151 research outputs found
Applications of genome editing tools in drug discovery and basic research
Since the discovery of the DNA double helix, major advances in biology have been; the development of recombinant DNA technology in the 1970s, methods to amplify DNA and gene targeting technology in the late 1980s. In organisms such as yeast and mice, the ability to accurately add or delete genetic information transformed biology, allowing an unmatched level of precision in studies of gene function. But, the ability to easily and specifically edit the genetic material of other cells and organisms remained impossible until recently for molecular biologists. The recent advent of programmable nucleases has dramatically changed the efficiency and speed of genome manipulation in several model organisms including cultured cells, as well as whole animals and plants. These tools opened up a powerful technique for biology research now called “genome editing” or “genome engineering” (Carroll, 2011; Hsu et al., 2014; Kim and Kim, 2014).
In the first half of my doctoral studies, I developed genome-editing strategies to discover drug targets for a rare genetic disease called Friedreich’s Ataxia. Friedreich’s Ataxia (FRDA) is a neurodegenerative disease caused by deficiency of the mitochondrial protein frataxin (FXN) (Campuzano et al., 1997). This deficiency results from an expansion of a trinucleotide GAA repeat in the first intron of the FXN gene (Campuzano et al., 1996; Durr et al., 1996). Therapeutics that reactivate FXN gene expression are expected to be beneficial to FRDA patients (Gottesfeld, 2007). However, high-throughput screening (HTS) for FXN activators has so far met with limited success because current cellular models do not accurately assess endogenous FXN gene regulation. Here I used genome-editing technologies to generate a cellular model in which a luciferase reporter is introduced into the endogenous FXN locus. Using this system in a high-throughput genomic screen, we discovered novel inhibitors of FXN-luciferase expression. I confirmed that reducing expression of one of these inhibitors, PRKD1, led to an increase in FXN expression in FRDA patient fibroblasts (Villasenor et al., 2015). We then used reprogramming technologies to create a disease-relevant situation and test small molecules that specifically modulate PRKD1. We found that WA-21-JO19, a chemical inhibitor of PRKD1, increases FXN expression levels in iPSC-derived FRDA patient neurons. This approach, developed at the interface between academic and pharmaceutical research, demonstrates how a combination of genome editing, cellular reprogramming, and high-throughput biology can generate an effective novel drug discovery platform.
In the second part of my doctoral work, we developed an interface between genome editing and proteomics to isolate native protein complexes produced from their natural genomic contexts. In many biological processes, proteins act as members of protein complexes. Understanding the molecular composition of protein complexes is a key task towards explaining their function in the cell. Conventional affinity purification followed by mass spectrometry analysis is a broadly applicable method to decipher molecular interaction networks and infer protein function. However, traditional affinity purification methods are limited by a number of factors such as antibody specificity and are sensitive to perturbations induced by overexpressed target proteins. Here, we combined genome editing with tandem affinity purification to circumvent current limitations. I uncovered subunits and interactions among well-characterized complexes and report the isolation of novel Mettl3-binding partners. The multi-protein complex composed of two active methyltransferases Mettl3 and Mettl14 mediates methylation of adenosines at position N6 on RNA molecules (Bokar et al., 1994; Bokar et al., 1997; Liu et al., 2014). N6-methyladenosine is the most abundant internal modification in eukaryotic mRNA and is often found on introns, which implies that methylation occurs co-transcriptionally (Fu et al., 2014). My work identified a set of nuclear RNA binding proteins, which specifically interact with the Mettl3-Mettl14 complex. We are currently testing the ability of these factors to function as “recruiters” of the Mettl3-Mettl14 complex to nascent mRNAs in the cell nucleus.
