Easured using realtime PCR. B. Electron transport system abnormalities are more
Easured using realtime PCR. B. Electron transport system abnormalities are more abundant in rats treated with the AMP kinase activator b-guanidinopropionic acid. Each Vastus lateralis skeletal muscle was stained for cytochrome C oxidase and succinate dehydrogenase every 60 microns for one millimeter. Regions of skeletal muscle fibers lacking COX activity 25033180 and hyper-reactive for SDH (the ETS abnormal phenotype) were counted. doi:10.1371/journal.pone.0059006.gDiscussionDespite the identification of the relationship between mitochondrial DNA deletion mutation BIBS39 accumulation and metabolic dysfunction, the specific mechanism(s) that originate and allow clonal accumulation of mtDNA deletion mutations with aging are enigmatic. The loss of electron transport activity in muscle fiber segments harboring intracellular clonal expansions of mtDNA deletion mutations suggests that many metabolic pathways, both anabolic and catabolic, would be affected by the inability to dispose of reducing equivalents generated by respiration. Since many of the enzymes in the citric acid cycle are susceptible to product (NADH) inhibition, the electron flux would decrease, and the central hub of metabolism would be compromised. This would have a direct effect on mitochondrial ATP synthesis, and result in the requirement for the use of inefficient compensatory biochemtransport and oxidative phosphorylation. To test the hypothesis, we stained for activated AMP kinase and overexpression of the peroxisome proliferator activated receptor alpha (ppara) using immunohistochemistry with antibodies specific to these factors, their cofactors and their target genes. AMP kinase was phosphorylated on threonine-172, an indication of its activation, in ETS abnormal fibers. Moreover, a primary downstream target of activated AMP kinase, acetyl-coA carboxylase, was phosphorylated on serine 79, inhibiting fatty acid synthesis, an energy intensive process, consistent with an increase in AMP concentration (Figure 2). Immunohistochemical analysis of ppara, pgc-1a (peroxisome proliferator activated receptor gamma coactivator 1 alpha) andMitobiogenesis Drives mtDNA Deletion MutationsFigure 4. 
Model of positive feedback loop in ETS abnormal fibers. Signal transduction pathways detect mitochondrial dysfunction and drive transcriptional activation leading to up-regulation of mitochondrial DNA replication and subsequent deletion mutation accumulation. Genes in green were up-regulated in ETS abnormal fibers. Proteins in blue were found to be up-regulated by immunohistochemistry in ETS abnormal fibers. Proteins in purple were detected by both purchase Castanospermine assays. wt: wild-type mitochondrial genomes, D deletion mutation containing 
mitochondrial genomes. doi:10.1371/journal.pone.0059006.gical pathways, depleting cellular ATP concentration. We tested whether the response to electron transport dysfunction induced by the expansion of mtDNA deletion mutations was non-adaptive and consistent with the proposed role for mitochondrial deletion mutations in sarcopenia. To better understand the molecular basis of sarcopenia, we profiled (Tables S1 and S2) muscle fibers containing intracellular expansions of deletion-mutation containing mitochondrial DNA. The profile obtained suggested that AMP kinase, the ubiquitous energy sensing molecule, was activated as was nuclear hormone signaling, a response indicating a program of mitochondrial biogenesis was activated, consistent with the observed mitochondrial dysfunction in deletio.Easured using realtime PCR. B. Electron transport system abnormalities are more abundant in rats treated with the AMP kinase activator b-guanidinopropionic acid. Each Vastus lateralis skeletal muscle was stained for cytochrome C oxidase and succinate dehydrogenase every 60 microns for one millimeter. Regions of skeletal muscle fibers lacking COX activity 25033180 and hyper-reactive for SDH (the ETS abnormal phenotype) were counted. doi:10.1371/journal.pone.0059006.gDiscussionDespite the identification of the relationship between mitochondrial