Monitoring protein denaturation using thermal conductivity probe

We propose a method for probing denaturation of proteins by measuring the thermal conductivity of the solution. We use the three-omega method with a microfabricated ac thermal sensor to measure the thermal conductivity of lysozyme, beta-lactoglobulin, and bovine serum albumin protein solutions over a range of temperature and pH. Results suggest that conformation transformation of the protein during denaturation changes the thermal network in protein solutions and thus changes the thermal conductivity for all the tested proteins. The proposed method of denaturation monitoring requires much simpler experimental setup than conventional methods such as differential scanning calorimetry and circular dichroism detection. We also demonstrate that the proposed analytical technique can detect the protein denaturation in real time. Consequently, it is expected to be useful in lab-on-a-chip (LoC) applications as the probe can be easily miniaturized for integration into LoC devices and allows real-time analysis. (C) 2012 Elsevier B.V. All rights reserved.X1177sciescopu

Development of a thermal sensor to probe cell viability and concentration in cell suspensions

This paper presents a novel biothermal sensor to probe cell viability and concentration of a cell suspension. The sensing technique exploits the thermophysical properties of the suspension, so no labeling of suspended cells is required. When the sensor is periodically heated, the amplitude and phase of the thermal signal are dependent on the thermal properties of the cell suspension, particularly the thermal conductivity k. We measured k of HeLa, hepatocyte, and NIH-3T3 J2 cell suspensions with various concentrations and viabilities. The results demonstrate that the k of a cell suspension has a strong correlation with its concentration and viability. Accordingly, k can be employed as an index of cell concentration and viability. Furthermore, without data processing to obtain k, the electric signal that reflects the thermal response of the sensor can be used as a tool to probe viability of a cell suspension in real time. The proposed thermal sensing technique offers label-free, non-invasive, long-term, and real-time means to probe the viability and concentration of cells in a suspension

Outer membrane vesicles derived from Escherichia coli up-regulate expression of endothelial cell adhesion molecules in vitro and in vivo.

Escherichia coli, as one of the gut microbiota, can evoke severe inflammatory diseases including peritonitis and sepsis. Gram-negative bacteria including E. coli constitutively release nano-sized outer membrane vesicles (OMVs). Although E. coli OMVs can induce the inflammatory responses without live bacteria, the effect of E. coli OMVs in vivo on endothelial cell function has not been previously elucidated. In this study, we show that bacteria-free OMVs increased the expression of endothelial intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule-1, and enhanced the leukocyte binding on human microvascular endothelial cells in vitro. Inhibition of NF-κB and TLR4 reduced the expression of cell adhesion molecules in vitro. OMVs given intraperitoneally to the mice induced ICAM-1 expression and neutrophil sequestration in the lung endothelium, and the effects were reduced in ICAM-1(-/-) and TLR4(-/-) mice. When compared to free lipopolysaccharide, OMVs were more potent in inducing both ICAM-1 expression as well as leukocyte adhesion in vitro, and ICAM-1 expression and neutrophil sequestration in the lungs in vivo. This study shows that OMVs potently up-regulate functional cell adhesion molecules via NF-κB- and TLR4-dependent pathways, and that OMVs are more potent than free lipopolysaccharide

Up-regulation of ICAM-1 expression and leukocyte adhesion on pulmonary endothelium by <i>E. coli</i> OMVs. (A-C)

<p>C57BL/6 wild-type mice were intraperitoneally administered with PBS or <i>E. coli</i> OMVs (15 µg in total protein/mouse; n  =  5). <b>A.</b> The number of neutrophils in BAL fluid after OMVs injection. <b>(B and C)</b> Six hours after the administration, the lungs were harvested. <b>B.</b> Immunohistochemistry with confocal microscopy of ICAM-1 (green), endothelial cell marker CD31 (red), and nuclei (blue) in the lungs (scale bars, 100 µm). White arrows indicate ICAM-1 positive endothelial cells. <b>C.</b> Hematoxylin and eosin staining of the lungs (scale bars, 100 µm). Black arrows indicate leukocytes on the pulmonary endothelium. <b>(D and E)</b> C57BL/6 wild-type and ICAM-1^-/- mice were intraperitoneally administered with PBS or <i>E. coli</i> OMVs (15 µg in total protein/mouse). Six hours after the administration, the lungs were harvested (n  =  3). <b>D.</b> Immunohistochemistry with confocal microscopy of a neutrophil marker NIMP-R14 (green) and nuclei (blue) in the lungs (scale bars, 50 µm). <b>E.</b> The number of neutrophils per field. *<i>P</i><0.05; **P<0.01; ***<i>P</i><0.001. Results are represented as means ± SEM.</p

Role of TLR4 in endothelial ICAM-1 expression and pulmonary neutrophil infiltration by <i>E. coli</i> OMVs

<p>. C57BL/6 wild-type and TLR4^-/- mice were intraperitoneally administered with PBS or <i>E. coli</i> OMVs (15 µg in total protein/mouse). Three hours after the administration, the lungs were harvested (n  =  3). <b>A.</b> Immunohistochemistry with confocal microscopy of ICAM-1 (green), endothelial cell marker CD31 (red), and nuclei (blue) in the lungs (scale bars, 50 µm). White arrows indicate ICAM-1 positive endothelial cells. <b>B.</b> The quantitative analysis of ICAM-1/CD31 co-localization. <b>C.</b> The number of neutrophils per field was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059276#pone-0059276-g003" target="_blank">Figure 3D and 3E</a>. **<i>P</i><0.01; ***<i>P</i><0.001. Results are represented as means ± SEM.</p

Role of LPS in inducing endothelial cell adhesion molecules by <i>E. coli</i> OMVs <i>in vitro</i>.

<p>HMVECs were treated as indicated in the figures for 12 hours in 5% FBS/EBM, and the expression of ICAM-1, E-selectin, and VCAM-1 was measured by Western blot of whole cell lysates (10 µg). <b>A.</b> The effect of various TLR agonists (HKLM (1×10⁷ cells/mL) for TLR2; ultrapure LPS from <i>E. coli</i> K-12 (100 ng/mL) for TLR4 (InvivoGen); flagellin (100 ng/mL) for TLR5) or <i>E. coli</i> OMVs (10 ng/mL in total protein). <b>B.</b> The effect of OMVs (10 ng/mL in total protein) derived from <i>E. coli</i> K-12 W3110 wild-type or <i>ΔmsbB</i> mutant (contain inactive LPS). <b>(C-E)</b> HMVECs were treated with <i>E. coli</i> OMVs (10 ng/mL in total protein), LPS (75 ng/mL) purified from the <i>E. coli</i> strain isolated from the peritoneal lavage fluid of cecal ligation and puncture-operated mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059276#pone.0059276-Park1" target="_blank">[28]</a>, or TNF-α (10 ng/mL). The effects of polymyxin B (1 µg/mL) and TLR4 antagonist (<i>R. sphaeroides</i> LPS, 10 µg/mL) are shown in <b>C</b> and <b>D</b>, respectively. <b>E.</b> The effect of absence or presence of FBS (5%) or CD14 (1 µg/mL) in EBM. The numbers under each blot indicate ratios calculated by dividing the densitometry values for ICAM-1, E-selectin, or VCAM-1 by those for actin.</p