Identification of PLK1 phosphorylation sites on Axin.

<p>(A) Mass spectrometry analysis using Axin immunoprecipitates shows that Ser-157 in Axin is one of the phosphorylation sites of PLK1. Upper panel: HA-Axin was immunoprecipitated from cells co-expressing PLK1-WT; lower panel: from cells co-expressing PLK1-DN. (B) Axin-3SA mutant no longer displays its mobility shift when co-expressed with PLK1. HA-tagged Axin-WT, Axin-S157A, Axin-3SA, Axin-S490A, and Axin-S798A, Axin-S490A/S798A mutants are co-expressed with Myc-PLK1-WT or Myc-PLK1-DN in HEK293T cells, respectively. At 24 h post-transfection, cell lysates were subjected to immunoblotting as indicated. (C) Phosphorylation of Axin by PLK1 <i>in vitro</i>. HEK293T cells were transfected with HA-Axin-WT or HA-Axin serine-to-alanine mutants, and cell extracts were immunoprecipitated with HA-antibody. The immunocomplexes were incubated with purified FLAG-PLK-WT or FLAG-PLK1-DN in the presence of [γ-³²P] ATP for the kinase assay. Following the assay, samples were subjected to Coomassie Blue staining and autoradiography as indicated.</p

Co-expression of PLK1 abolishes Axin-γ-tubulin co-localization.

<p>(A) GFP-Axin-WT or GFP-Axin-S157A was co-transfected with empty vector, FLAG-PLK1-WT or FLAG-PLK1-DN into HeLa cells. Cells were fixed and immuno-stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049184#s4" target="_blank">Materials and Methods</a>. Axin was visualized by GFP, PLK1 by using anti-FLAG (red), γ-tubulin by using anti-γ-tubulin (pink), and nuclei by DAPI staining. (B) Bar chart showing the quantification data for the co-localization rates between γ-tubulin and GFP-Axin-WT or GFP-Axin-S157A at γ-tubulin foci in the background of FLAG-PLK1 or FLAG-PLK1-DN overexpression as shown in (A). <i>n</i> = 30 (“<i>n</i>” indicates the number of the cells quantified), N.S.: not significant; **<i>P</i><0.01 (ANOVA followed by tukey). (C) The centrosome region was circled and analyzed using Volocity software. The co-localization rate was analyzed between 488 channel (Axin) and 642 channel (γ-tubulin). Statistical analysis was performed as in (B). (D) FLAG-PLK1-WT and FLAG-PLK1-DN were transfected respectively into HeLa cells. Goat anti-Axin, mouse anti-FLAG and rabbit anti-γ-tubulin were used for staining of endogenous Axin, PLK1 and γ-tubulin, respectively. The intensity of endogenous Axin and γ-tubulin staining along the γ-tubulin foci is shown by line profiles. A 2 µm line is drawn along the centrosome region and the Y axis shows the intensity of 488 channel (Axin) and 642 channel (γ-tubulin) along the distance of the line (X axis).</p

Altered centrosome number in Axin-S157A expressing mitotic cells.

<p>(A) HeLa cells were transfected with GFP-Axin-S157A. Cells were treated with thymidine-nocodazole, then fixed and stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049184#s4" target="_blank">Materials and Methods</a>. GFP staining for Axin, anti-α-tubulin staining for microtubule (red), anti-pericentrin staining for centrosome (pink), and nuclear staining by DAPI were performed. (B) Bar chart representing the percentage of the interphase or mitotic HeLa cells with multiple centrosomes. Cells were transfected with GFP vector (control), GFP-Axin-WT or GFP-Axin-S157A mutant. “<i>n</i>” indicates the number of the mitotic cells quantified. The differences among interphase groups are not significant, <i>P</i> = 0.528; the differences among mitosis groups are significant (<i>P</i> = 0.014, Vector^a; Axin-WT^a,b; Axin-S157A^b, Chi-Square Tests). (C) Centrosome numbers are not altered in Axin-WT or Axin-S157A expressing interphase cells. HeLa cells were transfected with FLAG-Axin-WT or FLAG-Axin-S157A. Axin was visualized by using anti-FLAG (red), pericentrin by anti-pericentrin (green), and nuclei by DAPI staining. (D) Axin-S157A overexpression delays the chromosome segregation. The cells were synchronized at metaphase and released in fresh medium followed by staining with DAPI.</p

Axin phosphorylation by PLK1.

