Frailty syndrome and end-stage kidney disease outcomes at a Latin American dialysis center

Introduction. Frailty syndrome generates a high risk of adverse outcomes and mortality, and its prevalence is elevated in patients with end-stage kidney disease. Few studies have reported the prevalence and outcomes of frailty in populations from less developed countries. Objective. To identify the clinical outcomes and factors associated with the frailty syndrome in patients with stage five chronic kidney disease who started renal replacement therapy - both hemodialysis and peritoneal dialysis- in a dialysis center in Bucaramanga, Colombia. Materials and methods. This was a prospective study of patients with end-stage kidney disease who initiated dialysis at a center in Colombia and had a twelve-month follow-up. Results. The overall frailty prevalence was 50.47% and two out of three patients older than 65 years had the syndrome. We found significantly higher followup mortality among patients with frailty: odds ratio of 2.95 (CI: 1.07- 8.13; p=0.036) in unadjusted analysis. Conclusions. Literature shows that compared to developed nations, Latin American adults are facing a higher prevalence of chronic diseases, and frailty syndrome is increasing. In this study, according to the FRAIL scale, having a frailty syndrome predicts a higher mortality; hypoalbuminemia and low creatinine levels at the beginning of dialysis could act as predictors of its diagnosis.

Introducción. El síndrome de fragilidad implica un alto riesgo de desenlaces adversos y mortalidad, y tiene una prevalencia elevada en pacientes con enfermedad renal en etapa terminal. Hay pocos estudios que investiguen la prevalencia y los desenlaces de este síndrome de fragilidad en las poblaciones de los países en desarrollo. Objetivo. Identificar los desenlaces clínicos y los factores asociados al síndrome de fragilidad en los pacientes con enfermedad renal crónica en estadio cinco que inician terapia de reemplazo renal -con hemodiálisis o diálisis peritoneal- en un centro de diálisis de Bucaramanga, Colombia. Materiales y métodos. Se trató de un estudio prospectivo de pacientes con enfermedad renal en etapa terminal que iniciaron diálisis en un centro de Colombia y a quienes se les hizo seguimiento durante doce meses. Resultados. La prevalencia global del síndrome de fragilidad fue del 50,47 % y dos de cada tres pacientes mayores de 65 años lo presentaban. Se encontró una mortalidad significativamente mayor entre los pacientes con síndrome de fragilidad: razón de probabilidad de 2,95 (IC:1,07-8,13; p=0,036) en el análisis no ajustado. Conclusiones. La literatura muestra que, en comparación con los países desarrollados, los adultos latinoamericanos presentan una mayor prevalencia de enfermedades crónicas y un aumento progresivo del síndrome de fragilidad. En este estudio, la fragilidad -según la escala FRAIL- predijo una mayor mortalidad. Además, la hipoalbuminemia y los niveles bajos de creatinina al inicio de la diálisis podrían actuar como elementos predictores de su diagnóstico.

Frailty syndrome and end-stage kidney disease outcomes at a Latin American dialysis center. / Desenlaces de enfermedad renal crónica y síndrome de fragilidad en un centro de diálisis de Latinoamérica

INTRODUCTION: Frailty syndrome generates a high risk of adverse outcomes and mortality, and its prevalence is elevated in patients with end-stage kidney disease. Few studies have reported the prevalence and outcomes of frailty in populations from less developed countries. OBJECTIVE: To identify the clinical outcomes and factors associated with the frailty syndrome in patients with stage five chronic kidney disease who started renal replacement therapy - both hemodialysis and peritoneal dialysis- in a dialysis center in Bucaramanga, Colombia. MATERIALS AND METHODS: This was a prospective study of patients with end-stage kidney disease who initiated dialysis at a center in Colombia and had a twelve-month follow-up. RESULTS: The overall frailty prevalence was 50.47% and two out of three patients older than 65 years had the syndrome. We found significantly higher followup mortality among patients with frailty: odds ratio of 2.95 (CI: 1.07- 8.13; p=0.036) in unadjusted analysis. CONCLUSIONS: Literature shows that compared to developed nations, Latin American adults are facing a higher prevalence of chronic diseases, and frailty syndrome is raising. In this study, according to the FRAIL scale, having a frailty syndrome predicts a higher mortality; hypoalbuminemia and low creatinine levels at the beginning of dialysis could act as predictors of its diagnosis.

