Iversity of kinetics better related to species ecology than phylogeny [4]. All

Iversity of kinetics better related to species ecology than phylogeny [4]. All eight residues shown under selection in Amaranthaceae using SLR and PAML models M2 and M8 were already shown to be under Darwinian selection in other groups of plants [6]. Five of these residues (145, 225, 262, 279 and 439) were among twenty most commonly selected Rubisco large subunit residues [6]. Findings in Amaranthaceae are in agreement with the previously described uneven distribution of putative fine-tuning residues in Rubisco [6]. Residues 43, 145, 225, 262 and 279 had only twoResults Phylogenetic analysisThe ML phylogenetic tree (Fig. 1) for rbcL sequences from 179 Amaranthaceae species was largely congruent with previously obtained phylogenies and accepted taxonomic subdivisions of the family [19,28,29,30,45,46,47,48]; however no statistical tests for topological similarity between our tree and previously published trees were performed because of different sizes and species compositions of datasets. A minimum of 16 independent origins of C4 photosynthesis were represented in the Amaranthaceae phylogeny if conservative approach for observed polytomies had been taken (Fig. 1), which is consistent with the PZ-51 web estimate by Sage et al. [16]. The other assumption of this estimate was that no reversals from C4 to C3 were allowed. Predominance of C4 gains over reversals to C3 is supported by both empirical data and theoretical work [49].Tests for positive selectionLikelihood ratio tests (LRTs) for variation in dN/dS ratios and for positive selection [33] were applied to the dataset of rbcL sequences from 179 C3 and C4 Amaranthaceae species. LRTs that were run using two different initial dN/dS values (0.1 and 0.4) to test for suboptimal local peaks produced identical results. LRTs for positive selection [33] showed that the models assuming positive selection (M2a and M8) fit the data better than the nested models without positive selection (M1a and M8a; p-value ,0.00001;Rubisco Evolution in C4 EudicotsTable 2. Characteristics of amino-acid replacements under positive selection in the C4 lineages of Amaranthaceae.AA No.aAA order 520-26-3 changes `C3’R`C4’Type of changesbDHcDPdDVeSAf ( )DGg (kJ/mol)RFPS ( ) hC3/ C4 species iLocation of residueStructural motifs ?within 5 AInteractionsj281A MR RS IHN R UP HN R HN22.6 2.1.1 20.0.4 3.0.00 8.DS (210.6) S (21.3)2.7 19.2.1/34.5 0.0/16.Helix 4 Strand FHelices 4, 5 Strand E; Helices F,DD IDAmino acid (AA) numbering is based on the spinach sequence after [63]. Side chain type changes. Types abbreviations: H ?hydrophobic; N ?nonpolar aliphatic; P ?polar uncharged; U ?hydrophilic (after [64]). Hydropathicity difference [65]. d Polarity difference [66]. e van der Waals volume difference [67]. f Solvent accessibility calculated using the spinach structure (pdb file 1RBO) by CUPSAT [44]. g Overall stability of the protein predicted using the spinach structure (pdb file 1RBO) by CUPSAT [44]. DS ?destabilizing, S ?stabilizing. h RFPS ?relative frequency of the particular residue to be under positive selection in C3 plants. Data from 112 rbcL datasets with detected positive selection from [6]. i Percentage of C3 and C4 species that have `C4′ amino acid among the 95 C3 species and 84 C4 species of Amaranthaceae analysed. j ?Interactions in which the selected residues and/or residues within 5 A of them are involved. ID ?intradimer interactions; DD ?dimer-dimer interactions (after [63]). doi:10.1371/journal.pone.0052974.tb caalternative amino acids.Iversity of kinetics better related to species ecology than phylogeny [4]. All eight residues shown under selection in Amaranthaceae using SLR and PAML models M2 and M8 were already shown to be under Darwinian selection in other groups of plants [6]. Five of these residues (145, 225, 262, 279 and 439) were among twenty most commonly selected Rubisco large subunit residues [6]. Findings in Amaranthaceae are in agreement with the previously described uneven distribution of putative fine-tuning residues in Rubisco [6]. Residues 43, 145, 225, 262 and 279 had only twoResults Phylogenetic analysisThe ML phylogenetic tree (Fig. 1) for rbcL sequences from 179 Amaranthaceae species was largely congruent with previously obtained phylogenies and accepted taxonomic subdivisions of the family [19,28,29,30,45,46,47,48]; however no statistical tests for topological similarity between our tree and previously published trees were performed because of different sizes and species compositions of datasets. A minimum of 16 independent origins of C4 photosynthesis were represented in the Amaranthaceae phylogeny if conservative approach for observed polytomies had been taken (Fig. 1), which is consistent with the estimate by Sage et al. [16]. The other assumption of this estimate was that no reversals from C4 to C3 were allowed. Predominance of C4 gains over reversals to C3 is supported by both empirical data and theoretical work [49].Tests for positive selectionLikelihood ratio tests (LRTs) for variation in dN/dS ratios and for positive selection [33] were applied to the dataset of rbcL sequences from 179 C3 and C4 Amaranthaceae species. LRTs that were run using two different initial dN/dS values (0.1 and 0.4) to test for suboptimal local peaks produced identical results. LRTs for positive selection [33] showed that the models assuming positive selection (M2a and M8) fit the data better than the nested models without positive selection (M1a and M8a; p-value ,0.00001;Rubisco Evolution in C4 EudicotsTable 2. Characteristics of amino-acid replacements under positive selection in the C4 lineages of Amaranthaceae.AA No.aAA changes `C3’R`C4’Type of changesbDHcDPdDVeSAf ( )DGg (kJ/mol)RFPS ( ) hC3/ C4 species iLocation of residueStructural motifs ?within 5 AInteractionsj281A MR RS IHN R UP HN R HN22.6 2.1.1 20.0.4 3.0.00 8.DS (210.6) S (21.3)2.7 19.2.1/34.5 0.0/16.Helix 4 Strand FHelices 4, 5 Strand E; Helices F,DD IDAmino acid (AA) numbering is based on the spinach sequence after [63]. Side chain type changes. Types abbreviations: H ?hydrophobic; N ?nonpolar aliphatic; P ?polar uncharged; U ?hydrophilic (after [64]). Hydropathicity difference [65]. d Polarity difference [66]. e van der Waals volume difference [67]. f Solvent accessibility calculated using the spinach structure (pdb file 1RBO) by CUPSAT [44]. g Overall stability of the protein predicted using the spinach structure (pdb file 1RBO) by CUPSAT [44]. DS ?destabilizing, S ?stabilizing. h RFPS ?relative frequency of the particular residue to be under positive selection in C3 plants. Data from 112 rbcL datasets with detected positive selection from [6]. i Percentage of C3 and C4 species that have `C4′ amino acid among the 95 C3 species and 84 C4 species of Amaranthaceae analysed. j ?Interactions in which the selected residues and/or residues within 5 A of them are involved. ID ?intradimer interactions; DD ?dimer-dimer interactions (after [63]). doi:10.1371/journal.pone.0052974.tb caalternative amino acids.


Xpressed as mean 6 SD (n = 4). *, P,0.05 versus WT. doi:10.1371/journal.pone.