DNA deletion mutation accumulation and metabolic dysfunction, the specific mechanism(s) that originate and allow clonal accumulation of mtDNA deletion mutations with aging are enigmatic. The loss of electron transport activity in muscle fiber segments harboring intracellular clonal expansions of mtDNA deletion mutations suggests that many metabolic pathways, both anabolic and catabolic, would be affected by the inability to dispose of reducing equivalents generated by respiration. Since many of the enzymes in the citric acid cycle are susceptible to product (NADH) inhibition, the electron flux would decrease, and the central hub of metabolism would be compromised. This would have a direct effect on mitochondrial ATP synthesis, and result in the requirement for the use of inefficient compensatory biochemtransport and oxidative phosphorylation. To test the hypothesis, we stained for activated AMP kinase and overexpression of the peroxisome proliferator activated receptor alpha (ppara) using immunohistochemistry with antibodies specific to these factors, their cofactors and their target genes. AMP kinase was phosphorylated on threonine-172, an indication of its activation, in ETS abnormal fibers. Moreover, a primary downstream target of activated AMP kinase, acetyl-coA carboxylase, was phosphorylated on serine 79, inhibiting fatty acid synthesis, an energy intensive process, consistent with an increase in AMP concentration (Figure 2). Immunohistochemical analysis of ppara, pgc-1a (peroxisome proliferator activated receptor gamma coactivator 1 alpha) andMitobiogenesis Drives mtDNA Deletion MutationsFigure 4. Model of positive feedback loop in ETS abnormal fibers. Signal transduction pathways detect mitochondrial dysfunction and drive transcriptional activation leading to up-regulation of mitochondrial DNA replication and subsequent deletion mutation accumulation. Genes in green were up-regulated in ETS abnormal fibers. Proteins in blue were found to be up-regulated by immunohistochemistry in ETS abnormal fibers. Proteins in purple were detected by both assays. wt: wild-type mitochondrial genomes, D deletion mutation containing mitochondrial genomes. doi:10.1371/journal.pone.0059006.gical pathways, depleting cellular ATP concentration. We tested whether the response to electron transport dysfunction induced by the expansion of mtDNA deletion mutations was non-adaptive and consistent with the proposed role for mitochondrial deletion mutations in sarcopenia. To better understand the molecular basis of sarcopenia, we profiled (Tables S1 and S2) muscle fibers containing intracellular expansions of deletion-mutation containing mitochondrial DNA. The profile obtained suggested that AMP kinase, the ubiquitous energy sensing molecule, was activated as was nuclear hormone signaling, a response indicating a program of mitochondrial biogenesis was activated, consistent with the observed mitochondrial dysfunction in deletio.
ensure the response had reached a steady-state. We conducted parallel studies, as described above; with the exception that fNADH instead of Rhod-2AM was imaged. The objective was to determine if the inhibition of the actinmyosin ATPase with 4.75 M of blebbistatin affected the dynamics of fNADH after administering DCA or pyruvate. Measurement of SR calcium load The effect of DCA and pyruvate on SR Ca2+ load was measured using 
reached a steady-state. We conducted parallel studies, as described above; with the exception that fNADH instead of Rhod-2AM was imaged. The objective was to determine if the inhibition of the actinmyosin ATPase with 4.75 M of blebbistatin affected the dynamics of fNADH after administering DCA or pyruvate. Measurement of SR calcium load The effect of DCA and pyruvate on SR Ca2+ load was measured using neonatal myocyte monolayers and a caffeine surge protocol. Neonatal rat ventricular myocytes were isolated and plated from a heterogeneous population of hearts, as previously described. Intracellular Ca2+ transients were imaged using Fluo-4 and confocal fluorescence microscopy. Cells were field stimulated at 0.2 Hz for 30 s, followed by an injection of 20 mM