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1.
RNA ; 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38942481

RESUMO

Direct methods for determining the fidelity of DNA polymerases are robust, with relatively little sample manipulation before sequencing. In contrast, methods for measuring RNA polymerase and reverse transcriptase fidelities are complicated by additional preparation steps that introduce ambiguity and error. Here, we describe a sequencing method, termed Roll-Seq, for simultaneously determining the individual fidelities of RNA polymerases and reverse transcriptases (RT) using Pacific Biosciences Single Molecule Real-Time sequencing. By employing reverse transcriptases with high rolling-circle activity, Roll-Seq generates long concatemeric cDNA from a circular RNA template. To discern the origin of a mutation, errors are recorded and determined to occur within a single concatemer (reverse transcriptase error) or all concatemers (RNA polymerase error) over the cDNA strand. We used Roll-Seq to measure the fidelities of T7 RNA polymerases, a Group II intron-encoded RT (Induro), and two LINE RTs (Fasciolopsis buski R2-RT and human LINE-1). Substitution rates for Induro and R2-RT are the same for cDNA and second strand synthesis while LINE-1 has 2.5-fold lower fidelity when performing second strand synthesis. Deletion and insertion rates increase for all RTs during second strand synthesis. In addition, we find that a structured RNA template impacts fidelity for both RNA polymerase and RT. The accuracy and precision of Roll-Seq enable this method to be applied as a complementary analysis to structural and mechanistic characterization of RNA polymerases and reverse transcriptases or as a screening method for RNAP and RT fidelity.

2.
Int J Pharm Pract ; 31(2): 218-224, 2023 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-36541698

RESUMO

OBJECTIVES: To examine the self-perceived knowledge, confidence and preparedness of undergraduate pharmacy students to provide palliative care. METHODS: A descriptive exploratory analysis was conducted in 2021 at an Australian university involving final-year pharmacy students (n = 200) who were provided with the opportunity to complete a survey on self-perceived knowledge, confidence and preparedness overall and with respect to a range of graduate capabilities which are essential to provide care in palliative care settings. Key capability areas include: communication, showing empathy, making clinical judgements and self-reflection. This was measured using the Palliative Care Curriculum for Undergraduates Questionnaire which was distributed electronically. Descriptive statistics were undertaken and Mann-Whitney U tests were used to explore any differences in outcomes with respect to factors related to demographics, personal experience and education. Thematic analysis was utilised for qualitative data. KEY FINDINGS: Forty-five percent of the student cohort (n = 89) responded, 70% of whom were female, and the median age for students was 22 years. Median scores (interquartile range) were modest for overall self-perceived knowledge: 5.0 (3.0-5.0), confidence: 4.0 (3.0-5.0) and preparedness: 4.0 (2.5-5.0). Students who had participated in learning about palliative care through clinical placements (n = 25, 28%), self-directed learning activities (n = 18, 20%) or case-/problem-based learning (n = 14, 16%) demonstrated a statistically significant increase in overall preparedness (P = 0.017), confidence with specific capabilities including evidence-based practice (P = 0.013), responding to medication queries (P < 0.05) and managing symptoms other than pain (P = 0.018). CONCLUSIONS: Findings suggest students were confident to manage symptoms and medication-related issues but less confident to address distress or discuss sensitive matters with patients and their families. There may be a need for greater exposure and practical experience in palliative care settings.


Assuntos
Cuidados Paliativos , Farmácia , Humanos , Feminino , Adulto Jovem , Adulto , Masculino , Cuidados Paliativos/métodos , Austrália , Estudantes , Currículo
3.
Palliat Support Care ; : 1-13, 2022 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-36503650

