Direct Kinetic Fingerprinting for High-Accuracy Single-Molecule Counting of Diverse Disease Biomarkers.

Methods for detecting and quantifying disease biomarkers in biofluids with high specificity and sensitivity play a pivotal role in enabling clinical diagnostics, including point-of-care tests. The most widely used molecular biomarkers include proteins, nucleic acids, hormones, metabolites, and other small molecules. While numerous methods have been developed for analyzing biomarkers, most techniques are challenging to implement for clinical use due to insufficient analytical performance, high cost, and/or other practical shortcomings. For instance, the detection of cell-free nucleic acid (cfNA) biomarkers by digital PCR and next-generation sequencing (NGS) requires time-consuming nucleic acid extraction steps, often introduces enzymatic amplification bias, and can be costly when high specificity is required. While several amplification-free methods for detecting cfNAs have been reported, these techniques generally suffer from low specificity and sensitivity. Meanwhile, the quantification of protein biomarkers is generally performed using immunoassays such as enzyme-linked immunosorbent assay (ELISA); the analytical performance of these methods is often limited by the availability of antibodies with high affinity and specificity as well as the significant nonspecific binding of antibodies to assay surfaces. To address the drawbacks of existing biomarker detection methods and establish a universal diagnostics platform capable of detecting different types of analytes, we have developed an amplification-free approach, named single-molecule recognition through equilibrium Poisson sampling (SiMREPS), for the detection of diverse biomarkers with arbitrarily high specificity and single-molecule sensitivity. SiMREPS utilizes the transient, reversible binding of fluorescent detection probes to immobilized target molecules to generate kinetic fingerprints that are detected by single-molecule fluorescence microscopy. The analysis of these kinetic fingerprints enables nearly perfect discrimination between specific binding to target molecules and any nonspecific binding. Early proof-of-concept studies demonstrated the in vitro detection of miRNAs with a limit of detection (LOD) of approximately 1 fM and >500-fold selectivity for single-nucleotide polymorphisms. The SiMREPS approach was subsequently expanded to the detection of rare mutant DNA alleles from biofluids at mutant allele fractions of as low as 1 in 1 million, corresponding to a specificity of >99.99999%. Recently, SiMREPS was generalized to protein quantification using dynamically binding antibody probes, permitting LODs in the low-femtomolar to attomolar range. Finally, SiMREPS has been demonstrated to be suitable for the in situ detection of miRNAs in cultured cells, the quantification of small-molecule toxins and drugs, and the monitoring of telomerase activity at the single-molecule level. In this Account, we discuss the principles of SiMREPS for the highly specific and sensitive detection of molecular analytes, including considerations for assay design. We discuss the generality of SiMREPS for the detection of very disparate analytes and provide an overview of data processing methods, including the expansion of the dynamic range using super-resolution analysis and the improvement of performance using deep learning algorithms. Finally, we describe current challenges, opportunities, and future directions for the SiMREPS approach.

Ultraspecific analyte detection by direct kinetic fingerprinting of single molecules.

The detection and quantification of biomarkers have numerous applications in biological research and medicine. The most widely used methods to detect nucleic acids require amplification via the polymerase chain reaction (PCR). However, errors arising from the imperfect copying fidelity of DNA polymerases, limited specificity of primers, and heat-induced damage reduce the specificity of PCR-based methods, particularly for single-nucleotide variants. Furthermore, not all analytes can be amplified efficiently. While amplification-free methods avoid these pitfalls, the specificity of most such methods is strictly constrained by probe binding thermodynamics, which for example hampers detection of rare somatic mutations. In contrast, single-molecule recognition through equilibrium Poisson sampling (SiMREPS) provides ultraspecific detection with single-molecule and single-nucleotide sensitivity by monitoring the repetitive interactions of a fluorescent probe with surface-immobilized targets. In this review, we discuss SiMREPS in comparison with other analytical approaches, and describe its utility in quantifying a range of nucleic acids and other analytes.

Qualitative Evaluation of a Role Play Bullying Simulation.