caffeine to induce total SR calcium release. The injection of caffeine was supplemented with 20 mM KCl and 1 mM verapamil to prevent rapid contractions. The average area under the curve of three baseline transients was compared to the area of the large transient induced by the caffeine surge. Arrhythmia scoring Pressure and electrogram signals were examined to identify premature ventricular contractions and episodes of non-sustained ventricular tachycardia. Arrhythmias were scored using a modified method from Jin et al., where hearts having 20 or less PVCs received a score of 0 and hearts having more than 20 PVCs or one episode of NSVT for less than 2 s received a score of 1. We did not observe any heart to have NSVT longer than 2 s. We also did not observe VF or other significant arrhythmias, so scores beyond 1 were not necessary. All hearts received arrhythmia scores of either 0 or 1. Statistics Statistical analyses were performed in R. Data are presented as meanstandard error of mean. Significance was defined by p<0.05, unless noted as p<0.01. One-way ANOVAs with Tukey post hoc tests were used to identify significant differences between groups. A threeway ANOVA with Tukey post hoc tests were used to compare calcium transient Author Manuscript Author Manuscript Author Manuscript Author Manuscript Pflugers Arch. Author manuscript; available in PMC 2016 January 06. Jaimes et al. Page 7 characteristics between baseline and treatments, pacing rates, and between treatments. All data were determined to be normal using the ShapiroWilk test. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Results Control studies with identical KH solutions in each side of the dual perfusion apparatus confirmed that perfusate switching did not cause artifacts or alter heart function. Perfusate switching introduced a maximum temperature variation of less than 1 C and a heart rate change of less than 5 %. No other changes in heart function were detected. HR changes of less than 5 % were also measured when administering DCA or pyruvate. LVDP and nNADH signals in all contracting heart studies consisted of three phases: a baseline phase, a transient phase, and a steady-state phase. The BP was the 10 min of baseline perfusion before a perfusate switch occurred. The TP was the period from the perfusate switch to when changes in LVDP or nNADH subsided. The SSP began when a given variable reached steady-state and corresponded to the time from the end of the TP to the end 
in the L4 and adult stages with abundant transcription. These genes encoded mainly cysteine-type, serine-type and metallopeptidases as well as some aspartate- and threoninetype peptidases. In addition, transcription of genes encoding 75 peptidase inhibitors was assessed. Many secreted peptidases likely to represent the `degradome’ and respective inhibitors are known 
to enable parasitic worms to invade, penetrate tissue 
to the formation of ketone bodies was minuscule relative to other carbon sources, which presumably were almost entirely fatty acids. Since serum levels of NEFAs were similar between DKO and wild-type mice, greater availability of fatty acids for oxidation does not explain the increase in ketone bodies. Fasting induces ketoacidosis and hypothermia 
pCO2 was significantly reduced in DKO mice. In addition to suffering from ketoacidosis, the DKO mice experienced hypothermia after 36 h of fasting, leading ultimately to their death. Expression of PDHK4 does not compensate for a lack of PDHK2 in PDHK2-KO mice and vice versa PDHK2 and PDHK4 were measured by Western blot analysis to assess whether altered expression of these proteins compensates for the lack of PDHK2 and PDHK4 in the corresponding KO mice. Protein levels of PDHK2 were not changed in the tissues of the PDHK4-KO mice compared with wild-type mice. Protein levels of PDHK4 were likewise similar in heart, liver and skeletal muscle of PDHK2-KO mice and wild-type mice. These findings suggest that, in the fast.Partially active in the fasted state in DKO mice, glucose should contribute more carbon to the synthesis of ketone bodies in these mice. This was examined by measuring the incorporation