Effects of RNA interference against <i>C. elegans</i> homologues of bile acid biosynthetic enzymes.

<p>To identify <i>C. elegans</i> genes involved in BA synthesis, we carried out Blast homology searches for the translated products of mouse genes known to be involved in the process. In one case, the Blast results of two mouse proteins (NP_663339 and NP_085114) identified the same in worm homologues. The most homologous genes (shown in the “<i>C. elegans</i> genes” column) were tested by treatment of <i>clk-1(qm30)</i> mutants with RNAi against these genes. All cytochrome P450s were screened as they are highly similar to each other. The defecation rates are given as means ± S.E.M.</p>#<p>The accession numbers of the proteins used for the search are given.</p><p>*The p-values were obtained by comparing to control <i>clk-1</i> mutants grown in parallel on HT115 bacteria harbouring only the empty pPD129.36 vector.</p

<i>qm179</i> is allelic to <i>tat-2</i>, which encodes a protein homologous to mammalian ATP8B1.

<p>(A) Both the <i>qm179</i> and <i>tat-2(tm1634)</i> deletion mutation show a short defecation cycle in the wild type background and suppress the slow defecation of <i>clk-1(qm30)</i> mutants in double mutant combinations. Furthermore the <i>tat-2(tm1634)</i> deletion mutations fails to complement <i>qm179</i> in both the wild type and <i>clk-1</i> backgrounds. The bars represent the mean defecation cycle of animals that have been scored for three consecutive defecation cycles each in the case of <i>clk-1(qm30)</i> mutants and for five consecutive defecation cycles for all other genotypes. The error bars represent S.E.M. (n≥20 animals for each genotype). The asterisks indicate that the data are significantly different from that of wild-type or <i>clk-1</i> mutants. All differences were significant at P<0.0001 by a t-test. (B) Schematic representation of the <i>tat-2</i> coding region and the lesions of the two alleles. TAT-2 is a membrane protein with 8 predicted transmembrane domains (red) and several consensus domains for all classes of P-type ATPases (blue) or specific for the P4 P-type ATPase (green). One of the P4 P-type ATPase-specific consensus domains harbors the residue that is changed in the <i>qm179</i> allele (alanine 665 to threonine). This residue (indicated by an asterisk) is absolutely conserved among mammalian ATP8B1, <i>C. elegans</i> TAT-1, and yeast DRS2. Residues that are not perfectly conserved in this region are underlined. (C) The suppression of <i>clk-1</i> produced by the <i>tat-2(qm179)</i> mutation can be partially rescued by transgenic expression of a cDNA coding for the mouse homologue ATP8B1 under the control of the endogenous <i>tat-2</i> promoter (P<i>tat-2::mAtp8b1</i>). Treating <i>clk-1(qm30); tat-2(qm179)</i> mutants that harbor the <i>Ptat-2:mAtp8b1</i> construct with RNAi against <i>mAtp8b1</i> abolished the rescue but had no effect by itself. The bars represent the mean defecation cycle of animals that have been scored for five consecutive defecation cycles. The error bars represent S.E.M. (n≥20 animals for each genotype). Differences were tested by a t-test; * represents P<0.05.</p

The activities of endogenous BA–like molecules are altered by mitochondrial ROS levels.

<p>The bars represent the mean defecation cycle lengths. As before (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002553#pgen-1002553-g002" target="_blank">Figure 2D</a>), we find that <i>clk-1</i> mutant lipid extracts contain more activity than the wild type. However, treatment with NAC lowers the activity level to that of the wild type extract, and the extract from <i>clk-1; sod-2</i> double mutants has much more activity than the extract from <i>clk-1</i> single mutants. All extracts are without effect on the wild type. The asterisks represent t-test comparisons to the control of 0.25% DMSO treatment (n≥25 animals). The error bars represent S.E.M. *** represents P<0.001, **represents P<0.01, *represents P<0.05.</p

<i>clk-1</i> mutants are sensitive to alterations in ROS metabolism.

<p>The bars represent the mean defecation cycle length of animals that were scored for one cycle. (A) RNAi mediated knock-down of the mitochondrial localized superoxide dismutases SOD-2 and SOD-3 enhance the slow defecation cycle phenotype of <i>clk-1</i> mutants (n≥6 for each genotype). (B) NAC (N-acetyl cysteine) supplementation suppresses the slow defecation of <i>clk-1</i> mutants at 8 and 10 mM concentrations (n≥22 for each genotype). (C) Treatment with 10 mM NAC is without effect on <i>tat-2; clk-1</i> double mutants (n≥6 for each genotype). The error bars represent S.E.M. *** represents P<0.001, **represents P<0.01, *represents P<0.05.</p

<i>clk-1</i> mutants are sensitive to the availability of endogenous BA–like molecules and to exogenous mammalian BAs.

