Inhibition of innate immunity with cycloheximide treatment.

<p>(A) PA5 and PA5Δ<i>fliC</i> were infiltrated at 2×10⁷ cfu/ml [or with 5 µg/ml cycloheximide (Cx)] in tobacco leaf and photographed at 2 days after inoculation. Inoculation buffer (-) or 5 µg/ml cycloheximide infiltrated alone was served as a control. (B) Electrolyte leakage from leaf areas inoculated with indicated inoculum. Significant differences (<i>p</i><0.05) were indicated by asterisk (*). n.s., not significant differences (<i>p</i>>0.05).The experiment was repeated at least three times with similar results.</p

<i>P. syringae</i> flagellins or nonglycosylated flagellins can impair the HR elicitation by PA5Δ<i>fliC</i>.

<p>(A) The macroscopic HR elicition on <i>Nicotiana benthamiana</i> leaves. <i>fliC</i> mutant (PA5Δ<i>fliC</i>) at 1×10⁷ cfu/ml was infiltrated simultaneously with 0.8 µM of <i>P. syringae</i> pv. <i>averrhoi</i> flagellin, nonglycosylated flagellin (NG-flagellin), <i>A</i>. <i>tumefaciens</i> flagellin (Agro-flagellin) and BSA proteins, respectively, compared to inoculation with <i>fliC</i> mutant (PA5Δ<i>fliC</i>) and complemented strain [PA5Δ<i>fliC</i> (pNCHU1039)]. The leaf inoculated at concentration of 3.2 µM flagellin by syringe-infiltration was served as a control. The photos were taken at 2 days post inoculation. pNCHU1039: pBBR1MCS5 carrying <i>fliC</i>. (B) The <i>P. syringae</i> pv. <i>averrhoi</i> flagellin protein was purified from PA5 by using ultracentrifugation as described in Materials and Methods and separated by SDS-12% PAGE. The molecular sizes of marker proteins are indicated on the left. (C) Electrolyte leakage from leaf areas inoculated with indicated inoculum. The conductivity values represent the mean and standard error of three different leaves. Significant differences (<i>p</i><0.05) were indicated by asterisk (*). All experiments were repeated at least three times with similar results.</p

Flagellin glycosylation and motility of wild type (PA5) and the <i>fgt1</i> mutant (PA5Δ<i>fgt1</i>).

<p>(A) Staining on glycosylated flagellin. Purified proteins separated with SDS-12%-PAGE were stained by Coomassie brilliant blue (CBB) (upper panel) and by using a GelCode glycoprotein staining kit (Pierce, Rockford, III) (lower panel). Soybean trypsin inhibitor was served as a negative control (N) and purified flagellin from <i>P. s.</i> pv. <i>tabaci</i> (Pta) was a positive control. (B) Motility assay. For swimming assays (upper), bacterial strains were incubated for 2 days at 23°C on 0.3% soft agar of <i>hrp</i>MM plate. For swarming assays (bottom), bacterial strains were inoculated on 0.5% SWM agar plate and were observed after 24 h at 28°C.</p

The motility and ability of hypersensitive response (HR) elicitation of <i>P</i>. <i>syringae</i> pv. <i>averrhoi</i> strains.

a<p>Data were adapted from the report by Lin et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041056#pone.0041056-Lin1" target="_blank">[23]</a>.</p>b<p>This swimming assay was performed on 0.3% soft nutrient gelatin agar (NGA) plate.</p>c<p>The scale shown with ±standard error is the mean diameter of swimming area and is calculated on triplicates.</p>d<p>The bacteria were inoculated in tobacco leaves at 10⁸ cfu/ml.</p>e<p>Symbol -, + and ++ denote respectively that bacteria are non-motile, swimming slowly and swimming well.</p>f<p>The time represents the appearance of necrosis symptom and was recorded within 48 h.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Bacterial growth in nonhost tobacco and host starfruit leaves.

<p>(A) Tobacco leaves were infiltrated with 1×10⁵ cfu/ml of indicated strains. <i>P. syringae</i> pv. <i>tabaci</i> 11528 is virulent to tobacco and was served as a host strain for a positive control. (B) Starfruit leaves were infiltrated with1×10⁵ cfu/ml of indicated strains. The populations were measured from three 0.6-cm-diameter leaf discs at indicated days post inoculation. Error bars indicate the standard error of populations measured from three leaf discs from each of two plants. The experiment was repeated three times with similar results.</p

Cloning of flagellum gene cluster and glycosylation island and construction of <i>fliC</i> and <i>fgt1</i> mutants.