In summary, our approach solidly establishes how a combination of genome editing and proteomics can simplify explorations of protein complexes as well as the study of post-translational modifications. In addition, this approach opens up new opportunities to study native protein complexes in a wide variety of cells and model organisms and will likely enable the systematic investigation of mammalian proteome function
Plan de acción comunitario para la gestión holística del territorio en Nueva Colonia, Tuapurie Santa Catarina Cuexcomatitlán, Mezquitic, Jalisco
Este Proyecto de Aplicación Profesional se llevó a cabo en la localidad de Nueva Colonia, Tuapurie Santa Catarina Cuexcomatitlán, Mezquitic, Jalisco, y tuvo el objetivo de impulsar un proyecto comunitario basado en la metodología de la permacultura y realizado en el contexto de la cultura local Wixárika
Main Cross-Cutting Training Contents of LEISURE and Free Time Schools: Acceptance of Groups Involved in the Leisure Time Instructor Courses
Time atomisation trends, leisure economy, and social and technological changes are causing
a reframe of the leisure and free-time industry. This study aims to analyse the assessment of nine
cross-cutting contents by the main agents involved in leisure-time instructor courses, and a group of
young subjects in Spain. The study sample consisted of 1049 individuals, including management
and technical teams, leisure and free-time schoolteachers, leisure and free-time school students
(receiving the leisure-time instructor course), and finally a group of external young subjects. An ad
hoc questionnaire was used, and the results were analysed through a correlational study using
contingency tables and chi-square and Somers’ D statistics, Spearman’s correlation to determine
within-population correlations, and the Kruskal–Wallis test to establish that these relationships were
not randomly established. The results show that all the analysed agents valued the training proposal
of cross-cutting contents as a consolidated item. This indicates that the nine cross-cutting contents
should be maintained in these courses. Social Skills content was crowned as the defining content of
this training, and there was dissonance in the ICT-Use content, which was not highly valued by main
agents but was highly valued by young people, leading to the need to review this content to adjust it
to the real needs of the young populatio
Can electro-bioremediation perform as a self-sustainable process?
This work studies the effect of treatment period on remediation efficiency in an oxyfluorfen-polluted soil through electro-bioremediation technology. Five lab-scale experimental plants were started up simultaneously, operated under an electric field of 1.0 V cm−1 with a polarity reversal frequency of 2 day−1 and disconnected at different times (2, 4, 6, 11 and 24 weeks); these plants underwent post mortem characterization after their operation period. Various parameters were monitored in the electrolyte wells during the experiments. The obtained results indicate that despite the low reproducibility of pH and conductivity in the wells (not in soil), the main conclusions that can be drawn for the different plants are sound and hence have acceptable reproducibility. Polarity reversal allowed suitable conditions for microbial life in terms of pH, but nutrients were also depleted in the soil, which leads to a decrease in the total population of microorganisms during treatment. For treatment periods of less than 10 weeks, there was an appreciable population of microorganisms in the soil, which reached oxyfluorfen removal levels of up to 40%. Longer reaction times were ineffective, and this was related to the much lower concentration of microorganisms. In comparing these results to those obtained in conventional soil bioremediation technology, the application of polarity-reversed electric current led to an increase in the average removal of oxyfluorfen from 0.11 to 0.17 mg kg−1 day−1 but a much higher decrease in the active microorganism population from 88.0 ± 9.0 to 41.0 ± 6.0% of the initial seeded value
Towards the optimization of electro-bioremediation of soil polluted with 2,4-dichlorophenoxyacetic acid
The aim of this work is to study the optimization of electro-bioremediation (EBR) treatment of a 2,4-dichlorophenoxyacetic acid (2,4-D) polluted clay soil. The influence of two different variables were evaluated through batch experiments in a bench-scale electrokinetic setup using previously acclimated microbial cultures for 2,4-D biodegradation. First, it was studied the influence of the frequency applied in polarity reversal (PR): frequencies under study were 1, 2 and 6 d−1, i.e., polarity changed every 24, 12 and 4 h respectively. The duration of experiments were 14 days and the electric field applied was 1.0 V cm−1 (20 V) at room temperature. The second variable under study was the operation time, and based on the previous results, the selected frequency of PR was 2 d-1 and three additional EBR experiments were conducted using different operation times (3, 7 and 10 days). Experiments without electric current (only biological contribution) for each operation time were simultaneously performed under the same experimental conditions as reference tests to check the influence of electrokinetics. Removal of 2,4-D from polluted clay soil was completed in 10 days. It was observed that solubility of the pollutant is a critical factor to ensure high removal efficiencies. Moreover, polarity reversal contributed to the successful results by maintaining correct pH values and reducing the removal of electrolytes from soil. By comparing the EBR results with the reference tests (without the contribution of EK phenomena), it was proved that the combination of bioremediation and electrokinetics has positive effects in the remediation of low permeable polluted soil
