MYH10 activation rescues contractile defects in arrhythmogenic cardiomyopathy (ACM).

The most prevalent genetic form of inherited arrhythmogenic cardiomyopathy (ACM) is caused by mutations in desmosomal plakophilin-2 (PKP2). By studying pathogenic deletion mutations in the desmosomal protein PKP2, here we identify a general mechanism by which PKP2 delocalization restricts actomyosin network organization and cardiac sarcomeric contraction in this untreatable disease. Computational modeling of PKP2 variants reveals that the carboxy-terminal (CT) domain is required for N-terminal domain stabilization, which determines PKP2 cortical localization and function. In mutant PKP2 cells the expression of the interacting protein MYH10 rescues actomyosin disorganization. Conversely, dominant-negative MYH10 mutant expression mimics the pathogenic CT-deletion PKP2 mutant causing actin network abnormalities and right ventricle systolic dysfunction. A chemical activator of non-muscle myosins, 4-hydroxyacetophenone (4-HAP), also restores normal contractility. Our findings demonstrate that activation of MYH10 corrects the deleterious effect of PKP2 mutant over systolic cardiac contraction, with potential implications for ACM therapy.

Sustained Elevated Blood Pressure Accelerates Atherosclerosis Development in a Preclinical Model of Disease.

The continuous relationship between blood pressure (BP) and cardiovascular events makes the distinction between elevated BP and hypertension based on arbitrary cut-off values for BP. Even mild BP elevations manifesting as high-normal BP have been associated with cardiovascular risk. We hypothesize that persistent elevated BP increases atherosclerotic plaque development. To evaluate this causal link, we developed a new mouse model of elevated BP based on adeno-associated virus (AAV) gene transfer. We constructed AAV vectors to support transfer of the hRenin and hAngiotensinogen genes. A single injection of AAV-Ren/Ang (1011 total viral particles) induced sustained systolic BP increase (130 ± 20 mmHg, vs. 110 ± 15 mmHg in controls; p = 0.05). In ApoE-/- mice, AAV-induced mild BP elevation caused larger atherosclerotic lesions evaluated by histology (10-fold increase vs. normotensive controls). In this preclinical model, atheroma plaques development was attenuated by BP control with a calcium channel blocker, indicating that a small increase in BP within a physiological range has a substantial impact on plaque development in a preclinical model of atherosclerosis. These data support that non-optimal BP represents a risk for atherosclerosis development. Earlier intervention in elevated BP may prevent or delay morbidity and mortality associated with atherosclerosis.

A thermostable DNA primase-polymerase from a mobile genetic element involved in defence against environmental DNA.

Primase-polymerases (Ppol) are one of the few enzymes able to start DNA synthesis on ssDNA templates. The role of Thermus thermophilus HB27 Ppol, encoded along a putative helicase (Hel) within a mobile genetic element (ICETh2), has been studied. A mutant lacking Ppol showed no effects on the replication of the element. Also, no apparent differences in the sensitivity to DNA damaging agents and other stressors or morphological changes in the mutant cells were detected. However, the mutants lacking Ppol showed an increase in two to three orders of magnitude in their transformation efficiency with plasmids and genomic DNA acquired from the environment (eDNA), independently of its origin and G + C content. In contrast, no significant differences with the wild type were detected when the cells received the DNA from other T. thermophilus partners in conjugation-like mating experiments. The similarities of this behaviour with that shown by mutants lacking the Argonaute (ThAgo) protein suggests a putative partnership Ppol-ThAgo in the DNA-DNA interference mechanism of defence, although other eDNA defence mechanisms independent of ThAgo cannot be discarded.

DNA interference by a mesophilic Argonaute protein, CbcAgo.

