Genomic profiling of fungal cell wall-interfering compounds: identification of a common gene signature

[Background]: The fungal cell wall forms a compact network whose integrity is essential for cell morphology and viability. Thus, fungal cells have evolved mechanisms to elicit adequate adaptive responses when cell wall integrity (CWI) is compromised. Functional genomic approaches provide a unique opportunity to globally characterize these adaptive mechanisms. To provide a global perspective on these CWI regulatory mechanisms, we developed chemical-genomic profiling of haploid mutant budding yeast cells to systematically identify in parallel those genes required to cope with stresses interfering the cell wall by different modes of action: β-1,3 glucanase and chitinase activities (zymolyase), inhibition of β-1,3 glucan synthase (caspofungin) and binding to chitin (Congo red). [Results]: Measurement of the relative fitness of the whole collection of 4786 haploid budding yeast knock-out mutants identified 222 mutants hypersensitive to caspofungin, 154 mutants hypersensitive to zymolyase, and 446 mutants hypersensitive to Congo red. Functional profiling uncovered both common and specific requirements to cope with different cell wall damages. We identified a cluster of 43 genes highly important for the integrity of the cell wall as the common >signature of cell wall maintenance (CWM)>. This cluster was enriched in genes related to vesicular trafficking and transport, cell wall remodeling and morphogenesis, transcription and chromatin remodeling, signal transduction and RNA metabolism. Although the CWI pathway is the main MAPK pathway regulating cell wall integrity, the collaboration with other signal transduction pathways like the HOG pathway and the invasive growth pathway is also required to cope with the cell wall damage depending on the nature of the stress. Finally, 25 mutant strains showed enhanced caspofungin resistance, including 13 that had not been previously identified. Only three of them, wsc1δ, elo2δ and elo3δ, showed a significant decrease in β-1,3-glucan synthase activity. [Conclusions]: This work provides a global perspective about the mechanisms involved in cell wall stress adaptive responses and the cellular functions required for cell wall integrity. The results may be useful to uncover new potential antifungal targets and develop efficient antifungal strategies by combination of two drugs, one targeting the cell wall and the other interfering with the adaptive mechanisms.This work was supported by grants BIO2010-22146, BIO2013-48136-P (MINECO, Spain) and S2010/BMD-2414 (Comunidad de Madrid) to J.A, and grant BIO2012-35372 (MINECO, Spain) to JCR.Peer Reviewe

Identification of Natural Killer (NK) Cells in Lesions of Human Cutaneous Graft-Versus-Host Disease: Expression of a Novel NK-Associated Surface Antigen (Kp43) in Mononuclear Infiltrates

We performed an immunohistochemical analysis of skin biopsies from 13 allogeneic bone marrow transplant (BMT) recipients, undergoing either acute graft-versus-host-disease (aGVHD, n = 8) or chronic GVHD (cGVHD, n = 5). A panel of different monoclonal antibodies (MoAb) was employed including anti-CD2, -CD3, -CD4, -CD8, -CD11b, -CD16, -CD56, and -CD57, as well as a recently described reagent (HP-3B1) specific for a novel natural killer (NK)-associated cell-surface antigen (Kp43). Our data indicate that in a GVHD lesions the proportions of CD2+ cells often exceeded those detected with anti-CD3 MoAb. Double labeling confirmed the presence of CD2+ CD3- lymphocytes and suggested the coexpression in some cells of CD2 and CD11b. When MoAb specific for non-lineage-restricted NK-associated markers were employed, anti-CD56 and -CD57 occasionally stained variable numbers of lymphocytes (x¯ = 14.6% of mononuclear cells in 0.05mm2, range <1-48% and x¯ = 10.3%, range 2–25%, respectively), whereas no CD16+lymphocytes were observed. In contrast, most samples consistently displayed substantial proportions of Kp43+cells (&xsline = 32.8%, range 12–63%), which appeared CD3-and were mainly located at the dermoepidermal junction. On the other hand, sections from most (four of five) cGVHD lichenoid lesions analyzed displayed lower proportions of Kp43 + and CD56 + cells. Our data point out the interest of the anti-Kp43 MoAb to identify NK cells in aGVHD lesions, suggesting their pathogenetic participation

