Geminiviral protein Rep interferes in PCNA sumoylation

Rep is a multifunctional protein essential for replication of geminivirus that interferes with the sumoylation of a key protein in the DNA replication, PCNA (Proliferating Cell Nuclear Antigen). It is known that Rep is capable of interacting with a plethora of plant proteins, including PCNA. Despite the biological significance remains unknown, it’s thought that this interaction should play a key role for generating new copies of the virus genome. Therefore, in order to characterize this interaction, we study which lysines are sumoylated in tomato PCNA (SlPCNA). Considering conservation, location and presence of sumoylation domain criteria, we have identified some candidate lysines and studied how its mutation affects this protein sumoylation in Escherichia coli assays. Finally, we plan to confirm and characterize the Rep interference on SlPCNA sumoylation and determine if this interference occurs in planta.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Estudio sobre la interacción planta geminivirus.

Los geminivirus son virus de plantas con genomas de DNA monocatenario formados por una (monopartitos) o dos (bipartitos) moléculas de DNA que codifican de seis a ocho proteínas. Estos virus causan importantes pérdidas económicas en cultivos comerciales de todo el mundo. La familia Geminiviridae comprende siete géneros (Mastrevirus, Curtovirus, Begomovirus, Topocuvirus, Becurtovirus, Eragrovirus y Turncurtovirus) en los que se encuadran los distintos geminivirus en función de su rango de hospedador y su insecto vector. En esta tesis nos centramos en virus del género Begomoviridae. Para que los geminivirus puedan establecer una infección exitosa su hospedador, deben ser capaces de ser transportados por su insecto vector, llegar al núcleo de las células del floema, replicar su DNA, expresar sus proteínas, contrarrestar los sistemas de defensa de la planta y moverse a células adyacentes o a otras distantes a través del sistema vascular de la planta para extender la infección. Todo esto no sería posible con tan solo seis u ocho genes sin la intervención de factores celulares de la planta. Los geminivirus son capaces de secuestrar los factores y mecanismos de la planta y usarlos en su beneficio. En esta tesis se abordan tres aspectos del estudio de estos virus: - Interferencia en la sumoilación de PCNA. - Generación de nuevas herramientas moleculares para la visualización in vivo de la infección por geminivirus. - Efecto de los estreses abióticos sobre la infección por geminivirus. Interferencia en la sumoilación de PCNA. PCNA es una proteína que interacciona con el DNA y con el replisoma, formado por diversos factores celulares implicados en la replicación, reparación y recombinación del mismo. Lleva a cabo su función proporcionándoles un anclaje para interactuar entre ellos y con el DNA. PCNA está finamente regulado mediante la adición y retirada de modificaciones post-traduccionales. Entre estas, destacan la ubiquitinación y la sumoilación, que determinan la maquinaria de replicación o reparación que se carga en el replisoma. En levaduras, cuando se desumoila la lisina 164 de PCNA, este cambia su abanico de interactores y carga polimerasas de DNA que tienden a cometer herrores y factores celulares que favorecen la recombinación homóloga. Rep es la proteína vírica responsable del inicio de la replicación. Interacciona con PCNA y con la maquinaria de sumoilación de la célula. En esta tesis se estudian varios aspectos de la interferencia de la proteína viral Rep con la sumoilación del factor celular PCNA. Generación de nuevas herramientas moleculares para la visualización in vivo de la infección por geminivirus. Las plantas 2IR son plantas de Nicotiana benthamiana transgénicas que contienen un casete de expresión de GFP (green fluorescent protein) flanqueado por dos repeticiones directas de la región intergénica de TYLCSV (Tomato yellow leaf curl Sardinia virus). La región intergénica es la parte del genoma de los geminivirus en la que se encuentran su origen de replicación y las secuencias reguladoras necesarias para la expresión de sus genes. Cuando un geminivirus infecta una célula que contiene la construcción 2IR, la proteína Rep expresada por el virus se une a las regiones intergénicas de la construcción y la escinde, formando una molécula extracromosómica que contiene el casete de expresión de GFP y una región intergénica, que permite que esta molécula extracromosómica se replique como un virus. El incremento de moléculas extracromosómicas conlleva un incremento sustancial en la expresión de GFP. Esto permite seguir el desarrollo de la infección en plantas vivas con una lámpara de luz ultravioleta sin necesidad de dañarlas. Estas plantas se han usado en varios estudios ya publicados en los que se han identificado factores celulares del hospedador implicados en la infección del virus o se ha indagado en las relaciones sinérgicas entre distintos virus de la misma familia. En el tercer capítulo de esta tesis se usan para estudiar el efecto de los estreses abióticos sobre la infección por geminivirus. Pese al enorme potencial de estas plantas, tienen limitaciones, como por ejemplo la enorme especificidad que tienen las proteínas Rep de los distintos geminivirus por la región intergénica de su virus. Esto impide usar las plantas 2IR para estudiar el desarrollo de la infección de TYLCV-IL (Tomato yellow leaf curl virus – Israel), el que se ha convertido en el virus con mayor incidencia en los últimos años. Otro problema es que la GFP fluoresce en N. benthamiana pero no en tomate, el hospedador de estos virus con mayor trascendencia económica. Durante el desarrollo de esta tesis se generaron nuevas versiones de las plantas 2IR con regiones intergénicas de TYLCV-IL y con fluoróforos que sí fluorescen en tomate. También se caracterizó el fenotipo de estas plantas en respuesta a la infección por los virus del complejo TYLCD. Efecto de los estreses abióticos sobre la infección por geminivirus. Durante la primera mitad del siglo XXI se van a consumir más alimentos que durante toda la historia de la humanidad. Producir estos alimentos es un reto que empeora teniendo en cuenta la situación de calentamiento global, desertización y salinización de los acuíferos que se da en muchas partes del globo. Además de estas fuentes de estrés, los cultivos deben sobrevivir a los virus y otros patógenos. Existen multitud de estudios en los que se exploran los efectos de estos factores sobre las plantas, pero apenas hay algunos que estudien la interacción de estos factores. En esta tesis se propone un sistema experimental en el que se pueden estudiar in vivo los efectos de temperatura, salinidad, sequía e infección por geminivirus sobre las plantas durante largos períodos de tiempo y sin necesidad de sacrificar las plantas, lo que permite hacer un seguimiento individualizado a lo largo del tiempo de un mismo proceso infectivo

