Estudio de las interacciones virus-hospedador: nuevas alternativas profilácticas frente a flavivirus

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 21-09-2017Las enfermedades infecciosas son causadas por microorganismos patógenos (bacterias, parásitos, hongos o virus) y pueden ser transmitidas directa o indirectamente de una persona a otra. Entre ellas destacan las enfermedades zoonóticas, que son aquellas que se transmiten entre animales vertebrados, incluidos los humanos. Éste es el caso del virus del Nilo Occidental (VNO), miembro del género Flavivirus (familia Flaviviridae), que es un patógeno neurotrópico que puede causar encefalitis y la denominada “fiebre del Nilo” en humanos. Como patógeno intracelular obligado, el VNO utiliza factores celulares para completar su ciclo de replicación, el cual está íntimamente asociado con el metabolismo lipídico celular. Así, se ha descrito que el VNO reorganiza las membranas intracelulares del retículo endoplasmático de las células infectadas para desarrollar plataformas adecuadas para una correcta replicación viral y su posterior expansión. Con estos antecedentes, el objetivo general de ésta tesis es profundizar en el conocimiento de los procesos celulares implicados en las interacciones patógeno-célula, y más concretamente en el papel de los lípidos celulares en la replicación vírica, con el fin de buscar dianas celulares para el desarrollo de estrategias antivirales innovadoras. Los resultados obtenidos indican que la manipulación farmacológica del metabolismo lipídico celular es una potente estrategia alternativa para combatir el VNO; y que enzimas como la acetil-Coenzima A carboxilasa o la esfingomielinasa neutra, así como la vía metabólica de las proteínas de unión de elementos reguladores del esterol celular (SREBPs), son objetivos apropiados para el desarrollo de antivirales. Es esperable que los conocimientos alcanzados contribuyan al estudio más detallado de las dinámicas de las interacciones lipídicas a lo largo del ciclo viral y, de este modo, al desarrollo de nuevas estrategias antivirales para combatir el VNO y otros flavivirus relacionados, tales como Usutu, dengue o Zika.Infectious diseases are caused by pathogenic microorganisms (bacteria, viruses, parasites or fungi) and can be spread, directly or indirectly, from one person to another. These include zoonotic diseases that are transmitted between vertebrates, including humans. This is the case of West Nile virus (WNV), a member of the genus Flavivirus (family Flaviviridae), which is a neurotropic pathogen and the causative agent of the so-called West Nile fever in humans and encephalitis. As an obligate intracellular pathogen, WNV uses cellular factors to complete its replication cycle, which is intimately associated to host cell lipid metabolism. Thus, it has been described that WNV rearranges intracellular membranes from the endoplasmic reticulum of infected cells to develop adequate platforms for an efficient viral replication and subsequent spread. Keeping this in mind, the general aim of this thesis was to achieve an accurate knowledge of the cellular processes involved in pathogen and host cell interactions, and more precisely in the role of the cellular lipids in viral replication, in order to search for cellular targets for the development of innovative antiviral strategies. The obtained results indicate that the pharmacological manipulation of cellular lipid metabolism is an alternative potential strategy to combat WNV, and that enzymes, as the acetyl-CoA carboxylase (ACC) or the neutral sphingomyelinase (nSMase), as well as the cellular sterol regulatory element binding proteins (SREBPs) metabolic pathway, are suitable targets for antiviral development. It is expected that the knowledge gained will contribute to a more detailed study of the dynamics of the lipids interactions throughout the viral cycle and, in this way, to the development of new antiviral strategies to fight WNV and other related flavivirus, such as Usutu, dengue, and Zika

Peroxynitrite induces F-actin depolymerization and blockade of myosin ATPase stimulation

Treatment of F-actin with the peroxynitrite-releasing agent 3-morpholinosydnonimine (SIN-1) produced a dose-dependent F-actin depolymerization. This is due to released peroxynitrite because it is not produced by ‘decomposed SIN-1’, and it is prevented by superoxide dismutase concentrations efficiently preventing peroxynitrite formation. F-actin depolymerization has been found to be very sensitive to peroxynitrite, as exposure to fluxes as low as 50–100 nM peroxynitrite leads to nearly 50% depolymerization in about 1 h. G-actin polymerization is also impaired by peroxynitrite although with nearly 2-fold lower sensitivity. Exposure of F-actin to submicromolar fluxes of peroxynitrite produced cysteine oxidation and also a blockade of the ability of actin to stimulate myosin ATPase activity. Our results suggest that an imbalance of the F-actin/G-actin equilibrium can account for the observed structural and functional impairment of myofibrils under the peroxynitrite-mediated oxidative stress reported for some pathophysiological conditions

Modification of the host cell lipid metabolism induced by hypolipidemic drugs targeting the acetyl coenzyme A carboxylase impairs West Nile virus replication