Xpressed as mean 6 SD (n = 4). *, P,0.05 versus WT. doi:10.1371/journal.pone.0050094.gFoxM1 Promotes Endothelial RepairFigure 2. Accelerated resolution of lung inflammation in FoxM1 Tg mice. (A) MPO activities in lung tissues. Lung tissues at indicated times post-CLP challenge were collected for MPO activity determination. Lung tissues from order Madrasin Sham-operated mice at 24 h post-surgery were collected as controls. Data are expressed as mean 6 SD (n = 3?). *, P,0.001 versus WT; **, P,0.05 versus WT. (B) Representative micrographs of H E staining of lung sections. At 24 h post-surgery, lungs were fixed for sectioning and H E staining. Arrows indicate perivascular leukocyte infiltration. Scale bar, 50 mm. doi:10.1371/journal.pone.0050094.gWe next determined whether FoxM1 expression in EC is indispensable for endothelial repair. As shown in Fig. 8A, both WT and FoxM1 CKO mice exhibited similar increases of lung vascular permeability at 18 h post-CLP challenge. At late time points (48 h and 72 h), lung vascular permeability in WT mice was markedly reduced whereas it remained elevated in FoxM1 CKO mice at levels similar to peak injury. Similarly, we observed a sustained increase of MPO activity in FoxM1 CKO lungs at 48 h and 72 h post-CLP challenge whereas MPO activity in WT lungs was drastically decreased at 48 h post-CLP challenge and returned to levels similar to baseline seen in sham-operated controls at 72 h post-CLP challenge (Fig. 8B).DiscussionWe have identified the necessary and sufficient role of FoxM1 in promoting endothelial repair and resolution of lung inflammation in a clinically relevant model of sepsis. 1480666 We showed that transgenicexpression of FoxM1 resulted in rapid recovery of vascular integrity and resolution of lung inflammation following CLPinduced polymicrobial sepsis. FoxM1 Tg mice exhibited a marked increase of survival. Mechanistically, we observed early induction of FoxM1 target genes essential for cell cycle progression and resulting endothelial proliferation in FoxM1 Tg lungs following CLP challenge. Additionally, selective deletion of FoxM1 in EC resulted in defective endothelial repair in FoxM1 CKO mice following CLP-induced lung vascular injury. Together, these data demonstrate the critical role of FoxM1 in mediating endothelial repair following lung vascular injury induced by sepsis. Endothelial repair requires endothelial regeneration and subsequent re-annealing of the endothelial cell-cell contacts to restore the characteristic restrictive endothelial barrier Docosahexaenoyl ethanolamide chemical information function [2,8,29]. Our previous studies have demonstrated the essential 1407003 role of FoxM1 in regulating endothelial proliferation and re-annealing of the endothelial adherens junctions complexes employing the FoxM1 CKO mice [18,19]. This study further demonstrated theFigure 3. Normalized expression of proinflammatory cytokines and adhesion molecule in FoxM1 Tg lungs at 24 h post-CLP. RNA were isolated from lungs collected at 24 h post-surgery and QRT-PCR analysis were employed to assess the expression levels. Data are expressed as mean 6 SD (n = 3?). *, P,0.05 versus WT-sham. doi:10.1371/journal.pone.0050094.gFoxM1 Promotes Endothelial RepairFigure 4. Increased survival of FoxM1 Tg mice following CLP challenge. 3 month old mice were monitored for 7 days to determine the survival rate following CLP challenge (n = 13 WT and 14 FoxM1 Tg). Sham-operated mice (n = 5 WT or FoxM1 Tg) were also monitored for survival. *, P,0.001 versus CLP-WT. Tg, FoxM1 Tg. doi:10.1371/.Xpressed as mean 6 SD (n = 4). *, P,0.05 versus WT. doi:10.1371/journal.pone.0050094.gFoxM1 Promotes Endothelial RepairFigure 2. Accelerated resolution of lung inflammation in FoxM1 Tg mice. (A) MPO activities in lung tissues. Lung tissues at indicated times post-CLP challenge were collected for MPO activity determination. Lung tissues from sham-operated mice at 24 h post-surgery were collected as controls. Data are expressed as mean 6 SD (n = 3?). *, P,0.001 versus WT; **, P,0.05 versus WT. (B) Representative micrographs of H E staining of lung sections. At 24 h post-surgery, lungs were fixed for sectioning and H E staining. Arrows indicate perivascular leukocyte infiltration. Scale bar, 50 mm. doi:10.1371/journal.pone.0050094.gWe next determined whether FoxM1 expression in EC is indispensable for endothelial repair. As shown in Fig. 8A, both WT and FoxM1 CKO mice exhibited similar increases of lung vascular permeability at 18 h post-CLP challenge. At late time points (48 h and 72 h), lung vascular permeability in WT mice was markedly reduced whereas it remained elevated in FoxM1 CKO mice at levels similar to peak injury. Similarly, we observed a sustained increase of MPO activity in FoxM1 CKO lungs at 48 h and 72 h post-CLP challenge whereas MPO activity in WT lungs was drastically decreased at 48 h post-CLP challenge and returned to levels similar to baseline seen in sham-operated controls at 72 h post-CLP challenge (Fig. 8B).DiscussionWe have identified the necessary and sufficient role of FoxM1 in promoting endothelial repair and resolution of lung inflammation in a clinically relevant model of sepsis. 1480666 We showed that transgenicexpression of FoxM1 resulted in rapid recovery of vascular integrity and resolution of lung inflammation following CLPinduced polymicrobial sepsis. FoxM1 Tg mice exhibited a marked increase of survival. Mechanistically, we observed early induction of FoxM1 target genes essential for cell cycle progression and resulting endothelial proliferation in FoxM1 Tg lungs following CLP challenge. Additionally, selective deletion of FoxM1 in EC resulted in defective endothelial repair in FoxM1 CKO mice following CLP-induced lung vascular injury. Together, these data demonstrate the critical role of FoxM1 in mediating endothelial repair following lung vascular injury induced by sepsis. Endothelial repair requires endothelial regeneration and subsequent re-annealing of the endothelial cell-cell contacts to restore the characteristic restrictive endothelial barrier function [2,8,29]. Our previous studies have demonstrated the essential 1407003 role of FoxM1 in regulating endothelial proliferation and re-annealing of the endothelial adherens junctions complexes employing the FoxM1 CKO mice [18,19]. This study further demonstrated theFigure 3. Normalized expression of proinflammatory cytokines and adhesion molecule in FoxM1 Tg lungs at 24 h post-CLP. RNA were isolated from lungs collected at 24 h post-surgery and QRT-PCR analysis were employed to assess the expression levels. Data are expressed as mean 6 SD (n = 3?). *, P,0.05 versus WT-sham. doi:10.1371/journal.pone.0050094.gFoxM1 Promotes Endothelial RepairFigure 4. Increased survival of FoxM1 Tg mice following CLP challenge. 3 month old mice were monitored for 7 days to determine the survival rate following CLP challenge (n = 13 WT and 14 FoxM1 Tg). Sham-operated mice (n = 5 WT or FoxM1 Tg) were also monitored for survival. *, P,0.001 versus CLP-WT. Tg, FoxM1 Tg. doi:10.1371/.


