Rhomboid family member 2 regulates cytoskeletal stress-associated Keratin 16.

Keratin 16 (K16) is a cytoskeletal scaffolding protein highly expressed at pressure-bearing sites of the mammalian footpad. It can be induced in hyperproliferative states such as wound healing, inflammation and cancer. Here we show that the inactive rhomboid protease RHBDF2 (iRHOM2) regulates thickening of the footpad epidermis through its interaction with K16. K16 expression is absent in the thinned footpads of irhom2-/- mice compared with irhom2+/+mice, due to reduced keratinocyte proliferation. Gain-of-function mutations in iRHOM2 underlie Tylosis with oesophageal cancer (TOC), characterized by palmoplantar thickening, upregulate K16 with robust downregulation of its type II keratin binding partner, K6. By orchestrating the remodelling and turnover of K16, and uncoupling it from K6, iRHOM2 regulates the epithelial response to physical stress. These findings contribute to our understanding of the molecular mechanisms underlying hyperproliferation of the palmoplantar epidermis in both physiological and disease states, and how this 'stress' keratin is regulated

Influence of gold nanoparticles on collagen fibril morphology quantified using transmission electron microscopy and image analysis

BACKGROUND: Development of implantable biosensors for disease detection is challenging because of poor biocompatibility of synthetic materials. A possible solution involves engineering interface materials that promote selfassembly and adhesion of autologous cells on sensor surfaces. Crosslinked type-I collagen is an acceptable material for developing engineered basement membranes. In this study, we used functionalized gold nanoparticles as the crosslinking agent. Functionalized nanoparticles provide sites for crosslinking collagen as well as sites to deliver signaling compounds that direct selfassembly and reduce inflammation. The goal of this study was to obtain a quantitative parameter to objectively determine the presence of crosslinks. METHODS: We analyzed TEM images of collagen fibrils by two methods: Run length analysis and topology analysis after medial axis transform. RESULTS: Run length analysis showed a significant reduction of the interfibril spaces in the presence of nanoparticles (change of 40%, P < 0.05), whereas the fibril thickness remained unchanged. In the topological network, the number of elements, number of branches and number of sides increased significantly in the presence of nanoparticles (P < 0.05). Other parameters, especially the number of loops showed only a minimal and nonsignificant change. We chose a ratiometric parameter of the number of branches normalized by the number of loops to achieve independence from gross fibril density. This parameter is lower by a factor of 2.8 in the presence of nanoparticles (P < 0.05). CONCLUSION: The numerical parameters presented herein allow not only to quantify fibril mesh complexity and crosslinking, but also to help quantitatively compare cell growth and adhesion on collagen matrices of different degree of crosslinking in further studies

Inhibition of gastric H,K-ATPase activity and gastric epithelial cell IL-8 secretion by the pyrrolizine derivative ML 3000

BACKGROUND: ML 3000 ([2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro-1H-pyrrolizine-5-yl]-acetic acid) is an inhibitor of both cyclooxygenase and 5-lipoxygenase in vitro, and shows promise as a novel non-steroidal anti-inflammatory drug (NSAID). Unlike conventional NSAIDs which are associated with gastric ulcerogenic effects, ML 3000 causes little or no damage to the gastric mucosa, even though it significantly depresses gastric prostaglandin synthesis. METHODS: As part of an effort to clarify mechanisms underlying the gastric sparing properties of ML 3000, we studied the effects of ML 3000 on H,K-ATPase activity in vitro, on acid accumulation in isolated gastric parietal cells, and on IL-8 secretion by gastric epithelial cells in culture. RESULTS: SCH28080-sensitive H,K-ATPase activity in highly-purified pig gastric microsomes was dose-dependently inhibited by ML 3000 (IC(50) = 16.4 μM). Inhibition was reversible, and insensitive to ML 3000 acidification in the pH range 2.0–8.0. In rabbit gastric parietal cells, ML 3000 dose-dependently inhibited histamine-stimulated acid accumulation (IC(50) = 40 μM) and forskolin-stimulated acid accumulation (IC(50) = 45 μM). Lastly, in human gastric adenocarcinoma (AGS) cells, ML 3000 dose-dependently inhibited both baseline and IL-1β-stimulated (20 ng/ml) IL-8 secretion with IC(50)s of 0.46 μM and 1.1 μM respectively. CONCLUSION: The data indicate that ML 3000 affects acid-secretory mechanisms downstream of cAMP mobilization induced by histamine H(2) receptor activation, that it directly inhibits H,K-ATPase specific activity, and that baseline gastric epithelial cell IL-8 secretory inhibition may be mediated by ML 3000 inhibition of 5-lipoxygenase activity. We conclude that these gastric function inhibitory data may underlie the gastric sparing properties of ML 3000

