Modulation of TLR expression in 661 W cells following S. aureus challenge.

<p>661W cells were stimulated with <i>S</i>. <i>aureus</i> (MOI 20:1) for the indicated time points. Total RNA was extracted, reverse transcribed, and subjected to semi-quantitative PCR for various TLRs (A). Band intensity was quantified by densitometric analysis using image J analysis software (NIH) and presented as the relative band intensity of TLRs vs. GAPDH (B). In a separate experiment, 661W cells were challenged with <i>S</i>. <i>aureus</i> (MOI 20:1) for 8 h and Immunostaining was performed using TLR2 specific antibody (C). TLR2 expression was further confirmed by Western blot analysis (D) and band intensity was quantified using Image J (E).</p

Western blot and IHC Analysis of TLR Expression in 661W cells.

<p>661W cells were either left untreated or challenged with the indicated TLR ligands (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119541#pone.0119541.g001" target="_blank">Fig. 1</a> legend) for 8h. Immunostaining was performed using specific antibodies to access TLR expression (A). In a separate experiment, cells were lysed using RIPA buffer and analyzed for TLRs expression by Western blot analysis using specific anti-TLR antibodies, with β-actin as a loading control (B). Band intensity was quantified by using Image J and presented as the relative band intensity of TLR vs. β-actin (C). Data points and error bars represent mean ± SD from two independent experiments.</p

Activation of TLR-downstream (NF-kB and MAPK) signaling following TLR agonist stimulation.

<p>In order to assess the activation of IkB, p38, and ERK signaling following stimulation with TLR agonists for 30 and 60 min., 661W cells were lysed in RIPA buffer and analyzed by Western blot analysis using antibodies against phospho-IkB (p-IkB), p-p38, and p-ERK. Antibodies against ERK, p38, and IkB were used to detect total protein levels (A-C). Band intensity was quantified using Image J and presented as the relative band intensity of the phosphorylated form vs. the total form for the respective proteins (A-C). To detect the nuclear translocation of NF-kB (p65), 661W cells were challenged for 60 min. with various TLR ligands. Cells were then fixed, permeablized, and immune-stained with antibodies against p65, a subunit of NF-kB (D). Statistical analysis was performed using one-way ANOVA (*, p<0.05), for comparisons of control versus 30 and 60 min. stimulation.</p

Effect of TLR agonists on secretion of inflammatory mediators in 661W cells.

<p>661W cells were challenged with the indicated TLR ligands for 8h and the secretion of inflammatory mediators was measured in conditioned media by ELISA. Data points and error bars represents mean ± SD of duplicates from three independent experiments. Statistical analysis was performed using one-way ANOVA (*, p<0.05; **, p<0.005; ***, p<0.005), for comparisons of control versus stimulated cells.</p

Effect of TLR2 neutralization on inflammatory responses towards S. aureus.

<p>661W cells were challenged with SA for 8 h following neutralization of TLR2 using anti-TLR2 neutralizing antibody. The expression of secretory cytokines and chemokines were measured by ELISA in the conditioned medium.Statistical analysis was performed using one-way ANOVA (**, p<0.005; ns, not significant).</p

S. aureus-triggered innate responses in 661W cells.

<p>661W cells were challenged with <i>S</i>. <i>aureus</i> (MOI 20:1) for the indicated time points and Western blot was performed for IkB, p38, and ERK using phosphorylated and non-phosphorylated antibodies (A). Band intensity was quantified using Image J (B). Immunostaining was performed to monitor the localization of NF-kB to the nucleus (C). qRT-PCR was performed for the quantification of induced mRNA expression of inflammatory mediators (IL-6, IL-1β, CXCL1, and CXCL2) and the results were expressed as relative fold changes with respect to a GAPDH control (D). The level of secretary cytokines/chemokines was measured by ELISA (E). Data points and error bars represents mean ± SD of triplicates from two independent experiments. Statistical analysis was performed using one-way ANOVA (*, p<0.05; **, p<0.005; ***, p<0.0005), for comparisons of control versus stimulated cells.</p

RT-PCR analysis of TLR expression in 661W cells.

<p>661W cells were either left untreated (control) or challenged with the indicated TLR ligands: Pam3CSK4 (10 μg/ml), Poly(IC) (10 μM), LPS (10 μg/ml), Flagellin (250 ng/ml), Poly(dT) (10 μM), or ODN (10 μg/ml) for 8h. Total RNA was extracted, reverse transcribed, and subjected to semi-quantitative RT-PCR using primers for specific TLRs, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the control (A). Band intensity was quantified by densitometric analysis using Image J analysis software (NIH) and presented as the relative band intensity of TLRs vs. GAPDH (B). Data points and error bars represent mean ± SD from two independent experiments.</p

Wheat-blast vulnerability indicators in Bangladesh, Pakistan and India.

<p>Wheat-blast vulnerability indicators in Bangladesh, Pakistan and India.</p

South Asia wheat-blast vulnerability map.

<p>Source: Authors’ own estimation.</p

Wheat-blast symptoms in Jhenaidah district, Bangladesh in February 2017.

<p><b>Panel A: Infected spikes; Panel B: Infected field.</b> Courtesy: Moksedul A. Arafat, Technical Officer, Jessore Hub, CIMMYT, Bangladesh, 2017.</p