Indirect ELISAs based on recombinant and affinity-purified glycoprotein E of Aujeszky's disease virus to differentiate between vaccinated and infected animals

Two indirect ELISAs for the detection of antibodies against glycoprotein E (gE) of Aujeszky's disease virus (ADV) in sera have been developed. The rec-gE-ELISA is based on theE. coli-expressed recombinant protein containing the N-terminal sequences of gE (aa 1-125) fused with the glutathione S-transferase fromSchistosoma japonicum. The affi-gE-ELISA is based on native gE, which was purified from virions by affinity chromatography. The tests were optimised and compared with each other, as well as with the recently developed blocking gE-ELISA (Morenkov et al., 1997b), with respect to specificity and sensitivity. The rec-gE-ELISA was less sensitive in detecting ADV-infected animals than the affi-gE-ELISA (sensitivity 80% and 97%, respectively), which is probably due to the lack of conformation-dependent immunodominant epitopes on the recombinant protein expressed inE. coli. The specificity of the rec-gE-ELISA and affi-gE-ELISA was rather moderate (90% and 94%, respectively) because it was necessary to set such cut-off values in the tests that provided a maximum level of sensitivity, which obviously increased the incidence of false positive reactions. Though the indirect ELISAs detect antibodies against many epitopes of gE, the blocking gE-ELISA, which detects antibodies against only one immunodominant epitope of gE, showed a better test performance (specificity 99% and sensitivity 98%). This is most probably due to rather high dilutions of the sera used in the indirect gE-ELISAs (1:30) as compared to the serum dilution in the blocking gE-ELISA (1:2). We conclude that the indirect gE-ELISAs are sufficiently specific and sensitive to distinguish ADV-infected swine from those vaccinated with gE-negative vaccine and can be useful, in particularly affi-gE-ELISA, as additional tests for the detection of antibodies to gE

Protealysin is not Secreted Constitutively

© 2019 Bentham Science Publishers. Background: Protealysin, a zinc metalloprotease of Serratia proteamaculans, is the prototype of a new group within the peptidase family M4. Protealysin-like proteases (PLPs) are widely spread in bacteria but are also found in fungi and archaea. The biological functions of PLPs have not been well studied, but published data showed the involvement of enzymes of this group in the interaction of bacteria with higher organisms, and most likely in the pathogenesis. Such functionality requires the release of the proteases from bacterial cells; however, the data on the cellular localization of PLPs are contradictory and no direct data of this kind have been published. Objective: Here, the protealysin cellular localization was studied for the first time using immunochemical methods. Methods and Results: We have produced polyclonal rabbit antibodies against the protealysin precursor. The enzyme was evaluated in cells and medium of periodic culture of S. proteamaculans 94 using Western blotting as well as the enzyme localization was analysed by immunoelectron microscopy. It was shown that more than 99% of the enzyme is in a cell-associated form. Protealysin is accumulated in cells as an inactive precursor. It matures only after the release from cells (after their lysis). Immunoelectron microscopy analysis of bacterial cells has revealed no specific localization of protealysin; it was evenly distributed in the cytoplasm. Conclusion: The data obtained suggest that S. proteamaculans protealysin and supposedly other protealysin-like proteases are not secreted constitutively and their release from bacteria is likely induced by a certain stimulus such as a contact with a eukaryotic cell. This finding is critical for further studies of the involvement of these enzymes in pathogenesis

Navigational features of dove flights in the gravity field of Ukraine

Results of test flights of domestic doves in the flat country of Ukraine characterized by partitioned gravity field (Buge anomalies from -30 to +70 mGal) have been considered. The length of flights from starting point to the place of permanent location was from 15 to 200 km. The routes were tracked trough weakly and strongly divided gravity field. Both local experienced birds and young ones specially bred in outlying gravity fields took part in the flights. GPS sensors were fastened on birds fixing coordinates of flight (ϕ, λ, H) in every 1 sec. Test results demonstrated with other factors (roads, rivers et al.) that gravity anomalies even small ones (±1-2 mGal) produce deviation of dove tracks from the straight line together. It is urgent to continue the experiment which will be corrected taking into account the experience obtained. Inertial-gravitational mechanism of animal navigation has been suggested

Deletion of the <i>dps</i> gene affects <i>rpoA</i> and <i>rpoB</i> expression.

