Cellular response to external signals in S. cerevisiae

En esta tesis, se utilizó como organismo modelo la levadura Saccharomyces cerevisiae para obtener conocimiento fundamental sobre una variedad de mecanismos moleculares utilizados por la célula para responder y adaptarse a los estímulos externos. En respuesta al aumento de la osmolaridad externa, la ruta de señalización MAPK High Osmolarity Glycerol (HOG) se activa para mediar cambios en diversas funciones celulares, incluyendo la reprogramación global de la transcripción y traducción, con el fin último de lograr la adaptación. Tras la detección de alta osmolaridad externa, la señal se transduce a través de dos ramas funcionalmente redundantes pero mecanísticamente distintas, SLN1 y SHO1, para finalmente activar el efector MAPK Hog1. Aunque múltiples interacciones entre sensores, proteínas adaptadoras y los componentes de señalización de la rama SHO1 se han descrito anteriormente, esta tesis caracteriza aún más la complejidad de los perfiles de las interacciones para dilucidar cómo se propaga la señal con eficacia y cómo se logra la fidelidad de la señal. En este sentido, hemos utilizado el nuevo método M-track que detecta tanto las interacciones entre proteínas de corta duración como las estables y hemos realizado varias observaciones interesantes. Una vez que se transduce la señal de respuesta a estrés, Hog1 activado entra en el núcleo de la célula y, entre sus más destacados funciones aguas abajo, modula los cambios de expresión génica global. El complejo de unión a la caperuza CBC (por cap-binding complex), que está formado por las proteínas de unión a RNA Cbc1 y Cbc2, se asocia con la caperuza en 5' del mensajero co-transcripcionalmente y se ha descrito su función en diversos aspectos de la vida de mensajero, incluyendo la transcripción. Utilizando técnicas de biología molecular y genómica, describimos Cbc1 como regulador de la transcripción global, tanto en condiciones de estrés y no estrés, para mediar la expresión génica de manera que se consigan niveles altos de una forma rápida. En respuesta a feromonas del tipo sexual contrario, la ruta de MAPK de feromonas de células de levadura haploides se activa para mediar una serie de cambios fisiológicos en preparación para el apareamiento, que incluyen la reprogramación de la expresión génica, la parada del ciclo celular, la formación de una proyección sexual denomina ¨shmoo¨, y en última instancia la fusión celular de los tipos de células de apareamiento contrario. El factor de traducción eIF5A, esencial y conservado evolutivamente, se ha descrito recientemente que funciona en la traducción de proteínas que contienen tres o más residuos de prolina consecutivos (polyPro), a través de su unión a ribosomas para aliviar el estancamiento del ribosoma durante la formación del enlace péptidico entre prolinas. La activación de eIF5A requiere una modificación post-traduccional única, la hipusinación, donde el residuo de hipusina se deriva de la espermidina, un factor esencial para la fertilidad de los mamíferos y que se requiere para el apareamiento de la levadura. Aquí se investigó eIF5A como regulador de la respuesta a feromonas a través de la traducción de proteínas polyPro con funciones en el apareamiento.In this thesis, we utilised the model organism the budding yeast Saccharomyces cerevisiae to gain fundamental knowledge on a variety of molecular mechanisms employed by the cell to respond and adapt to external stimuli. In response to increased external osmolarity, the yeast high osmolarity glycerol (HOG) MAPK pathway becomes activated to mediate changes to various cellular functions, including changes in glycerol accumulation, cell-cycle arrest, re-establishment of ion homeostasis and global reprogrammation of transcription and translation of the whole genome, in order to achieve adaptation. Upon detection of high external osmolarity, signal is transduced via two functionally redundant but mechanistically distinct branches, SLN1 and SHO1, to finally activate the effector MAPK Hog1. Although multiple interactions between osmosensors, adaptor proteins and signalling components of the SHO1 branch have been previously described, this thesis further characterises the complexity of their interaction profiles to elucidate how signal is effectively propagated and how signal fidelity is achieved. Here, we utilised the novel M-track method, which detects both short-lived and stable protein interactions, and made several interesting observations. Once the osmostress signal is transduced, activated Hog1 enters the cell nucleus and, amongst its most prominent downstream functions, modulates global gene expression changes. Within minutes of shock, global transcription rate rapidly decreases by 50 %, however the transcription machinery is reallocated to specific genes implicated in osmostress cellular protection and their transcription is strongly and rapidly induced. The cap-binding complex (CBC), consisting of mRNA-binding proteins Cbc1 and Cbc2, associates with the 5´ mRNA cap co-transcriptionally and has been described to function in various aspects of the mRNA life, including transcription. Utilising both genomic and molecular biology techniques, we describe Cbc1 as a global transcription regulator, both under stress and non-stress conditions, to mediate high and timely gene expression. In response to pheromones of the opposite mating type, the yeast pheromone MAPK pathway is activated to mediate a series of physiological changes in preparation for mating; including reprogrammation of gene expression, cell-cycle arrest, formation of a sexual projection termed ¨shmoo¨, and ultimately cell fusion of mating partners. The evolutionarily conserved and essential translation factor eIF5A has recently been described to promote translation of proteins containing three or more consecutive proline residues (polyPro) by binding to ribosomes and alleviating ribosome stalling during the formation of the Pro-Pro peptide bond. The activation of eIF5A requires the addition of a unique post-translational modification, a hypusine residue, which is derived from spermidine, an essential factor for mammalian fertility and required for yeast mating. Here we investigated eIF5A as a regulator of the pheromone response through the translation of polyPro proteins with roles in mating

