ABSTRACT
During the gene editing process mediated by CRISPR/Cas9, precise genome editing and gene knock-in can be achieved by the homologous recombination of double-stranded DNA (dsDNA) donor template. However, the low-efficiency of homologous recombination in eukaryotic cells hampers the development and application of this gene editing strategy. Here, we developed a novel CRISPR/Cas9-hLacI donor adapting system (DAS) to enhance the dsDNA-templated gene editing, taking the advantage of the specific binding of the LacI repressor protein and the LacO operator sequence derived for the Escherichia coli lactose operon. The codon-humanized LacI gene was fused as an adaptor to the Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus lugdunensis Cas9 (SlugCas9-HF) genes, and the LacO operator sequence was used as the aptamer and linked to the dsDNA donor template by PCR. The Cas9 nuclease activity after the fusion and the homology-directed repair (HDR) efficiency of the LacO-linked dsDNA template were firstly examined using surrogate reporter assays with the corresponding reporter vectors. The CRISPR/Cas9-hLacI DASs mediated genome precise editing were further checked, and we achieved a high efficiency up to 30.5% of precise editing at the VEGFA locus in HEK293T cells by using the CRISPR/SlugCas9-hLacI DAS. In summary, we developed a novel CRISPR/Cas9-hLacI DAS for dsDNA-templated gene editing, which enriches the CRISPR/Cas9-derived gene editing techniques and provides a novel tool for animal molecular design breeding researches.
Subject(s)
Humans , Animals , Gene Editing , CRISPR-Cas Systems/genetics , HEK293 Cells , Homologous Recombination , DNAABSTRACT
OBJECTIVES@#Patients with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) confront multiple difficulties during the disease adaptation process. Based on the comprehensive task-based adaptation model, this study aims to explore the process and experience of adapting to living with HIV among HIV/AIDS patients and to provide evidence for promoting the adaptation of this population.@*METHODS@#With the design of the phenomenon study, we purposefully recruited 43 HIV/AIDS patients and conducted semi-structural interviews. The qualitative data was analyzed by Van Manen method.@*RESULTS@#There were 1 307 significant quotes and 6 themes with 14 sub-themes. "The shadow comes along with the sunshine" was proposed to describe the process of adapting to life with HIV. Another 5 themes emerged to represent the tasks as follows: the direction of the mental anchor, the management of physical tasks, social network and support, the occupational dilemma and benefits, and the consideration of the future.@*CONCLUSIONS@#The adapting process possesses both common and personalized characteristics. Future intervention development should address the integrality and interaction of the adaptation tasks, contributing to the positive adaptation outcomes of HIV/AIDS patients.
Subject(s)
Humans , Acquired Immunodeficiency Syndrome , HIV , Patients , Physical ExaminationABSTRACT
En este estudio se evaluó la adaptación de una cepa compatible con Acidithiobacillus ferrooxidans a altas densidades de pulpa de calcopirita, esfalerita y galena, con dos distribuciones de tamaño de partícula, -200 y -325 serie Tyler de tamices. Los microorganismos fueron adaptados por la disminución gradual de la fuente principal de energía, sulfato ferroso, y el aumento en el contenido de mineral, para finalmente realizar un subcultivo sin la adición de fuente de energía externa. La realización de subcultivos en serie resultó ser una estrategia eficaz para la adaptación a altas densidades de pulpa de esfalerita, calcopirita y galena indicando que el protocolo empleado es adecuado. Los resultados muestran que la cepa compatible con Acidithiobacillus ferrooxidans es más resistente a altas concentraciones de esfalerita, seguido por calcopirita y finalmente por galena. El tamaño de partícula juega un papel fundamental en la adaptación de los microorganismos al mineral.
In this study the adaptation of Acidithiobacillus ferrooxidans-like to high concentrations of chalcopyrite, sphalerite and galena were evaluated with two mineral-particle sizes: 200 and 325 Tyler mesh. The strain was adapted using two simultaneous processes. The first one consisted in a gradual decreasing of the main energy source, ferrous sulphate. The second one consisted in a gradual increasing of the mineral content. Finally, a test was made without ferrous sulphate. The serial subculturing was found to be an efficient strategy to adapt Acidithiobacillus ferrooxidans-like to higher concentrations of chalcopyrite, sphalerite and galena. This indicates that a suitable protocol was employed. The results showed that Acidithiobacillus ferrooxidans-like is more resistant to high concentration of sphalerite, chalcopyrite and galena in descendant order. The particle size played an important role in the adaption of microorganism to the mineral.
Subject(s)
Adaptation, Biological/physiology , Adaptation, Biological/genetics , Adaptation, Biological/immunologyABSTRACT
Se realizó un estudio descriptivo de pacientes ingresados para determinar la justificación de los ingresos en el servicio de medicina interna como elemento para elaborar intervenciones que eleven la calidad y eficiencia de los servicios de salud. Se aplicó a 454 pacientes el Protocolo de Evaluación de la Adecuación (AEP). Se utilizó la técnica estadística de análisis de distribución de frecuencia, comparación de medias y pruebas de independencia para demostrar diferencias entre los grupos según la adecuación del ingreso. Se halló que el 18,7 % de los ingresos fueron considerados no adecuados (INA), los que tuvieron edades (53,5 + 18,2 años) inferiores que aquellos con ingresos adecuados(62,1 + 18,9 años) y estadías ligeramente superiores (9,8 vs. 8,5 d). Los pacientes con INA utilizaron el 20 % del total de días/pacientes del período (833 días/pacientes), fueron ingresados básicamente los lunes, martes, miércoles y viernes, y provenían, en general, de áreas de salud no asignadas al hospital. Se concluyó que el frecuente ingreso de pacientes de forma inadecuada requiere de la revisión de aspectos organizativos del servicio y la institución, que conduzcan a una mejor eficiencia de la gestión hospitalaria.
A descriptive study of patients admitted was conducted to determine the justification of admissions in the Internal Medicine service as an element to make interventions that increase the quality and efficiency of the health services. The Adapting Evaluation Protocol was applied to 454 patients. The statistical technique of frequency distribution analysis, mean comparison and independence tests was used to show the differences between the groups according to the admission adapting .It was found that 18.7% of the admissions were considered inadequate (INA). The individuals that were inadequately admitted were younger (53.5 ± 18.2 years old) than those with adequate admissions (62.1 ± 18.9 years old), and their lengths of stay were slightly higher (9.8 vs 8.5 days). Patients with inadequate admissions used 20 % of the total of days/patients of the period (833 days/patients), they were admitted basically on Monday, Tuesday, Wednesday and Friday, and they generally came from health areas that were not assigned to the hospital. It was concluded that the frequent inadequate admission of patients requires a review of organizational aspects of the service and institution leading to a better efficiency of hospital management.