Scanning Probe Microscopy Techniques for Studying the Cell Glycocalyx.

The glycocalyx is a brush-like layer that covers the surfaces of the membranes of most cell types. It consists of a mixture of carbohydrates, mainly glycoproteins and proteoglycans. Due to its structure and sensitivity to environmental conditions, it represents a complicated object to investigate. Here, we review studies of the glycocalyx conducted using scanning probe microscopy approaches. This includes imaging techniques as well as the measurement of nanomechanical properties. The nanomechanics of the glycocalyx is particularly important since it is widely present on the surfaces of mechanosensitive cells such as endothelial cells. An overview of problems with the interpretation of indirect data via the use of analytical models is presented. Special insight is given into changes in glycocalyx properties during pathological processes. The biological background and alternative research methods are briefly covered.

Protein Corona Attenuates the Targeting of Antitumor Sialyl Lewis X-Decorated Liposomes to Vascular Endothelial Cells under Flow Conditions.

Previously, we showed in the human umbilical vein endothelial cells (HUVECs) model that a liposome formulation of melphalan lipophilic prodrug (MlphDG) decorated with selectin ligand tetrasaccharide Sialyl Lewis X (SiaLeX) undergoes specific uptake by activated cells and in an in vivo tumor model causes a severe antivascular effect. Here, we cultured HUVECs in a microfluidic chip and then applied the liposome formulations to study their interactions with the cells in situ under hydrodynamic conditions close to capillary blood flow using confocal fluorescent microscopy. The incorporation of 5 to 10% SiaLeX conjugate in the bilayer of MlphDG liposomes increased their consumption exclusively by activated endotheliocytes. The increase of serum concentration from 20 to 100% in the flow resulted in lower liposome uptake by the cells. To elucidate the possible roles of plasma proteins in the liposome-cell interactions, liposome protein coronas were isolated and analyzed by shotgun proteomics and immunoblotting of selected proteins. Proteomic analysis showed that a gradual increase in SiaLeX content correlated with the overall enrichment of the liposome-associated proteins with several apolipoproteins, including the most positively charged one, ApoC1, and serum amyloid A4, associated with inflammation, on the one hand, and a decrease in the content of bound immunoglobulins, on the other. The article discusses the potential interference of the proteins in the binding of liposomes to selectins of endothelial cells.

Determination of glutathione in blood via capillary electrophoresis with pH-mediated stacking.

A new approach has been developed for the direct determination of reduced (glutathione [GSH]) and oxidized (glutathione disulfide [GSSG]) GSH in whole blood by means of capillary electrophoresis. Its features include GSH-stabilizing sample preparation, the use of an internal standard, and pH-mediated stacking. Blood stabilized with acid citrate and K3 EDTA was treated with acetonitrile with N-ethylmaleimide, and then the analytes were extracted with diethyl ether. The total analysis time was 8 min using a 50-µm (i.d.) by 32.5-cm (eff. length) silica capillary. The background electrolyte was 0.075-M citrate Na pH 5.8 with 200-µM cetyltrimethylammonium bromide and 5-µM sodium dodecyl sulfate, and the separation voltage was -14 kV. The quantification limit (S/N = 15) of the method was 1.5 µM for GSSG. The accuracy levels of GSH and GSSG analysis were 104% and 103%, respectively, and between-run precision levels were 2.6% and 3.2%, respectively. Analysis of blood samples from healthy volunteers (N = 24) showed that the levels of GSH and GSSG and the GSH/GSSG ratio in the whole blood were 1.05 ± 0.14 mM, 3.9 ± 1.25 µM, and 256 ± 94, respectively. Thus, the presented approach can be used in clinical and laboratory practice.

Dysregulation of lncRNA-miRNA-mRNA Interactome as a Marker of Metastatic Process in Ovarian Cancer.

Ovarian cancer (OC) is one of the most common types of cancer among malignancies of the female reproductive system. This pathology is asymptomatic until advanced stages and has a poor prognosis. Our study aimed to search for lncRNA-miRNA-mRNA competing triplets that promote ovarian tumorigenesis. For this purpose, we analyzed tumor samples from the TCGA database and verified the results experimentally in a set of 46 paired samples of tumor and matched histologically unchanged ovarian tissues from OC patients. The list of RNAs selected in silico for experimental studies included 13 mRNAs, 10 lncRNAs, and 5 miRNAs related to epithelial-mesenchymal transition and angiogenesis. We evaluated the expression of these RNAs by qRT-PCR and assessed the correlation between levels of miRNAs, mRNAs, and lncRNAs. Sixteen significant triplets were revealed, in some of which, e.g., OIP5-AS1-miR-203a-c-MET and OIP5-AS1-miR-203a-ZEB2, both lncRNA and mRNA had sites for miR-203a direct binding. Transfection of the OVCAR-3 and SKOV-3 cell lines with the miR-203a mimic was used to confirm the novel links of miR-203a with ZEB2 and c-MET in OC. These connections suggest that the interactomes have the potential for diagnostics of metastasis at early onset.

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity.

Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.