Influence of electric field on the remediation of polluted soil using a biobarrier assisted electro-bioremediation process
In this work, it was carried out the study of the electro-bioremediation of soil polluted with diesel by means of combined electrokinetic soil flushing and biobarriers, using bench scale setups and fourteen days-long tests. Two different biobarriers were evaluated: one of them was developed in the laboratory, with a culture of diesel-degrading microorganisms supported on gravel particles (BB1); the other was obtained by mixing directly clay soil with activated sludge obtained in a municipal wastewater treatment plant (BB2). Biobarriers were placed in a central point of the soil section to be treated, to prevent the negative consequences of pH fronts on microorganisms viability. Potential difference of 0.5 and 1.0 V cm−1 were applied. For the promotion in the transport of the diesel to the biobarrier, a surfactant solution was used as electrolyte (flushing fluid). Results confirm that negative influence of the extreme pH fronts on the microbial viability can be prevented using this electro-bioremediation approach. In the same way, uniform diesel removal was obtained all over the soil by using the surfactant solution. After fourteen days of treatment, in the experiments using the BB1, 19.36% and 27.36% of the total amount of diesel present in the soil were removed applying 0.5 and 1.0 V cm−1, respectively. In the experiments using BB2, 23.33% and 29.10% referred to the total amount of diesel were removed after the fourteen days tests at 0.5 and 1.0 V cm−1, respectively, indicating that the non-specific barrier is slightly more efficient despite not containing an acclimated culture.En este trabajo se llevó a cabo el estudio de la electro-biorremediación de suelos contaminados con diésel mediante lavado electrocinético combinado de suelos y biobarreras, utilizando montajes a escala de banco y ensayos de catorce días de duración. Se evaluaron dos biobarreras diferentes: una de ellas desarrollada en laboratorio, con un cultivo de microorganismos degradadores de diesel soportado en partículas de grava (BB1); el otro se obtuvo mezclando directamente tierra arcillosa con lodos activados obtenidos en una estación depuradora de aguas residuales municipales (BB2). Se colocaron biobarreras en un punto central de la sección del suelo a tratar, para prevenir las consecuencias negativas de los frentes de pH sobre la viabilidad de los microorganismos. Diferencia de potencial de 0,5 y 1,0 V cm −1se aplicaron. Para la promoción en el transporte del diésel a la biobarrera se utilizó una solución de surfactante como electrolito (líquido de lavado). Los resultados confirman que la influencia negativa de los frentes de pH extremos en la viabilidad microbiana se puede prevenir utilizando este enfoque de electro-biorremediación. De igual forma se obtuvo una remoción uniforme de diesel en todo el suelo al utilizar la solución de surfactante. Después de catorce días de tratamiento, en los experimentos con BB1, se eliminó el 19,36% y el 27,36% de la cantidad total de diésel presente en el suelo aplicando 0,5 y 1,0 V cm - 1 , respectivamente. En los experimentos con BB2, el 23,33 % y 29,10 % referido a la cantidad total de diésel se eliminó después de los catorce días de pruebas a 0,5 y 1,0 V cm −1, respectivamente, indicando que la barrera no específica es ligeramente más eficiente a pesar de no contener un cultivo aclimatado
Effect of the polarity reversal frequency in the electrokinetic-biological remediation of oxyfluorfen polluted soil. 2017, 177
This work studies the feasibility of the periodic polarity reversal strategy (PRS) in a combined electrokinetic-biological process for the remediation of clayey soil polluted with a herbicide. Five two-weeks duration electrobioremediation batch experiments were performed in a bench scale set-up using spiked clay soil polluted with oxyfluorfen (20 mg kg. Additionally, two complementary reference tests were done: single bioremediation and single electrokinetic. The microbial consortium used was obtained from an oil refinery wastewater treatment plant and acclimated to oxyfluorfen degradation. Main soil conditions (temperature, pH, moisture and conductivity) were correctly controlled using PRS. On the contrary, the electroosmotic flow clearly decreased as increased. The uniform soil microbial distribution at the end of the experiments indicated that the microbial activity remained in every parts of the soil after two weeks when applying PRS. Despite the adapted microbial culture was capable of degrade 100% of oxyfluorfen in water, the remediation efficiency in soil in a reference test, without the application of electric current, was negligible. However, under the low voltage gradients and polarity reversal, removal efficiencies between 5% and 15% were obtained, and it suggested that oxyfluorfen had difficulties to interact with the microbial culture or nutrients and that PRS promoted transport of species, which caused a positive influence on remediation. An optimalEste trabajo estudia la viabilidad de la estrategia de inversión periódica de polaridad (PRS) en un proceso combinado electrocinético-biológico para la remediación de suelos arcillosos