Theaflavins enhance intestinal barrier of Caco-2 Cell monolayers through the expression of AMP-activated protein kinase-mediated Occludin, Claudin-1, and ZO-1

<div><p>We investigated the effect of theaflavins (TFs) on membrane barrier of Caco-2 cells. For fluorescein-transport experiments, the apparent permeability (<i>P</i>_app) of fluorescein in Caco-2 cells pretreated with 20 μM TFs were significantly decreased compared with that in untreated cells. Although the respective monomeric catechins did not show any <i>P</i>_app reduction, purpurogallin pretreatment resulted in a significant <i>P</i>_app reduction similar to that of TF-3′-<i>O</i>-gallate (TF3′G) pretreatment. This indicates that the benzotropolone moiety may play a crucial role in the <i>P</i>_app reduction or tight junction (TJ)-closing effect induced by TFs. In TF-3′-<i>O</i>-gallate-pretreated Caco-2 cells, fluorescein transport was completely restored by compound C (AMPK inhibitor). In addition, TF3′G significantly increased both the mRNA and protein expression of TJ-related proteins (occludin, claudin-1, and ZO-1) as well as the phosphorylation of AMPK. It was, thus, concluded that TFs could enhance intestinal barrier function by increasing the expression of TJ-related proteins through the activation of AMPK in Caco-2 cells.</p></div

Characterization of H7 Influenza A Virus in Wild and Domestic Birds in Korea

<div><p>During surveillance programs in Korea between January 2006 and March 2011, 31 H7 avian influenza viruses were isolated from wild birds and domestic ducks and genetically characterized using large-scale sequence data. All Korean H7 viruses belonged to the Eurasian lineage, which showed substantial genetic diversity, in particular in the wild birds. The Korean H7 viruses from poultry were closely related to those of wild birds. Interestingly, two viruses originating in domestic ducks in our study had the same gene constellations in all segment genes as viruses originating in wild birds. The Korean H7 isolates contained avian-type receptors (Q226 and G228), no NA stalk deletion (positions 69–73), no C-terminal deletion (positions 218–230) in NS1, and no substitutions in PB2-627, PB1-368, and M2-31, compared with H7N9 viruses. In pathogenicity experiments, none of the Korean H7 isolates tested induced clinical signs in domestic ducks or mice. Furthermore, while they replicated poorly, with low titers (10 ^0.7–1.3EID₅₀/50 µl) in domestic ducks, all five viruses replicated well (up to 7–10 dpi, 10 ^0.7–4.3EID₅₀/50 µl) in the lungs of mice, without prior adaptation. Our results suggest that domestic Korean viruses were transferred directly from wild birds through at least two independent introductions. Our data did not indicate that wild birds carried poultry viruses between Korea and China, but rather, that wild-type H7 viruses were introduced several times into different poultry populations in eastern Asia.</p></div

Phylogenies of N7 (<i>n</i> = 148) genes.

<p>Tip and branch colors represent host origin (wild birds in green, domestic birds in red) of all of the Korean H7 viruses, and asterisks denote the Korean H7 viruses isolated in the present study. Phylogenetic trees were constructed using the maximum likelihood method with general time-reversible model with invariant sites and 4 gamma-distributed heterogeneous substitution rates (GTR+ I + Γ4 model) and 100 bootstrap replications (H7 I = 0.285 α = 1.092; N9 I = 0.416 α = 1.452; N7 I = 0.411 α = 1.528; N3 I = 0.269 α = 0.858; N8 I = 0.371 α = 1.162; N2 I = 0.417 α = 1.590; N6 I = 0.309 α = 0.962) in PhyML 3.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091887#pone.0091887-Guindon1" target="_blank">[11]</a>. Statistical support for the phylogenies was assessed by the approximate likelihood test using a Shimodaira-Hasegawa-like procedure in PhyML 3.0. The topology of trees was visualized in FigTree 1.4. Viruses from different hosts are indicated by: wild birds, green; poultry, orange; human, pink.</p

Phylogenies of N8 (<i>n</i> = 194) genes.

<p>Tip and branch colors represent host origin (wild birds in green, domestic birds in red) of all of the Korean H7 viruses, and asterisks denote the Korean H7 viruses isolated in the present study. Phylogenetic trees were constructed using the maximum likelihood method with general time-reversible model with invariant sites and 4 gamma-distributed heterogeneous substitution rates (GTR+ I + Γ4 model) and 100 bootstrap replications (H7 I = 0.285 α = 1.092; N9 I = 0.416 α = 1.452; N7 I = 0.411 α = 1.528; N3 I = 0.269 α = 0.858; N8 I = 0.371 α = 1.162; N2 I = 0.417 α = 1.590; N6 I = 0.309 α = 0.962) in PhyML 3.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091887#pone.0091887-Guindon1" target="_blank">[11]</a>. Statistical support for the phylogenies was assessed by the approximate likelihood test using a Shimodaira-Hasegawa-like procedure in PhyML 3.0. The topology of trees was visualized in FigTree 1.4. Viruses from different hosts are indicated by: wild birds, green; poultry, orange; human, pink.</p

Phylogenies of NP (<i>n</i> = 472) genes.