<p>(A) Total cell lysates (TCL) from cells expressing Axin or Axin2 with PLK1 or empty vector in HEK293T cells were analyzed by immunoblotting as indicated. (B) Axin interacts with PLK1 at its endogenous level. The HeLa cell lysates were immunoprecipitated with anti-Axin, mouse anti-PLK1 or control IgGs, respectively, followed by immunoblotting as indicated. (C) HA-Axin immunoprecipitates were subjected to CIP treatment and immunoblotting as indicated. (D) HeLa cells were mock treated or arrested in M-phase or early-S-phase by treatment with double-thymidine or thymidine-nocodazole. Axin immunoprecipitates were treated with CIP, followed by immunoblotting. (E, F) TCLs from cells expressing siRNA against <i>PLK1</i> (E) or were treated with 50 nM of PLK1 inhibitor 10 h before harvest (F) were analyzed by immunoblotting as indicated. (G) PLK1 phosphorylation on Axin at G2/M transition. HeLa cells were synchronized by double-thymidine block and released in fresh medium for indicated times. TCLs were analyzed by western blot and probed with antibodies as indicated.</p

Additional file 1 of Difference analysis and characteristics of incompatibility group plasmid replicons in gram-negative bacteria with different antimicrobial phenotypes in Henan, China

Supplementary Material 1: Antimicrobial susceptibility testing of 330 strain

The Brain Activity in Brodmann Area 17: A Potential Bio-Marker to Predict Patient Responses to Antiepileptic Drugs

<div><p>In this study, we aimed to predict newly diagnosed patient responses to antiepileptic drugs (AEDs) using resting-state functional magnetic resonance imaging tools to explore changes in spontaneous brain activity. We recruited 21 newly diagnosed epileptic patients, 8 drug-resistant (DR) patients, 11 well-healed (WH) patients, and 13 healthy controls. After a 12-month follow-up, 11 newly diagnosed epileptic patients who showed a poor response to AEDs were placed into the seizures uncontrolled (SUC) group, while 10 patients were enrolled in the seizure-controlled (SC) group. By calculating the amplitude of fractional low-frequency fluctuations (fALFF) of blood oxygen level-dependent signals to measure brain activity during rest, we found that the SUC patients showed increased activity in the bilateral occipital lobe, particularly in the cuneus and lingual gyrus compared with the SC group and healthy controls. Interestingly, DR patients also showed increased activity in the identical cuneus and lingual gyrus regions, which comprise Brodmann’s area 17 (BA17), compared with the SUC patients; however, these abnormalities were not observed in SC and WH patients. The receiver operating characteristic (ROC) curves indicated that the fALFF value of BA17 could differentiate SUC patients from SC patients and healthy controls with sufficient sensitivity and specificity prior to the administration of medication. Functional connectivity analysis was subsequently performed to evaluate the difference in connectivity between BA17 and other brain regions in the SUC, SC and control groups. Regions nearby the cuneus and lingual gyrus were found positive connectivity increased changes or positive connectivity changes with BA17 in the SUC patients, while remarkably negative connectivity increased changes or positive connectivity decreased changes were found in the SC patients. Additionally, default mode network (DMN) regions showed negative connectivity increased changes or negative changes with BA17 in the SUC patients. The abnormal increased in BA17 activity may be a key point that plays a substantial role in facilitating seizure onset.</p></div

Interface Engineering of Hollow CoO/Co₄S₃@CoO/Co₄S₃ Heterojunction for Highly Stable and Efficient Electrocatalytic Overall Water Splitting

The key to improve the performance of electrochemically water splitting and simplify the entire system is to develop a dual-functional catalyst, which can be applied to catalyze the process of HER and OER. Therefore, we synthesized a novel hollow CoO/Co4S3@CoO/Co4S3 heterojunction with a core–shell structure as an excellent dual-functional catalyst. This sample is composed of an outer hollow CoO/Co4S3 cubic thin shell and an inner hollow CoO/Co4S3 sphere, and it can provide abundant catalytic active sites while effectively promoting the flow of reactants, products, and electrolytes. Meanwhile, the O–Co–S bond in the heterojunction interface can promote both the CoO active site in OER and theCo4S3 active site in HER. Therefore, the overpotential of the hollow CoO/Co4S3@CoO/Co4S3 is only 190 mV (OER) and 81 mV (HER), respectively, at the current density of 10 mA cm–2. Moreover, the hollow CoO/Co4S3@CoO/Co4S3 showed the outstanding electrochemical stability in 60 h. In addition, in the two-electrode system assembled from the hollow CoO/Co4S3@CoO/Co4S3, only the potential of 1.48 V can achieve the current density of 10 mA cm–2. Impressively, the commercial solar panel is sufficient to drive the two-electrode electrolyzer consisting of hollow CoO/Co4S3@CoO/Co4S3. This finding offers a promising nonprecious metal-based catalyst that can be applied to catalyze the electrochemical overall water splitting

Spatial overlapping maps and scatter plots showing fALFF values of overlaps.