RESUMO

CONTEXT: The demand of palliative care is increasing due to the aging population and treatment hesitancy or intentional avoidance compromises symptom management. OBJECTIVES: To identify patient beliefs associated with medication hesitancy by using the theory of planned behavior (TPB) namely, attitudes, subjective norms, behavioral intention, and perceived behavioral control associated with medication hesitancy or intentional noncompliance by avoidance. METHODS: The Preferred Reporting Items for Systematic Reviews and Meta-Analysis guideline was followed to conduct a systematic literature search involving the CINAHL, Embase, MEDLINE, and PsycINFO databases from inception until March 2022. Hand-searched articles from reference lists and gray literature were included. Thematic analysis was conducted on qualitative data and triangulated with quantitative data. RESULTS: About 554 articles were retrieved from the literature search and 17 articles were included based on the eligibility criteria. Three subthemes that were identified under TPB constructs were attitude: negative attitude toward medications, passive attitude toward illness and inaccurate information about disease or medication; one subtheme was identified under subjective norms: perceived negative opinions from others; and one subtheme was identified under perceived behavioral control: perception of manageable symptoms. Quantitative data provided triangulation of qualitative findings related to fear of addiction and side effects, feelings of hopelessness, unclear direction and information, social stigma, endurable symptoms, and illness as determinants for medication avoidance. SIGNIFICANCE OF RESULTS: This systematic review highlighted some patient beliefs related to medication hesitancy or avoidance. Clinicians should take patient beliefs and concerns into consideration when creating treatment regimens for people receiving palliative care to optimize medication adherence and the quality of care.

4.
Curr Protoc ; 2(11): e595, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36374013

RESUMO

The formation and persistence of DNA damage can impact biological processes such as DNA replication and transcription. To maintain genome stability and integrity, organisms rely on robust DNA damage repair pathways. Techniques to detect and locate DNA damage sites across a genome enable an understanding of the consequences of DNA damage as well as how damage is repaired, which can have key diagnostic and therapeutic implications. Importantly, advancements in technology have enabled the development of high-throughput sequencing-based DNA damage detection methods. These methods require DNA enrichment or amplification steps that limit the ability to quantitate the DNA damage sites. Further, each of these methods is typically tailored to detect only a specific type of damage. RAre DAmage and Repair (RADAR) sequencing is a DNA sequencing workflow that overcomes these limitations and enables detection and quantitation of DNA damage sites in any organism on a genome-wide scale. RADAR-seq works by replacing DNA damage sites with a patch of modified bases that can be directly detected by Pacific Biosciences Single-Molecule Real Time sequencing. Here, we present three protocols that enable detection of thymine dimers and ribonucleotides in bacterial and archaeal genomes. Basic Protocol 1 enables construction of a reference genome required for RADAR-seq analyses. Basic Protocol 2 describes how to locate, quantitate, and compare thymine dimer levels in Escherichia coli exposed to varying amounts of UV light. Basic Protocol 3 describes how to locate, quantitate, and compare ribonucleotide levels in wild-type and ΔRNaseH2 Thermococcus kodakarensis. Importantly, all three protocols provide in-depth steps for data analysis. Together they serve as proof-of-principle experiments that will allow users to adapt the protocols to locate and quantitate a wide variety of DNA damage sites in any organism. © 2022 New England Biolabs. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Constructing a reference genome utilizing SMRT sequencing Basic Protocol 2: Mapping and quantitating genomic thymine dimer formation in untreated versus UV-irradiated E. coli using RADAR-seq Basic Protocol 3: Mapping and quantitating genomic ribonucleotide incorporation in wildtype versus ΔRNaseH2 T. kodakarensis using RADAR-seq.


Assuntos
Reparo do DNA , Dímeros de Pirimidina , Dímeros de Pirimidina/genética , Reparo do DNA/genética , Escherichia coli/genética , Dano ao DNA/genética , Ribonucleotídeos , Genoma Arqueal
5.
Artigo em Inglês | MEDLINE | ID: mdl-36429656

RESUMO

Many tribal populations are characterized by health disparities, including higher rates of infection, metabolic syndrome, and cancer-all of which are mediated by the immune system. Members of the Navajo Nation have suffered chronic low-level exposure to metal mixtures from uranium mine wastes for decades. We suspect that such metal and metalloid exposures lead to adverse health effects via their modulation of immune system function. We examined the relationships between nine key metal and metalloid exposures (in blood and urine) with 11 circulating biomarkers (cytokines and CRP in serum) in 231 pregnant Navajo women participating in the Navajo Birth Cohort Study. Biomonitored levels of uranium and arsenic species were considerably higher in participants than NHANES averages. Each biomarker was associated with a unique set of exposures, and arsenic species were generally immunosuppressive (decreased cellular and humoral stimulating cytokines). Overall, our results suggest that environmental metal and metalloid exposures modulate immune status in pregnant Navajo women, which may impact long-term health outcomes in mothers and their children.