<p>Tip and branch colors represent host origin (wild birds in green, domestic birds in red) of all of the Korean H7 viruses, and asterisks denote the Korean H7 viruses isolated in the present study. Phylogenetic trees were constructed using the maximum likelihood method with a general time-reversible model with invariant sites and 4 gamma-distributed heterogeneous substitution rates (GTR+ I + Γ4 model) and 100 bootstrap replications (PB2 I = 0.308 α = 0.749; PB1 I = 0.377 α = 0.899; PA I = 0.320 α = 0.773; NP 0.409 α = 0.874; M I = 0.146 α = 0.435; NS I = 0.161 α = 0.768) in PhyML 3.0 (Guindon et al., 2010). Statistical support for the phylogenies was assessed by the approximate likelihood test using a Shimodaira-Hasegawa-like procedure in PhyML 3.0. The topology of trees was visualized in FigTree 1.4.</p

Phylogenies of PB2 (<i>n</i> = 495) genes.

<p>Tip and branch colors represent host origin (wild birds in green, domestic birds in red) of all of the Korean H7 viruses, and asterisks denote the Korean H7 viruses isolated in the present study. Phylogenetic trees were constructed using the maximum likelihood method with a general time-reversible model with invariant sites and 4 gamma-distributed heterogeneous substitution rates (GTR+ I + Γ4 model) and 100 bootstrap replications (PB2 I = 0.308 α = 0.749; PB1 I = 0.377 α = 0.899; PA I = 0.320 α = 0.773; NP 0.409 α = 0.874; M I = 0.146 α = 0.435; NS I = 0.161 α = 0.768) in PhyML 3.0 (Guindon et al., 2010). Statistical support for the phylogenies was assessed by the approximate likelihood test using a Shimodaira-Hasegawa-like procedure in PhyML 3.0. The topology of trees was visualized in FigTree 1.4.</p

Phylogenies of M (<i>n</i> = 520) genes.

<p>Tip and branch colors represent host origin (wild birds in green, domestic birds in red) of all of the Korean H7 viruses, and asterisks denote the Korean H7 viruses isolated in the present study. Phylogenetic trees were constructed using the maximum likelihood method with a general time-reversible model with invariant sites and 4 gamma-distributed heterogeneous substitution rates (GTR+ I + Γ4 model) and 100 bootstrap replications (PB2 I = 0.308 α = 0.749; PB1 I = 0.377 α = 0.899; PA I = 0.320 α = 0.773; NP 0.409 α = 0.874; M I = 0.146 α = 0.435; NS I = 0.161 α = 0.768) in PhyML 3.0 (Guindon et al., 2010). Statistical support for the phylogenies was assessed by the approximate likelihood test using a Shimodaira-Hasegawa-like procedure in PhyML 3.0. The topology of trees was visualized in FigTree 1.4.</p

Antigenic analysis of H7 viruses isolated in Korea.

<p>*Values shown are HI titers. The titer of the homologous antigen group is shown in bold.</p><p>**r-value = (r¹×r²)1/2, r¹ = heterologous titer with virus 2/homologous titer with virus 1.</p><p>r² = heterologous titer with virus 1/homologous titer with virus.</p

Phylogenies of N8 (<i>n</i> = 194) genes.

<p>Tip and branch colors represent host origin (wild birds in green, domestic birds in red) of all of the Korean H7 viruses, and asterisks denote the Korean H7 viruses isolated in the present study. Phylogenetic trees were constructed using the maximum likelihood method with general time-reversible model with invariant sites and 4 gamma-distributed heterogeneous substitution rates (GTR+ I + Γ4 model) and 100 bootstrap replications (H7 I = 0.285 α = 1.092; N9 I = 0.416 α = 1.452; N7 I = 0.411 α = 1.528; N3 I = 0.269 α = 0.858; N8 I = 0.371 α = 1.162; N2 I = 0.417 α = 1.590; N6 I = 0.309 α = 0.962) in PhyML 3.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091887#pone.0091887-Guindon1" target="_blank">[11]</a>. Statistical support for the phylogenies was assessed by the approximate likelihood test using a Shimodaira-Hasegawa-like procedure in PhyML 3.0. The topology of trees was visualized in FigTree 1.4. Viruses from different hosts are indicated by: wild birds, green; poultry, orange; human, pink.</p

Pathotyping and replication of the selected H7 viruses in domestic ducks and mice.

<p>*Number of virus detected animals/number of virus inoculated animals. Virus titers inoculated intranasally with 10^6.5EID₅₀/0.1 ml of the virus.</p