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A reliable, scalable, cost-effective, and chromatography-free synthesis of 4-azido-l-phenylalanine beginning from l-phenylalanine is described. Investigations into the safety of the synthesis reveal that the Ullman-like Cu(I)-catalyzed azidation step does not represent a significant risk. The isolated 4-azido-l-phenylalanine product, however, exhibits previously undocumented explosive characteristics.
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Azidas/síntesis química , Fenilalanina/análogos & derivados , Azidas/química , Catálisis , Cobre/química , Estructura Molecular , Fenilalanina/síntesis química , Fenilalanina/químicaRESUMEN
Hypoxia-inducible factor pathway genes are linked to adaptation in both human and nonhuman highland species. EPAS1, a notable target of hypoxia adaptation, is associated with relatively lower hemoglobin concentration in Tibetans. We provide evidence for an association between an adaptive EPAS1 variant (rs570553380) and the same phenotype of relatively low hematocrit in Andean highlanders. This Andean-specific missense variant is present at a modest frequency in Andeans and absent in other human populations and vertebrate species except the coelacanth. CRISPR-base-edited human cells with this variant exhibit shifts in hypoxia-regulated gene expression, while metabolomic analyses reveal both genotype and phenotype associations and validation in a lowland population. Although this genocopy of relatively lower hematocrit in Andean highlanders parallels well-replicated findings in Tibetans, it likely involves distinct pathway responses based on a protein-coding versus noncoding variants, respectively. These findings illuminate how unique variants at EPAS1 contribute to the same phenotype in Tibetans and a subset of Andean highlanders despite distinct evolutionary trajectories.
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Adaptación Fisiológica , Altitud , Hematócrito , Pueblos Sudamericanos , Humanos , Adaptación Fisiológica/genética , Adaptación Fisiológica/fisiología , Pueblos del Este de Asia , Hipoxia/genética , Hipoxia/metabolismo , Mutación Missense/genética , Pueblos Sudamericanos/genéticaRESUMEN
The flexibility and precision of CRISPR-Cas9 and related technologies have made these genome editing tools increasingly popular in agriculture, medicine, and basic science research for the past decade. Genome editing will continue to be relevant and utilized across diverse scientific fields in the future. Given this, students should be introduced to genome editing technologies and encouraged to consider their ethical implications early on in precollege biology curricula. Furthermore, instruction on this topic presents an opportunity to create partnerships between researchers and educators at the K-12 levels that can strengthen student engagement in science, technology, engineering, and mathematics. To this end, we present a 3-day student-centered learning program to introduce high school students to genome editing technologies through a hands-on base editing experiment in Escherichia coli, accompanied by a relevant background lecture and facilitated ethics discussion. This unique partnership aims to educate students and provides a framework for research institutions to implement genome editing outreach programs at local high schools. We have included all requisite materials, including lecture slides, worksheets, experimental protocols, and suggestions on active learning strategies for others to reproduce our program with their local communities.
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Sistemas CRISPR-Cas , Edición Génica , Humanos , Sistemas CRISPR-Cas/genética , Estudiantes , Escherichia coli/genética , BiologíaRESUMEN
The flexibility and precision of CRISPR-Cas9 and related technologies have made these genome editing tools increasingly popular in agriculture, medicine, and basic science research over the past decade. Genome editing will continue to be relevant and utilized across diverse scientific fields in the future. Given this, students should be introduced to genome editing technologies and encouraged to consider their ethical implications early on in pre-college biology curricula. Furthermore, instruction on this topic presents an opportunity to create partnerships between researchers and educators at the K-12 levels that can strengthen student engagement in science, technology, engineering, and mathematics (STEM). To this end, we present a three-day student-centered learning program to introduce high school students to genome editing technologies through a hands-on base editing experiment in E. coli , accompanied by a relevant background lecture and facilitated ethics discussion. This unique partnership aims to educate students and provides a framework for research institutions to implement genome editing outreach programs at local high schools.
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Base editors (BEs) are genome editing agents that install point mutations with high efficiency and specificity. Due to their reliance on uracil and inosine DNA damage intermediates (rather than double-strand DNA breaks, or DSBs), it has been hypothesized that BEs rely on more ubiquitous DNA repair pathways than DSB-reliant genome editing methods, which require processes that are only active during certain phases of the cell cycle. We report here the first systematic study of the cell cycle-dependence of base editing using cell synchronization experiments. We find that nickase-derived BEs (which introduce DNA backbone nicks opposite the uracil or inosine base) function independently of the cell cycle, while non-nicking BEs are highly dependent on S-phase (DNA synthesis phase). We found that synchronization in G1 (growth phase) during the process of cytosine base editing causes significant increases in Câ¢G to Aâ¢T "byproduct" introduction rates, which can be leveraged to discover new strategies for precise Câ¢G to Aâ¢T base editing. We observe that endogenous expression levels of DNA damage repair pathways are sufficient to process base editing intermediates into desired editing outcomes, and the process of base editing does not significantly perturb transcription levels. Overall, our study provides mechanistic data demonstrating the robustness of nickase-derived BEs for performing genome editing across the cell cycle.
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This article presents a database which was obtained by acquiring measurements through a multisensory device called Electronic Nose (E-nose) based on a matrix of metal oxide sensors, in order to discriminate and classify a group of people affected by the respiratory disease Chronic Obstructive Pulmonary Disease (COPD), smokers and healthy control people through exhaled breath analysis. The database consists of 4 groups of measurements which were acquired through the E-nose system: 10 control samples (healthy people), 20 samples of people with COPD, 4 samples of smokers and 10 air samples, where in each group two samples of exhaled breath per person were acquired giving a total of 78 samples (40 from COPD, 20 from control, 8 from smokers and 10 from the air).
