RESUMEN
Molecular diagnostic methods to detect and quantify viral RNA in clinical samples rely on the purification of the genetic material prior to reverse transcription polymerase chain reaction (qRT-PCR). Due to the large number of samples processed in clinical laboratories, automation has become a necessity in order to increase method processivity and maximize throughput per unit of time. An attractive option for isolating viral RNA is based on the magnetic solid phase separation procedure (MSPS) using magnetic microparticles. This method offers the advantage over other alternative methods of making it possible to automate the process. In this study, we report the results of the MSPS method based on magnetic microparticles obtained by a simple synthesis process, to purify RNA from oro- and nasopharyngeal swab samples of patients suspected of COVID-19 provided by three diagnostic laboratories located in the Buenos Aires Province, Argentina. Magnetite nanoparticles of Fe3O4 (MNPs) were synthesized by the coprecipitation method and then coated with silica (SiO2) produced by hydrolysis of tetraethyl orthosilicate (TEOS). After preliminary tests on samples from the A549 human lung cell line and swabs, an extraction protocol was developed. The quantity and purity of the RNA obtained were determined by gel electrophoresis, spectrophotometry, and qRT-PCR. Tests on samples from naso- and oropharyngeal swabs were performed in order to validate the method for RNA purification in high-throughput SARS-CoV-2 diagnosis by qRT-PCR. The method was compared to the spin columns method and the automated method using commercial magnetic particles. The results show that the method developed is efficient for RNA extraction from nasal and oropharyngeal swab samples, and also comparable to other extraction methods in terms of sensitivity for SARS-CoV-2 detection. Of note, this procedure and reagents developed locally were intended to overcome the shortage of imported diagnostic supplies as the sudden spread of COVID-19 required unexpected quantities of nucleic acid isolation and diagnostic kits worldwide.
RESUMEN
Objetivo: Analizar las consideraciones éticas que surgen en situaciones donde las personas mayores con deterioro cognitivo delegan sus decisiones a terceros. Desarrollo: Trabajo de reflexión basado en una revisión narrativa desde diferentes fuentes de información. El envejecimiento de la población es un fenómeno donde se evidencian múltiples desafíos a atender. Chile se encuentra en una etapa de envejecimiento avanzado estimando una esperanza de vida sobre los 85 años para el 2050. La Organización Mundial de la Salud promueve el envejecimiento saludable mediante el fortalecimiento de la capacidad funcional como indicador de bienestar. Existen ocasiones donde las personas mayores delegan a un tercero su capacidad de decidir. Las decisiones por sustitución, que abordan dimensiones éticas y legales deben proteger la dignidad de la persona en todo momento, independientemente de su condición de salud y representar plenamente el derecho de autonomía cedido. Conclusiones. La discusión sobre las decisiones por sustitución y la protección de la autonomía, incluso en situaciones de limitaciones cognitivas, subraya la necesidad de tratar a las personas mayores con respeto y ética, desafiando los estereotipos negativos y evitando conductas discriminatorias y estigmatizantes. La importancia de promover la igualdad, inclusión, información y el respeto de los derechos de las personas mayores tiene un impacto directo en el bienestar y calidad de vida, elemento fundamental que la salud pública promueve bajo el contexto del envejecimiento activo, saludable y digno.
Objective: To analyze the ethical considerations in situations where older people with cognitive impairment delegate their decisions to third parties. Development: Reflective practice based on a narrative review of different sources of information. Population aging is a phenomenon where multiple challenges need to be addressed. Chile is at an advanced stage of population aging, with an estimated life expectancy of over 85 years to be reached by 2050. The World Health Organization (WHO) promotes healthy aging by strengthening functional capacity as an indicator of well-being. There are circumstances in which older people delegate their decision-making capacity to a third party. Surrogate decision-making, with its ethical and legal dimensions, should protect the person's dignity at all times, regardless of the state of their health, and fully represent their ceded right of autonomy. Conclusions: The discussion on surrogate decision-making and the protection of autonomy, even in the context of cognitive impairment, highlights the need to treat older people ethically and respectfully, challenging negative stereotypes and avoiding discriminatory and stigmatizing behaviors. Promoting equality, inclusion, information, and respect for the rights of older adults has a direct impact on their well-being and quality of life, which are fundamental elements that public health promotes in the context of active, healthy, and dignified aging.
Objetivo: Analisar as considerações éticas que surgem em situações em que pessoas idosas com deficiência cognitiva delegam as suas decisões a terceiros. Desenvolvimento: Trabalho de reflexão baseado numa revisão narrativa a partir de diferentes fontes de informação. O envelhecimento da população é um fenómeno onde existem múltiplos desafios a enfrentar. O Chile encontra-se numa fase avançada de envelhecimento, estimando uma esperança de vida superior a 85 anos até 2050. A Organização Mundial de Saúde promove o envelhecimento saudável através do fortalecimento da capacidade funcional como indicador de bem-estar. Há ocasiões em que os idosos delegam a sua capacidade de decisão a terceiros. As decisões de substituição, que abordam as dimensões éticas e legais, devem proteger a dignidade da pessoa em todos os momentos, independentemente do seu estado de saúde, e representar plenamente o direito concedido à autonomia. Conclusões: A discussão sobre as decisões de substituição e a proteção da autonomia, mesmo em situações de limitações cognitivas, sublinha a necessidade de tratar os idosos com respeito e ética, desafiando estereótipos negativos e evitando comportamentos discriminatórios e estigmatizantes. A importância da promoção da igualdade, da inclusão, da informação e do respeito pelos direitos dos idosos tem impacto direto no bem-estar e na qualidade de vida, elemento fundamental que a saúde pública promove no contexto do envelhecimento ativo, saudável e digno.
RESUMEN
Signaling by the kinase cascade composed of Raf, MEK, and ERK is critical for animal development and is often inappropriately activated in human malignancies. We sought to identify factors that control signaling mediated by the Caenorhabditis elegans Raf ortholog LIN-45. A genetic screen showed that the degradation of LIN-45 required the E3/E4 ubiquitin ligase UFD-2. Both UFD-2 and its partner, the ATP-dependent segregase CDC-48, were required for the developmental regulation of LIN-45 protein abundance. We showed that UFD-2 acted in the same pathway as the E3 ubiquitin ligase SCFSEL-10 to decrease LIN-45 abundance in cells in which Raf-MEK-ERK signaling was most highly active. UFD-2 also reduced the protein abundance of activated LIN-45 carrying a mutation equivalent to the cancer-associated BRAF(V600E) variant. Our structure-function studies showed that the disruption of LIN-45 domains that mediate protein-protein interactions, including the conserved cysteine-rich domain and 14-3-3 binding motifs, were required for UFD-2-independent degradation of LIN-45. We propose a model in which UFD-2 and CDC-48 act downstream of SCFSEL-10 to remove LIN-45 from its protein interaction partners and facilitate proteasomal targeting and degradation. These findings imply that UFD-2 and CDC-48 may be important for Raf degradation during normal and oncogenic Ras and MAPK signaling in mammalian cells.