Clinicopathological characteristics and prognosis of synchronous brain metastases from non-small cell lung cancer compared with metachronous brain metastases

PurposeBrain metastasis (BM) from non-small cell lung cancer (NSCLC) is a serious complication severely affecting patients’ prognoses. We aimed to compare the clinicopathological features and prognosis of synchronous and metachronous BM from NSCLC.MethodsClinical data of 461 patients with brain metastases from NSCLC who visited the Cancer Hospital of China Medical University from 2005 to 2017 were retrospectively collected. We analyzed the pathophysiological characteristics of synchronous and metachronous BM from NSCLC and survival rates of the patients. Propensity score matching analysis was used to reduce bias between groups. In addition, we used the Kaplan-Meier method for survival analysis, log-rank test to compare survival rates, and Cox proportional hazards regression model for multivariate prognosis analysis.ResultsAmong 461 patients with BM, the number of people who met the inclusion criteria was 400 cases, and after 1:2 propensity score matching,130 had synchronous BM and 260 had metachronous BM. The survival time was longer for metachronous BM in driver mutation-negative patients with squamous cell carcinoma than synchronous BM. Conversely, metachronous and synchronous BM with gene mutations and adenocarcinoma showed no differences in survival time. Multivariate analysis showed that metachronous BM was an independent prognostic factor for overall survival. Furthermore, the pathological type squamous cell carcinoma and Karnofsky Performance Status score <80 were independent risk factors affecting overall survival.ConclusionBM status is an independent factor influencing patient outcome. Moreover, synchronous and metachronous BM from NSCLC differ in gene mutation profile, pathological type, and disease progression and hence require different treatments

Ultrasonic augers for improved transport of granular materials

This paper explores the superposition of ultrasonic vibration on vertical rotating augers in a variety of granular media. Ultrasonic vibration is known to facilitate direct penetration of granular media, and it is anticipated that any related reduction in the torque requirements of a rotating system might improve augering performance. Experimental results suggest that, compared to the non-ultrasonic scenario, ultrasonically assisted augering significantly promotes the flow of granular media, while moderately reducing the torque required to operate the device. Furthermore, it was discovered that particle size, helix angle, and auger speed all affect the performance of the auger in different ways as the ultrasonic amplitude is adjusted

Evolutionary conserved role of eukaryotic translation factor eIF5A in the regulation of actin-nucleating formins

Elongation factor eIF5A is required for the translation of consecutive prolines, and was shown in yeast to translate polyproline-containing Bni1, an actin-nucleating formin required for polarized growth during mating. Here we show that Drosophila eIF5A can functionally replace yeast eIF5A and is required for actin-rich cable assembly during embryonic dorsal closure (DC). Furthermore, Diaphanous, the formin involved in actin dynamics during DC, is regulated by and mediates eIF5A effects. Finally, eIF5A controls cell migration and regulates Diaphanous levels also in mammalian cells. Our results uncover an evolutionary conserved role of eIF5A regulating cytoskeleton-dependent processes through translation of formins in eukaryotes