of carbon from glucose into ketone bodies in wild-type and DKO mice. As anticipated, greater hydroxybutyrate enrichment with two carbons was found in the plasma of the DKO mice, which, combined with the greater concentration of ketone bodies in the DKO mice, established that more ketone bodies were produced from glucose in the DKO mice than in the wild-type mice. This finding is consistent with greater flux through the PDH complex with subsequent conversion of acetyl-CoA into ketone bodies. However, the relative contribution of glucose carbon to the formation of ketone bodies was minuscule relative to other carbon sources, which presumably were almost entirely fatty acids. Since serum levels of NEFAs were similar between DKO and wild-type mice, greater availability of fatty acids for oxidation does not explain the increase in ketone bodies. Fasting induces ketoacidosis and hypothermia 
phosphorylation at T182 at centromeres/kinetochores is required for chromosome alignment in prometaphase is consistent with the known role of Polo in regulating 
phosphorylation at centromeres. Drosophila Polo T182 is preceded by a conserved stretch of basic residues resembling the consensus site for Aurora kinases . Indeed, Drosophila Aurora B complexed with a fragment of INCENP can directly phosphorylate Polo in vitro. A T182A mutation in the Polo used as a substrate reproducibly reduced its phosphorylation by about one half. Thus, Polo T182 is a major phosphorylation site for Aurora B. Similar results were obtained using human Aurora B on GST-PoloWT or GST-PoloT182D. Kinase inhibition studies suggest that Aurora B is responsible for PoloT182 phosphorylation in vivo. Binucleine 2 is the only specific Aurora B kinase inhibitor described to date that is effective in Drosophila cells. When DMel-2 cells were treated with 20 mM Binucleine 2 for 2 h, H3S10ph was undetectable in mitotic cells and INCENP and Aurora B were dispersed in clumps on the chromosomes. Both of these phenotypes are characteristic of the loss of Aurora B function. Aurora B kinase activity is required for Polo activation at kinetochores, and levels of kinetochore-associated PoloT182ph were greatly reduced in Binucleine 2-treated mitotic cells. In contrast, we observed no obvious differ.Total Polo localized normally to kinetochores following INCENP 
Cell proliferation in parental and subtoxic elisidepsin-treated cells. Cumulative numbers of cell divisions [shown as population doubling level (PDL)] are shown for MCF-7 and MiaPaCa-2 cells until passage 5. Proliferation of MCF-7 (IC50:0.4 mM) and MiaPaCa-2 (IC50:14 mM) cells was suppressed when elisidepsin was added to the culture at subtoxic doses (0.2 and 1 mM, respectively). The number of MiaPaCa-2 and MCF-7 seeded cells were 1.256105 and 1.46105, respectively. Each growth curve was performed at least twice with similar results, SDs are shown, 
Twist, vimentin and b-actin (loading control) were detected by western blot analysis using 50 mg of total protein. C) Basal levels of E-cadherin, bcatenin and vimentin were analyzed by IHC. Magnification 20x. Each experiment was performed at least in duplicate. doi:10.1371/journal.pone.0053645.gaffects sensitivity to elisidepsin in a.St three independent experiments. B) Cell proliferation in parental and subtoxic elisidepsin-treated cells. Cumulative numbers of cell divisions [shown as population doubling level (PDL)] are shown for MCF-7 and MiaPaCa-2 cells until passage 5. Proliferation of MCF-7 (IC50:0.4 mM) and MiaPaCa-2 (IC50:14 mM) cells was suppressed when elisidepsin was added to the culture at subtoxic doses (0.2 and 1 mM, respectively). The number of MiaPaCa-2 and MCF-7 seeded cells were 1.256105 and 1.46105, respectively. Each growth curve was performed at least twice with similar results, SDs are shown, 
NC basic residues are important for Gag assembly with a possible role in the timing and location of the initial Gag multimerization reaction Comparative studies on HIV-1 and MuLV Gag assembly indicate that MuLV Gag molecules start to interact at much later time after 
Wrote the paper: MM JLD. Assisted with manuscript preparation: CC BY.
