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How is a water-soluble globular protein able to spontaneously cross a cellular membrane? It is commonly accepted that it undergoes significant structural rearrangements on the lipid-water interface, thus acquiring membrane binding and penetration ability. In this study molecular dynamics (MD) simulations have been used to explore large-scale conformational changes of the globular viscumin A chain in a complex environment – comprising urea and chloroform/methanol (CHCl3/MeOH) mixture. Being well-packed in aqueous solution, viscumin A undergoes global structural rearrangements in both organic media. In urea, the protein is “swelling” and gradually loses its long-distance contacts, thus resembling the “molten globule” state. In CHCl3/MeOH, viscumin A is in effect turned “inside out”. This is accompanied with strengthening of the secondary structure and surface exposure of hydrophobic epitopes originally buried inside the globule. Resulting solvent-adapted models were further subjected to Monte Carlo simulations with an implicit hydrophobic slab membrane. In contrast to only a few point surface contacts in water and two short regions with weak protein-lipid interactions in urea, MD-derived structures in CHCl3/MeOH reveal multiple determinants of membrane interaction. Consequently it is now possible to propose a specific pathway for the structural adaptation of viscumin A with respect to the cell membrane – a probable first step of its translocation into cytoplasmic targets.
A cancer cell line originating from human epithelial colorectal adenocarcinoma (Caco-2 cells) serves as a high capacity model for a preclinical screening of drugs. Recent need for incorporating barrier tissue into multi-organ chips calls for inclusion of Caco-2 cells into microperfused environment. This article describes a series of systems biology insights obtained from comparing Caco-2 models cells grown as conventional 2D layer and in a microfluidic chip. When basic electrical parameters of Caco-2 monolayers were evaluated using impedance spectrometry and MTT assays, no differences were noted. On the other hand, the microarray profiling of mRNAs and miRNAs revealed that grows on a microfluidic chip leads to the change in the production of specific miRNA, which regulate a set of genes for cell adhesion molecules (CAMs), and provide for more complete differentiation of Caco-2 monolayer. Moreover, the sets of miRNAs secreted at the apical surface of Caco-2 monolayers grown in conventional 2D culture and in microfluidic device differ. When integrated into a multi-tissue platform, Caco-2 cells may aid in generating insights into complex pathophysiological processes, not possible to dissect in conventional cultures.
Regulatory T cells (Treg cells), whose differentiation and function are controlled by transcription factor Foxp3, express the closely related family member Foxp1. Here we explored Foxp1 function in Treg cells. We found that a large number of Foxp3-bound genomic sites in Treg cells were occupied by Foxp1 in both Treg cells and conventional T cells (Tconvcells). In Treg cells, Foxp1 markedly increased Foxp3 binding to these sites. Foxp1 deficiency in Treg cells resulted in their impaired function and competitive fitness, associated with markedly reduced CD25 expression and interleukin-2 (IL-2) responsiveness, diminished CTLA-4 expression and increased SATB1 expression. The characteristic expression patterns of CD25, Foxp3 and CTLA-4 in Treg cells were fully or partially rescued by strong IL-2 signaling. Our studies suggest that Foxp1 serves an essential non-redundant function in Treg cells by enforcing Foxp3-mediated regulation of gene expression and enabling efficient IL-2 signaling in these cells.
The necessary stage of epithelial-mesenchymal transformation (EMT) is the loss of apical-basal polarization by cells, in the formation of which an important role is played by the interaction of cells with the basement membrane and its laminins. The effect of the transition of colorectal cancer cell lines from the polarized state (2D) to the absence of apical-basal polarization (3D) on gene expression of epithelial and mesenchymal markers was investigated, and the role of laminins 332 and 411 (LM-332 and LM-411, respectively) in this process. The three studied cell lines, HT-29, HCT-116, and RKO, were found to exhibit different sensitivity to changes in cultivation conditions (2D / 3D) and the presence of laminins. One reason for this may be a difference in the initial state of the cells. It was shown that in the initial, 2D state, the lines under investigation were at different stages of EMT. In the HT-29 line, the expression profile characteristic of epithelial cells prevailed, in RKO, for mesenchymal, and in HCT-116, for the intermediate state. The most sensitive to the effects was the cell line HCT-116. No critical rearrangement of the profile of EMT genes was observed either during the transition from 2D to 3D cultivation or when laminins were added. However, for both effects, changes in the expression of the SNAI1 and ZEB1 genes, which encode transcription factors regulating the process of EMT, were recorded; moreover, the expression of SNAI1 increased in response to treatment with laminin in all three cell lines.
