Competition between bridged dinucleotides and activated mononucleotides determines the error frequency of nonenzymatic RNA primer extension

Nonenzymatic copying of RNA templates with activated nucleotides is a useful model for studying the emergence of heredity at the origin of life. Previous experiments with defined-sequence templates have pointed to the poor fidelity of primer extension as a major problem. Here we examine the origin of mismatches during primer extension on random templates in the simultaneous presence of all four 2-aminoimidazole-activated nucleotides. Using a deep sequencing approach that reports on millions of individual template-product pairs, we are able to examine correct and incorrect polymerization as a function of sequence context. We have previously shown that the predominant pathway for primer extension involves reaction with imidazolium-bridged dinucleotides, which form spontaneously by the reaction of two mononucleotides with each other. We now show that the sequences of correctly paired products reveal patterns that are expected from the bridged dinucleotide mechanism, whereas those associated with mismatches are consistent with direct reaction of the primer with activated mononucleotides. Increasing the ratio of bridged dinucleotides to activated mononucleotides, either by using purified components or by using isocyanide-based activation chemistry, reduces the error frequency. Our results point to testable strategies for the accurate nonenzymatic copying of arbitrary RNA sequences.

Competition between bridged dinucleotides and activated mononucleotides determines the error frequency of nonenzymatic RNA primer extension

ABSTRACTNonenzymatic copying of RNA templates with activated nucleotides is a useful model for studying the emergence of heredity at the origin of life. Previous experiments with defined-sequence templates have pointed to the poor fidelity of primer extension as a major problem. Here we examine the origin of mismatches during primer extension on random templates in the simultaneous presence of all four 2-aminoimidazole-activated nucleotides. Using a deep sequencing approach that reports on millions of individual template-product pairs, we are able to examine correct and incorrect polymerization as a function of sequence context. We have previously shown that the predominant pathway for primer extension involves reaction with imidazolium-bridged dinucleotides, which form spontaneously by the reaction of two mononucleotides with each other. We now show that the sequences of correctly paired products reveal patterns that are expected from the bridged dinucleotide mechanism, whereas those associated with mismatches are consistent with direct reaction of the primer with activated mononucleotides. Increasing the ratio of bridged dinucleotides to activated mononucleotides, either by using purified components or by using isocyanide-based activation chemistry, reduces the error frequency. Our results point to testable strategies for the accurate nonenzymatic copying of arbitrary RNA sequences.

A study to investigate the prevalence of device specific errors in inhaler technique in adults with airway disease (The SCORES Study): Protocol for a prevalence study (Preprint)

BACKGROUND It is a recurring theme in clinical practice that patients using inhaled medications via an inhaler do not use their device to a standard that allows for optimum therapeutic effect; with some studies showing that up to 90% of people do not use their inhalers properly. Observation and correction of inhaler technique by healthcare professionals is advised in both national and international guidelines and should be performed at every opportunity to ensure that the optimum inhaler technique is being achieved by the user. This study will deliver greater understanding of which technique errors are made most frequently by people using 13 different inhaler types. OBJECTIVE This study aims to identify and compare inhaler technique errors and their prevalence in adults, using device-specific checklists using manufacturers’ guidelines, for 13 specific inhaler types across all lung conditions and to correlate these errors with possible determinants of poor technique. It also aims to assess the error frequency at each step in the device-specific questionnaires and compare error rates between device types. METHODS In a single visit, participants using an inhaler included in the inclusion criteria will have their inhaler technique observed, recorded using device-specific checklists and then optimised. RESULTS The study is already underway, and it is anticipated that the results will be available by 2021. CONCLUSIONS The SCORES Study will ascertain the prevalence of device-specific inhaler technique errors at each step in the device-specific checklists, compare error rates between 13 device types and correlate these errors with possible determinants of poor technique. Future work will involve the clarification and classification of these errors into ‘critical’ and ‘non-critical’ categories. CLINICALTRIAL ClinicalTrials.gov; NCT04262271 https://clinicaltrials.gov/ct2/show/NCT04262271?term=NCT04262271&draw=2&rank=1