Introduction. Frailty syndrome generates a high risk of adverse outcomes and mortality, and its prevalence is elevated in patients with end-stage kidney disease. Few studies have reported the prevalence and outcomes of frailty in populations from less developed countries.Objective. To identify the clinical outcomes and factors associated with the frailty syndrome in patients with stage five chronic kidney disease who started renal replacement therapy ­both hemodialysis and peritoneal dialysis­ in a dialysis center in Bucaramanga, Colombia.Materials and methods. This was a prospective study of patients with end-stage kidney disease who initiated dialysis at a center in Colombia and had a twelve-month follow-up. Results. The overall frailty prevalence was 50.47% and two out of three patients older than 65 years had the syndrome. We found significantly higher followup mortality among patients with frailty: odds ratio of 2.95 (CI: 1.07- 8.13; p=0.036) in unadjusted analysis.Conclusions. Literature shows that compared to developed nations, Latin American adults are facing a higher prevalence of chronic diseases, and frailty syndrome is increasing. In this study, according to the FRAIL scale, having a frailty syndrome predicts a higher mortality; hypoalbuminemia and low creatinine levels at the beginning of dialysis could act as predictors of its diagnosis.

Single-molecule counting applied to the study of GPCR oligomerization.

Single-molecule counting techniques enable a precise determination of the intracellular abundance and stoichiometry of proteins and macromolecular complexes. These details are often challenging to quantitatively assess yet are essential for our understanding of cellular function. Consider G-protein-coupled receptors-an expansive class of transmembrane signaling proteins that participate in many vital physiological functions making them a popular target for drug development. While early evidence for the role of oligomerization in receptor signaling came from ensemble biochemical and biophysical assays, innovations in single-molecule measurements are now driving a paradigm shift in our understanding of its relevance. Here, we review recent developments in single-molecule counting with a focus on photobleaching step counting and the emerging technique of quantitative single-molecule localization microscopy-with a particular emphasis on the potential for these techniques to advance our understanding of the role of oligomerization in G-protein-coupled receptor signaling.

Estimating the dynamic range of quantitative single-molecule localization microscopy.

In recent years, there have been significant advances in quantifying molecule copy number and protein stoichiometry with single-molecule localization microscopy (SMLM). However, as the density of fluorophores per diffraction-limited spot increases, distinguishing between detection events from different fluorophores becomes progressively more difficult, affecting the accuracy of such measurements. Although essential to the design of quantitative experiments, the dynamic range of SMLM counting techniques has not yet been studied in detail. Here, we provide a working definition of the dynamic range for quantitative SMLM in terms of the relative number of missed localizations or blinks and explore the photophysical and experimental parameters that affect it. We begin with a simple two-state model of blinking fluorophores, then extend the model to incorporate photobleaching and temporal binning by the detection camera. From these models, we first show that our estimates of the dynamic range agree with realistic simulations of the photoswitching. We find that the dynamic range scales inversely with the duty cycle when counting both blinks and localizations. Finally, we validate our theoretical approach on direct stochastic optical reconstruction microscopy (dSTORM) data sets of photoswitching Alexa Fluor 647 dyes. Our results should help guide researchers in designing and implementing SMLM-based molecular counting experiments.

An expectation-maximization approach to quantifying protein stoichiometry with single-molecule imaging.

Motivation: Single-molecule localization microscopy (SMLM) is a super-resolution technique capable of rendering nanometer scale images of cellular structures. Recently, much effort has gone into developing algorithms for extracting quantitative features from SMLM datasets, such as the abundance and stoichiometry of macromolecular complexes. These algorithms often require knowledge of the complicated photophysical properties of photoswitchable fluorophores. Results: Here, we develop a calibration-free approach to quantitative SMLM built upon the observation that most photoswitchable fluorophores emit a geometrically distributed number of blinks before photobleaching. From a statistical model of a mixture of monomers, dimers and trimers, the method employs an adapted expectation-maximization algorithm to learn the protomer fractions while simultaneously determining the single-fluorophore blinking distribution. To illustrate the utility of our approach, we benchmark it on both simulated datasets and experimental datasets assembled from SMLM images of fluorescently labeled DNA nanostructures. Availability and implementation: An implementation of our algorithm written in Python is available at: https://www.utm.utoronto.ca/milsteinlab/resources/Software/MMCode/. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

FOCAL3D: A 3-dimensional clustering package for single-molecule localization microscopy.