<p>(A) Organization of partial flagellum gene cluster and glycosylation island. The black lines represent the PCR fragments amplified by indicated primer pairs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041056#pone.0041056.s003" target="_blank">Table S1</a>) as shown with arrows above the fragments. The constructs used to delete <i>fliC</i> and <i>fgt1</i> genes are aligned below the gene structure. (B) <i>fliC</i> mutant was screened by PCR using primers PC1 and PD2 as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041056#pone-0041056-g001" target="_blank">Figure 1A</a>. (C) <i>fgt1</i> mutant was screened by PCR using primers prLTail and prOR1–2 as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041056#pone-0041056-g001" target="_blank">Figure 1A</a>.</p

<i>fliC</i> mutants of Pav elicited stronger HR in nonhost tobacco and tomato leaves.

<p>(A) Tobacco leaves (<i>Nicotiana tabacum</i> L. cv. Van-Hicks) were infiltrated with 2.5×10⁷ cfu/ml of <i>P. syringae</i> pv. <i>averrhoi</i> strains PA5, HL2, and HL9 and 2×10⁵ cfu/ml of <i>P. s.</i> pv. <i>tomato</i> DC3000 and photographed at 6 days after inoculation. (B) Tomato leaf (<i>Solanum lycopersicum</i> cv. Moneymaker) was infiltrated with the indicated strains at a concentration of 1×10⁷cfu/ml and photographed at 1 day after inoculation.</p

Flagellar expression and motility of <i>P. syringae</i> pv. <i>averrhoi</i> strains.

<p>(A) TEM images of <i>P. syringae</i> pv. <i>averrhoi</i> strains HL1 (a), PA5 (b), PA5Δ<i>fliC</i> (c) grown for 24 h on KB plates, deposited on TEM grids. Bars represent 200 nm and the arrow indicates the flagella only presented in wild type PA5. (B) Swimming assay. Bacterial strains were incubated for 2 days at 23°C on soft agar of <i>hrp</i> minimal medium or LB containing 10 mM MgCl₂ plate (as indicated as LB). (C) Immunoblot analysis of flagellin expression in HL1, PA5, <i>fliC</i> mutant and complemented strain was detected by anti-FliC antiserum. Except for the flagellin of HL1 induced in <i>hrp</i> minimal medium (MM), the others were prepared from cultures in KB medium. pNCHU1039: pBBR1MCS5 carrying <i>fliC</i>.</p

DataSheet_1_Outcomes of “sandwich” chemoradiotherapy compared with chemotherapy alone for the adjuvant treatment of FIGO stage III endometrial cancer.pdf

ObjectiveTo analyze and compare outcomes of adjuvant chemoradiotherapy in patients with International Federation of Gynecology and Obstetrics (FIGO) stage III endometrial cancer (EC) patients using the “Sandwich” sequence and chemotherapy (CT) alone.MethodsFrom, 2005 to, 2019, we retrospectively reviewed 80 patients with FIGO stage III EC who received treatment at our institute. We analyzed 66 patients who had undergone complete surgical staging followed by adjuvant treatment with sandwich chemoradiotherapy (39 patients) and CT alone (27 patients). The 5-year overall survival (OS), progression-free survival (PFS), and disease-specific survival (DSS) were calculated using the Kaplan–Meier method. Additional prognostic factors were analyzed using Cox proportional hazards regression.ResultsHerein, the analysis was conducted using 66 patients with a median follow-up period of 50 and 85 months in the sandwich and CT-alone arms. Comparing the sandwich sequence and CT-alone groups, the 5-year OS and PFS were 87% vs. 70% (p = 0.097) and 77% vs. 65% (p = 0.209), respectively. The sandwich therapy conferred an improved 5-year DSS (92% vs. 70%, p = 0.041) and a lower local recurrence rate (0% vs. 11%, p = 0.031). In multivariable analyses, grade 3 histology and deep myometrial invasion were independent risk factors for 5-year OS and DSS. The sandwich sequence was a positive predictor for 5-year DSS (hazard ratio [HR] = 0.23, p = 0.029). The sandwich arm demonstrated higher acute hematologic toxicity than the CT-alone arm. CT dose delay/reduction and treatment completion rates were similar in both groups.ConclusionFor patients with stage III EC, postoperative sandwich chemoradiotherapy appears to offer a superior 5-year DSS and local control with tolerable toxicity when compared with CT alone.</p