La Influencia del personal branding en la construcción de la vida profesional

El Personal Branding es una estrategia usada para potencializar tu propia marca es decir la marca Yo, este término se volvió popular gracias a Thomas Peters en 1997. No es suficiente con tener una marca, actualmente el mercado requiere más que esto, partiendo de esa necesidad nace el Personal Branding. Se basa principalmente en resaltar esas cualidades que se poseen como ser humano, también se puede decir que es un proceso en el cual se aprende a conocer nuestras fuerzas y debilidades, de lo mencionado anteriormente podemos concluir que el Personal Branding busca definir las estra-tegias que se usaran para venderse como profesional

Clinical and Epidemiological Characteristics of Streptococcus suis Infections in Catalonia, Spain

Introduction: Streptococcus suis (S. suis) is a human zoonotic pathogen of occupational origin, with infection acquired through contact with live pigs or pig meat. Pig farming is one of Catalonia's biggest industries and as a result this region of Spain has one of the highest density pig populations per km2. The aim of our study was to describe the infections caused by S. suis occurring in that area over a 9-year period. Materials and Methods: A retrospective, multi-center study was carried out by searching records from 15 hospitals in Catalonia for the period between 2010 and 2019. Results: Over the study period altogether nine cases of S. suis infection were identified in five hospitals, with five of these cases occurring in the 2018-2019 period. The mean age of patients was 48 ± 8.9 years and all of them were males. Five patients (55.6%) worked in pig farms. The most frequent manifestation of infection was meningitis (5 cases; 55.6%) followed by septic arthritis (3 cases; 33.3%). None of the patients died at 30 days; nonetheless, 4 developed hearing loss as a long-term complication. Conclusion: The most commonly identified S. suis infection was meningitis. Over 50% of the episodes occurred in the last 2 years and have affected pig farm workers. Further surveillance is needed in order to know its prevalence

Clinical and Epidemiological Characteristics of Streptococcus suis Infections in Catalonia, Spain

Streptococcus suis (S. suis) is a human zoonotic pathogen of occupational origin, with infection acquired through contact with live pigs or pig meat. Pig farming is one of Catalonia's biggest industries and as a result this region of Spain has one of the highest density pig populations per km 2. The aim of our study was to describe the infections caused by S. suis occurring in that area over a 9-year period. A retrospective, multi-center study was carried out by searching records from 15 hospitals in Catalonia for the period between 2010 and 2019. Over the study period altogether nine cases of S. suis infection were identified in five hospitals, with five of these cases occurring in the 2018-2019 period. The mean age of patients was 48 ± 8.9 years and all of them were males. Five patients (55.6%) worked in pig farms. The most frequent manifestation of infection was meningitis (5 cases; 55.6%) followed by septic arthritis (3 cases; 33.3%). None of the patients died at 30 days; nonetheless, 4 developed hearing loss as a long-term complication. The most commonly identified S. suis infection was meningitis. Over 50% of the episodes occurred in the last 2 years and have affected pig farm workers. Further surveillance is needed in order to know its prevalence

Genome-Wide Analysis of Factors Affecting Transcription Elongation and DNA Repair: A New Role for PAF and Ccr4-Not in Transcription-Coupled Repair

RNA polymerases frequently deal with a number of obstacles during transcription elongation that need to be removed for transcription resumption. One important type of hindrance consists of DNA lesions, which are removed by transcription-coupled repair (TC-NER), a specific sub-pathway of nucleotide excision repair. To improve our knowledge of transcription elongation and its coupling to TC-NER, we used the yeast library of non-essential knock-out mutations to screen for genes conferring resistance to the transcription-elongation inhibitor mycophenolic acid and the DNA-damaging agent 4-nitroquinoline-N-oxide. Our data provide evidence that subunits of the SAGA and Ccr4-Not complexes, Mediator, Bre1, Bur2, and Fun12 affect transcription elongation to different extents. Given the dependency of TC-NER on RNA Polymerase II transcription and the fact that the few proteins known to be involved in TC-NER are related to transcription, we performed an in-depth TC-NER analysis of a selection of mutants. We found that mutants of the PAF and Ccr4-Not complexes are impaired in TC-NER. This study provides evidence that PAF and Ccr4-Not are required for efficient TC-NER in yeast, unraveling a novel function for these transcription complexes and opening new perspectives for the understanding of TC-NER and its functional interconnection with transcription elongation