Background: The search for putative enzymes that can facilitate gene editing has recently focused its attention on Argonaute proteins from prokaryotes (pAgos). Though they are structural homologues of human Argonaute protein, which uses RNA guides to interfere with RNA targets, pAgos use ssDNA guides to identify and, in many cases, cut a complementary DNA target. Thermophilic pAgos from Thermus thermophilus, Pyrococcus furiosus and Methanocaldococcus jasmanii have been identified and thoroughly studied, but their thermoactivity makes them of little use in mesophilic systems such as mammalian cells. Methods: Here we search for and identify CbcAgo, a prokaryotic Argonaute protein from a mesophilic bacterium, and characterize in vitro its DNA interference activity. Results: CbcAgo efficiently uses 5'P-ssDNA guides as small as 11-mers to cut ssDNA targets, requires divalent cations (preferentially, Mn 2+) and has a maximum activity between 37 and 42 °C, remaining active up to 55 °C. Nicking activity on supercoiled dsDNA was shown. However, no efficient double-strand breaking activity could be demonstrated. Conclusions: CbcAgo can use gDNA guides as small as 11 nucleotides long to cut complementary ssDNA targets at 37ºC, making it a promising starting point for the development of new gene editing tools  for mammalian cells.

Oxidative stress induces loss of pericyte coverage and vascular instability in PGC-1α-deficient mice.

Peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) is a regulator of mitochondrial oxidative metabolism and reactive oxygen species (ROS) homeostasis that is known to be inactivated in diabetic subjects. This study aimed to investigate the contribution of PGC-1α inactivation to the development of oxygen-induced retinopathy. We analyzed retinal vascular development in PGC-1α(-/-) mice. Retinal vasculature of PGC-1α(-/-) mice showed reduced pericyte coverage, a de-structured vascular plexus, and low perfusion. Exposure of PGC-1α(-/-) mice to hyperoxia during retinal vascular development exacerbated these vascular abnormalities, with extensive retinal hemorrhaging and highly unstructured areas as compared with wild-type mice. Structural analysis demonstrated a reduction in membrane-bound VE-cadherin, which was suggestive of defective intercellular junctions. Interestingly, PGC-1α(-/-) retinas showed a constitutive activation of the VEGF-A signaling pathway. This phenotype could be partially reversed by antioxidant administration, indicating that elevated production of ROS in the absence of PGC-1α could be a relevant factor in the alteration of the VEGF-A signaling pathway. Collectively, our findings suggest that PGC-1α control of ROS homeostasis plays an important role in the regulation of de novo angiogenesis and is required for vascular stability.

Regulation of endothelial dynamics by PGC-1α relies on ROS control of VEGF-A signaling.

UNLABELLED: Peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α) is a regulator of mitochondrial metabolism and reactive oxygen species (ROS) that is known to play a relevant role in angiogenesis. AIMS: This study aims to investigate the role of ROS on the regulation by PGC-1α of angiogenesis. METHODS AND RESULTS: We found that endothelial cells (ECs) from mice deleted for PGC-1α display attenuated adhesion to the extracellular matrix, together with slower and reversible spreading. Structural analysis demonstrates unstable formation of focal adhesions, defective cytoskeleton reorganization in response to cellular matrix adhesion, cell migration and cell-cell adhesion. Confluent cultures showed also a reduction of membrane bound VE-cadherin, suggesting defective inter-cellular junction formation. Functional consequences included impaired directional migration, and enhanced tip phenotype in aortic explants sprouting assays. At the molecular level, PGC-1α-deleted ECs exhibit a constitutive activation of the vascular endothelial growth factor-A (VEGF-A) signaling pathway and a defective response to VEGF-A. All these alterations are partially reversed by administration of the antioxidant EUK-189. The contribution of mitochondrial ROS and NOX activation was confirmed using a mitochondrial targeted antioxidant (MitoTEMPO) and a NOX inhibitor (VAS-2870). These results indicate that elevated production of ROS in the absence of PGC-1α is a key factor in the alteration of the VEGF-A signaling pathway and the capacity of endothelial cells to form stable interactions with other endothelial cells and with the extracellular matrix. Our findings show that PGC-1α control of ROS homeostasis plays an important role in the control of endothelial response to VEGF-A.

Control of endothelial function and angiogenesis by PGC-1α relies on ROS control of vascular stability.

