Papel de los reguladores moleculares Fbp1 y Bmh2 en la virulencia de Fusarium oxysporum

La rápida y específica degradación de proteínas por el sistema proteosomaubiquitina ha emergido como un importante mecanismo de regulación en eucariotas. Algunos de los procesos básicos celulares regulados por proteólisis dependiente de ubiquitina incluyen la progresión del ciclo celular, iniciación de la transcripción o transducción de señales. Las enzimas clave en el proceso de ubiquitinación son las ligasas de ubiquitina E3, puesto que son las que actúan como moduladores para el reconocimiento del sustrato y además son las que transfieren la ubiquitina activada a dicho sustrato. Las proteínas Skp1, Cul1 y la proteína F-box (SCF) son el grupo de ligasas E3 mejor conocido. Dada la importancia de dicho proceso, resultó de gran interés investigar el papel de las proteínas pertenecientes a la maquinaria de renovación proteica, en la patogénesis de Fusarium oxysporum f. sp. lycopersici sobre plantas de tomate. En este trabajo nos hemos centrado en el estudio de una proteína F-box, Fbp1, ortóloga de GRR1 en S. cerevisiae. Para estudiar el papel del gen fbp1 en la virulencia de F. oxysporum se ha llevado a cabo la interrupción dirigida del gen en la estirpe silvestre del hongo. La infección de plantas de tomate inoculadas con los mutantes deficientes en el gen mostró un retraso en el proceso de infección, lo que indica que Fbp1, aunque no es esencial para la patogenicidad, es necesaria para la completa virulencia de F.oxysporum. El mutante Δfbp1 presentó un retraso significativo en todas las fuentes de carbono y nitrógeno analizadas, además de un patrón de crecimiento ondulado de las hifas líder y una tinción del Spk muy abundante y deslocalizada. Mutantes de F. oxysporum en el gen fbp1 mostraron niveles reducidos de fosforilación de Fmk1 y compartían características fenotípicas con los mutantes Δ fmk1, tales como defectos en el crecimiento invasivo, en la colonización del tejido vivo de la planta y en la capacidad de fusión vegetativa. Por otro lado, los bajos niveles de fosforilación de la MAPK Mpk1 que exhibió el mutante nulo fbp1 junto con la sensibilidad a compuestos como SDS y CFW, sugieren que Fbp1 contribuye al mantenimiento de la integridad celular. Realizamos un análisis proteómico, en condiciones de crecimiento invasivo, para conocer las proteínas diferencialmente expresadas en la cepa mutante Δ fbp1 y poder así identificar posibles dianas del complejo SCFFbp1. De las 41 proteínas identificadas, 17 de ellas estaban sobre-expresadas en el mutantΔe fbp1 y relacionadas con funciones susceptibles de sufrir ubiquitinación: transporte, proteolisis, respuesta a estrés y organización de los componentes celulares...Fast and specific degradation proteins by the ubiquitin-proteasome system have recently emerged as a major regulatory mechanism of cellular function in eukaryotes. Some basic cellular processes regulated by ubiquitin proteasome system are cell cycle, signal transduction and transcription. The key enzymes in the ubiquitination process are the E3 ubiquitin ligases, because the function as the susbtrate recognition module of the system and are capable of transferring the activated ubiquitin to the substrate. Spk1, Cul1 and F-box protein (SCF) E3 ligases are amoung the best-understood ligases. Given the importance of this process, it was of great interest investigating the role of proteins belonging to the protein turnover machinery and their contribution to Fusarium oxysporum pathogenicity. In this study, we have focused in the study of an F-box protein called Fbp1 which encodes the orthologe of S. cerevisiae GRR1 gene. In order to investigate the role Fbp1 in pathogenicity of F. oxysporum we performed a targeted disruption of fbp1 gene. Disease progression on tomato plant inoculated with Δfbp1 mutant showed reduced virulence suggesting that Fbp1 is essential for full virulence in F.oxysporum. The Δfbp1 mutant showed reduced hyphal growth on all carbon and nitrogen sources analysed. In the mutant strain, leader hyphae displayed a zig-zag growth phenotype and an abundant and delocalized staining Spk. F. oxysporum strains lacking Δ fbp1 had a reduced phosphorylation levels of Fmk1 and shared characteristic phenotypes with Δfmk1mutants, including defects in hyphal growth under nutrient-limited conditions, invasion of the underlying substrate and colonization of living plant tissue. Interestingly, low phosphorylation levels of Mpk1 and hypersensitivity to cell wall targeting compounds like CFW and SDS showed Δ fbp1 mutant, suggesting that Fbp1 contributes to cell wall integrity via a MAPK Mpk1 pathway. In order to know differentially expressed proteins in Δfbp1 mutant invasiongrowth conditions, we carried out a proteomic approach to identify the target proteins of SCFFbp1 complex. 17 of the 41 proteins differentially expressed in the present work were up-regulated in the Δfbp1 mutant and they were related with process susceptible to be ubiquininated: transport, proteolysis, response to stress and cellular component organization. One of the most abundant protein in the Δfbp1 mutant was the FOXG_0146 gene, a 14-3-3 protein annotated in the Fusarium database like Bmh2. 14-3-3 proteins are dimeric and highly conserved proteins which are involved in a many cellular..

