Удосконалення комерційної діяльності як фактор підвищення конкурентоспроможності підприємства

Additional file 5. ELISA to assess the interaction between Campylobacter -specific nanobodies and purified MOMP. The saturation binding curve of the interaction between coated MOMP (1 µg/mL) and a His-tagged nanobody (1 × 10−6 to 1 × 102 µg/mL) was obtained via ELISA. The dose-dependent inhibitory effect of a strep-tagged nanobody (1 × 10−6 to 1 × 102 µg/mL) on the interaction between His-tagged Nb84 (5.10−2 µg/mL) and MOMP (1 µg/mL), is demonstrated in the competition binding curve. Inhibition by strep-tagged (A) Nb5, (B) Nb22, (C) Nb23, (D) Nb24, (E) Nb49, (F) 84, (G) Nb15, (H) Nb32, (I) Nb34, (J) Nb45, (K) Nb48 and (L) Nb63, was assessed. The ELISA was developed with mouse anti-Histidine tag monoclonal antibody and goat anti-mouse IgG conjugated to alkaline phosphatase. The error bars represent the standard deviations

The murine ApoL family.

<p>A. Phylogenetic tree of the murine <i>Apol</i> gene family, generated by the Gene Orthology/Paralogy prediction method available on the Ensemble server (<a href="http://www.ensembl.org/" target="_blank">http://www.ensembl.org</a>). Duplication nodes are indicated by black squares. Numbers represent the duplication confidence score. B. Organization of the mouse <i>Apol</i> genes cluster. Underlined regions (<i>Apol7b/9a</i> and <i>Apol7e/9b</i>) share 98% of nucleotide sequence identity and likely arose from a duplication event. C. Alignment of the human ApoL6 BH3 domain with the hypothetical BH3 domain of murine ApoL proteins. Two amino acids, <i>L</i> and <i>D</i>, shown in bold letters, are functionally conserved in all the BH3 domains identified. D. Structural organization of ApoL9 proteins. The putative BH3 domain (residues 81 to 89) is shown in dark gray. The white area (residues 128 to 144) corresponds to a potential transmembrane (TM) domain.</p

<i>Apol9</i> expression in mouse tissues and in L929 cells.

<p><i>Apol9</i> transcripts were quantified by RT-qPCR in mouse tissues and cells. A. <i>Apol9a</i> and <i>Apol9b</i> cDNA copies (mean and SD) detected per 10⁶ β-actin cDNA copies, in tissues of naive C57BL/6 mice (n = 3). The column indicated “+ IFN-α” gives the mean upregulation (x-fold) of <i>Apol9</i> expression observed in the presence of systemic IFN-α. This corresponds to the ratio between the number of <i>Apol9</i> cDNA copies detected in tissues of FVB/N mice electro-injected in the tibialis muscle with plasmid pcDNA3-IFN-α6T, which expresses IFN-α that circulates in the blood stream (n = 6) and that of control mice that were electro-injected with an empty pcDNA3 plasmid. N.D. = not determined. B. <i>Apol9b</i> expression in the central nervous system of FVB/N mice that were mock-infected (n = 3) or infected intracerebrally with 10⁶ PFU of the DA1 strain of TMEV (n = 5) for the indicated time. *: indicates a significant difference with the mock-infected mice at 7dpi. C. Time course of <i>Oasl2</i>, <i>Apol9a</i> and <i>Apol9b expression</i> in L929 cells treated with 5 units/ml of IFN-β (n = 3).</p

ApoL9 proteins are not-secreted, cytoplasmic proteins.