Nonalcoholic fatty liver disease (NAFLD) is the most common liver pathology. Here we propose tissue-cooperative, homeostatic model of NAFLD. During early stages of NAFLD the intrahepatic production of miR-122 falls, while the secretion of miRNA-containing exosomes by adipose increases. Bloodstream carries exosome to the liver, where their miRNA cargo is released to regulate their intrahepatic targets. When the deterioration of adipose catches up with the failing hepatic parenchyma, the external supply of liver-supporting miRNAs gradually tapers off, leading to the fibrotic decompensation of the liver and an increase in hepatic carcinogenesis. This model may explain paradoxical observations of the disease-associated decrease in intrahepatic production of certain miRNAs with an increase in their levels in serum. Infusions of miR-122 and, possibly, some other miRNAs may be efficient for preventing NAFLD-associated hepatocellular carcinoma. The best candidates for exosome-wrapped miRNA producer are adipose tissue-derived mesenchymal stem cells (MSCs), known for their capacity to shed large amounts of exosomes into the media. Notably, MSC-derived exosomes with no specific loading are already tested in patients with liver fibrosis. Carrier exosomes may be co-manufactured along with their cargo. Exosome-delivered miRNA cocktails may augment functioning of human organs suffering from a variety of chronic diseases.
Non-alcoholic fatty liver disease (NAFLD) is the worldwide most common cause of chronic liver pathology, which prevalence strongly correlates with the increasing incidence of diabetes, obesity and metabolic syndrome in the general population. Simple steatosis, the earliest NAFLD stage, usually remains asymptomatic, and appropriate changes in the lifestyle, as well as the diet, can reverse the affected liver into the healthy state. The potential of simple steatosis to progress into severe fibrotic stages and to facilitate carcinogenesis necessitates timely NAFLD detection and risk stratification in community-based healthcare settings. Since their initial discovery a decade ago, extracellular circulating miRNAs have been found in all human biological fluids including blood and shown to hold great promises as non-invasive biomarkers. Normally, intracellular miRNAs participate in the regulation of gene expression, but once released by dying/dead cells they remain highly stable in the extracellular environment for prolonged periods. Therefore, circulating miRNA profiles can reflect the ongoing pathogenic processes in body's tissues and organs, and enable highly sensitive non-invasive diagnosis of multiple disorders. A non-urgent character of the NAFLD-related decision-making justifies the use of chronic liver diseases as an excellent test case for examining the practical utility of circulating miRNAs as biomarkers for longitudinal monitoring of human health. In this review, we summarize the state-of-the-art in the field of early diagnosis of NAFLD using circulating blood miRNAs, and stress the necessity of additional experimental validation of their diagnostic potential. We further emphasize on the potential diagnostics promises of other cell-free RNA species found in human biological fluids.
