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Epigenetic modifications play a vital role in the preservation of genome integrity and in the regulation of gene expression. DNA methylation, one of the key mechanisms of epigenetic control, impacts growth, development, stress response and adaptability of all organisms, including plants. The detection of DNA methylation marks is crucial for understanding the mechanisms underlying these processes and for developing strategies to improve productivity and stress resistance of crop plants. There are different methods for detecting plant DNA methylation, such as bisulfite sequencing, methylation-sensitive amplified polymorphism, genome-wide DNA methylation analysis, methylated DNA immunoprecipitation sequencing, reduced representation bisulfite sequencing, MS and immuno-based techniques. These profiling approaches vary in many aspects, including DNA input, resolution, genomic region coverage, and bioinformatics analysis. Selecting an appropriate methylation screening approach requires an understanding of all these techniques. This review provides an overview of DNA methylation profiling methods in crop plants, along with comparisons of the efficacy of these techniques between model and crop plants. The strengths and limitations of each methodological approach are outlined, and the importance of considering both technical and biological factors are highlighted. Additionally, methods for modulating DNA methylation in model and crop species are presented. Overall, this review will assist scientists in making informed decisions when selecting an appropriate DNA methylation profiling method.
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Background: Managing the inflammatory response to SARS-Cov-2 could prevent respiratory insufficiency. Cytokine profiles could identify cases at risk of severe disease. Methods: We designed a randomized phase II clinical trial to determine whether the combination of ruxolitinib (5 mg twice a day for 7 days followed by 10 mg BID for 7 days) plus simvastatin (40 mg once a day for 14 days), could reduce the incidence of respiratory insufficiency in COVID-19. 48 cytokines were correlated with clinical outcome. Participants: Patients admitted due to COVID-19 infection with mild disease. Results: Up to 92 were included. Mean age was 64 ± 17, and 28 (30%) were female. 11 (22%) patients in the control arm and 6 (12%) in the experimental arm reached an OSCI grade of 5 or higher (p = 0.29). Unsupervised analysis of cytokines detected two clusters (CL-1 and CL-2). CL-1 presented a higher risk of clinical deterioration vs CL-2 (13 [33%] vs 2 [6%] cases, p = 0.009) and death (5 [11%] vs 0 cases, p = 0.059). Supervised Machine Learning (ML) analysis led to a model that predicted patient deterioration 48h before occurrence with a 85% accuracy. Conclusions: Ruxolitinib plus simvastatin did not impact the outcome of COVID-19. Cytokine profiling identified patients at risk of severe COVID-19 and predicted clinical deterioration. Trial registration: https://clinicaltrials.gov/, identifier NCT04348695.
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COVID-19 , Deterioro Clínico , Insuficiencia Respiratoria , Humanos , Femenino , Masculino , SARS-CoV-2 , Resultado del TratamientoRESUMEN
Most cellular proteins involved in genome replication are conserved in all eukaryotic lineages including yeast, plants and animals. However, the mechanisms controlling their availability during the cell cycle are less well defined. Here we show that the Arabidopsis genome encodes for two ORC1 proteins highly similar in amino acid sequence and that have partially overlapping expression domains but with distinct functions. The ancestral ORC1b gene, present before the partial duplication of the Arabidopsis genome, has retained the canonical function in DNA replication. ORC1b is expressed in both proliferating and endoreplicating cells, accumulates during G1 and is rapidly degraded upon S-phase entry through the ubiquitin-proteasome pathway. In contrast, the duplicated ORC1a gene has acquired a specialized function in heterochromatin biology. ORC1a is required for efficient deposition of the heterochromatic H3K27me1 mark by the ATXR5/6 histone methyltransferases. The distinct roles of the two ORC1 proteins may be a feature common to other organisms with duplicated ORC1 genes and a major difference with animal cells.