West Nile virus (WNV) is a neurotropic flavivirus transmitted by the bite of mosquitoes that causes meningitis and encephalitis in humans, horses, and birds. Several studies have highlighted that flavivirus infection is highly dependent on cellular lipids for virus replication and infectious particle biogenesis. The first steps of lipid synthesis involve the carboxylation of acetyl coenzyme A (acetyl-CoA) to malonyl-CoA that is catalyzed by the acetyl-CoA carboxylase (ACC). This makes ACC a key enzyme of lipid synthesis that is currently being evaluated as a therapeutic target for different disorders, including cancers, obesity, diabetes, and viral infections. We have analyzed the effect of the ACC inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA) on infection by WNV. Lipidomic analysis of TOFA-treated cells confirmed that this drug reduced the cellular content of multiple lipids, including those directly implicated in the flavivirus life cycle (glycerophospholipids, sphingolipids, and cholesterol). Treatment with TOFA significantly inhibited the multiplication of WNV in a dose-dependent manner. Further analysis of the antiviral effect of this drug showed that the inhibitory effect was related to a reduction of viral replication. Furthermore, treatment with another ACC inhibitor, 3,3,14,14-tetramethylhexadecanedioic acid (MEDICA 16), also inhibited WNV infection. Interestingly, TOFA and MEDICA 16 also reduced the multiplication of Usutu virus (USUV), a WNV-related flavivirus. These results point to the ACC as a druggable cellular target suitable for antiviral development against WNV and other flaviviruses.BIO2011-24351 (to F.S.), AGL2014-52395-C2-1-R (to F.S.), E-RTA2013-0013 (to J.-C.S. and F.S.), S2013/ABI-2906 (to J.-C.S. and F.S.), AGL2014-56518-JIN (to M.A.M.-A.), and CTQ2014-54743-R (to J.C.). Work at CBMSO was also supported by Fundación Ramón Areces.Peer Reviewe

The XMM-Newton wide angle survey (XWAS): the X-ray spectrum of type-1 AGN

Aims. We discuss the broad band X-ray properties of one of the largest samples of X-ray selected type-1 AGN to date (487 objects in total), drawn from the XMM-Newton Wide Angle Survey (XWAS). The objects presented in this work cover 2−10 keV (rest-frame) luminosities from ∼1042−1045 erg s−1 and are detected up to redshift ∼4. We constrain the overall properties of the broad band continuum, soft excess and X-ray absorption, along with their dependence on the X-ray luminosity and redshift.We discuss the implications for models of AGN emission. Methods. We fitted the observed 0.2−12 keV broad band spectra with various models to search for X-ray absorption and soft excess. The F-test was used with a significance threshold of 99% to statistically accept the detection of additional spectral components. Results. We constrained the mean spectral index of the broad band X-ray continuum to (Γ) = 1.96 ± 0.02 with intrinsic dispersion σ(Γ) = 0.27+0.01 −0.02. The continuum becomes harder at faint fluxes and at higher redshifts and hard (2−10 keV) luminosities. The dependence of Γ with flux is likely due to undetected absorption rather than to spectral variation. We found a strong dependence of the detection efficiency of objects on the spectral shape. We expect this effect to have an impact on the measured mean continuum shapes of sources at different redshifts and luminosities. We detected excess absorption in >∼3% of our objects, with rest-frame column densities ∼a few ×1022 cm−2. The apparent mismatch between the optical classification and X-ray properties of these objects is a challenge for the standard orientation-based AGN unification model. We found that the fraction of objects with detected soft excess is ∼36%. Using a thermal model, we constrained the soft excess mean rest-frame temperature and intrinsic dispersion to kT ∼ 100 eV and σkT ∼ 34 eV. The origin of the soft excess as thermal emission from the accretion disk or Compton scattered disk emission is ruled out on the basis of the temperatures detected and the lack of correlation of the soft excess temperature with the hard X-ray luminosity over more than 2 orders of magnitude in luminosity. Furthermore, the high luminosities of the soft excess rule out an origin in the host galaxy.We acknowledge Chris Done, Bozena Czerny, Gordon Stewart, Pilar Esquej and Ken Pounds for useful comments. We acknowledge the anonymous referee for a careful reading of the manuscript and for comments that improved the paper. S.M., M.W. and J.A.T. acknowledge support from the UK STFC research council. F.J.C. acknowledges financial support for this work from the Spanish Ministerio de Educación y Ciencia under project ESP2006-13608-C02-01. A.C. acknowledges financial support from the Spanish Ministerio de Educación y Ciencia fellowship and also from the MIUR and The Italian Space Agency (ASI) grants PRIN-MUR 2006-02-5203 and No. I/088/06/0. M.K. acknowledges support from the NASA grant NNX08AX50G and NNX07AG02G