A critical survey on the kinetic assays of DNA polymerase fidelity from a new theoretical perspective

The high fidelity of DNA polymerase is critical for the faithful replication of genomic DNA. Several approaches were proposed to quantify the fidelity of DNA polymerase. Direct measurements of the error frequency of the replication products definitely give the true fidelity but turn out very hard to implement. Two biochemical kinetic approaches, the steady-state assay and the transient-state assay, were then suggested and widely adopted. In these assays, the error frequency is indirectly estimated by using the steady-state or the transient-state kinetic theory combined with the measured kinetic rates. However, whether these indirectly estimated fidelities are equivalent to the true fidelity has never been clarified theoretically, and in particular there are different strategies to quantify the proofreading efficiency of DNAP but often lead to inconsistent results. The reason for all these confusions is that it’s mathematically challenging to formulate a rigorous and general theory of the true fidelity. Recently we have succeeded to establish such a theoretical framework. In this paper, we develop this theory to make a comprehensive examination on the theoretical foundation of the kinetic assays and the relation between fidelities obtained by different methods. We conclude that while the steady-state assay and the transient-state assay can always measure the true fidelity of exonuclease-deficient DNA polymerases, they only do so for exonuclease-efficient DNA polymerases conditionally (the proper way to use these assays to quantify the proofreading efficiency is also suggested). We thus propose a new kinetic approach, the single-molecule assay, which indirectly but precisely characterizes the true fidelity of either exonuclease-deficient or exonuclease-efficient DNA polymerases.

Morphological Error Analysis of Muhasabah Column at Sabili Islamic Magazine

This study aims to describe the morphological errors in the Muhasabah of Sabili Islamic Magazine March 10 edition which are caused by the wrong choice of affixes, wrong rephrasing, wrong composing of compound words, and wrong choice of word forms. The research method used in this research was a qualitative method. The steps taken by the writer in analyzing the data were as follows: (1) counting the number of sentences, (2) identifying sentences, (3) analyzing errors, (4) calculating the frequency of the error, (5) calculating the percentage of error, (6) interpreting the research results, and (7) concluding the research results. The result concludes that (1) the error frequency is 23 cases; (2) the percentage of errors is 43.40%; (3) medium error category (25% - 50%); (4) the morphological error was caused by the wrong choice of affixes, the wrong use of repeating words, the wrong formulation of compound words, and the wrong form; and (5) mistakes that are often made by language users should be reduced or completely eliminated if possible.

Electronic prescription: frequency and severity of medication errors

SUMMARY OBJECTIVE: To assess the frequency and severity of prescriptions errors with potentially dangerous drugs (heparin and potassium chloride for injection concentrate) before and after the introduction of a computerized provider order entry (CPOE) system. METHODS: This is a retrospective study that compared errors in manual/pre-typed prescriptions in 2007 (Stage 1) with CPOE prescriptions in 2014 (Stage 2) (Total = 1,028 prescriptions), in two high-complexity hospitals of Belo Horizonte, Brasil. RESULTS: An increase of 25% in the frequency of errors in Hospital 1 was observed after the intervention (p<0.001). In contrast, a decreased error frequency of 85% was observed in Hospital 2 (p<0.001). Regarding potassium chloride, the error rate remained unchanged in Hospital 1 (p>0.05). In Hospital 2, a significant decrease was recorded in Stage 2 (p<0.001). A reduced error severity with heparin (p<0.001) was noted, while potassium chloride-related prescription severity remain unchanged (p> 0.05). CONCLUSIONS: The frequency and severity of medication errors after the introduction of CPOE was affected differently in the two hospitals, which shows a need for thorough observation when the prescription system is modified. Control of new potential errors introduced and their causes for the adoption of measures to prevent these events must be in place during and after the implementation of this technology.