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Proteínas de Arabidopsis , Arabidopsis , Proteínas de Ciclo Celular , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Metiltransferasas , Complejo de Reconocimiento del Origen/genética , Fase S/genéticaRESUMEN
Climate change is increasing the frequency of extreme heat events that aggravate its negative impact on plant development and agricultural yield. Most experiments designed to study plant adaption to heat stress apply homogeneous high temperatures to both shoot and root. However, this treatment does not mimic the conditions in natural fields, where roots grow in a dark environment with a descending temperature gradient. Excessively high temperatures severely decrease cell division in the root meristem, compromising root growth, while increasing the division of quiescent center cells, likely in an attempt to maintain the stem cell niche under such harsh conditions. Here, we engineered the TGRooZ, a device that generates a temperature gradient for in vitro or greenhouse growth assays. The root systems of plants exposed to high shoot temperatures but cultivated in the TGRooZ grow efficiently and maintain their functionality to sustain proper shoot growth and development. Furthermore, gene expression and rhizosphere or root microbiome composition are significantly less affected in TGRooZ-grown roots than in high-temperature-grown roots, correlating with higher root functionality. Our data indicate that use of the TGRooZ in heat-stress studies can improve our knowledge of plant response to high temperatures, demonstrating its applicability from laboratory studies to the field.
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Ecosistema , Raíces de Plantas , Temperatura , Raíces de Plantas/metabolismo , Meristema , Calor , PlantasRESUMEN
Virulence, the effect of pathogen infection on progeny production, is a major determinant of host and pathogen fitness as it affects host fecundity and pathogen transmission. In plant-virus interactions, ample evidence indicates that virulence is genetically controlled by both partners. However, the host genetic determinants are poorly understood. Through a genome-wide association study (GWAS) of 154 Arabidopsis thaliana genotypes infected by Cucumber mosaic virus (CMV), we identified eight host genes associated with virulence, most of them involved in response to biotic stresses and in cell wall biogenesis in plant reproductive structures. Given that virulence is a main determinant of the efficiency of plant virus seed transmission, we explored the link between this trait and the genetic regulation of virulence. Our results suggest that the same functions that control virulence are also important for CMV transmission through seeds. In sum, this work provides evidence of a novel role for some previously known plant defense genes and for the cell wall metabolism in plant virus interactions.
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Nucleus-encoded plastid proteins are synthesized as precursors with N-terminal targeting signals called transit peptides (TPs), which mediate interactions with the translocon complexes at the outer (TOC) and inner (TIC) plastid membranes. These complexes exist in multiple isoforms in higher plants and show differential specificity and tissue abundance. While some show specificity for photosynthesis-related precursor proteins, others distinctly recognize nonphotosynthetic and housekeeping precursor proteins. Here we used TPs from four Arabidopsis thaliana proteins, three related to photosynthesis (chlorophyll a/b binding protein, Rubisco activase) and photo-protection (tocopherol cyclase) and one involved in the assimilation of ammonium into amino-acids, and whose expression is most abundant in the root (ferredoxin dependent glutamate synthase 2), to determine whether they were able to mediate import of a nuclear-encoded marker protein into plastids of different tissues of a dicot and a monocot species. In A. thaliana, import and processing efficiency was high in all cases, while TP from the rice Rubisco small chain 1, drove very low import in Arabidopsis tissues. Noteworthy, our results show that Arabidopsis photosynthesis TPs also mediate plastid import in rice callus, and in leaf and root tissues with almost a 100% efficiency, providing new biotechnological tools for crop improvement strategies based on recombinant protein accumulation in plastids by the expression of nuclear-encoded transgenes.
