Mitochondrial Na+ controls oxidative phosphorylation and hypoxic redox signalling

All metazoans depend on O2 delivery and consumption by the mitochondrial oxidative phosphorylation (OXPHOS) system to produce energy. A decrease in O2 availability (hypoxia) leads to profound metabolic rewiring. In addition, OXPHOS uses O2 to produce reactive oxygen species (ROS) that can drive cell adaptations through redox signalling, but also trigger cell damage1–4, and both phenomena occur in hypoxia4–8. However, the precise mechanism by which acute hypoxia triggers mitochondrial ROS production is still unknown. Ca2+ is one of the best known examples of an ion acting as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential and collaborating in ion transport10. Here we show that Na+ acts as a second messenger regulating OXPHOS function and ROS production by modulating fluidity of the inner mitochondrial membrane (IMM). We found that a conformational shift in mitochondrial complex I during acute hypoxia11 drives the acidification of the matrix and solubilization of calcium phosphate precipitates. The concomitant increase in matrix free-Ca2+ activates the mitochondrial Na+/Ca2+ exchanger (NCLX), which imports Na+ into the matrix. Na+ interacts with phospholipids reducing IMM fluidity and mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III, generating a redox signal. Inhibition of mitochondrial Na+ import through NCLX is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ import into the mitochondrial matrix controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences in cellular metabolism

Deep-sequencing reveals broad subtype-specific HCV resistance mutations associated with treatment failure

A percentage of hepatitis C virus (HCV)-infected patients fail direct acting antiviral (DAA)-based treatment regimens, often because of drug resistance-associated substitutions (RAS). The aim of this study was to characterize the resistance profile of a large cohort of patients failing DAA-based treatments, and investigate the relationship between HCV subtype and failure, as an aid to optimizing management of these patients. A new, standardized HCV-RAS testing protocol based on deep sequencing was designed and applied to 220 previously subtyped samples from patients failing DAA treatment, collected in 39 Spanish hospitals. The majority had received DAA-based interferon (IFN) a-free regimens; 79% had failed sofosbuvir-containing therapy. Genomic regions encoding the nonstructural protein (NS) 3, NS5A, and NS5B (DAA target regions) were analyzed using subtype-specific primers. Viral subtype distribution was as follows: genotype (G) 1, 62.7%; G3a, 21.4%; G4d, 12.3%; G2, 1.8%; and mixed infections 1.8%. Overall, 88.6% of patients carried at least 1 RAS, and 19% carried RAS at frequencies below 20% in the mutant spectrum. There were no differences in RAS selection between treatments with and without ribavirin. Regardless of the treatment received, each HCV subtype showed specific types of RAS. Of note, no RAS were detected in the target proteins of 18.6% of patients failing treatment, and 30.4% of patients had RAS in proteins that were not targets of the inhibitors they received. HCV patients failing DAA therapy showed a high diversity of RAS. Ribavirin use did not influence the type or number of RAS at failure. The subtype-specific pattern of RAS emergence underscores the importance of accurate HCV subtyping. The frequency of “extra-target” RAS suggests the need for RAS screening in all three DAA target regions

La ciclosporina A produce anión superóxido intracelular en el endotelio

5 p.-4 fig.Muchas enfermedades están asociadas con una sobreproducción de radicales libres del oxígeno generadores de daño celular. Entre estos radicales, el anión superóxido ha recibido especial atención por su participación en patologías con alta prevalencia, como por ejemplo, el daño por reperfusión (tras infarto miocárdico o accidente cerebrovascular) o los procesos inflamatorios como la artritis. La importancia del anión superóxido en términos de utilidad terapéutica queda reflejado en el esfuerzo, culminado ya al menos en animales1, por encontrar agentes mimetizadores de la actividad superóxido dismutasa (eliminación de anión superóxido) con estabilidad química y biológica in vivo. Hasta ahora se ha considerado que los efectos tóxicos de los fármacos inmunosupresores inhibidores de la calcineurina, como es la CsA, podían depender también de la inhibición de este enzima. Entre estos efectos tóxicos se encuentra el daño sobre el endotelio vascular, manifestado en ocasiones como hipertensión o como síndromes que recuerdan a la afectación vascular de la anemia hemolítica microangiopática (microangiopatía trombótica). Hasta la fecha no se ha demostrado relación entre la inhibición de la actividad fosfatasa de la calcineurina y estos efectos secundarios, por lo que se hace necesario estudiar otros mecanismos alternativos que permitan explicarlos.Javier Navarro Antolín es becario del Fondo de Investigación Sanitaria (FIS) (BEFI 98/9070). Los estudios comentados en este trabajo han sido financiados en parte por la Sociedad Española de Nefrología (AYUDA 2/98), el Plan Nacional de I+D (CICYT SAF 97-0035) y la Comunidad Autónoma de Madrid (CAM 08.4/0032/1998).Peer reviewe

