Sensing gastric cancer via point-of-care sensor breath analyzer.

BACKGROUND: Detection of disease by means of volatile organic compounds from breath samples using sensors is an attractive approach to fast, noninvasive and inexpensive diagnostics. However, these techniques are still limited to applications within the laboratory settings. Here, we report on the development and use of a fast, portable, and IoT-connected point-of-care device (so-called, SniffPhone) to detect and classify gastric cancer to potentially provide new qualitative solutions for cancer screening. METHODS: A validation study of patients with gastric cancer, patients with high-risk precancerous gastric lesions, and controls was conducted with 2 SniffPhone devices. Linear discriminant analysis (LDA) was used as a classifying model of the sensing signals obatined from the examined groups. For the testing step, an additional device was added. The study group included 274 patients: 94 with gastric cancer, 67 who were in the high-risk group, and 113 controls. RESULTS: The results of the test set showed a clear discrimination between patients with gastric cancer and controls using the 2-device LDA model (area under the curve, 93.8%; sensitivity, 100%; specificity, 87.5%; overall accuracy, 91.1%), and acceptable results were also achieved for patients with high-risk lesions (the corresponding values for dysplasia were 84.9%, 45.2%, 87.5%, and 65.9%, respectively). The test-phase analysis showed lower accuracies, though still clinically useful. CONCLUSION: Our results demonstrate that a portable breath sensor device could be useful in point-of-care settings. It shows a promise for detection of gastric cancer as well as for other types of disease. LAY SUMMARY: A portable sensor-based breath analyzer for detection of gastric cancer can be used in point-of-care settings. The results are transferrable between devices via advanced IoT technology. Both the hardware and software of the reported breath analyzer could be easily modified to enable detection and monitirng of other disease states.

A C-terminal CXCL8 peptide based on chemokine-glycosaminoglycan interactions reduces neutrophil adhesion and migration during inflammation.

Leucocyte recruitment is critical during many acute and chronic inflammatory diseases. Chemokines are key mediators of leucocyte recruitment during the inflammatory response, by signalling through specific chemokine G-protein-coupled receptors (GPCRs). In addition, chemokines interact with cell-surface glycosaminoglycans (GAGs) to generate a chemotactic gradient. The chemokine interleukin-8/CXCL8, a prototypical neutrophil chemoattractant, is characterized by a long, highly positively charged GAG-binding C-terminal region, absent in most other chemokines. To examine whether the CXCL8 C-terminal peptide has a modulatory role in GAG binding during neutrophil recruitment, we synthesized the wild-type CXCL8 C-terminal [CXCL8 (54-72)] (Peptide 1), a peptide with a substitution of glutamic acid (E) 70 with lysine (K) (Peptide 2) to increase positive charge; and also, a scrambled sequence peptide (Peptide 3). Surface plasmon resonance showed that Peptide 1, corresponding to the core CXCL8 GAG-binding region, binds to GAG but Peptide 2 binding was detected at lower concentrations. In the absence of cellular GAG, the peptides did not affect CXCL8-induced calcium signalling or neutrophil chemotaxis along a diffusion gradient, suggesting no effect on GPCR binding. All peptides equally inhibited neutrophil adhesion to endothelial cells under physiological flow conditions. Peptide 2, with its greater positive charge and binding to polyanionic GAG, inhibited CXCL8-induced neutrophil transendothelial migration. Our studies suggest that the E70K CXCL8 peptide, may serve as a lead molecule for further development of therapeutic inhibitors of neutrophil-mediated inflammation based on modulation of chemokine-GAG binding.

Multifactorial determinants that govern nanoparticle uptake by human endothelial cells under flow.

Vascular endothelium is a potential target for therapeutic intervention in diverse pathological processes, including inflammation, atherosclerosis, and thrombosis. By virtue of their intravascular topography, endothelial cells are exposed to dynamically changing mechanical forces that are generated by blood flow. In the present study, we investigated the interactions of negatively charged 2.7 nm and 4.7 nm CdTe quantum dots and 50 nm silica particles with cultured endothelial cells under regulated shear stress (SS) conditions. Cultured cells within the engineered microfluidic channels were exposed to nanoparticles under static condition or under low, medium, and high SS rates (0.05, 0.1, and 0.5 Pa, respectively). Vascular inflammation and associated endothelial damage were simulated by treatment with tumor necrosis factor-α (TNF-α) or by compromising the cell membrane with the use of low Triton X-100 concentration. Our results demonstrate that SS is critical for nanoparticle uptake by endothelial cells. Maximal uptake was registered at the SS rate of 0.05 Pa. By contrast, endothelial exposure to mild detergents or TNF-α treatment had no significant effect on nanoparticle uptake. Atomic force microscopy demonstrated the increased formation of actin-based cytoskeletal structures, including stress fibers and membrane ruffles, which have been associated with nanoparticle endocytosis. In conclusion, the combinatorial effects of SS rates, vascular endothelial conditions, and nanoparticle physical and chemical properties must be taken into account for the successful design of nanoparticle-drug conjugates intended for parenteral delivery.

L-plastin regulates polarization and migration in chemokine-stimulated human T lymphocytes.

