PES inhibits NF-κB nuclear translocation, IκB-α degradation, and NF-κB phosphorylation in LPS-stimulated macrophages.

<p>(A) Pretreatment with PES (20 µM) significantly reduced NF-κB nuclear translocation (left part: cytoplasm; right part: nucleus) in LPS (2 µg/mL)-stimulated macrophages. (B, C) Quantitative analysis of NF-κB expression in the cytoplasm and nucleus after PES administration, n = 3, **<i>P</i><0.01 vs. control or PES alone-treated group. (D) Quantitative analysis of the effect of PES on NF-κB binding to its promoter DNA in macrophage, n = 3, *<i>P</i><0.05, **<i>P</i><0.01 vs. control or PES alone-treated group. (E) Representative images showing that PES suppressed IκB-α degradation in LPS-stimulated macrophages. (F) Quantitative analysis of the effect of PES on IκB-α degradation in LPS-stimulated macrophage, n = 3, **<i>P</i><0.01 vs. control. (G) Upper: PES (20 µM) reduced the LPS (2 µg/mL)-induced NF-κB phosphorylation level. Lower: quantitative analysis of the effect of PES on LPS-induced NF-κB phosphorylation, n = 3, *<i>P</i><0.05, **<i>P</i><0.01 vs. control. All data were shown as mean ± SE. NS: no significance.</p

PES prevents NHE1 association to Hsp70 in LPS-stimulated macrophages.

<p>(A) Representative images showing that PES (20 µM) suppressed NHE1-Hsp70 association in LPS (2 µg/mL)-stimulated macrophages. (B) Quantitative analysis of NHE1-Hsp70 association in LPS- and/or PES-treated macrophages, n = 3, **<i>P</i><0.01 vs. control or PES alone group. (C) Summary data showing the effect of PES on NHE1 and Hsp70 expression in macrophages (n = 3). (D) Representative images showing that PES (5 mg/kg) suppressed the association of NHE1 to Hsp70 in LPS (0.25 mg/kg)-stimulated liver tissues. (E) Quantitative analysis of NHE1 and Hsp70 association in LPS- and/or PES-stimulated liver tissues, n = 6, **<i>P</i><0.01 vs. control or PES alone group. (F) Summary data showing the effect of PES on NHE1 and Hsp70 expression in liver (n = 6). All data were shown as mean ± SE. NS: no significance.</p

Proposed scheme for the influence of PES on the signaling pathways downstream of LPS-induced inflammatory gene expression in macrophages.

<p>Proposed scheme for the influence of PES on the signaling pathways downstream of LPS-induced inflammatory gene expression in macrophages.</p

PES reduces LPS-induced increase in serum ALT/AST activity and extent of apoptosis in the liver.

<p>LPS (0.25 mg/kg) and PES (1, 5, and 10 mg/kg) were injected intraperitoneally in C57BL/6 mice. All mice were sacrificed 8 h post injection. Serum ALT (A) and AST (B) activity as well as the extent of apoptosis (C, D) were measured as described in methods. Values were shown as mean ± SE, n = 8, **<i>P</i><0.01 vs. LPS alone-injected mice. The upper images in C were magnified at 200×. The boxed areas were magnified at 400×. Bar: 20 µm. Arrows: TUNEL-positive cells.</p

PES reduces the infiltration of inflammatory cells and iNOS induction in inflammatory cells.

<p>(A) Representative images showing that PES significantly reduced the infiltration of inflammatory cells in LPS-stimulated (0.25 mg/kg) liver. The HE images were magnified at 400×. Bar: 20 µm. Arrows: inflammatory cells. (B) Quantitative analysis of PES on the number of infiltrated cells in liver after LPS injection, n = 6, **<i>P</i><0.01 vs. control. All data were shown as mean ± SE. NS: no significance.</p

PES reduces LPS-induced increase in pro-inflammatory factors in liver.

<p>(A) Statistical data showing that pretreatment with PES (1 and 5 mg/kg, 1 h) reduced serum nitrite content after LPS injection (0.25 mg/kg), n = 8, **<i>P</i><0.01 vs. LPS alone-injected mice. (B) The expression (upper) and quantitative analysis (lower) of iNOS in LPS/PES-injected liver, n = 6, *<i>P</i><0.05, **<i>P</i><0.01 vs. LPS alone-injected mice. (C, D) Quantitative analysis of PES on serum TNF-α (C) and IL-6 (D) content after LPS injection, n = 8, **<i>P</i><0.01 vs. LPS alone-injected mice. All data were shown as mean ± SE.</p

PES inhibits NF-κB nuclear translocation, IκB-α degradation, and NF-κB phosphorylation in LPS-stimulated liver.