Antagonistic Potential of Native Trichoderma spp. against Phytophthora cinnamomi in the Control of Holm Oak Decline in Dehesas Ecosystems

Phytophthora root rot caused by the pathogen Phytophthora cinnamomi is one of the main causes of oak mortality in Mediterranean open woodlands, the so-called dehesas. Disease control is challenging; therefore, new alternative measures are needed. This study focused on searching for natural biocontrol agents with the aim of developing integrated pest management (IPM) strategies in dehesas as a part of adaptive forest management (AFM) strategies. Native Trichoderma spp. were selectively isolated from healthy trees growing in damaged areas by P. cinnamomi root rot, using Rose Bengal selective medium. All Trichoderma (n = 95) isolates were evaluated against P. cinnamomi by mycelial growth inhibition (MGI). Forty-three isolates presented an MGI higher than 60%. Twenty-one isolates belonging to the highest categories of MGI were molecularly identified as T. gamsii, T. viridarium, T. hamatum, T. olivascens, T. virens, T. paraviridescens, T. linzhiense, T. hirsutum, T. samuelsii, and T. harzianum. Amongst the identified strains, 10 outstanding Trichoderma isolates were tested for mycoparasitism, showing values on a scale ranging from 3 to 4. As far as we know, this is the first report referring to the antagonistic activity of native Trichoderma spp. over P. cinnamomi strains cohabiting in the same infected dehesas. The analysis of the tree health status and MGI suggest that the presence of Trichoderma spp. might diminish or even avoid the development of P. cinnamomi, protecting trees from the worst effects of P. cinnamomi root rot

Macro- and Microscopic Characterization of Components of Resistance against Puccinia striiformis f. sp. tritici in a Collection of Spanish Bread Wheat Cultivars

Yellow (stripe) rust, caused by the biotrophic fungus Puccinia striiformis f. sp. tritici (Pst), stands as the most serious wheat disease worldwide, affecting approximately 88% of world wheat production. Even though yellow rust generally develops in cool humid weather conditions, the expansion of new races adapted to warmer climates threatens zones where severe P. striiformis epidemics were infrequent, such as Andalusian wheat cropping areas. In order to characterize yellow rust resistance mechanisms in Spanish germplasm, our study evaluated 19 Spanish bread wheat cultivars against P. striiformis under controlled conditions for percentage of disease severity (DS) and infection type (IT). From this visual evaluation, 74% of evaluated cultivars showed resistant responses against P. striiformis infection with only five cultivars considered susceptible. Subsequently, macroscopic and microscopic components of resistance were identified through image analysis and histological studies, respectively, in six selected cultivars. Macroscopic parameters such as total pustule area and total affected area (%), together with microscopic parameters such as early-aborted and established microcolonies regarding plant cell death responses (%), and microcolony length (µm), were identified as capable of differentiating cultivars quantitatively. Thus, these parameters could be used as a basis for screening resistant responses in future breeding programs, complementary to physiology, genetic and biochemical studies of plant-Pst interaction. Finally, our study seems to be the first macroscopic and microscopic characterization of P. striiformis infection in a collection of Spanish bread wheat cultivars in controlled conditions

Fusarium oxysporum Casein Kinase 1, a Negative Regulator of the Plasma Membrane H+-ATPase Pma1, Is Required for Development and Pathogenicity

Like many hemibiotrophic plant pathogens, the root-infecting vascular wilt fungus Fusarium oxysporum induces an increase in the pH of the surrounding host tissue. How alkalinization promotes fungal infection is not fully understood, but recent studies point towards the role of cytosolic pH (pHc) and mitogen-activated protein kinase (MAPK) signaling. In fungi, pHc is mainly controlled by the essential plasma membrane H+-ATPase Pma1. Here we created mutants of F. oxysporum lacking casein kinase 1 (Ck1), a known negative regulator of Pma1. We found that the ck1Δ mutants have constitutively high Pma1 activity and exhibit reduced alkalinization of the surrounding medium as well as decreased hyphal growth and conidiation. Importantly, the ck1Δ mutants exhibit defects in hyphal chemotropism towards plant roots and in pathogenicity on tomato plants. Thus, Ck1 is a key regulator of the development and virulence of F. oxysporum