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Base-editing technologies enable the introduction of point mutations at targeted genomic sites in mammalian cells, with higher efficiency and precision than traditional genome-editing methods that use DNA double-strand breaks, such as zinc finger nucleases (ZFNs), transcription-activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) system. This allows the generation of single-nucleotide-variant isogenic cell lines (i.e., cell lines whose genomic sequences differ from each other only at a single, edited nucleotide) in a more time- and resource-effective manner. These single-nucleotide-variant clonal cell lines represent a powerful tool with which to assess the functional role of genetic variants in a native cellular context. Base editing can therefore facilitate genotype-to-phenotype studies in a controlled laboratory setting, with applications in both basic research and clinical applications. Here, we provide optimized protocols (including experimental design, methods, and analyses) to design base-editing constructs, transfect adherent cells, quantify base-editing efficiencies in bulk, and generate single-nucleotide-variant clonal cell lines. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Design and production of plasmids for base-editing experiments Basic Protocol 2: Transfection of adherent cells and harvesting of genomic DNA Basic Protocol 3: Genotyping of harvested cells using Sanger sequencing Alternate Protocol 1: Next-generation sequencing to quantify base editing Basic Protocol 4: Single-cell isolation of base-edited cells using FACS Alternate Protocol 2: Single-cell isolation of base-edited cells using dilution plating Basic Protocol 5: Clonal expansion to generate isogenic cell lines and genotyping of clones.
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Células Clonales , Edición Génica , Mutación Puntual , Sistemas CRISPR-Cas , Línea Celular , HumanosRESUMEN
Between September 1990 and December 2002, 511 cetacean carcasses stranded or caught in commercial fisheries in England and Wales were examined post mortem. Salmonella group B was isolated from 60 of 279 (21.51%) harbour porpoises (Phocoena phocoena), predominantly from lung tissue. Forty-three of the Salmonella group B isolates were subsequently serotyped and all found to have the antigenic structure O4,12:a:-. The annual proportion of harbour porpoises testing positive for Salmonella O4,12:a:- increased significantly from 6% in the early 1990s to 27% after 1999. The cause(s) of the increasing prevalence of Salmonella O4,12:a:- are not known, but may reflect natural variation in the epidemiological cycle of Salmonella O4,12:a:- in harbour porpoises. The probability of isolating Salmonella O4,12:a:- from harbour porpoises increased with age, suggesting that the mode of transmission is principally horizontal. There appeared to be a weak degree of seasonality in the probability of isolating Salmonella O4,12:a:- with a low proportion of cases in the months of April and May. Based on pathological findings from infected carcasses, Salmonella O4,12:a:- may be part of the normal commensal flora of the lungs of cetaceans with the potential to act as an opportunistic pathogen.
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Phocoena/microbiología , Salmonelosis Animal/microbiología , Salmonella/aislamiento & purificación , Animales , Inglaterra/epidemiología , Pulmón/microbiología , Prevalencia , Salmonella/clasificación , Salmonelosis Animal/epidemiología , Serotipificación , Gales/epidemiologíaRESUMEN
Introducción: El examen clínico objetivo estructurado (ECOE) se aplica desde la década de los setenta en la mayoría de las escuelas de medicina del mundo. Este examen consiste en una dinámica en la que los estudiantes rotan por estaciones, dotadas de todo lo necesario para realizar un procedimiento clínico en un tiempo determinado. Objetivo: Analizar la percepción del ECOE como instrumento de evaluación para estudiantes de tercer año de Odontología de la sede de Santiago de la Universidad San Sebastián de Chile en 2018. Sujetos y métodos: Se realizó una investigación exploratoria aplicando una encuesta que mide la percepción frente a la experiencia del ECOE por los estudiantes de tercer año de la carrera de Odontología. Resultados: Los resultados indicaron que los estudiantes percibieron que el ECOE estuvo bien implementado, la mayoría pensó que mide sus habilidades y conocimientos adquiridos en años anteriores, y consideran que lo evaluado es importante para los años posteriores de su carrera. Conclusión: Los estudiantes tienen una percepción adecuada del ECOE como instrumento evaluativo; se pudo obtener información para realizar mejoras en futuras implementaciones.(AU)
Introduction: The objective structured clinical examination (OSCE) has been used since the 1970s in most medical schools worldwide. It consists of students being tested at different workstations, where they must perform a clinical procedure. Each station is equipped with everything necessary to perform a clinical procedure within a certain time. Aim: The perception of the OSCE was analyzed as an evaluation instrument for third-year students of Dentistry at the San Sebastian University, Santiago, Chile in 2018. Subjects and methods: An exploratory research was carried out applying a survey that measures the perception of the experience of the OSCE by third-year students of the Dentistry career. Results: The results indicated that the students perceived was well implemented. The majority thought that the OSCE measured their skills and knowledge acquired in previous years and that what was asked was important for the later years of their careers. It was observed that they considered the fairest evaluation of the OSCE, the most difficult -the preclinical enabling tests. It was also reported that they preferred to be evaluated more frequently with the OSCE. Conclusion: The students have an adequate perception of the ECOE as an evaluation instrument and that information could be obtained to make improvements in future implementations.(AU)