Assuntos
Arsênio , Indígenas Norte-Americanos , Urânio , Gravidez , Criança , Humanos , Feminino , Estudos de Coortes , Urânio/análise , Arsênio/efeitos adversos , Citocinas , Inquéritos Nutricionais , Coorte de Nascimento , Biomarcadores
6.
Sci Rep ; 12(1): 13017, 2022 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-35906281

RESUMO

In vitro transcribed synthetic messenger RNAs (mRNAs) represent a novel therapeutic modality. To overcome the inherent immunogenicity, as well as to increase the therapeutic efficacy of the molecules, uridine analogs-such as pseudouridine (Ψ) and N1-methyl-pseudouridine (m1Ψ), are incorporated in the synthetic mRNA. To decipher the fidelity with which these modifications are incorporated during the in vitro transcription (IVT) process, we compared the incorporation fidelity of uridine analogs with different RNA polymerases. We demonstrate that m1Ψ is incorporated with higher fidelity than Ψ. The fidelity of nucleotide incorporation differs between RNA polymerases; however, the spectrum of mutations observed between the RNAPs is similar. We also show that the array of nucleotide misincorporation is not dependent on the template DNA sequence context and that the distribution of these misincorporated nucleotides is not localized to any specific region along the length of the RNA. Based on our findings, we introduce a novel method to improve uridine analog incorporation fidelity during IVT. Our proof-of-concept experiments for higher-fidelity incorporation of uridine analogs during IVT provide guidelines when choosing RNAPs for the generation of modified uridine-containing mRNAs in vitro.


Assuntos
Nucleotídeos , Pseudouridina , Sequência de Bases , Pseudouridina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Uridina/metabolismo
7.
J Phys Chem A ; 125(45): 9736-9756, 2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34731566

RESUMO

The charge density distribution in a novel cocrystal (1) complex of 1,3-dimethylxanthine (theophylline) and propanedioic acid (malonic acid) has been determined. The molecules crystallize in the triclinic, centrosymmetric space group P1̅, with four independent molecules (Z = 4) in the asymmetric unit (two molecules each of theophylline and malonic acid). Theophylline has a notably high hygroscopic nature, and numerous cocrystals have shown a significant improvement in stability to humidity. A charge density study of the novel polymorph has identified interesting theoretical results correlating the stability enhancement of theophylline via cocrystallization. Topological analysis of the electron density highlighted key differences (up to 17.8) in Laplacian (∇2ρ) between the experimental (EXP) and single-point (SP) models, mainly around intermolecular-bonded carbonyls. Further investigation via molecular electrostatic potential maps reaffirmed that the charge redistribution enhanced intramolecular hydrogen bonding, predominantly for N(2') and N(2) (61.2 and 61.8 kJ mol-1, respectively). An overall weaker lattice energy of the triclinic form (-126.1 kJ mol-1) compared to that of the monoclinic form (-133.8 kJ mol-1) suggests a lower energy threshold to overcome to initiate dissociation. Future work via physical testing of the novel cocrystal in both dissolution and solubility will further solidify the correlation between theoretical and experimental results.