Single-molecule localization microscopy (SMLM) is a powerful tool for studying intracellular structure and macromolecular organization at the nanoscale. The increasingly massive pointillistic data sets generated by SMLM require the development of new and highly efficient quantification tools. Here we present FOCAL3D, an accurate, flexible and exceedingly fast (scaling linearly with the number of localizations) density-based algorithm for quantifying spatial clustering in large 3D SMLM data sets. Unlike DBSCAN, which is perhaps the most commonly employed density-based clustering algorithm, an optimum set of parameters for FOCAL3D may be objectively determined. We initially validate the performance of FOCAL3D on simulated datasets at varying noise levels and for a range of cluster sizes. These simulated datasets are used to illustrate the parametric insensitivity of the algorithm, in contrast to DBSCAN, and clustering metrics such as the F1 and Silhouette score indicate that FOCAL3D is highly accurate, even in the presence of significant background noise and mixed populations of variable sized clusters, once optimized. We then apply FOCAL3D to 3D astigmatic dSTORM images of the nuclear pore complex (NPC) in human osteosaracoma cells, illustrating both the validity of the parameter optimization and the ability of the algorithm to accurately cluster complex, heterogeneous 3D clusters in a biological dataset. FOCAL3D is provided as an open source software package written in Python.

Sensitive Detection of Broad-Spectrum Bacteria with Small-Molecule Fluorescent Excimer Chemosensors.

Antibiotic resistance is a major problem for world health, triggered by the unnecessary usage of broad-spectrum antibiotics on purportedly infected patients. Current clinical standards require lengthy protocols for the detection of bacterial species in sterile physiological fluids. In this work, a class of small-molecule fluorescent chemosensors termed ProxyPhos was shown to be capable of rapid, sensitive, and facile detection of broad-spectrum bacteria. The sensors act via a turn-on fluorescent excimer mechanism, where close-proximity binding of multiple sensor units amplifies a red shift emission signal. ProxyPhos sensors were able to detect down to 10 CFUs of model strains by flow cytometry assays and showed selectivity over mammalian cells in a bacterial coculture through fluorescence microscopy. The studies reveal that the zinc(II)-chelates cyclen and cyclam are novel and effective binding units for the detection of both Gram-negative and Gram-positive bacterial strains. Mode of action studies revealed that the chemosensors detect Gram-negative and Gram-positive strains with two distinct mechanisms. Preliminary studies applying ProxyPhos sensors to sterile physiological fluids (cerebrospinal fluid) in flow cytometry assays were successful. The results suggest that ProxyPhos sensors can be developed as a rapid, inexpensive, and robust tool for the "yes-no" detection of broad-spectrum bacteria in sterile fluids.

Eficacia y seguridad del uso de hierro en el tratamiento de la falla cardiaca: revisión sistemática / Efficacy and safety of the use of iron in the treatment of heart failure: A systematic review

Resumen Introducción: La falla cardiaca asociada a deficiencia de hierro se relaciona directamente con disminución de parámetros funcionales y, por ende, con deterioro de la calidad de vida y pobre pronóstico de los pacientes que la padecen. Se ha encontrado que la corrección de la deficiencia de hierro mejora a corto plazo la clase funcional y otros parámetros de la enfermedad. Objetivo: Evaluar la eficacia y la seguridad del uso de hierro en el tratamiento de la falla cardiaca. Métodos: De seleccionaron ensayos clínicos que evaluaran el uso de hierro en administración intravenosa u oral vs. placebo en el contexto de pacientes con falla cardíaca y a su vez se eligieron pacientes que concomitantemente sufrieran anemia ferropénica. En la búsqueda se incluyeron bases de datos como MEDLINE (PubMed), Embase, Cochrane Central Register of Clinical trials (CENTRAL), LILACS y WPRIM. Resultados: De 10.729 títulos obtenidos 6 fueron elegibles con 835 pacientes, de los cuales 520 fueron tratados con terapia férrica y 315 con placebo o terapia convencional de falla cardiaca. Debido a la heterogeneidad de los resultados no fue posible hacer un metaanálisis. Conclusión: Se identificó mejoría significativa en múltiples parámetros evaluados, tales como el test de caminata de 6 minutos, clase funcional de la New York Heart Association, consumo máximo de oxígeno (VO2 máx.) y valoración global subjetiva. Por consiguiente, la terapia férrica es una opción segura y eficaz en el manejo de pacientes con falla cardiaca ya que mejora significativamente su capacidad funcional, calidad de vida y múltiples parámetros paraclínicos.