<p><b>A:</b> SUC vs. CON and DR vs. CON. Overlap regions account for 20% of SUC <i>vs</i>. controls, 41.7% of DR <i>vs</i>. controls, and 15.9% of total respectively. The yellow parts represents brain regions with common fALFF changes between SUC <i>vs</i>. controls and DR <i>vs</i>. controls including the bilateral lingual gyrus, cuneus, inferior occipital gyrus, middle occipital gyrus, right superior occipital gyrus, subcortical structure of left occipital lobe, subcortical structure of right temporal lobe, and left fusiform. The red parts show regions with fALFF differences from comparison of SUC vs. CON only. The blue parts show regions with fALFF differences from comparison of DR vs. CON only. <b>B:</b> SUC vs. SC and DR vs. WH. Overlap regions account for 27.2% of SUC <i>vs</i>. SC, 24.7% of DR <i>vs</i>. WH, and 14.9% of total respectively. The yellow parts represents brain regions with common fALFF changes between SUC <i>vs</i>. SC and DR <i>vs</i>. WH were found in the bilateral middle occipital gyrus, fusiform, lingual gyrus, the right superior occipital gyrus, cuneus and cerebellum posterior lobe. The red parts show regions with fALFF differences from comparison of SUC vs. SC only. The blue parts show regions with fALFF differences from comparison of DR vs. WH only. All comparisons were restrained in the ANOVA mask. <b>C:</b> fALFF values of overlap with common brain activity changes between SUC vs. CON and DR vs. CON. The error bar represents the standard deviation. * P< 0.001. <b>D:</b> fALFF values of overlap with common brain activity changes between SUC vs. SC and DR vs. WH. The error bar represents the standard deviation. * P = 0.001, * *P< 0.001.</p

Maps of fALFF differences.

<p><b>A:</b> SUC vs. control. Compared with healthy controls, the SUC patients showed significantly increased fALFF values in the warm color regions, including the bilateral cuneus, bilateral lingual gyrus, bilateral superior/middle/inferior occipital gyrus, and right posterior cingulate. <b>B:</b> SUC vs. SC. Compared with SC patients, the SUC patients showed significantly increased fALFF values in the warm color regions of the bilateral cuneus, bilateral lingual gyrus, bilateral middle temporal-occipital area, and right fusiform gyrus. <b>C:</b> DR vs. controls. The DR patients showed significantly increased fALFF values in the warm color regions of the bilateral cuneus, bilateral middle occipital gyrus, bilateral fusiform, and right middle temporal-occipital area. <b>D:</b> DR vs. WH. Compared with WH patients, the DR patients showed significantly increased fALFF values in the warm color regions of the left cuneus, bilateral fusiform, and right middle occipital gyrus. <b>E:</b> SC vs. CON. Compared with the healthy controls, the SC patients showed significantly increased fALFF values in the warm color region of the left inferior occipital gyrus. In contrast, the cold color regions in the right fusiform gyrus represent the area with decreased fALFF values in SC patients compared with controls. <b>F:</b> WH vs. CON. The WH patients showed only showed decreased fALFF values in the cold color region of the right fusiform gyrus. The statistical threshold was set at P < 0.05 with a cluster size > 351 mm³, which corresponded to a corrected P < 0.05.</p

Map of fALFF differences among the SUC, SC, DR, WH and control groups.

<p>There were significant fALFF differences among the five groups in the bilateral cuneus, lingual gyrus, inferior/middle occipital gyrus, calcarine, middle temporal-occipital area, fusiform, subcortical structure of left occipital lobe, subcortical structure of right temporal lobe, right posterior cingulated, and right cerebellum posterior lobe. The statistical threshold was set at P < 0.05 and a cluster size > 4158 mm³, which corresponded to a corrected P < 0.05.</p