Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in <Emphasis Type="Italic">Xenopus</Emphasis>

Background  

Blastomere injection of mRNA or antisense oligonucleotides has proven effective in analyzing early gene function in Xenopus. However, functional analysis of genes involved in neuronal differentiation and axon pathfinding by this method is often hampered by earlier function of these genes during development. Therefore, fine spatio-temporal control of over-expression or knock-down approaches is required to specifically address the role of a given gene in these processes.     

Circulation of Crimean-Congo Hemorrhagic Fever Virus in the Former Yugoslav Republic of Macedonia Revealed by Screening of Cattle Sera Using a Novel Enzyme-linked Immunosorbent Assay

BackgroundThere are only few assays available for the detection of Crimean-Congo Hemorrhagic Fever Virus (CCHFV)-specific antibodies in animals, and data about diagnostic sensitivity and specificity are incompletely documented for most of these tests. This is unfortunate since CCHFV antibodies in animals can be used as indicator for virus circulation in a geographic area and therewith potential risk of human exposure. This paper therefore reports on a novel ELISA for the detection of CCHFV-specific antibodies in cattle and on its application for testing ruminant sera from the Former Yugoslav Republic of Macedonia.ConclusionThis article describes a fully validated, highly sensitive and specific ELISA for the detection of CCHFV-specific IgG antibodies in cattle. Using this assay, CCHFV-specific antibodies were detected for the first time in cattle in the Former Yugoslav Republic of Macedonia, giving evidence for an active circulation of this virus in the country. Supporting this conclusion, the occurrence of the main vector of CCHFV was demonstrated in the present work for the first time in Former Yugoslav Republic of Macedonia.     

Computational analysis of position-dependent disorder content in DisProt database

A bioinformatics analysis of disorder content of proteins from the DisProt database has been performed with respect to position of disordered residues.Each protein chain was divided into three parts:N-and C-terminal parts with each containing 30 amino acid(AA) residues and the middle region containing the remaining AA residues.The results show that in terminal parts,the percentage of disordered AA residues is higher than that of all AA residues(17% of disordered AA residues and 11% of all).We analyzed the percentage of disorder for each of 20 AA residues in the three parts of proteins with respect to their hydropathy and molecular weight.For each AA,the percentage of disorder in the middle part is lower than that in terminal parts which is comparable at the two termini.A new scale of AAs has been introduced according to their disorder content in the middle part of proteins:CIFWMLYHRNVTAGQDSKEP.All big hydrophobic AAs are less frequently disordered,while almost all small hydrophilic AAs are more frequently disordered.The results obtained may be useful for construction and improving predictors for protein disorder.     

Tolerance of Listeria monocytogenes to Quaternary Ammonium Sanitizers Is Mediated by a Novel Efflux Pump Encoded by emrE

A novel genomic island (LGI1) was discovered in Listeria monocytogenes isolates responsible for the deadliest listeriosis outbreak in Canada, in 2008. To investigate the functional role of LGI1, the outbreak strain 08-5578 was exposed to food chain-relevant stresses, and the expression of 16 LGI1 genes was measured. LGI1 genes with putative efflux (L. monocytogenes emrE [emrE_Lm]), regulatory (lmo1851), and adhesion (sel1) functions were deleted, and the mutants were exposed to acid (HCl), cold (4°C), salt (10 to 20% NaCl), and quaternary ammonium-based sanitizers (QACs). Deletion of lmo1851 had no effect on the L. monocytogenes stress response, and deletion of sel1 did not influence Caco-2 and HeLa cell adherence/invasion, whereas deletion of emrE resulted in increased susceptibility to QACs (P < 0.05) but had no effect on the MICs of gentamicin, chloramphenicol, ciprofloxacin, erythromycin, tetracycline, acriflavine, and triclosan. In the presence of the QAC benzalkonium chloride (BAC; 5 μg/ml), 14/16 LGI1 genes were induced, and lmo1861 (putative repressor gene) was constitutively expressed at 4°C, 37°C, and 52°C and in the presence of UV exposure (0 to 30 min). Following 1 h of exposure to BAC (10 μg/ml), upregulation of emrE (49.6-fold), lmo1851 (2.3-fold), lmo1861 (82.4-fold), and sigB (4.1-fold) occurred. Reserpine visibly suppressed the growth of the ΔemrE_Lm strain, indicating that QAC tolerance is due at least partially to efflux activity. These data suggest that a minimal function of LGI1 is to increase the tolerance of L. monocytogenes to QACs via emrE_Lm. Since QACs are commonly used in the food industry, there is a concern that L. monocytogenes strains possessing emrE will have an increased ability to survive this stress and thus to persist in food processing environments.     