Equilibrium analysis in multi-echelon supply chain with multi-dimensional utilities of inertial players

In a supply chain, the importance of information elicitation from the supply chain players is vital to design supply chain network. The rationality and self-centredness of these players causes the information asymmetry in the supply chain and thus situation of conflict and non-participation of the players in the network design process. In such situations, it is required to analyse the supply chain players’ behaviour in order to explore potential for coordination through incentives. In this paper, a novel approach of social utility analysis is proposed to elicit the information for supply chain coordination among the supply chain players in a dyadic relationship – supplier and buyer. In principal, we consider a monopsony situation where buyer is a dominant player. With the objective of maximizing the social utility, efforts have been made to value behavioural issues in supply chain. On the other hand, the reluctance of player due to the information asymmetry is measured in the form of inertia – an offset to the supply chain profit. The suppliers’ behaviour is analysed with three distinct level of risk for two types of the product in procurement process. The useful insight from this paper is in supplier selection process where the reluctance of supplier offsets supply chain profit. The paper provides recommendations to supply chain managers for efficient decision-making ability in supplier selection process

Cholesterol Corrects Altered Conformation of MHC-II Protein in Leishmania donovani Infected Macrophages: Implication in Therapy

Previously we reported that Kala-azar patients show progressive decrease in serum cholesterol as a function of splenic parasite burden. Splenic macrophages (MΦ) of Leishmania donovani (LD) infected mice show decrease in membrane cholesterol, while LD infected macrophages (I-MΦ) show defective T cell stimulating ability that could be corrected by liposomal delivery of cholesterol. T helper cells recognize peptide antigen in the context of class II MHC molecule. It is known that the conformation of a large number of membrane proteins is dependent on membrane cholesterol. In this investigation we tried to understand the influence of decreased membrane cholesterol in I-MΦ on the conformation of MHC-II protein and peptide-MHC-II stability, and its bearing on the antigen specific T-cell activatio

Drug effects on the CVS in conscious rats: separating cardiac output into heart rate and stroke volume using PKPD modelling

Background and PurposePreviously, a systems pharmacology model was developed characterizing drug effects on the interrelationship between mean arterial pressure (MAP), cardiac output (CO) and total peripheral resistance (TPR). The present investigation aims to (i) extend the previously developed model by parsing CO into heart rate (HR) and stroke volume (SV) and (ii) evaluate if the mechanism of action (MoA) of new compounds can be elucidated using only HR and MAP measurements.Experimental ApproachCardiovascular effects of eight drugs with diverse MoAs (amiloride, amlodipine, atropine, enalapril, fasudil, hydrochlorothiazide, prazosin and propranolol) were characterized in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats following single administrations of a range of doses. Rats were instrumented with ascending aortic flow probes and aortic catheters/radiotransmitters for continuous recording of MAP, HR and CO throughout the experiments. Data were analysed in conjunction with independent information on the time course of the drug concentration following a mechanism-based pharmacokinetic-pharmacodynamic modelling approach.Key ResultsThe extended model, which quantified changes in TPR, HR and SV with negative feedback through MAP, adequately described the cardiovascular effects of the drugs while accounting for circadian variations and handling effects.Conclusions and ImplicationsA systems pharmacology model characterizing the interrelationship between MAP, CO, HR, SV and TPR was obtained in hypertensive and normotensive rats. This extended model can quantify dynamic changes in the CVS and elucidate the MoA for novel compounds, with one site of action, using only HR and MAP measurements. Whether the model can be applied for compounds with a more complex MoA remains to be establishe