<p>The bars represent the mean defecation cycle length of animals that had been scored for three consecutive cycles for <i>clk-1(qm30)</i> and <i>isp-1(qm150)</i> and for five consecutive defecation cycles for all other genotypes. (A) The BA-binding resin cholestyramine partially suppresses the slow defecation of <i>clk-1</i> mutants but is without effect on the wild type as well as on the mitochondrial mutant <i>isp-1(qm150)</i> (n≥50 for each genotype). (B) <i>clk-1(qm30)</i> mutants are sensitive to exogenous BAs, which enhance the <i>clk-1</i> phenotype (n≥20 animals). (C) Cholic acid (CA), a relatively hydrophilic BA, suppresses <i>clk-1</i> at very low concentration but enhances the phenotype at higher concentration. Chenodeoxycholic acid (CDCA) a relatively hydrophobic BA is without effect at 0.15 mM but enhances the phenotype at higher concentrations. All effects depend on the presence of cholesterol as they are totally or mostly (at 2.5 mM CDCA) abolished in the absence of cholesterol supplementation. The asterisks represent p values obtained by t-test comparing the defecation cycle after various treatments to that observed with 0.25% DMSO only. All treatments in the absence of cholesterol were indistinguishable from 0.25% DMSO except at 2.5 mM CDCA which was significantly slower at P<0.001; (n≥40 animals for each condition). (D) Ether extracts from the wild type or <i>clk-1</i> mutants enhance the defecation phenotype of <i>clk-1</i> mutants but not that of the wild type in a dose-dependent manner and extracts from <i>clk-1</i> mutants are approximately four times more active than extracts from the wild type. The asterisks represent t-test comparisons to the control of 0.25% DMSO treatment (n≥25 animals). (E) The defecation phenotype of <i>tat-2(qm179)</i> mutants can be rescued by exogenous BAs but this effect is abolished in the absence of cholesterol supplementation. In addition, the suppression of <i>clk-1</i> by <i>tat-2</i> is also suppressed by BA treatment, an effect which also requires cholesterol supplementation. BA treatment is without effect on the wild type and on <i>dsc-4</i> mutants. (n≥20 for each genotype and condition). The data presented in (B) and in (E) were generated simultaneously and the data for the wild type is the same in both panels. They are presented in separate panels for clarity. The error bars represent S.E.M. *** represents P<0.001, **represents P<0.01, *represents P<0.05.</p

Relief of endoplasmic reticulum stress enhances DNA damage repair and improves development of pre-implantation embryos

<div><p>Early-cleaving embryos are known to have better capacity to reach the blastocyst stage and produce better quality embryos compared to late-cleaving embryos. To investigate the significance of endoplasmic reticulum (ER) stress on early embryo cleavage kinetics and development, porcine embryos produced <i>in vitro</i> were separated into early- and late-cleaving groups and then cultured in the absence or presence of the ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Developing embryos were collected at days 3 to 7 of culture for assessment of ER stress status, incidence of DNA double-strand breaks (DSBs), development and total cell number. In the absence of TUDCA treatment, late-cleaving embryos exhibited ER stress, higher incidence of DNA DSBs, as well as reductions in development to the blastocyst stage and total embryo cell numbers. Treatment of late-cleaving embryos with TUDCA mitigated these effects and markedly improved embryo quality and development. These results demonstrate the importance of stress coping responses in early developing embryos, and that reduction of ER stress is a potential means to improve embryo quality and developmental competence.</p></div

Culture with TUDCA improves embryo quality and blastocyst rate.

