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Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3' ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.
Assuntos
DNA Complementar/biossíntese , Íntrons , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de RNA/métodos , Sequência de Bases , Clonagem Molecular , Sequência Conservada , DNA Complementar/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Biblioteca Gênica , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Células HeLa , Humanos , Células MCF-7 , MicroRNAs/genética , MicroRNAs/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Estabilidade Proteica , DNA Polimerase Dirigida por RNA/genética , Proteínas Recombinantes de Fusão/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TemperaturaRESUMO
Differential sensing using synthetic receptors as mimics of the mammalian senses of taste and smell is a powerful approach for the analysis of complex mixtures. Herein, we report on the effectiveness of a cross-reactive, supramolecular, peptide-based sensing array in differentiating and predicting the composition of red wine blends. Fifteen blends of Cabernet Sauvignon, Merlot and Cabernet Franc, in addition to the mono varietals, were used in this investigation. Linear Discriminant Analysis (LDA) showed a clear differentiation of blends based on tannin concentration and composition where certain mono varietals like Cabernet Sauvignon seemed to contribute less to the overall characteristics of the blend. Partial Least Squares (PLS) Regression and cross validation were used to build a predictive model for the responses of the receptors to eleven binary blends and the three mono varietals. The optimized model was later used to predict the percentage of each mono varietal in an independent test set composted of four tri-blends with a 15% average error. A partial least square regression model using the mouth-feel and taste descriptive sensory attributes of the wine blends revealed a strong correlation of the receptors to perceived astringency, which is indicative of selective binding to polyphenols in wine.
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Técnicas Biossensoriais/métodos , Peptídeos/química , Vitis/química , Vinho/análise , Análise dos Mínimos Quadrados , Modelos Teóricos , Odorantes/análise , Mapeamento de Peptídeos , Polifenóis/análise , Polifenóis/química , Olfato , Taninos/análise , Taninos/química , PaladarRESUMO
Introduction: The institutions (i.e., hubs) making up the National Institutes of Health (NIH)-funded network of Clinical and Translational Science Awards (CTSAs) share a mission to turn observations into interventions to improve public health. Recently, the focus of the CTSAs has turned increasingly from translational research (TR) to translational science (TS). The current NIH Funding Opportunity Announcement (PAR-21-293) for CTSAs stipulates that pilot studies funded through the CTSAs must be "focused on understanding a scientific or operational principle underlying a step of the translational process with the goal of developing generalizable solutions to accelerate translational research." This new directive places Pilot Program administrators in the position of arbiters with the task of distinguishing between TR and TS projects. The purpose of this study was to explore the utility of a set of TS principles set forth by NCATS for distinguishing between TR and TS. Methods: Twelve CTSA hubs collaborated to generate a list of Translational Science Principles questions. Twenty-nine Pilot Program administrators used these questions to evaluate 26 CTSA-funded pilot studies. Results: Factor analysis yielded three factors: Generalizability/Efficiency, Disruptive Innovation, and Team Science. The Generalizability/Efficiency factor explained the largest amount of variance in the questions and was significantly able to distinguish between projects that were verified as TS or TR (t = 6.92, p < .001) by an expert panel. Conclusions: The seven questions in this factor may be useful for informing deliberations regarding whether a study addresses a question that aligns with NCATS' vision of TS.
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Mobile group II introns, which are found in bacterial and organellar genomes, are site-specific retroelements hypothesized to be evolutionary ancestors of spliceosomal introns and retrotransposons in higher organisms. Most bacteria, however, contain no more than one or a few group II introns, making it unclear how introns could have proliferated to higher copy numbers in eukaryotic genomes. An exception is the thermophilic cyanobacterium Thermosynechococcus elongatus, which contains 28 closely related copies of a group II intron, constituting approximately 1.3% of the genome. Here, by using a combination of bioinformatics and mobility assays at different temperatures, we identified mechanisms that contribute to the proliferation of T. elongatus group II introns. These mechanisms include divergence of DNA target specificity to avoid target site saturation; adaptation of some intron-encoded reverse transcriptases to splice and mobilize multiple degenerate introns that do not encode reverse transcriptases, leading to a common splicing apparatus; and preferential insertion within other mobile introns or insertion elements, which provide new unoccupied sites in expanding non-essential DNA regions. Additionally, unlike mesophilic group II introns, the thermophilic T. elongatus introns rely on elevated temperatures to help promote DNA strand separation, enabling access to a larger number of DNA target sites by base pairing of the intron RNA, with minimal constraint from the reverse transcriptase. Our results provide insight into group II intron proliferation mechanisms and show that higher temperatures, which are thought to have prevailed on Earth during the emergence of eukaryotes, favor intron proliferation by increasing the accessibility of DNA target sites. We also identify actively mobile thermophilic introns, which may be useful for structural studies, gene targeting in thermophiles, and as a source of thermostable reverse transcriptases.