Peroxisome proliferator activated receptor g co-activator 1alpha (PGC-1α) is a regulator of oxidative metabolism and reactive oxygen species (ROS) homeostasis that has been show to play a relevant role in angiogenesis. PGC-1α KO mice show reduced vascular density in the retinas and KO primary vascular endothelial cells (ECs) migrate faster than the wild type, an effect that can be rescued by antioxidants, suggesting that excessive ROS levels might be relevant in PGC-1 α role in angiogenesis. This study aims to investigate the role of ROS homeostasis on the regulation by PGC-1 α of angiogenesis. We found that endothelial cells (ECs) from mice deleted for PGC-1 α display attenuated adhesion to the extracellular matrix, together with slower spreading, reduced formation of cellular junctions, a disorganized cytoskeleton and random motility, and a enhanced tip phenotype. Aditionally, PGC-1 α -deleted ECs exhibit an altered response to vascular endothelial growth factor-A (VEGF-A). In vivo, deletion of PGC-1 α results in addition to reduced retinal vascular density, sparse pericyte coverage. Exposure of PGC-1 α deleted mice to hyperoxia during retinal vascular development exacerbates these vascular abnormalities and mice show extensive retinal hemorrhaging, with highly unstructured areas and very poor perfusion, compared with wild-type mice. Structural analysis demonstrates a reduction of endothelial VE-cadherin, suggesting defective inter-cellular junctions. Interestingly, this hyperoxia-induced phenotype is partially reversed by antioxidant administration, indicating that elevated production of mitochondrial reactive oxygen species (ROS) in the absence of PGC-1 α is functionally important. Finally, in vitro studies show that antioxidant treatment improves VEGF-A signaling, suggesting that toxic effect of ROS may be caused by the alteration of the VEGF-A signaling pathway. In summary, our findings indicate that PGC-1 α control of ROS homeostasis plays an important role in the control of de novo angiogenesis, and is required for vascular stability.

SirT1 regulation of antioxidant genes is dependent on the formation of a FoxO3a/PGC-1α complex.

UNLABELLED: SirT1 is a class III histone deacetylase that has been implicated in metabolic and reactive oxygen species control. In the vasculature it has been shown to decrease endothelial superoxide production, prevent endothelial dysfunction and atherosclerosis. However, the mechanisms that mediate SirT1 antioxidant functions remain to be characterized. The transcription factor FoxO3a and the transcriptional coactivator peroxisome proliferator activated receptor γ-coactivator 1α (PGC-1α) have been shown to induce the expression of antioxidant genes and to be deacetylated by SirT1. AIMS: Here we investigated SirT1 regulation of antioxidant genes and the roles played by FoxO3a and PGC-1α in this regulation. RESULTS: We found that SirT1 regulates the expression of several antioxidant genes in bovine aortic endothelial cells, including Mn superoxide dismutase (MnSOD), catalase, peroxiredoxins 3 and 5 (Prx3, Prx5), thioredoxin 2 (Trx2), thioredoxin reductase 2 (TR2), and uncoupling protein 2 (UCP-2) and can be localized in the regulatory regions of these genes. We also found that knockdown of either FoxO3a or PGC-1α prevented the induction of antioxidant genes by SirT1 over-expression. Furthermore, SirT1 increased the formation of a FoxO3a/PGC-1α complex as determined by co-immunoprecipitation (IP) assays, concomitantly reducing H2O2-dependent FoxO3a and PGC-1α acetylation. Data showing that FoxO3a knockdown increases PGC-1α acetylation levels and vice versa, suggest that SirT1 activity on FoxO3a and PGC-1α may be dependent of the formation of a FoxO3a/PGC-1α complex. INNOVATION: A unifying mechanism for SirT1 activities is suggested. CONCLUSION: We show that SirT1 regulation of antioxidant genes in vascular endothelial cells depends on the formation of a FoxO3a/PGC-1α complex.

A real-time PCR assay for detection and quantification of 2-branched (1,3)-beta-D-glucan producing lactic acid bacteria in cider.

