19 research outputs found

    Near-Infrared Luciferase Complementation Assay with Enhanced Bioluminescence for Studying Protein–Protein Interactions and Drug Evaluation Under Physiological Conditions

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    Identification of protein–protein interactions (PPIs) that occur in various cellular processes helps to reveal their potential molecular mechanisms, and there is still an urgent need to develop the assays to explore PPIs in living subjects. Here, we reported a near-infrared split luciferase complementation assay (SLCA) with enhanced bioluminescence produced by cleaving a luciferase, Akaluc, for exploring and visualizing PPIs in living cells and live mice. Compared with the previously developed and widely used red SLCA based on split firefly luciferase (Fluc-SLCA), the signal intensities for PPI recognition in living cells and live mice of the Akaluc-SLCA increased by ∼3.79-fold and ∼18.06-fold in the measured condition, respectively. Additionally, the interactions between the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cellular RNA processing proteins were identified, and the drug evaluation assays were also performed in living cells using Akaluc-SLCA. This study provides a new tool in the near-infrared region for the identification of PPIs in living cells and in vivo and new information for the understanding and treatment of SARS-CoV-2

    Folding of the 5′ and 3′ NCRs of the DpCPV-MC genome segments.

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    <p>The RNAfold program was used to predict the secondary structures formed by the 16 DpCPV-MC genome segments. The panhandle structure was formed by base-pairing between the 5′ and 3′ ends, and the stem–loop structure was formed by either the 5′ or 3′ terminal sequence. The panhandle structure and the stem–loop structure are marked with long brackets.</p

    Electron micrographs of OBs of DpCPV-MC.

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    <p>(A) Transmission electron micrograph of ultrathin sections of OBs of DpCPV-MC. (B) Transmission electron micrograph of purified virions of DpCPV-MC.</p

    Median lethal concentrations (LC<sub>50</sub>) of DpCPV-MC and DpCPV-1 against second-instar <i>S. exigua</i> larvae.

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    a<p>Potency ratio was calculated by dividing the LC<sub>50</sub> of DpCPV-MC by that of DpCPV-1. Significant difference was based on whether the 95% confidence interval (CI) of the potency ratio included the value 1.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113201#pone.0113201-Robertson2" target="_blank">[47]</a>.</p><p>Median lethal concentrations (LC<sub>50</sub>) of DpCPV-MC and DpCPV-1 against second-instar <i>S. exigua</i> larvae.</p

    Neighbor-joining tree constructed from polyhedrin amino acid sequences of representative CPVs.

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    <p>Sequences were aligned with the multiple sequence alignment program ClustalX2. The neighbor-joining method was used to construct the phylogenetic tree of the derived polyhedrin protein sequences with the MEGA software version 5.2. The black triangle shows the position of DpCPV-MC. Bootstrap percentage values are indicated on the left. All reference sequences used for the construction of this tree were retrieved from GenBank with their corresponding accession numbers: <i>Antheraea assamensis cypovirus 4</i>, AY212275; <i>Antheraea mylitta cypovirus 4</i>, AY212273; <i>Antheraea proylei cypovirus 4</i>, AY212276; <i>Dendrolimus punctatus cypovirus 1</i>, AY204879; <i>Bombyx mori cypovirus 1</i>, D37770; <i>Culex restuans cypovirus 17</i>, DQ212785; <i>Choristoneura occidentalis cypovirus 16</i>, EU201043; <i>Euxoa scandens cypovirus 5</i>, J04338; <i>Heliothis assulta cypovirus 14</i>, DQ077914; <i>Heliothis armigera cypovirus 5</i>, DQ077912; <i>Lymantria dispar cypovirus 1</i>, AF389471; <i>Operophtera brumata cypovirus 18</i>, DQ192250; <i>Operophtera brumata cypovirus 19</i>, DQ192254; <i>Simulium ubiquitum cypovirus 20</i>, DQ834386; <i>Trichoplusia ni cypovirus 15</i>, NC_002565; <i>Uranotaenia sapphirina cypovirus 17</i>, AY876384; <i>Lymantria dispar cypovirus 14</i>, AF389461; <i>Choristoneura fumiferana cypovirus 16</i>, U95954.</p

    Southern blot analysis of genomic DNA of <i>S. exigua</i> larvae.