Assuntos
Cianobactérias/genética , Genoma Bacteriano , Íntrons , Sequência de Bases , DNA Bacteriano , Dados de Sequência Molecular , Fases de Leitura Aberta , Homologia de Sequência do Ácido NucleicoRESUMO
Patient satisfaction is an integral aspect of healthcare quality assessment, and it plays a crucial role in evaluating the effectiveness of healthcare services. This systematic review investigates patient satisfaction with dental services provided by public dental hospitals in rural and remote areas of Saudi Arabia. The study conducted a systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) standards. It involved a comprehensive search across multiple databases, including Medline, Cochrane, Embase, and CINAHL, with tailored search strategies for each database using MeSH terms and keywords. To ensure inclusivity, the search covered publications in both English and Arabic and included Google Scholar for gray literature. Inclusion criteria focused on empirical studies conducted in rural and remote public hospitals in Saudi Arabia, published between 2013 and January 2023, assessing patient satisfaction in oral or dental care for adult patients. Data screening and extraction followed a rigorous two-step process, and a narrative synthesis was used to analyze and summarize the findings. The findings reveal a complex landscape of patient satisfaction in these settings, with varying levels of contentment reported. While more than 50% of patients expressed satisfaction with the quality of dental care, significant challenges related to accessibility were evident. Patients residing in remote and rural areas often had to travel long distances to access dental clinics, resulting in dissatisfaction. Demographic factors, particularly education and age, were identified as significant influencers of patient satisfaction, with more educated individuals tending to be less satisfied. This study emphasizes the importance of continuous monitoring of patient satisfaction to enhance service delivery, particularly in public dental clinics serving remote and rural areas. Addressing issues related to access, availability, clinical quality, and effective communication is vital for improving patient satisfaction in these healthcare settings. The study concludes with recommendations for policymakers, including the development of quality assurance policies, cost mitigation strategies, and targeted interventions to address demographic disparities and enhance patient satisfaction with dental care services.
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Organophosphorus compounds include many synthetic, neurotoxic substances that are commonly used as insecticides. The toxicity of these compounds is due to their ability to inhibit the enzyme acetylcholine esterase. Some of the most toxic organophosphates have been adapted for use as chemical warfare agents; the most well-known are GA, GB, GD, GF, VX, and VR. All of these compounds contain a chiral phosphorus center, with the S(P) enantiomers being significantly more toxic than the R(P) enantiomers. Phosphotriesterase (PTE) is an enzyme capable of detoxifying these agents, but the stereochemical preference of the wild-type enzyme is for the R(P) enantiomers. A series of enantiomerically pure chiral nerve agent analogues containing the relevant phosphoryl centers found in GB, GD, GF, VX, and VR has been developed. Wild-type and mutant forms of PTE have been tested for their ability to hydrolyze this series of compounds. Mutant forms of PTE with significantly enhanced, as well as relaxed or reversed, stereoselectivity have been identified. A number of variants exhibited dramatically improved kinetic constants for the catalytic hydrolysis of the more toxic S(P) enantiomers. Improvements of up to 3 orders of magnitude relative to the value of the wild-type enzyme were observed. Some of these mutants were tested against racemic mixtures of GB and GD. The kinetic constants obtained with the chiral nerve agent analogues accurately predict the improved activity and stereoselectivity against the authentic nerve agents used in this study.
Assuntos
Compostos Organofosforados/química , Hidrolases de Triester Fosfórico/metabolismo , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Catálise , Substâncias para a Guerra Química/química , Hidrólise , Inseticidas/química , Organofosfatos/química , Hidrolases de Triester Fosfórico/química , EstereoisomerismoRESUMO
Course-based undergraduate research experiences (CUREs) provide a promising avenue to attract a larger and more diverse group of students into research careers. CUREs are thought to be distinctive in offering students opportunities to make discoveries, collaborate, engage in iterative work, and develop a sense of ownership of their lab course work. Yet how these elements affect students' intentions to pursue research-related careers remain unexplored. To address this knowledge gap, we collected data on three design features thought to be distinctive of CUREs (discovery, iteration, collaboration) and on students' levels of ownership and career intentions from â¼800 undergraduates who had completed CURE or inquiry courses, including courses from the Freshman Research Initiative (FRI), which has a demonstrated positive effect on student retention in college and in science, technology, engineering, and mathematics. We used structural equation modeling to test relationships among the design features and student ownership and career intentions. We found that discovery, iteration, and collaboration had small but significant effects on students' intentions; these effects were fully mediated by student ownership. Students in FRI courses reported significantly higher levels of discovery, iteration, and ownership than students in other CUREs. FRI research courses alone had a significant effect on students' career intentions.