<p>Evaluation of embryo quality, as indicated by mean total cell number, was performed in developing embryos and blastocysts cultured without or with TUDCA. Development to the blastocyst stage was also investigated. Percent development to the blastocyst stage (blastocyst rate) in each treatment group of early-cleaving embryos, n = 335 (A) and late-cleaving embryos, n = 585 (B). Mean total cell number for each treatment group of early-cleaving developing embryos, n = 42 (C) and late-cleaving developing embryos, n = 42 (D). Mean total cell number for each treatment group of early-cleaving blastocysts, n = 89 (E) and late-cleaving blastocysts, n = 65 (F). Representative images of early-cleaving and late-cleaving blastocysts cultured without or with TUDCA (G). Images were captured at 20X magnification. Developing embryos are defined as those collected at day 3 and 5 of culture. Data was collected from 5 replicates for blastocyst development and 7 replicates for blastocyst cell number. Late = late-cleaving embryos cultured without TUDCA; Late + TUDCA = late-cleaving embryos cultured with TUDCA; Early = early-cleaving embryos cultured without TUDCA; Early + TUDCA = early-cleaving embryos cultured with TUDCA. Statistically significant differences among groups are indicated by * (p < 0.05) and ** (p < 0.01).</p

ER stress in the developing embryo and blastocyst: Immunofluorescence.

<p>Comparison of ER stress marker immunofluorescence in embryos to evaluate ER stress status. Mean relative GRP78 immunofluorescence in the early-cleaving developing embryo, n = 19 (A) and late-cleaving developing embryo, n = 13 (B). Mean relative GRP78 immunofluorescence in the early-cleaving blastocyst, n = 9 (C) and late-cleaving blastocyst, n = 6 (D). Representative images of GRP78 immunofluorescence for each treatment group in developing embryos (E). Images were captured at 20X magnification. Developing embryos are defined as those collected at day 3 and 5 of culture. Data was collected from 5 and 4 replicates for developing embryo and blastocyst immunofluorescence, respectively. Late = late-cleaving embryos cultured without TUDCA; Late + TUDCA = late-cleaving embryos cultured with TUDCA; Early = early-cleaving embryos cultured without TUDCA; Early + TUDCA = early-cleaving embryos cultured with TUDCA. Statistically significant differences among groups are indicated by * (p < 0.05).</p

Inhibition of the Unfolded Protein Response Mechanism Prevents Cardiac Fibrosis

<div><p>Background</p><p>Cardiac fibrosis attributed to excessive deposition of extracellular matrix proteins is a major cause of heart failure and death. Cardiac fibrosis is extremely difficult and challenging to treat in a clinical setting due to lack of understanding of molecular mechanisms leading to cardiac fibrosis and effective anti-fibrotic therapies. The objective in this study was to examine whether unfolded protein response (UPR) pathway mediates cardiac fibrosis and whether a pharmacological intervention to modulate UPR can prevent cardiac fibrosis and preserve heart function.</p><p>Methodology/Principal Findings</p><p>We demonstrate here that the mechanism leading to development of fibrosis in a mouse with increased expression of calreticulin, a model of heart failure, stems from impairment of endoplasmic reticulum (ER) homeostasis, transient activation of the unfolded protein response (UPR) pathway and stimulation of the TGFβ1/Smad2/3 signaling pathway. Remarkably, sustained pharmacologic inhibition of the UPR pathway by tauroursodeoxycholic acid (TUDCA) is sufficient to prevent cardiac fibrosis, and improved exercise tolerance.</p><p>Conclusions</p><p>We show that the mechanism leading to development of fibrosis in a mouse model of heart failure stems from transient activation of UPR pathway leading to persistent remodelling of cardiac tissue. Blocking the activation of the transiently activated UPR pathway by TUDCA prevented cardiac fibrosis, and improved prognosis. These findings offer a window for additional interventions that can preserve heart function.</p></div

DNA damage in the developing embryo and blastocyst.

<p>Evaluation of H2AX139ph foci to assess DNA damage. Comparison of mean H2AX139ph foci per cell in early-cleaving developing embryos, n = 64 (A) and late-cleaving developing embryos, n = 55 (B). Comparison of proportion of cells positive for H2AX139ph foci in early-cleaving blastocysts, n = 52 (D) and late-cleaving blastocysts, n = 23 (E). Representative images of H2AX139ph immunofluorescence in developing embryos (C) and blastocysts (F), indicating nuclei (blue) and the H2AX139ph foci (red) used to assess DNA damage. Images were captured at 20X magnification. Developing embryos are defined as those collected at day 3 and 5 of culture. Late = late-cleaving embryos cultured without TUDCA; Late + TUDCA = late-cleaving embryos cultured with TUDCA; Early = early-cleaving embryos cultured without TUDCA; Early + TUDCA = early-cleaving embryos cultured with TUDCA. Statistically significant differences among groups are indicated by * (p < 0.05) and ** (p < 0.01).</p