Non-inferiority of dose reduction versusstandard dosing of TNF-inhibitors in axial spondyloarthritis

Objective: the objective was to determine if dose reduction is non-inferior to full-dose TNFi to maintain low disease activity (LDA) in patients already in remission with TNFi, in axial spondyloarthritis. Methods: randomized, parallel, non-inferiority, open-label multicentre clinical trial. Patients were eligible if they had axial spondyloarthritis and had been in clinical remission for ≥ 6 months with any available TNFi (adalimumab, etanercept, infliximab, golimumab) at the dose recommended by product labelling. Patients were randomized by automated central allocation to continue the same TNFi dose schedule, or to reduce the dose by roughly half according to the protocol. The main outcome was the proportion of subjects with LDA after 1 year. Serious adverse reactions or infections were recorded. Results: the trial stopped due to end of the funding period, after 126 patients were randomized; 113 patients (84.1% male, mean age (SD) 45.6 (13.0) years) were included in the main per-protocol subset. Non-inferiority was concluded for LDA at 1 year (47/55 (83.8%) patients in the full-dose and 48/58 (81.3%) patients in the reduced-dose arm, adjusted difference (95% CI) − 2.5% (− 16.6% to 11.7%)). Serious adverse reactions or infections were reported in 7/62 patients (11.3%) assigned to full dose and 2/61 patients (3.3%) assigned to reduced dose (p value = 0.164). Conclusion: in patients with ankylosing spondylitis in clinical remission for at least 6 months, dose reduction is non-inferior to full TNF inhibitor doses to maintain LDA after 1 year. Serious adverse events may be less frequent with reduced doses

Reducing Nitrogen Dosage in Triticum durum Plants with Urea-Doped Nanofertilizers

Nanotechnology is emerging as a very promising tool towards more efficient and sustainable practices in agriculture. In this work, we propose the use of non-toxic calcium phosphate nanoparticles doped with urea (U-ACP) for the fertilization of Triticum durum plants. U-ACP nanoparticles present very similar morphology, structure, and composition than the amorphous precursor of bone mineral, but contain a considerable amount of nitrogen as adsorbed urea (up to ca. 6 wt % urea). Tests on Triticum durum plants indicated that yields and quality of the crops treated with the nanoparticles at reduced nitrogen dosages (by 40%) were unaltered in comparison to positive control plants, which were given the minimum N dosages to obtain the highest values of yield and quality in fields. In addition, optical microscopy inspections showed that Alizarin Red S stained nanoparticles were able to penetrate through the epidermis of the roots or the stomata of the leaves. We observed that the uptake through the roots occurs much faster than through the leaves (1 h vs. 2 days, respectively). Our results highlight the potential of engineering nanoparticles to provide a considerable efficiency of nitrogen uptake by durum wheat and open the door to design more sustainable practices for the fertilization of wheat in fields.This research was funded by Fondazione CARIPLO (project no. 2016-0648: Romancing the stone: size-controlled HYdroxyaPATItes for sustainable Agriculture–HYPATIA) and the Spanish Ministerio de Ciencia, Innovación y Universidades (MCIU/AEI/FEDER) with the Projects NanoSmart (RYC-2016-21042) and NanoVIT (RTI-2018-095794-A-C22). GBRR also acknowledges the Spanish MICINN for her postdoctoral contract within the Juan de la Cierva Program (JdC-2017)

Monocyte Activation and Ageing Biomarkers in the Development of Cardiovascular Ischaemic Events or Diabetes in People with HIV

We investigated whether blood telomere length (TL), epigenetic age acceleration (EAA), and soluble inflammatory monocyte cytokines are associated with cardiovascular events or diabetes (DM) in people living with HIV (PLHIV). This was a case-control study nested in the Spanish HIV/AIDS Cohort (CoRIS). Cases with myocardial infarction, stroke, sudden death, or diabetes after starting antiretroviral therapy were included with the available samples and controls matched for sex, age, tobacco use, pre-ART CD4 cell count, viral load, and sample time-point. TL (T/S ratio) was analysed by quantitative PCR and EAA with DNA methylation changes by next-generation sequencing using the Weidner formula. Conditional logistic regression was used to explore the association with cardiometabolic events. In total, 180 participants (94 cases (22 myocardial infarction/sudden death, 12 strokes, and 60 DM) and 94 controls) were included. Of these, 84% were male, median (IQR) age 46 years (40-56), 53% were current smokers, and 22% had CD4 count ≤ 200 cells/mm3 and a median (IQR) log viral load of 4.52 (3.77-5.09). TL and EAA were similar in the cases and controls. There were no significant associations between TL, EAA, and monocyte cytokines with cardiometabolic events. TL and EAA were mildly negatively correlated with sCD14 (rho = -0.23; p = 0.01) and CCL2/MCP-1 (rho = -0.17; p = 0.02). We found no associations between TL, EAA, and monocyte cytokines with cardiovascular events or diabetes. Further studies are needed to elucidate the clinical value of epigenetic biomarkers and TL in PLHIV.This study was funded by an unrestricted and competitive grant from “The Fellowship Program” of Gilead Sciences (Exp. GLD16/00133). CoRIS is supported by the Instituto de Salud Carlos III through the Red Temática de Investigación Cooperativa en Sida (RD06/006, RD12/0017/0018 and RD16/0002/0006) as part of the Plan Nacional I + D + i and co-financed by Instituto de Salud Carlos III-Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER). The integrated HIV BioBank is supported by the Instituto de Salud Carlos III RD12/0017/0037.S

