A thermostable DNA primase-polymerase from a mobilegenetic element involved in defence againstenvironmental 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.post-print697 K

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.Ministry of Economy and Competitiveness [BIO2016-77031-R] to J. Berenguer. An institutional grant from Fundación Ramón Areces to the CBMS

DNA interference by a mesophilic argonaute protein, cbcago [version 1; peer review: 2 approved with reservations]

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) 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 37°C, making it a promising starting point for the development of new gene editing tools for mammalian cells.Spanish Ministry of Economy and Competitiveness [BIO2016-77031-R An institutional grant from Fundación Ramón Areces to the CBMSO is also acknowledge

Estudio translacional en pacientes con cancer de tiroides diferenciado para evaluar del valor pronostico de biomarcadores metabolicos sobre la progresión tumoral y el proceso angiogénico asociado

Resumen del póster presentado al 2nd Symposium on Biomedical Research: "Advances and perspectives in cancer", celebrado en Madrid el 17 de abril de 2015.-- et al.Obesity is considered the worldwide pandemia of the XXI century. It’s association with cardiovascular diseases and type 2 diabetes has been clearly established, more recently it has also being recognized a s relevant risk factor for tumor development, with levels of increased risk being particularly noticeable in some tumor types including: esophagus, pancreas, colon and rectum, breast, endometrium, kidney, thyroid, gallbladder, and hepatocarcinoma. One study, using NCI Surveillance, Epidemiology, and End Results (SEER) data, estimated that in 2007 in the United States, about 34,000 new cases of cancer in men (4 percent) and 50,500 in women (7 percent) were due to obesity. The percentage of cases attributed to obesity varied widely for different cancer types but was as high as 40 percent for some cancers, particularly endometrial cancer and esophageal adeno-carcinoma. Obesity not only increases the incidence, it also worsens prognosis, increasing the risk of metastasis and reducing the effectiveness of therapy. However, the mechanisms that link tumor development to metabolic control are still very poorly understood and the use of metabolic biomarkers or metabolism based therapeutical approaches is very limited in common medical practice. The most generally accepted hypothesis that aims to explain the link between metabolism and tumor development is based on the discoveries of the novel laureated Dr. Otto Warburg in the ‘50s, showing that proliferating cells make a very limited use of mitochondria as source of ATP and use mainly a glycolytic metabolism. This suppression of mitochondrial activity is also observed in obesity and other metabolic disorders, leading to the proposal that some metabolic regulators could also play a relevant physiological role as tumor suppressors. The thyroid is the main responsible for the control of whole body metabolic activity that is not directly regulated by diet, it is also a highly metabolically active organ heavily dependent on mitochondrial activity and with an elevated cellular turnover. Increasing weight has been found to be associated with an increase in the risk of thyroid cancer. However, the underlying mechanisms are still unclear. Thyroid cancer incidence has increased in the last years, but so far how metabolic dysfunction impacts on the origin and development of these tumors has not been investigated. Control of oxidative metabolism, mitochondrial activity and cellular antioxidant capacity are under the regulation of the transcriptional coactivator PGC-1α. PGC-1α activity is induced by caloric restriction and reduced in obesity, and would be expected to be reduced in tumor cells. Importantly, PGC-1α regulates the activity of the tumor suppressors p53 and TLS. PGC-1α activity is particularly high in metabolically active tissues like the thyroid. However, the possible role of PGC-1α activity in tumor suppression has not yet being investigated.Peer reviewe

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

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.This work was supported by grants from the Spanish ‘‘Ministerio de Economía y Competitividad’’ [Grant no. SAF2009-07599 & SAF2012-37693 to M.M and CSD 2007-00020 to M.M.]; and the ‘‘Comunidad Autónoma de Madrid’’ [Grant no. S2010/BMD-2361 to M.M.].Peer Reviewe

SirT1 regulation of antioxidant genes is dependent on the formation of a FoxO3a/PGC-1a complex in vascular endothelial cells