<p>A. Antiviral activity of N-terminally (FLAG-ApoL9b) and C-terminally (ApoL9b-FLAG) FLAG-tagged ApoL9b against TMEV. Histograms show the relative infection efficiency, of L929 cells that were transduced with theTM945 lentiviral vector expressing mCherry alone (vector), or with the MK82 or MK83 derivatives co-expressing mCherry and FLAG-ApoL9b or ApoL9b-FLAG, respectively. Infection efficiency was measured, by flow cytometry, after infection of cells for 10 hours with 0.5 PFU per cell of the GFP-expressing KJ26 virus. B. FLAG detection (green) and nuclear staining (blue) in HeLa-M cells transduced with the MK65 construct expressing FLAG-ApoL9b (scale bar: 10 um). Typical immunolabeling patterns are shown: left and central panels: one non-transduced cell (arrow) and transduced cells showing a diffuse cytoplasmic FLAG-ApoL9b localization; right panel: cells showing typical small and medium-sized FLAG-ApoL9b granules (arrows). C. Confocal images showing FLAG (green—left panel) and mitochondria (red—central panel) labelings and merged image (right panel). Nuclei were stained with Hoechst (Blue). D. Western blot detection of FLAG-tagged ApoL9b, ApoL9a and IFN-αA in 293T cells. Cells were mock-transfected, transfected with the empty pTM943 plasmid (vector), or with pTM943 derivatives coding for the indicated FLAG-tagged ApoL9 proteins. As a control cells were also transfected with plasmid pcDNA3-IFNαA-FLAG, which expresses the secreted FLAG-tagged mouse IFN-αA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133190#pone.0133190.ref012" target="_blank">12</a>]. 20 hours post-transfection, cells were mock-treated (-) or treated for 4 hours with 10ug/ml of Brefeldin A (+) prior to total protein extraction. FLAG and β-actin were detected by Western blot in cell lysates.</p

Recombinant viruses used in the study.

<p>1. virus DA1 is produced by reverse genetics from plasmid pTMDA1. Viruses KJ6 and KJ7 are produced from pKJ6 and pKJ7 respectively</p><p>2. L = leader protein of Theiler's virus; eGFP = enhanced GFP</p><p>Recombinant viruses used in the study.</p

Upregulation of ApoL9a/b expression in microarray data sets.

<p>Upregulation of ApoL9a/b expression in microarray data sets.</p

ApoL9 does not sensitize cells to apoptosis but acts on TMEV replication.

<p>A. Histograms show caspase 3 and 7 activities (mean and SD), measured in L929 cell populations that were transduced with the empty TM942 vector (white columns) or with the MK85 (light grey columns) and MK44 (dark grey columns) derivatives that express Apol9a and Apol9b respectively. These cells were mock-treated (Mock), infected with 1 PFU per cell of TMEV (KJ26) for 10 hours or treated with 1uM staurosporine for 7 hours (n = 3). B. Luciferase activity produced by a TMEV-luciferase replicon (TM973) transfected in ApoL9 expressing cells. Prior to replicon transfection, L929 cells were transduced with TM942 (vector-mCherry), MK85 (co-expression of mCherry and ApoL9a) or mK44 (co-expression of mCherry and ApoL9a). mCherry-positive cells were FACS sorted and subdivided in population of high (> 10⁴) and low (4.10³ to 10⁴) mCherry fluorescence. <i>In vitro</i> transcribed RNA of the TM973 replicon was transfected in these cells and luciferase activity was assayed at the indicated times post-transfection. Graphs show the luciferase activity normalized, for each construct, to the activity measured at two hours post-transfection. Results are representative of a typical experiment out of 3 performed in triplicate. * indicate the significance of the difference between cells expressing ApoL9 and the empty vector. C. Schematic representation of ApoL9 mutants. #BH3 refers to point mutations introduced in the putative BH3 domain. Δ50N and Δ50C refer to the deletion of the 50 N- and C-terminal amino acids, respectively. D. relative infection efficiency of L929 cells transduced either with the empty TM942 vector (vector) or with the same vector encoding wild type or mutant ApoL9 proteins (ApoL9a in light gray and ApoL9b in dark gray). Cells were infected for 8.5 hours with 0.5 PFU per cell of virus KJ7. GFP fluorescence was examined in cells that were gated for expression of the ApoL9 constructs (mCherry-positive). Histograms show the mean and SD, for a pool of 3 independent infection experiments performed in triplicates. For each experiment, the infection efficiency in ApoL9-expressing cells was normalized to that measured in cells carrying the empty vector. Stars indicate the difference significance between ApoL9-expressing cells and cells carrying the empty vector.</p

The murine ApoL family.