<p><b>A:</b> Profiles of the Dps binding sites obtained in two experiments (indicated) for the genomic region with three operons of ribosomal genes (running window of nine 35 bp bins). Genes are represented by blue horizontal arrows; magenta lines show ribosomal operons. Vertical arrows mark locations of inverted repeats (if longer than 7 bp). <b>B:</b> Band shift assays performed for indicated genomic loci. The regulatory region of the <i>dps</i> gene was used as a positive control for all band shift assays in this study. Fragment from the <i>lacZ</i> coding sequence was used as a reference gene for qRT-PCR experiments. Positioning of primers for amplification (<b>F</b> and <b>R</b>) is indicated in panel <b>A</b> of this figure, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182800#pone.0182800.g006" target="_blank">Fig 6A</a> (for the <i>dps</i> regulatory region) and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182800#pone.0182800.s003" target="_blank">S3 Fig</a> (for <i>rpoA</i> and <i>lacZ</i>). <b>C:</b> Changes in the expression efficiency of selected genes in response to <i>dps</i> deletion. Primers used for reverse transcription and consecutive PCR are designated as RT and PCR, respectively, here and all other figures. Expression levels were estimated based on 3 and 5 biological samples (3–18 technical repeats in each) for <i>rpoA</i> and <i>rpoD</i>, respectively. Error bars show an average deviation. Statistical significance was assessed using Student’s t-test.</p

Large-scale profiles of the Dps targets correlate with the landscape of direct and inverted repeats and the pattern of Fis binding sites.

<p><b>A:</b> Distribution of the Dps contact regions along the <i>E</i>. <i>coli</i> MG1655 genome identified by CLC GW in two experiments (the default settings). The areas covered by Dps are combined in 100,000 bp bins and plotted as percentage to the total length of all sites occupied by Dps. <b>B</b>: The same for the contact sites of Fis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182800#pone.0182800.ref049" target="_blank">49</a>], IHF [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182800#pone.0182800.ref051" target="_blank">51</a>], H-NS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182800#pone.0182800.ref049" target="_blank">49</a>] and RNAP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182800#pone.0182800.ref050" target="_blank">50</a>] from the cells grown in conditions similar to those used in our experiments. The plot for Dps shows the distribution of the sites from the combined set (<b>CS</b>) (Page 3, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182800#pone.0182800.s009" target="_blank">S3 Table</a>). <b>C:</b> The same for direct (5–24 bp separated by 1–15 bp) and inverted (5–18 bp separated by 3–20 bp) repeats collected from the genome of <i>E</i>. <i>coli</i> MG1655 using Unipro UGENE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182800#pone.0182800.ref053" target="_blank">53</a>].</p

Dps shares its binding sites with other proteins of bacterial nucleoid and has affinity to REP-elements and <i>promoter islands</i>.

<p>Intersection of Dps targets (<b>A</b>) and sites unbound by Dps (<b>B</b>) with structural and functional elements of bacterial genome was estimated as described above for repeated sequences and plotted as fold ratio to the expected values. Bent black arrows on the bottom schematically show areas occupied by CS or UR. Gray rectangles and numerals inside indicate the expected number of common base pairs if compared modules are independently distributed along the genome. Gray and colored bent arrows show registered overlap calculated in 1 bp resolution. Numerals in parenthesis indicate the size of compared sets. Genomic locations of REP elements were taken from KEGG DataBase (<a href="http://www.genome.jp/kegg/" target="_blank">http://www.genome.jp/kegg/</a>, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182800#pone.0182800.ref063" target="_blank">63</a>]), and fold ratios obtained for 302 REP-sequences containing 1–3 REP-modules (14–100 bp) were plotted. Analyzed ChIP-chip and ChIP-seq data sets were obtained from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182800#pone.0182800.ref046" target="_blank">46</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182800#pone.0182800.ref051" target="_blank">51</a>] for cells grown in LB medium (LB), M9 medium with fructose (M9) or MOPS minimal medium with glucose (GMM), harvested at early (EE), middle (ME) or late (LE) exponential phase or upon transition to the steady growth (TS).</p

Dps-binding sites are enriched with inverted repeats.

<p>The overlap between sequences of CS (colored box-plots) and UR (gray boxes) with direct or inverted repeats was characterized by the parameter <b>K</b>_<b>ij</b> as described in the text. Black dot on the right panel shows one outlier. Box-plots with statistically significant differences are provided with corresponding p-values. Regions of bound and unbound sets overlapping with repeated sequences of both types are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182800#pone.0182800.s009" target="_blank">S3 Table</a>.</p