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Here we describe the effects of a controlled, 30 min, high-intensity cycling test on blood rheology and the metabolic profiles of red blood cells (RBCs) and plasma from well-trained males. RBCs demonstrated decreased deformability and trended toward increased generation of microparticles after the test. Meanwhile, metabolomics and lipidomics highlighted oxidative stress and activation of membrane lipid remodeling mechanisms in order to cope with altered properties of circulation resulting from physical exertion during the cycling test. Of note, intermediates from coenzyme A (CoA) synthesis for conjugation to fatty acyl chains, in parallel with reversible conversion of carnitine and acylcarnitines, emerged as metabolites that significantly correlate with RBC deformability and the generation of microparticles during exercise. Taken together, we propose that RBC membrane remodeling and repair plays an active role in the physiologic response to exercise by altering RBC properties.

Aptamers are nucleic acid-based ligands identified through a process of molecular evolution named SELEX (Systematic Evolution of Ligands by Exponential enrichment). During the last 10-15 years, numerous aptamers have been developed specifically against targets present on or associated with the surface of human cells or infectious pathogens such as viruses, bacteria, fungi or parasites. Several of the aptamers have been described as potent probes, rivalling antibodies, for use in flow cytometry or microscopy. Some have also been used as drugs by inhibiting or activating functions of their targets in a manner similar to neutralizing or agonistic antibodies. Additionally, it is straightforward to conjugate aptamers to other agents without losing their affinity and they have successfully been used in vitro and in vivo to deliver drugs, siRNA, nanoparticles or contrast agents to target cells. Hence, aptamers identified against cell surface biomarkers represent a promising class of ligands. This review presents the different strategies of SELEX that have been developed to identify aptamers for cell surface-associated proteins as well as some of the methods that are used to study their binding on living cells.

Aptamer selection protocols, named cell-SELEX, have been developed to target trans-membrane proteins using whole living cells as target. This technique presents several advantages. (1) It does not necessitate the use of purified proteins. (2) Aptamers are selected against membrane proteins in their native conformation. (3) Cell-SELEX can be performed to identify aptamers against biomarkers differentially expressed between different cell lines without prior knowledge of the targets. (4) Aptamers identified by cell-SELEX can be further used to purify their targets and to identify new biomarkers. Here, we provide a protocol of cell-SELEX including the preparation of an oligonucleotide library, next-generation sequencing and radioactive binding assays. Furthermore, we also provide a protocol to purify and identify the target of these aptamers. These protocols could be useful for the discovery of lead therapeutic compounds and diagnostic cell-surface biomarkers.

BackgroundCell-SELEX is now widely used for the selection of aptamers against cell surface biomarkers. However, despite negative selection steps using mock cells, this method sometimes results in aptamers against undesirable targets that are expressed both on mock and targeted cells. Studying these junk aptamers might be useful for further applications than those originally envisaged.Methodology/Principal FindingsCell-SELEX was performed to identify aptamers against CHO-K1 cells expressing human Endothelin type B receptor (ETBR). CHO-K1 cells were used for negative selection of aptamers. Several aptamers were identified but no one could discriminate between both cell lines. We decided to study one of these aptamers, named ACE4, and we identified that it binds to the Annexin A2, a protein overexpressed in many cancers. Radioactive binding assays and flow cytometry demonstrated that the aptamer was able to bind several cancer cell lines from different origins, particularly the MCF-7 cells. Fluorescence microscopy revealed it could be completely internalized in cells in 2 hours. Finally, the tumor targeting of the aptamer was evaluated in vivo in nude mice xenograft with MCF-7 cells using fluorescence diffuse optical tomography (fDOT) imaging. Three hours after intravenous injection, the aptamer demonstrated a significantly higher uptake in the tumor compared to a scramble sequence.Conclusions/SignificanceAlthough aptamers could be selected during cell-SELEX against other targets than those initially intended, they represent a potential source of ligands for basic research, diagnoses and therapy. Here, studying such aptamers, we identify one with high affinity for Annexin A2 that could be a promising tool for biomedical application.

BACKGROUND: Endurance running events are known to cause inflammation and result in increased cytokine production. However, the effects of ultramarathons on cytokine profiles are not well characterized. OBJECTIVE: The aim of this study was to describe and compare the effects of a trail (40 km) race and an ultra-trail (171 km) race on leukocyte concentrations and cytokine profiles. METHODS: The study was conducted during the Ultra-Trail du Mont Blanc® ultra-marathon running event, and included 11 runners who completed the 40 km trail run and 12 runners who completed the 171 km ultra-trail. Blood samples were taken before and after the races. RESULTS: Leukocyte concentrations significantly increased after both races. Circulating levels of IL-6, IL-1β, MCP-1, and IFN-γ were significantly higher after the longer race compared to the shorter race. Furthermore, while both races resulted in significant increases in IL-6 and IL-8, only the longer race resulted in significant increases in MIP-1β, IL-7, IL-17a, and IL-4. CONCLUSIONS: These results illustrate that a 171 km ultra-trail race results in greater modulations in cytokine profiles than a traditional trail race.

Nucleic acid aptamers are promising ligands for analytical and preparative-scale affinity chromatography applications. However, a full industrial exploitation requires that aptamer-grafted chromatography media provide a number of high technical standards that remained largely untested. Ideally, they should exhibit relatively high binding capacity associated to a very high degree of specificity. In addition, they must be highly resistant to harsh cleaning/sanitization conditions, as well as to prolonged and repeated exposure to biological environment. Here, we present practical examples of aptamer affinity chromatography for the purification of three human therapeutic proteins from various sources: Factor VII, Factor H and Factor IX. In a single chromatographic step, three DNA aptamer ligands enabled the efficient purification of their target protein, with an unprecedented degree of selectivity (from 0.5% to 98% of purity in one step). Furthermore, these aptamers demonstrated a high stability under harsh sanitization conditions (100h soaking in 1M NaOH). These results pave the way toward a wider adoption of aptamer-based affinity ligands in the industrial-scale purification of not only plasma-derived proteins but also of any other protein in general.

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