Abstract Introduction: Heart failure combined with iron deficiency is directly related with the reduction in functional parameters and, as consequence, with a deterioration in the quality of life and poor prognosis in the patients that suffer from it. It has been found that correction of the iron deficiency improves the functional class and other parameters of the disease in the short-term. Objective: To evaluate the efficacy and safety of the use of iron in the treatment of heart failure. Methods: Clinical trials were selected that evaluated the use of administering intravenous or oral iron vs. placebo in the context of patients with cardiac failure and at the same time, patients were selected that concomitantly suffered from iron deficiency anaemia. The search was made in data bases such as MEDLINE (PubMed), Embase, Cochrane Central Register of Clinical trials (CENTRAL), LILACS and WPRIM. Results: Of the 10,729 articles obtained, only 6 were eligible with 835 patients, of which 520 were treated with iron therapy and 315 with a placebo or conventional heart failure therapy. Due to the heterogeneity of the results, it was not possible to perform a meta-analysis. Conclusion: A significant improvement was identified in several of the parameters evaluated, such as the 6-minute walk test, the New York Heart Association functional class, maximum oxygen uptake (VO2 max.), and an overall subjective evaluation. This showed that iron therapy is a safe and effective option in the management of patients with heart failure since there was a significant improvement in their functional capacity, quality of life, and several para-clinical parameters.

Revisión sistemática sobre factores de riesgo asociados a artralgia persistente en el paciente con fiebre del chikunguña / Risk factors associated with persistent arthralgia in chikungunya virus-infected patients - a systematic review

Antecedente: La fiebre del chikunguña es una enfermedad tropical infecciosa, caracterizada por la aparición de fiebre, erupción cutánea y artralgias, cuya incidencia ha aumentado en los últimos años, en Asia y Latinoamérica. Aunque es una enfermedad con baja mortalidad, se ha relacionado con limitación funcional a largo plazo, por su compromiso articular. Objetivo: Llevar a cabo una revisión sistemática de la literatura sobre los factores de riesgo asociados a artralgia persistente en el paciente con fiebre de chikunguña. Metodología: Se buscó información en bases de datos como PubMed, Embase, ScienceDirect, Scopus y Google Académico, con el descriptor de búsqueda (chikungunya fever OR chikungunya) AND (arthralgia). Resultados: Se obtuvieron 4281 estudios de los cuales al final se seleccionaron seis estudios de cohorte por su similitud metodológica en la evaluación de factores asociados y de los que se obtuvieron diferentes resultados en el análisis de datos. Conclusión: No existe información concluyente acerca de factores modificables que permitan predecir la persistencia de artralgias en pacientes infectados por el virus de la fiebre del chikunguña. Hacen falta estudios que permitan evaluar la presencia de dichos factores, fin de disminuir la morbilidad asociada a la artralgia persistente.

Background: Chikungunya fever is an infectious tropical disease characterized by the onset of fever, rash and arthralgia, whose incidence has increased in recent years in Asia and Latin America. Although it is a disease with low mortality, it has been associated with long-term functional limitation, by their joint involvement. Objective: To conduct a systematic review of the literature on risk factors associated with persistent arthralgia in patients with Chikungunya fever (CHKF). Methods: A search for information in databases such as Pubmed, Embase, ScienceDirect, Scopus and Google Scholar was performed, search the descriptor (OR chikungunya fever chikungunya) AND (arthralgia). Results: 4281 studies at the end of which six cohort studies for methodological similarity were selected in the evaluation of associated factors, obtaining different results in the analysis were obtained. Conclusion: There is no conclusive information about modifiable factors that predict persistent joint pain in patients infected CHKV. Studies are needed to assess the presence of these factors to decrease the morbidity associated with persistent arthralgia.

Molecular Counting with Localization Microscopy: A Bayesian Estimate Based on Fluorophore Statistics.

