Peroxidase proximity selection to identify aptamers targeting a subcellular location.

The efficient and specific delivery of functional cargos such as small-molecule drugs, proteins, or nucleic acids across lipid membranes and into subcellular compartments is a significant unmet need in nanomedicine and molecular biology. Systematic Evolution of Ligands by EXponential enrichment (SELEX) exploits vast combinatorial nucleic acid libraries to identify short, nonimmunogenic single-stranded DNA molecules (aptamers) capable of recognizing specific targets based on their 3D structures and molecular interactions. While SELEX has previously been applied to identify aptamers that bind specific cell types or gain cellular uptake, selection of aptamers capable of carrying cargos to specific subcellular compartments is challenging. Here, we describe peroxidase proximity selection (PPS), a generalizable subcellular SELEX approach. We implement local expression of engineered ascorbate peroxidase APEX2 to biotinylate naked DNA aptamers capable of gaining access to the cytoplasm of living cells without assistance. We discovered DNA aptamers that are preferentially taken up into endosomes by macropinocytosis, with a fraction apparently accessing APEX2 in the cytoplasm. One of these selected aptamers is capable of endosomal delivery of an IgG antibody.

Blood cultures in the emergency department evaluation of childhood pneumonia.

BACKGROUND: Blood cultures are frequently obtained in the emergency department (ED) evaluation of children with community-acquired pneumonia (CAP). OBJECTIVES: To determine the prevalence of bacteremia in children presenting to the ED with CAP, identify subgroups at increased risk for bacteremia, and quantify the effect of positive blood cultures on management. METHODS: This case-control study was nested within a cohort of children followed up at 35 pediatric practices. Patients from this cohort who were ≤18 years of age, evaluated in the ED in 2006-2007, and diagnosed with CAP were eligible. Cases were those with bacteremia. Controls included those with negative blood cultures and those without blood cultures performed. RESULTS: A total of 877 (9.6%) of 9099 children with CAP were evaluated in the ED. The mean age was 3.6 years; 53% were male. Blood cultures were obtained from 291 children (33.2%). Overall, the prevalence of bacteremia was 2.1% (95% confidence interval [CI]: 0.8%-4.4%). Bacteremia occurred in 2.6% (95% CI: 1.0%-5.6%) with an infiltrate on chest radiograph and in 13.0% (95% CI: 2.8%-33.6%) with complicated pneumonia. Streptococcus pneumoniae accounted for 4 of the 6 cases of bacteremia. Blood culture results altered management in 5 of the 6 bacteremic patients; 1 had an appropriate broadening and 4 had an appropriate narrowing of coverage. The contamination rate was 1.0% (95% CI: 0.2%-3.0%). CONCLUSION: Children presenting to the ED for evaluation of CAP are at low-risk for bacteremia. Although positive blood cultures frequently altered clinical management, the overall impact was small because of the low prevalence of bacteremia.