Putative genomic organization of TTV group 6 by using TW53A26 as representative isolate.

<p>ORFs are marked with black arrows. Predicted donor and acceptor sites are linked by dashed border. The dotted line indicated UTR that was not included in our ORF amplicon. ORF1 encoded for 736 amino acids (nt 445–2652), ORF2 for 159 amino acids (nt 152–628), ORF3 for 347 amino acids (nt 152–627 and 2155–2719), ORF4 for 160 amino acids (nt 152–627 and 2357–2360). The numbering of nt 1 (the first) and the nt 3211 (last nucleotide) of TW53A26 is according to nt 225 and nt 3336 of TA278, respectively. Numbers in bracket indicated the corresponding nucleotide position in TA278. The full length of TTV group 6 is about 3,932 nt calculated by assuming the UTR is conserved with Hel32, a strain with the same length of translated ORF1. *Since it is not included in consensus sequence, this group that a nonsense mutation of ORF1 at nt 523 of isolate TW53A26 is ignored here.</p

Frequency distribution plot of p-distance for TTVs.

<p>The uncorrected distance matrix is calculated from the same data set as that used for phylogenetic analysis. Y axis represents the frequency counts in each bin. X axis represents the value of uncorrected pairwise distance. Horizontal bars indicate the distribution ranges. The gray zone indicates the overlapping regions between the distribution of intragroup and intergroup distances. Rectangles indicate the distance distribution ranges of each group and divided into left and right regions. Left regions show the distribution range of which pairwise distances are calculated within each group (intragroup distances). Right regions show the distribution range of pairwise distances that compared to strains of other groups (intergroup distances). The slash lines indicate the overlapping gray area of each group. Rectangles of group 6 and group 7 have small left regions because only limited strains are available. The subgroups of group 3 (3a, 3b, and 3c) are indicated by dashed rectangles. Dashed rectangles are also divided into two parts as well as major groups, except the right regions showing the distribution range of pairwise distances that compared to strains from two other subgroups in group 3.</p

Count of amino acid substitutions^a for putative ORF1 protein within TW53A25 sub-clade.

<p>Count of amino acid substitutions<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149901#t003fn001" target="_blank">^a</a> for putative ORF1 protein within TW53A25 sub-clade.</p

Pairwise similarity table for ORF1 amino acid sequences by comparing representative strains to TTV prototype TA278.

<p>Pairwise similarity table for ORF1 amino acid sequences by comparing representative strains to TTV prototype TA278.</p

Comparison of the TTV’s prevalence detected by UTR region between eastern Taiwanese indigenous population and general population from other geographic regions.

<p>Comparison of the TTV’s prevalence detected by UTR region between eastern Taiwanese indigenous population and general population from other geographic regions.</p

Unrooted Bayesian inferred maximum clade credibility tree of TTV ORF1 well aligned region.

<p>Posterior probabilities are labeled at major branches. Branches with a posterior probabilities equal or above 0.95 are considered as strong supported. Isolates from this study are labeled in bold. The scale bar at the left bottom indicates evolutionary distance in unit of substitutions per site per generation. The semicircular symbols indicate seven major TTV phylogenetic groups. The dash lines indicate tentatively classifications. Tentatively classified strains are labeled for reference. The SANBAN, SAa-01, and SENVG are arbitrarily chosen as representative for showing subgroup 3a, 3b, and 3c respectively. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149901#pone.0149901.s001" target="_blank">S1 Fig</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149901#pone.0149901.s005" target="_blank">S1 Appendix</a> for detail taxa list. *Label TW53A25 including 8 isolates (TW53A25, TW53A27, TW53A28, TW53A29, TW53A32, TW53A34, TW53A35, and TW53A39). **Label TW53A26 including 3 isolates (TW53A26, TW53A30, and TW53A31).</p

Genetic profiling of young and aged endothelial progenitor cells in hypoxia

<div><p>Age is a major risk factor for diseases caused by ischemic hypoxia, such as stroke and coronary artery disease. Endothelial progenitor cells (EPCs) are the major cells respond to ischemic hypoxia through angiogenesis and vascular remodeling. However, the effect of aging on EPCs and their responses to hypoxia are not well understood. CD34⁺ EPCs were isolated from healthy volunteers and aged by replicative senescence, which was to passage cells until their doubling time was twice as long as the original cells. Young and aged CD34⁺ EPCs were exposed to a hypoxic environment (1% oxygen for 48hrs) and their gene expression profiles were evaluated using gene expression array. Gene array results were confirmed using quantitative polymerase chain reaction, Western blotting, and BALB/c female athymic nude mice hindlimb ischemia model. We identified 115 differentially expressed genes in young CD34⁺ EPCs, 54 differentially expressed genes in aged CD34⁺ EPCs, and 25 common genes between normoxia and hypoxia groups. Among them, the expression of solute carrier family 2 (facilitated glucose transporter), member 1 (SLC2A1) increased the most by hypoxia in young cells. Gene set enrichment analysis indicated the pathways affected by aging and hypoxia most, including genes “response to oxygen levels” in young EPCs and genes involved “chondroitin sulfate metabolic process” in aged cells. Our study results indicate the key factors that contribute to the effects of aging on response to hypoxia in CD34⁺ EPCs. With the potential applications of EPCs in cardiovascular and other diseases, our study also provides insight on the impact of ex vivo expansion might have on EPCs.</p></div

All differentially expressed genes.

<p>All differentially expressed genes.</p

GSEA results in differentially expressed genes (old only).

<p>GSEA results in differentially expressed genes (old only).</p

Determination of hypoxia time.

<p>(A) VEGF Western blot time course showed that VEGF was upregulated within 24 hours and reached a plateau in 48 hours. N = 3; * P< .01; # P< .05 (B) VEGF upregulation was confirmed by expression microarray in 3 EPC clones.</p