Ropiness in natural cider is a relatively frequent alteration, mainly found after bottling, leading to consumer rejection. It is derived from the production of exopolysaccharides (EPS) by some lactic acid bacteria most of which synthesize a 2-branched (1,3)-beta-D-glucan and belong to the genera Pediococcus, Lactobacillus and Oenococcus. This polysaccharide synthesis is controlled by a single transmembrane glycosyltransferase (GTF). In this work, a method based on quantitative PCR (qPCR) and targeting the gtf gene was developed for detection and quantification of these bacteria in cider. The newly designed primers GTF3/GTF4 delimit a 151bp fragment within the 417bp amplicon previously designed for conventional PCR. The inclusivity and exclusivity of the qPCR assay were assessed with 33 cider isolates belonging to genus Lactobacillus, Oenoccocus and Pedioccocus, together with reference strains of 16 species and five genera including beta-glucan, alpha-glucan and heteropolysaccharide (HePS) producing strains and non-EPS producers. The qPCR assay, followed by the melting curve analysis, confirmed the generation of a single PCR product from the beta-glucan producers with a T(m) of 74.28+/-0.08 and C(T) values (10ng DNA) ranging between 8.46 and 16.88 (average 12.67+/-3.5). Some EPS(-) LAB strains rendered C(T) values ranging from 28.04 to 37.75 but they were significantly higher (P(C(T)<28.54)=0.05) than those of the beta-glucan producers. The assay showed a wide quantification range of 5 log units using calibrated cell suspensions of Pediococcus parvulus 2.6 and Oenococcus oeni I4. The linearity was extended over 7 log orders when calibration curves were obtained from DNA. The detection limit for beta-glucan producing LAB in artificially contaminated cider was about 3x10(2)CFU per ml. The newly developed qPCR assay was successfully applied to monitor the cidermaking process, in 13 tanks from two cider factories, revealing a decrease in C(T) values derived from an increase in beta-glucan producing LAB populations. In addition, 8 naturally spoiled bottled cider were tested for the quantification of these organisms using the five standard curves constructed: P. parvulus 2.6 genomic DNA and gtf amplicon (417bp), calibrated cell suspensions of Pediococcus parvulus 2.6, Lactobacillus diolivorans G77 and Oenococcus oeni I4 and results were compared to LAB total counts on MRS. Levels obtained from the different approaches were within a log range and showed no significant differences. Therefore, the amplicon-derived standard curve is proposed for the routine estimation of gtf(+)populations in cider.

Inactivation of Foxo3a and subsequent downregulation of PGC-1 alpha mediate nitric oxide-induced endothelial cell migration.

In damaged or proliferating endothelium, production of nitric oxide (NO) from endothelial nitric oxide synthase (eNOS) is associated with elevated levels of reactive oxygen species (ROS), which are necessary for endothelial migration. We aimed to elucidate the mechanism that mediates NO induction of endothelial migration. NO downregulates expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 alpha), which positively modulates several genes involved in ROS detoxification. We tested whether NO-induced cell migration requires PGC-1 alpha downregulation and investigated the regulatory pathway involved. PGC-1 alpha negatively regulated NO-dependent endothelial cell migration in vitro, and inactivation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, which is activated by NO, reduced NO-mediated downregulation of PGC-1 alpha. Expression of constitutively active Foxo3a, a target for Akt-mediated inactivation, reduced NO-dependent PGC-1 alpha downregulation. Foxo3a is also a direct transcriptional regulator of PGC-1 alpha, and we found that a functional FoxO binding site in the PGC-1 alpha promoter is also a NO response element. These results show that NO-mediated downregulation of PGC-1 alpha is necessary for NO-induced endothelial migration and that NO/protein kinase G (PKG)-dependent downregulation of PGC-1 alpha and the ROS detoxification system in endothelial cells are mediated by the PI3K/Akt signaling pathway and subsequent inactivation of the FoxO transcription factor Foxo3a.

Activation of the diacetyl/acetoin pathway in Lactococcus lactis subsp. lactis bv. diacetylactis CRL264 by acidic growth.