Monium are produced by brain cells under the action of GA

Monium are produced by brain cells under the action of GA and 3-OHGA, suggesting a central liberation of ammonium in GA-I. Following the guidelines for GA-I, ammonium is not routinely determined during an acute illness [10,11], but could be worth to be measured in CSF. Ammonium is known to be toxic for brain cells causing reduced axonal elongation [16] as well as neuronal and oligodendrocytic cell death [15,18], which correlates with the brain atrophy and white matter changes observed in patients with primary hyperammonemias [20]. Its detection in brain cell cultures challenged with GA and 3-OHGA immediately raises the question of a potential role for ammonium in brain damage occurring in GA-I patients. As urea cycle is not active in central nervous system, ammonium produced during amino acid catabolism is mainly detoxified through amination of glutamate to Title Loaded From File glutamine by the enzyme glutamine synthetase. This enzyme is exclusivelyBrain Cell Damage in Glutaric Title Loaded From File Aciduria Type IFigure 5. Effects of GA and 3-OHGA on biochemical parameters measured in culture medium. Glucose (A), lactate (B), ammonium (C) and glutamine (D) were measured in the medium of cultures treated with protocols A (DIV 8) or B (DIV 14). Mean 6 SD of 7 replicate cultures assessed by Student’s t-test; *p,0.05, **p,0.01, *** p,0.001. doi:10.1371/journal.pone.0053735.gBrain Cell Damage in Glutaric Aciduria Type IFigure 6. Evaluation of cell death after treatment with GA and 3-OHGA. (A; left panel) Immunohistochemical staining for cleaved caspase-3 (red signal). Scale bar: 100 mm. (A; right panel) Representative western blots with data quantification of whole-cell lysates for full length caspase-3 and the large fragment of cleaved (e.g. activated) caspase-3 for protocol A (DIV 8, above) and protocol B (DIV 14, below). Actin was used as a loading control. The quantifications of cleaved caspase-3 are expressed as percentage of respective controls. The values represent the mean 6 SEM from 3 replicates taken from 2 independent experiments. (B) In situ cell death assay with TUNEL (green signal) and cleaved caspase-3 (red signal) on DIV 8 (protocol A). Merge of both signals leads to double-stained cells appearing in yellow. Scale bar: 100 mm. (C) LDH in culture medium of cultures from protocol A (DIV 8, above) and protocol B (DIV 14, below). Mean 6 SD of 7 replicate cultures assessed by Student’s t-test; **p,0.01, *** p,0.001. doi:10.1371/journal.pone.0053735.gBrain Cell Damage in Glutaric Aciduria Type Isupported by the observation of neuronal loss in the Gcdh2/2 mouse model [13]. Analysis of media from treated and control cultures on DIV 14 showed a marked increase in lactate with concomitant decrease in glucose concentrations. This combination can be observed in plasma of children with GA-I during acute encephalopathic crises. Underlying mechanisms may be the inhibition of the TCA cycle and/or respiratory chain with shift to lactate at the end of glycolysis, which is also supported by the 2-fold increase of the lactate/pyruvate ratio observed under 3-OHGA exposure. Lamp et al. have shown that 3-OHGA and GA inhibit astrocytic efflux and neuronal uptake of TCA cycle intermediates. These results suggest that elevated levels of 3-OHGA and GA may lead to neuronal injury and cell death via disruption of TCA cycle activity [21]. Direct effects on the respiratory chain have been reported controversially: While a recent report failed to prove changes on the activity of the different respi.Monium are produced by brain cells under the action of GA and 3-OHGA, suggesting a central liberation of ammonium in GA-I. Following the guidelines for GA-I, ammonium is not routinely determined during an acute illness [10,11], but could be worth to be measured in CSF. Ammonium is known to be toxic for brain cells causing reduced axonal elongation [16] as well as neuronal and oligodendrocytic cell death [15,18], which correlates with the brain atrophy and white matter changes observed in patients with primary hyperammonemias [20]. Its detection in brain cell cultures challenged with GA and 3-OHGA immediately raises the question of a potential role for ammonium in brain damage occurring in GA-I patients. As urea cycle is not active in central nervous system, ammonium produced during amino acid catabolism is mainly detoxified through amination of glutamate to glutamine by the enzyme glutamine synthetase. This enzyme is exclusivelyBrain Cell Damage in Glutaric Aciduria Type IFigure 5. Effects of GA and 3-OHGA on biochemical parameters measured in culture medium. Glucose (A), lactate (B), ammonium (C) and glutamine (D) were measured in the medium of cultures treated with protocols A (DIV 8) or B (DIV 14). Mean 6 SD of 7 replicate cultures assessed by Student’s t-test; *p,0.05, **p,0.01, *** p,0.001. doi:10.1371/journal.pone.0053735.gBrain Cell Damage in Glutaric Aciduria Type IFigure 6. Evaluation of cell death after treatment with GA and 3-OHGA. (A; left panel) Immunohistochemical staining for cleaved caspase-3 (red signal). Scale bar: 100 mm. (A; right panel) Representative western blots with data quantification of whole-cell lysates for full length caspase-3 and the large fragment of cleaved (e.g. activated) caspase-3 for protocol A (DIV 8, above) and protocol B (DIV 14, below). Actin was used as a loading control. The quantifications of cleaved caspase-3 are expressed as percentage of respective controls. The values represent the mean 6 SEM from 3 replicates taken from 2 independent experiments. (B) In situ cell death assay with TUNEL (green signal) and cleaved caspase-3 (red signal) on DIV 8 (protocol A). Merge of both signals leads to double-stained cells appearing in yellow. Scale bar: 100 mm. (C) LDH in culture medium of cultures from protocol A (DIV 8, above) and protocol B (DIV 14, below). Mean 6 SD of 7 replicate cultures assessed by Student’s t-test; **p,0.01, *** p,0.001. doi:10.1371/journal.pone.0053735.gBrain Cell Damage in Glutaric Aciduria Type Isupported by the observation of neuronal loss in the Gcdh2/2 mouse model [13]. Analysis of media from treated and control cultures on DIV 14 showed a marked increase in lactate with concomitant decrease in glucose concentrations. This combination can be observed in plasma of children with GA-I during acute encephalopathic crises. Underlying mechanisms may be the inhibition of the TCA cycle and/or respiratory chain with shift to lactate at the end of glycolysis, which is also supported by the 2-fold increase of the lactate/pyruvate ratio observed under 3-OHGA exposure. Lamp et al. have shown that 3-OHGA and GA inhibit astrocytic efflux and neuronal uptake of TCA cycle intermediates. These results suggest that elevated levels of 3-OHGA and GA may lead to neuronal injury and cell death via disruption of TCA cycle activity [21]. Direct effects on the respiratory chain have been reported controversially: While a recent report failed to prove changes on the activity of the different respi.


Regulator.Histone Methylation Dynamics in SeedsMajor changes in transcript abundance of

Regulator.Histone Methylation Dynamics in SeedsMajor changes in transcript abundance of the genes encoding regulators and markers of seed maturation and/or dormancy occurred during dormancy-termination per se (e.g. DOG1 and FLC), or once germination had been induced (e.g. ABI3 and LEC2) (Fig. 4). SOM expression was most strongly down-regulated upon the completion of germination (Fig. 4). The “marker” genes, RAB18 and 2S1, showed the greatest decline in abundance during germination (Fig. S2). The switch from activating H3K4me3- to repressive H3K27me3-deposition was associated with a change in transcript level of the dormancy regulators (Fig. 4). We are thus able to discriminate between genes that are required for germination and genes JSI124 chemical information involved in dormancy by their H3 methylation patterns. The former show a strong transcriptional up-regulation during germination that is associated with H3K4me3 deposition. This mark seems to be stable throughout further development and growth as it is also found in genome-wide H3K4me3 profiling studies using 10?0 day old seedlings [13,31,32]. The dormancy regulators were found to maintain Eliglustat site H3K27me3 throughout the subsequent seedling stage [13,33,34]. The transition to another life phase is directly reflected in a change at the chromatin level that is then maintained throughout further development. The cue for this life-cycle transition is the exposure of the imbibed seeds to low temperatures. The environmental temperature signal is therefore transduced to effect the observed chromatin changes. It is of interest to investigate whether the same patterns of histone modifications are transduced by other cues that effectively break seed dormancy such as afterripening. FLC deviates from the general pattern of a maintenance of repressive marks throughout the rest of the life cycle. Although this gene also showed a replacement of H3K4me3 by H3K27me3 during seed dormancy release by moist chilling and germination, FLC must be reset to an active state very soon after germination to fulfill its role as a negative regulator of flowering. However FLC has been tested both positive and negative for H3K27me3 in Arabidopsis plants, depending on natural variation, developmental state, and possibly growth conditions, respectively [28,34,35]. Recent work by 1317923 R.R. de Casas et al. [36] shows that moist chilling of seeds leads to earlier flowering in the resulting plants independently of the dormancy status of the seeds. It is thus possible that the appearance of H3K27me3 on FLC is caused by exposure to low temperatures, and not by the physiological process of dormancy breakage per se. The exposure of seeds to moist chilling might thereby lead to FLC repression on the chromatin level such that earlier flowering is promoted in the adult plants. A. Angel et al. [37] have described a nucleation process that takes place on the FLC-locus during induction of flowering competence through vernalization: H3K27me3 accumulates slowly over weeks of cold exposure in one segment of the FLC gene in the sampling population. When plants are returned to warm conditions, the mark spreads over the whole gene depending on the length of period of cold exposure, and the presence of the mark is quantitatively correlated with FLC expression [37]. Moreover, the quantity of initial H3K27me3 deposition and spreading over the gene body is linked to polymorphisms at the cislevel that reflects the different need for cold temperature exposure in different acce.Regulator.Histone Methylation Dynamics in SeedsMajor changes in transcript abundance of the genes encoding regulators and markers of seed maturation and/or dormancy occurred during dormancy-termination per se (e.g. DOG1 and FLC), or once germination had been induced (e.g. ABI3 and LEC2) (Fig. 4). SOM expression was most strongly down-regulated upon the completion of germination (Fig. 4). The “marker” genes, RAB18 and 2S1, showed the greatest decline in abundance during germination (Fig. S2). The switch from activating H3K4me3- to repressive H3K27me3-deposition was associated with a change in transcript level of the dormancy regulators (Fig. 4). We are thus able to discriminate between genes that are required for germination and genes involved in dormancy by their H3 methylation patterns. The former show a strong transcriptional up-regulation during germination that is associated with H3K4me3 deposition. This mark seems to be stable throughout further development and growth as it is also found in genome-wide H3K4me3 profiling studies using 10?0 day old seedlings [13,31,32]. The dormancy regulators were found to maintain H3K27me3 throughout the subsequent seedling stage [13,33,34]. The transition to another life phase is directly reflected in a change at the chromatin level that is then maintained throughout further development. The cue for this life-cycle transition is the exposure of the imbibed seeds to low temperatures. The environmental temperature signal is therefore transduced to effect the observed chromatin changes. It is of interest to investigate whether the same patterns of histone modifications are transduced by other cues that effectively break seed dormancy such as afterripening. FLC deviates from the general pattern of a maintenance of repressive marks throughout the rest of the life cycle. Although this gene also showed a replacement of H3K4me3 by H3K27me3 during seed dormancy release by moist chilling and germination, FLC must be reset to an active state very soon after germination to fulfill its role as a negative regulator of flowering. However FLC has been tested both positive and negative for H3K27me3 in Arabidopsis plants, depending on natural variation, developmental state, and possibly growth conditions, respectively [28,34,35]. Recent work by 1317923 R.R. de Casas et al. [36] shows that moist chilling of seeds leads to earlier flowering in the resulting plants independently of the dormancy status of the seeds. It is thus possible that the appearance of H3K27me3 on FLC is caused by exposure to low temperatures, and not by the physiological process of dormancy breakage per se. The exposure of seeds to moist chilling might thereby lead to FLC repression on the chromatin level such that earlier flowering is promoted in the adult plants. A. Angel et al. [37] have described a nucleation process that takes place on the FLC-locus during induction of flowering competence through vernalization: H3K27me3 accumulates slowly over weeks of cold exposure in one segment of the FLC gene in the sampling population. When plants are returned to warm conditions, the mark spreads over the whole gene depending on the length of period of cold exposure, and the presence of the mark is quantitatively correlated with FLC expression [37]. Moreover, the quantity of initial H3K27me3 deposition and spreading over the gene body is linked to polymorphisms at the cislevel that reflects the different need for cold temperature exposure in different acce.