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The seed is the propagule of higher plants and allows its dissemination and the survival of the species. Seed dormancy prevents premature germination under favourable conditions. Dormant seeds are only able to germinate in a narrow range of conditions. During after-ripening (AR), a mechanism of dormancy release, seeds gradually lose dormancy through a period of dry storage. This review is mainly focused on how chemical modifications of mRNA and genomic DNA, such as oxidation and methylation, affect gene expression during late stages of seed development, especially during dormancy. The oxidation of specific nucleotides produced by reactive oxygen species (ROS) alters the stability of the seed stored mRNAs, being finally degraded or translated into non-functional proteins. DNA methylation is a well-known epigenetic mechanism of controlling gene expression. In Arabidopsis thaliana, while there is a global increase in CHH-context methylation through embryogenesis, global DNA methylation levels remain stable during seed dormancy, decreasing when germination occurs. The biological significance of nucleic acid oxidation and methylation upon seed development is discussed.
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The generation of nitrogen fixing crops is considered a challenge that could lead to a new agricultural 'green' revolution. Here, we report the use of synthetic biology tools to achieve and optimize the production of active nitrogenase Fe protein (NifH) in the chloroplasts of tobacco plants. Azotobacter vinelandii nitrogen fixation genes, nifH, M, U and S, were re-designed for protein accumulation in tobacco cells. Targeting to the chloroplast was optimized by screening and identifying minimal length transit peptides performing properly for each specific Nif protein. Putative peptidyl-prolyl cis-trans isomerase NifM proved necessary for NifH solubility in the stroma. Purified NifU, a protein involved in the biogenesis of NifH [4Fe-4S] cluster, was found functional in NifH reconstitution assays. Importantly, NifH purified from tobacco chloroplasts was active in the reduction of acetylene to ethylene, with the requirement of nifU and nifS co-expression. These results support the suitability of chloroplasts to host functional nitrogenase proteins, paving the way for future studies in the engineering of nitrogen fixation in higher plant plastids and describing an optimization pipeline that could also be used in other organisms and in the engineering of new metabolic pathways in plastids.
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Nicotiana , Biología Sintética , Proteínas Bacterianas/metabolismo , Cloroplastos/metabolismo , Fijación del Nitrógeno , Nitrogenasa/metabolismo , Oxidorreductasas , Hojas de la Planta/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
Mitochondria fulfil essential functions in respiration and metabolism as well as regulating stress responses and apoptosis. Most native mitochondrial proteins are encoded by nuclear genes and are imported into mitochondria via one of several receptors that recognize N-terminal signal peptides. The targeting of recombinant proteins to mitochondria therefore requires the presence of an appropriate N-terminal peptide, but little is known about mitochondrial import in monocotyledonous plants such as rice (Oryza sativa). To gain insight into this phenomenon, we targeted nuclear-encoded enhanced green fluorescent protein (eGFP) to rice mitochondria using six mitochondrial pre-sequences with diverse phylogenetic origins, and investigated their effectiveness by immunoblot analysis as well as confocal and electron microscopy. We found that the ATPA and COX4 (Saccharomyces cerevisiae), SU9 (Neurospora crassa), pFA (Arabidopsis thaliana) and OsSCSb (Oryza sativa) peptides successfully directed most of the eGFP to the mitochondria, whereas the MTS2 peptide (Nicotiana plumbaginifolia) showed little or no evidence of targeting ability even though it is a native plant sequence. Our data therefore indicate that the presence of particular recognition motifs may be required for mitochondrial targeting, whereas the phylogenetic origin of the pre-sequences probably does not play a key role in the success of mitochondrial targeting in dedifferentiated rice callus and plants.
