containing an internal ribosome entry segment (IRES), which plays an important role in controlling viral translation and pathogenesis. The stem-loop V (SL-V) of this IRES contains a large lateral bulge loop which encompasses two conserved GNRA motifs. In this study, we analyzed the effects of point mutations within the GNRA motifs of the CVB3 IRES. We characterized in vitro virus production and translation efficiency and we tested in vivo virulence of two CVB3 mutants produced by site-directed mutagenesis. The GNAA1 and GNAA2 RNAs displayed decreased translation initiation efficiency when translated in rabbit reticulocyte lysates. This translation defect was correlated with reduced yields of infectious virus particles in HeLa cells in comparison with the wild type. When inoculated orally into Swiss mice, both mutant viruses were avirulent and caused neither inflammation nor necrosis in hearts. These results highlight the important role of the GNRA motifs within the SL-V of the IRES of CVB3, in directing translation initiation.     

High-performance liquid chromatography determination of flunitrazepam and its metabolites in plasma by use of column-switching technique: comparison of two extraction columns

A study, using on-line column-switching high-performance liquid chromatography, evaluated two different extraction columns for the determination of flunitrazepam and its major metabolites: 7-aminoflunitrazepam, 7-acetamidoflunitrazepam and desmethylflunitrazepam. The procedure was based on the enrichment of benzodiazepines on the extraction column, followed by transfer of the compounds to the analytical column. The two extraction columns were compared: the first column was a BioTrap 500 MS (hydrophobic polymer), 20×4 mm I.D., and the second was a LiChrospher RP-18 ADS, 25×4 mm I.D. The analytical column used was a LiChrospher select B RP-8, 125×3 mm I.D. with 5 μm particle size. The extraction conditions for the two pre-concentration columns, such as extraction temperature, buffer concentration, buffer pH, acetonitrile percentage and flow-rate, were studied for the extraction from plasma of flunitrazepam and its metabolites mentioned above. The mobile phase of the analytical column was isocratic and composed of acetonitrile–20 mM phosphate buffer at pH 2.1 (35:65, v/v) and at a flow-rate of 0.3 ml/min.     

Toxic and mutagenic properties of extracts from Tunisian traditional medicinal plants investigated by the neutral red uptake, VITOTOX and alkaline comet assays

We investigated the genotoxic properties of a number of extracts from Tunisian traditional medicinal plants with the bacterial VITOTOX test in Salmonella typhimurium and the alkaline comet assay in human C3A cells. Ethyl acetate and methanol extracts from Marrubium alysson L. and Retama raetam (Forsk.) Webb and methanol extracts from Peganum harmala L. were investigated. Toxicity was furthermore studied with the neutral red uptake test that served for dose-finding.All extracts showed antigenotoxic properties against 4-nitroquinoline-oxide (4-NQO) and benzo(α)pyrene in the VITOTOX test, except the methanol extracts from R. raetam where antigenotoxicity was not found against the mutagen 4-NQO (in the absence of S9). The ethyl acetate extract from R. raetam was found mutagenic with the VITOTOX test in the absence of S9, whereas both ethylacetate and methanol extracts of M. alysson L. induced DNA damage according to the alkaline comet assay in C3A cells.     

One-dimensional ladder like and two-dimensional polymorphs of heterometallic thiocyanate bridged copper(II) and mercury(II) coordination polymer: Syntheses, structural, vibration, luminescence and EPR studies

The 1D triclinic {CuHg(en)(μ-NCS-N,S)₄}_n (1) and 2D monoclinic {CuHg(en)(μ-NCS-N,S)₃(SCN)}_n (2) (en = ethylenediamine) heterometallic coordination polymers, as the two polymorphs of the {CuHg(en)(NCS)₄}_n, were synthesized at room temperature by the reaction of mercuric thiocyanate, potassium thiocyanate, copper(II) malonate and ethylenediamine using different reagent ratios. XRD on single crystals shows that the compound 1 consists of 1D ladder like zigzag ribbons extended along the b axis, whereas compound 2 shows a 2D wavy polymeric structure running parallel to the ab plane. In the crystal packing of the both polymorphs, the polymeric structures are further interlinked to each other via weak interactions and hydrogen bonding to afford a 3D network. Diagnostic ligand and metal-ligand bands in the IR, far-IR and Raman spectra are assigned for the studied compounds. While compound 1 shows no significant emission upon excitation at any wavelength in the UV-Vis region, compound 2 exhibits intense emission at around 410 nm. Moreover, the room temperature X-band EPR spectrum of a powdered sample of 1, shows a signal of rhombic symmetry with g₁ = 2.2637, g₂ = 2.0765 and g₃ = 2.0483. In contrast to this, 2 reveals an axial signal with g_⊥ = 2.0742; however, the g_|| is unresolved.     