Asis [5]. The only ubiquitination target of MGRN1 identified to date is

Asis [5]. The only ubiquitination target of MGRN1 identified to date is tumor susceptibility gene 101 (TSG101), a component of the endocytic trafficking machinery that sorts membrane proteins into multivesicular bodies [6,7]. Loss of MGRN1-dependent ubiquitination disrupts endo-lysosomal trafficking, leading to accumulation of activated epidermal growth factor receptor (EGFR) and alterations in the morphology of early endosomes, late endosomes and lysosomes. PrP is normally secreted and tethered to the plasma membrane by a GPI anchor, but ER stress and some pathogenic mutations in PRNP can induce AZP-531 mislocalization of PrP to the cytosol and induce non-transmissible neurotoxicity [8]. A recent study demonstrated that cytosolically exposed forms of PrP can bind to and sequester MGRN1 in HeLa cells [9], resulting in similar abnormalities in endo-lysosomal trafficking to those observed in cells in which Mgrn1 was knocked down by siRNA. Over-expressing MGRN1 rescued the trafficking defects. Reduced immunostaining for MGRN1 was observed in the brains of MedChemExpress AZP-531 transgenic mice expressing a transmembrane form of PrP, along with an age-dependent increase in lysosome size/number (based on Cathepsin D staining) in Purkinje cells. These data suggested that disrupted MGRN1dependent endo-lysosomal trafficking could be the cellularMGRN1 Levels Do Not Influence Prion DiseaseTable 1. Brain Mgrn1 expression.MiceMgrn1md-nc (null) mutant mice and Tg(Mgrn1I)C3Tmg transgenic (hereafter referred to as Tg+) mice, which express wild-type Mgrn1 isoform I from the human ?actin promoter, were described previously [4,13]. The Tg(Mgrn1I)C3Tmg transgenic line completely rescue all aspects of the Mgrn1 null mutant phenotype, including spongiform degeneration of the CNS. Mgrn1 null mutant (Mgrn1md2nc) mice are maintained by breeding heterozygotes with their homozygous mutant siblings. A wild-type control line was established by inbreeding +/+ animals that were generated by intercrossing Mgrn1md2nc/+ mice; this line is re-generated from Mgrn1 heterozygotes every 3 years. Tg+; Mgrn1 null mutant mice were backcrossed to wild-type mice to generate Tg+ and Tg2 Mgrn1md2nc/+ and wild-type (Mgrn1+/+) mice. Mice were genotyped for the Mgrn1md2nc mutation and the transgene as previously described [13].Genotype Tg2; Mgrn1md2nc/+ Tg2; Mgrn1+/+ Tg+; Mgrn1md2nc/+ Tg+; Mgrn1+/+aMgrn1 relative quantification value (range)0.42 (0.22?.79) 1.00 (0.82?. 21) 1.41 (0.76?.61)b 4.43 (1.59?2.32)p valuea 0.04 n/a 0.21 0.Student’s t-test against Tg2; Mgrn1+/+ value, p,0.05 significant. Student’s t-test against Tg2; Mgrn1mdnc/+ yields p = 0.04. doi:10.1371/journal.pone.0055575.tbmechanism underlying spongiform neurodegeneration in prion diseses. Cytosolically-exposed PrP has been proposed to contribute to the pathogenesis of inherited and transmissible spongiform encephalopathies [8]. Mutations in the hydrophobic domain of the prion gene that lead to increased production of a transmembrane form with its N-terminal domain exposed to the cytosol cause neurodegeneration with pathology reminiscent of prion disease in transgenic mice [10]. Similar transmembrane forms of prion protein have been detected in both genetic and transmitted prion diseases [10?2]. The relationship between cytosolic PrP and CNS vacuolation is unclear. We tested whether functional sequestration of MGRN1 by cytosolic PrP contributes to transmissible prion disease 1407003 by inoculating mice expressing reduced or elevated levels of Mgrn1 with Rock.Asis [5]. The only ubiquitination target of MGRN1 identified to date is tumor susceptibility gene 101 (TSG101), a component of the endocytic trafficking machinery that sorts membrane proteins into multivesicular bodies [6,7]. Loss of MGRN1-dependent ubiquitination disrupts endo-lysosomal trafficking, leading to accumulation of activated epidermal growth factor receptor (EGFR) and alterations in the morphology of early endosomes, late endosomes and lysosomes. PrP is normally secreted and tethered to the plasma membrane by a GPI anchor, but ER stress and some pathogenic mutations in PRNP can induce mislocalization of PrP to the cytosol and induce non-transmissible neurotoxicity [8]. A recent study demonstrated that cytosolically exposed forms of PrP can bind to and sequester MGRN1 in HeLa cells [9], resulting in similar abnormalities in endo-lysosomal trafficking to those observed in cells in which Mgrn1 was knocked down by siRNA. Over-expressing MGRN1 rescued the trafficking defects. Reduced immunostaining for MGRN1 was observed in the brains of transgenic mice expressing a transmembrane form of PrP, along with an age-dependent increase in lysosome size/number (based on Cathepsin D staining) in Purkinje cells. These data suggested that disrupted MGRN1dependent endo-lysosomal trafficking could be the cellularMGRN1 Levels Do Not Influence Prion DiseaseTable 1. Brain Mgrn1 expression.MiceMgrn1md-nc (null) mutant mice and Tg(Mgrn1I)C3Tmg transgenic (hereafter referred to as Tg+) mice, which express wild-type Mgrn1 isoform I from the human ?actin promoter, were described previously [4,13]. The Tg(Mgrn1I)C3Tmg transgenic line completely rescue all aspects of the Mgrn1 null mutant phenotype, including spongiform degeneration of the CNS. Mgrn1 null mutant (Mgrn1md2nc) mice are maintained by breeding heterozygotes with their homozygous mutant siblings. A wild-type control line was established by inbreeding +/+ animals that were generated by intercrossing Mgrn1md2nc/+ mice; this line is re-generated from Mgrn1 heterozygotes every 3 years. Tg+; Mgrn1 null mutant mice were backcrossed to wild-type mice to generate Tg+ and Tg2 Mgrn1md2nc/+ and wild-type (Mgrn1+/+) mice. Mice were genotyped for the Mgrn1md2nc mutation and the transgene as previously described [13].Genotype Tg2; Mgrn1md2nc/+ Tg2; Mgrn1+/+ Tg+; Mgrn1md2nc/+ Tg+; Mgrn1+/+aMgrn1 relative quantification value (range)0.42 (0.22?.79) 1.00 (0.82?. 21) 1.41 (0.76?.61)b 4.43 (1.59?2.32)p valuea 0.04 n/a 0.21 0.Student’s t-test against Tg2; Mgrn1+/+ value, p,0.05 significant. Student’s t-test against Tg2; Mgrn1mdnc/+ yields p = 0.04. doi:10.1371/journal.pone.0055575.tbmechanism underlying spongiform neurodegeneration in prion diseses. Cytosolically-exposed PrP has been proposed to contribute to the pathogenesis of inherited and transmissible spongiform encephalopathies [8]. Mutations in the hydrophobic domain of the prion gene that lead to increased production of a transmembrane form with its N-terminal domain exposed to the cytosol cause neurodegeneration with pathology reminiscent of prion disease in transgenic mice [10]. Similar transmembrane forms of prion protein have been detected in both genetic and transmitted prion diseases [10?2]. The relationship between cytosolic PrP and CNS vacuolation is unclear. We tested whether functional sequestration of MGRN1 by cytosolic PrP contributes to transmissible prion disease 1407003 by inoculating mice expressing reduced or elevated levels of Mgrn1 with Rock.


