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Chemokines are chemotactic cytokines comprised of 70-100 amino acids. The chemokines CXCL12 and CCL5 are the endogenous ligands of the CXCR4 and CCR5 G protein-coupled receptors that are also HIV co-receptors. Biochemical, structural and functional studies of receptors are ligand-consuming and the cost of commercial chemokines hinders their use in such studies. Here, we describe methods for the expression, refolding, purification, and functional characterization of CXCL12 and CCL5 constructs incorporating C-terminal epitope tags. The model tags used were hexahistidines and Strep-Tag for affinity purification, and the double lanthanoid binding tag for fluorescence imaging and crystal structure resolution. The ability of modified and purified chemokines to bind and activate CXCR4 and CCR5 receptors was tested in Xenopus oocytes expressing the receptors, together with a Kir3 G-protein activated K(+) channel that served as a reporter of receptor activation. Results demonstrate that tags greatly influence the biochemical properties of the recombinant chemokines. Besides, despite the absence of any evidence for CXCL12 or CCL5 C-terminus involvement in receptor binding and activation, we demonstrated unpredictable effects of tag insertion on the ligand apparent affinity and efficacy or on the ligand dissociation. These tagged chemokines should constitute useful tools for the selective purification of properly-folded chemokines receptors and the study of their native quaternary structures.

BackgroundDiabetic peripheral neuropathy (DPN) is a common complication of diabetes. Proactive treatment options remain limited, which is exacerbated by a lack of sensitive and convenient diagnostics, especially early in disease progression or specifically to assess small fiber neuropathy (SFN), the loss of distal small diameter axons that innervate tissues and organs.MethodsWe designed, fabricated, tested, and validated a first-of-its-kind medical diagnostic device for the functional assessment of transdermal small fiber nerve activity. This device, the Detecting Early Neuropathy (DEN), is an electrically conductive needle array designed to record nerve electrical activity in the skin and subdermal tissues, as a feature of a broader theragnostic platform.ResultsDEN recordings were validated across a time course of diet-induced PN in mice, using statistical and computational analyses and compared to other SFN measures. Based on these preclinical mouse data, the device design was adapted to obtain recordings in human with a flexible printed circuit board to mold to the leg or other skin regions. The DEN successfully recorded various types of neural activity in mouse and human, with or without stimulation, including validated action potentials and electromyography signals.ConclusionNew functional diagnostic tools like DEN offer a promising outlook for patients needing an earlier or more sensitive diagnosis of DPN/SFN, to allow for earlier and more effective treatment options, especially as more become available in the clinic in future years.