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    <p>A. Electrophoresis (left) and southern blot (right) of restriction endonuclease-digested genomic DNA of <i>S. exigua</i> larvae, detected by pheromone-binding protein 3 (PBP3) probe. Marker, phage λ DNA digested with <i>Eco</i>T14 I. XhoI, <i>Xho</i>I-digested genomic DNA of <i>S. exigua</i> larvae (2 µg). BamHI, <i>Bam</i>HI-digested genomic DNA of <i>S. exigua</i> larvae (2 µg). The arrows indicate hybridization bands in genomic DNA of <i>S. exigua</i> larvae. B. Electrophoresis (left) and southern blot (right) of restriction endonuclease-digested genomic DNA of <i>S. exigua</i> larvae, detected by probe mixture of six DpCPV-MC dsRNA segments (S6, S10, S11, S14, S15 and S16). The six plasmids containing full-length sequence of six segments was used as positive control. Marker, phage λ DNA digested with <i>Eco</i>T14 I. XhoI, <i>Xho</i>I-digested genomic DNA of <i>S. exigua</i> larvae (2 µg). BamHI, <i>Bam</i>HI-digested genomic DNA of <i>S. exigua</i> larvae (2 µg). pMD18-T-S6, pMD18-T vector containing full-length sequence of S6 (2 ng). pMD18-T-S10, pMD18-T vector containing full-length sequence of S10 (2 ng). pMD18-T-S11, pMD18-T vector containing full-length sequence of S11 (2 ng). pMD18-T-S14, pMD18-T vector containing full-length sequence of S14 (2 ng). pMD18-T-S15, pMD18-T vector containing full-length sequence of S15 (2 ng). pMD18-T-S16, pMD18-T vector containing full-length sequence of S16 (2 ng).</p

    Northern blot analysis of six dsRNA segments of DpCPV-MC.

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    <p>Total genomic dsRNA of DpCPV-MC was separated on 1% agarose gel, transferred to nylon membrane, and hybridized with the corresponding DIG-labeled probes derived from each of the six segment sequences. The left part is agar gel stained with ethidium bromide. M, phage λ DNA digested with <i>Eco</i>T14 I. The right six lanes are northern blot results.</p

    The most common social pathologigal traits of perpetrators of traffic accidents and their causes (based on the probes into their psyche in the book Fatal meetings by K. Havlík)

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    The aim of this thesis is to clear up and acquiant with psychological profile of failing drivers. There are dozer of drivers failing in these days, although we can suppose that we are not involved in this topic. But contrariwise, we are a part of this problematics as well, we could not be direct offenders of accidents or drivers with socially-pathological phenomenons, but we can be thein victims as well. That is why it is really important to pay attention to this problematics and to be interested in it and try to prevent it. So the aim of the thesis was to find out, what are the most usual phenomenonsof offenders of accidents, what are the causes of these phenomenons, where and in which situations are they springing up thein beginnings, that leads us to accidents afterwards. In kontent of thesis is analysis of risk factors in road traffic as well. The last charter is focused on preventive steps leading to limitation of accidents. The book by Karel Havlík "Osudová střetnutí" was a part of there search complex just as an interview with traffic psychologist. I have dividend this interview into eight questions, that were fulfilling theoretical problematics in the first part. I have found out by processing of the interview and by processing of the book "Osudová střetnutí" that usual socially-pathological phenomenons were the phenomenons, that have aggressive features in thein elements, from which it is springing up much more. On the contrary people with personality disorders are usually offenders of accidents as well. I have reached the conclusion that each human might chase anything in himself and in his behaving either in attitude to the others, but it is needed a lot of effort and persistence
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