Hacia una medicina de precisión en la dermatitis atópica mediante el uso de enfoques moleculares

Omics; Dermatitis atópica; Secuenciación de nueva generaciónOmics; Dermatitis atòpica; Seqüenciació de nova generacióOmics; Atopic dermatitis; Next generation sequencingLa dermatitis atópica es el trastorno inflamatorio de la piel crónico más común. Afecta hasta a 20% de los niños y a 10% de los adultos en países desarrollados. La fisiopatología de la dermatitis atópica es compleja e implica una fuerte predisposición genética e inflamación impulsada por células T. Aunque nuestra comprensión de la patología y las causas de esta enfermedad ha mejorado en los últimos años, aún existen lagunas de conocimiento en las vías inmunológicas involucradas. En consecuencia, los avances en nuevas tecnologías ómicas en la dermatitis atópica desempeñarán un papel clave en la comprensión de la patogénesis de esta enfermedad y podrían desarrollar estrategias preventivas y tratamientos personalizados. En esta revisión se discuten los últimos avances en genética, transcriptómica, epigenómica, proteómica y metagenómica, y entendemos cómo la integración de múltiples conjuntos de datos ómicos identificará posibles biomarcadores y descubrirá redes de asociaciones entre varios niveles moleculares.Atopic dermatitis is the most common chronic inflammatory skin disorder, affecting up to 20% of children and 10% of adults in developed countries. The pathophysiology of atopic dermatitis is complex and involves a strong genetic predisposition and T-cell driven inflammation. Although our understanding of the pathology and drivers of this disease has improved in recent years, there are still knowledge gaps in the immune pathways involved. Therefore, advances in new omics technologies in atopic dermatitis will play a key role in understanding the pathogenesis of this burden disease and could develop preventive strategies and personalized treatment strategies. In this review, we discuss the latest developments in genetics, transcriptomics, epigenomics, proteomics, and metagenomics and understand how integrating multiple omics datasets will identify potential biomarkers and uncover nets of associations between several molecular levelsThis work and the article processing charges were funded by IMIDomics, FIS grant (PI21/01139) and EADV project (PPRC 2021-22). The sponsors had no role in the design, execution, interpretation, or writing of the work

SIRT1/2 orchestrate acquisition of DNA methylation and loss of histone H3 activating marks to prevent premature activation of inflammatory genes in macrophages

Sirtuins 1 and 2 (SIRT1/2) are two NAD-dependent deacetylases with major roles in inflammation. In addition to deacetylating histones and other proteins, SIRT1/2-mediated regulation is coupled with other epigenetic enzymes. Here, we investigate the links between SIRT1/2 activity and DNA methylation in macrophage differentiation due to their relevance in myeloid cells. SIRT1/2 display drastic upregulation during macrophage differentiation and their inhibition impacts the expression of many inflammation-related genes. In this context, SIRT1/2 inhibition abrogates DNA methylation gains, but does not affect demethylation. Inhibition of hypermethylation occurs at many inflammatory loci, which results in more drastic upregulation of their expression upon macrophage polarization following bacterial lipopolysaccharide (LPS) challenge. SIRT1/2-mediated gains of methylation concur with decreases in activating histone marks, and their inhibition revert these histone marks to resemble an open chromatin. Remarkably, specific inhibition of DNA methyltransferases is sufficient to upregulate inflammatory genes that are maintained in a silent state by SIRT1/2. Both SIRT1 and SIRT2 directly interact with DNMT3B, and their binding to proinflammatory genes is lost upon exposure to LPS or through pharmacological inhibition of their activity. In all, we describe a novel role for SIRT1/2 to restrict premature activation of proinflammatory genes

Study of metabolic association between elevated fasting blood glucose and cognitive deterioration