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Núcleo Celular/metabolismo , Mitocondrias/metabolismo , Oryza/metabolismo , Fragmentos de Péptidos/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/metabolismo , Secuencias de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Núcleo Celular/genética , Proteínas Fluorescentes Verdes/metabolismo , Mitocondrias/genética , Oryza/genética , Fragmentos de Péptidos/genética , Proteínas de Plantas/genética , Señales de Clasificación de Proteína , Transporte de Proteínas , Proteínas Recombinantes/genética , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
DNA methylation is an important epigenetic mechanism for controlling innate immunity against microbial pathogens in plants. Little is known, however, about the manner in which viral infections interact with DNA methylation pathways. Here we investigate the crosstalk between epigenetic silencing and viral infections in Arabidopsis inflorescences. We found that tobacco rattle virus (TRV) causes changes in the expression of key transcriptional gene silencing factors with RNA-directed DNA methylation activities that coincide with changes in methylation at the whole genome level. Viral susceptibility/resistance was altered in DNA (de)methylation-deficient mutants, suggesting that DNA methylation is an important regulatory system controlling TRV proliferation. We further show that several transposable elements (TEs) underwent transcriptional activation during TRV infection, and that TE regulation likely involved both DNA methylation-dependent and -independent mechanisms. We identified a cluster of disease resistance genes regulated by DNA methylation in infected plants that were enriched for TEs in their promoters. Interestingly, TEs and nearby resistance genes were co-regulated in TRV-infected DNA (de)methylation mutants. Our study shows that DNA methylation contributes to modulate the outcome of viral infections in Arabidopsis, and opens up new possibilities for exploring the role of TE regulation in antiviral defence.
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Arabidopsis/genética , Arabidopsis/virología , Virus de Plantas/patogenicidad , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Metilación de ADN/genética , Metilación de ADN/fisiología , Elementos Transponibles de ADN/genética , Elementos Transponibles de ADN/fisiología , Epigénesis Genética/genética , Regulación de la Expresión Génica de las Plantas , Silenciador del GenRESUMEN
For more than 20 years, plant biologists have tried to achieve complete control of transgene expression. Until the techniques to target transgenes to safe harbor sites in the genome become routine, flanking transgenes with genetic insulators, DNA sequences that create independent domains of gene expression, can help avoid positional effects and stabilize their expression. We have, for the first time, compared the effect of three insulator sequences previously described in the literature and one never tested before. Our results indicate that their use increases transgene expression, but only the last one reduces variability between lines and between individuals. We have analyzed the integration of insulator-flanked T-DNAs using whole genome re-sequencing (to our knowledge, also for the first time) and found data suggesting that chiMARs can shelter transgene insertions from neighboring repressive epigenetic states. Finally, we could also observe a loss of accuracy of the RB insertion in the lines harboring insulators, evidenced by a high frequency of truncation of T-DNAs and of insertion of vector backbone that, however, did not affect transgene expression. Our data supports that the effect of each genetic insulator is different and their use in transgenic constructs should depend on the needs of each specific experiment.
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Regulación de la Expresión Génica , Genómica , Elementos Aisladores/genética , Transgenes , Animales , Arabidopsis/genética , Secuencia de Bases , Pollos/genética , Metilación de ADN/genética , ADN Bacteriano/genética , Luciferasas/genética , Regiones de Fijación a la Matriz/genética , Muramidasa/genética , Mutagénesis Insercional/genéticaRESUMEN
OBJECTIVE: Obtaining high and stable transgene expression is of vital importance for plant genetic engineering. A lot is known about the relationship between terminator efficiency and gene expression, but no studies have addressed the relationship between terminator usage and transgene expression stability or heritable gene silencing. In this paper, we aim to analyze if terminators are a determining factor in the establishment of promoter DNA methylation of plant transgenes. RESULTS: Our experiments comparing plants with a LUC reporter under the 35S CaMV promoter and good efficiency terminators (Thsp, T35S) show that the use of efficient terminator sequences does not avoid the accumulation of promoter DNA methylation and transgene silencing. However, Thsp lead to a higher reporter gene expression and lower promoter DNA methylation levels than T35S, supporting that terminator usage is indeed involved in the establishment of TGS by methylation of transgenes' promoters. In the case of a terminatorless construct, the PTGS initiated by the improperly terminated mRNAs is not followed by the establishment of heritable silencing in the form of strong promoter DNA methylation, like in the case of TAS genes and reactivated TEs (for the transgene DNA methylation levels remained below the 20%).
