Immunoreactivity characterisation of the three structural regions of the human coronavirus OC43 nucleocapsid protein by Western blot: Implications for the diagnosis of coronavirus infection

Previous studies have reported that a prokaryotic-expressed recombinant nucleocapsid protein (NP) is a suitable reagent for the epidemiological screening of coronavirus infection. In this study, soluble recombinant human coronavirus OC43 (HCoV-OC43) NP was produced to examine the antigenicity of the HCoV-OC43 NP of betacoronavirus. Using the purified recombinant NP as an antigen, a polyclonal antibody from rabbit serum with specificity for HCoV-OC43 NP was generated; this antibody reacts specifically with HCoV-OC43 NP and does not cross-react with other human CoV NPs (including those of SARS-CoV and HCoV-229E) by Western blot. Sera from 26 young adults, 17 middle-aged and elderly patients with respiratory infection, and 15 cord blood samples were also tested. Strong reactivity to the NPs of HCoV-OC43 was observed in 96%, 82%, and 93% of the serum samples from the young adults, respiratory patients, and cord blood samples, respectively. To identify the immunoreactivities of the three structural regions of the NP that are recognised by the rabbit polyclonal antibody and human serum, the antigenicities of three protein fragments, including the N-terminal domain (aa 1-173), the central-linker region (aa 174-300), and the C-terminal domain (aa 301-448), were evaluated by Western blot. The rabbit polyclonal antibody demonstrated greater immunoreactivity to the central-linker region and the C-terminal domain than to the N-terminal domain. Three different patterns for the immunoreactivities of the three structural regions of HCoV-OC43 NP were observed in human serum, suggesting variability in the immune responses that occur during HCoV-OC43 infection in humans. The central-linker region of the NP appeared to be the most highly immunoreactive region for all three patterns observed. The goal of this study was to offer insight into the design of diagnostic tools for HCoV infection

Identification of Novel Cdc7 Kinase Inhibitors as Anti-Cancer Agents that Target the Interaction with Dbf4 by the Fragment Complementation and Drug Repositioning ApproachResearch in context

Background: Cdc7-Dbf4 is a conserved serine/threonine kinase that plays an important role in initiation of DNA replication and DNA damage tolerance in eukaryotic cells. Cdc7 has been found overexpressed in human cancer cell lines and tumor tissues, and the knockdown of Cdc7 expression causes an p53-independent apoptosis, suggesting that Cdc7 is a target for cancer therapy. Only a handful Cdc7 kinase inhibitors have been reported. All Cdc7 kinase inhibitors, including PHA-767491, were identified and characterized as ATP-competitive inhibitors. Unfortunately, these ATP-competitive Cdc7 inhibitors have no good effect on clinical trial. Methods: Here, we have developed a novel drug-screening platform to interrupt the interaction between Cdc7 and Dbf4 based on Renilla reniformis luciferase (Rluc)-linked protein-fragment complementation assay (Rluc-PCA). Using drug repositioning approach, we found several promising Cdc7 inhibitors for cancer therapy from a FDA-approved drug library. Findings: Our data showed that dequalinium chloride and clofoctol we screened inhibit S phase progression, accumulation in G2/M phase, and Cdc7 kinase activity. In addition, in vivo mice animal study suggests that dequalinium chloride has a promising anti-tumor activity in oral cancer. Interestingly, we also found that dequalinium chloride and clofoctol sensitize the effect of platinum compounds and radiation due to synergistic effect. In conclusion, we identified non-ATP-competitive Cdc7 kinase inhibitors that not only blocks DNA synthesis at the beginning but also sensitizes cancer cells to DNA damage agents. Interpretation: The inhibitors will be a promising anti-cancer agent and enhance the therapeutic effect of chemotherapy and radiation for current cancer therapy. Fund: This work was supported by grants from the Ministry of Science and Technology, Ministry of Health and Welfare, and National Health Research Institutes, Taiwan. Keywords: Cdc7 inhibitor, Drug repositioning, Cancer therapy, Protein fragment complementation assay, Protein-protein interactio

Illustration of the signaling pathway involved in the effect of HYS-32 on microtubule catastrophes in rat astrocytes.

<p>HYS-32 induces microtubule catastrophes by causing EB1 dislodgement from microtubule plus ends and EB1 accumulation on the microtubule lattice through the modulation of the PI3K-GSK3β signaling pathway.</p

Removal of HYS-32 rescues microtubule catastrophes.

<p>(A) Control astrocytes (Con), astrocytes treated for 24 h with 5 μM HYS-32 (HYS), or astrocytes treated for 24 h with HYS-32 then replaced with normal culture medium for 1 to 24 h (R1 h, R2 h, R6 h, or R24 h) in the absence of HYS-32 were fixed in cold acetone and triple-stained for β-tubulin (green), N-cadherin (N-cad), and F-actin (blue). Arrowheads indicate the intercellular junctions. Double-arrows indicate the distance between microtubule tips and cell border (bars = 20 μm). (B) Quantification of the mean distances between microtubule tips and cell border. The results were collected from three independent experiments. *<i>p</i><0.01 compared to control, ^#<i>p</i><0.01 compared to HYS using one-way ANOVA with Dunnett’s post-hoc test.</p

SB415286 and LY294002 partially restore EB1 distribution on microtubule plus ends.

<p>Control astrocytes (Con) or astrocytes treated for 24 h with 5 μM HYS-32 (HYS), co-treated for 24 h with 5 μM HYS-32 and 20μM SB415286 (HYS+SB) or LY294002 (HYS+LY), or treated with 20 μM SB415286 (SB)or LY294002 (LY) were fixed in cold acetone and double-stained for β-tubulin (green) and EB1 (red) and subjected to confocal microscopy. Images were merged to show co-localization (Merged). Square areas were enlarged to show EB1 distribution (Enlarged). Arrowheads indicate microtubule tips. Double-arrowheads indicate microtubule tips without EB1 signals. Arrows indicate distribution of EB1 along the microtubules. Dashed lines mark the cell border (bars = 5μm).</p

SB415286 partially reverses the HYS-32-inhibited astrocyte migration.

<p>(A) Control astrocytes (Con) or astrocytes treated with 5 μM HYS-32 (HYS), co-treated for 24 h with 5 μM HYS-32 and 20 μM SB415286 (HYS+SB), or treated with 20 μM SB415286 (SB) were analyzed with migration assay culture inserts 0, 12, 24, and 36 h after treatment (bars = 500 μm). (B) The data for the astrocytes migration area were collected from three independent experiments. *<i>p</i><0.01 compared to HYS using one-way ANOVA with Dunnett’s post-hoc test. The dotted lines marked the migration starting edge.</p

Removal of HYS-32 restores EB1 comet-like streaks.

<p>Control astrocytes (Con) or astrocytes treated for 24 h with 5 μM HYS-32 (HYS), or treated for 24 h with 5 μM HYS-32 then replaced with normal culture medium for 15 min to 24 h (R15 min, R1 h, or R24 h) were fixed in cold acetone and double-stained for β-tubulin (green) and EB1 (red), and subjected to confocal microscopy. Images were merged to show co-localization (Merged). Square areas were enlarged to show EB1 distribution (Enlarged). Arrowheads indicate microtubule tips. Arrows indicate distribution of EB1 along the microtubules (bars = 5μm).</p