<p>A. Phylogenetic tree of the murine <i>Apol</i> gene family, generated by the Gene Orthology/Paralogy prediction method available on the Ensemble server (<a href="http://www.ensembl.org/" target="_blank">http://www.ensembl.org</a>). Duplication nodes are indicated by black squares. Numbers represent the duplication confidence score. B. Organization of the mouse <i>Apol</i> genes cluster. Underlined regions (<i>Apol7b/9a</i> and <i>Apol7e/9b</i>) share 98% of nucleotide sequence identity and likely arose from a duplication event. C. Alignment of the human ApoL6 BH3 domain with the hypothetical BH3 domain of murine ApoL proteins. Two amino acids, <i>L</i> and <i>D</i>, shown in bold letters, are functionally conserved in all the BH3 domains identified. D. Structural organization of ApoL9 proteins. The putative BH3 domain (residues 81 to 89) is shown in dark gray. The white area (residues 128 to 144) corresponds to a potential transmembrane (TM) domain.</p

The Interferon-Inducible Mouse Apolipoprotein L9 and Prohibitins Cooperate to Restrict Theiler’s Virus Replication

<div><p><i>Apolipoprotein L9b</i> (<i>Apol9b</i>) is an interferon-stimulated gene (ISG) that has antiviral activity and is weakly expressed in primary mouse neurons as compared to other cell types. Here, we show that both <i>Apol9</i> isoforms (<i>Apol9b</i> and <i>Apol9a</i>) inhibit replication of Theiler’s murine encephalomyelitis virus (TMEV) but not replication of vesicular stomatitis virus (VSV), Murid herpesvirus-4 (MuHV-4), or infection by a lentiviral vector. <i>Apol9</i> genes are strongly expressed in mouse liver and, to a lesser extent, in pancreas, adipose tissue and intestine. Their expression is increased by type I interferon and viral infection. In contrast to genuine apolipoproteins that are involved in lipid transport, ApoL9 has an intracytoplasmic localization and does not seem to be secreted. The cytoplasmic localization of ApoL9 is in line with the observation that ApoL9 inhibits the replication step of TMEV infection. In contrast to human ApoL6, ApoL9 did not sensitize cells to apoptosis, in spite of the presence of a conserved putative BH3 domain, required for antiviral activity. ApoL9a and b isoforms interact with cellular prohibitin 1 (Phb1) and prohibitin 2 (Phb2) and this interaction might contribute to ApoL9 antiviral activity. Knocking down <i>Phb2</i> slightly increased TMEV replication, irrespective of ApoL9 overexpression. The antiviral activity of prohibitins against TMEV contrasts with the pro-viral activity of prohibitins observed for VSV and reported previously for Dengue 2 (DENV-2), Chikungunya (CHIKV) and influenza H5N1 viruses. ApoL9 is thus an example of ISG displaying a narrow antiviral range, which likely acts in complex with prohibitins to restrict TMEV replication.</p></div

Recombinant lentiviral vectors used in the study.

<p>1. the names of plasmids used to produce the listed lentiviral vectors contain an extra p (e.g. pMK44 for lentivirus MK44)</p><p>2. pCCLsin = pCCLsin.PPT.hPGK.GFP.pre</p><p>3. Sequence of the FLAG-Apol9 junction is: MG-DYKDDDDK-GS-MASS… where GS is a linker and MASS are the N-terminal 4 residues of ApoL9.</p><p>4. Sequence of the Apol9-FLAG junction is: …YKTI-GS-DYKDDDDK where GS is a linker and YKTI are the C-terminal 4 residues of ApoL9.</p><p>5. L* = L star protein of Theiler’s virus; PGK = human phosphoglycerate kinase promoter; CMV = cytomegalovirus promoter; MCS = multi-cloning site; eGFP = enhanced GFP; Puro^R = puromycin resistance; Δ = deletion</p><p>Recombinant lentiviral vectors used in the study.</p