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Metilación de ADN , Silenciador del Gen , Genes Reporteros , Transgenes , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
BACKGROUND: There is a need for the development of synthetic biology methods and tools to facilitate rapid and efficient engineering of yeast that accommodates the needs of specific biotechnology projects. In particular, the manipulation of the mitochondrial proteome has interesting potential applications due to its compartmentalized nature. One of these advantages resides in the fact that metalation occurs after protein import into mitochondria, which contains pools of iron, zinc, copper and manganese ions that can be utilized in recombinant metalloprotein metalation reactions. Another advantage is that mitochondria are suitable organelles to host oxygen sensitive proteins as a low oxygen environment is created within the matrix during cellular respiration. RESULTS: Here we describe the adaptation of a modular cloning system, GoldenBraid2.0, for the integration of assembled transcriptional units into two different sites of the yeast genome, yielding a high expression level. We have also generated a toolkit comprising various promoters, terminators and selection markers that facilitate the generation of multigenic constructs and allow the reconstruction of biosynthetic pathways within Saccharomyces cerevisiae. To facilitate the specific expression of recombinant proteins within the mitochondrial matrix, we have also included in the toolkit an array of mitochondrial targeting signals and tested their efficiency at different growth conditions. As a proof of concept, we show here the integration and expression of 14 bacterial nitrogen fixation (nif) genes, some of which are known to require specific metallocluster cofactors that contribute to their stability yet make these proteins highly sensitive to oxygen. For one of these genes, nifU, we show that optimal production of this protein is achieved through the use of the Su9 mitochondrial targeting pre-sequence and glycerol as a carbon source to sustain aerobic respiration. CONCLUSIONS: We present here an adapted GoldenBraid2.0 system for modular cloning, genome integration and expression of recombinant proteins in yeast. We have produced a toolkit that includes inducible and constitutive promoters, mitochondrial targeting signals, terminators and selection markers to guarantee versatility in the design of recombinant transcriptional units. By testing the efficiency of the system with nitrogenase Nif proteins and different mitochondrial targeting pre-sequences and growth conditions, we have paved the way for future studies addressing the expression of heterologous proteins in yeast mitochondria.
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Clonación Molecular/métodos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Mitocondrias/genética , Proteínas Mitocondriales/genética , Plásmidos/genética , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Biología SintéticaRESUMEN
Background. Bloodstream infections (BSIs) are associated with considerable morbidity and mortality among inpatients. The aim of this study was to evaluate the impact of a stewardship program on clinical and antimicrobial therapy-related outcomes in patients with bacteraemia. Methods. Single-centre, before-and-after quasi-experimental study in adult inpatients. Over 1 January 2013 to 31 June 2013 all patients aged 18 years or older with a bacteraemia (interven-tion group, N=200) were compared to a historical cohort (1 Janu-ary 2012 to 31 December 2012) (control group, N=200). Results. Following blood culture results and adjusting for potential confounders, the stewardship program was associated with more changes to antibiotic regimens (adjusted odds ratio [ORa]: 4.6, 95% CI 2.9, 7.4), more adjustments to antimicrobial therapy (ORa: 2.4, 95% CI 1.5, 3.8), and better source control in the first five days (ORa 1.6, 95% CI: 1.0, 2.7). In the subgroup that initially received inappropriate empiric treatment (n=138), the intervention was associated with more antibiotic changes (OR: 3.9, 95% CI: 1.8, 8.5) and a better choice of definitive antimicrobial therapy (OR 2.3 95% CI: 1.2, 4.6). There were also more antibiotic changes in the subgroups with both Gram-negative (OR: 2.8, 95% CI: 1.6, 4.9; n=217) and Gram-positive (OR: 4.6, 95% CI: 1.8, 9.9; n=135) bacteraemia among those receiving the intervention, while the Gram-positive subgroup also received more appropriate definitive antimicrobial therapy (OR: 3.9, 95% CI: 1.8, 8.8). Conclusion. The stewardship program improved treatment of patients with bacteraemia and appropriateness of therapy (AU)
