Analysis of COP1/SPA signalling events in plant developmental responses

Plants are able to perceive the light cues and adjust its development according to the prevalent conditions. An essential core component of the light signaling is the CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) complex, that operates downstream of the photoreceptors to inhibit the light responses. Thus, the COP1/SPA complex is involved in the regulation of a variety of plant developmental processes, such as the tPlants are able to perceive the light cues and adjust its development according to the prevalent conditions. An essential core component of the light signaling is the CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) complex, that operates downstream of the photoreceptors to inhibit the light responses. Thus, the COP1/SPA complex is involved in the regulation of a variety of plant developmental processes, such as the timing of flowering and seedling growth. At the molecular level, COP1/SPA acts as an E3 ubiquitin ligase that promotes the selective degradation of its substrates, which mainly consist of transcription factors, i.e. CONSTANS (CO/BBX1). Since the spanQ and cop1 mutants exhibit severe growth and developmental compromises that cannot be explained by the targets described so far, this study aims to identify and characterize a novel COP1/SPA substrate. In addition, COP1 and SPAs have been shown to act in concert to regulate post-embryonic development. However, the degree at which COP1 and SPAs act independently of each other was not addressed so far. Furthermore, it was unknown whether COP1/SPA plays a role in the regulation of embryogenesis. These novel aspects of the COP1/SPAs co-action are addressed in part II of this thesis. Previously, a screening for interactors of COP1/SPA in vitro suggested that several B-Box transcription factors (BBX), that share similarity with CO, interact with COP1 and SPA1. This study confirms that several BBX proteins bind to COP1/SPA in planta, and focuses on the phenotypical and molecular characterization of CONSTANS-LIKE 12 (COL12/ BBX10). A domain mapping analysis indicated that the CO, CO-Like, TOC1 (CCT) domain of COL12 is important for binding to COP1 whereas the B-boxes are important for binding to SPA1, indicating that COP1 and SPAs might reinforce substrate binding. In agreement with the protein interactions, the COL12 protein undergoes proteolysis at the 26S proteasome in the darkness in a COP1-dependent manner. Both monochromatic red and blue light enhance COL12 protein stability, which contrasts with the destabilizing effects of red light on CO. Therefore, we conclude that COL12 is a novel COP1/SPA substrate. The phenotypical analysis reveals that overexpression of COL12 delays the plant flowering time and enhances branching. Importantly, COL12 repression of flowering is photoperiod-dependent indicating that the protein takes part in the photoperiodic pathway that controls flowering. At the center of this pathway, the CO-FT accumulation is restricted to the LD. The visible light has a prominent role as a positive regulator of CO protein levels. Here, biochemical analyses reveal that COL12 physically interacts with the master regulator of flowering CO, possibly antagonizing its function. In addition, FT mRNA accumulation is reduced in COL12 ox plants compared to WT. Altogether our experiments suggest that COL12 might constitute a fine-tuning mechanism thereby the visible light can enhance CO function by stabilization of the COL12 repressor. In the second part of this thesis, we isolated the quintuple cop1-5 spaQn mutant seedlings, demonstrating that COP1/SPA do not play a role in embryogenesis. We also isolated the cop1-4 spaQn mutant that is completely devoid in the conserved WD-repeats. These quintuple mutant plants resemble the null cop1-5 mutant. This result indicates that the WD- repeat domains provided by the SPA proteins can partially substitute for the lack of the COP1 WD-repeat domain in the COP1-4 protein. Therefore, the WD-repeats are essential for the signaling activity of COP1/SPA. Timing of flowering and seedling growth. At the molecular level, COP1/SPA acts as an E3 ubiquitin ligase that promotes the selective degradation of its substrates, which mainly consist of transcription factors, i.e. CONSTANS (CO/BBX1). Since the spanQ and cop1 mutants exhibit severe growth and developmental compromises that cannot be explained by the targets described so far, this study aims to identify and characterize a novel COP1/SPA substrate. In addition, COP1 and SPAs have been shown to act in concert to regulate post-embryonic development. However, the degree at which COP1 and SPAs act independently of each other was not addressed so far. Furthermore, it was unknown whether COP1/SPA plays a role in the regulation of embryogenesis. These novel aspects of the COP1/SPAs co-action are addressed in part II of this thesis. Previously, a screening for interactors of COP1/SPA in vitro suggested that several B-Box transcription factors (BBX), that share similarity with CO, interact with COP1 and SPA1. This study confirms that several BBX proteins bind to COP1/SPA in planta, and focuses on the phenotypical and molecular characterization of CONSTANS-LIKE 12 (COL12/ BBX10). A domain mapping analysis indicated that the CO, CO-Like, TOC1 (CCT) domain of COL12 is important for binding to COP1 whereas the B-boxes are important for binding to SPA1, indicating that COP1 and SPAs might reinforce substrate binding. In agreement with the protein interactions, the COL12 protein undergoes proteolysis at the 26S proteasome in the darkness in a COP1-dependent manner. Both monochromatic red and blue light enhance COL12 protein stability, which contrasts with the destabilizing effects of red light on CO. Therefore, we conclude that COL12 is a novel COP1/SPA substrate. The phenotypical analysis reveals that overexpression of COL12 delays the plant flowering time and enhances branching. Importantly, COL12 repression of flowering is photoperiod-dependent indicating that the protein takes part in the photoperiodic pathway that controls flowering. At the center of this pathway, the CO-FT accumulation is restricted to the LD. The visible light has a prominent role as a positive regulator of CO protein levels. Here, biochemical analyses reveal that COL12 physically interacts with the master regulator of flowering CO, possibly antagonizing its function. In addition, FT mRNA accumulation is reduced in COL12 ox plants compared to WT. Altogether our experiments suggest that COL12 might constitute a fine-tuning mechanism thereby the visible light can enhance CO function by stabilization of the COL12 repressor. In the second part of this thesis, we isolated the quintuple cop1-5 spaQn mutant seedlings, demonstrating that COP1/SPA do not play a role in embryogenesis. We also isolated the cop1-4 spaQn mutant that is completely devoid in the conserved WD-repeats. These quintuple mutant plants resemble the null cop1-5 mutant. This result indicates that the WD- repeat domains provided by the SPA proteins can partially substitute for the lack of the COP1 WD-repeat domain in the COP1-4 protein. Therefore, the WD-repeats are essential for the signaling activity of COP1/SPA