Ernal cells of the mechanosensory organs in order to commence pigmentation

Ernal cells of the mechanosensory organs in order to commence pigmentation, and in the second, after eclosion, a burst of pigmentation activity occurs that is controlled by a neuropeptide cascade, which is required for cuticular tanning and hardening ofTORC1 Controls Drosophila PigmentationFigure 4. Rheb activity Emixustat (hydrochloride) web drives increased TH levels in pupal epidermal cells. Western blot analysis reveals a robust increase in levels of TH protein, and more modest increase of Yellow protein, in Rheb overexpressing thoraces compared to pannier-Gal4 (pnr-G4) line alone (A). TH protein is expressed in a subset of anterior epidermal cells prior to the onset of pigmentation in the P10 stage pupal thorax (B). UAS-Rheb, pannier-Gal4 pupa showing increased numbers of TH protein expressing cells along the central dorsal region of the thorax (C), which is suppressed by either raptorRNAi (D), or s6k1RNAi (E). Overexpression of Rheb by pannier-Gal4 expands the expression of the TH4.1-LacZ reporter, as shown by b-gal labeling (gray, F, G). Genotypes of flies: Y/w, UAS-dicer2; pannier-Gal4/+ (A, B, G), Y/w, UAS-dicer2; UAS-Rheb/+; pannier-Gal4/+ (A, C, G), Y/w, UAS-dicer2; UAS-Rheb/+; pannier-Gal4/UAS-raptorRNAi(D), Y/w, UAS-dicer2; UAS-Rheb/UAS-s6k1RNAi; pannier-Gal4/+(E), Y/w, UAS-dicer2;+/TH4.1-LacZ, pannier-Gal4/+ and Y/w, UASdicer2; UAS-Rheb/TH4.1-LacZ; pannier-Gal4/+(F). doi:10.1371/journal.pone.0048720.gthe adult cuticle [15,19]. We show that Rheb promotes premature pigmentation of the mechanosensory bristles during the pupal stage, and also drives darkening of the posterior cuticle of the thorax after eclosion. It is unclear why this increased pigmentation is biased to the posterior region, known as the trident, but key pigmentation enzymes such as Yellow and Ebony, are expressed at different levels in this part of thorax, suggesting that this region may be more sensitive to changes in catecholamine levels. While our data indicates that Rheb activity increases TH protein levels, it is unclear whether this is through a transcriptional or post-transcriptional regulation. Previous studies have indicted that TH is translationally repressed during pupal eclosion, but the MedChemExpress JW-74 mechanism of this repression is not well understood [15]. TORC1, through the combined activities of both S6K and eIF4E activities,promotes recruitment of the initiation factor complex to mature mRNAs thereby increasing protein synthesis [24?6]. Although we saw increases in both protein levels of Yellow and TH when Rheb was overexpressed, TH levels were markedly higher, while its mRNA levels did not show an increase. These finding point to the possibility that TH translation may be limited by TORC1 activity in wildtype cells. High TORC1 activity promotes the unwinding of mRNAs with long and structured 59 UTRs by the helicase subunit of the initiation complex eIF4A [27]. The TH 59UTR is longer and predicted to be more structured than the yellow 59 UTR and knockdown of eIF4A blocks Rheb-induced hyperpigmentation (Fig. S2G, H). High Rheb levels could therefore increase translation rates of 11967625 TH without increasing the level of TH mRNA. We cannot exclude however that activation ofTORC1 Controls Drosophila PigmentationRheb may, directly or indirectly, also increase levels of transcription or stability of the TH mRNA that was not detected in our rtPCR experiments. The fact that we observe premature pigmentation in tsc1 clones is reminiscent of the precocious differentiation of tsc mutant photoreceptors.Ernal cells of the mechanosensory organs in order to commence pigmentation, and in the second, after eclosion, a burst of pigmentation activity occurs that is controlled by a neuropeptide cascade, which is required for cuticular tanning and hardening ofTORC1 Controls Drosophila PigmentationFigure 4. Rheb activity drives increased TH levels in pupal epidermal cells. Western blot analysis reveals a robust increase in levels of TH protein, and more modest increase of Yellow protein, in Rheb overexpressing thoraces compared to pannier-Gal4 (pnr-G4) line alone (A). TH protein is expressed in a subset of anterior epidermal cells prior to the onset of pigmentation in the P10 stage pupal thorax (B). UAS-Rheb, pannier-Gal4 pupa showing increased numbers of TH protein expressing cells along the central dorsal region of the thorax (C), which is suppressed by either raptorRNAi (D), or s6k1RNAi (E). Overexpression of Rheb by pannier-Gal4 expands the expression of the TH4.1-LacZ reporter, as shown by b-gal labeling (gray, F, G). Genotypes of flies: Y/w, UAS-dicer2; pannier-Gal4/+ (A, B, G), Y/w, UAS-dicer2; UAS-Rheb/+; pannier-Gal4/+ (A, C, G), Y/w, UAS-dicer2; UAS-Rheb/+; pannier-Gal4/UAS-raptorRNAi(D), Y/w, UAS-dicer2; UAS-Rheb/UAS-s6k1RNAi; pannier-Gal4/+(E), Y/w, UAS-dicer2;+/TH4.1-LacZ, pannier-Gal4/+ and Y/w, UASdicer2; UAS-Rheb/TH4.1-LacZ; pannier-Gal4/+(F). doi:10.1371/journal.pone.0048720.gthe adult cuticle [15,19]. We show that Rheb promotes premature pigmentation of the mechanosensory bristles during the pupal stage, and also drives darkening of the posterior cuticle of the thorax after eclosion. It is unclear why this increased pigmentation is biased to the posterior region, known as the trident, but key pigmentation enzymes such as Yellow and Ebony, are expressed at different levels in this part of thorax, suggesting that this region may be more sensitive to changes in catecholamine levels. While our data indicates that Rheb activity increases TH protein levels, it is unclear whether this is through a transcriptional or post-transcriptional regulation. Previous studies have indicted that TH is translationally repressed during pupal eclosion, but the mechanism of this repression is not well understood [15]. TORC1, through the combined activities of both S6K and eIF4E activities,promotes recruitment of the initiation factor complex to mature mRNAs thereby increasing protein synthesis [24?6]. Although we saw increases in both protein levels of Yellow and TH when Rheb was overexpressed, TH levels were markedly higher, while its mRNA levels did not show an increase. These finding point to the possibility that TH translation may be limited by TORC1 activity in wildtype cells. High TORC1 activity promotes the unwinding of mRNAs with long and structured 59 UTRs by the helicase subunit of the initiation complex eIF4A [27]. The TH 59UTR is longer and predicted to be more structured than the yellow 59 UTR and knockdown of eIF4A blocks Rheb-induced hyperpigmentation (Fig. S2G, H). High Rheb levels could therefore increase translation rates of 11967625 TH without increasing the level of TH mRNA. We cannot exclude however that activation ofTORC1 Controls Drosophila PigmentationRheb may, directly or indirectly, also increase levels of transcription or stability of the TH mRNA that was not detected in our rtPCR experiments. The fact that we observe premature pigmentation in tsc1 clones is reminiscent of the precocious differentiation of tsc mutant photoreceptors.


