RESUMEN
BACKGROUND: Children and adolescents recovering from burn injury are at heightened risk of psychosocial problems. An integrative form of psychosocial intervention is burn camp. However, evidence about burn camp effectiveness is equivocal. OBJECTIVES: This study examined the role of therapeutic camp experiences in the recovery journeys of children and adolescents who had experienced burn injury and been treated in a tertiary pediatric hospital in Brisbane, Australia. METHODS: Retrospective semi-structured interviews were conducted with youths and parents. Inductive reflexive thematic analysis was used with pooled interview data. RESULTS: The participants were eight youths who attended at least one burns camp (between 2009 and 2019) and 15 parents of youth campers. An overwhelming majority (96%) reported a positive experience of camp, that they would return, and that they recommended the camp to other youth with burns. The four strengths of the camp experience were fun, adventurous activities; social relatedness (friendships, socializing); camp setting and experience; and acceptance. The four impacts of the camp on youth campers were normalizing ("I'm not the only one", shared experience); social support (making new friendships, social confidence, mentoring others); psychological recovery (happier, mentally stronger, more resilient, independence building); and confidence (increased self-confidence, increased social confidence, leadership development). CONCLUSIONS: Although this is the first known research about burn camp in Australia, the findings are similar to a handful of other qualitative studies about burn camp experiences and impacts. Recommendations include future research on aspects of camp experiences that contribute to targeted outcomes, the role of staff and previous camp participants as mentors, and comparisons with other psychosocial interventions for youth burn survivors.
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Quemaduras , Acampada , Adolescente , Quemaduras/psicología , Quemaduras/terapia , Acampada/psicología , Niño , Estudios de Seguimiento , Humanos , Padres , Investigación Cualitativa , Estudios Retrospectivos , AutoimagenRESUMEN
The Target of Rapamycin kinase Complex I (TORC1) is the master regulator of cell growth and metabolism in eukaryotes. In the presence of pro-growth hormones and abundant nutrients, TORC1 is active and drives protein, lipid, and nucleotide synthesis by phosphorylating a wide range of proteins. In contrast, when nitrogen and/or glucose levels fall, TORC1 is inhibited, causing the cell to switch from anabolic to catabolic metabolism, and eventually enter a quiescent state. In the budding yeast Saccharomyces cerevisiae, TORC1 inhibition triggers the movement of TORC1 from its position around the vacuole to a single focus/body on the edge of the vacuolar membrane. This relocalization depends on the activity of numerous key TORC1 regulators and thus analysis of TORC1 localization can be used to follow signaling through the TORC1 pathway. Here we provide a detailed protocol for measuring TORC1 (specifically, Kog1-YFP) relocalization/signaling using fluorescence microscopy. Emphasis is placed on procedures that ensure: (1) TORC1-bodies are identified (and counted) correctly despite their relatively low fluorescence and the accumulation of autofluorescent foci during glucose and nitrogen starvation; (2) Cells are kept in log-phase growth at the start of each experiment so that the dynamics of TORC1-body formation are monitored correctly; (3) The appropriate fluorescent tags are used to avoid examining mislocalized TORC1.
RESUMEN
The target of rapamycin kinase complex 1 (TORC1) regulates cell growth and metabolism in eukaryotes. In Saccharomyces cerevisiae, TORC1 activity is known to be controlled by the conserved GTPases, Gtr1/2, and movement into and out of an inactive agglomerate/body. However, it is unclear whether/how these regulatory steps are coupled. Here we show that active Gtr1/2 is a potent inhibitor of TORC1-body formation, but cells missing Gtr1/2 still form TORC1-bodies in a glucose/nitrogen starvation-dependent manner. We also identify 13 new activators of TORC1-body formation and show that seven of these proteins regulate the Gtr1/2-dependent repression of TORC1-body formation, while the remaining proteins drive the subsequent steps in TORC1 agglomeration. Finally, we show that the conserved phosphatidylinositol-3-phosphate (PI(3)P) binding protein, Pib2, forms a complex with TORC1 and overrides the Gtr1/2-dependent repression of TORC1-body formation during starvation. These data provide a unified, systems-level model of TORC1 regulation in yeast.