Síndrome de Burnout en enfermeros del Hospital Universitario San José- Popayán, 2015.

El síndrome de Burnout constituye una respuesta al estrés laboral crónico, donde se  distinguen tres dimensiones: agotamiento emocional, despersonalización y realización personal. Los profesionales que tienen interacción directa y constante con otras personas, son los de mayor riesgo; los enfermeros, se consideran uno de los grupos más vulnerables para desarrollar el síndrome. Objetivo: Diagnosticar el Síndrome de Burnout e identificar las variables estadísticamente significativas relacionadas con el desarrollo del síndrome en enfermeros(as) del área clínica y administrativa del Hospital Universitario San José, Popayán. Materiales y Métodos: Estudio de tipo Descriptivo, enfoque cuantitativo, correlacional, de corte transversal, con participación voluntaria de 120 enfermeros(as), se aplicó un cuestionario de datos sociodemográficos y laborales y el Inventario de Maslach Burnout Inventory validado al español. Los datos se procesaron en el programa SPSS versión 23. Para el análisis estadístico se emplearon medidas de tendencia central y  para estimar la correlación entre las variables y las tres subescalas del síndrome. Resultados: La muestra estuvo constituida por 80,8% mujeres, 76,7% tenían vinculación a término indefinido, el 79,2% laboraban en el área asistencial, con una experiencia laboral mayor a 10 años del 52,5%. El 15,8% presentaron nivel alto en la subescala de Agotamiento Emocional, el 10,8% alta Despersonalización y el 9,2% nivel bajo en Realización Personal; la prevalencia del síndrome de Burnout fue del 1,7%. Conclusión: La presencia del síndrome de Burnout, en enfermeros que laboran en el Hospital Universitario San José, Popayán fue baja, el 65,8% presentaron alto nivel en realización personal.

Síndrome de Burnout en enfermeros del Hospital Universitario San José- Popayán, 2015.

El síndrome de Burnout constituye una respuesta al estrés laboral crónico, donde se  distinguen tres dimensiones: agotamiento emocional, despersonalización y realización personal. Los profesionales que tienen interacción directa y constante con otras personas, son los de mayor riesgo; los enfermeros, se consideran uno de los grupos más vulnerables para desarrollar el síndrome. Objetivo: Diagnosticar el Síndrome de Burnout e identificar las variables estadísticamente significativas relacionadas con el desarrollo del síndrome en enfermeros(as) del área clínica y administrativa del Hospital Universitario San José, Popayán. Materiales y Métodos: Estudio de tipo Descriptivo, enfoque cuantitativo, correlacional, de corte transversal, con participación voluntaria de 120 enfermeros(as), se aplicó un cuestionario de datos sociodemográficos y laborales y el Inventario de Maslach Burnout Inventory validado al español. Los datos se procesaron en el programa SPSS versión 23. Para el análisis estadístico se emplearon medidas de tendencia central y  para estimar la correlación entre las variables y las tres subescalas del síndrome. Resultados: La muestra estuvo constituida por 80,8% mujeres, 76,7% tenían vinculación a término indefinido, el 79,2% laboraban en el área asistencial, con una experiencia laboral mayor a 10 años del 52,5%. El 15,8% presentaron nivel alto en la subescala de Agotamiento Emocional, el 10,8% alta Despersonalización y el 9,2% nivel bajo en Realización Personal; la prevalencia del síndrome de Burnout fue del 1,7%. Conclusión: La presencia del síndrome de Burnout, en enfermeros que laboran en el Hospital Universitario San José, Popayán fue baja, el 65,8% presentaron alto nivel en realización personal.