Ultra Deep Sequencing of Circulating Cell-Free DNA as a Potential Tool for Hepatocellular Carcinoma Management

Biomarkers; cfDNA; Liquid biopsyBiomarcadores; cfDNA; Biopsia líquidaBiomarcadors; cfDNA; Biòpsia líquidaBackground: Cell-free DNA (cfDNA) concentrations have been described to be inversely correlated with prognosis in cancer. Mutations in HCC-associated driver genes in cfDNA have been reported, but their relation with patient’s outcome has not been described. Our aim was to elucidate whether mutations found in cfDNA could be representative from those present in HCC tissue, providing the rationale to use the cfDNA to monitor HCC. Methods: Tumoral tissue, paired nontumor adjacent tissue and blood samples were collected from 30 HCC patients undergoing curative therapies. Deep sequencing targeting HCC driver genes was performed. Results: Patients with more than 2 ng/µL of cfDNA at diagnosis had higher mortality (mean OS 24.6 vs. 31.87 months, p = 0.01) (AUC = 0.782). Subjects who died during follow-up, had a significantly higher number of mutated genes (p = 0.015) and number of mutations (p = 0.015) on cfDNA. Number of mutated genes (p = 0.001), detected mutations (p = 0.001) in cfDNA and ratio (number of mutations/cfDNA) (p = 0.003) were significantly associated with recurrence. However, patients with a ratio (number of mutations/cfDNA) above 6 (long-rank p = 0.0003) presented a higher risk of recurrence than those with a ratio under 6. Detection of more than four mutations in cfDNA correlated with higher risk of death (long-rank p = 0.042). Conclusions: In summary, cfDNA and detection of prevalent HCC mutations could have prognostic implications in early-stage HCC patientsThis research was funded by Instituto de Salud Carlos III (ISCIII) (PI18/00961) and (PI21/00714) to B.M. PI19/00301 by Instituto de Salud Carlos III (ISCIII) and Centro para el Desarrollo Tecnológico Industrial (CDTI) from the Spanish Ministry of Economy and Business, grant number IDI-20200297 to J.Q., E.V.-A. is a recipient of a predoctoral fellowship from Instituto de Salud Carlos III (ISCIII) (FI18/00027)

Decavanadate interactions with actin: inhibition of G-actin polymerization and stabilization of decameric vanadate

Decameric vanadate species (V10) inhibit the rate and the extent of G-actin polymerization with an IC50 of 68 ± 22 lM and 17 ± 2 lM, respectively, whilst they induce F-actin depolymerization at a lower extent. On contrary, no effect on actin polymerization and depolymerization was detected for 2 mM concentration of ‘‘metavanadate’’ solution that contains ortho and metavanadate species, as observed by combining kinetic with 51V NMR spectroscopy studies. Although at 25 C, decameric vanadate (10 lM) is unstable in the assay medium, and decomposes following a first-order kinetic, in the presence of G-actin (up to 8 lM), the half-life increases 5-fold (from 5 to 27 h). However, the addition of ATP (0.2 mM) in the medium not only prevents the inhibition of G-actin polymerization by V10 but it also decreases the half-life of decomposition of decameric vanadate species from 27 to 10 h. Decameric vanadate is also stabilized by the sarcoplasmic reticulum vesicles, which raise the half-life time from 5 to 18 h whereas no effects were observed in the presence of phosphatidylcholine liposomes, myosin or G-actin alone. It is proposed that the ‘‘decavanadate’’ interaction with G-actin, favored by the G-actin polymerization, stabilizes decameric vanadate species and induces inhibition of G-actin polymerization. Decameric vanadate stabilization by cytoskeletal and transmembrane proteins can account, at least in part, for decavanadate toxicity reported in the evaluation of vanadium (V) effects in biological systems

Quality of life with palbociclib plus fulvestrant versus placebo plus fulvestrant in postmenopausal women with endocrine-sensitive hormone receptor-positive and HER2-negative advanced breast cancer : patient-reported outcomes from the FLIPPER trial