Assuntos
Teofilina , Cristalização , Ligação de Hidrogênio , Solubilidade , Molhabilidade
8.
Sci Rep ; 11(1): 160, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420304

RESUMO

The BLL lectin from the edible Japanese "Kurokawa" mushroom (Boletopsis leucomelaena) was previously reported to bind to N-glycans harboring terminal N-acetylglucosamine (GlcNAc) and to induce apoptosis in a leukemia cell line. However, its gene has not been reported. In this study, we used a transcriptomics-based workflow to identify a full-length transcript of a BLL functional ortholog (termed BGL) from Boletopsis grisea, a close North American relative of B. leucomelaena. The deduced amino acid sequence of BGL was an obvious member of fungal fruit body lectin family (Pfam PF07367), a highly conserved group of mushroom lectins with a preference for binding O-glycans harboring the Thomsen-Friedenreich antigen (TF-antigen; Galß1,3GalNAc-α-) and having two ligand binding sites. Functional characterization of recombinant BGL using glycan microarray analysis and surface plasmon resonance confirmed its ability to bind both the TF-antigen and ß-GlcNAc-terminated N-glycans. Structure-guided mutagenesis of BGL's two ligand binding clefts showed that one site is responsible for binding TF-antigen structures associated with O-glycans, whereas the second site specifically recognizes N-glycans with terminal ß-GlcNAc. Additionally, the two sites show no evidence of allosteric communication. Finally, mutant BGL proteins having single functional bindings site were used to enrich GlcNAc-capped N-glycans or mucin type O-glycopeptides from complex samples in glycomics and glycoproteomics analytical workflows.


Assuntos
Basidiomycota/metabolismo , Proteínas Fúngicas/metabolismo , Lectinas/metabolismo , Agaricales/química , Agaricales/genética , Agaricales/metabolismo , Sequência de Aminoácidos , Basidiomycota/química , Basidiomycota/genética , Sítios de Ligação , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Lectinas/química , Lectinas/genética , Polissacarídeos/química , Polissacarídeos/metabolismo , Ligação Proteica , Alinhamento de Sequência
9.
J Pain Palliat Care Pharmacother ; 34(4): 225-236, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32730108

RESUMO

Evidence to support the use of antipsychotic medications for the management of delirium symptoms remains limited. The primary objective of this study was to compare the effect of antipsychotic and non-antipsychotic treatments for delirium symptoms among palliative care inpatients. Secondary outcomes were use of midazolam and overall survival. This involved retrospective analysis of medical records (November 2018 to April 2019) for adult palliative care patients diagnosed with delirium at an Australian tertiary hospital. NuDESC was used to assess symptoms daily from baseline to Day 3. All 65 patients (mean age 73.5 ± 13.7 years, 48% female, 59% with cancer) included received standard care which included management of underlying causes of delirium symptoms, of which 17 received additional treatment using antipsychotic medications. Forty-eight did not receive any antipsychotic medication. An absolute reduction in NuDESC score was observed in the group that did not receive additional treatment using antipsychotics (by 1.37 units, 95% CI 0.79-1.95, p < 0.0001). A significantly higher proportion of midazolam use (n = 9, 53% versus n = 2, 4%, p < 0.001) and shorter median survival (13 days versus 26 days, p = 0.03) was observed in the group of patients that received antipsychotics. The use of antipsychotic medications in addition to standard treatments targeting underlying precipitants did not lead to a significant improvement in delirium symptoms and was associated with a greater midazolam use and lower median duration of survival. Individualized treatment of underlying causes still appears to be essential in the management of delirium in patients receiving palliative care.


Assuntos
Antipsicóticos , Delírio , Adulto , Idoso , Idoso de 80 Anos ou mais , Antipsicóticos/uso terapêutico , Austrália , Delírio/tratamento farmacológico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Cuidados Paliativos , Estudos Retrospectivos
10.
Tob Control ; 29(5): 570-576, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-31462578