Resumen del trabajo presentado al International Symposium on Redox Signaling and Oxidative Stress in Health and Disease, IV Spanish and Portuguese Meeting on Free Radicals; celebrado en Valencia (España) del 5 al 7 de junio de 2012.Excessive production of reactive oxygen species (ROS) contributes to progression of atherosclerosis, at least in part by causing endothelial dysfunction and inflammatory activation. SirT1 is a class III histone deacetylase that has been implicated in metabolic and ROS control. In the vasculature, decreased endothelial SirT1 expression increases endothelial superoxide production, NF-κB signaling, and expression of adhesion molecules. However, the mechanisms that mediate SirT1 function on the endothelium remain to be characterized. FoxO3a and PGC-1a have been shown to be molecular targets of SirT1 and to control endothelial ROS production. Here we investigated SirT1 regulation of antioxidant genes in vascular endothelial cells and the role played by FoxO3a and PGC-1a in this regulation. We found that SirT1 regulates the expression of several antioxidant genes in BAEC, including MnSOD, catalase, Prx3, Prx5, UCP-2, Trx2 and TR, and can be localized in the regulatory regions of these genes. We also found that knockdown of either FoxO3a or PGC-1a prevented SirT1 antioxidant gene induction. Furthermore, SirT1 increased the formation of a FoxO3a PGC-1a complex as determined by co-IP assays, concomitantly reducing H2O2 dependent FoxO3a and PGC-1a acetylation. Data showing that FoxO3a knockdown increases PGC-1a acetylation levels and vice versa, suggests that SirT1 activity on FoxO3a and PGC-1a may be dependent of the formation of a FoxO3a/PGC-1a complex. In conclusion, we show that SirT1 regulation of antioxidant genes in vascular endothelial cells depends of the formation of a FoxO3a/PGC-1a complex.Peer Reviewe

Sistemas de utilización del citrato en bacterias ácido lácticas

38 p.-7 tab. + 13 fig.Numerosas especies bacterianas pueden utilizar el citrato como fuente de carbono o energía, tanto en condiciones de crecimiento aeróbicas como anaeróbicas. La fermentación del citrato puede producirse en presencia de oxígeno cuando las bacterias carecen del ciclo de Krebs o en anaerobiosis, cuando dicho ciclo no es completamente funcional. Como se describe en este capítulo, la fermentación del citrato puede generar diversos productos metabólicos dependiendo no sólo del género al que pertenecen las bacterias sino también de las condiciones de crecimiento de las mismas.En este capítulo se presentará una visión global de los mecanismos de utilización del citrato en bacterias para luego tratar específicamente esta ruta en bacterias ácido lácticas. Se realizará también una descripción general, tanto a nivel genético como bioquímico, de los transportadores del citrato y citrato liasas bacterianas. Además, se describirán las rutas del metabolismo del citrato en dos bacterias Gram-negativas Klebsiella pneumoniae y Escherichia coli, microorganismos modelo en el estudio de este proceso.Peer reviewe

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.This work was supported by grants from the Spanish ‘‘Ministerio de Economía y Competitividad’’ (Grant number SAF2009-07599 & SAF2012-37693 to M.M and CSD 2007-00020 to M.M.) and the ‘‘Comunidad de Madrid’’ (Grant Number S2010/BMD-2361 to M.M.).Peer Reviewe

Total or endothelium specific PGC-1α deficiency is associated with alterations in vascular function in mice

Resumen del trabajo presentado al 41 Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Santander del 10 al 13 de septiembre de 2018.Endothelial dysfunction is a common alteration in several cardiovascular diseases that is produced by increased oxidative stress and a vascular pro-inflammatory state. This condition is associated with an imbalance in the relative contribution of endothelium-derived relaxing factors such as nitric oxide (NO) and contracting factors such as endothelin-1 (ET-1) that results in alterations in vascular function. PGC-1α is a transcriptional coactivator that regulates oxidative metabolism and participates in the control of oxidative stress. Previous in vitro studies suggest that PGC-1α activity could prevent endothelial dysfunction induced by hyperglicaemia. The aim of this work was to analyze in vivo if PGC-1α deficiency results in alterations in vascular function. For that purpose, knockout mice for PGC-1α (KO) and mice with a specific deletion of PGC-1α in the vascular endothelium (Tg-TIE2-Cre/PGC-1af/-) were used. Aorta segments were set in an organ bath to perform vascular reactivity experiments in presence/absence of lipopolysaccharide (LPS). Aorta segments from PGC-1α KO mice showed increased vasoconstriction in response to noradrenaline (NA), angiotensin II (Ang II) and ET-1 in basal conditions and decreased relaxation in response to Ach after pre-incubation with LPS. Similarly, aorta segments from TIE mice showed increased vascular contraction in response to ET-1 and Ang II and decreased relaxation in response to Ach when LPS was present. The increased vasoconstrictor response was mediated by COX-2 activation as it was blocked in the presence of the COX-2 inhibitor meloxicam. The decreased relaxation in response to Ach in LPS-preincubated segments appears to be mediated by increased oxidative stress since it was attenuated in the presence of ROS scavengers. In conclusion, total or endothelium specific PGC-1α deficiency results in alterations in the vascular response of arterial segments to both vasoconstrictors and vasodilators, especially under an inflammatory challenge. Thus, PGC-1α might be a promising target of study against vascular diseases.Peer Reviewe

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

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.Ministerio de Economía y Competitividad (España)Comunidad de MadridDepto. de Biología CelularFac. de Ciencias BiológicasTRUEpu