Bullying against nurses is becoming a pervasive problem. In this article, a role play simulation designed for undergraduate nursing students is described. In addition, the evaluation findings from a subsample of students who participated in a role play simulation addressing bullying behaviors are reported. Focus group sessions were completed with a subset of eight students who participated in the intervention. Sessions were audiorecorded, transcribed verbatim, and analyzed using Colaizzi's procedural steps for qualitative analysis. Themes derived from the data were "The Experience of Being Bullied", "Implementation of the Program", "Desired Outcome of the Program", and "Context of Bullying in the Nursing Profession". Role play simulation was an effective and active learning strategy to diffuse education on bullying in nursing practice. Bullying in nursing was identified as a problem worthy of incorporation into the undergraduate nursing curriculum. To further enhance the learning experience with role play simulation, adequate briefing instructions, opportunity to opt out of the role play, and comprehensive debriefing are essential.

Relación entre el crecimiento estatural y la terapia con corticiodes en pacientes pediátricos con síndrome nefrótico tratados en el periodo de 2003 al 2009 en el Instituto Nacional de Salud del Niño / Relationship between growth and statural corticoids therapy in pediatric patients with nephrotic syndrome treated in the period 2003 to 2009 in the National Institute of Child Health

Objetivo: Conocer si existe relación entre el crecimiento estatural y el uso de corticoides en pacientes pediátricos con diagnóstico de Síndrome Nefrótico, atendidos en el Servicio de Nefrología del Instituto Nacional de Salud del Niño por un periodo de tres años, entre el 2003 y 2009. Materiales y Métodos: Se evaluó 70 historias de pacientes de Consultorio externo del Servicio de Nefrología del Instituto Nacional de Salud del Niño, con diagnóstico de Síndrome nefrótico, que tuvieran una evolución de tratamiento con corticoides de 3 años. Se realizó un estudio de tipo retrospectivo descriptivo, y se evaluaron las desviaciones estándar de la talla para la edad. Resultados: De un total de 70 pacientes en estudio se observo que el 62.9 por ciento eran de género masculino y 37.1 por ciento de género femenino. El promedio de edad de inicio de tratamiento fue de 6.6 años. El 56 por ciento de pacientes pertenecio al grupo II (4-6 cursos de corticoides), el 40 por ciento pertenecio al grupo I (1-3 cursos de corticoides), 3 por ciento fueron pacientes considerados en el grupo III(7-9 cursos de corticoides) y un 1 por ciento en el grupo IV(igual o mayor a 10 cursos). En el grupo I se observó que al inicio del tratamiento, presentó una desviación estándar (DS) promedio de - 1,8; en el primer control obtuvo una -1.2 DS, al segundo control y tercer control se encontraron valores de -1,1 en ambos casos; todos estos valores dentro de los rangos normales de crecimiento para la edad (-2DS a + 2DS). Los pacientes del Grupo 11, al inicio del tratamiento presentaron una OS de -1,57, en el primer control se encontró que el promedio fue de -1,63 OS, al segundo año se encontró que el promedio de OS fue de -1.59 y al tercer año de -1.54. Valores dentro del rango normal de crecimiento para la edad (-2DS a + 2DS)...

Objective: To determinate whether a relationship exists between the growing stature and the use of corticosteroids in pediatric patients diagnosed with nefrotic syndrome treated at the Nefrology Service of the National Institute of Child Health for a period of three years, between 2003 and 2009. Materials and Methods: We evaluated 70 patients stories outpatient c1inic of the Nephrology Department at the National Institute of Child Health, diagnosed with nephrotic syndrome that had an evolution of the treatment with corticosteroids for three years. We did a descriptive retrospective study and evaluated the standard deviations of height for age. Results: Of a total of 70 patients under study was observed that 62.9 per cent were male and 37.1 per cent were female. The average age at the beginning of the treatment was 6.6 years. A 56 per cent of the patients who belonged to group 11 (4-6 courses of corticosteroids), 40 per cent belonged to group I (1-3 corticoids courses), 3 per cent of the patients were considered in group 111 (7-9 courses of corticosteroids) and 1 per cent in group IV (more or equal to 10 courses). In group 1, was observed that at the beginning of the treatment, it presented a standard deviation (SD) averaged -1.8; in the first control, it obtained a -1.2 SD, in the second and third control, in both cases it obtained - 1,1; all of these values within the normal range for age growth (-205 a + 205). The patients in Group 11, at the beginning of the treatment showed DS of 1.57. The first control showed that the average was -1.63 DS, in the second year it was found that the average was -1.59 DS and in the third year was found an average of -1,54; all of these values within the normal range for age growth (-2 SD to + 2 SD)...