Introducción. Las bacteriemias están asociadas con una elevada morbilidad y mortalidad en pacientes hospitalizados. El objetivo del estudio fue evaluar el impacto de un programa de intervención clínica y de terapia antimicrobiana en pacientes con bacteriemia. Material. Estudio en un centro tipo cuasi-experimental pre y post-intervención en pacientes adultos hospitalizados. Desde 1 enero 2013 a 31 junio 2013, todos los pacientes mayores de 18 años en los que se identificaba una bacteriemia (grupo de intervención) se compararon con una cohorte histórica de bacteriemia (1 enero 2012 a 31 diciembre 2012) (grupo control). Resultados. Se incluyeron 200 pacientes en cada grupo. Después de ajustar por los posibles factores de confusión y tras conocer el resultado de los hemocultivos, el grupo de intervención tuvo más cambios de antibióticos (Odds ratio ajustada [ORa]: 4,6, intervalo de confianza [IC] 95%: 2,9-7,4), mayor adecuación del tratamiento antibiótico (ORa: 2,4, IC 95%: 1,5-3,8) y mayor control de la infección en los primeros cinco días (ORa 1,6, IC 95%: 1,0-2,7). En el subgrupo de pacientes que seguían un tratamiento inadecuado cuando se identificó el microorganismo en el hemocultivo (n =138), la intervención se asoció con un mayor cambio de antibiótico (OR: 3,9, IC 95%: 1,8-8,5) y una mejor elección final del antibiótico (OR: 2.3; IC 95%: 1.2-4.6). En el subgrupo de bacteriemia por gramnegativos (n=217), el programa de intervención en bacteriemia se asoció con un mayor cambio de antibiótico (OR: 2,8; IC 95%: 1,6-4,9) y en el subgrupo de bacteriemia por microorganismos grampositivos (n=135), el programa de intervención indujo un mayor cambio en el uso de antibióticos (OR: 4,6, IC 95%: 1,8-9,9) y una mejor elección final del tratamiento (OR: 3,9; 95% CI: 1,8-8,8). Conclusión. El programa de intervención en bacteriemia mejoró el tratamiento de los pacientes con bacteriemia y la adecuación del mismo (AU)
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Humanos , Evaluación de Resultados de Intervenciones Terapéuticas/métodos , Bacteriemia/tratamiento farmacológico , Antiinfecciosos/uso terapéutico , Hospitalización/estadística & datos numéricos , Indicadores de Morbimortalidad , Oportunidad Relativa , Bacterias Aerobias Gramnegativas , Bacterias Gramnegativas , Infecciones por Bacterias Gramnegativas/tratamiento farmacológicoRESUMEN
BACKGROUND & AIMS: Patients with decompensated cirrhosis show a marked innate immune response that shows a wide variability. The reasons for this fact have not been previously evaluated. This investigation was undertaken to study factors influencing the immune response intensity in both serum and ascitic fluid in patients with cirrhosis and ascites with presence of bactDNA. METHODS: 77 patients with cirrhosis and presence of bactDNA fragments in blood and ascitic fluid were included. Identification of bactDNA was evaluated by 16SrRNA gene PCR followed by nucleotide sequencing and by species-specific PCR. Concentration of amplified bacterial-DNA, bacteria identification, LPS, TNF-alpha, IFN-gamma, Interleukin 12 and nitric oxide in serum and ascitic fluid were evaluated as factors related to intensity of the immune response. RESULTS: Serum and AF levels of bactDNA, TNF-α, IFN-γ and nitric oxide concentration were higher in patients with presence of bactDNA from gram negative bacteria. Serum TNF-α levels showed a significant correlation with concentrations of bactDNA (r = 0.88; P = 0.001) and LPS (r = 0.28; P = 0.016). Serum nitric oxide levels were also significantly correlated with concentrations of bactDNA (r = 0.761; P = 0.001) but not with LPS levels. Levels of INF-γ and IL-12 were not significantly correlated with either bactDNA nor LPS levels. Plasmatic concentration of bactDNA was the most accurately correlated factor with the inflammatory response (ancova model included only levels of bactDNA (r(2) = 0.87, P = 0.047 for TNF-α; r(2) = 0.45, P = 0.03 for NOx). CONCLUSIONS: Bacterial-DNA concentration is the most influencing variable associated with serum TNF-α and nitric oxide response.