La proteína Rep/AL1 de geminivirus altera la sumoilación de PCNA

Los geminivirus, llamados así por la forma icosaédrica de su cápside, son una familia de virus patógenos de plantas que causan algunas de las enfermedades con mayor impacto económico a nivel mundial. Estos pequeños virus de ADN se replican en el núcleo de las células vegetales utilizando la maquinaria celular del hospedador, además de requerir la presencia de la proteína viral Rep. Rep es la única proteína del genoma del virus imprescindible para su replicación y es capaz de interaccionar con una gran variedad de proteínas del huésped. Trabajos previos de nuestro grupo demostraron que dos de esas proteínas son PCNA (Proliferating cell nuclear antigen), esencial en el metabolismo del DNA, y SCE (SUMO Conjugating Enzime), enzima que interviene en la sumoilación, uno de los principales mecanismos de modificación postraduccional que está implicado en la respuesta de la planta a estrés abiótico, en el desarrollo, el crecimiento y la respuesta a patógenos. Además, otros resultados que hemos obtenido demuestran que la expresión en Escherichia coli de la proteína Rep del begomovirus TGMV (Tomato golden mosaic virus) reduce la sumoilación de PCNA en plantas y que dicha interferencia no depende de la interacción de la proteína viral con SCE1. Para profundizar en el conocimiento de la interacción Rep-PCNA nos hemos propuesto identificar las lisinas de PCNA que se sumoilan. Teniendo en cuenta criterios de conservación, localización y presencia de dominio de sumoilación se han identificado varias lisinas candidatas y se ha estudiado cómo su mutación afecta a la sumoilación de la proteína. También nos hemos planteado confirmar y caracterizar la interferencia de Rep sobre la sumoilación de PCNA y determinar si dicha interferencia se produce en la planta.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Geminivirus replication protein decreases PCNA sumoylation at two acceptor sites

