Development of DNA aptamers for visualization of glial brain tumors and detection of circulating tumor cells.

Here, we present DNA aptamers capable of specific binding to glial tumor cells , , and for visualization diagnostics of central nervous system tumors. We selected the aptamers binding specifically to the postoperative human glial primary tumors and not to the healthy brain cells and meningioma, using a modified process of systematic evolution of ligands by exponential enrichment to cells; sequenced and analyzed ssDNA pools using bioinformatic tools and identified the best aptamers by their binding abilities; determined three-dimensional structures of lead aptamers (Gli-55 and Gli-233) with small-angle X-ray scattering and molecular modeling; isolated and identified molecular target proteins of the aptamers by mass spectrometry; the potential binding sites of Gli-233 to the target protein and the role of post-translational modifications were verified by molecular dynamics simulations. The anti-glioma aptamers Gli-233 and Gli-55 were used to detect circulating tumor cells in liquid biopsies.

Proteomics-Based Regression Model for Assessing the Development of Chronic Lymphocytic Leukemia.

The clinical course of chronic lymphocytic leukemia (CLL) is very ambiguous, showing either an indolent nature of the disease or having latent dangerous progression, which, if diagnosed, will require an urgent therapy. The prognosis of the course of the disease and the estimation of the time of therapy initiation are crucial for the selection of a successful treatment strategy. A reliable estimating index is needed to assign newly diagnosed CLL patients to the prognostic groups.

Aptamer-facilitated mass cytometry.

Mass cytometry is a novel cell-by-cell analysis technique, which uses elemental tags instead of fluorophores. Sample cells undergo rapid ionization in inductively coupled plasma and the ionized elemental tags are then analyzed by means of time-of-flight mass spectrometry. Benefits of the mass cytometry approach are in no need for compensation, the high number of detection channels (up to 100) and low background noise.

DNA Aptamers for the Characterization of Histological Structure of Lung Adenocarcinoma.

Nucleic acid aptamers are becoming popular as molecular probes for identification and imaging pathology and, at the same time, as a convenient platform for targeted therapy. Recent studies have shown that aptamers may be effectively used for tumor characterization and as commercially available monoclonal antibodies. Here we present three DNA aptamers binding to whole transformed lung cancer tissues, including tumor cells, connective tissues, and blood vessels.

Simultaneous analysis of enzyme structure and activity by kinetic capillary electrophoresis-MS.

To enable the detection of protein conformational isomers, their enzymatic activity and their inhibition in a single experiment, we developed a method based on kinetic capillary electrophoresis coupled on-line with UV detection and ion mobility mass spectrometry (CE-UV-IM-MS). Kinetic CE-UV separated protein conformers and monitored their interconversion dynamics in solution. Ion mobility mass spectrometry analyzed the conformer sizes, exact molecular weights, and structures of an enzyme and of its substrates, inhibitors and corresponding products.

Site-specific conjugation of the quencher on peptide's N-terminal for the synthesis of a targeted non-spreading activatable optical probe.

Optical imaging offers high sensitivity and portability at low cost. The design of 'smart' or 'activatable' probes can decrease the background noise and increase the specificity of the signal. By conjugating a fluorescent dye and a compatible quencher on each side of an enzyme's substrate, the signal remains in its 'off ' state until it reaches the area where a specific enzyme is expressed.

Carbohydrate-based ice recrystallization inhibitors increase infectivity and thermostability of viral vectors.

The inability of vaccines to retain sufficient thermostability has been an obstacle to global vaccination programs. To address this major limitation, we utilized carbohydrate-based ice recrystallization inhibitors (IRIs) to eliminate the cold chain and stabilize the potency of Vaccinia virus (VV), Vesicular Stomatitis virus (VSV) and Herpes virus-1 (HSV-1). The impact of these IRIs was tested on the potency of the viral vectors using a plaque forming unit assay following room temperature storage, cryopreservation with successive freeze-thaw cycles and lyophilization.

Conformational Dynamics of DNA G-Quadruplex in Solution Studied by Kinetic Capillary Electrophoresis Coupled On-line with Mass Spectrometry.

G-quadruplex-forming DNA/RNA sequences play an important role in the regulation of biological functions and development of new anticancer and anti-aging drugs. In this work, we couple on-line kinetic capillary electrophoresis with mass spectrometry (KCE-MS) to study conformational dynamics of DNA G-quadruplexes in solution. We show that peaks shift and its widening in KCE can be used for measuring rate and equilibrium constants for DNA-metal affinity interactions and G-quadruplex formation; and ion mobility mass spectrometry (IM-MS) provides information about relative sizes, absolute molecular masses and stoichiometry of DNA complexes.