Efecto de los inmunosupresores CsA y FK506 sobre vías de señalización intracelular y expresión génica en el endotelio

5 p.-5 fig.[EN]The primary action of CsA and FK506 is to block the expression of lymphokines produced by T-cells. Damage to the vascular endothelium is one of their most well known side effects. Little is known about the molecular changes in signal transduction and gene expression induced by CsA on the endothelium. A CsA calcidependent up-regulation of eNOS mRNA was detected in bovine aortic endothelial cells. A similar result was observed with FK506. CsA appears to increase eNOS mRNA mainly by increasing the rate of transcription. Since CsA and FK506 induce an increase in reactive oxygen species in the bovine endothelium, a role of peroxynitrite in the vascular damage by immunosuppressants should be ruled out. An implication of calcineurin and NF-AT in this toxic effect remains to be shown. A molecular description of the immunosuppressants associated toxic mechanisms should be helpful in the design of new immunosuppressive molecules.[ES]CsA y FK506, después de unirse a su correspondiente inmunofilina citosólica, inhiben la calcineurina y con ello la traslocación de NF-AT al núcleo. Este paso temprano en la activación del linfocito T es importante para la activación de la transcripción de genes que codifican citoquinas involucradas en la respuesta inmunológica. En la regulación transcripcional mediada por NF-AT suele participar AP-1. Además del efecto inmunosupresor, uno de los efectos tóxicos de estos fármacos es el daño sobre el endotelio vascular. Estudios in vivo e in vitro demuestran que la CsA modifica la regulación de algunas sustancias vasomotoras. Concretamente la CsA aumenta la producción de NO en el endotelio de aorta bovina. El aumento de la síntesis del NO puede tener un componente de regulación a nivel transcripcional. En el promotor del gen eNOS humano existen dos sitios putativos AP-1 y un sitio putativo NF-AT. La asociación del NO con radicales libres del oxígeno, que también son estimulados por la CsA en el endotelio de aorta bovina, podría dar lugar a la formación de especies reactivas con mayor capacidad de daño vascular, como sería la formación de peroxinitritos. Sólo un preciso conocimiento de los mecanismos moleculares por los que actúan los fármacos inmunosupresores, tanto en su acción inmunosupresora como en sus efectos tóxicos, permitirá un posterior desarrollo de nuevas moléculas con mejor resultado terapéutico.Los estudios comentados en este trabajo han sido financiados en parte por la CICYT (proyecto SAF 97-0035), la UE (proyecto BIOMED 2, BMH4-CT96-0979) y la CAM (proyecto 07/096/96).Peer reviewe

Transcriptional induction of endothelial nitric oxide gene by cyclosporine A. A role for activator protein-1