Geminiviruses are plant viruses with circular, single-stranded DNA (ssDNA) genomes that infect a broad range of plants causing substantial crop diseases worldwide. They replicate in nuclei of infected cells by using host DNA replication machinery and an essential protein encoded in their genome designated Rep (replication-associated protein). This multifunctional protein induces the accumulation of the host factors involved in replication and it is capable of interacting with a lot of plant proteins including PCNA (Proliferating Cell Nuclear Antigen), a processivity factor that coordinates a wide range of processes involved in maintenance, duplication and transmission of the genome, and the sumoylation enzyme that conjugates SUMO to target proteins (SUMO-conjugating enzyme- SCE). PCNA modification by SUMO, and also ubiquitin, has long been known to be of key importance for determining how DNA damage is processed by the replisome and for maintenance of overall genome integrity. In yeast, PCNA sumoylation has been associated to DNA repair involving homologous recombination (HR). Previously, we reported that Rep ectopic expression does not result in broad changes in the sumoylation pattern of plant cells, but it modifies the sumoylation state of selected host proteins. In this work, we show, using a reconstituted sumoylation system in Escherichia coli, that tomato PCNA is sumoylated at two residues, K254 and K164, and that co-expression of the Rep protein suppresses PCNA sumoylation at these lysines. Finally, we confirm that PCNA is sumoylated and that Rep also interferes with PCNA sumoylation in planta.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

The effect of an online exercise programme on bone health in paediatric cancer survivors (iBoneFIT): study protocol of a multi-centre randomized controlled trial

Background: New approaches on paediatric cancer treatment aim to maintain long-term health. As a result of radiotherapy, chemotherapy or surgery, paediatric cancer survivors tend to suffer from any chronic health condition. Endocrine dysfunction represents one of the most common issues and affects bone health. Exercise is key for bone mass accrual during growth, specifically plyometric jump training. The iBoneFIT study will investigate the effect of a 9-month online exercise programme on bone health in paediatric cancer survivors. This study will also examine the effect of the intervention on body composition, physical fitness, physical activity, calcium intake, vitamin D, blood samples quality of life and mental health. Methods: A minimum of 116 participants aged 6 to 18 years will be randomized into an intervention (n = 58) or control group (n = 58). The intervention group will receive an online exercise programme and diet counselling on calcium and vitamin D. In addition, five behaviour change techniques and a gamification design will be implemented in order to increase the interest of this non-game programme. The control group will only receive diet counselling. Participants will be assessed on 3 occasions: 1) at baseline; 2) after the 9 months of the intervention; 3) 4 months following the intervention. The primary outcome will be determined by dual energy X-ray absorptiometry (DXA) and the hip structural analysis, trabecular bone score and 3D-DXA softwares. Secondary outcomes will include anthropometry, body composition, physical fitness, physical activity, calcium and vitamin D intake, blood samples, quality of life and mental health. Discussion: Whether a simple, feasible and short in duration exercise programme can improve bone health has not been examined in paediatric cancer survivors. This article describes the design, rationale and methods of a study intended to test the effect of a rigorous online exercise programme on bone health in paediatric cancer survivors. If successful, the iBoneFIT study will contribute to decrease chronic health conditions in this population and will have a positive impact in the society.The iBoneFIT project is funded by a fellowship from 'la Caixa' Foundation (ID 100010434). The fellowship code is LCF/BQ/PR19/11700007. This study has been partially supported by the University of Granada, Plan Propio de Investigación 2016, Excellence actions: Units of Excellence; Unit of Excellence on Exercise and Health (UCEES), and by the Junta de Andalucía, Consejería de Conocimiento, Investigación y Universidades and European Regional Development Fund (ERDF), ref. SOMM17/6107/UGR