of mesenchymal cells were apparent at e11.5 along midline sagittal sections, the ventral vPCM, the dorsal dPCM and the internalCloaca Septation and Urogenital DevelopmentICM (Fig. 5). The caudal side of the cloaca was covered by the cloacal membrane, which was a composite of endoderm and ectoderm epithelia but devoid of any mesenchyme. At this stage, the distal end of ICM was juxtapositioned but not fused with dPCM and the cloacal membrane (Fig. 5C, asterisk), the likely site of the future anal canal. This unique juxtaposition separated the urogenital sinus and rectum, thereby serving as the first sign of separation between the urinary and the digestive tract (Fig. 5C). Asymmetric growth of these mesenchymal cells was likely involved in remodeling of the urogenital sinus to form the genital tubercle and the anal canal. In Six12/2;Six2+/2 mutants, the relative position of the cloacal mesenchyme, the cloacal membrane, and the unique juxtaposition were maintained (Fig. 5F). However, it was apparent that both the dPCM and the vPCM were hypoplastic, and that the size of the mutant genital tubercle was significantly smaller (Fig. 5D , and data not shown). These observations suggest that Six1 and Six2 may control the growth and/or expansion of these tissues. Since Six1 is required for the survival of renal and cardiac progenitors [12,16,22], we first used TUNEL assays to determine if survival of the PCM progenitors depended on Six.Enital distance in both wild type male and female pups (Figs. 4H , bracket). The same tissue in the Six12/2;Six2+/2 mutant washypoplastic, and the anogenital distance was significantly reduced (Fig. 4P and Q). Consistent with these gross defects, the mutant genital tubercles were hypoplastic. The Six1 and Six2 double null mutants exhibited a severe agenesis defect since the genital tubercle and the perineum were nearly absent (Fig. 4R and S). InFigure 3. An inducible genetic fate map of Six2-expressing PCM progenitors. Double Six2GCE/+;R26RLacZ pregnant females were treated with a single dose of tamoxifen at e11.5, e13.5, e14.5 and e15.5, and all embryos were collected and analyzed at e17.5 with X-gal staining (blue). (A, E, I and M) kidney sections; (B , F , J and N ) urogenital sections. CB, prospective corporal body; GT, genital tubercle; P, perineum; PF, preputial fold; PG, preputial gland; U, urethra. doi:10.1371/journal.pone.0055587.gCloaca Septation and Urogenital DevelopmentFigure 4. Genital urinary and anorectal defects of Six1;Six2 compound mutants. (A) A table of urogenital phenotypes of Six1;Six2 compound mutants. (B ) Gross ventral views of external urogenital structures. (H ) Hematoxylin and eosin (H E) staining of midline sagittal sections of urogenital structures from newborn pups. A, anus; B, bladder; GT, genital tubercle; T, tail; UM, urethral meatus; UC, umbilical cord; U, urethra; V, vagina. doi:10.1371/journal.pone.0055587.gaddition, the anal canal of the double null mutants was absent, resulting in a direct exposure of rectum epithelium (Fig 4, compare asterisk in M and S). Together, these findings suggest that Six1 and Six2 are required for the development of both digestive and urinary outlets.Survival and proliferation of PCM progenitors depend on Six1 and SixBecause of the rarity of obtaining double null mutants, we used Six12/2;Six2+/2 compound mutants to further characterize primary defects of digestive and urinary outlets during early embryogenesis. In wild type embryos, three populations of mesenchymal cells were apparent at e11.5 along midline sagittal sections, the ventral vPCM, the dorsal dPCM and the internalCloaca Septation and Urogenital DevelopmentICM (Fig. 5). The caudal side of the cloaca was covered by the cloacal membrane, which was a composite of endoderm and ectoderm epithelia but devoid of any mesenchyme. At this stage, the distal end of ICM was juxtapositioned but not fused with dPCM and the cloacal membrane (Fig. 5C, 
asterisk), the likely site of the future anal canal. This unique juxtaposition separated the urogenital sinus and rectum, thereby serving as the first sign of separation between the urinary and the digestive tract (Fig. 5C). Asymmetric growth of these mesenchymal cells was likely involved in remodeling of the urogenital sinus to form the genital tubercle and the anal canal. In Six12/2;Six2+/2 mutants, the relative position of the cloacal mesenchyme, the cloacal membrane, and the unique juxtaposition were maintained (Fig. 5F). However, it was apparent that both the dPCM and the vPCM were hypoplastic, and that the size of the mutant genital tubercle was significantly smaller (Fig. 5D , and data not shown). These observations suggest that Six1 and Six2 may control the growth and/or expansion of these tissues. Since Six1 is required for the survival of renal and cardiac progenitors [12,16,22], we first used TUNEL assays to determine if survival of the PCM progenitors depended on Six.