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Traslocación Bacteriana , ADN Bacteriano/sangre , Cirrosis Hepática/complicaciones , Cirrosis Hepática/inmunología , Óxido Nítrico/sangre , Factor de Necrosis Tumoral alfa/sangre , Anciano , Ascitis/inmunología , Ascitis/microbiología , Líquido Ascítico/microbiología , Femenino , Humanos , Inmunidad Innata , Interferón gamma/sangre , Interleucina-12/sangre , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Estudios Prospectivos , EspañaRESUMEN
Abscisic acid (ABA) is fundamental for plant development. Multiple factors have been identified that participate in the ABA signaling network, although a role of many proteins still await to be demonstrated. Here we have investigated the role of GEM (GL2 EXPRESSION MODULATOR), originally annotated as an ABA-responsive protein. GEM contains a GRAM domain, a feature shared with other eight Arabidopsis proteins for which we propose the name of GRE (GEM-RELATED) proteins. We found that (i) GEM expression responds to ABA, (ii) its promoter contains ABRE sites required for ABA response, and (iii) GEM expression depends on members of the ABA signaling pathway. This is consistent with the expression pattern of GEM during development in plant locations were ABA is known to play a direct role. We also found that GEM binds various phospholipids, e.g. mono and diphosphates and phosphatidic acid, suggesting a potential link of GEM with membrane-associated processes. Consistent with this, we found that the phosphoinositol-4-phosphate kinase PIP5K9 binds GEM in vivo. Finally, we demonstrated a role of GEM in seed dormancy. Together, our data led us to propose that GEM is an ABA-responsive protein that may function downstream of ABI5 as part of the ABA signaling pathway.
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Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Transducción de Señal , Proteínas de Arabidopsis/genética , Sitios de Unión , Regulación de la Expresión Génica de las Plantas , Péptidos y Proteínas de Señalización Intracelular/genética , Fosfolípidos/metabolismo , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
OBJECTIVE: Measure Yourself Concerns and Wellbeing (MYCaW) is a patient-centered questionnaire that allows cancer patients to identify and quantify the severity of their "concerns" and "wellbeing," as opposed to using a predetermined list. MYCaW administration is brief and aids in prioritizing treatment approaches. Our goal was to assess the convergent validity and responsiveness of MYCaW scores over time, the generalizability of the existing qualitative coding framework in different complementary and integrative oncology settings and content validity. METHODS: Baseline and 6-week follow-up data (n = 82) from MYCaW and FACIT-SpEx questionnaires were collected for a service evaluation of the Living Well with the Impact of Cancer course at Penny Brohn Cance Care. MYCaW convergent validity was determined using Spearman's rank correlation test, and responsiveness indices assessed score changes over time. The existing qualitative coding framework was reviewed using a new data set (n = 158) and coverage of concern categories compared with items of existing outcome measures. RESULTS: Good correlation between MYCaW and FACIT-SpEx score changes were achieved (r = -0.57, P ≥ .01). MYCaW Profile and Concern scores were highly responsive to change: standardized response mean = 1.02 and 1.08; effect size = 1.26 and 1.22. MYCaW change scores showed the anticipated gradient of change according to clinically relevant degrees of change. Categories, including "spirituality," "weight change," and "practical concerns" were added to the coding framework to improve generalizability. CONCLUSIONS: MYCaW scores were highly responsive to change, allowing personalized patient outcomes to be quantified; the qualitative coding framework appears generalizable across different integrative oncology settings and has broader coverage of patient-identified concerns compared with existing cancer-related patient-reported outcome measures.