Supernumerary centrosomes nucleate extra cilia and compromise primary cilium signaling

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Highlights? Supernumerary centrosomes nucleate cilia, resulting in superciliated cells (SCC) ? Ciliary proteins are reduced in concentration in SCC, compromising signaling ? Presence of extra cilia disrupts epithelial organization in 3D spheroid culture ? Extra cilia may contribute to disease phenotypes by disrupting signaling     

A Highlights from MBoC Selection: Ccdc11 is a novel centriolar satellite protein essential for ciliogenesis and establishment of left–right asymmetry

The establishment of left–right (L-R) asymmetry in vertebrates is dependent on the sensory and motile functions of cilia during embryogenesis. Mutations in CCDC11 disrupt L-R asymmetry and cause congenital heart disease in humans, yet the molecular and cellular functions of the protein remain unknown. Here we demonstrate that Ccdc11 is a novel component of centriolar satellites—cytoplasmic granules that serve as recruitment sites for proteins destined for the centrosome and cilium. Ccdc11 interacts with core components of satellites, and its loss disrupts the subcellular organization of satellite proteins and perturbs primary cilium assembly. Ccdc11 colocalizes with satellite proteins in human multiciliated tracheal epithelia, and its loss inhibits motile ciliogenesis. Similarly, depletion of CCDC11 in Xenopus embryos causes defective assembly and motility of cilia in multiciliated epidermal cells. To determine the role of CCDC11 during vertebrate development, we generated mutant alleles in zebrafish. Loss of CCDC11 leads to defective ciliogenesis in the pronephros and within the Kupffer’s vesicle and results in aberrant L-R axis determination. Our results highlight a critical role for Ccdc11 in the assembly and function of motile cilia and implicate centriolar satellite–associated proteins as a new class of proteins in the pathology of L-R patterning and congenital heart disease.     

Antimicrobial Activity of Scabiosa arenaria Forssk. Extracts and Pure Compounds Using Bioguided Fractionation

The emergence of multidrug resistant pathogens threatened the clinical efficacy of many existing antibiotics. This situation has been recognized globally as a serious concern and justifies further research to discover antimicrobial agents from natural origins including plant extracts. The aim of our work was to evaluate the antimicrobial activities of Scabiosa arenaria Forssk . extracts and pure compounds using a bioguided fractionation, and try to explain some traditional use of this genus. The best antimicrobial activity‐guided fractionation was obtained by BuOH fractions of flowers, fruits and (stems and leaves) against Escherichia coli, Pseudomonas aeruginosa and Candida albicans with minimum inhibitory concentration (MIC) values from 0.0195 to 5 mg/ml. Escherichia coli was the most affected bug, thus the MIC of fruits BuOH extract showed the best anti‐Escherichia coli activity (MIC = 0.0195 mg/ml), followed by the (stems and leaves) and flowers BuOH extracts; MIC = 0.078 and 0.15 mg/ml, respectively. Furthermore, the subfractions obtained from these three mixed fractions showed also an important antimicrobial activity against the three microorganisms, with MIC values between 0.0195 and 0.312 mg/ml. The fractionation of the aerial part BuOH fraction led to the isolation of oleanolic acid ( 1 ) and luteolin 7‐O‐glucopyranoside ( 2 ) which are reported here for the first time from S. arenaria. Both compounds showed good antimicrobial activities with MIC values ranging from 170 to 683 μm and 86 to 347 μm , respectively. These results support the use of the Scabiosa genus to inhibit the growth of tested pathogenic bacteria and yeasts which may reduce illnesses associated with their exposure.     

Effects of natamycin and potassium sorbate on growth and aflatoxin production in olives

Aspergillus flavus NRRL 6555 was inoculated onto whole olives and olive paste samples containing variable amounts of either natamycin or potassium sorbate and incubated at 15 degrees, 25 degrees, and 35 degrees C for 7, 14 and 21 days for whole olives and at 15 degrees and 25 degrees C for 8 and 16 days for olive pastes. The initiation time of growth was parallel to the concentrations of either preservatives applied. However, at 15 degrees C, natamycin at 160 and 320 micrograms/g (ppm) completely inhibited the growth of mold on whole olives for 21 days and olive paste for 7 and 15 days, respectively. All levels of potassium sorbate inhibited mold growth at 15 degrees C, but at 25 degrees C, 6000 micrograms/g (ppm) only, delayed growth for 15 days. The extent of growth at the end of the incubation periods was parallel to the temperatures of incubation. The analyses for aflatoxin B1 production in all samples at all levels of preservatives and control were negative.     

Selenium Distribution in Camel Blood and Organs After Different Level of Dietary Selenium Supplementation

Eight young female camels shared in four groups of two 2 years received a basal diet enriched respectively with 0, 2, 4, and 8 mg selenium under sodium selenite form for 64 days. Feed intake was assessed daily; blood samples were taken on weekly basis. One camel from each group was killed at the end of the experiment. Se concentration in serum was increased significantly in the supplemented groups with an average of 176.3 ± 18.0 ng/mL in the control group, 382.7 ± 107.6 in the group receiving 2 mg Se, 519.8 ± 168.4 in the group receiving 4 mg Se, and 533.4 ± 158.6 in the group receiving 8 mg Se daily. For glutathione peroxidase (GSH-Px) activity, the control group (51.0 IU/g Hb) and the group receiving 2 mg (50.5 IU/g Hb) were significantly different than groups receiving 4 and 8 mg (respectively, 65.9 and 76.1 IU/g Hb). No significant variation occurred for vitamin E (mean, 0.56 ± 0.23 ng/mL). Significant correlation between serum Se and GSH-Px was reported. Kidney was the richest organ in selenium followed by lung, spleen, and liver, but the increase in supplemented groups was more marked in liver and kidney. The hair seemed to be the best indicator of selenium intake in camel.