Lactococcus lactis subsp. lactis bv. diacetylactis strains are aroma-producing organisms used in starter cultures for the elaboration of dairy products. This species is essentially a fermentative microorganism, which cometabolizes glucose and citrate to yield aroma compounds through the diacetyl/acetoin biosynthetic pathway. Our previous results have shown that under acidic growth Lactococcus bv. diacetylactis CRL264 expresses coordinately the genes responsible for citrate transport and its conversion into pyruvate. In the present work the impact of acidic growth on glucose, citrate, and pyruvate metabolism of Lactococcus bv. diacetylactis CRL264 has been investigated by proteomic analysis. The results indicated that acid growth triggers the conversion of citrate, but not glucose, into alpha-acetolactate via pyruvate. Moreover, they showed that low pH has no influence on levels of lactate dehydrogenase and pyruvate dehydrogenase. Therefore, the influence of external pH on regulation of the diacetyl/acetoin biosynthetic pathway in Lactococcus bv. diacetylactis CRL264 has been analyzed at the transcriptional level. Expression of the als, aldB, aldC, and butBA genes encoding the enzymes involved in conversion of pyruvate into aroma compounds has been investigated by primer extension, reverse transcription-PCR analysis, and transcriptional fusions. The results support that this biosynthetic pathway is induced at the transcriptional level by acidic growth conditions, presumably contributing to lactococcal pH homeostasis by synthesis of neutral compounds and by decreasing levels of pyruvate.

Contribution of citrate metabolism to the growth of Lactococcus lactis CRL264 at low pH.

Lactococcus lactis subsp. lactis biovar diacetylactis CRL264 is a natural strain isolated from cheese (F. Sesma, D. Gardiol, A. P. de Ruiz Holgado, and D. de Mendoza, Appl. Environ. Microbiol. 56:2099-2103, 1990). The effect of citrate on the growth parameters at a very acidic pH value was studied with this strain and with derivatives whose citrate uptake capacity was genetically manipulated. The culture pH was maintained at 4.5 to prevent alkalinization of the medium, a well-known effect of citrate metabolism. In the presence of citrate, the maximum specific growth rate and the specific glucose consumption rate were stimulated. Moreover, a more efficient energy metabolism was revealed by analysis of the biomass yields relative to glucose consumption or ATP production. Thus, it was shown that the beneficial effect of citrate on growth under acid stress conditions is not primarily due to the concomitant alkalinization of the medium but stems from less expenditure of ATP, derived from glucose catabolism, to achieve pH homeostasis. After citrate depletion, a deleterious effect on the final biomass was apparent due to organic acid accumulation, particularly acetic acid. On the other hand, citrate metabolism endowed cells with extra ability to counteract lactic and acetic acid toxicity. In vivo 13C nuclear magnetic resonance provided strong evidence for the operation of a citrate/lactate exchanger. Interestingly, the greater capacity for citrate transport correlated positively with the final biomass and growth rates of the citrate-utilizing strains. We propose that increasing the citrate transport capacity of CRL264 could be a useful strategy to improve further the ability of this strain to cope with strongly acidic conditions.

Processing of as-48ABC RNA in AS-48 enterocin production by Enterococcus faecalis.

Enterocin AS-48 production and immunity characters are encoded by 10 genes (as-48ABCC(1)DD(1)EFGH) of the pMB2 plasmid from the Enterococcus faecalis S-48 strain. Among these, as-48A, encoding the AS-48 peptide, and the as-48BC genes constitute a cluster required for AS-48 biogenesis and full immunity. In this study, the levels of expression of this cluster have been altered by insertion and site-directed mutagenesis as well as by expression coupled to trans complementation. Phenotypic studies of the mutants have indicated cotranscription of the three genes and revealed that the inactivation of as-48B prevents the production of AS-48, thus confirming its essentiality in AS-48 biogenesis. These studies have also supported the involvement of as-48C in enterocin immunity. In addition, they established that the intergenic region between the as-48A and as-48B genes is decisive for AS-48 expression, since a 3-bp substitution, which should disrupt a potential 47-nucleotide complex secondary structure, resulted in a hypoproducing phenotype. Transcriptional analyses of the E. faecalis wild-type and mutant strains supports the possibility that the as-48ABC genes are transcribed from the P(A) promoter located upstream of as-48A. Moreover, analysis and bioinformatic predictions of RNA folding indicate that as-48ABC mRNA is processed at the secondary structure located between as-48A and as-48B. Thus, synthesis of the AS-48 peptide appears to be controlled at the posttranscriptional level and is uncoupled from as-48BC translation. This mechanism of genetic regulation has not been previously described for the regulation of bacteriocin expression in enterococci.