Superresolved localization microscopy has the potential to serve as an accurate, single-cell technique for counting the abundance of intracellular molecules. However, the stochastic blinking of single fluorophores can introduce large uncertainties into the final count. Here we provide a theoretical foundation for applying superresolved localization microscopy to the problem of molecular counting based on the distribution of blinking events from a single fluorophore. We also show that by redundantly tagging single molecules with multiple, blinking fluorophores, the accuracy of the technique can be enhanced by harnessing the central limit theorem. The coefficient of variation then, for the number of molecules M estimated from a given number of blinks B, scales like ∼1/Nl, where Nl is the mean number of labels on a target. As an example, we apply our theory to the challenging problem of quantifying the cell-to-cell variability of plasmid copy number in bacteria.

Tratamiento de la bacteriemia por enterococo resistente a vancomicina con daptomicina versus linezolid: revisión sistemática y metanálisis / Treatment of bacteremia by vancomycin-resistant Enterococcus with daptomycin versus linezolid: asystematic review and meta-analysese / Tratamento da bacteriemia por enterococus resistentea vancomicina com daptomicina versus linezolida: revisão sistemática e metanálise

Introducción: Para el tratamiento de las infecciones por Enterococcus resistente a vancomicina (ERV) se emplean fármacos de segunda línea como daptomicina y linezolid. Objetivo: hacer una revisión sistemática para evaluar el tratamiento de la bacteriemia por ERV, con daptomicina o linezolid. Metodología: búsqueda electrónica en las bases de datos de Pubmed, Embase, Scopus, ScienceDirect, CENTRAL, Lilacs y Google Académico, para identificar estudios anteriores a julio de 2015 que hayan comparado los tratamientos con daptomicina o linezolid de pacientes infectados por ERV. Resultados: se incluyeron 15 estudios de 1307 registros. No hubo diferencias entre daptomicina y linezolid con respecto a la mortalidad a 30 días. Con la daptomicina se logró más tempranamente el control microbiológico (OR: 0,64; IC95 %: 0,45-0,92). No hubo diferencias entre los dos antibióticos en cuanto a la mejoría clínica, la necesidad de admisión a la UCI o la aparición de efectos adversos como trombocitopenia, neutropenia e insuficiencia renal. Conclusiones: no encontramos diferencias entre daptomicina y linezolid en cuanto a la mortalidad de pacientes infectados por ERV, aunque con la daptomicina se logró una cura microbiológica más rápida.

Introduction: Second-line drugs such as linezolid and daptomycin are used for treatment of vancomycin-resistant Enterococcus (VRE) infections. Objective: A systematic review to evaluate treatment of VRE bacteremia with linezolid versus daptomycin. Methods: A search was done in the databases of Pubmed, Embase, Scopus, ScienceDirect, CENTRAL, Lilacs and Google Scholar to identify studies comparing treatment with daptomycin or linezolid of patients infected by VRE up to July 2015. Result: Only 15 studies were included of a total of 1.307 records. There were no differences between daptomycin and linezolid with respect to mortality at 30 days. Microbiological cure was better with daptomycin (OR: 0.64; 95 % CI: 0.45-0.92), whereas there was no difference between the two antibiotics with respect to clinical cure, need to ICU admission, and the occurrence of adverse effects such as thrombocytopenia, neutropenia and renal failure. Conclusions: No significant differences were observed between daptomycin and linezolid in reference to mortality of patients infected with VRE, although daptomycin treatment produced a faster microbiological cure.

Introdução: Para o tratamento das infecções por Enterococcusresistente a vancomicina (ERV) se empregam fármacos de segunda linha como daptomicina e linezolida. Objetivo: fazer uma revisão sistemática para avaliar o tratamento da bacteriemia por ERV, com daptomicina o linezolida. Metodologia: busca eletrônica nas bases de dados de Pubmed, Embase, Scopus, ScienceDirect, CENTRAL, Lilacs e Google Acadêmico, para identificar estudos anteriores a julho de 2015 que foram comparados os tratamentos com daptomicina ou linezolida de pacientes infectados por ERV. Resultados: se incluíram 15 estudos de 1.307 registros. Não houve diferenças entre daptomicina e linezolida com respeito à mortalidade a 30 dias. Com a daptomicina se conseguiu mais precoce o controle microbiológico (OR: 0,64; IC95 %: 0,45-0,92). Não houve diferenças entre os dois antibióticos em quanto à melhoria clínica, a necessidade de admissão à UTI ou a aparição de efeitos adversos como trombocitopenia, neutropenia e insuficiência renal. Conclusões: não encontramos diferenças entre daptomicina e linezolida em quanto à mortalidade de pacientes infectados por ERV, embora com a daptomicina se conseguiu uma cura microbiológica mais rápida.