Expression of p53 protein isoforms predicts survival in patients with multiple myeloma

Loss and/or mutation of the TP53 gene are associated with short survival in multiple myeloma, but the p53 landscape goes far beyond. At least 12 p53 protein isoforms have been identified as a result of a combination of alternative splicing, alternative promoters and/or alternative transcription site starts, which are grouped as α, β, γ, from transactivation domain (TA), long, and short isoforms. Nowadays, there are no studies evaluating the expression of p53 isoforms and its clinical relevance in multiple myeloma (MM). We used capillary nanoimmunoassay to quantify the expression of p53 protein isoforms in CD138‐purified samples from 156 patients with newly diagnosed MM who were treated as part of the PETHEMA/GEM2012 clinical trial and investigated their prognostic impact. Quantitative real‐time polymerase chain reaction was used to corroborate the results at RNA levels. Low and high levels of expression of short and TAp53β/γ isoforms, respectively, were associated with adverse prognosis in MM patients. Multivariate Cox models identified high levels of TAp53β/γ (hazard ratio [HR], 4.49; p < .001) and high‐risk cytogenetics (HR, 2.69; p < .001) as independent prognostic factors associated with shorter time to progression. The current cytogenetic‐risk classification was notably improved when expression levels of p53 protein isoforms were incorporated, whereby high‐risk MM expressing high levels of short isoforms had significantly longer survival than high‐risk patients with low levels of these isoforms. This is the first study that demonstrates the prognostic value of p53 isoforms in MM patients, providing new insights on the role of p53 protein dysregulation in MM biology

Areas of Interest and Social Consideration of Antidepressants on English Tweets: A Natural Language Processing Classification Study

Background: Antidepressants are the foundation of the treatment of major depressive disorders. Despite the scientific evidence, there is still a sustained debate and concern about the efficacy of antidepressants, with widely differing opinions among the population about their positive and negative effects, which may condition people’s attitudes towards such treatments. Our aim is to investigate Twitter posts about antidepressants in order to have a better understanding of the social consideration of antidepressants. Methods: We gathered public tweets mentioning antidepressants written in English, published throughout a 22-month period, between 1 January 2019 and 31 October 2020. We analysed the content of each tweet, determining in the first place whether they included medical aspects or not. Those with medical content were classified into four categories: general aspects, such as quality of life or mood, sleep-related conditions, appetite/weight issues and aspects around somatic alterations. In non-medical tweets, we distinguished three categories: commercial nature (including all economic activity, drug promotion, education or outreach), help request/offer, and drug trivialization. In addition, users were arranged into three categories according to their nature: patients and relatives, caregivers, and interactions between Twitter users. Finally, we identified the most mentioned antidepressants, including the number of retweets and likes, which allowed us to measure the impact among Twitter users. Results: The activity in Twitter concerning antidepressants is mainly focused on the effects these drugs may have on certain health-related areas, specifically sleep (20.87%) and appetite/weight (8.95%). Patients and relatives are the type of user that most frequently posts tweets with medical content (65.2%, specifically 80% when referencing sleep and 78.6% in the case of appetite/weight), whereas they are responsible for only 2.9% of tweets with non-medical content. Among tweets classified as non-medical in this study, the most common subject was drug trivialization (66.86%). Caregivers barely have any presence in conversations in Twitter about antidepressants (3.5%). However, their tweets rose more interest among other users, with a ratio 11.93 times higher than those posted by patients and their friends and family. Mirtazapine is the most mentioned antidepressant in Twitter (45.43%), with a significant difference with the rest, agomelatine (11.11%). Conclusions: This study shows that Twitter users that take antidepressants, or their friends and family, use social media to share medical information about antidepressants. However, other users that do not talk about antidepressants from a personal or close experience, frequently do so in a stigmatizing manner, by trivializing them. Our study also brings to light the scarce presence of caregivers in Twitter