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Comportamento Cooperativo , Laboratórios , Propriedade , Pesquisa/educação , Estudantes , Currículo , Feminino , Humanos , MasculinoRESUMO
It is becoming increasingly important to differentiate complex mixtures, especially in forensics. Cachaça, the most popular alcoholic beverage in Brazil, is made from distilled and fermented sugar cane juice. It contains a mixture of naturally occurring polyphenols known as tannins, whose composition is dictated by the type of wood used to age the beverage. These tannins can be differentiated in an Indicator Displacement Assay (IDA) using peptide-based ternary sensing ensembles. This investigation demonstrates a technique for fingerprinting the identity of the woods used to age cachaças. Unknown cachaça samples were tested against a training set of Brazilian woods in addition to oaks from different countries. Results obtained from the analysis showed that 62.5% of the samples were correctly identified. Furthermore, four samples anonymously added to the pool of unknowns from the training set were identified with 100% accuracy, emphasizing both the promising results obtained from this method of differentiation and the importance of analyzing same-age samples.
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Organophosphates have been widely used as insecticides and chemical warfare agents. The health risks associated with these agents have necessitated the need for better detoxification and bioremediation tools. Bacterial enzymes capable of hydrolyzing the lethal organophosphate nerve agents are of special interest. Phosphotriesterase (PTE) isolated from the soil bacteria Pseudomonas diminuta displays a significant rate enhancement and substrate promiscuity for the hydrolysis of organophosphate triesters. Directed evolution and rational redesign of the active site of PTE have led to the identification of new variants with enhanced catalytic efficiency and stereoselectivity toward the hydrolysis of organophosphate neurotoxins. PTE has been utilized to protect against organophosphate poisoning in vivo. Biotechnological applications of PTE for detection and decontamination of insecticides and chemical warfare agents are developing into useful tools. In this review, the catalytic properties and potential applications of this remarkable enzyme are discussed.
Assuntos
Substâncias para a Guerra Química/metabolismo , Compostos Organofosforados/metabolismo , Hidrolases de Triester Fosfórico/metabolismo , Pseudomonas/enzimologia , Catálise , Hidrolases de Triester Fosfórico/química , Conformação Proteica , Especificidade por SubstratoRESUMO
Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a nonspecific diesterase that enables Escherichia coli to utilize alkyl phosphodiesters, such as diethyl phosphate, as the sole phosphorus source. The catalytic properties of GpdQ were determined, and the best substrate found was bis(p-nitrophenyl) phosphate with a kcat/Km value of 6.7 x 10(3) M-1 s-1. In addition, the E. aerogenes diesterase was tested as a catalyst for the hydrolysis of a series of phosphonate monoesters which are the hydrolysis products of the highly toxic organophosphonate nerve agents sarin, soman, GF, VX, and rVX. Among the phosphonate monoesters tested, the hydrolysis product of rVX, isobutyl methyl phosphonate, was the best substrate with a kcat/Km value of 33 M-1 s-1. The ability of GpdQ to hydrolyze the phosphonate monoesters provides an alternative selection strategy in the search of enhanced variants of the bacterial phosphotriesterase (PTE) for the hydrolysis of organophosphonate nerve agents. This investigation demonstrated that the previously reported activity of GpdQ toward the hydrolysis of methyl demeton-S is due to the presence of a diester contaminant in the commercial material. Furthermore, it was shown that GpdQ is capable of hydrolyzing a close analogue of EA 2192, the most toxic and persistent degradation product of the nerve agent VX.