Ndy Hayes, Leo Zeef and Peter March in the Genomic Technologies

Ndy Hayes, Leo Zeef and Peter March in the Genomic Technologies, Bioinformatics and Bioimaging facilities, and Fiona Foster for advice; Alan 3-Bromopyruvic acid web Whitmarsh, Amanda O’Donnell and members of our laboratory for comments on the manuscript and stimulating discussions; and Charles Streuli’s lab for reagents.GABPA and Cell Migration ControlAuthor ContributionsConceived and designed the experiments: ZO ADS. Performed the experiments: ZO. Analyzed the data: ZO ADS. Contributed reagents/ materials/analysis tools: ZO. Wrote the paper: ZO ADS.
It is important to understand the specific response of somatic stem cells to genotoxic exposure, especially in comparison to the cell majority in tissues. Stem cell function is uniquely associated with regeneration, aging and wound repair responses, and these cells may serve as precursor cells during tumor development [1]. Various somatic stem cells have been tested for their response to genotoxic damage, including hematopoetic stem cells, neural stem cells, the epidermal stem cells of the follicular bulge, and melanocytes. In the examples studied to date, stem cells undergo a range of responses to genotoxic exposure, from resistance, to senescence, death by apoptosis, or differentiation. These responses likely illustrate the compromises that are made for each specific tissue to maximize success of the animal. Thus, the preservation of essential stem cells in tissues with a high turnover rate may come at the price of genetic integrity, and the resistance to tumor development offered by the elimination of mutant stem cells may be offset by premature aging [2,3,4,5,6,7,8]. In this study, we 1480666 evaluated the response of mammary stem cells to genotoxic exposure during juvenile development. The cellautonomous stem cell activity characterized (so far) for mammary gland copurifies with one of the two Castanospermine principal epithelial lineages, the basal(/myoepithelial) cell population [9,10]; thus afterdissociation of mammary epithelial cells from 1676428 the mammary ducts, a single basal cell can regenerate a whole mammary gland. Cells from the luminal population (responsible for milk secretion and the perception of the dominant estrogen growth signal) cannot reconstitute mammary gland, but this population does include progenitors that can generate limited outgrowths, and function as unipotent stem cells in vivo [11]. The overall frequency of ductal basal stem cells in mammary gland is at least 1/1600 (results from this study, these frequencies vary from strain to strain, and are tentative given that cell dissociation is likely to compromise functional activity). These cells cannot yet be recognized in situ, since there is no marker that can distinguish stem cells from the rest. There are two phases of growth in the mammary gland, one that establishes the ductal tree during peri-puberty, and another during pregnancy that serves to fill the space between the ducts with lobuloalveolar units. Neither basal nor luminal cells are “terminally differentiated” since both divide at about the same rate during these processes [12]. For this study, we tested the effect of genotoxic exposure during juvenile growth. The cells born during ductal outgrowth are long-lived, compared to the majority that are born and die during pregnancy and estrus. For this study, we selected a representative of the polycyclic aromatic hydrocarbons, DMBA (dimethylbenz[a]anthracene) asGenotoxins Inhibit Wnt-Dependent Mammary Stem Cellthe genotoxin. This group of compounds are e.Ndy Hayes, Leo Zeef and Peter March in the Genomic Technologies, Bioinformatics and Bioimaging facilities, and Fiona Foster for advice; Alan Whitmarsh, Amanda O’Donnell and members of our laboratory for comments on the manuscript and stimulating discussions; and Charles Streuli’s lab for reagents.GABPA and Cell Migration ControlAuthor ContributionsConceived and designed the experiments: ZO ADS. Performed the experiments: ZO. Analyzed the data: ZO ADS. Contributed reagents/ materials/analysis tools: ZO. Wrote the paper: ZO ADS.
It is important to understand the specific response of somatic stem cells to genotoxic exposure, especially in comparison to the cell majority in tissues. Stem cell function is uniquely associated with regeneration, aging and wound repair responses, and these cells may serve as precursor cells during tumor development [1]. Various somatic stem cells have been tested for their response to genotoxic damage, including hematopoetic stem cells, neural stem cells, the epidermal stem cells of the follicular bulge, and melanocytes. In the examples studied to date, stem cells undergo a range of responses to genotoxic exposure, from resistance, to senescence, death by apoptosis, or differentiation. These responses likely illustrate the compromises that are made for each specific tissue to maximize success of the animal. Thus, the preservation of essential stem cells in tissues with a high turnover rate may come at the price of genetic integrity, and the resistance to tumor development offered by the elimination of mutant stem cells may be offset by premature aging [2,3,4,5,6,7,8]. In this study, we 1480666 evaluated the response of mammary stem cells to genotoxic exposure during juvenile development. The cellautonomous stem cell activity characterized (so far) for mammary gland copurifies with one of the two principal epithelial lineages, the basal(/myoepithelial) cell population [9,10]; thus afterdissociation of mammary epithelial cells from 1676428 the mammary ducts, a single basal cell can regenerate a whole mammary gland. Cells from the luminal population (responsible for milk secretion and the perception of the dominant estrogen growth signal) cannot reconstitute mammary gland, but this population does include progenitors that can generate limited outgrowths, and function as unipotent stem cells in vivo [11]. The overall frequency of ductal basal stem cells in mammary gland is at least 1/1600 (results from this study, these frequencies vary from strain to strain, and are tentative given that cell dissociation is likely to compromise functional activity). These cells cannot yet be recognized in situ, since there is no marker that can distinguish stem cells from the rest. There are two phases of growth in the mammary gland, one that establishes the ductal tree during peri-puberty, and another during pregnancy that serves to fill the space between the ducts with lobuloalveolar units. Neither basal nor luminal cells are “terminally differentiated” since both divide at about the same rate during these processes [12]. For this study, we tested the effect of genotoxic exposure during juvenile growth. The cells born during ductal outgrowth are long-lived, compared to the majority that are born and die during pregnancy and estrus. For this study, we selected a representative of the polycyclic aromatic hydrocarbons, DMBA (dimethylbenz[a]anthracene) asGenotoxins Inhibit Wnt-Dependent Mammary Stem Cellthe genotoxin. This group of compounds are e.


