Comparison between BNP and NT-proBNP in pediatric populations.

BACKGROUND: B-type natriuretic peptide (BNP) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) are essential biomarkers for the evaluation of cardiac pathologies. However, pediatric reference intervals for BNP and NT-proBNP are not well defined and concordance between them in the evaluation of pediatric patients has been poorly described. METHODS: Paired BNP and NT-proBNP testing was performed on 311 specimens representing 175 pediatric patients. Pediatric BNP and NT-proBNP reference intervals derived from the literature were used to evaluate concordance of results based on age group and cardiac pathology. RESULTS: Deming regression analysis of BNP and NT-proBNP results revealed a slope of 13.63 (95% CI, 10.35-16.92) and y-intercept of -977.8 (-2063-107.2) with a positive Spearman correlation (r = 0.91). By age group, concordance kappa between BNP and NT-proBNP was 1.0 for 0-10 days, 0.23 (0-0.62) for 11-30 days, 0.82 (0.67-0.97) for 31 days-1 year, 0.81 (0.57-1.0) for 1-2 years and 0.73 (0.64-0.86) for 2-18 years. The ratio of NT-proBNP to BNP was lowest in heart transplant patients (ratio, 6.5 [95% CI, 5.1-8.1]) relative to those with heart disease (10.5 [8.8-13.7]) and pulmonary hypertension (14.2 [11.3-16.0]) but no differences in concordance were observed. For serial specimens, 21% displayed inverse, discordant changes in BNP and NT-proBNP results. Review of discordant serial results revealed that kinetics of changes was comparable and unlikely to be clinically significant. CONCLUSIONS: There is positive correlation and moderate concordance between BNP and NT-proBNP in the pediatric population studied.

Biomarkers and Clinical Laboratory Detection of Acute and Chronic Ethanol Use.

BACKGROUND: Ethanol use can lead to many health and socio-economic problems. Early identification of risky drinking behaviors helps provide timely clinical and social interventions. Laboratory testing of biomarkers of ethanol use supports the timely identification of individuals with risky drinking behaviors. This review provides an overview of the utility and limitations of ethanol biomarkers in the clinical laboratory. CONTENT: Direct assessment of ethanol in tissues and body fluids has limited utility due to the pharmacokinetics of ethanol. Therefore, the evaluation of ethanol use relies on nonvolatile metabolites of ethanol (direct biomarkers) and measurement of the physiological response to the toxic metabolites of ethanol (indirect biomarkers). Ethanol biomarkers help monitor both chronic and acute ethanol use. The points discussed here include the clinical utility of ethanol biomarkers, testing modalities used for laboratory assessment, the specimens of choice, limitations, and clinical interpretation of results. Finally, we discuss the ethical principles that should guide physicians and laboratorians when using these tests to evaluate alcohol use. SUMMARY: Indirect biomarkers such as carbohydrate-deficient transferrin, mean corpuscular volume, and liver enzymes activities may suggest heavy ethanol use. They lack sensitivity and specificity for timely detection of risky drinking behavior and have limited utility for acute ethanol use. Direct biomarkers such as ethyl glucuronide, ethyl sulfate, and phosphatidylethanol are considered sensitive and specific for detecting acute and chronic ethanol use. However, laboratory assessment and result interpretation lack standardization, limiting clinical utility. Ethical principles including respect for persons, beneficence, and justice should guide testing.

Aptamers with Tunable Affinity Enable Single-Molecule Tracking and Localization of Membrane Receptors on Living Cancer Cells.

Tumor cell-surface markers are usually overexpressed or mutated protein receptors for which spatiotemporal regulation differs between and within cancers. Single-molecule fluorescence imaging can profile individual markers in different cellular contexts with molecular precision. However, standard single-molecule imaging methods based on overexpressed genetically encoded tags or cumbersome probes can significantly alter the native state of receptors. We introduce a live-cell points accumulation for imaging in nanoscale topography (PAINT) method that exploits aptamers as minimally invasive affinity probes. Localization and tracking of individual receptors are based on stochastic and transient binding between aptamers and their targets. We demonstrated single-molecule imaging of a model tumor marker (EGFR) on a panel of living cancer cells. Affinity to EGFR was finely tuned by rational engineering of aptamer sequences to define receptor motion and/or native receptor density.

Modular cell-internalizing aptamer nanostructure enables targeted delivery of large functional RNAs in cancer cell lines.