Chemokines such as SDF-1α play a crucial role in orchestrating T lymphocyte polarity and migration via polymerization and reorganization of the F-actin cytoskeleton, but the role of actin-associated proteins in this process is not well characterized. In this study, we have investigated a role for L-plastin, a leukocyte-specific F-actin-bundling protein, in SDF-1α-stimulated human T lymphocyte polarization and migration. We found that L-plastin colocalized with F-actin at the leading edge of SDF-1α-stimulated T lymphocytes and was also phosphorylated at Ser(5), a site that when phosphorylated regulates the ability of L-plastin to bundle F-actin. L-plastin phosphorylation was sensitive to pharmacological inhibitors of protein kinase C (PKC), and several PKC isoforms colocalized with L-plastin at the leading edge of SDF-1α-stimulated lymphocytes. However, PKC ζ, an established regulator of cell polarity, was the only isoform that regulated L-plastin phosphorylation. Knockdown of L-plastin expression with small interfering RNAs demonstrated that this protein regulated the localization of F-actin at the leading edge of chemokine-stimulated cells and was also required for polarization, lamellipodia formation, and chemotaxis. Knockdown of L-plastin expression also impaired the Rac1 activation cycle and Akt phosphorylation in response to SDF-1α stimulation. Furthermore, L-plastin also regulated SDF-1α-mediated lymphocyte migration on the integrin ligand ICAM-1 by influencing velocity and persistence, but in a manner that was independent of LFA-1 integrin activation or adhesion. This study, therefore, demonstrates an important role for L-plastin and the signaling pathways that regulate its phosphorylation in response to chemokines and adds L-plastin to a growing list of proteins implicated in T lymphocyte polarity and migration.

Montelukast inhibition of resting and GM-CSF-stimulated eosinophil adhesion to VCAM-1 under flow conditions appears independent of cysLT(1)R antagonism.

Montelukast (MLK) is a cysteinyl leukotriene receptor-1 (cysLT(1)R) antagonist with inhibitory effects on eosinophils, key proinflammatory cells in asthma. We assessed the effect of MLK on resting and GM-CSF-stimulated eosinophil adhesion to recombinant human (rh)VCAM-1 at different flow rates using our novel microflow system. At 1 or 2 dyn cm(-2), shear-stress unstimulated eosinophils tethered immediately to rhVCAM-1, "rolled" along part of the channel until they tethered, or rolled without tethering. At flow rates greater than 2 dyn cm(-2), adherent eosinophils began to be displaced from rhVCAM-1. MLK (10 nM and 100 nM) gave partial ( approximately 40%) but significant (P<0.05) inhibition of unstimulated eosinophil adhesion to rhVCAM-1 at 1 or 2 dyn cm(-2) shear stress. Once adhered, unstimulated eosinophils did not exhibit morphological changes, and GM-CSF-stimulated eosinophil adhesion under flow was characterized by greater cell flattening with significant (P<0.05) inhibition of adherent cell numbers by 100 nM MLK observed. This effect appeared specific for MLK, as the analog (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid, sodium salt, had no significant effect on eosinophil adhesion to VCAM-1. The possibility that LTC(4), released from unstimulated or GM-CSF-treated eosinophils, contributed to their adhesion to VCAM-1 was excluded as the LT biosynthesis inhibitor 3-[1-(p-Chlorobenzyl)-5-(isopropyl)-3-t-butylthioindol-2-yl]-2,2-dimethylpropanoic acid had no inhibitory effect, and exogenously added LTC(4) did not enhance eosinophil adhesion. In contrast, LTD(4) enhanced eosinophil adhesion to VCAM-1, an effect blocked by MLK (10 and 100 nM). These findings demonstrate that MLK-mediated inhibition of unstimulated and GM-CSF-stimulated eosinophil adhesion to VCAM-1 under shear-stress conditions appears independent of cysLT(1)R antagonism.

A multidisciplinary approach to the study of T cell migration.

Active T cell locomotion depends on efficient repeated cycles of integrin receptor/ligand interactions mediating cell adhesion and detachment, intracellular signaling cascades orchestrating posttranslation modifications of interacting proteins, dynamic reassembly of participating cytoskeletal elements, and structural support of associated scaffolding molecules. Using an integrated approach based on novel cutting edge technologies of live cell imaging, cell transfection, proteomics, and nanotechnology, we provide here a detailed characterisation of crucial mechanisms involved in LFA-1 integrin-mediated T cell migration. Polarization and phenotypic changes associated with LFA-1-triggered T cell locomotion is largely dependent on the intact functioning of the microtubule cytoskeleton. Experiments utilizing 4-D (3-D over time) confocal live imaging of T cells, microinjected with fully functional constructs encoding protein kinase C beta (PKC-beta) isoenzyme tagged with enhanced green fluorescent protein (GFP), elucidate that LFA-1-induced activation is associated with translocation of PKC-beta to sites associated with centrosomes and tubulin cytoskeleton in locomotory T lymphocytes. We also provide here a characterization of a novel microfluidics-based multichannel platform enabling detailed analysis of leukocyte adhesion and migration under regulated shear stress conditions. Using precision machined surfaces, we demonstrate that the substrate topography can influence the motile response of the two different T cell types in different ways, and this can be quantified in terms of specified motility parameters. Finally, using an original in situ immunoprecipitation method, in which LFA-1 antibodies are utilized to induce intracellular association of proteins in the cytoskeletal/signaling complex, we demonstrate that this complex includes a number of structural and signaling proteins, which have been identified by 2-D electrophoresis and MALDI-TOF protein sequencing.