Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

Background: Visceral hypersensitivity has been evidenced in patients with irritable bowel syndrome (IBS) but its mechanisms remain poorly elucidated. We investigated the spinal transmission of nociceptive signals in IBS patients by analysing the effects of rectal distensions on electromyographic recordings of the somatic nociceptive flexion (RIII) reflex, an objective index of spinal nociceptive processes. Methods: Fourteen IBS and 10 healthy volunteers were included in the study. Slow ramp (40 ml/min) and rapid phasic (900 ml/min, 10, 20, 30, and 40 mm Hg) rectal distensions were randomly performed while the RIII reflex evoked by electrical stimulation of the sural nerve at the ankle was continuously recorded from the ipsilateral biceps femoris. Results: In healthy volunteers, significant progressive inhibition of the RIII reflex was observed during slow ramp distension (61 (13)% of control values) while biphasic effects (facilitation and inhibition) were observed during rapid distensions. In contrast, in IBS patients, the RIII reflex was significantly facilitated during slow ramp distension (139 (15)% of control values) and inhibitions induced by rapid distensions were significantly reduced. Volumes of distension and rectal compliance were similar in both groups. Conclusions: Our results provide direct evidence that a hyperexcitability of spinal nociceptive processes is present in a large subgroup of IBS patients.

Oxygen is an important component of the cellular microenvironment, mediating cell survival, differentiation, and function. Oxygen supply is a limiting factor during culture of highly metabolic cells such as hepatocytes. Here we present a simple formulation of a fluorocarbon-based oxygen carrier embedded in collagen gel that increases oxygen concentration in culture 6-fold. Rat hepatocytes cultured on oxygen carrier-collagen showed a significant increase in viability and function. Cytochrome P450IA1 activity was increased by 140% in serum-free cultures and by 820% in serum-containing cultures. The significantly higher hepatocellular function on oxygen carrier-collagen matrix persisted and increased during long-term culture. Long-term albumin secretion was increased by 350% in serum-free cultures and by 166% in serum-containing culture. Long-term urea secretion was increased by 79% in serum-free cultures and by 76% in serum-containing cultures. We conclude that oxygen supply may limit hepatocyte function in vitro. This limitation can be overcome by addition of an oxygen carrier to the extracellular matrix. Culture of hepatocytes on oxygen-carrying matrix mimics the oxygen-rich environment of the liver and provides a simple method for enhanced long-term function.

BACKGROUND-Steatosis decreases survival of liver grafts after transplantation due to poorly understood mechanisms. We examined the effect of steatosis on the survival of liver grafts in a rat liver transplantation model and the viability of cultured rat hepatocytes after hypoxia and reoxygenation.

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The use of biomarkers to infer drug response in patients is being actively pursued, yet significant challenges with this approach, including the complicated interconnection of pathways, have limited its application. Direct empirical testing of tumor sensitivity would arguably provide a more reliable predictive value, although it has garnered little attention largely due to the technical difficulties associated with this approach. We hypothesize that the application of recently developed microtechnologies, coupled to more complex 3-dimensional cell cultures, could provide a model to address some of these issues. As a proof of concept, we developed a microfluidic device where spheroids of the serous epithelial ovarian cancer cell line TOV112D are entrapped and assayed for their chemoresponse to carboplatin and paclitaxel, two therapeutic agents routinely used for the treatment of ovarian cancer. In order to index the chemoresponse, we analyzed the spatiotemporal evolution of the mortality fraction, as judged by vital dyes and confocal microscopy, within spheroids subjected to different drug concentrations and treatment durations inside the microfluidic device. To reflect microenvironment effects, we tested the effect of exogenous extracellular matrix and serum supplementation during spheroid formation on their chemotherapeutic response. Spheroids displayed augmented chemoresistance in comparison to monolayer culturing. This resistance was further increased by the simultaneous presence of both extracellular matrix and high serum concentration during spheroid formation. Following exposure to chemotherapeutics, cell death profiles were not uniform throughout the spheroid. The highest cell death fraction was found at the center of the spheroid and the lowest at the periphery. Collectively, the results demonstrate the validity of the approach, and provide the basis for further investigation of chemotherapeutic responses in ovarian cancer using microfluidics technology. In the future, such microdevices could provide the framework to assay drug sensitivity in a timeframe suitable for clinical decision making.