Duox2 belongs to the large family of NADPH-oxidase enzymes that are implicated in immune response, vasoregulation, hormone synthesis, cell growth and differentiation via the regulated synthesis of H2O2 and reactive oxygen species. We and others have shown that Duox2 and H2O2 are involved in platelet-derived growth factor (PDGF) induced migration of fibroblasts. Now, using the CRISPR/Cas9-mediated genome editing we demonstrate that the extreme C-terminal region of Duox2 is required for PDGF-stimulated activity of Duox2 and H2O2 production. We generated the fibroblast cells that stably co-express the wild-type or C-terminally modified Duox2 and fluorescent H2O2 probe Hyper. We found that nonsense substitution of the last 23 amino acids in Duox2 results in complete loss of PDGF stimulation of intracellular H2O2 and fibroblast migration, yet these mutations have no effects on the expression of Duox2 and other NADPH-oxidases in cells. These findings illustrate for the first time that the extreme C-terminus of Duox2 is required for the functional activity of the enzyme. Furthermore, the conservative nature of the C-terminus suggests its role for activity in other NADPH-oxidases
The endogenous opioid system (EOS) controls the processing of nociceptive stimuli and is a pharmacological target for opioids. Alterations in expression of the EOS genes under neuropathic pain condition may account for low efficacy of opioid drugs. We here examined whether EOS expression patterns are altered in the lumbar spinal cord of the rats with spinal nerve ligation (SNL) as a neuropathic pain model. Effects of the left- and right-side SNL on expression of EOS genes in the ipsi- and contralateral spinal domains were analysed. The SNL-induced changes were complex and different between the genes; between the dorsal and ventral spinal domains; and between the left and right sides of the spinal cord. Prodynorphin (Pdyn) expression was upregulated in the ipsilateral dorsal domains by each the left and right-side SNL, while changes in expression of μ-opioid receptor (Oprm1) and proenkephalin (Penk) genes were dependent on the SNL side. Changes in expression of the Pdyn and κ-opioid receptor (Oprk1) genes were coordinated between the ipsi- and contralateral sides. Withdrawal response thresholds, indicators of mechanical allodynia correlated negatively with Pdyn expression in the right ventral domain after right side SNL. These findings suggest multiple roles of the EOS gene products in spinal sensitization and changes in motor reflexes, which may differ between the left and right sides.
Most common drug development failures originate from either bioavailability problems, or unexpected toxic effects. The culprit is often the liver, which is responsible for biotransformation of a majority of xenobiotics. Liver may be modeled using "liver on a chip" devices, which may include established cell lines, primary human cells, and stem cell-derived hepatocyte-like cells. The choice of biological material along with its processing and maintenance greatly influence both the device performance and the resultant toxicity predictions. Impediments to the development of "liver on a chip" technology include the problems with standardization of cells, limitations imposed by culturing and the necessity to develop more complicated fluidic contours. Fortunately, recent breakthroughs in the development of cell-based reporters, including ones with fluorescent label, permits monitoring of the behavior of the cells embed into the "liver on a chip" devices. Finally, a set of computational approaches has been developed to model both particular toxic response and the homeostasis of human liver as a whole; these approaches pave a way to enhance the in silico stage of assessment for a potential toxicity.
The hypoxic response critically depends on the rapid stabilization of the Hypoxia-Inducible Factor (HIF). In normoxic conditions, HIF-prolyl hydroxylases mark α-subunits of HIF for degradation, while the hypoxia results in stabilization of HIF-α. Oxyquinoline derivatives suppress the activity of HIF-prolyl hydroxylases, leading to the stabilization of HIF. Here we show that the 24 hour incubation of the trophoblast model choriocarcinoma cells BeWo b30 line with the oxyquinoline derivative leads to a decrease in the transepithelial electrical resistance (TEER) of the cell monolayer, while the permeability of the monolayer for the FITC-dextran (70 kDa) remains unchanged. These observations indicate that the overall barrier function remain preserved, while the structure of intercellular tight junctions may undergo remodeling. Using Affymetrix Human Transcriptome Array 2.0, we show that the treatment with oxyquinoline derivative lead to a decrease in the expression of claudines 6 and 7 (CLDN6, CLDN7), occludin (OCLN), contact adhesion molecule 3 (JAM3) and angiotomin-like protein 1 (AMOTL1), which possibly explain the observed changes in TEER.
Hypoxia of trophoblast cells is an important regulating factor in the process of normal placenta development. However, the effect of hypoxia on the placenta in a number of pathological conditions, such as preeclampsia, leads to impaired cellular functions. A oxyquinoline derivative is capable of inhibiting HIF-prolyl hydroxylases, thereby stabilizing the transcription complex of HIF-1 and activating the cell response to hypoxia. BeWo b30 human choriocarcinoma cells are used to model trophoblast, which forms the basis for placenta barrier. Oxyquinoline effect leads both to an increased expression of a number of the genes that form the core response to hypoxia, and upregulated expression of NOS3, PDK1, and BNIP3 genes and downregulated expression of the PPARGC1B gene. This indicates the activation of mechanisms of metabolic cell reprogramming aimed at reducing oxygen consumption by reducing the number of mitochondria and switching from aerobic glucose metabolism to anaerobic. Possible applications of the obtained results is under discussion.