RESUMO

INTRODUCTION: American Indians and Alaska Natives face disproportionately high rates of smoking and secondhand smoke (SHS) exposure. The Cheyenne River Sioux Tribe (CRST) is among the few Tribal Nations controlling commercial tobacco exposures in public and work places. We had an opportunity to explore effects of the new commercial tobacco-free policy (implemented in 2015) in an environmental health study (2014-2016) that collected information about commercial tobacco use and SHS prevalence and examined predictor variables of serum cotinine concentrations. METHODS: Self-reported survey data were used in quantile regression statistical modelling to explore changes in cotinine levels, based on smoking status, smokeless tobacco consumption and SHS exposure. RESULTS: From enrolled 225 adults, 51% (N=114) were current smokers. Among 88 non-tobacco users, 35 (40%) reported current SHS exposure. Significant differences in cotinine median concentrations were found among participants with and without current SHS exposure. Extremely high cotinine concentrations (~100 times larger than the median) were detected in some non-tobacco users. After implementing the new smoke-free air Tribal policy, cotinine decreased in participants with intermediate (3-15 ng/mL, non-tobacco users with SHS exposure) and high (>15 ng/mL, mainly tobacco users) cotinine levels showing association with an abatement of opportunities for SHS exposure. Significant predictors of cotinine levels were sampling year, current smoking and tobacco chewing. No gender differences were observed in cotinine. CONCLUSIONS: Our results show decrease in cotinine concentrations in CRST participants since implementation of their 'Smoke-Free Clean Air Act' in 2015.


Assuntos
Indígena Americano ou Nativo do Alasca , Cotinina/sangue , Política de Saúde , Prevenção do Hábito de Fumar , Fumar/sangue , Poluição por Fumaça de Tabaco/efeitos adversos , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
11.
DNA Repair (Amst) ; 80: 36-44, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31247470

RESUMO

RAre DAmage and Repair sequencing (RADAR-seq) is a highly adaptable sequencing method that enables the identification and detection of rare DNA damage events for a wide variety of DNA lesions at single-molecule resolution on a genome-wide scale. In RADAR-seq, DNA lesions are replaced with a patch of modified bases that can be directly detected by Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing. RADAR-seq enables dynamic detection over a wide range of DNA damage frequencies, including low physiological levels. Furthermore, without the need for DNA amplification and enrichment steps, RADAR-seq provides sequencing coverage of damaged and undamaged DNA across an entire genome. Here, we use RADAR-seq to measure the frequency and map the location of ribonucleotides in wild-type and RNaseH2-deficient E. coli and Thermococcus kodakarensis strains. Additionally, by tracking ribonucleotides incorporated during in vivo lagging strand DNA synthesis, we determined the replication initiation point in E. coli, and its relation to the origin of replication (oriC). RADAR-seq was also used to map cyclobutane pyrimidine dimers (CPDs) in Escherichia coli (E. coli) genomic DNA exposed to UV-radiation. On a broader scale, RADAR-seq can be applied to understand formation and repair of DNA damage, the correlation between DNA damage and disease initiation and progression, and complex biological pathways, including DNA replication.


Assuntos
Dano ao DNA , Reparo do DNA , Genoma Arqueal , Genoma Bacteriano , Testes de Mutagenicidade/métodos , Análise de Sequência de DNA/métodos , Replicação do DNA , DNA Arqueal , DNA Bacteriano/efeitos da radiação , Escherichia coli/genética , Escherichia coli/efeitos da radiação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Dímeros de Pirimidina , Ribonucleotídeos , Thermococcus/genética , Raios Ultravioleta
12.
Front Mol Biosci ; 6: 28, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069234

RESUMO

A variant of 9°N DNA polymerase [Genbank ID (AAA88769.1)] with three mutations (D141A, E143A, A485L) and commercialized under the name "Therminator DNA polymerase" has the ability to incorporate a variety of modified nucleotide classes. This Review focuses on how Therminator DNA Polymerase has enabled new technologies in synthetic biology and DNA sequencing. In addition, we discuss mechanisms for increased modified nucleotide incorporation.

13.
ACS Synth Biol ; 7(11): 2665-2674, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30335370

RESUMO

Synthetic biology relies on the manufacture of large and complex DNA constructs from libraries of genetic parts. Golden Gate and other Type IIS restriction enzyme-dependent DNA assembly methods enable rapid construction of genes and operons through one-pot, multifragment assembly, with the ordering of parts determined by the ligation of Watson-Crick base-paired overhangs. However, ligation of mismatched overhangs leads to erroneous assembly, and low-efficiency Watson Crick pairings can lead to truncated assemblies. Using sets of empirically vetted, high-accuracy junction pairs avoids this issue but limits the number of parts that can be joined in a single reaction. Here, we report the use of comprehensive end-joining ligation fidelity and bias data to predict high accuracy junction sets for Golden Gate assembly. The ligation profile accurately predicted junction fidelity in ten-fragment Golden Gate assembly reactions and enabled accurate and efficient assembly of a lac cassette from up to 24-fragments in a single reaction.