N = the number of animals. doi:10.1371/journal.pone.0052058.tPlatelets, EPCs and

N = the number of animals. doi:10.1371/journal.pone.0052058.tPlatelets, EPCs and AtherosclerosisFigure 2. Representative immunoblots and densitometric data for the platelets from hamster groups: control (C), hypertensive-hypercholesterolemic (HH), prevention (HHin-EPCs), regression (HHfin-EPCs), HH 1326631 treated with PMPs (HH-PMPs) and HH treated with EPCs and PMPs (HH-EPCs-PMPs). (A): pFAK, FAK, (B): p85 subunit of PI3K, b- actin, (C): p-src, src. (*) Groups vs Control: p#0.05. (**) Groups vs HH: p#0.05. doi:10.1371/journal.pone.0052058.gtants. The results show that compared to C group, the values for VEGF were increased by ,1.26-fold in HH group, ,1.40-fold in HH-PMPs group and ,1.08-fold in HH-EPCs-PMPs group. The concentrations measured in HHin-EPCs and HHfin-EPCs groups were similar to de value in C group. Compared to HH group, in HHin-EPCs, HHfin-EPCs and HH-EPCs-PMPs groups, the values for VEGF were reduced by ,1.42-fold, ,1.34-fold and ,1.16-fold. In platelets isolated from HH+PMPs group the value for VEGF was enhanced by ,1.12-fold comparative with HH group (Table 2). Compared to C group, the analysis of PDGF-AB concentration in platelet supernatants isolated from HH, HH-PMPs and HHEPCs-PMPs groups revealed an augmentation of: ,1.46-fold, ,1.73-fold and ,1.66-fold, respectively (Table 2). In samples from HHin-EPCs and HHfin-EPCs, PDGF-AB values were comparable to these in C group. Compared to HH group, the values for platelet supernatant from these groups we reduced by ,1.47-fold in HHin-EPCs and ,1.41-fold in HHfin-EPCs. Conversely, the platelet supernatants in HH-PMPs and HHEPCs-PMPs groups displayed a slightly increase of PDGF-AB concentration by ,1.18-fold and ,1.14-fold respectively (Table 2). Platelets are the primary hematopoietic cell accumulating within a growing thrombus, where they release the TFPI, which is the main physiologic inhibitor of tissue factor, the initiator of blood coagulation. Measurement of TFPI concentration in platelet supernatants isolated from hamster groups showed a reduction compared to C group, of ,1.28-fold (for HH), ,1.39-fold (for HH-PMPs) and ,1.13-fold (for HH-EPCs-PMPs) (Table 2). Compared to C group, TFPI in HHin-EPCs and HHfin-EPCs groups is slightly increased i.e. ,1.19-fold, and ,1.10-fold, respectively. Compared to HH group, the enhancement observed in HHin-EPCs, HHfin-EPCs and HH-EPCs-PMPs group, was of ,1.52-fold, ,1.41-fold and ,1.135-fold, respectively. The platelet supernatant isolated from HH-PMPs group showed a slight decrease in TFPI of ,1.08-fold, compared to HH group. The above results indicate that EPC administration in hypertension associated with hypercholesterolemia reduces the levels of pro-inflammatory molecules SIS3 web secreted by activated platelets and improves the amount of TFPI released by platelets. Moreover, PMP administration induces a general augmentation of secreted molecules, except for TFPI level, that is diminished.Estimation of Cytokine/Chemokines 871361-88-5 web protein ExpressionThe results of immunoblotting experiments revealed that compared to C group (n = 6), in platelets isolated from HH group, protein expressions for SDF-1 and MCP-1 were increased by ,19.31-fold (n = 4) and ,2.59-fold, respectively (n = 8) (Fig. 3A). Compared to C group, in platelets isolated from the HHin-EPCs and HHfin-EPCs groups, SDF-1 expressions (n = 4) were unchanged, while MCP-1 expressions (n = 6) were slightly increased by ,1.36-fold, and 21.26-fold, respectively (Fig. 3A). Compared to HH group, bot.N = the number of animals. doi:10.1371/journal.pone.0052058.tPlatelets, EPCs and AtherosclerosisFigure 2. Representative immunoblots and densitometric data for the platelets from hamster groups: control (C), hypertensive-hypercholesterolemic (HH), prevention (HHin-EPCs), regression (HHfin-EPCs), HH 1326631 treated with PMPs (HH-PMPs) and HH treated with EPCs and PMPs (HH-EPCs-PMPs). (A): pFAK, FAK, (B): p85 subunit of PI3K, b- actin, (C): p-src, src. (*) Groups vs Control: p#0.05. (**) Groups vs HH: p#0.05. doi:10.1371/journal.pone.0052058.gtants. The results show that compared to C group, the values for VEGF were increased by ,1.26-fold in HH group, ,1.40-fold in HH-PMPs group and ,1.08-fold in HH-EPCs-PMPs group. The concentrations measured in HHin-EPCs and HHfin-EPCs groups were similar to de value in C group. Compared to HH group, in HHin-EPCs, HHfin-EPCs and HH-EPCs-PMPs groups, the values for VEGF were reduced by ,1.42-fold, ,1.34-fold and ,1.16-fold. In platelets isolated from HH+PMPs group the value for VEGF was enhanced by ,1.12-fold comparative with HH group (Table 2). Compared to C group, the analysis of PDGF-AB concentration in platelet supernatants isolated from HH, HH-PMPs and HHEPCs-PMPs groups revealed an augmentation of: ,1.46-fold, ,1.73-fold and ,1.66-fold, respectively (Table 2). In samples from HHin-EPCs and HHfin-EPCs, PDGF-AB values were comparable to these in C group. Compared to HH group, the values for platelet supernatant from these groups we reduced by ,1.47-fold in HHin-EPCs and ,1.41-fold in HHfin-EPCs. Conversely, the platelet supernatants in HH-PMPs and HHEPCs-PMPs groups displayed a slightly increase of PDGF-AB concentration by ,1.18-fold and ,1.14-fold respectively (Table 2). Platelets are the primary hematopoietic cell accumulating within a growing thrombus, where they release the TFPI, which is the main physiologic inhibitor of tissue factor, the initiator of blood coagulation. Measurement of TFPI concentration in platelet supernatants isolated from hamster groups showed a reduction compared to C group, of ,1.28-fold (for HH), ,1.39-fold (for HH-PMPs) and ,1.13-fold (for HH-EPCs-PMPs) (Table 2). Compared to C group, TFPI in HHin-EPCs and HHfin-EPCs groups is slightly increased i.e. ,1.19-fold, and ,1.10-fold, respectively. Compared to HH group, the enhancement observed in HHin-EPCs, HHfin-EPCs and HH-EPCs-PMPs group, was of ,1.52-fold, ,1.41-fold and ,1.135-fold, respectively. The platelet supernatant isolated from HH-PMPs group showed a slight decrease in TFPI of ,1.08-fold, compared to HH group. The above results indicate that EPC administration in hypertension associated with hypercholesterolemia reduces the levels of pro-inflammatory molecules secreted by activated platelets and improves the amount of TFPI released by platelets. Moreover, PMP administration induces a general augmentation of secreted molecules, except for TFPI level, that is diminished.Estimation of Cytokine/Chemokines Protein ExpressionThe results of immunoblotting experiments revealed that compared to C group (n = 6), in platelets isolated from HH group, protein expressions for SDF-1 and MCP-1 were increased by ,19.31-fold (n = 4) and ,2.59-fold, respectively (n = 8) (Fig. 3A). Compared to C group, in platelets isolated from the HHin-EPCs and HHfin-EPCs groups, SDF-1 expressions (n = 4) were unchanged, while MCP-1 expressions (n = 6) were slightly increased by ,1.36-fold, and 21.26-fold, respectively (Fig. 3A). Compared to HH group, bot.


With CAD also suffer from hyperlipidemia and take lipid-lowering drugs (mainly

With CAD also suffer from hyperlipidemia and take lipid-lowering drugs (mainly stains in our CAD patients) and do not take n-3 PUFAs or fish oil, which may partly explain these results. Statins are inhibitors of hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase. These drugs inhibitendogenous HMG-CoA reductase by competition and blocking the mevalonate metabolic pathway in cells, increasing the clearance of serum cholesterol. Therefore, the results do not truly reflect the situation of lipids in CAD patients. Besides, no significant difference was found in n-3/n-6 between controls and CAD patients. Our study had several limitations. First, no SNPs were evaluated in the SCD gene; thus there was no get 115103-85-0 information about the association of the SCD gene polymorphism with the composition of plasma fatty acids. Second, the concentrations of plasma fatty acids are influenced by both dietary intake and metabolic pathways. However, we did not obtain any information about energy intake. Overall, we firstly report that the rs174460 C allele is associated with a higher risk of CAD, and confirm that the rs174537 T allele is associated with a lower risk of CAD. Our results indicate that FADS gene polymorphisms are likely to influence plasma fatty acid concentrations and desaturase activities. Further investigations are needed to explore the potential mechanisms of rs174460 C allele and increased D6D, D9D activities and higher CAD risk.Supporting InformationFigure S1 Representative Chromatograms of plasma fatty acids by gas chromatography. (DOC)FADS Gene, Desaturase Activity and CADFigure S2 High-resolution melting curves of five studiedSNPs. (DOC)Table S1 Amplification primers utilized in the genotype.Wang Chun-Hong (School of Public Health, Wuhan University) and Dr. Xie Yan (School of Basic Medical Sciences, Wuhan University) for their guidance in statistical analysis.(DOC)Author ContributionsConceived and designed the experiments: SWL XZ SML. Performed the experiments: SWL KL PM. Analyzed the data: SWL SYL. Contributed reagents/materials/analysis tools: ZLZ YDZ. Wrote the paper: SWL XZ SML.AcknowledgmentsWe thank all of the participants of the study. Thanks to Wuhan Asia Heart Hospital for assistance with sample collection. We also thank Professor
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) following a high dose conditioning regimen has been the best treatment option for many young patients with hematological disorders. The antitumor activity of this approach is based not only on high dose chemo-radiotherapy given in the conditioning regimen but also on immune-mediated graft-versus-tumor effects [1,2]. These observations are the basis of the development of alloHSCT following nonmyeloablative conditioning, in which eradication of malignant cells depends on graft-versus-tumor effects [3?6]. Pentagastrin biological activity T-cell recovery after allo-HSCT following high-dose conditioning depends on both homeostatic peripheral expansion (HPE) of donor T cells contained in the graft, and T cell neo-production from donor hematopoietic stem cells (thymo-dependent pathway) [7?5]. In young patients given myeloablative allo-HSCT, most circulating T cells during the first months following HSCT are theprogeny of T cells infused with the grafts [16], while neogeneration of T cells by the thymus plays an increasing role in reconstituting the T cell pool beyond day 100 after allo-HSCT [17?2]. Since HPE allow the expansion of both NK cells and non-tolerant T cells, it is general.With CAD also suffer from hyperlipidemia and take lipid-lowering drugs (mainly stains in our CAD patients) and do not take n-3 PUFAs or fish oil, which may partly explain these results. Statins are inhibitors of hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase. These drugs inhibitendogenous HMG-CoA reductase by competition and blocking the mevalonate metabolic pathway in cells, increasing the clearance of serum cholesterol. Therefore, the results do not truly reflect the situation of lipids in CAD patients. Besides, no significant difference was found in n-3/n-6 between controls and CAD patients. Our study had several limitations. First, no SNPs were evaluated in the SCD gene; thus there was no information about the association of the SCD gene polymorphism with the composition of plasma fatty acids. Second, the concentrations of plasma fatty acids are influenced by both dietary intake and metabolic pathways. However, we did not obtain any information about energy intake. Overall, we firstly report that the rs174460 C allele is associated with a higher risk of CAD, and confirm that the rs174537 T allele is associated with a lower risk of CAD. Our results indicate that FADS gene polymorphisms are likely to influence plasma fatty acid concentrations and desaturase activities. Further investigations are needed to explore the potential mechanisms of rs174460 C allele and increased D6D, D9D activities and higher CAD risk.Supporting InformationFigure S1 Representative Chromatograms of plasma fatty acids by gas chromatography. (DOC)FADS Gene, Desaturase Activity and CADFigure S2 High-resolution melting curves of five studiedSNPs. (DOC)Table S1 Amplification primers utilized in the genotype.Wang Chun-Hong (School of Public Health, Wuhan University) and Dr. Xie Yan (School of Basic Medical Sciences, Wuhan University) for their guidance in statistical analysis.(DOC)Author ContributionsConceived and designed the experiments: SWL XZ SML. Performed the experiments: SWL KL PM. Analyzed the data: SWL SYL. Contributed reagents/materials/analysis tools: ZLZ YDZ. Wrote the paper: SWL XZ SML.AcknowledgmentsWe thank all of the participants of the study. Thanks to Wuhan Asia Heart Hospital for assistance with sample collection. We also thank Professor
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) following a high dose conditioning regimen has been the best treatment option for many young patients with hematological disorders. The antitumor activity of this approach is based not only on high dose chemo-radiotherapy given in the conditioning regimen but also on immune-mediated graft-versus-tumor effects [1,2]. These observations are the basis of the development of alloHSCT following nonmyeloablative conditioning, in which eradication of malignant cells depends on graft-versus-tumor effects [3?6]. T-cell recovery after allo-HSCT following high-dose conditioning depends on both homeostatic peripheral expansion (HPE) of donor T cells contained in the graft, and T cell neo-production from donor hematopoietic stem cells (thymo-dependent pathway) [7?5]. In young patients given myeloablative allo-HSCT, most circulating T cells during the first months following HSCT are theprogeny of T cells infused with the grafts [16], while neogeneration of T cells by the thymus plays an increasing role in reconstituting the T cell pool beyond day 100 after allo-HSCT [17?2]. Since HPE allow the expansion of both NK cells and non-tolerant T cells, it is general.