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Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Saccharomycetales/metabolismo , Modelos Biológicos , Dominios Proteicos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The Target of Rapamycin kinase Complex I (TORC1) is a master regulator of cell growth and metabolism in eukaryotes. Studies in yeast and human cells have shown that nitrogen/amino acid starvation signals act through Npr2/Npr3 and the small GTPases Gtr1/Gtr2 (Rags in humans) to inhibit TORC1. However, it is unclear how other stress and starvation stimuli inhibit TORC1, and/or act in parallel with the TORC1 pathway, to control cell growth. To help answer these questions, we developed a novel automated pipeline and used it to measure the expression of a TORC1-dependent ribosome biogenesis gene (NSR1) during osmotic stress in 4700 Saccharomyces cerevisiae strains from the yeast knock-out collection. This led to the identification of 440 strains with significant and reproducible defects in NSR1 repression. The cell growth control and stress response proteins deleted in these strains form a highly connected network, including 56 proteins involved in vesicle trafficking and vacuolar function; 53 proteins that act downstream of TORC1 according to a rapamycin assay--including components of the HDAC Rpd3L, Elongator, and the INO80, CAF-1 and SWI/SNF chromatin remodeling complexes; over 100 proteins involved in signaling and metabolism; and 17 proteins that directly interact with TORC1. These data provide an important resource for labs studying cell growth control and stress signaling, and demonstrate the utility of our new, and easily adaptable, method for mapping gene regulatory networks.
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Genoma Fúngico , Estudio de Asociación del Genoma Completo , Genómica , Complejos Multiproteicos/metabolismo , Presión Osmótica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Análisis por Conglomerados , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Técnicas de Inactivación de Genes , Redes Reguladoras de Genes , Genómica/métodos , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/genética , Mutación , Estrés Fisiológico/genética , Serina-Treonina Quinasas TOR/genéticaRESUMEN
The recent article "Evaluation of pK(a) Estimation Methods on 211 Druglike Compounds" ( Manchester, J.; et al. J. Chem Inf. Model. 2010, 50, 565-571 ) reports poor results for the program Epik. Here, we highlight likely sources for the poor performance and describe work done to improve the performance. Running Epik in the mode intended to calculate pK(a) values for sequentially adding/removing protons, as needed to reproduce the experimental conditions, improves the root mean squared error (RMSE) from 3.0 to 2.18 for the 85 public compounds available from the paper. Despite this improvement, there are still other programs in the Manchester paper that outperform Epik. The primary reason is that the public portion of the data set is not diverse and Epik is missing a few key functional groups in this data set that are heavily represented. We show that incorporation of these missing functional groups into the Epik training set improves the RMSE for the public compounds to 1.04. Furthermore, these enhancements help improve the overall performance of Epik on a large druglike test set.
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Fenómenos Químicos , Informática/métodos , Preparaciones Farmacéuticas/química , Bases de Datos Factuales , Reproducibilidad de los ResultadosRESUMEN
Generating the appropriate protonation states of drug-like molecules in solution is important for success in both ligand- and structure-based virtual screening. Screening collections of millions of compounds requires a method for determining tautomers and their energies that is sufficiently rapid, accurate, and comprehensive. To maximise enrichment, the lowest energy tautomers must be determined from heterogeneous input, without over-enumerating unfavourable states. While computationally expensive, the density functional theory (DFT) method M06-2X/aug-cc-pVTZ(-f) [PB-SCRF] provides accurate energies for enumerated model tautomeric systems. The empirical Hammett-Taft methodology can very rapidly extrapolate substituent effects from model systems to drug-like molecules via the relationship between pK(T) and pK(a). Combining the two complementary approaches transforms the tautomer problem from a scientific challenge to one of engineering scale-up, and avoids issues that arise due to the very limited number of measured pK(T) values, especially for the complicated heterocycles often favoured by medicinal chemists for their novelty and versatility. Several hundreds of pre-calculated tautomer energies and substituent pK(a) effects are tabulated in databases for use in structural adjustment by the program Epik, which treats tautomers as a subset of the larger problem of the protonation states in aqueous ensembles and their energy penalties. Accuracy and coverage is continually improved and expanded by parameterizing new systems of interest using DFT and experimental data. Recommendations are made for how to best incorporate tautomers in molecular design and virtual screening workflows.