7 p.-7 fig.We have previously shown that the immunosuppressant cyclosporine A (CsA) increases the activity, the protein level, and the steady-state levels of the mRNA of the endothelial nitric-oxide synthase (eNOS) gene in bovine aortic endothelial cells (BAEC). We have now investigated the mechanisms responsible for these effects. Preincubation with an inhibitor of RNA polymerase II abolished CsA-induced eNOS up-regulation. Nuclear run-on experiments demonstrated a 1.6-fold increase in the induction of eNOS gene by CsA. In agreement with these results, transient transfections showed that CsA augmented the transactivation of the eNOS promoter. Electrophoretic mobility shift assays showed an increase in the activator protein-1 (AP-1) DNA binding activity in BAEC treated with CsA. An increase in the level of c-fos mRNA and in the nuclear content of c-Fos protein was detected in BAEC treated with CsA. Site-directed mutagenesis of the AP-1 cis-regulatory element in the context of the human eNOS promoter resulted in the abrogation of the induction mediated by CsA. Hence, up-regulation ofeNOS mRNA by CsA is a transcriptional phenomenon involving the proximal AP-1 site in the 5′-regulatory region of the human eNOS gene. Furthermore, our data exemplify how immunosuppressive drugs may result in the regulation of specific genes involved in the homeostasis of endothelial function, such aseNOS. Whereas cyclosporine A (CsA)1 still stands as a cornerstone therapy for immunosuppression, its use encompasses serious side effects among which nephrotoxicity and hypertension are often encountered. The pathogenesis of their appearance has been investigated in clinical studies and animal models, but a complete understanding is still elusive. The partial reversibility of these effects suggests that the underlying disturbance has a functional component. Acute nephrotoxicity is associated with renal vasoconstriction and a reduced glomerular filtration rate. A significant amount of data support that the vasoconstrictor peptide, endothelin-1, is involved in this response (1, 2). Furthermore, transforming growth factor β (TGF-β) has been proposed as a link between CsA-enhanced endothelin synthesis and many of the pathological fibrotic lesions observed with chronic CsA toxicity (3). Although endothelin and other paracrine vasoconstrictors have been identified as mediators of the hypertensive response (1, 4, 5) associated with CsA toxicity, a less clear picture has emerged regarding the potential dysfunction of thel-arginine-NO-cGMP pathway. Several reports have described that there is a defect in the relaxation of blood vessels in response to endothelial agonists, but the precise step of diminished NO synthesis, enhanced degradation, or receptor abnormality has not been established (6-8). In previous work, using endothelial cells in culture treated with CsA, we found that NO synthesis is not only not reduced but moderately enhanced (9). These observations have been also confirmed in rats (10), in healthy volunteers, in whom venous antecubital infusion of CsA significantly decreased forearm blood flow (11), and recently in patients treated with CsA after heart transplantation (12). These data indicate that nitric oxide may constitute an important regulatory mechanism that protects against CsA-associated vasoconstriction in vivo. In support of this hypothesis we found that the expression of the eNOS gene is increased in bovine aortic endothelial cells (BAEC) treated with CsA (12-14). We have now investigated the mechanisms by whicheNOS is up-regulated by CsA.This work was supported by Grant SAF-97-0035 from the Comisión Interministerial de Ciencia y Tecnologia, Grant BMH4-CT96-0979 from European Union Program BIOMED-2, and Grant 08.4/0032/1998 from Comunidad Autonoma de Madrid.Peer reviewe

Influence of Media and Testing Methodology on Susceptibility to Tigecycline of Enterobacteriaceae with Reported High Tigecycline MIC ▿

The tigecycline susceptibility of six different Enterobacteriaceae strains with reported high tigecycline MICs was determined in quintuplicate by four methodologies using Mueller-Hinton agar and broth from six manufacturers. The MICs determined by Etest were a ≥1-fold dilution lower than those determined by broth microdilution and agar dilution, with the highest modal values given by agar dilution. The highest modal MICs were obtained using Oxoid medium, and the lowest inhibition zone values (disc diffusion) were obtained using Oxoid and bioMérieux media. The lowest MICs were obtained by Etest using Difco or Merck media

Consensus document on dog and cat allergy.

The prevalence of sensitization to dogs and cats varies by country, exposure time and predisposition to atopy. It is estimated that 26% of European adults coming to the clinic for suspected allergy to inhalant allergens are sensitized to cats and 27% to dogs. This document is intended to be a useful tool for clinicians involved in the management of people with dog or cat allergy. It was prepared from a consensus process based on the RAND/UCLA method. Following a literature review, it proposes various recommendations concerning the diagnosis and treatment of these patients, grounded in evidence and clinical experience. The diagnosis of dog and cat allergy is based on a medical history and physical examination that are consistent with each other and is confirmed with positive results on specific IgE skin tests. Sometimes, especially in polysensitized patients, molecular diagnosis is strongly recommended. Although the most advisable measure would be to avoid the animal, this is often impossible and associated with a major emotional impact. Furthermore, indirect exposure to allergens occurs in environments in which animals are not present. Immunotherapy is emerging as a potential solution to this problem, although further supporting studies are needed