Perfiles de interacción Químico-Genómicos en Saccharomyces cerevisiae: respuesta a agentes estresantes

[ES] El objetivo general del trabajo recogido en esta memoria es identificar los procesos celulares involucrados en la respuesta al estrés celular provocado por compuestos que inducen estrés a distintos niveles en la célula, caracterizar funcionalmente los genes involucrados en mecanismos de sensibilidad/resistencia frente a estos compuestos, y tratar de determinar el mecanismo de acción de los mismos. La aproximación empleada, que a continuación desarrollaremos en mayor detalle, ha consistido en realizar un análisis fenotípico cuantitativo de la respuesta a estrés celular ejercido por dichos compuestos en la colección completa de mutantes de deleción de la levadura Saccharomyces cerevisiae, generando así perfiles químico‐genómicos de sensibilidad y resistencia de cada uno de los agentes empleados. Los objetivos específicos que nos planteamos son: 1) Determinar los mecanismos de sensibilidad/resistencia a los distintos compuestos. 2) Identificar redes funcionales y rutas redundantes afectadas por los agentes estresantes. 3) Identificar dianas secundarias y determinar posibles efectos secundarios. 4) Elucidar la función de ORFs sin caracterizar. 5) Comparar el efecto, a nivel biológico, de cada uno de los compuestos. 6) Comparar los cambios inducidos por los compuestos en los perfiles de expresión génica con los efectos fenotípicos producidos por los mismos.[EN] The overall objective of the work contained in this report is to identify the cellular processes involved in the response to cellular stress caused by stress-inducing compounds at different levels in the cell, functionally characterize genes involved in mechanisms of sensitivity / resistance to these compounds, and try to determine the mechanism of their action. The approach used, which then develop in more detail, has been to make a quantitative phenotypic analysis of the response to cellular stress exerted by these compounds in the entire collection of deletion mutants of the yeast Saccharomyces cerevisiae, thus generating chemical genomic profiles sensitivity and resistance of each of the agents employed. The specific objectives that we consider are: 1) determine the mechanisms of sensitivity / resistance to various compounds. 2) to identify functional networks and redundant paths affected by the stressors. 3) Identify secondary targets and identify possible side effects. 4) To elucidate the function of uncharacterized ORFs. 5) Compare the effect on the biological level, each of the compounds. 6) Compare the compounds induced changes in gene expression profiles with the phenotypic effects produced by the same

New insights into the RNA-based mechanism of action of the anticancer drug 5'-Fluorouracil in eukaryotic cells

This is an open-access article distributed under the terms of the Creative Commons Attribution License.5-Fluorouracil (5FU) is a chemotherapeutic drug widely used in treating a range of advanced, solid tumours and, in particular, colorectal cancer. Here, we used high-density tiling DNA microarray technology to obtain the specific transcriptome-wide response induced by 5FU in the eukaryotic model Schizosaccharomyces pombe. This approach combined with real-time quantitative PCR analysis allowed us to detect splicing defects of a significant number of intron-containing mRNA, in addition to identify some rRNA and tRNA processing defects after 5FU treatment. Interestingly, our studies also revealed that 5FU specifically induced the expression of certain genes implicated in the processing of mRNA, tRNA and rRNA precursors, and in the post-transcriptional modification of uracil residues in RNA. The transcription of several tRNA genes was also significantly induced after drug exposure. These transcriptional changes might represent a cellular response mechanism to counteract 5FU damage since deletion strains for some of these up-regulated genes were hypersensitive to 5FU. Moreover, most of these RNA processing genes have human orthologs that participate in conserved pathways, suggesting that they could be novel targets to improve the efficacy of 5FU-based treatments.This work was supported by the Spanish Ministry of Economy and Competitiveness (BFU2011-23645 to MS and BFU2011-28274 to SM), the CONSOLIDER-INGENIO from the Spanish Ministry of Science and Innovation CSD2007-00015 (MS and SM), the Fundación Mutua Madrileña (MS) and an Institutional Grant from Fundación Ramón Areces to the Centro de Biología Molecular “Severo Ochoa”. Funding for LQ research come from the Spanish Ministry of Economy and Competitiveness (BFU2011-28804); LM was holder of a CONSOLIDER-INGENIO contract.Peer Reviewe