Síndrome de Burnout en enfermeros del Hospital Universitario San José- Popayán, 2015.

El síndrome de Burnout constituye una respuesta al estrés laboral crónico, donde se  distinguen tres dimensiones: agotamiento emocional, despersonalización y realización personal. Los profesionales que tienen interacción directa y constante con otras personas, son los de mayor riesgo; los enfermeros, se consideran uno de los grupos más vulnerables para desarrollar el síndrome. Objetivo: Diagnosticar el Síndrome de Burnout e identificar las variables estadísticamente significativas relacionadas con el desarrollo del síndrome en enfermeros(as) del área clínica y administrativa del Hospital Universitario San José, Popayán. Materiales y Métodos: Estudio de tipo Descriptivo, enfoque cuantitativo, correlacional, de corte transversal, con participación voluntaria de 120 enfermeros(as), se aplicó un cuestionario de datos sociodemográficos y laborales y el Inventario de Maslach Burnout Inventory validado al español. Los datos se procesaron en el programa SPSS versión 23. Para el análisis estadístico se emplearon medidas de tendencia central y  para estimar la correlación entre las variables y las tres subescalas del síndrome. Resultados: La muestra estuvo constituida por 80,8% mujeres, 76,7% tenían vinculación a término indefinido, el 79,2% laboraban en el área asistencial, con una experiencia laboral mayor a 10 años del 52,5%. El 15,8% presentaron nivel alto en la subescala de Agotamiento Emocional, el 10,8% alta Despersonalización y el 9,2% nivel bajo en Realización Personal; la prevalencia del síndrome de Burnout fue del 1,7%. Conclusión: La presencia del síndrome de Burnout, en enfermeros que laboran en el Hospital Universitario San José, Popayán fue baja, el 65,8% presentaron alto nivel en realización personal.

ADAMTS19-associated heart valve defects: Novel genetic variants consolidating a recognizable cardiac phenotype

Recently, ADAMTS19 was identified as a novel causative gene for autosomal recessive heart valve disease (HVD), affecting mainly the aortic and pulmonary valves. Exome sequencing and data repository (CentoMD) analyses were performed to identify patients with ADAMTS19 variants (two families). A third family was recognized based on cardiac phenotypic similarities and SNP array homozygosity. Three novel loss of function (LoF) variants were identified in six patients from three families. Clinically, all patients presented anomalies of the aortic/pulmonary valves, which included thickening of valve leaflets, stenosis and insufficiency. Three patients had (recurrent) subaortic membrane, suggesting that ADAMTS19 is the first gene identified related to discrete subaortic stenosis. One case presented a bi-commissural pulmonary valve. All patients displayed some degree of atrioventricular valve insufficiency. Other cardiac anomalies included atrial/ventricular septal defects, persistent ductus arteriosus, and mild dilated ascending aorta. Our findings confirm that biallelic LoF variants in ADAMTS19 are causative of a specific and recognizable cardiac phenotype. We recommend considering ADAMTS19 genetic testing in all patients with multiple semilunar valve abnormalities, particularly in the presence of subaortic membrane. ADAMTS19 screening in patients with semilunar valve abnormalities is needed to estimate the frequency of the HVD related phenotype, which might be not so rare

Characterization of the clinical and immunologic phenotype and management of 157 individuals with 56 distinct heterozygous NFKB1 mutations