(Fig. 2). These data suggest that both Leu64 and Lys65 are able to regulate a2A-AR cell-surface expression in different cell types.Effect of 
the residue Lys65 influences a2A-AR transport, GFP-tagged a2A-AR and its mutants K65A and K65R were co-localized with different intracellular markers. The mutant K65A was extensively colocalized with the ER marker DsRed2-ER (Fig. 5A), but not the Golgi marker GM130 (data not shown). In contrast, wild-type a2AAR and its mutant K65R did not clearly co-localize with DeRed2-Figure 2. Effects of mutating Leu64 and Lys65 residues on the subcellular distribution of a2A-AR. GFP-tagged wild-type (WT) a2A-AR and its mutants L64A, K65A and LK-AA were transiently expressed in HEK293 (upper panel) and HeLa cells (lower panel) and their subcellular distribution of the receptors was revealed by detecting GFP fluorescence by confocal microscopy. The data shown are representative images of at least three independent experiments. Green, GFP-tagged receptors; blue, DNA staining by DAPI (nuclei). Scale bar, 10 mm. doi:10.1371/journal.pone.0050416.ga2-AR Export and Cell-Surface ExpressionFigure 3. Effects of mutating Lys65 to Arg, Glu and Gln on the cell-surface expression and subcellular distribution of a2A-AR. (A) Quantification of the cell surface and total expression of a2A-AR and its Lys mutants. HEK293 cells were transfected with a2A-AR and its mutants. The cell-surface expression of the receptors was measured by intact cell binding assays using [3H]-RX821002 and total receptor expression by flow cytometry measuring the GFP signal as described in the legends of figure 1. (B) Quantification of the cell-surface expression of a2A-AR and its mutants by flow cytometry following staining with anti-HA antibodies in nonpermeabilized cells as described in the legends of figure 1. The data shown in (A) and (B) are percentages of the mean value obtained from cells transfected with wild-type (WT) a2A-AR and are presented as the mean 6 S.E. of four experiments. *, p,0.05 versus WT a2A-AR. (C) Effect of mutation of Lys65 on the subcellular distribution of a2A-AR. a.O Ala and Arg on the cellsurface expression of a2A-AR. These data also suggest that Lys65 in the ICL1 modulates not only receptor trafficking but also receptor signaling.to the results obtained in HEK293 cells, the a2A-AR mutants L64A, K65A and LK-AA displayed a strong intracellular distribution pattern in HeLa cells (Fig. 2). These data suggest that both Leu64 and Lys65 are able to regulate a2A-AR cell-surface expression in different cell types.Effect of Mutation of Lys65 to Arg, Glu and Gln on the Cell-surface Expression and Subcellular Distribution of a2A-AROur preceding data have revealed that Lys65 plays an important role in modulating a2A-AR cell-surface 
or vehicle control 
that the local metabolism of vitamin D in adipose tissue may regulate the conversion of preadipocytes to adipocytes and hence support the healthy remodeling of human adipose tissue. Addition of 1,25(OH)2D3 to the standard differentiation cocktail promoted the maturation of adipogenesis. Although 1,25(OH)2D3 did not affect the expression of C/EBPb, an early