Electrochemical aptasensor for human osteopontin detection using a DNA aptamer selected by SELEX

A DNA aptamer with affinity and specificity for human osteopontin (OPN), a potential breast cancer biomarker, was selected using the SELEX process, considering its homology rate and the stability of its secondary structures. This aptamer exhibited a satisfactory affinity towards OPN, showing dissociation constants lower than 2.5 nM. It was further used to develop a simple, label-free electrochemical aptasensor against OPN. The aptasensor showed good sensitivity towards OPN in standard solutions, being the square wave voltammetry (SWV), compared to the cyclic voltammetry, the most sensitive technique with detection and quantification limits of 1.4 ± 0.4 nM and 4.2 ± 1.1 nM, respectively. It showed good reproducibility and acceptable selectivity, exhibiting low signal interferences from other proteins, as thrombin, with 2.610 times lower current signals-off than for OPN. The aptasensor also successfully detected OPN in spiked synthetic human plasma. Using SWV, detection and quantification limits (1.3 ± 0.1 and 3.9 ± 0.4 nM) within the OPN plasma levels reported for patients with breast cancer (0.44.5 nM) or with metastatic or recurrent breast cancer (0.98.4 nM) were found. Moreover, preliminary assays, using a sample of human plasma, showed that the aptasensor and the standard ELISA method quantified similar OPN levels (2.2 ± 0.7 and 1.7 ± 0.1 nM, respectively). Thus, our aptasensor coupled with SWV represents a promising alternative for the detection of relevant breast cancer biomarkers.The authors acknowledge the financial support from the Strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), and from project BioTecNorte (project number NORTE-01-0145-FEDER-000004). This work was also financially supported by Project POCI-01–0145-FEDER-006984 – Associate Laboratory LSRE-LCM and by Project UID/QUI/00616/2013 – CQ-VR both funded by FEDER - Fundo Europeu de Desenvolvimento Regional through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) – and by national funds through FCT - Fundação para a Ciência e a Tecnologia, Portugal. S. Meirinho also acknowledges the research grant provided by Project UID/EQU/50020/2013.info:eu-repo/semantics/publishedVersio

Activation of tumor suppressor protein PP2A inhibits KRAS-driven tumor growth

Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A Aα scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins

Nucleic acid-based fluorescent probes and their analytical potential

It is well known that nucleic acids play an essential role in living organisms because they store and transmit genetic information and use that information to direct the synthesis of proteins. However, less is known about the ability of nucleic acids to bind specific ligands and the application of oligonucleotides as molecular probes or biosensors. Oligonucleotide probes are single-stranded nucleic acid fragments that can be tailored to have high specificity and affinity for different targets including nucleic acids, proteins, small molecules, and ions. One can divide oligonucleotide-based probes into two main categories: hybridization probes that are based on the formation of complementary base-pairs, and aptamer probes that exploit selective recognition of nonnucleic acid analytes and may be compared with immunosensors. Design and construction of hybridization and aptamer probes are similar. Typically, oligonucleotide (DNA, RNA) with predefined base sequence and length is modified by covalent attachment of reporter groups (one or more fluorophores in fluorescence-based probes). The fluorescent labels act as transducers that transform biorecognition (hybridization, ligand binding) into a fluorescence signal. Fluorescent labels have several advantages, for example high sensitivity and multiple transduction approaches (fluorescence quenching or enhancement, fluorescence anisotropy, fluorescence lifetime, fluorescence resonance energy transfer (FRET), and excimer-monomer light switching). These multiple signaling options combined with the design flexibility of the recognition element (DNA, RNA, PNA, LNA) and various labeling strategies contribute to development of numerous selective and sensitive bioassays. This review covers fundamentals of the design and engineering of oligonucleotide probes, describes typical construction approaches, and discusses examples of probes used both in hybridization studies and in aptamer-based assays

Redox Proteomics in Selected Neurodegenerative Disorders: From Its Infancy to Future Applications

Several studies demonstrated that oxidative damage is a characteristic feature of many neurodegenerative diseases. The accumulation of oxidatively modified proteins may disrupt cellular functions by affecting protein expression, protein turnover, cell signaling, and induction of apoptosis and necrosis, suggesting that protein oxidation could have both physiological and pathological significance. For nearly two decades, our laboratory focused particular attention on studying oxidative damage of proteins and how their chemical modifications induced by reactive oxygen species/reactive nitrogen species correlate with pathology, biochemical alterations, and clinical presentations of Alzheimer's disease. This comprehensive article outlines basic knowledge of oxidative modification of proteins and lipids, followed by the principles of redox proteomics analysis, which also involve recent advances of mass spectrometry technology, and its application to selected age-related neurodegenerative diseases. Redox proteomics results obtained in different diseases and animal models thereof may provide new insights into the main mechanisms involved in the pathogenesis and progression of oxidative-stress-related neurodegenerative disorders. Redox proteomics can be considered a multifaceted approach that has the potential to provide insights into the molecular mechanisms of a disease, to find disease markers, as well as to identify potential targets for drug therapy. Considering the importance of a better understanding of the cause/effect of protein dysfunction in the pathogenesis and progression of neurodegenerative disorders, this article provides an overview of the intrinsic power of the redox proteomics approach together with the most significant results obtained by our laboratory and others during almost 10 years of research on neurodegenerative disorders since we initiated the field of redox proteomics. Antioxid. Redox Signal. 17, 1610-1655