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Evaluación del Resultado de la Atención al Paciente , Calidad de Vida/psicología , Perfil de Impacto de Enfermedad , Encuestas y Cuestionarios , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/psicología , Adulto JovenRESUMEN
Recently, there has been an increase in the number of patients with Chagas disease outside of areas that are generally considered endemic. The aim of this investigation is to describe the clinical profile of a series of patients with Chagas disease in Alicante, Spain, which is a province located on the coast of the Mediterranean Sea. This study was performed at four general hospitals in Alicante between January 2002 and May 2011. A total of 128 patients from seven countries were diagnosed with Trypanosoma cruzi. The main country of origin of these patients was Bolivia (n5101; 78.9%), and the median of age of these patients was 35 years (range: 072 years). Four (3.3%) patients were children under 14 years of age, and 81 (63.3%) were female. Polymerase chain reaction (PCR) was used to analyze 106 patients, 66.0% of whom demonstrated positive PCR results. Visceral involvement was diagnosed in 26.8%: 24.1% demonstrated cardiac involvement, 0.9% demonstrated gastrointestinal involvement, 0.9% demonstrated cardiac and gastrointestinal involvement, and 0.9% demonstrated involvement of the central nervous system. Syncope was found to be associated with cardiomyopathy (28.0% versus 5.2%) (odds ratio: 6.5; 95% confidence interval: 1.527.1). Seventy-six patients received treatment with benznidazole, of whom 57 (75.0%) completed the treatment course without significant adverse events and 17.1% discontinued benznidazole due to adverse events. In total, 50% of patients experienced documented adverse reactions. Among patients with positive PCR results before treatment, all demonstrated negative PCR results following treatment. In conclusion, majority of our patients were female Bolivians immigrants, one of four of our patients demonstrated cardiac involvement, and treatment tolerance was poor. It is important to improve the clinical and epidemiological knowledge of Chagas disease in nonendemic with additional multicenter studies in order to determine the magnitude of this problem and provide improved public health and health resource planning.
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Enfermedad de Chagas/epidemiología , Enfermedad de Chagas/patología , Trypanosoma cruzi/aislamiento & purificación , Adolescente , Adulto , Anciano , Antiprotozoarios/uso terapéutico , Bolivia , Enfermedad de Chagas/complicaciones , Enfermedad de Chagas/tratamiento farmacológico , Niño , Preescolar , Emigración e Inmigración , Femenino , Hospitales Generales , Humanos , Lactante , Masculino , Persona de Mediana Edad , Nitroimidazoles/uso terapéutico , Embarazo , España/epidemiología , Resultado del Tratamiento , Adulto JovenRESUMEN
In eukaryotic cells, environmental and developmental signals alter chromatin structure and modulate gene expression. Heterochromatin constitutes the transcriptionally inactive state of the genome and in plants and mammals is generally characterized by DNA methylation and histone modifications such as histone H3 lysine 9 (H3K9) methylation. In Arabidopsis thaliana, DNA methylation and H3K9 methylation are usually colocated and set up a mutually self-reinforcing and stable state. Here, in contrast, we found that SUVR5, a plant Su(var)3-9 homolog with a SET histone methyltransferase domain, mediates H3K9me2 deposition and regulates gene expression in a DNA methylation-independent manner. SUVR5 binds DNA through its zinc fingers and represses the expression of a subset of stimulus response genes. This represents a novel mechanism for plants to regulate their chromatin and transcriptional state, which may allow for the adaptability and modulation necessary to rapidly respond to extracellular cues.