Cortical thinning over two years after first-episode psychosis depends on age of onset

First-episode psychosis (FEP) patients show structural brain abnormalities at the first episode. Whether the cortical changes that follow a FEP are progressive and whether age at onset modulates these changes remains unclear. This is a multicenter MRI study in a deeply phenotyped sample of 74 FEP patients with a wide age range at onset (15-35 years) and 64 neurotypical healthy controls (HC). All participants underwent two MRI scans with a 2-year follow-up interval. We computed the longitudinal percentage of change (PC) for cortical thickness (CT), surface area (CSA) and volume (CV) for frontal, temporal, parietal and occipital lobes. We used general linear models to assess group differences in PC as a function of age at FEP. We conducted post-hoc analyses for metrics where PC differed as a function of age at onset. We found a significant age-by-diagnosis interaction effect for PC of temporal lobe CT (d = 0.54; p = 002). In a post-hoc-analysis, adolescent-onset (≤19 y) FEP showed more severe longitudinal cortical thinning in the temporal lobe than adolescent HC. We did not find this difference in adult-onset FEP compared to adult HC. Our study suggests that, in individuals with psychosis, CT changes that follow the FEP are dependent on the age at first episode, with those with an earlier onset showing more pronounced cortical thinning in the temporal lobe

The effect of family environment and psychiatric family history on psychosocial functioning in first-episode psychosis at baseline and after 2 years

Abstract The aim of the present study was to evaluate the contribution of family environment styles and psychiatric family history on functioning of patients presenting first-episode psychosis (FEP). Patients with FEP and healthy controls (HC) were assessed at baseline and after 2 years. The Functional Assessment Short Test (FAST) was used to assess functional outcome and the Family Environment Scale (FES) to evaluate family environment. Linear regressions evaluated the effect that family environment exerts on functioning at baseline and at 2-year follow-up, when FEP patients were diagnosed according to non-affective (NA-PSYCH) or affective psychoses (APSYCH). The influence of a positive parents' psychiatric history on functioning was evaluated through one-way between-groups analysis of covariance (ANCOVA) models, after controlling for family environmental styles. At baseline, FEP patients presented moderate functioning impairment, significantly worse than HC (28.65±16.17 versus 3.25±7.92; p<0.001, g = 1.91). At 2-year follow-up, the functioning of NA-PSYCH patients was significantly worse than in A-PSYCH (19.92±14.83 versus 12.46±14.86; p = 0.020, g = 0.50). No specific family environment style was associated with functioning in FEP patients and HC. On the contrary, a positive psychiatric father's history influenced functioning of FEP patients. After 2 years, worse functioning in NA-PSYCH patients was associated with lower rates of active-recreational and achievement orientated family environment and with higher rates of moral-religious emphasis and control. In A-PSYCH, worse functioning was associated with higher rates of conflict in the family. Both family environment and psychiatric history influence psychosocial functioning, with important implications for early interventions, that should involve both patients and caregivers

Automatic Discrimination of Species within the Enterobacter cloacae Complex Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Supervised Algorithms

