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A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Wright-Patterson Air Force Base

The Henry M. Jackson Foundation

This study investigated the impact of work interfering with family (WIF) and family interfering with work (FIW) on women's organizational commitment and examined both the direct and moderating effects of their perceived organizational support. Participants were 143 professional employed mothers with at least 1 preschool-age child. The study found that WIF was positively related to continuance organizational commitment but unrelated to affective commitment, and FIW was not related to either form of organizational commitment. Results also indicated that perceived organizational support exhibited a main effect on both types of commitment.

Work-family conflict, perceived organizational support, and organizational commitment among employed mothers.

To document demographic characteristics and prevalence of infectious diseases in refugees and humanitarian entrants attending the Migrant Health Unit (MHU) in Perth for health assessment from 1 January 2003 to 31 December 2004.

Changing faces: A review of infectious disease screening of refugees by the Migrant Health Unit, Western Australia in 2003 and 2004.

Amlodipine toxicity vs. exposure in children.

We report a simple square capillary-based cell affinity chromatography device that utilizes a coating of aptamers for selective capture of target cancer cells from a flowing suspension. The device consists of a square capillary with an inner diameter of roughly five cell diameters, connected via Teflon tubing to a syringe. Aptamers are immobilized on the inner surface of the capillary through biotin-avidin chemistry, the extent of which can be controlled by adjusting the aptamer concentration. Introduction of different cell types into separate devices, as well as mixtures of target and non-target cells, demonstrated that aptamer-target cells can be captured in significantly higher concentrations compared to non-target cells. Once optimized, 91.1 ± 3.5% capture efficiency of target leukemia cells was reported, as well as 97.2 ± 2.8% and 83.6 ± 5.8% for two different colon cancer cell lines. In addition, cells captured in the device were imaged, and the square capillary exhibited better optical properties than standard cylindrical capillaries, leading to the detection of leukemia cells in blood samples. Compared to current microfluidic cell affinity devices, this capture device requires no complicated design or fabrication steps. By providing a simple means of detecting and imaging cancer cells in the blood, this work has potential to directly assist clinicians in determining disease prognosis and measuring therapeutic response.

Capturing cancer cells using aptamer-immobilized square capillary channels.

Bedside ultrasound is an extremely valuable and rapidly accessible diagnostic and therapeutic modality in potentially life- and limb-threatening situations in the emergency department. In this report, the authors discuss the role of ultrasound in quick assessment of pathologic conditions and its use to aid in diagnostic and therapeutic interventions.

Ultrasound-guided procedures in the emergency department-diagnostic and therapeutic asset.

Adverse drug reactions, including severe patient bleeding, may occur following the administration of anticoagulant drugs. Bivalirudin is a synthetic anticoagulant drug sometimes employed as a substitute for heparin, a commonly used anticoagulant that can cause a condition called heparin-induced thrombocytopenia (HIT). Although bivalrudin has the advantage of not causing HIT, a major concern is lack of an antidote for this drug. In contrast, medical professionals can quickly reverse the effects of heparin using protamine. This report details the selection of an aptamer to bivalirudin that functions as an antidote in buffer. This was accomplished by immobilizing the drug on a monolithic column to partition binding sequences from nonbinding sequences using a low-pressure chromatography system and salt gradient elution. The elution profile of binding sequences was compared to that of a blank column (no drug), and fractions with a chromatographic difference were analyzed via real-time PCR (polymerase chain reaction) and used for further selection. Sequences were identified by 454 sequencing and demonstrated low micromolar dissociation constants through fluorescence anisotropy after only two rounds of selection. One aptamer, JPB5, displayed a dose-dependent reduction of the clotting time in buffer, with a 20 µM aptamer achieving a nearly complete antidote effect. This work is expected to result in a superior safety profile for bivalirudin, resulting in enhanced patient care.

Selection of an aptamer antidote to the anticoagulant drug bivalirudin.

Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator that represents a promising target for the treatment of several metabolic diseases. Administration of recombinant wild type FGF21 to diabetic animals leads to a dramatic improvement in glycaemia and ameliorates other systemic measures of metabolic health. Here we report the pharmacologic outcomes observed in non-human primates upon administration of a recently described FGF21 analogue, LY2405319 (LY). Diabetic rhesus monkeys were treated subcutaneously with LY once daily for a period of seven weeks. The doses of LY used were 3, 9 and 50 mg/kg each delivered in an escalating fashion with washout measurements taken at 2, 4, 6 and 8 weeks following the final LY dose. LY therapy led to a dramatic and rapid lowering of several important metabolic parameters including glucose, body weight, insulin, cholesterol and triglyceride levels at all doses tested. In addition, we observed favorable changes in circulating profiles of adipokines, with increased adiponectin and reduced leptin indicative of direct FGF21 action on adipose tissue. Importantly, and for the first time we show that FGF21 based therapy has metabolic efficacy in an animal with late stage diabetes. While the glycemic efficacy of LY in this animal was partially attenuated its lipid lowering effect was fully preserved suggesting that FGF21 may be a viable treatment option even in patients with advanced disease progression. These findings support continued exploration of the FGF21 pathway for the treatment of metabolic disease.

LY2405319, an Engineered FGF21 Variant, Improves the Metabolic Status of Diabetic Monkeys.

Selection of aptamers that bind a specific ligand usually begins with a random library of RNA sequences, and many aptamers selected from such random pools have a simple stem-loop structure. We present here a computational approach for designing a starting library of RNA sequences with increased formation of complex structural motifs and enhanced affinity to a desired target molecule. Our approach consists of two steps: (1) generation of RNA sequences based on customized patterning of nucleotides with increased probability of forming a base pair and (2) a high-throughput virtual screening of the generated library to select aptamers with binding affinity to a small-molecule target. We developed a set of criteria that allows one to select a sequence with potential binding affinity from a pool of random sequences and designed a protocol for RNA 3D structure prediction. The proposed approach significantly reduces the RNA sequence search space, thus accelerating the experimental screening and selection of high-affinity aptamers.

Computational design of RNA libraries for in vitro selection of aptamers.

The "thoracic spine sign" is visualized when anechoic or hypoechoic fluid is present in the pleural space. Fluid serves as a medium through which the thoracic vertebral bodies are visualized above the diaphragm. We present three cases of emergency department patients with a thoracic spine sign identified on bedside ultrasound. These patients were subsequently diagnosed with pleural fluid accumulations on chest radiograph. Our findings suggest that the identification of the sonographic spine sign may aid the physician in the evaluation and diagnosis of emergency department patients with thoracic complaints.

The thoracic spine sign in bedside ultrasound. Three cases report.

The first-known aptamer for the stress biomarker cortisol was selected using a tunable stringency magnetic bead selection strategy. The capture DNA probe immobilized on the beads was systematically lengthened to increase the number of bases bound to the complementary pool primer regions following selection enrichment. This resulted in a single sequence (15-1) dominating the final round 15 pool, where the same sequence was the second-highest copy number candidate in the enriched pool with the shorter capture DNA probe (round 13). A thorough analysis of the next-generation sequencing results showed that a high copy number may only correlate with enhanced affinity under certain stringency and enrichment conditions, in contrast with prior published reports. Aptamer 15-1 demonstrated enhanced binding to cortisol (K(d) = 6.9 ± 2.8 μM by equilibrium dialysis; 16.1 ± 0.6 μM by microscale thermophoresis) when compared with the top sequence from round 13 and the negative control progesterone. Whereas most aptamer selections terminate at the selection round demonstrating the highest enrichment, this work shows that extending the selection with higher stringency conditions leads to lower amounts eluted by the target but higher copy numbers of a sequence with enhanced binding. The structure-switching aptamer was applied to a gold nanoparticle assay in buffer and was shown to discriminate between cortisol and two other stress biomarkers, norepinephrine and epinephrine, and a structurally analogous biomarker of liver dysfunction, cholic acid. We believe this approach enhances aptamer selection and serves as proof-of-principle work toward development of point-of-care diagnostics for medical, combat, or bioterrorism targets.

Jennifer A. Martin

711th Human Performance Wing,
Human Effectiveness Directorate,
Air Force Research Laboratory,
711th Human Performance Wing, Human Effectiveness Directorate, Air Force Research Laboratory

Wright-Patterson Air Force Base

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Jennifer A. Martin

711th Human Performance Wing,Human Effectiveness Directorate,Air Force Research Laboratory,711th Human Performance Wing, Human Effectiveness Directorate, Air Force Research Laboratory

Wright-Patterson Air Force Base

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

711th Human Performance Wing,
Human Effectiveness Directorate,
Air Force Research Laboratory,
711th Human Performance Wing, Human Effectiveness Directorate, Air Force Research Laboratory