Bioanalysis for biocatalysis: multiplexed capillary electrophoresis-mass spectrometry assay for aminotransferase substrate discovery and specificity profiling.

In this work, we introduce an entirely automated enzyme assay based on capillary electrophoresis coupled to electrospray ionization mass spectrometry termed MINISEP-MS for multiple interfluent nanoinjections-incubation-separation-enzyme profiling using mass spectrometry. MINISEP-MS requires only nanoliters of reagent solutions and uses the separation capillary as a microreactor, allowing multiple substrates to be assayed simultaneously. The method can be used to rapidly profile the substrate specificity of any enzyme and to measure steady-state kinetics in an automated fashion.

Assessment of energetic costs of AhR activation by β-naphthoflavone in rainbow trout (Oncorhynchus mykiss) hepatocytes using metabolic flux analysis.

Exposure to environmental contaminants such as activators of the aryl hydrocarbon receptor (AhR) leads to the induction of defense and detoxification mechanisms. While these mechanisms allow organisms to metabolize and excrete at least some of these environmental contaminants, it has been proposed that these mechanisms lead to significant energetic challenges. This study tests the hypothesis that activation of the AhR by the model agonist β-naphthoflavone (βNF) results in increased energetic costs in rainbow trout (Oncorhynchus mykiss) hepatocytes.

Real-time monitoring of protein conformational dynamics in solution using kinetic capillary electrophoresis.

Conformational analysis: Capillary electrophoresis (CE) allows for the rapid separation of slowly interconverting protein conformers. Kinetic analysis (k(open), k(closed), and K(d)) of electropherograms in the presence and absence of effector ligands allows the measurement of kinetic and thermodynamic constants associated with conformational changes and ligand binding.

Viral quantitative capillary electrophoresis for counting and quality control of RNA viruses.

The world of health care has witnessed an explosive boost to its capacity within the past few decades due to the introduction of viral therapeutics to its medicinal arsenal. As a result, a need for new methods of viral quantification has arisen to accommodate this rapid advancement in virology and associated requirements for efficiency, speed, and quality control. In this work, we apply viral quantitative capillary electrophoresis (viral qCE) to determine (i) the number of intact virus particles (ivp) in viral samples, (ii) the amount of DNA contamination, and (iii) the degree of viral degradation after sonication, vortexing, and freeze-thaw cycles.

Comparative study of three methods for affinity measurements: capillary electrophoresis coupled with UV detection and mass spectrometry, and direct infusion mass spectrometry.

We present affinity capillary electrophoresis and mass spectrometry (ACE-MS) as a comprehensive separation technique for label-free solution-based affinity analysis. The application of ACE-MS for measuring affinity constants between eight small molecule drugs [ibuprofen, s-flurbiprofen, diclofenac, phenylbutazone, naproxen, folic acid, resveratrol, and 4,4'-(propane-1,3-diyl) dibenzoic acid] and β-cyclodextrin is described. We couple on-line ACE with MS to combine the separation and kinetic capability of ACE together with the molecular weight and structural elucidation of MS in one system.

The collaborative role of molecular conformation and energetics in the binding of gas-phase non-covalent polymer/amine complexes.

A large series of similar non-covalent complexes were probed using ion mobility spectrometry, molecular mechanics/molecular dynamics (MM/MD), electrospray-tandem mass spectrometry (ESI-MS/MS) and RRKM theory in order to determine the effects of charge state and charge location upon the conformation, the 0 K activation energy (E(0)) and the entropy of activation (ΔS(‡)) of the dissociation of these complexes. The non-covalent complexes consisted of poly(methylmethacrylate) oligomers and singly and doubly charged diaminoalkanes of varying length. This allowed for control of the charge separation within the complexes, as well as the size of the complex.

Viral quantitative capillary electrophoresis for counting intact viruses.

The quantification of a virus plays an important role in vaccine development, clinical diagnostics, and environmental contamination assays. In all these cases, it is essential to calculate the concentration or number of intact virus particles (ivp) and estimate the degree of degradation and contamination of virus samples. In this work, we propose a cost-efficient, robust method for the quantification and characterization of intact viruses based on capillary zone electrophoresis.

Revealing equilibrium and rate constants of weak and fast noncovalent interactions.

Rate and equilibrium constants of weak noncovalent molecular interactions are extremely difficult to measure. Here, we introduced a homogeneous approach called equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM) to determine k(on), k(off), and K(d) of weak (K(d) > 1 μM) and fast kinetics (relaxation time, τ < 0.1 s) in quasi-equilibrium for multiple unlabeled ligands simultaneously in one microreactor.