Large RNAs and ribonucleoprotein complexes have powerful therapeutic potential, but effective cell-targeted delivery tools are limited. Aptamers that internalize into target cells can deliver siRNAs (<15 kDa, 19-21 nt/strand). We demonstrate a modular nanostructure for cellular delivery of large, functional RNA payloads (50-80 kDa, 175-250 nt) by aptamers that recognize multiple human B cell cancer lines and transferrin receptor-expressing cells. Fluorogenic RNA reporter payloads enable accelerated testing of platform designs and rapid evaluation of assembly and internalization. Modularity is demonstrated by swapping in different targeting and payload aptamers. Both modules internalize into leukemic B cell lines and remained colocalized within endosomes. Fluorescence from internalized RNA persists for ≥2 h, suggesting a sizable window for aptamer payloads to exert influence upon targeted cells. This demonstration of aptamer-mediated, cell-internalizing delivery of large RNAs with retention of functional structure raises the possibility of manipulating endosomes and cells by delivering large aptamers and regulatory RNAs.

Toward the Selection of Cell Targeting Aptamers with Extended Biological Functionalities to Facilitate Endosomal Escape of Cargoes.

Over the past decades there have been exciting and rapid developments of highly specific molecules to bind cancer antigens that are overexpressed on the surfaces of malignant cells. Nanomedicine aims to exploit these ligands to generate nanoscale platforms for targeted cancer therapy, and to do so with negligible off-target effects. Aptamers are structured nucleic acids that bind to defined molecular targets ranging from small molecules and proteins to whole cells or viruses. They are selected through an iterative process of amplification and enrichment called SELEX (systematic evolution of ligands by exponential enrichment), in which a combinatorial oligonucleotide library is exposed to the target of interest for several repetitive rounds. Nucleic acid ligands able to bind and internalize into malignant cells have been extensively used as tools for targeted delivery of therapeutic payloads both in vitro and in vivo. However, current cell targeting aptamer platforms suffer from limitations that have slowed their translation to the clinic. This is especially true for applications in which the cargo must reach the cytosol to exert its biological activity, as only a small percentage of the endocytosed cargo is typically able to translocate into the cytosol. Innovative technologies and selection strategies are required to enhance cytoplasmic delivery. In this review, we describe current selection methods used to generate aptamers that target cancer cells, and we highlight some of the factors that affect productive endosomal escape of cargoes. We also give an overview of the most promising strategies utilized to improve and monitor endosomal escape of therapeutic cargoes. The methods we highlight exploit tools and technologies that can potentially be incorporated in the SELEX process. Innovative selection protocols may identify aptamers with extended biological functionalities that allow effective cytosolic translocation of therapeutics. This in turn may facilitate successful translation of these platforms into clinical applications.

A Fluorescent Split Aptamer for Visualizing RNA-RNA Assembly In Vivo.

RNA-RNA assembly governs key biological processes and is a powerful tool for engineering synthetic genetic circuits. Characterizing RNA assembly in living cells often involves monitoring fluorescent reporter proteins, which are at best indirect measures of underlying RNA-RNA hybridization events and are subject to additional temporal and load constraints associated with translation and activation of reporter proteins. In contrast, RNA aptamers that sequester small molecule dyes and activate their fluorescence are increasingly utilized in genetically encoded strategies to report on RNA-level events. Split-aptamer systems have been rationally designed to generate signal upon hybridization of two or more discrete RNA transcripts, but none directly function when expressed in vivo. We reasoned that the improved physiological properties of the Broccoli aptamer enable construction of a split-aptamer system that could function in living cells. Here we present the Split-Broccoli system, in which self-assembly is nucleated by a thermostable, three-way junction RNA architecture and fluorescence activation requires both strands. Functional assembly of the system approximately follows second-order kinetics in vitro and improves when cotranscribed, rather than when assembled from purified components. Split-Broccoli fluorescence is digital in vivo and retains functional modularity when fused to RNAs that regulate circuit function through RNA-RNA hybridization, as demonstrated with an RNA Toehold switch. Split-Broccoli represents the first functional split-aptamer system to operate in vivo. It offers a genetically encoded and nondestructive platform to monitor and exploit RNA-RNA hybridization, whether as an all-RNA, stand-alone AND gate or as a tool for monitoring assembly of RNA-RNA hybrids.