Objective·To analyze and explore the influencing factors that lead to cognitive deterioration in individuals with elevated fasting blood glucose (FBG) and the metabolic clues associated with changes in the risk of cognitive deterioration.Methods·Data from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database were downloaded, and the samples with FBG and follow-up data were selected from the database. Clinical information, including age, gender, body mass index, education years, apolipoprotein E4 (APOE4) genotype and race, and corresponding metabolic indicator data, including amino acids, fatty acids, proteins and others were obtained. Based on the FBG levels and diagnosis of cognitive impairment stages in Alzheimer's disease, the subjects were categorized into four groups: normal FBG without/with cognitive deterioration, and elevated FBG without/with cognitive deterioration. The univariate analysis method, the Cox proportional hazards model, orthogonal projections to latent structures discriminant analysis (OPLSDA), and Spearman correlation analysis were employed for data analysis.Results·A total of 1 317 subjects were included, among which 1 153 had normal FBG level (>3.9 mmol/L and <6.1 mmol/L) and 164 had elevated FBG level (≥6.1 mmol/L). In the normal FBG group, 275 subjects showed cognitive deterioration, while in the elevated FBG group, 53 subjects showed cognitive deterioration. Univariate analysis revealed significant differences in gender and race between the normal FBG and elevated FBG group, and significant differences in age, gender, and APOE4 genotype between the groups with and without cognitive deterioration (all P<0.05). Cox regression analysis indicated that primary influencing factors for cognitive deterioration were APOE4 positivity, elevated FBG, and increasing age in order (HR=2.22,HR=1.38,HR=1.02; all P<0.05). In the analysis of baseline metabolic indicators in the groups without and with cognitive deterioration, as well as metabolic indicators before and after cognitive deterioration at different FBG levels, the results of the analysis of variance revealed that in the cognitively deteriorated population, the ratio of phospholipids carried by high-density lipoproteins (HDL) to total lipids was significantly higher; low-density lipoprotein (LDL) particle concentration and the lipids carried by LDL were significantly higher after cognitive deterioration. Correlation analysis showed that valine and leucine were significantly correlated not only with FBG level but also with phosphorylated tau (pTau) level in the plasma in the cognitively deteriorated population. Cholesterol and the ratio of phospholipids to total lipids carried by HDL were significantly correlated with pTau levels in cerebrospinal fluid (CSF).Conclusion·Compared to the individuals with normal FBG level, those with high FBG level have a significantly higher risk of cognitive deterioration. Additionally, different metabolic indicators show significant differences between the groups without and with cognitive deterioration, as well as metabolic indicators before and after cognitive deterioration at different FBG levels. Overall, LDL and its lipid content, and HDL-carried phospholipids show an increasing trend during cognitive deterioration, and the branched-chain amino acids valine and leucine are significantly correlated with pTau levels in CSF and plasma, suggesting that these metabolic markers may play an important role in cognitive deterioration

SIRT1/2 orchestrate acquisition of DNA methylation and loss of histone H3 activating marks to prevent premature activation of inflammatory genes in macrophages

Altres ajuts: CERCA Programme/Generalitat de Catalunya; [...].Sirtuins 1 and 2 (SIRT1/2) are two NAD-dependent deacetylases with major roles in inflammation. In addition to deacetylating histones and other proteins, SIRT1/2-mediated regulation is coupled with other epigenetic enzymes. Here, we investigate the links between SIRT1/2 activity and DNA methylation in macrophage differentiation due to their relevance in myeloid cells. SIRT1/2 display drastic upregulation during macrophage differentiation and their inhibition impacts the expression of many inflammation-related genes. In this context, SIRT1/2 inhibition abrogates DNA methylation gains, but does not affect demethylation. Inhibition of hypermethylation occurs at many inflammatory loci, which results in more drastic upregulation of their expression upon macrophage polarization following bacterial lipopolysaccharide (LPS) challenge. SIRT1/2-mediated gains of methylation concur with decreases in activating histone marks, and their inhibition revert these histone marks to resemble an open chromatin. Remarkably, specific inhibition of DNA methyltransferases is sufficient to upregulate inflammatory genes that are maintained in a silent state by SIRT1/2. Both SIRT1 and SIRT2 directly interact with DNMT3B, and their binding to proinflammatory genes is lost upon exposure to LPS or through pharmacological inhibition of their activity. In all, we describe a novel role for SIRT1/2 to restrict premature activation of proinflammatory genes