Caracterización clínica de infecciones de vías urinarias producidas por enterobacterias productoras de betalactamasas de espectro extendido en Duitama (Colombia), durante 2010-2015 / Clinical characterisation of urinary tract infections produced by extended-spectrum beta-lactamase-producing enterobacteria in Duitama (Colombia) from 2010-2015

Objetivo: Caracterizar las infecciones de vías urinarias (IVU) producidas por enterobacterias productoras de betalactamasas de espectro extendido (BLEE) en Duitama (Colombia) durante 2010-2015. Metodología: Se realizó un estudio descriptivo en 2 instituciones prestadoras de salud a partir de los aislamientos de patógenos BLEE asociados a IVU. Se tomaron variables sociodemográficas, comorbilidades, hospitalizaciones por IVU en el último año, agentes aislados, tratamiento empírico y dirigido, y respuesta clínica. Resultados: Se obtuvo un registro de 169 pacientes, con edad promedio de 66,01 ± 19,19; el 55,62% eran mayores de 65 años; el 59,2% eran de género femenino y el 73,6% provenían del área urbana. Las comorbilidades más frecuentes fueron enfermedad pulmonar obstructiva crónica (26%), diabetes (24,9%) y enfermedad renal crónica (16%), con un índice de Charlson de 4,43 ± 2,61. El 61,6% había sido hospitalizado en el último año a causa de IVU. Los agentes aislados más comunes fueron Escherichia coli (94,7%) y Klebsiella spp. (2,4%). Los tratamientos empíricos usados fueron ampicilina/sulbactam (15%), ciprofloxacino (29,6%) y nitrofurantoína (10,7%). Frente al tratamiento dirigido, el 36,7% no recibió ningún escalonamiento, el 32% fue tratado con ertapenem y el 8,9% con piperacilina/tazobactam. La mortalidad fue del 5,9% y la estancia hospitalaria fue en promedio de 7,24 ± 7,43 días. Conclusión: Los datos regionales son similares a los datos mundiales. Frente al tratamiento empírico se debe realizar una revaloración, ya que las guías actuales no recomiendan el uso de ciprofloxacino. También se debe hacer mejor seguimiento a las BLEE, ya que hay fallas en cuanto al tratamiento dirigido en gran porcentaje de las cepas.

Objective: To characterise epidemiologically urinary tract infections (UTI) caused by extended-spectrum betalactamase producing (ESBL)-producing Enterobacteriaceae in Duitama (Colombia) from 2010-2015. Methodology: A descriptive study was conducted on ESBL isolates of pathogens associated with UTI in 2 health institutions. Sociodemographic variables, comorbidities, hospitalisations in the last year for UTI, isolated agents, empirical and directed treatment, and clinical response were recorded. Results: A total of 169 patients were included, with an average age of 66.01 ± 19.19; 55.62% were over 65; 59.2% were female and 73.6% were from an urban area. The most frequent comorbidities were chronic obstructive pulmonary disease in 26%; 24.9% had diabetes and 16% had chronic kidney disease, with a Charlson index of 4.43 ± 2.61. Some 61.6% had been hospitalised in the last year due to UTIs. The most common isolated agents were Escherichia coli in 94.7% and Klebsiella spp. in 2.4%. The empirical treatments used were ampicillin/sulbactam in 15%, ciprofloxacin in 29.6% and nitrofurantoin in 10.7%. Regarding directed treatment, 36.7% do not have des-escalation, 32% of patients were treated with ertapenem and 8.9% were treated with piperacillin/tazobactam. Mortality was 5.9% and the average hospital stay was 7.24 ± 7.43 days. Conclusion: Regional data are similar to global data. Empirical treatment should be revaluated, since current guidelines do not recommend the use of ciprofloxacin. In addition, better tracking of ESLB is needed due to flaws in empirical treatment for a large percentage of the strains.