Assuntos
Enterobacter aerogenes/enzimologia , Compostos Organotiofosforados/química , Diester Fosfórico Hidrolases/química , Substâncias para a Guerra Química/química , Substâncias para a Guerra Química/metabolismo , Inibidores da Colinesterase/química , Inibidores da Colinesterase/metabolismo , Dissulfóton/química , Dissulfóton/metabolismo , Enterobacter aerogenes/genética , Enterobacter aerogenes/crescimento & desenvolvimento , Cinética , Viabilidade Microbiana/efeitos dos fármacos , Estrutura Molecular , Mutação , Nitrofenóis/química , Nitrofenóis/metabolismo , Organofosfatos/química , Organofosfatos/metabolismo , Organofosfonatos/química , Organofosfonatos/metabolismo , Compostos Organotiofosforados/metabolismo , Paraoxon/química , Paraoxon/metabolismo , Fenóis/farmacologia , Diester Fosfórico Hidrolases/biossíntese , Diester Fosfórico Hidrolases/metabolismo , Hidrolases de Triester Fosfórico/química , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
With the emergence of sequences and even structures for proteins of unknown function, structure-based prediction of enzyme activity has become a pragmatic as well as an interesting question. Here we investigate a method to predict substrates for enzymes of known structure by docking high-energy intermediate forms of the potential substrates. A database of such high-energy transition-state analogues was created from the KEGG metabolites. To reduce the number of possible reactions to consider, we restricted ourselves to enzymes of the amidohydrolase superfamily. We docked each metabolite into seven different amidohydrolases in both the ground-state and the high-energy intermediate forms. Docking the high-energy intermediates improved the discrimination between decoys and substrates significantly over the corresponding standard ground-state database, both by enrichment of the true substrates and by geometric fidelity. To test this method prospectively, we attempted to predict the enantioselectivity of a set of chiral substrates for phosphotriesterase, for both wild-type and mutant forms of this enzyme. The stereoselectivity ratios of the six enzymes considered for those four substrate enantiomer pairs differed over a range of 10- to 10,000-fold and underwent 20 switches in stereoselectivities for favored enantiomers, compared to the wild type. The docking of the high-energy intermediates correctly predicted the stereoselectivities for 18 of the 20 substrate/enzyme combinations when compared to subsequent experimental synthesis and testing. The possible applications of this approach to other enzymes are considered.
Assuntos
Algoritmos , Amidoidrolases/química , Simulação por Computador , Hidrolases de Triester Fosfórico/química , Amidoidrolases/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Bases de Dados de Proteínas , Modelos Químicos , Hidrolases de Triester Fosfórico/metabolismo , Estereoisomerismo , Especificidade por SubstratoRESUMO
An array of 16 enantiomeric pairs of chiral phosphate, phosphonate, and phosphinate esters was used to establish the breadth of the stereoselective discrimination inherent within the bacterial phosphotriesterase and 15 mutant enzymes. For each substrate, the leaving group was 4-hydroxyacetophenone while the other two groups attached to the phosphorus core consisted of an asymmetric mixture of methyl, methoxy, ethyl, ethoxy, isopropoxy, phenyl, phenoxy, cyclohexyl, and cyclohexoxy substituents. For the wild-type enzyme, the relative rates of hydrolysis for the two enantiomers ranged from 3 to 5.4 x 10(5). Various combinations of site-specific mutations within the active site were used to create modified enzymes with alterations in their enantioselective properties. For the single-site mutant enzyme, G60A, the stereoselectivity is enhanced relative to that of the wild-type enzyme by 1-3 orders of magnitude. Additional mutants were obtained where the stereoselectivity is inverted relative to the wild-type enzyme for 13 of the 16 pairs of enantiomers tested for this investigation. The most dramatic example was obtained for the hydrolysis of 4-acetylphenyl methyl phenyl phosphate. The G60A mutant preferentially hydrolyzes the SP-enantiomer by a factor of 3.7 x 10(5). The I106G/F132G/H257Y mutant preferentially hydrolyzes the RP-enantiomer by a factor of 9.7 x 10(2). This represents an enantioselective discrimination of 3.6 x 10(8) between these two mutants, with a total of only four amino acid changes. The rate differential between the two enantiomers for any given mutant enzyme is postulated to be governed by the degree of nonproductive binding within the enzyme active site and stabilization of the transition state. This hypothesis is supported by computational docking of the high-energy, pentavalent form of the substrates to modeled structures of the mutant enzyme; the energies of the docked transition-state analogues qualitatively capture the enantiomeric preferences of the various mutants for the different substrates. These results demonstrate that the catalytic properties of the wild-type phosphotriesterase can be exploited for the kinetic resolution of a wide range of phosphate, phosphonate, and phosphinate esters and that the active site of this enzyme is remarkably amenable to structural perturbations via amino acid substitution.