<p>(A) Pretreatment with PES (5 mg/kg) significantly reduced NF-κB nuclear translocation (left part: cytoplasm; right part: nucleus) in LPS-stimulated (0.25 mg/kg) liver. (B, C) Quantitative analysis of NF-κB expression in the cytoplasm (B) and nucleus (C) after PES administration, n = 8, *<i>P</i><0.05, **<i>P</i><0.01 vs. control or PES alone group. (D) Quantitative analysis of the effect of PES on NF-κB binding to its promoter DNA in liver, n = 6, *<i>P</i><0.05, **<i>P</i><0.01 vs. control or PES alone-treated group. (E) Representative images showing that PES suppressed IκB-α degradation in LPS-stimulated liver. (F) Quantitative analysis of the effect of PES (5 mg/kg) on IκB-α degradation in LPS-stimulated liver, n = 8, *<i>P</i><0.05, **<i>P</i><0.01 vs. control mice. (G) Upper: representative images showing that PES down-regulated LPS-induced NF-κB phosphorylation level. Lower: quantitative analysis of the effect of PES on LPS-induced NF-κB phosphorylation in liver, n = 8, *<i>P</i><0.05, vs. control mice. All data were shown as mean ± SE. NS: no significance.</p

PES reduces the increase in [Ca²⁺]_i and intracellular pH value (pH_i) in LPS-stimulated macrophages.

<p>(A) Upper: the effect of BAPTA-AM (20 µM) on iNOS expression in LPS (2 µg/mL)-stimulated macrophages. Lower: quantitative analysis of iNOS expression in macrophages pre-incubated with PES and treated with LPS. n = 3, **<i>P</i><0.01 vs. LPS alone-treated group. (B) Representative 340/380 nm ratio of macrophages showing the change of [Ca²⁺]_i in macrophages as induced by LPS (2 µg/mL) in the absence or presence of PES (20 µM). (C) Summary data (▵F/F) of LPS-induced [Ca²⁺]_i elevation, n = 25 for LPS group; n = 30 for LPS and PES-co-treated group, **<i>P</i><0.01 vs. control. (D) Summary data (▵F/F) of the change in pH_i of macrophages after LPS stimulation in the absence or presence of PES (20 µM). n = 28, **<i>P</i><0.01 vs. control. All data were shown as mean ± SE.</p

PES attenuates iNOS induction in both protein and mRNA levels in LPS-stimulated macrophages.

<p>(A) Upper: PES pretreatment (30 min) inhibited iNOS expression in LPS (2 µg/mL)-stimulated cells at different concentrations (1, 5, 10, 20 µM). Lower: quantitative analysis of iNOS expression in cells upon PES and/or LPS treatment (n = 3, *<i>P</i><0.05, **<i>P</i><0.01 vs. LPS alone treated-controls). (B) Upper: representative images showing the time-dependent (4, 8, 16, 24 h) effect of PES on LPS-induced iNOS expression. Lower: a time-course analysis of iNOS expression upon PES incubation (n = 3, **<i>P</i><0.01 vs. control). (C, D) Quantitative analysis of NO content (C) and iNOS gene expression (D) in LPS- and/or PES-stimulated macrophages (n = 3, **<i>P</i><0.01 vs. control). (E) Quantitative analysis of the cell viability of macrophages after PES (1, 10, and 20 µM) treatment (n = 8). All data were shown as mean ± S.E. NS: no significance.</p

Optimized double-digest genotyping by sequencing (ddGBS) method with high-density SNP markers and high genotyping accuracy for chickens

<div><p>High-density single nucleotide polymorphism (SNP) markers are crucial to improve the resolution and accuracy of genome-wide association study (GWAS) and genomic selection (GS). Numerous approaches, including whole genome sequencing, genome sampling sequencing, and SNP chips are able to discover or genotype markers at different densities and costs. Achieving an optimal balance between sequencing resolution and budgets, especially in large-scale population genetics research, constitutes a major challenge. Here, we performed improved double-enzyme digestion genotyping by sequencing (ddGBS) on chicken. We evaluated eight double-enzyme digestion combinations, and <i>Eco</i>R I- <i>Mse</i> I was chosen as the optimal combination for the chicken genome. We firstly proposed that two parameters, optimal read-count point (ORP) and saturated read-count point (SRP), could be utilized to determine the optimal sequencing volume. A total of 291,772 high-density SNPs from 824 animals were identified. By validation using the SNP chip, we found that the consistency between ddGBS data and the SNP chip is over 99%. The approach that we developed in chickens, which is high-quality, high-density, cost-effective (300 K, $30/sample), and time-saving (within 48 h), will have broad applications in animal breeding programs.</p></div