The Enterobacter cloacae complex (ECC) encompasses heterogeneous clusters of species that have been associated with nosocomial outbreaks. These species may have different acquired antimicrobial resistance and virulence mechanisms, and their identification is challenging. This study aims to develop predictive models based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) profiles and machine learning for species-level identification. A total of 219 ECC and 118 Klebsiella aerogenes clinical isolates from three hospitals were included. The capability of the proposed method to differentiate the most common ECC species (Enterobacter asburiae, Enterobacter kobei, Enterobacter hormaechei, Enterobacter roggenkampii, Enterobacter ludwigii, and Enterobacter bugandensis) and K. aerogenes was demonstrated by applying unsupervised hierarchical clustering with principal-component analysis (PCA) preprocessing. We observed a distinctive clustering of E. hormaechei and K. aerogenes and a clear trend for the rest of the ECC species to be differentiated over the development data set. Thus, we developed supervised, nonlinear predictive models (support vector machine with radial basis function and random forest). The external validation of these models with protein spectra from two participating hospitals yielded 100% correct species-level assignment for E. asburiae, E. kobei, and E. roggenkampii and between 91.2% and 98.0% for the remaining ECC species; with data analyzed in the three participating centers, the accuracy was close to 100%. Similar results were obtained with the Mass Spectrometric Identification (MSI) database developed recently (https://msi.happy-dev.fr) except in the case of E. hormaechei, which was more accurately identified with the random forest algorithm. In short, MALDI-TOF MS combined with machine learning was demonstrated to be a rapid and accurate method for the differentiation of ECC species

A longitudinal study of gene expression in first-episode schizophrenia; exploring relapse mechanisms by co-expression analysis in peripheral blood.

Little is known about the pathophysiological mechanisms of relapse in first-episode schizophrenia, which limits the study of potential biomarkers. To explore relapse mechanisms and identify potential biomarkers for relapse prediction, we analyzed gene expression in peripheral blood in a cohort of first-episode schizophrenia patients with less than 5 years of evolution who had been evaluated over a 3-year follow-up period. A total of 91 participants of the 2EPs project formed the sample for baseline gene expression analysis. Of these, 67 provided biological samples at follow-up (36 after 3 years and 31 at relapse). Gene expression was assessed using the Clariom S Human Array. Weighted gene co-expression network analysis was applied to identify modules of co-expressed genes and to analyze their preservation after 3 years of follow-up or at relapse. Among the 25 modules identified, one module was semi-conserved at relapse (DarkTurquoise) and was enriched with risk genes for schizophrenia, showing a dysregulation of the TCF4 gene network in the module. Two modules were semi-conserved both at relapse and after 3 years of follow-up (DarkRed and DarkGrey) and were found to be biologically associated with protein modification and protein location processes. Higher expression of DarkRed genes was associated with higher risk of suffering a relapse and early appearance of relapse (p = 0.045). Our findings suggest that a dysregulation of the TCF4 network could be an important step in the biological process that leads to relapse and suggest that genes related to the ubiquitin proteosome system could be potential biomarkers of relapse

Link between cognitive polygenic risk scores and clinical progression after a first-psychotic episode

Background: Clinical intervention in early stages of psychotic disorders is crucial for the prevention of severe symptomatology trajectories and poor outcomes. Genetic variability is studied as a promising modulator of prognosis, thus novel approaches considering the polygenic nature of these complex phenotypes are required to unravel the mechanisms underlying the early progression of the disorder. Methods: The sample comprised of 233 first-episode psychosis (FEP) subjects with clinical and cognitive data assessed periodically for a 2-year period and 150 matched controls. Polygenic risk scores (PRSs) for schizophrenia, bipolar disorder, depression, education attainment and cognitive performance were used to assess the genetic risk of FEP and to characterize their association with premorbid, baseline and progression of clinical and cognitive status. Results: Schizophrenia, bipolar disorder and cognitive performance PRSs were associated with an increased risk of FEP [false discovery rate (FDR) ⩽ 0.027]. In FEP patients, increased cognitive PRSs were found for FEP patients with more cognitive reserve (FDR ⩽ 0.037). PRSs reflecting a genetic liability for improved cognition were associated with a better course of symptoms, functionality and working memory (FDR ⩽ 0.039). Moreover, the PRS of depression was associated with a worse trajectory of the executive function and the general cognitive status (FDR ⩽ 0.001). Conclusions: Our study provides novel evidence of the polygenic bases of psychosis and its clinical manifestation in its first stage. The consistent effect of cognitive PRSs on the early clinical progression suggests that the mechanisms underlying the psychotic episode and its severity could be partially independent