Erm `opiate’ describes heroin, methadone, opium, poppy tea, and recreational use

Erm `opiate’ NT 157 price describes heroin, methadone, opium, poppy tea, and recreational use of codeine, oxycodeine, hydrocodeine, and/or morphine. The term `inhalant’ describes amyl nitrate, nitrous oxide, and/or glue. The term `sedative’ describes GHB/Fantasy, methaqualome, chelidonium majus, and recreational use of benzodiazepine, antidepressants, and antihistamine. doi:10.1371/journal.pone.0056438.tStimulant Drugs and Substantia Nigra MorphologyTable 3. Summary of lifetime use of stimulants and cannabis in the stimulant group.Subject 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Mean (SD)Total stimulants 3029 2967 2241 2059 1576 1396 875 833 670 387 367 332 247 234 209 204 139 86 79 57 36 32 27 19 19 16 14 13 12 7 7 6 6 6 3 3 506 (845)Amphetamines 3029 2651 2072 1851 1560 1034 719 832 520 327 211 228 244 231 208 164 14 13 35 5 10 12 23727046 26 8 1 1 9 1 3 7 1 1 4 0 0 0 486 (820)Ecstasy 0 317 169 208 16 362 156 1 150 60 156 104 3 4 1 40 125 73 44 52 26 20 1 11 18 15 5 12 9 0 6 5 2 6 3 3 64 (92)Cannabis 5475 5840 28 4745 15 8212 228 13 1140 54 4380 1251 7365 360 6570 33945 1104 128 11315 4380 474 832 270 6 15 20 10741 2555 72 4384 183 60 9855 260 104 15 3511 (6256)Single subject and mean data are presented (number of times used). The term `amphetamine’ describes amphetamine and amphetamine-like drugs such methamphetamine, purchase GSK -3203591 cocaine, dexamphetamine, RitalinH, and khat (1 subject). The term `ecstasy’ describes ecstasy, MDA (3,4-methylenedioxyamphetamine, 2 subjects), and MCAT (mephedrone, 1 subject). doi:10.1371/journal.pone.0056438.techogenicity is difficult in human drug users. We can conclude that the abnormality is not associated with the acute mechanism of action of stimulants because the average duration of abstinence was 263 years and subjects had a negative urine screen for stimulants, opiates, and benzodiazepines. The abnormality is also not associated with changes in memory, cognition, and gross brainvolume because all subjects passed neuropsychological screening and all subjects exhibited a normal ventricular system. The abnormality is also unlikely due to drug overdose because only 4 subjects reported experiencing such an event. However, beyond that one can only speculate due to methodological limitations associated with all studies on illegal stimulant use in humans. For example, no two people exhibit the same drug use pattern, lifestyle, or environment and there are challenges associated with self-reporting of lifetime drug use and difficulty in obtaining accurate information on the dose and composition of the substances used. Table 2 highlights another significant challenge, poly-drug use. In the current study, 94 of subjects in the stimulant group had used ecstasy, 81 had used methamphetamine, and 56 had used cocaine. Poly-stimulant use is well documented in the literature and is clearly evident in national drug surveys [54]. Cannabis use is also very common amongst stimulant users, with over 70 of stimulant users reporting concurrent cannabis use [54]. Furthermore, stimulant users consume more alcohol [55] and tobacco [56] than non-drug users. Thus, in humans, it is difficult to ascribe an observed abnormality to a specific drug but changes can be ascribed to a class of drug (e.g. stimulants) with careful experimental design and control measures. It is mechanistically plausible that use of each of the three illicit stimulants, methamphetamine, cocaine, and ecstasy, contributed to the a.Erm `opiate’ describes heroin, methadone, opium, poppy tea, and recreational use of codeine, oxycodeine, hydrocodeine, and/or morphine. The term `inhalant’ describes amyl nitrate, nitrous oxide, and/or glue. The term `sedative’ describes GHB/Fantasy, methaqualome, chelidonium majus, and recreational use of benzodiazepine, antidepressants, and antihistamine. doi:10.1371/journal.pone.0056438.tStimulant Drugs and Substantia Nigra MorphologyTable 3. Summary of lifetime use of stimulants and cannabis in the stimulant group.Subject 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Mean (SD)Total stimulants 3029 2967 2241 2059 1576 1396 875 833 670 387 367 332 247 234 209 204 139 86 79 57 36 32 27 19 19 16 14 13 12 7 7 6 6 6 3 3 506 (845)Amphetamines 3029 2651 2072 1851 1560 1034 719 832 520 327 211 228 244 231 208 164 14 13 35 5 10 12 23727046 26 8 1 1 9 1 3 7 1 1 4 0 0 0 486 (820)Ecstasy 0 317 169 208 16 362 156 1 150 60 156 104 3 4 1 40 125 73 44 52 26 20 1 11 18 15 5 12 9 0 6 5 2 6 3 3 64 (92)Cannabis 5475 5840 28 4745 15 8212 228 13 1140 54 4380 1251 7365 360 6570 33945 1104 128 11315 4380 474 832 270 6 15 20 10741 2555 72 4384 183 60 9855 260 104 15 3511 (6256)Single subject and mean data are presented (number of times used). The term `amphetamine’ describes amphetamine and amphetamine-like drugs such methamphetamine, cocaine, dexamphetamine, RitalinH, and khat (1 subject). The term `ecstasy’ describes ecstasy, MDA (3,4-methylenedioxyamphetamine, 2 subjects), and MCAT (mephedrone, 1 subject). doi:10.1371/journal.pone.0056438.techogenicity is difficult in human drug users. We can conclude that the abnormality is not associated with the acute mechanism of action of stimulants because the average duration of abstinence was 263 years and subjects had a negative urine screen for stimulants, opiates, and benzodiazepines. The abnormality is also not associated with changes in memory, cognition, and gross brainvolume because all subjects passed neuropsychological screening and all subjects exhibited a normal ventricular system. The abnormality is also unlikely due to drug overdose because only 4 subjects reported experiencing such an event. However, beyond that one can only speculate due to methodological limitations associated with all studies on illegal stimulant use in humans. For example, no two people exhibit the same drug use pattern, lifestyle, or environment and there are challenges associated with self-reporting of lifetime drug use and difficulty in obtaining accurate information on the dose and composition of the substances used. Table 2 highlights another significant challenge, poly-drug use. In the current study, 94 of subjects in the stimulant group had used ecstasy, 81 had used methamphetamine, and 56 had used cocaine. Poly-stimulant use is well documented in the literature and is clearly evident in national drug surveys [54]. Cannabis use is also very common amongst stimulant users, with over 70 of stimulant users reporting concurrent cannabis use [54]. Furthermore, stimulant users consume more alcohol [55] and tobacco [56] than non-drug users. Thus, in humans, it is difficult to ascribe an observed abnormality to a specific drug but changes can be ascribed to a class of drug (e.g. stimulants) with careful experimental design and control measures. It is mechanistically plausible that use of each of the three illicit stimulants, methamphetamine, cocaine, and ecstasy, contributed to the a.