Assuntos
DNA/metabolismo , Biologia Sintética/métodos , Pareamento de Bases , DNA/química , DNA Ligases/metabolismo , Óperon Lac/genética
14.
Cell Rep ; 24(5): 1290-1300, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-30067983

RESUMO

In vivo, replication forks proceed beyond replication-blocking lesions by way of downstream repriming, generating daughter strand gaps that are subsequently processed by post-replicative repair pathways such as homologous recombination and translesion synthesis (TLS). The way these gaps are filled during TLS is presently unknown. The structure of gap repair synthesis was assessed by sequencing large collections of single DNA molecules that underwent specific TLS events in vivo. The higher error frequency of specialized relative to replicative polymerases allowed us to visualize gap-filling events at high resolution. Unexpectedly, the data reveal that a specialized polymerase, Pol V, synthesizes stretches of DNA both upstream and downstream of a site-specific DNA lesion. Pol V-mediated untargeted mutations are thus spread over several hundred nucleotides, strongly eliciting genetic instability on either side of a given lesion. Consequently, post-replicative gap repair may be a source of untargeted mutations critical for gene diversification in adaptation and evolution.


Assuntos
Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Mutagênese , Replicação do DNA , Escherichia coli
15.
Nucleic Acids Res ; 46(13): e79, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-29741723

RESUMO

DNA ligases are key enzymes in molecular and synthetic biology that catalyze the joining of breaks in duplex DNA and the end-joining of DNA fragments. Ligation fidelity (discrimination against the ligation of substrates containing mismatched base pairs) and bias (preferential ligation of particular sequences over others) have been well-studied in the context of nick ligation. However, almost no data exist for fidelity and bias in end-joining ligation contexts. In this study, we applied Pacific Biosciences Single-Molecule Real-Time sequencing technology to directly sequence the products of a highly multiplexed ligation reaction. This method has been used to profile the ligation of all three-base 5'-overhangs by T4 DNA ligase under typical ligation conditions in a single experiment. We report the relative frequency of all ligation products with or without mismatches, the position-dependent frequency of each mismatch, and the surprising observation that 5'-TNA overhangs ligate extremely inefficiently compared to all other Watson-Crick pairings. The method can easily be extended to profile other ligases, end-types (e.g. blunt ends and overhangs of different lengths), and the effect of adjacent sequence on the ligation results. Further, the method has the potential to provide new insights into the thermodynamics of annealing and the kinetics of end-joining reactions.


Assuntos
DNA Ligases , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Pareamento Incorreto de Bases , Reparo do DNA por Junção de Extremidades
16.
Nucleic Acids Res ; 46(11): 5753-5763, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29750267

RESUMO

Ribonucleic acid (RNA) is capable of hosting a variety of chemically diverse modifications, in both naturally-occurring post-transcriptional modifications and artificial chemical modifications used to expand the functionality of RNA. However, few studies have addressed how base modifications affect RNA polymerase and reverse transcriptase activity and fidelity. Here, we describe the fidelity of RNA synthesis and reverse transcription of modified ribonucleotides using an assay based on Pacific Biosciences Single Molecule Real-Time sequencing. Several modified bases, including methylated (m6A, m5C and m5U), hydroxymethylated (hm5U) and isomeric bases (pseudouridine), were examined. By comparing each modified base to the equivalent unmodified RNA base, we can determine how the modification affected cumulative RNA polymerase and reverse transcriptase fidelity. 5-hydroxymethyluridine and N6-methyladenosine both increased the combined error rate of T7 RNA polymerase and reverse transcriptases, while pseudouridine specifically increased the error rate of RNA synthesis by T7 RNA polymerase. In addition, we examined the frequency, mutational spectrum and sequence context of reverse transcription errors on DNA templates from an analysis of second strand DNA synthesis.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , RNA/química , Proteínas Virais/metabolismo , Sequência de Bases , DNA Complementar/biossíntese , DNA Complementar/química , RNA/biossíntese , Transcrição Reversa , Ribonucleotídeos/química , Ribonucleotídeos/metabolismo , Transcrição Gênica
17.
ACS Chem Neurosci ; 8(11): 2374-2380, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28841278

