Rational engineering of an erythropoietin fusion protein to treat hypoxia.

Erythropoietin enhances oxygen delivery and reduces hypoxia-induced cell death, but its pro-thrombotic activity is problematic for use of erythropoietin in treating hypoxia. We constructed a fusion protein that stimulates red blood cell production and neuroprotection without triggering platelet production, a marker for thrombosis. The protein consists of an anti-glycophorin A nanobody and an erythropoietin mutant (L108A). The mutation reduces activation of erythropoietin receptor homodimers that induce erythropoiesis and thrombosis, but maintains the tissue-protective signaling. The binding of the nanobody element to glycophorin A rescues homodimeric erythropoietin receptor activation on red blood cell precursors. In a cell proliferation assay, the fusion protein is active at 10-14 M, allowing an estimate of the number of receptor-ligand complexes needed for signaling. This fusion protein stimulates erythroid cell proliferation in vitro and in mice, and shows neuroprotective activity in vitro. Our erythropoietin fusion protein presents a novel molecule for treating hypoxia.

Design of peptide nucleic acid probes on plasmonic gold nanorods for detection of circulating tumor DNA point mutations.

Here we present a gold nanorod-based platform for the sequence-specific detection of circulating tumor DNA (ctDNA) point mutations without the need for amplification or fluorescence labeling. Peptide nucleic acid probes complimentary to the G12V mutation in the KRAS gene were conjugated to gold nanorods, and the localized surface plasmon resonance absorbance through the sample was measured after exposure to synthetic ctDNA at various concentrations. Each step of the reaction was thoroughly controlled, starting from reagent concentrations and including conjugation, sonication, and incubation time. The platform was evaluated in both buffer and spiked healthy patient serum, demonstrating a linear working range below 125 nanograms of ctDNA per milliliter solution, and an effective limit of detection of 2 nanograms of ctDNA per milliliter. A clear distinction between mutant and wild type synthetic ctDNA was also found using this platform. In order to improve upon the selectivity of the sensor, a DNA hybridization simulation was performed to understand how the addition of mutations to the peptide nucleic acid probe could enhance the selectivity for capture of mutant over wild type sequences. The top candidate from the simulations, which had an additional mutation two base pairs away from the mutation of interest, had a significant impact on the selectivity between mutant and wild type capture. This paper provides a framework for sequence-specific capture of ctDNA, and a method of improving selectivity for desired point mutations through careful probe design.

Liquid biopsy on chip: a paradigm shift towards the understanding of cancer metastasis.

This comprehensive review serves as a guide for developing scalable and robust liquid biopsies on chip for capture, detection, and analysis of circulating tumor cells (CTCs). Liquid biopsy, the detection of biomarkers from body fluids, has proven challenging because of CTC rarity and the heterogeneity of CTCs shed from tumors. The review starts with the underlying biological mechanisms that make liquid biopsy a challenge before moving into an evaluation of current technological progress. Then, a framework for evaluation of the technologies is presented with special attention to throughput, capture rate, and cell viability for analysis. Technologies for CTC capture, detection, and analysis will be evaluated based on these criteria, with a focus on current approaches, limitations and future directions. The paper provides a critical review for microchip developers as well as clinical investigators to build upon the existing progress towards the goal of designing CTC capture, detection, and analysis platforms.