In the FLIPPER trial, palbociclib/fulvestrant significantly improved progression-free survival (PFS) compared with placebo/fulvestrant in postmenopausal women with HR+/HER2− advanced breast cancer (ABC). We assessed health-related quality of life (QoL) using patient-reported outcomes (PROs). In this phase II double-blinded study, PROs were assessed at baseline after every three cycles and at the end of the treatment using the European Organisation for Research and Treatment of Cancer QLQ-C30 and QLQ-BR23. Time to deterioration (TTD) in global health status (GHS)/QoL was defined as a decrease of ⩾10 points. Changes from baseline (CFB) and TTD were analysed using linear mixed-effect and Cox regression models, respectively. Of the 189 randomised (1:1) patients, 178 (94%) completed ⩾1 post-baseline assessment; 50% received ⩾22 cycles of study treatment, with a questionnaire compliance >90%. Mean baseline scores were comparable between arms. GHS/QoL scores were maintained throughout the palbociclib/fulvestrant treatment. CFB showed significant differences for GHS/QoL, appetite loss, constipation and systemic therapy side effect scores favouring placebo/fulvestrant. TTD in GHS/QoL was delayed in placebo/fulvestrant versus palbociclib/fulvestrant [30.3 versus 11.1 months; adjusted hazard ratio (aHR): 1.57, 95% CI: 1.03-2.39, p = 0.036]; this difference was not significant in patients with progressive disease (aHR: 1.2, 95% CI: 0.6-2.2, p = 0.658). No statistically significant differences in TTD were found for the other QLQ-C30 and QLQ-BR23 scales. Although TTD in GHS/QoL was prolonged with placebo/fulvestrant, no differences were observed on other functional or symptom scales. This finding and the improvement in PFS support the combination of palbociclib/fulvestrant as a beneficial therapeutic option for HR+/HER2− ABC. Sponsor Study Code: GEICAM/2014-12 EudraCT Number: 2015-002437-21 ClinTrials.gov reference: NCT0269048

Grupo español de cirugía torácica asistida por videoimagen: método, auditoría y resultados iniciales de una cohorte nacional prospectiva de pacientes tratados con resecciones anatómicas del pulmón

Introduction: our study sought to know the current implementation of video-assisted thoracoscopic surgery (VATS) for anatomical lung resections in Spain. We present our initial results and describe the auditing systems developed by the Spanish VATS Group (GEVATS). Methods: we conducted a prospective multicentre cohort study that included patients receiving anatomical lung resections between 12/20/2016 and 03/20/2018. The main quality controls consisted of determining the recruitment rate of each centre and the accuracy of the perioperative data collected based on six key variables. The implications of a low recruitment rate were analysed for '90-day mortality' and 'Grade IIIb-V complications'. Results: the series was composed of 3533 cases (1917 VATS; 54.3%) across 33 departments. The centres' median recruitment rate was 99% (25-75th:76-100%), with an overall recruitment rate of 83% and a data accuracy of 98%. We were unable to demonstrate a significant association between the recruitment rate and the risk of morbidity/mortality, but a trend was found in the unadjusted analysis for those centres with recruitment rates lower than 80% (centres with 95-100% rates as reference): grade IIIb-V OR=0.61 (p=0.081), 90-day mortality OR=0.46 (p=0.051). Conclusions: more than half of the anatomical lung resections in Spain are performed via VATS. According to our results, the centre's recruitment rate and its potential implications due to selection bias, should deserve further attention by the main voluntary multicentre studies of our speciality. The high representativeness as well as the reliability of the GEVATS data constitute a fundamental point of departure for this nationwide cohort

Long-Term Real-World Effectiveness and Safety of Ustekinumab in Crohn’s Disease Patients: The SUSTAIN Study

Background Large real-world-evidence studies are required to confirm the durability of response, effectiveness, and safety of ustekinumab in Crohn’s disease (CD) patients in real-world clinical practice. Methods A retrospective, multicentre study was conducted in Spain in patients with active CD who had received ≥1 intravenous dose of ustekinumab for ≥6 months. Primary outcome was ustekinumab retention rate; secondary outcomes were to identify predictive factors for drug retention, short-term remission (week 16), loss of response and predictive factors for short-term efficacy and loss of response, and ustekinumab safety. Results A total of 463 patients were included. Mean baseline Harvey-Bradshaw Index was 8.4. A total of 447 (96.5%) patients had received prior biologic therapy, 141 (30.5%) of whom had received ≥3 agents. In addition, 35.2% received concomitant immunosuppressants, and 47.1% had ≥1 abdominal surgery. At week 16, 56% had remission, 70% had response, and 26.1% required dose escalation or intensification; of these, 24.8% did not subsequently reduce dose. After a median follow-up of 15 months, 356 (77%) patients continued treatment. The incidence rate of ustekinumab discontinuation was 18% per patient-year of follow-up. Previous intestinal surgery and concomitant steroid treatment were associated with higher risk of ustekinumab discontinuation, while a maintenance schedule every 12 weeks had a lower risk; neither concomitant immunosuppressants nor the number of previous biologics were associated with ustekinumab discontinuation risk. Fifty adverse events were reported in 39 (8.4%) patients; 4 of them were severe (2 infections, 1 malignancy, and 1 fever). Conclusions Ustekinumab is effective and safe as short- and long-term treatment in a refractory cohort of CD patients in real-world clinical practice