contaminados con un herbicida. Se realizaron cinco experimentos por lotes de electrobiorremediación de dos semanas de duración en una instalación a escala de banco utilizando suelo arcilloso con púas contaminado con oxifluorfeno (20 mg kg. Adicionalmente, se realizaron dos pruebas complementarias de referencia: biorremediación simple y electrocinética simple . El consorcio microbiano utilizado fue obtenido de una planta de tratamiento de aguas residuales de una refinería de petróleo y aclimatado a la degradación del oxifluorfeno. Las principales condiciones del suelo (temperatura, pH, humedad y conductividad) se controlaron correctamente mediante PRS. Por el contrario, el flujo electroosmótico disminuyó claramente a medida que aumentado. La distribución microbiana uniforme del suelo al final de los experimentos indicó que la actividad microbiana permaneció en todas las partes del suelo después de dos semanas al aplicar PRS. A pesar de que el cultivo microbiano adaptado fue capaz de degradar el 100% del oxifluorfeno en el agua, la eficiencia de remediación en el suelo en una prueba de referencia, sin la aplicación de corriente eléctrica , fue insignificante. Sin embargo, bajo los gradientes de bajo voltaje y la inversión de polaridad, se obtuvieron eficiencias de remoción entre el 5% y el 15%, y sugirió que el oxifluorfeno tenía dificultades para interactuar con el cultivo microbiano o los nutrientes y que el PRS promovió el transporte de especies, lo que provocó una influencia positiva. sobre la remediación. Un óptim
Strategies for the electrobioremediation of oxyfluorfen polluted soils
This work compares three strategies for the in-situ remediation of oxyfluorfen polluted clay soil, based on the combination of biological processes and electrokinetics (EK): (i) EK-biostimulation, in which the action of microorganisms contained in soil is promoted without the use of biological permeable barriers, (ii) EK-bioaugmentation with fixed culture of microorganisms (BB1), which enhanced the natural bioremediation by including a microbial fixed biofilm reactor as permeable biobarrier, and (iii) EK-bioaugmentation with suspended culture of microorganisms (BB2), which improves the natural remediation by including a mixture of soil and a microbial suspension as permeable biobarrier. To do this, two-week batch electro-bioremediation tests were carried out using bench scale set-ups. In each case, a constant electric field (1 V cm−1) was applied, and electrode polarity was reversed periodically, every 12 h. In addition, a microbial consortium acclimated to oxyfluorfen biodegradation was used. The best oxyfluorfen removal efficiency (15%) was obtained using the option (i). Permeable biobarriers allowed the introduction of microorganism and nutrients into the soil, and the biological activity reached the whole soil positions. However, the insertion of these biobarriers caused an excessive decrease in the electro-osmotic flow (approximately 80%) which, in turn reduced the mobilization of the pollutants. Despite of that, results show that permeable reactive biobarriers may result in a successful alternative for in-situ EK-bioaugmentation, although higher retention times would be needed to obtain higher removal efficiencies
Effect of electric field on the performance of soil electro-bioremediation with a periodic polarity reversal strategy
In this work, it is studied the effect of the electric fields (within the range 0.0–1.5 V cm−1) on the performance of electrobioremediation with polarity reversal, using a bench scale plant with diesel-spiked kaolinite with 14-d long tests. Results obtained show that the periodic changes in the polarity of the electric field results in a more efficient treatment as compared with the single electro-bioremediation process, and it does not require the addition of a buffer to keep the pH within a suitable range. The soil heating was not very important and it did not cause a change in the temperature of the soil up to values incompatible with the life of microorganisms. Low values of water transported by the electro-osmosis process were attained with this strategy. After only 14 d of treatment, by using the highest electric field studied in this work (1.5 V cm−1), up to 35.40% of the diesel added at the beginning of the test was removed, value much higher than the 10.5% obtained by the single bioremediation technology in the same period.En este trabajo se estudia el efecto de los campos eléctricos (en el rango 0,0–1,5 V cm −1) sobre el desempeño de la electrobiorremediación con inversión de polaridad, utilizando una planta a escala de banco con caolinita enriquecida con diesel con pruebas largas de 14 días. Los resultados obtenidos muestran que los cambios periódicos en la polaridad del campo eléctrico dan como resultado un tratamiento más eficiente en comparación con el proceso único de electrobiorremediación, y no requiere la adición de un tampón para mantener el pH dentro de un rango adecuado. El calentamiento del suelo no fue muy importante y no provocó un cambio en la temperatura del suelo hasta valores incompatibles con la vida de los microorganismos. Con esta estrategia se lograron valores bajos de agua transportada por el proceso de electroósmosis. Después de solo 14 d de tratamiento, usando el campo eléctrico más alto estudiado en este trabajo (1.5 V cm −1), se eliminó hasta un 35,40% del diesel agregado al inicio de la prueba, valor muy superior al 10,5% obtenido por la tecnología de biorremediación sola en el mismo período
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