Background: An increasing number of NFKB1 variants are being identified in patients with heterogeneous immunologic phenotypes. Objective: To characterize the clinical and cellular phenotype as well as the management of patients with heterozygous NFKB1 mutations. Methods: In a worldwide collaborative effort, we evaluated 231 individuals harboring 105 distinct heterozygous NFKB1 variants. To provide evidence for pathogenicity, each variant was assessed in silico; in addition, 32 variants were assessed by functional in vitro testing of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-kappa B) signaling. Results: We classified 56 of the 105 distinct NFKB1 variants in 157 individuals from 68 unrelated families as pathogenic. Incomplete clinical penetrance (70%) and age-dependent severity of NFKB1-related phenotypes were observed. The phenotype included hypogammaglobulinemia (88.9%), reduced switched memory B cells (60.3%), and respiratory (83%) and gastrointestinal (28.6%) infections, thus characterizing the disorder as primary immunodeficiency. However, the high frequency of autoimmunity (57.4%), lymphoproliferation (52.4%), noninfectious enteropathy (23.1%), opportunistic infections (15.7%), autoinflammation (29.6%), and malignancy (16.8%) identified NF-kappa B1-related disease as an inborn error of immunity with immune dysregulation, rather than a mere primary immunodeficiency. Current treatment includes immunoglobulin replacement and immunosuppressive agents. Conclusions: We present a comprehensive clinical overview of the NF-kappa B1-related phenotype, which includes immunodeficiency, autoimmunity, autoinflammation, and cancer. Because of its multisystem involvement, clinicians from each and every medical discipline need to be made aware of this autosomal-dominant disease. Hematopoietic stem cell transplantation and NF-kappa B1 pathway-targeted therapeutic strategies should be considered in the future.Peer reviewe

The Global Alliance for Infections in Surgery : defining a model for antimicrobial stewardship-results from an international cross-sectional survey

Background: Antimicrobial Stewardship Programs (ASPs) have been promoted to optimize antimicrobial usage and patient outcomes, and to reduce the emergence of antimicrobial-resistant organisms. However, the best strategies for an ASP are not definitively established and are likely to vary based on local culture, policy, and routine clinical practice, and probably limited resources in middle-income countries. The aim of this study is to evaluate structures and resources of antimicrobial stewardship teams (ASTs) in surgical departments from different regions of the world. Methods: A cross-sectional web-based survey was conducted in 2016 on 173 physicians who participated in the AGORA (Antimicrobials: A Global Alliance for Optimizing their Rational Use in Intra-Abdominal Infections) project and on 658 international experts in the fields of ASPs, infection control, and infections in surgery. Results: The response rate was 19.4%. One hundred fifty-six (98.7%) participants stated their hospital had a multidisciplinary AST. The median number of physicians working inside the team was five [interquartile range 4-6]. An infectious disease specialist, a microbiologist and an infection control specialist were, respectively, present in 80.1, 76.3, and 67.9% of the ASTs. A surgeon was a component in 59.0% of cases and was significantly more likely to be present in university hospitals (89.5%, p <0.05) compared to community teaching (83.3%) and community hospitals (66.7%). Protocols for pre-operative prophylaxis and for antimicrobial treatment of surgical infections were respectively implemented in 96.2 and 82.3% of the hospitals. The majority of the surgical departments implemented both persuasive and restrictive interventions (72.8%). The most common types of interventions in surgical departments were dissemination of educational materials (62.5%), expert approval (61.0%), audit and feedback (55.1%), educational outreach (53.7%), and compulsory order forms (51.5%). Conclusion: The survey showed a heterogeneous organization of ASPs worldwide, demonstrating the necessity of a multidisciplinary and collaborative approach in the battle against antimicrobial resistance in surgical infections, and the importance of educational efforts towards this goal.Peer reviewe

Impact of hospital characteristics on implementation of a Pediatric Early Warning System in resource-limited cancer hospitals

BackgroundPediatric Early Warning Systems (PEWS) aid in identification of deterioration in hospitalized children with cancer but are underutilized in resource-limited settings. Proyecto EVAT is a multicenter quality improvement (QI) collaborative in Latin America to implement PEWS. This study investigates the relationship between hospital characteristics and time required for PEWS implementation.MethodsThis convergent mixed-methods study included 23 Proyecto EVAT childhood cancer centers; 5 hospitals representing quick and slow implementers were selected for qualitative analysis. Semi-structured interviews were conducted with 71 stakeholders involved in PEWS implementation. Interviews were recorded, transcribed and translated to English, then coded using a priori and novel codes. Thematic content analysis explored the impact of hospital characteristics and QI experience on time required for PEWS implementation and was supplemented by quantitative analysis exploring the relationship between hospital characteristics and implementation time.ResultsIn both quantitative and qualitative analysis, material and human resources to support PEWS significantly impacted time to implementation. Lack of resources produced various obstacles that extended time necessary for centers to achieve successful implementation. Hospital characteristics, such as funding structure and type, influenced PEWS implementation time by determining their resource-availability. Prior hospital or implementation leader experience with QI, however, helped facilitate implementation by assisting implementers predict and overcome resource-related challenges.ConclusionsHospital characteristics impact time required to implement PEWS in resource-limited childhood cancer centers; however, prior QI experience helps anticipate and adapt to resource challenges and more quickly implement PEWS. QI training should be a component of strategies to scale-up use of evidence-based interventions like PEWS in resource-limited settings