RESUMO

Adamantanyl benzamide 1 was identified as a potent P2X7R antagonist but failed to progress further due to poor metabolic stability. We describe the synthesis and SAR of a series of bioisosteres of benzamide 1 to explore improvements in the pharmacological properties of this lead. Initial efforts investigated a series of heteroaromatic bioisosteres, which demonstrated improved physicochemical properties but reduced P2X7R antagonism. Installation of bioisosteric fluorine on the adamantane bridgeheads was well tolerated and led to a series of bioisosteres with improved physicochemical properties and metabolic stability. Trifluorinated benzamide 34 demonstrated optimal physicochemical parameters, superior metabolic stability (ten times longer than lead benzamide 1), and an improved physicokinetic profile and proved effective in the presence of several known P2X7R polymorphisms.


Assuntos
Adamantano/análogos & derivados , Benzamidas/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X7/efeitos dos fármacos , Adamantano/farmacologia , Animais , Benzamidas/síntese química , Benzamidas/química , Benzamidas/farmacocinética , Biotransformação , Avaliação Pré-Clínica de Medicamentos , Estabilidade de Medicamentos , Humanos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Oxirredução , Polimorfismo de Nucleotídeo Único , Antagonistas do Receptor Purinérgico P2X/síntese química , Antagonistas do Receptor Purinérgico P2X/química , Antagonistas do Receptor Purinérgico P2X/farmacocinética , Ratos , Receptores Purinérgicos P2X7/genética , Relação Estrutura-Atividade
18.
PLoS One ; 12(7): e0181128, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28683110

RESUMO

[This corrects the article DOI: 10.1371/journal.pone.0169774.].

19.
PLoS One ; 12(1): e0169774, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28060945

RESUMO

Next-generation sequencing technology has enabled the detection of rare genetic or somatic mutations and contributed to our understanding of disease progression and evolution. However, many next-generation sequencing technologies first rely on DNA amplification, via the Polymerase Chain Reaction (PCR), as part of sample preparation workflows. Mistakes made during PCR appear in sequencing data and contribute to false mutations that can ultimately confound genetic analysis. In this report, a single-molecule sequencing assay was used to comprehensively catalog the different types of errors introduced during PCR, including polymerase misincorporation, structure-induced template-switching, PCR-mediated recombination and DNA damage. In addition to well-characterized polymerase base substitution errors, other sources of error were found to be equally prevalent. PCR-mediated recombination by Taq polymerase was observed at the single-molecule level, and surprisingly found to occur as frequently as polymerase base substitution errors, suggesting it may be an underappreciated source of error for multiplex amplification reactions. Inverted repeat structural elements in lacZ caused polymerase template-switching between the top and bottom strands during replication and the frequency of these events were measured for different polymerases. For very accurate polymerases, DNA damage introduced during temperature cycling, and not polymerase base substitution errors, appeared to be the major contributor toward mutations occurring in amplification products. In total, we analyzed PCR products at the single-molecule level and present here a more complete picture of the types of mistakes that occur during DNA amplification.

20.
Org Biomol Chem ; 15(3): 576-580, 2017 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-27991628

RESUMO

Preparation of N-(indol-2-yl)amides and N-(indol-3-yl)amides are scarce in the scientific literature due to unstable intermediates impeding current reported syntheses. We have employed cheap and readily available substrates in the Curtius rearrangement of indole-3-carboxazide to afford N-(indol-3-yl)amides. The reaction is observed for alkyl and aryl carboxylic acids and both N-substituted or 1H-indole derivatives are tolerated. This approach was extended to the preparation of N-(indol-2-yl)amides from the corresponding indole-2-carboxazides.

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