Identification of a metallothionein gene in honey bee Apis mellifera and its expression profile in response to Cd,Cu and Pb exposure

Metallothioneins are ubiquitous proteins important in metal homeostasis and detoxification. However, they have not previously been identified in honey bees or other Hymenoptera, where metallothioneins could be of ecophysiological and ecotoxicological significance. Better understanding of the molecular responses to stress induced by toxic metals could contribute to honey bee conservation. In addition, honey bee metallothionein could represent a biomarker for monitoring environmental quality. Here we identify and characterize a metallothionein gene in Apis mellifera (AmMT). AmMT is 1,680 bp long and encodes a 48 amino acids protein with 15 cysteines and no aromatic residues. A metal response element upstream of the start codon, coupled with numerous cis‐regulatory elements indicate the functional context of AmMT. Molecular modelling predicts several transition metal binding sites, and comparative phylogenetic analysis revealed five putative metallothionein proteins in three other hymenoptera species. AmMT was characterized by cloning the full‐length coding sequence of the putative metallothionein. Recombinant AmMT was found to increase metal tolerance upon overexpression in Escherichia coli supplemented with Cd, Cu or Pb. Finally, in laboratory tests on honey bees, gene expression profiles showed a dose‐dependant relationship between Cd, Cu and Pb concentrations present in food and AmMT expression, while field experiments showed induction of AmMT in bees from an industrial site compared to those from an urban area. These studies suggest that AmMT has metal binding properties in agreement with a possible role in metal homeostasis. Further functional and structural characterization of metallothionein in honey bees and other Hymenoptera are necessary.     

Climate change,biological invasions,and the shifting distribution of Mediterranean fishes: A large‐scale survey based on local ecological knowledge

Climate change and biological invasions are rapidly reshuffling species distribution, restructuring the biological communities of many ecosystems worldwide. Tracking these transformations in the marine environment is crucial, but our understanding of climate change effects and invasive species dynamics is often hampered by the practical challenge of surveying large geographical areas. Here, we focus on the Mediterranean Sea, a hot spot for climate change and biological invasions to investigate recent spatiotemporal changes in fish abundances and distribution. To this end, we accessed the local ecological knowledge (LEK) of small‐scale and recreational fishers, reconstructing the dynamics of fish perceived as “new” or increasing in different fishing areas. Over 500 fishers across 95 locations and nine different countries were interviewed, and semiquantitative information on yearly changes in species abundance was collected. Overall, 75 species were mentioned by the respondents, mostly warm‐adapted species of both native and exotic origin. Respondents belonging to the same biogeographic sectors described coherent spatial and temporal patterns, and gradients along latitudinal and longitudinal axes were revealed. This information provides a more complete understanding of the shifting distribution of Mediterranean fishes and it also demonstrates that adequately structured LEK methodology might be applied successfully beyond the local scale, across national borders and jurisdictions. Acknowledging this potential through macroregional coordination could pave the way for future large‐scale aggregations of individual observations, increasing our potential for integrated monitoring and conservation planning at the regional or even global level. This might help local communities to better understand, manage, and adapt to the ongoing biotic transformations driven by climate change and biological invaders.