Low-density lipoprotein (LDL) is an important carrier of plasma cholesterol and triglycerides whose concentration is regulated by the liver parenchymal cells. Abnormal LDL regulation is thought to cause atherosclerosis, while viral binding to LDL has been suggested to facilitate hepatitis C infection. Primary hepatocytes quickly lose the ability to clear LDL during in vitro culture. Here we show that the coculture of hepatocytes with liver sinusoidal endothelial cells (LSEC) significantly increases the ability of hepatocytes to uptake LDL in vitro. LDL uptake does not increase when hepatocytes are cocultured with other cell types such as fibroblasts or umbilical vein endothelial cells. We find that LSECs induce the hepatic expression of the LDL receptor and the epidermal growth factor receptor. In addition, while hepatocytes in single culture did not take up hepatitis C virus (HCV)-like particles, the hepatocytes cocultured with LSECs showed a high level of HCV-like particle uptake. We suggest that coculture with LSECs induces the emergence of a sinusoidal surface in primary hepatocytes conducive to the uptake of HCV-like particles. In conclusion, our findings describe a novel model of polarized hepatocytes in vitro that can be used for the study of LDL metabolism and hepatitis C infection. L ow-density lipoprotein (LDL) is a plasma-carried particle whose lipid component includes cholesterol and triglycerides. 1 LDL originates from very low-density lipoprotein (vLDL) synthesized by the liver with apoprotein B-100. The vLDL is converted to LDL by an endothelial lipase, which releases free fatty acids, increasing the density of the particle to form LDL. Excess LDL is then taken up by hepatocytes through LDL receptor (LDL-R)-mediated endocytosis. 1 Improper hepatic clearance of LDL results in elevated plasma levels of LDL which is a serious risk factor for the development of atherosclerosis. 2 One of the first steps in atherosclerosis is the passage of LDL into the vascular wall. Once trapped in the vessel wall, LDL can undergo oxidation. 3 Oxidized LDL is no longer a ligand for the LDL receptor. 4 Accumulation of oxidized LDL and LDL aggregates in the vessel wall stimulates an inflammatory response causing endothelial injury, macrophage recruitment, foam cell formation, and smooth muscle cell proliferation. 3 Current therapeutic intervention includes administration of statins, which block cholesterol production and increase expression of the LDL-R by the liver parenchymal cells, 2 removing LDL from circulation. 5 In addition, liver sinusoidal endothelial cells (LSECs) remove oxidized LDL from the circulation via the scavenger cell receptor. 5 LDL-and LDL-R-mediated pathways have also been shown to play a role in hepatitis C virus (HCV) infection. 6 The HCV surface receptors, glycoproteins E1 and E2, have been shown to associate with LDL and vLDL. 7 The virus particle itself was shown to associate with the

We have tried to understand the role of cellular tone (or internal tension mediated by actin filaments) and interactions with the microenvironment on cellular stiffness. For this purpose, we compared the apparent elasticity modulus of a 30-element tensegrity structure with cytoskeleton stiffness measured in subconfluent and confluent adherent cells by magnetocytometry, assessing the effect of changing cellular tone by treatment with cytochalasin D. Intracellular and extracellular mechanical interactions were analyzed on the basis of the non-dimensional relationships between the apparent elasticity modulus of the tensegrity structure normalized by Young's modulus of the elastic element versus: (i) element size, (ii) internal tension, and (iii) number of spatially fixed nodes, for small deformation conditions. Theoretical results and rigidity measurements in adherent cells consistently showed that higher cellular tone and stronger interdependencies with cellular environment tend to increase cytoskeleton stiffness. Visualization of the actin lattice before and after depolymerization by cytochalasin D tended to confirm the geometrical and mechanical assumptions supported by analysis of the present model.