Keratin 8/18 Regulation of Cell Stiffness-Extracellular Matrix Interplay through Modulation of Rho-Mediated Actin Cytoskeleton Dynamics

Cell mechanical activity generated from the interplay between the extracellular matrix (ECM) and the actin cytoskeleton is essential for the regulation of cell adhesion, spreading and migration during normal and cancer development. Keratins are the intermediate filament (IF) proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatic epithelial cell IFs are made solely of keratins 8/18 (K8/K18), hallmarks of all simple epithelia. Notably, our recent work on these epithelial cells has revealed a key regulatory function for K8/K18 IFs in adhesion/migration, through modulation of integrin interactions with ECM, actin adaptors and signaling molecules at focal adhesions. Here, using K8-knockdown rat H4 hepatoma cells and their K8/K18-containing counterparts seeded on fibronectin-coated substrata of different rigidities, we show that the K8/K18 IF-lacking cells lose their ability to spread and exhibit an altered actin fiber organization, upon seeding on a low-rigidity substratum. We also demonstrate a concomitant reduction in local cell stiffness at focal adhesions generated by fibronectin-coated microbeads attached to the dorsal cell surface. In addition, we find that this K8/K18 IF modulation of cell stiffness and actin fiber organization occurs through RhoA-ROCK signaling. Together, the results uncover a K8/K18 IF contribution to the cell stiffness-ECM rigidity interplay through a modulation of Rho-dependent actin organization and dynamics in simple epithelial cells

Prioritizing Risks and Uncertainties from Intentional Release of Selected Category A Pathogens

This paper synthesizes available information on five Category A pathogens (Bacillus anthracis, Yersinia pestis, Francisella tularensis, Variola major and Lassa) to develop quantitative guidelines for how environmental pathogen concentrations may be related to human health risk in an indoor environment. An integrated model of environmental transport and human health exposure to biological pathogens is constructed which 1) includes the effects of environmental attenuation, 2) considers fomite contact exposure as well as inhalational exposure, and 3) includes an uncertainty analysis to identify key input uncertainties, which may inform future research directions. The findings provide a framework for developing the many different environmental standards that are needed for making risk-informed response decisions, such as when prophylactic antibiotics should be distributed, and whether or not a contaminated area should be cleaned up. The approach is based on the assumption of uniform mixing in environmental compartments and is thus applicable to areas sufficiently removed in time and space from the initial release that mixing has produced relatively uniform concentrations. Results indicate that when pathogens are released into the air, risk from inhalation is the main component of the overall risk, while risk from ingestion (dermal contact for B. anthracis) is the main component of the overall risk when pathogens are present on surfaces. Concentrations sampled from untracked floor, walls and the filter of heating ventilation and air conditioning (HVAC) system are proposed as indicators of previous exposure risk, while samples taken from touched surfaces are proposed as indicators of future risk if the building is reoccupied. A Monte Carlo uncertainty analysis is conducted and input-output correlations used to identify important parameter uncertainties. An approach is proposed for integrating these quantitative assessments of parameter uncertainty with broader, qualitative considerations to identify future research priorities

Dynamic Modeling of Cell Migration and Spreading Behaviors on Fibronectin Coated Planar Substrates and Micropatterned Geometries

An integrative cell migration model incorporating focal adhesion (FA) dynamics, cytoskeleton and nucleus remodeling, actin motor activity, and lamellipodia protrusion is developed for predicting cell spreading and migration behaviors. This work is motivated by two experimental works: (1) cell migration on 2-D substrates under various fibronectin concentrations and (2) cell spreading on 2-D micropatterned geometries. These works suggest (1) cell migration speed takes a maximum at a particular ligand density (~1140 molecules/µm2) and (2) that strong traction forces at the corners of the patterns may exist due to combined effects exerted by actin stress fibers (SFs). The integrative model of this paper successfully reproduced these experimental results and indicates the mechanism of cell migration and spreading. In this paper, the mechanical structure of the cell is modeled as having two elastic membranes: an outer cell membrane and an inner nuclear membrane. The two elastic membranes are connected by SFs, which are extended from focal adhesions on the cortical surface to the nuclear membrane. In addition, the model also includes ventral SFs bridging two focal adhesions on the cell surface. The cell deforms and gains traction as transmembrane integrins distributed over the outer cell membrane bond to ligands on the ECM surface, activate SFs, and form focal adhesions. The relationship between the cell migration speed and fibronectin concentration agrees with existing experimental data for Chinese hamster ovary (CHO) cell migrations on fibronectin coated surfaces. In addition, the integrated model is validated by showing persistent high stress concentrations at sharp geometrically patterned edges. This model will be used as a predictive model to assist in design and data processing of upcoming microfluidic cell migration assays

Peroxiredoxin 3 Is a Redox-Dependent Target of Thiostrepton in Malignant Mesothelioma Cells

Thiostrepton (TS) is a thiazole antibiotic that inhibits expression of FOXM1, an oncogenic transcription factor required for cell cycle progression and resistance to oncogene-induced oxidative stress. The mechanism of action of TS is unclear and strategies that enhance TS activity will improve its therapeutic potential. Analysis of human tumor specimens showed FOXM1 is broadly expressed in malignant mesothelioma (MM), an intractable tumor associated with asbestos exposure. The mechanism of action of TS was investigated in a cell culture model of human MM. As for other tumor cell types, TS inhibited expression of FOXM1 in MM cells in a dose-dependent manner. Suppression of FOXM1 expression and coincidental activation of ERK1/2 by TS were abrogated by pre-incubation of cells with the antioxidant N-acetyl-L-cysteine (NAC), indicating its mechanism of action in MM cells is redox-dependent. Examination of the mitochondrial thioredoxin reductase 2 (TR2)-thioredoxin 2 (TRX2)-peroxiredoxin 3 (PRX3) antioxidant network revealed that TS modifies the electrophoretic mobility of PRX3. Incubation of recombinant human PRX3 with TS in vitro also resulted in PRX3 with altered electrophoretic mobility. The cellular and recombinant species of modified PRX3 were resistant to dithiothreitol and SDS and suppressed by NAC, indicating that TS covalently adducts cysteine residues in PRX3. Reduction of endogenous mitochondrial TRX2 levels by the cationic triphenylmethane gentian violet (GV) promoted modification of PRX3 by TS and significantly enhanced its cytotoxic activity. Our results indicate TS covalently adducts PRX3, thereby disabling a major mitochondrial antioxidant network that counters chronic mitochondrial oxidative stress. Redox-active compounds like GV that modify the TR2/TRX2 network may significantly enhance the efficacy of TS, thereby providing a combinatorial approach for exploiting redox-dependent perturbations in mitochondrial function as a therapeutic approach in mesothelioma

Multinationality and performance literature: a critical review and future research agenda

The literature on the relationship between the degree of multinationality (M) and performance (P) in the context of multinational enterprises (MNEs) has attracted a large volume of research in the past 50 years. Yet, the theoretical foundations and the empirical conclusions concerning the nature of M–P relationship vary greatly, thus call for a critical review and assessment. We examine 135 articles in 39 leading scholarly journals and classic books published during the period 1960–2015. We use an inductive approach and a qualitative content analysis methodology for our comprehensive and critical review of the literature. We incorporate international business, finance, and accounting perspectives in our analysis. We review the conceptualization and measurement of M, P, the findings on M–P relationships, methodologies, and geographic focus. We identify six key inconsistencies in the existing research, which cause ambiguity in the relevant findings. We make eight recommendations for future research to address these inconsistencies. Thus, our study contributes to the central debate in this research field